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A simultaneous ultra-performance liquid chromatography photodiode array detection method was developed and validated for Elvitegravir, Tenofovir and Emtricitabine and Cobicistat drugs in tablet dosage forms. Effective liquid chromatographic quantification was achieved using a Luna C18 (100 × 2.6 mm, 1.6 μ) column with an acetonitrile: 0.1% v/v trifluoro acetic acid (80:20% v/v) mobile phase and a photo diode array detector at 260 nm for a shorter run time of 3 min. The respective retention times for Elvitegravir, Tenofovir, Emtricitabine and Cobicistat were 0.311, 0.792, 1.379 and 2.045 min. The range of linearity was 37.50-225.00, 2.50-15.00, 50.00-300.00 and 37.50-225.00 μg/mL for Elvitegravir, Tenofovir, Emtricitabine and Cobicistat, respectively. A mass spectrometer was employed for quantitative analysis using electron spray ionization multiple reaction monitoring in positive mode. The developed method was validated for different parameters to meet the acceptance criteria as per ICH Q2 (R1) guidelines. Using UPLC-ESI-MS, 12 degradants were identified and their mechanisms were studied., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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 C 18 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-d 5 and TNF-d 7 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., (© 2021 John Wiley & Sons, Ltd.)

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.

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., (© 2021 John Wiley & Sons, Ltd.)

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., (© 2021 John Wiley & Sons, Ltd.)

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., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

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., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

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 (AUC 0-72h ) values for TFV-DP in peripheral blood mononuclear cells were similar in DMPA-treated (6991 fmol*h/10 6  cells) and untreated controls (5256 fmol*h/10 6  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., (Published by Elsevier B.V.)

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., Competing Interests: The authors have declared that no competing interests exist.

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 R 2 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., Competing Interests: 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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2020 John Wiley & Sons, Ltd.)

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.

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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.

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 C 18 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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objectives: Women likely require higher adherence than men to preexposure prophylaxis (PrEP) with tenofovir disoproxil fumarate (TDF)/emtricitabine (FTC) for similar efficacy. Pharmacologic metrics of adherence predict efficacy better than self-report, but expected drug levels (adherence benchmarks) must be established using directly observed therapy. We sought to evaluate whether tenofovir hair concentrations differ between women and men receiving directly observed TDF/FTC., Methods: We assessed tenofovir hair concentrations in HIV-uninfected volunteers randomized to receive 100%, 67%, or 33% of daily dosing of TDF/FTC for 12 weeks (DOT-DBS, NCT02022657). Hair samples were collected at dosing weeks 4, 8, and 12 and every 3 weeks during a 12-week washout. Tenofovir concentrations in the proximal 1.5 cm of hair (representing ∼6 weeks of exposure) were analyzed using liquid chromatography/tandem mass spectrometry. Linear regression was used to model tenofovir hair concentrations in terms of sex, doses over the prior 6 weeks, and number of days since last dose., Results: A total of 264 hair samples were analyzed from 23 female and 24 male participants. Female participants had similar tenofovir hair concentrations to men (estimated fold-difference 0.92, 95% CI 0.75-1.13, P = 0.43). The estimated fold-difference in tenofovir levels for female versus male participants did not appreciably change when age (0.93, 95% CI 0.76-1.15), weight (0.89, 95% CI 0.71-1.11), or race/ethnicity (0.95, 95% CI 0.77-1.17) were added to the model., Conclusion: Women and men have similar adherence benchmarks for tenofovir in hair samples. As pharmacokinetic metrics are increasingly used for PrEP monitoring, these findings provide guidance for assessing adherence via hair concentrations.

A liquid chromatography with triple quadrupole mass spectrometry method was developed and validated for the determination of tenofovir and tenofovir alafenamide concentrations in human plasma and cerebrospinal fluid. Tenofovir and tenofovir alafenamide were extracted from matrix by solid phase extraction. The dried extraction eluents were dissolved in water for LC-MS/MS analysis. Separation was achieved with a Phenomenex Synergi 4 μm Polar-RP 80A column (50 × 2 mm) with a gradient elution of 0.1% formic acid in water and acetonitrile. The total run time was 5 min. Detection of analytes was achieved using electrospray ionization (positive mode) and triple quadrupole selected reaction monitoring. Standard curve concentrations ranged from 0.5 to 500 ng/mL for the plasma assay and 0.1-50 ng/mL for the cerebrospinal fluid assay. The intra- and inter-day accuracy and precision were less than 12% in low, medium, and high quality control samples for both matrices. The validated methods were applied to the analysis of plasma and cerebrospinal fluid samples of a patient undergoing tenofovir therapy which involved the switch from Stribild ® (elvitegravir 150 mg/cobicistat 150 mg/emtricitabine 200 mg/tenofovir disoproxil fumarate 300 mg) to Genvoya ® (elvitegravir 150 mg/cobicistat 150 mg/emtricitabine 200 mg/tenofovir alafenamide 10 mg)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective: We describe the third case report of seroconversion with multidrug resistant (MDR)-HIV despite pre-exposure prophylaxis (PrEP) with emtricitabine (FTC) and tenofovir (TFV) disoproxil (TDF)., Design: Case report., Methods: PrEP adherence was assessed via self-report, pharmacy records, and measuring TFV/FTC levels with liquid-chromatography/tandem-mass-spectrometry in plasma and hair. Segmental hair analysis was performed to assess PrEP adherence over prior months. Genotypic resistance was assessed., Results: A 34 year-old white MSM started daily FTC/TDF in February 2016 after being provided 11 refills. In March 2017, he developed fevers, chills, myalgias and was assessed, but no HIV test was sent. In April 2017, an antigen/antibody HIV test was reactive (day 0). On day 2, HIV-1 RNA was 27 316 copies/ml, genotyping revealed M184V, K70T, K65R, and K103N mutations, plasma TFV and FTC concentrations were consistent with recent dosing. To evaluate adherence over preceding months, a hair sample was collected at day 27 and segmental analysis of TFV/FTC levels performed in one-centimeter segments from the scalp. Hair drug levels (0.0434-0.0520 ng TFV/mg hair) were commensurate with consistently high PrEP adherence over the prior 3 months., Conclusions: This study employs segmental analysis of PrEP drug levels in hair for the first time to assess adherence over preceding months in the setting of an HIV seroconversion on PrEP. High adherence over the period of likely acquisition makes MDR-HIV infection the most likely scenario in this case. Adequate adherence assessment when examining PrEP failures and ensuring best practices in PrEP prescribing and follow-up are important.

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., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Objective: The US preexposure prophylaxis (PrEP) Demonstration Project (U.S. Demo) evaluated MSM on PrEP postmarketing and found low seroconversion rates. The objective of this study is to examine hair levels as an adherence measure to PrEP., Design: Using an 'opt-in' design, participants of PrEP Demo were invited to enroll into a substudy where hair was collected quarterly., Methods: Tenofovir concentrations were measured in hair by liquid chromatography/tandem mass spectrometry. Hair levels consistent with ≥4 doses/week (protective in other studies) defined adequate adherence. Mixed effects multivariate logistic regression models examined factors associated with ≥4 doses/week. Separate mixed effects models evaluated the relationship between hair PrEP levels and changes in creatinine clearance (CrCl) over time., Results: Overall, 58% of U.S. Demo participants enrolled into this opt-in study; reasons for nonparticipation included insufficient hair (61%) and concerns about hairstyle (27%). Hair and dried blood spots levels consistent with ≥4 doses/week were highly concordant (84%). Hair levels showed adequate adherence in 87% of 875 person-visits (among 280 participants). Factors associated with adequate adherence in multivariate models were amphetamine use [adjusted odds ratio (aOR) 2.59 (0.97-6.9, P 0.06)], condomless receptive anal sex [aOR 2.28 (1.19-4.40, P 0.01)], and stable housing [aOR 2.63 (1.03-6.67), P 0.04]. Hair levels of tenofovir showed a monotonic relationship with decline in CrCl (P 0.01 for trend)., Conclusion: In this substudy of the U.S. PrEP demonstration project, hair and dried blood spots levels were highly concordant and hair concentrations demonstrated adequate adherence 87% of the time, with stable housing and high-risk behavior associated with higher adherence. Daily PrEP drug taking is associated with modest declines in CrCl.

Objectives: To present the validation and clinical application of a LC-MS/MS method for the quantification of lamivudine (3TC), emtricitabine (FTC) and tenofovir (TFV) in dried blood spots (DBS) and dried breast milk spots (DBMS)., Methods: DBS and DBMS were prepared from 50 and 30μL of drug-spiked whole blood and human breast milk, respectively. Following extraction with acetonitrile and water, chromatographic separation utilised a Synergi polar column with a gradient mobile phase program consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Detection and quantification was performed using a TSQ Quantum Ultra triple quadrupole mass spectrometer. The analytical method was used to evaluate NRTI drug levels in HIV-positive nursing mothers-infant pairs., Results: The assay was validated over the concentration range of 16.6-5000ng/mL for 3TC, FTC and TFV in DBS and DBMS except for TFV in DBMS where linearity was established from 4.2-1250ng/mL. Intra and inter-day precision (%CV) ranged from 3.5-8.7 and accuracy was within 15% for all analytes in both matrices. The mean recovery in DBS was >61% and in DBMS >43% for all three analytes. Matrix effect was insignificant. Median AUC 0-8 values in maternal DBS and DBMS, respectively, were 4683 (4165-6057) and 6050 (5217-6417)ngh/mL for 3TC, 3312 (2259-4312) and 4853 (4124-6691)ngh/mL for FTC and 1559 (930-1915) and 56 (45-80)ngh/mL for TFV. 3TC and FTC were quantifiable (>16.6ng/mL) in DBS from 2/6 and 1/6 infants respectively whereas TFV was undetectable in all infants., Conclusions: DBS and DBMS sampling for bioanalysis of 3TC, FTC and TFV is straightforward, robust, accurate and precise, and ideal for use in low-resource settings., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Antiretroviral-based strategies for HIV prevention have shown inconsistent results in women. We investigated whether vaginal microbiota modulated tenofovir gel microbicide efficacy in the CAPRISA (Centre for the AIDS Program of Research in South Africa) 004 trial. Two major vaginal bacterial community types-one dominated by Lactobacillus (59.2%) and the other where Gardnerella vaginalis predominated with other anaerobic bacteria (40.8%)-were identified in 688 women profiled. Tenofovir reduced HIV incidence by 61% ( P = 0.013) in Lactobacillus- dominant women but only 18% ( P = 0.644) in women with non- Lactobacillus bacteria, a threefold difference in efficacy. Detectible mucosal tenofovir was lower in non- Lactobacillus women, negatively correlating with G. vaginalis and other anaerobic bacteria, which depleted tenofovir by metabolism more rapidly than target cells convert to pharmacologically active drug. This study provides evidence linking vaginal bacteria to microbicide efficacy through tenofovir depletion via bacterial metabolism., (Copyright © 2017, American Association for the Advancement of Science.)

HIV-infected patients treated with certain nucleoside reverse transcriptase inhibitors (NRTIs) have experienced adverse effects due to drug-related mitochondrial toxicity. Tenofovir alafenamide (TAF) is a novel prodrug of the NRTI tenofovir (TFV) with an improved safety profile compared to tenofovir disoproxil fumarate (TDF). Prior in vitro studies have demonstrated that the parent nucleotide TFV has no significant effects on mtDNA synthesis. This study investigated whether clinically relevant TAF and TDF exposures affect mtDNA content in human lymphocytes. First, activated or resting peripheral blood mononuclear cells (PBMCs), as well as MT-2 and Jurkat T-cell lines, were continuously treated with ddC for 10 days to establish their susceptibility to mtDNA depletion. PBMCs had low sensitivity to NRTI-mediated mtDNA depletion in vitro. In contrast, ddC treatment of rapidly dividing MT-2 and Jurkat cells resulted in a dose-dependent decrease in mtDNA. Therefore, these two T-cell lines were selected for evaluating TAF and TDF treatment effects. MT-2 and Jurkat cells were pulse-treated with TAF or TDF every 24 h for 10 days to mimic pharmacologically relevant drug exposures. Pulse treatment of cells with 3.3 μM TAF or 1.1 μM TDF for 10 days resulted in 2- to 7-fold greater steady-state intracellular TFV-diphosphate (TFV-DP) levels than those observed clinically in TAF- or TDF-treated patients. At these concentrations, no significant TAF- (106.7% and 84.1% of control; p = 0.77 and 0.12 for MT-2 and Jurkat, respectively) or TDF- (100.6% and 91.0% of control; p = 0.91 and 0.37, respectively) associated reduction in mtDNA content was observed compared with untreated control cells. This study demonstrates that, despite delivering higher intracellular levels of TFV-DP than TDF, TAF does not inhibit mtDNA synthesis in vitro at concentrations exceeding the clinically relevant intracellular drug exposures. Thus, TAF has a low potential for mitochondrial toxicity in T-cells of HIV-infected patients., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Chlamydia trachomatis and Trichomonas vaginalis, two prevalent sexually transmitted infections, are known to increase HIV risk in women and could potentially diminish preexposure prophylaxis efficacy, particularly for topical interventions that rely on local protection. We investigated in macaques whether coinfection with Chlamydia trachomatis/Trichomonas vaginalis reduces protection by vaginal tenofovir (TFV) gel., Methods: Vaginal TFV gel dosing previously shown to provide 100 or 74% protection when applied either 30 min or 3 days before simian HIV(SHIV) challenge was assessed in pigtailed macaques coinfected with Chlamydia trachomatis/Trichomonas vaginalis and challenged twice weekly with SHIV162p3 for up to 10 weeks (two menstrual cycles). Three groups of six macaques received either placebo or 1% TFV gel 30 min or 3 days before each SHIV challenge. We additionally assessed TFV and TFV diphosphate concentrations in plasma and vaginal tissues in Chlamydia trachomatis/Trichomonas vaginalis coinfected (n = 4) and uninfected (n = 4) macaques., Results: Chlamydia trachomatis/Trichomonas vaginalis coinfections were maintained during the SHIV challenge period. All macaques that received placebo gel were SHIV infected after a median of seven challenges (one menstrual cycle). In contrast, no infections were observed in macaques treated with TFV gel 30 min before SHIV challenge (P < 0.001). Efficacy was reduced to 60% when TFV gel was applied 3 days before SHIV challenge (P = 0.07). Plasma TFV and TFV diphosphate concentrations in tissues and vaginal lymphocytes were significantly higher in Chlamydia trachomatis/Trichomonas vaginalis coinfected compared with Chlamydia trachomatis/Trichomonas vaginalis uninfected macaques., Conclusion: Our findings in this model suggest that Chlamydia trachomatis/Trichomonas vaginalis coinfection may have little or no impact on the efficacy of highly effective topical TFV modalities and highlight a significant modulation of TFV pharmacokinetics.

Millions of people worldwide live with human immunodeficiency virus (HIV) infection thus justifying the continuous search for new prevention and treatment strategies, including topical microbicide products combining antiretroviral drugs (ARVs) such as tenofovir (TFV) and efavirenz (EFV). Therefore, the aim of this work was to develop and validate a high performance liquid chromatography method coupled to triple quadrupole-tandem mass spectrometry (HPLC-MS/MS) for the quantification of TFV and EFV in biological matrices (mouse vaginal tissue, vaginal lavage and blood plasma). Chromatographic separation was achieved using a reversed phase C18 column (3μm, 100×2.1mm) at 45°C and elution in gradient mode using a combination of 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile at 0.35mLmin -1 . Total run time was 9min, with retention time of 2.8 and 4.1min for TFV and EFV, respectively. The MS was operated in positive ionization mode (ESI+) for TFV and in negative ionization mode (ESI-) for EFV detection. Data were acquired in selected reaction monitoring (SRM) mode and deuterated ARVs were employed as internal standards. Calibration curves were linear for ARV concentrations ranging from 4 to 500ngmL -1 with LOD and LOQ for both analytes ≤0.4 and ≤0.7ngmL -1 in sample extracts, respectively. The method was found to be specific, accurate (96.0-106.0% of nominal values) and precise (RSD<2.4%) in all matrices. Both TFV and EFV were found to be stable in all matrices after standing 24h at room temperature (20°C) or in the autosampler, and after three freeze-thawing cycles. Mean recovery values of ARVs spiked in mice tissues or fluids were ≥88.4%. Matrix effects were observed for EFV determination in tissue and plasma extracts but compensated by the use of deuterated internal standards. The proposed methodology was successfully applied to a pharmacokinetic study following intravaginal administration of both ARVs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this study, stress degradation behaviour of tenofovir alafenamide fumarate (TAF), a novel prodrug of tenofovir, was investigated and compared with currently used prodrug congener, tenofovir disoproxil fumarate (TDF), whose intrinsic stability was reported by us earlier [14]. Also, pH stability and gastrointestinal stability studies were conducted on both the drugs. High performance liquid chromatography (HPLC) analysis of stressed samples of TAF revealed formation of six degradation products (DPs) against twelve characterized earlier in the case of TDF (RSC Adv. 5(2015) 96117-96129). Like TDF, characterization of DPs of TAF was done by using sophisticated hyphenated liquid chromatography-high resolution mass spectrometry (LC-HRMS) and multistage mass spectrometry (MS n ) tools. pH-stability studies between pH 1.2-10 revealed greater stability of TAF, except in acidic conditions, where TAF was degraded extensively. Investigation of gastrointestinal stability in simulated gastric fluid (SGF), simulated intestinal fluid (SIF) and fed state simulated gastric fluid (FeSSGF) suggested that TAF must be administered in fed state, as the drug was practically stable in FeSSGF as compared to extensive loss at acidic pH and in SGF., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: As pre-exposure prophylaxis (PrEP) with tenofovir disoproxil fumarate and emtricitabine for the prevention of HIV infection is rolled out internationally, strategies to maintain effectiveness and to minimise adverse effects merit consideration. In this study, we aimed to assess reductions in renal function and predictors of renal toxicity in a large open-label study of PrEP., Methods: As part of the iPrEx open-label extension (OLE) study, men who have sex with men or transgender women aged 18-70 years who were HIV negative and had participated in three previous PrEP trials from Brazil, Ecuador, Peru, South Africa, Thailand, and the USA were enrolled into an open-label PrEP study. There were no restrictions on current renal function for enrolment into iPrEx OLE, in which participants were given combination tablets of tenofovir disoproxil fumarate (300 mg) and emtricitabine (200 mg) and advised to take one tablet per day. At follow-up sessions every 12 weeks, participants' creatinine clearance on PrEP was estimated and in a subset of participants, hair samples were collected to measure tenofovir and emtricitabine concentrations (a measure of adherence and exposure) via liquid-chromatography-tandem-mass-spectrometry. Reductions in creatinine clearance from baseline were calculated and predictors of decline were identified by use of multivariate models. iPrEx is registered with ClinicalTrials.com, number NCT00458393., Findings: Baseline characteristics were similar between all participants in iPrEx-OLE (1224 participants with 7475 person-visits) and those participating in the hair substudy (220 participants with 1114 person-visits). During a median of 72 weeks, the mean decline in creatinine clearance was -2·9% (95% CI -2·4 to -3·4; p trend <0·0001), but declines were greater for those who started PrEP at older ages: participants aged 40-50 years at baseline had declines of -4·2% (95% CI -2·8 to -5·5) and participants older than 50 years at baseline had declines of -4·9% (-3·1 to -6·8). In multivariate models, age and baseline creatinine clearance less than 90 mL/min predicted declines in renal function. We identified a monotonic association between percentage decrease in creatinine clearance and the number of doses of tenofovir disoproxil fumarate and emtricitabine taken per week, as estimated by hair concentrations of tenofovir and emtricitabine (p trend =0·008)., Interpretation: Our data suggest that the frequency of safety monitoring for PrEP might need to be different between age groups and that pharmacological measures can monitor for toxic effects as well as adherence., Funding: National Institutes of Health., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Accurate and sensitive liquid-chromatography tandem mass spectrometry method for the quantification of tenofovir and emtricitabine in seminal plasma has been developed and full validated. Molecules were separated by high-performance liquid chromatography on an Atlantis T3 C18 column using a gradient of deionized water and methanol, including 0.05% formic acid (250μl/min) and detected by electrospray ionisation/tandem mass spectrometry in positive ion mode. The method was validated over a clinical range of 3.13-1000ng/mL for tenofovir and 6.25-2000ng/mL for emtricitabine. Inter and intra-assay precisions were <9.37% for tenofovir and<10.88% for emtricitabine, and accuracies were between 0.48% and 8.43% for tenofovir, and between 0.64% and 13.87% for emtricitabine. The developed method was successfully applied for analysing tenofovir and emtricitabine concentrations in seminal plasma samples from a clinical study. The use of tandem mass spectrometry can be a suitable method for the analysis of this kind of matrices, providing high sensitivity and specificity to the analysis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A new simple, rapid stability indicating assay method has been developed and validated for the determination of emtricitabine, tenofovir disoproxil fumarate, elvitegravir and cobicistat using reverse-phase high-performance liquid chromatography in their pharmaceutical dosage form. The chromatographic separation was performed on an ODS column (250 × 4.6 mm, 5 µm) using mobile phase A (potassium dihydrogen orthophosphate, pH adjusted to 2.5) and mobile phase B (acetonitrile) in the ratio of 55:45% v/v at a flow rate of 1 mL/min. The analytes were detected at 250 nm. The method was found to be linear in the concentration range of 2-12 µg/mL for EMT, 3-18 µg/mL for TNDF, 1.5-9 µg/mL for ELV and COB, with the coefficient value (R(2)) of >0.9990. The accuracy was measured via recovery studies and found to be acceptable, and the percentage recoveries were found in the range of 99.93-100.08 ± 0.5%. Forced degradation studies were also conducted, and the drugs were subjected to various stress conditions such as acid hydrolysis, base hydrolysis, oxidative, photolytic and thermal degradation. The proposed method was successfully validated and applied for the quantitative estimation of these drugs in both bulk and tablet dosage forms., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., (© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

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., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Tenofovir disoproxil fumarate is a prodrug of tenofovir used in the treatment of HIV and HBV infections: it is the most used antiretroviral worldwide. Tenofovir is nucleotidic HIV reverse trascriptase inhibitor that showed excellent long-term efficacy and tolerability. However renal and bone complications (proximal tubulopathy, hypophosphatemia, decreased bone mineral density, and reduced creatinine clearance) limit its use. Tenofovir renal toxicity has been suggested as the consequence of drug entrapment in proximal tubular cells: measuring tenofovir urinary concentrations may be a proxy of this event and it may be used as predictor of tenofovir side effects. No method is currently available for quantifying tenofovir in this matrix: then, the aim of this work was to validate a new LC-MS method for the quantification of urinary tenofovir. Chromatographic separation was achieved with a gradient (acetonitrile and water with formic acid 0.05%) on an Atlantis 5 μm T3, 4.6 mm × 150 mm, reversed phase analytical column. Detection of tenofovir and internal standard was achieved by electrospray ionization mass spectrometry in the positive ion mode. Calibration ranged from 391 to 100,000 ng/mL. The limit of quantification was 391 ng/mL and the limit of detection was 195 ng/mL. Mean recovery of tenofovir and internal standard were consistent and stable, while matrix effect resulted low and stable. The method was tested on 35 urine samples from HIV-positive patients treated with tenofovir-based HAARTs and did not show any significant interference with antiretrovirals or other concomitantly administered drugs. All the observed concentrations in real samples fitted the calibration range, confirming the capability of this method for the use in clinical routine. Whether confirmed in ad hoc studies this method may be used for quantifying tenofovir urinary concentrations and help managing HIV-positive patients treated with tenofovir., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A liquid chromatography-tandem mass spectrometry assay for the determination of abacavir (ABC), tenofovir (TFV), darunavir (DRV), and raltegravir (RAL) in human plasma and saliva was developed and validated to investigate the applicability of saliva as an appropriate specimen for therapeutic drug monitoring. As internal standards, TFV was chosen for ABC, ABC was chosen for TFV, RAL for DRV, and DRV for RAL. Sample preparation involved protein precipitation with acetonitrile, evaporation of solvent using a centrifugal evaporator, and reconstitution by dissolving the residue in mobile phase. Liquid chromatography was performed on a C18 reverse phase column (1.5 × 50 mm, 5 μm) isocratically at a flow rate of 0.2 mL/min using 5mM formic acid-3% (v/v) acetonitrile as the mobile phase for ABC and TFV and 5mM formic acid-35% (v/v) acetonitrile as the mobile phase for DRV and RAL. The run time was 6 min, and the retention time was approximately 2.0 min for TFV, 2.5 min for RAL, and 4-4.5 min for ABC and DRV. Analytes were detected using tandem mass spectrometry in positive electrospray ionization mode. The precursor/product ion transitions (m/z) were 287.3/191.2 for ABC, 288.5/176.2 for TFV, 548.3/392.3 for DRV, and 445.3/109.5 for RAL, and were monitored on a triple-quadrupole mass spectrometer operated in the multiple reaction monitoring mode. The linearity of the assay was assessed in the range 1-10,000 ng/mL for all four drugs. Within-run and between-run mean accuracy, precision, and the extraction recovery for all drugs were -14.5-18.1%, 1.2-13.1%, and 86.0-111.1%, respectively. The proposed assay is sufficiently sensitive and accurate to quantify these drugs in plasma and saliva, and is suitable for investigating the relationship between drug concentrations in plasma and saliva., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Maternal tenofovir disoproxil fumarate (TDF) treatment among HIV-infected pregnant women results in fetal tenofovir (TFV) exposure. Fetal TFV toxicity was demonstrated in animals, but most clinical investigations have not observed toxicity in humans., Methods: We evaluated HIV-exposed, uninfected infants in the Surveillance Monitoring for Antiretroviral Therapy Toxicities cohort of the Pediatric HIV/AIDS Cohort Study whose mothers were prescribed TDF for ≥ 8 third trimester weeks. Infant dual-energy X-ray absorptiometry scans were obtained at 0-4 weeks to measure whole body bone mineral content. Meconium TFV concentrations were quantified by liquid chromatography-tandem mass spectrometry., Results: Fifty-eight TFV-exposed infants had meconium TFV quantified. Detectable concentrations were 11-48,100 ng/g; 3 infants had undetectable concentrations. Maternal TDF prescription duration ranged from 8 to 41 gestational weeks; infant gestational ages were 36-41 weeks. Meconium TFV concentrations were not correlated with TFV exposure duration or timing and did not vary by concomitant prescription of protease inhibitors. Increased meconium TFV concentrations were associated with greater gestational ages (ρ = 0.29, P = 0.03) and lower maternal plasma HIV RNA before delivery (ρ = -0.29, P = 0.04). Meconium TFV concentrations were not associated with infant weight, length (n = 58) or bone mineral content (n = 49)., Conclusions: For the first time, we explored associations between meconium TFV concentrations and infant growth and bone measurements; we did not observe a meconium concentration-dependent relationship for these infant outcomes. These findings support other clinical research failing to show dose-response relationships for growth and bone outcomes among intrauterine TFV-exposed infants. High meconium TFV concentrations correlated with low maternal viral load, suggesting maternal TDF adherence significantly contributes to meconium TFV concentrations.