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 a UPLC-MS method for determination of atazanavir sulfate by the "analytical quality by design" approach.

A UPLC-MS method for the estimation of atazanavir sulfate was developed using the "analytical quality by design" approach. The critical chromatographic quality attributes identified were retention time, theoretical plates and peak tailing. The critical method parameters established were percent of organic modifier, flow rate and injection volume. Optimization performed using Box-Behnken Design (BBD) established 10 % organic modifier, 0.4 mL min-1 flow rate and 6-µL injection volume as the optimum method conditions. Atazanavir sulfate eluted at 5.19 min without any interference. Method validation followed international guidelines. The method has proven linearity in the range of 10-90 µg mL-1. Recovery was between 100.2-101.0 % and precision within the accepted limits (RSD 0.2-0.7 %). LOD and LOQ were 2.68 and 8.14 µg mL-1, resp. Stress testing stability studies showed atazanavir sulfate to degrade under acidic and basic conditions. The suggested technique is simple, rapid and sustainable. It is, therefore, suggested for routine analysis of atazanavir sulfate.

Targeting HIV-1 Protease Autoprocessing for High-throughput Drug Discovery and Drug Resistance Assessment.

HIV-1 protease autoprocessing liberates the free mature protease from its Gag-Pol polyprotein precursor through a series of highly regulated autoproteolysis reactions. Herein, we report the development and validation (Z' ≥ 0.50) of a cell-based functional assay for high-throughput screening (HTS) of autoprocessing inhibitors using fusion precursors in combination with AlphaLISA (amplified luminescent proximity homogeneous assay ELISA). Through pilot screening of a collection of 130 known protease inhibitors, the AlphaLISA assay confirmed all 11 HIV protease inhibitors in the library capable of suppressing precursor autoprocessing at low micromolar concentrations. Meanwhile, other protease inhibitors had no impact on precursor autoprocessing. We next conducted HTS of ~23,000 compounds but found no positive hits. Such high selectivity is advantageous for large-scale HTS campaigns and as anticipated based on assay design because a positive hit needs simultaneously to be nontoxic, cell permeable, and inhibiting precursor autoprocessing. Furthermore, AlphaLISA quantification of fusion precursors carrying mutations known to cause resistance to HIV protease inhibitors faithfully recapitulated the reported resistance, suggesting that precursor autoprocessing is a critical step contributing to drug resistance. Taken together, this reported AlphaLISA platform will provide a useful tool for drug discovery targeting HIV-1 protease autoprocessing and for quantification of PI resistance.

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.

Hydroxyethylamine derivatives as HIV-1 protease inhibitors: a predictive QSAR modelling study based on Monte Carlo optimization.

Application of HIV-1 protease inhibitors (as an anti-HIV regimen) may serve as an attractive strategy for anti-HIV drug development. Several investigations suggest that there is a crucial need to develop a novel protease inhibitor with higher potency and reduced toxicity. Monte Carlo optimized QSAR study was performed on 200 hydroxyethylamine derivatives with antiprotease activity. Twenty-one QSAR models with good statistical qualities were developed from three different splits with various combinations of SMILES and GRAPH based descriptors. The best models from different splits were selected on the basis of statistically validated characteristics of the test set and have the following statistical parameters: r2 = 0.806, Q2 = 0.788 (split 1); r2 = 0.842, Q2 = 0.826 (split 2); r2 = 0.774, Q2 = 0.755 (split 3). The structural attributes obtained from the best models were analysed to understand the structural requirements of the selected series for HIV-1 protease inhibitory activity. On the basis of obtained structural attributes, 11 new compounds were designed, out of which five compounds were found to have better activity than the best active compound in the series.

Effects of liquid post-column addition in electrospray ionization performance in supercritical fluid chromatography-mass spectrometry.

In supercritical fluid chromatography coupled to atmospheric pressure ionization mass spectrometry (SFC-MS), the use of a make-up post-column is almost mandatory to avoid analyte precipitation, especially when using low percentage of modifier (<5%) in the mobile phase. Due to the specific nature of gaseous CO2, the tuning of the make-up conditions in electrospray becomes an important factor and can be used to tune analyte sensitivity. Neither a dilution effect (loss of signal) nor a relevant degradation of chromatographic performances is observed with the addition of a make-up at various flow-rates, up to 0.7mL/min. From supercritical conditions (1mL/min 40°C, 150bar) to gaseous state (room temperature, atmospheric pressure), the CO2 expands around 430 times, contributing to almost 5% of the nebulizing process. In positive mode, the presence of ammonium ions either in the mobile phase or in the make-up did significantly increase the MS signal, even at basic apparent pH. The ionization performance of electrospray is influenced by the acidic buffer power of the carbon dioxide, and was found to be restricted in the apparent pH range of 3.8-7.2 in the various conditions investigated. This may challenge sensitive detection in negative mode, as illustrated for bosentan. The use of DMSO as make-up additive (up to 30%) showed a simplification of the full scan spectrum regarding the adducts. Finally, the optimization of make-up composition leads to an enhancement up to a factor of 69 on the electrospray MS response signal, for the SFC-SRM/MS analysis of HIV protease inhibitors in plasma extracted from Dried Plasma Spots.

Multistage virtual screening and identification of novel HIV-1 protease inhibitors by integrating SVM, shape, pharmacophore and docking methods.

The HIV-1 protease has proven to be a crucial component of the HIV replication machinery and a reliable target for anti-HIV drug discovery. In this study, we applied an optimized hierarchical multistage virtual screening method targeting HIV-1 protease. The method sequentially applied SVM (Support Vector Machine), shape similarity, pharmacophore modeling and molecular docking. Using a validation set (270 positives, 155,996 negatives), the multistage virtual screening method showed a high hit rate and high enrichment factor of 80.47% and 465.75, respectively. Furthermore, this approach was applied to screen the National Cancer Institute database (NCI), which contains 260,000 molecules. From the final hit list, 6 molecules were selected for further testing in an in vitro HIV-1 protease inhibitory assay, and 2 molecules (NSC111887 and NSC121217) showed inhibitory potency against HIV-1 protease, with IC50 values of 62 µM and 162 µM, respectively. With further chemical development, these 2 molecules could potentially serve as HIV-1 protease inhibitors.

A rapid spin through oil results in higher cell-associated concentrations of antiretrovirals compared with conventional cell washing.

BACKGROUND: Determination of cell-associated antiretroviral drug concentrations is necessary for research into reservoirs of HIV. Variation exists in cell-associated drug concentrations among research groups. One cause for this may be washing cells during processing. We explored spinning cells through oil to minimize this variability. METHODS & RESULTS: Raltegravir, atazanavir, darunavir, efavirenz, lopinavir and ritonavir concentrations were assessed in CEM.ss T cells washed with HBSS and oil-spun cells. Oil-spun cells had significantly higher concentrations for all drugs compared with samples washed with HBSS. CONCLUSION: The decline in cell-associated drug concentrations with saline washes compared with a single spin through oil shows the utility of a spin through oil. Oil centrifugation results in high cell-associated drug concentrations, and can be done in a fast, efficient manner.

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.

A critical review of properties of darunavir and analytical methods for its determination.

Darunavir is a synthetic non-peptidic protease inhibitor that has been shown to be extremely potent against wild-type HIV, and it is an important component of highly active antiretroviral treatment (HAART), which is considered as one of the most significant advances in the field of HIV therapy. However, there are some concerns about darunavir quality control. Darunavir shows pseudo-polymorphism: in different ambient conditions one pseudo-polymorphic form can change to another. This behavior of darunavir is problematic because the dosage form is exposed to different ambient conditions around the world, since HIV/AIDS is prevalent globally. Issues around differences in the solubility and effects that different forms of darunavir can cause are of concern, and a more stable form is preferable. Important investigations of darunavir such as dissolution behavior, polymorphism, stability and degradation studies, and the impact of that on the quality of the product are being conducted by our working group. A cure for HIV/AIDS remains a long-term commitment, and there is much yet to achieve. This article discusses, by a critical review of the literature, the impact of the use of darunavir in the treatment of HIV-infected patients, its physical-chemical properties, the analytical methods to determine it, and challenges that remain in order to ensure the quality and stability of darunavir.

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.

RP-HPLC separation and characterization of unknown impurities of a novel HIV-protease inhibitor Darunavir by ESI-MS and 2D NMR spectroscopy.

A selective RP-HPLC method for separation and determination of darunavir from its process related substances has been developed and validated. The separation was accomplished on a Hiber, LiChrospher 60, RP-select B, C8 (250 mm × 4.6 mm, 5 µm) column connected to a photo diode array detector (PDA) using 10 mM phosphate buffer with 0.1% of triethylamine (pH: 4.0 adjusted with orthophosphoric acid)/acetonitrile as a mobile phase under gradient elution. Two unknown impurities of darunavir were isolated and characterized by ¹H, ¹³C, 2D-NMR and mass spectrometry. The method was validated in terms of accuracy, precision, linearity, robustness, LOD and LOQ.

Antibacterial, anti-HIV-1 protease and cytotoxic activities of aqueous ethanolic extracts from Combretum adenogonium Steud. Ex A. Rich (Combretaceae).

BACKGROUND: Records have shown that Combretum adenogonium Steud. Ex A. Rich (Combretaceae) is used in traditional medicine systems of several tribes in Tanzania. This study focused on the investigation of antibacterial activity, anti-HIV-1 protease activity, toxicity properties and classes of phytochemicals in extracts from C. adenogonium Steud. Ex A. Rich (Combretaceae) to evaluate potential of these extracts for development as herbal remedies. METHODS: Dried plant material were ground to fine powder and extracted using 80% aqueous ethanol to afford root, leaf and stem bark extracts. The extracts were assayed for anti-HIV-1 protease activities, antibacterial activities using microdilution methods and cytotoxicity using brine shrimps lethality assay. Screening for major phytochemical classes was carried out using standard chemical tests. RESULTS: All extracts exhibited antibacterial activity to at least one of the test bacteria with MIC-values ranging from 0.31-5.0 mg/ml. Two extracts, namely, root and stem bark exhibited anti-HIV-1 PR activity with IC50 values of 24.7 and 26.5 µg/ml, respectively. Stem bark and leaf extracts showed mild toxicity with LC50 values of 65.768 µg/ml and 76.965 µg/ml, respectively, whereas roots were relatively non-toxic (LC50 = 110.042 µg/ml). Phytochemical screening of the extracts indicated presence of flavonoids, terpenoids, alkaloids, tannins, glycosides and saponins. CONCLUSION: These results provide promising baseline information for the potential development of C. adenogonium extracts in treatment of bacterial and HIV/AIDS-related opportunistic infections.

Direct electrochemistry of hemoglobin immobilized in chitosan-room temperature ionic liquid film and application in its interaction with 3,4'-bis-(4-hydro-3-xycoumarin)-2,5-hexanediol.

The direct electrochemistry of hemoglobin (Hb) and application in its interaction with 3,4'-bis-(4-hydro-3-xycoumarin)-2,5-hexanediol (HCH) based on the Hb immobilized in chitosan-room temperature ionic liquid film were investigated by means of cyclic voltammetry, differential pulse voltammetry and amperometry. The binding ratio m and binding constant between HCH and Hb were estimated as 2 and 3.46 M(-1), respectively. The amperometric response showed a linear dependence on the concentration of HCH with the detection limit of 0.06 µM. In addition, the amperometry was a novel method in the field of binding studies and might be taken into account for future binding studies. At last a sensitive and convenient electrochemical method was proposed for the determination of HCH.

Targeting SVCT for enhanced drug absorption: synthesis and in vitro evaluation of a novel vitamin C conjugated prodrug of saquinavir.

In order to improve oral absorption, a novel prodrug of saquinavir (Saq), ascorbyl-succinic-saquinavir (AA-Su-Saq) targeting sodium dependent vitamin C transporter (SVCT) was synthesized and evaluated. Aqueous solubility, stability and cytotoxicity were determined. Affinity of AA-Su-Saq towards efflux pump P-glycoprotein (P-gp) and recognition of AA-Su-Saq by SVCT were studied. Transepithelial permeability across polarized MDCK-MDR1 and Caco-2 cells were determined. Metabolic stability of AA-Su-Saq in rat liver microsomes was investigated. AA-Su-Saq appears to be fairly stable in both DPBS and Caco-2 cells with half lives of 9.65 and 5.73 h, respectively. Uptake of [(3)H]Saquinavir accelerated by 2.7 and 1.9 fold in the presence of 50 µM Saq and AA-Su-Saq in MDCK-MDR1 cells. Cellular accumulation of [(14)C]AA diminished by about 50-70% relative to control in the presence of 200 µM AA-Su-Saq in MDCK-MDR1 and Caco-2 cells. Uptake of AA-Su-Saq was lowered by 27% and 34% in the presence of 5mM AA in MDCK-MDR1 and Caco-2 cells, respectively. Absorptive permeability of AA-Su-Saq was elevated about 4-5 fold and efflux index reduced by about 13-15 fold across the polarized MDCK-MDR1 and Caco-2 cells. Absorptive permeability of AA-Su-Saq decreased 44% in the presence of 5mM AA across MDCK-MDR1 cells. AA-Su-Saq was devoid of cytotoxicity over the concentration range studied. AA-Su-Saq significantly enhanced the metabolic stability but lowered the affinity towards CYP3A4. In conclusion, prodrug modification of Saq through conjugation to AA via a linker significantly raised the absorptive permeability and metabolic stability. Such modification also caused significant evading of P-gp mediated efflux and CYP3A4 mediated metabolism. SVCT targeted prodrug approach can be an attractive strategy to enhance the oral absorption and systemic bioavailability of anti-HIV protease inhibitors.

LC, LC-MS/TOF and MS(n) studies for the identification and characterization of degradation products of nelfinavir mesylate.

The objective of the present investigation was to separate, identify and characterize the major degradation products (DPs) of nelfinavir mesylate generated under hydrolytic, oxidative, photolytic and thermal stress conditions as advised in International Conference on Harmonization (ICH) guideline Q1A(R2). The drug was found to degrade under acidic, basic, oxidative and photolytic stress, while it was stable in neutral and thermal stress conditions. A total of three degradation products were formed, which were separated on a C-18 column employing a gradient HPLC method. A complete mass fragmentation pathway of the drug was first established with the help of multi-stage (MS(n)) and MS/TOF accurate mass studies. Then stressed samples were subjected to LC-MS/TOF studies, which provided their fragmentation pattern and accurate masses. The mass spectral data were employed to characterize the DPs and assign structures to them. The total information was also used to establish the degradation pathway of the drug. The degradation products were identified as 3-hydroxy-N-((2R,3R)-3-hydroxy-1-(phenylthio)butan-2-yl)-2-methylbenzamide and (3S,4aS,8aS)-N-tert-butyl-2-((2R,3R)-2-hydroxy-3-(3-hydroxy-2-methylbenzamido)-4-(phenylsulfinyl)butyl)decahydroisoquinoline-3-carboxamide.

CYP3A-mediated generation of aldehyde and hydrazine in atazanavir metabolism.

Atazanavir (ATV) is an antiretroviral drug of the protease inhibitor class. Multiple adverse effects of ATV have been reported in clinical practice, such as jaundice, nausea, abdominal pain, and headache. The exact mechanisms of ATV-related adverse effects are unknown. It is generally accepted that a predominant pathway of drug-induced toxicity is through the generation of reactive metabolites. Our current study was designed to explore reactive metabolites of ATV. We used a metabolomic approach to profile ATV metabolism in mice and human liver microsomes. We identified 5 known and 13 novel ATV metabolites. Three potential reactive metabolites were detected and characterized for the first time: one aromatic aldehyde, one α-hydroxyaldehyde, and one hydrazine. These potential reactive metabolites were primarily generated by CYP3A. Our results provide a clue for studies on ATV-related adverse effects from the aspect of metabolic activation. Further studies are suggested to illustrate the impact of these potential reactive metabolites on ATV-related adverse effects.

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.