RP-HPLC Method Development for Simultaneous Estimation of Oral Iron Chelator Deferiprone and its related Impurity

Shubhangi Sutar¹*, Puja Patil¹, Diptee Marchande¹, Sachinkumar Patil¹,

Sandip Bandgar¹, Ravindra Kumbhar²

¹Ashokrao Mane College of Pharmacy, Peth – Vadgaon.

²New College of Pharmacy, Kolhapur.

*Corresponding Author E-mail: shubhangi.sutar28@gmail.com

ABSTRACT:

Purpose: This study aims to develop and validate a RP-HPLC method for simultaneous estimation of deferiprone and its synthesized impurity. Method: Structure of impurity was confirmed by spectral analysis while their purity was confirmed by HPLC analysis. The chromatographic separation was achieved on Agilent Zorbax Bonus-RP (250 x 4.6mm, 5µ) using mobile phase of 20 ACN-80 0.1% TFA. Detection was carried out at 280 nm using PDA detector with flow rate of 1 ml/min and temperature of 30℃. Result: The method was validated as per ICH guidelines. The percent recovery was found to be within limit and %RSD for deferiprone and the impurity was found to be 0.09 and 0.48. The standard curve was linear over the concentration range of 60-140µg/ml and shows correlation coefficient (r2) of 1. The LOD and LOQ values for deferiprone were found to be 0.21µg/ml and 0.62µg/ml respectively. The method was found to be robust even by change in flow rate of ±0.2ml/min. Conclusion: Validation studies demonstrated that the proposed HPLC method is specific, reliable, rapid and reproducible. The developed method can be successfully employed for routine analysis for deferiprone in pharmaceutical dosage form.

KEYWORDS: Simultaneous Estimation, RP-HPLC, Deferiprone, Impurity, Validation.

1. INTRODUCTION:

Analytical system plays the significant role in identification of physical as well as chemical assets of the formulations¹. In current age, marketplace is flooded with several dosage forms. The present multi-component preparations due to patient suitability, increased effectiveness, multiple act and faster relief are gaining attention.^2,3. HPLC is the accurate analytical method use for quantitative and also for qualitative study of drug product and its stability⁴. Analytical method is significant part in the growth of pharmaceuticals. The goal line behind HPLC is to split and quantify the active drug, all synthetic material, impurities and degradants^5,6.

The most significant profit gain from the usage of HPLC method is that it helps in structure elucidation and quantitative determination of impurities⁷.

The simultaneous systematic analysis offers specificity plus assurance designed for the recognition of chemical entities present in pharmaceutical formulation⁸. Oral iron chelators are chief expansion that offering a possibility to recover compliance and patients' value of life. One basic clinical feature of iron chelator is the grade to which they are being absorbed from gastrointestinal tract.^9-10. The goal behind this study is to develop HPLC practice for simultaneous analysis among drug and its synthesized impurity¹¹. This could possibly improve the efficiency of the analysis and decrease laboratory supply costs¹². Chemical structure of deferiprone shown in Fig No. 1.Deferiprone drug is an oral iron chelator, basically used as second line agent in thalassemia disorder¹³. Basically thalassemias are form of genetic anemia which is generally due to deficiency in hemoglobin production.A literature study reveals that various techniques are reported for individual drug determination as UV, HPLC and LC-MS techniques, but there are no studies on simultaneous determination for drug along with its impurity by HPLC technique¹⁴.

Fig. 1 Structure of Deferiprone

2. MATERIAL AND METHODS:

2.1 Instruments and Chemicals:

IR Spectra was recorded on Bruker Agilent Cary 630; 1H-NMR spectra was recorded on Bruker Switzerland, Avance 300 Top Spin and their chemical shifts are reported in ppm units. The mass spectrum was recorded on Agilent 1200 series (Agilent Technologies, USA). Chromatography was performed on Agilent 1260 Infinity II with PDA detector. The separation was achieved using Agilent Zorbax Bonus-RP (250 x 4.6 mm, 5µ) with mobile phase of acetonitrile: 0.1% TFA (20:80) maintained at temperature of 30℃ and flow rate was 1ml/min. An analytically pure sample of Deferiprone was purchased from yarrow Chem Products, Mumbai.

2.2 Synthesis scheme for targeted impurity of deferiprone:

t- Butyl hypochlorite (12.96g, 0.12mol) was gradually added to solution of 1-(2-furyl) ethanol (4.48g, 0.04mol) in 35% aq. acetic acid (40ml) at -5- to -0^oC with stirring. Mixture was warmed at 25°C. Then after stirring the mixture at 25°C for 2 hrs, it was heated at 90°C for 4 hrs. Later solution was neutralized and also extracted with CHCl3.

2.3 Liquid Chromatographic Conditions:

Chromatographic separations were obtained by using mobile phase containing acetonitrile and 0.1% TFA water in the ratio of 20:80% (v/v) at a flow rate of 1ml/min through Agilent Zorbax Bonus-RP (250 x 4.6 mm, 5µ).

2.4 Method Development for RP-HPLC:

2.4.1 Preparation of Mobile Phase:

In 1000ml of water, place 1ml of Trifluroacetic acid through 0.45µ membrane filter, and then mix 800ml of 0.1% TFA water with 200ml of acetonitrile.

2.4.2 Preparation of standard solution:

a. Deferiprone Standard Stock Solution-I (DSSS-I)

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

b. Impurity Standard Stock Solution-I (ISSS-I)

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

c. Impurity Standard Stock Solution-II (ISSS-II)

Pipette out 1.0ml of ISSS-I in the 10ml volumetric flask and add 5ml diluent, mix for 2 minutes and make the volume to 10ml with diluent. (Conc. of impurity = 50µg/ml).

d. Mixture of DSSS-I and ISSS-II

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

2.4.3 Preparation of sample solution:

i. 10 Capsule were taken and Capsule content were weighed and average weight was calculated. And capsule content was crushed and mixed.

ii. 5mg Deferiprone was weighed in 10ml of volumetric flask and add 5ml of diluent and make the volume to 10ml with the diluent. Then Pipette out 1.0ml in the 10ml volumetric flaskand add 5ml diluents. Sonicate for 10 minutes and make the volume to 10ml with diluent. (Conc. of Deferiprone = 50µg/ml).

2.5 Validation of Proposed Method:

The proposed method was validated according to the ICH guidelines.

2.5.1 Specificity:

Solutions of standard and sample were prepared according to test method into chromatographic system.

2.5.2 Assay:

i. 10 Capsule were taken and Capsule content were weighed and average weight was calculated. And capsule content was crushed and mixed.

ii. 5mg Deferiprone was weighed in 10ml of volumetric flask and add 5ml of diluent and make the volume to 10ml with the diluent. Then Pipette out 1.0ml in the 10ml volumetric flask and add 5ml diluents. Sonicate for 10 minutes and make the volume to 10ml with diluent. (Conc. of Deferiprone = 50µg/ml).

2.5.3 Precision:

Add 1.0ml of DSSS-I and 1.0ml ISSS-II in 10ml volumetric flask and add 5ml diluent and vortex and make up the volume with diluent. (Conc. of Deferiprone =50µg/ml andImpurity = 5µg/ml).

2.5.4 System suitability:

A single sample was prepared and 5 injections were made from same sample and tested for system suitability.

2.5.5 Linearity:

i. 5 samples of varying concentrations ranging from 60-140% were made. Correlation coefficient should be not less than 0.9999% of y-intercept should be ±2.0. The concentrations range for deferiprone and impurity 3-7ug/ml respectively.

2.5.6 Accuracy:

i. Samples were prepared of 80%, 100% and 120% concentration by spiking the same amount of concentration given above for both Deferiprone and Impurity.

ii. Samples were injected in duplicate to calculate % RSD. iii. The mean % recovery of the deferiprone at each spike level should be NLT 98.0% and not more than 102.0%.

2.5.7 Robustness:

Standard was introduced into HPLC using flow rate of 0.8, 1.0 and 1.2ml/min.

2.5.8 Limit of Detection (LOD) and Limit of Quantitation (LOQ):

Calculate LOD and LOQ for deferiprone and Impurity based on standard deviation and slope of calibration Curve

3. RESULTS AND DISCUSSION:

3.1 Characterization of synthesized impurity:

Melting point of synthesized impurity was found to be 161 - 162 °C and Molecular Weight 126.11 g/mol.

IR(cm^-1) -1767, 3343, 1686, 1H-NMR -2.3(s,CH₃),6.4(d,CH), 7.9(d,CH),4.9(s,OH)

13C-NMR- 14.58(R-CH₃), 142(C=C), 114(C=C),175.81(C=O),155(C=C),155(C=C), MASS m/z -127.04, 149.02

The related impurity of deferiprone were synthesized and its structure shown in fig. 2.

Fig. 2 - Different impurities: a) IR Spectra for impurity b) H NMR spectra for impurity c)13 C NMR spectra for impurity d)LC-MS spectra for impurity

3.2 Method Development:

The separation of analyte was carried out on Agilent Zorbax Bonus-RP (250 x 4.6 mm, 5µ) column using 20 ACN-80 0.1% TFA as mobile phase and runtime of method was set at 08min. The detection of column effluent was monitored at a wavelength of 280 nm. The maximum absorption for drug was shown in fig. 3.

3.3 Validation of proposed method:

3.3.1 Specificity:

Specificity data is given in Table No: 2.

Table No: 2. Specificity data

Sample	Deferiprone	Maltol
	RT	RT
Deferiprone Std	2.33	-
Maltol Std	-	4.15
Mixture WS	2.33	4.15
Drug Product	2.34	4.15

3.3.2Assay:

The Assay of deferiprone was found to be 98.83%.

3.3.3 Precision:

The % relative standard deviation of individual deferiprone and maltol from the five units found to be 0.09 and 0.48. Test results are showing that the test method is Precise. The data is given below in Table No: 3.

Table No: 3. Precision data for Deferiprone and Maltol

	Deferiprone	Maltol
Reps	Area	Area
Rep 1	3155416	405614
Rep 2	3162367	410491
Rep 3	3160174	410024
Rep 4	3161998	409636
Rep 5	3161579	409264
Avg	3160307	409006
STDEV	2857.474182	1950.043384
RSD	0.09	0.48

3.3.4 System Suitability:

i. The %RSD for retention time of principal peak from five replicate injections of standard found NMT 2.0%.

ii. The %RSD for the peak area responses of principal peak from 5 replicate injection of each standard found NMT 2.0%.

iii. The number of theoretical plates peaks found NLT 2000. System suitability data is given in below in table no:4

Table No: 4. System Suitability Data for Deferiprone

Injection	RT		Asymmetry		Theoretical Plates		Resolution
	Deferiprone	Maltol	Deferiprone	Maltol	Deferiprone	Maltol	Deferiprone	Maltol
1	2.33	4.15	1.23	1.40	7138	11670	0.00	13.74
2	2.33	4.15	1.21	1.40	7191	11635	0.00	13.75
3	2.33	4.15	1.15	1.37	7198	11644	0.00	13.75
4	2.34	4.15	1.12	1.44	7119	11582	0.00	13.63
5	2.33	4.15	1.19	1.39	7179	11584	0.00	13.72
Avg	2.33	4.15
STDEV	0.004472136	0
RSD	0.19	0.00

Table no: 5 . Accuracy Data for Deferiprone and Maltol

% Level	Reps	Spiked Conc (ug/ml)	Area	Amount Recovered (ug/ml)	% Recovery	AVG	STDEV	RSD
80%	Rep 1	39.88	2521973	39.78	99.75	99.88	0.17910887	0.18
	Rep 2	39.88	2528377	39.88	100.01
100%	Rep 1	49.85	3155416	49.77	99.85	99.96	0.155526015	0.16
	Rep 2	49.85	3162367	49.88	100.07
120%	Rep 1	59.82	3793491	59.84	100.03	100.03	0.002908701	0.00
	Rep 2	59.82	3793647	59.84	100.03
Accuracy Data for Maltol
% Level	Reps	Spiked Conc (ug/ml)	Area	Amount Recovered (ug/ml)	% Recovery	AVG	STDEV	RSD
80%	Rep 1	3.99	324524	3.96	99.18	99.35	0.236635375	0.24
	Rep 2	3.99	325619	3.97	99.52
100%	Rep 1	4.99	405614	4.94	99.17	99.77	0.843156692	0.85
	Rep 2	4.99	410491	5.00	100.36
120%	Rep 1	5.98	490446	5.98	99.93	99.97	0.060221362	0.06
	Rep 2	5.98	490864	5.98	100.01

The mean % recovery of deferiprone and maltol at every spike level found NLT than 98.0 and NMT 102.0%.

Table no: 6. Robustness Data for Deferiprone and Maltol

Flow Rate (ml)	RT		Tailing factor		Standard Area
Flow Rate (ml)	Deferiprone	Maltol	Deferiprone	Maltol	Deferiprone	Maltol
0.8	2.33	4.15	1.23	1.41	3155416	405614
1.0	2.33	4.15	1.21	1.40	3162367	410491
1.2	2.34	4.15	1.12	1.37	3161998	410024
Avg	2.333333333	4.15			3159927	408709.6667
STDEV	0.005773503	0			39610.994886	2619.075312
RSD	0.24	0.00			0.12	0.65

The % RSD of retention time was found to be within limits for variation in flow rate.

3.3.7 Robustness:

Robustness data is given below in the table no. 6.

3.3.8 Limit of detection and limit of quantitation:

Limit of Detection and limit of Quantitation for Deferiprone were found to be 0.21 and 0.62µg/ml and for Maltol it is 0.12 and 0.36µg/ml.

4. CONCLUSION:

RP-HPLC method development was carried out using synthesized impurity and deferiprone drug. Characterization of synthesized impurity was done to assure the desired quality attributes. The results of method are well within the limit for quality control environment and also indicate that the method can be employed in routine quality analysis for estimation of titled drug in pharmaceutical dosage form. The method has been reported for RP-HPLC quantitation of synthesized impurity and drug Deferiprone. The reported method is simple, accurate and precise for estimation of deferiprone impurity and drug.

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Received on 29.11.2021 Modified on 10.03.2022

Research J. Pharm. and Tech 2023; 16(4):1890-1894.

DOI: 10.52711/0974-360X.2023.00310