D-1020
Determination of Apigenin by HPLC-UV
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1.0 Purpose This document describes the analytical procedure for the determination of Apigenin in raw materials and finished products. 2.0 Scope This procedure applies to the identification and quantification of Apigenin in raw materials and finished products. This method was validated under protocol PRTCL-23-0050. 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 MeOH — Methanol 4.3 DMSO -— Dimethylsulfoxide [SOP D-102 | Page 2 of 7] Standard Operating Procedure SOP No Rev Page 2 of 7 Determination of Apigenin by HPLC-UV D-1020 4.4 H3POs-— Phosphoric Acid 4.5 ACS— American Chemical Society 4.6 HPLC - High Performance Liquid Chromatography 4.7 UV-Vis — Ultraviolet-Visible (Detection) 4.8 SPP -—Superficially Porous Particle (Solid Core Column Stationary Phase) 5.0 References 5.1 PRTCL-23-0050, Protocol, Validation of an Analytical Method for the Determination of Apigenin by HPLC-UV 6.0 Supplies 6.1. Chemicals — All reagents are ACS grade or better 6.1.1 MeOH 6.1.2 DMSO 6.1.3 H3POs 6.1.4 Apigenin 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 6.2.3 Weigh paper 6.2.4 Syringes with 0.45p nylon syringe filters [SOP D-102 | Page 3 of 7] Standard Operating Procedure SOP No | Rev Determination of Apigenin by HPLC-UV D-1020 O.) | easpe ery 6.3. Equipment 6.3.1 Suitable gradient HPLC system consisting of a pump, autosampler, column compartment and UV-Vis detector with a chromatographic data handling system 6.3.2 Analytical Balance 6.3.3 Micro Analytical Balance 6.3.4 Wrist Action Shaker 7.0 Procedure 7.1 Mobile Phase, Extraction Solvent & Diluent Preparation 71.1 Mobile Phase 7.1.1.1 Mobile Phase A: Add 1000p] H3PO4 to 1000ml Milli-Q water and mix well. 7.1.1.2 Mobile Phase B: Use MeOH. 7.1.2 Extraction Solvent 7.1.2.1 Add equal volumes of MeOH and DMSO and mix well. 713 Diluent 7.1.3.1 Use MeOH. 7.2 Standard Preparation Tdsl Prepare standard stock at ~0.05 mg/mL analyte in Extraction Solvent. Mechanically shake stock for 15 minutes at ~ % volume then QS to volume. Shake vigorously then dilute stock 1:10 with Diluent. Shake vigorously then filter a SmL aliquot for analysis, discarding the first 3-4mL of filtrate. [SOP D-102 | Page 4 of 7] Standard Operating Procedure SOP No | Rev Determination of Apigenin by HPLC-UV D-1020 G:; |Rages oly 2 Sample Preparation Tel Specific sample testing details are provided in each product profile. If a specific testing details section is not available, follow preparation procedure as described below, maintaining concentration within the linear range of this method. 73.2 The validated linear range for the analytical method is 0.0030 — 0.0079 mg/mL. 1533 Pool at least 10 dosage units and homogenize as appropriate (e.g. grind tablets / capsule fill / powders / stick pack contents by mortar and pestle, cryogenically powder and dissolve gummies, etc.) Extract sufficient sample (based on the raw material manufacturer assay value / finished product profile) with Extraction Solvent in order to generate a sample stock that is ~0.05 mg/mL analyte. Mechanically shake stock for 15 minutes at ~ % volume then QS to volume. Shake vigorously then dilute stock 1:10 with Diluent. Shake vigorously then filter a 5mL aliquot for analysis, discarding the first 3-4mL of filtrate. 7.4 HPLC Parameters 7.4.1. Column: Waters Cortecs Shield RP18, 2.7m (SPP), 4.6 x 150mm 7.4.2 Column Temperature: 40°C 7.4.3 Flow rate: 0.7 mL/min That, tain 1 Jk — 7.4.4 Run Time: 15 minutes 0 55 45 3 55 45 7.4.5 Mobile Phase Gradient: 10 15 85 7.4.6 Wavelength: 339 nm 10.1 55 45 15 55 45 7.4.7 Injection Volume: 5 pL 74.8 Suggested 3-D Spectral Range (for Identification): 210nm - 450nm yee Recommended Sequence [SOP D-102 | Page 5 of 7] Standard Operating Procedure SOP No | Rev Determination of Apigenin by HPLC-UV D-1020 0 | PageS of 7 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. 7.5.4 Make a single injection of the Working Standard after every ten (10) sample injections or at the end of the run. 7.6 System Suitability Requirements 7.6.1. The %RSD of five (5) consecutive standard injections is NMT 2%. 7.6.2 The %RSD of all standard injections is NMT 3%. 7.7. Example calculations for determining % Assay: 7.7.1 % = Be x WeeX? x e! x 100x SS/LA R, Vai SA R,, Sample peak area R, Mean (All) standard peak area Wtea Weight of the reference standard, mg View Volume of the standard preparation accounting for dilutions, mL P Purity of the reference standard in decimal format SA Sample amount, mg Vet Volume of the sample preparation accounting for dilutions, mL SS Serving Size, mg (Use “1” for raw materials.) LA Label Amount, mg (Use “1” for raw materials.) 7.8 System Wash, Column Wash and Column Storage [SOP D-102 | Page 6 of 7] Standard Operating Procedure SOP No | Rev Determination of Apigenin by HPLC-UV D-1020 0 | Page 6 of 7 7.8.1 Wash system and column with 90% ACN. Store column on 50% ACN. 8.0 Chromatograms 8.1 Typical Diluent Chromatogram 4] Apigenin Run 07-17-2348 MeCH UMS. 40: mu a0 024 040+ 00+ 08} mnao 1T 0 2T0 3T 0 4T0 §T 0 4T0 7y0 8r0 9y0 10T 0 nTO 2T0 13T 0 14T0 150 8.2 Typical Standard Chromatogram 50. 5 PpigeninRun (7-17-2348 WsID UMS1WLS nfU no) 1-Prigerin- 40947 Zo 0 1804 100} 40) Q0+ q hin a0 10 20 30 40 80 40 70 ao 90 100 1.0 120 130 140 160 [SOP D-102 | Page 7 of 7] Standard Operating Procedure SOP No Rev Determination of Apigenin by HPLC-UV D-1020 Oc) Rage tots 8.3 Typical Finished Product Chromatogram ‘ApigeinRne 1720 isd UNS 1VULoMm nt 0) 1-Angrin- 10913 D0 20 220; 80} 100} [ W L_ A. | ane 4 nin o om » om 4 4%9© © 0m © 44© 1 fo0 | |) OK 9.0 Revision History | Rev | Date | Description of Changes | CCR # | By | |-----|----------|------------------------|-------|----| | 0 | 08/08/23 | New procedure. N/A C. Perry | - | - |