D-730
Determination of EPA and DHA by GC-FID
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1.0 Purpose The purpose of this procedure is to define the method for the determination of Eicosapentaenoic Acid (EPA), Docosahexaenoic Acid (DHA), and total Omega 3 fatty acids in raw materials and finished products by GC-FID. 2.0 Scope This procedure applies to the determination of EPA, DHA, and total Omega 3 fatty acids in raw materials and finished products by GC-FID 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 Management 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 4.4 CoA — Certificate of Analysis 4.5 EPA —Eicosapentaenoic Acid 4.6 DHA — Docosahexaenoic Acid [SOP Standard Operating Procedure SOP No | Rev Page Determination of EPA and DHA by GC-FID mane 1 2 of 11 5.0 References 5.1 PRTCL-20-0076, Protocol, Validation of an Analytical Method for the Determination of EPA and DHA by GC-FID 6.0 Supplies 6.1 Chemicals 6.1.1 Fish oil reference standard 6.1.2 Docosahexaenoic acid ethyl ester reference standard 6.1.3 Eicosapentaenoic acid ethyl ester reference standard 6.1.4 Methy] tricosanoate Docosahexaenoic acid methyl ester Tetracos-15-enoic acid (nervonic acid) methyl ester Boron trichloride (12%) in methanol Butylated hydroxytoluene (2,6-di-tert-butyl-4-methylphenol) 6.1.9 Sodium Hydroxide 6.1.10 Sodium Chloride 6.1.11 Sodium Sulfate Anhydrous 6.1.12 2,2,4-Trimethylpentane 6.1.13 Methanol 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 ] [SOP Standard Operating Procedure SOP No Rev Page Determination of EPA and DHA by GC-FID D-730 1 3 of 11 6.4 Equipment 6.4.1] Agilent 7890 GC with FID detector 6.4.2 Analytical Balance 7.0 GC Conditions Fl Column: Supelcowax 10, 30 m x 0.25 mm x 0.25 lum 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: ZI0 26 7.4 Detector Temp: 270 °C 7.5 Equilibration Time: 0.5 min 7.6 Flow Rate: 1 mL/min 7.7 Run Time: 35.3 min 7.8 Split ratio: 600:1 7.9 Septum purge: Off 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 0.3 wL 7.14 Injection Type Standard 7.15 Plunger Speed Fast 7.16 Wash Solvent 2,2,4-trimethylpentane vam Uy Temperature Ramp (Raw Materials and System Suitability Solution) Ramp Rate (°C/min) Temp (°C) Hold Time (min) N/A 170 2 3 240 10 [SOP Standard Operating Procedure SOP No | Rev Page Determination of EPA and DHA by GC-FID BEIM I 4 of 11 8.0 Antioxidant Solution 8.1 Accurately weigh and transfer about 10 mg of butylated hydroxytoluene to a suitable container. 8.2 Add 200 mL of 2,2,4-trimethylpentane 8.3 Mix well to dissolve. 9.0 Internal Standard Solution 9.1 Accurately weigh and transfer 350 mg of methyl tricosanoate to a 50-mL volumetric flask. 9.2 Dissolve in and dilute to volume with Antioxidant Solution. 10.0 Sodium Hydroxide Solution 10.1. Transfer 1 g of sodium hydroxide to a suitable container. 10.2. Add 50 mL of methanol, and sonicate to dissolve. 11.0 Saturated Sodium Chloride Solution 11.1 Transfer 20 g of sodium chloride to a suitable container. 11.2 Add 50 mL of H20. 11.3. Shake for at least 10 min. 11.4 Allow particulates to settle before use. 12.0 Sample Solution A Note: Do not use plastic pipets or containers for sample preparation. DHA, EPA and their methyl esters are extremely lipophilic, and will absorb strongly to plastic surfaces. 12.1 According to Table 1, transfer and accurately weigh the required amount of sample to a 10-mL volumetric flask. 12.2 Dissolve in and dilute to volume with Antioxidant Solution. T [SOP Standard Operating Procedure SOP No Rev Page Determination of EPA and DHA by GC-FID D-730 1 5 of 11 L223 If the test sample contains EPA and DHA in ethyl ester form, proceed to step 0. If the test sample contains EPA and DHA as triglycerides or the form is unknown, continue with step 12.4. 12.4 Transfer 2.0 mL of the resulting solution to a 10-mL headspace vial. 12.5 Evaporate the solvent under a gentle stream of nitrogen. 12.6 Add 1.5 mL of Sodium Hydroxide Solution. 12.7 Purge the container with argon or nitrogen gas, cap tightly, and mix. 12.8 Heat in a boiling water bath for 7 min, then allow to cool. 12.9 Remove the cap, and add 2.0 mL of boron trichloride in methanol. 12.10 Purge the container with argon or nitrogen gas, cap tightly, and mix. L2elel Heat in a boiling water batch for 30 min. 12.12 Allow to cool to room temperature, and remove the cap. 12.13 Add 5.0 mL of Saturated Sodium Chloride Solution. 12.14 Add 1.0 mL of 2,2,4-trimethylpentane, purge with argon or nitrogen gas, and cap tightly. 12.15 Mix on a vortex mixer or shake vigorously for at least 60 sec. 12.16 Allow the upper layer to become clear, remove the cap, and transfer the upper layer to a small glass vial. 12.17 Add another 1.0 mL of 2,2,4-trimethylpentane to the remaining bottom layer. 12.18 Purge the container with argon or nitrogen gas, and cap tightly. 12.19 Mix on a vortex mixer or shake vigorously for at least 15 sec. 12.20 Allow the upper layer to become clear, remove the cap, and combine the upper layer with the upper layer from the first extraction. 12.21 Add 1.0 mL of H20 to the combined extracts, purge the container with argon or nitrogen gas, cap, and shake well. L222 Allow the layers to separate, remove the cap, and discard the bottom layer. [SOP Standard Operating Procedure SOP No | Rev Page Determination of EPA and DHA by GC-FID ve I 6 of 11 1223 Add another 1.0 mL of HO to the combined extracts, purge the container with argon or nitrogen gas, cap, and shake well. 12.24 Allow the layers to separate, remove the cap, and discard the bottom layer. i229 Add 0.5 g of anhydrous sodium sulfate, and swirl gently for at least 15 seconds. 12.26 Transfer the solution to a 2-mL GC vial for analysis. Table 1: Sample Weight Required for EPA + DHA Determination Sum of EPA + DHA Sample Weight 30% - 50% 0.4¢-0.5¢ 50% - 70% 0.32 >70% 0:25 ¢ 13.0 Sample Solution B 13.1 According to Table 1, transfer and accurately weigh the required amount of sample to a 10-mL volumetric flask. 13.2 Dissolve in and dilute to volume with Internal Standard Solution. 13.3. If the test sample contains EPA and DHA in ethyl ester form, proceed to step 14.0. 13.4 Proceed as directed in steps 12.4 - 12.25. 14.0 Standard Solution A 14.1 Accurately weigh and transfer about 60 mg of DHA reference standard to a 10-ml. volumetric flask. 14.2 Dissolve in and dilute to volume with Jnternal Standard Solution. 14.3 If the test sample contains EPA and DHA in ethy] ester form, proceed to step 15.0. 14.4 If the test sample contains EPA and DHA in triglyceride form, proceed as directed in steps 12.4 - 12.25. 13.0 Standard Solution B ote| Accurately weigh and transfer about 90 mg of EPA reference standard to a 10-mL volumetric flask. 1:2 Dissolve in and dilute to volume with Jnternal Standard Solution. [SOP Standard Operating Procedure SOP No | Rey Page Determination of EPA and DHA by GC-FID laa I 7 of 11 15.3 If the test sample contains EPA and DHA in ethyl ester form, proceed to step Error! Reference source not found.. 15.4 If the test sample contains EPA and DHA in triglyceride form, proceed as directed in steps 12.4 - 12.25, 16.0 Standard Solution C 16.1 Accurately weigh and transfer 300 mg of Fish Oil reference standard to a 10-mL volumetric flask. 16.2 Dissolve in and dilute to volume with Antioxidant Solution. 16.3 Proceed as directed in steps 12.4 - 12.25. 17.0 System Suitability Solution Note: This solution is only required if the test sample is in triglyceride form and only if tetracos- 15-enoic acid methyl ester is not clearly observed in the chromatogram obtained from Test Solution 2. 17.1 Accurately weigh and transfer about 5.0 mg of tetracos-15-enoic acid (nervonic acid) methyl ester to a glass scintillation vial. 17.2 Add 10.0 mL of Antioxidant Solution, and swirl to dissolve. 17.3 Transfer about 55 mg of docosahexaenoic acid methy] ester to another glass scintillation vial. 17.4 Using a 20-mL glass pipet, transfer the solution containing nervonic acid methy] ester to the scintillation vial containing docosahexaenoic acid methy] ester. 17.5 Mix to dissolve. 18.0 Recommended Sequence 18.1 Make at least two injections of Internal Standard Solution. 18.2 Make one injection of Sample Solution A. 18.3 Make one injection of Sample Solution B. 18.4 Make one injection of Standard Solution A. [SOP Standard Operating Procedure SOPNo | Rev Page Determination of EPA and DHA by GC-FID Del I 8 of 11 18.5 | Make one injection of Standard Solution B. 18.6 Make one injection of Standard Solution C. 18.7. Make one injection of System Suitability Solution. 19.0 System Suitability Requirements 19.1 Resolution Note: Resolution is only required if the test sample is in triglyceride form. 19.1.1 From the injection of Sample Solution B or System Suitability Solution. £912 The resolution between the docosahexaenoic acid methyl ester and tetracos-15- enoic acid methyl ester is NLT 1.0. 20.0 Peak Identification 20.1 Approximate retention times for the acid methyl esters are given below. Use the injection of Standard Solution C, along with the certificate of analysis for the fish oil reference standard, to identify fatty acid ester peaks. 20.2 Use the injections of Sample Solution A and Sample Solution B to identify the internal standard peak. It should be the only peak present in Sample Solution B that is not present in Sample Solution A. Analyte Ret Time (min) C16:0 (Palmitic) 11.91 C18:0 (Stearic) 16.50 C18:3 n-3 19.76 C18:4 n-3 20.55 C20:0 (Arachidic) 21.34 C20:4 n-3 25.36 C20:5 n-3 (EPA) 25.98 C22:0 (Behenic) 26.16 C23:0 (Internal Std) 28.87 C21:5 n-3 28.93 C22:5 n-3 S22 C24:1 n-9 (Nervonic) 33.19 C22:6 n-3 (DHA) 33:39 [SOP Standard Operating Procedure SOPNo | Rev Page Determination of EPA and DHA by GC-FID vet I 9 of 11 21.0 Example Chromatograms 21.1 Internal Standard Solution INSTD STOLN 25- = ek. 104 es 5, — $3 ae =) ° : . 2 4 6 8 10 12 14 #16 18 20 22 24 26 28 a2: :Ba Time [min] 21.2 Standard Solution A STDA 25, |S bg ea 20: < On 10- = ees ee 0 — ae 2 4 6 8 10 12 14 16 18 20 22 24 26 28 #30 32 34 Time [min] 21.3. Standard Solution B STDB 25; & <5 20- Bs < 15 10- : ol. | rg a pee in 2 4 6 8 10 12 14 #16 «18 «20 22 «24 «26 ©«6280~«0302~=«32+~«éAS Time [min] 21.4 Standard Solution C STDC 257 20+ 15> Ap 10- etatimlaP lyhteM etaraetS lyhte APE DTS TNI> 3-n 5:220 » | M iu 3-n 3:81C 3-n 4:81C si etadihcarA !yhteM 3-n 4:02C - > dica eee a A BB4G JN - 12 14 16 18 20. 22 26 28 30 Time [min] .OW hN w sD )yot o ] [SOP Standard Operating Procedure SOP No Rev Page Determination of EPA and DHA by GC-FID oy I 10 of 11 21.5. Sample Solution A R45124-A 254 : z 20- > g qe 8 : a2 Olesen = a 2 BB IO 12 a AB 4 20 22 a 8. 98 0: ba ga 0 Time [min] 21.6 Sample Solution B R45124-B 25- : a 6B g = < shee : 3 é . f| 2 33 i 104 6 ois N 5 bs © O 3 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 «234 «36 Time [min] 21.7 System Suitability Solution SYS SUITB 251 20- 8 10- B ae aD 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 «36 Time [min} 22.0 Example Calculations 22.1 Ifthe sample contains DHA and EPA in triglyceride form: EPA or DHA (mg) fe x ist MP EW XM or mg) = Rst X [(% + Rp) — (1g = Ra)] Wty T+ Peak area of internal standard in Standard Solution A or B Ret Peak area for EPA or DHA in Standard Solution A or B Tp Peak area for internal standard in Sample Solution B ] [SOP Standard Operating Procedure SOP No | Rey Page Determination of EPA and DHA by GC-FID pat . 11 of 11 R, Peak area of EPA or DHA in Sample Solution B te Peak area of peak at the ret time of internal standard in Sample SD-730olution Ra Peak area of EPA or DHA in Sample Solution A Wt; | Weight of reference standard used to prepare Standard Solution A or B (mg) P Purity of reference standard from the CoA (% w/w) Wt, Weight of sample used to prepare Sample Solution B (mg) FW Theoretical fill/tablet weight (mg) M Factor to express as free fatty acids (EPA=0.915 and DHA=0.921) 22.2 If the sample contains EPA and DHA in ethyl ester form, calculate as outlined in section 22.1, but with M = 1.0. 22.3, To calculate Total Omega-3 Fatty Acids Total (mg) = EPA + DHA + [A,_3 X (EPA + DHA) + (Agpa + Apya)] EPA = content of EPA from Section 22.1 or 22.2 DHA = content of DHA from Section 22.1 or 22.2 Ay-3 = sum of the peak areas for C18:3 n-3, C18:4 n-3, C20:4 n-3, C21:5 n-3, and C22:5 n-3 in the chromatogram of Sample Solution A. Agpa = area of the EPA peak in the chromatogram of Sample Solution A. Apna = area of the DHA peak in the chromatogram of Sample Solution A 23.0 Revision History | Rev | Date | Description of Changes | CCR # | By | |-----|----------|------------------------|-------|----| | 0 | 09/17/20 | New N/A S. Sassman Remove requirement for System Suitability A (theoretical area | - | - | | 1 | 04/08/22 | percentages), update to reflect current lab practices, add example CC- chromatography. | 22-0166 | S. Sassman |