Accepted Manuscript Rapid and Sensitive Determination of Clenbuterol in Porcine Muscle and Swine Urine Using a Fluorescent Probe Xu Jing, Bing Bai, Chenxuan Zhang, Wenying Wu, Liming Du, Hailong Liu, Guojun Yao PII: DOI: Reference:
S1386-1425(14)01444-9 http://dx.doi.org/10.1016/j.saa.2014.09.086 SAA 12762
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
12 April 2014 19 September 2014 21 September 2014
Please cite this article as: X. Jing, B. Bai, C. Zhang, W. Wu, L. Du, H. Liu, G. Yao, Rapid and Sensitive Determination of Clenbuterol in Porcine Muscle and Swine Urine Using a Fluorescent Probe, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.09.086
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Rapid and Sensitive Determination of Clenbuterol in Porcine Muscle and Swine Urine Using a Fluorescent Probe a*
Xu Jing , Bing Bai , Chenxuan Zhang , Wenying Wu , Liming Du , Hailong Liu , Guojun Yao a
Department of Applied Chemistry, China Agricultural University, Beijing, 100193, P. R. China
Criminal Technology Department, Shanxi province Linfen City Public Security Bureau, Linfen, 041000, P. R. China c
Analytical and Testing Center, Shanxi Normal University, Linfen, 041000, P. R. China
*Corresponding author - [email protected]
, Tel.: +86 357 3337281; fax: +86 357 3337281 Abstract The feed additive Clenbuterol hydrochloric acid (CLB) is non-fluorescent, thus it is difficult to quantify through direct fluorescent method. Palmatine (PAL) can react with cucurbituril (CB) to form stable complexes as a fluorescent probe. Significant quenching of the fluorescence intensity of the CB-PAL complex was observed with the addition of CLB. Based on the significant quenching of the supramolecular complex fluorescence intensity, a novel spectrofluorimetric method with high convenience, selectivity and sensitivity was developed for the determination of CLB. The fluorescence quenching values (∆ F) showed good linear relationship with CLB concentrations from 0.011 µgg mL−1 to 4.2 µgg mL−1 with a detection limit 0.004 µgg mL−1. In this research, an ultrasound treatment replaced the former time-consuming shake method to form stable complexes. The proposed spectrofluorimetric method had been successfully applied to the determination of CLB in porcine muscle and swine urine with good precision and accuracy. The competing reaction and the supramolecular interaction mechanisms between the CLB and PAL as they fight for occupancy of the CB cavity were studied using spectrofluorimetry, 1H NMR, and molecular modeling calculations. Interestingly, results indicate that two stable CB-CLB complexes were formed. Keywords: Clenbuterol, Cucurbituril, Palmatine, Fluorescent probe Introduction
Cucurbit[n]uril (CB[n], n = 5−8, 10, Fig. 1) is a macrocyclic compound consisting of n glycoluril units connected by 2n methylene bridges. The symmetrical supermolecule hosts resemble a hollow barrel with hydrophobic cavities and restrictive polar portals lined with ureido carbonyl groups location [1-3]. These characteristics enable to form significant stable complexes with a variety of guest molecules in aqueous solution. The medical applications of the CB[n] has been developing rapidly, especially the drug release [4,5]. However, little attention has been devoted to their potential analytical applications of food safety, especially the determination of feed additive residues. (Fig. 1) Palmatine (PAL, Fig. 1) is a natural isoquinoline alkaloid . Aqueous solution of PAL exhibits weak native fluorescence. However, the fluorescence of PAL in aqueous solutions was observed to be greatly enhanced in the presence of CB . Clenbuterol (CLB, Fig. 1) is a member of β -adrenergic agonists, which enhances the lean meat/fat ratio and increases the efficiency of feed conversion by inhibiting fat synthesis, improving muscular mass, and decreasing adipose tissue deposition in livestock production. Thus, CLB is also called as a “leanness enhancer” and illegally used as a feed additive in meat industry . However, once the animals are fed with CLB, the residue may remain in the meat and liver for a long time as a result of its long half-life, so it may enter the body and distribute throughout the body and result in serious harmful health problems to human such as cardiovascular and central nervous diseases . Hence, many countries including China, the United States and most European countries have forbidden the use of CLB as feed additives . A number of assays have been reported for the determination of CLB in biological samples, including ELISA , HPLC , LC-MS , GC-MS , immunochromatographic , electrochemical limmunosensors , electrochemical biosensor , and fluorescence biosensor . Although these strategies exhibit promising results for sensitive detection of CLB, there are still some hindrances including expensive instrument, long operation times, and tedious sample preparation.Spectrofluorometry is considered one of the most convenient analytical technique, owing to its inherent simplicity, low cost, high sensitivity, and wide availability in most
quality control laboratories [18,19]. Therefore, a rapid, simple, and sensitive fluorescent method is required to monitor CLB in porcine muscle and swine urine samples. Considering that aqueous solutions of CLB have no native fluorescence, it cannot be directly determined through the normal fluorimetric method. Based on the significant quenching of the supramolecular complex fluorescence intensity, a spectrofluorimetric method of high convenience, sensitivity and selectivity was developed to determine CLB in aqueous solution. In the present research, we replaced the former time-consuming shake method and an improved ultrasound treatment was applied to accelerate the progress of quenching. This method was successfully used to determine CLB in real samples, and satisfactory assay results were obtained. Additionally, 1H NMR and molecular modeling calculations results indicate that two stable CB-CLB complexes were formed and co-exist in the solution. Experimental Apparatus Fluorescence spectra and intensity were obtained using an Agilent Technologies Cary Eclipse fluorescence spectrofluorometer (Agilent, Australia) equipped with a pulsed lamp. The slit widths of both excitation and emission monochromators were set to 5 nm. The fluorescence spectra were recorded at a scan rate of 600 nm min−1. All measurements were performed using a standard 10 mm path-length quartz cell at 25.0 °C ± 0.5 °C. The pH values were measured using a pHS-3 TC digital precision pH meter (Shanghai, China). 1H NMR spectra were obtained using a Bruker DRX-600MHz spectrometer (Switzerland) in D2O. Reagents PAL and CLB were obtained from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) without further treatment. CLB was dissolved in double-deionized water to prepare stock standard solutions of 100 µg mL−1. PAL was dissolved in double-deionized water to prepare stock solutions with final concentration of 1.0 mM. CB was prepared and characterized according to reported procedure . CB stock solution of 1.0 mM was
prepared by dissolving CB in double-deionized water. Stock standard solutions were stable for several weeks at room temperature. Standard working solutions were prepared by diluting the stock standard solutions with double-deionized water before use. All other chemicals were of analytical reagent grade, and double-deionized water was used throughout the procedure. Experimental procedure 0.7 mL solution of 0.2 mM CB was poured into a 10 mL colorimetric flask, to which 0.7 mL of the 0.2 mM PAL solution and 1.0 mL of 0.01 M hydrochloric acid were also added. Suitable amounts of CLB solution were sequentially added to the flask. The mixture was diluted to volume with doubledeionized water. Then an ultrasound treatment was used to accelerate the reaction rate. The fluorescence intensity values of the solution (FPAL-CB-CLB) and the blank solution (FPAL-CB) were measured at 495 nm using an excitation wavelength of 343 nm. Results and Discussion The fluorescence enhancement of PAL in the presence of CB CB is spectroscopically inert in aqueous solutions, the aqueous solution of PAL has weak native fluorescence, and the maximum excitation and emission wavelengths are at 235 nm and 370 nm, respectively. However, when CB was added to the aqueous solution of PAL, a significant increase in fluorescence intensity was observed, and accompanied by a red shift of emission wavelengths from 370 nm to 495 nm. The changes in the features of the fluorescence spectra of the solutions are attributed to the formation of an inclusion complex between PAL and CB . The fluorescence quenching of CB-PAL in the presence of CLB Significant quenching of fluorescence intensity of the CB-PAL complex with the addition of CLB was observed. The fluorescence spectra of the CB-PAL complex, in the presence of different concentrations of CLB, are shown in Fig. 2. Fluorescence intensity decreased with the increased CLB concentration, which is likely due to the competition between CLB and PAL molecules for occupancy of the CB cavity. Parts of the PAL molecule can be expelled from the cavity of CB by the
introduction of the CLB, thereby reducing the fluorescence intensity of CB-PAL because of the formation of a new inclusion complex between CLB and CB. (Fig. 2)
Effect of PAL concentration on the fluorescence intensity of the CB-PAL complex The effect of varying PAL concentrations on the fluorescence intensity of the CB-PAL complex was studied. The concentration of PAL was varied from 1.0 µΜ to 17.0 µΜ. The fluorescence intensity of the CB-PAL complex was gradually enhanced as PAL concentration increased until it reached the maximum inclusion equilibrium at CB saturation when the concentration of PAL was 14.0 µΜ. In the present paper, PAL served as a fluorescent probe; thus, determining the proper concentration was crucial. If the PAL concentration is too low, the sensitivity of the probe will also be low. Conversely, a very high concentration may not help determine the optimum detection limit of the analyte. Taking everything into consideration, the optimal PAL concentration was 14.0 µΜ.
Influence of pH In order to obtain the the maximum ∆F, the pH values were studied in the range of 1.0 to 7.0, adjusted by diluted HCl. The results indicate that ∆F is almost constant in the range. However, ∆F significantly decreased in aqueous alkali (PH>7).The reason is that the alkali cation lowers the rate constant of the ingress of organic guests . Hereby, pH of 3.0 was selected as optimal acidity for the further experiments.
Influence of temperature The effect of temperature on ∆F was examined within 10 °C to 80 °C. All formed complexes were stable up to 35 °C. Above 35 °C, the fluorescence intensity greatly decreased due to the dissociation of the complexes at high temperatures. Hence, all subsequent measurements were performed at room temperature.
Influence of reaction time Formal studies have shown that ∆F can reach a maximum after shaken for 15 min [7,21]. In the present research, we replaced the formal time-consuming method and an ultrasound treatment was applied to accelerate the formation of more stable complexes. The effect of reaction time on ∆F was examined within 10 s to 10 min. The results have shown that ∆F can reach a maximum within 1 min and remained constant for at least 5 h. The ultrasound treatment can significantly improve the efficiency of the determination, while the precision and accuracy was not affected. Hence, the standard reaction condition was set to room temperature for 1 min. The response mechanism of the fluorescent probe PAL exhibits weak fluorescence emission in aqueous solution because the isoquinoline and the substituted benzene rings in PAL are not on the same plane. This configuration prevents a conjugated system from being formed. When CB was added into the aqueous solution of PAL, the isoquinoline and the substituted benzene rings in PAL are nearly on the same plane. It cause the fluorescence of PAL molecule enhance. When CLB was added to the host-guest system of CB-PAL, the PAL and CLB competed to bind with CB. Some parts of the PAL molecule were expelled from the CB cavity with the introduction of the CLB. The addition of the CLB caused PAL to lose its protection in the CB hydrophobic cavity, thus resulting in reduced PAL fluorescence intensity. Molecular modeling calculations were optimized at the B3LYP/6-31G(d)  level of density functional theory  using the Gaussian 03 program. Interestingly, the molecular modeling calculations results indicate that two potential CB-CLB complexes were stable. In the previous literature, the stable form of CB-tested drugs complexs is unique [7,21,24]. In this research, calculations showed that the phenyl ring or tertiary butyl of CLB was embedded in the hydrophobic cavity of CB (Fig. 3). In the energy-minimized structures, the imino group is located in the vicinity of a carbonyl-laced portal in either case. Additionally, it is shown that the structure of CB was significantly changed in order to keep the inclusion complexes stable. (Fig. 3)
Fig. 4A shows the 1H NMR spectra of the CB-CLB complex solution. The results of the 1H NMR experiments confirmed the theoretical structures of the complex. Compared with the proton resonances of the unbound CLB molecules (Fig. 4B), the signals of Ha, Hc protons of the unbound CLB significantly shifted upfield and downfield at the same time. Upfield chemical shift is characteristic of this part of the molecule encapsulated in the CB cavity, while downfield chemical shifts is characteristic of the protons of this part of the molecule located just outside the carbonyl portal of the CB host . When tertiary butyl of CLB was embedded in the hydrophobic cavity of CB, the signals of Ha protons significantly shifted downfield to Ha’, and Hc protons markedly shifted upfield to Hc’. Contrastively, when phenyl ring of CLB was embedded, the signals of Ha protons shifted upfield to Ha’’, and Hc protons observably shifted downfield to Hc’’.What is more, the peak area ratio of Ha’/Ha’’ is equal to Hc’/Hc’’ (Fig. 4). These results are consistent with the theoretical structures of the complex. (Fig. 4) In summary, combination of hydrophobic interaction of the cavity of CB, ion-dipole interaction between the carbonyl portal of CB and N+ ion of CLB, and hydrogen bonding interaction leads to the formation of host-gust complex. Because of the excellent size and shape matches, CLB bound with CB more tightly than PAL.
Effect of interfering substances Prior to the application of the proposed spectrofluorimetric method to real samples, the effect of commonly substance in porcine muscle and swine urine on the determination of 0.2 µg mL–1 of CLB was studied under optimum experimental conditions. No interference was observed from commonly substance such as fat, protein, cholesterol, starch, cellulose and urea (100 µg mL–1), indicating good selectivity in the method used to test the animal feed additive in porcine muscle and swine urine samples. However, the components porcine muscle and swine urine samples, which are Na+, K+, Ca2+, cysteine, cystine, alanine, phenylalanine, and valine, may quench the fluorescence intensity of the CB-PAL complex to a certain degree (Table 1). Hence, it should be better to separate before
determination. The fluorescence intensity of the CB-PAL complex through the addition of ractopamine, a swine feed additive with similar functions, was also tested in the current work. The experimental results showed that no change in the fluorescence intensity of the CB-PAL complex was observed. The evidence also indicated that the proposed method had a high selectivity to CLB. (Table 1) Calibration graph and sensitivity Under the optimum experimental conditions described, the standard calibration curves of the complexes of the CLB with CB-PAL were drawn by plotting ∆F versus CLB concentration. The linear regression equation obtained was ∆F = 93.1 C + 1.1. The linear ranges were 0.011 µg mL−1 to 4.2 µg mL−1. The the limit of detection (LOD) and limit of quantification (LOQ) were determined according to the equation: 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 calibration curve. The LOD and LOQ were 0.004 µg mL− 1 and 0.011 µg mL− 1, respectively. The correlation coefficients were 0.9993, indicating good linearity. Analysis of spiked porcine muscle The proposed fluorescent probe was applied in the determination of CLB in porcine muscle. The sample treatment was done according to the following procedures: 15g of homogenized tissue matrix was transferred to a 50 mL polypropylene tube, and a certain quantity of CLB and 20 mL of ammonium acetate buffer solution was added followed by vortex mixing (3 min). After centrifugation at 3000 rpm for 3 min, the supernatant was decanted into another tube, and 20 mL of n-hexane was added followed by vortex mixing (3 min). After centrifugation at 3000 rpm for 3 min, the water layer was dried in a rotary evaporator. Then 1 mL double-deionized water was used to dissolve the residues and transfer them to a colorimetric flask for subsequent analysis. The recoveries were in the range of 96.05 to 97.21%. The results, which showed that the proposed method had satisfactory precision and accuracy, are presented in Table 2.
(Table 2) Analysis of spiked swine urine The proposed method was applied in the determination of the investigated feed additive in spiked samples of swine urine. Accuracy was assessed by investigating the recovery of CLB at five concentration levels covering the specified range (five replicates of each concentration). The results (Table 3) suggest that the probe can be used for the determination of CLB in biological fluids with satisfactory recoveries of 96.81 to 101.13% and an S.D. of less than 2.0, indicating both good accuracy and precision. (Table 3) Conclusions In conclusion, the new fluorescent probe system for CB-PAL complexes was devised for the determination of analytes. Considering the significant quenching observed in the fluorescence intensity of CB-PAL in the presence of CLB, the spectrofluorimetric method developed was of high sensitivity and selectivity for the determination of CLB. In this research, the ultrasound treatment replaced the former time-consuming shake method. The proposed method had been successfully applied to the determination of CLB in porcine muscle and swine urine. This method can be used in a fluorescence sensor for the detection of the animal feed additive. Related studies are in progress in our laboratory.
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Figure Captions Figure 1. The structures of CB, PAL, CLB Figure 2. Fluorescence spectra of CB-PAL in the presence of CLB in 1.0 mM HCl aqueous solution with λex = 343 nm. The concentrations of CLB (µg mL–1): (a) 0; (b) 0.8; (c) 1.6; (d) 2.4; (e) 3.2; (f) 4.0. CCB = CPAL = 14.0 µΜ Figure 3: Energy-minimized structure of CB-CLB complexes. Color codes: CLB, purple; CB, blue Figure 4. 1H NMR spectra (600 MHz) of CB-CLB complex(A) , CLB(B) in D2O
H 3 CO 7
H 2N Cl
Relative Fluoresence intensity
a 800 600
f 400 200 0 400
500 550 Wavelength (nm)
Table 1. Effect of interfering substances (tolerance error ± 5.0%) Tolerance ratio in mass
Cholesterol, Elastin, sucrose, lactose, glucose,
Urea, Uric Acid,
NH4+, K+, Na+,
Mg2+, Ca2+, Fe2+, Zn2+,
Cysteine, Cystine, Alanine, Phenylalanine, Valine
Table 2. Determination of CLB in spiked porcine muscle (n = 5) Samples
Recovery (%) ± S.D.
(µg kg-1) 0.20
(µg kg-1) 0.1921
96.05 ± 1.9
96.93 ± 1.8
97.21 ± 1.6
Table 3. Determination of CLB in spiked swine urine (n = 5) Samples
Recovery (%) ± S.D.
(µg mL-1) 0.05
(µg mL-1) 0.0484
96.81 ± 1.7
96.36 ± 1.5
97.10 ± 1.4
99.34 ± 0.97
101.13 ± 0.79
Highlights A novel spectrofluorimetric method determining the illegal feed additive was proposed. The method was applied for analysis of clenbuterol in porcine muscle and swine urine. It is a suitable method for an accurate, rapid and less expensive determination. The mechanism of the fl uorescence quenching of the supramolecular complex was discussed.