5. AOACSPDSMethods-2018AwardsV3

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1446 K oshy et al .: J ournal of AOAC I nternational V ol . 99, N o . 6, 2016 rotary evaporator until the volume is less than 40 mL. Cool the solution, transfer quantitatively to a 50 mL volumetric flask and bring to 50 mL with methanol. Filter through a 0.45 micron membrane filter.

( 3 )  Resolution .—Calculate between withanoside V and withaferin A peaks in the 50 μg/mL mixed standard preparation as follows: the resolution

(b)  Standardized (common) extract .—Accurately weigh a sample quantity of W. somnifera extract equivalent to 5 mg (~0.5 g is sufficient) of withanoside IV, withanosideV, withaferin A, 12-deoxywithastromonolide, withanolide A, and withanolide B in a 250 mL beaker. Extract the standardized extract with 100 mL methanol, boil in a 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 rotary evaporator until the volume is less than 40 mL. Cool the solution, transfer quantitatively to a 50 mL volumetric flask and bring to 50 mL with methanol. Filter through a 0.45 micron membrane filter.

= × − + R 2 T2 T1 W1 W2

where T1 and T2 are the retention times of withanoside V and withaferin A, respectively; and W1 and W2 are their peak widths measured at the baseline between tangents drawn to the sides of the peak. The resolution between withanoside V and withaferin A should be ≥3.0. ( 4 )  Tailing .—Calculate the tailing factor (F) for each withanolide in the 50 μg/mL mixed standard preparation as follows: L R L = + F 2 where L is the width (measured at 5% maximum peak height) from the front slope of the peak to the center line and R is the width (measured at 5% maximum peak height) from the center line to the back slope of the peak. The tailing factor must be ≤1.5 for all individual withanolides. ( 5 )  Coefficient of determination .—Plot peak area versus concentration for the mixed standard preparations in the range of 50 to 150 μg/mL. The r 2 for the regression line of peak area versus concentration for each withanolide must be ≥0.998. (d)  Calculation of withanolide content. —( 1 ) Calculate the percentage of withanoside IV, withanoside V, withaferin A, 12-deoxywithastramonolide, withanolide A, and withanolide B content from the mean peak areas of the duplicate test solution injections and the triplicate mixed standard preparation injections producing the most similar peak areas using the formula: ( ) ( ) ( ) ( ) ( ) ( ) = × × × Individual withanolide %w w Mean peak area of sample Mean peak area of standard Weight of standard mg Final standard preparation volume mL Final test solution volume mL Sample weight mg Purity of standard % ( 2 ) Alternatively, calculate the percentage of eachwithanolide from the mean peak areas of the duplicate test solution injections and the plots of peak area versus concentration from Section F(c)( 5 ) using the formula: Individual withanolide %w w Mean peak area of sample Final test solution volume mL Sample weight mg Purity of standard % b m ( ) ( ) ( ) = − × ×

F. Analysis

(a)  Chromatographic conditions .—( 1 )  Column .—Phenomenex Luna C18(2), 250×4.6 mm, 5 μm particle size (Part No. 00G- 4252- E0). ( 2 )  Temperature .—Maintained constant between 20 and 30°C (preferably 27°C). ( 3 )  Detector .—SPD-M 10Avp PDA or UV detector.

( 4 )  Wavelength .—227 nm. ( 5 )  Flow rate .—1.5 mL/min. ( 6 )  Run time .—45 min. ( 7 )  Injection volume .—20 μL. ( 8 )  Peak integration .—Base-to-base. ( 9 )  Gradient .— See Table 2015.17 .

(b)  Procedure .—( 1 ) Inject 20 μL mixed standard preparations in triplicate at three different concentrations: 50, 100, and 150 μg/mL. ( 2 ) Inject 20 μL of each test solution in duplicate. (c)  System suitability .—Verify that the following system suitability requirements are met with each run. If the system suitability requirements are not met, adjust the composition of the mobile phase or use a new LC column to meet system suitability before analyzing samples.—( 1 )  Repeatability .— The RSD of the peak areas from the triplicate injections of the 50 μg/mL mixed standard preparation must be ≤2.0% for each withanolide. ( 2 )  Retention times .—The relative retention times of the standards should be 0.70 for withanoside IV, 0.89 for withanoside V, 0.92 for withaferin A, 0.96 for 12-deoxywithastramonolide, 1.0 for withanolide A, and 1.15 for withanolide B.

Table 2015.17. Gradient Time, min

( )

Solvent A %

Solvent B %

0.01 18.0 25.0 28.0 35.0 40.0 45.0

95.0 55.0 20.0 20.0 55.0 95.0 95.0

5.0

where m is the slope of the plot and b is the y -intercept.

45.0 80.0 80.0 45.0

Specificity

Specificity was assessed by spectral similarity, by determining the resolution between each peak, and by checking peak purity using a PDAdetector. Reference standard solutions of withanoside IV, withanoside V, withaferin A, 12-deoxywithastramonolide,

5.0 5.0