INTRODUCTION:

Glucosamine is an amino sugar, a prominent precursor in the biochemical synthesis of glycolated proteins and lipids, commonly used in the treatment of osteoarthritis ¹. It is chemically (3R, 4R, 5S, 6R)-3-Amino-6-(hydroxymethoxy) oxane-2, 4, 5-triol. Vitamin C, an antioxidant, used in the treatment of Scurvy ², common cold and in the treatment of atrial septal defect (ASD) is chemically 2-oxo-L-threo-hexono-1,4- lactone-2,3-enediol. Vitamin E is a lipid soluble antioxidant which protects cell membranes from oxidation by reacting with lipid radicals produced in lipid peroxidation chain reaction ³, an anti-sterility vitamin chemically known as 2, 5, 7, 8- tetramethyl-2-(4, 8, 12-trimethyl tridecyl)-6-chromanol. Literature survey revealed that various analytical methods like spectrophotometric ⁴, HPLC ^5-17 and HPTLC ^18,19 have been reported for the determination of Glucosamine, Vitamin C and Vitamin E individually and combination with some other drugs. No HPTLC method for simultaneous estimation of Glucosamine, Vitamin C and Vitamin E in combined dosage forms has so far been reported. The review of literature prompted us to develop an accurate, precise and derivatized simultaneous method for the estimation of Glucosamine, Vitamin C and Vitamin E in combined dosage forms.

MATERIALS AND METHODS:

Chemicals and Equipment:

CARTIVIT Tablet used for the formulation analysis contains Glucosamine (500 mg), Vitamin C (50 mg) and Vitamin E (8 mg) and it is manufactured and marketed by Apex laboratories Ltd, Mumbai. Pure samples were procured from, Glucosamine – Culcruech pharma Ltd, Hyderabad, Vitamin C – S.D. fine chemicals, Mumbai and Vitamin E – Orchid pharmaceutical Ltd, Chennai. All the chemicals and reagents used were of analytical grade. A Camag HPTLC system comprising of Camag Linomat -5-applicator, Hamilton syringe, Camag twin trough chamber, Camag TLC scanner, and stationary phase pre coated with Silica gel 60F₂₅₄ were used.

Preparation of Standard Solutions:

The given standard Glucosamine and Vitamin C were dissolved in water to give 100 μg/ml and 10 μg/ml spot. Vitamin E was dissolved in ethanol to give 1μg/ml spot. Aliquots of standard solution having concentration ranging from 40 to 100 μg/ml spot of Glucosamine, 2-10 μg/ml spot of vitamin C and 0.2 to 1 μg/ml spot of vitamin E were applied on TLC plates.

Analysis of Tablet Formulation:

The given CARTIVIT twenty tablets were powdered using Pestle and Mortar to fine powder. From this, 250mg of powdered sample was extracted and dissolved in Methanol: water (1:1), centrifuged and the supernatant liquid was made-up to 10 ml in a volumetric flask with 50% Methanol-water and filtered through Whatman filter paper no 41. Aliquots of 40 μg of glucosamine and 4 μg of vitamin C and 600 ng of vitamin E were applied on the pre-coated silica gel 60F₂₅₄ plate. From the peak area obtained the amount of Glucosamine, vitamin C and vitamin E in formulation was simultaneously calculated using the respective calibration graph. The amount obtained per tablet and percentage label claim are shown in Table 1.

Table -1 RESULT OF ANALYSIS OF FORMULATION

Drug	Amount (mg/tablet)		% label claim^*	S.D^*
Drug	Labeled	Found^*	% label claim^*	S.D^*
Glucosamine	500	496.32	99.2	1.08
Vitamin C	50	49.26	98.5	1.26
Vitamin E	8	7.83	97.87	1.34

^*An average value ± relative standard deviation of 5 observations.

Development of Chromatograms:

The TLC plates were pre washed with methanol and activated by keeping at 115^° for about 30 min. The samples were spotted in the form of bands of width 5mm with 100 μl Hamilton syringe on the pre-coated silica gel 60F₂₅₄ plate (20×20cm) and the slit dimension was kept at 15 min respectively. The mobile phase used was Ethanol: acetic acid (9:1 v/v) in chamber and plate saturation time of 15 min, migration distance was allowed up to 95 mm, linear ascending development was carried out in (20×10cm) twin trough glass chamber. Subsequent to the development, TLC plates were dried in current of air and kept in photo documentation chamber. The images of developed plate were captured at white light, UV 254 nm and UV 366 nm using Camag – Reprostar -3 instrument. The developed plate was derivatized with iodine vapor and the images were done in white light using Camag – Reprostar -3 instrument. The derivatized plate was scanned at 500nm using Camag-TLC- scanner-3 instrument.

RESULTS AND DISCUSSION:

Validation Parameters:

The method was validated for linearity, accuracy, limit of detection, limit of quantification, inter-day and intra-day assay precision, repeatability of measurement and repeatability of sample application. Samples applied on the plate were developed with the mobile phase and the peak areas were noted. The mobile phase, ethanol: acetic acid (9:1 v/v), gave R_fvalue of 0.48 ± 0.03 for Glucosamine 0.69 ± 0.04 for vitamin C and 0.83 ± 0.03 for vitamin E (Fig.1).

Linearity and Regression:

A good linear relationship was obtained over the concentration range 20-100 μg/ml of Glucosamine, 2-10 μg/ml for vitamin C and 0.2-1 μg/ml of vitamin E respectively. The linear regression data showed a regression coefficient of 0.9954 for Glucosamine, 0.9994 for vitamin C and 0.9981 for vitamin E.

LOD and LOQ:

The LOD with signal/ noise ratio were found to be 300 ng, 90 ng and 60 ng/spot for Glucosamine, vitamin C and vitamin E respectively. The LOQ with signal/ noise ratio was found to be 1000 ng, 300 ng and 200 ng/spot for Glucosamine, vitamin C and vitamin E respectively.

Fig 1: Chromatogram of Glucosamine, Vitamin C and Vitamin E.

Chromatogram showing resolution of Glucosamine (R_f
= 0.48), Vitamin C (R_{f =} 0.69) and Vitamin E (R_{f =} 0.83).

Precision:

Intra-day assay precision was found by analysis of standard drug at three times on the same day. Inter-day assay precision was carried out using at three different days, and percentage relative standard deviation (%RSD) was calculated. The RSD was found to be less than 2 for both intra-day and inter-day precision. Repeatability of sample application was assessed by spotting 1 μl of drug solution, six times. From the peak areas, the percentage RSD was determined. The complete validation parameters are shown in Table 2.

TABLE -2 VALIDATION PARAMETERS

Parameters	Value
Parameters	Glucosamine	Vitamin C	Vitamin E
R_f	0.48 ± 0.03	0.69 ± 0.04	0.83 ±0.03
Linearity (μg/ml)	20-100μg	2-10μg	0.2-1μg
Correlation co efficient	0.9954	0.9994	0.9981
LOD (ng/spot)	300ng	90ng	60ng
LOQ (ng/spot)	1000ng	300ng	200ng
Precision (% RSD)
Inter-day	0.83	0.62	0.08
Intra-day	0.51	0.36	1.09
Repeatability (% RSD)	0.07	0.16	0.09

R_f- resolution factor, RSD- relative standard deviation, LOD – limit of detection, LOQ – limit of quantification.

Recovery Studies:

The recovery study was carried out at two levels, 50%, 100 %. To the powdered formulation, the standard drugs of Glucosamine, vitamin C and vitamin E were added at 50 % and 100 % levels, dilutions were made and analyzed by the method. The % recovery and % RSD were calculated and found to be within the limit, as listed in Table 3.

TABLE -3 RECOVERY DATA

Level	Amount added (mg)			Amount found (mg)^*			% Recovery^*			% RSD^*
Level	glu	vit C	vit E	glu	vit C	vit E	glu	vit C	vit E	glu	vit C	vit E
50%	250	25	4	252.42	25.5	4.08	100.96	102	102	0.94	1.03	1.12
100%	500	50	8	505	51.6	8.21	101	103.2	102.62	1.02	1.33	1.24

Glu=Glucosamine; vit C= vitamin C; vit E= vitamin E; ^*An average value ± relative standard deviation of 5 observations.

CONCLUSION:

Hence, the developed HPTLC method is precise, specific and accurate; the statistical analysis proved that the method is repeatable and selective for the simultaneous analysis of Glucosamine, vitamin C and vitamin E in bulk drugs and in pharmaceutical dosage forms without any interference from the excipients.

ACKNOWLEDGEMENTS:

The authors are grateful to the Management, RVS College of Pharmaceutical Sciences, Sulur, Coimbatore, and Dalmiah Research Centre, Coimbatore, for providing the required facilities.

REFERENCES:

1. Persiani S., Roda E., Rovati LC, Locatelli M., Giacovelli G and Roda A., Synovial and Plasma glucose concentrations in osteoarthritic patients following oral glucosamine sulphate at therapeutic dose, Osteoarthritis and Cartilage, 2007, 15, 764-772.

2. McCregar GP and Biesallski HK., Rationale and impact C in clinical nutrition, Curr. Opin.Clin. Nutr. Metab. Care, 2006, 9, 697-703.

3. Herrera, Vitamin E action metabolism and perspectives, Journal of Physiology and Biochemistry, 2001, 57(2) 43-56.

4. Priya G., Vineeta K., Rajani S and Chhaya D., Spectrophotometric method for determination of glucosamine in tablets, Ijpr, 2006, 68(1), 83-84.

5. Odriozola-Serrano I., Hernadez-Jover T., Martin-Bellosa O., Comparative evaluation of UV-HPLC methods and reducing to determine vitamin C in fruits, food chemistry, 2007, 105, 1151-1158.

6. Patric F., Tamara K and Olivier H., HPLC-UV determination of vitamin C in a wide range of fortified food products, food chemistry, 2006, 94 (4), 626-631.

7. Kitmani H., Simultaneous Pharmaceutical determination of vitamin C and saccharin by HPLC-UV, Kankyoto Sokutei Gijutsu, 2003, 30 (6), 32-36.

8. Romeu–nadal M., Morera-pons S., Castellote AI., lopez-sabater M.C., Rapid high performance liquid chromatographic method for determination of vitamin C in human milk versus an enzymatic method, J. Chromatogr., 2006, 830(1), 41-46.

9. Takahashi T., Iwase K., Koga S and Sasagawa K., Determination of vitamin E by HPLC, Reports of the Central Customs Laboratory, 1998, 38, 23-30.

10. Ryo Y., Hiroki N., Makoto S and Koji K., Analysis of vitamin E and its oxidation products by HPLC with electrochemical detection, Lipids, 2002, 37(5), 34-38.

11. Byung HH., Byoung-Sun C and Bong CC., Determination of vitamin E in procaine plasma after oral administration of vitamin E enriched feed stuffs using HPLC, Analytical Letters, 2001, 34(13), 2303-2310.

12. Ubaldi AD., Fusari A and Servant P., Quick HPLC method to determine vitamin E concentration in cow’s milk”, Ann.Fac. Medic.Vet, De.Pharm, 2005, 25, 101-110.

13. Wayne KW., Kathleeng G and Andrew GB., Determination of glucosamine in nutritional supplement by reverse-phase ion paring HPLC”, J.Liq. Chromatogr.Rt. Tech, 2000, 23(18), 2862-2871.

14. Ismaiel AT., Micheal CB., and Adrian CW., Optimized reverse phase HPLC method for evaluating percutaneous absorption of glucosamine hydrochloride, J. Pharm. Biomed. Anal., 2006, 41(2-3), 285-392.

15. Zohngmig L., James L., Abim BA., Mohamad A and Natalie D, Determination of glucosamine in raw materials, dosage forms and plasma, using pre-column derivatization with UV-HPLC, J. Pharm. Biomed. Anal., 2004, 35(3), 625-631.

16. Xiolan Z., Jibao C.,, Jun Y and Qinde S., Determination of glucosamine in impure chitin samples by RP- HPLC, Carbohydrate Research, 2005, 340(10), 1732- 1738.

17. Ali A., Saeed S., Franco P and Fakhraddin J., HPLC determination of glucosamine in rat plasma, J. Pham. Pharmaceut. Sci, 2002, 5(2), 176-180.

18. Sullivan C and Sharma J., Development and validation of an HPTLC –Densitometry method for assay for glucosamine of different form in dietary supplement tablets and capsules, Acta Chromatographica, 2005, 15, 119-130.

19. Aburajai T., Amro BI., Aiedesh K., Amurajai M and Al –khalil S., Second derivative UV and HPTLC for the simultaneous determination of vitamin C and dipyrone, Pharmazie, 2000, 55(10), 751-4.

Received on 26.11.2009 Modified on 19.01.2010

Asian J. Research Chem. 3(2): April- June 2010; Page 329-331