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©2003 Waters Corporation
Performance of an ultra low elutionvolume 96-well plate
Claude R. Mallet, Ziling Lu, Jeff R. Mazzeo, Uwe D. NeueWaters Corporation
PittCon 2003March 10-14 2003Orlando, Florida
©2003 Waters Corporation
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)
©2003 Waters Corporation
Method Development
Sample Preparation Chromatography
MassSpectrometry
Polarity:Silica- C18, C8, C4, C2Hybrid- C18, C8, C4, C2Polymer- C18, C8, C4, C2Embedded polar groupCyano, PhenylParticle size:2.5, 3.5, 5 or 7 µmInternal diameter:4.6, 3.9, 2.1, 1.0, 0.32 mm and 75 µmLength:150, 100, 50, 30, 20 mm
Source:ESIAPcINano-ESIMass analyzers:magnetic sectorselectric sectorstime of flightquadrupoleion trapFT-ICR
Raw sample:- CACO2, microsomes, P450,hepatocytes … etc- tissue, CSF, plasma, serumurine, tears … etc- water, sediment, food … etc
Extracted sampleFor LC/MS/MS
Ideally, the final sample should be as clean as a non extracted standard.
©2003 Waters Corporation
Extraction Techniques
“Many extractions practices are based on classical methodologiesof liquid-liquid or liquid-solid extraction using practices which have not changed for the last one hundred years”
Extraction Methods in Organic Analysis – A.J. Handley 1998
Protein precipitationLiquid-liquid extractionMembrane extractionSoxhlet
Solid phase extractionSolid phase micro extractionAccelerated fluid extractionSupercritical fluid extractionMicrowave-assisted extraction
Classical New technologies
©2003 Waters Corporation
Drug Development Overview
DRUG DISCOVERYDRUG DISCOVERY
DRUG DEVELOPMENT
DRUG DEVELOPMENT
DRUG MANUFACTURE
DRUG MANUFACTURE
Target ID, Lead Generation, Lead Optimization
Pre-Clinical, Clinical Trials, Pilot Plant
QA/QC, Patent Protection
Matrix: CaCO2, microsomes P450, hepatocytes, rat plasmaSample volume: 50 – 100 µLLinearity range: 5 – 250 ng/mL
Matrix: plasma/serum from rat, rabbit, dog, monkey, humanSample volume: 50 µL up to 1 mLLinearity range: 0.1 ng/mL – 500 ng/mL
©2003 Waters Corporation
96-Well Plates and Cartridges
96-well plate
barrel
©2003 Waters Corporation
Generic reversed phase SPE Method
Condition/Equilibrate500 µL methanol / 500 µL water
Load500 µL spiked sample solution
Wash500 µL 5% methanol in water
Elute500 µL methanol
Evaporate and Reconstitute
Prepare Sample Solution
Typical values for a 10 mg bed packing
Note: For larger bed packing (e.g. 30 or 60 mg), increase the condition, load, wash and elution volumesPre concentration step
©2003 Waters Corporation
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.
©2003 Waters Corporation
30 mg10 mg 60 mg5 mg
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
Waters two-Stage Well Designin 96-Well Extraction Plate
©2003 Waters Corporation
Oasis® µElution 96-well plate
©2003 Waters Corporation
µElution tip
Top ball frit
Bottom ball frit
Sorbent
750 µLreservoir
Plate tip
©2003 Waters Corporation
Fat LovingLipophilic monomer
NO
Water LovingHydrophilic monomer
Hydrophilic-Lipophilic Balanced copolymer
N-Vinyl-Pyrrolidone Di-Vinyl-Benzene
- Provides wetting properties- No impact of sorbent drying
-Provides reversed phase properties for analyte retention
Oasis® HLB Sorbent
©2003 Waters Corporation
Strong cation-exchange mode
Best retention at least 2 pH units below pKa
O
OH
NH
CH3
CH3
Propranolol
H+
Reversed-phase mode
Basic drug
NO
SO3HSO3H
Oasis® MCX Sorbent
©2003 Waters Corporation
Generic micro-elutionreversed phase SPE Method
Condition/Equilibrate200 µL methanol / 200 µL water
Load100 µL spiked sample solution
Wash200 µL 5% methanol in water
Elute25 µL ACN:IPA 40:60 + 2% FA
Dilute with 50 µl water
Prepare Sample Solution
Pre-concentration step is avoided
Smaller conditionvolumes
Dilute plasma1:1 ratio with water
HILIC
C18
©2003 Waters Corporation
Generic micro-elutionmixed mode SPE Method
Condition/Equilibrate200 µL methanol / 200 µL water
Load100 µL spiked sample solution
Prepare Sample Solution
Wash 2200 µL MeOH
Elute25 µL ACN:IPA 40:60 + 2% NH4OH
Dilute with 50 µl waterPre-concentration step is avoided
Smaller conditionvolume
Wash 1200 µL Water + 2 % FA Locks basic drug
on ion exchanger
Removes polar interferences
Dilute plasma1:1 ratio with water
©2003 Waters Corporation
Discovery Application
NCH3
CH3
ON
CH3CH3
HOHPropranolol
Antihypertensive
AmitriptylineAntidepressant
©2003 Waters Corporation.
LC/MS/MS conditions
MS: Micromass Quattro Ultima LC: Waters Alliance® 2795Ion source: ESI (+) Flow rate: 0.2 mL/minSource temperature: 150 °C Mobile phase A: Water + 0.5 % NH4OHGas cell: 2.0 e-3 bar Argon Mobile phase B: ACN + 0.5 % NH4OHDesolvation temperature: 350 °C Column: XTerra MS C18Capillary voltage: 3.5 kV 2.1 x 30 mm, 3.5 µmDrying gas flow: 500 L/hr LC conditions: 5% to 95% in 1 min. Cone gas flow: 50 L/hr Column temperature: ambientCone 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
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00Time2
100
%
7
100
%
16
100
%
MRM of 2 Channels ES+259.9 > 154.9
3.79
MRM of 2 Channels ES+259.9 > 154.93.83
MRM of 2 Channels ES+ 259.9 > 154.93.83
Ppt Propranolol 1 ng/mL
Reversed Phase Propranolol 1 ng/mL
Mixed Mode Propranolol 1 ng/mL
Area: 278
Area: 1329
Area: 13 534
Comparison of Ppt vs HLB vs MCX
©2003 Waters Corporation
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00Time2
100
%
4
100
%
17
100
%
MRM of 2 Channels ES+278.1 > 232.9
9.71e34.754.02
2.98
3.38
5.18
MRM of 2 Channels ES+ 278.1 > 232.9
3.62e44.67
MRM of 2 Channels ES+ 278.1 > 232.9
8.91e44.68
Ppt Amitriptyline 0.1 ng/mL
Reversed Phase Amitriptyline 0.1 ng/mL
Mixed Mode Amitriptyline 0.1 ng/mLArea: 5 904
Area: 1 624
Area: 434
Comparison of Ppt vs HLB vs MCX
©2003 Waters Corporation
Development Application
N
OH
OH
CH3CH3
CH3
TerfenadineAntihistaminic
N
OH
OH
CH3CH3
OH
N
OH
OH
CH3CH3
OHO
Terfenadine-alcohol metabolite
Terfenadine-carboxylate metabolite
©2003 Waters Corporation.
LC/MS/MS analysis of Terfenadine and Metabolites on mixed mode
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50Time0
100
%
0
100
%
0
100
%
0
100
%
MRM of 4 Channels ES+ 502.2 > 466.2
1.52e52.45
MRM of 4 Channels ES+488.2 > 452.2
2.97e52.61
MRM of 4 Channels ES+472.2 > 436.2
2.18e52.91
MRM of 4 Channels ES+263.9 > 190.8
2.20e62.45
Terfenadine 0.5 ng/mL
IS
Terfenadine-alcohol 0.5 ng/mL
Terfenadine-carboxylate 0.5 ng/mL
Area: 20 599
Area: 29 366
Area: 16 034
©2003 Waters Corporation.
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
Concentration (ng/mL)
Res
pons
e
Calibration curve of Terfenadine on mixed mode
Coefficient of Determination: 0.997836Calibration curve: 0.00190507 * x^2 + 0.617981 * x + -0.146974Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )Curve type: 2nd Order, Origin: Exclude, Weighting: 1/x^2, Axis trans: None
0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0 160.0 180.0 200.0-0.147
200
©2003 Waters Corporation.
Conc. ng/mL N=6
Average
Standard Deviation
RSD %
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
Concentration (ng/mL)
Res
pons
e
Calibration curve of Terfenadine-Carboxylate on mixed mode
Coefficient of Determination: 0.996306Calibration curve: 0.300987 * x + -0.0391382Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x^2, Axis trans: None
0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0 160.0 180.0 200.0-0.0391
63.5
©2003 Waters Corporation.
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
Concentration (ng/mL)
Res
pons
e
Calibration curve of Terfenadine-Alcohol on mixed mode
Coefficient of Determination: 0.993727Calibration curve: 0.854217 * x + -0.196977Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x^2, Axis trans: None
0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0 160.0 180.0 200.0-0.197
183
©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 naDay 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
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)
Long term stability of Terfenadine and Metabolite on mixed mode
©2003 Waters Corporation
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