Журнал аналитических и биоаналитических методов

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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

Eman Abdelsayed M, Sahar Maksoud A, Iman Sidhom, Mohamed Gad Z and Rasha Hanafi S

The need of a robust, sensitive HPLC method for the quantitation of 6-thioguaninenucleotides (6-TG) and 6-methylmercaptopurine (6-MMP) is indispensable to relate levels of these metabolites with emergence of signs of toxicity in patients undergoing treatment with 6-mercaptopurine (6-MP), paving the road to accurate dose calculations and thus providing a cost-effective treatment approach. Previously reported methods were either laborious, required special types of C18 columns, or had long run times. A Design of Experiments (DoE) approach targeting the shortest run time with greatest selectivity was adopted using a user friendly HPLC method development simulation software (DryLab®). Analytes eluted within 10 min, at 3.8, 4.2, 5.6 and 7.5 min for 6-TG, 6-MP, 6-MMP and Dithiothreitol (DTT) respectively. Excellent recovery percentages of 90.9 ± 14.4, 87.8 ± 6.7 and 92.1 ± 9.08, respectively were obtained. The method proved its validity and robustness according to the International Conference on Harmonization (ICH) guidelines. The LOD of 6-MP, 6-TG and 6-MMP were 6, 9 and 24 pmol/8 × 108 RBCs, respectively. Twenty-Two Acute Lymphocytic Leukemia (ALL) children recruited from 57357 Cancer Hospital (Cairo, Egypt) had their 6-MP metabolites measured using the developed method. A strong negative correlation was manifested between TG and RBCs count and hemoglobin (p=0.009 and 0.002 respectively). WBC and neutrophils showed a negative correlation to TG at Continuation 1 phase of treatment, confirming the association of TG with myelotoxicity. The significant correlation between MMP and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (p=0.030, 0.004) explained its potential hepatotoxicity.