Determination of Trimethylglycine (Betaine) by HPLC-UV

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1.0 Purpose 
 
 This document describes the analytical procedure for the determination of trimethylglycine
 
 (betaine) in raw materials and finished products. 
 
 2.0 Scope 
 
 This procedure applies to the identification and quantification of trimethylglycine in raw
 materials and finished products. 
 
 3.0 Responsibility 
 
 3.1 ‘It is the responsibility of QC 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 to
 
 keep this procedure current with the associated monographs and laboratory practices.
 
 4.0 Definitions 
 
 4.1 HPLC/UV — High Pressure Liquid Chromatography with Ultraviolet Detection
 
 4.2 QC - Quality Control 
 
 4.3. AD-— Analytical Development 
 
 4.4 TMG- Trimethylglycine (Betaine) 
 
 4.5 MeOH - Methanol 
 
 4.6 |NaH2PO4e2H20 — Sodium Phosphate Monobasic Dihydrate 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Trimethylglycine (Betaine) by D-784 2 7 aaa 
 HPLC/UV ° 
 
 4.7 H3PO4— 85% Phosphoric Acid 
 
 5.0 References 
 
 | MV-LAB-19-026, Protocol, Validation of an Analytical Method for the Determination
 of Trimethylglycine (Betaine) using HPLC/UV 
 
 5.2. D-793, SOP, Cryogenic Grinding of Chewable Gels 
 
 6.0 Supplies 
 
 6.1 Chemicals: All reagents are HPLC grade or better 
 
 6.1.1 MeOH 
 
 6.1.2 Sodium-1-nonanesulfonate 
 
 6.1.3. NaH2PO4*2H20 
 
 6.1.4 H3PO4 
 
 6.1.5 TMG Reference Standard 
 
 6.2 Supplies and Glassware 
 
 6.2.1 HPLC vials, 12 mm X 32 mm with screw cap enclosures w/ septa
 
 6.2.2 Class A Volumetric glassware and/or adjustable pipettes and tips
 
 6.2.3. Weigh paper or funnels 
 
 6.2.4 1.5 mL or 2.0 mL micro centrifuge tubes 
 
 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 
 
 6.3.3. Sonicator bath 
 
 6.3.4 Stir Plate 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Trimethylglycine (Betaine) by D-784 2 B ane 
 HPLC/UV i 
 
 6.3.5 Centrifuge 
 
 7.0 Preparation of Mobile Phase, Diluent, Samples and Standards Equipment 
 
 7.1. Mobile Phase Preparation 
 
 TLA Mobile Phase 
 
 7.1.1.1 Combine 1.09 g of sodium-1l-nonanesulfonate, 2.96 g of
 NaH2PO.°2H20, and 950 mL of water. Begin stirring and adjust the pH
 
 to 2.5 using H3PO4. Add 50 mL of MeOH and mix well. 
 
 Pla Diluent 
 
 7.1.2.1 Water 
 
 7.2 Standard Prep 
 
 7.2.1 Accurately weigh and transfer about 50 mg of TMG reference standard into a 100-
 
 mL volumetric flask. 
 
 7.2.2 Dissolve in and dilute to volume with Diluent. 
 
 7.2.3 The standard preparation may be scaled as required. 
 
 7.3. Sample Preparation 
 
 Specific sample testing details are provided in each products profile. If a specific
 Tool 
 testing details section is not available, then follow preparation procedure as
 described below, maintaining concentration within the linear range listed below.
 
 The linear range for TMG is 0.08 mg/mL — 0.80 mg/mL. The concentration of the
 Td.2 
 sample preparation must be within the linear range. 
 
 733 For solid and liquid dose finished products: at least 10 dosage units are pooled
 and ground by mortar and pestle. Based on the weight per dose and the label
 
 amount, weigh no less than 40 mg of the pooled dosages into a suitably sized
 volumetric flask to generate an analyte concentration that is within the validated
 
 linearity range. Dilute the sample to 2/3 of the flask volume with water and shake
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Trimethylglycine (Betaine) by D-784 2 pee 
 HPLC/UV 
 
 for 20 minutes to facilitate dissolution. Sonication for 10 minutes can also be used
 
 to assist dissolution. Equilibrate the sample to room temperature (if sonicated),
 
 and dilute to volume using water. 
 
 7.3.4 For liquid capsules: weigh no less than 5 dosage units and transfer them into a
 suitably sized volumetric flask to generate an analyte concentration that is within
 
 the validated linearity range. Fill with water to about 80% of the flask volume.
 Sonicate for 10 minutes or until the capsules are completely dissolved. Allow the
 
 sample to equilibrate to room temperature. Add water until the solution reaches
 
 the flask neck, and add a minimal volume of methanol to disperse frothing. Dilute
 to volume using water. 
 
 7.3.5 For raw materials: weigh no less than 40 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 for the analyte being tested. Fill with water to about
 
 80% of the flask volume, and sonicate for 10 minutes. Allow the sample to
 
 equilibrate to room temperature, and dilute to volume using water.
 
 7.3.6 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. Fill with water to about 80% of the flask volume, and sonicate for
 10 minutes. Allow the sample to equilibrate to room temperature, and dilute to
 
 volume using water. 
 
 7.3.7. To manage large volumes, the standard can be initially prepared at a higher
 concentration and further diluted into the linear range using Diluent. Equilibrate
 
 to room temperature prior to performing further dilution. Dilutions can be
 made using volumetric glassware and/or adjustable pipettes. Dilutions can be
 
 prepared in HPLC vials 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Trimethylglycine (Betaine) by D-784 2 fs aa 
 HPLC/UV 
 
 7.3.8 Centrifuge a portion of the sample at 10,000 rpm for 5 min to remove particulates
 
 prior to HPLC analysis. Alternatively, the sample may be filtered through a 0.45
 
 uum membrane discarding the first 3 - 4 mL. 
 
 8.0 HPLC Parameters 
 
 8.1 Column: Inertsil ODS-3, 5um, 4.6mm X 150mm 
 
 8.2 Column Temperature: 40 °C 
 
 8.3 Flow rate: 1.5 mL/min 
 
 8.4 Wavelength: 210 nm 
 
 8.5 Injection Volume: 10 wL 
 
 8.6 Run Time: 7 minutes 
 
 8.7 Recommended 3-D Spectral Range (for Identification)- 190nm to 400nm
 
 8.8 Recommended Sequence 
 
 8.8.1 Make at least 2 injections of the diluent. 
 
 8.8.2 Make five (5) injections of Standard Solution. 
 
 8.8.3. Make a single injection of each Sample Preparation. 
 
 8.8.4 Make a single injection of the Standard Solution after every ten (10) sample
 injections or at the end of a run. 
 
 8.9 System Suitability Requirements 
 
 8.9.1 The %RSD of the first five (5) standard injections is NMT 5.0%.
 
 8.9.2 The %RSD of all standard injections is NMT 5%. 
 
 8.9.3. 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 
 Page 
 Determination of Trimethylglycine (Betaine) by D-784 2 
 6 of 7 
 HPLC/UV 
 9.0 Example Calculation 
 
 Ru WtstaXP SS Vep1 
 9% assay = =R— X Vea xX =SAX TA XxX 100 
 
 Ry Sample peak area 
 
 Rg Mean standard peak area 
 
 Wtsta Weight of reference standard in mg 
 
 Veta Volume of the standard preparation accounting for dilutions in mL
 
 P Purity of the reference standard in decimal format 
 
 SA Sample amount in mg 
 
 SS Serving size: Weight of a single serving in mg, or 1 for raw materials.
 
 Vspl Volume of the sample preparation accounting for dilutions in mL 
 
 LA Label amount in mg (use | for raw materials) 
 
 10.0 Column Wash and Storage 
 
 10.1. Rinse the column with Water / MeOH (80/20). 
 
 10.2 Store the column in Water / MeOH (50/50). 
 
 11.0 Chromatograms 
 
 11.1 Typical Diluent Chromatogram 
 
 14.0 
 10.0 
 
 0.0 —y 
 2.0 7.00 731 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Trimethylglycine (Betaine) by D-784 2 . ae 
 HPLC/UV 
 
 11.2 Typical Working Standard Chromatogram 
 
14.0 mA

10.0 50

 00 —/ ’ 
 = 2 1.00 1.50 2.00 250 3.00 340 4.00 450 5.00 5.50 6.00 650 7.00 7H 
 
 11.3. Typical Raw Material Sample Chromatogram 
 
 14.0 mau \TMG - 2.943 
10.0 5.0 00j-~._, .
 
 ° ‘s 9 1.00 1.50 2.00 250 3.00 350 4.00 4.40 5.00 5.60 6.00 6.40 7.00 731 
 
 11.4 Typical Finished Product Sample Chromatogram 
 
14.0 mau (TMG - 2.963

10.0 $.0

 0.0 —/ min 
 4 9 1.00 1.50 2.00 2.50 3.00 3.50 4.00 450 6.00 6.50 6.00 6.50 7.60 734 
 
 12.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 06/26/19 | New N/A S. Sassman | - | - |
| 1 | 06/24/22 | Updated logo and format. CC- Minor edits for clarity, add instructions to follow test details | 22-0293 | K. Burris |
| 2 | 06/06/23 | containing product specific sample prep, add sample prep CC- instructions for gummies, remove salt correction from example t calculation. Update logo and format. | 23-0265 | % Sassman |