No-D quantitative ¹H Nuclear Magnetic Resonance spectroscopy method for the determination of ethanol in distilled spirits

Highlights

• ¹H qNMR analysis for ethanol without the use of deuterated solvent.

• Study provides full method validation for ethanol quantification using no-D qNMR.

• No-D qNMR analysis for ethanol shows good linearity, precision, and recoveries.

• The developed method is selective and sensitive with a low detection limit.

• No-D qNMR method for ethanol can be used as an alternative to GC-FID measurements.

Abstract

Quantitative NMR (qNMR) is an emerging analytical method that is recommended for food samples because of its simple and rapid analysis. Commonly prepared in deuterated solvents, the use of a non-deuterated solvent (no-D) for routine analysis was explored in this study. The amount of ethanol was determined in distilled spirits using Varian 500 MHz NMR spectrometer. Ethanol standards and samples were prepared in deionized water with acetonitrile as the internal standard. Using the optimized acquisition parameters, the ¹H NMR spectra of the standards and samples were obtained. The ethanol content of distilled spirit samples obtained from no-D qNMR experiments was validated using GC-FID and the results obtained were not significantly different (p > 0.05) using ANOVA. The developed and validated no-D qNMR method was fast, simple, accurate, non-destructive, and provides an alternative technique for the analysis of ethanol in distilled spirits.

Introduction

Distilled spirits are alcoholic beverages obtained from the distillation of fermented fruit juices or wines [1]. These contain at least 15 % by volume ethanol [2], which is higher compared to beers, ciders, and wines. Some of the distilled spirits are whiskey, gin, rum, and vodka [3], [4]. In 2018, about half of the global consumption of alcoholic beverages is attributed to distilled spirits. In the Western Pacific Region, which includes the Philippines, 58.8 % of the regional consumption corresponds to the distilled spirits [5]. The ethanol content of distilled spirits has a significant role in the aroma of alcoholic drinks [6]. Alcoholic drinks are labeled with the ethanol content either as alcohol by volume (ABV) or proof, which is ABV multiplied by a factor of two. The oral burning sensation intensifies with the increase in ethanol content of the alcoholic drink. Moreover, the amount of ethanol in an alcoholic drink determines the amount of excise tax paid by the manufacturer to the government. In the Philippines, the specific tax for distilled spirits is calculated per proof liter [7]. The determination of ethanol content is not only important in the quality control of alcoholic beverages but also the computation of the taxes. Hence, it is important to develop an analytical method for ethanol that is simple, rapid, and accurate.

The AOAC instrumental method for the analysis of ethanol in alcoholic beverages uses gas chromatography with a flame ionization detector, or GC-FID [8]. It can be introduced to the column by direct injection [9] or by headspace injection [10], [11]. Alternatively, ethanol can be quantified using gas chromatography coupled with mass spectrometry (GC–MS) [12]. The other methods of analysis for ethanol include HPLC [13], spectrophotometry [14], colorimetry [15] and Raman spectroscopy [16]. A recent method of analysis for ethanol uses enzymatic reactions and biosensors [17], [18], [19].

Nuclear Magnetic Resonance (NMR) spectroscopy is a highly selective technique in which the spectrum relates to the molecular structure of the compound. While the NMR analysis is generally known for qualitative analysis and structure elucidation of natural products and synthetic organic compounds, it can also be used for quantitative applications. There is a growing interest in the study of quantitative NMR or qNMR during the early 2000s [20]. The qNMR technique possesses the characteristics of a good analytical method. Similar to other analytical methods, qNMR provides both qualitative and quantitative data and is non-destructive of the sample. The technique is highly selective and sensitive, which is important in the analysis of food samples [21].

The basis of the analytical data is the relationship of the resonance signal of the qNMR to the number of resonance nuclei [22]. qNMR experiments were previously conducted on the purity determination of compounds [23], [24]. Also, this method was used for quantitative analysis of pharmaceutical samples [25], [26], and food samples [27]. The qNMR method was already previously used in the analysis of ethanol in alcoholic beverages [28], [29] wherein deuterated water was used as the solvent.

Commonly, samples for NMR analysis are dissolved in an appropriate deuterated solvent. This is to prevent the interference of the solvent peak with that of the analyte. Also, deuterated solvents are used to address the drift in the magnetic field which is apparent for long run times [30]. Gama et al. and Valim et al. used the no-D qNMR technique in their analyte solutions and both studies showed good analytical results [31], [32]. In both studies, however, a coaxial insertion tube was used where the deuterated solvent was placed.

In this study, a qNMR technique totally without a deuterated solvent (no-D) and in unlocked mode is applied in the analysis of the ethanol content of distilled spirits using acetonitrile as the internal standard. The integration area of the ethanol was used as a quantitative parameter. Minimal sample preparation and extensive method validation were performed. The results from the no-D qNMR experiments were validated using GC-FID measurements.