Simultaneous Determination of Glucosamine and its Derivative by HPLC/ELSD - Application in Quality Control and Biological Study Lab. of Food & Biomaterial Chemistry Han ji sung
CONTENTS Ⅰ. Introduction Ⅱ. Materials & Methods Ⅲ. Results Ⅳ. Conclusion
Introduction Glucosamine (GlucN) has been a focusing material for the improvement of osteoarthritis as well as N-Acetyl-D-glucosamine (GlucNAc) , which has attracted much attention owing to its therapeutic activity in osteoarthritis and been evaluated as a food supplement N-Acetyl-D-glucosamine Glucosamine hydrochloride
Glucose ↓ Glucose-6-Pi ↓ Glucosamine-6-Pi ←GlucN ↓ N-Acetylglucosamine-6-Pi ←GlucNAc ↓ N-Acetylglucosamine-1-Pi ↓ UDP-N-Acetylglucosamine ↓ ↓ Hyaluronic acid, chondroitin Metabolic pathways • Amino-monosaccharide naturally produced in humans • Biosynthesis of macromolecules that comprise articular cartilage, such as glycosaminoglycans, proteoglycans hyaluronic acid • Believed to play a role in cartiliage formation and repair
ELSD Detector proposes a new HPLC method for the separation and quantification of Glucosamine/N-acetylglucosamine using evaporative light scattering detection. This method is applicable for monitoring nutraceutical formulations, QC, and process monitoring.Improvements over current methods include:• Minimal sample preparation • Rapid analysis• High sensitivity• Quantitative and reproducible results
Nebulization: Inside the nebulizer, the column effluent passes through a needle, mixes with nitrogen gas, and forms a dispersion of droplets Detection: The sample particles pass through a flowcell where they are hit with a laser light beam. Light scattered by the sample particles is detected generating an electrical signal ELSD Detection Principles –Universal, Versatile, Sensitive Evaporative Light Scattering Detectors use a simple three-step process that produces a signal for any non-volatile sample component Mobile Phase Evaporation: The droplets pass through a heated “drift tube” where the mobile phase evaporates leaving a fine mist of dried sample particles in solvent vapor
Objective - Not contain a chromophore absorbing in a region useful for ultraviolet detection with HPLC - The high-cost, intricacy of experimental procedure, low sensitivity and unstability prevent using these method to determine of GlucN and GlucNAc for routine quality control in health/functional foods(HFFs) industry - ELSD is increasingly being used in as a quasi-universal detector eliminating the need for derivatization of non-absorbing analytes. - The objectives of our study is to develop and validate a new HPLC-ELSD for simultaneous determining and application in quality control and biological study
Classical methods of Glucosamine Spectrophotometry method Korea Food Additive Code Korea Helath/Funtional Food code HPLC/RI method
Spectrophotometry method Korea Food Additive Code 0.02g of GlucN is precisely weighed Dissolved in 20mL of water and diluted to 100ml with water(Test Solution). 1mL of Test Solution is transferred into a test tube 2mL of acetyl aceton is added. Mixed and heated Cooled in running water. 20mL of 96% alcohol is added 2mL of ρ-Dimethylaminobenzaldehyde is added Mixed and set aside for 1 hour at room temperature. Absorption at 535nm is measured.
Derivatization method by phenylisothiocyanate GlucN and GlucNAc solution along with 100 uL of Gal-HCl (40 ug/mL) were transferred into a 2 ml screw capped glass vial. 250 uL of 0.3 M phosphate buffer(pH 8.0) 200 uL of methanol 250 uL of PITC methanolic solution was added shaken and left for 15 min vortexed for 30 s and heated for 30 min at 96 ℃. cooled to 4 ◦C evaporation to dryness at 50 ◦C under either a vacuum or nitrogen. The residue was dissolved in 400 uL of HPLC mobile phase filtered through a PTFE syringe filter (0.45 um) HPLC analysis
ELSD(Glucosamine/N-acetylglucosamine) HPLC System:Shiseido HPLC nanospace/SI-1Detector:Alltech ELSD 2000Settings: Impactor “Off”, 90°C, 2.2 L/min, gain 1Column:Alltech Prevail™ Carbohydrate ES 250 x 4.6mmMobile Phase:Gradient initial 90:10(ACN:DW) ⇒ 15min 75:35(ACN:DW)Flow Rate:1.0 mL/minInjection Volume: 20µLSample: Dissolve an accurately weighed quantity of Standard in water 50mL Adding acetonitrile 50mL
Application in Biological Study N-acetylglucosamine : 160 mg/kg Glucosamine : 200 mg/kg
Application in Biological Study Divided into two groups Feed with GlucNAc and GlucN powder in capsule Blood samples collected from the marginal ear vein Separates from the blood 0.2 mL serum 200 uL of acetonitrile was added Mixed and centrifuged stored at –20 ℃ until analyzed
Classical Method_Glucosamine Derivatization methods - involve mixing, reacting, heating, cooling and extracting steps that are time consuming.Derivatives tend to build up on columns over time requiring frequent flushing. Many derivatization agents are toxic and can cause potentially expensive wastedisposal issues. - USP Method Glucosamine responds as two peaks and sensitivity is low.the response to 0.001N HCl is the same, although smaller,for the Glucosamine-HCl sample.
Classical Method_Glucosamine Glucosamine 1000(ug/mL) - RI detector • Sample preparation can be more difficult as the sample must be dissolved in the mobile phase to decrease solvent artifacts. • The RI detector can be time consuming to equilibrate. • RI tends to be much less sensitive, with 1-10mg/mL as a detectable range. Glucosamine 1000(ug/mL) - UV detector • Low sensitivity • Analysis at very low wavelength (195nm)
Method Validation - The HPLC method validation was established according to International Conference on Harmonization guideline, including specificity, linearity, detection and quantitation limit (LOQ),precision, accuracy, recovery, and robustness test Table 1. Performance of the proposed HPLC/ELSD method a Limit of detection. b Limit of quantitation.
Method Validation Table 2. Intra- and Inter-day precision and accuracy of HPLC-ELSD analysis of GlucN and GlucNAc a Precision of relative standard deviation. b Accuracy of relative error. c For 3 days (n=5)
(A) GlucNAc GlucN (B) GlucN (C) GlucN (D) GlucNAc [ mV ] Retention time(min) Method Validation Figure. 1. HPLC/ELSD chromatogram obtained from analysis of standards and Health /Functials Foods samples. (A) standard of GlucN (50.0 μg/mL) and GlucNAc (50.0 μg/mL ), (B) GlucN capsule containing multiple components, (C) GlucN tablet containing multiple active ingredients, (D) GlucNAc capsule containing single components.
Method Validation Table 3. Comparative determination of GlucN in Helath/Functional Foods by HPLC/ELSD and classical methods a Three different lots of health/functional foods formulations b Refractive index detection c p-Dimethylaminobenzaldehyde d USP requirements of glucosamine content upon the labeled amount e Data expressed with mean of three replicates
(a) GlucN (b) IS IS Method Validation Figure 2. HPLC method based on a reaction of phenylisothiocyanate with GlucN and GlucNAc (a) standard of GlucN (400 ug/mL) and IS (Galactosamine-HCl 40 ug/mL) (b) standard of GlucNAc (400 ug/mL) and IS (Galactosamine-HCl 40 ug/mL).
Table 4. Comparative analysis of raw materials by the HPLC/ELSD and HPLC/RI methods Quality Control - Problems with Refractive Index - Comparative analysis of raw materials
Quality Control Table 5. HPLC/ELSD assay of GlucN in Health/Functional Foods formulation (Single components) (Multi components)
GlucNAc Biological Study (A) (B) Figure 3. Allication in Biologycal study : (a) blank serum (b) serum sample 60 min after oral dosing of the rabbit with 160mg/kg of GlucN.
Conclusion - Simple and sensitive HPLC-ELSD method to simultaneous analyze GlucN and GlucNAc which applicable in routine quality control of Health/Functional Foods and biological study. - Fast and specific since two compounds can be analyzed in one run requiring only 15 min for determination. - Moreover this technique is very effective since it does not require expensive time-consuming stabilization or analytical procedures as other approaches for GlucN analysis in assay. - This assy can be used for content uniformity testing of dosage forms containing GlucN and GlucNAc, quality control of raw materials and bioavailability studies.