476
J. Sep. Sci. 2014, 37, 476–483
´ 1 N. Ferreiros S. Labocha1 J. El-Duweik1 C. Schlecker1 1,2 ¨ J. Lotsch G. Geisslinger1,2
Research Article
1 pharmazentrum
LC–MS/MS has been applied for the rapid determination of the nucleoside analogue ribavirin in human plasma and red blood cells. The incorporation of ribavirin to the erythrocytes has been assayed after in vitro incubation of the cells at different concentrations of the antiviral drug. After protein precipitation, samples were injected into a C8 column, achieving a complete separation of ribavirin from the endogenous isobaric compound uridine. Calibration ranges varied from 10 to 10 000 ng/mL in plasma and from 0.2 to 200 ng/cell pellet in red blood cells. Precision and accuracy values were always below 10 and 13%, respectively, in all assayed matrices. Ribavirin was demonstrated to remain unchanged after short and long time storage. No matrix effects could be assessed for the analyzed matrices. The developed method has been fully validated. Monitoring of ribavirin concentration in red blood cells in addition to the classic plasma monitoring of the drug could help to explain its efficacy and safety profiles in patients.
frankfurt/ ZAFES, Institute of Clinical Pharmacology, Goethe-University, Frankfurt, Germany 2 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology (TMP), Frankfurt, Germany Received October 29, 2013 Revised December 9, 2013 Accepted December 13, 2013
Quantitation of ribavirin in human plasma and red blood cells using LC–MS/MS
Keywords: Liquid chromatography / Red blood cells / Ribavirin / Tandem mass spectrometry / Validation DOI 10.1002/jssc.201301173
1 Introduction Ribavirin (1--D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a nucleoside analogue with antiviral activity against several RNA viruses. It is used, combined with interferon-␣, in the treatment of hepatitis C virus (HCV) [1]. The mechanism of action of ribavirin (RBV) has remained unclear. Ning et al. [2] suggested that RBV is a very potent inhibitor of virusinduced proinflammatory mediators and that its beneficial effects may owe to its ability to reduce macrophage activation and to diminish Th2 cytokine production while preserving Th1 cytokine production. Crotty et al. [1] described the mutagenic properties of RBV when incorporated into the viral genome by the viral RNA polymerase, and of its monophosphate derivative, as enhancer of this mutagenic/antiviral effect. RBV also potentiates the action of interferon during HCV therapy [3]. Following oral administration, the bioavailability of RBV is 35–65% due to extensive presystemic elimination in the gastrointestinal tract. RBV has a large volume of distribution due to its active uptake into cells by membrane transporters ´ Correspondence: Dr. Nerea Ferreiros, pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology, Goethe-University Frankfurt, Building 74 / 4th floor / room 4.106b, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany E-mail: [email protected] Fax: +49-69-6301-7331
Abbreviations: HCV, Hepatitis C Virus; IS, internal standard; i.u., instrument units; LLOQ, lower limit of quantitation; ME, matrix effects; PBS, phosphate buffered saline; RBC, red blood cell; RBV, ribavirin; RE, relative error
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
that are expressed in most cell types [4]. Specifically, RBV incorporation into the red blood cells (RBCs) is mediated by nucleoside transporters, in particular by high affinity concentrative nucleoside transporters 2 and 3 and by low affinity equilibrative nucleoside transporters 1 and 2 [5,6]. In the cells, the drug is reversibly phosphorylated. The phosphate derivatives of RBV (mono-, di-, and triphosphate-RBV) accumulate in RBCs because they cannot be exported [7]. Finally, RBV is eliminated mainly via the kidney as triazol metabolites or unchanged (5–15%) [8]. Despite the increasing availability of new HCV therapeutics, RBV seems to remain a component of anti-HCV therapy regimens [9]. Therefore, the necessity to monitor its concentrations persists. Until now, several methods to determine RBV in human biological matrices have been described, including the analysis of RBV alone [10–14] or in combination with other antiviral drugs [15, 16]. Owing to a dosedependency of both, the antiviral efficacy of RBV and also the severity of its most common side effect, hemolytic anemia [7], most described methods are applied to monitor the analyte in plasma or serum samples [10,12–16]. In addition, RBV tissue concentrations have been analyzed in monkey RBCs [17], liver [18] or plasma [19], and rat brain [20, 21]. Most analytical assays included reversed-phase chromatography despite the high polarity of RBV. This property allows the development of very fast chromatographic runs. The most widely used detection technique for determination of RBV in biological matrices is MS/MS although UV detection has also been employed [13]. Wei et al. described the utility of high-resolution MS to unequivocally differentiate RBV and its phosphorylated metabolites from the isobaric endogenous nucleoside uridine and phosphate derivatives
www.jss-journal.com
J. Sep. Sci. 2014, 37, 476–483
Liquid Chromatography
477
Figure 1. Molecular structures and MS/MS spectra of RBV and uridine.
using an anion-exchange material as stationary phase [22] (Fig. 1). This differentiation was often chromatographically resolved. A commonly used sample preparation procedure is protein precipitation with organic solvent although SPE has also been used [11]. The analytical methods were partially or fully validated. A full therapeutic drug monitoring requires the analysis of both, RBV plasma concentrations and its incorporation into RBCs. For this purpose, we developed an LC–MS/MS method for the determination of RBV in human plasma and RBCs, using protein precipitation and
J. Sep. Sci. 2014, 37, 476–483
´ 1 N. Ferreiros S. Labocha1 J. El-Duweik1 C. Schlecker1 1,2 ¨ J. Lotsch G. Geisslinger1,2
Research Article
1 pharmazentrum
LC–MS/MS has been applied for the rapid determination of the nucleoside analogue ribavirin in human plasma and red blood cells. The incorporation of ribavirin to the erythrocytes has been assayed after in vitro incubation of the cells at different concentrations of the antiviral drug. After protein precipitation, samples were injected into a C8 column, achieving a complete separation of ribavirin from the endogenous isobaric compound uridine. Calibration ranges varied from 10 to 10 000 ng/mL in plasma and from 0.2 to 200 ng/cell pellet in red blood cells. Precision and accuracy values were always below 10 and 13%, respectively, in all assayed matrices. Ribavirin was demonstrated to remain unchanged after short and long time storage. No matrix effects could be assessed for the analyzed matrices. The developed method has been fully validated. Monitoring of ribavirin concentration in red blood cells in addition to the classic plasma monitoring of the drug could help to explain its efficacy and safety profiles in patients.
frankfurt/ ZAFES, Institute of Clinical Pharmacology, Goethe-University, Frankfurt, Germany 2 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology (TMP), Frankfurt, Germany Received October 29, 2013 Revised December 9, 2013 Accepted December 13, 2013
Quantitation of ribavirin in human plasma and red blood cells using LC–MS/MS
Keywords: Liquid chromatography / Red blood cells / Ribavirin / Tandem mass spectrometry / Validation DOI 10.1002/jssc.201301173
1 Introduction Ribavirin (1--D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a nucleoside analogue with antiviral activity against several RNA viruses. It is used, combined with interferon-␣, in the treatment of hepatitis C virus (HCV) [1]. The mechanism of action of ribavirin (RBV) has remained unclear. Ning et al. [2] suggested that RBV is a very potent inhibitor of virusinduced proinflammatory mediators and that its beneficial effects may owe to its ability to reduce macrophage activation and to diminish Th2 cytokine production while preserving Th1 cytokine production. Crotty et al. [1] described the mutagenic properties of RBV when incorporated into the viral genome by the viral RNA polymerase, and of its monophosphate derivative, as enhancer of this mutagenic/antiviral effect. RBV also potentiates the action of interferon during HCV therapy [3]. Following oral administration, the bioavailability of RBV is 35–65% due to extensive presystemic elimination in the gastrointestinal tract. RBV has a large volume of distribution due to its active uptake into cells by membrane transporters ´ Correspondence: Dr. Nerea Ferreiros, pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology, Goethe-University Frankfurt, Building 74 / 4th floor / room 4.106b, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany E-mail: [email protected] Fax: +49-69-6301-7331
Abbreviations: HCV, Hepatitis C Virus; IS, internal standard; i.u., instrument units; LLOQ, lower limit of quantitation; ME, matrix effects; PBS, phosphate buffered saline; RBC, red blood cell; RBV, ribavirin; RE, relative error
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
that are expressed in most cell types [4]. Specifically, RBV incorporation into the red blood cells (RBCs) is mediated by nucleoside transporters, in particular by high affinity concentrative nucleoside transporters 2 and 3 and by low affinity equilibrative nucleoside transporters 1 and 2 [5,6]. In the cells, the drug is reversibly phosphorylated. The phosphate derivatives of RBV (mono-, di-, and triphosphate-RBV) accumulate in RBCs because they cannot be exported [7]. Finally, RBV is eliminated mainly via the kidney as triazol metabolites or unchanged (5–15%) [8]. Despite the increasing availability of new HCV therapeutics, RBV seems to remain a component of anti-HCV therapy regimens [9]. Therefore, the necessity to monitor its concentrations persists. Until now, several methods to determine RBV in human biological matrices have been described, including the analysis of RBV alone [10–14] or in combination with other antiviral drugs [15, 16]. Owing to a dosedependency of both, the antiviral efficacy of RBV and also the severity of its most common side effect, hemolytic anemia [7], most described methods are applied to monitor the analyte in plasma or serum samples [10,12–16]. In addition, RBV tissue concentrations have been analyzed in monkey RBCs [17], liver [18] or plasma [19], and rat brain [20, 21]. Most analytical assays included reversed-phase chromatography despite the high polarity of RBV. This property allows the development of very fast chromatographic runs. The most widely used detection technique for determination of RBV in biological matrices is MS/MS although UV detection has also been employed [13]. Wei et al. described the utility of high-resolution MS to unequivocally differentiate RBV and its phosphorylated metabolites from the isobaric endogenous nucleoside uridine and phosphate derivatives
www.jss-journal.com
J. Sep. Sci. 2014, 37, 476–483
Liquid Chromatography
477
Figure 1. Molecular structures and MS/MS spectra of RBV and uridine.
using an anion-exchange material as stationary phase [22] (Fig. 1). This differentiation was often chromatographically resolved. A commonly used sample preparation procedure is protein precipitation with organic solvent although SPE has also been used [11]. The analytical methods were partially or fully validated. A full therapeutic drug monitoring requires the analysis of both, RBV plasma concentrations and its incorporation into RBCs. For this purpose, we developed an LC–MS/MS method for the determination of RBV in human plasma and RBCs, using protein precipitation and
