Development and Validation of a Chromatographic Method for the Estimation of Rifampicin in Bulk and Pharmaceutical Formulations

 

Venkataramana N.V.¹, Nivedita R Desai¹, Sreenivasa S¹, Chaluvaraju K. C.², Aruna Kumar D. B.*¹

¹Department of Studies and Research in Chemistry, Tumkur University, Tumakuru-572103, India

²Deptartment of Pharmaceutical Chemistry, Government College of  Pharmacy, Bengaluru-560 027, India.

 

ABSTRACT:

In the present study arapid, simple, selective, linear, precise, accurate and robust reverse phase high performance liquid chromatography method was developed and validated for the qualitative and quantitative analysis of rifampicin in bulk and pharmaceutical formulations and their degradation studies such as acid, alkali and peroxide stressed were carried out. Gradient elution at a flow rate of 1.0 ml min^-1 was employed using symmetry syncronis C18 (250 x 4.6 mm 5µm), column at a temperature of 40⁰ C. The mobile phase used consisted of phosphate buffer (A) and acetonitrile (B) (100%) in the ratio of 50:50 v/v. The chromatograms were recorded at a wavelength of 236 nm using array detector. Linearity was observed in the concentration range of 25-125 µg/ml. The retention time of rifampicin was found to be 5.2 min. The method was validated as per the international conference on harmonization (ICH) guidelines and the proposed method can be successfully applied for the estimation of rifampicin in pharmaceutical formulations.   

 

 

 

INTRODUCTION:

Rifampicin (RIF) is chemically (7S,9E,11S,12R,13S,14R,15R,16R,17S,18S,19E,21Z)-2,15,17,27,29-pentahydroxy-11-methoxy-3,7,12,14,16,18,22-heptamethyl-26-[(1E)-[(4-methylpiperazin-1-yl)imino]methyl]-6,23-dioxo-8,30-dioxa-24-azatetracyclo[23.3.1.1⁴,⁷.0⁵,²⁸]triaconta-1(29),2,4,9,19,21,25,27-octaen-13-yl acetate^[1] having the structure (Fig. 1) with the molecular formula C₄₃H₅₈N₄O₁₂and  molecular weight of 822.9402 g / mol. Physically it appears as a Orange-brown to red-brown powder with a melting point of 183-188 °C and pKa value of 6.9 (Strongest acidic) and 7.53 (Strongest Basic) ^[2]. It is a well-known official anti-tubercular drug in IP, BP and USP ^[3,4,5].

 

Rifampicin belongs to a complex semisynthetic macrocyclic class of antibiotic obtained from Streptomycenmediterranei acts by inhibiting DNA-dependent RNA polymerase activity in susceptible cells. Specifically, it interacts with bacterial RNA polymerase but does not inhibit the mammalian enzyme. It is bactericidal and has a very good broad spectrum of activity against most gram-positive and gram-negative organisms including Pseudomonas aeruginosa and specifically Mycobacterium tuberculosis. Because of rapid emergence of resistant bacteria, its use is restricted to the treatment of mycobacterial infections and a few other indications. Rifampin acts via the inhibition of DNA-dependent RNA polymerase, leading to a suppression of RNA synthesis and cell death^[6].

 

Literature survey revealed that various analytical methods such as UV-Visible spectrophotometry^[7,8], spectroflurimetry^[9] and RP-HPLC^[10,11], LC-MS/MS^[12], were described for the quantitative analysis of rifampicin in combined dosage forms with other pharmaceutically important drugs such as isoniazid ^[13], piperine^[14] etc., But there is a scarcity of literature on the estimation of rifampicinalone in pharmaceutical formulation except HP-TLC^[15]. Hence, in the present study it was decided to develop and validate a reverse phase high performance liquid chromatographymethod for the estimation of rifampicin in bulk and tablet dosage forms with good accuracy, sensitivity and simplicity.

 

Fig.1

 

Table 1: The structure of rifampicin

SL.No.	Name	Fig No.
1	Blank	Rifampicin structure (Fig.1 )

 

MATERIALS AND METHODS:

The current developed method is reversed phase high performance liquid chromatography and the HPLC system used for the study was theroscientific ultimate 3000 diode array detector. The column used was Syncronis C18, 250 x 4.6 mm 5µm.

 

Reagents Used:

Rifampicin active ingredient was obtained as a gift sample from Hire Pharmaceuticals, Bangalore. The tablets used were collected from the marketed sample of BioconPvt. Ltd. HPLC grade water was used and obtained from milli-Q and all other chemicals used were of AR grade.

 

Chromatographic Conditions:

Column                : Syncronis C18, 250 x 4.6 mm 5µm.

Flow rate              : 1.0 ml/min

Wavelength         : 236 nm

Column Temperature        : 25⁰ C

Injection Volume                             : 10 µl

Needle wash                       : Acetonitrile

Elution                                : Mobile phase A: mobile phase B (50:50)

Retention time                   :5.2 min

 

PROCEDURES:

1.      Method description:

1.1.  Preparation of Buffer:

1.4 g of monobasic sodium phosphate was accurately weighed, dissolved in few ml of HPLC water in a 1000.0 ml volumetric flask, mixed well to dissolve and then made up the volume to the mark. pH was adjusted to 3.0 ±0.05 with dilute ortho phosphoric acid, filtered the solution through 0.45 µm nylon membrane filter and sonicated to degas for 5 minutes.

 

1.2.  Preparation of Mobile Phase:

Mobile phase A: buffer

Mobile phase B: 100% acetonitrile.

 

1.3.  Diluent preparation:

Mobile phase A and B in the ratio of 50:50 v/v were mixed and stirred well.

 

1.4.  Standard preparation:

Weighed and transferred accurately 12.5 mg of rifampicin into a 50.0 ml volumetric flask, added about 30.0 ml of diluent and sonicated for 3minutes to dissolve. The volume was made up to the mark with diluent and mixed well. Further, diluted 4 ml to 20ml with diluent and injected gradient (1), blank (diluent) (1), standard preparation (6 s) and checked the following system suitability parameters.

 

2.      Analytical Method Validation:

2.1.  System suitability:

The system suitability parameters are to be set to verify that the analytical system is working properly and gives accurate and precise results.

 

Table 2: Acceptance criteria of Rifampicin.

Sl. No.	Acceptance criteria	Results
1.	Tailing factor for Rifampicin peak in Standard Preparation should be not more than 2.0.	1.20
2.	Theoretical plate count for Rifampicin peak in Standard Preparation should be not less than 4000.	8004
3.	The RSD for Rifampicin peak from five replicates of Standard Preparation should be not more than 2.0%.	0.6 %

 

Data interpretation:

From the above results, it can be concluded that the system was suitable for the analytical method validation of Rifampicin.

 

2.2.  Specificity:

Specificity is the ability of an analytical method to assess the analyte unequivocally in the presence of components such as impurities and degraded products which may be expected to be present. The specificity parameters were performed by injecting diluent, standard preparation into the HPLC system and recorded the retention times of diluent, rifampicin peak from standard preparation.

 

Acceptance Criteria:

Diluent Peaks should not be interfered and interference should be less than or equal to 0.2 % with the main peak. The chromatogram peak of rifampicin should be pure.

 

Table 3: The chromatogram references to acceptance criteria of specificity.

SL.No.	Name	Retention time	Data file No.
1	Blank	--	Chromatogram No: 01 (Fig.3 )
2	Standard	12.3 min	Chromatogram No: 02 (Fig. 4)

 

Data interpretation:

From the above data, it was concluded that the diluents and rifampicin chromatogram peaks were not interfered with each other.

 

2.2.1. Specificity by degradation studies:

Forced degradation of rifampicin was carried out, to confirm that during stability studies or throughout the shelf life, if any degradation product found will not interfere with the main peak of rifampicin. In addition, the forced degradation study will also help to identify the stability indicating nature of the method.

 

Sample as such:

Transferred accurately 20.0 mg of rifampicin sample into a 50.0 ml volumetric flask and dissolved in few ml of the diluents, then made up to the mark, mixed well. Further, transferred 3.0 ml of the solution into a 25.0 ml volumetric flask, diluted up to the mark with diluent and mixed well.

 

Acid stressed sample: (0.1 N HCl)

Transferred accurately 19.85 mg of rifampicin sample in to a 50.0 ml volumetric flask andadded 2 ml of 0.1 N HCl and kept at room temperature for 30minutes, volume was made up to the mark and mixed well. Further, transferred 3.0 ml of the solution into a 25.0 ml volumetric flask, diluted up to the mark with diluent and mixed well.

 

Alkali stressed sample: (0.1 N NaOH)

Transferred accurately 20.12 mg of rifampicin sample in to 50.0 ml volumetric flask, added 2 ml of 0.1 N NaOH and kept at room temperature for 30 minutes, made up the volume to the mark with diluent and mixed well. Further, transferred 3.0 ml of the solution into a 25.0 ml volumetric flask, diluted up to the mark with diluent and mixed well.

 

 

Peroxide stressed sample: (3.0% w/v H₂O₂):

Transferred accurately 20.45 mg of rifampicin sample in to a 50.0 ml volumetric flask, added 2 ml of 3.0 % w/v H₂O₂and kept at room temperature for 30 minutes, made up the volume with the diluent and mixed well. Further transferred 3.0 ml of above the solution into a 25.0 ml volumetric flask, diluted up to the mark with diluent and mixed well.

 

Acceptance criteria:

The chromatogram peak of rifampicin should be pure. All unknown Impurities and degradation products if any should be well separated from rifampicin peak. The peak purity factor of rifampicin should be not less than 990. Any interference should be not more than 0.2% at the retention time of analyte.

 

Table 4: The acceptance criteria of specificity by forced degradation studies.

Degradation studies	Peak Purity**	Purity Match	% of Assay
Sample as Such	P	999	100.0%
Acid Stressed (0.1 N HCl )	P	997	55.6 %
Alkali Stressed (0.1 N NaOH)	P	999	99.6%
Peroxide stressed (3.0%w/v)	P	999	90.4%

 

Table 5: The chromatogram references to acceptance criteria of specificity.

SL.No.	Name	Data file No.
1	Acid Stressed (0.1 N HCl )	Chromatogram No: 03 (Fig.5 )
2	Alkali Stressed (0.1 N NaOH)	Chromatogram No: 04 (Fig. 6)
3	Peroxide stressed (3.0%w/v)	Chromatogram No: 05 (Fig. 7)

 

Where,

Peak Purity **:

‘P’ indicates Rifampicin peak is pure which was confirmed by diode array detector and Dionex Chrome Leon software.

 

Data interpretation:

The sample was found to be degraded more in alkali stressed conditions. The rifampicin peak was pure. Peak purity factor for rifampicin peak was more than 990. Hence, it can be concluded that the developed assay method is considered to be specific and stability indicating.

 

2.3. Precision:

The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple sampling of homogeneous sample. The precision of analytical method is usually expressed as the standard deviation or relative standard deviation (Coefficient of variation) of series of measurements.

 

2.3.1. Method precision:

In method precision, a homogeneous sample of a single batch was analyzed 6 times and it indicates that the method is giving consistent results of a single batch of sample.

Table 6: Results of method precision.

Set No.	Rifampicin (% Assay)
1	100.9
2	101.3
3	102.3
4	102.1
5	100.7
6	99.8
Mean	101.2
RSD	0.93%

 

Table 7: The chromatogram references to acceptance criteria of specificity.

SL.No.	Name	Data file No.
1	Precision	Chromatogram No: 06 (Fig.8 )

 

Data Interpretation:

The results were within prescribed limits and from the above results, it can be concluded that the method is highly precise.

 

2.4. Stability in analytical solution:

The stability in analytical solution was evaluated by injecting the standard preparation at regular intervals.

 

Acceptance Criteria:

The % difference of area response for the peak in standard preparation should be within ± 2.0% from initial area after a specified period.

 

Table 8: Stability in analytical solution of a standard preparation at 25°C

 

Data interpretation:

From the above data, it can be concluded that, the rifampicin standard preparation was stable up to 7 hrs (% difference is 0.66%) at room temperature (25°C).

 

2.5. Linearity:

The linearity of an analytical method is its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of analyte in samples within a given range.

 

The linearity for rifampicin standard was performed in the range of 30% to 120% of working concentration.

 

Linearity of stock solution:

Weighed and transferred 12.50 mg of rifampicin standard into a 50 ml volumetric flask dissolved and diluted to mark with diluent.

 

Table 9: Linearity of rifampicin standard

 

The area response at each level was recorded, calculated the correlation coefficient and regression coefficient

(R square).A graph of concentration (ppm) on X-axis and area response under the curve on Y-axis was plotted.

 

Acceptance Criteria:

Correlation coefficient and regression coefficient should be not less than 0.998.

 

Table 10: Correlation and regression coefficient.

Sl. No.	Conc. in ppm	Area response
1	13	280433
2	25	553166
3	38	848440
4	50	1115388
5	63	1405527
	Correlation Coefficient	0.9998
	Regression Coefficient	0.9999

 

Fig.2Linearity plot

 

Data Interpretation:

From the results of statistical analysis, the linearity data for rifampicin was found to be between 25 % and 125 % of the working concentration. The correlation coefficient and regression coefficients are more than 0.999.

 

2.6. Accuracy:

The accuracy of an analytical method is the closeness of the test results obtained by that method to the true value (Standard value). Spiked known quantity of rifampicin 75%, 100%, and 125% of working concentration into the placebo and analyzed these samples in triplicate for each level. From the results obtained the % recovery was calculated.

 

Acceptance criteria:

Mean % Recovery at each level should be between 97.0 % and 103.0 %.

 

Sample preparation:

Accurately weighed X mg of sample was transferred into 50 ml of volumetric flask, dissolved and made up to the mark with diluent. Further diluted 3 ml to 25 ml with diluent.

 

Table 11: preparation of samples at different levels.

Levels	Weight taken in mg (X mg)	Diluted to	Diluted	Made up to
Level -1	15.23	20ml	3 ml	25 ml
	15.02
	15.54
Level -2	21.24
	21.25
	20.52
Level -3	25.25
	25.13
	25.68

 

 

Table 12: Accuracy study results at different levels.

Sr. No	Level (about)	Area Response	*mg added	*mg Recovered	% Recovery	% Mean	% RSD
1	75 %	844998	15.23	15.61	102.5	101.7	1.1
2		830097	15.02	15.33	102.1
3		845181	15.54	15.61	100.5
1	100 %	1180207	21.24	21.80	102.6	101.3	1.7
2		1142948	21.25	21.11	99.3
3		1130754	20.52	20.89	101.8
1	125 %	1395096	25.25	25.77	102.1	102.0	0.3
2		1392249	25.13	25.72	102.3
3		1414197	25.68	26.12	101.7
Mean Recovery					101.7
RSD					1.1 %

 

Data interpretation:

From the above results, it can be concluded that the % recovery is well within the limit and hence the method is accurate.

 

Fig. 3. Representative Chromatogram of Blank

 

Chromatogram No: 02

 

Fig 4. Representative Chromatogram of Standard

 

Fig 5. Representative Chromatogram of Acid (0.1 N HCl) treated sample

 

 

 

Fig. 6. Representative Chromatogram of base stressed sample

 

Fig. 7. Representative chromatogram peroxide treated sample

 

Fig. 8. Representative chromatogram overlay of precision sample sample

 

2.7. Robustness study of rifampicin

The robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

 

Robustness parameters

Change in Column Temperature ± 20C.

Change in Flow rate ± 0.1 ml/min.                 

Change in Organic Phase ± 2%.

 

Acceptance criteria

The system suitability parameters should be passed for all the conditions.

 

 

 

 

Table 13.Results of analysis for the marketed samples of Rifampicin.

Acceptance criteria	Retention time (min)	Theoretical plates for rifampicin peak in standard solution should be not less than 4000.0	Tailing factor for rifampicin peak in standard solution should be not more than 2.0.
Increase  in Flow(-0.1ml/min)	4.800	7655	1.15
Decrease  in Flow(+0.1ml/min)	5.823	8398	1.19
Increase in Temperature(-5°C)	4.957	7082	1.18
Decrease  in Temperature(+5°C)	5.533	8365	1.14
Increase in Organic Phase  in MP-B	4.083	7914	1.15
Decrease in Organic Phase (-2%) in MP-A	7.940	7854	1.18

 

 

Data interpretation:

From the above results, it was concluded that, the method is robust.

 

2.8. Marketed sample analysis of rifampicin Procedure

Rifampicin tablets from Lupin limited were analysed using the validated method. An accurately weighed portion of the powdered tablets equivalent to about 12.5 mg of rifampicin was transferred to 50 ml volumetric flask and dissolved 30 ml of diluent, sonicated for 5 min

and filtered through 0.45µ Nylon filter paper, then volume was made upto the mark. Further diluted 4 ml to 20 ml with diluent and results are tabulated. 

 

Table 14: The chromatogram references to acceptance criteria of specificity.

Sr. No.	Acceptance criteria		Results
1	Tailing factor for Rifampicin peak in Standard Preparation should be not more than 2.0.		1.20
2	Theoretical plate count for Rifampicin peak  in Standard Preparation should be not less than 4000.		8004
3	The RSD for Rifampicin peak from five replicate s of Standard Preparation should be not more than 2.0%.		0.6%
% Assay
1	Sample 1	100.3%	% Average value
2	Sample 2	99.7%	100.0%

 

Table 15: Results of analysis for the marketed samples of Rifampicin.

SL.No.	Name	Data file No.
1	Marketed sample	Chromatogram No: 07 (Fig.9 )

 

Fig 9: Chromatogram of sample

 

SUMMARY AND CONCLUSIONS:

The proposed reverse phase high performance liquid chromatographic method for rifampicin is developed and validated. The validation parameters were established with acceptance criteria, the method is found to be specific and stability indicating, also linearity, robustness and accuracy of the method is established for rifampicin and the method is found to be precise and can be used for the routine quantitative, qualitative and stability sample analysis.

 

 

Table 16: Summarize results of the analysis.

Validation Parameter	Acceptance criteria	Results
System suitability	Tailing factor for rifampicin peak in standard preparation should be not more than 2.0.	1.20
	Theoretical plate count for rifampicin peak in standard preparation should be not less than 4000.	8004
	The RSD for rifampicin peak from five replicate s of standard preparation should be NMT 2.0%.	0.6%
Specificity	Diluent and placebo peaks should not interfere/interference should be less than or equal to 0.2% with main peak.	Diluent peaks were not interfered with Rifampicin Peak.
	Peak of rifampicin should be Pure.	Peak of Rifampicin was Pure.
Forced Degradation Study	All degradation products if any should be well separated from rifampicin peak.	All Degradation products were well separated from Rifampicin peak
	Peak purity factor for rifampicin peakshould be NLT 990.	Peak purity factor for Rifampicin peak was more than 999.
Method Precision	The results should be within specification limit.	The results were within specification limit.
	The RSD calculated on 6 determinations should be NMT 3.0%.	0.93%
Solution Stability	The % Difference of area response for the peak in standard preparationshould be within ± 2.0% from initial area after specified period.	Standard solution stable up to 7hrs.The % Difference of Area Response for the peak in Standard preparation is 0.66%
Linearity	Correlation coefficient and regression coefficient should be NLT 0.998.	Correlation coefficient	Regression Coefficient
		0.9998	0.9999
Accuracy	Mean % recovery at each level should be between 97.0 % and 103.0 %.	75%	101.7
		100%	101.3
		125%	102.0
	RSD obtained for all the accuracy level determinations should be NMT 3.0%.	1.1%

 

 

ACKNOWLEDGEMENT:

The authors are thankful to the chairperson Department of studies and research in chemistry, Tumkur University, Tumkur and Vinay Kumar Gupta, Assistance Manager INM Technologies Pvt. Ltd.  

 

REFERENCES:

1.       Lembke TL, Williams DA, Roche VF, Zito SW. Foyes principles of medicinal chemistry. 6^th ed. Lippincot Williams and wilkins; 2008.P.1127-1147.

2.       Patil JS, Suresh S. Physicochemical characterization, in-vitro release and permeation studies of respirable rifampicin-cyclodextrin inclusion complexes. 71(6):638-43.

3.       Indian Pharmacopoeia, Vol. III, New Delhi, The Controller Publication, Govt. of India; 2010: 2054-2065.

4.       British Pharmacopoeia, Vol. II, London, The British Pharmacopoeia Commission; 2010: 1844, 3063.

5.       The United State Pharmacopoeia, USP28 NF23, Rockville MD, United State Pharmacopoeial Convention, Inc; 2005: 3501 – 3507.

6.       Tripathi KD. Essentials of Medical Pharmacology. 7^thed. NewDelhi: Jaypee Brothers Medical Publishers (P) Ltd; 1985.P. 449-52.

7.       Begum ASK, Raju BD, Rao RN. Simultaneous estimation of rifampicin and isoniazid in combined dosage form by a simple UV spectrophotometric method. Der Pharmacia Lettre 2013; 5(3):419-26.

8.       Shah U, Jasani A. UV spectrophotometric and RP- HPLC methods for simultaneous estimation of isoniazid, rifampicin and piperine in pharmaceutical dosage form. International Journal of  Pharmacy and Pharmaceutical Sciences 2014;6(10):274-80.

9.       Maaboud IA, Mohamed, Mohamed FA, Atia NN, Botros SM. A novel spectrofluorimetric determination of four anti-TB drugs in their pure and pharmaceutical dosage forms by quenching effect on the fluorescence of NBS-phenothiazine product. Asian Journal of Biomedical and Pharmaceutical Sciences 2013;3(26):21-7.

10.     Sachin BS, Bhat V , Koul M, Sharma SC, Tikoo MK, Tikoo AK et al., Development and Validation of a RP-HPLC Method for the Simultaneous Determination of Rifampicin and a Flavonoid Glycoside - A Novel Bioavailability Enhancer of Rifampicin. Tropical Journal of Pharmaceutical Research 2009;8 (6): 531-7.

11.     Kumar SA, Debnath B, Seshagiri JVLN, Chaitanya B, Pavani AD, Ramya GP et al., rapid and sensitive RP-HPLC analytical method development and validation for estimation of rifampicin in bulk as well as in pharmaceutical formulation by using PDA detector. Indo American Journal of Pharmaceutical Research 2014;4(3):1561-72.

12.     Oswald S, Peters J, Venner M, Siegmund W. LC-MS/MS method for the simultaneous determination of clarithromycin, rifampicin and their main metabolites in horse plasma, epithelial lining fluid and broncho-alveolar cells. Journal of Pharmaceutical and Biomedical Analysis 2011;55(1):194-201.

13.     Leandro KC, Carvalho JMD, Giovanelli LF,  Moreira JC. Development and validation of an electroanalytical methodology for determination of isoniazid and rifampicin content in pharmaceutical formulations. Brazilian Journal of Pharmaceutical Sciences 2009;45(2):331-7.

14.     Kupuriya KG, Parmar PM, Topia HR, Faldu SD. Method development and validation of Rifampicin and piperin in their combined dosage form. International Bulletin of Drug Research 2012;1(2):71-80.

15.     Shishoo CJ, Shah SA, Rathod S, Savale SS, Vora MJ. Impaired bioavailability of rifampicin in presence of isoniazid from fixed dose combination (FDC) formulation. International Journal of Pharmaceutics 2001; 228: 53–67.

 

 

Accepted on 08.11.2016        © RJPT All right reserved

Research J. Pharm. and Tech 2016; 9(12):2191-2198.