AOAC 133rd Annual Meeting - Final Program

Presenter: Barry McCleary, Megazyme, Bray, Ireland, Email: barry@megazyme.com P-T-006 Markus Lacorn , Kathrin Endres , Rebecca Ziegler , Thomas Hektor , R-Biopharm AG, Darmstadt, Germany Enzymatic Quantification of Acetic Acid: Accuracy is Driven by Calibration According to EU regulation 606/2009, maximum levels for volatile acid (calculated as acetic acid) is 1.08 and 1.20 g/L in white and red wine, respectively. Besides the official method OIV-MA-AS313-02 (water steam distillation followed by titration), acetic acid alone is often measured as a surrogate for volatile acid by an enzymatic method. Beside its apparent simplicity and the possibility for automation, these methods are often characterized by non-accurate results, which are related to a missing end point of the reaction. In consequence, a non-linear calibration graph results. In consequence, any cali- bration for such a system needs more than two calibrators. Here, we present a true and precise enzymatic assay for quantification of acetic acid in wine and juice. It is based on an acetate kinase activity that converts acetate to acetylphosphate and in turn ATP to ADP followed by an indicator reaction that produces NADH. The assay consists of two ready-to-use reagents and four calibrators. We investigated a 2 nd order polynomial calibration model with four concentrations and tested the calibration stability under controlled temperature conditions. The measurement range is between 0.05 and 1.3 g/L. The assay can be used manually at 25°C and automated at 37°C. For a typical analytical setup, 2000 µL reagent 1 is incubated with 100 µL sample for 3 min at 25°C and measured at 340 nm afterwards. 500 µL of reagent 2 is added, incubated for 15 min and measured at 340 nm. Presenter: Markus Lacorn, R-Biopharm AG, Darmstadt, Germany, Email: m.lacorn@r-biopharm.de P-T-007 Jeffrey Shippar , Eurofins, Madison, WI, USA; Christophe Fuerer , Nestlé, Lausanne, Switzerland Detection of A1 Beta-Casein in Milk Powders by UHPLC-MS/MS Bovine milk is an important global food source that provides significant amounts of protein within the diet. Among the most common proteins found in milk is beta-casein, of which there are several variants. Two of the most prevalent variants include the A1-type and A2-type beta-caseins. These variants are found in different cow populations, where A1 and A2 beta-casein differ in protein sequence only by the amino acid present at posi- tion 67 (histidine vs. proline, respectively). The A2 beta-casein variant in milk has garnered significant attention over proposed health benefits in recent years, and “A2 only” milk products have been specifically marketed to contain only the A2 variant of beta-casein. It is therefore important to be able to differen- tiate the “A1” and “A2” variants of beta-casein found in milk from each other, but the small (one amino acid) difference in protein sequence presents many challenges. Here a method was developed to differentiate the A1 and A2 beta-casein content in multiple milk powders. The method uses surrogate peptide analysis of digested milk powder samples and UHPLC-MS/MS to detect A1 beta-casein and also to quantify total beta-casein

60 SEPTEMBER 6–12, 2019 SHERATON DENVER DOWNTOWN HOTEL (2016), an enzymatic phosphate detection step relying on the use of purine nucleoside phosphorylase (PNPase) and 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) was investigated to replace the existing molybdenum-based Pi detection procedure. The improvements of this method to deter- mine phytic acid content over the established methodology are clear; a more rapid protocol allowing for high-throughput analy- sis, an increased linear range and lower limit of detection (LOD), a defined biochemical transformation as the detection method with phosphate content directly proportional to the product of the enzymatic reaction, thereby removing the need for a phosphate calibration curve, and reduction in the handling of hazardous chemicals and heavy metal contaminated waste streams within laboratories. 4.72 mg/kg), even though data about Ni levels in vegetables are scarce worldwide. Presenter: Sabina Pederiva, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, CReAA, National Reference Centre for the Surveillance and Monitoring of Animal Feed, Turin, Italy, Email: sabina.pederiva@izsto.it P-T-005 Barry McCleary , Bridget Culleton , Claudio Cornaggia , Ruth Ivory , Tadas Kargelis , David Mangan , Vincent McKie , Megazyme, Bray, Ireland An Improved Method to Determine Phytic Acid Content of Cereals and Foodstuffs Monogastric animals lack the enzyme phytase, which hydro- lyses the phosphate esters in phytic acid, releasing inorganic phosphate (Pi) and making it available for absorption by these animals. To minimise environmental impact arising from the addition of inorganic phosphate to animal feed, manufacturers have turned to phytase supplementation to instead liberate the phosphorus required from phytic acid. A method for the quanti- tation of phytic acid in animal feed is therefore an important tool within agriculture. In a bid to further improve the phytase/alka- line phosphatase enzymatic method of McKie and McCleary