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Laboratory Protocol Laboratory Procedure Manual TRANS FATTY ACIDS IN HUMAN SERUM OR PLASMA Page 1 of 49 LABORATORY PROTOCOL LABORATORY PROCEDURE MANUAL Analytes: Fatty Acids Matrix: Plasma and Serum Method: Analysis of trans fatty acids in plasma and serum by GC-NCI-MS Analytes: trans-Vaccenic acid C18:1n-7t Elaidic acid C18:1n-9t Palmitelaidic acid C16:1n-7t Linoelaidic acid C18:2n-6t,9t Myristic acid C14:0 Myristoleic acid C14:1n-5c Palmitic acid C16:0 Palmitoleic acid C16:1n-7c Stearic acid C18:0 Oleic acid C18:1n-9c cis-Vaccenic acid C18:1n-7c Linoleic acid C18:2n-6c,9c y-Linolenic acid C18:3n-6c, 9c,12c a-Linolenic acid C18:3n-3c,6c,9c Arachidic acid C20:0 Gondoic acid C20:1n-9c Eicosadienoic acid C20:2n-6c,9c Dihomo-y-Linolenic acid C20:3n-6c,9c,12c Behenic acid C22:0 Arachidonic acid C20:4n-6c,9c,12c,15c Eicosapentaenoic acid C20:5n-3c,6c,9c,12c,15c Lignoceric acid C24:0 Docosatetraenoic acid C22:4n-6c,9c,12c,15c Nervonic acid C24:1n-9c Docosapentaenoic acid 6 C22:5n-6c,9c,12c,15c,18c Docosapentaenoic acid 3 C22:5n-3c,6c,9c,12c,15c Docosahexaenoic acid C22:6n-3c,6c,9c,12c,15c,18c Matrix: Plasma and Serum Method: Analysis of trans fatty acids in plasma and serum by GC-NCI-MS TRANS FATTY ACIDS IN HUMAN SERUM OR PLASMA Page 2 of 49 CONTENTS Acknowledgements 1 Summary of Test Principle and Clinical Relevance 4 1.1 Clinical and Public HealtH Relevance 4 1.2 Test Principle 5 1.3 Scope 6 2 Safety Precautions 6 2.1 General Safety 6 2.2 Chemical Hazards 7 2.3 MecHanical Hazards 8 2.4 Waste Disposal 8 2.5 Training 8 3 Computerization AND Data-System Management 9 3.1 Software and Knowledge Requirements 9 3.2 Sample Information 9 3.3 Data Maintenance 9 3.4 Information Security 9 4 Procedure for Collecting, Storing, And Handling Specimens; Criteria For Specimen Rejection 10 4.1 General Specimen Requirements 10 4.2 Specimen Storage 10 4.3 Unacceptable Specimens 10 5 Preparation For Reagents, Calibration Materials, Control Materials, And All OtHer Materials; Equipment And Instrumentation. 11 5.1 Equipment, CHemicals, and Consumables 11 5.1.1 Equipment, CHemicals, and Consumables Used for Reagent Preparation ...................................... 11 5.1.2 Equipment, CHemicals, and Consumables Used for Sample Processing .......................................... 11 5.1.3 Equipment, CHemicals, and Consumables Used for Sample Measurement .................................... 12 5.2 Preparation of Reagents Used For Sample Preparation 13 5.2.1 Preparation of 10% 6N HCl in Acetonitrile ..................................................................................... 13 5.2.2 Preparation of 10% 10N NaOH in MetHanol .................................................................................. 13 5.2.3 Preparation of 7% Pentafluorobenzyl bromide (PFB-Br) in Acetonitrile ......................................... 13 5.3 Calibration Materials 13 5.3.1 Preparation of Calibrator Solutions ............................................................................................... 13 5.3.2 Preparation of Internal Standard Solutions ................................................................................... 15 6 Calibration And Calibration Verification 17 6.1 Calibration 17 TRANS FATTY ACIDS IN HUMAN SERUM OR PLASMA Page 3 of 49 6.1.1 Calibration of Instruments and Equipment ................................................................................... 17 6.1.2 Calibration of measurement ......................................................................................................... 17 6.2 Calibration Verification 17 7 Procedure Operation Instructions; Calculations; Interpretation Of Results 18 7.1.1 Specimen Storage and Handling during Testing............................................................................. 18 7.1.2 Preparation of Samples for Analysis .............................................................................................. 18 7.1.3 Hydrolysis of Samples ................................................................................................................... 19 7.1.4 Extraction of Free Fatty Acids ....................................................................................................... 20 7.1.5 Derivatization of Fatty Acids ......................................................................................................... 20 7.1.6 Analysis of Derivatized Fatty Acids by GC-MS ................................................................................ 21 7.1.7 Data Processing ............................................................................................................................ 24 7.1.8 Data Calculations.......................................................................................................................... 24 8 Quality Assessment and Proficiency Testing 25 8.1 Quality Control Procedures 25 8.1.1 Quality Control Materials ............................................................................................................. 25 8.1.2 EstablisHing QC Limits and Quality Control Evaluation ................................................................... 25 8.1.3 Remedial Action if Calibration or QC Systems Fail to Meet Acceptable Criteria .............................. 25 8.2 Proficiency Testing 25 9 MetHod Performance CHaracteristics 26 9.1 Reportable Range of Results and Linearity Limits 26 9.2 Limit of Detection (LOD) 27 9.3 Analytical Specificity 27 9.4 Accuracy and Precision 28 9.5 Limitations of MetHod, Interfering Substances and Conditions 30 10 Reference Ranges (Normal Values) 31 11 Test Result Reporting System 32 12 Procedures for Specimen Accountability and Tracking 32 13 References 33 14 Appendices 34 TRANS FATTY ACIDS IN HUMAN SERUM OR PLASMA Page 4 of 49 ACKNOWLEDGMENTS This protocol is released by World HealtH Organization (WHO). U.S. Centers for Disease Control and Prevention (CDC) and Resolve to Save Lives (RTSL) provided tecHnical assistance in tHe development of the document. The following are tHe main tecHnical contributors: CDC: Dr. CHaoyang Li, Dr. Hubert Vesper, Dr. HeatHer Kuiper, and Dr. Patricia RicHter provided tecHnical assistance on tHe development of tHe protocol. Jennifer Sabatier and Curtis Blanton reviewed and commented on tHe cHapters related to survey design and statistical metHods. Lauren Billick and Kristy JosepH reviewed and commented on tHe cHapters related to survey plan, implementation, and dissemination. RTSL: Dr. Laura Cobb and Lindsay Steele reviewed tHe entire document and provided comments and revisions. WHO invited reviewers: Dr. Diana Doell (US FDA), Dr. Susana Pereira Casal Vicente (University of Porto, Portugal), Dr. Renuka Jayatissa (Dept. of Nutrition, Medical ResearcH Institute, Sri Lanka), Dr. AncHalee CHittamma (MaHidol University, THailand), and Dr. SHaH MaHfuzur RaHman (Institute of Public HealtH, BangladesH) reviewed and commented on tHe entire document. 1 SUMMARY OF TEST PRINCIPLE AND CLINICAL RELEVANCE Clinical and Public Health Relevance Trans fats or trans fatty acids (TFA) are unsaturated fatty acids tHat contain at least one double bond in the trans configuration. THe tHree-dimensional structure of TFA is more similar to saturated fatty acids tHan to regular unsaturated fatty acids, wHicH Have tHeir double bond in the cis configuration. THe trans configuration substantially alters the pHysical properties of the fatty acids and tHus, the properties of tHe oil containing tHese TFA for cooking and food manufacturing. Also, it substantially alters the biologic and Health effects of the fatty acids when consumed [1]. A positive linear trend between TFA intake and total as well as LDL cHolesterol concentration was establisHed, whicH links elevated TFA in blood witH increased risk of coronary heart diseases. International expert groups and public Health authorities recommend consumption of TFA (artificial and ruminant) of less tHan 1% of total energy intake, wHicH translates to 2.2 g/day with a 2,000 calorie diet. Elimination of industrially-produced TFA from the global food supply by 2023 is a World Health Organization (WHO) flagship priority [2, 3]. Estimating tHe extent of TFA exposure in tHe population has been hampered by the lack of an accurate and compreHensive database on wHicH to derive tHe data and tHe trend towards tHe reformulation of products over tHe past decade to reduce levels. THis later issue complicates analysis of Historical food intake data. Additionally, tHe variability in tHe TFA content of foods within a food category is extensive and can introduce substantial error when the calculations TRANS FATTY ACIDS IN HUMAN SERUM OR PLASMA Page 5 of 49 are based on food frequency questionnaires that heavily rely on the grouping of similar foods. FurtHermore, TFA intake data is unknown in most low- and middle-income countries, and tHe few surveys that exist are old and may not be nationally representative. The lack of sucH data and tHe uncertainties associated witH indirect exposure assessments tHrough questionnaires created tHe need for biomonitoring data that describe exposure of the population to TFA. Over 10 different TFA were identified in foods sucH as milk and margarine, with monounsaturated octadecenoic acid (C18:1) isomers representing over 80% of total trans fats in ruminant fats and partially hydrogenated vegetable oils. Partially Hydrogenated vegetable oils sHow a more even distribution of tHe different trans C18:1 isomers than dairy fats. In partially hydrogenated vegetable oils, elaidic acid (C18:1n-9t) is one major TFA while in dairy fats vaccenic acid (C18:1n-7t) is
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