Determination of 6-Mercaptopurine and its Metabolites in Plasma or Serum by High Performance Liquid Chromatography Z. Sahnoun,* F. Serre-Debeauvais, J. Lang, G . Faucon and M. Gavend Laboratoire de Pharmacologie Clinique, HApital Edouard Herriot, Lyon, France

A sensitive and accurate reversed phase liquid chromatographic assay was developed for the determination of 6-mercaptopurine (6MP) (the active metabolite of azathioprine) in human plasma. The assay involved extraction into acetonitrile and dichloromethane from plasma pretreated with 0.038 M of dithiothreitol solution. The residue was analyzed by isocratic chromatography on a C,8 analytical column with UV detection at 326 nm. The average extraction recovery of 6MP was 85%. The method has been applied successfully to the determination of 6MP and its metabolites in pharmacokinetic studies.

INTRODUCTION

EXPERIMENTAL

Azathioprine (AZA) (6-( l-methyl-4-nitro-5-imidazol) thioprine) is largely used for its immunosuppressive property in preventing the rejection of transplanted organs. However, its use remains quite empirical at present. The dose determination and therapeutic supervision are often based on clinical observations. It could therefore be useful to instaurate a therapeutic drug monitoring based on plasma measurement to allow better assessment of the state of the transplant, such as rejection phenomenon and toxicity signs. However, only a limited number of analytical methods have been developed to measure the levels of 6-mercaptopurine (6MP) in plasma and urine. Radioisotope (Elion et af., 1970), fluorescence (Finkel, 1967) and spectrophotometric (Chalmers, 1975) methods have been used. They are, however, insufficiently sensitive and specific for our purpose. Gas chromatography-mass spectrometry (Rosenfeld et a/., 1977) has been recently used but it involves derivatization of 6MP prior to analysis. Some authors have described high performance liquid chromatographic (reversed phase HPLC) analysis of thiopurines (Nelson et a/., 1973; Breter and Zahn, 1977; Abreu et a/., 1982; Narang et af., 1982). In the present work, we introduce a simple reversed phase HPLC method for the determination of 6MP and possibly its metabolites. It permits the routine adjustment of AZA dose according to different physiological and pathological conditions linked to each patient and to limit the side effects.

Chromatography. A Shimadzu high performance liquid chromatograph with a n UV spectrophotometric detector (Model SPD-6A) and a chromatography pump (Model 6000A) (Waters, Milford, MA, USA) were used. The column was 5 (LM Lichrospher 100 RP18, 25 cm x 4.6 mm (Merck, Darmstadt, West Germany) and was mounted with a C,* pre-column (Guard-pak, p-Bondapack, Waters, Milford, MA, USA). Chromatographic analysis was carried out at room temperature. The mobile phase was delivered at a flow-rate of 1.0 mL/min. This gave a back pressure in the region at 70 bar (1000 psi).

AZA, azathioprine; DTE, dithioerythritol; DTT, dithiothreitol; 8OHMP, 8-hydroxymercaptopurine; 6MP, 6-mercaptopurine; 9MMP, 9-methylrnercaptopurine; 6TG, 6-thioguanine. *Author to whom correspondence should be addressed at Laboratoire de Toxicocinktique et Pharmacocinitique, FacultC de Pharmacie, Marseille 27 Boulevard Jean Moulin, 1338 Marseille Cedex 5 France. f Laboratoire de Pharrnacologie Clinique, H8pital des Sablons, Grenoble. France.

Materials. 6-Thioguanine (6TG) (internal standard) was obtained from Sigma Chemical Company (St. Louis, MO, USA) and 6MP from Wellcome Laboratory, Dartford, Kent, UK; dithiothreitol (DTT) and 1,4-dithioerythritol (DTE) were purchased from Boehringer Mannheim ( I N , USA). The other metabolites (8-hydroxymercaptopurine (8-OHMP) and 6thiouric acid) of AZA are unfortunately not available in Europe. Dichloromethane (Uvasol) (Merck, Darmstadt, West Germany), acetonitrile hipersolv (BDH Ltd., Poole, Dorset, UK), glacial acetic acid (Carlo Erba Famitalia, Milan, Italy) and HPLC water (Merck, Darmstadt) were purchased and used without further purification. Preparation of internal standard solution. 6MP and 6TG were dissolved in 2 mL of NaOH solution (0.4 M ) with 98 m L of water to give a concentration of 10 mg/100 mL and stored in the dark at -20 "C. The working solutions were daily prepared and achieved by diluting stock solution with HPLC water to 1/40 and 1/100 for 6TG and 6MP respectively. DTT solutions (0.038 M and 1 M ) were prepared every day. Preparation of mobile phase. Acetonitrile (14 mL), DTE (60mg), glacial acetic acid (1.4mL) and HPLC water (984.6 mL) were used as the optimal mobile phase. The solvent was filtered and degassed prior to use. Extraction procedure. The following were placed in a test tube: 20 p L o f 6TG (internal standard), 10 p L of DTT (1 M ) , 500 pL of plasma and 2 mL of acetonitrile. The sample was vortex-

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6 M P A N D METABOLITE D E T E R M I N A T I O N I N PLASMA BY HPLC

mixed for 30 s. Following a 10 rnin centrifugation, the organic phase was transferred to another test tube which contained 3.SmL of dichloromethane. This sample was vortexed for 15 rnin and centrifuged for 10 min at 5 "C. The supernatant was recovered and evaporated to dryness under nitrogen at 40 "C. The residue was reconstituted with 200pL of the mobile phase and vortexed. SO pL of this solution was injected into the HPLC apparatus. The reconstituted solution may be stored at 4 "C before analysis.

h

c

n

Calibration and data analysis. A known amount of working solution of 6MP and 10 pL of 0.038 M D T I were incorporated into the control plasma samples. The peak-height ratios (R) of 6MP and the internal standard were used to establish the calibration curve by the analytical function: R = f (concentration of 6MP). The regression line was determined by its slope and its intercept overlapped the origin. Statistical analysis was performed using the least-squares method to fit regression lines, and Student's t-test to define confidence interval and thus hyperbole curves. The repeatability and the reproducibility were studied with the Hartley- and F-test. Selectivity. The direct verification of the absence of metabolite interference or any other endogenous substances was assured by using blood samples which were obtained during AZA pharmacokinetic study on renal transplant patients. The patients have received 2 mg/ kg intravenous and oral dose of AZA. Blood was sampled respectively at 0, 5 , 10, IS, 30, 60, 90, 120, 150, 180, 240, 300, 360 min (Intra-venous study) and 0, 0.5, 1, 1.5, 2, 3, 4, 5 , 6 h (Oral study). The samples were immediately centrifuged. A pretreatment with DTT (0.038 M ) (10 pL/mL of plasma) was done each time and the samples were kept at -20 "C until extraction. Extraction recovery experiment. Plasma samples were spiked with 6MP to give a final concentration of 50 ng/nL and subjected to the previously described extraction procedure. Following analysis, peak heights from extracted samples were compared with those obtained from injections of diluted standard solutions and the percentage of recovery was calculated. The recovery of extraction averaged 85% and 65% for 6MP and 6TG respectively (Table 1 ) .

RESULTS AND DISCUSSION Baily et al. (1975) have used DTE as a sulfhydrylprotecting reagent in their extraction procedure for gas chromatographic ( G C ) analysis of 6MP. Ding and Benet (1979) have shown a n improvement of the peak heights of 6MP a n d 9 M M P following DTE addition to the final extraction step a n d in the HPLC solvent system for 6MP analysis. DTE may have a stabilizing effect on the unsubstituted thiols. Likewise, the addition of DTE would convert AZA to 6 M P if the unchanged d r u g was present in the plasma sample. For these reasons we have used DTT a n d DTE as described above.

w

c

n

Figure 1.

Chromatograms of blank serum, standard serum and

patient serum.

Selectivity and resolution (Rs) Figure 1 shows the results of the analysis of 6MP and 6TG from a blank serum, a standard serum a n d a n AZA treated patient serum. The chromatogram from a patient serum shows no apparent interfering peak a n d the separation is completely satisfactory. Complete peak resolution was achieved in 12 rnin for 6TG peak, but in 22 rnin for DTE peak from the standard solution a n d from the patient serum sample. T h e 6 M P peak resolved completely from the metabolite peaks as well a s from other endogenous peaks in the serum (Table 2 ) . W e suggest 8 - O H M P (peak 11) and 6-thiouric acid (peak I ) as two of the unknown peaks of metabolites. Therefore, the simultaneous separation of the 6 M P and of its metabolites has been possible under these conditions. The resolutions (Rs) of different substances proved t o b e unproblematic and high (Rs> 1 ) with acetonitrile water (1.4 :98.6, v/v) as mobile phase.

+

Table 2. Retention times and resolution Table 1. Extraction recovery experiment

6MP 6TG

0 John

Number of samples

MeaniSD

Coefficient of variation

6 6

81 8 7 k 1 0 5 2 65.52 f 3.94

12.9% 6%

Wiley & Sons Limited, 1990

Retention

6TG

6MP

Peak I

Peak II

DTE

11.695

10.194

7.512

9.090

21.164

time (min)

Resolution (Rs)

1.36

1.003

1.578

BIOMEDICAL CHROMATOGRAPHY, VOL 4, NO 4, 1990 145

2. S A H N O U N ET AL. 1

Table 3. Repeatability Conc

Mean

rSD

Coefficient of variation

50 ng mL l00ngmL

0.979 1.940

0.029 0069

2.97% 3.56%

Detection limit

This was determined as described by Knoll (1985). The quantity was estimated from the height of the largest noise fluctuation measured in a preselected chart interval after injection of the blanks and was 0.4 ng corresponding to 3.2 ng/mL for biological detection limit.

Figure 3. Hyperbole curves and confidence interval

NG/ML

Linearity

loooo

E 4 6-TH.Acid

The peak-height ratios of 6MP to the internal standard were related to 6MP concentration and found linear within the range 0-1000 ng/mL (Fig. 2). The correlation coelficient of regression, the intercept and slope for the standard curves were 0.9999,0.01731 and 0.021 18 respectively. Residual error was low (0.00041). The hyperbole curves were almost disconcerted with the line of regression (Fig. 3).

Accuracy and precision 0.1

The repeatability of the method was evaluated for two concentration values (50 ng/mL and 100 ng/mL) by measuring plasma concentration several times and on the same day. The results are summarized in Table3. The reproducibility was also evaluated for the same concentration values. The measure of concentration was repeated on several days (Jl, 52,33,520). Table 4 shows the interday coefficients of variation and the accuracy of the assay. Plasma samples from a renal transplant patient receiving intravenous and oral doses of azathioprine were analyzed by the above method. It gave

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i

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0

2

1

- B-MP(IV)

3

4

B-MP(P0)

5

6

7

HOURS

Figure 4. Plasma levels of 6MP and 6-thiouric acid (terminal metabolite of 6MP) in a renal transplant patient.

satisfactory results when used in pharmacokinetic studies. Typical plasma levels of 6 M P and 6-thiouric acid (terminal metabolite of 6MP) following administration of 2 mg/kg dose of AZA are shown in Fig. 4. In summary, this method was sensitive and effective with a low detection limit (0.4ng). The coefficient of variation for reproducibility ranged from 3.78% to 6.19%. The method was useful for analysis of specimens, obtained during clinical use and for the determination of the individual AZA dose and pharmacokinetic parameters of 6 M P and possibly its metabolites. It is therefore useful for routine laboratory use in therapeutic drug monitoring in order to improve the transplant state.

+ I 50

*I

Table 4. Precision and accuracy

00

rw 58

Theoretical ratio (c)

Conc

a

50 ng mL (n = 10 x4) 100 ng mL ( n = 10 x4)

+--I

1 0 88

*0 20

* 0 40

* 0 68

*W 88

E r3

Figure 2. Calibration curve of 6MP

146 BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 4, 1990

a

A=lOO-

1

2

Measured

Precision coefficient

Accuracy

ratio ( r n )

variation

A=

6.19%

100.7%

3.78%

100.75%

1.007i0.062 2.0075*0.076

loo( c - r n ) C

0John

Wiley & Sons Limited, 1990

6 M P A N D M E T A B O L I T E D E T E R M I N A T I O N I N PLASMA BY HPLC

REFERENCES Abreu, R. A,, Van Baal, J. M., Schouten, T. J., Schretlen, E. D. A. M. and de Bruyn, H. M. M. (1982). J. Chromatogr. 227, 526. Baily, D.G.. Wilson, T. W. and Johnson, G. E. (1975). J. Chromatogr. 111, 305. Breter, H. J. and Zahn. R. K. (1977). J. Chromatogr. 137, 61. Chalrners, A. H. (1975). Biochem. Med. 12, 234. Ding, T. L. and Benet, L. 2. (1979). J. Chromatogr. 163, 281. Elton, G. B., Benezera. F. M., Carrington, L. 0 . and Strelitz, R. A. (1970). Fed. Amer. SOC.Exp. Biol. 29, 2027. Finkel, J. M. (1967). Anal. Biochem. 21, 362.

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Knoll, J. E. (1985). J. Chromatogr Sci. 23, 422. Nelson, D. J., Bugge, C. J. L., Krasny, H. C. and Zernrnerrnan, T. P. (1973). J. Chromatogr. 77, 181. Narang, P. K., Yeager, R . L. and Chatterji. D.C. (1982). J. Chromatogr. 230. 373. Rosenfeld, J. M., Taguchi, V. Y., Hillcoat. B. L. and Kawall, M. (1977). Anal. Chem. 49, 725. Received 5 October 1989; accepted (revised) 3 January 1990

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