Determination of Ro 23-7637 in dog plasma by multidimensional ion-exchange-reversed-phase high-performance liquid chromatography with ultraviolet detection.
Ro 23-7637 (I) is a new drug under development for the treatment of metabolic diseases. A high-performance liquid chromatographic-ultraviolet detection (HPLC-UV) analytical procedure for its analysis in dog plasma was developed and is reported here. Following C18 solid-phase extraction, the sample is applied to a strong cation-exchange column in the first dimension. The analyte and internal standard, Ro 24-4558 (II), are transferred to a base-deactivated C18 reversed-phase column in the second dimension (orthogonal separation mechanism), with UV detection at 254 nm. The reversed-phase solid-phase extraction provides a gross isolation of the drug, based on hydrophobicity. The first-dimension ion-exchange separation allows neutral species and anions to elute with the column void volume, while separating basic species according to pKa. The second dimension provides a high-resolution separation dependent upon the hydrophobicity of the sample species. The rationale for using orthogonal multidimensional chromatography was that an exhaustive examination of reversed-phase and normal-phase columns invariably resulted in co-elution of I with endogenous plasma components, which limited the sensitivity of the method. We have utilized C18 solid-phase extraction, followed by multidimensional HPLC-UV, to develop an accurate and precise analytical method whose limit of quantitation, 5 ng/ml using 0.5 ml of plasma, is determined by inherent detector sensitivity. Increased sensitivity can be readily achieved by using more sample or by using microbore HPLC on the second dimension.
Department of Drug Metabolism, Hoffmann-La Roche, Inc., Nutley, NJ 07110.
A J
AJ
Szuna
T E
TE
Mulligan
B A
BA
Mico
R W
RW
Blain
D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004285:Dogs; D010880:Piperidines / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D011725:Pyridines / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D012015:Reference Standards; D013056:Spectrophotometry, Ultraviolet
D010880:Piperidines; D011725:Pyridines; C083287:Ro 23-7637