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