key: cord-0918104-gs1328xu authors: Tse, Timothy J.; Nelson, Fina B.; Reaney, Martin J. T. title: Analyses of Commercially Available Alcohol-Based Hand Rubs Formulated with Compliant and Non-Compliant Ethanol date: 2021-04-04 journal: Int J Environ Res Public Health DOI: 10.3390/ijerph18073766 sha: cf2ff20714bfd75d35c621d5baa6639111df1f65 doc_id: 918104 cord_uid: gs1328xu The COVID-19 pandemic led to panic-buying of alcohol-based hand rubs (ABHRs). In response, governmental agencies (e.g., Health Canada) permitted the sale of ABHRs formulated with “technical-grade” ethanol to alleviate the growing demand. Technical-grade ethanol contains elevated concentrations of impurities (e.g., acetaldehyde, etc.), which may exhibit dose-dependent toxicity. In this study, a rapid solvent extraction was employed to analyze gelled ABHRs via gas chromatography with flame ionization detection. In total, 26 liquid and 16 gelled ABHRs were analyzed for nine common impurities to determine compliance with Health Canada interim guidelines. Of 42 samples analyzed, 11 ABHRs appear to be non-compliant with interim Health Canada guidelines. Non-compliant ABHRs exhibited elevated concentrations of acetaldehyde, with a maximal concentration observed of 251 ± 10 µL L(−1); 3.3× higher than currently permitted. Nonetheless, frequent testing of ABHRs should be routinely conducted to reduce the risk of consumer exposure to non-compliant ABHRs. Community transmission of infectious disease remains a significant concern [1], especially during the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; aka COVID- 19) pandemic. Many municipalities have enforced stay-at-home and workat-home orders to reduce and control community transmission of this respiratory disease. Hand hygiene is a key practice that reduces the incidence of infection [1] . Hand hygiene is best achieved by either handwashing with soap or by use of hand sanitizers. Both practices effectively reduce contaminants by removing or killing microorganisms [1] . Community education in proper hand hygiene is a key activity that can significantly reduce the spread of infectious disease through reduction of bacterial and viral counts [2] . Efficacy, followed by skin compatibility, were identified to be the most important attributes of a hand sanitizer [2] . Alcohol (e.g., ethanol) can be produced by fermentation of biomass or synthetically via direct or indirect ethylene hydration (e.g., petroleum-derived ethanol) [3, 4] . However, production of synthetic ethanol often requires expensive processes, such as high energy inputs to compress ethylene and water over phosphoric acid catalysts [3, 4] . Bioethanol processes are the predominant method for commercial ethanol production [5] due to their lower cost, renewability (e.g., cereal grains), and absence of toxic reagents (e.g., petroleum-based olefins). Alcohol content (e.g., 60-95%), type of alcohol (e.g., ethanol, isopropanol, or n-propanol), and presence of additives (e.g., H 2 O 2 ) [6] are critical in deriving an alcohol-based hand rub (ABHR) product with broad spectrum utility. Typically, alcohol concentrations between 60-95% (v/v) have superior antibacterial properties [6] with higher alcohol concentrations demonstrating better activity [7] . Concentrations higher than 95% are ineffective as water is required for protein denaturation, and preparations with higher alcohol concentrations evaporate quickly. Alcohol effectively kills all enveloped viruses [6] , including the novel COVID-19. Non-enveloped viruses may require higher alcohol concentrations to be effective (i.e., >70%) [6] . Alcohol-based hand rubs (ABHRs) can be available in liquid, gel, or foamable formats and are the first choice for hand hygiene in healthcare settings as they afford more effective antimicrobial activity than antiseptic soaps [8, 9] , improve convenience and compliance [10, 11] , are less time-consuming, and are less irritating than washing with soap and water [12] [13] [14] . Based largely on convenience ABHR in gel or foam formulations are typically favored, although liquid ABHRs typically leave less residue and dry more quickly [15] . The efficacy of ABHR is determined by the amount of hand sanitizer dispensed [16, 17] , the contact time [18, 19] , whether hands are soiled or contaminated with materials [20] , and hand hygiene procedures [1, 2] . ABHRs also do not eliminate all types of germs [16, 17] . Therefore, washing hands with mild soap for a minimum of 20 s, is a preferred method of decontamination [21, 22] , as it inactivates and removes a broad spectrum of bacterial or viral particles physically from the hand. Typically, raw materials destined for the pharmaceutical and food industry must meet specific standards for composition, such as those outlined in monographs available through the Food Chemicals Codex (FCC) and the United States Pharmacopeia (USP) [23, 24] . Compliance with these standardized testing protocols ensures that materials are pure and of quality. Compliance is verified and regulated by governmental agencies such as Health Canada in Canada, and the United States Food and Drug Administration (FDA). Recently, the global transmission of COVID-19 has led to panic-buying of common necessities [25] , including surface disinfectants and hand sanitizers, resulting in global shortages of both products and materials and further eroding supply and distribution chains. In response, governmental agencies (e.g., Health Canada and the USFDA) have relaxed regulations over the production of ABHRs, enabling many beverage distilleries and industrial ethanol plants to produce technical-grade alcohol to help alleviate growing demand. Furthermore, due to shortages in gelation agents, Health Canada has also eased some restrictions on substitutions for rheology modifiers [26] . This has resulted in increased availability of these alcohol-based products, from~370 (March 2020) to~5000 products presently (February 2021) [27] . However, many of these manufacturers possess different infrastructure and utilize different and variable feedstocks, thereby influencing the grade of the end-product alcohol and the type of impurities. Therefore, one must take extra precautions in ensuring compliance and efficacy of ABHR products which are currently available. Analyses of ABHRs, to ensure compliance and efficacy, is difficult especially if unknown impurities or materials are present (e.g., type of gelation agent in the product). Therefore, in this study, we sampled a broad variety of ABHRs purchased from local and online merchants to investigate both quality and compliance of the ethanol product. In the analyses of gelled ABHRs, we also developed a rapid method to precipitate gelation materials for downstream identification and quantification of ingredient materials using gas chromatography with flame ionization detection (GC-FID). Alcohol standards ( Table 1 ) are characteristic of a range of common alcohols (including fusel alcohols) produced during alcoholic fermentation and were obtained from Sigma Aldrich (Mississauga, ON, Canada). The internal and recovery standards were 3-pentanol (CAS number 584-02-1) and 4-methyl-2-pentanol (CAS number 108-11-2), purchased from Millipore Sigma (Mississauga, ON, Canada). Acetonitrile (HPLC grade), ethanol (HPLC grade), and dichloromethane (certified ACS) were obtained from Thermo Fisher (Ottawa, ON, Canada). A variety of 16 gelled and 26 liquid ABHRs were purchased locally or through online retailers between March 2020 and September 2020. A standard spiking solution of dichloromethane (DCM) containing the recovery standard, 4-methyl-2-pentanol (10 µL·L −1 ), was used as the extraction solvent for gelled ABHRs. Gelled ABHRs were vortexed for 20 sec with this solution at a ratio of 1:1 (sample:solvent; w/v; 1 g/mL), in 2 mL VWR microcentrifuge tubes (Mississauga, ON, Canada). The samples were then centrifuged at 10,000 rpm (Spectrafuge 24D, Labnet International Inc.; Edison, NJ, USA) for 5 min at room temperature to facilitate phase separation. The supernatant was then filtered using VWR 0.2 µm polytetrafluoroethylene (PTFE) syringe filters (Mississauga, ON, Canada) and spiked with 3-pentanol (10 µL·L −1 ), then analyzed directly via GC-FID. Liquid ABHRs were filtered with 0.2 µm PTFE syringe filters (Mississauga, ON, Canada), spiked with 4-methyl-2-pentanol (10 µL·L −1 ), and analyzed directly via GC-FID with no prior treatment. Each sample was measured in triplicate, with the exception of sample 26 (due to inadequate sample amount). Results are measured as mean ± standard deviation. Analyses of ABHR were conducted using an Agilent 7890 GC (Santa Clara, CA, USA) equipped with a flame ionization detector (FID). Samples were injected using an Agilent 7693A (Santa Clara, CA, USA) automatic liquid sampler with a 10 µL Hamilton cemented needle syringe (Reno, NV, USA). Volatile organic compounds were separated on a 30-m Agilent J&W DB-624 Ultra Inert column (ID 0.32 mm, 1.80 µm film). The FID was operated at 250 • C with flow rates of 400 mL min −1 of air, 30 mL min −1 of hydrogen, and 25 mL·min −1 nitrogen. Initial oven temperature was 40 • C and held for 5 min. The temperature was increased to 225 • C at 20 • C·min −1 and held for 2.5 min, giving a total run-time of 16.75 min. Helium was used as the carrier gas, with a flow rate of 6 mL·min −1 . The inlet was operated in split mode (40:1) at 140 • C and 1 µL of sample was injected. The syringe was thoroughly washed with acetonitrile between injections to avoid crosscontamination. A five-point calibration curve was derived for all the impurities listed in Table 1 , achieving an R 2 > 0.99 and an RSD ≤ 25%. The unprecedented impact of the COVID-19 pandemic has led to a global crisis resulting in widespread panic buying of essential items [28] including hand sanitizer and surface disinfectants. This demand led to shortages of commercial essential products and resulted in government regulatory agencies temporarily permitting the use of lower quality raw materials and substitutions in ABHRs [29] . A simplified licensing approach resulted in rapid manufacturing and the availability of over 5000 hand sanitizer products [27] , many of which were produced using non-USP-grade ethanol. These interim guidelines initially permitted acetaldehyde concentrations to be not more than (NMT) 1000 ppm. However, by June 2020, the concentration of permitted acetaldehyde was lowered to 400 ppm, and later reduced to 75 ppm by September 2020 [30] . This decrease in regulatory limits was attributed partly to improved distillation and purification processes at many manufacturing facilities [30] . Although, samples >75 ppm can be marketed and sold, they require additional health related labelling [30] . Typically, USP-compliant ethanol is required in the formulation of ABHRs and is limited to NMT 10 ppm of combined acetal and acetaldehyde [23] . Fortunately, many of the hand sanitizers examined in this study were compliant with Health Canada's interim guidelines for technical-grade ethanol. However, the main non-compliant contaminant appeared to be acetaldehyde. Typically, ABHRs are formulated using pharmaceutical-grade ethanol, examined against the USP ethanol monograph. This monograph specifically requires quantification of methanol, benzene, acetal (1,1-Diethoxyethane), and acetaldehyde in addition to a sum of all other organic compounds detected by GC. Traditionally benzene has been used as an entrainer in azeotropic distillation systems for ethanol dewatering [31] , however, due to its toxicity [32] this solvent has since been replaced with other less-hazardous solvents (e.g., cyclohexane and ethylene glycol) [33] . Currently, the most established drying methods are distillation and adsorption (e.g., molecular sieve) processes [33, 34] . Initial results demonstrated a compound elution at 3.90 min, which is typical of for benzene elution. This was later identified to be isobutanol and was confirmed through GC-MS. To be marketed as USP-grade ethanol, solutions must contain ≤200 µL·L −1 methanol, ≤2 µL·L −1 benzene, and ≤10 µL·L −1 combined acetal and acetaldehyde [23] , and the sum of all other impurities must also be ≤300 µL·L −1 [23] . Extraction of compounds of interest from gelled ABHRs using dichloromethane was successful, achieving a mean recovery of 105.7% ± 9.1%. However, 1-butanol co-eluted with the internal standard in many of the gelled samples. Therefore, to obtain an approximate concentration for 1-butanol, the average area of the internal standard was subtracted from the total peak area at the retention time of 5 min. An ethanol blank containing 10 ppm of 3-pentanol was injected every 10 samples to compensate for instrument drift. In total, 31 of the 42 samples complied with the interim guidelines outlined by Health Canada [30] . The remaining 11 ABHR samples (samples 4, 11, 13, 14, 15, 19, 21, 45, 25, 26, and 42) did not comply with current Health Canada legislation, primarily due to excess acetaldehyde content within the ABHRs. However, these products were purchased prior to the regulatory decrease in acetaldehyde concentrations issued by Health Canada. Methanol concentrations were below 200 µL L −1 in accordance with USP criteria, ranging from below limited of detection (LOD) to 47 ± 2 µL L −1 (Figure 1 ). Acetaldehyde appeared to be the most common impurity in ABHRs analyzed, with 28 samples containing acetaldehyde concentrations from 17 ± 0.5 µL·L −1 to 251 ± 10 µL·L −1 (Figure 2 ). Acetal concentrations were between