CHAPTER 3

SIMPLE AND RAPID SPECTROPHOTOMETRICDETERMINATIONS OF GLUCOSAMINE HYDROCHLORIDEAND GLUCOSAMINE SULPHATE IN PHARMACEUTICALSAND SINGLE CRYSTAL X-RAY STRUCTURE OFGLUCOSAMINE HYDROCHLORIDE

3. 1 INTRODUCTION

3. 2 ANALYTICAL CHEMISTRY

3. 3 EXPERIMENTAL

3. 4 RESULTS AND DISCUSSION

3. 5 SINGLE CRYSTAL XRD OF GLUCOSAMINE

HYDROCHLORIDE

3. 6 CONCLUSIONS

3. 7 REFERENCES

92

3. 1 INTRODUCTION

Osteoarthritis (OA) is a degenerative disease of the cartilage in the joints of

the human body and is generally characterized by pain or swelling in the affected

joint and has an increased occurrence in women and those who are overweight 1,2.

Chemically it is characterized by a change in the composition of the extra cellular

matrix of the joint. OA is the most common arthritic disease and its incidence

increases with age. As population demographics changes to include more elderly

individuals, this disease will have a serious impact in multiple ways. Along with the

cost for health care and lost work time, individuals with OA suffer from pain and

disability.

Glucosamine is an amino monosaccharide that is classified by food and drug

administration (FDA) as a dietary supplement and which has gained a great deal of

public interest as a potential treatment for OA 3,4. It is a natural amine sugar

extracted from the chitin in a sea shrimp and crab shell and is an intermediate

substrate involved in the biosynthesis of proteoglycans and glycosaminoglycan that

are found in gastrointestinal mucosal membranes, the articular cartilage matrix and

synovial fluid. It is a precursor of the disaccharide unit of glycosaminoglycan which

is the building blocks of the articular cartilage, the proteoglycans 5-9. However, the

ability to synthesize glucosamine in the body declines with age. This, in turn,

incapacitates the generation of proteoglycans and it is known that this incapacitation

results in senile osteoarthritis10. Therefore, glucosamine drew attention as a useful

substance for prevention and treatment of arthritis, especially glucosamine

hydrochloride (GLH) and glucosamine sulphate (GLS) 11,12. Chitin, together with

cellulose and starch are the three most abundant polysaccharides in nature. Chitosan,

the deacetylated form of chitin which is produced enzymatically or chemically, have

been widely used in pharmaceutical, agriculture and industrial fields for their

extraordinary properties and has been regarded as a ‘biomaterial in waiting’ in 21st

century. In vitro studies showed a positive effect of glucosamine on

glycosaminoglycan production in human chondrocyte cell cultures and the same

anabolic effect was found on bovine and rat explants. The antioxidant activity of

GLH was investigated employing various in vitro systems and has also been

reported in the literature13. The efficacy of GLH for the OA of the knee has been

reported in the literature14. Currently, there is no specific treatment to prevent or

93

retard the cartilage degradation in OA. Present treatments used for OA provide only

symptomatic relief from the pain. GLS, which has received attention as a putative

agent that may retard cartilage structural degradation in OA, has been investigated in

several OA trials15,16. The mechanism of retardation of cartilage degradation by

glucosamine is not known. Glucosamine has been demonstrated to have

immunosuppressive and tumor-inhibiting activity17,18. All these pleiotropic effects of

glucosamine may individually or collectively have a chondroprotective effect.

The importance of GLH and GLS for prevention and treatment of

osteoarthritis has been reported in the literature 19. It is notable that while both the

hydrochloride salt and glucosamine sulphate are used in pharmaceutical

preparations, glucosamine sulphate is thought to have higher biological activity due

to the presence of sulphate20. GLS is a water-soluble amine sugar that is extensively

used in the treatment of various conditions of arthritis and collagen deficiency.21-24

The crystal structure of glucosamine hydrochloride has been reported

earlier25-27. In view of the importance of glucosamine hydrochloride, redetermination

of the structure of glucosamine hydrochloride was performed along with the

spectrophotometric determination of glucosamine hydrochloride and glucosamine

sulphate.

3. 2 ANALYTICAL CHEMISTRY

Several techniques have been developed for the determination of

glucosamine, glucosamine hydrochloride (GLH) and glucosamine sulphate (GLS),

which rely upon sophisticated and also expensive chemicals 28-33.

Gaonkar et al.,34 reported a rapid and sensitive method for the determination

of glucosamine sulphate by uv-spectrophotometry using phenylisothiocyanate in

presence of base. Beer’s law obeyed in the range of 5-25 g mL-1. The method was

validated for linearity, precision, accuracy and specificity. The method has been

successfully applied for the quantification of glucosamine sulphate in formulations.

Roda et al.,35 developed a sensitive and specific HPLC-ESI- MS/MS method for the

direct determination of glucosamine. The method was applied for the determination

of glucosamine concentrations in human plasma samples.

94

Shao et al.,36 reported a stability-indicating high performance liquid

chromatographic (HPLC) method for the assay of glucosamine in bulk forms and

solid dosage formulations. The method was validated for specificity, linearity,

solution stability, accuracy, precision, limit of detection and limit of quantitation.

The detector response for glucosamine hydrochloride was linear over the selected

concentration range from 1.88-5.62 mg mL-1 with a correlation coefficient 0.9998.

The method was successfully used for the analysis of active-excipient compatibility

samples used for development of solid dosage formulations.

Sullivan and Sherma37 developed a quantitative method using silica gel

HPTLC-densitometry for the determination of glucosamine in nutritional

supplements. It has been demonstrated that the validation data meet the acceptance

criteria for accuracy, precision, linearity, detection and quantification limits set by

ICH for the assay of pharmaceutical products.

Liang et al.,38 reported a selective and specific HPLC method to quantitate

glucosamine hydrochloride in raw materials, dosage and plasma. The method was

found to have excellent linearity, accuracy and precision.

An optimized reverse-phase HPLC method has been developed by Tekko et

al.,39 for the determination of glucosamine hydrochloride based on the reaction of

phenylisothiocyanate with glucosamine in alkaline media. The method has been

validated for the determination of glucosamine hydrochloride permeating through

human skin in vitro.

Yamaguchi et al.,40 developed a method for the spectrophotometric

determination of glucosamine and its analogous amino sugars with o-

hydroxyhydroquinonephthalein and palladium (II). The method was based

on the fading of the palladium(II)-o-hydroxyhydroquinonephthalein-

hexadecyltrimethylammonium complex.

A simple, rapid, selective and specific HPLC method has been developed to

quantitate glucosamine and its (β-1-4)-D-polymeric form chitosan by El-Saharty and

Bary41. The method has been validated for the determination of glucosamine

sulphate and chitosan in raw materials and pharmaceutical formulations.

95

Aghazadeh-Habashi et al.,42 developed a high performance liquid

chromatographic method for the determination of glucosamine in rat plasma. The

assay was linear over the range of 1.25-400 µg mL-1 (CV<10%) with a detection

limit of 0.63 µg mL-1 and a limit of quantification of 1.25 µg mL-1.

The present investigation describes spectrophotometric method for the

determination of glucosamine hydrochloride and glucosamine sulphate in pure form

and in formulations. The method is simple, rapid, accurate and easy to apply in

routine analysis.

3. 3 EXPERIMENTAL

3. 3. 1 Apparatus

A Secomam Anthelie NUA 002 UV-VIS Spectrophotometer / Shimadzu

UV-2550 UV-VIS Spectrophotometer with 1cm matched quartz cells were used for

absorbance measurements. Bruker SMART CCD area detector diffractometer was

used for X-ray data collection. For absorption correction multi-scan SADABS and for

cell refinement SAINT were used. SHELXS97 and SHELXL97 programs were used

to solve structure and to refine the structure and for molecular graphics ORTEP-3

was used.43-45

3. 3. 2 Reagents and Solutions

All reagents used were of analytical reagent grade and distilled water was

used for the preparation of all solutions. A 1000 µg mL-1 standard drug solution of

glucosamine hydrochloride and glucosamine sulphate were prepared in distilled

water. The stock solution was diluted appropriately to get the working

concentration. Hydrochloric acid (2 M), chloramine-T (CAT) (0.02 M), xylene

cyanol FF (XCFF) (0.05 %) and crystal violet (CV) (0.01 %) were used.

3. 3. 3 Procedures

3. 3. 3. 1 Determination of glucosamine hydrochloride using xylene cyanol FF

as reagent

Different aliquots (0.2 – 1.6 µg mL-1) of GLH were transferred into a series

of 10 mL calibrated flasks by means of a micro burette. Then, 1mL of 2M HCl was

added followed by 0.5 mL of CAT solution. The contents were shaken well and

were set aside for 15 minutes with occasional shaking. Then, 0.5 mL of XCFF was

added to each flask, and the volume was adjusted up to the mark with distilled water

96

and mixed well. The absorbance of each solution was measured at 615 nm against

the corresponding reagent blank. Reagent blank was prepared by replacing the

analyte (drug) solution with distilled water. The absorbance corresponding to the

bleached color, which in turn corresponds to the drug concentration, was obtained

by subtracting the absorbance of the blank by that of the test solution.

3. 3. 3. 2 Determination of glucosamine hydrochloride using crystal violet as

reagent

Different aliquots (0.2 – 2.0 µg mL-1) of GLH were transferred into a series

of 10 mL calibrated flasks by means of a micro burette. Then, 1mL of 2M HCl was

added followed by 0.5 mL of CAT solution. The contents were shaken well and

were set aside for 15 minutes with occasional shaking. Then, 1mL of CV was added

to each flask, and the volume was adjusted up to the mark with distilled water and

mixed well. The absorbance of each solution was measured at 590 nm against the

corresponding reagent blank.

3. 3. 3. 3 Determination of glucosamine sulphate using xylene cyanol FF as

reagent

Different aliquots (0.2 – 1.6 µg mL-1) of GLS were transferred into a series

of 10 mL calibrated flasks by means of a micro burette. Then, 1mL of 2M HCl was

added followed by 1 mL of CAT solution. The contents were shaken well and were

set aside for 15 minutes with occasional shaking. Then, 0.5 mL of XCFF was added

to each flask, and the volume was adjusted up to the mark with distilled water and

mixed well. The absorbance of each solution was measured at 615 nm against the

corresponding reagent blank.

3. 3. 3. 4 Determination of glucosamine sulphate using crystal violet as reagent

Different aliquots (0.2 – 1.4 µg mL-1) of GLS were transferred into a series

of 10 mL calibrated flasks by means of a micro burette. Then, 1mL of 2M HCl was

added followed by 1 mL of CAT solution. The contents were shaken well and were

set aside for 15 minutes with occasional shaking. Then, 1mL of CV was added to

each flask, and the volume was adjusted up to the mark with distilled water and

mixed well. The absorbance of each solution was measured at 590 nm against the

corresponding reagent blank.

3. 3. 3. 5 Analysis of dosage forms

97

Three different GLS dietary supplement products were purchased and a

sample stock solution of each was prepared by grinding ten tablets using a mortar

and pestle. An amount of powdered tablets equivalent to 100 mg was transferred

into a 100 mL volumetric flask by washing with 70 mL deionized water. The

contents of the flask were stirred for about 30 minutes and sonicated for 15 minutes

for complete dissolution of the tablet. Then, the volume was made up to the mark

with water, mixed well, and filtered. The filtrate was diluted to get working

concentration for analysis by spectrophotometric methods.

3. 3. 3. 6 Preparation of crystals

Glucosamine hydrochloride (C6H14NO5+.Cl-) was obtained from Strides Arco

Labs (SeQuent Scientific Ltd.), Mangalore, India and recrystallized from water.

Mp:449-451K.

3. 4 RESULTS AND DISCUSSION

The determination of GLH and GLS are indirect and are based on the

determination of surplus CAT after the oxidation reaction of GLH / GLS by the

later. The method is based on the reaction of surplus CAT with the corresponding

dye solution in acidic medium, which bleaches the colored dye solution to colorless

leucoform, the decoloration being caused by the oxidative destruction of the dye,

which were measured at 590 and 615 nm for XCFF and CV respectively (Figure

III. 1 & III. 2) and the reaction mechanism are shown in Scheme 3. 3 and 3. 4.

3. 4. 1 Optimization of Experimental Conditions

The drug undergoes oxidation to the corresponding aldehyde according to

the reaction scheme given in Scheme 3. 1 and 3. 2, since the reaction stoichiometry

is found to be 1:1. The oxidation is found to be complete and quantitative in 10 -15

minutes. Formation of corresponding aldehyde is confirmed by performing

Borsche’s reagent test.

Many dyes are irreversibly destroyed to colorless products in acidic medium

by oxidizing agents and this has been exploited for the indirect spectrophotometric

determination of GLH and GLS. GLH and or GLS when added in increasing

concentration to a fixed concentration of CAT, consumes the later and there will be

a concomitant decrease in its concentration. A concomitant increase in the

98

concentration of dye resulted when a fixed concentration of the dye is added to

decreasing concentration of CAT.

Preliminary experiments are performed to fix the concentration of the dye

that could be measured spectrophotometrically and are found to be 0.05 % (0.5 mL)

and 0.01 % (1 mL) for XCFF and CV respectively. For both GLH and GLS, HCl

medium is found to be ideal and 1 mL of 2 M HCl in a total volume of 10 mL is

adequate for the oxidation process to take place. A 5 minute standing time is found

necessary for the complete bleaching of the dye color by CAT. Under these

conditions, the system is stable for a period of over 6 hrs.

3. 4. 2 Analytical Data

A linear correlation is found between absorbance and concentration of the

drug (Figure III. 3, III. 4, III. 5 & III. 6). The optical parameters such as molar

absorptivity, Beer’s law limit and Sandell’s sensitivity values are calculated and are

given in (Table 3. 1). Correlation coefficients, intercepts and slopes for the

calibration graphs are also compiled in (Table 3. 1). The limit of detection and

quantification calculated according to ICH guidelines are also given in (Table 3. 1).

3. 4. 3 Method Validation

To evaluate the accuracy and precision of the methods, pure drug within the

working limits are analyzed, each determination being repeated three times. The

relative error (%) and RSD (%) values are less than 3 and indicate good accuracy

and precision for the methods (Table 3. 2). For a better picture of reproducibility on

a day-to-day basis, a series is run in which standard drug solution at three levels is

determined each day for a week, preparing all solutions fresh. The relative standard

deviation values are in the range of 0.2- 2.0 % and represent the best appraisal of the

procedures in daily use.

The proposed method is applied to the determination of GLS in three

commercial dietary supplements (tablets) and the results are compiled in

(Table 3. 3). To a fixed amount of drug in dosage forms pure GLS solution at three

different levels (within the working limit) is analyzed, each being repeated three

times. The accuracy of the proposed method is checked by a thorough replicate

analysis of each spiked sample. The results are also compared statistically with those

99

of the reference method at 95 % confidence level34. The calculated student’s t- and

F-test values (Table 3. 3) did not exceed the tabulated value, indicating that there is

no significant difference between the proposed methods and the reference method in

respect to accuracy and precision.

3. 4. 4 Interference Study

In the pharmaceutical analysis, it is important to test the selectivity towards

the excipients and fillers added to the pharmaceutical preparations. Several species

which can occur in the real samples together with drug are investigated. The level of

interference is considered tolerable (Table 3. 4). From this study it is apparent that

the usual co-formulated substances seldom interfere in the proposed methods.

3. 5 SINGLE CRYSTAL XRD OF GLH

The previously reported crystal structures of GLH have been confirmed by

high precision, and the H atoms located, allowing the elucidation of hydrogen

bonding network. All the geometrical values (Figure III. 7) fall within the expected

range27 and the six membered ring is well described as a chair for atoms C1, C2, C4

and C5, r.m.s deviation from their mean plane is 0.021 Å, C3 and O1 deviate from the

plane by -0.652(2) Å and 0.6477(19) Å, respectively. The hydrogen atoms have

been located, allowing the hydrogen bonding scheme, involving combination of O-

H---O, N-H---O, O-H---Cl and N-H----Cl links (Table 3. 8). The interaction of N-

H---O and O-H---O bonds result in undulating (001) sheets (Figure III. 8). The O-

bound atoms are located in a different map and refined as riding in their as-found

relative positions with Uiso (H) = 1.2 Uiso (O). The other hydrogen atoms are

geometrically placed (C-H = 0.97 – 0.98 Å, N-H = 0.89 Å) and refined as riding

with Uiso (H) = 1.2 Ueq (carrier). The -NH3+ group is allowed to rotate, but not to tip,

to best fit the electron density.

The crystal data and other relevant parameters regarding data collection, data

reduction, structure solution and refinement are given in Table 3. 5. The atomic

coordinates with their equivalent displacement parameters, anisotropic displacement

parameters and hydrogen bonding geometry are compiled in Table 3. 6, Table 3. 7

and Table 3. 8 respectively.

100

FIGURE III. 7 - VIEW OF THE MOLECULAR STRUCTURE OF α-D- GLUCOSAMINE HYDROCHLORIDE, SHOWING 50% DISPLACEMENT ELLIPSOIDS (ARBITRARY SPHERES FOR THE H ATOMS). THE HYDROGEN BOND IS SHOWN AS A DOUBLE DASHED LINE.

FIGURE III. 8 - PART OF AN (001) SHEET OF CATIONS IN (I) LINKED BY O— H···O AND N—H···O HYDROGEN BONDS.

101

3. 6 CONCLUSIONS

Simple and facile titrimetric and spectrophotometric methods for the

determination of GLH and GLS have been developed and validated.

The method has been successfully applied for the determination of GLS in

tablets.

The ingredients usually present in the pharmaceutical formulations of these

drugs seldom interfere in the proposed method.

Hence the proposed method can be used for the routine determination of

GLH and GLS in pure and dosage forms.

Single crystal XRD of GLH has been performed and the structure has been

redetermined including the elucidation of hydrogen bond parameters.

102

TABLE 3. 1- ANALYTICAL PARAMETERS

ParametersGlucosamine Hydrochloride

(GLH)

Glucosamine Sulphate

(GLS)λ max (nm) 615 590 615 590

Beer’s law limits

(µg mL-1)

0.2 – 1.6 0.2 – 2.0 0.2 – 1.6 0.2 – 1.4

Molar absorptivity

(L mol-1 cm-1)

0.824 × 104 0.577 × 104 0.833 ×104 1.66 × 104

Sandell’s sensitivity

(µg cm -2)

3.64 × 10-2 8.04 × 10-2 8.19 × 10-2 5.12 × 10-2

Limit of detection**

(µg mL-1)

0.156 0.236 0.623 0.452

Limit of quantification**

(µg mL-1)

0.474 0.709 1.886 1.369

Regression equation* Y= 0.0113 +

0.0211X

Y= 0.0154 +

0.0141X

Y= 0.0157

+ 0.0053X

Y= 0.0325

+ 0.0073XSlope (b) 0.0211 0.0141 0.0053 0.0073Intercept (a) 0.0113 0.0154 0.0157 0.0325Correlation coefficient

(R)

0.9995 0.9980 0.9981 0.9989

* Y is the absorbance and X is the concentration in µg mL-1

** Calculated using ICH - Guidelines

TABLE 3. 2- EVALUATION OF ACCURACY AND PRECISION

103

Glucosamine Hydrochloride (Using Xylene Cyanol FF as Reagent)

Amount taken

(µg mL-1)

Amount found*

(µg mL-1)

RE

(%)

SD

(µg mL-1)

RSD

(%)

0.2 0.200 0.10 0.003 1.244

0.4 0.401 0.15 0.001 0.285

0.6 0.601 0.17 0.002 0.311

0.8 0.801 0.08 0.002 0.301

1.0 1.002 0.14 0.005 0.487

1.2 1.202 0.16 0.008 0.696

1.4 1.402 0.14 0.008 0.597

1.6 1.606 0.38 0.023 1.433

Glucosamine Hydrochloride (Using Crystal Violet as Reagent)

Amount taken

(µg mL-1)

Amount found*

(µg mL-1)

RE

(%)

SD

(µg mL-1)

RSD

(%)

0.2 0.201 0.20 0.002 0.906

0.4 0.401 0.20 0.002 0.480

0.6 0.602 0.23 0.002 0.252

0.8 0.801 0.13 0.002 0.249

1.0 1.003 0.28 0.007 0.699

1.2 1.202 0.18 0.015 1.232

1.4 1.401 0.11 0.011 0.771

1.6 1.601 0.08 0.007 0.456

1.8 1.805 0.26 0.011 0.619

2.0 2.004 0.20 0.023 1.149

Glucosamine Sulphate (Using Xylene Cyanol FF as Reagent)

104

Amount taken(µg mL-1)

Amount found*(µg mL-1)

RE(%)

SD(µg mL-1)

RSD(%)

0.2 0.202 1.0 0.002 0.990

0.4 0.401 0.15 0.002 0.518

0.6 0.603 0.50 0.002 0.262

0.8 0.802 0.25 0.002 0.249

1.0 1.003 0.28 0.007 0.699

1.2 1.202 0.18 0.015 1.232

1.4 1.405 0.34 0.011 0.796

1.6 1.604 0.25 0.006 0.353

Glucosamine Sulfate (Using Crystal Violet as Reagent)

Amount taken

(µg mL-1)

Amount found*

(µg mL-1)

RE

(%)

SD

(µg mL-1)

RSD

(%)

0.2 0.201 0.20 0.0018 0.906

0.4 0.401 0.05 0.0019 0.481

0.6 0.602 0.23 0.0015 0.252

0.8 0.801 0.10 0.0018 0.223

1.0 1.006 0.60 0.0195 1.937

1.2 1.204 0.37 0.0113 0.935

1.4 1.400 0.03 0.0021 0.148

* Mean value of five determinations

RE – Relative Error; SD- Standard Deviation; RSD- Relative Standard Deviation

TABLE 3. 3- RESULTS OF ASSAY OF FORMULATIONS BY THE PROPOSED

105

METHOD

Brand name oftablet

Labeledamount

(mg)

Found* ±SD

UsingXCFF(mg)

Found* ±SD

UsingCV(mg)

ReferenceMethod

Jointace – DN 750 752.00 ± 0.09 751.96 ± 0.03 -

Cartisafe – D 750 749.27 ± 0.02 749.00 ± 0.08 -

Cartilamine 500

499.66 ± 0.08at = 2.14 bF = 1.31

499.72 ± 0.05at =1.30 bF =1.96

499.8 ± 0.09

* Mean value of three determinations aTabulated t-value at 95% confidence level is 2.78

bTabulated F-value at 95% confidence level is 6.39

TABLE 3. 4 – AMOUNT OF TOLERANCE OF EXCIPIENTS

Excipients Amount (mg)

Lactose 20

Starch 30

Talc 30

Sucrose 10

Stearic acid 20

Fructose 15

Glucose 10

TABLE 3. 5 - CRYSTAL DATA AND STRUCTURE REFINEMENT

106

TABLE 3. 6 - ATOMIC COORDINATES WITH THEIR EQUIVALENT

107

DISPLACEMENT PARAMETERS

TABLE 3. 7- ATOMIC DISPLACEMENT AND GEOMETRIC PARAMETERS

108

TABLE 3. 8- HYDROGEN BOND GEOMETRY

TABLE 3. 8- HYDROGEN BOND GEOMERTY

109

Wavelength (nm)

450 500 550 600 650 700 750

Abs

orba

nce

0.0

0.1

0.2

0.3

0.4

0.5

FIGURE III. 1 – ABSORPTION SPECTRUM OF XYLENE CYANOL FF

110

Wavelength (nm)

500 520 540 560 580 600 620 640 660

Abs

orba

nce

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

FIGURE III. 2 – ABSORPTION SPECTRUM OF CRYSTAL VIOLET

Concentration (g mL-1)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Abs

orba

nce

FIGURE III. 3 – ADHERENCE OF BEER’S LAW FOR THE DETERMINATION

OF GLH USING XYLENE CYANOL FF AS A REAGENT

111

Concentration (g mL-1)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2

Abso

rban

ce

0.013

0.014

0.015

0.016

0.017

0.018

0.019

0.020

0.021

FIGURE III. 4 – ADHERENCE OF BEER’S LAW FOR THE DETERMINATION

OF GLH USING CRYSTAL VIOLET AS A REAGENT

0.0 0.5 1.0 1.5 2.0 2.5 3.0

0.014

0.016

0.018

0.020

0.022

0.024

0.026

Abs

orba

nce

Concentration (g mL-1)

FIGURE III. 5 – ADHERENCE OF BEER’S LAW FOR THE DETERMINATION

OF GLS USING XYLENE CYANOL FF AS A REAGENT

112

0.0 0.5 1.0 1.5 2.0 2.5 3.0

0.032

0.033

0.034

0.035

0.036

0.037

0.038

0.039

0.040

Abs

orba

nce

Concentration (g mL-1)

FIGURE III. 6 – ADHERENCE OF BEER’S LAW FOR THE DETERMINATION

OF GLS USING CRYSTAL VIOLET AS A REAGENT

SO3Na

SO3H

NH

CH3CH3

CH3

N

CH3

CAT/H+

SO3Na

SO3H

NH

CH3CH3

CH3

NH

CH3

Xylene Cyanol FF (Blue Color) Xylene Cyanol FF (Leucoform)

SCHEME 3. 3 - REACTION BETWEEN (GLH/GLS) -CAT SYSTEMS WITH

XCFF

113

N+

N NCH3

CH3 CH3

CH3

CH3CH3 N

N NCH3

CH3 CH3

CH3

CH3CH3

CAT/H+

Crystal Violet Crystal Violet (Leucoform)

SCHEME 3. 4 - REACTION BETWEEN (GLH/GLS) -CAT SYSTEM WITH CV

O

OHH

HH

OHOH

H NH3. Cl

H

OH

+

CH3

O2SN

Cl

H+

O

OHH

HH

OHOH

H NH3. Cl

H

CHO

+

CH3

O2SNH2

+ NaCl

+

-Na

+

+

-

-

SCHEME 3. 1 - REACTION OF GLH WITH CAT IN ACIDIC MEDIUM

114

O

OHH

HH

OHOH

H NH3

H

OH

+

CH3

O2SN

Cl

H+

O

OHH

HH

OHOH

H NH3

H

CHO

+

CH3

O2SN

H

H

+ NaCl

+

- Na+

+

SO42-.

2

2

. SO42-

SCHEME 3. 2 - REACTION OF GLS WITH CAT IN ACIDIC MEDIUM

3. 7 REFERENCES

[1]. R. Jurmain, Stories from the Skeleton, Behavioral Reconstruction in Human

Osteology, Gordon and Breach science publishers, Amsterdam, The

Netherlands (1999).[2]. N. Ishiguro, T. Kojima & A. R. Poole, Nagoya J. Med. Sci. (2002) 65, 73. [3]. M. O’Rourke, Nurse Pract. (2001) 26, 44.[4]. G. Hawker, Curr. Opin. Rheumatol. (1997) 9, 90.[5]. H. W. Greiling, W. Stuhlsatz & U. Tillmans, In Methods of Enzymatic

Analysis, Bergmeyer, H.U. (Ed.) 60 (1984).[6]. A. Conte, N. Volpi, L. Palimiera, I. Bahous, G. Ronca & F. Arzneim, Drug

Res. (1995) 45, 918. [7]. M. Huber & R. Bill, Compend. Contin. Educ. Pract. Vet. (1994) 16, 501.[8]. R. J. Todhunter & G. J. Lust, Am. Vet. Med. Assoc. (1994) 204, 1245.[9]. R. D. Altman, D. Dean, O. E. Muniz & D. Howell, Arthritis Rheum. (1989)

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