Quantification of short-chain fatty acids in human stool samples by LC-MS/MS following derivatization with aniline analogues.

The gut microbiome produces a range of short chain fatty acids (SCFA) crucially linked with diet and nutrition, metabolism, gastrointestinal health and homeostasis. SCFA are primarily measured using gas or liquid chromatography-mass spectrometry (LC/MS) after undergoing chemical derivatization. Here we assess the merits of a derivatization protocol using aniline and two aniline analogues (3-phenoxyaniline and 4-(benzyloxy)aniline) for the targeted LC-MS/MS quantification of nine SCFA (acetic, propionic, butyric, valeric, caproic acid, isobutyric, isovaleric, 2-methylbutyric, and 2-ethylbutyric acid). Evaluation of product ion spectra and optimization of MS detection conditions, provided superior detection sensitivity for 3-phenoxyaniline and 4-(benzyloxy)aniline compared to aniline. We developed a facile SCFA derivatization method using 3-phenoxyaniline under mild reaction conditions which allows for the simultaneous quantification of these SCFA in human stool samples in under eleven minutes using multiple reaction monitoring LC-MS/MS. The method was successfully validated and demonstrates intra- and inter-day accuracy (88.5-103% and 86.0-109%) and precision (CV of 0.55-7.00% and 0.33-9.55%) with recoveries (80.1-87.2% for LLOQ, 88.5-93.0% for ULOQ) and carry-over of (2.68-17.9%). Selectivity, stability and matrix effects were also assessed and satisfied validation criteria. Method applicability was demonstrated by analysing SCFA profiles in DNA-stabilized human stool samples from newly diagnosed colorectal cancer patients prior to surgery. The development of this improved method and its compatibility to measure SCFAs from DNA-stabilized stool will facilitate studies investigating the gut microbiome in health and disease.

High-Throughput Quantification of Monofluoroacetate (1080) in Milk as a Response to an Extortion Threat.

As a food defense measure against an extortion threat to poison infant formula with monofluoroacetate, a robust methodology for monofluoroacetate analysis in fluid milk and powdered dairy products was developed and optimized. Critical challenges posed by this situation required that the analytical methodology provide (i) high specificity, (ii) high throughput capable of analyzing thousands of samples of fluid milk per day, and (iii) trace-level detection of 1 ng/g or lower to achieve the maximum residue limit. Solid-phase extraction-purified acetone extracts of fluid milk were derivatized with aniline, and after ultrahigh-performance liquid chromatography using a Kinetex-C18 column packed with 1.3-µm shell particles, the resulting N-phenyl 2-fluoroacetamide could be determined by liquid chromatography-tandem mass spectrometry in a highly specific manner and with a limit of quantification of 0.5 ng/ml. By using 4-(4-chlorophenoxy)aniline as a derivatizing agent, the method could be extended to powdered dairy products with the same limit of quantification. Between January and July 2015, some 136,000 fluid milk samples were tested using this method. This analytical testing of fluid milk formed one element in a larger program of work by multiple agencies to ensure that consumers could continue to have confidence in the safety of New Zealand milk and dairy products.

Strawberry extract caused endothelium-dependent relaxation through the activation of PI3 kinase/Akt.

Polyphenolic compounds are vasodilators and help to lower the risk of cardiovascular diseases. We hypothesized that a freeze-dried strawberry powder that is rich in polyphenolic compounds would cause an endothelium-dependent relaxation (EDR) through the activation of phosphatidylinositol-3 (PI3)-kinase/protein kinase B (Akt) in rabbit aorta. The powder was prepared by freeze drying a homogenate of ripe California strawberry fruits. An aqueous extract of strawberry powder was applied to rabbit aortic rings suspended in organ baths containing Krebs-Henseleit buffer maintained at 37 degrees C. In aortic rings precontacted with norepinephrine, the extract produced a dose-dependent relaxation. The maximum relaxations elicited by the extract (73.1 +/- 1.0%) were similar to those elicited by acetylcholine (68.2 +/- 1.5%) ( n = 14 for each). The relaxation by strawberry extract was abolished by removal of the endothelium and by prior incubation with N omega-nitro- l-arginine methyl ester hydrochloride (L-NAME), confirming the essential role of endothelial nitric oxide synthase (eNOS). The responses to the strawberry were also abolished by incubation with wortmannin and LY294002, which are inhibitors of PI3 kinase. Using immunoblotting, we also demonstrated that the strawberry extract induced the phosphorylation of both Akt and eNOS in human umbilical vein endothelial cells (HUVECs) via PI3 kinase/Akt pathway. Taken together, our novel findings suggest that the EDR induced by the strawberry extract was mediated by activation of the PI3 kinase/Akt signaling pathway, resulting in phosphorylation of eNOS.

Quantitative analysis of ochratoxin A in wine and beer using solid phase extraction and high performance liquid chromatography-fluorescence detection.

A reliable and accurate method is described for the quantitative analysis of ochratoxin A (OTA) in wine and beer. The method involves the use of disposable non-polar polymeric and aminopropyl solid-phase extraction cartridges to isolate the mycotoxin from alcoholic beverages. Extracts were subsequently analysed using reverse-phase high-performance liquid chromatography-fluorescence detection with post column ammoniation to improve the limit of detection. The precision of the method determined at three levels in both wine and beer was less than 5% (RSD). Standard addition studies in both wine and beer showed that the recovery of OTA varied between 90 and 106% over a concentration range of 0.016-1.284 microg l-1. The detection and quantification limits were shown to be better than 0.004 (S/N = 3) and 0.016 microg l-1 (S/N = 10) for both beer and wine.

Fate of ochratoxin A during vinification of Semillon and Shiraz grapes.

Semillon and Shiraz grapes containing ochratoxin A (OA) were obtained by inoculation of bunches on the vine with Aspergillus carbonarius. Citric acid content was greater in the inoculated grapes than in healthy grapes. Samples were collected throughout vinification of these grapes and the OA content was quantified using a stable isotope dilution liquid chromatographic-tandem mass spectrometric method. The mass of processed and waste streams during vinification was also noted. Reduction in the amount of OA in juice and wine occurred at every solid-liquid separation stage. The OA concentration (microg/kg) in white and red wine after racking was 4% and 9%, respectively, of that in crushed grapes. This corresponds to 1% and 6% of the total OA content that was initially present in the inoculated grapes. The OA content was divided between solid and liquid phases at each stage of vinification. OA did not appear to be transformed either chemically or biologically by yeast during fermentation, rather was discarded with the marc, juice lees, and gross lees.

Analysis of acrylamide in coffee and cocoa by isotope dilution liquid chromatography-tandem mass spectrometry.

An accurate and precise method for the quantification of acrylamide using stable isotope dilution liquid chromatography-tandem mass spectrometry was developed and used to measure acrylamide in coffee and cocoa samples. The sample preparation involved extraction of the analyte and its internal standard, 13C3-acrylamide, into water and subsequent defatting of the aqueous extract with dichloromethane. An aliquot of the resulting aqueous extract was then azeotropically dried under reduced pressure and subsequently purified using an aminopropyl-bonded silica cartridge. The purified extracts were then chromatographed on a 5-microm 2.1 x 150 mm Hypercarb column, the effluent of which was monitored for the analyte and its internal standard using positive-ion APCI-selected reaction monitoring. The intra-laboratory reproducibility of the method, expressed as a relative coefficient of variation (%, n=5), was determined at four levels of concentration (12.3, 42.3, 139.3 and 464.8 microg kg(-1)) and was found to vary between 0.6-2.5%. The accuracy of the method was assessed using a reference sample of coffee. The average result obtained using our method differed from the assigned value of the reference material by less than 1%. An analysis of a cocoa sample revealed that the method is capable of precisely estimating acrylamide in challenging matrices down to a level of at least 12.3 microg kg(-1).