High-performance liquid chromatographic assay for the measurement of azathioprine in human serum samples

A quantitative, highly sensitive HPLC-based method for the direct measurement of azathioprine is described, introducing a newly synthesized 9-methyl derivative of this immunosuppressant as internal standard in combination with isocratic HPLC and UV-absorbance measurement at 285 nm. Analysis was performed on a RP18 select B column with acetonitrile-0.01 M potassium phosphate buffer (12:88, v/v) at pH 2.3 as mobile phase. Results of precision analysis from serum samples spiked with 3.125, 12.5, and 25 ng azathioprine, respectively, were (mean +/- S.D.): 3.148 +/- 0.259 ng (8.22%), 12.594 +/- 0.571 ng (4.53%), and 25.016 +/- 0.658 ng (2.63%) with C.V. values in parentheses for n = 5. The accuracy of the assay ranged from -7.6 to 0.7% (expressed as % bias) tested on five consecutive days. The limit of quantification was at 2.5 +/- 0.256 ng (C.V. 10.25%), thus allowing drug monitoring in long-term patients. The method can also be used to evaluate individual pharmacokinetic parameters of a single patient, as well as for drug monitoring of a cohort of patients who suffer from azathioprine-induced symptoms of toxicity. An example of the pharmacokinetic behaviour in an individual is given in this paper.