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.

Quality by design tool-evaluated stability-indicating ultra-performance liquid chromatography method for the determination of drugs (ritonavir and darunavir) used to treat the human immunodeficiency virus/acquired immunodeficiency syndrome.

Ritonavir and darunavir were examined using a ultra-performance liquid chromatography (UPLC) approach in pharmaceutical dosage forms. The small number of analytical studies that are currently available do not demonstrate the method's stability or nature. The study sought to assess both chemicals using a stability-indicating approach with a relatively short run time. The HSS C18 (100 × 2.1 mm), 2-mm column was used for the chromatographic separation, and isocratic elution was used to achieve this. In the mobile phase, methanol and 0.01 M phosphate buffer (pH 4.0) were included in a 60:40 (v/v) ratio. Throughout the analysis, the flow rate was kept at 0.2 mL min-1 , and a photodiode array detector set to 266 nm was used to find the major components. The proposed method showed a linear response (r2  > 0.999), and the accuracy was between 98.0% and 102.0%. The precision data showed relative standard deviation ≤1.0%. The UPLC method for quantification of ritonavir and darunavir in pharmaceutical dosage forms using a very short run time of under a minute is the subject of the proposed article. To meet current regulatory criteria, the quality by design idea was used in the method performance verification.

Functionalized graphene oxide tablets for sample preparation of drugs in biological fluids: Extraction of ritonavir, a HIV protease inhibitor, from human saliva and plasma using LC-MS/MS.

In this work, graphene oxide-based tablets (GO-Tabs) were prepared by applying a thin layer of functionalized GO on a polyethylene substrate. The GO was functionalized with amine groups (-NH2 ) by poly(ethylene glycol)bis(3-aminopropyl) terminated (GO-NH2 -PEG-NH2 ). The functionalized GO-Tabs were used for the extraction of ritonavir (RTV) in human saliva samples. RTV in plasma and saliva samples was analyzed using LC-MS/MS. Gradient LC system with MS/MS in the positive-ion mode [electrospray ionization (ESI+)] was used. The transitions m/z 721 → 269.0 and m/z 614 → 421 were used for RTV and the internal standard indinavir, respectively. This study determined the human immunodeficiency virus protease inhibitor RTV in human saliva samples using functionalized GO-Tab and LC-MS/MS, and the method was validated. The standard calibration curve for plasma and saliva samples was constructed from 5.0 to 2000 nmol L-1 . The limit of detection was 0.1 nmol L-1 , and the limit of quantification was 5.0 nmol L-1 in both plasma and saliva matrices. The intra- and inter-assay precision values were found to be between 1.5 and 5.8%, and the accuracy values ranged from 88.0 to 108% utilizing saliva and plasma samples. The extraction recovery was more than 80%, and the presented functionalized GO-Tabs could be reused for more than 10 extractions without deterioration in recovery.

Development and validation of RP-HPLC method for simultaneous estimation of docetaxel and ritonavir in PLGA nanoparticles.

OBJECTIVES: The main objective of the present study was to develop and validate simple, precise, sensitive and accurate RP-HPLC method for the simultaneous estimation of docetaxel (DTX) and ritonavir (RTV) in PLGA nanoparticles (PLGA-NPs). METHODS: The DTX and RTV co-loaded PLGA-NPs were developed by the nanoprecipitation technique. The RP-HPLC method was developed by using (Agilent Compact LC-1220) and Zorbax Eclipse plus C18 column (150×4.6mm, 3.5µm, Agilent). Finally, the developed method was validated according to the international conference on harmonization (ICH) guidelines. RESULTS: The chromatographic separations of DTX and RTV with good resolutions have been achieved by using the mobile phase Acetonitrile: Water (60:40 v/v) containing 0.1% v/v of orthophosphoric acid at a flow rate of 1.0mL/min, injection volume of 25µL, and at 239nm wavelengths. The validated method found to be linear in the range of 0.001-100µg/mL for DTX and RTV. Detection and quantification limits for DTX were found to be 0.7 and 2.31µg/mL respectively and for RTV it is 0.3 and 2.87µg/mL respectively. The % RSD was found to be less than 2% revealing the precision of the developed method. Besides, the recovery rate was observed close to 100% for both the drugs confirming the accuracy of the method. Minor alterations in the chromatographic conditions have revealed robustness and ruggedness of the developed method. CONCLUSION: The developed analytical method is simple, precise, sensitive, and reproducible which can be used for the simultaneous estimation of DTX and RTV in the PLGA-NPs.

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).

Investigating the Physicochemical Stability of Highly Purified Darunavir Ethanolate Extracted from PREZISTA® Tablets.

Understanding physicochemical stability of darunavir ethanolate is expected to be of critical importance for the development and manufacturing of high-quality darunavir-related pharmaceutical products. However, there are no enabling monographs for darunavir to illustrate its solid-state chemistry, impurity profile, and assay methods. In addition, the US Pharmacopeia reference standard of darunavir is still not commercially available. It has been also challenging to find reliable vendors to obtain highly purified darunavir ethanolate crystals to conduct the physicochemical stability testing. In the present research, we developed a straightforward and cost-effective approach to extract and purify darunavir ethanolate from PREZISTA® tablets using reverse-engineering and crystallization. Using these highly purified crystals, we thoroughly evaluated the potential risks of degradation and form conversions of darunavir ethanolate at stressed conditions to define the manufacturing and packaging specifications for darunavir-related products. Amorphization was observed under thermal storage caused by desolvation of darunavir ethanolate. The ethanolate-to-hydrate conversion of darunavir was observed at high relative humidity conditions. Moreover, acid/base-induced degradations of darunavir have been investigated herein to determine the possible drug-excipient compatibility issues in formulations. Furthermore, it is of particular interests to allow the production of high-quality darunavir-ritonavir fixed dose combinations for marketing in Africa. Thus, a validated HPLC method was developed according to ICH guideline to simultaneously quantify assays of darunavir and ritonavir in a single injection. In summary, the findings of this study provide important information for pharmaceutical scientists to design and develop reliable formulations and processings for darunavir-related products with improved stability.

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.

Simultaneous Determination of Impurities of Atazanavir and Ritonavir in Tablet Dosage Form by Using Reversed-Phase Ultra Performance Liquid Chromatographic Method.

A simple, rapid, selective and stability indicating reversed phase-ultra performance liquid chromatography method was developed and validated for the simultaneous quantification of process related and degradation impurities present in Atazanavir and Ritonavir tablets. The two proposed drug components and their respective impurities were separated using Acquity BEH C18 (100 mm × 2.1 mm), 1.7 µ column at a flow rate of 0.4 mL/min. Buffer used as Mobile phase-A which consists of 0.01 M monobasic potassium hydrogen phosphate adjusted the pH to 3.6 and acetonitrile is used as organic modifier (mobile phase-B). The detector wavelength of 240 nm was used for quantifying the impurities. Both the drug components along with their impurities were eluted within a runtime of 18 min. The performance of the developed method was checked by validating the method according to the requirements of International Conference on Harmonization for parameters such as specificity, precision, linearity, ruggedness, accuracy, sensitivity (limit of detection (LOD) and limit of quantitation (LOQ)) and robustness. Linearity and range were established from LOQ level to 150% level. Accuracy of the method was demonstrated from LOQ level to 150% level. The developed stability indicating method is capable for determination of impurities of Atazanavir and Ritonavir in combined tablet dosage form as well as individual dosage forms also. The reported method enables lesser solvent consumption and reduces time and cost of the analysis in quality control laboratory.

Development and validation of a UPLC-MS/MS method for the simultaneous determination of paritaprevir and ritonavir in rat liver.

AIM: Determination of paritaprevir and ritonavir in rat liver tissue samples. RESULTS: We successfully validated a UPLC-MS/MS method to measure paritaprevir and ritonavir in rat liver using deuterated internal standards (d8-paritapervir and d6-ritonavir). The method is linear from 20 to 20,000 and 5 to 10,000 pg on column for paritaprevir and ritonavir, respectively, and is normalized per milligram tissue. Interday and intraday variability ranged from 0.591 to 5.33% and accuracy ranged from -6.68 to 10.1% for quality control samples. The method was then applied to the measurement of paritaprevir and ritonavir in rat liver tissue samples from a pilot study. CONCLUSION: The validated method is suitable for measurement of paritaprevir and ritonavir within rat liver tissue samples for PK studies.

Preparation of ritonavir nanosuspensions by microfluidization using polymeric stabilizers: I. A Design of Experiment approach.

The objective of this study was to prepare ritonavir (RTV) nanosuspensions, an anti-HIV protease inhibitor, to solve its poor water solubility issues. The microfluidization method with a pre-treatment step was used to obtain the nanosuspensions. Design of Experiment (DoE) approach was performed in order to understand the effect of the critical formulation parameters which were selected as polymer type (HPMC or PVP), RTV to polymer ratio, and number of passes. Interactions between the formulation variables were evaluated according to Univariate ANOVA. Particle size, particle size distribution and zeta potential were selected as dependent variables. Scanning electron microscopy, X-ray powder diffraction, and differential scanning calorimetry were performed for the in vitro characterization after lyophilization of the optimum nanosuspension formulation. The saturation solubility was examined in comparison with coarse powder, physical mixture and nanosuspension. In vitro dissolution studies were conducted using polyoxyethylene 10 lauryl ether (POE10LE) and biorelevant media (FaSSIF and FeSSIF). The results showed nanosuspensions were partially amorphous and spherically shaped with particle sizes ranging from 400 to 600nm. Moreover, 0.1-0.4 particle size distribution and about -20mV zeta potential values were obtained. The nanosuspension showed a significantly increased solubility when compared to coarse powder (3.5 fold). Coarse powder, physical mixture, nanosuspension and commercial product dissolved completely in POE10LE; however, cumulative dissolved values reached ~20% in FaSSIF for the commercial product and nanosuspension. The nanosuspension showed more than 90% drug dissolved in FeSSIF compared to the commercial product which showed ~50% in the same medium. It was determined that RTV dissolution was increased by nanosuspension formulation. We concluded that DoE approach is useful to develop nanosuspension formulation to improve solubility and dissolution rate of RTV.

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.

Validation of simultaneous quantitative method of HIV protease inhibitors atazanavir, darunavir and ritonavir in human plasma by UPLC-MS/MS.

OBJECTIVES: HIV protease inhibitors are used in the treatment of patients suffering from AIDS and they act at the final stage of viral replication by interfering with the HIV protease enzyme. The paper describes a selective, sensitive, and robust method for simultaneous determination of three protease inhibitors atazanavir, darunavir and ritonavir in human plasma by ultra performance liquid chromatography-tandem mass spectrometry. MATERIALS AND METHODS: The sample pretreatment consisted of solid phase extraction of analytes and their deuterated analogs as internal standards from 50 µL human plasma. Chromatographic separation of analytes was performed on Waters Acquity UPLC C18 (50 × 2.1 mm, 1.7 µm) column under gradient conditions using 10 mM ammonium formate, pH 4.0, and acetonitrile as the mobile phase. RESULTS: The method was established over a concentration range of 5.0-6000 ng/mL for atazanavir, 5.0-5000 ng/mL for darunavir and 1.0-500 ng/mL for ritonavir. Accuracy, precision, matrix effect, recovery, and stability of the analytes were evaluated as per US FDA guidelines. CONCLUSIONS: The efficiency of sample preparation, short analysis time, and high selectivity permit simultaneous estimation of these inhibitors. The validated method can be useful in determining plasma concentration of these protease inhibitors for therapeutic drug monitoring and in high throughput clinical studies.

Role of P-glycoprotein in the distribution of the HIV protease inhibitor atazanavir in the brain and male genital tract.

The blood-testis barrier and blood-brain barrier are responsible for protecting the male genital tract and central nervous system from xenobiotic exposure. In HIV-infected patients, low concentrations of antiretroviral drugs in cerebrospinal fluid and seminal fluid have been reported. One mechanism that may contribute to reduced concentrations is the expression of ATP-binding cassette drug efflux transporters, such as P-glycoprotein (P-gp). The objective of this study was to investigate in vivo the tissue distribution of the HIV protease inhibitor atazanavir in wild-type (WT) mice, P-gp/breast cancer resistance protein (Bcrp)-knockout (Mdr1a-/-, Mdr1b-/-, and Abcg2-/- triple-knockout [TKO]) mice, and Cyp3a-/- (Cyp) mice. WT mice and Cyp mice were pretreated with a P-gp/Bcrp inhibitor, elacridar (5 mg/kg of body weight), and the HIV protease inhibitor and boosting agent ritonavir (2 mg/kg intravenously [i.v.]), respectively. Atazanavir (10 mg/kg) was administered i.v. Atazanavir concentrations in plasma (Cplasma), brain (Cbrain), and testes (Ctestes) were quantified at various times by liquid chromatography-tandem mass spectrometry. In TKO mice, we demonstrated a significant increase in atazanavir Cbrain/Cplasma (5.4-fold) and Ctestes/Cplasma (4.6-fold) ratios compared to those in WT mice (P<0.05). Elacridar-treated WT mice showed a significant increase in atazanavir Cbrain/Cplasma (12.3-fold) and Ctestes/Cplasma (13.5-fold) ratios compared to those in vehicle-treated WT mice. In Cyp mice pretreated with ritonavir, significant (P<0.05) increases in atazanavir Cbrain/Cplasma (1.8-fold) and Ctestes/Cplasma (9.5-fold) ratios compared to those in vehicle-treated WT mice were observed. These data suggest that drug efflux transporters, i.e., P-gp, are involved in limiting the ability of atazanavir to permeate the rodent brain and genital tract. Since these transporters are known to be expressed in humans, they could contribute to the low cerebrospinal and seminal fluid antiretroviral concentrations reported in the clinic.

Combined quantification of paclitaxel, docetaxel and ritonavir in human feces and urine using LC-MS/MS.

A combined assay for the determination of paclitaxel, docetaxel and ritonavir in human feces and urine is described. The drugs were extracted from 200 µL urine or 50 mg feces followed by high-performance liquid chromatography analysis coupled with positive ionization electrospray tandem mass spectrometry. The validation program included calibration model, accuracy and precision, carry-over, dilution test, specificity and selectivity, matrix effect, recovery and stability. Acceptance criteria were according to US Food and Drug Administration guidelines on bioanalytical method validation. The validated range was 0.5-500 ng/mL for paclitaxel and docetaxel, 2-2000 ng/mL for ritonavir in urine, 2-2000 ng/mg for paclitaxel and docetaxel, and 8-8000 ng/mg for ritonavir in feces. Inter-assay accuracy and precision were tested for all analytes at four concentration levels and were within 8.5% and <10.2%, respectively, in both matrices. Recovery at three concentration levels was between 77 and 94% in feces samples and between 69 and 85% in urine samples. Method development, including feces homogenization and spiking blank urine samples, are discussed. We demonstrated that each of the applied drugs could be quantified successfully in urine and feces using the described assay. The method was successfully applied for quantification of the analytes in feces and urine samples of patients.

Simultaneous determination of probe drugs, metabolites, inhibitors and inducer in human plasma by liquid chromatography/tandem mass spectrometry and its application to pharmacokinetic study.

Cytochrome P450 3A4 (CYP3A4) and UDP-glucuronosyltransferase 1A1 (UGT1A1) are important enzymes responsible for the metabolism of many xenobiotics. To investigate their induction and inhibition properties, administering probe drugs and monitoring their concentration in plasma under the effects of inducers/inhibitors is the gold standard method. A rapid and sensitive liquid chromatography-tandem mass spectrometry method was developed for simultaneous quantification of midazolam, raltegravir (probe drugs for CYP3A4 and UGT1A1), their major metabolites, 1'-hydroxymidazolam, 1'-hydroxymidazolam glucuronide and raltegravir glucuronide, rifampicin (inducer), ritonavir and ketoconazole (inhibitors). Analytes were extracted from 100µl of plasma using solid-phase extraction followed by chromatographic separation on a reversed-phase C18 column (50mm×2.1mm, particle size 1.8µm). The mass spectrometer was operated under positive ionization mode. Excellent linearity (r(2)≥0.995) was achieved for all. The method was validated and found to be accurate (88-111%), precise (CV%<13) and selective. Matrix effect was acceptable (88-118%) and analytes recovery was reproducible (60-95%). Analytes in plasma were also found to be stable in the autosampler (6°C for 48h) and after two freeze-thaw cycles. We have developed a robust analytical method to simultaneously quantify probes, inducer and inhibitor of important drug metabolism enzymes. The method was successfully applied in a clinical study to investigate the degree of induction and inhibition of CYP3A4 and UGT1A1 among ethnic groups in Singapore.

Development and validation of a systematic UPLC-MS/MS method for simultaneous determination of three phenol impurities in ritonavir.

A stability indicating gradient reverse phase UPLC-MS/MS method was developed and validated for the simultaneous determination of three phenol impurities in ritonavir drug substance. The chromatographic separation was performed on Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 µm) using gradient elution of 0.05% ammonia in methanol and 5.0 mM ammonium acetate buffer (30:70, v/v) at a flow rate of 0.2 mL/min. Both negative and positive electrospray ionization (ESI) modes were operated simultaneously for the quantification of three phenol impurities. The total run time was 11 min, within which ritonavir and its three impurities were well separated. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. The calibration curves showed a good linearity over the concentration range of 0.3-1.5 ppm for phenol and 0.1-1.5 ppm for both 4-nitrophenol and N-phenoxycarbonyl-L-valine (NPV). The determination coefficient obtained was >0.9998 in each case. The method had very low limit of detection (LOD) and limit of quantification (LOQ) and the accuracy lies between 97.8% and 103.2% for all the three phenol impurities. The developed method was successfully applied for five formulation batches of ritonavir to determine its phenol impurities.

Quantitative analysis of the effect of zidovudine, efavirenz, and ritonavir on insulin aggregation by multivariate curve resolution alternating least squares of infrared spectra.

Quantification of the effect of antiretroviral drugs on the insulin aggregation process is an important area of research due to the serious metabolic diseases observed in AIDS patients after prolonged treatment with these drugs. In this work, multivariate curve resolution alternating least squares (MCR-ALS) was applied to infrared monitoring of the insulin aggregation process in the presence of three antiretroviral drugs to quantify their effect. To evidence concentration dependence in this process, mixtures at two different insulin:drug molar ratios were used. The interaction between insulin and each drug was analysed by (1)H NMR spectroscopy. In all cases, the aggregation process was monitored during 45 min by infrared spectroscopy. The aggregates were further characterised by scanning electron microscopy (SEM). MCR-ALS provided the spectral and concentration profiles of the different insulin-drug conformations that are involved in the process. Their feasible band boundaries were calculated using the MCR-BANDS methodology. The kinetic profiles describe the aggregation pathway and the spectral profiles characterise the conformations involved. The retrieved results show that each of the three drugs modifies insulin conformation in a different way, promoting the formation of aggregates. Ritonavir shows the strongest promotion of aggregation, followed by efavirenz and zidovudine. In the studied concentration range, concentration dependence was only observed for zidovudine, with shorter aggregation time obtained as the amount of zidovudine increased. This factor also affected the aggregation pathway.

UPLC-MS/MS quantification of nanoformulated ritonavir, indinavir, atazanavir, and efavirenz in mouse serum and tissues.

Animal pharmacokinetic and tissue distribution assays of antiretroviral therapeutic drugs require accurate drug quantification in biological fluids and tissues. Here we report a simple, rapid, and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of commonly used antiretroviral drugs ritonavir (RTV), indinavir (IDV), atazanavir (ATV), and efavirenz (EFV) in mouse serum and tissues (liver, kidney, lung, and spleen). These antiretroviral drugs are currently the cornerstones of common therapeutic regimens for human immunodeficiency virus (HIV) infection. Chromatographic separation was achieved using a gradient mobile phase (5% acetonitrile in methanol and 7.5mM ammonium acetate (pH 4.0)) on an ACQUITY UPLC(®)BEH Shield RP 18 column. All compounds eluted within a 7 min run time. Lopinavir was used as an internal standard. Detection was achieved by dual positive and negative ionization modes on a quadrupole linear ion trap hybrid mass spectrometer with an electrospray ionization (ESI) source. The dynamic range was 0.2-1000 ng/mL for RTV, IDV, and ATV, and 0.5-1000 for EFV. The method was validated and showed high and consistent intra-day and inter-day accuracy and precision for all analytes. This method is used to support the preclinical development studies of targeted- and sustained-release combination ART (nanoART). The current data demonstrate a 1.5-4 fold increase in serum and tissue AUC of nanoformulated ATV, RTV, and EFV administered to mice when compared to native drug. In addition, the tested formulation enhanced exposure of the same anti-HIV drugs in mouse tissues.

Micellar electrokinetic chromatography method development for determination of impurities in ritonavir.

Ritonavir is a synthetic peptidomimetic human immunodeficiency virus (HIV) protease inhibitor employed in the treatment of AIDS since 1996. Synthetic precursors are potential impurities in the final product. In the present work a micellar electrokinetic chromatography (MEKC) method for the separation of Ritonavir from three available synthetic precursors was developed. The optimized separation is performed in a background electrolyte composed of sodium tetraborate (pH 9.6; 15mM) containing sodium dodecylsulfate (30mM) and acetonitrile (18%, v/v). Mass spectrometry was used to confirm the identity of the tested substances. Good repeatability was observed for migration time (RSD about 0.4%) and peak area (RSD about 0.8%). The limits of detection (LOD) obtained allow the determination of two of the impurities at levels as low as 0.005% m/m, and one at a level of 0.3% m/m.

LC-MS/MS studies of ritonavir and its forced degradation products.

Forced degradation of ritonavir (RTV), under the conditions of hydrolysis (acidic, basic and neutral), oxidation, photolysis and thermal stress as prescribed by ICH was studied using LC-MS/MS. Eight degradation products were formed and their separation was accomplished on Waters XTerra C(18) column (250 mm x 4.6 mm i.d., 5 microm) using water:methanol:acetonitrile as (40:20:40, v/v/v) mobile phase in an isocratic elution mode by LC. The method was extended to LC-MS/MS for characterization of the degradation products and the pathways of decomposition were proposed. No previous reports were found in the literature regarding the characterization of degradation products of ritonavir.