Development and Validation of RP HPLC Method for Estimation of Deferiprone and its Related Impurityin Pharmaceutical Dosage Form

 

Shweta Ubale1*, Mayur Bhosale2, S. K. Parajne3

1Student, Department of Pharmaceutical Chemistry, Pravara Rural College of Pharmacy,

Loni, Ahmednagar, Maharashtra, India

2Professor, Department of Pharmaceutical Chemistry, Pravara Rural College of Pharmacy,

Loni, Ahmednagar, Maharashtra, India

3Professor, Department of Pharmaceutical Chemistry, Pravara Rural College of Pharmacy,

Loni, Ahmednagar, Maharashtra, India

*Corresponding Author E-mail: shwetarubale97@gmail.com, mayur.bhosale@pravara.in

 

ABSTRACT:

Aim of this study is to develop a new, precise, sensitive, simple, efficient, selective, and accurate high-performance liquid chromatographic method for the separation and determination of Deferiprone and its impurity in the capsule dosage form. A wide-range of literature survey disclosed no method for estimation said as the above. The chromatographic separation was achieved on Agilent Zorbax Bonus-RP (250 x 4.6mm, 5) with a mobile phase of Methanol: 0.1% O-Phosphoric acid (10:90, % v/v) combination in 1000ml of Methanol: Water (50: 50, % v/v) using a diluent. The flow rate of 1mL/min and UV detection at 280nm use as wavelength. The developed method was validated as reported by ICH guidelines. The linearity of the calibration curve for deferiprone and its process-related impurity in the concentration range of 4.0-6.0μg/ml. There exists a good correlation between peak area and analyte concentration. The retention time for deferiprone was discovered to be 2.29 min and its impurity was 8.65min. Deferiprone's relative standard deviation value is 0.45 and its process-related impurity is 0.17. All the results tell that the proposed method was highly sensitive, simple, precise, accurate, and fast. A large number of samples can be analyzed in a shorter time due to shorter retention times, so it can be successfully applied for routine analysis of Deferiprone and related impurity (maltol) in pharmaceutical dosage forms.

 

KEYWORDS: Deferiprone, Maltol impurity, Method development, Validation, RP HPLC.

 

 


INTRODUCTION:

Deferiprone is in a class of medicines called iron chelators. Deferiprone is used for the controlling of Thalassemia major.

 

Thalassemia most important, when a child inherits mutated two genes, one from every parent. Most importantly, children born with thalassemia increase the chance of intense anaemia within the first year of life. They cannot produce normal hemoglobin and experience continual fatigue2. Deferiprone binds to iron in the blood. It treats to loss and helps to prevent too much iron in the bloodcaused by blood transfusions [8]. Deferiprone is an associate iron chelator that binds to metal ions (iron III) and forms a 3:1 (deferiprone: iron) stable complex and is then eliminated within the weewee. Deferiprone is a lot selective for iron during which alternative metals like zinc, copper, and place aluminum have a lower affinity for deferiprone.3,5 Deferiprone is chemically 3-hydroxy-1,2-dimethyl pyridine-4-one with molecular formula C7H9NO2.1,5.

 

Unwanted chemicals present within the formulation and active pharmaceutical ingredients which affect the quality, safety, and efficacy of the medicinal products are called impurity.4 A literature survey revealed that few analytical methods have been reported for the estimation of deferiprone alone or in combination with other drugs by ultraviolet (UV) spectrometry high-performance liquid chromatography (HPLC) and LC-mass spectrometry. However, there is no reported method for the separation and determination of deferiprone impurity. Hence, an attempt was made to develop a simple, accurate, precise, and sensitive HPLC method for the estimation of deferiprone in the presence of its above-mentioned impurity.

 

MATERIALS AND METHOD:

Chemicals and reagents:

Deferiprone and its impurity were obtained as a gift sample from Adhar life science Pvt. Ltd. Solapur. Kelfer capsules were purchased from a local pharmacy, Solapur, Maharashtra. Analytical grade Methanol, O-Phosphoric acid, and water were also available from Adhar life science Pvt.Ltd Solapur.


 

Table 1: Chemical name and structure of deferiprone and its related impurity 6,7

Sr. No.

Name of compound

Chemical Structure

Molecular formula

IUPAC name

1

Deferiprone

 

[C7H9NO2]

3-hydroxy-1,2-dimethyl pyridine-4-one

2

Impurity

[Maltol]

 

[C6H6O3]

3-Hydroxy-2-methyl-4H-pyran-4-one

 

 


Instrumentation and conditions:

The chromatographic separation the Agilent technologies 1260 infinity 2 instrument andAgilent Zorbax Bonus-RP (250 x 4.6mm, 5) column was used. The mobile phase used for the separation of both API and impurity was 0.1%ml of ortho-phosphoric acid and make the volume with 1000ml of Water: methanol (50: 50 v/v). Ambient temperature was maintained. Detection was made at a wavelength of 280.0nm. The validation study was carried out using the same optimized condition with suitable preparation of standard and sample solutions.

 

STANDARD PREPARATION:

Deferiprone Standard Stock Solution-I (DSSS-I):

Initially Prepare a Standard Stock Solution (SSS-I) by adding 5mg of Deferiprone in a 10ml volumetric flask and add 5ml diluent, mix for 2 minutes and make the volume to 10ml with diluent. (Conc. of Deferiprone = 500g/ml).

 

Impurity Standard Stock Solution-I (ISSS-I):

Then prepare a Standard Stock Solution (SSS-II) of impurity [Maltol] by adding 5mg in a 10ml volumetric flask and add 5ml diluent, mix for 2 minutes and make the volume to 10ml with diluent. (Conc. of Maltol = 500 g/ml).

Preparation of resolution solution:

Then add 1.0ml of DSSS-I and 1.0 ml ISSS-I in a 10ml volumetric flask and add 5ml diluent and vortex and make up the volume with diluent. (Conc. of Deferiprone =50g/ml and Maltol = 50g/ml).

 

Further, pipette out 1ml of above solution in 10ml volumetric flask and add 5ml diluent and vortex and make up the volume with diluent. (Conc. of Deferiprone =5g/ml and Maltol = 5g/ml).

 

Preparation of sample solution:

10 Capsule (Kelfer*) contents were weighed and average weight was calculated and power/granules were crushed and mixed in mortar and pestle. Powder weight equivalent to 5mg Deferiprone was weighed into 10ml volumetric flask and add 5ml diluent, sonicate for 10 minutes and make the volume to 10ml with diluent. (Conc. of Deferiprone =500g/ml). Further, pipette out 0.1ml of above solution in 10ml volumetric flask and add 5ml diluent and vortex and make up the volume with diluent. (Conc. of Deferiprone =5g/ml).

 

METHOD VALIDATION:

The method validation was performed as per the international conference on harmonization (ICH) guidelines8. The parameters such as specificity, linearity and range, precision, accuracy, limit of detection (LOD), and limit of quantitation (LOQ), were evaluated.

 

Specificity:

The specificity was demonstrated by injecting deferiprone standard solution, blank solution, and sample spiked with impurity solution, and the chromatograms were checked for interferences.9

 

Accuracy:

The accuracy of an analytical procedure is detected to observed the closeness of the test results to standard value. Accuracy is expressed as % recovery of the standard spiked to the already analyzed test sample of the capsule.10 It was measured in drug products by spiking known quantities (80%, 100%, and 120%) of the analyte into the analyzed powder and each concentration was injected into the column 3 times and the percent recovered was calculated.

 

System suitability:

System suitability studies form an integral part of method development and ensures good performance of chromatographic system. The standard solutions of deferiprone [5g/ml] and maltol impurity [5g/ml] of about 10μl were injected under suitable chromatographic conditions to obtain the suitability of the system.

 

Precision:

The precision was studied by repeatabilityand intermediate precision11. The precision was checked by injecting the sample solution spiked with impurities in 6 replicates, and the intermediate precision was evaluated. The %RSD of % total impurity was calculated.

 

Linearity and range:

Linearity was checked for standard solutions of drug and impurity at concentrations of 80%, 90%, 100%, 110% and 120%. Aliquot solutions of deferiprone and Maltol impurity were prepared in the range of 4.0-6.0μg/ml respectively and keeping the injection volume constant, i.e., 10μl.

 

Limit of detection and limit of quantitation:

LOD is defined as the lowest concentration of asubstance in the sample that can be detected with a specific analytical condition. (12). The LOQ is defined as the lowest concentration of an analyte in the sample that can be measured with acceptable accuracy, precision, and variability. The LOD and LOQ were calculated from the linearity curve by applying the formulae:

 

LOD= 3.3 S / σ

 

LOQ = 10 S / σ

 

Where,

σ is the standard deviation of the y-intercept and

S is the slope of the calibration plot.

 

Table 2: Optimized chromatographic conditions

Sr. No.

Parameters

Results

1

Dilution

Methanol: Water (50: 50, % v/v)

2

Mobile phase

Methanol: 0.1% O-Phosphoric acid (10:90, % v/v)

3

Column

Agilent Zorbax Bonus-RP (250 x 4.6 mm, 5 )

4

Flow rate

1 ml/min

5

Detection

280 nm

6

Injection volume

10l

7

Temperature

30℃

8

Retention time

2.29 min for deferiprone and 8.65 min for maltol impurity

9

Run time

11 minutes

 

RESULT:

Method development:

The 280nm wavelength was selected for the proposed HPLC method based on the UV absorption and literature survey of deferiprone. the separation of the impurity from the drug was achieved with Agilent Zorbax Bonus-RP (250 x 4.6mm, 5) column and with a mobile phase Methanol: 0.1% O-Phosphoric acid (10:90, % v/v), and the chromatogram of the drug and the impurity was good. The optimized parameters were listed in Table 2. A Chromatogram for standard solutions of deferiprone and maltol impurity was presented in Fig1

 

Fig.1: Chromatogram of resolution for deferiprone and impurity

 

Fig.2: Chromatogram of the blank solution..

 

Fig.3: Chromatogram for standard solution ofdeferiprone

 

Fig.4: Chromatogram for standard solution ofimpurity.

 

Specificity:

The specificity of the method was tested by comparing the chromatograms of blank (Fig. 2), deferiprone standard solution (Fig. 3), and impurity solution (Fig. 4). No interference peaks were observed at the deferiprone RT due to the blank, impurities.

 

Accuracy:

The accuracy of the proposed method was determined by analyzing the deferiprone sample solution spiked with impurities at 3 concentration levels of 80%, 100%, and 120% in triplicate. The mean percentage recovery was calculated and reported in Tables3 and 4.

 

Table 3: Accuracy study of deferiprone

Sr. no

% Level

Area

% Recovery

AVG

1

80%

238021

100.47

100.52

238154

100.53

238177

100.54

2

100%

298091

100.67

99.96

294473

99.44

295471

99.78

3

120%

359095

101.06

100.85

358045

100.76

357953

100.73

 

Table 4: Accuracy study of Impurity

Sr. No

% Level

Area

% Recovery

AVG

1

80%

316121

99.82

99.85

316275

99.87

316298

99.87

2

100%

395689

99.95

99.93

395712

99.96

395354

99.87

3

120%

473682

99.71

99.66

473183

99.61

473384

99.65

 

Table5: System suitability parameters.

Sr. No.

Parameters

Deferiprone

Impurity maltol

1

Retention time

2.29

8.65

2

Theoretical plate

7737

11765

3

Tailing factor

1.24

1.17

4

Resolution

0.00

30.03

 

System suitability studies:

system suitability parameters such as the number of (TP) theoretical plates, (T) tailing factor, and the resolution was calculated. The results are Reported in Table 5.

 

Precision:

Precision of the method by repeatability and intermediate precision was assessed by injecting sample solution spiked with impurities 6 times, and the results were expressed in terms of %RSD of the percentage of total impurities. The results are presented in Table 6, 7 and the chromatogram was presented in Figs. 5 and 6.

 

Linearity:

The linearity graph was plotted by the concentration versus peak area the calibration ranges tested for deferiprone and impurities. Results are shown in Table 8, and the linearity curves are shown in Figs. 7 and 8.

 

Limit of detection (LOD) and limit of quantitation (LOQ):

LOD and LOQ for deferiprone were found to be 0.27 ug/ml and 0.82ug/ml. LOD and LOQ for maltol impurity were found to be 0.24ug/ml and 0.74ug/ml respectively.

 

Table 6: Precision studies for standard solution of deferiprone. impurity

Sample ID

Area

RT

100%Rep 1

298091

2.29

100%Rep 2

294473

2.29

100%Rep 3

295471

2.29

100%Rep 4

297344

2.29

100%Rep 5

295478

2.29

100%Rep 6

295867

2.29

AVG

296121

2.29

STDEV

1341.119

4.86E-16

%RSD

0.45

0.00

 

Table 7: Precision studies for standard solutions of deferiprone. Impurity

Sample ID

Area

RT

100%Rep 1

395689

8.66

100%Rep 2

395712

8.66

100%Rep 3

395354

8.66

100%Rep 4

395121

8.66

100%Rep 5

396671

8.66

100%Rep 6

396713

8.66

AVG

395877

8.66

STDEV

668.875

1.95E-15

%RSD

0.17

0.00

 

Fig. 5: Precision chromatogram for standard solution of Deferiprone and Impurity Maltol

 

Fig. 6: precision Chromatogram for sample solution. (Kelfer*)

 

Table 8 Linearity studies for deferiprone and maltol impurity

Sr.No.

% Level

Concentration (ug/ml)

Area of deferiprone

Area of maltol

1

80

4.0

238021

316121

2

90

4.5

265596

355454

3

100

5.0

298091

395689

4

110

5.5

330319

438212

5

120

6.0

359095

473682

 

Fig.7: Calibration curve for deferiprone.

 

Fig.8: Calibration curve for maltol

 

DISCUSSION:

Various solvent system combinations for the determination of deferiprone and its related impurity (maltol)14,15 in pharmaceutical dosage forms were studied and by using mixture of 0.1% O-Phosphoric acid (10:90, % v/v) in 1000ml of water and methanol as mobile phase as it gave better resolution. The effect of inflow rate was studied at 1ml/min was preferred. The retention time (RT) was found to be 2.29 min for Deferiprone and 8.65 min for maltol impurity. The chromatographic system is considered suitable when it obtains the resolution between the peaks must be >2, the number of (TP) theoretical plates should be greater than 2000, and the (T) tailing factor must be lower than 2. as per ICH guidelines11 thisacceptance criteria.8, as no interference was observed from the blank at the RT of known impurities. Percent recovery was found to be 100.52, 99.96%, and 100.85% for drug and 99.85%, 99.93%, and 99.66% for related substance at 80%, 100%, and 120% respectively. All experimental results are within the acceptable criteria, i.e., 97-102% for drugs and 50-150% for related substances, and the method was found to be accurate. The % RSD values for the peaks were established within the limits, RSD ≤ 213 as shown in the results, so the method was precise. A calibration curve was met by plotting a graph between peak area and concentration. Excellent correlation was obtained between it with R2 = 0.9992 for deferiprone and 0.9993 for maltol impurity as per the limit R2 ˃ 0.99912. The LOD and LOQ were calculated from the slope of the result obtained was 0.27ug/ml and 0.82ug/ml for deferiprone and 0.24ug/ml and 0.74ug/ml for maltol impurity respectively. It is evident from the got data that all the peaks were well resolved, and the method is said to be specific.

 

CONCLUSION:

The method proposed for the analysis of deferiprone and related impurity in pharmaceutical dosage forms was established to be specific, precise, accurate, quick, and economical. The developed method was validated in terms of accuracy, linearity, LOD, LOQ, and precision by ICH Q2 R1 guidelines. Short (RT) retention time enabled analysis of deferiprone and maltol impurity with a minimal amount of mobile phase. The method was found to beprecise and accurate. Due to quantitation limits and low detection, the method was said to be sensitive. This method can be applied successfully for the determination of deferiprone and its related maltol impurity in pharmaceutical dosage forms.

 

AUTHORS CONTRIBUTION:

The research work, manuscript preparation, and grammar check using the software Grammarly were done by Ms. Shweta Ubale, the research work was guided by Dr. S. K. Parjane, and critical revision and final proofreading of the manuscript were done by Mrs. M. S. Bhosale.

 

ABBREVIATION:

LOD: Limit of detection; LOQ: Limit of quantitation; RSD: Relative standard deviation; DSSS-I: Deferiprone Standard Stock Solution-I; ISSS-I: Impurity Standard Stock Solution-I.

 

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest.

 

ACKNOWLEDGMENTS:

I thank Adhar life science Pvt.Ltd Solapur laboratories, for providing a gift sample of deferiprone and Maltol. I thank my guide and co-guide for supporting me and helping me and I also thank my family.

 

REFERENCES:

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Received on 24.04.2022 Modified on 28.09.2022

Accepted on 07.12.2022 Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2023; 13(1):1-6.

DOI: 10.52711/2231-5675.2023.00001