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Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC/ESIMS/ MS) Method For Quantitative Estimation of Moxifloxacin in Human Plasma
Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC/ESIMS/ MS) Method For Quantitative Estimation of Moxifloxacin in Human Plasma
Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC/ESIMS/ MS) Method For Quantitative Estimation of Moxifloxacin in Human Plasma
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http://www.jcps.ac.cn
Liquidchromatographyelectrosprayionizationtandemmassspectrometry
(LC/ESIMS/MS)methodforquantitativeestimationofmoxifloxacin
inhumanplasma
Vipul.M.Vaghela2,PrajeshPrajapati3*,HetalK.Patel1
1.ShreeSwaminarayanSanskarPharmacyCollege,Zundal382421,Gujarat,India
2.A.R.CollegeofPharmacyandG.H.PatelInstituteofPharmacy,VallabhVidhyanagar388120,Gujarat,India
3.InstituteofResearchandDevelopment,GujaratForensicSciencesUniversity,Gandhinagar382007,Gujarat,India
Abstract: Arapidandsensitiveliquidchromatographytandemmassspectrometry(LCMS/MS)methodhasbeendevelopedand
validatedforthedeterminationofmoxifloxacin(MOXI)inhumanplasma.Afterasimpleproteinprecipitationusingacetonitrile,
theposttreatmentsampleswereanalysedonaC18 columninterfacedwithaTripleQuadropoleTandemMassSpectrometer.Positive
electrospray ionizationwasemployedastheionizationsource.Themobilephaseconsistedof0.1%formicacidacetonitrile(60:40,
v/v).Ciprofloxacin(CIPRO)wasusedasaninternalstandard.Theanalyteandinternalstandard(CIPRO)weremonitoredinthe
multiplereaction monitoring mode (MRM). The masstransition ionpair has been followed asm/z402358.2 forMOXI and
332288.1forCIPRO.Themethodwaslinearintheconcentrationrangeof255000ng/mL.Thelowerlimitofquantification
was25ng/mL.Theintraandinterdayprecision(relativestandarddeviation)andaccuracy(relativeerror)valueswerewithin
12.4%.Eachplasmasamplewasanalyzedwithin3min.
Keywords: MoxifloxacinCiprofloxacinHumanplasmaLiquidchromatographytandemmassspectrometryElectrosprayionization
CLCnumber:R917
Documentcode:A
1.Introduction
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Copyright 2014 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University
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Vipul.M.Vaghelaetal./J.Chin.Pharm.Sci.2014,23(3),159164
(chemicalderivatizationorionpairseparation) ortwoor
morestepsforextractingMOXIfromtheplasma,andthe
useofmoresamplesfordetection.Inthepresentwork,
adifferentLCMS/MSmethodwasdevelopedtoanalyze
MOXI in human plasma using protein precipitation as
thesamplepretreatmentprocedure.
ESItandemmassspectrometry(MS/MS)providesarugged,
sensitiveandwidelyusedtechniquetoselectamassprecursor
andacharacteristicproduction ofananalyte,makingit
a highly specific method for the analysis of drugs in
humanplasma.Inthepresentstudy,proteinprecipitation
and onlineLC/ESIMS/MSusingciprofloxacin(CIPRO)
astheinternalstandardwasutilizedtoquantitateMOXIin
humanplasmainlessthan4minwithaLLOQof5ng/mL
isdescribed.
2.Experimental
2.1.Chemicalsandreagents
ThereferencestandardsofMOXI(99.98%)andCIPRO
(99.95%) were procured from Neuland Lab, Hyderabad.
HighpuritywaterwaspreparedinhouseusingaMilliQ
waterpurificationsystemobtainedfromMillipore(India)
Pvt. Ltd. HPLC grade methanol and acetonitrile were
obtainedfromJ.T.Baker(USA).Suprapure formic acid
(88%)waspurchasedfromMerck(Mumbai,India).Drug
free(blank)heparinizedhumanplasmawasobtainedfrom
Prathma Blood Centre (Ahmedabad, India) and was
storedat20Cpriortouse.
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2.2.Preparationofstocksolutions
2.3.Samplepreparation
Extractionofanalytefromplasmawasbasedonprotein
precipitation method. The calibration standards were
prepared by spiking 300 L of blank plasma in poly
propylenemicrocentrifugetubeswith30Loftheappro
priateMOXIworkingsolutions.Eachsample(calibration
standardandQC sample) wasalsospiked with 40L of
internalstandardworkingsolution(10g/mLofCIPRO)
andsubsequentlyprecipitatedwith0.5mLofacetonitrile.
All the tubes were then vortexed for 5 min, followed
by centrifugation for 15 min at 4500 r/min. Finally the
supernant was transferred into a glass vial and 10 L of
thissolutionwasinjectedintothecolumn.
2.4.Instrumentation
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ThestocksolutionofMOXIwaspreparedat1000g/mL
inmethanolwater(20:80,v/v)whilethestocksolutionof
internal standardCIPROwaspreparedat 1000g/mL in
methanolwater (50:50, v/v). Serial (working) dilutions
were prepared fromthese stocksolutionsforpreparation
of calibration curve standards and quality control (QC)
samplesbyusingmethanolwater(20:80,v/v)asdiluents.
Blankhumanplasmawasscreenedpriortospikingtoensure
it was free of endogenous interference at the retention
times of MOXI and internal standard CIPRO. An eight
point standard curve of MOXI was prepared by spiking
theblankplasmawithappropriateamountofMOXI.The
calibrationcurvewasplottedfrom25ng/mLto5000ng/mL
for MOXI. Quality control samples of three different
concentration levels, lower (LQC), medium (MQC) and
higher (HQC) were prepared at 71.21, 1177.48 and
4088.46 ng/mL, respectively. They were prepared in a
mannersimilartothepreparationofcalibrationstandards
fromthestocksolution.
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2.5.Validation
The methodhasbeenvalidatedforselectivity,sensitivity,
linearity, precision, accuracy, recovery and stability.
Copyright 2014 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University
http://www.jcps.ac.cn
Vipul.M.Vaghelaetal./J.Chin.Pharm.Sci.2014,23(3),159164
Selectivitywasperformedbyanalyzingtheblankplasma
samples from different sources (or donors) to test for
interference at the retention times of MOXIand internal
standard CIPRO. The intrarun and interrun accuracy
wasdeterminedbyreplicateanalysisofqualitycontrol
samplesandatLLOQthatwereextractedfromthesample
batch.
Accuracy is defined as the percent relative error (RE%)
andwascalculatedusingtheformulaRE%=(ET)100/T,
where E is the experimentally determined concentration
and T is the theoretical concentration. Assay precision
was calculated by using the formula percentage relative
standarddeviation(RSD%)=(SD/M)100,whereMis
themeanoftheexperimentallydeterminedconcentrationand
SDisthestandarddeviationofM.Thepercentrecoverywas
evaluatedbycomparingthepeakareaofextractedanalyte
tothepeakareaofnonextractedstandard.
Aspart of the method validation, stability was evaluated.
Analyteswere consideredstableifRE%ofthemeantest
responses were within 15% of appropriate controls.
Analytes were tested using the quality control samples
whenever appropriate. The stability of spiked human
plasma stored at room temperature (benchtop stability)
wasevaluatedfor6h.Theprocessedsamplestabilitywas
evaluatedbycomparingtheextractedplasmasamplesthat
wereinjectedimmediately(time0)withthesamplesthat
were injected after sitting in the autosampler at 6 C for
24h.Thefreezethawstabilitywasconductedbycomparing
thesamplesthathadbeenfrozenandthawedthreetimes,
with freshly spiked quality control samples. Six aliquots
of each low and high concentration were used for the
freezethawstabilityevaluation.Thelongtermstabilityof
spiked human plasma was evaluated by analyzing low,
mediumandhighqualitycontrolsamplesthatwerestored
at20Cfor14dtogetherwithfreshlyspikedcalibration
standardandqualitycontrolsamples.
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3.Resultsanddiscussions
3.1.Methoddevelopment
The goal of this work was to develop and validate a
simple,rapidandsensitivemethodforthe extractionand
quantificationofMOXI,suitablefordeterminingpharma
cokinetics of this compound in clinical studies. To achieve
thisgoal,duringmethoddevelopment,differentparameters
were evaluated to optimize sample extraction, detection
andchromatographicseparation.MOXIandCIPROwere
extracted from human plasma by protein precipitation
with acetonitrile. Electrospray ionization (ESI) and
atmospheric pressure chemical ionization (APCI) were
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Chromatogramsobtainedfromblankplasmaarepresented
in Figures 1 and 2 for MOXI and CIPRO, respectively.
Chromatograms obtained from plasma spiked with
LLOQ standard are represented in Figures 3 and 4 for
MOXI and CIPRO, respectively. No interfering peak of
endogenous compounds was observed at the retention
times of analytes in the blank plasma of five different
lots of heamolysed plasma. Utilization of predominant
product ions from each compound enhanced mass
spectrometric selectivity. The predominant product ions
of m/z 358.20 and 288.2 were specific for MOXI and
CIPRO,respectively.
3.3.Linearregressionanddetectionlimits
Thestandardcalibrationcurveswereproducedonthree
different days in spiked human plasma. The peak area
ratios of calibration standards were propotional to the
concentrationsofanalytesineachassayoverthenominal
concentration range of 255000 ng/mL for MOXI. The
calibrationcurves appeared linear(Fig. 5) andwere well
described by least squares lines. Aweighing factor of
1/concentration was chosen to achieve homogeneity of
variance. Thecorrelation coefficients were0.9998. The
meanSD slope of calibration curves for MOXI was
(0.0007880.0000145).Themeaninterceptofcalibration
curves for MOXI was (0.0031860.000845) (Table 1).
The LLOQ for MOXI was 25.0 ng/mL. This data is
tabulated in Table 2 for MOXI. The intraday precision
of the LLOQ plasma sample containing MOXI ranged
from 1.3% to 4.5% (n = 18). The intraday accuracy of
LLOQ plasma sample containing MOXI ranged from
9.7%to15.7%(n=18).
Copyright 2014 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University
http://www.jcps.ac.cn
Vipul.M.Vaghelaetal./J.Chin.Pharm.Sci.2014,23(3),159164
162
Intensity(cps)
1.31
300
Moxifloxacin
280
260
240
220
200
180
160
140
0.93
120
0.87
0.53
100 0.05 0.20
2.61 2.76 2.91
0.47 0.61 0.77
2.442.51
80
2.252.35
1.431.63 1.78
60
2.082.10
1.23
40
20
0
0.20.40.60.81.01.21.41.61.82.02.22.4
2.62.8
t(min)
Figure1.Representativechromatogramofblankplasmaformoxifloxacin.
Intensity(cps)
1.32
280
Ciprofloxacin
260
240
220
200
180
0.92
160
0.87
140
0.16
120
2.402.492.59
2.92
1.70
100 0.08 0.26 0.460.50
2.18 2.31
2.712.80
1.78 1.92
1.59
0.60
0.67
2.14 2.25
80
2.07
60
1.17
40
20
0
0.20.40.60.81.01.21.41.61.82.02.22.4
2.62.8
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t(min)
Intensity(cps)
Figure2.Representativechromatogramofblankplasmaforciprofloxacin.
1.31
6500
6000
5500
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
0
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Moxifloxacin
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0.20.40.60.81.01.21.41.61.82.02.22.4
2.62.8
t(min)
Intensity(cps)
Figure3. RepresentativechromatogramofplasmaspikedwithLLOQstandardformoxifloxacin.
1.30
2.6e5
2.4e5
2.2e5
2.0e5
1.8e5
1.6e5
1.4e5
1.2e5
1.0e5
8.0e4
6.0e4
4.0e4
2.0e4
0.0
Ciprofloxacin
0.20.40.60.81.01.21.41.61.82.02.22.4
2.62.8
t(min)
Analytearea/ISarea
Figure4. RepresentativechromatogramofplasmaspikedwithLLOQstandardforciprofloxacin.
4.0
3.6
3.2
2.8
2.4
2.0
1.6
1.2
0.8
0.4
0.0
Table1. Calibrationcurveparametersformoxifloxacin
Runnumber
01000
2000
3000
4000
Analyteconc./ISconc.
Figure5. Calibrationcurveofmoxifloxacin.
5000
Slope
Intercept
Rsquared
Day1
0.000805
0.00303
0.9998
Day2
0.000782
0.00410
0.9998
Day3
0.000778
0.00243
0.9998
MeanSD
0.0007880.0000145
0.0031860.000845
0.9998
18
18
18
N=No.ofrun.
Copyright 2014 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University
http://www.jcps.ac.cn
Vipul.M.Vaghelaetal./J.Chin.Pharm.Sci.2014,23(3),159164
3.4.Precisionandaccuracy
3.6.Stability
Bench top and process stabilities of MOXI were
investigated at LQC and HQC levels. The results revealed
thatMOXIwasstable inplasma for at least6 hat room
temperatureand24hattheautosampler.Itwasconfirmed
that repeated freeze and thaw (three cycles) of plasma
samples spiked with MOXI at LQC and HQC level did
not affect the stability of MOXI (Table 3). Long term
stability of MOXI in plasma at 70 C was performed at
LQC, MQC and HQC level. The results revealed that
MOXIwasstablefor14d(Table3).
3.5.Recovery
Sixreplicates at low,mediumandhighquality control
Table2. Intradayandinterdayprecisionandaccuracydataofmoxifloxacin
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Intradayprecisionandaccuracy(n =6)
QCID
LLOQ
Nominalconcentration
25.63(ng/mL)
31.3
LQC
HQC
71.21(ng/mL)
1177.47(ng/mL)
4088.46(ng/mL)
67.8
1240
4310
1260
4180
1220
4200
67.9
1170
4130
70.5
1240
4220
79.2
1230
4110
28.601.29
72.074.57
1226.6730.77
4191.6771.39
4.5
1.7
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28.2
30.8
27.4
28.5
MeanSD
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MQC
28.1
Day1
163
71.1
75.9
6.3
2.5
11.6
1.2
4.2
2.5
28.7
71.4
1180
4020
28.4
69.6
1230
4240
28.2
72.3
1250
4110
27.9
71.9
1220
4030
27.8
70.7
1220
4220
27.8
71.7
1240
4060
MeanSD
28.130.37
71.270.98
1223.324.22
4113.395.85
RSD(%)
1.3
1.4
2.0
2.3
RE(%)
9.7
0.1
3.9
0.6
28.9
71.7
1140
3950
29.3
72.6
1200
4210
31.3
72.1
1190
4110
28.9
72.8
1130
3930
29.4
73.2
1200
4230
30.2
72.4
1190
4070
29.670.93
72.470.53
1175.0031.46
4083.33126.28
3.1
0.7
2.7
3.1
15.7
1.8
0.2
0.1
RE(%)
Day2
Day3
MeanSD
RSD(%)
RE(%)
Interdayprecisionandaccuracy(n =18)
MeanSD
RSD(%)
RE(%)
LLOQ
LQC
MQC
HQC
28.811.09
71.932.60
1208.3336.50
4129.44105.35
3.8
3.6
3.0
2.6
12.4
1.0
2.6
1.0
Note:LLOQ:lowerlimitofquantification,LQC:lowerqualitycontrol,MQC:middlequalitycontrol,HQC:higherqualitycontrol.
Copyright 2014 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University
http://www.jcps.ac.cn
Vipul.M.Vaghelaetal./J.Chin.Pharm.Sci.2014,23(3),159164
164
Table3.StabilityresultsforMOXI
Stability
Spikedconcentration(ng/mL)
ObtainedconcentrationMeanSD(ng/mL, n=6)
RE(%)
Process
71.21
4088.46
73.101.80
4003.3087.10
2.60
2.10
Benchtop
71.21
4088.46
67.131.94
4118.30118.05
5.70
0.70
Freezeandthaw
71.21
4088.46
71.901.08
4151.7045.35
1.00
1.50
Longterm
71.21
1177.47
4088.46
66.240.62
1028.2648.12
3997.687.82
6.98
12.67
2.22
4.Conclusion
WedevelopedaproteinprecipitationandLC/ESIMS/MS
methodthatachievedtheaccurateestimationofMOXI
in human plasma and also provided sensitivity in the
low ng/mL range. The described method generated
quantitative extraction of analyte from plasma without
need of evaporation and reconstitution. In addition, it
was fast (run time less than 3 min), specific, rugged
andeasytouse.Highsampleturnovermakesthismethod
aneffective procedure inhighthroughput bioanalysis
ofMOXI.
[9]AVELOX(packageinsert),BayerCorporation,WestHaven,
CT,December.1999.
5.Acknowledgements
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