5. AOACSPDSMethods-2018AwardsV3

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K oshy et al .: J ournal of AOAC I nternational V ol . 99, N o . 6, 2016  1447

used for the W-V spike. The spiked materials were extracted and analyzed for total (endogenous + spike) withanolide content according to the candidate method. In a separate experiment, four materials (1714 mg WS/06Lot08, 5030 mg WS/05Lot20, 1640 mg RD/1045, and 4091 mg ERH-46) were extracted according to the candidate method. The extracts were spiked with pure standards using the third serial dilution of the stock standard solution from the linearity study. The spiked extracts were analyzed for total (endogenous + spike) withanolide content according to the candidate method. The ruggedness evaluation was conducted as an evaluation of intermediate precision. All eight materials were tested under two sets of conditions, including different analysts on different days using different LC equipment, column temperatures, and concentrations of H 3 PO 4 in the LC mobile phase (Table 5). Each material was extracted, and the extracts analyzed under each of the two conditions without replication. Ruggedness

withanolide A, and withanolide B were prepared in methanol at the concentrations shown in Table 4. Dietary ingredient samples, including raw material (plant root, Batch No. ERH-046, 4091 mg) and a standardized dried methanolic extract (Batch No. WS/06Lot10, 750 or 1520 mg) were extracted with hot methanol according to the method. Each reference standard, a mixed standard solution, and the sample solutions were analyzed according to the method on two different LC systems—a Shimadzu LC 2010A with a UV- Vis detector and a Shimadzu LC 10A with a PDA detector—on different days.

Linearity

The reference stock standard solution was prepared in methanol at ~1.2–1.5 mg/mL after correction for purity. Six 2-fold serial dilutions of the stock standard solution were then prepared in methanol. Five replicates of the stock and each dilution were analyzed.

Precision

Stability of Sample Solution

Peak areas and retention times— The data from the stock standard and serial dilutions for the linearity study were analyzed for repeatability based on the peak areas and retention times for each analyte. A standardized dried methanolic extract of W. somnifera (Batch No. WS/06Lot10, >2.5% total withanolides by the candidate LC method) was tested at three levels—754, 1503, and 2096 mg—each extracted and analyzed in duplicate according to the method. Content— Standards were prepared at ~0.2–0.4 mg/mL in methanol and analyzed in triplicate. Eight materials (1552 mg WS/06Lot08, 1536 mg WS/06Lot10, 5048 mg WS/05Lot20, 1527 mg WS/05Lot21, 1503 mg RD/1170, 1653 mg RD/1045, 4336 mg RD/1162, and 4229 mg ERH-46) were extracted and analyzed in triplicate. Withanolide content was calculated for each analyte. Precision

Material WS/05Lot21 (2613 mg) was extracted and filtered according to the method and placed into an autoinjector vial. The LC system and autosampler were in a room maintained at 25±2°C. The extracted sample and linearity standards were analyzed immediately and after 24 h. The difference in results between 0 and 24 h was determined.

System Suitability

In addition to the standards and samples injected with every run, a QC-check sample was developed for use during validation. Batch No. WS/06Lot10 was sampled (~500 g), homogenized, and stored in an airtight container for use throughout the validation study. An initial test portion was extracted according to the method and tested in duplicate by two analysts on different days on multiple days by multiple analysts to establish a “true” value. If the RSD of the obtained results was <2.5%, then the mean value of the obtained results was accepted as the true value. The QC- check sample was then included with every run during the validation study.

Recovery

Four materials (2613mgWS/05Lot21, 1520mgWS/06Lot10, 1633 mg RD/1170, and 4004 mg RD/1162) were spiked with pure standards (W-IV, WF-A, 12-D, W-A, and W-B) using the third serial dilution of the stock standard solution from the linearity study. A sample material containing 36% W-V was

Table 5. Conditions for ruggedness evaluation Parameter Condition 1

Condition 2

Analyst

A

B

Table 4. Reference standard concentrations

Day

1

2

Concn, μg/mL

HPLC

Shimadzu LC-2010A Shimadzu LC-10A

Reference standard

UV detector

PDA detector

Detector

UV-Vis

PDA

Withanoside IV Withanoside V

330.30 300.15 368.78 355.30 345.51 337.59

310.0 360.0 210.0 300.0 240.0 350.0

Merck LiChrospher 100 a Phenomenex Luna RP-18e (5 μm) Hibar RT 5 μm C18(2), 100 å 250×4.6 mm 250×4.6 mm

C18 column

Withaferin A

12-Deoxywithastramonolide

Column temperature, °C

25

30

Withanolide A Withanolide B

PO 4

concn

0.5 mL in 1000 mL 1.5 mL in 1000 mL

H 3

a  EMD Millipore, Billerica, MA.