Development and Validation of High-Performance Thin Layer Chromatography Method for Estimation of Rifabutin in Bulk and Formulation

Sachin Bhusari*, Irfan Ansari, Pravin Wakte

University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra, India

*Corresponding Author E-mail: chemtech.cdmpk@gmail.com

ABSTRACT:

A new, simple, rapid, accurate and precise high-performance thin layer chromatography (HPTLC) method has been developed and validated according to the guidelines of the International Conference on Harmonization (ICH Q2(R1) for the estimation of rifabutin in bulk and formulation. Optimized HPTLC mobile phase composition for rifabutin estimation was Chloroform: Methanol: n-Hexane (5:1:4 v/v/v). Spectro densitometric analysis of Rifabutin was carried out at 260nm and asymmetrical, well-resolved, well-defined peak was obtained at mean retardation factor (Rf) 0.84. The calibration plots were found to be linear in the range of 50 to 300 ng/spot and showed a good linear relationship with the coefficient of regression, R2=0.979 with respect to the peak area. The limit of detection and quantification of developed HPTLC method were 0.28 and 0.84ng/spot, respectively. The recovery study was carried out by standard addition method and the percentage recovery was found to be in between 99.68 to 100.35. Proposed HPTLC method can be applied for the quantitative determination of rifabutin in bulk drug and formulation.

KEYWORDS: Rifabutin, High Performance Thin Layer Chromatography, Method validation.

INTRODUCTION:

Rifabutin is an approved broad-spectrum antibiotic used for the treatment of tuberculosis^[1-2]. It has a molecular formula C₄₆H₆₂N₄O₁₁ and its molecular weight is 847.019g/mol^[3-4]. Rifabutin (Fig1) is a drug of choice in case of patients who cannot tolerate Rifampicin^[5-6]. Rifabutin has fewer drug interactions which makes it preferred anti-TB drug for the patients with HIV infection^[7-8].

Fig. 1: Chemical structure of Rifabutin

Extensive literature survey revealed that there are very few analytical methods that describe the estimation of rifabutin in bulk and formulation. Most of the rifabutin estimation methods are based on either spectrophotometric or reverse phase chromatographic techniques. Spectrophotometric methods have lack of selectivity whereas reverse phase chromatographic techniques are quite expensive. HPTLC has an advantage over these drawbacks by offering selectivity as well as cost effectiveness. Existing reported HPTLC methods of rifabutin estimation is based on use of highly volatile solvents like dichloromethane and acetone. Moreover, percentage of highly volatile solvents in mobile phase is more than 50 and the limit of quantification is towards higher side. Considering the disadvantages of existing HPTLC methods, it was envisaged that development of sensitive, accurate and precise HPTLC method for the estimation of rifabutin will be worth as it can be used for routine analysis of rifabutin.

MATERIALS AND METHODS:

Materials:

Rifabutin was obtained as gift sample from LUPIN Pharma Ltd, Aurangabad. Analytical grade reagent chloroform, methanol and n-hexane purchase from Merck (Mumbai, India). All the chemicals of analytical grade were used for the proposed study.

HPTLC Instrumentation:

The Solution of sample was applied to silica gel 60 F254 Plates (10cm _x10cm, 0.2mm thickness, Merck KGaA Darmstadt Germany) Using a Desaga Sarstedt-Grupee AS 30, equipped with a 10μl micro syringe. The sample were streaked in the form of bands of width 15mm and a constant application rate of 10μg/ml was employed and space between two band was 15mm. The mobile phase consisted of Chloroform: Methanol: n-Hexane (5:1:4 v/v/v). Development of the plates were carried out in a twin-trough glass chamber (12.5cm × 12.5cm × 5cm) saturated with mobile phase for up to 30 min as room temperature (25 ± 2◦C) and a relative humidity of 55 ± 5%. The migration distance was 15 mm. Densitometer CD 60 with Desaga software was use for densitometric scanning for the developed plates in the absorbance mode at 260nm. The slit dimension was kept at 0.40mm and 10mm/s scanning speed was employed. The source of radiation utilized was deuterium lamp emitting continuous UV spectrum in the range of 200–400nm.

Sample preparation and calibration:

A stock solution of 1mg/ml was prepared by dissolving 1ml methanol. Aliquots of the stock solutions were further diluted in methanol to achieve 100μg/ml and scanned in the wavelength range 800-200nm. The maximum wavelength (λmax) of Rifabutin was obtained at 260nm. stock solution was spotted in triplicate on TLC plate to obtain concentrations of 50, 100, 150, 200, 250, 300ng/spot of Rifabutin respectively. A calibration curve was obtained by plotting peak area against the corresponding concentration and linear least-square regression analysis was performed.

Analytical method development:

The method of analysis was validated as per the recommendations of ICH Q2 (R1)^[9] and USP^[10]for the parameters like accuracy, linearity, precision, detection limit, quantitation limit, recovery, and robustness.

Accuracy:

The rifabutin capsule formulation was first analyzed by the proposed method. The analyzed samples were spiked with 80%, 100%, and 120% of the standard drug and the mixture was reanalyzed. The experiment for each recovery sample was carried out six times to check the recovery of the drug at different levels in the formulations.

% RC = (SPS-S/SP) × 100

Where,

% RC = Percent recovery

SPS = Amount found in the spiked sample

SP = Amount added to the sample

S = Amount found in the sample

Precision:

Precision study was carried out using intra and inter-day method. In the intraday study, sample application and measurement of peak areas were carried out by analyzing three replicates of 50, 150 and 300 ng/spot on the same day whereas, for the inter-day study these three replicates were analyzed on the different days.

Limit of detection and quantification:

In order to determine detection and quantification limits (LOD and LOQ), rifabutin drug concentration in the lower part of calibration curve was used. Rifabutin solutions of 50, 100, 150, 200, 250 and 300μg/ml were prepared and applied in triplicate (10μl each). The amount of drug concentration versus average response (peak area) was plotted and the equation for this curve was determined. The standard deviations (SD) of responses were calculated. The average of standard deviations was calculated (ASD). Detection of limit was calculated by (3.3×ASD)/b and quantification limit was calculated by (10×ASD)/b, where b corresponds to the slope obtained in the linearity study of method.

Robustness:

Robustness of the developed method was determined by introducing small changes in the mobile phase composition, Mobile phases consisting of different composition chloroform: methanol:

n-hexane (4.8: 1.2: 4 v/v/v) and (5.2: 0.8: 4 v/v/v), the effect on the result was examined. Robustness of the method was evaluated 150ng/spot concentration.

Estimation of Rifabutin content in marketed formulation:

Developed and prevalidated high performance thin layer chromatography method was successfully used for estimation of Rifabutin content in marketed formulation. For the study, Ributin capsule were purchased from local market of Aurangabad and contents of capsule were collected and suitable dilution were made using pre-optimized co-solvent system. Prepared samples were analyzed using pre-validated HPTLC method and results were reported in terms of average percent assay.

RESULTS AND DISCUSSION:

Optimization of mobile phase:

The TLC procedure was optimized with a view to develop a method to quantify rifabutin API. Chloroform and methanol were selected as one of the components of mobile phase with acceptable resolution. However, the Rf value was too high, so the solvent strength was decreased by adding non-polar solvent, n-hexane was added to in chloroform, methanol and the chromatograms were developed. The mobile phase comprising of chloroform: methanol: n-hexane (7: 1: 2 v/v) showed good resolution with Rf = 0.71 for rifabutin but fronting was observed and the spot of rifabutin was slightly diffused. Addition of more quantity of non-polar solvent n-hexane improved the characteristics. The final mobile phase selected was a mixture of chloroform: methanol: n-hexane (5: 1: 4 v/v), which gave a well-defined symmetrical peak of rifabutin at Rf = 0.84 ± 0.02 (Fig 2). which was visible under short wavelength (260 nm) ultraviolet light (Fig 3).

Fig. 2: Densitometric Chromatogram of Rifabutin

Fig. 3: UV-visible spectra of Rifabutin

Calibration Curve and Linearity:

A good liner relationship over the concentration range 50 to 300ng/spot for Rifabutin was observed. The correlation coefficient was found to be 0.978 for rifabutin, Fig 3. The regression line equation is y = 11.009x + 80.995. The 3D chromatogram of calibration curve of rifabutin is shown in Table 1.

Table1: Results of calibration curve at 260 nm

Standard	Conc (μg/ml)	Peak Area±
CAL STD-1	50	549.12±0.0021
CAL STD-2	100	1182.45 ±0.0012
CAL STD-3	150	1965.20 ±0.0035
CAL STD-4	200	2240.76±0.0042
CAL STD-5	250	2628.93±0.0018
CAL STD-6	300	3479.43±0.0058

Fig. 3: Calibration curve for Rifabutin

METHOD VALIDATION:

Accuracy:

Accuracy by determined by standard addition method. The proposed method was applied for estimation of Rifabutin pharmaceutical dosage form. The recovery experiment was carried out in triplicate by spiking previously analyzed sample i.e. 150 ng/spot of Rifabutin with different concentration of standard drugs at 80%, 100% and 120%. At 80 % standard addition, mean recovery of Rifabutin was found to be 100.16% whereas at 100 and 120 % standard addition, it was found to be 100.35 and 99.68% respectively. % RSD was found to be less than 2 for the Rifabutin recovery studies as shown in Table 2.

Precision:

The repeatability of sample application and peak areas measured were expressed in terms of %RSD and the results revealed good system repeatability, intra- and inter-day precision in Table 3 and Table 4 respectively. The measurement of peak area at three different concentration levels (50, 150, and 300 ng/spot) The % RSD values if intra-day precision study were found to be in between 0.28 and 1.89 whereas those of inter-day precision study were in between 0.17 and 1.77. showed low values of %RSD for inter- and intra-day variations, suggesting that the method had excellent precision. This indicated that the system performance was very good and suitable for rifabutin analysis.

Table No 2: Accuracy data of HPTLC method for Rifabutin

Sr No.	Concentration (%)	Original level (µg/mL)	Amount added (µg/mL)	% Recovery	Mean % Recovery	% RSD
1	80	50	40	100.02	100.16	0.12
2	80	50	40	100.21
3	80	50	40	100.25
4	100	150	150	100.01	100.35	0.65
5	100	150	150	100.07
6	100	150	150	99.93
7	120	300	360	99.99	99.68	0.51
8	120	300	360	100.00
9	120	300	360	99.97

Table No 3: Intra-day precision data of HPTLC method for Rifabutin

S No.	Conc. (µg/mL)	Morning			Afternoon			Evening
S No.	Conc. (µg/mL)	Mean	% Assay	%RSD	Mean	% Assay	%RSD	Mean	% Assay	%RSD
1	50	557.02	99.46	1.03	554.46	99.01	1.24	553.30	98.80	1.04
2	150	2068.57	99.93	1.25	2047.03	98.89	0.28	2058.12	99.42	1.52
3	300	3520.73	101.60	1.80	3502.93	101.97	1.54	3499.31	101.87	1.89

Table No 4: Inter-day precision data of HPTLC method for Rifabutin

S No	Conc. (µg/mL)	Day 1			Day 2			Day 3
S No	Conc. (µg/mL)	Mean	% Assay	%RSD	Mean	% Assay	% RSD	Mean	% Assay	% RSD
1	50	554.92	99.09	0.45	563.12	98.79	1.77	559.09	98.08	1.60
2	150	2057.90	99.41	0.17	2023.20	99.17	1.52	2016.77	98.86	1.45
3	300	3501.15	101.48	0.86	3412.74	99.78	0.59	3395.19	99.27	0.24

Limit of detection and quantification:

LOD and LOQ were calculated by the method described previously. The calibration curve in this study was plotted between amount of analyte versus average response (peak area) and the regression equation was obtained (Y = 11.009x+ 80.995) with a regressioncoefficient of 0.979.

Therefore, LOD and LOQ were found 0.28 and 0.84 µg/mL respectively, which indicated adequate sensitivity of the method as introduce in Table 5.

Table No 5: LOD and LOQ data for HPTLC method for Rifabutin

1	LOD	0.28µg/mL
2	LOQ	0.84µg/mL

Table 6: Robustness data of HPTLC method for Rifabutin

Sr. No.

Concentration

(µg/mL)

Chloroform:

Methanol:

n-hexane

Absorbance

% RSD

150

(4.8: 1.2: 4 v/v)

2260.02

1.10

150

(5.2: 0.8: 4 v/v)

2131.03

1.04

Robustness:

The results of robustness was carried out at two different mobile phases, chloroform : methanol : n-hexane (4.8 : 1.2 :4 v/v/v) and chloroform : methanol : n-hexane (5.2 : 0.8 :4 v/v/v).The concentration level of 150ng/spot of rifabutin in triplicate with % RSD value of 1.10 and 1.04, the low values of % RSD obtained after introducing small changes in mobile phase composition indicated robustness of the method, shown in Table 6.

Estimation of Rifabutin:

The developed high-performance thin layer chromatography (HPTLC) method was successfully applied for the estimation of Rifabutin content in Ributin150mg USP. Average percent assay of Rifabutin capsule was found to be 100.31 %.

CONCLUSION:

The proposed HPTLC method was developed for estimation of rifabutin pharmaceutical dosage from. The % RSD of precision was found to be less than 2% and percentage recovery was found to be in range of 98-102% proves that the developed method is precise, specific, and accurate. Statistical analysis showed that the method is repeatable and selective for the analysis of rifabutin with no interference from excipients, this method can be used to determine the purity of drug.

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Received on 15.11.2019 Modified on 31.12.2019

Asian J. Pharm. Ana. 2020; 10(1):32-36.

DOI: 10.5958/2231-5675.2020.00007.1