key: cord-1009548-su74devj authors: Majorani, Costanza; Leoni, Claudia; Micheli, Laura; Cancelliere, Rocco; Famele, Marco; Lavalle, Roberta; Ferranti, Carolina; Palleschi, Luca; Fava, Luca; Draisci, Rosa; D’Ilio, Sonia title: Monitoring of alcohol-based hand rubs in SARS-CoV-2 prevention by HS-GC/MS and electrochemical biosensor: A survey of commercial samples date: 2022-02-26 journal: J Pharm Biomed Anal DOI: 10.1016/j.jpba.2022.114694 sha: 469d7fbc266c03bc08df6183806b434323b96642 doc_id: 1009548 cord_uid: su74devj [Figure: see text] Pag. 2 a 32 sanitiser products had the recommended average alcohol content, thus highlighting the need for analytical controls on this type of products. The spread of the Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2, is responsible for the global diffusion of the virus-related disease officially named COVID-19 (CoronaVIrusDisease-2019) [1] and has emerged as a serious public health issue [2] . To counter the spread of the virus, the WHO and the major public health agencies have recommended the use of adequate personal protective equipment (PPE) together with careful personal hygiene to be sought especially through frequent hand washing [3] and dedicated hand product use. Among these, hand sanitiser products/alcohol-based hand rubs (ABHRs) have found wide diffusion, becoming the most widespread means of obtaining rapid and effective hand hygiene [4] [5] [6] . The sanitising/disinfecting action of ABHR is due to the presence of alcohols, whose primary targets are the proteins in cell plasma membranes of pathogens, which have been shown to be active against a wide variety of viruses and bacteria [7] . Ethyl alcohol (EtOH), isopropyl alcohol (IPA) and n-propyl alcohol (n-PA) are, alone or in a combination, the most used alcohols in ABHR formulations. It is remarkable to highlight that n-PA is approved for the use as a biocide in the European Economic Area (EEA), but the U.S. Food and Drug Administration (FDA, Silver Spring, MD, USA) has limited its content in ABHRs to 0.1 % v/v since it is not listed as an active agent for hand antisepsis and surgical hand preparation in the United States [7] . According to the principal health agencies, ABHRs must contain at least 60 %v/v alcohol, to have an effect as disinfectants on pathogens, including SARS-CoV-2 [8] . Scientific literature has evidenced that, in addition to the alcohols concentration, other factors that contribute to sanitation must be taken into account, such as the minimum friction time and the amount of sanitiser applied on the hands [9, 10] . To cope with the new health emergency and limit infections, a wide variety of ABHRs has been placed on the market as cosmetics, biocidal products and galenic productions. Cosmetic hand sanitisers and galenic preparations are produced by Regulation (CE) N. 1223/2009 [11] and the European Pharmacopoeia protocols, respectively. Biocidal hand products must be authorized by the Italian Minister of Health [12] , before being placed on the market and the active substances therein contained must be approved as disinfectants in compliance with Regulation (CE) N. 528/2012 for Biocidal Products [13] . Different formulations are also available as solutions, foams and gels, with the latter ones widely diffused among the population because of their manageability and ease of handling. Acrylate acrylates (Carbopol™, carbomer, acrylates/c10-30, and tea-carbomer) or cellulose derivatives (hydroxypropyl methylcellulose, hydroxyethyl cellulose and polyquaternium-7) are examples of frequently used gelling agents [14] [15] . The sudden increase in the demand for ABHRs has favoured the spread of substandard products not aligned with the health agencies recommendations and in the literature, several analytical methods based on spectroscopy, spectrometry and flame ionization detection, have focused on the determination of alcohols in hand sanitisers [16] [17] [18] [19] [20] [21] . In 2020, the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA) has developed and evaluated different analytical methods (gas chromatography with flame ionization detection, liquid chromatography with ultraviolet absorbance detection, quantitative nuclear magnetic resonance spectroscopy, and attenuated total reflectance Fourier-transform infrared spectroscopy) for the quality control of ABHRs in terms of both alcohol concentration and impurities [20] . Indeed, depending on the purity of EtOH used for ABHRs production, the population may be exposed to harmful levels of substances not intended for use in ABHR that may be present as impurities [20] [21] [22] . In early 2020 FDA investigated methyl alcohol (MeOH) contamination in ABHRs and stated that it cannot be safely used as an ingredient, or as a denaturant, in hand sanitiser [7] . In Europe, Regulation (EU) 2020/1683 amended Annex III of Regulation 1223/2009/EC on Cosmetic Products [23] , setting a volume fraction limit of 5% for MeOH content in cosmetics, calculated as EtOH or IPA denaturant. Considering all these critical issues, we conducted an Italian survey, for the safeguard of consumers, on ninety ABHRs of different formulations (foam, liquid and gel), commercially available to the public during the first period of the pandemic. Quality assessment of collected alcohol-based hand sanitisers was performed by HS-GC/MS combined with an innovative approach based on the electrochemical biosensor. The simultaneous determination of EtOH, IPA, n-PA as active ingredients in hand sanitisers and of MeOH as impurity ruled by the Cosmetic Products Regulation [23] , was investigated with a specific analytical method. Considering that the target compounds are volatile, HS-GC/MS was chosen for the analysis of hand sanitisers because it reduces the equipment contamination caused by other ingredients [24] . The headspace conditions (as equilibration temperature and equilibration time) and acidification sample treatment were studied and optimised. Method validation parameters were evaluated according to ISO/IEC 17025 requirements [25] . In addition, a rapid and inexpensive screening was conducted on collected samples by an electrochemical biosensor, based on the immobilisation of Alcohol oxidase on screen-printed electrodes (SPEs). This tool is normally used for ethyl alcohol determination in food matrices such as cheese and wine [26] and the application for primary alcohol determination in ABHRs is described for the first time in this paper. Results obtained on commercially available hand sanitisers products by biosensors and by HS-GC/MS were finally compared. In this paper, the analyses of ninety commercial samples will evidence that quality assessment of these products is of primary importance for the safety of consumers. Furthermore, the study will show that the combination of HS-GC/MS and electrochemical biosensor is a fast and reliable tool, able to detect hand sanitisers with insufficient alcohol concentration. Methyl alcohol (98.7 %), isopropyl alcohol (99.8%), ethyl alcohol (99.7%) and n-propyl alcohol Ninety ABHR samples, which were randomly collected from several shops (supermarkets and pharmacies) in the city of Rome from April to November 2020, were stored at 25°C and subsequently analysed for this study. These ABHRs of different types and brands were selected and analysed to determine their alcohol content. About 82% of samples purchased were cosmetic products, 11% were biocidal products and 7% were galenic preparations. 81% of the samples collected were produced in Italy, 8% were made in Europe, 4% in other countries and 7% of samples had no indications on the label. Formulations consisted of 87% gel, 12% liquid and 1% foam. The Sample was sonicated at room temperature for 10 minutes and analysed by HS-GC/MS. A five-point calibration curve was obtained in a concentration range from 1% v/v to 80%v/v for EtOH, IPA, n-PA and MeOH. A non-alcohol based hand rub gel was used as a blank sample for the construction of the matrix-matched calibration curve for each analyte. The blank sample was subjected to all the sample processing steps. Calibration curves were determined by plotting the peak area ratio of the analytes to IS versus the analyte concentration. Quality control samples were prepared at concentrations of 50 %v/v. performed on capillary column Zebron™ ZB-WAXPLUS ™ (30 m x 250 µm x 0.25 µm) (Phenomenex, Torrance, CA, USA). The carrier gas was helium (99.999%). Before HS-GC/MS analysis, vials were placed in a headspace oven thermostated at 60 °C with vial shaking set to off. Different conditioning times were evaluated in order to maximize the partitioning of the volatile portion of the sample into the vial headspace. The time conditioning effect for each analyte was studied in four selected biocidal samples. Since EtOH was the only active substance in the selected biocides, they were fortified with the working solution in order to investigate the response of each analyte. The analyses were conducted in duplicate at the following conditioning times: 10, 20, 30 and 40 minutes. The gas-tight syringe, heated at 60°C, sampled and injected the steam (250 µL) in split mode (split ratio 40:1). Septum purge flow was 3 mL min -1 . The GC/MS oven temperature program was: 40 °C held for 1 min then ramped at 10°C min -1 up to 90 °C (run time: 6 minutes); carrier gas (helium) was kept at a constant flow rate of 1.3mLmin -1 . The electron impact energy was 70eV and the quadrupole, ionization source and injector temperatures were set at 150°C, 230°C and 90°C respectively. The mass analyser was set in the selected ion monitoring (SIM) mode and total scan (TIC) mode. Performance characteristics such as sensitivity, specificity, limit of detection (LoD), limit of quantification (LoQ), linearity, precision (repeatability and intermediate precision), accuracy and measurement uncertainty, were assessed according to well-established requirements of ISO/IEC 17025, Guide to the Expression of Uncertainty in Measurement (GUM) [27] and internal performance criteria. Screen-printed electrodes (SPEs) were home-made with a 245 DEK (High-performance multipurpose precision screen printer, Weymouth, UK) screen-printing machine. The electrodes, printed on a folding polyester film (Autostat HT5) obtained from Autotype Italia (Milan, Italy), were produced in foils of 48. Graphite-based ink (Elettrodag 421) from Acheson (Milan, Italy) was used to print the working and the counter electrode, while silver ink (Acheson Elettrodag 4038 SS) was used for the reference electrodes. The diameter of the SPE's working electrode was 0.3 cm resulting in an apparent geometric area of 0.07 cm 2 . The application of an insulating print (Argon Carbonflex 25.101S) defines the actual surface area. Screen-printed platforms were modified using Prussian Blue (PB, Fe 4 (Fe(CN) 6 ) 3 ) as a diffusional electrochemical mediator. This chemical deposition was carried out following an optimised procedure reported in previous works [28] . In particular, alcohol biosensors were obtained by immobilising alcohol oxidase (AOx) onto PB modified working electrodes. Specifically, a solution of AOx (1mgmL -1 ), Glutaraldehyde (1%) [29] and BSA (5%) in distilled water was prepared and cast. Pag. 6 a 32 The sensor presented in this work exploits the reaction reported below. The electrochemical measurement and therefore the current signal obtained from the hydrogen peroxide discharge is proportional to the concentration of alcohol present in the sample. Electrochemical experiments (Amperometry) were performed using a PalmSens (Palm Instruments BV, Electrochemical Sensor Interfaces, Houten, Netherlands), which is a hand-held battery-powered potentiostat instrument for use with electrochemical sensors or electrochemical cells. In particular, amperometric measurements were carried out applying on AOx-PB/SPE a 50mV potential for 40s. The hand rub samples were treated as follows: an equal amount of 50 mM phosphate buffer pH 7.4 was added to each sample (dilution 1:1 v/v); the mixed solutions were sonicated 60 minutes (50Hz, 35°C using a Hielscher UP100H) and then gently shaken overnight under controlled temperature in hermetic glass vials. For the analysis, the obtained solutions were directly analysed. The pH value of each sample was measured after a dilution with distilled water 1:1 v/v and stirring for 1h to avoid the matrix effect of the hydrogel. The instrument is pH8 + DHS (XS instruments, Carpi, Italy). Scanning Electron Microscopy (SEM) was used to investigate the morphology of hydrogel samples. The experiment was performed by using a field emission scanning electron microscope (FE-SEM) (SUPRATM 35, Carl Zeiss SMT, Oberkochen, Germany), using as operating parameters of the instrument 10 keV as gun voltage and a working distance of about 8 mm, while the detector used was the second electron one. Samples were previously metalised to allow electronic conduction on the sample surface. The metallisation, (1 min at 25 mA), was performed using a sputter coater (EMITECH K550X, Quorum Technologies Ltd., Laughton, UK) with a gold target. Microscope photos have been performed on a Celestron, Microcapture Pro apparatus (Celestron, Torrance, CA, USA) with 1600x magnification. In this study, an accurate and sensitive HS-GC/MS method for the simultaneous determination of EtOH, IPA, n-PA and MeOH in hand sanitiser products was developed. Headspace GC is a routinely used technique to investigate volatile analytes even though alcohols determination in several matrices may be carried out also using GC with direct sample injection [16, 20] . However, headspace analysis is particularly appropriate for ABHRs considering their formulations, since GC parts fouling is reduced and cleaner extracts are obtained ( Figure SI1 ) than with direct sample injection [24] . Since a Certified Reference Material (CRM) on this matrix was not commercially available, four biocidal ABHRs were spiked with a known amount of alcohol to assess the effectiveness of extraction because the alcohol content on the label of these products is mandatory [13] . Before the sample extraction, the effect of time was evaluated by plotting the ratio of the corresponding peak area obtained for each analyte to the IS peak area, versus different thermostatic times (10, 20, 30 and 40 minutes). The temperature of both the gas-tight syringe and headspace oven was constant at 60°C to avoid the volatilization of high boiling substances that would interfere with the analysis. Results revealed (data not shown) that n-PA and EtOH peak area ratios slightly increased in all samples as the equilibration time increased, though no substantial difference was observed between 30 and 40 minutes. MeOH and IPA peak area ratios were constant in all samples for all tested times. Therefore, 30 minutes was chosen as vials conditioning time before analysis, even though in literature shorter equilibrium times for HS-GC/MS methods are reported [17, 18] . As concerns MS conditions, the most prominent and characteristic fragment masses were selected from the Total Ion Current (TIC) mode spectrum of the pure analytical standard of each analyte. In particular, one quantifier ion and two qualifier ions were selected for each compound based on their selectivity and abundance. The fragment 31 m/z was chosen as a quantifier for MeOH, EtOH and n-PA because of its highest intensity; while the 45 m/z ions were selected as a quantifier for IPA. The same approach was adopted for IS quantifier ion selection. Table 1 shows the retention times and characteristic m/z ions selected for the acquisition in the SIM mode of analytes and IS. Analytes qualitative identification was assessed by the combination of chromatographic separation and mass spectrometry criteria. According to the first, the relative retention time (i.e. the ratio between the chromatographic retention times (t R ) of the analyte and the IS) of the analytes was compared with that obtained from the calibration curve of each analyte with a tolerance of 0.5%. As for the mass spectrometry criteria, the ratios between the quantifier ion and the two qualifiers, detected in SIM mode during sample analysis, were compared with those obtained from the standards in the calibration curve. [Please insert Once the instrumental conditions had been optimised, selected biocidal samples were analysed by HS-GC/MS, after being diluted in distilled water, added of IS, and sonicated. Results showed that EtOH content was lower than that reported on the label for all but one tested product, being this liquid while the other three samples were gels. Thus, the presence of gelling agents, which act as blockers to avoid alcohols evaporation that would compromise the sanitising properties of these products, required a different sample treatment to make the alcohol extraction effective. To break the polymer crosslinks, samples were acidified with HCl 0.1 M since, as reported in the literature, polymer crosslinking patterns are affected by pH [20, 30] . After this pre-treatment step, samples were analysed again keeping the other processing steps unchanged. The results showed that the alcohol contents, obtained by acidification of samples, were coherent with those reported on the products labels. It is noticeable that the liquid biocidal product was not affected by the acidification since, given its formulation, it did not contain polymers. Optimised sample treatment and instrumental conditions were then applied to the blank sample for the conduction of the validation studies, as well as to the samples collected from the market. Table 2 shows results obtained for the selected biocidal products at each different sample treatment. Table 2 . Results on biocidal products with different sample treatments. The performances of the analytical method were evaluated in terms of specificity, selectivity, detection limit, quantitation limit, linearity, precision, accuracy and measurement uncertainty. Validation studies were carried out by providing the optimised instrumental conditions and using a non-alcohol based hand rub as blank sample which was subjected to all the sample processing steps. Linearity was assessed through five-point matrix-matched calibration curves, prepared by spiking blank samples at analytes concentrations of 1, 10, 50, 70 and 80 %v/v and run on three different days. For each compound, the calibration curve was determined by plotting the ratio of the corresponding peak area to the IS peak area, versus the analyte concentration. The correlation between concentration and detector response for each analyte was determined by a linear regression model using the method of ordinary least squares. As shown in table 3, linear regressions were adequate as the correlation coefficients were not less than 0,999 for each compound. An ANOVA F-test was also applied to ensure the linearity of the method. The test confirmed that the method was linear for each compound in the concentration range selected as the observed values of F were greater than the critical value of F, deduced from the table at the significance level α = 0.05 and ν = 4 degrees of freedom ( Table 3 ). The equations of EtOH, IPA, n-PA and MeOH, obtained from the least-squares elaborations, were used to quantify these analytes in real samples (Figure SI To evaluate the applicability of the validated method to real samples, 90 ABHRs were selected and analysed. About 72 % of samples reported the presence of polymers on the label including acrylates/C10-30 alkylacrylatecrosspolymer, tea carbomer, polyacrylatecrossplymer-6, hydroxyethylcellulose, poly(methylmethacrylate) and polyquaternium-7. The morphological analysis of these samples showed similar behavior in function of the main component of the polymeric structure. The Scanning Electrone Microscope (SEM) images (Figure 2a) showed that the hand rub samples based on carbomer and hydroxyethylcellulose had a filamentous structure as confirmed by optical microscope analysis (Figure 2b) , where the lyophilised samples generated small white flakes [32] or uneven transparent film, typical of the derivate of cellulose [33] , respectively. For the other lyophilized polymers (acrylates/C10-30 alkylacrylatecrosspolymer, polyacrylatecrosspolymer-6, poly(methylmethacrylate) and polyquaternium-7) used for hand rubs, different behavior was observed in the function of the used cross linker as reported in the literature [34, 35] . Before measuring the alcohol content for all the samples at our disposal, a pH control was carried out, obtaining values ranging from 4.9 to 7.3. Ninety ABHRs, purchased from the Italian market, were analysed by HS-GC/MS and by biosensor. The results of seventy-four cosmetic products by HS-GC/MS analysis are shown in Figure 3 . Samples were plotted based on the average alcohol concentration, due to the contribution of all tested alcohols, expressed as %v/v. Concentrations ranged between 3.0 ± 0.1 %v/v and 80 ± 3 %v/v. The majority of samples (42%) had an alcohol concentration less than 49% v/v while 32% of samples were in the range 50 %v/v -59 %v/v. Only 26% of samples had average alcohol content greater or equal than 60 %v/v and, among these, only in 4% of samples, an alcohol concentration in the interval 70 %v/v -80 %v/v was measured. [Please insert figure] The most widely used alcohol for the production of selected cosmetic ABHRs was EtOH, which was found in 92% of the analysed samples while IPA was determined in 26% of samples alone or in combination with EtOH. MeOH and n-PA were below LoQ values (0.53 %v/v and 0.50 %v/v respectively) in all tested samples. Alcohol concentration was declared on 49% of cosmetic ABHRs labels but only 47% of them was coherent with the declared values. Analysis of 10 biocidal samples instead revealed that the average alcohol content was almost within the recommended range for these products, being 60 ± 2 %v/v the lowest determined alcohol concentration and 85.3 ± 3.8 %v/v the highest. Alcohol concentrations declared on the products labels were also confirmed. Among the biocidal products purchased for the study, one sample was collected from a public distributor, available to people, and analysed. The result obtained did not match with the 70 %v/v alcohol concentration declared on the label, as only 40 ± 1 %v/v was determined. A possible reason for this disagreement could be that the product was more exposed to spoilage, in terms of alcohol dispersion. EtOH was determined in all biocidal products and for three of them the bactericidal activity was due to a combination of EtOH and IPA. As for cosmetic products, MeOH and n-PA were below LoQ values in all tested samples. The smallest portion of the analysed samples consisted of six galenic preparations whose alcohol concentrations ranged between 55 ± 2 %v/v and 63 ± 3 %v/v, confirming the value on labels. EtOH was used in all the preparations tested, except for only one sample in which IPA was determined. Neither MeOH nor n-PA were determined. The survey pointed out that 61% of the analysed samples (74 cosmetic, 10 biocidal and 6 galenic products) collected in Italy contained alcohols below 60% v/v. This finding is in contrast with those of other studies conducted in the same period on hand sanitisers [17, 19, 20] and two explanations may be evaluated. The first consideration is attributable to the wide diffusion of substandard products not aligned with the health agencies recommendations in the first period of the pandemic, as also evidenced by Da Costa et al [17] . Otherwise, the manufacturing processes must be considered, in which losses of alcohols may occur leading to final concentrations lower than the expected values [16] . Even though the quality of analysed samples was questionable for active ingredients content, MeOH content was below LoQ value in all samples. in order to minimize the matrix effect on the electrochemical measurement. Moreover, the analytical performances of AOx-based biosensors in the above reported study were investigated. In particular, the screen-printed-based devices showed a good reproducibility (RSD <10%), sensitivity (LoD=27 %v/v) and reusability (the signal loss is <11%). This latter was evaluated up to 10 successive measurements with relative standard deviation (RSD) ranging from 10%. The alcohols concentrations (%v/v), in terms of mean and standard deviation, median, geometric mean, 5th and 95th percentiles, obtained with the two different techniques in 90 commercial hand sanitisers are reported in Table 4 . The two analytical methods gave results very similar to each other, with a slight trend of the electrochemical biosensor to overestimate with respect to HS-GC/MS. Using SigmaPlot ver 11, the 99% prediction interval for the percentage of alcohol content obtained with both methods is calculated using the following equation: where y 0 is the y value predicted for any X 0 , t value for (n-p-1) degrees of freedom, n is the number of the ), while the y-axis indicated the difference ( ). Horizontal lines represent the mean difference and the limits of the agreement are defined as the mean difference plus and minus 1.96 times the standard deviation of the differences. Information on the comparability of the two techniques is given by the position of the points in the graph. The plot in Figure 6 showed that the two techniques could be considered interchangeable as almost all points were between the limits of agreement. Furthermore, the differences between the two measurements had a slightly better agreement at lower alcohols mean concentration values. This allows considering the electrochemical biosensor as a complementary tool with screening purposes for the identification of hand sanitisers suspected to have low total alcohols content. Thus, rapid monitoring of commercial samples could be achieved as a more accurate and sensitive analytical technique, as the HS-GC/MS, might be used for confirmation analyses. (Turkey test) where, in both case, the differences in the median values among the treatment groups were not great enough to exclude the possibility that the difference is due to random sampling variability; there is not a statistically significant difference (p = 0.062). In this work, a novel electrochemical screen-printed based biosensor and an HS-GC/MS in-house validated (according to ISO/IEC 17025) method for the determination of alcohol content in ABHRs were reported. The analyses were conducted on ninety ABHRs (differing in formulation and brands) purchased on the Italian market from April to November 2020. All the analytical parameters and sample preparation steps were explored and optimised obtaining a sensitive and specific HS-GC/MS-based method for the simultaneous determination of EtOH, IPA, n-PA and MeOH. From the validation study, excellent trueness and good precision were assessed and the method can be considered as a valuable and reliable tool for quantifying the alcohols content in a diverse variety of commercial hand sanitisers. Moreover, encouraging results in terms of sensitivity (LoD 27%), reproducibility (RSD <10%) and reusability were obtained using an AOx-based biosensor. By comparing the results collected using the above-reported methods a good J o u r n a l P r e -p r o o f Pag. 14 a 32 correlation was observed (95%). In addition, it was observed that only 39% of the tested products had an average concentration of at least 60 %v/v of alcohol. Among the cosmetic sanitisers, the percentage of products containing the recommended alcohol levels was only 26%, while all biocidal products and galenic preparations analysed were aligned with the health agencies indications. This study highlighted that the combination of biosensor and HS-GC/MS would give a powerful tool for the fast analysis of hand sanitisers, in which the first method is directly usable on the market, lowering the analysis costs and avoiding consumer fraud. Furthermore, the survey confirmed the need to increase analytical controls as executive actions for this type of products, in order to protect the consumer from formulations in which the concentration of alcohol is not clearly stated on the label. Coronaviruses and SARS-CoV-2: A Brief Overview Coronavirus (COVID-19) Outbreak: What the Department of Radiology Should Know World Health Organization United Nations Children s Fund ( UNICEF), 2020 . Water, sanitation, hygiene, and waste management for the COVID-19 virus: interim guidance Show Me the Science -When & How to Use Hand Sanitizer in Community Settings Handwashing: Clean Hands Save Lives Hand sanitisers amid CoViD-19: A critical review of alcohol-based products on the market and formulation approaches to respond to increasing demand Temporary Policy for Preparation of Certain Alcohol-Based Hand Sanitizer Products During the Public Health Emergency (COVID-19) Guidance for Industry Inactivation of SARS-CoV-2 by commercially available alcohol-based hand sanitizers Chemical disinfectants and antiseptics -Hygienic handrub -Test method and requirements A large-scale investigation of alcohol-based handrub (ABHR) volume: hand coverage correlations utilizing an innovative quantitative evaluation system of the European Parliament and of the Council of Regolamento recante norme per la semplificazione dei procedimenti di autorizzazione alla produzione ed all'immissione in commercio di presidi medicochirurgici concerning the making available on the market and use of biocidal products Rheological characterization of topical carbomer gels neutralized to different pH The Influence of the Type and Concentration of Alcohol on the Rheological and Mucoadhesive Properties of Carpobol 940 Hydroalcoholic Gels A rapid and effective method for determination of ethanol content in hand sanitizers (alcohol gel) Quantifying Ethanol in Ethanol-Based Hand Sanitizers by Headspace Gas Chromatography with Flame Ionization Detector (HS-GC/FID) Development and validation of a headspace GC-MS method to evaluate the interconversion of impurities and the product quality of liquid hand sanitizers Inexpensive Portable Infrared Device to Detect and Quantify Alcohols in Hand Sanitizers for Public Health and Safety A Comparison of Measurement Methods for Alcohol-Based Hand Sanitizers Hidden threat lurking behind the alcohol sanitizers in COVID-19 outbreak Potential methanol toxicity and the importance of using a standardised alcohol-based hand rub formulation in the era of COVID-19 Commission Regulation (EU) 2020/1683 of 12 November 2020 amending Annexes II Optimization of Ethanol Detection by Automatic Headspace Method for Cellulose Insulation Aging of Oil-immersed Transformers IEC 17025 General Requirements for the Competence of Testing and Calibration Laboratories ISO Ethanol biosensors based on alcohol oxidase Jcgm Evaluation of measurement data -guide to the expression of uncertainty in measurement Vegetable waste scaffolds for 3D-stem cell proliferating systems and low cost biosensors Biochar from Brewers' Spent Grain: A Green and Low-Cost Smart Material to Modify Screen-Printed Electrodes The effect of pH on polymerization and volume change in PPy(DBS) Eurachem Guide: the Fitness for Purpose of Analytical Methods -A Laboratory Guide to Method Validation and Related Topics Viscoelastic properties and rheological characterization of carbomers Synthesis of hydroxyethyl cellulose from industrial waste using microwave irradiation Synthesis and Characterization of Poly Methyl Acrylate-Poly Ethyl Acrylate Copolymer Rheological Characterization of Hydrophylic Gels Costanza Majorani: writing -original draft, conceptualization, methodology, investigation writing -original draft, methodology, investigation, validation Laura Micheli: writing -original draft, methodology; investigation Rocco Cancelliere: writing -original draft, investigation, resources Marco Famele: writing -review & editing, resources Roberta Lavalle: writing -review & editing, resources Carolina Ferranti: writing -review and editing Luca Palleschi: writing -review and editing Luca Fava: writing -review and editing The authors wish to thank Dr. Pietro Mategazza for his valuable contribution to this manuscript. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.