Synephrine Determination by HPLC using UV-Vis Spectroscopy

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1.0 Purpose 
 
 The purpose of this procedure is to define a method for the quantitative analysis and/or
 identification of synephrine in complex matrices and raw materials using HPLC and UV/VIS
 
 spectrophotometry. 
 
 2.0 Scope 
 
 This procedure applies to the quantification and identification of synephrine. Some excipients
 
 and dietary ingredients used in finished products can interfere with the analysis of synephrine.
 
 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 QC Laboratory Management to implement this procedure and
 
 to ensure that the procedure is being followed. 
 
 4.0 Definitions 
 
 4.1 Synephrine - 4-[1-hydroxy-2-(methylamino)|epthehnyoll 
 
 4.2 UV/VIS — Ultraviolet and Visible Electromagnetic Spectrums 
 
 4.3 NaCl — Sodium Chloride 
 
 4.4 H3PO4 — Phosphoric Acid 
 
 4.5 CofA — Certificate of Analysis 
 
 4.6 RT — Room Temperature 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Synephrine Determination by HPLC using UV/VIS D-723 4 
 Page 2 of 8 
 Spectroscopy 
 
 4.7 H20 —Millipore water 
 
 48 STD- Standard 
 
 4.9 QC - Quality Control 
 
 5.0 References 
 
 5.1 MV-LAB-13-072, Protocol, Synephrine Determination by HPLC 
 
 6.0 Reagents, Supplies, Glassware and Equipment 
 
 6.1 Reagents: all reagents are HPLC or better. 
 
 6.1.1 Millipore Water 
 
 6.1.2 NaCl 
 
 6.1.3 H3PO4 
 
 6.1.4 Synephrine 
 
 6.2 Supplies and Glassware 
 
 6.2.1 HPLC vials, 12mm X 32mm with screw cap enclosures w/ septa 
 
 6.2.2 1L mobile phase container 
 
 6.2.3 10mL, 50mL, 100mL, 500mL, and 1L volumetric flasks 
 
 6.2.4 200uL, ImL, and 10mL pipette tips 
 
 6.2.5 10mL Plastic luer-lock syringes 
 
 6.2.6 0.2uM or 0.45uM 25mm Nylon syringe filters 
 
 6.2.7 22mL screw cap vials 
 
 6.2.8 1.5mL and 2.0mL micro centrifuge tubes 
 
 

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 Standard Operating Procedure SOP No | Rev 
 Synephrine Determination by HPLC using UV/VIS D-723 4 Page 3 of 8 
 Spectroscopy 
 
 6.2.9 Weigh paper and weigh boats 
 
 6.3. Equipment 
 
 6.3.1 Suitable HPLC system consisting of gradient capable pump, autosampler,
 column compartment, and diode array detector. 
 
 6.3.2 Phenomonex Luna 5uM SCX 100A, 4.6mm X 250mm or equivalent 
 
 6.3.3 Analytical Balance 
 
 6.3.4 Stir Plate 
 
 6.3.5 Wrist Action Shaker 
 
 6.3.6 Vortex 
 
 6.3.7 Sonicator Bath 
 
 6.3.8 200uL, 1ImL, and 10mL Pipettes- adjustable 
 
 6.4 Mobile Phase Preparation 
 
 6.4.1 Mobile Phase A (0.1% H3PO4 in H20) - prepared by adding 1mL H3PQOq to a 1L
 
 volumetric flask then diluting to 1L with H20. 
 
 6.4.2 Mobile Phase B (0.1% H3PO4 1M NaCl) - prepared by adding 1mL H3PQx, and
 
 58.44g NaCl to a 1L volumetric flask then diluting to 1L with H20.
 
 6.4.3 Dissolution buffer- Mobile Phase A 
 
 7.0 #Procedure 
 
 7.1 Standard Preparation 
 
 7.1.1 Use the actual purity from the CofA for synephrine in your calculations. The
 Standard Preparation reflects 100% of the label quantity for synephrine.
 
 

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 Standard Operating Procedure SOP No | Rev 
 Synephrine Determination by HPLC using UV/VIS D-723 4 Page 4 of 8 
 Spectroscopy 
 
 Synephrine standards need to be made fresh daily. 
 
 Example: Synephrine, 99.5% purity 
 
 Prepare 50mL of a 1mg/mL solution 
 
 1) Img/mL X 50mL = 50mg 
 
 2) 50mg/0.995 = 50.3mg 
 
 3) Dissolve 50.3mg up to 50mL = 1.0mg/mL 
 
 7.1.2 All standards are prepared by weighing no less than 50mg then bring up to two
 thirds their final volumes in an appropriate volumetric flask using Mobile Phase
 
 A. Mix on a wrist action shaker for 20 minutes then inspect to ensure complete
 
 dissolution. Sonication for 10 minutes can also be used to assist dissolution.
 Once the standard is fully dissolved, bring standard to final volume before
 
 beginning dilutions. 
 
 7.1.3 Standards and dilutions are prepared using Mobile Phase A. Dilutions can be
 
 made using volumetric flasks or using ImL and 200uL variable pipettes.
 Working standard concentrations will approximate the concentration expected to
 
 be found in the product being tested based on the sample dilution and calculated
 
 from the label. Final dilutions may be prepared directly in HPLC vials.
 
 7.2 Sample Preparation 
 
 7.2.1 The linear range of the method is 0.004 mg/mL — 0.4 mg/mL. All standards and
 samples to be injected must be within the linear range. 
 
 7.2.2 At least 20 dosage units are pooled and ground by mortar and pestle as
 
 necessary. 
 
 7.2.3 Based on the fill weight or tablet weight per dose weigh a portion of the pooled
 dosages to generate an analyte concentration that is within the validated linearity
 
 and solubility range for the analyte being tested. Raw materials samples can be
 
 

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 Standard Operating Procedure SOP No | Rev 
 Synephrine Determination by HPLC using UV/VIS D-723 4 Page 5 of 8 
 Spectroscopy 
 
 prepared using 100% as purity. 
 
 7.2.4 Samples can be dissolved in Mobile Phase A at any volume starting from 25mL.
 
 The volume chosen must be in the solubility range of synephrine. 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. See Attachment 1 for
 validated assay range. 
 
 7.2.5 No less than 50mg of sample can be weighed. 
 
 7.2.6 Dilute the sample to 2/3’s the calculated volume with Mobile Phase A, cap and
 
 shake for 20 minutes to facilitate dissolution. Sonication for 10 minutes can also
 
 be used to assist dissolution. Once the synephrine is completely dissolved, bring
 sample up to volume with Mobile Phase A before beginning dilutions.
 
 Fost If sonication is used allow sample to cool to RT before continuing.
 
 7.2.8 For filtration, using the final large scale diluted sample withdraw up to 10mL
 
 using a 10mL plastic syringe then filter and discard the first 0.5mL of sample
 before collecting. From the collected sample dilute as needed then add 1mL to
 
 an HPLC vial for analysis. 
 
 7.2.9 For centrifugation using the final large scale diluted sample, fill an even number
 
 of 1.5 or 2.0mL micro-centrifuge tubes and pellet insoluble matter for 5 minutes
 
 at 6000rpm. 
 
 7.2.10 For finished products or raw materials being analyzed for the first time using
 
 this method an in process validation is required to demonstrate spectral purity,
 baseline separation of peaks and extraction efficiency as a part of system
 
 suitability before data can be reported using this method. 
 
 7.3 Test Conditions 
 
 7.3.1 Standards, Pure Raw Materials, and Blanks 
 
 

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 Standard Operating Procedure SOP No | Rev 
 Synephrine Determination by HPLC using UV/VIS D-723 4 Page 6 of 8 
 Spectroscopy 
 
 7.3.1.1 Isocratic 55% Mobile Phase A : 45% Mobile Phase B 
 
 7.3.2 Finished Products and Complex Raw Materials 
 
 7.3.2.1 Time (min) %A %B 
 
 0.0 55 45 
 
 8.0 55 45 
 8.01 0 100 
 
 16.0 0 100 
 16.1 55 45 
 
 20.0 55 45 
 
 7.3.3, Column - Luna 5uM SCX 100A, 4.6mm X 250mm or equivalent 
 
 7.3.4 Flow Rate - 1.0 mL/min 
 
 7.3.5 UV Detection - 220nm 
 
 7.3.6 Injection Volume - 20uL 
 
 7.3.7 Temperature - 35°C 
 
 7.3.8 3-D Spectral Range- 190nm to 700nm 
 
 7.4 Recommended Sequence 
 
 7.4.1 Perform at least one injection of Diluent 
 
 7.4.2 Perform five injections of the Working Standard 
 
 7.4.3 Perform a single injection of each Sample Preparation 
 
 7.4.4 Perform a single injection of the Working Standard after every six samples and at
 
 the end of the run. 
 
 7.5 System Suitability 
 
 7.5.1 The %RSD of 5 injections of the Working Standard is NMT 3.0% 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Synephrine Determination by HPLC using UV/VIS D-723 Page 7 of 8 
 Spectroscopy 
 
 7.5.2 The %RSD of all injections of the Working Standard is NMT 3% 
 
 7.5.3. The spectral match is NLT 900. 
 
 7.5.4 The retention time of the sample is within 0.3 min of the standard.
 
 7.6 Calculations 
 
 % assay = R—y.4 x eWteraXPe .. Vs 100 
 Rg Vera SA 
 
 R Sample peak area 
 wa 
 R Mean standard peak area 
 & 
 Wt.., Weight of reference standard in mg 
 
 Vind Volume of the standard preparation accounting for dilutions in mL
 
 BP Purity of the reference standard in decimal format 
 
 SA Sample amount in mg (solids) or mL (liquids) 
 
 Vv, Volume of the sample preparation accounting for dilutions in mL
 sol 
 Ah) 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 | for raw materials 
 
 7.7. Column Wash and Storage 
 
 7.7.1 Rinse and store the column with Water / MeOH (70/30) 
 
 

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 Standard Operating Procedure SOP No | Rev 
 Synephrine Determination by HPLC using UV/VIS D-723 4 | Page 8 of8 
 Spectroscopy 
 
 8.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 2 | 04/01/16 | Biennial review: Updated SOP to current format | 16-0204 | J. Maignan |
| 3 | 09/03/19 | Scheduled review: update SOP with current laboratory practices. Update with current practices. Add bracketing standard as system | 19-0597 | I. Garrett |
| 4 | 03/23/22 | suitability requirement. Remove references to specific HPLC CC- systems. | 22-0119 | S. Sassman |