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Automated In-Injector Derivatization Combined with HighPerformance Liquid Chromatography−Fluorescence Detection for the Determination of Semicarbazide in Fish and Bread Samples Yinan Wang and Wan Chan* Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong S Supporting Information *

ABSTRACT: Semicarbazide (1) is a widespread genotoxic food contaminant originating as a metabolic byproduct of the antibiotic nitrofurazone used in fish farming or as a thermal degradation product of the common flour additive azodicarbonamide. The goal of this study is to develop a simple and sensitive high-performance liquid chromatography coupled with fluorescence detection (HPLC−FLD) method for the detection of compound 1 in food products. In comparison to existing methods for the determination of compound 1, the reported method combining online precolumn derivatization and HPLC−FLD is less labor-intensive, produces higher sample throughput, and does not require the use of expensive analytical instruments. After validation of accuracy and precision, this method was applied to determine the amount of compound 1 in fish and bread samples. Comparative studies using an established liquid chromatography coupled with tandem mass spectrometry method did not yield systematically different results, indicating that the developed HPLC−FLD method is accurate and suitable for the determination of compound 1 in fish and bread samples. KEYWORDS: semicarbazide, azodicarbonamide, nitrofurazone, high-performance liquid chromatography, fluorescence

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INTRODUCTION Semicarbazide (1) is a hydrazine derivative that can be detected as a contaminant in a wide variety of food products (Figure 1).1,2 Its formation has been demonstrated in multiple pathways, for example, during the bread-making process, the blowing agent azodicarbonamide is decomposed under heat treatments to form compound 1.3−5 Emerging evidence suggests that compound 1 can also be detected in foods that are packed in glass jars/bottles from the breakdown of azodicarbonamide in plastic gaskets used to seal the glass containers.5−7 It has also reported that compound 1 was produced as a protein-binding metabolite of the antibiotic nitrofurazone in poultry and aquatic animals.8−10 Semicarbazide can persist for up to 1 month in tissue samples of nitrofurazone-treated poultry; hence, compound 1 is an excellent marker molecule for monitoring the illegal use of nitrofurazone in livestock production.11−13 Despite being identified as a widespread food contaminant, the toxicity of compound 1 remains poorly understood.14−16 However, some studies have explored the potential genotoxicity and carcinogenicity of compound 1.17−19 We have also demonstrated recently the formation of a covalently bound DNA adduct of compound 1 with apurinic/apyrimidinic (AP) sites (unpublished results), which is one of the most abundant lesions in DNA. Therefore, investigations on the presence of compound 1 in food products are of significant importance for assessing the risk of human exposure to compound 1. Current methods for the determination of compound 1 are focused exclusively on liquid chromatography−tandem mass spectrometry (LC−MS/MS).20−22 In these studies, compound 1 was first released from proteins by acid hydrolysis and then derivatized with 2-nitrobenzaldehyde to increase its chromato© XXXX American Chemical Society

graphic retention on reversed-phase (RP) LC. Although these LC−MS/MS methods are highly sensitive, they suffer drawbacks of being tedious, time-consuming, and requiring expensive instruments. Simpler analytical methods are desirable to facilitate the routine surveillance of compound 1 in food products. The application of fluorescence detection (FLD), one of the most common and sensitive analytical techniques,23 to the direct analysis of compound 1 is hampered because this compound does not fluoresce. This issue has been overcome in a recently developed high-performance liquid chromatography (HPLC) method that combined 2-hydroxy-1-naphthaldehyde derivatization and FLD to detect compound 1 in shrimp samples.24 However, the hydrolysis and derivatization conditions, including reaction duration and temperature, have not yet been optimized. This current study is focused on developing a simple and accurate analytical method for the determination of compound 1 in food products. Fluorenylmethyloxycarbonyl chloride (Fmoc-Cl), is known to react efficiently with amines to yield fluorescent Fmoc derivatives;25,26 it has been used extensively to derivatize amino acids and amines with a fluorescent tag. Fmoc derivatives demonstrated enhanced chromatographic retention and analytical sensitivity when analyzed by HPLC−FLD.25,27,28 The application of Fmoc-Cl to the analysis of compound 1 has not yet been reported. Herein, this work demonstrated for the first time that compound 1 can react quantitatively with Fmoc-Cl, Received: February 6, 2016 Revised: March 16, 2016 Accepted: March 17, 2016

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DOI: 10.1021/acs.jafc.6b00651 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Formation of semicarbazide (1) in food products from thermal degradation of azodicarbonamide and from nitrofurazone metabolism, together with its reaction with Fmoc-Cl forming a fluorophoric derivative of enhanced chromatographic retention for HPLC−FLD analysis. optimized injector program. In brief, 8 μL of sample extract and 1 μL of borate buffer (1 M, pH 8.0) were loaded and mixed in the sampling loop. A total of 1 μL of Fmoc-Cl (15 mg/mL in acetonitrile) was then drawn into the sampling loop and combined with the sample extract, and the mixture was held for 5 min before being injected into a RPHPLC column for separation. The column used was a 150 × 2.0 mm inner diameter, 5 μm, GraceSmart C18 column (Grace, Deerfield, IL). The column was eluted with the following gradient of acetonitrile in 0.1% formic acid with a flow rate of 0.4 mL/min: 0−10 min, 30%; 10−15 min, 100%; and 15−20 min, 30%. The HPLC column was coupled with a fluorescence detector, and the excitation and emission wavelengths were set at 260 and 320 nm, respectively. Calibration. A stock solution of compound 1 at 500 μg/mL was prepared in methanol and stored at −20 °C until use. The calibration standard solutions of compound 1 (5, 10, 25, 50, 75, and 100 ng/mL) were prepared by serial diluting the stock solution, online derivatization with Fmoc-Cl, and HPLC analysis, as described above. Calibration curves were established by plotting the peak areas of the 1Fmoc derivative against the concentrations of compound 1 in the calibration standard solutions. Method Validation. The developed method was validated for accuracy, precision, and analytical sensitivity. The method accuracy was determined by spiking compound 1 (10, 50, and 100 μg/kg) to blank fish/bread samples, processed, and analyzed, as described above. In addition, the method precision was evaluated by analyzing the fish/ bread samples spiked with the three stated concentrations of compound 1 on the same day (n = 5) and over separate days within 1 month (n = 5). The limit of detection (LOD) and limit of quantitation (LOQ) were established as the amount of analyte that generated a signal that is 3 and 10 times the noise level, respectively.29,30 Method Comparison. To evaluate the performance of the developed HPLC−FLD method, the same fish and bread samples were also analyzed using the previously developed isotope dilution LC−MS/MS method.20 In brief, the samples were incubated overnight at 37 °C in 0.1 M HCl to release compound 1 from its protein-bonded form and in which compound 1 was also derivatized simultaneously with 2-nitrobenzaldehyde (50 mM). Isotope-labeled semicarbazide (13C,15N2-1) was added to the sample as an internal standard for the LC−MS/MS analysis. The mass spectrometer was operated in multiple reaction monitoring mode with the following m/z transitions for compound 1: 209/192 and 209/166; those for the internal standard were 212/195 and 212/168. The dwell time for each transition was set at 100 ms.

forming a fluorophore (1-Fmoc), which can be sensitively detected by HPLC−FLD using an automated online precolumn derivatization method (Figure 1).

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MATERIALS AND METHODS

Reagents. Semicarbazide (1), [13C,15N2]-semicarbazide (13C,15N21), nitrofurazone, 2-nitrobenzaldehyde, and Fmoc-Cl were purchased from Sigma-Aldrich (St. Louis, MO). HPLC-grade methanol, acetonitrile, and ethyl acetate were purchased from Tedia (Fairfield, OH). Deionized water was further purified by a Milli-Q Ultrapure water system (Millipore, Billerica, MA) and was used in all experiments. Zebra fish (Brachydanio rerio) and bread samples were obtained from local markets in Hong Kong. Instrumentation. HPLC−FLD analysis was performed on a 1200 series HPLC system equipped with a programmable autosampler and a fluorescence detector (Agilent, Palo Alto, CA). LC−MS/MS analysis was carried out on an Agilent 1100 HPLC system coupled with a 4000 QTRAP tandem mass spectrometer (Applied Biosystems, Foster City, CA). In addition, high-accuracy MS and MS/MS analyses were performed on a Xevo G2 Q-TOF LC−MS system (Waters, Milford, MA) with a standard electrospray ionization source operating in positive ionization mode. Fish Dosing. The protocol for animal experiments was approved by the Committee on Research Practice, The Hong Kong University of Science and Technology (HKUST). Zebra fish, each weighing ∼0.3 g, were acclimated for 1 week prior to conducting the study. After acclimation, fish were placed in separate aquaria containing nitrofurazone at 0.75, 1.5, 3, and 6 μg/mL. A control experiment was performed by keeping the acclimated fish in an unmedicated tank. Each treatment group typically contained 15−20 fish, which are sufficient for triplicate analysis. After 1 week of exposure, fish were removed from the aquaria, rinsed with fresh water, and then frozen immediately at −40 °C prior to analysis. Sample Preparation. After decapitation, the fish exposed to the same level of nitrofurazone were pooled, blot-dried, and then homogenized in a mini-blender. Approximately 1 g of the tissue homogenate was used for each experiment. The tissue homogenate was washed with 10 mL of normal saline (0.9% NaCl) and then with 10 mL of ethyl acetate. A total of 5 mL of 1% HCl was added to the washed tissue homogenates, and then the mixture was incubated at 50 °C for 3 h to release compound 1 from the tissue samples. After cooling to room temperature, the hydrolysates were centrifuged at 4200 relative centrifugal force (rcf) for 10 min to separate the coarse tissue fiber from the aqueous solution. A total of 8 mL of methanol was subsequently added to 2 mL of the supernatants; the mixture was mixed by vortex and then centrifuged at 4200 rcf for 10 min. The supernatant was collected and evaporated to dryness under a stream of nitrogen at 37 °C. The residue was reconstituted in 250 μL of methanol, centrifuged at 14 800 rcf for 5 min, and analyzed using HPLC−FLD analysis. Using the same protocol, the collected bread samples were processed for HPLC−FLD analysis. HPLC−FLD Analysis. The sample extract was drawn into a 100 μL sampling loop and then derivatized online with Fmoc-Cl using the

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RESULTS AND DISCUSSION Characterization of the Semicarbazide Fmoc Derivative. Compound 1 is non-fluorescent and demonstrated a poor chromatographic retention in RP-HPLC. Fmoc-Cl that has been extensively used as a fluorescent-tagging reagent in amino acid analysis was used to produce a nonpolar Fmoc moiety to B


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compound 1 proceeded rapidly at ambient temperature; an increasing reaction temperature had a negative impact on the reaction yield, which may be ascribed to the thermal instability of 1-Fmoc. Our analysis also showed that the reaction produced the best detector response within 5 min at ambient temperature (panels A and B of Figure 3). On the basis of the observation that derivatization of compound 1 with Fmoc-Cl proceeded rapidly at ambient temperature, the technique of online precolumn derivatization was adopted in the entire study. We then investigated the effects of the molar ratio of FmocCl to compound 1 on the reaction yield. From the variation of the concentration of Fmoc-Cl solution (5−20 mg/mL) used in the derivatization, it was found that the highest analytical signal was achieved with a concentration of 15 mg/mL Fmoc-C (Figure 3C). Therefore, Fmoc-Cl at 15 mg/mL was selected and used in all experiments. Borate buffer, one of the most commonly used derivatization buffers,32−34 was chosen as the reaction medium for the derivatization reaction. In contrast to other alkaline buffers, e.g., carbonate buffer, borate buffer produced a homogeneous solution for HPLC analysis. In addition to producing no insoluble deposit during the derivatization step,34 it was also observed that the incorporation of 1 mol/L borate buffer (pH 8.0) into the reaction system produced the best analytical signal (Figure 3D). Therefore, borate buffer was used as the derivatization medium in the entire study. Optimization of the Acid Hydrolysis Conditions. Using incurred tissue samples obtained from zebra fish exposed to high concentrations of nitrofurazone (6 μg/mL) as a model food sample, the effects of the temperature (25−70 °C), acid content (0.05−10% HCl), and acid hydrolysis time (1−7 h) on liberation of protein-bound compound 1 from food stuffs were investigated. Our results demonstrated that the efficiency of heat-assisted, acid-catalyzed hydrolysis increased linearly when the temperature and acid content were increased from 25 to 50 °C and from 0.05 to 1%, respectively (Figure 4). Further increases in the hydrolysis temperature resulted in a decrease in the analytical signal, and an acid content higher than 1% had no effect on the analytical signal. Therefore, 50 °C and 1% HCl were adopted in subsequent studies for hydrolyzing the protein-bound metabolite, compound 1, from the tissue protein. After optimization of the hydrolysis temperature and medium, we then investigated the effect of the incubation time on the yield of compound 1 from the fish muscle samples. Our results showed that the amount of compound 1 liberated from the acid hydrolysis plateaued after 3 h of incubation (Figure 4). A further increase in the incubation time weakened the analytical signal, which may be attributed to the thermal decomposition of compound 1 in a highly acidic environment. The hydrolysis condition was also found to be applicable to the efficient extraction of compound 1 from bread samples. A 3 h incubation at 50 °C was, therefore, adopted in subsequent experiments on fish and bread samples. In contrast to the 16 h hydrolysis/derivatization at 37 °C required in previous studies,24,35,36 the shortened hydrolysis time (3 h) in our study is anticipated to assist in increasing the sample throughput rate for large-scale surveillance projects. Calibration and Method Validation. Calibration curves were established using standard solutions of compound 1 in triplicate reactions, where the concentrations of compound 1 ranged from 5.0 to 100 ng/mL. The average peak areas of 1-

enhance the chromatographic behavior of compound 1 in RPHPLC.31,32 HPLC−FLD analysis of compound 1 derivatized with Fmoc-Cl revealed efficient formation of a fluorescent product at a chromatographic retention time of 5.9 min (Figure 2). Similar to the fluorometric properties of Fmoc-Cl, those of

Figure 2. Typical chromatograms obtained from HPLC−FLD analysis of semicarbazide: (A) standard, (B) fish, and (C) bread samples. The Fmoc derivative of semicarbazide was eluted at 5.9 min. Shown in the insets are the corresponding blank chromatograms.

1-Fmoc showed fluorescence excitation and emission maxima at 260 and 320 nm, respectively. For the sensitive detection of compound 1, the excitation and emission wavelengths of FLD were set at 260 and 320 nm, respectively. High-accuracy MS analyses of the HPLC eluates collected at a retention time of 5.9 min showed [M + Na]+ ions as the base peak at m/z 320.1031, which is in excellent agreement with the theoretical m/z value (320.1011; 6.2 ppm mass error). The eluates were further characterized using collision-induced dissociation (CID) MS/MS analysis, in which the characteristic fragment ions of the fluorenylmethyl moiety and compound 1 were detected as the major fragment ions. The close correlation between the theoretical and measured m/z values of the sodiated molecular ions and the characteristic fragmentation pattern obtained from the CID MS/MS analysis indicated that the fluorescent product was the Fmoc derivative of compound 1 (1-Fmoc, C16H15 N3O3). Optimization of the Derivatization Conditions. The derivatization reaction conditions, including the derivatization temperature, reaction time, and concentrations of the derivatization agent and borate buffer, were optimized to obtain the highest reaction yield of 1-Fmoc. Using a standard solution of compound 1 at 100 ng/mL, we investigated the effects of varying the derivatization temperature (25−60 °C) and reaction time (5−120 min) on the yield of 1-Fmoc. Our results showed that the reaction between Fmoc-Cl and C


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Figure 3. Influence of (A) derivatization time, (B) reaction temperature, (C) concentration of Fmoc-Cl, and (D) borate buffer concentration on the derivatization reaction yield of Fmoc-Cl derivative. The data represent the mean ± standard deviation (SD) for three independent experiments.

in nitrofurazone-treated zebra fish. Figure 2B shows a typical chromatogram obtained from HPLC−FLD analysis of a fish sample. As shown in Figure 5, a dose-dependent formation of compound 1 was observed in fish that were treated with nitrofurazone. Our results revealed that nitrofurazone exposure produced 10.2 μg/kg of compound 1 in fish muscle per μg/mL drug (r2 = 0.964). A similar concentration of compound 1 (12 μg/kg of muscle per μg/mL drug) formation was reported by Du et al.24 Our results are reasonably consistent with the formation levels of compound 1 (16 μg/kg of muscle per μg/ mL drug) in the study by Chu and Lopez,35 in consideration of the different exposure durations and aquatic organisms used. These authors used shrimp grown in seawater, whereas we used freshwater fish. In addition, Chu and Lopez35 adopted a 24 h exposure time, whereas we exposed fish to the drug for 7 days. Determination of Semicarbazide in Bread Samples. Semicarbazide is also produced when azodicarbonamide containing flour is heated during the baking process (Figure 1).5 In the present study, a total of eight bread samples were randomly collected from local supermarkets in Hong Kong and were analyzed using the developed method. Compound 1 was detected in five of the eight bread samples at concentrations ranging from 11.5 to 207.2 μg/kg (Table 2). These results are reasonably consistent with that reported by Becalski et al.,39 who detected compound 1 at concentrations ranged from 6.9 to 557 μg/kg in bread samples collected in Canada. Figure 2C shows a typical chromatogram obtained from the HPLC−FLD analysis of a bread sample. Comparison to the Isotope Dilution LC−MS/MS Method. The isotope dilution LC−MS/MS method that is known to be one of the most accurate analytical methods was also adopted in this study to quantitate compound 1 in fish and bread samples. Specifically, we compared the results obtained

Fmoc increased linearly within the stated concentration range, with r2 values higher than 0.9993. Figure 2A depicts a typical chromatogram obtained from the HPLC−FLD analysis of compound 1 at 15 ng/mL. The LOD and LOQ for the HPLC−FLD method, which are the concentration of compound 1 that generated a signal that is 3 and 10 times the noise level, were 1.5 and 5.0 pg, respectively. The LOD and LOQ correspond to the detection and quantification limit of 0.15 and 0.5 μg/kg of compound 1 in fish samples, respectively. The LOD is significantly lower than the minimum required performance level (1 μg/kg) set by the European Union and is similar to the existing LC−MS/MS (0.1−0.8 μg/kg) and other HPLC−FLD (0.2−0.3 μg/kg) methods.24,35,37,38 The accuracy of the method as determined by spiking compound 1 (10, 50, and 100 μg/kg) to blank fish and bread samples and analyzed by HPLC did not deviate from the true values by more than 3.4%. Moreover, the intra- and interday reproducibilities of the developed method were determined by analyzing fortified fish/bread samples spiked with the three stated concentrations of compound 1. The method precision expressed as the relative standard deviation from the mean values was less than 4.5 and 5.6% for intra- and interday measurements, respectively. Table 1 summarized the accuracy and reproducibility of the HPLC−FLD method. Data on high analytical accuracy and precision demonstrated the excellent performance of the developed HPLC−FLD method in determining compound 1 in fish and bread samples. Determination of Semicarbazide in NitrofurazoneTreated Fish Samples. Compound 1 is a protein-binding metabolite of the antibiotic nitrofurazone (Figure 1). Upon validation of the performance of the newly developed method, we subsequently applied the method to quantitate compound 1 D


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Figure 5. Formation of semicarbazide in nitrofurazone-exposed zebra fish. The data represent the mean ± SD of five independent experiments.

Table 2. Determination of Semicarbazide in Selected Bread Samples by the Developed HPLC−FLD and LC−MS/MS Methods semicarbazide (μg/kg) 1 2 3 4 5 6 7 8

butter roll satellite bun milk bread satellite bun thick sandwich twist bread light sandwich life bread

HPLC−FLDa

LC−MS/MSa

78.3 ± 5.9 NDb 11.5 ± 1.7 ND 12.9 ± 1.8 47.5 ± 3.2 207.2 ± 23.1 ND

77.9 ± 10.9 ND 9.8 ± 1.2 ND 11.4 ± 0.94 44.4 ± 2.9 198.5 ± 12.9 ND

a The data represent the mean ± SD for three independent determinations. bND = not detected.

0.99), with a data point difference not exceeding 18%. The average relative derivation of the sample analyses using the two methods was 7.1 and 9.6%, respectively. Considering the strong correlation between the two methods, we believe that the developed HPLC−FLD method can accurately determine the concentrations of compound 1 in both aquatic and bread samples. In summary, we have developed a new analytical method to determine compound 1 in fish and bread samples by combining online precolumn derivatization and HPLC−FLD analysis. In comparison to existing methods, our method has the advantages of being less labor-intensive and less timeconsuming and requiring no expensive analytical instruments. With semicarbazide being a widespread and potentially toxic food contaminant that is raising public concerns, we believe that the reported HPLC−FLD method will be an effective alternative protocol to survey compound 1 in different food products and to monitor the illegal use of the antimicrobial nitrofurazone in livestock production.

Figure 4. Effect of (A) incubation time, (B) temperature, and (C) HCl content on the extent of acid-induced hydrolysis of semicarbazide from the fish protein sample. The data represent the mean ± SD for three independent experiments.

Table 1. Accuracy and Intra- and Interday Precisions of the Developed Method for the Determination of Semicarbazide (1) in Fish and Bread Samples accuracy concentration of compound 1 spiked (μg/kg) fish musclea breadb

a

10 50 100 10 50 100

precision

concentration found (μg/kg)

recovery (%)

intraday (% RSD)a

interday (% RSD)a

± ± ± ± ± ±

96.6 98.7 98.6 97.9 100.4 98.9

3.5 2.0 0.7 4.5 2.0 0.7

4.9 2.7 1.2 5.6 2.5 1.0

10.0 50.5 99.8 10.1 49.7 99.1

0.3 0.4 0.6 0.2 0.4 0.6

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ASSOCIATED CONTENT

S Supporting Information *

n = 5. bn = 3.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b00651. Injector program for online derivatization (Table 1), fluorescence excitation and emission as well as highaccuracy MS and MS/MS spectra of the Fmoc derivative

by the HPLC−FLD analyses to those obtained using the LC− MS/MS method developed by Wang et al.9 A close correlation between the two methods was observed (A, r2 = 0.98; B, r2 = E


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of semicarbazide (Figures S1 and S2), effect of the incubation time, temperature, and acid content on extracting semicarbazide from bread samples (Figure S3), calibration curves from HPLC−FLD and LC−MS/ MS analyses of semicarbazide (Figure S4), mass spectra from ESI−MS/MS analysis (Figure S5), and LC−MS/ MS analyses (Figure S6) (PDF)

AUTHOR INFORMATION

Corresponding Author

*Telephone: 852-2358-7370. Fax: 852-2358-1594. E-mail: [email protected]. Funding

The authors express their sincere gratitude to The Hong Kong University of Science and Technology for a startup fund (Grant R9310). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors express their sincere gratitude to AB Sciex for providing the LC−MS/MS system for this research. REFERENCES

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