A simple and fast LC-MS/MS method for the simultaneous determination of tenofovir alafenamide and tenofovir in human plasma.

A simple and fast liquid chromatography-tandem mass spectrometry method was established and validated for the simultaneous determination of tenofovir alafenamide (TAF) and tenofovir (TNF) in human plasma. A simple protein precipitation procedure was employed to extract analytes from plasma. Chromatographic separation was performed on an Eclipse Plus C18 column utilizing a fast gradient elution starting with 2% of 2 mM ammonium acetate-formic acid (100/0.1, v/v) followed by increasing the percentage of acetonitrile. Detection was performed on a tandem mass spectrometer equipped with an electrospray ionization source operated in the positive ionization mode, using the transitions m/z 477.2 → m/z 346.1 for TAF and m/z 288.1 → m/z 176.1 for TNF. TAF-d5 and TNF-d7 were used as the internal standard of TAF and TNF, respectively. The method was validated in the concentration ranges 1.25-500 ng/mlfor TAF and 0.300-15.0 ng/ml for TNF with acceptable accuracy and precision.

Extent of In Utero Transfer of Tenofovir From Mother to Fetus: A Paired Analysis of Hair Specimens Collected at Birth From a Cohort in the United States.

Understanding in utero transfer of antiretrovirals is critical for interpreting safety. Hair levels measure cumulative exposure. We measured tenofovir (TFV) concentrations in hair at delivery among women living with human immunodeficiency virus receiving TFV disoproxil fumarate-based treatment and their infants, using liquid chromatography-tandem mass spectrometry. Among 103 mother-infant pairs, the mean log10 ratio of infant-to-maternal TFV levels was 1.08 (95% confidence interval, .97-1.20). TFV transfer was 60% lower from mothers who had preterm compared with term deliveries and 42% lower from mothers who had cesarean compared with vaginal deliveries. Like prior studies assessing transfer via short-term measures (plasma, cord blood, amniotic fluid), we found high cumulative transfer using hair.

A simple alternative and improved HPLC method for the estimation of doravirine, lamivudine, and tenofovir disoproxil fumarate in solid oral dosage form.

A novel method was developed for the simultaneous estimation of the doravirine, lamivudine, and tenofovir disoproxil fumarate in the pharmaceutical dosage form. The chromatogram was run through an Ascentis C18 column (150 × 4.6 mm, 2.7 µm), with the mobile phase consisting of a phosphate buffer and acetonitrile in the ratio of 50:50 (v/v). The mobile phase was pumped through the column at a flow rate of 1 mL/min. The column temperature was maintained at 30°C. The optimized wavelength for doravirine, lamivudine, and tenofovir disoproxil fumarate was 230.0 nm. The retention times for doravirine, lamivudine, and tenofovir disoproxil were 2.222, 2.764, and 3.403 min, respectively; the relative standard deviation (%) values of method precision for doravirine, lamivudine, and tenofovir disoproxil were 0.6, 0.6, and 0.1, respectively. The % recovery was 100.20%, 100.15%, and 100.36% for doravirine, lamivudine, and tenofovir disoproxil fumarate, respectively. The limit of detection and limit of quantification values were obtained from regression equations of doravirine, lamivudine, and tenofovir disoproxil fumarate, and were 0.24 and 0.73 ppm, 0.53 and 1.60 ppm, and 0.47 and 1.43 ppm, respectively. The regression equations of doravirine, lamivudine, and tenofovir disoproxil fumarate were y = 17,541x + 117,303, y = 15,555x + 10,791, and y = 15,250x + 31,663, respectively. The method developed was accurate, simple, precise, sensitive, and economical. Hence, it could be adopted for regular quality control for estimation of doravirine, lamivudine, and tenofovir disoproxil fumarate in pharmaceutical industries.

Rapid quantification of tenofovir in umbilical cord plasma and amniotic fluid in hepatitis B mono-infected pregnant women during labor by ultra-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: Tenofovir (TFV) is a first-line antiviral agent against hepatitis B virus (HBV) and is recommended for the prevention of mother-to-infant transmission of HBV. To study the distribution of TFV in umbilical cord plasma and amniotic fluid of HBV-infected pregnant women, a rapid and sensitive method for TFV determination was developed and validated. METHODS: The quantification method was developed using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). The analytes were separated on an Acquity UPLC HSS T3 column under gradient elution with methanol and 0.01% ammonia solution in 10 mM ammonium acetate/water. This is the first reported method for the determination of TFV using alkaline rather than acidic mobile phases. Linearity, accuracy, precision, limit of quantification, specificity and stability were assessed. RESULTS: Detection of TFV was achieved within 4 min. The calibration curves for TFV quantification showed excellent linearity in the range of 1-500 ng/mL. The intra- and interbatch precision and accuracy ranged from -4.35% to 6.92%. This method was successfully applied to determination of samples from 50 HBV mono-infected women undergoing tenofovir disoproxil fumarate therapy. The mean concentrations of TFV in the umbilical cord and amniotic fluid samples were 29.2 (4.6-86) and 470.9 (156-902) ng/mL, respectively, which showed a moderate positive correlation (r = 0.5299, P<0.001). CONCLUSIONS: A simple, rapid but sensitive bioanalytical method to determine TFV concentration in both umbilical cord plasma and amniotic fluid using LC/MS/MS was developed and applied to HBV-infected women during labor who were undergoing TDF therapy, which will help us understand the efficacy and safety of tenofovir during pregnancy.

Simple thin layer chromatography-ultraviolet spectrophotometric method for quality assessment of binary fixed-dose-combinations of lamivudine/tenofovir disoproxil fumarate and lamivudine/zidovudine in tablet formulations.

Antiretroviral fixed-dose-combination drugs are best assayed with high-performance liquid chromatography, or liquid chromatography-tandem mass spectrometry. However, most scientists in developing nations have no access to these expensive instruments. A more affordable quantitative technique is the use of ultraviolet-visible spectroscopy-where often the absorption spectra of these antiretrovirals are overlapping; thus complex derivative methodologies are required for quantification. A simple, rapid, and accurate thin layer chromatography-ultraviolet spectrophotometric method for the quantification of binary mixtures of lamivudine, zidovudine, and tenofovir-disoproxil-fumarate in tablet formulations was developed. Lamivudine/tenofovir-disoproxil-fumarate and lamivudine/zidovudine were extracted and separated on glass thin-layer chromatography plates. Drugs were identified in ultraviolet light at 254 nm and quantified in acidic medium using ultraviolet spectrophotometry. The retardation factors were 0.43, 0.79, and 0.81 for lamivudine, tenofovir-disoproxil-fumarate, and zidovudine, respectively, with corresponding absorption maxima at 270, 260, and 265 nm. Linearity ranged from 1 to 40 µg/mL for all drugs (R = 0.9998-0.9999), while recovery studies were 95.10-102.11% and amount in formulations ranged from 97.99 ± 0.63 to 101.47 ± 2.39%. The paired t-test (n = 5) indicated no significant difference between the proposed and high-performance liquid chromatography methods, hence comparable and can be used as an alternative method in routine quality determination of antiretroviral medicines.

Comparing self-reported medication adherence measures with hair antiretroviral concentration among people living with HIV in Guangxi, China.

BACKGROUND: Antiretroviral adherence is essential to HIV treatment efficacy. Various self-reported measures are commonly used for assessing antiretroviral adherence. Limited data are available regarding the validity of those self-reported measures in comparison with long-term objective biomarkers of adherence measures such as hair measures. METHODS: Self-reported adherence (frequency, percentage, and visual analog scale [VAS]) and hair tenofovir concentration were evaluated at a single time point from 268 people living with HIV in China. The responses to each of three self-reported measures were converted into percentage and then dichotomized as "optimal" (100%) vs. "suboptimal" (less than 100%) adherence. Two composite adherence scores (CAS) were created from the three self-reported measures: (1) an overall adherence was the average percentage of the three self-reported measures; (2) responses were termed optimal adherence if participants reporting optimal adherence in all three self-reported measures, while were termed suboptimal adherence. Hair tenofovir concentration was also dichotomized as "optimal" (above the limit of quantitation, 36 pg/mg) vs. "suboptimal" adherence (blow 36 pg/mg). Spearman correlation, kappa statistics, and logistic regression analysis were used to calculate the correlations, agreements, and predictions of self-reported measures with hair measure, respectively. RESULTS: Overall adherence, but any of the three self-reported adherence, was correlated with hair tenofovir concentration (r = 0.13, p < 0.05). Self-reported optimal adherence in VAS and CAS measures were agreed with and predicted optimal adherence assessed by hair measure (Kappa = 0.107, adjusted OR = 1.88, 95% CI 1.03-3.45; Kappa = 0.109, adjusted OR = 1.80, 95% CI 1.02-3.18; all p < 0.05, respectively). CONCLUSION: VAS may be a good individual self-reported measure for antiretroviral adherence, and CAS may be a good composite self-reported measure for antiretroviral adherence.

Comparison of Measures of Adherence to Human Immunodeficiency Virus Preexposure Prophylaxis Among Adolescent and Young Men Who Have Sex With Men in the United States.

Background: Young men-who-have-sex-with-men (MSM) are disproportionately impacted by human immunodeficiency virus (HIV). Preexposure prophylaxis (PrEP) could reduce HIV acquisition among youth, but suboptimal adherence threatens effectiveness. Optimal metrics of PrEP adherence among adolescents have remain undefined. Methods: The Adolescent Trials Network 110/113 studies provided daily oral PrEP with tenofovir (TFV) disoproxil fumarate/emtricitabine over 48 weeks to a diverse population of MSM (aged 15-22 years). Self-reported adherence was assessed and PrEP drug concentrations measured from hair and dried blood spot (DBS) samples; 23% of participants received Wisepill electronic monitoring devices. The average number of PrEP doses per week taken was estimated, and concordance between measures assessed. Results: Among 243 participants, hair samples were collected at 1186/1238 (96%) person-visits. The concordance of TFV levels in hair and TFV-diphosphate in DBS around thresholds consistent with taking ≥4 and 7 PrEP doses/week was high (76% and 80%). Hair and DBS concentrations correlated poorly with self-report and Wisepill metrics. Through week 12, 40%-60% of participants (by hair and DBS), ≤31% (Wisepill), and >85% (self-report) were estimated to have taken ≥4 PrEP doses/week (a threshold associated with protection among MSM). For all measures except self-report, adherence declined over time, with half of participants taking <2 doses/week by week 48. Conclusions: Among youth on PrEP, adherence waned over time. Self-report overestimated adherence, and use of Wisepill was limited. Hair collection was highly acceptable and provided similar interpretations to DBS. Incorporation of either metric in future PrEP studies among youth could identify suboptimal adherence and trigger interventions.

Impact of Sex on the Pharmacokinetics and Pharmacodynamics of 1% Tenofovir Gel.

BACKGROUND: Tenofovir (TFV) gel partially protected against human immunodeficiency virus (HIV) in one but not subsequent trials. The disappointing results were attributed largely to poor adherence. However, timing of gel application relative to sex may impact pharmacokinetics and contribute to outcomes. Thus, we conducted a single-dose pharmacokinetic study of TFV gel applied 1 or 24 hours before or 1 hour before and 1 hour after (BAT) sex and compared results with dosing without sex. METHODS: Twenty-four couples were enrolled; cervicovaginal lavage (CVL) and tissue were collected 2 hours after sex with matching timed collections at no sex visits and assayed for drug concentrations and CVL anti-HIV activity. RESULTS: Compared with dosing without sex, median TFV concentrations after sex decreased 72% and 78% (P < .001) in CVL, 75% and 71% (P < .001) in vaginal tissue, and 75% (P = .06) and 55% (P < .001) in cervical tissue with -1 hour and -24 hour dosing, respectively. Median concentration of TFV-diphosphate also decreased significantly in cervical tissue with -1 hour, dosing. BAT dosing resulted in drug levels at least as great as those in the absence of sex. Percent inhibition of HIV infection by post-coital CVL increased significantly from median (interquartile range) of 55% (54%) in the absence of gel to 99% (7%), 77% (57%), and 100% (0.4%) with -1 hour, -24 hour, or BAT dosing, respectively, and correlated significantly with drug concentration. CONCLUSIONS: Timing of TFV gel application relative to sex significantly impacts drug levels. BAT dosing or sustained delivery may be optimal for preexposure prophylaxis.

Strong Correlation Between Concentrations of Tenofovir (TFV) Emtricitabine (FTC) in Hair and TFV Diphosphate and FTC Triphosphate in Dried Blood Spots in the iPrEx Open Label Extension: Implications for Pre-exposure Prophylaxis Adherence Monitoring.

Self-reported adherence to pre-exposure prophylaxis (PrEP) has limitations, raising interest in pharmacologic monitoring. Drug concentrations in hair and dried blood spots (DBS) are used to assess long-term-exposure; hair shipment/storage occurs at room temperature. The iPrEx Open Label Extension collected DBS routinely, with opt-in hair collection; concentrations were measured with liquid chromatography/tandem mass spectrometry. In 806 hair-DBS pairs, tenofovir (TFV) hair levels and TFV diphosphate (DP) in DBS were strongly correlated (Spearman coefficient r = 0.734; P < .001), as were hair TFV/DBS emtricitabine (FTC) triphosphate (TP) (r = 0.781; P < .001); hair FTC/DBS TFV-DP (r = 0.74; P < .001); hair FTC/DBS FTC-TP (r = 0.587; P < .001). Drug detectability was generally concordant by matrix. Hair TFV/FTC concentrations correlate strongly with DBS levels, which are predictive of PrEP outcomes.

Detectable HIV RNA in late pregnancy associated with low tenofovir hair levels at time of delivery among women living with HIV in the United States.

OBJECTIVE: We evaluated peripartum tenofovir (TFV) exposure via hair measures among women living with HIV in the United States. DESIGN: Observational cohort study. METHODS: Hair samples were collected at or shortly after childbirth among mothers enrolled in the Surveillance Monitoring for Antiretroviral Therapy Toxicities Study of the Pediatric HIV/AIDS Cohort Study between 6/2014 and 7/2016. Among mothers receiving TFV disoproxil fumarate (TDF)-based regimens during pregnancy, TFV hair concentrations were analyzed using liquid chromatography/tandem mass spectrometry. Weight-normalized TFV concentrations were log10 transformed. Multivariable linear regression assessed correlates of TFV concentrations. RESULTS: Overall, 121 mothers on TDF-based antiretroviral therapy during pregnancy had hair specimens tested for TFV concentrations and were included in the analysis. Median age at delivery was 31 years [interquartile range (IQR) 26-36]; 71% self-identified as non-Hispanic black, and 10% had unsuppressed viral loads in late pregnancy (HIV RNA ≥ 400 copies/ml). Median time from birth to hair collection was 3 days (IQR 1-14) and median TFV hair concentration was 0.02 ng/mg (IQR 0.01-0.04). In multivariable models, an unsuppressed viral load in late pregnancy was associated with 80% lower adjusted mean peripartum TFV concentrations than pregnancies with viral suppression (95% confidence interval: -90% to -59%, P < 0.001). Use of TDF only in the first trimester and attaining high school graduation were also associated with lower TFV hair concentrations. CONCLUSION: Unsuppressed viral load during late pregnancy was strongly associated with lower maternal TFV hair concentrations at birth, though viremia was rare. Efforts to improve maternal virological outcomes and eliminate vertical HIV transmission could incorporate drug exposure monitoring using hair or other metrics.

"I wish to remain HIV negative": Pre-exposure prophylaxis adherence and persistence in transgender women and men who have sex with men in coastal Kenya.

BACKGROUND: Transgender women (TGW) and men who have sex with men (MSM) in sub-Saharan Africa have high HIV acquisition risks and can benefit from daily pre-exposure prophylaxis (PrEP). We assessed PrEP adherence by measuring tenofovir-diphosphate (TFV-DP) levels and explore motives for PrEP persistence in TGW and MSM. METHODS: Participants were enrolled in a one-year PrEP programme and made quarterly visits irrespective of whether they were still using PrEP. At their month 6 visit, participants provided a dried blood spot to test for TFV-DP levels; protective levels were defined as those compatible with ≥4 pills per week (700-1249 fmol/punch). Before TFV-DP levels were available, a sub-set of these participants were invited for an in-depth interview (IDI). Semi-structured IDI topic guides were used to explore motives to uptake, adhere to, and discontinue PrEP. IDI data were analyzed thematically. RESULTS: Fifty-three participants (42 MSM and 11 TGW) were enrolled. At month 6, 11 (20.7%) participants (8 MSM and 3 TGW) were lost to follow up or stopped taking PrEP. Any TFV-DP was detected in 62.5% (5/8) of TGW vs. 14.7% of MSM (5/34, p = 0.01). Protective levels were detected in 37.5% of TGW (3/8), but not in any MSM. Nineteen IDI were conducted with 7 TGW and 9 MSM on PrEP, and 1 TGW and 2 MSM off PrEP. Unplanned or frequent risky sexual risk behaviour were the main motives for PrEP uptake. Among participants on PrEP, TGW had a more complete understanding of the benefits of PrEP. Inconsistent PrEP use was attributed to situational factors. Motives to discontinue PrEP included negative reactions from partners and stigmatizing healthcare services. CONCLUSION: While MSM evinced greater adherence challenges in this PrEP programme, almost 40% of TGW were protected by PrEP. Given high HIV incidences in TGW these findings hold promise for TGW PrEP programming in the region.

Short Communication: Higher Tenofovir Concentrations in Hair Are Associated with Decreases in Viral Load and Not Self-Reported Adherence in HIV-Infected Adolescents with Second-Line Virological Treatment Failure.

Objective methods of measuring antiretroviral adherence are limited. We assessed the relationship between tenofovir disoproxil fumarate (TDF) hair concentrations, self-reported adherence, and virological outcomes in HIV-infected adolescents in Harare, Zimbabwe. HIV-infected adolescents on atazanavir/ritonavir-based second-line treatment for >6 months with viral load (VL) ≥1,000 copies/mL were randomized to either modified directly administered antiretroviral therapy (mDAART) or standard of care. Hair and VL samples were collected at baseline and after 90 days. Treatment outcome was defined as TDF concentrations in hair. Virological suppression was defined as VL <1,000 copies/mL. Thirty-four adolescents had TDF concentrations measured at baseline and follow-up. Mean (median); range age was 16 (16); 13-18 years and 53% were females. Nineteen (56%) were randomized to mDAART. Mean (SD); range TDF concentrations were 0.03 (0.04); 0-0.17 ng/mg hair and 0.06 (0.06); 0-0.3 ng/mg hair at baseline and follow-up, respectively. Higher TDF concentrations were associated with decreased VL [regression coefficient (RC) 0.8; 95% confidence interval (CI) 0.7-1.0; p = .008] and mDAART (RC 0.5; 95% CI 0.3-1.0; p = .04), but were not associated with self-reported adherence and virological suppression (VL <1,000 copies/mL). Higher TDF hair concentrations were observed with virological decrease and an adherence intervention. Hair antiretroviral concentrations could be useful in triggering adherence interventions among adolescents with second-line virological failure.

The effect of depot medroxyprogesterone acetate on tenofovir alafenamide in rhesus macaques.

Prevention of HIV infection and unintended pregnancies are public health priorities. In sub-Saharan Africa, where HIV prevalence is highest, depot medroxyprogesterone acetate (DMPA) is widely used as contraception. Therefore, understanding potential interactions between DMPA and antiretrovirals is critical. Here, we use a macaque model to investigate the effect of DMPA on the pharmacology of the antiretroviral tenofovir alafenamide (TAF). Female rhesus macaques received 30 mg of DMPA (n = 9) or were untreated (n = 9). Macaques received a human equivalent dose of TAF (1.5 mg/kg) orally by gavage. Tenofovir (TFV) and TFV-diphosphate (TFV-DP) were measured in blood, secretions, and tissues over 72 h. The median area under the curve (AUC0-72h) values for TFV-DP in peripheral blood mononuclear cells were similar in DMPA-treated (6991 fmol*h/106 cells) and untreated controls (5256 fmol*h/106 cells) (P = 0.174). Rectal tissue TFV-DP concentrations from DMPA+ animals [median: 20.23 fmol/mg of tissue (range: 4.94-107.95)] were higher than the DMPA- group [median: below the limit of quantification (BLOQ-11.92)], (P = 0.019). TFV-DP was not detectable in vaginal tissue from either group. A high-dose DMPA treatment in macaques was associated with increased rectal TFV-DP levels, indicating a potential tissue-specific drug-drug interaction. The lack of detectable TFV-DP in the vaginal tissue warrants further investigation of PrEP efficacy with single-agent TAF products. DMPA did not affect systemic TAF metabolism, with similar PBMC TFV-DP in both groups, suggesting that DMPA use should not alter the antiviral activity of TAF.

Tenofovir and emtricitabine concentrations in hair are comparable between individuals on tenofovir disoproxil fumarate versus tenofovir alafenamide-based ART.

Tenofovir disoproxil fumarate (TDF) in combination with emtricitabine (FTC) is the backbone for both human immunodeficiency virus (HIV) treatment and pre-exposure prophylaxis (PrEP) worldwide. Tenofovir alafenamide (TAF) with FTC is increasingly used in HIV treatment and was recently approved for PrEP among men-who-have-sex-with-men. TDF and TAF are both metabolized into tenofovir (TFV). Antiretrovirals in plasma are taken up into hair over time, with hair levels providing a long-term measure of adherence. Here, we report a simple, robust, highly sensitive, and validated high-performance liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS)-based analytical method for analyzing TFV and FTC from individuals on either TDF/FTC or TAF/FTC in small hair samples. TFV/FTC are extracted from ~5 mg hair and separated on a column using a gradient elution. The lower quantification limits are 0.00200 (TFV) and 0.0200 (FTC) ng/mg hair; the assay is linear up to 0.400 (TFV) and 4.00 (FTC) ng/mg hair. The intra-day and inter-day coefficients of variance (CVs) are 5.39-12.6% and 6.40-13.5% for TFV and 0.571-2.45% and 2.45-5.16% for FTC. TFV concentrations from participants on TDF/FTC-based regimens with undetectable plasma HIV RNA were 0.0525 ± 0.0295 ng/mg, whereas those from individuals on TAF/FTC-based regimens were 0.0426 ± 0.0246 ng/mg. Despite the dose of TFV in TDF being 10 times that of TAF, hair concentrations of TFV were not significantly different for those on TDF versus TAF regimens. Pharmacological enhancers (ritonavir and cobicistat) did not boost TFV concentrations in hair. In summary, we developed and validated a sensitive analytical method to analyze TFV and FTC in hair and found that hair concentrations of TFV were essentially equivalent among those on TDF and TAF.

Rapid and efficient chiral method development for lamivudine and tenofovir disoproxil fumarate fixed dose combination using ultra-high performance supercritical fluid chromatography: A design of experiment approach.

Fixed dose combination (FDC) of tenofovir disoproxil fumarate (TDF) and lamivudine (3TC) is one of the most preferred FDC for the treatment of acquired immunodeficiency syndrome (AIDS)/human immunodeficiency virus (HIV) infection. To the best of authors' knowledge there are no reported methods for chiral purity estimation of both drugs simultaneously from a FDC. The current study was focused on the development of a single chiral method uisng supercritical fluid chromatography (SFC) for separation of stereoisomers of TDF and 3TC combination employing design of experiment (DoE) approach. Method development was planned in three steps by using different experimental designs for each step. I-optimal, Taguchi orthogonal array and face-centred central composite designs (CCD) were employed for primary parameter selection, secondary parameter screening and final method optimization, respectively. All six stereoisomers were separated in a 10 minute run on Chiralpak IA column with carbon di-oxide /methanol (containing 0.5 % v/v n-butylamine) as mobile phase at 1.5 mL/min in gradient mode. The optimized method was verified for performance through establishing specificity, precision, linearity, accuracy, limit of quantification, and solution stability. Resolution between each isomeric pair was more than 1.5. The method was found to be linear from 1.5 µg/mL to 7.5 µg/mL for 3TC and 7.5 µg/mL to 37.5 µg/mL for TDF stereoisomers. The R2 values for all the linearity curves for undesired isomers were greater than 0.995. The method proved to be rapid, reproducible and efficient to quantify stereoisomers of both drugs in a single run.

Determination of selected antiretroviral drugs in wastewater, surface water and aquatic plants using hollow fibre liquid phase microextraction and liquid chromatography - tandem mass spectrometry.

This work describes a simple and sensitive method for the simultaneous isolation, enrichment, identification and quantitation of selected antiretroviral drugs; emtricitabine, tenofovir disoproxil and efavirenz in aqueous samples and plants. The analytical method was based on microwave extraction and hollow fibre liquid phase microextraction technique coupled with ultra-high pressure liquid chromatography-high resolution mass spectrometry. A multivariate approach via a half-fractional factorial design was used focusing on six factors; donor phase pH, acceptor phase HCl concentration, extraction time, stirring rate, supported liquid membrane carrier composition and salt content. The optimal enrichment factors for emtricitabine, tenofovir disoproxil and efavirenz from aqueous phase were 78, 111 and 24, respectively. The analytical method yielded recoveries in the range of 86 to 111%, and quantitation limits for emtricitabine, tenofovir disoproxil and efavirenz in wastewater were 0.033, 0.10 and 0.53 µg L-1, respectively. The drugs were detected in most samples with concentrations up to 37.6 µg L-1 recorded for efavirenz in wastewater effluent. Roots of the water hyacinth plant had higher concentrations of the investigated drugs ranging from 7.4 to 29.6 µg kg-1. Overall, hollow fibre liquid phase microextraction proved to be an ideal tool for isolating and pre-concentrating the selected antiretroviral drugs from environmental samples.

Tenofovir concentrations in hair strongly predict virologic suppression in breastfeeding women.

BACKGROUND: Antiretroviral treatment (ART) adherence is often suboptimal in the perinatal period. We measured hair tenofovir (TFV) concentrations as a metric of adherence in postpartum women to understand patterns and predictors of adherence throughout this critical period. In addition, we examined the association between hair TFV concentrations and virologic outcomes. METHODS: Between 12/2012 and 09/2016, hair samples were collected longitudinally from delivery through breastfeeding from women on ART in the Promoting Maternal and Infant Survival Everywhere study (NCT01061151) in sub-Saharan Africa. Hair TFV levels were measured using validated methods. Using generalized estimating equations, we estimated the association between hair TFV levels and virologic suppression (<400 copies/ml) over time and assessed predictors of hair TFV levels. RESULTS: Hair TFV levels were measured at 370 visits in 71 women from delivery through a median of 14 months (interquartile range 12-15) of breastfeeding. Levels ranged from below detection (0.002) to 1.067 ng/mg (geometric mean: 0.047). After at least 90 days on ART, 69 women had at least one viral load measured (median 5 measures, range 1-9); 18 (26%) experienced viremia at least once. Each doubling of TFV level more than doubled odds of concurrent virologic suppression [odds ratio 2.35, 95% confidence interval (CI): 1.44-3.84, P = 0.0006] and was associated with 1.43 times the odds of future suppression (95% CI: 0.75-2.73, P = 0.28). Relative to the first 3 months after delivery, hair levels were highest in months 6-12 (1.42-fold higher, 95% CI: 1.09-1.85, P = 0.01). CONCLUSION: Hair TFV levels strongly predicted concurrent virologic suppression among breastfeeding women. Objective adherence metrics can supplement virologic monitoring to optimize treatment outcomes in this important transition period.

Simultaneous determination of intracellular concentrations of tenofovir, emtricitabine, and dolutegravir in human brain microvascular endothelial cells using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Combination antiretroviral therapy (cART) regimens are recommended for HIV patients to better achieve and maintain plasma viral suppression. Despite adequate plasma viral suppression, HIV persists inside the brain, which is, in part thought to result from poor brain penetration of antiretroviral drugs. In this study, a simple and ultra-sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of tenofovir, emtricitabine, and dolutegravir in cell lysates of an immortalized human brain microvascular endothelial cell line (hCMEC/D3) was developed and validated. Analytes were separated on a reverse phase C18 column using water and 0.1% formic acid in acetonitrile as mobile phases. The analytes were detected using positive electrospray ionization mode with multiple reaction monitoring (MRM). The assay was linear in the concentration range of 0.1-100 ng mL-1 for all analytes. Intra- and inter-assay precision and accuracy were within ±13.33% and ±10.53%, respectively. This approach described herein was used to determine the intracellular accumulation of tenofovir, emtricitabine, dolutegravir simultaneously in hCMEC/D3 cells samples.

Guides concerning tenofovir exposure via breastfeeding: A comparison of drug dosages by developmental stage.

Tenofovir disoproxil fumarate (TDF) is a prodrug of tenofovir, and after being administered orally, it converts to tenofovir in the blood. With the increasing use of TDF in women for treatment and prevention of mother-to-child transmission (MTCT) of both human immunodeficiency virus (HIV) and hepatitis B virus (HBV), or the pre-exposure prophylaxis (PrEP) for HIV, many nursing mothers have to understand the risk of exposure to tenofovir via breastmilk and make the decision about breastfeeding while on TDF treatment. Despite the safety record of TDF in pregnancy, some guidelines recommend against its use during breastfeeding. In this paper, we compared the dosage levels of tenofovir exposure in fetuses, breastfed infants, and children receiving tenofovir treatment. We found that breastfed infants were exposed to only 0.5%-16% of the tenofovir dosage that fetuses experienced via placental transfer, and 0.01-0.04% of the recommended weight-adjusted therapeutic dose. The assessment of toxicity risk from the dose perspective is an important and natural way of addressing safety concerns about exposure to tenofovir via breastfeeding. Based on the safety data from fetuses and children with tenofovir exposure, and the comparatively negligible exposure dosage from breastfeeding, our study supports mothers on TDF treatment should be encouraged to breastfeed.

Electrochemical and spectroscopic studies of the interaction of antiviral drug Tenofovir with single and double stranded DNA.

In the present study, the electrochemical behavior of the antiviral drug tenofovir (tnf) on a boron-doped diamond electrode (BDDE) has been studied using square-wave voltammetry (SWV). The experimental conditions such as the supporting electrolyte composition and the SWV parameters were optimized. Under the optimized conditions, a simple and sensitive SWV procedure for tnf determination was developed in the concentration range of 5.0 × 10-6 to 1.0 × 10-4 mol L-1. The calculated limit of detection and limit of quantification were equal to 5.6 × 10-7 and 1.9 × 10-6 mol L-1, respectively. The biological significance of the developed method was demonstrated by a quantitative analysis of the pharmaceutical formulations Viread and Tenofovir disoproxil Teva. Moreover, both voltammetric and spectroscopic techniques were used to study the interaction between tnf and DNA. Changes in the electrochemical and spectroscopic behavior of tnf in the presence of DNA were used to calculate the binding constants of the formed complexes. The estimated values of Gibbs free energy revealed that the formation of the drug-DNA complexes was a spontaneous and favorable process. Moreover, for free and bound tenofovir, kinetic parameters such as heterogeneous rate constant, electron transfer coefficient, and diffusion coefficient were determined.