Development and Validation of Zero and First-Order Derivative Area under Curve Spectrophotometric Methods for the Determination of Pirfenidone in Bulk Material and Pharmaceutical Formulation
Mohammad Mujeeb G. Khan*, Pawara Pritam I., Prashant J. Chaudhari, Atul A. Shirkhedkar
Department of Pharmaceutical Chemistry, R. C. Patel Institute of Pharmaceutical Education and Research, Karwand Naka, Shirpur, 425405, Dist Dhule, Maharashtra, India
*Corresponding Author E-mail: mujeebgulzar@gmail.com
ABSTRACT:
The prospective methods explain simple, precise specific and accurate UV spectrophotometry methods for determination of Pirfenidone in bulk and pharmaceutical dosage form. Pirfenidone is a new broad-spectrum anti-fibrotic and anti-inflammatory agent. Four simple methods were established for determination of Pirfenidone by using double beam UV spectrophotometer (UV -2450, Shimadzu, Japan). Pirfenidone shows maximum absorbance (λ max) at 317 nm in methanol as solvent. PFD follows Beer-Lambert’s law over the concentration range of 15-40 μg/mL. The % recovery was found to be in the range of 98-102 %. Precision value less than 2 in terms of % RSD indicates precise nature of developed methods. Validation of the proposed methods was carried out for its accuracy, precision, specificity and ruggedness according to ICH guidelines. The depicted methods can be routinely be used for determination of PFD in bulk and pharmaceutical formulation.
UV spectrophotometry Derivative by AUC technique is still the choice since it is simple, sensitive, economical, rapid and more easily manageable analytical procedure.
The objective of present work is to develop and validate an UV spectrophotomertic by AUC technique method for the determination of Pirfenidone in bulk and solid dosage form. The present work emphasizes an economical, accurate, precise and reproducible UV spectrophotomertic method for determination of Pirfenidone in bulk material and pharmaceutical dosage form. Further, the developed method was validated according to the ICH guidelines19-29.
Fig.1.Chemical structure of Pirfenidone
MATERIALS AND METHOD:
Chemicals and Reagents:
Pharmaceutical grade working standards of PFD were obtained as gift sample from Cipla Pharmaceutical Ltd. (Mumbai). The pharmaceutical dosage form used in this study was Pirfenex Tab from Cipla Pharmaceutical Ltd., Mumbai, India), labeled to contain 200 mg of Pirfenex procured from the local market. Methanol purchased from Merck Ltd, Worli, and Mumbai, India, and double distilled water was used for analysis.
Instrumentation:
A double beam UV-VIS spectrophotometer (UV-2450, Shimadzu, Japan) connected to computer loaded with spectra manager software UV Probe 2.21 with 1 cm quartz cells was used. An electronic balance (Model Shimadzu AUX 120) was used for weighing purpose.
Selection of Solvents:
Methanol of analytical reagent grade was selected as common solvent for developing spectral characteristics of drug. The choice of the solvent was made after evaluating the solubility in different solvents.
Preparation of Stock Solution and Selection of Wavelength:
The stock standard solution of PFD was prepared by weighing accurately, 10 mg of PFD transferred in to 100ml volumetric flask and volume was made up to the mark with methanol, obtaining a concentration of 100 µg/ml. From the stock standard solution, 1 ml of solution was transferred into 10 ml of volumetric flask and volume was make up with the same to get concentration of 10 µg/ml. It is scanned through UV- region i.e. 200-400 nm. In zero order spectrum, PFD show absorbance maxima at 317 nm.
Fig 2: Zero Order Spectrum (a) and Area under Curve; (b) between selected wavelengths of Pirfenidone
Method A (Zero order derivative) and Method B (Zero order derivative-AUC):
For method A and method B the standard stock solutions of PFD (100 μg/ml) were further diluted to prepared different sets of standard solution of PFD ranging from (15-40 μg/mL). For this aliquots of 1.5 – 4.0 mL of standard stock solution were separately pipette out and transferred to series of volumetric flask having capacity upto 10 mL then volume was made upto the mark with methanol. For method A absorbance in zero order derivative spectrum was determined at 317 nm shown in (Fig. 2 (a). While for method B AUC in zero order spectrum was selected in between 302.60 nm and 329.00 nm shown in (Fig. 2(b)). The calibration curves of method A and method B were constructed by plotting concentration versus absorbance and AUC of zero order spectrum respectively, shown in (Fig. 3).
Fig. 3: Calibration Curve of Pirfenidone for Method A (a); and Method B (b)
Fig. 4: First Order Derivative Spectrum (a) and Area Under Curve; (b) between selected wavelengths of Pirfenidone
Fig. 5: Calibration Curve of Pirfenidone for Method C (a); and Method D (b)
Method C (First order derivative) and Method D (First order derivative-AUC):
For method C and method D spectra of previous solution derivatized into first order spectra using UV probe 2.21 version software with delta 2 and scaling factor 1. In method C amplitude was determined at 241.00 nm shown in (Fig. 4(a)). While for method D AUC in first order spectrum was selected in between 238.60 nm and 245.00 nm shown in (Fig. 4(b)). The calibration curves of method C and D were constructed by plotting concentration versus amplitude and AUC of first order spectrum respectively, shown in (Fig. 5).
Study of Marketed Formulation:
For analysis of marketed formulation twenty tablets of PFD (PIRFENIX having label claim 200 mg) were weighed and transferred to clean dry mortar then tablet grounded to fine powder by a pestle. Fine powder of tablet equivalent to 10 mg of PFD was transferred to 100 mL volumetric flask containing 70 mL methanol and sonicated for 10 min. After ultra-sonication volume was made upto the mark with methanol and filter through whatman filter paper (no. 41). From the filtrate sets of same concentration was separately transferred to 10 mL volumetric flask and volume was made upto the mark to get final concentration of 25 μg/mL. The absorbance recorded and concentrations in the sample were determined from linearity equation.
Method Validation:
The method was validated with respect to various parameters including linearity, limit of detection and quantification, precision and accuracy according to ICH guidelines
Linearity:
Aliquots 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mL of standard stock solution of PFD was transferred to series of 10 mL volumetric flask and volume was made up to the mark methanol. The calibration curve was obeyed in the concentration of range 15 - 40 μg/mL and the graph was plotted between concentrations versus absorbance, amplitude and AUC.
Accuracy/ Recovery studies:
To study the accuracy of the anticipated methods and to confirm the obstruction from excipients used in the dosage forms, recovery experiments were performed by the standard addition method. It was carried out by adding known amount of standard drug to the marketed tablet formulation at 80, 100 and 120 % level. It was then re-analyzed by the proposed methods. The % recovery was noted. This confirms that the proposed method was found to be accurate. Results of the recovery were given in table 2 for PFD.
Precision:
The precision of proposed method was determined in terms of intra-day and inter-day precision. Intra-day precision was resolute by examine the 20, 25 and 30 μg/mL of PFD for three times in the similar day. Inter-day precision were determine the concentration of 20, 25 and 30 μg/mL of PFD for three days.
Sensitivity:
For sensitivity measurement, solution of PFD analyzed by developed methods and estimated in terms of limit of detection (LOD) and limit of quantification (LOQ) which were calculated using formulae “LOQ =10 x N/B” and “LOD = 3.3 x N/B,” where “N” is the average standard deviation of the peak areas of the PFD (n=3), and B is the slope to calibration curve.
Ruggedness:
Ruggedness of the introduced methods was determined for 25 μg/mL concentration of PFD by analysis of aliquots from a homogenous slot by two analysts using same operational and environmental conditions. The results are in acceptable range that is % RSD values < 2 for all the methods.
Repeatability:
The repeatability was estimated by analyzing of 25 μg/mL solution of PFD for six times.
RESULT AND DISCUSSION:
Method Validation:
The method was validated with respect to various parameters including linearity, limit of detection and quantification, precision and accuracy according to ICH guidelines. The data of analysis is shown in (Table 1).
Table 1: Optical Characteristics of PFD
|
Parameters |
Method A |
Method B |
Method C |
Method D |
|
Beer-Lambert’s range (μg/mL) |
15-40 |
15-40 |
15-40 |
15-40 |
|
Lambda max |
317 |
302.60-329.00 |
241 |
238.60-245.00 |
|
Slope |
0.003 |
0.0093 |
0.0018 |
0.0005 |
|
Intercept |
0.0219 |
0.0723 |
0.002 |
0.0018 |
|
Correlation coefficient |
0.9994 |
0.9966 |
0.9986 |
0.9966 |
Linearity:
Linear regression data shows that for method calibration curve shows in fig (3) linear relationship over the concentration range of 15-40 μg/mL for PFD shown in (Table 1)
Accuracy:
To determine the accuracy of the proposed method, pure drug was added to the pre-analyzed sample solution at three different concentration levels; 80%, 100% and 120%. The result were shown in (table 2). The % recovery value indicates that the accuracy of the method was found to be satisfactory.
Precision
Intra-day:
For intra-day precision studied three triplicates of three different concentrations 20, 25, 30 μg/mL was analyzed at different time in same day. The % RSD data in (table 3)
Inter-day:
For inter-day precision three triplicates of three different concentrations 20, 25 and 30 μg/mL was analyzed in different day subsequently. The % RSD date in (table 3)
Sensitivity:
The LOD and LOQ of PFD by developed methods were found to be, for “method A” 2.40 μg and 7.29 μg, for “method B” 0.40 μg and 1.22 μg, for “method C” 0.39 μg and 1.2 μg and for “method D” 0.31 μg and 0.94 μg respectively.
Table 2: Accuracy studies
|
Drug |
Methods |
Initial amount (μg/mL) |
Amount added (μg/mL) |
Amount recovered (μg/mL, n=3) |
% Recovered |
% RSD |
|
PFD |
A |
15 |
12 |
27.23 |
100.86 |
0.005 |
|
|
15 |
15 |
30.14 |
100.48 |
0.019 |
|
|
|
15 |
18 |
32.74 |
99.21 |
0.008 |
|
|
B |
15 |
12 |
27.23 |
101.23 |
0.007 |
|
|
|
15 |
15 |
30.23 |
100.76 |
0.003 |
|
|
|
15 |
18 |
32.97 |
99.23 |
0.049 |
|
|
C |
15 |
12 |
26.65 |
98.70 |
0.002 |
|
|
|
15 |
15 |
30.01 |
100.33 |
0.011 |
|
|
|
15 |
18 |
32.75 |
98.30 |
0.005 |
|
|
D |
15 |
12 |
27.11 |
100.41 |
0.005 |
|
|
|
15 |
15 |
30.11 |
100.37 |
0.005 |
|
|
|
15 |
18 |
33.05 |
100.16 |
0.002 |
n= number of determinations
Table 3: Precision studies:
|
Drug |
Methods |
Concentration (μg/mL) |
Intra-day [n=3] |
% RSD |
Inter-day [n=3] |
% RSD |
|
PFD |
A |
20 |
20.05 |
0.010 |
20.06 |
0.006 |
|
|
25 |
24.78 |
0.009 |
24.80 |
0.007 |
|
|
|
30 |
29.48 |
0.015 |
30.09 |
0.028 |
|
|
B |
20 |
19.91 |
0.024 |
20.11 |
0.024 |
|
|
|
25 |
24.09 |
0.081 |
25.07 |
0.013 |
|
|
|
30 |
30.22 |
0.010 |
29.72 |
0.014 |
|
|
C |
20 |
20.20 |
0.0092 |
20.16 |
0.017 |
|
|
|
25 |
25.31 |
0.002 |
25.39 |
0.001 |
|
|
|
30 |
29.89 |
0.002 |
29.82 |
0.003 |
|
|
D |
20 |
19.83 |
0.009 |
19.87 |
0.012 |
|
|
|
25 |
25.14 |
0.0006 |
25.14 |
0.002 |
|
|
|
30 |
30.35 |
0.006 |
30.35 |
0.004 |
n= number of determination
Repeatability:
The Results of repeatability in terms of % RSD were less than 2 indicate precision nature of developed methods. Results are shown in (table 4)
Ruggedness:
The result of ruggedness were in acceptable range that is % RSD value not more than 2 for developed method as given in (table 5). The results prove that no statistical difference between analyst using same operational and environmental condition. Hence it proves that developed method is rugged in nature.
Table 4: Repeatability studies
|
Drug |
Methods |
Amount taken[μg/mL] |
Amount found[μg/mL] |
% Amount found [n=6] |
Mean ± SD |
% RSD |
|
PFD |
A |
25 |
24.72 |
98.91 |
98.91±0.74 |
0.007 |
|
B |
25 |
24.75 |
99.03 |
99.03±0.67 |
0.006 |
|
|
C |
25 |
25.37 |
101.5 |
101.5±0.92 |
0.009 |
|
|
D |
25 |
24.95 |
99.81 |
99.81±1.65 |
0.016 |
n= no of determinations
Table 5: Ruggedness studies
|
|
|
Analyst I |
Analyst II |
||
|
Drug |
Methods |
% Amount found± SD (n=6) |
%RSD |
% Amount found± SD (n=6) |
%RSD |
|
PFD |
A |
98.80±0.63 |
0.006 |
98.82 ± 0.65 |
0.006 |
|
B |
100.47±0.49 |
0.48 |
98.54±0.48 |
0.48 |
|
|
C |
102.03±1.05 |
0.010 |
101.83±0.62 |
0.006 |
|
|
D |
99.62±1.23 |
0.012 |
100.77 ± 2.40 |
0.023 |
|
n= no of determination
Analysis of marketed formulation:
From PFD tablet formulation amount of PFD estimated by using methods A, B, C and D were found to be 101.68%, 101.47%, 101.83%, and 99.59%, respectively. The % amount found from tablet formulation shows that there was no interference from excipients present in tablet formulation.
CONCLUSION:
The methods mentioned above all are developed for the quantitative determination of PFD in bulk and Tablet formulation by incorporating derivative spectroscopic technique and AUC technique of UV-spectrophotometry. After obtaining the result, we can say that all methods were follows the conditions given by ICH guideline Q2 (R1) for analytical methods. The outcome of the experiment suggests that developed methods are simple, precise and accurate and can be implicated for quantitative and routine analysis of PFD in bulk and pharmaceutical formulation.
ACKNOWLEDGEMENT:
The authors are thankful to Dr. S. J. Surana Principal R.C. Patel Institute of Pharmaceutical Education and Research, Shirpur (M.S.), India for providing the necessary facilities to carry out this research work
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Received on 08.04.2019 Modified on 20.05.2019
Accepted on 02.06.2019 ©AJRC All right reserved
Asian J. Research Chem. 2019; 12(3): 129-135.
DOI: 10.5958/0974-4150.2019.00027.0