Systematic LC‐MS/MS bioanalytical method development that incorporates plasma phospholipids risk avoidance, usage of incurred sample and well thought‐out chromatography

Abstract

This treatise summarizes the underlying principle and the road map for systematic LC-MS/MS bioanalytical method development. The three themes that have recently emerged as central to building quality during method development-phospholipids, incurred sample and sound chromatographic considerations-are the main focus of this article. In order to reduce the bioanalytical risk associated with plasma phospholipids, a dual approach involving extraction and chromatography is recommended. The use of incurred sample during method development is essential to avoid interference arising from drug-related components. This is different from the current practice of incurred sample reanalysis, which tests reproducibility during method application. LC column/mobile phase optimization is needed to achieve appropriate peak shape, sensitivity and the separation of the analyte from interfering metabolites and phospholipids. Related to sound chromatographic considerations, we lay out facts and myths related to UPLC, vis-à-vis HPLC. Incorporation of quality during method development avoids the costly experience of finding out by chance about the invalidity of a method after it has been used in support of a number of pivotal clinical and non-clinical studies. To this end, we put forth an outline of a protocol for a systematic LC-MS/MS bioanalytical method development.

Quantification of Glucuronide Metabolites in Biological Matrices by LC-MS/MS

“…This is why Jemal et al suggest to use an incurred plasma sample (plasma after dose which still contains metabolites) during the method development in order to ascertain that the developed method for a drug is not compromised by their presence either by chromatographic co-elution and channel cross-talk or by deconjugation during the sample handling (Jemal, Ouyang et al 2010). The glucuronides often prove to be difficult analytes for HPLC separations.…”

“…Sometimes, a liquid-liquid extraction (LLE) may be preferable over a solid phase extraction (see paragraph 3.3) because of its greater simplicity and applicability to almost all laboratories and low cost (Jemal, Ouyang et al 2010). However, the extraction of glucuronides into a lipophilic organic solvent can be difficult because of the glucuronide's highly polar and hydrophilic nature (both the glucuronic acid and hydroxyl groups can be ionized).…”

“…The choice of the extraction solvents may have a strong impact on the matrix effects later in LC-MS analysis. Methyl-tert-butyl ether (MTBE) and n-butyl chloride seem to extract only negligible amounts of lyso-phosphatidyl choline, which is the matrix component that elutes early from a reverse phase (RP) column and tends to interfere with ionization of drugs and their metabolites (Jemal, Ouyang et al 2010). However, both solvents are highly non-polar and the recovery of glucuronides is expected to be low.…”

“…Typical values of dwell times are 1 to 250 ms (Lee, Zhu et al 2011). However, with modern UHPLC columns which can produce very high and narrow peaks (often less than 5 s wide at half height) (Jemal, Ouyang et al 2010), the data acquisition rate should be fast enough to provide at least 10-15 points per peak. Not only the dwell time, also the cycle time (the summation of dwell times of all MRMs and pause times between them) is extremely important for data acquisition rate.…”

Incurred sample stability of ASP3258 in the presence of its acyl glucuronide

“…To prevent back-conversion of AG to its aglycone, it is generally recommended that samples are handled under acidic pH at lower temperatures [15,16]. In accordance with this general recommendation, blood samples were cooled in an ice-water bath immediately after collection.…”