Green Photoluminescent Methodology for Aluminium Traces Quantification in 24-Hour Urine of Subjects with Different Exposition to Tobacco Smoke

The aim of this work has been to evaluate the aluminium (Al(III)) traces contents in 24-hour urine samples from subjects with different tobacco smoke expositions using a new methodology with 1,4-dihydroxy-9, 10-anthraquinone (Quinizarine, QZ) as a fluorosphore. Biological samples were tested using commercial reagent strips and clinical parameters. Al(III) was determined complexing with QZ followed by a solid phase extraction step using Nylon membranes as a solid support. The analyte was subsequently quantified by solid surface fluorescence (SSF, λem= 573, λexc= 490) with a detection limit of 0.88 μg L-1 and quantification limit of 2.69 μg L-1. The calibration curve was linear from 2.69 to 499.13 μg L-1 Al(III) (R2 = 0.9973). Urine samples were successfully analysed with an average recovery close to 100%. Solid phase extraction step showed efficacy to eliminate foreign ions and the highly fluorescent matrix own of urine. Results were validated by electrothermal atomic absorption spectrometry (ETAAS) with an adequate concordance. The new methodology has low operation cost with simple instrumentation and without organic solvent.


Introduction
Aluminium is a human non-essential metal that makes up about 8% of the How to cite this paper: Santarossa, D.G., Talio If the levels of aluminium exposure surpass the capacity of natural detoxification by the part of the organism, it will be accumulated, mainly in the bones, the liver and the brain, being considering as possible cause of renal osteodystrophy, Alzheimer's disease and Parkinson's disease [7] [8] [9]. Monitoring aluminium levels is of paramount importance in the field of clinical chemistry to prevent diseases associated with this metal. Between other biological fluids, urine is particularly attractive because the same patient can obtain the sample by non-invasive sampling procedure.
Tobacco consuming is one of the worst threatens to public health world has faced, due to it is one of the main risk factors of chronic diseases like cancer and pulmonary and cardiovascular diseases [10]. In Argentina, every year more than 40,000 of smokers die because of diseases related to tobacco. Although facts reveal that tobacco use has decreased in the latest 8 years, its effects continue to be alarming which conduct specialists to carry on working to dismiss cigarette consumption [11].
Many studies have been done related to the harmful effects of carbon monoxide, nicotine, tar, irritants and other damaging gases in the tobacco smoke [12].
However, possible effects of cigarettes heavy metals on human biochemical processes have been less studied [13] [14] [15].
However, due to the low level of metal concentration in biological fluids, the introduction of a preconcentration step prior to instrumental detection results indispensable. The traditional methods of preconcentration and separation for metal ions are liquid-liquid extraction, coprecipitation and ion exchange, between others. These methods often require large amounts of high purity organic solvents, which are harmful to health and cause environmental problems [22] [23] [24] [25] [26].
Solid phase extraction (SPE) is being widely used for the analytes preconcentration or separation showing advantages such as high enrichment factors, minimum costs due to low reagent consumption, flexibility and easy automati- zation [27]. Different solid materials such as filter papers, silica gel, exchange resins, aluminium oxides, C 18 membranes, and cyclodextrins, among others have   been successfully employed as supports for SPE. Recently, Nylon membranes   have proved to be a suitable support for the luminescent detection of organic   compounds showing efficacy in the elimination of highly fluorescent matrix [28] [29] [30]. The experimental results showed that this support has good selectivity, low background signal and can be used without previous treatment.
In a previous work, our research group determined Al(III) traces contents present in drink and tap waters of San Luis city, with the aim to know exposition levels to this metal [31], using a CPE-fluorescence combined method. In this opportunity, attending to aluminium is one of the 4700 chemical compounds contained in cigarette smoke, a new methodology is proposed for the study of the tobacco incidence in Al(III) levels in urine samples of population of the same geographic zone. The separation/chemisorption of Al(III) complexed with QZ on Nylon membranes is proposed for the subsequent quantification by solid surface fluorescence (SSF) in 24-hour urine samples of subjects with different levels of tobacco exposure. The experimental variables that affect the sensitivity and precision of the proposed method were investigated and optimized in detail. A centrifuge equipment (ROLCO SRL, Buenos Aires, Argentine) with an angle rotor (6-place, 3500 × g) was used for urine samples processing.

Reagents
Standard solution of 1000 mg L −1 Al (III) was prepared dissolving appropriate amounts of Al(NO 3 ) 3 •9 H 2 O (E-Merck, Darmstadt, Germany) in ultrapure water.
The standard stock solution was stored in a glass bottle at 4˚C in the dark. Lower All chemicals used were analytical grade and ultrapure water was used throughout.

Biological Samples
Per regulation, all participants of the present research signed a written informed consent.
Urine samples of 24 h of healthy subjects with different levels of tobacco addiction were collected in polystyrene bottles without the addition of chemical stabilizers. The volume of each sample was measured (diuresis) in order to calculate the real concentration of Al(III). Then, approximately 10 mL of each sample were centrifuged at 3500 × g during 20 minutes. After that, supernatants were separated, frozen at −18˚C, and reserved for analysis.
Biological samples were physically characterized, namely colour, odor and appearance, presence of sediment, blood and mucus, in order to establish variables that could affect the obtained results. Additionally, urine samples were tested using commercial reagent strips and clinical parameters (pH, urobilinogen, glucose, ketones, bilirubin and proteins, among others) were determined.
Processed samples can be mainly considered within the normal physical and clinical parameters.
In order to assure the obtaining on of representative samples, subjects received detailed information about the collection protocol: • Do not intake vitamin or mineral aggregated 36 h before urine collection.
• Do not drink tap water during 24 h previous to sample collection.
• Samples must be directly remitted to laboratory for analysis; if it is not possible, they must be preserved at 4˚C until analysis.

Treatment of Samples to Remove Calcium
A volume of 2 mL of urine sample, 2 mL of water ultrapure and 1 mL of 4%

Interferences Study
Different amounts of foreign ions, which may be present in samples, (1/1, 1/10, 1/50 and 1/100 Al (III)/interferent ratio) were added to the test solution containing 24.95 µg L −1 Al(III) and the General Procedure was applied.

Dilution Test
In order to establish the proper volume of each urine sample for realizing Al(III) determination, several sample volumes were assayed. The adequate dilution for each sample was that signal which intensities fall into the linearity range of the developed methodology. Dilution test was of 100 µL for subjects with minor exposition and of 0.025 µL for the most exposed subjects. These dilution factors were adopted for the following studies. Al(III) contents were determined by the proposed methodology, employing the obtained volume samples through test dilution.

Accuracy Study
Volumes of 0.1 mL of urine samples were spiked with increasing amounts of Al (III) (24.95 and 49.91 µg L −1 ). Aluminium contents were determined by proposed methodology.

Precision Study
The repeatability (within-day precision) of the method was tested for urine replicate samples (n = 3) spiked with 24.95 and 49.91 µg L −1 of Al(III) and metal contents were determined by proposed methodology.

Validation
Al(III) contents in water samples were determined by ETAAS, using operational conditions previously consigned in apparatus item.

Results and Discussion
Quinizarine (4-dihydroxy-9,10-anthraquinone, QZ) is an organic dye derived from anthraquinone characterized because it forms neutral chelates with metal ions. The formation of a highly fluorescent pink-red complex between Al(III) and QZ will depend on the nature of the solvent, the molar ratio and the medium pH [32] [33] [34]. The experimental parameters that influence the SPE procedure and the SSF determination were studied and optimized.
Systems were prepared containing QZ solution and increasing concentrations of Al(III) at pH 5 using acetic acid/acetate buffer; they were filtered through solid support, dried at to room temperature and SSF signal of each system was determined using a solid sampler holder. It was evidenced that the presence of Al(III) exalted the SSF of the fluorophore (Figure 2).
Retention of the QZ-Al (III) complex was studied using different solid supports. Some of studied solid supports were not effective for the QZ-Al (III) retention; in Table 1, the obtained results are shown. The retention levels for each analyzed supports were verified by measuring SSF intensity at λ em = 573 nm, using a λ exc = 490. The best result was obtained when Nylon membranes were used.     Table 2 summarizes the studied experimental variables, the optimal values for Conditions: λ em = 570 nm; λ exc = 490 nm; C QZ = 1.75 × 10 −6 mol L −1 ; C buffer acetic/acetate = 5 × 10 −2 mol L −1 ; pH = 5; C Al(III) = 24.95 µg L −1 . Other experimental conditions are described under procedure.

Interferences Study
The effect of foreign ions on the recovery of Al(III) was tested. An ion was considered as interferent when it caused a variation in the SSF signal of the analyte greater than ±5%. The assayed ions for interferences study were selected considering the nature of the sample analysed and the possible presence because of exposure to tobacco smoke. Figure 6 shows the obtained results for assayed ions.
At optimal working conditions an adequate tolerance was obtained for interferent/Al(III) 100/1 ratio, at exception made of Ca(II), that it must be removed as it has been detailed in 2.3.2 Treatment of samples to remove calcium.

Applications
To evaluate the usefulness of the developed methodology in the determination of Al(III) traces, samples of 24-hour urine from subjects with different exposition to tobacco were studied.
Attending to smoking habits, the studied subjects can be described as follow: Sample 1-3: Non-smokers subjects. Once in the laboratory, urine samples were observed and characterized respect to physical appearance in order to establish variables that could interfere with Al(III) determinations. All processed samples can be namely considered within the normal physical parameters.
Samples were centrifuged for 10 min at 1000 × g. Supernatants were reserved for Al(III) examination. Urine samples were tested using commercial reagent strips and clinical parameters pH, urobilinogen, glucose, ketones, bilirubin, proteins, nitrite, blood, specific gravity and leucocytes were determined. Processed samples can be mainly considered within the normal clinical parameters.
The accuracy of the methodology was performed using the standard addition method. Diluted urine samples (100 μL, n = 3) were spiked with increasing amounts of Al(III). The repeatability (within-day precision) of the method was evaluated carrying out the proposed methodology, 3 times for each sample.
Obtained results showed satisfactory agreement with adequate precision and recovery. Table 3 shows the recovery results achieved for each urine sample.

D. G. Santarossa et al. American Journal of Analytical Chemistry
As there were not available Certified Materials, in order to check the accuracy of the proposed method, a comparative analysis by electrothermal atomic absorption spectrometry (ETAAS) was carried out using the conditions published in previous works [31].
important exposition sources which contribute to Al(III) found; between others, it can be mentioned foods and cooking modes (use of aluminum cookware). traces allowed its determination by SSF. It constitutes a green alternative of conventional preconcentration methods with additional advantages including low cost, safety, using non-polluting solvents. Considering the results obtained by our research group in reference to Al(III) traces determination, it can be concluded that the San Luis studied population is exposed to multiple sources of exposure to this toxical metal. So it would be important its control and monitoring in order to reduce the exposition sources, especially those related to tobacco consumption in all different forms.