High-performance liquid chromatographic assay for hydroxychloroquine and metabolites in blood and plasma, using a stationary phase of poly(styrene divinylbenzene) and a mobile phase at pH 11, with fluorimetric detection.

A sensitive rapid high-performance liquid chromatographic assay for hydroxychloroquine and three major metabolites, desethylhydroxychloroquine, desethylchloroquine and bisdesethylchloroquine, has been developed. An ion-suppression separation technique is used with a macroporous polymer, poly(styrene divinylbenzene), stationary phase and a mobile phase of methanol--water with triethylamine, pH 11. A single ether extraction from alkalinized plasma or whole blood, with chloroquine added as an internal standard, is used. Using fluorescence detection, with excitation at 337 nm, the limit of sensitivity is 1 ng/ml. Some anti-inflammatory drugs which may be used concurrently for rheumatoid disorders are shown not to interfere with the assay. Samples of whole blood and plasma obtained from rheumatoid patients contained hydroxychloroquine and all three metabolites.