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Background: Polymorphic thiopurine S-methyltransferase (TPMT) is a major determinant of thiopurine toxicity., Methods: We extracted 6-thioguanine nucleotides (6-TGNs) and 6-methylmercaptopurine nucleotides (6-MMPNs) from erythrocytes with perchloric acid and converted them to 6-thioguanine (6-TG) and a 6-methylmercaptopurine (6-MMP) derivative during a 60-min acid hydrolysis step. The liquid chromatography system consisted of a C(18) column with an ammonium acetate-formic acid-acetonitrile buffer. 8-Bromoadenine was the internal standard. Analytes were measured with positive ionization and multiple reaction monitoring mode. With PCR-restriction fragment length polymorphism analysis and TaqMan allelic discrimination, common TPMT alleles (*1, *2, *3A, *3B, *3C) were determined in 31 792 individuals. We used perchloric acid extraction, acid hydrolysis, and HPLC with ultraviolet detection to measure erythrocyte 6-TG and 6-MMP nucleotide concentrations in 6189 patients with inflammatory bowel disease receiving azathioprine/6-mercaptopurine therapy., Results: Intra- and interday imprecision were <10% at low and high analyte concentrations. The conversion of 6-TG and 6-MMP nucleoside mono-, di-, and triphosphates was complete after hydrolysis. Allelic frequency for TPMT variant alleles ranged from 0.0063% (*3B) to 3.61% (*3A). Compared with wild types, TPMT heterozygotes had an 8.3-fold higher risk for 6-TGNs >450 pmol/8 x 10(8) erythrocytes (concentration associated with increased risk for leukopenia), but an 8.2-fold lower risk for 6-MMPNs >5700 pmol/8 x 10(8) erythrocytes (concentration associated with increased risk for hepatotoxicity)., Conclusions: The liquid chromatography-tandem mass spectrometry method can be applied to the routine monitoring of thiopurine therapy. The association between TPMT genotype and metabolite concentrations illustrates the utility of pharmacogenetics in the management of patients undergoing treatment with thiopurines.

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.

6-thioguanine (6-TGN) and methyl 6-mercaptopurine nucleotides (Me6-MPNs) are the two major metabolites found in erythrocytes after administration of azathioprine. In an attempt to understand the role of these metabolites in the pharmacologic and toxic activity of thiopurines, we have developed a HPLC method for the simultaneous determination of 6-TGNs and Me6-MPNs in erythrocytes. A simple and rapid treatment procedure based on deproteinization by perchloric acid with dithiothreitol is described. The nucleotides were hydrolyzed to their own bases by heating the sample for 45 min at 100 degrees C. During acid hydrolysis Me6-MP was converted into a compound analyzed on a Purospher RP18-e column with 0.02 mol/L dihydrogenophosphate buffer-methanol as eluents. With this procedure, mean recoveries of 73.1% and 84.0% for 6-TGN and Me6-MPN derivatives, respectively, were found.

We developed an isocratic, selective, and very sensitive HPLC method for the determination of ketamine and its two main metabolites in plasma. The compounds were extracted from plasma by a liquid-liquid extraction with a dichloromethane:ethyl acetate mixture followed by an acidic back-extraction. Separation was achieved on a new stationary phase, Purospher RP-18 endcapped, with a mobile phase containing acetonitrile:0.03 mol/L phosphate buffer (23:77 by vol) adjusted to pH 7.2. Because of the high column efficiency and the significant improvement of peak symmetry, the quantification limit could be down to 5 micrograms/L for ketamine and norketamine (NK). The intraday and interday CVs ranged from 1.7% to 5.8% and 3.1% to 10.2% for all compounds respectively. The method is sensitive enough for monitoring ketamine, NK, and dehydroketamine in plasma during pharmacokinetic studies after an intravenous bolus of a low dose of ketamine.

Using HPLC methods, we measured the concentrations of nucleosides and nucleotides for a patient with no purine nucleoside phosphorylase (PNP; EC 2.4.2.1) enzymatic activity. Concentrations of inosine and guanosine were abnormally high in urine and plasma, whereas guanosine diphosphate (GDP) and guanosine triphosphate (GTP) concentrations in erythrocytes were depleted. The unusual presence of deoxyribonucleosides (deoxyinosine and deoxyguanosine) and deoxyribonucleotides (dGDP and dGTP) was also notable. Thus, HPLC represents an accurate and useful tool for the study of purine metabolic disorders.

In this rapid, specific, and sensitive high-performance liquid-chromatographic method of analysis for homogentisic acid in biological fluids, homogentisic acid is separated on a column of Nucleosil CN. This method, which we applied to the diagnosis of three cases of alcaptonuria, represents a suitable analytical tool for the diagnosis of alcaptonuria.

In this anion-exchange "high-performance" liquid-chromatographic method of analysis for purine nucleotides, the nucleotides are separated with high efficiency and selectivity on a weak anion exchanger (Hypersil APS 2, 3-micron particle size) by elution with a gradient of eluent pH and concentration. Applying this method to analysis for these compounds in human blood cells, we determined them in a patient with adenosine deaminase deficiency who was treated with a bone-marrow transplantation, finding that the transplantation did not entirely correct the patient's abnormalities of purine metabolism.