Stability Indicating RP-HPLC-PDA Method for Simultameous Quantification of Glucosamine, Methyl Sulphonyl Methane and Diacerein Tablets

 

Mohammed Danish, Bharath Rathna Kumar. P*

Department of Pharmaceutical Analysis, Anwarul Uloom College of Pharmacy, Hyderabad,Telangana., India.

 

ABSTRACT:

A simple, fast, accurate and specific RP-HPLC-PDA method has been developed for the simultaneous quantification of, glucosamine, diacerein and methyl sulphonyl methane and in bulk and tablet dosage form. The chromatographic separation was performed on a reverse phase Altima C18 Column (150×4.6mm, 5µm particle size) consisting mobile phase of potassium dihydrogen ortho phosphate buffer: acetonitrile (65:35 v/v), with a flow rate 1ml/min, temperature 30˚C and UV detection wavelength 254nm. The retention times of glucosamine, diacerein and methyl sulphonyl methane were observed as 2.13min, 3.48min and 4.16 min respectively. The developed method was validated by validation parameters such as linearity, range, accuracy, precision and robustness. The results obtained for validation parameters are within the limits as per ICH guidelines. The linearity of the drugs were obtained in the range of 37.5 ppm-225 ppm for Glucosamine, 12.5 ppm -75 ppm methyl sulphonyl methane and 2.5 ppm -15 ppm for diacerein. %RSD from precision studies were 0.8, 0.6 and 0.8, mean percentage recovery from accuracy studies were found to be 99.53%, 99.83% and 100.09% for Glucosamine, methyl sulphonyl methane and diacerein, respectively. LOD, LOQ values obtained from regression equations of Glucosamine, Methyl sulfonyl methane and diacerein were 0.47ppm, 0.53ppm, 0.14ppm, and 1.42ppm 1.60ppm, 0.41ppm respectively. The method designed and validated can be successfully used for the regular quantification of Glucosamine, Methyl sulfonyl methane and diacerein in tablet and bulk forms.

 

 

 

INTRODUCTION:

Glucosamine (GLU) (Fig.1) is chemically (3R,4R,5S)-3-amino-6(hydroxymethyl)oxane-2,4,5-triol. Molecular formula of glucosamine is C₆H₁₃NO_5. Molecular formula is 179.1g/mol. Glucosamine is commonly used for the treatment of osteoarthritis. It is an amino sugar and prominent precursor in the biochemical synthesis of glycosylated proteins and lipids. Glucosamine is a precursor for glycosaminoglycans. Glycosaminoglycans are a major component of joint cartilage, supplemental glucosamine may help to rebuild cartilage and treat arthritis.

 

 

Literature review reveals that only one method is developed so far for the simultaneous quantification of Glucosamine, methyl sulphonyl methane and diacerein in triple drug combination¹. There are few methods reported for the estimation of Glucosamine with other drugs by HPLC^2-5. Methyl sulfonyl methane (MSM) (Fig.2) is a organo sulphur compound with molecular formula (CH₃)₂SO₂ and molecular weight 94.1 g/mol. It is chiefly used for the treatment of oxidative stress and osteoarthritis. There are many methods for the estimation of diacerein with other drugs by spectrophotometric^6-8, HPLC^9-11. Diacerein (DIA) (Fig.3) is chemically 4, 5-diacetyloxy-9, 10-dioxo-anthracene-2-carboxylic acid. Molecular formula and molecular weight of Diacerein are C₁₉H₁₂O₈ and 368.2g/mol respectively. It acts by blocking the action of interleukin-1 beta a protein involved in degenerative joint disease. Diacerein is under investigation for the treatment of diabetes mellitus 2 and diabetes related complications. Hence the authors developed a simple, fast, precise, accurate, robust and stability indicating method for simultaneous assay of Glucosamine, methyl sulphonyl methane and diacerein, in tablet dosage form.

 

 

Fig.1. Structure of glucosamine

 

 

Fig.2. Sructure of methyl sulphonyl methane

 

 

Fig.3. Sructure of diacerein

 

Experimental:

Chemicals and reagents:

Glucosamine, methyl sulphonyl methane, diacerein standards were obtained from Spectrum Pharma research solutions, Hyderabad. Glucosamine, methyl sulphonyl methane, diacerein, with brand name GLUCERINE manufactured by Torainse Pharma, Chandighar, India was purchased from local pharmacy. Acetonitrile, methanol, were procured from Thermo Fischer Scientific India Pvt. Ltd. Milli-Q water was used throughout the analysis for buffer preparation. WATERS HPLC system with Empower 2 software was used for chromatographic separation

 

Chromatographic Conditions:

Chromatographic separation was carried out by emloying Altima C18 Column (150mm×4.6µm particle size) using 0.01N potassium dihydrogen ortho phosphate buffer and acetonitrile in the ratio 65:35%v/v as mobile phase. The flow rate was adjusted to 1ml/min with run time 8min. The coloumn temperature was maintained at 30˚C. The column effluents were detected at isobestic point 254nm and injection volume was 10µL.

Procedure for preparation of 0.1N KH₂PO₄ buffer solution:

13.6 gm of KH₂PO₄ was weighed and transferred in to a 1000ml of volumetric flask and dissolved in 900ml of water and diluted with water upto 1000ml, filtered through 0.45µ membrane filter.

 

Procedure for mobile phase preparation:

650ml of 0.1N KH₂PO₄ buffer solution and 350ml of acetonitrile mixture was used as mobile phase. The mobile phase was filtered through 0.45µ membrane filter and sonicated by means of ultrasonication to remove dissolved gases.

 

Procedure for preparation of diluent:

Water and acetonitrile mixture in the ratio 50:50v/v was used as diluent for sample and standard solution preparation.

 

Standard solution preparation:

75mg of Glucosamine, 25mg of Methyl sulfonyl methane 5mg of Diacerein and working Standards were weighed and transferred in to 50ml clean, dry volumetric flask. Then add about 30ml of diluent to the flask and dissolve by means of ultrasonication, make up the volume by adding the diluent. From the above solution 1ml was pipetted out and transferred in to 10ml clean and dry volumetric flask to prepare the concentration of 150ppm, 50ppm and 10ppm of Glucosamine, Methyl sulfonyl methane and Diacerein respectively.

 

Sample solution preparation:

Sample solution was prepared by weighing and powdering 20 tablets. An amount equivalent to 75mg of Glucosamine, 25mg of Methyl sulfonyl methane and 5mg of Diacerein was ransferred in to a 500ml volumetric flask. The contents of the flask were dissolved in 50 ml of diluent, sonicated for 25min and made up to the final volume with diluent. 1ml of above solution was pipetted and transferred in to 10ml volumetric flask and the final volume was made up to the mark using diluent. The final concentrations of Glucosamine, Methyl sulfonyl methane and Diacerein sample solution was found to be 150ppm, 50ppm and 10ppm of respectively.

 

Method development and validation:

The present method was developed as per ICH guidelines Q2(R1)¹². Validation parameters employed for this study are selectivity, linearity, accuracy, precision, robustness, LOD and LOQ.

 

Specificity and Selectivity¹³:

As shown in Fig.4 it was noticeable that there was no interference due to tablet excipients at the retention time of analyte peaks as well as stress degradation products

 

 

Fig.4.Standard Chromatogram of Glucosamine, diacerein, methyl sulphonyl methane

 

Linearity:

Linearity of the analytes were determined by injecting 6 different concentrations of working standard solutions of glucosamine (37.5 ppm-225 ppm), methyl sulphonyl methane (12.5 ppm -75 ppm), diacerein (2.5 ppm -15 ppm) into HPLC system. The calibration curves were shown in fig 5 to fig 7. Linearity data for glucosamine, methyl sulfonyl methane and diacerein was shown in Table 1.

 

TABLE-1 :Linearity Results For Glucosamine, Methyl Sulphonyl Methane And Diacerein

Glucosamine		Methyl sulfonyl methane		Diacerein
Conc (ppm)	Peak area	Conc (ppm)	Peak area	Conc (ppm)	Peak area
0	0	0	0	0	0
37.5	204986	12.5	71448	2.5	23480
75	405264	25	140510	5	46560
112.5	618838	37.5	215695	7.5	69644
150	810513	50	278772	10	93844
187.5	1035654	62.5	341085	12.5	116545
225	1216150	75	415633	15	138965

 

 

Fig.5 Linearity curve for Glucosamine

 

 

Fig.6 Linearity curve for Methyl sulfonyl methane

 

 

Fig 7: Linearity curve for Diacerein

 

Accuracy:

Standard addition method¹⁴ was employed for the accuracy studies. Accuracy of the developed assay method was evaluated in triplicate at three concentration levels (50%, 150% and 150%) and the percentage recoveries were calculated. The percentage recovery was calculated and enlisted in Table 2.

 

Precision:

System precision was established by injecting six different solutions of Glucosamine having concentration equal to 150ppm, methyl sulphonyl methane having concentration equal to 50ppm and diacerein having concentration equal to 10ppm. The mean and %RSD values of peak area and retention time were presented in Table-3 and Table-4.

 

Robustness:

Robustness of the developed analytical method refers to its ability to remain unaffected due to small but deliberate variations in the method parameters (flow rate, mobile phase ratio and column temperature) which indicates reliability of the method for routine analysis. The flow rates selected for study were 0.8ml/min, 1ml/min and 1.2ml/min. The column temperature was changed from 25˚C to 30˚C and 35˚C. The sample solution was injected in to the HPLC system in triplicate and the peak areas were recorded and shown in Table-5

 

Table 2: Accuracy Results

Drugs	Spiked concentration (ppm)		% Recovery
Glucosamine	75	50%	100.25
	150	100%	99.53
	225	150%	99.45
Methyl Sulphonyl Methane	25	50%	99.30
	50	100%	99.83
	75	150%	99.77
Diacerein	5	50%	98.89
	10	100%	100.09
	15	150%	100.33

 

Table. 3: System precision values for GLU, MSM and DIA standard solutions

S.No	Average area*			Rt(min)*
	GLU	MSM	DIA	GLU	MSM	DIA
1	816694	276726	93875	2.11	3.46	4.13
2	810225	277608	93246	2.12	3.47	4.15
3	814141	278100	95381	2.12	3.48	4.16
4	813558	278057	93789	2.13	3.48	4.16
5	801897	274823	93336	2.13	3.48	4.17
6	802742	273983	93570	2.13	3.49	4.18
Mean	808513	276550	93866	2.12	3.47	4.16
SD	6211.5	1754.8	781.6	0.0	0.0	0.0
% RSD	0.8	0.6	0.8	0.4	0.3	0.4

*(n=6)

 

Table.4: Method precision values for GLU, MSM and DIA tablet sample solution

S.No	Average area*			Rt(min)*
	GLU	MSM	DIA	GLU	MSM	DIA
1	812342	275175	93399	2.09	3.42	4.08
2	815297	279376	93876	2.10	3.43	4.10
3	809674	276954	93999	2.10	3.43	4.10
4	810861	275403	92949	2.10	3.43	4.10
5	809773	277680	93671	2.10	3.43	4.10
6	809332	272914	93989	2.11	3.45	4.12
Mean	811213	276250	93647	2.10	3.43	4.10
SD	2282.9	2250.1	410.5	0.00	0.00	0.01
% RSD	0.3	0.8	0.4	0.3	0.3	0.3

 *(n=6)

 

Table-5: Results of Robustness Study

Chromatographic conditions	Rt(min)			Average area
	GLU	MSM	DIA	GLU	MSM	DIA
Buffer: Acetonitrile 70:30(v/v)	2.03	3.15	4.64	810062	266666	94424
Buffer: Acetonitrile 65:35(v/v)	2.13	3.48	4.16	808513	276550	93866
Buffer: Acetonitrile 60:40(v/v)	2.17	3.87	4.90	806642	271488	93428
Flow rate (0.8 mL/min)	2.35	3.40	4.11	814226	269675	94056
Flow rate (1.0 mL/min)	2.13	3.48	4.16	808513	276550	93866
Flow rate (1.2 mL/min)	2.17	3.11	3.87	814692	275498	93370
Temperature 25°C	2.13	3.71	4.60	803921	267951	90732
Temperature 30˚C	2.13	3.48	4.16	808513	276550	93866
Temperature 35°C	2.04	3.25	3.81	814148	270243	92742

 

RESULTS AND DISCUSSION:

A new analytical method was developed and validated for simultaneous estimation of Glucosamine, methyl sulphonyl methane and diacerein, in tablet dosage form. Different coloumns were tried and the final column chosen was Altima C18 Column (150mm×4.6µm) which gave satisfactory resolution and run time. Many mobile phase ratios were tried to resolve the three chromatographic peaks such as methanol water but broadness of the peaks were observed with methanol and water as mobile phase. To improve the sharpness of the peaks mobile phase was made slightly acidic, and produced symmetric peaks. The retention times of the Glucosamine, methyl sulphonyl methane and diacerein were 2.13 min, 3.48min and 4.16min respectively(Fig 4). The best fit for the calibration curve could be achieved by separate linear regression equations, which were y=5436.x + 1280 (GLU), y= 5498.x + 2830 (MSM), y= 9290.x + 186.5 (DIA). The percentage recoveries of Glucosamine, methyl sulphonyl methane and diacerein were 99.63%, 99.83% and 100.09% respectively. LOD, LOQ values obtained from regression equations of Glucosamine, Methyl sulfonyl methane and Diacerein were 0.47ppm, 0.53ppm, 0.14ppm, and 1.42ppm 1.60ppm, 0.41ppm respectively. The analytical method developed was validated according to ICH Q2R1 guidelines. Validation parameters according to ICH Q2R1 guidelines were Specificity, linearity, precision, accuracy, robustness, limit of detection and limit of quantification.

 

Forced degradation studies:

Glucosamine, methyl sulphonyl methane and diacerein sample solution was subjected for stress degradation studies as per ICH guidelines entitled stability testing of new drug substances and products to elucidate the internal stability of the main analytes¹⁵. Standard test procedures were followed for the stress conditions like acid, base, peroxide, heat, water and UV light^16-17. The three drugs were degraded in the selected stress conditions and showed 2-6% degradation in acidic, alkaline, peroxide and heat conditions. Slight degradation was observed in UV light and water degradation (Table 6). Though degradants peaks were observed in the chromatograms, no de degradants peas were interfering the retention times of Glucosamine, methyl sulphonyl methane and diacerein peaks, hence the method was proved to be stability indicating.

 

Table-6: Results of Forced Degradation Study

Stress conditions	% Assay of active ingredients
	GLU	% Degradation	MSM	% Degradation	DIA	% Degradation
Acid, 2N HCl,	95.56	4.44	93.75	6.25	94.34	5.66
Base, 2N NaOH	96.11	3.89	94.76	5.24	95.55	4.45
H2O2 (20%,v/v)	97.14	2.86	95.69	4.31	97.22	2.78
Dry heat (105 ºC)	97.57	2.43	97.68	2.32	97.64	2.36
UV	98.76	1.24	98.49	1.51	98.51	1.49
Water	99.24	0.76	99.22	0.78	99.32	0.68

 

CONCLUSION:

The developed RP-HPLC-PDA method was found to be suitable for the analysis of glucosamine, methyl sulphonyl methane and diacerein in tablet dosage form and was found to be simple , reliable, economical and precise. Therefore, this RP-HPLC method for estimation of glucosamine, methyl sulphonyl methane and diacerein can be used in various laboratories for its quantitative determination in bulk and pharmaceutical tablet dosage form.

 

ACKNOWLEDGEMENT:

The authors are grateful to, Dr.A.Venkateshwar Reddy, Principal, Anwarul Uloom College of Pharmacy, Hyderabad, and Spectrum Pharma Research Solutions, Hyderabad for providing lab facilities to carry out this work.

 

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Accepted on 02.11.2018       © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(1): 103-107.