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. Author manuscript; available in PMC: 2021 Aug 1.
Published in final edited form as: Clin Biochem. 2019 Dec 13;82:66–72. doi: 10.1016/j.clinbiochem.2019.12.005

Validation and implementation of an ultrasensitive liquid chromatographic-tandem mass spectrometric (LC-MS/MS) assay for dapivirine quantitation in breast milk

Madhuri Manohar a, Mark A Marzinke a,b,*
PMCID: PMC6944438  NIHMSID: NIHMS1547121  PMID: 31843664

Abstract

Background

The non-nucleoside reverse transcriptase inhibitor dapivirine has been evaluated as a topical microbicidal agent to prevent HIV-1 acquisition. Several clinical trials have evaluated the pharmacokinetics of dapivirine when administered as a 25-mg intravaginal ring. Recent studies have focused on the distribution of dapivirine into breast milk. Drug distribution during lactation and breastfeeding can have implications in terms of infant drug exposure. Thus, sensitive bioanalytical tools are required to characterize the pharmacokinetics of dapivirine in breast milk.

Methods

Whole breast milk was spiked with dapivirine and internal standard. Lipid content was disrupted via pre-treatment with n-hexane, and supernatants were subjected to solid phase extraction. Extracted materials were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. Separation occurred using a Waters BEH C8, 50 × 2.1 mm UPLC column with a 1.7 μm particle size and dapivirine was detected on an API 5000 mass analyzer. Methods were validated in accordance with FDA Bioanalytical Method Validation recommendations.

Results

The analytical method was optimized for dapivirine extraction from breast milk. The analytical measuring range of the assay was 10 – 1000 pg/mL. Calibration curves were generated via weighted linear regression of standards. Intra- and inter-assay precision and accuracy studies demonstrated %CVs ≤ 14.6% and %DEVs ≤ ± 12.7%. Stability and matrix effects studies were also conducted and deemed acceptable. The method was applied to a previously reported phase 1 clinical trial and demonstrated appropriate performance in the quantitation of dapivirine in breast milk samples from lactating women.

Conclusions

An ultrasensitive LC-MS/MS assay has been developed and validated for the quantitation of dapivirine in breast milk. The described method meets validation acceptance criteria and has been applied to a phase 1 clinical trial.

Keywords: Antiretroviral, LC-MS/MS, Breast Milk, Dapivirine, HIV

Introduction

HIV remains a public health concern; currently, the global incidence of HIV is nearly 1.7 million new infections annually, and there are approximately 37 million individuals living with the disease worldwide [1]. The primary modality for the treatment of HIV is the use of antiretroviral therapies (ART), which target the various stages of HIV pathogenic life cycle. Antiretroviral agents can target viral entry, RNA reverse transcription, integration of viral cDNA into the host cell genome, or the assembly and release of viral progeny for propagation. Consequently, these drugs are classified as entry inhibitors, nucleoside/nucleotide (NRTI/NtRTI) and non-nucleoside reverse transcriptase inhibitors (NNRTI), integrase strand transfer inhibitors (INSTI) and protease inhibitors (PI), respectively. There are now more than 25 antiretroviral agents approved by the United States Food and Drug Administration (FDA) for disease management in both single and combinatorial formulations [2]. In addition to their application in HIV management, antiretrovirals have also shown success in preventing HIV, both as pre-exposure prophylactic and treatment as prevention strategies [3–5]. Currently, only oral formulations for HIV treatment and prevention are FDA-approved; however, alternative drug delivery systems, including long-acting injectables, implants, and topical microbicides, have been investigated as potential routes of drug administration [6–10]. Additionally, several new drugs are currently under investigation for HIV treatment and/or prevention.

The NNRTI dapivirine (TMC120) is a highly non-polar diarylpyrimidine analog that non-competitively inhibits the enzyme responsible for reverse transcription of viral RNA into cDNA [11,12]. While the drug has previously been shown to exhibit poor oral bioavailability, it is currently being pursued as a topical agent for HIV prevention in women [13]. In two phase 3 clinical trials, when administered as a 25-mg intravaginal ring, dapivirine was shown to reduce a woman’s relative risk of HIV-1 acquisition as compared to placebo [7,9]. It has been further demonstrated that efficacy is linked to product use, and women who were most adherent to the prevention method were better protected against HIV acquisition [14]. Thus, topical administration of dapivirine as an intravaginal ring is a promising HIV prevention option for women. Safety and pharmacokinetic studies of the product have also been conducted in several sub-populations, including post-menopausal and lactating women; findings show that dapivirine is well tolerated and the drug is consistently released and maintained in plasma and vaginal fluid after product insertion[15,16].

A critical component in the characterization of dapivirine for HIV prevention is understanding the systemic and compartmentalized distribution of the drug at sites of potential seroconversion. We have previously described the development, validation, and implementation of liquid chromatographic-tandem mass spectrometric (LC-MS/MS) assays for dapivirine quantitation in plasma and cervicovaginal fluid to assess systemic and localized drug exposure, respectively. However, as the antiretroviral agent may be used in women who become pregnant and breastfeed post-partum, understanding the pharmacokinetics of dapivirine in breast milk during lactation is an important consideration. Of note, both high fertility rates as well as extended periods of breastfeeding are common in regions with high HIV incidence [17]. It has also been demonstrated that there is increased risk for HIV-infection in women in the post-partum period[17,18]. Thus, it is plausible for women to use dapivirine as a topical microbicide during the post-partum, breastfeeding period. Understanding overall drug distribution and infant exposure are important pharmacokinetic variables to characterize this newer antiretroviral agent.

Breast milk contains proteins, lipids, carbohydrates, as well as nutrients and vitamins. The composition of breast milk changes over time and throughout lactation [19]. During the first few days after delivery, IgA-enriched fluid, called colostrum, is produced, aiding in coating the neonatal gastrointestinal tract. This ultimately matures into breast milk that is produced in the subsequent weeks and months of breastfeeding. Approximately 5 – 7% of the milk is composed of carbohydrates, and approximately 5% is fat. Drugs that may selectively partition into the aqueous portion of milk or into the fat globules can be absorbed by the infant gastrointestinal tract. With regards to antiretroviral agents, drug penetration into the matrix has been well described in other studies evaluating the distribution of both PIs and NRTIs [20]. Based on pharmacokinetic analyses conducted in plasma and breast milk from the Breastfeeding, Antiretroviral and Nutrition (BAN) study, it was demonstrated that water-soluble NRTIs concentrated in breast milk, whereas the more hydrophobic PIs did not accumulate in the matrix 20.

In order to understand the distribution of the novel NNRTI dapivirine into breast milk, we developed and validated an LC-MS/MS assay for drug quantitation. The LC-MS/MS assay was applied to a phase 1 clinical trial evaluating the pharmacokinetics of a dapivirine intravaginal ring used for 14 days in lactating women who were not breastfeeding (MTN-029/IPM 039; ClinicalTrials registration no. NCT02808949) [16]. Also, the assay is currently being employed in an ongoing phase 3 clinical trial evaluating dapivirine drug exposure in breastfeeding mother-infant pairs. Here, we describe the development of an ultrasensitive LC-MS/MS assay for dapivirine quantitation in breast milk, the validation of the assay in accordance with bioanalytical recommendations, as well as its application to a clinical trial.

Methods

Chemicals

Dapivirine (TMC120 (4-[[4-[(2,4,6-trimethylphenyl)-amino]-2-pyrimidinyl]amino]benzonitrile)) was purchased from Toronto Research Chemicals (North York, ON, Canada) in powdered form. The deuterated internal standard 2H4-dapivirine was obtained from the International Partnership for Microbicides (IPM; Silver Spring, MD, USA) in powdered form (Figure 1). Drug-free human breast milk was purchased from BioIVT (Westbury, NY, USA). Optima-grade acetonitrile, methanol, water, 0.1% formic acid in water and certified ACS plus grade ammonium hydroxide were purchased from Fisher Scientific (Fairlawn, NJ, USA). ACS reagent grade formic acid was purchased from Sigma Aldrich (St. Louis, MO, USA). HPLC grade n-Hexane was purchased from Alfa Aesar (Haverhill, MA, USA).

Fig. 1.

Fig. 1.

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Structure of the non-nucleoside reverse transcriptase inhibitor dapivirine (C20H19N5). * denotes positions of deuterium atoms for the deuterated 2H4-dapivirine internal standard.

Preparation of Standards and Quality Controls

Stock solutions of dapivirine and 2H4-dapivirine were independently prepared in acetonitrile at final concentrations of 1 mg/mL and 2.084 mg/mL, respectively. Dapivirine stock solutions were diluted with acetonitrile to generate working solutions of 10000, 1000, 100, 10 and 1 ng/mL; the internal standard was diluted with acetonitrile to a concentration of 5 μg/mL, and to a final working stock of 750 pg/mL. For dapivirine quantitation in breast milk, calibration standards were prepared by spiking drug-free human breast milk with appropriate volumes of working stock solutions. Final breast milk concentrations were as follows: 10, 25, 50, 100, 250, 500, 750, and 1000 pg/mL. Quality control (QC) master and working stocks were independently weighed and prepared in acetonitrile as described above. QC materials were generated by spiking human breast milk with working stock solutions. Concentrations at the lower limit of quantitation (LLOQ), low, mid, and high QCs were 10, 30, 200, and 850 pg/mL, respectively.

Sample preparation

Dapivirine was ultimately isolated from whole breast milk by solid phase extraction. As an initial pre-treatment step, 0.2 mL breast milk was combined with 0.15 mL 2H4-dapivirine in acetonitrile and 0.7 mL n-hexane to disrupt fat globules. Following mixing, samples were incubated at room temperature for 30 min, and then subjected to centrifugation at 2000 x g for 10 min. Supernatants were removed and added to a methanol and water containing 1.5% formic acid pre-conditioned Oasis MCX 96-well solid phase extraction plate (30 mg, 10 μm; Waters Corporation, Milford MA). The mixture was then subjected to vacuum pressure. Retained materials were washed with 1 mL water containing 1.5% formic acid, followed by 1 mL methanol, and ultimately eluted in 0.75 mL acetonitrile containing 10% ammonium hydroxide and evaporated to dryness. Post-evaporation, materials were reconstituted in 0.075 mL of a 1:1 solution mixture (water containing 0.1% formic acid:acetonitrile containing 0.1% formic acid); 0.01 mL was subjected to chromatographic separation and mass spectrometric analysis.

Analyte Separation and Instrument Acquisition Parameters

Chromatographic separation of dapivirine and 2H4-dapivirine was achieved using a Waters BEH C8, 50 × 2.1 mm UPLC column with a 1.7 μm particle size, on a Waters Acquity Ultra-High Performance Liquid Chromatography (UPLC) system (Waters Corporation, Milford, MA, USA). The mobile phase system used for separation consisted of water containing 0.1% formic acid (mobile phase A) and acetonitrile containing 0.1% formic acid (mobile phase B). The chromatographic method included a gradient elution from 30% to 50% mobile phase B over 2.0 min; dapivirine and its internal standard eluted at 1.35 min. The gradient was extended to 95% mobile phase B for 0.2 min, held at 95% mobile phase B for 0.4 min and the re-equilibrated to 30% mobile phase B over 0.1 min. The flow rate throughout the analytical run was 0.5 mL/min, and the analytical run time was 3.0 min.

Detection of dapivirine was performed using an API 5000 triple quadrupole mass analyzer (SCIEX, Redwood City, CA) using an ESI source operated in positive ionization and selective reaction monitoring (SRM) modes. Optimized declustering potential (170 V), collision energy (48 V) and exit potential (9 V) was determined for the analyte. Ion transitions monitored for dapivirine and 2H4-dapivirine were 330.2→158.1 m/z and 334.3→119.0 m/z, respectively.

Data analysis

Data were acquired using Analyst® 1.6.2 software (SCIEX). Calculations for validation assessment, which included precision, accuracy, stability, and matrix effects, were performed using Microsoft Office Excel 2013. Outliers were defined by Grubbs’ Outlier Test.

Method Validation

The LC-MS/MS method for dapivirine quantitation in breast milk was validated in accordance with the Food and Drug Administration (FDA) Guidance for Industry, Bioanalytical Method Validation recommendations. Intra (within) and inter (between) assay precision and accuracy, linearity, selectivity, stability, matrix effects, and carryover were all evaluated. The validated method was also applied to a phase 1, open-label clinical trial evaluating the pharmacokinetics of dapivirine in lactating women when administered as a 25-mg intravaginal ring for 14 days (MTN-029) [16].

Precision and Accuracy

Intra-assay precision was evaluated through the analysis of six samples containing dapivirine in breast milk at the aforementioned LLOQ, low, mid, and high QC concentrations. Inter-assay precision was assessed through the testing of QC materials in replicates of six across three independent runs. Observed means, standard deviations (SDs) and coefficients of variation (%CV) were calculated for all QC levels. Intra- and inter-assay accuracy (also known as recovery) was also evaluated at established QC levels; accuracy was characterized by the percent deviation (%DEV), which is a measure of the difference between mean observed and theoretical QC concentrations divided by the theoretical concentration and multiplied by 100.

Linearity and Dilutional Integrity

Standard curves were calculated using the ratio of the peak area of the analyte to isotopically labeled internal standard using 1/x2 weighted linear regression. Extended linearity was also evaluated through the preparation of breast milk controls containing drug concentrations at three times the upper limit of quantitation (ULOQ; 1000 pg/mL) of the assay. Controls were diluted four-, eight-, and 16-fold with drug-free breast milk to contain 750, 375, and 187.5 pg/mL dapivirine, respectively. Precision and accuracy were assessed by setting theoretical values at calculated diluted concentrations. To evaluate the ability to dilute samples within the primary linearity of the assay, two- and four-fold dilutions of mid and high QCs were prepared; precision and accuracy were evaluated in the same manner as QC materials prepared above the ULOQ.

Stability Challenges

Stability studies were performed under several conditions. Freeze-thaw stability studies were performed using QC materials that were frozen at ≤−70°C and thawed through three cycles. Sample matrix stability was assessed by incubating QC samples at room temperature (21–25°C) for 3 days prior to extraction and analysis. Stability was evaluated by comparing observed values to freshly tested materials. Re-injection stability studies were performed by extracting QC samples and immediately measuring them; samples were then re-analyzed six days after being maintained at 4–8°C. Long-term stability studies were performed by analyzing QC materials at low, mid, and high concentrations stored at ≤70°C for one year against freshly prepared and analyzed quality controls. For the aforementioned challenges, stability was evaluated by the calculation of a percent difference (%DIF), which assesses the difference between a stability-challenged sample and a non-challenged QC specimen divided by the non-challenged prepared QC sample; this result is then multiplied by 100. For all stability challenges, acceptability was determined as a %DIF ≤15% between observed and reference QC value.

Selectivity

Selectivity was performed by analyzing six independent lots of drug-free breast milk. Samples were prepared as previously described, and chromatographic review was performed to evaluate any potentially interfering substances at the expected retention times or ion transitions.

Matrix Effects Characterization

The effects of ion suppression or enhancement were assessed for dapivirine and 2H4-dapivirine following experiments previously described by Matuszewski and colleagues [21]. Un-extracted materials were prepared at low, mid, and high QC concentrations in the absence of the breast milk matrix. Post-extracted materials were prepared by spiking post-extracted breast milk samples with dapivirine at low, mid and high QC concentrations. Pre-extracted sets were analyzed following the previously described sample preparation conditions. Pre- and post-extracted sets were evaluated using independent lots of breast milk (n=6). Raw peak areas for analyte and internal standard were used to determine overall matrix effects (a comparison of post-extracted samples to un-extracted samples), extraction efficiency (a comparison of pre-extracted samples to post-extracted samples) and processing efficiency (a comparison of pre-extracted samples to un-extracted samples).

Analysis of Clinical Trial Samples

The described method was applied to a phase 1 open-labeled trial assessing the pharmacokinetics of dapivirine in human plasma and breastmilk of lactating women when using dapivirine as a 25-mg intravaginal ring for 14 days [16]. Study participants provided written informed consent to participate in the study, and the clinical protocol was approved by the Institutional Review Boards at the two clinical sites (Pittsburgh, PA, and Birmingham, AL). Breast milk was collected within the clinical unit or at home in polypropylene tubes; for home collection, samples were delivered to the clinical unit, where milk was aliquoted to cryovials and stored at ≤−70°C prior to shipment to the testing laboratory. Breast milk was collected prior to intravaginal ring insertion, as well as 3 h, 6 h, 24 h, 7 d, and 14 d after insertion, as well as 48 h after ring removal.

Results

Method development

We previously developed and validated methods for dapivirine quantitation in plasma, tissue, and luminal fluids [22–24]. However, while mass spectrometric transitions and conditions were conserved across analytical matrices, sample processing and chromatographic parameters were optimized for isolation and chromatographic separation of dapivirine quantitation in breast milk, respectively. Dapivirine is a highly hydrophobic diarylpyrimidine analog. Given the high lipid content and presence of fat globules in breast milk, full disruption of the matrix was required to achieve reproducible dapivirine recovery. It was demonstrated that direct extraction of dapivirine from the solid phase extraction plate led to inconsistent isolation of dapivirine from the breast milk, yielding irreproducible results. This was attributed to the presence of lipids and proteins displaced during sample extraction and the inability to wash wells or elute the analyte from the plate. Therefore, sample pre-treatment was required for downstream analysis. Several disruption solvents were evaluated to ensure lysis of all fat globules and to produce a matrix that was amenable for downstream solid phase extraction. Breast milk was combined with methanol, acetonitrile, or n-hexane and incubated for various times to optimize matrix disruption. Signal to noise ratios and peak shapes were compared for the aforementioned approaches, and initial disruption with n-hexane for 30 min yielded negligible background signal and optimal peak shape (data not shown). The pre-treatment of breast milk with n-hexane prior to analysis via solid phase extraction was then utilized for downstream method validation.

Due to the hydrophobicity of dapivirine, an octyl column was employed for separation, similar to our approaches with other matrices, including plasma[22]. However, as the targeted LLOQ of dapivirine (10 pg/mL) in breast milk was two-fold lower as compared to plasma, a prolonged hold at 95% mobile phase B was added to the analytical run time. The addition of this hold minimized carryover, and ultimately resulted in a more streamlined chromatographic method. The modified chromatography allowed for additional throughput without compromising result integrity.

Method Validation

Precision, accuracy, and linearity assessment

Intra- and inter-assay precision and accuracy of dapivirine measurements in breast milk were assessed by analyzing controls prepared at the aforementioned described LLOQ, low, mid, and high QC concentrations. Intra-and inter-assay precision and accuracy ranges from 2.08% to 14.6%, and −11.1% to 12.7%, respectively. Observed values demonstrate %CVs and %DEVs ≤± 20% at the LLOQ and ≤± 15% at other QC levels, and are within the specifications recommended by the FDA Guidelines, Guidance for Industry [25]. A comprehensive summary of intra- and inter-assay precision and accuracy is described in Table 1. Representative chromatograms for dapivirine at the LLOQ and its isotopically labeled are shown in Figure 2.

Table 1.

Intra- and inter-assay precision accuracy of dapivirine quantitation in breast milk.

QC Level Intra - Assay Precision and Accuracya
 Inter - Assay Precision and Accuracyb
Mean [pg/mL] SD [pg/mL] %CV %DEV Mean [pg/mL] SD [pg/mL] %CV %DEV
LLOQ [10 pg/mL] 9.70 0.422 4.36 −3.12 9.59 1.11 11.6 −4.06
Low [30 pg/mL] 28.7 1.71 5.94 −4.22 30.4 3.37 11.1 1.19
Mid [200 pg/mL] 200 6.28 3.14 0.167 203 6.24 3.08 1.44
High [850 pg/mL] 923 25.2 2.73 8.53 937 27.1 2.89 10.3
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b

n=17 for LLOQ and mid QC levels; n=18 for low and high QC levels; inter-assay precision and accuracy is from three analytical runs

a

n=6 for each level of QC; representative data from a single analytical run

Fig. 2.

Fig. 2.

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Representative chromatogram of (a) dapivirine and (b) 2H4-dapivirine extracted from breast milk. The concentration of dapivirine is 10 pg/mL, the lower limit of quantitation of the assay. The retention time for both drug and internal standard is 1.35 min.

Linearity and Dilutional Analysis

Breast milk calibration curves were generated to encompass the primary linearity of the assay. Calibration curves were generated using 1/x2 weighted linear regression. Precision, and accuracy, was performed for calibrators across three analytical runs. Using acceptance criteria described for QCs, performance of standards was acceptable across the analytical range of the assay for all analytes. A representative calibration curve for dapivirine is shown in Figure 3. Dilutional studies were performed both within the analytical measuring range as well as above the primary linearity of the assay. Dapivirine QC samples prepared at 200, 850, and 3000 pg/mL were diluted with blank breast milk to yield dilution controls at final concentrations of 50, 100, 187.5, 212.5, 375, 425, and 750 pg/mL. Spiking studies demonstrate that breast milk samples may be diluted for dapivirine quantitation, and precision and accuracy studies demonstrate %CVs and %DEVs ≤ 4.78% and ≤ ±8.58%, respectively.

Fig. 3.

Fig. 3.

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Representative calibration curve of dapivirine for quantitation in breast milk. The curve is fit to matrix-based calibrators with linear regression using 1/x2 weighing. The slope of the line is y = 0.00436x + 0.014, with an r2 value of 0.9989.

Stability Challenges

Dapivirine stability in breast milk was assessed under a variety of conditions, including re-injection stability, sample matrix stability, and three freeze-thaw cycles. Dapivirine stability was assessed by comparing stability-challenged QCs against freshly prepared or analyzed QCs. In accordance with FDA recommendations, a %DIF ≤ ±15% was considered acceptable when compared to a freshly prepared or tested sample. Injection matrix stability, which assessed dapivirine stability post-extraction and maintained in reconstitution buffer for six days, showed a %DIF ≤ ±11.2%. Sample matrix studies, in which dapivirine-spiked breast milk was maintained at room temperature for 3 days, yielded a %DIF ≤ ±11.3%. Dapivirine was also stable in breast milk for three freeze-thaw cycles. Injection matrix, sample matrix, and freeze-thaw stability study results are described in Table 2. Further, long-term stability studies showed that dapivirine is stable in breast milk stored at ≤−70°C for at least one year; the %DIF when compared to freshly prepared and tested QCs was ≤ ±9.88%.

Table 2.

Stability challenges for dapivirine in breast milk.

QC Level Freeze-Thaw Stability (n=6) (<−70°C, 3 cycles)
 Sample Matrix Stability (n=6) (21–25°C, 3 days)
 Injection Matrix Stability (n=6) (4–8°C, 6 days)
Control Mean (Pg/mL) (%CV) Challenged Mean (pg/mL) (%CV) %DIF Control Mean (Pg/mL) (%CV) Challenged Mean (pg/mL) (%CV) %DIF Control Mean (Pg/mL) (%CV) Challenged Mean (pg/mL) (%CV) %DIF
Low QC [30 pg/mL] 29.3 (4.5) 30.2 (5.8) 2.90 29.3 (4.5) 30.8 (5.0) 5.17 27.6 (4.6) 30.7 (6.5) 11.2
Mid QC [200 pg/mL] 209 (2.1) 199 (1.6) −4.84 209 (2.1) 200 (3.3) −4.04 200 (9.1) 208 (7.8) 4.01
High QC [850 pg/mL] 932 (2.7) 884 (2.2) −5.07 932 (2.7) 827 (5.3) −11.3 943 (2.8) 860 (4.9) −8.83
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Selectivity and matrix effects

Assay selectivity was evaluated by analyzing 6 blank lots of breast milk. Based on peak area, extracted ion currents showed minimal background noise in SRM transitions for dapivirine and 2H4-dapivirine (<20% and <5%, respectively; data not shown). Matrix effects were evaluated quantitatively by comparing average peak areas of post-extracted breast milk samples to unextracted specimens. Dapivirine showed moderate ion suppression; the average ion suppression for dapivirine and 2H4-dapivirine were 19.1% and 18.3%, respectively. Although moderate matrix effects were observed, relative matrix effects were negligible. In addition to matrix effects, overall recovery and processing efficiencies were assessed by comparing ratios of pre-extracted to post-extracted or un-extracted sample sets, respectively. Overall efficiencies were comparable between dapivirine and its internal standard, and a summary of these results were included in Table 3. To further evaluate reproducibility of dapivirine across matrix lots, each set of quality controls in the un-extracted, pre-extracted and post-extracted preparations were assigned as calibrators, and the slope of the line was recorded. The %CVs across the three preparation conditions was ≤2.70% and the %DIF was ≤±1.80%.

Table 3.

Matrix effects, recovery efficiency, and processing efficiency of dapivirine in breast milk.

QC Level Matrix Effects (ME; %)a
 Recovery Efficiency (RE; %)b
 Processing Efficiency (PE; %)c
Analyte Internal Standard Analyte Internal Standard Analyte Internal Standard
Low QC [30 pg/mL] 86.6 84.1 69.9 67.6 60.6 56.8
Mid QC [200 pg/mL] 79.0 81.4 67.8 67.4 53.5 54.9
High QC [850 pg/mL] 77.3 79.6 73.3 73.5 56.6 58.5
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a

ME% = Peak area of (post-extracted samples/un-extracted samples) * 100

b

RE% = Peak area of (pre-extracted samples/post-extracted samples) * 100

c

PE% = Peak area of (pre-extracted samples/un-extracted samples) * 100

Application to clinical trials samples

To evaluate the fidelity of the described LC-MS/MS assay, the method was applied to a phase 1, open label, clinical trial to evaluate the pharmacokinetics of dapivirine transfer into breast milk of lactating women using a 25-mg dapivirine intravaginal ring [16]. The study was registered at ClinicalTrials.gov under registration no. NCT02808949. Breast milk from 16 women were evaluated over 14 days of intravaginal ring use. Dapivirine was quantifiable in all breast milk samples from study participants. 2% (2/98) of breast milk samples evaluated were above the primary linearity of the assay and required dilutional analysis. Aggregate median drug concentration across all study visits was 300 pg/mL (IQR 166.5–512 pg/mL). Drug concentrations were quantifiable 3 h post-ring insertion, and increased until ring-removal. The lowest quantifiable measurement was 11.9 pg/mL. Post-ring insertion, maximum drug concentrations (Cmax) were higher in breast milk as compared to blood plasma (breast milk: plasma ratio of 2.2). A full description of the study findings, including pharmacokinetic parameters, may be found in Noguchi et al. [16].

Discussion

The described LC-MS/MS method has been developed and validated to facilitate dapivirine quantitation in breast milk. The aforementioned assay is sensitive, specific, and has a streamlined analytical run time of 3.0 min. Specimen preparation has been optimized to maximize dapivirine recovery from whole breast milk. Thus, the described assay is appropriate to support clinical trials and has been applied to a phase 1, open-label clinical study focused on dapivirine compartmentalization during lactation.

Drug measurement in alternative specimen sources should be optimized for both the compound’s target analytical measuring range as well as extraction from the matrix. We have previously described optimized methods for dapivirine quantitation in plasma, as well as alternative specimen sources such as cervicovaginal fluid and rectal, cervical, and vaginal tissue [22–24]. LC-MS/MS assays were developed and optimized to take into account the route of drug administration, expected drug concentrations, and matrix composition. Each of these criteria was considered during the development of the described LC-MS/MS assay.

Dapivirine is an antiretroviral agent that is currently being pursued as a topical microbicide, and may be administered vaginally or rectally [7,9,26]. Following topical administration, drug concentrations were expected to be higher in proximal environments, such as the vaginal epithelium and cervicovaginal fluid, as compared to plasma or breast milk. Therefore, when developing an assay to measure dapivirine in breast milk, we aimed for an analytical measuring range that was comparable to plasma. Due to the lack of data on dapivirine distribution into breast milk, we validated an ultrasensitive assay with a LLOQ of 10 pg/mL to ensure quantifiable antiretroviral drug measurement in the matrix. The LLOQ for dapivirine measurement in breast milk is two-fold lower than our previously established assay for drug measurement in plasma. Lastly, breast milk has intermittently high lipid content during lactation, and thus the composition of the specimen source differs from plasma [19]. Dapivirine is a lipophilic molecule that may accumulate in fat depots or globules; in order to measure total dapivirine concentrations in breast milk, a method to disrupt fat globules was required. Therefore, n-hexane was used to facilitate complete disruption of the breast milk matrix prior to solid phase extraction of dapivirine for downstream quantitation. The specimen processing event resulted in a mean recovery efficiency from breast milk of 70.3% (Table 3). Thus, based on both drug and matrix characteristics, the bioanalytical assay was optimized for dapivirine quantitation in breast milk. This was capitulated via its successful application to clinical trials samples.

Two phase 3, randomized, placebo-controlled trials have been completed to evaluate the efficacy of a 25-mg intravaginal ring for HIV prevention [7,9]. In these studies, dapivirine was shown to reduce a woman’s relative risk of HIV acquisition, and its protective capacity was linked to continued product use. Data from these trials demonstrated permeation of the drug into systemic circulation. Additional studies also demonstrated the measurement of high drug concentrations in vaginal tissue and fluids [15,16]. As continued dapivirine release over time can lead to drug compartmentalization into plasma, and as recently described, in breast milk, the extent of infant exposure during breastfeeding should be considered. Drug exposure to nursing infants is currently being evaluated in a phase 3b clinical trial evaluating breastfeeding mothers.

Conclusions

An ultrasensitive LC-MS/MS method has been developed, validated and implemented for the quantitation of dapivirine in breast milk. The method utilizes the same mass spectrometric parameters employed for other matrices, and exploits a sample preparation schema optimized for extraction and measurement from breast milk. The methods described herein highlights a roadmap for the development and optimization of bioanalytical methods in alternative specimen sources.

Highlights.

Development of an ultrasensitive LC-MS/MS method for dapivirine quantitation in breast milk

Validation of a dapivirine breast milk assay in accordance with bioanalytical guidelines

In vivo measurement dapivirine in an open-label phase I clinical trial focused on dapivirine distribution during lactation

Acknowledgements

The MTN-029 phase 1 trial was designed and implemented by the Microbicide Trials Network (MTN) funded by the National Institute of Allergy and Infectious Diseases through individual grants (grants number UM1AI068633, UM1AI068615, and UM1AI106707), with co-funding from the Eunice Kennedy Shriver National Institute of Child Health and Human Development and the National Institute of Mental Health, all components of the U.S. National Institutes of Health (NIH). This work presented here was funded by NIH grant UM1AI106707. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Footnotes

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References

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