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at room temperature protected from direct light and, if possible, in desiccators.

Table 4. Approximate retention time of the six major ginsenosides

Ginsenoside

Retention time, min

Method

Rg1

16.0–16.4

Principle This method is suitable for the determination of the major ginsenosides in raw materials and select finished product containing P. ginseng (Asian) and P. quinquefolius (North American ginseng). These ginsenosides include Rg 1 , Re, Rb 1 , Rc, Rb 2 , and Rd. Ginsenosides are extracted from the matrixes with aqueous methanol (70%, v/v) by sonication. For unprocessed test samples (root materials) that contain malonyl ginsenosides, an aliquot of this extract is hydrolyzed with an aqueous potassium hydroxide solution (5%, w/v) to convert the acidic ginsenosides into their neutral forms. The resulting solutions are then subjected to RP-HPLC analysis using a C 18 column and UV absorbance detection. Quantification is performed using an external seven-point calibration curve constructed using the mixed ginsenoside standard. ( a )  HPLC system .—Suitable HPLC system equipped with a binary pump capable of gradient elution, autosampler, and UV absorbance detector. The HPLC operating conditions are as follows: column temperature: 25°C, mobile phase flow rate: 1.5 mL/min, mobile phase A (MPA): HPLC-grade or nanopure filtered water, mobile phase B (MPB): aqueous acetonitrile (80%, v/v), mobile phase gradient program as shown in Table 2, injection volume: 10 µL and detection wavelength: 203 nm. ( b )  HPLC column .—Phenomenex (Torrance, CA) Luna C 18 , 150×4.6 mm id, 5 µm particle size, 100 Å pore size, equipped with guard column, or equivalent. ( c )  Analytical balance .—Readability, ± 0.1 mg. ( d )  Ultrasonication bath . ( e )  Centrifuge .—Capable of centrifuging 50 mL conical tubes at 3500 rpm. ( f )  Microcentrifuge tubes .—Polypropylene, 2 mL. ( g )  Vortex mixer. ( h )  Conical tubes .—Polypropylene, 50 mL. ( i )  Volumetric flasks .—10, 25, 50, and 100 mL. ( j )  Graduated cylinders .—10, 500, and 1000 mL. ( k )  Syringe filters .—PTFE, 0.45 µm pore size, 25 mm diameter. ( l )  Automatic pipets .—10, 100, 200 and 1000 µL. ( m )  HPLC vials .—2 mL clear and amber with Teflon ® - coated caps. ( n )  HPLC vial inserts .—200 µL. ( o )  Syringes .—3 mL Luer-lok ® . ( p )  Reagent bottles .—Clear or amber glass, 1000 and 2000 mL. ( q )  Beakers .—Clear glass, 100 and 500 mL. Apparatus Note: Equivalent apparatus may be substituted.All volumetric pipets and flasks are Class A grade.

Re

16.5–16.9

Rb1

28.7–29.0

Rc

29.0–30.1

Rb2

31.3–31.7

Rd

34.4–34.8

control test material consisted of maltodextran (99%) and magnesium stearate (1%).

Standards

A batch of amber ampules containing the MGRS was provided by Anthony Windust from the NRC. Two ampules of the MGRS containing the six major ginsenosides at varying concentrations were provided to each laboratory. Laboratories were instructed to dilute the standards in 100% methanol at different concentration levels to provide a seven-point calibration curve. One ampule was used for the analysis of the practice samples and the other was used for the test samples. A representative chromatogram of the calibration standard is shown in Figure 1. The MGRSs were shipped accompanied with cooler packs to prevent degradation during transit. Laboratories were instructed to transfer the MGRS to –20°C storage upon receipt. Whole root, powdered extract, and spiked matrix blank samples were shipped at ambient temperature and sealed in individual glass vials, while tablets and capsules were shipped in ZipLoc ® plastic bags. Laboratories were instructed to store the samples Preparation and Shipment of Standards and Samples

Table 5. HorRat values for the individual ginsenosides in the ginseng test materials

GIN-GCS a Rg 1

Matrix

Re Rb 1

Rc Rb 2

Rd

1, 2 1.7 1.0 1.3 1.2 4.1 1.0

P.q. powdered whole root b P.q. powdered extract

3, 4 1.4 1.2 1.4 1.0 3.2 0.8

P.q. powdered whole root tablet

5

1.7 1.3 3.1 5.3 7.8 2.0

6, 7 1.6 1.5 2.0 1.6 1.0 1.0

P.g. powdered whole root c P.g. powdered extract

8, 9 1.3 1.7 2.0 1.8 1.6 1.3

P.g. powdered whole root capsule P.g. spiked matrix blank

10

1.5 1.6 3.0 2.3 1.9 2.0

11, 12

1.1

1.2

1.6

1.8 2.3 1.5

Reagents

a GIN-GCS = Ginsenoside-glucocorticoids; ginseng collaborative sample. b P.q. = Panax quinque folius . c P.g. = P. ginseng.

Chemicals from certified suppliers that meet the method specification may be used. Acetonitrile and methanol are