Simple and rapid low-cost assays to investigate ethylene glycol and diethylene glycol contamination in raw materials and medicinal syrups

Simple and rapid low-cost assays to investigate ethylene glycol and diethylene glycol contamination in raw materials and medicinal syrups | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Simple and rapid low-cost assays to investigate ethylene glycol and diethylene glycol contamination in raw materials and medicinal syrups Benediktus Yohan Arman, Isabelle Legge, John Walsby-Tickle, Tehmina Bharucha, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6683642/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract There have been hundreds of child deaths due to contamination of medicinal syrups with diethylene glycol (DEG) and ethylene glycol (EG). Detection of DEG and EG is usually performed by gas chromatography, a method that is costly, laborious, time-consuming, and the device is not readily available in many low- and middle-income countries (LMICs). Thin-layer chromatography is relatively lower cost and is portable but, as with gas chromatography, requires time and trained personnel. Alternative rapid, low-cost and simple methods to determine DEG/EG contamination are desirable. We tested the suitability of enzymatic, chemical and antibody-based assays to determine DEG/EG. Assays using alcohol dehydrogenase and aldehyde dehydrogenase alone as well as in combination with glycolate oxidase could determine EG in raw materials and at less than 0.1% m/m in some finished products. Saliva and breast milk alcohol test strips containing alcohol oxidase and costing $ 1 could determine EG with a detection limit of 0.5 to 2% m/m in under 2 minutes. Disposable breathalysers also costing only $ 1 could determine both DEG and EG from other alcohols in only 10 seconds. The methods described provide simple, rapid and low-cost assays to help determine DEG and EG. By repurposing the breathalysers and alcohol test strips, these disposable tests could have helped to prevent many of the hundreds of infant deaths in 2022 and offer low-cost and rapid approaches for LMICs to screen for DEG and EG. Health sciences/Health care/Drug regulation Health sciences/Health care/Public health Biological sciences/Drug discovery/Drug regulation Biological sciences/Drug discovery/Drug safety Biological sciences/Drug discovery/Pharmaceutics Biological sciences/Drug discovery/Toxicology Biological sciences/Biological techniques/Analytical biochemistry/Biochemical assays Biological sciences/Biochemistry/Enzymes substandard medical products diethylene glycol ethylene glycol contamination rapid test falsified syrup Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Substandard and falsified (SF) medical products have been found worldwide, although they are more prevalent in low- and middle-income countries (LMICs) with, often, under-resourced national medicine regulatory authorities (NMRAs) 1 – 3 . Substandard medical products do not meet their quality standards and/or quality specifications and originate from poor manufacturing practices or are degraded due to inappropriate storage on the supply chain 1 , 4 . In contrast, falsified medical products ‘deliberately and fraudulently misrepresent their identity, composition or source’ 1 , 4 . Medicinal syrups often contain excipients such as propylene glycol (PG) and glycerol as a base to sweeten and thicken the medicine. These must be of pharmaceutical grade so that they meet stringent purity standards approved for human consumption. Diethylene glycol (DEG) and ethylene glycol (EG) are industrial solvents often used in antifreeze and vehicle brake fluid and should never be used as an excipient in medicinal syrups since they are metabolised to the toxic acids 2-hydroxyethoxyacetic acid (HEAA), glycolic acid and calcium oxalate which can lead to neurological damage, renal failure, metabolic acidosis, hyperosmolality and death 5 . These toxic metabolites originate from the enzymatic reactions involving alcohol dehydrogenase and aldehyde dehydrogenase 5 , 6 (Fig. 1 ). DEG and EG contamination in medical products may result from accidental or deliberate mislabelling of barrels since both industrial solvents are much less expensive than that of pharmaceutical-grade PG and glycerol. Industrial-grade PG and glycerol are also cheaper and should not be used either 7 . The ingestion of DEG can lead to serious, potentially lethal, health effects due to metabolic acidosis caused by the metabolite HEAA which contributes to renal and neurological toxicity 7 . The metabolites from EG, glycolic acid and oxalic acid, are toxic to patients and they contribute to significant metabolic acidosis 8 – 11 . The first reported case of contaminated syrup intoxication occurred in 1937 in the United States of America with the antibiotic product Elixir Sulfanilamide 12 and multiple outbreaks have occurred in different parts of the world since then 5 . From October 2022 to March 2025, whilst this research was being carried out, over a dozen medical product alerts were issued by the WHO and medicine regulators for more than 40 over-the-counter (OTC) medicines contaminated with DEG and/or EG in The Gambia 13 , Indonesia 14 , Uzbekistan and Cambodia 15 , the Federated States of Marshall Islands and Micronesia 16 , Cameroon 17 , the Republic of Iraq 18 , the Maldives and Pakistan 19 , 20 , Thailand 21 , India 22 , Kenya, Nigeria, Rwanda, South Africa, Tanzania, and Zimbabwe 23 . It was estimated in 2022 that there were at least 300 fatalities in children worldwide 24 . In Gambia, the adulteration of antihistamine, cough and cold syrups led to the deaths of 70 children, while in Indonesia the Indonesian Ministry of Health reported 324 cases of acute kidney injury leading to 199 child deaths due to the fraudulent use of EG/DEG as a solvent in liquid medicines 25 , 26 . In 2022, the Indonesian Food and Drug Authority (Badan Pengawas Obat dan Makanan Republik Indonesia/BPOM RI) identified barrels labelled as PG but were contaminated with 4.69 to 99.09% EG 27 . In another report, BPOM RI found cases of PG contaminated with 33.46% EG and 5.94% DEG. In addition, 1.28 to 443.66 mg/mL (equivalent to 0.13 to 44.37% m/m) of EG and DEG contamination was detected in finished syrup products 28 . The minimum lethal dose of DEG/EG intoxication in humans is uncertain with a wide toxicity range 29 . Syrup manufacturers should test that all barrels of PG and glycerol contain less than 0.1% m/m DEG and EG, the maximum allowable limit in raw ingredients 30 . However, there may be a failure of adhering to this manufacturing quality control procedure 31 . The definitive detection and quantitation of DEG/EG in syrups is by gas chromatography (GC), which is costly, laborious, time-consuming and often not available in many LMICs 34 , 35 . The use of thin-layer chromatography (TLC) provides a lower-cost and portable method to screen DEG/EG in medical products. However, TLC has failed to detect contaminants in cough syrup 36 and, although is easier to perform than GC, requires personnel with adequate laboratory skills 37 , 35 . The WHO has drafted a chapter for inclusion in The International Pharmacopoeia which includes protocols for both TLC and GC with over a dozen steps for each method including preparing both syrups and reference solutions in alcohols with volumetric flasks 37 . Although TLC is a lower-cost approach compared to GC, the method requires several consumables including silica gel TLC plates, a chromatographic tank, a hairdryer, solvents (which are toxic, flammable, corrosive and hazardous), a visualisation solution containing potassium permanganate (which is very toxic, corrosive and harmful) and a laboratory fume hood. Alternative rapid, low-cost, portable and simpler methods to determine DEG/EG contamination are therefore desirable, which do not require harmful chemicals/solvents or as many consumables as TLC and GC, so that they could be easily performed by inspectors even outside of a scientific laboratory. To our knowledge, the only low-cost and portable test which can successfully differentiate glycerol from DEG is by using an mbira and mobile phone 38 . However, the mbira has not been tested for PG and EG and therefore it is unknown if this could have helped to identify the barrels in Indonesia and Pakistan which were recently labelled as PG but had 96–100% EG 19,39 . Furthermore, the limit of detecting DEG in glycerol using an mbira has not been investigated and it is unlikely that it would be able to detect low levels of DEG or EG near the toxic and fatal threshold levels. As a low-cost approach, we initially replicated how EG is metabolised in the body by converting it using alcohol dehydrogenase and aldehyde dehydrogenase to form glycolic acid which could then be detected using glycolate oxidase and a substrate (Fig. 1 ). By analysing enzyme kinetics prior to the addition of glycolate oxidase, we observed that the conversion of EG was far higher than the other alcohols. We hypothesised that EG may also convert quickly with alcohol oxidase and therefore could be used in a low-cost colorimetric rapid test. This led us to test a simple, rapid and low-cost assay to determine EG by repurposing rapid diagnostic tests, commonly used for detecting alcohol in human saliva and breast milk which use alcohol oxidase and a colorimetric substrate. For DEG, we investigated if a polyethylene glycol (PEG) antibody, which recognises the polyether chain of PEG, could preferentially recognise the ether in DEG. Disposable breathalysers contain an oxidiser that changes colour with alcohols and by exploring whether the alcohols oxidise at different rates, we could successfully use them to differentiate both DEG and EG from the other alcohols rapidly and at very low-cost. Unlike GC and TLC, these novel methods do not require any harmful chemicals or solvents and can be used as rapid screening tests in field settings with minimal training. The tests could also provide useful preliminary indicators of DEG and EG prior to further testing using more selective and sensitive techniques. Results Determining EG in raw materials using enzymatic assays EG was successfully differentiated from the other alcohols (glycerol, PG and DEG) using alcohol dehydrogenase, aldehyde dehydrogenase and a plate reader to record the formation of NADH at 340 nm (Fig. 2 a). By additionally using glycolate oxidase, a fluorogenic substrate, and recording the fluorescence with a plate reader, it was possible to more specifically determine EG and differentiate it from other glycols by analysing end-point absorbance (Fig. 2 b) and relative fluorescence units (Fig. 2 c). More importantly, it was possible to successfully identify EG simply by visual observation of the oxidised fluorogenic substrate as pink colour (Fig. 2 d) without the need for a plate reader. Different concentrations of EG spiked into PG were also tested and EG could be confidently determined down to 2% m/m using alcohol dehydrogenase and aldehyde dehydrogenase (Fig. 3 a) and 1% m/m when additionally tested using the glycolic acid assay (Fig. 3 b). Determining EG in medicinal syrups using enzymatic assays EG was determined down to 0.1% and 0.5% m/m in paediatric Calprofen and Dimetapp syrups, respectively using alcohol dehydrogenase and aldehyde dehydrogenase (Fig. 4 a). By additionally using the glycolic acid assay, significant differences in fluorescence were detected down to 0.01% m/m for both paediatric syrups (Fig. 4 b), although only small absorbance differences were observed in the syrups with EG spiked below 0.1% m/m. The assays did not perform well to determine EG in some ethanol containing syrups suitable for teenagers and adults (Beechams and Benylin Chesty), but they did work successfully for ethanol-containing Covonia down to below 0.1% m/m (Supplementary Fig. 1). Alcohol strip tests to determine EG in raw materials Five different brands of alcohol strip tests designed to detect alcohol in saliva and breast milk were tested and all could successfully and rapidly (within 2 minutes) differentiate EG from glycerol and PG (Supplementary Fig. 2, Fig. 5 a). These strips did not show any colour change with undiluted EG and only worked with EG diluted in water with the optimal dilution being 5% v/v EG in water (data not shown). A sixth brand (Wondfo One Step Alcohol Saliva Test, Guangzhou, China) showed similar results (data not shown). The Surescreen alcohol saliva strip could also determine EG down to 0.5% m/m when spiked into PG (Fig. 5 b). A slight colour change was observed for DEG although was not easily noticeable (Fig. 5 a). Alcohol strip tests to determine EG in medicinal syrups In a similar way to raw materials, medicinal syrups were found to work best when diluted 5× in water before being applied to the pad (dilution optimisation data not shown). No change in the colour of the saliva strips was observed for Benylin Infant and Piriteze syrups both of which are alcohol-free paediatric syrups (Fig. 6 and Table 1 ). Dimetapp, known to be free from ethanol, did not show a blue colour change but the pad was slightly coloured due to the syrup colour. Calprofen and Covonia, both known to contain ethanol, showed no blue colour on the pad of the strips although for Covonia the pad was coloured due to the syrup colour. All other syrups known to contain ethanol (Beechams, Benylin Chesty and Paratussin) generated a blue colour on the pad of the strips. Visualisation of the blue colour change was more noticeable in colourless syrup samples (Fig. 6 ). Table 1 Medicinal syrups bought from registered pharmacies in the UK, US and Indonesia. Ethanol is shown in bold to highlight the syrups containing this excipient. Brand Manufacturer Intended use Active Pharmaceutical Ingredients Excipients Beechams All in One GlaxoSmithKline, Brentford, UK Adults and children aged 16 years and over Paracetamol, guaifenesin, phenylephrine hydrochloride Ethanol (19% v/v) , sodium propylene glycol, sorbitol, glycerol Benylin Chesty Coughs non-drowsy McNeil, High Wycombe, UK Adults and children aged 12 years and over Guaifenesin, levomenthol Sucrose, liquid glucose, ethanol , glycerol, sodium citrate, saccharin sodium, citric acid monohydrate, sodium benzoate Benylin Infant's Cough Syrup McNeil, High Wycombe, UK Children aged 3 months to 5 years Glycerol Maltitol liquid, Sodium, Sodium benzoate, Propylene glycol Bodrexin Flu & Batuk PE PT. Tempo Scan Pacific, Tbk., Bekasi, Indonesia Children below 12 years Paracetamol, phenylephrine hydrochloride, guaifenesin, bromhexine hydrochloride, chlorphenamine maleate No information Calprofen McNeil, UK Babies and children aged 3 months to 12 years Ibuprofen Glycerol, xanthan gum, polysorbate 80, flavouring agent (contains propylene glycol and ethanol ), maltitol, saccharin sodium, citric acid monohydrate, sodium methyl hydroxybenzoate, sodium propylhydroxybenzoate Covonia Thornton and Ross, Huddersfield, UK Adults and children aged over 1 year Honey, capsicum tincture, menthol, peppermint oil, anise oil, liquorice extract Glycerol, ethanol (in capsicum tincture), citric acid, glucose, flavouring agent (contains ethanol), propylene glycol Dimetapp Cold and cough Foundation Consumer Brands, LLC., USA Children 6 to under 12 years, children 12 years and older and adults Brompheniramine maleate, dextromethorphan HBr, Phenylephrine HCl. Anhydrous citric acid, glycerin, propylene glycol, sodium benzoate, sodium citrate, sorbitol solution, sucralose OBH Combi Anak Batuk plus flu Combiphar, Bandung, Indonesia Children aged 2 to 12 Paracetamol, succus liquiritiae, ammonium chloride, pseudoephedrine hydrochloride, chlorphenamine maleate No information Paratusin PT. Darya Varia Laboratories, Tbk., Bogor, Indonesia Adults and children aged 2 to 12 Paracetamol, pseudoephedrine hydrochloride, noscapine, chlorphenamine maleate, guaifenesin, succus liquiritiae Ethanol (10% v/v) Piriteze Children’s Hayfever & Allergy Syrup (GSL) Haleon, Weybridge, Surrey, UK Children 2 years and above Cetirizine hydrochloride Sorbitol Termorex Plus Flu dan batuk PT. Konimex, Sukoharjo, Indonesia Children aged 2 to 12 Paracetamol, pseudoephedrine hydrochloride, guaifenesin, chlorphenamine maleate No information Alcohol strip tests to determine EG spiked into infant medicinal syrups Benylin infant and Piriteze syrups were spiked with various concentrations of EG. Alcohol test strips for both saliva and breast milk were successfully used to determine EG (Fig. 7 ). The lowest detectable levels of EG in the spiked syrups, for both alcohol strips were 1% and 2% m/m EG in Benylin infant and Piriteze syrups, respectively. Breathalysers to help determine DEG and EG Disposable breathalysers could help to determine both DEG and EG by observing the rate of colour change of the crystals after the addition of the alcohols. Using a 1% v/v dilution in water, both glycerol and PG samples rapidly changed the colour of the crystal in test tubes from white to dark brown from as early as 10 seconds whereas DEG showed no colour change at all and EG showed a very pale pink colour (Fig. 8 ). The diluted EG sample turned dark brown after two minutes whereas the DEG sample did not change the crystal colour even after two minutes (Fig. 8 ). The breathalysers worked best with the alcohols diluted to 1% v/v in water. When using a higher 5% v/v DEG in water, a faint pink colour change was observed after two minutes, although the reaction was not rapid (Fig. 8 ) and the pink colour intensity was considerably weaker than the dark brown observed for the glycerol and PG samples even when used at only 1% v/v. The breathalysers were tested using 0.1% v/v alcohols in water and they were also able to successfully differentiate DEG from the other alcohols (Supplementary Fig. 3). PEG ELISA for DEG determination The competitive PEG ELISA showed very weak preferential binding of DEG due to the lowest absorbance compared to the other three alcohols although the difference was very minor and not reliable enough to confidently differentiate DEG from the other alcohols (Supplementary Fig. 4). The competitive PEG ELISA showed very weak preferential binding of DEG due to the lowest absorbance compared to the other three alcohols although the difference was very minor and could not reliable enough to confidently differentiate DEG from the other alcohols (Supplementary Fig. 4). Discussion GC and TLC are the two recommended tests for the detection of DEG and EG in raw ingredients and medicinal syrups 37 . In this study, we have explored alternative rapid, low-cost and portable methods to determine DEG and EG in both raw materials and finished products. These tests use enzymatic, chemical and antibody-based assays which unlike GC and TLC do not require any harmful chemicals. The alcohols, glycerol and PG, used in syrups are converted by alcohol dehydrogenase and aldehyde dehydrogenase to acids which are safe. While DEG and EG themselves are not toxic, they are converted by the same two enzymes into very toxic metabolites (Fig. 1 ). We used alcohol dehydrogenase and aldehyde dehydrogenase enzymes in vitro to copy the metabolism which occurs in vivo for individuals who have consumed these alcohols. In the case of EG, it is metabolised to glycolic acid for which an oxidase, glycolate oxidase, exists allowing it to be detected simply with a chromogenic or fluorogenic substrate (Fig. 2 b-d). We are unaware of an oxidase for HEAA or any other metabolites for DEG. Also, an oxidase for HEAA may not help since we observed little conversion of DEG with alcohol dehydrogenase and aldehyde dehydrogenase (Fig. 2 a). Therefore, we have not explored enzymatic assays for DEG. We successfully show that alcohol dehydrogenase and aldehyde dehydrogenase could be used to determine EG and differentiate it from other glycols (Table 2 ) and, when used in combination with a glycolic acid assay, could determine EG as low as 1% m/m in PG (Fig. 3 b) and down to < 0.1% m/m in some medicinal syrups (Fig. 4 b). The glycolate oxidase used in this study was expected to be specific for glycolic acid, an alpha hydroxy acid. Although, the additional use of this enzyme did not work well with teenage/adult syrups containing ethanol, they did work successfully for ethanol-containing Covonia down to below 0.1% m/m (Supplementary Fig. 1). This is most likely due to Covonia having low levels of ethanol (Fig. 6 ) since it is suitable for children over 1 year old in addition to teenagers and adults. Ethanol was not expected to interfere in the assay since it would have converted to acetaldehyde with alcohol dehydrogenase and then oxidised by aldehyde dehydrogenase to acetic acid which is not an alpha hydroxy acid and not an expected substrate for glycolate oxidase. Table 2 Summary of the assays repurposed to help determine ethylene glycol (EG)/diethylene glycol (DEG) Assay Initial intended use Repurposed for DEG/EG LoD (% m/m in matrix) Proposed use Ethanol assay Ethanol EG 2.0% (in PG); 0.1–0.5% (in syrup) Raw materials and ethanol-free paediatric syrups Glycolic acid assay Glycolic acid Glycolic acid from EG 1.0% (in PG); 0.1% (in syrup) Alcohol strip tests (saliva or breast milk) Ethanol EG 0.5% (in PG); 1.0–2.0% (in syrup) Alcohol breath test Ethanol DEG and EG ~ 0.1 and 1.0%, respectively (in water) Raw materials only PEG ELISA PEG DEG Only 5.0% tested (in water) Custom antibody required DEG, diethylene glycol; EG, ethylene glycol; ELISA, enzyme-linked immunosorbent assay; LoD, limit of detection; PG, propylene glycol; PEG, polyethylene glycol These assays could potentially be used in NMRA laboratories, hospitals, or testing laboratories. Although a plate reader is required to measure the absorbance of NADH at 340 nm, we show that EG could also be determined without the need for a plate reader since the reduced fluorescent substrate was seen visually by eye as a pink colour (Fig. 2 d). It may be possible for this enzymatic test to be developed into a low-cost and rapid pad-based strip test using a cocktail of four enzymes (alcohol dehydrogenase, aldehyde dehydrogenase, glycolate oxidase and a peroxidase) and a chromogenic substrate such as tetramethylbenzidine to visualise a colour change. While this should work for raw materials, a potential problem is the testing of finished products since some syrups are coloured (Fig. 6 ) which may hinder the visualisation of the colour change. To overcome this problem, a fluorogenic substrate could be used instead since syrups are free from fluorescent excipients and this approach potentially has the advantage of greater sensitivity. Rapid tests using florescence have successfully been visualised simply using a low-cost UV torch (e.g. Hough COVID-19 Home test, Burleigh West, Australia). Due to the resource confines of this study, we were unable to explore the possibility of developing such a rapid test. Conversion of EG was far higher than the other alcohols when using only alcohol dehydrogenase and aldehyde dehydrogenase (Fig. 2 a). Since EG converted with these enzymes more like ethanol than the other alcohols, we hypothesised that EG may also convert faster than the other alcohols with alcohol oxidase. This led us to evaluate alcohol rapid test strips which use alcohol oxidase, a peroxidase and a chromogenic substrate which turns blue in the presence of alcohol. By repurposing these alcohol test strips, we show that EG could be rapidly and simply determined and easily differentiated from glycerol and PG which yielded negative results (Fig. 5 a). This successfully worked with three different brands of saliva strips and three brands of breast milk strips (five shown in Supplementary Fig. 3) suggesting that this approach may work with any brand of alcohol rapid test strips for testing raw materials. These rapid (2 minutes) and inexpensive (less than $ 1) strips do not require instrumentation and are easy to use with minimal training since they are designed for public use. The strips would be useful for syrup manufacturers for additional testing of incoming raw materials and checking for EG barrels which have been mislabelled, such as the reported incidents in Indonesia 39 and Pakistan 19 , where barrels containing 96–99% 39 and up to 100% EG 19 , respectively, were mislabelled as PG. In Indonesia, hundreds of children died due to EG contamination and simply testing raw ingredients with these rapid alcohol strips could have helped avoid many of these deaths. The rapid alcohol strips were able to determine levels as low as 0.5% m/m EG in PG (Fig. 5 b). While this does not meet the 0.1% m/m WHO regulatory threshold, it may help to prevent deaths and/or toxicity. This is only the case if 0.5% m/m is below the fatal/toxic concentration limits although there is no published evidence on these clinical limits. In a similar way as discussed earlier, it may be possible to improve the sensitivity down to 0.1% m/m if a more sensitive fluorogenic substrate was used but it was not possible to investigate this further. The strips were less sensitive when testing EG spiked into syrups (Fig. 7 ) where detection was down to around 1–2% EG. Again, while this is above the regulatory limit, it would have helped to determine major EG contamination such as in Indonesia and therefore could have prevented toxicity and/or deaths. These test strips were only tested with glycerol and PG as excipients used in syrups since these alcohols are commonly used in relatively large amounts and recent cases of contamination have been with PG 19 , 39 . However, the US FDA do additionally recommend testing maltitol and sorbitol solutions for DEG and EG 30 . We show that the alcohol strips show no sign of blue colour change with the maltitol-containing syrups, Benylin Infant and Calprofen, and the sorbitol-containing syrup, Dimetapp (Table 1 , Fig. 6 ). This suggests that not only can these strips determine EG in PG and glycerol but it could also be used to determine EG in both maltitol and sorbitol solutions received by syrup manufacturers. Furthermore, we show that the alcohol strips could successfully determine the presence of EG down to 1% m/m in a maltitol-containing syrup, Benylin Infant (Fig. 7 ). Disposable breathalysers were repurposed to determine DEG and EG (Fig. 8 ). They contain a proprietary oxidiser which changes from white to pink/brown with alcohols. Some disposable breathalysers contain potassium dichromate which is orange in colour and changes to green in the presence of alcohols. We did not test potassium dichromate since it is extremely toxic and harmful and therefore not feasible to be used in the field to determine DEG or EG. The advantage of the disposable breathalysers used is that it is safe to use since the chemical oxidiser is contained inside the plastic case of the breathalyser and would not come into contact with an inspector using it to determine DEG/EG. DEG was less reactive to the oxidiser in the breathalyser compared to glycerol and PG allowing DEG to be successfully determined. EG did oxidise but at a lower rate allowing EG to be determined. While the breathalysers are low-cost (around $ 1) and the reaction is rapid (10 seconds), since a positive result was observed with glycerol and PG and the test relies on a negative result for DEG (and slower positive for EG), the breathalysers can only be used for testing raw materials testing and cannot be used in finished products. Glycerol and PG oxidise to result in a dark brown colour whereas when the rate of colour change is lower or almost none, a suspicion of probable EG or DEG contamination, respectively, could be raised. This method may be beneficial to prevent cases of DEG and EG contamination in medical products. Substandard Naturcold cough syrup was contaminated with 28.6% DEG in Cameroon 17 and with such a high contamination it is highly possible that the barrels of raw material used contained neat DEG since typically 20–30% glycerol and/or PG are used in medicinal syrups. Furthermore, these breathalysers could have helped to determine EG in Indonesia and Pakistan for the barrels which contained 96–100% EG 14,39 . 14,39 We also explored the potential use of a competitive PEG ELISA in determining DEG (Supplementary Fig. 4). The antibody in the kit recognises the polyether chain of PEG and was tested to see if it could recognise the ether in DEG. There was some weak preferential binding to DEG (lowest absorbance) compared to the other alcohols although the difference was so minor that it could not be used to differentiate DEG from the other alcohols and the breathalysers were considerably better to help determine DEG. The poor performance of the PEG ELISA was not surprising since it uses an antibody specific for PEG instead of DEG. A custom antibody or aptamer against DEG, due to its ether group and differences in branching/topology, may improve binding and DEG detection. While it is possible to develop custom antibodies and aptamers for small molecules, it may be challenging to develop them to be specific for DEG especially when levels of other alcohols such as glycerol and PG could be present as excipients in the syrups at much higher concentrations. But if a custom antibody can recognise DEG, then the antibody may not bind to glycerol and PG as well due to minor differences in branching and such an antibody could potentially be used in a rapid test. While such an antibody or aptamer could be used in a low-cost test, this was not investigated due to the initial expensive development costs. Conclusions Enzymatic assays are useful in determining DEG and EG in both raw materials and finished products. By repurposing alcohol rapid test strips, we show that we can simply, inexpensively (less than $ 1) and rapidly (in under 2 minutes) determine EG. These strips could have been used to save the lives of the hundreds of children who died in Indonesia where the problem was mainly with EG. We showcase the repurposing of assays to determine EG with the LoD ranging from 0.1 to 2% m/m. Breathalysers are also simple, inexpensive (~ $ 1) and can rapidly (in just 10 seconds) identify both DEG and EG mislabelling of raw materials. The novel approaches are considerably easier and safer to carry out than GC and TLC with only a few simple steps and could therefore be performed by inspectors outside of a scientific laboratory and with minimal training. Methods Chemicals and sample preparation Ethylene glycol (≥ 99%, Sigma-Aldrich Cat. No. 102466), diethylene glycol (for synthesis, Sigma-Aldrich Cat. No. 8.03131), propylene glycol (Ph. Eur. grade, Sigma-Aldrich Cat. No. 16033), and glycerol (Ph. Eur. grade, Sigma-Aldrich Cat. No. 49779) were used in the study. Each of these four alcohols was prepared as 5% v/v solutions by weighing 0.5 mL of the alcohols and adding ultrapure distilled water (Milli-Q, Merck Millipore) up to 10.0 mL. EG-spiked PG samples were prepared by weighing EG in an amount to generate a percentage of 10.0, 5.0, 2.0, 1.0, 0.5, 0.1, and 0.05% m/m in PG. Finished medical products Eleven medicinal syrups were purchased OTC from local pharmacies in the UK, US and Indonesia (Table 1 ). EG-spiked samples of representative OTC syrups were prepared by gravimetrically adding EG to the syrup matrix, generating 10.0, 5.0, 2.0, 1.0, 0.5, 0.1, 0.05 and 0.01% m/m solutions. The products include adult syrups containing ethanol (Beechams, Covonia, and Benylin Chesty) and paediatric syrups without ethanol (Dimetapp, Benylin Infant’s, and Piriteze). Converting the alcohols with alcohol dehydrogenase and aldehyde dehydrogenase Alcohol dehydrogenase and aldehyde dehydrogenase in an ethanol assay kit (Megazyme, Cat. No. K-ETOH, Wicklow, Ireland) were used to convert the alcohols. In a clear-flat 96-well microplate (Greiner Bio-One, Stonehouse, UK), 10 µL of the sample was mixed with 200 µL of Milli-Q water, 20 µL of buffer, 20 µL of nicotinamide adenine dinucleotide (NAD + ), and 5 µL of alcohol dehydrogenase. A blank using 10 µL of buffer and a sample diluent control with 10 µL Milli-Q water were also prepared. The samples in the microplate were kept at ambient room temperature (RT, recorded as 20 ± 1°C) for 2 minutes and the first absorbance (A1) was measured at 340 nm on a Clariostar Plus microplate reader (BMG Labtech, Ortenberg, Germany). Without delay, 2 µL of aldehyde dehydrogenase enzyme was added to the reaction mix, incubated for 5 minutes at RT, and measured for the second absorbance at 340 nm (A2; absorbance peak for NADH, the reduced form of NAD + ). Absorbances were measured every minute for 15 minutes. The final absorbance was achieved from the subtraction of A2 and A1. The resulting absorbances were then blank-subtracted. The acids formed in the wells of the plate were then used for the glycolic acid assay. Glycolic acid assay A fluorometric glycolic acid assay kit (Abcam Cat. No. 282915, Cambridge, UK) was used according to the manufacturer’s protocol. The samples used were the samples produced after using alcohol dehydrogenase and aldehyde dehydrogenase and were diluted 10× in assay buffer. These diluted samples (50 µL) were added into wells of a 96-well black plate for fluorescence with a flat bottom (Corning, UK). For each sample, a reaction mix was prepared consisting of 44 µL of assay buffer, 2 µL of detection reagent, 2 µL of enzyme mix containing glycolate oxidase and 2 µL of fluorogenic probe. This reaction mix (50 µL) was then added to each diluted sample in the 96-well black plate and the fluorescence was recorded in 30-second intervals for 90 minutes at RT with the excitation/emission set to 535/587 nm. The fluorescence for the blank was subtracted from the fluorescence measurements for each sample. Alcohol strip tests Glycerol, PG, DEG and EG diluted in ultrapure water to 5% v/v were tested by adding 20 µL of the diluted alcohol onto the pad of three brands of alcohol rapid test strips for saliva (Surescreen Diagnostics, Eagle Park, UK; One Step distributed by Home Health UK, Bushey, UK; AllTest distributed by UK Drug Testing, Aylsham, UK) and two brands of rapid alcohol test strips for breast milk (Frida, Miami, Florida, USA; Easy@Home, Burr Ridge, Illinois, USA). Spiked samples prepared in PG and cough syrup samples were diluted in water before being added to the pad and the best dilution was found to be a 5× dilution in Milli-Q water. After 2 minutes, the intensity of the resultant blue colour was analysed visually by two individuals. Alcohol breathalysers Disposable breathalysers (One Step alcohol breath test manufactured by Test&Drive, Ploty, Poland and distributed by Home Health UK, Bushey, UK) were repurposed to test glycerol, PG, DEG, and EG diluted to 1% and 0.1% v/v in Milli-Q water. The protective foil of the breathalyser was pierced by pressing firmly inwards on the protective caps on both ends according to the manufacturer’s instructions. The diluted alcohols (75 µL) were pipetted into the blowing end of the tube and flicked twice to ensure that the liquid reaches the white crystals. The breathalysers were left at RT for 2 minutes and any colour changes observed visually with the crystals were recorded at 10 seconds and 2 minutes by two individuals. Polyethylene glycol (PEG) ELISA A competitive PEG ELISA kit (Abcam Cat. No. ab215546, Cambridge, UK) was used according to the manufacturer’s protocol. A mixture of 50 µL sample and 50 µL of 1× PEG-HRP was prepared and 50 µL of the mixture was then added to the anti-PEG antibody-coated well of a strip in the kit. The reaction was incubated for 45 minutes at RT on a plate shaker set to 400 rpm. Following the incubation, the sample mix was aspirated and the well was washed three times with 1× Wash Buffer. A volume of 100 µL of TMB substrate was then added to the well and incubated for 15 minutes at RT in the dark with shaking, prior to the addition of 100 µL of stop solution and absorbance was read at 450 nm. Declarations Conflict of interest P.M declares consultancy for Agilent Technologies, of which R.S is an employee. All other authors have no competing interests. The authors alone are responsible for the views expressed in this and they do not necessarily represent the views, decisions or policies of the institutions with which they are affiliated. Competing Interests P.M declares consultancy for Agilent Technologies, of which R.S is an employee. All other authors have no competing interests. The authors alone are responsible for the views expressed in this and they do not necessarily represent the views, decisions or policies of the institutions with which they are affiliated. Author Contribution B.G, N.Z, P.N.N, C.C, J.M and P.M designed the study. B.Y.A, I.L and B.G performed the experiments and/or data analysis. B.Y.A prepared the original draft. B.Y.A, I.L, J.W-T, T.B, J.G, G.G, M.D, S.B, R.S, P.M, J.M, C.C, P.N.N, N.Z and B.G contributed to the writing, reviewing, and editing of the manuscript. P.N.N, N.Z, J.M and P.M were involved in funding acquisition. B.G, N.Z, P.N.N, C.C, J.M and P.M were involved in project management. All authors read and approved the final manuscript. Acknowledgement We are very grateful to the Bill & Melinda Gates Foundation for the funding of this study. Furthermore, we would like to thank Cathrin Hauk, Kerlijn Van Assche and Raymond A. Dwek of Oxford University, Tony Cass and Danny O'Hare of Imperial College London and Tim James of Oxford University Hospitals NHS Foundation Trust for their generous support of this project and expert advice. M.D, C.C and P.N.N are supported by the Wellcome Trust (222506/Z/21/Z). B.Y.A is funded by the Indonesian Education Scholarship (Beasiswa Pendidikan Indonesia) from the Ministry of Higher Education, Science and Technology of the Republic of Indonesia (Kemendiktisaintek) within a funding scheme from Indonesia Endowment Fund for Education (LPDP). B.G was partly supported by the Oxford Glycobiology Endowment. This research was funded in part, by the Wellcome Trust [220211/Z/20/Z, 222506/Z/21/Z]. For the purpose of Open Access, the author has applied for a CC BY public copyright license to any Author Accepted Manuscript versions arising from this submission. The funder played no role in the study design, data collection, analysis and interpretation of data, or the writing of this manuscript. Data Availability All data generated or analysed during this study are included in this published article and its supplementary information files. References Pyzik, O. Z. & Abubakar, I. Fighting the fakes: tackling substandard and falsified medicines. Nat. Rev. Dis. Primers . 8 , 1–2 (2022). Newton, P. N. & Bond, K. C. Oxford Statement signatories. Global access to quality-assured medical products: the Oxford Statement and call to action. Lancet Glob Health . 7 , e1609–e1611 (2019). Medicine Quality Research Group, University of Oxford. Oxford Statement following the MQPH 2018 Conference. https://www.tropicalmedicine.ox.ac.uk/events/medicine-quality/mqph2018/oxford-statement World Health Organization. WHO Global Surveillance and Monitoring System for Substandard and Falsified Medical Products (World Health Organization, 2017). Kraut, J. A. & Kurtz, I. 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(2024). https://www.who.int/news/item/15-04-2024-medical-product-alert-n-1-2024--falsified-(contaminated)-usp-ep-propylene-glycol World Health Organization. Medical Product Alert N°8/2023: Substandard (contaminated) syrup and suspension medicines. (2023). https://www.who.int/news/item/07-12-2023-medical-product-alert-n-8-2023--substandard-(contaminated)-syrup-and-suspension-medicines Bangkok Post. Toxin found in 15 syrup products for children. (2024). https://www.bangkokpost.com/thailand/general/2807819/toxin-found-in-15-syrup-products-for-children Singh, R. Over 100 Indian cough syrup samples fail quality tests, linked to deaths. (2024). Wasswa, H. African countries recall batch of Johnson and Johnson cough syrup because of toxicity concerns. BMJ 385 , q923 (2024). World Health Organization. Diethylene Glycol (DEG) and Ethylene Glycol (EG) contamination - analytical methods developed for testing paediatric medicines. 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A Selective Gas Chromatography–Tandem Mass Spectrometry Method for Quantitation of Ethylene and Diethylene Glycol in Paediatric Syrups. Heliyon 10 , e27559 (2024). Singh, J. et al. Diethylene glycol poisoning in Gurgaon, India, 1998. Bull. World Health Organ. 79 , 88–95 (2001). World Health Organization. Tests for diethylene glycol and ethylene glycol in liquid preparations for oral use. Chapter for inclusion in The International Pharmacopoeia. (2023). Bhakta, H. C., Choday, V. K. & Grover, W. H. Musical Instruments As Sensors. ACS Omega . 3 , 11026–11032 (2018). Widianto, S. Deadly Indonesian cough syrup was almost pure toxin, court papers show. (2023). https://www.reuters.com/business/healthcare-pharmaceuticals/deadly-indonesian-cough-syrup-was-almost-pure-toxin-court-papers-show-2023-10-13/ Jähnke, R. W. O. & Dwornik, K. A. Concise Quality Control Guide on Essential Drugs and other Medicines: Special edition 2024 for the testing of toxic impurities in liquids for oral use. (2024). Additional Declarations Competing interest reported. P.M declares consultancy for Agilent Technologies, of which R.S is an employee. All other authors have no competing interests. The authors alone are responsible for the views expressed in this and they do not necessarily represent the views, decisions or policies of the institutions with which they are affiliated. Supplementary Files ArmanBYetal.DEGEGenzymaticassaySupplementaryInformation.docx Cite Share Download PDF Status: Published Journal Publication published 03 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 04 Jul, 2025 Reviews received at journal 03 Jul, 2025 Reviewers agreed at journal 21 Jun, 2025 Reviews received at journal 10 Jun, 2025 Reviewers agreed at journal 01 Jun, 2025 Reviewers invited by journal 30 May, 2025 Editor assigned by journal 30 May, 2025 Editor invited by journal 30 May, 2025 Submission checks completed at journal 30 May, 2025 First submitted to journal 16 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6683642","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":470237821,"identity":"b600685e-a29a-47a5-b1ea-318206e7dcb4","order_by":0,"name":"Benediktus Yohan Arman","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Benediktus","middleName":"Yohan","lastName":"Arman","suffix":""},{"id":470237822,"identity":"6face255-0cb7-4339-8e74-81f13dbc029c","order_by":1,"name":"Isabelle Legge","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Isabelle","middleName":"","lastName":"Legge","suffix":""},{"id":470237823,"identity":"ec310e34-98e0-4a16-a027-59dd9f909d32","order_by":2,"name":"John Walsby-Tickle","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"Walsby-Tickle","suffix":""},{"id":470237824,"identity":"4ad64381-8cd3-4f02-bcb6-f5110079b5c6","order_by":3,"name":"Tehmina Bharucha","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Tehmina","middleName":"","lastName":"Bharucha","suffix":""},{"id":470237825,"identity":"154364aa-1b48-4955-85d4-c4cd8989daaa","order_by":4,"name":"Julia Gabel","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Gabel","suffix":""},{"id":470237826,"identity":"b18d0cb1-7050-4a17-a758-595eb78890a1","order_by":5,"name":"Gesa Gnegel","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Gesa","middleName":"","lastName":"Gnegel","suffix":""},{"id":470237827,"identity":"054750de-b1a0-4590-b85a-8a1bdda08101","order_by":6,"name":"Michael Deats","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Deats","suffix":""},{"id":470237828,"identity":"57a33e81-becb-48b7-bb9f-5bfba7a3f0fa","order_by":7,"name":"Sneha Banerjee","email":"","orcid":"","institution":"STFC Rutherford Appleton Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Sneha","middleName":"","lastName":"Banerjee","suffix":""},{"id":470237829,"identity":"bbee3ef6-f303-44ee-ba06-818415101c19","order_by":8,"name":"Robert Stokes","email":"","orcid":"","institution":"Agilent Technologies LDA UK","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Stokes","suffix":""},{"id":470237830,"identity":"daec28e7-ac23-4eb7-aac3-59d1a214267a","order_by":9,"name":"Pavel Matousek","email":"","orcid":"","institution":"Science and Technology Facilities Council, Rutherford Appleton Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Pavel","middleName":"","lastName":"Matousek","suffix":""},{"id":470237831,"identity":"30bf9d52-9962-40e8-9471-fdc68fb9a7af","order_by":10,"name":"James McCullagh","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"","lastName":"McCullagh","suffix":""},{"id":470237832,"identity":"84d713be-749d-41cd-9d16-332aa0d346fc","order_by":11,"name":"Céline Caillet","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Céline","middleName":"","lastName":"Caillet","suffix":""},{"id":470237833,"identity":"cfc028d4-9e91-48bb-9d36-d6b1e27a1872","order_by":12,"name":"Paul N. 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NADH was measured at 340 nm over 15 minutes. The dotted line shows the blank absorbance limit. \u003cstrong\u003eb\u003c/strong\u003e By additionally using glycolate oxidase to detect glycolic acid, EG could be determined more specifically. End-point absorbances were measured at 571 nm. \u003cstrong\u003ec\u003c/strong\u003eThe detection of glycolic acid in alcohol samples, measured as the relative fluorescence unit (RFU). \u003cstrong\u003ed\u003c/strong\u003e Successful identification of EG simply by visual observation of the oxidised fluorogenic substrate as pink colour without the need for a plate reader. No pink colour was observed for the other alcohols. Error bars show the standard deviations of two replicates.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6683642/v1/0392bcc478dd003140ea3faa.png"},{"id":84666068,"identity":"abb49468-614d-4709-9361-b08e871c4edb","added_by":"auto","created_at":"2025-06-16 05:42:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":587934,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetermining EG in PG using enzymatic assays.\u003c/strong\u003e PG was spiked with different percentages of EG. \u003cstrong\u003ea\u003c/strong\u003e End-point 340 nm absorbance readings of NADH after using alcohol dehydrogenase and aldehyde dehydrogenase. \u003cstrong\u003eb\u003c/strong\u003e Relative fluorescence unit (RFU) readings after additionally using glycolate oxidase in the glycolic acid assay. Milli-Q water was used as a no-matrix control. The dotted line shows the limit of neat PG. Error bars show the standard deviations of two replicates.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6683642/v1/0e6a7c5dde735077a36bcf39.png"},{"id":84666069,"identity":"fa3ff9c4-d6b9-4f5e-859f-d22c347a0899","added_by":"auto","created_at":"2025-06-16 05:42:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":215857,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnzymatic assay results for two paediatric syrups (Calprofen and Dimetapp) spiked with different percentages of EG.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e End-point 340 nm absorbance readings of NADH after using alcohol dehydrogenase and aldehyde dehydrogenase. \u003cstrong\u003eb\u003c/strong\u003e End-point 571 nm absorbance readings after additionally using glycolate oxidase in the glycolic acid assay. Error bars show the standard deviations of two replicates.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6683642/v1/6a030ec0385d5164666f6191.png"},{"id":84666087,"identity":"1749daac-f4f5-4ae7-b628-6823743ba905","added_by":"auto","created_at":"2025-06-16 05:42:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":101044,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetermining EG using Surescreen alcohol saliva strips.\u003c/strong\u003e Photos show zoomed-in images of the pad on the strips. \u003cstrong\u003ea\u003c/strong\u003eSuccessful conversion of EG with the alcohol oxidase in the pad of the strip (blue colour) and differentiation from glycerol, PG and DEG using 5% v/v of the alcohols in water. \u003cstrong\u003eb\u003c/strong\u003e Different percentages of EG spiked into neat PG. Limit of detection ~0.5% EG m/m.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6683642/v1/9247727ced85d6d73151d4b1.png"},{"id":84666073,"identity":"1252ee95-50ac-4f8a-bfe5-87665c595bc9","added_by":"auto","created_at":"2025-06-16 05:42:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":155561,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInvestigating if ethanol and the colour of syrups interfere with the alcohol saliva strip test results.\u003c/strong\u003e \u003cem\u003eUpper \u003c/em\u003erow\u003cem\u003e:\u003c/em\u003e Eleven neat medicinal syrups were applied to the pad of the strips and compared to a 5% v/v PG solution in water. Photos show the zoomed images of the pads on the strips. A blue colour on the pad was observed for Beechams, Benylin Chesty, Paratusin (all ethanol-containing) whereas alcohol-free paediatric syrups (Benylin Infant and Piriteze) and alcohol-containing paediatric syrups (Calprofen) did not show any colour change. There was no blue development colour on the pads for some syrups although they were coloured due to the colour of the syrups (Covonia, OBH Combi and Dimetapp). \u003cem\u003eLower \u003c/em\u003erow\u003cem\u003e:\u003c/em\u003e The syrups were pipetted into transparent plastic tubes and the colours were noted. The photos show zoomed-in images of the bottom of the tubes. Coloured syrups, such as Covonia and Dimetapp, contribute to the colour observed on the pads of the strips and therefore could interfere with the visualisation of any blue colour if EG were to be present in similarly coloured syrups.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6683642/v1/c6493c90288515121e42b1ed.png"},{"id":84666078,"identity":"c2ace00c-725d-434d-8bf1-cd824cd46210","added_by":"auto","created_at":"2025-06-16 05:42:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":155581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetermining EG spiked into medicinal syrups.\u003c/strong\u003e Zoomed-in images of the pads on the strips are shown. The Surescreen alcohol saliva and Frida mom breast milk strips could determine the presence of EG down to 1% and 2% m/m for Benylin infant and Piriteze, respectively. The red boxes show where the blue colour on the pad could be seen when visually observed.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6683642/v1/095d8483241e789a99631d0b.png"},{"id":84666076,"identity":"2a7b2651-aa4d-4c4a-a0f0-667a2190281b","added_by":"auto","created_at":"2025-06-16 05:42:01","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":102864,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDisposable breathalysers can successfully indicate if glycerol or PG raw materials have been substituted with DEG or EG.\u003c/strong\u003eZoomed-in images of the crystals seen through the viewing window of the breathalyser are shown. All alcohols were diluted to 1% v/v except for DEG which was additionally tested at 5% v/v. In only 10 seconds, glycerol and PG used in raw materials changed the crystals in the breathalyser from white to dark brown whereas DEG showed no change in colour and EG showed a very faint pink colour. There was also no change in the colour of the crystals after 10 seconds when DEG was tested at the 5× higher concentration. At 2 minutes, there was still no colour change for DEG. The crystals changed to dark brown for EG suggesting that the white crystals do oxidise EG but at a slower rate compared to glycerol and PG, both of which remained dark brown at 2 minutes. For DEG, there was a slight change in the colour of the crystals to pink after 2 minutes when tested at the 5× higher concentration although was still a lot less intense than glycerol and PG when tested after 10 seconds.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6683642/v1/a406f97de6e7adb1e3086c6a.png"},{"id":97724581,"identity":"64d70fca-3331-4514-83ea-a7a70223eb4a","added_by":"auto","created_at":"2025-12-08 16:12:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3211407,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6683642/v1/c3157f1c-6b66-433b-bc66-1760837488bf.pdf"},{"id":84666637,"identity":"4bd477f7-066b-4ed1-aab0-116aac665075","added_by":"auto","created_at":"2025-06-16 05:50:01","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1605445,"visible":true,"origin":"","legend":"","description":"","filename":"ArmanBYetal.DEGEGenzymaticassaySupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6683642/v1/a7a888e97b250280551db46e.docx"}],"financialInterests":"Competing interest reported. P.M declares consultancy for Agilent Technologies, of which R.S is an employee. All other authors have no competing interests. The authors alone are responsible for the views expressed in this and they do not necessarily represent the views, decisions or policies of the institutions with which they are affiliated.","formattedTitle":"Simple and rapid low-cost assays to investigate ethylene glycol and diethylene glycol contamination in raw materials and medicinal syrups","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSubstandard and falsified (SF) medical products have been found worldwide, although they are more prevalent in low- and middle-income countries (LMICs) with, often, under-resourced national medicine regulatory authorities (NMRAs)\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Substandard medical products do not meet their quality standards and/or quality specifications and originate from poor manufacturing practices or are degraded due to inappropriate storage on the supply chain\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In contrast, falsified medical products \u0026lsquo;deliberately and fraudulently misrepresent their identity, composition or source\u0026rsquo;\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMedicinal syrups often contain excipients such as propylene glycol (PG) and glycerol as a base to sweeten and thicken the medicine. These must be of pharmaceutical grade so that they meet stringent purity standards approved for human consumption. Diethylene glycol (DEG) and ethylene glycol (EG) are industrial solvents often used in antifreeze and vehicle brake fluid and should never be used as an excipient in medicinal syrups since they are metabolised to the toxic acids 2-hydroxyethoxyacetic acid (HEAA), glycolic acid and calcium oxalate which can lead to neurological damage, renal failure, metabolic acidosis, hyperosmolality and death\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These toxic metabolites originate from the enzymatic reactions involving alcohol dehydrogenase and aldehyde dehydrogenase\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDEG and EG contamination in medical products may result from accidental or deliberate mislabelling of barrels since both industrial solvents are much less expensive than that of pharmaceutical-grade PG and glycerol. Industrial-grade PG and glycerol are also cheaper and should not be used either\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The ingestion of DEG can lead to serious, potentially lethal, health effects due to metabolic acidosis caused by the metabolite HEAA which contributes to renal and neurological toxicity\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The metabolites from EG, glycolic acid and oxalic acid, are toxic to patients and they contribute to significant metabolic acidosis\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The first reported case of contaminated syrup intoxication occurred in 1937 in the United States of America with the antibiotic product Elixir Sulfanilamide\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and multiple outbreaks have occurred in different parts of the world since then\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. From October 2022 to March 2025, whilst this research was being carried out, over a dozen medical product alerts were issued by the WHO and medicine regulators for more than 40 over-the-counter (OTC) medicines contaminated with DEG and/or EG in The Gambia\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, Indonesia\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, Uzbekistan and Cambodia\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, the Federated States of Marshall Islands and Micronesia\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, Cameroon\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, the Republic of Iraq\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, the Maldives and Pakistan\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, Thailand\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, India\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, Kenya, Nigeria, Rwanda, South Africa, Tanzania, and Zimbabwe\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. It was estimated in 2022 that there were at least 300 fatalities in children worldwide\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In Gambia, the adulteration of antihistamine, cough and cold syrups led to the deaths of 70 children, while in Indonesia the Indonesian Ministry of Health reported 324 cases of acute kidney injury leading to 199 child deaths due to the fraudulent use of EG/DEG as a solvent in liquid medicines\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn 2022, the Indonesian Food and Drug Authority (Badan Pengawas Obat dan Makanan Republik Indonesia/BPOM RI) identified barrels labelled as PG but were contaminated with 4.69 to 99.09% EG\u003csup\u003e27\u003c/sup\u003e. In another report, BPOM RI found cases of PG contaminated with 33.46% EG and 5.94% DEG. In addition, 1.28 to 443.66 mg/mL (equivalent to 0.13 to 44.37% m/m) of EG and DEG contamination was detected in finished syrup products\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The minimum lethal dose of DEG/EG intoxication in humans is uncertain with a wide toxicity range\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Syrup manufacturers should test that all barrels of PG and glycerol contain less than 0.1% m/m DEG and EG, the maximum allowable limit in raw ingredients\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. However, there may be a failure of adhering to this manufacturing quality control procedure\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe definitive detection and quantitation of DEG/EG in syrups is by gas chromatography (GC), which is costly, laborious, time-consuming and often not available in many LMICs\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. The use of thin-layer chromatography (TLC) provides a lower-cost and portable method to screen DEG/EG in medical products. However, TLC has failed to detect contaminants in cough syrup\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e and, although is easier to perform than GC, requires personnel with adequate laboratory skills\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. The WHO has drafted a chapter for inclusion in The International Pharmacopoeia which includes protocols for both TLC and GC with over a dozen steps for each method including preparing both syrups and reference solutions in alcohols with volumetric flasks\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Although TLC is a lower-cost approach compared to GC, the method requires several consumables including silica gel TLC plates, a chromatographic tank, a hairdryer, solvents (which are toxic, flammable, corrosive and hazardous), a visualisation solution containing potassium permanganate (which is very toxic, corrosive and harmful) and a laboratory fume hood. Alternative rapid, low-cost, portable and simpler methods to determine DEG/EG contamination are therefore desirable, which do not require harmful chemicals/solvents or as many consumables as TLC and GC, so that they could be easily performed by inspectors even outside of a scientific laboratory. To our knowledge, the only low-cost and portable test which can successfully differentiate glycerol from DEG is by using an mbira and mobile phone\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. However, the mbira has not been tested for PG and EG and therefore it is unknown if this could have helped to identify the barrels in Indonesia and Pakistan which were recently labelled as PG but had 96\u0026ndash;100% EG\u003csup\u003e19,39\u003c/sup\u003e. Furthermore, the limit of detecting DEG in glycerol using an mbira has not been investigated and it is unlikely that it would be able to detect low levels of DEG or EG near the toxic and fatal threshold levels.\u003c/p\u003e \u003cp\u003eAs a low-cost approach, we initially replicated how EG is metabolised in the body by converting it using alcohol dehydrogenase and aldehyde dehydrogenase to form glycolic acid which could then be detected using glycolate oxidase and a substrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). By analysing enzyme kinetics prior to the addition of glycolate oxidase, we observed that the conversion of EG was far higher than the other alcohols. We hypothesised that EG may also convert quickly with alcohol oxidase and therefore could be used in a low-cost colorimetric rapid test. This led us to test a simple, rapid and low-cost assay to determine EG by repurposing rapid diagnostic tests, commonly used for detecting alcohol in human saliva and breast milk which use alcohol oxidase and a colorimetric substrate. For DEG, we investigated if a polyethylene glycol (PEG) antibody, which recognises the polyether chain of PEG, could preferentially recognise the ether in DEG. Disposable breathalysers contain an oxidiser that changes colour with alcohols and by exploring whether the alcohols oxidise at different rates, we could successfully use them to differentiate both DEG and EG from the other alcohols rapidly and at very low-cost.\u003c/p\u003e \u003cp\u003eUnlike GC and TLC, these novel methods do not require any harmful chemicals or solvents and can be used as rapid screening tests in field settings with minimal training. The tests could also provide useful preliminary indicators of DEG and EG prior to further testing using more selective and sensitive techniques.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDetermining EG in raw materials using enzymatic assays\u003c/h2\u003e \u003cp\u003eEG was successfully differentiated from the other alcohols (glycerol, PG and DEG) using alcohol dehydrogenase, aldehyde dehydrogenase and a plate reader to record the formation of NADH at 340 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). By additionally using glycolate oxidase, a fluorogenic substrate, and recording the fluorescence with a plate reader, it was possible to more specifically determine EG and differentiate it from other glycols by analysing end-point absorbance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) and relative fluorescence units (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). More importantly, it was possible to successfully identify EG simply by visual observation of the oxidised fluorogenic substrate as pink colour (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed) without the need for a plate reader. Different concentrations of EG spiked into PG were also tested and EG could be confidently determined down to 2% m/m using alcohol dehydrogenase and aldehyde dehydrogenase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) and 1% m/m when additionally tested using the glycolic acid assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetermining EG in medicinal syrups using enzymatic assays\u003c/h3\u003e\n\u003cp\u003eEG was determined down to 0.1% and 0.5% m/m in paediatric Calprofen and Dimetapp syrups, respectively using alcohol dehydrogenase and aldehyde dehydrogenase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). By additionally using the glycolic acid assay, significant differences in fluorescence were detected down to 0.01% m/m for both paediatric syrups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), although only small absorbance differences were observed in the syrups with EG spiked below 0.1% m/m. The assays did not perform well to determine EG in some ethanol containing syrups suitable for teenagers and adults (Beechams and Benylin Chesty), but they did work successfully for ethanol-containing Covonia down to below 0.1% m/m (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAlcohol strip tests to determine EG in raw materials\u003c/h3\u003e\n\u003cp\u003eFive different brands of alcohol strip tests designed to detect alcohol in saliva and breast milk were tested and all could successfully and rapidly (within 2 minutes) differentiate EG from glycerol and PG (Supplementary Fig.\u0026nbsp;2, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). These strips did not show any colour change with undiluted EG and only worked with EG diluted in water with the optimal dilution being 5% v/v EG in water (data not shown). A sixth brand (Wondfo One Step Alcohol Saliva Test, Guangzhou, China) showed similar results (data not shown). The Surescreen alcohol saliva strip could also determine EG down to 0.5% m/m when spiked into PG (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). A slight colour change was observed for DEG although was not easily noticeable (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAlcohol strip tests to determine EG in medicinal syrups\u003c/h3\u003e\n\u003cp\u003eIn a similar way to raw materials, medicinal syrups were found to work best when diluted 5\u0026times; in water before being applied to the pad (dilution optimisation data not shown). No change in the colour of the saliva strips was observed for Benylin Infant and Piriteze syrups both of which are alcohol-free paediatric syrups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Dimetapp, known to be free from ethanol, did not show a blue colour change but the pad was slightly coloured due to the syrup colour. Calprofen and Covonia, both known to contain ethanol, showed no blue colour on the pad of the strips although for Covonia the pad was coloured due to the syrup colour. All other syrups known to contain ethanol (Beechams, Benylin Chesty and Paratussin) generated a blue colour on the pad of the strips. Visualisation of the blue colour change was more noticeable in colourless syrup samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMedicinal syrups bought from registered pharmacies in the UK, US and Indonesia. Ethanol is shown in bold to highlight the syrups containing this excipient.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBrand\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eManufacturer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIntended use\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eActive Pharmaceutical Ingredients\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExcipients\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBeechams All in One\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlaxoSmithKline, Brentford, UK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAdults and children aged 16 years and over\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eParacetamol, guaifenesin, phenylephrine hydrochloride\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eEthanol (19% v/v)\u003c/b\u003e, sodium propylene glycol, sorbitol, glycerol\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBenylin Chesty Coughs non-drowsy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMcNeil, High Wycombe, UK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAdults and children aged 12 years and over\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGuaifenesin, levomenthol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSucrose, liquid glucose, \u003cb\u003eethanol\u003c/b\u003e, glycerol, sodium citrate, saccharin sodium, citric acid monohydrate, sodium benzoate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBenylin Infant's Cough Syrup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMcNeil, High Wycombe, UK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChildren aged 3 months to 5 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlycerol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMaltitol liquid, Sodium, Sodium benzoate, Propylene glycol\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBodrexin Flu \u0026amp; Batuk PE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePT. Tempo Scan Pacific, Tbk., Bekasi, Indonesia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChildren below 12 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eParacetamol, phenylephrine hydrochloride, guaifenesin, bromhexine hydrochloride, chlorphenamine maleate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo information\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCalprofen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMcNeil, UK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBabies and children aged 3 months to 12 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIbuprofen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGlycerol, xanthan gum, polysorbate 80, flavouring agent (contains propylene glycol and \u003cb\u003eethanol\u003c/b\u003e), maltitol, saccharin sodium, citric acid monohydrate, sodium methyl hydroxybenzoate, sodium propylhydroxybenzoate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCovonia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThornton and Ross, Huddersfield, UK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAdults and children aged over 1 year\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHoney, capsicum tincture, menthol, peppermint oil, anise oil, liquorice extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGlycerol, \u003cb\u003eethanol\u003c/b\u003e (in capsicum tincture), citric acid, glucose, flavouring agent (contains ethanol), propylene glycol\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDimetapp Cold and cough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFoundation Consumer Brands, LLC., USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChildren 6 to under 12 years, children 12 years and older and adults\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBrompheniramine maleate, dextromethorphan HBr, Phenylephrine HCl.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAnhydrous citric acid, glycerin, propylene glycol, sodium benzoate, sodium citrate, sorbitol solution, sucralose\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOBH Combi Anak Batuk plus flu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCombiphar, Bandung, Indonesia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChildren aged 2 to 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eParacetamol, succus liquiritiae, ammonium chloride, pseudoephedrine hydrochloride, chlorphenamine maleate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo information\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParatusin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePT. Darya Varia Laboratories, Tbk., Bogor, Indonesia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAdults and children aged 2 to 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eParacetamol, pseudoephedrine hydrochloride, noscapine, chlorphenamine maleate, guaifenesin, succus liquiritiae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eEthanol (10% v/v)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePiriteze Children\u0026rsquo;s Hayfever \u0026amp; Allergy Syrup (GSL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHaleon, Weybridge, Surrey, UK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChildren 2 years and above\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCetirizine hydrochloride\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSorbitol\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTermorex Plus Flu dan batuk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePT. Konimex, Sukoharjo, Indonesia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChildren aged 2 to 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eParacetamol, pseudoephedrine hydrochloride, guaifenesin, chlorphenamine maleate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo information\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eAlcohol strip tests to determine EG spiked into infant medicinal syrups\u003c/h3\u003e\n\u003cp\u003eBenylin infant and Piriteze syrups were spiked with various concentrations of EG. Alcohol test strips for both saliva and breast milk were successfully used to determine EG (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The lowest detectable levels of EG in the spiked syrups, for both alcohol strips were 1% and 2% m/m EG in Benylin infant and Piriteze syrups, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBreathalysers to help determine DEG and EG\u003c/h2\u003e \u003cp\u003eDisposable breathalysers could help to determine both DEG and EG by observing the rate of colour change of the crystals after the addition of the alcohols. Using a 1% v/v dilution in water, both glycerol and PG samples rapidly changed the colour of the crystal in test tubes from white to dark brown from as early as 10 seconds whereas DEG showed no colour change at all and EG showed a very pale pink colour (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The diluted EG sample turned dark brown after two minutes whereas the DEG sample did not change the crystal colour even after two minutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The breathalysers worked best with the alcohols diluted to 1% v/v in water. When using a higher 5% v/v DEG in water, a faint pink colour change was observed after two minutes, although the reaction was not rapid (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) and the pink colour intensity was considerably weaker than the dark brown observed for the glycerol and PG samples even when used at only 1% v/v. The breathalysers were tested using 0.1% v/v alcohols in water and they were also able to successfully differentiate DEG from the other alcohols (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePEG ELISA for DEG determination\u003c/h3\u003e\n\u003cp\u003eThe competitive PEG ELISA showed very weak preferential binding of DEG due to the lowest absorbance compared to the other three alcohols although the difference was very minor and not reliable enough to confidently differentiate DEG from the other alcohols (Supplementary Fig.\u0026nbsp;4). The competitive PEG ELISA showed very weak preferential binding of DEG due to the lowest absorbance compared to the other three alcohols although the difference was very minor and could not reliable enough to confidently differentiate DEG from the other alcohols (Supplementary Fig.\u0026nbsp;4).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eGC and TLC are the two recommended tests for the detection of DEG and EG in raw ingredients and medicinal syrups\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. In this study, we have explored alternative rapid, low-cost and portable methods to determine DEG and EG in both raw materials and finished products. These tests use enzymatic, chemical and antibody-based assays which unlike GC and TLC do not require any harmful chemicals.\u003c/p\u003e \u003cp\u003eThe alcohols, glycerol and PG, used in syrups are converted by alcohol dehydrogenase and aldehyde dehydrogenase to acids which are safe. While DEG and EG themselves are not toxic, they are converted by the same two enzymes into very toxic metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We used alcohol dehydrogenase and aldehyde dehydrogenase enzymes \u003cem\u003ein vitro\u003c/em\u003e to copy the metabolism which occurs \u003cem\u003ein vivo\u003c/em\u003e for individuals who have consumed these alcohols. In the case of EG, it is metabolised to glycolic acid for which an oxidase, glycolate oxidase, exists allowing it to be detected simply with a chromogenic or fluorogenic substrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d). We are unaware of an oxidase for HEAA or any other metabolites for DEG. Also, an oxidase for HEAA may not help since we observed little conversion of DEG with alcohol dehydrogenase and aldehyde dehydrogenase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Therefore, we have not explored enzymatic assays for DEG. We successfully show that alcohol dehydrogenase and aldehyde dehydrogenase could be used to determine EG and differentiate it from other glycols (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and, when used in combination with a glycolic acid assay, could determine EG as low as 1% m/m in PG (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) and down to \u0026lt;\u0026thinsp;0.1% m/m in some medicinal syrups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The glycolate oxidase used in this study was expected to be specific for glycolic acid, an alpha hydroxy acid. Although, the additional use of this enzyme did not work well with teenage/adult syrups containing ethanol, they did work successfully for ethanol-containing Covonia down to below 0.1% m/m (Supplementary Fig.\u0026nbsp;1). This is most likely due to Covonia having low levels of ethanol (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) since it is suitable for children over 1 year old in addition to teenagers and adults. Ethanol was not expected to interfere in the assay since it would have converted to acetaldehyde with alcohol dehydrogenase and then oxidised by aldehyde dehydrogenase to acetic acid which is not an alpha hydroxy acid and not an expected substrate for glycolate oxidase.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of the assays repurposed to help determine ethylene glycol (EG)/diethylene glycol (DEG)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAssay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInitial intended use\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRepurposed for DEG/EG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLoD (% m/m in matrix)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProposed use\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEthanol assay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEthanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.0% (in PG); 0.1\u0026ndash;0.5% (in syrup)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eRaw materials and ethanol-free paediatric syrups\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlycolic acid assay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlycolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGlycolic acid from EG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.0% (in PG); 0.1% (in syrup)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlcohol strip tests (saliva or breast milk)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEthanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5% (in PG); 1.0\u0026ndash;2.0% (in syrup)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlcohol breath test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEthanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDEG and EG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e~\u0026thinsp;0.1 and 1.0%, respectively (in water)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRaw materials only\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG ELISA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePEG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDEG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOnly 5.0% tested (in water)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCustom antibody required\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eDEG, diethylene glycol; EG, ethylene glycol; ELISA, enzyme-linked immunosorbent assay; LoD, limit of detection; PG, propylene glycol; PEG, polyethylene glycol\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThese assays could potentially be used in NMRA laboratories, hospitals, or testing laboratories. Although a plate reader is required to measure the absorbance of NADH at 340 nm, we show that EG could also be determined without the need for a plate reader since the reduced fluorescent substrate was seen visually by eye as a pink colour (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). It may be possible for this enzymatic test to be developed into a low-cost and rapid pad-based strip test using a cocktail of four enzymes (alcohol dehydrogenase, aldehyde dehydrogenase, glycolate oxidase and a peroxidase) and a chromogenic substrate such as tetramethylbenzidine to visualise a colour change. While this should work for raw materials, a potential problem is the testing of finished products since some syrups are coloured (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) which may hinder the visualisation of the colour change. To overcome this problem, a fluorogenic substrate could be used instead since syrups are free from fluorescent excipients and this approach potentially has the advantage of greater sensitivity. Rapid tests using florescence have successfully been visualised simply using a low-cost UV torch (e.g. Hough COVID-19 Home test, Burleigh West, Australia). Due to the resource confines of this study, we were unable to explore the possibility of developing such a rapid test.\u003c/p\u003e \u003cp\u003eConversion of EG was far higher than the other alcohols when using only alcohol dehydrogenase and aldehyde dehydrogenase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Since EG converted with these enzymes more like ethanol than the other alcohols, we hypothesised that EG may also convert faster than the other alcohols with alcohol oxidase. This led us to evaluate alcohol rapid test strips which use alcohol oxidase, a peroxidase and a chromogenic substrate which turns blue in the presence of alcohol. By repurposing these alcohol test strips, we show that EG could be rapidly and simply determined and easily differentiated from glycerol and PG which yielded negative results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). This successfully worked with three different brands of saliva strips and three brands of breast milk strips (five shown in Supplementary Fig.\u0026nbsp;3) suggesting that this approach may work with any brand of alcohol rapid test strips for testing raw materials. These rapid (2 minutes) and inexpensive (less than \u003cspan\u003e$\u003c/span\u003e1) strips do not require instrumentation and are easy to use with minimal training since they are designed for public use. The strips would be useful for syrup manufacturers for additional testing of incoming raw materials and checking for EG barrels which have been mislabelled, such as the reported incidents in Indonesia\u003csup\u003e \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e \u003c/sup\u003e and Pakistan\u003csup\u003e \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e \u003c/sup\u003e, where barrels containing 96\u0026ndash;99%\u003csup\u003e39\u003c/sup\u003e and up to 100% EG\u003csup\u003e19\u003c/sup\u003e, respectively, were mislabelled as PG. In Indonesia, hundreds of children died due to EG contamination and simply testing raw ingredients with these rapid alcohol strips could have helped avoid many of these deaths.\u003c/p\u003e \u003cp\u003eThe rapid alcohol strips were able to determine levels as low as 0.5% m/m EG in PG (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). While this does not meet the 0.1% m/m WHO regulatory threshold, it may help to prevent deaths and/or toxicity. This is only the case if 0.5% m/m is below the fatal/toxic concentration limits although there is no published evidence on these clinical limits. In a similar way as discussed earlier, it may be possible to improve the sensitivity down to 0.1% m/m if a more sensitive fluorogenic substrate was used but it was not possible to investigate this further. The strips were less sensitive when testing EG spiked into syrups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) where detection was down to around 1\u0026ndash;2% EG. Again, while this is above the regulatory limit, it would have helped to determine major EG contamination such as in Indonesia and therefore could have prevented toxicity and/or deaths. These test strips were only tested with glycerol and PG as excipients used in syrups since these alcohols are commonly used in relatively large amounts and recent cases of contamination have been with PG\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. However, the US FDA do additionally recommend testing maltitol and sorbitol solutions for DEG and EG\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. We show that the alcohol strips show no sign of blue colour change with the maltitol-containing syrups, Benylin Infant and Calprofen, and the sorbitol-containing syrup, Dimetapp (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This suggests that not only can these strips determine EG in PG and glycerol but it could also be used to determine EG in both maltitol and sorbitol solutions received by syrup manufacturers. Furthermore, we show that the alcohol strips could successfully determine the presence of EG down to 1% m/m in a maltitol-containing syrup, Benylin Infant (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDisposable breathalysers were repurposed to determine DEG and EG (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). They contain a proprietary oxidiser which changes from white to pink/brown with alcohols. Some disposable breathalysers contain potassium dichromate which is orange in colour and changes to green in the presence of alcohols. We did not test potassium dichromate since it is extremely toxic and harmful and therefore not feasible to be used in the field to determine DEG or EG. The advantage of the disposable breathalysers used is that it is safe to use since the chemical oxidiser is contained inside the plastic case of the breathalyser and would not come into contact with an inspector using it to determine DEG/EG. DEG was less reactive to the oxidiser in the breathalyser compared to glycerol and PG allowing DEG to be successfully determined. EG did oxidise but at a lower rate allowing EG to be determined. While the breathalysers are low-cost (around \u003cspan\u003e$\u003c/span\u003e1) and the reaction is rapid (10 seconds), since a positive result was observed with glycerol and PG and the test relies on a negative result for DEG (and slower positive for EG), the breathalysers can only be used for testing raw materials testing and cannot be used in finished products. Glycerol and PG oxidise to result in a dark brown colour whereas when the rate of colour change is lower or almost none, a suspicion of probable EG or DEG contamination, respectively, could be raised. This method may be beneficial to prevent cases of DEG and EG contamination in medical products. Substandard Naturcold cough syrup was contaminated with 28.6% DEG in Cameroon\u003csup\u003e \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e \u003c/sup\u003e and with such a high contamination it is highly possible that the barrels of raw material used contained neat DEG since typically 20\u0026ndash;30% glycerol and/or PG are used in medicinal syrups. Furthermore, these breathalysers could have helped to determine EG in Indonesia and Pakistan for the barrels which contained 96\u0026ndash;100% EG\u003csup\u003e14,39\u003c/sup\u003e.\u003csup\u003e14,39\u003c/sup\u003e \u003c/p\u003e \u003cp\u003eWe also explored the potential use of a competitive PEG ELISA in determining DEG (Supplementary Fig.\u0026nbsp;4). The antibody in the kit recognises the polyether chain of PEG and was tested to see if it could recognise the ether in DEG. There was some weak preferential binding to DEG (lowest absorbance) compared to the other alcohols although the difference was so minor that it could not be used to differentiate DEG from the other alcohols and the breathalysers were considerably better to help determine DEG. The poor performance of the PEG ELISA was not surprising since it uses an antibody specific for PEG instead of DEG. A custom antibody or aptamer against DEG, due to its ether group and differences in branching/topology, may improve binding and DEG detection. While it is possible to develop custom antibodies and aptamers for small molecules, it may be challenging to develop them to be specific for DEG especially when levels of other alcohols such as glycerol and PG could be present as excipients in the syrups at much higher concentrations. But if a custom antibody can recognise DEG, then the antibody may not bind to glycerol and PG as well due to minor differences in branching and such an antibody could potentially be used in a rapid test. While such an antibody or aptamer could be used in a low-cost test, this was not investigated due to the initial expensive development costs.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eEnzymatic assays are useful in determining DEG and EG in both raw materials and finished products. By repurposing alcohol rapid test strips, we show that we can simply, inexpensively (less than \u003cspan\u003e$\u003c/span\u003e1) and rapidly (in under 2 minutes) determine EG. These strips could have been used to save the lives of the hundreds of children who died in Indonesia where the problem was mainly with EG. We showcase the repurposing of assays to determine EG with the LoD ranging from 0.1 to 2% m/m. Breathalysers are also simple, inexpensive (~\u003cspan\u003e$\u003c/span\u003e1) and can rapidly (in just 10 seconds) identify both DEG and EG mislabelling of raw materials. The novel approaches are considerably easier and safer to carry out than GC and TLC with only a few simple steps and could therefore be performed by inspectors outside of a scientific laboratory and with minimal training.\u003c/p\u003e "},{"header":"Methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003eChemicals and sample preparation\u003c/h2\u003e \u003cp\u003eEthylene glycol (\u0026ge;\u0026thinsp;99%, Sigma-Aldrich Cat. No. 102466), diethylene glycol (for synthesis, Sigma-Aldrich Cat. No. 8.03131), propylene glycol (Ph. Eur. grade, Sigma-Aldrich Cat. No. 16033), and glycerol (Ph. Eur. grade, Sigma-Aldrich Cat. No. 49779) were used in the study. Each of these four alcohols was prepared as 5% v/v solutions by weighing 0.5 mL of the alcohols and adding ultrapure distilled water (Milli-Q, Merck Millipore) up to 10.0 mL. EG-spiked PG samples were prepared by weighing EG in an amount to generate a percentage of 10.0, 5.0, 2.0, 1.0, 0.5, 0.1, and 0.05% m/m in PG.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFinished medical products\u003c/h2\u003e \u003cp\u003eEleven medicinal syrups were purchased OTC from local pharmacies in the UK, US and Indonesia (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). EG-spiked samples of representative OTC syrups were prepared by gravimetrically adding EG to the syrup matrix, generating 10.0, 5.0, 2.0, 1.0, 0.5, 0.1, 0.05 and 0.01% m/m solutions. The products include adult syrups containing ethanol (Beechams, Covonia, and Benylin Chesty) and paediatric syrups without ethanol (Dimetapp, Benylin Infant\u0026rsquo;s, and Piriteze).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eConverting the alcohols with alcohol dehydrogenase and aldehyde dehydrogenase\u003c/h2\u003e \u003cp\u003eAlcohol dehydrogenase and aldehyde dehydrogenase in an ethanol assay kit (Megazyme, Cat. No. K-ETOH, Wicklow, Ireland) were used to convert the alcohols. In a clear-flat 96-well microplate (Greiner Bio-One, Stonehouse, UK), 10 \u0026micro;L of the sample was mixed with 200 \u0026micro;L of Milli-Q water, 20 \u0026micro;L of buffer, 20 \u0026micro;L of nicotinamide adenine dinucleotide (NAD\u003csup\u003e+\u003c/sup\u003e), and 5 \u0026micro;L of alcohol dehydrogenase. A blank using 10 \u0026micro;L of buffer and a sample diluent control with 10 \u0026micro;L Milli-Q water were also prepared. The samples in the microplate were kept at ambient room temperature (RT, recorded as 20\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) for 2 minutes and the first absorbance (A1) was measured at 340 nm on a Clariostar Plus microplate reader (BMG Labtech, Ortenberg, Germany). Without delay, 2 \u0026micro;L of aldehyde dehydrogenase enzyme was added to the reaction mix, incubated for 5 minutes at RT, and measured for the second absorbance at 340 nm (A2; absorbance peak for NADH, the reduced form of NAD\u003csup\u003e+\u003c/sup\u003e). Absorbances were measured every minute for 15 minutes. The final absorbance was achieved from the subtraction of A2 and A1. The resulting absorbances were then blank-subtracted. The acids formed in the wells of the plate were then used for the glycolic acid assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGlycolic acid assay\u003c/h2\u003e \u003cp\u003eA fluorometric glycolic acid assay kit (Abcam Cat. No. 282915, Cambridge, UK) was used according to the manufacturer\u0026rsquo;s protocol. The samples used were the samples produced after using alcohol dehydrogenase and aldehyde dehydrogenase and were diluted 10\u0026times; in assay buffer. These diluted samples (50 \u0026micro;L) were added into wells of a 96-well black plate for fluorescence with a flat bottom (Corning, UK). For each sample, a reaction mix was prepared consisting of 44 \u0026micro;L of assay buffer, 2 \u0026micro;L of detection reagent, 2 \u0026micro;L of enzyme mix containing glycolate oxidase and 2 \u0026micro;L of fluorogenic probe. This reaction mix (50 \u0026micro;L) was then added to each diluted sample in the 96-well black plate and the fluorescence was recorded in 30-second intervals for 90 minutes at RT with the excitation/emission set to 535/587 nm. The fluorescence for the blank was subtracted from the fluorescence measurements for each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAlcohol strip tests\u003c/h2\u003e \u003cp\u003eGlycerol, PG, DEG and EG diluted in ultrapure water to 5% v/v were tested by adding 20 \u0026micro;L of the diluted alcohol onto the pad of three brands of alcohol rapid test strips for saliva (Surescreen Diagnostics, Eagle Park, UK; One Step distributed by Home Health UK, Bushey, UK; AllTest distributed by UK Drug Testing, Aylsham, UK) and two brands of rapid alcohol test strips for breast milk (Frida, Miami, Florida, USA; Easy@Home, Burr Ridge, Illinois, USA). Spiked samples prepared in PG and cough syrup samples were diluted in water before being added to the pad and the best dilution was found to be a 5\u0026times; dilution in Milli-Q water. After 2 minutes, the intensity of the resultant blue colour was analysed visually by two individuals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAlcohol breathalysers\u003c/h2\u003e \u003cp\u003eDisposable breathalysers (One Step alcohol breath test manufactured by Test\u0026amp;Drive, Ploty, Poland and distributed by Home Health UK, Bushey, UK) were repurposed to test glycerol, PG, DEG, and EG diluted to 1% and 0.1% v/v in Milli-Q water. The protective foil of the breathalyser was pierced by pressing firmly inwards on the protective caps on both ends according to the manufacturer\u0026rsquo;s instructions. The diluted alcohols (75 \u0026micro;L) were pipetted into the blowing end of the tube and flicked twice to ensure that the liquid reaches the white crystals. The breathalysers were left at RT for 2 minutes and any colour changes observed visually with the crystals were recorded at 10 seconds and 2 minutes by two individuals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePolyethylene glycol (PEG) ELISA\u003c/h2\u003e \u003cp\u003eA competitive PEG ELISA kit (Abcam Cat. No. ab215546, Cambridge, UK) was used according to the manufacturer\u0026rsquo;s protocol. A mixture of 50 \u0026micro;L sample and 50 \u0026micro;L of 1\u0026times; PEG-HRP was prepared and 50 \u0026micro;L of the mixture was then added to the anti-PEG antibody-coated well of a strip in the kit. The reaction was incubated for 45 minutes at RT on a plate shaker set to 400 rpm. Following the incubation, the sample mix was aspirated and the well was washed three times with 1\u0026times; Wash Buffer. A volume of 100 \u0026micro;L of TMB substrate was then added to the well and incubated for 15 minutes at RT in the dark with shaking, prior to the addition of 100 \u0026micro;L of stop solution and absorbance was read at 450 nm.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eP.M declares consultancy for Agilent Technologies, of which R.S is an employee. All other authors have no competing interests. The authors alone are responsible for the views expressed in this and they do not necessarily represent the views, decisions or policies of the institutions with which they are affiliated.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eP.M declares consultancy for Agilent Technologies, of which R.S is an employee. All other authors have no competing interests. The authors alone are responsible for the views expressed in this and they do not necessarily represent the views, decisions or policies of the institutions with which they are affiliated.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eB.G, N.Z, P.N.N, C.C, J.M and P.M designed the study. B.Y.A, I.L and B.G performed the experiments and/or data analysis. B.Y.A prepared the original draft. B.Y.A, I.L, J.W-T, T.B, J.G, G.G, M.D, S.B, R.S, P.M, J.M, C.C, P.N.N, N.Z and B.G contributed to the writing, reviewing, and editing of the manuscript. P.N.N, N.Z, J.M and P.M were involved in funding acquisition. B.G, N.Z, P.N.N, C.C, J.M and P.M were involved in project management. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe are very grateful to the Bill \u0026amp; Melinda Gates Foundation for the funding of this study. Furthermore, we would like to thank Cathrin Hauk, Kerlijn Van Assche and Raymond A. Dwek of Oxford University, Tony Cass and Danny O'Hare of Imperial College London and Tim James of Oxford University Hospitals NHS Foundation Trust for their generous support of this project and expert advice. M.D, C.C and P.N.N are supported by the Wellcome Trust (222506/Z/21/Z). B.Y.A is funded by the Indonesian Education Scholarship (Beasiswa Pendidikan Indonesia) from the Ministry of Higher Education, Science and Technology of the Republic of Indonesia (Kemendiktisaintek) within a funding scheme from Indonesia Endowment Fund for Education (LPDP). B.G was partly supported by the Oxford Glycobiology Endowment. This research was funded in part, by the Wellcome Trust [220211/Z/20/Z, 222506/Z/21/Z]. For the purpose of Open Access, the author has applied for a CC BY public copyright license to any Author Accepted Manuscript versions arising from this submission. The funder played no role in the study design, data collection, analysis and interpretation of data, or the writing of this manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePyzik, O. Z. \u0026amp; Abubakar, I. Fighting the fakes: tackling substandard and falsified medicines. \u003cem\u003eNat. Rev. Dis. Primers\u003c/em\u003e. \u003cb\u003e8\u003c/b\u003e, 1\u0026ndash;2 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNewton, P. N. \u0026amp; Bond, K. C. Oxford Statement signatories. 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(2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"substandard, medical products, diethylene glycol, ethylene glycol, contamination, rapid test, falsified, syrup","lastPublishedDoi":"10.21203/rs.3.rs-6683642/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6683642/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThere have been hundreds of child deaths due to contamination of medicinal syrups with diethylene glycol (DEG) and ethylene glycol (EG). Detection of DEG and EG is usually performed by gas chromatography, a method that is costly, laborious, time-consuming, and the device is not readily available in many low- and middle-income countries (LMICs). Thin-layer chromatography is relatively lower cost and is portable but, as with gas chromatography, requires time and trained personnel. Alternative rapid, low-cost and simple methods to determine DEG/EG contamination are desirable. We tested the suitability of enzymatic, chemical and antibody-based assays to determine DEG/EG. Assays using alcohol dehydrogenase and aldehyde dehydrogenase alone as well as in combination with glycolate oxidase could determine EG in raw materials and at less than 0.1% m/m in some finished products. Saliva and breast milk alcohol test strips containing alcohol oxidase and costing \u003cspan\u003e$\u003c/span\u003e1 could determine EG with a detection limit of 0.5 to 2% m/m in under 2 minutes. Disposable breathalysers also costing only \u003cspan\u003e$\u003c/span\u003e1 could determine both DEG and EG from other alcohols in only 10 seconds. The methods described provide simple, rapid and low-cost assays to help determine DEG and EG. By repurposing the breathalysers and alcohol test strips, these disposable tests could have helped to prevent many of the hundreds of infant deaths in 2022 and offer low-cost and rapid approaches for LMICs to screen for DEG and EG.\u003c/p\u003e","manuscriptTitle":"Simple and rapid low-cost assays to investigate ethylene glycol and diethylene glycol contamination in raw materials and medicinal syrups","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-16 05:41:56","doi":"10.21203/rs.3.rs-6683642/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-04T17:20:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-03T06:57:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"265349711906672568204154270857710864598","date":"2025-06-21T15:16:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-11T02:06:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"30852049120606662462428840223525838360","date":"2025-06-01T21:07:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-30T14:05:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-30T14:04:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-30T13:43:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-30T08:32:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-16T22:31:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9fb064b9-516f-11e9-9e20-12b504df345a","owner":[],"postedDate":"June 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":49932230,"name":"Health sciences/Health care/Drug regulation"},{"id":49932231,"name":"Health sciences/Health care/Public health"},{"id":49932232,"name":"Biological sciences/Drug discovery/Drug regulation"},{"id":49932233,"name":"Biological sciences/Drug discovery/Drug safety"},{"id":49932234,"name":"Biological sciences/Drug discovery/Pharmaceutics"},{"id":49932235,"name":"Biological sciences/Drug discovery/Toxicology"},{"id":49932236,"name":"Biological sciences/Biological techniques/Analytical biochemistry/Biochemical assays"},{"id":49932237,"name":"Biological sciences/Biochemistry/Enzymes"}],"tags":[],"updatedAt":"2025-12-08T16:09:26+00:00","versionOfRecord":{"articleIdentity":"rs-6683642","link":"https://doi.org/10.1038/s41598-025-26670-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-03 15:56:56","publishedOnDateReadable":"December 3rd, 2025"},"versionCreatedAt":"2025-06-16 05:41:56","video":"","vorDoi":"10.1038/s41598-025-26670-1","vorDoiUrl":"https://doi.org/10.1038/s41598-025-26670-1","workflowStages":[]},"version":"v1","identity":"rs-6683642","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6683642","identity":"rs-6683642","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}