Simultaneous Determination of Glucosamine and its DerivatiSimultaneous Determination of Glucosamine and its Derivati

ve by HPLC/ELSD - Application in Quality Control and Biolve by HPLC/ELSD - Application in Quality Control and Biol

ogical Studyogical Study

Lab. of Food & Biomaterial ChemistryLab. of Food & Biomaterial Chemistry

Han ji sungHan ji sung

Ⅰ. Introduction

Ⅱ. Materials & Methods

Ⅲ. Results

Ⅳ. Conclusion

CONTENTSCONTENTS

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

-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

Metabolic pathways

Glucose ↓

Glucose-6-Pi ↓

      Glucosamine-6-Pi ←GlucN ↓

N-Acetylglucosamine-6-Pi ←GlucNAc↓

N-Acetylglucosamine-1-Pi ↓

UDP-N-Acetylglucosamine ↓ ↓

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

ELSD Detector

Evaporative Light Scattering Detectors use a simple three-step

process that produces a signal for any non-volatile sample component

ELSD Detection Principles –Universal, Versatile, Sensitive

Nebulization: Inside the nebulizer,

the column effluent passes through a

needle, mixes with nitrogen gas, and

forms a dispersion of dropletsMobile 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

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

- 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

Objective

Materials & Methods

Classical methods of Glucosamine

Korea Helath/Funtional Food codeKorea Helath/Funtional Food code

HPLC/RI method

Korea Food Additive CodeKorea Food Additive Code

Spectrophotometry method

Absorption at 535nm is

measured.

2mL of acetyl aceton is added.

20mL of 96% alcohol is added2mL of ρ-Dimethylaminobenzaldehyde is added

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

Mixed and heated

Cooled in running water.

Mixed and set aside for 1 hour at room temperature.

Korea Food Additive CodeKorea Food Additive Code

Spectrophotometry method

Derivatization method by phenylisothiocyanate

250 uL of 0.3 M phosphate buffer(pH 8.0)200 uL of methanol

250 uL of PITC methanolic solution was added

GlucN and GlucNAc solution along with 100 uL of Gal-HCl (40 ug/mL) were transferred into a 2 ml screw capped glass vial.

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.

filtered through a PTFE syringe filter (0.45 um)

HPLC analysis

The residue was dissolved in 400 uL of HPLC mobile phase

HPLC System:HPLC System: Shiseido HPLC nanospace/SI-1

Detector:Detector: Alltech ELSD 2000Settings: Impactor “Off”, 90°C, 2.2 L/min, gain 1

Column:Column: Alltech Prevail™ Carbohydrate ES 250 x 4.6mm

Mobile Phase:Mobile Phase: Gradient

initial 90:10(ACN:DW) 15min 75:35⇒ (ACN:DW)

Flow Rate:Flow Rate: 1.0 mL/min

Injection Volume: 20µL

Sample: Dissolve an accurately weighed quantity of Standard in water 50mL Adding acetonitrile 50mL

ELSD(Glucosamine/N-acetylglucosamine)

Compound Experimentation Dose Detector Derivatization reference

Glucosamine Rat 350 mg/kg UV 1-naphtyl isothiocyanateAghazadeh-Habashi., et al 200

2

Glucosamine Dog 133 mg/kg UV PhenylisothiocyanateLiang., et al 19

99

N-butyryl glucosamine

Rat 233 mg/kg UV1-phenyl-3-methyl-5-Pyra

zolone

Aghazadeh-Habashi., et al 200

5

Glucosamine Human 1500mg HPLC/MS NoRoda., et al 200

6

Glucosamine Human 1500mg FLD9-fluorenylmethoxy carbo

nyl chlorideZhang., et al

2006

N-acetylglucosamine : 160 mg/kg

Glucosamine : 200 mg/kg

Application in Biological Study

200 uL of acetonitrile was added

Application in Biological Study

Divided into two groups

Feed with GlucNAc and GlucN powder in capsule

Blood samples collected from the marginal ear vein

0.2 mL serum

Mixed and centrifuged

stored at –20 ℃ until analyzed

Separates from the blood

Results

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 wastepotentially expensive wastedisposal issues.disposal issues.

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

- USP Method

• 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)

Classical Method_Glucosamine

- RI detector

Glucosamine 1000(ug/mL)

Glucosamine 1000(ug/mL)

Table 1. Performance of the proposed HPLC/ELSD method

AnalytesConcentration ra

nge (mg/mL)Correlation

coefficient (r2)LOD a

(µg)LOQ b

(µg)

Glucosamine 0.050 – 0.250 0.9987 1.0 5.0

N-acetyl glucosamine

0.025 – 0.100 0.9992 0.5 2.0

a Limit of detection.

b Limit of quantitation.

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

Analyte N-acetyl glucosamine glucosamine

Prepared conc.(µg/mL)

Measured conc. (%)

(mean ± S.D)%R.S.Da %R.E b

Measured conc. (%)

(mean ± S.D)%R.S.Da %R.E b

Intra-day

80 99.54 ± 1.52 1.90 - 0.054 100.71 ± 1.65 1.92 - 0.341

100 99.81 ± 1.47 1.15 - 0.189 98.29 ± 0.39 0.72 - 1.284

120 98.53 ± 1.80 1.52 - 0.750 96.79 ± 1.28 0.82 - 2.254

Inter-day c

80 100.69 ± 1.54 2.06 0.614 100.41 ± 1.98 2.48 0.014

100 99.94 ± 0.72 0.42 0.031 98.99 ± 0.82 0.84 - 0.601

120 97.04 ± 1.07 1.11 0.043 98.99 ± 1.25 0.92 - 0.849a Precision of relative standard deviation. b Accuracy of relative error.c For 3 days (n=5)

Table 2. Intra- and Inter-day precision and accuracy of HPLC-ELSD analysis of GlucN and GlucNAc

Method Validation

GlucN

GlucNAc

Retention time(min)

[ m

V ]

(A)

(B)

(C)

(D)

GlucN

GlucN

GlucNAc

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.

Figure. 1.

Method Validation

Samplesa HPLC-ELSD amount (%)

HPLC-RI b

amount (%)Spectrophotometry c

amount (%)Specifications d

(assay %)

Single capsule 99.5 e 108.9 100.5 90.0 - 110.0

Multi-capsule 102.1 103.2 107.8 90.0 - 120.0

Multi- tablet 99.8 97.2 100.8 90.0 - 120.0

a Three different lots of health/functional foods formulations b Refractive index detectionc p-Dimethylaminobenzaldehyded USP requirements of glucosamine content upon the labeled amounte Data expressed with mean of three replicates

Table 3. Comparative determination of GlucN in Helath/Functional Foods

by HPLC/ELSD and classical methods

Method Validation

IS IS

GlucN (a) (b)

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).

Method Validation

Table 4. Comparative analysis of raw materials by the HPLC/ELSD and HPLC/RI methods

List RI ELSD

Time consuming to Equilibrate

6 hours 15 min

Sensitivity 1 mg – 10 mg/mL 1 ug – 1 mg/mL

Solvent Mobile phase Selectivity

Gradient Impossibility Possibility

- Comparative analysis of raw materials

AnalytesAssay

RI (%) ELSD (%)

R-1 (GlucN HCl) 100 103

R-2 (GlucN HCl) 98 98

R-3 (GlucN HCl) 100 102

R-4 (GlucN HCl) 103 97

R-5 (GlucN HCl) 102 102

R-6 (GlucN HCl) 102 103

R-7 (GlucN HCl) 100 99

R-8 (GlucN Sulfate) 104 102

- Problems with Refractive IndexQuality Control

Table 5. HPLC/ELSD assay of GlucN in Health/Functional Foods formulation (Single components)

SampleLabeled amount

(mg)HPLC amount (mg) Assasy(%)

S-1(sulfate) 275mg/500mg 276mg/500mg 100.5

S-2(HCl) 456mg/570mg 459mg/570mg 100.8

S-3(HCl) 440mg/550mg 442mg/550mg 100.6

S-4(HCl) 440mg/550mg 465mg/550mg 105.8

S-5(HCl) 800mg/1000mg 812mg/1000mg 101.6

S-6(HCl) 440mg/550mg 446mg/550mg 101.4

S-7(HCl) 440mg/550mg 454mg/550mg 103.3

S-8(HCl) 400mg/500mg 418mg/500mg 104.7

S-9(HCl) 440mg/500mg 445mg/500mg 101.2

S-10(HCl) 320mg/400mg 328mg/400mg 102.7

S-11(HCl) 440mg/550mg 450mg/550mg 102.4

S-12(HCl) 400mg/500mg 419mg/500mg 104.8

S-13(Sulfate) 300mg/550mg 302mg/550mg 100.9

S-14(HCl) 880mg/1100mg 923mg/1100mg 104.9

S-15(HCl) 440mg/550mg 474mg/550mg 107.9

S-16(HCl) 360mg/450mg 377mg/450mg 104.8

S-17(HCl) 480mg/600mg 517mg/600mg 107.9

S-18(HCl) 440mg/550mg 447mg/550mg 101.6

S-19(Sulfate) 302.5mg/550mg 331mg/550mg 109.5

S-20(HCl) 440mg/550mg 451mg/550mg 102.6

Quality Control

SampleLabeled amount

(mg)HPLC amount

(mg)Assasy(%)

M-1(HCl) 250mg/800mg 265mg/800mg 106.1

M-2(Sulfate) 440mg/2000mg 427mg/2000mg 97.1

M-3 500mg/1300mg 485mg/1300mg 96.9

M-4 120mg/600mg 125mg/600mg 104.5

M-5(Sulfate) 202mg/1010mg 212mg/1010mg 105.0

M-6(HCl) 515mg/1009mg 519mg/1009mg 100.7

M-7(HCl) 400mg/2000mg 436mg/2000mg 109.1

M-8(HCl) 600mg/1050mg 612mg/1050mg 102.1

M-9(HCl) 375mg/1000mg 375mg/1000mg 99.9

(Multi components)

GlucNAc

Biological Study

Figure 3. Allication in Biologycal study : (a) blank serum (b) serum sample 6

0 min after oral dosing of the rabbit with 160mg/kg of GlucN.

(B)

(A)

- Simple and sensitive HPLC-ELSD method to simultaneous analyze Gluc

N and GlucNAc which applicable in routine quality control of Health/Fun

ctional Foods and biological study.

- Fast and specific since two compounds can be analyzed in one run requir

ing only 15 min for determination.

- Moreover this technique is very effective since it does not require expensi

ve time-consuming stabilization or analytical procedures as other approac

hes for GlucN analysis in assay.

- This assy can be used for content uniformity testing of dosage forms cont

aining GlucN and GlucNAc, quality control of raw materials and bioavaila

bility studies.

Conclusion

Thank you for your attention !!