Development of analytical methods for Catecholamines, Metanephrines and related metabolites in urine and plasma matrices

Development of analytical methods for Catecholamines, Metanephrines and related metabolites in urine and plasma matrices From sample preparation to LC/MS/MS Linda Côté Christophe Deckers Agilent Technologies

Outline Introduction Why a unified approach? Description of each method Sample preparation Chromatography Figures of merits Sample preparation Why Which sample preparation technique to choose Conclusions Questions and Answers 2

Which ones and in which matrix urine or plasma? 7 different methods Free catecholamines in urine Total metanephrines in urine Free catecholamines in plasma Free metanephrines in plasma VMA in urine HVA in urine 5-HIAA in urine 3

Why a unified approach? Challenge: Many analysis to perform with One LC/MS/MS system 4

Which ones and in which matrix urine or plasma? Webinar April 2013 Free catecholamines and metanephrines in urine Total catecholamines and metanephrines in urine Free catecholamines in plasma (in progress) Free metanephrines in plasma (in progress) VMA in urine (in progress) HVA in urine (in progress) 5-HIAA in urine (in progress) 5

Catecholamines and related metabolites in urine or plasma 4 methods with a unified approach Free catecholamines and metanephrines in urine Total catecholamines and metanephrines in urine Free catecholamines in plasma Free metanephrines in plasma VMA in urine HVA in urine 5-HIAA in urine 2 4 3 1 6

Unified approach in method development Matrix Linear range Sample prep Chromatography Mobile phase LC/MS/MS 1. Free and Total Cats/Mets 2. VMA, HVA, 5- HIAA 3. Mets 4. Cats 7

Unified approach in method development 1. Free and Total Cats/Mets 2. VMA, HVA, 5- HIAA 3. Mets 4. Cats Matrix Urine Urine Plasma Plasma Linear range 1.56-3000 ng/ml Sample prep Chromatography Mobile phase LC/MS/MS 0.08-100 mg/l 15.6-10000 pg/ml 5-2500 pg/ml 8

Unified approach in method development 1. Free and Total Cats/Mets 2. VMA, HVA, 5- HIAA 3. Mets 4. Cats Matrix Urine Urine Plasma Plasma Linear range 1.56-3000 ng/ml Sample prep Boronate complex/ Bond Elut Plexa SPE 0.08-100 mg/l 15.6-10000 pg/ml 5-2500 pg/ml Dilution WCX SPE Captiva ND Lipids / BondElut PBA SPE Chromatography Pursuit-PFP Pursuit-PFP Pursuit-PFP Pursuit-PFP Mobile phase 0.2% formic acid - Methanol 0.2% formic acid - Methanol 0.2% formic acid - Methanol 1 mm ammonium fluoride - Methanol LC/MS/MS 1290/6460 1290/6460 1290/6460 1290/6460 9

Catecholamines and metanephrines in a single run 16

Catecholamines and metanephrines in a single run One SPE cartridge is used to recover both catecholamines and metanephrines SPE Bond Elut Plexa was chosen for best recoveries Simple acid elution for direct injection into LC-MS/MS ph control for stabilization of catecholamines Metanephrines are also retained under the same conditions, even though they are methylated and do not contain the cis-diol moiety for the covalent linkage binding mechanism Studies have shown that metanephrines do have some affinity for this sorbent [1] Ann Clin Biochem 2009; 46: 129 136. DOI: 10.1258/acb.2008.008180 17

Sample preparation Calibrators are prepared with clean urine matrix from Golden West Biologicals Isotopically labelled Internal standards 24 hours collection of urine Native urine for free catecholamines and metabolites (typically for catecholamines) Acid-hydrolysed urine for total (typically for metanephrines) Solid phase extraction (SPE) is used to cleanup urine 18

Compound structures HO HO OH HO OH HO NH 2 HO NH 2 HO HN CH 3 Dopamine (D) C8H11NO2 Neutral Mass: 153.08 Norepinephrine (NE) C8H11NO3 Neutral Mass: 169.07 Epinephrine (E) C9H13NO3 Neutral Mass: 183.09 CH 3 CH 3 OH CH 3 OH O O O HO NH 2 HO NH 2 HO HN CH 3 3-Methoxytyramine (3-MT) C9H13NO2 Neutral Mass: 167.09 Normetanephrine (NMN) C9H13NO3 Neutral Mass: 183.09 Metanephrine (MN) C10H15NO3 Neutral Mass: 197.11 19

Chromatography challenge Need to keep resolution between E and NMN Also between MN and 3-MT 20

Catecholamines and metanephrines in a single run Chromatography E/NMN and MN/3-MT Resolution is Critical 1. Norepinephrine (NE) 2. Epinephrine (E) 3. Normetanephrine (NMN) 4. Dopamine (D) 5. Metanephrine (MN) 6. 3-Methoxytyramine (3-MT) E D MN 3-MT NE NMN 21

Calibration curves 22

Results Summary of Analyte Performance Compound R 2 Concentration Concentration Dopamine 0.9997 Accuracy (%) Intraday CV (%) Interday CV (%) (ng/ml) (nmol/l) n = 3 n = 3 n = 5 1.56 10.2 107.5 1.0 2.7 62.5 408.0 99.1 1.7 2.0 1000 6528.3 101.3 0.1 0.3 Norepinephrine 0.9999 1.56 9.2 102.9 0.9 5.4 62.5 369.4 101.1 3.5 4.0 1000 5910.9 101.1 0.6 0.6 Epinephrine 0.9998 1.56 8.5 101.6 4.3 2.7 62.5 341.2 100.9 2.5 2.0 1000 5458.4 100.3 0.4 0.3 Note: Signal to noise ratios and CVs indicate that LLOQs are lower than measured here for all analytes 23

Results Summary of Analyte Performance Compound R 2 Concentration Concentration 3-Methoxytyramine 0.9999 Accuracy (%) Intraday CV (%) Interday CV (%) (ng/ml) (nmol/l) n = 3 n = 3 n = 5 4.69 28 95.7 1.1 3.6 187.5 1121.4 102.9 0.9 2.0 3000 17942.1 100.0 0.2 0.3 Normetanephrine 0.9999 4.69 25.6 100.1 1.5 3.2 187.5 1023.45 102.0 1.1 2.5 3000 16375.2 100.7 0.2 0.2 Metanephrine 0.9999 4.69 23.8 100.5 0.3 2.8 187.5 950.7 102.0 0.5 2.2 3000 15210.6 100.8 0.1 0.2 Note: Signal to noise ratios and CVs indicate that LLOQs are lower than measured here for all analytes 24

Results Inter-run Over 3 Days for Commercial QC (BioRad Lyphocheck) Level 1 Level 2 Compound Free/Total Range (HPLC) Measured CV (%) Range (HPLC) Measured CV (%) Dopamine Free 44.4-75.0 61.4 3.4 377 629 509 2.8 Norepinephrine Free 31.3 51.6 38.4 5.8 156 239 192 4.8 Epinephrine Free 9.62 19.1 14.3 5.3 67.8 104 86.7 2 3-Methoxytyramine Total 28.6 48.7 44.7 3.8 381 572 557.7 2.2 Normetanephrine Total 220-366 300.7 2.4 1084 1630 1379.2 2.8 Metanephrine Total 69.0-116 91.2 2 434-655 612 2.5 All measurements are in ng/ml Bio-Rad QC material was used. Ranges provided were for free catecholamines and total metanephrines 25

Results Inter-run Over 3 Days for Commercial QC (BioRad Lyphocheck) Level 1 Level 2 Compound Free/Total Range (HPLC) Measured CV (%) Range (HPLC) Measured CV (%) Dopamine Free 290-490 401 3.4 2465-4105 3323 2.8 Norepinephrine Free 185-305 227 5.8 920-1410 1135 4.8 Epinephrine Free 52.5-104 78 5.3 370-570 473 2 3-Methoxytyramine Total 171-291 267 3.8 2280-3420 3335 2.2 Normetanephrine Total 1200-2000 1641 2.4 5920-8900 7528 2.8 Metanephrine Total 350-590 462 2 2200-3320 3103 2.5 All measurements are in nmol/l Bio-Rad QC material was used. Ranges provided were for free catecholamines and total metanephrines 26

Results Recoveries Observed Using SPE Procedure Compound Absolute Recoveries %* (n = 9) Relative recoveries % With ISTDs corrections** (n = 9) Average SD Range Average SD Dopamine 73.5 2.4 95.0-103.4 100.0 2.5 Norepinephrine 112.5 4.9 99.0-102.4 100.0 1.0 Epinephrine 90.3 3.6 94.5-104.4 100.0 2.9 3-Methoxytyramine 53.2 3.6 94.4-102.5 100.0 3.0 Normetanephrine 88.7 7.5 97.3-102.0 100.0 2.0 Metanephrine 93.9 3.6 97.2-103.9 100.0 2.2 * ISTDs peak areas spiked in formic acid subjected to SPE compared with spiked formic acid without SPE ** Calculated concentrations with ISTDs peak area ratios corrections (with SPE) versus theoretical concentrations 27

Results Matrix Effects Observed Using SPE Procedure Compound Matrix effects %* (n = 9) Accuracies % With ISTDs corrections** (n = 9) Average SD Range Average SD Dopamine 102.1 3.9 95.4-102.5 100.0 2.3 Norepinephrine 30.1 5.2 94.7-104.4 100.0 3.6 Epinephrine 107.1 3.2 95.2-104.6 100.0 3.7 3-Methoxytyramine 88.2 8.5 95.4-104.0 100.0 3.1 Normetanephrine 93.8 3.1 96.4-101.8 100.0 2.8 Metanephrine 103.0 1.3 95.3-101.3 100.0 2.8 * ISTDs peak areas spiked in urine subjected to SPE compared with spiked formic acid subjected to SPE ** Calculated concentrations with ISTD corrections (urine with SPE) versus theoretical concentrations 28

Compound structures Vanillyl mandelic acid (VMA) C9H10O5 Neutral Mass: 198.05 5-Hydroxyindole-3-acetic acid (5-HIAA) C10H9NO3 Neutral Mass: 191.06 Homovanillic acid (HVA) C9H10O4 Neutral Mass: 182.06 30

Sample preparation Calibrators are prepared with clean urine matrix from Golden West Biologicals Isotopically labelled Internal standards 24 hours collection of urine Dilution of urine (vortex and centrifuge) 1 in 10 with 0.2% formic acid in water 31

Example chromatogram 2 1. VMA 2. 5-HIAA 3. HVA 1 3 32

Calibration curves 33

Results Summary of Analyte Performance Compound R 2 Concentration Concentration VMA 0.9999 Accuracy (%) Intraday CV (%) Interday CV (%) (mg/l) (µmol/l) n = 3 n = 3 n = 5 0.078 0.39 106.8 3.9 3.9 12.5 63.1 100.1 1.3 1.7 100 504.6 100.5 0.6 0.5 5-HIAA 0.9998 0.078 0.41 109.7 3.4 3.4 12.5 65.4 99.1 2.2 1.7 100 523.1 100.8 0.1 0.1 HVA 0.9999 0.078 0.43 102.2 4.2 3.2 12.5 68.6 99.9 1.7 1.3 100 548.9 100.5 0.4 0.3 Note: Signal to noise ratios and CVs indicate that LLOQs are lower than measured here for all analytes 34

Results Inter-run Over 3 Days for Commercial QC (BioRad Lyphocheck) Compound Range (HPLC) Level 1 Level 2 Measured CV (%) Range (HPLC) Measured CV (%) VMA 2.1-3.1 2.5 2.3 11.2-16.8 14.6 2.4 5-HIAA 2.2-3.4 2.8 2.0 20.8-31.2 27.6 2.8 HVA 1.00-1.40 1.3 5.8 13.0-19.6 15.8 3.9 All measurements are in mg/l (n=5) Bio-Rad QC material was used. Ranges provided were for HPLC method 35

Results Matrix Effects Observed Compound Matrix effects %* (n = 10) Accuracies % With ISTDs corrections** (n = 10) Average SD Range Average SD VMA 91.5 6.4 95.6-108.3 100.0 3.9 5-HIAA 93.2 3.3 94.6-115.2 100.0 5.8 HVA 91.3 1.5 92.9-103.9 100.0 3.1 Measurements done at 10 different concentrations ranging from 0.078 to 100 mg/l * Peak areas from urine spiked compared with 0.2% formic acid-h2o solutions spiked ** Calculated concentrations of urine spiked with ISTD corrections versus theoretical concentrations 36

Sample preparation Calibrators are prepared with clean plasma matrix from Golden West Biologicals Isotopically labelled Internal Standards Solid phase extraction (SPE) is used to cleanup plasma 38

Plasma calibrator chromatogram with extra wash 1. Normetanephrine 2. Metanephrine 3. 3-Methoxytyramine 3 2 1 39

Calibration curves 40

Results Summary of analyte performance Compound R 2 Concentration Concentration Accuracy (%) Intraday CV (%) Interday CV (%) (pg/ml) (nmol/l) n = 3 n = 3 n = 5 15.63 0.09 115.4 2.4 1.9 3-Methoxytyramine 0.9997 78.13 0.47 97.6 1.8 1.6 1250 7.5 100.4 1.1 1.8 10000 59.8 100.0 0.4 1.0 15.63 0.09 117.3 2.7 4.9 Normetanephrine 0.9996 78.13 0.43 100.0 2.4 2.5 1250 6.8 97.1 2.5 1.8 10000 54.6 100.4 0.4 0.8 15.63 0.08 116.6 1.7 1.9 Metanephrine 0.9998 78.13 0.40 96.5 2.5 2.2 1250 6.3 100.2 1.1 0.9 10000 50.7 100.3 0.6 0.6 41

Results Inter-run over 3 days for commercial QC materials (ChromSystems) Level 1 (n=3) Level 2 (n=3) Compound Measured (pg/ml) CV (%) Measured (pg/ml) CV (%) 3-Methoxytyramine -- -- 1768.9 0.8 Normetanephrine 95.9 5.1 7369.9 3.0 Metanephrine 85.0 2.2 1620.8 3.2 42

Results SPE extraction procedure recoveries with spiked formic acid solutions with and without SPE Compound Absolute Recoveries %* (n = 9) Relative recoveries % With ISTDs corrections** (n = 9) Average SD Range Average SD 3-Methoxytyramine 75.5 19.4 94.4-115.4 100.0 5.7 Normetanephrine 28.0 8.2 95.4-117.3 100.0 5.1 Metanephrine 74.5 14.7 96.4-116.6 100.0 2.9 *: Peak areas spiked in formic acid subjected to SPE compared with spiked formic acid without SPE **: Calculated concentrations with ISTDs peak area ratios corrections (with SPE) versus theoretical concentrations 43

Results Matrix effect and Recovery efficiency Compound Matrix effects % (n = 9) Recovery efficiency % (n = 9) Average SD Average SD 3-Methoxytyramine 78.3 7.2 99.1 16.9 Normetanephrine 36.0 13.7 87.7 27.0 Metanephrine 73.0 9.0 104.4 17.3 A: neat standard solutions B: plasma extracted then spiked (post-ext) C: spiked plasma then extracted (pre-ext) Matrix effect % = B/A * 100 Recovery efficiency % = C/B * 100 44

Sample preparation Challenge: Need to measure low pg/ml in plasma, therefore must cleanup and concentrate Calibrators are prepared with clean plasma matrix from Golden West Biologicals Isotopically labelled Internal Standards Catecholamines are readily oxidized and unstable in whole blood and plasma. Stabilizers should be added to plasma. Step 1: Protein precipitation and lipids removal with Captiva ND Lipids filter cartridges Step 2: Solid phase extraction (SPE) is used to further cleanup plasma 46

Sample Preparation Plasma pretreatment Pretreatment of samples: Plasma should be kept frozen at -80 C until sample analysis Stabilizer solutions: Sodium metabisulfite: 317 mg/ml of sodium metabisulfite in water EDTA 0.5M For sample plasma, calibrators and QCs Add 2% v/v of each stabilizer solution 47

Method LC Conditions (1290) Column: Analytical: Guard: Pursuit 3 PFP, 2 x 150 mm, 3 µm Pursuit 3 PFP MetaGuard 2 mm Column Temp: 30 C Injection volume: 20 µl Needle Wash: 1:1:1:1 MeOH:ACN:IPA:H 2 O + 0.1% formic acid 20 seconds Injector Temp: 4 C Mobile Phase: Flow rate: 1290 Pump Gradient: A: 1 mm Ammonium Fluoride in Water B: Methanol 0.3 ml/min. Time (min) %B 0.0 0 3.8 0 4.0 95 10 95 Stop time: 10 min. Requilibration time: 3 min. 48

Plasma Catecholamines calibrator chromatogram 3 1. Norepinephrine 2. Epinephrine 3. Dopamine 1 2 49

Calibration curves 50

Results Summary of analyte performance Compound R 2 Concentration Concentration Accuracy (%) Inter-day CV (%) n = 3 (pg/ml) (nmol/l) n = 3 n = 3 5 0.03 107.6 4.7 Norepinephrine 0.9999 20 0.12 95.4 1.2 250 1.5 98.6 1.4 2500 14.8 100.2 0.3 5 0.03 108.4 2.1 Epinephrine 0.9998 20 0.11 96.5 1.1 250 1.4 97.5 1.6 2500 13.6 100.6 0.3 5 0.03 108.7 3.2 Dopamine 0.9997 20 0.13 98.8 2.6 250 1.6 98.1 0.9 2500 16.3 99.6 1.1 51

Results Intra and Inter-day over 3 days for commercial QC materials (ChromSystems) Compound QC Level Measured Value Intra-day (n=3) pg/ml - nmol/l Intra-day CV% (n=3) Measured Value Inter-day (n=3) pg/ml - nmol/l Inter-day CV% (n=3) Norepinephrine 0010 0020 240 1756 1.42 10.4 3.6 0.7 242 1767 1.43 10.4 3.0 0.8 Epinephrine 0010 0020 93.4 451 0.51 2.46 1.6 0.7 92.3 449 0.50 2.45 1.2 0.4 Dopamine 0010 0020 164 595 1.07 3.88 0.9 0.5 162 597 1.06 3.90 0.8 0.9 52

Results Matrix effect and Recovery efficiency Compound Matrix effects % (n = 3) Recovery efficiency % (n = 3) Average SD Average SD Norepinephrine 42.3 1.9 56.3 6.3 Epinephrine 70.1 6.6 56.5 2.4 Dopamine 118.5 21.5 58.7 4.3 A: neat standard solutions B: plasma extracted then spiked (post-ext) C: spiked plasma then extracted (pre-ext) Matrix effect % = B/A * 100 Recovery efficiency % = C/B * 100 53

Sample preparation Why Which sample preparation technique to choose 54

Objectives of Sample Preparation Removal of interferences that affect detection and accuracy Reach lower detection limits Increase method robustness Decrease overall operating costs 55

Types of Interferences in Biological Samples Major causes of matrix effects: Salts generally elute early in the run Abundant Proteins most prominent interference Lipids, phospholipids, and lysophosphatidylcholines difficult to remove Surfactants, dosing agents, excipients Phthalates and plasticizers from plastic ware 56

Instrumentation affects selection of Sample Preparation Techniques More Specific Instrument Separation and Detection Specificity Less Specific Less Specific Sample Preparation Specificity More Specific Sample Prep Technique Interference Removed Dilute & Shoot Filtration Liquid/Liquid Extractions Supported Liquid Extractions (SLE) Dried Matrix Spotting Precipitation QuEChERS Lipid Removal Hybrid' Filtration Solid Phase Extraction Lipids No No No Some No No Yes Yes Yes Oligomeric Surfactants No No No No No No No Yes Yes Particulates No Yes No Some No Yes Yes Yes Yes Pigments No No No Some No No Yes No Yes Polar Organic Acids No No Yes Yes No No Yes No Proteins No No Yes Yes Yes Yes Yes Yes Yes Salts No No Yes Yes No No No No Yes Suggested Agilent Product Agilent Autosampler Vials Captiva Syringe Filters Chem Elut Captiva ND Bond Elut QuEChERS Captiva ND LIPIDS Bond Elut Silica and Polymeric SPE 57

Why did we use different sample preparation techniques? 1. Free and Total Cats/Mets 2. VMA, HVA, 5- HIAA 3. Mets 4. Cats Matrix Urine Urine Plasma Plasma Linear range 1.56-3000 ng/ml Sample prep Boronate complex/ Bond Elut Plexa SPE 0.08-100 mg/l 15.6-10000 pg/ml 5-2500 pg/ml Dilution WCX SPE Captiva ND Lipids / BondElut PBA SPE Analyte Dependant HO CH 3 O OH HO NH 2 HO HN CH 3 Sample Dependant 58

Why did we use different sample preparation techniques? Consideration 1: Sample type (Urine vs Plasma) The dirtier the sample, the better sample cleanup you need to remove interferences If the sample matrix is not removed you may have: carry-over, lipid build-up, ion suppression, low signal to noise ratio A dirty sample increases operational costs, impacting instrument maintenance, sample re-runs, column lifetime Consideration 2: Analyte characteristics and LLOQs To reach pg/ml levels, you need a sample prep technique that concentrates the analyte The analyte s chemical properties are unique (Polarity, Log P, pka, solubility) Some analytes have stability limitations 59

What is Solid Phase Extraction (SPE) Four steps to Selective cleanup and Elution Green = Blue and Yellow Blue is more non polar than yellow Blue is retained Sample loading Retention Rinsing Elution 60

Catecholamines and metanephrines in urine Solid Phase Extraction (SPE) Prepare complexed samples: 0.5 ml urine*, calibrators, QCs* Add 40 µl of internal standards mix Add 0.8 ml of Diphenyl-Boronate complexing agent Verify ph, must be between 7.5-9.5. If necessary adjust to ph 8.5 with NH4OH Step 1: Step 2: Step 3: Step 4: Condition SPE cartridge (Bond Elut Plexa, 30 mg, 3 ml) with: 1 ml of MeOH 1 ml of wash buffer 0.2 M NH4Cl-NH4OH Add complexed samples Wash with 1 ml of 5% MeOH wash buffer 0.2 M NH4Cl-NH4OH Dry at full vacuum for 5 minutes Elute with 1 ml of 5% formic acid in water. Apply vacuum 5 Hg for 30 seconds Transfer to autosampler vial * Native for free catecholamines, hydrolyzed for total metanephrines (add 25 µl HCl 6N, incubate at 90 deg. C for 25 min., cool at RT) [1] Ann Clin Biochem 2009; 46: 129 136. DOI: 10.1258/acb.2008.008180 [2] Talwar et al., Journal of Chromatography B, 769 (2002) 341 349 61

Diphenyl boronate-catecholamine complex The diphenyl boronate forms a stable negatively charged complex (Fig. 1) with cishydroxyl groups of catecholamines, which is strongly retained on a C18 extraction sorbent when operating in alkali media. This allows for column washing with methanol-buffer solutions to remove interfering compounds without the loss of the catecholamines which are eluted by disrupting the complex under acid conditions. [2] Talwar et al., Journal of Chromatography B, 769 (2002) 341 349 62

Metanephrines in plasma Pretreatment of samples: 0.5 ml plasma, calibrators, QCs Add 50 µl of internal standards mix Add 0.5 ml of 10 mm NH4H2PO4 buffer ph 6.5 Step 1: Step 2: Step 3: Step 4: Condition SPE cartridge (SampliQ WCX, 30 mg, 1 ml) with: 1 ml of MeOH 1 ml of 10 mm NH4H2PO4 buffer ph 6.5 Add samples Wash with 1 ml H2O, 1 ml Methanol, 1 ml 0.2% formic acid in acetonitrile Dry at full vacuum for 5 minutes Elute with 2 x 250 µl of 2% formic acid in acetonitrile. Apply vacuum 5 Hg for 60 seconds Evaporate under nitrogen flow at 40 deg. C Reconstitute with 100 µl of 0.2% formic acid in water 65

Catecholamines in plasma Step 1 Protein precipitation and lipids removal Add 1.5 ml of 0.5% formic acid in acetonitrile to a Captiva ND Lipids cartridge (3 ml, PN: A5300635) or plate Add 50 µl of internal standard solution Add 750 µl of pretreated sample plasma or calibrators or QCs Mix 3-5 times with a 1.5 ml empty pipette tip Wait 5-10 minutes Place under vacuum at 7 Hg for 2 minutes, then at 15 Hg until dry Use filtrate for SPE 66

Captiva ND Lipids Lipid removal and filtration principles Features: - Captiva particulate filter removes protein interferences - Proprietary Lipid Stripping Media removes lipids - Non-Drip Membrane ease of use 67

Catecholamines in plasma Step 2 Solid Phase Extraction (SPE) Pretreatment of samples: Use filtrate from step 1 Add 2 ml of 100 mm NH4H2PO4 buffer ph 10 1: Condition SPE cartridge (BondElut PBA, 100mg, 3 ml, PN: 12102127) with: 1 ml acetonitrile 1 ml 5% formic acid in methanol 1 ml 100 mm NH4H2PO4 buffer ph 10 2: Add pretreated samples 3: Wash with: 2 ml 1% NH4OH in 95% methanol 2 ml 1% NH4OH in 95% acetonitrile 2 ml 1% NH4OH in 30% acetonitrile Dry at full vacuum for 5 minutes 4: Elute with: 3 x 500 µl of 5% formic acid in methanol Apply vacuum 5 Hg for 60 seconds Evaporate under nitrogen flow at 35 deg. C Reconstitute with 100 µl of 0.1% formic acid in water 68

Conclusions A unified approach where the same chromatography principles and the same modern LC/MS/MS instrumentation were used to analyze different compounds and matrices Novel and selective sample preparation techniques were adapted to the matrix and to the desired LLOQ Four analytical methods that are robust, sensitive and precise were developed 69

More information 5991-6194EN 5991-6053EN 5991-6531EN 5991-6530EN 70

Questions Thank You! For more information on Agilent s Clinical Research Solutions, visit www.agilent.com/chem/clinicalresearch 71