Research ArticleFast and Simple LC-MSMS Method for RifampicinQuantification in Human Plasma

Cane Temova Rakuša1 Robert Roškar1 Anita KlanIar Andrejc1 Tina Trdan Lušin1

Nataša Faganeli12 Iztok Grabnar1 Aleš Mrhar 1 Albin Kristl1 and Jurij Trontelj 1

1University of Ljubljana Faculty of Pharmacy Ljubljana Slovenia2Valdoltra Orthopaedic Hospital Ankaran Slovenia

Correspondence should be addressed to Jurij Trontelj jurijtronteljffauni-ljsi

Received 30 November 2018 Revised 28 December 2018 Accepted 8 January 2019 Published 3 February 2019

Academic Editor Stig Pedersen-Bjergaard

Copyright copy 2019 Zane Temova Rakusa et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

A simple fast and cost-effective LC-MSMS method for quantification of rifampicin in human plasma was developed and fullyvalidated The plasma samples containing rifampicin and isotopically labelled internal standard rifampicin D8 were cleaned upusing a Captiva ND Lipids filtration plate Chromatographic separation was achieved on an 1290 Infinity liquid chromatographcoupled to 6460 Triple Quadrupole operated in positive mode on a core-shell Kinetex C18 column (50 times 21 mm 26 120583m) bygradient elution using 01 formic acid in water and acetonitrile as a mobile phase The proposed method is the fastest methodpublished by now both in terms of sample preparation (approximately one minute per sample) and chromatographic analysis(total run time 24 min) Another key benefit is the outstanding sensitivity and wide analytical range (5-40000 120583gL) with goodlinearity accuracy and precisionThemethod showed almost complete recovery (92) and absence of any significant matrix effectas demonstrated by uniform responses from QC samples prepared in blood plasma from 6 volunteers (RSD lt5) The proposedmethod was successfully applied to rifampicin quantification in 340 patientsrsquo plasma samples thus demonstrating its suitability forboth therapeutic drug monitoring and pharmacokinetic analysis

1 Introduction

Tuberculosis is an aggressive infectious disease caused byMycobacterium tuberculosis According to WHO it is cur-rently the leading cause of death from a single infectiousagent with a reported mortality of 16 million in 2017[1] Rifampicin (RIF) is highly effective bacteriostatic andbactericidal macrocyclic antibiotic primarily used as oneof the first line drugs in the treatment of tuberculosis [2]It is also indicated in the treatment of leprosy meningitisosteomyelitis and staphylococcal endocarditis [3] Further-more the role of RIF is well-defined in patients with pros-thetic joint infections [4] RIF pharmacokinetics has beenreported as complex with significant interindividual vari-ability and plasma concentrations below the recommendedtherapeutic range (8000-24000 120583gL) in majority of adultpatients [5ndash8]The latter is tightly associated with therapeutic

failure and development of drug resistance Furthermore ithas been reported that the pediatric population differs inRIF pharmacokinetic parameters requiring almost twofoldhigher dosage in mgkg body weight compared to adultsto reach equivalent plasma concentrations [5] Consideringits pharmacokinetic variability therapeutic drug monitoring(TDM) is a useful and valuable tool for improvement of RIFeffectiveness by individual dose adjustment and consequentlyprevention of therapeutic failure and drug resistance [9] RIFTDM may be also beneficial in the sense of preventing orreducing the incidence of potential toxicity and side effectsincluding hepatotoxicity which is the most common seriousadverse effect reported in 13-36 of the patients [10] RIFdetermination in patientsrsquo plasma is further recommendedin multidrug therapies since it is a potent inducer of drugtransporters and metabolizing enzymes As such RIF may

HindawiInternational Journal of Analytical ChemistryVolume 2019 Article ID 4848236 7 pageshttpsdoiorg10115520194848236

2 International Journal of Analytical Chemistry

interact and thus reduce the plasma concentration of otherdrugs including other tuberculosis drugs which are oftenused in combination with RIF [11]

For routine monitoring of RIF plasma concentration asimple selective robust and cost-effectivemethod is neededThere are several reported methods for RIF quantification inhuman plasma based primarily on HPLC-UV [12ndash24] andLC-MSMS [9 10 25ndash33] However all of them are associatedwith certain disadvantages to their widespread use in clinicalpractice The most commonly encountered issues includelengthy (ge20 min) andor complex extraction procedures [912ndash19 21ndash24 28 30 31] and relatively long analysis run time(ge10 min) [12ndash19 21ndash24 27 31] making them less suitablefor routine use HPLC-UV methods are in general lesssensitive and require relatively large sample volumes (ge200120583L) [12ndash19 21 23 24] limiting their applicability in pediatricstudies Such methods are applicable for TDM of RIF butnot for its pharmacokinetic studies due to insufficient limitsof quantification [12 13 15ndash19 21ndash24] Sufficient sensitivityand efficiency can be achieved with methods based on LC-MSMS However the reported LC-MSMS methods aremostly associated with lack of isotopically labelled inter-nal standard [9 28ndash31] persistent carry-over effect [29]and inadequately implemented matrix effect determinationmostly in terms of lack of relative matrix effect determination[9 10 25 26 28ndash31]

Therefore the aim of this work was to develop andvalidate a simple fast and sensitive LC-MSMS method forrifampicin quantification in human plasma The proposedmethod is undoubtedly appropriate for routine use in bothTDM and pharmacokinetic studies which was confirmed on340 samples from 56 different patients

2 Experimental

21 Chemicals and Reagents Rifampicin (gt970) was pur-chased from Fluka Honeywell (Morris Plains NJ USA)Stable-isotope labelled rifampicin D8 used as internalstandard (IS) was purchased from Alsachim (Illkirch-Graffenstaden France) HPLC grade methanol and acetoni-trile as well as LC-MS grade acetonitrile were obtainedfrom Honeywell (Morris Plains NJ USA) Formic acid wasobtained from Merck (Darmstadt Germany) Milli-Q waterwas generated by anAdvantage A10 water purification system(Millipore Corp Billerica USA)

22 Instrumentation and Chromatographic Conditions Thepretreated samples were analyzed by an Agilent 1290 Infinityliquid chromatograph coupled to 6460 Triple Quadrupoledetector (Agilent Technologies Santa Clara USA) equippedwith a Jetstream electrospray interfaceThe chromatographicseparation was achieved on a core-shell Kinetex C18 column(50times 21mm 26120583m) (Phenomenex Torrance USA) at 50∘Cby gradient elution using 01 formic acid in Milli-Q water(mobile phase A) and 998 acetonitrile 02 Milli-Q water(mobile phase B) with the following gradient (min B flowrate in mLmin) (00 30 0800) (025 35 0750) (05 450650) (10 55 0500) (125 65 0500) (15-17 95 0800)(1930 0900) and (200 30 0800) The injection volume

was 1 120583L Total run time including reequilibration was 24min The mass spectrometer was operated in positive modeusing the following parameters of the ion source gas flow(and temperature) 5 Lmin (275∘C) nebulizer pressure 310kPa and sheath gas flow (and temperature) 11 Lmin (400∘C)and the capillary voltage was 4000 VThe fragmentor voltagewas 80 V Both quadrupoles were set at the widest massresolution (25 amu) with the followingmz transitions 8234997888rarr 1071 at 61 eV and 8234 997888rarr 1631 at 41 eV for qualifierand quantifier ion of rifampicin respectively For IS the mztransition 8315997888rarr 1052 at collision energy of 85 eVwas usedInstrument control and data acquisition and processing wereperformed with Mass Hunter Workstation software B0600(Agilent Technologies Santa Clara USA)

23 Preparation of Calibration Standards and Quality ControlSamples Individual stock solutions of IS and RIF (primarystock solution PS) with concentration 1000 mgL wereprepared by dissolving appropriate amounts in methanolRIF secondary tertiary and quaternary stock solutions wereprepared with appropriate PS dilutions with a mixture ofmethanol and water (11 vv) to obtain solutions with 50050 and 5 mgL respectively These four stock solutions werefurther diluted with the same solvent to obtain eight workingsolutions in the range 01-800 mgL Aliquots (50 120583L) of theindividual working solutions were diluted 20-fold with blankplasma to obtain calibration standards with the followingRIF concentrations 5 25 50 100 1000 5000 15000 and40000 120583gL Quality control (QC) samples were prepared onsix levels (15 75 750 3000 10000 and 30000 120583gL) fromfreshly prepared RIF PS following the same protocol as forcalibration standards

The extraction procedure combined protein precipitationwith solid-phase extraction (SPE) and was as follows a 100120583L aliquot of plasma sample was mixed with 300 120583L of icecold acetonitrile containing IS (25mgL) After precipitationthe sample mixture was filtered through the Captiva NDLipids filtration plate (Agilent Technologies Santa ClaraUSA) using vacuum manifold The collected filtrates weretransferred to the autosampler and subjected to LC-MSMSanalysis Unless otherwise noted RIF concentration wasdetermined based on RIFIS ratio

24 Sample Collection and Preparation A significant numberof blood samples (340) were obtained from 56 patientsreceiving 450mg RIF every 12 hoursThe study was approvedby the Republic of Slovenia National Medical Ethics Com-mittee (approval no 480611) Samples were collected atdifferent predetermined time points schedule according tothe site of the infection and were properly treated to obtainplasma samples Blank plasma samples were obtained fromsix healthy volunteers Plasma samples were stored at -80∘C and thawed prior to analysis The sample preparationprocedure was exactly the same as for the calibrator and QCsamples All patient samples were processed in at least twoparallels

25 Method Validation The proposed method was validatedaccording to the EMA guideline on bioanalytical method

International Journal of Analytical Chemistry 3

validation [34] Recovery relative and absolute matrix effectwere evaluated in accordancewithMatuszewski et al [35 36]

Method selectivity was assessed by the analysis of plasmasamples from six different lots which were evaluated forinterference with RIF or IS Furthermore RIF identity wasconfirmed when the deviation of qualifier to quantifier ionratio was less than 20

The lower limit of quantification (LLOQ)was determinedas the lowest calibration standard which reached acceptableaccuracy (100 plusmn 20) and precision (RSD below 20)Additionally the RIF signal in LLOQ was checked to be atleast 5-fold greater compared to the response from blanksample

Method linearity was evaluated on eight nonzero calibra-tion standards in the concentration range from 5 to 40000120583gL for three consecutive days of the validation Linearitywas determined based on the least-square linear regressionThe acceptance criterion was a correlation coefficient of morethan 099

Carry-over was assessed by injecting blank sample afterthe highest calibration standard (40000 120583gL) and highconcentration samples The acceptance criterion was carry-over of less than 20 of the LLOQ and 5 for the IS

Accuracy and precision of the method were determinedon QC samples at six concentration levels covering thecalibration range and were prepared fresh on each of thethree consecutive days of the validationWithin-run accuracyand precision were evaluated on QC samples prepared infive replicates and analyzed in a single run Between-runaccuracy and precision were evaluated on QC samples (n=6)prepared and analyzed on each of the three consecutive daysof the validation Accuracy was acceptable when the meanconcentration was within plusmn 15 of the nominal value for allQC samples except at the LLOQ where plusmn 20 deviation wasaccepted Acceptance criterion for precision expressed as thecoefficient of variation (CV) was plusmn 15 for the QC samplesexcept at the LLOQ (plusmn 20)

Recovery absolute and relative matrix effect were eval-uated on four QC levels (15 750 10000 and 30000 120583gL)each prepared in triplicate Relative matrix effect (RME) wasdetermined on QC samples prepared from blank plasmafrom six individual donors and was expressed as CV ()of RIF responses at each concentration level CV () valueof standard line slopes obtained from six different plasmalots was also calculated Absolute matrix effect (AME) wasdetermined at each QC concentration level as the ratiobetween blank plasma spiked after extraction with RIF andIS (B) and standard RIF and IS solution in neat solvent (A)AME = BA times 100 Recovery (RE) was determined as theratio of RIF responses of extracted QC samples and blankplasma spiked after extraction with the same nominal RIFand IS concentration as in the QC samples (B) RE = QCB times100

RIF stability was evaluated on four QC levels (15 75010000 and 30000 120583gL) each prepared in triplicate Stabilitystudies included freeze and thaw stability (after three cycles)short-term stability (after 4h at room temperature) of RIF inhuman plasma and autosampler stability of RIF in extractedsamples (reanalyzed after 24 hours at 4∘C) Deviations in

mean RIF concentration at each QC level of less than 15were considered acceptable

3 Results and Discussion

31 Method Optimization Mass spectrometry ion sourceconditions were optimized to achieve the highest possibleresponses for both RIF and its isotopically labelled IS (RIF-D8) The optimal MRM transitions were obtained by auto-mated method development software (MassHunter Opti-mizer Agilent Technologies) The most abundant fragmention was chosen as quantifier and the second most abundantas qualifier the ratio of qualifier to quantifier ions was usedto confirm the RIF identity For IS only one mass transitionwas used in order to increase the sensitivity of the methodtowards RIF

The chromatographic method utilizing a core-shell C18column was optimized to provide fast and robust RIF separa-tion from backgroundmatrix with low system back-pressuremaking it suitable not only for UHPLC but for ordinaryHPLC systems as well

Sample preparation procedure for RIF quantitation wasbased on a method described by Srivastava et al [31] whichwas further optimized in terms of substantial time reductionwhich is especially important when large sample batchesneed to be analyzed Based on literature findings [37] aswell as previous practical experience with plasma proteinprecipitation acetonitrile was chosen as the optimal organicsolvent Typical plasma samples preparation proceduresinclude vigorous vortexmixing to allow protein precipitationfollowed by centrifugation to remove the precipitates Theseconventional and most time consuming steps in bioanalysiswere upgraded to in-well protein precipitation and cleanupby Captiva ND Lipids filtration plate With the introductionof this fast and simplified workflow sample preparationtime was reduced from approximately 30 min to 1 minuteper sample with high recovery and lipid matrix removalSuch sample preparation is cost-effective and can be fullyautomated for high-throughput sample analysis

32 Method Validation Method selectivity was confirmed asno interference from endogenous compounds was observedat the retention time of RIF or IS in the individual blankplasma from six different sources Representative chro-matograms of six blank plasma samples and LLOQ sampleare presented in Figure 1 Rifampicin qualifier to quantifierion intensity ratio (average 069) was within the acceptancecriterion (plusmn 20) in all tested samples

Linearity of the method was confirmed over a very wideconcentration range 5-40000 120583gL which completely coversthe therapeutic range of rifampicin (8000-24000 120583gL) Theobtained calibration curve from the first validation dayplotted as response ratio of RIF to IS on y-axis versus RIFconcentration on the x-axis of using weighting of 1c wasy=0003220x-0004509 The corresponding determinationcoefficient (r2) was 09993 The methodrsquos LLOQ was thelowest calibration standard (5 120583gL) based on precision accu-racy and LLOQblank sample signal ratio The method wastherefore found sufficiently sensitive for RIF determination

4 International Journal of Analytical Chemistry

Table 1 Accuracy and precision data for RIF

QC (120583gL) Accuracy () Precision (CV)Within-run Between-run Within-run Between-run

15 10504 10047 195 67075 9923 9793 125 395750 8721 8867 063 7923000 9843 9809 127 23510000 9823 10180 352 64330000 10387 10645 297 532

+ESI MRM Frag=800V CID410 (8234 -gt 1631) 2UM 5dNoise (Peak to Peak) = 1756 SNR (109min) = 684

5

48

46

109

1201

44

42

4

38

36

34

32

3

28

26

24

22

2

18

16

14

12

1

08

06

04

02

0

09 095 1 105 11 115 12 125 13 135 14 145

Counts vs Acquisition Time (min)

times102

Figure 1 Representative LC-MSMS chromatograms of six blankplasma samples and LLOQ sample Retention time of RIF is 11 min

in bioequivalence and pharmacokinetic studies The wideconcentration range enables RIF quantification in varioussamples without the need of any additional sample processing(eg dilution)

Carry-over effect has previously been reported as a trou-blesome part of RIF LC-MSMS analysis [29 38] Howeverthe analysis of blank solutions immediately after the highestcalibration standard andhigh concentration samples revealedno quantifiable carry-over

The obtained results for within-run and between-runaccuracy and precision are within the acceptance criterionas presented in Table 1

The evaluation of recovery and matrix effect is a veryimportant part of bioanalytical LC-MSMS method valida-tion It is however somewhat neglected or undervaluedaspect due to incomplete experimental protocol by theFDA and EMA guidelines in terms of relative matrix effectevaluation [34 39]The latter is a crucial parameter in bioan-alytical assays validationThe greatest drawback of previouslyreported methods is RIF quantification in human plasmafrom different patients without appropriate demonstrationthat the relativeMSMS response is not affected by thematrixElimination of relative matrix effect uncertainty is thus

Table 2 Relative matrix effect data for RIF

Relative matrix effect (CV)QC (120583gL) IS correction Without IS correction15 475 1200750 297 133410000 326 110330000 365 266slope 271 294

Table 3 Stability data for RIF

Stability ()QC (120583gL) Freeze and thaw Short-term Autosampler15 303 506 -988750 -1023 -189 ND10000 300 -380 ND30000 161 224 -218ND = not determined

essential in order to obtain reliable and useful results forfurther pharmacokinetic analysis Therefore relative matrixeffectwas quantitatively assessed as proposed byMatuszewskiet al [35] The obtained results for relative matrix effect(CV lt5) in combination with the low CV of standardline slopes obtained from six different plasma lots (Table 2)are an excellent demonstration of the absence of relativematrix effect The obtained average absolute matrix effect plusmnSE for RIFIS ratio was 10189 plusmn 195 Based on the obtainedresults for both relative and absolute matrix effect it can beconcluded that the proposed method is free from any majorion suppression or enhancement The mean recoveries (RE plusmnSE) for both RIF (9248 plusmn 397) and IS (8839 plusmn 307) wereconsistent and reproducible thus confirming the suitabilityof the extraction procedure

The results for freeze and thaw stability short-termstability and autosampler stability expressed as of changefrom the initial sample are summarized in Table 3 Thestability of RIF in all QC samples was acceptable (plusmn 15change) under all tested conditions Long-term stability wasnot assessed since RIF has previously been shown stablein plasma stored at -20∘C for at least 1 month [9] andfor 4 months after storage at -80∘C [2] Patientsrsquo plasmasamples were stored at -80∘C and were analyzed as soon aspossible

International Journal of Analytical Chemistry 5

+ESI MRM Frag=1700V CID850 (8315 -gt 1052) 25R 13-4 IId

+ESI MRM Frag=800V CID610 (8234 -gt 1071) 25R 13-4 IId

+ESI MRM Frag=800V CID410 (8234 -gt 1631) 25R 13-4 IId

4

3

3

2

2

3

3

1

1

21

21

0

times103

times102

times104

8

6

4

2

0

14

12

1

08

06

04

02

0

06 07 08 09 1 11 12 13 14 15 16 17 18

Counts vs Acquisition Time (min)

Figure 2 LC-MSMS chromatogram of a real plasma sample from a patient receiving 450 mg RIF every 12 hours The determined RIFconcentration is 283 120583gL Top trace represents IS middle trace represents RIF qualifier ion and bottom trace represents RIF quantifier ion

33 Application and Prospect of the Method The applicabilityof the proposed method was confirmed on 340 plasmasamples from 56 patients with orthopedic endoprosthesisinfections receiving 450 mg RIF every 12 hours The deter-mined RIF concentrations ranged between 592 and 21447120583gL Calculated mean RIF concentration (with standarddeviation) in the assayed samples with RIF concentrationsabove LLOQ was found 2433 plusmn 3324 120583gL An example ofa real plasma sample chromatogram with determined RIFconcentration 283 120583gL is shown in Figure 2The analysis ofsuch a number of plasma samples was facilitated by the fastand simple sample preparation which took approximatelyone minute per sample Sample preparation time can befurther reduced by the use of a fully automated approachThis in combination with the fast LC-MSMS method withtotal run time of 24 min is the most important advantagecompared to other reported methods for RIF quantificationAdditional advantages include low LLOQ andwide analyticalrange making it suitable for routine analysis of plasma sam-ples with diverse RIF concentrations Due to the small plasmavolume (100 120583L) the proposed method is also suited forpediatric studies where sample volumes are commonly quite

limited This assay is therefore undoubtedly applicable forpharmacokinetic bioequivalence or bioavailability studies ofRIF as well as for TDM

4 Conclusions

The developed LC-MSMS method was fully validated interms of selectivity linearity accuracy precision dilutionintegrity carry-over recovery matrix effect and stability Itssuitability for routine analyses was confirmed by rifampicinquantification in 340 patientsrsquo plasma samples The fastsimple and efficient sample preparation along with the shortLC-MSMS run time enables the analysis of a large numberof plasma samples in a short time thus providing a fastreliable and cost-effective analytic tool for RIF therapeuticmonitoring and pharmacokinetic studies

Data Availability

The data used to support the findings of this study areincluded within the article

6 International Journal of Analytical Chemistry

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Acknowledgments

This research was financially supported by SlovenianResearch Agency (ARRS) [P1-0189]

References

[1] WHO (WorldHealthOrganization)Global Tuberculosis Report2018 Geneva Switzerland 2018 httpwwwwhointtbpubli-cationsglobal reporttb18 ExecSum web 4Oct18pdfua=1

[2] K Govender J H Adamson and P Owira ldquoThe developmentand validation of a LC-MSMS method for the quantitation ofmetformin rifampicin and isoniazid in rat plasma using HILICchromatographyrdquo Journal of Chromatography B vol 1095 pp127ndash137 2018

[3] R Jogi and S K Tyring ldquoTherapy of nontuberculous mycobac-terial infectionsrdquo Dermatologic Therapy vol 17 no 6 pp 491ndash498 2004

[4] T F Moriarty R Kuehl T Coenye et al ldquoOrthopaedic device-related infection current and future interventions for improvedprevention and treatmentrdquo EFORT Open Reviews vol 1 no 4pp 89ndash99 2016

[5] P Donald J Maritz and A Diacon ldquoThe pharmacokineticsand pharmacodynamics of rifampicin in adults and children inrelation to the dosage recommended for childrenrdquoTuberculosisvol 91 no 3 pp 196ndash207 2011

[6] L Aıt Moussa O El Bouazzi S Serragui D Soussi TananiA Soulaymani and R Soulaymani ldquoRifampicin and isoniazidplasma concentrations in relation to adverse reactions in tuber-culosis patients a retrospective analysisrdquoTherapeutic Advancesin Drug Safety vol 7 no 6 pp 239ndash247 2016

[7] F Fahimi P Tabarsi F Kobarfard et al ldquoIsoniazid rifampicinand pyrazinamide plasma concentrations 2 and 6 h post dosein patients with pulmonary tuberculosisrdquo The InternationalJournal of Tuberculosis and LungDisease vol 17 no 12 pp 1602ndash1606 2013

[8] L Van Tongeren S Nolan V J Cook J M FitzGerald and JC Johnston ldquoTherapeutic drug monitoring in the treatment oftuberculosis a retrospective analysisrdquoThe International Journalof Tuberculosis and LungDisease vol 17 no 2 pp 221ndash224 2013

[9] S Gao Z Wang X Xie et al ldquoRapid and sensitive methodfor simultaneous determination of first-line anti-tuberculosisdrugs in human plasma by HPLC-MSMS Application totherapeutic drug monitoringrdquo Tuberculosis vol 109 pp 28ndash342018

[10] K H Hee J J Seo and L S Lee ldquoDevelopment and validationof liquid chromatography tandem mass spectrometry methodfor simultaneous quantification of first line tuberculosis drugsand metabolites in human plasma and its application in clinicalstudyrdquo Journal of Pharmaceutical and Biomedical Analysis vol102 pp 253ndash260 2015

[11] G Maartens M Boffito and C W Flexner ldquoCompatibility ofnext-generation first-line antiretrovirals with rifampicin-basedantituberculosis therapy in resource limited settingsrdquo CurrentOpinion in HIV and AIDS vol 12 no 4 pp 355ndash358 2017

[12] S Goutal S Auvity T Legrand et al ldquoValidation of a simpleHPLC-UV method for rifampicin determination in plasmaApplication to the study of rifampicin arteriovenous concen-tration gradientrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 123 pp 173ndash178 2016

[13] Z Zhou L Chen P Liu M Shen and F Zou ldquoSimultaneousDetermination of Isoniazid Pyrazinamide Rifampicin andAcetylisoniazid inHuman Plasma byHigh-Performance LiquidChromatographyrdquo Analytical Sciences vol 26 no 11 pp 1133ndash1138 2010

[14] R Panchagnula A Sood N Sharda K Kaur and C KaulldquoDetermination of rifampicin and itsmainmetabolite in plasmaand urine in presence of pyrazinamide and isoniazid by HPLCmethodrdquo Journal of Pharmaceutical and Biomedical Analysisvol 18 no 6 pp 1013ndash1020 1999

[15] L Melo R Queiroz and M Queiroz ldquoAutomated determina-tion of rifampicin in plasma samples by in-tube solid-phasemicroextraction coupled with liquid chromatographyrdquo Journalof Chromatography B vol 879 no 24 pp 2454ndash2458 2011

[16] D Fox R OrsquoConnor P Mallon and G McMahon ldquoSimulta-neous determination of efavirenz rifampicin and its metabolitedesacetyl rifampicin levels in human plasmardquo Journal of Phar-maceutical and Biomedical Analysis vol 56 no 4 pp 785ndash7912011

[17] B Louveau C Fernandez N Zahr et al ldquoDetermination ofrifampicin in human plasma by high-performance liquid chro-matography coupled with ultraviolet detection after automa-tized solid-liquid extractionrdquo Biomedical Chromatography vol30 no 12 pp 2009ndash2015 2016

[18] AAllansonMCotton J Tettey andA Boyter ldquoDeterminationof rifampicin in human plasma and blood spots by highperformance liquid chromatography with UV detection Apotential method for therapeutic drug monitoringrdquo Journal ofPharmaceutical and Biomedical Analysis vol 44 no 4 pp 963ndash969 2007

[19] A Walubo P Smith and P I Folb ldquoComprehensive assayfor pyrazinamide rifampicin and isoniazid with its hydrazinemetabolites in human plasma by column liquid chromatog-raphyrdquo Journal of Chromatography B Biomedical Sciences andApplications vol 658 no 2 pp 391ndash396 1994

[20] M Ishii and H Ogata ldquoDetermination of rifampicin and itsmainmetabolites in human plasma by high-performance liquidchromatographyrdquo Journal of Chromatography B BiomedicalSciences and Applications vol 426 pp 412ndash416 1988

[21] H Seifart P Kruger D Parkin P van Jaarsveld and P DonaldldquoTherapeutic monitoring of antituberculosis drugs by direct in-line extraction on a high-performance liquid chromatographysystemrdquo Journal of Chromatography B Biomedical Sciences andApplications vol 619 no 2 pp 285ndash290 1993

[22] B Ratti R R Parenti A Toselli and L F Zerilli ldquoQuan-titative assay of rifampicin and its main metabolite 25-desacetylrifampicin in human plasma by reversed-phase high-performance liquid chromatographyrdquo Journal of Chromatogra-phy B Biomedical Sciences and Applications vol 225 no 2 pp526ndash531 1981

[23] P J Smith J van Dyk and A Fredericks ldquoDetermination ofrifampicin isoniazid and pyrazinamide by high performanceliquid chromatography after their simultaneous extraction fromplasmardquo International Journal of Tuberculosis and Lung Diseasevol 3 no 11 Suppl 3 Article ID 10593712 pp S325ndashS328 1999

International Journal of Analytical Chemistry 3

validation [34] Recovery relative and absolute matrix effectwere evaluated in accordancewithMatuszewski et al [35 36]

Method selectivity was assessed by the analysis of plasmasamples from six different lots which were evaluated forinterference with RIF or IS Furthermore RIF identity wasconfirmed when the deviation of qualifier to quantifier ionratio was less than 20

The lower limit of quantification (LLOQ)was determinedas the lowest calibration standard which reached acceptableaccuracy (100 plusmn 20) and precision (RSD below 20)Additionally the RIF signal in LLOQ was checked to be atleast 5-fold greater compared to the response from blanksample

Method linearity was evaluated on eight nonzero calibra-tion standards in the concentration range from 5 to 40000120583gL for three consecutive days of the validation Linearitywas determined based on the least-square linear regressionThe acceptance criterion was a correlation coefficient of morethan 099

Carry-over was assessed by injecting blank sample afterthe highest calibration standard (40000 120583gL) and highconcentration samples The acceptance criterion was carry-over of less than 20 of the LLOQ and 5 for the IS

Accuracy and precision of the method were determinedon QC samples at six concentration levels covering thecalibration range and were prepared fresh on each of thethree consecutive days of the validationWithin-run accuracyand precision were evaluated on QC samples prepared infive replicates and analyzed in a single run Between-runaccuracy and precision were evaluated on QC samples (n=6)prepared and analyzed on each of the three consecutive daysof the validation Accuracy was acceptable when the meanconcentration was within plusmn 15 of the nominal value for allQC samples except at the LLOQ where plusmn 20 deviation wasaccepted Acceptance criterion for precision expressed as thecoefficient of variation (CV) was plusmn 15 for the QC samplesexcept at the LLOQ (plusmn 20)

Recovery absolute and relative matrix effect were eval-uated on four QC levels (15 750 10000 and 30000 120583gL)each prepared in triplicate Relative matrix effect (RME) wasdetermined on QC samples prepared from blank plasmafrom six individual donors and was expressed as CV ()of RIF responses at each concentration level CV () valueof standard line slopes obtained from six different plasmalots was also calculated Absolute matrix effect (AME) wasdetermined at each QC concentration level as the ratiobetween blank plasma spiked after extraction with RIF andIS (B) and standard RIF and IS solution in neat solvent (A)AME = BA times 100 Recovery (RE) was determined as theratio of RIF responses of extracted QC samples and blankplasma spiked after extraction with the same nominal RIFand IS concentration as in the QC samples (B) RE = QCB times100

RIF stability was evaluated on four QC levels (15 75010000 and 30000 120583gL) each prepared in triplicate Stabilitystudies included freeze and thaw stability (after three cycles)short-term stability (after 4h at room temperature) of RIF inhuman plasma and autosampler stability of RIF in extractedsamples (reanalyzed after 24 hours at 4∘C) Deviations in

mean RIF concentration at each QC level of less than 15were considered acceptable

3 Results and Discussion

31 Method Optimization Mass spectrometry ion sourceconditions were optimized to achieve the highest possibleresponses for both RIF and its isotopically labelled IS (RIF-D8) The optimal MRM transitions were obtained by auto-mated method development software (MassHunter Opti-mizer Agilent Technologies) The most abundant fragmention was chosen as quantifier and the second most abundantas qualifier the ratio of qualifier to quantifier ions was usedto confirm the RIF identity For IS only one mass transitionwas used in order to increase the sensitivity of the methodtowards RIF

The chromatographic method utilizing a core-shell C18column was optimized to provide fast and robust RIF separa-tion from backgroundmatrix with low system back-pressuremaking it suitable not only for UHPLC but for ordinaryHPLC systems as well

Sample preparation procedure for RIF quantitation wasbased on a method described by Srivastava et al [31] whichwas further optimized in terms of substantial time reductionwhich is especially important when large sample batchesneed to be analyzed Based on literature findings [37] aswell as previous practical experience with plasma proteinprecipitation acetonitrile was chosen as the optimal organicsolvent Typical plasma samples preparation proceduresinclude vigorous vortexmixing to allow protein precipitationfollowed by centrifugation to remove the precipitates Theseconventional and most time consuming steps in bioanalysiswere upgraded to in-well protein precipitation and cleanupby Captiva ND Lipids filtration plate With the introductionof this fast and simplified workflow sample preparationtime was reduced from approximately 30 min to 1 minuteper sample with high recovery and lipid matrix removalSuch sample preparation is cost-effective and can be fullyautomated for high-throughput sample analysis

32 Method Validation Method selectivity was confirmed asno interference from endogenous compounds was observedat the retention time of RIF or IS in the individual blankplasma from six different sources Representative chro-matograms of six blank plasma samples and LLOQ sampleare presented in Figure 1 Rifampicin qualifier to quantifierion intensity ratio (average 069) was within the acceptancecriterion (plusmn 20) in all tested samples

Linearity of the method was confirmed over a very wideconcentration range 5-40000 120583gL which completely coversthe therapeutic range of rifampicin (8000-24000 120583gL) Theobtained calibration curve from the first validation dayplotted as response ratio of RIF to IS on y-axis versus RIFconcentration on the x-axis of using weighting of 1c wasy=0003220x-0004509 The corresponding determinationcoefficient (r2) was 09993 The methodrsquos LLOQ was thelowest calibration standard (5 120583gL) based on precision accu-racy and LLOQblank sample signal ratio The method wastherefore found sufficiently sensitive for RIF determination

4 International Journal of Analytical Chemistry

Table 1 Accuracy and precision data for RIF

QC (120583gL) Accuracy () Precision (CV)Within-run Between-run Within-run Between-run

15 10504 10047 195 67075 9923 9793 125 395750 8721 8867 063 7923000 9843 9809 127 23510000 9823 10180 352 64330000 10387 10645 297 532

+ESI MRM Frag=800V CID410 (8234 -gt 1631) 2UM 5dNoise (Peak to Peak) = 1756 SNR (109min) = 684

5

48

46

109

1201

44

42

4

38

36

34

32

3

28

26

24

22

2

18

16

14

12

1

08

06

04

02

0

09 095 1 105 11 115 12 125 13 135 14 145

Counts vs Acquisition Time (min)

times102

Figure 1 Representative LC-MSMS chromatograms of six blankplasma samples and LLOQ sample Retention time of RIF is 11 min

in bioequivalence and pharmacokinetic studies The wideconcentration range enables RIF quantification in varioussamples without the need of any additional sample processing(eg dilution)

Carry-over effect has previously been reported as a trou-blesome part of RIF LC-MSMS analysis [29 38] Howeverthe analysis of blank solutions immediately after the highestcalibration standard andhigh concentration samples revealedno quantifiable carry-over

The obtained results for within-run and between-runaccuracy and precision are within the acceptance criterionas presented in Table 1

The evaluation of recovery and matrix effect is a veryimportant part of bioanalytical LC-MSMS method valida-tion It is however somewhat neglected or undervaluedaspect due to incomplete experimental protocol by theFDA and EMA guidelines in terms of relative matrix effectevaluation [34 39]The latter is a crucial parameter in bioan-alytical assays validationThe greatest drawback of previouslyreported methods is RIF quantification in human plasmafrom different patients without appropriate demonstrationthat the relativeMSMS response is not affected by thematrixElimination of relative matrix effect uncertainty is thus

Table 2 Relative matrix effect data for RIF

Relative matrix effect (CV)QC (120583gL) IS correction Without IS correction15 475 1200750 297 133410000 326 110330000 365 266slope 271 294

Table 3 Stability data for RIF

Stability ()QC (120583gL) Freeze and thaw Short-term Autosampler15 303 506 -988750 -1023 -189 ND10000 300 -380 ND30000 161 224 -218ND = not determined

essential in order to obtain reliable and useful results forfurther pharmacokinetic analysis Therefore relative matrixeffectwas quantitatively assessed as proposed byMatuszewskiet al [35] The obtained results for relative matrix effect(CV lt5) in combination with the low CV of standardline slopes obtained from six different plasma lots (Table 2)are an excellent demonstration of the absence of relativematrix effect The obtained average absolute matrix effect plusmnSE for RIFIS ratio was 10189 plusmn 195 Based on the obtainedresults for both relative and absolute matrix effect it can beconcluded that the proposed method is free from any majorion suppression or enhancement The mean recoveries (RE plusmnSE) for both RIF (9248 plusmn 397) and IS (8839 plusmn 307) wereconsistent and reproducible thus confirming the suitabilityof the extraction procedure

The results for freeze and thaw stability short-termstability and autosampler stability expressed as of changefrom the initial sample are summarized in Table 3 Thestability of RIF in all QC samples was acceptable (plusmn 15change) under all tested conditions Long-term stability wasnot assessed since RIF has previously been shown stablein plasma stored at -20∘C for at least 1 month [9] andfor 4 months after storage at -80∘C [2] Patientsrsquo plasmasamples were stored at -80∘C and were analyzed as soon aspossible

International Journal of Analytical Chemistry 5

+ESI MRM Frag=1700V CID850 (8315 -gt 1052) 25R 13-4 IId

+ESI MRM Frag=800V CID610 (8234 -gt 1071) 25R 13-4 IId

+ESI MRM Frag=800V CID410 (8234 -gt 1631) 25R 13-4 IId

4

3

3

2

2

3

3

1

1

21

21

0

times103

times102

times104

8

6

4

2

0

14

12

1

08

06

04

02

0

06 07 08 09 1 11 12 13 14 15 16 17 18

Counts vs Acquisition Time (min)

Figure 2 LC-MSMS chromatogram of a real plasma sample from a patient receiving 450 mg RIF every 12 hours The determined RIFconcentration is 283 120583gL Top trace represents IS middle trace represents RIF qualifier ion and bottom trace represents RIF quantifier ion

33 Application and Prospect of the Method The applicabilityof the proposed method was confirmed on 340 plasmasamples from 56 patients with orthopedic endoprosthesisinfections receiving 450 mg RIF every 12 hours The deter-mined RIF concentrations ranged between 592 and 21447120583gL Calculated mean RIF concentration (with standarddeviation) in the assayed samples with RIF concentrationsabove LLOQ was found 2433 plusmn 3324 120583gL An example ofa real plasma sample chromatogram with determined RIFconcentration 283 120583gL is shown in Figure 2The analysis ofsuch a number of plasma samples was facilitated by the fastand simple sample preparation which took approximatelyone minute per sample Sample preparation time can befurther reduced by the use of a fully automated approachThis in combination with the fast LC-MSMS method withtotal run time of 24 min is the most important advantagecompared to other reported methods for RIF quantificationAdditional advantages include low LLOQ andwide analyticalrange making it suitable for routine analysis of plasma sam-ples with diverse RIF concentrations Due to the small plasmavolume (100 120583L) the proposed method is also suited forpediatric studies where sample volumes are commonly quite

limited This assay is therefore undoubtedly applicable forpharmacokinetic bioequivalence or bioavailability studies ofRIF as well as for TDM

4 Conclusions

The developed LC-MSMS method was fully validated interms of selectivity linearity accuracy precision dilutionintegrity carry-over recovery matrix effect and stability Itssuitability for routine analyses was confirmed by rifampicinquantification in 340 patientsrsquo plasma samples The fastsimple and efficient sample preparation along with the shortLC-MSMS run time enables the analysis of a large numberof plasma samples in a short time thus providing a fastreliable and cost-effective analytic tool for RIF therapeuticmonitoring and pharmacokinetic studies

Data Availability

The data used to support the findings of this study areincluded within the article

6 International Journal of Analytical Chemistry

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Acknowledgments

This research was financially supported by SlovenianResearch Agency (ARRS) [P1-0189]

References

[1] WHO (WorldHealthOrganization)Global Tuberculosis Report2018 Geneva Switzerland 2018 httpwwwwhointtbpubli-cationsglobal reporttb18 ExecSum web 4Oct18pdfua=1

[2] K Govender J H Adamson and P Owira ldquoThe developmentand validation of a LC-MSMS method for the quantitation ofmetformin rifampicin and isoniazid in rat plasma using HILICchromatographyrdquo Journal of Chromatography B vol 1095 pp127ndash137 2018

[3] R Jogi and S K Tyring ldquoTherapy of nontuberculous mycobac-terial infectionsrdquo Dermatologic Therapy vol 17 no 6 pp 491ndash498 2004

[4] T F Moriarty R Kuehl T Coenye et al ldquoOrthopaedic device-related infection current and future interventions for improvedprevention and treatmentrdquo EFORT Open Reviews vol 1 no 4pp 89ndash99 2016

[5] P Donald J Maritz and A Diacon ldquoThe pharmacokineticsand pharmacodynamics of rifampicin in adults and children inrelation to the dosage recommended for childrenrdquoTuberculosisvol 91 no 3 pp 196ndash207 2011

[6] L Aıt Moussa O El Bouazzi S Serragui D Soussi TananiA Soulaymani and R Soulaymani ldquoRifampicin and isoniazidplasma concentrations in relation to adverse reactions in tuber-culosis patients a retrospective analysisrdquoTherapeutic Advancesin Drug Safety vol 7 no 6 pp 239ndash247 2016

[7] F Fahimi P Tabarsi F Kobarfard et al ldquoIsoniazid rifampicinand pyrazinamide plasma concentrations 2 and 6 h post dosein patients with pulmonary tuberculosisrdquo The InternationalJournal of Tuberculosis and LungDisease vol 17 no 12 pp 1602ndash1606 2013

[8] L Van Tongeren S Nolan V J Cook J M FitzGerald and JC Johnston ldquoTherapeutic drug monitoring in the treatment oftuberculosis a retrospective analysisrdquoThe International Journalof Tuberculosis and LungDisease vol 17 no 2 pp 221ndash224 2013

[9] S Gao Z Wang X Xie et al ldquoRapid and sensitive methodfor simultaneous determination of first-line anti-tuberculosisdrugs in human plasma by HPLC-MSMS Application totherapeutic drug monitoringrdquo Tuberculosis vol 109 pp 28ndash342018

[10] K H Hee J J Seo and L S Lee ldquoDevelopment and validationof liquid chromatography tandem mass spectrometry methodfor simultaneous quantification of first line tuberculosis drugsand metabolites in human plasma and its application in clinicalstudyrdquo Journal of Pharmaceutical and Biomedical Analysis vol102 pp 253ndash260 2015

[11] G Maartens M Boffito and C W Flexner ldquoCompatibility ofnext-generation first-line antiretrovirals with rifampicin-basedantituberculosis therapy in resource limited settingsrdquo CurrentOpinion in HIV and AIDS vol 12 no 4 pp 355ndash358 2017

[12] S Goutal S Auvity T Legrand et al ldquoValidation of a simpleHPLC-UV method for rifampicin determination in plasmaApplication to the study of rifampicin arteriovenous concen-tration gradientrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 123 pp 173ndash178 2016

[13] Z Zhou L Chen P Liu M Shen and F Zou ldquoSimultaneousDetermination of Isoniazid Pyrazinamide Rifampicin andAcetylisoniazid inHuman Plasma byHigh-Performance LiquidChromatographyrdquo Analytical Sciences vol 26 no 11 pp 1133ndash1138 2010

[14] R Panchagnula A Sood N Sharda K Kaur and C KaulldquoDetermination of rifampicin and itsmainmetabolite in plasmaand urine in presence of pyrazinamide and isoniazid by HPLCmethodrdquo Journal of Pharmaceutical and Biomedical Analysisvol 18 no 6 pp 1013ndash1020 1999

[15] L Melo R Queiroz and M Queiroz ldquoAutomated determina-tion of rifampicin in plasma samples by in-tube solid-phasemicroextraction coupled with liquid chromatographyrdquo Journalof Chromatography B vol 879 no 24 pp 2454ndash2458 2011

[16] D Fox R OrsquoConnor P Mallon and G McMahon ldquoSimulta-neous determination of efavirenz rifampicin and its metabolitedesacetyl rifampicin levels in human plasmardquo Journal of Phar-maceutical and Biomedical Analysis vol 56 no 4 pp 785ndash7912011

[17] B Louveau C Fernandez N Zahr et al ldquoDetermination ofrifampicin in human plasma by high-performance liquid chro-matography coupled with ultraviolet detection after automa-tized solid-liquid extractionrdquo Biomedical Chromatography vol30 no 12 pp 2009ndash2015 2016

[18] AAllansonMCotton J Tettey andA Boyter ldquoDeterminationof rifampicin in human plasma and blood spots by highperformance liquid chromatography with UV detection Apotential method for therapeutic drug monitoringrdquo Journal ofPharmaceutical and Biomedical Analysis vol 44 no 4 pp 963ndash969 2007

[19] A Walubo P Smith and P I Folb ldquoComprehensive assayfor pyrazinamide rifampicin and isoniazid with its hydrazinemetabolites in human plasma by column liquid chromatog-raphyrdquo Journal of Chromatography B Biomedical Sciences andApplications vol 658 no 2 pp 391ndash396 1994

[20] M Ishii and H Ogata ldquoDetermination of rifampicin and itsmainmetabolites in human plasma by high-performance liquidchromatographyrdquo Journal of Chromatography B BiomedicalSciences and Applications vol 426 pp 412ndash416 1988

[21] H Seifart P Kruger D Parkin P van Jaarsveld and P DonaldldquoTherapeutic monitoring of antituberculosis drugs by direct in-line extraction on a high-performance liquid chromatographysystemrdquo Journal of Chromatography B Biomedical Sciences andApplications vol 619 no 2 pp 285ndash290 1993

[22] B Ratti R R Parenti A Toselli and L F Zerilli ldquoQuan-titative assay of rifampicin and its main metabolite 25-desacetylrifampicin in human plasma by reversed-phase high-performance liquid chromatographyrdquo Journal of Chromatogra-phy B Biomedical Sciences and Applications vol 225 no 2 pp526ndash531 1981

[23] P J Smith J van Dyk and A Fredericks ldquoDetermination ofrifampicin isoniazid and pyrazinamide by high performanceliquid chromatography after their simultaneous extraction fromplasmardquo International Journal of Tuberculosis and Lung Diseasevol 3 no 11 Suppl 3 Article ID 10593712 pp S325ndashS328 1999

4 International Journal of Analytical Chemistry

Table 1 Accuracy and precision data for RIF

QC (120583gL) Accuracy () Precision (CV)Within-run Between-run Within-run Between-run

15 10504 10047 195 67075 9923 9793 125 395750 8721 8867 063 7923000 9843 9809 127 23510000 9823 10180 352 64330000 10387 10645 297 532

+ESI MRM Frag=800V CID410 (8234 -gt 1631) 2UM 5dNoise (Peak to Peak) = 1756 SNR (109min) = 684

5

48

46

109

1201

44

42

4

38

36

34

32

3

28

26

24

22

2

18

16

14

12

1

08

06

04

02

0

09 095 1 105 11 115 12 125 13 135 14 145

Counts vs Acquisition Time (min)

times102

Figure 1 Representative LC-MSMS chromatograms of six blankplasma samples and LLOQ sample Retention time of RIF is 11 min

in bioequivalence and pharmacokinetic studies The wideconcentration range enables RIF quantification in varioussamples without the need of any additional sample processing(eg dilution)

Carry-over effect has previously been reported as a trou-blesome part of RIF LC-MSMS analysis [29 38] Howeverthe analysis of blank solutions immediately after the highestcalibration standard andhigh concentration samples revealedno quantifiable carry-over

The obtained results for within-run and between-runaccuracy and precision are within the acceptance criterionas presented in Table 1

The evaluation of recovery and matrix effect is a veryimportant part of bioanalytical LC-MSMS method valida-tion It is however somewhat neglected or undervaluedaspect due to incomplete experimental protocol by theFDA and EMA guidelines in terms of relative matrix effectevaluation [34 39]The latter is a crucial parameter in bioan-alytical assays validationThe greatest drawback of previouslyreported methods is RIF quantification in human plasmafrom different patients without appropriate demonstrationthat the relativeMSMS response is not affected by thematrixElimination of relative matrix effect uncertainty is thus

Table 2 Relative matrix effect data for RIF

Relative matrix effect (CV)QC (120583gL) IS correction Without IS correction15 475 1200750 297 133410000 326 110330000 365 266slope 271 294

Table 3 Stability data for RIF

Stability ()QC (120583gL) Freeze and thaw Short-term Autosampler15 303 506 -988750 -1023 -189 ND10000 300 -380 ND30000 161 224 -218ND = not determined

essential in order to obtain reliable and useful results forfurther pharmacokinetic analysis Therefore relative matrixeffectwas quantitatively assessed as proposed byMatuszewskiet al [35] The obtained results for relative matrix effect(CV lt5) in combination with the low CV of standardline slopes obtained from six different plasma lots (Table 2)are an excellent demonstration of the absence of relativematrix effect The obtained average absolute matrix effect plusmnSE for RIFIS ratio was 10189 plusmn 195 Based on the obtainedresults for both relative and absolute matrix effect it can beconcluded that the proposed method is free from any majorion suppression or enhancement The mean recoveries (RE plusmnSE) for both RIF (9248 plusmn 397) and IS (8839 plusmn 307) wereconsistent and reproducible thus confirming the suitabilityof the extraction procedure

The results for freeze and thaw stability short-termstability and autosampler stability expressed as of changefrom the initial sample are summarized in Table 3 Thestability of RIF in all QC samples was acceptable (plusmn 15change) under all tested conditions Long-term stability wasnot assessed since RIF has previously been shown stablein plasma stored at -20∘C for at least 1 month [9] andfor 4 months after storage at -80∘C [2] Patientsrsquo plasmasamples were stored at -80∘C and were analyzed as soon aspossible

International Journal of Analytical Chemistry 5

+ESI MRM Frag=1700V CID850 (8315 -gt 1052) 25R 13-4 IId

+ESI MRM Frag=800V CID610 (8234 -gt 1071) 25R 13-4 IId

+ESI MRM Frag=800V CID410 (8234 -gt 1631) 25R 13-4 IId

4

3

3

2

2

3

3

1

1

21

21

0

times103

times102

times104

8

6

4

2

0

14

12

1

08

06

04

02

0

06 07 08 09 1 11 12 13 14 15 16 17 18

Counts vs Acquisition Time (min)

Figure 2 LC-MSMS chromatogram of a real plasma sample from a patient receiving 450 mg RIF every 12 hours The determined RIFconcentration is 283 120583gL Top trace represents IS middle trace represents RIF qualifier ion and bottom trace represents RIF quantifier ion

33 Application and Prospect of the Method The applicabilityof the proposed method was confirmed on 340 plasmasamples from 56 patients with orthopedic endoprosthesisinfections receiving 450 mg RIF every 12 hours The deter-mined RIF concentrations ranged between 592 and 21447120583gL Calculated mean RIF concentration (with standarddeviation) in the assayed samples with RIF concentrationsabove LLOQ was found 2433 plusmn 3324 120583gL An example ofa real plasma sample chromatogram with determined RIFconcentration 283 120583gL is shown in Figure 2The analysis ofsuch a number of plasma samples was facilitated by the fastand simple sample preparation which took approximatelyone minute per sample Sample preparation time can befurther reduced by the use of a fully automated approachThis in combination with the fast LC-MSMS method withtotal run time of 24 min is the most important advantagecompared to other reported methods for RIF quantificationAdditional advantages include low LLOQ andwide analyticalrange making it suitable for routine analysis of plasma sam-ples with diverse RIF concentrations Due to the small plasmavolume (100 120583L) the proposed method is also suited forpediatric studies where sample volumes are commonly quite

limited This assay is therefore undoubtedly applicable forpharmacokinetic bioequivalence or bioavailability studies ofRIF as well as for TDM

4 Conclusions

The developed LC-MSMS method was fully validated interms of selectivity linearity accuracy precision dilutionintegrity carry-over recovery matrix effect and stability Itssuitability for routine analyses was confirmed by rifampicinquantification in 340 patientsrsquo plasma samples The fastsimple and efficient sample preparation along with the shortLC-MSMS run time enables the analysis of a large numberof plasma samples in a short time thus providing a fastreliable and cost-effective analytic tool for RIF therapeuticmonitoring and pharmacokinetic studies

Data Availability

The data used to support the findings of this study areincluded within the article

6 International Journal of Analytical Chemistry

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Acknowledgments

This research was financially supported by SlovenianResearch Agency (ARRS) [P1-0189]

References

[1] WHO (WorldHealthOrganization)Global Tuberculosis Report2018 Geneva Switzerland 2018 httpwwwwhointtbpubli-cationsglobal reporttb18 ExecSum web 4Oct18pdfua=1

[2] K Govender J H Adamson and P Owira ldquoThe developmentand validation of a LC-MSMS method for the quantitation ofmetformin rifampicin and isoniazid in rat plasma using HILICchromatographyrdquo Journal of Chromatography B vol 1095 pp127ndash137 2018

[3] R Jogi and S K Tyring ldquoTherapy of nontuberculous mycobac-terial infectionsrdquo Dermatologic Therapy vol 17 no 6 pp 491ndash498 2004

[4] T F Moriarty R Kuehl T Coenye et al ldquoOrthopaedic device-related infection current and future interventions for improvedprevention and treatmentrdquo EFORT Open Reviews vol 1 no 4pp 89ndash99 2016

[5] P Donald J Maritz and A Diacon ldquoThe pharmacokineticsand pharmacodynamics of rifampicin in adults and children inrelation to the dosage recommended for childrenrdquoTuberculosisvol 91 no 3 pp 196ndash207 2011

[6] L Aıt Moussa O El Bouazzi S Serragui D Soussi TananiA Soulaymani and R Soulaymani ldquoRifampicin and isoniazidplasma concentrations in relation to adverse reactions in tuber-culosis patients a retrospective analysisrdquoTherapeutic Advancesin Drug Safety vol 7 no 6 pp 239ndash247 2016

[7] F Fahimi P Tabarsi F Kobarfard et al ldquoIsoniazid rifampicinand pyrazinamide plasma concentrations 2 and 6 h post dosein patients with pulmonary tuberculosisrdquo The InternationalJournal of Tuberculosis and LungDisease vol 17 no 12 pp 1602ndash1606 2013

[8] L Van Tongeren S Nolan V J Cook J M FitzGerald and JC Johnston ldquoTherapeutic drug monitoring in the treatment oftuberculosis a retrospective analysisrdquoThe International Journalof Tuberculosis and LungDisease vol 17 no 2 pp 221ndash224 2013

[9] S Gao Z Wang X Xie et al ldquoRapid and sensitive methodfor simultaneous determination of first-line anti-tuberculosisdrugs in human plasma by HPLC-MSMS Application totherapeutic drug monitoringrdquo Tuberculosis vol 109 pp 28ndash342018

[10] K H Hee J J Seo and L S Lee ldquoDevelopment and validationof liquid chromatography tandem mass spectrometry methodfor simultaneous quantification of first line tuberculosis drugsand metabolites in human plasma and its application in clinicalstudyrdquo Journal of Pharmaceutical and Biomedical Analysis vol102 pp 253ndash260 2015

[11] G Maartens M Boffito and C W Flexner ldquoCompatibility ofnext-generation first-line antiretrovirals with rifampicin-basedantituberculosis therapy in resource limited settingsrdquo CurrentOpinion in HIV and AIDS vol 12 no 4 pp 355ndash358 2017

[12] S Goutal S Auvity T Legrand et al ldquoValidation of a simpleHPLC-UV method for rifampicin determination in plasmaApplication to the study of rifampicin arteriovenous concen-tration gradientrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 123 pp 173ndash178 2016

[13] Z Zhou L Chen P Liu M Shen and F Zou ldquoSimultaneousDetermination of Isoniazid Pyrazinamide Rifampicin andAcetylisoniazid inHuman Plasma byHigh-Performance LiquidChromatographyrdquo Analytical Sciences vol 26 no 11 pp 1133ndash1138 2010

[14] R Panchagnula A Sood N Sharda K Kaur and C KaulldquoDetermination of rifampicin and itsmainmetabolite in plasmaand urine in presence of pyrazinamide and isoniazid by HPLCmethodrdquo Journal of Pharmaceutical and Biomedical Analysisvol 18 no 6 pp 1013ndash1020 1999

[15] L Melo R Queiroz and M Queiroz ldquoAutomated determina-tion of rifampicin in plasma samples by in-tube solid-phasemicroextraction coupled with liquid chromatographyrdquo Journalof Chromatography B vol 879 no 24 pp 2454ndash2458 2011

[16] D Fox R OrsquoConnor P Mallon and G McMahon ldquoSimulta-neous determination of efavirenz rifampicin and its metabolitedesacetyl rifampicin levels in human plasmardquo Journal of Phar-maceutical and Biomedical Analysis vol 56 no 4 pp 785ndash7912011

[17] B Louveau C Fernandez N Zahr et al ldquoDetermination ofrifampicin in human plasma by high-performance liquid chro-matography coupled with ultraviolet detection after automa-tized solid-liquid extractionrdquo Biomedical Chromatography vol30 no 12 pp 2009ndash2015 2016

[18] AAllansonMCotton J Tettey andA Boyter ldquoDeterminationof rifampicin in human plasma and blood spots by highperformance liquid chromatography with UV detection Apotential method for therapeutic drug monitoringrdquo Journal ofPharmaceutical and Biomedical Analysis vol 44 no 4 pp 963ndash969 2007

[19] A Walubo P Smith and P I Folb ldquoComprehensive assayfor pyrazinamide rifampicin and isoniazid with its hydrazinemetabolites in human plasma by column liquid chromatog-raphyrdquo Journal of Chromatography B Biomedical Sciences andApplications vol 658 no 2 pp 391ndash396 1994

[20] M Ishii and H Ogata ldquoDetermination of rifampicin and itsmainmetabolites in human plasma by high-performance liquidchromatographyrdquo Journal of Chromatography B BiomedicalSciences and Applications vol 426 pp 412ndash416 1988

[21] H Seifart P Kruger D Parkin P van Jaarsveld and P DonaldldquoTherapeutic monitoring of antituberculosis drugs by direct in-line extraction on a high-performance liquid chromatographysystemrdquo Journal of Chromatography B Biomedical Sciences andApplications vol 619 no 2 pp 285ndash290 1993

[22] B Ratti R R Parenti A Toselli and L F Zerilli ldquoQuan-titative assay of rifampicin and its main metabolite 25-desacetylrifampicin in human plasma by reversed-phase high-performance liquid chromatographyrdquo Journal of Chromatogra-phy B Biomedical Sciences and Applications vol 225 no 2 pp526ndash531 1981

[23] P J Smith J van Dyk and A Fredericks ldquoDetermination ofrifampicin isoniazid and pyrazinamide by high performanceliquid chromatography after their simultaneous extraction fromplasmardquo International Journal of Tuberculosis and Lung Diseasevol 3 no 11 Suppl 3 Article ID 10593712 pp S325ndashS328 1999

International Journal of Analytical Chemistry 5

+ESI MRM Frag=1700V CID850 (8315 -gt 1052) 25R 13-4 IId

+ESI MRM Frag=800V CID610 (8234 -gt 1071) 25R 13-4 IId

+ESI MRM Frag=800V CID410 (8234 -gt 1631) 25R 13-4 IId

4

3

3

2

2

3

3

1

1

21

21

0

times103

times102

times104

8

6

4

2

0

14

12

1

08

06

04

02

0

06 07 08 09 1 11 12 13 14 15 16 17 18

Counts vs Acquisition Time (min)

Figure 2 LC-MSMS chromatogram of a real plasma sample from a patient receiving 450 mg RIF every 12 hours The determined RIFconcentration is 283 120583gL Top trace represents IS middle trace represents RIF qualifier ion and bottom trace represents RIF quantifier ion

33 Application and Prospect of the Method The applicabilityof the proposed method was confirmed on 340 plasmasamples from 56 patients with orthopedic endoprosthesisinfections receiving 450 mg RIF every 12 hours The deter-mined RIF concentrations ranged between 592 and 21447120583gL Calculated mean RIF concentration (with standarddeviation) in the assayed samples with RIF concentrationsabove LLOQ was found 2433 plusmn 3324 120583gL An example ofa real plasma sample chromatogram with determined RIFconcentration 283 120583gL is shown in Figure 2The analysis ofsuch a number of plasma samples was facilitated by the fastand simple sample preparation which took approximatelyone minute per sample Sample preparation time can befurther reduced by the use of a fully automated approachThis in combination with the fast LC-MSMS method withtotal run time of 24 min is the most important advantagecompared to other reported methods for RIF quantificationAdditional advantages include low LLOQ andwide analyticalrange making it suitable for routine analysis of plasma sam-ples with diverse RIF concentrations Due to the small plasmavolume (100 120583L) the proposed method is also suited forpediatric studies where sample volumes are commonly quite

limited This assay is therefore undoubtedly applicable forpharmacokinetic bioequivalence or bioavailability studies ofRIF as well as for TDM

4 Conclusions

The developed LC-MSMS method was fully validated interms of selectivity linearity accuracy precision dilutionintegrity carry-over recovery matrix effect and stability Itssuitability for routine analyses was confirmed by rifampicinquantification in 340 patientsrsquo plasma samples The fastsimple and efficient sample preparation along with the shortLC-MSMS run time enables the analysis of a large numberof plasma samples in a short time thus providing a fastreliable and cost-effective analytic tool for RIF therapeuticmonitoring and pharmacokinetic studies

Data Availability

The data used to support the findings of this study areincluded within the article

6 International Journal of Analytical Chemistry

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Acknowledgments

This research was financially supported by SlovenianResearch Agency (ARRS) [P1-0189]

References

[1] WHO (WorldHealthOrganization)Global Tuberculosis Report2018 Geneva Switzerland 2018 httpwwwwhointtbpubli-cationsglobal reporttb18 ExecSum web 4Oct18pdfua=1

[2] K Govender J H Adamson and P Owira ldquoThe developmentand validation of a LC-MSMS method for the quantitation ofmetformin rifampicin and isoniazid in rat plasma using HILICchromatographyrdquo Journal of Chromatography B vol 1095 pp127ndash137 2018

[3] R Jogi and S K Tyring ldquoTherapy of nontuberculous mycobac-terial infectionsrdquo Dermatologic Therapy vol 17 no 6 pp 491ndash498 2004

[4] T F Moriarty R Kuehl T Coenye et al ldquoOrthopaedic device-related infection current and future interventions for improvedprevention and treatmentrdquo EFORT Open Reviews vol 1 no 4pp 89ndash99 2016

[5] P Donald J Maritz and A Diacon ldquoThe pharmacokineticsand pharmacodynamics of rifampicin in adults and children inrelation to the dosage recommended for childrenrdquoTuberculosisvol 91 no 3 pp 196ndash207 2011

[6] L Aıt Moussa O El Bouazzi S Serragui D Soussi TananiA Soulaymani and R Soulaymani ldquoRifampicin and isoniazidplasma concentrations in relation to adverse reactions in tuber-culosis patients a retrospective analysisrdquoTherapeutic Advancesin Drug Safety vol 7 no 6 pp 239ndash247 2016

[7] F Fahimi P Tabarsi F Kobarfard et al ldquoIsoniazid rifampicinand pyrazinamide plasma concentrations 2 and 6 h post dosein patients with pulmonary tuberculosisrdquo The InternationalJournal of Tuberculosis and LungDisease vol 17 no 12 pp 1602ndash1606 2013

[8] L Van Tongeren S Nolan V J Cook J M FitzGerald and JC Johnston ldquoTherapeutic drug monitoring in the treatment oftuberculosis a retrospective analysisrdquoThe International Journalof Tuberculosis and LungDisease vol 17 no 2 pp 221ndash224 2013

[9] S Gao Z Wang X Xie et al ldquoRapid and sensitive methodfor simultaneous determination of first-line anti-tuberculosisdrugs in human plasma by HPLC-MSMS Application totherapeutic drug monitoringrdquo Tuberculosis vol 109 pp 28ndash342018

[10] K H Hee J J Seo and L S Lee ldquoDevelopment and validationof liquid chromatography tandem mass spectrometry methodfor simultaneous quantification of first line tuberculosis drugsand metabolites in human plasma and its application in clinicalstudyrdquo Journal of Pharmaceutical and Biomedical Analysis vol102 pp 253ndash260 2015

[11] G Maartens M Boffito and C W Flexner ldquoCompatibility ofnext-generation first-line antiretrovirals with rifampicin-basedantituberculosis therapy in resource limited settingsrdquo CurrentOpinion in HIV and AIDS vol 12 no 4 pp 355ndash358 2017

[12] S Goutal S Auvity T Legrand et al ldquoValidation of a simpleHPLC-UV method for rifampicin determination in plasmaApplication to the study of rifampicin arteriovenous concen-tration gradientrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 123 pp 173ndash178 2016

[13] Z Zhou L Chen P Liu M Shen and F Zou ldquoSimultaneousDetermination of Isoniazid Pyrazinamide Rifampicin andAcetylisoniazid inHuman Plasma byHigh-Performance LiquidChromatographyrdquo Analytical Sciences vol 26 no 11 pp 1133ndash1138 2010

[14] R Panchagnula A Sood N Sharda K Kaur and C KaulldquoDetermination of rifampicin and itsmainmetabolite in plasmaand urine in presence of pyrazinamide and isoniazid by HPLCmethodrdquo Journal of Pharmaceutical and Biomedical Analysisvol 18 no 6 pp 1013ndash1020 1999

[15] L Melo R Queiroz and M Queiroz ldquoAutomated determina-tion of rifampicin in plasma samples by in-tube solid-phasemicroextraction coupled with liquid chromatographyrdquo Journalof Chromatography B vol 879 no 24 pp 2454ndash2458 2011

[16] D Fox R OrsquoConnor P Mallon and G McMahon ldquoSimulta-neous determination of efavirenz rifampicin and its metabolitedesacetyl rifampicin levels in human plasmardquo Journal of Phar-maceutical and Biomedical Analysis vol 56 no 4 pp 785ndash7912011

[17] B Louveau C Fernandez N Zahr et al ldquoDetermination ofrifampicin in human plasma by high-performance liquid chro-matography coupled with ultraviolet detection after automa-tized solid-liquid extractionrdquo Biomedical Chromatography vol30 no 12 pp 2009ndash2015 2016

[18] AAllansonMCotton J Tettey andA Boyter ldquoDeterminationof rifampicin in human plasma and blood spots by highperformance liquid chromatography with UV detection Apotential method for therapeutic drug monitoringrdquo Journal ofPharmaceutical and Biomedical Analysis vol 44 no 4 pp 963ndash969 2007

[19] A Walubo P Smith and P I Folb ldquoComprehensive assayfor pyrazinamide rifampicin and isoniazid with its hydrazinemetabolites in human plasma by column liquid chromatog-raphyrdquo Journal of Chromatography B Biomedical Sciences andApplications vol 658 no 2 pp 391ndash396 1994

[20] M Ishii and H Ogata ldquoDetermination of rifampicin and itsmainmetabolites in human plasma by high-performance liquidchromatographyrdquo Journal of Chromatography B BiomedicalSciences and Applications vol 426 pp 412ndash416 1988

[21] H Seifart P Kruger D Parkin P van Jaarsveld and P DonaldldquoTherapeutic monitoring of antituberculosis drugs by direct in-line extraction on a high-performance liquid chromatographysystemrdquo Journal of Chromatography B Biomedical Sciences andApplications vol 619 no 2 pp 285ndash290 1993

[22] B Ratti R R Parenti A Toselli and L F Zerilli ldquoQuan-titative assay of rifampicin and its main metabolite 25-desacetylrifampicin in human plasma by reversed-phase high-performance liquid chromatographyrdquo Journal of Chromatogra-phy B Biomedical Sciences and Applications vol 225 no 2 pp526ndash531 1981

[23] P J Smith J van Dyk and A Fredericks ldquoDetermination ofrifampicin isoniazid and pyrazinamide by high performanceliquid chromatography after their simultaneous extraction fromplasmardquo International Journal of Tuberculosis and Lung Diseasevol 3 no 11 Suppl 3 Article ID 10593712 pp S325ndashS328 1999

6 International Journal of Analytical Chemistry

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Acknowledgments

This research was financially supported by SlovenianResearch Agency (ARRS) [P1-0189]

References

[1] WHO (WorldHealthOrganization)Global Tuberculosis Report2018 Geneva Switzerland 2018 httpwwwwhointtbpubli-cationsglobal reporttb18 ExecSum web 4Oct18pdfua=1

[2] K Govender J H Adamson and P Owira ldquoThe developmentand validation of a LC-MSMS method for the quantitation ofmetformin rifampicin and isoniazid in rat plasma using HILICchromatographyrdquo Journal of Chromatography B vol 1095 pp127ndash137 2018

[3] R Jogi and S K Tyring ldquoTherapy of nontuberculous mycobac-terial infectionsrdquo Dermatologic Therapy vol 17 no 6 pp 491ndash498 2004

[4] T F Moriarty R Kuehl T Coenye et al ldquoOrthopaedic device-related infection current and future interventions for improvedprevention and treatmentrdquo EFORT Open Reviews vol 1 no 4pp 89ndash99 2016

[5] P Donald J Maritz and A Diacon ldquoThe pharmacokineticsand pharmacodynamics of rifampicin in adults and children inrelation to the dosage recommended for childrenrdquoTuberculosisvol 91 no 3 pp 196ndash207 2011

[6] L Aıt Moussa O El Bouazzi S Serragui D Soussi TananiA Soulaymani and R Soulaymani ldquoRifampicin and isoniazidplasma concentrations in relation to adverse reactions in tuber-culosis patients a retrospective analysisrdquoTherapeutic Advancesin Drug Safety vol 7 no 6 pp 239ndash247 2016

[7] F Fahimi P Tabarsi F Kobarfard et al ldquoIsoniazid rifampicinand pyrazinamide plasma concentrations 2 and 6 h post dosein patients with pulmonary tuberculosisrdquo The InternationalJournal of Tuberculosis and LungDisease vol 17 no 12 pp 1602ndash1606 2013

[8] L Van Tongeren S Nolan V J Cook J M FitzGerald and JC Johnston ldquoTherapeutic drug monitoring in the treatment oftuberculosis a retrospective analysisrdquoThe International Journalof Tuberculosis and LungDisease vol 17 no 2 pp 221ndash224 2013

[9] S Gao Z Wang X Xie et al ldquoRapid and sensitive methodfor simultaneous determination of first-line anti-tuberculosisdrugs in human plasma by HPLC-MSMS Application totherapeutic drug monitoringrdquo Tuberculosis vol 109 pp 28ndash342018

[10] K H Hee J J Seo and L S Lee ldquoDevelopment and validationof liquid chromatography tandem mass spectrometry methodfor simultaneous quantification of first line tuberculosis drugsand metabolites in human plasma and its application in clinicalstudyrdquo Journal of Pharmaceutical and Biomedical Analysis vol102 pp 253ndash260 2015

[11] G Maartens M Boffito and C W Flexner ldquoCompatibility ofnext-generation first-line antiretrovirals with rifampicin-basedantituberculosis therapy in resource limited settingsrdquo CurrentOpinion in HIV and AIDS vol 12 no 4 pp 355ndash358 2017

[12] S Goutal S Auvity T Legrand et al ldquoValidation of a simpleHPLC-UV method for rifampicin determination in plasmaApplication to the study of rifampicin arteriovenous concen-tration gradientrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 123 pp 173ndash178 2016

[13] Z Zhou L Chen P Liu M Shen and F Zou ldquoSimultaneousDetermination of Isoniazid Pyrazinamide Rifampicin andAcetylisoniazid inHuman Plasma byHigh-Performance LiquidChromatographyrdquo Analytical Sciences vol 26 no 11 pp 1133ndash1138 2010

[14] R Panchagnula A Sood N Sharda K Kaur and C KaulldquoDetermination of rifampicin and itsmainmetabolite in plasmaand urine in presence of pyrazinamide and isoniazid by HPLCmethodrdquo Journal of Pharmaceutical and Biomedical Analysisvol 18 no 6 pp 1013ndash1020 1999

[15] L Melo R Queiroz and M Queiroz ldquoAutomated determina-tion of rifampicin in plasma samples by in-tube solid-phasemicroextraction coupled with liquid chromatographyrdquo Journalof Chromatography B vol 879 no 24 pp 2454ndash2458 2011

[16] D Fox R OrsquoConnor P Mallon and G McMahon ldquoSimulta-neous determination of efavirenz rifampicin and its metabolitedesacetyl rifampicin levels in human plasmardquo Journal of Phar-maceutical and Biomedical Analysis vol 56 no 4 pp 785ndash7912011

[17] B Louveau C Fernandez N Zahr et al ldquoDetermination ofrifampicin in human plasma by high-performance liquid chro-matography coupled with ultraviolet detection after automa-tized solid-liquid extractionrdquo Biomedical Chromatography vol30 no 12 pp 2009ndash2015 2016

[18] AAllansonMCotton J Tettey andA Boyter ldquoDeterminationof rifampicin in human plasma and blood spots by highperformance liquid chromatography with UV detection Apotential method for therapeutic drug monitoringrdquo Journal ofPharmaceutical and Biomedical Analysis vol 44 no 4 pp 963ndash969 2007

[19] A Walubo P Smith and P I Folb ldquoComprehensive assayfor pyrazinamide rifampicin and isoniazid with its hydrazinemetabolites in human plasma by column liquid chromatog-raphyrdquo Journal of Chromatography B Biomedical Sciences andApplications vol 658 no 2 pp 391ndash396 1994

[20] M Ishii and H Ogata ldquoDetermination of rifampicin and itsmainmetabolites in human plasma by high-performance liquidchromatographyrdquo Journal of Chromatography B BiomedicalSciences and Applications vol 426 pp 412ndash416 1988

[21] H Seifart P Kruger D Parkin P van Jaarsveld and P DonaldldquoTherapeutic monitoring of antituberculosis drugs by direct in-line extraction on a high-performance liquid chromatographysystemrdquo Journal of Chromatography B Biomedical Sciences andApplications vol 619 no 2 pp 285ndash290 1993

[22] B Ratti R R Parenti A Toselli and L F Zerilli ldquoQuan-titative assay of rifampicin and its main metabolite 25-desacetylrifampicin in human plasma by reversed-phase high-performance liquid chromatographyrdquo Journal of Chromatogra-phy B Biomedical Sciences and Applications vol 225 no 2 pp526ndash531 1981

[23] P J Smith J van Dyk and A Fredericks ldquoDetermination ofrifampicin isoniazid and pyrazinamide by high performanceliquid chromatography after their simultaneous extraction fromplasmardquo International Journal of Tuberculosis and Lung Diseasevol 3 no 11 Suppl 3 Article ID 10593712 pp S325ndashS328 1999

International Journal of Analytical Chemistry 7

[24] M S Balbao C BertucciMM Bergamaschi et al ldquoRifampicindetermination in plasma by stir bar-sorptive extraction and liq-uid chromatographyrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 51 no 5 pp 1078ndash1083 2010

[25] H Kim K Seo H Kim et al ldquoSimple and accurate quanti-tative analysis of 20 anti-tuberculosis drugs in human plasmausing liquid chromatographyndashelectrospray ionizationndashtandemmass spectrometryrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 102 pp 9ndash16 2015

[26] R Hartkoorn S Khoo D Back et al ldquoA rapid and sensitiveHPLCndashMS method for the detection of plasma and cellularrifampicinrdquo Journal of Chromatography B vol 857 no 1 pp 76ndash82 2007

[27] W A Korfmacher J Bloom M I Churchwell E B HansenT A Getek and K T McManus ldquoCharacterization of threerifamycins via electrospray mass spectrometry and hplc-thermospray mass spectrometryrdquo Journal of ChromatographicScience (JCS) vol 31 no 12 pp 498ndash501 1993

[28] L Baietto A DrsquoAvolio F G De Rosa et al ldquoDevelopmentand validation of a simultaneous extraction procedure forHPLC-MSquantification of daptomycin amikacin gentamicinand rifampicin in human plasmardquo Analytical and BioanalyticalChemistry vol 396 no 2 pp 791ndash798 2010

[29] F de Velde J C Alffenaar A M Wessels B Greijdanus andD R Uges ldquoSimultaneous determination of clarithromycinrifampicin and their main metabolites in human plasma byliquid chromatographyndashtandem mass spectrometryrdquo Journal ofChromatography B vol 877 no 18-19 pp 1771ndash1777 2009

[30] L Baietto A Calcagno I Motta et al ldquoA UPLC-MS-MSmethod for the simultaneous quantification of first-line anti-tuberculars in plasma and in PBMCsrdquo Journal of AntimicrobialChemotherapy vol 70 no 9 pp 2572ndash2575 2015

[31] A SrivastavaDWaterhouse AArdrey and SAWard ldquoQuan-tification of rifampicin in humanplasma and cerebrospinal fluidby a highly sensitive and rapid liquid chromatographicndashtandemmass spectrometric methodrdquo Journal of Pharmaceutical andBiomedical Analysis vol 70 pp 523ndash528 2012

[32] J B Prahl M Lundqvist J M Bahl et al ldquoSimultaneousquantification of isoniazid rifampicin ethambutol and pyrazi-namide by liquid chromatographytandemmass spectrometryrdquoAPMIS-Acta Pathologica Microbiologica et Immunologica Scan-dinavica vol 124 no 11 pp 1004ndash1015 2016

[33] S H Song S H Jun K U Park et al ldquoSimultaneousdetermination of first-line anti-tuberculosis drugs and theirmajor metabolic ratios by liquid chromatographytandemmassspectrometryrdquo Rapid Communications in Mass Spectrometryvol 21 no 7 pp 1331ndash1338 2007

[34] Committee for Medicinal Products for Human Use(CHMP) Guideline on Bioanalytical Method ValidationEuropean Medicines Agency (EMA) London UK 2011httpswwwemaeuropaeucontact

[35] B Matuszewski ldquoStandard line slopes as a measure of a relativematrix effect in quantitative HPLCndashMS bioanalysisrdquo Journal ofChromatography B vol 830 no 2 pp 293ndash300 2006

[36] B K Matuszewski M L Constanzer and C M Chavez-EngldquoStrategies for the assessment of matrix effect in quantitativebioanalytical methods based on HPLC-MSMSrdquo AnalyticalChemistry vol 75 no 13 pp 3019ndash3030 2003

[37] C Polson P Sarkar B Incledon V Raguvaran and RGrant ldquoOptimization of protein precipitation based uponeffectiveness of protein removal and ionization effect in liquid

chromatographyndashtandem mass spectrometryrdquo Journal of Chro-matography B vol 785 no 2 pp 263ndash275 2003

[38] D Vu R Koster A Wessels B Greijdanus J Alffenaar and DUges ldquoTroubleshooting carry-over of LCndashMSMS method forrifampicin clarithromycin and metabolites in human plasmardquoJournal of Chromatography B vol 917-918 pp 1ndash4 2013

[39] US FDA Guidance for Industry Bioanalytical MethodValidation 2018 httpwwwfdagovDrugsGuidanceCompli-anceRegulatoryInformationGuidancesdefaulthtm

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