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Differences in nucleotide hydrolysis contribute to the differences between erythrocyte 6-thioguanine nucleotide concentrations determined by two widely used methods.

Thiopurine drugs [6-mercaptopurine, 6-thioguanine (6-TG), [1] and azathioprine] are widely used in both the treatment of acute lymphoblastic leukemia and autoimmune disorders and in immunosuppressive regimens for the prevention of acute rejection after solid organ transplantation. The active metabolites of these drugs, the 6-thioguanine nucleotides (6-TGNs), are considered to exert their therapeutic effect through incorporation into DNA with subsequent disruption of cell replication (1). The metabolism and bioavaility of thiopurines are subject to wide inter-and intraindividual variation (2-5) as a result of both genetic and environmental factors (6-8). There is accumulating evidence that individualized therapy is necessary for optimization of the therapeutic response to these agents (6-10). Because 6-TGN concentrations in erythrocytes have been found to correlate with the extent of 6-TGN incorporation in peripheral blood leukocyte DNA (11,12), their routine determination as a surrogate marker has been advocated to assess the effect of thiopurine therapy. High concentrations of erythrocyte 6-TGNs have been associated with the risk of myelosuppression (2,13-17) and related complications, such as septicemia and consecutive multiple organ failure. On the other hand, low 6-TGN concentrations may lead to suboptimal treatment, as shown in several clinical studies involving patients suffering from acute lymphoblastic leukemia (18) and Crohn disease (19) as well as recipients of kidney (9) or heart (2) transplants.

Chromatographic methods are exclusively used for the determination of erythrocyte 6-TGN concentrations. Since the synthesis of the first thiopurine drug, 6-mercaptopurine, by Elion in 1951 (20), numerous HPLC procedures have been reported. The method developed by Lennard in 1987 (21) has been the most widely used method in those clinical studies that have served as the basis for establishing treatment-related therapeutic ranges for 6-TGN. This method has been routinely used in our laboratory for many years (2, 7). However, the sample preparation procedure for the Lennard assay is relatively laborious and time-consuming and uses the neurotoxic reagent phenylmercuric acetate (PMA) for the extraction. Furthermore, to allow the simultaneous determination of 6-TGNs and 6-methylmercaptopurine in a single sample, the method was modified, primarily through an alteration of the pH for extraction (22). This is a compromise that is accompanied by suboptimal analytical conditions for both analytes. We therefore sought an alternative HPLC method that would be more rapid and easier to perform and that allowed the simultaneous measurement of 6-methylmercaptopurine with comparable analytical reliability. Dervieux and Boulieu (23) have published a method that appeared to meet these requirements. Surprisingly, we found that the 6-TGN concentrations determined with the new method were always considerably higher than those measured with the Lennard (21) method. The presence of such a difference has major implications for the application of this new method to the monitoring of thiopurine therapy because the therapeutic ranges established with the Lennard method are not valid if the Dervieux-Boulieu procedure is used. In addition, the comparison of results from clinical studies derived with these different analytical procedures is impossible. We therefore performed a detailed comparison of the sample preparation of these two methods.

Materials and Methods

DRUGS AND REAGENTS

All reagents used were of the highest available purity. 6-TG (2-amino-6-mercaptopurine), 5-bromouracil (5-BU), D,L-dithiothreitol (DTT), PMA, and Hanks' balanced salt solution (HBSS) were purchased from Sigma; isoamyl alcohol was from J.T. Baker; and toluene, sulfuric acid, hydrochloric acid, phosphoric acid, sodium hydroxide, potassium dihydrogen phosphate, perchloric acid, and methanol (HPLC grade) were from Merck. The 6-TG (0.1 g/L) and 5-BU (1 g/L) stock solutions were prepared in 0.1 mol/L sodium hydroxide and stored at -80 [degrees]C. The toluene-isoamyl alcohol-PMA solution used in the Lennard procedure and the DTT solutions were prepared fresh each day.

ERYTHROCYTE PREPARATION

Erythrocytes were obtained from ammonium heparinateor EDTA-anticoagulated whole blood by centrifugation at 1000g for 10 min. Platelet-rich plasma and the buffy coat, together with the upper layer of erythrocytes, were discarded. Two washing steps were performed with HBSS under the same conditions. Cells were finally re-suspended in HBSS to yield a hematocrit of ~0.40, and the exact hematocrit and red blood cell count for calculation of 6-TGN concentration were determined with an automatic hematologic cell counting device (AC.T 5diff; Beckman Coulter). The isolated erythrocytes were portioned into 250-[micro]L aliquots and stored at -80 [degrees]C until analysis. Erythrocytes obtained from venous blood of healthy volunteers were processed as above and used for preparation of quality-control samples and calibrators.

Only anonymous excess material from blood samples sent to the laboratory for routine analysis was used to perform all experiments, as well as to prepare the in-house controls. Therefore, according to the guidelines of the local ethics committee, Institutional Review Board approval and/or donor written informed consent were not required.

SAMPLE PREPARATION

Lennard method (21). The procedure was performed as originally described by Lennard (21) and included two steps. The first step consisted of simultaneous denaturation of the erythrocyte proteins and hydrolysis (100 [degrees]C for 1 h) of the 6-TGN to 6-TG by sulfuric acid (final concentration, 0.5 mol/L) in the presence of 2 mmol/L DTT to protect thiol groups from oxidation. In the second step, the 6-TG was extracted by formation of the phenylmercury adduct in toluene at alkaline pH. Back-extraction of this organic phase with 0.1 mmol/L hydrochloric acid hydrolyzed the adduct and liberated the free thiopurine into the acid layer.

Erythrocytes (0.8 X [10.sup.9] cells in 200 [micro]L) were added to 800 [micro]L of DTT (3.75 mmol/L) in a 10-mL glass tube with screw cap. After the addition of 500 [micro]L of 1.5 mol/L sulfuric acid, tubes were incubated at 100 [degrees]C for 1 h in a heating block. After cooling, 500 [micro]L of 5 mol/L sodium hydroxide was added to each tube, followed by 8 mL of toluene containing 170 mmol/L isoamyl alcohol and 1.3 mmol/L PMA. The tubes were gently mixed for 10 min and then centrifuged at 900g (5 min). A 6-mL portion of the upper toluene layer was transferred to a new tube and back-extracted with 0.2 mL of 0.1 mol/L hydrochloric acid three times (20 s each time) in a multitube vortexmixer. The tubes were centrifuged as described above, and 80 [micro]L of the lower acid layer was injected onto the chromatographic column. Whereas the original method described by Lennard (21) used external standard mode for quantification, we used DTT as internal standard in our measurements.

Dervieux-Boulieu method (23). The procedure was performed as published by Dervieux and Boulieu (23) and included sample deproteinization using 1 mol/L perchloric acid in the presence of 60 mmol/L DTT. This was followed by hydrolysis (100 [degrees]C for 45 min) of the 6-TGNs in the separated supernatant to release the 6-TG. There was no further extraction or any other pretreatment of the sample before chromatography. In our laboratory, this procedure was conducted with minor modifications, consisting of the use of an internal standard, 5-BU, and a 50% reduction in the reaction volume. Briefly, we mixed 250 [micro]L of erythrocytes with 20 [micro]L of internal standard (5-BU; 314 [micro]mol/L), 20 [micro]L of DTT (1.1 mol/L), and 50 [micro]L of water for 30 s by vortex-mixing in a 1.5-mL polypropylene tube. To this mixture we added 34 [micro]L of perchloric acid (700 mL/L) and vortex-mixed the tube at the highest frequency; the tube was then centrifuged for 15 min at 30008. The supernatant (~220 [micro]L) was transferred to brown glass tubes with screw caps, which were then heated for 45 min at 100 [degrees]C to hydrolyze the thiopurine nucleotides. After cooling, a 80-[micro]L aliquot was injected into the column.

CHROMATOGRAPHIC CONDITIONS

For chromatographic separation of the free 6-TG after both sample preparation procedures described above, we used a modification (7) of the Lennard chromatographic method (21). A Hypersil [C.sub.18] column [25 cm X 4.6 mm (i.d.); particle size, 5 [micro]m; Mz Analysentechnik] was used as the stationary phase. The mobile phase consisted of solution A [50 mL of methanol and 950 mL of potassium dihydrogen phosphate, pH 3.0 (final concentration, 20 mmol/L), containing 100 mmol/L triethylamine and 0.5 mmol/L DTT] and solution A [230 mL of methanol and 770 mL of potassium dihydrogen phosphate, pH 3.0 (final concentration, 20 mmol/L), containing 100 mmol/L triethylamine and 0.5 mmol/L DTT]. The analytes were eluted at a flow rate of 1.1 mL/min with the following gradient: 0-1 min, 0% B; 1-4 min, 0-20% B; 4-5.5 min, 20-100% B; 5.5-10 min, 100% B; 10-10.5 min, 100-0% B. The column was maintained at 42 [degrees]C. The HPLC system consisted of a chromatographic pump (M480), an automatic injector (GINA 50), a diode array detector (UVD 3405), and a computer interface system controller linked to a PC (Dionex-Gynkotek). 6-TGN concentrations were determined by absorbance at 345 nm in the internal standard mode. Under routine conditions, the assay was calibrated using two-point calibration, 0 and 300 pmol/ 0.8 X [10.sup.9] erythrocytes. Deviation from full-range historic calibration curves was always <10%. // The DTT used as internal standard for the Lennard procedure was detected at 322 nrn, whereas the 5-BU used as internal standard for the Dervieux-Boulieu procedure was detected at 280 nm. Internal standardization was used to achieve better precision because the samples are kept for 45-60 min at 100 [degrees]C, which may cause evaporation. Chromeleon software (Dionex), Ver. 6.3, was used for recording and calculating the data. Ultraviolet spectra of known (calibrators) and unknown peaks were visually compared to exclude possible interferents.

The quality-control samples for both methods [in-house drug-free erythrocyte lysates to which 6-TG (100 and 700 pmol/0.8 X [10.sup.9] erythrocytes) had been added] were analyzed in each run. The allowed deviation from target values was [+ or -] 10%. In addition, pooled erythrocytes from patients undergoing azathioprine therapy constituted a further precision control (accepted range mean [+ or -] 2 SD). Thioguanine concentrations were expressed as pmol/0.8 X [10.sup.9] erythrocytes.

ASSESSMENT OF PERFORMANCE CHARACTERISTICS

The detection limit for 6-TG was calculated for both methods on the basis of a signal-to-noise ratio of 3. For this purpose, the baseline noise signal was obtained from a segment of the respective chromatograms that preceded the 6-TG peak. The lower limit of quantification was set at a 6-TG concentration for which an acceptable within-run imprecision (CV<15%; n = 12) could be obtained. The linearity of the methods was established by constructing calibration curves (n = 3) using drug-free erythrocyte lysates to which increasing 6-TG concentrations (25, 30, 100, 500, 1000, 2000, 3000, and 10 000 pmol/0.8 x [10.sup.9] erythrocytes) were added. Within- and between-run imprecision and extraction efficiency were studied with drug-free erythrocyte lysates to which 6-TG was added to yield final concentrations of 100, 300, and 700 pmol/0.8 X [10.sup.9] erythrocytes. The extraction efficiencies for the internal standards 5-BU and DTT were investigated at the concentrations used in the hydrolysis mixture (16.8 [micro]mo1/L and 2 mmol/L, respectively). The extraction efficiency was calculated by comparing peak areas obtained for the extracted erythrocyte samples containing 6-TG and DTT or 6-TG and 5-BU with peak areas obtained for aqueous solutions containing the same amount of the compound, which were injected directly on the column without extraction after incubation at 100 [degrees]C for 60 or 45 min, respectively. Possible chromatographic interference was evaluated by separate analysis of 50 patient specimens free of 6-TG. The analytical recovery for each method was determined by adding known amounts of 6-TG (100, 300, and 700 pmol/0.8 X [10.sup.9] erythrocytes) to drug-free erythrocyte lysates. The recovery was calculated by comparing the measured concentrations with the expected concentrations.

EFFECT OF HYDROLYSIS CONDITIONS AND DURATION OF HYDROLYSIS ON BETWEEN-METHOD DIFFERENCES MEASURED IN 6-TGN CONCENTRATIONS

There are three major differences between the two methods with respect to the hydrolysis step: (a) the type and concentration of the acid used; (b) the concentration of the DTT used to prevent thiol oxidation; and (c) the duration of hydrolysis. To investigate whether these could be responsible for the observed differences in measured values, we performed the following experiments:

We analyzed 6-TGNs in erythrocyte preparations from 50 blood specimens obtained from patients on azathioprine therapy according to the two analytical procedures described above. In the first set of experiments, the sulfuric acid (0.5 mol/L) typically used in the Lennard method (21) was replaced by perchloric acid (1 mol/L), which is used in the Dervieux-Boulieu method (23). The hydrolyzed samples were then processed without further modifications according to Lermard: i.e., extraction of the phenylmercury adduct into toluene at basic pH and back-extraction with hydrochloric acid.

In a second set of experiments, we investigated the effect of several DTT concentrations (2, 5, 10, 25, and 60 mmol/L) on 6-TG recovery with both methods, using pooled erythrocytes from patients on azathiprine therapy.

In a third set of experiments, we carefully investigated the effect of the duration of hydrolysis (15, 30, 45, 60, and 75 min at 100 [degrees]C in both protocols. In addition, we repeated time course studies with the Lennard method using perchloric acid (1 mol/L) instead of sulfuric acid. For these experiments, we used pooled erythrocytes from patients on azathioprine therapy.

STATISTICAL METHODS

Method comparisons were performed using the nonparametric regression procedure of Passing and Bablok (24) as well as the procedure described by Bland and Altman (25). For correlation analyses, we used the Spearman rank correlation test. Passing-Bablok calculations were performed with EVAPAK (Ver. 2.08; Boehringer Mannheim). Bland-Altman comparisons and Spearman rank correlation analyses were performed using MedCalc computer software (MedCalc Software).

Results

Shown in Fig. 1 are representative chromatograms of erythrocyte preparations derived from a patient on azathioprine therapy, which were processed according to either Lennard (Fig. 1A) or Dervieux and Boulieu (Fig. 1B). The chromatographic analyses for samples prepared by both preparation protocols were free of interferences, and there was baseline separation between peaks. The performance characteristics of both procedures, as established in our laboratory (Table 1), were almost identical to those published by the respective authors in their original publications (21,23). Our observed extraction efficiency for 6-TG with the Lennard procedure was ~40% compared with the 64% reported by Lennard (21). Linear regression analyses yielded slopes of 0.00047-0.00051, y-intercepts of 0.019-0.047, and correlation coefficients of 0.997-0.999 (range; n = 3) for the Lennard method, and slopes of 0.00331-0.00338, y-intercepts of -0.011 to -0.026), and correlation coefficients of 0.999-1.00 (range; n = 3) for the Dervieux-Boulieu procedure. The extraction efficiencies for both internal standards (DTT and 5-BU) were constant over a hydrolysis time up to 75 min and independent of increasing 6-TG concentrations. In addition, the extraction efficiency for 5-BU was independent of increasing DTT concentrations in the hydrolysis step. The extraction efficiencies for all experiments described here did not vary considerably. The CVs for the signals obtained with the internal standards within different batches were 5.4% [+ or -] 0.53% for DTT and 5.9% [+ or -] 0.35% for 5-BU.

[FIGURE 1 OMITTED]

Although both methods were calibrated with the same calibrator and the target values of the quality-control samples (also identical) were met with both procedures, the 6-TGN concentrations in patient samples measured with the Dervieux-Boulieu procedure [median (range), 236.5 (37-5210) pmol/0.8 X [10.sup.9] erythrocytes; n = 50] were ~2.6-fold higher than the corresponding values obtained with the Lennard procedure [115.0 (30-1599) pmol/0.8 X 109 erythrocytes; n = 50]. Despite this large difference, there was an excellent and highly significant correlation between the two methods [r = 0.99; N <0.001; y = 2.64(2.31-2.88)x - 54.73(-82.77 to -22.62) pmol/0.8 X [10.sup.9] erythrocytes; 68% median distance = 20.89; [S.sub.y|x] = 50.38; n = 50]. To improve the graphic resolution, Fig. 2A shows the Passing-Bablok comparison between the two methods after exclusion of the highest value. As can be seen, inclusion of the highest value did not greatly impact the regression result because we used Passing-Bablok regression analysis, which is known to be very robust against outliers (24). A difference plot (Fig. 2B) revealed that the absolute difference between the two methods increased with the magnitude of the measurement and that this relationship was highly significant (r = 0.96; P <0.001).

Replacement of the sulfuric acid (0.5 mol/L) originally used for hydrolysis in the Lennard procedure (21) with perchloric acid (1 mol/L) reduced the difference to 1.4-fold [r = 0.99; y = 1.37(1.24-1.53)x - 34.85(-64.95 to -19.05) pmol/0.8 X [10.sup.9] erythrocytes; 68% median distance = 30.04; [S.sub.y|x] = 50.88; n = 50]. For the sake of clarity, Fig. 3A shows the Passing-Bablok comparison between the two methods after exclusion of the highest value. Again the Bland-Altman difference plot (Fig. 3B) revealed that the absolute difference between the two methods increased with the magnitude of the measurement, and the relationship remained significant (r = 0.70; P <0.001).

Because the two methods use different concentrations of DTT (2 mmol/L for the Lennard procedure and 60 mmol/L for the Dervieux-Boulieu procedure), we investigated the influence of the DTT concentration on the recovery of 6-TG. For the Dervieux-Boulieu method (23), increasing the DTT concentrations from 2 to 60 mmol/L increased the recovery of 6-TG (Fig. 4). The results also remained similar when external standard mode was used for quantification (data not shown). However, in the case of the Lennard procedure (21), DTT concentrations >2 mmol/L led to extremely low 6-TG recoveries, presumably because of poor recoveries in the extraction step, which precluded quantification.

[FIGURE 2 OMITTED]

We next investigated the effect of duration of hydrolysis at 100 [degrees]C in both protocols, using times ranging from 15 to 75 min. The method-dependent 6-TG concentration-time curves are presented in Fig. 5, A and B. A minimum hydrolysis time of 30 min is necessary for complete hydrolysis of the 6-TGN in the Dervieux-Boulieu method (Fig. 5A). In the original publication, the authors used a hydrolysis time of 45 min. In the Lennard procedure, a continual increase in 6-TG values was observed in the time periods investigated, which was rapid up to a hydrolysis time of ~60 min and slower thereafter. Although the experiment was extended up to 4 h (data not shown), the curve still did not reach a plateau. The time course studies were repeated with the Lennard method using perchloric acid (1 mol/L) instead of sulfuric acid (0.5 mol/L). As shown in Fig. 5C, after this modification, the 6-TG concentration-time curve reached a plateau in a time period comparable to that observed with the Dervieux-Boulieu procedure. The results also remained similar when external standard mode was used for quantification (data not shown).

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Discussion

Determination of erythrocyte 6-TGN concentrations can assist clinicians in optimizing therapeutic response to therapy with thiopurine drugs. Multiple assays have been established to monitor 6-TGN concentrations. These assays differ in the matrices used, such as isolated erythrocytes (21-23,26-31), whole blood (32), isolated lymphocytes (33, 34), leukocyte DNA (11, 35), and plasma (36-39). For routine drug monitoring of 6-TGNs, methods based on isolated erythrocytes are predominantly used. Some of these methods measure individual 6-TGN mono-, di-, and triphosphates (27, 29). However, more commonly, free 6-TG is determined after hydrolysis of 6-TGNs at increased temperatures and acidic pH (21-23,26,28,33). These approaches differ in sample preparation. The Lennard method uses NIA adduct formation in toluene (21,22) after the acid hydrolysis, whereas the Dervieux-Boulieu method uses only deproteinization (23). Other authors have applied extraction steps using mercurial cellulose resin and 2-mercaptoethanol (29), aluminum ion complexation (37), or ethyl acetate-dichloromethane (33). The extracted samples are subjected to reversed-phase chromatography with either isocratic or gradient elution. The analyte 6-TG is then monitored by ultraviolet detection (21-23, 26, 29, 36, 37) or fluorescence detection after derivatization with either potassium permanganate (27, 28, 32) or monobromobimane (35). Finally, the results of 6-TGN measurements have been reported in various units, such as pmol/0.8 X [10.sup.9] erythrocytes (21, 22), mci1/mmol of hemoglobin (40), pmol/ 100 erythrocytes (31), or pmol/25 mg of hemoglobin (26). This wide variety in technical details and reporting of the results makes it extremely difficult to compare the published data derived from studies. In this report, we have concentrated on two widely used methods that use the same matrix, chromatography conditions, detection mode, and units. Despite good correlation between the methods, there was a 2.6-fold difference in the measured 6-TGN concentrations in patient samples. These findings illustrate the importance of the sample pretreatment step in 6-TGN determination. The methods were calibrated with the same calibrator, and each method met the target values for the identical quality-control samples. The principal difference between calibrator/ quality-control samples and patients samples is that free 6-TG is added to the former samples, whereas in the latter samples the 6-TGNs must first be hydrolyzed to free 6-TG before quantification. We therefore presumed that the differences between hydrolysis protocols of the two methods could be of particular importance for the differences found.

[FIGURE 5 OMITTED]

From the results of our experiments, it seems that the procedure of Dervieux and Boulieu (23) produces more complete conversion of erythrocyte 6-TGNs to 6-TG, leading to higher measured 6-TG concentrations. Multiple factors may be responsible for this observation. Of particular importance are the acid used and the DTT concentration. Furthermore, the hydrolysis step must be allowed to reach completion. We presume that the use of perchloric acid produces more complete protein precipitation, leading to faster liberation of the 6-TGNs from the cell lysates to become available for hydrolysis, than does the use of sulfuric acid. Alternatively, it could be also true that perchloric acid reacts more readily and completely with 6-TGNs to form 6-TG than does sulfuric acid. The observation that increasing the DTT concentrations reduced the extraction efficiency for 6-TG in the Lennard method may be attributable to a competition between DTT and 6-TG for adduct formation with PMA.

In support of our results indicating the importance of the hydrolysis step for 6-TGN determination, Lowry et al. (41) have recently reported an -1.6-fold difference between 6-TGN concentrations measured with a modified version the Lennard method and a modification of an assay published by Erdmann et a1. (28), with the values according to Erdmann being lower. However, in line with our investigation, the authors found a high correlation between the results obtained with both methods (41). Lowry et a1. (41) did not investigate the reasons for this difference in more detail but suggested a role of hydrolysis time. In fact, the Erdmann procedure uses a hydrolysis time of 45 min, whereas the Lennard method has a hydrolysis time of 60 min (28, 32). In addition, the concentration of sulfuric acid used for the hydrolysis is lower in the Erdmann assay (28, 32). This may further contribute to incomplete hydrolysis, leading to lower measured 6-TG concentrations.

As a consequence of these methodologic differences, the putative therapeutic ranges are method dependent. To date, little attention has been given to this fact. To interpret results properly, method-specific therapeutic ranges should be considered, which are not available for the Dervieux-Boulieu procedure. Efforts should be made to standardize the analytical procedures for the determination of 6-TGNs.

We gratefully acknowledge the skillful technical assistance of Tanja Schneider and Melanie Fischer. We thank Dr. E. Schidtz for fruitful discussions.

Received August 2, 2002; accepted November 13, 2002.

References

(1.) Lennard L. The clinical pharmacology of 6-mercaptopurine. Eur J Clin Pharmacol 1992;43:329-39.

(2.) Schutz A, Gummert J, Mohr FW, Armstrong VW, Oellerich I. Should 6-thioguanine nucleotides be monitored in heart transplant recipients given azathioprine? Ther Drug Monit 1996;18:228-33.

(3.) Ohlman S, Albertioni F, Peterson C. Day-to-day variability in azathioprine pharmacokinetics in renal transplant recipients. Clin Transplant 1994;8:217-23.

(4.) Bergan S, Rugstad HE, Bentdal 0, Endresen L, Stokke 0. Kinetics of mercaptopurine and thioguanine nucleotides in renal transplant recipients during azathioprine treatment. Ther Drug Monit 1994; 16:13-20.

(5.) Chan GL, Erdmann GR, Gruber SA, Matas AJ, Canafax DM. Azathioprine metabolism: pharmacokinetics of 6-mercaptopurine, 6-thiouric acid and 6-thioguanine nucleotides in renal transplant patients. J Clin Pharmacol 1990;30:358-63.

(6.) Armstrong VW, Oellerich I. New developments in the immunosuppressive drug monitoring of cyclosporine, tacrolimus, and azathioprine. Clin Biochem 2001;34:9-16.

(7.) Schutz A, Gummert J, Armstrong VW, Mohr FW, Oellerich I. Azathioprine pharmacogenetics: the relationship between 6-thioguanine nucleotides and thiopurine methyltransferase in patients after heart and kidney transplantation. Eur J Clin Chem Clin Biochem 1996;34:199-205.

(8.) Weinshilboum R. Thiopurine pharmacogenetics: clinical and molecular studies of thiopurine methyltransferase. Drug Metab Dispos 2001;29:601-5.

(9.) Bergan S, Rugstad HE, Bentdal 0, Sodal G, Hartmann A, Leivestad O, et al. Monitored high-dose azathioprine treatment reduces acute rejection episodes after renal transplantation. Transplantation 1998;66:334-9.

(10.) Lennard L. Therapeutic drug monitoring of cytotoxic drugs [Review]. Br J Clin Pharmacol 2001;52(Suppl 1):75S-87S.

(11.) Cuffari C, Seidman EG, Latour S, Theoret Y. Quantitation of 6-thioguanine in peripheral blood leukocyte DNA in Crohn's disease patients on maintenance 6-mercaptopurine therapy. Can J Physiol Pharmacol 1996;74:580-5.

(12.) Bergan S, Bentdal O, Sodal G, Brun A, Rugstad HE, Stokke O. Patterns of azathioprine metabolites in neutrophils, lymphocytes, reticulocytes, and erythrocytes: relevance to toxicity and monitor ing in recipients of renal allografts. Ther Drug Monit 1997;19: 502-9.

(13.) Bergan S, Rugstad HE, Bentdal O, Stokke O. Monitoring of azathioprine treatment by determination of 6-thioguanine nucleotide concentrations in erythrocytes. Transplantation 1994;58: 803-8.

(14.) Lennard L, Lilleyman JS, Van Loon J, Weinshilboum RM. Genetic variation in response to 6-mercaptopurine for childhood acute lymphoblastic leukaemia. Lancet 1990;336:225-9.

(15.) Lennard L, Rees CA, Lilleyman JS, Maddocks JL. Childhood leukaemia: a relationship between intracellular 6-mercaptopurine metabolites and neutropenia. Br J Clin Pharmacol 1983;16:359-63.

(16.) Innocenti F, Danesi R, Favre C, Nardi M, Menconi MC, Di Paolo A, et al. Variable correlation between 6-mercaptopurine metabolites in erythrocytes and hematologic toxicity: implications for drug monitoring in children with acute lymphoblastic leukemia. Ther Drug Monit 2000;22:375-82.

(17.) Evans WE, Horner M, Chu YQ, Kalwinsky D, Roberts WM. Altered mercaptopurine metabolism, toxic effects, and dosage requirement in a thiopurine methyltransferase-deficient child with acute lymphocytic leukemia. J Pediatr 1991;119:985-9.

(18.) Lilleyman JS, Lennard L. Mercaptopurine metabolism and risk of relapse in childhood lymphoblastic leukaemia. Lancet 1994;343: 1188-90.

(19.) Dubinsky MC, Lamothe S, Yang HY, Targan SR, Sinnett D, Theoret Y, et al. Pharmacogenomics and metabolite measurement for 6-mercaptopurine therapy in inflammatory bowel disease. Gastroenterology 2000;118:705-13.

(20.) Elion GB. Historical background of 6-mercaptopurine. Toxicol Ind Health 1986;2:1-9.

(21.) Lennard L. Assay of 6-thioinosinic acid and 6-thioguanine nucleotides, active metabolites of 6-mercaptopurine, in human red blood cells. J Chromatogr 1987;423:169-78.

(22.) Lennard L, Singleton HJ. High-performance liquid chromatographic assay of the methyl and nucleotide metabolites of 6-mercaptopurine: quantitation of red blood cell 6-thioguanine nucleotide, 6-thioinosinic acid and 6-methyl mercaptopurine metabolites in a single sample. J Chromatogr 1992;583:83-90.

(23.) Dervieux T, Boulieu R. Simultaneous determination of 6-thioguanine and methyl 6-mercaptopurine nucleotides of azathioprine in red blood cells by HPLC. Clin Chem 1998;44:551-5.

(24.) Passing H, Bablok. A new biometrical procedure for testing the equality of measurements from two different analytical methods. Application of linear regression procedures for method comparison studies in clinical chemistry. Part E. J Clin Chem Clin Biochem 1983;21:709-20.

(25.) Bland JM, Altman DG. Comparing methods of measurement: why plotting difference against standard method is misleading. Lancet 1995;346:1085-7.

(26.) Mawatari H, Kato Y, Nishimura S, Sakura N, Ueda K. Reversed-phase high-performance liquid chromatographic assay method for quantitating 6-mercaptopurine and its methylated and non-methylated metabolites in a single sample. J Chromatogr B Biomed Sci Appl 1998;716:392-6.

(27.) Keuzenkamp-Jansen CW, De Abreu RA, Bokkerink JP, Trijbels JM. Determination of extracellular and intracellular thiopurines and methylthiopurines by high-performance liquid chromatography. J Chromatogr B Biomed Appl 1995;672:53-61.

(28.) Erdmann GR, France LA, Bostrom BC, Canafax DM. A reversed phase high performance liquid chromatography approach in determining total red blood cell concentrations of 6-thioguanine, 6-mercaptopurine, methylthioguanine, and methylmercaptopurine in a patient receiving thiopurine therapy. Biomed Chromatogr 1990;4: 47-51.

(29.) Lavi LE, Holcenberg JS. A rapid and sensitive high-performance liquid chromatographic assay for 6-mercaptopurine metabolites in red blood cells. Anal Biochem 1985;144:514-21.

(30.) Dooley T, Maddocks JL. Assay of an active metabolite of 6-thioguanine, 6-thioguanosine 5'-monophosphate, in human red blood cells. J Chromatogr 1982;229:121-7.

(31.) Weller S, Thurmann P, Rietbrock N, Gossmann J, Scheuermann EH. HPLC analysis of azathioprine metabolites in red blood cells, plasma and urine in renal transplant recipients. Int J Clin Pharmacol Ther 1995;33:639-45.

(32.) Pike MG, Franklin CL, Mays DC, Lipsky JJ, Lowry PW, Sandborn WJ. Improved methods for determining the concentration of 6-thioguanine nucleotides and 6-methylmercaptopurine nucleotides in blood. J Chromatogr B Biomed Sci Appl 2001;757:1-9.

(33.) Erdmann GR, Steury JC, Carleton BC, Stafford RJ, Bostrom BC, Canafax DM. Reversed-phase high-performance liquid chromatographic approach to determine total lymphocyte concentrations of 6-thioguanine, methylmercaptopurine and methylthioguanine in humans. J Chromatogr 1991;571:149-56.

(34.) Dervieux T, Chu Y, Su Y, Pui CH, Evans WE, Relling MV. HPLC determination of thiopurine nucleosides and nucleotides in vivo in lymphoblasts following mercaptopurine therapy. Clin Chem 2002; 48:61-8.

(35.) Warren DJ, Slordal L. A high-performance liquid chromatographic method for the determination of 6-thioguanine residues in DNA using precolumn derivatization and fluorescence detection. Anal Biochem 1993;215:278-83.

(36.) Su Y, Hon YY, Chu Y, Van de Poll ME, Relling MV. Assay of 6-mercaptopurine and its metabolites in patient plasma by high-performance liquid chromatography with diode-array detection. J Chromatogr B Biomed Sci Appl 1999;732:459-68.

(37.) Lin KT, Varin F, Rivard GE, Leclerc JM. Isolation of 6-mercaptopurine in human plasma by aluminum ion complexation for highper-formance liquid chromatographic analysis. J Chromatogr 1991;536:349-55.

(38.) Andrews PA, Egorin MJ, May ME, Bachur NR. Reversed-phase high-performance liquid chromatography analysis of 6-thioguanine applicable to pharmacologic studies in humans. J Chromatogr 1982;227:83-91.

(39.) Dooley T, Maddocks JL. Assay of 6-thioguanine in human plasma. Br J Clin Pharmacol 1980;9:77-82.

(40.) Bruunshuus I, Schmiegelow K. Analysis of 6-mercaptopurine, 6-thioguanine nucleotides, and 6-thiouric acid in biological fluids by high-performance liquid chromatography. Scand J Clin Lab Invest 1989;49:779-84.

(41.) Lowry PW, Franklin CL, Weaver AL, Pike MG, Mays DC, Tremaine WJ, et al. Measurement of thiopurine methyltransferase activity and azathioprine metabolites in patients with inflammatory bowel disease. Gut 2001;49:665-70.

MARIA SHIPKOVA,* VICTOR WILLIAM ARMSTRONG, EBERHARD WIELAND, and MICHAEL OELLERICH

Department of Clinical Chemistry, Georg-August-University Gottingen, D-37075 Gottingen, Germany.

*Address correspondence to this author at: Abteilung Klirusche Chemie, Zentrum Innere Medizin, Georg-August-Universitat, Robert Koch Strasse 40, D-37075 Gottingen, Germany. Fax 49-551-3912503; e-mail maria.shipkova@ med.uni-goettingen.de.

[1] Nonstandard abbreviations: 6-TG, 6-thoguanine; 6-TGN, 6-thioguanine nucleotide; NIA, phenylmercuric acetate; 5-BU, 5-bromouracil; DTT, dithiothreitol; and HBSS, Hanks' balanced salt solution.
Table 1. Performance characteristics of the Lennard (21) and
Dervieux-Boulieu (23) methods for determination of erythrocyte
6-TGN concentrations as established in our laboratory. (a)

Characteristic Lennard method

LLQ (b, c) 30
Detection limit (c) 9.0
Linearity (c) 30-3000
 (n = 3) (r = 0.999)
Extraction efficiency for 6-TG, (d) % 34.9-44.9
 (n = 3 for each 6-TG
 concentration evaluated)
Extraction efficiency for IS, (e) % 40.0 [+ or -] 2.2 (DTT)
 (n = 10)
Imprecision (CV), %
Within-run (d) 5.3-8.0
 (n = 12)
Between-run (d) 6.4-8.8
 (n = 12)
Within-run at the LLQ 13.5
 (n = 12)
Analytical recovery, % 97.7-105.8
 (n = 5)
Selectivity No known interferences

Characteristic Dervieux-Boulieu method

LLQ (b, c) 20
Detection limit (c) 4.5
Linearity (c) 20-10 000
 (n = 3) (r = 0.999)
Extraction efficiency for 6-TG, (d) % 78.7-83.3
 (n = 3 for each 6-TG
 concentration evaluated)
Extraction efficiency for IS, (e) % 72.8 [+ or -] 4.4 (5-BU)
 (n = 10)
Imprecision (CV), %
Within-run (d) 2.7-3.4
 (n = 12)
Between-run (d) 3.9-5.9
 (n = 12)
Within-run at the LLQ 12.6
 (n = 12)
Analytical recovery, % 100.6-104.2
 (n = 5)
Selectivity No known interferences

(a) For more details, see Materials and Methods.

(b) LLQ, lower limit of quantification; IS, internal standard.

(c) As pmol/0.8 x [10.sup.9] erythrocytes.

(d) Determined in the working range 100-700 pmol/0.8 x [10.sup.9]
erythrocytes.

(e) Concentrations of the internal standards In the hydrolysis
mixture: 16.8 [micro] mol/L 5-BU, 2 mmol/L DTT.
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Title Annotation:Drug Monitoring and Toxicology
Author:Shipkova, Maria; Armstrong, Victor William; Wieland, Eberhard; Oellerich, Michael
Publication:Clinical Chemistry
Date:Feb 1, 2003
Words:5501
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