Trace analysis of fumagillin in honey by liquid chromatography-diode array-electrospray ionization mass spectrometry

In this work a new liquid chromatography with diode array detection and electrospray ionization mass spectrometry (LC-DAD-ESI-MS) method has been developed for the determination of fumagillin residues in honey. This procedure involves a solid-phase extraction on polymeric cartridges for the isolation of fumagillin from diluted honey. Chromatographic separation of fumagillin was performed in isocratic mode, on a C(18) column (150 mm x 4.60mm i.d., 5 microm), the mobile phase consisted of a mixture of ammonium formate 20mM in water and acetonitrile (61/39, v/v), at 35 degrees C and the flow rate was set at 1.0 mL/min. Average analyte recoveries, influenced by the botanical origin were from 88 to 96% in replica sets of fortified honey samples. The detection limits of the LC-DAD-ESI-MS method were between 24 and 1 microg/kg for clear honeys (rosemary) and between 45 and 4 microg/kg for dark honeys (heather). The developed method has been applied to the analysis of fumagillin residues in honey samples collected from veterinary treated beehives, infected by Nosema ceranae and fed with the technical product at different doses.     

气相色谱-质谱法分析蜂蜜中的多种农药残留

开展了蜂蜜中23种农药残留的气相色谱-电子轰击离子源质谱(GC-EI/MS)分析方法的研究,并对其中3种农药的EI/MS碎片离子的断裂机理与结构进行了初步解析。探讨了蜂蜜试样前处理条件的优化与选择。将蜂蜜试样用乙酸乙酯提取剂超声提取、Florisil硅藻土色谱柱净化和正己烷-乙酸乙酯(体积比为7∶3)混合洗脱剂洗脱后,以PCB103为内标物,采用选择离子监测(SIM)方式下的GC-EI/MS分析。当试样的加标浓度为50,100和200 μg/kg时,加标回收率为82%～120%,相对标准偏差小于11.0%。23种农药的检测限都小于10.0 μg/kg,线性范围为10～500 μg/kg,相关系数都大于0.995。此分析方法已成功地应用于蜂蜜中23种痕量农药残留的分析。     

Multiresidue determination of pesticides in honey by matrix solid-phase dispersion and gas chromatography with electron-capture detection

A multiresidue method was developed for the determination of 15 pesticides (organochlorines, organophosphorus compounds, pyrethroids, and other acaricides) in various commercial honeys (eucalyptus, lavender, orange, rosemary, and multifloral). The analytical procedure is based on the matrix solid-phase dispersion of honey in a mixture of Florisil and anhydrous sodium sulfate; the mixture is placed in small plastic columns and extracted with hexane-ethyl acetate (90 + 10, v/v). The pesticide residues are determined by capillary gas chromatography with electron-capture detection. Recoveries with the method at concentrations between 0.15 and 1.5 microg/g ranged from 80 to 113%, and relative standard deviations were <10% for all the pesticides studied. The pesticide detection limits were within the range 0.5-5 microg/kg for organochlorines, around 3 microg/kg for the chlorinated organophosphorus pesticides studied, near 15 microg/kg for fluvalinate, and about 3 microg/kg for the other pyrethroids.     

Determination of polycyclic aromatic hydrocarbons in honey by matrix solid-phase dispersion and gas chromatography/mass spectrometry

A multiresidue method was developed for the de termination of 16 polycyclic aromatic hydrocarbons (PAHs) in unifloral and multifloral honeys. The analytical procedure is based on the matrix solid-phase dispersion of honey on a mixture of Florisil and anhydrous sodium sulfate in small glass columns and extraction with hexane-ethyl acetate (90 + 10, v/v) with assisted sonication. The PAH residues are determined by gas chromatography with mass spectrometric detection using selected-ion monitoring. Average recoveries for all the PAHs studied were in the range of almost 80 to 101%, with relative standard deviations of 6 to 15%. The limits of detection ranged from 0.04 to 2.9 microg/kg. The simultaneous extraction and cleanup of samples makes this method simple and rapid, with low consumption of organic solvents     

Determination of insecticides in honey by matrix solid-phase dispersion and gas chromatography with nitrogen-phosphorus detection and mass spectrometric confirmation

A multiresidue method was developed for the determination of 12 organophosphorus insecticides (diazinon, parathion methyl, fenitrothion, pirimiphosmethyl, malathion, fenthion, chlorpyrifos, quinalphos, methidathion, ethion, azinphosmethyl, coumaphos), one carbamate (pirimicarb), and one amidine (amitraz) in unifloral and multifloral honeys. The analytical procedure was based on the matrix solid-phase dispersion of honey on a mixture of Florisil and anhydrous sodium sulfate in small glass columns and subsequent extraction with a low volume of hexane-ethyl acetate (90 + 10, v/v), assisted by sonication. The insecticide residues were determined by capillary chromatography with nitrogen-phosphorus detection and confirmed by mass spectrometry. Average recoveries at the 0.05-0.5 microg/g levels were >80% for organophosphorus insecticides and about 60% for the other insecticides, pirimicarb and amitraz, with relative standard deviations <10%. The detection limit for the different insecticides ranged between 6 and 15 microg/kg. The main advantages of the proposed method are that extraction and cleanup are performed in a single step with a low volume of organic solvent. The method is simple, rapid, and less laborious than conventional methods. Several Spanish honeys were analyzed with the proposed method and no residues of the studied insecticides were found.     

Detection of streptomycin and dihydrostreptomycin residues in milk,honey and meat samples using an optical biosensor

Immuno-biosensor inhibition assays for the detection of streptomycin and dihydrostreptomycin residues in whole cows' milk, honey, pig kidney and pig muscle are reported. The antibody showed high cross-reactivity with dihydrostreptomycin in various foodstuffs (buffer 103%, milk 96%, honey 84%, kidney extract 129% and muscle extract 98%). There was no significant cross-reaction with other aminoglycosides or commonly used antibiotics. A streptomycin derivative was used to prepare a stable, reusable sensor chip surface. The assay allowed the direct analysis of bovine whole milk (fat content approximately 3.5%). Honey samples required dilution with buffer, while kidney and muscle samples from pigs were homogenized in an aqueous extraction buffer and clarified by centrifugation. The limit of detection for each assay was determined from known streptomycin-free samples (n = 20; mean - (3 x standard deviation)) and the results were as follows: milk 30 microg kg(-1), honey 15 microg kg(-1), kidney 50 microg kg(-1) and muscle 70 microg kg(-1). Repeatability (or relative standard deviation) between runs were calculated (n = 3) at the respective Community maximum residue limits (MRL) and 0.5 x MRL with the exception of honey since no European MRL exists at present. Results were determined as 4.3% (200 microg kg(-1)) and 2.8% (100 microg kg(-1)) in milk, 13.3% (40 microg kg(-1)) and 9.5% (20 microg kg(-1)) in honey, 7.1% (1000 microg kg(-1)) and 7.6% (500 microg kg(-1)) in kidney and 7.1% (500 microg kg(-1)) and 11% (250 microg kg(-1)) in muscle.     

Development of a rapid and sensitive method for the simultaneous determination of 1,2-dibromoethane, 1,4-dichlorobenzene and naphthalene residues in honey using HS-SPME coupled with GC-MS

A new method for the simultaneous determination of 1,4-dichlorobenzene (p-DCB), naphthalene and 1,2-dibromoethane (1,2-DBE) residues in honey has been developed. Analysis is carried out using gas chromatography-mass spectrometry (GC/MS) in selected ion monitoring mode (SIM), after extraction and preconcentration of target analytes by headspace solid-phase microextraction (HS-SPME), with a 100 microm film thickness polydimethylsiloxane (PDMS) fiber. Several parameters affecting the extension of the adsorption process (i.e., addition of salt, extraction time, extraction temperature) were studied. The optimal conditions for the determination of these analytes were established. The proposed HS-SPME method showed good sensitivity, without carryover between the samples. Linearity was studied from 5 to 2500 microg kg(-1) for p-DCB, 0.5 to 500 microg kg(-1) for naphthalene and 5 to 500 microg kg(-1) honey for 1,2-DBE with correlation coefficients (r(2)) ranging from 0.9901 to 0.9999. Precision was assessed and both intra and inter-day R.S.D.s (%) were below 6.3%. The detection limits were found to be 1, 0.1 and 2 microg kg(-1) honey for p-DCB, naphthalene and 1,2-DBE, respectively. The percentage recoveries that were evaluated with the proposed HS-SPME method and the standard addition calibration technique gave values among 72.8 and 104.3% for measurements in samples spiked with one target analyte or mixtures of the three. This method has been applied for the analysis of unknown honey samples. The results showed an excellent applicability of the proposed method for the determination of the target compounds in honey samples.     

高效液相色谱-串联质谱法测定荔枝花粉和花蜜中腈菌唑和苯醚甲环唑残留

采用改良的QuEChERS-高效液相色谱-串联质谱（HPLC-MS/MS）技术,建立了荔枝花粉和花蜜中2种主要杀菌剂腈菌唑和苯醚甲环唑的测定方法。花粉和花蜜样品均由乙腈提取,花粉样品经0.9 g无水硫酸镁、0.15 g N-丙基乙二胺（PSA）和0.15 g十八烷基键合硅胶（C₁₈）吸附剂净化,花蜜样品由0.9 g无水硫酸镁和0.15 g PSA净化。采用Poroshell-120 EC-C₁₈色谱柱分离,以0.1%（v/v）甲酸水溶液-乙腈（25∶75,v/v）为流动相等度洗脱,在电喷雾离子（ESI）源、正离子扫描和选择离子监测模式下进行检测,基质匹配标准溶液法定量。结果显示：腈菌唑和苯醚甲环唑在1~100 μg/L范围内线性关系良好,相关系数（r²）均大于0.9990;腈菌唑和苯醚甲环唑的检出限（LOD）分别为0.25 μg/kg和0.50 μg/kg,定量限（LOQ）分别为0.83 μg/kg和1.7 μg/kg;腈菌唑和苯醚甲环唑在荔枝花粉和花蜜样品中的平均加标回收率分别为87.0%~95.2%和90.1%~96.4%,相对标准偏差（RSD）分别为1.2%~3.6%和0.7%~4.1%。该法快速、简便、灵敏,可用于荔枝花粉和花蜜样品中腈菌唑和苯醚甲环唑的痕量测定,可为蜜蜂等授粉昆虫的暴露性风险评估提供技术支持。     

Determination of antibiotics such as macrolides,ionophores and tiamulin in liquid manure by HPLC–MS/MS

A method for the analysis of several macrolide and ionophore antibiotics as well as tiamulin in liquid manure was developed. Reversed-phase liquid chromatography and atmospheric pressure chemical ionisation (APCI) tandem mass spectrometry was used for detection.High-performance liquid chromatographic (HPLC) separation of the antibiotics was achieved in 35 min. The analytes were extracted with ethyl acetate and the extracts were cleaned up by solid-phase extraction on a diol SPE cartridge. Recovery experiments with spiked liquid manure concentrations varying from 6 to 2,000 microg kg(-1) gave constant recovery rates. The recovery rates for the macrolides erythromycin, roxithromycin and oleandomycin were 75-94%, that for the ionophore salinomycin was 119%, while that for the pleuromutilin tiamulin was 123%, when using a macrolide internal standard. The relative standard deviation was found to be 15-36% and the limits of detection were 0.4-11.0 micro g kg(-1).The maximum concentrations found in manure samples were 43 micro g kg(-1) for tiamulin and 11 micro g kg(-1) for salinomycin.     

Application of matrix solid-phase dispersion and high-performance liquid chromatography for determination of sulfonamides in honey

A novel method for simultaneous determination of 8 sulfonamide residues (sulfathiazole, sulfapyridine, sulfadiazine, sulfamerazine, sulfamonome-thoxine, sulfachloropyridazine, sulfamethoxazole, and sulfadimethoxine) in honey samples by high-performance liquid chromatography (HPLC) has been developed on the basis of precolumn derivatization with 9-fluorenylmethyl-chloroformate (FMOC-Cl). Sulfonamide residues in honey samples were extracted and purified by matrix solid-phase dispersion with C18 as the solid support. The residues were derivatized by FMOC-CI, and the FMOC-sulfonamide derivatives were further purified by solid-phase extraction with silica gel as the solid support prior to HPLC analysis. The average recoveries for most sulfonamide compounds at different spiking levels (from 10 to 250 microg/kg) were > 70% with relative standard deviations < 16%, and their limits of detection were 4.0 microg/kg. The established analytical method has high sensitivity and repeatability and can be applicable for determining the sulfonamide residues in various honey matrixes.     

高效液相色谱-电喷雾串联质谱法测定蜂蜜中的林可胺类抗生素残留

建立了蜂蜜中林可胺类抗生素林可霉素和氯林可霉素的高效液相色谱-电喷雾串联质谱(HPLC/ESI-MS/MS)检测方法。样品经固相萃取提取净化、反相液相色谱分离后进行质谱分析,在选择反应监测模式(SRM) 下进行特征母-子离子对信号采集。根据保留时间、母离子和两个特征子离子信息进行定性分析,以共同的基峰离子m/z 126进行定量。两种抗生素的检测限(S/N=3) 为 0.1 μg/kg,定量限为 0.5 μg/kg,在1.0～200 μg/L时峰强度与质量浓度的线性关系良好(r2>0.996)。在1.0,5.0,20.0 μg/kg 3个添加水平,两种抗生素的平均回收率范围为80%～110%,日内测定结果的相对标准偏差小于8%,日间测定结果的相对标准偏差小于15%。结果表明,该法简单、灵敏,特异性强,适用于蜂蜜中林可胺类抗生素残留的分析确证。     

固相萃取-液相色谱-质谱/质谱法同时测定蜂蜜中的多类药物残留

建立了采用固相萃取-液相色谱-质谱/质谱(SPE-LC-MS/MS)对蜂蜜中磺胺类、硝基咪唑类、喹诺酮类、大环内酯类、林可酰胺类和吡喹酮共计6大类54种药物残留同时测定的方法。蜂蜜经磷酸盐缓冲溶液(pH 8)稀释,Oasis HLB固相萃取柱净化后,通过液相色谱-质谱联用技术进行检测(正离子方式,多反应监测模式)。采用同位素稀释内标法或外标法进行定量,线性关系良好,相关系数大于0.992。方法的定量限(LOQ,以信噪比(S/N)大于10计)分别为磺胺类和硝基咪唑类药物1.0 μg/kg,喹诺酮类和林可酰胺类药物2.0 μg/kg,大环内酯类药物3.0 μg/kg,吡喹酮0.3 μg/kg。总体回收率为32.6%～114%,相对标准偏差为1.3%～28.9%。该方法的定量限满足目前国内外药物的最大残留限量要求,可作为蜂蜜中相关药物残留量的筛选检测方法。     

Determination of streptomycin and dihydrostreptomycin in milk and honey by liquid chromatography with tandem mass spectrometry

Two liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed for the determination of streptomycin (STR) and its derivative dihydrostreptomycin (DHSTR) in milk and honey. These aminoglycoside antibiotics are used as veterinary drugs. In the EU, the presence of dihydro- and streptomycin residues in honey is forbidden, the maximum residue level (MRL) in milk is 200 microg/kg. The methods were optimised with regard to sensitivity and chromatographic efficiency, and validated by a procedure consistent with EU directive 2002/657. Average recoveries and accompanying standard deviations were satisfactory. The limit of quantification of STR was 2 microg/kg in honey and 10 microg/kg in milk, of DHSTR it was a factor two lower. The precision of the milk analysis was improved by using STR as the internal standard for DHSTR and vice versa. In a survey of 186 honeys available on the Dutch market, 26% of the honeys of foreign origin were positive for (DH)STR. This occurence rate was consistent with previous surveys, but lower concentrations were found.     

Determination of closantel residues in milk and animal tissues by HPLC with fluorescence detection and SPE with oasis MAX cartridges

A liquid chromatographic method for the determination of closantel residues in milk and tissues is developed and validated. An acetonitrile-acetone solution (80:20, v/v) is used for the extraction of closantel residues from milk and animal tissues, and the extract is purified by solid-phase extraction with Oasis MAX cartridges and a mixture of formic acid-acetonitrile (5:95, v/v) as the elution solution. A C(18) bonded silica column is used for chromatographic separation. The mobile phase consists of acetonitrile-water (85:15, v/v) containing 0.05% triethylamine at pH 2.5, adjusted with phosphoric acid with the flow-rate set at 1.0 mL/min. Using the fluorescence emission of closantel at lambda(ex) = 335 nm and lambda(ex) = 510 nm, the calibration curve is linear, with a correlation coefficient of 0.9999 over the concentration range of 10-5000 microg/kg for the tissue sample and 10-5000 microg/L for the milk sample. The detection limit (s/n = 3) is 3 microg/kg for tissue sample and 3 microg/L for milk sample. The intra- and inter-day repeatabilities are between 3.35-7.66% and 4.04-8.67%, respectively. The proposed method enables the quantitative determination of closantel residues at levels as low as 10 microg/kg in animal tissue samples and 10 microg/L in milk samples.     

Multi-screening approach to monitor and quantify 42 antibiotic residues in honey by liquid chromatography-tandem mass spectrometry

A multi-screening approach for monitoring potential chemical contaminants in honey by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has been developed. A total of 42 veterinary drugs (5 tetracyclines, 7 macrolides, 3 aminoglycosides, 8 beta-lactams, 2 amphenicols and 17 sulfonamides) were surveyed with the ultimate goal of unambiguously confirmed and quantified these analytes at a concentration level of 20 microg/kg. A basic sample preparation including four subsequent liquid/liquid extraction steps was necessary to adequately extract the compounds of interest from the honey. The four extracts were injected into the LC-ESI-MS/MS using a stacking injection procedure. Validation of the entire procedure was carried out according to the European Union directive 2002/657/EC at three concentration levels, i.e., 10, 20 and 30 microg/kg. Good performance data were obtained for 37 analytes, out of the 42 studied. Limit of compliance and detection limit were calculated based on an internal limit set at 20 microg/kg for all the compounds and ranged between 24-30 and 27-80 microg/kg, respectively. A limited survey on honeys of different geographical origins has demonstrated that positive honey samples were often contaminated by more than one class of drugs, thus highlighting the usefulness of such multi-screening approach to ensure and warrants the quality of honey.     

Validation of a high-performance liquid chromatography method for the determination of oxytetracycline,tetracycline, chlortetracycline and doxycycline in bovine milk and muscle

High-performance liquid chromatography with diode-array detection (HPLC-DAD) was optimised and validated for the determination of tetracyclines in bovine milk and tissues. Milk and tissue samples were extracted and purified using a solid-phase extraction HLB Oasis cartridge and analysed using HPLC-DAD set at 365 nm. The analyses were carried out using the mobile phase of 0.01 M oxalic acid-acetonitrile-methanol (60:25:15, v/v/v) on a C8 column (250 x 4.6 mm I.D., 5 microm). Recoveries of tetracyclines from spiked samples at the three concentrations (0.5, 1 and 1.5) of the maximum residues limits (corresponding to 100 microg/kg for milk and the muscle) were higher than 81.1% in milk and 83.2% in muscle. The method was successfully validated for bovine milk and muscle in compliance with requirements set by draft SANCO/ 1805/ 2000 European Decision. The decision limit (CCalpha) was in the range 113.2-127.2 microg/kg and 107.7-129.9 micro/kg for all compounds in milk and muscle, respectively. The detection capability (CCbeta) was in the range 117.2-131.3 microg/kg and 114.9-133.1 microg/kg for all compounds in milk and muscle, respectively.     

Trace analysis of antibacterial tylosin A, B, C and D in honey by liquid chromatography-electrospray ionization-mass spectrometry

A new LC-ESI-MS method was developed for the determination of residues of the antibacterial tylosins A, B, C and D in honey. The procedure employed an SPE on polymeric cartridges for the isolation of tylosins from diluted honey. Chromatographic separation of the tylosins was performed on a C18 column (150 x 4.60 mm2 ID, 5 microm) using a ternary gradient made of formic acid 1% in water (solvent A), methanol (solvent B) and ACN (solvent C) as mobile phase, at 30 degrees C and at a flow rate of 0.8 mL/min. Average analyte recoveries for the studied compounds ranged from 89 to 106% in replica sets of fortified honey samples. The detection limits for the four drugs studied were between 2 and 3 microg/kg. The developed method has been applied to the analysis of tylosin residues in honey from veterinarian treated beehives fed with the technical product, which contains the four compounds and is a new candidate antibiotic to treat American foulbrood disease of honey bee colonies.     

Validated method for determination of ultra-trace closantel residues in bovine tissues and milk by solid-phase extraction and liquid chromatography-electrospray ionization-tandem mass spectrometry

A liquid chromatographic-electrospray ionisation-tandem mass spectrometry method (LC-ESI-MS/MS) with solid extraction was developed and validated for the detection and determination of closantel residues in bovine tissues and milk. An acetonitrile-acetone mixture (80:20, v/v) was used for one-stage extraction of closantel residues in bovine tissues and milk samples, and the extract was cleaned by solid phase extraction with Oasis MAX cartridges. The mass spectrometer was operated in multiple reactions monitoring mode with negative electrospray interface. The limits of detection in different matrices were in the range of 0.008-0.009 microg/kg. The overall recoveries for bovine muscle, liver, kidney and milk samples spiked at four levels including MRL were in the range of 76.0-94.3%. The overall relative standard deviations were in the range of 3.57-8.61%. The linearity is satisfactory with a correlation coefficient (r(2)) of 0.9913-0.9987 at both concentration ranges of 0.02-100 microg/kg and 200-5000 microg/kg. The method is capable of identifying closantel residues at > or =0.02 microg/kg levels and was applied in the determination of closantel residues in animal origin foods.     

高效液相色谱-串联质谱法测定食品中多杀菌素A和D的残留量

建立了食品中多杀菌素(spinosyn) A和D残留的液相色谱-串联质谱(HPLC-MS/MS)检测方法。甘蓝等食品样品经乙腈-水(50:50, v/v)提取、HLB固相萃取柱净化后,采用HPLC-MS/MS进行检测分析,利用外标法定量。质谱分析采用电喷雾电离,正离子扫描,多反应监测模式。结果表明,柱净化后无明显的基质效应,多杀菌素在1～20 μg/L内具有较好的线性关系,其相关系数可达0.9999;样品中添加1～10 μg/kg的多杀菌素,其回收率为76.2%～114.0%,相对标准偏差(n=10)小于10%;检出限(信噪比（S/N）=3): 多杀菌素A为0.2 μg/kg,多杀菌素D为0.5 μg/kg;定量限(S/N=10): 多杀菌素A为0.5 μg/kg,多杀菌素D为1.0 μg/kg。在最佳实验条件下,对来自福建厦门的969份食品样品进行了检测,其中15份样品检测结果呈阳性。实验结果表明,该方法提取效果好,具有良好的灵敏度、回收率和重复性。     

气相色谱-负化学源质谱法测定蜂蜜和王浆中4种杀虫剂的残留

建立了气相色谱-负化学源质谱（GC-NCI/MS）测定蜂蜜和王浆中4种杀虫剂残留量的方法。蜂蜜样品由乙酸乙酯提取、乙二胺-N-丙基硅烷（PSA）净化,而王浆样品经乙腈-水（1：1,v/v）提取、C18固相萃取柱净化,采用GC-NCI/MS测定,外标法定量。结果表明：在50~500 μg/L范围内4种农药的线性良好;所有农药的LOD在0.12~5.0 μg/kg之间,LOQ在0.40~16.5 μg/kg之间;在10、15、20 μg/kg 3个添加水平下,4种农药的平均回收率在78.2%~110.0%之间,且RSD均小于14%。所有农药的测定均没有出现干扰峰。该方法简单、快速,准确度、精密度和选择性高,抗干扰能力强,可用于蜂蜜和王浆中这4种农药的快速检测。