Assessment of quality of alcohol-based hand sanitizers used in johannesburg area during the covid‐19 pandemic

ABSTRACT Since the outbreak of the Coronavirus Disease 2019 (CoViD-19), the World Health Organization has recommended that, in absence of soap and water, alcohol-based hand sanitizer can be

used to prevent the transmission of coronaviruses. Unfortunately, many media and anecdotal reports indicate that many alcohol-based hand sanitizers sold in South Africa are substandard and

some contain potentially toxic ingredients. The study aimed to identify hand sanitizers used in the Johannesburg area during the CoViD-19 pandemic that do not contain the recommended alcohol

concentration of at least 70% propanol or 60% ethanol, and contain traces of toxic ingredients. Hand sanitizers randomly collected from various traders around Johannesburg were analyzed

using Agilent auto sampler coupled to a gas chromatograph utilizing flame ionisation detection. Of the 94 hand sanitizer samples collected, three preparations contained no alcohol, whereas

the rest contained either ethanol, 2-propanol or 1-propanol or a combination of two alcohols. Of the alcohol-containing hand sanitizers, 37 (41%) contained less than 60% alcohol. Ethyl

acetate, isobutanol and other non-recommended alcohols (methanol and 3-methyl-butanol) were also identified. Consumers are therefore warned that among the many brands of hand sanitizers

found around Johannesburg, there are some substandard preparations and some that contain traces of toxic ingredients. SIMILAR CONTENT BEING VIEWED BY OTHERS DETERMINATION OF ETHANOL,

ISOPROPYL ALCOHOL AND METHANOL IN ALCOHOL-BASED HAND SANITISER TO ENSURE PRODUCT QUALITY, SAFETY AND EFFICACY Article Open access 10 June 2023 QUALITY AND SAFETY INVESTIGATION OF COMMONLY

USED TOPICAL COSMETIC PREPARATIONS Article Open access 31 October 2022 ADULT AND CHILDREN’S USE OF HAND SANITIZER DURING A PANDEMIC – AN OBSERVATIONAL STUDY Article Open access 24 September

2022 INTRODUCTION The gold standard for hand hygiene and prevention of the spread of non-airborne infectious diseases is regarded as washing with warm water and soap, because water and soap

remove oils from hands that can harbour pathogens1. However, in the absence of water, hand sanitizers are recommended2,3. The transmission of respiratory pathogens spread by droplet or

airborne routes is limited through respiratory hygiene/cough etiquette and physical space infection prevention measures4,5. Since the outbreak of SARS-CoV-2, CoViD-19 (coronavirus), it is

recommended by the World Health Organisation (WHO) that, in absence of water, the use of alcohol-based hand sanitizers can prevent the transmission of coronavirus6. Consequently, the demand

for hand sanitizers has increased worldwide including South Africa, resulting in a surge in the trade of hand sanitizers and initially leading to shortages in their supply. Hand sanitizer

formulations exist in the form of liquids, gels and foams. Depending on the active ingredient used, hand sanitizers can be classified as one of two types: alcohol-based and alcohol-free.

Alcohol-based hand sanitizers are recommended for general use, whereas the alcohol-free ones are not7,8. Hand sanitizers with less than the recommended alcohol content (60–95% alcohol) have

been found not to work well for many types of pathogens, in that they may merely reduce their growth rate and hence reduce their numbers rather than kill them outright9,10. Alcohol-based

hand sanitizers are available in the form of rinses (liquid) and rubs (gel, foam and cream), and both are effective agents for reducing the number of viable pathogens, including coronavirus,

on hands. Alcohol-based hand sanitizers may contain a variety of alcohols [e.g., isopropyl alcohol (isopropanol, 2-propanol), ethanol (ethyl alcohol), _n_-propanol (1-propanol)] or a

combination of two of these, including other ingredients11,12,13,14. For alcohol-based hand sanitizers, the US Centres for Disease Control and Prevention (CDC) recommends a concentration of

60–95% ethanol or 2-propanol mixed with distilled water15. Alcohol acts on microbes in the presence of water by making the organism cell membrane permeable leading to cytoplasm leakage,

denaturing of proteins and eventually, cell lysis12. At higher concentrations (> 95%) alcohol is not effective since microbial denaturing of proteins only takes place in the presence of

water16. Alcohols with four carbons and more are hence, not recommended to be used as hand sanitizers since they are less soluble in water2. Ethanol has been shown to be effective against a

variety of enveloped viruses, beginning at concentrations of 42.6%17. Addition of acids to ethanol can substantially improve the virucidal activity against most viruses17. For example, a

formulation with low alcohol content and citric acid was found to inactivate all enveloped and non-enveloped viruses18. Several studies demonstrate that 2-propanol is considerably less

effective compared to ethanol against viruses17. Some studies have also shown that ethanol gel formulations, unless they have been specially formulated and tested are less efficacious than

ethanol solution formulations19, even though this has not yet been proven for SARS-CoV-2. As previously indicated the global medical crisis as a result of the CoViD-19 pandemic has resulted

in a great surge in the trade of hand sanitization products. This emergent situation is expected to continue for a considerable period of time until more efficient infection preventive

measures become available, hence hand sanitizer demand will remain for an extended time. Unfortunately, many hand sanitizers in South Africa have not been verified to meet the regulators’

recommendations or that they are manufactured under the stipulated regulatory conditions20,21. In addition, the regulator [South African Bureau of Standards (SABS)] lacks verifiable

information to ascertain the methods being used to prepare hand sanitizers at homes and to determine if these sanitizers are safe for use on human skin. As part of public awareness campaign

and contribution to assist during the CoViD-19 pandemic, the project aimed to identify sanitizers available and used in the Johannesburg area that do not contain the recommended quality and

alcohol content. In South Africa, alcohol-based hand sanitizers must comply with the standard SANS 490 as recommended by SABS20. The standard specifies that a minimum of 70% alcohol content

is required if; alcohol, such as ethanol, isopropanol or _n_-propanol is the main ingredient; and that 60% alcohol content is required if there are other active ingredients. Solvents such as

acetone (propanone), methanol, methylated spirits or other spirits are not allowed to be used. MATERIALS AND METHODS COLLECTION OF HAND SANITIZER SAMPLES Ninety-four (94) samples of hand

sanitizer sold in retail stores, spaza shops (informal convenience shop business in South Africa) and by street vendors, were randomly collected around Johannesburg during the period March

to June 2020. The products were purchased ensuring not to buy repeat products/brands. Where two products of the same brand were included in the study it was so that one represents a gel and

the other a liquid hand sanitizer. The hand sanitizer (HS) samples were labelled as HS1 to HS94 (Table 1). PREPARATION OF INTERNAL STANDARD (2% ACETALDEHYDE) Two millilitres (2 ml) of

acetaldehyde (Sigma-Aldrich, Germany) were added to a 100 ml volumetric flask. Deionized water was added to make up the volume to the mark. PREPARATION OF 2% STOCK STANDARDS Two millilitres

of each reagent (Sigma-Aldrich) were added to a 100 ml volumetric flask. Deionized water was added to make up the volume to the mark. A stock of each of the following reagents was prepared;

methanol, ethanol, 1-propanol, 2-propanol, isobutanol, 3-methyl-butanol and ethyl acetate. PREPARATION OF CALIBRATION STANDARDS A calibration standard was prepared in a range of 0.1–1.8% by

diluting the stock solution with deionized water. The standards were each prepared in a 10 ml headspace vial, capped and mixed well on a vortex mixer. The standards were then immediately

placed onto the headspace auto sampler tray for analysis. PREPARATION OF QUALITY CONTROLS A 2% quality control stock solution was prepared by adding 2 ml of alcohol (Sigma-Aldrich, Germany)

to a 100 ml volumetric flask and filling up to the mark with deionized water. Three quality controls at low (QC 1, 0.2%), medium (QC 2, 1.0%) and high level concentration (QC 3, 1.6%) were

prepared from the stock solution. Each QC was prepared in a 10 ml headspace vial, capped with septa and aluminium crimp cap and mixed well on a vortex mixer. All 3 QCs were prepared in

duplicate and positioned on the auto sampler tray for analysis after calibration standards and after every 5 duplicate samples. PREPARATION OF HAND SANITIZER SAMPLES PREPARATION OF LIQUID

HAND SANITIZER SAMPLES In a sterile polypropylene cup (urine container) was pipetted 350 µl sanitizer to which was added 25.65 ml deionized water. Then 900 µl of this solution was

transferred to a 10 ml headspace vial to which 100 µl of internal standard was also added. The vial was capped and contents mixed thoroughly on a vortex mixer before analysis. PREPARATION OF

GEL HAND SANITIZER SAMPLES In a sterile polypropylene cup on a weighing balance 10 g of deionized water was measured and 0.350 g of gel hand sanitizer was also added. The urine container

was filled up with more deionized water until a mass of 25 g was reached. The cup was capped and shaken to mix contents well. Then 900 µl of this solution was pipetted into a 10 ml headspace

vial to which was also added 100 µl of internal standard. The vial was capped and contents mixed on a vortexer before analysis. ANALYSIS OF SAMPLES BY HEADSPACE GAS CHROMATOGRAPHY CONNECTED

TO A FLAME IONISATION DETECTOR (HS-GC/FID) Following sample preparation samples were immediately placed onto the Agilent G1888 headspace auto sampler tray (Agilent Technologies, USA) for

analysis. The samples were analysed using a 6890N Agilent gas chromatograph (Agilent Technologies, USA) utilizing a flame ionisation detector. The column of choice was a SUPELCOWAX column (L

 = 30 m, ID = 0.25 mm and film thickness = 0.5 µl) purchased from Sigma-Aldrich. DATA ACQUISITION AND PROCESSING Quantitation was performed using the Agilent OpenLab CDS ChemStation Edition

C.01.05 integration software for GC Systems, accompanying the GC system22. A determination coefficient (r2) of more than 0.999 was obtained for the calibration curves. Method accuracy,

precision and repeatability were assessed by calculating the standard deviation (SD) of replicate measurements, the standard error of the mean (SEM) and the coefficient of variation (CV %)

(see Tables S2–S6, in Supplementary Material). DATA ANALYSIS Results were analyzed using Microsoft Excel. Descriptive statistics using tables, mean and percentage was used to describe the

data obtained. RESULTS Ninety-four (94) samples of hand sanitizer sold in retail stores, spaza shops and by street vendors, were randomly collected around Johannesburg during the period

March to June 2020. The samples consisted of fifty (50) gels and forty-four (44) liquids as presented in Fig. 1. Forty of the sanitizers (14 liquids and 26 gels) did not have their alcohol

content stated on the container and only one sample was clearly indicated as alcohol-free (Table 1). This sample set represents most of the hand sanitizer brands available and/or sold in

retail stores, spaza shops and by individuals, that are in use in different households and various workplaces around Johannesburg during the CoViD-19 pandemic. Of the 94 hand sanitizer

samples collected, three sanitizer preparations were found to contain no alcohol, whereas the rest contained either ethanol or 2-propanol or a combination of these two (Table S1). Only one

hand sanitizer sample contained solely 1-propanol. By comparison, liquid formulations had on average less alcohol (56.38 ± 26.74%) than the gel formulations (66.14 ± 20.95%). Of the

alcohol-containing sanitizers, 37 (41%) contained less than 60% alcohol. Toxic alcohol denaturants (ethyl acetate and isobutanol) and other non-recommended alcohols (methanol and

3-methyl-butanol) were also identified in 17% of these preparations (Fig. 2). Results from this study indicate that there are about similar number of gel hand sanitizers in existence around

Johannesburg as the liquid formulations (50 gels versus 44 liquids). However, there is no certainty that this is a true representation of the then available alcohol-based hand sanitizing

products as no statistical techniques were employed to collect these hand sanitizer samples. By comparison, liquid formulations (56.38 ± 27%) had on average less alcohol than the gel

formulations (66.14 ± 20.95%). To assess the quality of the results, the following criteria were examined so as to confirm the accuracy and robustness of the analytical method23. All peaks

including ethanol and isopropanol which eluted very close to each other, had baseline separation. Peaks were sharp with narrow baseline width. The results were repeatable and reproducible as

shown in Tables S2 and S3, passing the repeatability and reproducibility tests with the difference between two results chosen randomly being less than 2.83 × SD. The average and standard

deviation of the retention times for all the compounds identified were calculated. Results showed acceptable performance within run and between runs. Data for the internal standard stability

and reliability are provided in Table S4. Data were generated on different days and by two analysts. There was minimal drift in retention times for all three levels of quality control

throughout all the analytical runs. Linearity for the responses was assessed by examining the correlation coefficients for the calibration for all analytical runs. It is evident as shown in

Table S5 that there was strong positive association (linear response) between the concentrations and the signal responses for all the analytes. Furthermore, the deviations in linearity

between the analytical runs were minimal with all correlations remaining at 99% positive linear association. Two quality controls of each of the three levels were run immediately after

calibration and thereafter after every 10 samples and at the end of the analytical run. Box and Whisker plots were used to identify and remove all outliers in the data sets. Results for the

quality controls were acceptable as can be seen in Table S6. Average recoveries (Av Recovery %) were acceptable. As this was a non-standard in-house developed method, Guidelines for Standard

Method Performance Requirements AOAC Official Methods of Analysis24 was used to estimate acceptable recoveries. The recoveries were deemed acceptable for this work. DISCUSSION While more

(56%) brands of hand sanitizer in this study contained the recommended concentration of alcohol, there were also many (44%) substandard and possibly subpotent preparations. Unfortunately,

tests to determine if any of the analyzed hand sanitizers with lower alcohol content than is recommended had any of the virucidal activity enhancing ingredients, such as acids, were beyond

the scope of this study. The study also found that only 30% (10 gels and 9 liquids) of the analyzed hand sanitizers contained ≥ 80% alcohol. Even though alcohol concentrations higher than

80% are known to be less potent against bacteria because proteins are not easily denatured in the absence of water, this bodes well for disinfection against SARS-CoV-2 as ethanol at ≥ 80% is

highly effective against enveloped viruses19. Moreover, it was found that some hand sanitizers contained, in addition to the acceptable alcohols (ethanol, 1-propanol and 2-propanol), some

toxic ingredients, such as ethyl acetate, 3-Methyl-1-butanol and methanol. This is worrying because even if a hand sanitizer contains enough alcohol as recommended or contains ingredients

that enhance its virucidal activity in case of low alcohol content (< 60%), the presence of toxic ingredients renders the preparation harmful and unfit for human use. It is for this

reason that it is recommended that all consumers (workplaces and the public in general) be aware of untrustworthy brands of hand sanitizer supplying substandard and possibly sub-potent

sanitizer preparations or sanitizers with toxic ingredients. The tendency for unscrupulous manufacturers in South Africa is to mislead the public by labelling their products as “SABS

Approved” yet not carrying the SABS Mark Scheme number. The SABS provides on its website the information that must be available on every container of approved hand sanitizer sold in South

Africa21. Unknowingly, using a hand sanitizer with no virucidal activity against SARS-CoV-2 may give one a false sense of security, while those using sanitizers containing toxic ingredients

are likely to suffer from the associated risks. For example, exposure to methanol through both ingestion and transdermal absorption, if left untreated, can be extremely dangerous, leading to

significant disability and death25. Even if toxic substances are just traces, the typical frequent use of hand sanitizer products throughout the day can result in very high total exposure

with consequent adverse health effects. The US FDA is therefore continually adding certain hand sanitizers found to contain toxic ingredients to import alerts, to stop these products from

legally entering the U.S. market26, while the South African Bureau of Standards (SABS) has also warned consumers about some unscrupulous manufacturers that are making false claims that their

products are SABS-approved27. CONCLUSION Just like several other countries around the world, South Africa (SA) has relaxed legislation to make it easier for local businesses to rapidly

produce alcohol-based hand sanitizers to meet the great surge in demand for hand sanitization products during the SARS-CoV-2 outbreak. However, those producing hand sanitizers are still

advised to follow both the WHO and SABS guidelines, and avoid using poor alcohol quality which is likely to contain toxic substances. The SA public is also advised to remain alert to media

reports that continually keep surfacing about hand sanitizer brands in violation of the SABS guidelines21, by producing sanitizer preparations that are subpotent or contain toxic substances.

It is also worth noting that the presence or addition of other pharmaceutical ingredients (e.g., chlorhexidine, triclosan, iodine/iodophores and benzalkonium chloride) may assist in

instances where the alcohol-based hand sanitizers may fall short against certain bacteria and viruses11,12. However, though the presence of these other ingredients may impart additional

antiviral and antimicrobial properties to the alcohol-based hand sanitizers, they may as well possibly exhibit some toxicity to humans. For example, although iodine is effective against most

viruses and bacteria, it is also believed to cause skin irritation and discolouring, thus its presence may be harmful to humans12,18. DATA AVAILABILITY The datasets used and/or analysed

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2020. Download references ACKNOWLEDGEMENTS The authors would like to thank the National Institute for Occupational Health (NIOH), a Division of the National Health Laboratory Service (NHLS),

for supporting this research. Also, greatly acknowledged are the NIOH Analytical Services team members (Angela Mawela, Lesiba Sethosa, Jane Mulaudzi and Sesitjie Moremi) for assisting in

the collection of the hand sanitizer samples. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Analytical Services, National Institute for Occupational Health, National Health Laboratory

Service, P.O. Box 4788, Johannesburg, South Africa Puleng Matatiele, Bianca Southon, Boitumelo Dabula, Talulani Marageni, Poobalan Poongavanum & Boitumelo Kgarebe Authors * Puleng

Matatiele View author publications You can also search for this author inPubMed Google Scholar * Bianca Southon View author publications You can also search for this author inPubMed Google

Scholar * Boitumelo Dabula View author publications You can also search for this author inPubMed Google Scholar * Talulani Marageni View author publications You can also search for this

author inPubMed Google Scholar * Poobalan Poongavanum View author publications You can also search for this author inPubMed Google Scholar * Boitumelo Kgarebe View author publications You

can also search for this author inPubMed Google Scholar CONTRIBUTIONS P.M. wrote the manuscript text. B.S., T.M. and B.D. performed data collection and analysis. P.P. assisted with analysis

and results interpretation. B.K. reviewed and edited the manuscript, and supervised the team. All authors reviewed the final manuscript. CORRESPONDING AUTHOR Correspondence to Puleng

Matatiele. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER'S NOTE Springer Nature remains neutral with regard to

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quality of alcohol-based hand sanitizers used in Johannesburg area during the CoViD‐19 pandemic. _Sci Rep_ 12, 4231 (2022). https://doi.org/10.1038/s41598-022-08117-z Download citation *

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