Validated Degradation studies for the estimation of Pyrazinamide, Ethambutol, Isoniazid and Rifampacin in a fixed dose combination by UPLC

 

Sri Lakshmi D¹, Jane T Jacob²

¹Department of Pharmaceutical Chemistry, Aditya Pharmacy College, Surampalem, Andhra Pradesh India.

 

ABSTRACT:

Objectives: The intended study was to develop simple, reproducible, reliable and less time consuming analytical methods for estimation of pyrazinamide, ethambutol ,isoniazid and rifampicin in FDC by UPLC and its validation Materials and Methods: RP-UPLC method for estimation of Pyrazinamide, Isoniazid, Ethambutol and Rifampicin has been developed and validated for determination of compounds in commercial tablet dosage form. The compounds were well separated isocratically on a X Bridge 3x50mm, 3.7 μm C₁₈ column using a mobile phase consisting of Triethylamine and Potassium Dihydrogen Ortho Phosphate pH – 7.4) [Sol A]: Methanol and Acetonitrile in a ratio of 85:15 v/v [ Sol B] in the ratio 90:10,0.5 ml/min flow rate with PDA detector. Results and Conclusions: Retention time for Pyrazinamide, Isoniazid, Ethambutol and Rifampicin was found to be 2.012, 3.120, 4.026, 5.926 min respectively. The method was validated in accordance with ICH guidelines. The study showed that the reverse phased liquid chromatography was sensitive and selective for detecting Pyrazinamide,  Isoniazid,  Ethambutol,  Rifampicin using the single mobile phase.

 

 

INTRODUCTION^1,3:

The rationale behind the work is to develop new analytical methods which are economical, less time consuming and reliable. Tuberculosis being one of the most common infectious diseases that affects mankind and India happens to be on the top of the list people suffering from tuberculosis.

 

Pyrizanamide, isoniazide, ethambutol and rifampacin are the drugs used in the first stage of treatment in TB. Rationale for recommending four drugs FDC is that it simplifies both treatment and management of drug supply, and prevent the emergence of drug resistance.

 

Pyrazinamide^3,4:

Chemically pyrazinamide is Pyrazine-2-carboxamide. A pyrazine that is used therapeutically as an antitubercular agent. Pyrazinamide diffuses into M. tuberculosis, where the enzyme pyrazinamidase converts pyrazinamide to the active form pyrazinoic acid. Under acidic conditions, the pyrazinoic acid that slowly leaks out converts to the protonated conjugate acid, which is thought to diffuse easily back into the bacilli and accumulate. The net effect is that more pyrazinoic acid accumulates inside the bacillus at acid pH than at neutral pH. Pyrazinoic acid has also been shown to bind to the ribosomal protein S1 (RpsA) and inhibit trans-translation. This may explain the ability of the drug to kill dormant mycobacteria.

 

 

 

Fig 1 Structure of Pyrazinamide

 

Isoniazid^5,6:

Chemically Isoniazid is isonicotinic acid hydrazide. Antibacterial agent used primarily as a tuberculostatic. It remains the treatment of choice for tuberculosis. Isoniazid is a prodrug and must be activated by bacterial catalase. Specifically isoniazid inhibits InhA, the enoyl reductase from Mycobacterium tuberculosis, by forming a covalent adduct with the NAD cofactor. It is the INH-NAD adduct that acts as a slow, tight-binding competitive inhibitor of InhA.

 

 

Fig 3 Structure of Isoniazid

 

Ethambutol^7,8:

Chemically ethambutol is (S,S)-N,N’-ethylenebis(2-aminobutan-1-ol)dihydrochloride. An antitubercular agent that inhibits the transfer of mycolic acids into the cell wall of the tubercle bacillus. It may also inhibit the synthesis of spermidine in mycobacteria. Ethambutol inhibits arabinosyl transferases which is involved in cell wall biosynthesis. By inhibiting this enzyme, the bacterial cell wall complex production is inhibited. This leads to an increase in cell wall permeability.

 

 

Figure 4 Ethambutol

 

Rifampicin^9,10:

Chemically rifampicin  is (12Z,14E,24E)-(2S,16S,17S,18R,19R,20R,21S,22R,23S)-1,2-dihydro-5,6,9,17,19-penahydroxy-23-methoxy-2,4,12,16,18,20,22-heptamethyl-8-(4-methyl-piperazin-1-yliminomethyl)-1,11-dioxo-2,7-(epoxypentadeca-1,11,13-rienimino)naphtha[2,1-b]furan-21-yl acetate. A semisynthetic antibiotic produced from Streptomyces mediterranei. It has a broad antibacterial spectrum, is an antibiotic that inhibits 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 broad spectrum of activity against most gram-positive and gram-negative organisms (including Pseudomonas aeruginosa) and specifically Mycobacterium tuberculosis.

 

 

Fig 2 Structure of Rifampicin

 

New drugs are introduced into market at an alarming rate, so development of new analytical methods plays a vital role in the estimation of dosage forms. Since there is a time lag for introduction of drugs into official books such as pharmacopeias there is a great necessity to develop analytical methods which are less time consuming, precise, and accurate and at the same time should be reproducible and repeatable. The quality of the drug entering into the market can be monitored by performing the stability indicating studies.

 

Medicaments containing two or more active ingredients in a fixed proportion are known as combination products/FDCs. Combination products are creating daunting challenges to the chemists in the analytical department who are responsible for validation of analytical methods which are developed.

Main criteria for analytical method development is

The drug or drug products are not official in the pharmacopeia,

Patent regulations on the drugs will not give the adequate literature for proper analytical procedure, 

·        Due to the interference caused by the formulation excipients a method may not be available for the estimation of drug in the formulation, 

·        Analytically quantitation of the drug in biological fluids may not be available, 

·        Analytical method with particular drug combination may be not available,

·        As the available methods may require expensive reagents and solvents and also the procedure involved is cumbersome and process is complicated and also may not be reliable.

 

FDC is quite popular among, anti-tuberculosis, anti diabetic, antiretroviral, bronchial asthma, anti-cancer and anti-hypertensive preparations. In a meta-analysis of studies in patients with anti-retroviral, tuberculosis, hypertension and diabetes, FDC resulted in a 26% decrease in the risk of noncompliance compared with free-drug component regimen.

 

Literature  survey  of  Pyrazinamide, Isoniazid, Rifampicin and Ethambutol revealed no method for simultaneous estimation of all the four drugs.

 

Experimental^11,12:

Materials and Methods: Reagents required Acetonitrile : UPLC grade, Water : UPLC grade, Methanol : UPLC grade, Potassium dihydrogen ortho phosphate: AR grade, Triethylamine: AR grade.

 

Drugs used:

Pure drug samples of Pyrazinamide, Rifampicin, Isoniazid and Ethambutol were used and the marketed formulation containing Pyrazinamide 750 mg, Isoniazid 300 mg, Ethambutol 800 mg and Rifampicin 450 mg was used.

 

Preparation of solutions for mobile phase:

Solution-A is composed of Buffer pH 7.5 adjusted with Ortho phosphoric acid (10 mL of Triethylamine and 20 mg of Potassium Dihydrogen Ortho Phosphate into 1000 mL of HPLC water)

Solution –B Mixture of Methanol and Acetonitrile in a ratio of 85:15 v/v

 

Mobile phase Preparation:

Mobile phase used is a mixture of solution - A and solution - B in the ratio of 90:10 v/v

 

Optimized chromatographic conditions

Analytical column	X bridge C18 (50 mm x 1.7mm, 3 mm)
Mobile Phase	Mobile phase used is a mixture of solution-A and solution - B in the ratio of 90:10 v/v
Temperature	Ambient
Wavelength	290 nm
Flow rate	0.5 mL / min
Injection Volume	6 µL
Run Time	10 minutes
Rt (Pyrazinamide)	2.065 min
Rt (Isoniazid)	3.026 min
Rt (Ethambutol)	3.956 min
Rt (Rifampicin)	5.847 min

 

Diluent selection:

The diluent used in throughout the method was mobile phase in which the drugs are soluble.

 

Standard Solution Preparation:

Standard solution of Pyrazinamide, Ethambutol, Isoniazid and Rifampacin were prepared by dissolving 75 mg, 30 mg, 80 mg and 45 mg of each drug into 100 mL volumetric flask. Then dilution was made by adding 70 mL of the Diluent solution to 100 mL standard flask and making up the volume with the diluent. The final concentration of each drug was found to be 750, 300, 800, 450 µg/mL respectively.

 

Working solution:

Working solution of Pyrazinamide, Ethambutol, Isoniazide and Rifampacin was prepared by pipette 1.0 mL of standard solution to 10 mL of standard flask add diluent and made the volume with diluent. The final concentration of the individual was 75, 30, 80, 45 µg/mL respectively.

 

Sample preparation:

The developed procedure was extended to formulation of pyrazinamide, isoniazid, ethambutol and rifampicin. Average weight of twenty tablets were taken and make powder, powder containing 750 mg metformin was transferred to 100 mL of volumetric flask and add diluent to sonicate and filter through whatmann filter paper 0.22µ make up to mark with diluent. Procedure as mentioned for the pure drug was followed for the formulation. The concentrations of all pyrazinamide, isoniazid, ethambutol and rifampicin were determined by measuring peak area at 290 nm. (Table No 1.1 and Figure no 4)

 

Method validation¹³:

Accuracy:

Accuracy was performed at three different levels by adding a known concentration of standard to the sample at three different levels. Each level was repeated three times (n=3). (Table No 1.2-1.5)

 

Precision:

Standard solution of 75 µg/mL, 30 µg/mL, 80 µg/mL, and 45 µg/mL of pyrazinamide isoniazid, ethambutol and rifampicin was injected for five times and measured the area for all six replicate solutions in UPLC. %RSD was calculated for the area of six replicate injections. (Table No 1.6-1.9)

 

Linearity:

Linearity was observed in the range of 18.75-93.75 µg/mL, 7.5-37.5 µg/mL, 20-100 µg/mL, 11.25-56.25 µg/mL for pyrazinamide, isoniazid, ethambutol and rifampicin. Correlation coefficient was found to be 0.999 and slope was calculated. (Table No 1.10 and Figure no 6-9)

 

Detection limit (LOD) and Quantification limit (LOQ):

LOD and LOQ were determined as signal to noise ratio. LOD of pyrazinamide, isoniazid, ethambutol and rifampicin was calculated by using the concentrations of 0.12 µg/mL, 0.015 µg/mL, 0.12 µg/mL, and 0.015 µg/mL respectively. LOQ was calculated by using the concentrations of 0.42 µg/mL of pyrazinamide, 0.05 µg/mL of isoniazid, 0.42 µg/mL ethambutol and 0.05 µg/mL of rifampicin. (Table No 1.11)

 

Robustness:

Small changes were done in the optimized conditions like change in the flow rate and wavelength and it was observed that there is no change in the response of the drugs regarding peak area of individual drugs. (Table No 1.12-1.16).

 

Degradation studies:

Sample solution was prepared by transferring the powder equivalent to the weight of 750, 300, 800, and 450 mg of pyrazinamide, isoniazid, ethambutol and rifampicin into 100 ml flask. Dilutions were made using the mobile phase to get the concentration equivalent to 56.25, 22.5, 60, 33.75 μg/mL for pyrazinamide, isoniazide, ethambutol and rifampicin respectively. (Table 1.16)

 

Acid Degradation:

Sample solution of 3 mL was mixed with 3 mL of 1N hydrochloric acid and heated at 60°C for 10 hrs in a water bath. After 10 hrs the solution was neutralized with 3 mL of 1N sodium hydroxide, volume was made to 10 mL with diluent and analyzed in UPLC.

 

Base Degradation:

Sample solution and 1N sodium hydroxide each of 3 mL was mixed and heated for 10 hrs on a water bath. After 10 hrs solution was neutralized by adding 3mL of 1N hydrochloric acid make to 10 mL with diluent and analyzed in UPLC.

 

Oxidative Degradation:

The oxidative degradation was performed by 3 mL of sample solution was mixed with 3mL of 30% v/v aqueous hydrogen peroxide solution and kept for 10 hrs. After 10 hrs made the volume upto the mark with diluent and analyzed in UPLC. 

 

Thermal degradation:

Solid drug samples were taken and exposed to 80°C for 15 min to 60 min at 220°C for 2-5 min.

 

RESULTS:

 

Fig 5: Standard chromatograph

 

Table 1.1: Assay of formulation pyrizanamide, isoniazide, ethambutol and rifampicin

Sl. No.	Drug	Labeled Amount (mg )	% Recovery	%RSD (n=5)
1	Pyrizanamide	275 mg	99.18	0.4
2	Isoniazide	100 mg	99.95	0.4
3	Ethambutol	150 mg	99.94	0.3
4	Rifampacin	500 mg	99.99	0.33

 

Accuracy:

Table 1.2: Accuracy of pyrizanamide

%Concentration (at specification Level)	Area	Amount Added (µg)	Amount Found (µg)	% Recovery	Mean Recovery
50%	5013801	60	60.97	101.62%	101.20%
100%	6175537	75	75.10	101.14%
150%	7537449	90	91.66	101.85%

Table 1.3: Accuracy of isoniazide

%Concentration (at specification Level)	Area	Amount Added (µg)	Amount Found (µg)	% Recovery	Mean Recovery
50%	4694180	24	24.06	100.25%	99.99%
100%	5851253	30	29.99	99.97%
150%	7005518	36	35.90	99.74%

 

Table 1.4: Accuracy of ethambutol

%Concentration (at specification Level)	Area	Amount Added (µg)	Amount Found (µg)	% Recovery	Mean Recovery
50%	10568258	64	63.51	99.25%	99.33%
100%	13216091	80	79.43	99.29%
150%	15882411	96	95.46	99.44%

 

Table 1.5: Accuracy of rifampacin

%Concentration (at specification Level)	Area	Amount Added (µg)	Amount Found (µg)	% Recovery	Mean Recovery
50%	4784837	36	35.88	99.68%	99.75%
100%	5972865	45	44.79	99.54%
150%	7201414	54	54.00	100.01%

 

Method precision:

Table 1.6 Precision results of Pyrazinamide

Injection	Area
Average	6155129
Standard Deviation	45544
%RSD	0.7

 

Table 1.7Precision results of Isoniazid

Injection	Area
Average	680653
Standard Deviation	5623342
%RSD	0.8

 

Table 1.8 Precision results of Ethambutol

Injection	Area
Average	13171988
Standard Deviation	1028810
%RSD	0.8

 

Table1.9 Precision results of Rifampicin

Injection	Area
Average	5963277
Standard Deviation	43524
%RSD	0.7

 

 

 

Table 1.10 Linearity results of pyrizanamide, isoniazide, ethambutol and rifampacin

S.No	PYZ  (µg/ml)	Area (mV.s)	INZ (µg/ml)	Area (mV.s)	EMB (µg/ml)	Area (mV.s)	RFP (µg/ml)	Area (mV.s)
1	18.75	3138072	7.5	2896958	20	6707719	11.25	3065340
2	37.5	4635650	15	4590749	40	10592507	22.5	4801909
3	56.25	6203864	22.5	5668945	60	13186987	33.75	5996742
4	75.0	7659663	30.0	6822783	80	15938931	45.0	7238830
5	93.75	9293330	37.5	8495871	100	18819497	56.25	8995607

 

 

 

Fig 6 Linearity of Pyrizanamide

 

Fig 7 Linearity of Isoniazide

 

Fig 8 Linearity of Ethambutol

 

 

Fig 9 Linearity of Rifampacin

 

 

 

Table 1.11 LOD and LOQ pyrizanamide, isoniazide, ethambutol and rifampicin

	Pyrizanamide	Isoniazide	Ethambutol	Rifampacin
LOD	2.9	3.0	2.9	3.0
LOQ	10	10.03	10	10.03

 

 

Table 1.12 Robustness effects of wavelength pyrizanamide, isoniazide, ethambutol and rifampacin

S. No	Change in the wavelength (nm)	USP Plate Count
		Pyrizanamide	Isoniazide	Ethambutol	Rifampacin
1	260	1361	2080	2632	5309
2	290	2063	2937	5409	17961
3	320	1361	2080	2632	5309

 

 

 

Table 1.13 Robustness effects of wavelength pyrizanamide, isoniazide, ethambutol and rifampacin

S. No	Change in the wavelength (nm)	USP Tailing
		Pyrizanamide	Isoniazide	Ethambutol	Rifampacin
1	260	1.50	1.60	1.63	1.74
2	290	1.19	1.14	1.17	1.14
3	320	1.50	1.60	1.63	1.74

 

 

 

Table 1.14 Robustness effects of flow rate pyrizanamide, isoniazide, ethambutol and rifampacin

S.No	Change in the flow rate	USP Plate Count
		Pyrizanamide	Isoniazide	Ethambutol	Rifampacin
1	0.45	1332	2146	3004	8170
2	0.50	2063	2937	5409	17961
3	0.55	1332	2146	3004	8170

 

 

 

Table 1.15 Robustness effects of flow rate pyrizanamide, isoniazide, ethambutol and rifampacin

S.No	Change in the flow rate	USP Tailing
		Pyrizanamide	Isoniazide	Ethambutol	Rifampacin
1	0.45	1.43	1.46	1.46	1.39
2	0.50	1.19	1.14	1.17	1.14
3	0.55	1.43	1.46	1.46	1.39

 

 

 

Table 1.16 Degradation data pyrizanamide, isoniazide, ethambutol and rifampacin

Drug	Degradation	Area	% Recovered	% Degraded
Pyrizanamide	Acid	5923111	96.23	3.77
	Base	5629000	91.46	8.54
	Peroxide	5593215	90.87	9.13
	Thermal	5694158	92.51	7.49
Isoniazide	Acid	5208183	89.16	10.84
	Base	5476831	93.76	6.24
	Peroxide	5427091	92.91	7.09
	Thermal	5329637	91.24	8.76
Ethambutol	Acid	12964165	97.60	2.40
	Base	12851430	96.75	3.25
	Peroxide	12038705	90.63	9.37
	Thermal	12624626	95.04	4.96
Rifampacin	Acid	5610702	93.69	6.31
	Base	5734912	95.77	4.23
	Peroxide	5293003	88.39	11.61
	Thermal	5605456	93.61	6.39

 

 

 

 

DISCUSSION:

Stability indicating UPLC method was used for the estimation of this combination has not been reported yet. Validation parameters such as precision, accuracy, linearity, LOD, LOQ and ruggedness were within the limits. By using simple mobile phase the retention time obtained is 2.06, 3.02, 3.65 and 5.84 for pyrazinamide, isoniazid, ethambutol and rifampicin respectively.  Degradation studies were performed for the drugs by exposing to extreme conditions but very less degradation was observed and no method was reported for the estimation of all the four drugs simultaneously.

 

CONCLUSION:

Simple accurate precise repeatable and reproducible stability indicating analytical method have been developed for the estimation of pyrazinamide, isoniazide, ethambutol and rifampicin. The proposed UPLC method for estimation of Pyrazinamide, isoniazide, ethambutol and rifampacin was isocratic with simple mobile phase. The method gives good resolution between the compounds with a short analysis time.

 

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Accepted on 26.04.2018                             © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(7): 2869-2875.