BACKGROUND: A high-throughput bioanalytical methodology for analysis and quantification of lipophilic pharmaceutical compounds in plasma using liquid-liquid extraction (LLE) was developed. RESULTS: A fast and robust alternative to the widely used protein precipitation of plasma samples is sometimes required in order to avoid matrix effects in MS detection. LLE is known to produce clean extracts and hence reduce levels of matrix components that cause ion suppression. The proposed sample preparation was automated LLE using 96-well plates and a Tecan GenMate 96-tips liquid handling robot. With direct injection of the organic phase (methyl tert-butyl ether: iso-hexane 50:50 v/v) onto a reversed-phase column and without evaporation of the organic phase and reconstitution of the sample, the LLE was no more time consuming than standard protein precipitation, furthermore, matrix effects were minimized. The small injection volume (5 µl) when used with lipophilic compounds and a rapid gradient elution made it possible to inject the organic phase with maintained chromatographic performance. Good chromatographic behavior was confirmed for eight commercially available lipophilic compounds. CONCLUSIONS: The proposed method of LLE with injection of the organic phase onto a reversed-phase column in LC-MS/MS is no more time consuming than standard protein precipitation, and matrix effects were minimized, thus making it suitable as a high-throughput bioanalytical methodology for use in drug discovery.
BACKGROUND: A high-throughput bioanalytical methodology for analysis and quantification of lipophilic pharmaceutical compounds in plasma using liquid-liquid extraction (LLE) was developed. RESULTS: A fast and robust alternative to the widely used protein precipitation of plasma samples is sometimes required in order to avoid matrix effects in MS detection. LLE is known to produce clean extracts and hence reduce levels of matrix components that cause ion suppression. The proposed sample preparation was automated LLE using 96-well plates and a Tecan GenMate 96-tips liquid handling robot. With direct injection of the organic phase (methyl tert-butyl ether: iso-hexane 50:50 v/v) onto a reversed-phase column and without evaporation of the organic phase and reconstitution of the sample, the LLE was no more time consuming than standard protein precipitation, furthermore, matrix effects were minimized. The small injection volume (5 µl) when used with lipophilic compounds and a rapid gradient elution made it possible to inject the organic phase with maintained chromatographic performance. Good chromatographic behavior was confirmed for eight commercially available lipophilic compounds. CONCLUSIONS: The proposed method of LLE with injection of the organic phase onto a reversed-phase column in LC-MS/MS is no more time consuming than standard protein precipitation, and matrix effects were minimized, thus making it suitable as a high-throughput bioanalytical methodology for use in drug discovery.