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Abstract INTRODUCTION: Phospholipids are major constituents of plasma membranes and have been shown to cause severe ion suppression or enhancement in the analysis of biological samples by liquid chromatography with electrospray ionization mass spectrometry (LC-ESI-MS). Additionally, phospholipids tend to accumulate on reversed-phase columns, causing a decrease in column performance and reduced life time.
OBJECTIVE: In this study we have developed an innovative inline sample preparation (ILSP) technique to remove phospholipids from protein precipitated plasma samples.
METHODS: Protein precipitation of human plasma was performed by adding acetonitrile containing 1% formic acid to blank or fortified plasma in a (3:1) ratio, respectively. After vortexing for 30 seconds at 3000 rpm, samples were centrifuged for 10 min at 4000 rpm/10°C. All data was collected on a Shimadzu Nexera UHPLC equipped with an additional binary pump and 6-port switching valve. Analysis was conducted using a 5x2.1 mm ILSP cartridge followed by a Raptor Biphenyl 2.7 µm, 100 x 2.1 mm analytical column. Phospholipids and target analytes were monitored in SIM and scan modes using a Shimadzu 2020 MS with electrospray ionization in positive ion mode.
RESULTS: Accurately timed valve switching is critical for a successful inline phospholipid removal method to facilitate washing of phospholipids from the ILSP cartridge while maintaining target analytes. Timing is dependent on the degree of phospholipid retention on the ILSP cartridge in conjunction with the hydrophilic or hydrophobic properties of the analytes. Once the timing of the valve switch is determined, an analytical column is added and the gradient is optimized for speed. For initial experiments, the Raptor Biphenyl 2.7 µm, 100 x 2.1 mm column was chosen. The time program was optimized to allow for simultaneous flushing of phospholipids from the ILSP cartridge, while gradient analysis proceeds on the analytical column. The final analysis with phospholipid removal was completed in <7 minutes. Utilizing this method, lifetime of the ILSP cartridge was demonstrated by successfully performing 500 consecutive injections of protein precipitated fortified human plasma with consistent analyte response (≤2.2% RSD) and retention time (≤0.24% RSD).
CONCLUSIONS: Phospholipid removal by ILSP results in very clean sample extracts and improved signal to noise. A comparison study was performed where plasma samples were fortified with ketoprofen, sulfadiazine, amphetamine methadone, nortriptyline, and prednisolone. Samples were extracted using 36 wells of a representative 96-well plate offline phospholipid removal product and compared to 36 ILSP injections following protein precipitation. Average analyte signal to noise ratios increased by 15 – 189% on average for the ILSP technique, with the exception of amphetamine, which showed a marginal decreased of 23%.
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= Discovery stage.
= Translation stage.
= Clinically available.
