Chapter-IV 4.1. GLUCOSAMINE HYDROCHLORIDE

Transcription

1 4.. GLUCOSAMINE HYDROCHLORIDE Glucosamine hydrochloride (Figure-IV.) is commonly used for arthritis (). 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. : 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 220

2 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 (0-) CHONDROITIN SULFATE Chondroitin (Figure-IV.2) is a molecule that occurs naturally in the body (2). 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 (3). Chondroitin sulfate is a chemical that is normally found in cartilage around joints in the body (4). 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 (5-6). Now, scientists believe that, overall, chondroitin sulfate may 22

3 reduce arthritis pain slightly (7-8). 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.) Table-IV. : 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 (9). Dosage: Doses of milligrams by mouth twice to three times daily, or 800-,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. 222

4 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, milligrams as a single daily injection or divided into two daily injections has been used. Medical supervision is recommended. Children (younger than 8 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 (2-23) 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 223

5 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 (3-32) REVIEW OF LITERTURE 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. 224

6 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 Literature summary: Abimbola O (4) 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 signifi- cantly 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 5%. Products with a retail price of less than or equal to one dollar per 200 mg of chondroitin sulfate were found to be seriously deficient in meeting label claim (less than 0% 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 225

7 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 2.4% of the label claim for glucosamine and 77.69% to over % 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 00 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 -naphthyl isothiocyanate. Sample cleanup included passage through an anion exchange cartridge. Analysis was accomplished by injection of 0. ml of the sample solution into an isocratic HPLC system consisting of a C8 column, a mobile phase of acetonitrile: water: acetic acid: triethylamine (4.5: 95.5:0.: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 mg/ml (CV<0%) with a detection limit of 0.63 μ g/ml and a limit of quantification of.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 226

8 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 00% and 50% of the declared amount were 99.0% with an RSD of 2.% and 0% 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.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 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 00.0% and 0.5%, respectively. Repeatability for one sample, which was analyzed six times on a single plate, was.72% relative standard deviation (RSD). The intermediate precision was.20% RSD for a sample analyzed in duplicate once per plate on five different days over a sevenday 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 227

9 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 mg (r = ) was constructed. The recovery rate of the developed method was % (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 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 QUANTIFICATION OF GLUCOSAMINE IMPURITIES -METHOD DEVELOPMENT & METHOD VALIDATIION 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). 228

10 Method of Analysis Buffer Preparation: 0.8 gram of phosphoric acid (85%),.0 ml of Triethylamine and.2 gram of Octane sulfonic acid sodium salt were transferred into 000 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 00-5 C8; ( ) mm; 5 µm Column temperature : 25 C Flow rate : 0.5 ml / minute Injection volume : 0 µl. Detector Wavelength : 95 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 0 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 00 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 00 ml 229

11 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 0 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 0 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 N Acetyl Glucosamine Theoretical Plate Count for 5 Hydroxymethyl 2 Furaldehyde peak from first injection of Pyrazine diluted standard solution. 2 Furaldehyde Pyrrole 2 Carboxaldehyde N Acetyl Glucosamine % RSD for peak area from 5 Hydroxymethyl 2 Furaldehyde five replicate injections of Pyrazine diluted standard solution. 2 Furaldehyde Pyrrole 2 Carboxaldehyde Tailing factor for peak from N Acetyl Glucosamine first injection of diluted 5 Hydroxymethyl 2 Furaldehyde 2 Furaldehyde standard solution. Pyrrole 2 Carboxaldehyde Tailing factor for Pyrazine peak from first injection of diluted standard solution. Resolution between Pyrazine and 5 Hydroxymethyl 2 Furaldehyde peaks from first injection of diluted standard solution. NLT 2000 NMT 5.0 NMT 2.0 NMT 3.5 NLT

12 Table- IV.3 : RRT values S. No. Impurity Name RRT N Acetyl Glucosamine.00 2 Pyrazine Hydroxymethyl 2 Furaldehyde Furaldehyde Pyrrole 2 Carboxaldehyde 8.9 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 Calculation: i) % of N-Acetyl-Glucosamine Impurity AIUIT WS 5 00 P AW = x x x x x x00 ADSP WT 00 LC ii) % of 5-Hydroxymethyl-2-Furaldehyde (5-HMF) Impurity AIUIT2 WS P2 AW = x x x x x x00 ADSP WT 00 LC iii) % of Pyrazine Impurity AIUIT3 WS P3 AW = x x x x x x00 ADSP WT 00 LC iv) % of 2-Furaldehyde Impurity AIUIT4 WS P4 AW = x x x x x x00 ADSP WT 00 LC 23

13 v) % of Pyrrole 2-Carboxaldehyde Impurity AIUIT5 WS P5 AW = x x x x x x00 ADSP WT 00 LC vi) % of Maximum Single unknown Impurity AIUIT WS 5 00 P AW = x x x x x x00 ADSP WT 00 LC % of Total unknown Impurities ATIT WS 5 00 P AW = x x x x x x00 ADSP WT 00 LC % of Total Impurities = (% of all known impurities + % of total unknown Impurities) Where, WS = Weight of N-Acetyl-Glucosamine standard taken in the preparation of diluted standard solution. 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. ADSP = Average area of N-Acetyl-Glucosamine peak from diluted standard injections. ADSP2 = Average area of 5-Hydroxymethyl-2-Furaldehyde peak from diluted standard injections. 232

14 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. AIUIT = 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. P = 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 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% 233

15 METHOD DEVELOPMENT (46-47) 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 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 0.0% it indicates method was highly sensitive in quantification of impurities. Injection volume of 0 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 5 ml of diluent was added sonicate for 5 minutes to dissolve the material and made up the volume with diluent and mixed well. ml 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 500 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. 234

16 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 00-5C8 (300 X 4.0 mm, 5 µm) column. Mixture of the Buffer and Acetonitrile in the ratio of 90:0 (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 25 o C and the wavelength was monitored at 95 nm. The injection volume was 0 µl, diluent HPLC grade water. Note: - Buffer preparation gm of phosphoric acid (85 %),.0 ml of Triethylamine and.2 gm of Octane sulphonic acid salt were dissolved in 000 ml of HPLC grade water and mixed well. Column section: Based on molecule nature started the development by using conventional C8 column of Zorbax SB C8 ( ) 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 5%. 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( ) mm; 5 µm. and with this resolution found satisfactory. This column was with the carbon load of 9%, high pure silica with endcapped so that peak shape was good. For the further improvement purpose finalized the ( ) mm; 5 µm. System suitability establishment By considering USP (48) chapter <62> 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 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 235

17 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 95nm 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 METHOD VALIDATION (49) SYSTEM SUITABILITY & SPECIFICITY 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 236

18 Figure- IV.5: Chromatogram from Diluted Standard Solution Table- IV.5 : System suitability results System suitability parameters Observed value N Acetyl Glucosamine 5758 Theoretical Plate Count 5 Hydroxymethyl 2 for peak from first Furaldehyde 399 injection of diluted Pyrazine 238 standard solution. 2 Furaldehyde 4032 Pyrrole 2 Carboxaldehyde 468 % Relative Standard Deviation for peak area from five replicate injections of diluted standard solution. Tailing factor for peak from first injection of diluted standard solution. N Acetyl Glucosamine 0. 5 Hydroxymethyl 2 Furaldehyde 0. Pyrazine 0. 2 Furaldehyde 0.3 Pyrrole 2 Carboxaldehyde 0. N Acetyl Glucosamine.2 5 Hydroxymethyl 2 Furaldehyde. 2 Furaldehyde. Pyrrole 2 Carboxaldehyde. Tailing factor for Pyrazine peak from first injection of diluted standard solution. Resolution between Pyrazine and 5 Hydroxymethyl 2 Furaldehyde peaks from first injection of diluted standard solution Acceptance criteria NLT 2000 NMT 5.0 NMT 2.0 NMT 3.5 NLT

19 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) 238

20 Table- IV.6 : Placebo interference Interference found (Yes/No) Sample N Common Placebo With Common Placebo Chondroitin Sulfate 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 to6. Figure- IV.8 : Chromatogram from impurity solution (n acetyl glucosamine) 239

21 Figure- IV.9 : Chromatogram from impurity solution (pyrazine) Figure- IV.0 : Chromatogram from impurity solution (5 hydroxymethyl 2 furaldehyde) Figure- IV. : Chromatogram from impurity solution (2 furaldehyde) 240

22 Figure- IV.2 : Chromatogram from impurity solution (pyrrole 2 carboxaldehyde) Figure- IV.3 : Chromatogram from impurity blend solution Figure- IV.4 : HPLC chromatogram from impurity spiked test solution 24

23 Figure- IV.5 : Purity plot for glucosamine: Figure- IV.6 : 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 N Acetyl Glucosamine Pyrazine Hydroxymethyl 2 Furaldehyde Furaldehyde Pyrrole 2 Carboxaldehyde

24 Table- IV.8 : Peak purity results S. No. Impurity Name Purity Purity Angle Threshold Glucosamine N Acetyl Glucosamine PRECISION 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 Response Injection N N Acetyl 5 Hydroxymethyl Glucosamine 2 Furaldehyde Pyrazine Mean Standard deviation % RSD Table- IV.9b : System precision results (Contd.,): Injection N Response 2 Furaldehyde Pyrrole 2 Carboxaldehyde Mean Standard deviation % RSD

25 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.7. The individual results were reported in the Table-IV.0a&b below together with mean value, the standard deviation and relative standard deviation for Glucosamine impurities and total impurities. Figure- IV.7 : HPLC Chromatogram From Impurity Spiked Test Solution Sample N Table- IV.0a : Method precision results Glucosamine Impurities N Acetyl 5 Hydroxymethyl Pyrazine Glucosamine 2 Furaldehyde % % % Mean Standard deviation % RSD

26 Table- IV.0b : Method precision results (Contd.,): Glucosamine Impurities Sample N 2 Furaldehyde Pyrrole 2 Carboxaldehyde Total Impurities % % % Mean Standard deviation % RSD INTERMEDIATE PRECISION: Study conducted as like method precision by varying the system, column and analyst. Obtained values were presented in Table-IV. &2a,b,c,d,e,f. 245

27 Table- IV. : Intermediate precision system suitability results (Analyst to Analyst, System to System and Column to Column Variation): System suitability parameters Theoretical Plate Count for peak from first injection of diluted standard solution. % Relative Standard Deviation for peak area from five replicate injections of diluted standard solution. Tailing factor for peak from first injection of diluted standard solution. Observed value Analyst - I Analyst - II N Acetyl Glucosamine Hydroxymethyl 2 Furaldehyde Pyrazine Furaldehyde Pyrrole 2 Carboxaldehyde N Acetyl Glucosamine Hydroxymethyl 2 Furaldehyde Pyrazine Furaldehyde Pyrrole 2 Carboxaldehyde N Acetyl Glucosamine Hydroxymethyl 2 Furaldehyde.. 2 Furaldehyde.. Pyrrole 2 Carboxaldehyde.. Tailing factor for Pyrazine peak from first injection of diluted standard solution. Resolution between Pyrazine and 5 Hydroxymethyl 2 Furaldehyde peaks from first injection of diluted standard solution Acceptance criteria NLT 2000 NMT 5.0 NMT 2.0 NMT 3.5 NLT

28 Table- IV.2a : Intermediate precision results (Analyst to Analyst, System to System and Column to Column Variation): Sample N Analyst - I N Acetyl Glucosamine Analyst - II % % Mean Standard deviation % RSD.3.4 Mean difference 0.00 Sample N Table- IV.2b : Intermediate precision results (Contd.,): 5 Hydroxymethyl 2 Furaldehyde Analyst - I Analyst - II % % Mean Standard deviation % RSD Mean difference

29 Table- IV.2c : Intermediate precision results (Contd.,): Pyrazine Sample N Analyst - I Analyst - II % % Mean Standard deviation % RSD Mean difference 0.00 Table- IV.2d : Intermediate precision results (Contd.,): 2 Furaldehyde Sample N Analyst - I Analyst - II % % Mean Standard deviation % RSD.3.5 Mean difference

30 Table- IV.2e : Intermediate precision results (Contd.,): Pyrrole 2 Carboxaldehyde Sample N Analyst - I Analyst - II % % Mean Standard deviation % RSD Mean difference 0.0 Table-IV.2f : Intermediate precision results (Contd.,): Total Impurities Sample N Analyst - I Analyst - II % % Mean Standard deviation % RSD Mean difference

31 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%, 00%, 50% 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.3 to IV.7 Table- IV.3 : Accuracy for N Acetyl Glucosamine Series LOQ 50% 80% 00% 50% 200% Sample N Theoretical content (µg/ml) Calculated content (µg/ml) % recovery Mean % Recovery % RSD

32 Table- IV.4 : Accuracy for 5 Hydroxymethyl 2 Furaldehyde: Series LOQ 50% 80% 00% 50% 200% Series LOQ 50% 80% 00% 50% 200% Sample N Theoretical content (µg/ml) Calculated content (µg/ml) % recovery Table- IV.5 : Accuracy for Pyrazine: Sample N Theoretical content (µg/ml) Calculated content (µg/ml) % recovery Mean % Recovery % RSD Mean % Recovery % RSD

33 Table- IV.6 : Accuracy for 2 Furaldehyde: Series LOQ 50% 80% 00% 50% 200% Series LOQ 50% 80% 00% 50% 200% Sample N Theoretical content (µg/ml) Calculated content (µg/ml) % recovery Mean % Recovery Table- IV.7 : Accuracy for Pyrrole 2 Carboxaldehyde: Sample N Theoretical content (µg/ml) Calculated Content (µg/ml) % recovery % RSD Mean % Recovery % RSD

34 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.8 to22 given below. Linearity plots were mentioned in Figure-IV.8 to22. Figure- IV.8 : N Acetyl Glucosamine 253

35 Figure- IV.9 : Pyrazine Figure- IV.20 : 5 Hydroxymethyl 2 Furaldehyde 254

36 Figure- IV.2 : 2 Furaldehyde Figure- IV.22 : Pyrrole 2 Carboxaldehyde 255

37 Table- IV.8 : Linearity for N Acetyl Glucosamine Solution No. Concentration % Concentration µg/ml Response LOQ % % % % % Slope Y-Intercept Correlation Coefficient Table- IV.9 : Linearity for 5 Hydroxymethyl 2 Furaldehyde: Solution No. Concentration % Concentration µg/ml Response LOQ % % % % % Slope Y-Intercept Correlation Coefficient

38 Table- IV.20 : Linearity for Pyrazine: Solution No. Concentration % Concentration µg/ml Response LOQ % % % % % Slope Y-Intercept Correlation Coefficient Table- IV.2 : Linearity for 2 Furaldehyde: Solution No. Concentration % Concentration µg/ml Response LOQ % % % % % Slope Y-Intercept Correlation Coefficient

39 Table- IV.22 : Linearity for Pyrrole 2 Carboxaldehyde: Solution No. Concentration % Concentration µg/ml Response LOQ % % % % % Slope Y-Intercept Correlation Coefficient 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 0. 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. 258

40 Table- IV.23 : LOD & LOQ Values Name % of Impurity Signal to Noise Ratio LOD LOQ LOD LOQ N Acetyl Glucosamine Hydroxymethyl 2 Furaldehyde Pyrazine Furaldehyde Pyrrole 2 Carboxaldehyde Table- IV.24a : Precision & Accuracy at Limit of Quantitation level: Name Injection N LOQ (%) % Recovery at LOQ N Acetyl Glucosamine Mean Standard Deviation %RSD Table- IV.24b : Precision & Accuracy at Limit of Quantitation level: contd., Name Injection N LOQ (%) % Recovery at LOQ 5 Hydroxymethyl 2 Furaldehyde Mean Standard Deviation %RSD

41 Table- IV.24c : Precision & Accuracy at Limit of Quantitation level: contd., Name Injection N LOQ (%) % Recovery at LOQ Pyrazine Mean Standard Deviation %RSD Table- IV.24d : Precision & Accuracy at Limit of Quantitation level: contd., Name Injection N LOQ (%) % Recovery at LOQ 2 Furaldehyde Mean Standard Deviation %RSD Table- IV.24e : Precision & Accuracy at Limit of Quantitation level: contd., Name Injection N LOQ (%) % Recovery at LOQ Pyrrole 2 Carboxaldehyde Mean Standard Deviation %RSD

42 RUGGEDNESS 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 Similarity factor Hours N Acetyl Glucosamine 5 Hydroxymethyl Pyrazine Time in Similarity factor Hours 2 Furaldehyde Pyrrole 2 Carboxaldehyde Table- IV.26a : Stability of test preparation at ambient temperature about (25ºC) Time in hours N Acetyl Glucosamine % Imp Difference Glucosamine Impurities 5 Hydroxymethyl 2 Furaldehyde % Imp Difference Spl- Spl-2 from Initial Spl- Spl-2 from Initial Spl- Spl-2 Pyrazine % Imp Difference from Initial Initial NA NA NA Table- IV.26b : Stability of test preparation at ambient temperature about (25ºC) Time in hours 2 Furaldehyde % Imp Difference Glucosamine Impurities Pyrrole 2 Carboxaldehyde % Imp Difference Total Impurities Spl- Spl-2 from Initial Spl- Spl-2 from Initial Spl- Spl-2 % Imp Difference from Initial Initial NA NA NA

43 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) Similarity factor Time in N Acetyl 5 Hydroxymethyl Hours Pyrazine Glucosamine 2 Furaldehyde Time in Similarity factor Hours 2 Furaldehyde Pyrrole 2 Carboxaldehyde Table- IV.28 : Stability of test preparation at refrigerator temperature about (8ºC) Glucosamine Impurities 5 Hydroxymethyl Time in N Acetyl Glucosamine Pyrazine 2 Furaldehyde hours % Imp Difference % Imp Difference % Imp Difference Spl- Spl-2 from Initial Spl- Spl-2 from Initial Spl- Spl-2 from Initial Initial NA NA NA

44 Table- IV.29 : Stability of test preparation at refrigerator temperature about (8ºC) Glucosamine Impurities 2 Furaldehyde Time in hours % Imp Differenc Pyrrole 2 Total Impurities Carboxaldehyde % Imp Difference % Imp e from from Spl- Spl-2 Spl- Spl-2 Spl- Spl-2 Initial Initial Difference from Initial Initial NA NA NA Bench top Stability of Mobile phase: Stability of mobile phase at ambient temperature about (25ºC) was conducted at initial, after 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.3 263

45 Table- IV.30 : Bench top stability of Mobile phase system suitability results Observed value System suitability parameters Initial Day- Day-2 Theoretical Plate Count for peak from first injection of diluted standard solution. % Relative Standard Deviation for peak area from five replicate injections of diluted standard solution. Tailing factor for peak from first injection of diluted standard solution. N Acetyl Glucosamine Hydroxymethyl 2 Furaldehyde Pyrazine Furaldehyde Pyrrole 2 Carboxaldehyde N Acetyl Glucosamine Hydroxymethyl 2 Furaldehyde Pyrazine Furaldehyde Pyrrole 2 Carboxaldehyde N Acetyl Glucosamine Hydroxymethyl 2 Furaldehyde Furaldehyde..2.2 Pyrrole 2 Carboxaldehyde..2.2 Tailing factor for Pyrazine peak from first injection of diluted standard solution Resolution between Pyrazine and 5 Hydroxymethyl 2 Furaldehyde peaks from first injection of diluted standard solution. Acceptance criteria NLT 2000 NMT 5.0 NMT 2.0 NMT 3.5 NLT