L-Citrulline Determination by HPLC with UV-Vis Detection

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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 L-citrulline in finished products and raw materials using HPLC coupled with
 
 UV/VIS detection. 
 
 2.0 Scope 
 
 This procedure has been validated to quantify and identify L-citrulline. Some excipients and
 
 dietary ingredients used in the finished products can interfere with the analysis of L-citrulline.
 Amax was used for the validation, but other wavelengths can be used to measure area if
 
 interferences are present. 
 
 3.0 Responsibility 
 
 Fe 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. 
 
 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 ACN — Acetonitrile 
 
 4.2 | H3PQ4-— Phosphoric Acid 
 
 4.3. HeO- Millipore Water, 18.2Q 
 
 44 QC -— Quality Control 
 
 4.5 CofA — Certificate of Analysis 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 L-Citrulline Determination by HPLC coupled with D-736 S Page 2 of 7 
 
 UV/VIS Detection 
 
 4.6 RT — Room Temperature 
 
 5.0 References 
 
 5.1 MV-LAB-13-043, Protocol, L-Citrulline Determination by HPLC 
 
 6.0 Reagents, Supplies, Glassware and Equipment 
 
 6.1 Reagents: all reagents are HPLC grade or better unless otherwise noted.
 
 6.1.1 H2O- Millipore Water 
 
 6.1.2 ACN 
 
 6.1.3 Phosphoric acid- ACS Grade 
 
 6.1.4 L-citrulline traceable standard 
 
 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, lmL, and 10mL pipette tips 
 
 6.2.5 10mL Plastic luer-lock syringes 
 
 6.2.6 0.2uM or 0.45uM Nylon syringe filters 
 
 6.2.7 22mL screw cap vials 
 
 6.2.8 1.5mL and 2.0mL micro centrifuge tubes 
 
 6.2.9 Weigh paper and weigh boats 
 
 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 Column- Acclaim 120-C18, 5u, 120A, 4.6 X 250mm 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 
 L-Citrulline Determination by HPLC coupled with D-736 Page 3 of 7 
 UV/VIS Detection 
 
 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, lmL, and 10mL Pipettes- adjustable 
 
 7.0 Preparation of Mobile Phase, Diluent, Samples and Standards 
 
 7.1 Mobile Phase A - 0.1% H3PO4 in H20 
 7.1.1 Transfer 1000 mL H20 to a 1-L bottle. 
 
 7.1.2 Add 1.0 mL H3POs, and mix well. 
 
 7.2 Mobile Phase B - 0.1% H3PO4 in ACN 
 
 7.2.1 Transfer 1000 mL ACN to a 1-L bottle. 
 
 7.2.2 Add 1.0 mL H3POq4, and mix well. 
 
 7.3 Diluent 
 
 7.3.1 Use Mobile Phase A 
 
 7.4 Stock Standard Preparation 
 
 7.4.1 Accurately weigh and transfer about 25 mg of reference standard into a 50-mL
 volumetric flask. 
 
 7.4.2 Dissolve in and dilute to volume using Diluent. 
 
 7.5 Working Standard Preparation 
 
 (ei | Transfer 5.0 mL of Stock Standard into a 50-mL volumetric flask.
 7.5.2. Dilute to volume using Diluent. 
 
 7.5.3 Alternate standard preparations are acceptable provided that the Working
 Standard concentration is within the linear range listed below.
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 L-Citrulline Determination by HPLC coupled with D-736 S Page 4 of 7 
 
 UV/VIS Detection 
 
 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 0.0005 mg/mL — 0.6 mg/mL. The concentration
 
 of the Sample Preparation must be within this range. 
 
 7.6.3 10 dosage units can be pooled and ground by mortar and pestle if necessary.
 
 7.6.4 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
 
 prepared using 100% as purity. 
 
 7.6.5 Dilute the sample to the calculated volume with diluent, cap, and sonicate for 10
 minutes to facilitate dissolution. Samples can be shaken on a wrist action shaker
 
 for 30 minutes at RT in two thirds their initial volume then brought up to final
 
 volume. 
 7.6.6 Remove sample from the sonicator and allow it to cool to RT. (Skip step if using
 
 wrist action shaker). 
 
 7.6.7 Samples can be dissolved in diluent at any volume starting from 10 mL. 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. The final diluted sample must
 be filtered or centrifuged before analyzing by HPLC. 
 
 7.6.8 For filtration, using the final large scale diluted sample withdraw up to 10 mL
 using a 10 mL plastic syringe then filter and discard at least the first 0.5 mL of
 
 sample before collecting. From the collected sample dilute as needed then add 1
 mL to an HPLC vial for analysis. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 L-Citrulline Determination by HPLC coupled with D-736 5 Page 5 of 7 
 
 UV/VIS Detection 
 
 7.6.9 For centrifugation using the final large scale diluted sample, fill an even number
 of 1.5 or 2.0 mL micro-centrifuge tubes and pellet insoluble matter for 5 minutes
 
 at 10,000rpm. 
 
 7.7. Test Conditions 
 
 7.7.1 Gradient-Isocratic 
 
 7.7.1.1. Column- Acclaim 120-C18, 5p, 120A, 4.6 X 250mm, or equivalent
 
 7.71.2 Flow Rate- 1.0mL/min 
 
 7.71.3 Time %A %B Gradient type 
 
 0.00 98 2 0 
 
 6.00 98 2 0 
 
 7.71.4 UV detection- 195nm 
 
 7.71.5 Injection volume- 20uL 
 
 7.7.1.6 Column Temperature- 40°C 
 
 7.7.1.7 Recommended 3-D Spectral Range- 200nm to 700nm 
 
 7.8 Recommended Sequence 
 
 7.8.1 Make at least 2 injections of a Blank (Diluent). 
 
 7.8.2 Make five injections of the Working Standard. 
 
 7.8.3 Make a single injection of each Sample Preparation. 
 
 7.8.4 Make a single injection of the Working Standard after every six samples and at
 the end of the run. 
 
 7.9 System Suitability 
 
 7.9.1 The %RSD of five consecutive injections of the Working Standard is NMT 5.0%.
 
 7.9.2 The %RSD of all standard injections is NMT 5%. 
 
 7.10 Column Wash and Storage 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 L-Citrulline Determination by HPLC coupled with D-736 S Page 6 of 7 
 
 UV/VIS Detection 
 
 7.10.1 Rinse the column with at least 15 mL of H20 / ACN (90/10). 
 
 7.10.2 Rinse the column with at least 10 mL of H20 / ACN (50/50). 
 
 7.10.3 Store the column with H20 / ACN (50/50). 
 
 7.11 Example calculations for determining finished product % label or raw material % purity
 
 7.11.1 y% assay =»R R , W—t V sse t r ada x P .x. V c—s a p2i x.. i SS a= x 100
 
 Ry Sample peak area 
 
 Rg Mean standard peak area 
 
 Wtsig Weight of reference standard in mg 
 
 Vsta 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) 
 
 Vp 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 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 D-736 5 
 L-Citrulline Determination by HPLC coupled with Page 7 of 7 
 UV/VIS Detection 
 
 8.0 Example Chromatograms 
 
 8.1 Working Standard 
 
 DADI1A,Sig=195,4 Ref=off 
 500- 
 450- 
 400+ 
 350+ 
 ~ 3004 
 = 250- 
 2004 
 150- 
 100- 
 50- 
 gnillurtiC 
 | 
 0.25 0.5 0.75 1 195 151.75 2 22525275 3 325353.75 4 42545 4.75 5 5.25555.75 6
 Time [min] 
 8.2 Sample 
 DAD1A,Sig=195,4 Ref=off 
 600- 
 550- 
 500- 
 450- 
 400+ 
 ~ 350+ 
 = 300- 
 250- 
 200- 
 150- 
 100+ 
 50- 
116.2 es

 enillurtiC 
 182.4 665.54 
 0.25 0.5 0.75 1 42515175 2 22525275 3 3.25353.75 4 42545475 5 52555 5.75 6
 Time [min] 
 10.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 3 | 01/29/19 | Scheduled review. Update responsibilities and sample prep Update to reflect current practices. Simplify mobile phase 4 el erqepuaerantieoin.e, Asdecdt iroen cwoimtmh esnydsetedm s seqaulentcey .se cUtpiodna.t Ree Splaatciens inl Be SAAD calculation for consistency with current methods. Simplify standard preparation, add instruction to check the | 19-0114 | J. Maignan |
| 5 | 02/22/23 | product profile for test details, remove language requiring in- CC- process validation for new products. | 23-0097 | S. Sassman |