5. AOACSPDSMethods-2018AwardsV3

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AOAC Official Method 2018.09 Ginsenoside Content in Panax ginseng C.A. Meyer and Panax quinquefolius L. Root Materials and Finished Products High-Performance Liquid Chromatography with Ultraviolet Absorbance Detection First Action 2018 Caution : Acetonitrile and methanol are flammable organic solvents; keep away from any sources of heat and open flames and use a fume hood during sample processing. A. Principle 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. B. Apparatus Note: Equivalent apparatus may be substituted. All volumetric pipets and flasks are Class A grade. ( a )  HPLC system .—Suitable HPLC system equipped with a binary pump capable of gradient elution, autosampler, and UV absorbance detector. 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 2018.09A , injection volume: 10 µL and detection wavelength: 203 nm. ( b )  HPLC column .—Phenomenex (Torrance, CA, USA) 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.

Table 2018.09A. HPLC gradient elution timetable for determination of ginsenosides

Time, min

MPA, % a

MPB, % a

0 8

76 76 68 60 52

24 24 32 40 48

18 25 42 43 44 45

0 0

100 100

76

24

a  MP = Mobile phase.

C. Reagents Chemicals from certified suppliers that meet method specification may be used. ( a )  Solvents .—Acetonitrile, HPLC grade; methanol, HPLC grade; water, HPLC grade or equivalent. ( b )  Potassium phosphate monobasic (KH 2 PO 4 ) .—≥99% ACS reagent grade or equivalent. ( c )  Potassium hydroxide (KOH) .—≥99% ACS reagent grade or equivalent. ( d )  Extraction solvent .—For every 1000 mL of extraction solvent, mix 700 mL HPLC grade methanol and 300 mL HPLC grade water. Sonicate for 5 min. ( e )  MPA .—For every 1000 mLMPA, mix 1000 mL HPLC grade water. Sonicate for 5 min. ( f )  MPB .—For every 1000 mL MPB, mix 800 mL HPLC grade acetonitrile and 200 mL HPLC grade water. Sonicate for 5 min. ( g )  Basic hydrolyzing agent (5% KOH, w/v) .—For every 100 mL basic hydrolyzing agent, weigh approximately 5 g (±0.1 g) KOH and dilute with 100 mL HPLC grade water. Mix well. ( h )  Neutralizing agent (14% KH 2 PO 4 , w/v).— For every 100 mL neutralizing agent, weigh approximately 14 g (±0.1 g) KH 2 PO 4 and dilute with 100 mL HPLC grade water. Mix well. ( i )  Reference standard .—A batch of mixed ginsenoside reference standard (MGRS) in HPLC grade methanol supplied by the National Research Council of Canada (NRC) in the form of individual 0.5 mL amber glass ampules or alternative certified reference material can be used as standards. These standards are intended to be diluted to appropriate concentrations to serve as

Table 2018.09B. Concentration of each ginsenoside for each linearity (Lin) solution in the seven-point calibration curve

Concn, µ g/mL Lin 1 Lin 2 Lin 3 Lin 4 Lin 5 Lin 6 Lin 7

Analyte

Rg1

394

131

77

45

15 46

9

5

Re

1188 2981

396 231 135

27 16

Rb1

994 580 338 115 67 39

Rc

499 406 600

166 135

97 79

57 46 68

19 16 23

11

7 5 8

Rb2

9

Rd

200 117

14

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