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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.

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slide1

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

slide2

CONTENTS

Ⅰ. Introduction

Ⅱ. Materials & Methods

Ⅲ. Results

Ⅳ. Conclusion

slide3

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

slide4

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
slide5

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

slide6

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

slide7

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

slide9

Classical methods of Glucosamine

Spectrophotometry method

Korea Food Additive Code

Korea Helath/Funtional Food code

HPLC/RI method

slide10

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.

slide11

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

slide12

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

slide13

Application in Biological Study

N-acetylglucosamine : 160 mg/kg

Glucosamine : 200 mg/kg

slide14

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

slide16

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.

slide17

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)

slide18

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.

slide19

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)

slide20

(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.

slide21

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

slide22

(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).

slide23

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

slide24

Quality Control

Table 5. HPLC/ELSD assay of GlucN in Health/Functional Foods formulation

(Single components)

(Multi components)

slide25

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.

slide26

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.