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Dietary Supplement Testing and Analysis: Quality Control

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Dietary Supplement Testing and Analysis: Quality Control

Dietary supplements are subject to FDA requirements for good manufacturing practices (cGMP) and quality control in the United States. cGMP require specifications for each ingredient and finished dietary supplement. The specifications list parameters for identity, purity, potency and other requirements for regulatory compliance. Each parameter on the specification must be tested with a scientifically valid method.

NaturPro Scientific, as an UnLab, partners with expert analytical laboratories to conduct specific testing on dietary supplements. Testing typically includes:

  • Physical characteristics (visual, color, odor, taste, density, mesh size)
  • Identity (matching an ingredient in a pass/fail fashion to a particular species of botanical or herb, or a chemical purity test)
  • Potency (concentration of active or marker compounds)
  • Purity (absence of impurities such as moisture, microbiology, pathogens, heavy metals, residual solvents, pesticides, mycotoxins)

The following are analytical principles or instruments that may be used for dietary supplement testing:

  • Karl Fischer
  • Ro-tap and particle size analysis
  • Titration
  • Gravimetry
  • Thin Layer Chromatography (TLC or HP-TLC)
  • High Performance Liquid Chromatography (HPLC)
  • Gas Chromatography with Flame Ionization Detection (GC-FID)
  • Gas Chromatography with Mass Spectrometry (GC-MS of GC-MS-MS)
  • Inductively Coupled Plasma Mass Spectrometry (ICP-MS)
  • Total Aerobic Plate Count
  • Pathogens (Salmonella, E. Coli, Staph)

The following is a list of documentation and regulations requiring testing under cGMPs:

  • Documentation of the specifications established (21 CFR 111.95(b)(1))
  • Documentation of your qualification of a supplier for the purpose of relying on the supplier’s certificate of analysis (21 CFR 111.95(b)(2))
  • Documentation for why meeting in-process specifications, in combination with meeting component specifications, helps ensure that the dietary supplement meets the specifications for identity, purity, strength, and composition; and for limits on those types of contamination that may adulterate or may lead to adulteration of the finished batch of the dietary supplement (21 CFR 111.95(b)(3))
  • Documentation for why the results of appropriate tests or examinations for the product specifications that you selected for testing ensure that the dietary supplement meets all product specifications (21 CFR 111.95(b)(4))
  • Documentation for why any component and in-process testing, examination, or monitoring, and any other information, will ensure that a product specification that is exempted under 21 CFR 111.75(d) is met without verification through periodic testing of the finished batch, including documentation that the selected specifications tested or examined under 21 CFR 111.75 (c)(1) are not able to verify that the production and process control system is producing a dietary supplement that meets the exempted product specification and there is no scientifically valid method for testing or examining such exempted product specification at the finished batch stage (21 CFR 111.95(b)(5))

There are a number of sources of information for developing specifications and test methods for analysis of dietary supplements. The below is a partial list of references and resources:

  1. Dietary Supplement Ingredient Database,
  2. Dietary Supplement Label Database,
  3. Dietary supplement laboratory quality assurance program: the first five exercises. Phillips MM, Rimmer CA, Wood LJ, Lippa KA, Sharpless KE, Duewer DL, Sander LC, Betz JM.  J AOAC Int 2011;94:803-14.
  4. Heavy metals: analysis and limits in herbal dietary supplements,
  5. Pesticide Analytical Manual, Vol I, FDA. Source:
  6. Pesticide Analytical Manual, Vol II, FDA. Source:
  7. Quality assurance of cultivated and gathered medicinal plants. Mathe and Mathe, Source:
  8. Quality control methods for medicinal plant materials (1998) World Health Organization
  9. Recommendations for microbial limits in herbal products, American Herbal Products Association,
  10. Standardization of herbal medicines – A review. Kunle O.F. et al, (2012) Int. J Biodiv and Conserv. 4(3) 101-112. Source:
  11. USP Food Fraud Mitigation Guidance,


Turmeric Supplement Testing — Curcumin Products

by NaturPro in Uncategorized Comments: 0

Laboratory testing of turmeric supplements and curcumin products is important for quality, safety, dosage and bioavailability. NaturPro Scientific offers testing and analysis consulting for turmeric, and works with expert research and quality control testing laboratories.

A number of analytical methods and monographs have been developed for turmeric to ensure bioavailability, consistency, potency and purity of curcumin products.

Turmeric Supplement Testing — Curcumin Products

We recommend all turmeric products have routine and/or periodic independent testing for the following parameters:

  1. Curcuminoids (curcumin) by HPLC
  2. Biological activity
  3. Bioavailability
  4. Heavy metals
  5. Microbiology and pathogens
  6. Residual solvents
  7. Pesticides
  8. Natural source by carbon radioisotope (if labeled as ‘turmeric’)
  9. Food allergens
  10. Sudan dyes

Traditional dosage forms listed by the EU Community Herbal Monograph include the following herbal preparations:

  1. Powdered herbal substance
  2. Comminuted herbal substance
  3. Tincture (Ratio of herbal substance to extraction solvent 1:10), extraction solvent ethanol 70% (v/v)
  4. Dry extract (DER 13-25:1), extraction solvent ethanol 96% (v/v)
  5. Dry extract (DER 5.5-6.5:1), extraction solvent ethanol 50% (v/v)
  6. Tincture (Ratio of herbal substance to extraction solvent 1:5), extraction solvent ethanol 70% (v/v)
    Other solvents are commonly used to extract curcuminoids.

The JECFA has developed a monograph on turmeric oleoresin:

“Obtained by solvent extraction of turmeric (Curcuma longa L.). Only the following solvents may be used in the extraction: acetone, dichloromethane, 1,2-dichloroethane, methanol, ethanol, isopropanol and light petroleum (hexanes).

The selection of a turmeric oleoresin of a particular composition is based on the intended use in food. In general, all turmeric oleoresins contain colouring matter and most contain flavouring matter but some oleoresins are processed to remove aromatic compounds. Commercial products include oleoresins (per se) and formulations in which oleoresin is diluted in carrier solvents and which may contain emulsifiers and antioxidants. Purified extracts of turmeric containing more than 90% total colouring matter are subject to specifications for “Curcumin”.

Turmeric Oleoresins are sold on the basis of “colour value” or “curcumin content”, which generally means the total content of the curcuminoid substances: (I) curcumin, (II) demethoxycurcumin and (III) bis- demethoxycurcumin.

The principle colouring components are:
I. 1,7-Bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene- 3,5-dione
II. 1-(4-Hydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-hepta-1,6-diene- 3,5-dione
III. 1,7-bis(4-hydroxyphenyl)hepta-1,6-diene-3,5-dione

Turmeric Oleoresins, per se, are deep brownish-orange viscous oily fluids, pasty semisolids or hard amorphous solids containing 37-55% curcuminoids and up to 25% volatile oil. Diluted turmeric oleoresin formulations are, generally yellow solutions containing 6-15% curcuminoids and nil to 10% volatile oil.

Residual solvents limits:

Acetone : Not more than 30 mg/kg
Methanol: Not more than 50 mg/kg
Ethanol: Not more than 50 mg/kg
Isopropanol: Not more than 50 mg/kg
Dichloromethane and 1,2-dichloroethane: Not more than 30 mg/kg, singly or in combination

Light petroleum (hexanes): Not more than 25 mg/kg

The WHO monograph for medicinal plants for turmeric is excerpted below:

Rhizome (root) of Curcuma Longa L. (turmeric)


Rhizoma Curcumae Longae is the dried rhizome of Curcuma longa L. (Zingiberaceae) (1).

Dried rhizomes of Curcuma wenyujin Y.H. Lee et C. Ling, C. kwangsiensis S. Lee et C.F. Liang. and C. phaeocaulis Val. are also official sources of Radix Curcumae or Turmeric Root-Tuber in China (2).


Curcuma domestica Valeton., C. rotunda L., C. xanthorrhiza Naves, Amomum curcuma Jacq. (3–5).

Selected vernacular names

Acafrao, arqussofar, asabi-e-safr, avea, cago rerega, chiang-huang, common tumeric, curcum, curcuma, dilau, dilaw, Gelbwurzel, gezo, goeratji, haladi, haldi, haldu, haku halu, hardi, haridra, huang chiang, hsanwen, hurid, Indian saffron, jiânghuang, kaha, kakoenji, kalo haledo, khamin chan, khaminchan, kilunga kuku, kitambwe, kiko eea, koening, koenit, koenjet, kondin, kooneit, kunyit, kurcum, kurkum, Kurkumawurzelstock, luyang dilaw, mandano, manjano, manjal, nghe, nisha, oendre, pasupu, rajani, rame, renga, rhizome de curcuma, saffran vert, safran, safran des indes, skyer-rtsa, tumeric, tumeric root, tumeric rhizome, turmeric, ukon, ul gum, wong keong, wong keung, yellow root, yii-chin, zardchob (13, 6–14).


Perennial herb up to 1.0 m in height; stout, fleshy, main rhizome nearly ovoid (about 3 cm in diameter and 4 cm long). Lateral rhizome, slightly bent (1cm × 2–6cm), flesh orange in colour; large leaves lanceolate, uniformly green, up to 50cm long and 7–25cm wide; apex acute and caudate with tapering base, petiole and sheath sparsely to densely pubescent. Spike, apical, cylindrical, 10– 15cm long and 5–7 cm in diameter. Bract white or white with light green upper half, 5–6 cm long, each subtending flowers, bracteoles up to 3.5 cm long. Pale yellow flowers about 5cm long; calyx tubular, unilaterally split, unequally toothed; corolla white, tube funnel shaped, limb 3-lobed. Stamens lateral, petaloid, widely elliptical, longer than the anther; filament united to anther about the middle of the pollen sac, spurred at base. Ovary trilocular; style glabrous. Capsule ellipsoid. Rhizomes orange within (1, 4, 6, 15).

Plant material of interest: dried rhizome

General appearance

The primary rhizome is ovate, oblong or pear-shaped round turmeric, while the secondary rhizome is often short-branched long turmeric; the round form is about half as broad as long; the long form is from 2–5cm long and 1–1.8cm thick; externally yellowish to yellowish brown, with root scars and annulations, the latter from the scars of leaf bases; fracture horny; internally orangeyellow to orange; waxy, showing a cortex separated from a central cylinder by a distinct endodermis (1, 9, 13).

Organoleptic properties

Odour, aromatic; taste, warmly aromatic and bitter (1, 9, 13). Drug when chewed colours the saliva yellow (9).

Microscopic characteristics

The transverse section of the rhizome is characterized by the presence of mostly thin-walled rounded parenchyma cells, scattered vascular bundles, defi- nite endodermis, a few layers of cork developed under the epidermis and scattered oleoresin cells with brownish contents. The cells of the ground tissue are also filled with many starch grains. Epidermis is thin walled, consisting of cubical cells of various dimensions. The cork cambium is developed from the subepidermal layers and even after the development of the cork, the epidermis is retained. Cork is generally composed of 4–6 layers of thin-walled brickshaped parenchymatous cells. The parenchyma of the pith and cortex contains curcumin and is filled with starch grains. Cortical vascular bundles are scattered and are of collateral type. The vascular bundles in the pith region are mostly scattered and they form discontinuous rings just under the endodermis. The vessels have mainly spiral thickening and only a few have reticulate and annular structure (1, 8, 9).

Powdered plant material

Coloured deep yellow. Fragments of parenchymatous cells contain numerous altered, pasty masses of starch grains coloured yellow by curcumin, fragments of vessels; cork fragments of cells in sectional view; scattered unicellular trichomes; abundant starch grains; fragments of epidermal and cork cells in surface view; and scattered oil droplets, rarely seen (1, 13).

Geographical distribution

Cambodia, China, India, Indonesia, Lao People’s Democratic Republic, Madagascar, Malaysia, the Philippines, and Viet Nam (1, 13, 16). It is exten- sively cultivated in China, India, Indonesia, Thailand and throughout the tropics, including tropical regions of Africa (1, 7, 13, 16).

General identity tests

Macroscopic and microscopic examinations; test for the presence of curcuminoids by colorimetric and thin-layer chromatographic methods (1).

Purity tests


The test for Salmonella spp. in Rhizoma Curcumae Longae products should be negative. The maximum acceptable limits of other microorganisms are as follows (17–19). For preparation of decoction: aerobic bacteria-not more than 107/g; fungi-not more than 105/g; Escherichia coli-not more than 102/g. Preparations for internal use: aerobic bacteria-not more than 105/g or ml; fungi-not more than 104/g or ml; enterobacteria and certain Gram-negative bacteria-not more than 103/g or ml; Escherichia coli-0/g or ml.

Foreign organic matter

Not more than 2% (1, 9).

Total ash

Not more than 8.0% (1, 15).

Acid-insoluble ash

Not more than 1% (1, 9, 15).

Water-soluble extractive

Not less than 9.0% (1).

Alcohol-soluble extractive

Not less than 10% (1).


Not more than 10% (1).

Pesticide residues

To be established in accordance with national requirements. Normally, the maximum residue limit of aldrin and dieldrin in Rhizoma Curcumae Longae is not more than 0.05 mg/kg (19). For other pesticides, see WHO guidelines on quality control methods for medicinal plants (17) and guidelines for predicting dietary intake of pesticide residues (20).

Heavy metals

Recommended lead and cadmium levels are not more than 10 and 0.3mg/kg, respectively, in the final dosage form of the plant material (17).

Radioactive residues

For analysis of strontium-90, iodine-131, caesium-134, caesium-137, and plutonium-239, see WHO guidelines on quality control methods for medicinal plants (17).

Other purity tests

Chemical tests to be established in accordance with national requirements.

Chemical assays

Not less than 4.0% of volatile oil, and not less than 3.0% of curcuminoids (1). Qualitative analysis by thin-layer and high-performance liquid chromatography (1, 21) and quantitative assay for total curcuminoids by spectrophotometric (1, 22) or by high-performance liquid chromatographic methods (23, 24).

Major chemical constituents

Pale yellow to orange-yellow volatile oil (6%) composed of a number of monoterpenes and sesquiterpenes, including zingiberene, curcumene, α- and β- turmerone among others. The colouring principles (5%) are curcuminoids, 50–60% of which are a mixture of curcumin, monodesmethoxycurcumin and bisdesmethoxycurcumin (1, 6, 25). Representative structures of curcuminoids are presented below.

Dosage forms

Powdered crude plant material, rhizomes (1, 2), and corresponding preparations (25). Store in a dry environment protected from light. Air dry the crude drug every 2–3 months (1).

Medicinal uses

Uses supported by clinical data

The principal use of Rhizoma Curcumae Longae is for the treatment of acid, flatulent, or atonic dyspepsia (26–28).

Uses described in pharmacopoeias and in traditional systems of medicine

Treatment of peptic ulcers, and pain and inflammation due to rheumatoid arthritis (2, 11, 14, 29, 30) and of amenorrhoea, dysmenorrhoea, diarrhoea, epilepsy, pain, and skin diseases (2, 3, 16).

Uses described in folk medicine, not supported by experimental or clinical data

The treatment of asthma, boils, bruises, coughs, dizziness, epilepsy, haemorrhages, insect bites, jaundice, ringworm, urinary calculi, and slow lactation (3, 7, 8–10, 14).




Echinacea Supplement Testing — Echinacea purpurea, E. angustifolia

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Echinacea Supplement Testing — Echinacea purpurea, E. angustifolia

Echinacea testing is critical to determining the quality, identity and potency of an echinacea material. NaturPro Scientific offers testing consulting for echinacea supplements.

Echinacea analysis and testing is based mainly on the WHO  monograph on Echinacea that is excerpted in part below, and the USP monograph.


Herba Echinaceae Purpureae


Herba Echinaceae Purpureae consists of the fresh or dried aerial parts of Echinacea purpurea (L.) Moench harvested in full bloom (Asteraceae) (1).


Brauneria purpurea (L.) Britt., Echinacea intermedia Lindl., E. purpurea (L.) Moench f., E. purpurea (L.) Moench var. arkansana Steyerm., E. speciosa Paxt., Rudbeckia purpurea L., R. hispida Hoffm., R. serotina Sweet (2, 3).

Asteraceae are also known as Compositae.

Selected vernacular names

Coneflower, purple coneflower herb, purpurfarbener Igelkopf, purpurfarbene Kegelblume, purpurfarbener Sonnenhut, red sunflower, roter Sonnenhut (48).


A hardy, herbaceous perennial. Stems erect, stout, branched, hirsute or glabrous, 60–180 cm high; basal leaves ovate to ovate-lanceolate, acute, coarsely or sharply serrate, petioles up to 25 cm long, blades to 20 cm long and 15cm wide, blade abruptly narrowing to base, often cordate, decurrent on petiole, 3–5 veined; cauline leaves petiolate below, sessile above, 7–20 cm long, 1.5–8cm broad, coarsely serrate to entire, rough to the touch on both surfaces; phyllaries linear-lanceolate, attenuate, entire, pubescent on outer surface, ciliate, passing into the chaff; heads 1.5–3cm long and 5–10mm broad, purplish; pales 9– 13mm long, awn half as long as body; disc corollas 4.5–5.5mm long, lobes 1mm long; achene 4–4.5 mm long, pappus a low crown of equal teeth; pollen grains yellow, 19–21µm in diameter; haploid chromosome number n = 11 (2).

Plant material of interest: fresh or dried aerial parts

General appearance

The macroscopic characteristics of Herba Echinaceae Purpureae are as described above under Description. An abbreviated description is currently unavailable.

Organoleptic properties

Mild, aromatic odour; initially sweet taste that quickly becomes bitter.

Microscopic characteristics

A description of the microscopic characteristics of a cross-section of the aerial parts of the plant is currently unavailable.

Powdered plant material

A description of the powdered plant material is currently unavailable.

Geographical distribution

Echinacea purpurea is native to the Atlantic drainage area of the United States of America and Canada, but not Mexico. Its distribution centres are in Arkansas, Kansas, Missouri, and Oklahoma in the United States of America (2). Echinacea purpurea has been introduced as a cultivated medicinal plant in parts of north and eastern Africa and in Europe (9).

General identity tests

Macroscopic examination (2) and thin-layer chromatography and highperformance liquid chromatography (4, 10–13) of the lipophilic constituents and chicoric acid in methanol extracts.

Purity tests


The test for Salmonella spp. in Herba Echinaceae Purpureae should be negative. The maximum acceptable limits of other microorganisms are as follows (1416). For preparation of decoction: aerobic bacteria-not more than 107/g; fungi-not more than 105/g; Escherichia coli-not more than 102/g. Preparations for internal use: aerobic bacteria-not more than 105/g or ml; fungi-not more than 104/g or ml; enterobacteria and certain Gram-negative bacteria-not more than 103/g or ml; Escherichia coli-0/g or ml. Preparations for external use: aerobic bacteria-not more than 102/g or ml; fungi-not more than 102/g or ml; enterobacteria and certain Gram-negative bacteria-not more than 101/g or ml.

Pesticide residues

To be established in accordance with national requirements. Normally, the maximum residue limit of aldrin and dieldrin in Herba Echinaceae Purpureae is not more than 0.05 mg/kg (16). For other pesticides, see WHO guidelines on quality control methods for medicinal plants (14) and guidelines for predicting dietary intake of pesticide residues (17).

Heavy metals

Recommended lead and cadmium levels are no more than 10 and 0.3mg/kg, respectively, in the final dosage form of the plant material (14).

Radioactive residues

For analysis of strontium-90, iodine-131, caesium-134, caesium-137, and plutonium-239, see WHO guidelines on quality control methods for medicinal plants (14).

Other purity tests

Chemical tests and tests for acid-insoluble ash, dilute ethanol-soluble extractive, foreign organic matter, moisture, total ash, and water-soluble extractive to be established in accordance with national requirements.

Chemical assays

For essential oil (0.08–0.32%); chicoric acid (1.2–3.1%) (4). Quantitative analysis of echinacoside, chicoric acid, isobutylamides, and other constituents by high-performance liquid chromatography (4). Quantitative analysis of alkamides and caffeic acid derivatives by thin-layer chromatography and highperformance liquid chromatography (4, 12).

Major chemical constituents

A number of chemical entities have been identified, including alkamides, polyalkenes, polyalkynes, caffeic acid derivatives, and polysaccharides (3, 5–9).

The volatile oil contains, among other compounds, borneol, bornyl acetate, pentadeca-8-(Z)-en-2-one, germacrene D, caryophyllene, and caryophyllene epoxide.

Isobutylamides of C11–C16 straight-chain fatty acids with olefinic or acetylenic bonds (or both) are found in the aerial parts of Herba Echinaceae Purpureae, with the isomeric dodeca-(2E,4E,8Z,10E/Z)-tetraenoic acid isobutylamides.

The caffeic acid ester derivative chicoric acid is the major active compound of this class found in the aerial parts of Echinacea purpurea, with a concentration range of 1.2–3.1%. Chicoric acid methyl ester and other derivatives are also present.

Polysaccharide constituents from Herba Echinaceae Purpureae are of two types: a heteroxylan of average relative molecular mass about 35 000 (e.g. PS-I), and an arabinorhamnogalactan of average relative molecular mass about 45000 (e.g. PS-II).

Other constituents include trace amounts of pyrrolizidine alkaloids (tussilagine (0.006%) and isotussilagine). At these concentrations, the alkaloids are considered to be non-toxic (8). Furthermore, because these alkaloids lack the 1,2-unsaturated necine ring of alkaloids such as senecionine (structure in box) from Senecio species, they are considered to be non-hepatotoxic (3).

Dosage forms

Powdered aerial part, pressed juice and galenic preparations thereof for internal and external use (1, 3).

Medicinal uses

Uses supported by clinical data

Herba Echinaceae Purpureae is administered orally in supportive therapy for colds and infections of the respiratory and urinary tract (1, 3, 5, 7, 8, 18). Beneficial effects in the treatment of these infections are generally thought to be brought about by stimulation of the immune response (3, 5, 7). External uses include promotion of wound healing and treatment of inflammatory skin conditions (1, 3, 5, 7, 8, 9, 19).

Uses described in pharmacopoeias and in traditional systems of medicine


Uses described in folk medicine, not supported by experimental or clinical data

Other medical uses claimed for Herba Echinaceae Purpureae include treatment of yeast infections, side-effects of radiation therapy, rheumatoid arthritis, blood poisoning, and food poisoning (1, 5, 7, 9).

The following summarizes some current methods for identifying  from a published review on echinacea:

“Alkamides, caffeic acid derivatives, and polysaccharides have been considered important constituents of the plant. A number of studies revealed that alkamides are involved in the immunomodulatory properties of Echinacea extracts in vitroand in vivo.[4,5] Additionally, caffeic acid is found in some species of Echinacea and could be applied toward authentication and quality control of the plant extracts. The polysaccharides play an important role in the anti-inflammatory effect of Echinacea preparations.[6] Taxonomic, chemical, pharmacological, and clinical characteristics of some species of the Echinacea genus including E. angustifolia, E. pallida, and E. purpurea were reviewed in previous papers.[1,7] Medicinal properties of the plant were also considered in a review paper, which suggested that more research is required for more definitive medicinal recommendations.[8] This paper is a review about E. purpurea: Its phytochemical contents and its pharmacological and biological activities, along with common methods of plant extract analysis. In addition, the psychoactive and mosquitocidal effects of the plant are mentioned in this paper….

Alkamides have been analyzed with reverse-phase HPLC coupled with different detectors including UV spectrophotometric, coulometric electrochemical, and electrospray ionization mass spectrometric.[83,84] Furthermore, caffeic acid derivatives have been determined using reverse-phase HPLC or capillary electrophoresis (CE) with photodiode array (FDA) UV spectrophotometric detection.[85,86,87] Phenolic acids were analyzed by micellar benzoic acid electrokinetic chromatography (MEKC), both charged and uncharged analytes, based on the use of sodium deoxycholate (SDC), a surfactant in borate buffer (pH 9.2), as well as in the E. purpurea extract.[88] However, determination methods for both caffeic acid derivatives and alkamides have been developed in single analysis. Although it is a difficult process to separate these diverse constituents in one analysis, methods for the concurrent determination of caffeic acid derivatives and alkamides have the advantages of reduced time and sample size needed for the analysis.[85] Gradient elution on reverse-phase HPLC has been employed for concurrent analysis of caffeic acid derivatives and alkamides from E. purpurea using various detectors such as FDA UV spectrophotometric and electrospray ionization mass spectrometry (EIMS).[79,85] Simultaneous analysis of both mentioned derivatives has also been performed by electrophoresis with FDA UV spectrophotometric detector, together with sodium dodecyl sulfate and hydroxypropyl-β-cyclodextrin in Britton Robinson buffer (10 mM, pH 8.0).[89]”

Source: Pharmacogn Rev. 2015 Jan-Jun; 9(17): 63–72 (

Single Laboratory Validation of Ethanol in Kombucha Tea

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Single Laboratory Validation of Ethanol in Kombucha Tea by Gas Chromatography with Flame Ionization Detection

The objective of this study was to ensure the validity of test results of ethanol in kombucha tea by performing single laboratory validation (SLV) of a method using gas chromatography with flame ionization detection (GC-FID).


SLV Study of Ethanol in Kombucha

Research News: SLV Study of Ethanol in Kombucha



Why Verify, Then Trust?

by NaturPro in Uncategorized Comments: 0

“Verification” is the 2016 buzzword for food and supplements, due to the sequence of food safety crises that arguably started with salmonella in peanut butter in the early 2000s.  Recently, FSMA and the “Identity Crisis” for botanical ingredients in supplements have renewed the requirement for verification of quality and safety practices in the supply chain: raw materials, manufacturing practices and test methods being three big areas of focus.

“Trust But Verify” is attributed to President Reagan and later FDA and quality assurance folks. Although it is a well meaning mantra, doesn’t it make verification seem optional?  Shouldn’t we verify BEFORE trusting?

We do know that trust disappears soon after a failure to verify becomes apparent. From Salmonella in peanut butter, to misidentified plant extracts, to pesticides in cannabis, verification is how trust is ensured.

So while trust is the ultimate goal, verification comes first.


First published on LinkedIn, April 2016

2-Minute Tip: 6 Ways Ingredients Communicate Value

by NaturPro in Uncategorized Comments: 0

Product development is an increasingly painful process, taking weeks and months to sort through and evaluate ingredients.

That’s because the evaluation process involves cutting through the marketing fluff and understanding (and communicating) the core value of your product.  This makes it a difficult and time-consuming task for your customers.

Marshmallow fluff GinnyWhy should your customer pick your product or ingredient over all the others?  Because they are able to communicate it’s value.

Effective customer education  is one great way to help customers navigate the pitfalls of the product development process, and keep your product top of mind.  The results often include higher customer conversion and less wasted activity.

 Here’s a 2-Minute Tip listing a few things to be sure to include in your customer education materials:

2-Minute Tip: Six Ways Ingredients Communicate Value



What do supplement testing and Star Wars have in common?

by NaturPro in Uncategorized Comments: 0

Star Wars and science fiction fans know that technology is a double-edged sword. On one hand, advances in science offer us fantastic powers to solve difficult problems (space travel, light sabers). On the other hand, the potential for catastrophe is also greater. With better technology comes a greater responsibility to prevent its misuse.

Early botanical scientists understood both the power and limitations of science to describe a complex natural world. Carl Linnaeus, who developed the original system to classify plants and animals, recognized that all organisms are not discrete species necessarily, but exist on a continuous spectrum of life.

Five ways NaturPro helps to ensure scientific validity


Today, scientists in academia work to identify and quantify the diverse array of chemical constituents in botanical products, while industry works to ensure a safe, effective and consistent product. At our disposal are alphabet soups of various analytical technologies that offer increasingly better detection of constituents, even down to the picogram, which relative to a gram can be visualized as a drop of water in a thousand swimming pools.

But with picoscale resolution comes a lot of noise (one trillion per gram, to be exact) and even more responsibility to reliably separate a signal from it. Even at the parts-per-million (ppm) level—equivalent to a cup of water in a swimming pool—we often observe unexplainable results that defy logic.

How our “UnLab” approach controls for shoddy methods and unexplainable results… 

For example, only today’s best and most expensive instruments, such as multiple mass spectrometers linked to a chromatograph, such as LC/MS/MS (also known as tandem-MS, which means two mass spectrometers are hooked to each other; the first MS removes a lot of the “junk” that can interfere with the result from the second MS), are able to account for matrix effects that occur when testing complex mixtures. The reason complex mixtures are so difficult to examine is they contain so many different compounds, and therefore the chances are relatively high that one of these is observed at the same retention time (or peak) on the chromatogram as the compound a scientist is trying to quantify. Also, because the sample is being injected into super-heated, high-pressure instruments, there are often chemical reactions create new interfering compounds. Matrix effects can falsely change results in a significant way that cannot be resolved without further work. Results should always be questioned and replicated, and ultimately, investments in the development of methods are required to generate confidence.

FDA Supplement FactsValidation of matrix-specific methods across multiple laboratories address these challenges, however few methods have been validated to the extent required to be confident in the results. An example from the nutrition field: the inherent challenges in quantification of vitamin D (a pure compound and age-old vitamin, no less!)

Both the best and worst thing about good science is that with each answer comes another question. There is always more work to be done to achieve the greater goal: reproducible results. Needless to say, rigorous analysis of complex mixtures such as botanical products is often not straightforward. Unfortunately, the aims of science often oppose the aims of high-throughput lab testing.
How do you know whether a lab is focused on getting the right results? Here are some criteria to help decide whether or not to work with an independent laboratory:

  • Is it transparent? Does it share methods, chromatograms, observations, historical data and control charts?
  • Does it perform validation? Does it verify methods using appropriate controls such as calibration curves and spike recovery? What steps are taken when it initially sets up a method?
  • Does it have a process for dealing with out-of-specification results, and will it share that process? Does it have an internal recordkeeping system that tracks method precision and alerts them when a method or system is out of calibration?
  • Does it run internal control samples? Does it run samples in triplicate or duplicate at least, and does it report statistical analysis on the certificate of analysis (CoA), such as standard deviation from multiple runs?
  • How does it validate the purity of reference standards? When it gets a new batch of reference standard, does it run it against an internal control sample? How often does it make fresh reference standard solution?
  • Is it a proactive communicator, for example how often does it advise on the best methods to use, and alert their customers on new developments in methods?


Not all testing needs to be expensive or high-tech, but every method needs to be rigorous enough to provide results that are reproducible in another lab. For example, thin layer chromatography (TLC) is not high-tech, but it can be valid to determine botanical identity with the right mix of expertise, a rigorous and validated set of reference standards, and enough trial and error to develop the method and be confident in reproducibility of results. High-performance liquid chromatography (HPLC) is great when actual validation of the method and reference standards have been certified for their purity.

MicroscopeThe true test of scientific validity is when multiple labs running different methods achieve the same result, especially when they are blinded as to the expected result.

Despite all of the challenges in quality control (QC) testing of botanicals, the world is changing, and our industry is rapidly improving. With scientific validity mandated by supplement GMPs (good manufacturing practices), and increasing demands for transparency and validity from all stakeholders, everyone is upping their game. Good science, not science fiction, provides reproducible results we can all be confident in.

Learn about reproducible results through our UnLab…

By: Blake Ebersole

This article appears with revisions, and was originally published in the March 2014 issue of Natural Products Insider.

Eight Steps to Developing Research Relationships

by NaturPro in Uncategorized Comments: 0

Developing relationships with scientists is much like any other; the first step is in understanding scientists’ challenges and needs. Sensitivity to the ways of the scientific research world, especially academia, is one of the best ways to get the most out of your research investment.

As for what else a supplement manufacturer needs to do:

Show an interest in the science. Like anyone, scientists can sense if you’re more interested in doing great science or just the marketing benefits from it. Offer solutions that boost both scientific and business objectives. Add to the debate and question assumptions.

Try to discover something new. There are thousands of questions to be answered and thousands of different study designs. To be industry-relevant, adopt “standard” methods used widely—but allow some space for new discoveries. Also, test some new hypothesized bioactivity or clinical effect.  One-hundred percent “me-too” science just isn’t very interesting to scientists or consumers. Plus, new findings are more likely to go viral.

Decide on a budget and be realistic. Most research costs money, unless you can get into a study funded by someone like the NIH. But government funding is decreasing every year, while grant applications have multiplied exponentially. Performing strong research often requires expensive labor and materials, and the coordination of many different shared resources.

Offer unrestricted grants for basic research. Research seeking to understand mechanisms of action often best developed step-by-step, making long-term planning difficult. Unrestricted grants that don’t guarantee a specific study plan allow you to support critical shared resources, and they prevent you from painting yourself into a corner at the beginning of your scientific journey.

Agree to milestones for projects, but anticipate delays. University-based, public-funded research requires the alignment of many parts, so some projects hit snags. Plan in advance to prevent potential troubles with approval, recruitment, testing, or finances. Add a “delay buffer” to your timeline for a more realistic expectation.

Decide whether to publish research results and, if so, where. Agree early on who owns the data and who has final decision on whether to publish results. Deciding this early on is a good idea because it sets the standard for the rigor of study design. It’s not necessary to always publish in a patent application or journal. Consider the fact that by publishing, you are likely helping both humankind and your competition. Decide which one outweighs the other.

Presentations at research conferences are sometimes a good idea because you can “publish” data that is somewhat peer-reviewed, and isn’t widely available to the public.

Scrutinize everything. Analyze all methods, data, and reports closely; question them to the best of your ability. Form an internal peer review panel of experts from related disciplines. Be sure to give yourself and other sufficient time to review and discuss revisions.

License technology. Many universities have inventions or start-ups that quietly clamor for attention and funding. Look for available technologies that are scalable and offer a new benefit for humankind.

By: Blake Ebersole

First published in Natural Products Insider, December 8, 2015

Keys for Meeting Supplement GMP Testing Requirements

by NaturPro in Uncategorized Comments: 0

A core concept across GMPs for many industries is scientific validity, and this is also one of the necessary requirements of the dietary supplement GMPs. For example, the purpose of an ingredient specification is to disclose scientifically valid methods and results for the tests, and these methods and results are used to verify the quality and identity of the material being sold.

Scientific validity means that tests must be suitable for what they are intended to measure. In a rapidly evolving industry, scientific validity is a core principle guiding our efforts to ascertain the identity, safety, and label claims of the material that millions of people take to support their health.

Here’s some ways NaturPro helps to ensure scientific validity

To apply scientific principles to the measurement means that we develop a foundation of confidence in test results that accumulates only through repeated testing of viable hypotheses. During the process, we understand that like with many scientific measurements, sources of error exist which tend to increase with complexity. For example, complex samples containing thousands of chemical constituents (e.g., botanical extracts), and instrumentation methods that have a lot of variables all contribute to our bank of “known unknown” and “unknown unknowns.”

Testing using any single method can be an educated guess as an answer to a different question, especially for labs that may only sporadically test a given matrix with a single type of test.

gel electrophoresisToday’s analytical technology to measure analytes in complex mixtures is way ahead of the not-too-distant past, but now we understand a mitigating factor: that with greater power and resolution comes an increasing number of factors that may cause test results to be inaccurate or imprecise.

For example, it can be difficult to account for systematic error associated with dirty chromatography columns or non-optimal instrument conditions. Inaccurate purity data on reference standards (due to either inaccurate standard purity values, or unaccounted-for degradation during storage) are also a common sources of error — when we are simply trying to figure out the “actual” composition of a material. Another source of error arises from the calculation of the results; for example, moisture can account for a certain amount of the measured weight of both samples and standards, which is often simply estimated, even if it is accounted for.

What more does supplement testing and Star Wars have in common?

Other sources of error in testing can be chalked up to incomplete extraction and isolation during the sample preparation.  The subject of dissolution is an interesting one. For example, it is a common assumption that when a sample “dissolves” during HPLC sample prep, then it is fully “ionized” and thus is not strongly bonded to any solid particles (which then often get caught on the filter and not pass into the detector).

If both standard and sample dissolve to the same degree, no problem!  But (unknown unknown) error due to lower than expected ‘percent recovery’  creeps in when your sample is prepared with heat and time, becoming different compounds and binding differently to the protein-fat-and-sugar matrix of a biological product.  So the analyte that you are trying to extract into another phase is often a lot easier using the pure, unbound. chemical reference standard — leading to a difference in percent recovery.  So chemical reference standards are best complemented in testing with an additional control being the original, authentic botanical reference — yes a whole plant part, taken from the same source as the raw material in question.  Sounds easy, but its actually not for a lot of people. 14963749580_49e4e7ed8a_k

Then compound the sample preparation challenges with the high heat and pressure applied by an analytical instrument like HPLC, where more chemical reactions can happen in the complex sample to degrade what you are measuring, all while your pure reference standard survives nicely to the detector. (Theoretically, this scenario can also happen the other way around, where the matrix stabilizes the analyte better than the standard solution under the HPLC conditions.)

Good-Manufacturing-PracticesExciting stuff, all this mystery, which we eventually find answers to through validation and repetitious testing.  While it’s difficult to predict analytical uncertainty, the point is to control it to the extent possible, hopefully to within 5-10% of your expected result — not bad compared to the 20% tolerance limit required by pharmaceuticals.

The practical question facing suppliers and manufacturers is how to ensure your specification accounts for testing variance?  One solution commonly opted for in the short term is surprisingly simple: add the testing variance to the label or spec requirement, to ensure a high statistical probability that the material won’t fail due to inherent imprecision of the test.

The implications of an imprecise test often means that manufacturers are forced to add an ‘overage’ of material, which essentially makes the cost of the material 10% more expensive for every 10% difference in test results. 

Scientific validity in QC testing for supplement all too often is discussed not on a daily basis, but when the cost of “mistakes” has finally sunk in.  Many a product formulator saw hours and months of work go down the drain due to quality testing failures, and everyone involved in product development can testify to the measurable waste of time and resources that result from testing failures, which can include both the approval of bad material, as well as the rejection of good material.

Five ways NaturPro helps to ensure scientific validity

Here is a short list of some practices that QC units can perform to achieve scientific validity as per GMPs:

–Review your lab’s methods for their suitability for the intended purpose. There are good independent labs out there that will share method information, and answer your questions. Always ask whether the sample is being tested in triplicate and request to receive the individual values.

–Review the documentation on the reference standard, specifically the methods and results of the testing used to determine its purity. When was the standard made, when was its purity last tested, and how was it stored in between?

–Blind your sample so your lab does not know what value to expect.

–Test control samples (samples that do not contain the suspected analyte, OR samples that you previously sent to the same lab).

–Work with labs that can demonstrate having worked to some basic degree to optimize/validate the method.

Sounds like costly work, but not so much when put in perspective of the potential costs. With transparency among customer, supplier, and lab together, a little teamwork goes a long way to reduce the costs and maximize the benefits of quality systems.

By: Blake Ebersole

This article was first published in Natural Products Insider in June 2013