Determination of Mifepristone in Human Plasma by Automated ...€¦ · mifepristone in human plasma...
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Determination of Mifepristone in Human Plasma by Automated On-Line SPE Combined with MS/MS David Lewiston, Bruce Babson, David Beyerlein; MicroConstants, Inc., 9050 Camino Santa Fe, San Diego, CA 92121 Methods Instrumentation Spark Holland Symbiosis SPE-LC system coupled to a Micromass Quattro Micro tandem quadrupole mass spectrometer Analyte Mifepristone Internal Standard Mifepristone-d 3 Sample Volume 0.05 mL Sample Preparation Method Plasma proteins are precipitated with 2 mL of 1% ammonium formate in methanol. The samples are vortexed, centrifuged, and a 0.7 mL aliquot is transferred to a well plate. A 0.005 mL aliquot was injected on the Symbiosis SPE-LC system. Analytical Column N/A SPE Cartridge HySphere C18 HD (Spark Holland, The Netherlands) Mass Transitions Mifepristone: 430.30 > 372.20 I.S. (Mifepristone-d 3 ): 433.30 > 375.20 On-line SPE Conditions Condition: 1 mL methanol Equilibrate: 1 mL Water:Acetonitrile:Formic Acid (950:50:1, v/v/v) Load: 1 mL Water:Acetonitrile:Formic Acid (950:50:1, v/v/v) Wash: 0.5 mL Water:Acetonitrile:Ammonium Formate:Formic Acid (700:300:1.25:0.8, v/v/w/v) Elute: Standard mode (HPLC solvent stream is the eluting solvent) 0.45 mL Methanol:Water:Ammonium Formate:Formic Acid (700:300:0.75:0.6, v/w/v) Retention Times Mifepristone, I.S.: ~0.4 min Injection to Injection Cycle Time 2.03 minutes Curve Range 100 to 10,000 ng/mL Introduction Mifepristone is a synthetic steroid compound that has been studied in Phase II clinical trials for many medical uses, including: oral contraception, uterine fibroids, endometriosis, major depression with psychotic features, glaucoma, meningiomas, Cushing’s syndrome, and various cancers. Mifepristone can be detected by LC/MS/MS following sample clean-up by various methods, including: protein precipitation, solid phase extraction, or liquid/liquid extraction. However, automated on-line SPE circumvents the need for an analytical HPLC column and provides a rapid, highly selective method of sample clean-up, allowing detection at levels relevant for therapeutic drug monitoring (100 ng/mL) in human plasma. ASMS Conference on Mass Spectrometry and Allied Topics | May 20 - 24, 2012 | Vancouver, Canada | Poster # ThP18 Accuracy and Precision Interday Analytical Run Replicate Concentration (ng/mL) 100 300 1,000 3,000 8,000 AR01 1 104 325 1,040 3,250 7,920 2 104 311 1,060 3,110 8,010 3 104 313 1,040 3,110 8,020 4 107 308 1,040 3,150 8,070 5 102 308 1,030 3,190 7,800 6 106 306 1,070 3,190 7,890 AR02 1 99.8 299 979 3,090 7,650 2 103 315 1,060 3,130 7,730 3 102 306 1,020 3,160 7,660 4 101 296 1,000 3,090 7,710 5 98.3 305 989 2,980 7,600 6 98.5 296 988 3,080 7,860 AR03 1 102 294 1,030 2,980 7,530 2 95.0 307 1,020 3,160 8,030 3 107 329 1,020 3,100 8,020 4 105 291 1,050 3,180 7,830 5 91.7 314 1,050 3,160 7,840 6 110 311 1,020 3,040 7,450 AR04 1 97.7 327 992 3,090 7,890 2 101 297 1,060 3,150 7,940 3 102 315 1,000 3,000 7,750 4 108 322 1,000 2,970 7,880 5 99.9 301 1,020 3,050 7,860 6 104 294 1,060 2,970 8,100 Mean 102 308 1,030 3,100 7,840 %CV 4.09 3.52 2.63 2.53 2.21 %DEV 2.00 2.67 3.00 3.33 -2.00 Interday accuracy and precision were determined by analyzing replicate QC samples in human plasma at five concentrations over the course of four separate analyses. Impact of Matrix Effect Theoretical Concentration (ng/mL) Plasma Lot Number Replicate Reported Concentration (ng/mL) %DEV Lot Acceptance 100 BC11810PM2 1 99.3 -0.700 Pass 2 95.6 -4.40 3 95.2 -4.80 BC11810PM3 1 97.2 -2.80 Pass 2 101 1.00 3 98.2 -1.80 BC11810PM4 1 95.8 -4.20 Pass 2 97.3 -2.70 3 95.1 -4.90 BC11810PM6 1 92.6 -7.40 Pass 2 93.5 -6.50 3 91.6 -8.40 BC040710PM1 1 95.5 -4.50 Pass 2 97.5 -2.50 3 91.5 -8.50 BC040710PM3 1 97.1 -2.90 Pass 2 98.1 -1.90 3 104 4.00 Overall Acceptance: 100% The impact matrix effect on mifepristone was determined by calculating the %DEV of three replicates for each lot spiked at the LLOQ standard concentration level. HPLC Carry-Over Evaluation for Mifepristone in Human Plasma Analytical Run Lowest LLOQ Peak Height Replicate Peak Height Carry-Over Percentage of LLOQ Peak Height* Carry-Over ULOQ Carry-Over Peak Height MB01 3,813 1 397,797 0 0.00% 2 406,438 0 0.00% 3 449,103 0 0.00% 4 429,762 0 0.00% 5 428,220 0 0.00% 6 508,032 0 0.00% * Following injection of a unique ULOQ sample HPLC carryover was evaluated by injecting a precipitation solution blank sample immediately following a unique ULOQ sample not included in the calibration curve. A mean response less than 20.0% of the LLOQ peak height indicates that there is no significant carry-over effect between injections. Conclusions • A robust, specific, and simple assay for the analysis of mifepristone in human plasma has been validated. • On-line SPE is an effective clean-up step to achieve specificity for mifepristone. • The method is suitable to quantify human plasma in therapeutic drug monitoring studies. Representative Chromatograms for Mifepristone and Internal Standard in Human Plasma MRM of 2 channels,ES+ 433.3 > 375.2 6.564e+001 AR01r022 matrix blank 100 % 0 0.01 0.18 0.25 0.49 0.64 0.83 0.96 1.18 min 0.20 0.40 0.60 0.80 1.00 1.20 1.40 MRM of 2 channels,ES+ 430.3 > 372.2 5.500e+001 AR01r022 matrix blank 100 % 0 min 0.09 0.28 0.49 0.82 1.18 1.32 0.68 Plasma Blank 100 % 0 MRM of 2 channels,ES+ 430.3 > 372.2 5.376e+003 AR01r026 LLOQ QC 100 ng/mL Mifepristone 0.41 min min MRM of 2 channels,ES+ 433.3 > 375.2 1.966e+004 AR01r026 LLOQ QC 100 ng/mL 100 % 0 I.S. 0.40 0.20 0.40 0.60 0.80 1.00 1.20 1.40 LLOQ Representative Calibration Curve (100 to 10,000 ng/mL) for Mifepristone in Human Plasma 100.0000 Log (Concentration (ng/mL)) Log (Peak Height Ratio) 10.0000 1.0000 0.1000 100.0 1000.0 10000.0 Structure Representative Chromatograms Showing Separation of Mifepristone and its Major Metabolites (Not Analyzed) on the SPE Cartridge MRM of 5 Channels ES+ 430.3 > 372.2 3.61e3 Mifepristone mateff117 100 % 0 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 MRM of 5 Channels ES+ 433.3 > 375.2 1.64e4 Mifepristone-d 3 mateff117 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 100 % 0 MRM of 5 Channels ES+ 416.3 > 358.2 2.39e5 Desmethylmifepristone mateff117 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 100 % 0 MRM of 5 Channels ES+ 402.3 > 344.2 1.33e5 Didesmethylmifepristone mateff117 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 100 % 0 MRM of 5 Channels ES+ 446.3 > 388.2 1.31e5 Hydroxymifepristone mateff117 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 100 % 0 0.46 0.43 0.32 0.27 0.24
Transcript of Determination of Mifepristone in Human Plasma by Automated ...€¦ · mifepristone in human plasma...
Determination of Mifepristone in Human Plasma by Automated On-Line SPE Combined with MS/MSDavid Lewiston, Bruce Babson, David Beyerlein; MicroConstants, Inc., 9050 Camino Santa Fe, San Diego, CA 92121
MethodsInstrumentation Spark Holland Symbiosis SPE-LC
system coupled to a Micromass Quattro Micro tandem quadrupole mass spectrometer
Analyte Mifepristone
Internal Standard Mifepristone-d3
Sample Volume 0.05 mL
Sample Preparation Method Plasma proteins are precipitated with 2 mL of 1% ammonium formate in methanol. The samples are vortexed, centrifuged, and a 0.7 mL aliquot is transferred to a well plate. A 0.005 mL aliquot was injected on the Symbiosis SPE-LC system.
Analytical Column N/A
SPE Cartridge HySphere C18 HD (Spark Holland, The Netherlands)
Mass TransitionsMifepristone: 430.30 > 372.20
I.S. (Mifepristone-d3): 433.30 > 375.20
On-line SPE ConditionsCondition: 1 mL methanol
Equilibrate: 1 mL Water:Acetonitrile:Formic Acid (950:50:1, v/v/v)
Load: 1 mL Water:Acetonitrile:Formic Acid (950:50:1, v/v/v)
Wash: 0.5 mL Water:Acetonitrile:Ammonium Formate:Formic Acid (700:300:1.25:0.8, v/v/w/v)
Elute: Standard mode (HPLC solvent stream is the eluting solvent) 0.45 mL Methanol:Water:Ammonium Formate:Formic Acid (700:300:0.75:0.6, v/w/v)
Retention TimesMifepristone, I.S.: ~0.4 min
Injection to Injection Cycle Time 2.03 minutes
Curve Range 100 to 10,000 ng/mL
Introduction
Mifepristone is a synthetic steroid compound that has been studied in Phase II clinical trials for many medical uses, including: oral contraception, uterine fibroids, endometriosis, major depression with psychotic features, glaucoma, meningiomas, Cushing’s syndrome, and various cancers. Mifepristone can be detected by LC/MS/MS following sample clean-up by various methods, including: protein precipitation, solid phase extraction, or liquid/liquid extraction. However, automated on-line SPE circumvents the need for an analytical HPLC column and provides a rapid, highly selective method of sample clean-up, allowing detection at levels relevant for therapeutic drug monitoring (100 ng/mL) in human plasma.
ASMS Conference on Mass Spectrometry and Allied Topics | May 20 - 24, 2012 | Vancouver, Canada | Poster # ThP18
Accuracy and PrecisionInterday
Analytical Run Replicate Concentration (ng/mL)100 300 1,000 3,000 8,000
AR01 1 104 325 1,040 3,250 7,9202 104 311 1,060 3,110 8,0103 104 313 1,040 3,110 8,0204 107 308 1,040 3,150 8,0705 102 308 1,030 3,190 7,8006 106 306 1,070 3,190 7,890
AR02 1 99.8 299 979 3,090 7,6502 103 315 1,060 3,130 7,7303 102 306 1,020 3,160 7,6604 101 296 1,000 3,090 7,7105 98.3 305 989 2,980 7,6006 98.5 296 988 3,080 7,860
AR03 1 102 294 1,030 2,980 7,5302 95.0 307 1,020 3,160 8,0303 107 329 1,020 3,100 8,0204 105 291 1,050 3,180 7,8305 91.7 314 1,050 3,160 7,8406 110 311 1,020 3,040 7,450
AR04 1 97.7 327 992 3,090 7,8902 101 297 1,060 3,150 7,9403 102 315 1,000 3,000 7,7504 108 322 1,000 2,970 7,8805 99.9 301 1,020 3,050 7,8606 104 294 1,060 2,970 8,100
Mean 102 308 1,030 3,100 7,840%CV 4.09 3.52 2.63 2.53 2.21
%DEV 2.00 2.67 3.00 3.33 -2.00
Interday accuracy and precision were determined by analyzing replicate QC samples in human plasma at five concentrations over the course of four separate analyses.
Impact of Matrix EffectTheoretical Concentration
(ng/mL)Plasma Lot
NumberReplicate
Reported Concentration (ng/mL)
%DEV Lot Acceptance
100 BC11810PM2 1 99.3 -0.700Pass2 95.6 -4.40
3 95.2 -4.80
BC11810PM3 1 97.2 -2.80Pass2 101 1.00
3 98.2 -1.80
BC11810PM4 1 95.8 -4.20Pass2 97.3 -2.70
3 95.1 -4.90
BC11810PM6 1 92.6 -7.40Pass2 93.5 -6.50
3 91.6 -8.40
BC040710PM1 1 95.5 -4.50Pass2 97.5 -2.50
3 91.5 -8.50
BC040710PM3 1 97.1 -2.90Pass2 98.1 -1.90
3 104 4.00
Overall Acceptance: 100%
The impact matrix effect on mifepristone was determined by calculating the %DEV of three replicates for each lot spiked at the LLOQ standard concentration level.
HPLC Carry-Over Evaluation for Mifepristone in Human Plasma
Analytical RunLowest LLOQ Peak Height
ReplicatePeak Height
Carry-Over Percentage of LLOQ Peak Height*Carry-Over ULOQ
Carry-Over Peak Height
MB01 3,813 1 397,797 0 0.00%2 406,438 0 0.00%3 449,103 0 0.00%4 429,762 0 0.00%5 428,220 0 0.00%6 508,032 0 0.00%
* Following injection of a unique ULOQ sample
HPLC carryover was evaluated by injecting a precipitation solution blank sample immediately following a unique ULOQ sample not included in the calibration curve. A mean response less than 20.0% of the LLOQ peak height indicates that there is no significant carry-over effect between injections.
Conclusions• A robust, specific, and simple assay for the analysis of
mifepristone in human plasma has been validated.• On-line SPE is an effective clean-up step to achieve specificity for
mifepristone.• The method is suitable to quantify human plasma in therapeutic
drug monitoring studies.
Representative Chromatograms for Mifepristone and Internal Standard in
Human Plasma
MRM of 2 channels,ES+433.3 > 375.2
6.564e+001
AR01r022matrix blank
100
%
0
0.01
0.18 0.25
0.49
0.64
0.83
0.96
1.18
min0.20 0.40 0.60 0.80 1.00 1.20 1.40
MRM of 2 channels,ES+430.3 > 372.2
5.500e+001
AR01r022matrix blank
100
%
0 min
0.09
0.28
0.49
0.82
1.18
1.320.68
Plasma Blank
100
%
0
MRM of 2 channels,ES+430.3 > 372.2
5.376e+003
AR01r026LLOQ QC 100 ng/mL
Mifepristone0.41
min
min
MRM of 2 channels,ES+433.3 > 375.2
1.966e+004
AR01r026LLOQ QC 100 ng/mL
100
%
0
I.S.0.40
0.20 0.40 0.60 0.80 1.00 1.20 1.40
LLOQ
Representative Calibration Curve (100 to 10,000 ng/mL) for Mifepristone
in Human Plasma100.0000
Log (Concentration (ng/mL))
Log
(Pea
k H
eig
ht R
atio
)
10.0000
1.0000
0.1000100.0 1000.0 10000.0
Structure
Representative Chromatograms Showing Separation of Mifepristone and its Major Metabolites
(Not Analyzed) on the SPE Cartridge
MRM of 5 Channels ES+430.3 > 372.2
3.61e3
Mifepristone
mateff117
100
%
00.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80
MRM of 5 Channels ES+433.3 > 375.2
1.64e4
Mifepristone-d3
mateff117
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80
100
%
0
MRM of 5 Channels ES+416.3 > 358.2
2.39e5
Desmethylmifepristone
mateff117
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80
100
%
0
MRM of 5 Channels ES+402.3 > 344.2
1.33e5
Didesmethylmifepristone
mateff117
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80
100
%
0
MRM of 5 Channels ES+446.3 > 388.2
1.31e5
Hydroxymifepristone
mateff117
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80
100
%
0
0.46
0.43
0.32
0.27
0.24
