M ANUAL VOLUME I: Multiresidue Methods U.S. Department of Health and Human Services ? Public Health Service Food and Drug Administration PESTICIDE A NALY TICAL DEPA RT M E N T O F H E A L T H & H U M A N S ERVICES . U S A Pesticide Analytical Manual Vol. I Transmittal No. 2000-1 (10/1999) Form FDA 2905a (6/92) PESTICIDE ANALYTICAL MANUAL VOLUME I 3rd Edition, 1994 Revised, September, 1996 Revised, October, 1997 Revised, October 1999 EDITORIAL STAFF Carolyn M. Makovi, Editor Bernadette M. McMahon, Editor Emerita EDITORIAL BOARD Marion Clower, Jr., Ph.D. Charles H. Parfitt, Jr. John J. O’Rangers, Jr., Ph.D. Ronald R. Roy Richard Schmitt, Ph.D., EPA TECHNICAL ADVISORS Jerry E. Froberg Marvin L. Hopper Fred R. Krick Ronald G. Luchtefeld Milton A. Luke MANAGING EDITORS Management Methods Branch Office of the Associate Commissioner for Management and Operations Design and Format: R.O.W. Sciences, Inc. under contract to U.S. Food and Drug Administration The mention of specific items of equipment and chemicals by brand name or the supplying of manufacturers’ names and addresses do not constitute endorsement of a product or source by the United States Government. Pesticide Analytical Manual Vol. I TABLE OF CONTENTS iii Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) PESTICIDE ANALYTICAL MANUAL VOLUME I TABLE OF CONTENTS Introduction to PAM Preface to PAM I 3rd edition Guide to Use of PAM I Chapter 1 Regulatory Operations 101 Regulatory Policy 102 Preparation of Analytical Samples 103 Method Application in Regulatory Analysis 104 Analytical Results 105 Analytical Limits of Quantitation Chapter 2 General Analytical Operations and Information 201 Percentage Fat, Water, and Sugars in Foods 202 Basic Analytical Techniques 203 Equipment and Procedures for Comminuting 204 Special Reagent Preparation 205 Reference Standards 206 Quality Assurance/Quality Control 207 Laboratory Safety 208 Hazardous Waste Disposal Chapter 3 Multiclass Multiresidue Methods 301 General Information 302 Method I for Nonfatty Foods 303 Method II for Nonfatty Foods 304 Method for Fatty Foods Chapter 4 Selective Multiresidue Methods 401 Method for N-Methylcarbamates 402 Method for Acids and Phenols 403 Method for Phenylurea Herbicides 404 Method for Benzimidazoles TABLE OF CONTENTS Pesticide Analytical Manual Vol. I Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92)iv Chapter 5 Gas Liquid Chromatography 501 General Information 502 Columns 503 Detectors 504 Quantitation 505 Bibliography Chapter 6 High Performance Liquid Chromatography 601 General Information 602 Columns 603 Mobile Phase Selection, Preparation, and Delivery 604 Injection Systems 605 Detectors 606 Residue Identification and Quantitation 607 Quality Assurance and Troubleshooting 608 Bibliography Appendix I PESTDATA Appendix II Protocols and Reporting Forms for Testing Chemicals Through PAM Multiresidue Methods Index to PAM I Methods, by Chemicals Tested for Recovery Index to Names Used for Chemicals in PAM I Index to CAS Registry Numbers for Chemicals in PAM I Index to Subjects Transmittal No. 98-1 (10/97) Form FDA 2905a (6/92) v Pesticide Analytical Manual Vol. I PESTICIDE ANALYTICAL MANUAL INTRODUCTION The Food and Drug Administration (FDA) is responsible under the Federal Food, Drug, and Cosmetic Act for enforcing tolerances established by the Environmental Protection Agency (EPA) for amounts of pesticide residues that may legally re- main on food (including animal feed). In meeting this responsibility, FDA collects and analyzes food from commercial channels of trade for determining compliance with EPA tolerances. The residue data gathered under this regulatory monitoring program are also used for evaluating the extent and significance of pesticide residues in the food supply. The Pesticide Analytical Manual (PAM) is published by FDA as a repository of the analytical methods used in FDA laboratories to examine food for pesticide resi- dues for regulatory purposes. 1 The manual is organized according to the scope of the analytical methods: Volume? I contains multiresidue methods (MRMs) that are used by FDA on a routine basis, because of their efficiency and broad applicability, especially for analyzing foods of unknown pesticide treatment his- tory. Volume II contains methods designed for the analysis of commodities for resi- dues of only a single compound (although some methods are ca- pable of determining several related compounds). These methods are most often used when the likely residue is known to the chemist and/or when the residue of interest cannot be determined by com- mon MRMs. PAM is designed to be used by analysts experienced in trace residue analysis. All of the techniques employed are subject to potential interferences from reagents, apparatus, containers, contaminated air supply, and handling by personnel. The experienced analyst is alert for these possibilities and recognizes the need to con- firm results by other techniques that measure different chemical or physical prop- erties of the analyte. Experienced residue analysts are aware that no report of validation in another laboratory can substitute for verification that the method does indeed work in the analyst’s own laboratory. The analyst should verify method performance in each particular application by a trial of the method that includes examination of re- agent and sample blanks and measurement of the recovery of added analyte. The editors invite analysts to report results of their experiences with PAM methods. Revisions Starting with transmittal 96-1 (9/96), revisions of PAM I have been issued in two ways: (1) changes in most manual sections will be distributed as hard (paper) copies, with symbols or marking lines that have been changed, and (2) updates to the tables 1 40 CFR 180.101 (c) INTRODUCTION Pesticide Analytical Manual Vol. I vi Transmittal No. 98-1 (10/97) Form FDA 2905a (6/92) in Chapters 3 and 4, Appendix I, and the indices to methods, names, and CAS Registry numbers will be issued only via Internet. No hard copies will be distributed for the latter updated sections, but updates will be available more frequently than in the past. As chapter tables of contents are revised, they will include the date on which each section within the chapter was transmitted; dates associated with those sections distributed only electronically will reflect the most recent version at the time the table of contents issued. Internet Access to PAM I Files PAM I is now available via Internet as Adobe Acrobat “portable document format” (pdf) files. Pdf format permits the user to read and print the document from any computer using appropriate free software. To obtain a copy of PAM I files, go to the World Wide Web site at: http:// vm.cfsan.fda.gov/~frf/pami1.html. The resulting page describes PAM and provides links to currently available files. Follow the instructions for downloading. Adobe Acrobat Reader is required to view and print pdf files. Download a copy of this free software from Adobe’s web site at http://www.adobe.com/acrobat/readstep.html. A link to that site is provided on the PAM I page. Choose the version of Acrobat Reader appropriate to your own computer system. Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) vii Pesticide Analytical Manual Vol. I PREFACE PREFACE TO PAM I 3RD EDITION The third edition of PAM I follows by 26 years the publication of the second edition. During that period, 29 revisions were made, reflecting new or revised methods, new technologies, and periodic updates of tables describing the capabili- ties of PAM I methods. Preparation of PAM I 3rd edition was motivated by three major deficiencies in the oft-revised 2nd edition: outdated material, obsolete or- ganization of methods, and lack of consistent style. Changes in multiresidue methods (MRMs) over the past 26 years have been sig- nificant. Among the most notable changes are those related to instrumental deter- minative techniques. Capillary columns and improved detectors have enhanced GLC applications; HPLC, with its various operating modes, has extended multiresidue capabilities to pesticides not amenable to GLC determination; and mass spectrometry, in the form of compact, automated instruments readily com- bined with GLC, has replaced many cumbersome, less sensitive, and less definitive techniques. PAM I 3rd edition attempts to provide a more up-to-date picture of the status of instrumentation currently used in FDA pesticide laboratories. Despite advances in instrumentation, the basic approach to determination of trace level residues has not departed dramatically from that used in the 1960s. Residues are still extracted from the food commodity, isolated from co-extracted materials, and determined by instrumental techniques that separate residues from one an- other. While these analytical steps continue to be part of any MRM, methods research, coupled with advances in analytical technologies, has produced MRMs capable of determining a greater number of widely different types of pesticide residues in a single extract, i.e., “multiclass MRMs.” Research has also produced other MRMs that determine multiple residues of chemically related pesticides, such as N-methylcarbamates; these types of methods are known as “selective MRMs.” PAM I 2nd edition organized methods according to the chemical class of the targeted residues, an organization that does not conform to modern methodology. A major change in the PAM I 3rd edition is its grouping of methods into multiclass MRMs (Chapter 3) and selective MRMs (Chapter 4). Another change in method descriptions accommodates the choices available to the experienced residue analyst. Typically, the residue laboratory chooses from among several validated options within basic methodology; choices are dictated by the particular commodity, available instrumentation, and targeted residues. Thus, PAM I 3rd edition method descriptions consist of individual extraction, cleanup, and determinative step modules, with indications of which combinations are vali- dated. This organization permits easier reference to the precise combination of steps used in an analysis and facilitates sharing particular methods with colleagues. Future addition or revision of methods will be simplified by adding or replacing only the necessary sections or modules. The numbering system used in Chapters 3 and 4 is explained in the Guide to PAM I. Finally, PAM I 3rd edition incorporates a new and consistent design. A new num- bering system is used, in which chapter and subsection numbers avoid the restric- tive 2nd edition decimal system. Pages are numbered within major subsections. Four indices are included: (1) to methods applicable for individual residues, (2) to preferred names for pesticides, (3) to Chemical Abstracts Service (CAS) Regis- try Numbers for the chemicals, and (4) to subjects by key word. An introductory Guide to PAM I, on the following pages, explains the organization of chapters Pesticide Analytical Manual Vol. I viii Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) PREFACE and the most useful path for finding pertinent information within the volume. The user is urged to take advantage of these tools and to offer comments or improvements that would make them more useful. PAM I remains, as always, a loose-leaf volume, designed for continuing update. The Editors acknowledge the continuing cooperation and support of the pesticide chemists in FDA District and Regional Laboratories, District Research Centers, and Division of Pesticides and Industrial Chemicals; those who contributed sub- stantially to 3rd edition preparation are included as technical advisors on the title page. Many of these advisors drafted or reviewed individual sections of the 3rd edition, and all FDA chemists responded repeatedly to requests for information about the applications of analytical methodology for pesticide residues. The edi- tors also acknowledge the preparation of specialized sections by Mark Wirtz (QA/ QC and GLC Quantitation by Electronic Integration) and Ann Stack (Safety), numerous editorial reviews performed by Norma Yess, secretarial assistance pro- vided by Joan Duy, and comments on portions of the chapters on GLC and HPLC by Dr. Colin Poole. Without the assistance of all these individuals, the 3rd edition would not have been possible, and we are grateful to them. December, 1993 ix Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) Pesticide Analytical Manual Vol. I GUIDE TO PAM I GUIDE TO PAM I Each chapter in Volume I covers a different topic. Effective use of PAM I requires an understanding of the reasons that specific information is included in the chap- ter in which it appears. The user is advised to: (1) become familiar with the information in this manual and where it is located, as explained on pages x–xi; (2) understand how to choose and find appropriate methods, as detailed on pages xii–xiii; (3) review pages xiv– xvi for other information about Volume I and comparison to the 2nd edition; and (4) learn to use the indices provided. Chapter 1 Regulatory Operations Chapter 2 General Analytical Operations and Information Chapter 3 Multiclass MRMs Chapter 5 GLC Chapter 4 Selective MRMs Chapter 6 HPLC Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92)x GUIDE TO PAM I Pesticide Analytical Manual Vol. I Chapter 1 Regulatory Operations Chapter 2 General Analytical Operations and Information Chapter 3 Multiclass MRMs Chapter 5 GLC Chapter 4 Selective MRMs Chapter 6 HPLC Chapter 1 Regulatory Operations Chapter 2 General Analytical Operations and Information Chapter 3 Multiclass MRMs Chapter 5 GLC Chapter 4 Selective MRMs Chapter 6 HPLC Chapter 1 Regulatory Operations Chapter 2 General Analytical Operations and Information Chapter 3 Multiclass MRMs Chapter 5 GLC Chapter 4 Selective MRMs Chapter 6 HPLC Chapter 1 provides information and directions that reflect FDA regu- latory policy. PAM I is not the authoritative source for publication of FDA policy, but policy decisions that directly affect the application of pesticide analytical methodology are included here as a service to the manual user. Chapter 3 includes multiclass multiresidue methods (MRMs), those that are capable of determining pesticide residues of many chemical types. The actual number and identity of the residues determinable by the methods are usually dependent on the number and variety of different determinative steps used to examine the extract. Each method in this chapter is presented as a series of modules for the extraction, cleanup, and determinative steps; a complete method is defined as a combination of one or more modules from each step. Complete methods that have been validated in interlaboratory studies, including collaborative studies performed under the aus- pices of AOAC International, are listed; these are sanctioned for use in regulatory analyses. Other combinations of modules must be treated as experimental methodology; additional supporting data for the validity of the analysis are required when such combinations are used in regulatory analyses. Chapter 2 is a collection of data and directions on a variety of unrelated topics, each of which pro- vides background information needed to perform methods of Chapters 3 and 4. Where information in Chapters 1 and 2 appears to overlap (e.g., Sec- tions 102, Preparation of Analytical Samples; and 203, Equipment and Procedures for Comminuting), the material in Chapter 1 reflects the agency policy that must be followed for enforcement of regula- tions, and Chapter 2 provides information and hints found useful by FDA pesticide chemists. xi Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) Pesticide Analytical Manual Vol. I GUIDE TO PAM I Chapter 1 Regulatory Operations Chapter 2 General Analytical Operations and Information Chapter 3 Multiclass MRMs Chapter 5 GLC Chapter 4 Selective MRMs Chapter 6 HPLC Chapter 1 Regulatory Operations Chapter 2 General Analytical Operations and Information Chapter 3 Multiclass MRMs Chapter 5 GLC Chapter 4 Selective MRMs Chapter 6 HPLC Chapter 4 includes descriptions of selective MRMs, i.e., methods designed to determine a limited number of residues related by chemical structure. Selective MRMs target residues that are not amenable to determination by the multiclass MRMs of Chapter 3. In actual practice, FDA laboratories often examine an extract from a multiclass MRM with determinative step(s) from selec- tive MRMs to broaden the scope of the analysis. When such uses have been validated in interlaboratory studies, the determina- tive steps are included as modules in Chapter 3 methodology. When no interlaboratory validation has occurred, such combi- nations are treated as experimental methodology, with the same requirements noted above for use in regulatory analyses. Tables of data are provided for each method in Chapters 3 and 4 to describe the analytical behavior of each chemical tested through the method. Data are available only for methods sanctioned for use by virtue of previous interlaboratory studies. Chapter 5, GLC, and Chapter 6, HPLC, provide background information about the two major determinative steps used in MRMs. Basic information about the techniques is included, as well as specific directions for implementing use of the instruments based on experiences in FDA laboratories. Manual users attempting to employ the determinative steps defined in Chapters 3 and 4 may need to refer to Chapters 5 and 6 for additional information and advice. Appendix I combines information on GLC behavior of particular chemicals with recoveries of the chemicals through Chapter 3 methods. Appendix II defines the steps of protocols that were used to develop such method behavior data, for use in continuing testing. Chapter 1 Regulatory Operations Chapter 2 General Analytical Operations and Information Chapter 3 Multiclass MRMs Chapter 5 GLC Chapter 4 Selective MRMs Chapter 6 HPLC Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92)xii GUIDE TO PAM I Pesticide Analytical Manual Vol. I STEP-BY-STEP SAMPLE ANALYSIS PAM I provides information and/or directions to facilitate performance of each step in sample analysis. Use these sections for information needed at each step: REVIEW Secs. 101, Appropriate procedures for regulatory PROCEDURES 103 analyses PREPARE Sec. 102 Portion of commodity to include in analytical SAMPLE sample, regulatory requirements for subsamples Sec. 203 How to comminute or homogenize various commodities CHOOSE Based on commodity type: METHOD Based on targeted residues: REVIEW Sec. 201 Percentage fat, water, and sugars in commodity BACKGROUND INFORMATION Sec. 202 Detailed directions for column (as needed) chromatography, solvent concentration Sec. 204 Directions for preparing frequently used reagents Sec. 205 Reference standards Sec. 206 Quality assurance/quality control Sec. 207 Safety Sec. 208 Hazardous waste disposal Chap. 5 Operation of GLC systems Chap. 6 Operation of HPLC systems PERFORM Chap. 3 Method directions ANALYSIS Chap. 4 IDENTIFY Tables, Details of behavior of chemicals tested RESIDUES Appendix I through methods, for tentative identification QUANTITATE Secs. 504, Quantitation for GLC, HPLC RESIDUES 606 CONFIRM Sec. 103 Approach to confirmation of residue RESIDUES identity REPORT Secs. 104, FDA procedures RESIDUES 105 xiii Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) Pesticide Analytical Manual Vol. I GUIDE TO PAM I (fruits and vegetables, grains, etc.) t t CHOICE OF METHOD BASED ON COMMODITY TYPE PRODUCTS >2% FAT Sec. 304 Appropriate extraction for product; relatively (dairy, meat, fish, nonpolar residues; actual residues covered oilseeds, etc.) depends on determinative step(s) included Sec. 401 N-methylcarbamate residues Sec. 402 Acidic and phenolic residues NONFATTY PRODUCTS Sec. 302 Nonpolar and polar residues, if no cleanup (<2% fat) is used; actual residues covered depends on determinative step(s) included Sec. 303 Relatively nonpolar residues; actual residues covered depends on determinative step(s) included Sec. 401 N-methylcarbamate residues Sec. 402 Acidic and phenolic residues Sec. 403 Phenylurea herbicide residues Sec. 404 Benomyl (as MBC), thiophanate-methyl, allophanate, and thiabendazole EGGS, EGG WHITES Sec. 303 E2; relatively nonpolar residues DRIED EGG WHITES Sec. 303 E3; relatively nonpolar residues CHOICE OF METHOD BASED ON TARGETED RESIDUES SPECIFIC RESIDUE(S) Use Index to Methods to find method(s) applicable to targeted residue(s). N-METHYL- Sec. 302 Use appropriate extraction + C3 or C4 + CARBAMATES DL1. Confirm residues with Sec. 401. ACIDS, PHENOLS Sec. 402 Confirm residues by use of additional appropriate GLC systems. PHENYLUREAS Sec. 403 Confirm residues as directed in method. BENZIMIDAZOLES Sec. 404 Confirm residues as directed in method. NO TARGET Sec. 301 Use scheme for multiclass MRM, Figure 301-a. Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92)xiv GUIDE TO PAM I Pesticide Analytical Manual Vol. I NOTES ON TERMINOLOGY Within PAM I, abbreviations are explained the first time they are used within any major subsection of a chapter (Section 101, 204, etc.). Subsequent use within that major subsection is not explained. Certain common abbreviations are used without explanation throughout the volume; these include units of length, weight, volume, time, and concentration. PAM I alphabetized tables of data use the following sequence: [space] ! “ # $ % & ’ () * + , - . / 0 1 2 3 4 5 6 7 8 9 : ; < = > ? @ A B C D E F G H I J K L M N O P Q R S T U V W X Y Z [\]. Because commas precede hyphens in this sequence, chemical names that start with combinations of numbers, hyphens, and commas may not appear where expected, e.g., 2,4,5,-T precedes 2-chloroethyl caprate. "LIB" references refer to FDA’s in-house Laboratory Information Bulletins, issued by Office of Regulatory Affairs, Division of Field Science, 5600 Fishers Lane, Rockville, MD 20857. LIB references are used only for material that was not subsequently published in the scientific literature; the latter reference is used whenever available. NUMBERING OF METHODS MODULES Methods described in Chapters 3 and 4 of this volume are presented as a series of extraction, cleanup, and determination modules. This organization offers flexible combination of modules as appropriate to the commodity being analyzed and/or the residues being targeted. The analyst and laboratory are responsible for assuring that the combination is valid. Each method in Chapters 3 and 4 is treated as a major subsection, i.e., it is numbered consecutively with a whole number: 301, 302, etc., 401, 402, etc. Within those subsections, modules are numbered according to the following scheme: Extraction E, followed by a number. “E” module numbers are repeated in different methods (i.e., E1 of Section 302 is not the same as E1 of Section 303) so both the section number and the module number must be included in a reference. For example, 302 E1 defines an extraction step, but E1 does not. Cleanup C, followed by a number. As above, “C” module numbers are repeated in different methods and both the section number and the module number must be referenced. Determination DG, followed by a number, for GLC determinative steps. DL, followed by a number, for HPLC determinative steps. Unlike E and C numbers, there is no repetition of DG or DL numbers, because the same determinative steps are used to exam- ine cleaned-up extracts from many different methods. DG1 always refers to the same GLC system, no matter what section of Chapter 3 (or 4) it is combined with. xv Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) Pesticide Analytical Manual Vol. I GUIDE TO PAM I COMPARISON TO SECOND EDITION Where PAM I 2nd edition material appears in the 3rd edition, section numbers are different. The following list of equivalent references applies: 2nd Edition Description 3rd Edition Methods 211.13a-k/231.1 (Mills) method for fatty foods extractions 304 E1-E5 211.14a Petroleum ether-acetonitrile partitioning 304 C1-C4 211.14b Petroleum ether-acetonitrile “backwash” not in 3rd ed 211.14c Partition chromatography not in 3rd ed 211.14d Florisil cleanup with ethyl ether/petr ether 304 C1, C3 211.15a-c Supplemental cleanups not in 3rd ed 211.15d Alkaline hydrolysis supplemental cleanup part of 304 C7 212.13a-d/232.1 (MOG) method for nonfatty foods extractions 303 E1-E5 212.14 Cross-reference to Florisil with ethers 303 C1 212.2 (Luke) method for nonfatty foods with 302 E1+C5 Florisil cleanup 221.1 (Hopper) method for chlorophenoxy acids, 402 phenols 232.2 Sweep co-distillation method for OPs not in 3rd ed 232.3 (Storherr) method for OPs not in 3rd ed 232.4/242.1 (Luke) method for nonfatty foods, no cleanup 302 E1 242.2 (Krause) method for N-methylcarbamates 401 242.3 (PICRC) method for benzimidazoles 404 242.4 (Luchtefeld) method for substituted ureas 403 251.1 Silicic acid separation of PCBs and pesticides not in 3rd ed 251.2 Derivatization and separation of PCBs, pesticides not in 3rd ed 252.1 Florisil elution with methylene chloride 303 C2, 304 C2, C4 253 Exhaustive extraction of organochlorine residues not in 3rd ed Gas Chromatography (Chapter on GLC has been revised extensively; references to equivalent sections reflect the same topic but not necessarily the same information.) 300 Application of GLC to pesticide residue analysis 501, 504 301 Columns 502 310 Detectors 503 311 Electron capture detector 503 B 312 Microcoulometric detector not in 3rd ed 313 Potassium chloride thermionic detector not in 3rd ed 314 Flame photometric detector 503 C 315 Electrolytic conductivity detector 503 D 316 N/P detector 503 E 320 Multiple detectors not in 3rd ed 330 GLC parameters and data tables 302 DG1-DG23, Appendix I HPLC (Chapter on HPLC has undergone only minor revision, but chapter and sections are renumbered.) 500 General information 601, 608 510 HPLC columns 602 520 Mobile phase selection, preparation, delivery 603 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92)xvi GUIDE TO PAM I Pesticide Analytical Manual Vol. I 530 Injection systems 604 540 Detectors 605 550 Data handling 606 560 Quality assurance and troubleshooting 607 Tables 201 Chemicals tested through specific methods tables follow method sections 202 Percentage Fat, Water, and Sugar in Foods 201 Appendix I PESTDATA lists of GLC data and recoveries Appendix I Other Information 110, 120 [Combined] lists of apparatus and reagents not in 3rd ed 130 Standards 205 141-142 Preparation of analytical portion, compositing 102, 203 143.1 Reporting analytical results 104 B 143.2 Analytical limit of quantitation 105 230 Organophosphate residues not in 3rd ed Chapter 4 TLC not in 3rd ed Chapter 6 Confirmation not in 3rd ed, except 103 E