Antimony (Sb), as an element, has been known since ancient times and has been used by many civilizations for different purposes. It is classified as a heavy metal, since it has a specific density of more than 5 gr/cm³ [1], and it has adverse effects on the health and physiology of living organisms [2]. Sb is a metalloid that is often combined with other metals to form hardened alloys used in lead-acid batteries, solder, sheet metal, pipes, metal bearings, castings, and munitions Furthermore, exposure to Sb can come from air, water (tap water, bottled water, and contaminated natural water sources), soil, as well as from cosmetics and older medications (Figure 1Figure 1). Various factors, such as environmental persistence and mobility, cause this element to accumulate in the environment, endangering ecosystems and sources of drinking water for extended periods.

Figure 1. Main sources of exposure to Sb(III).

Commonly, it forms compounds with a valence state of (III) or (V) that have applications in products including polyethylene terephthalate water bottles and fire retardants applied to fabrics [3]. It exists in four valence states: 0, −3, +3, and +5. Most absorbed Sb is excreted rapidly through the urine and feces. Elimination and the route of excretion depend on the type of Sb compound. Urinary excretion is higher for pentavalent than for trivalent Sb compounds, whereas gastrointestinal excretion is higher for trivalent than for pentavalent Sb. Some data on humans, as well as on animals, indicate that a small part of absorbed and retained Sb may have a long biological half-life, especially in the lung. After acute or chronic oral or parenteral exposure to Sb, the highest concentrations are found in the thyroid, adrenals, liver, and kidney [4][5].

Both heavy metals and metalloids pose a particular danger to human health as they are non-biodegradable and therefore prone to accumulate in biological systems. In recent years, concern about Sb exposure has focused on potential mutagenic and carcinogenic risks [4].

It is for this reason that it is necessary to have simple and precise analytical methodologies that allow the quantification of Sb in complex samples. Today, the most commonly analytical methods for Sb are atomic spectroscopies: atomic fluorescence spectrometry [6], inductively coupled plasma mass spectrometry [7]-[9] and inductively coupled plasma optical emission spectroscopy [9]. Although these methodologies are characterized by high precision and reproducibility, the equipment used is expensive and requires high maintenance. Among the less common methodologies are radiochemical methods [10] and UV-vis spectrophotometry; the former are difficult to access due to the need for highly complex security laboratories; the latter, while widely useful in routine laboratories due to their safety and relatively low instrument cost, often prove to be less selective for the analysis of complex samples, being necessary to introduce a previous cleaning treatment [11][12].

In this sense, molecular fluorescence offers interesting analytical characteristics due to its high sensitivity and adequate selectivity, combined with its instrumental availability. Although not all substances possess native fluorescence, its applicability is feasible if an appropriate derivatization reaction is used [13][14].

This work proposes the Sb(III) traces determination by solid surface fluorescence. The QZ fluorosphore in used as chelant reagent of analyte; the product is retained on filter paper treated with HTAB cationic surfactant and the solid support is presented to spectrofluorimeter for spectrocopic measure by solid phase fluorescence. The experimental variables that influence on preconcentration and measurement steps will be studied and optimized. The new methodology will be applied to Sb(III) traces quantification present in argentinien water samples. Given the toxicity of Sb(III), we have focused our determination on this valence state. However, our goal in the future is to adapt the new method for the analysis of total antimony (e.g., after a pre-reduction step).

To evaluate the presence of antimony in natural and bottled drinking water samples from the specific region under study, a complex was formed, and given its low concentrations, a separation/preconcentration step was necessary. A solid-phase extraction (SPE) stage was introduced before the instrumental determination of Sb(III) using SSF. SPE offers a dual benefit: firstly, it allows for the preconcentration of the analyte, thanks to its retention in a small area of the solid support, and secondly, it improves selectivity by isolating the analyte from the complex matrix of the sample, eliminating potential interferences.

To establish the best conditions to analyte quantification, the experimental parameters that influence the SPE procedure and the SSF determination were studied and optimized.

Quinizarin is an organic compound, specifically a dihydroxyanthracenone (1,4-dihydroxyanthracenone), primarily used as a dye [15]. It has a deep orange color and various applications, including as an intermediate in the synthesis of other dyes, coloring fuels, and in research on organic electronic devices, as well as a pH indicator. Its planar structure allows it to absorb visible light, making it a fluorescent compound used as a sensor to detect and quantify certain ions [16]. In this case, it is used as a fluorophore for the quantification of Sb(III).

The concentration of the chelating agent was also studied by keeping the metal concentration constant and varying the QZ concentration between 1 × 10⁻¹⁰ and 3 × 10⁻⁹ mol∙L⁻¹. The concentration of 1 × 10⁻¹⁰ mol∙L⁻¹ was selected as optimal, since it is high enough to guarantee an excess of QZ with respect to the expected Sb(III) content in the studied samples (Table 2 and Figure 3Figure 3).

Figure 3. Optimization of Quinizarin (QZ) concentration.

Systems containing QZ solution and increasing concentrations of Sb(III) at pH 7.5 were prepared using potassium phosphate buffer. These systems were filtered through a solid support, dried at room temperature, and the SSF signal of each was determined using a solid sample holder. The SSF of the fluorophore was observed to increase in the presence of Sb(III) as the metal concentration increased.

The retention of the Sb(III)/QZ complex was studied using different solid supports. No retention of the Sb(III)/QZ complex or insufficient signal was observed on any of the supports tested (Table 2). The retention levels for each support analyzed were verified by measuring the SSF intensity at λ_em = 575 nm, using λ_exc = 490 nm. The best results in terms of sensitivity and reproducibility were obtained with blue ribbon filter paper. Therefore, this support was selected for subsequent tests.

Surfactants play a crucial role in analytical chemistry, where they are used to modify the physical and chemical properties of solutions and interfaces, enabling improved performance in various analytical techniques. In spectroscopy (UV-Vis, fluorescence), their use in analyte solubilization and method sensitivity is particularly noteworthy. In our experience, we have previously used surfactants of different natures to improve the separation, extraction, and/or preconcentration of analytes of diverse nature in matrices of varying complexity, achieving satisfactory quantification [17]-[20]. In this particular case, tests were performed with two surfactants: SDS (anionic surfactant) and HTAB (cationic surfactant).

Hexadecyltrimethylammonium bromide, also known as Cetyltrimethylammonium bromide, [21] is a quaternary ammonium compound widely used in various industrial and laboratory applications. It is an amphiphilic compound, meaning it has a polar “head” and a long, nonpolar “tail”. This structure allows it to reduce surface tension between different phases. It possesses a hydrophilic group with a net positive charge, which gives it a strong affinity for negatively charged surfaces.

In this case, it was the surfactant chosen to pretreat the FP, as it significantly improved the sensitivity of the proposed method. The concentration range studied was 1 × 10⁻⁵ mol∙L⁻¹ to 2 × 10⁻⁴ mol∙L⁻¹ with the optimal condition being 5 × 10⁻⁵ mol∙L⁻¹ M. The blue ribbon filter paper, when treated with the cationic surfactant HTAB, showed a 5-fold improvement in the method’s sensitivity. The optimal surfactant concentration is shown in Table 2 and Figure 4Figure 4. As a possible hypothesis for the enhancement of the fluorescent signal caused by the surfactant, we propose that the interaction of the filter paper/HTAB/Sb(III) complex increases structural rigidity, favoring the enhancement of the fluorescent emission.

Therefore, the blue band filter paper was immersed in a solution of HTAB for 2 minutes, allowed to dry at room temperature, and then used for subsequent tests.

Figure 4. Optimization of HTAB concentration.

The pH is a critical factor in the formation and stability of metal complexes, as it determines the speciation of both the metal ion and the ligand in aqueous solution. Precise pH adjustment is essential to control and optimize the fluorescent intensity and achieve the quantification of Sb(III).

The pH value of the aqueous systems containing a constant concentration of Sb(III) was adjusted between 3.5 and 10.5 by adding a suitable buffer solution. Figure 5Figure 5 shows the results of this study. The highest emission for Sb(III)/QZ was obtained at pH 7.5. Subsequently, the potassium phosphate buffer concentration was tested from 1 × 10⁻⁵ mol∙L⁻¹ to 2 × 10⁻⁵ mol∙L⁻¹ to obtain the maximum fluorescent signal. A buffer concentration of 2 × 10⁻⁵ mol∙L⁻¹ was chosen as optimal (Table 2 and Figure 6Figure 6).

Figure 5. Influence of pH on the signal emission fluorescent of Sb(III) quantification.

Figure 6. Optimization of phosphate buffer concentration.

Among the potential challenges of this approach are adapting it for applicability to more complex matrices (e.g., industrial wastewater) and studying the long-term stability of the analyzed complex on filter paper.

Table 2 summarizes the studied experimental variables, the optimal values for separation/determination of Sb(III)/QZ on FP preptratated with HTAB and analytical parameters for the new proposed methodology. The limit of detection (LOD) was calculated as 3.3 s/m [22], where s is the standard deviation of 10 successive means of the blank and m is the slope of the calibration curve (calibration sensitivity). The limit of quantification (LOQ) was calculated as 10 s/m. The range of linearity was evaluated by checking the linear regression coefficient (R²) of the calibration curve. The linearity of the calibration curve was considered acceptable when R² > 0.9983.

Table 2. Analytical parameters and Figures de Merit for Sb(III) determination.

The analytical quality parameters obtained present advantages to highlight compared to other methods [6]-[9]: wide linearity range (5 orders of magnitude), LOD on the order of ppt, recovery percentages close to 100% which demonstrates good sensitivity, adequate selectivity and wide dynamic range without generating large economic costs or operational complexity as demanded by other equipment.

The proposed method was applied to natural, tap, and commercially bottled water samples from northern and central Argentina to evaluate the population’s potential exposure to this highly toxic substance. Increasing amounts of Sb(III) (from 0.26 to 0.58 µg∙L⁻¹) were added to different sample aliquots. The results showed adequate precision (see Table 4 and Table 5).

The repeatability of the assay was evaluated by repeating the analysis six times for each sample. Table 4 and Table 5 shows the recovery results. The results indicate that the proposed method is suitable for determining Sb(III) in the studied samples.

Table 4. Recovery studies Sb(III) determination in Tap Water sample.

1—(UNSL Campus-San Luis); 2—Northern Zone of the city of San Luis; 3—Southern Zone of the city of San Luis; 4—Potrero de los Funes-San Luis; 5—Nogoli-San Luis; 6—Salta Capital-Central Zone; 7—Salta-Cafayate; 8—Salta-Cachi; 9—Jujuy-Humahuaca; 10—Jujuy-Purmamarca; 11—San Juan Capital; 12—La Rioja Capital; 13—Tucumán-San Miguel de Tucumán; 14—Catamarca Capital; 15—Catamarca-Tinogasta.

Table 5. Recovery studies Sb(III) determination in Mineral Water sample.

1—Bottled Mineral Water-Bottled in San Salvador de Jujuy; 2—Bottled Mineral Water-Brand Bottled in Salta; 3—Bottled Mineral Water-Brand Bottled in Catamarca; 4—Bottled Mineral Water-Brand Bottled in Santiago del Estero; 5—Bottled Mineral WaterBrand Bottled in Tucumán; 6—Bottled Mineral Water-Brand Bottled in Mendoza; 7—Bottled Mineral Water-Brand Bottled in Córdoba 1; 8—Bottled Mineral Water-Brand Bottled in Córdoba 2; 9—Bottled Mineral Water-Brand Bottled in San Luis 1; 10—Bottled Mineral Water-Brand Bottled in San Luis 2. ^aMean ± standard deviation for six determinations. ^b% Recovery = 100 * (analyte concentration in fortified sample – analyte concentration in the unfortified sample)/analyte concentration added in the unfortified sample. ^cCoefficient variation = (SD/mean). ^d%RE (Percent relative error) = 100 × (|measured value − actual value|)/actual value. Measured value: Sb(III) concentration obtained by applying the developed methodology. Actual value: Sb(III) concentration found by ETAAS.

Since certified materials were unavailable, to verify the accuracy of the proposed method, the samples studied were analyzed using electrothermal atomic absorption spectrometry (ETAAS), under the instrumental conditions indicated in Table 1. The results obtained for the replicated samples (n = 6) with the proposed method and the ETAAS technique were statistically compared (t-test), and no significant differences were observed (p = 0.05).

As previously mentioned, exposure to antimony can cause both acute and chronic toxic effects in the population, which is why drinking water is regulated in several countries worldwide. However, the maximum permissible concentration of antimony in mineral bottle water in Argentina is not explicitly established in the main regulations, as is the case for other contaminants. Nevertheless, a guideline value can be inferred indirectly through the National Guideline Levels for Water Quality, which indicate a limit of 1.5 µg∙L⁻¹ for filtered water samples from surface sources and 1.2 µg∙L⁻¹ for unfiltered water samples from groundwater sources [23].

On the other hand, in Argentina, the Argentine Food Code (CAA) establishes a maximum permissible limit for antimony in drinking water, but actual concentrations in the distribution network can vary. The Maximum Permissible Limit: The Argentine Food Code (Chapter XII, “Hydraulic Beverages”) establishes a maximum limit of 0.02 mg/L (milligrams per liter) for antimony in drinking water [24]. All the samples analyzed are below this value; therefore, we can say that the current regulations are being met in our country.

The developed methodology proposes the determination of trace antimony based on the formation of the Sb(III)/QZ complex, employing solid-phase extraction as a preconcentration/separation strategy. This method has demonstrated effectiveness in real water samples from different sources: tap and mineral bottled. It constitutes an environmentally friendly alternative to traditional preconcentration methods, offering advantages such as lower cost (thanks to the use of filter paper as a solid support for analytical retention), ease of use in laboratories with limited resources (since the equipment is inexpensive), availability for installation in toxicological and environmental control laboratories, safety for the operator and the environment, the use of non-polluting solvents, and operational simplicity. The level of sensitivity achieved was comparable to that of atomic spectroscopies. The good tolerance to common components suggests high selectivity and versatility of the new method. Precision and accuracy were evaluated with good agreement. Given the toxicity of the metal studied, the need to minimize its presence in drinking water, and the lack of legislation regulating its content, we emphasize the importance of informing and raising awareness among the population about the precautions that must be taken to minimize exposure. Consuming water from reliable sources, protecting soil and air, and ensuring safety in the workplace make health a priority that must be addressed from various perspectives.

This work was financed with Instituto de Quimica de San Luis (INQUISAL CONICET), Project PIP 11220130100605CO and Universidad Nacional de San Luis (PROICO 02-1120).

This work was supported by Instituto de Química San Luis-Consejo Nacional de Investigaciones Científicas y Tecnológicas (INQUISAL CONICET, Project PIP 11220200101596CO) and Universidad Nacional de San Luis (Project PROICO 02-1120).