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,Iman Sidhom,Rasha Hanafi S,Mohamed Gad Z,Sahar Maksoud A

doi:10.4172/2155-9872.1000358

Eman Abdelsayed M, Iman Sidhom + Show 3 more

Open Access

https://doi.org/10.4172/2155-9872.1000358

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Abstract

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.

Highlights

Acute Lymphocytic Leukemia (ALL) is a widespread disease, mostly affecting childhood
The method has a short run time as well as utilizes a cheap, readily available, conventional C18 column, and implements an already used reagent in the procedure as an internal standard
High MMP levels correlated with hepatotoxicity, while high TGN levels correlated with myelosuppression

Summary

Introduction

Acute Lymphocytic Leukemia (ALL) is a widespread disease, mostly affecting childhood. 6-Mercaptopurine (6-MP) is an analog of hypoxanthine nucleoside. Treatment is divided into 3 major phases: induction, consolidation and maintenance, each having its own regimens. It is an antimetabolite anticancer drug used in the treatment of ALL in consolidation and remission phases. The drug belongs to the thiopurines family of anticancer drugs, which includes thioguanine (TG) and azathioprine (AZA). These drugs are used for the treatment of inflammatory bowel disease (IBD) and as immune-suppressants in organ transplants, severe rheumatoid arthritis, and autoimmune hepatitis, since more than 5 decades [1]. Measuring plasma levels of 6-MP is of no clinical value, since only 16% of the drug remains in the systemic circulation, with the rest being extensively metabolized [2]

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