Instrumentation and conditions:

The chromatographic separation the Agilent technologies 1260 infinity 2 instrument andAgilent Zorbax Bonus-RP (250 x 4.6mm, 5µ) 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 = 500µg/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 µg/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 =50µg/ml and Maltol = 50µg/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 =5µg/ml and Maltol = 5µg/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 =500µg/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 =5µg/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 [5µg/ml] and maltol impurity [5µg/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 µ)
4	Flow rate	1 ml/min
5	Detection	280 nm
6	Injection volume	10µl
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, 5µ) 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:

1. P. Vamsi Reddy, et.al., Stability indicating method development and validation of Deferiprone in pharmaceutical dosage form by RP-HPLC, Journal of Innovations in Pharmaceutical and Biological Sciences, Vol 4 (2), 53-57, 2017.

2. Thalassemia foundation of Canada.Thalassemia Major « Thalassemia. 2022.

3. FDA Approved Drug Products: Ferriprox (deferiprone) oral tablets;1-16;2011.https://www.accessdata.fda.gov/drugsatfda_docs/label/2021/021825s010lbl.pdf.

4. Federal Register International Conferences on Harmonization. Draft Revised guidance on Impurities in New Drug Substances, Q3A(R); 2000. p. 45085-90

5. Manzoor Ahmed, Vinod Kumar Jangade, et. al., RP-HPLC method development and validation for estimation of deferiprone in capsule dosage form. World Journal of Pharmacy and Pharmaceutical Science. 2015;831-838.

6. K. Sai Sireesha, et.al., A new RP-HPLC method development and validation of deferiprone in bulk and its pharmaceutical dosage form. International Journal of Advance Research. 2016;2174-2179.

7. Introduction to Food Toxicology A volume in Food Science and Technology Book; Chapter 7 - Food Contaminants from Industrial Wastes;1Pages 117-140.

8. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use. Validation of Analytical Procedures: Text and Methodology ICH Q2 (R1); 2005.

9. Daksh S, Goyal A, Pandiya CK. Validation of analytical methodsstrategies and significance. Int J Res Dev Pharm Life Sci 2015; 4:1489-97

10. Guidance Document, Validation of Analytical Methods, Document ID IPC/GD/04 Version 1.0 Issue Date 16th September 2021;1-13.

11. ICH guidelines validation of analytical procedures; Text and methodology Q2 R1; 2005.

12. David A Armbruster. Limit of Blank, Limit of Detection and Limit of Quantitation; PubMed; National Library of Medicine; 2008 Aug; 29(Suppl 1): S49–S52.

13. United States Pharmacopeia (USP) 34 (NF 29), Chapter 621, Edition 2011.

14. European Medical Agency withdrawal assessment report of deferiprone; 2020;13-18; "UPKANZ, INN-deferiprone" (europa.eu).

15. Deferiproneimpurity, Pharmaffiliates.

https://www.pharmaffiliates.com/en/118-71-8-deferiprone-impurity-b-pa3177020.html

Received on 24.04.2022 Modified on 28.09.2022

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

DOI: 10.52711/2231-5675.2023.00001