An Overview of the Analytical Methods for the Determination of Organic Ultraviolet Filters in Cosmetic Products and Human Samples

UV filters are a group of compounds commonly used in different cosmetic products to absorb UV radiation. They are classified into a variety of chemical groups, such as benzophenones, salicylates, benzotriazoles, cinnamates, p-aminobenzoates, triazines, camphor derivatives, etc. Different tests have shown that some of these chemicals are absorbed through the skin and metabolised or bioaccumulated. These processes can cause negative health effects, including mutagenic and cancerogenic ones. Due to the absence of official monitoring protocols, there is an increased number of analytical methods that enable the determination of those compounds in cosmetic samples to ensure user safety, as well as in biological fluids and tissues samples, to obtain more information regarding their behaviour in the human body. This review aimed to show and discuss the published studies concerning analytical methods for the determination of organic UV filters in cosmetic and biological samples. It focused on sample preparation, analytical techniques, and analytical performance (limit of detection, accuracy, and repeatability).


Introduction
In recent decades, there has been a progressive increase in UV radiation due to the depletion of the stratospheric ozone layer. This promotes an increase in the number of harmful effects on human health such as skin burns, skin photoaging, damage to the skin's immunological system, pterygium, or skin cancer [1,2]. Accordingly, the number of personal care products containing UV filters has increased rapidly to protect human skin from damaging exposure to sunlight. The currently estimated volume production of UV filters reaches 26.9 million tons [3]. UV filters are frequently added to all types of personal care products such as lotions, shampoos, creams, aftershave products, make-up products, etc. [4][5][6].
The European Union (EU) Regulation 1223/2009-Cosmetics Regulation defines UV filters as "substances which are exclusively or mainly intended to protect the skin against certain UV radiation by absorbing, reflecting or scattering UV radiation" [7]. UV filters are classified into two groups: organic (chemical) UV filters, which absorb UV light, as well as inorganic (physical) UV filters, which reflect and scatter UV radiation. Chemical UV filters are organic molecules capable of absorbing high UV-A and UV-B range radiation. The UV filters have one or more benzene rings and sometimes are conjugated with carbonyl groups [8]. They can be classified into different groups according to their chemical structure: benzophenone derivatives, p-aminobenzoic acid and its derivatives, salicylates, cinnamates, camphor derivatives, triazine derivatives, benzotriazole derivatives, benzimidazole derivatives, and others (Table 1) [9]. One of the most widely used family of UV filters are benzophenones, in particular BP-3, which in 2012 was classified by the US Environmental Protection Agency (US EPA) as "high production volume chemical" [3]. The scale of the problem of the existence of UV filters in the environment was presented by Astle et al. [3], who performed research among Swiss sunbathers on the use of UV filters during one tourist season. On their basis, it was estimated that about 1249 kg of ethylhexyl methoxycinnamate, 152 kg of octocrylene, 145 kg of 4-MBC, and 122 kg of avobenzene were released into Lake Zürich. Therefore, these compounds are the most frequently determined UV filters.
To protect consumers' health, the substances that can be used as UV filters in personal care products and their maximum allowed concentrations are strictly defined in each country [8]. The European Union regulations permit the use of 29 UV filters in cosmetics in concentrations ranging from 2 to 25% (Table 1). However, only two are inorganic (titanium dioxide and zinc oxide) [7]. Organic UV filters have a hydrophilic or lipophilic character and most of them are classified as water-resistant [8].
Despite the limitations on their use in UV filters, there are no established official analytical methods for the determination of these compounds in cosmetics products. However, to maintain the safety and adequate effectiveness of products containing UV filters, analytical methods should be developed to control the content of UV filters in them [10].
Moreover, due to the daily use of cosmetics containing UV filters, such compounds are absorbed through the skin into the body, where they can be metabolized and eventually bioaccumulated and/or excreted. The dermal absorption may result in harmful health effects like dermatitis but also more serious effects, such as mutagenic, cancerogenic, and/or estrogenic activity [11]. Therefore, because of the adverse effects of UV filters on human health and their potential bioaccumulation, such biological samples as urine, plasma, breast milk, semen, or tissues must be checked for their presence.
In this context, this review aimed to provide a comprehensive overview of the developments related to the determination of UV filters in cosmetic samples and biological fluids and tissues, with special emphasis on sample preparation and analytical techniques, as well as the achieved detection limits, accuracy, and repeatability.

Sample Preparation
Cosmetic sample preparation depends on sample type, target analytes, and the technique that is to be used. In general, the preparation of a cosmetic sample does not require a complex pre-treatment sample. This is because the UV filter content in the cosmetic samples is at a sufficiently high level for the sample treatment not to require the extraction and concentration steps. Additionally, in most cases (approximately 90%), liquid chromatography is used for analysis, which enables direct analysis of matrices such as cosmetics. It was alleged that in recent decades the methods of determining UV filters in cosmetics have not been modified too much [11,12].
Cadena-Aizaga M.I. et al. [39]. b Solubility in water at 25 °C.  These procedures are aimed at completely dissolving the sample or leaching the target analytes (e.g., in case of difficult-to-dissolve samples such as wax-balms, lipsticks, or foundations containing insoluble compounds). The achieved high recoveries (Table 2), amounting from 80 to 113%, confirm the effectiveness of these procedures.   Despite the UV filters being the basic components of the samples, no special extraction techniques are needed. However, some authors proposed the use of extraction techniques such as pressurised liquid extraction [35,38], cloud point extraction [14], dispersive liquidliquid microextraction [27], or hollow fibre liquid-phase microextraction [19].

Analytical Techniques
Since the UV filters are part of the cosmetic products, their determination by direct measurement without a prior separation step is impossible. As such, chromatography methods are typically used. The most common chromatographic technique for determining UV filters is liquid chromatography; this is because UV filters have very high boiling points. In the majority of publications, the reversed-phase liquid chromatography coupled with a UV/Vis spectrometry detector with a single wavelength or with a diode-array is commonly used for this purpose. The application of a diode-array detector makes it possible to receive the whole UV spectrum for all peaks. The most used stationary phase is the traditional octadecylsilica type (C18), but octysilica (C8) and amide (C16) have been used as well [9]. In the case of reversed-phase separations, the most used solvents include water, methanol, tetrahydrofuran, acetonitrile, or their mixtures. The more environmentally friendly analytical methods include using the ethanol-water mixture in the mobile phase [6,12,19,22]. Isocratic or gradient elution modes are practised as well. Some substances can be added to the eluent to cut back peak tailing, such as acetic acid in the case of BP-3 [14,35]. Such reagents as phosphate, sodium acetate, and ammonium acetate are used for buffering. Hydroxypropyl-β-cyclodextrin is used as a mobile phase modifier to improve the resolution between varied analytes [6]. Therefore, gas chromatography is used in derivatization procedures with silylating reagents that can increase UV filter volatility, as well as sensitivity. Some publications [32,35] describe the use of gas chromatography coupled with mass spectrometry with electron impact, with N,O-Bis(trimethylsilyl) trifluoroacetamide and acetic anhydrite used as the derivatizing reagents.
Apart from liquid and gas chromatography, there are also a few other separation techniques. One of them is micellar electrokinetic chromatography [14,24,25,27], which utilises uncoated silica capillaries and sodium dodecyl sulphate as a surfactant. Others include thin-layer chromatography [22,36,37,41], supercritical fluid chromatography [30,42], and square wave voltammetry [20]. Table 2 shows the published reports on the determination of individual UV filters, including the sample preparation step and the analytical methodology, as well as the results obtained in terms of the limits of quantification, recovery method, and its precision.

Analytical Methods for UV Filter Determination in Biological Samples
Upon classifying published studies dealing with the determination of UV filters in human samples according to the studied matrix (Tables 3-5), it is clearly visible that the most studied biological matrix is urine (~61%), followed by blood, plasma, or serum (~20%). Other matrices such as milk (~7%), tissues (~5%), and nail, semen, or saliva (~8%) have only been analysed intermittently (Figure 1). analytical methodology, as well as the results obtained in terms of the limits of quantification, recovery method, and its precision.

Analytical Methods for UV Filter Determination in Biological Samples
Upon classifying published studies dealing with the determination of UV filters in human samples according to the studied matrix (Tables 3-5), it is clearly visible that the most studied biological matrix is urine (~61%), followed by blood, plasma, or serum (~20%). Other matrices such as milk (~7%), tissues (~5%), and nail, semen, or saliva (~8%) have only been analysed intermittently (Figure 1).
To date, most research work is focused on the analysis of BP-3 and its metabolites, which have been widely determined in all types of biological samples. Other UV filters that have been analysed, albeit less often, include EMC, OMC, PABA, BDM, EDP, ES, HS, TDS, etc.
In the last decades, a gradual increase in the use of microextraction methods for the isolation and enrichment of analytes in the tested samples has been observed. In the work of Jiménez-Díaz et al. from 2014 [43] on methods for determining UV filters in human samples, the contribution of microextraction methods was only about 7%. Microextraction techniques include the dispersive liquid-liquid microextraction (DLLME) (~10%), as well as the less frequently employed air-assisted liquid-liquid microextraction (AALLME), bar adsorptive microextraction (BAμE), hollow-fibre liquid-phase microextraction (HFLPME), microextraction by packed sorbent (MEPS), stir bar sorptive extraction (SBSE), single-drop microextraction (SDME), solid-phase microextraction (SPME), microextraction using a monolithic stirring extraction unit (MUMSEU), and vortex-assisted dispersive liquid-liquid microextraction (VADLLME) (each of them accounts for ~2%) (Figure 3). To date, most research work is focused on the analysis of BP-3 and its metabolites, which have been widely determined in all types of biological samples. Other UV filters that have been analysed, albeit less often, include EMC, OMC, PABA, BDM, EDP, ES, HS, TDS, etc.

Sample Preparation
To determine UV filters in biological samples, the extraction (~75%) and microextraction (~25%) techniques have been used (Figure 2). Extraction techniques include liquid-liquid extraction (LLE) (~28%), solid-phase extraction (SPE) (~28%), fabric phase sorptive extraction (FPSE) (~5%), as well as the less frequently used accelerated solvent extraction (ASE); microwave-assisted digestion/extraction (MAE); microporous membrane liquid-liquid extraction (MMLLE); matrix solid-phase dispersion (MSPD); sequential injection solid-phase extraction (SI SPE); Quick, Easy, Cheap, Effective, Rugged, and Safe Extraction (QuEChERSExtraction); solid-liquid extraction (SLE); ultrasound-assisted extraction (UAE); and ultrasound-assisted dispersive solid-phase extraction (USAD-SPE) (each~2%).  Urine is the most frequently analysed sample. In urine, the compounds usually occur in free and conjugated forms; hydrolysis is often required to determine their total content (free plus conjugated). Without the hydrolysis step, it is only possible to determine the content of the free ones. The difference between free and conjugated content gives the total conjugated content. Older studies typically used 6 M hydrochloric acid to hydrolyse the bounded compounds [44,45]. Today, enzymatic hydrolysis is achieved by incubating a urine sample with β-glucuronidase or with β-glucuronidase/sulfatase (under specific conditions such as pH, temperature, and time) . After enzymatic hydrolysis, the enzyme is denatured by treated with cold acetonitrile, methanol, or acetic acid to stop the reaction and then separated by centrifugation. The supernatant undergoes the next sample preparation step.
When examining plasma or serum, blood must undergo additional treatment to isolate them (Table 4). Plasma also includes large proteins such as albumin or immunoglobulin. Such treatment consists in the centrifugation of fresh blood with the addition of an anticoagulant. Serum, however, is prepared by centrifuging blood samples without anticoagulant. To determinate the total compound content, the hydrolysis step must be performed with either acid [81] or an enzyme solution [82][83][84][85]. In the case of blood, serum, or plasma samples, protein precipitation is commonly used to reduce matrix interferences. This is performed by mixing the sample with such organic solvents as acetonitrile [60,63,86], methanol [73,81], acetone [83], or formic acid [84,85]. Proteins are denatured, precipitated, and separated through centrifugation.    Total content [97] The most popular extraction technique in the case of plasma, serum, or blood samples is liquid-liquid extraction with the use of such organic solvents as acetonitrile [60,63,86], as well as a methyl tert-butyl ether [81,87] (Table 4). Another technique is dispersive liquid-liquid microextraction (DLLME) with the use of acetone as the disperser solvent and trichloromethane as the extraction solvent [82,83] or acetone as the disperser solvent and chloroform as the extraction solvent [45]. Solid-phase extraction with the C18 sorbent [84,85] and fabric phase sorptive extraction (FPSE) [73] have also been employed.
In the case of milk, semen, and silva samples, determination takes place in the same way as for urine and plasma samples, and as such, the first step is the acid or enzymatic hydrolysis [69,[88][89][90]. Afterwards, acetonitrile [88,90], formic acid [56], isopropanol [89], or methanol [91] is added to precipitate proteins. Finally, in the case of other biological samples such as placenta, nail, or epidermal membrane tissue, homogenisation takes place as well. The samples are shaken and mixed to enable tissue break up ( Table 5).

Analytical Techniques
Even if an exhaustive initial sample treatment is performed to eliminate possible interfering compounds from the sample, an adequate analytical separation technique must still be selected to improve analyte determination. Tables 3-5 present the most used analytical techniques for the detection and quantification of UV filters in biological samples. Liquid chromatography and gas chromatography coupled with MS or MS/MS is the most frequent choice. The choice of either GC or LC is mainly based on the physicochemical properties of the target compounds. GC is usually employed to determinate volatile analytes, whereas LC is applied to quantify both more polar and less volatile compounds.
Liquid chromatography has been used most widely for the determination of UV filters in biological samples. LC coupled with mass spectrometry detectors in tandem is the preferable option. Various ionisation sources have also been used. The most frequently used ionisation mode has been electrospray ionisation (ESI) [45,46,51,55,59,[63][64][65][81][82][83]87,90,92,[94][95][96][97]. Moreover, it was found that ESI + has better efficiency than ESI − [56]. It is a soft ionisation technique suitable for polar and mildly non-polar compounds. Nevertheless, since ion suppression or improvement in the complex matrix may occur, atmospheric pressure chemical ionisation (APCI) [47][48][49]75,77] and atmospheric pressure photoionisation (APPI) [84,85] have also been used. In all mentioned cases, the determination was carried by multiple reaction monitoring (MRM) mode of the most intense transition, with another one employed to confirm the presence of UV filters in biological matrices at very low concentration levels. Yet another type of detector coupled to liquid chromatography is based on UV/Vis spectroscopy. It is often used due to the fact that UV filters exhibit a high absorbance in the UV range of the electromagnetic spectrum [44,52,60,70,[75][76][77]80,86]. Liquid chromatography coupled with a fluorometric detector has been scarcely used because most UV filters do not exhibit fluorescence properties. LC-FL was only used twice-in determining PBSA [71], as well as PEG-25 and PABA [72] in urine samples.
While gas chromatography has been used less often, in most cases it is coupled with mass spectrometry with electron impact [53,54,62,74,79]. In the case of UV filters, a derivatisation step is required before the GC analysis. UV filters have been typically derivatized by using such silylating reagents as N,O-Bis (trimethylsilyl) trifluoroacetamide with trimethylchlorosilane (BSTFA-TMCS) [62] or N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) [54].
Lastly, despite comprehensive sample preparation and the use of carefully select analytical techniques, it must be noted that final results may sometimes be affected by the "matrix effect." This phenomenon may impact quantitative recoveries when using external calibration. As such, it may cause differences in the behaviour of the analytes with the accompanying matrix compounds that one can use to enhance or decrease the signal (e.g., ion suppression in the mass spectrum) or affect the extraction efficiency when the extraction technique is used. This negative effect has been adjusted for by using a matrix-matched calibration (the use of the same matrix without analytes to prepare the standard calibration solutions). In other cases, the standard addition calibration method or an isotopic internal standard was used.

Accuracy and Sensitivity
Tables 3-5 show information about achieved results for different analytical methods used for the determination of UV filters in biological samples.
The analytical methods presented in it resulted in recoveries enabling exhaustive quantification of the target UV filters in the biological matrices, using external or matrixmatched or standard addition calibration. Thus, in the case of urine samples, the greatest recoveries have been achieved for BP-2 (118%) using microextraction by packed sorbent [78] and for EHS (113%) using liquid-liquid extraction [63]. In the case of blood, plasma, and serum samples, the best recoveries have been obtained for BP-1 (146.4%) using liquidliquid extraction [81]. In milk samples, the highest-level recoveries have been achieved for BP-3 (112%) by using salt-assisted liquid-liquid extraction coupled with dispersive solid-phase extraction [88]. The recoveries in the case of the determination of OMC in placenta tissue by using ultrasound-assisted extraction amounted up to 112% [97].
In terms of sensitivity, the published methods (Tables 3-5) enable the determination of UV filters in the low pg mL −1 range.
In the urine samples, the lowest limit of detection (LOD) has been achieved for BP-3 (5 pg mL −1 ) using hollow-fibre liquid-phase microextraction [42]. The LOD for BP-3, 4-MBC, OC, and HS (0.47-0.59 pg mL −1 ) was obtained by using accelerated solvent extraction coupled with solid-phase extraction [62]. In the plasma sample, the LOD was at a level of 0.8 pg mL −1 for BP; it was determined using liquid-liquid extraction in conjunction with solid-phase extraction [87].
In the milk sample, the best LOD has been achieved for BP-6 and BP-1 (0.1 ng mL −1 ) using salt-assisted liquid-liquid extraction coupled with dispersive solid-phase extraction [69]. In the determination of 4-OH-BP in the tissue sample, the LOD of 0.02-10 ng mL −1 has been obtained using solid-liquid extraction [95].
The low levels achieved in the determination of UV filters in biological samples have been influenced by the use of sensitive analytical techniques (e.g., MS/MS), as well as such enrichment techniques as LLE, SPE, MALLE, SPME, SBSE, SDME, HF-LPME, and MALLME.

Conclusions
Organic UV filters are a family of cosmetic ingredients most widely used in a common variety of cosmetic products to protect consumers from UV solar radiation. Since compounds belonging to this group can be metabolised, excreted, and/or bioaccumulated, UV filters may be harmful to the human body. This has made analysing UV filters both in cosmetics products and biological samples a necessity.
Liquid chromatography with MS or UV detection is the dominant method for the determination of UV filters. The large majority of published works used conventional C18 or C8 separation columns. Due to the low level of UV filters in the biological samples (e.g., urine, blood, milk), it is necessary to perform the extraction and clean-up steps before the determination procedure to improve the detection limits. LLE and SPE are the most widely used sample preparation and enrichment methods among all those used. However, these conventional techniques present some drawbacks, such as the consumption of large volumes of sample and often toxic organic solvents, but they are time consuming. Nonetheless, such modern microextraction techniques as MEPS, SPME, SBSE, or DLLME are used as well. However, they are only used in 25% of analytical procedures. Due to the trends of modern analytical techniques towards "Green Analytical Chemistry," they should in the future replace the classic methods of preparing samples for research. This is because of their many advantages, i.e., time-consuming and labour intensity, and above all because they are solvent-free methods.
This review paid special attention to the analytical performance, e.g., limits of detection, accuracy, and repeatability for developed and validated analytical methods. Organic UV filters have been determined to be prevalent in all kinds of biological matrices and are associated with specific markers connected to metabolism, physiological development, and harmful effects in the human body. ultrasound-assisted extraction UHPLC: ultra-high-performance liquid chromatography UHPSFC: ultra-high performance supercritical fluid chromatography UPLC: ultra-performance liquid chromatography USAD-SPE: ultrasound-assisted dispersive solid phase extraction UV/Vis: ultraviolet/visible spectrometry VADLLME: vortex-assisted dispersive liquid-liquid microextraction