Linearity of the standard curves in the *Artemia* culture medium and *Artemia* metanauplii (Table 2) was excellent, as indicated by the R2 values of 0.999 for BPA and t-OCT. The Relative Bias estimated as the percentage of ((calculated- nominal)/nominal]) was within the accepted range of ±10%. Intra-day precision was calculated by three analyses performed on the same day, while inter-day precision was calculated by performing six analyses over three days and estimated as % RSD, never exceeding 5.0% for all analytes. Our results indicate that the method is precise and repeatable with excellent recovery rates. The nominal concentrations of 1.0 μg/mL BPA and 1.0 μg/mL t-OCT in the culture medium were below the calculated LOD values (Table 3) and were, thus, excluded from the estimation of recovery results described above. The values for LOD and LOQ calculation were based on the response and the slope of the calibration curve.

**Table 3.** LOD, LOQ, and RT values for BPA and t-OCT.

The value of a LOD of 0.21 μg/mL and 0.17 μg/mL were estimated for BPA and t-OCT, respectively, in *Artemia* tissue corresponding to 10.5 ng/g dry weight and 8.6 ng/g dry weight, respectively, and of 0.63 μg/mL and 0.41 μg/mL for *Artemia* culture medium (Table 3). Although LOD values of 0.07 μg/kg were reported for BPA in mussels' tissue [80], estimations were made with σ taken as the standard deviation of three samples spiked at the experimental estimated LOQ, and S as the slope of the corresponding calibration curve. On the other hand, LOD values of 5.6 μg/mL and 6.3 μg/mL, were reported following liquid–liquid extraction of water samples [81]. The previously reported LOQ values in zooplankton amounted to 2 ng/g dry weight for BPA and 0.8 ng/g dry weight for t-OCT, and to 5 and 1 ng/L, respectively, for water samples, determined as a tenfold signal-to-noise ratio for a sample with a very low analyte content [27], employing different methods for the biological and water samples. Variable results were obtained from the computation of LOD and LOQ values, depending on the statistical approach used. The residual standard deviation or error of intercept of calibration line was recommended instead of the mean blank signal value, as it is considered that it provides a more accurate estimate [82].

Since the LOD and LOQ values are calculated based on the calibration curve, they greatly depend on its range and lower values are expected when a range of lower concentrations are used. However, the range of the calibration curve should extend over the range of expected analytes' concentrations in the samples [83]. In the present work, the expected concentrations of Bisphenol-A and t-octylphenol were within the range of the LC50 values. These values were acquired from the toxicity experiments that allowed the estimation of the amount that could be administered to *Artemia* metanauplii without leading to high mortality rates.

Typical HPLC chromatograms of *Artemia* and culture medium samples spiked with BPA, t-OCT, and n-OCT are presented in Figure 1. The peaks of the analytes BPA, t-OCT, and of the internal standard n-OCT, showed with a retention time (Rt) of 15.200 ± 0.12, 21.876 ± 0.11 26.241 ± 0.1 min, respectively. The peak appearing from 0 to 6 min, in all samples, possibly corresponds to the solvents, while the peak appearing at approximately 7.0 ± 0.5 min only in the medium samples possibly corresponds to medium constituents. Similar peaks also appear in the blank samples without, however, interfering with analysis (Figure S1).

#### *3.3. Administration of Pollutants*

BPA was administered at a concentration of 10 μg/mL, while t-OCT was administered at 0.5 μg/mL, since their LOD values amounted to15.6 and 0.73 μg/mL, respectively (Figure S2). The simultaneous administration of the two pollutants at LC50 levels was not applicable, since it resulted in higher mortality values, possible due to synergistic effects. Hence, each pollutant, namely BPA or t-OCT, was administered separately. Acute toxicity and inhibition of growth of the early developmental stages of *Artemia* nauplii (Instar I-II) have been reported after a 24 h exposure to BPA with an LC50 of 44.8 mg/L [43] for *Artemia franciscana*. The LC50 values for *Artemia sinica* nauplii amounted to 47.5 mg/L for BPA and to 2.27 mg/L for t-OCT [84]. On the other hand, the LC50 values of 70.1 μg/L and 7.7 μg/L were reported for adult (15 days old) *Artemia sinica* individuals exposed to BPA and n-hexylphenol [45], illustrating that the advanced developmental stages present greater sensitivity to these pollutants. The increased sensitivity of subsequent developmental stages

to pollutants was attributed to the fact that the organisms appear more ravenous compared to the earlier developmental stages, during which the animals depend on reserved yolk for their energy requirements [85]. In addition to the importance of the developmental stage employed, the origin of cysts and abiotic parameters regarding, among others, the pH, hatching temperature, photoperiod duration, and saltwater treatments are also of great importance [42]. Considering that bacteria are capable of BPA biotransformation [18], the use of axenic cultures in the autoclaved medium were used in the present study. Representative chromatograms of the pollutants following administration of 10 μg/mL BPA or 0.5 μg/mL t-OCT to *Artemia*, that are below their estimated LC50 values, are presented in Figure 2 for *Artemia*, and in Figure 3 for its culture medium.

**Figure 2.** HPLC chromatograms of *Artemia* tissue following administration of (**A**) 10 μg/mL BPA; and (**B**) 0.5 μg/mL t-OCT.

The peaks appearing at 12.5 ± 0.18, 13.1 ± 0.08 and 13.8 ± 0.2 and apparent following administration of either pollutant (Figure 2A,B) might correspond to products produced during their biotransformation by *Artemia*. Similar peaks were observed following BPA microbial biotransformation corresponding to 4-Hydroxyacetophenone, 4-Hydroxybenzoic acid, and hydroquinone [18]. Moreover, biodegradation of tert-octylphenol by the fungus *Thielavia* sp. HJ22 has been reported to result in the formation of 4-hydroxybenzoic acid hydroquinone [86]. Although UV spectra presented a good match with standard compounds, the identity of these peaks was not confirmed by MS in the present work. The peak that appears at 19.7 (Figures 2B and 3B) is evident only following administration of t-OCT and might correspond to a photodegradation product, since similar peaks were observed during the photodegradation experiment (Figure S3D). Photodegradation via photolysis has been confirmed for BPA [25,27] and for t-OCT [87], and the products of solar transformation presented higher toxicity than the parent compound [88]. The feeding experiments were conducted under normal light and, hence, photodegradation losses were estimated to examine whether any pollutant concentration decline is due to photolysis. In the photolysis experiments, the initial BPA and t-OCT concentration were calculated at 49.15 μg/mL and 9.81 μg/mL, respectively, and their concentration following a 24 h incubation amounted to 44.63 μg/mL and 7.87 μg/mL, while after 48 h BPA amounted

to 37.94 and t-OCT to only 1.7 μg/mL. Hence, the reduction in the analyte concentration induced by photodegradation was 9.2% for BPA and 19.7% for t-OCT at 24 h (Table 4).

**Figure 3.** HPLC chromatograms of culture medium following the administration of (**A**) 10 μg/mL BPA; and (**B**) 0.5 μg/mL t-OCT.

**Table 4.** Bisphenol A and t-octylphenol levels in administration experiments.

These values are in accordance with those reported for bisphenol A degradation [89] but higher than the previously reported absence of t-OCT degradation under one hour of direct solar irradiation [90]. Although part of the pollutants is degraded via photolysis, this does not compensate for the observed reduction in the total levels of both pollutants (Table 4). The amounts detected in the processed medium samples were 66.8 and 3.0 μg and the total amounts in a 1000 mL culture corresponded for BPA to 66.8% and for t-OCT to 63.8% of the initially added amounts. In addition, the amounts detected in *Artemia* tissue amounted to 4.1% and 0.7% of the initially added amounts for BPA and t-OCT, respectively, and a reduction of up to approximately 32% in total BPA and of 35% in total t-OCT was observed in our experiments. The accumulation of BPA in zooplankton has been confirmed in an eco-system bioreactor during incubation of phytoplankton (*Nannochloropsis* sp.) and zooplankton (*Artemia* sp. or *Brachinous* sp.) in the presence of the pollutant [91].

The occurrence of high levels of BPA and alkylphenols in the marine environment has been documented in aquaculture facilities which are in the vicinity of industrial or urban activity [92]. The BPA values amounted to 37 ng/L in mussels cultured in Thailand [92], to 1.5 ng/g d. w. in fish cultured in Taiwan [93], to 4.2 ng/g in the muscle

of fish cultured in Malaysia [94], or 272 ng/g d. w. in the North Atlantic Ocean [24]. The relevant t- octylphenol concentration was 16 ng/ g d. w. in mussels cultured in Malaysia [94], while the occurrence of the alkylphenol nonylphenol has been documented in the sediments of the Northern Aegean, indicating a risk for the organisms that live in it [95]. The cultured fish fry fed on *Artemia* have a weight of 15 mg and consume about 300 nauplii per day [96], corresponding to a wet weight of 5 mg, indicating that these pollutants, if present in *Artemia*, may accumulate and possibly pose a health risk to the fish larvae. Recently the accumulation of bisphenols in farmed seabream muscle following exposure to microplastics, amounting to 0.3 μg/g muscle, was reported and the authors consider aquaculture equipment as a source of such microplastics [97]. Considering the TDI for BPA amounting to 0.04 ng/kg b. w. daily and the suggested TDI for t-OCT for men of 0.067 ng/kg b. w. daily (Table 1), it becomes apparent that the exposure of cultured organisms to the pollutants might also pose a possible hazard to human health. The use of marine phytoplanktons and zooplanktons has been suggested for the recovery of BPA from seawater [91]. The uncompensated decline in both BPA and t-OCT levels observed in the present study indicates that, although these compounds might accumulate in *Artemia*, their biotransformation is possible. Since the toxicity of the possible biotransformation products has not been investigated and *Artemia* is used as a live feed for cultured marine larvae, the monitoring of its pollutants levels is of extreme importance.

#### **4. Conclusions**

The developed analytical method provides a simple approach for the simultaneous detection and quantification of bisphenol A and t-octylphenol in the live fish feed *Artemia* and in its culture medium. The presented extraction protocol and the use of 4-n-octylphenol as the internal standard provided a detection limit of 0.21 for BPA and 0.17 ng/μL for t-OCT. The method was applied in the analysis following administration of the pollutants to *Artemia* metanauplii at levels close to their corresponding LC50. The administration experiments revealed that *Artemia* is capable to uptake bisphenol and t-octylphenol from the culture medium. Experimental efforts to reduce the pollutant load of *Artemia* are currently under investigation. Further experiments are needed to evaluate the transfer of the pollutants to the fish larvae that feed on *Artemia*.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/mps5030038/s1, Figure S1. HPLC chromatograms of blank *Artemia* tissue (A) and culture medium (B) samples; Figure S2. Linear Regression analysis for the determination of LC50 values of BPA and t-OCT; Figure S3. Photodegradation of BPA and t-OCT. HPLC chromatograms of medium with (A)BPA at 0 h; (B) BPA at 48 h; (C) t-OCT at 0 h; (D) t-OCT at 48 h.

**Author Contributions:** M.T. and V.F.S. conceived and designed the experiments; M.T., writing original draft preparation; V.F.S., review and editing; D.G., formal analysis in BPA experiments; K.D., formal analysis in the t-OCT experiments. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** No additional funding was received in support of the present research work. The authors wish to thank INVE HELLAS S.A., for the provision of *Artemia* cysts and Sorgeloos for the provision of yeast.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Allergens and Other Harmful Substances in Hydroalcoholic Gels: Compliance with Current Regulation**

**Ana Castiñeira-Landeira 1, Lua Vazquez 1 , Thierry Dagnac 2 , Maria Celeiro 1,\* and María Llompart 1,\***

**Abstract:** Hydroalcoholic gels or hand sanitisers have become essential products to prevent and mitigate the transmission of COVID-19. Depending on their use, they can be classified as cosmetics (cleaning the skin) or biocides (with antimicrobial effects). The aim of this work was to determine sixty personal care products frequently found in cosmetic formulations, including fragrance allergens, synthetic musks, preservatives and plasticisers, in hydroalcoholic gels and evaluate their compliance with the current regulation. A simple and fast analytical methodology based on solid-phase microextraction followed by gas chromatography–tandem mass spectrometry (SPME-GC-MS/MS) was validated and applied to 67 real samples. Among the 60 target compounds, 47 of them were found in the analysed hand sanitisers, highlighting the high number of fragrance allergens (up to 23) at concentrations of up to 32,458 μg g−1. Most of the samples did not comply with the labelling requirements of the EU Regulation No 1223/2009, and some of them even contained compounds banned in cosmetic products such as plasticisers. Method sustainability was also evaluated using the metric tool AGREEPrep, demonstrating its greenness.

**Keywords:** hydroalcoholic gels; personal care products; solid phase microextraction; gas chromatography; tandem mass spectrometry

#### **1. Introduction**

Due to the COVID-19 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) infectious disease that was declared a pandemic by the World Health Organization (WHO) in March 2020, promoting hand hygiene by means of alcohol-based hand rub (ABHR) has been considered the primary strategy to mitigate its transmission and infection [1]. In this sense, the production and consumption of hand sanitisers drastically increased worldwide (by up to 561% in Italy, one of the most affected European countries) [2,3]. These products were massively placed in public areas such as shopping centres, schools, banks, supermarkets for public use. Hand sanitisers can be defined as 'borderline products' since their classification as a biocide or as a cosmetic product is not clear; this depends on the presence of an active substance and the product's main purpose [4]. Those designed to disinfect hands, eliminating the microorganisms and their possible transmission, are considered as biocides and are subject to Biocidal Products Regulation (EU) No 528/2012 [5]. On the other hand, if their main purpose is cleaning or cleansing the skin, notably in the absence of water rinsing, then they are considered cosmetics, and probably do not protect through biocidal action. In this case, they must comply with the Cosmetics Regulation (EU) No 1223/2009 [6].

To homogenise their formulation and fabrication, ensuring their antimicrobial properties in the context of the COVID-19 pandemic, the WHO published a protocol, and two

**Citation:** Castiñeira-Landeira, A.; Vazquez, L.; Dagnac, T.; Celeiro, M.; Llompart, M. Allergens and Other Harmful Substances in Hydroalcoholic Gels: Compliance with Current Regulation. *Methods Protoc.* **2023**, *6*, 95. https://doi.org/ 10.3390/mps6050095

Academic Editors: Victoria Samanidou, Verónica Pino and Natasa Kalogiouri

Received: 31 July 2023 Revised: 28 September 2023 Accepted: 30 September 2023 Published: 7 October 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

formulations were established, containing: (i) ethanol (96%), hydrogen peroxide (3%) and glycerol (98%) and (ii) isopropyl alcohol (99.8%), hydrogen peroxide (3%) and glycerol (98%) [7]. This protocol also strongly recommended that no ingredients other than those specified above be added to the formulation, whilst the addition of fragrances was not recommended because of the risk of allergic reactions. Although it is well known that fragrances are the main responsible of allergic contact dermatitis (ACD), they are usually added to cosmetics with the intention of providing a pleasant scent. Other compounds such as preservatives and plasticisers can also be present in cosmetics and daily care products. Preservatives are added to protect products and consumers against microbial growth, whereas plasticisers (mainly phthalates and adipates) are added to fix and dissolve fragrances, although some can be transferred from the plastic containers to the product and then to consumers, causing health problems, since many of them are catalogued as endocrine disruptors [8–11].

Different studies have demonstrated that SARS-CoV-2 was efficiently inactivated by WHO-recommended formulations, reducing the viral load to a background level within 30 s, whereas formulations containing other additives present a lower effectivity against the virus [12–14].

Therefore, the main objective of this work was the validation and application of a sensitive analytical method to simultaneously determine fragrances (allergens and synthetic musks) and other potentially harmful substances, such as plasticisers and preservatives, including 60 compounds, in a broad range of hand sanitiser samples. In general, the analysis of cosmetics and personal care products is a challenge for analysts, as ingredients may be present at % concentrations, or at trace levels in the case of impurities. A suitable option for the multianalyte analysis of these matrices is the use of solid-phase microextraction (SPME), a simple, fast and environmentally friendly extraction technique, followed by gas chromatography–tandem mass spectrometry (GC-MS/MS). This combination has been reported as a reliable and sensitive tool to analyse a high number of personal care products (PCPs) in different cosmetic products, including hydroalcoholic gels [15–18]. The method was employed to conduct a survey of hand sanitisers for personal use (personal use) and placed in local shops or public areas (public use) to verify compliance with applicable legislation. A labelling study was also carried out to check whether the information for consumers was correct and complete. To the best of our knowledge, there are no analytical studies dedicated to researching this type of samples used daily by a large part of the world's population since the COVID-19 pandemic.

#### **2. Materials and Methods**

#### *2.1. Chemicals, Reagents and Materials*

The 60 target compounds, their CAS numbers, molecular mass, European legislation restrictions, retention times and MS/MS transitions are shown in Table S1.

Ultrapure water and methanol, both MS grade, were purchased from Scharlau (Barcelona, Spain) and acetone from Sigma Aldrich Chemie GmbH (Steinheim, Germany). Moreover, 50/30 μm of commercial divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/ PDMS) fibre housed in a manual SPME holder was obtained from Supelco (Bellefonte, PA, USA). Prior to the first use, the fibre was conditioned as recommended by the manufacturer by inserting it into the GC injector with carrier gas flow at 270 ◦C for 30 min.

The target compounds were selected, including 23 fragrance allergens, 11 synthetic musks, 16 plasticisers and 10 preservatives. Individual stock solutions were prepared in methanol or ethyl acetate, followed by further dilutions in acetone. Working solutions were prepared weekly. All of them were stored in amber glass vials and protected from light at −20 ◦C.

#### *2.2. Samples and Sampling*

Sixty-seven hydroalcoholic gel samples were collected from different establishments in Galicia (Northwest Spain), including banks, restaurants, pharmacies, universities, local markets in 15 mL amber vials. The samples were kept at room temperature and protected from light until analysis. Sampling details about the establishments where they were collected, and the composition indicated in the label, are summarised in Table S2.

#### *2.3. Sample Preparation Procedure: SPME*

Herein, 10 mg of hydroalcoholic gel and 10 mL of ultrapure water (1:1000, *w*/*v* dilution) were placed in a 20 mL glass vial, which was sealed with an aluminium cap furnished with PTFE-faced septa. The vial with the sample was immersed in a water bath at 100 ◦C. First, the sample was conditioned during 5 min and then the DVB/CAR/PDMS fibre was exposed for 20 min to the headspace over the sample (HS-SPME mode). Agitation under magnetic stirring was employed during the extraction procedure. After the extraction time, the SPME fibre was desorbed at 270 ◦C for 5 min in the GC injection port and the GC-MS/MS analysis was carried out.

In some cases, the high concentration of some of the analytes in the samples made it necessary to perform a further dilution of the hydroalcoholic gel–water mixture in ultrapure water, followed by the corresponding SPME GC-MS/MS analysis.

Since one of the studied families are plasticisers, and to avoid contamination and overestimation in the results, the plastic material was replaced with glass and metallic. All material was also maintained at 230 ◦C for 12 h before use. In addition, fibre blanks and procedure blanks using 10 mL of ultrapure water were carried out with the aim of avoiding false-positive results.

#### *2.4. GC-MS/MS Analysis*

The GC-MS/MS analysis was performed employing a Thermo Scientific Trace 1310 gas chromatograph coupled to a triple-quadrupole mass spectrometer (TSQ 8000) from Thermo Scientific (San Jose, CA, USA).

Separation was performed on a Zebron ZB-Semivolatiles (30 m × 0.25 mm i.d. × 0.25 μm film thickness) obtained from Phenomenex (Torrance, CA, USA). Helium (purity 99.999%) was used as carrier gas at a constant column flow of 1 mL min−1. The GC oven temperature was programmed from 60 ◦C (held 1 min), to 100 ◦C at 8 ◦C min−1, to 150 ◦C at 20 ◦C min−1, to 200 ◦C at 25 ◦C min−1 (held 5 min), to 220 ◦C at 8 ◦C min−1, and finally, to 290 ◦C at 30 ◦C min−1 (held 7 min). The total run time was 30 min. Split/splitless mode was used for injection (200 kPa, held 1.2 min) and the injector temperature was kept at 270 ◦C.

The mass spectra detector (MSD) was operated in the electron impact (EI) ionisation positive mode (+70 eV). The temperatures of the transfer line and the ion source were established at 290 and 350 ◦C, respectively. The filament was set at 25 μA and the electron multiplier was set at a nominal value of 1800 V. Two or three transitions were monitored per compound working in the selected reaction monitoring (SRM) acquisition mode, as can be seen in Table S1. The system was operated by Xcalibur 2.2, and Trace FinderTM 3.2 software.

#### **3. Results and Discussion**

#### *3.1. SPME-GC-MS/MS—Method Validation*

The SPME experimental parameters, including fibre coating, extraction mode, temperature and dilution factor were previously optimised [17], and the most suitable conditions are described in Section 2.

The miniaturised method, employing only 10 mg of sample, was validated in terms of linearity, repeatability, accuracy and reproducibility. Instrumental detection limits (IDLs) and limits of detection (LODs) were also calculated. The results are summarised in Table S3. External calibration was assessed by preparing the standards in ultrapure water and following the procedure detailed in Section 2.3 covering a concentration range between 0.02 and 5 μg L−1 (see specific ranges for each target analyte in Table S3). The method showed coefficients of determination (R2) higher than 0.9901, demonstrating a direct proportional relationship between the amount of the target compound with the corresponding chromatographic response (area counts).

Precision was evaluated within a day (*n* = 4) and among several days (*n* = 6) for all the calibration levels. Table S3 shows relative standard deviation (RSD) values for 1 μg L−1. As can be seen, they were lower than 13% and 16% for repeatability and reproducibility, respectively, in most cases. Accuracy was verified using an hydroalcoholic gel sample free of the target compounds which was fortified at two levels (0.2 μg g−1 and 2 μg g−1). Mean recoveries between 76 and 117% and precision (RSD%) < 10% were obtained, as can be seen in Figure S1.

IDLs were calculated as the compound concentration giving a signal-to-noise ratio of three (S/N = 3), employing ultrapure water standards containing low concentrations of the target analytes. LODs were calculated employing a real sample spiked with the target compounds. For the plasticisers that were detected in the procedure blanks (DEP, DBP, DEHP, DPhP, and DEHA), IDLs and LODs were calculated as the mean concentration corresponding to the signal of the blanks plus three times its standard deviation. Results are summarised in Table S3 and they were at the low ng g−1 level for all compounds. In this case, it should be kept in mind that the gel samples are diluted by a factor of 1/1000, which explains the difference between IDL and LOD values. If higher sensitivity were required, a low dilution factor could be applied.

#### *3.2. Greenness Assessment*

In recent years, the development of extraction methodologies fulfilling green analytical chemistry (GAC) and green sample preparation (GSP) principles is increasing. These procedures include the use of safe solvents, reagents and materials minimising the experimental steps and reducing waste generation and energy consumption, allowing high sample throughput. In 2022, a metric tool, AGREEprep [19] was proposed for assessing the greenness of the sample preparation stage of an analytical procedure. The metric is based on ten principles of GSP, assigning weights at each criteria, depending on its significance in the analytical method. These weights range from 1, indicating low importance, to 5, representing high importance. Once the ten principles are evaluated, the tool is recalculated to the 0–1 scale to reflect the sample preparation greenness. The colour of the inner circle also provides the overall sample preparation greenness performance.

The sustainability of the SPME-GC-MS/MS method for extracting 60 personal care products from hydroalcoholic gels was calculated and results are depicted in Figure 1.

**Figure 1.** Evaluation of the degree of greenness. Pictogram obtained for SPME-GC-MS/MS under optimised conditions.

As can be seen, a value of 0.80 as well as a green label were obtained showing the greenness of the proposed SPME method. Considering each criteria, the developed method procedure is in-line/in situ since the SPME technique enables the integration of sample preparation and analysis (criteria 1); no toxic materials are used (criteria 2); the employed SPME fibre can be used several times (criteria 3); no waste is generated because the samples are hydroalcoholic gels (criteria 4); the sample amount is 0.01 g (criteria 5); the duration of the sample preparation stage is around 20 min; three samples per hour (criteria 6); the procedure consists of two steps, extraction and desorption, and it is a fully automated system (criteria 7); the energy consumption is more than 183 Wh due to the heating magnetic stirrer (criteria 8); GC-MS/MS is used as analytical instrument (criteria 9); and, finally, no hazards are associated with the procedure (criteria 10). The weights of each criteria were, in general, not modified (default weights given by AGREEprep).

#### *3.3. Analysis of Real Samples*

The SPME-GC-MS/MS method was applied to analyse the target compounds in 67 hydroalcoholic gels, collected in different public places (see Section 2.2), in which 47 of the 60 target compounds were found (see Figure 2).

Most of the analysed samples do not comply with the WHO recommendations due to the presence of the target compounds (fragrances allergens, synthetic musks, plasticisers and preservatives) in the formulations. In this context, if the sample should be considered a cosmetic product, it must comply the European Cosmetics Regulation (EC) No 1223/2009 [6] in order to ensure product safety. This study highlights that 61% of the hydroalcoholic gels contain at least one compound which is prohibited by the Cosmetic Products Regulation, as can be seen in Figure 3.

**Figure 3.** Percentage of banned compounds in the analysed samples and distribution by families: (**a**) phthalates and (**b**) parabens.

A description of the compliance with this regulation is presented below for each studied family. Table 1 summarises the concentration range, mean and median of the target compounds in the analysed samples, and the specific quantification results for the studied compounds in each sample are summarised in Table 2.

**Table 2.** Individual concentration (μg g−1) of the fragrance allergens detected in 67 hydroalcoholic gel samples and total

 content.

**Table 2.** *Cont.*

**Table 2.** *Cont.*

#### 3.3.1. Fragrance Allergens

As can be seen in Tables 1 and 2, all studied fragrance allergens were detected in the real analysed samples. Limonene, benzyl alcohol and benzyl benzoate were found in most samples in 53, 52 and 41 out of the 67 hand sanitisers, respectively. Other fragrances such as linalool (37/67), citronellol (35/67), hexyl cinnamal (34/67), methyl-2-octynoate (32/67), citral and geraniol (31/67) were found in 50% of the samples. The presence of this family of cosmetic ingredients is remarkable since even the least frequently detected fragrances such as eugenol, anise alcohol and methyl eugenol, were found in 20% of the samples (see Figure 2).

Regarding the number of compounds per sample, 7 of the analysed samples contained around 17–19 of the 23 target fragrance allergens (G36, G43, G50, G51, G53, G65 and G66). In contrast, only two were free of these substances, samples G12 and G47, and only one sample (G16) contained one fragrance allergen (citronellol).

It should be underlined that the high levels found for some allergens in some samples reach concentrations of parts per hundred (up to 3%) as amyl cinnamal (32,458 μg g−1 in sample G51) and farnesol (28,737 μg g−1 in sample G24).

#### Regulatory Issues

According to EC Cosmetic Regulation No. 1223/2009, fragrance allergens can cause allergic skin reactions and other adverse effects especially at high concentrations. Among the 23 studied fragrance allergens, 20 (limonene, benzyl alcohol, linalool, methyl-2-octynoate, citronellol, citral, geraniol, cinnamaldehyde, anise alcohol, cinnamyl alcohol, eugenol, isoeugenol, *α*-isomethylionone, amyl cinnamal, amylcinnamyl alcohol, farnesol, hexyl cinnamal, benzyl salicylate and benzyl cinnamate) should appear on the label of cosmetic products when the concentration exceeds 0.001% (*w*/*w*, 10 μg g−1) in leave-on products, as is the case for hand sanitisers [6]. Among the 67 analysed samples, 33 (49%) exceed this limit for several compounds; in addition, most of they are under-labelled since the corresponding fragrances are not included in the product label. In contrast, some hydroalcoholic gels were over-labelled, as they claimed to contain more allergens than they did (over-labelling is not included in the regulation). For example, sample G24 indicates the presence of benzyl benzoate, which is over-labelled since it does not surpass its legal limit (0.001%, 10 μg g−1). In addition, two out of the studied fragrances present a maximum permitted concentration in final products as methyl-2-octynoate with 0.01% and isoeugenol with 0.02%. The sample G36 is the only one which surpassed the isoeugenol limit with a concentration of 739 μg g−1.

Some fragrance allergens are prohibited, such as lilial, which has been banned since March 2022 because it damages fertility and it is suspected of damaging the unborn child, in addition to causing skin irritation [20]. Lilial was present in around the 37% (Figure 2) of the analysed hydroalcoholic gels (25/67) at concentrations below 0.001% (*w*/*w*) (10 μg g−1) in 22 samples. Three samples (G10, G24 and G29) contained between 7 and 15 μg g−1 of lilial, highlighting one sample which presented a very high value (517 μg g−1, G20). However, it should be pointed out that some hydroalcoholic gel samples were taken after the lilial legislation had changed.

#### 3.3.2. Synthetic Musks

Regarding the synthetic musks, as can be seen in Table 3, 7 of the 11 targets were found in the analysed hydroalcoholic gels, highlighting the presence of galaxolide in 63% of the samples (42/67), followed by cashmeran in 36% (24/67).

**Table 3.** Individual concentration (μg g−1) of the synthetic musks detected in 67 hydroalcoholic gel samples and total content.

#### **Table 3.** *Cont.*

Only 20% of the samples were free of these compounds (16/67). The highest number of synthetic musks (galaxolide, cashmeran, phantolide and tonalide) were detected in sample G33. Although several of these PCPs such as galaxolide were found at concentrations above 10 μg g−1 (e.g., 56 μg g−1 in G33), in most cases, these compounds were presented at concentrations below 1 μg g−1. An exception is the musk xylene which was detected in two samples at extraordinarily high concentrations, 2356 μg g−1 in G65 and 1840 μg g−1 in G66; this being the synthetic musk with the highest concentrations followed by cashmeran at 74 μg g−1 in G67.

#### Regulatory Issues

The regulation of cosmetic products states that the synthetic musks ambrette, tibetene and moskene are prohibited. These chemicals were not detected in any of the analysed hydroalcoholic gels. Nevertheless, 51 samples contained at least one target synthetic musk, so the terms "parfum" or "aroma" must appear on their label. However, only 17 samples (see Table S2) were labelled with the word 'parfum', which indicates that 34 of 51 are under-labelled, although in general, the concentrations for this family of fragrances were quite low as it was commented. Some substances such as tonalide, phantolide and musk xylene must be mentioned when their concentration surpass 1%, 2% and 0.03%, respectively. Musk xylene was detected in two samples at very high concentrations, surpassing the regulation's limit, and its presence was not indicated in the label of any sample. In the case of phantolide and tonalide, they were detected in four and six samples, respectively, but none of them surpassed the regulation's limit, so they did not have to be indicated, as it is the case. As was mentioned, galaxolide was the most frequently found (63% of the

analysed samples) and is under assessment as a persistent, bioaccumulative and endocrine disruptor [21].

3.3.3. Preservatives

All target preservatives were found in the analysed samples, as can be seen in Table 4.

**Table 4.** Individual concentration (μg g−1) of the preservatives detected in 67 hydroalcoholic gel samples and total content.

#### **Table 4.** *Cont.*

The most frequently found were EtP in half of the samples (52%) and PhEtOH in 37% (25/67). The antioxidant BHT and the parabens MeP and iBuP were found in more than 20% of the samples and TCS and iBuP were detected in more than 10 samples.

The highest number of preservatives found in the same sample was six in G29, G51 and G53. Nevertheless, more than the 56% of the samples contained between 1 (25/67) and 2 (13/67) of the target preservatives. In six samples, none of the target preservatives were present (G14, G26, G40, G47, G60 and G64).

In most cases, the concentrations were at the low μg g−1 (Table 4), excluding PhEtOH which was found at very high concentrations of up to 13,735 μg g−1. The median concentration value of this substance in the analysed samples was 20 μg g−1, which is the highest value with a noticeable difference with the rest of the preservatives. Regarding the detected parabens, PrP was presented at concentrations of up to 150 μg g−1, EtP, iBuP and BzP up to 60 μg g−1 and MeP up to 23 μg g−1. BHT was detected at concentrations of up to 170 μg g−1 and TCS and BHA only appear at very low concentrations below 1 μg g−1.

#### Regulatory Issues

The Annex V of the EC Regulation No 1223/2009 comprised the preservatives allowed in cosmetic products, among which some of the target substances are included (PhEtOH, MeP, EtP and PrP).

The established maximum concentration in ready-for-use preparation for PhEtOH, a substance which is harmful if ingested, causing serious eye damage and may cause respiratory irritation, is 1.0% (10,000 μg g−1). However, samples G49 and G22 surpassed this limit since they contained 1.4 and 1.0% (*w*/*w*) of the sample, respectively. The labels of these hydroalcoholic gels in addition to G35, G37 and G44 show that this compound is present at 2.1% (*w*/*w*). As described in Table S2, these samples comply with UNE-EN standards and, although all samples have the same labelling, the brand name is different, so they were analysed individually. The appearance of this compound in the product labelling in such high concentrations is due to these hydroalcoholic gels being considered biocides and that ECHA has endorsed the approval of PhEtOH as an active substance in type 1 biocidal product, which are those intended for human hygiene [5,22]. Nevertheless, they contained other substances such as fragrances, so they should be considered cosme-tics, although some of them would not comply with this regulation.

As regard the parabens EtP and MeP, PrP were detected at concentrations lower than the permitted limits, 0.4% (EtP and MeP) and 0.14% (PrP). The prohibited parabens in cosmetic products, as iPrP, iBuP and BzP, were found in around 9%, 21% and 6%, respectively, of the analysed hand sanitisers at concentrations of up to 60 μg g−1 (iBuP in G4 and BzP in G56).

The antioxidant BHT, which has been very recently (July 2023) included in the Annex III of Regulation EC 1223/2009—substances in cosmetics products must not contain except subject to the restrictions laid down—is regulated for leave-on and rinse-off products at a maximum concentration of 8000 μg g−1, but none of the hydroalcoholic gels surpassed this value since the highest concentration was 170 μg g−1 in G24. In contrast, the triclosan is regulated but it does not have a limit for this type of cosmetic products.

#### 3.3.4. Plasticisers

Considering plasticisers, four phthalates and three adipates were present in the analysed hydroalcoholic gels: DMP (8/67), DEP (24/67), DPP (3/67), DEHP (15/67) and DMA (7/67), DEA (10/67), DEHA (2/67), as can be seen in Table 5.

**Table 5.** Individual concentration (μg g−1) of plasticisers detected in 67 hydroalcoholic gel samples and total content.

**Table 5.** *Cont.*

Regarding the number of compounds, in most samples, two or three of the target compounds were found. One sample presented five of the compounds (G51) followed by the sample G39 with four compounds (Table 5). Almost half of the samples (31) were free of the target plasticisers.

In general, the concentrations were below 2 μg g−1 but it should be underlined that there is the high concentration of DEP in some samples with concentrations between 1800 and 8240 μg g−1 (0.2–1%). DMP reached a concentration of 108 μg g−1 in sample G28, although this value is much lower than the previous values indicated for DEP.

#### Regulatory Issues

Some of the analysed plasticisers are banned by the EC Regulation No 1223/2009. Among the detected ones, DEHP and DPP are prohibited in cosmetics (see Figure 3), and they were detected in 3 and 15 samples, respectively. Both substances are toxic for reproduction and DEHP is also considered an endocrine disruptor. The presence of these phthalates may be due to migration from the plastic container to the hydroalcoholic gel. Previous studies have demonstrated the transfer of certain substances from the applicators to cosmetic products [23]. Other prohibited substances such as DBP, DMEP, DIPP, BBP, DIBP, DIHP, DPhP, DnOP and DCHP were not found in any of the analysed samples. On the other hand, it is important to note that DEP, the plasticiser found in the highest concentrations (up to 8238 μg g−1), is under evaluation as an endocrine disruptor, even though it is not legislated in cosmetic products.

#### **4. Conclusions**

Sixty-seven hydroalcoholic gels collected in different establishments were analysed by SPME-GC-MS/MS, a validated methodology which allows a complete analysis of the hydroalcoholic gel samples. Sixty personal care products, including fragrance allergens, synthetic musks, preservatives and plasticisers were targeted. The results revealed the presence of 48 out of these 60 target compounds in the analysed hydroalcoholic gels. Only one sample complied with the WHO recommendations for hand sanitiser formulations. The highest concentrations were observed for fragrance allergens and many of the samples did not comply with the legislation for these substances as, despite not being labelled, they contained some above the 0.001% limit in leave-on products. Furthermore, some prohibited compounds (iPrP, iBuP, BzP, DEHP and DPP) were detected in some cases. In addition, some hydroalcoholic gels are considered biocidal, although after the analysis of their composition, they should be considered cosmetic products. In this context, PhEtOH sometimes exceeds the limits set for cosmetics, while it is allowed for biocides for human hygienic use. Therefore, results demonstrated that greater control over the formulations of these frequently used cosmetic products is necessary to ensure consumer safety without causing undesirable side effects.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/mps6050095/s1, Table S1: Target compounds. CAS number, molecular mass, retention time, MS/MS transitions and restrictions for leave-on cosmetics (EC 1223/2009 regulation) [6]; Table S2: Information for the analysed samples including the composition indicated on the label; Figure S1: Comparison of the recoveries obtained for fortified hydroalcoholic gel samples free of target compounds (except DEP) at two levels: 0.2 μg g−1 and 2 μg g−1. Table S3: SPME-GC-MS/MS performance. Linearity, precision, recoveries, instrumental detection limits (IDLs) and limits of detection (LODs).

**Author Contributions:** Conceptualisation, M.L.; validation, A.C.-L. and L.V.; formal analysis, A.C.-L. and L.V.; investigation, A.C.-L. and L.V.; resources, M.L. and T.D.; data curation, A.C.-L. and L.V.; writing—original draft preparation, A.C.-L. and M.C.; writing—review and editing, M.C. and M.L.; supervision, M.L.; project administration, M.L.; funding acquisition, M.L. and T.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the projects ED431 2020/06, IN607B 2022/15 (Consolidated Research Groups Program, Xunta de Galicia). This study was based upon work from the Sample Preparation Study Group and Network, supported by the Division of Analytical Chemistry of the European Chemical Society. The authors are members of the National Network for Sustainability in Sample Preparation, RED2022-134079-T (Ministry of Science, Innovation and Universities, Spain). These programmes are co-funded by FEDER (EU).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data are available in the manuscript and Supplementary Material.

**Acknowledgments:** A.C.-L. acknowledges the Xunta de Galicia predoctoral contract (ED481A and IN606A). M.C. and M.L. acknowledge the IUPAC project 2021-015-2-500: Greenness of official standard sample preparation methods.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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## *Article* **An Efficient, Simultaneous Electrochemical Assay of Rosuvastatin and Ezetimibe from Human Urine and Serum Samples**

**Leyla Karadurmus 1, Sevinc Kurbanoglu 2, Bengi Uslu 2 and Sibel A. Ozkan 2,\***

**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** The drug combination of rosuvastatin (ROS) and ezetimibe (EZE) is used to treat hypercholesterolemia. In this work, a simultaneous electrochemical examination of ROS and EZE was conducted for the first time. The electrochemical determination of ROS and EZE was carried out using adsorptive stripping differential pulse voltammetry (AdSDPV) on a glassy carbon electrode (GCE) in 0.1 M H2SO4. The effects of the pH, scan rate, deposition potential, and time on the detection of ROS and EZE were analyzed. Under optimum conditions, the developed sensor exhibited a linear response between 1.0 × 10−6 M and 2.5 × 10−5 M for EZE and 5.0 × 10−6 M, and 1.25 × 10−5 M for ROS. The detection limits for ROS and EZE were 3.0 × 10−7 M and 2.0 × 10−6 M, respectively. The developed sensor was validated in terms of linear range, accuracy, precision, the limit of determination (LOD), and the limit of quantification (LOQ), and it was evaluated according to ICH Guidelines and USP criteria. The proposed method was also used to determine ROS and EZE in human urine and serum samples, which are reported in terms of recovery studies.

**Keywords:** rosuvastatin; ezetimibe; glassy carbon electrode; adsorptive stripping differential pulse voltammetry

#### **1. Introduction**

Rosuvastatin (ROS) is a hypercholesterolemia drug that lowers plasma cholesterol levels (Scheme 1a) [1]. ROS has a structure that is similar to most other synthetic statins, but unlike other statins, it contains sulfur. ROS is a competitive inhibitor of the enzyme HMG-CoA reductase [2–4]. Ezetimibe (EZE) is a drug that the FDA has confirmed as curing hypercholesterolemia (Scheme 1b). EZE is the first lipid-lowering drug that reduces the amount of lipoprotein cholesterol by preventing the absorption of cholesterol at the brush-border level of the intestine. It prevents the intestinal uptake of dietary and bile cholesterol [4,5].

**Scheme 1.** The chemical structures of (**a**) rosuvastatin and (**b**) ezetimibe.

**Citation:** Karadurmus, L.; Kurbanoglu, S.; Uslu, B.; Ozkan, S.A. An Efficient, Simultaneous Electrochemical Assay of Rosuvastatin and Ezetimibe from Human Urine and Serum Samples. *Methods Protoc.* **2022**, *5*, 90. https:// doi.org/10.3390/mps5060090

Academic Editors: Victoria Samanidou, Verónica Pino and Natasa Kalogiouri

Received: 29 September 2022 Accepted: 27 October 2022 Published: 1 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Statins and EZE have different lipid-lowering mechanisms of action, and combining them can obtain the strongest impact on lowering lipids and stabilizing plaque areas [6]. In the literature, it has been found that the combination of ROS and EZE further lowers total cholesterol and LDL cholesterol, clearly lowering triglyceride levels, and potentiating the lipid-lowering effects. The combination of ROS and EZE decreases lipid levels and the plaque burden. The combination of a statin and EZE has a greater effect on coronary plaque regression in patients with acute coronary syndrome [6,7]. Adding EZE to ROS significantly improves many more lipid parameters than does doubling the ROS dose [8]. The literature includes descriptions of patients who received 5, 10, 20, or 40 mg of ROS every day, and the average plasma concentration for ROS was 1.6 ng/mL, 3.5 ng/mL, 6.3 ng/mL, and 9.8 ng/mL, respectively [9]. For patients taking one dose of 10 mg of ezetimibe, average ezetimibe peak plasma concentrations (Cmax) of 3.4 to 5.5 ng/mL were acquired within 4 to 12 h [10].

The two major fields of the natural sciences, chemistry and electrical science, came together in the 19th century to form electrochemistry [11]. Electrochemical techniques are extensively used in drug analysis. Among all of the electrochemical methods, stripping analysis is one of the most sensitive electrochemical techniques, and it is therefore used in quantitative determinations, especially in drug analysis. In recent years, stripping voltammetry has been used in the analysis of many drug substances [12]. The reason for this great sensitivity is the combination of an efficient accumulation phase with advanced measurement processes that produce an excellent signal [13,14]. The adsorptive accumulation is intended to deposit the analyte present in the solution on an electrode surface with a small surface area. Stripping voltammetry is also used in clinical practice and allows the conduct of various analyses of human blood, urine, and tissues [15].

The literature reveals some analytical techniques for the simultaneous detection of ROS and EZE. These methods are reverse-phase high-performance liquid chromatography [16,17], micellar liquid chromatography [18], high-performance column liquid chromatography, high-performance thin-layer chromatography [19], spectrophotometry [20,21], and liquid chromatography/mass spectrometry [22,23]. In this work, EZE and ROS were electrochemically analyzed using the AdSDPV technique at GCE. The efficacy of the electrochemical method was fully analyzed for the detection of ROS and EZE in commercial human serum and in urine samples, and we report on it in terms of recovery studies.

#### **2. Experimental Design**

#### *2.1. Materials*

Different supporting electrolytes of H2SO4 solutions (0.1 and 0.5 M), acetate (pH 3.7–5.7), and phosphate (pH 2.0–8.0) buffers were prepared for electrochemical measurements. AdS-DPV voltammogram recordings were obtained after the addition of each aliquot. Drug-free human serum from male AB plasma was purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetic acid, acetonitrile, methanol, phosphoric acid, sodium acetate trihydrate, sodium dihydrogen phosphate dihydrate, sodium hydroxide, sodium phosphate monobasic, sodium phosphate, and sulfuric acid were purchased from Sigma-Aldrich. All reagents were of analytical grade and were used without pre-processing. All measurements were realized at room temperature; all solutions were kept from light and used within 24 h to prevent degradation.

#### *2.2. Equipment*

A Bioanalytical Systems (BAS 100W) electrochemical analyzer with a standard threeelectrode system was used for the voltammetric measurements. The three-electrode system included a platinum-wire counter electrode, an Ag/AgCl-saturated KCl reference electrode, and a GCE (GC, BAS; 3 mm, diameter), which served as a working electrode. The surface of the GCE was polished with an aqueous slurry of alumina powder (Φ: 0.01 μm) on a damp, smooth polishing cloth just before each experiment. The pH was checked using a pH meter Model 538 (Weilheim, Germany). Operating conditions for AdSDPV were as

follow: pulse amplitude, 50 mV; deposition time, 15 s; scan rate, 20 mV/s; pulse width, 50 ms; sensitivity, 10 μA/V; sample width, 17 ms; pulse period, 200 ms; quiet time, 10 s.

#### **3. Procedures**

#### *3.1. Standards and Sample Preparation*

The 1 × 10−3 M stock solution of ROS and EZE was prepared in methanol and kept in a refrigerator (+4 ◦C). The solutions of ROS and EZE for the voltammetric measurements were prepared by direct dilution of the stock solution with 0.1 M H2SO4, and they included a constant amount of methanol (20%, *v*:*v*). Analytical curves were obtained by adding aliquots of the stock solutions of ROS and EZE into the electrochemical cell containing 10.0 mL of the 0.1 M H2SO4 with a constant amount of methanol.

#### *3.2. Biological Sample Preparation*

The applicability of the developed procedure to human urine samples was also investigated. Drug-free urine samples were collected from a healthy laboratory employee on the day of the experiment. To prepare a stock urine solution, 5.4 mL of acetonitrile, 3.6 mL of the drug-free urine samples, and 1 mL of the ROS/EZE stock solution (1 × 10−3 M) were placed in a 10 mL centrifuge tube. First, the mixture was vortexed for 10 min, and then it was centrifuged at 3500 rpm for 30 min. The supernatant part was carefully transferred to a distinct, clean tube. In this procedure, acetonitrile acted as a precipitating agent. A ROS/EZE-free sample of the same urine was used as a blank solution. All measurements were performed at least in triplicate, and the standard addition technique was performed for the determination of ROS/EZE.

Synthetic human serum was kept frozen at −20 ◦C in a freezer until analysis. For the preparation of a stock serum sample, a standard procedure was followed. Quantities of 1 mL of ROS/EZE, 5.4 mL of acetonitrile, and 3.6 mL of synthetic human serum were added to a centrifuge tube to prepare a stock serum solution. First, it was vortexed for 10 min and then centrifuged at 3500 rpm for 30 min, and later, the supernatant was taken. Here, acetonitrile was used to precipitate serum proteins. The supernatant was diluted with 0.1 M H2SO4 to prepare certain concentrations for the recovery measurements. All of the experiments were performed at least three times for calibration and five times for the recovery experiments.

Analytical curves were obtained by adding aliquots of the stock solutions of ROS and EZE from synthetic human serum or human urine into the electrochemical cell containing 10.0 mL of the 0.1 M H2SO4 with a constant amount of methanol.

#### **4. Results and Discussion**

#### *4.1. Voltammetric Behavior of ROS and EZE*

The voltammetric behavior of ROS and EZE was examined on a GCE in detail. In the first step, the behavior of ROS and EZE was investigated by CV studies to characterize their electrochemical oxidation behavior in the range of 0 V to 1.6 V. The CV results indicated the irreversible nature of the oxidation process of ROS and EZE. Moreover, the adsorptive stripping differential pulse voltammetric (AdSDPV) technique was further used, and the anodic oxidation was observed until reaching a potential of about 0.9 V, and 1.2 V; there was a single well-defined and sharp oxidation peak for EZE and ROS, respectively, using the AdSDPV technique on a GCE in 0.1 M H2SO4 (Figure 1).

**Figure 1.** The AdSDPV voltammograms of 5.0 × 10−6 M EZE and 7.5 × 10−6 M ROS in 0.1 M H2SO4 (stripping conditions: accumulation potential of 0.0 V and accumulation time of 15 s).

#### *4.2. Influence of the pH*

The electrochemical behavior of ROS and EZE was studied within a wide pH range (pH 0.3–7.0) using the DPV technique on a GCE. With the DPV method, the maximum current occurred in the 0.1 M H2SO4 medium. The following equation followed the effect of pH on the peak potential. The *Ep*-pH plots indicated that a pH increase caused the shifting of peak potentials to less positive values (Figure 2).

*Ep* (mV) = 1354.24 − 22.79 pH; R2 = 0.997 for ROS

$$E\_p \text{ (mV)} = 998.49 - 50.99 \text{ pH/R}^2 = 0.998 \text{ for EZE}$$

**Figure 2.** Plot of *Ip* vs. pH of 1 × 10−4 M ROS and EZE solution using the DPV.

#### *4.3. Influence of the Scan Rate*

Scan rate experiments were performed to understand the electrochemical oxidation/reduction mechanisms, such as adsorption or diffusion. The influence of the scan rate between 5 and 1000 mV/s on the peak current and potential was investigated in 0.1 M H2SO4 using CV, where the highest peak was obtained in pH studies using a GC electrode.

The plot of *Ep* vs. log *v* was linear; this attitude is coherent with the EC nature of the reaction in which the electrode reaction is coupled with an irreversible follow-up chemical step in CV. According to [24], *Ep* can be defined by the following equation;

$$E\_p = E^{0'} - \frac{2.303RT}{\alpha nF} \log \frac{RTk^0}{\alpha nF} + \frac{2.303RT}{\alpha nF} \log v\_0$$

where *E*0 is the formal potential, *R* is the gas constant, *T* is the temperature, *k*0 is the standard heterogeneous rate constant, α is the transfer coefficient of the oxidation of ROS and EZE, *v* is scan rate, *F* is the Faraday constant, and n is the number of electrons that are involved in the electrooxidation of ROS and EZE [22].

In general, *α* is used as 0.5 for irreversible processes. Since *α* is 0.5 for irreversible systems, n can be calculated from

*Ep* (V) = 0.046 log *v* (V·s−1) + 1.312 (r = 0.997) (0.1 × 10−3 M ROS), and n is found to be 2.36 for ROS, and

*Ep* (V) = 0.049 log *v* (V·s−1) + 1.066 (r = 0.997) (0.1 × 10−3 M EZE), and n was calculated as being 2.38 for EZE (Figure 3a,b).

**Figure 3.** Results of scan rate studies: (**a**) *Ep* vs. log *v*, (**b**) log *Ip* vs. log *v* for 0.1 mM ROS, (**c**) *Ep* vs. log *v*, and (**d**) log *Ip* vs. log *v* for 0.1 mM EZE in H2SO4.

Moreover, the logarithm of peak current vs. the logarithm of scan rate gives more detailed information about the electrochemical mechanisms. When these graphs were plotted, for EZE, from the slope of the equation log (*Ip*) = 0.783 log *v* − 1.186 (r = 0.998), it can be concluded that the reaction is adsorption-controlled since the slope was close to 1. Thus, as a result of the scan rate experiments, in the 0.1 M H2SO4 medium, the electrochemical behavior of EZE was found to be adsorption-controlled (Figure 3d).

For ROS, the slope of the equation log (*Ip*) = 0.582 log *v* − 1.008 (r = 0.992), and the electrochemical behavior of ROS was found to be diffusion-controlled (Figure 3c). As we aimed to determine these two drug-active compounds simultaneously, we applied the adsorptive stripping method, which enabled us to assess ROS and EZE precisely.

In the literature, the electrochemical determination of ROS and its possible oxidation mechanism have been studied. The authors suggested an electrooxidation mechanism involving a Kolbe electrolysis reaction of the carboxylic acid group localized at the dihydroxyhept-6-enoic acid portion of the rosuvastatin calcium molecule [25–27]. In the literature, the electrochemical behavior and possible oxidation mechanism of EZE was also reported by the authors as being due to the inductive effect of the fluoride group in the aromatic rings of the EZE molecule; oxidation takes place in the hydroxyl group of phenol (EC mechanism) and the main voltammetric behavior of aromatic hydroxyl derivatives, which are structurally related to the mechanism of oxidation of EZE, may be postulated by the oxidation of the hydroxyl group on the aromatic ring [28,29].

#### *4.4. Effect of Deposition Time and Potential*

Parameters, such as deposition time and potential, significantly affect the AdSDPV peaks of the analytes. Hence, these parameters as related to AdSDPV were optimized to obtain the best results for the determination of ROS and EZE. The effect of the deposition time on stripping peak current was studied in the range of 0 s to 50 s, with 0 V deposition potential. It was observed that the peak current increased between 0 and 15 s (Figure 4). However, after 15 s, a decrease was observed in the peak current. As a result, 15 s was selected as the optimum deposition time. The effect of deposition potential, which is another important parameter, on stripping peak currents was studied in deposition potentials ranging from −0.1 V to +0.1 V, with a constant accumulation time of 15 s (Figure 4). A decrease in stripping peak currents was observed after the 0 V deposition potential, with an accumulation time of 15 s. A deposition time of 15 s and a deposition potential of 0 V, at which the maximum peak current was observed, were used in all subsequent experiments (Figure 4).

**Figure 4.** Effects of deposition potential (**a**) and (**b**) time.

#### *4.5. Analytical Characterization and Validation*

Under optimum deposition potential and time conditions using the AdSDPV, samples with increasing concentrations of EZE and ROS were prepared. Analytical characterization in terms of LOD and LOQ based on 3 s/m and 10 s/m, respectively, were achieved using linear curves; where m is the slope of the related calibration curves and s is the standard deviation of the peak currents of the lowest concentration of the analyte. EZE was determined in the linear range between 1.0 × 10−6 M to 2.5 × 10−5 M, with a LOD of 3.0 × 10−7 M and a LOQ of 1.0 × 10−6 M. ROS was determined in the linear range between 5 × 10−6 M to 1.25 × 10−5 M, with a LOD of 2.0 × 10−6 M and a LOQ of 6.6 × 10−6 M. For the validation of the developed method, accuracy and precision were investigated by analyzing five replicate experiments between days and within days. Relative standard deviations (RSD%) were determined to control the precision of the technique. As summarized in Table 1, the results after statistical evaluation indicate that the technique is analytically acceptable (Figure 5 and Table 1).

**Table 1.** Statistical assessment of the calibration data for determination of ROS and EZE by the AdS-DPV method in 0.1 M H2SO4 (stripping conditions: accumulation potential of 0.0 V and accumulation time of 15 s).

\* Average of the five values.

**Figure 5.** AdSDPV of sensor in various concentrations of (**a**) EZE and (**b**) ROS using the AdSDPV technique in 0.1 M H2SO4 (stripping conditions: accumulation potential of 0.0 V and accumulation time of 15 s).

#### *4.6. Determination of Ezetimibe and Rosuvastatin in Biological Samples*

In optimized conditions, the electrochemical method was also applied for the detection of EZE and ROS in buffer, spiked human serum, and urine samples, and reported in terms of recovery. Using the suggested method, the purified samples were used for the simultaneous determination of EZE and ROS. Recovery studies were performed by adding ROS and EZE in certain amounts to the human urine samples and serum samples by the proposed technique. The recovery studies of ROS and EZE were assessed based on the data given in Table 1. The proposed technique of RSD% and the average recovery results confirmed suitable accuracy and precision. The applicability of the developed method was indicated by constituting calibration graphs for ROS and EZE in the presence of spiked urine and serum samples. The developed technique was used for the accurate determination of ROS and EZE in biological samples without any pretreatment procedure. The outcomes of the calibration calculations and related parameters obtained in human urine and serum samples are given in Table 1. Recovery results of ROS and EZE were controlled with the corresponding calibration equations, obtained in human urine and serum samples, and found acceptable (Table 2). All results indicated the potential applicability of the developed method for evaluating human urine and serum samples.

**Table 2.** Recovery results obtained from the analysis of ROS and EZE in human urine and serum samples by AdSDPV method in 0.1 M H2SO4 (stripping conditions: accumulation potential of 0.0 V and accumulation time of 15 s).

\* Average of the five values.

#### **5. Conclusions**

In this study, the electrochemical behavior of ROS and EZE was studied simultaneously for the first time. AdSDPV was used for the reliable detection of ROS and EZE in a 0.1 M H2SO4 solution with commercial deproteinated human serum samples and human urine samples using a GCE, and results were reported in terms of recovery. The developed simple and low-cost method showed high sensitivity, a low limit of detection, good repeatability, and good linearity. In the proposed technique, we monitored linear relationships varying from 1.0 × 10−6 M to 2.5 × 10−5 M for EZE concentrations and from 5.0 × 10−6 M to 1.25 × 10−5 M for ROS concentrations. LOD values were found for ROS and EZE as 3.0 × 10−7 M and 2.0 × 10−6 M, respectively. As is stated in the literature, for patients taking 40 mg ROS daily, the average plasma concentration of ROS (Cmax) was 9.8 ng/mL (0.0098 μg/mL) [9]. Furthermore, after one dose of EZE, average EZE peak plasma concentrations (Cmax) of 3.4 to 5.5 ng/mL (0.0055 μg/mL) were obtained within 4 to 12 h (Tmax) [10]. These values are higher than our limit of detection value, indicating that the proposed method can be used to detect ROS and EZE in real samples.

**Author Contributions:** Conceptualization, S.K., B.U. and S.A.O.; methodology, L.K. and S.K.; software, L.K.; validation, L.K. and S.K.; formal analysis, L.K.; investigation L.K. and S.K.; resources, L.K. and S.K.; data curation, L.K.; writing—original draft preparation, L.K. and S.K.; writing—review and editing, S.K., B.U. and S.A.O.; visualization, S.A.O.; supervision, S.A.O.; project administration, S.A.O.; funding acquisition, L.K., S.K., B.U. and S.A.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Ankara University BAP 17B0237002.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Determination of UV Filters in Waste Sludge Using QuEChERS Method Followed by In-Port Derivatization Coupled with GC–MS/MS**

**Cemile Yücel 1,2 , Ilgi Karapinar 1,\*, Serenay Ceren Tüzün 1, Hasan Erta¸s 2 and Fatma Nil Erta¸s 2**

**Abstract:** UV filters (UVFs) are widely used in personal care and in industrial products for protection against photodegradation. In recent years, their potential toxicological and environmental effects have received growing attention. Due to their excessive use, their residue levels in the environment are gradually increasing and they tend to accumulate on biological wastewater treatment sludge. The utilization of sludge as fertilizer could be one of the main routes of UVF contamination in the environment. Therefore, the development of a reliable and sensitive method of analyzing their trace level residues in waste sludge samples is of great importance. The success of the method largely depends on the sample preparation technique in such complex matrices. This study presents a rapid, sensitive and green analysis method for eight UVFs in sludge samples, selected for their rather low no-observed-effect concentrations (NOEC). For this purpose, the QuEChERS methodology was coupled with in-port derivatization for subsequent detection of the targeted UVFs via GC–MS/MS. The analysis time was substantially shortened using this method, and reagent utilization was also reduced. The method was validated in the sludge samples, and high recovery (66–123%) and low RSD values (<25.6%) were obtained. In addition, major contributing uncertainty sources and expanded uncertainties were determined.

**Keywords:** UVFs; QuEChERS; in-port derivatization; waste sludge

#### **1. Introduction**

UVFs are the general name for the chemical group that absorbs ultraviolet light, through which the adverse effects of UV light are eliminated. UVFs can be grouped as inorganic, such as TiO2 or ZnO [1], and organic, mostly used in personal care products, as well as in plastics, automobile paints and rubber industries to increase resistance towards UV light degradation [2]. These compounds can reach to water bodies through industrial and domestic effluents. Since their utilization in personal care products and industrial applications are widespread, the UVF residue levels in the environment are increasing extensively [3].

Organic UVFs are a wide range of compounds that differ in their structures and properties. These highly persistent compounds in the environment tend to accumulate in water [3–5], suspended particles, soil [6], sediment [7] and sludges [8–10]. Organic UVFs can also accumulate in biota and disrupt the endocrine systems of aquatic organisms by increasing their estrogen-induced cell proliferation [11]. It has been clearly stated that UVFs affect different hormonal targets as well as estrogenic activity in mammals and fish, and are therefore known as endocrine-disrupting chemicals (EDCs) [12,13]. Consequently, one of these compounds has recently been included in the Watch List by The European Water Framework Directive as a future priority pollutant [14]. Although the use of these compounds in cosmetics is legally restricted [15], there is no limit to their concentration in water and sewage sludge matrices. However, studies on the presence of UVFs in waste

**Citation:** Yücel, C.; Karapinar, I.; Tüzün, S.C.; Erta¸s, H.; Erta¸s, F.N. Determination of UV Filters in Waste Sludge Using QuEChERS Method Followed by In-Port Derivatization Coupled with GC–MS/MS. *Methods Protoc.* **2022**, *5*, 92. https://doi.org/ 10.3390/mps5060092

Academic Editors: Victoria Samanidou, Verónica Pino and Natasa Kalogiouri

Received: 11 October 2022 Accepted: 20 November 2022 Published: 23 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

sludge have revealed that their concentration range from a few to thousands of μg/g in dry mass [16]. The use of this sludge as fertilizer is another concern due to the widespread distribution of UVF residues in agricultural soils and the possible contamination of crops.

Since their concentrations are very low, an appropriate preparation technique must be applied to the samples to isolate and preconcentrate these filters. Current trends in the determination of organic UVFs in environmental water samples based on microextraction techniques have been reviewed [3,17,18]. However, their determination in sludge samples faces difficulties due to the complexity of the matrices and their impact on the environment. To date, one of the techniques used for UVF extraction from sewage sludge comprises liquid–liquid extraction (LLE) followed by solid-phase extraction (SPE) [19–22] where the excessive use of solvent is the main drawback of the method. The other technique is pressurized liquid extraction (PLE), applied alone or coupled with SPE [23–29] or gel permeation chromatography (GPC) [30]. Despite the advantage of using a smaller solvent volume in the PLE technique, special equipment is required to conduct this method.

In the last few decades, method development studies have been devoted to modern sample preparation techniques based on shorter analytical periods and the minimization of organic solvent utilization for a wide range of pollutants. One of these modern techniques, called QuEChERS (which is an acronymic for quick, easy, cheap, effective, rugged and safe) has been well-established to improve laboratory efficiency and throughput. The main advantage of this method is its small quantities of solvent utilization. The method has already been tested for UVFs in human milk [31] and seafood samples [32], followed by liquid chromatography systems coupled with mass detectors (LC-MS) and sludge samples, and then, by detection using gas chromatography (GC–MS) [16,21–23]. LC-MS systems are preferred for the analysis of polar UVFs, and no derivatization step is required in accordance with the physicochemical properties of UVFs [33–36]. GC–MS has been used for rather non-polar and volatile UV filters. Although the GC method provides high separation efficiency, high selectivity and good sensitivity, it displays some disadvantages such as the derivatization step, in which more reagents, greater reaction time and more labor are required. Fortunately, the in-port derivatization technique is a practical and environmentally friendly solution to these issues wherein the reaction takes place in the injection block rather than in an off-line interaction. The analysis time can be substantially reduced using this method and the selectivity can be improved since the technique enables extra purification for the matrix effect.

In the present study, we aimed to detect eight UVFs in sludge samples. Benzophenone-3 (BP-3), 3-benzylidene camphor (3BC), 2-ethyl hexyl-4-(dimethyl amino) benzoate (EDP), 2-ethyl-hexyl-4-trimethoxy cinnamate (EHMC), ethylhexyl salicylate (EHS), homosalate (HMS), isoamyl p-methoxycinnamate (IAMC) and 4-methylbenzylidene camphor (4-MBC) were selected according to their no-observed-effect concentration (NOEC) values. Their chemical structures and NOEC limits are presented in Table 1. Considering the hydrophobicity (logKow: 3.79–6.16) of the analytes, the QuEChERS method was adopted; however, due to the low volatility of these analytes along with their weak acidity (pKa: 7.56–8.13), the analytes were derivatized using an in-port silylation technique prior to subsequent quantification using the GC–MS/MS system. To the best of our knowledge, this is the first report in the literature about the combination of QuEChERS with in-port derivatization followed by GC–MS/MS for a wide variety of UVF analyses in waste sludge. This method extensively decreases solvent or chemical consumption and shortens the analysis time compared to off-line derivatization. The method validation parameters, major contributing uncertainty sources and expanded uncertainties were determined.

#### **Table 1.** Chemical Structures, IUPAC names and NOEC concentration of selected UVFs.

#### **2. Materials and Method**

#### *2.1. Reagents and Standard Solutions*

The chemical standards of 2-ethylhexyl salicylate (EHS), 2-hydroxy-4-methoxybenzophenone (benzophenone-3, BP-3) >98%, 3,3,5-trimethylciclohexylsalicylate (homosalate, HMS) >98%, 4-methylbenzylidene camphor (4-MBC) 99%, 2-ethyl-hexyl-4-trimethoxy cinnamate (EHMC) 99%, and 2-ethyl hexyl-4-(dimethylamino) benzoate (EDP) 99% were obtained from Dr. Ehrenstorfer (Augsburg, Germany). 3-benzylidene camphor (3BC) 99%, isoamyl p-methoxycinnamate (IAMC) and % were acquired from Sigma-Aldrich (St. Louis, MO, USA), Toronto Research Chemicals (Toronto, ON, Canada) and Chemservice (West Chester, PA, USA), respectively.

LC-MS-grade methanol and acetonitrile, GC–MS-grade ethyl acetate (EtAC) and acetone, sodium chloride, sodium sulfate and orthophosphoric acid were purchased from Merck (Darmstadt, Germany). Bis(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane (BSTFA + TMCS; 99:1, *v*/*v*) from Macherey-Nagel was used as derivatization reagent. This reagent was stable for only 3 h during analysis. 2-dodecanol, 2-dodecanone, and 1-undecanol

from Sigma were used as extraction solvents. Anhydrous magnesium sulphate (MgSO4), primary and secondary amine exchange bonded silica sorbent (PSA) and octadecylsilan (C18) were obtained from Supelco (Bellefonte, PA, USA) for the extraction step.

The stock solutions of individual UVFs (1000 μg mL−1) and mixtures of UVFs (50 μg mL−1) were prepared in ethyl acetate to optimize the injection port derivatization conditions in methanol to validate the QuEChERS method. These solutions of 1000 μg mL−1 and 50 μg mL−1 were stable for about 5 months and five days at −20 ◦C, respectively. The working aqueous solutions were prepared daily from standards in methanol at different concentrations using ultrapure water and environmental water. The sludge samples were collected from a domestic wastewater treatment plant in Izmir, Turkey. Sludges were air-dried and stored in the dark at −20 ◦C until analysis.

#### *2.2. Sample Extraction*

The procedure for extracting UVFs from sludge is a modified version of the method reported previously [16]. As shown in Figure 1, a 0.5 g sewage sludge sample was dried at room temperature and a spiked sample was transferred into a conical-bottom 15 mL polypropylene tube containing 10 mL of ACN, vortexed for 2.5 min, and then, left in an ultrasonic bath (J.P. Selecta, Barcelona, Spain) for 15 min. Then, the organic phase was separated via centrifugation at 3500 rpm for 15 min and transferred to a conical-bottom 15 mL polypropylene tube containing 500 mg MgSO4, 410 mg C18 and 315 mg PSA. The extract was then vortexed for 2.5 min and centrifuged for 15 min. After the supernatant was transferred to a 10 mL tube, it was evaporated to dryness under N2 gas. Then, the extract was dissolved in 1000 μL EtAC, instead of hexane as performed by Ramos et al. [16], for the in-port derivatization step as previously applied to a surface water sample by our research group [37]. The extract dissolved in EtAC was filtered through a 13 mm, 0.22 μm PTFE filter, and then, it was transferred to a 1.5 mL amber vial. Finally, 2 μL of BSTFA and 2 μL of extract were derivatized in the injection port using the sandwich technique. In this technique, two aliquots of 2 μL BSTFA and 2 μL extract in EtAc, separated with an air gap, are drawn into the microsyringe of a PAL autosampler, and then, injected to the GC–MS/MS system.

**Figure 1.** Schematic illustration of the whole procedure.

#### *2.3. GC–MS/MS Analysis*

GC–MS/MS analysis was performed using an Agilent 7890 B gas chromatograph coupled with a triple quadrupole mass spectrometer (MS 7000C) and a PAL autosampler (GC Sampler 80). For derivatization, the injection port temperature was held at 70 ◦C for 3 min, then, increased to 300 ◦C at a rate of 400 ◦C min−1 in splitless mode with a purge-off time of 4.5 min. The oven temperature started at 70 ◦C for 4 min, increased to 180 ◦C at 25 ◦C min−1, then, increased to 230 ◦C at a rate of 5 ◦C min−1 and to 300 ◦C at a rate of 25 ◦C min−1; it was held for 10 min at this temperature. The injector was operated using programmed temperature evaporation (PTV).

Separation was performed on 5% phenyl-arylene/95%-dimethylpolysiloxane HP-5MS (30 m × 0.25 mm i.d. 0.25 μm film thickness) supplied by Phenomenex (Torrance, CA, USA). Helium (99.999% purity) was used as the carrier gas at a constant flow of 1 mL min−1. In MS/MS analysis, the temperatures of the ion source and the transfer line were 280 and 300 ◦C, respectively. The multiple reaction monitoring (MRM) technique was applied, and electron ionization (EI) mode was used. The retention times (Rt) obtained, the optimized MRM transitions and the collision energies (CE) for each UVF are given in Table 2. The bold parent and product ions in the table show the quantification transitions.

**Table 2.** Experimental GC–MS/MS parameters of UVFs.

#### *2.4. Validation Studies*

The matrix match method was used for the calibration curves. In this method, the curves were constructed by subtracting the peak area values of the real sludge sample from the spiked extract and plotting against the concentration of the UV filter added into the real sludge sample.

The linear range, intra-day and inter-day repeatability, Limit of Detection (LOD), Limit of Quantitation (LOQ) and recovery parameters were determined, and measurement uncertainties of the method applied for UVFs were calculated. For the linearity of UVFs, sludge samples were spiked with 40, 80, 200, 600 and 1200 ng g−1 UVF standards. The extracts were evaporated to dryness under nitrogen gas, and they were diluted to 1000 μL using ethyl acetate. The LOQ and LOD were calculated according to S/N = 10 and

S/N = 3, respectively. Intra-day and inter-day repeatability studies were performed at low (80 ng g−1), medium (300 ng g−1), and high (600 ng g−1) concentrations, with 3 replicates.

The selected test material was analyzed repeatedly under different conditions such as on different days, using different analysts and different equipment, etc. The total variation in the whole cluster can be represented as the combination of variances (s2) between (S*between*) and within groups (Sr). The repeatability (intra-day) and intermediate precision (inter-day) values were calculated via ANOVA [38]. The standard deviation of Sr was calculated by taking the square root of the within-group mean square term, as shown in Equation (1), and the contribution of the grouping factor to the total variation was obtained from Equation (2). In Equations (1) and (2), MSw is the within-group mean square term and *MSb* is the between-group mean square term.

Then, intermediate precision (SI) was calculated by combining the within-group and between-group variance components, as shown in Equation (3).

$$S\_r = \sqrt{MS\_w} \tag{1}$$

$$s\_{between} = \sqrt{\frac{MS\_b - MS\_w}{n}}\tag{2}$$

$$S\_I = \sqrt{S\_r^2 + S\_{between}^2} \tag{3}$$

The intra-day (*n* = 3) and inter-day (*n* = 2) relative standard deviations (RSD%) of the QuEChERS followed by GC–MS/MS were obtained using spiked solutions of the analytes at different concentration levels. According to the 2015/1787 directive, if the RSD% value of the applied method is less than 25%, the precision of this method is acceptable for organic compounds.

Recovery studies to determine the accuracy of the method were carried out by adding the UVF standards to the sewage sludge samples at concentrations of 80, 300 and 600 ng g−1. The recovery percentages were calculated from Equation (4) where, C*pre* − *Ext* and C*post* − *Ext* are the concentrations of sludges in which analytes were added before and after extraction, respectively. *Csample* is the concentration of UVFs in sludges without the addition of the analyte.

$$\text{Recovery }\%= \frac{\text{C} \, pre-\,\text{Ext}-\,\text{C} \, sample}{\text{C} \, post-\,\text{Ext}-\,\text{C} \, sample} \times 100\tag{4}$$

The measurement uncertainty is a parameter that was included with the measured result and characterizes the distribution of values that can correspond to the measurand. Knowing the uncertainty means increased confidence in the accuracy of the measurement result. This value is very important in comparing the measurement results of two different methods and deciding whether the results are within the defined limits. Measurement uncertainty consists of many components. Some of these components are derived from the statistical distribution of the results of repeated measurement series to obtain the standard deviations. Combined standard uncertainty (*u*(*c*)) is the standard uncertainty that considers contributions from all important uncertainty sources by combining the relevant uncertainty components, and it was calculated as shown in Equation (5). The expanded uncertainty provides the range of an analyte concentration believed to be spread at a higher confidence level. The expanded uncertainty (*U*) is calculated by multiplying the combined standard uncertainty by "*k*", which is equal to 2 at 95% confidence level [39]. In this study, the uncertainty sources were defined first; then, the uncertainty of each parameter was determined, and finally, the combined *U*c(y) and expanded uncertainties (*U*) were calculated.

$$
\mu(c) = \sqrt{\mathfrak{u}\_{calibration}^2 + \mathfrak{u}\_{SI}^2 + \mathfrak{u}\_{Recovery}^2} \tag{5}
$$

#### **3. Results and Discussion**

#### *3.1. Optimization Studies*

For the extraction of targeted UVFs from sludge samples, the QuEChERS methodology was adopted for the in-port derivatization and the extract obtained was dissolved in 1000 μL ethyl acetate (EtAc) instead of hexane. In a previous study carried out in this lab, in-port derivatization conditions were optimized for a wide range of UVFs extracted from surface water via vortex-assisted dispersive liquid–liquid microextraction based on the solidified floating organic droplet (VA-DLLME-SFOD) technique. It was determined that the injection temperature was a statistically significant factor and the optimal temperature was determined to be 260 ◦C [37]. The effect of injection temperature for the studied UVFs in sludge samples was further studied to see any deviation from the optimal conditions determined. The temperature varied between 260–320 ◦C, and the peak areas are given in Figure 2.

**Figure 2.** The effect of injection temperature on peak areas of UVFs.

As can be deduced from the figure, even though the mean peak areas of the UVFs are not substantially different from each other for the studied injection temperatures, a slight decrease was observed for 300 ◦C. However, this injection temperature was chosen for further studies since the carry-over effect occurred in samples up to 290 ◦C. The sharp peaks in UVFs with injection port derivatization, after QuEChERS, of the spiked sludge sample were obtained, as shown in Figure 3.

#### *3.2. Validation Studies*

The linearity and linear range of a method should unequivocally be determined for any analytical method. It is a fact that the lowest concentration of the calibration curve should be very close to the LOQ value for the accurate analysis of analytes with known precision at an LOQ-level concentration. Table 3 depicts the linear equations for the working range of 40–1200 ng g−1 with correlation coefficients close to unity (R2 > 0.9970).

**Figure 3.** Chromatograms of the UV filters in sewage sludge spiked with UVFs at 300 ng g−1.

**Table 3.** Analytical merits of the GC–MS/MS method coupled with QuEChERS for UVF determination in sludge samples.

\* Y = ax + b.

LOQ is defined as the lowest concentration that can be measured with acceptable precision (20% RSD) and accuracy [40–45]. Three different methods are used to determine the LOQ value. The most common approach for LOQ calculation in chromatographic analysis is the signal-to-noise ratio (S/N) [45]. This ratio can be defined as the difference between the height of the analyte peak (signal) and the highest and lowest points of the baseline (noise) in each area around the signal. For LOQ, S/N usually needs to be at least equal to 10. In the second method, a specific calibration curve should be studied using samples containing an analyte in the range of the LOQ. The residual standard deviation of a regression line or the standard deviation of the y-intercepts of regression lines may be used as the standard deviation [39]. The third approach is the concentration corresponding to a response 10 times greater than the SD of the analysis at the minimum concentration [37,41,45].

The LOD and LOQ values were calculated based on the S/N = 3 approach and S/N = 10, respectively. The LOQ values calculated using this method were found to be in agreement with previous studies on the analysis of UVFs in sludge [17,24,26]. In-port derivatization provides lower detection limits by converting polar analytes to more volatile compounds, as reported earlier [46–48]. For most of the compounds, the LOQ is close to or less than the lowest concentration level of the calibration curve. The RSD% values obtained in this study are below 25% as seen in Table 4.

**Table 4.** Intra-day (*n* = 3) and inter-day (*n* = 2) relative standard deviations (RSD%) calculated for the UVFs.

The recoveries were in the range of 66%–23% as depicted in Table 5. Although, the recoveries are almost the same as the results obtained in other studies [17,24,26,31], our method provides certain advantages such as the lack of special apparatus and comparatively shorter analysis times.

**Table 5.** Recoveries of UVFs from sludge samples spiked with different concentrations.

#### *3.3. Measurement Uncertainty*

Here, the main uncertainty components such as *ucalibration*, *uSI* and *uRecovery* were calculated for the analysis of two different UVF concentrations, as given in Table 6. The uncertainty of the QuEChERS-GC–MS/MS method for UVFs is between 13.2–47.4% at a concentration of 300 ng g−1 and between 6.9–43.6% at a concentration of 600 ng g−1, which are quite satisfactory in such a complex matrix. Uncertainties regarding the recovery and calibration curve were the largest source contributing to measurement uncertainty.

**Table 6.** Expanded uncertainties of QuEChERS followed by the in-port derivatization method for studied UVFs.

**Table 6.** *Cont.*

#### *3.4. Comparison with Other Methods*

The recovery values obtained for all analytes are similar to those of studies with laborious and expensive techniques. In some studies, recovery values at a single concentration were determined [21,22,27,36], while in others, recovery values at different concentrations were investigated [16,19,23,33]. Our results indicated that for 4-MBC, only at a high concentration (600 ng g−1) was the recovery value lower than that of the reported ones. Fortunately, good recovery values were obtained at lower concentrations. In addition, to the best of our knowledge, method validation for the analysis of 3-BC, EHS, HMS and IAMC in sludge was attained successfully. Analytes (EHS, HMS, BP-3) were derivatized using the in-port derivatization technique, which is fast, reliable and eco-friendly without being affected by the complexity of the matrix.

The LOD values of the selected UVFs in the spiked sludge sample ranged from 8 ng g−1 to 12.1 ng g−1. These limits are quite successful when compared to other studies (Table 7). The LOD values obtained for UVFs were lower than previous studies for 4-MBC, EHMC [23,27], BP-3 [23,36] and EDP [16]. On the other hand, the LOD values for 4-MBC [21,22,33], EHMC [16,21,22], BP-3 [19,27,33] and EDP [23] were higher.

**Table 7.** Comparison of QuEChERS method with previous microextraction studies in sludge samples.

1H-BT: 1H-benzotriazole, 5Me-1H-BT: 5-methyl-1H-benzotriazole, TBHPBT: 2-(5-t-butyl-2-hydroxyphenyl) benzotriazole, 4-OH-HB: 4-hydroxy benzophenone. \* LOQ values.

The developed method was applied to sludge samples collected from a domestic wastewater treatment plant. The concentrations of UV filters were found to be 66.9, 161.9, 54.5, <LOQ and 79.9 ng g−1 for EHS, HMS, BP-3, EDP and EHMC, respectively. These results indicate that even in domestic wastewater sludge, high concentrations of UVFs could be found, and the sludge used for soil conditioning or fertilization purposes could raise environmental concerns.

#### **4. Conclusions**

In this study, eight different UVFs were extracted from sludge samples using a wellestablished QuEChERS methodology, and then, in-port derivatization was applied for more polar and less volatile analytes. The method combining QuEChERS and in-port derivatization, which ensures extra sensitivity, was shown to be accurate, reproducible and sensitive. This method also provided substantially reduced analysis time and solvent or chemical consumption compared to off-line derivatization. Method validation resulted in high recovery of UVFs (66–123%), meaning good accuracy; moreover, low inter-day RSD% (10.1–25.6) indicates high precision, and low values of LOD (<2.1 ng g−1) and LOQ (<40 ng g−1) show the sensitivity of the analysis for a matrix as complex as waste sludge. This study can be extended to the analysis of UVF degradation metabolites in sludge samples using QuEChERS with in-port derivatization followed by GC–MS/MS.

**Author Contributions:** C.Y. carried out the experiments, evaluation of results and preparation of the manuscript. F.N.E. and I.K. guided the experiments, edited the manuscript and evaluated the results. H.E. designed the experiments and guided the analysis. Research Assistant S.C.T. contributed to the analysis and experiments. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Dokuz Eylül University Scientific Research Projects (BAP) Coordination Unit] grant number [2021.KB.FEN.008].

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We would like to thank Dokuz Eylül University Scientific Research Projects Coordination Unit (project number 2021.KB.FEN.008) for their financial support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **On-Site Multisample Determination of Chlorogenic Acid in Green Coffee by Chemiluminiscent Imaging**

**Sergi Mallorca-Cebria, Yolanda Moliner-Martinez \*, Carmen Molins-Legua and Pilar Campins-Falcó**

MINTOTA Research Group, Departament de Química Analítica, Facultad de Química, Universitat de Valencia, C/Doctor Moliner 50, 46100 Burjassot, Spain

**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** The potential of antioxidants in preventing several diseases has attracted great attention in recent years. Indeed, these products are part of a multi-billion industry. However, there is a lack of scientific information about safety, quality, doses, and changes over time. In the present work, a simple multisample methodology based on chemiluminiscent imaging to determine chlorogenic acid (CHLA) in green coffee samples has been proposed. The multi-chemiluminiscent response was obtained after a luminol-persulfate reaction at pH 10.8 in a multiplate followed by image capture with a charge-coupled device (CCD) camera as a readout system. The chemiluminiscent image was used as an analytical response by measuring the luminescent intensity at 0 ◦C with the CCD camera. Under the optimal conditions, the detection limit was 20 μM and precision was also adequate with RSD < 12%. The accuracy of the proposed system was evaluated by studying the matrix effect, using a standard addition method. Recoveries of chlorogenic acid ranged from 93–94%. The use of the CCD camera demonstrated advantages such as analysis by image inspection, portability, and easy-handling which is of particular relevance in the application for quality control in industries. Furthermore, multisample analysis was allowed by one single image saving time, energy, and cost. The proposed methodology is a promising sustainable analytical tool for quality control to ensure green coffee safety through dosage control and proper labelling preventing potential frauds.

#### **1. Introduction**

The increasing consumption of antioxidants raises public health concerns about their efficiency, safety, and real benefits for human health. Antioxidants are widely available and commonly used, especially in the women's health market [1]. However, there is a lack of information about the composition, doses, potential degradation, and function of antioxidants [2]. Mainly, the benefits of antioxidant supplements are associated with their interaction with free radicals and hence protect health against oxidative stress [3]. The interaction mechanism depends on the chemical nature and reactivity. These compounds are classified as direct and indirect antioxidants and they mediate oxidative stress through different mechanisms. Nevertheless, there are controversial results and conclusions about the real level and function of these compounds [4].

What is certain, is that the antioxidant nutraceutical market is now a multi-billion dollar industry experiencing a continuous increase, and this has led to regulations that ensure safety, proper labelling, and more information about the ingredients, which means there is a real need to ensure the concentration of the antioxidant compounds, and that they are within safety limits, in particular in quality control studies [5].

Focusing on direct antioxidants, chlorogenic acid (CHLA) is an abundant watersoluble antioxidant in the human diet. Indeed, it is available from a wide variety food and beverage sources. In particular, CHLA is linked to green coffee and green coffee extract-based products' health benefits. That fact, together with the relevance of the global

**Citation:** Mallorca-Cebria, S.; Moliner-Martinez, Y.; Molins-Legua, C.; Campins-Falcó, P. On-Site Multisample Determination of Chlorogenic Acid in Green Coffee by Chemiluminiscent Imaging. *Methods Protoc.* **2023**, *6*, 20. https:// doi.org/10.3390/mps6010020

Academic Editors: Victoria Samanidou, Verónica Pino and Natasa Kalogiouri

Received: 16 December 2022 Revised: 6 February 2023 Accepted: 10 February 2023 Published: 14 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

coffee market, has led to a growing interest in the development of analytical methods for the quantification of this antioxidant [6].

In this context, the development of analytical strategies that facilitates the determination of CHLA, in commercial products is mandatory to help the regulation, labelling, and warming of potential side effects [7]. Basically, the main methods to determine this compound are based on separation techniques such as HPLC and UPLC, coupled to different detectors such as DAD, luminescent, and MS [8–12]. Their performance has been demonstrated, yet they required specialized laboratory instrumentation, trained personnel, and a large instrumental investment. Thus, more recently simple, cost-effective, rapid, easy-handling, and portable analytical procedures have been proposed.

These new strategies are principally based on optical readouts with the added value of providing visual responses that allow semiquantitative determination by visual or image inspection and on-site determination [13]. Colorimetric-based sensors using paper-based devices and a metal nanooxide array have been recently published [14,15].

Among the different on-site strategies, chemiluminiscence (CL) is attracting attention since it allows for the achievement of sensitivity in short analysis times [16]. Going a step forward, chemiluminiscent imaging is a powerful alternative since it combines the typical CL properties with visual inspection and portability [12,17]. In this case, the use of charge-couple device (CCD) cameras has been demonstrated to be a feasible detector for this aim [18]. Fundamentally, the photons produced in the hemiluminescent reaction are captured by the CCD camera and subsequently, an image is displayed based on the light generated, the intensity measurement can be correlated with the target analyte. The key parameters in the application of these systems are mainly based on the control of experimental variables since the CL efficiency depends on the kinetic emission CCD camera response time. From the analytical point of view, there is scarce knowledge of the potential application of CCD cameras as analytical response read-out systems. This strategy has been proposed for environmental analysis [18], however, other application areas and in particular for quality control of human consumption products, are still unexplored.

The objective of the present work was to demonstrate the utility of chemiluminiscent imaging employing the CCD camera to estimate CHLA in natural products, in particular green coffee samples. The novelty relies on the combination of an on-site portable device with high-performance analytical parameters for quality control of commercial products. Optimization of the reaction parameters was aided by the use of a portable luminometer. For this aim, the instrumental parameters were optimized and the analytical properties were established. Finally, the practical application and validation of the proposed methodology were evaluated by analysing commercial samples.

#### **2. Experimental Section**

#### *2.1. Reagents*

Sodium hypochlorite 10% was obtained from Panreac (Barcelona, Spain). Sodium persulfate, Trolox, CHLA, and luminol were purchased from Sigma-Aldrich (Taufkirchen, Germany). Sodium carbonate and sodium hydroxide were from VWR Chemicals (Radnor, PA, USA).

Standard solutions of sodium persulfate (50 mM) in carbonate buffer 0.3 M (pH 10.8) were prepared weekly. Trolox (1.1 mM), luminol (20 mM), and CHLA (2.0 mM) were prepared in carbonate buffer (0.3 M, pH 10.8). Working standard solutions of Trolox and CHLA were prepared by adequate dilution in carbonate buffer.

#### *2.2. Equipment*

The CL response was measured by using a 338L Mono CCD Camera themoelectrically cooled at 5 ◦C (Atik Cameras, Norwich, UK. In addition, an LB 9509 portable tube luminometer (Berthold Technologies, Bad Wildbad, Baden-Württemberg, Germany) was also used to determine the optima chemiluminiscent reaction conditions.

#### *2.3. Procedure*

#### 2.3.1. Optimization and Measurement of the CL Response

In the preliminary studies, the optimization of the reaction parameters, such as oxidant, concentration, pH, and analysis time was carried out using the luminometer and CHLA analytical grade standards (see Section 2.1). Oxidants such as sodium persulfate and sodium hypochlorite were evaluated. For this aim, 100 μL of luminol was mixed with 100 μL of persulfate or hypochlorite of luminol and the response was registered every 5 s, for 1 min. The study of persulfate and luminol concentrations were performed in the range of 0.7–10 mM, and 1.4–4.8 mM, respectively, by measuring the signal at 60 s. Finally, the influence of pH was studied in the interval 10.0–11.5, using persulfate (10 mM) and luminol (4.8 mm).

Once the optimal reaction conditions were established, the CCD camera instrumental parameters were studied to monitor the CL imaging response.

#### 2.3.2. CCD Measurements

The CCD camera was studied as a readout system. In this case, contrast, temperature, and exposition time were previously optimized. Under the different conditions, the images were processed by the software Artemis Capture, and the luminescent intensity (I) for each spot was registered. Standards and samples were deposited on a multiplate and placed inside the CCD camera dark chamber for its measurement. The device used to perform the CCD measurements is shown in Figure 1. It should be noted that exposition distance was optimized in previous work [18].

**Figure 1.** Schematic diagram of the CCD camera dark chamber used for the determination of CHLA.

The measurement was performed in a multiplate by mixing luminol (100 μL) and persulfate (100 μL). After 5 min, 100 μL of the blank, working standards, or samples, prepared in a carbonate buffer (0.3 M, pH 10.8), were added, and the image was registered. The working concentration interval was up to 500 μM for CHLA. Trolox was also measured as another example of the antioxidant analyte.

#### *2.4. Analysis of Commercial Green Coffee Samples*

The application of the proposed method was validated by the analysis of three commercial green coffee samples acquired in a local store. Sample preparation was carried out as follows: a capsule of each sample was dissolved in ultrapure water and sonicated during 5 min, to extract CHLA. Subsequently, the extract was diluted in carbonate buffer (0.3 M, pH 10.8). Samples were measured following the proposed procedure. The analysis was performed in triplicate. In addition, the accuracy of the proposed method was evaluated by using the standard addition method (SAM). For this aim, the calibration curve was performed following the procedure described in Section 2.3 and spiking the sample with known concentrations of CHLA standards.

#### **3. Procedure**

#### *3.1. Study of Chemiluminiscent Imaging as an Analytical Response*

In the previous experiments, the CL response was optimised considering the hypothesis that the reaction between luminol and an oxidant in basic media, induces the generation of a luminescent signal that can be monitored, and the presence of antioxidants such as chlorogenic acid may inhibit the CL signal as a function of the concentration. Hence, the first step was the optimization of the CL reaction, taking into account that the CCD camera will be the readout system, which means that detection time is a key parameter. Figure 2 shows the variation of the CL response as a function of the experimental parameters (measurements done with the portable luminometer).

**Figure 2.** (**A**) Variation of the luminescent signal (RLU) produced by luminol using 0.25 mM hypochlorite (blue) and 0.7 mM persulfate (red); Luminol = 1.4 mM, carbonate buffer 0.3 M, pH 10.8. (**B**) Variation of the RLU as a function of luminol concentration, (**C**) Variation of the RLU as a function of persulfate concentration, (**D**) Variation of the RLU signal in presence of CHLA (50 μM, pH 10.8) for blank (1, grey), luminol-persulfate, CHLA addition at t = 5 min (2, red) and persulfate-CHLA luminol addition at t = 10 min (3, blue). Conditions: 10 mM persulfate, 1.4 mM luminol.

As can be seen in Figure 2A, the use of hypochlorite as an oxidant resulted in a fast reaction kinetic with a signal decay in 10 s. Meanwhile, persulfate provided a constant CL signal up to 60 s. Therefore, persulfate was selected as an oxidant, taking into account the exposition time that subsequently will be needed for the measurement in the CCD camera. Regarding luminol and persulfate concentrations (see Figure 2B,C), 4.8 mM, and 10 mM were the optimum concentration, respectively, since they maximized the CL signal, and hence the sensitivity of the target analytes by inhibition of this CL response. It is well known the dependence of CL reactions with pH, thus, this parameter was also studied. NaOH and carbonate buffer were studied. The results indicated that carbonate buffer 0.3 M and pH 10.8, provided satisfactory intensity, taking into account the concretion level of the samples of interest.

Under the above-mentioned conditions, the influence of the addition of antioxidants was evaluated. Previous reports indicated that antioxidants give rise to a decrease in the CL response and the mechanism involved is not yet clear, since some authors pointed to CL quenching in addition to the inhibition of luminol oxidation by the antioxidants that has also been reported [17]. In this scenario, different strategies were evaluated. In the first case, persulfate and CHLA were mixed, and after 10 min, luminol was added. In the second experiment, luminol and persulfate were mixed, and after 5 min, CHLA was added. Figure 1D shows the results obtained. As can be seen, in the second condition, a decrease of the CL signal was observed with the addition of CHLA. Hence, that was the strategy selected for further experiments.

By another hand, the variation of the CL decrease as a function of CHLA concentration was also studied in order to establish the potential use as a quantitative methodology. Firstly, the reaction time between persulfate and luminol, before the addition of the antioxidant was studied. For this aim, additional times of 180 and 300 s were studied. Figure 3A shows the variation of the response as a function of time, and at two antioxidant addition times. The addition of the target analytes at 300 s was advantageous since the stability of the response was higher than at 180 s, more likely due to a slower kinetic of the antioxidants since, persulfate in the reaction media is lower at 300 s than at 180 s. This stability was a key parameter, since, the CCD camera required a stability interval to properly register the image. The inclusion criteria, in this case, was that the signal was stable for up to 30 s. Figure 3B shows the variation of the kinetics at different concentration levels. By measuring the signal at 30 s after the addition of chlorogenic acid, the CL decrease could be linearly correlated with the concentration level (sensitivity = −1400 ± 50 μM−1, R2 = 0.9930), hence the quantitative potential was demonstrated under these conditions with a detection limit, LOD = 2 μM.

**Figure 3.** (**A**) Variation of the RLU in presence of CHLA (500 μM) after 180 s (blue) and 300 s (red). (**B**) Variation of the RLU over CHLA concentration under the optima reaction conditions.

Remarkably, it should be considered that in presence of other antioxidant compounds, the decrease in the luminescent signal would be a result of the total antioxidant content extracted under the extraction conditions described in Section 2.4. In order to demonstrate this, the RLU signal for trolox was also measured. The results indicated the same behaviour as CHLA, however, the decrease of the luminescent signal was slightly higher, achieving a detection limit of 0.7 μM. Hence, in this case, the luminescent signal would provide a measurement of the total content of CL active compounds under the extraction conditions.

Once the reaction parameters were optimized, the CCD camera instrumental parameters, the contrast parameters, exposition time, and temperature were studied. The CL response was established by capturing the image and processing that image with Artemis Capture software. Figure 4 shows the schematic representation of the set-up and the images obtained for different blanks measured at different times under the contrast parameters white 2000, black 450, and log 1.5 for CHLA. It should be noted that the image shown in Figure 4 was a real image, where the responses were specific dots for each blank. In this case, the analytical response was the luminescent intensity (I) of each sample, and hence the balance between black and white intensity and grey contrast was fundamental to obtain adequate image quality. As can be seen, the profile of the CL image signal was similar to that measured with the luminometer. Hence, it was expected, that the addition of CHLA to the plate would result in a decrease in the CL that can be monitored by the CCD camera.

**Figure 4.** Schematic set-up of the CCD camera to measure CL image, the real image of the variation of the bank signal as a function of time, and the representation of the luminescent intensity for each image.

To demonstrate the response as a function of CHLA concentration, the target analyte was added and the experiment was performed at two temperatures. The results indicated that the temperature of the CCD camera was a key parameter. Mainly at high a temperature, the background noise or dark current gave rise to a low-resolution image. Meanwhile, working at 0 ◦C, the resolution was satisfactory, and the difference in the blank signal compared with the CHLA standard could be monitored by the image. Figure 5A shows the CL images carried out at 0 and 23 ◦C for CHLA compared with blanks at the same temperature.

**Figure 5.** (**A**) Effect of the CCD camera temperature on the chemiluminiscent image (CHLA 100 μM), (**B**) Variation of the chemiluminiscent intensity over CHLA concentration, and (**C**) Correlation of the CL intensity as a function of CHLA concentration.

The variation of the luminescent intensity with the exposition time can be seen in Figure 3, the readout measurement is stable up to 30 s due to the chemistry of the reaction. Hence, exposition times from 5 to 20 s were studied. The results indicated, that at times lower than 10 s, the image resolution was poor. At exposition times higher than 10 s, the resolution was satisfactory, however, there was no improvement of the longer times (20 s) used. Therefore, 10 s was selected for further experiments.

Finally, and under the above-mentioned instrumental parameters, the variation of the CCD camera response was studied as a function of the concentration of CHLA. Figure 5B shows the image obtained. These results clearly demonstrated that the decrease of the CL signal as a function of the concentration of CHLA was linearly correlated (Figure 5C). It should be noted that the image parameter black and white, which enhanced the intensity of black and white, and the grey contrast up to 1.5 (log parameter) gave rise to an adequate image quality that allowed the determination of the target analyte up to 400 μM.

#### *3.2. Analytical Parameters of the Procedure*

Table 1 summarizes the analytical parameters such as sensitivity as the slope of the calibration curve, linearity, and detection limit. Moreover, intra-and interday precision was also evaluated as the relative standard deviation (%RSD)

**Table 1.** Analytical parameters and precision (%RSD) values for the determination of CHLA by the CCD camera.

a: intercept; sa: standard deviation of a; b: slope, sb: standard deviation of b; LOD: detection limit, RSD: relative standard deviation.

The results indicated that when using a chemiluminiscent image as an analytical response, the working interval was up to 400 μM, and the limit of detection was 20 μM. This LOD was satisfactory for the application in dietary products, however, a more sensitive determination will require the use of other analytical responses, such as the use of a portable luminometer. Intraday and interday precision was also evaluated. In both cases, satisfactory RSD values were achieved for practical applications.

As mentioned before, previous methods based on HPLC have been proposed, and the comparison reveals that, in general, chromatographic methods provided LOD slightly lower than the LOD obtained with the CCD method, recoveries were also satisfactory, and, obviously, selectivity was better when using separation methods [19]. Capillary electrophoresis with CL detection has resulted in an adequate method for the analysis of natural products, however, the LOD was higher than that obtained with the proposed method [20]. NIR has also been proposed for CHLA determination, with successful results. However, multivariate calibration was necessary [21]. Regarding the in situ-based analytical systems, sensors and biosensors have also been studied in depth. A review focused on this topic has been recently published by Munteanu and Apetrei [6]. Recent advances in sensitive materials for electrochemical sensors and biosensors have led to methodologies, with low μM detection limits, which is an advantage if the content of this type of antioxidant is at a trace level [22]. However, precision depends on the accuracy and expertise in the preparation of the sensitive material. Therefore, the proposed methodology shows some advantages over these previously proposed methods; since the determination of CHLA can be performed by image inspection, a semiqualitative analysis for sample screening is possible. In addition, quantitative analysis can be also carried out for samples with contents higher than 20 μM. By another hand, cost of the CCD is lower than chromatographic electrophoretic methods.

#### *3.3. Procedure Validation: Application to the Analysis of Green Coffee Samples*

In order to validate the proposed procedure for its practical application, the analysis of three different green coffee samples, being CHLA the active compound. In addition, and in order to evaluate the matrix effect and the potential interference of other ingredients present in the sample composition, the standard addition method (SAM) and a recovery study were carried out and the slopes of the calibration curves were compared with external calibration. Figure 6 shows the images of both calibration strategies. As was expected, the luminosity intensity of the blank in the external calibration was higher than in the first sample of the standard addition method due to the presence of CHLA, and in both cases, there was a decrease in the CL intensity over the CHLA concentration.

The slopes of the SAM calibration were −0.49 ± 0.3, −0.37 ± 0.02, −0.37 ± 0.01 μM−1 for samples 1, 2, and 3, respectively. The statistical analysis of the SAM slope and the slope of the external calibration (see Table 1) demonstrated that the sample matrix may influence the luminescent intensity, and hence, it may be necessary for SAM as a calibration method. In the samples analysed in this work, only sample 1 was not affected by the matrix effect, since there were no statistical differences in the slope of the calibration graph. However, that influence depended on the commercial product, the matrix effect was observed in samples 2 and 3. The calculated CHLA contents were 145 ± 10, 220 ± 12, and 204 mg CHLA/ capsule for sample 1, sample 2, and sample 3, respectively. These results were then compared with the labels of the commercial products. Sample 1 and sample 2 were labelled with a CHLA content of 157 mg/capsule and 234 mg/capsule, respectively. The statistical comparison with the quantitative analysis carried out with the proposed methodology revealed no statistical differences, and hence, accuracy was demonstrated. In the case of sample 3, the content of CHLA was not labelled. However, the analysis of the sample determined 207 mg/capsule, expressed as CHLA. Recovery values were in the range between 93 ± 8 and 94 ± 9 %. These results indicated that the proposed methodology can be a relevant methodology to evaluate the content of CHLA in the analysed samples, since commercially, some of these products are not properly labelled, and that matrix effect has to be evaluated as a function of the analysed samples. It has been demonstrated that standard addition methods provided satisfactory results in the case of the matrix effect.

**Figure 6.** Comparison of the CL image for external calibration and standard addition method in sample 1.

#### **4. Conclusions**

In this work, an on-site methodology based on chemiluminscent imaging has been proposed to estimate the concentration of CHLA in green coffee samples. The fundamentals of the proposed strategy relied on the use of the CL to decrease the presence of antioxidants, in particular CHLA, produced in the reaction luminol-persulfate under basic conditions. Under the optima chemical conditions, the results indicated that the addition of CHLA induced a stable analytical response that can be used as an analytical signal for the CCD camera as the detection system. The image taken by the CCD camera was processed. At aT=0 ◦C and an exposition time of 10 s, the decrease in the luminiscent intensity was linearly correlated with CHLA concentration, with a LOD of 20 μM and RSD values up to 12%. The satisfactory analytical performance was validated by applying the proposed strategy for the analysis of commercial green coffee samples. The results demonstrated that the matrix effect should be considered when analysing real samples. SAM was adequate for the calibration since the sample matrix may influence the luminosity intensity, however, that effect depended on the sample. The analysis of samples was validated by comparing the results with the labelled products, indicating that satisfactory accuracy was obtained. Hence, it can be concluded that the proposed strategy can be a potential on-site tool to estimate the concentration of antioxidants in dietary products. The analysis of the chemiluminiscent image in the set-up allowed the multisample detection resulting in a costand energy-efficient protocol with reagent consumption at μM level. That demonstrated that the proposed method is a promising sustainable analytical tool for quality control to ensure supplement safety and proper labelling, preventing potential fraud.

**Author Contributions:** S.M.-C.: investigation, methodology, validation. Y.M.-M.: conceptualization, funding acquisition, supervision, revision. C.M.-L.: conceptualization, supervision. P.C.-F.: conceptualization, project administration, funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research has been funded by the EU and the Gobierno de España MCI-AEI (PID2021- 124554NB-I00), the Generalitat Valenciana (PROMETEO Program 2020/078 and EU FEDER- Generalitat Valenciana (ID-FEDER/2018/049).

**Data Availability Statement:** Data is contained within the article.

**Acknowledgments:** Authors acknowledge the EU and the Gobierno de España MCI-AEI (PID2021- 124554NB-I00) and the Generalitat Valenciana (PROMETEO Program 2020/078 and EU FEDER-Generalitat Valenciana (ID-FEDER/2018/049).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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## *Article* **pH and NaCl Optimisation to Improve the Stability of Gold and Silver Nanoparticles' Anti-Zearalenone Antibody Conjugates for Immunochromatographic Assay**

**Thasmin Shahjahan 1,†, Bilal Javed 1,2,3,\*,† , Vinayak Sharma 2 and Furong Tian 1,2**

**Abstract:** The aim of this research is to define optimal conditions to improve the stability of gold and silver nanoparticles' anti-zearalenone antibody conjugates for their utilisation in lateral flow immunochromatographic assay (LFIA). The Turkevich–Frens method was used to synthesise gold nanoparticles (AuNPs), which were between 10 and 110 nm in diameter. Silver nanoparticles (AgNPs) with a size distribution of 2.5 to 100 nm were synthesised using sodium borohydride as a reducing agent. The onset of AuNP and AgNP aggregation occurred at 150 mM and 80 mM NaCl concentrations, respectively. Stable Au and Ag nanoparticle–antibody conjugates were achieved at 1.2 mM of K2CO3 concentration, which corresponds to the pH value of ≈7. Lastly, the highest degree of conjugation between Au and Ag nanoparticles and anti-zearalenone antibodies was at 4 and 6 μg/mL of antibody concentrations. The optimisation of the conjugation conditions can contribute to better stability of nanoparticles and their antibody conjugate and can improve the reproducibility of results of bioreporter molecules in biosensing lateral flow devices.

#### **1. Introduction**

The detection of zearalenone via lateral flow immunochromatographic assays plays a significant role in food safety and agriculture. Zearalenone is a mycotoxin, which is produced by certain fungal species and poses significant risks to both human and animal health when present in food and feed beyond a certain level. Rapid detection of zearalenone through LFIA provides a cost-effective, user-friendly, and on-site screening device for farmers, food processors, and regulatory authorities. It enables the timely identification of contaminated crops and products, which facilitates immediate interventions to mitigate health risks and economic losses. Moreover, the portability and simplicity of the assay make it an essential tool in monitoring and ensuring the safety of our food supply chain.

In the LFIA, the signal labels are generally conjugated with a detection agent, such as antibodies for the specific binding to the target analyte, like mycotoxins. This allows a visible signal to be generated by the recognition elements to indicate the presence of the antigen. The challenge in immobilising antibodies onto AuNPs or AgNOs is avoiding aggregation and ensuring that the antibodies are orientated correctly to maintain the functionality and the accessibility of their paratopes to conjugate with the antigens or analyte [1]. In order for the signal labels to precisely and accurately detect zearalenone, they must fulfil a range of criteria, which include having high stability, exhibiting little or no non-specific binding, being cost-effective, and forming reproducible and efficient conjugates without compromising the functionality and activity of the detection molecule [2].

**Citation:** Shahjahan, T.; Javed, B.; Sharma, V.; Tian, F. pH and NaCl Optimisation to Improve the Stability of Gold and Silver Nanoparticles' Anti-Zearalenone Antibody Conjugates for Immunochromatographic Assay. *Methods Protoc.* **2023**, *6*, 93. https:// doi.org/10.3390/mps6050093

Academic Editors: Victoria Samanidou, Verónica Pino and Natasa Kalogiouri

Received: 18 August 2023 Revised: 21 September 2023 Accepted: 27 September 2023 Published: 3 October 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).