An improved HPLC method for the quantitation of 6-mercaptopurine and its metabolites in red blood cells

Abstract

A procedure is described for the rapid determination of the intraerythrocyte concentration of 6-mercaptopurine (6-MP) and its metabolites, 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP). Erythrocytes (8 x 10 8 cells) in 350 µl Hanks solution containing 7.5 mg dithiothreitol were treated with 50 µl 70% perchloric acid. The precipitate was removed by centrifugation (13,000 g) and the supernatant hydrolyzed at 100°C for 45 min. After cooling, 100 µl was analyzed directly by HPLC using a Radialpack Resolve C18 column eluted with methanol-water (7.5:92.5, v/v) containing 100 mM triethylamine. 6-TG, 6-MP and the hydrolysis product of 6-MMP, 4-amino-5-(methylthio)carbonyl imidazole, were monitored at 342, 322 and 303 nm using a Shimadzu SPD-M10A diode array UV detector. The analytes eluted at 5.3, 6.0 and 10.2 min, respectively. The calibration curves were linear (r 2 > 0.998), and the analytical recoveries were 73.2% for 6-TG, 119.1% for 6-MP and 97.4% for 6-MMP. The intra-and inter-assay variations were highest for 6-MP (9

HPLC Determination of the Levels of 6-Mercaptopurine Metabolites Suitable for the Clinical Risk Assessment of its Toxicity among Egyptian Children with Acute Lymphocytic Leukemia

“…The mean extraction efficiency was 87.8% for 6-MP, 90.9% for 6-TG and 92.1% for 6-MMP. Such recoveries are higher than that obtained by previous methods [1,[12][13][14][15][16][17], due to applying some modifications to the extraction process as advised by Hawwa et al [11]. Recovery seemed to increase with increasing drug concentrations, and performing a oneway ANOVA test revealed a statistically significant difference (p<0.05) between recovery % of low and high concentrations of the 3 analytes, however with no significant difference between the low/intermediate/ high concentrations.…”

“…Nevertheless, further experiments need to be carried out to exactly determine the causative factor. It is expected that such differences do not affect method performance as even lower recoveries are acceptable and mean values are better than other reported methods [1,[12][13][14][15][16][17].…”

“…The developed method offers the shortest retention times for analytes elution, compared to all methods measuring the drug and its metabolites simultaneously [1,11,15,16], with all peaks of interest eluting within 8 minutes. On top of that, the use of a derivatized (capped with polar groups) C18 column is inevitable if isocratic elution mode was to be performed for the separation of these analytes, as shown in previous methods [1,11,12,14,15]. This is probably explained as thiopurine compounds are highly polar and tend to co-elute with the use of a conventional C18 column.…”

“…LC-MS/MS and HPLC-UV are the two chromatographic techniques reported to measure 6-MP and its metabolites levels in blood [1,[11][12][13][14][15][16][17][18][19][20][21][22]. Each of the reported methods had its advantages and drawbacks, many of which were overcome by the present method, which measures 6-MP, 6-TG and 6-MMP by HPLC-UV in one sample using a single wavelength and a conventional C18 column.…”

Optimization and validation of an RP-HPLC method for the estimation of 6-mercaptopurine in bulk and pharmaceutical formulations

“…Since 6-MP is widely used in chemotherapy, it is important to develop and validate analytical methods for its determination in pharmaceutical dosage forms. Review of literature has revealed that all methods reported for the estimation of 6-MP are useful in estimating 6-MP in biological samples (Sorouraddin et al, 2011;Hawwa et al, 2009;Umrethia et al, 2006;Oliveira et al, 2004;Su et al, 1999;Mawatari et al, 1998;Sahnoun et al, 1990;Erdmann, Chan, Canafax, 1988;Rudy et al, 1988;Migulla, Prumke, Huller, 1987). So far no systematic HPLC method has been reported for determination of 6-MP in pharmaceutical formulations.…”