An Isocratic RP-HPLC Method for Simultaneous Analysis of Ilaprazole And Domperidone in Pharmaceutical Formulation

 

Tandel Jinal N.1*, Pumbhadiya Dilipkumar A.1, Chauhan Payal P1, Shah Samir K2

1Department of Quality Assurance, Sardar Patel College of Pharmacy, Gujarat Technological University, Bakrol, Gujarat

2Department of Pharmacology, Sardar Patel College of Pharmacy, Gujarat Technological University, Bakrol, Gujarat

*Corresponding Author E-mail: jinaltandel1202@gmail.com

 

ABSTRACT:

A simple, sensitive and precise high performance liquid chromatographic method for the analysis of Ilaprazole (ILA) and Domperidone (DOM) in their combine dosage form has been developed and validated. The compounds were well separated an isocratically on a C18 Hyperchrom ODS-BP column (250 mm 4.6 mm, 5 µm) utilizing a mobile phase consisting of methanol: phosphate buffer pH 3.5 (35:65 % v/v,) at a flow rate of 1.0 ml/min with UV detection at 281.1 nm. The retention time of ILA and DOM was found to be 3.84 min and 6.55 min. respectively. The proposed method is linear over the concentration ranges 5–15 μg/ml and 15–45 μg/ml for ILA and DOM respectively. Accurate with 99.80-100.09 % recovery for ILA and 99.40-99.77 % recovery for DOM and precise (% RSD < 2 %).The LOD were 3.38 and 4.28 μg/ml and LOQ were 10.24 and 12.99 μg/ml for ILA and DOM respectively. The method has been used to determine potency of commercial product and potency was found within limit. The method is applicable  for the analysis of Ilaprazole and Domperidone in marketed formulation.

 

KEYWORDS: Domperidone, Ilaprazole, Validation, RP-HPLC., LUPILA-D.

 

 


INTRODUCTION:

Ilaprazole is a substituted benzimidazole which is antiulcerous compound known for decreasing gastric acid secretion. This compound, also known as proton pump inhibitor (PPI) is commonly indicated for the treatment of gastric ulcer, peptic ulcer, duodenal ulcers, erosive or ulcerative GERD (Gastro esophageal reflux disease), symptomatic GERD, pathological hypersecretory conditions.

 

The chemical structures of ilaprazole is [-[(4-methoxy-3-methyl-pyridin-2-yl) methylsulfinyl]-6-pyrrol-1-yl-1H. (Figure.1)(1)

 

 

Figure 1- Chemical Structures of Ilaprazole

 

Domperidone,5-chloro-1-[1-[3-(2,3-dihydro-2-oxo-1Hbenzimidazol-1-yl)propyl]-4-piperidinyl]-1, 3-dihydro-2Hbenzimidazol-2-one (Figure 2)(2) , is a potent dopamine antagonist used for treatment of nausea and vomiting . Domperidone does not cross the blood-brain barrier and therefore has fewer adverse CNS effects than other dopamine antagonists.(3)

 

 

Figure 2- Chemical Structures of Domperidone.

 

Literature survey reveals that many analytical methods have been reported for determination of ilaprazole  individually and in combination with other drug (4-6) and for the determination of Domperidone various methods like, UV(7-9), HPLC(10-11) Stability indicting HPLC(12) HPTLC(13) and LC/MS(14) No single method was reported for the combination of these two. So attempt was taken to develop and validate an economic, rapid reversed phase high performance liquid chromatographic method for the quality control of ILA and DOM in pharmaceutical preparations with lower solvent consumption along with the short analytical run time that leads to an environmentally friendly chromatographic procedure and will allow the analysis of a large number of samples in a short period of time. The method was validated and found to be simple, accurate, and          precise. (15)

 

MATERIAL AND METHOD:

Ilaprazole was procured from Lupin Ltd and domperidone was procured from Torrent pharmaceuticals, Gujarat. High Performance Liquid Chromatography (HPLC) grade acetonitrile, methanol and water were obtained from Chemdyes, Rajkot, Gujarat. Potassium dihydrogen phosphate, ortho Phaosphoric acid and methanol of Analytical Reagent (AR) grade were obtained from Chemdyes, Rajkot, Gujarat. Lupila- D capsules were purchased from local market in Gujarat.

 

Chromatographic conditions:

The instrument used was an Analytical Technologies with Rheodyne Injector, 2203 UV-Visible detector and the data recorded using Alchrome A 2000 software. Hyperchrome Octa DecylSilane (ODS) C18, (250mm × 4.6mm, 5µm) was used utilizing a mobile phase consisting of methanol: phosphate buffer (35:65, v/v, pH 3.5) at a flow rate of 1.0 ml/min with UV detection at 281.1 nm.

 

Preparation of standard stock solution:

10 mg and 30 mg of standard ilaprazole (ILA) and domperidone(DOM) was weighed accurately and transferred to two separate 100 ml volumetric flasks respectively. Both the drugs were dissolved in 50 ml of mobile phase with shaking and then volume was made up to the mark with mobile phase to get 100μg/ml and 300μg/ml of standard stock solution of ILA and DOM respectively. Several aliquots of standard solutions of ILA and DOM were taken in different 10 ml volumetric flasks and diluted up to the mark with mobile phase to get five different concentrations 5, 7.5, 10, 12.5, 15μg/ml and 15, 22.5, 30, 37.5,45μg/ml  of ILA and DOM respectively.

 

Preparation of sample solution:

Sample solution containing both the drugs was prepared by dissolving capsule powder into mobile phase. Twenty ILA and DOM capsules were weighed separately. Their average weights were determined. Powder of capsules equivalent to 10 mg of ILA and 30 mg of DOM were weighed and taken in a 100 ml volumetric flask, dissolved in mobile phase and sonicated for about 10 min then filtered through filter paper. An aliquot (1 ml) of filtered solution was further diluted in 10 ml volumetric flask with mobile phase to make the final concentration of working sample solution (10μg/ml ILA and 30μg/ml DOM).

 

Development and validation of HPLC method: Present study was conducted to obtain a new, affordable, cost-effective and convenient method for HPLC determination of ILA and DOM in capsule dosage form. The method was validated for the parameters like system suitability, selectivity, linearity, accuracy, and precision.

 

System suitability:

System suitability study of the method was carried out and various chromatographic parameters such as retention time, peak area tailing factor, theoretical plates of the column and resolution between the peaks were determined and the method was evaluated by analyzing these parameters.

 

Selectivity:

Selectivity test determines the effect of excipients on the assay result. To determine the selectivity of the method, standard sample of ILA and DOM were injected first then commercial product solution were run in the instrument one after another.

 

Linearity:

Linearity of the method was determined by constructing calibration curves. Standard solution of ILA and DOM of different concentration levels 5-15μg/ml and 15- 45μg/ml of ILA and DOM respectively. The peak areas of the chromatograms were plotted against the concentrations to obtain the calibration curves and correlation coefficients.

 

Accuracy:

The accuracy of the method was tested based on % Recovery by the assay of known and added amount of analyte. The recovery experiments were carried out in triplicate by spiking previously analyzed samples of the capsules (ILA 5μg/ml and DOM 15μg/ml) with three different concentrations of standards (ILA 4, 5,6μg/ml and DOM 12,15, 18μg/ml)

 

Precision:

Intra-day precision was determined by performing three repeated analysis of the three standard solutions 5, 10 and 15μg/ml and 15, 30, 45μg/ml of ILA and DOM, respectively on the same day. On the other hand inter-day precision of the method was assessed by carrying out the analysis of standard solutions 5, 10 and 15μg/ml and 15, 30, 45μg/ml of ILA and DOM, respectively on three different days in the same laboratory. The relative standard deviation (% RSD) was calculated.

 

Limit of detection (LOD) and Limit of quantification (LOQ):

The LOD is defined as the smallest level of analyte that gives a measurable response. Limit of quantification LOQ is defined as the lowest concentration at which the precision expressed by relative standard deviation (RSD) is less than 2% and accuracy expressed by relative difference in the measured and true value is also less than 2%.

 

The LOD and LOQ for both ILA and DOM were determined according to ICH guideline Q2B1. The LOD and LOQ were estimated from the standard calibration curve. The residual standard deviation of regression line or standard deviation of y intercepts of regression lines used to calculate LOD and LOQ.

 

Here, LOD=3.3* D/S and LOQ=10*D/S.

Where, D is the standard deviation of y intercept of regression line and S is the slope of calibration curves.

 

RESULTS AND DISCUSSION:

Results of system suitability study of the standard solution showed uniform retention time, theoretical plate count, tailing factor and resolution for both the drugs which indicate a good system for analysis (Table I).

 

TABLE I-Result of system suitability tests of ILA and DOM.

Parameters

ILA

DOM

Retention time (min)

3.86

6.58

Tailing factor

1.40

1.38

Resolution factor

11.05

Theoretical plate

6822

7411

 

The analytical characteristics of the proposed method derived from the calibration curves are as shown (Table II).

TABLE II-Analytical characteristics of the proposed method.

Parameter

ILA

DOM

Linear Range (μg/ml)

5-15

15-45

Mean of Slope

10.08

15.17

S.D. of Intercept

10.3302

19.7149

Limit of Detection (μg/ml)

3.3819

4.2886

Limit of Quantification (μg/ml)

10.2482

12.9960

Regression coefficient (r2)

0.9997

0.9997

 

Chromatograms explain that retention time for standard sample and commercial product of ILA and DOM are same. This proves that, excipients have no effect on the analytical method. On the other hand, blank peak did not overlap drug peak. So the method is highly selective (Figure 3 and 4).

 

A linear relationship between peak areas versus concentrations was observed from 5-15μg/ml and 15-45μg/ml for ILA and DOM respectively. Correlation coefficient was 0.999 for both the drugs which prove that the method is linear (Figures 5 and 6).

 

 

Figure 3-Chromatogram of standard Ilaprazole and Domperidone

 

 

Figure 4-Chromatogram of sample and standard solution of Ilaprazole and Domperidone

 

Figure 5-Calibration curve of Ilaprazole

 

Figure 6-Calibration curve of Domperidone

 

Results of accuracy study were obtained by recovery test. Spiked amount of both the drug were compared against the recovery amount. % Recovery was 99.80-100.09 % for ILA and 99.40-99.77 % for DOM. All the results indicate that the method is highly accurate (Table III).

 

 

 

 


TABLE III-Accuracy (% Recovery) results of ILA and DOM.

Level of % recovery

Amount present

(μg/ml)

Amount of standard drug  added (μg/ml)

Total amount recovered (μg/ml) (n=3)

% Recovery

ILA

DOM

ILA

DOM

ILA

DOM

ILA

DOM

80%

5

15

4

12

4.0037

11.9730

100.0944

99.7751

100%

5

15

5

15

4.9903

14.9327

99.8069

99.5519

120%

5

15

6

18

5.9883

17.8571

99.8065

99.4060

 

 


Intra-day variability was done from 9.00 am to 6.00 pm on the same day. % RSD of peak areas was calculated for various run. The method is highly precise as % RSD of peak area was less than 2 % in all tests (Tables IV and V).


 

TABLE IV-Intraday and inter day precision result of ILA.

Conc. μg/ml

Intra-day Peak Area Mean ± Std. Deviation (n=3)

% RSD

Inter-day Peak Area Mean ± Std. Deviation (n=3)

% RSD

5

481.48466 ± 2.78579

0.57858

479.0683 ± 5.6606

1.18159

10

969.8600 ± 10.0702

1.03831

967.1427 ± 12.17039

1.25838

15

1454.32133 ± 13.3960

0.92111

1452.6520 ± 12.3806

0.85228

 

TABLE V-Intraday and inter day precision result of DOM.

Conc. μg/ml

Intra-day Peak Area Mean ± Std. Deviation (n=3)

% RSD

Inter-day Peak Area Mean ± Std. Deviation (n=3)

% RSD

15

915.3236 ± 10.2098

1.11543

914.2053 ± 9.4772

1.0366

30

1849.7546 ± 18.5217

1.00130

1849.2300 ± 13.9903

0.7565

45

2777.0893 ± 19.1634

0.69005

2765.9240 ± 15.4274

0.5577

The result of assay of was obtained and found to be in range 98%-102%. (Table VI).

 

Table VI- Amount of ILA and DOM in LUPILA-D Capsule by Proposed HPLC Method.

Amount present in (mg/cap)

Amount obtained in (mg/cap) (n=5)

Label Claim %

ILA

DOM

ILA

DOM

ILA

DOM

10

30

9.8829

29.7633

98.9680

99.2367

 


CONCLUSION:

The proposed high-performance liquid chromatographic method has been evaluated for the accuracy, precision and linearity. The measured signals were shown to be precise, accurate and linear over the concentration range tested (5-15μg/ml of ILA and 15-45μg/ml of DOM) with a correlation coefficient of 0.999. In this method, there was no interference from matrix sources. Moreover, the lower solvent consumption along with the short analytical run time of 10 min leads to an environmentally friendly chromatographic procedure that allows the analysis of a large number of samples in a short period of time. Therefore, this HPLC method can be used as a routine sample analysis.

 

ACKNOWLEDGEMENT:

The author is thankful to Lupin Ltd. and Torrent Pharmaceutical for providing gift sample and Sardar Patel College of Pharmacy for providing the laboratory facilities and other requirements.

 

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Received on 16.05.2017          Accepted on 15.09.2017

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2018; 8(1): 01-05.

DOI: 10.5958/2231-5691.2018.00001.1