Determination of B-Caryophyllene by GC-FID

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1.0 Purpose 
 
 The purpose of this procedure is to define the method for the quantification and/or
 identification of B-Caryophyllene in raw materials and finished products by GC using flame
 
 ionization detection. 
 
 2.0 Scope 
 
 This procedure applies to the quantification and identification of B-Caryophyllene in raw
 
 materials and finished products by 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 GC — Gas Chromatography 
 
 4.2. FID-— Flame Ionization Detection 
 
 4.3 CofA — Certificate of Analysis 
 
 4.4 BCP -—B-Caryophyllene 
 
 4.5 IPA — Isopropanol 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev Page 
 Determination of B-Caryophyllene by GC/FID D-779 : 2 of5 
 
 5.0 References 
 
 5.1 MV-LAB-19-042, Protocol, Validation of a Method for the Determination of B-
 Caryophyllene and a-Humulene by GC-FID 
 
 6.0 Reagents, Supplies Glassware and Equipment 
 
 6.1 Chemicals: All reagents are GC grade or better. 
 
 61.1.2 BCP 
 
 6.1.2 IPA 
 
 6.2 Compressed Gases 
 
 6.2.1 Hydrogen 
 
 6.2.2 Helium 
 
 6.2.3 Air 
 
 6.2.4 Nitrogen 
 
 6.3. Glassware 
 
 6.3.1 Volumetric glassware as required by standard and sample preparations
 
 6.4 Equipment 
 
 6.4.1 Agilent 7890 GC 
 
 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: 160 °C 
 
 7.4 Detector Temp: 260 °C 
 
 7.5 Equil Time: 0.5 min 
 
 ES Flow Rate: 1.2 mL/min 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev Page 
 Determination of B-Caryophyllene by GC/FID D-779 | 3 of5 
 
 7.7 Run Time: 13.4 min 
 
 7.8 Split ratio: 20:1 
 
 7.9 Septum purge: 2 mL/min 
 
 7.10 Air flow: 400 mL/min 
 
 7.11 Hydrogen flow: 40 mL/min 
 
 7.12 Makeup flow: 25 mL/min (column + makeup = constant) 
 
 7.13. Temperature Ramp: 
 
 Ramp Rate 0 Hold Time 
 (°C/min) temp’) (min) 
 N/A 60 0.3 
 50 130 0 
 1.5 143 
 
 50 245 I 
 8.0 Standard Preparation 
 
 8.1 Use the actual purity from the CofA or the standard certification for the reference
 
 material in calculations. 
 
 8.2 Stock Standard: Use a pipet to transfer about 250 mg of BCP directly into a 100-mL
 
 volumetric flask. Dilute to volume with IPA and mix well. 
 
 8.3. Working Standard: Transfer 2.0-mL of Stock Standard to a 100-mL volumetric flask.
 Dilute to volume with IPA and mix well. 
 
 8.4 Alternatively, a commercially pre-prepared Stock Standard may be used. Alternative
 
 dilution schemes may be used. 
 
 9.0 Sample Preparation 
 
 9.1 The validated linear range of the method is 2 ug/mL — 200 ug/mL. The BCP content of
 
 the sample preparation must be within the linear range. 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev Page 
 Determination of B-Caryophyllene by GC/FID D-779 1 4 of5 
 
 9.2 Place a suitably sized volumetric flask of no less than 25 mL on the balance and press
 Tare. Based on the label claim, weigh no less than 250 mg of sample directly into the
 
 volumetric flask to generate a concentration of BCP that is within the linear range of the
 method. Record the sample weight. Dilute to volume with IPA and mix thoroughly.
 
 Perform further dilutions as required using IPA. 
 
 9.3. For raw materials or finished products being analyzed for the first time using this
 
 method, in-process verification is required to demonstrate specificity and extraction
 efficiency before the method can be implemented. 
 
 10.0 Recommended Sequence 
 
 10.1 Make 2 injections of the Blank (IPA). 
 
 10.2. Make five (5) injections of the Working Standard. 
 
 10.3. Make a single injection of each Sample Preparation. 
 
 10.4 Make a single injection of the Working Standard after every ten (10) sample injections
 and at the end of a run. 
 
 11.0 System Suitability Requirements 
 
 11.1 The%RSD of the first five (5) standard injections is NMT 3.0%. 
 
 11.2 The %RSD of all standard injections is NMT 3%. 
 
 11.3. No significant (>0.5%) interfering peaks are present in the blank (IPA) injection.
 
 12.0 Retention Times 
 
 12.1 The retention time of BCP is about 9.7 min. 
 
 13.0 Calculations 
 
 13.1 Calculation for determining finished product % label or raw material % purity
 
 13.1.1 ‘% assay y = —R Rs x — Vst S d x S Spl E wt x LA 
 
 Ru Sample peak area 
 
 R, Mean working standard peak area 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev Page 
 Determination of B-Caryophyllene by GC/FID ae I aut 
 
 Wteta Weight of reference standard in mg (correct for moisture if required)
 
 Vsta Volume of the standard preparation accounting for dilutions in mL
 
 P Purity of the reference standard in percent 
 
 Splwet Sample weight in mg 
 
 Vspl Volume of the sample preparation accounting for dilutions in mL
 
 LA Label amount in percent (use 100 for raw materials) 
 
 14.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 06/26/19 | New procedure. N/A S. Sassman 07/21/22 Scheduled review: updated logo and format. CC-22-0292 K. Burris | - | - |