Abstract
Azathioprine is the main thiopurine drug used in the treatment of immune-based inflammations of gastrointestinal tract. For the purpose of therapy control and optimization, effective and reliable analytical methods for a rapid drug monitoring in biological fluids are essential. Here, we developed a separation method based on the capillary electrophoresis (CE) hyphenated with tandem mass spectrometry (MS/MS) for the simultaneous determination of azathioprine and its selected metabolites (6-thioguanine, 6-mercaptopurine, and 6-methylmercaptopurine) as well as other co-medicated drugs (mesalazine, prednisone, and allopurinol). The optimized CE-MS/MS conditions provided a very efficient and stable system for the separation and sensitive detection of these drugs in human urine matrices. The developed method was successfully applied for the assay of the targeted drugs and their selected metabolites in urine samples collected from patients suffering from inflammatory bowel disease (IBD) and receiving azathioprine therapy. The developed CE-MS/MS method, due to its reliability, short analysis time, production of complex clinical profiles, and favorable performance parameters, evaluated according to FDA guidelines for bioanalytical method validation, is proposed for routine clinical laboratories to optimize thiopurine therapy, estimate enzymatic activity, and control patient compliance with medication and co-medication.
Highlights
Crohn’s disease (CD) along with ulcerative colitis belong to the group of inflammatory bowel diseases (IBDs) characterized by chronic inflammation of gastrointestinal tract (GIT) based on immune-mediated reaction
Ammonium acetate adjusted at pH 9 by 3% (v/v) ammonium hydroxide and including a 5% (v/v) methanol addition was used as an optimum background electrolyte (BGE)
A 10 mM ammonium acetate adjusted at pH 9 by 3% (v/v) ammonium hydroxide and including a 5% (v/v) methanol addition was used as an optimum background electrolyte (BGE)
Summary
Crohn’s disease (CD) along with ulcerative colitis belong to the group of inflammatory bowel diseases (IBDs) characterized by chronic inflammation of gastrointestinal tract (GIT) based on immune-mediated reaction. Even though there is currently no cure for Crohn’s disease, a wide range of treatment options is available that can help to control and reduce the symptoms and complications and achieve and maintain a remission [1]. Thiopurines, namely azathioprine (AZA) and its main active metabolites (6-mercaptopurine, 6-MP, and 6-thioguanine, 6-TG), represent the main group of therapeutics used for Crohn’s disease treatment. Their pharmacological effect is based on the cytotoxic and immune suppressive function through various active metabolites that are created during complex enzymatic reactions [2].
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