Rapid bioanalytical determination of dextromethorphan in canine plasma by dilute-and-shoot preparation combined with one minute per sample LC-MS/MS analysis to optimize formulations for drug delivery

The determination of dextromethorphan in canine plasma is used to demonstra te the high throughput bioanalytical approach of automated dilute-and-shoot (DAS) sample preparation followed by a 1 min isocratic liquid chromatograp hy tandem mass spectrometry (LC-MS/MS) analysis. Dilute-and-shoot preparati on is commonly used for the determination of drugs in several biological ma trices such as urine and saliva, but is not typically used with plasma samp les because the amount of protein present in plasma can lead to a variety o f problems including column failure. As a result, plasma sample preparation usually removes protein by precipitation, extraction or filtration; howeve r, the dilute-and-shoot approach solubilizes proteins throughout the chroma tographic portion of the assay. The attributes of this approach are compare d with a previously validated liquid/liquid extraction procedure for determ ination of dextromethorphan in plasma. Accuracy and precision of both metho ds are similar. The lower limit of quantitation (LLOQ) of the dilute-and-sh oot approach is much higher at 2 ng/ml versus 5 pg/ml with the liquid/liqui d extraction; however, the sample throughput of the preparation portion of the dilute-and-shoot approach is more than 50-fold greater. The ruggedness of the dilute-and-shoot method was thoroughly investigated because of the p roblems traditionally associated with the direct injection of diluted plasm a onto an LC-MS/MS instrument. With the optimal conditions, greater than 10 00 injections of diluted plasma have been successfully performed on a singl e column in less than 19 h making this technique an excellent approach for the rapid preparation and high throughput of plasma samples containing drug levels in the ng/ml range or higher. Application of this methodology to me asure the levels of dextromethorphan in canine plasma to evaluate drug deli very from various formulations is also presented. (C) 2000 Elsevier Science B.V. All rights reserved.