A simplified protein precipitation/mixed‐mode cation‐exchange solid‐phase extraction, followed by high‐speed liquid chromatography/mass spectrometry, for the determination of a basic drug in human plasma

Abstract

A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction (PPT/SPE) procedure has been investigated. A mixture of acetonitrile and methanol along with formic acid was used to precipitate plasma proteins prior to selectively extracting the basic drug. After vortexing and centrifugation, the supernatants were directly loaded onto an unconditioned Oasis MCX microElution 96-well extraction plate, where the protonated drug was retained on the negatively charged sorbent while interfering neutral lipids, steroids or other endogenous materials were washed away. Normal wash steps were deemed unnecessary and not used before sample elution. The sample extracts were analyzed under both conventional and high-speed liquid chromatography/tandem mass spectrometry (LC/MS/MS) conditions to examine the feasibility of the PPT/SPE procedure for human plasma sample clean-up. For the conventional LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 50 mm column with gradient elution (k' = 5.5). The mobile phase contained 0.1% formic acid in water and 0.1% formic acid in acetonitrile. For the high-speed LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 10 mm guard column with gradient elution (k' = 2.2, Rt = 0.26 min). The mobile phase contained 0.1% formic acid in water and 0.001% trifluoroacetic acid in acetonitrile. Detection for both conventional and high-speed LC/MS/MS methods was by positive ion electrospray tandem mass spectrometry on a ThermoElectron Finnigan TSQ Quantum Ultra, where enhanced resolution (RP 2000; 0.2 amu) was used for high-speed LC/MS/MS. The standard curve, ranging from 0.5 to 100 ng/mL, was fitted to a 1/x weighted quadratic regression model. This combined PPT/SPE procedure effectively eliminated time-consuming sorbent conditioning and wash steps, which are essential for a conventional mixed-mode SPE procedure, but retained the advantages of both PPT (removal of plasma proteins) and mixed-mode SPE (analyte selectivity). The validation results demonstrated that this PPT/SPE procedure was well suited for both conventional and high-speed LC/MS/MS analyses. In comparison with a conventional mixed-mode SPE procedure, the simplified PPT/SPE process provided comparable sample extract purity. This simple sample clean-up procedure can be applied to other basic compounds with minor modifications of PPT solvents.

Determination of remifentanil in human plasma by liquid chromatography–tandem mass spectrometry utilizing micro extraction in packed syringe (MEPS) as sample preparation

“…However, these methods require long time for sample preparation and instrumental analysis. The use of LC with MS detection for remifentanil analysis in plasma [1], blood [4] and urine [19] made it possible to decrease the instrumental analysis time but the time for sample preparation was still long.…”

Recent advances in high-throughput quantitative bioanalysis by LC–MS/MS

“…Xue et al [19] investigated a simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction (PPT/SPE) procedure. A mixture of acetonitrile and methanol along with formic acid was used to precipitate plasma proteins prior to selectively extracting the basic drug.…”

Determination of Levocetirizine in Human Plasma by LC–MS-MS: Validation and Application in a Pharmacokinetic Study

“…Protein precipitation using conventional protein precipitants such as acetonitrile and methanol is not usually suitable for basic drugs with high protein binding including levocetirizine (10). Trichloroacetic acid has been shown to remove proteins efficiently (15) and an acidic protein precipitant is usually suitable for plasma determination of a basic drug with high protein binding (16,17). Levocetirizine contains a strongly basic amino group and has high plasma protein binding, and thus trichloroacetic acid is likely to improve the extraction efficiency compared with conventional protein precipitating solvents such as methanol and acetonitrile (16,17).…”

“…Trichloroacetic acid has been shown to remove proteins efficiently (15) and an acidic protein precipitant is usually suitable for plasma determination of a basic drug with high protein binding (16,17). Levocetirizine contains a strongly basic amino group and has high plasma protein binding, and thus trichloroacetic acid is likely to improve the extraction efficiency compared with conventional protein precipitating solvents such as methanol and acetonitrile (16,17). Unlike conventional protein precipitants, trichloroacetic acid in acetonitrile achieves plasma protein removal and enhances the solubility of the analyte in the precipitating solution.…”