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JournalofChinesePharmaceuticalSciences

159

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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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ArticleID: 10031057(2014)315906

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Respiratory infection is a major cause of morbidity.


Various antibiotics are used to treat these infections, which
include penicillins, cephalosporins, macrolide antibiotics,
tetracyclines and quinolones[1]. The widespread use of
earlier generation of quinolones has led to pathogen
resistanceandtreatmentfailures,especiallywithisolates
of Streptococcus pneumoniae. Moxifloxacin (MOXI),
1cyclopropyl7(2,8diazobicyclo [4.3.0] nonane)6fluoro
8methoxy1,4dihydro4oxo3quinoline carboxylic acid,
isanewfourthgenerationquinolone,whichdemonstrates
effectiveness against acute bacterial sinusitis, acute
bacterialexacerbationofchronicbronchitisandcommunity
acquired pneumonia[28].Inadditionto enhancedactivity
againstmanyclinicalisolates,MOXIhasbeenchemically
engineered to optimize its safety and pharmacokinetic
profiles. The absence of a halide at the C 8 position
minimizesthepotentialforphotosensitivityreactionsand
amethoxygroupatthepositiontheoreticallymay confer
enhancedactivity againstresistant grampositive bacteria
andreducedselectionofresistantmutants[9,10].MOXIalso
has a diazobicyclononyl ring moiety at the 7 position,
Received: 20131007Revised: 20131029Accepted: 20131107.
*
Correspondingauthor.Tel.:+919825318996Fax:07923247465
Email:prajeshprajapati@gmail.com
http://dx.doi.org/10.5246/jcps.2014.03.020

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which enhances the potency, spectrum of activity and


halflife[11]. The drug is rapidly absorbed with peak plasma
concentrationsreachedwithin14hafteradministrationand
a long halflife (11.415.6 h), making it suitable for
oncedaily administration. MOXI appears promising for
the treatment of respiratory tract infections caused by
commonbacterialspecies[12].
Liquidchromatographyiswidelyusedforthequantitative
determination of pharmaceutical compounds with UV,
fluorescence or electrochemical detection. For the deter
mination of pharmaceutical compounds for clinical and
preclinical studies, LCMS with electrospray ionization
(ESI)oratmosphericpressurechemicalionization(APCI)
isestablishedasapowerfulanalyticaltool[1317].
Quinolonesarenormallyquantitatedbyreversedphase
highperformanceliquid chromatography(RPHPLC)with
UV detection orfluorometricdetection[18].For determination
of MOXI, HPLC[1921] and capillary electrophoresis[22]
methodshavebeenreported.Recently,aspectrofluorometric
method was reported for the determination of MOXI in
human serum and urine[23]. Vishwanathan K. et al has
reportedtheonlyLCMS/MSmethodforthedetermination
of MOXI in human plasma using solid phase extraction
sample preparation method[24]. Different HPLC and spectro
scopicmethods have been published for MOXIassay in
biological fluids and some presented several disadvantages:
lackofsensitivity(lowlimitofquantitation(LLOQ)above
50 ng/mL), use of complex sample preparation procedures

Copyright 2014 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University

http://www.jcps.ac.cn

160

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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Chromatographicseparation of the analytewascarried


out on a Shimadzu HPLC with HYPURITY C18 column
(5 m particle size, 50 mm4.6 mm) (Thermo Electron
Corporation,UK).Anisocraticmobilephaseof0.1%formic
acidacetonitrile(60:40,v/v)wasdeliveredwithaflowrateof
0.5mL/min.Tenmicrolitresoftheextractwereinjectedonto
the HPLCsystem for analysis. Autosamplerwasmaintained
at6Cwhilethecolumntemperaturewaskeptat40C.
Thetotalruntimeforeachsamplewas3.0min.
The HPLC system was coupled with API4000 (Applied
Biosystem)triplequadrupolemassspectrometerequipped
with electron spray ionization source. Sample was
introduced and ionized by electrospray ionization in the
positive mode. The measurement was made at 525 C
sourcetemperatureand5000Vionsprayvoltageforboth
MOXIandCIPRO.Declusteringpotentialwas60eVand
50 eV for MOXI and CIPRO, respectively. Collision
energy was 26 eV and 24 eV for MOXI and CIPRO,
respectively. Nitrogen gas was used as collision gas.
Theassaywasbasedonmonitoringthe[M+H]+ ionswith
m/z 402 and 332 for the analyte and internal standard,
respectively,inthefirstquadrupoleandtheircorresponding
product ions with m/z 358.2 and 288.2 for MOXI and
CIPRO, respectively, in the third quadrupole with dwell
time of 100 ms. Multiple reaction monitoring (MRM)
chromatographic data were collected using ANALYST
1.4.2software.Forquantification,thepeakarearatiosof
thetargetionsofthedrugtothoseoftheinternalstandard
werecomparedwithweighted(1/C)leastsquarescalibration
curves in which the peak area ratios of the calibration
standardswereplottedversustheirconcentrations.

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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

161

evaluated to obtain better response of analytes. It was


foundthatthebestsignalwasachievedwithESIpositive
ion mode. APCI did notshow any advantages over ESI.
Furthermore, optimization of chromatographic condition
increased signal of analytes. A mobile phase containing
0.1% formic acid (pH = 2.50.2) in combination with
acetonitrile resulted in improved signal. Use of short
Hypurity C 18 (50 mm4.6 mm, 5 m) column resulted
in reduced run time as low as 3 min. The column oven
temperature was optimized to 40 C in order to obtain
symmetric analyte peaks. The resulted high signal under
optimized chromatographic conditions and detection
parameters eliminated the laborious extraction steps of
evaporation of eluent and reconstitution involved in
generic SPE method and liquidliquid extraction method
withoutcompromisingthesensitivity.
3.2.Selectivity

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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

y =0.000782x 0.0041(r =0.9998)

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

concentrations for the MOXIwere preparedfor recovery


determination.ThemeanrecoveryforMOXIwas98.90%.
ThemeanrecoveryforCIPROwas99.98%.

The intra day (n = 6) and interday (n = 18) precision


and accuracy calculated from QC samples analyzed on
threedaysforMOXIatconcentrationofLQC,MQCand
HQC are tabulated in Table 2. The intraday precision
was2.3%forMOXI,andtheintradayaccuracywas9.7%
forMOXI(Table2).Theintradayprecisionandaccuracy
weredeterminedbypoolingallindividualassayresults
ofreplicate(n= 6)qualitycontrol overthethreeseparate
batch runs. The interday precision was3.8% for MOXI,
and the interday accuracy was 12.4% for MOXI
(Table2).

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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RSD(%)

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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

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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

[8] Donati, M. Rodriguez, F.M. Olmo, A. Apote, L.D.


Ceben,R. Antimicrob.AgentsChemother. 1999, 43,825827.

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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