Anal Bioanal Chem (2017) 409:2255–2260 DOI 10.1007/s00216-017-0191-3
RAPID COMMUNICATION
Identification and quantification of nitrofurazone metabolites by ultraperformance liquid chromatography–quadrupole time-of-flight high-resolution mass spectrometry with precolumn derivatization Shuai Zhang 1,2 & PeiPei Li 1 & Zhongyong Yan 1 & Ju Long 1 & Xiaojun Zhang 1
Received: 13 September 2016 / Revised: 6 December 2016 / Accepted: 5 January 2017 / Published online: 26 January 2017 # Springer-Verlag Berlin Heidelberg 2017
Abstract An ultraperformance liquid chromatography–quadrupole time-of-flight high-resolution mass spectrometry method was developed and validated for the determination of nitrofurazone metabolites. Precolumn derivatization with 2,4-dinitrophenylhydrazine and pdimethylaminobenzaldehyde as an internal standard was used successfully to determine the biomarker 5-nitro-2furaldehyde. In negative electrospray ionization mode, the precise molecular weights of the derivatives were 320.0372 for the biomarker and 328.1060 for the internal standard (relative error 1.08 ppm). The matrix effect was evaluated and the analytical characteristics of the method and derivatization reaction conditions were validated. For comparison purposes, spiked samples were tested by both internal and external standard methods. The results show high precision can be obtained with p-dimethylaminobenzaldehyde as an internal standard for the identification and quantification of nitrofurazone metabolites in complex biological samples. Keywords Nitrofurazone metabolites . p-Dimethylaminobenzaldehyde . 5-Nitro-2-furaldehyde . Ultraperformance liquid chromatography–quadrupole time-of-flight high-resolution mass spectrometry . 2,4-Dinitrophenylhydrazine Electronic supplementary material The online version of this article (doi:10.1007/s00216-017-0191-3) contains supplementary material, which is available to authorized users. * Xiaojun Zhang
[email protected] 1
Key Lab of Mariculture & Enhancement, Marine Fisheries Research Institute of Zhejiang Province, Zhoushan 316000, China
2
Marine and Fisheries Research Institute, Zhejiang Ocean University, Zhejiang Zhoushan 316000, China
Introduction Nitrofurans are a group of broad-spectrum antibiotics that all contain the 5-nitro-2-furaldehyde (NF) structure. The most frequently used nitrofurans are furazolidone, furaltadone, nitrofurazone (NFZ), and nitrofurantoin [1]. Nitrofurans are rapidly metabolized with a half-life of several hours. Studies in animals have shown that the amount of the parent drug remaining after 1 day is very low. The rapid metabolism of these drugs can be attributed to their low stability and photosensitivity. It is very difficult to monitor the amount of a parent drug in a living subject, and test results cannot be used to assess short-term drug abuse. However, metabolites of these drugs that bind to proteins are stable and will be present in the body for a long time, with some studies showing they can be detected for up to 56 days after treatment with NFZ [2–4]. Consequently, the side chains of these metabolites have long been considered as biomarkers for determination of NFZ abuse. However, the presence of semicarbazide (SEM) in a sample is not solely attributable to the use of NFZ, and this often leads to false positives. In recent years, the issue of SEM-positive samples has received global attention. The European Commission has issued a notification to member states concerning the presence of nitrofurans in a wide variety of food products [5–14]. NFZ is used as an antibiotic but has carcinogenic properties. For genotoxic carcinogens that damage DNA, Annex IV to European Union Regulation 2377/90 recommends zero tolerance [15]. Therefore, new biomarkers for NFZ in complex biological specimens are required. In our previous research [16, 17], the new biomarker NF was used for confirmation of NFZ abuse after precolumn derivatization with 2,4-dinitrophenylhydrazine (DNPH). The present study aimed to develop a simple and reliable internal standard calibration method using electrospray ionization (ESI) and quadrupole time-of-flight high-resolution mass spectrometry (QTOF-
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HRMS) to identify and quantify NF and pdimethylaminobenzaldehyde (PDAB) derivatives in aqueous samples.
Materials and methods Chemicals and reagents Analytical-grade PDAB was purchased from Sigma-Aldrich (St Louis, MO, USA). NF standard (CAS Registry Number 698-635, molecular weight 141.08, declared purity greater than 97%) was obtained from Tokyo Chemical Industry Co. (Tokyo, Japan). NFZ reference material (purity greater than 98.5%) was purchased from Dr. Ehrenstorfer (Augsburg, Germany). NF-DNPH [purity greater 98.5%, checked by highperformance liquid chromatography (LC) with photodiode array detection] was synthesized and purified by our group. ACSgrade DNPH and hydrochloric acid were obtained from Sinopharm Chemical Reagent Co. (Shanghai, China). LCgrade methanol, absolute ethanol, and acetonitrile were obtained from Merck (Darmstadt, Germany). Hydrophilic–lipophilic-balanced copolymer cartridges (Oasis PRiME HLB) were used for sample purification (Waters, Milford, MA, USA). Polytetrafluoroethylene syringe filters (13 mm × 0.2 μm) were purchased from Thermo Fisher Scientific (Shanghai, China). Ultrapure water (18.2 MΩ•cm, 25 °C, produced by a Direct-Q water purification system; Millipore, Billerica, MA, USA) was used throughout the study. Instrumentation Chromatographic analysis was performed with an ACQUITY® UPLC® system (Waters, Milford, MA, USA) composed of a sample manager, autosampler, column heater, and binary solvent manager. For ultraperformance LC (UPLC), an Athena C 18 column (2.1 mm × 150 mm, 1.8-μm particle size) was used along with a Athena C 1 8 precolumn (2.1 × 20 mm) (ANPEL laboratory technoogies, Shanghai Inc.). Mass spectrometry detection was performed with a Xevo G2-S quadrupole time-of-flight high-resolution mass spectrometer (Waters, Manchester, UK) equipped with an ESI source. Sample preparation used an Eppendorf (Germany) centrifuge 5810 and a BUCHI (Switzerland) Rotavapor R-210. A Thermo Scientific (Finland) Finnpipette F3 was used for the preparation of standard solutions. Preparation of standard solutions and derivatizing reagent Biological samples, standard solutions, and the derivatizing reagent were prepared by a modification of an established method
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[15]. Briefly, the edible parts of shrimp, crab, fish, and turtle samples were retained, and the viscera, shell, and scales were discarded. The samples were then minced and stored at –18 °C before use. We prepared the derivatizing reagent by dissolving DNPH (100 mg) in hydrochloric acid (1 mL) to form a solution with a concentration of 12 mol L–1, and then diluted this with absolute ethanol to a final volume of 100 mL to give a concentration of 1.0 mg L–1. Working solutions of NF and PDAB, both at 100 ng mL–1, were prepared in acetonitrile–water (7:3, v/v). The PDAB solution was used as an internal standard at a concentration of 10.0 ng mL–1. The standard solution of NF was derivatized with DNPH to produce NF-DNPH. A 40 mg L–1 stock solution of NF-DNPH (10.0 mg) was prepared in 250 mL of acetonitrile–water (7:3, v/v). This stock solution was diluted stepwise with acetonitrile–water (7:3, v/v) to prepare a series of NF-DNPH working solutions with concentrations of 1.0, 2.0, 5.0, 10, and 20 ng mL–1. The working solutions were stored at 4 °C before use. For quantification, internal and external calibration curves were constructed by our analyzing the five standard solutions in triplicate. Excellent linearity was obtained with the optimized parameters described later for the concentration range from 1.0 to 20 ng mL–1 for NF-DNPH. The regression equations and correlation coefficients for the calibration curves were y = 297.4x + 35.5 and r2 = 0.994 for the internal standard curve and y = 4724x + 10015 and r2 = 0.996 for the external standard curve. The concentration of any NF-DNPH present in the samples was calculated with use of the internal and external standard curves. Derivatization, purification, and UPLC–QTOF-HRMS analysis The derivatization procedure was performed as previously described [16]. More specifically, 1.0 ± 0.1 g of a biological sample, the standard solution, hydrochloric acid (10 mL of 0.2 mol L–1 solution), DNPH (100 μL of a 1.0 mg L–1 solution), and PDAB (100 μL of 100 ng mL–1 solution) were added sequentially to a 50-mL polypropylene tube. After thorough mixing, a derivatization reaction was conducted at 60 °C for 20 min in a water bath in the dark. The sample was then centrifuged at 11,500 g for 5 min, and the supernatant was decanted into a clean tube. An aliquot (1 mL) of the supernatant was loaded onto a solid-phase extraction (SPE) cartridge (Oasis PRiME HLB, Waters, Milford, MA, USA) without solvent equilibration or washing. The sample flow rate was one drop per second. All of the effluent was collected and then concentrated to less 0.5 mL under a stream of nitrogen at 40 °C. The sample was then reconstituted to 1.00 mL with use of the initial mobile phase, and filtered through a 0.2-μm polytetrafluoroethylene syringe filter before UPLC analysis. The mobile phase for UPLC was a mixture of ultrapure water (solvent A, 30%) and acetonitrile (solvent B, 70%).
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Chromatographic separation was achieved with a mobile phase flow rate of 0.2 mL min–1. The column temperature was fixed at 45.0 °C. QTOF-HRMS detection was performed in negative ESI mode. Nitrogen (99.9%) and argon (99.9999%) were used as the cone and collision gases. The QTOF-HRMS parameters were optimized as follows: cone gas flow rate, 30 L h–1; desolvation gas flow rate, 800 L h–1; collision gas flow rate, 0.15 mL min–1; sampling cone voltage, 40 V; source offset, 80 V; capillary voltage, 2.5 kV; source temperature, 140 °C; and desolvation temperature, 450 °C. MassLynx version 4.1 (Waters) was used for data management. For NF-DNPH and PDAB-DNPH detection, the Xevo G2-S mass spectrometer with IntelliStart™ and Step Wave™ technology was calibrated with leucine enkephalin (error less than 1.0 ppm), and the analyses were performed in resolution mode. Data were collected for each sample from 100 to 1200 Da with use of the MSE method with a scan time of 0.1 s and processed by a workstation equipped with MassLynx and QuanLynx version 4.1.
Results and discussion Selection of internal standard For use as an internal standard, a compound should be structurally or chemically similar to the analyte, well separated from the target analyte and other peaks, and be able to be used to correct any variation other than that related to the amount of analyte present in a sample, such as variability in dilution, evaporation, degradation, derivatization, injection, and mass spectrometry detection [18, 19]. Normally, deuterated compounds are chosen as internal standards for precise qualitative and quantitative analysis of biological samples. However, the method using NF as the metabolite relies on an external calibration for quantification, rather than an isotopically labelled internal standard. Unfortunately, our group could not find a source of isotopically labelled NF. For the analysis of NFZ, PDAB, which has a similar structure and undergoes similar fragmentation on ionization to NF, was used as the internal standard. The mass spectra (Fig. 1) of NF and the PDAB derivatives at the same concentration were similar. Experiments showed that PBAB performed well, and gave good recovery, accuracy, and low relative standard deviation (RSD) in quantitative analysis. QTOF-HRMS of PDABDNPH (Fig. 2) showed the response of the internal standard was stable (RSD 7.36%, n = 5) with changes in the target analyte concentration. Selection of SPE purification strategy Many laboratories use a Bdilute and shoot^ procedure for determination of target analytes in biological samples. However, the presence of phospholipids, fats, proteins, salts, and other
Fig. 1 High-resolution mass spectra of analytes and internal standard: m/z 320.0372 and m/z 328.1060 for 5-nitro-2-furaldehyde–2,4dinitrophenylhydrazine (NF-DNPH) and p-dimethylaminobenzaldehyde– 2,4-dinitrophenylhydrazine (PDAB-DNPH) in negative electrospray ionization (ES-) mode. TOF MS time-of-flight mass spectrometry
substances in the sample can result in excessive matrix effects, greatly reduce identification and quantification accuracy of the method, and increase instrument downtime resulting from contaminant buildup on ESI sources in QTOF-HRMS. The use of SPE for sample preparation provides cleaner samples than most other techniques, making it an ideal method for obtaining accurate results. Typically, SPE purification involves solvent equilibration, sample loading, washing, and elution steps. Consequently, traditional SPE is often perceived as complex or time-consuming. However, judicious choice of the method can greatly simplify this process. In the present study, the waterwettable nature of the Oasis PRiME HLB reversed-phase sorbent reduced the four-step purification process to a single step by eliminating the equilibration and conditioning steps without any loss in recovery or reproducibility for NF and PDAB derivatives. Meanwhile, fast and even flow through the cartridges with minimal plugging and fast overall processing make this method suitable for high-throughput analysis. The performance results of Oasis PRiME HLB cartridges for removal of phospholipids, fats, and colored substances are available in the electronic supplementary material.
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Fig. 2 Precursor ion chromatograms obtained by ultraperformance liquid chromatography–electrospray ionization high-resolution mass spectrometry of standard, spiked, and representative real samples. ES- negative electrospray ionization, TOF MS time-of-flight mass spectrometry
Identification and quantification According to Council Directive 96/23/EC [20], guidance document SANCO/12571/2013 [21], and a US Food and Drug Administration memorandum on the criteria for the confirmation of the identity of chemical residues [22], UPLC–QTOF-HRMS provides more accurate results than high-performance LC–tandem mass spectrometry with respect to the signal intensity requirement, retention time, number of structurally significant ions, and mass accuracy. The derivatives of NF-DNPH and PDAB-DNPH were characterized by QTOF-HRMS in negative ESI mode. The precise molecular weights were 320.0372 for the biomarker and 328.1060 for the internal standard, and were associated with specific molecular formulas (Fig. 1; relative error 1.08 ppm).
The method based on NF as a marker metabolite has been applied successfully to detect illegal use of NFZ in fish, crab, and shrimp [16], in large part because, when combined with LC–tandem mass spectrometry, the European Union’s minimum requited performance limit of 1 ng g–1 was readily achievable. However, use of QTOF-HRMS has not been investigated since modern instrumentation capable of achieving a minimum requited performance limit of 1 ng g–1 became available. The limit of quantification was defined as the lowest concentration that could be determined with 80–120% accuracy and with an analyte response that was at least five times that of the blank. The limits of detection were calculated on the basis of the concentration giving a signal-to-noise ratio of 3 at the corresponding retention time of each analyte. Under the optimized conditions for UPLC–QTOF-HRMS and with a 10-μL injection volume, the limit of detection and the limit of
Identification and quantification of nitrofurazone metabolites Table 1
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Comparison of average recoveries, precision, and accuracy of internal and external standard methods from different biological matrices
Sample types
Identity confirmation
Litopenaeus vannamei
Negative
Swimming crab
Negative
Turbot
Negative
Soft-shelled turtle
Positive
Spiked amount (ng g-1)
Internal standard (%)
External standard (%)
Recovery
RSD
Recovery
RSD
1 5
102.3 98.2
6.01 4.35
95.4 90.1
7.73 6.56
10
103.1
3.98
110.5
6.04
1 5
91.7 94.1
5.11 4.73
82.5 85.8
6.35 6.12
10
101.2
4.15
109.1
5.14
1 5
86.3 90.3
6.02 5.53
80.9 88.4
7.38 6.51
10 –
95.2 Found 1.53
4.14 4.69
89.5 Found 0.45 ng g-1
4.43 7.60
ng g-1 RSD relative standard deviation
quantification of NF-DNPH were 0.1 and 0.2 ng g–1 respectively. Three different types of blank samples (Litopenaeus vannamei, swimming crab, and turbot) and one SEMpositive sample (soft-shelled turtle) were used to evaluate the specificity of the method. Samples were spiked with 1– 10 ng g–1, and each spiked sample was analyzed five times. The mean recoveries ranged between 86.3% and 103.1% (RSD < 6.02%, n = 5) for the internal standard method, and between 80.9% and 110.5% (RSD < 7.73% n = 5) for the external standard method. For the SEM-positive sample, a structurally significant ion at 320.0372 confirmed the presence of SEM (Fig. 2; relative error 1.08 ppm). With the internal and external standard methods the concentrations were 1.53 (RSD 4.69) and 0.45 ng g–1 (RSD 7.60), respectively. These results showed that the internal standard method performed well, and had better recovery and lower RSDs than the external standard method in quantitative analysis (Table 1). UPLC–QTOF-HRMS can be used for identification and quantification analysis of NFZ metabolites. Advancements in SPE sorbent technology minimize the biological sample matrix effects by removing interferences, fats, and phospholipids. The experimental results suggest that the method developed has high sensitivity, excellent recovery, and excellent precision, is suitable for high-throughput use, and could be used in a wide range of applications.
Association for Ethical Studies and were approved by the Institutional Animal Care and Use Committee of Zhejiang Ocean University. Conflict of interest The authors declare that they have no conflict of interest.
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Acknowledgements This research was supported by Zhejiang Provincial Natural Science Foundation of China (LY17C200009). The authors thank the Scientific Research Foundation (SRF, Q1435) and Zhejiang Ocean University and Science and Technology Plan of Zhejiang province (2016 F30022) for supporting the research.
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Compliance with ethical standards Experimental protocols complied with the Guide for the Care and Use of Laboratory Animals of the China
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