5. AOACSPDSMethods-2018AwardsV3

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K oshy et al .: J ournal of AOAC I nternational V ol . 99, N o . 6, 2016  1445

sample matrix using methanol and separation of the compounds using gradient LC on a C18 column with UV detection at 227 nm.

Table 1. Performance requirements for analytical range and LOQ Parameter Total glycosides Aglycones Analytical range, ppm 10–250000 10–20000 LOQ, ppm 10

B. Apparatus

(a)  LC system .—Shimadzu HPLC system equipped with an LC10A pump with an SPD-M 10Avp photodiode array (PDA) or UV detector in combination with CLASS-VP software (Shimadzu) or an LC-2010A and LC-2010HT integrated system (Shimadzu) equipped with a quaternary gradient and autoinjector in combination with laboratory solution software; or any other suitable HPLC system with a similar configuration. (b)  Column .—Phenomenex Luna C18(2), 250×4.6 mm, with 5 μm particle size (Part No. 00G-4252- E0; Phenomenex, Torrance, CA; http://www.phenomenex.com); or equivalent. (c)  Analytical balance .—Accuracy to 0.1 mg. (d)  Filtration apparatus .—0.45 μm nylon filter. (e)  Ultrasonic bath . (f)  Syringe filter .—0.45 μm polyethersulfone filter. (a)  Degassed mobile phase .—( 1 )  Solvent A .—Dissolve 0.136 g anhydrous potassium dihydrogen orthophosphate (KH 2 PO 4 ) in 900 mL HPLC grade water (Milli-Q Water purification system; Millipore) and add 0.5 mL orthophosphoric acid. Dilute to 1000 mLvolume with water, filter through 0.45 μm membrane, and degas in a sonicator for 3 min (solvent A). ( 2 )  Solvent B .—Acetonitrile. (b)  Diluent .—Methanol. (c)  Individual withanolide standards .—Natural Remedies Private Ltd (Bangalore, India; www.naturalremedy.com), or other suppliers.—( 1 )  Withanoside IV .—CAS No. 362472-81-9. ( 2 )  Withanoside V .—CAS No. 256520-90-8. ( 3 )  Withaferin A .—CAS No. 5119-48-2. ( 4 )  12-Deoxywithastromonolide .—CAS No. 60124-17-6. Accurately weigh 10 mg each of withanoside IV, withanoside V, withaferin A, 12-deoxywithastromonolide, withanolide A, and withanolide B reference standards in a 50 mL volumetric flask, dissolve in 10 mL methanol with the aid of gentle heating, cool, and dilute to a 50 mL volume with methanol to yield 200 μg/mL of each standard. Suitably prepare three additional concentrations of withanolides to obtain concentrations of 150, 100, and 50 μg/mL. (a)  Raw material .—Accurately weigh a sample quantity of W. somnifera raw material equivalent to 5 mg (~2.5 g is sufficient) of withanoside IV, withanoside V, withaferin A, 12-deoxywithastromonolide, withanolide A, and withanolide B in a 250 mL beaker. Extract raw material with 100 mL methanol on a boiling water bath for 10–15 min, and repeat this procedure three to four times until the raw material has been completely extracted or the extracts become colorless. Combine all extracts and evaporate the methanol on a water bath or by using a vacuum E. Preparation of Test Solutions C. Reagents ( 5 )  Withanolide A .—CAS No. 32911-62-9. ( 6 )  Withanolide B .—CAS No. 56973-41-2. D. Preparation of Mixed Standards

Table 2. Performance requirements for recovery, repeatability, and reproducibility

Range, ppm 10–100 >100–1000 >1000–10000 >10000

Parameter

Recovery, % 80–110 90–107

95–105

97–103

Repeatability, % ≤7 Reproducibility, % ≤10

≤6 ≤9

≤4 ≤6

≤1 ≤2

Table 3. Sample materials used in the validation study Sample Standardized to Batch No. Raw material Total withanolides >0.2% by HPLC RD/1162 Raw material Total withanolides >0.2% by HPLC ERH-046 Water extract Total withanolides >0.15% by HPLC WS/05Lot20 Hydroalcoholic extract Total withanolides >1.0% by HPLC WS/05Lot21 Methanolic extract Total withanolides >1.5% by HPLC RD/1045 Methanolic extract Total withanolides >2.5% by HPLC WS/06Lot08 Methanolic extract Total withanolides >2.5% by HPLC WS/06Lot10 Methanolic extract Total withanolides >2.5% by HPLC RD/1170 AOAC Official Method 2015.17 Withanolides (Withanoside IV, Withanoside V, Withaferin A, 12-Deoxywithastromonolide, Withanolide A, and Withanolide B) in Withania somnifera LC First Action 2015 [Applicable to the determination of withanoside IV, withanoside V, withaferin A, 12-deoxywithastromonolide, withanolide A, and withanolide B content in raw material (roots) and dried extracts.] Caution : This method uses common-use solvents and

reagents. Refer to an adequate manual or Material Safety Data Sheets (MSDSs) to ensure that safety guidelines are applied before using chemicals. Store solvents in a flammable-liquid storage cabinet. The solvents and reagents used herein are harmful if inhaled, swallowed, or absorbed through the skin. Use appropriate personal protective equipment, such as a laboratory coat, safety glasses, rubber gloves, and a fume hood. Dispose of all materials according to federal, state, and local regulations.

A. Principle

Withanolides are compounds specific to Withania somnifera . This method comprises extraction of withanolides from the