Simple and specific reversed-phase liquid chromatographic method with diode-array detection for simultaneous...

This paper describes a simple, specific, and sensitive high-performance liquid chromatographic (HPLC) method using ion-pair reversed-phase chromatography with photodiode-array detection for the simultaneous determination of hydroxychloroquine (HCQ) and chloroquine (CQ) in serum samples from rheumatoid arthritis patients receiving either HCQ sulphate or CQ diphosphate. The assay is also applicable to the simultaneous determination of corticosteroids. The method consisted of two diethyl ether extractions of 1.0 ml of serum, to which two internal standards (2,3-diaminonaphthalene and 18-hydroxy-11-deoxycorticosterone) and 1.0 ml of 0.25 M sodium hydroxide had been added. After the organic phase was evaporated to dryness at 30-40 degrees C under a stream of nitrogen, the extract was reconstituted with a 1:1 mixture of 0.1 M perchloric acid and methanol, an aliquot of which was injected on to the system. Peak-height ratios at different wavelengths (A245/343, A245/256, A245/265 and A245/275) were utilized as a method of assessing peak homogeneity. Some anti-inflammatory drugs which may be used for rheumatic disorders were shown not to interfere with the assay. The method provides selectivity by using diode-array detection at several wavelengths. The use of two internal standards not only compensates for losses during the sample manipulation but also prevents erroneous results in case of interference.