Yohimbine Alkaloid Determination by HPLC and UV-Vis Spectroscopy

Original Document

Scanned document (image-only PDF)

Extracted Text

Searchable text extracted from PDF

1.0 Purpose 
 
 The purpose of this procedure is to define the method for the quantitation and/or identification
 
 of individual yohimbe alkaloids in raw materials and finished products using HPLC coupled with
 UV/VIS spectrophotometry. 
 
 2.0 Scope 
 
 This procedure applies to the identification and quantitation of yohimbe alkaloids in raw
 materials and finished products in the QC laboratory at Ion Labs. Yohimbe alkaloids are
 
 determined as Yohimbine equivalents by comparison against a Yohimbine HCl reference
 
 standard without regard to their individual relative response factors. 
 
 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 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 UV/VIS — Ultraviolet and Visible Electromagnetic Spectrums 
 
 4.2 H20 — Water 
 
 4.3 QC — Quality Control 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Yohimbine Alkaloid Determination by HPLC and D-750 3 Page 2 of 9 
 UV/VIS Spectroscopy 
 
 5.0 References 
 
 5.1 MV-LAB-15-043, Protocol, Total Yohimbine Alkaloids Determination using HPLC
 
 coupled with UV/VIS and Mass Spectroscopy. 
 
 5.2. D-793, SOP, Cryogenic Grinding of Chewable Gels 
 
 6.0 Procedure 
 
 6.1 Chemicals: All reagents are HPLC grade or better. 
 
 6.1.1 H20 © 18.2 MQ-cm) 
 
 6.1.2 Triethylamine 
 
 6.1.3. Methanol 
 
 6.1.4 Yohimbine HCl reference standard 
 
 6.2 Glassware 
 
 6.2.1 HPLC vials, 12mm x 32mm with screw cap enclosures with septa
 
 6.2.2 Scintillation vials 
 
 6.2.3. Mobile phase containers 
 
 6.2.4 Volumetric glassware as required by standard and sample preparations
 
 6.3. Disposables 
 
 6.3.1 Tips for adjustable pipettes 
 
 6.3.2 Micro-centrifuge tubes 
 
 6.3.3. 16mL Test Tubes 
 
 6.3.4 Disposable plastic luer lock syringes 
 
 6.3.5 Nylon syringe filters, 0.45um 
 
 6.3.6 Weigh paper 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Yohimbine Alkaloid Determination by HPLC and D-750 3 | Page 3 of9 
 UV/VIS Spectroscopy 
 
 6.4 Equipment 
 
 6.4.1 Suitable gradient HPLC system consisting of a pump, autosampler, column oven
 and UV detector with a chromatographic data handling system 
 
 6.4.2 Analytical balance 
 
 6.4.3 Shaker 
 
 6.4.4 Vortex 
 
 6.4.5 Stir Plate 
 
 6.4.6 Eppendorf centrifuge 
 
 6.4.7 Adjustable pipettes 
 
 7.0 Preparation of Mobile Phase, Diluent, Samples, and Standards 
 
 Tad Mobile Phase A (0.5% triethylamine in H20) 
 
 7.1.1. Transfer 1000 mL H20 to a mobile phase bottle. 
 
 7.1.2 Add 5.0 mL of triethylamine, and mix well. 
 
 7.2. Mobile Phase B (0.5% triethylamine in methanol) 
 
 7.2.1. Transfer 1000 mL methanol to a mobile phase bottle. 
 
 7.2.2 Add 5.0 mL of triethylamine, and mix well. 
 
 7.3 Diluent (Methanol/H20 50/50) 
 
 7.3.1 Transfer 500 mL methanol to a mobile phase bottle. 
 
 7.3.2 Add 500 mL H20, and mix well 
 
 7.3.3 Equilibrate to room temperature before use. 
 
 7.4 Stock Standard Preparation 
 
 7.4.1. Accurately weigh and transfer about 25 mg of Yohimbine HCI reference standard
 
 into a 100-mL volumetric flask. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Yohimbine Alkaloid Determination by HPLC and D-750 3 | Page 4 of 9 
 UV/VIS Spectroscopy 
 
 7.4.2 Dissolve in and dilute to volume using Diluent. 
 
 7.5 Working Standard Preparation 
 
 7.5.1 Transfer 5.0 mL of Stock Standard to a 50-mL volumetric flask.
 
 7.5.2 Dilute to volume using Diluent. 
 
 7.6 Sample Preparation 
 
 7.6.1 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.
 
 7.6.2 The linear range of the method is 2.5 — 750 wg/mL. The concentration of the
 
 sample preparation must be within the linear range of the method. If the potency
 is given only for total alkaloids, assume Yohimbine is the only alkaloid present
 
 to calculate sample weight and volume. 
 
 7.6.3 For raw materials: Based on the expected Yohimbine content, weigh a portion
 
 into a suitably sized volumetric flask to generate an analyte concentration of about
 25 ug/mL. Dilute the sample to 2/3 of the flask volume with Diluent, sonicate for
 
 5 min with occasional shaking, shake mechanically for 30 minutes, and dilute to
 volume using Diluent. 
 
 7.6.4 For solid and liquid dose finished products: Combine and homogenize at least 10
 
 dosage units. Based on the weight per dose and the label amount, weigh a portion
 
 of the homogenized sample into a suitably sized volumetric flask to generate a
 Yohimbine concentration of about 25 g/mL. Dilute the sample to 2/3 of the flask
 
 volume with Diluent, sonicate for 5 min with occasional shaking, shake
 mechanically for 30 minutes, and dilute to volume using Diluent.
 
 7.6.5 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 suitably sized beaker. Use
 
 several small portions of diluent to completely transfer the sample into a suitably
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Yohimbine Alkaloid Determination by HPLC and D-750 
 Page 5 of 9 
 UV/VIS Spectroscopy 
 sized volumetric flask to generate a Yohimbine concentration of about 25 ng/mL.
 
 Dilute the sample to 2/3 of the flask volume with Diluent, sonicate for 5 min with
 
 occasional shaking, shake mechanically for 30 minutes, and dilute to volume
 using Diluent. 
 
 7.6.6 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 analyte concentration into the linear range of measurement.
 
 7.6.7 The final sample must be filtered or centrifuged before analyzing by HPLC.
 
 7.6.7.1. 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. 
 
 7.6.7.2 For centrifugation: centrifuge for 5 min at 10,000 rpm.
 
 8.0 Test Conditions 
 
 8.1 Gradient (Blanks and Standards) 
 
 Time %A %B Gradient Type 
 
 0.00 69% 31% 0 
 
 35.00 69% 31% 0 
 
 8.2 Gradient (Samples) 
 
 Time %A %B Gradient Type 
 
 0.00 69% 31% 0 
 
 80.00 69% 31% 0 
 
 80.10 0% 100% step 
 
 85.00 0% 100% 
 
 85.10 69% 31% step 
 
 90.0 69% 31% 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Yohimbine Alkaloid Determination by HPLC and D-750 Page 6 of 9 
 UV/VIS Spectroscopy 
 
 8.3 Column — Kinetex EVO C18 100 A Sp, 150mm x 3.0mm 
 
 8.4 Flow Rate — 0.7mL/min 
 
 8.5 UV Detection — 282nm 
 
 8.6 Recommended 3-D Spectral Range — 220nm — 400nm 
 
 8.7 Injection Volume - 20uL 
 
 8.8 Column Temperature — 30°C 
 
 8.9 Relative Retention Times 
 
 8.9.1 Ajmaline — 0.31 
 
 8.9.2 Unknownl-— 0.69 
 
 8.9.3 Yohimbine — 1.00 
 
 8.9.4 Coryanthine— 1.13 
 
 8.9.5 Unknown2- 1.38 
 
 8.9.6 Rauwolscin— 2.19 
 
 8.9.7 Ajmalicine — 3.94 
 
 8.9.8 Reserpine — 5.13 
 
 8.10 Recommended Sequence 
 
 8.10.1 Make at least 2 injections of a Blank (Diluent). 
 
 8.10.2 Make five injections of the Working Standard. 
 
 8.10.3 Make a single injection of each Sample Preparation. 
 
 8.10.4 Make a single injection of the Working Standard after every six samples and at
 the end of the run. 
 
 8.11 System Suitability 
 
 8.11.1 The %RSD of five consecutive standard injections is NLT 5.0%.
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Yohimbine Alkaloid Determination by HPLC and D-750 3 | Page 7 of9 
 UV/VIS Spectroscopy 
 
 8.11.2 The %RSD of all standard injections is NLT 5%. 
 
 8.12 Column Wash and Storage 
 
 8.12.1 Rinse and store the column with H20 / methanol (50/50). 
 
 9.0 Calculations 
 
 9.1 %% assayy = R R—s xX w — t VS o t tS a a X — P x V S s A pl x ~ x 100
 
 Ru Sample peak area 
 
 Rs Mean standard peak area 
 
 Wtstq Weight of reference standard in mg 
 
 Vseq 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 Wolume 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 | for raw materials 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Yohimbine Alkaloid Determination by HPLC and D-750 3 | Page 8 of9 
 UV/VIS Spectroscopy 
 
 10.0 Example Chromatography 
 
 10.1 Diluent 
 Blank 
 2.2% 
 24 
 1.8 
 1.65 
 1.44 
 1.24 
 44 
 0.84 
 0.67 
 0.44 
 0.24 
 UAm 
 Ajmiaine 
 -0.2- 5 jo. 15. 20 25 30 35 40 #45 #50 55 60 65 70 75 80 85 90 
 Time [min] 
 10.2 Standard 
 21AS051 30 ug/mL 
 200 
24.543 180+ Yohimbine HCL

 160- 
 140+ 
 = 1207 
 = 100+ 
 80+ 
 60- 
 40+ 
 20- p-587 
 2.24 
 
 1.84 
 1.67 
 1.44 
 1.24 
 14 
 0.84 
 0.67 
 0.44 
 0.24 
 04 
 UAm 
 7 > 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
 Time [min] 
 10.3. Sample 
 i 
 22014 
 e 
 5 re 
5.267 Ik 3

24.288 re HCL

 i — ee 
 -0.24 10. 15 20.25 30 35 40 445 + «50 #55 60 65 70 75 80 85 90 
 Time [min] 
 ew 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Yohimbine Alkaloid Determination by HPLC and D-750 3 | Page 9 of 9 
 UV/VIS Spectroscopy 
 
 11.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 09/28/15 | New procedure | 15-0961 | J. Maignan |
| 1 | 01/14/19 | Scheduled review: updated format to match current Ion practices. Update for consistency with current methods, simplify mobile phase preparation, simplify standard and sample preparation, add short ane. : DbOMe2 gradient for standards and blanks, add column wash and storage, add CO 220299 1S. Sassman example chromatography. Update for consistency with current methods, add instruction to | 19-0039 | J. Maignan |
| 3 | 05/10/23 | follow test details, add specific sample preparation for different CC- dosage forms. | 23-0248 | S. Sassman |