5. AOACSPDSMethods-2018AwardsV3

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be stored in freezer at –20 ± 2°C for up to 50 days (stability data not shown). Prepare 6-point calibration curve by diluting standard stock solution. D. Optimization of Extraction To optimize extraction conditions, ginger rhizome powder sample was used as study material. Extraction solvents [methanol; methanol–water (80 + 20%, v/v); methanol–water (50 + 50%, v/v); ethanol, ethyl acetate; and dichloromethane–methanol (50 + 50%, v/v)] were tested for extraction efficiency optimization using 1 h (sonicate 30 min, vortex 5 s, sonicate another 30 min) sonication. After determining the extraction solvent, acidic environment and enzyme application in extraction solvent were also studied using the same ginger rhizome powder sample. The treatments included: ( a ) Control.— 80% Methanol–20% water was used as extraction solvent. ( b ) Acidic extraction.— Water pH was adjusted to 5 using citric acid solution before mixing with methanol to make the 80% methanol–20% water diluent. The rest of the sample preparation was the same as in ( a ) Control. ( c ) Published enzymatic method (1) .—0.5% (v/w) cellulase solution was used for each gram of ginger powder, and reaction occurs in pH 5 water. Sample was incubated for 1 h in 50°C water bath before adding methanol for further extraction. Final methanol– water ratio was 80 + 20%. The rest of the sample preparation was the same as in ( a ) Control. ( d ) Optimal enzymatic method .—Reaction conditions were designed based on the enzyme manufacturer’s recommendations (email communication with Sigma-Aldrich). 0.5% (v/w) cellulase solution was used for each gram of ginger powder, and the reaction occurs in pH 8 water. Sample was incubated for 1 h in 60°C water bath before adding methanol for further extraction. Final methanol– water ratio was 80 + 20%. The rest of sample preparations was the same as in ( a ) Control. To further evaluate the impacts of changing factors [sonication time and temperature, vortex time before sonication, filter type, pH of water fraction of the diluent (citric acid), glassware, and sample (ginger root powder/diluent ratio)] in the analytical procedure, a Youden ruggedness trial (2) was conducted. Eight aliquots of the ginger rhizome powder sample were extracted and tested using different factor combinations. E. Sample Preparation In dietary supplements and ingredients samples, estimated levels of total nonvolatile ginger constituents may vary from 0.03 to 50% (w/w). They were determined from the dietary supplement formulation or dietary ingredient supplier’s certificate of analysis. Because of the large range and importance of sample/diluent ratio, the amount to weigh for each sample (Table 2018.04C ) was adjusted based on the expected level and the mid-level of the calibration curve. To ensure peak area within the curve, sample weight was calculated to target levels 2–4 of the calibration solution’s concentration. Maximum sample weight was 1000 mg for all the matrices to avoid the oversaturation of diluent. ( a ) Rhizome powder and rhizome dry extract .—For example, for ginger rhizome powder that has an expected level of 1% total nonvolatile ginger constituents, 60 ± 6 mg (Table 2018.04C ), accurately weigh sample and transfer into a 20 mL amber VOAvial. Before weighing, mix the sample thoroughly until homogenized. Grind samples if necessary.

AOAC Official Method 2018.04 Select Nonvolatile Ginger Constituents in Dietary Ingredients and Dietary Supplements HPLC–UV First Action 2018 [Applicable for determination of nonvolatile ginger constituents: 6-, 8-, and 10-gingerols; 6-, 8-, and 10-shogaols; 6-paradol; and zingerone in a variety of dietary ingredients and dietary supplements matrices, including dry extract, powder, tablet, capsule, liquid capsule, softgel capsule, and oleoresin.] See Tables 2018.04A and B for results of method performance study supporting acceptance of the method. A. Principle HPLC with UV-Vis detection is used for determination of nonvolatile ginger constituents by following AOAC Guidelines for Single-Laboratory Validation . Sample is accurately weighed and diluted with acidified water-methanol mixture. Sample solution is then sonicated and filtered through a PTFE filter and analyzed under a linear gradient scheme instrument condition. A reverse- phase superficially porous particle (SPP) C18 column and an absorption wavelength of 230 nm are used for analyte separation and determination. The method was demonstrated to be selective, linear (R 2 > 0.999), specific, accurate (91.1–103.2% spike recovery rate), and precise (RSD r < 5%, RSD R < 8%), and, therefore, met all AOAC Standard Method Performance Requirements (SMPR® 2017.012) criteria. With a relatively short run time (12 min) and optimized extraction solvent system, the method was validated to simultaneously determine nonvolatile ginger constituents in a variety of dietary ingredients and dietary supplements matrices including dry extract, powder, tablet, capsule, liquid capsule, softgel capsule, and oleoresin. B. Reagents and Materials ( a ) Standard 6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol, and 10-shogaol .—ChromaDex (Irvine, CA, USA). ( b ) Zingerone, cellulase (cellulase from Aspergillus sp.; 1200 IU/mL; Enzyme Commission (EC) No. 3.2.1.4; Sigma Product No. C2605), and citric acid (reagent grade) .—Sigma-Aldrich (St. Louis, MO, USA). ( c ) 6-Paradol .—Donated by Dalton Research (Toronto, Canada). ( d ) Methanol and acetonitrile .—HPLC grade (Fisher Scientific, Pittsburgh, PA, USA). ( e ) Samples .—Ten samples in the forms of both dietary supplements (tablet, capsule, softgel, etc.) and dietary ingredients (rhizome powder, dry extract, and oleoresin) were assigned random identification codes and used for validation studies. Some samples were donations from manufacturers. Other samples were purchased from www.iHerb.com. Aginger rhizome powder sample (Botanical Reference Materials 30290-5) was obtained from ChromaDex. Samples were stored at room temperature prior to testing. C. Preparation of Standard Solution Standard stock solution .—Accurately weigh and transfer 2.5 ± 0.3 mg 6-gingerol, 0.75 ± 0.08 mg 8-gingerol, 1.0 ± 0.1 mg 10-gingerol, 1.0 ± 0.1 mg 6-shogaol, 0.75 ± 0.08 mg 8-shogaol, 1.0 ± 0.1 mg 10-shogaol, 0.5 ± 0.1 mg 6-paradol, and 1.0 ± 0.1 mg zingerone to a 20 mL amber VOAvial, and then dilute with 20.0 mL methanol. Store stock solution in freezer until use. Solution may

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