INTRODUCTION:

Around 10 million human beings become debilitated with tuberculosis, and more over 1·2 million individuals die from the illness each year. World Health Organization has proposed a few aggressive focuses in their End TB Strategy, and global leaders committed to reaching these targets within the next decade. As these time limit approaches, the rhetoric has increased but has produced apparently little progress, creating an air of desperation among all cadres engaged in the battle towards tuberculosis.

Pretomanid used to be first recognized in 2000, in a series of 100 nitroimidazopyran derivatives manufactured and studied for antitubercular activity, by Patho Genesis (now a subsidiary of Novartis).

Pretomanid (Fig. 1) is a nitroimidazole, a class of novel anti-bacterial agents as per the U.S. Food and Drug Administration to treatment of pulmonary extensively drug resistant (XDR), or treatment-intolerant or nonresponsive multidrug-resistant (MDR) tuberculosis (TB), in combination with bedaquiline and linezolid as part of the BPal regimen^1-3. Pretomanid is a nonproprietary name for PA-824. The “preto” part of the compound’s name honors Pretoria, South Africa, the home of a TB Alliance clinical development office where much of the drug’s development took place, while the “-manid” stem designates compounds with similar chemical structures^4,5. The most common side effects include nerve damage, acne, vomiting, headache, low blood sugar, diarrhea, and liver inflammation^6,7.

Figure 1: Chemical structure of the Pretomanid drug.

Various reliable studies have published methods for the quantification of Pretomanid, including validated analytical methods from rat plasma^8–10, a method for determination from pharmaceutical formulations¹¹, A validated liquid chromatography tandem mass spectrometry assay method from human plasma¹², and a brief summary of a methodused for the determination of Pretomanid from human plasma¹³. However, up till now, no stability indicating RP-HPLC Assay method has been reported for quantification of Pretomanid drug in pharmaceutical dosage forms, hence a validated stability indicating RP-HPLC method for its quantification was required. The objective of the current analytical study was to develop a stability indicating RP-HPLC method to quantify the Pretomanid drug in formulation.

MATERIALS AND METHODS:

Chemicals and Reagents:

Pharmaceutical grade of Pretomanid (PMD) was obtained as a gift sample. The tested Pretomanid tablet formulation containing 200mg dosage was purchased from local medical store. HPLC grade solvents like Acetonitrile, Methanol and water were purchased from Merck chemicals, Mumbai, India. All other reagents and buffers are analytical grade chemicals purchased from Merck chemicals, Mumbai, India and prepared with double distilled water.

Instruments:

An Agilent 1100 HPLC system (Santa Clara, USA) used for method development and validation which was equipped with a pump (model G1311), an auto sampler (ALS) with capacity of 0.1 to 1500µl (model G 1329A), COLCOM G1316A column temperature control with thermostat and G-1314 A model variable wavelength UV detector. Xtimate C18 column (250mm×4.6mm; 5µm) column (Paisley, UK) used for separation. Chemstation Software (Version Rev B.04.03 (16)) was used for data processing, analysis and reporting.

Preparation of Standard PMD Solution:

A stock standard solution of PMD was prepared by dissolving an accurately weighed 10mg of PMD in 100mL methanol in to 100mL volumetric flask and volume made up to mark with methanol (100µg/mL). Different working standard solutions of PMD (10-200µg/mL) were prepared by serial dilutions of the stock solution by the same solvent and were stored at 4°C. All the standard stock and working solutions were daily prepared.

Preparation of Sample Solution:

Marketed formulation of PMD (Pretomanid-200mg) tablet was purchased from local market and were weighed, triturated in porcelain mortar, and mixed, and the average weight of tablet was calculated. About 25 mg of PMD tablet powder was accurately weighed amount and transferred completely to a 100mL volumetric flask, 50mL of the methanol was added later sonicated for 30 minutes. The volume was completed to mark by the methanol solvent to obtain a solution of PMD with a concentration of 20µg/mLi.e prepared solution was filtered through 0.45µm membrane filters.

Method development:

The chromatographic separation was performed using isocratic elution at ambient temperature (25°C). In order to develop the analytical method, methanol was chosen as major solvent as the standard PMD are found highly dissolved in methanol. The composition of methanol and acetonitrile was optimized in order to achieve symmetrical shape peak complying the system suitable conditions. Different compositions of the mobile phase, with different solvents methanol, acetonitrile, and water in varying combination were tried as mobile phase and low response was observed. The pH modifiers like Ammonium phosphate, were added enhance the response of separation and sensitivity of method. The separation was carried on different configurations of stationary phases, and eluents were recorded using a UV detector. The conditions that produce valid system suitability were studied for validation.

Method Validation:

This method validation carry out as per ICH guidelines, and the validation in terms of the following parameters; Specificity, Linearity, sensitivity (LOQ and LOD), precision, accuracy, and stability of analytical solutions.

Specificity:

Specificityof the method was evaluated by determining the Interference from solvents and endogenous matrix components was investigated by injecting of blank samples as well as PMDsample by the proposed method. It is evaluated by measuring system suitability parameters such as plate count and tailing factors.

Linearity and Range:

To evaluate the linearity and range of the method, five independent levels of calibration curve in range of 10-50 µg/mL for PMD was analyzed. The linearity range of PMD is expressed in term of correlation coefficient of linear regression analysis. The calibration curves were obtained by plotting graph of peak area vs. concentration and linear regression analysis was used to evaluate the linearity of the calibration curve by using the least square linear regression method.

Sensitivity:

The sensitivity of the proposed method was estimated in terms of Limit of detection (LOD)/limit of quantification (LOQ). Limit of detection and limit of quantification were defined based on signal to noise ratio of 3:1 and 10:1, respectively. Limit of quantification was taken as lowest concentration of PMD that could be quantitatively determined with acceptable accuracy and precision results.

Precision:

The method precision (repeatability) and system precision of the proposed methods were determined by several measurements of standard solution and sample solution, respectively. The intraday precision was assessed by analyzing the standard PMD solution at 20 µg/mL concentrations within the same day. The inter-day precision was determined by analyzing of six replicates of PMD solution at 20µg/mL on the three different days. The RSD of the obtained results was calculated and precision of the method was expressed as the relative standard deviation (%RSD).

Ruggedness and Accuracy:

The ruggedness of the method was evaluated by injecting the PMD standard solution at 20µg/mL by different chemists in different days. The accuracy of the proposed method was evaluated by recovery studies at the three concentration levels (50%, 100% and 150%), i.e., 15, 20 and 25µg/mL.To a fixed amount of pre analyzed sample (10µg/mL), increasing aliquots of PMD working standard solution (5, 10 and 15µg/mL) were added respectively and diluted to mark with methanol and injected. The percentage recovery of added PMD and RSD were calculated for each of the replicate samples.

Robustness:

Robustness of the proposed method was verified by applying minor and deliberate changes in the experimental parameters like flow rate (±0.2mL/min), mobile phase composition (±5%) and UV detector wavelength (±5). Change was made to evaluate its effect on the method. For each case, the percentage of recovery and %RSD of the obtained results calculated to determine the effect of change on the method. The stability of the sample and standard solution has been evaluated by analyzed the solution in different time intervals and % of recovery was calculated.

Forced Degradation:

Forced degradation studies^14-35 for Pretomanid can be used to determine the degradation pathways and degradation products that could form during storage, and facilitate during formulation, development, manufacturing and packaging. To evaluate the stability of the Pretomanid drug, various stress conditions are applied to the drug like acid, base, peroxide, sunlight, UV light etc. Degradation test is performed by incubating the standard for 48hours in different conditions.

Light (Normal and UV light):

To demonstrate the degradation of the sample, kept in open petri-dish at Lab light and UV light. The sample was further analyzed after 24hours exposed sample at Lab light and UV light.

Thermal:

The Pretomanid sample kept in a Petri dish and keep in oven at 80°C up to 24hours. After expose of the samples and prepare sample solution for injection.

Acid:

100mg of Pretomanid sample was incubated for 24 hours in 20mL of 0.1N hydrochloric acid solution. Then 5mL of acid hydrolyzed sample solution was neutralized with 5mL of 0.1N sodium hydroxide solution and make up with diluents in 25mL of volumetric flask. The above solutions inject once after system suitability solution and evaluate the degradants in chromatogram and compare with standard values.

Base:

100mg of Pretomanid sample added in 20mL of 0.1N Sodium Hydroxide solution. After 48 hours to take 5mL of Base hydrolyzed sample solution in 25mL volumetric flask and neutralize with 5mL of 0.1 N Hydrochloric acid solutions and make up with diluent. The above solutions inject once after system suitability solution and evaluate the degradants in chromatogram and compare with without base hydrolysis values.

Hydrogen Peroxide:

For peroxide degradation 100mg Pretomanid sample is incubated for in 20mL of 3% Hydrogen Peroxide for 24hours. 5mL of oxidized sample solution taken into 25mL volumetric flask and make up with diluent. The above solutions inject once after system suitability solution and evaluate the degradants in chromatogram and compare with without oxidized (Initial) values.

RESULT AND DISCUSSION:

After several trails of optimization in method conditions, chromatogram of PMD drug complying the all-systemsuitability parameters was achieved. Final optimized chromatography conditions for estimation of PMD includes isocratic elution at ambient temperature (25°C) by employing Xtimate C18 column (250mm×4.6mm; 5.0µm id) with UV detection at 262nm. The mobile phase was composed of the mixture of Methanol: Acetonitrile: Ammonium phosphate buffer in the ratio of 55:40:05 (v/v/v) with pH maintained at 5.7. The mobile phase flow rate was set at 1.0mL/min, injection volume was 20µl, and the run time was 10minutes. The chromatography conditions of the proposed method are presented in Table1.

Table1: Final optimized chromatographic conditions

S. No.	Condition	Results
1	Mobile phase	Methanol: Acetonitrile: Ammonium phosphate buffer in the ratio of 55:40:05 (v/v/v).
2	Pump mode	Isocratic
3	pH	5.7
4	Diluents	Mobile phase
5	Column	Xtimate C18 column (250mm×4.6mm; 5µm)
6	Column Temperature	Ambient
7	Wavelength	262 nm.
8	Injection Volume	20μl.
9	Flow rate	1.0mL/min.
10	Run time	10min.

Method validation:

The specificity of the method was evaluated by analyzing the blank sample. The standard chromatogram of the PMD is sharp and symmetrical in shape and meets all the system suitable conditions like tailing factor (below 1), theoretical plates (above 5000). The blank chromatogram shows no interference of the solvent or impurities. The chromatograms of blank and standards of the PMD are presented in Figure 2 and Figure3.

Figure 2:The blank chromatogram of Pretomanid.

Figure 3:The standard chromatogram of Pretomanid.

The linearity study was found to be in the range of 10-40µg/mL. The linearity results are presented in Table 2. The mean peak area obtained from the HPLC was plotted against corresponding concentration, to obtain the calibration graph for concentrations of PMD standard solution (10-50µg/mL) is shown in Fig.4. Correlation co-efficient for calibration curve of PMD was achieved 0.999 which meet the analytical method validation acceptance criteria. The linear equation obtained from the calibration curve was y = 7879.2x –10007.

Table 2: Results of linearity study.

Level	Concentration in µg/mL	Peak Area
L 1	5	30653.1
L 2	10	68621.3
L 3	15	107356.4
L 4	20	146534.1
L 5	25	186352.5
L 6	30	228647.8
L 7	35	263491.7
L 8	40	306541.5

Slope: 7879

Intercept: -10007

Correlation Coefficient: 0.999

Figure 4: Calibration graph of Pretomanid Standard.

The limit of detection (LOD) is the lowest amount of analyte in a sample that can be detected, but not necessarily quantitated. The LOD of the proposed method was 0.3µg/mL. The limit of quantification (LOQ) is the lowest amount of analyte in a sample that can be quantitatively determined with suitable precision. The lowest concentration on calibration curve that can be reproducibly quantified with acceptable accuracy and precision, 5µg/mL, was experimentally considered as LOQ of the assay method. Limit of detection (LOD) and limit of quantification (LOQ) studies chromatograms were depicted in fig. 5 and fig. 6.

Figure 5. Limit of detection Chromatogram of Pretomanid

Figure 6. Limit of quantification Chromatogram of Pretomanid.

For intraday precision, replicate analysis (n = 6) 20 μg/mL of PMD standard solution in same day and for inter day precision, 20μg/mL of PMD standard solution was injected for six times in in three different days. The relative standard deviation (%RSD) of area values calculated and the results of precision studies are presented in Table3. The %RSD of precision studies was found to be 0.83 for intraday precision and 0.60 for inter day precision respectively.

Table3: Results of precision study and ruggedness study.

S. No.	Peak Area
S. No.	Intra-day Precision	Inter-day Precision	Ruggedness
1	145521.5	146825.7	143216.2
2	146753.4	145962.1	145965.1
3	143981.2	144931.2	144783.4
4	144326.3	145627.4	145637.2
5	146235.1	146916.5	146204.7
6	146743.9	144937.9	145863.5
% RSD	0.83	0.60	0.77

The ruggedness of the proposed LC method was evaluated by replicate analysis (n = 6) 20μg/mL of PMD standard solution by different analyst in different days. The relative standard deviation (%RSD) of area values calculated and the results of ruggedness are presented in Table 3. The %RSD of ruggedness study was found to be 0.77. % of RSD values of this developed method for precision and ruggedness studies were not more than 2.0% as recommended by ICH guideline.

The accuracy of an analytical procedure expresses the closeness of results obtained by that method to the true value. The accuracy of method was assessed by recovery studies. The recovery at the three concentration levels (50%, 100% and 150%), i.e., 15, 20 and 25µg/mL prepared and injected for three times. The percent recovery of all obtained results were calculated and summarized in above Table 4. The good recovery values have been achieved within the range from 98.67 to 99.96% which indicates the applicability of the method for routine drug analysis.The robustness of the analytical method robustness was tested by evaluating the influence of minor modifications in flow rate, mobile phase composition and UV detector wavelength on system suitability parameters of the proposed method. The percent of recovery was analyzed in order to determine the change in the area. Results of robustness study are summarized in Table5 and it was observed that the percent of recovery was within acceptable limits and the %RSD is within limit of not more than 2.0%. The tailing factors and number of theoretical plates were found within acceptable limits as well.

Table 4: Results of recovery study.

Level	Concentration in µg/mL			Peak Area observed	Amount of Recovered (µg/mL)	% Recovery
Level	Target	Spiked	Total	Peak Area observed	Amount of Recovered (µg/mL)	% Recovery
50 %	10	5	15	106229.2	14.84	98.95
	10	5	15	106562.0	14.89	99.26
	10	5	15	107313.5	14.99	99.96
100%	10	10	20	144585.2	19.73	98.67
	10	10	20	145361.8	19.84	99.20
	10	10	20	146397.6	19.98	99.91
150%	10	15	25	185131.3	24.84	99.34
	10	15	25	185582.3	24.90	99.59
	10	15	25	186024.3	24.96	99.82

Table 5.Results of robustness study

S. No.	Parameters Changes	Change	Peak Area	% Change
1	Optimized	-	146534.1	-
2	MP Change 1	Methanol: Acetonitrile: Ammonium phosphate buffer in the ratio of 50:45:5 (v/v/v)	146251.1	0.19
3	MP Change 2	Methanol: Acetonitrile: Ammonium phosphate buffer in the ratio of 60:35:5 (v/v/v)	144587.9	1.33
4	pH Change 1	5.6	145634.5	0.61
5	pH Change 2	5.8	144271.3	1.54
6	WL Change 1	267 nm	145789.2	0.51
7	WL Change 2	257 nm	145267.9	0.86

Forced Degradation studies:

Forced degradation study of Pretomanid was carried under different stress conditions. From the peroxide degradation study of Pretomanid, it was found that two additional peaks were observed. In this condition the % recovery was found to be 95.23% and only 4.77% drug was degraded in this conditions. The results of acidic hydrolysis showed 3 degradation peaks along with the drug peak. The peak area showed that 5.13% of degradation of the drug occurred when the drug was kept in 0.1M HCl up to 24 h. Pretomanid upon alkaline degradation in 0.1M NaOH at 80°C up to 24 h. underwent degradation showing 2 degradation peak in the chromatogram. The peak area of the drug showed that the percentage degradation is 4.35% in the above condition. Pretomanid upon UV light degradation up to 24h underwent degradation showing 3 degradation peaks in the chromatogram and no degradation peaks observed in light degradation study. For the thermal degradation of Pretomanid at 80°C up to 24 h underwent degradation showing no degradation peak in the chromatogram. The degradation results of Pretomanid were given in table 6. The forced degradation chromatograms was given in figure 7-12 for Acidic, Base, Light, Peroxide, Thermal and UV conditions respectively.

Table 6: Forced degradation results.

S. No.	Condition	No additional peaks observed	Peak Area	% Obtained	% degradation
1	Standard	......	146534.1	100	0.0
2	Acidic	3	139011.3	94.87	5.13
3	Base	2	140162.2	95.65	4.35
4	Light	0	145012.1	98.96	1.04
5	Peroxide	2	139538.3	95.23	4.77
6	Thermal	0	145009.7	98.96	1.04
7	UV	3	141241.9	96.39	3.61

Figure 7: Degradation chromatogram of Pretomanid under Acidic condition.

Figure 8: Degradation chromatogram of Pretomanid under Base condition.

Figure 9: Degradation chromatogram of Pretomanid under Light condition.

Figure 10: Degradation chromatogram of Pretomanid under Peroxide condition.

Figure 11: Degradation chromatogram of Pretomanid under Thermal condition.

Figure 12: Degradation chromatogram of Pretomanid under UV condition.

The solution stability was evaluated by injecting 20µg/mL of standard solution in different time intervals. The percent of recovery was found within the range of 98.0% to 102.0%, indicating a good stability of the standardsolutions for 24hr at both conditions. The market formulation of Pretomanid tablet (200mg) dosage was injected under proposed condition. About 20µg/mL sample concentration was injected and 19.8µg/mL was recovered with 98.90% assay indicates accuracy in estimation of PMD drug in formulation. The results of formulation analysis were presented in Table-7.

CONCLUSION:

On the basis of obtained results, it was concluded that the developed method of assay for Pretomanid drug was validated as per the ICH guidelines with the following objectives.

a) A simple, precise, accurate and effective analytical assay of Pretomanid drug had been developed by using RP-HPLC method. (The shorter time run enables rapid determination of Pretomanid drug having a symmetrical shape peak complying with the system suitable conditions).

b) The developed method has been validated as per ICH guidelines, and it meets all the accepted criteria given in ICH guidelines. (From the experimental result the data: For the sensitivity study LOD and LOQ were estimated as 0.30µg/mL and 5.0µg/mL, for Accuracy was established between 98-102%, in the precision % RSD was found to be < 2, for linearity study Correlation co-efficient was 0.999, for Robustness study % of recovery was < 2.0%.)

c) Here on the basis of the results of forced degradation study it was proved that no any kind of degradation peak was merged with that of the drug. (The results of stress testing reveal that the method is stability-indicating. The proposed method has the ability to separate the Pretomanid drug from their degradation products found in tablet dosage forms and can be applied to the analysis of samples obtained during accelerated stability experiments)

A simple, rapid, accurate, precise and stability-indicating HPLC analytical method has been developed and validated for the routine analysis of Pretomanid in API and tablet dosage forms.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interests regarding the publication of this article.

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33. M. T. Mohite, V. V. Dudhabale,K. V. Chandgude. Validation of Stability Indicating RP-HPLC, Method of Analysis for assay of Ivabradine HCl in SR Tablet. Asian J. Pharm. Ana. 2019; 9(3):133-137.doi: 10.5958/2231-5675.2019.00025.5

34. S. Muneer, Hindustan Abdul Ahad, Chandra SekharKothapalliBonnoth. A Novel Stability Indicating RP-HPLC Assay Method Development and Validation for the Quantification of Cyamemazine Tartrate in bulk and its Pharmaceutical Dosage Form. Asian J. Pharm. Ana. 2018; 8(3): 169-173. doi: 10.5958/2231-5675.2018.00031.5

35. B. Bhavya, P. Nagaraju, V. Mounika, G. Indira Priyadarshini. Stability Indicating RP-HPLC Method Development and Validation forSimultaneous Estimation of Albendazole and Ivermectin in Pharmaceutical Dosage form. Asian J. Pharm. Ana. 2017; 7(1): 6-14.doi: 10.5958/2231-5675.2017.00002.3

Received on 13.06.2022 Modified on 16.10.2022

Research J. Pharm. and Tech 2023; 16(5):2385-2392.

DOI: 10.52711/0974-360X.2023.00393

S. No.	Brand Name	Form	Dosage	Amount Prepared	Amount Found	% Assay
1	Pretomanid	Tablet	200mg	20µg/mL	19.78µg/mL	98.90