Clinical, Forensic and Pharmaceutical Applications

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1 Clinical, Forensic and Pharmaceutical Applications

2 Page 4 Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS Page 11 Determination of 9 -tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation Page 17 Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation Page 23 Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS Page 29 Simultaneous screening and quantitation of amphetamines in urine by on-line SPE-LC/MS method Page 36 Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin Page 42 Development and validation of direct analysis method for screening and quantitation of amphetamines in urine by LC/MS/MS Page 48 Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS Page 54 Application of a sensitive liquid chromatographytandem mass spectrometric method to pharmacokinetic study of telbivudine in humans Page 60 Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry Page 66 Highly sensitive quantitative analysis of felodipine and hydrochlorothiazide from plasma using LC/MS/MS Page 73 Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS Page 80 Development of 2D-LC/MS/MS method for quantitative analysis of 1a,25-Dihydroxylvitamin D3 in human serum Page 86 Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer Page 93 A rapid and reproducible Immuno-MS platform from sample collection to quantitation of IgG Page 99 Simultaneous determinations of 20 kinds of common drugs and pesticides in human blood by GPC-GC-MS/MS Page 103 Low level quantitation of loratadine from plasma using LC/MS/MS

3 P-CN1452E Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS ASMS 2014 ThP 672 Miho Kawashima 1, Satohiro Masuda 2, Ikuko Yano 2, Kazuo Matsubara 2, Kiyomi Arakawa 3, Qiang Li 3, Yoshihiro Hayakawa 3 1 Shimadzu Corporation, Tokyo, JAPAN, 2 Kyoto University Hospital, Kyoto, JAPAN, 3 Shimadzu Corporation, Kyoto, JAPAN

4 Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS Introduction Method development for therapeutic drug monitoring (TDM) is indispensable for managing drug dosage based on the drug concentration in blood in order to conduct a rational and efficient drug therapy. Liquid chromatography coupled with tandem quadrupole mass spectrometry is increasingly used in TDM because it can perform selective and sensitive analysis by simple sample pretreatment. The UHPLC method scouting system coupled to tandem quadrupole mass spectrometer used in this study can dramatically shorten the total time for optimization of analytical conditions because this system can make enormous combinatorial analysis methods and run batch program automatically. In this study, we developed a high-speed and sensitive method for measurement of seventeen antiepileptics in plasma by UHPLC coupled with tandem quadrupole mass spectrometer. N NH 2 Carbamazepine N NH 2 Carbamazepine- 10,11-epoxide - N + Cl H N N Clonazepam Cl N N CH 3 Diazepam H 3 C NH CH 3 Ethomuximide H 2 N Felbamate NH 2 N H 2 H Cl Cl N N NH 2 N NH 2 N CH 3 NH 2 N + - H N N H 3 C H N N H HN NH Gabapentin Lamotrigine Levetiracetam Nitrazepam Phenobarbial Phenytoin C H 3 H N NH H 3 C S CH 3 S N H CH 3 H 3 C S H 2 N CH 3 CH 3 H 2 C NH 2 H N CH S 3 Primidone Tiagabine Topiramate Vigabatrin Zonisamide Figure 1 Antiepileptic drugs used in this assay Experimental Instruments UHPLC based method scouting system (Nexera X2 Method Scouting System, Shimadzu Corporation, Japan) is configured by Nexera X2 UHPLC modules. For the detection, tandem quadrupole mass spectrometer (LCMS-8050, Shimadzu Corporation, Japan) was used. The system can be operated at a maximum pressure of 130 MPa, and it enables to automatically select up to 96 unique combinations of eight different mobile phases and six different columns. A dedicated software was newly developed to control the system (Method Scouting Solution, Shimadzu Corporation, Japan), which provides a graphical aid to configure the different type of columns and mobile phases. The software is integrated into the LC/MS/MS workstation (LabSolutions, Shimadzu Corporation, Japan) so that selected conditions are seamlessly translated into method files and registered to a batch queue, ready for analysis instantly. 2

5 Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS Figure 2 Nexera Method Scoutuing System and LCMS-8050 triple quadrupole mass spectrometer Calibration standards and QC samples The main standard mixture was prepared in methanol from individual stock solutions. The calibration standards were prepared by diluting the standard mixture with methanol. QC sample was prepared by adding 4 volume of acetonitrile to 1 volume of control plasma, thereby precipitating proteins, and subsequently adding the standard mixture to the supernatant to contain plasma concentration equivalents stated in Table 4. The QC samples were further diluted 100 times (10 μl sample added to 990μL methanol) before injection. Next step of preparation procedure was divided into three groups by the intensity of each compound. For ethomuximide, phenobarbial and phenytoin, the supernatant was used for the LC/MS/MS analysis without further dilution. For zonisamide, 10 μl supernatant was further diluted with 990 μl methanol. For others, 100 μl supernatant was further diluted with 900 μl methanol. The diluted solutions were used for the LC/MS/MS analysis. Result MRM condition optimization The MS condition optimization was performed by flow injection analysis (FIA) of ESI positive and negative ionization mode, and the compound dependent parameters such as CID and pre-bias voltage were adjusted using automatic MRM optimization function. The transition that gave highest intensity was used for quantification. The MRM transitions used in this assay are listed in Table 1. 3

6 Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS Table 1 Compounds, Ionization polarity and MRM transition Compound Retaintion (min) Polarity Precursor m/z Product m/z Carbamazepine Carbamazepine-10,11-epoxide Clonazepam Diazepam Ethomuximide Felbamate Gabapentin Lamotrigine Levetiracetam Nitrazepam Phenobarbial Phenytoin Primidone Tiagabine Topiramate Vigabatrin Zonisamide UHPLC condition optimization 36 analytical conditions, comprising combinations of 9 mobile phase and 4 columns, were automatically investigated using Method Scouting System. Schematic representation of scouting system was shown in Figure 3. From the result of scouting, the combination of 10 mm ammonium acetate water and methanol for mobile phase and Inertsil-DS4 for separation column were selected. Using this combination of mobile phase and column, the gradient condition was further optimized. The final analytical condition was shown in Table 2. Kinetex XB-C18 (Phenomenex) 2.1 x 50 mm Pump A Kinetex PFP (Phenomenex) 2.1 x 50 mm InertsilDS-4 (GL Science) 2.1 x 50 mm Discovery HS F5-5 (SPELC) 2.1 x 50 mm LPGE Unit Auto Sampler LCMS-8050 Column ven Pump B (A) (B) 1 10mM Ammonium Acetate 2 10mM Ammonium Formate 3 0.1%FA - 10mM Ammonium Acetate 1 Methanol 2 Acetonitrile 3 Methanol/Acetonitrile=1/1 Figure. 3 Schematic representation and features of the Nexera Method Scouting System. 4

7 Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS Table.2 UHPLC analytical conditions Column Mobile phase Binary gradient Flow Rate Injection vol. Column Temp. : Inertsil DS-4 (50 mml. x 2.1mmi.d., 2um) : A) 10mM Ammonium Acetate B) Methanol : B conc. 3% (0.65 min) 40% (0 min) 85% (5.00 min) 100% ( min) 3% ( min) : 0.4 ml/min : 1 μl : 40 deg. C Precision, accuracy and linearity of AEDs Figure 4 shows MRM chromatograms of the 17 AEDs. It took only 10 minutes per one UHPLC/MS/MS analysis, including column rinsing. Vigabatrin >71.15(+) Felbamate >117.20(+) Carbamazepine >194.20(+) min Gabapentin >154.25(+) min Lamotrigine >215(+) min Nitrazepam >236.20(+) min Levetiracetam >126.15(+) min Phenobarbial 230>42.05(-) min Clonazepam >269.55(+) min Ethomuximide 140>42.00(-) min Topiramate >78.00(-) min Tiagabine >111.15(+) min Zonisamide >132.10(+) min Carbamazepine-10,11-epoxide >180.15(+) min Diazepam > min Primidone >162.15(+) min Phenytoin 250>208.20(-) min min min Figure. 4 Chromatogram of 17 AEDs calibration standards 5

8 Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS Table 3 illustrates linearity of 17 AEDs and Table 4 illustrates accuracy and precision of the QC samples at three concentration levels. Determination coefficient (r 2 ) of all calibration curves was larger than 0.995, and the precision and accuracy were within +/- 15%. Excellent linearity, accuracy and precision for all 17 AEDs were obtained at only 1 μl injection volume. Table.3 Linearity of 17 AEDs QC sample Compound Linarity (ng/ml) r 2 Carbamazepine Carbamazepine-10,11-epoxide Clonazepam Diazepam Ethomuximide Felbamate Gabapentin Lamotrigine Levetiracetam Nitrazepam Phenobarbial Phenytoin Primidone Tiagabine Topiramate Vigabatrin Zonisamide

9 Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS Table.4 Accuracy and precision of 17 AEDs QC sample Compound Plasma concentration equivalents (µg/ml) Precision (%) Accuracy (%) Low Middle High Low Middle High Low Middle High Carbamazepine Carbamazepine-10,11-epoxide Clonazepam Diazepam Ethomuximide Felbamate Gabapentin Lamotrigine Levetiracetam Nitrazepam Phenobarbial Phenytoin Primidone Tiagabine Topiramate Vigabatrin Zonisamide Conclusions We could select the most suitable combination of mobile phase and column from 36 analytical condition without time-consuming investigation. We have measured plasma sample as it is after ,000 times dilution by methanol without making tedious sample pretreatment. Excellent linearity, precision and accuracy for all 17 AEDs were obtained at only 1 ul injection volume. First Edition: June, For Research Use nly. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 2014

10 P-CN1446E Determination of Δ 9 -tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation ASMS 2014 ThP600 Sylvain DULAURENT 1, Mikaël LEVI 2, Jean-michel GAULIER 1, Pierre MARQUET 1,3 and Stéphane MREAU 2 1 CHU Limoges, Department of Pharmacology and Toxicology, Unit of clinical and forensic toxicology, Limoges, France ; 2 Shimadzu France SAS, Le Luzard 2, Boulevard Salvador Allende, Marne la Vallée Cedex 2 3 Univ Limoges, Limoges, France

11 Determination of Δ 9 -tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation Introduction In France, as in other countries, cannabis is the most widely used illicit drug. In forensic as well as in clinical contexts, 9 -tetrahydrocannabinol (THC), the main active compound of cannabis, and two of its metabolites [11-hydroxy- 9 -tetrahydrocannabinol (11-H-THC) and 11-nor- 9 -tetrahydrocannabinol-9-carboxylic acid (THC-CH)] are regularly investigated in biological fluids for example in Driving Under the Influence of Drug context (DUID) (figure 1). Historically, the concentrations of these compounds were determined using a time-consuming extraction procedure and GC-MS. The use of LC-MS/MS for this application is relatively recent, due to the low response of these compounds in LC-MS/MS while low limits of quantification need to be reached. Recently, on-line Solid-Phase-Extraction coupled with UHPLC-MS/MS was described, but in our hands it gave rise to significant carry-over after highly concentrated samples. We propose here a highly sensitive UHPLC-MS/MS method with straightforward QuEChERS sample preparation (acronym for Quick, Easy, Cheap, Effective, Rugged and Safe). CH 3 H H H C C H 3 H 3 THC H H 2 C H H H H H H C C H 3 H 3 H C C H 3 H 3 11-H-THC THC-CH Figure 1: Structures of THC and two of its metabolites Methods and Materials Isotopically labeled internal standards (one for each target compound in order to improve method precision and accuracy) at 10 ng/ml in acetonitrile, were added to 100 µl of sample (urine, whole blood or plasma) together with 50 mg of QuEChERS salts (MgS 4 /NaCl/Sodium citrate dehydrate/sodium citrate sesquihydrate) and 200 µl of acetonitrile. Then the mixture was shaken and centrifuged for 10 min at 12,300 g. Finally, 15 µl of the upper layer were injected in the UHPLC-MS-MS system. The whole acquisition method lasted 3.4 min. 2

12 Determination of Δ 9 -tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation UHPLC conditions (Nexera MP system) Column : Kinetex C18 50x2.1 mm 2.6 µm (Phenomenex) Mobile phase A : 5mM ammonium acetate in water B : CH 3 CN Flow rate : 0.6 ml/min Time program : B conc. 20% ( min) - 90% ( min) - 20% ( min) Column temperature : 50 C MS conditions (LCMS-8040) Ionization : ESI, negative MRM mode Ion source temperatures : Desolvation line: 300 C Heater Block: 500 C Gases : Nebulization: 2.5 L/min Drying: 10 L/min MRM Transitions: Compound MRM Dwell time (msec) THC > (Quan) > (Qual) > (Qual) 60 THC-D > (Quan) > (Qual) 5 11-H-THC > (Quan) > (Qual) > (Qual) H-THC-D > (Quan) > (Qual) 5 THC-CH > (Quan) > (Qual) > (Qual) > (Qual) 50 THC-CH-D > (Quan) > (Qual) 5 Pause time Loop time : 3 msec : 0.4 sec (minimum 20 points per peak for each MRM transition) 3

13 Determination of Δ 9 -tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation Results Chromatographic conditions A typical chromatogram of the 6 compounds is presented in figure 1. Figure 1: Chromatogram obtained after an injection of a 15 µl whole blood extract spiked at 50 µg/l Extraction conditions As described by Anastassiades et al. J. AAC Int 86 (2003) , the combination of acetonitrile and QuEChERS salts allowed the extraction/partitioning of compounds of interest from matrix. This extraction/partitioning process is not only obtained with whole blood and plasma-serum where deproteinization occurred and allowed phase separation, but also with urine as presented in figure 2. A B Figure 2: influence of QuEChERS salts on urine extraction/partitioning: A: acetonitrile with urine sample lead to one phase / B: acetonitrile, QuEChERS salts and urine lead to 2 phases. 4

14 Determination of Δ 9 -tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation Validation data ne challenge for the determination of cannabinoids in blood using LC-MS/MS is the low quantification limits that need to be reached. The French Society of Analytical Toxicology proposed 0.5 µg/l for THC et 11-H-THC and 2.0 µg/l for THC-CH. With the current application, the lower limit of quantification was fixed at 0.5 µg/l for the three compounds (3.75 pg on column). The corresponding extract ion chromatograms at this concentration are presented in figure 3. THC-CH 11-H-THC THC Figure 3: Chromatogram obtained after an injection of a 15 µl whole blood extract spiked at 0.5 µg/l (lower limit of quantification). The upper limit of quantification was set at 100 µg/l. Calibration graphs of the cannabinoids-to-internal standard peak-area ratios of the quantification transition versus expected cannabinoids concentration were constructed using a quadratic with 1/x weighting regression analysis (figure 4). THC-CH 11-H-THC Figure 4: Calibration curves of the three cannabinoids THC Contrary to what was already observed with on-line Solid-Phase-Extraction no carry-over effect was noted using the present method, even when blank samples were injected after patient urine samples with concentrations exceeding 2000 µg/l for THC-CH. 5

15 Determination of Δ 9 -tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation Conclusions Quick sample preparation based on QuEChERS salts extraction/partitioning, almost as short as on-line Solid Phase Extraction. Low limit of quantification compatible with determination of DUID. No carry over effect noticed. First Edition: June, For Research Use nly. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 2014

16 P-CN1445E Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation ASMS 2014 ThP599 Sylvain DULAURENT 1, Mikaël LEVI 2, Jean-michel GAULIER 1, Pierre MARQUET 1,3 and Stéphane MREAU 2 1 CHU Limoges, Department of Pharmacology and Toxicology, Unit of clinical and forensic toxicology, Limoges, France ; 2 Shimadzu France SAS, Le Luzard 2, Boulevard Salvador Allende, Marne la Vallée Cedex 2 3 Univ Limoges, Limoges, France

17 Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation Introduction The determination of drugs of abuse (opiates, amphetamines, cocaine) in biological fluids is still an important issue in toxicology, in cases of driving under the influence of drugs (DUID) as well as in forensic toxicology. At the end of the 20th century, the analytical methods able to determine these three groups of narcotics were mainly based on a liquid-liquid-extraction with derivatization followed by GC-MS. Then LC-MS/MS was proposed, coupled with off-line sample preparation. Recently, on-line Solid-Phase-Extraction coupled with UHPLC-MS/MS was described, but in our hands it gave rise to significant carry-over after highly concentrated samples. We propose here another approach based on the QuEChERS (acronym for Quick, Easy, Cheap, Effective, Rugged and Safe) sample preparation principle, followed by UHPLC-MS/MS. Methods and Materials This method involves 40 compounds of interest (13 opiates, 22 amphetamines, as well as cocaine and 4 of its metabolites) and 18 isotopically labeled internal standards (designed with *) (Table1). Table 1: list of analyzed compounds with their associate internal standard (*) Cocaine and metabolites Anhydroecgonine methylester Benzoylecgonine* Cocaethylene* Cocaine* Ecgonine methylester* Amphetamines or related compounds 2-CB 2-CI 4-MTA Ritalinic acid Amphetamine* BDB Ephedrine* MBDB m-cpp MDA* MDEA* MDMA* MDPV Mephedrone Metamphetamine* Methcathinone Methiopropamine Methylphenidate Norephedrine Norfenfluramine Norpseudoephedrine Pseudoephedrine piates 6-monoacetylmorphine* Dextromethorphan Dihydrocodeine* Ethylmorphine Hydrocodone Hydromorphone Methylmorphine* Morphine* Naloxone* Naltrexone* Noroxycodone* xycodone* Pholcodine 2

18 Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation To 100 µl of sample (urine, whole blood or plasma) were added isotopically labeled internal standards (in order to improve method precision and accuracy) at 20 µg/l in acetonitrile (20 µl), and 200 µl of acetonitrile. After a 15 s shaking, the mixture was placed at -20 C for 10 min. Then approximately 50 mg of QuEChERS salts (MgS 4 /NaCl/Sodium citrate dehydrate/sodium citrate sesquihydrate) were added and the mixture was shaken again for 15 s and centrifuged for 10 min at g. The upper layer was diluted (1/3; v/v) with a 5 mm ammonium formate buffer (ph 3). Finally, 5 µl were injected in the UHPLC-MS/MS system. The whole acquisition method lasted 5.5 min. UHPLC conditions (Nexera MP system, figure 1) Column : Restek Pinnacle DB PFPP 50x2.1 mm 1.9 µm Mobile phase A : 5mM Formate ammonium with 0.1% formic acid in water B : 90% CH 3 H/ 10% CH 3 CN (v/v) with 0.1 % formic acid Flow rate : ml/min Time program : B conc. 15% ( min) - 20% (1.77 min) - 90% (2.20 min) 100% (4.00 min) 15% ( min) Column temperature : 50 C MS conditions (LCMS-8040, figure 1) Ionization : ESI, Positive MRM mode Ion source temperatures : Desolvation line: 300 C Heater Block: 500 C Gases : Nebulization: 2.5 L/min Drying: 10 L/min MRM Transitions : 2 Transitions per compounds were dynamically scanned for 1 min except pholcodine (2 min) Pause time : 3 msec Loop time : sec (minimum 17 points per peak for each MRM transition) Figure 1: Shimadzu UHPLC-MS/MS Nexera-8040 system 3

19 Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation Results Chromatographic conditions The analytical conditions allowed the chromatographic separation of two couples of isomers: norephedrine and norpseudoephedrine; ephedrine and pseudoephedrine (figure 2). A typical chromatogram of the 58 compounds is presented in figure 3. A B Figure 2: Chromatograms obtained after an injection of a 5 µl whole blood extract spiked at 200 µg/l. rder of retention - A: norephedrine and norpseudoephedrine / B: ephedrine and pseudoephedrine Figure 3: Chromatogram obtained after an injection of a 5 µl whole blood extract spiked at 200 µg/l 4

20 Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation Extraction conditions As described by Anastassiades et al. J. AAC Int 86 (2003) , the combination of acetonitrile and QuEChERS salts allowed the extraction/partitioning of compounds of interest from matrix. This extraction/partitioning process is not only obtained with whole blood and plasma-serum where deproteinization occurred and allowed phase separation, but also with urine as presented in figure 4. A B Figure 4: influence of QuEChERS salts on urine extraction/partitioning: A: acetonitrile with urine sample lead to one phase / B: acetonitrile, QuEChERS salts and urine lead to 2 phases. Validation data Among the 40 analyzed compounds, 38 filled the validation conditions in term of intra- and inter-assay precision and accuracy were less than 20% at the lower limit of quantification and less than 15% at the other concentrations. Despite the quick and simple sample preparation, no significant matrix effect was observed and the lower limit of quantification was 5 µg/l for all compounds, while the upper limit of quantification was set at 500 µg/l. The concentrations obtained with a reference (GC-MS) method in positive patient samples were compared with those obtained with this new UHPLC-MS/MS method and showed satisfactory results. Contrary to what was already observed with on-line Solid-Phase-Extraction, no carry-over effect was noted using the present method, even when blank samples were injected after patient urine samples with analytes concentrations over 2000 µg/l. 5

21 Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation Conclusions Separation of two couples of isomers with a run duration less than 6 minutes and using a 5 cm column. Quick sample preparation based on QuEChERS salts extraction/partitioning, almost as short as on-line Solid Phase Extraction. Lower limit of quantification compatible with determination of DUID. No carry over effect noticed. First Edition: June, For Research Use nly. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 2014

22 P-CN1442E Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS ASMS 2014 ThP-592 Toshikazu Minohata 1, Keiko Kudo 2, Kiyotaka Usui 3, Noriaki Shima 4, Munehiro Katagi 4, Hitoshi Tsuchihashi 5, Koichi Suzuki 5, Noriaki Ikeda 2 1 Shimadzu Corporation, Kyoto, Japan 2 Kyushu University, Fukuoka, Japan 3 Tohoku University Graduate School of Medicine, Sendai, Japan 4 saka Prefectural Police, saka, Japan 5 saka Medical Collage, Takatsuki, Japan

23 Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS Introduction In Forensic Toxicology, LC/MS/MS has become a preferred method for the routine quantitative and qualitative analysis of drugs of abuse. LC/MS/MS allows for the simultaneous analysis of multiple compounds in a single run, thus enabling a fast and high throughput analysis. In this study, we report a developed analytical system using ultra-high speed triple quadrupole mass spectrometry with a new extraction method for pretreatment in forensic analysis. The system has a sample preparation utilizing modified QuEChERS extraction combined with a short chromatography column that results in a rapid run time making it suitable for routine use. Methods and Materials Sample Preparation Whole blood sample preparation was carried out by the modified QuEChERS extraction method (1) using Q-sep QuEChERS Sample Prep Packets purchased from RESTEK (Bellefonte, PA). 1) Add 0.5 ml of blood and 1 ml of distilled water into the 15 ml centrifugal tube and agitate the mixture using a vortex mixer. 2) Add two 4 mm stainless steel beads, 1.5 ml of acetonitrile and 100 µl of acetonitrile solution containing 1 ng/µl of Diazepam-d5. Then agitate using the vortex mixer. 3) Add 0.5 g of the filler of the Q-sep QuEChERS Extraction Salts Packet. 4) Vigorously shake the tube by hand several times, agitate well using the vortex mixer for approximately 20 seconds. Then centrifuge the tube for 10 minutes at 3000 rpm. 5) Move the supernatant to a different 15 ml centrifugal tube and add 100 µl of 0.1 % TFA acetonitrile solution. Then, dry using a nitrogen-gas-spray concentration and drying unit or a similar unit. 6) Reconstitute with 200 µl of methanol using the vortex mixer. Then move it to a microtube, and centrifuge for 5 minutes at 10,000 rpm. 7) Transfer 150 µl of the supernatant to a 1.5 ml vial for HPLC provided with a small-volume insert. [ ref.] (1) Usui K et al, Legal Medicine 14 (2012), Water 1 ml ACN 1.5 ml Diazepam-d5 (IS) 100ng Stainless-Steel Beads (4mm x 2) Transfer supernatant Add 100uL of 0.1% TFA Dry Sample 0.5 ml Q-sep QuEChERS Extraction Salts (MgS 4,NaAc) [Shake] [Centrifuge] Reconstitution with 200 ul MeH LC/MS/MS analysis Figure 1 Scheme of the modified QuEChERS procedure 2

24 Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS LC-MS/MS Analysis Treated samples were analyzed using a Nexera UHPLC system coupled to a LCMS-8050 triple quadrupole mass spectrometer (Shimadzu Corporation, Japan) with LC/MS/MS Rapid Tox. Screening Database. The Database contains product ion scan spectra for 106 forensic and toxicology-related compounds of Abused drugs, Psychotropic drugs and Hypnotic drugs etc (Table 1) and provides Synchronized Survey Scan parameters (product ion spectral data acquisition parameters based on the MRM intensity as threshold) optimized for screening analysis. Samples were separated on a YMC Triart C18 column. A flow rate of 0.3 ml/min was used together with a gradient elution. Analytical Conditions HPLC (Nexera UHPLC system) Column : YMC Triart C18 (100x2mm, 1.9μm) Mobile Phase A : 10 mm Ammonium formate - water Mobile Phase B : Methanol Gradient Program : 5%B (0 min) - 95%B (10 min - 13min) - 5%B (13.1 min - 20 min) Flow Rate : 0.3 ml / min Column Temperature : 40 ºC Injection Volume : 5 ul Mass (LCMS-8050 triple quadrupole mass spectrometry) Ionization : heated ESI Polarity : Positive & Negative Probe Voltage : +4.5 kv (ESI-Positive mode); -3.5 kv (ESI-Negative mode) Nebulizing Gas Flow : 3 L / min Drying Gas Pressure : 10 L / min Heating gas flow : 10 L / min DL Temperature : 250 ºC BH Temperature : 400 ºC MRM parameter : Analytes Ret. Time Q1 m/z Q3 m/z Collision Energy Analytes Ret. Time Q1 m/z Q3 m/z Collision Energy Diazepam-d Risperidone Alprazolam Triazolam Atropine Amobarbital (neg) Estazolam Barbital (neg) Ethyl loflazepate Phenobarbital (neg) Etizolam Thiamylal (neg) Haloperidol

25 Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS positive negative Figure 2 LCMS-8050 triple quadrupole mass spectrometer Results and Discussion Alprazolam Etizolam Risperidone Triazolam (x10 3 ) >281.10(+) 2.0 (x10 3 ) >314.10(+) (x10 3 ) >195(+) (x10 2 ) >315.00(+) 1 ng/ml S/N 39.5 S/N S/N S/N 18.8 (x10 4 ) >281.10(+) (x10 4 ) >314.10(+) (x10 4 ) >195(+) (x10 3 ) >315.00(+) 0.1 ng/ml Area Ratio r 2 = Conc. Ratio Area Ratio (x0.1) Area Ratio Area Ratio (x0.1) r 2 = r 2 = r 2 = Conc. Ratio Conc. Ratio Conc. Ratio Conc. Area Accuracy %RSD ,004 8,288 9,519 75,236 75,983 74, , , , Conc. Area Accuracy %RSD ,865 5,109 4,321 48,038 49,152 54, , , , Conc. Area Accuracy %RSD ,832 32,436 30, , , ,172 3,826,373 3,718,854 3,705, Conc. Area Accuracy %RSD ,047 3,064 3,356 27,991 25,542 26, , , ,

26 Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS Amobarbital (neg) Barbital (neg) Phenobarbital (neg) Thiamylal (neg) ng/ml (x10 2 ) >42.00(-) (x10) >42.10(-) (x10 2 ) >42.20(-) (x10 2 ) >58.10(-) 5.0 S/N 40.2 S/N 15.3 S/N 38.2 S/N (x10 3 ) 10 ng/ml >42.00(-) (x10 2 ) >42.10(-) (x10 3 ) >42.20(-) 0.5 (x10 3 ) >58.10(-) Area Ratio (x0.1) r 2 = Area Ratio (x1) r 2 = Area Ratio (x0.1) 0 r 2 = Area Ratio (x0.1) 4.0 r 2 = Conc. Ratio Conc. Ratio Conc. Ratio Conc. Ratio Conc. Area Accuracy %RSD ,837 1,862 2,041 21,685 22,169 20, , , , Conc. Area Accuracy %RSD ,078 5,033 5,424 55,420 55,658 53, Conc. Area Accuracy %RSD ,909 8,564 7,939 81,987 83,274 82, Conc. Area Accuracy %RSD ,520 2,192 2,288 30,808 29,623 31, , , , Figure 3 Results of 8 drugs spiked in human whole blood using LCMS-8050 In this experiment, two different matrices consisting of human whole blood and urine were prepared and 18 drugs were spiked into extract solution. Calibration curves constructed in the range from 1 to 1 ng/ml for 12 drugs (Alprazolam, Aripiprazole, Atropine, Brotizolam, Estazolam, Ethyl loflazepate, Etizolam, Flunitrazepam, Haloperidol, Nimetazepam, Risperidone and Triazolam) and from 1 to 100 ng/ml for 6 drugs (Bromovalerylurea, Amobarbital, Barbital, Loxoprofen, Phenobarbital and Thiamylal). All calibration curves displayed linearity with an R2 > and excellent reproducibility was observed for all compounds (CV < 12%) at low concentration level. 5

27 Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS Amobarbital (neg) Barbital (neg) Phenobarbital (neg) Thiamylal (neg) (x10 2 ) >42.00(-) (x10 2 ) >42.10(-) (x10 2 ) >42.20(-) (x10 2 ) >58.10(-) ng/ml 5.0 S/N 14.7 S/N 9.4 S/N 18.3 S/N (x10 3 ) >42.00(-) (x10 2 ) >42.10(-) (x10 3 ) >42.20(-) (x10 3 ) >58.10(-) ng/ml Area Ratio (x0.1) Area Ratio (x0.1) Area Ratio (x0.1) Area Ratio (x0.1) 3.0 r 2 =0.999 r 2 =0.999 r 2 =0.999 r 2 = Conc. Ratio Conc. Ratio Conc. Ratio Conc. Ratio Conc. Area Accuracy %RSD ,468 1,233 1,245 17,241 20,546 18, , , , Conc. Area Accuracy %RSD ,989 5,613 5,443 55,392 69,481 66, Conc. Area Accuracy %RSD ,656 6,632 6,384 71,965 88,685 82, Conc. Area Accuracy %RSD ,142 3,470 3,153 27,257 34,377 32, , , , Figure 4 Results of 4 drugs spiked in human urine using LCMS-8050 Conclusions The validated sample preparation protocol can get adequate recoveries in quantitative works for all compounds ranging from acidic to basic. The combination of the modified QuEChERS extraction method and high-speed triple quadrupole LC/MS/MS with a simple quantitative method enable to acquire reliable data easily. First Edition: June, For Research Use nly. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 2014

28 P-CN1460E Simultaneous Screening and Quantitation of Amphetamines in Urine by n-line SPE-LC/MS Method ASMS 2014 ThP587 Helmy Rabaha 1, Lim Swee Chin 1, Sun Zhe 2, Jie Xing 2 & Zhaoqi Zhan 2 1 Department of Scientific Services, Ministry of Health, Brunei Darussalam; 2 Shimadzu (Asia Pacific) Pte Ltd, Singapore, SINGAPRE

29 Simultaneous Screening and Quantitation of Amphetamines in Urine by n-line SPE-LC/MS Method Introduction Amphetamines belong to stimulant drugs and are also controlled as illicit drugs worldwide. The conventional analytical procedure of amphetamines in human urine includes initial immunological screening followed by GCMS confirmation and quantitation [1]. With new SAMHSA guidelines effective in ct 2010 [2], screening, confirmation and quantitation of illicit drugs including amphetamines were allowed to employ LC/MS and LC/MS/MS, which usually does not require a derivatization step as used in the GCMS method [1]. The objective of this study was to develop an on-line SPE-LC/MS method for analysis of five amphetamines in urine without sample pre-treatment except dilution with water. The compounds studied include amphetamine (AMPH), methamphetamine (MAMP) and three newly added MDMA, MDA and MDEA by the new SAMHSA guideline (group A in Table 1). Four potential interferences (group B in) and PMPA (R) as a control reference were also included to enhance the method reliability in identification of the five targeted amphetamines from those structurally similar analogues which potentially present in forensic samples. Experimental The test stock solutions of the ten compounds (Table 1) were prepared in the toxicology laboratory in the Department of Scientific Services (MH, Brunei). Five urine specimens were collected from healthy adult volunteers. The urine samples used as blank and matrix to prepare spiked amphetamine samples were not pre-treated off-line by any means except dilution of 10 times with pure water. An on-line SPE-LC/MS was set up on the LCMS-2020, a single quadrupole system, with a switching valve and a trapping column kit (Shimadzu Co-Sense configuration) installed in the column oven and controlled by the LabSolutions workstation. The analytical column used was Shim-pack VP-DS 150 x 2mm (5um) and the trapping column was Synergi Polar-RP 50 x 2mm (2.5um), instead of a normal SPE cartridge. The injected sample first passed through the trapping column where the amphetamines were trapped, concentrated and washed by pure water for 3 minutes followed by switching to the analytical flow line. The trapped compounds were then eluted out with a gradient program: 1min, valve at position 0 & B=5%; 3 min, valve at position 1; min, B=5% 15%; min, B=65%; 12.1 min, B=5%; 14 min stop, valve to position 0. The mobile phases A and B were water and MeH both with 0.1% formic acid and mobile C was pure water. The total flow rates of the trapping line and analytical line are 0.6 and 0.3 ml/min, respectively. The injection volume was 20uL in all experiments. 2

30 Simultaneous Screening and Quantitation of Amphetamines in Urine by n-line SPE-LC/MS Method Table 1: Amphetamines & relevant compounds No Name Abbr. Name Formula Structure A1 Amphetamine AMPH C 9 H 13 N A2 Methampheta-mine MAMP C 10 H 15 N A3 3,4-methylene-dioxyamphetamine MDA C 10 H 13 N 2 A4 3,4-methylene-dioxymetham phetamine MDMA C 11 H 15 N 2 A5 3,4-methylene dioxy-n-ethyl amphetamine MDEA C 12 H 17 N 2 B1 Nor pseudo-ephedrine Nor pseudo-e C 9 H 13 N B2 Ephedrine Ephe C 10 H 15 N B3 Pseudo-Ephedrine Pseudo-E C 10 H 15 N B4 Phentermine Phent C 10 H 15 N R Propyl-amphetamine PAMP C 12 H 19 N Pump A Mixer SPE Trapping Column Manual injector Analytical column LCMS Waste Pump B Switching Valve Auto sampler Pump C Figure 1: Schematic diagram of on-line SPE-LC/MS system 3

31 Simultaneous Screening and Quantitation of Amphetamines in Urine by n-line SPE-LC/MS Method Results and Discussion Development of on-line SPE-LC/MS method With ESI positive SIM and scan mode, all of the 10 compounds formed protonated ions [M+H] + which were used as quantifier ions. The scan spectra were used for confirmation to reduce false positive results. Mixed standards of the ten compounds in Table 1 spiked in urine was used for method development. An initial difficulty encountered was that the normal reusable SPE cartridges (10-30 mml) for on-line SPE could not trap all of the ten compounds. With using a 50mmL C18-column to replace the SPE cartridge, the ten compounds studied were trapped efficiently. Furthermore, the trapped compounds were well-separated and eluted out in 8~13 minutes as sharp peaks (Figure 2) by the fully automated on-line SPE-LC/MS method established. 2.0 (x1,000,000) 2:136.10(+) 2:150.10(+) 2:178.10(+) 2:180.10(+) 2:194.10(+) 1.5 2:208.20(+) 2:166.10(+) 2:152.10(+) (a) Urine blank 2.0 (x1,000,000) 2:136.10(+) 2:150.10(+) 2:178.10(+) 2:180.10(+) 2:194.10(+) 1.5 2:208.20(+) 2:166.10(+) 2:152.10(+) (b) spiked samples Ephedrine Pseudo AMPH MAMP MDA MDMA MDEA PAMP Phent Norpseudo min min Figure 2: SIM chromatograms of urine blank (a) and five amphetamines and related compounds (125 ppb each) spiked in urine (b) by on-line SPE-LC/MS. Calibration curves of the on-line SPE-LC/MS method were established using mixed standard samples with concentrations from 2.5 ppb to 500 ppb. Linear calibration curves with R 2 > were obtained for every compound (Figure 3 & Table 2) Area (x1,000,000) AMPH Area (x10,000,000) MAMP 0.5 Area (x10,000,000) 2.0 Area (x10,000,000) Area (x10,000,000) 3.0 MDA MDMA MDEA Conc. Area (x1,000,000) Nor pseudo-e Conc Conc. Area (x10,000,000) Ephedrine Conc Conc Area (x10,000,000) Pseudo-E Conc Conc. Area (x10,000,000) 0.5 Phent Conc Conc. 2.0 Area (x10,000,000) PAMP Conc. Figure 3: Calibration curves of five amphetamines and five related compounds with concentrations from 2.5 ppb to 500 ppb by on-line SPE-LC/MS method 4

32 Simultaneous Screening and Quantitation of Amphetamines in Urine by n-line SPE-LC/MS Method Table 2: Peak detection, retention, calibration curves and method performance evaluation Name SIM ion (+) RT (min) Conc. range (ppb) Linearity (r 2 ) Rec. % (62.5ppb) M.E % (62.5ppb) RSD%(n=6) (62.5ppb) S/N (2.5ppb) LD/LQ (ppb) Norpseudo-E /2.17 Ephe /0.76 Pseudo-E /0.88 AMPH /1.46 MAMP /0.80 MDA /1.36 MDMA /0.70 MDEA /0.57 Phent /2.01 PAMP (Ref) /0.66 Performance evaluation of on-line SPE-LCMS method The trapping efficiency of the on-line SPE is critical and must be evaluated first, because it determines the recovery of the method. In this study, the recovery of the on-line SPE was determined by injecting a same mixed standard sample from a manual injector installed before the analytical column (by-pass on-line SPE) and also from the Autosampler (See Figure 1). The peaks areas obtained by the two injections were used to calculate recovery value of the on-line SPE method. As shown in Table 2, the recovery obtained with 62.5 ppb mixed standards are at 69.5% ~ 97.3%. The recovery with 250 ppb and 500 ppb mixed samples were also determined and similar results were obtained. Matrix effect was determined with 62.5 ppb and 250 ppb levels of mixed samples in clear solution and in urine. The results (Table 2) show a variation between 69.3% and 116% with compounds. The matrix effect with different urine specimens did not show significant differences. Repeatability was evaluated with spiked mixed samples of 62.5 ppb and 250 ppb. The results of 62.5 ppb is shown in Table 2, RSD between 0.41% and 5.3%. The sensitivity of the on-line SPE-LC/MS method was evaluated with spiked sample of 2.5 ppb level. The SIM chromatograms are shown in Figure 4. The S/N ratios obtained ranged 11.3~42, which were suitable to determine LQ (S/N = 10) and LD (S/N = 3). Since the urine samples were diluted for 10 times with water before injection, the LD and LQ of the method for source urine samples were at 1.9~7.1 and 5.7~21.7 ng/ml, respectively. The confirmation cutoff values of the five targeted amphetamines (Group A) in urine enforced by the new SMAHSA guidelines are 250 ng/ml [2]. The on-line SPE-LC/MS method established has sufficient allowance in terms of sensitivity and confirmation reliability for analysis of actual urine samples. (x10,000) 6.0 2:136.10(+) 2:150.10(+) 2:178.10(+) 2:180.10(+) 5.0 2:194.10(+) 2:208.20(+) 2:166.10(+) 4.0 2:152.10(+) Norpseudo Ephedrine Pseudo AMPH MAMP MDA MDMA MDEA PAMP Phent min Figure 4: SIM chromatograms of 10 compounds with 2.5 ppb each by on-line SPE-LC/MS method. 5

33 Simultaneous Screening and Quantitation of Amphetamines in Urine by n-line SPE-LC/MS Method Durability of on-line SPE trapping column The durability of the trapping column was tested purposely by continuous injections of spiked urine samples (125 ppb) for 200 times in a few days. Figure 5 shows the chromatograms of the first and 200 th injections of a same spiked sample. The results show that the variations of peak area and retention time of the 200 th injection compared to the 1 st injection were at 89.5%~117.8% and 89.5%~99.8% respectively. (x1,000,000) 2:136.10(+) 2.0 2:150.10(+) 2:178.10(+) 1 st injection 2:180.10(+) 2:194.10(+) spiked mixed std 125ppb in urine 1.5 2:208.20(+) 2:166.10(+) inj vol: 20 µl 2:152.10(+) 0.5 Norpseudo Ephedrine Pseudo AMPH MDA MAMP MDMA MDEA Phent PAMP (x1,000,000) 2:136.10(+) 2.0 2:150.10(+) 2:178.10(+) 200 th injection 2:180.10(+) 2:194.10(+) spiked mixed std 125ppb in urine 1.5 2:208.20(+) 2:166.10(+) inj vol: 20 µl 2:152.10(+) 0.5 Norpseudo Ephedrine Pseudo MAMP MDMA AMPH MDA PAMP MDEA Phent min min Figure 5: Durability test of on-line SPE-LC/MS method, comparison of 1 st and 200 th injections. Confirmation Reliability Confirmation reliability of LC/MS and LC/MS/MS methods must be proven to be equivalent to the GCMS method according to the SMAHSA guidelines [2]. Validation of confirmation reliability of the on-line SPE-LC/MS method has not be carried out systematically. The high sensitivity of MS detection in SIM mode is a key factor to ensure no false-negative and the scan spectra acquired simultaneously is used for excluding false-positive. In this work, the confirmation reliability was evaluated using five different urine specimens as matrix to prepare spiked samples of 2.5 ppb (correspond 25 ng/ml in source urine) and above. The results show that false-positive and false negative results were not found. Conclusions A novel high sensitivity on-line SPE-LC/MS method was developed for screening, conformation and quantification of five amphetamines: AMPH, MAMP, MDMA, MDA and MDEA in urines. The recovery of the on-line SPE by employing a 50mmL Synergi Polar-RP column was at 72%~86% for the five amphetamines, which are considerably high if comparing with conventional on-line SPE cartridges. The method performance was evaluated thoroughly with urine spiked samples. The results demonstrate that the on-line SPE-LC/MS method is suitable for direct analysis of the amphetamines and relevant compounds in urine samples without off-line sample pre-treatment. 6

34 Simultaneous Screening and Quantitation of Amphetamines in Urine by n-line SPE-LC/MS Method References 1. Kudo K, Ishida T, Hara K, Kashimura S, Tsuji A, Ikeda N, J Chromatogr B, 2007, 855, SAMHSA Manual for urine laboratories, National laboratory certification program, ct 2010, US Department of Health and Human Services. First Edition: June, For Research Use nly. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 2014

35 P-CN1481E Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin ASMS 2014 MP762 Alan J. Barnes 1, Carrie-Anne Mellor 2, Adam McMahon 2, Neil J. Loftus 1 1 Shimadzu, Manchester, UK 2 WMIC, University of Manchester, UK

36 Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin Introduction Dried plasma sample collection and storage from whole blood without the need for centrifugation separation and refrigeration opens new opportunities in blood sampling strategies for quantitative LC/MS/MS bioanalysis. Plasma samples were generated by gravity filtration of a whole blood sample through a laminated membrane stack allowing plasma to be collected, dried, transported and analysed by LC/MS/MS. This novel plasma separation card (PSC) technology was applied to the quantitative LC/MS/MS analysis of warfarin, in blood samples. Warfarin is a coumarin anticoagulant vitamin-k antagonist used for the treatment of thrombosis and thromboembolism. As a result of vitamin-k recycling being inhibited, hepatic synthesis is in-turn inhibited for blood clotting factors as well as anticoagulant proteins. Whilst the measurement of warfarin activity in patients is normally measured by prothrombin time by international normalized ratio (INR) in some cases the quantitation of plasma warfarin concentration is needed to confirm patient compliance, resistance to the anticoagulant drug, or diet related issues. In this preliminary evaluation, warfarin concentration was measured by LC/MS/MS to evaluate if PSC technology could complement INR when sampling patient blood. Materials and Methods Sample preparation Warfarin standard was dissolved in water containing 50% ethanol + 0.1% formic acid, spiked (60uL) to whole human blood (1mL) and mixed gently. 50uL of spiked blood was deposited onto the PSC. After 3 minutes, the primary filtration overlay was removed followed by 15 minutes air drying at room temperature. The plasma sample disc was prepared directly for analysis after drying. LC/MS/MS sample preparation involved vortexing the sample disk in 40uL methanol, followed by centrifugation 16,000g 5 min. 20uL supernatant was added directly to the LCMS/MS sample vial already containing 80uL water (2uL analysed). Control plasma comparison was prepared by centrifuging remaining blood at 1000g for 10min. 2.5uL supernatant plasma was taken, 40uL methanol added, and prepared as PSC samples. LCMS/MS sample injection volume, 2uL. LC-MS/MS analysis Warfarin was measured by MRM, positive negative switching mode (15msec). LC/MS/MS System : Nexera UHPLC system + LCMS-8040 Shimadzu Corporation Flow rate : 0.4mL/min (0-7.75min), 0.5mL/min (7.5-14min), 0.4mL/min (15min) Mobile phase : A= Water + 0.1% formic acid B= Methanol + 0.1% formic acid Gradient : 20% B (0-0.5 min), 100% B (8-12 min), 20% B ( min) Analytical column : Phenomenex Kinetex XB C x 2.1mm 1.7um 100A Column temperature : 50ºC Ionisation : Electrospray, positive, negative switching mode Desolvation line : 250ºC Drying/Nebulising gas : 10L/min, 2L/min Heating block : 400ºC 2

37 Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin Design of plasma separator technology Control Spot: [Determines whether enough blood was placed on the card]. Spreading Layer [Lateral spreading layer rapidly spreads blood so it will enter the filtration layer as a front while adding buffers and anticoagulants. The lateral spreading rate is 150um/sec]. Filtration Layer [Filtration layer captures blood cells by a combination of filtration and adsorption. The average linear vertical migration rate is approximately 1um/sec]. Isolation Screen [Precludes lateral wicking along the card surface]. Collection Layer [Loads with a specific aliquot of plasma onto a 6.35mm disc]. Although flow through the filtration membrane is unlikely to be constant throughout the plasma extraction process, the average loading rate of the Collection Disc was 13 nl/sec. This corresponds to a volumetric flow rate into the Collection Disc of 400 pl/mm 2 /sec. Plasma separation workflow The collection disc is removed from the card and is ready for extraction for LC-MS/MS analysis. A NoviPlex card is removed from foil packaging. Approximately 50uL of whole blood is added to the test area. After 3 minutes, the top layer is completely removed (peeled back). The collection disc contains 2.5uL of plasma. Card is air dried for 15 minutes. Figure 1. Noviplex workflow. 3

38 Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin Figure 2. Applying a blood sample, either as a finger prick or by accurately measuring the blood volume, to the laminated membrane stack retains red cells and allows a plasma sample to be collected. The red cells are retained by a combination of adsorption and filtration whilst plasma advances through the membrane stack by capillary action. After approximately three minutes the plasma Collection Disc was saturated with an aliquot of plasma and was ready for LC/MS/MS analysis. Results Comparison between plasma separation cards (PSC) and plasma (x100,000) Plasma separation card 2.00 Positive ion Warfarin m/z > Q1 (V) Collision energy Q3 (V) ug/mL 0.75 Calibration standard ug/mL 0.25 Calibration standard min (x100,000) Plasma separation card Negative ion Warfarin m/z > Q1 (V) 14 Collision energy 19 Q3 (V) ug/mL Calibration standard 0.4ug/mL Calibration standard min (x100,000) min (x100,000) Plasma Positive ion Warfarin m/z > Q1 (V) -22 Collision energy -15 Q3 (V) ug/mL Calibration standard 0.4ug/mL Calibration standard Plasma Negative ion Warfarin m/z > 165 Q1 (V) 14 Collision energy 19 Q3 (V) ug/mL Calibration standard 0.4ug/mL Calibration standard min Figure 3. Comparison between the warfarin response in both positive and negative ion modes for warfarin calibration standards at 2.5ug/mL and 0.4ug/mL extracted from the plasma separation cards and a conventional plasma sample. There is a broad agreement in ion signal intensity between the 2 sample preparation techniques. 4

39 Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin 800, , , , , , , ,000 Plasma separation card Positive ion Warfarin m/z > Replicate calibration points at 2.5ug/mL and 0.4ug/mL (n=3) Linear regresson analysis y = x R² = Blood concentration ( ug/ml) Plasma separation card Negative ion Warfarin m/z > Replicate calibration points at 2.5ug/mL and 0.4ug/mL (n=3) Linear regression analysis y = x R² = Blood concentration ( ug/ml) Figure 4. In both ion modes, the calibration curve was linear over the therapeutic range studied for warfarin extracted from PSC s (calibration range 0-3ug/mL, single point calibration standards at each level with the exception of replicate calibration points at 2.5ug/mL and 0.4ug/mL (n=3); r2>0.99 for PSC analysis [r2>0.99 for a conventional plasma extraction]). (x10,000) Matrix blank comparison Positive ion Plasma separation card matrix blank Plasma matrix blank min (x10,000) Matrix blank comparison Negative ion Plasma separation card matrix blank Plasma matrix blank min Figure 5. Matrix blank comparison. In both ion modes, the MRM chromatograms for PSC and plasma are comparable. Warfarin ion signals were not detected in the any PSC or plasma matrix blank. Plasma separation card comparison The drive to work with smaller sample volumes offers significant ethical and economical advantages in pharmaceutical and clinical workflows and dried blood spot sampling techniques have enabled a step change approach for many toxicokinetic and pharmacokinetic studies. However, the impressive growth of this technique in the quantitative analysis of small molecules has also discovered several limitations in the case of sample instability (some enzyme labile compounds, particularly prodrugs, analyte stability can be problematic), hematocrit effect and background interferences of DBS. DBS also shows noticeable effects on many lipids dependent on the sample collection process. To compare PSC to plasma lipid profiles the same blood sample extraction procedure applied for warfarin analysis was measured by a high mass accuracy system optimized for lipid profiling. 5

40 Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin Monoacylglycerophosphoethanolamines Monoacylglycerophosphocholines Ceramide Diacylglycerophosphocholines phosphocholines Plasma separation card sample Positive ion LCMS-IT-TF Lipid profiling Conventional plasma sample Positive ion LCMS-IT-TF Lipid profiling min min Figure 6. Lipid profiles from the same human blood sample extracted using a plasma separation card (left hand profile) compared to a conventional plasma samples (centrifugation). Both lipid profiles are comparable in terms of distribution and the number of lipids detected (the scaling has been normalized to the most intense lipid signal). Conclusions In this limited study, plasma separation card (PSC) sampling delivered a quantitative analysis of warfarin spiked into human blood. PSC generated a linear calibration curve in both positive and negative ion modes (r2>0.99; n=5); The warfarin plasma results achieved by using the PSC technique were in broad agreement with conventional plasma sampling data. The plasma generated by the filtration process appears broadly similar to plasma derived from conventional centrifugation. Further work is required to consider the robustness and validation in a routine analysis. References Jensen, B.P., Chin, P.K.L., Begg, E.J. (2011) Quantification of total and free concentrations of R- and S-warfarin in human plasma by ultrafiltration and LC-MS/MS. Anal Bioanal Chem., 401, Radwan, M.A., Bawazeer, G.A., Aloudah, N.M., Aluadeib, B.T., Aboul-Enein, H.Y. (2012) Determination of free and total warfarin concentrations in plasma using UPLC MS/MS and its application to patient samples. Biochemical Chromatography, 26, 6-11 First Edition: June, For Research Use nly. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 2014

41 P-CN1462E Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS ASMS 2014 MP535 Zhaoqi Zhan 1, Zhe Sun 1, Jie Xing 1, Helmy Rabaha 2 and Lim Swee Chin 2 1 Shimadzu (Asia Pacific) Pte Ltd, Singapore, SINGAPRE; 2 Department of Scientific Services, Ministry of Health, Brunei Darussalam

42 Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS Introduction Amphetamines are among the most commonly abused drugs type worldwide. The conventional analytical procedure of amphetamines in human urine in forensic laboratory involves initial immunological screening followed by GCMS confirmation and quantitation [1]. The new guidelines of SAMHSA under U.S. Department of Health and Human Services effective in ct 2010 [2] allowed use of LC/MS/MS for screening, confirmation and quantitation of illicit drugs including amphetamines. ne of the advantages by using LC/MS/MS is that derivatization of amphetamines before analysis is not needed, which was a standard procedure of GCMS method. Since analysis speed and throughput could be enhanced significantly, development and use of LC/MS/MS methods are in Experimental The stock standard solutions of amphetamines and related compounds as listed in Table 1 were prepared in the Toxicology Laboratory in the Department of Scientific Services (MH, Brunei). Five urine specimens were collected from healthy adult volunteers. The urine samples used as blank and spiked samples were not pre-treated by any means except dilution of 10 times with Milli-Q water. An LCMS-8040 triple quadrupole coupled with a Nexera UHPLC system (Shimadzu Corporation) was used. The analytical column used was a Shim-pack XR-DS III UHPLC column (1.6 µm) 50mm x 2mm. The mobile phases used demand and many such efforts have been reported recently [3]. The objective of this study is to develop a fast LC/MS/MS method for direct analysis of amphetamines in urine without sample pre-treatment (except dilution with water) on LCMS-8040, a triple quadrupole system featured as ultra fast mass spectrometry (UFMS). The compounds studied include amphetamines (AMPH), methamphetamine (MAMP) and three newly added MDMA, MDA and MDEA by the new SAMHSA guidelines, four potential interferences as well as PMPA as a control reference (Table 1). Very small injection volumes of 0.1uL to 1uL was adopted in this study, which enabled the method suitable for direct injection of untreated urine samples without causing significant contamination to the ESI interface. were water (A) and MeH (B), both with 0.1% formic acid. A fast gradient elution program was developed for analysis of the ten compounds: 0-1.6min, B=2%->14%; min, B=70%; 2.4min, B=2%; end at 4min. The total flow rate was 0.6 ml/min. Positive ESI ionization mode was applied with drying gas flow of 15 L/min, nebulizing gas flow of 3 L/min, heating block temperature of 400 ºC and DL temperature of 250 ºC. Various injection volumes from 0.1 ul to 5 ul were tested to develop a method with a lower injection volume to reduce contamination of untreated urine samples to the interface. Results and Discussion Method development of direct injection of amphetamines in urine MRM optimization of the ten compounds (Table 1) was performed using an automated MRM optimization program with LabSolutions workstation. Two MRM transitions were selected for each compound, one for quantitation and second one for confirmation (Table 1). The ten compounds were separated and eluted in 0.75~2.2 minutes as sharp peaks as shown in Figure 1. In addition to analysis speed and detection sensitivity, this method development was also focused on evaluation of small to ultra-small injection volumes to develop a method suitable for direct injection of urine samples without any pre-treatment while it should not cause significant contamination to the interface. The Nexera SIL-30A auto-sampler enables to inject as low as 0.10 ul of sample with excellent precision. Figure 1 shows a few selected results of direct injection of urine blank (a) and mixed standards spiked in urine with 1 ul (c and d) and 0.1 ul (b) injection. It can be seen that all compounds (12.5 ppb each in urine) could be detected with 0.1uL injection except MDA and Norpseudo-E. With 1uL injection, all of them were detected. 2

43 Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS Table 1: MRMs of amphetamines and related compounds Cat. B1 B2 B3 A1 A2 A3 A4 A5 B4 R Compound Abbr. RT (min) MRM 152>134 Nor pseudo ephedrine Nor pseudo-e > >148 Ephedrine Ephe >91 166>148 Pseudo ephedrine Pseudo-E 1 166>91 136>91 Amphetamine AMPH > >91 Methampheta-mine MAMP > >163 3,4-methylenedi oxyamphetamine MDA > >163 3,4-methylene dioxymeth amphetamine MDMA > >163 3,4-methylene dioxy-n-ethyl amphetamine MDEA > >91 Phentermine Phent > >91 Propyl amphetamine PAMP >65 CE (V) (x10,000) (a) Urine blank, 1 ul inj 3.0 (x10,000) (b) 12.5ppb in urine, 0.1uL inj PAMP Norpseudo Ephedrine Pseudo AMPH MAMP MDA MDMA Phent MDEA min (x100,000) 3.0 (c) 12.5ppb, 1uL inj PAMP min (x1,000,000) 1.5 (d) 62.5ppb in urine, 1uL inj PAMP 2.0 Norpseudo Ephedrine Pseudo AMPH MAMP MDMA MDA Phent MDEA min min Figure 1: MRM chromatograms of urine blank (a) and spiked samples of amphetamines and related compounds in urine by LC/MS/MS method with 1uL and 0.1uL injection volumes. Norpseudo Ephedrine Pseudo AMPH MAMP MDMA MDA Phent MDEA 3

44 Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS Calibration curves with small and ultra-small injection volumes Linear calibration curves were established for the ten compounds spiked in urine with different injection volumes: 0.1, 0.2, 0.5, 1, 2 and 5 ul. Good linearity of calibration curves (R2>0.999) were obtained for all injection volumes including 0.1uL, an ultra-small injection volume. The calibration curves with 0.1 ul injection volume are shown in Figure 2. The linearity (r2) of all compounds with 0.1 ul and 1 ul injection volumes are equivalently good as shown in Table Area (x100,000) AMPH Area (x1,000,000) MAMP Area (x100,000) Area (x100,000) Area (x100,000) 5.0 MDA 7.5 MDMA MDEA Conc Conc Conc Conc Conc. Area (x100,000) 3.0 Nor pseudo-e 5.0 Area (x100,000) Ephedrine 5.0 Area (x100,000) Pseudo-E 7.5 Area (x100,000) Phent 1.5 Area (x1,000,000) PAMP Conc Conc Conc Conc Conc. Figure 2: Calibration Curves of amphetamines spiked in urine with 0.1uL injection Performance validation Repeatability of peak area was evaluated with a same loading amount (6.25 pg) but with different injection volumes. The RSD shown in Table 2 were 1.6% ~ 7.9% and 1.6 ~ 7.8% for 0.1uL and 1uL injection, respectively. It is worth to note that the repeatability of every compounds with of 0.1uL injection is closed to that of 1uL injection as well as 5uL injection (data not shown). Matrix effect of the method was determined by comparison of peak areas of mixed standards in pure water and in urine matrix. The results of 62.5ppb with 1uL injection were at % except norpseudoephedrine (79%) as shown in Table 2. Accuracy and sensitivity of the method were evaluated with spiked samples of low concentrations. The results of LD and LQ of the ten compounds in urine are shown in Table 3. Since the working samples (blank and spiked) were diluted for 10 times with water before injection, the concentrations and LD/LQ of the method described above for source urine samples have to multiply a factor of 10. Therefore, the LQs of the method for urine specimens are at ng/ml for AMPH, PAMP, MDMA and MDEA and 53 ng/ml for MDA. The LQs for the potential interferences (Phentermine, Ephedrine, Pseudo-Ephedrine and Norpseudo-Ephedrine) are at ng/ml, 2.4 ng/ml for the internal reference MAMP. The sensitivity of the direct injection LC/MS/MS method are significantly higher than the confirmation cutoff (250 ng/ml) required by the SAMHSA guidelines. 4

45 Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS Table 2: Method Performance with different inj. volumes Name Norpseudo-E Ephe Pseudo-E AMPH MAMP MDA MDMA MDEA Phent PAMP (ppb) Calibration curve, R2 (0.1uL) (1uL) : Measured with mixed stds of 62.5 ppb in clear solution and spiked in urine 2: For 0.1uL injection, the lowest conc. is 2.5 or 12.5 ppb RSD% area (n=6) (0.1uL) (1uL) M.E. % 1 (1uL) Table 3: Method performance: sensitivity & accuracy (1uL) Name Norpseudo-E Ephe Pseudo-E AMPH MAMP MDA MDMA MDEA Phent PAMP Conc. (ppb) Accuracy Sensitivity (ppb) Prep. Meas. (%) S/N LD LQ Method operational stability The method operational stability with 1uL injection was tested with spiked samples of 25 ppb in five urine specimens, corresponding to 250 ng/ml in the source urine samples. Continuous injections of accumulated 120 times was carried out in about 10 hours. The purpose of the experiment was to evaluate the operational stability against the ESI source contamination by urine samples without pre-treatment. Figure 3 shows the first injection and the 120 th injection of the same spiked sample (S1) as well as other spiked samples (S2, S3, S4 and S5) in between. Decrease in peak areas of the compounds occurred, but the degree of the decrease in average was about 17% from the first injection to the last injection. This result indicates that it is possible to carry out direct analysis of urine samples (10 times dilution with water) by the high sensitivity LC/MS/MS method with a very small injection volume. 5

46 Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS (x100,000) (x100,000) (x100,000) 7.5 S1 (1 st inj) PAMP 7.5 S2 (11 th inj) PAMP 7.5 S3 (21 st inj) PAMP Norpseudo Ephedrine Pseudo AMPH MAMP MDMA MDA Phent MDEA Norpseudo Ephedrine Pseudo AMPH MAMP MDMA MDA Phent MDEA Norpseudo Ephedrine Pseudo AMPH MAMP MDMA MDA Phent MDEA 2.0 min (x100,000) S4 (31 st inj) Norpseudo Ephedrine Pseudo AMPH MAMP MDA MDMA Phent MDEA PAMP 2.0 min 2.0 min (x100,000) S5 (41 st inj) Norpseudo Pseudo Ephedrine AMPH MAMP MDA MDMA Phent MDEA 2.0 min PAMP 2.0 min (x100,000) S1 (110 th inj) Norpseudo Ephedrine Pseudo AMPH MAMP MDMA MDA Phent MDEA PAMP 2.0 min Figure 3: Selected chromatograms of continuous injections of spiked samples (25 ppb) with 1 µl injection. Five urine specimens S1, S2, S3, S4 and S5 were used to prepare these spiked samples. Conclusions In this study, we developed a fast LC/MS/MS method for direct analysis of five amphetamines and related compounds in human urine for screening and quantitative confirmation. Very small injection volumes of 0.1~ ul were adopted to minimize ESI contamination and enhance operational stability. The good performance results observed reveals that screening and confirmation of amphetamines in human urine by direct injection to LC/MS/MS is possible and the method could be an alternative choice in forensic and toxicology analysis. References 1. Kudo K, Ishida T, Hara K, Kashimura S, Tsuji A, Ikeda N, J Chromatogr B, 2007, 855, Mandatory guidelines for Federal Workplace Drug Testing Program, 73 FR , Nov. 25, Huei-Ru Lina, Ka-Ian Choia, Tzu-Chieh Linc, Anren Hu,, Journal of Chromatogr B, 2013, 929, First Edition: June, For Research Use nly. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 2014

47 P-CN1482E Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS ASMS 2014 WP641 Alan J. Barnes 1, Carrie-Anne Mellor 2, Adam McMahon 2, Neil Loftus 1 1 Shimadzu, Manchester, UK 2 2WMIC, University of Manchester, UK

48 Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS Introduction Plasma extraction technology is a novel technique achieved by applying a blood sample to a laminated membrane stack which allows plasma to flow through the asymmetric filter whilst retaining the cellular components of the blood sample. Plasma separation card technology was applied to the quantitative analysis of temozolomide (TMZ); an oral imidazotetrazine alkylating agent used for the treatment of Grade IV astrocytoma, an aggressive form of brain tumour. Under physiological conditions TMZ is rapidly converted to 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC) which in-turn degrades by hydrolysis to 5-aminoimidazole-4-carboxamide (AIC). Storage of plasma has previously shown that both at -70C and 4C degradation still occurs. In these experiments, whole blood containing TMZ standard was applied to NoviPlex plasma separation cards (PSC). The aim was to develop a robust LC/MS/MS quantitative method for TMZ. Materials and Methods Plasma separation TMZ spiked human blood calibration standards (50uL) were applied to the PSC as described below in figure The collection disc is removed from the card and is ready for extraction for LC-MS/MS analysis. A NoviPlex card is removed from foil packaging. Approximately 50uL of whole blood is added to the test area. After 3 minutes, the top layer is completely removed (peeled back). The collection disc contains 2.5uL of plasma. Card is air dried for 15 minutes. Figure 1. Noviplex plasma separation card workflow 2

49 Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS Control Spot: [Determines whether enough blood was placed on the card]. Spreading Layer [Lateral spreading layer rapidly spreads blood so it will enter the filtration layer as a front while adding buffers and anticoagulants. The lateral spreading rate is 150um/sec]. Filtration Layer [Filtration layer captures blood cells by a combination of filtration and adsorption. The average linear vertical migration rate is approximately 1um/sec]. Isolation Screen [Precludes lateral wicking along the card surface]. Collection Layer [Loads with a specific aliquot of plasma onto a 6.35mm disc]. Although flow through the filtration membrane is unlikely to be constant throughout the plasma extraction process, the average loading rate of the Collection Disc was 13 nl/sec. This corresponds to a volumetric flow rate into the Collection Disc of 400 pl/mm 2 /sec. Figure 1. Noviplex plasma separation card workflow (Cont'd) Figure 2. Applying a blood sample, either as a finger prick or by accurately measuring the blood volume, to the laminated membrane stack retains red cells and allows a plasma sample to be collected. The red cells are retained by a combination of adsorption and filtration whilst plasma advances through the membrane stack by capillary action. After approximately three minutes the plasma Collection Disc was saturated with an aliquot of plasma and was ready for LC/MS/MS analysis. Sample preparation TMZ was extracted from the dried plasma collection discs by adding 40uL acetonitrile + 0.1% formic acid, followed by centrifugation 16,000g for 5 min. 30uL supernatant was added directly to the LC/MS/MS sample vial for analysis. As a control, conventional plasma samples were prepared by centrifuging the human blood calibration standards at 1000g for 10min. TMZ was extracted from 2.5uL of plasma using the same extraction protocol as applied for PSC. 3

50 Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS LC/MS/MS analysis Ionisation : Electrospray, positive mode MRM > CE -10 Desolvation line : 300ºC Drying/Nebulising gas : 10L/min, 2L/min Heating block : 400ºC HPLC : HILIC Nexera UHPLC system Flow rate : 0.5mL/min (0-7min), 1.8mL/min (7.5min-17.5min) Mobile phase : A= Water + 0.1% formic acid B= Acetonitrile + 0.1% formic acid Gradient : 95% B 30%% B (6.5 min), 30% B (7.5 min), 95% B (18 min) Analytical column : ZIC HILIC 150 x 4.6mm 5um 200ª Column temperature : 40ºC Injection volume : 10uL Reverse Phase Nexera UHPLC system 0.4mL/min A= Water + 0.1% formic acid B= methanol + 0.1% formic acid 5% B 100%% B (3 min), 100% B (7 min), 5% B (10 min) Phenomenex Kinetex XB C x 2.1mm 1.7um 100A 50ºC 2µL Results HILIC LC/MS/MS Temozolomide is known to be unstable under physiological conditions and is converted to 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC) by a nonenzymatic, chemical degradation process. Previous studies have used HILIC to analyze the polar compound and to avoid degradation in aqueous solutions. (x10,000) 5.0 Plasma separation card HILIC analysis TMZ m/z > Q1 (V) -20 Collision energy -10 Q3 (V) Peak Area Plasma separation card HILIC analysis TMZ Single point calibration standards Calibration curve ug/mL ug/mL calibn std 0.5ug/mL calibn std min Linear regression analysis y = 64578x R² = Blood Concentration (ug/ml) Figure 3. HILIC LC/MS/MS chromatograms for PSC TMZ analysis at 0.5 and 8ug/mL. The PSC calibration curve was linear between ug/mL (r2>0.99). HILIC was considered in response to previous published data and to minimize potential stability issues. However, to reduce sample cycle times a reverse phase method was also developed. 4

51 Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS Reversed Phase LC/MS/MS (x10,000) Plasma separation card RP analysis TMZ m/z > Q1 (V) -20 Collision energy -10 Q3 (V) -12 Peak Area 800, , ,000 Plasma separation card RP analysis TMZ calibration curve Replicate calibration points at 0.5ug/mL and 8ug/mL (n=3) , ug/mL Calibration standard 0.5ug/mL Calibration standard 400, , , ,000 Linear regression analysis y = 72219x R² = min Blood Concentration (ug/ml) Figure 4. Reverse phase LC/MS/MS chromatograms for PSC TMZ analysis at 0.5 and 8ug/mL. The PSC calibration curve was linear between ug/mL (r2>0.99; replicate samples were included in the liner regression analysis at 0.5 and 8ug/mL; n=3). Due to the relatively long cycle time (18 min), a faster reversed phase method was developed (10 min). Sample preparation was modified with PSC sample disk placed in 40uL methanol + 0.1% formic acid, followed by centrifugation 16,000g 5 min. 20uL supernatant was added directly to the LC/MS sample vial plus 80uL water + 0.1% formic acid. In addition to reversed phase being faster, the sample injection volume was reduced to just 2uL as a result of higher sensitivity from narrower peak width (reversed phase,13 sec; HILIC, 42 sec). Comparison between PSC and plasma (x100) Matrix blank comparison MRM >67.05 Plasma separation card matrix blank Plasma matrix blank (x1,000) ng/mL comparison MRM >67.05 Plasma separation card 500ng/mL calibration standard Plasma 500ng/mL calibration standard min min Figure 5. Human blood TMZ calibration standards were prepared using PSC and conventional plasma. Using the confirmatory ion transition >67.05 both the PSC and plasma sample are in broad agreement with regard to matrix ion signal distribution. 5

52 Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS (x10,000) Matrix peak Matrix blank comparison MRM > Plasma separation card matrix blank Plasma matrix blank (x10,000) Matrix peak 500ng/mL comparison MRM > Plasma separation card 500ng/mL calibration standard Plasma 500ng/mL calibration standard TMZ TMZ Rt 1.7mins min min Figure 6. Human blood TMZ calibration standards were prepared using PSC and conventional plasma. Using the quantitation ion transition > both the PSC and plasma sample are in broad agreement in signal distribution and intensity including the presence of a matrix peak at 2.4mins. Conclusions This technology has the potential for a simplified clinical sample collection by the finger prick approach, with future work aimed to evaluate long term sample stability of PSC samples, stored at room temperature. Quantitation of drug metabolites MTIC and AIC also could help provide a measure of sample stability. References Andrasia, M., Bustosb, R., Gaspara,A., Gomezb, F.A. & Kleknerc, A. (2010) Analysis and stability study of temozolomide using capillary electrophoresis. Journal of Chromatography B. Vol. 878, p Denny, B.J., Wheelhouse, R.T., Stevens, M.F.G., Tsang, L.L.H., Slack, J.A., (1994) NMR and molecular modeliing investigation of the mechanism of activation of the antitumour drug temozolomide and its Interaction with DNA. Biochemistry, Vol. 33, p First Edition: June, For Research Use nly. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 2014

53 P-CN1475E Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans ASMS 2014 WP 629 Bicui Chen 1, Bin Wang 1, Xiaojin Shi 1, Yuling Song 2, Jinting Yao 2, Taohong Huang 2, Shin-ichi Kawano 2, Yuki Hashi 2 1 Pharmacy Department, Huashan Hospital, Fudan University, 2 Shimadzu Global CE, Shimadzu (China) Co., Ltd.

54 Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans Introduction Telbivudine is a synthetic L-nucleoside analogue, which is phosphorylated to its active metabolite, 5 -triphosphate, by cellular kinases. The telbivudine 5 -triphosphate inhibits HBV DNA polymerase (a reverse transcriptase) by competing with the natural substrate, dttp. Incorporation of 5 -triphosphorylated-telbivudine into viral DNA obligates DNA chain termination, resulting in inhibition of HBV replication. The objectives of the current studies were to develop a selective and sensitive LC-MS/MS method to determine of telbivudine in human plasma. Method Sample Preparation (1) Add 100 μl of plasma into the polypropylene tube, add 40 μl of internal standard working solution (33 µg/ml, with thymidine phosphorylase) to all other tubes. (2) Incubate the tubes for 1 h at 37 ºC in dark. (3) Add 200 μl of acetonitrile to all tubes, seal and vortex for 1 minutes. (4) Centrifuge the tubes for 5 minutes at rpm. (5) Transfer 200 μl supernatant to a clean glass bottle and inject into the HPLC-MS/MS system. LC-MS/MS Analysis The analysis was performed on a Shimadzu Nexera UHPLC instrument (Kyoto, Japan) equipped with LC-30AD pumps, CT-30A column oven, DGU-30A 5 on-line egasser, and SIL-30AC autosampler. The separation was carried out on GL Sciences InertSustain C18 column (3.0 mmi.d. x 100 mml.) with the column temperature at 40 ºC. A triple quadruple mass spectrometer (Shimadzu LCMS-8050, Kyoto, Japan) was connected to the UHPLC instrument via an ESI interface. Analytical Conditions HPLC (Nexera UHPLC system) Column : InertSustain (3.0 mmi.d. x 100 mml., 2 μm, GL Sciences) Mobile Phase A : water with 0.1% formic acid Mobile Phase B : acetonitrile Gradient Program : as shown in Table 1 Flow Rate : 0.4 ml/min Column Temperature : 40 ºC Injection Volume : 2 µl Table 1 Time Program Time (min) Module Command Value 0 Pumps Pump B Conc Pumps Pump B Conc Pumps Pump B Conc Controller Stop 2

55 Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans MS (LCMS-8050 triple quadrupole mass spectrometer) Ionization : ESI Polarity : Positive Ionization Voltage : +0.5 kv (ESI-Positive mode) Nebulizing Gas Flow : 3.0 L/min Heating Gas Flow : 8.0 L/min Drying Gas Flow : 12.0 L/min Interface Temperature : 250 ºC Heat Block Temperature : 300 ºC DL Temperature : 350 ºC Mode : MRM Table 2 MRM Parameters Compound Precursor m/z Product m/z Dwell Time (ms) Q1 Pre Bias (V) CE (V) Q3 Pre Bias (V) Telbivudine Telbivudine-D Results and Discussion Human plasma samples containing telbivudine ranging from to ng/ml were prepared and extracted by protein precipitation and the final extracts were analyzed by LC-MS/MS. MRM chromatograms of telbivudine (1 ng/ml) and deuterated internal standard are presented in Fig. 1 (blank) and Fig. 2 (spiked). The linear regression for telbivudine was found to be > The calibration curve with human plasma as the matrix were shown in Fig. 3. Excellent precision and accuracy were maintained for four orders of magnitude, demonstrating a linear dynamic range suitable for real-world applications. LLQ for telbivudine was ng/ml, which met the criteria for bias (%) and precision within ±15% both within run and between run. The intra-day and inter-day precision and accuracy of the assay were investigated by analyzing QC samples. Intra-day precision (%RSD) at three concentration levels (3, 30, and 8000 ng/ml) were below 2.5% and inter-day precision (%RSD) was below 2.5%. The recoveries of telbivudine were 100.6±2.5 %, 104.5±1.5% and 104.3±1.6% at three concentration levels, respectively. The stability data showed that the processed samples were stable at the room temperature for 8 h, and there was no significant degradation during the three freeze/thaw cycles at -20 ºC. The reinjection reproducibility results indicated that the extracted samples could be stable for 72 h at 10 ºC. 3

56 Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans 4.0 (x100) 1:Telbivudine >127.10(+) CE: (x1,000) 2:Telbivudine-D >130.10(+) CE: min min Figure 1 Representative MRM chromatograms of blank human plasma (left: transition for telbivudine, right: transition for internal standard) (x100) 1:Telbivudine >127.10(+) CE: -1 Telbivudine (x1,000,000) :Telbivudine-D >130.10(+) CE: Telbivudine-D min min Figure 2 Representative MRM chromatograms of telbivudine (left, 1 ng/ml) and internal standard (right) in human plasma Area Ratio Conc. Ratio Figure 3 Calibration curve of telbivudine in human plasma 4

57 Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans Compound Calibration Curve Linear Range (ng/ml) Accuracy (%) r Telbivudine Y = ( )X + ( ) 1~ ~116.6% Table 3 Accuracy and precision for the analysis of amlodipine in human plasma (in pre-study validation, n=3 days, six replicates per day) Added Conc. (ng/ml) Intra-day Precision (%RSD) Inter-day Precision (%RSD) Accuracy (%) ~ ~ ~101.3 QC Level LQC MQC HQC Table 4 Recovery for QC samples (n=6) Concentartion (ng/ml) Recovery (%) Table 5 Matrix effect for QC samples (n=6) QC Level Added Conc. (ng/ml) Matrix Factor IS-Normalized Matrix Factor LQC % 99.0% MQC % 10% HQC % 101.5% 3.0 (x10,000) 1:Telbivudine >127.10(+) CE: -1 (x1,000,000) 2:Telbivudine-D >130.10(+) CE: min min Figure 4 Representative MRM chromatograms of real-world sample 5

58 Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans Conclusion Results of parameters for method validation such as dynamic range, linearity, LLQ, intra-day precision, inter-day precision, recoveries, and matrix effect factors were excellent. The sensitive LC-MS/MS technique provides a powerful tool for the high-throughput and highly selective analysis of telbivudine in clinical trial study. First Edition: June, For Research Use nly. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 2014

59 P-CN1449E Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry ASMS 2014 WP628 Natsuyo Asano 1, Tairo gura 1, Kiyomi Arakawa 1 1 Shimadzu Corporation. 1, Nishinokyo Kuwabara-cho, Nakagyo-ku, Kyoto , Japan

60 Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry Introduction Immunosuppressants are drugs which lower or suppress activity of the immune system. They are used to prevent the rejection after transplantation or treat autoimmune disease. To avoid immunodeficiency as adverse effect, it is recommended to monitor blood level of therapeutic drug with high throughput and high reliability. There are several analytical technique to monitor drugs, LC/MS is superior in terms of cross-reactivity at low level and throughput of analysis. Therefore, it is important to analyze these drugs in blood by using ultra-fast mass spectrometer to accelerate monitoring with high quantitativity. We have developed analytical method for four immunosuppressants (Tacrolimus, Rapamycin, Everolimus and Cyclosporin A) with two internal standards (Ascomycin and Cyclosporin D) using ultra-fast mass spectrometer. H H H N H H H N H H N H H Tacrolimus MW: Rapamycin MW: Everolimus MW: N N N H N H N H N N H H N H N N N H H N H H H HN N N N H N H N N H H N N N N Cyclosporin A MW: Ascomycin (IS) MW: Cyclosporin D (IS) MW: Figure 1 Structure of immunosuppressants and internal standards (IS) 2

61 Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry Methods and Materials Standard samples of each compound were analyzed to optimize conditions of liquid chromatograph and mass spectrometer. Whole blood extract was prepared based on liquid-liquid extraction described bellow. 2.7 ml of Whole blood and 20.8 ml of Water Vortex for 15 seconds Add 36 ml of MTBE/Cyclohexane (1:3) Vortex for 15 seconds and Centrifuge with 3000 rpm at 20 ºC for 10 minutes Extract an rganic phase Evaporate and Dry under a Nitrogen gas stream Redissolve in 1.8 ml of 80 % Methanol solution with 1 mmol/l Ammonium acetate Vortex for 1 minute and Centrifuge with 3000 rpm at 4 ºC for 5 minutes Filtrate and Transfer into 1 ml glass vial Table 1 Analytical conditions UHPLC Liquid Chromatograph : Nexera (Shimadzu, Japan) Analysis Column : YMC-Triart C18 (30 mml. 2 mmi.d.,1.9 μm) Mobile Phase A : 1 mmol/l Ammonium acetate - Water Mobile Phase B : 1 mmol/l Ammonium acetate - Methanol Gradient Program : 60 % B. (0 min) 75 % B. (0.10 min) 95 % B. ( min) 60 % B. ( min) Flow Rate : 0.45 ml/min Column Temperature : 65 ºC Injection Volume : 1.5 µl MS MS Spectrometer : LCMS-8050 (Shimadzu, Japan) Ionization : ESI (negative) Probe Voltage : -4.5 ~ -3 kv Nebulizing Gas Flow : 3.0 L/min Drying Gas Flow : 5.0 L/min Heating Gas Flow : 15.0 L/min Interface Temperature : 400 ºC DL Temperature : 150 ºC HB Temperature : 390 ºC 3

62 Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry Result Immunosuppressants, which we have developed a method for monitoring of, has been often observed as ammonium or sodium adduct ion by using positive ionization. In general, protonated molecule (for positive) or deprotonated molecule (for negative) is more preferable for reliable quantitation than adduct ions such as ammonium, sodium, and potassium adduct. In this study, each compound was detected as deprotonated molecule in negative mode by using heated ESI source of LCMS-8050 (Table 2). The separation of all compounds was achieved within 1.8 min, with a YMC-Triart C18 column (30 mml. 2 mmi.d.,1.9 μm) and at 65 ºC of column oven temperature. (x100,000) min Figure 2 MRM chromatograms of immnosuppresants in human whole blood (50 ng/ml) Table 2 MRM transitions Peak No. Compound Porality Precursor ion (m/z) Product ion (m/z) 1 Ascomysin (IS) neg Tacrolimus neg Rapamycin neg Everolimus neg Cyclosporin A neg Cyclosporin D (IS) neg

63 Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry a) Tacrolimus 0.5 ng/ml Ascomycin 40 ng/ml ng/ml b) Rapamycin 0.5 ng/ml Ascomycin 40 ng/ml ng/ml c) Everolimus 0.5 ng/ml Ascomycin 40 ng/ml ng/ml d) Cyclosporin A 0.5 ng/ml Cyclosporin D 100 ng/ml ng/ml Figure 3 MRM chromatograms at LLQ and ISTD (left), and calibration curves (right) for four immnosuppresants in human whole blood 5

64 Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry Figure 3 illustrates both a calibration curve and chromatogram at the lowest calibration level for all immunosuppressants analyzed. Table 3 lists both the reproducibility and accuracy for each immunosuppressant that has been simultaneously measured in 1.8 minutes. Table 3 Reproducibility and Accuracy Compound Concentration CV % (n = 6) Accuracy % Tacrolimus Low (0.5 ng/ml) Low-Mid (2 ng/ml) High (1000 ng/ml) Rapamycin Low (0.5 ng/ml) Low-Mid (5 ng/ml) High (500 ng/ml) Everolimus Low (0.5 ng/ml) Low-Mid (5 ng/ml) High (100 ng/ml) Cyclosporin A Low (0.5 ng/ml) Low-Mid (10 ng/ml) High (1000 ng/ml) In high speed measurement condition, we have achieved high sensitivity and wide dynamic range for all analytes. Additionally, the accuracy of each analyte ranged from 88 to 110 % and area reproducibility at the lowest calibration level of each analyte was less than 20%. Conclusions Monitoring with negative mode ionization permitted more sensitive, robust and reliable quantitation for four immunosuppressants. A total of six compounds were measured in 1.8 minutes. The combination of Nexera and LCMS-8050 provided a faster run time without sacrificing the quality of results. Even with a low injection volume of 1.5 μl, the lower limit of quantitation (LLQ) for all compounds was 0.5 ng/ml. In this study, it is demonstrated that LCMS-8050 is useful for the rugged and rapid quantitation for immunosuppressants in whole blood. Acknowledgement We appreciate suggestions from Prof. Kazuo Matsubara and Assoc. Prof. Ikuko Yano from the department of pharmacy, Kyoto University Hospital, and Prof. Satohiro Masuda from the department of pharmacy, Kyusyu University Hospital. First Edition: June, For Research Use nly. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 2014

65 P-CN1468E Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS ASMS 2014 TP497 Shailendra Rane, Rashi Kochhar, Deepti Bhandarkar, Shruti Raju, Shailesh Damale, Ajit Datar, Pratap Rasam, Jitendra Kelkar Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh Chambers, Makwana Road, Marol, Andheri (E), Mumbai , Maharashtra, India.

66 Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS Introduction Felodipine is a calcium antagonist (calcium channel blocker), used as a drug to control hypertension [1]. Hydrochlorothiazide is a diuretic drug of the thiazide class that acts by inhibiting the kidney s ability to retain water. It is, therefore, frequently used for the treatment of hypertension, congestive heart failure, symptomatic edema, diabetes insipidus, renal tubular acidosis and the prevention of kidney stones [2]. Efforts have been made here to develop high sensitive methods of quantitation for these two drugs using LCMS-8050 system from Shimadzu Corporation, Japan. Presence of heated Electro Spray Ionization (ESI) probe in LCMS-8050 ensured good quantitation and repeatability even in the presence of a complex matrix like plasma. Ultra high sensitivity of LCMS-8050 enabled development quantitation method at low ppt level for both Felodipine and Hydrochlorthiazide. Felodipine Figure 1. Structure of Felodipine Felodipine is a calcium antagonist (calcium channel blocker). Felodipine is a dihydropyridine derivative that is chemically described as ± ethyl methyl 4-(2,3-dichlorophenyl)1,4-dihydro-2,6-dimethyl-3,5-pyridin edicarboxylate. Its empirical formula is C 18 H 19 Cl 2 N 4 and its structure is shown in Figure 1. Hydrochlorothiazide Figure 2. Structure of Hydrochlorothiazide Hydrochlorothiazide, abbreviated HCTZ (or HCT, HZT), is a diuretic drug of the thiazide class that acts by inhibiting the kidney s ability to retain water. Hydrochlorothiazide is 6-chloro-1,1-dioxo-3,4-dihydro-2H-1,2,4-benzothiadiazine- 7-sulfonamide.Its empirical formula is C 7 H 8 ClN 3 4 S 2 and its structure is shown in Figure 2. Method of Analysis Preparation of matrix matched plasma by protein precipitation method using cold acetonitrile To 100 µl of plasma, 500 µl of cold acetonitrile was added for protein precipitation then put in rotary shaker at 20 rpm for 15 minutes for uniform mixing. It was centrifuged at rpm for 15 minutes. Supernatant was collected and evaporated to dryness at 70 ºC and finally reconstituted in 200 µl Methanol. 2

67 Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS Preparation of matrix matched plasma by liquid-liquid extraction method using diethyl ether and hexane mixture (1:1 v/v) To 500 µl plasma, 100 µl sodium carbonate (0 mol/l) and 5 ml of diethyl ether : hexane (1:1 v/v) was added. It was placed in rotary shaker at 20 rpm for 15 minutes for uniform mixing and centrifuged at rpm for 15 minutes. Supernatant was collected and evaporated to dryness at 60 ºC. It was finally reconstitute in 1000 µl Methanol. Preparation of calibration standards in matrix matched plasma Response of Felodipine and Hydrochlorothiazide were checked in both above mentioned matrices. It was found that cold acetonitrile treated plasma and diethyl ether: hexane (1:1 v/v) treated plasma were suitable for Felodipine Calibration Std : 5 ppt, 10 ppt, 50 ppt, 100 ppt, 500 ppt, 1 ppb and 10 ppb HCTZ Calibration Std : 2 ppt, 5 ppt, 10 ppt, 50 ppt, 100 ppt, and 500 ppt Felodipine and Hydrochlorothiazide molecules respectively. Calibration standards were thus prepared in respective matrix matched plasma. Figure 3. LCMS-8050 triple quadrupole mass spectrometer by Shimadzu Figure 4. Heated ESI probe LCMS-8050 triple quadrupole mass spectrometer by Shimadzu (shown in Figure 3), sets a new benchmark in triple quadrupole technology with an unsurpassed sensitivity (UFsensitivity), Ultra fast scanning speed of 30,000 u/sec (UFscanning) and polarity switching speed of 5 msecs (UFswitching). This system ensures highest quality of data, with very high degree of reliability. In order to improve ionization efficiency, the newly developed heated ESI probe (shown in Figure 4) combines high-temperature gas with the nebulizer spray, assisting in the desolvation of large droplets and enhancing ionization. This development allows high-sensitivity analysis of a wide range of target compounds with considerable reduction in background. LC/MS/MS analysis Compounds were analyzed using Ultra High Performance Liquid Chromatography (UHPLC) Nexera coupled with LCMS-8050 triple quadrupole system (Shimadzu Corporation, Japan), The details of analytical conditions are given in Table 1 and Table 2. 3

68 Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS Table 1. LC/MS/MS conditions for Felodipine Column : Shim-pack XR-DS (75 mm L x 3 mm I.D.; 2.2 µm) Flow rate : 0.3 ml/min ven temperature : 40 ºC Mobile phase : A: 10 mm ammonium acetate in water B: methanol Gradient program (%B) : 3.0 min 90 (%); min (%); min 100 (%); min (%) min 90 (%) Injection volume : 10 µl MS interface : ESI Nitrogen gas flow : Nebulizing gas 1.5 L/min; Drying gas 10 L/min; Zero air flow : Heating gas 10 L/min MS temperature : Desolvation line 200 ºC; Heating block 400 ºC Interface 200 ºC Table 2. LC/MS/MS conditions for Hydrochlorothiazide Column : Shim-pack XR-DS (100 mm L x 3 mm I.D.; 2.2 µm) Flow rate : 0.2 ml/min ven temperature : 40 ºC Mobile phase : A: 0.1% formic acid in water B: acetonitrile Gradient program (%B) : min 80 (%); 3.5 min (%); min 100 (%); min (%) min 90 (%) Injection volume : 25 µl MS interface : ESI Nitrogen gas flow : Nebulizing gas 2.0 L/min; Drying gas 10 L/min; Zero air flow : Heating gas 15 L/min MS temperature : Desolvation line 250 ºC; Heating block 500 ºC Interface 300 ºC Results LC/MS/MS analysis results of Felodipine LC/MS/MS method for Felodipine was developed on ESI positive ionization mode and > MRM transition was optimized for it. Checked matrix matched plasma standards for highest (10 ppb) as well as lowest concentrations (5 ppt) as seen in Figure 5 and Figure 6 respectively. Calibration curves as mentioned with R 2 = were plotted (shown in Figure 7). Also as seen in Table 3, % Accuracy was studied to confirm the reliability of method. Also, LD as 2 ppt and LQ as 5 ppt was obtained. 4

69 Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS 5.0 (x100,000) >338.25(+) 2.0 (x1,000) >338.25(+) 2.5 FELDIPINE FELDIPINE Figure 5. Felodipine at 10 ppb in matrix matched plasma Figure 6. Felodipine at 5 ppt in matrix matched plasma Table 3: Results of Felodipine calibration curve Sr. No. Standard Nominal Concentration (ppb) Measured Concentration (ppb) % Accuracy (n=3) % RSD for area counts (n=3) 1 STD-FEL STD-FEL STD-FEL STD-FEL STD-FEL STD-FEL STD-FEL Area (x1,000,000) Conc. Area (x10,000) Conc. Figure 7. Calibration curve of Felodipine LC/MS/MS analysis results of Hydrochlorothiazide LC/MS/MS method for Hydrochlorothiazide was developed on ESI negative ionization mode and > MRM transition was optimized for it. Checked matrix matched plasma standards for highest (500 ppt) as well as lowest (2 ppt) concentrations as seen in Figures 8 and 9 respectively. Calibration curves as mentioned with R 2 =0.998 were plotted (shown in Figure 10). Also as seen in Table 4, % Accuracy was studied to confirm the reliability of method. Also, LD as 1 ppt and LQ as 2 ppt were obtained. 5

70 Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS 1.5 (x10,000) >204.90(-) HCTZ (x100) >204.90(-) HCTZ Figure 8. Hydrochlorothiazide at 500 ppt in matrix matched plasma Figure 9. Hydrochlorothiazide at 2 ppt in matrix matched plasma Table 4. Results of Hydrochlorothiazide calibration curve Sr. No. Standard Nominal Concentration (ppb) Measured Concentration (ppb) % Accuracy (n=3) % RSD for area counts (n=3) 1 STD-HCTZ STD-HCTZ STD-HCTZ STD-HCTZ STD-HCTZ STD-HCTZ Area (x100,000) Area (x10,000) Conc Conc. Figure 10. Calibration curve of Hydrochlorothiazide Conclusion Highly sensitive LC/MS/MS method for Felodipine and Hydrochlorothiazide was developed on LCMS-8050 system. LD of 2 ppt and LQ of 5 ppt was achieved for Felodipine and LD of 1 ppt and LQ of 2 ppt was achieved for Hydrochlorothiazide by matrix matched methods. Heated ESI probe of LCMS-8050 system enables drastic augment in sensitivity with considerable reduction in background. Hence, LCMS-8050 system from Shimadzu is an all rounder solution for bioanalysis. 6

71 Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS References [1] YU Peng; CHENG Hang, Chinese Journal of Pharmaceutical Analysis, Volume 32, Number 1, (2012), 35-39(5). [2] Hiten Janardan Shah, Naresh B. Kataria, Chromatographia, Volume 69, Issue 9-10, (2009), First Edition: June, For Research Use nly. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 2014

72 P-CN1467E Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS ASMS 2014 TP496 Shruti Raju, Deepti Bhandarkar, Rashi Kochhar, Shailesh Damale, Shailendra Rane, Ajit Datar, Pratap Rasam, Jitendra Kelkar Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh Chambers, Makwana Road, Marol, Andheri (E), Mumbai , Maharashtra, India.

73 Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS Introduction The toxicological assessment of Genotoxic Impurities (GTI) and the determination of acceptable limits for such impurities in Active Pharmaceutical Ingredients (API) is a difficult issue. As per European Medicines Agency (EMEA) guidance, a Threshold of Toxicological Concern (TTC) value of 1.5 µg/day intake of a genotoxic impurity is considered to be acceptable for most pharmaceuticals [1]. Dronedarone is a drug mainly used for indications of cardiac arrhythmias. GTI of this drug has been quantitated here. Method has been optimized for simultaneous analysis of DRN-IA {2-n-butyl-3-[4-(3-di-n-butylamino-propoxy)benzoyl]-5-nitro benzofuran}, DRN-IB {5-amino-3-[4-(3-di-n-butylamino-propoxy)benzoyl}-2-n-but yl benzofuran} and BHBNB {2-n-butyl-3-(4-hydroxy benzoyl)-5-nitro benzofuran}. Structures of Dronedarone and its GTI are shown in Figure 1. As literature references available on GTI analysis are minimal, the feature of automatic MRM optimisation in LCMS-8040 makes method development process less tedious. In addition, the lowest dwell time and pause time and ultrafast polarity switching of LCMS-8040 ensures uncompromised and high sensitive quantitation. C 4 H 9 C 4 H 9 N C 4 H 9 N C 4 H 9 NHS 2 Me N 2 C 4 H 9 C 4 H 9 Dronedarone C 4 H 9 DRN-IA N C 4 H 9 H N 2 NH 2 C 4 H 9 DRN-IB BHBNB C 4 H 9 Figure 1. Structures of Dronedarone and its GTI 2

74 Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS Method of Analysis Sample Preparation Preparation of DRN-IA and DRN-IB and BHBNB stock solutions 20 mg of each impurity standard was weighed separately and dissolved in 20 ml of methanol to prepare stock solutions of each standard. Preparation of calibration levels GTI mix standards (DRN-IA, DRN-IB and BHBNB) at concentration levels of 0.5 ppb, 1 ppb, 5 ppb, 10 ppb, 40 ppb, 50 ppb and 100 ppb were prepared in methanol using stock solutions of all the three standards. Preparation of blank sample 400 mg of Dronedarone powder sample was weighed and mixed with 20 ml of methanol. Mixture was sonicated to dissolve sample completely. Preparation of spiked (at 12 ppb level) sample 400 mg of sample was weighed and spiked with 60 µl of 1 ppm stock solution. Solution was then mixed with 20 ml of methanol. Mixture was sonicated to dissolve sample completely. LC/MS/MS Analytical Conditions Analysis was performed using Ultra High Performance Liquid Chromatography (UHPLC) Nexera coupled with LCMS-8040 triple quadrupole system (Shimadzu Corporation, Japan), shown in Figure 2. Limit of GTI for Dronedarone is 2 mg/kg. However, general dosage of Dronedarone is 400 mg, hence, limit for GTI is 0.8 µg/400 mg. This when reconstituted in 20 ml system, gives an effective concentration of 40 ppb. For analytical method development it is desirable to have LQ at least 30 % of limit value, which in this case corresponds to 12 ppb. The developed method gives provision for measuring GTI at much lower level. However, recovery studies have been done at 12 ppb level. Figure 2. Nexera with LCMS-8040 triple quadrupole system by Shimadzu 3

75 Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS Below mentioned table shows the analytical conditions used for analysis of GTI. Table 1. LC/MS/MS analytical conditions Column : Shim-pack XR-DS II (75 mm L x 3 mm I.D.; 2.2 µm) Mobile phase : A: 0.1% formic acid in water B: acetonitrile Flow rate : 0.3 ml/min ven temperature : 40 ºC Gradient program (B%) : 2.0 min 35 (%); min (%); min (%); min (%); min 100 (%); 1 11 min (%); min 35 (%) Injection volume : 1 µl MS interface : Electro Spray Ionization (ESI) MS analysis mode : MRM Polarity : Positive and negative MS gas flow : Nebulizing gas 2 L/min; Drying gas 15 L/min MS temperature : Desolvation line 250 ºC; Heat block 400 ºC Note: Flow Control Valve (FCV) was used for the analysis to divert HPLC flow towards waste during elution of Dronedarone so as to prevent contamination of Mass Spectrometer. Results LC/MS/MS analysis LC/MS/MS method was developed for simultaneous quantitation of GTI mix standards. MRM transitions used for all GTI are given in Table 2. No peak was seen in diluent (methanol) at the retention times of GTI for selected MRM transitions which confirms the absence of any interference from diluent (shown in Figure 3). MRM chromatogram of GTI mix standard at 5 ppb level is shown in Figure 4. Linearity studies were carried out using external standard calibration method. Calibration graphs of each GTI are shown in Figure 5. LQ was determined for each GTI based on the following criteria (1) % RSD for area < 15 %, (2) % Accuracy between % and (3) Signal to noise ratio (S/N) > 10. LQ of 0.5 ppb was achieved for DRN-IB and BHBNB whereas 1 ppb was achieved for DRN-IA. Results of accuracy and repeatability for all GTI are given in Table 3. Table 2: MRM transitions selected for all GTI Name of GTI MRM transition Retention time (min) Mode of ionization DRN-IB > Positive ESI DRN-IA > Positive ESI BHBNB > Negative ESI 4

76 Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS :DRA-IB >170.15(+) CE: :DRA-IA >114.10(+) CE: -4 3:BHBNB >244.05(-) CE: min Figure 3. MRM chromatogram of diluent (methanol) 1:DRA-IB >170.15(+) CE: :DRA-IA >114.10(+) CE: -4 3:BHBNB >244.05(-) CE: DRN-IB > DRN-IA > BHBNB > min Figure 4. MRM chromatogram of GTI mix standard at 5 ppb level Area Area Area DRN-IB R DRN-IA R BHBNB R Conc Conc Conc. Figure 5. Calibration graphs for GTI 5

77 Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS Table 3: Results of accuracy and repeatability for all GTI Sr. No. Name of GTI Standard concentration (ppb) Calculated concentration from calibration graph (ppb) (n=6) % Accuracy (n=6) % RSD for area counts (n=6) DRN-IB DRN-IA BHBNB Recovery studies For recovery studies, samples were prepared as described previously. MRM chromatogram of blank and spiked samples are shown in Figures 6 and 7 respectively. Results of recovery studies have been shown in Table 4. Recovery could not be calculated for DRN-IB as blank sample showed higher concentration than spiked concentration :DRA-IB >170.15(+) CE: :DRA-IA >114.10(+) CE: -4 3:BHBNB >244.05(-) CE: DRN-IB > DRN-IA > BHBNB > min Figure 6. MRM chromatogram of blank sample 6

78 Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS :DRA-IB >170.15(+) CE: :DRA-IA >114.10(+) CE: -4 3:BHBNB >244.05(-) CE: DRN-IB > DRN-IA > BHBNB > min Figure 7. MRM chromatogram of spiked sample Table 4. Results of the recovery studies Name of Impurity Concentration of GTI mix standard spiked in blank sample (ppb) Average concentration obtained from calibration graph for blank sample (ppb) (A) (n=3) Average concentration obtained from calibration graph for spiked sample (ppb) (B) (n=3) % Recovery = (B-A)/ 12 * 100 DRN-IB NA NA DRN-IA BHBNB Conclusion A highly sensitive method was developed for analysis of GTI of Dronedarone. Ultra high sensitivity, ultra fast polarity switching (UFswitching) enabled sensitive, selective, accurate and reproducible analysis of GTI from Dronedarone powder sample. References [1] Guideline on The Limits of Genotoxic Impurities, (2006), European Medicines Agency (EMEA). First Edition: June, For Research Use nly. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 2014

79 P-CN1470E Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum ASMS 2014 WP449 Daryl Kim Hor Hee 1, Lawrence Soon-U Lee 1, Zhi Wei Edwin Ting 2, Jie Xing 2, Sandhya Nargund 2, Miho Kawashima 3 & Zhaoqi Zhan 2 1 Department of Medicine Research Laboratories, National University of Singapore, 6 Science Drive 2, Singapore Customer Support Centre, Shimadzu (Asia Pacific) Pte Ltd, 79 Science Park Drive, #02-01/08, Singapore Global Application Development Centre, Shimadzu Corporation, 1-3 Kanda Nishihiki-cho, Chiyoda-ku, Tokyo , Japan

80 Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum Introduction Developments of LC/MS/MS methods for accurate quantitation of low pg/ml levels of 1α,25-dihydroxy vitamin D2/D3 in serum were reported in recent years, because their levels in serum were found to be important indications of several diseases associated with vitamin D metabolic disorder in clinical research and diagnosis [1]. However, it has been a challenge to achieve the required sensitivity directly, due to the intrinsic difficulty of ionization of the compounds and interference from matrix [2,3]. Sample extraction and clean-up with SPE and immunoaffinity methods were applied to remove the interferences [4] prior to LC/MS/MS analysis. However, the Experimental High purity 1α,25-dihydroxyl Vitamin D3 and deuterated 1α,25-dihydroxyl-d6 Vitamin D3 (as internal standard) were obtained from Toronto Research Chemicals. Charcoal-stripped pooled human serum obtained from Bioworld was used as blank and matrix to prepare spiked samples in this study. A 2D-LC/MS/MS system was set up on LCMS-8050 (Shimadzu Corporation) with a column switching valve installed in the column oven and controlled by LabSolutions workstation. The details of columns, mobile phases and gradient programs of 1 st -D and 2 nd -D LC amount of serum required was normally rather high from 0.5mL to 2mL, which is not favourite in the clinical applications. Direct analysis methods with using smaller amount of serum are in demand. Research efforts have been reported in literatures to enhance ionization efficiency by using different interfaces such as ESI, APCI or APPI and ionization reagents to form purposely NH3 adduct or lithium adduct [4,5]. Here, we present a novel 2D-LC/MS/MS method with APCI interface for direct analysis of 1α,25-diH-VD3 in serum. The method achieved a detection limit of 3.1 pg/ml in spiked serum samples with 100 ul injection. separations and MS conditions are compiled into Table 1. The procedure of sample preparation of spiked serum samples is shown in Figure 1. It includes protein precipitation by adding ACN-MeH solvent into the serum in 3 to 1 ratio followed by vortex and centrifuge at high speed. The supernatant collected was filtered before standards with IS were added (post-addition). The clear samples obtained were then injected into the 2-D LC/MS/MS system. Table 1: 2D-LC/MS/MS analytical conditions LC condition MS Interface condition Column 1 st D: FC-DS (2.0mml.D. x 75mm L, 3μm) 2 nd D: VP-DS (2.0mmI.D. x 150mm L, 4.6μm) Interface MS mode Heat Block & DL Temp. CID Gas Nebulizing Gas Flow Drying Gas Flow APCI, 400ºC Positive, MRM 300ºC & 200ºC Ar (270kPa) N2, 2.5 L/min N2, 7.0 L/min Mobile Phase of 1 st D A: Water with 0.1% formic acid B: Acetontrile Mobile Phase of 2 nd D C: Water with 0.1% formic acid D: MeH with 0.1% formic acid 1 st D gradient program & flow rate B: 40% (0 to 0.1min) 90% (5 to 7.5min) 15% (11 to 12min) 40% (14 to 25min); Total flow rate: 0.5mL/min 2 nd D gradient program & flow rate D: 15% (0min) 80% (20 to 22.5min) 15% (23 to 25min); Peak cutting: 3.15 to 3.40; Total flow rate: 0.5 ml/min ven Temp. Injection Vol. 45ºC 100 ul 2

81 Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum 150µL of serum 450µL of ACN/MeH (1:1) Shake and Vortex 10mins Centrifuge for 10 minutes at 13000rpm 480µL of Supernatant 0.2µm nylon filter 400µL of filtered protein precipitated Serum 50µL of of Std stock 50µL of IS stock 500µL of calibrate Figure 1: Flow chart of serum sample pre-treatment method Results and Discussion Development of 2D-LC/MS/MS method An APCI interference was employed for effective ionization of 1α,25-diH-VitD3 (C 27 H 44 3, MW 416.7). A MRM quantitation method for 1α,25-diH-VitD3 with its deuterated form as internal standard (IS) was developed. MRM optimization was performed using an automated MRM optimization program with LabSolutions workstation. Two MRM transitions for each compound were selected (Table 2), the first one for quantitation and the second one for confirmation. The parent ion of 1α,25-diH-VitD3 was the dehydrated ion, as it underwent neutral lost easily in ionization with ESI and APCI [2,3]. The MRM used for quantitation (399.3>381.3) was dehydration of the second H group in the molecule. Table 2: MRM transitions and CID parameters of 1α,25-diH-VitD3 and deuterated IS Name RT 1 (min) Transition (m/z) Q1 Pre Bias CID Voltage (V) CE Q3 Pre Bias 1α,25-dihydroxyl Vitamin D > > α,25-dihydroxyl-d6 Vitamin D3 (IS) > > , Retention time by 2D-LC/MS/MS method 3

82 Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum The reason to develop a 2-D LC separation for a LC/MS/MS method was the high background and interferences occurred with 1D LC/MS/MS observed in this study and also reported in literatures. Figure 2 shows the MRM chromatograms of 1D-LC/MS/MS of spiked serum sample. It can be seen that the baseline of the quantitation MRM (399.3>381.3) rose to a rather high level and interference peaks also appeared at the same retention time. The 2-D LC/MS/MS method developed in this study involves cutting the targeted peak in the 1 st -D separation precisely (3.1~3.4 min) and the portion retained in a stainless steel sample loop (200 ul) was transferred into the 2 nd -D column for further separation. The operation was accomplished by switching the 6-way valve in and out by a time program. Both 1 st -D and 2 nd -D separations were carried out in gradient elution mode. The organic mobile phase of 2 nd -D (MeH with 0.1% formic acid) was different from that of 1 st -D (pure ACN). The interference peaks co-eluted with the analyte in 1 st -D were separated from the analyte peak (22.6 min) as shown in Figure 3. 1:H2D >381.30(+) CE: :H2D >157.00(+) CE: :H2D >105.00(+) CE: H2-VD min 700 2:H2D3-D >383.30(+) CE: :H2D3-D >366.30(+) CE: min Peak cutting (125 ul) in 1 st D separation and transferred to 2 nd D LC H2-VD3-D3 Figure 2: 1D-LC/MS/MS chromatograms of 22.7 pg/ml 1α,25-diH-VitD3 (top) and 182 pg/ml internal standard (bottom) in serum (injection volume: 50uL) Calibration curve (IS), linearity and accuracy Two sets of standard samples were prepared in serum and in clear solution (diluent). Each set included seven levels of 1α,25-diH-VitD3 from 3.13 pg/ml to 200 pg/ml, each added with 200 pg/ml of IS (See Table 3). The chromatograms of the seven spiked standard samples in serum are shown in Figure 3. A linear IS calibration curve (R2 > 0.996) was established from these 2D-LC/MS/MS analysis results, which is shown in Figure 4. It is worth to note that the calibration curve has a non-zero Y-intercept, indicating that the blank (serum) contains either residual 1 α,25-dih-vitd3 or other interference which must be deducted in the quantitation method. The peak area ratios shown in Table 3 are the results after deduction of the background peaks. The accuracy of the method after this correction is between 92% and 117%. Area Ratio α,25-diH-VitD3 5.0 R2 = min min Figure 3: verlay of 2 nd -D chromatograms of 7 levels from 3.13 pg/ml to 200 pg/ml spiked in serum. Non-zero intercept Conc. Ratio Figure 4: Calibration curves of 1α,25-diH VD3 in serum by IS method. 4

83 Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum Table 3: Seven levels of standard samples for calibration curve and performance evaluation Conc. Level of Std. 1α,25-diH VD3 (pg/ml) Conc. Ratio 1 (Target/IS) Area Ratio 2 (in serum) Area Ratio 2 (in clear solu) Accuracy 3 (%) Matrix Effect (%) L L L L L L L , Target = 1α,25-diH VD3; 2, Area ratio = area of target / area of IS; 3, Based on the data of spiked serum samples Matrix effect, repeatability, LD/LQ and specificity Matrix effect of the 2D-LC/MS/MS method was determined by comparison of peak area ratios of standard samples in diluent and in serum at the seven levels. The results are compiled into Table 3. The matrix effect of the method are between 58% and 95%. It seems that the matrix effect is stronger at lower concentrations than at higher concentrations. Repeatability of peak area of the method was evaluated with L2 and L3 spiked serum samples for both target and IS. The Results of RSD (n=6) are displayed in Table 4. The MRM peaks of 1α,25-diH VD3 in clear solution and in serum are displayed in pairs (top and bottom) in Figure 5. It can be seen from the first pair (diluent and serum blank) that a peak appeared at the same retention of 1α,25-diH VD3 in the blank serum. As pointed out above, this peak is from either the residue of 1α,25-diH VD3 or other interference present in the serum. Due to this background peak, the actual S/N ratio could not be calculated. Therefore, it is difficult to determine the LD and LQ based on the S/N method. Tentatively, we propose a reference LD and LQ of the method for 1α,25-diH VD3 to be 3.1 pg/ml and 10 pg/ml, respectively. The specificity of the method relies on several criteria: two MRMs (399>381 and 399>157), their ratio and RT in 2 nd -D chromatogram. The MRM chromatograms shown in Figure 5 demonstrate the specificity of the method from L1 (3.1 pg/ml) to L7 (200 pg/ml). It can be seen that the results of spiked serum samples (bottom) meet the criteria if compared with the results of samples in the diluent (top) :399.30>381.30(+) 1:399.30>157.00(+) Diluent :399.30>381.30(+) 1:399.30>157.00(+) :399.30>381.30(+) 1:399.30>157.00(+) 1:399.30>381.30(+) :399.30>157.00(+) H2VD3/ :399.30>381.30(+) :399.30>157.00(+) L1 L3 L5 L7 H2VD3/ H2VD3/ H2VD3/ :399.30>381.30(+) 1:399.30>157.00(+) H2VD3/ Serum blank :399.30>381.30(+) 1:399.30>157.00(+) H2VD3/ :399.30>381.30(+) 1:399.30>157.00(+) H2VD3/ :399.30>381.30(+) 1:399.30>157.00(+) H2VD3/ :399.30>381.30(+) :399.30>157.00(+) L1 L3 L5 L7 H2VD3/ Figure 5: MRM peaks of 1α,25-diH-VitD3 spiked in pure diluent (top) and in serum (bottom) of L1, L3, L5 and L7 (spiked conc. refer to Table 3) 5

84 Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum Table 4: Repeatability Test Results (n=6) Sample Compound Spiked Conc. (pg/ml) %RSD L2 1α,25-diH VD3 IS L3 1α,25-diH VD3 IS Conclusions A 2D-LC/MS/MS method with APCI interface has been developed for quantitative analysis of 1α,25-dihydroxylvitamin D3 in human serum without offline extraction and cleanup. The detection and quantitation range of the method is from 3.1 pg/ml to 200 pg/ml, which meets the diagnosis requirements in clinical applications. The performance of the method was evaluated thoroughly, including linearity, accuracy, repeatability, matrix effect, LD/LQ and specificity. The results indicate that the 2D-LC/MS/MS method is sensitive and reliable in detection and quantitation of trace 1α,25-dihydroxylvitamin D3 in serum. Further studies to enable the method for simultaneous analysis of both 1α,25-dihydroxylvitamin D3 and 1α,25-dihydroxylvitamin D2 are needed. References 1. S. Wang. Nutr. Res. Rev. 22, 188 (2009). 2. T. Higashi, K. Shimada, T. Toyo oka. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. (2010) 878, J. M. El Khoury, E. Z. Reineks, S. Wang. Clin. Biochem DI: /jssc Chao Yuan, Justin Kosewick, Xiang He, Marta Kozak and Sihe Wang, Rapid Commun. Mass Spectrom. 2011, 25, Casetta, I. Jans, J. Billen, D. Vanderschueren, R. Bouillon. Eur. J. Mass Spectrom. 2010, 16, 81. For Research Use nly. Not for use in diagnostic procedures. First Edition: June, For Research Use nly. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 2014

85 P-CN1450E Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer ASMS 2014 WP 182 William Hedgepeth, Kenichiro Tanaka Shimadzu Scientific Instruments, Inc., Columbia MD

86 Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer Introduction Polysorbate 80 is commonly used for biotherapeutic products to prevent aggregation and surface adsorption, as well as to increase the solubility of biotherapeutic compounds. A reliable method to quantitate and characterize polysorbates is required to evaluate the quality and stability of biotherapeutic products. Several methods for polysorbate analysis have been reported, but most of them require time-consuming sample pretreatment such as derivatization and alkaline hydrolysis because polysorbates do not have sufficient chromophores. Those methods also require an additional step to remove biotherapeutic compounds. Here we report a simple and reliable method for quantitation and characterization of polysorbate 80 in biotherapeutic products using two-dimensional HPLC. Materials Reagents and standards Reagents: Tween 80 (Polysorbate 80), IgG from human serum, potassium phosphate monobasic, potassium phosphate dibasic, and ammnonium formate were purchased from Sigma-Aldrich. Water was made in house using a Millipore Milli-Q Advantage A10 Ultrapure Water Purification System. Isopropanol was purchased from Honeywell. Standard solutions: 10 mmol/l phosphate buffer (ph 6.8) was prepared by dissolving 680 mg of potassium phosphate monobasic and 871 mg of potassium phosphate dibasic in 1 L of water. Polysorbate 80 was diluted with 10 mmol/l phosphate buffer (ph 6.8) to 200, 100, 50, 20, 10 mg/l and transferred to 1.5 ml vials for analysis. Sample solutions: A model sample was prepared by dissolving 2 mg of IgG in 0.1 ml of a 100 mg/l polysorbate 80 standard solution. The sample was centrifuged and transferred to a 1.5 ml vial for analysis. H z w H x H y w+x+y+z=approx. 20 CH 3 Fig.1 Typical structure of polysorbate 80 2

87 Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer System The standard and sample solutions were injected into a Shimadzu Co-Sense for BA system consisting of two LC-20AD pumps and a LC-20AD pump equipped with a solvent switching valve, DGU-20A5R degassing unit, SIL-20AC autosampler, CT-20AC column oven equipped with a 6-port 2-position valve, and a CBM-20A system controller. Polysorbate 80 was detected by a LCMS-2020 single quadrupole mass spectrometer or a LCMS-8050 triple quadrupole mass spectrometer because polysorbates do not have any chromophores and are present at low concentrations in antibody drugs. A SPD-20AV UV-VIS detector was used to check protein removal. Fig. 2 shows the flow diagram of the Co-Sense for BA system. In step 1, a sample pretreatment column Shim-pack MAYI-DS traps polysorbate 80 in the sample. Proteins (antibody) cannot enter the pore interior that is blocked by a hydrophilic polymer bound on the outer surface. ther additives and excipients such as sugars, salts, and amino acids cannot be retained by the DS phase of the inner surface due to their polarity. In step 2, the trapped polysorbate 80 is introduced to the analytical column by valve switching. Step 1 : Protein removal Mobile phase C Mobile phase D Pump 2 Valve (Position 0) Analytical column Autosampler Mass spectrometer Mobile phase A (solution for sample injection) Protein, Salts, Amino acids, Sugars UV-VIS detector Polysorbate 80 Pump 1 Step 2 : Analyzing the trapped polysorbate Sample pretreatment column Mobile phase B (solution for rinse) Polysorbate 80 Mass spectrometer Mobile phase C Mobile phase D Pump 2 Valve (Position 1) Analytical column Autosampler Mobile phase A (solution for sample injection) UV-VIS detector Pump 1 Sample pretreatment column Mobile phase B (solution for rinse) Fig.2 Flow diagram of Co-Sense for BA 3

88 Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer Results Quantitation method A fast analysis for quantitation will be shown here. Table 1 shows the analytical conditions and Fig. 3 shows the TIC chromatogram of a 100 mg/l polysorbate 80 standard solution and the mass spectrum of the peak at 4.4 min. Polysorbates contain many by-products, so several peaks appeared on the TIC chromatogram. The peak at 4.4 min was identified as polyoxyethylene sorbitan monooleate (typical structure of polysorbate 80) based on E. Hvattum et al The ion at 783 was used as a marker for detection in selected ion mode (SIM). This ion is attributable to the 2NH 4 + adduct of polyoxyethylene sorbitan monooleate containing 25 polyoxyethylene groups. Fig. 4 shows the SIM chromatogram of the model sample (20 g/l of IgG, 100 mg/l of polysorbate 80 in 10 mmol/l phosphate buffer ph6.8). Polysorbate 80 in the model sample was successfully analyzed. The peak at 4.4 min was used for quantitation. Six replicate injections for the model sample were made to evaluate the reproducibility. The relative standard deviations of retention time and peak area were 34 % and 1.11 %, respectively. The recovery ratio was obtained by comparing the peak area of the model sample and a 100 mg/l polysorbate 80 standard solution and was 99 %. Five different levels of polysorbate 80 standard solutions ranging from 10 to 200 mg/l were used for the linearity evaluation. The correlation coefficient (R 2 ) of determination was higher than Table 1 Analytical Conditions System : Co-Sense for BA equipped with LCMS-2020 [Sample Injection] Column : Shim-pack MAYI-DS (5 mm L. x 2.0 mm I.D., 50 μm) Mobile Phase : A: 10 mmol/l ammonium formate in water B: Isopropanol Solvent Switching : A (0-1.5 min), B ( min), A (3.5-9 min) Flow Rate : 0.6 ml/min Valve Position : 0 (0-1 min, 7-9 min), 1 (1-7 min) Injection Volume : 1 µl [Separation] Column : Kinetex 5u C18 100A (50 mm L. x 2.1 mm I.D., 5 μm) Mobile Phase : A: 10 mmol/l ammonium formate in water B: Isopropanol Time Program : B. Conc 5 % (0-1 min) % (6-7 min) -5 % ( min) Flow Rate : 0.3 ml/min Column Temperature : 40 ºC [UV Detection] Detection : 280 nm Flow Cell : Semi-micro cell [MS Detection] Ionization Mode : ESI Positive Applied Voltage : 4.5 kv Nebulizer Gas Flow : 1.5 ml/min DL Temperature : 250 ºC Block Heater Temp. : 400 ºC Scan : m/z SIM : m/z 783 4

89 Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer min Inten.(x100,000) Triply charged ions Doubly charged ions m/z Fig.3 TIC Chromatogram of 100 mg/l polysorbate 80 standard solution and mass spectrum of the peak at 4.4 min min Fig.4 SIM chromatogram of the model sample Characterization method An analysis for characterization will be shown here. Table 2 shows the analytical conditions and Fig. 5 shows the TIC chromatogram of the model sample and mass spectra of the peaks from 10 to 30 min. A longer column and gradient were applied to obtain better resolution. Polysorbate 80 consists of not only monooleate (typical structure of polysorbate 80), but also many by-products such as polyoxyethylene, polyoxyethylene sorbitan, polyoxyethylene isosorbide, dioleate, trioleate, tetraoleate and others. The peaks on the TIC chromatogram are assumed to correspond to those by-products. For example, the peaks from 10 to 22 min correspond to polyoxyethylene and polyoxyethylene isosorbide and the peaks from 22 to 30 min correspond to polyoxyethylene sorbitan. This method is helpful for characterization as well as checking degradation such as auto-oxidation and hydrolysis. 5

90 Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer Table 2 Analytical Conditions System : Co-Sense for BA equipped with LCMS-8050 [Sample Injection] Column : Shim-pack MAYI-DS (5 mm L. x 2.0 mm I.D., 50 μm) Mobile Phase : A: 10 mmol/l ammonium formate in water B: Isopropanol Solvent Switching : A (0-1.5 min), B ( min), A (3.5-9 min) Flow Rate : 0.6 ml/min (0-10 min, min), 0.1 ml/min (11-95 min) Valve Position : 0 (0-3 min, min), 1 (3-100 min) Injection Volume : 5 µl [Separation] Column : Kinetex 5u C18 100A (100 mm L. x 2.1 mm I.D., 5 μm) Mobile Phase : A: 10 mmol/l ammonium formate in water B: Isopropanol Time Program : B. Conc 5 % % (0-3min) 35% (15min) 100% (100min) 5% ( min) Flow Rate : 0.2 ml/min Column Temperature : 40 ºC [UV Detection] Detection : 280 nm Flow Cell : Semi-micro cell [MS Detection] Ionization Mode : ESI Positive Applied Voltage : 4.5 kv Nebulizer Gas Flow : 2 ml/min Drying Gas Flow : 10 ml/min Heating Gas Flow : 10 ml/min Interface Temperature : 300 ºC DL Temperature : 250 ºC Block Heater Temp. : 400 ºC Q1 Scan : m/z (x100,000,000) 1:TIC(+) (x10,000,000) 1:TIC(+) min min Inten.(x100,000) m/z Inten.(x100,000) m/z H y H z Polyoxyethylene isosorbide H H x Polyoxyethylene H z H w H x H y Polyoxyethylene sorbitan Fig.5 TIC chromatogram of the model sample 6

91 Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer Confirmation of protein removal Fig. 6 shows the chromatogram of elution from the sample pretreatment column. Protein (IgG) was successfully removed from the sample by using the MAYI-DS column. uv uL injection of model sample 1uL injection of model sample min Fig.6 Chromatogram of elution from the sample pretreatment column Conclusions 1. Co-Sense for BA system automatically removed protein from the sample and enabled quantitation and characterization of polysorbate 80 in a protein formulation. 2. The quantitation method was successfully applied to the model sample with excellent reproducibility and recovery. 3. The high-resolution chromatogram was obtained by the characterization method. This method is helpful for characterization as well as checking degradation such as auto-oxidation and hydrolysis. Reference E. Hvattum, W.L. Yip, D. Grace, K. Dyrstad, Characterization of polysorbate 80 with liquid chromatography mass spectrometry and nuclear magnetic resonance spectroscopy: Specific determination of oxidation products of thermally oxidized polysorbate 80, J Pharm Biomed Anal 62, (2012) 7-16 First Edition: June, For Research Use nly. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 2014

92 P-CN1457E A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG ASMS 2014 WP161 Rachel Lieberman 1, David Colquhoun 1, Jeremy Post 1, Brian Feild 1, Scott Kuzdzal 1, Fred Regnier 2, 1 Shimadzu Scientific Instruments, Columbia, MD, USA 2 Novilytic L.L.C, North Webster, IN, USA

93 A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG Novel Aspect Using rapid, automated processing, coupled to the speed and sensitivity of the LCMS-8050 allows for improved analysis of Immunoglobulin G. Introduction Dried blood spot analysis (DBS) has provided clinical laboratories a simple method to collect, store and transport samples for a wide variety of analyses. However, sample stability, hematocrit effects and inconsistent spotting techniques have limited the ability for wide spread adoption in clinical applications. Dried plasma spots (DPS) offer new opportunities by providing stable samples that avoid variability caused by the hematocrit. This presentation focuses on an ultra-fast-immuno-ms platform that combines next generation plasma separator cards (Novilytic L.L.C., North Webster, IN) with fully automated immuno-affinity enrichment and rapid digestion as an upfront sample preparation strategy for mass spectrometric analysis of immunoglobulins. Sample Workflow Plasma Generation Affinity Selection Buffer Exchange Enzyme Digestion Desalting LC/MS/MS Noviplex TM Card Perfinity Workstation LCMS-8050 Triple Quadrupole MS Rapid plasma extraction technology from whole blood (~ 18 minutes) ul of plasma collected (3 min) - Air dry for 15 minutes - Extract plamsa disc for analysis Automates and integrates key proteomic workflow steps: - Affinity Selection (15 min) - Trypsin digestion (1-8 min) - nline Desalting - Reversed phase LC Exceptional reproducibility (CV less than 10%) - Ultrafast MRM methods - Up to 555 MRM transitions per run - Heated electrospray source - Scan speeds up to 30,000 u/sec - Polarity switching 5 msec 2

94 A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG Methods IgG was weighed out and then diluted in 500 μl of 0.5% BSA solution. Approximately15 ul of IgG standard was spiked into mouse whole blood and processed using the Noviplex card. The resulting plasma collection disc was extracted with 30 ul of buffer and each sample was reduced and alkylated to yield a total sample volume of 100 ul. IgG standards and QC samples were directly injected onto the Perfinity-LCMS-8050 platform for affinity pulldown with a Protein G column followed by trypsin digestion and LC/MS/MS analysis. Level Conc. (μg/ml) Amount on column (μg) Amount on column (pmol) Time (min) %B %B Time (minutes) IgG concentrations for calibration levels. LCMS gradient conditions. Compound Name Transitions +/- Q1 Rod Bias (V) CE (V) Q3 Rod Bias (V) TTPPVLDSDGSFFLYSK > > VVSVLTVLHQDWLNGK > MRM transitions on LCMS-8050 for two IgG peptides monitored. Noviplex Cards (2) (3) (4) (1) Approximately 50 ul of the spiked whole blood was pipetted onto the Noviplex card test area (1). The spot was allowed to dry for 3 minutes (2). The top layer of the card was then peeled back (3) to reveal the plamsa collection disc. The plasma collection disc was allowed to dry for an additional 15 minutes. nce the disc was dry (4), it was placed into an eppendorf tube for solvent extraction. 3

95 A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG Results - Chromatograms min Total Ion Chromatogram for IgG ptimization of Collision Energies for peptides of interest Range CE: -50 to -10 V TTPPVLDSDGFFLYSK min Inten [M+2H] TTPPVLDSDGSFFLYSK VVSVLTVLHQDWLNGK [P2+2H] +2 [P1+2H] m/z min MRM Chromatogram for Level 4 standard of spiked IgG in whole blood. Carryover Assessment Control - Mouse blood Blank Injection min min 4

96 A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG Results - Calibration Curves Calibration Curve and MS Chromatograms TTPPVLDSDGSFFLYSK VVSVLTVLHQDWLNGK >805.70(+) >836.25(+) >836.25(+) >805.70(+) >723.95(+) Level >723.95(+) Level Level Level Area r 2 = Area r 2 = Conc Conc. Results - Tables and Replicates QC data and Calculations for IgG Peptides VVSVLTVLHQDWLNGK Sample Ret. Time Area Calc. Conc. Std. Conc. % Accuracy QC , QC , QC , QC , TTPPVLDSDGSFFLYSK Sample Ret. Time Area Calc. Conc. Std. Conc. % Accuracy QC , QC , QC , QC ,

97 A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG Skyline Data - Retention Time Replicates VVSVLTVLHQDWLNGK TTPPVLDSDGSFFLYSK 6.65 y y Retention Time AM_ L AM_ L AM_ L AM_ L AM_ L P M_ L P M_ L P M_ L AM_ L AM_ L AM_ L P M_ L P M_ L Retention Time 1433 P M_ L Replicate Replicate Integration of Skyline Software into LabSolutions allows for further interrogation of data. Here are representative figures showing the retention time reproducibility for each peptide monitored during the analysis. Conclusions Combining the sample collection technique of next generation plasma separator Noviplex cards for quick plamsa collection from whole blood, with the automated affinity selection and trypsin digestion of the Perfinity workstation coupled to LCMS-8050, provides a very rapid and reproducible Immuno-MS platform for quantitation of IgG peptides. Furthermore, this rapid immuno-ms platform can be applied to many other peptide/protein applications. First Edition: June, For Research Use nly. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 2014

98 P-CN1473E Simultaneous Determinations of 20 kinds of common drugs and pesticides in human blood by GPC-GC-MS/MS ASMS 2014 TP 757 Qian Sun, Jun Fan, Taohong Huang, Shin-ichi Kawano, Yuki Hashi, Shimadzu Global CE, Shanghai, China

99 Simultaneous Determinations of 20 kinds of common drugs and pesticides in human blood by GPC-GC-MS/MS Introduction n-line gel permeation chromatography-gas chromatography/mass spectrometry (GPC-GC-MS) is a unique technique to cleanup sample that reduce the time of sample preparation. GPC can efficiently separates fats, protein and pigments from samples, due to this advantage, on-line GPC is widely used for pesticide analysis. Meanwhile, compared to widely used GC-MS, GC-MS/MS techniques provide much better selectivity thus significantly lower detection limits. In this work, a new method was developed for rapid determination of 20 common drugs and pesticides in human blood by GPC-GC-MS/MS. The modified QuEChERS method was used for sample preparation. Experimental The human blood samples were extracted with acetonitrile, then was purified by PSA, C18 and MgS 4 to remove most of the fats, protein and pigments in samples, then after on-line GPC-GC-MS/MS analysis which further removed macromolecular interference material, such as protein and cholesterol, the background interference brought about by the complex matrix in samples was effectively reduced. Sample pretreament human blood 2 ml CH 3 CN vortex PSA/C18/MgS 4 vortex centrifuge supernatant evaporate set volume using moblie phase GPC-GC-MS/MS Figure 1 Schematic flow diagram of the sample preparation 2

100 Simultaneous Determinations of 20 kinds of common drugs and pesticides in human blood by GPC-GC-MS/MS Instrument GPC Mobile phase : acetone/cyclohexane (3/7, v/v) Flow rate : 0.1mL/min Column : Shodex CLNpak EV-200 (2 mmi.d. x 150 mml.) ven temperature : 40 ºC Injection volume : 10 μl GCMS-TQ8030 Column : deactivated silica tubing [0.53 mm(id) x 5 m(l)] +pre-column Rtx-5ms [0.25 mm(id) x 5 m(l)] Rtx-5ms [0.25mm(ID) x 30 m(l), Thickness: 0.25 μm] Injector : PTV Injector time program : 120 ºC(4.5min)-(80 ºC/min)-280 ºC(33.7 min) ven temperature program : 82 ºC(5min)-(8 ºC/min)-300 ºC(7.75 min) Linear velocity : 48.8 cm/sec Ion Source temperature : 210 ºC Interface temperature : 300 ºC Results For all of analytes, recoveries in the acceptable range of 70~120% and repeatability (relative standard deviations, RSD) 5% (n=3) were achieved for matrices at spiking levels of 1 µg/ml. The limitis of detection were 3~4.4 µg/l. The method is simple, rapid and characterized with acceptable sensitivity and accuracy to meet the requirements for the analysis of common drugs and pesticides in the human blood. (x10,000,000) Figure 2 MRM chromatograms of standard mixture 3

101 Simultaneous Determinations of 20 kinds of common drugs and pesticides in human blood by GPC-GC-MS/MS Table 1 Results of method validation for drugs and pesticides (Concentration range: μg/l, LDs: S/N 3, LQs: S/N 10, RSDs: n=3) No. Compound Name t R (min) Correlation Coefficient* LD (µg/l) LQ 1 µg/ml (µg/l) Recovery (%) RSD (%) 1 Dichlorvos Methamidophos Barbital Sulfotep Dimethoate Malathion Chlorpyrifos Phenobarbital Parathion Triazophos Zopiclone deg Diazepam Midazolam Zolpidem Clonazepam Estazolam Clozapine Alprazolam Zolpidem Triazolam Conclusion A very quick, easy, effective, reliable method in human blood based on modified QuEChERS method was developed using GPC-GCMS-TQ8030. The performance of the method was very satisfactory with results meeting validation criteria. The method has been successfully applied for determination of human blood samples and ostensibly has further application opportunities, e.g. biological samples. First Edition: June, For Research Use nly. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 2014

102 P-CN1466E Low level quantitation of Loratadine from plasma using LC/MS/MS ASMS 2014 TP498 Shailesh Damale, Deepti Bhandarkar, Shruti Raju, Rashi Kochhar, Shailendra Rane, Ajit Datar, Pratap Rasam, Jitendra Kelkar Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh Chambers, Makwana Road, Marol, Andheri (E), Mumbai , Maharashtra, India.

103 Low level quantitation of Loratadine from plasma using LC/MS/MS Introduction Loratadine is a histamine antagonist drug used for the treatment of itching, runny nose, hay fever and such other allergies. Here, an LC/MS/MS method has been developed for high sensitive quantitation of this molecule from plasma using LCMS-8050, a triple quadrupole mass spectrometer from Shimadzu Corporation, Japan. Presence of heated Electro Spray Ionization (ESI) interface in LCMS-8050 ensured good quantitation and repeatability even in the presence of a complex matrix like plasma. Ultra high sensitivity of LCMS-8050 enabled development of a low ppt level quantitation method for Loratadine. Loratadine Ethyl 4- (8-chloro-5, 6-dihydro-11H-benzo [5, 6] cyclohepta [1, 2-b] pyridin-11-ylidene) -1-piperidinecarboxylate Figure 1. Structure of Loratadine Loratadine, a piperidine derivative, is a potent long-acting, non-sedating tricyclic antihistamine with selective peripheral H1-receptor antagonist activity. It is used for relief of nasal and non-nasal symptoms of seasonal allergies and skin rashes [1,2,3]. Due to partial distribution in central nervous system, it has less sedating power compared to traditional H1 blockers. Loratadine is given orally, is well absorbed from the gastrointestinal tract, and has rapid first-pass hepatic metabolism; it is metabolized by isoenzymes of the cytochrome P450 system, including CYP3A4, CYP2D6, and, to a lesser extent, several others. Loratadine is almost totally (97 99 %) bound to plasma proteins and reaches peak plasma concentration (T max ) in ~ 1 2 h [4,5]. Method of Analysis This bioanalytical method was developed for measuring Loratadine in therapeutic concentration range for the analysis of routine samples. It was important to develop a simple and accurate method for estimation of Loratadine in human plasma. Preparation of matrix matched plasma by protein precipitation method using cold acetonitrile To 100 µl of plasma 500 µl cold acetonitrile was added for protein precipitation. It was placed in rotary shaker at 20 rpm for 15 minutes for uniform mixing. This solution was centrifuged at rpm for 15 minutes. Supernatant was taken and evaporated to dryness at 70 ºC. The residue was reconstituted in 200 µl Methanol. Preparation of calibration standards in matrix matched plasma 1 ppt, 5 ppt, 50 ppt, 100ppt, 500 ppt, 1 ppb, 5 ppb and 10 ppb of Loratadine calibration standards were prepared in cold acetonitrile treated matrix matched plasma. 2

104 Low level quantitation of Loratadine from plasma using LC/MS/MS LC/MS/MS analysis LCMS-8050 triple quadrupole mass spectrometer by Shimadzu Corporation, Japan (shown in Figure 2A), sets a new benchmark in triple quadrupole technology with an unsurpassed sensitivity (UFsensitivity) with Scanning speed of 30,000 u/sec (UFscanning) and polarity switching speed of 5 msecs (UFswitching). This system ensures highest quality of data, with very high degree of reliability. In order to improve ionization efficiency, the newly developed heated ESI probe combines high-temperature gas with the nebulizer spray, assisting in the desolvation of large droplets and enhancing ionization. This development allows high-sensitivity analysis of a wide range of target compounds with considerable reduction in background. Presence of heated Electro spray interface in LCMS-8050 (shown in Figure 2B) ensured good quantitative sensitivity even in presence of a complex matrix like plasma. The parent m/z of giving the daughter m/z of in the positive mode was the MRM transition used for quantitation of Loratadine. MS voltages and collision energy were optimized to achieve maximum transmission of mentioned precursor and product ion. Gas flow rates, source temperature conditions and collision gas were optimized, and linearity graph was plotted for 4 orders of magnitude. Figure 2A. LCMS-8050 triple quadrupole mass spectrometer by Shimadzu Figure 2B. Heated ESI probe Table 1. LC conditions Table 2. LCMS conditions Column Shim-pack XR-DS (100 mm L x 2.0 mm ID ; 2.2 µm) MS Interface ESI Mobile Phase A : 0.1% formic acid in water B : acetonitrile Polarity Nebulizing Gas Flow Positive 2.0 L / min (nitrogen) Gradient Program Time (min) A conc. (%) B conc. (%) Stop Drying Gas Flow Heating Gas Flow Interface Temp. Desolvation Line Temp. Heater Block Temp. MRM Transition 1 L / min (nitrogen) 15.0 L / min (zero air) 300 ºC 250 ºC 400 ºC > Flow Rate 0.15 ml/min ven Temperature 40 ºC Injection Volume 20 µl 3

105 Low level quantitation of Loratadine from plasma using LC/MS/MS Results LC/MS/MS Analysis LC/MS/MS method for Loratadine was developed on ESI +ve ionization mode and > MRM transition was optimized for Loratadine. Checked matrix matched plasma standards for highest (10 ppb) as well as lowest (01 ppb) concentrations as seen in Figures 4A and 4B respectively. ptimized MS method to ensure no plasma interference at the retention time of Loratadine (Figure 5). Calibration curve was plotted for Loratadine concentration range. Also as seen in Table 3, % Accuracy was studied to confirm the reliability of method. Linear calibration curves were obtained with regression coefficients R 2 > % RSD of area was within 15 % and accuracy was within % for all calibration levels. 3.5 (x1,000,000) >337.10(+) LRATADINE/ (x10,000) >337.10(+) LRATADINE/ Figure 4A. Mass chromatogram 10 ppb Figure 4B. Mass chromatogram 01 ppb Specificity and interference (x10,000) 1.2 1:LRATIDINE >337.10(+) CE: LRA_PLASMA_003.lcd 1:LRATIDINE >337.10(+) CE: LRA_PLASMA_002.lcd LQ Level Blank min Figure 5. verlay chromatogram 4

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