3. 800-675-8375 • covance.com/nutri 1
INTRODUCTION
Advancements in the way food and dietary supplements are developed, packaged and prepared require flexibility, innovation
and a broad scientific understanding of those implications on analytical data. Covance has over 80 years of experience in food
analysis and has built a reputation for scientific and regulatory expertise.
Understanding the key drivers in analytical testing—placing the customer first, creating processes to drive efficiency and
providing high-quality data—has positioned Covance as a leader in outsourced food and dietary supplement testing services.
This directory provides a description of our analytical chemistry assays, quality processes, service descriptions and ordering
information. We invite you to visit our facilities, speak with our staff and experience a level of personal commitment to
helping you reach your product development goals while remaining focused on regulatory compliance and food safety.
TABLE OF CONTENTS
Quick Reference..............................................................................................................................................................................2–19
Assays...............................................................................................................................................................................................20–81
Ordering Information.................................................................................................................................................................82–84
Experience New Potential with SampleKinect®.............................................................................................................. 83
Sampling........................................................................................................................................................................................ 84
Importing Samples to Covance............................................................................................................................................. 84
ORDERING
INFORMATION
Sample Submittal/Shipping
Please order via your SampleKinect account
at samplekinect.covance.com or our Analysis
Request Form available from your Customer
Service Associate.
5. 3800-675-8375 • covance.com/nutri
NL-Proximate (moisture, ash, protein)
Calories (by calculation)
Calories from fat (by calculation)
*Requires fat, moisture, protein and ash testing
Total fat [sum of fatty acids (saturated, monounsaturated,
polyunsaturated, trans)]
Cholesterol
Total carbohydrates (by calculation)
*Requires fat, moisture, protein and ash testing
Total dietary fiber
Sugars
HPLC
GC
Either HPLC or GC will be used, depending on the sample matrix.
Vitamin A
beta Carotene
If there is a source of beta carotene, the assay for beta carotene
should be used instead of (or in addition to) vitamin A to obtain
total vitamin A activity for nutrition labels.
Vitamin C
Calcium, iron, sodium (ICP)
Mandatory Nutrient Package
This package includes all nutrients, with sugars assayed by
GC, minerals by ICP and total fat as the sum of the fatty acids
calculated as triglycerides.
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NUTRITION LABELING MANDATORY NUTRIENT LIST
Regulatory initiatives such as the Nutrition Labeling and Education Act have had a profound impact on the food industry. The
adoption of standardized content and format guidelines for nutrition labeling have provided consumers with the tools to plan
a balanced diet, as well as given producers data to distance their product from the competition.
6. 4 Pricing available from a Covance representative. For updates to assays, refer to our online catalog at covance.com/nutri.
Astaxanthin
Biotin
Carotenoids
Option 1: alpha Carotene, beta Carotene, Lycopene
Option 2: Cryptoxanthin, Lutein, Zeaxanthin
Option 3: Lutein only
Choline (chemical)
Choline (enzymatic)
Cyanocobalamin (vitamin B12)
Folate (vitamin B9)
Inositol
L-Ascorbyl-2-Phosphate
Niacin (vitamin B3)
Pantothenic acid (vitamin B5)
Pyridoxine (vitamin B6)
Riboflavin (vitamin B2)
Thiamin (vitamin B1)
Tocopherols (alpha, beta, gamma and delta)
Tocotrienols (alpha, beta, gamma and delta)
Vitamin A modified USP for dietary supplements (HPLC)
Vitamin A, retinol
Vitamin B profile (HPLC)
Vitamin B profile (HPLC-MS/MS)
Vitamin C
Vitamin D
Vitamin E (alpha tocopherol)
Vitamin E (in feeds and forages)
Vitamin K1
VITAMIN ANALYSIS
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Cholesterol
Conjugated linoleic acid (CLA)
Fatty acid profile (C4 - C24) (including Omega-3 and
Omega-6 isomers, and trans fatty acids)
Fatty acid profile (C4 - C24) (including Omega-3 and
Omega-6 isomers, not including fatty acids)
Free fatty acids (total by titration)
Free fatty acids (by GC)
Glycerol
Hexanal
Iodine value
p-Anisidine
Peroxide value
Sterols
TBA value
Acid detergent fiber
Aspartame, Acesulfame K, DKP and saccharine
beta Glucan
Crude fiber
Galactooligosaccharides (GOS) content in Infant Formula
Galactooligosaccharides (GOS) content in Raw Material
Insoluble and soluble fiber (Lee Method)
Insoluble, soluble and total dietary fiber (CODEX
definition) by enzymatic-gravimetric method and liquid
chromatography (McCleary)
Inulin (fructan), HPLC
Inulin (fructan), spectrographic
Lactulose
Lignin
Neutral detergent fiber (enzymatic)
Polydextrose
Resistant starch
Starch
Sucralose (finished products)
Sucralose (packets, pure material) (USP Method)
Sugar alcohols
Sugar profile after acid hydrolysis
Sugar profile by GC
Sugar profile, low levels
Sugar profile by HPLC
Sugar profile by IC
Total dietary fiber (CODEX definition) by
enzymatic-gravimetric method and liquid
chromatography (McCleary)
Total dietary fiber containing supplemented resistant
maltodextrin (Fibersol)
Total dietary fiber (Lee or Prosky)
LIPIDS
CARBOHYDRATES
8. 6 Pricing available from a Covance representative. For updates to assays, refer to our online catalog at covance.com/nutri.
Acid insoluble ash
Ash
Available carbohydrates (direct determination)
Bomb calorimetry
Density
Fat extractions
Acid hydrolysis
Methanol/chloroform
Roese-Gottlieb
Soxhlet
Fat (sum of fatty acids)
Loss on drying (LOD)
Moisture (Karl Fischer)
Moisture (vacuum oven)
pH
Protein (Dumas)
Protein (Kjeldahl)
Proximate package (moisture, ash, protein and fat)
Calories (by calculation)
Total carbohydrates (by calculation)
Residue on ignition (ROI)
Titratable acidity
Water activity
Alcohol (added)
Alcohol (residual)
BHA, BHT and TBHQ (antioxidants)
Caffeine, theobromine and theophylline
Capsacin/capsacinoid (Scoville heat)
Dissolution
Ethanol and Methanol
Nucleosides
Nucleotides
Osmolality
Parabens (methyl, ethyl, propyl, butyl, isopropyl, isobutyl)
Polysorbates
Residual solvents
Soy isoflavones (genistein, daidzein, glycitein)
Species Identification by ELISA (beef, poultry or pork)
Sulfites (Monier-Williams)
TBHQ
Viscosity (Brookfield or Bostwick)
Allergens
Almond
Brazil Nut
Casein
Cashew Kernel
Celery
Coconut
Crustaceae Shellfish
Egg
Gluten
Hazelnut
Lupine
Macadamia Nut
Mustard
Peanut
Pistachio
Sesame
Soy Allergen
Total Milk
Walnut
Other Contaminants
3-MCPD (3-Chloro-1,2-Propanediol)
4-MEI
Acrylamide by LC-MS/MS
Aflatoxins by HPLC (B1, B2, G1, G2)
Aflatoxins (B1, B2, G1, G2), Ochratoxin A
and Zearalenone by HPLC
Aflatoxins M1 by HPLC
Arsenic Speciation
Bisphenol A (BPA)
Cyanuric acid by LC-MS/MS
Heavy metals by ICP (arsenic, cadmium, lead, mercury)
Heavy metals by ICP-MS (arsenic, cadmium, lead, mercury)
Melamine by LC-MS/MS
Melamine and analogs by UHPLC-MS/MS
Mycotoxins in infant formula (Aflatoxin B1, B2, G1, G2, M1,
M2, deoxynivalenol, Ochratoxin A, Zearalenone,
Fumonisin B1, B2, HT-2, T-2 toxin)
Polycyclic aromatic hydrocarbons (PAH)
Residual solvents
PROXIMATE
OTHER ASSAYS
NATURAL CONTAMINANTS
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ICP
Aluminum
Barium
Beryllium
Boron
Cadmium
Calcium
Chromium
Cobalt
Copper
Iron
Magnesium
Manganese
Molybdenum
Nickel
Phosphorus
Potassium
Sodium
Strontium
Vanadium
Zinc
ICP-MS
Antimony
Arsenic
Barium
Beryllium
Bismuth
Cadmium
Caesium
Calcium
Chromium
Cobalt
Copper
Gallium
Germanium
Iodine
Iron
Lanthanum
Lead
Lithium
Magnesium
Manganese
Mercury
Molybdenum
Nickel
Palladium
Platinum
Rubidium
Ruthenium
Selenium
Silver
Strontium
Sulfate*
Sulfur
Thallium
Tin
Titanium
Tungsten
Vanadium
Zinc
INDIVIDUAL ELEMENTS
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INORGANIC ANALYSIS
The method used for the analysis of various elements in food and dietary supplement samples is dependent on both the
concentration and the sample matrix. For most elements the precision using any of these techniques is less than 5% RSD.
Inductively Coupled Plasma Spectroscopy (ICP) is used for macro-levels inorganic constituents in all matrices. Usually 10g
of sample is either dry or wet ashed, or samples are analyzed directly. The limit of quantitation is based on the individual
analyte and matrix, but generally yields detection in the PPM range. A common scan is for calcium, iron and sodium used for
nutritional labeling.
Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) is the method of choice when analyzing trace level analytes. It is
applicable to all matrices. Usually 0.5 g of sample is wet ashed in a closed-vessel microwave digestion, although other ashing
techniques are used. Limits of quantitation are lower than ICP and are dependent on the analyte and matrix, but generally
yield detection in the PPB range. A common scan is for arsenic, cadmium, lead and mercury used for heavy metal analysis.
Atomic Absorption Spectrospcopy (AA) is used for single analyte measurements using flame, hydride-generation or graphite
furnace techniques. This technology, in general, has been surpassed by the advances made in ICP and ICP-MS techniques.
Please see the details in the assay section of the catalog for specifics.
Flame AA
Silicon
Titanium
Other analytes available on request include calcium,
chromium (in fortified supplements), cobalt, copper, iron,
lead, magnesium, manganese, molybdenum, potassium,
selenium, sodium and zinc.
Graphite Furnace AA
Aluminum
Other analytes, such as chromium, available on request
Inorganic Speciation IC-ICP-MS
Arsenic
Miscellaneous AA
Selenium (hydride)
* Calculated from total sulfur by ICP-MS
10. 8 Pricing available from a Covance representative. For updates to assays, refer to our online catalog at covance.com/nutri.
Inorganics by ICP Emission Spectrometry
Packages
Option 1: ICP3–calcium, iron, sodium (for nutrition
labeling)
Option 2: ICPL–calcium, copper, iron, magnesium,
manganese, phosphorus, potassium, sodium, zinc
Option 3: ICPN–Option 2 plus aluminum, barium, boron,
chromium*, molybdenum*, strontium
Option 4: ICPS–Option 3 plus beryllium, cadmium, cobalt,
nickel, vanadium
* May need to perform an alternate test for certain matrices.
Inorganics by Wet Chemistry
Chloride
Fluoride (chemical)
Fluoride (ISE)
Iodine (chemical)
Iodine (ISE)
Nitrate/nitrite by HPLC
Nitrate/nitrite by IC and VIS
Heavy metals as lead by USP 231
INORGANIC ANALYSIS
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MICROBIOLOGY SERVICES
Feeling challenged by the demands for increased food safety in today’s market place? Covance is ready to help you
produce safe products and keep you compliant and competitive with our broad array of services that address consumer
and regulatory requirements.
In anticipation of your needs, we have dedicated two ISO accredited laboratories (Battle Creek, Michigan and Madison,
Wisconsin) to delivering microbiological results you can use to base food safety decisions. Along with testing capabilities,
Covance offers microbiology consulting, customized research and training services.
Contact Us Today
(1.855.83MICRO or 1.855.33MICRO)
Discover why Covance is your optimum partner in food safety services
CONSULTING
• Investigations to help
identify, isolate and eliminate
microbial harborage points
• Responses to regulatory
actions
• Expert witness services
• Food safety program
development and review
• GFSI audit preparation
RESEARCH
• White paper, laboratory and
in-plant process validations
• Customized challenge studies
based on NACMCF guidelines
• Pathogen- and spoilage-based
microbial shelf-life evaluations
• Confirmation and/or validation of
cooking/baking instructions
TRAINING
General education sessions or
customized courses, held in our
classrooms or delivered at your facility
• International HACCP Alliance
accredited training classes
and instructors
• Prerequisite programs
• Internal audits
• Sampling technique
• Sampling site selection
• Laboratory techniques
• Other topics upon request
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12. 10 Pricing available from a Covance representative. For updates to assays, refer to our online catalog at covance.com/nutri.
MICROBIOLOGY ASSAYS
Food, Ingredients and Environmental Swabs
Quantitative – Counts or MPN
Aerobic plate Lactic Acid Bacteria
Bacillus cereus Lactobacillus - Heterofermentative
Chronobacter sakazakii Mesophilic aerobic spores
Coliform Mesophilic anaerobic spores
Coliform MPN Staphylococcus species
E. coli Staphylococcus species – Coagulase (+)
E. coli MPN Yeasts and molds
Enterobacteriaceae Yeasts and molds – Osmophilic
Additional charges for samples requiring dilution beyond 1/1000.
Qualitative – Presence/Absence
E. coli O157:H7 Listeria species
E. coli – STEC Listeria monocytogenes
Salmonella species
Additional charges for sample sizes 100 g and for compositing of individual samples.
Confirmation/Identification
Gram stain Biochemical – VITEK and API
Dietary Supplements and Nutritional Products
USP
Verification of assay suitability Clostridia species
Total aerobic microbial count (TAMC) E. coli
Total combined yeasts and molds count (TYMC) Salmonella species
Bile-tolerant Gram negative bacteria Staphylococcus aureus
Candida albicans
Additional charges for dilutions beyond 1/10 and sample neutralization.
Non-USP
Probiotic enumerations Enumeration of microencapsulated microorganisms
Shelf-life viability Simulated gastric dissolution
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CONTAMINANTS IN FOODS, FEEDS AND DIETARY SUPPLEMENTS
Global distribution of food, feed and dietary supplement
products into new regions requires a broad understanding of
the interplay between growers, manufacturers and regulators.
First-hand experience developing new testing methods, global
regulatory knowledge and the scientific expertise to develop
an appropriate testing program is critical to meeting your
specific product needs. Our scientists have designed and
managed hundreds of testing programs for heavy metals,
acrylamide, melamine and pesticide residues in support of
Proposition 65, response to economic adulteration, routine
supplier verification and Infant Formula EU Directive
2006/141/EC, respectively.
Our Center of Excellence for Contaminants in Greenfield,
Indiana, offers a broad portfolio of contaminant testing
services. The 10,000 sq ft state-of-the-art facility in Greenfield
provides clients the most up-to-date analysis of chemical
residues and contaminants as well as the development of new
analytical methods.
We approach the analysis of contaminants based on
analytical quantitation using highly selective technology
including ICP-MS, LC- and GC-MS/MS. As a true partner
in food and dietary supplement analysis, we have the
resources to meet your requirements for quality, flexibility,
value and customer service.
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Multi-Residue Analysis (MRA) by GC-MS/MS and LC-MS/MS (300+ compounds)
Botanicals, Spices and Other Specialty Commodities and Products
All analyses reported to 10 ppb or LOQ, whichever is higher.
Finished Food and Raw Ingredients
All analyses reported to 10 ppb or LOQ, whichever is higher.
This offering includes:
▶ Fruits and vegetables
▶ Grains and cereals
▶ Fats and oils
▶ High-fat food products
▶ Finished processed foods, including meals
Infant Formula
All analytes are reported to 10 ppb (or lower if required). EU Directive 2006/141/EC (10 ng/g or less) offering is available.
Matrix-Based Quantitation
Additional analyses (after initial analysis) to perform Method of Standard Addition, which provides the most
accurate quantitation.
Individual Pesticides or Subgroups
Individual pesticides, subgroups of or additions to the MRA.
Carbamates by LC-MS/MS
14. 12 Pricing available from a Covance representative. For updates to assays, refer to our online catalog at covance.com/nutri.
USP/EP Analysis (99 Analytes)
USP 561 USP/EP Pesticide Full Reporting Package
Report at the USP/EP limits. Full multipoint calibration for all analytes by GC-MS/MS and LC-MS/MS. Includes EBDCs
(dithiocarbamates) by GC, inorganic bromide by GC and matrix-based quantitation.*
*Matrix-Based Quantitation: additional analyses (after initial analysis) to perform Method of Standard Addition, which provides
the most accurate quantitation.
Ala Cart Analysis
GC-MS/MS and LC-MS/MS Multi-Residue Analysis (select 1 to 90+ analytes)
Matrix-based quantitation
EBDCs (dithiocarbamates) by GC
Bromide, inorganic
COMPENDIAL TESTING SERVICES
Raw Material/Ingredient Analysis
At Covance, we have been performing monograph analyses of raw materials and ingredients for over 30 years. In accordance
with many global regulations, our database of over 400 different raw materials and capacity to set up more provides you with
the analytical services you need for your raw materials testing.
To expedite your analysis, please provide the following information in advance:
▶ Quarterly or yearly list of raw materials
▶ Notice of shipment
▶ Analysis request form (available online or from Client Services)
This testing is compliant with GMP regulations (21 CFR Part 111) for ingredients in dietary supplements. For assays
compliant with 21 CFR Part 211 for human drug ingredients, please contact us.
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CONTAMINANTS IN FOODS, FEEDS AND DIETARY SUPPLEMENTS
15. 13800-675-8375 • covance.com/nutri
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STABILITY STUDIES: STABILITY AND SHELF-LIFE EVALUATION
Covance provides comprehensive analytical services related to stability and shelf-life of products for the food and dietary
supplement industries. Stability is defined as the extent to which a product retains, within specified limits throughout its
period of storage and use, the same properties and characteristics possessed at the time of packaging. Understanding product
stability is an essential measure of the quality of a food or dietary supplement. Covance can custom design your stability study
from start to finish.
Study Design - Essential Information
for Stability Studies
▶ Packaging materials
▶ Region the product is being sold
▶ Product specifications
▶ Type of study (accelerated or real time)
▶ Shelf-life anticipated
▶ Regulatory requirements (if applicable)
▶ Method applicability
Testing Services for Stability Studies
▶ Chemical analysis (nutrients, active compounds)
▶ Physical properties (moisture, water activity, disintegration,
hardness, etc.)
▶ Organoleptic evaluation (appearance, taste, smell, color)
▶ Microbiological evaluation (safety assessment,
antimicrobial preservative effectiveness)
▶ Monograph testing (USP or International)
Goals of the Stability Study
▶ Ensure the product is stable over its shelf-life
▶ Establish or verify the shelf-life of the product
▶ Aid in product research and development
▶ Provide data to support the quality of the product over time
▶ Fulfill any regulatory requirements
Due to the custom nature of designing a stability study, please
inquire on pricing and storage availability.
Typical Stability Studies
Testing Intervals
Storage Study Condition (ICH guidelines)
Real Time 25°C/60% RH 3, 6, 9, 12, 18, 24 months
Accelerated 40°C/75% RH 3, 6 months
Alternate 30°C/65% RH 6, 9, 12 months
PHYTOCHEMICALS
The terms phytochemicals and phytonutrients have been adopted as descriptors for certain organic components of plants
that are thought to promote human health. Unlike the traditional nutrients, these compounds are not “essential” for
life from a traditional standpoint but are being increasingly recognized as having beneficial health characteristics. As
the popularity of these products has increased with mainstream consumers, the use of a variety of dietary supplements,
herbals/botanicals and functional foods has expanded and resulted in an intense and competitive marketplace. The ability
to scientifically substantiate claims for quality, purity, consistency, stability, safety and efficacy is an essential marketing tool
and frequently a regulatory requirement.
16. 14 Pricing available from a Covance representative. For updates to assays, refer to our online catalog at covance.com/nutri.
METHOD DEVELOPMENT, VALIDATION AND TRANSFER
Responding to changes in market or regulatory requirements demands new or improved high-quality scientific methods
engineered for greater efficiency, lower limits of detection and improved reproducibility. As a partner in developing
solutions to method challenges, Covance offers the services of our Technical Development team on a contract basis. You
can leverage the team’s expertise across a wide range of matrices and technologies, including LC-MS/MS and UHPLC, to
address your analytical challenges. Projects are priced on a time/materials or project basis.
Typical projects we have completed include:
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SPECIAL SERVICES
Colors
Artificial Colors
FDC Red No. 2 (Amaranth)
FDC Red No. 3 (Erythrosin B)
FDC Red No. 4 (Ponceau SX)
FDC Red No. 40 (Allura Red)
FDC Green No. 3 (Fast Green FCF)
FDC Blue No. 1 (Erigluacine)
FDC Blue No. 2 (Indigo Carmine)
Acid Orange 12 (Crocein Orange G)
FDC Yellow No. 5 (Tartrazine)
FDC Yellow No. 6 (Sunset Yellow FCF)
FDC Yellow No. 10 (Quinoline Yellow)
▶ New method development
▶ Compendial/journal method setup
▶ Matrix extensions (incl. validation)
▶ Client method validation
▶ Existing method modernization
▶ New technology application
▶ Client method variability resolution
Japanese Export Testing
Sennosides (UV)
Fenfluramine, N-nitroso-fenfluramine (HPLC)
p-Hydroxybenzoates (methyl, ethyl, propyl, butyl, isoproyl, isobutyl)
Thryoxine, triiodothyronine (HPLC)
Benzoic and sorbic acids
Prices for these tests are all product-dependent—specific pricing for your supplement product can be obtained
by calling us at 800-675-8375.
17. 15800-675-8375 • covance.com/nutri
BOTANICALS
For centuries, people have used herbs and other botanicals for their healthful effects, possibly due to their phytochemical
composition, and in recent years, the use of these products has experienced a rise in popularity among consumers. This
has resulted in corresponding regulatory initiatives and the accelerated need for analytical methods for botanical products.
Several regulatory initiatives, including changes to current Good Manufacturing Practices (cGMP), address identification
testing for botanical ingredients.
FOOD CONTACT MATERIALS (MIGRATION COMPLIANCE)
The potential for migration of indirect additives and contaminants from food contact material is a concern for both
food products and dietary supplements. As the design and use of innovative packaging methods change, there is a
corresponding effect on the regulatory and experimental guidelines for determining the impact of packaging components
on the safety of food and dietary products. In the case of dietary supplements and botanicals, migration could potentially
have a negative effect on the efficacy of the active ingredient. When evaluating the safety of packaging materials there are
three key aspects to be considered: the source of the materials, the nature of the manufacturing process and the conditions
of use.
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▶ Total and chloroform-soluble extractables from
food packaging
▶ Recycled and degradable packaging
▶ Retort pouch barrier integrity
▶ Pigment and dye migration from consumer products
▶ Benzene in polymeric packaging materials
▶ Microwave packaging
▶ End use testing verifying compliance with
21 CFR Parts 172-181
▶ Conditions of use A-J
▶ Experience with FCN and EU recommendations
For guidance, please refer to: www.packaginglaw.com
18. 16 Pricing available from a Covance representative. For updates to assays, refer to our online catalog at covance.com/nutri.
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DIETARY SUPPLEMENTS
Covance has over 80 years of experience in food analysis and has built a reputation for scientific and regulatory expertise,
responsiveness to customer needs and the capacity to turn around samples quickly and efficiently. As a full-service
laboratory, we analyze food, dietary supplement and biotechnology products.
Product/Analyte........................................................................................................................................ Testing Method
5-Hydroxytryptophan (5-HTP)...........................................................................................................................................HPLC
Acetyl Cysteine....................................................................................................................................................................HPLC
Allicin (garlic)......................................................................................................................................................................HPLC
alpha Lipoic Acid (USP).....................................................................................................................................................HPLC
Anthocyanins......................................................................................................................................................................HPLC
Anthocyanins (bilberry, elderberry, cranberry, fruits)........................................................................................................ VIS
Astaxanthin.........................................................................................................................................................................HPLC
beta-Glucan........................................................................................................................................................................... VIS
Black Currant Oil (linoleic acid and GLA)...........................................................................................................................GC
Caffeine, Theobromine, Theophylline..............................................................................................................................HPLC
Capsaicin/Capsaicinoid (Scoville Heat)............................................................................................................................HPLC
Catechins (EGCG, GCG, catechin, EGC, EC, ECG, GC, CG) + gallic acid.....................................................................HPLC
Chlorogenic Acid................................................................................................................................................................HPLC
Chondroitin Sulfate by CPC (USP).................................................................................................................................Titration
Citrus Bioflavanoids...........................................................................................................................................................HPLC
Coenzyme Q10....................................................................................................................................................................HPLC
Conjugated Linoleic Acid (CLA)...........................................................................................................................................GC
Cranberry (quinic, malic, citric acids)...............................................................................................................................HPLC
Creatine...............................................................................................................................................................................HPLC
Cryptoxanthin.....................................................................................................................................................................HPLC
DHEA or 7-KETO DHEA
(Dehydroepiandrosterone) or (3-acetyl-7-oxo-dehydropiandrosterone).......................................................................HPLC
Dong Quai (ferulic acid).....................................................................................................................................................HPLC
Echinacea spp. (caftaric, chlorogenic, echinacoside, chicoric)..........................................................................................HPLC
Echinacea spp. [total phenolics as gallic acid equivalents (GAE)]....................................................................................... VIS
Elagic Acid...........................................................................................................................................................................HPLC
Ephedrine Alkaloids
Ephedra or Ma Huang.................................................................................................................................................LC-MS/MS
Evening Primrose Oil (linoleic acid and GLA)....................................................................................................................GC
Fo-ti Powder (trans-resveratrol).........................................................................................................................................HPLC
Fructan (AOAC 997.08)................................................................................................................................................ HPAEC-PAD
Fructan (AOAC 999.03)........................................................................................................................................................ VIS
Furfural...............................................................................................................................................................................HPLC
gamma Aminobutyric Acid (GABA).................................................................................................................................HPLC
gamma Linoleic Acid (GLA)..................................................................................................................................................GC
19. 17800-675-8375 • covance.com/nutri
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DIETARY SUPPLEMENTS (CONTINUED)
Product/Analyte........................................................................................................................................ Testing Method
Garcinia cambogia (hydroxycitric acid)..............................................................................................................................HPLC
Garlic (allicin)......................................................................................................................................................................HPLC
Ginkgo Biloba Flavonoids..................................................................................................................................................HPLC
Ginkgo Biloba Terpenoids..................................................................................................................................................HPLC
Ginseng, Panax or Korean (ginsenosides)........................................................................................................................HPLC
Glucosamine........................................................................................................................................................... HPLC/HPAEC-PAD
Glycerol (glycerine)................................................................................................................................................................GC
Goldenseal (hydrastine and berberine).............................................................................................................................HPLC
Grape Seed Extract
Catechins..........................................................................................................................................................................HPLC
Total Polyphenols (Folin-Ciocalteu Method) .................................................................................................................. VIS
Proanthocyandins (polyphenols - catechins)............................................................................................................. VIS/HPLC
trans-Resveratrol..............................................................................................................................................................HPLC
Green Tea Catechins...........................................................................................................................................................HPLC
Griffonia Seed Extract (5-Hydroxytryptophan) 5-HTP......................................................................................................HPLC
Guarana (theobromine, theophylline, caffeine)................................................................................................................HPLC
Hydroxycitric Acid (garcinia extract).................................................................................................................................HPLC
Isoflavones (daidzein, glycitein, genistein, daidzin, glycitin, genistin)..........................................................................HPLC
Kudzu (isoflavones)............................................................................................................................................................HPLC
Lutein...................................................................................................................................................................................HPLC
Lycopene..............................................................................................................................................................................HPLC
Marine Lipids (EPA/DHA)....................................................................................................................................................GC
Methylsulfonylmethane (MSM)............................................................................................................................................GC
Milk Thistle (silychristin, silydianin, silibibin A B)......................................................................................................HPLC
Omega-3 Fatty Acids..............................................................................................................................................................GC
ORAC, Total (includes hydrophilic and lipophilic).............................................................................................................FL
Oxalic acid...........................................................................................................................................................................HPLC
PABA (para-aminobenzoic acid)........................................................................................................................................HPLC
Phenolic Acid (Phenylproponoids) Caffeic, p-Coumaric, Sinapic, and Ferulic Acids....................................................HPLC
Phospholipids (Lecithin Phospholipids)
Phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol....HPLC
Phytic Acid...........................................................................................................................................................................HPLC
Phytosterols (stigmasterol, beta sistosterol, campesterol, brassicasterol).........................................................................GC
Policosanol (octacosanol, triacontanol, tetracosanol, hexacosanol)...................................................................................GC
Polydextrose............................................................................................................................................................ HPLC/HPAEC-PAD
Polygonum cuspidatum (trans-resveratrol).........................................................................................................................HPLC
Polyphenols (Total Polyphenols) Folin-Ciocalteu Method................................................................................................. VIS
Polyphenols (Total) with % Solids
Tea and tea products for Tea Association members only............................................................................................... VIS
Pueraria (isoflavones).........................................................................................................................................................HPLC
Punicalagins........................................................................................................................................................................HPLC
20. DIETARY SUPPLEMENTS (CONTINUED)
Product/Analyte........................................................................................................................................ Testing Method
Pygeum (beta sitosterol)........................................................................................................................................................GC
Pyruvate (pyruvic acid).......................................................................................................................................................HPLC
Quercetin.............................................................................................................................................................................HPLC
Rose Hips (ascorbic acid)......................................................................................................................................................FL
Royal Jelly 10-HDA (10-hydroxy-2-decanoic acid).............................................................................................................HPLC
Rutin....................................................................................................................................................................................HPLC
SAMe (S-adenosylmethionine)..........................................................................................................................................HPLC
Saw Palmetto (phytosterols + fatty acids).............................................................................................................................GC
Scoville Heat (capsaicin and capsaicinoid)........................................................................................................................HPLC
Soy Isoflavones....................................................................................................................................................................HPLC
Stinging Nettles (5-hydroxytryptophan)............................................................................................................................HPLC
Theobroma cacoa (caffeine, theobromine, theophylline)..................................................................................................HPLC
Tocotrienols (alpha, beta, gamma, delta)..........................................................................................................................HPLC
Tomato (lycopene)..............................................................................................................................................................HPLC
trans-Resveratrol.................................................................................................................................................................HPLC
Ubiquinone (Coenzyme Q10)............................................................................................................................................HPLC
Valerian Extract (valerenic acid).........................................................................................................................................HPLC
Yerba Mate (caffeine, theobromine, theophylline)...........................................................................................................HPLC
Zeaxanthin..........................................................................................................................................................................HPLC
18 Pricing available from a Covance representative. For updates to assays, refer to our online catalog at covance.com/nutri.
QUICK REFERENCE
23. 800-675-8375 • covance.com/nutri
ASSAYS
3-CHLORO-1,2-PROPANEDIOL (3-MCPD)
Purpose: Applicable for the determination of 3-MCPD in
hydrolyzed vegetable protein, soups and stocks, stock cubes,
soy sauce, malt extract, salami, fish, cheese, flour, starch,
cereals and bread.
Method facts:
▶ Sample size: 25 g
▶ Limit of quantitation: 0.025 ppm for most matrices
▶ Precision: Varies with matrix
▶ Method reference: AOAC 2000.01
Description: An internal standard is added to the sample
followed by a salt solution. This is then blended to
homogenize, sonicated, then purified. A portion of the purified
extract is derivitized and analyzed by gas chromatography with
mass spectrometric detection (GC-MS).
4-METHYLIMIDAZOLE (4-MEI) AND
2-METHYLIMIDAZOLE (2-MEI)
Purpose: Applicable for the determination of 4-MeI and 2-MeI
in beverages and water soluble samples.
Method facts:
▶ Sample size: 25 g
▶ Limit of quantitaion: Varies with matrix, as low as 1 ppb for
4-MeI and 2 ppb for 2-MeI
▶ Precision: Varies with matrix
▶ Method reference: Simultaneous quantitation of 2- and
4-methylimidazoles in carbonated drinks containing
caramel coloring by ultrahigh-performance liquid
chromatography tandem mass spectrometry, by Wang, J.;
Liu, X.; Pohl, C. and Schnute, W., Food Quality Magazine,
June/July 2011, 34-38.
Solid-phase extraction of 4(5)-methylimidazole (4MeI)
and 2-acetyl-4(5)-1,2,3,4-tetrahydroxylbutyl-imidazole
(THI) from foods and beverages with subsequent liquid
chromatographic-electrospray mass spectrometric
quantification. J. Sep. Sci. 2006, 29, 378-384.
Reversed-phase high-performance liquid chromatographic/
mass spectrometric method for separation of
4-methylimidazole and 2-acetyl-4-tetrahydroxybutyl
imidazole at pg levels. Analytic Chimica Acta 477 (2003)
49-58.
Description: Sample is weighed and an internal standard is
added. Sample is diluted with purified water and analyzed
using HPLC-MS/MS.
5-HYDROXYTRYPTOPHAN (5-HTP)
Analyzed with free amino acid procedure
ACESULFAME K
Analyzed with aspartame: see Aspartame
ACETIC ACID
see Organic acid profiles — option 2
ACID DETERGENT FIBER
see Detergent fiber, acid
ACRYLAMIDE
Purpose: Applicable for the determination of acrylamide in
various foods.
Method facts:
▶ Sample size: 5 g
▶ Limit of quantitation: 10 ppb
▶ Precision: Varies with matrix
▶ Method reference: United States Food and Drug
Administration, Center for Food Safety and Applied
Nutrition Office of Plant Dairy Foods and Beverages,
“Detection and Quantitation of Acrylamide in
Foods” (2002)
Description: Acrylamide is extracted from food samples with
0.1% formic acid. If product contains fat, petroleum ether is
used prior to extraction with 0.1% formic acid. The extract
is purified through a solid phase extraction (SPE cartridge).
Acrylamide in the sample extract is determined using
LC-MS/MS. The acrylamide is determined using least square
linear regression with 13
C3-labeled acrylamide as an internal
standard. Ions monitored for acrylamide are m/z 55, 44
and 27 and for the internal standard m/z 58. The ratio of
peak areas for m/z 55 (acrylamide) and m/z 58 (internal
standard) are compared to those for standards over the
standard curve range.
AFLATOXINS
Purpose: Applicable for the determination of aflatoxins B1,
B2, G1 and G2 in a wide range of matrices including
premixes, raw materials, food ingredients and finished feeds.
Method facts:
▶ Sample size: 50 g
▶ Limit of quantitation: 0.5 ppb for each aflatoxin compound
21
24. 22 Pricing available from a Covance representative. For updates to assays, refer to our online catalog at covance.com/nutri.
▶ Precision: NA
▶ Method reference: AOAC 991.31
Description: The sample is extracted with a mixture of
methanol:water. The extract is diluted with water and a
portion is applied to an antibody affinity column. The column
is washed first with water to remove major interferences.
The aflatoxins are eluted with acetonitrile and the eluent is
then dried under a stream of nitrogen. The aflatoxins are
derivitized with acid to form the highly fluorescent hemi-acetal
compounds. The sample extract is then quantified utilizing
HPLC by comparison to standards of known concentration.
AFLATOXIN (M1)
Purpose: This method is applicable to the analysis of aflatoxin
M1 in fluid milk, infant formulas and other matrixes.
Method facts:
▶ Sample size: 20 g of powder or 100 mL of liquid
▶ Limit of Quantitation: 0.1 ppb for M1 compound
▶ Precision: NA
▶ Method reference: Official Methods of Analysis for
AOAC International 2000.08
Description: The sample is extracted with a buffer solution.
The extract is diluted and a portion is applied to an antibody
affinity column. The column is washed first with water
to remove major interferences. The aflatoxins are eluted
with acetone and the eluent is then dried under a stream of
nitrogen. The aflatoxins are derivatized acid to form the highly
fluorescent hemi-acetal compounds. The sample extract is then
quantified utilizing high performance liquid chromatography
by comparison to standards of known concentration.
AFLATOXIN, OCHRATOXIN A AND
ZEARALENONE BY HPLC
Purpose: Applicable for the analysis of aflatoxins B1, B2, G1,
G2, ochratoxin A and zearalenone in a wide range of foods,
feeds and nutriceutical products.
Method facts:
▶ Sample size: 50 g
▶ Limit of quantitation: 0.5 ppb for each aflatoxin compound,
1.0 ppb for ochratoxin A and 10 ppb for zearalenone
▶ Precision: NA
▶ Method reference: AOAC 2005.08, AOAC 993.17, AOAC
999.07
Description: The mycotoxins are extracted from the sample
with a methanol/sodium bicarbonate mix. Following
extraction, the samples are cleaned with liquid-liquid partition
and antibody affinity solid phase extraction columns. The
mycotoxins are determined by HPLC-fluorescence detection,
utilizing post column photochemical derivitization to
determine aflatoxin B1 and G1.
ALACHLOR
see USP 561 and EP pesticides
ALDRIN AND DIELDRIN
see USP 561 and EP pesticides
ALLICIN (GARLIC)
see Garlic
ALMOND PROTEIN BY ELISA
Purpose: Allergens are proteins in food that can create an
immune response in sensitive individuals. Clear ingredient
labeling of food products, by manufacturers, aids in
protection from accidental ingestion by those individuals.
Testing for the presence of these proteins ensures the food is
free of the allergen at a potentially harmful level.
Method facts:
▶ Sample size: 50 g
▶ Limit of quantitation: 2.5 ppm
▶ Method reference: Veratox®
Quantitative Almond Allergen
Kits Neogen Corporation
Description: Allergen protein is extracted with a buffered salt
solution, allowed to settle then placed in an antibody (capture)
coated microwell to bind if present. All unbound protein is
washed away before a detector antibody (enzyme labeled) is
added. The detector antibody then binds to bound protein if
present. After another wash, and the addition of a substrate,
color develops as a result of bound detector antibodies. After
the addition of a stop reagent, the test is read with a Stat Fax
reader. Optical densities of the controls form a standard curve
which samples are plotted against to obtain concentrations of
allergen protein in ppm.
ALPHA CAROTENE
see beta Carotene
ASSAYS
25. ASSAYS
23800-675-8375 • covance.com/nutri
ALPHA LIPOIC ACID (USP)
Purpose: Applicable for the determination of alpha lipoic acid
in tablets, capsules, premixes and raw material.
Method facts:
▶ Sample size: 5 g
▶ Limit of quantitation: Most matrices - 0.025%
▶ Precision: On a drink preparation, the RSD is 6.37%
▶ Method reference: The United States Pharmacopoeia
Description: Sample extraction is performed by mechanical
shaking. A portion of the extract is analyzed by HPLC and UV
detection, then quantitated by comparing the response to a
standard curve of known concentration.
ALPHA TOCOPHEROL
Purpose: Applicable to the determination of tocopherols and
tocotrienols in foods, feeds and nutritional products. This
method may also be used at the supervisor’s discretion for
matrices outside of the intended scope.
Method facts:
▶ Sample size: 30 g in food products, 10 g in supplements
and premixes, 50 g in feeds
▶ Limit of quantitation: 0.500 mg/100 g, but may vary with
sample matrix
▶ Precision: On an infant formula matrix, the RSD is 6.10%
▶ Method reference: Speek, A.J., Schijver, J. and Schreurs,
W.H.P., Journal of Food Science, 50: 121-124 (1985)
(modified)
Cort, W.M., Vincente, T.S., Waysek, E.H. and Williams,
B.D., Journal of Agricultural Food Chemistry, 31: 1330-1333
(1983) (modified)
McMurray, C.H., Blanchflower, W.J. and Rice, D.A.,
Journal of the Association of Official Analytical Chemists,
63: 1258-1261 (1980) (modified)
Description: The product is typically saponified to break
down the fat and release the vitamins. The digest is then
extracted with organic solvent. Additionally, alternate
extraction procedures which do not require saponification
may be utilized for specified matrices. The tocopherols and
tocotrienols are quantitated by ultra or high performance
liquid chromatography (UHPLC or HPLC) using fluorescence
detection.
ALUMINUM (ICP)
see Inorganic analysis by ICP
ALUMINUM (GRAPHITE FURNACE)
Purpose: Applicable for the determination of aluminum in
foods, feeds, dietary supplements and biological materials.
Method facts:
▶ Sample size: 10 g
▶ Limit of quantitation: Most matrices - 0.5 ppm
▶ Precision: On a rice flour sample matrix with a certified
value of 4.4 ppm, the RSD is 5.85%
▶ Method reference: Standard Methods for the Examination
of Water and Wastewater, 20th Ed., Method 3113:
3-20-3-27, American Public Health Association,
Washington, D.C. (1998) (modified)
Description: An appropriately sized sample is wet-ashed with
nitric acid, hydrofluoric acid, and 30% hydrogen peroxide
using open vessel microwave digestion. The amount of
aluminum is determined by comparing the signal of the
unknown sample, measured by the graphite furnace atomic
absorption spectrophotometer, with the signal of the standard
solutions. A rhodium/magnesium matrix modifier is used in
the analysis.
AMINO ACID PROFILE (AOAC), TOTAL
Purpose: Applicable to all samples for the following amino
acids: Alanine, arginine, aspartic acid (including asparagine),
cystine, glutamic acid (including glutamine), glycine,
histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine, threonine, tyrosine,
tryptophan, valine. (An accurate protein value is
recommended to perform these analyses.)
Method facts:
▶ Sample size: 5 g
▶ Limit of quantitation: Most matrices - 0.1 mg/g
▶ Precision: On an infant formula matrix, examples of
typical RSDs are as follows: leucine - 1.9%, aspartic - 1.6%,
glutamic - 1.5%, alanine - 1.7%
▶ Method reference: AOAC 982.30
Description: The sample is hydrolyzed in hydrochloric acid
(HCl) and adjusted to pH 2.2 for all amino acids except
tryptophan. Tryptophan samples are hydrolyzed in sodium
hydroxide and adjusted to pH 5.2. Individual amino acids are
determined using an automated amino acid analyzer.
Cystine and methionine (AOAC) only
Tryptophan (AOAC) only
26. ASSAYS
24 Pricing available from a Covance representative. For updates to assays, refer to our online catalog at covance.com/nutri.
AMINO ACID PROFILE (AOAC), FREE BY AAA
Purpose: Applicable to all samples for the following amino
acids: alanine, arginine, aspartic acid (including asparagine),
cystine (including cysteine), glutamic acid (including
glutamine), glycine, histidine, hydroxyproline, isoleucine,
leucine, lysine, methionine, phenylalanine, proline, serine,
threonine, tyrosine, tryptophan and valine.
Method facts:
▶ Sample size: 10 g
▶ Limit of quantitation: Most matrices - 0.1 mg/g
▶ Precision: NA
▶ Method reference: AOAC Method 982.30
Description: The sample is extracted in a mild acid.
Determination is by an amino acid analyzer.
AMINO ACID PROFILE, FREE BY HPLC
Purpose: Applicable to all samples for the following amino
acids: L-aspartic acid, L-glutamic acid, L-serine, L-histidine,
glycine, L-threonine, L-arginine, L-alanine, L-tyrosine,
L-cystine, L-valine, L-methionine, L-phenylalanine,
L-isoleucine, L-leucine, L-lysine, proline, L-asparagine,
L-glutamine, L-citrulline, L-tryptophan and hydroxyproline.
Method facts:
▶ Sample size: 10 g
▶ Limit of quantitation: Most matrices - 0.1 mg/g
▶ Precision: On a corn sample, examples of typical RSDs
are as follows: proline 4.8%, glutamic acid 5.0%, histidine
4.6%, aspartic acid 3.1%.
▶ Method reference: Henderson, J.W., Ricker, R.D.,
Bidlingmeyer, B.A., Woodward, C., “Rapid, Accurate,
Sensitive, and Reproducible HPLC Analysis of Amino
Acids, Amino Acid Analysis Using Zorbax Eclipse-AAA
columns and the Agilent 1100 HPLC,” Agilent Publication,
2000
R. Schuster, “Determination of Amino Acids in Biological,
Pharmaceutical, Plant and Food Samples by Automated
Precolumn Derivitization and HPLC,” J. Chromatogr., 1988,
431, 271-284
Description: The sample is extracted in acid. Determination
is by high-performance liquid chromatography (HPLC) with
fluorescence or diode array detection. The primary amino
acids are derivitized with fluorenylmethyl chloroformate
before injection.
AMINO ACID PROFILE (HPLC), TOTAL
Purpose: Applicable to all samples for the following amino
acids: alanine, arginine, aspartic acid (including asparagine),
cystine (including cysteine), glutamic acid (including
glutamine), glycine, histidine, hydroxyproline, isoleucine,
leucine, lysine, methionine, phenylalanine, proline, serine,
threonine, tyrosine, tryptophan and valine.
Method facts:
▶ Sample size: 10 g
▶ Limit of quantitation: Most matrices - 0.1 mg/g
▶ Precision: On an infant formula, examples of typcial RSDs
are as follows: leucine 1.4%, aspartic acid 1.8%, tyrosine
2.5%, methionine 2.9%.
▶ Method reference: Henderson, J.W., Ricker, R.D.,
Bidlingmeyer, B.A., Woodward, C., “Rapid, Accurate,
Sensitive, and Reproducible HPLC Analysis of Amino
Acids, Amino Acid Analysis Using Zorbax Eclipse-AAA
columns and the Agilent 1100 HPLC,” Agilent Publication,
2000
Barkholt and Jenson, “Amino Acid Analysis: Determination
of Cystine plus Half-Cystine in Proteins after Hydrochloric
Acid Hydrolysis with a Disulfide Compound as Additive,”
Analytical Biochemistry, 1989, 177, 318-322
R. Schuster, “Determination of Amino Acids in Biological,
Pharmaceutical, Plant and Food Samples by Automated
Precolumn Derivitization and HPLC,” J. Chromatogr., 1988,
431, 271-284
Description: The samples are hydrolyzed in 6 N hydrochloric
acid for 24 hours at approximately 110°C. Phenol is added
to the 6 N hydrochloric acid to prevent halogenation
of tyrosine. Cystine and cysteine are converted to S-2-
carboxyethylthiocysteine by the addition of dithiodipropionic
acid. Tryptophan is hydrolyzed from proteins by heating at
approximately 110°C in 4.2 N sodium hydroxide.
The samples are analyzed by HPLC after pre-injection
derivitization. The primary amino acides are derivitized with
o-phthalaldehyde (OPA) and the secondary amino acids are
derivitized with fluorenylmethyl chloroformate (FMOC)
before injection.
P-ANISINIDE VALUE
Purpose: Applicable for the determination of p-anisinide
value in all normal fats and oils. This method determines the
amount of aldehydes in animal and vegetable oils.
27. ASSAYS
25800-675-8375 • covance.com/nutri
Method facts:
▶ Sample size: 20 g
▶ Limit of quantitation: NA
▶ Precision: NA
▶ Method reference: AOCS Cd 18-90
Description: The sample is diluted in isooctane and read on a
spectrophotometer. The sample is then spiked and
re-read on a spectrophotometer to determine the amount of
aldehydes in the sample.
ANTHOCYANINS, TOTAL
Purpose: Applicable for the determination of total
anthocyanin content in blueberry, cranberry, bilberry and
other fruit extracts in tablets, capsules, premixes and raw
material.
Method facts:
▶ Sample size: 5 g
▶ Limit of quantitation: 0.01%
▶ Precision: Varies with matrix
▶ Method reference: Givisti, M.M., Molar Absorptivity
and Color Characteristics of Acylated and Non-Acylated
Pelrgonidin-based Anthocyanins, J. Agri and Food
Chemistry, 47(11):4631-4637 (1999)
Description: Sample extraction is performed with water and
sonication. Portions of that extract are subjected to buffers
with differing pH. Aliquots are analyzed for absorbance on
a spectrophotometer. The difference in absorbance and the
molar absorbtivity coefficient of cyanidin-3-glucoside
determine total anthocyanin content.
ANTHOCYANINS BY HPLC
Purpose: Applicable for the determination of anthocyanin
content in blueberry, cranberry, bilberry and other fruit
extracts in tablets, capsules, premixes and raw material.
Method facts:
▶ Sample size: 5 g
▶ Limit of quantitation: Most matrices- 10 ppm
▶ Precision: Varies with matrix
▶ Method reference: Indena Method 29/04/LRA1-00
Description: Samples are extracted in an acidic methanol
solution with sonication. Separation is achieved with reversed-
phase HPLC and a C18 column with detection set at 535 nm.
Twenty-one anthocyanins and anthocyanidins are identified
and quantified by comparing to cyanidin chloride and
cyanidin-3-O-glucoside, respectively. The final result for each
compound is then converted by the molecular weight ratio.
ARSENIC
see Inorganic analysis by ICP-MS
ARSENIC SPECIATION
Purpose: This method is applicable to the determination of
organic and inorganic arsenic, as well as the most common
individual species in rice, rice products and juice. Both of the
highly toxic inorganic species arsenite (As(+3)) and arsenate
(As(+5)), and the organic species dimethylarsinic acid (DMA)
and monomethylarsonic acid (MMA) can be determined.
Of the less common organic species in rice and juice,
arsenocholine (AsC) is able to be determined individually,
while arsenobetaine (AsB) and trimethylarsine oxide (TMAO)
both elute in the void volume together and are not able to be
determined individually*.
* Note: While the sum of organic arsenic will include the
presence of any species that elute at this time, they will be
calculated using AsB as the reference standard.
Method facts:
▶ Sample size: 2 g
▶ Limit of quantitation: 10.0 ppb in rice and products
containing rice 2.00 ppb in juices
▶ Precision: In rice products the RSD is 4.8%. In juices the
RSD is 3.3%.
▶ Method reference: FDA Elemental Analysis Manual
[Internet]. Silver Spring (MD): Food and Drug
Administration (US); Section 4.11 [Version 1.1; 2012
November] Arsenic Speciation in Rice and Rice Products
Using High Performance Liquid Chromatography-
Inductively Coupled Plasma-Mass Spectrometric
Determination
Kutscher, D., McSheehy, S., Wills, J., Jensen, D. IC-ICP-MS
Speciation Analysis of As in Apple Juice using the Thermo
Scientific iCAP Q ICP-MS. Thermo Scientific Application
Note 43099, 2012
Description: Various arsenic species are extracted from
samples in plastic vessels with dilute nitric acid using oven
digestion and then filtered prior to analysis. The amount
of any given arsenic species is determined using an ion
chromatography - inductively coupled plasma - mass
spectrometer (IC-ICP-MS) by comparing the peak area
generated for the samples to those generated by standards of
known concentration.
28. ASSAYS
ASCORBIC ACID
see Vitamin C
L-ASCORBYL-2-PHOSPHATE
(2-phospho-L-ascorbic acid)
Purpose: Applicable to the quantitation of L-ascorbyl-2-
phosphate in foods, feeds and premixes. Also applicable to
the various salts of the acid. This method does not determine
the di- or tri-phosphate forms of ascorbic acid.
Method facts:
▶ Sample size: 25 g
▶ Limit of quantitation: 50 mg/kg
▶ Precision: 2.25% on a dry dog food control
▶ Method reference: Simultaneous HPLC Analysis of
L-Ascorbic Acid, L-Ascorbyl-2-Sulfate, and L-Ascobyl-2-
Polyphosphate, Journal of Liquid Chromatography and
Related Technology, 19(19):3105-3118 (1996), (modified)
Description: The sample is extracted with an aqueous
phosphate buffer solution. The extract is then filtered, pH
adjusted and injected onto an HPLC system equipped with
a Phenomenex, Inertsit ODS-2, 250 mm x 4.6 mm, 5 µL
column, held in an oven set at 30.0°C. The analyte is eluted
with a phosphate buffer, acetonitrile and ethanol mobile
phase. The amount of L-ascorbyl-2-phosphate is detected
using an ultraviolet absorbance detector.
ASH
Purpose: Applicable for the determination of ash in most
foods and feeds.
Method facts:
▶ Sample size: 5 g
▶ Limit of quantitation: Most matrices - 0.1%
▶ Precision: On a dog food matrix, the RSD is 5.0%
▶ Method reference: AOAC 923.03
Description: Organic matter is burned off by igniting the
sample at 550°C in an electric furnace. The remaining
material is determined gravimetrically and referred to as ash.
ASPARTAME AND ACESULFAME K, DKP
AND SACCHARINE
Purpose: Applicable for the determination of aspartame,
diketopiperazine (DKP), acesulfame K and saccharine in food
and beverages. It may not be applicable for samples with a
high protein content.
Method facts:
▶ Sample size: 25 g
▶ Limit of quantitation: Most matrices - 50 ppm
▶ Precision: Varies with matrix
▶ Method reference: Prodolliet, J., and Bruelhart, M.,
Determination of Aspartame and its Major Decomposition
Products in Foods. J. of AOAC, 76(2) (1993)
Description: Samples are weighed, then dissolved in a
phosphate buffer/methanol solution. The solutions are
injected on an HPLC equipped with a UV detector. Samples
are calculated against standards of known concentration.
ASTAXANTHIN
Purpose: This method is applicable to the determination
of free or total astaxanthin (sum of free and esterified) in
nutritional supplements and fish.
Method facts:
▶ Sample size: 10 g
▶ Limit of quantitation: 0.200 mg/100 g for softgels, and
0.600 mg/100 g for salmon flesh
▶ Method reference: Britton, G., Liaaen-Jensen, S., Pfander,
H. (1995). Carotenoids Volume 1A: Isolation and Analysis.
Basel, Switzerland: Birkhäuser Verlag
Fuji Chemical Industry Co., LTD., “Spectrophotometric
and HPLC Analysis Method for Determining Astaxanthin
Content in AstaREAL®
-P2AF” [online] Ver. June 1918,
Http://www.fujihealthscience.com, Fuji Health Science
Inc., (accessed 29 Dec. 2011)
Description: Esterified astaxanthin is hydrolyzed
(de-esterified) by an enzymatic procedure to yield all free
astaxanthin. The sample is then injected on a normal phase
HPLC (high-performance liquid chromatograph) system with
visible (VIS) light detection. Results are quantified using an
internal standard and linear regression analysis.
26 Pricing available from a Covance representative. For updates to assays, refer to our online catalog at covance.com/nutri.
29. ASSAYS
BHA, BHT AND TBHQ (ANTIOXIDANTS)
Purpose: Applicable for the determination of Butylated
Hydroxyanisole (BHA)/Butylated Hydroxytoluene
(BHT)/tert-Butylhydroquinone.
Method facts:
▶ Sample size: 25 g
▶ Limit of quantitation: Varies with matrix
▶ Precision: 5% for most matrices
▶ Method reference: Official Methods of Analysis of AOAC
INTERNATIONAL (2000) 17th Ed., AOAC
INTERNATIONAL, Gaithersburg, MD, USA, Official
Method 968.17 (modified)
Description: The lipid is extracted from food and feed
samples and diluted. BHA, BHT and TBHQ are quantified by
gas liquid chromatography (GLC).
BENZENE
Purpose: Applicable for the determination of benzene in most
matrices.
Method facts:
▶ Sample size: 10 g
▶ Limit of quantitation: Most matrices - 0.02 mg/100 g
▶ Precision: Varies with matrix
▶ Method reference: Bertsch, Brian, “Developing One
Universal Method for Residual Solvents Using the New
Teledyne Tekmar HT3 Headspace Sample Introduction
System,” Application Note (Teledyne Tekmar), Document
#HT3-001, September 2005
Description: The samples are introduced to a gas
chromatograph-mass spectrometer (GC-MS) via a Tekmar
HT3 purge-and-trap headspace autosampler.
BENZOIC ACID (SODIUM BENZOATE), SORBIC
ACID (POTASSIUM SORBATE)
see Organic acid profiles - option 1
BERBERINE
see Goldenseal
BETA CAROTENE
Purpose: Applicable for the determination of alpha and beta
carotene in food, feeds and dietary supplements.
Method facts:
▶ Sample size: 10 g
▶ Limit of quantitation: Most matrices - 0.02 mg/100 g
▶ Precision: On an infant formula matrix, the RSD is 7.4%
▶ Method reference: AOAC 941.15
Quackenbush, F. W., Journal of Liquid Chromatography,
10:643-653 (1987)
Description: Food products are de-esterified with potassium
hydroxide and extracted with hexane. Juice products are
blended with alcohol and extracted. Nutraceutical products
are enzymatically treated and then extracted. The extracts are
injected on a reverse-phase HPLC system equipped with UV
detection and compared to a standard curve.
BETA-GLUCAN
Purpose: Applicable for the determination of beta-glucan
in food products, with the exception of yeast and mushroom
products.
Method facts:
▶ Sample size: 2 g
▶ Limit of quantitation: Most matrices - 0.5%
▶ Precision: On a cereal sample matrix, the RSD is 4.81%
▶ Method reference: AOAC 995.16
Description: beta-Glucan is hydrolyzed to oligosaccharides by
lichenase. These oligosaccharides are reduced to glucose by
beta glucosidase. The glucose is reacted with a glucose
oxidase/peroxidase reagent mixture, and the reaction product
is determined spectrophotomerically at 510 nm.
BILBERRY
see Anthocyanins, total
see Anthocyanins by HPLC
BIOTIN
Purpose: Applicable for the determination of biotin in most
foods, feeds and dietary supplements.
Method facts:
▶ Sample size: 2 g
▶ Limit of quantitation: Most matrices - 0.005 mcg/g
▶ Precision: On an infant formula matrix, the RSD is 7.1%
27800-675-8375 • covance.com/nutri
30. ASSAYS
▶ Method reference: Wright Skeggs, Procedures of the
Society of Experimental Biology and Medicine, 56:95 (1944)
Schiner Deritter, “Biotin Content of Feed Stuffs,” Journal
of Agricultural Food Chemistry, 23:1157-1162 (1975)
Methods of Analysis for Infant Formulas, Infant Formula
Council (1985)
Journal of the AOAC, 49:882, (1996)
Description: The sample is extracted with either water, dilute
sodium hydroxide (NaOH), or sulfuric acid (H2
SO4
). The
amount of biotin is determined by comparing the growth
response of the sample, using the bacteria Lactobacillus
plantarum, with the growth response of a biotin standard.
This response is measured turbidimetrically.
BISPHENOL A
Purpose: Applicable to analysis of Bisphenol A (BPA) in infant
formula, infant formula concentrates, baby food and various
food matrices. BPA as part of a packaging material analysis is
available upon request.
Method facts:
▶ Sample size: 10 g
▶ Limit of quantitation: 0.5 ppb in infant formula;
LOQ varies by sample matrix
▶ Precision: NA
▶ Method reference: Determination of Bisphenol A in Liquid
Infant Formula by Solid Phase Extraction with Acetic
Anhydride Derivatization and Gas Chromatography-Mass
Spectrometry, Minister of Health Canada (2008).
Description: The sample is diluted with water and
acetonitrile, then mixed and centrifuged. The supernatant
is diluted with a phosphate buffer and passed through a
solid phase extraction column (Varian 1210-2052, 500 mg
C18, 6 mL). The column is eluted with an aqueous solution
of acetonitrile and the extract concentrated. The sample is
derivatized and extracted into iso-octane and MTBE, then
concentrated to near dryness and diluted with toluene. The
BPA is quantified and confirmed by GC/MS on an HP-5ms
column.
BLACK CURRANT OIL (LINOLENIC ACID AND GLA)
see Fatty acid profiles
BOMB CALORIMETRY
Purpose: Applicable for the determination of total calories in
foods and feeds.
Method facts:
▶ Sample size: 2 g for solids, 7 g for liquids
▶ Limit of quantitation: Most matrices - 5calories/g
▶ Precision: On a benzoic acid matrix, the RSD is 0.67%
▶ Method reference: Metals Energy Mining Agriculture
Geology, AC-350 Instruction Manual, LECO Corporation,
St. Joseph, MI, pp 5-26 through 5-29 (2002)
Description: Calories are determined by burning a weighed
sample in an atmosphere of oxygen inside a bomb submerged
in a measured quantity of water. The temperature rise of the
water, resulting from the combustion of the sample, is used to
calculate the number of calories liberated.
BROMIDE, INORGANIC
Bromine containing fumigants determined as total inorganic
bromide
Purpose: Applicable to the determination of inorganic
bromide in dietary supplements (raw materials, ingredients
and soft gels), high fat matrices and plant materials.
Method facts:
▶ Sample size: 25 g
▶ Limit of quantitation: Dry samples - 5 μg/g, moist
or liquid samples - 1 μg/g
▶ Method reference: Community Reference Laboratory for
Single Residue Methods, CVUA, Stuttgart, Schaflandstr.
3/2, 70736 Fellbach, Germany
T. Stijve, Gas Chromatographic Determination of
Inorganic Bromide Residues - a Simplified Procedure,
Dtsch. Lebenm. Rundsch. 77 99-101 (1981)
Deutsche Forschungsgemeinschaft (DFG), Manual of
Pesticide Residue Analysis, Volume I, by Verlag Chemie,
1987. The bromide method has the code S 18. ISBN
3-527-27010-8
Description: The samples are homogenized and suspended
in an acidified aqueous solution of propylene oxide, with
bromide being simultaneously extracted and derivatized into
1-bromo-2-propanol and 2-bromo-1-propanol. The derivatives
are partitioned into ethyl acetate and determined by GC-ECD.
28 Pricing available from a Covance representative. For updates to assays, refer to our online catalog at covance.com/nutri.
31. ASSAYS
CADMIUM (ICP OR ICP-MS)
see Inorganic analysis by ICP or ICP-MS
CAFFEIC ACID
see Phenolic acid
CAFFEINE, THEOBROMINE, THEOPHYLLINE
Purpose: Applicable for the determination of caffeine,
theobromine and theophylline in various food and
supplements.
Method facts:
▶ Sample size: 25 g
▶ Limit of quantitation: Most matrices - 50 ppm
▶ Precision: On a cocoa powder matrix, the RSD is 5.42% for
caffeine and 3.62% for theobromine
▶ Method reference: Journal of Food Science,
48:745-747 (1983)
Description: The sample is extracted with boiling water.
A portion of the extract is injected onto an HPLC system with
UV detection set at 272 nm. The areas of the peaks
are determined and compared with those obtained from
injected standards.
CALCIUM (ICP)
see Inorganic analysis by ICP
CALORIES, BY CALCULATION
Purpose: Used to calculate calories in foods and feeds.
Method facts:
▶ Sample size: NA
▶ Limit of quantitation: 1.7 calorie
▶ Precision: NA
▶ Method reference: 21 CFR 101.9
Description: Calories = (Protein(g) x 4) +
(Carbohydrates(g) x 4) + (Fat(g) x 9) or calculated by specific
Atwater factors or other corrections by request.
CALORIES FROM FAT, BY CALCULATION
Purpose: Used to calculate calories from fat.
Method facts:
▶ Sample size: NA
▶ Limit of quantitation: 0.9 calorie from fat
▶ Precision: NA
▶ Method reference: 21 CFR 101.9
Description: Calories from fat = Fat(g) x 9
CAPSAICIN/CAPSAICINOID (SCOVILLE HEAT)
Purpose: Applicable for the determination of pungency levels
in capsicums and their oleoresins.
Method facts:
▶ Sample size: 30 g
▶ Limit of quantitation: Most matrices ~10 ppm
▶ Precision: On a powder matrix, the RSD is 5.40%
▶ Method reference: AOAC 995.03
Description: The sample is extracted in warm ethanol.
The extract is filtered and then injected using HPLC
equipped with an ultraviolet detector. Results are typically
expressed in Scoville units.
CARBAMATE PESTICIDES (LC-MS/MS)
Purpose: Applicable but not limited to, the analysis of selected
carbamate pesticides and related metabolites in food and
dietary supplement products and raw materials.
Method facts:
▶ Sample size: 25 g
▶ Limit of quantitation: Listed in the table to the right
▶ Precision: Varies with matrix
▶ Method reference:
1
AOAC Official Method 2007.01, Pesticide Residues in
Foods by Acetonitrile Extraction and Partitioning with
Magnesium Sulfate
2
CEN Standard Method EN 15662: Food of plant origin –
Determination of pesticide residues using GC-MS and/or
LC-MS/MS following acetonitrile extraction/partitioning
and clean-up by dispersive SPE – QuEChERS method
3
Anastassiades, M.; Lehotay, S.J.; Stajnbaher, D.; Schenck,
F.J. Fast and easy multiresidue method employing
acetonitrile extraction/partitioning and “dispersive
solid-phase extraction” for the determination of pesticide
residues in produce. Journal of AOAC INTERNATIONAL
2003, 86, 412-431
4
Lehotay, S.J.; Mastovska, K; Lightfield, A.R. Use of
buffering and other means to improve results of
problematic pesticides in a fast and easy method for
residue analysis of fruits and vegetables. Journal of AOAC
INTERNATIONAL 2005, 88, 615-629
29800-675-8375 • covance.com/nutri
32. ASSAYS
5
Lehotay, S.J.; Mastovska, K.; Yun, S. J. Evaluation of two
fast and easy methods for pesticide residue analysis in
fatty food matrixes. Journal of AOAC INTERNATIONAL
2005, 88, 630-638
6
Mastovska, K.; Dorweiler, K.; Lehotay, S.J.; Wegscheid,
J.; Szpylka, K. Pesticide multiresidue analysis in cereal
grains using modified QuEChERS method combined with
automated direct sample introduction GC-TOFMS and
UPLC-MS/MS techniques. J. Agric. Food Chem., 2010,
58, 5959-5972
Description: The sample extraction and clean-up are based on
the QuEChERS method.1-6
The final extract is analyzed using
liquid chromatography-tandem mass spectrometry
(LC-MS/MS). Typical reporting limits are listed in the table
below. Other reporting limits (at or above LOQ for a given
carbamate-matrix combination) can be used based on client
requests:
CARBOHYDRATES, TOTAL, BY CALCULATION
Purpose: Applicable for the determination of carbohydrates in
foods and feeds.
Method facts:
▶ Sample size: NA
▶ Limit of quantitation: Most matrices - 0.4%
▶ Precision: NA
▶ Method reference: 21 CFR 101.9
Description: Carbohydrate = 100 - [protein(g) + fat(g) + ash(g)
+ moisture(g)]
L-CARNITINE
Purpose: Applicable for the quantitation of L-carnitine in
infant formulas, powders and premixes.
Method facts:
▶ Sample size: 25 g
▶ Limit of quantitation: 0.50 mg/100 g
▶ Precision: 4.15% on an infant formula control
▶ Method reference: Starey et al.; Journal of AOAC
INTERNATIONAL vol. 91, No. 1, 2008 (modified)
Description: The sample is diluted in water and a deuterated
analog of carnitine is added as an internal standard. It is
then filtered and injected onto a Waters Symmetry C8, 50
mm x 2.1 mm, 3.5 µm column, held in an oven set at 30.0°C.
Three mobile phases are used in a gradient consisting of
0.1% heptafluorobutyric acid (HFBA) in water, 0.1% HFBA
in methanol and 80% water/20% methanol. The amount of
L-carnitine is determined using HPLC-MS/MS.
CAROTENOIDS
Option 1: alpha Carotene, beta Carotene, Lycopene
Option 2: Cryptoxanthin, Lutein, Zeaxanthin
Option 3: Lutein only
Purpose: Applicable to the determination for the following
carotenoids in foods, feeds, premixes, pharmaceutical and
nutrition supplements: lycopene, α and β-carotene, free
lutein, lutein esters, β-cryptoxanthin, or zeaxanthin.
Vitamin A from carotenes may be calculated.
Method facts:
▶ Sample size: 25 g for food or feed products; 2 g for
supplements and premixes
Carbamate Pesticide LOQ (mg/kg)
Aldicarb 0.01
Aldicarb sulfone 0.01
Aldicarb sulfoxide 0.01
Bendiocarb 0.01
Butocarboxim 0.02
Butoxycarboxim 0.01
Carbaryl 0.01
Carbofuran 0.01
Carbofuran, 3-hydroxy- 0.01
Chlorpropham 0.01
Dioxacarb 0.01
Ethiofencarb 0.01
Fenobucarb 0.01
Fenoxycarb 0.01
Indoxacarb 0.02
Iprovalicarb 0.01
Isoprocarb 0.01
Methiocarb 0.01
Methiocarb sulfone 0.01
Methiocarb sulfoxide 0.01
Methomyl 0.02
Metolcarb 0.01
Oxamyl 0.01
Promecarb 0.01
Propham 0.01
Propoxur 0.01
Thiofanox 0.05
30 Pricing available from a Covance representative. For updates to assays, refer to our online catalog at covance.com/nutri.
33. ASSAYS
▶ Limit of quantitation: 0.0200 mcg/100 g, but can vary by
sample size
▶ Precision: Varies with matrix
▶ Method reference: Official Methods of Analysis of AOAC
INTERNATIONAL, Current Ed., Method 2005.07, AOAC
INTERNATIONAL, Gaithersburg, MD, USA (modified)
Official Methods of Analysis of AOAC INTERNATIONAL,
Current Ed., Methods 941.15, 2005.07, AOAC
INTERNATIONAL, Gaithersburg, MD, USA (modified)
Quackenbush, F. W., Reverse Phase HPLC Separation of
cis- and trans-Carotenoids and its Application to
Beta Carotenes in Food Materials,” Journal of Liquid
Chromatography, 10: 643-653 (1987) (modified)
Description: Low-fat samples are extracted with alcohol
and/or tetrahydrofuran. Samples with a higher level of fat
are saponified and extracted with hexane. Each sample is
then injected on a reverse phase high-performance liquid
chromatography system (HPLC) with ultraviolet (UV)
detection. Quantitation is achieved with a linear regression
analysis.
CATECHINS
Epigallocatechin, catechin, epigallocatechin gallate,
gallocatechin gallate, gallic acid, epicatechin, epicatechin
gallate, gallocatechin, catechin gallate.
Purpose: Applicable for the determination of catechins
in various matrices including extracts (e.g. grape seed), tea,
fortified products and premixes.
Method facts:
▶ Sample size: 5 g
▶ Limit of quantitation: Most matrices-100 ppm, liquids-1 ppm
▶ Precision: On a green tea matrix, the RSD is 4.09%
▶ Method reference: Sakakibara, H., Honda, Y., Nakagawa,
S., Ashida, H., and Kanazawa, K., “Simultaneous
Determination of All Polyphenols in Vegetables, Fruits, and
Teas,” Journal of Ag Food Chem., 51(3):572-580 (2003)
Description: Sample extraction is performed using a
buffered solution. A portion of that extract is analyzed on
a HPLC using UV detection. Analytes are then quantitated
by comparing the response of a standard of known
concentration.
CHLORIDE
Purpose: Applicable for the determination of acid-soluble
chloride in feeds, plants, food products and tissues.
Method facts:
▶ Sample size: 10 g
▶ Limit of quantitation: Most matrices - 200 ppm
▶ Precision: On a cereal matrix, the RSD is 1.01%
▶ Method reference: AOAC 963.05, 969.10, 971.27 (modified)
Description: Samples are weighed, double-deionized water is
added and the solution is mixed thoroughly and made acidic
with nitric acid. Chloride is determined potentiometrically
by titrating with a silver nitrate solution to a predetermined
endpoint.
CHLOROGENIC ACID
Purpose: Applicable for the determination of chlorogenic acid
in raw material, extracts and dietary supplement preparations.
Method facts:
▶ Sample size: 5 g
▶ Limit of quantitation: 50 ppm, 1 ppm for liquids
▶ Precision: 5.02%
▶ Method reference: Journal of Ag Food Chem, 51(3):572-580
Description: The sample is extracted in a variety of ways
depending upon the matrix. A portion of the extract is
injected into an HPLC using UV detection. Chlorogenic acid
is quantified by comparing the response to a known standard.
CHLOROPHYLL
Purpose: Applicable to the determination of total chlorophyll
content derived from plants.
Method facts:
▶ Sample size: 25 g
▶ Limit of quantitation: Most matrices - 0.01 mg/g
▶ Precision: NA
▶ Method reference: AOAC Official Methods of Analysis
of AOAC International (2006) 18th Edition, AOAC
INTERNATIONAL, Gaithersburg, MD, USA, Official
Method 942.04. (modified)
31800-675-8375 • covance.com/nutri
34. ASSAYS
Description: Chlorophyll is extracted from the sample with
acetone. The sample extract is then washed with ethyl ether
in a separatory funnel. The chlorophyll migrates to the ethyl
ether layer and the remaining acetone is removed through
a series of water rinses. The ethyl ether extract is then read
at 642.5 and 660 nm on a UV-VIS spectrophotometer to
determine the total chlorophyll content.
CHOLESTEROL
Purpose: Applicable for the determination of cholesterol in
most matrices including foods, feeds and fecal samples.
Method facts:
▶ Sample size: 5 g
▶ Limit of quantitation: 1 mg/100 g
▶ Precision: On a ground waffle matrix, the RSD is 6.0%
▶ Method reference: AOAC 994.10
Description: The sample is saponified using ethanolic
potassium hydroxide. The unsaponifiable fraction that
contains cholesterol and other sterols is extracted with
toluene. The toluene is evaporated to dryness and the
residue is dissolved in dimethylformamide (DMF). The
samples are derivitized to form trimethylsilyl ethers. The
derivitized cholesterol is quantitatively determined by gas
chromatography using 5 a-cholestane as an internal standard.
CHOLINE (CHEMICAL)
Purpose: Applicable for the determination of choline in
premixes, infant formula, dietary supplements and feeds.
Method facts:
▶ Sample size: 10 g
▶ Limit of quantitation: Most matrices - 15.1 mg/100 g;
liquid samples - 3.3 mg/100 g
▶ Precision: On a powdered infant formula matrix, the
RSD is 6.31%
▶ Method reference: Glick, Journal of Biological Chemistry,
156:643 (1944)
Description: The sample is hydrolyzed with mild acid.
The resulting solution is purified on an ion exchange
column. The concentration of choline is determined
spectrophotometrically from a reineckate coupling reaction.
CHOLINE (ENZYMATIC)
Purpose: Applicable for the determination of choline in milk,
infant formula, foods and beverages.
Method facts:
▶ Sample size: 5 g
▶ Limit of quantitation: Most matrices - 25 mg/100 g
▶ Precision: On an infant formula matrix, the RSD is 3.78%
▶ Method reference: AOAC 999.14
Description: The product is hydrolyzed at 70°C to release
bound choline. Following pH adjustment, residual choline
phospholipids are cleaved with phospholipase D and free
choline is subjected to choline oxidase with liberation of
peroxide. In the presence of peroxidase, phenol is oxidized and a
quinoneimine chromiphore is formed with 4-aminoantipyrine.
Absorbance is measured and choline content calculated by
interpolation from a multilevel calibration.
CHONDROITIN SULFATE BY CPC
Purpose: Applicable for the determination of chondroitin
sulfate in tablets, premixes, capsules and other dietary
supplements. Sources may include porcine, bovine or shark.
Method facts:
▶ Sample size: 5 g
▶ Limit of quantitation: 25 mg/g
▶ Precision: On a tablet, the RSD is 1.87%
▶ Method reference: USP
Description: Sample extraction is performed with water and
agitation. Extracts are analyzed on an auto-titrator equipped
with a light emitting phototriode. Samples are calculated
against a standard curve of known concentrations.
CHROMIUM (ATOMIC ABSORPTION)
Purpose: Applicable for the determination of chromium
in feeds, animal tissues, food products, plants, dietary
supplements, water, soils and minerals.
Method facts:
▶ Sample size: 15 g
▶ Limit of quantitation: Most matrices - 2 ppm
▶ Precision: Based on a powdered drink control, the RSD is
1.90%
32 Pricing available from a Covance representative. For updates to assays, refer to our online catalog at covance.com/nutri.
35. ASSAYS
▶ Method reference: Analytical Methods for Atomic
Absorption Spectrophotometry, Perkin-Elmer: Norwalk, CT
(2000)
AOAC 974.27
Description: The sample can either be dry-ashed, wet-ashed
or read directly. If dry-ashed, the sample is ashed at 500°C
±50° until ashing is complete. The resulting ash is treated
with concentrated hydrochloric acid, dried and redissolved
in hydrochloric acid solution. If wet-ashed, the sample is
digested on a hot plate with nitric acid, hydrochloric acid
and/or hydrogen peroxide.
The amount of chromium is determined by comparing the
signal of the unknown sample, measured by the atomic
absorption (AA) spectrophotometer, with the signal of the
standard solutions.
CHROMIUM (ICP OR ICP-MS)
see Inorganic analysis by ICP or Inorganic analysis by
ICP-MS
CITRIC ACID
see Organic acid profiles - option 2
CITRUS BIOFLAVONOIDS
Purpose: This method is applicable to the determination
of common citrus bioflavonoids: rutin, narirutin, naringin,
hesperidin, neohesperidin, quercetin, naringenin, hesperetin,
nobiletin and tangeretin in dietary supplements (tablets,
capsules and powders).
Method facts:
▶ Sample size: 10g
▶ Limit of quantitation: 1.2 mg/g
▶ Method reference: Covance developed method
Description: Each sample is sonicated in dimethyl sulfoxide,
methanol is added and the mixture is again sonicated.
The extract is analyzed by ultra high performance liquid
chromatography (UHPLC) with variable wavelength
detection.
COBALT (ICP OR ICP-MS)
see Inorganic analysis by ICP or Inorganic analysis by
ICP-MS
COENZYME Q10
Purpose: Applicable for the determination of coenzyme
Q10 in dietary supplements and raw material.
Method facts:
▶ Sample size: 10 g
▶ Limit of quantitation: Most matrices - 5 mg/100 g
▶ Precision: Based on a powdered drink control, the RSD is
1.90%
▶ Method reference: AOAC 2008.07
Description: Coenzyme Q10 is extracted using alcohol and
hexane. The extract is then injected on a reverse-phase HPLC
system using UV detection for quantitation.
COLORS, ARTIFICIAL
Purpose: This method is designed to demonstrate the
absence of food coal tar colorants:
▶ FDC Red No. 2 (Amaranth)
▶ FDC Red No. 3 (Erythrosin B)
▶ FDC Red No. 4 (Ponceau SX)
▶ FDC Red No. 40 (Allura Red)
▶ FDC Green No. 3 (Fast Green FCF)
▶ FDC Blue No. 1 (Erigluacine)
▶ FDC Blue No. 2 (Indigo Carmine)
▶ Acid Orange 12 (Crocein Orange G)
▶ FDC Yellow No. 5 (Tartrazine)
▶ FDC Yellow No. 6 (Sunset Yellow FCF)
▶ FDC Yellow No. 10 (Quinoline Yellow)
Method facts:
▶ Sample size: 10 g
▶ Limit of quantitation: 5 ppm
▶ Precision: NA
▶ Method reference: Canadian Food Inspection Agency;
HPLC Identification and Quantification of Synthetic Food
Colours in Aqueous Media; LCAQ-016-05; 2009 (modified)
Description: Samples are extracted in a basic solution and
then injected on an HPLC equipped with a UV detector
capable of reading at multiple wavelengths. Samples are
then calculated against standards of a known concentration.
Analytes detected: FDC Red No. 2, FDC Red No. 3, FDC
33800-675-8375 • covance.com/nutri
36. ASSAYS
Red No. 4, FDC Red No. 40, FDC Green No. 3, FDC Blue
No. 1, FDC Blue No. 2, Acid Orange 12, FDC Yellow No. 5,
FDC Yellow No. 6, FDC Yellow No. 10.
CONJUGATED LINOLEIC ACID (CLA)
Purpose: This procedure is used to quantitate conjugated
linoleic acids in foods and feeds.
Applicable for foods and feed containing at least 0.2% lipids.
The method detects fatty acids having 8 to 22 carbon atoms
as methyl esters. The conditions specified in this method are
not suitable for determining epoxy or oxidized fatty acids that
have been polymerized.
Method facts:
▶ Sample size: 4 g, 1 g if oil
▶ Limit of quantitation: Most matrices - 0.1%
▶ Precision: The RSD value for 18:1 conjugated in an oil
control sample is 2.76%.
▶ Method reference: AOAC 996.06/AOCS Ce 1k-07/
Ce 2-66
Description: The lipid is extracted, saponified with 0.5 N
methanolic sodium hydroxide, and methylated with 14%
BF3-methanol. The resulting methyl esters of the fatty acids
are extracted with heptane containing an internal standard.
The methyl esters of the fatty acids are analyzed by gas
chromatography using external standards for quantitation.
COPPER (ICP OR ICP-MS)
see Inorganic analysis by ICP or ICP-MS
COUMARIC ACID
see Phenolic acids
CRANBERRY (QUINIC, MALIC, CITRIC ACIDS)
see Organic acid profiles
CRANBERRY
see Anthocyanins, total
see Anthocyanins by HPLC
CREATINE
Purpose: Applicable for the determination of creatine and
creatinine in dietary supplements, sports nutrition products,
etc.
Method facts:
▶ Sample size: 25 g
▶ Limit of quantitation: 0.5%
▶ Precision: On a creatine capsule, the RSD is 1.87%
▶ Method reference: Analytical Biochemistry, 214, pp278-283
Description: The sample is extracted with water. The extract
is injected into an HPLC using UV detection. Results are
calculated against a standard of known concentration.
CRYPTOXANTHIN
see Carotenoids
CYANOCOBALAMIN
see Vitamin B12
CYANURIC ACID (LC-MS/MS)
Purpose: Applicable for the determination of cyanuric acid
using LC-MS/MS in food products.
Method facts:
▶ Sample size: 25 g
▶ Limit of quantitation: 0.5 ppm Cyanuric Acid
▶ Precision: 5% RSD at 0.5 ppm
▶ Method reference: United States Food and Drug
Administration, Interim Method for Determination of
Melamine and Cyanuric Acid Residues in Food using
LC-MS/MS: Version 1.0 Laboratory Information Bulletin
No. 4422 (October 2008)
Description: Cyanuric acid is extracted from tissue and
infant formula with a 50:50 acetonitrile:water extraction
solution, followed by centrifugation and SPE cleanup. The
compound is analyzed using a zwitterionic HILIC LC column.
Electrospray ionization is used in the negative ion (cyanuric
acid) mode. This procedure has been validated by the FDA
for the determination of cyanuric acid in tissues. The method
has been validated in infant formula powder by Covance.
34 Pricing available from a Covance representative. For updates to assays, refer to our online catalog at covance.com/nutri.
37. ASSAYS
Two multiple reaction monitoring (MRM) transitions are
monitored for the compound. Isotope internal standards are
used to correct for any matrix effects. Fortified test portions
were within 85-105% recovery for the infant formula powder
validation.
CYSTEINE, FREE
see Amino acid profile, free by AAA
CYSTINE (AOAC)
see Amino acid profile (AOAC)
CYSTINE, FREE
see Amino acid profile, free
DHEA OR 7-KETO DHEA
Dehydroepiandrosterone or
3-acetyl-7-oxo-dehydropiandrosterone
Purpose: Applicable for the determination of DHEA or
7 KETO DHEA in premixes and dietary supplements.
Method facts:
▶ Sample size: 5 g
▶ Limit of quantitation: 50 ppm
▶ Precision: 4.2%
▶ Method reference: Journal of AOAC INTERNATIONAL,
83(4):847-857
Description: Extraction is conducted by sonicating with
methanol. Samples are analyzed by HPLC and quantified by
comparison with a known standard.
DENSITY
Purpose: Applicable for the determination of specific gravity
in liquid samples.
Method facts:
▶ Sample size: 100 mL
▶ Limit of quantitation: NA
▶ Precision: On a water matrix, the RSD is 0.3%
▶ Method reference: USP 841
Description: A known volume of sample is weighed. The
weight of the sample per unit volume is calculated.
DETERGENT FIBER, ACID (ADF)
Purpose: Applicable for the determination of acid detergent
fiber in forages and feeds with low carbohydrate contents.
Method facts:
▶ Sample size: 5 g
▶ Limit of quantitation: Most matrices - 0.1%
▶ Precision: On a dog food matrix, the RSD is 14.3%
▶ Method reference: United States Department of Agriculture
Forage and Fiber Analysis, Handbook #379.8, United States
Department of Agriculture, Washington, D.C. (1970)
Description: The protein, carbohydrate and ash content is
removed by treating samples with a boiling detergent solution
and filtering. The fats and pigments are removed via an
acetone wash leaving lignocellulose fraction in a frit, which
is determined gravimetrically.
DETERGENT FIBER, NEUTRAL (NDF)
Purpose: Applicable for the determination of neutral
detergent fiber in forages and feeds.
Method facts:
▶ Sample size: 5 g
▶ Limit of quantitation: 0.1%
▶ Precision: On commercial dog food matrix, the RSD is
11.1%
▶ Method reference: AACC 32.20
United States Department of Agriculture, Forage and Fiber
Analysis, Agriculture Handbook #379.8, United States
Department of Agriculture, Washington, D.C. (1970)
Description: The protein, carbohydrate, enzyme, and ash
content is dissolved by a boiling detergent solution and
filtered off. The fats and pigments are removed via an
acetone wash leaving hemicellulose, cellulose, and lignin
fractions in a frit and determined gravimetrically.
DIGESTIBLE FAT
Purpose: Applicable to fat and saturated fat in Olestra
products, pure Olestra and blended Olestra.
Method facts:
▶ Sample size: 5 g
▶ Limit of quantitation: 0.01%
35800-675-8375 • covance.com/nutri
38. ASSAYS
▶ Precision: On a potato chip the RSD is 9.28%
▶ Method reference: “Capillary Gas Chromatographic
Determination of Fat in Olestra Savory Snack Products,”
Guererra III, Frank P., Pharmline Inc., personal
correspondence, April 1996
Description: Samples are extracted with chloroform-methanol
solution, yielding a lipid extract that contains the total fat and
olestra. The extracted lipid is hydrolyzed by lipase, yielding
unreacted olestra and fatty acid from the fat. The fatty acids
are precipitated as calcium soaps. Olestra is extracted from
the insoluble soaps with hexane and discarded. The isolated
soaps are converted back to fatty acids with hydrochloric acid
and extracted into hexane. These fatty acids are converted to
methyl esters with boron trifluoride-methanol solution and
quantified by gas chromatography using an internal standard.
DISSOLUTION PREPARATION (USP)
Purpose: This test is provided to determine compliance with
the dissolution requirements where stated in the individual
monograph for a tablet or capsule dosage form, except where
the label states that the tablets are to be chewed.
Method facts:
▶ Sample size: 25 tablets or capsules
▶ Method reference: United States Pharmacopeia, 2040,
United States Pharmacopeial Convention, Inc.: Rockville,
Maryland (current edition)
Description: Unless otherwise stated in the individual
monograph, 6 dosage units are tested as directed under
Dissolution 2040. A stated volume of Dissolution Medium
is placed into each vessel of the dissolution apparatus
specified in the individual monograph. Once the Dissolution
Medium is equilibrated to 37 ± 0.5°C, 1 tablet or 1 capsule is
placed into each of 6 vessels. The apparatus is immediately
operated at the rate specified in the individual monograph.
Within the time interval specified, a specimen is withdrawn
from a specific zone of each vessel. Equal volumes from each
vessel are filtered and pooled into a single container.
DONG QUAI (FERULIC ACID)
see Phenolic acids
EBDCs (DITHIOCARBAMATES)
Purpose: Applicable for the determination of EBDC residues
in milk, eggs, nuts, feeds, crops and formulations. This
method screens for EBDC compounds using standards from
mancozeb, maneb, metiram, thiram, nabam, ziram and
zineb.
Method facts:
▶ Sample size: 50 g
▶ Limit of quantitation: Most matrices - 2000 ppb
▶ Precision: Based on spike recovery; most matrices are
70-120%
▶ Method reference: The Analyst, 6:782-787 (July 1981)
Description: EBDCs are quantitatively decomposed to
carbon disulfide using a headspace solvent layer procedure.
The sample is weighed into a 125 mL serum bottle and a
hydrochloric acid-stannous chloride solution is added.
Iso-octane is volumetrically pipetted onto the sample and
the serum bottle is sealed with a crimp top cap fitted with
a Teflon-coated silicone septum. The sample is heated at
approximately 80°C in a water bath for 1 hour and allowed
to cool to room temperature. The sample is then centrifuged
to separate it from the iso-octane layer. The iso-octane layer
is then pipetted off the top of the sample and analyzed on a
gas chromatograph using a flame photometric detector in
sulfur mode.
ECHINACEA SPP.
Caftaric, chlorogenic, echinacoside, chicoric
Purpose: Applicable for the determination of echinacosides in
raw material or dietary supplements.
Method facts:
▶ Sample size: 10 g
▶ Limit of quantitation: 50 ppm (1 ppm for liquids)
▶ Precision: Varies with matrix
▶ Method reference: Journal of Ag Food Chem., 51(3): 572-580
Description: Extraction technique may vary with matrix.
The extract is injected into an HPLC with UV detection.
The echinacosides are quantified by comparison to known
standards.
36 Pricing available from a Covance representative. For updates to assays, refer to our online catalog at covance.com/nutri.