Cholecalciferol (Vitamin D) Determination by HPLC using UV-Vis Spectroscopy

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1.0 Purpose 
 The purpose of this procedure is to describe a method for the quantitative analysis and
 
 identification of cholecalciferol in finished products and raw materials using HPLC coupled with
 
 UV/VIS detection. 
 
 2.0 Scope 
 
 This procedure applies to the quantification and identification of cholecalciferol in raw materials
 
 and finished products in the QC laboratory at on Nutritional Labs. 
 
 3.0 Responsibility 
 
 3) It is the responsibility of QC and Analytical Chemists to follow this procedure.
 
 362 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 to
 keep this procedure current with latest lon Nutritional Labs practices.
 
 4.0 Definitions 
 
 4.] UV/VIS — Ultraviolet/visible light 
 
 4.2 ACN - Acetonitrile 
 
 4.3 EtOH — Ethanol 
 
 4.4 CofA — Certificate of Analysis 
 
 4.5 Cholecalciferol Reference Standard — also known as Vitamin D or Vitamin D3
 
 4.6 IPA — Isopropyl Alcohol 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Cholecalciferol (Vitamin D) Determination by HPLC D-721 8 Page 2 of 10 
 using UV/VIS Spectroscopy 
 
 4.7 MeOH — Methanol 
 
 4.8 DMSO - Dimethylsulfoxide 
 
 4.9 HCl — Hydrochloric Acid 
 
 4.10 QC — Quality Control 
 
 4.11 HPLC — High Performance Liquid Chromatography 
 
 5.0 References 
 
 5.1 MV-LAB-13-008, Protocol, Cholecalciferol Determination by HPLC 
 
 5.2 NCR-23-0008, Product Specific Method Optimization for Vitamin D in SGM00379
 
 (Notebook 200 Pg 105-106, 111-113) 
 
 5.3. PRTCL-24-0035, Supplemental Validation of D-721 for Determination of
 Cholecalciferol in Chewable Gels by HPLC-UV 
 
 6.0 Reagents, Supplies, Glassware and Equipment 
 
 6.1 Reagents: all reagents are ACS grade or better 
 
 6.1.1 ACN 
 
 6.1.2 DMSO 
 
 6.1.3 MeOH 
 
 6.1.4 Ethanol denatured with 5% IPA and 5% MeOH 
 
 6.1.5 Cholecalciferol, traceable standard 
 
 6.1.6 2.7N HCl 
 
 6.1.7 Deionized Water (> 18.2 MQ-cm) 
 
 6.1.8 IPA 
 
 6.2 Supplies and Glassware 
 
 6.2.1 HPLC vials, 12mm X 32mm with screw cap enclosures w/ septa 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Cholecalciferol (Vitamin D) Determination by HPLC D-721 Page 3 of 10 
 using UV/VIS Spectroscopy 
 
 6.2.2 Mobile phase containers 
 
 6.2.3 Volumetric flasks 
 
 6.2.4 Pipette tips 
 
 6.2.5 Plastic luer-lock syringes 
 
 6.2.6 0.45um 25mm PTFE syringe filters 
 
 6.2.7. Erlenmeyer flasks 
 
 6.2.8 Microcentrifuge tubes 
 
 6.2.9 Weigh Paper and/or weigh boats 
 
 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 Acclaim 120 C18, 5um, 120A, LC column, 250mm X 4.6mm, or equivalent
 
 6.3.3 Analytical Balance 
 
 6.3.4 Micro Analytical Balance 
 
 6.3.5 Wrist Action Shaker 
 
 6.3.6 Sonicator 
 
 6.3.7 Vortex Mixer 
 
 6.3.8 Stir Plate 
 
 6.3.9 Adjustable Pipette(s) 
 
 6.3.10 Microcentrifuge 
 
 7.0 Procedure 
 
 TA Mobile Phase A — 100% ACN 
 
 Ta Mobile Phase B — Ethanol, denatured with 5% IPA and 5% MeOH 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Cholecalciferol (Vitamin D) Determination by HPLC D-721 8 Page 4 of 10 
 using UV/VIS Spectroscopy 
 
 73 Extraction Solvent / Diluent — Mobile Phase B 
 
 Toul Note: Microencapsulated D3 requires a pre-extraction in H20. 
 ee Note: Use 1:1 MeOH/DMSO when analyzing gummies. 
 
 7.4 Standard Preparation 
 
 7.4.1 Use the actual purity from the CofA for cholecalciferol in your calculations.
 
 7.4.2 All Standards are prepared by weighing no less than the minimum weight of the
 analytical balance. 
 
 7.4.3 Accurately weigh and transfer the reference standard into a suitably sized
 volumetric flask. 
 
 7.4.4 Add Diluent to two thirds final volume and place on the wrist action shaker for
 
 15 minutes before bringing up to final volume using Diluent. 
 
 7.4.5 Dilutions are prepared using diluent. Dilutions can be made using volumetric
 glassware or using 10mL, ImL and 200uL variable pipettes. Specific standard
 
 concentrations will approximate the concentration expected to be found in the
 
 product being tested based on the sample dilution and calculated from the label.
 Final dilutions may be prepared directly in HPLC vials. 
 
 7.4.6 Alternative standard preparations are acceptable as long as the preparations are
 
 within the linear range of this method, which is listed below.
 
 7.5 Sample Preparation 
 
 1.51 Specific sample testing details are provided in each product profile. If a specific
 
 testing details section is not available, then follow preparation procedure as
 described below, maintaining concentration within the linear range of this
 
 method. 
 
 Tae The linear range of the method is listed below. Samples must be prepared within
 this range. 
 
 7.5.2.1 Agilent 1260 with 60 mm flow cell: 0.025 ug/mL — 10 pg/mL.
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Cholecalciferol (Vitamin D) Determination by HPLC D-721 8 Page 5 of 10 
 using UV/VIS Spectroscopy 
 
 7.5.2.2 All other instruments: 0.5 pg/mL — 10 wg/mL. 
 
 7.5.3. 10 or more dosage units can be pooled and ground by mortar and pestle as
 
 necessary. (When analyzing gummies, prepare as a cryogrind as per D-793 and
 extract / dilute using 1:1 DMSO/MeOH as described below in 7.5.10.)
 
 7.5.4 Based on the label claim & dosage form weight, weigh a portion of the pooled
 
 dosages to generate an analyte concentration that is within the validated linearity
 and solubility range for the analyte being tested. 
 
 7.5.5 Sample pretreatment to remove encapsulation from encapsulated forms of
 
 cholecalciferol: 
 
 7.5.5.1 Many solid or powder forms of cholecalciferol are micro encapsulated
 
 and require a pretreatment to release the vitamin from the substrate
 before preparing the sample. The primary method to remove the
 
 encapsulation matrix is to pretreat the powdered sample with a volume
 of H2O equal to 10% of the flask volume and vortex making sure all
 
 water is either absorbed onto the powder or all the powder comes into
 
 contact with the water. 
 
 7.5.5.2 Other extraction methods may be required based on the encapsulation
 chemistry. 
 
 7.5.6 Sample pretreatment for Gel caps containing cholecalciferol: 
 
 7.5.6.1 Adda volume of 2.7N HCL equal to 5% - 10% of the flask volume, and
 
 shake on a wrist-action shaker for 30 minutes. 
 
 7.5.7 After any necessary pre-treatment steps, add diluent equal to about 1/3 the volume
 
 of the flask and shake on the wrist action shaker for 15 minutes.
 
 7.5.8 Next, further dilute to 2/3 the final volume using diluent and mix again on the
 wrist action shaker for an additional 15 minutes before diluting to final volume.
 
 7.5.9 Samples can be dissolved in diluent at any volume starting from 10mL. To
 
 manage large volumes the sample can be initially dissolved in a smaller volume
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Cholecalciferol (Vitamin D) Determination by HPLC D-721 Page 6 of 10 
 using UV/VIS Spectroscopy 
 
 that is within the solubility range and a portion further diluted to bring the analyte
 
 concentration into the linear range of measurement. 
 
 7.5.10 Example for ~3g gummy containing 50ug Ds: 
 
 7.5.10.1 Transfer ~3g cryogrind to 125ml Erlenmeyer flask. Add stir bar and
 ~35ml of 1:1 DMSO/MeOH then stir on a stir plate for 30 minutes
 (protected from light). 
 
 7.5.10.2 Quantitatively transfer the slurry to a 5Oml (low actinic) volumetric
 flask. QS to volume and sonicate for 10 minutes. Filter as described
 
 below using a PTFE syringe filter. 
 7.5.11 The final sample must be filtered or centrifuged before analyzing by HPLC.
 
 7.5.11.1 For filtration, using the final large scale diluted sample, withdraw up to
 
 10 mL using a 10 mL plastic syringe. Filter though a 0.45 um nylon,
 PVDF or PTFE membrane, discarding at least 0.5 mL of sample before
 
 collecting a portion for analysis. From the collected sample dilute as
 
 needed then add 1mL to an HPLC vial for analysis. 
 
 7.5.11.2 For centrifugation, using the final large scale diluted sample, fill an even
 number of 1.5 or 2.0 mL microcentrifuge tubes and pellet insoluble
 
 matter for 5 minutes at 10,000 rpm. 
 
 7.6 Test Conditions 
 
 7.6.1. Gradient- Isocratic 
 
 Time HA %B 
 
 0.00 50 50 
 
 10.00 50 50 
 
 7.6.2. Column- Acclaim 120 C18, 5um, 120A, 250mm X 4.6mm or equivalent.
 
 7.6.3 Flow Rate- 1.0 mL/min 
 
 7.6.4 UV Detection- 265 nm 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Cholecalciferol (Vitamin D) Determination by HPLC D-721 8 Page 7 of 10 
 using UV/VIS Spectroscopy 
 
 7.6.5 Injection Vol.- 20 wh 
 
 7.6.6 Temperature- 35°C 
 
 7.6.7 Run Time — at least 10 minutes 
 
 7.6.8 Retention Time — about 8.5 minutes 
 
 7.6.9 Recommended 3-D Spectral Range- 210nm to 320nm 
 
 7.7 Recommended Sequence 
 
 7.7.1 Make at least 2 injections of the diluent. 
 
 7.7.2 Make five (5) injections of Standard Solution. 
 
 7.7.3. Make a single injection of each Sample Preparation. 
 
 7.7.4 Make a single injection of the Standard Solution after every six (6) samples and
 at the end of the run. 
 
 7.8 System Suitability Requirements 
 
 7.8.1 The %RSD of the first five (5) standard injections is NMT 5.0%.
 
 7.8.2 The %RSD of all standard injections is NMT 5%. 
 
 7.9 Example calculations for determining finished product % label or raw material % purity
 
 Ruy x Wtstq XP Vspl SS 
 7.9.1 0% assay Lu= Veea Se wae 100 
 Ry Sample peak area 
 
 Re Mean standard peak area 
 
 Wtstq Weight of reference standard in mg 
 
 Veg 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) 
 
 Vent Volume of the sample preparation accounting for dilutions in mL
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Cholecalciferol (Vitamin D) Determination by HPLC D-721 8 Page 8 of 10 
 using UV/VIS Spectroscopy 
 
 55 Serving size: Average Weight of a single dosage unit in mg for tablets,
 
 capsules, and gummies. Single serving from the theoretical formula in
 
 mL for liquids or mg for powders, or 1 for raw materials.
 
 LA Label amount in mg per dose or | for raw materials 
 
 7.10 Column Wash and Storage 
 
 7.10.1 Column rinse is not required. 
 
 7.10.2 Store the column with Mobile Phase A/B (50/50). 
 
 7.11 Example Chromatography 
 
 7.11.1 Blank (Diluent) 
 
 DILUENT 
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12.5 5 10
 
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 ie + 18 225-3 Seo ade SER. q eT TT eo Rk ee Bae 4T0 
 Time [min] 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Cholecalciferol (Vitamin D) Determination by HPLC | 2-72! 8 Page 9 of 10 
 using UV/VIS Spectroscopy 
 
 7.11.2 Working Standard 
 
 21AS071 0.5MCG/ML 
 16; 
 149 
 
 12; 
 10; 
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 ON 
 oa 
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 165“ 
 05 1 15 2 25 3 35 4 45 5 55 65 7 75 8 85 9 95 10 
 Time [min] 
 
 7.11.3 Sample 
 
 200342 
 ry 
 2001 +O 
 
 150: 
 = 
 £100} 
 
 501 
 
 0 T tT T tae q T qT LU T T T LU LJ qT T 1 q q Lj 
 
 05 1 15 2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 95 10 
 Time [min] 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Cholecalciferol (Vitamin D) Determination by HPLC D-721 8 | Page 10 of 10 
 using UV/VIS Spectroscopy 
 
 8.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 01/20/12 | New - - Revalidated method, changed mobile phase to ACN:ETOH 50:50, | - | - |
| 1 | 05/24/13 | changed calculations to % label, added information on extraction and release conditions | 13-405 | B. Johns |
| 2 | 09/09/15 | ae review: Updated SOP format. Updated HPLC method | 15-0864 | B. Johns |

 Scheduled review: Changed Responsibilities, Added to sample 
 3 02/04/19 | preparation to make it easier to follow, Updated stability 19-0120 J. Maignan
 requirements. 
 Update to reflect current practices, simplify standard and sample
 preparation, add recommended sequence section, replace 
 4 04/11/22 | requirements section with system suitability, update example CC-22-0170 | S.Sassman
 calculation for consistency with current methods, narrow the range
 for spectral match, add example chromatography. 
 5 12/20/22 | Added test details section. Minor edits. CC-22-0475 | J. Sassman
 6 03/09/23 | Change section 7.6.7 from 10 minutes to “at least 10 minutes”. CC-23-0115 | J. Sassman
 7 09/08/23 Extend lower linearity range when using 60mm flow cell, clarify CC-23-0452 | S. Sassman
 amount of water to use for encapsulated forms of vitamin D. 
 8 05/15/24 | Added details for analysis of finished product gummies. CC-24-0174 C. Perry