Determination of Tributyrin by GC-FID

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1.0 Purpose 
 
 The purpose of this procedure is to define the method for the determination of tributyrin in raw
 
 materials and finished products by GC-FID. 
 
 2.0 Scope 
 
 This procedure applies to the determination of tributyrin in raw materials and finished products
 
 in the QC Laboratory at Ion Labs. 
 
 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 
 
 4.2 GC-—Gas Chromatography 
 
 4.3. FID —Flame Ionization Detection 
 
 44 CoA-—Certificate of Analysis 
 
 4.5 TBT —Tributyrin 
 
 4.66 DGM-— Diethylene glycol methyl ether 
 
 4.7 THF — Tetrahydrofuran 
 
 
 

[SOP 

 Standard Operating Procedure SOPNo | Rev 
 Page 
 Determination of Tributyrin by GC-FID D-729 2 2 of8 
 
 5.0 References 
 
 5.1 | PRTCL-20-0041, Protocol, Validation of an Analytical Method for the Determination of
 Tributyrin by GC-FID 
 
 5.2 RPT-21-0024, Report, D-729 Estimation of Uncertainty 
 
 5.3. D-793, SOP, Cryogenic Grinding of Chewable Gels 
 
 6.0 Supplies 
 
 6.1 Chemicals 
 
 6.1.1 TBT Reference Standard (>97%) 
 
 6.1.2 DGME (ACS reagent grade or better) 
 
 6.1.3 THF (Not stabilized, GC 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 1.0-mL gas-tight syringe 
 
 6.3.2 Volumetric glassware as required for standard and sample preparation
 
 6.4 Equipment 
 
 6.4.1 Agilent 7890 GC with FID detector 
 
 6.4.2 Analytical Balance 
 
 7.0 GC Conditions 
 
 7.1 Column: Agilent HP-5, 30 m x 0.32 mm x 0.25 ym or equivalent 
 
 
 

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 7.2 Inlet Liner: Restek, 4.0 mm ID x 6.3 mm OD x 78.5 mm length straight liner with glass
 
 wool or equivalent 
 
 7.3. Injector Temp: 220 °C 
 
 7.4 Detector Temp: 280 °C 
 
 7.5 Equilibration Time: 0.5 min 
 
 7.6 Flow Rate: 2 mL/min 
 
 7.7 Run Time: 11 min 
 
 7.8 Split ratio: 10:1 
 
 7.9 Septum purge: 3 mL/min 
 
 7.10 Air flow: 350 mL/min 
 
 7.11. Hydrogen flow: 30 mL/min 
 
 7.12 Makeup flow: 30 mL/min (column + makeup = constant) 
 
 7.13 Injection Volume: 1 pL 
 
 7.14 Injection Type: Standard 
 
 7.15 Plunger Speed: Fast 
 
 7.16 Wash Solvent: THF 
 
 7.17 Temperature Ramp (Raw Materials and System Suitability Solution) 
 
 R ( a ° m C/ p m R i a n) t e Temp ( — °C) Hold Ti . me (mi : n)
 
 N/A 50 1 
 20 250 
 
 8.0 Diluent Preparation 
 
 8.1 For 1 L: Mix 1.0 mL of DGME with 1000 mL of THF. 
 
 9.0 Standard Preparation 
 
 9.1 Use the actual purity from the CoA for the reference material in calculations.
 
 
 

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 Standard Operating Procedure SOP No | Rev Page 
 
 Determination of Tributyrin by GC-FID D-729 2 4 of 8 
 
 92 Working Standard: Draw about 0.05 mL of reference standard into a 1-mL gas-tight
 
 syringe, and wipe the outside of the needle with a Kimwipe. Place a 25-mL volumetric
 
 flask on a balance, and press Tare. Transfer about 25 mg of reference standard (about 5
 drops) into the flask, taking care to direct the standard directly to the bottom of the flask.
 
 Record the weight of standard added. Dilute to volume with Diluent, and mix well. The
 standard preparation may be scaled up as desired. Prepare a second standard (Std B) as
 
 a standard recovery check. 
 
 10.0 Sample Preparation 
 
 10.1 Specific sample testing details are provided in each products profile. If a specific testing
 
 details section is not available, then follow preparation procedure as described below,
 
 maintaining concentration within the linear range listed below. 
 
 10.2 The validated linear range of the method is 0.8 — 1.2 mg/mL. The content of the sample
 preparation must be within the linear range. Flask size used cannot be less than 50mL.
 
 10.3 Ensure that the sample is thoroughly homogenized prior to weighing. It is not
 
 recommended to use a mortar and pestle to grind the sample because tributyrin is a
 viscous liquid. For most solid and liquid samples, transfer the entire sample to a container
 
 of suitable size so that the sample takes up less than half of the container volume, and
 homogenize by turning end-over-end for at least one minute. For gummies, homogenize
 
 as outlined in D-793 Cryogenic Grinding of Chewable Gels. 
 
 10.4 The use of glass pipets is recommended for transfers and dilutions. TBT may absorb to
 
 plastics, and THF is not compatible with automatic pipets. 
 
 10.5 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 volume, and sonicate for 10 min. 
 
 10.6 For solid or liquid dose finished products: Combine and homogenize no less than ten
 dosage units. Based on the label claim and fill weight (capsules), serving size (powders)
 
 or tablet weight per dose, weigh no less than 50 mg of the pooled dosages into a suitably
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev Page 
 
 Determination of Tributyrin by GC-FID D-729 2 5 of8 
 
 sized volumetric flask of no less than 25 mL to generate an analyte concentration that is
 
 within the validated linear range. Add Diluent to volume, and sonicate for 10 min.
 
 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 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 volume, and sonicate for 10 min. 
 
 10.8 To manage large volumes, the sample can be initially prepared at a higher concentration
 
 and further diluted into the linear range. Equilibrate the stock sample to room
 temperature prior to performing further dilution. 
 
 10.9 If particulates remain in the final sample preparation, a portion may be centrifuged at
 
 10,000 rpm for 5 min prior to HPLC analysis. Alternatively, the sample may be filtered
 
 through a 0.45 um membrane discarding the first 3 - 4 mL. 
 
 11.0 Recommended Sequence 
 
 11.1 Make two injections of Diluent 
 
 11.2 Make five injections of Working Standard A 
 
 11.3 Make two injections of Working Standard B 
 
 11.4 Make a single injection of each Sample Preparation 
 
11.5 Make a single injection of Working Standard A after every six samples and at the end of

 the run 
 
 12.0 System Suitability Requirements 
 
 12.1 Samples and Standards should be prepared and analyzed on the same day.
 
 12.2 No significant (>0.5%) interfering peaks are present in the blank (Diluent) injection.
 
 The %RSD of the peak area ratio in five consecutive injections of the Working Standard
12.3 is NMT 2.0%.

 
 

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 Standard Operating Procedure SOP No | Rev Page 
 Determination of Tributyrin by GC-FID D-729 2 6 of 8 
 
 12.4 The % recovery of Working Standard A, using Working Standard B is 98-102%.
 
 12.5 The %RSD of the peak area ratio for all injections of Working Standard A is NMT 2%.
 
 13.0 Retention Times 
 
 13.1 DGME = 4.0 min (elutes as a doublet, use the second peak) 
 
 13.2: TBI =9.9 min 
 
 14.0 Example Calculations 
 
 Ry, Wty XP. V, FW 
 % Assay = x as x se x 5z x 100 
 
 Ry Sample peak area ratio 
 
 Re Mean Working Standard A peak area ratio (5 injections) 
 
 Wtsta Weight of reference standard used to prepare Working Standard A (mg)
 
 P Purity of reference standard from the CoA (% w/w) 
 
 Veta Volume of Working Standard A (mL) 
 
 Vept Volume of Sample Solution (mL) 
 
 Splwe Sample weight (mg) 
 
 FW Theoretical fill/tablet weight (mg, use 1 for raw materials)
 
 LA Label amount (mg, use 1 for raw materials) 
 
 15.0 Reporting Results 
 
 15.1. The expanded uncertainty for Tributyrin is 1.0%, with a coverage factor of 2
 
 15.2 Report results along with the expanded uncertainty and coverage factor in the following
 format: 
 
 15.2.1 102% of Label Claim, U=+ 1.0% k=2 
 
 
 

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 Standard Operating Procedure SOPNo | Rev Page 
 
 Determination of Tributyrin by GC-FID D-729 2 7 of8 
 
 16.0 Example Chromatography 
 
 16.1 Blank (Internal Standard Solution) 
 Diluent 
 550+ = os 
 5004 ~= m ( h $8 
 450+ 
 400+ 
 350+ 
 <= 300-4 
 250+ «© 
 2004 < 
 1 1 0 5 0 0 + + 7 LIS5VSsee © = ld e888 
 o5 1 #15 2 25 3 35 4 45 5 55 6 65 75 8 85 9 95 10 105 11 
 Time [min] 
 
 16.2 Standard 
 
 22AS101 
 + 
 ~ w 
 1000+ 
 
 800+ — w 
 << foe] 
 600+ Baa 
 foe] 
 400+ N 
 a Hesee 
 wayi 
 3 2 8 28 
 Pay 
 es 8 
 S 
 0 ee ee ~ © gr oe i 
 05 1 #15 2 25 3 35 4 45 5 55 6 65 75 8 85 9 95 10 105 11 
 Time [min] 
 16.3 Finished Product Sample 
 230106-06 
 1400+ ¥ or 
 = Se 
 12007 
 10004 
 a 800+ mets w 
 600+ Bo. 
 roe) 
 4 a 0 i 0 i 7 Regge S <] B 8888 & g oe. ..81 2s S e) 3 = 
 é Gets Soe? + g og Ss ay 
 o5 1 #415 2 25 3 35 4 45 5 55 6 65 75 8 85 9 95 10 105 11 
 Time [min] 
 

[SOP 

 Standard Operating Procedure SOP No | Rev Page 
 Determination of Tributyrin by GC-FID D-729 2 8 of 8 
 
 17.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 05/29/20 | New Document N/A S. Sassman | - | - |
| 1 | 11/04/21 | Updated to reflect ISO 17025:2017 requirements CC- Add instruction to refer to test details for product specific sample | 21-0407 | J. Sassman |
| 2 | 05/16/23 | . PreP> add specific sample prep for different dosage forms, add CC- example chromatography, add reference to cryogenic grinding SOP for gummies, | 23-0227 | S. Sassman |