Monitoring Mass Spectrometry Method Research Articles

Diamine Oxidase as a Therapeutic Enzyme: Study of Germination from Vegetal Sources and Investigation of the Presence of β-N-Oxalyl-L-α,β-diaminopropionic Acid (β-ODAP) Using LC-MS/MS.

Vegetal diamine oxidase (vDAO), an enzyme proposed to relieve symptoms of histaminosis, shows better reactivity with histamine and aliphatic diamines, as well as higher enzymatic activity than DAO of animal origin. The objective of this study was to evaluate the enzyme activity of vDAO from germinating grains from Lathyrus sativus (grass pea) and Pisum sativum (pea), and to verify the presence of a neurotoxin, β-N-Oxalyl-L-α,β-diaminopropionic acid (β-ODAP), in the crude extract obtained from their seedlings. A targeted liquid chromatography-multiple-reaction monitoring mass spectrometry method was developed and used to quantify β-ODAP in the analysed extracts. An optimized sample preparation procedure, involving protein precipitation with acetonitrile followed by mixed-anion exchange solid-phase extraction, allowed for high sensitivity and good peak shape for β-ODAP detection. The Lathyrus sativus extract exhibited the highest vDAO enzyme activity of the extracts, followed by the extract from pea cultivar Amarillo from the Crop Development Centre (CDC). The results have also shown that even though β-ODAP was present in the crude extract from L. sativus, its content was far below the toxicity threshold (300 mg of β-ODAP/kg body/day). CDC Amarillo showed 5000-fold less β-ODAP than the undialysed L. sativus extract. It was concluded that both species can be considered as convenient sources of vDAO for potential therapeutic use.

Analysis of Endogenous Peptides Released from Osteoarthritic Cartilage Unravels Novel Pathogenic Markers*[S

Osteoarthritis (OA) is a pathology characterized by the loss of articular cartilage. In this study, we performed a peptidomic strategy to identify endogenous peptides (neopeptides) that are released from human osteoarthritic tissue, which may serve as disease markers. With this aim, secretomes of osteoarthritic and healthy articular cartilages obtained from knee and hip were analyzed by shotgun peptidomics. This discovery step led to the identification of 1175 different peptides, corresponding to 101 proteins, as products of the physiological or pathological turnover of cartilage extracellular matrix. Then, a targeted multiple reaction monitoring-mass spectrometry method was developed to quantify the panel of best marker candidates on a larger set of samples (n = 62). Statistical analyses were performed to evaluate the significance of the observed differences and the ability of the neopeptides to classify the tissue. Eight of them were differentially abundant in the media from wounded zones of OA cartilage compared with the healthy tissue (p < 0.05). Three neopeptides belonging to Clusterin and one from Cartilage Oligomeric Matrix Protein showed a disease-dependent decrease specifically in hip OA, whereas two from Prolargin (PRELP) and one from Cartilage Intermediate Layer Protein 1 were significantly increased in samples from knee OA. The release of one peptide from PRELP showed the best metrics for tissue classification (AUC = 0.834). The present study reveals specific neopeptides that are differentially released from knee or hip human osteoarthritic cartilage compared with healthy tissue. This evidences the intervention of characteristic pathogenic pathways in OA and provides a novel panel of peptidic candidates for biomarker development.

Development of a liquid chromatography/tandem mass spectrometry method for monitoring of long-term exposure to parabens.

Parabens, the alkyl esters of 4-hydroxybenzoic acid, are a family of compounds widely used as preservatives in cosmetic products, including for children, and some are permitted in foods. Parabens are known to be weak endocrine disruptors because they interfere with the function of endogenous hormones through binding to estrogen receptors. Therefore, the levels of parabens in biological samples indicate endocrine-disruptive exposure. In particular, hair samples can provide information on accumulated exposure to parabens. For monitoring of long-term exposure to parabens, an improved analytical method for rapid and direct determination in hair sample was developed involving ultra-performance liquid chromatography-tandem mass spectrometry using on-line extraction. Five parabens (methyl-, ethyl-, propyl-, butyl- and benzylparaben) were separated within 10 min after incubation with 1 N HCl. Parabens were separated using a Waters BEH C18 column (2.1 mm × 100 mm, 1.7 μm) and a mobile phase consisting of 10 mM ammonium acetate in water and acetonitrile with a gradient program at a flow rate of 300 μL/min. The analysis of the separated parabens was monitored with electrospray negative ionization tandem mass spectrometry. The linearity of the method was demonstrated by r2 ≥0.994. The limits of detection as defined by a signal-to-noise ratio of 3 were 1-5 ng/g. The mean concentration of the five parabens in hair of human subjects was measured to be 55.6 ± 24.3 to 136.9 ± 48.5 ng/g. The levels of parabens in hair samples may play an important role in understanding probable endocrine-disruptive exposure, and the described method could be used to evaluate and monitor long-term exposure to parabens as endocrine disruptors.

Liquid chromatography – high resolution mass spectrometry method for monitoring of 17 mycotoxins in human plasma for exposure studies

Mycotoxins are secondary metabolites produced by filamentous fungi. Primary route of human exposure to mycotoxins is the intake of the contaminated food. Minimizing mycotoxin exposure is important for population health, as their chronic toxic effects have been associated with kidney and liver diseases, some types of cancer and immunosuppression. The objective of this work was to develop and validate a multi-class mycotoxin method suitable for exposure monitoring of mycotoxins in human plasma. A sensitive liquid chromatography – mass spectrometry method was developed for 17 mycotoxins: nivalenol (NIV), deoxynivalenol, fusarenon X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, T-2 toxin, HT-2 toxin, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, zearalenone, α-zearalenol (α-ZOL), β-zearalenol, zearalanone, α-zeranol and, and β-zeranol. The method relies on three-step liquid–liquid extraction with ethyl acetate to eliminate the need for immunoaffinity extraction and minimize ionization matrix effects. Chromatographic separation of mycotoxins, including all isomers, was achieved with pentafluorophenyl column and water/methanol mobile phase. Mycotoxin detection and quantitation were performed using high-resolution mass spectrometry on LTQ Velos Orbitrap, in both positive and negative electrospray ionization (ESI(+) and (ESI(−)). The use of 0.02% acetic acid as mobile phase additive for ESI(−) resulted in significant increase in ionization efficiency ranging from 1.7 to 26 times for mycotoxins that ionize better in ESI(−). The optimized method was validated according to FDA guidance procedures. LOQs of all mycotoxins ranged from 0.1 to 0.5 ng/ml, except NIV which resulted in LOQ of 3 ng/ml because of low extraction recovery of this highly polar mycotoxin. Mean intra-day accuracy ranged from 85.8% to 116.4%, and intra-day precision (n = 6) ranged from 1.6% to 12.5% RSD for all mycotoxins except α-ZOL where mean accuracy ranged from 72.9% to 97.2%. Inter-day accuracy and precision were 85.6%–111.5% and 2.7–15.6% RSD respectively, showing good analytical performance of the method for biomonitoring.

Improvement and validation of a high-performance liquid chromatography in tandem mass spectrometry method for monitoring of omeprazole in plasma.

Omeprazole (OME) is a proton pump inhibitor with a 58% bioavailability after a single oral dose. It is subject to marked interindividual variations and significant drug-drug interactions. The authors developed a simple and rapid method based on liquid chromatography in tandem with mass spectrometry with solid phase extraction and isotope-labeled internal standard to monitor plasma levels of OME in pharmacokinetics and drug-drug interaction studies. OME and its internal standard (OME-D3) were eluted with a Zorbax Extend C-18 rapid resolution column (4.6 × 50 mm, 3.5 μm) at 25°C, under isocratic conditions through a mobile phase consisting of 1 mM ammonium acetate, pH 8.5 (55%), and acetonitrile (45%). The flow rate was 0.8 mL/min, and the chromatogram run time was 1.2 minutes. OME was detected and quantified by liquid chromatography in tandem with mass spectrometry with positive electrospray ionization, which operates in multiple-reaction monitoring mode. The method was linear in the range of 1.5-2000 ng/mL for OME. The validation assays for accuracy and precision, matrix effect, extraction recovery, and stability of the samples for OME did not deviate more than 20% for the lower limit of quantification and no more than 15% for other quality controls. These findings are consistent with the requirements of regulatory agencies. The method enables rapid quantification of OME concentrations and can be used in pharmacokinetic and drug-drug interaction studies.

Determination and Validation of a Solid-phase Extraction Gas Chromatography-mass Spectrometry for the Quantification of Methadone and Its Principal Metabolite in Human Plasma.

This study aimed to develop a solid-phase extraction gas chromatography-selected ion monitoring-mass spectrometry method for the determination of methadone (MDN) and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in human plasma. The linear response was obtained over the concentration range from 10 to 2000 ng/mL for MDN and EDDP. The absolute recoveries of MDN and EDDP were 95.9%–98.9% and 94.8%–102.4%, with relative standard deviation (RSD) ranging from 1.8% to 2.7% and 1.8% to 3.9%, respectively. The intra- and interday precisions were found to be less than 5% for both analytes. The limits of detection of MDN and EDDP were 4 and 5 ng/mL, respectively. The presented method was convenient for therapeutic drug monitoring and pharmacokinetic studies in patients on heroin-assisted MDN therapy.

Evolution of the Krebs cycle during the prepubertal‐pubertal transition in rat mammary gland

In rat models of mammary carcinogenesis, the highest incidence of carcinomas occurs when the animals are exposed to carcinogens during puberty (typically postnatal day 50). Gene expression analysis of rat mammary gland in Sprague‐Dawley rats revealed a consistent down‐regulation of Krebs cycle genes in tissues from 50‐day old animals relative to 21‐day animals. This result was confirmed at the protein level by Western blot analysis and assessment of specific activity of selected Krebs cycle enzymes; succinate dehydrogenase (SDH)‐B, fumarase (FH), NAD+ isocitrate dehydrogenase (IDH3A) were significantly reduced at late puberty (day 50) compared with prepubertal (day 21) in the mammary gland. A nanoLC‐multiple reaction ion monitoring‐mass spectrometry method was developed that measures each of these proteins in a single assay in tissue and mitochondrial preparations. The results reported here support the gene expression data obtained by microarray analysis and suggest that the increased vulnerability of the rat mammary gland at late puberty to breast cancer‐inducing carcinogens is correlated with an overall down‐regulation of aerobic metabolism. Supported by NCI U54 CA100949.

In vitro stability of triclosan in dentifrice under simulated use condition

Triclosan has been formulated into a dentifrice at a 0.3% level to enhance the antibacterial function of the dentifrice, to improve oral health and to decrease the daily malodor inside the mouth cavity. The hypothesis that chloroform may be generated from triclosan when contacted with chlorinated drinking water has challenged our guarantee of safe use of triclosan in oral care products, especially in Colgate Total toothpaste. Currently, there was no available analytical method to detect chloroform levels under the use conditions expected during daily tooth brushing. To fill this gap and to continue guaranteeing that our customers can safely use Colgate Total toothpaste products, a gas chromatography-single ion monitoring-mass spectrometry method for detecting chloroform in artificial saliva media has been developed. The limit of detection (LOD) and limit of quantitation are about 41 and 130 ppb, respectively. This LOD level is lower than the current Environmental Protection Agency trihalomethanes contamination limit, which is required for our daily drink water. Our in vitro study indicated that Colgate Total does not form detectable chloroform levels (41 ppb) over the range of expected consumer-brushing times while using normal chlorinated drinking water.

Effects of Dietary Habits on the Serum Isoflavones Levels

Purpose: Recent studies have suggested that isoflavones have an inverse correlation with the risk of prostate cancer. In addition, the serum isoflavones levels are thought to be decided not only by the level of intake of isoflavones, but also by the metabolic processes or the genetic abilities required for ingestion of isoflavones. So, we conducted this study to determine the effects of dietary habits on the serum isoflavones levels. Materials and Methods: One hundred age- and community-matched healthy men between the ages of 10 and 59 years were interviewed using a food frequency questionnaire that was developed and validated for Korean populations. The individuals’ dietary habits during the previous one-year period and the foods they ingested during the previous one-week period before blood sampling were assessed. The serum concentrations of isoflavones were analyzed by the reversed-phase high performance liquid chromatography-multiple reaction ion monitoring-mass spectrometry method(HPLC-MS), (SRL Co. Tokyo, Japan). Results: The genistein and daidzein levels were significantly correlated with age(p=0.026 and p=0.016, respectively), but the equol level was not (p=0.091). The foods associated with the genistein level were unmilled rice, beans, garlic and etc. The foods significantly related to the daidzein level were unmilled rice, garlic and etc. Of the foods ingested during the previous one-week period, fermented soybean soup, garlic, strawberries and mung-bean pancakes were associated with the equol level. Conclusions: The genistein and daidzein levels were associated with usual dietary habits, but the equol level was related to the short-period food consumption. Changes in dietary habit might induce significant changes in the genistein and daidzein levels.