同时测定鱼肉中氯霉素和甲砜霉素残留量的毛细管气相色谱法

研究了鱼肉中氯霉素和甲砜霉素残留量的气相色谱测定方法。鱼肉中的待测物用乙酸乙酯提取,浓缩至干,溶于水,用正己烷脱脂,水层过 C18小柱,用甲醇洗脱后,用 N, O - 双（三甲基硅烷基）三氟乙酰胺（ BSTFA）＋ 1％ (φ )三甲基氯硅烷（ TMCS）衍生,加入甲苯并用水灭活衍生过程。用外标法定量。当添加水平（ w）为 5× 10－ 9～ 20× 10－ 9时,回收率为 80％～ 108％。相对标准偏差为 4.5％～ 11％;线性相关系数 r>0.997。     

Comparsion of an immunochromatographic strip with ELISA for simultaneous detection of thiamphenicol,florfenicol and chloramphenicol in food samples

Rapid and sensitive indirect competitive enzyme‐linked immunosorbent assays (ic‐ELISA) and gold nanoparticle immunochromatographic strip tests were developed to detect thiamphenicol (TAP), florfenicol (FF) and chloramphenicol (CAP) in milk and honey samples. The generic monoclonal antibody for TAP, FF and CAP was prepared based on a hapten [D ‐threo‐1‐(4‐aminophenyl)‐2‐ dichloroacetylamino‐1,3‐propanediol], and the haptenwas linked to a carrier protein using the diazotization method. After the optimization of several parameters (coating, pH, sodium chloride content and methanol content), the ic‐ELISA was established. The quantitative working range for TAP was 0.11–1.36 ng/mL, with an IC₅₀ of 0.39 ng/mL. The optimized ELISA showed cross‐reactivity to CAP (300%) and FF (15.6%), with IC₅₀ values of 0.13 and 2.5 ng/mL, respectively. The analytical recovery of TAP, FF and CAP in milk and honey samples in the ic‐ELISA ranged from 81.2 to 112.9%. Based on this monoclonal antibody, a rapid and sensitive immunochromatographic test strip was also developed. This strip had a detection limit of 1 ng/mL for TAP, FF and CAP in milk and honey samples. Moreover, the test was completed within 10 min. Our results showed that the proposed ic‐ELISA and immunochromatographic test strip method are highly useful screening tools for TAP, FF and CAP detection in milk and honey samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous determination of florfenicol and florfenicol amine in fish, shrimp, and swine muscle by gas chromatography with a microcell electron capture detector

A rapid and sensitive gas chromatography method was developed for the simultaneous determination of florfenicol (FF) and its metabolite florfenicol amine (FFA) in fish, shrimp, and swine muscle. The extracted samples were defatted with hexane and cleaned up by solid-phase extraction using Oasis MCX cartridges. The eluate was evaporated to dryness, and residues were derivatized and determined by gas chromatography with a microcell electron capture detector. Overall average recoveries ranged from 81.7 to 109.7% for fish, 94.1 to 103.4% for shrimp, and 71.5 to 91.4% for swine muscle. The detection limit was 0.5 ng/g for FF and 1 ng/g for FFA, respectively. The method was validated for the determination of incurred swine muscle samples in an actual residue study.     

Characterisation and application of molecularly imprinted polymers for group-selective recognition of antibiotics in food samples

Group-selective molecularly imprinted polymers (MIPs) for amphenicol antibiotics, including chloramphenicol (CAP), thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA), were developed for the first time using TAP as the template molecule. The characteristics of the obtained MIPs were systematically evaluated by chromatographic methods and frontal analysis, demonstrating that the MIPs had excellent chromatographic behaviors, good selectivity, and high-binding capability. A molecularly imprinted solid-phase extraction (MISPE) procedure was developed based on the chromatography results. The MIPs exhibited better group selectivity for CAP, TAP, FF, and FFA than non-imprinted polymers (NIPs) under the optimized washing conditions of 10% acetonitrile in PBS buffer (25 mmol L(-1), pH = 5). Compared with conventional solid-phase extraction, significant recoveries ranging from 92.4% to 98.8% with lower relative standard deviation values in the range of 3.2-7.3% for both intraday- and interday-assays were obtained. The limits of detection (LODs) of MISPE for CAP, TAP, FF, and FFA in shrimp were found to be 0.016, 0.093, 0.102, and 0.029 μg kg(-1), respectively. The results acquired in this study contribute to the strategic development of MIPs and MISPE methods for the multi-residual recognition of antibiotics from complex matrices.     

气相色谱-质谱联用法测定动物组织中氯霉素、氟甲砜霉素和甲砜霉素的残留量

建立了气相色谱-负离子化学电离源质谱同时测定动物组织中氯霉素(CAP)、甲砜霉素(TAP)和氟甲砜霉素(FF)残留量的方法。样品用乙酸乙酯提取,正己烷分配去脂肪,再用Florisil柱进一步净化,甲苯作为反应介质,用N,O-双(三甲基硅基)三氟乙酰胺(BSTFA)-三甲基氯硅烷(TMCS)(体积比为99∶1)进行硅烷化处理,用间硝基氯霉素(m-CAP)作为内标进行测定。CAP的检测限可达到0.03 μg/kg,TAP和FF的检测限可达到0.2 μg/kg;上述3种药物的标准曲线的线性相关系数均大于0.99。CAP,FF和TAP的批内测定的精密度(以相对标准偏差表示)依次为5.5%,10.4%和8.8%;批间测定的精密度依次为7.4%,20.7%和19.1%。回收率为80.0%~111.5%,相对标准偏差为1.2%~15.4%。该方法前处理步骤简单,处理后杂质干扰少,灵敏度高,适用性强,可用于猪肉及禽类、水产品等多种动物组织中氯霉素类药物残留的检测。     

Determination of residues of azamethiphos in salmon tissue by liquid chromatography with fluorescence detection.

A liquid chromatographic (LC) method with fluorescence detection (FLD) is described for determining residues of the pesticide azamethiphos (AZA) in salmon tissue. The sample is extracted with ethyl acetate, centrifuged, dehydrated with anhydrous sodium sulfate, evaporated, reconstituted in water, and defatted with hexane. The aqueous phase is passed through a C18 solid-phase extraction (SPE) column. The SPE column is eluted with methanol, and the eluate is evaporated to dryness and then taken up in 10% acetonitrile (ACN) in water. The analyte is determined by LC using a C18 column, ACN-H2O (32 + 68) mobile phase, and FLD with excitation at 230 nm and emission at 345 nm. Composited salmon tissues were fortified with AZA at 5, 10, 21, 42, and 83 ng/g or ppb (target level, X = 10 ng/g). Overall recoveries were 86%, with between-day variability of 5.3%. The method detection limit was calculated as 1.2 ppb AZA based on a 5 g sample. The limit of quantitation as determined empirically by this method is the lower limit of the standard curve, approximately 5 ppb.     

改良的QuEChERS与HPLC-HESI/MS/MS同时测定中华鳖组织中氯硝柳胺和酰胺醇类药物及其代谢物的残留量

建立了中华鳖(Trionyx sinensis)组织(血浆、肌肉、裙边、肝脏和肾脏)中氯硝柳胺、氯霉素、甲砜霉素、氟苯尼考和氟苯尼考胺同时测定的高效液相色谱-加热电喷雾电离源串联质谱法(HPLC-HESI/MS/MS)。样品经改进的QuEChERS方法提取净化,以氨化乙腈为提取剂,十八烷基硅烷键合硅胶(C18)粉为净化剂,甲醇-水为流动相,流速为0.3 mL/min,以Waters Symmetry~ C_(18)(2.1 mm×100 mm,3.5μm)为色谱分离柱,采用正负离子分段扫描和多反应监测模式(MRM)检测。氯霉素、甲砜霉素、氟苯尼考和氟苯尼考胺采用内标标准曲线法定量,氯硝柳胺采用基质匹配标准曲线外标法定量。结果表明,在0.3~100μg/L范围内,5种待测物均呈良好的线性关系,相关系数(r2)均不小于0.998 7。在1~20μg/kg加标水平下,中华鳖空白血浆、肌肉、裙边、肝脏和肾脏的加标回收率为77.9%~105.3%(n=6),相对标准偏差为2.7%~10.5%(n=6),方法的检出限分别为0.5、0.1、0.5、0.5、0.5μg/kg,定量下限分别为1.0、0.3、1.0、1.0、1.0μg/kg。该方法操作简便、准确、灵敏度高,适用于中华鳖组织中氯硝柳胺、氯霉素、甲砜霉素、氟苯尼考和氟苯尼考胺残留量的同时测定。     

Simultaneous determination of residues of chloramphenicol,thiamphenicol, florfenicol,and florfenicol amine in farmed aquatic species by liquid chromatography/mass spectrometry

A liquid chromatographic (LC)/mass spectrometric (MS) method was developed for determining the residues of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine in a number of aquatic species. The phenicols are extracted with acetone, the extracts are partitioned with dichloromethane, the aqueous layer is removed, and the organic layer is evaporated to dryness. The residue is dissolved in dilute acid and defatted with hexane, and the aqueous layer is prepared for analysis by LC. The phenicols are determined by reversed-phase LC by using a Hypersil C18-BD column with a water-acetonitrile gradient and MS detection using selected-ion recording. Calibration curves were linear for all analytes between 0.015 and 0.425 ng injected. The relative standard deviations for measurements by the proposed method were < 10% for all of the analytes studied, with recoveries ranging from 71% for florfenicol amine to 107% for florfenicol in salmon tissue spiked at the 2 ng/g level. Detection limits of 0.1 ng/g for florfenicol and chloramphenicol, 0.3 ng/g for thiamphenicol, and 1.0 ng/g for florfenicol amine are easily obtainable. The operational errors, interferences, and recoveries for spiked samples compare favorably with those obtained by established LC methodology. The proposed method is simple, rapid, and specific for monitoring residues of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine in a number of aquatic species.     

肉食品中常见4种抗生素残留的高效液相色谱分析

建立了同时测定肉食品中泰乐菌素、替米考星、氯霉素、氟苯尼考的高效液相色谱方法,样品经乙腈提取,正己烷脱脂, HLB小柱净化后,以C18反相色谱柱为分离柱,甲醇－磷酸二氢钠缓冲溶液(pH 3.0,含体积比为10％的甲醇溶液）为流动相进行梯度洗脱,检测波长λ为275 nm.泰乐菌素、替米考星、氯霉素、氟苯尼考的线性范围是0.1～20.0 mg/L,相关系数分别为0.9987、0.9992、0.9985、0.9970.其平均回收率为75％～87%,相对标准偏差为1.35％～5.41％,泰乐菌素、替米考星、氯霉素、氟苯尼考的检出限分别为20、32、19、16μg/kg.方法满足肉食品中泰乐菌素、替米考星、氯霉素、氟苯尼考的残留量测定.     

Confirmation of azaperone and its metabolically reduced form, azaperol, in swine liver by gas chromatography/mass spectrometry.

The method described confirms the use of the tranquilizer azaperone by detecting the parent compound and the metabolically reduced form, azaperol. Both are confirmed in swine liver at a target concentration of 10 ppb by gas chromatography/mass spectrometry (GC/MS) with electron ionization in the selected-ion-monitoring mode. Swine liver tissue is ground with dry ice. Acetonitrile is added to extract the drug from the tissue. Sodium chloride buffer is added to the extract in preparation for solid-phase extraction (SPE). The aqueous extract is loaded onto an SPE cartridge designed to extract acidic and neutral drug residues from biological matrixes. The cartridge is washed with methanol and conditioned with sodium phosphate buffer. Azaperone and azaperol residues are eluted with a 2% ammonium hydroxide in ethyl acetate. The extracts are evaporated to dryness under a stream of nitrogen and reconstituted in ethyl acetate for GC/MS analysis. A DB-1 analytical column is used to separate the compounds prior to electron ionization. The parent ion, the base peak ion, and one diagnostic fragment ion are monitored for both compounds. The method was validated with fortified tissue samples containing both azaperone and azaperol. Azaperone-incurred tissues also were analyzed, and the presence of the parent drug and the metabolically reduced form, azaperol, was confirmed.     

Synchronized separation,concentration and determination of trace chloramphenicol,thiamphenicol and florfenicol in food by using polyoxyethylene cetyl ether-salt aqueous two-phase system coupled with high-performance liquid chromatography

As a novel and green pretreatment technique to trace samples, polyoxyethylene cetyl ether (POELE20)–(NH₄)₂SO₄ aqueous two-phase extraction system was coupled with high-performance liquid chromatography to analyse synchronously chloramphenicol (CAP), thiamphenicol (TAP) and florfenicol (FF) in chicken and pork samples. It was found that the extraction efficiency (E%) and enrichment factor (F) of the three antibiotics were influenced by the types of salts, the concentration of salt, the concentration of POELE20, system temperature and pH. The final optimal condition was as following: the phase-forming salt is (NH₄)₂SO₄, the concentration of (NH₄)₂SO₄ is 0.141 g mL^?1, the concentration of POELE20 is 0.03 g mL^?1, the temperature is 298.15 K, and the system pH is 4.5. This POELE20–(NH₄)₂SO₄ ATPS was applied to separate and enrich three antibiotics in real sample under the optimal conditions, and it was found that the recovery was 97.20–102.00 % with a RSD of 0.61–4.85 %. The limit of detection for CAP, TAP and FF were 0.10, 0.50 and 0.50 μg kg^?1, and the limit of quantitation for CAP, TAP and FF was 0.15, 1.50 and 1.50 μg kg^?1. Seven times the experiments were used to verify the repeatability and veracity of this method, and the RSD for the intra-day and inter-day were 1.13–3.22 and 1.74–4.72 %.     

Determination of benzimidazole anthelmintics in animal-derived biological matrices

This paper describes an easy multiresidue procedure for the determination of 8 benzimidazole anthelmintics (Albendazole, Albendazole sulphone, Albendazole sulphoxide, Ox-bendazole, Ossibendazole, p-OH-Fenbendazole, Fenbendazole, Flubendazole) in foodstuffs of animal origin for human consumption. According to the proposed procedure, 10 g of homogenised sample were extracted with acetonitrile in ultrasonic bath, the organic layer was evaporated to dryness and the residue dissolved in HCl 0.005 M. After an initial washing with hexane to remove fats, the aqueous layer was purified on SPE C2 column and the analytes were eluted with methanol, evaporated to dryness and reconstituted with phosphate buffer/acetonitrile (50/50). The solution obtained was analysed by HPLC with diode array detector (DAD); this detector provides useful information about the peaks present in routine samples, because it gains not only retention times of analytes, as well as their UV-spectra. Benzthiazuron was used as internal standard and the method was validated in the range 10-100 ppb, as requested by the imposed limits. The mean recoveries in spiked samples ranged between 70% and 95%, for 10 and 100 ppb respectively. The developed method resulted sensitive, simple and fast. It is particularly suitable for laboratories that execute screening analysis on a great number of samples, observing the EC regulations that establish the benzimidazole Maximum Residue Limits (MRLs) in food.     

Atmospheric pressure ionization–tandem mass spectrometry of the phenicol drug family

In this work, the mass spectrometry behaviour of the veterinary drug family of phenicols, including chloramphenicol (CAP) and its related compounds thiamphenicol (TAP), florfenicol (FF) and FF amine (FFA), was studied. Several atmospheric pressure ionization sources, electrospray (ESI), atmospheric pressure chemical ionization and atmospheric pressure photoionization were compared. In all atmospheric pressure ionization sources, CAP, TAP and FF were ionized in both positive and negative modes; while for the metabolite FFA, only positive ionization was possible. In general, in positive mode, [M + H]⁺ dominated the mass spectrum for FFA, while the other compounds, CAP, TAP and FF, with lower proton affinity showed intense adducts with species present in the mobile phase. In negative mode, ESI and atmospheric pressure photoionization showed the deprotonated molecule [M–H]^?, while atmospheric pressure chemical ionization provided the radical molecular ion by electron capture. All these ions were characterized by tandem mass spectrometry using the combined information obtained by multistage mass spectrometry and high‐resolution mass spectrometry in a quadrupole‐Orbitrap instrument. In general, the fragmentation occurred via cyclization and losses or fragmentation of the N‐(alkyl)acetamide group, and common fragmentation pathways were established for this family of compounds. A new chemical structure for the product ion at m/z 257 for CAP, on the basis of the MS³ and MS⁴ spectra is proposed. Thermally assisted ESI and selected reaction monitoring are proposed for the determination of these compounds by ultra high‐performance liquid chromatography coupled to tandem mass spectrometry, achieving instrumental detection limits down to 0.1 pg. Copyright © 2013 John Wiley & Sons, Ltd.     

Rapid determination of five antibiotic residues in swine wastewater by online solid-phase extraction–high performance liquid chromatography–tandem mass spectrometry

A simple and quick online solid-phase extraction (SPE) coupled to liquid chromatography (LC)/tandem mass spectrometry (MS/MS) for the determination of the five antibiotics (florfenicol, FF; lincomycin, LCM; oxytetracyclin, OTC; tylosin, TS; valnemulin, VLM) in swine wastewater has been developed. After filtration, aliquots (100 μl) of wastewater samples were directly injected to a column-switching LC system. Some matrix interference was removed by washing up SPE column with 0.2% formic acid solution and acetonitrile. Antibiotics eluted from SPE column were separated on analytical column by converting switching valve and were detected by MS/MS. Calibration curves using the method of standard addition had very good correlation coefficients (r > 0.99) in the range of 0.1 to 2 ng/ml. The intra-day precision of the method was less than 12% and the inter-day precision was between 6 to 17%. The detection limits were 0.01–0.1 ng/ml. When this method was applied to wastewater samples in swine facilities, four compounds (LCM, OTC, TS, and VLM) were detected.     

Determination of residual clopidol in chicken muscle by capillary gas chromatography/mass spectrometry

Clopidol was extracted from chicken with acetonitrile, and the extract was evaporated to about 5 mL. The concentrated solution was centrifuged and applied to a glass column filled with basic alumina. The column was eluted with methanol and the methanol was evaporated to dryness. The residue was derivatized with Sylon BFT and analyzed by gas chromatography/mass spectrometry (GC/MS). Mass characteristics of trimethylsilyl derivative of clopidol were interpreted manually. The selected-ion monitoring mode was performed at m/z 191, 248, 263, and 265. A sensitive, reproducible GC/MS method was developed for monitoring residues of clopidol in chickens. All recoveries of the drug from chicken muscle fortified at 1, 10, and 100 ng/g were > 65%, and relative standard deviations were < 15%. The detection limit was about 0.5 ng/g, and the quantitation limit was the lower limit of the standard curve, which was about 1 ng/g.     

Determination of chloramphenicol residues in milk, eggs, and tissues by liquid chromatography/mass spectrometry

A new liquid chromatography/mass spectrometry (LC/MS) method is presented for the determination of chloramphenicol (CAP) residues in milk, eggs, chicken muscle and liver, and beef muscle and kidney. CAP is extracted from the samples with acetonitrile and defatted with hexane. The acetonitrile extracts are then evaporated, and residues are reconstituted in 10mM ammonium acetate--acetonitrile mobile phase and injected into the LC system. CAP is determined by reversed-phase chromatography using an Inertsil ODS-2 column and MS detection with negative ion electrospray ionization. Calibration curves were linear between 0.5-5.0 ng/g for all matrixes studied. The relative standard deviations for measurements by this method were generally <12%, and average recoveries ranged from 80 to 120%, depending on the matrix involved. The method detection limits of CAP ranged from 0.2 to 0.6 ng/g, which are comparable to previously reported results. The proposed method is rapid, simple, and specific, allowing a single analyst to easily prepare over 40 samples in a regular working day.     

Improved high-performance liquid chromatographic assay for the determination of ethionamide in serum

A solid-phase extraction (SPE) method was developed to simplify the preparation of human serum prior to high-performance liquid chromatography of ethionamide (ETA). Octadecyl SPE columns were used. Serum constituents were removed from the column with water, and ETA was eluted with methanol. Samples were evaporated to dryness, reconstituted in mobile phase, and assayed. The method is reproducible, with a recovery of ETA of 64%, comparable to the more tedious liquid-liquid extraction method for ETA.     

Multiresidue determination of fluoroquinolones in shrimp by liquid chromatography-fluorescence-mass spectrometry

An efficient multiresidue method for analysis of fluoroquinolones in shrimp has been developed in which quantitation by fluorescence and confirmation by Multiple Stage Mass Spectrometry (MS) is achieved simultaneously. In this method, shrimp tissue is extracted with ammoniacal acetonitrile and the extract is defatted and then evaporated. After dissolution in basic phosphate buffer, fluoroquinolones in the extract are separated by liquid chromatography and quantitated, taking advantage of their intense fluorescence. Eluate from the fluorescence detector enters the MS, which allows for confirmation by monitoring ratios of 2 prominent product ions in the MS3 or MS2 spectrum. Using this method, 8 fluoroquinolones have been analyzed in shrimp samples fortified at 10, 25, 50, or 100 ppb levels. Recoveries for desethyleneciprofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, orbifloxacin, sarafloxacin, and difloxacin ranged from 75 to 92%, with relative standard deviation values of <6%. The limits of quantitation ranged from 0.1 to 1 ng/g. Enrofloxacin and ciprofloxacin were also successfully determined in enrofloxacin-incurred shrimp using this method.     

Determination of two oxy-pyrimidine metabolites of diazinon in urine by gas chromatography/mass selective detection and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry

An analytical method was developed for the determination in urine of 2 metabolites of diazinon: 6-methyl-2-(1-methylethyl)-4(1H)-pyrimidinone (G-27550) and 2-(1-hydroxy-1-methylethyl)-6-methyl-4(1H)-pyrimidinone (GS-31144). Two of the urine sample preparation procedures presented rely on gas chromatography/mass selective detection (GC/MSD) in the selected ion monitoring mode for determination of G-27550. For fast sample preparation and a limit of quantitation (LOQ) of 1.0 ppb, urine samples were purified by using ENV+ solid-phase extraction (SPE) columns. For analyte confirmation at an LOQ of 0.50 ppb, classical liquid/liquid partitioning was used before further purification in a silica SPE column. An SPE sample preparation procedure and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry (LC/ESI/MS/MS) were used for both G-27550 and GS-31144. The limit of detection was 0.01 ng for G-27550 with GC/MSD, and 0.016 ng when LC/ESI/MS/MS was used for both G-27550 and GS-31144. The LOQ was 0.50 ppb for G-27550 when GC/MSD and the partitioning/SPE sample preparation procedure were used, and 1.0 ppb for the SPE only sample preparation procedure. The LOQ was 1.0 ppb for both analytes when LC/ESI/MS/MS was used.     

Peer validation of a method to confirm chloramphenicol in honey by liquid chromatography-tandem mass spectrometry

Chloramphenicol (CAP) is extracted from an aqueous dilution of honey using ethyl acetate. The extracts are evaporated and redissolved in water. CAP is then extracted from the aqueous solutions using reversed-phase solid-phase extraction cartridges. CAP is eluted from the reversed-phase cartridges with acetonitrile-water and re-extracted into ethyl acetate. The ethyl acetate is evaporated, and the residue is reconstituted in an aqueous solution. Extracts are chromatographed using a reversed-phase column and analyzed by electrospray negative mode tandem mass spectrometry. Four product ions of precursors m/z 321 or 323 are monitored. The method meets confirmation criteria recommended by the U.S. Food and Drug Administration and 4-point identification criteria established by the European Union. With slight modifications to accommodate different equipment, the method was validated in 2 laboratories.