LC-MS Development Strategies for Quantitative Bioanalysis

Abstract

Although quantitative bioanalysis using liquid chromatography in conjunction with atmospheric pressure ionization tandem mass spectrometry (LC-MS/MS) has been in use for approximately fifteen years, new concepts and technologies are continuously being introduced to enhance the multiple steps of quantitative LC-MS/MS bioanalysis. In this review article, we have focused on concepts and technologies that have recently been introduced to achieve further improvements in biological sample collection/storage and extraction, chromatography and mass spectrometric detection. Under these major headings, a number of specific topics are presented, summarizing the most recent findings in these areas. Included among the topics discussed are: off-line plasma extraction, on-line plasma extraction, enhanced mass resolution, atmospheric pressure photoionization, high-field asymmetric waveform ion mobility spectrometry, electron capture atmospheric pressure chemical ionization, enhancing MS detection via formation of anionic and cationic adducts, chemical derivatization, ultra-performance chromatography, hydrophilic interaction chromatography, and MS-friendly ion-pair reversed-phase chromatography. In the end, we discuss potential pitfalls in LC-MS/MS bioanalysis and the means to avoid them. Such pitfalls may occur due to mass spectral interference from metabolites or prodrugs, due to the use of inappropriate calibration standard and quality control samples for analysis involving unstable drugs or metabolites, and due to the wild card phenomenon commonly known as the matrix effect.

Analytical Methods for Quantification of Drug Metabolites in Biological Samples

“…RAM columns may be used either in single column mode, being extraction (SPE) and analytical column at same time, or extraction column in combination with second analytical column. Single column mode approach shows simplicity but is limited due to chromatographic separation power [79]. Monolith phases as extraction sorbents for sample treatment looks promising and has been reviewed recently [77].…”

“…An example is shown in Figure 1. Additionally, unstable metabolites, such as N-oxides or glucuronides may be converted to parent drug by in-source dissociation or thermal degradation [79] or in collision cell (ion channel cross-talk). Interferences with endogenous compounds should also be avoided as matrix effect may appear (see 9.1.).…”

“…The formed neutral ion pars increase retention and also improve peak shape. Among different ion-paring reagents trifluoroacetic acid and other perfluorated acids for basic analytes and for instance nucleoside phosphates for acidic analytes are appropriate for LC-MS/MS analyses [79]. These additives, especially trifluoroacetic acid, should be used at low concentrations because they cause ion suppression.…”

“…PGC chromatography commonly employs water, acetonitrile and methanol as the mobile phase but provides markedly greater retention and selectivity for polar analytes than reversed phase columns. For analyte elution PGC normally requires larger organic content in the mobile phase than reversed phase chromatography what consequently results in favorable sensitivity with MS detection [79,83,89].…”

“…Reducing the particle diameter from 5.0 µm to 1.7 µm will, in principle, result in a 3-fold increase of efficiency, 1.7-fold increase in resolution, a 1.7-fold in sensitivity, and 3-fold increase in speed [79]. For fast analyses using sub-2µm particle column dimensions are typically 50x2 mm.…”