Food Chemistry 201 (2016) 270–274 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Analytical Methods Detecting ethanol and acetaldehyde by simple and ultrasensitive fluorimetric methods in compound foods ⇑ M. Zachut, F. Shapiro, N. Silanikove Biology of Lactation Laboratory, Department of Ruminant Sciences, Institute of Animal Sciences, Agricultural Research Organization, Volcani Center, Bet Dagan 50250, Israel article info abstract Article history: There is a need for simple, accurate, and rapid analysis of ethanol (Eth) and acetaldehyde (AA) in a wide Received 15 July 2015 variety of beverages and foods. A novel enzymatic assay coupled to formation of fluorescent chro- Received in revised form 20 October 2015 mophore is presented. Eth detection was further improved by adding semicarbazide to the reaction mix- Accepted 19 January 2016 ture, which interacts with AA and prevents its inhibitory effect on Eth oxidation. The limits of detection of Available online 21 January 2016 Eth (0.5 mg/L) and AA (0.9 mg/L) are comparable with the performance of modern gas chromatography techniques. The repeatability of Eth and AA detection in various foods (9% on average) was lower than Keywords: that with commercial kits (23%). The high sensitivity of the developed method enables detection of AA Ethanol in common foods [e.g., bio-yogurt (12.2 mg/L), and the existence of endogenous Eth (1.8 mg/L) and AA Acetaldehyde Fluorimetric assay (2.0 mg/L) in bacteria-free non-fermented bovine milk], which could not measured so far by enzymatic methods. Ó 2016 Elsevier Ltd. All rights reserved. 1. Introduction instruments (HPLC). GC/FID has been used for the determination of Eth and AA and in saliva and alcohol beverages without sample Ethanol (Eth) and acetaldehyde (AA) are metabolites, which are preparation; however, it requires relatively high sample volumes produced during fermentation processes and are commonly pre- ( 450 ll) and dedicated equipment (Homann et al., 1997; sent in fermented beverages and foods. They may therefore be pre- Linderborg, Salaspuro, & Väkeväinen, 2011). A particular problem sent in humans’ biological fluids. In mammals, AA is the main with the GC/FID method is formation of artificial AA due to Eth oxi- product of Eth oxidation in the liver (Hipólito, Sánchez, Polache, dations, which requires particular procedures to account for the & Granero, 2007). Accurate measurements of Eth and AA are there- problem (Fukunaga, Silanaukee, & Eriksson, 1993). Consequently, fore required in a variety of matrices, such as alcoholic beverages, the availability of an analytical method that is simple, rapid, foodstuffs, cosmetics, and pharmaceuticals. Moreover, measure- cost-effective and accurate for the determination of Eth and AA is ment of Eth and AA in the blood plasma and other biological fluids desirable. AA is a toxic substance, a class 1 carcinogen and muta- is of particular importance for the diagnosis and treatment of genic at concentrations of 50–100 lM (2.2–4.4 mg/L) (Seitz & alcohol-use disorders, as biomarkers for several diseases, in Stickel, 2007). Recently, mutagenic levels of AA have been reported acute intoxications, and in forensic settings (Schlatter, Chiadmi, in various foods (Lachenmeier & Sohnius, 2008; Uebelacker & Gandon, & Chariot, 2014). Lachenmeier, 2011). Although the detection limit of modern GC Many methods, such as gas-diffusion flow-injection analysis methods is sufficient to detect mutagenic levels of AA in foods (FIA), electroanalysis, FIA-electroanalytical detection, infrared (IR) (Lachenmeier & Sohnius, 2008; Pontes et al., 2009), this method spectroscopy, direct injection gas chromatography (GC)/flame ion- is quite cumbersome for routine analyses. Conversely, current ization detection (FID), headspace injection GC/FID, high- enzymatic analyses, with detection limits above 10 mg/L (Beutler, performance liquid chromatography (HPLC)/Fourier transform 1988), are not suitable for detecting mutagenic levels of AA. Thus, (FT) and others have been developed for analyses of Eth and AA the need for a fast and versatile method to determine low levels of (Jain & Cravey, 1972a, 1972b; Ramdzan, Mornane, McCullough, AA in various beverages and foods is of particular importance. Mazurek, & Kolev, 2013; Schlatter et al., 2014). However, some of Enzymatic methods that utilize alcohol dehydrogenase (ADH) these methods are not sufficiently accurate (e.g., older versions of and AA dehydrogenase (AADH) are well-known and frequently GC; Schlatter et al., 2014), some require complex and expensive used to analyze Eth and AA in biological specimens (Beutler, 1988; Redetzki & Dees, 1976). ADH oxidizes Eth to AA and AADH ⇑ Corresponding author. oxidizes AA to acetic acid. Both enzymes use nicotinamide adenine + E-mail address: [email protected] (N. Silanikove). dinucleotide (NAD ) as coenzyme, which is reduced during the http://dx.doi.org/10.1016/j.foodchem.2016.01.079 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved. M. Zachut et al. / Food Chemistry 201 (2016) 270–274 271 reaction to form NADH. NADH formation is stoichiometrically 2.3. Reaction mixtures linked to the oxidation of Eth and AA. Thus, the concentration of NADH in specimens can be used to monitor the concentrations of All reagent solutions were prepared fresh once a week with metabolites formed by NAD+ dependent dehydrogenases spec- double-distilled water. For Eth and AA determinations according to trophotometrically or fluorometrically (Beutler, 1988; Redetzki & Option 1 (Fig. 1), the reaction mixture was composed of 1 mM Dees, 1976; Shapiro, Shamay, & Silanikove, 2002). However, mea- NAD+,48lM resazurin, 1U/mL, diaphorase, 100 mM KCl, 0.0004% suring NADH in foods and other matrices is frequently difficult (w/v) Triton X-100, and 50 lL ADH (10 kU/mL) and 10 lL AADH and problematic because: (i) these substances contain fat droplets (75 U/mL) dissolved in 50 mM potassium phosphate buffer, pH 7.6. of varying sizes that scatter light in an unpredictable way; (ii) as a For the separate determination of AA, addition of ADH was omitted . result of their opaque and colloidal properties, they scatter and InOption2 (Fig.1), thestocksolutionforAAwaspreparedwithout absorb light; (iii) they frequently contain intense colorants that ADH and contained 75 mM semicarbazide and 130 mM glycine. interfere with the monochromatic absorbance. NADH can be deter- mined by fluorometric means, which are free of these limitations. 2.4. Standard curves However, because additional indigenous biological substances emit light in the same range, fluorometric determination of NADH Stock solutions were prepared by dissolving 100 mg/mL of Eth is associated with considerable background noise, which reduces or AA in double-distilled water. Standards were prepared by seri- the sensitivity of the method (Shapiro & Silanikove, 2010, 2011; ally diluting the stock solutions of the test substances in distilled Shapiro et al., 2002). water to yield concentrations of 1, 2.5, 5, 10, 25, 50, 100, 250, A general solution for measuring metabolites that are involved 500 and 1000 mg/L. in reactions of NAD+-coupled dehydrogenases is to combine the reaction to another set of coupling reactions: diaphorase (EC 2.5. Reaction procedures 1.6.99.1) oxidizes NADH to NAD+, and this reaction can be coupled to the conversion of non-fluorometric resazurin to the highly fluo- All procedures were carried out in the wells of a 96-well micro- rochromophoric substance resorufin (Shapiro & Silanikove, 2010, plate suitable for fluorometric reading. Reaction mixture (100 lL) 2011; Shapiro et al., 2002). To date, this methodology has been and test solution (10 lL) (standard or test samples) were incubated found useful for accurate determination of D- and L-lactate, lactose, together in the wells for 30 min at room temperature. The plates galactose citrate, malate pyruvate and oxaloacetate in milk, were read in a fluorometer (ELx800, BioTek Instruments, Winooski, yogurts and colored drinks, such as red wine and beer, without VT, USA) at excitation and emission wavelengths of 540 and the need for pretreatments (Shapiro & Silanikove, 2010, 2011). 590 nm, respectively. Oxidation of Eth is particularly sensitive to inhibition by its pro- duct, AA (Kristoffersen, Skuterud, Larssen, Skurtveit, & Smith- Kielland, 2005; Kristoffersen & Smith-Kielland, 2005). A potential 2.6. Biological and food samples solution to this problem is to force the reaction toward completion, thereby overcoming product inhibition. When semicarbazide was Milk was sampled from the commingled milk of six cows with added to a reaction solution containing AADH and NAD+, it reacted bacteria-free udders. Bacteria-free samples were defined as the with AA to form semicarbazone, which does not inhibit the reaction rate (Kristoffersen & Smith-Kielland, 2005; Kristoffersen et al., 2005). In that modification, NADH was determined spectrophoto- metrically, suggesting that the sensitivity and range of biological sources without sample preparation might be improved by apply- ing the fluorometric determination of resorufin, as already noted. Hence, the objective of this study was to apply the above- described modifications to improve the detection of Eth and AA in compound beverages and foods. 2. Materials and methods 2.1. Chemicals The following chemicals were obtained from Sigma (Rehovot, Israel): ADH (EC 1.1.1.1), AADH (EC 1.2.1.5), diaphorase from Pseu- domonas fluorescens (100 U/L), AA, Eth (AA and Eth standards are