11.4 Nutrient Analysis

• 11.4.1 Water Soluble Vitamins (Chemical Analysis)
• 11.4.2 Water Soluble Vitamins (Microbiological Analysis)
• 11.4.3 Fat Soluble Vitamins (HPLC)
• 11.4.4 Proximate Analysis
• 11.4.5 Metals Analysis - Graphite Furnace, ICP, Atomic Absorption/Hydride Generation

11.4.1 Water Soluble Vitamins (Chemical Analysis)

I. Thiamine (B1)

A. Objective

The analyst will become familiar with Thiamine (B1) through thiamine reading materials and exercise questions. The analyst will be able to perform thiamine procedure as written in the AOAC.

B. References

1. Methods of vitamin assay (3rd ed., chap. 6, pp. 123-142). Interscience Publishers
2. Sebrell and Harris. The vitamins (Vol. V, chap. 15 on Thiamine).
3. Combs, G. F., Jr. The vitamins (chaps. 1, 3, 10, 19).
4. Eitenmiller, R. R., Ph.D., and Landen, W. O. (1999). Vitamin analysis for the health and food sciences. Boca Raton, FL: CRC Press.
5. AOAC official methods of analysis (current ed.). Gaithersburg, MD: Association of Official Analytical Chemists International.
6. Other reading material that analyst can find through Internet and library searches.
   
   Websites for Thiamine Information
   www.britannica.com
   www.sciencekomm.at/journals/food.html 

C. Pre-Assay Questions

1. What is a vitamin?
2. Name examples of fat-soluble and water-soluble vitamins.
3. What are the recommended daily allowances of thiamine in adults, infants, and lactating and pregnant women?
4. What are other names for thiamine?
5. List some natural food products that are considered rich sources of thiamine.
6. What form of thiamine is present in animal and plant products? 
7. What is the function of thiamine in the body?
8. What are the effects of thiamine deficiency in man?
9. Are there any toxicity effects when high dosages of thiamine are taken? Is so what are the effects?
10. Discuss the solubility of thiamine in water, alcohol, and organic solvents.
11. Discuss the stability of thiamine with respect to light, pH, oxidizing and reducing agents.
12. Draw a structure of the thiamine molecule.

D.  Exercise

Analyst will read pertinent information pertaining to thiamine and answer questions on thiamine. The analyst will perform thiamine procedure on Standard Reference Material (SRM1846) and a practice sample.

E.  Post Assay Questions

1. In the procedure for thiamine what are possible sources of error? Could substances within the sample matrix interfere with the thiamine determination?
2. What precautions should one take during the procedure to protect the analyte and analyst?
3. What is the final concentration of the sample extract in the procedure?
4. What are some stopping points where the procedure can be interrupted and stored overnight without affecting the results?  

II. Riboflavin (B2)

A. Objective

The analyst will become familiar with riboflavin (Vitamin B2) through reading materials and exercise questions.  The analyst will be able to perform the riboflavin method as written in the AOAC.

B. References

1. Methods of vitamin assay (3rd ed., chap 7, pp. 147-166). Interscience Publishers.
2. Sebrell and Harris. The vitamins (Vol. V, chap. 14 on Riboflavin).
3. Combs, G. F., Jr. The vitamins (chaps. 1, 3, 10, 19).
4. Eitenmiller, R. R., Ph.D., and Landen, W. O. (1999). Vitamin analysis for the health and food sciences. Boca Raton, FL: CRC Press.
5. AOAC official methods of analysis (current edition). Gaithersburg, MD: Association of Official Analytical Chemists International.
6. Other references that analyst finds through library and Internet searches.
   
   Websites for Riboflavin Information:
   www.bodywise.com/products/ingredients/riboflavin.htm
   www.britannica.com
   www.sciencekomm.at/journals/food.html

C. Pre-Assay Questions

1. Is Riboflavin a water-soluble or fat-soluble vitamin? 
2. What are the recommended daily allowances of riboflavin in adults, infants, and lactating and pregnant women?
3. What are other names for riboflavin?
4. List some natural food products that are considered rich sources of riboflavin.
5. What form of riboflavin in present in animal and plant products?
6. What is the function of riboflavin in the body?
7. What are the effects of riboflavin deficiency in man?
8. Are there any toxicity effects when high dosages of riboflavin are taken? Is so, what are the effects?
9. Discuss the solubility of riboflavin in water, alcohol, and organic solvents.
10. Discuss the stability of riboflavin with respect to light, pH, oxidizing and reducing agents.
11. Draw a structure of the riboflavin molecule.

D. Exercise

The analyst will read pertinent information pertaining to riboflavin and answer questions. Analyst will perform riboflavin procedure on Standard reference Material (SRM1846) and a practice sample.

E. Post Assay Questions

1. In the procedure for riboflavin what are possible sources of error? Could substances within the sample matrix interfere with the riboflavin determination?
2. What precautions should one take during the procedure to protect the analyte and analyst?
3. What is the final concentration of the sample extract in the procedure?
4. What are some stopping points where the procedure can be interrupted and stored overnight without affecting the results?

III. Vitamin C

A. Objective

This training module is designed to train the employee to analyze vitamin C (ascorbic acid). This module covers two methods. These methods include the following:

1. 2,6-dicloroindophenol titrimetric method, and
2. total vitamin C microfluorometric method. 

The employee will read background material on vitamin C, answer questions, and receive the methodology training.

B. References

1. Deutsch, M. J., and Weeks, C. E. (1965). Microfluorometric assay for vitamin C. Journal Association of Official Analytical Chemists, 48, 1248.
2. Deutsch, M. J. (1967). Assay for vitamin C: a collaborative study. Journal of Association of Official Analytical Chemists, 50, 798.
3. Horowitz, W. (Ed.). (2000). AOAC official methods of analysis. (17th ed., sects. 50.1.09 (985.33), and 45.1.15 (967.22). Gaithersburg, MD: Association of Official Analytical Chemists International.
4. Eitenmiller, R. R., Ph.D., and Landen, W. O. (1999). Vitamin analysis for the health and food sciences (chap. 6, p. 223). Boca Raton: CRC Press.
5. Combs, G.F. (1992). The vitamins (chap. 9, p. 225). New York: Academic Press Inc.

C. Pre-Assay Questions

1. What is the %DV for vitamin C in adults, children, and infants?
2. What is disease related to vitamin C deficiency?
3. (a) What is the trivial name for vitamin C?
   (b) What is the definition of vitamin C?
   (c) What are the primary dietary sources of vitamin C?
   (d) What are the 2 main biologically active forms of vitamin C?
4. What is the stability of L-ascorbic acid in solution?

D. Exercise

There are two commonly used methods for vitamin C in ACNA:

1. AOAC 17th ed. 50.1.09 (985.33) for infant formulas and medical foods, and 
2. AOAC 17th ed. 45.1.15 (967.22) for all other matrixes.

The methods differ at the quantitation step and the forms of ascorbic acid that is measured. The trainee will read and make an outline of each procedure. The trainer will demonstrate each method. The trainer and trainee will perform each of the methods with various sample matrixes and controls. (In house controls such as infant formula, medical food, dietary supplement, and SRM 1846 are usable, as well as, samples received in the laboratory that has been previously analyzed. These samples or controls may be spiked at different levels unknown to the trainee.). The trainee will perform the method and meet the criteria of the method. When proficiency is demonstrated, the trainee will run official samples.

E. Post-Assay Questions

1. What are the two main analytical procedures for vitamin C in ACNA? What form of vitamin C does each method measure?
2. (a) What is the purpose of Norit?
   (b) What is the purpose of 0-phenylenediamine?
3. What are the stopping points in each method?
4. What are the sources of interference in each method, and how does an analyst avoid them?

11.4.2 Water Soluble Vitamins (Microbiological Analysis)

I.  Folic Acid

A. Objective

This module is designed to train the employee to analyze folic acid. This module covers three methods. These methods include the following:

1. a single enzyme digestion for the analysis of medical foods and infant formula,
2. a trienzyme digestion for all other food products, and
3. an alkaline digestion for dietary supplements. 

The employee will read background material on folic acid, answer questions, and receive the methodology training.

B. References

1. U.S. Food & Drug Administration, Atlanta Center for Nutrient Analysis, Southeast Regional Laboratory. Memo of analysis for total folate analysis (internal document). 
2. Horowitz, W. (Ed.). (2000). AOAC Official Methods of Analysis (17th ed., sections 50.1.21 (992.05), 45.2.03 (944.12), and 45.2.01 (960.46). Gaithersburg, MD: Association of Official Analytical Chemists International. 
3. U.S. Food & Drug Administration, Southeast Regional Laboratory. (2000, August 2). Total diet program: folate in foods. Standard Operating Procedure N/AM/4/1/94.
4. Martin, J., Landen, W. O., Soliman, A., and Eitenmiller R. R., Ph.D. (1990). Application of trienzyme extraction for total folate determination in foods. Journal of Association of Official Analytical Chemists, 73, 805.
5. Rader, J., Weaver, C., and Angyal, G. (1998). Use of microbiological assay with trienzyme extraction for measurement of pre-fortification levels of folates in enriched cereal-grain products. Food Chemistry, 62, 451.
6. Souza, S. and Eitenmiller, R. R., Ph.D. (1990). Effects of different enzyme treatments on extraction of total folate from various foods prior to microbiological assay and radioassay. Journal of Micronutrient Analysis, 7, 37.
7. Aiso, K. and Tamura, T. (1998). Trienzyme treatment for food folate analysis: optimal pH and incubation time for alpha-amylase and protease treatments. Journal Nutr. Sci. Vitaminol., 44, 361.
8. Handout: Structures and Properties of Folic Acid.
9. Handout: Microbiological Determination of Vitamins.
10. Mills, J. (2000). Fortification of foods with folic acid - how much is enough? New England Journal of Medicine, 342, 1442.
11. Williams, R. (1994, May). FDA proposes folic acid fortification. FDA Consumer. 
12. U.S. Food and Drug Administration. (1999, March-April). Folic acid awareness. FDA Consumer. 
13. Eitenmiller, R. R., Ph.D. and Landen W. O., W. 1999. Vitamin Analysis for the Health and Food Sciences. Boca Raton: CRC Press.
14. Combs, G. (1992). The vitamins. San Diego, CA: Academic Press Inc. 
15. Spallholz, J., Boylan, L., and Driskell, J. Nutrition chemistry and biology (2nd ed.). Boca Raton, FL: CRC Press
16. Glenn, C. (1997). Putting folates to work. Food Formulating, p. 47.
17. Rader, J., Weaver, C., and Angyal, G. (2000). Total folate in enriched cereal-grain products in the United States following fortification. Food Chemistry, 70, 275.
18. U.S. Food & Drug Administration. (2000). Letter regarding dietary supplement health claim for folic acid with respect to neural tube defects. Docket No.: 91N-100H.
19. U.S. Food and Drug Administration, Office of Public Affairs. (2000). History of FDA’s total diet study. 
20. U.S. Food and Drug Administration. (1996). Folic acid to fortify U.S. food products to prevent birth defects. HHS News, P96-3.
21. U.S. Food and Drug Administration. (1997). FDA announces name changes for lower-fat milks and folic acid fortification for bakery products. HHS News, P97-47.
22. U.S. Food and Drug Administration, Office of Public Affairs. (1996). Folic acid fortification.

C. Pre-Assay Questions

1. Why did the Food and Drug Administration decide to fortify cereal-grain products with folic acid?
2. What is the %DV (folic acid) for adults, infants, and children?
3. What are the four predominant forms of naturally occurring folates?
4. What is the stability of folic acid?
5. What are the symptoms/consequences of folic acid deficiency?
6. What are the consequences of too much folic acid in the diet?
7. What are good sources of folic acid in foods? 

D. Exercise

There are three commonly used methods for folic acid in ACNA:

1. Memo of Analysis: total folate analysis (AOAC 17th ed. 50.1.21(992.05),
2. Folic acid in vitamin preparations: AOAC 17th ed. 45.2.03(944.12), and
3. Folate in foods: SOP N/AM/4/1/94.

The three methods differ in the digestion procedures, the microbiology portion of the assays are similar. The trainee will read and make an outline of the three different procedures. The trainer will then demonstrate each method. The trainee and trainer will perform each of the methods with various sample matrixes and controls. (In-house controls such as an infant formula, dietary supplements, and SRM 1846 are usable, as well as, samples received in the laboratory that have been previously analyzed.) The trainee will then demonstrate proficiency in the methods by performing them independently with proper samples and controls. After proficiency is established, the trainee may be requested to analyze official samples. The trainer will also demonstrate operation of the automated dilutors, pipettors, and the Autoturb II reader. 

E. Post-Assay Questions

1. What are the analytical procedures used in ACNA for determining folic acid? How do they differ? 
2. Explain the use of the three enzymes in the SOP N/AM/4/1/94 procedure.
3. What two microorganisms are used to assay folic acid and what are the concentrations of their respective standard curves? Which one is used for dietary supplements?
4. What are the stopping points in each method?
5. What are the precautions used in each method to preserve the integrity of the samples/vitamin?
6. What are the hazardous materials and dangers in each method?

II. Niacin, Biotin, Vitamin B6 (Pyridoxine), Vitamin B12 (Cyanocobalamin) and Pantothenic Acid

A. Objective

The purpose of this training is to provide the analyst training to analyze water-soluble vitamins by AOAC microbiological methods and memo of analysis. Trainee will familiarize with Media (Broth and Agar) preparation, inoculum preparation, and instruments, reagents, and supplies used in the procedures.

B. Assignment

1. Review references and answer questions.
2. Review laboratory chemical hygiene plan, hazardous waste plan, and SRL Quality Assurance Plan.
3. Perform each assay using SRM 1846 and Infant Formula Control-198 (Milk based) along with training sample [see Exercises below].
4. Measure at least one recovery by spiking the sample with known amount of standard.
5. Read samples on Autoturb and Spectronic 20.
6. Calculate results manually (graph) and by using computer program.
7. Familiarize with weekly microorganism transfers to maintain cell line, Agar to Agar, and Agar to broth.

C. References

1. Combs, G. F. The vitamins: fundamental aspects.  Nutrition & Health.  
2. Eitenmiller, R. R., Ph.D., and Landen, W. O., Jr. Vitamin analysis for the health & food sciences.
3. Angyal, G. Method for the microbiological analysis of selected nutrients.
4. Horowitz, W. (Ed.). (2000). AOAC official methods of analysis (17th ed.). Gaithersburg, MD: Association of Official Analytical Chemists International.
5. Journal of Association of  Official Analytical Chemist articles.
6. Any text which covers “Vitamins” & “Vitamin analysis” in the lab and library.
7. Handouts from trainer.
8. Merck Index.

D. Questions

1. Discuss the stability and solubility of each vitamin with respect to light, pH, oxidation, reduction, water, and organic solvents.
2. Name the diseases caused by the deficiency of each vitamin. Discuss symptoms of the diseases caused by each vitamin.
3. List some natural food products that are considered rich sources for each vitamin.
4. What is the recommended concentration of each vitamin in the final sample assay extract?
5. Indicate the stopping points, where an analyst can interrupt the procedure for overnight storage without affecting the results.
6. Identify the test organisms listed below and indicate the vitamins assayed by each.
   (a) L.l. (b) S. f. (c) L.p. (d) L.c. (e) S.u.
7. How are the test organisms kept viable?
8. What are an inoculated and an uninoculated blank?
9. Define heat treatment and sterilization?
10. Why is lactic acid bacteria widely used in microbiological assays?
11. Why it is possible to use a test organism to assay more than one vitamin?
12. Why transfer bacteria weekly?
13. What is Lactobacillus agar, Lactobacillus broth, agar slants, agar stabs, 0.9% Saline, vitamin B6 broth, and rinse and define the purpose of each.
14. How long do stab cultures stay active?
15. How often does one transfer stab cultures?
16. How long does one grow most bacteria for an inoculum?
17. What is the incubating temperature range for most assays? Is temperature critical to an assay?
18. What is the growth time for microbiological assays? Is the growth time critical to an assay?
19. What is the recommended temperature for storing agar, slants stabs?
20. Discuss the purpose of basal media in the assay.
21. What are the criteria of an official assay?
22. What is the advantage of an eight point standard curve versus a five point standard curve?
23. What precautions should be taken when performing microbiological assays to avoid contamination and invalidating the assays?
24. What are the differences between titrimetric and turbidimetric assays?
25. How does one determine if a sterilization cycle is complete and has rendered the items inside as sterile?
26. What are the three major factors that affect the growth of microorganisms?
27. How does one determine when  the standard has reached its maximum growth?

E. Exercise

The trainee will read each method and make an outline of procedure. The trainer will demonstrate and discuss different aspects of each method with trainee. The trainee will perform each method with or without trainer help, but under his/her supervision. The trainee will run SRM 1846 and a training Sample. Upon satisfactory completion of training, trainee will run official sample.

The methods routinely used in ACNA are as follows:

Pantothenic Acid: Memo of Analysis.

Biotin: Memo of Analysis.

Niacin: Memo of Analysis for Biotin (Same extract can be used to assay niacin).   

• AOAC 17th Ed  50.1.19 (985.34) RTF milk based IF.
• AOAC 17th Ed  45.2.04 (944.13) Vitamin Preparation.

Vitamin B6 

• AOAC 17th Ed 50.1.18 (985.32) RTF milk based IF.
• AOAC 17th Ed 45.2.08 (961.15) Food products.

Vitamin B12 

• AOAC 17th Ed 50.1.20 (986.23) RTF milk based IF.
• AOAC 17th Ed 45.2.02 (952.20) Vitamin Preparation.

Outline of training program is as follows: 

Week # 1: Training assignment

• Review references and answer questions
• Review laboratory chemical hygiene plan, hazardous waste plan, and SRL Quality Assurance Plan.

Week # 2: Niacin (SRM & Duplicate Training Sample)

• Extraction
• Set up tubes
• Preparation of inoculum, media
• Reading sample & standard on Spectronic 20 & Autoturb II
• Calculation by regression analysis manually (graph)      
• Calculation by computer program
• Complete worksheets

Week # 3: Vitamin B6  (SRM & Duplicate Training Sample)

• Extraction
• Set up tubes
• Preparation of inoculum, media
• Reading sample & standard on Autoturb II
•  Calculation by computer program.
• Complete worksheets
• Discuss extraction procedure for Biotin & Pantothenic Acid.

Week # 4: Vitamin B12 (SRM & Duplicate Training Sample)

• Extraction-, Set up tubes,Preparation of inoculum, media
• Reading sample , standard on Autoturb II
• Calculation by computer program.
• Complete worksheets.

Week # 5: To determine dry cell weight for one vitamin 

• Use niacin or folic acid or any other vitamin
• Calibrate Autoturb or Spectronic 20
• Check contamination during assay
• Draw a curve plotting % transmittance reading for each level of diluted cell suspension against cell content (mg dry wt) of respective tubes).

11.4.3 Fat Soluble Vitamins (HPLC)

I.  Vitamin K

A. Objective

This training module is designed to train the employee to analyze Vitamin K. This method covers the AOAC 999.15 procedure, which is the official procedure used in ACNA. The employee will read background material on Vitamin K, answer questions, and receive the methodology training.

B. References

1. Horowitz, W. (Ed.). AOAC official methods of analysis. (2000). (17th ed., sect. 50.1.25 (999.15). Gaithersburg, MD: Association of Official Analytical Chemist International.          
2. Indyk, H. E., and Woollard, D.C. (2000). Determination of vitamin K in milk and infant formula by liquid chromatography: collaborative study.  Journal of Association of Official Analytical Chemists  International, 83(1), 121-130.
3. Indyk, H. E., Woodllard, D. C. (1997, May). Vitamin K in milk and infant formulas: determination and distribution of phylloquinone and menaquinone-4.  Analyst, 122, 465-469.
4. Simplified Vitamin K Assay Procedure.
5. HP 1100 Operating Instructions for Vitamin K.
6. Misc. attachments and e-mail messages.
7. Horowitz, W. (Ed.). (2000). AOAC official methods of analysis. (17th ed., sect. 50.1.06 (previous method used in Atlanta Center for Nutrient Analysis). Gaithersburg, MD: Association of Official Analytical Chemists International
8. Hewlett Packard books for the HP1100 ChemStation (particularly Understanding Your ChemStation) (Note: Hewlett Packer is now Agilent).
9. Eitenmiller, R. R., Ph.D., and Landen, W. O. (1999). Vitamin analysis for the health and food sciences. Boca Raton: CRC Press.
10. Combs, G. (1992). The vitamins. San Diego, CA: Academic Press Inc.

C. Pre-Assay Questions

1. What is the %DV (Vitamin K) for adults, infants, and children?
2. What are the forms of vitamin K and were do they come from (source)?
3. What is the stability of Vitamin K?
4. What are the symptoms/consequences of too much or too little vitamin K in the diet for adults and children or infants?
5. What are good sources of vitamin K in foods?

D. Exercise

The method to be used is the AOAC 999.15. The trainer will demonstrate the assay procedure, then the trainee and the trainer will perform the assay including the High Performance Liquid Chromatography (HPLC) set-up and run. (In house controls such as the SRM1846 Infant Formula and a blank are included, as well as samples received in the laboratory that have been previously analyzed.) The trainee will then demonstrate proficiency by performing the assay independently with proper samples and controls.

E. Post-Assay Questions

1. What are the differences in the new and old procedures?
2. What is the purpose of the buffer, the Lipase, the alcohol solution, the K2CO3, and the hexane?
3. Explain the post column reductor. What precautions are needed in the use of this piece of equipment? 
4. What is the purity factor and how is it determined?
5. Why would an analyst use a slightly elevated column temperature instead of ambient temperature?
6. Explain the system suitability test.
7. What does using a “forced zero” in the linear regression curve mean?
8. If a sample calculates to have a fluorescence value above the highest standard point value, how can one get it on the curve?
9. What are the stopping points in the assay?
10. What are the precautions used in the method to preserve the integrity of the sample/vitamin?
11. What are the hazardous materials and dangers in the method?

11.4.4 Proximate Analysis (Various Methods)

I.  Cholesterol (HPLC)

A. Objective

This training module is designed to train analysts in the cholesterol assay published in Journal of AOAC International Vol. 77, No 5, 1994. The method is usable for a wide variety of food matrixes. Under the Nutrition Labeling and Education Act (NLEA) of 1990, the amount of cholesterol is mandatory on the label if present in more than 2 mg/serving in products regulated by FDA. At least two health claims linking cholesterol to disease are approved. For regulatory compliance with the nutrition label, ACNA frequently receives samples for cholesterol analysis. The trainee will read references, answer questions, and receive methodology training. Upon satisfactory completion of training, trainee will demonstrate proficiency by running official samples.

B. References

1. Hamill, T. and Soliman, A. (1994). Determination of cholesterol by p-nitrobenzoate derivatization and liquid chromatography. Journal Association of Official Analytical Chemists, 77(5).
2. Simplified Cholesterol assay procedure and notes by analyst.
3. High Performance Liquid Chromatography Troubleshooting guides from Supelco.
4. High Performance Liquid Chromatography columns use and care by ANSYS Technologies Inc.
5. Attachments and Email messages regarding cholesterol waste.

C. Questions

1. What is cholesterol and where it is found (source)?
2. What are the health claims approved by FDA linking cholesterol to disease?
3. Describe toxicity of pyridine and benzene. What are the waste codes and what they mean?
4. How does one dispose the above two reagents?
5. What are the stopping points in the assay?
6. What is the purpose of benzene, p-nitrobenzoyl chloride, pyridine, and 20% aq KOH during the derivatization process?

D. Exercise

The method used for cholesterol assay in ACNA is Thomas Hamill's paper, which is published in Journal of AOAC International Vol. 77, No 5, 1994. Read the method thoroughly during training and make notes. The trainer will demonstrate the assay procedure. Trainee will watch the demonstration and then perform the assay independently. Trainer and trainee will run SRM 1845 (NIST), Training sample and Standard.

11.4.5 Metals Analysis (Graphite Furnace, Inductively Coupled Atomic Plasma (ICAP), Atomic Absorption/Hydride Generation)

I. Iron, Calcium, Phosphorus, Manganese, Magnesium, Zinc, Copper, Sodium, Potassium, Selenium, Chromium, Molybdenum

A. Objective

This module provides training in the four metal analysis methods. These methods include the analysis of different metal elements using the IACP technique, the Hydride Generator, and the Graphite Furnace Atomic Absorption (GFAA). The employee will read background material on metals, answer questions, and receive the methodology training.

B. References

1. Instrumental Manual for the Perkin-Elmer 2100 and TJA ICAP 61E.
2. National Research Council. (1989). Recommended dietary allowance (10th ed.). Washington, DC: National Academy Press. 
3. Perkin-Elmer. Analytical methods for furnace Atomic Absorption Spectroscopy (AAS).
4. Stewart and Whitaker. (1984). Modern methods of food analysis. CT: AVI Publishing Co., Inc. 
5. Willard, Merritt, Dean, and Settle. Instrumental methods of analysis (6th ed., pp.154-176).
6. SOP’s on elemental analysis using Inductively Coupled Atomic Plasma and Hydride Generation.
7. AOAC references listed after elemental analysis of infant formula.
8. U.S. Food & Drug Administration, Center for Food Safety and Applied Nutrition. Compliance program guidance manual. Compliance Program 7321.006 Infant Formula Survey and 7321.005/007 Domestic/Import NLEA, Nutrient Sample/Analysis & General Food Labeling
9. Code of Federal Regulation. (2003). Title 21, Pt. 101.9-Nutrition labeling of food (c) and (g). Washington DC: Office of Federal Register National Archives and Records Administration.
10. Code of Federal Regulation. (2003). Title 21, Pts. 136-Bakery Products, 139-Macaroni and Noodle Products. Washington DC: Office of Federal Register National Archives and Records Administration.
11. United States Pharmacopoeia XXI (p. 189).

C. Questions

Background

1. How is sensitivity, sensitivity check and detection limits defined?
2. How are limits of detection (LOD) and limits of quantitation (LOQ) defined?
3. When dry ashing a sample, why is it needed to char samples before the ashing step? How critical is temperature in the ashing procedures? What are good stopping places in the ashing procedure?
4. What is the biochemical function of iron and sodium in the body?

21 CFR and Compliance Program

1. What are the RDI values for iron, calcium, sodium, and selenium in adults? What are the Infant Formula Act requirements for these same elements?
2. List three foods that have standard requirements for minerals. Cite the requirements with the respective references.
3. Distinguish/define: sodium-free, low sodium, and reduced sodium.
4. A sample of crackers is labeled as low sodium with the sodium content declared at 35 milligrams per serving. The serving size is 30 grams. Is this product labeled correctly for sodium? The sample is found to contain 70 milligrams sodium per serving. Is the sample a violation for sodium according to the CP 7321.005/007 for NLEA?

Elemental Analysis

1. What precaution is to be taken for perchloric acid digestions?
2. Why expose the Se digestion in HCl after the wet digestion?
3. What is the principle of the Hydride Generation method for Se analysis?
4. What is the primary difference between ICAP and AAS technology?
5. What are some advantages of using the ICAP technology verses the GFAA technology for elemental analysis?

D. Exercise

This training exercise will consist of performing all determinations in duplicate. The employee is to assay the SRM 1846, any other assigned samples, and 2 method blanks.

1. Assay the samples for Calcium, Phosphorous, Magnesium, Iron, Copper, Manganese, Zinc, Sodium, and Potassium using the ICP technique. (Method: AOAC 17th ed. 50.1.15 (984.27)).
2. Assay the samples for Selenium. See Selenium SOP procedure.
3. Assay a medical food sample for Chromium and Molybdenum using GFAA. See memo of analysis for procedure.