Performance of an ultra low elution volume 96-well plate Claude R. Mallet, Ziling Lu, Jeff R. Mazzeo, Uwe D. Neue Waters Corporation PittCon 2003 March 10-14 2003 Orlando, Florida
Today s Challenges Faced by Analytical Chemists in Methods Development Sample Process Faster methods development Generic and simple extraction protocol Increased sensitivity and selectivity Instrumentation Faster analysis (1000-analysis-per-day barrier)
Method Development Sample Preparation Raw sample: - CACO2, microsomes, P450, hepatocytes etc - tissue, CSF, plasma, serum urine, tears etc - water, sediment, food etc Extracted sample For LC/MS/MS Ideally, the final sample should be as clean as a non extracted standard. Chromatography Polarity: Silica- C 18, C 8, C 4, C 2 Hybrid- C 18, C 8, C 4, C 2 Polymer- C 18, C 8, C 4, C 2 Embedded polar group Cyano, Phenyl Particle size: 2.5, 3.5, 5 or 7 µm Internal diameter: 4.6, 3.9, 2.1, 1.0, 0.32 mm and 75 µm Length: 150, 100, 50, 30, 20 mm Mass Spectrometry Source: ESI APcI Nano-ESI Mass analyzers: magnetic sectors electric sectors time of flight quadrupole ion trap FT-ICR
Extraction Techniques Many extractions practices are based on classical methodologies of liquid-liquid or liquid-solid extraction using practices which have not changed for the last one hundred years Classical Protein precipitation Liquid-liquid extraction Membrane extraction Soxhlet New technologies Solid phase extraction Solid phase micro extraction Accelerated fluid extraction Supercritical fluid extraction Microwave-assisted extraction Extraction Methods in Organic Analysis A.J. Handley 1998
Drug Development Overview DRUG DISCOVERY Target ID, Lead Generation, Lead Optimization Matrix: CaCO2, microsomes P450, hepatocytes, rat plasma Sample volume: 50 100 µl Linearity range: 5 250 ng/ml DRUG DEVELOPMENT DRUG MANUFACTURE Pre-Clinical, Clinical Trials, Pilot Plant Matrix: plasma/serum from rat, rabbit, dog, monkey, human Sample volume: 50 µl up to 1 ml Linearity range: 0.1 ng/ml 500 ng/ml QA/QC, Patent Protection
96-Well Plates and Cartridges 96-well plate barrel
Generic reversed phase SPE Method Prepare Sample Solution Condition/Equilibrate 500 µl methanol / 500 µl water Typical values for a 10 mg bed packing Load 500 µl spiked sample solution Pre concentration step Wash 500 µl 5% methanol in water Elute 500 µl methanol Evaporate and Reconstitute Note: For larger bed packing (e.g. 30 or 60 mg), increase the condition, load, wash and elution volumes
Choice of Sorbent Weight Based on Sample Size Sorbent per Well Maximum Mass Capacity Typical Sample Volume Typical Elution Volume 5 mg 0.15 to 1 mg 10 to 100 µl < 150 µl 10 mg 0.35 to 2 mg 50 to 400 µl < 250 µl 30 mg 1 to 5 mg 100 µl to 1 ml > 400 µl 60 mg 2 to 10 mg 200 µl to 2 ml > 800 µl With decreasing limit of quantification (LOQ s), it will be necessary to include an evaporation and reconstitution step, which is extremely time consuming.
Waters two-stage Well Design in 96-Well Extraction Plate Enabling technology for 96-well plates - Oasis sorbent in the amount required to meet your capacity and elution volume needs. Designed for Optimal Flow Property 5mg 30 mg 10 mg 60 mg
Oasis µelution 96-well plate
µelution tip 750 µl reservoir Top ball frit Plate tip Sorbent Bottom ball frit
Oasis HLB Sorbent Hydrophilic-Lipophilic Balanced copolymer N O N-Vinyl-Pyrrolidone Water Loving Hydrophilic monomer - Provides wetting properties - No impact of sorbent drying Di-Vinyl-Benzene Fat Loving Lipophilic monomer -Provides reversed phase properties for analyte retention
Oasis MCX Sorbent O N Strong cation-exchange mode SO 3 H SO 3 H Best retention at least 2 ph units below pka CH3 Reversed-phase mode O OH H N + H CH3 Propranolol Basic drug
Generic micro-elution reversed phase SPE Method Prepare Sample Solution Condition/Equilibrate 200 µl methanol / 200 µl water Smaller condition volumes Dilute plasma 1:1 ratio with water Load 100 µl spiked sample solution Wash 200 µl 5% methanol in water Elute 25 µl ACN:IPA 40:60 + 2% FA HILIC Pre-concentration step is avoided Dilute with 50 µl water C 18
Generic micro-elution mixed mode SPE Method Prepare Sample Solution Condition/Equilibrate 200 µl methanol / 200 µl water Smaller condition volume Dilute plasma 1:1 ratio with water Removes polar interferences Load 100 µl spiked sample solution Wash 1 200 µl Water + 2 % FA Wash 2 200 µl MeOH Elute 25 µl ACN:IPA 40:60 + 2% NH 4 OH Locks basic drug on ion exchanger Pre-concentration step is avoided Dilute with 50 µl water
Discovery Application O OH N CH 3 CH 3 H Propranolol Antihypertensive N CH 3 CH 3 Amitriptyline Antidepressant
LC/MS/MS conditions MS: Micromass Quattro Ultima LC: Waters Alliance 2795 Ion source: ESI (+) Flow rate: 0.2 ml/min Source temperature: 150 C Mobile phase A: Water + 0.5 % NH 4 OH Gas cell: 2.0 e-3 bar Argon Mobile phase B: ACN + 0.5 % NH 4 OH Desolvation temperature: 350 C Column: XTerra MS C18 Capillary voltage: 3.5 kv 2.1 x 30 mm, 3.5 µm Drying gas flow: 500 L/hr LC conditions: 5% to 95% in 1 min. Cone gas flow: 50 L/hr Column temperature: ambient Cone voltage: 35 volts MRM transition: Metoclopramide (IS) m/z 299.8 226.7 Propranolol m/z 259.9 154.9 Amitriptyline m/z 278.1 232.9 2003. Waters Corporation
Comparison of Ppt vs HLB vs MCX 100 MRM of 2 Channels ES+ 259.9 > 154.9 % Ppt Propranolol 1 ng/ml 3.79 Area: 278 16 100 3.83 MRM of 2 Channels ES+ 259.9 > 154.9 % Reversed Phase Propranolol 1 ng/ml Area: 1329 7 100 3.83 MRM of 2 Channels ES+ 259.9 > 154.9 % Mixed Mode Propranolol 1 ng/ml Area: 13 534 2 Time 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Comparison of Ppt vs HLB vs MCX 100 Ppt Amitriptyline 0.1 ng/ml 4.02 4.75 MRM of 2 Channels ES+ 278.1 > 232.9 9.71e3 % 2.98 3.38 5.18 Area: 434 17 100 % Reversed Phase Amitriptyline 0.1 ng/ml 4.67 MRM of 2 Channels ES+ 278.1 > 232.9 3.62e4 Area: 1 624 4 100 Mixed Mode Amitriptyline 0.1 ng/ml 4.68 MRM of 2 Channels ES+ 278.1 > 232.9 8.91e4 % Area: 5 904 2 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 Time
Development Application H 3 C CH 3 N CH 3 Terfenadine Antihistaminic OH N OH OH H 3 C CH 3 OH Terfenadine-alcohol metabolite OH H 3 C CH 3 N OH O OH Terfenadine-carboxylate metabolite OH
LC/MS/MS analysis of Terfenadine and Metabolites on mixed mode 100 % Terfenadine-carboxylate 0.5 ng/ml 2.45 Area: 16 034 MRM of 4 Channels ES+ 502.2 > 466.2 1.52e5 0 100 % Terfenadine-alcohol 0.5 ng/ml 2.61 Area: 29 366 MRM of 4 Channels ES+ 488.2 > 452.2 2.97e5 0 100 2.91 MRM of 4 Channels ES+ 472.2 > 436.2 2.18e5 % Terfenadine 0.5 ng/ml Area: 20 599 0 100 % 2.45 IS MRM of 4 Channels ES+ 263.9 > 190.8 2.20e6 0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 Time 2003. Waters Corporation
Calibration curve of Terfenadine on mixed mode Conc. ng/ml N=6 Average Standard Deviation RSD % 0.5 1.0 5.0 10.0 20.0 100.0 200.0 0.50 0.96 4.99 10.11 19.26 103.25 197.18 0.008 0.048 0.25 0.51 0.38 3.68 1.92 2 5 5 5 2 4 1 200 Coefficient of Determination: 0.997836 Calibration curve: 0.00190507 * x^2 + 0.617981 * x + -0.146974 Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area ) Curve type: 2nd Order, Origin: Exclude, Weighting: 1/x^2, Axis trans: None Response -0.147 0.0 20.0 40.0 60.0 80.0 100.0 120.0 Concentration (ng/ml) 140.0 160.0 180.0 200.0 2003. Waters Corporation
Calibration curve of Terfenadine-Carboxylate on mixed mode Conc. ng/ml N=6 Average Standard Deviation RSD % 63.5 0.5 1.0 5.0 10.0 20.0 100.0 200.0 0.49 1.02 5.05 10.12 19.71 101.69 196.39 0.013 0.052 0.26 0.49 0.46 3.25 4.41 3 5 5 5 2 3 2 Coefficient of Determination: 0.996306 Calibration curve: 0.300987 * x + -0.0391382 Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x^2, Axis trans: None Response -0.0391 0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0 160.0 180.0 200.0 Concentration (ng/ml) 2003. Waters Corporation
Calibration curve of Terfenadine-Alcohol on mixed mode Conc. ng/ml N=6 Average Standard Deviation RSD % 0.5 1.0 5.0 10.0 20.0 100.0 200.0 0.51 0.97 5.19 10.24 19.80 102.05 196.55 0.008 0.041 0.27 0.44 0.77 3.15 7.92 2 4 5 4 4 3 4 183 Coefficient of Determination: 0.993727 Calibration curve: 0.854217 * x + -0.196977 Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x^2, Axis trans: None Response -0.197 0.0 20.0 40.0 60.0 80.0 100.0 120.0 Concentration (ng/ml) 140.0 160.0 180.0 200.0 2003. Waters Corporation
Conc. (ng/ml) 0.5 1.0 5 10 20 100 200 Terfenadine Day 1 (N=6) (C.V.) 0.50 (3) 0.97 (8) 4.96 (4) 9.93 (2) 19.89 (4) 105.29 (4) 195.87 (2) Day 2 (N=6) 0.51 (2) 0.92 (2) 5.29 (2) 10.51 (4) 19.41 (3) 100.56 (4) 200.70 (1) Day 3 (N=6) 0.50 (3) 0.98 (5) 5.09 (6) 9.64 (5) 19.22 (3) 106.38 (3) 194.47 (2) Day 4 (N=6) 0.51 (2) 0.89 (1) 5.21 (3) 9.94 (5) 19.20 (3) 107.04 (2) 197.1 (1) Terfenadine-carboxylate Day 1 (N=6) (C.V.) 0.50 (2) 0.96 (5) 4.88 (6) 10.45 (5) 20.74 (6) na na Day 2 (N=6) 0.50 (3) 1.01 (6) 4.79 (1) 9.68 (4) 21.11 (3) 97.12 (5) 204.06 (4) Day 3 (N=6) 0.45 (3) 0.99 (5) 5.21 (4) 10.08 (5) 20.79 (4) 98.36 (4) 194.85 (4) Day 4 (N=6) 0.50 (2) 1.00 (4) 5.10 (6) 10.35 (3) 20.34 (3) 96.02 (5) 192.70 (3) Terfenadine-alcohol Long term stability of Terfenadine and Metabolite on mixed mode Day 1 (N=6) (C.V.) 0.52 (1) 0.92 (2) 4.99 (2) 10.30 (4) 20.53 (2) 96.74 (4) 195.23 (5) Day 2 (N=6) 0.51 (1) 0.94 (2) 5.01 (4) 10.03 (4) 20.55 (3) 100.83 (3) 191.65 (3) Day 3 (N=6) 0.50 (2) 0.97 (5) 4.99 (5) 9.88 (2) 20.27 (5) 99.34 (5) 204.10 (3) Day 4 (N=6) 0.50 (2) 1.00 (5) 5.03 (4) 10.36 (4) 20.23 (6) 97.43 (7) 196.04 (3)
Conclusions Wide volume range for loading (50 to 800 µl) Low elution volume (25 50 µl) Generic protocols for reversed phase and mixed mode Pre-concentration factor up to 10x Sub ng/ml quantitation limits 96-well plate format for high throughput No evaporation and reconstitution step needed