Development and Validation of Zero and First-Order Derivative Area under Curve Spectrophotometric Methods for the Determination of Deferiprone in Bulk Material and Capsules
Aamir Malik1, Sandip Firke2*, Ravindra Patil2, Atul Shirkhedkar2, Mohan Kalaskar
1Research Scholar, Department, R. C. Patel Institute of Pharmaceutical Education and Research, Shipur, Dhule (425405)
2Professor, Pharmaceutical Chemistry Department, R. C. Patel Institute of Pharmaceutical Education and Research, Shipur, Dhule (425405)
*Corresponding Author E-mail: sandipfirke@rediffmail.com
ABSTRACT:
A simple, accurate and precise spectrophotometric method including area under curve has been developed for the estimation of Deferiprone (DEF) in bulk material and capsule dosage form. Deferiprone is an iron chelating agent, used in the treatment of thalassemia syndromes when iron overload due to blood transfusions occurs. Four simple UV Spectrophotometric methods were established for determination of Deferiprone, using double beam UV spectrophotometer (UV-2450, Shimadzu, Japan). Deferiprone has absorbance maxima at 278 nm with distilled water used as a solvent. Deferiprone obeys Beer-Lambert’s law in the concentration range of 2-10 μg/mL. The % recovery by proposed methods was found to be 98-101%. The Precision studies show less than 2 in terms of %RSD indicates precise nature of proposed method. All methods were validated as per the ICH guidelines, Q2 (R1). The statistical data and results prove that developed methods are simple, accurate and precise and can be used for the routine analysis of DEF in bulk and pharmaceutical formulation.
KEYWORDS: Deferiprone (DEF), AUC, UV-spectrophotometer, Derivative spectroscopy.
INTRODUCTION:
Deferiprone is chemically known as 3-hydroxy-1, 2-dimethylpyridin-4(1H)-one (Fig.1)[1]. It is one of pyridine derivative. It is sparingly soluble in methanol and water and having slight solubility in ethanol as well as in chloroform. The molecular weight is 139.152 g/mol with C7H9NO2 molecular formula and λmax is 279nm[2]. DEF is an iron chelating agent, administered orally, used in the treatment of thalassemia syndromes when iron overload due to blood transfusions occurs.
It forms steady complex with ferric ions in 3:1 (deferiprone: iron) ratio and get excreted in urine, thus reduce the blood iron level. It is used as a second line agent for treatment of beta-thalassemia, an iron burden that leads to the impaired production of RBCs in body. Deferiprone generally known for life-saving agent in beta-thalassemia patient to remove excess iron from heart[3].
Extensive literature survey reveals that very few analytical methods has been proposed for the estimation of DEF. HPLC in bulk and pharmaceuticals as well as in biologicals has been reported[2,4,5]. Also there is one paper on LC-MS/MS[6]and two on simple UV-spectrophotometric method is present[7,8]. However, till date there is no UV-spectroscopy method have been developed for estimation of DEF in bulk and pharmaceuticals using AUC[9], derivative spectroscopy[10,11] and amplitude technique[12].
The aim of the practical was to develop simple, accurate and precise spectrophotometric zero order[13], first order derivative method usingAUC and Amplitude technique for the estimation of DEF in bulk material and pharmaceuticals. Further, developed methods were validated according to the ICH guidelines, Q2 (R1)[14].
Fig. 1: Deferiprone
MATERIAL AND METHODS:
Chemicals: Pure DEF was obtained as a gift sample from Cipla pharmaceutical Ltd., Mumbai India. The marketed formulation (Kelfer) 250mg was purchased from local market. Distilled water used as a diluent for this analysis.
Instrumentation:
A double beam UV-VIS spectrophotometer (Model- UV-2450, Shimadzu, Japan) equipped with 10mm matched quartz cell with UV probe version 2.21 software was utilized in the current research work for all absorbance measurement. For weighingan electronic weighing balance (Model- AUX 120, Shimadzu, Japan) was used.
Selection of Solvent:
The solubility of DEF was checked in different solvent. It is found that DEF is soluble in both distilled water and methanol and as well as inan aqueous acid after sonication for 5 minute. For ease distilled water was selected throughout the method development and validation process. There was no any degradation occur in this solvent.
Preparation of standard stock solution and determination of lambda (λ max):
Standard stock solution (100µg/mL) was prepared was dissolving accurately weighed 10 mg of DEF in 30 mL distilled water in 100 mL volumetric flask and after sonication for 5 min. and 100 mL volume was made up with same solvent.
From the standard stock solution 1 mL was pipette out and transferred to a 10 mL volumetric flask. Further it was diluted up to the mark by distilled water to obtained final concentration 10µg/mL. It is scanned through UV- region i.e. 200-400 nm. In zero order spectrum, DEF show absorbance maxima at 278 nm.
Method A (Zero order derivative) and Method B (Zero order derivative-AUC):
For both these methods standard stock solution (100µg/mL) was further diluted to prepare different standard concentration of DEF ranging from (2-10 µg/mL). For this aliquots, 0.2-1 mL of stock solutions were pipette out separately in 10 mL volumetric flask and diluted up to 10 mL with diluent to achieve final concentration. For method A absorbance in zero order derivative spectrum was determined at 278 nm as in (Fig. 2(b)). While for method B AUC of DEF in zero order spectrum was selected in between 266.20 nm and 288.20 nm as in (Fig. 2(a)). 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).
Method C (First order derivative) and Method D (First order derivative-AUC): For method C and method D zero order spectra of previous solutions derivatized into first order spectra using UV probe 2.21 version software with delta value 2 and scaling factor 1. In method C amplitude was determined at 291.40 nm shown in (Fig. 4(a)). While for method D AUC in first order spectrum was selected in between 280.40 nm and 299.60 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).
METHOD VALIDATION:[12]
The developed method was validated as per the ICH guideline, Q2 (R1). The validation of the method was done by performing parameters, i.e. Linearity, Accuracy, Precision, Repeatability and Limit of detection (LOD) and Limit of Quantification (LOQ).
Linearity:
For method A, B, C and D, linearity of DEF was performed by using concentration range 2-10 µg/mL. The aliquots were prepared by pipette out 0.2-1 mL of standard stock solution in10 mL volumetric flask and volume is made up by using distilled water. DEF obeys Beer-Lambert’s law in the concentration range of 2-10 μg/mL. The graph was plotted between concentration versus absorbance, AUC and amplitude.
Repeatability:
Repeatability in “all methods” was determine by analyzing 6 μg/mL solution of DEF for six times.
a b
Fig. 2: Zero Order Derivative Spectrum (a) and Area under Curve; (b) between selected wavelengths of Deferiprone
a b
Fig. 3: Calibration curve of DEF for Method A (a) and Method B (b)
a b
Fig. 4: First Order Derivative Spectrum (a) and Area under Curve; (b) between selected wavelengths of Deferiprone
a b
Fig no. 5: Calibration curve of DEF for Method C (a) and Method D (b)
Precision:
Precision study was carried out in the form of intra-day and inter-day variations. For all methods precision was found out by analyzing the 4, 6, and 8 μg/mL of DEF solution separately for intra-day and inter-day variations.
Accuracy:
The accuracy of all methods was evaluated in terms of recovery. To the pre-analyzed DEF aliquot (4 μg/mL in all method), known amount of stock standard solution were added at different levels, i.e., 80%, 100%, and 120%. The experiments were repeated for three times at each level in all methods.
Sensitivity:
For sensitivity measurement, specific calibration curve of DEF plotted and sensitivity was determine in terms of limit of detection (LOD) and limit of quantification (LOQ) which were calculated using formulae “LOQ = 10 × σ/S” and “LOD = 3.3 × σ/S,” where “σ” is standard deviation of y-intercepts of regression lines of the absorbance, amplitudes or peak areas of the DEF (n = 3), taken as a measure of noise, and “S” is the slope related to calibration curve.
Ruggedness:
For all methods ruggedness study for developed method was carried out by analyzing 6 μg/mL of DEF solutions (n = 6) by two different analyst using identical operational and environmental conditions.
RESULT AND DISCUSSION:
Method validation:
DEF was validated as per the following parameters linearity, accuracy, precision, sensitivity, LOD, LOQ, and ruggedness. The results obtained were lies in the acceptable range as per the ICH guideline. The data of regression analysis was shown in table1.
Linearity:
After performing linear regression analysis it was found that for “methods A, B, C, and D” calibration curves shown in (Fig. 3 & 5) are in linear relationship over the concentration range of 2-10 μg /mL for DEF shown in (Table 1).
Table 1: Linearity of Deferiprone
Parameters |
Method A |
Method B |
Method C |
Method D |
Beer-Lambert’s range(μg/mL) |
2-10 (μg/mL) |
2-10 (μg/mL) |
2-10 (μg/mL) |
2-10 (μg/mL) |
Lambda Max (nm) |
278 |
266.20-288.20 |
291.40 |
280.40-299.60 |
Slope |
0.4592 |
0.0897 |
0.0075 |
0.0641 |
Intercept |
0.2559 |
0.0723 |
0.0029 |
0.0188 |
Correlation coefficient |
0.9975 |
0.9986 |
0.9975 |
0.9988 |
Repeatability:
Repeatability performed and calculated in terms of % RSD for “methods A, B, C, and D,” and it was found to be less than 2 which indicate precise nature of developed methods. Results are shown in (Table 2).
Precision:
Intra-day:
For intraday precision studies three replicates of three different concentration that is, 4, 6 and 8 μg/mL were analyzed at different time in same day. Results are shown in (Table. 3).
Table 2: Repeatability data
Drug |
Method |
Amount taken (μg/mL) |
Amount found (μg/mL) |
% Amount found (n = 6) |
Mean ± SD |
% RSD |
Deferiprone |
A |
6 |
6.0756 |
101.26 |
0.5699 |
0.5628 |
B |
6 |
6.0573 |
101.02 |
0.6277 |
0.6214 |
|
C |
6 |
6.0244 |
100.40 |
0.6350 |
0.6324 |
|
B |
6 |
6.0633 |
101.05 |
0.3765 |
0.3726 |
n- Number of determinants
Table 3: Precision data
Drug |
Method |
Concentration (μg/mL) |
INTRA-DAY Mean ± SD (n= 3) |
% RSD |
INTER-DAY Mean ± SD (n= 3) |
% RSD |
Deferiprone
|
A |
4 |
0.4435 ± 0.0049 |
1.10 |
0.4282 ± 0.0056 |
1.31 |
6 |
0.6147 ± 0.0027 |
0.44 |
0.6173 ± 0.0021 |
0.33 |
||
8 |
0.7796 ± 0.0097 |
1.23 |
0.7899 ± 0.0081 |
1.03 |
||
B |
4 |
2.1654 ± 0.0239 |
1.10 |
2.0886 ± 0.0328 |
1.57 |
|
6 |
3.0049 ± 0.0182 |
0.60 |
3.0164 ± 0.0152 |
0.50 |
||
8 |
3.7444 ± 0.0524 |
1.40 |
3.8562 ± 0.0400 |
1.03 |
||
C |
4 |
0.0348 ± 0.0005 |
1.29 |
0.0345 ± 0.0003 |
1.05 |
|
6 |
0.0483 ± 0.0003 |
0.71 |
0.0473 ± 0.0002 |
0.42 |
||
8 |
0.0614 ± 0.0006 |
1.07 |
0.0564 ± 0.0007 |
1.27 |
||
D |
4 |
0.2858 ± 0.0034 |
1.19 |
0.2859 ± 0.0031 |
1.10 |
|
6 |
0.3966 ± 0.0031 |
0.78 |
0.3937 ± 0.0019 |
0.50 |
||
8 |
0.5089 ± 0.0063 |
1.24 |
0.4696 ± 0.0063 |
1.34 |
n- Number of determinants
Intra-day:
For interday precision studies same concentrations as of intraday, that is, 4, 6 and 8 μg/mL were analyzed for three consecutive days. Results are shown in (Table. 3).
Accuracy:
To find out the accuracy of the developed methods, pure bulk drug solutions were added in pre-tested sample solutions at three different concentration levels; 80 %, 100 % and 120 %. The results were summarized in (Table. 4). The % Recovery values depictthat the accuracy of the methods was found to be agreeable.
Ruggedness:
For ruggedness study, aliquots of three different concentrations were prepared and analyzed by two different analyst while experimental conditions were kept same.
Table. 4: Accuracy study of Deferiprone
Drug |
Method |
Initial amount (μg/mL) |
Amount added (μg/mL) |
Amount found [μg/mL, n = 3] |
% Amount found |
% RSD |
Deferiprone |
A |
4 |
3.2 |
7.2479 |
100.66 |
0.46 |
4 |
4 |
8.0445 |
100.55 |
0.81 |
||
4 |
4.8 |
8.9297 |
100.83 |
0.52 |
||
B |
4 |
3.2 |
7.1606 |
99.45 |
0.45 |
|
4 |
4 |
8.0451 |
100.56 |
0.78 |
||
4 |
4.8 |
8.9061 |
101.20 |
0.78 |
||
C |
4 |
3.2 |
7.28 |
101.11 |
0.18 |
|
4 |
4 |
8.12 |
101.27 |
0.25 |
||
4 |
4.8 |
8.98 |
100.70 |
0.62 |
||
D |
4 |
3.2 |
7.1464 |
99.25 |
0.42 |
|
4 |
4 |
8.0036 |
100.04 |
0.39 |
||
4 |
4.8 |
8.8390 |
100.44 |
0.33 |
RSD= Relative Standard Deviation
Table. 5: Ruggedness data
|
Analyst-I |
Analyst-II |
|||
Drug |
Method |
% Amount found ± SD (n = 6) |
% RSD |
% Amount found ± SD (n = 6) |
% RSD |
Deferiprone |
A |
101.19 ± 0.53 |
0.53 |
101.09 ± 1.03 |
1.01 |
B |
100.94 ± 0.6902 |
0.68 |
100.99 ± 0.7430 |
0.73 |
|
C |
99.62 ± 0.50 |
0.50 |
100.74 ± 1.42 |
1.41 |
|
D |
101.42 ± 0.3971 |
0.39 |
101.10 ± 0.0604 |
1.05 |
SD= Standard Deviation
Sensitivity:
The LOD and LOQ of DEF by developed methods were found to be, for “method A” 0.22 μg and 0.65 μg, for “method B” 0.34 μg and 0.77 μg, for “method C” 0.14 μg and 0.47 μg and for “method D” 0.33 μg and 0.89 μg respectively.
Analysis of Pharmaceutical:
The amount of drug estimated from DEF capsule formulation by methods A, B, C and D were found to be 100.89%, 100.40%, 101.02%, and 99.91%, respectively. The % amount recovered from capsule formulation indicates that there was no any interference by excipients present in the formulation.
CONCLUSION:
The methods mentioned above all are developed for the quantitative determination of DEF in bulk and capsule formulation by incorporating derivative spectroscopic technique and AUC technique of UV-spectrophotometry. After obtaining the result, we can say that all methods were mimic the conditions given by ICH guideline Q2 (R1) for analytical methods. The outcome of the experiment suggest that developed methods are simple, precise and accurate and can be implicated for quantitative and routine analysis of DEF in bulk and pharmaceutical formulation.
ACKNOWLEDGEMENT:
Authors are grateful to Dr. S. J. Surana, Principal R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Dist: Dhule (MS) for providing needed laboratory facility.
CONFLICT OF INTEREST:
The author declares no conflict of interest.
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Received on 14.02.2019 Accepted on 04.04.2019
© Asian Pharma Press All Right Reserved
Asian J. Pharm. Ana. 2019; 9(2):49-54.
DOI: 10.5958/2231-5675.2019.00011.5