Quality assessment of oral antimalarial and antiretroviral medicines used by public health systems in Sahel countries.

Malaria and Human Immunodeficiency Virus infections are among the top 10 causes of death in low income countries. Furthermore, many medicines used in these treatment areas are substandard, which contributes to the high death rate. Using a monitoring system to identify substandard and falsified medicines, the study aims to evaluate the quality of antimalarial and antiretroviral medicines in Sahel countries, assessing site conditions, compliance of medicines with pharmacopoeia tests, formulation equivalence with a reference medicine, and the influence of climate on quality attributes. Ultra Performance Liquid Chromatography methods for eight active pharmaceutical ingredients were validated following the International Conference for Harmonization guideline for its detection and quantification. Quality control consists of visual inspections to detect any misinformation or imperfections and pharmacopeial testing to determine the quality of pharmaceutical products. Medicines which complied with uniformity dosage units and dissolution tests were stored under accelerated conditions for 6 months. Artemether/Lumefantrine and Lopinavir/Ritonavir formulations failed uniformity dosage units and disintegration tests respectively, detecting a total of 28.6% substandard medicines. After 6 months stored under accelerated conditions (40 °C // 75% relative humidity) simulating climatic conditions in Sahel countries, some medicines failed pharmacopeia tests. It demonstrated the influence of these two factors in their quality attributes. This study emphasizes the need of certified quality control laboratories as well as the need for regulatory systems to maintain standards in pharmaceutical manufacturing and distribution in these countries, especially when medicines are transported to rural areas where these climatic conditions are harsher.

Comprehensive micropollutant characterization of wastewater during Covid-19 crisis in 2020: Suspect screening and environmental risk prioritization strategy.

Micropollutants monitoring in wastewater can serve as a picture of what is consuming society and how it can impact the aquatic environment. In this work, a suspect screening approach was used to detect the known and unknown contaminants in wastewater samples collected from two wastewater treatment plants (WWTPs) located in the Basque Country (Crispijana in Alava, and Galindo in Vizcaya) during two weekly sampling campaigns, which included the months from April to July 2020, part of the confinement period caused by COVID-19. To that aim, high-resolution mass spectrometry was used to collect full-scan data-dependent tandem mass spectra from the water samples using a suspect database containing >40,000 chemical substances. The presence of > 80 contaminants was confirmed (level 1) and quantified in both WWTP samples, while at least 47 compounds were tentatively identified (2a). Among the contaminants of concern, an increase in the occurrence of some compounds used for COVID-19 disease treatment, such as lopinavir and hydroxychloroquine, was observed during the lockdown. A prioritization strategy for environmental risk assessment was carried out considering only the compounds quantified in the effluents of Crispijana and Galindo WWTPs. The compounds were scored based on the removal efficiency, estimated persistency, bioconcentration factor, mobility, toxicity potential and frequency of detection in the samples. With this approach, 33 compounds (e.g. amantadine, clozapine or lopinavir) were found to be considered key contaminants in the analyzed samples based on their concentration, occurrence and potential toxicity. Additionally, antimicrobial (RQ-AR) and antiviral (EDRP) risk of certain compounds was evaluated, where ciprofloxacin and fluconazole represented medium risk for antibiotic resistance (1 > RQ-AR > 0.1) in the aquatic ecosystems. Regarding mixture toxicity, the computed sum of toxic unit values of the different effluents (> 1) suggest that interactions between the compounds need to be considered for future environmental risk assessments.

Simultaneous quantitation of zidovudine, efavirenz, lopinavir and ritonavir in human hair by liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry.

Nowadays, zidovudine, efavirenz, lopinavir and ritonavir are important components of the second-line antiretroviral therapeutic regimen of National Free Antiretroviral Treatment Program in China. The measurement of antiretroviral drugs in hair will facilitate for the evaluation of the long-term adherence to highly active antiretroviral therapy. Nevertheless, no study illustrated simultaneous quantitation of the four drugs in hair. The study intended to develop a simple, sensitive and selective method for simultaneous quantitation of the four drugs in hair. The detection employed high liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization in positive mode. Hair samples were incubated in methanol at 40 °C for 16 h. The method showed the limit of detection at 18, 8, 5 and 6 pg/mg for zidovudine, efavirenz, lopinavir and ritonavir, respectively. The linear range (R2 > 0.99) was 36-5000 pg/mg for zidovudine, 16-5000 pg/mg for efavirenz, 10-50,000 pg/mg for lopinavir and 12-12,500 pg/mg for ritonavir. The intra-day and inter-day coefficients of variation were <15% and the recovery varied from 88.1 to 110.5%. The population analysis revealed that patients with high adherence showed significantly higher drug concentrations in hair than those with low adherence for both AZT and EFV (ps < 0.05). There was high association in drug contents in hair between AZT and EFV or LPV and RTV (ps < 0.05).

Simultaneous quantification of four antiretroviral drugs in breast milk samples from HIV-positive women by an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method.

The primary strategy to avoid mother-to-child transmission of human immunodeficiency virus (HIV) through breastfeeding is administration of highly active antiretroviral therapy (HAART) to HIV-positive pregnant women. Because significant changes in the pharmacokinetics of antiretroviral (ARV) drugs occur during pregnancy, quantifying HAART and the viral load in breast milk in this population is essential. Here, we developed an analytical assay for the simultaneous quantification of four ARV drugs in breast milk using ultra-performance liquid chromatography coupled to tandem mass spectrometry. We validated this method following Mexican and international guidelines. ARV drugs. We extracted the ARV drugs from 200 µL samples of breast milk and detected these drugs in a triple quadrupole mass spectrometer with positive electrospray ionization. The validated concentration ranges (ng/mL) for zidovudine, lamivudine, lopinavir, and ritonavir were 12.5-750, 50-2500, 100-5000 and 5 to 250, respectively. Additionally, the absolute recovery percentages (and matrix effects) were 91.4 (8.39), 88.78 (28.75), 91.38 (11.77) and 89.78 (12.37), respectively. We determined that ARV drugs are stable for 24 h at 8°C and 24°C for 15 days at -80°C. This methodology had the capacity for simultaneous detection; separation; and accurate, precise quantification of ARV drugs in human breast milk samples according to Mexican standard laws and United States Food and Drug Administration guidelines.

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.

Random lopinavir concentrations predict resistance on lopinavir-based antiretroviral therapy.

Considering that most patients who experience virological failure (VF) on lopinavir-based antiretroviral therapy (ART) fail due to poor adherence rather than resistance, an objective adherence measure could limit costs by rationalising the use of genotype antiretroviral resistance testing (GART) in countries with access to third-line ART. A cross-sectional study was conducted in a resource-limited setting at two large clinics in Kwazulu-Natal, South Africa, in patients experiencing VF (HIV-RNA > 1000 copies/mL) on lopinavir-based ART who had undergone GART. Associations between major protease inhibitor (PI) resistance mutations and random plasma lopinavir concentrations were explored. A total of 134 patients, including 31 children, were included in the analysis. The prevalence of patients with major PI resistance mutations was 20.9% (n = 28). A random lopinavir concentration above the recommended minimum trough of 1 µg/mL [adjusted odds ratio (aOR) = 5.81, 95% confidence interval (CI) 2.04-16.50; P = 0.001] and male sex (aOR = 3.19, 95% CI 1.22-8.33; P = 0.018) were predictive of the presence of at least one major PI resistance mutation. Random lopinavir concentrations of <1 µg/mL had a negative predictive value of 91% for major PI resistance mutations. Random lopinavir concentrations are strongly associated with the presence of major PI resistance mutations. Access to costly GART in patients experiencing VF on second-line ART could be restricted to patients with lopinavir concentrations above the recommended minimum trough of 1 µg/mL or, in areas where GART is unavailable, could be used as a criterion to empirically switch to third-line ART.

Using Lopinavir Concentrations in Hair Samples to Assess Treatment Outcomes on Second-Line Regimens Among Asian Children.

We conducted a prospective monitoring study to determine whether antiretroviral (ARV) levels in hair of Asian children on second-line protease inhibitor-based ARV therapy (ART) are associated with virologic failure (VF), compared to plasma drug levels and self-reported adherence. HIV-infected Asian children on second-line ART regimens were enrolled into a longitudinal cohort. Traditional adherence measures, plasma, and hair samples were collected 24 weeks after study enrollment. Hair ARV levels were determined via liquid chromatography/tandem mass spectrometry. Among 149 children on lopinavir/ritonavir-based regimens, 47% were female; the median [interquartile range (IQR)] age was 10.3 (7.9-13.3) years. The median CD4% was 26% (IQR 21.7-32.1%) and the median CD4 cell count 754 (IQR 596-1,013) cells/mm(3). The median duration of lopinavir-based ART prior to week 24 of the study was 2.9 (IQR 1.6-4.2) years. Adherence was >95% in 91% (135/148) by visual analogue scale and 89% (129/145) by pill count. The median lopinavir hair concentrations were 5.43 (IQR 3.21-9.01) ng/mg in children with HIV RNA >1,000 copies/ml and 9.96 (IQR 6.51-12.31) ng/mg in children with HIV RNA <1,000 copies/ml (p = 0.003). Plasma trough and lopinavir hair concentrations were not statistically significantly correlated (Pearson's correlation coefficient 0.20; p = 0.13). Increasing lopinavir hair concentrations in quartiles were strongly associated with virologic success (odds ratios ≥4.0, overall p = 0.02), while self-reported adherence, pill count, and plasma lopinavir levels were not. Based on this first report of hair ARV concentrations and virologic outcomes in children, ARV hair concentrations, representing longer-term adherence, may be useful to identify children at risk for VF.

Hair concentrations of antiretrovirals predict viral suppression in HIV-infected pregnant and breastfeeding Ugandan women.

OBJECTIVE: Hair concentrations are a noninvasive measure of cumulative antiretroviral exposure and the strongest predictor of viral suppression in large cohorts of nonpregnant patients. We examined hair concentrations of antiretrovirals in relation to virologic outcomes in pregnant and breastfeeding women for the first time. DESIGN AND METHODS: The Prevention of Malaria and HIV Disease in Tororo trial (NCT00993031) enrolled HIV-infected pregnant Ugandan women at 12-28 weeks gestation who were randomized to lopinavir or efavirenz-based antiretroviral therapy (ART). Small hair samples were collected at 30-34 weeks gestation and 10-25 weeks postpartum. Efavirenz and lopinavir hair concentrations were measured via liquid chromatography/tandem mass spectrometry. Multivariate logistic regression models examined predictors of viral suppression (HIV-1 RNA ≤400 copies/ml) at delivery and 24 weeks postpartum. RESULTS: Among 325 women, median CD4 cell count was 366 cells/µl (interquartile range 270-488) at ART initiation. Mean self-reported 3-day adherence was greater than 97% in each arm. Viral suppression was achieved by 98.0% (efavirenz) and 87.4% (lopinavir) at delivery. At 24 weeks postpartum, 92.5% (efavirenz) and 90.6% (lopinavir) achieved viral suppression; 88% of women were breastfeeding. In multivariate models including self-reported adherence and pretreatment HIV-1 RNA, antiretroviral hair concentrations were the strongest predictor of viral suppression at delivery [efavirenz: adjusted odds ratio (aOR) 1.86 per doubling in concentration, 95% confidence interval (CI) 1.14-3.1, P = 0.013; lopinavir: aOR 1.90, 95% CI 1.33-2.7, P = 0.0004] and 24 weeks postpartum (efavirenz: aOR 1.81, 95% CI 1.22-2.7, P = 0.003; lopinavir: aOR 1.53, 95% CI 1.05-2.2, P = 0.026). CONCLUSION: Antiretroviral hair concentrations represent an innovative tool that strongly predicts viral suppression among HIV-infected childbearing women during the critical periods of delivery and breastfeeding.

Simultaneous determination of lopinavir and three ester prodrugs by LC-MS/MS in lysates of BeWo cells.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with positive electrospray ionization (ESI) mode has been validated for the simultaneous determination of lopinavir (LPV) and three prodrugs, succinic acid ester of lopinavir (SLPV), glutaric acid ester of lopinavir (GLPV), and diglycolic acid ester of lopinavir (DLPV) in BeWo cell lysate. A methanolic precipitation was employed after addition of an internal standard ritonavir (RTV), followed by centrifugation, separation of the supernatant liquid, evaporation, and reconstitution. A reversed-phase Phenomenex C18 column was used for separation of lopinavir and three prodrugs with a mobile phase which comprises methanol (solvent A), acetonitrile (solvent B), and 50mM ammonium acetate buffer adjusted to pH 5.5 with acetic acid (solvent C), with isocratic elution (A:B:C=34:34:32, v/v). The detection of target compounds was conducted using multiple reaction monitoring (MRM) with the following transitions of m/z 729→447, 745→563, 743→429, 629→447 and 721→296 to measure SLPV, DLPV, GLPV, LPV and RTV (I.S.), respectively. The calibration ranges were determined using quadratic regression over concentration ranges of 7-743 ng/mL (DLPV), 7.5-745 ng/mL (GLPV), 7-729 ng/mL (SLPV) and 6-189 ng/mL (LPV). The reverse calculated residuals for LPV and these prodrugs were all less than 15% different from nominal (range). The method we established was fully validated for accuracy (≤15% different from nominal) and precision (≤15% RSD). The results proved that the method is accurate and specific, and also this method has been successfully applied to test the uptake of lopinavir and prodrugs into BeWo cells.

Quantification of cell-associated atazanavir, darunavir, lopinavir, ritonavir, and efavirenz concentrations in human mononuclear cell extracts.

An ultrasensitive assay utilizing high-pressure liquid chromatography and mass spectrometry detection was developed and validated for the quantification of the antiretrovirals atazanavir (ATV), darunavir (DRV), lopinavir (LPV), ritonavir (RTV), and efavirenz (EFV) in human mononuclear cell (MNC) extracts. The assay utilizes 20 µl of cellular extract that contains as few as 50,000 MNCs. The analytical range of the assay is 0.0200 to 10.0 fmol/µl for ATV, 0.0500 to 25.0 fmol/µl for DRV, LPV, and RTV, and 0.200 to 100 fmol/µl for EFV. The assay has proven to be a clinically useful tool for investigating antiretroviral drug concentrations in virologic sanctuaries where harvested cell numbers are extremely low. The assay provides a tool for investigators to explore the clinical pharmacology of strategies for prevention, treatment, and cure in pathophysiologically relevant sites.

Preparation and evaluation of metastable solid-state forms of lopinavir.

In this work, we present the preparation and evaluation of previously unreported metastable forms of the antiretroviral drug, lopinavir. By maintaining the chemical structure, physicochemical properties like the glass transition temperature (T(g)), dissolution and solubility can be readily attributed to the stability of the system. Commercially-available lopinavir was used to prepare partially amorphous crystals, semicrystalline needles, resins and glasses. The physicochemical properties of each were investigated using differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR) and powder X-ray diffraction (PXRD). Each sample's thermal and spectroscopic analyses, as well as dissolution and solubility studies were performed one month after sample preparation, for better comparability. Glass transition temperature, activation energy for global molecular mobility (deltaE(Tg)), and activation energy for local molecular mobility (deltaE(beta)) were assessed as primary indicators for structural stability of the systems. Relating these properties to aqueous solubility revealed that each metastable form possessed its own unique equilibrium solubility. Cumulative dissolved fractions (alpha) were fitted against deceleratory kinetics models, and from the data hereby obtained the dissolution process was determined to followed first-order kinetics (R2 = 0.998). From the rate constants, the activation energy for dissolution (deltaE(Diss)) of each sample was calculated. The results suggest that multiple metastable solid-state forms of lopinavir can exist under similar conditions, depending on the preparation conditions.