Abstract-Overview UV-visible scanning spectroscopy is seeing a resurgence as a general method for food analysis and towards a rapid authentication of alcoholic beverages. Distilled spirits have, up until now, been the main focus of the application of scanning of alcoholic beverages in the UV and visible regions of the electro-magnetic spectrum. Such analyses extending analytical capabilities to those lacking more expensive and complex NIR instruments; the latter mode of spectroscopy also seeing application with respect to beverages and foods in academic and larger beverage manufacturer operations. A number of spectrophotometers have been developed dedicated to authentication or “trueness to type identification” of distilled spirits. Publications, from academia, dealing with uv-visible analyses of distilled spirits and wine analyses have appeared within the last decade or so with a couple dealing with beer analyses; though only a handful in total - yet the benefits for analyses in the beverage industry are enormous in terms of quality, safety and economic returns. The methods are, however, only just now beginning to see acceptance and adoption within the alcohol beverage industry. Coupled with chemometrics analyses, and through the building up of libraries of beverage spectral data, authentication of beverages and testing for adulterated products is seen to be possible. However, we propose the method also as a rapid fingerprinting - or blunt force - approach for monitoring quality and consistency of beverage production and for beverage formulation especially for small-scale production facilities; craft brewers, craft distillers and wineries.

Some instruments are designed for field work and are really only dedicated to this type of application requiring, as discussed below, some manipulation of

instrument parameters by the operator. Other units are only applicable in the laboratory setting. We noted benefits to using microplate reading technology using economical approaches for the analyses as described below, while still allowing the flexibility of using such instruments for many other types of analyses. If performed on a scanning micro-plate reading spectrophotometer the method is rapid, precise and cost effective with low sample volume permitting light transmission through the short path-lengths required for recording data from darker-colored beverages. The use of small volume additions to multi-well plates obviates the need, in many instances, for dilution of the beverages which can affect the overall spectral characteristics of the samples while still maintaining suitable detector response. Multiple samples are tested in minutes in near real-time in the laboratory as could be the case also in the production and bottling facilities.

Brewers, winemakers and distillers are finding more uses for spectroscopy in routine analyses such as for color determinations, bitterness analyses of beers, free amino nitrogen (FAN) nutrient analyses for efficient fermentation, polyphenols, antioxidant activities, enzymatic analyses of alcohol, sugars, acids and other analytes of interest (described in appropriate methods of analyses manuals and by regulatory bodies dealing with their interests and specific areas and not further referenced here). It may be of benefit for those in these beverage areas to consider scanning instruments and microplate reading technologies for analytical flexibility especially as the cost of such instruments has come down dramatically within the last two decades. This brief review focuses on some new types of testing to support product development and quality, authenticity, safety, and consistency. It is intended as

BDAS, LLC WPSP#1 - 2015

Scanning UV-Visible Spectroscopy and Beverage Quality, Consistency and Authentication: Preliminary Fingerprinting Application in the Analysis of a

Wide Variety of Alcoholic Beverages – A Brief Application Note

Gary Spedding, Ph.D.

[Brewing and Distilling Analytical Services, LLC, 1141 Red Mile Road, Suite 202, Lexington, KY 40504.] Tel. (859) 278-2533. Website: www.alcbevtesting.com.

Correspondence: gspedding@alcbevtesting.com

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a short introduction only to a rapidly developing area of potential quality control analysis for the alcohol beverage industry.

Introduction This short review covers developments in the UV-visible scanning of and analysis of alcoholic beverages (1-8). The craft brewing and distilled beverage industries are creating new products at a rapid pace while at the same time being under a constant attack from counterfeiters and those who would clone-copy, dilute or adulterate beverages for their own economic gain (3, 5, and 8). It is estimated that the distilled spirits industry alone incurs losses of hundreds of millions of dollars annually based on counterfeit production around the world (5). As a result of such economic and regulatory issues distilled spirits – Tequila and Whisky in particular, have been the focus of fingerprinting or “true-identity” evaluations to ensure authentication and to look for adulterations of product in trade (1-8). Such investigations have included rapid analysis (and later application of chemometric evaluation; 1-4, 7, 9) by means of various types of spectroscopy including UV-visible scanning analysis. Studies to discriminate wines (10, 11) and specialty beers (12) have also been undertaken. Academic studies make use of sophisticated and expensive broad-range application instruments whereas a number of simpler and dedicated spectrophotometers are reaching the market for laboratory use within the industry and even, with miniaturized instruments, for off-site or field analyses such as in bars and restaurants, etc. (5, 6).

Specialized field-based instruments are available from a number of manufacturers often following on from their development in academic settings (5, 6). Usually instruments rely on single sample analysis at a time to build up a library of “fingerprints” which can be compared with fresh samples to ensure either product batch consistency or authentication as to brand. With such analysis undiluted samples are pumped into the spectrophotometer, one at a time and through variable path-length cells (often interchangeable) prior to a complete scan, and examined typically from 200 to 500 nanometers (or up to 1000 nm) to obtain the “profile” of the sample (2,4-9, 12). We recently took advantage of micro-plate technology to scan multiple samples or replicates with low volume sample use providing the short path-lengths needed to examine undiluted samples. The method has proven reliable, fast and economical and can return data on many samples within a few minutes (17). This can provide for near real-time analysis of production batches as well as for evaluation of suspected adulterated samples obtained from the trade. For distilled spirits the samples can be tested in as low of a volume as 50 microliters without the need for dilution which can affect the spectral properties of chemically complex beverages. Beer and sherry samples (for example) do unfortunately often need to be diluted but do give decent results; for beer complex polymeric compounds responsible for the color of the product breakdown upon dilution thus changing the spectral properties (see 13 for a review of the complexities of measuring color in beer) and such

Figure 1: A selection of distilled spirits: Top down – Aged tequila, 3 Bourbon brands, Canadian whiskey, a Blended Scotch whisky and a Brandy.

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might be the case with other components changing the overall fingerprint profile upon sample dilution; alcoholic beverages composed of several hundred constituents providing a complex matrix for such analyses. The issues with respect to sample dilution with beer samples was also discussed elsewhere (12). Nevertheless, either with or without sample dilution it seems the method can be applied to a wide range of alcoholic beverages and provide solid and reliable results as to quality – consistency, brand authentication and potential adulteration of the spirit, liqueur, wine or beer. The method may be used alone or in conjunction with powerful chemometric evaluations of the spectral data to further discriminate different brands or types/styles of beverages (1-4, 7, 9, and 10). Recent developments in chemometric analyses are, in our opinion, expected to play a greater role in this arena as more testing of alcoholic beverages takes place. Our lab in collaboration with a British University team is looking into the application of chemometrics to further discriminate samples and map styles.

Experimental Protocols and Systems Assessment Samples of most distilled spirits, 1:10 distilled water-diluted sherry samples or beer/malt beverages were loaded as 50 or 100 microliter additions into micro-wells (Costar Corning #3635 UV transparent 96-well plates) avoiding bubbles; typically up to eight wells per sample were examined in each run to test for any variance in data through optical transmission through the base of the plate and within the individual cells

and usually no blank wells needed to be scanned; thus maximizing sample testing. Three wells per sample are sufficient, in our opinion, once a library of data has been established for a product to assist in subsequent testing of newer or suspect batches of the same beverage. Carbonated beverages were first degassed. Plates were then read in a BMGLABTECH SPECTROStar Nano scanning spectrophotometer in 1 nm increments from 220 to 1000 nm; path-length correction was set to “off ”. Averaged data from the individual sets were evaluated and plotted using spreadsheet programs (either internal or external to the manufacturers’ software). Simple Z-score statistical evaluations were made to look for occasional outlier data from wells measured from within a set. Deviation may be caused by slight imperfections in the plastic micro-plates, if the wells are soiled or otherwise contaminated, if the correct volume from multi-pipette additions are not even across the range of wells or if any air bubbles are present after pipetting such low volumes. Only occasionally do we see any such issues. The method and testing should be amenable to newly trained non-laboratory personnel (including beverages sales teams) without the need to perform statistical analyses for routine quality evaluations in the beverage plant or the field. While many instruments on the market are useful for such analyses, as described herein, we believe that many benefits are offered through the use of a scanning plate-reading instrument. As the preferred instrument in our own facility (used in other studies, 18 and 19) we discuss such advantages in more detail elsewhere

Figure 2: Five sherry samples with different aging periods and characteristics.

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(17) though provide references here detailing the use of other instruments including those dedicated simply to distilled spirits authenticity testing and product quality evaluations. One key advantage with micro-well plate reading is the ability to ensure a narrow path length via small volume sample addition. In other instruments there is sometimes a need to switch path-length devices which may slow down analysis time. In addition only a single sample can be tested via pumping sample through such units.

Regardless as to single or multiple analyses at one time the ability to test many types and styles of beverages is the main aim of our short discourse here; providing what we feel to be an important introduction to the industry. The reader will be able to evaluate the advantages and disadvantages of each type of instrument as it applies to their specific needs and situation. The literature on this topic is limited and largely represented in our reference list. This literature will, however, hopefully assist both the beverage manufacturer in assessing the scope of this methodology and the manufacturers of instruments applicable to this field of testing. Such manufacturers are now reaching out to the beverage industries in regards to authenticity issues and routine evaluations. Furthermore, an understanding of the power of the technique should lead to an expansion of the database of beverage information. So far tequila, whiskey (1-8), a few wines (10, 11), and a specialized beer style (12) have been evaluated and detailed in the literature. We have expanded on beer analyses and hope to apply some chemometric evaluations in an ongoing collaboration

with another research group in the near future. The evaluation and assessment of many more beverages are now possible through the use of UV-visible spectroscopy. Such results will impact quality, safety, the collection of taxes and a reduction in economic losses to the industry through detection of fake and adulterated products in the marketplace and possibly via “authentic seals of approval” via regulatory testing bodies. Application of the technique in analyzing competitor samples and product formulation are also possible through this methodology. Rapid aging for measuring deterioration of quality or rapid aging/maturation of spirits may also be examined via “fingerprinting” and monitoring changes in Maillard reaction components (for example) and “bleaching” of samples as noted by specific regional peak and shoulder changes (unpublished observations and see below).

Results and Conclusions Typical data for a range of distilled spirits – white or raw un-aged spirits and aged (usually exhibiting color derived during maturation) are shown in Figure 1, five sherry samples in Figure 2, several Bourbon barrel aged beers in Figure 3 and Pilsner beers in Figure 4. The data show clear discrimination between different spirit types, different brewer’s beers and differentially (naturally) aged sherry samples. Multiple repeat measurements on each sample and in several cases replicates (e.g., different bottle and canned batches of samples) showed little variability both between runs and within runs (see Fig. 4). Figure 1 shows the power of the method in discriminating between different classes of distilled spirit and between

Figure 3: A selection of Bourbon barrel aged ales and stouts.

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three different brands of the same style, namely Bourbon whiskey. For all alcoholic beverages there is a broad band centered around 280 nanometers. Though the actual peak maxima and the amplitudes in this region differ (as seen upon close inspection of the figures) between different classes and styles of beverage (1-4, 8, 9 with beer perhaps centered at closer to 270 nanometers, 12 and see Figures 3 and 4). The constituents represented under this peak region have been investigated closely in the case of tequila (4, 9) and are seen to be complex Maillard reaction products such as furfural although further work is needed for other alcoholic beverages; some specific chemical constituent information is ascribed to spectral regions in the case for certain wines (10). Figure 2 shows the profiles of imported sherry examples which had seen different regimens and or times of aging. One well aged example was so dark that it was off-scale over the region of 220-350 nm and was excluded from the present data set – though showed a clear distinction from the others. Comparisons should always be made on undiluted samples whenever possible or on similarly diluted samples and with the same sample volume loaded into the micro-wells. For the sherry samples there are slight shifts away from 280 nanometers as peak maxima especially with the more “robust” or longer aged examples. Figure 3 shows the profiles for a selection of Bourbon barrel aged ales and stouts. The examples are of more or less robustly flavored products with closely similar profiles here on two brands (which showed clear organoleptic differences) and discrimination

against other barrel aged beers. Figure 4 shows data for three samples of the same Pilsner-style beer. Obtained from a major US brewery two canned batches and 1 bottled batch show the expected consistency of product – with closely overlapping “fingerprints”. A craft-brewed example Pilsner is shown for comparison and shows a higher amplitude fingerprint across the spectrum. Thus discrimination of brands of different types of alcoholic beverages are possible using UV-visible spectroscopy alone. Such fingerprinting may also be used to study batch to batch consistency and possibly formula changes. Furthermore, (data not shown) we have been able to illustrate differences between forced aged vs. un-aged beverages and the presence of spirit grade caramel when added to vodka samples. Other additives or adulterants (with unique spectral properties) might also be detected using the scanning methods outlined here (5, 8); artificial colors (used in some modern distilled spirits and cocktails) for example can be discriminated through spectral analysis (14, 15). Most of the useful data are collected between 220 and 550 nanometers and profiles tend to coalesce closer and closer to the zero baseline from 550 to 1000 nanometers though the water and alcohol activities are seen in the 950-1000 nanometer region (16). We do note that some instruments can read down to the 200 nanometer region and can provide some additional useful information pertaining to the composition and profiling of beverages in the 200-220 nanometer region of the UV spectrum. This region is not adequately covered by the instruments we currently

Figure 4: Three batches of a US Pilsner beer - 2 canned and 1 bottled version in comparison with (upper curve) a craft-brewed Pilsner

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use. Those laboratories also using Near (and Mid-range) Infrared spectroscopies can extend analyses in the direction beyond 1000 nm (or wavenumbers).

The method has proven to be fast, reliable and economical for our testing of various beverages on a daily basis and should prove useful for quality consistency testing requiring little operator effort in the modern production environment. We, therefore, feel there is a lot of potential for re-evaluating beverages via scanning UV-visible spectroscopy and especially with more rapid analyses possible with plate-reading scanning spectrophotometers such as used in our laboratory (17). The application of chemometric analyses and vector support machines has been noted (1-4, 7, 9, 10) in conjunction with UV-visible spectroscopic data and could also find wider application in this type of work. With thus far limited studies it seems the time is ripe for further investigations and testing for routine quality control using UV-visible and possibly other types of spectroscopy within a growing field of beverage production. We would be happy to discuss further the applications of this older though still very useful technology.

References(1) O. Barbosa-Garcia, G. Ramos-Ortiz, J.L. Maldonado, J.L. Pichardo-Molina, M.A. Meneses-Nava, J.E.A. Landgrave, and J. Cervantes-Martinez. UV-vis absorption spectroscopy and multivariate analysis as a method to discriminate tequila. Spectrochimica Acta Part A. 66, 129-134 (2007).

(2) O. Barbosa-Garcia, G. Ramos-Ortiz, J. Pichardo Molina, J.L. Maldonado, M.A. Meneses Nava, and J.E.A. Landgrave. UV-Vis absorption spectroscopy and chemometrics to discriminate between the two basic categories and types of tequila. Proc. of SPIE: Sixth Symposium Optics in Industry. J.C. Gutierrez-Vega, J. Davila-Rodriguez, and C. Lopez-Mariscal, (Eds). 6422, 64221W1-W6 (2007).

(3) U. Contreras, O. Barbosa-Garcia, J.L Pichardo-Molina, G. Ramos-Ortiz, J.L. Maldonado, M.A.Meneses-Nava, N.E. Ornelas-Soto, and P.L Lopez-de-Alba. Screening method for identification of adulterate and fake tequilas by using UV-VIS spectroscopy and chemometrics. Food Research International. 43 (10), 2356-2362 (2010).

(4) A.C. Munoz-Munoz, O. Barbosa-Garcia, G. Ramos-Ortiz, J.L. Pichardo-Molina, J.L Maldonado, M.A. Meneses-Nava, and P.L. Lopez de Alba. UV-vis Spectroscopy and Multivariate Calibration (PLS) as a Tool for Identification and Classification of Tequilas. In: Proceedings 3rd International Workshop on Alcoholic Beverages Authentication. C. Guillou, and J. Ryder (Eds). European Commission, Joint Research Center. 71-79 (2007).

(5) J. Ferguson, K. Reid, and J. Gilchrist. Current Developments in in-bar Authentication of Spirits. In: Proceedings 3rd International Workshop on Alcoholic Beverages Authentication. C. Guillou, and J. Ryder (Eds). European Commission, Joint Research Center. 149- 156 (2007).

(6) W.M. MacKenzie and R.I. Aylott. Analytical strategies to confirm Scotch whisky authenticity. Analyst.129, 607-612 (2004).

(7) G. R. Jones, A.G. Deakin, and J.W. Spencer. Chromatic signatures of broadband optical spectra for liquor discrimination. Meas. Sci. Technol. 20, 1-10 (2009). 025304 (http://iopscience.iop.org/0957-0233/20/2/025304).

(8) E.C.L Nascimento, M.C.U. Araujo, and R.K.H. Galvao. A Flow-Batch Analyzer for UV-Vis Spectrophotometric Detection of Adulteration in Distilled Spirits. J. Braz. Chem. Soc. 22 (6), 1061- 1067 (2011).

(9) A.C. Munoz-Munoz, J.L. Pichardo-Molina, G. Ramos-Ortiz, O. Barbosa-Garcia, J.L. Maldonado, M.A. Meneses-Nava, N.E. Ornelas-Soto, A. Escobedo and P.L. Lopez-de-Alba. Identification and Quantification of Furanic Compounds in Tequila and Mezcal using Spectroscopy and Chemometric Methods. J. Braz. Chem. Soc. 21 (6), 1077-1087 (2010).

(10) F. J. Acevedo, J. Jimenez, S. Maldonado, E. Dominguez, and A. Narvaez. Classification of Wines Produced in Specific Regions by UV-Visible Spectroscopy Combined with Support Vector Machines. J. Agric. Food. Chem. 55, 6842-6849 (2007).

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(11) M.J. Martelo-Vidal and M. Vazquez. Evaluation of Ultraviolet, Visible, and Near Infrared Spectroscopy for the Analysis of Wine Compounds. Czech J. Food Sci. 32 (1), 37-47 (2014).

(12) J. Weeranantanaphan and G. Downey. Identity Confirmation of a Branded, Fermented Cereal Product by UV Spectroscopy: A Feasibility Study Involving a Trappist Beer. J. Inst. Brew. 116 (1), 56– 61 (2010).

(13) A. J. deLange. The Standard Reference Method of Beer Color Specification as the Basis for a New Method of Beer Color Reporting. J. Am. Soc. Brew. Chem. 66 (3), 143-150 (2008).

(14)http://chemw ik i.ucdav is .e du/Organic_C h e m i s t r y / O r g a n i c _ C h e m i s t r y _ Wi t h _ a _B i o l o g i c a l _ E m p h a s i s / C h a p t e r _ 0 4 % 3 A _S t r u c t u r e _ D e t e r m i n a t i o n _ I /S e c ti on_4.3%3A_U ltr av i ol et_and_v isibl e_spectroscopy (last accessed March 8, 2015).

(15)http://www.rsc.org/learn-chemistry/resource/res00000941/spectroscopy-in-a-suitcase-uv-vis-teacher-resources?cmpid=CMP00000328 (last accessed March 8, 2015).

(16)http://www.a-a-inc.com/documents/AA_AN012_Measuring-Proof-and-Color-in-Whiskey.pdf (last accessed March 8, 2015).

(17) G. Spedding and C. Peters. Authentication and Quality Testing of Distilled Spirits Using the SPECTROstar Nano – Application Note 277. Rev 01/2015. http://www.bmglabtech.com/media/35216/1308715.pdf (last accessed March 8, 2015).

(18) G. Spedding. The World’s Most Popular Assay? A Review of the Ninhydrin-Based Free Amino Nitrogen Reaction (FAN Assay) Emphasizing the Development of Newer Methods and Conditions for Testing Alcoholic Beverages. J. Am. Soc. Brew. Chem. 71 (2), 83-89 (2013).

(19) M. R. Schmitt and Budde, M. Wort Free Amino Nitrogen Analysis Adapted to a Microplate Format. J. Am. Soc. Brew. Chem. 70 (2), 95-102 (2012).

Gary Spedding. Ph.D. is managing owner at Brewing and Distilling Analytical Services, LLC (BDAS, LLC) in Lexington KY specializing in a field where various modes of spectroscopy find application in the evaluation of alcoholic beverages and beverage raw materials. BDAS, LLC tests beer, wine, distilled spirits and beverage raw materials including, hops, malt and water. Performing routine and specialized chemical, physical, microbiological and sensory testing and providing certificates for export. BDAS, LLC is a laboratory certified by the US agency - the TTB.

Please direct any correspondence to:gspedding@alcbevtesting.com.

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