Abstract
Antiretroviral therapy (ART) is highly effective for the treatment of HIV-1 infection. ART previously consisted of concomitant administration of many drugs, multiple times per day. Currently, ART generally consists of two- or three-drug regimens once daily as fixed-dose combinations. Drug monitoring may be necessary to ensure adequate concentrations are achieved in the plasma over the dosing interval and prevent further HIV resistance formation. Additionally, nonadherence remains an issue, highlighting the need to ensure sufficient ART exposure. Towards this effort, we developed and validated a highly selective ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of a panel of nine antiretrovirals: abacavir, bictegravir, cabotegravir, dolutegravir, doravirine, emtricitabine, lamivudine, raltegravir, and tenofovir in human plasma. Using only 50 μL of plasma, a simple protein precipitation with acetonitrile with internal standards followed by reconstitution in 50uL (high) or 400 uL (low)was performed. Analyte separation was achieved using a multistep UPLC gradient mixture of (A: 0.1% formic acid in water and B: acetonitrile) and a Waters CORTECS T3 (2.1x100 mm) column. The method was comprehensively validated according to the United States Food and Drug Administration Bioanalytical Guidelines over two clinically relevant ranges (1-250 ng/mL and 100-5000 ng/mL) with excellent linearity (R2 > 0.99 for all). The assay run time was 7.5 minutes. This method achieves acceptable performance of trueness (89.7-104.1%), repeatability, and precision (CV <15%), and allows for simultaneous quantification of guideline-recommended ART regimens. This method can be utilized for the therapeutic monitoring of antiretrovirals in human plasma.
Keywords: Antiretrovirals, Concentrations, HIV, UPLC-MS/MS, Therapeutic drug monitoring, Clinical pharmacology
1. Introduction
According to the World Health Organization, over 38 million people live with HIV (PLWH).[1] Antiretroviral therapy (ART) has transformed HIV infection from a deadly illness into a manageable chronic condition.[2,3] ART typically consists of two or three active therapeutic agents from different drug classes. While ART is effective, adherence remains an important concern; non-adherence can lead to treatment failure and subsequent viral resistance.[4,5] One objective assessment of ART adherence is plasma drug concentrations.[6]
Multiple assays are generally utilized to quantify an entire ART regimen. By employing multiple assays, larger plasma volumes are necessary, thereby requiring greater allocation of biological resources and decreasing analytical efficiency.
Our objective was to develop a simple, efficient, and highly sensitive LC-MS/MS assay to simultaneously quantify a panel of nine contemporarily used antiretroviral medications prescribed per the United States Department of Health and Human Services Guidelines.[7] Specifically, we targeted the following antiretrovirals: abacavir, bictegravir, cabotegravir, dolutegravir, doravirine, emtricitabine, lamivudine, raltegravir, and tenofovir.
Towards this goal, this article describes an ultra-high performance liquid chromatography-tandem mass spectrometry analytical method that was developed and comprehensively validated across two dynamic ranges to quantify antiretroviral medication concentrations in plasma. These dynamic ranges represent the therapeutic concentrations over the respective dosing intervals for these antiretrovirals. To our knowledge, there is no published LC-MS/MS assay that can measure these nine contemporary antiretroviral medications simultaneously. This method can be used extensively and efficiently for antiretroviral concentration quantification in multi-site clinical trials.
2. Materials and methods
2.1. Chemicals and reagents
Abacavir, abacavir-d4, bictegravir, cabotegravir, cabotegravir-d5, dolutegravir, dolutegravir-d5, emtricitabine, lamivudine, raltegravir, raltegravir-d4, tenofovir, and tenofovir-d6, were purchased from Cayman Chemical (Ann Arbor, MI, USA). [13C2H215N]-bictegravir, doravirine, [13C6]-doravirine, [2H315N]-emtricitabine, [13C2H215N2]-lamivudine, were purchased from Alsachim (Illkirch-Graffenstaden, France). The chemical structures of all analytes and internal standards are displayed in Figure 1. Optima LC–MS grade 2-propanol, acetonitrile, methanol, and water were purchased from Fisher Scientific (Pittsburgh, PA, USA). Formic Acid, LC-MS (98%) was purchased from Fisher Scientific (Pittsburgh, PA, USA). Ultra-pure nitrogen gas (>99.9%) was produced by a Parker N2-35 Nitrogen Generation System (Haverhill, MA), and ultra-pure argon gas (>99.9%) was obtained from Matheson (Basking Ridge, NJ, USA). Blank human plasma was received from the Central Blood Bank (Pittsburgh, PA, USA).
Figure 1.
The chemical structures of the nine antiretrovirals and their respective isotopically-labeled compounds.
2.2. Equipment and UPLC-MS/MS conditions
Ultra-performance liquid chromatography (UPLC) was performed with a Waters Acquity UPLC I-class which consisted of a binary solvent manager and a sample manager (Waters, Milford, MA, USA). A 50 mm and 100 mm C18 column were tested but poor retention and incomplete separation with these columns were observed. The next column that was tested was a Waters CORTECS T3 (2.1 x 50 mm; 1.6 μm), which is optimized for more polar compounds. All the analytes retained on this column, but several of the analytes had similar retention times. Therefore, we opted for a longer column (100 mm) to achieve optimal separation and retention of all nine ARVs. The chromatographic separation of the analytes was achieved with a Waters CORTECS T3 (2.1 × 100 mm; 1.6 μm particle size) along with CORTECS T3 Vanguard pre-column (2.1 x 5 mm, 1.6 μm particle size) at 55°C (Waters, Milford, MA, USA). The sample manager was kept at 10°C during all analyses.
For the mobile phases, methanol was initially selected as mobile phase B, but broad peaks were observed. Acetonitrile was tried next and provided sharp, narrow, and reproducible peaks. Initially, isocratic conditions were tested, but due to poor separation and broader peaks, a gradient was utilized. During sample preparation, various reconstitution solvents and volumes of the solvents were investigated to determine which yielded optimal peak intensity and sharpness.
The solvent flow rate was 0.300 mL/min, and the mobile phases were 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). The gradient started with 97% A and 3% B for 0-1.0 minutes; followed by a linear gradient to 80% A from 1.0-1.5 minutes; then decreased to 15% A from 1.5-4.0 minutes; then increasing to 50% A from 4.0-5.0 minutes before returning to initial conditions from 5.0-7.0 minutes. The gradient then was held at 97% A from 7.0-7.5 minutes. The total run time was 7.5 minutes.
A Thermo TSQ Quantis Plus mass spectrometer (Thermo Scientific, San Jose, CA, USA) equipped with a HESI source for ionization was used to perform MS/MS analyses. The optimized spray voltage was determined to be 3.0 kV, and the vaporizer and ion transfer capillary temperatures were both set to 350°C. The optimized sheath and auxiliary gas settings were 50 and 10 (arbitrary units), respectively. The pressure of the collision gas was set to 1.5 mTorr. The full peak width at half maximum of 0.7 m/z for quadrupole one (Q1) and of 1.2 m/z for quadrupole three (Q3) was used.
Mass spectrometer settings were optimized, and higher intensity ions were observed in positive ion mode than in negative ion mode. An issue that was observed for the high curve was that bictegravir and doravirine ionized well which led to detection saturation when using the most intense fragment in the MS/MS spectra. To remedy this, the second most intense fragment was used to provide adequate sensitivity of the curve on the high end while eliminating detection saturation. For the low curve, the most intense fragment was then used to achieve optimal sensitivity as saturation was not observed.
All antiretrovirals were detected in positive ionization mode with selected reaction monitoring (SRM). The ion transitions monitored, collision energies, and retention times are listed in Table 1 for the high curve and Table S1 in the Supplementary Material for the low curve. The mass spectrometer signals were detected and processed with Xcalibur software 5.474.0 (Thermo Scientific, San Jose, CA, USA).
Table 1.
MS/MS parameters and typical retention times of the nine antiretrovirals and their respective stable isotopically-labeled compounds
| Compound | ESI polarity (+/−) | Precursor ion (m/z) | Product ion (m/z) | Collision Energy (eV) | RF Lens (V) | Retention time (min) |
|---|---|---|---|---|---|---|
| Abacavir | + | 287.10 | 190.96 | 18.60 | 111 | 2.53 |
| Abacavir-d4 | + | 291.10 | 194.06 | 19.25 | 112 | 2.53 |
| Bictegravir | + | 450.06 | 144.80 | 40.00 | 181 | 3.81 |
| [13C,2H2,15N]-Bictegravir | + | 454.05 | 288.96 | 28.90 | 183 | 3.81 |
| Cabotegravir | + | 406.01 | 126.88 | 34.00 | 167 | 3.63 |
| Cabotegravir-d5 | + | 411.03 | 267.96 | 24.00 | 166 | 3.62 |
| Dolutegravir | + | 420.04 | 126.83 | 33.70 | 170 | 3.74 |
| Dolutegravir-d5 | + | 425.03 | 131.83 | 33.70 | 170 | 3.73 |
| Doravirine | + | 425.96 | 111.96 | 23.80 | 133 | 3.89 |
| [13C6]-Doravirine | + | 431.94 | 111.96 | 23.80 | 133 | 3.89 |
| Emtricitabine | + | 248.00 | 129.80 | 5.00 | 110 | 2.43 |
| [2H3,15N]-Emtricitabine | + | 251.98 | 131.96 | 11.70 | 110 | 2.43 |
| Lamivudine | + | 229.96 | 111.96 | 8.00 | 110 | 1.61 |
| [13C,2H2,15N2]-Lamivudine | + | 234.96 | 114.96 | 12.50 | 112 | 1.61 |
| Raltegravir | + | 445.10 | 360.96 | 17.10 | 135 | 3.73 |
| Raltegravir-d4 | + | 449.06 | 364.96 | 17.10 | 142 | 3.73 |
| Tenofovir | + | 287.96 | 175.96 | 24.00 | 139 | 1.30 |
| Tenofovir-d6 | + | 294.05 | 181.96 | 25.00 | 139 | 1.30 |
2.3. Preparation of the calibration standards and quality control (QC) samples
For stock solutions, abacavir was prepared in methanol (1.0 mg/mL), bictegravir and doravirine were prepared in dimethyl sulfoxide (DMSO; 1 mg/mL), cabotegravir and dolutegravir were prepared in DMSO (2.5 mg/mL), emtricitabine and lamivudine were prepared in water (1 mg/mL), raltegravir was prepared in water (0.5 mg/mL), and tenofovir was prepared in water (2.5 mg/mL). These stock solutions were diluted with 80:20 water:methanol to prepare two intermediate stock solutions (high curve; 10 μg/mL and 50 μg/mL) or three intermediate stock solutions (low curve; 10 μg/mL, 50 μg/mL, and 1 μg/mL). Two sets of intermediate stocks were prepared separately, one set was used for the calibration standards and the other for the QCs. The intermediate stock solutions were spiked into blank human plasma to produce calibration standards at concentrations of 100, 250, 500, 1000, 2500, 5000 ng/mL. Lower limit of quantitation, low, medium, and high QCs (LLOQ, LQC, MQC, and HQC) were prepared by spiking the intermediate stocks into blank human plasma to provide concentrations of 100, 300, 900, and 4000 ng/mL. For the low curve, calibration standards were prepared at 1, 2.5, 5, 10, 50, 100, and 250 ng/mL; LLOQ and QCs were prepared at 1, 3, 45, and 200 ng/mL. Working internal standard solution was made by diluting internal standard stock solutions to 40 ng/mL in acetonitrile. All samples, stock solutions, intermediate stocks, calibration standards, and QCs were stored at −80°C until further testing.
2.4. Sample preparation
Varying combinations of methanol, acetonitrile, water, and formic acid were tested, and the best results for all analytes was with 400 μL of water. For the protein precipitation solvent, methanol was initially used but after repeated testing, acetonitrile provided the most reproducible and sharper peak shapes for the chromatography of all the analytes. Utilizing protein precipitation allowed for high-throughput sample preparation while also being cost-effective compared to more complex extraction techniques.
100 μL of internal standard solution was added to 50 μL of the calibration standards, QCs, or unknown plasma samples. The samples were then vortexed for 10 seconds and centrifuged at 10,000 × g for 10 minutes. 100 μL of the supernatant was transferred to 12x75 mm borosilicate glass tubes and dried down under nitrogen at 40°C. The samples were then reconstituted with 50 μL of water (high curve) or 400 μL (low curve) and were then added to UPLC vials for analysis. 6 μL of sample was injected for UPLC-MS/MS analyses.
2.5. Assay validation
The assay panels were validated in accordance with the United States Food and Drug Administration (FDA) Guidance for Bioanalytical Method Validation.[8]
2.5.1. Calibration and linearity
Six (high curve) and seven (low curve) standard concentrations for the antiretrovirals were used to construct calibration curves. For three days, the lower limit of quantification (LLOQ) was run in triplicate while the other calibration standards were run in duplicate. The LLOQ was determined to have a signal-to-noise ratio (S:N) greater than 10:1. For each standard curve, the absolute peak-area ratios of each antiretroviral to the isotopically-labeled internal standard were calculated and plotted against the nominal analyte concentration with 1/X weighting.
2.5.2. Accuracy and precision
Precision and accuracy were determined by the analysis of six replicate LLOQ, LQC, MQC, and HQC samples for 2 consecutive days followed by 12 replicate QC samples (n = 24 of each QC) on a third day. The 12 replicates on the third day determined the intra-day accuracy and precision while all 24 replicate QC samples were used to calculate the inter-day precision and accuracy. The calculated mean concentration relative to the spiked concentration was used to express accuracy (% deviation). Means, standard deviations and coefficients of variation were calculated from the QC values and used to estimate the intra- and inter-day precision.
2.5.3. Matrix effect and recovery studies
The matrix effect of antiretrovirals from plasma was assessed by the analysis of plasma samples spiked at the three QC concentration levels compared to methanol samples spiked at the same concentrations that were not extracted. Three replicate ‘control’ methanol samples and three spiked samples of each QC level were analyzed using standard curves generated from plasma-based standards as described in Section 2.3. Measured concentrations of the non-extracted methanol QC samples were defined as 100%.
Recovery was determined by comparing the post-extraction spiked QC samples with the extracted plasma-based QC samples. Measured concentrations of the post-extracted spiked plasma QC samples were defined as 100%. Means, standard deviations and coefficients of variation were calculated for both matrix and recovery studies.
2.5.4. Carryover
Carry-over was evaluated by placing vials of blank mobile phase at several locations, including after the highest standard in the analysis set. Carryover percentages of ≤20% of the respective LLOQs were considered acceptable.
2.5.5. Stability studies
The stability of stored samples was tested by analyzing three replicates of samples at two different QC levels (LQC and HQC) that were left out on the bench top at room temperature (RT) under normal fluorescent light for 4 hours and compared with the recovery from samples that were freshly prepared. The results found from the freshly prepared samples were defined as 100%. The results of the later time points were expressed in terms of percent of the first measurement. Results within 15% of the first value were regarded as stable on the bench top for up to 4 hours.
Stability of the processed samples in the autosampler was also investigated. The samples were processed (LQC and HQC) in triplicate and left in the autosampler for a period of either 24 h or 72 h. Recovery at these times were compared with results from freshly processed samples, which were defined as 100%. The results from the later time points were expressed in terms of percentage of the first measurement. The analytes were considered to be stable under these conditions if the results differ ≤15% from the first value.
Freeze/thaw cycle stability was also assessed. Quality control samples at two concentration levels (LQC and HQC) were prepared in triplicate and stored at −80°C before being subjected to three consecutive freeze/thaw cycles and analyzed. The results of the later time points were expressed in terms of percentage of the first measurement (freshly prepared). The analytes were considered stable after undergoing up to three freeze/thaw cycles if the results differ by ≤15% from the freshly processed sample values.
Long-term stability was assessed by comparing freshly prepared standard curves and QCs (LQC and HQC) to those following 51 days of storage at −80°C. For stock solution stability, fresh QCs (LQC and HQC) were prepared from stock solutions prepared 7 months prior and stored at −20°C on the original calibration curve. The analytes were considered stable under these conditions if the results differ ≤15%.
2.5.6. Dilution analysis
Antiretroviral samples were prepared at a concentration that was two-fold greater than the highest calibration standard (10000 and 500 ng/mL for the high and low curves, respectively). Aliquots of the samples were then diluted 2, 3 and 5-fold prior to analysis. Each dilution level was processed 5 times each and back calculated against duplicate standard curves to determine if the results were within 15% of the nominal concentration.
2.5.7. Clinical application
The validated antiretroviral panel assays were applied to analyze patient samples from the University of Pittsburgh Medical Center. The sample procurement protocol was approved by the University of Pittsburgh Institutional Review Board (STUDY 22110116) and written informed consent was obtained from each study participant. Demographics and time after last dose were recorded. Opportunistic blood samples were collected in EDTA tubes on ice and centrifuged within 1 hour of collection at 1700 x g at 4°C for 10 minutes. The plasma fraction was aliquoted and stored at −80°C until analysis.
3. Results
Simple and high-throughput UPLC-MS/MS methods were developed and validated for the quantification of nine antiretrovirals in human plasma.
3.1. Mass Spectrometry, Chromatographic Separation, and Sample Preparation
In the current study, the use of a Waters CORTECS T3 (2.1 × 100 mm; 1.6 μm) allowed for excellent separation with a total run time of 7.5 min. Example chromatograms of nine antiretrovirals at the LLOQs are shown in Figure 2 (high curve), and the chromatograms of the internal standards are shown in Figure S1 in the Supplementary Material. The corresponding chromatograms for the LLOQs of the low curve are shown in Figure S2, of the Supplementary Material.
Figure 2.
Representative chromatograms of LLOQ samples of Abacavir (A), Bictegravir (B), Cabotegravir (C), Dolutegravir (D), Doravirine (E), Emtricitabine (F), Lamivudine (G), Raltegravir (H), and Tenofovir (I).
3.2. Assay Validation
The current antiretroviral panel assays were successfully validated per the published FDA guidelines.[8]
3.2.1. Calibration and Linearity
The standard curves for the antiretrovirals were analyzed over three separate runs with each standard curve analyzed in duplicate (lowest standard in triplicate) using plasma-based standards. Linear calibration curves were obtained for all antiretrovirals over the concentration ranges, as shown in Table 2 and Table S2 in the Supplementary Material. The coefficient of determination (R2) was 0.9950 or greater during the three days of validation. The LLOQ was determined to be 100 and 1 ng/mL for the two curves, respectively. Precision and accuracy data obtained from back calculated calibration standards demonstrated the suitability of the calibration method. All values were within 15% of the nominal concentration.
Table 2.
Antiretroviral panel assay validation results
| Analyte | LLOQ (ng/mL) | Linear range (ng/mL) | Slope (n=3) | Intercept (n=3) | Coefficient of determination (R2, n=3) |
|---|---|---|---|---|---|
| Abacavir | 100 | 100-5000 | 0.0011 | −0.0254 ± 0.0006 | 0.9983 ± 0.0004 |
| Bictegravir | 0.0024 | −0.0242 ± 0.0052 | 0.9976 ± 0.0007 | ||
| Cabotegravir | 0.0010 | −0.0160 ± 0.0015 | 0.9988 ± 0.001 | ||
| Dolutegravir | 0.0013 | −0.0257 ± 0.0015 | 0.9994 ± 0.0002 | ||
| Doravirine | 0.0013 | −0.0020 ± 0.0068 | 0.9979 ± 0.0002 | ||
| Emtricitabine | 0.0006 | −0.0002 ± 0.0034 | 0.9967 ± 0.0012 | ||
| Lamivudine | 0.0014 | −0.0688 ± 0.0051 | 0.9967 ± 0.0012 | ||
| Raltegravir | 0.0007 | −0.0079 ± 0.0013 | 0.9982 ± 0.0017 | ||
| Tenofovir | 0.005 | −0.0063 ± 0.0075 | 0.9961 ± 0.0019 |
The intercept and correlation coefficient are presented as mean ± standard deviation.
3.2.2. Accuracy and Precision
The accuracy and precision of the method was determined by the analysis of antiretroviral QC samples at three concentration levels over a three-day period. The mean inter- and intra-day precision and accuracy values were all within ±15% for QCs and for ±20% LLOQ (Table 3 and Table S3 in the Supplementary Material). The data showed that the antiretroviral assays were both accurate and precise.
Table 3.
Intra- and inter-day accuracy and precision for the nine antiretroviral LLOQ and QC plasma samples
| Compound | Level | Nominal Concentration (ng/mL) | Intra-daya | Inter-dayb | ||
|---|---|---|---|---|---|---|
| Deviation (%) | CV% | Deviation (%) | CV% | |||
| Abacavir | LLOQ | 100 | 12.5 | 4.3 | 12.2 | 4.8 |
| LQC | 300 | −8.3 | 2.9 | −10.2 | 3.3 | |
| MQC | 900 | 0.4 | 3.3 | 2.2 | 4.4 | |
| HQC | 4000 | 2.8 | 4.5 | 2.6 | 4.6 | |
| Bictegravir | LLOQ | 100 | 11.9 | 4.0 | 11.0 | 4.8 |
| LQC | 300 | −2.6 | 5.2 | −3.7 | 5.3 | |
| MQC | 900 | −12.1 | 2.6 | −9.8 | 4.3 | |
| HQC | 4000 | −0.5 | 4.7 | 1.0 | 5.0 | |
| Cabotegravir | LLOQ | 100 | 13.7 | 3.5 | 12.6 | 3.6 |
| LQC | 300 | −13.3 | 1.7 | −13.0 | 1.6 | |
| MQC | 900 | −2.6 | 2.7 | −0.1 | 4.9 | |
| HQC | 4000 | 1.2 | 4.7 | 2.3 | 4.9 | |
| Dolutegravir | LLOQ | 100 | 14.0 | 3.7 | 14.7 | 3.2 |
| LQC | 300 | −12.6 | 1.1 | −12.9 | 1.3 | |
| MQC | 900 | −3.2 | 3.1 | −1.3 | 4.4 | |
| HQC | 4000 | 0.7 | 5.3 | 0.8 | 5.1 | |
| Doravirine | LLOQ | 100 | 7.5 | 5.4 | 5.8 | 5.6 |
| LQC | 300 | −3.5 | 4.6 | −4.4 | 5.2 | |
| MQC | 900 | −10.8 | 2.9 | −6.5 | 7.0 | |
| HQC | 4000 | 0.5 | 7.1 | 2.5 | 6.4 | |
| Emtricitabine | LLOQ | 100 | 3.2 | 6.2 | 0.9 | 8.9 |
| LQC | 300 | 0.6 | 7.9 | −1.5 | 9.0 | |
| MQC | 900 | −11.3 | 4.6 | −7.5 | 8.4 | |
| HQC | 4000 | −4.5 | 5.1 | −2.8 | 5.7 | |
| Lamivudine | LLOQ | 100 | 17.5 | 1.3 | 17.1 | 2.0 |
| LQC | 300 | −13.8 | 0.9 | −13.3 | 1.4 | |
| MQC | 900 | −11.2 | 2.7 | −8.2 | 4.5 | |
| HQC | 4000 | 1.5 | 4.1 | 2.2 | 4.4 | |
| Raltegravir | LLOQ | 100 | 8.5 | 3.9 | 8.3 | 4.3 |
| LQC | 300 | −0.3 | 4.1 | −1.9 | 4.6 | |
| MQC | 900 | −8.9 | 3.6 | −5.2 | 6.0 | |
| HQC | 4000 | −0.3 | 5.2 | 2.0 | 5.9 | |
| Tenofovir | LLOQ | 100 | −3.0 | 11.1 | 3.8 | 11.0 |
| LQC | 300 | 3.5 | 6.9 | −1.4 | 8.3 | |
| MQC | 900 | −6.8 | 8.5 | −6.4 | 7.9 | |
| HQC | 4000 | 8.0 | 5.9 | 2.2 | 8.9 | |
Data are presented as the mean values for the intra-day and inter-day deviation percentages.
12 replicates for all levels.
24 replicates for all levels.
3.2.3. Recovery and matrix effect
The average recoveries of all antiretrovirals at all three concentrations ranged from 86.9-113.4% (Table 4) and 93.1-110.0% (Table S4 in the Supplementary Material), demonstrating excellent recovery and minimal loss of the analytes. The measured plasma concentrations varied by −13% to 11% from the neat samples, thus the matrix effects were negligible.
Table 4.
Recovery of antiretrovirals from human plasma (n = 3)
| Compound | QC Level | Nominal Concentration (ng/mL) | Recovery | Matrix Effect | ||
|---|---|---|---|---|---|---|
| % | CV% | % | CV% | |||
| Abacavir | LQC | 300 | 91.0 | 3.5 | 87.0 | 2.0 |
| MQC | 900 | 102.1 | 5.0 | 102.2 | 2.2 | |
| HQC | 4000 | 113.4 | 0.9 | 99.3 | 1.9 | |
| Bictegravir | LQC | 300 | 93.7 | 2.3 | 103.5 | 5.3 |
| MQC | 900 | 90.7 | 5.7 | 101.0 | 1.1 | |
| HQC | 4000 | 93.2 | 4.7 | 103.7 | 6.0 | |
| Cabotegravir | LQC | 300 | 95.8 | 2.6 | 96.4 | 0.7 |
| MQC | 900 | 92.9 | 4.1 | 108.2 | 5.5 | |
| HQC | 4000 | 109.3 | 4.5 | 100.3 | 4.4 | |
| Dolutegravir | LQC | 300 | 93.3 | 2.6 | 98.6 | 0.6 |
| MQC | 900 | 90.9 | 6.1 | 108.8 | 5.5 | |
| HQC | 4000 | 108.7 | 4.1 | 100.1 | 5.1 | |
| Doravirine | LQC | 300 | 94.4 | 3.1 | 100.1 | 2.3 |
| MQC | 900 | 86.9 | 4.0 | 101.6 | 7.7 | |
| HQC | 4000 | 91.8 | 1.7 | 104.3 | 7.6 | |
| Emtricitabine | LQC | 300 | 101.2 | 2.6 | 105.3 | 7.2 |
| MQC | 900 | 95.7 | 6.4 | 98.4 | 2.4 | |
| HQC | 4000 | 97.3 | 3.2 | 93.1 | 9.6 | |
| Lamivudine | LQC | 300 | 91.6 | 3.2 | 87.3 | 1.2 |
| MQC | 900 | 90.3 | 4.4 | 100.0 | 2.2 | |
| HQC | 4000 | 110.4 | 2.9 | 94.4 | 2.3 | |
| Raltegravir | LQC | 300 | 92.4 | 6.0 | 109.4 | 3.9 |
| MQC | 900 | 90.9 | 4.2 | 101.0 | 1.1 | |
| HQC | 4000 | 97.9 | 3.6 | 103.7 | 6.0 | |
| Tenofovir | LQC | 300 | 104.2 | 3.2 | 103.9 | 3.2 |
| MQC | 900 | 94.9 | 2.8 | 96.9 | 4.7 | |
| HQC | 4000 | 97.4 | 5.9 | 106.6 | 5.6 | |
Data are presented as the mean values for the percent recovery and matrix effect.
3.2.4. Carryover
Carryover was evaluated by placing mobile phase at six random locations throughout a sequence. Carryover of antiretrovirals from previous runs for samples containing mobile phase were consistently below 20% of the LLOQ for each analyte. For the high curve, average carryover was 3.8% (maximum 10.4%). For the low curve, average carryover was 5.9% (maximum 11.4%). Carryover was acceptable for both concentration ranges. In addition, there were no endogenous analytes in the blank plasma lots that affected the signal for the antiretrovirals or their respective internal standards.
3.2.5. Stability studies
The stability of the plasma samples was tested prior to processing at room temperature, post-processing with storage in the autosampler at 10°C, and after three consecutive freeze/thaw cycles. The mean averaged concentrations for each type of stability samples were within ≤15% of the freshly prepared samples, demonstrating sample stability under all testing conditions (Table 5 and Table S5 in the Supplementary Material).
Table 5.
Stability studies
| Compound | Nominal concentration (ng/mL) | Freeze/Thaw between −80°C and RT | Autosampler at 10°C | Benchtop at RT | Longterm stability | Stock solution stability | |||
|---|---|---|---|---|---|---|---|---|---|
| 1 F/T | 2 F/T | 3 F/T | 24h | 72h | 4h | 51 days | 7 months | ||
| Abacavir | 300 | 100.9 (2.6) | 101.2 (3.9) | 101.8 (4.5) | 101.4 (1.4) | 105.4 (0.6) | 94.0 (8.3) | 90.6 (5.6) | 112.8 (3.5) |
| 4000 | 99.8 (4.9) | 106.2 (2.3) | 108.0 (2.3) | 100.0 (0.2) | 103.8 (0.5) | 95.3 (4.6) | 93.7 (9.3) | 105.3 (2.0) | |
| Bictegravir | 300 | 100.7 (5.2) | 98.6 (8.1) | 96.6 (3.5) | 104.1 (5.3) | 103.2 (1.7) | 90.2 (5.0) | 94.6 (1.1) | 106.7 (4.6) |
| 4000 | 100.3 (8.4) | 99.2 (5.2) | 97.3 (5.2) | 101.8 (2.6) | 101.0 (1.2) | 90.0 (5.6) | 103.4 (5.5) | 98.2 (4.6) | |
| Cabotegravir | 300 | 99.7 (1.6) | 100.3 (1.8) | 101.1 (1.2) | 100.4 (0.9) | 103.2 (2.4) | 90.1 (3.4) | 93.9 (9.7) | 108.5 (6.5) |
| 4000 | 98.3 (5.3) | 109.0 (3.0) | 107.8 (3.0) | 102.7 (1.4) | 107.3 (1.5) | 94.1 (5.1) | 102.2 (3.2) | 107.5 (2.4) | |
| Dolutegravir | 300 | 98.2 (0.6) | 99.4 (1.9) | 98.9 (1.4) | 99.9 (0.7) | 101.9 (0.7) | 92.5 (5.3) | 96.0 (3.8) | 108.0 (7.2) |
| 4000 | 100.1 (3.7) | 107.8 (3.9) | 108.7 (1.1) | 101.5 (0.6) | 104.5 (1.5) | 93.4 (3.4) | 94.8 (8.3) | 101.6 (2.0) | |
| Doravirine | 300 | 98.6 (9.8) | 98.6 (7.7) | 99.0 (1.7) | 101.1 (2.6) | 102.5 (0.3) | 91.9 (11.2) | 96.9 (7.8) | 98.2 (4.1) |
| 4000 | 97.1 (5.7) | 105.4 (3.2) | 104.0 (3.2) | 97.8 (0.1) | 101.5 (1.9) | 93.0 (4.4) | 93.5 (9.9) | 99.2 (2.2) | |
| Emtricitabine | 300 | 101.7 (7.5) | 94.4 (2.3) | 104.4 (10.8) | 99.1 (4.6) | 108.1 (2.8) | 95.2 (12.1) | 92.5 (5.4) | 101.7 (8.0) |
| 4000 | 99.4 (8.2) | 98.3 (1.4) | 108.4 (6.0) | 101.9 (4.0) | 111.3 (3.5) | 97.9 (6.9) | 98.2 (8.1) | 106.3 (3.0) | |
| Lamivudine | 300 | 99.7 (1.1) | 103.0 (4.9) | 107.0 (5.9) | 101.2 (0.8) | 103.1 (1.9) | 89.4 (3.2) | 97.0 (1.5) | 100.1 (2.1) |
| 4000 | 100.5 (4.5) | 104.9 (3.1) | 111.6 (3.1) | 104.1 (1.7) | 110.5 (0.6) | 90.5 (4.4) | 93.9 (9.6) | 101.8 (2.4) | |
| Raltegravir | 300 | 98.6 (5.5) | 98.6 (4.8) | 98.9 (4.3) | 101.1 (0.2) | 103.4 (1.3) | 93.8 (6.6) | 93.2 (7.7) | 103.2 (6.1) |
| 4000 | 99.9 (5.2) | 109.7 (3.5) | 108.1 (3.5) | 99.6 (1.3) | 103.1 (1.1) | 94.0 (3.4) | 90.3 (8.9) | 101.9 (3.5) | |
| Tenofovir | 300 | 106.1 (2.4) | 103.3 (6.7) | 112.3 (2.9) | 109.4 (1.4) | 111.5 (3.9) | 93.5 (4.5) | 94.4 (7.7) | 93.1 (1.1) |
| 4000 | 93.3 (1.0) | 101.1 (5.0) | 109.1 (4.9) | 100.1 (5.0) | 93.1 (5.7) | 98.9 (5.9) | 99.5 (3.6) | 90.6 (2.7) | |
Data are mean percent recoveries (CV%). N=3 for each stability level. N=5 for long-term stability studies.
3.2.6. Dilution analysis
The mean accuracy and precision obtained for all antiretrovirals at all dilution levels were well within ±15% (Table 6 and Table S6 in the Supplementary Material) of the human plasma samples before analysis and did not affect accuracy and precision.
Table 6.
Dilution Studies
| Compound | Dilution | Diluted Concentration (ng/mL) | Nominal Concentration before dilution (ng/mL) | Resulting Concentration (ng/mL) | % Deviation from nominal concentration before dilution | CV% |
|---|---|---|---|---|---|---|
| Abacavir | 2x | 5214.0 | 10000 | 10428.0 | 4.3 | 1.5 |
| 3x | 3419.0 | 10257.0 | 2.6 | 3.5 | ||
| 5x | 2152.9 | 10764.5 | 7.6 | 1.3 | ||
| Bictegravir | 2x | 5351.2 | 10702.4 | 7.0 | 2.1 | |
| 3x | 3318.2 | 9954.6 | −0.5 | 3.1 | ||
| 5x | 2015.9 | 10079.5 | 0.8 | 2.7 | ||
| Cabotegravir | 2x | 5407.8 | 10815.6 | 8.2 | 2.3 | |
| 3x | 3461.5 | 10384.5 | 3.8 | 0.8 | ||
| 5x | 2148.9 | 10744.5 | 7.4 | 0.8 | ||
| Dolutegravir | 2x | 5171.5 | 10343.0 | 3.4 | 1.8 | |
| 3x | 3498.4 | 10495.2 | 5.0 | 3.6 | ||
| 5x | 2108.0 | 10540.0 | 5.4 | 1.5 | ||
| Doravirine | 2x | 5205.6 | 10411.2 | 4.1 | 2.3 | |
| 3x | 3523.4 | 10570.2 | 5.7 | 1.0 | ||
| 5x | 2019.6 | 10098.0 | 1.0 | 3.5 | ||
| Emtricitabine | 2x | 5256.0 | 10512.0 | 5.1 | 2.3 | |
| 3x | 3626.1 | 10878.3 | 8.8 | 2.0 | ||
| 5x | 2050.1 | 10250.5 | 2.5 | 0.8 | ||
| Lamivudine | 2x | 5318.9 | 10637.8 | 6.4 | 1.6 | |
| 3x | 3565.8 | 10697.4 | 7.0 | 4.1 | ||
| 5x | 2117.2 | 10586.0 | 5.9 | 2.2 | ||
| Raltegravir | 2x | 5163.2 | 10326.4 | 3.3 | 2.3 | |
| 3x | 3564.2 | 10692.6 | 6.9 | 0.6 | ||
| 5x | 2096.6 | 10483.0 | 4.8 | 1.5 | ||
| Tenofovir | 2x | 5194.2 | 10388.4 | 3.9 | 3.3 | |
| 3x | 3577.4 | 10732.2 | 7.3 | 5.3 | ||
| 5x | 1976.4 | 9882.0 | −1.2 | 3.7 |
Data are presented as the mean values. N=5 for each level of dilution.
3.2.7. Clinical application
The antiretroviral assays were applied to patients receiving antiretroviral therapy The representative chromatograms are shown in Figure 3, and the pharmacokinetic data are shown in Table 7.
Figure 3.
Representative chromatograms of plasma samples from patients receiving antiretroviral therapy.
Table 7.
Clinical application of antiretroviral assays
| Analyte | Time after dose (hours) | Curve Utilized | Concentration (ng/mL) |
|---|---|---|---|
| Abacavira | 1.8 | High | 4866 |
| Bictegravirb | 10.8 | High | 4825 |
| Cabotegravir | 984 | High | 2421 |
| Dolutegravira | 1.8 | High | 3072 |
| Doravirine | 11.6 | High | 1072 |
| Emtricitabineb | 10.8 | High | 1268 |
| Lamivudinea | 1.8 | High | 2236 |
| Tenofovirb | 10.8 | Low | 11.5 |
Superscript letters (a and b) represent samples from patients receiving combination antiretroviral therapy.
Discussion
A sensitive UPLC-MS/MS method for the simultaneous quantification of nine antiretrovirals in human plasma was developed and comprehensively validated using only 50 μL of plasma. The sample preparation procedure with protein precipitation was high-throughput, cost-effective, and sample sparing; sample preparation only requires 50 μL of plasma for analysis. The panel of antiretrovirals covers all the contemporary and current United States Department of Health and Human Services guideline-recommended first-line therapy options for the treatment of HIV-1 infection, as of the September 2022 guideline update.[7], which improves upon previously established antiretroviral assays.[9,10] Furthermore, by incorporating all these antiretrovirals into one analytical method, we are able to quantify an entire contemporarily prescribed, first-line antiretroviral medication regimen.
We applied our method to plasma samples from patients prescribed combination antiretroviral therapy. Based on the time after dose data, the concentrations described herein are aligned with concentrations reported based on pharmacokinetic data previously reported, either observed or through pharmacokinetic modeling results.[11–17] For instance, the concentrations reported for abacavir, dolutegravir, and lamivudine – which represents a recommended regimen for initial treatment – were quantified from a patient sample obtained 1.8 hours after the dose. This timepoint represents the time of the maximum concentration during the dosing interval, and the concentrations are aligned with the maximum concentrations reported previously.[11]
The low curve was utilized for tenofovir from a patient receiving antiretroviral therapy consisting of tenofovir alafenamide, a prodrug of tenofovir, and aligns with the concentration ranges observed in previous studies.[18] However, nonadherence remains an important concern for PLWH, which significantly affect transmission rates.[19] Our low curve antiretroviral assay allows for the quantification of antiretroviral medication concentrations following consecutively missed doses; obtaining these concentrations provides the opportunity to understand adherence patterns and facilitate more targeted patient and health care provider discussions. Furthermore, as efforts to re-formulate contemporarily used antiretrovirals into ultra-long-acting formulations to achieve lower concentrations over a longer dosing interval, there will be a need to ensure effective and therapeutic concentrations.[20]
In summary, the validated UPLC-MS/MS methods for the nine antiretrovirals described herein have been included in the routine analyses within the Small Molecule Biomarker Core at the University of Pittsburgh School of Pharmacy. The inclusion of these methods allows for opportunities to perform clinical and translational studies within the HIV/AIDS research space and improve patient care.
Supplementary Material
Highlights.
Recommended antiretroviral therapy consists of multiple therapeutic agents
Nonadherence remains a concern in those living with HIV
Quantifying concentrations in plasma ensures adequate antiretroviral exposure
A UPLC-MS/MS method was validated to quantify nine antiretrovirals in human plasma
Funding:
This work used the services of the University of Pittsburgh Small Molecule Biomarker Core, which was partially funded by NIH through S10RR023461 and S10OD028540 as well as the Rustbelt Center for AIDS Research Clinical Sciences Core (P30AI036219). The content is solely the responsibility of the authors and does not necessarily represent the official views of National Institutes of Health.
Conflicts of Interest:
S.A.R. has received research grants from Gilead Sciences and Merck and Co., Inc. The other authors have no conflicts of interest to disclose.
Footnotes
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CRediT authorship contribution statement
Raymond West 3rd: Conceptualization, Methodology, Formal analysis, Investigation, Resources, Data curation, Writing – original draft, Writing – review & editing, Visualization, Supervision, Validation. Patrick Oberly: Investigation, Resources, Data Curation, Writing – review & editing, Validation. Sharon Riddler: Resources, Writing – Review & Editing. Thomas Nolin: Writing – Review & Editing, Supervision, Project administration. Aaron Devanathan: Formal analysis, Resources, Writing – Original Draft, Writing – Review & Editing, Supervision, Project administration.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Sharon A. Riddler reports a relationship with Gilead Sciences Inc that includes: funding grants. Sharon A. Riddler reports a relationship with Merck & Co Inc that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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