Simultaneous determination of nine kinds of preservatives in foods by HPLC

A simple and rapid HPLC method was developed for the simultaneous determination of nine kinds of preservatives, benzoic acid (BA), sorbic acid (SOA), dehydroacetic acid (DHA), methyl p-hydroxybenzoate (PHBA-Me), ethyl p-hydroxybenzoate (PHBA-Et), isopropyl p-hydroxybenzoate (PHBA-isoPu), propyl p-hydroxybenzoate (PHBA-Pu), isobutyl p-hydroxybenzoate (PHBA-isoBu) and butyl p-hydroxybenzoate (PHBA-Bu), in foods. For solid foods, the preservatives were extracted with methanol. After addition of 5 mmol/L citrate buffer to the extract, the extract solution was cleaned up on an Oasis HLB cartridge. The cartridge was washed with 5 mmol/L citrate buffer and methanol-5 mmol/L citrate buffer (4:6). Then, nine kinds of preservatives were eluted with methanol. The eluent was used for BA, SOA and DHA determination by HPLC. Furthermore, a part of the eluent was cleaned up on a Bond Elut PSA cartridge for p-hydroxybenzoate esters determination by HPLC. Liquid foods were cleaned up after addition of 5 mmol/L citrate buffer without the extraction process, and the subsequent procedure was the same as for solid foods. The recoveries of p-hydroxybenzoate esters from ten kinds of foods fortified at levels of 0.01 and 0.10 g/kg each were 91.5 to 107.4%, and those of BA, SOA and DHA were 76.4 to 104.8%. The quantitation limits of the preservatives in foods were 0.005 g/kg. (Received March 20, 2006)     

超高效液相色谱- 质谱联用测定食品中叶酸含量

建立食品中叶酸含量的超高效液相色谱质谱联用测定方法。样品提取后以Acquity UPLC BEH C18柱(2.1mm×50mm,1.7μm)为色谱柱分离,以电喷雾电离串联质谱在正离子选择反应监测模式下进行测定。以乙腈-0.1%甲酸溶液(5:95,V/V)为流动相、流速0.25mL/min、色谱柱温度40℃、进样量2μL,母离子m/z 442.3、定量子离子m/z 295.1、定性子离子m/z 176.0,碰撞能量为14eV。对空白试样进行3个添加水平4个重复的添加结果表明：回收率80.7%～89.7%,相对标准偏差2.90%～3.85%。该方法检出限1ng/mL,线性范围0.001～1.000μg/mL。该方法具有样品预处理简单、检测周期短、灵敏度较高等优点。     

Determination of nine preservatives in processed foods using a modified QuEChERS extraction and quantified by HPLC-PDA

ABSTRACT

In this study, a new method was developed for simultaneously determining nine preservatives, that is, benzoic acid (BA), sorbic acid (SOA), dehydroacetic acid (DHA) and PHBAs (methyl p-hydroxybenzoate [PHBA-me], ethyl p-hydroxybenzoate [PHBA-et], isopropyl p-hydroxybenzoate [PHBA-ipro], propyl p-hydroxybenzoate [PHBA-npro]), isobutyl p-hydroxybenzoate [PHBA-ibut] and butyl p-hydroxybenzoate [PHBA-nbut]), in processed foods, employing liquid chromatography (LC). This procedure accelerated sample preparation and improved efficiency by employing modified quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction without clean-up. Samples were prepared with 20 mL of acetonitrile/water (1:1) with the assistance of a ceramic stone. The extract solutions were diluted 10 times or according to the detection amount and then injected into an LC-PDA. This method showed good linearity, and the LOQs were 10 mg/kg for BA, SOA and DHA and 5?mg/kg for the PHBAs. When validating this method, the recoveries of the nine preservatives were in the range 77.0–99.6%, RSD_r values were in the range 0.7–5.3% and those of RSD_wr were in the range 2.3–8.4%. These results suggest that this new method is highly reproducible.     

Analysis of thyroid hormones in health foods by LC/MS

A method was developed for analysis of thyroid hormones, 3,5,3'-triiodo-L-thyronine (T3) and L-thyroxine (T4), in health foods by liquid chromatography-electrospray ionization-mass spectrometry (LC/ESI-MS). In order to release T3 and T4 from thyroglobulin, samples were first hydrolyzed enzymatically using protease at a temperature of 37 +/- 1 degrees C for 28 h. The T3 and T4 were extracted with ethyl acetate and then the ethyl acetate layer was evaporated. The residue was dissolved in the mobile phase and analyzed by LC/MS. The LC separation was carried out on a ODS-3 column (2.1 x 150 mm) using H2O/CH3CN/CH3COOH (650/350/5) as the mobile phase. LC/ESI-MS was performed in the selected ion monitoring (SIM) mode using [M + H]+ ions at m/z 652 for T3 and m/z 778 for T4. The detection limit of both T3 and T4 released from thyroglobulin was 0.1 microgram/g in health foods. The present method was applied to analysis of three health foods which were labeled as foods for dieting. T3 and T4 were detected in two of the samples, and their contents were 16 and 29 micrograms/g and 31 and 90 micrograms/g, respectively.     

Application of ion-trap LC/MS/MS for determination of acrylamide in processed foods

Ion-trap LC/MS/MS was evaluated for use in the determination of acrylamide (AA) in processed foods. Multiple reaction monitoring (MRM) analysis of a series of AA standard solutions containing deuterium-labeled acrylamide (AA-d3) as an internal standard was performed. A linear relationship between the concentration of AA and the ratio of peak area (AA/AA-d3) in the extracted ion chromatogram (m/z 55, 58 derived from m/z 72, 75, respectively) was obtained over a wide range of 2-20,000 ng/mL. The quantification limit of AA was 2 ng/mL. In analyses of 37 commercial foods, AA was detected in a potato snack at the maximum value of 3,570 ng/g and found in 23 foods prepared or cooked at high temperature. The samples were analyzed in triplicate and the relative standard deviations (RSD) were less than 15% in many processed foods.     

Simultaneous Determination of Ractopamine,Chloramphenicol, and Zeranols in Animal-Originated Foods by LC-MS/MS Analysis with Immunoaffinity Clean-up Column

A novel analytical method employing immunoaffinity column (IAC) clean-up coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of ractopamine, chloramphenicol, and zeranols (α-zearalanol, β-zearalanol, zearalanone, α-zearalenol, β-zearalenol, and zearalenone) in animal-originated foods. The sample was first digested by β-glucuronidase/sulfatase and then extracted with ethyl acetate-diethyl ether (9:1, v/v). The extracted solution was evaporated to dryness and then the residue was dissolved by 2 mL of 50% acetonitrile solution. After filtration, 1 mL filtrate was diluted to 10 mL with PBS. The reconstituted solution was cleaned up with immunoaffinity column and then analyzed by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The established method was shown to be sensitive efficient and reliable as indicated by the linearity (r ² ≥ 0.9994), precision (RSD ≤ 1.7%), average recovery (72.3–103.2%), and the limit of detection (0.05–0.10 μg/kg). The method can be used for determination of trace residues of ractopamine, chloramphenicol, and zeranols in animal-originated foods.     

高效液相色谱法同时测定食品中18种食品添加剂

建立高效液相色谱同时测定食品中18种食品添加剂的高通量分析方法。对于不含油脂或油脂含量低的样品采用含抗坏血酸棕榈酸酯的正己烷饱和乙腈-6mol/L HCl溶液-饱和氯化钠混合溶液一次提取净化,对于油脂样品采用含抗坏血酸棕榈酸酯的正己烷饱和乙腈-乙腈饱和正己烷液液萃取。采用Ecosil C18色谱柱(250mm×4.6mm,5μm),乙腈-0.6%乙酸溶液作为流动相,梯度洗脱,用紫外检测器检测,检测波长280nm,外标法峰面积定量。18种食品添加剂在1.0～25mg/L范围内线性良好,相关系数r均大于0.99,样品在10、25mg/kg和50mg/kg三个添加水平的平均回收率为88.9%～99.9%之间,相对标准偏差为2.43%～11.7%(n=6),方法的定量限为10mg/kg。方法简便、准确,适用于食品中18种食品添加剂高通量的检测。     

Analysis of nine kinds of sweeteners in foods by LC/MS

A simple and rapid method for the simultaneous determination of nine kinds of sweeteners (acesulfame potassium, AK; sucralose, SUC; saccharin, SA; cyclamate, CYC; aspartame, APM; dulcin, DU; glycyrrhizic acid, GA; stevioside, STV; rebaudioside A, REB) in various foods by high-performance liquid chromatography-electrospray mass spectrometry (LC/ESI-MS) was developed. The LC separation was performed on a ZORBAX Eclipse XDB-C18 (2.1 mm x 150 mm) with a mobile phase of 5 mmol/L dibutylammonium acetate (DBAA) and acetonitrile-water (8: 2). Mass spectral acquisition was done in the negative ion mode by applying selected ion monitoring (SIM). The sweeteners were extracted from foods with 0.08 mol/L phosphate buffer (pH 7.0)- ethanol (1:1), and the extract was cleaned up on a Sep-pak Vac C18 cartridge after the addition of tetrabutylammonium bromide and phosphate buffer (pH 3.0). The recovery of the nine kinds of sweeteners from five kinds of foods fortified at the level 0.01 g/kg, 0.05 g/kg and 0.20 g/kg was 75.7-109.2%, and the between-day SD values were 0.5-10.9%. The quantification limits of AK, SA, CYC, APM and STV were 0.001 g/kg, and those of SUC, DU, GA and REB were 0.005 g/kg. A recovery test from each cleaned-up sample solution was necessary to detect ionization suppression.     

Simultaneous determination of quinolones in foods by LC/MS/MS

A simple method was developed for the simultaneous determination of seven quinolones (enoxacin, ofloxacin, ciprofloxacin, danofloxacin, lomefloxacin, enrofloxacin and sarafloxacin) in foods using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The seven quinolones were extracted with acetonitrile containing 0.2% formic acid, and the extracted solution was cleaned up on a C18 cartridge. The extract was diluted with 5 mmol/L IPCC-MS3 for injection into the LC-ESI-MS/MS. The LC separation was carried out on an ODS column with gradient elution of 5 mmol/L IPCC-MS3-acetonitrile as the mobile phase. Mass spectral acquisition was done in the positive ion mode by applying selected reaction monitoring (SRM). The recoveries of the seven quinolones were mostly greater than 60% from foods fortified at 10 ng/g. The detection limits in foods were 2 ng/g for enoxacin and ciprofloxacin, and 1 ng/g for the other drugs. Twenty cattle muscle, 7 swine muscle, 9 chicken muscle, 16 milk, 19 prawn and 20 broiled eel samples from retail markets were analyzed by this method. Enrofloxacin and its metabolite ciprofloxacin were detected in 9 broiled eel at the level of trace (tr)-34 ng/g and tr-10 ng/g, respectively.     

UPLC-MS/MS同时检测婴幼儿配方乳粉中氯酸盐和高氯酸盐残留

该实验建立了超高效液相色谱-串联质谱法（UPLC-MS/MS）同时测定婴幼儿配方乳粉中氯酸盐和高氯酸盐残留量的方法。样品经甲酸水提取,乙腈沉淀蛋白,乙腈饱和正己烷除去脂肪,采用Anionic Polar Pesticide色谱柱（2.1 mm×150 mm,5 μm）分离,以0.9%甲酸+50 mmol/L甲酸铵水溶液和乙腈为流动相梯度洗脱,在电喷雾离子源负离子（ESI-）多反应监测模式条件下,对氯酸盐和高氯酸盐进行定性和定量分析。结果表明,高氯酸盐和氯酸盐分别在0.5～50.0 μg/L和1.0～100.0 μg/L范围内线性关系良好,高氯酸盐和氯酸盐的定量限（LOQ）分别为7.5 μg/kg和15.0 μg/kg。高氯酸盐和氯酸盐的加标回收率为82.5%～98.5%,精密度试验结果的相对标准偏差（RSD）为2.0%～8.7%;该方法稳定、准确、灵敏度高,可用于婴幼儿配方乳粉中氯酸盐和高氯酸盐残留量检测。     

Confirmatory method for the analysis of carbadox and olaquindox in porcine feedingstuffs using LC-electrospray MS-MS.

A method is described for the quantitative determination of the two feed additives carbadox and olaquindox in porcine feedingstuffs. The use of these agents in feedingstuffs was prohibited in the European Union as a result of concerns about their toxicity. Regulatory laboratories are required to have suitably validated analytical methods to ensure compliance with the ban. The analytes were extracted from finished feedingstuffs into acetonitrile:chloroform (1:1, v/v), and aliquots (1.0 ml) of the extract were dried down under a stream of nitrogen at 65 degrees C. All residues were re-dissolved in HPLC mobile phase containing acetonitrile/water/formic acid. Analysis was based on LC coupled to positive-ion electrospray MS-MS, with daughter ions for carbadox at m/z 231 and 90, and for olaquindox at m/z 212 and 143 being monitored. The method was validated by analysing feed samples fortified with carbadox and olaquindox at 0.5, 2.5 and 5 mg kg(-1) on three separate occasions. Sample preparation was simple, thus allowing the confirmation of these compounds in large numbers of samples.     

HPLC-MS法测定食品中生物碱的研究

建立了一种高效液相色谱电喷雾质谱(HPLC-ESI-MS)法同时测定食品中六种生物碱的方法。采用ZORBAX SB-C8(2.1mm×150mm,3.5μm)色谱柱,乙腈(A)和pH5的4mmol/L乙酸铵溶液(B)为流动相,梯度洗脱使所有分析物均达到基线分离。采用ESI+一级质谱扫描模式得到甜菜碱、胆碱、苦参碱、阿托品、马钱子碱和乌头碱的定性离子,分别为m/z118.2、104.2、249.5、290.3、395.4和646.4,结合质谱保留时间检测六种生物碱。结果表明,采用1.0mg/mL混合标准溶液,准确性好,RSD<0.59%。该方法应用于检测实际样品荒漠肉苁蓉、山药、宁夏枸杞,添加回收率分别为81.7%~112.9%、80.8%~117.5%、96.9%~119.3%,说明建立的方法可以快速准确的检测实际样品中的生物碱成分。     

食品中杀草强残留量的检测方法研究

建立了一套检测食品中杀草强的液相色谱及液相色谱- 质谱分析方法,采用高效液相色谱荧光检测器(LCFLD),在激发波长380nm、发射波长484nm 条件下,高效液相色谱串联四级杆质谱配大气压电离源(LC-MS/MSAPCI),在多反应监测(MRM)模式下,母离子m/z 85、定量子离子m/z 43、定性子离子m/z 57、碰撞能量m/z85 ＞ m/z 57 为56.9/11eV、m/z 85 ＞ m/z 43 为58.1/11eV、裂解电压100V 条件下,分别对杀草强残留量进行检测,其中液相色谱荧光检测器法杀草强的检出限0.01mg/kg;液相色谱串联质谱法杀草强的检出限为0.005mg/kg。该方法简便、快速,分析结果准确、可靠,两种仪器条件可灵活选择,适用于食品中杀草强残留量的快速检测。     

超高效液相色谱-串联质谱法检测辅助降血脂保健食品中6 种非法添加化学药物

建立超高效液相色谱-串联质谱法同时快速检测辅助降血脂类保健食品中6 种降血脂药物（烟酸、普伐他汀钠、氟伐他汀钠、美伐他汀、洛伐他汀、辛伐他汀）的方法。样品经甲醇提取,Waters Acquity BEH-C18色谱柱分离,0.1%甲酸-乙腈溶液梯度洗脱,流速0.2?mL/min,电喷雾离子源正离子模式扫描,多反应监测模式测定,外标法定量。结果表明：在实验条件下4?min内可快速、准确地定性定量分析6?种降血脂化学药物,分离度良好,方法检出限为4～39?μg/kg,加样回收率为80.2%～106.1%。利用本方法检测20?批降血脂类保健食品,其中3?批分别含有烟酸和洛伐他汀。本方法线性关系良好、快速、简便、准确,可作为辅助降血脂类保健食品中非法添加化学药物的定性定量检测方法。     

腌腊肉制品中13-HODE、9,10-DHODE、9,10-EPODE、9,10,13-THODE的HPLC-MS/MS检测

采用高效液相色谱-串联质谱法对腌腊肉制品中13-羟基-9,11-十八碳二烯酸（13-hydroxy-9,11-octadecadienoic acid,13-HODE）、9,10-二羟基-12-十八碳烯酸（9,10-dihydroxy-12-octadecenoic acid,9,10-DHODE）、9,10-环氧-12-十八碳烯酸（9,10-epoxyoctadec-12-enoic acid,9,10-EPODE）及9,10,13-三羟基-11-十八碳烯酸（9,10,13-trihydroxy-11-octadecenoic acid,9,10,13-THODE）进行检测。腌腊肉制品中13-HODE、9,10-DHODE、9,10-EPODE、9,10,13-THODE用甲醇提取,经固相萃取柱去净化浓缩,然后以0.1%甲酸与乙腈为流动相,在XBridge色谱柱上进行梯度分离,采用电喷雾源负离子多反应监测模式进行分析。结果表明,13-HODE、9,10-DHODE、9,10-EPODE、9,10,13-THODE分别在质量浓度为0.05～2.0、0.01～0.5、0.05～1.0、0.01～0.5 μg/mL的范围内具有较好的线性关系（R2＞0.999）;检出限分别为0.120、0.008、0.200、0.016 μg/g;不同添加水平的平均回收率分别为95.1%、85.2%、86.8%、86.2%。对26 种腌腊肉制品含量分析表明,所有样本都含有13-HODE、9,10-DHODE、9,10,13-THODE,含量分别在1.4～100.7、0.1～3.9、0.4～10.2 μg/g范围内,21 种样本中含有9,10-EPODE,含量范围为0.8～6.9 μg/g。结果表明,腌腊肉制品中存在目标分析物的异构体,羟基脂肪酸实际含量可能大大高于检测的量。     

Sorbic and benzoic acid in non-preservative-added food products in Turkey

Sorbic acid (SA) and benzoic acid (BA) were determined in yoghurt, tomato and pepper paste, fruit juices, chocolates, soups and chips in Turkey by using high-pressure liquid chromatography (HPLC). Levels were compared with Turkish Food Codex limits. SA was detected only in 2 of 21 yoghurt samples, contrary to BA, which was found in all yoghurt samples but one, ranging from 10.5 to 159.9?mg/kg. Both SA and BA were detected also in 3 and 6 of 23 paste samples in a range of 18.1–526.4 and 21.7–1933.5?mg/kg, respectively. Only 1 of 23 fruit juices contained BA. SA was not detected in any chips, fruit juice, soup, or chocolate sample. Although 16.51% of the samples was not compliant with the Turkish Food Codex limits, estimated daily intake of BA or SA was below the acceptable daily intake.     

Determination of acrylamide in processed foods by LC/MS using column switching

An LC/MS method was developed for the determination of acrylamide (AA) in processed or cooked foods. AA was extracted with a mixture of water and acetone from homogenized food samples after the addition of 13C-labeled acrylamide (AA-1-(13)C) as an internal standard. The extract was concentrated, washed with dichloromethane for defatting, and cleaned up on Bond Elut C18, PSA and ACCUCAT cartridge-columns, and then AA was determined by LC/MS in the selected ion recording (SIR) mode. For the LC/MS analysis, four LC columns were connected in-line and the flow of the mobile phase was switched according to a time-program. Monitoring ions for AA were m/z 72 and 55, and those for AA-1-(13)C were m/z 73 and 56. AA and AA-1-(13)C were determined without interference from the matrices in all samples. The recoveries of AA from potato chips, corn snack, pretzel and roasted tea spiked at the level of 500 ng/g of AA were 99.5-101.0% with standard deviations (SD) in the range from 0.3 to 1.6%. The limits of detection and quantification of the developed method were 9 and 30 ng/g for AA in samples, respectively. The method was applied to the analysis of AA in various processed or cooked food samples purchased from retail markets. High levels of AA were found in potato chips and French-fried potato (467-3,544 ng/g). Fried and sugar-coated dough cakes (karinto) contained 374 and 1,895 ng/g. Corn snacks contained 117-535 ng/g of AA. Roasted foods (such as roasted sesame seed, roasted barley (mugi-cha), roasted tea (hoji-cha), coffee beans and curry powder) contained 116-567 ng/g of AA. Foods made from fish, egg and meat contained lower levels of AA than the plant-based foods. Foods containing much water showed a tendency to have low levels of AA compared with dry foods. The proposed method was applicable to the analysis of AA in variety of processed foods.     

海产品贝毒素大田软海绵酸ELISA及HPLC-MS/MS检测方法的研究

为了检测海产品贝毒大田软海绵酸,保障其食用安全,利用活泼酯法将小分子OA与载体蛋白偶联,免疫BALB/c小鼠,细胞融合技术建立分泌抗OA的杂交瘤细胞株,并对其各种特性进行分析;小鼠腹水法大量生产抗体,纯化后建立ELISA方法,同时建立OA的高效液相色谱-串联质谱(HPLC-MS)检测法,并对部分市售海产品进行了实际检测,ELISA方法标准曲线为y=－34.212x+83.49,相关系数为0.9784,线性范围0.4～25μg/L,灵敏度0.18μg/L;HPLC-MS/MS检测方法标准曲线y=193.07x－780.6(Q1/Q3：m/z 827.4～m/z 723.5)和y=83.021x－335.6(Q1/Q3：m/z 827.4～m/z 809.5),R2 均为0.9991,线性范围10～800μg/L,灵敏度小于2μg/L,平均RSD为4.34%。在检测的实际样品中两种样品ELISA呈阳性反应,其中一种经过了HPLC-MS/MS的验证。所建立的ELISA及HPLC-MS检测方法均可用于海产品腹泻性贝毒OA限量标准检测,为进出口海产品OA标准方法的建立提供实验基础。     

超高效液相色谱-串联质谱法测定奶粉中的三聚氰酸

建立奶粉中的三聚氰酸的超高效液相色谱-四极杆串联质谱(UPLC-MS/MS)检测方法。样品经透析袋净化、反相液相色谱分离后进行质谱分析,在多反应监测模式(MRM)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,以离子对m/z 42.0进行定量。三聚氰酸的最低检测限(RSN=3)为100.0μg/kg。在100.0～2000.0μg/L时峰强度与质量浓度的线性关系良好(r2≥0.9999)。在100.0、500.0、1000.0μg/kg 3个添加水平,三聚氰酸的回收率范围为87.5%～98.8%,相对标准偏差小于9%。结果表明,该法简单、灵敏,适用于奶粉中三聚氰酸的分析确证。     

高通量全自动仪器法测定植物源性食品中甲基托布津残留量

建立高效的应用高通量全自动均质仪、全自动净化在线浓缩仪及液相色谱-串联质谱仪测定植物源性食品中甲基托布津残留量的检测方法。试样经高通量全自动均质仪,用乙腈均质提取,经全自动净化在线浓缩仪(凝胶色谱柱、固相萃取柱)净化、在线浓缩后,采用液相色谱-串联质谱仪定量定性分析,外标法定量,液相流动相为甲醇-水,采用电喷雾离子源,定性离子对为m/z 343.0＞151.1、m/z 343.0＞192.1,定量离子对为m/z 343.0＞151,添加0.01～10.0mg/kg的甲基托布津标准品时的回收率为88.4%～97.9%,相对标准偏差(RSD)在1.3%～2.9%之间,最低检出限为0.01mg/kg。该方法特异性强、敏感性高、高通量、全自动、快速高效,可应用于植物源性食品的甲基托布津残留量检测。