Comparison of turbulent-flow chromatography with automated solid-phase extraction in 96-well plates and liquid-liquid extraction used as plasma sample preparation techniques for liquid chromatography-tandem mass spectrometry.
Turbulent flow chromatography (TFC) combined with the high selectivity and sensitivity of tandem mass spectrometry (MS-MS) is a new technique for the fast direct analysis of drugs from crude plasma. TFC in the 96-well plate format reduces significantly the time required for sample clean-up in the laboratory. For example, for 100 samples the workload for a technician is reduced from about 8 h by a manual liquid-liquid extraction (LLE) assay to about 1 h in the case of TFC. Sample clean-up and analysis are performed on-line on the same column. Similar chromatographic performance and validation results were achieved using HTLC Turbo-C18 columns (Cohesive Technologies) and Oasis HLB extraction columns (Waters). One 96-well plate with 96 plasma samples is analyzed within 5.25 h, corresponding to 3.3 min per sample. Compared to this LLE and analysis of 96 samples takes about 16 h. Two structurally different and highly protein bound compounds, drug A and drug B, were analyzed under identical TFC conditions and the assays were fully validated for the application to toxicokinetics studies (compliant with Good Laboratory Practices-GLP). The limit of quantitation was 1.00 microg/l and the linear working range covered three orders of magnitude for both drugs. In the case of drug A the quality of analysis by TFC was similar to the reference LLE assay and slightly better than automated solid-phase extraction in 96-well plates. The accuracy was -3.1 to 6.7% and the precision was 3.1 to 6.8% in the case of drug A determined for dog plasma by TFC-MS-MS. For drug B the accuracy was -3.7 to 3.5% and the precision was 1.6 to 5.4% for rat plasma, which is even slightly better than what was achieved with the validated protein precipitation assay.
Zimmer
D
D
Bayer AG, Preclinical Pharmacokinetics, Wuppertal, Germany.
Pickard
V
V
Czembor
W
W
Müller
C
C
D000818:Animals; D001331:Automation; D001774:Blood Chemical Analysis; D002853:Chromatography, Liquid / Q000379:methods*; D004285:Dogs; D013058:Mass Spectrometry / Q000379:methods*; D010599:Pharmacokinetics; D051381:Rats; D012015:Reference Standards; D015203:Reproducibility of Results