AOAC 2018 Methods

Table 2018.04B. AOAC SMPR 2017.012 criteria and HPLC method results a

Result

Parameter

Requirement

6-Gingerol

8-Gingerol

6-Shogaol

10-Gingerol

Analytical range b , % w/w

0.05-50

0.02–50

LOQ, % w/w Recovery, %

≤0.05

0.0103

0.0045

0.0022

0.0015

90-107

95.9–99.6

98.3–103.3

98.7–101.1

93.7–101.0

RSD r , % RSD R , %

≤5 ≤8

≤2 ≤6

≤3 ≤7

≤2 ≤4

≤2

≤7 a  Table only summarizes the results of AOAC SMPR 2017.12 required analytes (6-, 8-, 10-gingerols and 6-shoganol) in required matrices (rhizome powder, rhizome dry extract, tablet, and capsules). b  Analytical range is calculated from total select nonvolatile ginger constituents contents.

( b ) Tablet and capsule .—Composite 10 tablets or capsules (remove shells), and grind samples until homogenized. For example, for a tablet that has 3% ginger powder and an expected level of 0.03% total nonvolatile ginger constituents, 1000 ± 100 mg (Table 2018.04C ), weigh into a 20 mL amber VOA vial. ( c ) Liquid capsule, softgel capsule, and oleoresin.— Use Pasteur pipet, positive displacement pipet, or other appropriate tool to transfer sample (for softgel liquid capsule, mix and composite the content of five capsules before sample transfer). For example, for ginger oleoresin that has an expected level of 50% total nonvolatile ginger constituents, 1.5 ± 0.2 mg (Table 2018.04C ), weigh into a 20 mL amber VOA vial using a microbalance. After weighing, dilute each sample with 10.0 mL 80% methanol–20% water (water is acidified with citric acid, pH 5), vortex for 1 ± 0.1 min, sonicate for 30 ± 3 min in cold water (if the sonicator has no temperature control function, use ice bags to keep the temperature low, ≤30°C), vortex for 5 s, sonicate for another 30 ± 3 min in cold water, and filter through a 0.45 µm PTFE filter into an amber autosampler vial for future analysis. F. HPLC Analysis ( a ) HPLC system .—Equipped with a binary pump and a diode- array detector (190–400 nm), Agilent (Santa Clara, CA, USA). ( b ) Column .—Kinetex C18 (5 µm, 150 × 3 mm) with a SecurityGuard Ultra Cartridge (Phenomenex, Torrance, CA, USA); for chromatographic separation. ( c ) Instrument conditions .—Optimal instrument conditions are injection volume, 5.0 μL; column temperature, 30°C; flow rate, 1.1 mL/min; and detection wavelength, 230 nm. Gradient program with water as mobile phase A and acetonitrile as mobile phase B is

as follows: 0–1.5 min, hold 35% B; 1.5–1.8 min, from 35 to 60% B; 1.8–5 min, hold 60% B; 5–6.5 min, from 60 to 100% B; 6.5–9 min, hold 100% B; 9–9.1 min, 100–35% B; 9.1–12 min, hold 35% B. Total run time is 12.0 min. G. Single-Laboratory Validation Parameters ( a ) Selectivity.— For major target matrices (ginger rhizome dietary ingredient, and tablet and capsule dietary supplements), method selectivity was demonstrated by running “placebo” samples expected to be free of nonvolatile ginger constituents. Galangal (Kaempferia galangal, Aromatic Ginger) root was used as a placebo of ginger rhizome matrix because of their morphological similarities. Galangal root is also considered as an adulterant of ginger rhizome (3). An excipient blend placebo was formulated (50% maltodextrin, 42% hydroxypropyl methyl cellulose, 5% stearic acid, 2% magnesium stearate, and 1% silicon dioxide) and tested to evaluate potential chromatographic interferences from the filler ingredients used in common tablet and capsule dietary supplements. ( b )  System suitability.— To demonstrate the overall chromatographic system suitability, five replicate injections of the reference material solution were performed for all eight nonvolatile ginger constituents. Peak retention time, peak area, and peak shape were analyzed. %RSD (relative standard deviation) of peak area, %RSD of retention time, USP tailing factor, and relative retention time of each analyte to 6-gingerol were calcuated. As the quality control criteria for system suitability, reference material peak area %RSD must be ≤2.5. Reference material retention time %RSD must be ≤2.5. USP tailing factor of the reference material peak must be <2.0 for all analyte peaks. ( c ) Linearity.— Six calibration solutions were injected at the beginning of each injection sequence. Calibration curves of the method cover the range approximately 0.5–50 µg/mL for zingerone; 1.2–120 µg/mL for 6-gingerol; 0.35–35 µg/mL for 8-gingerol; 0.5–50 µg/mL for 6-shogaol; 0.25–25 µg/mL for 6-paradol; 0.5– 50 µg/mL for 10-gingerol; 0.35–35 µg/mL for 8-shogaol; and 0.5–50 µg/mL for 10-shogaol. Correlation coefficients, calibration equation slopes, and y -intercepts are automatically generated by the HPLC data processing software (e.g., Agilent ChemStation). The blank was not considered as part of the calibration curve. Each calibration curve was made up of six data points, and the resulting R 2 coefficients for all curves must exceed the requirement of NLT 0.999. ( d ) Limits of detection (LOD) and quantitation (LOQ).— The International Union of Pure and Applied Chemistry (IUPAC)

Table 2018.04C. Sample preparation for samples with different matrices and expected levels

Rhizome powder, rhizome dry extract

Liquid capsule, softgel capsule, and oleoresin

Example estimated level, % w/w

Tablet, capsule

Example sample’s weight, mg

0.03

NA NA

1000 ± 100 1000 ± 100

1000 ± 25 250 ± 25

0.3

1 5

60 ± 6 12 ± 3

300 ± 30

75 ± 8 15 ± 2

60 ± 6 6 ± 0.6

50

1.2 ± 0.3

1.5 ± 0.2

© 2018 AOAC INTERNATIONAL