Optimizing removal of antiretroviral drugs from tertiary wastewater using chlorination and AI-based prediction with response surface methodology.

Chemical and pharmaceutical chemicals found in water sources create substantial risks to human health and the environment. The presence of pharmaceutical contaminants in water can cause antibiotic resistance development, toxicity to aquatic organisms, and endocrine disruption. Hence, the elimination of chemicals and other contaminants from wastewater prior to its release is a burgeoning concern in the domains of engineering and science. The use of treatment technologies in wastewater treatment plants can remove pharmaceutical contaminants through the oxidation process. However, many traditional wastewater treatment plants lack the advanced monitoring tools required to detect low concentrations of pharmaceuticals. Without the ability to detect these compounds, it's challenging to treat them effectively. The goal of this study was to use Response Surface Methodology (RSM) and Artificial Neural Networks (ANN) algorithms to model and improve how Nevirapine and Efavirenz break down in different chlorination conditions. The RSM analysis revealed statistically significant models (F-values: Nevirapine, pH-t: 108.15, T-t: 76.55, ICC-t: 110.84), indicating a strong correlation between operational parameters (pH, temperature, and initial chlorine concentration) and degradation behavior. The ANN model accurately predicted the degradation of both Nevirapine and Efavirenz under various chlorination conditions, as confirmed by analyzing actual-predicted graphs, residual plots, and Mean Squared Error (MSE) values. The ANN model using ICC-t achieved the highest MOD value of 31.31 % for Nevirapine. The ANN model based on ICC-t yielded a maximum MOD value of 16.06 % for Efavirenz. These findings provide valuable insights into optimizing chlorination processes for better removal of these pharmaceutical contaminants from water.

Occurrence and environmental risks of contaminants of emerging concern across the River Athi Basin, Kenya, in dry and wet seasons.

Globally, the environmental occurrence of Contaminants of Emerging Concern (CECs) including pharmaceuticals (PhACs), personal care products (PCPs) and modern polar pesticides has raised ecological and human health awareness. However, as the developed world races against time to establish regulatory measures to mitigate their effects, developing nations including Kenya are lagging behind, partly due to unavailability of adequate data. In this work, a multi-residue analysis of 86 CECs was carried out on 198 surface water and 18 effluent samples collected at 24 sites across the River Athi basin area, Kenya, in both dry and rainy seasons. Overall, 57 CECs comprising 31 PhACs (0.4 ng L-1-142 µg L-1), 6 PCPs (0.7-570 ng L-1) and 20 pesticides (0.3 ng L-1-8.3 µg L-1) were detected. The maximum loads varied from 217 g day-1 (PCPs) to 46 kg day-1 (PhACs). Individually, carbamazepine, nevirapine, sulfamethoxazole and DEET were the most ubiquitous CECs, with detection frequencies (DF) higher than 80 %. The highest concentrations were observed at river sites that are heavily impacted by informal settlements, highlighting the critical role of slums in urban rivers pollution. At least 8 CECs including acetamiprid, alachlor, atrazine, diuron, nevirapine and paracetamol show potential risk to algae, Daphnia magna and fish, as exemplified by Risk Quotients (RQ) up to 174. Similarly, potential risk of antibiotic resistant bacteria development is evident (RQ up to 64), being driven by metronidazole, sulfamethoxazole and trimethoprim. Ultimately, further studies on the occurrence and distribution of antibiotic resistant bacteria within the basin and among the communities consuming untreated river water for drinking is merited.

Methods optimization and application: Solid phase extraction, ultrasonic extraction and Soxhlet extraction for the determination of antiretroviral drugs in river water, wastewater, sludge, soil and sediment.

The continuous release of antiretroviral drugs into the environmental has resulted in the interest to assess their occurrence in various environmental matrices. Their presence has led to antiretroviral drugs being considered the pollutants of concern due to their possible alterations of the ecosystem as well as the antiviral resistance that may develop upon their unintentional consumption. Therefore, in this work, solid phase extraction (SPE), ultrasonic extraction (UE), Soxhlet extration (SE) and liquid chromatography coupled to photodiode array detector (LC-PDA) methods have been optimized and validated. They were then applied for the simultaneous determination of abacavir, nevirapine and efavirenz antiretroviral drugs in wastewater, river water, sludge, soil and sediments. The percentage recoveries ranged from 71% to 112% for SPE, 88 - 108% for SE and 61 - 104% for UE. Good precision with a relative standard deviation less than 20% in all compounds for all methods was obtained. The LODs and LOQs ranged between 0.68 and 0.77 µg/L and 2.1-2.4 µg/L for SPE; 0.8-0.9 µg/kg and 2.3-2.8 µg/kg for SE and 1.6-2.8 µg/kg and 4.9 - 7.0 µg/kg for UE, respectively. The concentrations ranged from

Nevirapine hair and plasma concentrations and HIV-1 viral suppression among HIV infected ante-partum and post-partum women attended in a mother and child prevention program in Maputo city, Mozambique.

INTRODUCTION: Prevention of mother to child transmission of HIV (PMTCT) is frequently challenged by irregular access to more effective anti-retroviral therapy. Nevirapine single dose (sdNVP), sdNVP+AZT+3TC for MTCT prophylaxis and NVP+ AZT+3TC for treatment and PMTCT were withdrawn due to low genetic resistance barrier and low efficacy. However current PMTCT lines in Mozambique include DTG+3TC+TDF, TDF+3TC+EFV, DTG +ABC+3TC, and AZT + NVP syrup prophylaxis for exposed babies. We assessed NVP hair and plasma concentrations and association with HIV-1RNA suppression among HIV+ ante-partum and post-partum women under PMTCT in Maputo, Mozambique. METHODS: From December 2013 to November 2014, prospectively were enrolled 200 HIV+ ante-partum women on 200mg nevirapine and zidovudine 300 plus lamivudine 150mg twice daily at least with 3 months treatment and seen again at 24 weeks post-partum. Self-reported pill-taking adherence, NVP concentrations in hair, plasma, hemoglobin, CD4 cell count, HIV-1 RNA load was evaluated. NVP concentration in hair and plasma was analyzed as categorical quartile variable based on better data fit. NVP concentration was set between ≤3.77 ng/ml in plasma and ≤17,20 ng/mg in hair in quartile one to ≥5.36 ng/ml in plasma and ≥53.21 ng/mg in hair in quartile four. Logistic regression models for repeated measures were calculated. Following the World Health Organization (WHO) guidelines we set viral suppression at HIV-1RNA < 1000 c/mL. Outcome was HIV-1 RNA<1000 copies/ml. Predictor was NVP concentration in hair categorized in quartiles. RESULTS: In total 369 person-visits (median of 1.85) were recorded. Self-reported adherence was 98% (IQR 97-100%) at ante-partum. In 25% person visits, NVP concentrations were within therapeutic levels (3.77 ng/ml to 5.35 ng/ml) in plasma and (17.20 ng/mg to 53.20 ng/mg) in hair. In 50% person visits NVP concentrations were above 5.36 ng/ml in plasm and 53.21 ng/mg in hair. HIV-1 RNA suppression was found in 34.7% of women with two viral loads, one at enrollment and another in post-partum. Odds of HIV-1 RNA suppression in quartile 4, was about 6 times higher than in quartile 1 (p-value = 0.006) for NVP hair concentration and 7 times for NVP plasma concentration (p-value = 0.012). CONCLUSIONS: The study results alert for potential low efficacy of current PMTCT drug regimens in use in Mozambique. Affordable means for individual monitoring adherence, ART plasma and hair levels, drug resistant and HIV-1 RNA levels monitoring are recommended for prompt identification of inadequate drug regimens exposure patterns and adjust accordingly.

Effect on growth of exposure to maternal antiretroviral therapy in breastmilk versus extended infant nevirapine prophylaxis among HIV-exposed perinatally uninfected infants in the PROMISE randomized trial.

BACKGROUND: Malnutrition is highly prevalent in HIV-exposed perinatally uninfected infants (HEUs) increasing the risk of morbidity and mortality throughout the life course. We set out to compare the effect of postnatal exposure to maternal antiretroviral therapy (mART) in breastmilk versus infant Nevirapine prophylaxis (iNVP) on somatic growth of HEUs in the randomized PROMISE trial. METHODS AND FINDINGS: We randomized 2431 mothers with HIV and their 2444 HEUs from six African countries and India 6-14 days after delivery to mART or iNVP for prevention of breastmilk HIV transmission. The mART regimen contained tenofovir/emtricitabine (99%) plus lopinavir/ritonavir. Infant growth parameters were compared at postnatal week 10, 26, 74 and 104 using World Health Organization (WHO) z-scores for length-for-age (LAZ), weight-for-age (WAZ), and head circumference-for-age (HCAZ). Week 26 LAZ was the primary endpoint measure. Student T-tests compared mean LAZ, WAZ, and HCAZ; estimated mean and 95% confidence interval (CI) are presented. Maternal and infant baseline characteristics were comparable between study arms. The estimated median breastfeeding duration was 70 weeks. After a mean follow-up of 88 weeks, mean LAZ and WAZ were below the WHO reference population mean at all timepoints, whereas mean HCAZ was not. The mART and iNVP arms did not differ for the primary outcome measure of LAZ at week 26 (p-value = 0.39; estimated mean difference (95%CI) of -0.05 (-0.18, 0.07)) or any of the other secondary growth outcome measures or timepoints (all p-values≥0.16). Secondary analyses of the primary outcome measure adjusting for week 0 LAZ and other covariates did not change these results (all p-values≥0.09). However, infants assigned to mART were more likely to have stunting compared to iNVP infants at week 26 (odds ratio (95% CI): 1.28 (1.05, 1.57)). CONCLUSIONS: In HEUs, growth effects from postnatal exposure to mART compared to iNVP were comparable for measures on length, weight and head circumference with no clinically relevant differences between the groups. Despite breastfeeding into the second year of life, length and weight were below reference population means at all ages in both arms. Further investment is needed to optimize postnatal growth of infants born to women with HIV. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov number NCT01061151.

LC-MS/MS Quantification of Nevirapine and Its Metabolites in Hair for Assessing Long-Term Adherence.

The adherence assessment based on the combination of nevirapine (NVP) and its two metabolites (2-hydroxynevirapine and 3-hydroxynevirapine) would more comprehensively and accurately reflect long-term adherence than that of a single prototype. This study aimed to develop a specific, sensitive and selective method for simultaneous detection of the three compounds in hair and explore whether there was consistency among the three compounds in assessing long-term adherence. Furthermore, 75 HIV-positive patients who were taking the NVP drug were randomly recruited and divided into two groups (high-and low-adherence group). All participants self-reported their days of oral drug administration per month and provided their hair strands closest to the scalp at the region of posterior vertex. The concentrations of three compounds in the hair were determined using a developed LC-MS/MS method in multiple reaction monitoring. This method showed good performances in limit of quantification and accuracy with the recoveries from 85 to 115% and in precision with the intra-day and inter-day coefficients of variation within 15% for the three compounds. The population analysis revealed that patients with high-adherence showed significantly higher concentrations than those with low-adherence for all three compounds. There were significantly moderate correlations of nevirapine with 2-hydroxynevirapine and 3-hydroxynevirapin and high correlation between 2-hydroxynevirapine and 3-hydroxynevirapin. The two NVP's metabolites showed high consistency with NVP in evaluating long-term adherence.

Solid-phase extraction combined with dispersive liquid-liquid microextraction/HPLC-UV as a sensitive and efficient method for extraction, pre-concentration and simultaneous determination of antiretroviral drugs nevirapine, efavirenz and nelfinavir in pharmaceutical formulations and biological samples.

Therapeutic drug monitoring (TDM) of antiretroviral drugs requires accurate and precise analysis of their trace amounts in plasma samples. Solid-phase extraction (SPE) coupled with dispersive liquid-liquid microextraction based on solidification of floating organic drop (DLLME-SFO) was introduced as a simple and sensitive method for extraction, pre-concentration and simultaneous determination of efavirenz (EFV), nelfinavir (NFV) and nevirapine (NVP) in human plasma and pharmaceutical formulations by using high performance liquid chromatography (HPLC) with UV detection. A response surface methodology (RSM) based on central composite design (CCD) was used for optimizing the main variables in the extraction procedure. Under the optimum experimental conditions, the calibration curves were linear in the range of 0.1-400 ng mL-1 with correlation coefficients from 0.9982 to 0.9997 and limits of detection (at S/N = 3) of 0.03 to 0.07 ng mL-1. Moreover the intra-day and inter-day precision (RSD%) were in the range of 2.2-4.2% and 3.1-5.2%, respectively. The enrichment factors and relative recoveries of the anti-HIV drugs were 459-1507 and 93.7-105.4%. The method was successfully applied to the simultaneous determination of the trace amounts of the antiretroviral drugs in blood plasma and pharmaceutical formulations.

Automated high throughput analysis of antiretroviral drugs in dried blood spots.

For therapeutic drug monitoring in remote settings, dried blood spots (DBS) are particularly advantageous, as blood sample collection and handling is uncomplicated. The aim of this study was to develop and validate an automated extraction method for the analysis of nevirapine, efavirenz and lopinavir in DBS samples. Automated extraction was performed with methanol : water (70 : 30 v/v), using a DBS-MS 500 autosampler coupled to a liquid chromatography tandem mass spectrometry system. The autosampler used digital images of each DBS to position the extraction head, sprayed 10 µl of internal standard onto each DBS and extracted a 4-mm disc (Ø) from the centre of each spot by unilateral flow using 25-µl extraction solvent. The analytes were baseline separated on a pentafluorophenyl column and analysed by using electrospray ionization with multiple reaction monitoring in positive polarity mode for nevirapine and lopinavir and in negative mode for efavirenz. The method was linear between 10 and 10 000 ng/ml for all analytes. Automated sample extraction resulted in consistent recoveries (nevirapine: 70 ± 6%, efavirenz: 63 ± 11% and lopinavir: 60 ± 10%) and matrix effects between different donors and concentration levels. Intra-day and inter-day accuracy and precision deviations were ≤15%. Manual and automated extractions of DBS samples collected within the framework of an adherence assessment study in rural Tanzania showed good agreements with deviations of less than 10%. Our study highlights that therapeutic drug monitoring samples obtained in the resource-constrained setting of rural Africa can be reliably determined by automated extraction of DBS. Overall, automatization improved method sensitivity and facilitates analysis of large sample numbers. Copyright © 2017 John Wiley & Sons, Ltd.

Determination of endocrine disrupting chemicals and antiretroviral compounds in surface water: A disposable sorptive sampler with comprehensive gas chromatography - Time-of-flight mass spectrometry and large volume injection with ultra-high performance liquid chromatography-tandem mass spectrometry.

Many rural dwellers and inhabitants of informal settlements in South Africa are without access to treated water and collect untreated water from rivers and dams for personal use. Endocrine disrupting chemicals (EDCs) have been detected in surface water and wildlife of South Africa. EDCs are often present in complex environmental matrices at ultra-trace levels complicating detection thereof. We report a simplified multi-residue approach for the detection and quantification of EDCs, emerging EDCs, and antiretroviral drugs in surface water. A low cost (less than one US dollar), disposable, sorptive extraction sampler was prepared in-house. The disposable samplers consisted of polydimethylsiloxane (PDMS) tubing fashioned into a loop which was then placed in water samples to concentrate EDCs and emerging pollutants. The PDMS samplers were thermally desorbed directly in the inlet of a GC, thereby eliminating the need for expensive consumable cryogenics. Comprehensive gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOFMS) was used for compound separation and identification. Linear retention indices of EDCs and emerging pollutants were determined on a proprietary Crossbond® phase Rtx®-CLPesticides II GC capillary column. In addition, large volume injection of surface water into an ultra-performance liquid chromatograph tandem mass spectrometer (UPLC-MS/MS) was used as complementary methodology for the detection of less volatile compounds. Large volume injection reduced tedious and costly sample preparation steps. Limits of detection for the GC method ranged from 1 to 98pg/l and for the LC method from 2 to 135ng/l. Known and emerging EDCs such as pharmaceuticals, personal care products and pesticides, as well as the antiretroviral compounds, efavirenz and nevirapine, were detected in surface water from South Africa at concentration levels ranging from 0.16ng/l to 227ng/l.

Stability-indicating UHPLC method for determination of nevirapine in its bulk form and tablets: identification of impurities and degradation kinetic study.

Nevirapine (NVP), a non-nucleoside reverse transcriptase inhibitor, is a drug widely used in the treatment of Acquired Immunodeficiency Syndrome (AIDS). The evaluation of NVP stability is of fundamental importance in order to guarantee drug product efficacy, safety and quality. In this study, NVP active pharmaceutical ingredient (API) and tablets were subjected to a detailed study of forced degradation, employing several degrading agents (acid, alkaline, water, metal ions, humidity, heat, light and oxidation agents). In order to determine NVP and the degradation products formed, a stability-indicating UHPLC method using fused core column was developed and validated. The separation was carried out using a Poroshell 120C18 column (100×2.1mm i.d.; 2.7µm particle size) and the mobile phase was composed of acetonitrile and water in a gradient elution, at a flow rate of 0.2ml/min. Chemical structures and mechanisms for the formation of three degradation products were proposed by means of LC/MS-MS. Also, NVP degradation kinetic was studied and its order of degradation evaluated. NVP was degraded in acidic and oxidative conditions and the degradation profile for NVP tablets and API were similar. The stability-indicating method proved to be selective for NVP and its degradation products. Calibration curve was linear in the range of 8-48µg/ml and the method showed to be precise, accurate and robust for both NVP API and tablets, with detection and quantification limits of 0.092µg/ml and 0.174µg/ml, respectively.

Calibration transfer from powder mixtures to intact tablets: A new use in pharmaceutical analysis for a known tool.

Calibration transfer is commonly used for spectra obtained in different spectrometers or other conditions. This paper proposed the use of calibration transfer between spectra recorded for the same samples in different physical forms. A new method was developed for the direct determination of nevirapine in solid pharmaceutical formulations based on diffuse reflectance near infrared spectroscopy (NIRS) and partial least squares (PLS). This method was developed with 50 powder mixtures and then, successfully extended to the quantification in intact tablets by using calibration transfer with double window piecewise direct standardization (DWPDS). This chemometric strategy provided good results with a small number of tablet transfer samples, only seven, prepared out of the narrow range of active principle ingredients (API) content around the nominal value of the formulation (100%). The method was fully validated in the working range of 83.0-113.9% of nevirapine and the use of DWPDS allowed to significantly decreasing the root mean square error of prediction (RMSEP) from 4.8% (tablets predicted by a model built with only powder samples) to 2.6%. The range of relative errors decreased from -5.1/8.7% to -4.6/3.3%. Considering that the amount of raw materials demanded for preparing tablets is up to ten times higher than for powder mixtures, this type of application is of particular interest in pharmaceutical analysis. In the context of process analytical technology (PAT), the use of the same multivariate model in different steps of the production is very advantageous, saving time and labor.

Assessment of HIV antiretroviral therapy adherence by measuring drug concentrations in hair among children in rural Uganda.

Current tools for measuring medication adherence have significant limitations, especially among pediatric populations. We conducted a prospective observational study to assess the use of antiretroviral (ARV) drug levels in hair for evaluating antiretroviral therapy (ART) adherence among HIV-infected children in rural Uganda. Three-day caregiver recall, 30-day visual analog scale (VAS), Medication Event Monitoring System (MEMS), and unannounced pill counts and liquid formulation weights (UPC) were collected monthly over a one-year period. Hair samples were collected quarterly and analyzed for nevirapine (NVP) levels, and plasma HIV RNA levels were collected every six months. Among children with at least one hair sample collected, we used univariable random intercept linear regression models to compare log transformed NVP concentrations with each adherence measure, and the child's age, sex, and CD4 count percentage (CD4%). One hundred and twenty-one children aged 2-10 years were enrolled in the study; 74 (61%) provided at least one hair sample, and the mean number of hair samples collected per child was 1.9 (standard deviation [SD] 1.0). Three-day caregiver recall, VAS, and MEMS were found to be positively associated with increasing NVP concentration in hair, although associations were not statistically significant. UPC was found to have a nonsignificant negative association with increasing hair NVP concentration. In conclusion, NVP drug concentrations in hair were found to have nonsignificant, although generally positive, associations with other adherence measures in a cohort of HIV-infected children in Uganda. Hair collection in this population proved challenging, suggesting the need for community education and buy-in with the introduction of novel methodologies.

Effect of combination of acrylic polymers on the release of nevirapine formulated as extended release matrix pellets using extrusion and spheronization technique.

The aim of the present research work was to formulate and evaluate the extended release matrix pellets of nevirapine using extrusion and spheronization technique which will be an alternative technique for making extended release dosage forms and to compare the drug release profiles of the formulations with the reference product. In vitro dissolutions were carried out in 0.04M Phosphate buffer pH 6.8 with 2% w/v SLS (sodium lauryl sulphate) for 24 hours with USP type I apparatus at 75rpm. The drug release from the optimised formulation was comparable to that of the reference product and follows first order kinetics followed by non-fickian transport mechanism of drug release which confirms the drug release pattern involves complex mixture of diffusion and erosion. The similarity factor, f2 value of optimised formulation was found to be 70, which shows that the developed formulation was comparable to that of the reference product.

Antiretroviral concentrations in small hair samples as a feasible marker of adherence in rural Kenya.

Antiretroviral hair levels objectively quantify drug exposure over time and predict virologic responses. We assessed the acceptability and feasibility of collecting small hair samples in a rural Kenyan cohort. Ninety-five percentage of participants (354/373) donated hair. Although median self-reported adherence was 100% (interquartile range, 96%-100%), a wide range of hair concentrations likely indicates overestimation of self-reported adherence and the advantages of a pharmacologic adherence measure. Higher nevirapine hair concentrations observed in women and older adults require further study to unravel behavioral versus pharmacokinetic contributors. In resource-limited settings, hair antiretroviral levels may serve as a low-cost quantitative biomarker of adherence.

Short communication: A low-cost method for analyzing nevirapine levels in hair as a marker of adherence in resource-limited settings.

The measurement of antiretroviral concentrations in hair is emerging as an important technology to objectively quantify adherence to combination antiretroviral therapy. Hair levels of antiretrovirals are the strongest independent predictor of virologic success in large prospective cohorts of HIV-infected patients and surpass self-report in predicting outcomes. Hair is easy to collect and store, but validated methods to analyze antiretroviral levels in hair using liquid chromatography tandem mass spectrometry (LC-MS/MS) are expensive. We report here on the development of a thin-layer chromatography (TLC) assay for the semiquantitative analysis of nevirapine in hair. TLC assay results from 11 samples were consistent with results using LC-MS/MS [Spearman correlation coefficient 0.99 (95% CI 0.95-0.996)]. This simple, low-cost method of analyzing nevirapine concentrations in hair may provide a novel monitoring tool for antiretroviral adherence in resource-limited settings and merits further study in clinical settings.

Use of dried blood spots for the determination of plasma concentrations of nevirapine and efavirenz.

OBJECTIVES: Plasma concentrations are frequently used for therapeutic drug monitoring of antiretroviral drugs. Dried blood spot sampling offers a patient-friendly and easy alternative to plasma sampling. However, dried blood spot concentrations are not necessarily equal to plasma concentrations and therefore the objective of this work was to establish the relationship between nevirapine and efavirenz dried blood spot and plasma concentrations to facilitate clinical implementation of dried blood spot sampling. METHODS: Paired dried blood spot and plasma samples were obtained from 40 HIV-infected patients on nevirapine and 40 on efavirenz treatment. All samples were analysed using validated HPLC-tandem mass spectrometry methods for the two matrices. Theoretical plasma concentrations were calculated from dried blood spot concentrations using the formula [dried blood spot concentration/(1 - haematocrit)] × fraction bound to plasma proteins = plasma concentration. Linear regression and Bland-Altman analysis were used to compare the two methods. RESULTS: Dried blood spot and plasma concentrations of nevirapine and efavirenz correlated well (r(2) = 0.867 and 0.972, respectively), although efavirenz dried blood spot concentrations were 39.8% (SD 7.1%) lower than plasma concentrations. Theoretical plasma concentrations (using patient-specific haematocrit) of nevirapine and efavirenz were similar to measured plasma concentrations, with a mean difference between the two methods of 0.29 mg/L (SD 1.35 mg/L) and 0.08 mg/L (SD 0.31 mg/L), respectively. CONCLUSIONS: Dried blood spot concentrations of nevirapine and efavirenz were equal to plasma concentrations after correction for haematocrit and compound-specific plasma protein binding and can therefore be used in clinical practice.

Microanalysis of the antiretroviral nevirapine in human hair from HIV-infected patients by liquid chromatography-tandem mass spectrometry.

Sufficient drug exposure is crucial for maintaining durable responses to HIV treatments. However, monitoring drug exposure using single blood samples only provides short-term information and is highly subject to intra-individual pharmacokinetic variation. Drugs can accumulate in hair over a long period of time, so hair drug levels can provide drug exposure information over prolonged periods. We now report on a specific, sensitive, and reproducible liquid chromatography-tandem mass spectrometry method for measuring nevirapine (NVP), a widely used antiretroviral drug, levels in human hair using even a single short strand of hair. Hair samples are cut into small segments, and the drug is extracted in methanol/trifluoroacetic acid (v/v, 9:1) shaken at 37 °C in a water bath overnight, followed by liquid-liquid extraction under alkaline conditions. The extracted samples are then separated on a BDS-C(18) column with a mobile phase composed of 50% acetonitrile containing 0.15% acetic acid and 4 mM ammonium acetate with an isocratic elution for a total run time of 3 min and detected by triple quadrupole electrospray multiple reaction mode at precursor/product ion at 267.0 > 225.9 m/z. Deuterated nevirapine-d5 was used as an internal standard. This method was validated from 0.25 to 100 ng/mg using 2 mg hair samples. The accuracies for spiked NVP hair control samples were 98-106% with coefficients of variation (CV) less than 10%. The CV for incurred hair control samples was less than 7%. The extraction efficiency for incurred control hair samples was estimated at more than 95% by repeated extractions. This method has been successfully applied to analyze more than 1,000 hair samples from participants in a large ongoing cohort study of HIV-infected participants. We also showed that NVP in human hair can easily be detected in a single short strand of hair. This method will allow us to identify drug non-adherence using even a single strand of hair.

Evaluation of saliva as an alternative matrix for monitoring plasma Zidovudine, Lamivudine and nevirapine concentrations in Rwanda.

Saliva may provide interesting advantages as matrix for compliance measurements, pharmacokinetic studies and therapeutic drug monitoring in resource limited countries. We investigated the feasibility of using saliva for compliance monitoring of zidovudine (ZDV), lamivudine (3TC) and nevirapine (NVP) in 29 HIV-1 infected patients from Rwanda. ZDV, 3TC and NVP drug levels were quantified by an LC/MS-MS method in plasma and stimulated saliva samples and compared using Bland-Altman analysis. Seven patients demonstrated undetectable saliva ZDV levels while five out of these seven also showed no 3TC salivary concentrations. For the other samples, we observed a good agreement between salivary and plasma concentrations of each antiretroviral drug. A significant relation between the difference in saliva and plasma ZDV concentrations and the average ZDV concentration in the two matrices was deduced as follow: y = -380.15 + 1.79 x. The log saliva and plasma concentration difference of both 3TC and NVP was consistent across the range of average log concentration. Overall, we showed large agreement limits suggesting a wide inter patient variability that may result to non-reliable plasma level predictions from saliva drug measurements. Therefore, our results indicate that saliva may serve as a valuable tool only for NVP compliance testing because of its high salivary concentration.

Good performance of an immunoassay based method for nevirapine measurements in human breast milk.

BACKGROUND: Understanding the distribution of antiretro-virals in breastfeeding HIV-positive mothers is essential, both for prevention of mother-to-child HIV transmission and for research on the development of drug resistance. The ARK nevirapine (NVP)-test is an immunoassay method for nevirapine measurements, developed and validated for plasma use. In this study, the ARK NVP-test was evaluated for measurement of nevirapine concentrations in breast milk. High performance liquid chromatography (HPLC) is the method currently used to determine nevirapine in breast milk. This method, however, requires complicated extraction techniques. The ARK method employs an immunoassay technology and requires a small sample volume (40 µL) and no pre-treatment of the samples. METHODS: Commercial enzyme and antibody were used and calibration standards and quality controls were prepared from pooled breast milk from HIV-uninfected women. Clinical samples from HIV-infected women receiving a single-dose of nevirapine were analyzed. RESULTS: Precision and accuracy were evaluated with two concentrations of quality control materials analyzed in three replicates on four different days and was <4%, and between 96.5% and 104.6%, respectively. Clinical samples were analyzed and CVs ranged from 0.0% to 11.1%. The median nevirapine concentration in breast milk 1 week post-partum was 0.29 µg/mL (range 0.11-0.90 µg/mL) in women treated with a single-dose of nevirapine. CONCLUSIONS: The ease of use and small sample volume makes the ARK assay an attractive alternative to HPLC analyses for determinations of nevirapine concentrations in breast milk.

Automatic nevirapine concentration interpretation system using support vector regression.

Follow-up of human immunodeficiency virus (HIV) patients treated with Nevirapine (NVP) is a necessary process to evaluate the drug resistance and the HIV mutation. It is also usually tested by immunochromatographic (IC) strip test. However, it is difficult to estimate the amount of drug the patient gets by visually inspection of color. In this paper, we propose an automatic interpretation system using a commercialized optical scanner. Several IC strips can be placed at any direction as long as they are on the scanner plate. There are three steps in the system, i.e., light intensity normalization, image segmentation and NVP concentration interpretation. We utilized the Support Vector Regression to interpret the NVP concentration. From the results, we found out the performance of the system is promising and better than that of the linear and nonlinear regression.