Assessment of matrix effects and determination of niacin in human plasma using liquid–liquid extraction and liquid chromatography–tandem mass spectrometry

Abstract

A simple, sensitive and rapid liquid-liquid extraction method for the analysis of nicotinic acid (niacin) and its labeled internal standard nicotinic acid-d4 (niacin-d4) in human plasma was developed and validated. The analyte and its internal standard were isolated from acidified plasma using a single liquid-liquid extraction procedure with methyl-t-butyl ether. The extracted samples were analyzed by liquid chromatography-tandem mass spectrometry in positive electrospray ionization mode with multiple reaction monitoring. The calibration curves were linear in the measured range between 5 and 1000 ng/mL and the limit of detection was calculated as 122 pg/mL. The method required 250 microL of human plasma and the total run time between injections was 3.5 min. Matrix effects were assessed by post-column infusion experiments, phospholipids monitoring and post-extraction addition experiments. The extraction of phospholipids and niacin from plasma was studied under acidic, neutral and basic conditions. Acidic conditions were optimal for both the recovery of niacin and the removal of phospholipids; the degree of matrix effects for niacin was determined to be 2.5%. It was concluded that effective removal of matrix components can overcome low recovery issues associated with liquid-liquid extractions of polar analytes.

Bioanalytical assay validation parameters re‐visited: perspectives on stability studies

“…If this information is known before hand, one may be cautious in not attempting to analyze the NCE beyond third F/T cycle; however, it should be noted that it is not often that compounds will be analyzed beyond the third F/T cycle. As observed from the recent literature on potentially dubious data generation due to matrix effects (Srinivas 2009;Peoples et al, 2008;Chambers et al, 2007), it is unknown subjecting the matrix to multiple F/T cycles further aggravates the situation of the observed matrix effects. There has not been a specific evaluation of this effect performed and reported in the literature.…”

Dodging matrix effects in liquid chromatography tandem mass spectrometric assays—compilation of key learnings and perspectives

“…Piecing together the work of several researchers, it appears that phospholipids as a single source may maximally contribute to the so-called matrix effects and effective removal of the same would be a great strategy to avoid matrix effects. Accordingly, acidic pH conditions that favour retention of phospholipids in the aqueous layer have been recommended, which in turn would translate into a cleaner organic phase containing the compound in question (Chambers et al, 2007;Ismaiel et al, 2007;Peoples et al, 2008). Alongside these developments, there have been suggestions to monitor phospholipids under identical assay conditions; such data would perhaps help in further optimization of the assay if matrix effects continue to pose problems (Chambers et al, 2007).…”

“…Current thinking in getting ahead of matrix effects has evolved and includes the monitoring of phospholipids as evidenced by the recent assays developed by several researchers (Peoples et al, 2008;Ismaiel et al, 2007;Chambers et al, 2007;Little et al, 2006). Piecing together the work of several researchers, it appears that phospholipids as a single source may maximally contribute to the so-called matrix effects and effective removal of the same would be a great strategy to avoid matrix effects.…”

“…By maintaining a low acidic pH, it was possible to better extract zwitter-ionic niacin (whose recovery appeared to be poor in basic and/or neutral pH conditions); however, under these pH conditions matrix interference was reduced since the non-drug matrix components did not partition in to the organic solvent. Therefore, the final extract for injection had negligible matrix contribution, while the recovery of niacin appeared to be more than optimal (Peoples et al, 2008). Mei et al (2003) were the first to systematically investigate both the exogenous and endogenous contributions relating to matrix effects in LC/MS/MS assays.…”

“…Recently, the work of Peoples et al (2008) suggested that, in spite of insufficient recovery, adequate sensitivity was possible for the determination of niacin in human plasma because the authors had figured out a strategy to remove the matrix interference components from the plasma. By maintaining a low acidic pH, it was possible to better extract zwitter-ionic niacin (whose recovery appeared to be poor in basic and/or neutral pH conditions); however, under these pH conditions matrix interference was reduced since the non-drug matrix components did not partition in to the organic solvent.…”