A highly sensitive and selective assay of doxycycline by dualwavelength overlapping resonance Rayleigh scattering

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 237–242

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A highly sensitive and selective assay of doxycycline by dualwavelength overlapping resonance Rayleigh scattering Jinghui Zhu, Shaopu Liu, Zhongfang Liu, Yuanfang Li, Jing Tian, Xiaoli Hu ⇑ Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 A rapid assay of doxycycline was

A dual-wavelength overlapping resonance Rayleigh scattering (DWO-RRS) methodology based on the specific multi-site coordination between lanthanum(III) and doxycycline (DOTC) has been successfully designed (rather than accidently encountered) for highly sensitive and selective assay of doxycycline in several meat samples.

performed.  Dual-wavelength overlapping resonance Rayleigh scattering was employed. 1  The detection limit (1.1 nmol L ) was lower than or comparable to most of the reported methods.  The generating mechanisms of multiresponse RRS signals were proposed.  A semi-empirical principle was established for better design of multiresponse RRS probes.

a r t i c l e

i n f o

Article history: Received 4 November 2013 Received in revised form 27 December 2013 Accepted 30 December 2013 Available online 13 January 2014 Keywords: Dual-wavelength overlapping Resonance Rayleigh scattering Doxycycline La3+/Lanthanum(III)

a b s t r a c t A dual-wavelength overlapping resonance Rayleigh scattering (DWO–RRS) method was developed and validated for highly sensitive and selective assay of doxycycline residues in several meat samples. The response signals were dependent on the specific multi-site coordination between lanthanum(III) and doxycycline (DOTC). And La(III)–DOTC complex would further aggregate to form [La(III)–DOTC]n nanoparticles, resulting in the occurrence of two new scattering peaks. Notably, with the addition of DOTC, the increments of both of these two wavelengths were proportional to the concentration of DOTC over the ranges of 3.9–4.0  103 nmol L1 (1.7–1.8  103 lg/kg). The detection limit of DWO–RRS was 1.1 nmol L1 (0.5 lg/kg), which was lower than or comparable to most of the published methods. Additionally, the generating mechanisms of multi-response RRS signals were discussed and a semi-empirical principle was established for better design of multi-response RRS probes. Ó 2014 Elsevier B.V. All rights reserved.

Introduction Currently, the frequency of food safety problems caused by antibiotic residues in animal-origin foods has stimulate active ⇑ Corresponding author. Tel.: +86 023 68252360; fax: +86 023 68254000. E-mail address: [email protected] (X. Hu). 1386-1425/$ - see front matter Ó 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.12.114

research in food and chemical toxicology [1,2]. The testing and analysis of multifarious pollutants in foodstuff becomes one of the hot issues of food chemistry [3–7]. A great deal of endeavors have been made directly toward the toxicity, pharmacology, biocompatibility, bioavailability as well as analytical methodology of various food contaminants. The contaminants include toxic metals, polychlorinated biphenyls, dioxins, pesticides, animal drugs and

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other agrochemicals. Among these chemical hazards, doxycycline (DOTC) is especially touchable and has received special attention owing to its universal use in freshwater aquaculture [8], poultry [9] and grass-based animal husbandry [10]. DOTC plays a significant role in the prevention or control of bacterial infections and animal diseases. However, exposure to high levels of DOTC has harmful effects on human health, such as allergic reactions, liver damage, yellowing of teeth, and gastrointestinal disturbance [11]. Despite the fact that China and European Union [12] has set maximum residue limits for DOTC (100 lg/kg in muscle, 600 lg/kg in kidney, 300 lg/kg in liver and 200 lg/kg in egg), the quality of some of the presently marketed products is still questionable. Some meats, for example, may be contaminated due to excess of DOTC in feed. Thus, to establish an international approach for the administration of DOTC is essential for its surveillance, ensuring the safety of food for consumers. To date, instrumental techniques that have been employed to detect DOTC mainly include liquid chromatography–mass spectrometry [13], voltammetry [14], enzyme-linked immunosorbent assay [12,15] and capillary electrophoresis [16]. Although chromatographic methodology offers sensitive and specific multi-analytical results, it is time consuming and dependent on highly skilled personnel and expensive equipments. Electrochemical technique is used because of advantages like low-cost, simplicity, sensitivity, selectivity and mechanical stability, yet it may take a long time to fabricate functionalized electrodes. Immunosorbent assay provides relatively lower detection limit, but it involves several complicated procedures so as to obtain the antibody. Capillary electrophoresis is rapid and effective, yet it lacks of reproducibility. All things considered, those methods are not suitable for routine, rapid analysis of large numbers of samples. There is therefore an urgent need for a rapid, sensitive, specific, high-capacity and inexpensive method for DOTC detection. Resonance Rayleigh scattering (RRS) [17] is a potent method to investigate aggregate systems owing to its high sensitivity and simplicity. And this technique has been widely applied to the study and determination of metal ions [18], non-metallic inorganic substances [19], surfactants [20], biomacromolecules [21], and pharmaceuticals [22]. Besides, this technology has been used for the determination of some physicochemical parameters such as the critical micelle concentration of surfactant [23] and the inclusion constant of b-cyclodextrin [24]. Into a typical RRS system, the output signals are commonly restricted to single-wavelength responses, which suffer from variable factors such as the apparatus readouts, probe concentrations, and ambient interferences. In order to compensate for these defaults, Hao et al. reported a triple wavelength overlapping resonance Rayleigh scattering method (TWO–RRS) [25] for the detection of nanogram dextran sulfate, providing much better flexibility and higher sensitivity than the single-wavelength method. Despite these advantages, the multi-response signals can only be encountered by accident rather than through scientific design. As a consequence, the generating mechanisms of multi-response RRS signals remain elusive and the theory and application of this advantageous technique is poorly investigated [26,27]. Thus, it would be of great challenge to develop a semi-empirical or well-founded principle to elucidate this phenomenon, promoting a revolutionary new era for the progress of RRS techniques. Here in this paper we have successfully designed (rather than accidently encountered) a dual-wavelength overlapping resonance Rayleigh scattering (DWO–RRS) methodology for highly sensitive and selective assay of doxycycline in several meat samples. This methodology not only effectively overcomes the deficiency of single-wavelength scattering method, but also extensively enrichs the research contents of RRS.

Experimental Instrumentation A Hitachi F-2500 spectrofluorophotometer (Tokyo, Japan) was used for recording scattering spectra. A UV-2450 spectrophotometer (Shimadzu, Japan) was employed to acquire absorption spectra. A Hitachi S-4800 scanning electron microscope (SEM) (Tokyo, Japan) was used to observe the morphology. A pHS-3D pH meter (Shanghai Scientific Instruments Company, China) was used to measure the pH values. Chemicals All of the materials were purchased from commercial suppliers and used without further purification. Stock solutions of 4.0  104 mol L1 doxycycline (DOTC, Aladdin Reagent (Shanghai) Co., Ltd., China) and La(NO3)3 (Aladdin Reagent (Shanghai) Co., Ltd., China) were prepared and kept at 4 °C. Tris–HCl buffer solutions with different pH values were prepared by mixing 0.2 mol L1 Tris(Hydroxymethyl)aminomethane with 0.2 mol L1 HCl in proportion, and the pH values were adjusted with a pH meter. Aqueous solutions were prepared using deionized water (Milli–Q system). Recommended procedure for RRS determination of DOTC A suitable aliquot of DOTC, 1.0 mL of 0.2 mol L1 Tris–HCl buffer solution and 1.0 mL of 4.0  105 mol L1 La(NO3)3 solution were added into a 10.0 mL calibrated flask. The mixture was diluted to the mark with doubly distilled water. The contents of each flask  was mixed well at room temperature (25  5 C) and the RRS was measured against the reagent blank with synchronous scanning at kex = kem (i.e. Dk = 0 nm).

Results and discussion RRS spectra Fig. 1 showed the RRS spectra of DOTC, La(III) and their complex at pH 7.8 from 250 to 650 nm. It can be seen that RRS intensity of either DOTC or La(III) in the whole scanning wavelength region is negligible weak. However, when they reacted with each other, the interaction between DOTC and La(III) by virtue of multidentate coordination occurred, which significantly enhanced the RRS intensity with two new peaks appearing at 335 and 451 nm. All these peaks positively increased upon addition of DOTC, suggesting that the system can be used for the quantification of DOTC.

Fig. 1. RRS spectra of La(III)–DOTC system. C(La3+) = 4.0  106 mol L1, C(DOTC)/ (1–5, 106 mol L1): 0.8, 1.6, 2.4, 3.2, 4.0. pH 7.8.

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pH of Tris–HCl buffer solution. It could be seen from Fig. 2 that the optimum pH range was 7.6–8.2. When the acidity was higher or lower than the optimum range, the relative intensities (DI) decreased. Therefore, pH 7.8 was selected for further studies.

Fig. 2. Effects of acidity. 1: La3+; 2: La(III)–DOTC. C(La3+) = 4.0  106 mol L1, C(DOTC) = 4.0  106 mol L1.

Effect of the La(III) concentration The effects of La(III) concentration on the RRS intensity were studied and the results were presented in Fig. 3. When the concentration of La(III) was low, the scattering intensity of La(III)–DOTC system was faint owing to the incomplete reaction; upon addition of La(III), the scattering intensity of the system enhanced gradually and reached a maximum at the concentration of 4.0  106 mol L1 and then when the amounts of La(III) increased further, the scattering intensity decreased gradually. So we chose 4.0  106 mol L1 as the best La(III) concentration for the further study. Effect of adding orders The effect of the adding order of the reagents on the RRS intensity was studied. The result (Table 1S) showed that mixed buffer solution and DOTC solution first and then added La(III) could result in a higher RRS intensity than any other adding sequences. This could be explained as that mixing of buffer and DOTC solution first would promote the ionization of DOTC drastically, which was beneficial to the reaction of DOTC with La(III). So the best addition mode for the system was to add DOTC solution, buffer, and La(III) in order.

Fig. 3. Effects of La(III) concentration. C(DOTC) = 4.0  106 mol L1, pH 7.8.

Optimum reaction conditions The test revealed that the RRS intensity of both of the two peaks possessed the same fluctuating trend toward all kinds of influencing factors; therefore, the maximum scattering peak at 451 nm was selected to optimize the proposed method. Effect of acidity The effect of the solution acidity on the scattering intensity of the system was investigated. The results were achieved by keeping the DOTC and La(III) concentrations constant while changing the

Effect of temperature and the stability of the system The effect of the temperature on the RRS intensity of the system was also examined. The results showed the RRS intensity reached the maximum value and kept constant in the range of 10–30 °C (Table 2S). Therefore, the experiment could be processed at room temperature. And the scattering intensity reached the maximum in 5 min and remained stable for 6 h at least. Therefore, the scattering signals could relatively keep stable. Selectivity of the method The influences of foreign coexisting substances on the determination of 2.0  106 mol L1 DOTC were investigated by pre-mixing DOTC with foreign substances and the results were listed in Table 1. The tolerance limit was taken as the maximum concentration of the foreign substances which caused an approximately ±5%

Table 1 Effects of coexisting substances. Foreign substance

Concentration (lg mL1)

Relative error (%)

Foreign substance

Concentration (lg mL1)

Relative error (%)

NHþ 4 Na+ + K Ni2+ Co2+ Cu2+ Mn2+ Zn2+ Mg2+ Ca2+ Fe2+ Fe3+ Al3+ NO 2

400 2000 1800 225 300 80 320 300 65 420 280 70 58 250 125

4.3 4.9 4.7 4.9 4.6 4.1 4.3 4.7 4.8 4.5 4.7 4.8 4.7 4.6 4.8

Cholesterol Stearate Lactose Glucose Starch Urea L-Leucine L-Proline L-Tryptophan Vitamin A Vitamin B12 Vitamin K Vitamin B1 Ascorbic acid Nicotinic acid

90 120 55 200 80 75 20 35 100 70 20 30 140 25 130

–4.4 4.7 4.3 4.1 4.6 4.6 4.1 4.9 4.3 4.7 4.7 4.1 4.6 5.0 4.6

12 15 350 380

4.7 4.8 5.0 4.6

Ethanol Acetone Triton X-100 Tween 80

25* 25* 280 260

4.6 4.9 4.3 4.7

C2 O2 4 SDS SDBS CTAB CPC

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*

Muscle samples

Found (lg/kg)

Spiked (lg/kg)

Average found (lg/kg)

Recovery (%)

RSD* (%, n = 5)

1 2 3

0 0 0

5.0 500.0 1500.0

4.78 514.52 1489.95

95.6 102.9 99.3

–4.3 2.8 0.7

RSD: relative standard deviation.

Analytical application of the method

Fig. 4. Calibration graph of La(III)–DOTC system.

relative error in the determination. It could be seen that common metal ions, sugars, amino acids and some vitamins in meats can be allowed with relatively high concentration. We also tested the influence of ethanol, acetone, and surfactants on the determination. The experimental results showed that low amount (<25%, v/ v) of ethanol and acetone would not interfere with the reaction. This may attribute to the fact that doxycycline is only slightly soluble in ethanol and acetone and thus low amount (<25%, v/v) of ethanol and acetone would not affect the formation of the hydrophobic interface. Besides, cationic and non-ionic surfactants can be allowed with relatively high concentration, while anionic surfactants may influence the determination, it should be avoid.

Calibration curve Under the optimum conditions, the scattering intensities of the La(III)–DOTC complex were measured. Calibration graphs of IRRS against the concentrations of DOTC were depicted (Fig. 4). The results of the SW–RRS and dual-wavelength overlapping resonance Rayleigh scattering (TWO–RRS) methods were listed in Table 2. Additionally, the comparison of several published methods with the proposed method was listed in Table 3.

As reported [28], three shares (2 mg per share) of chicken muscle samples was firstly mixed with 10.0 mL double distilled water and homogenised. Afterward, 0.5 mL of this mixture was spiked with adequate amount of DOTC as an internal standard, then for protein precipitation, 3.5 mL of acetonitrile was added to this mixture and centrifuged at 1000 rpm for 10 min and the supernatant was used for determination. The corresponding results were listed in Table 4. Generating mechanisms of multi-response RRS signals and the reasons for RRS enhancement Generating mechanisms of multi-response RRS signals It is universally acknowledged that Rayleigh scattering was next to the molecular absorption band [25–27], when the molecule of the complex absorbed the radiation wave of specific wavelength and the electron was excitated to the higher vibrational energy level of a certain electron excitated singlet state, it fleetly gave birth to a vibrational laxation and the redundant vibrational energy was diverted into medium and the molecule dropped to the lowest vibrational energy level that finally transfer to the ground state. And thus the resonance scattering generated. However, this elucidation was insufficient because it was incapable to explain the reason why some systems possessed two or three absorption bands with only one scattering peak [29]. After reviewing numerous published works, we found for the first time that, a multi-site interaction might be the second

Table 2 Analytical parameters of SW-RRS and DWO-RRS methods. Method

k (nm)

Linear equation (c, lmol L1)

Linear range (C, nmol L1)

R

Detection limit (nmol L1)

RRS RRS DWO-RRS

335 451 335 + 451

DI = 255.6 + 876.5c DI = 125.0 + 1713.7c DI = 130.6 + 2590.2c

11.4–4000 72.9–4000 3.9–4000

0.9991 0.9989 0.9987

3.4 1.8 1.1

Table 3 Analytical features of some typical methods employed for DOTC determination. Method*

Linearity (nmol L1)

Detection limit (nmol L1)

Remarks

LC–UV [13] LC–MS [13]

34.0–NG 4.5–NG

22.5 2.3

Voltammetry [14]

14.1  104–NG

43.5  103

ELISA [12] ELISA [15] MSPD-CZE [16] DWO-RRS (Present work)

1.1–NG 0.7–NG 606.1–NG 3.9–4.0  103

0.3 0.2 181.8 1.1

Accurate and sensitive yet time consuming; require highly skilled personnel and expensive equipments; using toxic organic solvents. Low–cost, simple, stable, yet involving several complicated and tedious steps to fabricate functionalized electrodes. Low detection limit, selective, but involving sophisticated procedures to obtain the antibody. Rapid, effective, but lacking of reproducibility. Sensitive, selective, simple, rapid, and free from interference, no need for organic solvents.

NG: not given. LCUV: liquid chromatography–spectrophotometry; LCMS: liquid chromatography–tandem mass spectrometry; ELISA: enzyme linked immunosorbent assay; MSPD– CZE: matrix solid-phase dispersion extraction and capillary zone electrophoresis.

*

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Fig. 7. SEM image of [La(III)–DOTC]n nanoparticles.

Scheme 1. Reaction of La(III) with DOTC.

The reasons for RRS enhancement The possible reasons for typical RRS enhancement could be explained [25–27] as resonance enhanced scattering effect, enlargement of the molecular volume as well as formation of the hydrophobic interface, so did La(III)–DOTC system. Additionally, La(III)–DOTC complex would further aggregate to form [La(III)– DOTC]n nanoparticles. The size of [La(III)–DOTC]n nanoparticles was observed by scanning electron microscope (SEM) (Fig. 7). As shown, the average size was about 272 nm (3lm/11  272 nm). Conclusions

Fig. 5. Absorption spectra of La(III)–DOTC system. C(La3+) = 4.0  106 mol L1, C(DOTC) = 4.0  106 mol L1. pH 7.8.

In this paper we have successfully investigated the determination of DOTC with La(III) by DWO–RRS. DWO–RRS is based on the addition of RRS intensity of the two single wave-lengths, so it could counteract a series of positive and negative common influences at the three wavelengths such as the pH, dye concentration and ionic strength, etc. The DWO–RRS could get more reliable information of the system, and is a promising method to enlarge the applications of RRS. Additionally, the generating mechanisms of multi-response RRS signals were discussed and a semi-empirical principle was established for better design of multi-response RRS probes. Acknowledgements The authors gratefully acknowledge financial support for this study by grants of the National Natural Science Foundation of China (Grant No. 21175109) and the Special Fund of Chongqing Key Laboratory (CSTC).

Fig. 6. An overlapping of absorption (a) and RRS (b) spectra of La(III)–DOTC system. C(La3+) = 4.0  106 mol L1, C(DOTC) = 4.0  106 mol L1. pH 7.8.

requirement of multi-response RRS signals. For example, the interaction of dextran with crystal violet [25] could be regarded as the interaction of several sulfonic group (the microenvironment of each sulfonic group is different) with crystal violet. Another example might be the interaction DNA with thionine [30], which can be viewed as a multi-site binding of thionine to the phosphate backbone of DNA. With respect to La(III)–DOTC system, on the one hand, the dual wavelength scattering peaks may be derived from the multidentate coordination between La(III) and DOTC (Scheme 1), just as reported by Karthikeyan et al. [31]. On the other hand, multiresponse RRS signals were also attributed to the binary absorption band (Figs. 5 and 6).

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