Determination of Ethanol Content in Alcoholic Products by LF-NMR

Abstract

Nuclear magnetic resonance (NMR) is a technique used for many years by chemists for elucidation of molecular structure. Technological progress over the years has enabled this technique, making it easier to use. Thus, the LF-NMR (low-field NMR) was introduced as a side technique, characterized by low management costs and shorter analysis time than its main counterpart. The application of ¹H LF-NMR for the quantification of ethyl alcohol in different alcoholic matrices is herein described, comparing the results obtained with this technique with those determined by a reference gas chromatographic method.

1. Introduction

According to The Global Health Observatory, almost all countries all over the world applied an excise duty to the production and consumption of alcohol and alcoholic beverages. An excise duty exemption is applicable through a denaturing process, which consists in the addition of certain substances which make the alcohol unsuitable for human consumption.

In the European Union, excise duties are regulated by Directive 92/83/EEC [1] on the harmonization of the structures of excise duties on alcohol and alcoholic beverages, and by Regulation (EC) 3199/93 [2] (and its amendments), which establishes the mutual recognition of procedures for the complete denaturing of alcohol.

Ethanol content determination has a relevant role in the process of the complete denaturation of alcohols (CDA) in the European Union. As the majority of the denaturing formulas are expressed in litres or grams of substance per hectolitre of absolute ethanol, it is necessary to have an exact quantification of the ethanol content in alcoholic products which are subjected to denaturing processes. In particular, the European Customs Agencies’ task is to check the ethanol content in alcoholic products both before and after the denaturation process, respectively, to find the correct denaturation formula or possible frauds. For these reasons, a good method of quantification must be able to determine the correct amount of alcohol in different kind of matrices: denatured, crude alcohols, and generic ethyl alcohols of agricultural or synthetic origin. Generally, this determination is carried out by a gas chromatographic technique with a flame ionization detector (GC-FID), necessary to separate and quantify ethanol from other substances (i.e., methanol, or other interfering volatile substances).

Customs Laboratories European Network (CLEN) has developed an analytical method for this purpose: CLEN/ILIADe 143:2023. It is a GC-FID method that determines ethanol content by a calibration curve using an internal standard, 15 mL of methanol to dilute 1 mL of the sample and about 20 min of a chromatographic course. A quality control sample must be prepared every day to check the calibration curve validity for each analytical sequence, and a calibration procedure should be performed frequently to achieve sufficient accuracy in analytical results. All these aspects can be considered disadvantages of the GC technique compared to the use of ¹H LF-NMR spectroscopy for the same determination; see discussion below.

Nuclear magnetic resonance (NMR) is a non-destructive technique that recognizes the chemical structures of molecules selectively, because each molecule has its own spectrum. Among different nuclei, the proton is the most studied for this purpose. The capability of NMR for quantitation (qNMR) to simultaneously detect several compounds in complex mixtures without separation proves to be advantageous as a method to use for analyses of natural products [3,4] including food, fruit juices, or alcoholic beverages [5,6,7]. In particular, ¹HNMR methodologies have been applied to determine chemical constituents (phenolics, sugars, and organic acids) and to measure the amount of alcohol for authenticating the quality of wine [8,9,10,11]. The LF-NMR is a technique characterized by the use of a low-intensity field (80 MHz in this study), which involves a reduction in costs and maintenance procedures, as well as the felling of the samples’ analysis time. As reported in the recent literature, benchtop LF-NMR instruments seem to be suitable for metabolic fingerprinting and targeted quantification of specific metabolites, fitting the needs of clinical applications [12,13,14,15]. Indeed, applications of chemometric/metabolomics approaches to the multivariate analysis of complex mixtures have been demonstrated in fields such as materials science [14], forensic chemistry [15], and biomedical sciences in general [16,17]. As a disadvantage, the low field could be reflected on a lower signal sensitivity, but this is offset by the high ethyl alcohol content in some alcoholic products (>80%vol). The purpose of this article was to investigate the use of a low-field NMR spectrometer to determine the ethanol content of different alcoholic products, especially of crude and denatured alcohols, verifying validation parameters and comparing the results with those obtained by the reference GC-FID method, in accordance with the ILIADe 143:2021|CLEN Method “Determination of Ethanol in Alcoholic Products by GC-FID” Version 2 February 2021 [18].

2. Materials and Methods

2.1. Samples

For this study, 25 real alcohol samples were analysed by the Customs Laboratory of Bologna; to perform a repeatability test, a reference material of the “Proficiency test on completely denatured alcohol (Burning Alcohol)” was organized by CLEN to verify trueness, and some alcoholic solutions containing different common denaturants of ethyl alcohol were used to study specificity. Solvents and reagents were all commercially available and used without further purification (Merck, KGaA, Darmsdadt, Germany).

2.2. Chemical Analysis

All samples were analysed using chromatographic, densimetric, and spectroscopic (LF-NMR) methods, according to the ILIADe 143:2021|CLEN Method “Determination of Ethanol in Alcoholic Products by GC-FID” Version 2 February 2021 and Reg CE 2870/2000 19 December 2000 GU CE L333 29 December 2000 All I App II Met B + Reg UE 383/2023 16 February 2023 GU UE L53 21 February 2023 All, for the first two methods, respectively. The NMR spectroscopic analysis is described below.

2.2.1. Chromatographic Method

Chromatographic analyses, according to the CLEN method mentioned above, accredited by the Italian Customs and Monopolies Agency (ADM)—Laboratory of Bologna, were carried out by a GC-FID Shimadzu GC 2030 with autosampler (Shimadzu Corporation, Kyoto, Japan) equipped with a GC column DB-624, fused silica capillary 60 m × 0.25 mm i.d., and 1.40 mm film thickness (Agilent, Santa Clara, CA, USA). The carrier gas was hydrogen, at a flow rate of 50 mL min⁻¹, with a flow of nitrogen and air, respectively, of 30 mL min⁻¹ and 400 mL min⁻¹. The injection volume was 0.8 mL with a split ratio of 1:150; the injection temperature T_inj and the FID temperature T_det were, respectively, 200 °C and 230 °C. The column temperature was as follows: 37 °C held for 3 min, then to 90 °C (rate of 4.50 °C min⁻¹), and finally to 220 °C (rate of 40 °C min ⁻¹). The total analysis time was around 20 min. The ethanol content in the sample is expressed in %v/v. For sample preparation, 15 mL of methanol was mixed with 0.5 mL of internal standard (4-methyl-2-pentanol) and 1.0 mL of the sample. Quantification of ethanol content was based on a calibration curve, performed every 4 months (verifying an R² > 0.99). A quality control (QC) by a 99.9% vol ethanol CRM (certified reference material) was carried out in duplicate before each sequence analysis to verify the reliability if analysis.

2.2.2. Densimetric Method

Alcohol content (%v/v) in anhydrous ethyl alcohol samples and hydroalcoholic mixture solutions was determined by an Anton Paar DMA 5000M electronic densimeter with Xsample 520 autosampler (Anton Paar S.r.l., Rivoli, Italy).

2.2.3. Spectroscopic (LF-NMR) Method

¹H LF-NMR spectroscopy analyses were carried out by a Spinsolve 80 Multi-X Ultra benchtop NMR spectrometer, characterized by a magnetic field strength of 80 MHz (FKV S.r.l., Torre Boldone, Italy). The sample was introduced into a classic NMR glass tube, and for the analysis, the operators used the pre-installed sequence 1D PROTON (1 min). The parameters of the sequence were settled directly by the instrument, after performing an inversion recovery experiment to determine the relaxation time T1 of each proton of interest. Indeed, T1 inversion recovery experiments were the basis to select a repetition time (relaxation delay) of 15 s, being approximately 5 times the longest T1 highlighted (related to the hydrogen of IS aldehyde group). The general scheme of this sequence is reported below in Figure 1.

The setup experimental parameters of 1D PROTON protocol are the following:

-

pulse angle: 90°.

-

dwell time: 200 µs;

-

acquisition time: 6.4 s (for each scan);

-

n. of scans: 4;

-

repetition time (relaxation delay): 15 s;

Therefore, the experiment lasts 1 min overall. The data report was created using the Spinsolve 2.2.4 version software and the data were elaborated using Mestrenova software v.14 by Mestrelab Research S.L.

A mixture of about 450 μL of internal standard (salicylaldehyde 99.0% w/w) and 150 μL of alcoholic sample weighed in a GC-vial using an analytical balance with a sensitivity of 0.1 mg was transferred to an NMR glass tube and analysed. The ethanol content was expressed in terms of % w/w, calculated as reported in Equation (1):

$$C_x={\displaystyle \frac{I_x}{I_{STD}}}\ast {\displaystyle \frac{N_{STD}}{N_x}}\ast {\displaystyle \frac{M_x}{M_{STD}}}\ast {\displaystyle \frac{m_{STD}}{m_x}}\ast P_{STD}$$

(1)

where C_x is the ethanol content in w/w%, and I_x, I_std, N_STD, N_x, M_x, M_std, m_std, m_x, and P_std are, respectively, the integral area, proton’s number-generating peaks, molecular mass, sample and standard weights (in grams), and purity related to ethanol (x) and IS (std). Integrated signals refer to the methylene (or methyl) group of ethanol peaks and the aldehydic group of salicylaldehyde (IS) peaks.

A typical spectrum of the mixture consisting of the alcohol sample and salicylaldehyde is shown in Figure 2.

The quantification was done using the aldehyde’s group integral at 9.4 ppm and both ethanolic peaks at 3.9 ppm for CH₂ and 0.8 ppm for CH₃ groups. The expression of ethanol content in %v/v was performed by multiplying the result obtained in %w/w by the ratio of the sample’s and ethanol’s densities.

3. Results and Discussion

The validation parameters studied in this article for ethanol content determination by ¹H LF-NMR content were the following:

• Precision data;
• Trueness;
• Specificity;
• Application field;
• Linearity;
• Robustness.

3.1. Precision Data (Repeatabilty and Uncertainty of Measurement)

The method’s precision was estimated by eleven independent repeatability determinations on four different kinds of alcoholic matrices, as explained in

Table 1. Matrices studied for repeatability tests.

Matrix	EtOH Content Determined by GC-FID
Anhydrous ethyl alcohol	EtOH: 99.9% v/v
Denatured ethyl alcohol (euro DG)	EtOH: 88.5% v/v
Crude alcohol	EtOH: 92.7% v/v
Hydroalcoholic mixture ethyl alcohol/water 80/20% v/v	EtOH: 82.7% v/v

.

The matrices 1, 2, and 3 represent the majority of samples analysed by the ADM Laboratory of Bologna. The last matrix was studied to evaluate the solubility of the internal standard in samples with a higher water content than typical alcohol samples. In fact, water may cause accuracy problems if the internal standard used does not completely dissolve. Thus, a higher water content could determine an overlap of the -OH peak with -CH₂ peaks of ethanol.

The repeatability of laboratory r_LAB was calculated as reported in Equation (2):

$$r_{LAB}=t\ast s_r\ast \sqrt{2}$$

(2)

where t is the t of Student and s_r is the repeatability standard deviation. Considering that there are no precision data regarding this method, the Horwitz equation was used [19]. Using this approach, the reproducibility standard deviation was calculated from ethanol content (1), then multiplied by two to obtain the expanded uncertainty, as reported in Equation (3a,b):

$${\sigma }_R=0.02\ast C^{0.8495};$$

(3a)

$$U_H=2\ast {\sigma }_R$$

(3b)

where σ_R is the reproducibility standard deviation calculated with Horwitz’s approach, C is the average of the concentrations of ethyl alcohol expressed in w/w, and U_H is the expanded uncertainty of measurement. Refer to the Supporting Information for further details. The summary of the results obtained for repeatability and measurement uncertainty (expressed in %v/v) is shown in

Table 2. Summary of results obtained for the evaluation of method accuracy data.

Matrix	rLAB (%v/v) -CH3 Signal	rLAB (%v/v) -CH2 Signal	UH (%v/v)
Anhydrous ethyl alcohol	1.64	0.74	2.00
Denatured ethyl alcohol (euro DG)			1.84
Crude alcohol			1.90
Hydroalcoholic mixture ethyl alcohol/water 80/20% v/v			1.76

.

3.2. Trueness

The trueness of the method was evaluated through the following approaches:

• Comparison with a PT sample (interlaboratory circuit residual);
• Comparison between results obtained by NMR spectroscopic and reference chromatographic methods, using both standards and real samples.

3.2.1. Comparison with a PT Sample

The method is considered reliable if the analysis on a PT sample residual (Burning Alcohol, organized by CLEN) yields a result such that the z-score is ±2. The sample of the “Proficiency test on completely denatured alcohol 27 June 2019- Burning Alcohol” of CLEN was used as reference material. The analysis was performed in triplicate. The z-score was defined as reported in Equation (4):

$$z={\displaystyle \frac{x-x_{pt}}{s_{pt}}}$$

(4)

where x is the result obtained by the laboratory with the spectroscopic method; x_pt is the reference value of the Proficiency test and s_pt is the reproducibility standard deviation of the circuit.

The results are reported in

Table 3. Comparison between laboratory and Proficiency test results and acceptability criteria.

Matrix	X (%v/v)	Xpt (%v/v)	spt	Z-Score	Acceptability (−2 ≤ z ≤ +2)
PT CLEN 2019-Burning Alcohol (-CH3)–Repetition 1	86.76	89.62	0.65	−4.40	NO
PT CLEN 2019-Burning Alcohol (-CH3)–Repetition 2	87.61			−3.09	NO
PT CLEN 2019-Burning Alcohol (-CH3)–Repetition 3	86.31			−5.09	NO
PT CLEN 2019-Burning Alcohol (-CH2)–Repetition 1	89.25			−0.57	YES
PT CLEN 2019-Burning Alcohol (-CH2)–Repetition 2	90.04			0.65	YES
PT CLEN 2019-Burning Alcohol (-CH2)–Repetition 3	88.78			−1.30	YES

.

As highlighted, it can be inferred that the method used provides accurate results using the methylene group (-CH₂) signal, unlike the underestimated outcome obtained by the methyl group (-CH₃) signal. It is noteworthy that results were reliable on a denatured alcohol sample notwithstanding the presence of denaturants that could interfere, based on their concentration (see Specificity below).

3.2.2. Comparison between Results Obtained by NMR Spectroscopic and Reference Chromatographic Methods

Another check on trueness was performed by comparing the results of the ¹H-NMR method with those obtained by gas chromatographic quantification.

For this purpose, the same four matrices (anhydrous ethyl alcohol, denatured ethyl alcohol: euro DG, crude, and hydroalcoholic mixture ethyl alcohol-water 80% v/v) were also analysed by the reference GC-FID method, carrying out six determinations for each kind of sample.

As reported in the literature [20], this approach is used when certified reference material is not available, as in our case for crude alcohol.

For both methods, a normal distribution of data was verified, standard deviations were calculated, and the homogeneity of variances was tested by the F-Test. Therefore, the two methods were compared and considered statistically equal if Equation (5) was satisfied:

$$|\Delta |=\left|{\overline{x}}_A-{\overline{x}}_N\right|\le t_{p=1-\alpha , v\Delta }\ast s_{\Delta }$$

(5)

where ${\overline{x}}_A$ and ${\overline{x}}_N$ are, respectively, the average ethanol content values determined by the NMR (alternatively using -CH₂ and -CH₃ signals) and GC-FID methods, t is the Student’s t, and $s_{\Delta }$ is a combination of standard deviations, as defined in full in Supporting Information. The results are reported in

Table 4. Comparison between NMR and GC methods.

	${\overline{\mathit{x}}}_{\mathit{N}}$	${\overline{\mathit{x}}}_{\mathit{A}}$  -CH3 Signal	$\left|{\overline{\mathit{x}}}_{\mathit{A}}-{\overline{\mathit{x}}}_{\mathit{N}}\right|$ -CH3 Signal	$\mathit{t}\ast {\mathit{s}}_{\mathbf{\Delta }}$ -CH3 Signal	Acceptability	${\overline{\mathit{x}}}_{\mathit{A}}$  -CH2 Signal	$\left|{\overline{\mathit{x}}}_{\mathit{A}}-{\overline{\mathit{x}}}_{\mathit{N}}\right|$ -CH2 Signal	$\mathit{t}\ast {\mathit{s}}_{\mathbf{\Delta }}$ -CH2 Signal	Acceptability
Anhydrous ethyl alcohol	100.88	99.49	1.39	0.16	NO	100.03	0.85	0.14	NO
Denatured ethyl alcohol (euro DG)	88.08	89.07	1.00	0.26	NO	88.28	0.20	0.40	YES
Crude alcohol	92.73	92.76	0.03	0.56	YES	92.92	0.19	0.65	YES
Hydroalcoholic mixture ethyl alcohol/water 80/20% v/v	82.72	81.32	1.40	0.25	NO	82.99	0.26	0.33	YES

.

The results highlighted that NMR and GC methods provide statistically comparable results using the methylene group (-CH₂) signal, unlike using the methyl group (-CH₃) signal, which tends to underestimate the outcomes, as reported in the previous paragraph.

As an exception, it also emerged that the comparison of absolute ethyl alcohol samples was negative. However, this was not due to the non-accuracy of the NMR method determination, but rather to the overestimation obtained by the gas chromatographic method (100.88% v/v). Indeed, NMR provided an average value of 100.03% v/v, certainly more accurate considering the type of sample (stated to be 99.9% v/v by the supplier). To further support the trueness of anhydrous ethyl alcohol, the sample was characterized by a densimeter and considered as a reference. Therefore, to value the NMR result, the Equation (6) (t-test) was used:

$${\displaystyle \frac{\left|\overline{x}-x_{rif}\right|}{\sqrt{u_{\overline{x}}^2+u_{rif}^2}}}\le t_{p,v}$$

(6)

where $\overline{x}$ and $x_{rif}$ are the average values obtained with NMR and a densimeter; and $u_{\overline{x}}$ and u_rif are the uncertainties of the laboratory associated with the mean values obtained by the two techniques, respectively.

The results are reported in

Table 5. Trueness test for anhydrous ethyl alcohol.

	CH3 $\frac{\left|\overline{\mathit{x}}-{\mathit{x}}_{\mathit{r}\mathit{i}\mathit{f}}\right|}{\sqrt{{\mathit{u}}_{\overline{\mathit{x}}}^2+{\mathit{u}}_{\mathit{r}\mathit{i}\mathit{f}}^2}}$	Acceptability	CH2 $\frac{\left|\overline{\mathit{x}}-{\mathit{x}}_{\mathit{r}\mathit{i}\mathit{f}}\right|}{\sqrt{{\mathit{u}}_{\overline{\mathit{x}}}^2+{\mathit{u}}_{\mathit{r}\mathit{i}\mathit{f}}^2}}$	Acceptability
Anhydrous ethyl alcohol—CRM	0.47	YES	0.11	YES

.

Moreover, the comparison between the NMR and GC methods was carried out on 25 real crude heads-tails alcohol samples, with an 85–96% vol range alcohol content, analysed in duplicate by each technique.

Firstly, the repeatability condition for each duplicate determination was verified for all samples, both for NMR and GC analysis, according to Equation (7):

$${\Delta }_m\le r_{LAB}$$

(7)

where ${\Delta }_m$ is the difference of duplicate results obtained by NMR (both with -CH₃ and -CH₂ group signals) or the GC technique.

Hence, the comparison between NMR and GC analysis was evaluated by Equation (6) for each analysed sample and by the correlation graph reported in Figure 3.

Once more, it was shown that NMR analyses based on the -CH₂ group signal provide results that are more accurate (precise and true) and definitely comparable with GC outcomes.

Indeed, even the graphs highlighted that by the NMR -CH₂ group signal, besides a higher degree of correlation (as indicated by the R² value), the slope and the intercept values are not significantly different from one and zero, respectively, excluding constant or proportional systematic errors and confirming the perfect comparability of the two methods, as reported in

Table 6. Correlation equations of NMR and GC methods. The intercept and slope values based on -CH₂ signal showed no significant differences between the two methods, as both are, respectively, different from zero and one.

Equation -CH3 Signal	a (Intercept) ± s(a) -CH3 Signal	b (Slope) ± s(b) -CH3 Signal	Equation -CH2 Signal	a (Intercept) ± s(a) -CH2 Signal	b (Slope) ± s(b) -CH2 Signal
y = 0.803x + 17.047	17.0 ± 16.1	0.80 ± 0.17	y = 0.9234x + 7.2033	7.20 ± 8.22	0.92 ± 0.09

.

Details and results of calculations are reported in Supporting information (Tables S5 and S6).

3.3. Specificity

The LF-NMR spectroscopy technique has several advantages, such as a short time analysis as well as easy sample preparation. Despite many advantages, this technique may suffer specificity problems, since it does not provide, unlike the GC technique, a preventive separation of the components of a sample. The main and potential interferents for the alcohol matrices of this study are the following substances, which are sometimes naturally contained but are typically added as denaturants in ethyl alcohol samples: methanol, acetone, 2-propanol tert-butanol, n-hexane, n-propanol, ethyl-methyl ketone (MEK), ethyl acetate, cyclohexane, isopropyl acetate, toluene, ethylene glycol, eucalyptol, and diethyl phtalate. The addition of the denaturants listed above is regulated both at National and European levels, at the latter by Reg. UE 2017/2236 [21]. In crude heads-tails alcohol samples, this problem may be even more relevant, due to the possible presence of different volatile substances. To verify possible interferences in ethanol determination by LF-NMR, specificity tests were carried out on the following solutions:

• Ethanol standard solution containing all 14 denaturants mentioned above;
• Ethanol standard solution containing the two most common volatile substances generally found in crude alcohol: methanol and ethyl acetate;
• Ethanol standard solutions containing, singularly, common or critical denaturants as received by our laboratory.

3.3.1. Analysis of Ethanol Standard Solution Containing 14 Denaturants

A 55% ethanol standard solution containing 5 l/hl a.a. of each of the 14 denaturants listed above, prepared from a 99.9% v/v absolute ethyl alcohol as the reference material, was analysed in triplicate. The interference assessment was based on the recovery calculation of ethanol content compared to the nominal value, defined as in Equation (8):

$$Recovery={\displaystyle \frac{C_{experimental}}{C_{theoretical}}}\ast 100$$

(8)

Equation (8)—Equation for ethanol recovery calculation.

A recovery within 3% (equal to $\sqrt{2}$U) is to be considered acceptable.

The LF-NMR spectra are reported below in Figure 4:

Comparing the two spectra, interferences due to denaturants are already shown by the shape of the methylene or methyl group of ethanol, as confirmed by the recovery calculation reported in

Table 7. Specificity of ethanol standard solution containing 14 denaturants.

	Cexperimental -CH3 Signal	Cexperimental -CH2 Signal	Ctheoretical	Recovery -CH3 Signal	Recovery -CH2 Signal
Ethanol standard solution containing 14 denaturants	98.65% w/w	103.41% w/w	54.99% w/w	179.39%	188.06%

.

Therefore, we established that, in general, denaturants may interfere with ethanol determination; further tests were carried out on the most common denaturants to determine the critical amount beyond which ethanol content determination is not reliable.

3.3.2. Analysis of Ethanol Standard Solution Containing Methanol and Ethyl Acetate

An 80% ethanol standard solution containing 10% methanol and 10% ethyl acetate was prepared and analysed in triplicate, since methanol and ethyl acetate represent the two most common volatile substances generally found, even at high concentrations, in crude alcohol.

As in the previous paragraphs, the interference assessment was based on the recovery calculation of ethanol content compared to the nominal value, as reported in

Table 8. Specificity of ethanol standard solution containing methanol and ethyl acetate.

	Cexperimental -CH3 Signal	Cexperimental -CH2 Signal	Ctheoretical	Recovery -CH3 Signal	Recovery -CH2 Signal
Ethanol std + methanol & ethyl acetate	83.13% w/w	81.31% w/w	80.26% w/w	103.58%	101.31%

.

It was highlighted that the quantification of ethanol content in the mixture met the acceptability criterion for specificity if based on the -CH₂ group signal.

3.3.3. Analysis of Ethanol Standard Solutions Containing, Singularly, Common or Critical Denaturants

Five standard solutions containing 85–90% ethanol and, singularly, 10–15% of other common or critical denaturants were prepared and analysed, since these compounds in particular produce signals that may overlap with those of ethanol. They are acetone, ethyl-methyl ketone (MEK), 2-propanol, ethylene glycol, and n-propanol. The results are reported in

Table 9. Specificity of ethanol standard solution containing, singularly, different denaturants.

	Cexperimental -CH3 Signal	Cexperimental -CH2 Signal	Ctheoretical	Recovery -CH3 Signal	Recovery -CH2 Signal
Ethanol std + acetone	88.52	91.43	90.10	98.25	101.47
Ethanol std + MEK	89.99	90.53	89.99	100.60	101.75
Ethanol std + 2-propanol	103.69	92.69	90.25	114.89	102.70
Ethanol std + ethylene glycol	85.67	83.41	85.67	97.37	124.80
Ethanol std + n-propanol	89.54	90.95	89.54	101.58	109.98

.

Considering the acceptability criterion of 3%, only ethylene glycol and n-propanol produced interferences, respectively, at concentrations of 15% and 10%, when using the -CH₂ group signal. Although representing critical substances, n-propanol is generally present in traces in crude alcohol and ethylene glycol is a specific denaturant which is not used in a such high concentration. Quantification based on the -CH₃ group signal failed the recovery test even using 2-propanol.

Since, generally, the denaturants added did not exceed 3 L/hL a.a. for completely denatured alcohol (see Reg. UE 2017/2236), the LF-NMR method may be considered definitely reliable.

In case of doubt, in particular whenever altered peaks’ shapes are observed, qualitative or semiquantitative GC screening is suggested, to evaluate if ethanol content determination may be affected by interference.

3.4. Application Field

The matrices analysed in this study allowed us to set an application field ranging between 80 and 100% v/v of ethanol in hydroalcoholic, even denatured, samples.

3.5. Linearity

Calibration is not necessary since quantification is completed by Equation (1).

Linearity was investigated in the concentration range of the application field (80.0–100.0% w/w of ethanol) by four ethanol standard solutions, using both the -CH₃ and CH₂ group signals. As expected, a linearity of R² > 0.95 was obtained, as reported in Figure 5.

3.6. Robustness

Robustness represents the capacity of an analytical procedure to produce unbiased results when small changes in the experimental conditions are made voluntarily. Considering the proposed method, some procedural changes may occur only during sample preparation; therefore, different ratios of the sample an internal standard were tested for robustness, using the OFAT (one-factor-at-time) approach.

Sample preparation consisted of weighing a mixture of an internal standard (salicylaldehyde) and an alcohol sample in a 3:1 ratio. It was tested at 2:1 and a 4:1 ratios.

As reported in Supporting Information, the method was robust for the 2:1 ratio, but not for the 4:1 ratio.

4. Conclusions

In this paper, a quantitative, reliable, rapid, and cost-effective determination of ethyl alcohol in alcoholic matrices by ¹H LF-NMR was proposed.

Indeed, ethanol content determination in alcohol products has a relevant role in the process of the complete denaturation of alcohol (CDA) in the European Union as denaturing formulas are expressed in litres or grams of denaturing agents (denaturants) per hectolitre of absolute ethanol.

The method herein discussed was evaluated for accuracy (precision and trueness), specificity, application field, linearity, and robustness. Expanded measurement uncertainty was estimated by Horwitz’s approach, proving to be approximately 2%.

The results of the experiments show that the ¹H LF-NMR technique, if based on the ethanol CH₂ group signal for quantification, is reliable and comparable with the gas chromatographic method, with the advantage of being faster and even more precise in the set application field (80–100% vol). Instead, the results of the quantification based on the ethanol CH₃ group signal lack accuracy. Further study could probably explain these small differences.

About specificity, this study showed that ethanol NMR determination may suffer from the presence of some interfering substances, naturally occurring in crude alcohol or added as denaturants, if their content is above 10% vol. However, typically, only methanol and ethyl acetate are found in crude heads-tails alcohol, and they may interfere with the NMR signal if they exceed such a concentration. In case of doubt, also deduced from the shape of the reference signals of NMR spectra, a qualitative chromatography is recommended to evaluate the presence of any interferents.

Finally, robustness tests suggest to maintain the weight ratio of the internal standard and the sample between 2:1 and 3:1 during sample preparation.