Determination of Lipid Soluble Vitamins - Retinyl Acetate, Tocopheryl Acetate, Tocopheryl Succinate, Menoquininone-7

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1.0 Purpose 
 
 This document describes the analytical procedure for the determination of the lipid soluble
 vitamins: Tocopheryl Acetate, Tocopheryl Succinate, Retinyl Acetate, and Menoquinone 7 in
 
 raw materials and finished products. 
 
 2.0 Scope 
 
 This procedure applies to the identification and quantification of the lipid soluble vitamins, listed
 
 above, in raw materials and finished products. This method was validated under Protocol MV-
 LAB-19-122. 
 
 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 Vit Ara — Retinyl Acetate; Vitamin A Acetate 
 
 4.4 Wit Era — Tocopheryl Acetate; Vitamin E Acetate 
 
 4.5 Vit Ets — Tocopherol Succinate; Vitamin E Succinate 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Determination of Lipid Soluble Vitamins: Retinyl Acetate, D-786 Page 2 of 8 
 
 Tocopheryl Acetate, Tocopheryl Succinate, Menoquinone-7 
 
 4.6 Vit K2mx7 — Menoquinone 7; MQ7; MK7 
 
 4.7 ACN — Acetonitrile 
 
 4.8 Ethanol — Ethanol 
 
 4.9 HPLC — High Performance Liquid Chromatography 
 
 4.10 UV/Vis — Ultraviolet & Visible Electromagnetic Spectra 
 
 5.0 References 
 
 5.1 MV-LAB-19-122, Protocol, Lipid Soluble Vitamin Determination Using HPLC with
 UV/Vis Spectroscopy 
 
 6.0 Reagents, Supplies, Glassware, and Equipment 
 
 6.1 Chemicals — All reagents are HPLC grade or better 
 
 6.1.1 Miulli-Q Water 
 
 6.1.2 ACN 
 
 6.1.3 EtOH 
 
 6.1.4 Appropriate 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 or funnels 
 
 6.2.4 10ml Syringes with 17mm x 0.45u 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 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Lipid Soluble Vitamins: Retinyl Acetate, D-786 1 | Page 3 of 8
 
 Tocopheryl Acetate, Tocopheryl Succinate, Menoquinone-7 
 
 6.3.3 Sonicator bath 
 
 6.3.4 Wrist Action Shaker 
 
 7.0 Procedure 
 
 a Mobile Phase, Extraction Solvent & Diluent Preparation 
 
 7.1.1. Mobile Phase A — 100% ACN 
 
 7.1.2 Mobile Phase B —- EtOH 
 
 7.1.3 Diluent - EtOH 
 
 ce Standard Prep 
 
 7.2.1 Accurately weigh and transfer reference standard into a 50-mL volumetric flask.
 Add 5mL of DI H20 and vortex ensuring all solid has come into contact with the
 
 water. Sonicate for 2min, cool, then add about 15 mL of EtOH and put on wrist
 
 action shaker for 10min. Add about 15 more mL of EtOH and shake for another
 10min. QS with EtOH. 
 
 7.2.2 Dilute reference standard into linear range for the analyte being run to obtain
 
 “Working Standard” used for the assay. 
 
 Ted If more than one analyte is being run, standards can be mixed and run
 simultaneously (be sure to ensure all standards are within the linear range for that
 
 analyte). 
 
 7.2.4 Other flask sizes may be used however ratio of water to EtOH should remain
 
 constant. 
 
 7.2.5 Alternative standard preparations are acceptable as long as the preparations are
 within the linear range of this method. 
 
 7.3 Sample Preparation 
 
 73,1 Specific sample testing details are provided in each products profile. If a specific
 
 testing details section is not available, then follow preparation procedure as
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Lipid Soluble Vitamins: Retinyl Acetate, D-786 1 | Page 4 of 8
 
 Tocopheryl Acetate, Tocopheryl Succinate, Menoquinone-7 
 
 described below, maintaining concentration within the linear range of this
 
 method. 
 
 me The validated range is as follows for each analyte: 
 
 7.3.2.1 Vit Ara is 2.64ng — 264 ng (0.000528 — 0.0528mg/mL for 5uL
 injections) on column. 
 
 7.3.2.2 Vit Era is 22ng — 220ng (0.0044- 0.044mg/mL for 5uL injections) on
 column 
 
 7.3.2.3 Vit Ers is 32.6ng — 326ng (0.00652 — 0.0652mg/mL for 5uL injections)
 
 on column 
 
 7.3.2.4 MK7 is 23.6ng — 236ng (0.00472 — 0.0472mg/mL for SuL injections)
 on column 
 
 7.3.3 For finished products, at least 10 dosage units are pooled and/or ground by mortar
 
 and pestle. Based on the fill weight (for capsules) or tablet weight per dose and
 
 the label claim, weigh the pooled dosages into a suitably sized volumetric flask
 to generate an analyte concentration that is within the validated linearity range for
 
 the analyte being tested. 
 
 7.3.4 Prepare raw materials like standards. (However, be sure to consult the
 specification for expected potency, as raw material samples may not be 100%.)
 
 feBe To the flask, containing the finished product or raw material, add DI H2O equal
 
 to 10% of the volume of the flask and vortex to ensure all powder has come into
 
 contact with the water. Sonicate for 2min, cool, then add about 15 mL of EtOH
 and put on wrist action shaker for 10min. Add about 15 more mL of EtOH and
 
 shake for another 10min. QS with EtOH. 
 
 7.3.6 Filter a portion for use in subsequent dilutions. 
 
 7.3.7 Perform further dilutions as required using Diluent. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 
 Determination of Lipid Soluble Vitamins: Retinyl Acetate, D-786 1 | Page 5 of 8
 Tocopheryl Acetate, Tocopheryl Succinate, Menoquinone-7 
 
 7.3.8 Samples can be extracted in diluent at any volume starting from 25mL. 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 analyte
 
 concentration into the linear range. 
 
 7.4 HPLC Parameters 
 
 7.4.1 Column: Thermo Scientific AcclaimTM 120 C18, 4.6 x 250mm, 5um
 
 7.4.2 Column Temperature: 35°C 
 
 7.4.3 Flow rate: 2 mL/min 
 
 74.4 Wavelength: 270 nm 
 
 7.4.5 Injection Volume: 5 nL 
 
 7.4.6 Run Time: 12 minutes. 
 
 7.4.7 Recommended 3-D Spectral Range (for Identification) - 210nm to 550nm
 
 7.4.8 Mobile Phase: Isocratic 50:50 ACN:EtOH 
 
 7.5 Recommended Sequence 
 
 7.5.1 Make an injection 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 5.0%
 
 FOe2 The %RSD of all standard injections is NMT 6.0%. 
 
 1,65 If present, any interference in the diluent should be subtracted out of the sample
 
 and standard peak areas. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Lipid Soluble Vitamins: Retiny! Acetate, D-786 1 | Page 6 of8
 
 Tocopheryl Acetate, Tocopheryl Succinate, Menoquinone-7 
 
 7.7 Example calculations for determining finished product % label or raw material % purity
 
 7.7.1 % Analyte = = x en x Sx 2 x 100 
 
 Ri Sample peak area 
 
 Re Mean (n=5) standard peak area 
 
 Wteia Weight of the reference standard in mg (corr. for water if applicable)
 
 Vsta Volume of the standard preparation accounting for dilutions in mL
 
 P Purity of the reference standard in decimal format 
 
 SA Sample amount in mg (solids) or mL (liquids) 
 
 Vspl Volume of the sample preparation accounting for dilutions in mL
 
 SS Serving size: Average weight in mg for tablets, capsules, and
 gummies. Mass of a single serving in mg for powders, volume of a
 
 single serving from the theoretical formula in mL for liquids, or 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 50:50 ACN / Milli-Q Water. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Lipid Soluble Vitamins: Retinyl Acetate, D-786 1 Page 7 of 8
 
 Tocopheryl Acetate, Tocopheryl Succinate, Menoquinone-7 
 
 8.0 Chromatograms 
 
 8.1 Typical Diluent Chromatogram 
 
 Su | 
 : P o O o A et Sclctle tary ns accuracy Seaciic h Prec UMS FWLe om 
 
 mrt 
 
 8.2 Typical Working Standard Chromatogram 
 
 3 T-31-t9 Lies Setutie Vitemina Accuracy Soeciserty PCR OE ee Tt Les 
 
 Hamin K2 MK? - 3.207 
 | | fi act tA . Le Tacopnerot Acetat- e4 B5Q7 - $436 
 { ; i j 5 -Tocephercl Succinate-} 677 = 
 ft jt it .3-2 680! 4 ‘ 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 
 Determination of Lipid Soluble Vitamins: Retinyl Acetate, D-786 [| Page 8 of 8
 Tocopheryl Acetate, Tocopheryl Succinate, Menoquinone-7 
 
 9.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 09/11/19 | New N/A J. Maignan i 12/20/22 Added Test Details. Minor edits. CC-22-0475 J. Sassman | - | - |