Determination of Dehydroepiandrosterone and Piperine by HPLC using UV-VIS Spectroscopy

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1.0 Purpose 
 
 The purpose of this procedure is to define the method for the quantification and/or identification
 of Dehydroepiandrosterone and Piperine in raw materials and finished products by HPLC using
 
 UV-Vis Spectroscopy. 
 
 2.0 Scope 
 
 This procedure applies to the identification and quantification of Dehydroepiandrosterone &
 
 Piperine in raw materials and finished products. This method was validated under Protocol MV-
 LAB-19-137. 
 
 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 QC Laboratory Management to implement this procedure and
 
 to ensure that the procedure is being followed. 
 
 3.3 It is the responsibility of QC Laboratory Management and/or Analytical Development
 Personnel to keep this procedure current with the associated monographs and laboratory
 
 practices. 
 
 4.0 Definitions 
 
 4.1 QC — Quality Control 
 
 4.2 H3POa — Phosphoric Acid 
 
 4.3 MeOH — Methanol 
 
 4.4 DHEA — Dehydroepiandrosterone 
 
 
 

[SOP D-787 | Page 2 of 8]

 Standard Operating Procedure SOP No | Rev 
 Dehydroepiandrosterone and Piperine Determination by | -787 2 Page 2 of 8 
 HPLC using UV-VIS Spectroscopy 
 
 4.5 HO — Deionized Water 
 
 4.6 PIP —Piperine 
 
 4.7 HPLC — High Performance Liquid Chromatography 
 
 48 UV/Vis — Ultraviolet & Visible Electromagnetic Spectra 
 
 5.0 References 
 
 5.1 | MV-LAB-19-137, Protocol, Dehydroepiandrosterone and Piperine Determination Using
 
 HPLC with UV/Vis Spectroscopy 
 
 6.0 Supplies 
 
 6.1 Chemicals — All reagents are ACS grade or better 
 
 6.1.1 H2O0 
 
 6.1.2 MeOH 
 
 6.1.3 H3PO,4 
 
 6.1.4 DHEA Reference Standard 
 
 6.1.5 PIP 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 
 
 6.2.2.1 CAUTION: Piperine solutions are not light stable. Minimize exposure
 
 to light during transfers and only use red or foil-wrapped glassware!
 
 6.2.3 Weigh paper or funnels 
 
 6.2.4 10ml Syringes with 0.45u Nylon Syringe Filters 
 
 6.3 Equipment 
 
 
 

[SOP D-787 | Page 3 of 8]

 Standard Operating Procedure SOP No Rev 
 Dehydroepiandrosterone and Piperine Determination by | 2-787 2 Page 3 of 8 
 HPLC using UV-VIS Spectroscopy 
 
 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 
 
 6.3.4 Wrist Action Shaker 
 
 7.0 Procedure 
 
 | Mobile Phase, Diluent & Diluent Preparation 
 
 Tell Mobile Phase A — 0.1% H3PO4 
 
 7.1.1.1 Combine 1 mL of H3P04 with 1000 mL of H20. Mix well. 
 
 FA2 Mobile Phase B - MeOH 
 
 7.1.3 Diluent - MeOH 
 
 7.1.4 Preparations may be scaled as necessary 
 
 Tee Standard Prep 
 
 Tid 
 Accurately weigh and transfer about 25 mg each of DHEA and PIP reference
 standards into a 100-mL volumetric flask. Add 50mL of Diluent. Sonicate for
 5min, equilibrate to room temperature, then QS to volume with Diluent.
 
 Dilute 1:10 w/ Diluent for a Working Standard concentration of 25 pg/ml.
 eee 
 7.3 Sample Preparation 
 
 Specific sample testing details are provided in each products profile. If a specific
7.3.1 testing details section is not available, then follow preparation procedure as

 described below, maintaining concentration within the linear range listed below.
 
 The validated range for the analytical method is 5 —- 40 mcg/mL for DHEA and 6
7.3.2 —117 mcg/mL for PIP.

 For raw materials: weigh no less than 25 mg into a suitably sized volumetric flask
 To 
 of no less than 25 mL volume to generate an analyte concentration that is within
 
 
 

[SOP D-787 | Page 4 of 8]

 Standard Operating Procedure SOP No | Rev 
 Dehydroepiandrosterone and Piperine Determination by | -787 2 Page 4 of 8 
 HPLC using UV-VIS Spectroscopy 
 
 the validated linearity range. Fill the flask to about 50% of the calculated volume
 
 with Diluent and shake mechanically for 15 minutes. Sonicate for 5 minutes,
 
 equilibrate to room temperature and bring up to volume with Diluent.
 
 7.3.4 For solid and liquid dose finished products: Combine and homogenize no less
 than ten dosage units. Based on the label claim and fill weight (capsules), serving
 
 size (powders and liquids) or tablet weight per dose, weigh no less than 100 mg
 
 of the pooled dosages into a suitably sized volumetric flask of no less than 25 mL
 to generate an analyte concentration that is within the validated linear range. Fill
 
 the flask to about 50% of the calculated volume with Diluent and shake
 mechanically for 15 minutes. Sonicate for 5 minutes, equilibrate to room
 
 temperature and bring up to volume with Diluent. 
 
 13D For chewable gels (gummies), homogenize at least 10 dosage units according to
 the procedure outlined in D-793 Cryogenic Grinding of Chewable Gels. Quickly
 
 weigh no less than 400 mg of the pooled and homogenized dosages into a beaker.
 
 Use several small portions of Diluent to completely transfer the sample into a
 suitably sized volumetric flask of no less than 50 mL to generate an analyte
 
 concentration that is within the validated linear range. Fill the flask to about 50%
 of the calculated volume with Diluent and shake mechanically for 15 minutes.
 
 Sonicate for 5 minutes, equilibrate to room temperature and bring up to volume
 
 with Diluent. 
 
 73:6 To manage large volumes, the sample can be initially prepared at a higher
 concentration and further diluted into the linear range using Diluent. Dilutions
 
 can be made using volumetric glassware and/or adjustable pipettes. Dilutions can
 be prepared in HPLC vials 
 
 Dod Centrifuge an aliquot of the final sample at 10,000 rpm for 5 min to remove
 particulates. Alternatively, the sample may be filtered through a 0.45 um
 membrane discarding the first 3 - 4 mL before collecting a portion for analysis.
 
 7.4 HPLC Parameters 
 
 
 

[SOP D-787 | Page 5 of 8]

 Standard Operating Procedure SOP No | Rev 
 Dehydroepiandrosterone and Piperine Determination by | 2-787 2 Page 5 of 8 
 HPLC using UV-VIS Spectroscopy 
 
 7.4.1 Column: Thermo Scientific Accucore Polar Premium, 3.0 x 100mm, 2.6u
 
 7.4.2 Column Temperature: 45°C 
 
 7.4.3 Flow rate: 0.5 mL/min 
 
 7.4.4 Wavelength: DHEA @ 210 nm, PIP @ 343 nm 
 
 7.4.5 Injection Volume: 5 pL 
 
 7.4.6 Run Time: 12 minutes. 
 
 7.4.7 Recommended 3-D Spectral Range (for Identification) - 200nm to 400nm
 
 7.4.8 Mobile Phase Gradient — Isocratic: 50% A /50% B 
 
 7.5 Recommended Sequence 
 
 7.5.1 Make at least 2 injections of the Diluent. 
 
 7.5.2 Make five (5) injections of Working Standard. 
 
 7.5.3. Make a single injection of each Sample Preparation. 
 
 7.5.4 Make a single injection of the Working Standard after every ten (10) sample
 
 injections or at the end of a run. 
 
 7.6 System Suitability Requirements 
 
 7.6.1 The %RSD of the first five (5) standard injections is NMT 2.0%
 
 7.6.2 The %RSD of all standard injections is NMT 3.0%. 
 
 7.6.3 If present, any interference in the diluent should be subtracted out of the sample
 
 and standard peak areas. 
 
 Tad Example calculations for determining finished product % label or raw material % purity
 
 % Analyte = a R x o Wtsig o X P x oSSR Vo. a 
 x 100 
 s std 
 Ru Sample peak area 
 
 R, Mean (n=5) standard peak area 
 
 
 

[SOP D-787 | Page 6 of 8]

 Standard Operating Procedure SOP No Rev 
 D-787 
 Dehydroepiandrosterone and Piperine Determination by Page 6 of 8 
 HPLC using UV-VIS Spectroscopy 
 
 Wt.tq Weight of the reference standard in mg (corr. for water if applicable)
 
 Vseq Volume of the standard preparation accounting for dilutions in mL
 
 P Purity of the reference standard in decimal format 
 
 SA Sample amount in mg 
 
 Vs». | Volume of the sample preparation accounting for dilutions in mL
 
 SS Serving size: Weight of a single dosage unit in mg (use 1 for raw materials)
 
 LA Label amount in mg of analyte (use 1 for raw materials) 
 
 7.8 Column Wash and Storage 
 
 7.8.1. Wash and store the column in 75:25 ACN / Milli-Q Water. 
 
 8.0 Example Chromatograms 
 
 8.1. Typical DHEA Diluent Chromatogram 
 
 
 

[SOP D-787 | Page 7 of 8]

 Standard Operating Procedure SOP No | Rev 
 Dehydroepiandrosterone and Piperine Determination by | 2-787 2 Page 7 of 8 
 HPLC using UV-VIS Spectroscopy 
 
 8.2. Typical DHEA Working Standard Chromatogram 
 
 i 
 i 
 { 
 
 | \ 
 | avon 
 | /\ | \ 
 = J L J| XX _— ae Fe{ OR | 
 
 i 
 ij 
 | 
 8.3. Typical DHEA Sample Chromatogram 
 
 | | | 
 
 | \ | | 
 4 \ io 
 \ | \ 
 \ yi| \\ [\ 
 | a J YN 
 
 8.4 Typical PIP Diluent Chromatogram 
 
 400: N 
 
 300 | 
 6.280 | 
 208 | 
 
 ves 
 en 
 
 : 
 
 
 

[SOP D-787 | Page 8 of 8]

 Standard Operating Procedure SOP No Rev 
 Dehydroepiandrosterone and Piperine Determination by | 2-787 2 Page 8 of 8 
 HPLC using UV-VIS Spectroscopy 
 
 8.5 Typical PIP Working Standard Chromatogram 
 
 ae ie 
 600 | 
 50.0 | 
 | | 
 400 | | 
 300 
 
 \ 
 100 | 
 
 8.6 Typical PIP Sample Chromatogram 
 
 | | 
 
 wo | | 
 | | 
 | | 
10.0 0 } |

 H \ 
 ; | 
 100 a J { \ aes a a } { \\ 
 
 9.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 09/11/19 | New procedure. N/A C. Perry | - | - |
| 1 | 07/21/22 | Scheduled review: updated logo and format. CC- Minor edits for consistency with current methods, add instruction | 22-0292 | K. Burris |
| 2 | 06/06/23 | to follow test details for sample preparation, add specific CC- instruction for different dosage forms. Update logo and format. | 23-0276 | S. Sassman |