Formulations containing Rifampicin, Isoniazid and Pyrazinamide are widely available in the market. To perform an assay of these formulations various methods have been developed, however after going through various research articles it has been observed that a method for simultaneous estimation of the combination of these three drugs by UV-VIS spectrophotometry has not been developed yet.

Rifampicin¹ chemically known as (7S,9E, 11S,12R, 13S,14 R,15R, 16R, 17S, 18S, 19E, 21Z) -2, 15, 17, 27, 29-pentahydroxy- 11-methoxy-3, 7, 12, 14, 16, 18, 22-heptamethyl -26- [(E)- (4-methylpiperazin-1-yl) iminomethyl]- 6,23- dioxo-8, 30-dioxa -24-azatetracyclo [23.3.1.1^4,7.0^5,28] triaconta-1(29),2,4,9, 19, 21, 25, 27-octaen-13-yl acetate having molecular formula C₄₃H₅₈N₄O₁₂ and molecular weight of 822.9 g/mol.

Fig 1: Structure of Rifampicin

Rifampicin or rifampin is a semisynthetic derivative of Rifamycin B obtained from Streptomyces mediterranei. It is bactericidal to M. tuberculosis and commonly used in combination with other agents as therapy of tuberculosis. M. lapreae is highly sensitive but some are moderately susceptible. Rifampicin is an antibiotic that inhibits DNA-dependent RNA polymerase which is encoded by rpo B gene and blocks its polymerizing function. Specifically, it interacts with bacterial RNA polymerase but does not inhibit the mammalian enzyme. It is bactericidal and has a very broad spectrum of activity against most gram-positive and gram-negative organisms and specifically Mycobacterium tuberculosis.

Isoniazid² chemically known pyridine-4-carbohydrazide having chemical formula of C₆H₇N₃O with a molecular weight of 137.139g/mol.

Fig 2: Structure of Isoniazid

Isoniazid is the most reliable and most commonly used medication for tuberculosis. Isoniazid is mycobactericidal in nature. Isoniazid is a carbohydrazide obtained by formal condensation between pyridine-4-carboxylic acid and hydrazine.It derives from an isonicotinic acid. Isoniazid blocks the synthesis of mycolic acids, major components of the mycobacterial cell wall. Isoniazid is a pro drug which gets converted to a reactive metabolite by the help of enzyme catalase peroxidase (encoded by katG gene). This free radical inhibits Enoyl ACP reductase enzyme which is responsible for synthesis of mycolic acid thus a cell wall deficient mycobacterium is formed, another minor mechanism by which it inhibits enzyme DHFRase. So, Isoniazid is a mycobactericidal drug.

Pyrazinamide³ is chemically known as pyrazine-2-carboxamide having molecular formula C₅H₅N₃O and molecular weight of 123.113g/mol.

Fig 3: Structure of Pyrazinamide

Pyrazinamide is a first line antituberculotic medication, but is used only in combination with other antituberculotic medications such as isoniazid or rifampicin. It is weakly mycobactericidal but gets converted to a strong one in acidic medium. It is more active against intracellular bacilli. Pyrazine carboxamide is a monocarboxylic acid amide resulting from the formal condensation of the carboxy group of pyrazinoic acid (pyrazine-2-carboxylic acid) with ammonia It has a role as an antitubercular agent and a prodrug. Pyrazinamide kills or stops the growth of certain bacteria that cause tuberculosis (TB). It is used with other drugs to treat tuberculosis. It is a highly specific agent and is active only against Mycobacterium tuberculosis. The drug is active only at a slightly acidic pH. Pyrazinamide gets activated to Pyrazinoic acid in the bacilli where it interferes with fatty acid synthase FAS I. This interferes with the bacterium ability to synthesize new fatty acids, required for growth and replication. It has a good sterilizing activity. Pyrazinamide is particularly active against slowly multiplying intracellular bacilli (unaffected by other drugs) by an unknown mechanism of action. Its bactericidal action is dependent upon the presence of bacterial pyrazinamidase, which removes the amide group to produce active pyrazinoic acid. Pyrazinamide is an important component of multidrug therapy for tuberculosis.

2. MATERIAL AND METHODS:

2.1. Chemicals and Reagents:

Isoniazid, Rifampicin and Pyrazinamide were used. Marketed pharmaceutical dose Rifater tablet was used manufactured by Sanofi India Ltd. containing Isoniazid 50mg, Rifampicin 120mg, Pyrazinamide 300mg. Methanol of analytical grade were used as solvent

2.2. Instrumentation:

The proposed work was carried out on a Shimadzu UV-visible spectrophotometer (model UV-1700 series), which possesses a double beam double detector configuration with a1 cm quartz matched cell. All weighing was done on electronic balance.

2.3. Solubility:

The drugs Isoniazid, Rifampicin and Pyrazinamide are soluble in methanol. Thus, methanol was chosen as the solvent for developing the medium.

2.4. Introduction to UV Method Development: Spectrophotometry is largely favoured particularly by little scale businesses as the expense of the gear is less and the support issues are insignificant. UV spectrophotometer principle follows the Beer-Lambert Law. This law states that whenever a beam of monochromatic light is passed through a solution with an absorbing substance, the decreasing rate of the radiation intensity along with the thickness of the absorbing solution is actually proportional to the concentration of the solution and the incident radiation.

2.5. Preparation of Stock Solution:

Standard stock solution of Isoniazid, Rifampicin and Pyrazinamide was prepared by dissolving 10mg of Isoniazid in 10ml of methanol to produce a concentration of 1000µg/ml. 1ml of this stock solution was taken and then diluted up to 10ml by using methanol to produce a concentration of 100µg/ml which is the standard stock solution. Again, 1ml of this stock solution was taken and then diluted up to 10ml by using methanol to produce a concentration of 10µg/ml which is the standard stock solution.

2.6. Preparation of Working Standard Solution:

2.7. From the above stock solution of concentration 10 µg/ml of Isoniazid, 0.75ml was diluted to 10ml with methanol to prepare a concentration of 0.75µg/ml. Similarly, 1.5ml, 2.25ml, 3ml and3.75ml were diluted to 10ml with methanol to prepare concentrations of 1.5µg/ml,2.25µg/ml, 3µg/ml and 3.75µg/ml respectively.

From the above stock solution of Rifampicin of concentration 10µg/ml,1.5ml was diluted to 10ml with methanol to prepare a concentration of1.5µg/ml. Similarly, 3ml, 4.5ml, 6ml and 7.5ml were diluted to 10ml with methanol to prepare concentrations of 3µg/ml, 4.5µg/ml, 6µg/ml and 7.5µg/ml respectively.

From the above stock solution of concentration 10µg/ml of Pyrazinamide 4ml was diluted to 10ml with methanol to prepare a concentration of 4µg/ml. Similarly,8ml was diluted to 10ml with methanol to prepare concentrations of 8 µg/ml. Then from the above stock solution of concentration 100µg/ml of Pyrazinamide 1.2ml was diluted to 10ml with methanol to prepare a concentration of 12µg/ml. Similarly, 1.6ml and 2ml, were diluted to 10ml with methanol to prepare concentrations of 16µg/ml and 20µg/ml.

2.8. Determination of zero crossing point:

All the prepared solution of the drugs of different concentrations were scanned in UV-VIS Spectrophotometer in the range 800-200nm using methanol as a blank. After scanning of the samples, the zero-crossing point for the respective drugs were determined from the graphs obtained after the scanning by the UV-VIS Spectrophotometer Zero crossing point is the point of the graph where the sample shows zero absorbance. We check for points where two drug samples show zero absorbance but the third drug sample shows absorbance at this point. The zero-order graph of Isoniazid was transformed to first order derivative graph and the point selected 721nm. For Rifampicin the zero-order graph was transformed to first order derivative graph and the point selected was 322nm. For Pyrazinamide the zero-order graph was transformed to first order derivative graph and the point selected was 256 nm.

Overlay spectra of the drugs:

Overlay of first order derivative graph of Isoniazid, Rifampicin and Pyrazinamide

2.9. Preparation of Calibration Curve of Isoniazid: The calibration curve was prepared by taking the above solutions of concentration ranging from 0.75-3.75µg/ml. Then, the calibration curve was plotted by taking concentration on x-axis and absorbance from the first order derivative graph obtained after UV-VIS spectrophotometric scanning of the solutions on the y- axis. The curve showed linearity in the concentration range of 0.75-3.75µg/ml. The correlation coefficient (r²) was found to be 0.9983

2.10. Preparation of Calibration Curve of Rifampicin:

The calibration curve was prepared by taking the above solutions of concentration ranging from 1.5-7.5µg/ml. Then, the calibration curve was plotted by taking concentration on x-axis and absorbance from the first order derivative graph obtained after UV-VIS spectrophotometric scanning of the solutions on the y- axis. The curve showed linearity in the concentration range of 1.5-7.5µg/ml. The correlation coefficient (r²) was found to be 0.9967

2.11. Preparation of Calibration Curve of Pyrazinamide:

The calibration curve was prepared by taking the above solutions of concentration ranging from 4-20µg/ml. Then, the calibration curve was plotted by taking concentration on x-axis and absorbance from the second order derivative graph obtained after UV-VIS spectrophotometric scanning of the solutions on the y- axis. The curve showed linearity in the concentration range of 4-20µg/ml. The correlation coefficient (r²) was found to be 0.9927.

2.12. Method Validation⁴:

Validation is a process of establishing documented evidence, which provides a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its predetermined specifications and quality characteristics. The validation for UV method development was performed using parameters like Linearity, Accuracy, Precision, Robustness, Ruggedness, and Limit of detection (LOD), Limit of quantification (LOQ) (Table no. 1).

2.12.1. Linearity:

Various aliquots were prepared form the stock solution of Isoniazid ranging from 0.75-3.75µg/ml, Rifampicin ranging from 1.5-7.5µg/ml and Pyrazinamide 4-20µg/ml. The samples were scanned in UV-VIS Spectrophotometer using methanol as blank. It was found that the selected drug shows linearity between 0.75-3.75µg/ml in case of Isoniazid, Rifampicin between 1.5-7.5µg/ml and Pyrazinamide between 4 - 20µg/ml. (Table 8)

2.12.2. Accuracy:

The accuracy of the method was determined by preparing solutions of different concentrations that is 80%, 100% and 120% in which the amount of marketed formulation was kept constant and the amount of pure drug was varied respectively. The solutions were prepared in triplicates and the accuracy was indicated by % recovery. (Table 2)

Table no.2: Accuracy Readings of Isoniazid, Rifampicin and Pyrazinamide

OBSERVATION/RESULT
No. of preparations	Conc. (µg/ml)						% Recovery
No. of preparations	Formulation			Pure Drug			% Recovery
Drug	INZ	RIF	PYZ	INH	RIF	PYZ	INH	RIF	PYZ
S₁: 80%	3.75	7.5	20	3	6	16	102.3	100.8	100.4
S₂: 80%	3.75	7.5	20	3	6	16	101.08	100.6	100.02
S₃: 80%	3.75	7.5	20	3	6	16	100.01	100.5	100.4
S₄: 100%	3.75	7.5	20	3.75	7.5	20	99.7	99.9	99.8
S₅: 100%	3.75	7.5	20	3.75	7.5	20	99.9	100.2	100.1
S₆: 100%	3.75	7.5	20	3.75	7.5	20	100.3	100.1	99.9
S₇: 120%	3.75	7.5	20	4.5	9	24	100.2	100.2	100.1
S₈: 120%	3.75	7.5	20	4.5	9	24	100.3	99.7	100.3
S₉: 120%	3.75	7.5	20	4.5	9	24	99.5	99.8	100.1

Table no.2: Cont……

	OBSERVATION/RESULT
No. of preparations	Statistical Result
No. of preparations	Mean			SD			%RSD
Drug	INH	RIF	PYZ	INH	RIF	PYZ	INH	RIF	PYZ
S₁: 80%
S₂: 80%	101. 1	100.9 7	100.2 9	1.1 5	0.4 7	0.2 0	1.1 3	0.4 6	0 2 0
S₃: 80%
S₄: 100%
S₅: 100%	99.9 7	100.0 6	99.93	0.3 0	0.1 5	0.1 5	0.3 0	0.1 5	0.1 5
S₆: 100%
S₇: 120%
S₈: 120%	100. 2	99.93	100.1 7	0.4 3	0.3 2	0.1 1	0.4 3	0.3 2	0.1 1
S₉: 120%

2.12.3. Precision:

Precision of the technique was exhibited by intraday and interday variety ponders. In intraday variety contemplate, 6 distinct arrangements of same fixation that is 2.25µg/ml were set up if there should arise an occurrence of INH, 6 unique arrangements of RIF of focus 4.5µg/ml were readied, 6 unique arrangements of PYZ of 12 µg/ml and broke down multiple times in multi day i.e. morning, evening and evening and the absorbances were noted. The outcome was demonstrated by % RSD (Table 3). In the interday variety think about, arrangements of same fixations were arranged and examined multiple times for three continuous days and the absorbances were noted. The outcome was shown by % RSD (Table 3).

2.12.4. Robustness:

Two different analysts performed the scanning of the samples at different conditions of temperature and the % recovery followed by % RSD was obtained. (Table 4)

2.12.5. Ruggedness:

Two different analysts performed the scanning of the samples and after obtaining the % recovery, %RSD was calculated. (Table 4)

2.12.6. Limit of Detection (LOD):

LOD was determined by signal is to noise ration which is termed as S/N is 10/3. Therefore, according to the formula 3.3LOD= LOQ.LOD for Isoniazid was found to be 0.006µg/ml, for Rifampicin0.047µg/ml and for Pyrazinamide, 0.0301µg/ml. (Table 5)

2.12.7. Limit of Quantification (LOQ):

LOQ was determined by taking the least concentration where the machine shows sensitivity i.e. three times to that of the base line and noted as the LOQ. LOQ was found to 0.0198µg/ml for Isoniazid 0.15µg/ml for Rifampicin and Pyrazinamide 0.099µg/ml was recorded. These are minimum concentration in which these three drugs can be quantitated. (Table 5)

2.13. Assay of INH, RIF and PYZ tablets (RIFATER®):

A quantity of powder equivalent to 3.75mg of INH, 7.5mg of RIF and 20mg of PYZ was taken in a 100ml volumetric flask and it was dissolved and diluted up to the mark with methanol. The resultant solution was ultrasonicated for 15 minutes. The solution was then filtered using Whatmann filter paper No.40. From the filtrate, appropriate dilutions were made in ethanol to obtain the desired concentration. This solution was then analysed in UV and the result was indicated by % recovery given in table 5.

Table no. 3: Intraday Precision and Inter day precision

Conc. (µg/ml)			Recovered Conc. (µg/ml) 1			Recovered Conc. (µg/ml) 2			Recovered Conc. (µg/ml) 3			Avg %RSD
INH	RIF	PYZ	INH	RIF	PYZ	INH	RIF	PYZ	INH	RIF	PYZ	INH		RIF	PYZ
2.25	4.5	12	2.21	4.41	11.96	2.23	4.42	11.98	2.21	4.45	12.01
2.25	4.5	12	2.21	4.42	11.96	2.23	4.42	12.01	2.23	4.43	11.99
2.25	4.5	12	2.21	4.41	12.01	2.22	4.43	12.01	2.20	4.45	11.99
2.25	4.5	12	2.22	4.42	11.97	2.23	4.42	11.96	2.22	4.5	12.03
2.25	4.5	12	2.20	4.42	11.99	2.21	4.42	11.96	2.23	4.5	11.97
2.25	4.5	12	2.22	4.42	11.99	2.23	4.42	11.97	2.23	4.43	12.05
%RSD			0.34 %	0.20 %	0.16 %	0.40 %	0.10 %	0.19 %	0.57 %	0.72 %	0.25 %	0.43 %		0.34 %	0.2 %
Inter day precision
Conc. (µg/ml)			Day 1		Day 2				Day 3			%RSD
INH	RIF	PYZ	INH	RIF	PYZ	INH	RIF	PYZ	INH	RIF	PYZ	INH	RIF		PYZ
2.25	4.5	12	2.25	4.45	11.96	2.29	4.6	12.05	2.24	4.41	12.1	0.74%	0.79%		0.38%

Table 4: Results Showing Robustness and Ruggedness of Method

Room Temp.									Temp. 18°
Conc. (µg/ml)			Recovered Conc. (µg/ml)			Statistical Analysis (%RSD)			Recovered Conc. (µg/ml)			Statistical Analysis (%RSD)
INZ	RIF	PYZ	INZ	RIF	PYZ	INZ	RIF	PYZ	INZ	RIF	PYZ	INZ	RIF	PYZ
2.25	4.5	12	2.29	4.42	11.99	1.23 %	0.32 %	0.19	2.24	4.45	11.96	0.74%	0.79%	0.38%
2.25	4.5	12	2.21	4.45	12.03				2.29	4.6	12.05
2.25	4.5	12	2.22	4.41	11.096				2.25	4.41	12.1

Table No.5: Summary of validation

PARAMETER	RESULT
PARAMETER	ISONIAZID	RIFAMPICIN	PYRAZINAMIDE
Linearity indicated by correlation coefficient	0.9983	0.9967	0.9927
Precision indicated by %RSD	0.94%	1.33%	0.551%
Accuracy indicated by % recovery	102.3	100.8	100.4
Limit of Detection	0.006 µg/ml	0.047 µg/ml	0.0301µg/ml
Limit of Quantification	0.0198 µg/ml	0.15µg/ml	0.099µg/ml
Range(µg/mL)	0.75-3.75	1.5-7.5	4-20
Linear regression equation	y=0.0255x-0.0089	y=0.0282x+ 0.0348	y=0.0228x+ 0.029
Robustness indicated by %RSD	0.74%	0.79%	0.38%
Assay indicated by % recovery for tablet	101.6	100.8	101.2

Fig 5: first order spectra of marketed formulation

3. RESULTS AND DISCUSSION:

The developed method was found to be precise as the %RSD values for intra-day and inter-day were found to be less than 2%. Good recoveries (100.4% to 102.3%) of the drug were obtained at each added concentration, indicating that the method was accurate. The LOD and LOQ were found to be in sub-microgram level indicating the sensitivity of the method. The method was also found to be robust and rugged as indicated by the %RSD values which are less than 2%. The results of Assay show that the amount of drug was in good agreement with the label claim of the formulation as indicated by % recovery (101.6%, 100.8% and 101.2%). Summary of validation parameters of proposed spectrophotometric method is shown in table 5.

From the above study we can conclude that the proposed method was accurate, precise, simple, sensitive, robust and cost effective and can be applied successfully for the estimation of Isoniazid, Rifampicin and Pyrazinamide in bulk and marketed formulation. Till date not a suitable method has been developed for the simultaneous estimation of these three drugs.

4. ACKNOWLEDGEMENT:

The authors are grateful to the management of Dr. B. C. Roy College of Pharmacy and A.H.S. for providing solvents, apparatus, instrument and suitable facilities that was required to carry out the project work. We would also like to extend our gratitude to the management of The Mission Hospital, Durgapur, for providing the required pharmaceutical products for our project.

5. REFERENCES:

1. https://www.drugbank.ca/drugs/DB00951 accessed on 16/9/2019.

2. https://www.drugbank.ca/drugs/DB01045 accessed on 16/9/2019.

3. https://www.drugbank.ca/drugs/DB00339 accessed on 16/9/2019.

4. ICH, Q2 (R1) validation of analytical procedures: text and methodology, International conference on harmonization; Nov.1996.Tripathi KD. Essentials of Medical Pharmacology.7th ed. New Delhi: Jaypee Brothers; 2013: 765-769.

Received on 01.12.2019 Modified on 18.01.2020

Asian J. Research Chem. 2020; 13(2):117-122.

DOI: 10.5958/0974-4150.2020.00024.3

Concentration (µg/ml) (INZ)	Abs(INZ)	Conc. (µg/ml) (RIF)	Abs (RIF)	Conc. (µg/ml) (PYZ)	Abs(PYZ)
0.75	0.0112	1.5	0.0811	4	0.1062
1.5	0.0291	3	0.1176	8	0.2313
2.25	0.0465	4.5	0.1561	12	0.3018
3	0.0687	6	0.2054	16	0.39
3.75	0.0872	7.5	0.2488	20	0.4824