Development and validation of an HPLC-UV method for quantifying nevirapine and its main phase I metabolites in human blood

Marinho, Aline T.; Godinho, Ana L.A.; Novais, David A.; Antunes, Alexandra M. M.; Marques, M. Matilde; Ramos, T. Dorta; Dias, Clara G.; Monteiro, Emília C.; Pereira, Sofia A.

doi:10.1039/c3ay41911h

Cited by 9 publications

(4 citation statements)

References 23 publications

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“…Establishment of an efficient bioanalytical method is highly useful for analysis of the drug molecules in rat and human plasma during pharmacokinetic, bioequivalence and toxicological studies. For estimation of nevirapine, several literature reports have been documented for chromatographic method analysis and quantification of nevirapine in biological samples employing high-performance liquid chromatography (Hamrapurkar et al, 2010;Nandi et al, 2012;Sahoo et al, 2013), and tandem mass liquid chromatography (LC-MS/MS; Chi et al, 2003;Hollanders et al, 2000;Marinho et al, 2014;Pav et al, 1999;van Heeswijk et al, 1998). These chromatographic methods are associated with several intricacies and inadequacies, like the use of high-cost solvents, buffers and guard columns, and the need for close monitoring of flow rate, injection volume, flow gradient, column oven temperature, pH, etc., which invariably are critical for consistent method performance during routine analysis (Burhenne, 2012;Tiwari and Tiwari, 2010).…”

Section: Introductionmentioning

confidence: 99%

QbD‐oriented development and validation of a bioanalytical method for nevirapine with enhanced liquid–liquid extraction and chromatographic separation

Beg

Chaudhary

Sharma

et al. 2015

Biomedical Chromatography

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The present studies describe the systematic quality by design (QbD)-oriented development and validation of a simple, rapid, sensitive and cost-effective reversed-phase HPLC bioanalytical method for nevirapine in rat plasma. Chromatographic separation was carried out on a C18 column using isocratic 68:9:23% v/v elution of methanol, acetonitrile and water (pH 3, adjusted by orthophosphoric acid) at a flow rate of 1.0 mL/min using UV detection at 230 nm. A Box-Behnken design was applied for chromatographic method optimization taking mobile phase ratio, pH and flow rate as the critical method parameters (CMPs) from screening studies. Peak area, retention time, theoretical plates and peak tailing were measured as the critical analytical attributes (CAAs). Further, the bioanalytical liquid-liquid extraction process was optimized using an optimal design by selecting extraction time, centrifugation speed and temperature as the CMPs for percentage recovery of nevirapine as the CAA. The search for an optimum chromatographic solution was conducted through numerical desirability function. Validation studies performed as per the US Food and Drug Administration requirements revealed results within the acceptance limit. In a nutshell, the studies successfully demonstrate the utility of analytical QbD approach for the rational development of a bioanalytical method with enhanced chromatographic separation and recovery of nevirapine in rat plasma. Copyright © 2015 John Wiley & Sons, Ltd.

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Section: Introductionmentioning

confidence: 99%

QbD‐oriented development and validation of a bioanalytical method for nevirapine with enhanced liquid–liquid extraction and chromatographic separation

Beg

Chaudhary

Sharma

et al. 2015

Biomedical Chromatography

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“…The data for D31 is presented in Supplementary Figure S2. Phase I and Phase II NVP metabolites were analyzed and quantified by HLPC as described previously [41]. Briefly, for the quantification of Phase I and Phase II NVP metabolites, 15 mL of cell culture supernatants were incubated for 24 h at 37 • C in the absence (unconjugated/free metabolites) or presence of either sulfatase (type H-1 from Helix pomatia, E.C.…”

Section: Quantification Of Nevirapine Metabolitesmentioning

confidence: 99%

“…The pH of all samples was readjusted to 7.0 before extraction of the free metabolites with dichloromethane (2 × 15 mL). Upon evaporation, the organic extracts were dissolved in 150 µL H 2 O/methanol (1:1) and analyzed on an Agilent 1100 Series HPLC-UV system (Agilent Technologies Inc., Santa Clara, CA, USA) as described in Marinho et al [41]. The metabolite levels were normalized to the cell number and expressed as ng/10 6 cells.…”

Section: Quantification Of Nevirapine Metabolitesmentioning

confidence: 99%

Nevirapine Biotransformation Insights: An Integrated In Vitro Approach Unveils the Biocompetence and Glutathiolomic Profile of a Human Hepatocyte-Like Cell 3D Model

Cipriano

Pinheiro

Sequeira

et al. 2020

IJMS

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The need for competent in vitro liver models for toxicological assessment persists. The differentiation of stem cells into hepatocyte-like cells (HLC) has been adopted due to its human origin and availability. Our aim was to study the usefulness of an in vitro 3D model of mesenchymal stem cell-derived HLCs. 3D spheroids (3D-HLC) or monolayer (2D-HLC) cultures of HLCs were treated with the hepatotoxic drug nevirapine (NVP) for 3 and 10 days followed by analyses of Phase I and II metabolites, biotransformation enzymes and drug transporters involved in NVP disposition. To ascertain the toxic effects of NVP and its major metabolites, the changes in the glutathione net flux were also investigated. Phase I enzymes were induced in both systems yielding all known correspondent NVP metabolites. However, 3D-HLCs showed higher biocompetence in producing Phase II NVP metabolites and upregulating Phase II enzymes and MRP7. Accordingly, NVP-exposure led to decreased glutathione availability and alterations in the intracellular dynamics disfavoring free reduced glutathione and glutathionylated protein pools. Overall, these results demonstrate the adequacy of the 3D-HLC model for studying the bioactivation/metabolism of NVP representing a further step to unveil toxicity mechanisms associated with glutathione net flux changes.

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“…All included individuals were HIV-infected patients treated with NVP for at least one month. The plasma concentrations of NVP and its phase I metabolites were quantified by HPLC [ 7 ]. ApoA1 levels were assessed by an immunoturbidimetric assay.…”

mentioning

confidence: 99%

Sex differences in apolipoprotein A1 and nevirapine‐induced toxicity

Marinho¹,

Dias²,

Antunes³

et al. 2014

Journal of the International AIDS Society

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Nevirapine (NVP) is associated with severe liver and skin toxicity through sulfotransferase (SULT) bioactivation of the phase I metabolite 12-hydroxy-NVP [1–3]. The female sex, a well-known risk factor for NVP-induced toxicity, is associated with higher SULT expression [4] and lower plasma levels of 12-hydroxy-NVP [3]. Interestingly, apolipoprotein A1 (ApoA1) increases SULT2B1 activity and ApoA1 synthesis is increased by NVP [5, 6]. Herein, we explore the effect of ApoA1 levels on NVP metabolism and liver function. The study protocol was firstly approved by the hospitals’ Ethics Committees. All included individuals were HIV-infected patients treated with NVP for at least one month. The plasma concentrations of NVP and its phase I metabolites were quantified by HPLC [7]. ApoA1 levels were assessed by an immunoturbidimetric assay. Forty-nine HIV-infected patients on NVP were included (53% men, 59% Caucasian). NVP plasma levels were correlated with HDL-cholesterol (Spearman r=0.2631; p=0.0441) and ApoA1 (Spearman r=0.3907; p=0.0115). Women had higher ApoA1 levels than men (Student's t Test; p=0.0051). In both sexes, 12-hydroxy-NVP levels were negatively correlated with ApoA1 (male: Spearman r=−0.3810; p=0.0499 female: Spearman r=−0.5944; p=0.0415). In men, ApoA1 was positively correlated with aspartate aminotransferase (AST, Spearman r=0.5507; p=0.0413), while in women ApoA1 was associated (Spearman r=0.6408; p=0.0056) with alanine aminotransferase (ALT). These results show sex differences in NVP-induced ApoA1 synthesis. The higher ApoA1 levels in women might stabilize SULT2B1 [6]. This would explain the lower levels of 12-hydroxy-NVP [3] and the higher hepatotoxicity found in women, due to increased sulfonation of this metabolite. These data support a role for ApoA1 in the sex dimorphic mechanism leading to NVP-induced toxicity.

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Development and validation of an HPLC-UV method for quantifying nevirapine and its main phase I metabolites in human blood

Cited by 9 publications

References 23 publications

QbD‐oriented development and validation of a bioanalytical method for nevirapine with enhanced liquid–liquid extraction and chromatographic separation

QbD‐oriented development and validation of a bioanalytical method for nevirapine with enhanced liquid–liquid extraction and chromatographic separation

Nevirapine Biotransformation Insights: An Integrated In Vitro Approach Unveils the Biocompetence and Glutathiolomic Profile of a Human Hepatocyte-Like Cell 3D Model

Sex differences in apolipoprotein A1 and nevirapine‐induced toxicity

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