Studies on Antiretroviral Drug Concentrations in Breast Milk: Validation of a Liquid Chromatography–Tandem Mass Spectrometric Method for the …

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  Studying the pharmacokinetics of antiretroviral drugs in breast milk has important implications for the health of both the mother and the infant, particularly in resource-poor countries. Breast milk is a highly complex biological matrix, yet it is
   NUMBER 1 OF 1 AUTHOR QUERIES DATE 8/18/2008JOB NAME FTDARTICLE FTD200483QUERIES FOR AUTHORS Rezk et al THIS QUERY FORM MUST BE RETURNEDWITH ALL PROOFS FOR CORRECTIONS AU1) Please provide the short running head introduced.AU2) Please define ‘‘BM’’ and ‘‘BP’’ in Table 4. Also note that ‘‘NA’’ has been defined as ‘‘not available.’’AU3) Please check whether the level head ‘‘Experimental’’should be changed to ‘‘Experimental Procedures.’’AU4) Please provide corresponding ‘‘g values’’ for ‘‘12,000 rpm’’ in the sentence ‘‘All tubes were . .’’AU5) Please check the use of ‘‘high organic’’ in the sentence ‘‘After this wash . .’’AU6) Please note that the reference citation ‘‘Taylor et al.’’ in the sentence ‘‘Possible matrix . ’’ has been changed to ‘‘Taylor’’ as per the reference list. Please check.AU7) Please define ‘‘MRM’’ in the sentences ‘‘d4T (222.9 / 42.0) . ’’ and ‘‘The first period  . .’’AU8) Please spell out ‘‘CV’’ in the sentence ‘‘At the LLOQ . ’’ and in Table 3.AU9) Please note that the table citation ‘‘Table 5’’ has been changed to ‘‘Table 4.’’ Please check.AU10) Please note that ‘‘PI’’ in the sentence ‘‘To accommodate . ’’ has been spelled out as ‘‘protease inhibitor.’’ Please check.Also spell out ‘‘PFPA’’ in the same sentence.AU11) Please provide the accessed date for Ref. 7.AU12) Please provide article title for Ref. 10.AU13) Please provide author name for Ref. 2.AU14) Please provide volume number and page range for Ref. 5.AU15) We are unable to fix figure 2B within single page. is it ok?  O RIGINAL  A RTICLE Studies on Antiretroviral Drug Concentrations in BreastMilk: Validation of a Liquid Chromatography–TandemMass Spectrometric Method for the Determination of 7 Anti-Human Immunodeficiency virus Medications  Naser L. Rezk, PhD,* Nicole White, BS,* Arlene S. Bridges, PhD,* Mohamed F. Abdel-Megeed, PhD,† Tarek M. Mohamed, PhD,‡ Said S. Moselhy, PhD,§ and Angela D. M. Kashuba, PharmD* Abstract:  Studying the pharmacokinetics of antiretroviral drugs in breast milk has important implications for the health of both themother and the infant, particularly in resource-poor countries. Breast milk is a highly complex biological matrix, yet it is necessary todevelop and validate methods in this matrix, which simultaneouslymeasure multiple analytes, as women may be taking any number of drug combinations to combat human immunodeficiency virusinfection. Here, we report a novel extraction method coupled tohigh-performance liquid chromatography and tandem mass spec-trometry for the accurate, precise, and specific measurement of 7 antiretroviral drugs currently prescribed to infected mothers. Using200  m L of human breast milk, simultaneous quantification of lamivudine (3TC), stavudine (d4T), zidovudine (ZDV), nevirapine(NVP), nelfinavir (NFV), ritonavir, and lopinavir was validated over the range of 10–10,000 ng/mL. Intraday accuracy and precision for all analytes were 99.3% and 5.0 %, respectively. Interday accuracyand precision were 99.4 % and 7.8%, respectively. Cross-assayvalidation with UV detection was performed using clinical breast milk samples, and the results of the 2 assays were in good agreement (  P   = 0.0001, r = 0.97). Breast milk to plasma concentration ratiosfor the different antiretroviral drugs were determined as follows:3TC = 2.96, d4T = 1.73, ZDV = 1.17, NVP = 0.82, and NFV = 0.21. Key Words:  breast milk, HIV, mother-to-child transmission,antiretroviral, nucleoside analogue reverse transcriptase inhibitor,nonnucleoside reverse transcriptase inhibitor, protease inhibitors,LC-MS/MS, chromatography( Ther Drug Monit   2008;0:000–000) INTRODUCTION Women represent 50% of the human immunodeficiencyvirus (HIV)–infected population worldwide. Mother-to-child transmission of HIV is the most significant source of HIVinfection in children younger than 10 years. 1 In resource-poor settings, more than 40% of all HIV-1 transmission frommother to infant occurs during breastfeeding. 2,3 In thesesettings, women must breast-feed their babies due to suchissues as inadequate financial resources, lack of clean water supplies, and stigma. Recent reports also suggest that breastfed  babies of HIV-infected mothers have a significantly lower rateof mortality than formula-fed infants. 4 Therefore, a variety of antiretroviral strategies are currently being investigated for  prevention of HIV transmission during breastfeeding. 3,5 Tosupport this effort, it is necessary to accurately characterize theexposure of antiretrovirals in breast milk to understand boththe pharmacodynamics of HIV replication in breast milk and the pharmacology of antiretrovirals in the breast feedinginfant.Because human milk is a highly complex biologicalmatrix, extracting drugs to accurately quantify total concen-trations is extremely challenging. Milk contains large amountsof proteins and fats, and the proportions of these vary over time. This complexity necessitates a sophisticated extractionmethod for accurate and reproducible results. There area number of methods for isolation of drugs from breast milk described in the literature: dialysis, ultrafiltration, liquid– liquid extraction (LLE), solid phase extraction (SPE), and immunoaffinity extraction. The most successful of those are based on LLE or SPE principles. 6 However, the validation of these analytical methods is incomplete, primarily due todifficulties in achieving the requisite precision and accuracy. 7 For this investigation, we modified our established multiplex liquid chromatography and tandem mass spectrom-etry (LC-MS/MS) method for measuring antiretroviral agentsin blood plasma 8 and combined it with a minor modification toour highly efficient extraction technique for breast milk  9 toexpand the range of analytes that can be measured in 1 assay.This article presents the validation of a high-performanceliquid chromatography and tandem mass spectrometry(HPLC-MS/MS) assay for the simultaneous quantificationof lamivudine (3TC), zidovudine (ZDV), stavudine (d4T),nevirapine (NVP), nelfinavir (NFV), lopinavir (LPV), and  Received for publication April 16, 2008; accepted July 4, 2008.From the *Clinical Pharmacology/Analytical Chemistry Core, Center for AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, NC; Departments of   † Chemistry (Organic Chemistry Section), and  ‡ Chemistry (Biochemistry Section), Faculty of Science, Tanta University,Tanta, Egypt; and §Department of Biochemistry, Faculty of Science, AinShams University, Cairo, Egypt.Correspondence: Naser L. Rezk, PhD, 3324 Kerr Hall, CB 7360, Universityof  North Carolina at Chapel Hill, Chapel Hill, NC 27599 (e-mail:     2008 by Lippincott Williams & Wilkins Ther Drug Monit     Volume 0, Number 0, Month 2008  1  ritonavir (RTV) in human breast milk. This method was used to determine the extent of drug penetration in breast milk compared with blood plasma. EXPERIMENTAL AU3 Materials 3TC, ZDV, d4T, NVP, NFV, RTV, and LPV wereobtained from the NIH AIDS Research & Reference Reagent Program (McKesson HBOC BioServices, Rockville, MD).The internal standard (IS) cimetidine (CMI) was purchased from Sigma Chemical Company (St Louis, MO). HPLC gradewater and methanol were purchased from Fisher Scientific(Norcross, GA). Purified compressed nitrogen gas wasobtained from National Welder’s Supply (Charlotte, NC).Breast milk was purchased from Biological Specialty Corpora-tion (Colmar, PA). Three drug-free pooled human breast milk samples were obtained from multiple healthy nursing mothers,1–6 months after delivery. Instrumentation A Shimadzu solvent delivery system (Columbia, MD)and a Leap HTC Pal thermostated autosampler (Carrboro, NC)connected to an Applied Biosystems API 4000 triple quadru- pole mass spectrometer with a TurboSpray ion source (Applied Biosystems,FosterCity, CA)were usedfor this analyticalstudy.Tuning, operation, integration, and data analysis were per-formed using Analyst software v.1.4.1 (Applied Biosystems). Calibration Standards and QualityControl Samples A stock solution for each anti-HIV drug (analyte) was prepared at a concentration of 1 mg/mL in a specific ratio of HPLC grade water to methanol. ZDVand NVP required 50%methanol, NFV and RTV required 60% methanol, and LPVrequired 70% methanol. The water-soluble compounds 3TCand d4T were dissolved in 100% HPLC water. The master stock solution was prepared as a composite of 7 analytes (1 mLeach), adjusted in a volumetric flask to the final concentrationof 100 m g/mL in 10 mL using 3 mL of 50% methanol in water.This 100  m g/mL stock solution was used to prepare 7 inter-mediate solutions (100, 250, 500, 1000, 5000, 10,000, and 50,000 ng/mL) in 50% methanol in water. The calibrationcurve (10, 25, 50, 100, 500, 1000, 5000, and 10,000 ng/mL)was prepared in drug-free pooled breast milk. Quality control(QC) samples were prepared at 80, 200, 800, 2000, and 8000ng/mL from a separate 100 m g/mL master stock solution.The IS chosen for this method was CMI. CMI 5 mg wasweighed and dissolved in HPLC grade water to achieve a finalconcentration of 1 mg/mL (IS stock solution). To make theworking IS solution, this stock solution was diluted further in HPLC grade water to a final working concentration of 2 m g/mL. Sample Preparation Whole breast milk was transferred to clean cryovialsand stored at   2 70  C until analysis. Before extraction, allsamples, including blank breast milk samples, clinicalsamples, calibration standards, and QC samples, were brought to room temperature.Disruption of fat globules in breast milk with protein precipitation was modified for an mass spectrometry (MS)application from a previously published method. 9 To each 200 m L sample of human breast milk (blank, calibrator, QC, or  patient sample), 100 m L of normal hexane was added to assist in disruption of the fat globules. The tubes were capped and vortex mixed for 1.0 minute, sonicated for 1.0 minute, and then mixed with 125  m L of 15% pentafluoropropionic acid and 700  m L of CMI working IS (2  m g/mL water). All tubeswere  AU4 capped and vortex mixed for 0.5 minutes and centrifuged at 12,000 rpm for 5 minutes at room temperature. The hexanemixed with the fat formed a solid disk at the top of the solutionand allowed the supernatant to be poured into a conditioned SPE column while leaving the disk behind. 9 Solid Phase Extraction SPE columns (1 mL, 100 mg BOND ELUTE-C18;Varian, Harbor City, CA) were placed in a vacuum elutionmanifold (20-SPE system; Waters, Milford, MA). Cartridgeswere conditioned with 1 mL of HPLC grade methanol and equilibratedwith1.0mLof100mMammoniumacetate(pH7).The clear sample supernatant was carefully transferred tothe conditioned SPE column and allowed to pass through thecolumn bed with minimal suction (2–5 mm Hg). The columnwas further washed with 1 mL water. Finally, the analytes wereeluted with 500 m L of 90 % methanol in HPLC water.The sample eluant was evaporated to dryness under a nitrogen stream at 40  C, and the residue was reconstituted with 100 m L of a 50:50 mixture of HPLC grade water:HPLCgrade methanol with 0.1% (vol/vol) formic acid. The resultingsolution was vortexed for 0.5 minutes and centrifuged at 18,000  g   for 3 minutes. The supernatant was transferred to200  m L HPLC polypropylene microvials with a support foot (Agilent Technologies), and 10  m L (for negative mode) or 3 m L (for positive mode) of the supernatant was injected intothe HPLC column. High-Performance Liquid Chromatographyand MS/MS Detection All samples were analyzed by LC-MS/MS using anAquasil C18, 50 3 2.1 mm column, with a 5- m m particle size(Thermo Electron, San Jose, CA), based on a modification of a previously validated sensitive and specific LC-MS/MSmethod for measuring antiretroviral agents in blood plasma. 8 Samples were injected twice, first for negative-mode quanti-fication and second for positive-mode quantification. Mobile Phase for Negative-Mode Analysis For negative ion mode using reverse-phase chromatog-raphy, the flow rate was 0.75 mL/min. Initial conditions for mobile phase A (mobile phase A neg ) were 100% 10 mMammonium acetate. These conditions were held for 0.7 minuteand the eluant was directed to waste. From 0.7 to 2 minutes,the mobile phase composition increased linearly to 95%methanol (mobile phase B neg ), and the eluant directed to themass spectrometer. The flow was held at 95% mobile phaseB neg  until 2.3 minutes. Finally, the column was equilibrated  2  q 2008 Lippincott Williams & Wilkins Rezk et al   Ther Drug Monit     Volume 0, Number 0, Month 2008  with 100% mobile phase A neg . Total runtime, includingequilibration, was 3 minutes per injection. Mobile Phase for Positive-Mode Analysis For positive ion mode using reverse-phase chromatog-raphy, the flow rate was 0.5 mL/min. Initial conditions for mobile phase A (mobile phase A  pos ) were 100% of 0.1%formic acid in water. This was held for 0.7 minutes and theeluant was directed towaste. From 0.7 to 3 minutes, the mobile phase composition increased linearly to 7% of 0.1% formicacid in methanol (mobile phase B  pos ), and the eluant directed to the mass spectrometer. From 3 to 9 minutes, the mobile phase composition increased linearly to 15% mobile phaseB  pos . At 9 minutes, the second period of acquisition started.From 9 to 15 minutes, the mobile phase composition increased linearly to 95% mobile phase B  pos . The flow was held at 95%B  pos  for 30 seconds. After t  AU5  his wash with high organic, thecolumn was equilibrated with 100% A  pos . Total runtime,including equilibration, was 18 minutes per injection. Linearity Linearity was assessed using 3 calibration curves run onseparate days. For validation, each point on the calibrationcurve was run in duplicate (2 separate extractions). After validation, single calibration curves were used for routineclinical sample testing. Standard curve equations for eachanalyte were derived using weighted (1/   x 2 ) linear least-squaresregression analysis. Limits of Detection and QuantificationAccuracy and Precision The lower limit of quantification was defined as theconcentration for which both the relative standard deviation(RSD, also called the coefficient of variation) and the percent deviation from the nominal concentration were less than 20%.Additionally, the lower limit of quantification had to havea signal to noise ratio above 5. The upper limit of quantitationwas defined as the concentration for which both the RSD and the percent deviation from the nominal concentration were lessthan 15%. 10 The lower limit of detection was defined asa signal to noise ratio of 3:1.Accuracy and precision were quantified using 5 con-centrations of QC samples covering the expected clinicalconcentrations: 80, 200, 800, 2000 and 8000 ng/mL. Thecalculated concentrations were compared with these nominalconcentrations. Specificity, Selectivity, Recovery, Matrix Effect,and Stability Six blank human breast milk samples were prepared todetermine the specificity, selectivity, recovery, and matrixeffect of the analytical method. One half of the samples(‘‘blank breast milk,’’ n = 3) were spiked with IS only, and onehalf of the samples (‘‘double blank breast milk,’’ n = 3) werenot spiked with anything. The double blank breast milk samples received 700  m L of IS-free HPLC grade water tomaintain sample composition. With the exception of theaddition of IS, all 6 samples were prepared and analyzed asdescribed above. The results of the double blank breast milk samples were investigated for peaks that might interfere either with the detection of the 7 drugs of interest or with the IS.Peaks with an area . 200 units and a signal to noise ratio . 1:3at the retention time of any of the 7 analytes or of the IS wereconsidered to be interfering.To determine analyte recovery, peak areas of spiked  breast milk (test) replicates were compared with those of spiked neat reconstitution solution (control) replicates of equalnominal concentration. Test and control replicates at200 ng/mL(n = 6 each) and 2000 ng/mL (n = 6 each) were prepared. Test samples were extracted and analyzed as described above.Possible matrix  AU6 effects were investigated according tothe method of Taylor. 11 First, 18 double blank breast milk (test)samples were extracted as described above. At the reconsti-tution step, solutions containing 80 ng/mL, 200 ng/mL (n = 5),2000 ng/mL (n = 5), or 8000 ng/mL (n = 5) of all 7 drugs of interest were used. After analysis, peak areas of these recon-stituted test samples were compared with those of unextracted neat control samples at equal concentrations. Possible matrixeffects were calculated as percent deviation from valuesobserved with standard solutions.Samples were tested for stability in breast milk at roomtemperature for 24 hours, in breast milk at 4  C for 72 hours,and in the reconstitution solution at room temperature in theHPLC autosampler for 24 hours. Analysis of Clinical Samplesand Cross-Validation The clinical samples were obtained from the Breastfeed-ing, Antiretroviral, and Nutrition study. 3,12 The study wasapproved by the Institutional Review Boards at the Universityof North Carolina at Chapel Hill and the National HealthScience Research Committee in Malawi, and all subjects gaveinformed consent before enrolling. This study was designed to evaluate antiretroviral and nutrition interventions toreduce MTCT of HIV during breastfeeding. Clinical sampleswere obtained from women who had been randomized toreceive antiretroviral therapy while breastfeeding. Thesesamples were analyzed on 2 different days using 2 different standard curves. Data in both runs were compared to evaluatereproducibility of the methodology, performed as a requisite part of the validation process to ensure optimal interday LC-MS/MS detection. To confirm the quality of the data obtained  by HPLC-MS/MS, these results were also compared (usingSpearman rho) with those obtained from avalidated LC–diodearray detection assay. 9 RESULTSLC-MS/MS Analysis Negative-Mode Analysis To improve signal intensity of negatively charged drugs,a separate injection using a mobile phase more amenable to producing negative ions was used. d4T (222.9 ! 42.0)  AU7 , ZDV(266.0 ! 223.0), and CMI neg  (IS, 250.3 ! 156.9) were moni-tored (MRM) in negative ion mode using reverse-phasechromatography at 0.75 mL/min. As source fragmentation of ZDV produced apparent d4T, care was taken to ensure AU1 q 2008 Lippincott Williams & Wilkins  3 Ther Drug Monit     Volume 0, Number 0, Month 2008  Studies on Antiretroviral Drug Concentrations in Breast Milk   chromatographic separation between ZDV and d4T. Aninjection of 10  m L of reconstituted sample was required for an adequate d4T signal. Chromatograms for the negativeanalysis mode for the low QC are shown in F1  Figure 1. Positive-Mode Analysis To improve signal intensity of positively charged drugs,a separate injection using a mobile phase more amenable to producing positive ions was used. In addition, a 2-period acquisition was used to reduce duty cycle as a means offurther increasing signal intensity. The first period monitored the tran-sitions (MRM) for 3TC (230.1 ! 112.1), ZDV (268.3 ! 127.1),and CMI  pos  (IS, 253.1 ! 159.3). The second period monitored the transitions for NVP (267.2 ! 226.1), NFV (568.3 ! 467.2),LPV (629.4 ! 447.3), and RTV (721.0 ! 268.0). An injectionof 3 m L was required. Chromatograms for the 2-period anal-ysis are shown in F2  Figure 2 using the low QC. All transitions for the 7 analytes are summarized in T1  Table 1. Assay Validation Linearity and Limitsof Quantification Standard curves over 3 days for d4T and ZDV werequantified using the peak area ratios (analyte peak area to IS neg  peakarea)ofthespikedcalibrationstandards(10–10,000ng/mL).Standard curves over 3 days for 3TC, ZDV, NVP, NFV, RTV,and LPV were quantified using the peak area ratios (analyte peak area to IS  pos  peak area) of the spiked calibration standards(10–10,000 ng/mL). A weighted (1/   x 2 ) least-squares re-gression analyses were used for all analytes.All drugs were linear over 10–10,000 ng/mL. Standard curve data are summarized in  T2 Table 2. The accuracy was between80%and120%forall levels (n= 3)andLLOQ(n=5).At the LLOQ  AU8 , accuracy and CV% were 117%–88% and 1.3%–8.4%, respectively, for all analytes. Calibration standard curves yielded correlation coefficients consistently $ 0.990 for all analytes across 3 different days. Concentrations rangingfrom 10 to 10,000 ng/mL were sufficient to quantify 3 nucle-oside analogue reverse transcriptase inhibitors (3TC, d4T, and ZDV), 1 nonnucleoside reverse transcriptase inhibitor (NVP),and 3 protease inhibitors (NFV, RTV, and LPV). Assay Accuracy and Precision Accuracy and precision of the analytical procedure wereevaluated using 3 concentrations of QC samples assayed in3 replicates on 3 different days. The concentrations of the QCsamples were back-calculated from daily calibration curves.Intra- and interday accuracy and precision for these concen-trations are presented in  T3 Table 3. Accuracy for all analytes waswithin the acceptance limits ( 6 15% of the nominal) specified  by Shah et al. 10 The  # 15% RSD criteria for repeatability FIGURE1. Chromatogram of low QC (80ng/mL) in negative mode. Thefigure illustrates peaks for ZDV (266.0/223.0), d4T (22.9/42.0), and CMI (250.3/156.9) ion pair. 4  q 2008 Lippincott Williams & Wilkins Rezk et al   Ther Drug Monit     Volume 0, Number 0, Month 2008
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