Testing categories
Dietary Supplements

As of June 2007, FDA - DSHEA (Dietary Supplement Health and Education Act) is fully implemented and enforces cGMP (current Good Manufacturing Practice) requirements on Dietary Supplements Manufacturing.

DSHEA 1994 definition:
A dietary supplement is a product taken by mouth that contains a "dietary ingredient" intended to supplement the diet. The "dietary ingredient" must be one or any combination of the following substances:
• a vitamin,
• a mineral,
• an herb or other botanical,
• an amino acid, enzymes, or
• a concentrate, metabolite, constituent or extract.
FDA /USDA set a maximum daily dose for essential vitamin and nutrient as part of the nutritional label claim.

List of Dietary Supplement (Nutritional Supplement) Testing Services:
• Vitamin Testing (Oil Soluble Vitamin, Water Soluble Vitamin)
• Mineral Testing (such as Calcium Gluconate, Calcium Citrate, etc.)
• Nutrient (Glucosamine, Chondroitin Sulfate, Co-Q10, etc.)
• Antioxidant Activity in Natural Products.
• Natural Products: Fruit, Berries, Vegetable (Anthocyanin).
• Amino Acid (Essential, Non-essential) Essential Fatty Acid (DHA, EPA, CLA, etc.)
• Essential Fatty Acid (DHA, EPA, CLA, etc.)
• Dietary Enzymes (Amylase, Lipase, B-galactosidase or Lactase, Papain, Bromelein, etc.)
• Botanical, Phytochemistry, Herbal Active Testing.
• USP <561>, Contaminant and Foreign Substance.

Vitamin Analysis

Ullmann Laboratory's Dietary Supplement Lab has the utmost capability on testing vitamin actives in raw materials, tablets, capsules, solutions, or powder mixes in the form of proteins such as milks or soy matrices. The test analyses are performed using state-of-the-art instruments such as HPLC (high performance liquid chromatography), and GC (gas chromatography). There are two main class of vitamins: water soluble vitamins and fat (oil) soluble vitamins. Ullmann's test vitamin raw materials and finished products using official USP methods under the USP Dietary Supplement Chapter and other published validated methods such as AOAC, etc.

For regulatory compliance according to 2007 DSHEA, all vitamin raw materials used in supplements and food products must be tested for identity by infrared spectrometer and assay for potency percentage. The manufacturer must assure the quality and purity of each raw material before use in production.

The vitamin‘s finished product can be in the form of capsules, tablets, powder mixes or oral liquid solutions. The USP (United States Pharmacopeia) has set acceptable allowable ranges based on individual active label claims for vitamins in finished products. Vitamin potency that is less than minimum range in the finished product is called sub-potency.

Based on GMP regulation, if the sample fails the specification it must be rejected and disposed of properly, and under no circumstances be sold to consumers. The Dietary Reference Intake (DRI) table published from the Institute of Medicine (IOM) and National Academy of Science provides a guideline to dietary supplement manufacturers on vitamin and mineral daily dosages.

Dietary Minerals

Dietary minerals are the chemical compounds required by living organisms, including humans, for normal metabolic function. These are in the form of metals with organic compounds. There are more than eleven chemical elements required to support human biochemical processes by serving structural and functional roles. The function of minerals in our body range from building bones, making hormones to regulating heartbeat.

The suggested mineral RDI (Daily Reference Intake):

There is a wide variety of foods containing these minerals, including fruits, meats, vegetables or supplements. Ullmann Laboratory's Nutritional Department routinely performs mineral raw material by following USP methods. The table below lists partially the USP mineral raw material acceptable specification requirements.

Essential Nutrients

Ullmann Laboratories' has state of the art equipment such as HPLC, GC, and spectrophotometery, fluorescence, UV/VIS and potentiometric titration for performing the quantitation of nutrient active claims.

List of single ingredient as nutrient testing by HPLC, GC, or spectrophotometry:

Natural Products Antioxidant Activity

Ullmann laboratory " has the capability for testing antioxidant, free radical scavenging activity on natural products such as fruit, berries, vegetable, and herbal products.

List of ULPL Antioxidant Activity Services:
1. Total Anthocyanin as Cyanidin
2. Total Polyphenols as Gallic Acid Equivalent (GAE)
3. Total Flavonoid as Catechin Equivalent
4. Total Proanthocyanidin
5. FRAP Ferric Reducing Antioxidant Activity
8a. Lipophilic (Oil Soluble)
8b. Hydrophilic (Water Soluble)

Total Anthocyanins
Anthocyanins are natural colorant flavonoids of fruits and vegetables that have deep colors. Examples of anthocyanins are the acai berry, grapes skin, grapeseed, bilberry, maqui berry, blueberry, cranberry, or pomagranate juice extract. Anthocyanin are very powerful antioxidants that benefit the entire body by protecting the cells from free radicals. The most common anthocyanin groups are aglycone of cyanidin, delphinidin, malvidin, petunidin, and pelargonidin.

Determination of anthocyanidins is performed by alcohol extraction of leaves, petals, seeds, berries, or herbs. A measurement of absorbance is taken from two difference pH solutions and measured at two different wavelengths; this is known as the pH differential method.The total anthocyanins are reported as cyn 3-glu per 100 grams.

Only the natural color anthocyanins from fruit or vegetable sources are pH sensitive in acidic conditions. An rise in spectrophotometry absorbance is observed when there is a change in acidity in the fruit or vegetable during the anthocyanin test. On the other hand, any artificial red or purple color may be discolored when extracted with alcohol in acidic conditions, and may show no change in color or no response to the anthocyanin test.Total Phenolic Determination as Gallic Acid (TP Method).

Phenolic compounds in natural products are one or multiple hexagonal rings with three double bonds inside the ring, and more than one hydroxyl or OH group attached to it.

Examples lude chlorogenic acid, quercetin, gallic, flavonoid, etc.
The TP method uses gallic acid as phenolic standard, gallic acid ug/mL at different concentrations ug/mL, and for linearity curve and extracted samples, are determined simultaneously by colorimetry with the Folin–Ciocalteu method. Results are expressed as gallic equivalent per gram sample.

Total Flavonoid as Catechin Equivalent
The flavonoids from fruit and vegetable products are extracted, and using catechin as a standard, the total flavonoids in food products are measured by aluminum chloride reduction reaction. The absorbance is measured against a reagent blank at visible spectrometry. Total flavonoid content is expressed as a percentage of catechin equivalent per 100 gram sample mass.

Total Proanthocyanidin (PAC)
Proanthocyanidins or oligomeric proanthocyanidins (OPCs) are phenolic organic clusters of dimers (2), trimers (3), tetramers (4) and pentamer natural compounds in fruits and vegetables. OPCs are determined by AOAC–USDA DMAC method. Using procyanidin B2 as a standard, reported result as mg of proanthocyanidin per gram sample.

Ferric Reducing Antioxidant Activity (FRAP)
Sample and sample blank are treated with Fe+3, TPTZ, presence of antioxidants show a reduction of Fe+3 to Fe+2 show rease of blue ferrous form. Change in absorbance is proportional to reducing/ antioxidant power(FRAP), and expressed as micromole trolox equivalent per gram sample mass (umol TE/g).

(2,2 Diphenyl-1 Picrylhydrazyl)-Radical Scavenging Activity (DPPH)
DPPH measures the ability of bioactive to act as free radical scavengers, or hydrogen donors, using trolox as reference standard. The odd electron in the DPPH free radical gives a strong absorption purple color. Upon reacting with the bioactive chemical, the odd electron in the DPPH radical becomes paired with the hydrogen from the free radical scavenger antioxidant, and the color turns from a purple to yellow. The more discolorized of DPPH solution after reaction, the higher the antioxidant scavenger or umole-trolox unit per 100 grams.

Trolox Equivalent Antioxidant Capacity (TEAC)
TEAC method is based on the suppression of the absorbance of radical probe (ABTS 2,2'-azinobis (3-ethylbenzothiazoline 6-sulfonate)) by antioxidant sample, when ABTS is ubated with peroxidase and H2O2.

Oxygen Radical Antioxidant Capacity (ORAC)
Hydrophilic ORAC measures watersoluble antioxidant activity in the natural product sample such a fruitor vegetable. Using AAPH as peroxy radicals generator and fluoresceinas reaction proble observe the decay of the fluorescence emissioncurve, using Trolox as standard. Lipophilic ORAC measures oilsoluble antioxidant activity. Samples are extracted with buffer, andhexane. The hexane layer is used for L-ORAC. Using pAAPH as peroxyradical generator, fluorescein as as reaction probe, observed the oilsoluble decay under fluorescen emission curve, using Trolox asstandard.

Natural Products: Fruit, Berries, Vegetables (Anthocyanidin)

Ullmann Laboratories' tests the natural colorants from fruits and vegetables, such as purple yams or purple cabbage for anthocyanins using state of art instrumentation. Fruits and berries such as black currant berries, blueberries, pomagrante fruit, acai berries, grape skin extract, blackberry extracts, and raspberry extracts are high in anthocyanins.

Anthocyanins are water soluble vacuolar pigments that appear red, purple or blue, and are affected by different pH. These are the 3-glycoside of the anthocyanidins. The most common group of anthocyanidins are cyanidin, delphinidin, malvidin, petunidin, pelargonidin. Researchers have found that fruits and vegetables rich in red, purple or blue color pigments are high in anthocyanins.

This chemical group provides potential health effects against aging, neurological diseases, and inflamamation. There are several methods on testing an anthocyanin pigment against spiked artificial colors or those diluted with other synthethic colors.

Thin layer chromatography (TLC): compared to natural marker compound acid condition separation.
High performance liquid chromatography (HPLC): using 3D DAD, HPLC determines the fingerprint and identification of the natural colorant.

Example of the Anthocyadinin in different colorful fruit and vegetable:

Amino Acid Testing by HPLC

Ullmann labs are capable of testing the amino acid as either basic amino acids or those formed from protein hydrolysis. Proteins are composed of groups of amino acids of different types and percentages. The protein samples are subject to acid hydrolysis and break down into individual amino acids, which react with derivatized chemical agents and convert to stable-chromophore amino acids. These are separated and analyzed by HPLC.

Amino acids are molecules containing an amine NH2-group, a carboxylic COOH-group and a side chain that varies between different amino acids. These groups of amino acids are the basic building blocks in the human body. Amino acids are classified into two main groups: essential and non-essential. An essential amino acid is an amino acid that cannot be synthesized by our body and therefore must be supplied in the diet.

There are eight essential amino acids in the group:
• Phenylalanine
• Valine
• Threonine
• Tryptophan
• Isoleucine
• Methionine
• Leucine
• Lysine

Non-essential amino acids are amino acids that can be produced by our body. Their uses and functions in our body are equally important as the limiting amino acids.

There are 13 non-essential amino acids:
• Alanine
• Arginine
• Cysteine
• Cystine
• Glutamine
• Glutathione
• Glycine
• Histidine
• Serine
• Tyrosine
• Hydroxyproline
• Asparagine
• Proline

The US Pharmacopeia Dietary Supplement Chapter lists detailed test methods on each individual amino acid ingredient as follows:
• Identification perform by FTIR
• Chromatographic impurities performed by TLC–ninhydrin method
• Potency of raw material assay are performed by titration

Summary on Finished Goods Amino Acid Testing
The amino acids in finished goods such as tablets, capsules, powders, liquid syrups are hydrolyzed to break down into amino acids and are analyzed by high performance liquid chromatography (HPLC).

List of raw material dietary supplement nutrient and amino acid in USP monograph identification and potency:

Dietary Supplement: Fatty Acid Analysis

ullmann Lab routinely analyzes fatty acid from fish oil, CLA, saw palmetto, borage oil, etc. The most common method used on fatty acids is done by gas chromatography, where oils are extracted by hexane or heptane. These are subjected then to a Boron Trifluoride reaction in the presence of methanol, where the free fatty acid is converted into methyl ester fatty acid, which is analyzed by gas chromatography FID. This method is derived from the AOAC-USP method.

Some of the common essential fatty sources are:
• Fish oil containing DHA, EPA, DPA
• Saw palmetto; 70 to 85% fatty acid
• Conjugated linonleic acid (CLA) found in meat and dairy products
• Mono-saturated, polyunsaturated fatty acids from seeds and nuts
• Flax seeds
• Evening primrose
• Borage oil

Identification of different types of USP, FCC, EP, or BP oils can be done by thin layer chromatography and the fingerprint of a few of the fatty acid compositions. Assays and quantitation of the dietary supplement, gas chromatography and methyl ester fatty acid distribution are required by European Pharmacopeia.

Dietary Enzymes

Dietary enzymes are protein chemicals that act as catalyzers (moving the metabolic reaction faster or slower), or aids in digesting protein, carbohydrates, or fatty lipids.

The process of enzyme reaction involves three steps. Molecules serves ¬ substrate are fed to enzyme proteins, the temperature is controlled and minute of reaction. The enzyme converts the substrate into different molecules called end products. The potency of the enzyme is calculated as a unit of substrate converted or digested into end product per minute or second. Based on the Food Chemical Codex listing, food enzymes are grouped as carbohydrates, proteins, and lipid enzymes.

Here is a tabulation of the FCC most common enzymes:

Phytochemicals Testing

Another essential component of ULPL's testing expertise is herbal supplement, botanical supplement and natural product source testing. We routinely analyze natural products for identification, potency, marker quantitation, trace impurities, and organic volatile impurities, as well as many other analyses.

"PHYTOCHEMICALS" are defined as chemical actives that are derived from plants, and also sometimes called botanical supplements. These botanical actives may be a variation of flavonoid, alkaloid, coumarin, isoflavone, fatty acid, phenolic, saponin, vitamin or phytosterol. These may be present as more than one active as a combination of the aforementioned actives.

Herbal supplements are dietary supplements that are derived from plant sources. An herb is derived from plant parts (such as roots, bark, leaves, flowers, or seeds) and is used for its flavor, scent, and/or therapeutic properties to help promote body energy or as preventie supplements. The term "botanical" is often used as a synonym for "herb." An herbal supplement may contain a single herb or mixtures of groups of herbs in the form of capsules, powders or drinks.

ULPL tests herbal, botanical and dietary supplement analysis products following the guidelines of only highly respected publications, such as British Herbal Compendium, China Pharmacopoeia, USP dietary supplement herbs, AOAC method, British Pharmacopeia (BP), European Pharmacopeia (EP), Indian Herbal Pharmacopeia (IP), and other recognized publication. We test everything from aloe vera to yohimbe. *Common herbs in USA, see table on summary of herbal list from USP, BP, EP, and IP monographs.

Below are partial herb lists that Ullmann Laboratories tests. If the herb of interest is not in the following list, please contact us. We are continuously developing and validating new methods.

Common Indian Herbal Pharmacopeia

Contaminants and Foreign Substances

Aflatoxins are toxic metabolites produced by fungus in foods and feeds. Aflatoxins are potentially hazardous if they contaminate the food source. Toxins can cause illness and even death to humans and animals. There are four major aflatoxins: B1, B2, G1, G2. Aflatoxins are detected in milk, eggs, cheese, corn, peanuts, cottonseed, nuts, almonds, figs, spices, and a variety of other foods and feeds. Highest at risk of aflatoxin contamination are corn, peanuts, and cottonseed.

Aflatoxins can be analyzed by immune-solid phase extraction and HPLC fluorescence methods. The samples are ground into powder, dissolved in the AOAC suggested solvent, and passed through isolation step (i.e.antibody immune column) and the antibody in the imunocolumn will attract all the aflatoxin and wash out the proteins, sugars or lipids. Then andeluate in sample with very limited collecting solvent. The method is very sensitive and can detect aflatoxin concentrations as low as 5 ppb. The FDA has set maximum limits of aflatoxin as no more than 20 ppb for grain and food, but less than 0.5 ppb for M1 species in milk. FDA Website for Contaminants and Pesticides.

Fumonisin B1 is the most prevalent toxin, produced by Fusarium molds and found in maize, wheat and other cereals. The FDA/USDA Working Group on Fumonisin advises less than 4 ppm fpr products for human consumption, less than 5 mg/kg in horse feed, less than 10 mg/kg for swine feed, and less than 50 mg/kg for cattle feed. Mycotoxinfumonisins are analyzed by immune-affinity column extraction, HPLC quantitation, and ppb detection limits.

Ochratoxin A, B are mycotoxins produced by Aspergillus ochraceus or Penicillium viridicatum. Ochratoxin A is known to occur in commodities like cereals, coffee, dried fruit and red wine. It is considered a human carcinogen. AOAC approved methods are to test by collecting sample into imnuno-affinity column and analyzing by HPLC. Sensitivity is 0.5 ppb. Citrinin is a mycotoxin originally isolated from Penicillium citrinum. It has se been found to be produced by a variety of other fungi which are used in the production of human foods such as grain, cheese, sake and red pigments. Citrinin can be analyzed by liquid extraction and imunoaffinity isolation column, and HPLC flourescense and a sensitivity of 10 ppb.

Patulin is a mycotoxin produced by a variety of molds, particularly Aspergillus and Penicillium. It is commonly found in rotting apples. The World Health Organization (WHO) recommends a maximum concentration of 50 µg/L in apple juice. In the European Union, the limit is set to 50 micrograms per kilogram (µg/kg) in both apple juice and cider. The AOAC validated method is by using a mycotoxin isolation column, extracting the petulin in buffer, and separation by HPLC.
Zearalenone (ZEA), also known as RAL and F-2 mycotoxin, is a potent estrogenic metabolite. Zearalenone is the primary toxin causing infertility, abortion or other breeding problems, especially in swine. Higher concentrations (50-100 ppm) can interfere with conception, ovulation, implantation, fetal development, and the viability of newborn animals. Zearalenone is heat-stable and is found worldwide in a number of cereal crops, such as maize, barley, oats, wheat, rice, and sorghum [2] and also in bread. Testing on Zearalenone is based on the AOAC–FDA approved method of imuno-affinity HPLC method. Sensitivity is detected at 0.1 to 5 ppm.