Validation of a column-switching high-performance liquid chromatographic (HPLC) method for determination of ML-1035 and its five metabolites in plasma.
A fully automated column-switching HPLC procedure has been developed and validated for quantitation of ML-1035 and its five metabolites in plasma employing direct injection. Plasma samples were injected onto a CN extraction column (4 x 4.6 mm, 5 microns) for micellar cleanup with 0.5% sodium dodecyl sulfate (SDS) in 50 mM phosphate. The proteinaceous components were solubilized and flushed out. The extracted compounds were then eluted by forward flush onto a C8 analytical column (150 x 4.6 mm, 5 microns) for further analysis using fluorescence detection (excitation, 308 nm; emission, 350 nm). After the subsequent washing and reequilibration with a sequence of three solvent mixtures, the extraction column was ready for the next injection. The limit of quantitation for all compounds of interest was about 10 to 15 ng/ml using 100 microliters of plasma. Excellent precision, accuracy, and linearity were obtained for all compounds over a range of 10 to 1500 ng/ml. The practicality of the HPLC method was also validated with plasma samples from dogs receiving ML-1035. Longevity for both extraction and analytical columns is excellent. Micellar cleanup coupled with the column-switching technique is a promising HPLC procedure when using direct injection of biological fluids.
Kuo
B S
BS
Drug Metabolism Section, Marion Merrell Dow Research Inc., Kansas City, Missouri 64134.
Poole
J C
JC
Hwang
K K
KK
D001331:Automation; D001549:Benzamides / Q000097:blood*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D008787:Metoclopramide / Q000031:analogs & derivatives*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001549:Benzamides; C071737:ML 1035; D008787:Metoclopramide