Determination of Squalene by GC-FID

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1.0 Purpose 
 
 The purpose of this procedure is to define the method for the determination of squalene in raw
 
 materials and finished products by GC-FID. 
 
 2.0 Scope 
 
 This procedure applies to the determination of squalene 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/Analytical Development to keep
 
 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 TMP -— 2,2,4-trimethylpentane 
 
 5.0 References 
 
 5.1 PRTCL-21-0059, Protocol, Validation of an Analytical Method for the Determination of
 Squalene by GC-FID 
 
 
 

[SOP D-100 | Page 2 of 6]

 Standard Operating Procedure SOP No Rev Page 
 Determination of Squalene by GC-FID D-1004 0 2 of 6 
 
 6.0 Supplies 
 
 6.1 Chemicals 
 
 6.1.1 | Squalene reference standard (>95%) 
 
 6.1.2 Methyl tricosanoate reference standard (>97%) 
 
 6.1.3. TMP (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 Disposable glass transfer pipet 
 
 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 um or equivalent
 
 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: 300 °C 
 
 7.4 Detector Temp: 3262C 
 
 7.5 Equilibration Time: 0.5 min 
 
 7.6 Flow Rate: 1.8 mL/min 
 
 tal Run Time: 16 min 
 
 

[SOP D-100 | Page 3 of 6]

 Standard Operating Procedure SOP No Rev Page 
 Determination of Squalene by GC-FID D-1004 0 3 of 6 
 
 7.8 Split ratio: 50:1 
 
 19 Septum purge: 3 mL/min 
 
 7.10 Aur flow: 350 mL/min 
 
 (eel Hydrogen flow: 30 mL/min 
 
 7.12 Makeup flow: 30 mL/min (column + makeup = constant) 
 
 7.13 Injection Volume 1 ul 
 
 7.14 Injection Type Standard 
 
 7.15 Plunger Speed Fast 
 
 7.16 Wash Solvent TMP 
 
 717 Temperature Ramp (Raw Materials and System Suitability Solution) 
 
 Ramp Rate | Temp Hold Time 
 
 (°C/min) (°C) (min) 
 
 N/A 150 0 
 
 10 300 i 
 
 8.0 Internal Standard Solution Preparation 
 
 8.1 Accurately weigh and transfer about 75 mg of methyl tricosanoate into a suitable
 
 container. 
 
 8.2 Add 250 mL of TMP, and mix to dissolve. 
 
 9.0 Standard Preparation 
 
 9.1 Use glass pipets for transfers and dilutions. Squalene may absorb to plastics, and
 
 TMP is not compatible with automatic pipets. 
 
 92 Place a 20-mL scintillation vial on the balance and press TARE. 
 
 Accurately weigh about 30 mg of squalene reference standard into the vial.
 
 9.4 Use small portions of Internal Standard Solution to dissolve and quantitatively transfer
 the reference standard into a 100-mL volumetric flask. 
 
 

[SOP D-100 | Page 4 of 6]

 Standard Operating Procedure SOP No Rev Page 
 Determination of Squalene by GC-FID D-1004 0 4 of 6 
 
 FS) Dilute to volume with Jnternal Standard Solution. 
 
 10.0 Sample Preparation 
 
 10.1 The linear range of the method is 0.1 — 0.5 mg/mL. The content of the sample preparation
 
 must be within the linear range. 
 
 10.2 For finished products, pool at least 10 dosage units. Based on the dosage weight (for
 
 powders), fill weight (for capsules) or tablet weight and the label claim, weigh no less
 than 50 mg of the pooled dosages into a suitably sized volumetric flask to generate an
 
 analyte concentration that is within the linear range. For viscous oils, weigh the sample
 into a vial and use small portions of Internal Standard Solution to quantitatively transfer
 
 the sample to the volumetric flask. Add Internal Standard Solution to about 2/3 of the
 flask volume and sonicate for 5 minutes, shake for 20 minutes, and dilute to volume with
 
 Internal Standard Solution. 
 
 10.3 For raw materials, based on the expected potency, weigh no less than 25 mg of sample
 
 into a suitably sized volumetric flask to generate an analyte concentration that is within
 the validated linear range. For viscous oils, weigh the sample into a vial and use small
 
 portions of Internal Standard Solution to quantitatively transfer the sample to the
 volumetric flask. Add Internal Standard Solution to about 2/3 of the flask volume and
 
 sonicate for 5 minutes. Shake for 20 minutes, and dilute to volume with Jnternal Standard
 
 Solution. 
 
 10.4 If particulates remain in sample preparations, remove them by allowing the particulates
 to settle. 
 
 11.0 Recommended Sequence 
 
 11.1 Make two injections of Internal Standard Solution. 
 
 11.2 Make five injections of Working Standard. 
 
 11.3 Make a single injection of each Sample Preparation. 
 
 11.4 After every six samples and at the end of the run, make a single injection of Working
 Standard. 
 
 

[SOP D-100 | Page 5 of 6]

 Standard Operating Procedure SOP No Rev Page 
 Determination of Squalene by GC-FID D-1004 0 S of 6 
 
 12.0 System Suitability Requirements 
 
 12.1 No significant (>0.5%) interfering peaks are present in the blank (Diluent) injection.
 
 12.2. The %RSD of the peak area ratio in five consecutive injections of the Working Standard
 
 is NMT 5.0%. 
 
 12.3. The %RSD of all injections of the Working Standard is NMT 5%. 
 
 13.0 Retention Times 
 
 13.1 Methyl Tricosanoate (internal standard) ~ 12.2 min 
 
 13.2 Squalene ~ 13.7 min 
 
 14.0 Example Chromatograms 
 
 14.1. Blank (internal Standard Solution) 
 
 INT STD 
 7110 le 
 100- 
 — 
 90- 
 8&o- 
 7O- 
 60- 
 912.21 dtS 
 tnI 
 <x 
 = 50-4 
 40+ 
 30- 
 20- T3Ms 
 eee 
 0 : 2 > ' T ’ t La t i” a t Ls , ' t t 
 4 2 3 4 5 6 7 8 9 40 41 12 13 414 415 16 
 Time {min} 
 14.2 Working Standard 
 VVSTD 
 s 11 e 0 q— 2rc . Sew5z 
 9g0- , 
 80- 
 70-4 
 <_< &o 
 = 50- 
 40- 
 30-4 
 20- 
 10- 
 _ 
 0 4 2 3. 4. 2 5cl 6a >' 8, 9.g 4+0 4r1 1.2 1, 3 1L%4 1.5 126 
 Time [min] 
 

[SOP D-100 | Page 6 of 6]

 Standard Operating Procedure SOP No Rev Page 
 Determination of Squalene by GC-FID D-1004 0 6 of 6 
 
 14.3. Raw Material Sample 
 
 +10 R44129 
 Ko 5 
 
 4 2 3 a § 8 7 et ime 410 41 12 13 44 1S 16 
 
 15.0 Example Calculation 
 
 R, Wtsg XP Vsy1 TMFW 
 % Label (FP) or % Assay (RM) = — x —— Px —— x 100 
 Rs Vsta Splwe LA 
 Ry Sample peak area ratio 
 
 Rg Mean Working Standard peak area ratio (5 injections) 
 
 Wttq Weight of reference standard used to prepare Working Standard (mg)
 
 P Purity of reference standard from the CoA (% w/w) 
 
 Vstq Volume of Working Standard (mL) 
 
 Vsn. | Volume of Sample Solution including dilutions (mL) 
 
 Splwt Sample weight (mg) 
 
 FW _ Theoretical fill/tablet weight (mg, use 1 for raw materials) 
 
 LA Label amount (mg, use | for raw materials) 
 
 16.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 02/16/22 | New procedure. N/A S. Sassman | - | - |