D-720
Caffeine, Theacrine, and Theobromine Determination using HPLC and UV-Vis Detection
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1.0 Purpose The purpose of this procedure is to define a process for the quantitative analysis and/or identification of caffeine, theacrine and theobromine in finished products and raw materials using HPLC with UV/VIS detection 2.0 Scope This procedure applies to the quantification and identification of caffeine, theacrine and theobromine. 3.0 Responsibility 3.1 It is the responsibility of QC and Analytical Chemists to follow this procedure. 3.2 It is the responsibility of Analytical Development and 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 to keep this procedure aligned with current practices. 4.0 Definitions 4.1 Caffeine — 1,3,7 Trimethylxanthine 4.2 Theacrine — 1,3,7,9 tetramethyluric acid 4.3 Theobromine — 3,7-dimethylxanthine 4.4 ACN — Acetonitrile 4.5 | KH2PO«4— Potassium Phosphate Monobasic 4.6 | H3PO4 — Phosphoric Acid 4.7. STD - Standard [SOP D-720 | Page 2 of 10] Standard Operating Procedure SOP No Rev Caffeine, Theacrine, and Theobromine Determination D-720 Page 2 of 10 using HPLC coupled with UV/VIS Spectroscopy 48 QC - Quality Control 5.0 References 5.1 MV-LAB-049, Protocol, Method Validation Caffeine (2011) 5.2. MV-LAB-18-065, Protocol, Method Validation Theacrine 5.3 MV-LAB-18-123, Protocol, Method Validation Theobromine 5.4 RPT-21-0025, Report, D-720 Estimation of Uncertainty 6.0 Reagents, Supplies, Glassware and Equipment 6.1 Reagents: all reagents are HPLC grade or better unless otherwise noted. 6.1.1 H20- Millipore Water (> 18 MQ-cm) 6.1.2 ACN 6.1.3. KH2PO04 6.1.4 H3PO04 6.1.5 Caffeine (traceable reference standard with uncertainty of no more than 0.2%) 6.1.6 Theacrine reference standard 6.1.7 Theobromine reference standard 6.2 Supplies and Glassware 6.2.1 HPLC vials with screw cap enclosures 6.2.2 1L mobile phase container 6.2.3. Volumetric glassware 6.2.4 10mL Plastic luer-lock syringes 6.2.5 0.2uM or 0.45uM 25mm Nylon syringe filters 6.2.6 1.5mL and 2.0mL micro centrifuge tubes 6.2.7. Weigh paper and weigh boats [SOP D-720 | Page 3 of 10] Standard Operating Procedure SOP No Rev Caffeine, Theacrine, and Theobromine Determination D-720 Page 3 of 10 using HPLC coupled with UV/VIS Spectroscopy 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 6.3.3. Wrist Action Shaker 6.3.4 Vortex 6.3.5 Sonicator Bath 6.4 Mobile Phase Preparation 6.4.1 Buffer (0.1M KH2POs in H20, pH 3.2-3.3) 6.4.1.1 Transfer 13.609g of KH2P04 to a 1-L bottle. 6.4.1.2 Add 950mL H20. 6.4.1.3. Adjust the pH to 3.2 - 3.3 using H3PO4. 6.4.1.4 Dilute to volume using H20. 6.4.2. Mobile Phase 6.4.2.1. Transfer 250 mL of ACN to a 1-L bottle. 6.4.2.2 Add 750 mL of Buffer. 6.4.2.3 Filter through 0.45 um membrane. 6.4.3 Diluent 6.4.3.1 Use H20 as the Diluent. 7.0 Procedure 7.1 Standard Preparation 7.1.1. Use the actual purity from the CofA for caffeine, theacrine, or theobromine standard in the calculations. 7.1.2 All standards are prepared in duplicate (Std A and Std B). [SOP D-720 | Page 4 of 10] Standard Operating Procedure SOP No | Rev Caffeine, Theacrine, and Theobromine Determination | 0-720 8 | Page 4 of 10 using HPLC coupled with UV/VIS Spectroscopy 7.1.3 Theacrine and theobromine standard preparations may be scaled down within the working range of the analytical balance. 7.1.4 Transfer 25 mg of reference standard into a 250-mL volumetric flask. Falad Dissolve in and dilute to volume using diluent. For theobromine, sonicate for 20 minutes after diluting to volume to facilitate dissolution. 7.2. Sample Preparation Tau 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. 7.2.2 The sample preparation must be within the linear range of the method: 7.2.2.1 Caffeine: 2-200 mcg/mL 7.2.2.2 Theacrine: 10 — 300 mcg/mL 7.2.2.3 Theobromine: 10 — 100 mcg/mL 723 Theobromine has a limited solubility and should be prepared at a concentration of 0.3mg/mL or less. It should also be sonicated for at least 20 minutes to ensure solubility before further dilutions or analysis. 7.2.4 Some samples, especially those containing extended release or botanically sourced caffeine materials, may require sonication for up to 30 minutes for complete extraction. Alternatively, such samples may be heated at up to 90°C with stirring to extract extended release caffeine. Toe For raw materials: Accurately weigh and transfer a portion 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. Dilute to volume with Diluent, and sonicate for 10 min. 7.2.6 For solid dose finished products: Combine and homogenize no less than 20 dosage units. Based on the label claim and fill weight (for capsules) or tablet [SOP D-720 | Page 5 of 10] Standard Operating Procedure SOP No | Rev Caffeine, Theacrine, and Theobromine Determination | 0-720 8 | Page 5 of 10 using HPLC coupled with UV/VIS Spectroscopy weight per dose, accurately weigh and transfer a portion of the homogenized sample into a suitably sized volumetric flask of no less than 25 mL to generate an analyte concentration that is within the validated linearity range. Dilute to volume with Diluent, and sonicate for 10 min. Tay For liquid dose finished products: Use a TC pipet to transfer no less than 2.0 mL of the product into a suitably sized volumetric flask of no less than 25 mL to generate an analyte concentration that is within the validated linearity range. Wipe the outside of the pipet, and rinse the pipet three times with Diluent collecting the rinses in the volumetric flask. Dilute to volume using Diluent. 7.2.8 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 homogenized sample into a volumetric flask of no less than 25 mL to generate an analyte concentration that is within the validated linearity range. Dilute to volume with Diluent, and sonicate for 10 minutes. 129 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 to bring the analyte concentration into the linear range of measurement. Ensure that the stock sample is equilibrated to room temperature prior to performing further dilution. 7210 The final sample must be filtered or centrifuged before analyzing by HPLC. 7.2.10.1 For filtration: filter a portion through a 0.45um nylon membrane discarding the first 2-3 mL of filtrate before collecting an aliquot for analysis. 7.2.10.2 For centrifugation: centrifuge for about 5 min at about 10,000 rpm. 13 HPLC Test Conditions- Isocratic Tit Time- 7.0 minutes Vive Flow Rate- 1.0 mL/min [SOP D-720 | Page 6 of 10] Standard Operating Procedure SOP No | Rev Caffeine, Theacrine, and Theobromine Determination | 0-729 8 | Page 6 of 10 using HPLC coupled with UV/VIS Spectroscopy 7.3.3. UV detection- 275nm Caffeine and Theobromine; 254nm Theacrine 7.3.4 Injection Volume- 20uL 7.3.5 Column Temperature- 35°C 7.3.6 Column- AcclaimTM 120 C18 S5uM 120A, 4.6mm X 250mm 7.3.7 Recommended 3-D Spectral Range- 200nm to 700nm. 7.4 Approximate Retention Times 7.4.1. Caffeine 3.9 min 7.4.2 Theacrine 3.7 min 7.4.3 Theobromine3.2 min 7.5 Recommended Sequence 7.5.1. Make at least 2 injections of the diluent. 7.5.2 Make five (5) injections of Working Std A. 7.5.3. Make two (2) injections of Working Std B. 7.5.4 Make a single injection of each Sample Preparation. 7.5.5 Make a single injection of Working Std A after every ten (10) sample injections or at the end of a run. 7.6 System Suitability Requirements 7.6.1. The %RSD of the first five (5) standard injections is NMT 3.0% 7.6.2 The % recovery of Working Standard A, using Working Standard B is 97-103%. 7.6.3 The %RSD of all Working Std A injections is NMT 3%. 7.7. Example calculations for determining finished product % label or raw material % purity hdeh 9Y assay =_ R Re x aWtc t V qs ettda’X PE x pV a slla p eLl gk SeS x 1000 Ry Sample peak area [SOP D-720 | Page 7 of 10] Standard Operating Procedure SOP No | Rev Caffeine, Theacrine, and Theobromine Determination | 0-720 8 | Page 7 of 10 using HPLC coupled with UV/VIS Spectroscopy Rg Mean standard peak area Wtstq Weight of reference standard in mg Veta Volume of the standard preparation accounting for dilutions in mL P Purity of the reference standard in decimal format SA Sample amount in mg (solids) or mL (liquids) Vsp1 Volume of the sample preparation accounting for dilutions in mL SS Serving size: Weight of a single dosage unit in mg for tablets and capsules, volume of a single serving from the theoretical formula in mL for liquids, or 1 for raw materials. LA Label amount in mg per dose or 1 for raw materials 7.8 Reporting Results for Caffeine 7.8.1 The expanded uncertainty of the method for caffeine is 2.9% with a coverage factor of 2. 7.8.2 Results should include the expanded uncertainty of the method along with the coverage factor in the following format: 7.8.2.1 105% oflabel claim, U =+ 2.9% k =2 8.0 Example Chromatography and Spectra 8.1 Blank 1200+ 1100+ 1000+ 900+ 800+ 700+ 2 600+ £ 500+ 400+ 300+ 2004 100+ (e) 0.5 1 1.5 2 2.5 3 3.5 4 45 5 5.5 6 6.5 7 Time [min] [SOP D-720 | Page 8 of 10] Standard Operating Procedure SOP No Rev Caffeine, Theacrine, and Theobromine Determination D-720 Page 8 of 10 using HPLC coupled with UV/VIS Spectroscopy 8.2 Caffeine Working Standard 23AS017 A 1100+ 1000+ 900+ 800+ 7004 2 600+ 5004 400+ 300+ 200+ 100+ 43 enieffac 0.5 2.5 3 3.5 4 45 6.5 7 Time [min] 8.3 Caffeine Finished Product Sample 230181 1200 1100+ 1000+ 9004 800+ 700- E=S 650000++ 4004 3004 200+ 100+ 734 gnieffac 792,4} 0.5 2.5 3 3.5 4 45 Time [min] 8.4 Theacrine Working Standard 23AS026 STDA 1400+ 12004 1000+ 2 8004 6004 4004 2004 913.3 644.3 enircaehT 0.5 2.5 3 3.5 4 45 6.5 7 Time [min] 8.5 Theacrine Finished Product Sample 230104 140074 1200+ 100074 > 8004 6007 40074 2004 413 344.3 enircaehT 347.3 \ “ 3.5 4 45 65 7 Time [min] [SOP D-720 | Page 9 of 10] Standard Operating Procedure SOP No | Rev Caffeine, Theacrine, and Theobromine Determination | 0-720 8 | Page 9 of 10 using HPLC coupled with UV/VIS Spectroscopy 8.6 | Theobromine Working Standard 19AS112 0.01mg/ml 2004 é : 1507 4 E £1007 50+ 0 0.5 1 1:5 2 2:5 = 3.5 4 45 § 5:5 6.5 7 Time [min] 8.7. Theobromine Finished Product Sample 200127 Theobro 0.01mg/ml 250; — 2007 2 8 Z 150] = 100- 507 0 0.5 1 1.5 2 2.6 3 35 4 4.5 § 55 6.5 7 Time [min] 8.8 Caffeine UV Spectrum caffeine - 3.921 (2023-03-24 15-39-16-04-00-05.dx) 100 904 80+ \ } \ 704 \ 60+ \ # 450044) / 2\ 230044 of \ 104 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 am 8.9 Theacrine UV Spectrum Theacrine ~ 3.444 (2023-03-15 19-23-06-04-00-03-1003.dx) 100: fos 907 78004+ / /Fea\ # 504 ‘ a \ \ 404 \ 304 \ 20+ 10+ 0 20t 0 220 24t 0 26t 0 28T 0 30T 0 32T 0 e y 34T 0 36T 0 38T 0 40t 0 42T 0 44T 0 46T0 48T 0 50t 0 52T 0 54t0 56T 0 58T0 60T0 nm [SOP D-720 | Page 10 of 10] Standard Operating Procedure SOP No | Rev Caffeine, Theacrine, and Theobromine Determination | 0-720 8 | Page 10 of 10 using HPLC coupled with UV/VIS Spectroscopy 8.10 Theobromine UV Spectrum ‘en Theobromine - 3.34 (2020-02-26 13-41-25-05-00-15.dx) 90+ 80- \ i x 504 2300+4 \SoS \ 107 Me o700 220 -240~«280~«280~-«900+~«S20=«dO.”=«C«00 «88D ~«OO~CO aaa «dO «800-520 +540 860 580 600 9.0 Revision History | Rev | Date | Description of Changes | CCR # | By | |-----|----------|------------------------|-------|----| | 0 | 01/20/12 | New 7 2 Added Attachment 1, adjusted flow rate for sensitivity, added linear range of sensitivity for Amax, simplified calculations, | - | - | | 1 | 03/07/13 | : . - - separated mobile phase components, added spectral analysis, updated format, changed title. Added HPLC, added equation, content adjustments, | - | - | | 4 | 09/03/13 | standardized HPLC format, dita Benne | - | - | | 3 | 01/05/16 | Updated SOP to reflect current method formatting. | 16-0017 | N. Zhang | | 4 | 07/03/18 | Updated SOP and added Theacrine to method. | 18-0180 | J. Maignan | | 5 | 08/10/18 | Updated SOP and added Theobromine to method. Updated sample preparation section to include large serving size . baat instructions. Minor clarifications throughout document. niin Sasouan | 19-0004 | J. Maignan | | 7 | 02/08/22 | Revised to include ISO 17025 requirements CC- Add instruction to follow test details in the product profile for standard and sample preparation, modify sample preparation OR: : ee section to outline specific instructions for different sample Gee2-0N"? er Sascinat types, add example chromatography and spectrum. | 22-0058 | S. Sassman |