Clinical Chemistry 44: 2511-2515, 1998;
(Clinical Chemistry. 1998;44:2511-2515.)
© 1998 American Association for Clinical Chemistry, Inc.
|
Drug Monitoring and Toxicology |
Identification of 6-methylmercaptopurine derivative formed during acid hydrolysis of thiopurine nucleotides in erythrocytes, using liquid chromatography–mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance assay
Thierry Dervieux and
Roselyne Boulieua
a Address correspondence to this author at: Université Claude Bernard Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques, Département de Pharmacie Clinique, de Phamacocinétique et d'Evaluation du Médicament, 8 avenue Rockefeller, 69373 Lyon Cedex 08, France. Fax 04 78 77 71 58.
 |
Abstract
|
|---|
6-Thioguanine and 6-methylmercaptopurine (Me6-MP) nucleotides are the
two major thiopurine metabolites of azathioprine found in erythrocytes.
During the acid hydrolysis required for the conversion of thiopurine
nucleotides into their free bases, Me6-MP was converted into a compound
that could be analyzed on a Purospher RP18-e column with dihydrogen
phosphate-methanol buffer as eluent. The pH of the acid extract
strongly influenced the conversion of Me6-MP into its derivative. The
Me6-MP derivative was identified using liquid chromatography–mass
spectrometry and infrared and nuclear magnetic resonance spectrometric
methods. During the acid hydrolysis of thiopurine nucleotides in
erythrocytes, Me6-MP undergoes degradation, leading to
4-amino-5-(methylthio)carbonyl imidazole.
|
Introduction
|
|---|
Azathioprine is a thiopurine drug coadministered with cyclosporine
and corticosteroids to prevent allograft rejection after
transplantation (1). The immunosuppressive properties are
related to intracellular conversion into thiopurine nucleotides via
hypoxanthine guanine phosphoribosyl transferase, leading to
6-thioguanine nucleotides
(6-TGNs),1
and methylation of 6-thioinosine monophosphate via thiopurine
methyltransferase, leading to methyl 6-methylmercaptopurine (Me6-MP)
nucleotides (2). However, the relationship between
thiopurine nucleotides and the immunosuppressive and/or
myelosuppressive effect of azathioprine remains unclear in vivo. Some
studies have implicated high 6-TGN concentrations in erythrocytes in
the myelosuppressive effect of azathioprine when the homozygote allele
for thiopurine methyl transferase deficiency is expressed
(3); another study, however, did not found any relationship
between 6-TGNs in erythrocytes and myelosuppression induced by
azathioprine (4).
Few HPLC methods have been developed for the simultaneous determination
of nonmethylated and methylated thiopurine nucleotides of azathioprine
in erythrocytes (5)(6)(7). In those methods, the bases were
liberated from the nucleotide moiety by acid hydrolysis in sulfuric
(5)(6) or perchloric acid (7).
In the method described by Lennard and Singleton (5) Me6-MP
liberated from the nucleotide moiety was converted into a compound that
could be extracted by phenyl mercury acetate adduct formation like
6-thiopurines. The Me6-MP derivative formed was extracted with
recoveries <40%.
We previously reported (7) a reversed-phase HPLC method for
the analysis of 6-TGNs and Me6-MP nucleotides in erythrocytes. The
sample treatment procedure based on deproteinization by perchloric acid
is simple and exhibits mean analytical recoveries of 84%. Here we
propose to investigate the structure of the Me6-MP derivative formed
during the acid hydrolysis step using liquid chromatography–mass
spectrometry (LC–MS) and infrared (IR) and nuclear magnetic resonance
(NMR) spectrometric methods.
|
Materials and Methods
|
|---|
reagents and stock solutions
Me6-MP, 6-methylmercaptoribonucleoside (Me6-MPR), and sodium
bicarbonate were obtained from Sigma Chemical Co. Methanol, potassium
dihydrogen phosphate, and perchloric acid were obtained from Merck. For
experiments, stocks solutions of Me6-MP and Me6-MPR were prepared in
0.1 mol/L hydrochloric acid and stored at -80 °C.
apparatus and chromatographic conditions for analysis of
Me6-MP AND THE Me6-MP DERIVATIVE
The liquid chromatograph consisted of a model 510 pump connected
to a model 960 photodiode array detector and a Wisp 715 solvent
delivery system, all from Waters.
Me6-MPR and Me6-MP and its derivative were analyzed using a
reversed-phase HPLC method described previously (7).
The separation was achieved on a Purospher RP18-e (Merck) with a linear
gradient elution mode using 0.02 mol/L KH2PO4,
pH 3.5, and 400 mL/L 0.01 mol/L KH2PO4, pH
3.5–600 mL/L methanol. The concentration of methanol rose from 0 to
200 mL/L over a period of 10 min. The flow rate was 1.2 mL/min, and the
detector was set at 291 nm.
influence of the pH OF THE ACID SUPERNATANT ONMe6-MP DERIVATIVE FORMATION
Lysed erythrocytes (500 µL) to which Me6-MPR was added were
rapidly transferred into a tube containing 5 mg of dithiothreitol and
deproteinized by addition of 50 µL of 700 mL/L perchloric acid. The
deproteinized sample was centrifuged at 3000g for 15
min at 4 °C. The supernatant was then removed, and the pH was
adjusted to 0–1.0 with 70 µL of 1 mol/L sodium bicarbonate, pH 13.
Aliquots (80 µL) taken after acidification but before the sample was
heated, and after acid hydrolysis at 100 °C for 45 min were
injected.
preparation of the Me6-MP DERIVATIVE
For IR and NMR experiments, the Me6-MP derivative was prepared
according to the following procedure: Me6-MP was dissolved in 1 mol/L
hydrochloric acid, and the solution was heated for 45 min at 100 °C.
After cooling, the solution was evaporated to dryness at 50 °C. The
dry residue was dissolved in water and injected into the HPLC column.
The conversion of Me6-MP into its derivative was confirmed through peak
retention times and spectral library matching by comparison of
unknown peaks to the reference spectrum of a synthesized standard.
lc–ms
LC–MS analysis was carried out using an HP 59980 B Particle Beam
coupled to a HP 5989 mass detector, both from Hewlett Packard.
The LC system consisted of a Purospher RP18-e, with 400 mL/L
methanol–600 mL/L water as eluent. The flow rate was set at 0.3
mL/min. The carrier gas was helium, and the desolvation room was heated
at 60 °C under 400 psi of helium.
The MS conditions were as follows: electron energy was 70 and 200 eV,
and the trap current was 300 and 200 µA for electron ionization and
chemical ionization, respectively.
1
h and c nmr spectroscopy
The NMR spectrometer consisted of a Brucker model AC 200 (400
MHz). The spectrum was run after addition of D20
and tetramethylsilane as reference signals. The operating frequencies
were at 200 and 50 MHz for 1
H and C NMR,
respectively.
ir spectroscopy
IR spectroscopy was carried out on a Nicolet 20 SXC. Scans were
run from 4000 to 600 cm-1 at a scan rate of 1
cm-1 per second. The spectrum was recorded at a resolution
of 4 cm-1.
|
Results and Discussion
|
|---|
formation of the Me6-MP DERIVATIVE
Because there are no standards for Me6-MP nucleotides, Me6-MPR was
used to evaluate the conversion into the Me6-MP derivative. The
percentage of conversion of Me6-MPR into Me6-MP or the Me6-MP
derivative during acid hydrolysis was determined by comparison of the
peak height of Me6-MPR before heating but after acidification with
those obtained after the hydrolysis step.
The conversion of Me6-MP into its derivative appears to be strongly
influenced by the pH of the acid extract (Fig. 1
). The formation of Me6-MP derivative was 100% under the acidic
conditions required for the conversion of thiopurine nucleotides into
their free bases.
The chromatographic separation of Me6-MP and the Me6-MP derivative, and
the location of Me6-MPR are shown in Fig. 2
. The ultraviolet spectra of Me6-MP and its derivative,
determined by the photodiode array detector with 1.2-nm resolution, are
shown in Fig. 3
.
View larger version (21K):
[in this window]
[in a new window]
|
Figure 2. Chromatographic separation of Me6-MP and the Me6-MP
derivative.
DTT, dithiothreitol; AU, absorbance units.
|
|
identification of the Me6-MP DERIVATIVE
LC–MS results.
The molecular weight of the derivative was
found to be 157 from the [M + H+] ion
(m/z 158) and adduct ions [M + 29] (m/z 186) in
chemical ionization mode and the M+ ion (m/z
157) observed in electron ionization mode (Fig. 4
). The mechanism of fragmentation is described Fig. 5
.
NMR results.
The 1
H NMR spectrum showed only two
peaks, at 2.3 ppm (3 H) and 8.20 ppm (1 H), which were assigned
respectively to the methyl group and the proton substituted on the
imidazole ring (Fig. 6
).
The C NMR spectrum showed five peaks: at 12.76,
111.13, 133.04, 150.63, and 184.21, which were assigned to
CH3, S
C
O, and C at positions 2, 4, and 5 of the
imidazole ring, respectively.
IR results.
The IR spectrum of the Me6-MP derivative is shown
in Fig. 7
. The spectrum indicated a thioester group (absorption band,
1662 cm-1). The vibration bands at 1552 and 1451
cm-1 can be attributed to the imidazole group, with a band
at 850 cm-1 corresponding to CH of the imidazole ring.
|
Conclusion
|
|---|
LC–MS analysis suggests that the Me6-MP derivative corresponds to
4-amino-5-(methylthio)carbonyl imidazole. This structure was fully
consistent with NMR and IR data. This proposition is in accordance with
the action of acid on the 6-methylthio derivatives of purines described
by Albert (8) in 1969.
Thus, our results show that Me6-MP liberated from the nucleoside in
erythrocytes during the hydrolysis step undergoes a degradation under
acidic conditions, leading to 4-amino-5-(methylthio)carbonyl imidazole.
This last compound is stable under the acidic conditions required for
the hydrolysis of thiopurine nucleotides into their free bases
(7).
Using the hydrolysis step described above, we have determined the
Me6-MP derivative in erythrocytes from organ transplant patients under
azathioprine therapy. In all erythrocyte samples analyzed, only the
Me6-MP derivative was recovered on the chromatograms, which confirms
the complete conversion of Me-6MP into its derivative.
4-Amino-5-(methylthio)carboxy imidazole can be analyzed easily on a
Purospher RP18-e column as reported previously (7), using a
simple sample treatment procedure with recoveries >84% .
|
Acknowledgments
|
|---|
We thank A. Alamercery, M. Desage, and J. Vatton for technical
assistance. We also thank J.J. Barieux, D. Page, and L. Legleut for
helpful discussions.
|
Footnotes
|
|---|
Département de Pharmacie Clinique, de Phamacocinétique et d'Evaluation du Médicament, Université Claude Bernard Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques, 8 avenue Rockefeller, 69373 Lyon Cedex 08, France, and Hôpital Neuro-Cardiologique, Pharmacie, 59 boulevard Pinel, 69394 Lyon Cedex 03, France.
1 Nonstandard abbreviations: 6-TGN, 6-thioguanine nucleotide; Me6-MP, 6-methylmercaptopurine; LC–MS, liquid chromatography–mass spectrometry; IR, infrared; NMR, nuclear magnetic resonance; and Me6-MPR, 6-methylmercaptoribonucleoside. 
|
References
|
|---|
-
Simmonds RL, Canafax DM, Fryd SF, Asher NL, Payne WD, Sutherland DR, Narajan JS. New immunosuppressive drug combination for mismatched related and cadaveric renal transplantation. Transplant Proc 1986;18:76-81.
[Medline]
[Order article via Infotrieve]
-
Lennard L. The clinical pharmacology of 6-mercaptopurine. Eur J Clin Pharmacol 1992;43:329-339.
[ISI][Medline]
[Order article via Infotrieve]
-
Anstey A, Lennard L, Mayou SC, Kirby JD. Pancytopenia related to azathioprine–an enzyme deficiency caused by a common genetic polymorphism: a review. J R Soc Med 1991;85:752-776.
[ISI][Medline]
[Order article via Infotrieve]
-
Boulieu R, Lenoir A, Mornex JF, Bertocchi M. Intracellular thiopurine nucleotides and azathioprine myelotoxicity in organ transplant patients. Br J Clin Pharmacol 1997;43:116-118.
[Medline]
[Order article via Infotrieve]
-
Lennard L, Singleton H. High-performance liquid chromatographic assay of the methyl and nucleotide metabolites of 6-mercaptopurine: quantification of red blood cell 6-thioguanine nucleotide, 6-thioinosinic acid and methylmercaptopurine metabolites in a single sample. J Chromatogr 1992;58:383-390.
-
Erdman G, France L, Bostrom B, Canafax M. A reversed phase high performance liquid chromatographic approach in determining total red blood cell concentrations of 6-thioguanine, 6-mercaptopurine, methylthioguanine and methylmercaptopurine nucleotides in a patient receiving thiopurine therapy. Biomed Chromatogr 1990;4:47-51.
[ISI][Medline]
[Order article via Infotrieve]
-
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;44:551-555.
[Abstract/Free Full Text]
-
Albert A. 1,2,3,4,6-Penta-azaindens (8-azapurines). Part VII. Degradation by acid of the 6-methyl-thio-derivatives of 8-azapurines and purines to thiol esters such as 4-amino-5-(methyl thio) carbonyl-1,2,3-triazole and the corresponding imidazole. J Chem Soc 1969;6:2379-2385.
The following articles in journals at HighWire Press have cited this article:
|
|
|
|
 
T. Dervieux, G. Meyer, R. Barham, M. Matsutani, M. Barry, R. Boulieu, B. Neri, and E. Seidman
Liquid Chromatography-Tandem Mass Spectrometry Analysis of Erythrocyte Thiopurine Nucleotides and Effect of Thiopurine Methyltransferase Gene Variants on These Metabolites in Patients Receiving Azathioprine/6-Mercaptopurine Therapy
Clin. Chem.,
November 1, 2005;
51(11):
2074 - 2084.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Dervieux, Y. Chu, Y. Su, C.-H. Pui, W. E. Evans, and M. V. Relling
HPLC Determination of Thiopurine Nucleosides and Nucleotides in Vivo in Lymphoblasts following Mercaptopurine Therapy
Clin. Chem.,
January 1, 2002;
48(1):
61 - 68.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
) into its derivative (
) during the hydrolysis step.
