Kuo B S, Poole J C, Hwang K K
Drug Metabolism Section, Marion Merrell Dow Research Inc., Kansas City, Missouri 64134.
Pharm Res. 1992 Jan;9(1):119-25. doi: 10.1023/a:1018948314823.
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.
已开发并验证了一种全自动柱切换高效液相色谱法，用于直接进样定量测定血浆中的ML-1035及其五种代谢物。将血浆样品注入CN萃取柱（4×4.6 mm，5微米），用50 mM磷酸盐中的0.5%十二烷基硫酸钠（SDS）进行胶束净化。蛋白质成分被溶解并冲洗掉。然后将萃取的化合物通过正向冲洗洗脱到C8分析柱（150×4.6 mm，5微米）上，使用荧光检测（激发波长308 nm；发射波长350 nm）进行进一步分析。在用三种溶剂混合物依次进行后续洗涤和重新平衡后，萃取柱即可用于下一次进样。使用100微升血浆时，所有目标化合物的定量限约为10至15 ng/ml。在10至1500 ng/ml范围内，所有化合物均具有出色的精密度、准确度和线性。还使用接受ML-1035的犬的血浆样品验证了该高效液相色谱法的实用性。萃取柱和分析柱的使用寿命都非常出色。胶束净化与柱切换技术相结合，在直接进样生物流体时是一种很有前景的高效液相色谱法。
