Response normalized liquid chromatography nanospray ionization mass spectrometry

Abstract

The widely different LC-MS response observed for many structurally different compounds limits the use of LC-MS in full scan detection mode for quantitative determination of drugs and metabolites without using reference standard. The recently introduced nanospray ionization (NSI) technique shows comparable MS response for some compounds under non-LC-MS conditions. However, in the presence of numerous endogenous compounds commonly associated with biological samples such as urine, plasma, and bile, LC-MS is required to separate, detect, identify, and measure individual analytes. An LC-NSI-MS system was devised and the MS response obtained in this system for a variety of pharmaceutical drugs and their metabolites. The set-up involves two high-performance liquid chromatography (HPLC) systems, a chip-based NSI source and a quadrupole-time-of-flight (Q-TOF) mass spectrometer. Herein this is referred to as the response normalized-liquid chromatography NSI-MS (RNLC-NSI-MS) system. One HPLC unit performs the analytical separation, while the other unit adds solvent post-column with an exact reverse of the mobile phase composition such that the final composition entering the NSI source is isocratic throughout the entire HPLC run . LC-MS/MS techniques for quantitation require reference and internal standard(s), so that LC-MS/MS responses from unknown quantities of relevant drug-related peaks can be related back to responses obtained using known quantities of each analyte. Usually a standard curve is generated for each and every analyte to be quantified [6]. Although reference standards may be available for the parent drug, it is very unlikely to have standards for metabolites available in early stage of drug discovery and development thereby making conventional LC-MS/MS based quantitation of metabolites impractical.Nanospray ionization (NSI), introduced by Wilm and Mann in the 1990s [8], has been used to identify proteins [9 -13] and drug metabolites [14], quantify pharmaceutical drugs [15,16], study absorption, distribution, metabolism and excretion (ADME) properties of drugs [17], and employed in various other applications [12, 13, 15, 16, 18 -34]. NSI-MS has the following advantages over conventional flow ESI-MS: (1) higher sensitivity, (2) lower rate of sample consumption, and (3) improved signal-to-noise ratio (S/N). Although both on-and off-line NSI-MS have been developed, off-line NSI-MS methods have been more frequently used because of the technical difficulties associated with interfacing conventional flow LC systems with NSI and

Small Molecule Quantification by Liquid Chromatography-Mass Spectrometry for Metabolites of Drugs and Drug Candidates

“…These observations indicate that the signal of a metabolite can be higher or lower than that of the substrate and that none of the different metabolic transformations give a consistently better agreement in the slopes. This result does not appear to be the case for carboxylic acid metabolites (Hop et al, 2005;Valaskovic et al, 2006;Ramanathan et al, 2007) or for the loss of an amine by N-dealkylation (Benetton et al, 2003), both of which consistently show a lower signal for the metabolite. Indeed, the fact that all the compounds tested have multiple nitrogen atoms and no other ioniz- b Slope (average) of the standard curve for metabolite versus internal standard divided by slope (average) of the standard curve for substrate versus internal standard using nanospray ionization.…”

“…In this regard, other methodologies show promise. For example, a number of investigators have used nanospray LC-MS to decrease the differences between the signal from parent drug and metabolites (Benetton et al, 2003;Hop et al, 2005;Valaskovic et al, 2006;Ramanathan et al, 2007). Furthermore, a number of other methods to estimate exposure to a metabolite using corrected responses in LC-MS/MS (Vishwanathan et al, 2009;Gao et al, 2010;Ma et al, 2010;Tong et al, 2010;Mutlib et al, 2011;Walker et al, 2011) or NMR based methods (Vishwanathan et al, 2009;Mutlib et al, 2011;Walker et al, 2011) have been developed.…”

Investigation of Figopitant and Its Metabolites in Rat Tissue by Combining Whole-Body Autoradiography with Liquid Extraction Surface Analysis Mass Spectrometry

“…(Hop et al, 2005). Differences in ionization efficiency of compounds with different physicochemical characteristics are in general reduced by the application of chip-based NSI (Hop et al, 2005;Hop, 2006;Valaskovic et al, 2006;Wickremsinhe et al, 2006;Ramanathan et al, 2007). Recently, the accuracy of nano-electrospray ionization MS response of drug compounds and their respective metabolites from biological matrices compared with accurate radiometric quantification was evaluated, and it has been demonstrated that nanoelectrospray mass spectrometry can be used for semiquantitation of metabolites (Schadt et al, 2011).…”

“…Samples obtained from the freeze-dried, 30-m whole-body sections were analyzed using a linear ion trap LTQ/ Orbitrap hybrid mass spectrometer (Thermo Scientific) (Hu et al, 2005) equipped with a TriVersa NanoMate nanospray ion source (Schultz et al, 2000;Ramanathan et al, 2007). For sample extraction from the tissue sections, 1 to 2 l of solvent (ACN/water 50:50 with 0.1% FA) were used.…”

“…The samples were analyzed by LC nano-electrospray ionization MS (NSI-MS) in the positive ion mode using a linear ion trap/Orbitrap hybrid mass spectrometer (Thermo Scientific, Bremen, Germany) (Hu et al, 2005) equipped with a TriVersa NanoMate nanospray ion source (Advion BioSciences, Ithaca, NY) (Schultz et al, 2000;Ramanathan et al, 2007). The instrument was coupled to a Berthold LB 509 (Berthold Technologies, Bad Wildbad, Germany) radioactivity detection system.…”