The Effect of Storage Time on the Levels of Phthalates in Extemporaneous Preparations in Ghana

The Effect of Storage Time on the Levels of Phthalates in Extemporaneous Preparations in Ghana | 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 Research Article The Effect of Storage Time on the Levels of Phthalates in Extemporaneous Preparations in Ghana Abenaa Owusuwaa Adu, Isaac Ayensu, Abena Amponsaa Brobbey, Samuel Oppong Bekoe, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4572478/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The safety and quality of pharmaceutical products can be affected by chemical contaminants that migrate into them, depending on the type and composition of the packaging material. The migration of phthalates during manufacturing of plastics poses potential significant health concerns. This research aims to provide insight into factors driving phthalate migration and propose strategies for reducing its occurrence in extemporaneous preparations in Ashanti region, Ghana. Factors such as storage time and migration kinetics over two years were investigated to understand their impact on phthalate levels in Mist Senna Co (MSC), Mist Potassium Citrate (MPC), Mist Magnesium Trisilicate (MMT) and Mist Expectorant Sedative (MES). The samples were analysed using reverse phase – High Pressure Liquid Chromatography (RP-HPLC) with Photodiode array (PDA) detection. A gradient elution using acetonitrile and 0.1% formic acid at a detection wavelength of 235 nm and a flow rate of 1 mL/min was employed. As the storage time increased, the levels of the phthalates increased. The migration of phthalates was influenced by the duration the polyethylene terephthalate (PET) bottle was in contact with the content of the bottle. The highest migration rate per week for all the phthalates was observed in diethylhexyl phthalate with the highest level being 19 µg/L in MMT, 16 µg/L in MSC, 12 µg/L in MES and 8.3 µg/L in MPC. The finding emphasizes the significance of implementing optimized storage time in reducing phthalate exposure in extemporaneous preparations in Ghana. This study could contribute to enhancing the safety and quality of pharmaceutical products in Ghana. extemporaneous preparations phthalates high pressure liquid chromatography photodiode array polyethylene terephthalate Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 INTRODUCTION Urbanization and industrialization have led to a rise in the use of plastic packaging materials in recent years. In the food industry, over 30 different types of plastics are used (Zhang and Wen, 2014). With the increasing demand for industrialized products, flexible plastic packaging has been widely used due to their versatility and the possible increase in a product’s shelf life (Parker, 2006). Most of these plastics are made of high-density polyethylene, or polyethylene terephthalate (PET). PET is classified as a type of polyester thermoplastic polymer that is obtained from the combination of monoethylene glycol and dimethyl terephthalate or terephthalic acid (Mihucz and Záray, 2016). Regardless of their benefits and advantages: lightness, flexibility, transparency, and barrier to gas transmission, there are concerns about the possibility of leaching of harmful chemical compounds from the packaging materials (Askar, Al-Kamaki and Hassan, 2023). This is because, these compounds are not chemically bonded to the polymer structure from production processes and have the potential to be released, when in contact with water, pharmaceutical products and food, thereby posing a risk to human health (Guerreiro et al. , 2018). Prior studies have demonstrated that these phthalates, used as additives in plastic packaging, have the ability to migrate from packaging materials under specific conditions (Howdeshell et al. , 2003; Bošnir et al. , 2007; Mihucz and Záray, 2016). A wide range of phthalate esters are classified to disrupt endocrine and metabolic functions. Prolonged exposure to these compounds can lead to a range of adverse effects and disorders, including breast cancer, prostate cancer (Pinto and Reali, 2009; Sax, 2010), disruptions in androgenic hormone production and function (Ceretti et al. , 2010), infertility, attention deficit hyperactivity disorder (ADHD) (Chopra et al. , 2014), obesity, liver damage, increased levels of free radicals, allergic reactions, asthma, as well as potential genetic and developmental abnormalities (Guerreiro et al. , 2018). In a study by Blout et al ., (2000), urine samples were collected from adults participating in the Third National Health and Nutrition Examination Survey. From these samples, 289 individuals were randomly chosen and evaluated for phthalate monoesters. According to researchers at the Centres for Disease Control and Prevention (CDC) (Blount et al. , 2000), all 289 individuals tested positive for DBP in their bodies. Scientists suspect that the primary toxic component of DBP is its initial breakdown product, monobutyl phthalate, which has been linked to adverse effects on the male reproductive system, particularly leading to birth defects in laboratory animals, especially male offspring (Kopelovich et al. , 2015). The use of phthalates in consumer items has been outlawed or severely regulated by responsible regulatory bodies in the USA, the EU, Australia, Canada, and UK (Marsh and Bugusu, 2007). This has raised concerns and attracted great attention since significant number of plastics are used for packaging worldwide, highlighting the possible leaching of phthalates into food products consumed (Rastkari et al. , 2017). Investigations have shown that leaching of phthalates is triggered by various factors such as pH, temperature, storage time, volume, light, colour, etc (Rastkari et al. , 2017). Findings by Mihucz et al. , (2016) showed that the concentration of DEPH increased as the storage time was prolonged. Reimann et al. , (2012). demonstrated that the concentration of DiBP, DBP, BBP, and DEHP leaching was about four times higher than its initial concentration when stored at elevated temperature (40–80 o C). Additionally, a study by Lertsirisopon et al. , (2009) examined the non-biological breakdown of BBP, DBP, and DEHP in relation to pH and temperature. They suggested that the degradation of short alkyl chain phthalates like BBP and DBP is reduced at lower pH levels, whereas for DEHP, hydrolysis becomes significant within the pH range of 5 to 9 (Lertsirisopon et al. , 2009). Extemporaneous formulation describes the use of traditional compounding techniques to manipulate various drugs and chemical ingredients to produce suitable drugs when commercial forms are not available. These techniques are widely used in the practice of pediatric pharmacy (Alemón-Medina et al. , 2015). There are various extemporaneous preparations on the Ghanaian market that make use of plastic packaging. These include Mist Magnesium Trisilicate, Ferric ammonium chloride, Mist potassium citrate, Mist Expectorant sedative, Mist Senna Co, among others. These drugs are readily available over the counter and is commonly used by patients. MMT, MPC, MES and MSC are over-the-counter medicines used to treat a variety of disorders including gastrointestinal, genitourinary, respiratory, and constipation disorders respectively thus, they are frequently used for their respective antacid, urinary alkalinizing, antitussive and laxative effects. The Ghana Standards Authority (GSA) is the organization tasked with establishing the requirements for plastics used in Ghana for different purposes. GSA provides no specific phthalate levels within the bottles according to the PET preform specification (GS 1239:2018) and specification for plastic containers for pharmaceutical use other than parenteral and ophthalmic preparations (GS 290:2018). Individuals who purchase products from such bottles run a risk of toxicity as a result of phthalate exposure. Extemporaneous preparation in Ghana is regulated by the Food and Drugs authority (FDA) under small scale manufacturing (Food and Drugs Authority, 2019). Up until now, there has been no study on the migration kinetics of phthalates in extemporaneous preparations globally and on the Ghanaian market. In the present study, the effect of storage time on the levels of phthalates and migration kinetics of phthalates in extemporaneous preparations in Ashanti region of Ghana were investigated. MATERIALS AND METHODS Reagents and chemicals All chemicals and solvents used were of HPLC grade. Acetonitrile (CAS #: 75-05-8, purity ≥ 99.9%), hexane (CAS #: 110-54-3, purity 99.9%), petroleum ether (CAS #: 8032-32-4, purity 99%), methanol (CAS #:67-56-1, purity ≥ 99.8%), dichloromethane (purity 99.9%), formic acid (CAS #:64-18-6, purity 98%). A certified mixed reference standards of phthalates in isooctane [1000 µg/mL each of the dimethyl phthalate (purity 99.5%), diethyl phthalate (purity 98.9%), dibutyl phthalate (purity 99.5%), benzyl butyl phthalate (purity 98.7), diethyl hexyl phthalate ( purity 99.5%), Di-n-octyl phthalate (purity 99.5%) ] was obtained from Chem Service INC . STUDY AREA The research primarily took place in the Ashanti region at coordinates 6⁰53’08.9” N 1⁰31’14.7”. The Ashanti region was chosen as the main study area because the area presents a more representative population sample for research purposes. Ashanti region provides a good variation in demographics, which could be used to generalize the population in Ghana. This decision is supported by a recent study conducted by Ampomah et al. , (2022) on integrated healthcare and the experiences of individuals in Ghana’s Ashanti region. SAMPLING The extemporaneous preparations considered were MMT, MES, MSC and MPC. The extemporaneous formulations, including MMT, MES, MSC and MPC were freshly prepared, and some were stored in plastics, while others were stored in glass bottles under the same conditions. Over a period of two years following their initial preparation, samples from each plastic and glass bottle of these formulations were analysed at weekly intervals. Throughout this period, all products were stored at room temperature under normal atmospheric conditions, without exposure to sunlight. Depending on the specific storage duration being evaluated, samples corresponding to the storage time were selected and subjected to analysis using the established and validated HPLC method. The product stored in glass bottles within the same period (storage time) were used as control. The plastic bottles used were purchased from factories that sell to most companies that produce extemporaneous preparations. To ensure that the samples were kept in comparable conditions prior to the experiment and that the PET-bottle materials were the source of phthalate esters, the bottles were obtained directly from the factories where they were manufactured. Triplicate analyses of the samples were performed. EXTRACTION The extraction method was a modification of the method reported in literature (EPA, 2000; Adegunwa, Adegunwa and Oyatoyinbo, 2022; Angnunavuri et al. , 2022). Extraction of the phthalates from the various extemporaneous preparations was done using liquid-liquid extraction. After several trials, a mixture of dichloromethane: petroleum ether (20:80) was used as the extracting solvent. 50 ml of the sample and 75 ml of the extracting solvent were mixed and shaken vigorously. The two phases were agitated by shaking, to bring about significant physical mixing in a separatory funnel. A quantity of the mixed reference standard (12 µL) corresponding was added. The sample and the extracting solvent were in a ratio of 2:3. After agitation, the phases were allowed to separate. The maximum extraction of phthalates present in the samples was achieved after 48 hours of contact between the sample and the extracting solvent. For maximum extraction and efficient recovery, the extraction was repeated twice. The extracts were combined and dried over anhydrous sodium sulphate. They were evaporated to dryness on a rotary evaporator at 40 ℃ and under a flow of Nitrogen gas. The extraction procedure was validated through recovery analysis. The percentage recovery was calculated to evaluate the effectiveness of the extraction procedure as well as the instrumental analytical procedure. The percentage recovery (%R) = \(\frac{A-B}{C}\) X 100% ………………….. ( 1 ) Where, A is the concentration of phthalate in the spiked sample, B is the concentration of phthalate in the unspiked sample, and C is the concentration used for spiking. INSTRUMENTATION Method Development Employing C18 column (Phenomenex LC Column 150 x 4.6mm, 5 µm C18 110A; Germany), a reverse phase HPLC method was selected based on the physicochemical properties of the analytes (Sircar et al. , 2006; Zaater, Tahboub and Al Sayyed, 2014; Ranganadham, 2017). The HPLC used was Perkin Elmer Flexar LC; Germany with a PDA detector. The wavelength of detection was 235 nm at a flow rate of 1 mL/min. A gradient elution program was employed with mobile phases; acetonitrile: 0.1% formic acid (70: 30 v/v) as illustrated in Table I . Table I . Gradient elution program with mobile phase of 0.1% formic acid(A): acetonitrile(B) Step Step type Step time (min) %A %B 0 Equilibration 1 30.00 70.00 1 Run 5 30.00 70.00 2 Run 1 0.00 100.00 3 Run 5 0.00 100.00 4 Run 1 30.00 70.00 5 Run 5 30.00 70.00 Validation The method was validated according to the ICH recommendations (Conference et al. , 1994). For linearity, 20 µl of each of the concentrations (0.5–64 µg/ml) was injected and loaded three times onto the column. The mean peak areas were plotted against their corresponding concentrations to obtain a calibration curve. The coefficient of correlation was obtained from the graph and the limits of detection (LOD) and quantification (LOQ) were calculated ( Table II ). For accuracy, freshly prepared samples each of MMT, MES, MPC and MSC already established not to contain any of the phthalates were each spiked with three different concentrations of the mixed reference standard 6.4 µg/ml (80%), 8.0µg/ml (100%) and 9.6 µg/ml (120%). Percentage recoveries at different concentrations of the working concentration were also estimated (20%, 30% and 50%). The solutions were taken through the extraction and clean-up protocol and then analyzed using the stated HPLC chromatographic conditions. Solutions were injected in triplicate and percentage recoveries estimated. MIGRATION STUDIES (KINETICS) To estimate the rate of leaching for the difference phthalates being investigated, migration samples from MES, MMT, MSC and MPC were prepared and analyzed over the two year period. The data obtained were modelled using linear regression to estimate the rate of leaching. Linear models obtained from least squares regression were of the form: $$y=mx+c$$ 2 ………………………… The gradient ‘m’ of the linear models was taken as the constant rate value for the migration of phthalates. Data Management and Statistical Analyses Data collected was entered, cleaned, and coded using Microsoft Excel 2021. Statistical analyses were conducted using the R language for statistical computing version 4.2.3. Categorical variables were presented as frequencies and percentages. Parametric continuous variables were represented as means and standard deviations and non-parametric continuous variables as median and interquartile ranges. Kruskal-Wallis H-test and post-hoc test by Bonferroni pairwise comparison test was used to compare the levels of DMP, DEP, DBP, BBP, DEHP, and DnOP between the different products. Spearman’s Rho correlation analysis correlations and a linear regression model were performed to determine the rate of DMP, DEP, DBP, BBP, DEHP, DnOP leaching and shelf life of the different products. Moreover, Friedman’s ANOVA and post-hoc test by Bonferroni pairwise comparison test was used to compare the levels between DMP, DEP, DBP, BBP, DEHP, and DnOP within the study samples. A p-value of < 0.05 was considered as statistically significant. RESULTS IDENTIFICATION OF PURE REFERENCE COMPOUNDS USING HPLC Table II. RETENTION TIME OF THE PTHALATES COMPOUND RETENTION TIME (MINUTES) DMP 2.589 ± 0.05508 DEP 3.325 ± 0.010214 DBP 7.270 ± 0.02121 BBP 7.790 ± 0.06364 DEHP 12.606 ± 0.0707 DnOP 12.972 ± 0.01732 Table II Linearity range and R 2 values of the reference standards REFERENCE STANDARD LINEARITY RANGE (μg/mL) CORRELATION COEFFICIENT(R 2 ) LOD (μg/L) LOQ (μg/L) DMP 0.5 – 64 0.9962 0.1231 0.3704 DEP 0.5 – 64 0.9993 0.1317 0.3991 DBP 0.5 – 64 0.9982 0.1682 0.5096 BBP 0.5 – 64 0.9978 0.1735 0.5258 DEHP 0.5 – 64 0.9976 0.2567 0.7778 DnOP 0.5 – 64 0.9976 0.2672 0.8096 Comparison of Levels of DMP, DEP, DBP, BBP, DEHP, and DnOP between MPC, MMT, MSC and MES controlled samples This study observed significant differences in levels of DMP, DEP, DBP, BBP, DEHP, and DnOP between MPC, MMT, MSC and MES migration samples ( p < 0.0001). In a post-hoc pairwise comparison test, except for the non-significant differences in the levels of DMP, DEHP and DnOP between MMT and MSC (Figure 1) , there was significant differences in the pairwise comparison in the levels of DMP, DEP, DBP, BBP, DEHP, and DnOP between MPC, MMT, MSC and MES migration samples ( p < 0.05) (Figure 1) . DMP, DEP, DBP, BBP, DEHP, DnOP leaching in controlled samples over two years [Figures 3-6] Rate of DMP, DEP, DBP, BBP, DEHP, DnOP Leaching in controlled samples In a linear regression model [Figures 7-12] DISCUSSION The established method was validated following the ICH protocol Q2 (R1) (Conference et al. , 1994). The validation process confirmed the method's specificity, precision and accuracy. From the results, as the storage time prolonged, regardless of the type of extemporaneous preparation, the concentration of the phthalates increased (Figs. 3 , 4 , 5 , 6 ). The migration of the phthalates clearly is affected by the duration of the exposure between the PET bottle and the contents of the bottle (Jeddi et al. , 2015). This observation was similar to the findings from other studies (Rastkari et al. , 2017; Luo et al. , 2020). A study by Rastkari et al. , (2017) also reported the effect of storage time (2, 4 and 6 months) under different conditions. The migration of the phthalates was in the order DEHP > DEP > DBP and DEP > DEHP > DBP in HDPE and PET bottles, respectively. It was also realized the speed and amount of phthalate migration was much faster and larger in the initial storage period than the later period. This observation was similar to a study conducted by Luo et al. , (2020). It has been established from literature that lower polarity enhances the release of phthalates from plastic bottles (Amiridou and Voutsa, 2011). In a post-hoc analyses, MMT and MSC random samples had significantly higher levels of DMP, DEP, DBP, BBP, DEHP, and DnOP levels than compared to MES and MPC ( p < 0.0001). Similarly, MES had higher levels of DMP, DEP, DBP, BBP, DEHP, and DnOP levels than compared to MPC ( p < 0.0001). There were significant differences in the pairwise comparison in the levels of DMP, DEP, DBP, BBP, DEHP, and DnOP between MPC, MMT, MSC and MES in the control samples ( p < 0.05). This observation can be attributed to the difference in the polarities of the extemporaneous preparations studied. MMT, which is likely the most non-polar extemporaneous preparation among the four studied had the highest level of phthalates, followed by MSC, then MES and finally MPC. MPC, which is reported to be more polar compared to the other extemporaneous preparations had the least concentration of phthalates recorded, confirming this theory. These results obtained are similar to the findings of previous studies carried out on edible oils by Luo et al. , (2020). From the results, there was a non-significant difference in the levels of DMP, DEHP and DnOP between MMT and MSC (Fig. 1 ). This is because both MMT and MSC are relatively non-polar. The levels of phthalates reported in this study are higher than the phthalate levels recorded in water in Ghana and other parts of the world, all substantiating the evidence that lower polarity increases the migration of phthalates (Angnunavuri et al. , 2022; Khishdost et al. , 2023). Our findings suggest MMT, which has the highest pH showed the highest leaching of phthalates with the highest recorded phthalate levels; implying a higher pH enhances the migration of phthalates. This assertion from our findings is congruent with research investigated by Rastkari et al. , (2017). The research by Rastkari et al. , (2017) supports the theory that a higher pH increases the rate of migration of phthalates. From the results of this study, it can be inferred that when polarity and acidity act in tandem, it appears that polarity has a stronger influence than acidity. From the linear regression models, for DMP migration, the rate of migration was 13 µg/L, 13 µg/L, 9.31 µg/L and 7.6µg/L per week for MMT, MSC, MES and MPC respectively. A similar observation was made in DEP migration where the highest migration rate per week was recorded in MMT (12 µg/L), followed by MSC (11 µg/L), then MES (9.1 µg/L) and MPC (7.1 µg/L). DBP and BBP migration per week also followed the same trend with the highest rate of migration (12 µg/L) recorded in MMT and the least rate of migration (5.9 µg/L and 4.2 µg/L in DBP and BBP respectively) recorded in MPC. It could be seen that the lower the polarity of the extemporaneous preparation, the greater the rate constant and the greater the initial release rate (Fig. 6 ). The results show a larger release rate in MMT compared to the other products, confirming literature findings. Similar findings have been reported by research work on alkylphenols and phthalates in bottled water (Amiridou and Voutsa, 2011). The levels of phthalates recorded in this study is however significantly higher than the levels reported in bottled water globally and in Ghana (Zaater, Tahboub and Al Sayyed, 2014; Jeddi, 2018). This trend could be attributed to the difference in the polarities of the various extemporaneous preparations. The higher the polarity, the lower the phthalate migration (Amiridou and Voutsa, 2011; Luo et al. , 2020). CONCLUSION The kinetics of the migration of phthalates into extemporaneous preparations has been studied over a period of two ( 2 ) years. Phthalates levels increased as the storage time increased. For the first time, the migration kinetics of phthalates in extemporaneous preparation in Ghana has been studied. The rate of migration for DMP was 13 µg/L, 13 µg/L, 9.30 µg/L and 7.6 µg/L per week for MMT, MSC, MES and MPC respectively. DEP migration kinetics followed a similar pattern where the highest migration rate per week was recorded in MMT (12 µg/L), followed by MSC (11 µg/L), then MES (9.1 µg/L) and MPC (7.1 µg/L). DBP and BBP migration per also showed the highest rate of migration (12 µg/L) recorded in MMT and the least rate of migration (5.9 µg/L and 4.2 µg/L in DBP and BBP respectively) recorded in MPC. The highest migration rate per week for all the phthalates was observed in DEHP with the highest level being 19 µg/L in MMT, 16 µg/L in MSC, 12 µg/L in MES and 8.3 µg/L in MPC. The rate and concentration of phthalates from the plastic bottles were much faster and larger in the initial storage period than the later period. This implies that the longer the storage time of the product, the higher the rate of phthalate migration, resulting in elevated health risk. Declarations Funding There was no funding Ethics approval and consent to participate Not applicable Competing interests All authors declared that no competing interests exited References Adegunwa, A.O., Adegunwa, A.K. and Oyatoyinbo, R.M. (2022) ‘Toxicological exposure to phthalates mixtures and human health risk assessment of drinking water and fruit drinks packaged in plastics and tetra pak cartons’, Bulletin of the National Research Centre , 46(1). Available at: https://doi.org/10.1186/s42269-022-00878-x . Alemón-Medina, R. et al. 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(2014) ‘Determination of phthalates in Jordanian bottled water using GC-MS and HPLC-UV: Environmental study’, Journal of Chromatographic Science , 52(5), pp. 447–452. Available at: https://doi.org/10.1093/chromsci/bmt059 . Zhang, H. and Wen, Z.G. (2014) ‘The consumption and recycling collection system of PET bottles: A case study of Beijing, China’, Waste Management , 34(6), pp. 987–998. Available at: https://doi.org/10.1016/j.wasman.2013.07.015 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-4572478","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":316548903,"identity":"016d9177-c2b2-43de-a3ee-8a3585ed9183","order_by":0,"name":"Abenaa Owusuwaa Adu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYDACCTgrgfEBkOThI0ULswFICxspWtjAbIJa+Gc3H3vwo4ZBjr89x6zya46dDBsD88NHN/BZcudYumHPMQZjiTNvzG7LbksGOozN2DgHnzU3cswkgMoSG4CM25LbmIFsHjZpfFrkb+R/k/zzjyFxPlBLseS2esJaDG7ksEnztjEkbgBqYfy47TBhLYZ3jplJy/ZJGBueeVYszbjtOA8bMwG/yN1ufib55puNnNzx5I0ff26rtudnb374GK/3IQASO8w8YJKwcgRg/EGK6lEwCkbBKBgxAAAWIkN7LmblIwAAAABJRU5ErkJggg==","orcid":"","institution":"Kwame Nkrumah University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Abenaa","middleName":"Owusuwaa","lastName":"Adu","suffix":""},{"id":316548904,"identity":"f7324b0b-43d4-45b4-8381-486a190c5f58","order_by":1,"name":"Isaac Ayensu","email":"","orcid":"","institution":"Kwame Nkrumah University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Isaac","middleName":"","lastName":"Ayensu","suffix":""},{"id":316548905,"identity":"98292842-198f-4f34-ae03-ec3b7a6f0e0b","order_by":2,"name":"Abena Amponsaa Brobbey","email":"","orcid":"","institution":"Kwame Nkrumah University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Abena","middleName":"Amponsaa","lastName":"Brobbey","suffix":""},{"id":316548906,"identity":"abaff092-5671-4975-95f7-7f9ef1b537fa","order_by":3,"name":"Samuel Oppong Bekoe","email":"","orcid":"","institution":"Kwame Nkrumah University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Samuel","middleName":"Oppong","lastName":"Bekoe","suffix":""},{"id":316548909,"identity":"44a58599-8f9c-443e-a0b4-f25ed987b68a","order_by":4,"name":"David Azanu","email":"","orcid":"","institution":"Kwame Nkrumah University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Azanu","suffix":""},{"id":316548912,"identity":"0e5244f0-ce38-4c0e-85c9-cb0c7b572960","order_by":5,"name":"Joshua Boateng","email":"","orcid":"","institution":"University of Greenwich","correspondingAuthor":false,"prefix":"","firstName":"Joshua","middleName":"","lastName":"Boateng","suffix":""}],"badges":[],"createdAt":"2024-06-12 21:23:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4572478/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4572478/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59056930,"identity":"5bcdbe56-8e27-48fb-81d7-57a8f8fd394b","added_by":"auto","created_at":"2024-06-25 21:53:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":51436,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC chromatography of DMP, DEP, DBP, BBP, DEHP and DnOP at a concentration of 64 μg/ml\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4572478/v1/05b7554c85756cdd3f996534.png"},{"id":59056939,"identity":"621060e3-789d-434c-8188-9f014951da8d","added_by":"auto","created_at":"2024-06-25 21:53:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":200181,"visible":true,"origin":"","legend":"\u003cp\u003eBox and whiskers plot depicting comparison of comparison DMP, DEP, DBP, BBP, DEHP, and DnOP Between MPC, MMT, MSC and MES migration samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote: \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-values computed by Kruskal-Wallis H-test and post-hoc test by Bonferroni pairwise comparison test, ns: not significant, *: \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.05, **: \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.01, ***: \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.001, ****: \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt; 0.0001\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4572478/v1/ac70e687176fae6d8ff6ff43.png"},{"id":59057131,"identity":"b48aa857-d34e-4c39-b963-638e4ee109b2","added_by":"auto","created_at":"2024-06-25 22:01:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":68823,"visible":true,"origin":"","legend":"\u003cp\u003eDMP, DEP, DBP, BBP, DEHP, DnOP leaching in Weeks from MES controlled samples\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4572478/v1/5541bf864c3295fa7b4e0537.png"},{"id":59056938,"identity":"1b7aacf2-5e5a-4055-85e7-4b60350bcc7f","added_by":"auto","created_at":"2024-06-25 21:53:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":66097,"visible":true,"origin":"","legend":"\u003cp\u003eDMP, DEP, DBP, BBP, DEHP, DnOP leaching and Weeks in MPC controlled samples\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4572478/v1/535f216e9970c42553d6ed8e.png"},{"id":59056928,"identity":"b75a1ae4-9fa6-46f6-834b-19ed131ec0ef","added_by":"auto","created_at":"2024-06-25 21:53:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":62959,"visible":true,"origin":"","legend":"\u003cp\u003eDMP, DEP, DBP, BBP, DEHP, DnOP leaching and Weeks in MMT controlled samples\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4572478/v1/468b84e0bc83d6eeefda5d06.png"},{"id":59056927,"identity":"595d42bc-60d4-4896-9d17-e584a1a383da","added_by":"auto","created_at":"2024-06-25 21:53:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":63452,"visible":true,"origin":"","legend":"\u003cp\u003eDMP, DEP, DBP, BBP, DEHP, DnOP leaching and Weeks in MSC controlled samples\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4572478/v1/117d86764118856a989f8609.png"},{"id":59056925,"identity":"c7dcdb35-deb8-45c4-8ec0-9587c575fe59","added_by":"auto","created_at":"2024-06-25 21:53:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":92245,"visible":true,"origin":"","legend":"\u003cp\u003eRate of DMP migration in MES, MMT, MPC and MSC controlled samples\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNote: R: Spearman’s Rho correlation value, R\u003csup\u003e2\u003c/sup\u003e: R square change, y= m*x + c* represents linear regression model equation where variables change respectively\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4572478/v1/67e9991271ecc865dbc65d92.png"},{"id":59056922,"identity":"5355ff4d-1c8a-49b3-8e4f-c20c1d3d6e65","added_by":"auto","created_at":"2024-06-25 21:53:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":86204,"visible":true,"origin":"","legend":"\u003cp\u003eRate of DEP migration in MES, MMT, MPC and MSC controlled samples\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNote: R: Spearman’s Rho correlation value, R\u003csup\u003e2\u003c/sup\u003e: R square change, y=m*x + c* represents linear regression model equation where variables change respectively\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4572478/v1/346df624bcb2eed3a43088ef.png"},{"id":59056926,"identity":"89a80bb0-54f2-4bb0-b405-96b78ee635bb","added_by":"auto","created_at":"2024-06-25 21:53:32","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":94858,"visible":true,"origin":"","legend":"\u003cp\u003eRate of DBP migration in MES, MMT, MPC and MSC Samples\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNote: R: Spearman’s Rho correlation value, R\u003csup\u003e2\u003c/sup\u003e: R square change, y=m*x + c* represents linear regression model equation where variables change respectively\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4572478/v1/acaf6f13baf5ebc5a0c51108.png"},{"id":59056923,"identity":"89f2ea3c-b43e-4aee-befd-4aa6c140985e","added_by":"auto","created_at":"2024-06-25 21:53:31","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":87293,"visible":true,"origin":"","legend":"\u003cp\u003eRate of BBP migration in MES, MMT, MPC and MSC Samples\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNote: R: Spearman’s Rho correlation value, R\u003csup\u003e2\u003c/sup\u003e: R square change, y=m*x + c* represents linear regression model equation where variables change respectively\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4572478/v1/40608368535dd12f5f7fb2a4.png"},{"id":59056924,"identity":"a6b71968-3e31-47cb-88c5-bb7fc5a3c19d","added_by":"auto","created_at":"2024-06-25 21:53:32","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":90829,"visible":true,"origin":"","legend":"\u003cp\u003eRate of DEHP migration in MES, MMT, MPC and MSC Samples\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNote: R: Spearman’s Rho correlation value, R\u003csup\u003e2\u003c/sup\u003e: R square change, y=m*x + c* represents linear regression model equation where variables change respectively\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-4572478/v1/7eb617c66f8d3827287c45a0.png"},{"id":59056937,"identity":"59b3e03d-f2a2-42ef-a779-b8cd7784c064","added_by":"auto","created_at":"2024-06-25 21:53:33","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":95347,"visible":true,"origin":"","legend":"\u003cp\u003eRate of DnOP migration in MES, MMT, MPC and MSC samples\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNote: R: Spearman’s Rho correlation value, R\u003csup\u003e2\u003c/sup\u003e: R square change, y=m*x + c* represents linear regression model equation where variables change respectively\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-4572478/v1/4f24611567bec54e54b2049d.png"},{"id":67213977,"identity":"6fdcd529-56d8-4421-a5c6-ed62e387761e","added_by":"auto","created_at":"2024-10-22 12:54:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1452370,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4572478/v1/30ad865e-0153-40cc-b3ee-fc43de329f8c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eThe Effect of Storage Time on the Levels of Phthalates in Extemporaneous Preparations in Ghana\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eUrbanization and industrialization have led to a rise in the use of plastic packaging materials in recent years. In the food industry, over 30 different types of plastics are used (Zhang and Wen, 2014). With the increasing demand for industrialized products, flexible plastic packaging has been widely used due to their versatility and the possible increase in a product\u0026rsquo;s shelf life (Parker, 2006). Most of these plastics are made of high-density polyethylene, or polyethylene terephthalate (PET).\u003c/p\u003e \u003cp\u003e PET is classified as a type of polyester thermoplastic polymer that is obtained from the combination of monoethylene glycol and dimethyl terephthalate or terephthalic acid (Mihucz and Z\u0026aacute;ray, 2016). Regardless of their benefits and advantages: lightness, flexibility, transparency, and barrier to gas transmission, there are concerns about the possibility of leaching of harmful chemical compounds from the packaging materials (Askar, Al-Kamaki and Hassan, 2023). This is because, these compounds are not chemically bonded to the polymer structure from production processes and have the potential to be released, when in contact with water, pharmaceutical products and food, thereby posing a risk to human health (Guerreiro \u003cem\u003eet al.\u003c/em\u003e, 2018). Prior studies have demonstrated that these phthalates, used as additives in plastic packaging, have the ability to migrate from packaging materials under specific conditions (Howdeshell \u003cem\u003eet al.\u003c/em\u003e, 2003; Bošnir \u003cem\u003eet al.\u003c/em\u003e, 2007; Mihucz and Z\u0026aacute;ray, 2016).\u003c/p\u003e \u003cp\u003eA wide range of phthalate esters are classified to disrupt endocrine and metabolic functions. Prolonged exposure to these compounds can lead to a range of adverse effects and disorders, including breast cancer, prostate cancer (Pinto and Reali, 2009; Sax, 2010), disruptions in androgenic hormone production and function (Ceretti \u003cem\u003eet al.\u003c/em\u003e, 2010), infertility, attention deficit hyperactivity disorder (ADHD) (Chopra \u003cem\u003eet al.\u003c/em\u003e, 2014), obesity, liver damage, increased levels of free radicals, allergic reactions, asthma, as well as potential genetic and developmental abnormalities (Guerreiro \u003cem\u003eet al.\u003c/em\u003e, 2018).\u003c/p\u003e \u003cp\u003eIn a study by Blout \u003cem\u003eet al\u003c/em\u003e., (2000), urine samples were collected from adults participating in the Third National Health and Nutrition Examination Survey. From these samples, 289 individuals were randomly chosen and evaluated for phthalate monoesters. According to researchers at the Centres for Disease Control and Prevention (CDC) (Blount \u003cem\u003eet al.\u003c/em\u003e, 2000), all 289 individuals tested positive for DBP in their bodies. Scientists suspect that the primary toxic component of DBP is its initial breakdown product, monobutyl phthalate, which has been linked to adverse effects on the male reproductive system, particularly leading to birth defects in laboratory animals, especially male offspring (Kopelovich \u003cem\u003eet al.\u003c/em\u003e, 2015). The use of phthalates in consumer items has been outlawed or severely regulated by responsible regulatory bodies in the USA, the EU, Australia, Canada, and UK (Marsh and Bugusu, 2007). This has raised concerns and attracted great attention since significant number of plastics are used for packaging worldwide, highlighting the possible leaching of phthalates into food products consumed (Rastkari \u003cem\u003eet al.\u003c/em\u003e, 2017).\u003c/p\u003e \u003cp\u003eInvestigations have shown that leaching of phthalates is triggered by various factors such as pH, temperature, storage time, volume, light, colour, etc (Rastkari \u003cem\u003eet al.\u003c/em\u003e, 2017). Findings by Mihucz \u003cem\u003eet al.\u003c/em\u003e, (2016) showed that the concentration of DEPH increased as the storage time was prolonged. Reimann \u003cem\u003eet al.\u003c/em\u003e, (2012). demonstrated that the concentration of DiBP, DBP, BBP, and DEHP leaching was about four times higher than its initial concentration when stored at elevated temperature (40\u0026ndash;80\u003csup\u003eo\u003c/sup\u003eC). Additionally, a study by Lertsirisopon \u003cem\u003eet al.\u003c/em\u003e, (2009) examined the non-biological breakdown of BBP, DBP, and DEHP in relation to pH and temperature. They suggested that the degradation of short alkyl chain phthalates like BBP and DBP is reduced at lower pH levels, whereas for DEHP, hydrolysis becomes significant within the pH range of 5 to 9 (Lertsirisopon \u003cem\u003eet al.\u003c/em\u003e, 2009).\u003c/p\u003e \u003cp\u003eExtemporaneous formulation describes the use of traditional compounding techniques to manipulate various drugs and chemical ingredients to produce suitable drugs when commercial forms are not available. These techniques are widely used in the practice of pediatric pharmacy (Alem\u0026oacute;n-Medina \u003cem\u003eet al.\u003c/em\u003e, 2015). There are various extemporaneous preparations on the Ghanaian market that make use of plastic packaging. These include Mist Magnesium Trisilicate, Ferric ammonium chloride, Mist potassium citrate, Mist Expectorant sedative, Mist Senna Co, among others. These drugs are readily available over the counter and is commonly used by patients. MMT, MPC, MES and MSC are over-the-counter medicines used to treat a variety of disorders including gastrointestinal, genitourinary, respiratory, and constipation disorders respectively thus, they are frequently used for their respective antacid, urinary alkalinizing, antitussive and laxative effects.\u003c/p\u003e \u003cp\u003eThe Ghana Standards Authority (GSA) is the organization tasked with establishing the requirements for plastics used in Ghana for different purposes. GSA provides no specific phthalate levels within the bottles according to the PET preform specification (GS 1239:2018) and specification for plastic containers for pharmaceutical use other than parenteral and ophthalmic preparations (GS 290:2018). Individuals who purchase products from such bottles run a risk of toxicity as a result of phthalate exposure.\u003c/p\u003e \u003cp\u003eExtemporaneous preparation in Ghana is regulated by the Food and Drugs authority (FDA) under small scale manufacturing (Food and Drugs Authority, 2019). Up until now, there has been no study on the migration kinetics of phthalates in extemporaneous preparations globally and on the Ghanaian market. In the present study, the effect of storage time on the levels of phthalates and migration kinetics of phthalates in extemporaneous preparations in Ashanti region of Ghana were investigated.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\n\u003cp\u003eReagents and chemicals\u003c/p\u003e \u003cp\u003eAll chemicals and solvents used were of HPLC grade. Acetonitrile (CAS #: 75-05-8, purity\u0026thinsp;\u0026ge;\u0026thinsp;99.9%), hexane (CAS #: 110-54-3, purity 99.9%), petroleum ether (CAS #: 8032-32-4, purity 99%), methanol (CAS #:67-56-1, purity\u0026thinsp;\u0026ge;\u0026thinsp;99.8%), dichloromethane (purity 99.9%), formic acid (CAS #:64-18-6, purity 98%). A certified mixed reference standards of phthalates in isooctane [1000 \u0026micro;g/mL each of the dimethyl phthalate (purity 99.5%), diethyl phthalate (purity 98.9%), dibutyl phthalate (purity 99.5%), benzyl butyl phthalate (purity 98.7), diethyl hexyl phthalate ( purity 99.5%), Di-n-octyl phthalate (purity 99.5%) ] was obtained from Chem Service \u003csub\u003eINC\u003c/sub\u003e.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSTUDY AREA\u003c/h2\u003e \u003cp\u003eThe research primarily took place in the Ashanti region at coordinates 6⁰53\u0026rsquo;08.9\u0026rdquo; N 1⁰31\u0026rsquo;14.7\u0026rdquo;. The Ashanti region was chosen as the main study area because the area presents a more representative population sample for research purposes. Ashanti region provides a good variation in demographics, which could be used to generalize the population in Ghana. This decision is supported by a recent study conducted by Ampomah \u003cem\u003eet al.\u003c/em\u003e, (2022) on integrated healthcare and the experiences of individuals in Ghana\u0026rsquo;s Ashanti region.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSAMPLING\u003c/h2\u003e \u003cp\u003eThe extemporaneous preparations considered were MMT, MES, MSC and MPC. The extemporaneous formulations, including MMT, MES, MSC and MPC were freshly prepared, and some were stored in plastics, while others were stored in glass bottles under the same conditions. Over a period of two years following their initial preparation, samples from each plastic and glass bottle of these formulations were analysed at weekly intervals. Throughout this period, all products were stored at room temperature under normal atmospheric conditions, without exposure to sunlight. Depending on the specific storage duration being evaluated, samples corresponding to the storage time were selected and subjected to analysis using the established and validated HPLC method.\u003c/p\u003e \u003cp\u003eThe product stored in glass bottles within the same period (storage time) were used as control. The plastic bottles used were purchased from factories that sell to most companies that produce extemporaneous preparations. To ensure that the samples were kept in comparable conditions prior to the experiment and that the PET-bottle materials were the source of phthalate esters, the bottles were obtained directly from the factories where they were manufactured. Triplicate analyses of the samples were performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEXTRACTION\u003c/h2\u003e \u003cp\u003eThe extraction method was a modification of the method reported in literature (EPA, 2000; Adegunwa, Adegunwa and Oyatoyinbo, 2022; Angnunavuri \u003cem\u003eet al.\u003c/em\u003e, 2022). Extraction of the phthalates from the various extemporaneous preparations was done using liquid-liquid extraction. After several trials, a mixture of dichloromethane: petroleum ether (20:80) was used as the extracting solvent. 50 ml of the sample and 75 ml of the extracting solvent were mixed and shaken vigorously. The two phases were agitated by shaking, to bring about significant physical mixing in a separatory funnel. A quantity of the mixed reference standard (12 \u0026micro;L) corresponding was added.\u003c/p\u003e \u003cp\u003eThe sample and the extracting solvent were in a ratio of 2:3. After agitation, the phases were allowed to separate. The maximum extraction of phthalates present in the samples was achieved after 48 hours of contact between the sample and the extracting solvent. For maximum extraction and efficient recovery, the extraction was repeated twice. The extracts were combined and dried over anhydrous sodium sulphate. They were evaporated to dryness on a rotary evaporator at 40 ℃ and under a flow of Nitrogen gas. The extraction procedure was validated through recovery analysis. The percentage recovery was calculated to evaluate the effectiveness of the extraction procedure as well as the instrumental analytical procedure.\u003c/p\u003e \u003cp\u003eThe percentage recovery (%R) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{A-B}{C}\\)\u003c/span\u003e\u003c/span\u003e X 100% \u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;.. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eWhere, \u003cb\u003eA\u003c/b\u003e is the concentration of phthalate in the spiked sample, \u003cb\u003eB\u003c/b\u003e is the concentration of phthalate in the unspiked sample, and \u003cb\u003eC\u003c/b\u003e is the concentration used for spiking.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eINSTRUMENTATION\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eMethod Development\u003c/h2\u003e \u003cp\u003eEmploying C18 column (Phenomenex LC Column 150 x 4.6mm, 5 \u0026micro;m C18 110A; Germany), a reverse phase HPLC method was selected based on the physicochemical properties of the analytes (Sircar \u003cem\u003eet al.\u003c/em\u003e, 2006; Zaater, Tahboub and Al Sayyed, 2014; Ranganadham, 2017). The HPLC used was Perkin Elmer Flexar LC; Germany with a PDA detector. The wavelength of detection was 235 nm at a flow rate of 1 mL/min.\u003c/p\u003e \u003cp\u003eA gradient elution program was employed with mobile phases; acetonitrile: 0.1% formic acid (70: 30 v/v) as illustrated in \u003cb\u003eTable I\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable I\u003c/b\u003e. Gradient elution program with mobile phase of 0.1% formic acid(A): acetonitrile(B)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStep\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStep type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStep time (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e%A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e%B\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEquilibration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e70.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRun\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e70.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRun\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRun\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRun\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e70.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRun\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e70.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eValidation\u003c/h2\u003e \u003cp\u003eThe method was validated according to the ICH recommendations (Conference \u003cem\u003eet al.\u003c/em\u003e, 1994). For linearity, 20 \u0026micro;l of each of the concentrations (0.5\u0026ndash;64 \u0026micro;g/ml) was injected and loaded three times onto the column. The mean peak areas were plotted against their corresponding concentrations to obtain a calibration curve. The coefficient of correlation was obtained from the graph and the limits of detection (LOD) and quantification (LOQ) were calculated (\u003cb\u003eTable II\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eFor accuracy, freshly prepared samples each of MMT, MES, MPC and MSC already established not to contain any of the phthalates were each spiked with three different concentrations of the mixed reference standard 6.4 \u0026micro;g/ml (80%), 8.0\u0026micro;g/ml (100%) and 9.6 \u0026micro;g/ml (120%). Percentage recoveries at different concentrations of the working concentration were also estimated (20%, 30% and 50%). The solutions were taken through the extraction and clean-up protocol and then analyzed using the stated HPLC chromatographic conditions. Solutions were injected in triplicate and percentage recoveries estimated.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eMIGRATION STUDIES (KINETICS)\u003c/h2\u003e \u003cp\u003eTo estimate the rate of leaching for the difference phthalates being investigated, migration samples from MES, MMT, MSC and MPC were prepared and analyzed over the two year period. The data obtained were modelled using linear regression to estimate the rate of leaching.\u003c/p\u003e \u003cp\u003eLinear models obtained from least squares regression were of the form:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$y=mx+c$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u003c/p\u003e \u003cp\u003eThe gradient \u003cb\u003e\u0026lsquo;m\u0026rsquo;\u003c/b\u003e of the linear models was taken as the constant rate value for the migration of phthalates.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData Management and Statistical Analyses\u003c/h2\u003e \u003cp\u003eData collected was entered, cleaned, and coded using Microsoft Excel 2021. Statistical analyses were conducted using the R language for statistical computing version 4.2.3. Categorical variables were presented as frequencies and percentages. Parametric continuous variables were represented as means and standard deviations and non-parametric continuous variables as median and interquartile ranges. Kruskal-Wallis H-test and post-hoc test by Bonferroni pairwise comparison test was used to compare the levels of DMP, DEP, DBP, BBP, DEHP, and DnOP between the different products. Spearman\u0026rsquo;s Rho correlation analysis correlations and a linear regression model were performed to determine the rate of DMP, DEP, DBP, BBP, DEHP, DnOP leaching and shelf life of the different products.\u003c/p\u003e \u003cp\u003eMoreover, Friedman\u0026rsquo;s ANOVA and post-hoc test by Bonferroni pairwise comparison test was used to compare the levels between DMP, DEP, DBP, BBP, DEHP, and DnOP within the study samples. A p-value of \u0026lt;\u0026thinsp;0.05 was considered as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eIDENTIFICATION OF PURE REFERENCE COMPOUNDS USING HPLC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Aptos\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eTable II.\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;RETENTION TIME OF THE PTHALATES\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border: none;width:450.8pt;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 225.4pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid black;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003eCOMPOUND\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 225.4pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid black;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003eRETENTION TIME (MINUTES)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 225.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003eDMP\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 225.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003e2.589 \u0026plusmn; 0.05508\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 225.4pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003eDEP\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 225.4pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003e3.325 \u0026plusmn; 0.010214\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 225.4pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003eDBP\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 225.4pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003e7.270 \u0026plusmn; 0.02121\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 225.4pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003eBBP\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 225.4pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003e7.790 \u0026plusmn; 0.06364\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 225.4pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003eDEHP\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 225.4pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003e12.606 \u0026plusmn; 0.0707\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 225.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003eDnOP\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 225.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003e12.972 \u0026plusmn; 0.01732\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Aptos\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Aptos\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Aptos\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eTable II\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eLinearity range and R\u003csup\u003e2\u003c/sup\u003e values of the reference standards\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88.25pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eREFERENCE STANDARD\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.3pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eLINEARITY RANGE (\u0026mu;g/mL)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107.5pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eCORRELATION\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eCOEFFICIENT(R\u003csup\u003e2\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80.35pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eLOD (\u0026mu;g/L)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80.4pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eLOQ (\u0026mu;g/L)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88.25pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eDMP\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.5 \u0026ndash; 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64\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107.5pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.9978\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80.35pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.1735\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80.4pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.5258\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88.25pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eDEHP\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.3pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.5 \u0026ndash; 64\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107.5pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.9976\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80.35pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.2567\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80.4pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.7778\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88.25pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eDnOP\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.5 \u0026ndash; 64\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.9976\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80.35pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.2672\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.8096\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Aptos\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparison of Levels of DMP, DEP, DBP, BBP, DEHP, and DnOP between MPC, MMT, MSC and MES controlled samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study observed significant differences in levels of DMP, DEP, DBP, BBP, DEHP, and DnOP between MPC, MMT, MSC and MES migration samples (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001). In a post-hoc pairwise comparison test, except for the non-significant differences in the levels of DMP, DEHP and DnOP between MMT and MSC \u003cstrong\u003e(Figure 1)\u003c/strong\u003e, there was significant differences in the pairwise comparison in the levels of DMP, DEP, DBP, BBP, DEHP, and DnOP between MPC, MMT, MSC and MES migration samples (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05) \u003cstrong\u003e(Figure 1)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDMP, DEP, DBP, BBP, DEHP, DnOP leaching in controlled \u0026nbsp;samples \u0026nbsp; over two years\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e[Figures 3-6]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRate of DMP, DEP, DBP, BBP, DEHP, DnOP Leaching in controlled samples \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn a linear regression model\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e[Figures 7-12]\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe established method was validated following the ICH protocol Q2 (R1) (Conference \u003cem\u003eet al.\u003c/em\u003e, 1994). The validation process confirmed the method's specificity, precision and accuracy.\u003c/p\u003e \u003cp\u003eFrom the results, as the storage time prolonged, regardless of the type of extemporaneous preparation, the concentration of the phthalates increased (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e,\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e,\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The migration of the phthalates clearly is affected by the duration of the exposure between the PET bottle and the contents of the bottle (Jeddi \u003cem\u003eet al.\u003c/em\u003e, 2015). This observation was similar to the findings from other studies (Rastkari \u003cem\u003eet al.\u003c/em\u003e, 2017; Luo \u003cem\u003eet al.\u003c/em\u003e, 2020). A study by Rastkari \u003cem\u003eet al.\u003c/em\u003e, (2017) also reported the effect of storage time (2, 4 and 6 months) under different conditions. The migration of the phthalates was in the order DEHP\u0026thinsp;\u0026gt;\u0026thinsp;DEP\u0026thinsp;\u0026gt;\u0026thinsp;DBP and DEP\u0026thinsp;\u0026gt;\u0026thinsp;DEHP\u0026thinsp;\u0026gt;\u0026thinsp;DBP in HDPE and PET bottles, respectively. It was also realized the speed and amount of phthalate migration was much faster and larger in the initial storage period than the later period. This observation was similar to a study conducted by Luo \u003cem\u003eet al.\u003c/em\u003e, (2020).\u003c/p\u003e \u003cp\u003eIt has been established from literature that lower polarity enhances the release of phthalates from plastic bottles (Amiridou and Voutsa, 2011). In a post-hoc analyses, MMT and MSC random samples had significantly higher levels of DMP, DEP, DBP, BBP, DEHP, and DnOP levels than compared to MES and MPC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Similarly, MES had higher levels of DMP, DEP, DBP, BBP, DEHP, and DnOP levels than compared to MPC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). There were significant differences in the pairwise comparison in the levels of DMP, DEP, DBP, BBP, DEHP, and DnOP between MPC, MMT, MSC and MES in the control samples (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This observation can be attributed to the difference in the polarities of the extemporaneous preparations studied. MMT, which is likely the most non-polar extemporaneous preparation among the four studied had the highest level of phthalates, followed by MSC, then MES and finally MPC. MPC, which is reported to be more polar compared to the other extemporaneous preparations had the least concentration of phthalates recorded, confirming this theory. These results obtained are similar to the findings of previous studies carried out on edible oils by Luo \u003cem\u003eet al.\u003c/em\u003e, (2020).\u003c/p\u003e \u003cp\u003eFrom the results, there was a non-significant difference in the levels of DMP, DEHP and DnOP between MMT and MSC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This is because both MMT and MSC are relatively non-polar. The levels of phthalates reported in this study are higher than the phthalate levels recorded in water in Ghana and other parts of the world, all substantiating the evidence that lower polarity increases the migration of phthalates (Angnunavuri \u003cem\u003eet al.\u003c/em\u003e, 2022; Khishdost \u003cem\u003eet al.\u003c/em\u003e, 2023).\u003c/p\u003e \u003cp\u003eOur findings suggest MMT, which has the highest pH showed the highest leaching of phthalates with the highest recorded phthalate levels; implying a higher pH enhances the migration of phthalates. This assertion from our findings is congruent with research investigated by Rastkari \u003cem\u003eet al.\u003c/em\u003e, (2017). The research by Rastkari \u003cem\u003eet al.\u003c/em\u003e, (2017) supports the theory that a higher pH increases the rate of migration of phthalates. From the results of this study, it can be inferred that when polarity and acidity act in tandem, it appears that polarity has a stronger influence than acidity.\u003c/p\u003e \u003cp\u003eFrom the linear regression models, for DMP migration, the rate of migration was 13 \u0026micro;g/L, 13 \u0026micro;g/L, 9.31 \u0026micro;g/L and 7.6\u0026micro;g/L per week for MMT, MSC, MES and MPC respectively. A similar observation was made in DEP migration where the highest migration rate per week was recorded in MMT (12 \u0026micro;g/L), followed by MSC (11 \u0026micro;g/L), then MES (9.1 \u0026micro;g/L) and MPC (7.1 \u0026micro;g/L). DBP and BBP migration per week also followed the same trend with the highest rate of migration (12 \u0026micro;g/L) recorded in MMT and the least rate of migration (5.9 \u0026micro;g/L and 4.2 \u0026micro;g/L in DBP and BBP respectively) recorded in MPC. It could be seen that the lower the polarity of the extemporaneous preparation, the greater the rate constant and the greater the initial release rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The results show a larger release rate in MMT compared to the other products, confirming literature findings. Similar findings have been reported by research work on alkylphenols and phthalates in bottled water (Amiridou and Voutsa, 2011).\u003c/p\u003e \u003cp\u003eThe levels of phthalates recorded in this study is however significantly higher than the levels reported in bottled water globally and in Ghana (Zaater, Tahboub and Al Sayyed, 2014; Jeddi, 2018). This trend could be attributed to the difference in the polarities of the various extemporaneous preparations. The higher the polarity, the lower the phthalate migration (Amiridou and Voutsa, 2011; Luo \u003cem\u003eet al.\u003c/em\u003e, 2020).\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe kinetics of the migration of phthalates into extemporaneous preparations has been studied over a period of two (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) years. Phthalates levels increased as the storage time increased.\u003c/p\u003e \u003cp\u003eFor the first time, the migration kinetics of phthalates in extemporaneous preparation in Ghana has been studied. The rate of migration for DMP was 13 \u0026micro;g/L, 13 \u0026micro;g/L, 9.30 \u0026micro;g/L and 7.6 \u0026micro;g/L per week for MMT, MSC, MES and MPC respectively. DEP migration kinetics followed a similar pattern where the highest migration rate per week was recorded in MMT (12 \u0026micro;g/L), followed by MSC (11 \u0026micro;g/L), then MES (9.1 \u0026micro;g/L) and MPC (7.1 \u0026micro;g/L). DBP and BBP migration per also showed the highest rate of migration (12 \u0026micro;g/L) recorded in MMT and the least rate of migration (5.9 \u0026micro;g/L and 4.2 \u0026micro;g/L in DBP and BBP respectively) recorded in MPC. The highest migration rate per week for all the phthalates was observed in DEHP with the highest level being 19 \u0026micro;g/L in MMT, 16 \u0026micro;g/L in MSC, 12 \u0026micro;g/L in MES and 8.3 \u0026micro;g/L in MPC.\u003c/p\u003e \u003cp\u003eThe rate and concentration of phthalates from the plastic bottles were much faster and larger in the initial storage period than the later period. This implies that the longer the storage time of the product, the higher the rate of phthalate migration, resulting in elevated health risk.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was no funding\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and \u0026nbsp; consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll \u0026nbsp;authors declared that no competing interests exited\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdegunwa, A.O., Adegunwa, A.K. and Oyatoyinbo, R.M. 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(2014) \u0026lsquo;The consumption and recycling collection system of PET bottles: A case study of Beijing, China\u0026rsquo;, \u003cem\u003eWaste Management\u003c/em\u003e, 34(6), pp. 987\u0026ndash;998. Available at: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.wasman.2013.07.015\u003c/span\u003e\u003cspan address=\"10.1016/j.wasman.2013.07.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"extemporaneous preparations, phthalates, high pressure liquid chromatography, photodiode array, polyethylene terephthalate","lastPublishedDoi":"10.21203/rs.3.rs-4572478/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4572478/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe safety and quality of pharmaceutical products can be affected by chemical contaminants that migrate into them, depending on the type and composition of the packaging material. The migration of phthalates during manufacturing of plastics poses potential significant health concerns. This research aims to provide insight into factors driving phthalate migration and propose strategies for reducing its occurrence in extemporaneous preparations in Ashanti region, Ghana. Factors such as storage time and migration kinetics over two years were investigated to understand their impact on phthalate levels in Mist Senna Co (MSC), Mist Potassium Citrate (MPC), Mist Magnesium Trisilicate (MMT) and Mist Expectorant Sedative (MES). The samples were analysed using reverse phase \u0026ndash; High Pressure Liquid Chromatography (RP-HPLC) with Photodiode array (PDA) detection. A gradient elution using acetonitrile and 0.1% formic acid at a detection wavelength of 235 nm and a flow rate of 1 mL/min was employed. As the storage time increased, the levels of the phthalates increased. The migration of phthalates was influenced by the duration the polyethylene terephthalate (PET) bottle was in contact with the content of the bottle. The highest migration rate per week for all the phthalates was observed in diethylhexyl phthalate with the highest level being 19 \u0026micro;g/L in MMT, 16 \u0026micro;g/L in MSC, 12 \u0026micro;g/L in MES and 8.3 \u0026micro;g/L in MPC. The finding emphasizes the significance of implementing optimized storage time in reducing phthalate exposure in extemporaneous preparations in Ghana. This study could contribute to enhancing the safety and quality of pharmaceutical products in Ghana.\u003c/p\u003e","manuscriptTitle":"The Effect of Storage Time on the Levels of Phthalates in Extemporaneous Preparations in Ghana","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-25 21:53:18","doi":"10.21203/rs.3.rs-4572478/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7ce8ccaa-1e45-453d-8fb0-d7054a702233","owner":[],"postedDate":"June 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-22T12:54:07+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-25 21:53:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4572478","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4572478","identity":"rs-4572478","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}