HIV combination drug therapies: development and validation of an LC–MS-MS method for simultaneous quantitation of abacavir, dolutegravir, and lamivudine in rat matrices in support of toxicology studies

Abstract

Abacavir (ABC), Dolutegravir (DTG), and Lamivudine (3TC) are part of a fixed-dose combination medication for the treatment of HIV. The three drugs offer different but complementary mechanisms of action by inhibiting reverse transcriptase and integrase, and ultimately inhibiting HIV replication. Due to the lack of information regarding long-term safety following in utero exposure, we are evaluating potential toxicity to offspring following in utero exposure to this combination therapy in Hsd:Sprague Dawley®SD® (HSD) rats, including cardiovascular toxicity and neurotoxicity. Generating internal exposure data are integral to putting toxicological findings into context. The objective of this work was to develop and validate a method to simultaneously quantitate ABC, DTG, and 3TC in rat matrices following exposure to this combination. The method used protein precipitation of plasma, fetal, placental, brain, or heart homogenate, followed by ultra-performance liquid chromatography-tandem mass spectrometry. In adult Sprague Dawley rat plasma, the method was linear (r ≥ 0.99) over the range 10/15/5 to 10,000/15,000/5000 ng/mL for ABC/DTG/3TC and recovery was ≥92% for all three analytes at all concentration levels. The limits of detection were 2.22, 3.69, and 0.978 ng/mL for ABC, DTG, and 3TC, respectively. Intra- and inter-day precision was ≤8.7% relative standard deviation (RSD), and relative error (RE) ≤±12.0% for standards prepared at 20/30/10, 400/600/200, and 5000/7500/2500 ng/mL. Matrix standards as high as 40/60/20 µg/mL could be diluted into the calibration range (RE≤±3.5% and RSD ≤2.4%). The method was evaluated for HSD rat maternal plasma and fetal, placental, brain, and heart homogenates (mean RE ≤±15.0% and RSD ≤8.6%). Analyte stability was demonstrated in extracted plasma for 2 days at different temperatures, and in various matrices stored at −80°C for at least 32 days (80–113% of Day 0 concentrations). These data demonstrate that this simple and efficient method is suitable for quantitation of ABC, DTG, and 3TC in rat matrices generated from toxicology studies. The method can easily be adapted to other biological matrices and species (e.g. human).

Introduction

Abacavir (ABC), dolutegravir (DTG), and lamivudine (3TC) are drugs in a fixed-dose combination medication sold under the brand name Triumeq® for the treatment of HIV infection in adults and children. DTG, a second-generation integrase-inhibitor, plus the two nucleoside reverse transcriptase inhibitors, offers different but complementary mechanisms of action that ultimately inhibit HIV replication. Summaries available from preclinical and clinical studies indicate that high exposure to ABC can result in an increase in the incidence of tumors in rats and mice [1]. All three drugs exhibit high placental transfer and are excreted in the breast milk [2–4], and ABC was shown to cause embryonic and fetal toxicity, including malformations and increased incidence of stillbirth in rats [5]. An increased risk for cardiovascular diseases and myocardial infarction has also been reported for ABC [6]. DTG was shown to cause an increase in neural-tube defects in humans [7], and evidence of DTG-related neurotoxicity, especially when combined with ABC, has been reported [8].

Due to a lack of information following exposure to the combination therapy, we are investigating the potential toxicity to offspring following in utero exposure of Hsd:Sprague Dawley®SD® (HSD) rats, including cardiovascular toxicity and neurotoxicity. As such, a method to simultaneously quantitate all three drugs in various complex matrix types is needed. Internal exposure data are critical for interpreting toxicity data and translating animal toxicity findings to predict human outcomes. The objective of this work was to develop and validate an analytical method to simultaneously quantitate ABC, DTG, and 3TC in rodent plasma, fetus, placenta, brain, and heart tissues in support of planned toxicity studies. Published methods for the analysis of ABC, DTG, and 3TC in plasma have used liquid chromatography–tandem mass spectrometry (LC–MS-MS) [9–17], but these methods often required complex sample extraction (e.g. solid phase extraction), large sample volume (e.g. 200 µL), or only one of the drugs of interest was quantified in a single analysis. The ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS-MS) method described herein allows simultaneous quantitation of all three analytes in several complex HSD rat matrices using a small sample size (i.e. 50 µL). The method can be easily adapted to measure these analytes in other species and/or matrices.

Experimental

Materials

Abacavir sulfate (lot ASE-1 710 002) and lamivudine (lot 18-4045-807) were purchased from Tecoland Corporation (Irvine, CA), dolutegravir sodium (lot 0122803) was from Gojira Fine Chemicals (Bedford Heights, OH), and internal standards (IS) abacavir-d₄ (lot 2-YJK-108-1), dolutegravir-d₄ (lot 8-GBH-101-2), and lamivudine-¹⁵N₂,¹³C (lot 8-MMS-176-4) were from Toronto Research Chemicals (Ontario, Canada). Structures of the analytes and ISs are shown in Fig. 1. Adult male Sprague Dawley (SD) rat plasma, female SD rat placenta, and male and female SD rat hearts used for analytical method validation were purchased from BioIVT (Hicksville, NY). HSD rat gestational day (GD) 18 maternal plasma and fetuses, post-natal day (PND) 4 pup hearts, and PND 23 brains were obtained internally from Southern Research (Birmingham, AL). All other reagents were purchased from commercial sources.

Figure 1.

Structures of ABC Sulfate, DTG Sodium, and 3TC and ISs.

Method validation approach

UPLC–MS-MS conditions were optimized with respect to chromatographic performance, sensitivity, selectivity, and dynamic range. The optimized method was validated in adult male SD rat plasma according to the guidelines of the FDA [18]. Calibration standards and quality control (QC) samples were in replicates over multiple analysis days to demonstrate linearity, selectivity, sensitivity, recovery, intra- and inter-day precision and accuracy, and reproducibility over the desired concentration ranges. In anticipation of study samples from animals dosed with a broad range of ABC/DTG/3TC combination, the calibration range was 10/15/5 to 10,000/15,000/5000 ng/mL for ABC/DTG/3TC in plasma. Selectivity was assessed by analyzing six matrix blanks (without IS) and six method blanks (matrix blanks with IS) for background interferences at the analyte retention times. Accuracy was evaluated as percent relative error (%RE); precision was evaluated as percent relative standard deviation (%RSD), also known as coefficient of variation. The lower limit of quantitation (LLOQ) was the lowest matrix standard that could be accurately quantitated within 20% of the nominal value and at which six replicates could be reproduced within 20% RSD. The limit of detection (LOD) was defined as three times the standard deviation of the LLOQ response expressed as concentration [19]. Recovery was expressed as the percentage of the analyte response (peak area ratio) for matrix standards fortified before and after extraction. Intra-day precision and accuracy were evaluated at three concentration levels in plasma using three independent QC samples prepared on the same day. Inter-day precision and accuracy were evaluated at the same three concentration levels, using multiple sets of independent QC samples prepared and analyzed over multiple analysis days. Carryover was evaluated using three method blanks run immediately following the highest calibration standard for ABC, DTG, and 3TC. The response in each of these blanks was compared to the mean response for the method blanks run at the beginning of the sample set. Instrument drift (reinjection reproducibility) was assessed by preparing a set of calibration standards and running them at the beginning and end of the sample set, with ∼40 samples run in-between. The first analysis of the matrix calibration standards was used for calibration; the second, re-injected set was analyzed as an independent QC set to assess drift. The method was evaluated in secondary matrices by preparing blanks and QC samples in secondary matrix and quantitating using a calibration curve prepared in primary matrix (male SD rat plasma). The secondary matrices were GD18 maternal plasma, fetus, placenta, brain, and heart.

Preparation of stock solutions

Three replicate standard stock solutions (A, B, and C) of each individual analyte were prepared in methanol at one concentration: 0.5 mg/mL ABC, 0.3 mg/mL DTG, and 0.5 mg/mL 3TC. Using alternate stock solutions (A and B), eight combined spiking solutions were prepared over the range 50/75/25–50,000/75,000/25,000 ng/mL ABC/DTG/3TC by diluting in methanol. The third stock solution (C) was used to prepare three additional combined spiking solutions at 100/150/50, 2000/3000/1000, and 25,000/37,500/12,500 ng/mL, to be used for preparation of QC samples. The combined IS contained 500 ng/mL ABC-d₄, 5000 ng/mL DTG-d₄, and 2000 ng/mL 3TC-¹⁵N₂,¹³C in methanol. All solutions were stored refrigerated when not in use.

Preparation of plasma calibration standards and QC samples

Eight matrix calibration standards (10/15/5, 20/30/10, 100/150/50, 400/600/200, 1000/1500/500, 2000/3000/1000, 5000/7500/2500, and 10,000/15,000/5000 ng/mL ABC/DTG/3TC) and QC samples (20/30/10, 400/600/200, and 5000/7500/2500 ng/mL) were prepared by spiking 50 µL of male SD rat plasma with 10 µL of the appropriate spiking solution and 10 µL of IS. For method blanks, 10 µL of methanol was substituted for the spiking solution. To each sample 400 µL of acetonitrile were added, the vials mixed briefly, and centrifuged at ∼5°C at ∼20,000 × g for 10 min. A 30 µL aliquot of each supernatant was diluted with 270 µL of Mobile Phase A (0.1% formic acid in water) and vortexed briefly prior to the analysis by UPLC–MS-MS as described below. To estimate extraction recovery, extracts of blanks were spiked with the analytes post-extraction (i.e. to represent 100% recovery). These post-extraction matrix standards were prepared at the same concentrations and in the same manner as the matrix standards, except that 10 µL of spiking solution and 10 µL of IS were added to the supernatant after extraction, rather than before.

Tissue homogenization

All tissue homogenates were prepared using 3 mL water added per 1 g tissue. Fetus, placenta, brain, and adult heart tissues were homogenized using a Brinkmann polytron (Kinematica AG, Malters, Switzerland), ∼30 s initially to break up the tissue, then again after the water was added until the samples were completely homogenized. Due to their small size, pup hearts were homogenized with a Geno/Grinder (SPEX, Metuchen, NJ), using 1.5 mm stainless steel beads at twice the weight of each tissue. Each sample was homogenized at 1750 rpm for 1 min, enough times to break up the tissue, then again after the water was added until the samples were completely homogenized. Concentrations in all tissue homogenate (ng/mL) were therefore converted to ng/g tissue using a dilution factor of four.

Secondary matrix evaluation

The secondary matrices evaluated were HSD rat maternal plasma, fetal, placental, brain, and heart homogenates. Six replicate matrix samples were prepared at 20/30/10 ng/mL ABC/DTG/3TC in each secondary matrix and analyzed using a primary (adult male SD rat plasma) calibration curve. Six matrix blanks (no IS) and six method blanks (with IS) were prepared in each secondary matrix to evaluate selectivity.

Dilution verification

In anticipation of samples with concentrations higher than the validated range, a dilution verification was conducted to demonstrate that samples with ABC, DTG, or 3TC concentrations greater than the upper limit of the validated range could be quantified accurately by diluting into the validated range. Triplicate matrix samples were prepared at 40/60/20 µg/mL for ABC/DTG/3TC in male SD rat plasma. The extracts were diluted by a factor of 10 with extracted blank matrix containing the IS prior to analysis.

Stability

Stability of ABC, DTG, and 3TC in extracted samples during the analysis period was determined by preparing plasma QC samples at three concentration levels (20/30/10, 400/600/200, and 5000/7500/2500 ng/mL), extracting them, and then storing the extracts at ambient and refrigerated conditions for 2 days. To determine analyte stability during study sample storage conditions and duration, QC samples were prepared in study sample matrices (secondary matrices) at two concentration levels (20/30/10 and 5000/7500/2500 ng/mL) and stored at −80°C for at least 30 days. The stability samples were analyzed according to the validated method. Determined concentrations were compared to the mean concentration of freshly prepared Day 0 samples, expressed as percent of Day 0.

LC–MS-MS analysis and analyte quantitation

The UPLC–MS-MS system consisted of a Waters (Milford, MA) Acquity UPLC coupled to a 4000 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer with a TurboIonSpray (electrospray) source (Sciex, Framingham, MA). Chromatographic separation was performed using a Waters Acquity UPLC BEH C18 column (50 mm × 2.1 mm i.d., 1.7 μm particle size) and a Waters Acquity UPLC BEH C18 guard column (5 mm × 2.1 mm i.d, 1.7 μm particle size). A 7.5 µL volume of sample was injected onto the column maintained at 30°C, and elution was achieved using a binary gradient and a flow rate of 0.3 mL/min. The mobile phases consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in methanol. The gradient was 0% B for 1 min, ramp to 100% B in 4 min, hold at 100% B for 3 min. The total run time was 10 min. The electrospray ion source was operated in positive ion mode with an ionspray voltage of 3500 V. The mass spectrometer source temperature was 750°C and the curtain gas was 10 psi. The nebulizer gas was 30 psi, the heater gas was 80 psi, and the interface heater was on. The multiple reaction monitoring ion transitions and optimized compound-dependent parameters for the analytes are shown in Table 1. Analyst software version 1.6.2 (Sciex) was used for data acquisition and analysis.

Table 1.

Instrument parameters for analysis of ABC, DTG, and 3TC

Compound	RT (min)	Q1 (Da)	Q3 (Da)	DP (V)a	EP (V)b	CE (V)c	CXP (V)d
3TC	2.3	230.0	112.1	46	10	17	4
3TC-15N2,13C	2.3	233.0	115.1	85	10	17	15
ABC	3.0	287.1	191.2	100	10	35	10
ABC-d4	3.0	291.1	195.3	100	10	37	10
DTG	4.4	420.0	277.2	101	10	37	16
DTG-d4	4.4	424.1	279.2	121	10	37	16

^aDP = declustering potential.

^bEP = entrance potential.

^cCE = collision energy.

^dCXP = collision cell exit potential.

For ABC, DTG, and 3TC, the ratio of the analyte peak area to the corresponding IS peak area was calculated. Weighted (1/x²) linear regression equations were computed by plotting the peak area ratio for at least six matrix standards against corresponding concentration. The concentration of each analyte in the standards and QC samples were determined from the peak area ratio, linear regression equation, and dilution factor when applicable. Plasma, fetal homogenate, placental homogenate, brain homogenate, and heart homogenate concentrations are given as ng analyte per mL matrix; concentrations in the fetus, placenta, brain, and heart were converted to ng analyte per g tissue using a dilution factor of four.

Results and Discussion

Method development and validation

The UPLC–MS-MS method to simultaneously quantitate ABC, DTG, and 3TC in rat plasma, fetus, brain, placenta, and heart was developed using a standard C18 column, mobile phases consisting of water and methanol, and electrospray ionization in positive ion mode. A sample diluent containing at least 90% aqueous solution was needed to achieve suitable chromatographic peak shape for 3TC. Therefore, in order to use a simple acetonitrile protein crash, the supernatant was diluted with Mobile Phase A (0.1% formic acid in water) to achieve a 90/10 water/acetonitrile composition. Calibration curves were linear using 50 μL of male rat plasma from ∼ 2 to 2000 ng/mL for each analyte; the ranges were then adjusted and extended to accommodate the expected concentrations and ratios from toxicology studies. MS source parameters were optimized for DTG, which was the least responsive analyte. However, in order to achieve a linear response over the target calibration range for ABC, compound-specific parameters were detuned for lower response. Labeled ABC, DTG, and 3TC were commercially available, so they were incorporated as the respective ISs.

Method validation data are summarized in Table 2. The quantitation range of the validated method is 10–10,000 ng/mL for ABC, 15–15,000 ng/mL for DTG, and 5–5000 ng/mL for 3TC, with LODs of 2.22, 3.69, and 0.978 ng/mL, respectively. No matrix interferences were detected in the matrix blanks at the retention times of the analytes, demonstrating adequate selectivity. Representative chromatograms for the analytes in primary matrix (adult male SD rat plasma) are presented in Fig. 2. Sensitivity could certainly be improved significantly if needed; however, the ranges were shown to be optimal for planned toxicology studies. For ABC, the optimal collision energy and declustering potential were found to be lower, but these parameters were detuned such that a higher calibration curve could be used that would correspond to the anticipated concentrations in study samples. Furthermore, since an aqueous composition is needed for the final sample diluent, the acetonitrile extracts could be dried down and reconstituted rather than diluted prior to sample injection. Other anti-HIV drugs could also be analyzed using this method. For example, in our laboratory we have developed and validated a method for simultaneous quantitation of emtricitabine (FTC), tenofovir (TEN; administered as a prodrug of tenofovir disoproxil fumarate or tenofovir alafenamide fumarate), and efavirenz (EFV) in mouse tissues using the same column, mobile phases, and gradient (manuscript in preparation). The analytes elute at 2.2 min (TEN) and 2.6 min (FTC), which can be analyzed with 3TC (2.3 min), ABC (3.0 min), and DTG (4.4 min), before switching the instrument to negative ion mode for EFV (5.1 min). Sample extraction using either acetonitrile or 0.5% formic acid in methanol could be used, with the final extract being diluted or reconstituted to a final ≥ 90% aqueous composition.

Table 2.

Method validation data for ABC, DTG, and 3TC

Validation parameter	ABC	DTG	3TC
Adult male SD rat plasma
Matrix concentration range (ng/mL)	10.0–10,000	15.0–15,000	5.00–5000
LOD (ng/mL)a	2.22	3.69	0.978
LOQ (ng/mL)b	10.0	15.0	5.00
Correlation coefficient (r)	≥0.99	≥0.99	≥0.99
Recovery (%)	92.0–110	97.2–115	102–106
Precision and accuracyc
Intra-day precision (%RSD)d	≤6.8	≤5.1	≤6.0
Intra-day accuracy (Mean %RE)e	≤ ±5.9	≤ ±5.3	≤ ±12.0
Inter-day precision (%RSD)	≤8.7	≤7.6	≤7.4
Inter-day accuracy (Mean %RE)	≤ ±6.9	≤ ±2.8	≤ ±6.9
Dilution verificationf
Precision (%RSD)	2.4	1.4	2.0
Accuracy (Mean %RE)	1.8	1.8	3.5
Secondary matrix evaluationg
GD18 maternal rat plasma
Precision (%RSD)	8.6	5.9	4.2
Accuracy (Mean %RE)	8.8	−2.3	2.0
GD18 fetal homogenate
Precision (%RSD)	6.5	4.2	3.3
Accuracy (Mean %RE)	7.8	−4.7	1.0
Placental homogenate
Precision (%RSD)	2.4	3.3	2.3
Accuracy (Mean %RE)	−1.5	10.7	−2.2
Brain homogenate
Precision (%RSD)	2.1	3.1	1.1
Accuracy (Mean %RE)	3.5	11.3	−1.8
Adult heart homogenate
Precision (%RSD)	5.5	2.9	3.9
Accuracy (Mean %RE)	−2.5	15.0	−5.3
Pup heart homogenate
Precision (%RSD)	5.6	6.1	6.2
Accuracy (Mean %RE)	7.0	4.7	0.0

^aLOD = limit of detection. Defined as three times the standard deviation of the LOQ, expressed as concentration.

^bLOQ = lower limit of quantitation; lowest standard at which RE ≤ ±20% and RSD ≤ 20% for n = 6.

^cPrecision and accuracy determined for triplicate QCs at three levels in plasma; n = 3 for intra-day; n = 9 for inter-day.

^d%RSD = percent relative standard deviation.

^e%RE = percent relative error.

^fPrecision and accuracy determined for triplicate QCs in plasma at 40 µg/mL ABC, 60 µg/mL DTG, and 20 µg/mL 3TC, diluted into range.

^gPrecision and accuracy for secondary matrices determined for six replicate QCs in HSD rat GD 18 maternal plasma and fetal, placental, brain, and heart homogenates. QC level was 20 ng/mL ABC, 30 ng/mL DTG, and 10 ng/mL 3TC.

Figure 2.

Representative UPLC–MS-MS chromatograms for ABC, DTG, and 3TC in rat matrices: blank rat plasma with ISs; 10/15/5 ng/mL ABC/DTG/3TC in plasma; and 20/30/10 ng/mL ABC/DTG/3TC in fetal homogenate.

The calibration curves in plasma were linear with correlation coefficients r ≥ 0.99. The intra- and inter-day precisions for QC samples prepared at 20/30/10, 400/600/200, and 5000/7500/2500 ng/mL were ≤ 8.7%, and accuracies were within ±12.0%RE. Extraction recovery was determined at all calibration levels; the mean recoveries were 105% for ABC, 103% for DTG, and 104% for 3TC. To assess instrument drift over an analytical run sequence, a set of matrix calibration standards was prepared in plasma and injected at the start and the end of the run. The relative differences between the determined concentrations for the second set compared to the calibration set were within ±6.3% for all three analytes, indicating minimal instrument drift over a typical batch. QC samples prepared at 40/60/20 µg/mL in plasma and diluted into the validated range using extracted matrix had RE ≤ ±3.5% and RSD ≤ 2.4%, demonstrating the ability to dilute samples into the range of the calibration curve. Study samples were diluted in this same manner, using extracted matrix.

Carryover was evaluated by assessing the response in three method blanks run immediately following the highest calibration standard of 10,000/15,000/5000 ng/mL ABC/DTG/3TC and comparing them to the mean response for the method blanks run at the beginning of the sample set. Carryover was present for all three analytes, but for ABC and 3TC the levels were <30% of the LLOQ, suggesting that there would be no impact on the validated range. However, carryover for DTG was more substantial; it is recommended to run at least three solvent blanks after running high level samples and before any low-level samples are run, if the anticipated concentration of analytes in samples is known. The method was validated to apply to samples coming from animal studies evaluating toxicity where doses used were high, but carryover is not anticipated to be a challenge for studies investigating exposure from low doses.

Evaluation of method in study matrices (maternal plasma and fetal, placental, brain, and heart homogenates)

The method validated for ABC, DTG, and 3TC in adult rat plasma was evaluated in anticipated study matrices—HSD rat GD18 maternal plasma and fetal, placental, brain, and heart (adult and pup) homogenates. A representative chromatogram in HSD rat fetal homogenate is presented in Fig. 2 (bottom pane). Secondary matrix samples were prepared at 20/30/10 ng/mL and quantitated against the SD rat plasma calibration curve; RE and RSD were ≤ ±15.0% and ≤ 8.6%, respectively. No matrix interferences were detected in the matrix blanks at the retention times of the analytes, demonstrating adequate selectivity. However, it is interesting to note that the intensity of the DTG peak relative to the ABC and 3TC peak intensities was slightly different in the different matrices, but using stable-isotope-labeled internal standards compensated for any matrix effects. These data demonstrated that the method can be used to quantitate ABC, DTG, and 3TC in HSD rat maternal plasma and fetal, placental, brain, and heart homogenates.

Stability

Studies were performed to evaluate the stability of ABC, DTG, and 3TC in both the extracted sample during the analysis period and in matrix during study sample storage. The determined concentrations of the stability samples were compared to those of freshly prepared samples (Day 0), and data are presented in Table 3. Analytes in extracts were 101 to 110% of Day 0, demonstrating that ABC, DTG, and 3TC were stable up to 2 days at ambient and refrigerated temperatures.

Table 3.

Stability data for ABC, DTG, and 3TC in plasma, fetal, brain, and heart homogenate

	Mean % of Day 0 (% RSD)
Stability endpoint	ABC	DTG	3TC
Extract stabilitya
Refrigerated extracts (4°C, 2 d)	107 to 110 (≤5.5%)	101 to 107 (≤5.5%)	102 to 107 (≤4.6%)
Ambient extracts (2 d)	108 to 109 (≤6.1%)	103 to 106 (≤4.8%)	103 to 106 (≤4.5%)
Frozen matrix stability (−80°C)b
GD18 maternal rat plasma (199 d)	79.7 to 92.5 (≤7.9%)	82.6 to 95.1 (≤10.4%)	89.6 to 90.2 (≤8.7%)
GD18 fetal homogenate (199 d)	99.0 to 105 (≤7.2%)	96.3 to 113 (≤8.9%)	99.6 to 112 (≤6.8%)
Brain homogenate (36 d)	97.3 to 103 (≤4.6%)	97.3 to 101 (≤5.5%)	96.9 to 97.3 (≤3.0%)
Brain homogenate (191 d)	108 to 109 (≤8.6%)	107 to 113 (≤7.3%)	109 to 111 (≤10.4%)
Heart homogenate (32 d)	87.3 to 90.0 (≤11.4%)	80.5 to 81.8 (≤9.3%)	99.3 to 107 (≤10.7%)
Heart homogenate (141 d)	69.0 to 70.0 (≤11.6%)	79.2 to 80.4 (≤8.4%)	94.7 to 105 (≤10.7%)

^aAnalysis period stability (ambient and refrigerated extracts) determined for triplicate QCs at three levels in male SD rat plasma.

^bFrozen matrix stability determined for triplicate QCs at two levels in GD18 maternal plasma and fetal, brain, and heart homogenates.

ABC, DTG, and 3TC were stable in maternal plasma, fetal, and brain homogenates stored frozen (−80°C) for at least 191 days; mean values in each matrix were ∼80–113% of Day 0. It should be noted that the low concentration sample for ABC in maternal plasma had a percent of Day 0 of 79.7%, which was due to a slightly elevated Day 0 result compared to nominal and did not appear to be a stability issue. 3TC was stable in adult heart homogenate after 32 days of storage, and again when it was evaluated after 141 days of storage (94.7–107% of Day 0). For DTG, some initial loss of analyte was observed at both concentrations in heart homogenate (Mean % of Day 0 ∼81%), but the decline did not continue beyond the initial 32 days. All three analytes were added from the same spiking solution for the stability samples, so the fact that similar results were not observed for ABC and 3TC or other matrices suggests that the results were not due to a preparation error and were unique to DTG in that matrix. For ABC, a steady decline was observed in heart homogenate (Mean % of Day 0 ∼92% at 32 days, then ∼70% at 141 days), indicating that ABC may not be stable over long periods of time in this matrix.

Conclusion

A method was developed and validated using a simple protein precipitation and UPLC–MS-MS analysis to simultaneously quantitate ABC, DTG, and 3TC in rat plasma, fetus, brain, placenta, and heart. The method was simple and efficient with a total run time of 10 min. The linear range was 10–10,000 ng/mL for ABC, 15–15,000 ng/mL for DTG, and 5–5000 ng/mL for 3TC, with the ability to dilute into range samples as high as 40 µg/mL for ABC, 60 µg/mL for DTG, and 20 µg/mL for 3TC. The LLOQ was 10 ng/mL for ABC, 15 ng/mL for DTG, and 5 ng/mL for 3TC, with corresponding LODs 2.22, 3.69, and 0.978 ng/mL, respectively. Analytes were stable in extracts at ambient and refrigerated temperatures for 2 days, and in frozen matrix for at least 32 days. This method is suitable for quantitation of ABC, DTG, and 3TC in rats following exposure to this combination therapy in support of toxicology studies and can easily be extended to other species and matrices.

Funding

This work was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences, Intramural Research projects ZIC ES103391-01, and performed for the Division of Translational Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, US Department of Health and Human Services under contract HHSN273201400022C (RTI International, RTP, NC).

Data availability

The data underlying this article are available in the article.