Journal of Pharmaceutical and Biomedical Analysis 90 (2014) 119–126
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Development and validation of a useful UPLC–MS/MS method for quantification of total and phosphorylated-ribavirin in peripheral blood mononuclear cells of HCV+ patients夽 Danilo Agnesod 1 , Amedeo De Nicolò 1 , Marco Simiele, Adnan Mohamed Abdi, Lucio Boglione, Giovanni Di Perri, Antonio D’Avolio ∗ Laboratory of Clinical Pharmacology and Pharmacogenetic; Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Turin, Italy
a r t i c l e
i n f o
Article history: Received 21 September 2013 Received in revised form 21 November 2013 Accepted 22 November 2013 Available online 1 December 2013 Keywords: PBMC HCV Intracellular Ribavirin Phosphorylated drugs
a b s t r a c t The current standard-of-care therapy in HCV consists in ribavirin (RBV) plus pegylated-interferon-␣ 2a or 2b and, for HCV-1 infected patients, also directly acting antivirals (DAAs). Despite the increase in the number of patients who reach sustained virological response (SVR) for HCV-1, a great inter-individual variability in the response to therapy remains. Whether new drugs are available in combination with RBV and Peg-IFN for HCV-1, the treatment of the other viral genotypes remains the same: this issue highlights the lasting importance of RBV and Peg-IFN in anti-HCV treatment. Moreover, a strong limiting factor to the usefulness of anti-HCV treatment remains the occurrence of adverse events, first of all hemolytic anemia, which have increased with the addition of DAAs, but is mainly an RBV-dependent effect. For these reasons, the monitoring of RBV exposure in the various compartments should be important. Since the routinely determination of RBV in the target cells as the hepatocytes is impracticable for of its invasiveness, the quantification in easier to obtain cells could be a good choice. In this work, we developed and validated an ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) assay method to quantify RBV concentrations in peripheral blood mononucleated cells (PBMCs). QCs were prepared with RBV and RBV-monophosphate (RMP). Each sample was divided into two aliquots, which undergone the same extraction procedure: one was treated with acid phosphatase to convert RBV phosphorylated metabolites into free RBV, the other one was not-treated. The extracts were analyzed with reverse-phase column with UPLC–MS/MS. Calibration curves fitted a least squares model (weighed 1/X) for ribavirin levels in a range from 0.1 ng to 200 ng (mean r2 = 0.9993). Accuracy, intra-day and inter-day precision of the methods were in accordance with FDA guidelines. Moreover, phosphorylated QCs were used to assess the correct determination of total RBV concentration. We tested this method by monitoring RBV concentrations in PBMCs from 20 HCV+ patients, receiving alpha interferon-plus RBV combination therapy. This method showed to be reliable, precise, accurate and suitable for evaluation of intracellular RBV concentrations. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Hepatitis C virus (HCV) represents a major cause of chronic hepatitis worldwide and the leading cause of liver transplantation in
夽 UNI EN ISO 9001:2008 Certificate Laboratory; Certificate No. IT-64386; Certification for: “Design, development and application of determination methods for anti-infective drugs. Pharmacogenetic analysis.” www.tdm-torino.org ∗ Corresponding author at: Laboratory of Clinical Pharmacology and Pharmacogenetic; Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Corso Svizzera 164 – 10149, Turin, Italy. Tel.: +39 011 4393979; fax: +39 011 4393882. E-mail address: [email protected] (A. D’Avolio). 1 Both authors contributed equally to this work. 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.11.027
developed countries, with a global prevalence of 2.8%, representing 170 million infected people and 350,000 deaths occur each year due to HCV-related diseases. Standard of care (SOC) therapy for chronic hepatitis C (CHC), even with the introduction of directly acting antivirals (DAAs) for HCV-1, is the combination of pegylated-interferon-␣ (Peg-IFN␣) and ribavirin (RBV). Despite its effectiveness and the recent addition of new DAA drugs Telaprevir (TEL) and Boceprevir (BOC), substantial differences in reaching sustained virological response (SVR) among patients remain: a major cause of treatment failure is RBV-induced hemolytic anemia, which occurs in about 30% of patients in therapy without DAAs and around 50% with DAAs [1,2], leading to the need of supportive care with erythropoietin, which inevitably increases therapy cost [3].
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D. Agnesod et al. / Journal of Pharmaceutical and Biomedical Analysis 90 (2014) 119–126
Other factors involved in virological response and toxicity might be related to genetic polymorphisms of IL28B and ITPA genes [4–9], respectively, but also to pharmacokinetics [5,6,10–12]. The correlation between toxicity, outcome and RBV levels is already reported in literature [4–6,10,11,13], but the accumulation rate of RBV and phosphorylated-RBV (RBV-P) within the nucleate cells is not still described, probably due to methodological limitations [14–16]. Moreover, the probability to achieve SVR has been related to erythrocytic RBV concentrations [17,18]. Actually, despite these pharmacokinetic and pharmacogenetic data, excluding liver biopsy, we have no possibility to get information about the RBV concentration in the hepatocytes, the real target cell of anti-HCV therapy, and to evaluate correlation with clinical outcome. Because of the obvious difficulty to sample patients hepatocytes with biopsy, we chose to investigate the RBV concentrations in the most easy-to-reach nucleated cells: peripheral blood mononuclear cells (PBMCs). Moreover, previous preliminary studies on PBMCs showed a correlation between efficacy and toxicity with ribavirin intracellular concentrations [19–21]. For these reasons, basing on our previous experience on erythrocytes [22], we aimed to develop a new ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method to quantify the intra-PBMCs concentrations of total RBV and not-phosphorylated RBV in HCV+ patients. These data allowed us to indirectly determine the RBV-P concentrations in PBMCs too. 2. Materials and methods 2.1. Chemicals Acetonitrile HPLC grade and methanol HPLC grade were purchased from J.T. Baker (Deventer, Holland). HPLC grade water was produced with Milli-DI system coupled with a Synergy 185 system by Millipore (Milan, Italy). Blank PBMC were taken from buffy coat of healthy donors, kindly supplied by the Blood Bank of the Maria Vittoria Hospital (Turin, Italy). Ribavirin mono-phosphate (RMP) was purchased from Toronto Research Chemicals Inc. (North York, Canada). RBV (1--d-ribofuranosyl-1,2,4-triazole-3-carboxamide), the Internal Standard (IS) deoxy-thymidine (5 -amino-5 -deoxythymidine), acid phosphatase type 4 from sweet potato (1.8 M ammonium sulfate containing 10 mM MgCl2 , pH 5.3), Tris–HCl buffer, sodium acetate, KOH powder and all other chemicals were purchased from Sigma–Aldrich Corp. (Milan, Italy). The enzyme, RBV and deoxy-thymidine powders were stored in a refrigerator at 4 ◦ C, RMP powder and blank PBMCs were stored in a refrigerator at −80 ◦ C to prevent degradation, until the use. All other chemicals were stored at room temperature. 2.2. UPLC–MS/MS instruments and chromatographic conditions The chromatographic system was an Acquity UPLC, consisting in a binary pump, a refrigerated sample manager and a triple quadrupole detector (TQD) (Waters, Milan, Italy). Chromatographic separation was performed using an Acquity UPLC HSS T3 1.8 m (150 mm × 2.1 mm, Waters, Milan, Italy) column, protected by a pre-coloumn frit (0.2 m × 2.1 mm) and heated at 40 ◦ C using a column thermostat. For the quantification of RBV and IS, the mass spectrometer was settled in the positive ion mode (ES+), with a capillary voltage of 3.50 kV, a source temperature of 150 ◦ C and a desolvation temperature of 500 ◦ C. The nitrogen gas flow was 800 L/h and 50 L/h for desolvation and cone, respectively. The cone voltage and collision energy were, respectively, 14 and 10 for Ribavirin, 22 and 11 for IS and 15 and 12 for uridine. The mass transitions
Table 1 Chromatographic gradient: mobile phase A (H2 O + 0.05% formic acid) and mobile phase B (acetonitrile + 0.05% formic acid). Time (min)
Flow (mL/min)
Mobile phase A (%)
Mobile phase B (%)
0.00 0.10 1.25 2.55 3.30 3.31 4.49 4.50 7.00
0.400 0.400 0.400 0.400 0.400 0.400 0.400 0.400 0.400
100.0 100.0 97.0 85.0 50.0 5.0 5.0 100.0 100.0
0.0 0.0 3.0 15.0 50.0 95.0 95.0 0.0 0.0
were 245.10 > 113.05, 242.14 > 116.05 and 245.10 > 113.02 for RBV, IS and uridine, respectively. The run was performed with a gradient (Table 1) of two different mobile phases: mobile phase A (H2 O + 0.05% formic acid) and mobile phase B (acetonitrile + 0.05% formic acid). 2.3. Stock solutions, standards, and quality controls Stock solutions of RBV, RMP and IS were made in pure water at final concentration of 1 mg/mL and were stored at 4 ◦ C (for RBV and IS, no more than 1 month) or −80 ◦ C (for RMP, no more of 3 months). Single aliquots of solutions at the final concentrations of 1 ng/mL, 5 ng/mL, 10 ng/mL, 50 ng/mL, 250 ng/mL, 500 ng/mL and 2000 ng/mL for standards (STDs) and 2 ng/mL, 20 ng/mL, 200 ng/mL and 800 ng/mL for quality controls (QCs) were prepared in water and stored at −80 ◦ C. Each aliquot was then thawed before the analysis and 100 L was used to spike 500 L-blank-PBMC aliquots in order to obtain 7 STD points of the calibration curve and 4 QCs. Then, the RBV amounts in the standard samples were: 0.1 ng, 0.5 ng, 1 ng, 5 ng, 25 ng, 50 ng and 200 ng, from STD 1 to STD 7, respectively. Similarly, the amounts of RBV in QC were 80 ng, 20 ng, 2 ng and 0.2 ng for High (H), Medium (M), Low (L) and Low–Low (LL) levels, respectively. Phosphorylated QCs were prepared in the same way with RMP amounts corresponding to 20 ng and 2 ng of RBV for QC-H and QC-L, respectively. For each working session, IS solution was prepared by diluting 50 L of a stock solution of deoxy-thymidine (1 mg/mL) in 3950 L of water–methanol (50:50) solution, with a final concentration of 12.5 g/mL. 2.4. Specificity and selectivity As requested by FDA guidelines [23], interference from endogenous compounds was investigated by the analysis of six different blank PBMCs samples. Moreover, we focused the possible interference of endogenous uridine, which in MS/MS spectrometry presents the same mass transition of RBV, as previously described in other papers [24,25]. In particular, we investigated uridine retention time by the addition of a standard to a solution containing RBV at the same concentration; then, we monitored its presence and retention time in each validation session. Since the new therapy for HCV-1 is based on the administration of one HCV protease inhibitor, as BOC or TEL, in association with RBV and Peg-IFN, we have also evaluated the interference of these two new possible concomitant co-administered drugs, using the same procedure used for uridine. 2.5. Enzyme digestion and extraction procedure Each 500 L sample of STDs and QCs (one batch for the quantification of phosphorylated and another one for the quantification of not-phosphorylated fraction) has been spiked with 40 L of IS
D. Agnesod et al. / Journal of Pharmaceutical and Biomedical Analysis 90 (2014) 119–126
solution, while each 1000 L patients sample has been separated into two aliquots of 500 L (transferred in PTFE tubes) and added each with 40 L of IS solution. Then, each STD and QC was spiked with the correct amount of RBV, as described before (point 2.3). Patients’ samples were “mock” spiked with pure water, to reach the same volume of STDs and QCs. Each sample was then centrifuged at 6800 × g for 5 min at 4 ◦ C, and the supernatants were transferred in other PTFE tubes. The remaining pellet was washed with 200 L of pure methanol in order to dissolve cellular membranes and to extract all the content, then vortexed and centrifuged again at 21,000 × g for 5 min at 4 ◦ C. At this point, the new methanolic supernatant was transferred in the same tubes already used at the previous step. All samples were then placed in vacuum-centrifuge at 60 ◦ C. After samples being completely dried, they were treated with a dephosphorylation protocol already published [22,26–28], and partially modified for PBMC. 200 L of an acid buffer solution (pH4; Tris–HCl buffer 30 mM pH 8.0, pure water and acetate buffer 1 M pH4, 3:1:0.25, v:v:v, respectively) was added to each sample. One half of the aliquots from each patient and a dedicated batch of STDs and QCs were added with 0.5 units of acid phosphatase for the quantification of total RBV, which is the sum of free RBV and phosphorylated-RBV (RBV-P, generic for mono-, di- and tri-phosphorylated-RBV); the other “not-treated” samples, STDs and QCs aliquots were used to quantify the amount of notphosphorylated-RBV. The only difference between the samples treated and nontreated lies in the fact that in those treated was added the phosphatase enzyme and buffers. Samples were then incubated in a water bath at 37 ◦ C for 1 h in order to obtain optimal dephosphorylation conditions. After incubation, 2.5 L of KOH 10 N was added to alkalize the solution, ensuring the rapid deactivation of acid phosphatase. At this point, 1 mL of acetonitrile was added in order to achieve protein precipitation. Then, after being vortexed for 10 , they were centrifuged at 21,000 × g for 10 min at 4 ◦ C. After centrifugation, the supernatant was transferred to glass shots and dried in a vacuum-centrifuge at 60 ◦ C. Dried pellets were then resuspended in 100 L of pure water, transferred in maximum recovery vials (Waters, Milan, Italy) and loaded in the autosampler. 5 L was injected in the UPLC system. 2.6. Accuracy, precision, and limit of quantification Intra-day and inter-day accuracy and precision were determined by performing 10 validation sessions, both for treated and not treated QCs. RBV QCs were analyzed in duplicate, both for treated and not treated samples. Accuracy was calculated as the mean percent deviation from the nominal concentrations. Inter-day and intra-day precision were performed following FDA guidelines and were expressed as the relative standard deviation at each QC concentration. The calibration curve was obtained using the standard points described at point 2.3. Limit of detection (LOD) was considered as the concentration that yields a signal-to-noise ratio of 3. Percentage of deviation from the nominal concentration (measure of accuracy) and relative standard deviation (measure of precision) at the concentration considered as the limit of quantification (LOQ) was 113.0.
substances were observed in the chromatogram for blank PBMC samples (Fig. 3).
and not treated sample was 71.57% (R.S.D. = 4.87%) and 85.0% (R.S.D. = 7.08%), respectively.
3.2. Accuracy, precision, and limit of quantification
3.4. Matrix effect
The results of method validation for both untreated and treated samples are shown in Table 2A and B, respectively. Accuracy (R.S.D.) of phosphorylated QCs was 2.01% for QC-H and -2.72% for QC-L. LOD and LOQ were 0.05 and 0.1 ng, respectively (Fig. 3). Calibration curves (range 0.1–200 ng) were calculated with weighted least squares regression (weighing factor 1/X), with mean r2 = 0.9993 for not-treated curve, and mean r2 = 0.9987 for treated curve.
The average mean percent deviations of the peak areas at the four RBV QCs’ amounts was 18.48% (R.S.D. = 15.71%) and −24.93% (R.S.D. = 15.69%) for treated and not treated samples, respectively. About the experiments on the possible interference on RBV deriving from different cells number in different samples, the mean percent deviation of peak areas at the four amounts corresponding to those present in the QCs was comparable and was −19.8%, −22.0% and −23.9% for samples containing 2, 4 and 8 million cells, respectively. Concerning IS, the average mean percent deviations of the peak areas calculated in the QCs was −20.18% and −6.34% for treated and not treated samples, respectively. The analysis of the IS peak areas at the four QCs amounts at different cells concentration, seems to show a link between cell number increase and peak area decrease: the mean percent
3.3. Recovery Mean recovery of treated RBV QCs was 91.54% (R.S.D. = 4.86% among different QCs), while in not treated RBV QCs was 74.0% (R.S.D. = 7.40% among different QCs). Mean recovery of IS in treated
Fig. 3. Overlapped chromatograms of RBV LOQ (0.1 ng), RBV LOD (0.05 ng) peaks and blank plasma extracted samples.
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deviation of the peak areas was −5.96%, −9.72% and −29.79% for samples containing 2, 4 and 8 million cells, respectively. 3.5. Other additional experiments The injection and analysis of pure water containing increasing concentrations of acid phosphatase showed that there was not any peak interfering with RBV one. Regarding the analysis of possible signal differences between samples treated with active or inactive enzyme, the results for both concentrations showed no differences in signal yield. All samples containing the same amount of RBV, spiked with increasing concentration of phosphatase, presented a meaningful increase in RBV peak areas, directly proportional to the amount of phosphatase solution added. Therefore, we compared peak areas resulting from the direct injection of samples (all containing the same amount of RBV) at different pH conditions. This experiment showed a progressive increase of peak areas related to the progressive acidification of samples. Finally, the experiments that occurred the effects of buffers, showed their important role in the final signals yields. The comparison between the peak areas of water-suspended molecules directly injected and samples containing buffers, demonstrated the following data: for RBV, the samples containing only the mixed buffer presented a mean decrease at the four different QCs amounts of 9.44% in the peak areas, while those containing only the buffer in which the phosphatase is suspended presented a mean increase in the peak areas of 24.13%; differently, the samples added with both kind of buffer showed a variation in the peak areas that was a sort of sum of the two opposite effects. Instead, concerning IS, the samples containing only the mixed buffer presented a mean decrease of the peak areas (at the four different QCs amounts) of 0.94%, while those containing only the phosphatase buffer and the mixture of the two different buffers presented a mean decrease of 24.28% and 21.95%, respectively. 3.6. Analysis of patients’ samples The here reported method was applied to calculate intra-PBMC RBV concentration after the first month of treatment of twenty HCV+ patients, using the above mentioned formula. The median collected number of cells from patients samples was 1,476,700 (IQR 10,22,400–1,717,100). PBMC samples were obtained at a median of 12.15 h (IQR 11.75–12.9) after last drug intake (Ctrough ). Average of total RBV and not-phosphorylated RBV amount in PBMC samples were 21.2 ng and 1.9 ng, respectively. Maximum and minimum amounts observed as results of the analysis sessions were 64.1 ng and 6.6 ng for total RBV and 4.9 ng and 0.5 ng for not-phosphorylated RBV, corresponding to concentrations of 111,516 ng/mL and 71351 ng/mL for total RBV, while of 7393 ng/mL and 2012 ng/mL for notphosphorylated RBV, respectively. The concentrations, expressed in ng/mL, were calculated applying the formula described at point 2.10, and so are referred to specific PBMC samples, with particular absolute number of PBMCs and MCV. Median and IQR values for the different RBV forms referring to samples from patients are shown in Table 3. Maximum and minimum RBV amounts described above explain the choice of the RBV concentration range used for the standard points of the calibration curve (from 0.1 to 200 ng). 4. Discussion Recently the anti-HCV-1 therapy has been rendered more effective with the introduction of the DAAs [1,2]. Therefore, several
Table 3 Median concentrations of RBV forms determined in patient samples (Interquartile Range) after 4 weeks of standard therapy. RBV-forms
4 Weeks [ng/mL] (IQR)
Total RBV (treated) Not-phosphorylated RBV (not-treated) Phosphorylated RBV (total − not-phosphorylated) Ratio (total/not-phosphorylated) Ratio (phosphorylated/not-phosphorylated)
49,391 (33,739–64,859) 4162 (3407–6551) 45,033 (29,629–59,402) 12.09 (10.20–13.74) 11.09 (9.20–12.74)
methods have been studied for the dosing of boceprevir and telaprevir [33–38], but ribavirin remains an essential drug in the treatment of HCV infection from all genotypes [4,6,39], although causing toxic effects such as anemia [5,7,8,21,40]. Moreover, recent studies indicate that the DAAs may increase intracellular and plasma ribavirin concentrations through a not yet identified mechanism [19,20,41]. Several assays for the quantification of ribavirin in plasma have been validated to the time [32,42,43], while there is a lower number of papers describing assays for the dosage into erythrocytes [22,26,44–46]. Here we report for the first time a work that evaluates the concentrations of ribavirin within PBMCs. The validation procedure here reported showed for this method good precision and accuracy, with both CV and mean inaccuracy
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Development and validation of a useful UPLC–MS/MS method for quantification of total and phosphorylated-ribavirin in peripheral blood mononuclear cells of HCV+ patients夽 Danilo Agnesod 1 , Amedeo De Nicolò 1 , Marco Simiele, Adnan Mohamed Abdi, Lucio Boglione, Giovanni Di Perri, Antonio D’Avolio ∗ Laboratory of Clinical Pharmacology and Pharmacogenetic; Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Turin, Italy
a r t i c l e
i n f o
Article history: Received 21 September 2013 Received in revised form 21 November 2013 Accepted 22 November 2013 Available online 1 December 2013 Keywords: PBMC HCV Intracellular Ribavirin Phosphorylated drugs
a b s t r a c t The current standard-of-care therapy in HCV consists in ribavirin (RBV) plus pegylated-interferon-␣ 2a or 2b and, for HCV-1 infected patients, also directly acting antivirals (DAAs). Despite the increase in the number of patients who reach sustained virological response (SVR) for HCV-1, a great inter-individual variability in the response to therapy remains. Whether new drugs are available in combination with RBV and Peg-IFN for HCV-1, the treatment of the other viral genotypes remains the same: this issue highlights the lasting importance of RBV and Peg-IFN in anti-HCV treatment. Moreover, a strong limiting factor to the usefulness of anti-HCV treatment remains the occurrence of adverse events, first of all hemolytic anemia, which have increased with the addition of DAAs, but is mainly an RBV-dependent effect. For these reasons, the monitoring of RBV exposure in the various compartments should be important. Since the routinely determination of RBV in the target cells as the hepatocytes is impracticable for of its invasiveness, the quantification in easier to obtain cells could be a good choice. In this work, we developed and validated an ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) assay method to quantify RBV concentrations in peripheral blood mononucleated cells (PBMCs). QCs were prepared with RBV and RBV-monophosphate (RMP). Each sample was divided into two aliquots, which undergone the same extraction procedure: one was treated with acid phosphatase to convert RBV phosphorylated metabolites into free RBV, the other one was not-treated. The extracts were analyzed with reverse-phase column with UPLC–MS/MS. Calibration curves fitted a least squares model (weighed 1/X) for ribavirin levels in a range from 0.1 ng to 200 ng (mean r2 = 0.9993). Accuracy, intra-day and inter-day precision of the methods were in accordance with FDA guidelines. Moreover, phosphorylated QCs were used to assess the correct determination of total RBV concentration. We tested this method by monitoring RBV concentrations in PBMCs from 20 HCV+ patients, receiving alpha interferon-plus RBV combination therapy. This method showed to be reliable, precise, accurate and suitable for evaluation of intracellular RBV concentrations. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Hepatitis C virus (HCV) represents a major cause of chronic hepatitis worldwide and the leading cause of liver transplantation in
夽 UNI EN ISO 9001:2008 Certificate Laboratory; Certificate No. IT-64386; Certification for: “Design, development and application of determination methods for anti-infective drugs. Pharmacogenetic analysis.” www.tdm-torino.org ∗ Corresponding author at: Laboratory of Clinical Pharmacology and Pharmacogenetic; Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Corso Svizzera 164 – 10149, Turin, Italy. Tel.: +39 011 4393979; fax: +39 011 4393882. E-mail address: [email protected] (A. D’Avolio). 1 Both authors contributed equally to this work. 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.11.027
developed countries, with a global prevalence of 2.8%, representing 170 million infected people and 350,000 deaths occur each year due to HCV-related diseases. Standard of care (SOC) therapy for chronic hepatitis C (CHC), even with the introduction of directly acting antivirals (DAAs) for HCV-1, is the combination of pegylated-interferon-␣ (Peg-IFN␣) and ribavirin (RBV). Despite its effectiveness and the recent addition of new DAA drugs Telaprevir (TEL) and Boceprevir (BOC), substantial differences in reaching sustained virological response (SVR) among patients remain: a major cause of treatment failure is RBV-induced hemolytic anemia, which occurs in about 30% of patients in therapy without DAAs and around 50% with DAAs [1,2], leading to the need of supportive care with erythropoietin, which inevitably increases therapy cost [3].
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D. Agnesod et al. / Journal of Pharmaceutical and Biomedical Analysis 90 (2014) 119–126
Other factors involved in virological response and toxicity might be related to genetic polymorphisms of IL28B and ITPA genes [4–9], respectively, but also to pharmacokinetics [5,6,10–12]. The correlation between toxicity, outcome and RBV levels is already reported in literature [4–6,10,11,13], but the accumulation rate of RBV and phosphorylated-RBV (RBV-P) within the nucleate cells is not still described, probably due to methodological limitations [14–16]. Moreover, the probability to achieve SVR has been related to erythrocytic RBV concentrations [17,18]. Actually, despite these pharmacokinetic and pharmacogenetic data, excluding liver biopsy, we have no possibility to get information about the RBV concentration in the hepatocytes, the real target cell of anti-HCV therapy, and to evaluate correlation with clinical outcome. Because of the obvious difficulty to sample patients hepatocytes with biopsy, we chose to investigate the RBV concentrations in the most easy-to-reach nucleated cells: peripheral blood mononuclear cells (PBMCs). Moreover, previous preliminary studies on PBMCs showed a correlation between efficacy and toxicity with ribavirin intracellular concentrations [19–21]. For these reasons, basing on our previous experience on erythrocytes [22], we aimed to develop a new ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method to quantify the intra-PBMCs concentrations of total RBV and not-phosphorylated RBV in HCV+ patients. These data allowed us to indirectly determine the RBV-P concentrations in PBMCs too. 2. Materials and methods 2.1. Chemicals Acetonitrile HPLC grade and methanol HPLC grade were purchased from J.T. Baker (Deventer, Holland). HPLC grade water was produced with Milli-DI system coupled with a Synergy 185 system by Millipore (Milan, Italy). Blank PBMC were taken from buffy coat of healthy donors, kindly supplied by the Blood Bank of the Maria Vittoria Hospital (Turin, Italy). Ribavirin mono-phosphate (RMP) was purchased from Toronto Research Chemicals Inc. (North York, Canada). RBV (1--d-ribofuranosyl-1,2,4-triazole-3-carboxamide), the Internal Standard (IS) deoxy-thymidine (5 -amino-5 -deoxythymidine), acid phosphatase type 4 from sweet potato (1.8 M ammonium sulfate containing 10 mM MgCl2 , pH 5.3), Tris–HCl buffer, sodium acetate, KOH powder and all other chemicals were purchased from Sigma–Aldrich Corp. (Milan, Italy). The enzyme, RBV and deoxy-thymidine powders were stored in a refrigerator at 4 ◦ C, RMP powder and blank PBMCs were stored in a refrigerator at −80 ◦ C to prevent degradation, until the use. All other chemicals were stored at room temperature. 2.2. UPLC–MS/MS instruments and chromatographic conditions The chromatographic system was an Acquity UPLC, consisting in a binary pump, a refrigerated sample manager and a triple quadrupole detector (TQD) (Waters, Milan, Italy). Chromatographic separation was performed using an Acquity UPLC HSS T3 1.8 m (150 mm × 2.1 mm, Waters, Milan, Italy) column, protected by a pre-coloumn frit (0.2 m × 2.1 mm) and heated at 40 ◦ C using a column thermostat. For the quantification of RBV and IS, the mass spectrometer was settled in the positive ion mode (ES+), with a capillary voltage of 3.50 kV, a source temperature of 150 ◦ C and a desolvation temperature of 500 ◦ C. The nitrogen gas flow was 800 L/h and 50 L/h for desolvation and cone, respectively. The cone voltage and collision energy were, respectively, 14 and 10 for Ribavirin, 22 and 11 for IS and 15 and 12 for uridine. The mass transitions
Table 1 Chromatographic gradient: mobile phase A (H2 O + 0.05% formic acid) and mobile phase B (acetonitrile + 0.05% formic acid). Time (min)
Flow (mL/min)
Mobile phase A (%)
Mobile phase B (%)
0.00 0.10 1.25 2.55 3.30 3.31 4.49 4.50 7.00
0.400 0.400 0.400 0.400 0.400 0.400 0.400 0.400 0.400
100.0 100.0 97.0 85.0 50.0 5.0 5.0 100.0 100.0
0.0 0.0 3.0 15.0 50.0 95.0 95.0 0.0 0.0
were 245.10 > 113.05, 242.14 > 116.05 and 245.10 > 113.02 for RBV, IS and uridine, respectively. The run was performed with a gradient (Table 1) of two different mobile phases: mobile phase A (H2 O + 0.05% formic acid) and mobile phase B (acetonitrile + 0.05% formic acid). 2.3. Stock solutions, standards, and quality controls Stock solutions of RBV, RMP and IS were made in pure water at final concentration of 1 mg/mL and were stored at 4 ◦ C (for RBV and IS, no more than 1 month) or −80 ◦ C (for RMP, no more of 3 months). Single aliquots of solutions at the final concentrations of 1 ng/mL, 5 ng/mL, 10 ng/mL, 50 ng/mL, 250 ng/mL, 500 ng/mL and 2000 ng/mL for standards (STDs) and 2 ng/mL, 20 ng/mL, 200 ng/mL and 800 ng/mL for quality controls (QCs) were prepared in water and stored at −80 ◦ C. Each aliquot was then thawed before the analysis and 100 L was used to spike 500 L-blank-PBMC aliquots in order to obtain 7 STD points of the calibration curve and 4 QCs. Then, the RBV amounts in the standard samples were: 0.1 ng, 0.5 ng, 1 ng, 5 ng, 25 ng, 50 ng and 200 ng, from STD 1 to STD 7, respectively. Similarly, the amounts of RBV in QC were 80 ng, 20 ng, 2 ng and 0.2 ng for High (H), Medium (M), Low (L) and Low–Low (LL) levels, respectively. Phosphorylated QCs were prepared in the same way with RMP amounts corresponding to 20 ng and 2 ng of RBV for QC-H and QC-L, respectively. For each working session, IS solution was prepared by diluting 50 L of a stock solution of deoxy-thymidine (1 mg/mL) in 3950 L of water–methanol (50:50) solution, with a final concentration of 12.5 g/mL. 2.4. Specificity and selectivity As requested by FDA guidelines [23], interference from endogenous compounds was investigated by the analysis of six different blank PBMCs samples. Moreover, we focused the possible interference of endogenous uridine, which in MS/MS spectrometry presents the same mass transition of RBV, as previously described in other papers [24,25]. In particular, we investigated uridine retention time by the addition of a standard to a solution containing RBV at the same concentration; then, we monitored its presence and retention time in each validation session. Since the new therapy for HCV-1 is based on the administration of one HCV protease inhibitor, as BOC or TEL, in association with RBV and Peg-IFN, we have also evaluated the interference of these two new possible concomitant co-administered drugs, using the same procedure used for uridine. 2.5. Enzyme digestion and extraction procedure Each 500 L sample of STDs and QCs (one batch for the quantification of phosphorylated and another one for the quantification of not-phosphorylated fraction) has been spiked with 40 L of IS
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solution, while each 1000 L patients sample has been separated into two aliquots of 500 L (transferred in PTFE tubes) and added each with 40 L of IS solution. Then, each STD and QC was spiked with the correct amount of RBV, as described before (point 2.3). Patients’ samples were “mock” spiked with pure water, to reach the same volume of STDs and QCs. Each sample was then centrifuged at 6800 × g for 5 min at 4 ◦ C, and the supernatants were transferred in other PTFE tubes. The remaining pellet was washed with 200 L of pure methanol in order to dissolve cellular membranes and to extract all the content, then vortexed and centrifuged again at 21,000 × g for 5 min at 4 ◦ C. At this point, the new methanolic supernatant was transferred in the same tubes already used at the previous step. All samples were then placed in vacuum-centrifuge at 60 ◦ C. After samples being completely dried, they were treated with a dephosphorylation protocol already published [22,26–28], and partially modified for PBMC. 200 L of an acid buffer solution (pH4; Tris–HCl buffer 30 mM pH 8.0, pure water and acetate buffer 1 M pH4, 3:1:0.25, v:v:v, respectively) was added to each sample. One half of the aliquots from each patient and a dedicated batch of STDs and QCs were added with 0.5 units of acid phosphatase for the quantification of total RBV, which is the sum of free RBV and phosphorylated-RBV (RBV-P, generic for mono-, di- and tri-phosphorylated-RBV); the other “not-treated” samples, STDs and QCs aliquots were used to quantify the amount of notphosphorylated-RBV. The only difference between the samples treated and nontreated lies in the fact that in those treated was added the phosphatase enzyme and buffers. Samples were then incubated in a water bath at 37 ◦ C for 1 h in order to obtain optimal dephosphorylation conditions. After incubation, 2.5 L of KOH 10 N was added to alkalize the solution, ensuring the rapid deactivation of acid phosphatase. At this point, 1 mL of acetonitrile was added in order to achieve protein precipitation. Then, after being vortexed for 10 , they were centrifuged at 21,000 × g for 10 min at 4 ◦ C. After centrifugation, the supernatant was transferred to glass shots and dried in a vacuum-centrifuge at 60 ◦ C. Dried pellets were then resuspended in 100 L of pure water, transferred in maximum recovery vials (Waters, Milan, Italy) and loaded in the autosampler. 5 L was injected in the UPLC system. 2.6. Accuracy, precision, and limit of quantification Intra-day and inter-day accuracy and precision were determined by performing 10 validation sessions, both for treated and not treated QCs. RBV QCs were analyzed in duplicate, both for treated and not treated samples. Accuracy was calculated as the mean percent deviation from the nominal concentrations. Inter-day and intra-day precision were performed following FDA guidelines and were expressed as the relative standard deviation at each QC concentration. The calibration curve was obtained using the standard points described at point 2.3. Limit of detection (LOD) was considered as the concentration that yields a signal-to-noise ratio of 3. Percentage of deviation from the nominal concentration (measure of accuracy) and relative standard deviation (measure of precision) at the concentration considered as the limit of quantification (LOQ) was 113.0.
substances were observed in the chromatogram for blank PBMC samples (Fig. 3).
and not treated sample was 71.57% (R.S.D. = 4.87%) and 85.0% (R.S.D. = 7.08%), respectively.
3.2. Accuracy, precision, and limit of quantification
3.4. Matrix effect
The results of method validation for both untreated and treated samples are shown in Table 2A and B, respectively. Accuracy (R.S.D.) of phosphorylated QCs was 2.01% for QC-H and -2.72% for QC-L. LOD and LOQ were 0.05 and 0.1 ng, respectively (Fig. 3). Calibration curves (range 0.1–200 ng) were calculated with weighted least squares regression (weighing factor 1/X), with mean r2 = 0.9993 for not-treated curve, and mean r2 = 0.9987 for treated curve.
The average mean percent deviations of the peak areas at the four RBV QCs’ amounts was 18.48% (R.S.D. = 15.71%) and −24.93% (R.S.D. = 15.69%) for treated and not treated samples, respectively. About the experiments on the possible interference on RBV deriving from different cells number in different samples, the mean percent deviation of peak areas at the four amounts corresponding to those present in the QCs was comparable and was −19.8%, −22.0% and −23.9% for samples containing 2, 4 and 8 million cells, respectively. Concerning IS, the average mean percent deviations of the peak areas calculated in the QCs was −20.18% and −6.34% for treated and not treated samples, respectively. The analysis of the IS peak areas at the four QCs amounts at different cells concentration, seems to show a link between cell number increase and peak area decrease: the mean percent
3.3. Recovery Mean recovery of treated RBV QCs was 91.54% (R.S.D. = 4.86% among different QCs), while in not treated RBV QCs was 74.0% (R.S.D. = 7.40% among different QCs). Mean recovery of IS in treated
Fig. 3. Overlapped chromatograms of RBV LOQ (0.1 ng), RBV LOD (0.05 ng) peaks and blank plasma extracted samples.
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deviation of the peak areas was −5.96%, −9.72% and −29.79% for samples containing 2, 4 and 8 million cells, respectively. 3.5. Other additional experiments The injection and analysis of pure water containing increasing concentrations of acid phosphatase showed that there was not any peak interfering with RBV one. Regarding the analysis of possible signal differences between samples treated with active or inactive enzyme, the results for both concentrations showed no differences in signal yield. All samples containing the same amount of RBV, spiked with increasing concentration of phosphatase, presented a meaningful increase in RBV peak areas, directly proportional to the amount of phosphatase solution added. Therefore, we compared peak areas resulting from the direct injection of samples (all containing the same amount of RBV) at different pH conditions. This experiment showed a progressive increase of peak areas related to the progressive acidification of samples. Finally, the experiments that occurred the effects of buffers, showed their important role in the final signals yields. The comparison between the peak areas of water-suspended molecules directly injected and samples containing buffers, demonstrated the following data: for RBV, the samples containing only the mixed buffer presented a mean decrease at the four different QCs amounts of 9.44% in the peak areas, while those containing only the buffer in which the phosphatase is suspended presented a mean increase in the peak areas of 24.13%; differently, the samples added with both kind of buffer showed a variation in the peak areas that was a sort of sum of the two opposite effects. Instead, concerning IS, the samples containing only the mixed buffer presented a mean decrease of the peak areas (at the four different QCs amounts) of 0.94%, while those containing only the phosphatase buffer and the mixture of the two different buffers presented a mean decrease of 24.28% and 21.95%, respectively. 3.6. Analysis of patients’ samples The here reported method was applied to calculate intra-PBMC RBV concentration after the first month of treatment of twenty HCV+ patients, using the above mentioned formula. The median collected number of cells from patients samples was 1,476,700 (IQR 10,22,400–1,717,100). PBMC samples were obtained at a median of 12.15 h (IQR 11.75–12.9) after last drug intake (Ctrough ). Average of total RBV and not-phosphorylated RBV amount in PBMC samples were 21.2 ng and 1.9 ng, respectively. Maximum and minimum amounts observed as results of the analysis sessions were 64.1 ng and 6.6 ng for total RBV and 4.9 ng and 0.5 ng for not-phosphorylated RBV, corresponding to concentrations of 111,516 ng/mL and 71351 ng/mL for total RBV, while of 7393 ng/mL and 2012 ng/mL for notphosphorylated RBV, respectively. The concentrations, expressed in ng/mL, were calculated applying the formula described at point 2.10, and so are referred to specific PBMC samples, with particular absolute number of PBMCs and MCV. Median and IQR values for the different RBV forms referring to samples from patients are shown in Table 3. Maximum and minimum RBV amounts described above explain the choice of the RBV concentration range used for the standard points of the calibration curve (from 0.1 to 200 ng). 4. Discussion Recently the anti-HCV-1 therapy has been rendered more effective with the introduction of the DAAs [1,2]. Therefore, several
Table 3 Median concentrations of RBV forms determined in patient samples (Interquartile Range) after 4 weeks of standard therapy. RBV-forms
4 Weeks [ng/mL] (IQR)
Total RBV (treated) Not-phosphorylated RBV (not-treated) Phosphorylated RBV (total − not-phosphorylated) Ratio (total/not-phosphorylated) Ratio (phosphorylated/not-phosphorylated)
49,391 (33,739–64,859) 4162 (3407–6551) 45,033 (29,629–59,402) 12.09 (10.20–13.74) 11.09 (9.20–12.74)
methods have been studied for the dosing of boceprevir and telaprevir [33–38], but ribavirin remains an essential drug in the treatment of HCV infection from all genotypes [4,6,39], although causing toxic effects such as anemia [5,7,8,21,40]. Moreover, recent studies indicate that the DAAs may increase intracellular and plasma ribavirin concentrations through a not yet identified mechanism [19,20,41]. Several assays for the quantification of ribavirin in plasma have been validated to the time [32,42,43], while there is a lower number of papers describing assays for the dosage into erythrocytes [22,26,44–46]. Here we report for the first time a work that evaluates the concentrations of ribavirin within PBMCs. The validation procedure here reported showed for this method good precision and accuracy, with both CV and mean inaccuracy
