Determination of Glucosamine in Raw materials and Dietary Supplements Containing Glucosamine Sulfate and/or Glucosamine Hydrochloride by HPLC with FMOC-Su Derivatization: Collaborative Study

 

Joseph Zhou, Ted Waszkuc, and Felicia Mohammed

NOW Natural Foods, Inc., Methods Development Laboratory, 395 S. Glen Ellyn Rd, Bloomingdale, IL 60108

 

Collaborators: Chuck Ray; Randy Buren; Wendi Wang; Hao Nguyen; Darryl Sullivan; Jack Jabusch; Xiaolan Kou; Qiuping Yang; Aniko Solyom; Jonathan Wang; Tang S. Peng; Mike Blumhorst; Mythili Nagarajan; Brandon Podhola; Li Huang; Cathy Shevchuk; Rupa Das; Kevin Orellana; Klaus Reif.

 

A collaborative study was conducted on the method for the determination of glucosamine in raw materials and dietary supplements containing glucosamine sulfate and/or glucosamine hydrochloride by HPLC with N-(9-fluorenylmethoxycarbonyloxy) succinimide (FMOC-Su) derivatization. Thirteen blind duplicates of materials consisting of various commercial products including tablets, capsules, drink mix and liquid products as well as raw materials, blanks, and spike recovery products were tested by twelve collaborating laboratories. The tests with the blank samples and the samples with glucosamine spiked showed good specificity of the method. The average spike recoveries at the spike levels of 100% and 150% of the declared amount were 99.0% with an RSD of 2.1% and 101% with an RSD of 2.3%, respectively. The test results between laboratories on each commercial product were reproducible with all of RSD no more than 4.0%, and the results were repeatable in the same laboratory with an average RSD of 0.7%.HORRAT values ranged from 0.5 to 1.7 on both tests of spike recovery and reproducibility between laboratories on commercial products. The average determination coefficient of the calibration curves from the laboratories was 0.9995 with an RSD of 0.03%. None of the results from the collaborating laboratories was outlier, partly indicating the robustness of the method. It is recommended that the method be accepted by AOAC as Official First Action.

 

The glucosamine HPLC method with FMOC-Su derivatization (1-3) was selected by an AOAC expert review panel (ERP) as the most appropriate method to recommend for further laboratory validation. The results of the subsequent single lab validation study (SLV) when subjected to peer review by the ERP and selected members of the AOAC Methods committee on Dietary Supplements indicated the method was suitable for a full collaborative study (4).

The collaborative study is to evaluate the method’s accuracy and precision based on its intra- and inter- laboratory performance (5-6). In this study, thirteen blind duplicates of glucosamine test materials consisting of various commercial products as well as blanks and spike recovery samples were analyzed by twelve collaborating laboratories.

Glucosamine product is one of the most popular dietary supplements in the United States, and its effectiveness has been clinically proved for the treatment of osteoarthritis (7). Glucosamine HCl and glucosamine sulfate are the two most important glucosamine salt forms used in these products and also claimed on the product labels.  The establishment of an official glucosamine method will facilitate its product quality control and regulatory compliance.

 

Collaborative Study

Study Design

The study was conducted on twelve different test materials. One of them was split into two identical samples to test repeatability of the analytical results in the same laboratory. The identity or content of these thirteen samples was not released to the collaborators, and random identification numbers were assigned to each of the test materials. The blind duplicates of these materials and glucosamine reference standard were supplied to each of the laboratories for the study. Practice samples were also provided to ensure that each participant could successfully follow the method and to optimize their instruments before proceeding with the actual tests. The study director was available for consultation. Table 1 lists these thirteen test samples and their ingredients and potencies.

Collaborators

 Twelve laboratories participated in the study. Three of them were industrial dietary supplement finished product manufacturers, five were raw material vendors, three were commercial testing laboratories, and one was the university laboratory. Geographically, ten of them were from the United States, one from Canada, and one from Germany.

Study Procedures

Product Types.æ There were basically five types of the test materials analyzed: i) tablets; ii) capsules; iii) drink mix powder; iv) raw materials; and v) liquid products. At least one product of each type was chosen to represent the group. These representative samples also covered different material sources, e.g., glucosamine from shellfish or glucosamine from vegetable source; different salt forms: glucosamine hydrochloride and glucosamine sulfate; and different combination products with chondroitin, methylsulfonylmethane (MSM), S-adenosyl-L-methionine (SAMe), Vitamin C, etc.

Sample Potency Range.æ Because of the large variety of test materials covered in this study, the glucosamine concentrations differed greatly. As shown in Table 1, for example, 12.6 g of the commercial drink-mix powder contains only 1.5 g of glucosamine HCl. However 12.6 g of glucosamine HCl raw material with min 98.5% purity contains 12.4 g of glucosamine HCl. In addition, between glucosamine HCl and glucosamine sulfate, because the industrial glucosamine sulfate raw material has a complex empirical formula as 2GlucosamineFreeBase·H2SO4·2KCl, 1 g of such a material only contains 0.59 g of glucosamine free base (GFB), but 1 g of glucosamine HCl contains 0.83 g of GFB. As result, depending on which salt form to use and how much to use (limited by total physical sizes of the tablets or capsules, etc.), the GFB potency in the final products varies significantly. These differences in reality make it difficult in the method to use the same sampling size in weight for all the samples. Therefore, to ensure the best accuracy of tests, the method has classified different sample categories based on the material types for sampling. 

Preparation of Test Samples.æ i) Commercial Products: Labels were removed from the original commercial packages. Tablets were ground into powder and capsule contents were emptied out from the shells. All the powders including raw materials and drink-mix were well mixed to prevent sample non-uniformity problems. For shipment 20-30 g powder was packed into a film bag. in a quantity of 20-30 g. The liquid products were mixed thoroughly and re-packed into 60-mL plastic bottles for shipment.

ii) Blank Powder: A combination blank powder for tablets and capsules was prepared based on the weight percentages shown in Table 1, and treated as a tablet powder for analysis.

iii) Spike Recovery Samples: The two samples for spike recovery levels I&II were initially made as a liquid using the blank powder and glucosamine standard reference to avoid homogeneity problems, but those products decomposed from microbial action. It was later found that 1% sodium benzoate can be used as a preservative. Because of time limitation and stability concerns on the other products, the collaborators were asked to prepare G1 and G2 (the two spike recovery samples) with glucosamine HCl standard and G12 (the blank powder) provided. But the identity of G1, G2 and content of G12 (marked as a tablet powder) were kept unknown to collaborators all time. The parent products were made by accurately mixing 360 ± 10 mg of glucosamine HCl standard with 70 ± 10 mg of G12, and mixing 240 ± 10 mg of glucosamine HCl standard with 190 ± 10 mg of G12, respectively. All of the samples were processed under a very clean and controlled condition to avoid contamination. Glucosamine in solid state is, in general, chemically stable, but under special cases such as hot temperature and chemical reactions as well as microorganism, it may degrade.

Shipment.æ Samples and standard were shipped to collaborators at ambient temperature. Each of the test bags or bottles was labeled for identification (G1, G2…) and product type, e.g., tablet powder or capsule powder to assist collaborators to differentiate the products and find right categories. Collaborators were required to return a receipt acknowledgement forms to indicate receipt and condition of the shipped items. They were also directed to store samples and standard at room temperature.

Practice Samples.æ The practice samples were prepared, also randomly coded (P1, P2…), shipped and analyzed as if they were the actual samples. Collaborators were required to analyze the samples and report the results to acquire study director’s approval before proceeding with the actual sample study.

Analysis.æ Collaborators are required to prepare new calibration solutions and curve each test day. For test material analysis, single preparation and single injection are required.

Data Reporting.æ  Collaborators were asked to report the linearity of each calibration curve, the corresponding concentrations of the calibration solutions, and the percentages of glucosamine free base (GFB) found in each of the thirteen test samples using the data reporting sheets. They were also asked to report any important observations and significant deviations to the method.

Expected Values and Validation Data of the Test Materials.æ Table 2 lists the expected values (e.g., the GFB values calculated from the label claims) and validation data of the test materials. The validation data were obtained by the Study Director’s laboratory at NOW Foods using the same method and on the same materials as sent to the collaborating laboratories in this study.

 

The Method

 

Determination of Glucosamine in Raw Materials and Dietary Supplements Containing Glucosamine Sulfate and/or Glucosamine Hydrochloride by

HighPerformance Liquid Chromatography of FMOC-Su Derivatives

 

A.     Principle

Glucosamine sulfate/hydrochloride finished products or raw materials are dissolved in water.  The glucosamine free base is released by adding triethylamine to the solution to neutralize the H2SO4/HCl salts, and derivatized with 9-fluorenylmethoxycarbonyl succinimide (FMOC-Su). The derivative is separated by HPLC and measured with UV detection. Glucosamine has two natural steroisomers (a and b), and the interconversion of these two in aqueous solution is not preventable, resulting in two peaks in the chromatogram. The sum of the areas of these two peaks is used for the quantification of the glucosamine free base.

 

B.  Apparatus

(a)    LC system (Equivalent equipment meeting the specification 5 may be used).æAgilent HPLC 1100 series with pump, degasser, autosampler, thermostatted column compartment, photodiode array detector, and Chemstation software for system control and data acquisition (Agilent Technologies, Inc., Palo Alto, CA; www.agilent.com). Operate the LC system under the following conditions: mobile phase flow rate, 0.8 mL/min; detection wavelength, 265 nm; column compartment temperature, 30 °C; and injection volume, 10 mL.

(b)    LC column.æ Phenomenex Prodigy (MidBore™) ODS-3 100 Å, 5m, 150 x 3.2mm, Phenomenex order #: 00F-4097-R0 (Phenomenex, Torrance, CA; www.phenomenex.com).

(c)    LC guard column.æ Phenomenex Prodigy SecurityGuard™ Cartridges ODS-3 100 Å, 4 x 3.0mm, Phenomenex order No: AJO-4287 (Phenomenex,).

(d)    Analytical balance.æ Ohaus AS60; readability, ± 0.0001 g (Ohaus, Florham Park, NJ; www.ohaus.co).

(e)    Sonicator (As described or equivalent).æ Branson 8210 ultrasonic cleaner (Branson Ultrasonic Corporation, Danbury, CT; www.bransonultrasonics.com).

(f)     Vortex (As described or equivalent).æ Type 16700 mixer (Barnstead International, Dubuque, Iowa; www.barnsteadthermolyne.com).

(g)    pH meter.æ Beckman f40 (Beckman Instruments, Inc., Irvine, CA; www.beckman.com).

(h)    Grinder.æ One-Touch Coffee Grinder (General Electric Company, Fairfield, CT; www.gehousewares.com) Model No. 106854.

(i)       Volumetric flasks.æ 5 and 100 mL, class A.

(j)      Volumetric pipettors.æ 10 mL, class A. (This is needed only for liquid sample analysis.)

(k)    LC solvent filters.æ 0.45μm nylon membrane.

(l)      Syringe filters.æ PTFE, 0.45μm x 13mm (Restek, Bellefonte, PA; www.restekcorp.com), and 0.45μm x 25mm (Fisher Scientific Pittsburgh, PA; www.fisherscientific.com).

(m)  Syringe.æ Luer-Lok™, 3 mL, from Becton, Dickinson and Company through VWR International (South Planfield, NJ; www.vwrsp.com).

(n)    Eppendorf variable volume pipettors and tips.æ 50-200μL (accuracy: ± 1.0-0.6%, precision: £ 0.3-0.2%) and 500-2500μL (accuracy: ± 1.5-0.6%, precision: £ 0.3-0.2%). Both are available from VWR International (South Planfield, NJ; www.vwrsp.com). Note: make sure both pipettors are properly calibrated.

(o)   HPLC injection vials.æ Screw cap vials with Teflon coated caps (Agilent Technologies, Inc., Palo Alto, CA). (As described or equivalent).

 

C.  Reagents

(a)    Reference standard (chemicals from other suppliers meeting the specifications may also be used).æ D(+)-Glucosamine Hydrochloride, minimum 99% pure, available from Sigma (St. Louis, MO; www.sigmaaldrich.com) Product No: G4875, 25g.

(b)    Derivatization reagent.æ N-(9-Fluorenylmethoxycarbonyloxy)succinimide (FMOC-Su),  97% pure, available from Lancaster (Windham, NH;  www.lancastersynthesis.com)  Cat. No: 6908.

(c)    Solvents.æ Acetonitrile, HPLC Grade; Trifluoroacetic Acid (TFA), min 99.0% pure; Triethylamine (TEA), min 99% pure; Water, HPLC grade. All from Fisher Scientific (Pittsburgh, PA).

(d)    FMOC-Su derivatization solution. –– 15 mM.  Dissolve 50±1.0 mg of FMOC-Su in 10 mL of acetonitrile. Prepare this solution fresh for each test.

(e)    Mobile phase.æ Mobile Phase A: water containing 0.05% trifluoroacetic acid, pH 2.4. Mobile Phase B: pure acetonitrile.

(f)     Derivatization quench solution.æ Mixture of mobile phases A/B (1/1, v/v).

 

D.  Preparation of Test Solutions

Accurately weigh or measure the amount, as indicated in Table 3, into separate 100 mL volumetric flasks. For tablets, find and record the mean weight of 20 tablets, grind, mix and weigh. For capsules, empty and record the mean fill weight of 20 capsules, grind the contents, mix and weigh.  For liquid products: shake well before taking the test portion.

Add 90 mL of water to the test portion, vortex for 1 min and sonicate for 5 min or until all solids dissolve (Note: some of the products’ excipients, e.g., silicon dioxide, may never dissolve). Pipette 750 mL of triethylamine into each of the flasks to neutralize HCl or H2SO4, and dilute to volume with water. Filter about 1.5 mL of each solution through a0.45 mm x 25 mm PTFE filter into an HPLC injection vial.

 

E.   Derivatization Procedures

Note: Both standards and test solutions must be derivatized simultaneously in 5 mL volumetric flasks.

Pipet the exact amount, as specified below, of the filtered solutions from the HPLC injection vials into separate 5 mL volumetric flasks: (i) Glucosamine Standard Working Solutions (three-point calibration): 50, 125(or 100), and 200 mL respectively; and (ii) for all other products: 125(or 100) mL.   Add 500 mL of 15 mM FMOC-Su solution to each flask. Cap the flasks tightly with Teflon stoppers, mix well with vortex, and sonicate all the flasks in the sonicator water bath at 50 °C for 30 min.  Remove the flasks from the bath, let cool to room temperature, and dilute the flasks to volume with the mixture of mobile phases A/B (1/1, v/v). Mix well with vortex. Filter each solution through 0.45 mm x 13 mm PTFE filter into an HPLC vial for injection.

 

F.   Determination

(a)    System suitability tests.æ Equilibrate the HPLC system with the mobile phase for at least 30 min. Make 5 replicate injections of the second (mid-concentration) glucosamine HCl working standard. The typical retention time (t) of glucosamine anomer peak 1(the earlier eluted peak) should not be less than 4 minutes, and the relative retention times (Rr) of glucosamine anomers peak 2 to peak 1 should be 1.2 (Rr = t2/t1). The peak tailing factor (T) should not be more than 2.0 (T = W0.05/2f, where W0.05 is width of the peak measured at a point 5% of the peak height from the baseline; and f is horizontal distance from the vertical line at the peak maximum to the point on leading edge of the peak at 5% height). The relative standard deviation (RSD) of the sums of peak area of glucosamine peaks 1 and 2 from the 5 injections should not be more than 2.0%.

(b)    Mobile phase gradient  program.æ Elute the analytes with the following gradient mode of mobile phases A and B. 0.0-6.0 min: held isocratic at 70A:30B; 6.0-11.0 min: change to 0A:100B; 11.0-13.0 min: to 70A:30B; and 13.0-15.0 min: held isocratic at 70A:30B.

(c)    Run time.æ 15 min.

(d)    Injection.æ Make single injection of each standard working solution and unknown solution.

 

G.  Calculation

(a)    Concentrations of glucosamine working standard solutions.æCalculate the concentrations of glucosamine free base (GFB) in working standard solutions, after FMOC derivatization:

STD n, GFB, mg/mL = 0.83091x d x W x F 

Where: n =1, 2, 3 for three different standard working solutions; 0.83091 is the conversion factor from glucosamine HCl to glucosamine free base: 179.17/215.63; d = the dilution factor: v/(100 mL x 5 mL), with v = 0.050, 0.125 (or 0.100), and 0.200 mL for STD1, STD2, STD3, respectively; W = the amount of glucosamine HClstandard weighed, mg; and F = the purity factor of glucosamine HCl standard used.

(b)  Percentage of Glucosamine Free Base in All Glucosamine Contained Materials.æ Calculate the % glucosamine free base in all glucosamine contained materials:

% mg/mg GFB = (P – b) x 100 / (a x D x W)

Where: P = the sum of peak area of glucosamine peaks 1 and 2 of the unknown test sample; a = slope of the calibration curve; b = intercept of the calibration curve; D is the dilution factor: v/(100 mL x 5 mL), with v = 0.125 (or 0.100) mL; W = the amount of unknown test portion weighed, mg.

For liquid sample:

mg/mL GFB = (P – b) /  (a x D x V)

Where: V = the amount of liquid unknown product used, mL.

(c)  Percentage of Glucosamine HCl in Finished Products or Raw Materials.æ Calculate the % glucosamine HCl in glucosamine HCl finished products or raw materials:

% mg/mg G×HCl = (P – b) x 100 / (0.83091 x a x D x W)

For liquids:

mg/mL G×HCl = (P – b) /  (0.83091 x a x D x V)

 (d) Percentage of Glucosamine Sulfate (2GlucosamineFreeBase·H2SO4) in Finished Products or Raw Materials.æ Calculate the % glucosamine sulfate in glucosamine sulfate finished products or raw materials:

% mg/mg G×Sulfate =   (P – b) x 100 /  (0.78511 x a x D x W)

Where:             0.78511 is the conversion factor from glucosamine sulfate (2GlucosamineFreeBase·H2SO4) to glucosamine free base: (2x179.17)/456.418

For liquid samples:

mg/mL G×Sulfate = (P – b) /  (0.78511 x a x D x V)

 (e) Amount of Glucosamine Sulfate (or HCl) Per Product Unit.æCalculate Glucosamine Sulfate (or HCl) in mg per Tablet (or Capsule), or Glucosamine Sulfate (or HCl) in mg per Serving Volume (mL):

mg G×Sulfate (HCl) per Tablet (Capsule) = %w/w G×Sulfate (HCl) x 100 x mgOneTab (Cap)

or:         mg G×Sulfate (HCl) per Serving Volume = mg/mL G×Sulfate (HCl) x mLOneServing

Where: mgOneTab (Cap) = the average weight of one tablet or the average fill weight of one capsule, in mg; and mLOneServing = the serving volume for liquid products, in mL.

 

H.     Typical Chromatogram

 

 


 

 


Results and Discussion

      Collaborative Study Results

The analyses were completed in all twelve collaborating laboratories in two weeks. The sample identification, which was randomly assigned to the test samples, was decoded after the test results were received, and the names of the participating laboratories were coded from L1 to L12 for their data presentation in this report.

Table 4 shows the complete set of data submitted from the collaborating laboratories in the percentages of glucosamine free base for eight commercial products and two different blanks. These data indicate the method performance on reproducibility of the analytical results between laboratories.  Table 4 also shows the results on two identical test samples (G10 and G13), that not only indicate the method performance on the repeatability of the analytical results in the same lab, also reproducibility between laboratories. Table 5 shows the concentrations of calibration solutions and linearity of calibration curves reported from each laboratory. Table 6shows the spike recovery results of two spike levels from all the laboratories. The percentages of spike recovery were calculated by dividing the percent glucosamine free base found in the test in each test sample by the percent GFB fortified in the test sample and multiplying by 100.  The data indicate both the method accuracy and method precision between-laboratories.

The results in Table 4 were used to generate the statistical data presented in Tables 7 and 8 respectively for reproducibility between laboratories and repeatability in the same laboratory.

The HORRAT value is the ratio of the relative standard deviation, expressed as a percent (%RSD) to the predicated relative standard deviation, expressed as a percent (%PRSD), i.e., HORRAT = %RSD / %PRSD, where %PRSD = 2C-0.1505 and C = the estimated mean concentration. For the spike recovery results presented in Table 6, the true concentrations of glucosamine free base (%GFB spiked) was used to calculate %PRSD for HORRAT values. But for the statistical data shown in Tables 7 and 8, the experimental concentration values of GFB were used to calculate %PRSD. 

 

Collaborators’ Comments

Most of the collaborators found the method was easy to follow and perform. Reasonable modifications of the method (as shown below) without significantly affecting the test results and also with the Study Director’s permission were allowed to show the method’s robustness.

Laboratory 2 increased the post gradient equilibration time from 2 to 7 min before next injection for a total run time of 20 min. The results from the other laboratories were obtained using the conditions per method.

Laboratory 5 observed a small peak around a retention time of 4.1 min when the analyst uses water as a blank for test. The laboratory also observed this little peak in some glucosamine sample analysis, and suspected it had merged with peak 1 of glucosamine in other tests. The analyst also found in some tests another small peak between peaks 1 and 2 of glucosamine. It is not sure that the peaks were due to some impurities in the particular lot of the reagents. However since the size of these peaks were tiny the quantitation results from the laboratory were not affected as shown in Tables 4-8.

Laboratory 6 reported that during a replicate injection of 5, the area of glucosamine peak 1 slightly increased while the area of peak 2 slightly decreased, but the total area of peaks 1 and 2 remained the same (RSD = 0.19% and 0.33% with 5 injections for each of 2 trials).

Laboratory 7 analyzed the samples after being prepared and stored in HPLC vials for 4 days at ambient temperature. The results are adequate, indicating good stability of the glucosamine-FMOC-Su derivatives.   

Laboratory 10 prepared all the samples by using a half of the recommended amount for sampling, and 50 mL volumetric flasks instead of the 100 mL per method. For derivatization, they used 10 mL volumetric flasks instead of 5 mL, and all recommended volumes for sample and reagents were doubled. Some of the differences in sampling weight were shown in Table 5 (Amounts of the blank powder and glucosamine HCl standard used). However all of their test results are acceptable.

Laboratory 11 performed the tests without a guard column.

Laboratory 12 noticed insoluble residues in the sample preparations of G1, G2, G8, G10, G12 and G13.

None of the collaborating laboratories reported any system suitability problems in the study.

 

Performance Characteristic of the Method

In summary of the data presented in Tables 4-8, the tests with the blank samples and the samples with glucosamine spiked showed good specificity of the method. The average spike recoveries at the spike levels of 100% and 150% of the declared amount were 99.0% with an RSD of 2.1% and 101% with an RSD of 2.3%, respectively. The test results between laboratories on each commercial product were reproducible with all of RSD no more than 4.0%. The results were repeatable in the same laboratory on two identical samples, with an average RSD of 0.68% for all laboratories.  The average determination coefficient of the calibration curves from the laboratories was 0.9995 with an RSD of 0.03%.

HORRAT values showed 1.1 and 0.9 for the spike recovery analysis at the levels of 100% and 150% respectively. For the tests of reproducibility between laboratories on each commercial product, HORRAT values ranged from 0.5 to 1.7. The tests of repeatability in the same laboratory on two blind duplicates showed HORRAT values of 0 - 1.2 for all laboratories.  The zero and near zero values for HORRAT are the results of some laboratories finding and reporting the exactly same or similar %GFB on two identical tablet samples (G10 and G13), which diminished RSD in the numerator for calculation of HORRAT value. The fact that collaborating laboratories found the same or very similar results on two blind duplicates among 13 test materials demonstrated well the reliability of the method.

The method is also rugged and robust (6, 8 for definitions). The method has been tested by twelve collaborating laboratories on various test materials. Although the same method was followed, the actual use conditions in different laboratories may vary greatly.   It is important to note that all of the twelve laboratories succeed in the study, andnone of their results was outlier.

Since glucosamine concentration in commercial products varies significantly with product manufacturer, type (tablets, capsules, raw materials, etc.) and glucosamine salt form used (glucosamine hydrochloride and glucosamine sulfate), the tests may be more accurate if sampling amount is based on its label claims. 

 

Recommendations  

Based upon the results of the collaborative study it is recommended that the method be accepted by AOAC as Official First Action.

 

Acknowledgments

 

We appreciate AOAC Methods Committee K and ERP members for their valuable guidance, comments and suggestions, as well as AOAC staff’s hard working and strong support to this collaborative study. We thank the following collaborators for their participation:

Chuck Ray and Randy Buren, Cargill, Inc., Eddyville, IA

Wendi Wang, Hao Nguyen, Advanced Botanical Consulting & Testing, Inc., Tustin, CA

Darryl Sullivan and Jack Jabusch, Covance Laboratories, Inc., Madison, WI

Xiaolan Kou and Qiuping Yang, Nature’s Sunshine Products, Inc., Provo, UT

Aniko Solyom, The University of Arizona, Tucson, AZ

Jonathan Wang, Nature’s Way Products, Inc., Springville, UT

Tang S. Peng, Pure World Botanicals, South Hackensack, NJ

Mike Blumhorst, ArcherDaniels Midland Company, Decatur, IL

Mythili Nagarajan and Brandon Podhola, Enzymatic Therapy, Inc., Green Bay, WI

Li Huang, Cathy Shevchuk, JR Laboratories Inc., Burnaby, Canada

Rupa Das and Kevin Orellana, BI Nutraceuticals, Long Beach, CA

Klaus Reif, PhytoLabs, Vesternbergsgreuth, Germany

This study is the result of a contract between the Center for Food Safety and Applied Nutrition, FDA and the Office of Dietary Supplements, NIH with the AOAC INTERNATIONAL. The purpose of the contract is to provide the FDA, as well as other government agencies, and the dietary supplements industry with AOAC Official Methods, applicable to commercial available dietary supplements and their raw materials.