Methylcobalamin Determination by HPLC using UV-Vis Spectroscopy

Original Document

Scanned document (image-only PDF)

Extracted Text

Searchable text extracted from PDF

1.0 Purpose 
 
 The purpose of this procedure is to define the method for the determination and/or identification
 
 of Methylcobalamin in finished products and raw materials using HPLC and UV/VIS
 
 spectroscopy. 
 
 2.0 Scope 
 
 This procedure applies to methylcobalamin quantification and identification. Methylcobalamin
 is quantified at 351 nm. Alternate wavelengths may be used with justification to avoid
 
 interferences. 
 
 3.0 Responsibility 
 
 3.1 It is the responsibility of QC and analytical development personnel 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 Analytical Development
 personnel to keep this procedure current with latest Ion Labs practices.
 
 4.0 Definitions 
 
 4.1 UV/VIS — Ultraviolet and Visible Electromagnetic Spectrums 
 
 4.2. H3PO4-— Phosphoric Acid 
 
 4.3 KH2PO4-— Potassium phosphate monobasic 
 
 4.4 ACN — Acetonitrile 
 
 4.5 KOH — Potassium Hydroxide 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Page 
 Methylcobalamin Determination by HPLC using D-713 
 2 of 8 
 UV/VIS Spectroscopy 
 4.6 HO — Water 
 
 4.7 QC - Quality Control 
 
 5.0 References 
 
 5.1 HPLC Application ID# 2581 by Phenomenex, 2014 
 
 5.2 MV-LAB-15-004, Protocol, Methylcobalamin Determination using HPLC and UV/VIS
 
 Spectroscopy. 
 
 53 Novel RP-HPLC method for the Simultaneous Estimation of Thiamine Mononitrate,
 Calcium Methylcobalamin, L-Cystine and Para Amino Benzoic Acid in Multi Vitamin
 
 Dosage Forms. Tamma Narendra Kumar et al., IJSID 2011, 1 (20), 226-242.
 
 5.4 D-793, SOP, Cryogenic Grinding of Chewable Gels 
 
 6.0 Reagents, Supplies, Glassware and Equipment 
 
 6.1 Chemicals: All reagents are HPLC grade or better. 
 
 6.1.1 H2O 
 
 6.1.2 ACN 
 
 6.1.3 KH2PO4 
 
 6.1.4 H3POs 
 
 6.1.5 KOH 
 
 6.1.6 Methylcobalamin, Reference Standard, MW 1344.40, CAS No: 13422-55-4
 
 6.2 Glassware 
 
 6.2.1. Low Actinic HPLC vials, 12mm x 32mm with screw cap enclosures with septa
 
 6.2.2 Scintillation Vials 
 
 6.2.3 Mobile Phase Containers 
 
 6.2.4 25mL, 50mL, 100ml Low Actinic Volumetric Flasks 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Methylcobalamin Determination by HPLC using D-713 4 a ve 
 UV/VIS Spectroscopy ° 
 
 6.3 Disposables 
 
 6.3.1 Pipette Tips 
 
 6.3.2 Microfuge tubes 
 
 6.3.3. Disposable Plastic Luer Lock Syringe — 3mL, 6mL, or 10mL 
 
 6.3.4 Nylon Syringe Filters, 0.45um 
 
 6.3.5 Weigh paper 
 
 6.4 Equipment 
 
 6.4.1 Suitable HPLC system consisting of a pump, autosampler, column compartment,
 
 and diode array detector. Analytical Balance 
 
 6.4.2 Vortex 
 
 6.4.3 Stir Plate 
 
 6.4.4 Wrist-action mechanical shaker 
 
 6.4.5 Eppendorf Centrifuge 
 
 6.4.6 Adjustable Pipettes 
 
 7.0 Preparation of Mobile Phase, Diluent, Samples, and Standards 
 
 7.1. Mobile Phase and Buffer Preparation 
 
 7.1.1. Mobile Phase A — Prepared by dissolving 680.5mg KH2PO4 , in 950ml of
 
 Millipore water adjusted pH to 5.6 with phosphoric acid or potassium hydroxide,
 then bring up to 1| liter 
 
 7.1.2 Mobile Phase B — 100% ACN 
 
 7.1.3. Diluent— 95:5 (Mobile A : Mobile B) 
 
 7.2 Standard Preparation 
 
 Note: Methylcobalamin is especially susceptible to degradation in solution. Ensure the
 
 autosampler chiller is set to 4°C, and place the working sample and/or standard
 
 
 

[SOP 

 Methylcob S a t l a a n m d i a n r d D O e p t e e r r a m t i i n n a g t P i r o o n c e b d y u H re P LC using S D O - P 71 N 3 o | Re 4 v pace
 
 UV/VIS Spectroscopy 
 
 into a vial in the autosampler quickly to avoid degradation. Ensure that it is not
 
 exposed to light/heat sources and samples should be analyzed soon after
 
 preparation. 
 
 7.2.1 The standard is prepared by weighing no less than the scale’s limit at the time of
 
 use, then bringing up to two thirds the final volume in an appropriate low actinic
 volumetric flask using Diluent then shaking for up to 20 minutes (until no solid
 
 material remains) and bringing up to final volume with Diluent.
 
 7.2.2 Dilutions can be made using low actinic volumetric flasks, volumetric glass
 
 pipettes, and/or adjustable 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. Dilutions can be
 prepared in low actinic HPLC vials. 
 
 7.3 Sample Preparation 
 
 Note: Avoid exposure to light during sample preparation. Samples should be prepared
 
 in low actinic (red) glassware, placed in the chilled autosampler, and used within
 
 2 hours. 
 
 7.3.1 The linear range of the method is 0.01 — 0.5 mg/mL. All standards and samples
 
 to be injected must be within this range. 
 
 7.3.2 Specific sample testing details are provided in each products profile. If a specific
 testing details section is not available, then follow preparation procedure as
 
 described below, maintaining concentration within the linear range listed above.
 
 7.3.3 For raw materials: weigh no less than 20 mg into a suitably sized volumetric flask
 of no less than 25 mL volume to generate an analyte concentration that is within
 
 the validated linearity range. Add Diluent to 2/3 of the flask volume, shake
 
 mechanically for 10 min, dilute to volume with Diluent, and mix well.
 
 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
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Methylcobalamin Determination by HPLC using D-713 4 z mse 
 UV/VIS Spectroscopy 
 
 size (powders) or tablet weight per dose, weigh no less than 50 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. Add Diluent to
 2/3 of the flask volume, shake mechanically for 10 min, dilute to volume with
 
 Diluent, and mix well. 
 
 130 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 a portion 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 to generate an analyte concentration that is within the validated
 
 linear range. Add Diluent to 2/3 of the flask volume, shake mechanically for 10
 min, dilute to volume with Diluent, and mix well. 
 
7.3.6 When the sample matrix is found to cause greater instability to methylcobalamin,

 vigorous shaking for 1 minute can be used in place of mechanical shaking to
 reduce degradation. 
 
 To manage large volumes, the standard can be initially prepared at a higher
 Tabet 
 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 
 Filter an aliquot of the sample through a 0.45 um membrane discarding the first
7.3.8 3-4 mL before collecting a portion for analysis.

 8.0 Test Conditions 
 
 8.1 Gradient 
 
 Time %A %B Gradient Type 
 
 0.00 85 15 Linear 
 
 5.00 85 15 Linear 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Methylcobalamin Determination by HPLC using D-713 4 eee 
 UV/VIS Spectroscopy 
 
 10.00 75 Zo Linear 
 
 11.50 55 45 Linear 
 
 11.60 50 50 Linear 
 
 14.00 90 10 Linear 
 
 8.2 Column — Phenomenex Luna C8 (2), 5um, 100A, LC column, 150mm x 4.6mm
 
 8.3 Flow Rate — 1.0 mL/min 
 
 8.4 UV Detection —351nm 
 
 8.5 Injection Volume - 20uL 
 
 8.6 Column Temperature — 30°C 
 
 8.7 Peltier Sample Chiller - 4°C, equilibrated before adding test samples
 
 8.8 Retention Time — about 9 min 
 
 8.9 Recommended Sequence 
 
 8.9.1 Perform 2 Blank (Diluent) injections. 
 
 8.9.2 Perform 5 injections of the Working Standard. 
 
 8.9.3. Perform a single injection of each sample preparation. 
 
 8.9.4 Perform 1 injection of the Working Standard after every 6 samples and at the end
 
 of the run. 
 
 8.10 System Suitability 
 
 8.10.1 %RSD of 5 injections of working standard is NMT 3%. 
 
 8.10.2 %RSD of all injections of working standard is NMT 5%. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Page 
 Methyleobalamin Determination by HPLC using 
 D-713 
 7 of 8 
 UV/VIS Spectroscopy 
 9.0 Example Calculation 
 
 R Wteg XP V. SS 
 0 cen x std x spl ey 
 % assay R, Vea CA TA 100 
 Ry Sample peak area 
 
 Rg Mean standard peak area 
 
 Wtsq Weight of reference standard in mg 
 
 Vstqa Wolume 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) 
 
 Vsp_ Volume of the sample preparation accounting for dilutions in mL
 
 So Serving size: Weight of a single dosage unit in mg for tablets and capsules,
 volume of a single serving from the theoretical formula in mL for liquids, or 1 for
 
 raw materials. 
 
 LA Label amount in mg per dose or 1 for raw materials 
 
 10.0 Example Chromatography and Spectrum 
 
 10.1 Standard 
 
 22AS019 
 & 
 404 5 
 357 3 
 307 > 
 257 \ 2 
 > 207 
 Fs 154 o 3 
 10+ = = 2 
 5 < 
 O47 
 5+ 
 -10+ OS 1 15 2 25 5 354455556 65 7 75 8 8S 9 95 10105 11 11.5 12 12.5 13 13.5 14
 Time [min] 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev ‘ 
 Methylcobalamin Determination by HPLC using D-713 4 i 
 8 of 8 
 UV/VIS Spectroscopy 
 
 10.2. UV Spectrum 
 
 é methylcobalamin - 8.908 (2023-01-24 12-20-35-05-00-03.dx) 
 90- 
 80+ 
 707 
 60- 
 504 
 407 
 304 
 205 ee 
 10: ee ae —- 
 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 
 nm 
 
 11.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 08/21/15 | New | 15-0800 | X. Shao |
| 1 | 04/19/17 | . Updated testing procedure to meet current practices. Scheduled review: Added information about the lack of stability of — pea Methylcobalamin. Removed release requirements ac aadl Wis ieaiacinite | 17-0435 | B. Johns |
| 3 | 03/23/22 | Updated to match current practices. Updated format. Removed CC- references to specific HPLC systems. Removed unnecessary information and align with current SOP 4 apaiZ= A fo d r d m e at d , s A pe d c d i e fi d c i s n a s m tr p u l c e t i p o r n e t p o f f o o r l d l i o f w f e p r r e o n d t u d c o t s s a p g e e c i f f o i r c m t s e . s t A d d e d ta e i d ls . COs T en e 022h, Se BAssihan example chromatography and spectrum. Changed logo. | 22-0113 | S. Sassman |