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Determination of Norgestrel and 17-Deacetylnorgestimate in Human Plasma by Methoxyamine Hydrochloride Derivatization Followed by Automated On-Line SPE with LC/MS/MS
David Lewiston, Bruce Babson, David Beyerlein and Gilbert Lam, Ph.D.; MicroConstants, Inc., 9050 Camino Santa Fe, San Diego, CA 92121
Conclusions• Arobust,specific,andsimpleassayfortheanalysisofnorgestreland
17-deacetylnorgestimate in human plasma has been validated.
• On-lineSPEisaneffectivecleanupsteptoachievespecificityformethoxyaminehydrochloride derivitized norgestrel and 17-deacetylnorgestimate.
• Themethodissuitabletoquantifyhumanplasmainpharmacokineticstudies.
Introduction
Norgestrel(NG)and17-deacetylnorgestimate(NGMN)arebio-activeprogestins.AnalysisofNGischallengingduetolow
concentrationsinplasmaandmediocreionizationinLC-MSutilizingeitherelectrosprayorAPCI.DerivatizationofNGwith
methoxyaminehydrochlorideconvertsthe3-ketogrouptoa3-methoxyiminogroupenhancingionizationinelectrospray
MS(NGMN,alreadycontaininga3-hydroxyiminogroupisunaffectedbythederivatizationprocedure).Unfortunatelythe
derivatization procedure is carried out in pyridine, complicating the ability to introduce the sample to HPLC. Automated on-line
SPEprovidesasimpleandhighlyselectivemethodofsampleclean-up,allowingdetectionatrelevantlevels
(40 pg/mL NG, 20 pg/mL NGMN) in human plasma.
MethodsInstrumentation: SparkHollandSymbiosisSPE-LCsystemcoupledtoaMicromassQuattroMicrotandem
quadrupole mass spectrometer
Analytes: Norgestrel (NG) and 17-deacetylnorgestimate (NGMN)
Internal Standards: Norgestrel-d6 and 17-deacetylnorgestimate-d
6
SampleExtractionVolume: 0.25 mL
SampleExtractionMethod: Solventextractionwith4mLof40%ethylacetateinhexane.Theorganicphasewasdried,andderivatizedwithmethoxyaminehydrochloride.Thederivatizedextractswerequenchedwithacetone,dilutedwithwater,transferredtoinjectionvialsanda0.05mLaliquotwasinjectedontheSymbiosisSPE-LCsystem.
Analytical Column: BetaBasic-450X2.1mm,3μm,analyticalcolumn(Thermo,Wahlford,MA)
SPE Cartridge: HySphereC2SE(SparkHolland,TheNetherlands)
Mass Transitions: NGMN: 328.05>123.45
NGMN, I.S. 335.05>128.45NG: 342.05>137.45
NG, I.S. 348.05>142.45
On-line SPE Conditions: Condition: 1 mL methanol
Equilibrate: 1mL0.5%FormicAcidinWater:Acetonitrile(93:7v/v)
Load: 1mL0.5%FormicAcidinWater:Acetonitrile(93:7v/v)
Wash: 0.5mL0.1%FormicAcidinWater:Acetonitrile(85:15v/v)
Elute: Focusmode(blendselutingsolventwithHPLCsolventstream)0.35mL0.1%FormicAcidinWater:Acetonitrile(50:50v/v)
HPLC solvent system: Solvent A: 0.1%FormicAcidinWaterSolvent B: 0.1%FormicAcidinAcetonitrile
HPLC Gradient: Pump Time,
min%
Solvent A%
Solvent BFlowRate,mL/minute
FlowRate,SPE Elution
%Acetonitrileon Column
FlowRateon Column
00:01 80 20 0.300 0.300 35 0.600
01:00 80 20 0.300 0.300 35 0.600
04:02 25 75 0.300 N/A 75 0.300
05:00 25 75 0.300 N/A 75 0.300
05:02 0 100 0.300 N/A 100 0.300
06:02 0 100 0.300 N/A 100 0.300
06:15 80 20 0.300 N/A 20 0.300
08:00 80 20 0.300 N/A 20 0.300
RetentionTimes: NGMN, I.S.: ~3.7minNG, I.S.: ~4.6 min
CurveRanges: NGMN (20.0 to 10,000 pg/mL), NG (40.0 to 20,000 pg/mL)
Sensitivity (LLOQ)
Theoretical Concentration
(pg/mL)Replicate
NG Found Concentration
(pg/mL)
NGMN Found Concentration
(pg/mL)
4.00/2.00 1 43.8 21.4
2 43.5 21.9
3 41.6 21.3
4 46.6 20.9
5 42.1 18.5
6 42.2 19.8
Mean 43.3 20.6%CV 4.22 6.12
%DEV 8.25 3.00
Structure of 17-Deacetylnorgestimate
Representative Chromatograms for NGMN and the NG - Methoxyamine Complex in Human Plasma
SixreplicatesofthelowestNGandNGMNcalibrationstandard(40.0/20.0pg/mL)wereincludedwithoneofthevalidationrunstodetermineprecisionandaccuracyatthelowerlimitofquantitation.
Interday Accuracy and PrecisionNG Theoretical Concentration (pg/mL)
120 1,200 16,000
Mean 125 1,260 17,000
%CV 5.88 4.37 4.34
%DEV 4.17 5.00 6.25
NGMN Theoretical Concentration (pg/mL)
60.0 600 8,000
Mean 64.9 648 8,150
%CV 4.31 4.08 4.07
%DEV 8.17 8.00 1.88
InterdayaccuracyandprecisionweredeterminedbyanalyzingreplicateQCsamplesinhumanplasmaatthreeconcentrationsoverthecourseoffourseparateanalyses.
Impact of Matrix Effect
Theoretical Concentration 40.0 pg/mL NG, 20.0 pg/mL NGMN
Plasma Lot Number
Mean Reported NG Concentration
(pg/mL)%DEV
Mean Reported NGMN Concentration
(pg/mL)%DEV
BC011909-1 43.5 8.7 22.6 13.0
BC011909-3 40.9 2.3 22.0 10.0
BC011909-5 39.3 -1.8 21.3 6.5
BC011909-6 43.2 8.0 21.8 9.0
BC011909-7 37.6 -6.0 19.3 -3.5
BC011909-8 41.4 3.5 21.9 9.5
TheimpactmatrixeffectonNGandNGMNwasdeterminedbycalculatingthe%DEVofthreereplicatesforeachlotspikedattheLLOQstandardconcentrationlevel.
Plasma Blank LLOQ
Norgestrel Derivatization Scheme
PyridineCH
3ONH
2
Representative Calibration Curve (20.0 to 10,000 pg/mL) for NGMN
in Human Plasma
Representative Calibration Curve (40.0 to 20,000 pg/mL) for NG
in Human Plasma
17-Deacetylnorgestimate Concentration (pg/mL)
PeakHeigh
tRatio
PeakHeigh
tRatio
Norgestrel Concentration (pg/mL)
AR02022MatrixBlankPool
F1:MRMof2Channels,ES+328.05>123.45
1.629e+002
1.852.46 3.29
3.63 3.89
100
%
0min
AR02022MatrixBlankPool
F2:MRMof2Channels,ES+342.05>137.45
3.260e+002
4.35
4.48
4.61
4.874.95
5.60
6.006.18
7.396.63 7.64
100
%
0min
AR02020Std.20.0/40.0pg/mLW1
F1:MRMof2Channels,ES+328.05>123.45
4.943e+00217-deacetylnorgestimate_
3.7966.12439
2.43 2.70 3.28
100
%
0 min
AR02020Std.20.0/40.0pg/mLW1
F2:MRMof2Channels,ES+342.05>137.45
1.338e+003Norgetrel_
4.6098.411206
4.99 5.60
100
%
0min
AmericanSocietyforMassSpectrometry(ASMS)ConferenceJune2011•Denver,CO•Poster#797