An Automated Dynamic BioAnalytical Sample Preparation Routine for Integration with Liquid Handlers

1 2 1 1
Charles E. Taylor , Qiner Yang , John P. Walsh and Robyn A. Rourick
1 2
Kalypsys, Inc. Pharmaceutical Sciences Department and Informatics 10420 Wateridge Circle, San Diego, CA 92121

Abstract Experimental Results

The Analytical Chemistry department at Kalypsys has developed and implemented Plasma Vol of Prev Vol of Total Remaining Vol Stock Solution C57 Male Mouse Plasma Sprague-Dawley Male Rat Plasma Rat Manual Rat Automatic
Blank Vol Conc. R1 R2 R3 AVG CV CV AVG R1 R2 R3 Conc.
an automated sample preparation method for its routine BioAnalytical work. The Conc. Manual Preparation Automated Preparation Manual Preparation Automated Preparation
• 200ug/mL of Analyte in 50:50 4 93.1% 102% 97.8% 97.63% 4.56% 100.92% 231.83% 97.9% 502% 95.6% 4
(ng/mL) (uL) (uL) (uL) (uL) 100807_Trial7_Manual_Mouse.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000244 x + 0.00143 (r = 0.9957)�� 100807_Trial17_FullAuto_Mouse.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000299 x + 0.00112 (r = 0.9977)�� 100507_Trial3_Manual_Rat.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000471 x + 0.00225 (r = 0.9958)�� 101207_Trial22_FullAuto_Rat.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000232 x + 0.00116 (r = 0.9983)��

purpose of this system was to assume the work effort of protein precipitation sample acetonitrile:water
101407_Trial24_Manual_Mouse.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000501 x + 0.004 (r = 0.9989)��
101407_Trial30_Manual_Mouse.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000511 x + 0.0162 (r = 0.9986)��
101507_Trial36_FullAuto_Mouse.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000295 x + 0.00198 (r = 0.9975)��
101507_Trial42_FullAuto_Mouse.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000276 x + 0.00187 (r = 0.9971)��
101407_Trial25_Manual_Rat.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000498 x + 0.00391 (r = 0.9969)��
101407_Trial31_Manual_Rat.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000511 x + 0.0126 (r = 0.9990)��
101507_Trial37_FullAuto_Rat.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000307 x + 0.00293 (r = 0.9980)��
101507_Trial43_FullAuto_Rat.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000284 x + 0.00217 (r = 0.9965)��
10 115% 86.9% 104% 101.97% 13.89% 6.96% 105.17% 104% 98.5% 113% 10
10000 35 of stock 665 700 250
preparation, reducing the FTE time required per study. Designed to process sample 5.0
Curve R2 Slope
100807_Trial15_Manual_Human.rdb
3.9 (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000243 x + 0.00118 (r = 0.9910)
Curve R2 Slope
5.0
Curve R2 Slope
100807_Trial15_Manual_Human.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000243 x + 0.00118 (r = 0.9910)
3.5 Curve R2 Slope 20 120% 112% 100% 110.67% 9.1% 7.94% 99.77% 101% 107% 91.3% 20
7500 450 150 600 240 • Vortexed and sonicated 4.0 1st .9957 .000244
3.5
2.0 1st 0.9977 .000299 1st .9958 .000471 2.0 1st .9983 .000232 50 110% 103% 108% 107% 3.37% 10.65% 101.03% 104% 89.1% 110% 50
4.0 3.0
plates with any combination of matrices and layouts into methodical injection plates, 3.0

Analyte Area / IS Area

Area/ IS Area

Area/ IS Area
Analyte Area / IS Area
2nd .9989 .000501 2nd 0.9975 .000295 2nd .9969 .000498 2nd .9980 .000307
5000 360 180 540 290 Internal Standard (IS) 2.5 2.5 100 125% 97.4% 103% 108.47% 13.45% 2.82% 100.47% 101% 97.4% 103% 100
3.0 3rd .9986 .000511 3rd 0.9971 .000276 3.0 3rd .9990 .000511 3rd .9965 .000284
the system is able to achieve much of the sample arrangement that a human analyst 2500 250 250 500 250
1.5
2.0
1.5
2.0 250 101% 113% 101% 105% 6.6% 3.91% 103.33% 101% 108% 101% 250

Area / ISArea

Area / ISArea
• 500ng/mL deuterated Analyte (Analyte-D6) 2.0 1.5 2.0 1.5 500 107% 103% 98.6% 102.87% 4.08% 2.5% 98.4% 101% 98.1% 96.1% 500
would provide. The system depends on proper plate map registration; a tool was 1000 250 375 625 375

Analyte Analyte

Analyte Analyte
1.0
1.0 1.0
1.0
500 250 250 500 250 in acetonitrile 1.0 1.0 1000 103% 96.6% 97% 98.87% 3.63% 2.16% 95.8% 95.2% 98.1% 94.1% 1000
developed to allow users to populate the database with a platemap containing 0.5 0.5
2500 97.1% 96.3% 105% 99.47% 4.83% 5.12% 98.5% 94.1% 104% 97.4% 2500
250 250 250 500 250 Vortexed and sonicated 0.0 0.0
0.5 0.0 0.0
0.5
animal number, compound number, dose type, matrix, model, time point and • 0.0 2000.0 4000.0 6000.0
Analyte Conc. / IS Conc.
8000.0 1.0e4 0.0 2000.0 4000.0 6000.0
Analyte Conc. / IS Conc.
8000.0 1.0e4 0.0 2000.0 4000.0 6000.0
Analyte Conc. / IS Conc.
8000.0 1.0e4 0.0 2000.0 4000.0 6000.0
Analyte Conc. / IS Conc.
8000.0 1.0e4 5000 91.2% 97.1% 95.3% 94.53% 3.2% 6.14% 96.3% 91.8% 103% 94.1% 5000
100 250 375 625 375 Figure 2: Automated Liquid Handler Generated 7500 92% 95.7% 94.6% 94.1% 2.02% 2.19% 95.2% 96.3% 96.5% 92.8% 7500
concentration for each well of a plate. A server based program crafts a liquid handler CS0 Controls Figure 1: Automated Liquid Handler Generated 0.0
2000.0 4000.0
Figure 3: Automated Liquid Handler Generated Figure
0.0
4: Automated Liquid Handler Generated
50 250 250 500 250 Calibration Curve for Mouse Plasma Calibration Curve for 6000.0
Analyte Conc. / IS Conc.
8000.0
Mouse Plasma 1.0e4
Calibration Curve for Rat Plasma
2000.0 4000.0
Calibration Curve forConc.
Analyte Rat
6000.0
/ ISPlasma
Conc.
8000.0 1.0e4
10000 90.2% 95.7% 96.2% 94.03% 3.54% 4.52% 115% 112% 121% 112% 10000
script that constructs the corresponding injection plate(s) from the sample plate(s), 20 250 375 625 375 • 25uL of blank plasma was combined with 0.20 0.08
0.14 0.08
Table 5: The back-calculated concentrations of the standard curve plasma samples were made by entering the analyte peak
once the desired calibration curve is chosen on the web based user interface. The 0.06 0.12 area into the calibration curve, which those samples were used to construct. The tolerability of the standard curve
10 250 250 500 250 100uL of IS and placed in the same 0.15
0.06

A naly te A rea / IS A rea

A naly te A rea / IS A rea

A naly te A rea / IS A rea

A naly te A rea / IS A rea

0.04 0.10
0.04 point to be included in the curve is measured by it’s statistical difference from the theoretical concentration.
resulting script is a standalone file, not a work list, which is recognized and executed 4 250 375 625 625 precipitation plate as the standard 0.10
0.02 0.08
0.02 Exclusion from the standard curve occurs when the point is outside of the 85% to 115% tolerability range; the LLOQ is
0.00 0.06

by Tecan’s Gemini software. The script generator optimizes the liquid handling by Table 1: Serial dilution used in the construction of the concentration samples. 0.05
-0.02
0.04
0.00

-0.02
allowed 80% to 120% tolerability. The red cells denote excluded data points. The rat model is the most pertinent at
0.02 Kalypsys, which is why it is displayed here. The results confirm that the automated method of preparing rat plasma
mimicking the logic a human analyst would use in injection plate construction. One standard curves in each plasma type. -0.04
-0.04
0.00
0.00 samples is consistent in producing tight standard curves with minimal point exclusion due to intolerability.
0 20 40 60 80 100 120 140 160 180 200 220 240 260 0 20 40 60 80 100 120 140 160 180 200 220 240 260 0 20 40 60 80 100 120 140 160 180 200 220 240 260 0 20 40 60 80 100 120 140 160 180 200 220 240 260
representation is the ordering of samples into the densest arrangement. This allows, Analyte Conc. / IS Conc. Analyte Conc. / IS Conc. Analyte Conc. / IS Conc. Analyte Conc. / IS Conc.

Double Blank Controls Figure 1a: Expanded view of Mouse Plasma LLOQ Figure 2a: Expanded view of Mouse Plasma LLOQ Figure 3a: Expanded view of Rat Plasma LLOQ Figure 4a: Expanded view of Rat Plasma LLOQ
in some cases, multiple sample plates to be condensed into one injection plate.
• 25uL of blank plasma was combined with 100uL of acetonitrile 10000
9500

Another is the detection of multiple compounds in the plate, which triggers the
9000

Male Rabbit Plasma Male Beagle Plasma 8500

8000
7500

Peak Area
generator to program the construction of corresponding calibration curves and CS0 • Placed in the same precipitation plate as the standard concentration samples. 7000
6500
6000
Manual Preparation Automated Preparation Manual Preparation Automated Preparation 5500
5000
4500
100807_Trial9_Manual_Rabbit.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000247 x + 0.0011 (r = 0.9978)�� 100807_Trial18_FullAuto_Rabbit.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000285 x + 0.00107 (r = 0.9971)�� 101207_Trial11b_Manual_Beagle.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.00034 x + 0.00237 (r = 0.9977)�� 100807_Trial19_FullAuto_Beagle.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000276 x + 0.00138 (r = 0.9995)��
samples. With the capabilities in place, the ruggedness testing and performance Plasma Standard Curve Generation 101407_Trial26_Manual_Rabbit.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.0005 x + 0.00472 (r = 0.9987)��
101407_Trial32_Manual_Rabbit.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.00046 x + 0.0133 (r = 0.9992)��
101507_Trial38_FullAuto_Rabbit.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000304 x + 0.000751 (r = 0.9969)��
101507_Trial44_FullAuto_Rabbit.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000264 x + 0.00164 (r = 0.9994)��
101407_Trial27_Manual_Beagle.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000504 x + 0.00544 (r = 0.9962)��
101407_Trial33_Manual_Beagle.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000522 x + 0.00987 (r = 0.9951)��
101507_Trial39_FullAuto_Beagle.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000277 x + 0.000817 (r = 0.9949)��
101507_Trial45_FullAuto_Beagle.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000281 x + 0.00205 (r = 0.9972)��
4000
3500
3000
2500
2000
1500

trials were initiated. The system met specification for standard curve construction • Serial dilution of a concentrated, spiked solution was performed in blank plasma 5.0
Curve R2 Slope 3.5 Curve R2 Slope 5.0 Curve R2 Slope
100807_Trial15_Manual_Human.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000243 x + 0.00118 (r = 0.9910)
3.5 Curve R2 Slope
1000
500
0

1st .9978 .000247 3.0 1st .9971 .000285 1st .9977 .00034 2.0 1st .9995 .000276
and consistency of internal standard concentrations in multiple matrices. Tractability • Each level was thoroughly mixed via aspiration/dispensation repetitions
4.0
4.0
3.0

Analyte Area / IS Area

Analyte Area / IS Area

Analyte Area / IS Area

Area/ IS Area
2nd .9987 .0005 2.5 2nd .9969 .000304 2nd .9962 .000504 2nd .9949 .000277
2.5
of preparation error is achieved by monitoring the liquid handling error log files, and 3.0 3rd .9992 .00046
2.0
3rd .9994 .000264
3.0
3rd .9951 .000522
1.5
3rd .9972 .000281
Plasma Preparation 2.0

Area / ISArea
1.5
making database tags of any samples that initiated a liquid handling error. Expansion 2.0 2.0 1.5

Analyte Analyte
• 25µL of each spiked standard concentration was transferred to a deep well plate. 1.0 1.0
1.0

1.502
1.485
1.468
1.452
1.435
1.418
1.402
1.385
1.368
1.352
efforts are targeted for the system and include: automated SPE, hemolysis tracking,

1.335
1.318
1.0

1.302
1.285
1.0

1.268
1.252
1.235
1.218
1.202
1.185
1.168
1.152
1.135
1.118
1.102
1.085
0.5

1.068
0.5

1.052
1.035
1.018
1.002
Stud
liquid-liquid extraction, matrix expansion and high density plate use. These efforts • 100µL aliquot of the internal standard was added to each of the wells in the plate 0.0 0.0 0.0 0.0
0.5
Time (min.)
ies
0.0 2000.0 4000.0 6000.0 8000.0 1.0e4 0.0 2000.0 4000.0 6000.0 8000.0 1.0e4 0.0 2000.0 4000.0 6000.0 8000.0 1.0e4 0.0 2000.0 4000.0 6000.0 8000.0 1.0e4

look to capture a larger portion of the workflow and position this system as a to track the extraction, analysis and to act as a precipitating agent. Figure 5: Automated Liquid Handler Generated
Analyte Conc. / IS Conc.
Figure 6: Automated Liquid Handler Generated
Analyte Conc. / IS Conc.
Figure 7: Automated Liquid Handler Generated
Analyte Conc. / IS Conc.
Figure
Analyte Conc. / IS Conc.
8: Automated Liquid Handler Generated Figure 14a Figure 14b
0.0
2000.0 4000.0 6000.0 8000.0 1.0e4
Calibration Curve for Rabbit Plasma Calibration Curve for Rabbit Plasma Calibration Curve for Beagle Plasma Calibration Curve forConc.
Mouse
/ IS Conc.Plasma Figure 14: The analyte peaks were consistent in retention time, with the higher concentrations maintaining good peak shape
multipurpose BioAnalytical platform. Precipitation Plate Preparation Analyte
0.070 0.076 (14a). The IS trends for the studies are shown in Figure 14b, with the colors corresponding to the replicate as
0.060 0.080
• CS0 Controls and the Double Blank Controls added to the plate 0.25 0.060
shown in the calibration graph section. The attenuation in intensity (Figure 14b) is due to the presence of two IS
Objectives 0.040 0.040

A naly te A rea / IS A rea

A naly te A rea / IS A rea

A naly te A rea / IS A rea

A naly te A rea / IS A rea

0.20 0.060 batches. The crosses in Figure 14b correspond to manually prepared trials; the circles represent automated trials.
• Mixed thoroughly on a multitube vortexer. 0.15
0.020 0.020

Conclusion
0.000 0.040 0.000

• Plasma samples for this study are to be prepared • Centrifuged at 35000rpm for 20min and 0.10
-0.020
0.020
-0.020
0.05
by protein precipitation and monitored with an Injection Plate Preparation
-0.040 -0.040
• Automated construction of standard curves by the liquid handler for different animal model
Equipment List 0.00
0 20 40 60 80 100 120 140 160 180 200 220 240 260 0 20 40 60 80 100 120 140 160 180 200 220 240 260
0.000
0 20 40 60 80 100 120 140 160 180 200 220 240 260 0 20 40 60 80 100 120 140 160 180 200 220 240 260

internal standard Analytical Balance Mettler Toledo AX26

Analyte Conc. / IS Conc. Analyte Conc. / IS Conc. Analyte Conc. / IS Conc. Analyte Conc. / IS Conc.
plasmas was successful.
• 50µL of each supernatant from Figure 5a: Expanded view of Rabbit Plasma LLOQ Figure 6a: Expanded view of Rabbit Plasma LLOQ Figure 7a: Expanded view of Beagle Plasma LLOQ Figure 8a: Expanded view of Mouse Plasma LLOQ
• Quantification is to be performed through the Liquid Handler Tecan Freedom EVO 100 precipitation plate transferred to a • Standard curves had greater than 6 points with weighted regression values exceeding 99%
generation of a standard curve Tip Configuration 8 Disposable Tip
deep-well microtitre plate. Male Cynomolgus Monkey Plasma Human Plasma • Data points within these curves passed tolerability for inclusion using the 85% to 115% rule
Mounts (DiTi) Manual Preparation Automated Preparation Manual Preparation Automated Preparation
• Liquid handler is to use file generated from the 200 µL DiTi Tips • 50µL Milli-Q water was added 101407_Trial34_Manual_Cyno.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.00103 x + 0.0122 (r = 0.9974)�� 101407_Trial23_FullAuto_Cyno.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000363 x + 0.000573 (r = 0.9983)�� 101407_Trial35_Manual_Human.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000544 x + 0.00965 (r = 0.9964)�� 100807_Trial21_FullAuto_Human.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000292 x + 0.00119 (r = 0.9979)��
• The lower limits of quantification (LLOQ) for these automated prep samples were very
Tip Sizes Used 100807_Trial13_Manual_Cyno.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000231 x + 0.00366 (r = 0.9952)�� 101507_Trial40_FullAuto_Cyno.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000298 x + 0.00228 (r = 0.9982)�� 100807_Trial15_Manual_Human.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000233 x + 0.00187 (r = 0.9953)�� 101507_Trial41_FullAuto_Human.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.00031 x + 0.000469 (r = 0.9997)��

server based script generator 1000 µL DiTi Tips 101407_Trial28_Manual_Cyno.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000556 x + 0.00374 (r = 0.9973)�� 101507_Trial46_FullAuto_Cyno.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000287 x + 0.000583 (r = 0.9980)�� 101407_Trial29_Manual_Human.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000465 x + 0.00446 (r = 0.9983)�� 101507_Trial47_FullAuto_Human.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000276 x + 0.00183 (r = 0.9994)��
linear, proving the ability for the method to carry out a precise serial dilution in viscous
HPLC System
• 15µL injected according to the 100807_Trial15_Manual_Human.rdb (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000243 x + 0.00118 (r = 0.9910)
5.0 100807_Trial15_Manual_Human.rdb
3.5 (K932961): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000243 x + 0.00118 (r = 0.9910)

2 Curve R2 Slope 4.0 Curve R2 Slope Curve R2 Slope Curve R2 Slope medium
• Curves are to exceed R values of 0.99 Pump Agilent HP1100 listed analysis method. 8.0 1st .9974 .0122 2.0 1st .9983 .000363
4.0
1st .9964 .000544
3.0
2.0 1st .9979 .000292

Analyte Area / IS Area

2.5 Server based liquid handler file generator is able to create production capable files

Area/ IS Area

Area/ IS Area
Analyte Area / IS Area
2nd .9952 .00366 3.0 2nd .9982 .000298 2nd .9953 .000233 2nd .9997 .000310 •
• Tolerability of data points included in the curve Binary Pump
MS Parameters 6.0
3rd .9973 .00374
1.5
3rd .9980 .000287 3.0 3rd .9983 .000465 2.0
1.5
3rd .9994 .000276
This ability for an automated platform to produce injection ready samples from raw

Area / ISArea

Area / ISArea
•
are to be between 85% and 115% Degasser Agilent HP1100 Source Type Turbospray 4.0
2.0
1.5
2.0

Analyte Analyte

Analyte Analyte
Autosampler CTC PAL Ionization ESI+ (positive) 1.0
1.0
1.0
1.0 materials positions this method as a front running technology that is ready to be utilized in
• Minimum of 6 points in a calibration curve 2.0 1.0 0.5
the Kalypsys BioAnalytical workflow.
Table 2: Equipment used in the preparation and analysis. Source Temperature 500
0.0
0.5 0.0
0.5
• LLOQ for every curve is not to exceed CS20 after HPLC Conditions Spray Voltage 5.5 kV
0.0 0.0

Collision Gas (CAD) Medium

2000.0 4000.0 6000.0
Analyte Conc. / IS Conc.
8000.0 1.0e4 0.0 2000.0 4000.0 6000.0
Analyte Conc. / IS Conc.
8000.0 1.0e4 2000.0 4000.0 6000.0
Analyte Conc. / IS Conc.
8000.0 1.0e4 0.0 2000.0 4000.0 6000.0
Analyte Conc. / IS Conc.
8000.0 1.0e4
Acknowledgements
data exclusion Flow Rate 1000µL/min Figure 9: Automated Liquid Handler Generated Figure
0.0
10: Automated Liquid Handler Generated
2000.0 4000.0 6000.0 8000.0 1.0e4
Figure 11: Automated Liquid Handler Generated Figure
0.0
12: Automated Liquid Handler Generated
2000.0 4000.0 6000.0 8000.0 1.0e4
Mobile Phase A: 0.1% Formic Acid in Water Curtain Gas 10 Calibration Curve for Cyno Monkey Plasma Calibration Curve
Analyte Conc.for Cyno Monkey Plasma
/ IS Conc. Calibration Curve for Human Plasma Calibration Curve for /Human
Analyte Conc. IS Conc. Plasma
Ion Gas 1 50 0.09 The authors would like to thank the following individuals for their input and contributions:
B: 0.1% Formic Acid in Acetonitrile 0.25
0.08 0.120

• Replicates need to be consistent Ion Gas 2 60 0.06

0.25
0.100 Nahid Yazdani, Michael Herman and Tiffany Chea.
Gradient 0.0 – 1.0 min: 10%B to 95%B
A naly te A rea / IS A rea

A naly te A rea / IS A rea

A naly te A rea / IS A rea

A naly te A rea / IS A rea

0.20 0.20
0.04
and inter trial tolerability CV for a Entrance Potential 22 0.080
Document Reference Subject Product Reference Manufacturer
1.0 – 1.9 min: 95%B 0.15 0.02 0.15
0.060
Scan Type Multiple Reaction 0.00
AC-MethDev-2007-015v1 BioA Tecan Sample Prep Freedom EVO 100 Tecan AG
given concentration should no 2.0 – 2.7 min: 10%B 0.10
-0.02
0.10
0.040
Monitoring (MRM) 0.05 0.05
Legend exceed 15% Injection Volume 5 µL -0.04 0.020
Costar Deep Well Plate Corning
Table 4: Mass spectrometer parameters used in the 0.00 0.00 0.000
Manually Prepared Liquid Handler Prepared Column 0 20 40 60 80 100 120 140 160 180 200 220 240 260 0 20 40 60 80 100 120 140 160 180 200 220 240 260 0 20 40 60 80 100 120 140 160 180 200 220 240 260 0 20 40 60 80 100 120 140 160 180 200 220 240 260 Poster Contact Email
Phenomenex Polar-RP 2.0x30mm, 4µm MS/MS detection of the analyte and inter- Analyte Conc. / IS Conc. Analyte Conc. / IS Conc. Analyte Conc. / IS Conc. Analyte Conc. / IS Conc. Costar Shallow V-Well Plate Corning
Decision Point Data Compilation Table 3: HPLC parameters used in the analysis of the compounds. nal standard. Figure 9a: Expanded view of Cyno Monkey Plasma LLOQ Figure 10a: Expanded view of Cyno Monkey Plasma LLOQ Figure 11a: Expanded view of Human Plasma LLOQ Figure 12a: Expanded view of Human Plasma LLOQ Charles Taylor ctaylor@kalypsys.com