Simultaneous HPLC assay for quantification of indinavir, nelfinavir, ritonavir, and saquinavir in human plasma

Rory P. Remmel, Sagar P. Kawle, Dennis Weller, Courtney V. Fletcher

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

Background: HIV protease inhibitors are recommended as part of combination antiretroviral therapy. Dual protease inhibitor therapy is also being used clinically. Consequently, a simultaneous assay for indinavir, nelfinavir, ritonavir, and saquinavir was developed. Methods: Indinavir, nelfinavir, ritonavir, and saquinavir were extracted from plasma (250 μL) with methyl-t-butyl ether at basic pH after addition of an internal standard (A-86093). The compounds were separated on a Keystone BetaBasic C4 column (250 x 3 mm i.d.) at 40 °C with a mobile phase of acetonitrile-50 mmol/L ammonium formate buffer, pH 4.1 (52:48, by volume) at a flow rate of 0.5 mL/min. Indinavir, nelfinavir, ritonavir, and the internal standard (A-86093) were detected at 218 nm, and saquinavir was detected at 235 nm. The method was validated by analysis of five triplicate analyses of calibrators along with quality-control samples at three different concentrations prepared in human plasma. Results: The extraction recovery was 87-92%. Within-run accuracy for quality-control samples was 6-8%, with CVs of 2-8%. Limits of quantification were 40-50 μg/L for indinavir, nelfinavir, and ritonavir, and 20 μg/L for saquinavir. Cross-validation with a liquid chromatography-mass spectroscopy method for saquinavir and nelfinavir was conducted with patient samples. Regression analysis revealed a good correlation (r2 >0.94) between methods. Larger variations at concentrations >4000 μg/L were observed with nelfinavir. Interference with drugs commonly used in AIDS patients was not observed. Pharmacokinetic profiles for two patients on dual protease therapy were determined. Conclusions: A reliable and rugged simultaneous HPLC assay for four HIV protease inhibitors was developed. The assay method is convenient for clinical laboratories involved in therapeutic drug monitoring for HIV protease inhibitors. The assay has enough sensitivity to conduct pharmacokinetic studies in patients taking more than one HIV protease inhibitor along with other antiretroviral medications. (C) 2000 American Association for Clinical Chemistry.

Original languageEnglish (US)
Pages (from-to)73-81
Number of pages9
JournalClinical chemistry
Volume46
Issue number1
StatePublished - Feb 23 2000

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Nelfinavir
Saquinavir
Indinavir
Plasma (human)
Ritonavir
HIV Protease Inhibitors
Assays
High Pressure Liquid Chromatography
formic acid
Pharmacokinetics
Quality Control
Quality control
Clinical laboratories
Drug Monitoring
Liquid chromatography
Protease Inhibitors
Regression analysis
Liquid Chromatography
Pharmaceutical Preparations
Mass Spectrometry

Cite this

Simultaneous HPLC assay for quantification of indinavir, nelfinavir, ritonavir, and saquinavir in human plasma. / Remmel, Rory P.; Kawle, Sagar P.; Weller, Dennis; Fletcher, Courtney V.

In: Clinical chemistry, Vol. 46, No. 1, 23.02.2000, p. 73-81.

Research output: Contribution to journalArticle

Remmel, Rory P. ; Kawle, Sagar P. ; Weller, Dennis ; Fletcher, Courtney V. / Simultaneous HPLC assay for quantification of indinavir, nelfinavir, ritonavir, and saquinavir in human plasma. In: Clinical chemistry. 2000 ; Vol. 46, No. 1. pp. 73-81.
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abstract = "Background: HIV protease inhibitors are recommended as part of combination antiretroviral therapy. Dual protease inhibitor therapy is also being used clinically. Consequently, a simultaneous assay for indinavir, nelfinavir, ritonavir, and saquinavir was developed. Methods: Indinavir, nelfinavir, ritonavir, and saquinavir were extracted from plasma (250 μL) with methyl-t-butyl ether at basic pH after addition of an internal standard (A-86093). The compounds were separated on a Keystone BetaBasic C4 column (250 x 3 mm i.d.) at 40 °C with a mobile phase of acetonitrile-50 mmol/L ammonium formate buffer, pH 4.1 (52:48, by volume) at a flow rate of 0.5 mL/min. Indinavir, nelfinavir, ritonavir, and the internal standard (A-86093) were detected at 218 nm, and saquinavir was detected at 235 nm. The method was validated by analysis of five triplicate analyses of calibrators along with quality-control samples at three different concentrations prepared in human plasma. Results: The extraction recovery was 87-92{\%}. Within-run accuracy for quality-control samples was 6-8{\%}, with CVs of 2-8{\%}. Limits of quantification were 40-50 μg/L for indinavir, nelfinavir, and ritonavir, and 20 μg/L for saquinavir. Cross-validation with a liquid chromatography-mass spectroscopy method for saquinavir and nelfinavir was conducted with patient samples. Regression analysis revealed a good correlation (r2 >0.94) between methods. Larger variations at concentrations >4000 μg/L were observed with nelfinavir. Interference with drugs commonly used in AIDS patients was not observed. Pharmacokinetic profiles for two patients on dual protease therapy were determined. Conclusions: A reliable and rugged simultaneous HPLC assay for four HIV protease inhibitors was developed. The assay method is convenient for clinical laboratories involved in therapeutic drug monitoring for HIV protease inhibitors. The assay has enough sensitivity to conduct pharmacokinetic studies in patients taking more than one HIV protease inhibitor along with other antiretroviral medications. (C) 2000 American Association for Clinical Chemistry.",
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T1 - Simultaneous HPLC assay for quantification of indinavir, nelfinavir, ritonavir, and saquinavir in human plasma

AU - Remmel, Rory P.

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AU - Fletcher, Courtney V.

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N2 - Background: HIV protease inhibitors are recommended as part of combination antiretroviral therapy. Dual protease inhibitor therapy is also being used clinically. Consequently, a simultaneous assay for indinavir, nelfinavir, ritonavir, and saquinavir was developed. Methods: Indinavir, nelfinavir, ritonavir, and saquinavir were extracted from plasma (250 μL) with methyl-t-butyl ether at basic pH after addition of an internal standard (A-86093). The compounds were separated on a Keystone BetaBasic C4 column (250 x 3 mm i.d.) at 40 °C with a mobile phase of acetonitrile-50 mmol/L ammonium formate buffer, pH 4.1 (52:48, by volume) at a flow rate of 0.5 mL/min. Indinavir, nelfinavir, ritonavir, and the internal standard (A-86093) were detected at 218 nm, and saquinavir was detected at 235 nm. The method was validated by analysis of five triplicate analyses of calibrators along with quality-control samples at three different concentrations prepared in human plasma. Results: The extraction recovery was 87-92%. Within-run accuracy for quality-control samples was 6-8%, with CVs of 2-8%. Limits of quantification were 40-50 μg/L for indinavir, nelfinavir, and ritonavir, and 20 μg/L for saquinavir. Cross-validation with a liquid chromatography-mass spectroscopy method for saquinavir and nelfinavir was conducted with patient samples. Regression analysis revealed a good correlation (r2 >0.94) between methods. Larger variations at concentrations >4000 μg/L were observed with nelfinavir. Interference with drugs commonly used in AIDS patients was not observed. Pharmacokinetic profiles for two patients on dual protease therapy were determined. Conclusions: A reliable and rugged simultaneous HPLC assay for four HIV protease inhibitors was developed. The assay method is convenient for clinical laboratories involved in therapeutic drug monitoring for HIV protease inhibitors. The assay has enough sensitivity to conduct pharmacokinetic studies in patients taking more than one HIV protease inhibitor along with other antiretroviral medications. (C) 2000 American Association for Clinical Chemistry.

AB - Background: HIV protease inhibitors are recommended as part of combination antiretroviral therapy. Dual protease inhibitor therapy is also being used clinically. Consequently, a simultaneous assay for indinavir, nelfinavir, ritonavir, and saquinavir was developed. Methods: Indinavir, nelfinavir, ritonavir, and saquinavir were extracted from plasma (250 μL) with methyl-t-butyl ether at basic pH after addition of an internal standard (A-86093). The compounds were separated on a Keystone BetaBasic C4 column (250 x 3 mm i.d.) at 40 °C with a mobile phase of acetonitrile-50 mmol/L ammonium formate buffer, pH 4.1 (52:48, by volume) at a flow rate of 0.5 mL/min. Indinavir, nelfinavir, ritonavir, and the internal standard (A-86093) were detected at 218 nm, and saquinavir was detected at 235 nm. The method was validated by analysis of five triplicate analyses of calibrators along with quality-control samples at three different concentrations prepared in human plasma. Results: The extraction recovery was 87-92%. Within-run accuracy for quality-control samples was 6-8%, with CVs of 2-8%. Limits of quantification were 40-50 μg/L for indinavir, nelfinavir, and ritonavir, and 20 μg/L for saquinavir. Cross-validation with a liquid chromatography-mass spectroscopy method for saquinavir and nelfinavir was conducted with patient samples. Regression analysis revealed a good correlation (r2 >0.94) between methods. Larger variations at concentrations >4000 μg/L were observed with nelfinavir. Interference with drugs commonly used in AIDS patients was not observed. Pharmacokinetic profiles for two patients on dual protease therapy were determined. Conclusions: A reliable and rugged simultaneous HPLC assay for four HIV protease inhibitors was developed. The assay method is convenient for clinical laboratories involved in therapeutic drug monitoring for HIV protease inhibitors. The assay has enough sensitivity to conduct pharmacokinetic studies in patients taking more than one HIV protease inhibitor along with other antiretroviral medications. (C) 2000 American Association for Clinical Chemistry.

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