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diffuse reflectance spectroscopy to detect doping in sport
Microchemical Journal 109 (2013) 68–72
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Rapid determination of furosemide by combined spot test/diffuse reflectance spectroscopy to detect doping in sport Vitor Hugo Marques Luiz, Leonardo Pezza, Helena Redigolo Pezza ⁎ Instituto de Química, Universidade Estadual Paulista “Júlio de Mesquita Filho”, UNESP, R. Prof. Francisco Degni 55, P.O. Box 355, 14800-900, Araraquara, SP, Brazil
a r t i c l e
i n f o
Article history: Received 17 November 2011 Received in revised form 31 March 2012 Accepted 6 April 2012 Available online 15 April 2012 Keywords: Furosemide Doping Forensic analysis Diffuse reflectance spectroscopy
a b s t r a c t This paper describes the development and application of a simple, cheap, and clean method for the quantification of furosemide in urine samples from athletes, to detect doping, using a combined spot test/diffuse reflectance spectroscopy procedure. The method is based on the complexation reaction of furosemide (5(aminosulfonyl)-4-chloro-2-((furanylmethyl)amino)benzoic acid, dissolved in ethanol, with FeCl3 and the surfactant dodecyltrimethylammonium bromide (DTAB) in aqueous solution, yielding a colored compound on the surface of a filter paper. The reagent concentrations were optimized using a chemometric experimental design. The reflectometric measurements of the complex formed were carried out at 477 nm. The linear range obtained was 1.65–9.00 × 10 − 3 mol L− 1 of furosemide (R = 0.997), and the detection and quantification limits were 4.9 × 10− 4 and 1.62 × 10 − 3 mol L− 1, respectively. The proposed method was successfully applied in the analysis of furosemide in spiked urine, demonstrating that it is a reliable alternative method for the detection of furosemide doping in sport. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Spot tests [1,2] have broad applications in chemical analyses, and play an important role in forensic science because of the wide variety of chemical problems encountered. Forensic chemistry is based on the application of chemical knowledge to the analysis of materials including drugs, blood, urine, and corpses, amongst others [3], during police, workplace, environmental, and sports investigations. In official doping analyses, samples are analyzed for around 300 drugs and metabolites. This requires the availability of fast, portable, and simple methods that should also be sensitive, selective, and in conformity with the principles of Green Chemistry. Doping in sport is the use of drugs or other illicit substances to enhance athletic performance. The analyst's role is to help control the use of these forbidden substances. Control of doping is stipulated by the International Olympic Committee (IOC), which accredits laboratories to perform the analyses [4]. The principal objectives are to avoid unfair advantages, and to maintain athletes' health [5] since many of the substances used can cause serious side effects. Notably, the use of anabolic steroids in men can lead to impotence and to the production of abnormal levels of female hormones. Urine is one of the most common substances used for detection of doping, since large volumes are available and collection is not
⁎ Corresponding author. Fax: + 55 16 33222308. E-mail address: [email protected] (H.R. Pezza). 0026-265X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2012.04.002
invasive [4]. The main method of analysis is chromatography coupled with mass spectrometry. There are many prohibited substances [4,5], including stimulants (which increase levels of motor and cognitive activity), analgesics (for pain relief), diuretics (for loss of weight and as masking agents), and anabolic steroids (which promote cell growth and division, resulting in the development of various tissues, especially muscle and bones) [5,6]. In addition to the use of chemical substances, there are other techniques that are also considered to be doping [6,7], such as increased oxygen transfer, chemical and physical manipulation, and genetic doping. Furosemide (5-(aminosulfonyl)-4-chloro-2-((furanylmethyl)amino) benzoic acid), also known as frusemide or fursemide [8], a derivative of anthranilic acid, is one of the most commonly used diuretics. It is a loop diuretic, and is frequently employed in the treatment of congestive heart failure, chronic renal failure, edemas, and hypertension. Furosemide acts by blocking salt and liquid absorption in the loop of Henle and, to a lesser extent, in the kidney tubules [9,10], resulting in a lower loss of water by osmosis and, consequently, an accumulation of water in the kidney so that a larger quantity of water is excreted. Furosemide is therefore often used to lose weight. Diuretics in general, in addition to causing weight loss, mask the use of other doping compounds by decreasing their concentrations in the urine. This is a result of increasing the pH of the urine, which reduces the excretion of other doping agents. Furosemide is absorbed rapidly following ingestion, and a large fraction (>90%) is excreted unchanged in urine, with the remainder being excreted as metabolites [10]. Numerous techniques have been reported over the years for the determination of furosemide in pharmaceutical preparations and
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biological samples (especially urine), including electroanalytical [11–15], reflectometric [16], spectrophotometric [17–23], and chromatographic [24–32] methods. Several of these methods display good detection limits and accuracy [11,13,15,19,20,22,24,26–32]. However, most of them are time-consuming [20,23], involve a heating step [16,21,23], some extraction [20,23,24,28,29,32], solvent-usage intensive [18,20,24–26,28–30,32] and/or require expensive equipment [13–15,19,22,24–32]. The development of rapid, simple and inexpensive analytical methods is of growing interest, especially since fast decisions are often needed in the forensic chemistry field. In addition, in the development of a new analytical method the amounts and toxicities of the reagents used, and the wastes produced, are as important as any other analytical feature. There is therefore a great need for the development of methods that are less harmful to humans and to the environment, and which are in accordance with the 12 principles of Green Chemistry [33]. Spot tests using filter papers, coupled with diffuse reflectance spectroscopy, is a technique that uses very small quantities of reagents and solvents, making waste treatment easier and reducing costs, in accordance with the principles of Green Chemistry. The fact that spot tests are especially rapid and simple, and that they minimize use of reagents/solvents makes them very useful for the development of analytical methods that address the notion of Green Chemistry, which aims to develop methods and techniques that reduce or eliminate the use and generation of substances hazardous to human health or to the environment [33]. The procedure is very simple, practical, and fast, and can be performed using portable equipment (which can be homemade) for in situ analyses. Filter paper provides a good surface for spot tests because it is composed of cellulose fibers, is very inexpensive, and is manufactured in nearly every part of the world from renewable and recyclable resources [34]. This paper describes the development and application of a simple, portable, and environmentally friendly method for the rapid determination of furosemide in urine. The proposed method is based on the complexation reaction of furosemide, dissolved in ethanol, with FeCl3 in the presence of dodecyltrimethylammonium bromide (DTAB) in aqueous solution, yielding a colored compound on the surface of a filter paper (λmax = 477 nm). Experimental design methodologies were used to optimize the measurement conditions. 2. Experimental 2.1. Instruments The reflectance measurements were made using a handheld integrating sphere (ISP-REF, Ocean Optics, Dunedin, USA) connected to a fiber optic mini-spectrometer (USB2000, Ocean Optics). The USB2000 mini-spectrometer was fitted with a 2048 pixels Sony ILX511 CCD array detector. SpectraSuite software (Ocean Optics) was used for acquisition and storage of spectra. Eppendorf (10–100 μL) and Brand (100–1000 μL) micropipettes were used to deliver measured volumes in the experiments. 2.2. Materials, reagents and solutions Whatman No. 1 qualitative filter paper was used as the solid support. All reagents employed were analytical grade and were used without any prior purification. Ultrapure water (18 MΩ cm, Milli-Q system, Millipore) was used to prepare the solutions. The chromogenic reagent solution consisted of a mixture of iron (III) chloride (FeCl3, Merck, 99%) and dodecyltrimethylammonium bromide (DTAB, Sigma, ~99%), at concentrations of 5.7 × 10− 4 mol L − 1 and 2.7 × 10− 4 mol L− 1, respectively, in aqueous medium. A stock standard solution of 9.0 × 10 − 3 mol L − 1 of furosemide (Purifarma, >99%) was prepared daily in distilled ethanol. Working
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solutions of furosemide were prepared daily by appropriate dilutions of the stock solution with distilled ethanol. 2.3. Samples Seven urine samples were used to evaluate the performance of the proposed method applied to biological material. Three synthetic urine samples (A, B, C) were prepared according to procedures described in the literature [18,35]. Four samples of human urine was collected from three healthy male (D, E, F) and a healthy female (G) donors shortly before analysis. 2.4. Procedure 2.4.1. Spot test reaction For the spot test reaction, the solutions were spotted onto 2.25 cm2 filter papers (Whatman No. 1). 20 μL of the chromogenic reagent solution was spotted, followed immediately afterwards by addition of 20 μL of the furosemide solution (1.65–9.00× 10− 3 mol L − 1). The solutions were spotted onto the center of the filter paper using a micropipette fixed in a holder, according to the procedure described by Tubino [36]. The reflectance measurements were then carried out at 477 nm. A blank was prepared using 20 μL of the chromogenic reagent solution and 20 μL of deionized water. 2.4.2. Optical stability study The optical stability over time of the colored product of the reaction on the filter paper was determined by measuring the AR values at 477 nm every five minutes during one hour. 2.4.3. Study of interferences An interference study was carried out using synthetic urine prepared with urea and a mixture of ions (derived from the compounds CaCl2, NaCl, Na2SO4, KH2PO4, KCl, and NH4Cl). Three different concentrations (50, 100, and 200% greater than that of furosemide) of the ionic compounds were used. 2.4.4. Sample preparation The urine samples were spiked with furosemide and passed through a C18 solid-phase extraction column (Bond Elut JR, 500 mg, Varian) [37]. The column was conditioned by the addition of 3 mL of ethanol followed by 3 mL of deionized water. 20 mL of sample were then added to the column, followed by washing with 10 mL of deionized water and elution with 1 mL of ethanol (resulting in a 20fold pre-concentration). For the application of the proposed method, three samples of synthetic urine (A, B, C), and four of human urine (D, E, F, G), were spiked with furosemide, including 2.0 × 10 − 4 mol L − 1, equivalent about to 90% unchanged excretion of 80 mg of furosemide in one liter of urine (approximately 90% of a furosemide dose is excreted into the urine without being metabolized [18]). The results were compared with a comparative method (Method A) described in the literature [20]. The comparative method consists of the extraction of furosemide from the urine samples using diethyl ether and spectrophotometric detection at 520 nm. 2.4.5. Standard addition and recovery Two representative samples (synthetic and human urine) were spiked with different concentrations of furosemide stock solution. An aliquot of these solutions (20 μL) was taken for the spot test reaction and analyzed by diffuse reflectance at 477 nm. 3. Results and discussion This study describes the development of a combined spot test/ diffuse reflectance technique, using a filter paper surface, for detection
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Table 1 Matrix obtained from the coordinates of the central composite design points. Experiment
1 2 3 4 5 6 7 8 9 10 11 12 13 a
Factors
Sample
[FeCl3] (mol L− 1)a
[DTAB] (mol L− 1)a
0.040 0.090 0.040 0.090 0.030 0.100 0.065 0.065 0.065 0.065 0.065 0.065 0.065
0.016 0.016 0.044 0.044 0.030 0.030 0.010 0.050 0.030 0.030 0.030 0.030 0.030
(−) (+) (−) (+)pffiffiffi (−pffiffiffi2) (þ 2) (0) (0) (0) (0) (0) (0) (0)
Table 2 Recoveries of furosemide added to urine samples.
(−) (−) (+) (+) (0) (0)pffiffiffi (−pffiffiffi2) (þ 2) (0) (0) (0) (0) (0)
Coded values are shown in parentheses.
of furosemide doping in sport, based on the complexation reaction of furosemide with FeCl3 in a micellar dodecyltrimethylammonium bromide (DTAB) medium. The use of this medium increases the sensitivity of spectrophotometric detection [38]. Reproducible quantitative analysis by reflectance measurements requires consideration of several factors that can influence the uniformity and intensity of the spot test reaction. Investigation of several surfactants revealed that use of DTAB generated the best AR signal, with no observable precipitation when in contact with the Fe(III) salt. The order of reagent addition was studied, and tests were performed to identify any need to acidify the solution or adjust the ionic strength (no such adjustments were necessary). Optimum signal and spot homogeneity were achieved when 20 μL of the chromogenic reagent (containing FeCl3 and DTAB) were added prior to addition of 20 μL of furosemide solution. Three solvents for furosemide were tested: ethanol, acetone, and 1:1 acetone/ethanol. Although furosemide dissolved better in acetone and in 1:1 acetone/ethanol, the AR signals obtained were almost half of those achieved when the compound was dissolved in ethanol, which was therefore the solvent chosen to carry out the subsequent experiments. No changes in the uniformity of the colored spot were observed when different volumes of the solutions were added to the filter papers. In view of this, it was decided to use 20 μL of the chromogenic reagent solution, followed by 20 μL of furosemide solution. These volumes produced a spot of sufficient diameter to be measured by the reflectometer.
104 × [FUR]added (mol L− 1)
Synthetic (C)
urine 1.47 2.15 2.84 3.52 Human urine (D) 2.67 3.35 4.04 4.72
104 × [FUR]found (mol L− 1)
Recovery (%)
1.49 2.27 2.97 3.59 2.90 3.49 4.35 5.00
101.4 ± 1.2 105.6 ± 0.8 104.6 ± 0.6 102.0 ± 0.5 108.6 ± 1.9 104.2 ± 2.2 107.7 ± 1.7 105.9 ± 2.0
The optical stability measurements showed that the colored product was stable for at least 60 minutes. 3.1. Optimization of variables After identification of the significant parameters, the variables were optimized by multivariate analysis using central composite design [39] in order to obtain the best analytical conditions for the spot reaction on the filter paper. All the experiments were carried out using a fixed furosemide concentration of 7.50 × 10 − 3 mol L − 1. The statistical calculations were performed using Statistics 6.0 software. The factors studied were the FeCl3 and DTAB concentrations. pffiffiffi The points of a central composite design are coded unities 2distant from the central point (coded pffiffiffi as zero point); hence, all points lie on a circumference of radius 2. Table 1 shows the matrix of the central composite design. The experiment corresponding to the central point was carried out using five replicates. Fig. 1 illustrates the threedimensional response surface graph obtained from the fit of the experimental data. The quadratic regression model could be described by: 2
2
z ¼ −0:017 þ 2:366x−21:020x þ 6:601y−123:125y þ 2:143xy where z is the response factor corresponding to the AR value, x is the concentration of FeCl3, and y is the concentration of DTAB. The darker region within the response surface represents the maximum values of AR, obtained under optimum reaction conditions. The statistical treatments showed that the optimum concentrations were 5.7 × 10 − 4 mol L − 1 (FeCl3) and 2.7 × 10 − 4 mol L − 1 (DTAB). 3.2. Determination of the figures of merit
[DTAB] / mol L-1
The proposed method was validated by considering the linear dynamic range, repeatability, limit of detection (LOD), limit of quantification (LOQ), precision, interferences, and recovery. The analytical curve, which was linear in the range 1.65 to 9.00 mmol L − 1, was constructed by appropriate dilutions of the
Table 3 Results for the determination of furosemide in urine samples.
[FeCl3] / mol L-1
Sample
Proposed method [FUR] × 104 (mol L− 1)
Comparative method [20] [FUR] × 104 (mol L− 1)
t-testc (4, 303)
Aa Ba Ca Db Eb Fb Gb
2.19 ± 0.02 2.09 ± 0.04 2.24 ± 0.03 1.41 ± 0.04 1.59 ± 0.06 2.34 ± 0.01 1.98 ± 0.12
2.18 ± 0.02 2.05 ± 0.02 2.32 ± 0.01 1.37 ± 0.08 1.65 ± 0.02 2.30 ± 0.05 2.00 ± 0.04
1.8 × 10− 4 3.20 3.53 0.82 1.421 1.525 0.484
a
Fig. 1. Central composite design response surface obtained for AR values as a function of FeCl3 and DTAB concentrations.
b c
Synthetic urine. Human urine. Critical values of t at 95% confidence level.
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Table 4 Comparison of some spectrophotometric methods for the determination of furosemide in urine with reflectometric methods. Reagent/reaction
Range mol L− 1
LOD mol L− 1
Remark
Ref.
Complexation with Fe(III)ions in ethanolic medium. λmax = 513 nm Method A: diazotized furosemide coupled with (naphthyl)ethylenediamine (NED). λmax = 520 nm Method B: diazoted furosemide coupled with chromotropic acid (CA). λmax = 500 nm Furosemide reacts with p-dimethylaminocinnamaldehyde. λmax = 585 nm Reaction between furosemide and Fe(III) in micellar media. λmax = 477 nm
1.00 × 10− 4 to 1.00 × 10− 2
3.00 × 10− 5
[18]
5.29 × 10− 6 to 6.35 × 10− 5
8.46 × 10− 7
Requires flow injection set up, organic solvent. Sample: synthetic urine Involves organic solvent extraction, time-consuming Sample: human urine
Involves a heating step. Sample: pharmaceutical formulations Involves extremely low consumption of samples, reagents/solvents and does not require heating step. Sample: synthetic and human urine
[16]
7.56 × 10
−6
7.56 × 10
−3
to 9.07 × 10
−5
3.32 × 10
to 6.05 × 10
−2
2.49 × 10− 3
1.65–9.00 × 10− 3
furosemide stock solution. A blank was prepared containing 20 μL of the chromogenic reagent and 20 μL of ethanol. The regression equation obtained was: AR ¼ −0:0084 þ 0:01903½FUR ðR ¼ 0:997Þ The LOD and LOQ were determined according to IUPAC recommendations [40]: LOD = 3 (s/S), and LOQ = 10 (s/S), where s is the standard deviation of measurements of the blank (n = 10), and S is the slope of the linear dynamic range. The LOD and LOQ were 4.9 × 10 − 4 mol L − 1 and 1.62 × 10 − 3 mol L − 1, respectively. Precision is expressed as the relative standard deviation (RSD) of an analytical response. In order for a method to be considered precise, the RSD should be less than 2.0%. Here, the precision of the technique was assessed by repetition of points of the analytical curve at different times on one day (intra-day), and on different days (inter-day). The coefficients of variation obtained were 1.8% and 1.1%, respectively. 3.3. Interferences study A variation of the signal exceeding ±5.0% in the determination of furosemide, due to the presence of other ions or compounds in urine, was considered to be indicative of interference. Given this criterion, only urea caused interference in the analysis. Urea was therefore eliminated using solid-phase extraction, since it (and all the other ions) could be easily eluted with water, while furosemide (which is insoluble in water) was subsequently eluted using ethanol, during the pre-concentration step. 3.4. Standard addition and recovery Possible matrix interferences were investigated using standard additions, and the accuracy of the technique was determined using the recovery of furosemide. Recovery tests were performed by spiking synthetic and human urine with known amounts of standard solutions, followed by analyses using the proposed method. The results obtained are presented in Table 2. The recoveries ranged from 101.4% to 108.6%. These results show that there were no significant matrix effects in the reflectometric measurements. 3.5. Application The new technique was applied using three samples of synthetic urine and four human urine, all of which were spiked with furosemide. The results obtained by the proposed method were compared statistically (using t-tests at a 95% confidence level) with those obtained using the comparative method [20], and showed good agreement (Table 3). The calculated t values did not exceed the critical values, indicating that there was no significant difference between the two methods in terms of precision and accuracy.
[20]
−7
4.9 × 10− 4
Present method
A comparison of spectrophotometric methods for the determination of furosemide in urine samples with reflectometric methods; their features and drawbacks are enumerated in Table 4. Spectrophotometric procedures [20] for furosemide determination based on diazo-coupling reaction resulting in dye formation are characterized by high sensitivity, but often have drawbacks of pH dependence, coupling time and diazotation temperature. Besides, these procedures use large volumes of carcinogenic reagent(s) or organic solvents, which make it outside of the standards of green chemistry. The other methods [16,18] are not applied to real urine samples. 4. Conclusions This study demonstrates the feasibility of employing diffuse reflectance spectroscopy for the detection of furosemide doping in sports, using a spot test on a filter paper surface. The developed method represents an advantageous alternative to other traditional methods for detection of furosemide in urine because it is inexpensive, simple, portable, precise, accurate, allows rapid determination at low operating costs and requires minimum amounts of samples and reagents/solvents (environmentally friendly analytical method). Acknowledgements We would like to thank CNPq for financial support. References [1] F. Feigl, Spot Tests in Organic Analysis, 7th ed. Elsevier, Amsterdam, 1966. [2] L. Pezza, M. Tubino, C.B. Melios, H.R. Pezza, Anal. Sci. 16 (3) (2000) 313–315. [3] T.L. Bohan, Strengthening forensic science: a way station on the journey to justice, J. Forensic Sci. 55 (1) (2010) 5–7. [4] Doping: a química vai ao Pan, Quím. Hoje 07 (January-March 2007) 14–16. [5] Available at: www.cob.org.br/pesquisa_estudo/antidoping.asp. Accessed on February 3, 2010. [6] Available at: www.wada-ama.org/en. Accessed on February 3, 2010. [7] Informações sobre o uso de medicamentos no esporte. Online booklet available by Comitê Olímpico Brasileiro (COB) at: http://www.cob.org.br/pesquisa_estudo/pdfs/ Livreto_doping_2010.pdf. Accessed on February 3, 2010. [8] The Merck Index, 14th ed. Whitehouse Stations, NJ, USA, 2006. [9] M.E. Bosch, A.J.R. Sanchez, F.S. Rojas, C.B. Ojeda, Recent developments in analytical determination of furosemide, J. Pharm. Biomed. Anal. 48 (2008) 519–532. [10] J. Caslavska, W. Thormann, Rapid analysis of furosemide in human urine by capillary electrophoresis with laser-induced fluorescence and electrospray ionization-ion trap mass spectrometric detection, J. Chromatogr. B 770 (2002) 207–216. [11] A.O. Santini, H.R. Pezza, R. Sequinel, J.L. Rufino, L. Pezza, Potentiometric sensor for furosemide determination in pharmaceuticals, urine, blood serum and bovine milk, J. Braz. Chem. Soc. 1 (2009) 64–73. [12] I. L., T. Dias, G.O. Neto, D.C. Vendramini, C. Sommer, J.L.S. Martins, L.T. Kubota, A poly(vinyl chloride) membrane electrode for the determination of the diuretic furosemide, Anal. Lett. 37 (1) (2004) 35–46. [13] M.B. Barroso, R.M. Alonso, R.M. Jiménez, Electrochemical determination of the loop diuretics piretadine and furosemide in pharmaceutical formulations and urine, Anal. Chim. Acta 305 (1–3) (1995) 332–339. [14] A. Yang, Electrochemistry property and the voltammetric determination of furosemide, Yaowu Fenxi Zazhi 30 (5) (2010) 915–918.
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Microchemical Journal journal homepage: www.elsevier.com/locate/microc
Rapid determination of furosemide by combined spot test/diffuse reflectance spectroscopy to detect doping in sport Vitor Hugo Marques Luiz, Leonardo Pezza, Helena Redigolo Pezza ⁎ Instituto de Química, Universidade Estadual Paulista “Júlio de Mesquita Filho”, UNESP, R. Prof. Francisco Degni 55, P.O. Box 355, 14800-900, Araraquara, SP, Brazil
a r t i c l e
i n f o
Article history: Received 17 November 2011 Received in revised form 31 March 2012 Accepted 6 April 2012 Available online 15 April 2012 Keywords: Furosemide Doping Forensic analysis Diffuse reflectance spectroscopy
a b s t r a c t This paper describes the development and application of a simple, cheap, and clean method for the quantification of furosemide in urine samples from athletes, to detect doping, using a combined spot test/diffuse reflectance spectroscopy procedure. The method is based on the complexation reaction of furosemide (5(aminosulfonyl)-4-chloro-2-((furanylmethyl)amino)benzoic acid, dissolved in ethanol, with FeCl3 and the surfactant dodecyltrimethylammonium bromide (DTAB) in aqueous solution, yielding a colored compound on the surface of a filter paper. The reagent concentrations were optimized using a chemometric experimental design. The reflectometric measurements of the complex formed were carried out at 477 nm. The linear range obtained was 1.65–9.00 × 10 − 3 mol L− 1 of furosemide (R = 0.997), and the detection and quantification limits were 4.9 × 10− 4 and 1.62 × 10 − 3 mol L− 1, respectively. The proposed method was successfully applied in the analysis of furosemide in spiked urine, demonstrating that it is a reliable alternative method for the detection of furosemide doping in sport. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Spot tests [1,2] have broad applications in chemical analyses, and play an important role in forensic science because of the wide variety of chemical problems encountered. Forensic chemistry is based on the application of chemical knowledge to the analysis of materials including drugs, blood, urine, and corpses, amongst others [3], during police, workplace, environmental, and sports investigations. In official doping analyses, samples are analyzed for around 300 drugs and metabolites. This requires the availability of fast, portable, and simple methods that should also be sensitive, selective, and in conformity with the principles of Green Chemistry. Doping in sport is the use of drugs or other illicit substances to enhance athletic performance. The analyst's role is to help control the use of these forbidden substances. Control of doping is stipulated by the International Olympic Committee (IOC), which accredits laboratories to perform the analyses [4]. The principal objectives are to avoid unfair advantages, and to maintain athletes' health [5] since many of the substances used can cause serious side effects. Notably, the use of anabolic steroids in men can lead to impotence and to the production of abnormal levels of female hormones. Urine is one of the most common substances used for detection of doping, since large volumes are available and collection is not
⁎ Corresponding author. Fax: + 55 16 33222308. E-mail address: [email protected] (H.R. Pezza). 0026-265X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2012.04.002
invasive [4]. The main method of analysis is chromatography coupled with mass spectrometry. There are many prohibited substances [4,5], including stimulants (which increase levels of motor and cognitive activity), analgesics (for pain relief), diuretics (for loss of weight and as masking agents), and anabolic steroids (which promote cell growth and division, resulting in the development of various tissues, especially muscle and bones) [5,6]. In addition to the use of chemical substances, there are other techniques that are also considered to be doping [6,7], such as increased oxygen transfer, chemical and physical manipulation, and genetic doping. Furosemide (5-(aminosulfonyl)-4-chloro-2-((furanylmethyl)amino) benzoic acid), also known as frusemide or fursemide [8], a derivative of anthranilic acid, is one of the most commonly used diuretics. It is a loop diuretic, and is frequently employed in the treatment of congestive heart failure, chronic renal failure, edemas, and hypertension. Furosemide acts by blocking salt and liquid absorption in the loop of Henle and, to a lesser extent, in the kidney tubules [9,10], resulting in a lower loss of water by osmosis and, consequently, an accumulation of water in the kidney so that a larger quantity of water is excreted. Furosemide is therefore often used to lose weight. Diuretics in general, in addition to causing weight loss, mask the use of other doping compounds by decreasing their concentrations in the urine. This is a result of increasing the pH of the urine, which reduces the excretion of other doping agents. Furosemide is absorbed rapidly following ingestion, and a large fraction (>90%) is excreted unchanged in urine, with the remainder being excreted as metabolites [10]. Numerous techniques have been reported over the years for the determination of furosemide in pharmaceutical preparations and
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biological samples (especially urine), including electroanalytical [11–15], reflectometric [16], spectrophotometric [17–23], and chromatographic [24–32] methods. Several of these methods display good detection limits and accuracy [11,13,15,19,20,22,24,26–32]. However, most of them are time-consuming [20,23], involve a heating step [16,21,23], some extraction [20,23,24,28,29,32], solvent-usage intensive [18,20,24–26,28–30,32] and/or require expensive equipment [13–15,19,22,24–32]. The development of rapid, simple and inexpensive analytical methods is of growing interest, especially since fast decisions are often needed in the forensic chemistry field. In addition, in the development of a new analytical method the amounts and toxicities of the reagents used, and the wastes produced, are as important as any other analytical feature. There is therefore a great need for the development of methods that are less harmful to humans and to the environment, and which are in accordance with the 12 principles of Green Chemistry [33]. Spot tests using filter papers, coupled with diffuse reflectance spectroscopy, is a technique that uses very small quantities of reagents and solvents, making waste treatment easier and reducing costs, in accordance with the principles of Green Chemistry. The fact that spot tests are especially rapid and simple, and that they minimize use of reagents/solvents makes them very useful for the development of analytical methods that address the notion of Green Chemistry, which aims to develop methods and techniques that reduce or eliminate the use and generation of substances hazardous to human health or to the environment [33]. The procedure is very simple, practical, and fast, and can be performed using portable equipment (which can be homemade) for in situ analyses. Filter paper provides a good surface for spot tests because it is composed of cellulose fibers, is very inexpensive, and is manufactured in nearly every part of the world from renewable and recyclable resources [34]. This paper describes the development and application of a simple, portable, and environmentally friendly method for the rapid determination of furosemide in urine. The proposed method is based on the complexation reaction of furosemide, dissolved in ethanol, with FeCl3 in the presence of dodecyltrimethylammonium bromide (DTAB) in aqueous solution, yielding a colored compound on the surface of a filter paper (λmax = 477 nm). Experimental design methodologies were used to optimize the measurement conditions. 2. Experimental 2.1. Instruments The reflectance measurements were made using a handheld integrating sphere (ISP-REF, Ocean Optics, Dunedin, USA) connected to a fiber optic mini-spectrometer (USB2000, Ocean Optics). The USB2000 mini-spectrometer was fitted with a 2048 pixels Sony ILX511 CCD array detector. SpectraSuite software (Ocean Optics) was used for acquisition and storage of spectra. Eppendorf (10–100 μL) and Brand (100–1000 μL) micropipettes were used to deliver measured volumes in the experiments. 2.2. Materials, reagents and solutions Whatman No. 1 qualitative filter paper was used as the solid support. All reagents employed were analytical grade and were used without any prior purification. Ultrapure water (18 MΩ cm, Milli-Q system, Millipore) was used to prepare the solutions. The chromogenic reagent solution consisted of a mixture of iron (III) chloride (FeCl3, Merck, 99%) and dodecyltrimethylammonium bromide (DTAB, Sigma, ~99%), at concentrations of 5.7 × 10− 4 mol L − 1 and 2.7 × 10− 4 mol L− 1, respectively, in aqueous medium. A stock standard solution of 9.0 × 10 − 3 mol L − 1 of furosemide (Purifarma, >99%) was prepared daily in distilled ethanol. Working
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solutions of furosemide were prepared daily by appropriate dilutions of the stock solution with distilled ethanol. 2.3. Samples Seven urine samples were used to evaluate the performance of the proposed method applied to biological material. Three synthetic urine samples (A, B, C) were prepared according to procedures described in the literature [18,35]. Four samples of human urine was collected from three healthy male (D, E, F) and a healthy female (G) donors shortly before analysis. 2.4. Procedure 2.4.1. Spot test reaction For the spot test reaction, the solutions were spotted onto 2.25 cm2 filter papers (Whatman No. 1). 20 μL of the chromogenic reagent solution was spotted, followed immediately afterwards by addition of 20 μL of the furosemide solution (1.65–9.00× 10− 3 mol L − 1). The solutions were spotted onto the center of the filter paper using a micropipette fixed in a holder, according to the procedure described by Tubino [36]. The reflectance measurements were then carried out at 477 nm. A blank was prepared using 20 μL of the chromogenic reagent solution and 20 μL of deionized water. 2.4.2. Optical stability study The optical stability over time of the colored product of the reaction on the filter paper was determined by measuring the AR values at 477 nm every five minutes during one hour. 2.4.3. Study of interferences An interference study was carried out using synthetic urine prepared with urea and a mixture of ions (derived from the compounds CaCl2, NaCl, Na2SO4, KH2PO4, KCl, and NH4Cl). Three different concentrations (50, 100, and 200% greater than that of furosemide) of the ionic compounds were used. 2.4.4. Sample preparation The urine samples were spiked with furosemide and passed through a C18 solid-phase extraction column (Bond Elut JR, 500 mg, Varian) [37]. The column was conditioned by the addition of 3 mL of ethanol followed by 3 mL of deionized water. 20 mL of sample were then added to the column, followed by washing with 10 mL of deionized water and elution with 1 mL of ethanol (resulting in a 20fold pre-concentration). For the application of the proposed method, three samples of synthetic urine (A, B, C), and four of human urine (D, E, F, G), were spiked with furosemide, including 2.0 × 10 − 4 mol L − 1, equivalent about to 90% unchanged excretion of 80 mg of furosemide in one liter of urine (approximately 90% of a furosemide dose is excreted into the urine without being metabolized [18]). The results were compared with a comparative method (Method A) described in the literature [20]. The comparative method consists of the extraction of furosemide from the urine samples using diethyl ether and spectrophotometric detection at 520 nm. 2.4.5. Standard addition and recovery Two representative samples (synthetic and human urine) were spiked with different concentrations of furosemide stock solution. An aliquot of these solutions (20 μL) was taken for the spot test reaction and analyzed by diffuse reflectance at 477 nm. 3. Results and discussion This study describes the development of a combined spot test/ diffuse reflectance technique, using a filter paper surface, for detection
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Table 1 Matrix obtained from the coordinates of the central composite design points. Experiment
1 2 3 4 5 6 7 8 9 10 11 12 13 a
Factors
Sample
[FeCl3] (mol L− 1)a
[DTAB] (mol L− 1)a
0.040 0.090 0.040 0.090 0.030 0.100 0.065 0.065 0.065 0.065 0.065 0.065 0.065
0.016 0.016 0.044 0.044 0.030 0.030 0.010 0.050 0.030 0.030 0.030 0.030 0.030
(−) (+) (−) (+)pffiffiffi (−pffiffiffi2) (þ 2) (0) (0) (0) (0) (0) (0) (0)
Table 2 Recoveries of furosemide added to urine samples.
(−) (−) (+) (+) (0) (0)pffiffiffi (−pffiffiffi2) (þ 2) (0) (0) (0) (0) (0)
Coded values are shown in parentheses.
of furosemide doping in sport, based on the complexation reaction of furosemide with FeCl3 in a micellar dodecyltrimethylammonium bromide (DTAB) medium. The use of this medium increases the sensitivity of spectrophotometric detection [38]. Reproducible quantitative analysis by reflectance measurements requires consideration of several factors that can influence the uniformity and intensity of the spot test reaction. Investigation of several surfactants revealed that use of DTAB generated the best AR signal, with no observable precipitation when in contact with the Fe(III) salt. The order of reagent addition was studied, and tests were performed to identify any need to acidify the solution or adjust the ionic strength (no such adjustments were necessary). Optimum signal and spot homogeneity were achieved when 20 μL of the chromogenic reagent (containing FeCl3 and DTAB) were added prior to addition of 20 μL of furosemide solution. Three solvents for furosemide were tested: ethanol, acetone, and 1:1 acetone/ethanol. Although furosemide dissolved better in acetone and in 1:1 acetone/ethanol, the AR signals obtained were almost half of those achieved when the compound was dissolved in ethanol, which was therefore the solvent chosen to carry out the subsequent experiments. No changes in the uniformity of the colored spot were observed when different volumes of the solutions were added to the filter papers. In view of this, it was decided to use 20 μL of the chromogenic reagent solution, followed by 20 μL of furosemide solution. These volumes produced a spot of sufficient diameter to be measured by the reflectometer.
104 × [FUR]added (mol L− 1)
Synthetic (C)
urine 1.47 2.15 2.84 3.52 Human urine (D) 2.67 3.35 4.04 4.72
104 × [FUR]found (mol L− 1)
Recovery (%)
1.49 2.27 2.97 3.59 2.90 3.49 4.35 5.00
101.4 ± 1.2 105.6 ± 0.8 104.6 ± 0.6 102.0 ± 0.5 108.6 ± 1.9 104.2 ± 2.2 107.7 ± 1.7 105.9 ± 2.0
The optical stability measurements showed that the colored product was stable for at least 60 minutes. 3.1. Optimization of variables After identification of the significant parameters, the variables were optimized by multivariate analysis using central composite design [39] in order to obtain the best analytical conditions for the spot reaction on the filter paper. All the experiments were carried out using a fixed furosemide concentration of 7.50 × 10 − 3 mol L − 1. The statistical calculations were performed using Statistics 6.0 software. The factors studied were the FeCl3 and DTAB concentrations. pffiffiffi The points of a central composite design are coded unities 2distant from the central point (coded pffiffiffi as zero point); hence, all points lie on a circumference of radius 2. Table 1 shows the matrix of the central composite design. The experiment corresponding to the central point was carried out using five replicates. Fig. 1 illustrates the threedimensional response surface graph obtained from the fit of the experimental data. The quadratic regression model could be described by: 2
2
z ¼ −0:017 þ 2:366x−21:020x þ 6:601y−123:125y þ 2:143xy where z is the response factor corresponding to the AR value, x is the concentration of FeCl3, and y is the concentration of DTAB. The darker region within the response surface represents the maximum values of AR, obtained under optimum reaction conditions. The statistical treatments showed that the optimum concentrations were 5.7 × 10 − 4 mol L − 1 (FeCl3) and 2.7 × 10 − 4 mol L − 1 (DTAB). 3.2. Determination of the figures of merit
[DTAB] / mol L-1
The proposed method was validated by considering the linear dynamic range, repeatability, limit of detection (LOD), limit of quantification (LOQ), precision, interferences, and recovery. The analytical curve, which was linear in the range 1.65 to 9.00 mmol L − 1, was constructed by appropriate dilutions of the
Table 3 Results for the determination of furosemide in urine samples.
[FeCl3] / mol L-1
Sample
Proposed method [FUR] × 104 (mol L− 1)
Comparative method [20] [FUR] × 104 (mol L− 1)
t-testc (4, 303)
Aa Ba Ca Db Eb Fb Gb
2.19 ± 0.02 2.09 ± 0.04 2.24 ± 0.03 1.41 ± 0.04 1.59 ± 0.06 2.34 ± 0.01 1.98 ± 0.12
2.18 ± 0.02 2.05 ± 0.02 2.32 ± 0.01 1.37 ± 0.08 1.65 ± 0.02 2.30 ± 0.05 2.00 ± 0.04
1.8 × 10− 4 3.20 3.53 0.82 1.421 1.525 0.484
a
Fig. 1. Central composite design response surface obtained for AR values as a function of FeCl3 and DTAB concentrations.
b c
Synthetic urine. Human urine. Critical values of t at 95% confidence level.
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Table 4 Comparison of some spectrophotometric methods for the determination of furosemide in urine with reflectometric methods. Reagent/reaction
Range mol L− 1
LOD mol L− 1
Remark
Ref.
Complexation with Fe(III)ions in ethanolic medium. λmax = 513 nm Method A: diazotized furosemide coupled with (naphthyl)ethylenediamine (NED). λmax = 520 nm Method B: diazoted furosemide coupled with chromotropic acid (CA). λmax = 500 nm Furosemide reacts with p-dimethylaminocinnamaldehyde. λmax = 585 nm Reaction between furosemide and Fe(III) in micellar media. λmax = 477 nm
1.00 × 10− 4 to 1.00 × 10− 2
3.00 × 10− 5
[18]
5.29 × 10− 6 to 6.35 × 10− 5
8.46 × 10− 7
Requires flow injection set up, organic solvent. Sample: synthetic urine Involves organic solvent extraction, time-consuming Sample: human urine
Involves a heating step. Sample: pharmaceutical formulations Involves extremely low consumption of samples, reagents/solvents and does not require heating step. Sample: synthetic and human urine
[16]
7.56 × 10
−6
7.56 × 10
−3
to 9.07 × 10
−5
3.32 × 10
to 6.05 × 10
−2
2.49 × 10− 3
1.65–9.00 × 10− 3
furosemide stock solution. A blank was prepared containing 20 μL of the chromogenic reagent and 20 μL of ethanol. The regression equation obtained was: AR ¼ −0:0084 þ 0:01903½FUR ðR ¼ 0:997Þ The LOD and LOQ were determined according to IUPAC recommendations [40]: LOD = 3 (s/S), and LOQ = 10 (s/S), where s is the standard deviation of measurements of the blank (n = 10), and S is the slope of the linear dynamic range. The LOD and LOQ were 4.9 × 10 − 4 mol L − 1 and 1.62 × 10 − 3 mol L − 1, respectively. Precision is expressed as the relative standard deviation (RSD) of an analytical response. In order for a method to be considered precise, the RSD should be less than 2.0%. Here, the precision of the technique was assessed by repetition of points of the analytical curve at different times on one day (intra-day), and on different days (inter-day). The coefficients of variation obtained were 1.8% and 1.1%, respectively. 3.3. Interferences study A variation of the signal exceeding ±5.0% in the determination of furosemide, due to the presence of other ions or compounds in urine, was considered to be indicative of interference. Given this criterion, only urea caused interference in the analysis. Urea was therefore eliminated using solid-phase extraction, since it (and all the other ions) could be easily eluted with water, while furosemide (which is insoluble in water) was subsequently eluted using ethanol, during the pre-concentration step. 3.4. Standard addition and recovery Possible matrix interferences were investigated using standard additions, and the accuracy of the technique was determined using the recovery of furosemide. Recovery tests were performed by spiking synthetic and human urine with known amounts of standard solutions, followed by analyses using the proposed method. The results obtained are presented in Table 2. The recoveries ranged from 101.4% to 108.6%. These results show that there were no significant matrix effects in the reflectometric measurements. 3.5. Application The new technique was applied using three samples of synthetic urine and four human urine, all of which were spiked with furosemide. The results obtained by the proposed method were compared statistically (using t-tests at a 95% confidence level) with those obtained using the comparative method [20], and showed good agreement (Table 3). The calculated t values did not exceed the critical values, indicating that there was no significant difference between the two methods in terms of precision and accuracy.
[20]
−7
4.9 × 10− 4
Present method
A comparison of spectrophotometric methods for the determination of furosemide in urine samples with reflectometric methods; their features and drawbacks are enumerated in Table 4. Spectrophotometric procedures [20] for furosemide determination based on diazo-coupling reaction resulting in dye formation are characterized by high sensitivity, but often have drawbacks of pH dependence, coupling time and diazotation temperature. Besides, these procedures use large volumes of carcinogenic reagent(s) or organic solvents, which make it outside of the standards of green chemistry. The other methods [16,18] are not applied to real urine samples. 4. Conclusions This study demonstrates the feasibility of employing diffuse reflectance spectroscopy for the detection of furosemide doping in sports, using a spot test on a filter paper surface. The developed method represents an advantageous alternative to other traditional methods for detection of furosemide in urine because it is inexpensive, simple, portable, precise, accurate, allows rapid determination at low operating costs and requires minimum amounts of samples and reagents/solvents (environmentally friendly analytical method). Acknowledgements We would like to thank CNPq for financial support. References [1] F. Feigl, Spot Tests in Organic Analysis, 7th ed. Elsevier, Amsterdam, 1966. [2] L. Pezza, M. Tubino, C.B. Melios, H.R. Pezza, Anal. Sci. 16 (3) (2000) 313–315. [3] T.L. Bohan, Strengthening forensic science: a way station on the journey to justice, J. Forensic Sci. 55 (1) (2010) 5–7. [4] Doping: a química vai ao Pan, Quím. Hoje 07 (January-March 2007) 14–16. [5] Available at: www.cob.org.br/pesquisa_estudo/antidoping.asp. Accessed on February 3, 2010. [6] Available at: www.wada-ama.org/en. Accessed on February 3, 2010. [7] Informações sobre o uso de medicamentos no esporte. Online booklet available by Comitê Olímpico Brasileiro (COB) at: http://www.cob.org.br/pesquisa_estudo/pdfs/ Livreto_doping_2010.pdf. Accessed on February 3, 2010. [8] The Merck Index, 14th ed. Whitehouse Stations, NJ, USA, 2006. [9] M.E. Bosch, A.J.R. Sanchez, F.S. Rojas, C.B. Ojeda, Recent developments in analytical determination of furosemide, J. Pharm. Biomed. Anal. 48 (2008) 519–532. [10] J. Caslavska, W. Thormann, Rapid analysis of furosemide in human urine by capillary electrophoresis with laser-induced fluorescence and electrospray ionization-ion trap mass spectrometric detection, J. Chromatogr. B 770 (2002) 207–216. [11] A.O. Santini, H.R. Pezza, R. Sequinel, J.L. Rufino, L. Pezza, Potentiometric sensor for furosemide determination in pharmaceuticals, urine, blood serum and bovine milk, J. Braz. Chem. Soc. 1 (2009) 64–73. [12] I. L., T. Dias, G.O. Neto, D.C. Vendramini, C. Sommer, J.L.S. Martins, L.T. Kubota, A poly(vinyl chloride) membrane electrode for the determination of the diuretic furosemide, Anal. Lett. 37 (1) (2004) 35–46. [13] M.B. Barroso, R.M. Alonso, R.M. Jiménez, Electrochemical determination of the loop diuretics piretadine and furosemide in pharmaceutical formulations and urine, Anal. Chim. Acta 305 (1–3) (1995) 332–339. [14] A. Yang, Electrochemistry property and the voltammetric determination of furosemide, Yaowu Fenxi Zazhi 30 (5) (2010) 915–918.
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