INTRODUCTION•Mometasonefuroateisapotentsyntheticcorticosteroidwithminimalbioavailabilitywhenadministeredtopically(dermatitis)orviainhalation(allergy,asthma)
•Maximumplasmaconcentrationsnear50pg/mLareexpectedfollowinginhalationof100-400µg,andasensitivemethodisrequiredtomeasureanadequatenumberofdatapointsforcalculatingpharmacokineticparameters
• Apost-extractionderivatizationprocedurecoupledwithadual-columnLC-MS/MSset-upprovidedsufficientsensitivitytovalidateanLLOQof0.250pg/mL
Figure 1. Mometasone furoate and hydrazone reaction products
ThetargetLLOQof0.25pg/mLwaschallengingtoachieveastheneutralsteroidtendstoformadductsinpositiveESIandthesignalisdilutedbythepresenceof2chlorineatoms.Derivatizationwithseveralhydrazinecompoundsordansylchloridewasinvestigatedtoincreaseandstabilizetheresponse.Twoisomericproductswereoftenobservedwithreversedphasechromatography.
Figure 2. Reversed phase chromatography of derivatised mometasone furoate.
Thereactionproductspresentedthemselvesasasinglepeakusingcationexchangechromatographywithmoderateretention.
A Sub-Picogram LC-MS/MS Method for the Analysis of Mometasone Furoate in Human Plasma.Lapko, V; Dzerk, A; Linderholm, K; Coe, R; Retke, B; Merrill, M; Sheldon, CCelerion, Lincoln, NE, USA
Figure 3. Mometasone furoate derivative SCX chromatography
SAMpLE pREpARATIONSeveralsolventsweretestedacrossthepHrangeandn-butylchlorideat alkaline pHwas chosen to provide the cleanest baseline andadequaterecoveryfrom1.0mLofEDTAplasmapriortoderivatization.
1mL Sample /IS + 1mL 2% NH4OH + 4.5 mL ButylChloride
Mix/Separate & evaporate organic
Derivatize/Evaporate/reconstitute
Severematrixsuppressionwasobservedforallderivativesincludingseveralhighlyretainedspeciesthatnearlyeliminatedthesignalfromsubsequentinjections.
Figure 4. Post column infusion experiment, mometasone furoate retention time ca. 1.6 minutes under typical (not final) SCX conditions.
INSTRUMENTATIONAdual-columnback-flushmethodwasadopted.Inthisapproach,2columnsareusedunderisocraticconditionswithonebeingrinsedofflineinthereversedirectionwhiletheanalysiscolumniselutingtothemassspec.Alternatingcolumnsviaavalveswitcheliminatesdownfieldelutionofsuppressivecompounds.•MobilePhase:Acetonitrile:Methanol:15mMammoniumformate,pH2.5;AnalyticalFlowRate0.9mL/min,Back-flushFlowRate1.0mL/min
• Column:SCX3x50mm5µm,50°C• Detection:Electrosprayionization(ESI)datawasacquiredbymultiplereaction-monitoring(MRM)inpositivemodeonanABSCIEX5500Q-TRAP®massspectrometer.Acquisitiontime2.0minutes
Figure 5. Dual column backflush LC-MS/MS design.
Mometasonechromatographyunderthefinalconditions,LLOQ,BlankandPostColumnInfusion. Figure 6. Final conditions for mometasone furoate derivative
O
CH3
HO
Cl
H
H
CH3
OCl
O
O
O
CH3
NN
CH3
Cl
H
H
CH3HO
OCl
O
O
O
R
H
CH3
N NH2
H
R
N
CH3
Cl
H
H
CH3HO
OCl
O
O
O
CH3
N
R
H
H+
Mometasone d3-37Cl-Mometasone
MS
Autosampler
Position 1
MS
Position 2
PC COLUMN
COLUMNPC
Waste
PC COLUMN
PUMP B
WASTE
MassSpec
Pump A
Position 1
Position 2
PC Column A
PC COLUMNPC Column A
Autosampler
Pump A
Column BPC
PC Column B
Pump B
Waste
Pump B
MassSpec
RESULTS• Thevalidatedanalyticalrangewas0.25to25.0pg/mLofmometasonefuroateusing1.0mLofhumanEDTAplasma
• Theinter-batchprecision(%C.V.)andaccuracy(%Bias)ofqualitycontrolsamplesisshowninTable1
• Theextractionrecoveryofmometasonefuroatewas87–93%• SelectivityandmatrixeffectdatawereacceptableforhealthyandhemolyzedhumanEDTAplasma
•MometasonefuroatewasstableinhumanEDTAplasmathrough6freeze/thawcycles,inclusiveof53hoursatambienttemperatureunderwhitelightingandforupto57dayswhenstoredat-20°C
•Derivatizedmometasonefuroateintegritywasmaintainedupto78hrspostextraction
Table 1. Validation Interday Reproducibility
Abioequivalencestudyofa200ugmometasonenasalspraygeneratedover3000samplesforanalysis.Reanalysisof262samplesconfirmed97%oftheoriginalresultsusingestablishedindustry-widecriteriaforincurredsamplereproducibility.Theaveragedifferencebetweenallreassayresultswas4.1%.
CONCLUSIONSArobustmethodforanalysisofmometasonefuroateinhumanplasmahasbeendevelopedandvalidated.Despitesub-picogramsensitivityrequirements,therelativelysimplemethodovercomesextremematrixeffectstoaccuratelyandreproduciblyquantifymometasonefuroateforpharmacokineticcharacterizationofverylowdosageintranasalandtopicalformulations.
ACKNOwLEDgMENTTheauthorsthankAlyssaPerry,ChrisKafonek,MarzukiMohamed,andJosephSilvafortheircontributions.
a. Lower Limit of Quantitation 0.25 pg/mL
b. Blank Control Plasma
c. Post Column Infusion
Quality Control Samples Precision (% CV) Accuracy (% Bias) Inter-Batch LLOQ 0.25 pg/mL
Low 0.75 pg/mL Medium 5.0 pg/mL High 20.0 pg/mL
16.9 7.6 2.3 2.3
4.4 2.8 5.8 1.5
