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Paper platform for determination of bumetanide in human urine samples to detect doping in sports using digital image analysis
Microchemical Journal 147 (2019) 43–48
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Microchemical Journal journal homepage: www.elsevier.com/locate/microc
Paper platform for determination of bumetanide in human urine samples to detect doping in sports using digital image analysis
T
Vitor Hugo Marques Luiz1, Liliane Spazzapam Lima1, Eduardo Luiz Rossini, 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
ARTICLE INFO
ABSTRACT
Keywords: Paper platform Digital image analysis Bumetanide Doping Urine
Development and application of a new, simple, inexpensive, and eco-friendly method is presented for the quantification of bumetanide in human urine samples for the detection of doping in sports. The method involves the use of a paper platform and digital image detection and is based on the reaction between bumetanide, pdimethylaminocinnamaldehyde (p-DAC), and HCl in a methanolic medium, yielding a colored compound in a delimited area on a qualitative filter paper. The concentrations of p-DAC and HCl were optimized by a chemometric experimental design. After the addition of the reagents to the paper, digital images were obtained by scanning in a multifunctional printer and analyzed using the RGB pattern. The linear bumetanide concentration range was 6.90 × 10−5–1.37 × 10−3 mol L−1 with R2 = 0.993 and the limits of detection (LOD) and quantification (LOQ) were 3.60 × 10−4 and 1.09 × 10−4 mol L−1, respectively. A SPE clean-up step for the samples were required to eliminate the interference of urinary urea. The recoveries obtained varied from 94% to 105%, indicating the absence of significant matrix effects or interferences in urine samples after clean-up step. The proposed method was successfully applied to the analysis of bumetanide in human urine samples.
1. Introduction Non-medical use of therapeutic drugs to alter the performance of athletes in sports competitions is referred to as doping, which is typically identified by the presence of prohibited substances or their metabolites in athletes' body fluids [1–5]. In order to tackle these problems in organized sports, the World Anti-Doping Agency (WADA) was established in 1999 as an independent international agency composed and funded equally by the sports organizations and governments around the world [1,2]. WADA is responsible for the publication and revision of the Prohibited List [2], which includes all the controlled doping substances and methods. However, it is the International Olympic Committee that is responsible for the control of doping and accredits laboratories to undertake the required analysis in order to avoid unfair advantage and to monitor the health of athletes [6]. Some examples of the substances commonly used in doping are diuretics such as bumetanide, furosemide, and triamterene. Diuretics, in addition to helping with weight-loss, mask the use of other doping compounds by decreasing their concentration in urine. This happens because, by increasing the pH of urine, they reduce the excretion of
other doping agents [7]. Urine is one of the most commonly used samples for the detection of doping because it is available in large volumes and can be obtained non-invasively, although it is also possible to analyze sanguine plasma [2]. Bumetanide (3-butylamino-4-phenoxy-5-sulfamoylbenzoic acid) (Fig. 1) is classified as a strong loop diuretic and acts by inhibiting sodium and water reabsorption in the kidneys. It is 40–60 times more potent than furosemide, considering potency by mass of the diuretic drug, its therapeutic dose being 0.5–2.0 mg per day [4]. Its excretion occurs 65% renally and 35% by the metabolic pathway [8]. In addition to being a potent diuretic, it is also used in the treatment of edema associated with heart failure and kidney problems. The common methods described in the literature for the analysis of bumetanide include voltammetry [9], capillary electrophoresis–amperometric detection [10], spectrophotometry [11], diffuse reflectance spectroscopy [12], spectrofluorimetry [3,13], and chromatography [14–20], while the main method of identification is the use of chromatography with mass spectrometry [1,6,17–19]. However, some of these analytical methods [11,12] were employed to investigate bumetanide only in pharmaceutical samples and others have the
Corresponding author. E-mail address: [email protected] (H.R. Pezza). 1 These authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.microc.2019.03.006 Received 30 October 2018; Received in revised form 27 February 2019; Accepted 1 March 2019 Available online 02 March 2019 0026-265X/ © 2019 Elsevier B.V. All rights reserved.
Microchemical Journal 147 (2019) 43–48
V.H.M. Luiz, et al.
Drug-free urine samples were collected from healthy volunteers two no smoker women (25 and 35 years old), one smoker woman (35 years old), one no smoker man (40 years old) - and stored at −20 °C without additives. Before analysis, they were thawed to room temperature. 2.2. Digital image quantification The digital image was used as detection method in the present paper. The software Image J® was used to obtain the color intensity values of the digitalized spot. First, the region of interest was analyzed through the color histogram using a circular selection (w = 135 and l = 135). The mean intensity of the green color channel was used since it is the nearest complementary color to the wavelength of the compound formed in the analysis (520 nm), which guarantees an increase in sensitivity [23,24]. The effective intensity of the green color channel (AG) was calculated as AG = −log(GS/GB), as defined by Abbaspour [24], where GS and GB are the green color intensities of the sample and the blank, respectively. All conversions were carried out in the same manner. All experiments were performed in triplicate.
Fig. 1. Structure of diuretic bumetanide.
disadvantage of the employment of large volumes of organic solvents [14–16], which generate chemical wastes that contribute to environmental pollution, or require expensive and sophisticated instruments [14–20]. Development of portable doping-detection devices based on paper platforms for screening analysis could help to obtain fast responses while avoiding the laborious analysis of many samples. In addition, the advantages of this type of platform over conventional methods of analysis are numerous, including low reagent consumption, small sample volumes, simplicity, portability, and disposability. In 2009, the use of wax as a hydrophobic barrier in a microfluidic platform was proposed by Carrilho et al. [21,22] for analytical applications, and only in 2015 were portable paper-based analytical devices fabricated using wax printing proposed for forensic determination of illicit drugs [22]. To the best of our knowledge, there is no report in the literature on the use of a paper platform for the determination of bumetanide in urine for doping analysis. In the present work, a paper platform was constructed using a wax as a hydrophobic barrier for the determination of bumetanide in human urine to identify doping in sports. The proposed method is based on the reaction between bumetanide, p-dimethylaminocinnamaldehyde (pDAC), and HCl, yielding a violet compound in a delimited area on the paper platform, which was detected by digital image. The use of wax to delimit a circular area on the surface of the qualitative filter paper affords advantages similar to those of microfluidic paper-based analytical devices (μPADs), such as an increase in the sensitivity of the method, low cost, simplicity, and portability. The proposed analytical device can be used to evaluate bumetanide doping in human urine samples.
2.3. Procedure 2.3.1. Spot-test reaction Preliminary tests were performed initially in order to identify the best order of addition of the reagents and the analyte. Based on the findings of the preliminary tests, all subsequent analyses were performed with the addition of 10 μL of the HCl solution to the enclosed area of the filter paper. After it had dried, 10 μL of the p-DAC solution was added and after that had completely dried, 10 μL of the bumetanide solution was added. The solutions were spotted onto the center of the circle on the filter paper using a micropipette. The blank was prepared as described above, but with the bumetanide solution replaced with methanol. The paper platform was digitally scanned and the intensity of the green color (RGB format) was determined using ImageJ®. The effective intensities were used to quantify bumetanide using analytical curves. 2.3.2. Stability study To evaluate the stability of the colored reaction product on the filter paper over time, monitoring of the signal intensity values was conducted every 5 min for 1 h. 2.3.3. Optimization of the variables After identification of the significant parameters, the variables were optimized by multivariate analysis using a central composite design [25] to obtain the best analytical conditions for the spot reaction on the filter paper. All statistical calculations were performed using Statistica 7.0 software. The factors studied were the HCl and p-DAC concentrations in the range of 0.10–1.0 mol L−1 and 0.2–0.6%, respectively. The points of the central composite design were coded unities 2 distant from the central point (coded as the zero point); thus, all points lie on a circumference with 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 and the experiments were analyzed through the RGB color pattern.
2. Experimental 2.1. Apparatus, reagents, solutions, and samples Eppendorf (10–100 μL) and Brand (100–1000 μL) micropipettes were used to deliver measured volumes in the experiments. Scanned images were obtained using an HP PSC 1315 All-in-One multifunction printer, and the images were analyzed using the free software ImageJ®. A Xerox ColorQube 8580 printer with wax-based ink was used to create hydrophobic barriers on Whatman No. 1 qualitative filter paper, which was used as a solid support. Wax was printed on the filter papers to obtain a circular area with a diameter of 1.5 cm and an edge thickness of 0.75 mm before heating. After printing, the papers were placed in an oven at 120 °C for 2 min in order to melt the wax so that it impregnated into the pores of the filter paper. The chromogenic reagent solution consisted of p-dimethylaminocinnamaldehyde (p-DAC, Aldrich, 98%) and hydrochloric acid (HCl, Merck, 37%) at concentrations of 0.60% (w/v) and 0.26 mol L−1, respectively, in a methanolic medium, the solution was stable for 1 week when stored in darkness at 4 °C. Solutions of bumetanide (Deg, > 99%) was prepared daily in methanol.
2.3.4. Study of interferents The interference study was carried out using the possible compounds found in urine such as CaCl2, NaCl, Na2SO4, KH2PO4, KCl, NH4Cl, and, uric acid, ascorbic acid, creatinine, glucose, urea, and albumin. 2.3.5. Sample preparation Before analysis, drug-free urine samples were spiked with 44
Microchemical Journal 147 (2019) 43–48
V.H.M. Luiz, et al.
Table 1 Central Composite Design Matrix for the determination of bumetanide. Experiment
Factors [p-DAC] (%)a
1 2 3 4 5
6 7
8
9 10 11 12 13 a
0.26 0.54 0.26 0.54
[HCl] (mol L−1)a
(−) (+) (−) (+)
0.23 (−) 0.23 (−) 0.87 (+) 0.87 (+) 0.55 (0)
0.139 0.185 0.097 0.134 0.117
0.10 (− 2 )
0.179
0.20 (− 2 ) 0.60 (+ 2 ) 0.40 (0) 0.40 (0) 0.40 0.40 0.40 0.40 0.40
(0) (0) (0) (0) (0)
AG
0.55 (0)
1.00 (+ 2 ) 0.55 (0) 0.55 (0) 0.55 (0) 0.55 (0) 0.55 (0)
0.183
Fig. 2. Spot test of bumetanide using hydrophobic barriers (left) and direct in the paper platform (right). (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
0.117 0.149 0.144 0.134 0.157 0.163
The stability of the colored product was evaluated as described in Section 2.3.2, and it was observed that the effective intensity measurement remained constant for at least 1 h at room temperature (25 °C).
The coded values are shown in parenthesis.
100 × 10−6 mol L−1 of bumetanide. It was used a C18 solid-phase extraction cartridge (Bond Elut, Agilent) [15]. Column conditioning was performed by the addition of 1 mL of methanol followed by 1 mL of deionized water before the addition of 1 mL of the sample. Washing was performed with 5 aliquot of 1 mL of deionized water followed by elution with 1 mL of methanol. For the standard addition and recovery study, the urine sample (100 × 10−6 mol L−1) was spiked with 50.0 × 10−6, 100 × 10−6 and 150 × 10−6 mol L−1 of bumetanide. For the validation of the proposed method, four drug-free urine sample were spiked with 100 × 10−6 mol L−1 of bumetanide and analyzed by the proposed method.
3.1. Optimization of variables Fig. 3 presents the level curve graphic obtained from the fit of the experimental data, which was performed as indicated in Table 1. The level surface graphic indicates that increasing the concentration of pDAC and decreasing the concentration of HCl lead to higher analytical response, and the optimum conditions would be at higher concentration of p-DAC without HCl, although experimentally was found that the HCl was necessary to proceed the reaction. Therefore, other optimizations were not made, and the best conditions for this experiment within the utilized range involved concentrations of 0.6% of p-DAC and 0.26 mol L−1 of HCl. The statistical analysis of the generated surface using Statistica 7.0 that could be described by the quadratic regression equation: AG = 0.0871 + 0.273[HCl] − 0.110[HCl]2 − 0.0150[p-DAC] − 0.0325[p-DAC]2 − 0.0502[HCl][p-DAC].
3. Results and discussion The reaction of secondary aromatic amines with p-DAC in an acidic medium is assumed to take place through condensation of the secondary amino group with the protonated carbonyl group of the reagent to produce an iminium salt, a Schiff base. This reaction has been used for the spectrophotometric determination of secondary amines in acidic media in the presence of surfactants or with heating [26]. In this study, bumetanide (Fig. 1) reacts with p-DAC in acidic medium to form instant violet colored product/compound in a delimited area on the surface of a paper platform, with detection based on analysis of a digital image. For quantitative analysis with reproducible results some points must be taken into account as they can influence the uniformity and the intensity of the spot test. It is important emphasize, when hydrophobic barriers are used, the use of surfactants or pure organic solvents are not possible as it allows the added solutions on the filter paper to pass through the barrier, but the colored product of the reaction was confined by the barriers even it was used organic solvents, as demonstrated in Fig. 2; it produced spots more homogeneous and intense with smaller standard deviation. A study on the order of addition of reagents was performed to verify whether there was any change in the uniformity of the spot or the signal obtained from the digital image of the formed compound. To this, was added in the10 μL of each reagent (HCl and p-DAC) and 10 μL of bumetanide solution were added to the qualitative filter paper in different orders, taking care that each solution had completely dried before the subsequent one was added to be certain that none of the solutions passed over the edges of the polymeric resin printed on the filter paper. It was observed that when the analyte was added before any of the reagents and when p-DAC was added before HCl, the spot formed was not homogeneous. Thus, the best order of addition was found to be 10 μL of HCl, followed by 10 μL of p-DAC and then 10 μL of bumetanide solution.
3.2. Determination of the figures of merit The proposed method was developed considering the linear dynamic range, repeatability, limit of detection (LOD), limit of quantification (LOQ), precision, interference, and recovery. The analytical curve, linear in the concentration range from 6.90 × 10−5 to 1.37 × 10−3 mol L−1, was constructed by preparation
Fig. 3. Response surface for the measurements of the digital image in the green channel in function of [HCl] in mol L−1 and [p-DAC] in % (w/v). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 45
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Table 2 Standard addition and recovery for human urine sample. [BMT] addeda
[BMT] founda
Recoveryb (%)
– 50.0 100 150
104 54.5 96.8 141
– 109 ± 1 96 ± 3 94 ± 2
a b
All values are expressed in 10−6 mol L−1. Average of three determinations.
Table 3 Validation of the proposed method for human urine samplesa. Urine sample 1c 2c 3c 4d
Fig. 4. Color palette for semi-quantitative visual determination of bumetanide. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
a
of appropriate dilutions of a stock solution of bumetanide for measurements using the RGB color pattern. The regression equation obtained was AG = (2.04 ± 0.09) × 10−2[BMT] mol L−1 + (0.05 ± 0.01) with correlation determination R2 = 0.993. The spot-tests for the analytical curve are illustrated in Fig. 4. Additionally, a semi-quantitative visual determination can be performed by comparison with that color palette, when no scanner is available. The LOD and LOQ were determined according to IUPAC recommendations [27]: LOD = 3SDBlank/B and LOQ = 10SDBlank/B, where SDBlank is the standard deviation of measurements of the blank (n = 10) and B is the slope of the linear dynamic range. The calculated LOD and LOQ were 1.19 × 10−5 and 3,98 × 10−5 mol L−1, respectively. The repeatability of the method was assessed by the repetition of some points of the analytical curve at different times on the same day (intra-day) and on different days (inter-day). The coefficients of variation obtained were 2.21% and 1.38% (intra-day), and 2.44% and 2.98% (inter-day) for a 3.60 × 10−4 and 1.09 × 10−4 mol L−1, respectively. The results were all below 10%, demonstrating that the proposed method was repeatable and that the analytical device could be used for the determination of bumetanide in urine.
b c d
[BMT] addeda
[BMT] founda
Recoveryb (%)
100 100 100 100
94.0 99.9 101 105
94 ± 2 100 ± 3 101 ± 2 105 ± 2
All values of concentration are expressed in 10−6 mol L−1. Average of three determinations. No smoker samples. Smoker sample.
3.5. Application The proposed method was applied using four human urine samples spiked with bumetanide at a concentration of 100 × 10−6 mol L−1. The results obtained using the proposed method are illustrate in Table 3. It was analyzed four samples, 1, 2 and 3 from a no smoker donor and sample 4 from a smoker donor. Fig. 5 shows that was retained much more impurities in the cartridge from the sample 4 (smoker urine sample) after the SPE procedure, indicating that some others compounds that could be found in urine sample are retained in the cleanup step and don't interfere in the analysis. The recovery obtained were between 94 and 105% indicating the accuracy of the developed method. The information presented in Table 4 shows a comparison between the developed method and previously methods described for the determination of bumetanide in urine. The proposed method is safer for the operator and the environment, minimizing the use of organic
3.3. Interference study Variation of the analyte signal exceeding ± 5.0% in the determination of bumetanide was considered to be indicative of interference because of the presence of other ions or compounds present in urine. Urea was the only interferent found in this analysis to have a significant effect on the signal. The elimination of urea was performed using solid-phase extraction since urea and all the other ions can be easily eliminated with water, whereas bumetanide is insoluble in water and can be collected after washing using methanol. Preconcentration was not necessary but could also be performed for low concentration bumetanide samples. 3.4. Standard addition and recovery The possibility of matrix interference was investigated using standard additions, and the accuracy of the technique was determined based on the recovery of bumetanide. Recovery tests were performed by spiking human urine samples with known amounts of standard solutions, followed by analysis using the proposed method. The results obtained are given in Table 2. The recoveries obtained varied from 94% to 109%, indicating the absence of significant matrix effects or interferences after the clean-up procedure.
Fig. 5. C18 SPE cartridge after the cleanup procedure for the four samples analyzed. Samples 1, 2 and 3 are from a non-smoker donor and sample 4 is from a smoker donor. 46
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V.H.M. Luiz, et al.
Table 4 Comparison of parameters of the proposed and previously reported methodologies for the determination of bumetanide. Analytical method Voltammetry Capillary electrophoresis–amperometric detection Diffuse reflectance spectroscopy Spectrofluorimetry Liquid chromatography–tandem mass spectrometry Liquid chromatography–diode array detector Spot test-digital image
Observations
LOQ (mol L−1)
Matrix
- Uses modified electrodes with reduced graphene oxide - Samples were centrifuged; - 12 min rinse between runs - Spot-test in paper platform without hydrophobic barriers - Samples were centrifuged; - Determination includes several steps - SPE extraction procedure; - Use large volume of methanol - Solid bar microextraction; - Use large volume of acetonitrile - SPE extraction procedure
solvents such as methanol; it is cheaper than other methodologies because doesn't uses expensive equipment as chromatograph, electrophoresis system, or spectrofluorimeter; uses paper that is a low cost platform to perform analysis; and is portable because the spot test and the digital image steps are simple and easy-to-use. The SPE sample clean-up is a widely used and common pre-treatment technique that is used in several application and has the advantage that sample clean up could be conducted in the field and then the cartridge can be mailed to the laboratory [28] or use portable systems to perform the extraction [29].
[7]
[8] [9] [10] [11]
4. Conclusions We have described the development of a paper platform for the analysis of bumetanide using digital images and successfully applied it in the determination of bumetanide in human urine samples to detect doping in sports. The hydrophobic barriers used in this work increased the sensitivity and the repeatability of the method without any need for heating or the use surfactants. The proposed method presented advantages including simplicity, portability, high sensitivity, repeatability, low reagent consumption, minimal generation of waste, and low cost.
[12] [13]
[14]
[15]
Acknowledgements
[16]
We would like to thank CAPES (CAPES, Grant #001) and the São Paulo Research Foundation (FAPESP, Grant #2015/21733-1) for financial support.
[17]
Author contributions
[18]
V.H.M.L. and L.S.L. contributed equally to this article. [19]
Compliance with ethical standards The authors declare that they have no competing interests.
[20]
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Paper platform for determination of bumetanide in human urine samples to detect doping in sports using digital image analysis
T
Vitor Hugo Marques Luiz1, Liliane Spazzapam Lima1, Eduardo Luiz Rossini, 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
ARTICLE INFO
ABSTRACT
Keywords: Paper platform Digital image analysis Bumetanide Doping Urine
Development and application of a new, simple, inexpensive, and eco-friendly method is presented for the quantification of bumetanide in human urine samples for the detection of doping in sports. The method involves the use of a paper platform and digital image detection and is based on the reaction between bumetanide, pdimethylaminocinnamaldehyde (p-DAC), and HCl in a methanolic medium, yielding a colored compound in a delimited area on a qualitative filter paper. The concentrations of p-DAC and HCl were optimized by a chemometric experimental design. After the addition of the reagents to the paper, digital images were obtained by scanning in a multifunctional printer and analyzed using the RGB pattern. The linear bumetanide concentration range was 6.90 × 10−5–1.37 × 10−3 mol L−1 with R2 = 0.993 and the limits of detection (LOD) and quantification (LOQ) were 3.60 × 10−4 and 1.09 × 10−4 mol L−1, respectively. A SPE clean-up step for the samples were required to eliminate the interference of urinary urea. The recoveries obtained varied from 94% to 105%, indicating the absence of significant matrix effects or interferences in urine samples after clean-up step. The proposed method was successfully applied to the analysis of bumetanide in human urine samples.
1. Introduction Non-medical use of therapeutic drugs to alter the performance of athletes in sports competitions is referred to as doping, which is typically identified by the presence of prohibited substances or their metabolites in athletes' body fluids [1–5]. In order to tackle these problems in organized sports, the World Anti-Doping Agency (WADA) was established in 1999 as an independent international agency composed and funded equally by the sports organizations and governments around the world [1,2]. WADA is responsible for the publication and revision of the Prohibited List [2], which includes all the controlled doping substances and methods. However, it is the International Olympic Committee that is responsible for the control of doping and accredits laboratories to undertake the required analysis in order to avoid unfair advantage and to monitor the health of athletes [6]. Some examples of the substances commonly used in doping are diuretics such as bumetanide, furosemide, and triamterene. Diuretics, in addition to helping with weight-loss, mask the use of other doping compounds by decreasing their concentration in urine. This happens because, by increasing the pH of urine, they reduce the excretion of
other doping agents [7]. Urine is one of the most commonly used samples for the detection of doping because it is available in large volumes and can be obtained non-invasively, although it is also possible to analyze sanguine plasma [2]. Bumetanide (3-butylamino-4-phenoxy-5-sulfamoylbenzoic acid) (Fig. 1) is classified as a strong loop diuretic and acts by inhibiting sodium and water reabsorption in the kidneys. It is 40–60 times more potent than furosemide, considering potency by mass of the diuretic drug, its therapeutic dose being 0.5–2.0 mg per day [4]. Its excretion occurs 65% renally and 35% by the metabolic pathway [8]. In addition to being a potent diuretic, it is also used in the treatment of edema associated with heart failure and kidney problems. The common methods described in the literature for the analysis of bumetanide include voltammetry [9], capillary electrophoresis–amperometric detection [10], spectrophotometry [11], diffuse reflectance spectroscopy [12], spectrofluorimetry [3,13], and chromatography [14–20], while the main method of identification is the use of chromatography with mass spectrometry [1,6,17–19]. However, some of these analytical methods [11,12] were employed to investigate bumetanide only in pharmaceutical samples and others have the
Corresponding author. E-mail address: [email protected] (H.R. Pezza). 1 These authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.microc.2019.03.006 Received 30 October 2018; Received in revised form 27 February 2019; Accepted 1 March 2019 Available online 02 March 2019 0026-265X/ © 2019 Elsevier B.V. All rights reserved.
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Drug-free urine samples were collected from healthy volunteers two no smoker women (25 and 35 years old), one smoker woman (35 years old), one no smoker man (40 years old) - and stored at −20 °C without additives. Before analysis, they were thawed to room temperature. 2.2. Digital image quantification The digital image was used as detection method in the present paper. The software Image J® was used to obtain the color intensity values of the digitalized spot. First, the region of interest was analyzed through the color histogram using a circular selection (w = 135 and l = 135). The mean intensity of the green color channel was used since it is the nearest complementary color to the wavelength of the compound formed in the analysis (520 nm), which guarantees an increase in sensitivity [23,24]. The effective intensity of the green color channel (AG) was calculated as AG = −log(GS/GB), as defined by Abbaspour [24], where GS and GB are the green color intensities of the sample and the blank, respectively. All conversions were carried out in the same manner. All experiments were performed in triplicate.
Fig. 1. Structure of diuretic bumetanide.
disadvantage of the employment of large volumes of organic solvents [14–16], which generate chemical wastes that contribute to environmental pollution, or require expensive and sophisticated instruments [14–20]. Development of portable doping-detection devices based on paper platforms for screening analysis could help to obtain fast responses while avoiding the laborious analysis of many samples. In addition, the advantages of this type of platform over conventional methods of analysis are numerous, including low reagent consumption, small sample volumes, simplicity, portability, and disposability. In 2009, the use of wax as a hydrophobic barrier in a microfluidic platform was proposed by Carrilho et al. [21,22] for analytical applications, and only in 2015 were portable paper-based analytical devices fabricated using wax printing proposed for forensic determination of illicit drugs [22]. To the best of our knowledge, there is no report in the literature on the use of a paper platform for the determination of bumetanide in urine for doping analysis. In the present work, a paper platform was constructed using a wax as a hydrophobic barrier for the determination of bumetanide in human urine to identify doping in sports. The proposed method is based on the reaction between bumetanide, p-dimethylaminocinnamaldehyde (pDAC), and HCl, yielding a violet compound in a delimited area on the paper platform, which was detected by digital image. The use of wax to delimit a circular area on the surface of the qualitative filter paper affords advantages similar to those of microfluidic paper-based analytical devices (μPADs), such as an increase in the sensitivity of the method, low cost, simplicity, and portability. The proposed analytical device can be used to evaluate bumetanide doping in human urine samples.
2.3. Procedure 2.3.1. Spot-test reaction Preliminary tests were performed initially in order to identify the best order of addition of the reagents and the analyte. Based on the findings of the preliminary tests, all subsequent analyses were performed with the addition of 10 μL of the HCl solution to the enclosed area of the filter paper. After it had dried, 10 μL of the p-DAC solution was added and after that had completely dried, 10 μL of the bumetanide solution was added. The solutions were spotted onto the center of the circle on the filter paper using a micropipette. The blank was prepared as described above, but with the bumetanide solution replaced with methanol. The paper platform was digitally scanned and the intensity of the green color (RGB format) was determined using ImageJ®. The effective intensities were used to quantify bumetanide using analytical curves. 2.3.2. Stability study To evaluate the stability of the colored reaction product on the filter paper over time, monitoring of the signal intensity values was conducted every 5 min for 1 h. 2.3.3. Optimization of the variables After identification of the significant parameters, the variables were optimized by multivariate analysis using a central composite design [25] to obtain the best analytical conditions for the spot reaction on the filter paper. All statistical calculations were performed using Statistica 7.0 software. The factors studied were the HCl and p-DAC concentrations in the range of 0.10–1.0 mol L−1 and 0.2–0.6%, respectively. The points of the central composite design were coded unities 2 distant from the central point (coded as the zero point); thus, all points lie on a circumference with 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 and the experiments were analyzed through the RGB color pattern.
2. Experimental 2.1. Apparatus, reagents, solutions, and samples Eppendorf (10–100 μL) and Brand (100–1000 μL) micropipettes were used to deliver measured volumes in the experiments. Scanned images were obtained using an HP PSC 1315 All-in-One multifunction printer, and the images were analyzed using the free software ImageJ®. A Xerox ColorQube 8580 printer with wax-based ink was used to create hydrophobic barriers on Whatman No. 1 qualitative filter paper, which was used as a solid support. Wax was printed on the filter papers to obtain a circular area with a diameter of 1.5 cm and an edge thickness of 0.75 mm before heating. After printing, the papers were placed in an oven at 120 °C for 2 min in order to melt the wax so that it impregnated into the pores of the filter paper. The chromogenic reagent solution consisted of p-dimethylaminocinnamaldehyde (p-DAC, Aldrich, 98%) and hydrochloric acid (HCl, Merck, 37%) at concentrations of 0.60% (w/v) and 0.26 mol L−1, respectively, in a methanolic medium, the solution was stable for 1 week when stored in darkness at 4 °C. Solutions of bumetanide (Deg, > 99%) was prepared daily in methanol.
2.3.4. Study of interferents The interference study was carried out using the possible compounds found in urine such as CaCl2, NaCl, Na2SO4, KH2PO4, KCl, NH4Cl, and, uric acid, ascorbic acid, creatinine, glucose, urea, and albumin. 2.3.5. Sample preparation Before analysis, drug-free urine samples were spiked with 44
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Table 1 Central Composite Design Matrix for the determination of bumetanide. Experiment
Factors [p-DAC] (%)a
1 2 3 4 5
6 7
8
9 10 11 12 13 a
0.26 0.54 0.26 0.54
[HCl] (mol L−1)a
(−) (+) (−) (+)
0.23 (−) 0.23 (−) 0.87 (+) 0.87 (+) 0.55 (0)
0.139 0.185 0.097 0.134 0.117
0.10 (− 2 )
0.179
0.20 (− 2 ) 0.60 (+ 2 ) 0.40 (0) 0.40 (0) 0.40 0.40 0.40 0.40 0.40
(0) (0) (0) (0) (0)
AG
0.55 (0)
1.00 (+ 2 ) 0.55 (0) 0.55 (0) 0.55 (0) 0.55 (0) 0.55 (0)
0.183
Fig. 2. Spot test of bumetanide using hydrophobic barriers (left) and direct in the paper platform (right). (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
0.117 0.149 0.144 0.134 0.157 0.163
The stability of the colored product was evaluated as described in Section 2.3.2, and it was observed that the effective intensity measurement remained constant for at least 1 h at room temperature (25 °C).
The coded values are shown in parenthesis.
100 × 10−6 mol L−1 of bumetanide. It was used a C18 solid-phase extraction cartridge (Bond Elut, Agilent) [15]. Column conditioning was performed by the addition of 1 mL of methanol followed by 1 mL of deionized water before the addition of 1 mL of the sample. Washing was performed with 5 aliquot of 1 mL of deionized water followed by elution with 1 mL of methanol. For the standard addition and recovery study, the urine sample (100 × 10−6 mol L−1) was spiked with 50.0 × 10−6, 100 × 10−6 and 150 × 10−6 mol L−1 of bumetanide. For the validation of the proposed method, four drug-free urine sample were spiked with 100 × 10−6 mol L−1 of bumetanide and analyzed by the proposed method.
3.1. Optimization of variables Fig. 3 presents the level curve graphic obtained from the fit of the experimental data, which was performed as indicated in Table 1. The level surface graphic indicates that increasing the concentration of pDAC and decreasing the concentration of HCl lead to higher analytical response, and the optimum conditions would be at higher concentration of p-DAC without HCl, although experimentally was found that the HCl was necessary to proceed the reaction. Therefore, other optimizations were not made, and the best conditions for this experiment within the utilized range involved concentrations of 0.6% of p-DAC and 0.26 mol L−1 of HCl. The statistical analysis of the generated surface using Statistica 7.0 that could be described by the quadratic regression equation: AG = 0.0871 + 0.273[HCl] − 0.110[HCl]2 − 0.0150[p-DAC] − 0.0325[p-DAC]2 − 0.0502[HCl][p-DAC].
3. Results and discussion The reaction of secondary aromatic amines with p-DAC in an acidic medium is assumed to take place through condensation of the secondary amino group with the protonated carbonyl group of the reagent to produce an iminium salt, a Schiff base. This reaction has been used for the spectrophotometric determination of secondary amines in acidic media in the presence of surfactants or with heating [26]. In this study, bumetanide (Fig. 1) reacts with p-DAC in acidic medium to form instant violet colored product/compound in a delimited area on the surface of a paper platform, with detection based on analysis of a digital image. For quantitative analysis with reproducible results some points must be taken into account as they can influence the uniformity and the intensity of the spot test. It is important emphasize, when hydrophobic barriers are used, the use of surfactants or pure organic solvents are not possible as it allows the added solutions on the filter paper to pass through the barrier, but the colored product of the reaction was confined by the barriers even it was used organic solvents, as demonstrated in Fig. 2; it produced spots more homogeneous and intense with smaller standard deviation. A study on the order of addition of reagents was performed to verify whether there was any change in the uniformity of the spot or the signal obtained from the digital image of the formed compound. To this, was added in the10 μL of each reagent (HCl and p-DAC) and 10 μL of bumetanide solution were added to the qualitative filter paper in different orders, taking care that each solution had completely dried before the subsequent one was added to be certain that none of the solutions passed over the edges of the polymeric resin printed on the filter paper. It was observed that when the analyte was added before any of the reagents and when p-DAC was added before HCl, the spot formed was not homogeneous. Thus, the best order of addition was found to be 10 μL of HCl, followed by 10 μL of p-DAC and then 10 μL of bumetanide solution.
3.2. Determination of the figures of merit The proposed method was developed considering the linear dynamic range, repeatability, limit of detection (LOD), limit of quantification (LOQ), precision, interference, and recovery. The analytical curve, linear in the concentration range from 6.90 × 10−5 to 1.37 × 10−3 mol L−1, was constructed by preparation
Fig. 3. Response surface for the measurements of the digital image in the green channel in function of [HCl] in mol L−1 and [p-DAC] in % (w/v). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 45
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Table 2 Standard addition and recovery for human urine sample. [BMT] addeda
[BMT] founda
Recoveryb (%)
– 50.0 100 150
104 54.5 96.8 141
– 109 ± 1 96 ± 3 94 ± 2
a b
All values are expressed in 10−6 mol L−1. Average of three determinations.
Table 3 Validation of the proposed method for human urine samplesa. Urine sample 1c 2c 3c 4d
Fig. 4. Color palette for semi-quantitative visual determination of bumetanide. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
a
of appropriate dilutions of a stock solution of bumetanide for measurements using the RGB color pattern. The regression equation obtained was AG = (2.04 ± 0.09) × 10−2[BMT] mol L−1 + (0.05 ± 0.01) with correlation determination R2 = 0.993. The spot-tests for the analytical curve are illustrated in Fig. 4. Additionally, a semi-quantitative visual determination can be performed by comparison with that color palette, when no scanner is available. The LOD and LOQ were determined according to IUPAC recommendations [27]: LOD = 3SDBlank/B and LOQ = 10SDBlank/B, where SDBlank is the standard deviation of measurements of the blank (n = 10) and B is the slope of the linear dynamic range. The calculated LOD and LOQ were 1.19 × 10−5 and 3,98 × 10−5 mol L−1, respectively. The repeatability of the method was assessed by the repetition of some points of the analytical curve at different times on the same day (intra-day) and on different days (inter-day). The coefficients of variation obtained were 2.21% and 1.38% (intra-day), and 2.44% and 2.98% (inter-day) for a 3.60 × 10−4 and 1.09 × 10−4 mol L−1, respectively. The results were all below 10%, demonstrating that the proposed method was repeatable and that the analytical device could be used for the determination of bumetanide in urine.
b c d
[BMT] addeda
[BMT] founda
Recoveryb (%)
100 100 100 100
94.0 99.9 101 105
94 ± 2 100 ± 3 101 ± 2 105 ± 2
All values of concentration are expressed in 10−6 mol L−1. Average of three determinations. No smoker samples. Smoker sample.
3.5. Application The proposed method was applied using four human urine samples spiked with bumetanide at a concentration of 100 × 10−6 mol L−1. The results obtained using the proposed method are illustrate in Table 3. It was analyzed four samples, 1, 2 and 3 from a no smoker donor and sample 4 from a smoker donor. Fig. 5 shows that was retained much more impurities in the cartridge from the sample 4 (smoker urine sample) after the SPE procedure, indicating that some others compounds that could be found in urine sample are retained in the cleanup step and don't interfere in the analysis. The recovery obtained were between 94 and 105% indicating the accuracy of the developed method. The information presented in Table 4 shows a comparison between the developed method and previously methods described for the determination of bumetanide in urine. The proposed method is safer for the operator and the environment, minimizing the use of organic
3.3. Interference study Variation of the analyte signal exceeding ± 5.0% in the determination of bumetanide was considered to be indicative of interference because of the presence of other ions or compounds present in urine. Urea was the only interferent found in this analysis to have a significant effect on the signal. The elimination of urea was performed using solid-phase extraction since urea and all the other ions can be easily eliminated with water, whereas bumetanide is insoluble in water and can be collected after washing using methanol. Preconcentration was not necessary but could also be performed for low concentration bumetanide samples. 3.4. Standard addition and recovery The possibility of matrix interference was investigated using standard additions, and the accuracy of the technique was determined based on the recovery of bumetanide. Recovery tests were performed by spiking human urine samples with known amounts of standard solutions, followed by analysis using the proposed method. The results obtained are given in Table 2. The recoveries obtained varied from 94% to 109%, indicating the absence of significant matrix effects or interferences after the clean-up procedure.
Fig. 5. C18 SPE cartridge after the cleanup procedure for the four samples analyzed. Samples 1, 2 and 3 are from a non-smoker donor and sample 4 is from a smoker donor. 46
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Table 4 Comparison of parameters of the proposed and previously reported methodologies for the determination of bumetanide. Analytical method Voltammetry Capillary electrophoresis–amperometric detection Diffuse reflectance spectroscopy Spectrofluorimetry Liquid chromatography–tandem mass spectrometry Liquid chromatography–diode array detector Spot test-digital image
Observations
LOQ (mol L−1)
Matrix
- Uses modified electrodes with reduced graphene oxide - Samples were centrifuged; - 12 min rinse between runs - Spot-test in paper platform without hydrophobic barriers - Samples were centrifuged; - Determination includes several steps - SPE extraction procedure; - Use large volume of methanol - Solid bar microextraction; - Use large volume of acetonitrile - SPE extraction procedure
solvents such as methanol; it is cheaper than other methodologies because doesn't uses expensive equipment as chromatograph, electrophoresis system, or spectrofluorimeter; uses paper that is a low cost platform to perform analysis; and is portable because the spot test and the digital image steps are simple and easy-to-use. The SPE sample clean-up is a widely used and common pre-treatment technique that is used in several application and has the advantage that sample clean up could be conducted in the field and then the cartridge can be mailed to the laboratory [28] or use portable systems to perform the extraction [29].
[7]
[8] [9] [10] [11]
4. Conclusions We have described the development of a paper platform for the analysis of bumetanide using digital images and successfully applied it in the determination of bumetanide in human urine samples to detect doping in sports. The hydrophobic barriers used in this work increased the sensitivity and the repeatability of the method without any need for heating or the use surfactants. The proposed method presented advantages including simplicity, portability, high sensitivity, repeatability, low reagent consumption, minimal generation of waste, and low cost.
[12] [13]
[14]
[15]
Acknowledgements
[16]
We would like to thank CAPES (CAPES, Grant #001) and the São Paulo Research Foundation (FAPESP, Grant #2015/21733-1) for financial support.
[17]
Author contributions
[18]
V.H.M.L. and L.S.L. contributed equally to this article. [19]
Compliance with ethical standards The authors declare that they have no competing interests.
[20]
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