Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS

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1 Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS TIAFT 204 Toshikazu Minohata, Keiko Kudo 2, Kiyotaka Usui 3, Noriaki Shima 4, Munehiro Katagi 4, Hitoshi Tsuchihashi 5, Koichi Suzuki 5, Noriaki Ikeda 2 Shimadzu Corporation, Kyoto, Japan 2 Kyushu University, Fukuoka, Japan 3 Tohoku University Graduate School of Medicine, Sendai, Japan 4 Osaka Prefectural Police, Osaka, Japan 5 Osaka Medical Collage, Takatsuki, Japan

2 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 () using Q-sep QuEChERS Sample Prep Packets purchased from RESTEK (Bellefonte, PA). ) Add ml of blood and ml of distilled water into the 5 ml centrifugal tube and agitate the mixture using a vortex mixer. 2) Add two 4 mm stainless steel beads,.5 ml of acetonitrile and 00 µl of acetonitrile solution containing ng/µl of Diazepam-d5. Then agitate using the vortex mixer. 3) Add 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 0 minutes at 3000 rpm. 5) Move the supernatant to a different 5 ml centrifugal tube and add 00 µl of 0. % 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 0,000 rpm. 7) Transfer 50 µl of the supernatant to a.5 ml vial for HPLC provided with a small-volume insert. [ ref.] () Usui K et al, Legal Medicine 4 (202), Water ml ACN.5 ml Diazepam-d5 (IS) 00ng Stainless-Steel Beads (4mm x 2) Transfer supernatant Add 00uL of 0.% TFA Dry Sample ml Q-sep QuEChERS Extraction Salts (MgSO 4,NaOAc) [Shake] [Centrifuge] Reconstitution with 200 ul MeOH LC/MS/MS analysis Figure Scheme of the modified QuEChERS procedure 2

3 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 06 forensic and toxicology-related compounds of Abused drugs, Psychotropic drugs and Hypnotic drugs etc (Table ) 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 C8 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 C8 (00x2mm,.9μm) Mobile Phase A : 0 mm Ammonium formate - water Mobile Phase B : Methanol Gradient Program : 5%B (0 min) - 95%B (0 min - 3min) - 5%B (3. 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 : 0 L / min Heating gas flow : 0 L / min DL Temperature : 250 ºC BH Temperature : 400 ºC MRM parameter : Analytes Ret. Time Q m/z Q3 m/z Collision Energy Analytes Ret. Time Q m/z Q3 m/z Collision Energy Diazepam-d Risperidone Alprazolam Triazolam Atropine Amobarbital Estazolam Barbital Ethyl loflazepate Phenobarbital Etizolam Thiamylal Haloperidol

4 positive negative Figure 2 LCMS-8050 triple quadrupole mass spectrometer Results and Discussion Alprazolam Etizolam Risperidone Triazolam (x0 3 ) 309.0>28.0(+) (x0 3 ) >34.0(+) (x0 3 ) 4.20>9.05(+) (x0 2 ) >30(+).0 S/N S/N 45.5 S/N 07.6 S/N 8.8 (x0 4 ) 309.0>28.0(+) (x0 4 ) >34.0(+) (x0 4 ) 4.20>9.05(+) (x0 3 ) >30(+) Area Ratio Conc. Ratio Area Ratio (x0.) Area Ratio Area Ratio (x0.) Conc. Ratio Conc. Ratio Conc. Ratio 0. 9,004 8,288 9,59 75,236 75,983 74, ,59 83, , ,865 5,09 4,32 48,038 49,52 54, ,640 58, , ,832 32,436 30,46 335, , ,72 3,826,373 3,78,854 3,705, ,047 3,064 3,356 27,99 25,542 26,37 288, , ,

5 Amobarbital Barbital Phenobarbital Thiamylal (x0 2 ) 225.5>40(-) (x0) 83.0>4(-) (x0 2 ) 23.0>42.20(-) (x0 2 ) >58.0(-) S/N 40.2 S/N S/N 38.2 S/N 67.9 (x0 3 ) >40(-) (x0 2 ) 83.0>4(-) (x0 3 ) 23.0>42.20(-).0 (x0 3 ) >58.0(-) Area Ratio (x0.) Area Ratio (x) Area Ratio (x0.) Area Ratio (x0.) Conc. Ratio 2 5 Conc. Ratio Conc. Ratio Conc. Ratio 0 00,837,862 2,04 2,685 22,69 20, , , , ,078 5,033 5,424 55,420 55,658 53, ,909 8,564 7,939 8,987 83,274 82, ,520 2,92 2,288 30,808 29,623 3,379 38,233 37,24 33, 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 8 drugs were spiked into extract solution. Calibration curves constructed in the range from to for 2 drugs (Alprazolam, Aripiprazole, Atropine, Brotizolam, Estazolam, Ethyl loflazepate, Etizolam, Flunitrazepam, Haloperidol, Nimetazepam, Risperidone and Triazolam) and from to 00 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 < 2%) at low concentration level. 5

6 Amobarbital Barbital Phenobarbital Thiamylal (x0 2 ) 225.5>40(-) (x0 2 ) 83.0>4(-) (x0 2 ) 23.0>42.20(-) (x0 2 ) >58.0(-) S/N 4.7 S/N 9.4 S/N 8.3 S/N (x0 3 ) 225.5>40(-) (x0 2 ) 83.0>4(-) (x0 3 ) 23.0>42.20(-) (x0 3 ) >58.0(-) Area Ratio (x0.) Area Ratio (x0.) Area Ratio (x0.) Area Ratio (x0.) Conc. Ratio 2 5 Conc. Ratio 2 5 Conc. Ratio 2 5 Conc. Ratio ,468,233,245 7,24 20,546 8,689 2,97 25, , ,989 5,63 5,443 55,392 69,48 66, ,656 6,632 6,384 7,965 88,685 82, ,42 3,470 3,53 27,257 34,377 32, ,563 43, , 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: November, For Research Use Only. 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, 204

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