Withanolides Determination by HPLC with UvVis Spectroscopy

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1.0 Purpose 
 
 This document describes the analytical procedure for the determination of Withanolides in raw
 materials. Withanolides are calculated as the sum of Withanolide Aglycones, calculated as
 Withanolide A, and Withanolide Glycosides, calculated as Withanoside IV. The USP
 designates the following species as comprising the Withanolide Aglycones: Withaferin A,
 Deoxywithastramonolide, Withanolide A, Withanone and Withanolide B. The USP designates
 
 the following species as comprising the Withanolide Glycosides: | Withanoside IV,
 Withanoside V and Withanoside VI. (Note: Withanosides V & VI coelute). Raw material
 samples vary, and some of the Withanolides mentioned above may be present in small
 quantities - or may be totally absent. The sample is considered compliant if the sum of the
 Withanolides present is NLT the stated specification. 
 
 2.0 Scope 
 
 This procedure applies to the identification and quantification of Withanolides in raw materials.
 This method was validated under protocol PRTCL-20-0105 and reported in RPT-20-0053.
 
 3.0 Responsibility 
 
 3.1 It is the responsibility of QC and Analytical chemists who have verified their ability to
 execute this procedure to follow this procedure. 
 
 3.2 It is the responsibility of the QC Laboratory Management to implement this procedure and
 to ensure that the procedure is being followed. 
 
 3.3 It is the responsibility of the QC Laboratory Management and AD Personnel to keep this
 procedure current with the associated monographs and laboratory practices.
 
 4.0 Definitions 
 4.1 QC - Quality Control 
 
 4.2 AD - Analytical Development 
 
 4.3. ACN —- Acetonitrile 
 4.4 MeOH — Methanol 
 
 4.5 DMSO — Dimethylsulfoxide 
 
 
 

[SOP 

 Standard Operating Procedure 
 Withanolides Determination by HPLC with UV/Vis S D O - P 7 5 N 5 o Re 0 v Page 2 of 7
 Spectroscopy 
 
 4.6 H3PO,4-— Phosphoric Acid 
 
 4.7 KH 2POs, — Potassium Phosphate Monobasic 
 
 4.8 ACS — American Chemical Society 
 4.9 HPLC — High Performance Liquid Chromatography 
 
 4.10 UV — Ultraviolet Electromagnetic Spectra 
 53.0 References 
 
 5.1 PRTCL-20-0105 — Verification of an Analytical Method for the Determination of
 Withanolides by HPLC-UV 
 
 5.2 RPT-20-0053 — Verification of an Analytical Method for the Determination of
 Withanolides by HPLC-UV 
 
 5.3. USP Monograph — Ashwagandha Root Dry Extract 
 6.0 Supplies 
 
 6.1 Chemicals — All reagents are ACS grade or better. 
 
 6.1.1 Milli-Q Water 
 6.1.2 ACN 
 
 6.1.3 MeOH 
 6.1.4 H3PO, 
 
 6.1.5 KH»PO, 
 6.1.6 Withanoside IV Reference Standard 
 
 6.1.7 Withanolide A Reference Standard 
 6.2 Supplies and Glassware 
 
 6.2.1 HPLC vials, 12mm X 32mm with screw cap enclosures w/ septa 
 
 6.2.2 Volumetric glassware and/or adjustable pipettes and tips 
 6.2.3 Weigh paper and/or funnels 
 
 6.2.4 Syringes with 0.45 Nylon Syringe Filters 
 
 6.3 Equipment 
 6.3.1 Suitable gradient HPLC system consisting of a pump, autosampler, column
 
 oven and UV detector with a chromatographic data handling system
 6.3.2 Analytical Balance 
 
 6.3.3 Sonicator Bath 
 
 

[SOP 

 Standard Operating Procedure 
 Withanolides Determination by HPLC with UV/Vis S D O - P 7 5 N 5 o Re 0 v Page 3 of 7
 Spectroscopy 
 
 7.0 Procedure 
 
 7.1 Mobile Phase & Diluent Preparation 
 
 7.1.1 Mobile Phase 
 
 7.1.1.1 | Mobile Phase A: Add 0.14g KH>PO, + 500 uL of H3PQO, to 1000
 mL of water and mix well. 
 
 7.1.1.2 Mobile Phase B: ACN 
 7.1.2 Extraction Solvent = Diluent = MeOH 
 
 7.1.3 Preparations may be scaled as necessary 
 7.2 Standard Preparation 
 
 7.2.1 Accurately weigh and transfer about 25 mg of Withanolide A reference
 standard into a 25 mL volumetric flask. Add ~20 mL of Diluent and sonicate
 until dissolved. (Note: Withanolide A has poor solubility and must be
 
 transferred and dissolved with utmost care! When necessary, quantitatively
 transfer the contents of small vials with careful DMSO (1X) & MeOH (5X)
 rinses and calculate the mass transferred to the flask by difference.)
 
 7.2.2 Cool to ambient then QS to volume with Diluent and mix well — this is the
 Withanolide A Stock. 
 
 7.2.3 Accurately weigh and transfer about 10 mg of Withanoside IV reference
 standard into a 100 mL volumetric flask. Add ~50 mL of Diluent and swirl to
 dissolve. (When necessary, quantitatively transfer the contents of small vials
 with careful DMSO (1X) & MeOH (5X) rinses and calculate the mass
 transferred to the flask by difference.) 
 7.2.4 Using a glass volumetric pipet, transfer 10.0 mL of the Withanolide A Stock to
 
 the 100 mL flask containing the Withanoside IV. QS to volume with Diluent
 and mix well — this is the Working Standard. 
 7.2.5 It is permissible to prepare the Working Standard using only Withanoside IV
 or only Withanolide A. Relative response factors are supplied in the
 calculation section enabling expression of one standard in terms of the other.
 
 7.3 Sample Preparation 
 7.3.1 The validated range for the analytical method is 0.0595 — 0.1388 mg/mL
 
 Withanoside IV and 0.0527 — 0.1230 mg/mL Withanolide A. However, the
 calculation of Total Withanolides is based on several species.
 
 7.3.2 Therefore, extract sufficient sample (based on the manufacturer assay value)
 with Diluent in order to generate a concentration that is 2.5 times the
 concentration of the Working Standard. 
 
 

[SOP 

 Standard Operating Procedure 
 SOP No Rev 
 Withanolides Determination by HPLC with UV/Vis D-755 0 Page 4 of 7 
 Spectroscopy 
 7.3.3 Samples can be extracted in Diluent at any volume starting from 10mL. The
 
 volume chosen must be in the solubility range (validated at ~0.25 mg/ml). To
 manage large volumes, the sample can be initially dissolved in a smaller
 volume that is within the solubility range and a portion further diluted to bring
 the concentration into the linear range. 
 7.3.4 Fill the flask to about 70% of the chosen volume with Diluent then sonicate for
 25 minutes. Cool to ambient, perform further dilutions as required using
 
 Diluent then filter a 5ml aliquot for analysis, discarding the first 3-4ml of
 filtrate. 
 
 7.3.5 For raw materials being analyzed for the first time using this method, an in
 process validation is required to demonstrate spectral purity, baseline
 separation of peaks, and extraction efficiency as a part of system suitability
 before data can be reported using this method. 
 
 7.4 HPLC Parameters 
 
 7.4.1 Column: Restek Raptor ARC-18, 4.6 x 150mm, 2.7m (Or Equivalent)
 7.4.2 Column Temperature: 45°C 
 
 7.4.3 Flow rate: 1.2 mL/min 
 
 7.4.4 Mobile Phase: Gradient 
 7.4.4.1 Time, min %B (ACN) 
 0 5 
 18 45 
 25 80 
 28 80 
 
 30 5 
 40 5 
 7.4.5 Wavelength: 227 nm 
 
 7.4.6 Injection Volume: 5 pL 
 7.4.7 Run Time: 40 minutes 
 
 7.4.8 3-D Spectral Range (for Identification) - 210nm to 350nm 
 
 7.49 Retention Times: Withanoside IV ~ 14.0 min, Withanolide A ~ 19.7 min
 7.4.10 Assign the Withanolide Aglycone / Glycoside peaks using the approximate
 relative retention times provided in the table below: 
 
 Analyte Relative Retention Time 
 Withanoside IV 0.71 
 Withanoside V & VI 0.90 
 Withaferin A 0.93 
 Withastramonolide 0.96 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Withanolides Determination by HPLC with UV/Vis D-755 0 Page 5 of 7 
 Spectroscopy 
 
 Withanolide A 1.00 
 Withanone 1.01 
 Withanolide B 1.10 
 
 7.5 Recommended Sequence 
 
 7.5.1. Make at least 2 injections of the Diluent. 
 
 7.5.2 Make at least five (5) injections of Working Standard. 
 7.5.3 Make a single injection of each Sample Preparation. 
 
 7.6 System Suitability Requirements 
 
 7.6.1 The %RSD of five (5) consecutive standard injections is NMT 5.0%.
 7.6.2 The average asymmetry of the peak(s) present in the standard injections is
 
 NMT 1.5. 
 7.6.3 If present, any interference in the diluent should be subtracted out of the
 sample and standard peak areas. 
 
 7.7 Example calculations for determining raw material % assay: 
 
 7.7.1 % Withanolide Glycosi, des = R —cosidies_ x WtstaXP 1xV! x 100
 Withanoside IV Vstd SA 1 
 Rglycosides Sum of sample peak areas for Withanolide Glycoside peaks
 
 Rwithanosiaew Average peak area for Withanoside IV reference standard injections
 Wtsta Weight of the Withanoside IV reference standard 
 
 Vista Volume of the standard preparation accounting for dilutions in mL
 
 Pp Purity of the reference standard in decimal format 
 SA Sample amount 
 
 Vso Volume of the sample preparation accounting for dilutions in mL
 
 7.7.2 | % Withanolide Aglycones = Raglycones _ y WtstaXP 1 x2V.! x 100
 Rwithanolide A Vstd SA 1 
 Raglycones Sum of sample peak areas for Withanolide Aglycone peaks
 
 Rwithanoiaea AVerage peak area for Withanolide A reference standard injections
 Wtsta Weight of the Withanolide A reference standard 
 
 V sta Volume of the standard preparation accounting for dilutions in mL
 
 P Purity of the reference standard in decimal format 
 
 SA Sample amount 
 
 

[SOP 

 Standard Operating Procedure 
 SOP No Rev 
 Withanolides Determination by HPLC with UV/Vis D-755 0 Page 6 of7 
 Spectroscopy 
 
 Vsp, Volume of the sample preparation accounting for dilutions in mL
 7.7.3 % Withanolides = % Withanolide Glycosides + % Withanolide Aglycones
 
 7.7.3.1 | When standardizing the quantitation using Withanoside IV reference
 standard alone, divide the % Withanolide Aglycones term in the
 sum in 7.7.3 by 3.1126. 
 7.7.3.2 | When standardizing the quantitation using Withanolide A reference
 standard alone, divide the % Withanolide Glycosides term in the
 sum in 7.7.3 by 0.3213. 
 
 7.8 Column Wash and Storage 
 
 7.8.1 Wash and store the column with 90% ACN. 
 
 8.0 Chromatograms 
 
 8.1 Typical Diluent Chromatogram 
 
 ‘3 Withanolides (USP) Run 11-18-20 #3 
 
 8 
 8 
 yerp 
 
 ; 4.0 6.0 8.0 1 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0
 
 ang. Withanoldes (USP) Run 11-18-2808 WSTO UV_VIS_1 WVL227 nm
 7} mAU 
 220 4 
 2004 
 190 4 2 -- WWiitt hanolide A -“ 1919.660 | 
 j 
 160 + 
 140 4 
 120 + 
 100 + 
 805 1 - Withanoside IV - 14.000 
 60- 
 404 
 204 | | 
 0- T T 
 32 
 0.0 20 40 60 80 10.0 120 140 16.0 18.0 20.0 20 24.0 26.0 28.0 30.0 320 34.0 36.0 38.0 40.0
 

[SOP 

 Standard Operating Procedure 
 Withanolides Determination by HPLC with UV/Vis SOP No Rev Page 7 of 7 
 D-755 
 Spectroscopy 
 8.3 Typical Raw Material Chromatogram 
 
 3} Withanolides (USP) Run 11-18-20 #17 Precision-1 UV_VIS_1 WVL:227 nm
 mAU 
 
300.41 -

 VI edisonahtiW 
 
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765.41 - &

076.41 - 6

358.41 -

 D nilu7g8a7s.y4h1P 
 -- 
010.51 -9

 354.51-24 
085.51 -

342.61 - 54

023.61 - 64

785.61 - 74 SB

038.61 - 845

300.71 - 94

027.71 - IV &

 V 
 edisonahtiW 
 - 42 
023.81 - A

 nirefahtiW 
 - 82 
064.81 - 925

098.81 -

 edilonomartsa0h2t8i.W8 
 1- 2-34 
 389.81-38I 322..997= 708634.9.19 
 1- - 63 
 784 
777.91 - 83

309.91 - enopaehtiyY

 = SG 
776.12 -B

 ei 
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 aS ae] Ra Da ET SID ca aS lr eT BE ee ee Ce fs ge 
 9.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 04/14/21 | New N/A C. Perry | - | - |