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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.
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
Ⅱ. Materials & Methods
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
Hyaluronic acid, chondroitin
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
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
- 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
Korea Food Additive Code
Korea Helath/Funtional Food code
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.
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 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
N-acetylglucosamine : 160 mg/kg
Glucosamine : 200 mg/kg
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
- 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.
- 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.
- UV detector
• Low sensitivity
• Analysis at very low wavelength (195nm)
- 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.
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)
[ mV ]
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.
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
d USP requirements of glucosamine content upon the labeled amount
e Data expressed with mean of three replicates
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).
and HPLC/RI methods
- Problems with Refractive Index
- Comparative analysis of raw materials
Table 5. HPLC/ELSD assay of GlucN in Health/Functional Foods formulation
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.
- 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.