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Chapter-IV

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4.1. GLUCOSAMINE HYDROCHLORIDE

Glucosamine hydrochloride (Figure-IV.1) is commonly used for arthritis (1).

Glucosamine hydrochloride is a naturally occurring chemical found in the human

body. It is in the fluid that is around joints (2). Glucosamine is also found in other

places in nature. For example, the glucosamine hydrochloride that is put into dietary

supplements is often harvested from the shells of shellfish (3-5). Glucosamine

hydrochloride used in dietary supplements does not always come from natural

sources.

Glucosamine is an amino sugar produced from the shells of chitin (shellfish)

and a key component of cartilage. Glucosamine works to sti-mulate joint function and

repair. It has been proven effective in numerous scientific trials for easing

osteoarthritis pain, aiding in the rehabilitation of cartilage, renewing synovial fluid,

and repairing joints that have been da-maged from osteoarthritis. Over the years,

people have tried glucosamine for a variety of other uses. For example, it has been

tried for glaucoma and for weight loss. But glucosamine has not been adequately

studied for these uses.

Figure-IV.1 : Glucosamine chemical structure

Glucosamine is not a stable compound in its free-base form, and there are

numerous more-stable forms of glucosamine available to consumers (6). There are

different forms of glucosamine including glucosamine sulfate, glucosamine

hydrochloride, and N-acetyl-glucosamine. These different chemicals have some

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similarities; however, they may not have the same effects when taken as a dietary

supplement.

Glucosamine is also in some skin creams used to control arthritis pain. These

creams usually contain camphor and other ingredients in addition to glucosamine (7).

Researchers believe that any pain relief people may experience from these creams is

due to ingredients other than glucosamine.

Glucosamine hydrochloride is a chemical found in the human body. It is used

by the body to produce a variety of other chemicals that are involved in building

tendons, ligaments, cartilage, and the thick fluid that surrounds joints (8). Joints are

cushioned by the fluid and cartilage that surround them. In some people with

osteoarthritis, the cartilage breaks down and becomes thin. This results in more joint

friction, pain, and stiffness (9). Researchers think that taking glucosamine supplements

may either increase the cartilage and fluid surrounding joints or help prevent

breakdown of these substances, or maybe both (10-11).

4.2. CHONDROITIN SULFATE

Chondroitin (Figure-IV.2) is a molecule that occurs naturally in the body (12). It

is a major component of cartilage -- the tough, connective tissue that cushions the

joints. Commercial chondroitin is derived from natural sources, such as shark and

bovine cartilage, or synthetic production. Chondroitin helps keep cartilage healthy by

absorbing fluid (particularly water) into the connective tissue. It may also block

enzymes that break down cartilage, and it provides the building blocks for the body to

produce new cartilage (13).

Chondroitin sulfate is a chemical that is normally found in cartilage around

joints in the body (14). Chondroitin sulfate is manufactured from animal sources, such

as cow cartilage. Chondroitin sulfate is used for osteoarthritis. It is often used in

combination with other products, including manganese ascorbate, glucosamine

sulfate, glucosamine hydrochloride, or N-acetyl glucosamine. Research from a couple

of decades ago showed that chondroitin sulfate helped arthritis pain when taken with

conventional medicines, such as aspirin, for pain and swelling. But later research

wasn’t so positive (15-16). Now, scientists believe that, overall, chondroitin sulfate may

http://www.webmd.com/osteoarthritis/default.htm

http://arthritis.webmd.com/default.htm

http://www.webmd.com/drugs/mono-3-ASPIRIN+-+ORAL.aspx?drugid=1082&drugname=Aspirin+Oral

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reduce arthritis pain slightly (17-18). Some people use chondroitin sulfate for heart

disease, weak bones (osteoporosis), and high cholesterol. Chondroitin sulfate is also

used in a complex with iron for treating iron-deficiency anemia. Chondroitin sulfate is

available as an eye drop for dry eyes. In addition, it is used during cataract surgery,

and as a solution for preserving corneas used for transplants. It is approved by the

FDA for these uses.

Figure- IV.2 : Chondroitin chemical structure

Chondroitin sulfate was originally isolated well before the structure was

characterised, leading to changes in terminology with time. Early researchers

identified different fractions of the substance with letters. (Table-IV.1)

Table-IV.1 : Chondroitin therapeutic use

Letter identification Site of sulfation Systematic name

Chondroitin sulfate A carbon 4 of the N-acetylgalactosamine

(GalNAc) sugar chondroitin-4-sulfate

Chondroitin sulfate C carbon 6 of the GalNAc sugar chondroitin-6-sulfate

Chondroitin sulfate D carbon 2 of the glucuronic acid and 6 of

the GalNAc sugar chondroitin-2,6-sulfate

Chondroitin sulfate E carbons 4 and 6 of the GalNAc sugar chondroitin-4,6-sulfate

"Chondroitin sulfate B" is an old name for dermatan sulfate, and it is no longer

classified as a form of chondroitin sulfate (19).

Dosage:

Doses of 200-400 milligrams by mouth twice to three times daily, or 800-1,200

milligrams once daily have been used in studies (20). Higher doses (up to 2,000

milligrams) appear to have similar efficacy. In the treatment of osteoarthritis, full

effects may take several weeks to occur.

http://www.webmd.com/heart-disease/default.htm

http://www.webmd.com/heart-disease/default.htm

http://www.webmd.com/osteoporosis/default.htm

http://www.webmd.com/cholesterol-management/default.htm

http://www.webmd.com/a-to-z-guides/understanding-anemia-basics

http://www.webmd.com/eye-health/picture-of-the-eyes

http://www.webmd.com/fitness-exercise/tc/life-after-transplant-safe-activities-exercise

http://en.wikipedia.org/wiki/File:Chondroitin_Sulfate_Structure_NTP.png

http://en.wikipedia.org/wiki/Dermatan_sulfate

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It is not clear what dose is optimal when used in combination with

glucosamine or whether the combination is as effective as or more effective than

either agent alone.

For osteoarthritis, 50-100 milligrams as a single daily injection or divided into

two daily injections has been used. Medical supervision is recommended.

Children (younger than 18 years)

There is no proven effective dose for chondroitin in children. Alternatively, adults can

take 600 mg of this supplement twice daily. As the safety of chondroitin sulfate has

not been studied in children, do not give this supplement to a child (21-23).

4.3. Impurities

Impurities and respective chemical structures of the impurities present in

Glucosamine HCl are given in the Figure- IV.3

Structures of Impurities:

Chemical name Structure

N – Acetyl –

Glucosamine

Pyrazine

5 – Hydroxymethyl – 2

– Furaldehyde

2 – Furaldehyde

Pyrrole – 2 –

Carboxaldehyde

Figure- IV.3 : Chemical structure

http://en.wikipedia.org/wiki/File:N-Acetylglucosamine.svg

http://en.wikipedia.org/wiki/File:Hydroxymethylfurfural.png

http://en.wikipedia.org/wiki/File:Furfural_structure.png

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4.4. Dosage forms

In the Sustain Release form only tablets oral dosage form is available.

Glucosamine and chondroitin sustained release tablets have been widely

promoted as a treatment for Osteoarthritis (OA) (24-28). Glucosamine, an amino sugar,

is for the formation and repair of cartilage. Chondroitin, a carbohydrate, is a cartilage

component that is to promote water retention and elasticity and to inhibit the

enzymes that break down cartilage (29-30). Both compounds are manufactured by the

body. Glucosamine supplements are derived from shellfish shells; chondroitin

supplements are generally made from cow cartilage (31-32).

4.5. REVIEW OF LITERTURE

4.5.1. Introduction

Osteoarthritis is the most common arthritis in the world. It affects millions of

people with age being the greatest risk factor for developing the disease. The burden

of disease will worsen with the aging of the world’s population. The disease causes

pain and functional disability.

Chondroitin sulfate consists of repeating chains of molecules called

glycosaminoglycans. It is a major constituent of cartilage, tendons and ligaments,

providing structure, holding water and nutrients, and allowing other molecules to

move through cartilage — an important property, as there is no blood supply to

cartilage. This nutrient will work by acting as a building block for proteoglycan

molecules, and also have anti-inflammatory properties. Taking orally does lead to

effective absorption.

The indirect costs include work absences and lost wages. Many studies have

sought to find a therapy to relieve pain and reduce disability.

Glucosamine hydrochloride and chondroitin Sulfate is a mucopolysaccharide found in

cartilage, tendons and ligaments, where it is bound to proteins such as collagen and

elastin. In our joints, it contributes to strength, flexibility and shock absorption.

Recent research indicates that supplementation may help maintain proper joint

function.

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Glucosamine have the reported methods like pre-derivatization, spectroscopy

methods (33-37). Chondroitin also have the reported methods like HPLC, UV, and

chemical methods (38-40) and there is no stability indicating methods for the

determination of glucosamine and chondroitin. The present research objective is to

develop a stability indicating method for the determination of both drug products

along with the impurities.

4.5.2. Literature summary:

Abimbola O (41) et al., Objective: The purpose of this report is to evaluate and

present the results of analysis of actual contents of several products in the

marketplace containing glucosamine and/or chondroitin sulfate and to determine if

they significantly deviate from label claim. In addition, the study examined the

intestinal transport of several marketed sources of chondroitin sulfate. Methods: A

total of fourteen products containing glucosamine hydrochloride or sulfate and eleven

products containing chondroitin sulfate were evaluated using a UVHPLC method. In

addition, a total of 32 products containing chondroitin sulfate were tested using a

titration method. The permeability of various marketed sources of raw materials of

chondroitin sulfate across Caco-2 cell monolayers were assessed. This analysis was an

attempt to evaluate whether different suppliers of chondroitin sulfate use different

grades of material. Results and conclusions: The amounts of glucosamine and

chondroitin found after analysis were significantly different from the label claim in

some products, with deviations from label claims ranging from as low as 0% to over

115%. Products with a retail price of less than or equal to one dollar per 1200 mg of

chondroitin sulfate were found to be seriously deficient in meeting label claim (less

than 10% of label claim). The permeability of the different molecular weight

chondroitin sulfates was found to be significantly different (p<0.05), with the

permeability coefficient increasing with decreasing molecular weight. This suggests

that molecular weight of chondroitin sulfate could be a possible predictor of

permeability. Investigation on Counterfeit Glucosamine and Chondroitin Products in

Iranian Pharmaceutical Markets Massoud Amanlou. The purpose of this report is to

present the results of analysis of actual glucosamine and/or chondroitin contents of

several such products in the market place and to determine if they significantly

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deviate from their label claim. A total of fourteen products containing glucosamine

sulfate and nine products containing chondroitin sulfate were evaluated. The

amounts of glucosamine and chondroitin were found to be significantly different from

the label claim in one product, ranging from as low as 59.00% to over 112.14% of the

label claim for glucosamine and 77.69% to over 94.86 % for chondroitin. Retail price

of the product did not appear to be related to the quantity of active ingredients. The

overall results of this study show that famous brands are better candidates for

counterfeiting than expensive ones.

Ali Aghazadeh-Habashi (42) et al., Purpose: A high performance liquid

chromatographic method was developed for the determination of glucosamine (GlcN)

in rat plasma. Method: Internal standard, galactosamine, was added to 100 mL of

plasma containing GlcN followed by precipitation of plasma proteins with acetonitrile.

Evaporation of the decanted supernatant solution was accelerated by the addition of

methanol. GlcN was derivatized by addition of a solution containing 1-naphthyl

isothiocyanate. Sample cleanup included passage through an anion exchange

cartridge. Analysis was accomplished by injection of 0.1 mL of the sample solution

into an isocratic HPLC system consisting of a C18 column, a mobile phase of

acetonitrile: water: acetic acid: triethylamine (4.5: 95.5:0.1:0.05), a flow rate of 0.9

mL/min, and a UV detector set at 254 nm. Results: Galactosamine and GlcN appeared

26 and 29 min post-injection, respectively. The assay was linear over the range of

1.25-400 mg/mL (CV<10%) with a detection limit of 0.63 μ g/mL and a limit of

quantification of 1.25 mg/mL. The method was applied to the determination of GlcN

in rat plasma after oral administration of 350 mg/kg of GlcN hydrochloride.

Conclusion: The present assay is specific, sensitive, precise, and accurate and is

suitable for pharmacokinetic studies.

Joseph Zhou (43) study was conducted on the method for the determination of

glucosamine in raw materials and dietary supplements containing glucosamine

sulfate and/or glucosamine hydrochloride by HPLC with N-(9-

fluorenylmethoxycarbonyloxy) succinimide (FMOC-Su) derivatization. Thirteen blind

duplicates of materials consisting of various commercial products including tablets,

capsules, drink mix and liquid products as well as raw materials, blanks, and spike

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recovery products were tested by twelve collaborating laboratories. The tests with

the blank samples and the samples with glucosamine spiked showed good specificity

of the method. The average spike recoveries at the spike levels of 100% and 150% of

the declared amount were 99.0% with an RSD of 2.1% and 101% with an RSD of

2.3%, respectively. The test results between laboratories on each commercial product

were reproducible with all of RSD no more than 4.0%, and the results were repeatable

in the same laboratory with an average RSD of 0.7%.HORRAT values ranged from 0.5

to 1.7 on both tests of spike recovery and reproducibility between laboratories on

commercial products. The average determination coefficient of the calibration curves

from the laboratories was 0.9995 with an RSD of 0.03%. None of the results from the

collaborating laboratories was outlier, partly indicating the robustness of the method.

It is recommended that the method be accepted by AOAC as Official First Action.

C. Sullivan (44) et al., A quantitative method using silica gel HPTLC plates,

automated bandwise sample application, detection with ninhydrin chromogenic

reagent solution, and automated visible mode densitometry has been developed for

determination of glucosamine in nutritional supplements containing a variety of other

active and inactive ingredients. Accuracy was validated by analysis of spiked blank

and standard addition samples and precision by performing replicate analysis on a

single day and different days. Recoveries of glucosamine hydrochloride from the

spiked blank and standard addition samples were 100.0% and 101.5%, respectively.

Repeatability for one sample, which was analyzed six times on a single plate, was

1.72% relative standard deviation (RSD). The intermediate precision was 1.20% RSD

for a sample analyzed in duplicate once per plate on five different days over a seven-

day period. A survey was made of free glucosamine content compared to the label

values for nine commercial supplement products using the new method, which is

shown to be suitable for routine use in nutritional supplement analysis for

manufacturing quality control or governmental regulatory purposes.

Yunqi Wu (45) a spectrophotometric method for determination of glucosamine

release from sustained release (SR) hydrophilic matrix tablet based on reaction with

ninhydrin is developed, optimized and validated. The purple color (Ruhemann

purple) resulted from the reaction was stabilized and measured at 570 nm. The

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method optimization was essential as many procedural parameters influenced the

accuracy of determination including the ninhydrin concentration, reaction time, pH,

reaction temperature, purple color stability period, and glucosamine/ninhydrin ratio.

Glucosamine tablets (600 mg) with different hydrophilic polymers were formulated

and manufactured on a rotary press. Dissolution studies were conducted (USP 26)

using deionized water at 37 ± 0.2 C with paddle rotation of 50 rpm, and samples were

removed manually at appropriate time intervals. Under given optimized reaction

conditions that appeared to be critical, glucosamine was quantitatively analyzed and

the calibration curve in the range of 0.202–2.020 mg (r = 0.9999) was constructed.

The recovery rate of the developed method was 97.8–101.7% (n = 6). Reproducible

dissolution profiles were achieved from the dissolution studies performed on

different glucosamine tablets. The developed method is easy to use, accurate and

highly cost-effective for routine studies relative to HPLC and other techniques.

4.5.3. Objective of this chapter

The objective of this chapter was to provide the stability indicating HPLC

method by using UV detector for the quantification of impurities related to

glucosamine in Glucosamine Hydrochloride 500 mg and Chondroitin Sulfate 400 mg

SR Tablets.

4.6. QUANTIFICATION OF GLUCOSAMINE IMPURITIES -METHOD

DEVELOPMENT & METHOD VALIDATIION

4.6.1. Reagents and chemicals:

Tablets and Standards of glucosamine hydrochloride impurities namely N-

Acetyl –D-Glucosamine impurity (99.0%), 5-Hydroxymethyl-2-Furaldehyde impurity

(99.3%), Pyrazine impurity (99.2%), 2-Furaldehyde impurity (99.8%), were supplied

by Genovo Development Services Limited, Bengaluru, India. All solvents were HPLC

grade and High purity water was prepared by using Millipore Milli-Q plus water

purification system (Millipore, Milford, MA, USA).

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4.6.2. Method of Analysis

Buffer Preparation:

0.8 gram of phosphoric acid (85%), 1.0 ml of Triethylamine and 1.2 gram of

Octane sulfonic acid sodium salt were transferred into 1000 ml of HPLC grade water,

and mixed well.

Mobile phase:

Buffer and Acetonitrile were taken in the ratio of 95:5 v/v respectively then

filtered through 0.45 µm membrane filter and degassed it.

Chromatographic system:

Column : Kromasil 100-5 C18; (300 × 4.0) mm; 5 µm

Column temperature : 25°C

Flow rate : 0.5 ml / minute

Injection volume : 10 µl.

Detector Wavelength : 195 nm

Diluent : Mobile phase

Run time : For Diluted Standard solution about 45 minutes,

Blank and test sample solution about 60 minutes

Impurities standard Stock Solutions:

25 mg of each impurity standards of N-acetyl-Glucosamine, 5-

Hydroxymethyl-2-Furaldehyde, Pyrazine, 2- Furaldehyde and Pyrrole-2-

Carboxaldehyde were transferred into individual each 50 ml volumetric flasks, added

35 ml of diluent into each flasks, sonicate for 10 minutes to dissolve the material and

made up the volume up to mark with diluent and mixed well to get the identical

solution.

Diluted Standard solution (Resolution solution):

5mL of each impurities standard stock solutions were transferred into 100 ml

volumetric flask and made up the volume up to mark with diluent and mixed well.

Sample Preparation:

Twenty tablets were taken and the weight was noted down. Crushed the 20

tablets in to fine powder with mortar pestle, then transferred an accurately weighed

quantity equivalent to 500 mg of Glucosamine Hydrochloride into a 100 ml

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Volumetric flask, added 70 ml of diluent, sonicate for 30 minutes with intermediate

shaking by maintaining sonicator bath temperature below at 28°C and made up the

volume up to the mark with diluent and mixed well.

Test solution was centrifuged by closing the centrifuge tube tightly with stopper or

Para film (to avoid solvent evaporation) at 3000 RPM for 10 minutes. Supernatant

solution was used for filtration through 0.45 µm PVDF filter.

Procedure:

Diluent as blank, five replicate injections of diluted Standard solution and one

injection of Sample solution were injected (each 10 µl) into HPLC. Recorded the

chromatograms and measured the peak responses.

Details of system suitability parameters and RRT values were given in Table IV.2 &3.

Table- IV.2 : System suitability limits

System suitability

parameters Observed value

Acceptance

criteria

Theoretical Plate Count for

peak from first injection of

diluted standard solution.

N – Acetyl – Glucosamine

Should be

NLT 2000

5 – Hydroxymethyl – 2 – Furaldehyde

Pyrazine

2 – Furaldehyde

Pyrrole – 2 – Carboxaldehyde

% RSD for peak area from

five replicate injections of



Should be

NMT 5.0


Pyrazine

2 – Furaldehyde


Tailing factor for peak from

first injection of diluted

standard solution.

N – Acetyl – Glucosamine Should be

NMT 2.0


2 – Furaldehyde


Tailing factor for Pyrazine peak from first injection of diluted standard

solution.

Should be

NMT 3.5

Resolution between Pyrazine and 5 – Hydroxymethyl – 2 –

Furaldehyde peaks from first injection of diluted standard solution.

Should be

NLT 2.0

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Table- IV.3 : RRT values

S. No. Impurity Name RRT

1 N – Acetyl – Glucosamine 1.00

2 Pyrazine 2.30

3 5 – Hydroxymethyl – 2 – Furaldehyde 3.11

4 2 – Furaldehyde 4.41

5 Pyrrole – 2 – Carboxaldehyde 8.19

Note:- (i) Glucosamine and Chondroitin peaks will elute like “ ” shape integrate

together. (ii) Mobile phase was very sensitive with respective to organic phase of

solvent, above mentioned RRT’s will vary slightly, so can be identified with the diluted

standard (Resolution solution) injection.

4.6.3. Calculation:

i) % of N-Acetyl-Glucosamine Impurity

AIUIT1 WS1 5 100 P1 AW

= -------------x-------------x-------------x-------------x-------------x-------------x100

ADSP1 50 100 WT 100 LC

ii) % of 5-Hydroxymethyl-2-Furaldehyde (5-HMF) Impurity


= -------------x-------------x-------------x-------------x-------------x-------------x100

ADSP2 50 100 WT 100 LC

iii) % of Pyrazine Impurity


= -------------x-------------x-------------x-------------x-------------x-------------x100

ADSP3 50 100 WT 100 LC

iv) % of 2-Furaldehyde Impurity


= -------------x-------------x-------------x-------------x-------------x-------------x100

ADSP4 50 100 WT 100 LC

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v) % of Pyrrole 2-Carboxaldehyde Impurity


= -------------x-------------x-------------x-------------x-------------x-------------x100

ADSP5 50 100 WT 100 LC

vi) % of Maximum Single unknown Impurity

AIUIT WS1 5 100 P1 AW

= -------------x-------------x-------------x-------------x-------------x-------------x100

ADSP1 50 100 WT 100 LC

% of Total unknown Impurities

ATIT WS1 5 100 P1 AW

= -------------x-------------x-------------x-------------x-------------x-------------x100

ADSP1 50 100 WT 100 LC

% of Total Impurities = (% of all known impurities + % of total unknown

Impurities)

Where,

WS1 = Weight of N-Acetyl-Glucosamine standard taken in the preparation of


WS2 = Weight of 5-Hydroxymethyl-2-Furaldehyde standard taken in the

preparation of diluted standard solution.

WS3 = Weight of Pyrazine standard taken in the preparation of diluted

standard solution.

WS4 = Weight of 2-Furaldehyde standard taken in the preparation of diluted

standard solution.

WS5 = Weight of Pyrrole 2- Carboxaldehyde standard taken in the preparation

of diluted standard solution.

ADSP1 = Average area of N-Acetyl-Glucosamine peak from diluted standard

injections.

ADSP2 = Average area of 5-Hydroxymethyl-2-Furaldehyde peak from diluted

standard injections.

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ADSP3 = Average area of Pyrazine peak from diluted standard injections.

ADSP4 = Average area of 2-Furaldehyde peak from diluted standard injections.

ADSP5 = Average area of Pyrrole 2- Carboxaldehyde peak from diluted standard

injections.

WT = Weight of test sample taken in mg for test preparation.

AIUIT1 = Area of N-Acetyl-Glucosamine impurity obtained from test preparation.

AIUIT2 = Area of 5-Hydroxymethyl-2-Furaldehyde impurity obtained from test

preparation.

AIUIT3 = Area of Pyrazine impurity obtained from test preparation.

AIUIT4 = Area of 2-Furaldehyde impurity obtained from test preparation.

AIUIT5 = Area of Pyrrole 2- Carboxaldehyde impurity obtained from test

preparation

AIUIT = Area of maximum individual unknown impurity obtained in test

preparation.

ATIT = Sum of areas of Glucosamine unknown impurities from test solution.

P1 = Purity of N-Acetyl Glucosamine standard.

P2 = Purity of 5-Hydroxymethyl-2-Furaldehyde standard.

P3 = Purity of Pyrazine standard.

P4 = Purity of 2-Furaldehyde standard.

P5 = Purity of Pyrrole 2- Carboxaldehyde standard

Allowable limits of Glucosamine impurities were presented in Table IV.4.

Table- IV.4 : Specification limits

S. No. Impurity Name Specification Level

1 N – Acetyl – Glucosamine 0.5%

2 Pyrazine 0.5%

3 5 – Hydroxymethyl – 2 – Furaldehyde 0.5%

4 2 – Furaldehyde 0.5%

5 Pyrrole – 2 – Carboxaldehyde 0.5%

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4.6.4. METHOD DEVELOPMENT (46-47)

4.6.4.1 Buffer Selection:

Phosphoric acid and triethylamine mixed buffer solution was taken so that pH

was about 4.0. This buffer solution was unable to retain the active peaks and those

were eluted below 1 minute. To retain the peaks; introduced ion pair reagent of

octane sulfonic acid and from this achieved the retention time of about 2.0 minutes.

Impurities were well separated. To avoid the interference of peaks at early eluted

peaks decreased the flow of column to 0.5 so that actives were eluted at about 3.5

minutes. Reproducibility was achieved and finalized the buffer solution with ion pair

reagent.

Selection of std/sample concentration:

Concentration was optimized by considering the impurity quantification level.

Specification level at 0.5% and achieved LOQ was less than 0.01% it indicates method

was highly sensitive in quantification of impurities. Injection volume of 10 microliters

was selected to avoid the volume overloading on column.

Impurity standard solution prepared at specification level with respective to the test

concentration.

Preparation of Diluted Standard:

25 mg of N-Acetyl-D-Glucosamine, 5-Hydroxymethyl-2-Furaldehyde, Pyrazine

and 2-Furaldehyde standard was transferred into a 25 ml volumetric flask then 15 ml

of diluent was added sonicate for 5 minutes to dissolve the material and made up the

volume with diluent and mixed well. 1ml of the above stock solution was pipetted and

transferred in to a 50 ml with diluent and mixed well. Finally a stock solution of

20µg/ml with four impurities was prepared.

Preparation of sample solutions

Twenty glucosamine hydrochloride 1500 mg tablets were weighed,

transferred to a clean and dry mortar and grinded into a fine powder. Tablet powder

equivalent to 500 mg of glucosamine hydrochloride was transferred to a 50 ml

volumetric flask, 35 ml of diluent was added, sonicated to about 30 minutes with

occasional shaking and made up to volume with diluent and filtered through a 0.45

µm pore size Nylon 66 membrane filter.

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4.6.4.2. Chromatographic conditions

The Shimadzu UFLC system (Shimadzu, Kyoto, Japan) used consists of a pump,

auto sampler and a UV detector. The output signal was monitored and processed by

using Empower-2 software.

The method was developed using Kromasil 100-5C18 (300 X 4.0 mm, 5 µm)

column. Mixture of the Buffer and Acetonitrile in the ratio of 90:10 (v/v) ratios was

used as a mobile phase. Then filtered the solution through 0.45 µm membrane filter

and degassed. The flow rate of the mobile phase was 0.5 ml/min. The column

temperature was maintained at 25oC and the wavelength was monitored at 195 nm.

The injection volume was 10 µl, diluent HPLC grade water.

Note: - Buffer preparation - 0.8 gm of phosphoric acid (85 %), 1.0 ml of Triethylamine

and 1.2 gm of Octane sulphonic acid salt were dissolved in 1000 ml of HPLC grade

water and mixed well.

Column section:

Based on molecule nature started the development by using conventional C18

column of Zorbax SB C18 (250 × 4.6) mm; 5 µm. Peaks shapes were not good and

separation between Pyrazine and 5 – Hydroxymethyl – 2 – Furaldehyde peaks were

not satisfactory. To overcome the resolution issue selected Inertsil ODS3 brand

column with carbon load of 15%. Injected the impurities mixed solution and

evaluated the chromatogram and concluded that resolution was not satisfactory but

better when compared to Zorbax brand.

Resolution improvement purpose selected Kromasil brand column(250 × 4.6)

mm; 5 µm. and with this resolution found satisfactory. This column was with the

carbon load of 19%, high pure silica with endcapped so that peak shape was good. For

the further improvement purpose finalized the (300 × 4.0) mm; 5 µm.

System suitability establishment

By considering USP (48) chapter <621> extensively established the system

suitability to ensure produced data was reliable and precise. Theoretical plate count

for all the impurities at specification level was established and fixed as minimum

2000. Tailing factor for all impurities except Pyrazine was controlled and fixed as not

more than 2.0 & for Pyrazine fixed as not more than 3.5. To ensure the reproducibility

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of the system established the %rsd for impurities and fixed as not more than 5. Due to

close elution of Pyrazine and 5 – Hydroxymethyl – 2 – Furaldehyde peaks resolution

was established and fixed as minimum 2.

Establishment of RRF

By considering the quantification at 195nm relative response factor was not

established. Quantification selected by the external standard method in which

impurities calculated against 0.5% of impurity mixture standard area responses.

Unknown peaks were quantified by using N-acetyl Glucosamine.

Establishment of RRT

Relative retention times were established with respective to the N-acetyl

Glucosamine.

4.6.5. METHOD VALIDATION (49)

4.6.5.1. . SYSTEM SUITABILITY & SPECIFICITY

4.6.5.1.1. SYSTEM SUITABILITY:

Procedure:

Diluted Standard solution was prepared as per test procedure and injected into

the HPLC system as per test method. Chromatograms were presented below Figure

IV.4&5. Evaluated the system suitability parameters obtained results were

summarized in Table-IV.5.

Figure-IV.4 : Chromatogram from Blank Solution

Chapter-IV

237

Figure- IV.5: Chromatogram from Diluted Standard Solution

Table- IV.5 : System suitability results

System suitability parameters Observed

value Acceptance

criteria

Theoretical Plate Count for peak from first injection of diluted standard solution.

N–Acetyl–Glucosamine 5758

Should be NLT 2000

5–Hydroxymethyl–2–Furaldehyde

13199

Pyrazine 2381 2–Furaldehyde 14032 Pyrrole–2–Carboxaldehyde 14681

% Relative Standard Deviation for peak area from five replicate injections of diluted standard solution.

N–Acetyl–Glucosamine 0.1

Should be NMT 5.0


0.1

Pyrazine 0.1 2–Furaldehyde 0.3 Pyrrole–2–Carboxaldehyde 0.1

Tailing factor for peak from first injection of diluted standard solution.

N–Acetyl–Glucosamine 1.2

Should be NMT 2.0


1.1

2–Furaldehyde 1.1 Pyrrole–2–Carboxaldehyde 1.1

Tailing factor for Pyrazine peak from first injection of diluted standard solution.

2.5 Should be NMT 3.5

Resolution between Pyrazine and 5–Hydroxymethyl–2–Furaldehyde peaks from first injection of diluted standard solution.

5.2 Should be NLT 2.0

Chapter-IV

238

4.6.5.1.2. SPECIFICITY

PLACEBO INTERFERENCE:

A study to establish the interference of placebo was conducted. Samples were

prepared in duplicate, by taking the Common placebo and Common placebo with

Chondroitin Sulfate equivalent to the amount of weight present in portion of test

preparation as per the test method (4.6.2) and injected into the HPLC system and the

results were presented in Table-IV.6. Chromatograms were presented below

(Figure-IV.6 & 7).

Figure- IV.6 : Chromatogram from placebo solution – (common placebo)

Figure- IV.7 : Chromatogram from placebo solution – (placebo with chondroitin

sulfate)

Chapter-IV

239

Table- IV.6 : Placebo interference

Sample N°

Interference found (Yes/No)

Common Placebo Common Placebo With

Chondroitin Sulfate

1 NO NO

2 NO NO

IMPURITY INTERFERENCE:

A study was conducted on the impurity interference of Glucosamine impurities

Preparation individual, mixed solution and spiked sample solution of Glucosamine

Impurities (at spec level i.e. 0.5%) was injected into the HPLC. For Identification

purpose 0.5% of each Glucosamine individual impurity solution injected in to the

HPLC and obtained retention time and relative retention time values were mentioned

in Table-IV.7. Peak purity values were mentioned in Tale-IV.8. Chromatograms and

peak purity spectra’s were mentioned from Figure-IV.8 to16.

Figure- IV.8 : Chromatogram from impurity solution – (n – acetyl – glucosamine)

Chapter-IV

240

Figure- IV.9 : Chromatogram from impurity solution – (pyrazine)

Figure- IV.10 : Chromatogram from impurity solution – (5 – hydroxymethyl – 2 –

furaldehyde)

Figure- IV.11 : Chromatogram from impurity solution – (2 – furaldehyde)

Chapter-IV

241

Figure- IV.12 : Chromatogram from impurity solution – (pyrrole – 2 –

carboxaldehyde)

Figure- IV.13 : Chromatogram from impurity blend solution

Figure- IV.14 : HPLC chromatogram from impurity spiked test solution

Chapter-IV

242

Figure- IV.15 : Purity plot for glucosamine:

Figure- IV.16 : Purity plot for n – acetyl – glucosamine:

Table- IV.7 : Retention time and relative retention time values from spiked sample

S. No. Impurity Name RT RRT

1 N – Acetyl – Glucosamine 4.572 1.00

2 Pyrazine 10.545 2.31

3 5 – Hydroxymethyl – 2 – Furaldehyde 13.960 3.05

4 2 – Furaldehyde 19.887 4.35

5 Pyrrole – 2 – Carboxaldehyde 36.526 7.99

Chapter-IV

243

Table- IV.8 : Peak purity results

S. No. Impurity Name Purity Angle

Purity Threshold

1 Glucosamine 12.815 17.188 2 N – Acetyl – Glucosamine 1.355 1.631

4.6.5.2. PRECISION

4.6.5.2.1. SYSTEM PRECISION:

Procedure:

The Diluted Standard solution was prepared as per test procedure and made six

replicate injections of diluted standard solution into the HPLC system. Results were

reported in the Table –IV.9a&b below together with mean value, the standard

deviation, the relative standard deviation.

Table- IV.9a : System precision results

Injection N° Response



Pyrazine

01 1030394 1507936 1637946 02 1030511 1508725 1635440 03 1031327 1509653 1632559 04 1031798 1510314 1637480 05 1031968 1510440 1637388 06 1031142 1509409 1633463

Mean 1031190 1509413 1635713 Standard deviation 647 957 2280

% RSD 0.1 0.1 0.1

Table- IV.9b : System precision results (Contd.,):

Injection N° Response

2 – Furaldehyde Pyrrole – 2 – Carboxaldehyde 01 828119 1381801 02 826841 1383213 03 827305 1384136 04 825909 1384423 05 823057 1382290 06 821785 1382817

Mean 825502 1383113 Standard deviation 2524 1026

% RSD 0.3 0.1

Chapter-IV

244

4.6.5.2.2. METHOD PRECISION:

The Method precision of test method was evaluated by analysing six test

preparations by spiking test preparation with Glucosamine impurities blend solution

to get 0.5% of each impurity with respect to test concentration and analyzed as per

test method (4.6.2). Representative chromatogram presented as Figure-IV.17. The

individual results were reported in the Table-IV.10a&b below together with mean

value, the standard deviation and relative standard deviation for Glucosamine

impurities and total impurities.

Figure- IV.17 : HPLC Chromatogram From Impurity Spiked Test Solution

Table- IV.10a : Method precision results

Sample N°

Glucosamine Impurities



Pyrazine

% % %

1 0.498 0.489 0.506

2 0.509 0.499 0.498 3 0.511 0.499 0.495 4 0.511 0.496 0.493 5 0.516 0.501 0.499 6 0.516 0.500 0.488

Mean 0.510 0.497 0.497 Standard deviation 0.00662 0.00441 0.00609

% RSD 1.3 0.9 1.2

Chapter-IV

245

Table- IV.10b : Method precision results (Contd.,):

Sample N°


2 – Furaldehyde Pyrrole – 2 –

Carboxaldehyde Total Impurities

% % %

1 0.488 0.483 2.744

2 0.505 0.493 2.795 3 0.505 0.493 2.785 4 0.502 0.494 2.776 5 0.503 0.493 2.802 6 0.501 0.489 2.783

Mean 0.501 0.491 2.781 Standard deviation 0.00641 0.00422 0.02025

% RSD 1.3 0.9 0.7

4.6.5.2.3. INTERMEDIATE PRECISION:

Study conducted as like method precision by varying the system, column and analyst.

Obtained values were presented in Table-IV.11 &12a,b,c,d,e,f.

Chapter-IV

246

Table- IV.11 : Intermediate precision system suitability results

(Analyst to Analyst, System to System and Column to Column Variation):

System suitability parameters

Observed value Acceptance

criteria Analyst - I Analyst - II

Theoretical Plate Count for peak from first injection of diluted standard solution.

N–Acetyl–Glucosamine

5758 6746

Should be NLT 2000


13199 17244

Pyrazine 2381 2686

2–Furaldehyde 14032 18449

Pyrrole–2–Carboxaldehyde

14681 19556

% Relative Standard Deviation for peak area from five replicate injections of diluted standard solution.


0.1 0.2

Should be NMT 5.0


0.1 0.1

Pyrazine 0.1 0.4

2–Furaldehyde 0.3 0.4


0.1 0.1

Tailing factor for peak from first injection of diluted standard solution.


1.2 1.2

Should be NMT 2.0


1.1 1.1

2–Furaldehyde 1.1 1.1


1.1 1.1

Tailing factor for Pyrazine peak from first injection of diluted standard solution.

2.5 2.6 Should be NMT 3.5

Resolution between Pyrazine and 5–Hydroxymethyl–2–Furaldehyde peaks from first injection of diluted standard solution.

5.2 5.7 Should be NLT 2.0

Chapter-IV

247

Table- IV.12a : Intermediate precision results (Analyst to Analyst, System to System and Column to Column Variation):

Sample N°


Analyst - I Analyst - II

% %

1 0.498 0.496

2 0.509 0.516

3 0.511 0.511

4 0.511 0.514

5 0.516 0.511

6 0.516 0.514

Mean 0.510 0.510

Standard deviation 0.00662 0.00728

% RSD 1.3 1.4

Mean difference 0.00

Table- IV.12b : Intermediate precision results (Contd.,):

Sample N°



% %

1 0.489 0.486

2 0.499 0.503

3 0.499 0.503

4 0.496 0.504

5 0.501 0.507

6 0.500 0.506

Mean 0.497 0.502


% RSD 0.9 1.5


Chapter-IV

248

Table- IV.12c : Intermediate precision results (Contd.,):

Sample N°

Pyrazine


% %

1 0.506 0.491

2 0.498 0.499

3 0.495 0.501

4 0.493 0.494

5 0.499 0.497

6 0.488 0.499

Mean 0.497 0.497


% RSD 1.2 0.7


Table- IV.12d : Intermediate precision results (Contd.,):

Sample N°

2 – Furaldehyde


% %

1 0.488 0.474

2 0.505 0.487

3 0.505 0.492

4 0.502 0.486

5 0.503 0.495

6 0.501 0.491

Mean 0.501 0.488


% RSD 1.3 1.5


Chapter-IV

249

Table- IV.12e : Intermediate precision results (Contd.,):

Sample N°



% %

1 0.483 0.499

2 0.493 0.506

3 0.493 0.508

4 0.494 0.503

5 0.493 0.509

6 0.489 0.505

Mean 0.491 0.505


% RSD 0.9 0.7


Table-IV.12f : Intermediate precision results (Contd.,):

Sample N°

Total Impurities


% %

1 2.744 2.698

2 2.795 2.787

3 2.785 2.802

4 2.776 2.820

5 2.802 2.821

6 2.783 2.820

Mean 2.781 2.791


% RSD 0.7 1.7

Mean difference 0.0

Chapter-IV

250

4.6.5.3. ACCURACY:

Accuracy study for Glucosamine impurities from spiked test preparation was

conducted. Samples were prepared in triplicate at each level by spiking test

preparation with LOQ, 50%, 80%, 100%, 150% and 200% of target concentration

(i.e., 0.5% of each impurity) of Glucosamine impurities.

The individual values, the % recovery, the % relative standard deviation for %

recovery of samples at each concentration level were reported in Table IV.13 to IV.17

Table- IV.13 : Accuracy for N – Acetyl – Glucosamine

Series Sample

N°

Theoretical content (µg/ml)

Calculated content (µg/ml)

% recovery

Mean % Recovery

% RSD

LOQ 1

0.0198 0.0202 102.0

101.5 1.5 2 0.0204 102.7 3 0.0198 99.8

50% 1

12.4245 13.2726 106.8

107.0 1.6 2 13.0948 105.4 3 13.5174 108.8

80% 1

19.8792 20.8787 105.0

104.6 0.4 2 20.8204 104.7 3 20.7056 104.2

100% 1

24.8490 25.4545 102.4

102.8 0.4 2 25.5572 102.9 3 25.6147 103.1

150% 1

37.2735 37.9705 101.9

101.7 0.3 2 37.8072 101.4 3 37.9373 101.8

200% 1

49.6980 50.3036 101.2

101.3 0.2 2 50.3416 101.3 3 50.4430 101.5

Chapter-IV

251

Table- IV.14 : Accuracy for 5 – Hydroxymethyl – 2 – Furaldehyde:

Series Sample

N°



% recovery

Mean % Recovery

% RSD

LOQ 1

0.0298 0.0308 103.4

103.9 1.2 2 0.0314 105.3 3 0.0307 102.9

50% 1

12.5622 11.9370 95.0

96.0 1.4 2 12.2572 97.6 3 11.9952 95.5

80% 1

20.0995 19.1779 95.4

96.2 0.7 2 19.3632 96.3 3 19.4520 96.8

100% 1

25.1244 24.4501 97.3

97.5 0.2 2 24.5271 97.6 3 24.5092 97.6

150% 1

37.6866 37.0868 98.4

98.1 0.3 2 36.8960 97.9 3 36.9350 98.0

200% 1

50.2488 49.1010 97.7

97.8 0.2 2 49.0921 97.7 3 49.2652 98.0

Table- IV.15 : Accuracy for Pyrazine:

Series Sample

N°



% recovery

Mean % Recovery

% RSD

LOQ 1

0.0300 0.0294 97.9

98.6 1.1 2 0.0294 98.0 3 0.0299 99.8

50% 1

12.4347 12.3991 99.7

103.0 2.9 2 12.9290 104.0 3 13.1161 105.5

80% 1

19.8997 19.8829 99.9

99.0 2.0 2 19.9412 100.2 3 19.2485 96.7

100% 1

24.8747 24.0923 96.9

96.8 0.8 2 24.2541 97.5 3 23.8709 96.0

150% 1

37.3120 36.1118 96.8

96.2 1.3 2 36.1914 97.0 3 35.3870 94.8

200% 1

49.7493 50.2702 101.0

101.7 0.6 2 50.7600 102.0 3 50.7226 102.0

Chapter-IV

252

Table- IV.16 : Accuracy for 2 – Furaldehyde:

Series Sample

N°



% recovery

Mean % Recovery

% RSD

LOQ 1

0.0597 0.0618 103.4

101.0 2.7 2 0.0607 101.6 3 0.0586 98.1

50% 1

12.5615 11.5798 92.2

91.9 1.2 2 11.6642 92.9 3 11.3904 90.7

80% 1

20.0983 18.4774 91.9

92.7 0.9 2 18.5939 92.5 3 18.8000 93.5

100% 1

25.1229 23.0087 91.6

92.6 0.5 2 23.6587 94.2 3 23.1001 91.9

150% 1

37.6844 35.5494 94.3

94.2 0.2 2 35.5153 94.2 3 35.4292 94.0

200% 1

50.2458 46.8246 93.2

93.2 0.5 2 46.6162 92.8 3 47.0940 93.7 Table- IV.17 : Accuracy for Pyrrole – 2 – Carboxaldehyde:

Series Sample

N°


Calculated Content (µg/ml)

% recovery

Mean % Recovery

% RSD

LOQ 1

0.0718 0.0701 97.7

98.5 0.9 2 0.0707 98.4 3 0.0714 99.4

50% 1

12.6124 11.6264 92.2

94.2 3.3 2 12.3400 97.8 3 11.6796 92.6

80% 1

20.1798 18.5498 91.9

93.7 1.7 2 19.0136 94.2 3 19.1601 94.9

100% 1

25.2248 24.4802 97.0

97.4 0.4 2 24.6743 97.8 3 24.5548 97.3

150% 1

37.8371 37.2663 98.5

98.2 0.3 2 37.0358 97.9 3 37.1272 98.1

200% 1

50.4495 49.3313 97.8

97.9 0.3 2 49.2950 97.7 3 49.5386 98.2

Chapter-IV

253

4.6.5.4. LINEARITY:

Linearity was established by plotting a graph between concentration versus peak

area and the correlation coefficient was determined. A series of solutions of

Glucosamine impurities with concentrations ranging from LOQ% to 200% of the

target concentration (0.5%) was prepared and injected into the HPLC system. The

results were summarized in the table-IV.18 to22 given below. Linearity plots were

mentioned in Figure-IV.18 to22.

Figure- IV.18 : N – Acetyl – Glucosamine

Chapter-IV

254

Figure- IV.19 : Pyrazine

Figure- IV.20 : 5 – Hydroxymethyl – 2 – Furaldehyde

Chapter-IV

255

Figure- IV.21 : 2 – Furaldehyde

Figure- IV.22 : Pyrrole – 2 – Carboxaldehyde

Chapter-IV

256

Table- IV.18 : Linearity for N – Acetyl – Glucosamine

Solution No. Concentration % Concentration

‘µg/ml’ Response

1 LOQ 0.020 823

2 50% 12.400 504749

3 80% 19.840 794211

4 100% 24.800 992360

5 150% 37.199 1478074

6 200% 49.599 1978813

Slope 39764.312

Y-Intercept 4771.251

Correlation Coefficient 0.999978

Table- IV.19 : Linearity for 5 – Hydroxymethyl – 2 – Furaldehyde:

Solution No. Concentration % Concentration

‘µg/ml’ Response

1 LOQ 0.030 1812

2 50% 12.422 741508

3 80% 19.876 1167607

4 100% 24.845 1441049

5 150% 37.267 2171077

6 200% 49.690 2908244

Slope 58315.955

Y-Intercept 4367.801


Chapter-IV

257

Table- IV.20 : Linearity for Pyrazine:

Solution No. Concentration % Concentration ‘µg/ml’ Response

1 LOQ 0.030 1916

2 50% 12.502 753741

3 80% 20.004 1222085

4 100% 25.005 1515836

5 150% 37.507 2295992

6 200% 50.010 3142757

Slope 62627.183

Y-Intercept -25370.299


Table- IV.21 : Linearity for 2 – Furaldehyde:


1 LOQ 0.060 1939

2 50% 12.442 377989

3 80% 19.908 622719

4 100% 24.885 760360

5 150% 37.327 1150295

6 200% 49.769 1545097

Slope 31014.546



Chapter-IV

258

Table- IV.22 : Linearity for Pyrrole – 2 – Carboxaldehyde:


1 LOQ 0.072 3513

2 50% 12.827 645086

3 80% 20.523 1021680

4 100% 25.654 1267129

5 150% 38.481 1915566

6 200% 51.309 2566378

Slope 49923.249



4.6.5.5. LIMIT OF QUANTITATION AND LIMIT OF DETECTION:

A study to establish the Limit of Detection and Limit of Quantification of

Glucosamine impurities was conducted.

Limit of Detection and Limit of Quantitation were established based on signal

to noise ratio. A series of injections of blank solution were injected and average noise

was calculated. Limit of Detection for each impurity was established by identifying

the concentration which gives signal to noise ratio about 3. Limit of Quantitation was

established by identifying the concentration which gives signal to noise ratio

about 10.

Precision of Glucosamine impurities at about Limit of Quantitation level was

conducted. Six test preparations having impurities at the concentration level of about

Limit of Quantitation in presence of placebo were prepared and injected into HPLC

system.

The % mean recovery of Glucosamine impurities was calculated and summarized in

the Table-IV.23 & 24a,b,c,d,e.

Chapter-IV

259

Table- IV.23 : LOD & LOQ Values

Name % of Impurity Signal to Noise Ratio

LOD LOQ LOD LOQ

N – Acetyl – Glucosamine 0.0001 0.0005 3.5 9.8

5 – Hydroxymethyl – 2 –

Furaldehyde 0.0002 0.0005 3.6 10.1

Pyrazine 0.0003 0.0012 3.0 9.3

2 – Furaldehyde 0.0003 0.0014 3.1 9.8

Pyrrole – 2 – Carboxaldehyde 0.0004 0.0014 2.9 9.7

Table- IV.24a : Precision & Accuracy at Limit of Quantitation level:

Name Injection N° LOQ (%) % Recovery at LOQ

N – Acetyl –

Glucosamine

1 0.0004 102.0

2 0.0004 102.7

3 0.0004 99.8

4 0.0004 102.3

5 0.0004 106.4

6 0.0004 103.5

Mean 0.0004 102.8

Standard Deviation 0.0 2.1609

%RSD 0.0 2.1

Table- IV.24b : Precision & Accuracy at Limit of Quantitation level: contd.,


5 – Hydroxymethyl –

2 – Furaldehyde

1 0.0006 103.4

2 0.0006 105.3

3 0.0006 102.9

4 0.0006 102.8

5 0.0006 103.1

6 0.0006 105.8

Mean 0.0006 103.9


%RSD 0.0 1.3

Chapter-IV

260

Table- IV.24c : Precision & Accuracy at Limit of Quantitation level: contd.,


Pyrazine

1 0.0006 97.9

2 0.0006 98.0

3 0.0006 99.8

4 0.0006 99.7

5 0.0006 102.1

6 0.0006 99.4

Mean 0.0006 99.5


%RSD 0.0 1.5

Table- IV.24d : Precision & Accuracy at Limit of Quantitation level: contd.,


2 – Furaldehyde

1 0.0012 103.4

2 0.0012 101.6

3 0.0012 98.1

4 0.0012 101.2

5 0.0012 103.7

6 0.0012 104.4

Mean 0.0012 102.1


%RSD 0.0 2.3

Table- IV.24e : Precision & Accuracy at Limit of Quantitation level: contd.,


Pyrrole – 2 –

Carboxaldehyde

1 0.0014 97.7

2 0.0014 98.4

3 0.0014 99.4

4 0.0014 99.5

5 0.0014 98.4

6 0.0014 96.5

Mean 0.0014 98.3


%RSD 0.0 1.1

Chapter-IV

261

4.6.5.6 RUGGEDNESS

4.6.5.6.1 Bench top Stability of diluted standard solution and Test preparation:

Stability of Glucosamine Hydrochloride test preparation spiked with Glucosamine

impurities at target concentration level (i.e., 0.5%) at ambient temperature about

(25ºC) was conducted at initial and at 30 hours.

Stability of Glucosamine diluted standard preparation at ambient temperature about

(25ºC) was conducted at initial and at 34 hours. Results were reported in the

table-IV.25 & IV.26a,b.

Table- IV.25 : Stability of Diluted standard preparation at ambient temperature about

(25ºC)

Time in

Hours

Similarity factor

N – Acetyl – Glucosamine 5 – Hydroxymethyl –

2 – Furaldehyde

Pyrazine

34 1.01 1.00 0.98

Time in

Hours

Similarity factor

2 – Furaldehyde Pyrrole – 2 – Carboxaldehyde

34 0.97 1.00

Table- IV.26a : Stability of test preparation at ambient temperature about (25ºC)

Time in

hours



2 – Furaldehyde Pyrazine

% Imp Difference

from Initial

% Imp Difference

from Initial

% Imp Difference

from Initial Spl-1 Spl-2 Spl-1 Spl-2 Spl-1 Spl-2

Initial 0.496 0.516 NA 0.486 0.503 NA 0.491 0.499 NA

30 0.525 0.545 0.03 0.03 0.485 0.495 0.00 0.01 0.483 0.507 0.01 0.01

Table- IV.26b : Stability of test preparation at ambient temperature about (25ºC)

Time in

hours




% Imp Difference

from Initial

% Imp Difference

from Initial

% Imp Difference


Initial 0.474 0.487 NA 0.499 0.506 NA 2.698 2.787 NA

30 0.499 0.467 0.03 0.02 0.496 0.510 0.00 0.00 2.655 2.752 0.0 0.0

Chapter-IV

262

4.6.5.6.2 Refrigerator Stability of diluted standard solution and Test

preparation:

Stability of Glucosamine Hydrochloride test preparation spiked with Glucosamine

impurities at target concentration level (i.e., 0.5%) at refrigerator temperature about

(8ºC) was conducted at initial and at 32 hours.

Stability of Glucosamine diluted standard preparation at refrigerator temperature

about (8ºC) was conducted at initial and at 35 hours. Results were reported in the

Table-IV.27 to29.

Table- IV.27 : Stability of Diluted standard preparation at refrigerator temperature

about (8ºC)

Time in

Hours

Similarity factor

N – Acetyl –

Glucosamine

5 – Hydroxymethyl –


35 1.01 1.00 1.00

Time in

Hours

Similarity factor

2 – Furaldehyde Pyrrole – 2 – Carboxaldehyde

35 1.01 1.00

Table- IV.28 : Stability of test preparation at refrigerator temperature about (8ºC)

Time in

hours




% Imp Difference

from Initial

% Imp Difference

from Initial

% Imp Difference


Initial 0.496 0.516 NA 0.486 0.503 NA 0.491 0.499 NA

32 0.522 0.528 0.03 0.01 0.500 0.502 0.01 0.00 0.517 0.499 0.03 0.00

Chapter-IV

263

Table- IV.29 : Stability of test preparation at refrigerator temperature about (8ºC)

Time in

hours




% Imp Differenc

e from

Initial

% Imp Difference

from

Initial

% Imp Difference


Initial 0.474 0.487 NA 0.499 0.506 NA 2.698 2.787 NA

32 0.510 0.526 0.04 0.0

4 0.512 0.513 0.01 0.01 2.788 2.814 0.1 0.0

4.6.5.6.3 Bench top Stability of Mobile phase:

Stability of mobile phase at ambient temperature about (25ºC) was conducted at

initial, after 1 day and 2 days by performing Glucosamine impurities spiked test

preparation in duplicate using same lot of mobile phase each time. System suitability

values presented in Table- IV.30 & Impurities RRT values were mentioned in

Table-IV.31

Chapter-IV

264

Table- IV.30 : Bench top stability of Mobile phase system suitability results

System suitability parameters Observed value Acceptance

criteria Initial Day-1 Day-2

Theoretical Plate

Count for peak

from first

injection of

diluted standard

solution.

N–Acetyl–Glucosamine 6756 6457 6594

Should be

NLT 2000

5–Hydroxymethyl–2–

Furaldehyde 17244 16966 17451

Pyrazine 2686 2543 2579

2–Furaldehyde 18449 18068 18619

Pyrrole–2–

Carboxaldehyde 19556 19636 20498

% Relative

Standard

Deviation for

peak area from

five replicate

injections of

diluted standard

solution.

N–Acetyl–Glucosamine 0.2 0.1 0.1

Should be

NMT 5.0


Furaldehyde 0.1 0.0 0.0

Pyrazine 0.4 0.2 0.1

2–Furaldehyde 0.4 0.2 0.3

Pyrrole–2–

Carboxaldehyde 0.1 0.1 0.1

Tailing factor for

peak from first

injection of

diluted standard

solution.


Should be

NMT 2.0




Pyrrole–2–


Tailing factor for Pyrazine peak from first

injection of diluted standard solution. 2.6 2.6 2.6

Should be

NMT 3.5

Resolution between Pyrazine and 5–

Hydroxymethyl–2–Furaldehyde peaks from

first injection of diluted standard solution.

5.7 5.6 5.6 Should be

NLT 2.0

Chapter-IV

265

Table- IV.31 : Bench top stability of Mobile phase RRT’s

Time in

Days


N–Acetyl–

Glucosamine

5–Hydroxymethyl–


2-

Furaldehyde

Pyrrole–2–

Carboxaldehyde

RRT’s

Initial NA 3.02 2.23 4.32 7.87

1 NA 3.02 2.23 4.33 7.89

2 NA 2.99 2.22 4.30 7.75

4.6.5.6.4 FILTER VALIDATION:

A study to establish the suitability of filters was conducted by using two different

filters namely, 0.45 µm Millipore PVDF filter and 0.45 µm Nylon filter.

The test preparation was prepared in duplicate by spiking Glucosamine impurities

blend solution and then different portions of the above solution were filtered through

different filters. A portion of the above solution was centrifuged. The centrifuged and

filtered solutions were injected into the HPLC system and the difference between

centrifuged sample and filtered samples for % of individual impurities and total

impurities were calculated and results were reported.

Two diluted standard preparations were made and filtered through above mentioned

filters. Area ratio was calculated for filtered diluted standard solutions against

unfiltered diluted standard solution and reported (Table-IV.32 to 37).

Table- IV.32 : Details of filters used in the study

Filter description Filters

PVDF Nylon

Manufacturer Name Millipore Millex HV Axiva Sichem Biotech

Lot No. R0MA79683K SF0460311

Size 0.45 µm 0.45 µm

Chapter-IV

266

Table- IV.33 : Diluted Standard Solution results:


Filter Area Similarity Factor

STD -1 STD -2 STD -1 STD -2

Unfiltered 1003434 1003321 NA NA

0.45 µm PVDF 997673 998745 0.99 1.00

0.45 µm Nylon 977193 977150 0.97 0.97





0.45 µm PVDF 1463080 1464855 0.99 0.99

0.45 µm Nylon 1435515 1435609 0.97 0.97

Pyrazine




0.45 µm PVDF 1501379 1508558 0.98 0.99

0.45 µm Nylon 1483857 1483510 0.97 0.98

Table- IV.34 : Diluted Standard Solution results contd.,

2 – Furaldehyde




0.45 µm PVDF 749973 750825 0.99 0.99

0.45 µm Nylon 743818 741235 0.98 0.98

Chapter-IV

267

Table- IV.35 : Diluted Standard Solution results contd.,





0.45 µm PVDF 1278387 1278307 0.99 1.01

0.45 µm Nylon 1257992 1259840 0.97 1.00

Table-IV.36 : Diluted Standard Solution results contd.,


Filter % Imp Difference

1 2 1 2

Centrifuge 0.490 0.497 NA NA

0.45 µm

PVDF 0.487 0.496 0.00 0.00

0.45 µm

Nylon 0.487 0.498 0.00 0.00

Chapter-IV

268

For Test solution:

Table- IV.37 : Filter validation results



1 2 1 2


0.45 µm PVDF 0.520 0.521 0.00 0.00

0.45 µm Nylon 0.518 0.526 0.00 0.00

Pyrazine


1 2 1 2 Centrifuge 0.514 0.514 NA NA

0.45 µm PVDF 0.507 0.518 0.01 0.00 0.45 µm Nylon 0.495 0.501 0.02 0.01

2 – Furaldehyde


1 2 1 2 Centrifuge 0.503 0.492 NA NA

0.45 µm PVDF 0.483 0.502 0.02 0.01 0.45 µm Nylon 0.489 0.491 0.01 0.00



1 2 1 2 Centrifuge 0.498 0.504 NA NA

0.45 µm PVDF 0.502 0.508 0.00 0.00 0.45 µm Nylon 0.497 0.509 0.00 0.01

Total impurities


1 2 1 2


0.45 µm PVDF 2.744 2.780 0.0 0.0

0.45 µm Nylon 2.736 2.780 0.0 0.0

Conclusion:

The 0.45 µm Nylon and 0.45 µm PVDF filters were suitable for performing related

substances test sample preparation.

Chapter-IV

269

4.6.5.7 ROBUSTNESS:

4.6.5.7.1 Effect of Variation in Flow Rate:

The effect of variation in flow rate was conducted. Diluted standard solution and

Glucosamine Hydrochloride test solution was prepared as per the test method and

injected into HPLC system with flow rate 0.45 ml/min and 0.60 ml/min. System

suitability parameters were evaluated and the results were mentioned in

Table-IV.38 & 39.

Chapter-IV

270

Table - IV.38: Flow variation results

System suitability parameters

Observed value Acceptance

criteria 0.45

ml/min

0.50

ml/min

0.60

ml/min

Theoretical Plate

Count for peak

from first

injection of

diluted standard

solution.


Should be

NLT 2000


Furaldehyde 20072 19821 19079

Pyrazine 2676 2749 2731

2–Furaldehyde 22081 22406 21902

Pyrrole–2–


% Relative

Standard

Deviation for

peak area from

five replicate

injections of

diluted standard

solution.


Should be

NMT 5.0





Pyrrole–2–


Tailing factor for

peak from first

injection of

diluted standard

solution.


Should be

NMT 2.0




Pyrrole–2–



injection of diluted standard solution. 2.6 2.7 2.7 2.6




5.7 5.8 5.8 5.8

Chapter-IV

271

Table- IV.39 : Flow variation results (RRT values)

S. No. Impurity Name

RRT

0.45

ml/min

0.50

ml/min

0.60

ml/min

1 N – Acetyl – Glucosamine NA NA NA

2 Pyrazine 2.26 2.27 2.26

3 5 – Hydroxymethyl – 2 –


4 2 – Furaldehyde 4.35 4.37 4.37

5 Pyrrole – 2 – Carboxaldehyde 7.94 8.00 8.00

4.6.5.7.2 Effect of Variation in Column Oven temperature:

The effect of variation in column oven temperature was conducted. Diluted standard

solution and Glucosamine Hydrochloride test solution was prepared as per the test

method and injected into HPLC system at column oven temperature 20°C and 30°C.

System suitability parameters were evaluated and the results were mentioned in

Table IV.40 & 41.

Chapter-IV

272

Table- IV.40 : Temperature variation results


criteria 20°C 25°C 30°C

Theoretical Plate

Count for peak

from first

injection of

diluted standard

solution.


Should be

NLT 2000


Furaldehyde 18975 19750 20393

Pyrazine 2153 2739 3326

2–Furaldehyde 21584 22517 22803

Pyrrole–2–


% Relative

Standard

Deviation for

peak area from

five replicate

injections of

diluted standard

solution.


Should be

NMT 5.0





Pyrrole–2–


Tailing factor for

peak from first

injection of

diluted standard

solution.


Should be

NMT 2.0




Pyrrole–2–




Should be

NMT 3.5




5.5 5.8 6.0 Should be

NLT 2.0

Chapter-IV

273

Table- IV.41 : Temperature variation results (RRT values)


20°C 25°C 30°C


2 Pyrazine 2.32 2.27 2.22

3 5 – Hydroxymethyl – 2 – Furaldehyde 3.17 3.06 2.95

4 2 – Furaldehyde 4.46 4.36 4.27


4.6.5.7.3 Effect of Variation in Mobile phase composition (Acetonitrile):

The effect of variation in mobile phase composition was conducted by using

two mobile phases, one containing 90% and other containing 110% of the method

organic phase (Acetonitrile) composition. Diluted standard solution and Glucosamine

Hydrochloride test solution were prepared as per the test method and injected into

HPLC system. System suitability parameters were evaluated and the results were

presented in Table-IV.42 & 43.

Chapter-IV

274

Table- IV.42 : System suitability results


criteria 90% 100% 110%

Theoretical

Plate Count for

peak from first

injection of

diluted

standard

solution.


Should be

NLT 2000


Furaldehyde 20258 19995 19799

Pyrazine 2661 2673 3025

2–Furaldehyde 22341 22421 22325

Pyrrole–2–


% Relative

Standard

Deviation for

peak area from

five replicate

injections of

diluted

standard

solution.


Should be

NMT 5.0





Pyrrole–2–


Tailing factor

for peak from

first injection of

diluted

standard

solution.


Should be

NMT 2.0




Pyrrole–2–




Should be

NMT 3.5


Hydroxymethyl–2–Furaldehyde peaks

from first injection of diluted standard

solution.

5.8 5.8 5.9 Should be

NLT 2.0

Chapter-IV

275

Table- IV.43 : Effect of Variation in Mobile phase composition (Acetonitrile)


90% 100% 110%


2 Pyrazine 2.31 2.26 2.21

3 5 – Hydroxymethyl – 2 –


4 2 – Furaldehyde 4.43 4.36 4.28


Acceptance criteria

The system suitability results should be within the limits as per test method (4.6.2).

The relative retention times of Glucosamine impurities should be comparable with

the method flow rate.

Conclusion: From the above data the allowable variation in flow rate was from 0.45

ml/min to 0.60 ml/min.

From the above data the allowable variation in column oven temperature was from

20°C to 30°C.

From the above data the allowable variation in mobile phase composition

(Acetonitrile) was from 90% to 110%.

4.7. RESULTS AND DISCUSSION

The following was the summary of method validation study conducted for related

substances of Glucosamine Hydrochloride in Glucosamine Hydrochloride 500 mg and

Chondroitin Sulfate 400 mg SR Tablets.

System suitability was established as mentioned in the test method.

The test method was specific for the estimation of Glucosamine Hydrochloride

impurities.

System suitability, System Precision, Method Precision and Intermediate

precision were established for test method.

Chapter-IV

276

The test method was accurate for the quantification of Glucosamine

Hydrochloride impurities.

The detector response was linear with a correlation coefficient of 0.999 for

Glucosamine Hydrochloride impurities.

The method was found linear, precise and accurate.

The Diluted Standard was stable for a period of 1 day (34 hours) and test

solution was stable for a period of 1 day (30 hours) at room temperature (i.e.

about 250C).

The Diluted Standard was stable for a period of 1 day (35 hours) and test

solution was stable for a period of 1 day (32 hours) at refrigerator

temperature (i.e. about 80C).

The mobile phase was stable for a period of 2 days on bench top.

Allowable variation in flow rate was from 0.45 ml/min to 0.60 ml/min.

Allowable variation in mobile phase composition (Acetonitrile) was from 90%

to 102%.

Allowable variation in column oven temperature was from 20°C to 30°C.

The 0.45 µm Nylon and 0.45 µm PVDF filters were suitable for performing

related substances test sample preparation.

Chapter-IV

277

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