Development of a low volume plasma sample precipitation procedure for liquid chromatography/tandem mass spectrometry assays used for drug discovery applications

Abstract

The demand for high sensitivity bioanalytical methods has dramatically increased in the drug discovery stage; in addition, there has been a growing trend of reducing the sample volume that is required for these assays. A sensitive high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) procedure has been developed and tested to meet these needs. The assay requires only a low plasma sample volume (10 microL) and employs a protein precipitation procedure using a 1:6 plasma/acetonitrile ratio. The supernatant is injected directly into the LC/MS/MS system using the selected reaction monitoring (SRM) procedure for detection. A generic HPLC gradient based on a methanol/water mobile phase with a flow rate set to 0.8 mL/min was used. The test method showed very good linearity between 0.1-1000 ng/mL (R2 = 0.9737), precision (%RSD = 6-9), accuracy (%RE = -2) and reproducibility (%RSD = 11). A drug discovery IV/PO study was assayed using both the new low volume method and our standard volume (50 microL) method. The correlation of the two sets of data from the two methods was excellent (R2 = 0.9287). This new assay procedure has been successfully used in our laboratory for over 100 different rat or mouse discovery PK studies.

‘LC‐electrolyte effects’ improve the bioanalytical performance of liquid chromatography/tandem mass spectrometric assays in supporting pharmacokinetic study for drug discovery

“…MeOH, EtOH and CH 3 CN) can be effective in removing plasma proteins at a P‐P ratio of 2:1 (v/v),23 Xu et al . reported that lowering the plasma sample volume and increasing the P‐P ratio (6:1, v/v) resulted in a significant reduced level of noise background for analytes and an evidently lower limit of detection, as compared to results obtained using a procedure with a lower P‐P ratio (3:1, v/v) 24. In this study, we found that using an increased P‐P ratio (4:1, v/v; relative to our conventionally used P‐P ratio 2:1, v/v) also reduced the matrix effect window.…”

Increasing Speed and Throughput When Using HPLC-MS/MS Systems forDrug Metabolism and Pharmacokinetic Screening

“…The automation of front-end sample preparation with microplate technology prior to HPLC-MS/MS analyses has played a key role in the high-throughput bioanalytical process [54,109,[121][122][123][124][125]. A microplate is a flat plate composing of multiple sample vials (known as "wells") arranged in rows.…”

Use of trifluoroacetic acid to quantify small, polar compounds in rat plasma during discovery-phase pharmacokinetic evaluation

“…In the past, this has been time consuming, and DMPK labs are evaluating new automation techniques and generic LC/MS methods to keep turnaround in pace with requests [1][2][3][4][5]. Low molecular weight, polar compounds may be important in drug discovery because of the good physical properties they possess, including bioavailability [6][7][8][9][10][11].…”