D-1019
Determination of Vitamin K1 by HPLC-UV
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1.0 Purpose This document describes the analytical procedure for the determination of Vitamin K1 in raw materials. 2.0 Scope This procedure applies to the identification and quantification of Vitamin K1 in raw materials. This method was validated under protocol PRTCL-23-0019. 3.0 Responsibility Jul 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 ACN — Acetonitrile 4.3. EtOH — Ethanol [SOP D-101 | Page 2 of 6] Standard Operating Procedure SOP No Rev Determination of Vitamin K1 by HPLC-UV D-1019 I ds 4.4 ACS -— American Chemical Society 4.5 HPLC — High Performance Liquid Chromatography 4.6 UV-Vis — Ultraviolet-Visible (Detection) 5.0 References 3.1 PRTCL-23-0019, Protocol, Validation of an Analytical Method for the Determination of Vitamin K1 by HPLC-UV 6.0 Supplies 6.1 Chemicals — All reagents are ACS grade or better 6.1.1 ACN 6.1.2 EtOH 6.1.3 Vitamin K1 (Phytonadione) Reference Standard 6.2 Supplies and Glassware (Use Red Glassware!) 6.2.1 HPLC vials, 12mm X 32mm with screw cap enclosures w/ septa 6.2.2 Red Volumetric glassware 6.2.3 Weigh paper 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 compartment and UV-Vis detector with a chromatographic data handling system 6.3.2 Analytical Balance [SOP D-101 | Page 3 of 6] Standard Operating Procedure SOP No | Rev Determination of Vitamin K1 by HPLC-UV D1019),| 0% | Pane deh 6.3.3. Wrist Action Shaker 6.3.4 Sonicator 7.0 Procedure 7.1. Mobile Phase and Extraction Solvent / Diluent Preparation 7.1.1 Mobile Phase 7.1.1.1 ACN (lsocratic) 7.1.2 Extraction Solvent / Diluent 7.1.2.1 Extraction Solvent = Diluent = EtOH 7.2 Standard Preparation (Use Red Glassware!) 7.2.1. Prepare standard stock at ~0.6 mg/mL analyte in Extraction Solvent. Mechanically shake stock for 15 minutes at ~ 2 volume then QS to volume. Sonicate for 10 minutes then let cool to room temperature. Shake vigorously then dilute stock 2:50 with Diluent. Shake vigorously then filter a SmL aliquot for analysis, discarding the first 3-4mL of filtrate. 7.3. Sample Preparation (Use Red Glassware!) 7.3.1 The validated linear range for the analytical method is 0.0097 — 0.0341 mg/mL. 7.3.2. Extract sufficient sample (based on the raw material manufacturer assay value) with Extraction Solvent in order to generate a sample stock that is ~0.6 mg/mL analyte. Mechanically shake stock for 15 minutes at ~ % volume then QS to volume. Sonicate for 10 minutes then let cool to room temperature. Shake vigorously then dilute stock 2:50 with Diluent. Shake vigorously then filter a 5mL aliquot for analysis, discarding the first 3-4mL of filtrate. 7.4 HPLC Parameters [SOP D-101 | Page 4 of 6] Standard Operating Procedure SOP No | Rev Determination of Vitamin K1 by HPLC-UV D-1019 | 0 | Page 4of6 7.4.1. Column: Supelco Ascentis Express RP-Amide, 2.7um (SPP), 4.6 x 150mm 7.4.2 Column Temperature: 30°C 7.4.3. Flow rate: 1.5 mL/min 7.4.4 Run Time: 10 minutes 7.4.5 Mobile Phase: Isocratic 7.4.6 Wavelength: 248 nm 7.4.7 Injection Volume: 10 uL 7.4.8 Suggested 3-D Spectral Range (for Identification): 210nm - 400nm 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. 7.5.4 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: 77 Of = Ru y WhaaXP y W“wel x 100 R, Vesa SA [SOP D-101 | Page 5 of 6] Standard Operating Procedure SOP No | Rev Determination of Vitamin K1 by HPLC-UV D-1019 | 0 | Page saf6 Ry Sample peak area R, Mean (All) standard peak area Wt..a Weight of the reference standard, mg Vos 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 7.8 System Wash, Column Wash and Column Storage 7.8.1 Wash and store the column in 100% ACN. 8.0 Chromatograms 8.1 Typical Diluent Chromatogram FRIRNGB28 BCH UWS 1WL28nm mu 0} 80} 40; 40) 204 Oo -204 404 nin 60 a 0 rT as 10 15 20 25 30 35 40 45 5 T 0 L AISLE § 5 JU RA NL 6 G 0 L A 65 O L 70 1S 80 85 a0 a5, aC [SOP D-101 | Page 6 of 6] Standard Operating Procedure SOP No | Rev Determination of Vitamin K1 by HPLC-UV D-lgl? | 0... | Page0f6 8.2 Typical Standard Chromatogram 0. BKIRnGOH VsID U/MS 1WiL28rm neu 04 )1-K1-6020 20 205 180} 1004 604 Q0; 1 40; ~100- nin a0 as 40 15 20° 25 do 35 40 45 60 60 65 70 7580 85 20 a” “ac 8.3. Typical Raw Material Chromatogram nn EKIRNGBBH7 RMI ULVS 1WiL28rm nu 1754 \1-KI1-6047 1804 25 1005 75 504 25, 00} r 25) 40} 75, n, nin a0 a5 40 15 20 25 40 5 40 45 50 60 és 70 5 a0 85 go a5 ac 9.0 Revision History | Rev | Date | Description of Changes | CCR # | By | |-----|----------|------------------------|-------|----| | 0 | 05/22/23 | New procedure. N/A C. Perry | - | - |