D-734
Determination of Phytosterols by GC-FID
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1.0 Purpose The purpose of this procedure is to define the method for the determination of phytosterols in raw materials and finished products by GC-FID. 2.0 Scope This procedure applies to the determination of phytosterols in raw materials and finished products in the QC Laboratory. 3.0 Responsibility 3.1 It is the responsibility of QC Chemists to follow this procedure. 3.2 It is the responsibility of QC Laboratory Management to ensure that this procedure is being followed. 3.3 It is the responsibility of QC Laboratory Management and/or Analytical Development to keep this procedure aligned with current practices. 4.0 Definitions 4.1 QC — Quality Control 42 GC-FID — Gas Chromatography with Flame Ionization Detection 4.3 PTFE - Polytetrafluoroethylene [SOP Standard Operating Procedure SOP No | Rev Page Determination of Phytosterols by GC-FID Deis 1 2 of 9 5.0 References 5.1 PRTCL-20-0113, Protocol, Validation of an Analytical Method for the Determination of Phytosterols by GC-FID 5.2 D-793, SOP, Cryogenic Grinding of Chewable Gels 6.0 Supplies 6.1 Chemicals 6.1.1 Stigmasterol Reference Standard (>97%) 6.1.2 Tetrahydrofuran (ACS reagent grade or better) 6.1.3 5a-Cholestane (ACS reagent grade or better) 6.2 Compressed Gases (use ultra-high purity gases) 6.2.1 Hydrogen 6.2.2 Helium 6.2.3 Air 6.2.4 Nitrogen 6.3. Supplies and Glassware 6.3.1 Volumetric glassware as required for standard and sample preparation 6.3.2 2-mL GC vials with PTFE lined closures 6.4 Equipment 6.4.1. Agilent 7890 GC with FID detector [SOP Standard Operating Procedure SOP No Rev Page D-734 Determination of Phytosterols by GC-FID 3 of 9 6.4.2 Analytical Balance 7.0 GC Conditions 7.1 Column: Agilent HP-5, 30 m x 0.32 mm x 0.25 um or equivalent Tl Inlet Liner: Restek, 4.0 mm ID x 6.3 mm OD x 78.5 mm length straight liner with glass wool or equivalent 12 Injection Volume 1 pL 7.4 Oven Temp: 270 °C (isothermal) Ta Injector Temp: 300 °C 7.6 Detector Temp: 320 °C 7.7 Equilibration Time: 0.5 min 7.8 Flow Rate: 1.2 mL/min ve Run Time: 20 min 7.10 Split ratio: 50:1 7.11 Septum purge: Off PAZ Air flow: 350 mL/min 7.13 Hydrogen flow: 30 mL/min 7.14 Makeup flow: 30 mL/min (column + makeup = constant) 7.15 Injection Type Standard 7.16 Plunger Speed Fast 7.17 Wash Solvent THF [SOP Standard Operating Procedure SOP No | Rev Page Determination of Phytosterols by GC-FID oe 4 of 9 8.0 Internal Standard Solution Preparation 8.1 Accurately weigh and transfer about 50 mg of 5a-cholestane to a suitable container. 8.2. Add 200-mL of tetrahydrofuran. 8.3. Mix until completely dissolved. 9.0 Working Standard Preparation 9.1 Use the actual purity from the CoA for the reference material in calculations. 9.2 Accurately weigh and transfer about 25 mg of stigmasterol to a 100-mL volumetric flask. 9.3 Dissolve in and dilute to volume with Internal Standard Solution. 10.0 Sample Preparation 10.1 Specific sample testing details are provided in each products profile. Ifa specific testing details section is not available, then follow preparation procedure as described below, maintaining concentration within the linear range of this method. 10.2 The validated linear range of the method is 0.01 — 1.0 mg/mL. The content of the three most abundant phytosterols in the sample preparation must be within the linear range. 10.3. Ensure that the sample is thoroughly homogenized prior to weighing. 10.4 The use of glass pipets is recommended for transfers and dilutions. Phytosterols may absorb to plastics, and THF is not compatible with automatic pipets. 10.5 For finished products, pool at least 10 dosage units and homogenize. Based on the dosage weight (for powders), fill weight (for capsules) or tablet weight and the label claim, weigh no less than 20 mg of the pooled dosages into a suitably sized volumetric flask to generate concentrations for the three most abundant phytosterols that are within the validated [SOP Standard Operating Procedure SOP No | Rey Page Determination of Phytosterols by GC-FID D734 1 5 of9 linear range, add Internal Standard Solution to about 70% of the flask volume and shake for at least 10 minutes. Dilute to volume with Internal Standard Solution, and mix well. 10.6 For raw materials, based on the expected potency, weigh no less than 20 mg of sample into a suitably sized volumetric flask to generate concentrations for the three most abundant phytosterols that are within the validated linear range, add Internal Standard Solution to about 70% of the flask volume and shake for at least 10 minutes. Dilute to volume with Internal Standard Solution, and mix well. 10.7 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 no less than 200 mg of the pooled and homogenized dosages into a suitably sized beaker. Add a volume of Internal Standard Solution equivalent to 50% of the desired flask volume, add a stir bar, cover the top of the beaker, and stir until dissolved. Transfer the solution to a volumetric flask of size suitable to generate concentrations for the three most abundant phytosterols that are within the validated linear range. Use several small portions of Internal Standard Solution to rinse any remaining residue from the beaker into the volumetric flask ensuring complete transfer, and dilute to volume using Internal Standard Solution. 10.8 To manage large volumes, the sample can be initially dissolved in a smaller volume and a portion further diluted using Internal Standard Solution to bring the analyte concentration into the linear range. 10.9 If particulates remain in sample preparations, remove them by allowing the particulates to settle, filtration, or centrifugation. For filtration, use a 0.45 um membrane discarding the first 2-3 mL before collecting a portion for analysis. For centrifugation, spin at 10,000 rpm for 5 min. 11.0 Recommended Sequence Lil Make two injections of Internal Standard Solution [SOP Standard Operating Procedure SOP No | Rev Page Determination of Phytosterols by GC-FID oe ; 6 of 9 11.2. Make five injections of Working Standard A 11.3. Make a single injection of each Sample Preparation 11.4 Make a single injection of Working Standard A after every 10 injections and at the end of the run. 12.0 System Suitability Requirements t24 No significant (>0.5%) interfering peaks are present in the injection of Internal Standard Solution. 12.2 The %RSD of the peak area ratio in five consecutive injections of the Working Standard is NMT 2.0%. 12.3 The %RSD of the peak area ratio in all injections of the Working Standard is NMT 3.0%. 12.4 The Tailing Factor for the stigmasterol peak in the injection of the Working Standard is within the range 0.8 — 1.2. 12:5 The USP resolution between the three most abundant phytosterol peaks and any adjacent peak is NLT 1.0. 13.0 Retention Times 13.1 5a-cholestane 7.12 min ee Cholesterol 11.50 min 13.3 Brassicasterol 12.61 min 13.4 Campesterol 14.20 min 13.5 Campestanol 14.42 min 13.6 Stigmasterol 15.10 min [SOP Standard Operating Procedure SOPNo | Rev Page Determination of Phytosterols by GC-FID D-734 1 7 of 9 13.7 Sitosterol 16.91 min 13.8 Sitostanol 17.25 min 13.9 Avenasterol 17.52 min 14.0 Relative Response Factors 14.1. Cholesterol 0.909 14.2 Brassicasterol 1.000 14.3. Campesterol 0.951 14.4 Campestanol 0.925 14.5 Stigmasterol 1.000 14.6 Sitosterol 0.915 14.7 Sitostanol 0.942 14.8 Avenasterol 1.000 15.0 Example Calculations % Label = = x Vera Spl: x TA x RRF x 100 Ry Sample peak area ratio R, Mean Working Standard A peak area ratio (all injections) Wtsta Weight of reference standard used to prepare Working Standard (mg) P Purity of reference standard from the CoA (% w/w) Vsea Volume of Working Standard (mL) [SOP Standard Operating Procedure SOPNo | Rev Page Determination of Phytosterols by GC-FID ele . 8 of 9 Veni Volume of Sample Solution (mL) Splwt Sample weight (mg) FW Theoretical fill/tablet weight (mg, use 1 for raw materials) LA Label amount (mg, use 1 for raw materials) RRF Relative Response Factor 16.0 Example Chromatography 16.1. Blank (Internal Standard Solution) Intl Std Sol 0 471.1 100+ 904 80+ 704 60+ & 504 40+ 304 207 105 : 0 eee 298.1 850.2 093.5 enatseiohC | 2 3 4 5S 6 7 8 9 4 41 «+12 «13 #44 «45 «16 «17 «218 «19~+=«20 Time [min] 16.2 Working Standard 21AS126 0 100+ 907 804 707 607 901.4 880.5 enatselohC 40+ 307 207 107 : 098.1 497.01 — }oretsamgitS +B 6 10 41 12 «113 14 #15 #16 «17 «+18 «+19 ~«20 Time [min] = )sL )ser 4b no oO [SOP Standard Operating Procedure SOP No | Rev Page Determination of Phytosterols by GC-FID Wl u 9 of 9 16.3. Raw Material Sample YH23ZC0818 40+ 5 = n o 35+ 3 De 2 30} is 5 § 6 2 8 25+ & 20) 5 2 3S wn 154 § Ss F ES 5 5 5 2 fe) ns i TERSteOsaes ash mN ok A aoe ; 2 8 4 6 6 7 O8. 8. 40 Mo 42 AS 1418 es 17 618 19) 20 Time [min] 17.0 Revision History | Rev | Date | Description of Changes | CCR # | By | |-----|----------|------------------------|-------|----| | 0 | 01/11/21 | New N/A S. Sassman Add instruction to follow product specific test details if available, | - | - | | 1 | 04/03/24 | add specific sample preparation instructions for gummies, and CC- add example chromatography. | 24-0133 | S. Sassman |