Determination of Total Tocotrienols by HPLC-UV

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1.0 Purpose 
 
 This document describes the analytical procedure for the determination of Total Tocotrienols in
 
 raw materials and finished products. Tocotrienols occur as four vitamers: D-Alpha, D-Beta, D-
 Gamma and D-Delta - which differ from one another in the number and position of methyl groups
 
 substituted on the chromanol ring. The D-Beta and D-Gamma forms both possess dimethylated
 
 benzopyrane ring structures, and are generally unable to be separated by reverse phase HPLC.
 However, given that both species possess the same chromophore and molecular weight, D-
 
 Gamma-Tocotrienol will (somewhat arbitrarily - based on its predominate occurrence in the raw
 materials utilized) be used to standardize the quantitation of the sum of D-Gamma and D-Beta
 
 Tocotrienol. Total Tocotrienols is calculated as the sum of D-Alpha, D-Beta, D-Gamma and D-
 
 Delta Tocotrienol. 
 
 2.0 Scope 
 
 This procedure applies to the identification and quantification of Tocotrienols in raw materials
 and finished products. The method lacks specificity regarding the identification and quantitation
 
 of D-Beta-T3 in the presence of D-Gamma-T3. The method is, however, specific for the
 
 identification and quantification D-Alpha-T3 and D-Delta-T3 in the presence of D-Beta-T3 and
 D-Gamma-T3. The method is suitable for its intended purpose, which is the determination of
 
 Total Tocotrienols in raw materials and finished products. This method was validated under
 protocol PRTCL-20-0123 and reported in RPT-20-0056. 
 
 3.0 Responsibility 
 
 3.1 It is the responsibility of QC and Analytical chemists who have verified their ability to
 execute this procedure to follow this procedure. 
 
 
 

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 Standard Operating Procedure SOP No | Rev 
 Determination of Total Tocotrienols by HPLC-UV Dea? : Fased of 10 
 
 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
 
 personnel to keep this procedure current with the associated monographs and laboratory
 practices. 
 
 4.0 Definitions 
 
 4.1 QC — Quality Control 
 
 4.2. AD-— Analytical Development 
 
 4.3. ACN - Acetonitrile 
 
 4.4 EtOH — Ethanol 
 
 4.5 DMSO - Dimethylsulfoxide 
 
 4.6 MeOH - Methanol 
 
 4.7. BHT -—Butylated Hydroxy-Toluene (2,6-di-tert-butyl-4-methylphenol)
 
 4.8 T3- Tocotrienol 
 
 4.9 ACS — American Chemical Society 
 
 4.10 HPLC — High Performance Liquid Chromatography 
 
 4.11 UV-— Ultraviolet Electromagnetic Spectra 
 
 5.0 References 
 
 D.1 PRTCL-20-0123, Protocol, Validation of an Analytical Method for the Determination of
 
 Total Tocotrienols by HPLC-UV 
 
 ae RPT-20-0056, Report, Validation of an Analytical Method for the Determination of Total
 
 Tocotrienols by HPLC-UV 
 
 6.0 Supplies 
 
 6.1 Chemicals — All reagents are ACS grade or better. 
 
 
 

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 6.1.1 ACN 
 
 6.1.2 EtOH 
 
 6.1.3 MeOH 
 
 6.1.4 BHT 
 
 6.1.55 DMSO 
 
 6.1.6 D-Alpha-T3 Reference Standard, 50 mg Vial (Preferred Mass) 
 
 6.1.7 D-Gamma-T3 Reference Standard, 50 mg Vial (Preferred Mass) 
 
 6.1.8 D-Delta-T3 Reference Standard, 50 mg Vial (Preferred Mass) 
 
 6.2 Supplies and Glassware 
 
 6.2.1. Amber HPLC vials, 12mm X 32mm with screw cap enclosures w/ septa
 
 6.2.2 Volumetric glassware (Use Red Glassware!) 
 
 6.2.3. Volumetric pipets and/or adjustable pipettes and tips 
 
 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 
 
 7.0 Procedure 
 
 7.1 Mobile Phase & Diluent Preparation 
 
 7.1.1 Mobile Phase 
 
 7.1.1.1. Mobile Phase A: EtOH 
 
 7.1.1.2 Mobile Phase B: ACN 
 
 7.1.2 Diluent: 100 mg/L BHT in EtOH 
 
 7.1.3 Preparations may be scaled as necessary 
 
 7.2 Standard Preparation 
 
 
 

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 7.2.1 Use red glassware and minimize material exposure to air and light! Prepare
 
 Tocotrienol stock standards at ~1 mg/mL by transferring approximately 50 mg of
 
 each into separate 50 mL volumetric flasks. Add ~25 mL of Diluent and swirl
 thoroughly until completely dissolved. QS to volume with Diluent and mix well.
 
 Note: Tocotrienols are waxy oils that do not lend themselves to simple weigh-offs. As
 
 such, quantitatively transfer the contents of scrupulously clean, pre-weighed vials
 
 by means of careful DMSO (1X) & Diluent (5X) rinses into the 50 mL volumetric
 flasks. Carefully rinse the evacuated vials with MeOH (3X) then dry thoroughly
 
 prior to obtaining the empty vial masses. Calculate the masses of the standards
 transferred to the volumetric flasks by difference. 
 
 7.2.2 Use the D-Gamma-T3 stock as the D-Gamma-T3 working standard (WS-1).
 
 7.2.3. Combine 7 parts of the D-Alpha-T3 stock standard with 3 parts of the D-Delta-
 
 T3 stock standard (WS-2) and mix well. 
 
 7.2.4 Alternate standard preparation is acceptable provided that the final working
 
 standard concentrations are within the linear range listed below.
 
 7.3 Sample Preparation 
 
 7.3.1 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.
 
 7.3.2 Use red glassware and minimize material exposure to air and light! The
 validated linear ranges for the analytical method are 0.575 — 1.341 mg/mL D-
 
 Gamma-T3, 0.426 — 0.994 mg/mL D-Alpha-T3 and 0.166 — 0.386 mg/mL D-
 
 Delta-T3. 
 
 7.3.3 Extract sufficient sample (based on the raw material manufacturer assay value /
 finished product label claim) with Diluent in order to generate analyte
 
 concentrations within their respective validated ranges. 
 
 7.3.4 Samples can be extracted in Diluent at any volume starting from 50mL. The
 volume chosen must be in the solubility range (validated at ~1 mg/mL D-Gamma-
 
 
 

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 D-747 Page 5 of 10 
 Determination of Total Tocotrienols by HPLC-UV 
 T3, ~0.3 mg/mL D-Delta-T3 & ~0.7 mg/mL D-Alpha-T3). To manage large
 
 volumes, the sample can be initially dissolved in a smaller volume that is within
 
 the solubility range and a portion further diluted using Diluent to bring the
 concentration into the linear range. 
 
 7.3.5 Fill the flask to about 50% of the chosen volume with Diluent then swirl
 
 thoroughly until completely dissolved. QS to volume with Diluent and mix well.
 
 74 HPLC Parameters 
 
 7.4.1. Column: Thermo Scientific Acclaim 120 Cis, 4.6 x 250mm, 5um (Or Equiv.)
 
 7.4.2 Column Temperature: 35°C 
 
 7.4.3 Flow rate: 2 mL/min 
 
 7.4.4 Mobile Phase: Gradient 
 
 7.4.4.1 Short Run (Standards) Time, min __%B (ACN) 
 
 0.00 95 
 
 2.00 95 
 
 8.00 85 
 
 8.01 95 
 
 12.00 95 
 
 7.4.4.2 Long Run (Samples) Time, min _%B (ACN) 
 
 0.00 95 
 
 2.00 95 
 
 14.00 75 
 
 17.00 75 
 
 17.10 95 
 
 20.00 95 
 
 
 

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 7.4.5 Wavelength: 295 nm 
 
 7.4.6 Injection Volume: 5 wL 
 
 7.4.7 Run Time: 12 min (Short), 20 min (Long) 
 
 7.4.8 Recommended 3-D Spectral Range (for Identification): 210 nm - 550 nm
 
 7.5 | Recommended Sequence 
 
 7.5.1. Make at least two (2) injections of the Diluent. 
 
 7.5.2 Make five (5) injections of the Working Standard 1 (WS-1). 
 
 7.5.3 Make five (5) injections of Working Standard 2 (WS-2). 
 
 7.5.4 Make a single injection of each Sample Preparation. 
 
 7.5.5 Make a single injection of Working Standard 1 (WS-1) after every ten (10)
 
 samples and at the end of the run. 
 
 7.5.6 Make a single injection of Working Standard 2 (WS-2) after every ten (10)
 samples and at the end of the run. 
 
 7.6 System Suitability Requirements 
 
 7.6.1 The %RSD of five (5) consecutive standard injections is NMT 3.0%.
 
 7.6.2 The %RSD of all standard injections is NMT 3%. 
 
 7.6.3 If present, any interference in the diluent should be subtracted out of the sample
 
 and standard peak areas. 
 
 7.7 Example Calculations: 
 
 7.7.1 %D-—(Gamma + Beta) —T3 = Rp—-2—- GammatBeta—=t s y WteA'tstdqTM XP x =ss _x 2V.! x 100
 Rp-Gamma-T3 Vsta SA LA 
 Rp-(Gamma+Beta)-T3 Sample D-(Gammat+Beta)-T3 Peak Area 
 
 Rp-camma-t3. Average D-Gamma-T3 Peak Area for the Standard Injections
 
 Wtsig Weight of the D-Gamma-T3 reference standard 
 
 Veta Volume of the standard preparation accounting for dilutions in mL
 
 
 

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 Standard Operating Procedure SOP No | Rev 
 Determination of Total Tocotrienols by HPLC-UV 1a Nae a 
 
 P Purity of the reference standard in decimal format 
 
 SS Serving Size (Enter “1” for Raw Materials) 
 
 SA Sample Amount 
 
 Vept Volume of the sample preparation accounting for dilutions in mL
 
 LA Label Amount (Enter “1” for Raw Materials) 
 
 Todvax % D — Alpha —T3 = = Rp- sea. x WtestqxP ess x Vspl xX 100 
 D—Alpha-—T3(Std) std 
 
 Rp-aipha-r3 Sample D-Alpha-T3 Peak Area 
 
 Rp-alpha-Tacsta) Average D-Alpha-T3 Peak Area for the Standard Injections
 
 Wtsta Weight of the D-Alpha-T3 reference standard 
 
 Vista Volume of the standard preparation accounting for dilutions in mL
 
 P Purity of the reference standard in decimal format 
 
 SS Serving Size (Enter “1” for Raw Materials) 
 
 SA Sample Amount 
 Vspt Volume of the sample preparation accounting for dilutions in mL
 
 LA Label Amount (Enter “1” for Raw Materials) 
 
 % D — Delta—T3 = oo x eat x Sx “2! x 100 
7.7.3 Rp-alpha-rz Sample D-Delta-T3 Peak Area
 
 Rp-alpha-Tacsta) Average D-Delta-T3 Peak Area for the Standard Injections
 
 Wtsta Weight of the D-Delta-T3 reference standard 
 
 Veg Volume of the standard preparation accounting for dilutions in mL
 
 : Purity of the reference standard in decimal format 
 
 SS Serving Size (Enter “1” for Raw Materials) 
 
 
 

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 Standard Operating Procedure SOPNo | Rev 
 Bi ; D-747 | Page 8 of 10 
 Determination of Total Tocotrienols by HPLC-UV 
 
 SA Sample Amount 
 
 Vspt Volume of the sample preparation accounting for dilutions in mL
 
 LA Label Amount (Enter “1” for Raw Materials) 
 
 7.7.4 Total Tocotrienols = % D-(GammatBeta)-T3 + % D-Alpha-T3 +% D-Delta-T3
 
 7.8 Column Wash and Storage 
 
 7.8.1 Wash and store the column using 100% ACN. 
 
 8.0 Chromatograms 
 
 8.1 Typical Diluent Chromatogram 
 
 seal 
 
 JN 
 
 min} 
 
 
 

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 Standard Operating Procedure SOP No Rev 
 D-747 Page 9 of 10 
 Determination of Total Tocotrienols by HPLC-UV 
 
 8.2 Typical Working Standard Chromatograms 
 
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 I A 41) 4859263 | updo sma Pasi ai oecess ecre 
 Vv t t Tr TT bea aah 
 i 
 
 pb Alpho-T3 
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 + Data-T3 - 4.617 (| 
 
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 | [| | | 
 \\ j Fy | _ 2 SampeReazleyas 626-660 
 at T port ae os 3 
 
 
 

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 Standard Operating Procedure SOP No | Rev 
 Determination of Total Tocotrienols by HPLC-UV Dae 1 Hane tof 10 
 
 8.3. Typical Raw Material Chromatogram 
 
 neg} TocotrienotV¥alidation 12-17-20819 
 2 mA 
 
 8.4 ypical Finished Product Chromatogram 
 cobrignol Validation 12-17-20 812 
 
 10-6510 
 aC OAC 
 
 9.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 04/14/21 | New N/A C. Perry Added option for alternate standard preparation. Added | - | - |
| 1 | 05/22/23 | instruction to follow product specific test details. Added CC- requirement to run bracketing standard. Updated logo. | 23-0253 | S. Sassman |