D-1018
Determination of 5-Hydroxytryptophan, Tryptophan & Melatonin by HPLC-UV
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1.0 Purpose This document describes the analytical procedure for the determination of 5- Hydroxytryptophan, Tryptophan and Melatonin in raw materials and finished products. 2.0 Scope This procedure applies to the identification and quantification of 5-Hydroxytryptophan, Tryptophan and Melatonin in raw materials and finished products. This method was validated under protocol PRTCL-23-0013. 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. 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 ACN-Acetonitrile 4.3 H2O- Milli-Q Water [SOP D-101 | Page 2 of 8] Standard Operating Procedure SOP No Rev Determination of 5-Hydroxytryptophan, Tryptophan D-1018 Page 2 of 8 & Melatonin by HPLC-UV 4.4 | H3PO«4-— Phosphoric Acid 4.5 ACS -— American Chemical Society 4.6 HPLC - High Performance Liquid Chromatography 4.7 UV-Vis — Ultraviolet-Visible (Detection) 5.0 References 5.1 PRTCL-23-0013, Validation of an Analytical Method for the Determination of 5- Hydroxytryptophan, Tryptophan and Melatonin by HPLC-UV 6.0 Supplies 6.1 Chemicals — All reagents are ACS grade or better 6.1.1 Milli-Q Water 6.1.2 ACN 6.1.3 H3PO, 6.1.4 5-Hydroxytryptophan Reference Standard 6.1.5 Tryptophan Reference Standard 6.1.6 Melatonin Reference Standard 6.2 Supplies and Glassware 6.2.1 HPLC vials, 12mm X 32mm with screw cap enclosures w/ septa 6.2.2 Volumetric glassware (Adjustable pipets may also be used.) 6.2.3. Weigh paper [SOP D-101 | Page 3 of 8] Standard Operating Procedure SOP No Rev Determination of 5-Hydroxytryptophan, Tryptophan | D-1018 Page 3 of 8 & Melatonin by HPLC-UV 6.2.4 Syringes with 0.45 nylon syringe filters 6.3 Equipment 6.3.1 Suitable gradient HPLC system consisting of a pump, autosampler, column compartment and UV-Vis detector with a chromatographic data handling system 6.3.2 Analytical Micro Balance 6.3.3. Analytical Balance 6.3.4 Wrist Action Shaker 7.0 Procedure 7.1. Mobile Phase, Extraction Solvent & Diluent Preparation 7.1.1. Mobile Phase 7.1.1.1 Mobile Phase A: Combine 1000mL H20 + 1000uL H3PO4 and mix well. 7.1.1.2 Mobile Phase B: Combine 1000mL ACN + 1000yL H3PO4 and mix well. 7.1.2 Extraction Solvent 7.1.2.1. Combine 600mL ACN + 400mL H20 and mix well. Allow solution to warm to ambient temperature prior to use. 7.1.3 Diluent 7.1.3.1 Use 20. 7.2 Standard Prep [SOP D-101 | Page 4 of 8] Standard Operating Procedure SOP No | Rev Determination of 5-Hydroxytryptophan, Tryptophan D-1018 0 Page 4 of 8 & Melatonin by HPLC-UV 7.2.1 Prepare standard stock at ~0.1 mg/mL analyte in Extraction Solvent. Shake stock for 15 minutes at ~ % volume then QS to volume. Shake vigorously then dilute stock 1:10 with Diluent. Shake vigorously then filter a SmL aliquot for analysis, discarding the first 3-4mL of filtrate. 7.3 Sample Preparation 7.3.1 Specific sample testing details are provided in each product profile. Ifa specific testing details section is not available, follow preparation procedure as described below, maintaining concentration within the linear range of this method. 7.3.2 The validated linear ranges for the analytical method are 0.00614 — 0.0143 mg/mL 5-Hydroxytryptophan, 0.00622 -— 0.0145 mg/mL Tryptophan and 0.00614 — 0.0143 mg/mL Melatonin. 7.3.3 Pool at least 10 dosage units and homogenize as appropriate (e.g. grind tablets / capsule fill / powders / stick pack contents by mortar and pestle, cryogenically powder and dissolve gummies, etc.) Extract sufficient sample (based on the raw material manufacturer assay value / product profile) with Extraction Solvent in order to generate a sample stock that is ~0.1 mg/mL analyte. Shake for 15 minutes at ~ % volume then QS to volume. Dilute stock 1:10 with Diluent. Shake vigorously then filter a 5mL aliquot for analysis, discarding the first 3- 4mL of filtrate. 7.4 HPLC Parameters 7.4.1 Column: Supelco Ascentis Express Cg, 2.7m (SPP), 90A, 4.6 x 100mm 7.4.2 Column Temperature: 30°C 7.4.3 Flow rate: 0.5 mL/min 7.4.4 Run Time: 20 minutes [SOP D-101 | Page 5 of 8] Standard Operating Procedure SOP No | Rev Determination of 5-Hydroxytryptophan, Tryptophan | D-1018 0 | Page 5 of8 & Melatonin by HPLC-UV 7.4.5 Mobile Phase: Gradient Time, min | %A | %B 0 95 5 7.4.6 Wavelength: 220 nm > 95 5 7.4.7 Injection Volume: 5 uL 15 50 50 7.4.8 Suggested 3-D Spectral Range (for Identification): re 2 210nm - 350nm 20 95 5 7.5 Recommended Sequence 7.5.1 Make at least 2 injections of the Diluent. 7.5.2 Make at least five (5) injections of Working Standard. 7.5.3 Make a single injection of each Sample Preparation. 7.5.4 Make a single injection of the Working Standard after every ten (10) sample injections or at the end of the run. 7.6 System Suitability Requirements 7.6.1 The %RSD of five (5) consecutive standard injections is NMT 2%. 7.6.2 The %RSD of all standard injections is NMT 3%. Tt Example calculations for determining % Assay / LC: 1.71 % = Be x WisaXP y Yet x 100x SS/LA R, Veta SA R, Sample peak area R, Mean (All) standard peak area Wt..a Weight of the reference standard, mg Viva Volume of the standard preparation accounting for dilutions, mL [SOP D-101 | Page 6 of 8] Standard Operating Procedure SOP No | Rev Determination of 5-Hydroxytryptophan, Tryptophan D-1018 0 Page 6 of 8 & Melatonin by HPLC-UV P Purity of the reference standard in decimal format SA Sample amount, g Vevt Volume of the sample preparation accounting for dilutions, mL SS Serving Size, g (Use “1g” for Raw Materials.) LA Label Amount, mg (Use “lmg” for Raw Materials.) 7.8 System Wash, Column Wash and Column Storage 7.8.1 Wash and store the column in 60% ACN. 8.0 Chromatograms 8.1 Typical Diluent Chromatogram 7 SHIP TP&MIAR6N23G48 Wier UMS 1WWL22rm meu [SOP D-101 | Page 7 of 8] Standard Operating Procedure SOP No Rev Determination of 5-Hydroxytryptophan, Tryptophan D-1018 Page 7 of 8 & Melatonin by HPLC-UV 8.2 Typical Standard Chromatogram 3 HIP TP8MBLARNB BH VeID OAS 1VL2rm nf 2-P- 0617 3-MIA- 14913 &5) 1-SHIP-367 8.3. Typical Raw Material Chromatograms 7 HIP TP&MAARNB- B23 FHIP-1 UNS 1VW20Mm mu Ea 1-5HIP-3647 [SOP D-101 | Page 8 of 8] Standard Operating Procedure SOP No Rev Determination of 5-Hydroxytryptophan, Tryptophan D-1018 Page 8 of 8 & Melatonin by HPLC-UV 400. HIP TP&MAARNBBBH2 UMS AWiL22rm mu 1-TP- 10607 180} 125} 00} 5} 04 5, 04 Kies 0} R54 004 -25, -20- "40 80 0 70 80 9T0 80 FIP PSNMAAR6N2G34-5 MaA-1 UMS 1WL22nm 3 8.8 88B = 1-MELA- 14913 2- 18: 40. 80 6. 70 20 9.0 Revision History | Rev | Date | Description of Changes | CCR # | By | |-----|----------|------------------------|-------|----| | 0 | 03/28/23 | New procedure. N/A C. Perry | - | - |