Determination of Chondroitin, N-Acetylglucosamine, and Glucosamine by HPLC-UV

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1.0 Purpose 
 
 This document describes the analytical procedure for the determination of chondroitin, N-
 acetylglucosamine and glucosamine using HPLC coupled with UVspectrophotometry.
 
 2.0 Scope 
 
 This procedure applies to the identification and quantification of chondroitin, N-
 acetylglucosamine and glucosamine in raw materials and finished products.
 
 2.1 Two separate methods are described. 
 
 2.1.1 See Sections 7.0 —8.0 for analysis of chondroitin and N-acetylglucosamine.
 
 2.1.2 See Sections 9.0 — 10.0 for analysis of glucosamine. 
 
 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 associated monographs and laboratory
 
 practices. 
 
 4.0 Definitions 
 
 4.1 H2O — Water (18.2 MQ-cm) 
 
 4.2 ACN — Acetonitrile 
 
 4.3 ACS — American Chemical Society 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Chondroitin, N-acetylglucosamine D-733 6 Page 2 of 12 
 and Glucosamine by HPLC-UV 
 
 4.4 H3PO4 — Phosphoric Acid 
 
 4.5 K2HPOsg — Potassium Phosphate Dibasic 
 
 4.6 NH4OH — Ammonium Hydroxide 
 
 4.7 HCl — Hydrochloride 
 
 4.8 QC — Quality Control 
 
 4.9 CofA — Certificate of Analysis 
 
 4.10 HPLC — High Performance Liquid Chromatography 
 
 4.11 UV — Ultraviolet (Detection) 
 
 5.0 References 
 
 MV-LAB-13-010, Protocol, Validation: Glucosamine Determination 
5.1 MV-LAB-18-083, Protocol, Validation: Chondroitin Determination

5.2 MV-LAB-18-179, Protocol, Validation: N-acetylglucosamine Determination

5.3 PRTCL-22-0067, Protocol, Validation of an Analytical Method for the Determination of

5.4 Glucosamine by HPLC-UV

 6.0 Reagents, Supplies, Glassware and Equipment 
 
 6.1 Reagents: all reagents are ACS grade or better. 
 
 6.1.1 H20 
 
 6.1.2 ACN 
 
 6.1.3 H3PQO4 
 
 6.1.4 K2HPO, 
 
 6.1.5 NHs4OH 
 
 6.1.6 Glucosamine HCl reference standard 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Determination of Chondroitin, N-acetylglucosamine D-733 Page 3 of 12 
 and Glucosamine by HPLC-UV 
 
 6.1.7 N-acetylglucosamine reference standard 
 
 6.1.8 Chondroitin sulfate reference 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 containers 
 
 6.2.3 Volumetric Glassware 
 
 6.2.4 50mL and 100mL beakers 
 
 6.2.5 200uL, ImL, 10mL pipette tips 
 
 6.2.6 Plastic luer lock syringes 
 
 6.2.7 0.45um, 25mm Nylon syringe filters 
 
 6.2.8 22mL screw cap vials 
 
 6.2.9 Micro centrifuge tubes 
 
 6.2.10 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 Analytical Balance 
 
 6.3.3. Vortex Mixer 
 
 6.3.4 Stir Plate 
 
 6.3.5 Wrist action shaker 
 
 6.3.6 200uL, lmL and 10mL pipettes 
 
 6.3.7. Micro-centrifuge 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Chondroitin, N-acetylglucosamine D-733 6 | Page 4 of 12 
 and Glucosamine by HPLC-UV 
 
 7.0 Preparation of Mobile Phase, Diluent, Standards, and Samples for 
 
 Determination of Chondroitin and N-acetylglucosamine 
 
 7.1 Mobile Phase and Buffer Preparation 
 
 Halal Mobile Phase A (0.1% H3PO4 in H2O) 
 
 7.1.1.1. Transfer 1000mL of H20 to a mobile phase bottle. 
 
 7.1.1.2 Add 1.0mL of H3PO,, and mix well. 
 
 Fale Mobile Phase B (0.1% H3PO4 in ACN) 
 
 7.1.2.1 Transfer 1000mL of ACN to a mobile phase bottle. 
 
 7.1.2.2 Add 1.0mL of H3PO4, and mix well. 
 
 7.1.3 Diluent (50:50 H20:ACN) 
 
 7.1.3.1. Transfer 500mL of ACN to a mobile phase bottle. 
 
 7.1.3.2 Add 500mL of H20, and mix well. 
 
 7.1.3.3. Allow to equilibrate to room temperature before using.
 
 hee Standard Preparation 
 The linear range of the method is listed below. All standard and sample
 Tak 
 preparations must be within the linear range of the method. If the analyte form is
 not the same as listed below, the linear range may need to be corrected for analyte
 
 form. 
 
 7.2.1.1. Chondroitin sulfate sodium —0.1mg/mL to 1.0mg/mL 
 
 7.21.2 N-acetylglucosamine — 0.01mg/mL to 0.1mg/mL 
 
 7.2.2 Use the actual purity from the CofA in your calculations. 
 
 All Standards are prepared by weighing no less than the minimum weight of the
 Tass 
 analytical balance. Transfer to a suitably sized volumetric flask. Dissolve in and
 dilute to volume using Diluent. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Chondroitin, N-acetylglucosamine | 2-733 | © | Page5 of 12
 and Glucosamine by HPLC-UV 
 
 7.2.4 Dilutions are prepared using Diluent. Dilutions can be made using volumetric
 flasks or using 10mL, 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 can be prepared directly in HPLC vials. 
 
 te Sample Preparation 
 
 Specific sample testing details are provided in each products profile. If a specific
 TA 
 testing details section is not available, then follow preparation procedure as
 described below, maintaining concentration within the linear range of this
 
 method. 
 
 For finished products, 20 or more dosage units can be pooled and ground by
 Dead 
 mortar and pestle as necessary. 
 Based on the fill or tablet weight per dose for finished products or raw material
7.3.3 potency, weigh a portion of the pooled dosages to generate an analyte

 concentration that is within the validated linear range. 
 
 Samples can be dissolved in Diluent at any volume starting from 50mL. To
7.3.4 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
7.3.5 HPLC. Filter using a 0.45um nylon syringe filter, discarding at least 0.5mL of

 filtrate before collecting the filtered sample in a vial for analysis.
 
 For centrifugation using the final large scale diluted sample, fill an even number
7.3.6 of 1.5 or 2.0mL microcentrifuge tubes and pellet insoluble matter for 5 minutes

 at 6000rpm. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Chondroitin, N-acetylglucosamine | D-755 6 | Page 6 of 12 
 and Glucosamine by HPLC-UV 
 
 8.0 Test Conditions for Determination of Chondroitin and N-acetylglucosamine
 
 8.1 Gradient — Isocratic 60% A : 40% B 
 
 Column — Acclaim C18, 5u, 120A, 4.6 X 250mm 
8.2 8.3 Flow Rate — 0.6mL/min

 8.4 UV detection — 195nm 
 
 8.5 Injection volume — 20uL 
 
 8.6 Column Temperature — 35°C 
 
 Recommended 3-D Spectral Range — 190nm to 300nm 
8.7 8.8 Recommended Sequence
 
 8.8.1 Make at least 2 injections of a Blank (Diluent). 
 
 8.8.2 Make at least 5 injections of the Working Standard. 
 
 8.8.3. Make a single injection of each Sample Preparation 
 
 8.8.4 Makea single injection of the Working Standard after every 6 samples and at the
 
 end of the run. 
 
 8.9 System Suitability 
 
 891 The %RSD of 5 consecutive injections of Working Standard is NMT 5.0%
 
 89.2 The %RSD of all injections of Working Standard is NMT 5%. 
 
 8.10 Column Wash and Storage 
 8 10.1 Rinse the column with H2O / ACN (50/50) at 1 mL/min for at least 15 min.
 
 8.10.2 Store the column with H2O0 / ACN (50/50). 
 
 9.0 Preparation of Mobile Phase, Diluent, Standards and Samples for 
 
 Determination of Glucosamine 
 
 Mobile Phase and Buffer Preparation 
 | 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Chondroitin, N-acetylglucosamine D-733 6 | Page 7 of 12 
 and Glucosamine by HPLC-UV 
 
 ve Mobile Phase A 
 
 9.1.1.1 Dissolve ~3.5g K2HPOs in 1000mL of water, then add 0.25mL NHsOH
 and pH to 7.5 with H3PQO4. 
 
 9.1.2 Mobile Phase B 
 
 9.1.2.1 Use ACN. 
 
 Diluent (50:50 H20:ACN) 
 
 9.1.3.1 Transfer 500mL of ACN to a mobile phase bottle. 
 
 9.1.3.2 Add 500mL of H20, and mix well. 
 
 9.1.3.3 Allow to equilibrate to room temperature before using.
 
 92 Standard Preparation 
 
 O21 The linear range of the method is listed below. All standard and sample
 preparations must be within the linear range of the method. If the analyte form is
 
 not the same as listed below, the linear range may need to be corrected for analyte
 
 form. 
 
 9.2.1.1 Glucosamine HCl: 2.25 —5.26mg/mL 
 
 9.2.2 Use the actual purity from the CofA in your calculations. 
 
 All Standards are prepared by weighing no less than the minimum weight of the
9.2.3 analytical balance. Transfer to a suitably sized volumetric flask. Dissolve in and

 dilute to volume using Diluent. 
 
 Dilutions are prepared using Diluent. Dilutions can be made using volumetric
9.2.4 flasks or using 10mL, 1mL 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 can be prepared directly in HPLC vials. 
 
 9.3 Sample Preparation 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Chondroitin, N-acetylglucosamine D-733 6 | Page 8 of 12 
 and Glucosamine by HPLC-UV 
 
 9.3.1 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 of this
 method. 
 
 9.3.2 For finished products, 20 or more dosage units can be pooled and ground by
 
 mortar and pestle as necessary. 
 
 9.3.3. Based on the fill or tablet weight per dose for finished products or raw material
 potency, weigh a portion of the pooled dosages to generate an analyte
 
 concentration that is within the validated linear range. 
 
 9.3.4 Samples can be dissolved in Diluent at any volume starting from 50mL. 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. 
 
 9.3.5 The final diluted sample must be filtered or centrifuged before analyzing by
 
 HPLC. Filter using a 0.45ym nylon syringe filter, discarding at least 0.5mL of
 filtrate before collecting the filtered sample in a vial for analysis.
 
 9.3.6 For centrifugation using the final large scale diluted sample, fill an even number
 
 of 1.5 or 2.0mL microcentrifuge tubes and pellet insoluble matter for 5 minutes
 
 at 6000rpm. 
 
 10.0 Test Conditions for Determination of Glucosamine 
 
 10.1. Gradient —Isocratic 40% A : 60% B 
 
 10.2 Column — HyperSil GOLD Amino, 5p, 175A, 4.6 X 150mm 
 
 10.3. Flow Rate — 1.0mL/min 
 
 10.4 UV detection — 195nm 
 
 10.5 Injection volume — 5pL 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Chondroitin, N-acetylglucosamine D-733 6 Page 9 of 12 
 and Glucosamine by HPLC-UV 
 
 10.6 Column Temperature — 35°C 
 
 10.7. Recommended 3-D Spectral Range — 190nm to 260nm 
 
 10.8 Recommended Sequence 
 
 10.8.1 Make at least 2 injections of a Blank (Diluent). 
 
 10.8.2 Make at least 5 injections of the Working Standard. 
 
 10.8.3 Make a single injection of each Sample Preparation 
 
 10.8.4 Make a single injection of the Working Standard after every 6 samples and at
 
 the end of the run. 
 
 10.9 System Suitability 
 
 10.9.1 The %RSD of 5 consecutive injections of Working Standard is NMT 5.0%
 
 10.9.2 The %RSD of all injections of Working Standard is NMT 5%. 
 
 10.10 Column Wash and Storage 
 
 10.10.1 Rinse column with 60% ACN at 1 mL/min for at least 1 hour.
 
 10.10.2 Store column on 100% ethanol. 
 
 11.0 Example Calculation 
 
 11.1 %%as say= y — R Rs X Wt — ot V q sS X ta P x V—S s A pl x -- x 100
 
 Ruy Sample peak area 
 
 Rg Mean standard peak area 
 
 Wt.tq Weight of reference standard in mg 
 
 Vetq Wolume 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) 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Chondroitin, N-acetylglucosamine D-733 Page 10 of 12 
 and Glucosamine by HPLC-UV 
 
 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 
 
 12.0 Example Chromatography 
 
 12.1 Chondroitin / N-acetylglucosamine Blank 
 
 DILUENT 
 200 
 180- 
 160- 
 140- 
 = 120 
 < 100+ 
 80+ 
 607 
 40- 
 20: #.199 
 0.5 ; 15 2 25 3 3.5 4 45 5 55 6 6.5 
 Time [min] 
 12.2 N-acetylglucosamine Working Standard 
 
 21AS011 0.075MG/ML 
 meal T\ 3.584 
 180- N-acetyl-glucosamine 
 160° 
 140- 
 100: 
 80- 
 60- 
 40- 
 20- ¥ 197 
 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5.5 
 Time [min] 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Determination of Chondroitin, N-acetylglucosamine D-733 Page 11 of 12 
 and Glucosamine by HPLC-UV 
 
 12.3. Glucosamine Diluent 
 
 50.6 % Slucosantine Run 12-02-22 33 Dévent UU _vIS_ TAYE 185 nn
 506 mAU 
 
 10 
 
 5 vg 0 4 ene oe ad P oe ee ——— atl Oe rs ~ i eal cate ia eee Me a = ami wes ie OE Re an ee all Pe —_ \ \ i ald ; Sas al ite i a ~_
 
 ran 
4256.00 5 40 ; 20 2.50 30 3.50 4 $50 6.09 8.00

 12.4 Glucosamisne Standard 
 azn __ 2 Glucosamine Run 12-02-22 st WSTD 
 " , 
 OH mau 
 4 
 300 
 i 
 | , 1 - Glucosamine - 3.940 
 ff \\ 
 | i | i; \ 
 j i \ 
 Po j \ a ; | S / \ e —— eee 
 mm 
 
 12.5 Glucosami° ne Raw Materi*al 
 
 an SawwlOs2 é¢ Run 1 1 Ue SI YL 195 am 
 #0 mals 
 | 
 225 4 it 
 200 | | 
 1356 | | 
 12 | i 1 1- Glucosamme - 3.937 
 i 
 
 25 e 4 ee CTae RR ee \ fA e h e, = | 4 a , H| \ C en eee Ld / / i \ \Sede ee eee a _ _ eS ee
 254 
 5 i 152 248 2 5: 3.00 56 5 5 £0 59 6.0% 759 mmras 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Chondroitin, N-acetylglucosamine D-733 6 | Page 12 of 12 
 and Glucosamine by HPLC-UV 
 
 12.6 Glucosamine Finished Product 
 
 | 
 
 LA | \1-2627 \ 
 : _ _ V NJ L \ /\ 2-2ny 4-3527/ \ 5 ; sdoie eH 
 
 13.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 4 | 01/02/19 | MV-LAB- Update for consistency with current methods, add recommended | 18-179 | . 19-0001 J. Maignan |
| 5 | 06/21/22 | Sequence section, replace requirements with system suitability CC- section, add column wash and storage, add example chromatography. Update logo and format. Added new glucosamine procedure. Added reference to product | 22-0279 | S. Sassman |
| 6 | 01/20/23 | profile regarding specific testing details. Removed first time CC- validation statement. | 23-0003 | C. Perry |