Negative-ion Electrospray Tandem Mass Spectrometry Research Articles

Liquid chromatography/tandem mass spectrometry methods for quantitation of mevalonic acid in human plasma and urine: method validation, demonstration of using a surrogate analyte, and demonstration of unacceptable matrix effect in spite of use of a stable isotope analog internal standard.

Selective, accurate, and reproducible liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the determination of mevalonic acid, an intermediate in the biosynthesis of cholesterol and therefore a useful biomarker in the development of cholesterol lowering drugs, in human plasma and urine. A hepta-deuterated analog of mevalonic acid was used as the internal standard. For both methods, calibration standards were prepared in water, instead of human plasma and urine, due to unacceptably high levels of endogenous mevalonic acid. The lower quality control (QC) samples were prepared in water while the higher QC samples were prepared in the biological matrices. For the isolation/purification of mevalonic acid from the plasma and urine matrices, the samples were first acidified to convert the acid analyte into its lactone form. For the plasma samples, the lactone analyte was retained on and then eluted off a polymeric solid-phase extraction (SPE) sorbent. For the urine method, the sample containing the lactone analyte was passed through a C-18 SPE column, which did not retain the analyte, with the subsequent analyte retention on and then elution off a polymeric SPE sorbent. Chromatographic separation was achieved isocratically on a polar-endcapped C-18 analytical column with a water/methanol mobile phase containing 0.5 mM formic acid. Detection was by negative-ion electrospray tandem mass spectrometry. The standard curve range was 0.500-20.0 ng/mL for the plasma method and 25.0-1,000 ng/mL for the urine method. Excellent accuracy and precision were obtained for both methods at all concentration levels tested. It was interesting to note that for certain batches of urine, when a larger sample volume was used for analysis, a high degree of matrix effect was observed which resulted not only in the attenuation of the absolute response, but also in a change of analyte/internal standard response ratio. This demonstrated that, under certain conditions, the use of a stable isotope analog internal standard does not, contrary to conventional thinking, guarantee the constancy of the analyte/internal response ratio, which is a prerequisite for a rugged bioanalytical method. On the other hand, under conditions where the sample matrix does not have such a deleterious effect, we have found that a stable isotope analog could serve as a surrogate (substitute) analyte. Thus, we have shown that using calibration standards prepared by spiking plasma with tri-deuterated or tetra-deuterated mevalonic acid, instead of mevalonic acid itself (the analyte), plasma QC samples that contain mevalonic acid can be successfully analyzed for the accurate and precise quantitation of mevalonic acid. The use of a surrogate analyte provides the opportunity to gauge the daily performance of the method for the low concentration levels prepared in the biological matrix, which otherwise is not achievable because of the endogenous concentrations of the analyte in the biological matrices.

Ranking of gas-phase acidities and chloride affinities of monosaccharides and linkage specificity in collision-induced decompositions of negative ion electrospray-generated chloride adducts of oligosaccharides

Negative ion electrospray-tandem mass spectrometry has been employed to study chloride adducts of saccharide molecules. Decompositions of [M + Cl] − obtained under identical low-energy collision conditions allow the approximate ranking of chloride affinities and gas-phase acidities of a series of isomeric monosaccharides. The ketohexoses are found to be more acidic than the aldohexoses. Chloride adduct decompositions are examined for a glucopyranosyl fructose and a glucopyranosyl glucose series. For each disaccharide series, the linkage position is shown to markedly influence the favored pathways of [M + Cl] − decompositions, initiated either by loss of neutral HCl to form [M − H] − and possibly leading to further (consecutive) decompositions, or by loss of M to form Cl −. Upon formation of [M − H] −, both cross-ring cleavages and glycosidic bond decompositions were observed in varying degrees for the two series of disaccharides. Remarkably, for three non-reducing polysaccharides that each contain a terminal sucrose group at the “downstream” end, chlorine-containing product ions arising from cleavage of the Glcα–2Fru linkage have been observed. Apart from Cl −, chlorine-containing product ions are not observed for any of the other disaccharides investigated, and they appear to be specifically diagnostic of a terminal Glcα–2Fru linkage. Their appearance is rationalized based upon a substantially reduced tendency for HCl loss from these non-reducing polysaccharides.

Determination of folates in human plasma using hydrophilic interaction chromatography-tandem mass spectrometry.

Folic acid is an essential nutrient, and folate deficiency is associated with a variety of disorders including neural tube defects (during pregnancy) and heart disease. A fast, sensitive, and robust HPLC-tandem mass spectrometry (LC-MS-MS) method was developed for the quantification of free folic acid, tetrahydrofolate, 5'-methyltetrahydrofolate, and 5'-formyltetrahydrofolate in human plasma. Sample preparation required only acetonitrile precipitation of proteins followed by filtration instead of solid-phase extraction or solvent-solvent extraction as in other methods. The rapid and streamlined sample handling procedure minimized degradation of the highly unstable folate species. Hydrophilic interaction chromatography was used for additional sample cleanup on-line, and baseline separation and detection of all four folate species was achieved in less than 30 min. The folate species were detected using negative ion electrospray-tandem mass spectrometry with multiple reaction monitoring of the diagnostic fragment ions of each deprotonated molecule. The predominately organic (hydrophobic) solvent system combined with the microbore flow rate (50 microL/min) used for the chromatography resulted in enhanced electrospray signal response compared to reversed-phase HPLC using a wider bore column. The recovery of all folate species (from spiked plasma) was >97% over a concentration range from 300 pg/L to 12 mg/L with intraday precision (RSD, n = 5) of 3.7-6.5%. Stability studies were carried out for spiked samples in order to define storage and handling conditions. The folic acid limit of quantification (LOQ) in human plasma was 80 pmol/L +/- 10%, and the limit of detection (LOD) was 37.5 pmol/L. The LOQ and LOD for tetrahydrofolate, 5'-methyltetrahydrofolate, and 5'-formyltetrahydrofolate were 1250, 400, and 360 pmol/L of plasma and 425, 165, and 140 pmol/L of plasma, respectively.

Quantitative analysis of mevinolinic acid in human plasma by high-performance liquid chromatography coupled with negative-ion electrospray tandem mass spectrometry.

Mevinolinic acid (MVA), the major active metabolite of Lovastatin in human blood, is analysed by microbore high-performance liquid chromatography coupled to electrospray tandem mass spectrometry. Quantification is carried out by using methylmevinolinic acid (MMVA) as internal standard. Positive- and negative-ion mass spectra of these compounds are shown. Because of the higher sensitivity obtainable, the negative-ion mode is selected for the analysis. Solid-phase extraction cartridges are used off-line to prepurify and concentrate the sample and a microbore (1 x 100 mm) reversed-phase column is used for chromatography. Tandem mass spectrometry is carried out in the precursor-ion mode using a selected-reaction monitoring procedure. The [M-H]- precursor ions for MVA and MMVA are selected in the first quadrupole analyser and the second analyser is focused on the common product ion at m/z 319. Detection and quantification limits are ca. 50 pg/mL and ca. 200 pg/mL respectively. An example of the application of the method to the routine analysis of samples from a bioequivalence study is also shown.

Identification of Monohydroxy Fatty Acids by Electrospray Mass Spectrometry and Tandem Mass Spectrometry

Negative-ion electrospray mass spectrometry (ESI–MS) and tandem mass spectrometry (ESI–MS/MS) were used to characterize saturated and unsaturated monohydroxy fatty acids and fatty acid metabolites formed following incubation with soybean lipoxygenase. Ions corresponding to [M-H]−of eicosanoids were readily observed using ESI–MS, but double bond migration precluded the use of MS to localize double bonds or the position of hydroxyl moieties; however, by following MS analysis with negative-ion ESI–MS/MS of precursor ions, the position of oxygenation could be determined for picogram quantities of underivatized monohydroxy fatty acids. Loss of 46 mass units from the precursor ion of saturated monohydroxy compounds was explained in some cases by resonance stabilization of enolate ions, but this product ion was found in spectra of compounds where resonance was not possible. Spectra of deuterated analogs supported charge-driven vinylic processes as the most common mechanism of fragmentation. The utility of low-collision-energy ESI–MS/MS to examine biological samples was shown by examining the products formed by the metabolism of linoleic (18:2ω6) and arachidonic (20:4ω6) acids by soybean lipoxygenase using aerobic and anaerobic incubation conditions that generated increasingly complex mixes of metabolites.

Quantitation of Cyclic Nucleotides in Biological Samples by Negative Electrospray Tandem Mass Spectrometry Coupled to Ion Suppression Liquid Chromatography

Measurement of cyclic nucleotides by electrospray ionization mass spectrometry constitutes an unequivocal quantitative and qualitative technique superior to the established estimation methods of UV spectroscopy, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and other mass spectrometric ionization techniques such as electron impact, thermospray and fast-atom bombardment. Optimal conditions for negative-ion electrospray tandem mass spectrometry showed a linear detection range using solutions with a concentration ranging from 10−6 and 5×10−9 M with an absolute detection limit of 100 fmol injected. Measurement of diagnostic product ions derived from low-energy collision-activated dissociation of the [M−H]− ions provided unambiguous identification. Quantitation of cyclic nucleotides present in biological samples in the femtomole region, needed further purification using on-line ion suppression high-performance liquid chromatography in order to avoid reduced sensitivity caused by matrix quenching.