Liquid Chromatography Assay for Routine Monitoring of Cellular Ribavirin Levels in Blood

Ribavirin-induced hemolytic anemia is one cause for cessationof combination therapy with alpha interferon 2b and ribavirinfor hepatitis C infection. Determining cellular ribavirin levelsin blood, including the levels of its phosphorylated metabolites,might be useful for predicting ribavirin-induced anemia, becausethe metabolites accumulate in erythrocytes. We simplified anassay method developed previously to make it suitable for routinemonitoring of cellular ribavirin. Whole blood diluted with asixfold volume of ice-cold distilled water was subjected toacid phosphatase digestion to convert phosphorylated ribavirinmetabolites to free ribavirin. The resulting mixture, spikedwith an internal standard, was treated by phenyl boronic acidcolumn extraction, followed by reverse-phase high-performanceliquid chromatography analysis. The calibration curve for ribavirinlevels in whole blood was linear at concentrations of 5.3 to1,024 µM (r² = 0.9999). Validation coefficients of variationfor intra- and interday assays were 2.9 to 5.8% and 4.3 to 8.3%,respectively. We tested this method by monitoring blood ribavirinconcentrations in two hepatitis C patients receiving alpha interferon2b-plus-ribavirin combination therapy.

INTRODUCTION

Top

Introduction

Ribavirin, a guanosine analog broad-spectrum antiviral agent,is used for hepatitis C virus (HCV) elimination in combinationwith alpha interferon 2b (2, 3, 13). Polyethyleneglycol-alphainterferon 2a combined with oral ribavirin also brings substantialbenefit to HCV patients (12, 14). Despite the strong beneficialeffects of these combination therapies in severe HCV infection,loss of hemoglobin occurs in a substantial population of HCVpatients. Progressive loss of hemoglobin leads to anemia, whichis counteracted by reducing the ribavirin dose or prematurelydiscontinuing the combination therapy (1, 5, 12). Current studiessuggest that the excessive accumulation of ribavirin in erythrocytesis responsible for the anemia (8, 11). Once incorporated intoerythrocytes, ribavirin is converted into phosphorylated metabolitesby intracellular phosphorylation (9). The phosphorylated metabolitesdecrease intracellular ATP levels, resulting in the reductionof erythrocyte integrity, which is followed by extravascularhemolysis via the reticuloendothelial system (4). It shouldtherefore be possible to predict the occurrence of ribavirin-inducedanemia by determining the ribavirin concentrations in bloodcells and plasma.

A high-performance liquid chromatography (HPLC) method was previouslydeveloped to determine ribavirin levels in order to assess thedisposition of ribavirin in erythrocytes (7). Since phosphorylatedmetabolites are the main form of intracellular ribavirin (7,8), whole-blood samples were treated with acid phosphatase priorto column extraction and analysis. However, the dephosphorylationprocedure used in a previous study (7) was too tedious to usefor routine monitoring of cellular ribavirin. In the presentstudy, we simplified the dephosphorylation procedure. The modifiedmethod was tested for therapeutic drug monitoring of cellularribavirin levels in two patients during the first 8 weeks ofHCV treatment by combination therapy with ribavirin and alphainterferon 2b.

MATERIALS AND METHODS

Top

Materials and Methods

Chemicals and instruments. All chemicals were of HPLC or reagent grade and were obtainedfrom Wako Pure Chemicals Industries (Osaka, Japan) or Sigma-AldrichCorp. (St. Louis, Mo.). Ribavirin (1-ß-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide),3-methylcytidine methosulfate (internal standard), and acidphosphatase type 4 prepared from sweet potato were purchasedfrom Sigma. Phenyl boronic acid (PBA) cartridges with a highspecificity for cis-diol-containing compounds (100 mg; BondElute PBA), which were used for solid-phase extraction, wereobtained from Varian (Palo Alto, Calif.).

The HPLC system used in this study was the Tosoh (Tokyo, Japan)model 8020 system equipped with a UV detector, an autosampler,and a pump. A C₁₈ reversed-phase column (4.6 [inner diameter]by 150 mm; TSK-Gel ODS-80Ts, Tosoh) was used, and UV absorbancewas monitored at 225 nm. The mobile-phase solvent, 10 mM ammoniumphosphate buffer (pH 2.5), was pumped through at a flow rateof 1.0 ml min^–1.

Reference sample preparation. A stock ribavirin solution (4.1 mM) was prepared by dissolvingribavirin in distilled water. Reference samples were preparedby diluting the stock solution with a human plasma alternative(Twin-Consera H; Nissui, Tokyo, Japan) or with drug-free wholeblood collected in heparinized tubes from a healthy volunteer.The concentrations of reference samples were 0.25, 0.5, 1, 5,10, and 20 µM in the plasma alternative and 5.3, 21, 85,341, 683, and 1,024 µM in whole blood. The concentrationranges of the reference samples were established in a previousstudy (8). Reference samples were stored at –30°Cuntil use. An internal standard was prepared as a 250 µMsolution of 3-methylcytidine methosulfate in distilled water,and the solution was stored at 4°C.

Enzyme digestion and column extraction. A 20-µl sample of whole blood was supplemented with 120µl of ice-cold distilled water and vortexed intenselyfor 20 s to accomplish complete hemolysis. The hemolysate wastreated with acid phosphatase to hydrolyze the phosphorylatedmetabolites, according to the method of Homma et al. (7). Thereaction mixture, consisting of the hemolysate (140 µl),30 µM Tris-HCl (200 µl), 1 M sodium acetate (20µl), and 2 U of acid phosphatase, was incubated for 1h at 37°C. The reaction was terminated by neutralizing thereaction mixture with 10 M KOH. That dephosphorylation of thephosphorylated metabolites of ribavirin went to completion wasconfirmed by monitoring the peak height ratio after variousincubation times (30, 60, 90, and 120 min); a plateau was reachedat 30 min. Furthermore, addition of another 2 U of enzyme preparationto the reaction mixture after 120 min of incubation did notlead to any change in peak height.

The dephosphorylated samples were supplemented with 20 µlof the internal standard and 500 µl of 250 mM ammoniumphosphate buffer (pH 8.5). After being centrifuged at 1,500x g for 5 min, the supernatants were loaded onto PBA cartridgesthat had been pretreated with 1 ml of 100 mM formic acid, followedby 5 ml of 250 mM ammonium phosphate buffer (pH 8.5). The cartridgeswere positioned in a 12-port manifold (Alltech, Lexington, Ky.)and washed with 5 ml of 250 mM ammonium phosphate buffer (pH8.5). Ribavirin and the internal standard were subsequentlyeluted with 1 ml of 100 mM formic acid into glass tubes. Therecovery rate was as almost the same as that described in aprevious study (7). The effluents were evaporated to drynessat 40°C under a nitrogen gas flow. The samples were reconstitutedwith 200 µl of mobile-phase solution, and a 20-µlportion was injected into the HPLC system.

For quantification of plasma ribavirin, a plasma sample (200µl) was supplemented with 20 µl of the internalstandard and 500 µl of 250 mM ammonium phosphate buffer(pH 8.5) and subjected to PBA column extraction. Plasma sampleswere not treated with acid phosphatase, because phosphorylatedribavirin was not found in plasma in our preliminary study.

Assessment of cellular ribavirin disposition in blood. Our modified assay method was tested by assessing cellular ribavirinconcentrations in two HCV patients treated with alpha interferon2b plus ribavirin. The daily dose of ribavirin was 800 mg throughoutthe study, in accordance with the standard dosing instructionsfor HCV patients treated in Japan. The daily dose of alpha interferon2b started at 10 million units intramuscularly for 14 days,followed by 6 million units three times weekly. Kidney functionin both patients was normal throughout the treatment. A 10-mlsample of blood was collected in heparinized tubes at 0, 1,2, 7, 14, 21, 28, 56, and 84 days after the start of the combinationtherapy. Blood samples (5 ml each) drawn from patients wereimmediately divided into two portions: one whole-blood portionto be treated by enzyme digestion, and the other portion tobe used to obtain plasma by centrifugation.

The concentration of cellular ribavirin was calculated withthe formula C_c = [C_w – C_p (1 – Ht)]/Ht, where C_cis the ribavirin concentration in blood cells, C_w is the concentrationin whole blood, C_p is the concentration in plasma, and Ht isthe hematocrit. Hematocrit includes many blood cell types suchas erythrocytes, lymphocytes, and neutrophils. Most ribavirinin blood cells is located in erythrocytes, because ribavirinand its phosphorylated metabolites could not be detected inthe fraction of white blood cells in our preliminary experiment(data not shown). In this study, therefore, C_c is essentiallyidentical to ribavirin concentration in erythrocytes. The hematologicalparameters (hemoglobin and hematocrit values) were also measuredon each sampling day. Informed consent was obtained from allpatients, and the study was approved by the ethics committeeof our university.

RESULTS

Top

Results

A typical HPLC chromatogram for determination of ribavirin levelsin phosphatase-digested whole blood samples is shown in Fig.1B. Peaks representing ribavirin and the internal standard wereobserved at retention times of 4.7 and 6.1 min, respectively.No interfering peaks generated from endogenous substances wereobserved on the chromatogram for blank (i.e., untreated) wholeblood (Fig. 1A). Similar chromatograms were obtained from plasmasamples (data not shown).

View larger version (27K):
[in this window]
[in a new window]
FIG. 1. HPLC chromatograms of blank whole blood (A) and whole blood spiked with 1,024 µM ribavirin (RBV) and an internal standard (IS) (B).

Calibration curves for ribavirin levels determined at concentrationsof 0.25 to 20 µM for plasma and 5.3 to 1,024 µMfor whole blood were linear. The equations for the lines calculatedby regression analysis were y = 0.0387x + 0.0037 (r² = 1.0000)for plasma and y = 0.0034x + 0.0026 (r² = 0.9999) for wholeblood, where y is the peak height ratio of ribavirin/internalstandard and x the concentration of ribavirin.

The analytical precision of determination of ribavirin whole-bloodlevels was evaluated with whole-blood reference samples containingribavirin concentrations of 21, 341, and 1,024 µM. Validationcoefficients of variation for intra- and interday assay precisionwas assessed five times in a single run and five times in separateruns, respectively (Table 1). Coefficients of variation forintra- and interday assays were 2.9 to 5.8% and 4.3 to 8.3%,respectively.

View this table:
[in this window]
[in a new window]
TABLE 1. Validation coefficients of variation for intra- and interday assay precision

We tested our new method by using it to determine ribavirincellular levels for two HCV patients. No interfering peaks wereobserved on the chromatograms despite various medications thatwere administrated concomitantly, such as an analgesic (acetaminophen),an antacid (Sucralfate), and a cholagogue (ursodesoxycholicacid). A decrease in hemoglobin levels was observed for bothpatients within the first 3 weeks of combination treatment,as the concentration of cellular ribavirin increased (Fig. 2).Cellular ribavirin reached steady-state levels within 3 to 4weeks after the combination therapy was begun. Steady-statelevels of cellular ribavirin were 146 times higher than plasmaribavirin levels for patient 1 and 150 times higher than plasmaribavirin levels for patient 2 (Table 2). Around 90% of cellularribavirin consisted of phosphorylated metabolites, which werenot detected in plasma (Table 2). The difference in hemoglobindrop between the two patients appeared to correspond to theircellular ribavirin levels. That is, patient 1, who showed markedhemoglobin reduction, had a higher cellular ribavirin concentration(1,662 µM) than did patient 2 (929 µM).

View larger version (15K):
[in this window]
[in a new window]
FIG. 2. Changes in hemoglobin levels ( ${circ}$ ) and ribavirin concentrations in blood cells (•) during combination treatment of hepatitis C patients with alpha interferon 2b and ribavirin.

View this table:
[in this window]
[in a new window]
TABLE 2. Steady-state average of plasma and cellular ribavirin concentrations and maximal drop in hemoglobin

DISCUSSION

Top

Discussion

In this study, we succeeded in simplifying the quantificationof ribavirin in whole-blood samples. The advantage of the presentmethod is the elimination of the handling required for precipitationof whole blood and the tedious pH adjustment prior to enzymedigestion (both of which were required with the previous method).Since ribavirin concentrations in whole blood were extremelyhigh (>1,000 µM at steady state), we could decreasethe sample volume, which enabled us to carry out hemolysis withice-cold water instead of by acid precipitation. Another modificationwas the use of a TSK-Gel ODS-80Ts column (4.6 [inner diameter]by 150 mm) instead of a Novapak column (3.9 [inner diameter]by 300 mm) as the analytical C₁₈ reverse-phase column. The TSK-GelODS-80Ts column achieved baseline separation of the ribavirinand internal standard peaks from those of other components moreefficiently, even though the detection wavelength of 225 nmwas lower and retention times were shorter than in a previouslydescribed method (4, 7). The limit of detection was also improvedfrom 2.4 to 1.2 ng in terms of the amounts injected. This correspondsto ribavirin concentrations of 0.2 to 0.1 µM. And thesignal-to noise ratio of >3 was higher than by the previousmethod.

We tested the revised method by determining ribavirin concentrationsin plasma and blood cell samples of two HCV patients receivingalpha interferon 2b and ribavirin in combination. Cellular ribavirinconcentrations reached steady-state levels 2 to 3 weeks afterthe patients started combination therapy, when hemoglobin reductionalso bottomed out (Fig. 2). Interestingly, changes in cellularribavirin concentration were inversely related to changes inthe levels of hemoglobin. Patient 1, with 1,662 µM cellularribavirin, showed a marked decrease in hemoglobin (–4.8g dl^–1) compared with patient 2, whose cellular ribavirinlevel was only 929 µM. These observations are consistentwith the hypothesis that marked accumulation of intracellularribavirin in erythrocytes accelerates hemolysis by reducingerythrocyte life spans (5, 8). We should mention that interferoncan also lead to a decrease in hemoglobin.

Cellular/plasma ratios of ribavirin were 146 and 150 in patients1 and 2, respectively. The ratios were much higher than thatreported for a previous study (i.e., 60) by Lertora et al. (11).This discrepancy is due to differences between the present HPLCassay method and the previous radioimmunoassay method. Sincethe radioimmunoassay procedure did not include a dephosphorylationstep, it could not correctly determine the concentrations ofphosphorylated metabolites, leading to underestimation of totalcellular ribavirin levels.

Concerning the antiviral effects of ribavirin, normalizationof the level of alanine aminotransferase and undetectable amountsof HCV RNA were observed with samples from patient 1 but notwith samples from patient 2 (data not shown). Jen et al. (10)suggested that higher plasma ribavirin concentrations in treatmentweek 4 were associated with a higher virologic response rate.However, discussion of the antiviral effects of ribavirin inthe present study would be inappropriate because of the smallsample size of two patients. We intend to investigate this hypothesisfurther by studying a larger number of patients.

In conclusion, our HPLC method can be used for routine monitoringof cellular ribavirin disposition in blood. The impact of measuringcellular ribavirin levels to evaluate the efficacy and toxicityof treatment needs to be clarified in future clinical studieswith a large number of patients.

FOOTNOTES

* Corresponding author. Mailing address: Department of Pharmaceutical Sciences, Institute of Clinical Medicine, University of Tsukuba, Ten-nodai 1-1-1, Tsukuba, Ibaraki 305-8575, Japan. Phone: 81-298-53-3859. Fax: 81-298-53-7025. E-mail: masatoh{at}md.tsukuba.ac.jp.

REFERENCES

Top