Analysis of the extreme diversity of salivary alpha-amylase isoforms generated by physiological proteolysis using liquid chromatography-tandem mass spectrometry

Saliva is a crucial biofluid for oral health and is also of increasing importance as a non-invasive source of disease biomarkers. Salivary alpha-amylase is an abundant protein in saliva, and changes in amylase expression have been previously associated with a variety of diseases and conditions. Salivary alpha-amylase is subject to a high diversity of post-translational modifications, including physiological proteolysis in the oral cavity. Here we developed methodology for rapid sample preparation and non-targeted LC-ESI-MS/MS analysis of saliva from healthy subjects and observed an extreme diversity of alpha-amylase proteolytic isoforms. Our results emphasize the importance of consideration of post-translational events such as proteolysis in proteomic studies, biomarker discovery and validation, particularly in saliva. (C) 2012 Elsevier B.V. All rights reserved.

Analysis of Daphnane Orthoesters in Poisonous Australian Pimelea Species by Liquid Chromatography-Tandem Mass Spectrometry.

Cattle grazing in arid rangelands of Australia suffer periodic extensive and serious poisoning by the plant species Pimelea trichostachya, P. simplex, and P. elongata. Pimelea poisoning (also known as St. George disease and Marree disease) has been attributed to the presence of the diterpenoid orthoester simplexin in these species. However, literature relating to previous studies is complicated by taxonomic revisions, and the presence of simplexin has not previously been verified in all currently recognized taxa capable of inducing pimelea poisoning syndrome, with no previous chemical studies of P. trichostachya (as currently classified) or P. simplex subsp. continua. We report here the isolation of simplexin from P. trichostachya and the development of a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method to measure simplexin concentrations in pimelea plant material. Simplexin was quantified by positive-ion atmospheric pressure chemical ionization (APCI) LC-MS/MS with selected reaction monitoring (SRM) of the m/z 533.3 > 253.3 transition. LC-MS/MS analysis of the four poisonous taxa P. trichostachya, P. elongata, P. simplex subsp. continua, and P. simplex subsp. simplex showed similar profiles with simplexin as the major diterpenoid ester component in all four taxa accompanied by varying amounts of related orthoesters. Similar analyses of P. decora, P. haematostachya, and P. microcephala also demonstrated the presence of simplexin in these species but at far lower concentrations, consistent with the limited reports of stock poisoning associated with these species. The less common, shrubby species P. penicillaris contained simplexin at up to 55 mg/kg dry weight and would be expected to cause poisoning if animals consumed sufficient plant material.

Sensitive Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Olanzapine in Human Urine

A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method was developed to determine olanzapine (OLZ) in human urine. After solid-phase extraction with SPE cartridge, the urine sample was analysed on a C-18 column (Symmetry 3.5 mu m, 50 x 4.6 mm i.d) interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of ammonium acetate (pH 7.8)-acetonitrile (10:90, v/v). The method was linear over a concentration range of 1-100 ngml(-1). The lower limit of quantitation was 1 ngml(-1). The intra-day and inter-day relative standard deviation across three validation runs over the entire concentration range was < 11.5 %. The accuracy determined at three concentrations (8.0, 50.0 and 85.0 ngml(-1) OLZ) was within +/- 1.21 % in terms of relative errors.

Determinations of MC-LR and [Dha(7)] MC-LR Concentrations and Physicochemical Properties by Liquid Chromatography-Tandem Mass Spectrometry

A liquid chromatography electrospray mass spectrometry (LC/ESI/MS) method working in multiple reactions monitoring mode for the determination of trace amounts of microcystin variants (MC-LR and [Dha(7)] MC-LR) in waters was developed. The limit of quantification was 0.05 mu g/L and the limit of detection was 0.015 mu g/L for MC-LR and [Dha(7)] MC-LR, respectively. Recoveries for MCs were in the range of 68%-81%. MC-LR and [Dha(7)] MC-LR were chemically stable with similar physiochemical behavior.

Simultaneous determination of microcystin-LR and its glutathione conjugate in fish tissues by liquid chromatography-tandem mass spectrometry

A sensitive and selective liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantitative determination of microcystin-LR (MC-LR) and its glutathione conjugate (MC-LR-GSH) in fish tissues. The analytes were extracted from fish liver and kidney using 0.01 M EDTA-Na-2-5% acetic acid, followed by a solid-phase extraction (SPE) on Oasis HLB and silica cartridges. High-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry, operating in selected reaction monitoring (SRM) mode, was used to quantify MC-LR and its glutathione conjugate in fish liver and kidney. Recoveries of analytes were assessed at three concentrations (0.2, 1.0, and 5 mu g g(-1) dry weight [DW]) and ranged from 91 to 103% for MC-LR, and from 65.0 to 75.7% for MC-LR-GSH. The assay was linear within the range from 0.02 to 5.0 mu g g(-1) DW, with a limit of quantification (LOQ) of 0.02 mu g g(-1) DW. The limit of detection (LOD) of the method was 0.007 mu g g(-1) DW in both fish liver and kidney. The overall precision was determined on three different days. The values for within- and between-day precision in liver and kidney were within 15%. This method was applied to the identification and quantification of MC-LR and its glutathione conjugate in liver and kidney of fish with acute exposure of MC-LR. (c) 2007 Elsevier B.V. All rights reserved.

Rapid identification of saponins in plant extracts by electrospray ionization multi-stage tandem mass spectrometry and liquid chromatography/tandem mass spectrometry

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn) and liquid chromatography coupled with on-line mass spectrometry (LC/MS/MS) were applied to characterize saponins in crude extracts from Panax ginseng. The MSn data of the [M - H](-) ions of saponins can provide structural information on the sugar sequences of the saccharide chains and on the sapogins of saponins. By ESI-MSn, non-isomeric saponins and isomeric saponins with different aglycones can be determined rapidly in plant extracts. LC/MS/MS is a good complementary analytical tool for determination of isomeric saponins. These approaches constitute powerful analytical tools far rapid screening and structural assignment of saponins in plant extracts. Copyright (C) 2000 John Wiley & Sons, Ltd.

High-performance liquid chromatography-tandem mass spectrometry for identification of isoflavones and description of the biotransformation of kudzu root

High-performance liquid chromatography-tandem mass spectrometry has been used to identify isoflavone aglycones and glycosides in kudzu root. Fourteen isoflavones were detected. Among these, six were identified by comparison with authentic standards. Tentative identifications of the other isoflavones are based on UV spectra, mass spectra of protonated and deprotonated molecules, and MS-MS data. Several are reported for the first time in kudzu root. The bioactivity and bioavailability of isoflavone aglycones are usually greater than those of their glycosides. To improve the bioavailability of kudzu root isoflavones, crude beta-glycosidases prepared from microbes were used to hydrolyze the isoflavone glycosides. Several MS modes are combined not only to identify the isoflavones in kudzu root, but also to describe the biotransformation of kudzu root isoflavone glycosides. It is also proved that crude beta-glycosidases have high selectivity toward the O-glycosides of isoflavones.

Rapid confirmatory method for the determination of sixteen synthetic growth promoters and bisphenol A in bovine milk using dispersive solid-phase extraction and liquid chromatography-tandem mass spectrometry

A rapid liquid chromatographic-tandem mass spectrometric (LC-MS/MS) multi-residue method for the simultaneous quantitation and identification of sixteen synthetic growth promoters and bisphenol A in bovine milk has been developed and validated. Sample preparation was straightforward, efficient and economically advantageous. Milk was extracted with acetonitrile followed by phase separation with NaCl. After centrifugation, the extract was purified by dispersive solid-phase extraction with C18 sorbent material. The compounds were analysed by reversed-phase LC-MS/MS using both positive and negative ionization and operated in multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from each of the chosen precursor ions for unambiguous confirmation. Total chromatographic run time was less than 10 min for each sample. The method was validated at a level of 1 mu g L-1. A wide variety of deuterated internal standards were used to improve method performance. The accuracy and precision of the method were satisfactory for all analytes. The confirmative quantitative liquid chromatographic tandem mass spectrometric (LC-MS/MS) method was validated according to Commission Decision 2002/657/EC. The decision limit (CC alpha) and the detection capability (CC beta) were found to be below the chosen validation level of 1 mu g L-1 for all compounds. (C) 2010 Elsevier B.V. All rights reserved.

Studies on the persistence of estradiol benzoate and nortestosterone decanoate in hair of cattle following treatment with growth promoters, determined by ultra-high-performance liquid chromatography-tandem mass spectrometry

Measurement of steroid esters in bovine hair samples, using sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS), provides a powerful tool for identifying animals treated illicitly with growth promoters. The successful application of such testing requires appropriate sampling of hair from treated animals. This paper describes the results of hair analysis by LC-MS/MS for two animal studies in which animals were treated with estradiol-3-benzoate and nortestosterone decanoate. The results from the first animal study indicate that animals treated with these anabolic steroids may not always be identified from analysis of hair samples; positive test results occur sporadically and only for some of the treated animals. The results from the second animal study identify conditions attaching to positive hair samples, such as, that concentrations of steroid esters in hair are related to distance of sampling from point of injection and to time post-treatment, that concentrations of steroid esters in hair are related to dose given to the animal but that this relationship may vary over time post-treatment, and that steroid esters may be measured in regrowth hair taken some weeks after treatment. Steroid esters are determined along the length of the hair, confirming that accumulation of steroid esters into hair occurs from various sources, including blood, sweat and sebum. The reported research provides some useful insights into the mechanisms governing the persistence of steroid esters in bovine hair following illicit treatment with growth promoters. (C) 2009 Elsevier B.V. All rights reserved.

Surveillance study of a number of synthetic and natural growth promoters in bovine muscle samples using liquid chromatography-tandem mass spectrometry

A simple, new method permitting the simultaneous determination and confirmation of trace residues of 24 different growth promoters and metabolites using liquid chromatography-mass spectrometry was developed and validated. The compounds were extracted from bovine tissue using acetonitrile; sodium sulphate was also added at this stage to aid with purification. The resulting mixture was then evaporated to approximately 1 ml and subsequently centrifuged at high speed and an aliquot injected onto the LC-MS/MS system. The calculated CC values ranged between 0.11 and 0.46 mu g kg-1; calculated CC were in the range 0.19-0.79 mu g kg-1. Accuracy, measurement of uncertainty, repeatability and linearity were also determined for each analyte. The analytical method was applied to a number of bovine tissue samples imported into Ireland from third countries. Levels of progesterone were found in a number of samples at concentrations ranging between 0.28 and 30.30 mu g kg-1. Levels of alpha- and beta-testosterone were also found in a number of samples at concentrations ranging between 0.22 and 8.63 mu g kg-1 and between 0.16 and 2.08 mu g kg-1 respectively.

Screening and Quantitative Confirmatory Method for the Analysis of Glucocorticoids in Bovine Milk Using Liquid Chromatography-Tandem Mass Spectrometry

An LC/MS/MS method was developed and validated for the simultaneous identification, confirmation, and quantification of 12 glucocorticoids in bovine milk. The method was validated in accordance with the criteria defined in Commission Decision 2002/657/EC. The developed method can detect and confirm the presence of dexamethasone, betamethasone, prednisolone, flumethasone, 6 alpha-methylprednisolone, fluorometholone, triamcinolone acetonide, prednisone, cortisone, hydrocortisone, clobetasol propionate, and clobetasol butyrate in bovine milk. Milk samples are extracted with acetonitrile; sodium chloride is subsequently added to aid partition of the milk and acetonitrile mixture. The acetonitrile extract is then subjected to liquid-liquid purification by the addition of hexane. The purified extract is evaporated to dryness and reconstituted in a water acetonitrile mixture, and determination is carried out by LC/MS/MS. The method permits analysis of up to 30 samples in 1 day.

Development of a rapid, multi-class method for the confirmatory analysis of anti-inflammatory drugs in bovine milk using liquid chromatography tandem mass spectrometry

A rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous identification, confirmation and quantitation of seven licensed anti-inflammatory drugs (AIDS) in bovine milk. The method was validated in accordance with the criteria defined in Commission Decision 2002/657/EC. Two classes of AIDS were investigated, corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs). The developed method is capable of detecting and confirming dexamethasone (DXM), betamethasone (BTM), prednisolone (FRED), tolfenamic acid (TV), 5-hydroxy flunixin (5-OH-FLU). meloxicam (MLX) and 4-methyl amino antipyrine (4-MAA) at their associated maximum residue limits (MRLs). These compounds represent all the corticosteroids and NSAIDs licensed for use in bovine animals producing milk for human consumption. These compounds have never been analysed before in the same method and also 4-methyl amino antipyrine has never been analysed with the other licensed NSAIDs. The method can be considered rapid as permits the analysis of up to 30 samples in one day. Milk samples are extracted with acetonitrile; sodium chloride is added to aid partition of the milk and acetonitrile mixture. The acetonitrile extract is then subjected to liquid-liquid purification by the addition of hexane. The purified extract is finally evaporated to dryness and reconstituted in a water/acetonitrile mixture and determination is carried out by LC-MS/MS. Decision limit (CC alpha) values and detection capability (CC beta) values have been established for each compound. (C) 2009 Elsevier B.V. All rights reserved.

Confirmatory method for the determination of various acetylgestagens in animal kidney fat using liquid chromatography-tandem mass spectrometry

A confirmatory method has been developed and validated that allows for the simultaneous detection of medroxyprogesterone acetate (MPA), megestrol acetate (MGA), melengestrol acetate (MLA), chlormadinone acetate (CMA) and delmadinone acetate (DMA) in animal kidney fat using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The compounds were extracted from kidney fat using acetonitrile, defatted using a hexane wash and subsequent saponification. Extracts were then purified on Isolute CN solid-phase extraction cartridges and analysed by LC-MS/MS. The method was validated in animal kidney fat in accordance with the criteria defined in Commission Decision 2002/657/EC. The decision limit (CC) was calculated to be 0.12, 0.48, 0.40, 0.63 and 0.54 g kg-1, respectively, for MPA, MGA, MLA, DMA and CMA, with respective detection capability (CC) values of 0.20, 0.81, 0.68, 1.07 and 0.92 g kg-1. The measurement uncertainty of the method was estimated at 16, 16, 19, 27 and 26% for MPA, MGA, MLA, DMA and CMA, respectively. Fortifying kidney fat samples (n = 18) in three separate assays showed the accuracy of the method to be between 98 and 100%. The precision of the method, expressed as % RSD, for within-laboratory reproducibility at three levels of fortification (1, 1.5 and 2 g kg-1 for MPA, 5, 7.5 and 10 g kg-1 for MGA, MLA, DMA and CMA) was less than 5% for all analytes.