Evaluating the chemical and metal contamination of commercial Rakı, a grape-based alcoholic beverage from Turkey

Turkish Rakı is one of the most consumed alcoholic beverages in Turkey. The chemical and metal contamination of alcoholic beverages is a threat to human health and lowers the quality of the product. Ethyl carbamate and furfural are carcinogenic to animals, and have been classified as Group 2A and 3 agents respectively by the International Agency for Research on Cancer. Factors, such as raw materials, process time and storage, affect metal concentrations in beverages. Limits for chemicals and metals in Rakı have not yet been established by the Turkish agency. The present study aimed to evaluate the chemical and metal contamination of commercial Turkish Rakı. Thirty-seven different types of Turkish Rakı were purchased from local markets in Turkey. Ethyl carbamate (EC) and furfural (FR) levels were measured by Gas Chromatography-Mass Spectroscopy (GC-MS). Arsenic, copper, lead, and zinc levels were measured by Inductively Coupled Plasma–Optical Emission Spectrometry (ICP-OES). None of the Rakı samples contained EC. Furfural was not detected in commercial Rakı samples but only in illegal Rakı samples. Ethanol and methanol levels complied with Turkish regulation. Our data shows that commercial Rakı from Turkey was contaminated by very low amounts of arsenic (<LOD–0.02 mg/L), copper (0.05–0.41 mg/L), lead (< LOD– 0.03 mg/L) and zinc (0.01–0.39 mg/L).


INTRODUCTION
Rakı is a double distilled, traditional aniseed spirit, one of the most consumed alcoholic beverages produced in Turkey (Darici et al., 2019). Rakı was accorded Protected Designation of Origin (PDO) by the Turkish Patent and Trademark Office in 2009 (TPI, 2009). In accordance with this PDO document and the Turkish Food Codex Communiqué on Distilled Alcoholic Beverage (TFC, 2017), Rakı is produced by distilling Suma (grape distillate) with aniseed (only Pimpinella anisum) in a traditional copper pot still. Suma is a distillate of grapes/raisins, which are distilled to up to 94.5 % v/v alcohol by column still distillation with the purpose of retaining the flavour and smell of grapes. Rakı is also one of the most adulterated alcoholic beverages in Turkey. It is often mixed with ethyl alcohol, methanol, water, sugar, and aniseed aroma to increase the profit margin. Adulteration with methanol specifically can cause severe health issues, such as blindness, and can even result in death (Ghadirzadeh et al., 2019). In 2020, 33 people lost their lives in Turkey due to consumption of counterfeited Rakı adulterated with methanol. While the addition of methanol to alcoholic beverages can cause many health problems, other types of chemical and metal contamination can be carcinogenic to humans, and their high consumption can cause the same or worse problems as methanol does (Lachenmeier et al., 2011).
Ethyl carbamate (EC) (C 2 H 5 OCONH 2 ) has been classified by the International Agency for Research on Cancer (IARC) as a Group 2A agent, being carcinogenic to animals and possibly to humans (IARC, 2007). Ethyl carbamate can be formed through multiple pathways, and it forms naturally in fermented products including distilled spirits ( Figure 1). The most common pathway for EC formation is the reaction between ethanol and N-carbamyl compounds, such as urea produced from arginine metabolism by yeast or lactic acid bacteria (Choi et al., 2018). EC can also be formed during distillation and 48 hours after the distillation is complete (Bruno et al., 2007). Because of its carcinogenicity, EC has attracted attention in many countries, and its concentrations in alcoholic beverages have been limited (Table 1). However, the Turkish regulatory agency has not yet established the limits for ethyl carbamate, and no data is available for Turkish Rakı. Rakı is one of the most consumed distilled spirits in Turkey and the most prone to adulteration; therefore, the determination of EC levels in Rakı is needed. EC is commonly determined by GC-MS, UPLC-MS and HPLC with a fluorescence detector (Riachi et al., 2014). To our best knowledge, the present study contains the first reported data for EC levels in Rakı.  Group 3 since no data is available for human  studies, and not enough tests on animals have been  made to be accepted in Group 2A (IARC, 2014). Furfural is a volatile compound toxic to humans and is formed from pentoses in fruits. Alcoholic beverages containing high concentrations of furfural threaten human health; for instance, their consumption may result in pain, diarrhea, headaches and excessive vomiting. Furfural is generally formed when distillation is not carried out properly, especially when applying high heat (Coldea et al., 2014). Homemade alcohols and illegal products can have a high concentration of furfural, because direct high heat is applied, thus producing harmful compounds, a caramel colour and a burnt taste (Coldea et al., 2017). Determination of furfural is generally carried out by using spectroscopy (UV-Vis) and chromatography (GC, GC-MS) techniques (Barbosa-Garcia et al., 2007). Furfural levels in Tequila from Mexico and Pisco from Peru are limited to a maximum of 40 mg/L and 50 mg/L of anhydrous alcohol respectively. The Turkish Food Codex does not limit furfural levels in Rakı; however, it forbids any furfural in the suma. Due to its toxicity, furfural levels in Rakı need to be determined.
The contamination of alcoholic beverages with metals is a threat to human health and can impact the quality of the final product; for example, homemade rum can cause human paralysis because of equipment containing Pb, and metals in alcoholic beverages can cause turbidity, colour change and the formation of congeners (Pohl, 2007;Ibanez et al., 2008). Conversely, alcoholic beverages can be a source of metals beneficial for human health: for example, the consumption of wine can provide many essential minerals, such as Cu, Fe and Zn (Ibanez et al., 2008). Many factors, such as distillation type, raw materials and storage equipment, can affect the levels of metals in the final product (Pohl, 2007). The limits for metal concentrations in alcoholic beverages vary depending on the product; for example, the maximum limit for copper in cachaca from Brazil and Pisco from Peru is 5 mg/L (Menevseoglu, 2019). However, the Turkish Food Codex has not set limits for metal levels in Rakı.
The objective of this study was to evaluate the chemical and metal contamination of commercial Rakı from Turkey. To the best of our knowledge, the data in this study on ethyl carbamate, furfural, and metal concentrations in Turkish Rakı are the first to be reported.

Materials
Commercial Rakı samples (n = 34), and counterfeited Rakı (n = 3) were purchased from local Turkish markets and obtained from manufacturers. Detailed information about the Rakı samples is given in Table 2.

Determination of ethyl carbamate and furfural by GC-MS
For chemical contamination, a Gas Chromatography Mass Spectroscopy (GC-MS) technique was used following a method modified from Bortoletto and Alcarde (2016). A standard curve for ethyl carbamate (EC) and furfural (FR) was established to quantify the concentration of both these chemicals ( Figure 2). Concentrations of 5-350 µg/L EC in 40 % ethanol in water solutions were used. Similarly, to create a standard curve for FR concentrations in Rakı, 1-30 mg furfural/1000 mL in 40 % ethanol in water solutions were used. An Agilent Technologies 7820A GC was used with a 5877B MSD (Santa Clara, CA, USA); this is a single quadrupole mass spectrometry instrument that uses electrospray ionisation to ionise the sample. A DB-WAX UI column (Agilent Technologies, Santa Clara, CA, USA) was used; it was 30 m long and 0.250 mm wide with an inner diameter of 0.25 µm. The initial oven temperature was set at 90 °C for 2 min, increased at a rate of 10 °C/ min to 150°C at which it was held for 6 min, and then increased at a rate of 40 °C/min to a final temperature of 230 °C with 2 min of hold time. The injection volume for the analysis was 2 µL. The injection method was a split (20:1) method with an injection port temperature of 220 °C. The pressure of the injection port was set at 8.743 psi. The mass spectrometer source temperature was set at 240 °C and the quadrupole temperature set at 180 °C. The electron energy of the MS was set at 70 eV, and the acquisition type was set to SIM and SCAN mode. For EC and FR, 62 m/z and 95 m/z ions respectively were measured and used for quantification. The validation parameters for EC and FR are given in Table 3.  The determination of ethanol volume in Rakı was carried out using a NIR spectrometer Anton Paar Alcolyzer (DMA 4500M-Alcolyzer ME). This NIR instrument comprises a LED light source, a multi-lens and a detector array. It has a temperature-controlled unit; the temperature of the samples were automatically adjusted to 20 °C. It can also be programmed to give the alcohol volume as a 2-digit percentage; the method was adapted from Darici and Cabaroglu (2018).

Methanol
An Agilent Technologies 7820A GC was used with a 5877B MSD (Santa Clara, CA, USA). A DB-WAX UI column (Agilent Technologies, Santa Clara, CA, USA) was used; it was 30 m long and 0.250 mm wide with an inner diameter of 0.25 µm. The initial oven temperature was set at 50 °C for 3 min, increased at a rate of 4 °C/min to 80 °C for 5 min, and then increased at a rate of 15 °C/min to a final temperature of 200 °C with 1 min of hold time. The injection volume for the analysis was 0.3 μL. The injection method was a split (20:1) method with an injection port temperature of 220 °C. The pressure of the injection port was set at 8.743 psi. The mass spectrometer source temperature was set at 240 °C and the quadrupole temperature set at 180 °C. The electron energy of the MS was set at 70 eV and the acquisition type was set to SIM and SCAN mode. For methanol, 31 m/z ions were measured and used for quantification. The method was adapted from Menevseoglu (2019).   (Correa et al., 2014). For instance, in whisky, only 1 % of EC can be formed during the second distillation (Riffkin et al., 1989). Moreover, high concentrations of EC are generally found in stone-fruit alcoholic beverages produced from, for example, sour cherry and plum, because the process of breaking stones can initiate the formation of amygdalin-derived ethyl carbamate (Deak et al., 2010). Brazilian distilled spirit Cachaça produced from sugar cane was reported to have a high level of EC: as much as 5589 µg/kg, which is above the permitted limit of 150 µg/kg (Andrade-Sobrinho et al., 2002). A distilled spirit in China, Luzhou-flavour, had EC concentrations of between 17 and 151 µg/kg (Fang et al., 2018).

RESULTS AND DISCUSSION
Furfural (FR) levels in commercial Rakı are shown in Table 4. The range of FR levels was between undetectable and 3.97 mg/L, which is below the generally permitted limit (50 mg/L). Furfural contamination is found when the distillation continued after the tail part of the wine is reached. Therefore, carrying out the production steps carefully may help to reduce contamination. Commercial Rakı samples did not contain furfural, but three counterfeited Rakı samples had low amounts. In literature, many distilled spirits have been evaluated for their furfural concentrations; a b Regression equation: y = ax + b, where y is the peak area and x is the concentration c R 2 , correlation coefficient. d Limit of detection: calculated by signal-to-noise ratio. e Identification. MS identified based on mass spectral library.

Compound
Calibration curve (n = 7) Linearity range Slope (   Similarly, the methanol levels of the commercial Rakı complied with regulations. Only sample R35, a counterfeit Rakı, did not contain methanol since it was produced using an agricultural ethanol purified from some other alcohol. Table 5 shows the metal contamination of Rakı samples. None of the samples contained arsenic, except for samples R34 and R35, which contained 0.018 mg/L and 0.022 mg/L respectively; these arsenic concentrations can be considered negligible. The absence of arsenic may be due to the arsenic-free copper alembics that was used during the production process. While arsenic is not common in distilled spirits, copper is problematic; it can, for example, catalyse the formation of ethyl carbamate, and the consumption of a high concentration of copper can produce toxic effects, such as diarrhea, excessive vomiting and liver damage (Silva et al., 2020). In the present study, the concentrations of copper in the Rakı samples were low: between 0.046 and 0.414 mg/L, which is also below the limits for distilled spirits (i.e., 5 mg/L). Navarro (2007) reported copper concentrations in whisky, gin, rum, liquor, brandy, wine, and beer as being 1.01 mg/L, 0.1 mg/L, 2.34 mg/L, 0.59 mg/L, 8.01 mg/L, 0.39 mg/L and 0.39 mg/L respectively. Another toxic metal, Pb, was found at ppb levels in the commercial Rakı, most likely due to lead-free copper alembics.
The permissible limit of Pb in alcoholic spirits in some European countries is in the range of 0.2-0.3 mg/L (Kostic et al., 2010). Zinc concentrations of the Rakı was in the range of 0.01-0.39 mg/L. The permissible limit for zinc is 5 mg/L, thus Zn concentrations in the commercial Rakı were below the limit. The absence or a very low amount of zinc in the copper alembics could explain these results.

CONCLUSIONS
This study aimed to determine the chemical and metal contamination of commercial Rakı. Our results showed that neither ethyl carbamate nor furfural were detected in the commercial Rakı. The arsenic, copper, lead and zinc levels of the commercial Rakı were either below the limits or undetectable. In conclusion, the chemical and metal contamination of commercial Rakı is very low.