AOCS Ce 1i-07 Determination of Fatty Acids in Marine and Other Oils by Capillary GC鱼油的检测方法2

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超高效合相色谱―质谱法快速检测工业油酸中5种常见的脂肪酸[权威资料]

超高效合相色谱―质谱法快速检测工业油酸中5种常见的脂肪酸本文档格式为WORD,感谢你的阅读。

摘要采用超高效合相色谱-质谱（UPC2-MS）技术，建立了工业油酸中5种常见的脂肪酸（软脂酸、硬脂酸、油酸、亚油酸和亚麻酸）的快速检测方法。

样品用正己烷溶解，采用超临界CO2-甲醇/乙腈（1∶1， V/V）梯度洗脱，经Acquity UPC2BEH 2-EP色谱柱（100 mm × 2.1 mm，1.7 μm）分离，通过质谱检测器在负离子电喷雾模式下对目标化合物进行分析，外标法定量。

通过对UPC2-MS条件的优化，5种脂肪酸在3 min内实现有效分离，目标物在0.5～100 mg/L 范围内具有良好的线性（相关系数大于0.9985）; 在3个添加水平下，5种脂肪酸的回收率在89.3%～106.7%之间，相对标准偏差为0.8%～3.0%; 方法检出限（S/N≥3）为0.07～0.26 mg/L。

实际样品分析结果表明，本方法不但简单快速，分离效果好，而且无需对脂肪酸样品进行衍生化，同时为UPC2在油脂类相关领域的研究与开发提供一种快速有效的检测方法。

关键词超高效合相色谱-质谱法; 工业油酸; 未衍生化; 脂肪酸1 引言工业油酸是一种重要的化工中间体，可用于生产脂肪酸甲酯、生物柴油等，市场需求较大lt;supgt;[1]lt;/supgt;。

它是以植物油脂、皂脚为原料，通过水解、精馏而得到的一种混合脂肪酸，主要含有5种脂肪酸，其中以油酸（C18∶1）、亚油酸（C18∶2）为主，还含有少量的软脂酸（C16∶0）、硬脂酸（C18∶0）和亚麻酸（C18∶3）等lt;supgt;[2]lt;/supgt;。

原料和生产方法的差异会造成工业油酸中5种脂肪酸含量的不同lt;supgt;[2]lt;/supgt;，而脂肪酸的不同比例将造成工业油酸的品类不同，因此建立快速、准确地检测这5种脂肪酸含量的方法是区分工业油酸品类的必然选择。

国际食品法典检测、鉴定和量化食品中特定基因序列和蛋白质的执行

国际食品法典检测、鉴定和量化食品中特定基因序列和蛋白质*的执行标准和方法确认准则CAC/GL 74-2010*应用于来自现代生物技术、食品认证、食品生产和其他目的的食品。

1引言分子和免疫学分析方法是目前公认的测定食品中基因和蛋白质分析物的检测工具。

然而,为了不同实验室通过这种方法获得的结果能被广泛接受和认可，这就需要分析方法满足特定的质量准则。

本指南提供适当的标准来验证用来测定食品中特定基因序列或特定蛋白质分析方法的可靠性。

本指南的第一部分给出了关于验证特定基因序列或特定蛋白质分析方法的一般原则。

附件提供的内容包括定量聚合酶链式反应（PCR）方法的验证，定性PCR方法的验证和基于蛋白质方法的验证。

1.1目的和目标本准则的目标是为分子和免疫学检测方法的建立提供支持，食品中特定基因序列和特定蛋白质定量的验证，确保不同实验室的结果具有类似的再现性。

本指南的目的是为通过定义适当的检验标准如何建立方法来检测和验证食品中特定基因序列和蛋白质提供指导，一个方法是否遵守这些标准是以该方法的性能特点为基础的。

本指南详细说明相关标准，并解释如何理解这些标准，即：-通过提供标准的基本原理-通过展示怎样找出一个方法是否满足给定的标准要求。

1.2适用范围本指南提供的食品分析方法的验证标准信息涉及可能出现在食品中的特定基因序列和特定蛋白质的检测、鉴定和定量，其中包括这些食品所含的来自现代生物技术的原料。

这些分子和免疫学方法应用范围很广，例如用于测定食品中的生物标志物,其中包括这些来源于现代生物技术和食品认证技术，以及用于实验室进行食品分析。

2方法验证食品法典委员会（CAC）强调任何一个被接受的分析方法必须进行国际合作试验，同时遵守根据ISO 5725:1994 或AOAC/IUPAC的国际协定。

因此，这就可能需要在没有合作试验数据时采取单一实验室确证的临时措施。

然而，用于基因序列和蛋白质的分析方法必须能够适用于多个实验室。

毛细管气相色谱法测定食用油中15种反式脂肪酸的含量

毛细管气相色谱法测定食用油中15种反式脂肪酸的含量祝伟霞;杨冀州;王彩娟;储晓刚;袁萍;刘亚风【摘要】提出了毛细管气相色谱法测定橄榄油、芝麻油和花生油中的15种反式脂肪酸.样品用异辛烷溶解,与2 mol·L-1氢氧化钾的甲醇溶液进行酯化反应,生成的脂肪酸甲酯经强极性聚二氰丙基硅氧烷毛细管气相色谱柱SP-2560分离,用氢火焰离子化检测器检定.在优化的试验条件下,15种反式脂肪酸甲酯的质量浓度在一定范围内与其峰面积呈线性关系,方法的检出限(3S/N)和测定下限(10S/N)分别为0.012％(以脂肪计)和0.024％(以脂肪计).对空白样品进行加标回收试验,回收率在86.6％～109％之间,测定值的相对标准偏差(n=10)在2.0％～4.2％之间.方法用于分析市售橄榄油、芝麻油和花生油中的15种反式脂肪酸,结果与标准方法的测定值相符.【期刊名称】《理化检验-化学分册》【年(卷),期】2015(051)003【总页数】6页(P375-380)【关键词】气相色谱法;反式脂肪酸;食用油【作者】祝伟霞;杨冀州;王彩娟;储晓刚;袁萍;刘亚风【作者单位】河南出入境检验检疫局,郑州450003;河南出入境检验检疫局,郑州450003;河南出入境检验检疫局,郑州450003;中国检验检疫科学研究院,北京100123;河南出入境检验检疫局,郑州450003;河南出入境检验检疫局,郑州450003【正文语种】中文【中图分类】O657.7反式脂肪酸（TFAs）是一种含有反式双键的不饱和脂肪酸，食品中TFAs主要来自于植物油的氢化、精炼加工等过程，以反式油酸（C18：1t）、反式亚油酸（C18：2t）、反式亚麻酸（C18：3t）为主［1－2］。

研究表明：长期食用含TFAs的食品易引起糖尿病、心血管、心脏等疾病，还可影响婴幼儿的生长发育［3－4］。

为保护人类的身体健康，美国食品药物管理局（FDA）、我国卫生部均强制要求含反式脂肪酸的食品在标签中标注，同时联合国粮农组织和世界卫生组织规定TFAs每日最大摄入量不得超过总能量的1%。

AOCS脂肪酸检测方法

AOCS脂肪酸检测方法SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS AOCS Official Method Ce 1f-96Reapproved 1997 ? Revised 2001 Determination of cis-and trans-Fatty Acids in Hydrogenated and Refined Oils and Fats by Capillary GLCDEFINITIONThis method consists of the gas–liquid chromatography (GLC) conditions optimized to identify and quantify the trans fatty acid isomers in vegetable oils and fats (References, 1). The fatty acid methyl esters (FAME) of the sample are separated on a capillary gas chromatography column having a high-ly polar stationary phase, according to their chain length (CL), degree of (un)saturation, and geome-try and position of the double bonds [DB(s)].SCOPEThis method is specially designed to evaluate, by a single capillary GLC procedure, the level of trans isomers as formed during (high-temperature) refining or during hydrogenation of vegetable oils or fats (see Notes, 1 and 2). The method may also be used to report all other fatty acids, for example to obtain saturated fatty acid (SAFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA) levels from the same sample and same analysis.APPARATUS1.Gas ch ro m at ograph—equipped with a cap i l l a ry injectionsystem (pre fe rably split mode, operated at a split ratio of ap p r ox i m a t e l y 1:100) and flame ionization detector (FID), capable of meeting the fo l l owing re q u i re m e n t s: injection port temperat u re, 250°C; detector temperat u re, 250°C; oven temperat u re conditions as given in Table 1.Typical results with these described conditions are show n in example ch ro m at ograms (Fi g u res 1–5).2.Column—highly polar stationary phase, such as one ofthe following:(a)CP?-Sil 88, 100 or 50 m×0.25 mm i.d., 0.20 μmfilm (Chrompack, Middelburg, The Netherlands).(b)SP-2650, 100 m ×.025 mm i.d., 0.20 μm fi l m(Supelco Inc., Bellefonte, PA, USA).(c)SP-2340, 60 m× 0.25 mm i.d., 0.2 μm fi l m(Supelco Inc.).(d)BPX-70, 120 m or 50 m×0.22 mm i.d., 0.25 μmfilm (SGE Inc., Austin, TX, USA).3.Recording instrument.4.Electronic integrator or chromatography software. REAGENTSUnless otherwise stat e d, use only re agents as specified in ISO 6353 (parts 2 and 3) (Refe rences, 2) if listed there; if not, then use re agents of re c og n i zed analytical grade and water of at least grade 3 as defined in ISO 3696 (Refe rences, 3).1.Carrier gas—helium, nitrogen, or hydrogen, GC quali-ty, dried, and oxygen removed by suitable filters.2.Internal standard (for calculating fatty acid data as mgper g oil)—tridecanoin, 5.0 mg/mL in chloroform. This solution is stable up to 1 week if stored in refrigerator in well sealed amber bottle. (See Notes, 3). PROCEDURE1.Sample preparation—(a)P r ep a r e the methyl esters from the tri g l y c e r i d e sf r om the oils or fats to be analy z e d, using theb o r on tri fl u o ride method as descri b e d, for ex a m-ple, in AOCS Official Method Ce 2-66 or IUPAC2.301 (References, 5 and 6).(b) B e f o r e test portions are taken from samples, thesamples should be mixed thoro u g h ly. Solid samplesshould be melted to ensure proper mixing.2.C h ro m at ograp hy—(a)Set up the gas ch ro m at ograph with the temperat u reand column as described in Table 1. Measure theave r age carrier gas linear velocity as indicated inTable 1, with a split ratio of ap p rox i m at e ly 1:100.(b)Inject 0.5 to 1 μL of the methyl esters (concentra-tion approximately 7 mg/mL) from the test sampleinto the gas ch r o m a t o graph. Compare the re s u l twith the example chromatograms (Figures 1–5). Ifthe sep a r ation obtained is not identical to theTable 1Proposed optimal GLC conditions for identification and quantification of trans isomers in refined and hydrogenated veg-etable oil samples (see References, 3).Stationary phase SP-2340SP-2560CP?-Sil 88BPX-70 Temperature conditions Isotherm 192°C Isotherm 170°C Isotherm 175°C Isotherm 198°C Column head pressure (kPa)125125130155Linear velocity of carrier gas (He)15 cm/sec16 cm/sec19 cm/sec17 cm/secPage 1of 6Page 2of 6SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS Ce 1f-96 ? Determination of cis-and trans-Fatty Acids in Hydrogenated and Refined Oils and Fatsexamplech r o m a t o grams, small ch a n g es in ove n t e m p e r at u r e may be re q u i r e d . If so, decrease or i n c r ease the oventemperat u r e with subsequent s t e ps of 1°C u ntil a good sep a r ation is obtained.These small corrections might be re q u i r ed to correct for batch differences between columns and i n s t r ument temperat u r e control and ge n e r a l l y fa l l within a ra n g e of only a few degrees (plus or minus) at maximum from the indicated value. The 20:1c peak will elute earlier relative to 18:3ccc if the oven temperature is increased (see Notes,4).3.Performance check—(a)If the GLC system is set up properly, the separa-tion obtained should allow identifi c a tion of the small amount of the nat u r a l l y present 18:1 11c i s isomer next to the 18:1 9cis peak in (high-temper-at u r e) re f ined oils such as soybean oil. The two 1 8:1c i s o m e r s should be cl e a r ly sep a r ated (see Figures 1–5).(b)The 20:1 nat u r al isomer should be positionedexactly between the last eluting 18:3 trans isomer (t r ans, cis, cis ) and the 18:3c c c (linolenic acid)peak in (high-temperature) refined oils.(c)If the separation is sufficient for this type of analy-sis, in (high-temperature) refined oils a small peak for the 18:1 t r a n s i s o m e r , two ap p r ox i m a t e l y e q u a l l y sized 18:2 t r a n s i s o m e r s, and 4 (some-times 5) 18:3 t r a n s i s o m e r s should be obtained (see Figures 1–5).(d)For partially hydrogenated oils and fats, the sepa-ration of the 18:1 13t r a n s and the 18:1 9c i s i s o m e r s should be visible on the ch r o m a t o gra m .This is required for an accurate peak split between cis and trans .4.Peak identification—(a )For (high-temperat u r e) re f ined oils and fats, thet ra n s i s o m e rs are limited in number because only ge o m e t r ical isomers, with the DB(s) on the same n a t u r al position,are fo r m e d . For C 1 8fatty acids these specific isomers are 18:1 9t ; 18:2 9c 1 2t and 9t 1 2c ; and 18:3 t c t , c c t , c t c , t c c 9, 12, 15-i s o m e rs (in some samples the 18:2 9t 12t and 18:3t t c i s o m e rs are found as well in ve ry small amounts).(b)For part i a l l y hy d r oge n a ted oils and fats the t ra n sDB-containing isomers are identified using the e q u i valent chain length (ECL) concept (Refe r -ences, 7; see Table 2). For accurate peak identifi-cation with this system, the ECL values have to be determined after suitable calibration with available cis and trans fatty isomer standards.SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS Ce 1f-96 ? Determination of cis-and trans- Fatty Acids in Hydrogenated and Refined Oils and FatsPage 3of 6Figure 4.Chromatogram of methyl esters from a physicall y refined rapeseed oil sample using 50 m ×0.25 mm ×0 .20μm CP?-Sil 88 column (Chr o m p a c k). The t r a n s fatty acid isomers are indicated in the chromatogram.Figure 5.Chromatogram of methyl esters from a high-tem -perature-refined rapeseed oil sa mple, using 50 m ×0.22 mm ×0.25 μm BPX-70 (SGE). The t ra n s fatty acid isomers are indicated in the chromatogram.SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS Ce 1f-96 ? Determination of cis-and trans- Fatty Acids in Hydrogenated and Refined Oils and Fats ArrayPage 4of 6SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS Ce 1f-96 ? Determination of cis-and trans- Fatty Acids in Hydrogenated and Refined Oils and FatsSAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS Ce 1f-96 ? Determination of cis-and trans- Fatty Acids in Hydrogenated and Refined Oils and Fats2.D u r ing (high-temperat u r e) re f i n i n g, only ge o m e t r i c a li s o m e r s of the mono- and poly u n s a t u r ated fatty acidsare formed; that is, the DBs remain on the same, natur-al position. During hy d r oge n a tion, on the other hand, both positional and geometrical isomers are formed.3.If quantitation of fatty acids is re q u i r ed (mg/g), thei n t e r nal standard must be added prior to methy l a t i o n.The addition of a known quantity will allow the calcu-lation of fatty acid content by simple proportions. If ac o m p l e x mat e r ial is being examined for indiv i d u a lfatty acid content for labeling purposes, the intern a l s t a n d a r d should be added to the test sample befo r e extraction commences.4.The elution profile of the BPX-70 column [Apparatus,2(c)] is somewh a t diffe r ent; the 20:1c peak alway s elutes after the 18:3ccc peak using these conditions. REFERENCES1.This method parap h r ases one submitted by Dr. GuusS.M.J.E. Duch ateau of Unilever Research Lab o rat o ri e s,V l a a rd i n gen, The Netherlands, November 1995.2.ISO 6353, Reagents for Chemical Analysis, Pa r t 2(1983) and 3 (1987); Specifications.3.ISO 3696, Water for Analytical Lab o r at o r y Use—Specifications and Test Methods (1987).4.D u c h a teau, G. S. M.J.E., H.J. van Oosten, and M.A.Vasconcellos, Analysis of c i s- and t r a n s- F atty AcidI s o m e rs with Cap i l l a ry GLC in Hydroge n ated and Refi n e dV egetable Oils, J. Am. Oil Chem. Soc. 73:275 (1996).5.AOCS Official Method Ce 2-66, Preparation of MethylEsters of Long-Chain Fatty Acids.6.I U P AC, S t a n d a r d Methods for Analysis of Oils, Fat sand Derivat i ve s,B l a c k w ell Scientific Publ i c a t i o n s;IUPAC Method 2.301.7.J. Am. Oil Chem. Soc. 58:662 (1981).8.Th i r d Unilever interl a b o r at o r y test on the determ i n a-tion of low trans levels by capillary GC. Visser, R.G., P.A. Zandbelt, Y.S.J. V eldhuizen.9.G a r f i e l d, F.M., Quality Assurance Principles fo rAnalytical Laboratories, AOAC International, 1995.Page 6of 6。

反式脂肪酸(TFA)膳食暴露评估及红细胞膜TFA分析检测研究进展

反式脂肪酸(TFA)膳食暴露评估及红细胞膜TFA分析检测研究进展SOUCHINDA THILAPHONG;张嘉峻;高留飞;徐芳;单淑晴;许莎莎;殷建忠【摘要】TFA是食品安全中比较敏感且备受关注的话题.对TFA风险评估、TFA膳食暴露评估、红细胞膜TFA分析检测的研究进展进行综述,对中国TFA标准的制定和食品质量安全控制都有着积极的理论和现实意义.【期刊名称】《卫生软科学》【年(卷),期】2017(031)002【总页数】3页(P35-37)【关键词】反式脂肪酸;膳食暴露;红细胞膜;检测方法【作者】SOUCHINDA THILAPHONG;张嘉峻;高留飞;徐芳;单淑晴;许莎莎;殷建忠【作者单位】昆明医科大学公共卫生学院,云南昆明650500;昆明医科大学公共卫生学院,云南昆明650500;昆明医科大学公共卫生学院,云南昆明650500;昆明医科大学公共卫生学院,云南昆明650500;昆明医科大学公共卫生学院,云南昆明650500;昆明医科大学公共卫生学院,云南昆明650500;昆明医科大学公共卫生学院,云南昆明650500【正文语种】中文【中图分类】R151反式脂肪酸(trans fatty acids，TFA)是食品安全中比较敏感且备受关注的话题。

开展TFA风险评估对中国TFA的国家标准及其他相关标准的制修订工作提供参考依据。

基于科学的TFA风险评估结果有助于政策决策者在更大社会范围内将其风险降低到可以接受的水平[1]。

中国的风险评估还处于初级发展阶段，很多机制还不完善，还缺乏系统的TFA数据和各种食品消费量数据以及易感人群如妇女、儿童和老人等群体和少数民族人群的膳食模式资料。

因此，评估中国不同人群TFA的膳食暴露状况具有重要的公共卫生学意义。

在研究TFA摄入状况时，必须依据膳食中主要食物品种及其消费量进行综合评价。

目前，中国对TFA组成和含量的系统研究报道较少，尚缺乏可直接借鉴的数据。

中国和多省份食物成分表中，未列出TFA含量的基础数据，故日常基础膳食中TFA暴露水平无法评估。

AOCS脂肪酸检测方法

SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILSAOCS Official Method Ce 1f-96Reapproved 1997 • Revised 2001 Determination of cis-and trans-Fatty Acids in Hydrogenated and Refined Oils and Fatsby Capillary GLCDEFINITIONThis method consists of the gas–liquid chromatography (GLC) conditions optimized to identify and quantify the trans fatty acid isomers in vegetable oils and fats (References, 1). The fatty acid methyl esters (FAME) of the sample are separated on a capillary gas chromatography column having a high-ly polar stationary phase, according to their chain length (CL), degree of (un)saturation, and geome-try and position of the double bonds [DB(s)].SCOPEThis method is specially designed to evaluate, by a single capillary GLC procedure, the level of trans isomers as formed during (high-temperature) refining or during hydrogenation of vegetable oils or fats (see Notes, 1 and 2). The method may also be used to report all other fatty acids, for example to obtain saturated fatty acid (SAFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA) levels from the same sample and same analysis.APPARATUS1.Gas ch ro m at ograph—equipped with a cap i l l a ry injectionsystem (pre fe rably split mode, operated at a split ratio of ap p r ox i m a t e l y 1:100) and flame ionization detector (FID), cap able of meeting the fo l l owing re q u i re m e n t s: injection port temperat u re, 250°C; detector temperat u re, 250°C; oven temperat u re conditions as given in Table 1.Typical results with these described conditions are show n in example ch ro m at ograms (Fi g u res 1–5).2.Column—highly polar stationary phase, such as one ofthe following:(a)CP™-Sil 88, 100 or 50 m×0.25 mm i.d., 0.20 µmfilm (Chrompack, Middelburg, The Netherlands).(b)SP-2650, 100 m ×.025 mm i.d., 0.20 µm fi l m(Supelco Inc., Bellefonte, PA, USA).(c)SP-2340, 60 m× 0.25 mm i.d., 0.2 µm fi l m(Supelco Inc.).(d)BPX-70, 120 m or 50 m×0.22 mm i.d., 0.25 µmfilm (SGE Inc., Austin, TX, USA).3.Recording instrument.4.Electronic integrator or chromatography software. REAGENTSUnless otherwise stat e d, use only re agents as specified in ISO 6353 (parts 2 and 3) (Refe rences, 2) if listed there; if not, then use re agents of re c og n i zed analytical grade and water of at least grade 3 as defined in ISO 3696 (Refe rences, 3).1.Carrier gas—helium, nitrogen, or hydrogen, GC quali-ty, dried, and oxygen removed by suitable filters.2.Internal standard (for calculating fatty acid data as mgper g oil)—tridecanoin, 5.0 mg/mL in chloroform. This solution is stable up to 1 week if stored in refrigerator in well sealed amber bottle. (See Notes, 3). PROCEDURE1.Sample preparation—(a)P r ep a r e the methyl esters from the tri g l y c e r i d e sf r om the oils or fats to be analy z e d, using theb o r on tri fl u o ride method as descri b e d, for ex a m-ple, in AOCS Official Method Ce 2-66 or IUPAC2.301 (References, 5 and 6).(b) B e f o r e test portions are taken from samples, thesamples should be mixed thoro u g h ly. Solid samplesshould be melted to ensure proper mixing.2.C h ro m at ograp hy—(a)Set up the gas ch ro m at ograph with the temperat u reand column as described in Table 1. Measure theave r age carrier gas linear velocity as indicated inTable 1, with a split ratio of ap p rox i m at e ly 1:100.(b)Inject 0.5 to 1 µL of the methyl esters (concentra-tion approximately 7 mg/mL) from the test sampleinto the gas ch r o m a t o graph. Compare the re s u l twith the example chromatograms (Figures 1–5). Ifthe sep a r ation obtained is not identical to theTable 1Proposed optimal GLC conditions for identification and quantification of trans isomers in refined and hydrogenated veg-etable oil samples (see References, 3).Stationary phase SP-2340SP-2560CP™-Sil 88BPX-70 Temperature conditions Isotherm 192°C Isotherm 170°C Isotherm 175°C Isotherm 198°C Column head pressure (kPa)125125130155Linear velocity of carrier gas (He)15 cm/sec16 cm/sec19 cm/sec17 cm/secPage 1of 6Page 2of 6SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILSCe 1f-96 • Determination of cis-and trans-Fatty Acids in Hydrogenated and Refined Oils and Fatsexamplech r o m a t o grams, small ch a n g es in ove n t e m p e r at u r e may be re q u i r e d . If so, decrease or i n c r ease the oven temperat u r e with subsequent s t e ps of 1°C until a good sep a r ation is obtained.These small corrections might be re q u i r ed to correct for batch differences between columns and i n s t r ument temperat u r e control and ge n e r a l l y fa l l within a ra n g e of only a few degrees (plus or minus) at maximum from the indicated value. The 20:1c peak will elute earlier relative to 18:3ccc if the oven temperature is increased (see Notes, 4).3.Performance check—(a)If the GLC system is set up properly, the separa-tion obtained should allow identifi c a tion of the small amount of the nat u r a l l y present 18:1 11c i s isomer next to the 18:1 9cis peak in (high-temper-at u r e) re f ined oils such as soybean oil. The two 1 8:1c i s o m e r s should be cl e a r ly sep a r ated (see Figures 1–5).(b)The 20:1 nat u r al isomer should be positionedexactly between the last eluting 18:3 trans isomer (t r ans, cis, cis ) and the 18:3c c c (linolenic acid)peak in (high-temperature) refined oils.(c)If the separation is sufficient for this type of analy-sis, in (high-temperature) refined oils a small peak for the 18:1 t r a n s i s o m e r , two ap p r ox i m a t e l y e q u a l l y sized 18:2 t r a n s i s o m e r s, and 4 (some-times 5) 18:3 t r a n s i s o m e r s should be obtained (see Figures 1–5).(d)For partially hydrogenated oils and fats, the sepa-ration of the 18:1 13t r a n s and the 18:1 9c i s i s o m e r s should be visible on the ch r o m a t o gra m .This is required for an accurate peak split between cis and trans .4.Peak identification—(a )For (high-temperat u r e) re f ined oils and fats, thet ra n s i s o m e rs are limited in number because only ge o m e t r ical isomers, with the DB(s) on the same n a t u r al position, are fo r m e d . For C 1 8fatty acids these specific isomers are 18:1 9t ; 18:2 9c 1 2t and 9t 1 2c ; and 18:3 t c t , c c t , c t c , t c c 9, 12, 15-i s o m e rs (in some samples the 18:2 9t 12t and 18:3t t c i s o m e rs are found as well in ve ry small amounts).(b)For part i a l l y hy d r oge n a ted oils and fats the t ra n sDB-containing isomers are identified using the e q u i valent chain length (ECL) concept (Refe r -ences, 7; see Table 2). For accurate peak identifi-cation with this system, the ECL values have to be determined after suitable calibration with available cis and trans fatty isomer standards.SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILSCe 1f-96 • Determination of cis-and trans- Fatty Acids in Hydrogenated and Refined Oils and FatsPage 3of 6Figure 4.Chromatogram of methyl esters from a physicall y refined rapeseed oil sample using 50 m ×0.25 mm ×0 .20µm CP™-Sil 88 column (Chr o m p a c k). The t r a n s fatty acid isomers are indicated in the chromatogram.Figure 5.Chromatogram of methyl esters from a high-tem -perature-refined rapeseed oil sample, using 50 m ×0.22 mm ×0.25 µm BPX-70 (SGE). The t ra n s fatty acid isomers are indicated in the chromatogram.SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILSCe 1f-96 • Determination of cis-and trans- Fatty Acids in Hydrogenated and Refined Oils and Fats ArrayPage 4of 6SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS Ce 1f-96 • Determination of cis-and trans- Fatty Acids in Hydrogenated and Refined Oils and FatsSAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILSCe 1f-96 • Determination of cis-and trans- Fatty Acids in Hydrogenated and Refined Oils and Fats2.D u r ing (high-temperat u r e) re f i n i n g, only ge o m e t r i c a li s o m e r s of the mono- and poly u n s a t u r ated fatty acidsare formed; that is, the DBs remain on the same, natur-al position. During hy d r oge n a tion, on the other hand, both positional and geometrical isomers are formed.3.If quantitation of fatty acids is re q u i r ed (mg/g), thei n t e r nal standard must be added prior to methy l a t i o n.The addition of a known quantity will allow the calcu-lation of fatty acid content by simple proportions. If ac o m p l e x mat e r ial is being examined for indiv i d u a lfatty acid content for labeling purposes, the intern a l s t a n d a r d should be added to the test sample befo r e extraction commences.4.The elution profile of the BPX-70 column [Apparatus,2(c)] is somewh a t diffe r ent; the 20:1c peak alway s elutes after the 18:3ccc peak using these conditions. REFERENCES1.This method parap h r ases one submitted by Dr. GuusS.M.J.E. Duch ateau of Unilever Research Lab o rat o ri e s,V l a a rd i n gen, The Netherlands, November 1995.2.ISO 6353, Reagents for Chemical Analysis, Pa r t 2(1983) and 3 (1987); Specifications.3.ISO 3696, Water for Analytical Lab o r at o r y Use—Specifications and Test Methods (1987).4.D u c h a teau, G. S. M.J.E., H.J. van Oosten, and M.A.Vasconcellos, Analysis of c i s- and t r a n s- F atty AcidI s o m e rs with Cap i l l a ry GLC in Hydroge n ated and Refi n e dV egetable Oils, J. Am. Oil Chem. Soc. 73:275 (1996).5.AOCS Official Method Ce 2-66, Preparation of MethylEsters of Long-Chain Fatty Acids.6.I U P AC, S t a n d a r d Methods for Analysis of Oils, Fat sand Derivat i ve s,B l a c k w ell Scientific Publ i c a t i o n s;IUPAC Method 2.301.7.J. Am. Oil Chem. Soc. 58:662 (1981).8.Th i r d Unilever interl a b o r at o r y test on the determ i n a-tion of low trans levels by capillary GC. Visser, R.G., P.A. Zandbelt, Y.S.J. V eldhuizen.9.G a r f i e l d, F.M., Quality Assurance Principles fo rAnalytical Laboratories, AOAC International, 1995.Page 6of 6。

反式脂肪酸分析方法的研究进展

反式脂肪酸分析方法的研究进展宋志华单良王兴国(江南大学，教育部食品科学与安全重点实验室)【摘要】本文综述了反式脂肪酸的来源、结构和分析方法，详细介绍了红外光谱法、气相色谱法、银离子高效液相色谱法在反式脂肪酸研究中的应用，并对各种分析方法的优缺点进行了比较。

【关键词】反式脂肪酸；来源；分析方法；研究进展反式脂肪酸(trans fatty acids–TFA)是指在不饱和脂肪酸碳链上存在反式构型双键的脂肪酸。

TFA 主要来源于植物油脂的选择性氢化，天然存在的不饱和脂肪酸的双键一般都是顺式构型，只有在反刍动物体内会存在一些少量的TFA。

TFA 能升高人体血清中低密度脂蛋白(LDL)胆固醇含量，同时能降低高密度脂蛋白(HDL)胆固醇含量，增加患心血管疾病的危险；升高人体内胰岛素水平，降低红细胞对胰岛素的反应，导致患II 型糖尿病的危险；影响δ脱饱和酶的功能，导致必需脂肪酸的缺乏，影响- 6生长发育。

丹麦政府依据该国营养委员会对TFA 潜在危害性的研究结论，于2003 年6月制定了严格的规定，成为世界上第一个对食品中TFA设立法规进行限制的国家。

美国食品和药品监督管理局(FDA)在2003年7月作出规定：自2006年1月1日起，食品营养标签中必须标注产品的饱和脂肪酸含量及TFA的含量。

近年来，人们对TFA 问题日益关注，TFA 成为研究的热点。

本文综述了TFA 的来源、存在形式及分析方法方面的研究进展。

1 TFA 的来源及其存在形式1.1 反刍动物(如牛、羊等) 脂肪及其乳制品反刍动物体脂及乳制品中的TFA 约占总脂肪酸含量的1%~8%，主要来源于饲料中不饱和脂肪酸在反刍动物肠腔中丁酸弧菌属菌群的酶促生物氢化。

双键所在的位置有Δ4~Δ16位，其中以Δ11t- 18：1为主。

1.2 植物油脂精炼过程中的高温脱臭油脂脱臭过程中形成的TFA 为总脂肪酸含量的3%左右，主要来源于亚油酸和亚麻酸的顺反异构，以单反式多不饱和脂肪酸为主，反式单不饱和脂肪酸的含量极少。

美国油脂检测标准AOCS Ca 5a-40

Page 1 of 2SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILSAPPARATUS 1. Oil sample bottles—115 or 230 mL (4 or 8 oz), or 250 mL Erlenmeyer flasks.REAGENTS 1. Ethyl alcohol, 95%—USSD formulas 30 and 3A are permitted (see Notes, 1). The alcohol must give a definite, distinct and sharp end point with phenolphthalein and must be neutralized with alkali to a faint, but permanent pink color just before using. 2. Phenolphthalein indicator solution—1% in 95% alcohol. 3. Sodium hydroxide solution—accurately standardized. See AOCS Specification H 12-52. See Table 1 for the appropriate normality of the sodium hydroxide solution, depending on the expected free fatty acid concentration range in the sample.PROCEDURE 1. T est samples must be well mixed and entirely liquid before weighing; however, do not heat the sample more than 10°C over the melting point. 2. Use Table 1 to determine the test portion weight for various ranges of fatty acids. Weigh the designated sample size into an oil sample bottle or Erlenmeyer flask (see Notes, 2). 3. Add the specified amount of hot neutralized alcohol and 2 mL of indicator. 4. Titrate with standard sodium hydroxide, shaking vigorously until the appearance of the first permanent pink color of the same intensity as that of the neutralized alcohol before the addition of the sample. The color must persist for 30 seconds.CALCULATIONS 1. The percentage of free fatty acids in most types of fats and oils is calculated as oleic acid, although in coconut and palm kernel oils it is frequently expressed as lauric acid and in palm oil in terms of palmitic acid.(a) Free fatty acids as oleic, % = mL of alkali M 28.2mass,g of test port ××i on(b) Freefatty acids as lauric, % = mL of alkali M 20.0mass,g of test port ××i on (c) Free fatty acids as palmitic, % = mL of alkali M 25.6mass,g of test port ××i on2. The free fatty acids are frequently expressed in terms of acid value instead of percentage free fatty acids. The acid value is defined as the number of milligrams of KOH necessary to neutralize 1 g of sample. To convert percentage free fatty acids (as oleic) to acid value, multiply the percentage free fatty acids by 1.99.PRECISION Precision data for refined, bleached and deodorized oils are shown in Table 2. Precision data for crude oils are shown in Table 3.NOTES 1. Isopropanol, 99%, may be used as an alternate solvent with crude and refined vegetable oils. 2. Cap bottle and shake vigorously for 1 min if oil has been blanketed with carbon dioxide gas.REFERENCES See J. Assoc. Off. Anal. Chem. 59:658 (1976) regarding the ruggedness of this method.Page 2 of 2SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS Ca 5a-40 • Free Fatty AcidsTable 1Free fatty acid range, alcohol volume and strength of alkali a FFA range (%) Test portion (g) Alcohol (mL) Strength of alkali 0.00 to 0.2 56.4 ± 0.2 50 0.1 M 0.2 to 1.0 28.2 ± 0.2 50 0.1 M 1.0 to 30.0 7.05 ± 0.05 75 0.25 M 30.0 to 50.0 7.05 ± 0.05 100 0.25 or 1.0 M 50.0 to 100 3.525 ± 0.001 100 1.0 M a FFA, free fatty acid; N, normality.Table 2The average, expected between-laboratory variation (standard deviation of reproducibility, S R ) for the determination of free fatty acids in refined, bleached and deodorized oils a Approximate FFA value (%) SR 0.007 0.010 0.046 0.073RSD (CV , %) 33.93 12.73 9.90 4.75R (95%) = 2.8 × S R 0.02 0.03 0.13 0.20a Values obtained from the AOCS Laboratory Proficiency Program.Table 3The average, expected between-laboratory variation (standard deviation of reproducibility, S R ) for the determination of free fatty acids in crude oils a Approximate FFA value (%) 0.1–1.0 1.0–2.0SR 0.077 0.156RSD (CV , %) 14.57 9.84R (95%) = 2.8 × S R 0.22 0.44a Values obtained from the AOCS Laboratory Proficiency Program.。

Solus E. coli O157 ELISA - AFNOR 认证的E. coli O157检测

Insert Number:38 Issue Number: 2.0 Date of Issue:April 2020 Product Code(s):EC-048SThis method is certified by AFNOR Certification for the detection of E. coli O157, including H7, in raw beef meat products (seasoned or not) raw milk and dairy products, vegetablesand environmental samples (validation ref. no. SOL 37/03-10/15).SOL 37/03-10/15ALTERNATIVE ANALYTICAL METHODS FOR AGRIBUSINESShttp///en1. INTRODUCTIONSolus E. coli O157 ELISA provides a negative or a presumptive positive result from a single enrichment within 18 to 22 hours, including the assay time.2. INTENDED USESolus E. coli O157 ELISA is for the detection of E. coli O157 in selected foods and production environmental samples. The test method is easy to perform, however it requires laboratory facilities plus qualified and trained personnel. Basic training is recommended to first time users and is given by Solus Scientific Solutions Ltd.Using the method includes compliance with Good Laboratory Practices (refer to EN ISO 7218).3. REAGENTS PROVIDEDMost kit components are supplied stabilised and ready to use at working concentration with only the Washing Buffer concentrate requiring dilution. Each kit contains sufficient material for 1 x 93 determinations, plus controls. The kit expiry date is displayed on each product label.4. MATERIALS AND EQUIPMENT REQUIRED BUT NOT PROVIDEDRefrigerator at 2-8°C TimerDeionised or distilled (DI) water Incubator at 37±1°CModified Tryptone Soya Broth + novobiocin (20mg/L)Incubator at 41.5±1°CMeasuring cylinders for various volumes (e.g. 250 ml, 1L)Heating apparatus (e.g. heat block) capable of heating to85-100°CSterile 10ml test tubes suitable for selective enrichmentHomogeniser (or similar apparatus) and filter bags Pipettes and tips (1ml; 0.1ml)3ml transfer pipettes (sterile)Dynex DS2 or Microplate washer and microplate readerwith 450nm filterVortex mixerTubes for sample boiling (e.g. 5ml polypropylene rimlessAutoclave for decontamination of samplestubes 12x75mm)5. REAGENT PREPARATION5.1 Wash Buffer:Add the contents of a Wash buffer bottle (10ml) to 240ml of deionised water to prepare the wash buffer at assay concentration. Dispense and label as appropriate. The prepared solution can be stored for a maximum of 4 weeks if kept at 2-8°C.5.2 Culture Broth (growth medium):Prepare the modified Tryptone Soya Broth following manufacturer’s instructions. Allow to cool to ambient temperature (18-30°C) before adding the novobiocin supplement at 20mg/L (mTSBn).6. SAMPLE PREPARATION AND ENRICHMENT- standard methodHomogenise 25g of the sample to be tested, if necessary by homogeniser, in 225ml Modified Tryptone Soya Broth (mTSB) + novobiocin (20mg/L) and incubate for 16-20 hours at 41.5±1°C. In the context of NF VALIDATION, test portions weighing more than 25g have not been tested.7. POST ENRICHMENT HEAT INACTIVATION7.1. When the sample incubation period is completed, transfer 1-2ml aliquot (avoiding particulates) to a sampleboiling tube (e.g. 5ml polypropylene tube).7.2. Heat the aliquot to 85-100°C for 15-20 minutes in the tube. After heating, allow the sample to cool toambient temperature (18-30°C). This may be accelerated by placing the tubes in cold tap water for ~5 minutes.7.3. To prevent pipetting issues, especially on the Dynex DS2 instrument, add a frit filter directly into each sampletube and gently push down to allow pipetting of a relatively clear sample from above the level of the frit.NOTE - The frit should only be inserted after the heating stage, and once the tube has cooled back to ambient temperature (18-30°C).The non-heat-inactivated samples should be kept for verification until ELISA results are obtained. These samples should be kept at 41.5±1°C if the ELISA test is to be carried out within 2 hours. If this is notpossible, keep the broths for up to 72 hours at 2-8°C prior to the ELISA test.8. ELISA ASSAY PROCEDURE8.1. Take test kit from storage at least one hour before use to allow the components to reach ambienttemperature (18-30°C). Determine the number of wells required for the test. Take required number of strips from the pouch and fit them to the frame provided. Unused strips should be returned to the pouch andstored at 2-8°C.8.2. Prepare Wash Buffer as detailed in section 5.1.8.3. Leave the first well in the strip empty to serve as a ‘blank’ for measuring the absorbance of the substrate.8.4. Pipette 0.1ml of Negative Control (Green label) into the second well.8.5. Pipette 0.1ml of Positive Control (Red label) into the third well.8.6. Pipette 0.1ml of each heat-inactivated sample separately into consecutive wells in the strip. If there are wells left over at the end of a test strip the Positive or Negative Controls may be repeated. †8.7. Incubate the plate (containing the strips) at 37±1°C for 30-35 minutes.8.8. After incubation, aspirate the contents of the wells, removing as much of the liquid as possible. Wash the wells 5 times with wash buffer ensuring complete filling and emptying of the wells through each wash cycle. The washing technique is critical to assay performance, hence it is recommended to use a microplatewasher instrument.8.9. Pipette 0.1ml of Conjugate (Orange label) into all wells except the ‘blank’.8.10. Incubate the plate at 37±1°C for 30-35 minutes.8.11. Repeat the wash cycles as detailed in section 8.88.12. Pipette 0.1ml of Substrate (Blue label) into all wells, including the ‘blank’ well. 8.13. Incubate the plate at ambient temperature (18-30°C) for 10 minutes in the dark.8.14. After incubation, stop the reaction by adding 0.1ml of Stop Solution (Yellow label) to all wells including the ‘blank’ well. The stop solution will cause any blue colour in wells to change to yellow.8.15. Read the optical densities of wells within 10 minutes in a plate reader using a 450nm filter. Before reading, inspect the wells for air bubbles and, if present, burst with a needle. The reader should be zeroed against the ‘blank’ well before the other wells are read. Do not use reference filter. The use of automatic ELISAequipment is preferred and should be set up and validated to this protocol.†If using the Dynex instrumentation, care must be taken to avoid bubbles in the sample and reagent tubes, or films forming across the tube above the level of the liquid. It is essential to check that the system has successfully pipetted samples into the assay plate before proceeding.9. INTERPRETATION OF RESULTSResults are expressed as optical density (OD 450) measurements using a microplate reader. Acceptance criteria:The value of the blanking well (usually A1 when processing manually) should always be subtracted. Should the value of Negative or Positive controls not meet these criteria, the test is not considered valid and must be performed again.Negative Control OD 450< 0.100Positive Control OD 450> 0.500Samples with OD450 readings of < 0.200 are considered negative in which case the analysis is complete, the resultsmay be reported and the corresponding non-heat-inactivated samples may be discarded following local regulations/ guidelines.Sample wells with OD450 ≥ 0.200 are considered presumptive positive for E. coli O157. Presumptive positive resultsmust be verified using a recognised culture method.10. CONFIRMATION OF POSITIVE RESULTS FROM E. COLI O157 ELISAIn the context of NF VALIDATION, all samples identified as positive by the alternative method must be confirmed by one of the following tests:• Using the conventional tests described in the standardised methods by CEN or ISO. The confirmation step must start from the non-heat-inactivated mTSB+n broth stored at 41.5°C or 2-8°C. (See ISO 16654:2001 Horizontal method for the detection of Escherichia coli O157).• Streaking mTSB+n (10μL) onto CT-SMAC and a chromogenic agar plate (such as CHROMagar O157) and incubate the plates at 37°C±1° for 18-24 hours. Perform a serological identification of characteristic colonies, with or without a purification step, using an O157 latex test (Microgen E. coli O157 M44) and after a purification step using H7 latex test (Wellcolex E. coli O157:H7 R30959601).In the event of discordant results (presumptive positive ELISA results and a negative culture result) an IMS step must be used as a confirmation step by taking 1 ml of mTSB+n (non-heated) and follow the method as described in ISO 16654:2001 prior to streaking the magnetic particles onto both agar plates. This method is more sensitive than direct plating and can help to confirm samples that contain lower levels of target organism.In the event of discordant results (presumptive positive ELISA result non-confirmed by one of the means described above and in particular for the latex tests) the laboratory must follow the necessary steps to ensure the validity of the result obtained.11. KIT STORAGE AND EXPIRYThe kit and any unused kit components should be stored at 2-8°C. DO NOT FREEZE. The kit expiry date is displayed on the kit box plus all of the kit components within the box. Any unused diluted wash buffer can be stored for up to 4 weeks if kept at 2-8°C. Any unused microplate strips should be returned to the foil pouch with the desiccant sachet and the seal closed completely, then stored at 2-8°C.12. SAFETYWhile the procedures detailed are simple and easy to perform, they require laboratory facilities with qualified personnel trained for the handling of potentially pathogenic organisms. Training is recommended to first time users and is provided by Solus Scientific Solutions Ltd.• The Stop Solution contains sulphuric acid which is corrosive. Wash immediately with large quantities of water if the solution comes into contact with skin or mucous membranes.As a guide, the following precautions should be taken as a minimum:• Protective clothing should be worn including lab coat, safety glasses, mask and gloves where appropriate.• Do not pipette by mouth.• Avoid contact with the skin.• Do not eat, drink or apply cosmetics in the laboratory.• Follow all applicable local, state/provincial, and/or national regulations on disposal of biological wastes.13. PRECAUTIONS• Reagents are provided at fixed working concentration. Optimum sensitivity and specificity will be reduced if reagents are modified or not stored under the recommended conditions.• Do not mix different lots of reagents.• Avoid microbial contamination of opened reagent bottles.• Ensure that no cross contamination occurs between wells.• It is essential for proper performance of the test that the enzyme-conjugated antibody is not allowed to contaminate other reagents and equipment.• Ensure that kit components are not exposed to temperatures greater than 40°C.• Solutions containing sodium azide should not be used for cleaning of equipment, especially washing devices (the peroxidase enzyme used in the kit is inactivated by sodium azide).• Do not use for diagnostic purposes of medical specimens.14. MSDS INFORMATIONMaterial safety data sheets (MSDS) are available for this test on request.15. WARRANTYAccurate results depend on the proper use of the kit by following the instructions for use carefully. If the kit fails to perform according to specification, please contact:Solus Scientific Solutions Ltd9 Mansfield NetworkcentreMillennium Business ParkConcorde WayMansfieldNottinghamshireNG19 7JZTel - +44 (0)1623 429701Fax - +44 (0)1623 620977Email:************************Manufactured at:Solus Scientific Solutions Ltd.9 Mansfield Networkcentre,Millennium Business Park, Concorde Way,Mansfield, Nottinghamshire NG19 7JZ UKTel - +44 (0)1623 429701PerkinElmer, Inc 940 Winter Street,Waltham, MA 02451 USA P: (800) 762-4000 or (+1) For a complete listing of our global offices, visit /ContactUsCopyright InformationThis document, including all photographs and illustrations, contains proprietary information that is protected by copyright. No part of this publication may be reproduced in any form whatsoever or translated into any language without the prior, written permission of PerkinElmer. ©2020 PerkinElmer, Inc. All rights reserved.TrademarksPerkinElmer is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners.Summary of changesNOTE: Minor changes (e.g. formatting, grammar, correcting typographical errors) are not included in the summary of changes.For more information visit 。

AOCS Cd (单甘酯测定方法)11b-91-2009英文版

SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILSAPPARATUS1. Gas chromatograph—with split injection or on-column injection (see Notes, 1), oven temperature programming and flame-ionization detector.2. Recording potentiometer.3. Electronic integrator.4. Column—capillary, glass or fused silica, surface fully deactivated by silylation agent (see Notes, 2), 15–25 m, 0.25–0.35 mmi.d., coating SE-54 (or other phase with similar polarity), film thickness 0.1–0.2 µm (see Notes, 3).5. Operating conditions:—split injection (split ratio 1:10–1:50)—direct injection (splitless, hold for 1 min)—injection port 320°C (or for on-column injection 60°C)—column initial 80°C (or for on-column 60°C)—program rate 10°C/min—final temperature 360°C, hold 15 min (see Notes, 3)—detector 350°C—carrier gas flow 5 mL He/min (at 80°C)—injection volume 1–5 µL6. An automatic sampler is advantageous (see Notes, 4).7. Screw-cap vials—2.5 mL; or crimp-top vials for auto sampler (e.g., 2.0 mL), with Teflon tm-faced septa (see Notes, 3).8. Heating device for vials—70°C.9. Balance—analytical, 200-g capacity with ±0.0001 g sensitivity.REAGENTS1. N,N-bis(trimethylsilyl)trifluoroacetamide (BSTFA).2. T rimethylchlorsilane (TMCS).3. Pyridine—analytical reagent grade, kept over KOH (see Notes, Caution).4. n-T etradecane—analytical reagent grade (minimum 99%).5. n-Hexane—analytical reagent grade (see Notes, Caution).6. Reference materials—glycerol, palmitic acid, 1-palmitoyl glycerol, 1-stearoyl glycerol, 1,2-dipalmitoyl glycerol, 1,3-dipalmi-toyl glycerol, 1,2-distearoyl glycerol.7. Internal standard solution—prepared by accurately weighing approximately 100 mg internal standard n-tetradecane into10-mL volumetric flask and diluting to volume with pyridine.8. Reference solution—prepared by accurately weighing approximately 100 mg of reference substance (e.g., glycerol, fatty acid,mono- and diacyl glycerol) and accurately weighing approximately 100 mg of n-tetradecane into the same 10 mL volumetric flask and diluting to volume with pyridine; or weigh approximately 100 mg of a mixture containing several (e.g., 5) reference materials and n-tetradecane, each component being present in about the same quantity, into a 2 mL volumetric flask and dilute to volume with pyridine (see Notes, 5).PROCEDURE1. T est solution—Accurately weigh approximately 10 mg of homogenized test sample of emulsifier concentrates, or 50 mg ofoils and fats containing emulsifiers, into a 2.5 mL screw-cap vial with Teflon tm-faced septa. Add 0.2 mL BSTFA and 0.1 mLPage 1 of 5SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILSCd 11b-91 • Determination of Mono- and DiglyceridesTMCS, and then 0.1 mL of internal standard solution (Reagents, 7) containing 1 mg n-tetradecane to the test sample (see Notes, 4). Moisture must be strictly excluded. Close vial and shake vigorously. Heat the reaction mixture in heating device at 70°C for approximately 20 min. Inject 1–5 µL of the reaction mixture into the gas chromatograph showing a stable base line. Avoid delay of GC analysis. The reaction is carried out two times, and two injections are made per reaction (see Notes, 1 and 2).2. Reference solution—Add 0.10 mL of reference solution (Reagents, 8) into vials and add the silylating agents, 0.2 mL BSTFAand 0.1 mL TMCS (no internal standard solution is added). Heat the reaction mixture and inject into the gas chromato-graph as described above (see Notes, 4). Use a concentration range of reference standards similar to the range of the com-ponents to be quantified in the test solution. A plot of response vs. concentration of reference substances may be useful to check linearity (see Notes, 1 and 2).3. Response factors—Check response factors periodically. Response factors should be above approximately 0.5. Lower responsefactors indicate some loss or decomposition. Use a concentration range of 0.5–10 mg/mL of components in both the r eference and test solutions. See Calculations, 1, for calculation of response factors.IDENTIFICATION1. Analyze reference solution under the same operation conditions as test solution. Identify peaks by comparison of retentiontime with known substances, or apply coupled GC/MS. See Figure 1.CALCULATIONS AND EXPRESSION OF RESULTS1. Response factor—Calculate response factor of the reference substance vs. internal standard using the reference solutionchromatogram. The value of the response factor is given by the formula:R x= (m is/m x) × (A x/A is)Where—R x= response factor of reference substance xm is = mass of internal standard, in mgm x= mass of reference substance x, in mgA x= peak area of reference substance xA is= peak area of internal standard2. Calculation of test portion component content—Calculate percentage of mass content of component x in the test portionby the formula:m'x (%) = 1/R x× (m'is/m's) × (A'x/A'is) × 100%Where—m'x = percentage of mass of component x in test portionR x = response factor of component x in test portionm'is=mass of internal standard in test portion, in mgm's = mass of test portion, in mgA'x = peak area of component x in test portionA'is = peak area of internal standard in test portion3. Typical chromatograms—See Figure 1 for typical chromatograms of reference standards and mono- and diglycerides. Thesilylation procedure, column specifications, operating conditions and peak identification relating to Figure 1 are as follows:(a) Silylation—T est sample size: 10 mg; reagents: 0.1 mL pyridine containing 1.0 mg n-tetradecane, 0.2 mL BSTFA, 0.1mL TMCS; reaction time: 20 min at 70°C.(b) Column—Fused silica capillary, 25 m × 0.31 mm (i.d.); film thickness, 0.17 µm, consisting of 5% phenylmethyl sili-con, Ultra #2 (Hewlett-Packard, Palo Alto, CA, USA).(c) Operating conditions—Injector 320°C, column initial 80°C, program 10°C/min, final 360°C, hold 15 min, detector350°C; carrier gas, helium at 5 mL/min (at 80°C) (see Apparatus, 5).(d) Peak identification—IS (internal standard), tetradecane; 1, glycerol; 2, diglycerol; 3, hexadecanoic acid; 4, octa-decanoate acid; 5, glycerol 1-tetradecanoate; 6, glycerol 2-hexadecanoate; 7, glycerol 1-hexadecanoate; 8, glycerol 2-octadecanoate; 9, glycerol 1-octadecanoate; 10, glycerol 1-icosanoate; 11, glycerol 1-docosanoate; 12, glycerol 1-tetradecanoate-3-hexadecanoate; 13, glycerol 1,2-dihexadecanoate; 14, glycerol 1,3-dihexadecanoate; 15, glycerol 1-hexadecanoate-2-octadecanoate; 16, glycerol 1-hexadecanoate-3-octadecanoate; 17, glycerol 1,2-dioctadecanoate; 18, glycerol 1,3-dioctadecanoate; 19, triglyceride C48; 20, triglyceride C50; 21, triglyceride C52; 22, triglyceride C54. PRECISION1. Repeatability—When the mean of the values obtained from two single determinations, carried out in rapid succession by thesame operator using the same apparatus under the same conditions for the analysis of the same test sample, lies within the range of the mean values cited in Tables 1, 2 and 3, the difference between the two values obtained should not be greater than the repeatability value (r), which can generally be deduced by linear interpolation from Tables 1, 2 and 3 (References, 2).Page 2 of 5SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILSCd 11b-91 • Determination of Mono- and Diglycerides2. Reproducibility—When the values for the final result, obtained by operators in different laboratories, using different appa-ratus under different conditions for the analysis of the same laboratory sample, lie within the range of mean values cited in Tables 1, 2 and 3, the difference between the values for the final result obtained by those operators should not be greater than the reproducibility value (R), which can generally be deduced by linear interpolation from Tables 1 and 2.NOTESCautionPyridine is flammable and a dangerous fire risk. The explosive limits in air are 1.8–12.4%. It is toxic by ingestion and inhala-tion. The TLV is 5 ppm in air. The danger from crude pyridine is greater than from pure pyridine, the associated homologs and impurities being even more toxic than pyridine.Hexane is flammable and a dangerous fire risk. The TLV is 50 ppm in air. The Occupational Safety and Health Administration recommends that exposure not exceed 350 ng per cubic meter for a time-weighted average. Hexane vapor causes lung irritation and produces neurotoxic effects. A fume hood should be used at all times when using hexane.NUMBERED NOTES1. For on-column injection, or direct injection, dilute 50 µL of reaction mixture (Procedure, 1 and 2) with 1 mL hexane andinject (1 µL). In order to lengthen lifetime of the columns, when applying on-column injections, a precolumn is useful.On-column injection gives better response factors.2. Use length of columns required to separate mono- or diglycerides. Individual unsaturated mono- and diglycerides may notbe separated from the saturated or less-unsaturated mono- or diglycerides. TLC on silica gel impregnated with boric acid, immediately prior to derivatization, can be used to resolve 2-mono-glycerides from 1-monoglycerides (References, 3).3. The final column temperature of 360°C is not compatible with the maximum operating temperature of 320°C to 325°Crecommended for SE-54. The detector should be 10°C to 20°C higher than the maximum column temperature. Therefore, it is recommended to use a maximum column temperature of 325°C to 330°C (apparently this will not affect the separation of the mono- and diglycerides), with a detector temperature of 335°C to 350°C. These proposed changes have not been validated by collaborative study.4. For automatic samplers with 2-mL crimp-top vials, it is convenient to double the amount of test sample and reagents.5. One or more reference solutions (Reagents, 8) can also be prepared without adding n-tetradecane to the solution. Silylationof the reference solutions (Procedure, 2) is then carried out as described for the test solution (Procedure, 1) after addition of the internal standard solution (Reagents, 7) and silylating reagents.REFERENCES1. Standard Methods for the Analysis of Oils, Fats and Derivatives, International Union of Pure and Applied Chemistry, 7th edn.,Blackwell Scientific Publications, 1987, IUPAC Method 6.002 (ex. 2.326).2. Pure Appl. Chem. 63:1153 (1991).3. Christie, W.W., Lipid Analysis, 2nd edn., Pergamon Press, New York, 1982, pp. 101, 159.Table 1Statistical analysis of results for mono- and diglyceride concentrates (expressed as percent of mass of test portion).1-Palmitate-1-Myristate 1-Palmitate 1-Stearate 1,3-Dipalmitate 3-stearate 1,3-Distearate No. of laboratories 8 8 8 8 8 8 No. of results 16 16 16 16 16 16 No. of laboratories retained afterelimination of outliers a7 8 8 7 7 8 No. of accepted results 14 16 16 14 14 16 Mean value, % 1.7 27.2 60.1 0.2 0.8 1.1 Repeatability standard deviation (S r) 0.05 0.9 2.1 0.01 0.05 0.07 Repeatability co-efficient ofvariation (RSD r) 3.0 3.3 3.5 4.6 6.0 6.8 Repeatability value (r) [2.83 × S r] 0.14 2.61 5.91 0.031 0.14 0.20 Reproducibility standarddeviation (S R) 0.1 2.4 6.4 0.06 0.1 0.3 Reproducibility co-efficient ofvariation (RSD R) 5.7 8.9 10.7 30.0 17.8 24.8 Reproducibility value (R)[2.83 × S R] 0.3 6.8 18.1 0.2 0.4 0.8a Eliminated using Dixon and Cochran test methods.Page 3 of 5SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILSCd 11b-91 • Determination of Mono- and DiglyceridesTable 2Statistical analysis of results for mono- and diglycerides in oil (expressed as percent of mass of test portion).1-Palmitate 1-Stearate 1,2-Dipalmitate 1,3-Dipalmitate 1,2-Distearate No. of laboratories 8 8 8 8 8 No. of results 16 16 16 16 16 No. of laboratories retained afterelimination of outliers a8 8 8 8 8 No. of accepted results 16 16 16 16 16 Mean value, % 0.96 0.98 0.97 0.93 0.97 Repeatability standard deviation (S r) 0.03 0.03 0.04 0.02 0.06 Repeatability co-efficient ofvariation (RSD r) 3.3 3.4 4.0 2.5 6.2 Repeatability value (r) [2.83 × S r] 0.08 0.08 0.11 0.06 0.17 Reproducibility standarddeviation (S R) 0.12 0.14 0.24 0.19 0.19 Reproducibility co-efficient ofvariation (RSD R) 12.0 13.8 24.4 20.2 19.8 Reproducibility value (R)[2.83 × S R] 0.34 0.40 0.68 0.54 0.54a Eliminated by the Dixon and Cochran test methods.Table 3Statistical analysis of results for mono- and diglycerides in sunflower oil (blind duplicates, expressed as percent of mass of test portion).1-Palmitate 1-Stearate 1,2-Dipalmitate 1,3-Dipalmitate 1,2-Distearate No. of laboratories 8 8 8 8 8 No. of results 30 30 30 30 30 No. of laboratories retained afterelimination of outliers 7 8 7 7 7 No. of accepted results 26 30 26 26 30 Mean value, % 0.75 1.4 1.1 1.3 2.6 Repeatability standard deviation (S r) 0.07 0.24 0.09 0.07 0.30 Repeatability co-efficient ofvariation (RSD r) 10.0 17.0 8.4 5.3 11.8 Repeatability value (r) [2.83 × S r] 0.21 0.67 0.25 0.48 0.86 Reproducibility standarddeviation (S R) 0.14 0.33 0.11 0.12 0.94 Reproducibility co-efficient ofvariation (RSDR) 18.5 23.4 10.4 9.2 36.3 Reproducibility value (R)[2.83 × SR] 0.39 0.92 0.32 0.28 2.6Page 4 of 5Page 5 of 5SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILSCd 11b-91 • Determination of Mono- and DiglyceridesFigure 1. Typical chromatograms of trimethylsilylether derivatives of mono- and diglycerides: A, reference standards and B, mono- and diglyceride emulsifier. The silylation procedure, column specifications, operating conditions, and peak identifica-tion are as follows: (a) Silylation —Test sample size, 10 mg; reagents, 0.1 mL pyridine containing 1.0 mg n-tetradecane, 0.2 mL BSTFA, 0.1 mL TMCS; reaction time, 30 min at 70°C. (b) Column —25 m ë 0.31 mm i.d. fused silica; 0.17 µm film thickness (5% phenylmethyl silicon, Ultra #2, Hewlett-Packard). (c) Operating conditions —Injector, 320°C; hold 15 min; detector, 350°C; carrier gas, He, 5 mL/min, 80°C. (d) Peak identification —IS, n-tetradecane (internal standard); 1, glycerol; 2, diglycerol; 3, hexadecanoic acid; 4, octadecanoic acid; 5, glycerol 1-tetradecanoate; 6, glycerol 2-hexadecanoate; 7, glycerol 1-hexadec-anoate; 8, glycerol 2-octadecanoate; 9, glycerol 1-octadecanoate; 10, glycerol 1-icosanoate; 11, glycerol 1-docosanoate; 12, glycerol 1-tetradecanoate-3-hexadecanoate; 13, glycerol 1,2-dihexadecanoate; 14, glycerol 1,3-dihexadecanoate; 15, glyc-erol 1-hexadecanoate-2-octadecanoate; 16, glycerol 1-hexadecanoate-3-octadecanoate; 17, glycerol 1,2-dioctadecanoate; 18, glycerol 1,3-dioctadecanoate; 19, triglyceride C48; 20, triglyceride C50; 21, triglyceride C52; and 22, triglyceride C54.D e t e c t o r r e s p o n s e1020304010203040IS791413171ISRetention time (min)43791416181519202122AB17131211108652。

ANKOM纤维仪通过AOCS、AOAC认证(A200)

AOCS Approved Procedure Ba 6a-05 01-11-08 ANKOM Technology Method 7Crude Fiber Analysis in FeedsBy Filter Bag Technique(For A200, A200I, A220)DEFINITIONThis method determines crude fiber which is the organic residue remaining after digesting with 0.255N H2SO4 and 0.313N NaOH. The compounds removed are predominantly protein, sugar, starch, lipids and portions of both the structural carbohydrates and lignin.SCOPEThis method is applicable for all feed materials such as grains, meals, pet foods, mixed feeds, forages and the following oilseeds: corn and soybeans.APPARATUS1.Analytical Balance—capable of weighing down to0.1 mg.2.Oven—capable of maintaining a temperature of102±2°C.3.Electric muffle furnace—with rheostat control andpyrometer that will maintain a temperature of600±15°C4.Digestion instrument—capable of performing thedigestion at 100±0.5°C and maintaining a pressureof 10-25 psi. The instrument must also be capableof creating a similar flow around each sample toensure uniformity of extraction. (ANKOM200w/65 rpm agitation, ANKOM Technology).5.Filter bags—constructed from chemically inertand heat resistant filter media, capable of beingheat sealed closed and able to retain 25 micronparticles while permitting rapid solution penetration (F57 or F58, ANKOM Technology,see Numbered Notes 1).6.Heat sealer—sufficient for sealing the filter bagsclosed to ensure complete closure (1915,ANKOM Technology).7.Desiccator pouch—collapsible sealable pouchwith desiccant inside that enables the removal ofair from around the filter bags (MoistureStopWeigh Pouch, ANKOM Technology).8.Marking pen—solvent and acid resistant (F08,ANKOM Technology).REAGENTS1.Sulfuric acid solution—0.255±0.005N. 1.25 gH2SO4/100 mL. Concentration must be checkedby titration (see Notes, Caution).2.Sodium hydroxide solution—0.313±0.005N. 1.25g NaOH/100 mL. Concentration must be checkedby titration (see Notes, Caution). PREPARATION OF SAMPLEGrind samples through a centrifugal mill with a 2 mm screen or cutter type (Wiley) mill with a 1 mm screen. Samples ground finer may show particle loss from the filter bags and result in low values.PROCEDUREe a solvent and acid resistant marker to labelthe filter bags. Weigh filter bag (W1) and zerobalance.Note—Do not pre-dry filter bags; any moisture will be accounted for by the blank bagcorrection.2.Weigh 0.95-1.00 g of prepared sample (W2)directly in filter bag. Avoid placing the sample onthe upper 4 mm of the bag.ing a heat sealer, completely seal the upperedge of the filter bag within 4 mm of the top.Note—Use sufficient heat to completely seal the filter bag and allow enough cool time (2sec) before removing the bag from the heat sealer. 4.Weigh one blank bag and include in run todetermine blank bag correction (C1, see NumberNote 2).5.Extract fat from samples by placing all bags into a250 mL container. Add enough petroleum etherto cover bags and soak for 10 min. Pour offsolvent and allow bags to air-dry. Spread sampleuniformly inside the filter bag by shaking andflicking the bag to eliminate clumping.6.Place a maximum of 24 bags into the BagSuspender. All nine trays are used regardless ofthe number of bags being processed. Place threebags per tray and then stack trays on center postwith each level rotated 120 degrees. Insert theBag Suspender with bags into the fiber analyzervessel and place the Bag Suspender weight on topof the empty 9th tray to keep it submerged.Note—Prior to inserting the Bag Suspender, if the vessel temperature is warm froma previous run, add cool water and exhaust.7.When processing 24 sample bags, pour 1900-2000 mL of ambient temperature acid (0.255NH2SO4) solution into the fiber analyzer vessel. If。

Investigation of Out of Specification (OOS) Laboratory Results-May

方的合规和向顾客报告结果。
• “When investigation by a contract laboratory does not determine an assignable cause,
all test results should be reported to the customer on the certificate of analysis ” 当由合同实验室进行的调查不能确定原因时，所有的测试结果都应该在分析报告中向顾客报告
5 | Presentation Title | Presenter Name | Date | Subject | Business Use Only
Highlights of Court Findings and Guidance
审计发现项和法规指南
“Drug products failing to meet specifications will be rejected” 不能满足标准的产品将会被
超标调查简史
Food Drug and Administration Warning Letters
食品及药物管理局警告信
OOS Laboratory Investigation Process 超标实验室调查过程 CAPA System CAPA 系统 The Ineffective CAPA Circle 无效的CAPA 循环 Ten Opportunities of the CAPA System CAPA 系统中的十个机会 Effective CAPA Process 有效CAPA 过程
(Deviation System) 在阶段和等级中的实验室调查中应与生产调查（偏差系统）有及时联系
Priority of laboratory investigation in the laboratory routine 实验室调查在实验室日常工作

FCC-V附录VII-脂肪和相关物质[P110]

APPENDIX VII:FATS AND RELATED SUBSTANCESACETYL VALUE(Based on AOCS Method Cd4-40)The acetyl value is defined as the number of mg of potassium hydroxide required to neutralize the acetic acid obtained by saponifying1g of the acetylated sample.Acetylation Boil50mL of the oil or melted fat with50 mL of freshly distilled acetic anhydride for2h under a reflux condenser.Pour the mixture into a beaker containing500mL of water,and boil for15min,bubbling a stream of nitrogen or carbon dioxide through the mixture to prevent bumping. Cool slightly,remove the water,add another500mL of water, and boil again.Repeat for a third time with another500-mL portion of water,and remove the wash water,which should be neutral to litmus.Transfer the acetylated fat to a separator, and wash with two200-mL portions of warm water,separating as much as possible of the wash water each time.Transfer the washed sample to a beaker,add5g of anhydrous sodium sulfate,and let stand for1h,agitating occasionally to assist drying.Filter the oil through a dry filter paper,preferably in an oven at100°to110°,and keep the filtered oil in the oven until it is completely dry.The acetylated product should be a clear,brilliant oil.Saponification Weigh accurately from2to2.5g each of the acetylated oil and of the original,untreated sample into separate250-mL Erlenmeyer flasks.Add to each flask25.0 mL of0.5N alcoholic potassium hydroxide,and continue as directed in the Procedure under Saponification Value,in this Appendix,beginning with‘‘Connect an air condenser....’’Record the saponification value of the untreated sample as S, and that of the acetylized oil as S′,then calculate the acetyl value of the sample by the formula(S′–S)/(1.000–0.00075S).ACID VALUE(Based on AOCS Methods Te1a-64and Cd3d-63) The acid value is defined as the number of mg of potassium hydroxide required to neutralize the fatty acids in1g of the test substance.Method I(Commercial Fatty Acids)Unless otherwise directed,weigh accurately about5g of the sample into a500-mL Erlenmeyer flask,and dissolve it in 75to100mL of hot alcohol,previously boiled and neutralized to phenolphthalein TS with sodium hydroxide.Agitation and further heating may be necessary to effect complete solution of the sample.Add0.5mL of phenolphthalein TS,and titrate immediately,while shaking,with0.5N sodium hydroxide to the first pink color that persists for at least30s.Calculate the acid value by the formula56.1V×N/W,in which V is the volume,in mL,and N is the normality, respectively,of the sodium hydroxide solution;and W is the weight,in g,of the sample taken.Method II(Animal Fats and Vegetable and Marine Oils) Prepare a solvent mixture consisting of equal parts,by volume, of isopropyl alcohol and toluene.Add2mL of a1%solution of phenolphthalein in isopropyl alcohol to125mL of the mixture,and neutralize with alkali to a faint but permanent pink color.Weigh accurately the appropriate amount of well-mixed liquid sample indicated in the table below,dissolve it in the neutralized solvent mixture,warming if necessary,and shake vigorously while titrating with0.1N potassium hydrox-ide to the first permanent pink color of the same intensity as that of the neutralized solvent before mixing with the sample. Calculate the acid value by the formula56.1V×N/W,in which V is the volume,in mL,and N is the normality, respectively,of the potassium hydroxide solution;and W is the weight,in g,of the sample taken.Acid Value Sample Weight(g)0–1201–4104–15 2.515–750.575and over0.1CHLOROPHYLL(Based on AOCS Method Cc13d-55)Use a reliable spectrophotometer with a sample holder equili-brated at44°Ϯ3°to obtain absorbance values at630,670, and710nm.Calculate the concentration of chlorophyll(C) using the following equation:C=[A670–(A630/2)–(A710/2)]/(K×b),in which C is the concentration of chlorophyll,in mg/kg;A is the absorbance at the wavelength indicated by the subscript; K is the constant for the specific spectrophotometer being used and is equal to0.1016for the Beckman Model DU;and b is the optical pathlength through the sample,in cm.COLD TEST(Based on AOCS Method Cc11-53)Filter a sample(200to300mL),and transfer to a clean,dry bottle.Fill the bottle completely,and insert a cork stopper. Seal with paraffin,and equilibrate at25°in a water bath so that it is completely covered.Next,immerse the bottle in an ice and water bath so it is completely covered.Monitor the bath during the test and replenish the ice frequently to keep the bath at0°.After5.5h remove the bottle from the bath.The sample must be clear;fat crystals or cloudiness must be totally absent. COLOR(AOCS-Wesson)(Based on AOCS Method Cc13b-45)Apparatus Use a Lovibond tintometer or the equivalent and a set of color comparison glasses that conform to the AOCS-Wesson Tintometer Color Scale(available from the National Institute of Standards and Technology).A minimum set of glasses consists ofRed0.10.20.30.40.50.60.80.91.02.0 2.53.0 3.54.05.06.07.07.68.09.010.011.012.016.020.0Yellow 1.0 2.0 3.0 5.010.015.020.035.050.070.0For making color comparisons,use color tubes of clear,color-less glass with a smooth,flat,polished bottom(length154 mm;id19mm;od22mm),and marked to indicate liquid columns of25.4and133.35mm.Procedure Add0.1g of diatomaceous earth to a60-g sam-ple,agitate for2.5min at room temperature(or10°to15°above the melting point if the sample is not liquid),and filter. Adjust the temperature to25°to35°(or not more than100 above the melting point),and fill the color tube to the desired mark.Place the tube in the tintometer(in a dark booth or cabinet),and match the sample color as closely as possible with a standard glass.FATTY ACID COMPOSITION(Based on AOCS Methods Ce1-62,Ce1b-89,Ce 1e-91)Apparatus Use a suitable gas chromatograph(see Appendix IIA)equipped with a flame ionization detector(FID)and containing either a3.05-m×2-or4-mm id glass column packed with preconditioned10%,by weight,DEGS-PS on 100-to120-mesh diatomaceous earth(Chromosorb WHP,or equivalent)or a30-m×0.20-to0.35-mm id capillary fused silica column,or equivalent,containing a suitable station-ary phase.Operating Conditions The operating conditions may vary with the instrument used,but a suitable chromatogram may be obtained using a temperature program180°to215°;inlet temperature(injector),300°;detector,300°;and a suitable carrier gas flow.Standard Solutions Run through the chromatograph a com-mercially available standard containing a mixture of fatty-acid methyl esters.Fatty acids and methyl esters with a wide range of carbon numbers and double-bond configurations can be purchased.The calculated concentration should compare to that claimed withinϮ2␴,where␴is the standard deviation calculated from at least10replicate determinations,preferably made over a period of several days.Determine that the system is functioning properly:inject into the chromatograph a suitable number of samples of the standard to ensure that the resolution factor,R,defining the efficiency of the separation between methyl stearate and methyl oleate is0.9or greater.Calculate R by the equationR=2(t2–t1)/(w2+w1),in which t2and t1are the retention times of peak2and peak 1,respectively,and w2and w1are the corresponding widths of the bases of the peaks obtained by extrapolating relatively straight sides of the peaks to the baseline.Baseline separation of the various components in both the standard and the sample preparations is desirable.Sample Preparation(for fats and oils)(Based on AOCS Method Ce2-66)Introduce100to1000mg of the fat into a50-or125-mL reaction flask.Add4to10mL of0.5N methanolic sodium hydroxide,and add a boiling chip.Attach a condenser,and heat the mixture on a steam bath until the fat globules go into solution.This step should take5to10 min.Add5to12mL of12.5%boron fluoride–methanol reagent(this reagent contains125g of boron fluoride per L of methanol and is available commercially)through the condenser,and boil for2min.Add2to5mL of heptane through the condenser,and boil for1min longer.Remove from heat,remove condenser,and add about15mL of satu-rated sodium chloride solution.Stopper the flask,and shake vigorously for15s.Transfer about1mL of the heptane solution into a test tube and add a small amount of anhydrous sodium sulfate.The dry heptane solution may then be injected directly into a gas chromatograph.The methyl esters should be analyzed as soon as possible. They may be kept in an atmosphere of nitrogen in a screw-cap vial at2°for24h.For longer storage,they should be sealed in a glass ampule,subjected first to a vacuum and then backfilled with nitrogen and stored at−20°(freezer).Procedure Inject an appropriate volume(0.1␮L to1.0␮L) of sample into the chromatograph.If an automated system is used,follow the manufacturer’s instructions;if calculations are to be done manually,proceed as follows:Calculate the area percent of each component(C N)by the equationC N=[A N/T S]×100,in which A N is the area of the peak corresponding to component C N,and T S is the total area for all detected components [T S=⌺A N].FREE FATTY ACIDS(Based on AOCS Method Ca5a-40)Unless otherwise directed,accurately weigh the appropriate amount of the sample,indicated in the table below,into a 250-mL Erlenmeyer flask or other suitable container.Add2 mL of phenolphthalein TS to the specified amount of hot alcohol,neutralize with alkali to the first faint,but permanent, pink color,and then add the hot,neutralized alcohol to the sample container.Titrate with the appropriate normality of sodium hydroxide,shaking vigorously,to the first permanent pink color of the same intensity as that of the neutralized alcohol.The color must persist for at least30s.Calculate the percentage of free fatty acids(FFA)in the sample by the formulaVNe/W,in which V is the volume and N is the normality of the sodium hydroxide used;W is the weight of the sample,in g;and e is the equivalence factor given in the monograph.FFA Range Grams of Milliliters of Strength (%)Sample Alcohol of NaOH 0.00–0.256.4Ϯ0.2500.1N0.2–1.028.2Ϯ0.2500.1N1.0–30.07.05Ϯ0.05750.25N 30.0–50.07.05Ϯ0.051000.25–1.0N 50.0–100 3.525Ϯ0.001100 1.0N FREE GLYCERIN OR PROPYLENE GLYCOL(Based on AOCS Method Ca14-56)Reagents and Solutions Use the Periodic Acid Solution, Potassium Iodide Solution,and Chloroform as described under 1-Monoglycerides,in this Appendix.Procedure To the combined aqueous extracts obtained as directed under1-Monoglycerides,add50.0mL of Periodic Acid Solution.Run two blanks by adding50.0mL of this reagent solution to two500-mL glass-stoppered Erlenmeyer flasks,each containing75mL of water.Continue as directed in the Procedure under1-Monoglycerides,beginning with ‘‘...and allow to stand for at least30min but no longer than 90min.’’Calculation Calculate the percentage of free glycerin in the original sample by the formula(b–S)×N×2.30/W,or calculate the percentage of free propylene glycol by the formula(b–S)×N×3.81/W,in which b is the number of mL of sodium thiosulfate con-sumed in the blank determination;S is the number of mL required in the titration of the aqueous extracts from the sample;N is the exact normality of the sodium thiosulfate; W is the weight,in g,of the original sample taken;2.30is the molecular weight of glycerin divided by40;and3.81is the molecular weight of propylene glycol divided by20.Note:If the aqueous extract contains more than20mgof glycerin or more than30mg of propylene glycol,dilute the extract in a volumetric flask and transfer asuitable aliquot into a500-mL glass-stoppered Erlen-meyer flask before proceeding with the test.The weightof the sample should be corrected in the calculation. HEXANE-INSOLUBLE MATTERIf the sample is plastic or semisolid,soften a portion by warming it at a temperature not exceeding60°,and then mix it thoroughly.Transfer100g of well-mixed sample into a 1500-mL wide-mouth Erlenmeyer flask,add1000mL of sol-vent hexane,and shake until the sample is dissolved.Filter the resulting solution through a600-mL Corning‘‘C’’porosity,or equivalent,filtering funnel that previously has been dried at 105°for1h,cooled in a desiccator,and weighed.Wash the flask with two successive250-mL portions of solvent hexane, and pass the washings through the filter.Dry the funnel at 105°for1h,cool to room temperature in a desiccator,and weigh.From the gain in weight of the funnel,calculate the percentage of the hexane-insoluble matter in the sample. HYDROXYL VALUE(Based on AOCS Methods Cd4-40and Cd13-60) The hydroxyl value is defined as the number of mg of potas-sium hydroxide equivalent to the hydroxyl content of1g of the unacetylated sample.Method IProceed as directed under Acetyl Value,in this Appendix,but calculate the hydroxyl value by the formula(S′–S)/(1.000–0.00075S′).Method IIUnless otherwise directed,accurately weigh the appropriate amount of the sample indicated in the table below,transfer it into a250-mL glass-stoppered Erlenmeyer flask,and add 5.0mL of pyridine–acetic anhydride reagent(mix3volumes of freshly distilled pyridine with1volume of freshly distilled acetic anhydride).Hydroxyl Value Sample Weight(g)0–201020–50550–1003100–1502150–200 1.50200–250 1.25250–3001300–3500.75Pipet5mL of the pyridine–acetic anhydride reagent into a second250-mL flask for the reagent blank.Heat the flasks for1h on a steam bath under reflux condensers,then add10 mL of water through each condenser,heat for10min longer, and allow the flasks to cool to room temperature.Add15mL of n-butyl alcohol,previously neutralized to phenolphthalein TS with0.5N alcoholic potassium hydroxide,through the condenser,then remove the condensers,and wash the sides of the flasks with10mL of n-butyl alcohol.To each flask add1mL of phenolphthalein TS,and titrate to a faint pink endpoint with0.5N alcoholic potassium hydroxide,recording the mL required for the sample as S and that for the blank as B.To correct for free acid,mix about10g of the sample, accurately weighed,with10mL of freshly distilled pyridine, previously neutralized to phenolphthalein,add1mL of phe-nolphthalein TS,and titrate to a faint endpoint with0.5N alcoholic potassium hydroxide,recording the mL required as A.Calculate the hydroxyl value by the formula[B+(WA/C)–S]×56.1N/W,in which W and C are the weights,in g,of the samples taken for acetylation and for the free acid determination,respec-tively;and N is the exact normality of the alcoholic potassium hydroxide.IODINE VALUE(Based on AOCS Method Cd1d-92)The iodine value is a measure of unsaturation and is expressed as the number of g of iodine absorbed,under the prescribed conditions,by100g of the test substance.Modified Wijs Method(Acetic Acid/Cyclohexane Method) Wijs Solution Dissolve13g of resublimed iodine in1000 mL of glacial acetic acid.Pipet10.0mL of this solution into a250-mL flask,add20mL of potassium iodide TS and100 mL of water,and titrate with0.1N sodium thiosulfate,adding starch TS near the endpoint.Record the volume required as A.Set aside about100mL of the iodine–acetic acid solution for future use.Pass chlorine gas,washed and dried with sulfu-ric acid,through the remainder of the solution until a10.0-mL portion requires not quite twice the volume of0.1N sodium thiosulfate consumed in the titration of the original iodine solution.A characteristic color change occurs when the desired amount of chlorine has been added.Alternatively, Wijs Solution may be prepared by dissolving16.5g of iodine monochloride,ICl,in1000mL of glacial acetic acid.Store the solution in amber bottles sealed with paraffin until ready for use,and use within30days.Total Halogen Content Pipet10.0mL of Wijs Solution into a500-mL Erlenmeyer flask containing150mL of recently boiled and cooled water and15mL of potassium iodide TS. Titrate immediately with0.1N sodium thiosulfate,recording the volume required as B.Halogen Ratio Calculate the I/Cl ratio by the formulaA/(B–A).The halogen ratio must be between1.0and1.2.If the ratio is not within this range,the halogen content can be adjusted by adding the original solution or by passing more chlorine through the solution.Note:Wijs Solution is commercially available. Procedure The appropriate weight of the sample,in g,is calculated by dividing the number25by the expected iodine value.Melt the sample,if necessary,and filter it through a dry filter paper.Transfer the accurately weighed quantity of sample into a clean,dry,500-mL glass-stoppered bottle or flask containing20mL of glacial acetic acid/cyclohexane, 1:1,v/v,and pipet25.0mL of Wijs Solution into the flask. The excess of iodine should be between50%and60%of the quantity added,that is,between100%and150%of the quan-tity absorbed.Swirl,and let stand in the dark for1.0h where the iodine value is<150and for2.0h where the iodine value is≥150.Add20mL of potassium iodide TS and100mL of recently boiled and cooled water,and titrate the excess iodine with0.1N sodium thiosulfate,adding the titrant gradually and shaking constantly until the yellow color of the solution almost disappears.Add starch TS,and continue the titration until the blue color disappears entirely.Toward the end of the titration,stopper the container and shake it violently so that any iodine remaining in solution in the glacial acetic acid/ cyclohexane,1:1,solution may be taken up by the potassium iodide solution.Concomitantly,conduct two determinations on blanks in the same manner and at the same temperature. Calculate the iodine value by the formula(B–S)×12.69N/W,in which B–S represents the difference between the volumes of sodium thiosulfate required for the blank and for the sample,respectively;N is the normality of the sodium thiosulfate;and W is the weight,in g,of the sample taken.MELTING RANGEFats of animal and vegetable origin do not exhibit a sharp melting point.For the purpose of this test,melting range is defined as the range of temperature in which the sample becomes a perfectly clear liquid after first passing through a stage of gradual softening,during which it may become opalescent.Apparatus Use any suitable commercial or other apparatus. Use melting-point capillary tubes—id,1mm;od,2mm; length,50to80mm;and open at both ends.ProcedureCapillary Method(Based on AOCS Method Cc1-25) Melt the sample and filter it through filter paper;the sample must be absolutely dry.Dip three capillary tubes in the liquid sample so that the oil stands approximately10mm high in the tubes,and fuse the end of the tube containing the sample without burning it.Place the tubes containing the liquid sample in a beaker,and equilibrate them at least16h at4°to10°in a refrigerator.Determine the melting range,using a temperature increase of0.5°per min when within10°of the anticipated melting point.The melting ranges of the three samples should be no more than0.5°apart.1-MONOGLYCERIDES(Based on AOCS Method Cd11-57)Reagents and SolutionsPeriodic Acid Solution Dissolve5.4g of periodic acid, H5IO6,in100mL of water,add1900mL of glacial acetic acid,and mix.Store in a light-resistant,glass-stoppered bottle or in a clear,glass-stoppered bottle protected from light. Chloroform Use chloroform meeting the following test: To each of three500-mL flasks add50.0mL of Periodic Acid Solution,then add50mL of chloroform and10mL of water to two of the flasks and50mL of water to the third.To each flask add20mL of potassium iodide TS,mix gently,and continue as directed in the Procedure,beginning with ‘‘...allow to stand at least1min....’’The difference be-tween the volume of0.1N sodium thiosulfate required in the titrations with and without the chloroform is not greater than 0.5mL.Procedure Melt the sample,if not liquid,at a temperature not higher than10°above its melting point,and mix thor-oughly.Transfer an accurately weighed portion of the sample,equivalent to about150mg of1-monoglycerides,into a100-mL beaker(or weigh a sample equivalent to20mg of glycerin or30mg of propylene glycol if only Free Glycerin or Propyl-ene Glycol is to be determined),and dissolve in25mL of chloroform.Transfer the solution,with the aid of an additional 25mL of chloroform,into a separator,wash the beaker with 25mL of water,and add the washing to the separator.Stopper the separator tightly,shake vigorously for30to60s,and allow the layers to separate.(Add1to2mL of glacial acetic acid to break emulsions formed due to the presence of soap.) Collect the aqueous layer in a500-mL glass-stoppered Erlen-meyer flask,and extract the chloroform solution again using two25-mL portions of water.Retain the combined aqueous extracts for the determination of Free Glycerin or Propylene Glycol(in this Appendix).Transfer the chloroform to a500-mL glass-stoppered Erlenmeyer flask,and add50.0mL of Periodic Acid Solution to this flask and to each of two blank flasks containing50mL of chloroform and10mL of water. Swirl the flasks during the addition of the reagent,and allow to stand for at least30min,but no longer than90min.To each flask,add20mL of potassium iodide TS,and allow to stand at least1min,but no longer than5min,before titrating. Add100mL of water,and titrate with0.1N sodium thiosul-fate,using a magnetic stirrer to keep the solution thoroughly mixed,to the disappearance of the brown iodine color,then add2mL of starch TS and continue the titration to the disap-pearance of the blue color.Calculate the percentage of1-monoglycerides1in the sample by the formula(B–S)×N×17.927/W,in which B is the number of mL of sodium thiosulfate con-sumed in the blank determination;S is the number of mL required in the titration of the sample;N is the exact normality of the sodium thiosulfate;W is the weight,in g,of the sample taken;and17.927is the molecular weight of glyceryl monoste-arate divided by20.TOTAL MONOGLYCERIDESPreparation of Silica Gel Place about10g of100-to200-mesh silica gel of a grade suitable for chromatographic work in a tared weighing bottle,cap immediately,and weigh accu-rately.Remove the cap,dry at200°for2h,cap immediately, and cool for30min.Raise the cap momentarily to equalize the pressure,then weigh again,reheat for5min at200°, cool,and reweigh.Repeat this5-min drying cycle until two consecutive weights agree within10mg.Calculate the per-centage of water in the original silica gel(A)by the formula(loss in wt/sample wt)×100,1The monoglyceride may be calculated to some monoester other than glyceryl monostearate by dividing the molecular weight of the monoglyc-eride by20and substituting the value so obtained for17.927in the formula,using17.80,for example,in calculating to the monooleate.then calculate the amount of water required to adjust the water content to5%by the formulaW×(5–A)/95,in which W is the weight,in g,of the undried sample to be used. Accurately weigh the appropriate amount of the undried silica gel to be used in the determination,transfer to a suitable blender or mixer,and add the calculated amount of water to give a final water content of5%Ϯ0.1%.Blend for1h to ensure complete water distribution,and store in a sealed container.Determine the water content of the adjusted silica gel as directed above,and readjust if necessary.Note:Each new lot of silica gel should be checked forsuitability by the analysis of a monoglyceride of knowncomposition.Sample Preparation(Caution:To avoid rearrangement of partial glycerides,use extreme caution in applying heat to samples,and do not heat above50°.)Samples Melting Below50°Melt the sample,if necessary, by warming for short periods below50°,not exceeding a total of30min.Samples Melting Above50°Grind about10g in a mortar and pestle,chilling solid samples,if necessary,in carbon dioxide.Weigh accurately about1g of the prepared sample into a 100-mL beaker,add15mL of chloroform,and warm,if necessary,to effect e only minimal heat,and do not heat above40°.Preparation of Chromatographic Column Connect a19-×290-mm chromatographic tube,equipped with an outer19/ 22standard-taper joint at the top and a coarse,fritted-glass disk and inner19/22standard-taper joint at the bottom,with an adapter consisting of an outer19/22joint connected to a Teflon stopcock.Do not grease the joints.Weigh30g of the prepared silica gel into a150-mL beaker,add50to60mL of petroleum ether,and stir slowly with a glass rod until all air bubbles are expelled.Transfer the slurry to the column through a powder funnel,and open the stopcock,allowing the liquid level to drop to about2cm above the silica gel. Transfer any silica gel slurry remaining in the beaker into the column with a minimum amount of petroleum ether,then rinse the funnel and sides of the column.Drain the solvent through the stopcock until the level drops to2cm above the silica gel,and remove the powder funnel.Procedure Carefully add the Sample Preparation to the prepared column.Open the stopcock,and adjust the flow rate to about2mL/min,discarding the eluate.Rinse the sample beaker with5mL of chloroform,and add the rinsing to the column when the level drops to2cm above the silica gel. Never allow the column to become dry on top,and maintain a flow rate of2mL/min throughout the elution.Avoid inter-ruptions during elution as they may cause pressure buildup and result in leakage through the stopcock or cracks in the silica gel packing.Attach a250-mL reservoir separator,provided with a Teflon stopcock and a19/22standard-taper drip tip inner joint,to the column.Add200mL of benzene,elute,and discard the eluate,which contains the triglycerides fraction.When the level of benzene drops to2cm above the silica gel,add200 mL of a1:10mixture of ether in benzene,elute,and discard the eluate,which contains the diglycerides and the free fatty acid fraction.When all of the ether–benzene solvent has been added from the separator and the level in the column drops to2cm above the silica gel,add from250to300mL of ether,and collect the monoglyceride fraction in a tared flask. Rinse the tip of the column into the flask with a few mL of ether,and evaporate to dryness on a steam bath under a stream of nitrogen or dry air.Cool for at least15min,weigh,then reheat on the steam bath for5min in the same manner. Cool,reweigh,and repeat the5-min evaporation,cooling,and reweighing procedures until two consecutive weights agree within2mg.The weight of the residue represents the total monoglycerides in the sample taken. OXYETHYLENE DETERMINATION Apparatus The apparatus for oxyethylene group determina-tion is shown in Fig.35.It consists of a boiling flask,A,fitted with a capillary side tube to provide an inlet for carbon dioxide and connected by a condenser with trap B,which contains an aqueous suspension of red phosphorus.The first absorption tube,C,contains a silver nitrate solution to absorb ethyl iodide. Absorption tube D is fitted with a1.75-mm spiral rod(23 turns,8.5-mm rise per turn),which is required to provide a longer contact of the evolved ethylene with the bromine solu-tion.A standard-taper adapter and stopcock are connected to tube D to permit the transfer of the bromine solution into a titration flask without loss.A final trap,E,containing a potas-sium iodide solution,collects any bromine swept out by the flow of carbon dioxide.Dimensions of the apparatus not readily determined from Fig.35are as follows:carbon dioxide inlet capillary,1-mm id;flask A,28-mm diameter,12/18standard-taper joint;con-denser,9-mm id;inlet to trap B,2-mm id;inlet to trap C,7/ 15standard-taper joint,2-mm id;trap C,14-mm id;trap D, inner tube,8-mm od,2-mm opening at bottom of spiral;outer tube,approximately12.5-mm id;side arm7cm from top of inserted spiral,3.5-mm id,2-mm opening at bottom. ReagentsHydriodic Acid Use special-grade hydriodic acid suitable for alkoxyl determinations,or purify reagent-grade as follows: Distill over red phosphorus in an all-glass apparatus,passing a slow stream of carbon dioxide through the apparatus until the distillation is terminated and the receiving flask has com-pletely cooled.Caution:Use a safety shield,and conduct the distilla-tion in a hood.Silver Nitrate Solution Dissolve15g of silver nitrate in 50mL of water,mix with400mL of alcohol,and add a few drops of nitric acid.。

Antibiotic 107891, its Factors A1 and A2, pharmace

专利名称：Antibiotic 107891, its Factors A1 and A2, pharmaceutically acceptable salts andcompositions, and use thereof发明人：FRANCO PARENTI,ENRICO SELVA,LUCIANO GASTALDO,DANIELE LOSI,GIANPAOLOCANDIANI,FLAVIA MARINELLI,ISMAELACICILIATO,AMERIGA LAZZARINI申请号：AU2005324536申请日：20050119公开号：AU2005324536A1公开日：20060720专利内容由知识产权出版社提供摘要：The invention relates to an antibiotic substance of microbial origin, arbitrarily denominated antibiotic 107891 which is produced by fermentation of Microbispora sp. ATCC PTA-5024, the pharmaceutically acceptable salts and compositions thereof, and their use as an antibacterial agent having inhibitory activity versus susceptible microbes. Antibiotic 107891, which is a complex comprising two Factors, denominated Factors Al and A2, has a peptide structure containing lanthionine and methyllanthionine as constituents which are typical characteristics of the antibiotics of the lantibiotics group. Antibiotic 107891 and its Factors Al and A2 show a good antibacterial activity against Gram-positive bacteria including methicillin resistant and van comycin resistant strains, and is active also against some Gram-negative bacteria such as M. catharralis, Neisseria species and H. influenzae and Mycobacteria.申请人：VICURON PHARMACEUTICALS INC.更多信息请下载全文后查看。

Esters of fatty acids and juvenoid alcohols, and a

专利名称：Esters of fatty acids and juvenoid alcohols, and a method of their preparation and use 发明人：Zden{haeck over (e)}k Wimmer,JelenaKuldová,Ivan Hrdý,Blanka Bennettová申请号：US10182218申请日：20020725公开号：US06734316B2公开日：20040511专利内容由知识产权出版社提供摘要：Esters derived from fatty acids and juvenoid alcohols of the general formula (I), in which X means the oxygen atom or the NH group, Y means the NH group or the oxygen atom, R means methyl, ethyl, 1-propyl, 2-methylethyl or propargyl and R′ means saturated or unsaturated alkyl with 4 to 22 carbon atoms. Esters derived from fatty acids and juvenoid alcohols are produced by a reaction of a juvenoid alcohol of the general formula (II), in which X means the oxygen atom or the NH group, Y means the NH group or the oxygen atom and R means methyl, ethyl, 1-propyl, 2-methylethyl or propargy] with a fatty acid chloride of the general formula (III): R′COC1 in which R′ means saturated or unsaturated alkyl with 4 to 22 carbon atoms, under the continuous stirring, at 0 to 70°C., using a convenient solvent, as toluene, xylene or benzene and under the presence of a base catalyst, as triethylamine, pyridine or quinoline. The compounds claimed in the claim 1 of the general formula (I) are applied for the insect population density control.申请人：INSTITUTE OF ORGANIC CHEMISTRY AND BIOCHEMISTRY ASCR代理机构：Notaro & Michalos P.C.更多信息请下载全文后查看。

美国油脂检测标准AOCS Ca 5a-40

Page 1 of 2SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS APPARATUS 1. Oil sample bottles—115 or 230 mL (4 or 8 oz), or 250 mL Erlenmeyer flasks.REAGENTS 1. Ethyl alcohol, 95%—USSD formulas 30 and 3A are permitted (see Notes, 1). The alcohol must give a definite, distinct and sharp end point with phenolphthalein and must be neutralized with alkali to a faint, but permanent pink color just before using. 2. Phenolphthalein indicator solution—1% in 95% alcohol. 3. Sodium hydroxide solution—accurately standardized. See AOCS Specification H 12-52. See Table 1 for the appropriate normality of the sodium hydroxide solution, depending on the expected free fatty acid concentration range in the sample.PROCEDURE 1. T est samples must be well mixed and entirely liquid before weighing; however, do not heat the sample more than 10°C over the melting point. 2. Use Table 1 to determine the test portion weight for various ranges of fatty acids. Weigh the designated sample size into an oil sample bottle or Erlenmeyer flask (see Notes, 2). 3. Add the specified amount of hot neutralized alcohol and 2 mL of indicator. 4. Titrate with standard sodium hydroxide, shaking vigorously until the appearance of the first permanent pink color of the same intensity as that of the neutralized alcohol before the addition of the sample. The color must persist for 30 seconds.CALCULATIONS 1. The percentage of free fatty acids in most types of fats and oils is calculated as oleic acid, although in coconut and palm kernel oils it is frequently expressed as lauric acid and in palm oil in terms of palmitic acid.(a)(b) Freefatty acids as lauric, % = mL of alkali M 20.0mass,g of test port i on (c) Free fatty acids as palmitic, % = mL of alkali M 25.6mass,g of test port ××i on2. The free fatty acids are frequently expressed in terms of acid value instead of percentage free fatty acids. The acid value is defined as the number of milligrams of KOH necessary to neutralize 1 g of sample. To convert percentage free fatty acids (as oleic) to acid value, multiply the percentage free fatty acids by 1.99.PRECISION Precision data for refined, bleached and deodorized oils are shown in Table 2. Precision data for crude oils are shown in Table 3.NOTES 1. Isopropanol, 99%, may be used as an alternate solvent with crude and refined vegetable oils. 2. Cap bottle and shake vigorously for 1 min if oil has been blanketed with carbon dioxide gas.REFERENCES See J. Assoc. Off. Anal. Chem. 59:658 (1976) regarding the ruggedness of this method.Page 2 of 2SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS Ca 5a-40 • Free Fatty Acids Table 1Free fatty acid range, alcohol volume and strength of alkali a FFA range (%) Test portion (g) Alcohol (mL) Strength of alkali 0.00 to 0.2 56.4 ± 0.2 50 0.1 M 0.2 to 1.0 28.2 ± 0.2 50 0.1 M 1.0 to 30.0 7.05 ± 0.05 75 0.25 M 30.0 to 50.0 7.05 ± 0.05 100 0.25 or 1.0 M 50.0 to 100 3.525 ± 0.001 100 1.0 M a FFA, free fatty acid; N, normality.Table 2The average, expected between-laboratory variation (standard deviation of reproducibility, S R ) for the determination of free fatty acids in refined, bleached and deodorized oils a Approximate FFA value (%) 0–0.05 0.05–0.1 0.1–1.0 1.0–2.0SR 0.007 0.010 0.046 0.073RSD (CV , %) 33.93 12.73 9.90 4.75R (95%) = 2.8 × S R 0.02 0.03 0.13 0.20a Values obtained from the AOCS Laboratory Proficiency Program.Table 3The average, expected between-laboratory variation (standard deviation of reproducibility, S R ) for the determination of free fatty acids in crude oils a Approximate FFA value (%) 0.1–1.0 1.0–2.0SR 0.077 0.156RSD (CV , %) 14.57 9.84R (95%) = 2.8 × S R 0.22 0.44a Values obtained from the AOCS Laboratory Proficiency Program.。

美国油脂检测标准AOCS Ca 5a-40

AOCSCe1k-07

行业文档APPARATUS1.Gas chromatographic system—see AOCS method Ce1h-05.2.Flasks—50-mL round flat-bottom with 24/40 joints orequivalent.3.Teflon flask-neck sleeves (size 24/40).4.Stopper—Hollow, full-length (size 24/40), barrel-shaped head.5.Hot plates—Explosion proof or equivalent (Notes, 1).6.Condensers—Water-cooled or equivalent (Notes, 1).7.Autosampler vials—2-mL amber write-on.8.Autosampler vial Teflon-lined caps.9.V olumetric flask—500-mL and 1-L, Class A.10.V olumetric pipettes—0.5, 1, 2, or 5-mL, Class A.11.Crimper12.Spatulas13.V ortex mixer14.Transfer pipettes15.Pipetman—5 mL.16.Pipette tips—5 mL.17.Funnel—glass powder.18.50-mL Beaker.19.Analytical balance—capable of reading to ±0.0001 mg20.Weighing funnels 21.Flask—1-L transparent polymethylpentene screw-top.22.Nalgene bottle—500-mL.23.Porous boiling chips24.Stir plate—magnetic.25.Stir bars—magnetic.b timer27.Disposable glovesREAGENTS1.Sodium hydroxide—0.5N, Analytical reagent grade.Add 20 g of NaOH to a 1-L polymethylpentene flask.Add methanol and mix well to dissolve before diluting to volume. Refrigerate when not in use.2.Methanol—high purity gas chromatographic grade.3.Sodium chloride—analytical reagent grade, saturated.Dissolve 36 g NaCl in 100 mL distilled water.4.n-Heptanes or n-Heptane—high purity gas chromato-graphic grade.5.Chloroform—high purity gas chromatographic grade.6.Boron trifluoride—14%, in Methanol (Notes, 2).7.Sodium sulfate—anhydrous, reagent grade.8.Pyrogallol—ACS reagent.9.Internal standard—21:0, Triheneicosanoin (P/N T-175,Nu-Chek-Prep, Inc. or equivalent) for all samples otherPage 1of 4SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILSCe 1k-07 • Direct Methylation of Lipids for the Determination of Total FatPage 2of 4SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS Ce 1k-07 • Direct Methylation of Lipids for the Determination of Total FatPage 3of 42.BF 3/MeOH should be refrigerated when not in use.3.If the sample has been dried, calculate the fat content on a dry basis and use the appropriate sample amount to achieve a fat concentration of between 15–20 mg oil per mL of n -hexanes or n -heptane.REFERENCES1.IUPAC, Standard Methods for Analysis of Oils, Fats and Derivatives , Blackwell Scientific Publications IUPAC Method2.301.2.AOCS Official Method Ce 1h-05, Determination of cis -, trans -, Saturated, Monounsaturated and Polyunsat-urated Fatty Acids in Vegetable or Non-Ruminant Animal Oils and Fats by Capillary GLC.3.Barnes, Jr., P.C. and C.E. Holaday, Rapid Preparation of Fatty Acid Esters Directly from Ground Peanuts, J. of Chromat. Sci. 10, 181–183, 1972.4.Hougen, F.W. and V. Bodo, Extraction and Methanolysis of Oil from Whole or Crushed Rapeseed for Fatty Acid Analysis, JAOCS 50 230–234, 1973.5.Dahmer, M.L., P.D. Fleming, G.B. Collins, and D.F.Hildebrand, A Rapid Screening Technique for Determining the Lipid Composition of Soybean Seeds,JAOCS 66(4), 543–548, 1989.6.Ulberth, F. and M. Henninger, One-Step Extraction/Methylation for Determining the Fatty Acid Composition of Processed Foods, JAOCS, 69(2), 174–177, 1992.7.Rule, D.C., Direct Transesterification of Total Fatty Acids of Adipose Tissue, and of Freeze-dried Muscle and Liver with Boron Trifluoride in Methanol, Meat Science , V ol. 46, No. I, 23–32, 1997.Page 4of 4SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILSCe 1k-07 • Direct Methylation of Lipids for the Determination of Total Fat。

AOCS Tg 1a-64-2009

INDUSTRIAL OILS AND DERIVATIVESAPPARATUS1. Glass-stoppered bottles or iodine flasks or wide mouth Erlenmeyer flask—500 mL.2. Glass-stoppered volumetric flask—conforming to National Institute of Standards & Technology (NIST) tolerances andaccurately calibrated to contain 1000 mL.3. Pipets—5, 20, and 25 mL.4. Filter paper—Whatman no . 41H, or equivalent.REAGENTS (See Numbered Notes)1. Glacial acetic—ACS grade. The permanganate test should be applied to be sure that this specification is met.(a) Permanganate test—dilute 2 mL of the acid with 10 mL of distilled water and add 0.1 mL of 0.2 M N KMnO4. Thepink color must not be entirely discharged within 2 hr.2. Potassium iodide—ACS or AR grade.3. Chlorine—99.8% (see Notes, Caution). Satisfactory commercial grades are available in cylinders, but gas must not be driedby passing through sulfuric acid (sp. gr. 1.84) before introducing into the iodine solution.Note—Prepared Wijs (see Notes, Caution and Numbered Notes, 2) solution is available commercially and may be used in place of laboratory-prepared solution.4. Soluble starch—tested for sensitivity. Make a paste with 1 g of starch and a small amount of cold distilled water. Add, whilestirring, 200 mL of boiling water. Place 5 mL of this solution in 100 mL of water and add 0.05 mL of 0.1 M iodine solution.The deep blue color produced must be discharged by 0.05 mL of 0.1 M sodium thiosulfate.5. Potassium dichromate—ACS grade. The potassium dichromate should be finely ground and dried to constant weight atabout 110°C before using.Note—A standard sample of potassium dichromate with a certificate of analysis may be obtained from the NIST in Washington, D.C. This sample, or equivalent, is strongly recommended for the primary standard for this method. T reat as directed in the certificate of analysis accompanying the sample.6. Sodium thiosulfate (Na2S2O3 · 5H2O)—ACS grade.7. Iodine—ACS grade.8. Cyclohexane-acetic acid solution—1:1, v/v. Make fresh daily (see Recommendations).9. Wijs solution, AR grade (see Notes, Caution).SOLUTIONS1. Potassium iodine solution—dissolve 150 g in distilled water and make up to 1 L.2 Starch indicator solution—make a homogeneous paste of 10 g of potato starch in cold distilled water. Add to this 1 L ofboiling distilled water, stir rapidly and cool. Salicylic acid (1.25 g/L) may be added to preserve the indicator. If long storage is required, the solution must be kept in a refrigerator at 4–10°C (40–50°F). Fresh indicator must be prepared when the end point of the titration from blue to colorless fails to be sharp. If stored under refrigeration, the starch solution should be stable for about 2–3 wk.3. Sodiu.m thiosulfate solution, 0.1 M—dissolve 24.9 g of sodium thiosulfate in distilled water and dilute to 1 L.(a) Standardization—weigh 0.16–0.22 g of finely ground and dried potassium dichromate into a 500-mL flask or bottleby difference from a weighing bottle. Dissolve in 25 mL of water, add 5 mL of hydrochloric acid, 20 mL of potassium iodine solution and rotate to mix. Allow to stand for 5 min and then add 100 mL of distilled water. Titrate with sodium thiosulfate solution, shaking continuously until yellow color has almost disappeared. Add 1–2 mL of starch indicator and continue the titration, adding the thiosulfate solution slowly until the blue color has just disappeared. The strength of the sodium thiosulfate solution is expressed in terms of its molarity.Molarity of Na2S2O3 solution =×20.394mass of K Cr O,g volume,mL of227s odium thiosulfate solutionPage 1 of 4Page 2 of 4INDUSTRIAL OILS AND DERIVATIVES Tg 1a-64 • Iodine Value of Fatty Acids, Wijs Method4. Wijs solution (see Notes, Caution ) can be purchased (see Reagents, 3, 9) or prepared in the laboratory. Dissolve 13.0 g of iodine in 1 L glacial acetic acid. Gentle heating may be necessary to promote solution. Cool, remove a small quantity (100–200 mL) and set aside in a cool place for future use. Pass dry chlorine gas into the iodine solution until the original titration is not quite double. A characteristic color change takes place in the Wijs solution when the desired amount of chlorine has been added. This may be used to assist in judging the end point. A convenient procedure is to add a small excess of chlorine and immediately bring back to the desired titration by addition of some of the original iodine solution which was taken out at the beginning. The original solution and finished Wijs solution are both titrated with Na 2S 2O 3 solution as directed in Procedure, 6 and 7. The Wijs solution may be prepared from commercial iodine monochloride as follows: (a) Stock solution—Add 317 ± 0.1 g of iodine monochloride to 1 L of glacial acetic acid and filter through Whatman no. 41H filter paper or equivalent into a clean and dry actinic glass bottle. Filter rapidly to prevent contamination with moisture and store in a cool place. Discard the solution if a precipitate forms on standing. (b) Wijs solution—Using a graduated cylinder pour 117.0 ± 0.1 mL of the stock solution into a standard 5 lb bottle of glacial acetic acid and mix well by shaking.5. The I/Cl ratio of the Wijs solution shall be within the limits of 1.10 ± 0.1. The procedure for determining the ratio is as follows: (a) Iodine content— (1) Pour 150 mL of saturated chlorine water into a 500 mL iodine flask and add a few glass beads. (2) Pipet 5 mL of the Wijs solution into the flask containing the saturated chlorine water. Shake, and heat to boiling. (3) Boil briskly for 10 min, cool and add 30 mL of 2% sulfuric acid and 15 mL of 15% potassium iodide solution. (4) Mix well, and titrate immediately with 0.1 M sodium thiosulfate solution to a starch end point. (b) T otal halogen content— (1) Pour 150 mL of recently boiled distilled water into a clean, dry 500-mL iodine flask. (2) Add 15 mL of 15% potassium iodine solution. (3) Pipet 20 mL of Wijs solution into the flask, and mix well. (4) Titrate with 0.1 M sodium thiosulfate solution to a starch end point.PROCEDURE 1. Melt the test sample, if it is not already liquid, and filter through filter paper to remove any solid impurities and the last traces of moisture. The temperature during melting and filtering should not exceed the melting point of the test sample by more than 10°C. The test sample must be absolutely dry. Note —All glassware must be absolutely clean and completely dry. 2. Weigh the test sample (see Notes, 3) accurately into a 500 mL flask or bottle to which has been added 15 mL of cyclohexane:acetic acid, 1:1, v/v. The weight of the test sample must be such that there will be an excess of Wijs solution of 100–150% over the amount absorbed. Table 1 is a guide to the size of the test sample to weight. 3. Pipet the 25 mL of Wijs solution into the flask containing the test sample, stopper the flask and swirl to ensure an intimate mixture. 4. Prepare and conduct one blank determination with each group of test samples simultaneously and similar in all respects to the test samples. 5. Store the flasks in a dark place for 30 min at a temperature of 25 ± 5°C (see Notes, 4). 6. Remove the flasks from storage and add 20 mL of KI solution, followed by 100 mL of distilled water added to the lip of the iodine flasks to absorb any iodine that may leak past the stoppers while they are waiting to be titrated. 7. Titrate with 0.1 M Na 2S 2O 3 solution, adding it gradually and with constant and vigorous shaking (see Notes, 5). Continue the titration until the yellow color has almost disappeared. Add 1–2 mL of starch indicator solution and continue the titra-tion until the blue color has just disappeared.CALCULATIONIodine value =−××(B S)M 12.691mass of test portions s ,gWhere— B = volume of titrant, mL of blankS = volume of titrant, mL of sampleM = molarity of Na 2S 2O 3 solutionINDUSTRIAL OILS AND DERIVATIVESTg 1a-64 • Iodine Value of Fatty Acids, Wijs MethodPRECISIONAt a confidence level of 95%:Fatty acidsSoybean TallowMaterials: Linseed CoconutRange of values: 130–200 7–551. T wo single determinationsperformed in one laboratoryshould not differ from the meanvalue by more than: ± 1.8% ± 4.6%2. Single determinations performedin different laboratories shall notdiffer from the mean valueby more than: ± 2.1% ± 5.2%NOTESCautionChlorine is a poisonous gas. The TLV is 1 ppm in air. It is a strong oxidizing agent and should not be allowed to come in contact with organic materials, hydrogen, powdered metals and reducing agents. A fume hood should be used at all times when using chlorine.Wijs solution causes severe burns and vapors can cause lung and eye damage. Use of a fume hood is recommended. Wijs solution without carbon tetrachloride is available commercially.RECOMMENDATIONSCyclohexane has been proposed as a replacement for carbon tetrachloride in the iodine value method—see AOCS Recommended Practice Cd 1b-87 and JAOCS 65:745 (1988). Cyclohexane-acetic acid, 1:1, v/v has been shown to be a suitable replacement for carbon tetrachloride—see AOCS Official Method Cd 1d-92.NUMBERED NOTES1. When the iodine value is determined on materials having conjugated systems, the result is not a measure of total unsatura-tion, but rather is an empirical value indicative of the amount of unsaturation present. Reproducible results are obtained which afford a comparison of total unsaturation. When the iodine value is required on the fatty acids of natural and synthetic drying oils, the preparation and separation are performed as directed in AOCS Official Method Cd 6-38.2. All Wijs solutions are sensitive to temperature, moisture and light. Store in a cool and dark place and never allow to come toa temperature above 25–30°C.3. In the case of dehydrated castor oil fatty acids, weigh 0.11–0.13 g.4. Store test portions with an iodine value greater than 150 for 1 hr in the dark at 25 ± 5°C.5. Mechanical stirring is satisfactory for agitating during the addition of thiosulfate.6. 1,1,1-T richloroethane (TCE) has been suggested as a replacement for chloroform in this method. Although TCE is a halo-genated hydrocarbon, the TLV (350 ppm) is less than those of chloroform, carbon tetrachloride, and methylene chloride.Cyclohexane and isooctane may also be considered as alternate solvents. These solvents have not been collaboratively stud-ied by the AOCS technical committees. This recommendation does not represent official approval by the AOCS Uniform Methods Committee.Page 3 of 4INDUSTRIAL OILS AND DERIVATIVESTg 1a-64 • Iodine Value of Fatty Acids, Wijs MethodTABLE 1Test portion mass, gNormal oils Conjugated oilsIodine 100–150% excess 115–135% excessV alue of reagent of reagent< 3 10 –103 8.4613–10.5765 5.0770–6.346010 2.5384–3.173020 0.8461–1.586540 0.6346–0.793560 0.4321–0.528880 0.3175–0.3969 0.3377–0.369190 0.2822–0.3528 0.3002–0.3281100 0.2540–0.3175 0.2702–0.2953110 0.2309–0.2886 0.2456–0.2684120 0.2117–0.2646 0.2252–0.2461130 0.1954–0.2442 0.2078–0.2271140 0.1814–0.2268 0.1930–0.2109150 0.1693–0.2116 0.1801–0.1969160 0.1587–0.1984 0.1689–0.1846170 0.1494–0.1868 0.1589–0.1737180 0.1411–0.1764 0.1501–0.1640190 0.1337–0.1671 0.1422–0.1554200 0.1270–0.1587 0.1351–0.1476210 0.1210–0.1547 0.1287–0.1406220 0.1155–0.1443 0.1228–0.1374Page 4 of 4。

反式脂肪酸的危害及其检测方法_赖晓英

反式脂肪酸的危害及其检测方法赖晓英，武德银，伍飏，褚小菊，代汉慧，唐英章(中国检验检疫科学研究院食品安全研究所，北京100025)摘要：随着美国肯德基停止使用反式脂肪酸决定的公布，反式脂肪酸又回到了人们的视野。

本文对反式脂肪酸问题的始末，反式脂肪酸的性质、危害、来源，各国采取的控制措施、检测方法等多方面情况进行了介绍。

关键词：反式脂肪酸；危害；控制措施；检测方法中图分类号：TS201.2+2；文献标识码：A；文章篇号: 1673-9078(2007)02-0073-04Hazards and Determination of Trans Fatty AcidsLAI Xiao-ying, WU De-yin, WU Y ang, CHU Xiao-ju, DAI Han-hui, TANG Ying-zhang(Chinese Academy of Inspection and Quarantine, Beijing 100025) Abstract: Much attention has been paid to the Trans fatty acids (FTA) in recent years, especially after the announcement by KFC that they will stop to use FTA in its products in the USA. In this paper, its characteristic, hazards, origin, determination methods, control strategies, etc., were introduced.Key words: trans fatty acids (TFA); hazards; control measure; testing methods2006年10月30日，美国5500家肯德基加盟店作出“重大决定”，将在美国所有肯德基连锁店停止使用人造反式脂肪酸。