alexa Simultaneous Determination of Omeprazole, Tinidazole and Clarithromycin in Bulk Powder and Helicure Tablets by HPLC | Open Access Journals
ISSN: 2157-7064
Journal of Chromatography & Separation Techniques
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Simultaneous Determination of Omeprazole, Tinidazole and Clarithromycin in Bulk Powder and Helicure Tablets by HPLC

Hesham Salem1, Safa M Riad2, Mamdouh R Rezk2, Kholoud Ahmed1*

1Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, October University for Modern Sciences and Arts, Egypt

2Analytical Chemistry Department, Faculty of Pharmacy-Cairo University, Kasr El-Aini Street, Egypt

*Corresponding Author:
Kholoud Ahmed
Pharmaceutical Analytical Chemistry Department
Faculty of Pharmacy, October University for Modern Sciences and Arts, Egypt
Tel: +20 1226483727
E-mail: [email protected]

Received date: February 21, 2014; Accepted date: April 22, 2014; Published date: April 24, 2014

Citation: Salem H, Riad SM, Rezk MR, Ahmed K (2014) Simultaneous Determination of Omeprazole, Tinidazole and Clarithromycin in Bulk Powder and Helicure® Tablets by HPLC. J Chromatograph Separat Techniq 5:221. doi:10.4172/2157-7064.1000221

Copyright: © 2014 Salem H, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Sensitive and precise chromatographic method was developed and validated for simultaneous determination of omeprazole (OMP), tinidazole (TND) and clarithromycin (CLR) in bulk powder, laboratory prepared mixture and pharmaceutical preparation. The technique adopted for quantification is HPLC. A mixture of acetonitrile, methanol, phosphate buffer at pH 3.5 (33: 17: 50, v/v/v) was used as a mobile phase. The stationary phase used was (150 mm×4.6 mm, 10μm) C8 LichrosorbTM analytical column. The method was linear in the range of 0.2-250 μg mL-1, 0.5-250 μg mL-1 and 75-2000 μg mL-1 for OMP, TND and CLR respectively. The selectivity of the proposed method was checked using laboratory prepared mixtures. The proposed method was successfully applied to the analysis of OMP, TND and CLR in their mixture and in pharmaceutical dosage form without interference from other additives

Keywords

Omeprazole; Tinidazole; Clarithromycin; HPLC.

Introduction

Omeprazole (OMP), is 6-methoxy-2-[[(4-methoxy-3,5-dimethyl- 2-pyridinyl) methyl] sulphinyl]-1H-benzimidazole [1], (Figure 1). It is the first member of the “proton pump inhibitors” that are widely used for the prophylaxis and treatment of both gastroduodenal ulcers and symptomatic gastro-esophageal reflux. It is highly effective in the treatment of Zollinger-Ellison syndrome [2]. Tinidazole (TND) is 1-[2-(ethyl sulphonyl) ethyl]-2-methyl-5- nitro-1H-imidazole, [1] (Figure 2). It is used as antiprotozoal agent. Clarithromycin (CLR), is (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)- 4-[(2,6-Dideoxy-3-C-methyl-3-O-methyl-a-L-ribohexopyranosyl) oxy]-14-ethyl-12,13-dihydroxy-7-methoxy-3,5,7,9,11,13-hexamethyl- 6-[[3,4,6-trideoxy-3-(dimethylamino)-b-D-xylo hexopyranosyl] oxy] oxacyclotetradecane-2,10-dione (6-O-methylerythromycin A), [1] (Figure 3). CLR is semi-synthetic macrolide antibacterial agent [1].

chromatography-separation-techniques-structure-Omeprazole

Figure 1: Chemical structure of Omeprazole (OMP).

chromatography-separation-techniques-structure-Tinidazole

Figure 2: Chemical structure of Tinidazole (TND).

chromatography-separation-techniques-structure-Clarithromycin

Figure 3: Chemical structure of Clarithromycin (CLR).

The literature survey reveals several analytical methods for quantitative estimation of OMP alone in body fluids and in pharmaceutical formulations these methods include spectrophotometry [3-14], electrochemical methods [15], HPLC [16-21], liquid chromatography-electrospray ionization tandem mass spectrometry [22] and electrophoresis [23]. Tinidazole was estimated in body fluids and in pharmaceutical formulations by spectrophotometry [24-29], potentiometry [29], HPLC methods [29-31], polarography [32,33] and resonance light scattering technique [34]. Clarithromycin has been reported to be estimated in body fluids and in pharmaceutical formulations by spectrophotometry [35], HPLC methods [36-44]. Omeprazole, Tinidazole and Clarithromycin were simultaneously determined by spectrophotometry [45,46].

Up to our knowledge, there is no isocratic HPLC method was described for the simultaneous determination of the three studied drugs in their laboratory prepared mixtures and in the pharmaceutical dosage form without prior derivatisation. The present work aimed to develop an isocratic HPLC method for simultaneous determination of OMP, TND and CLR in laboratory prepared mixtures and pharmaceutical dosage form. The proposed method has advantage of being cheap, simple, rapid and time saving (one run in less than 7 minutes).

Experimental

Instruments

A liquid chromatograph consisted of an quaternary pump (Agilent model G1316 A/G1316 B), a diode array multiple wavelength detector (model G1316 C/D and G1365C/D, Agilent 1200 Series), Standard and preparation autosamplers (Agilent 1200 series) equipped vacuum degasser, Agilent. Stationary phase (150 mm×4.6 mm, 10 μm) C8 Lichrosorb TM analytical column. Mobile phase; acetonitrile, methanol, buffer at pH 3.5 (33: 17: 50, v/v/v). The mobile phase was filtered through a 0.45 μm Millipore membrane filter and was degassed for 15 min in an ultrasonic bath prior to use. UV-detection was done at 210 nm. The samples were filtered also through a 0.45 μm membrane filter.

Standards, solvents, and pharmaceutical preparation

Reference omeprazole (OMP), reference tinidazole (TND) and reference clarithromycin (CLR) were kindly donated by EGYPHAR Pharmaceuticals Co. The potency was found to be 100.30%, 100.13% and 100.16% for OMP, TND and CLR, respectively. Pharmaceutical dosage form (Heli-cure tablets were kindly supplied by EGYPHAR and were claimed to contain 20 mg of OMP, 500 mg TND and 250 mg of CLR per tablet. Acetonitrile, methanol (HPLC grade) and phosphate buffer adjusted to pH 3.5.

Standard solutions

OMP, TND standard solutions (each 0.5 mg mL-1) and CLR standard solution (2 mg mL-1) were prepared in mobile phase for the suggested HPLC method. The standards solutions were freshly prepared on the day of analysis and stored in a refrigerator to be used within 24 hr.

Procedures

Linearity: Portions of OMP, TND standard solutions (each 0.5 mg mL-1) and CLR standard solution (2 mg mL-1) were transferred separately into a series of 10-mL volumetric flasks and completed with mobile phase. Several dilutions were done and the content of each flask was completed to volume with the mobile phase to get the concentrations of 0.2-250 μg mL-1 OMP, 0.5-250 μg mL-1 TND and 75- 2000 μg mL-1 CLR. The samples were then chromatographed using the following chromatographic condition. Stationary phase (150 mm×4.6 mm,10 μm) C8 lichrosorbTM. Many mobile phases such as methanol and acetonitrile (50:50, 60:40, 65:35, by volume), methanol, acetonitrile and phosphate buffer adjusted at pH 3.5 (30: 20:50, 40: 30: 30) by volume and different other ratios but the mobile phase which give the best separation and peaks shape was found to be a mixture of acetonitrile, methanol, buffer at pH 3.5 (33: 17: 50, v/v/v). The mobile phase was filtered through a 0.45 μm millipore membrane filter and was degassed for about 15 min in an ultrasonic bath prior to use, flow rate; 0.7 mL min-1 [isocratically at temperature (35°C)], with UV-detection at 210 nm, the detection wavelength was set regarding the UV absorption spectra of the drugs (Figure 5) and their relative concentrations within the pharmaceutical formulation. Whereas TNZ is nominally 2 and 25 times more concentrated than CLR and OMP, respectively. The drugs have strong contributions in the overall UV region (200-375 nm). This is why an optimum detection wavelength was set at 210 nm during the chromatographic separation, favoring the quantification of both CLR and OMP, which represent the less concentrated components of this ternary mixture. In addition, this chosen detection wavelength can greatly improve the sensitivity of the proposed method for the CLR determination because it exhibits absorption maxima (at 210 nm). The samples were filtered also through a 0.45 μm membrane filter. To reach good equilibrium, the analysis was usually performed after passing 50- 60 mL of the mobile phase, just for conditioning and pre-washing of the stationary phase. The relative peak area ratios were then plotted versus the corresponding concentrations of OMP, TND and CLR to get the calibration graphs and to compute the corresponding regression equations.

chromatography-separation-techniques-Absorption-spectra

Figure 5: Absorption spectra of OMP (2 μg/mL) ( —), TND (50 μg/mL ) (-.-.-) and CLR (25 μg/mL) (…..) &( 250 μg/mL ) (- - - -) in ethanol.

Analysis of laboratory prepared mixtures containing different ratios of OMP, TND and CLR: Aliquots of each standard solution were mixed to prepare different mixtures containing different ratios (3: 4:90, 1:0.2:26, 7:4:130, 2:0.2:23, 0.5:50:336, 30:12.5:168, 6:2.5:124, 0.5:1:144) of OMP, TND and CLR, respectively. The concentrations were calculated from the corresponding regression equations.

Assay of pharmaceutical formulations (Heli-cure tablets): Twenty tablets were powdered well and homogeneously mixed in a morter. A mass of the powdered tablets equivalent to 20 mg of OMP, 250 mg of CLR and 500 mg of TND was weighed and transferred to a 100-ml volumetric flask. The powder was extracted by shaking with 3×30 mL mobile phase with vigorous shaking for 15 minutes then filtered. The volume was completed to the mark with the mobile phase. Several portions 0.5-2 mL of aliquot were transferred separately to 10- mL volumetric flasks, the volumes were completed to the mark with mobile phase and chromatographed under the previous mentioned conditions.

Results and Discussion

High-performance liquid chromatography

A simple isocratic high-performance liquid chromatographic method was developed for the determination of OMP, TND and CLR in pure form and in pharmaceutical preparation using (150 mm×4.6 mm, 10 μm) C8 lichrosorbTM analytical column. The mobile phase consisted of acetonitrile, methanol, buffer at pH 3.5 (33: 17: 50, v/v/v). The mobile phase was chosen after several trials to reach the optimum stationary/mobile-phase matching. The average retention times under the conditions described are 2.06 min for OMP, 1.36 min for TND and 5.44 for CLR (Figure 4). One sample can be chromatographed in less than 6 min.

chromatography-separation-techniques-Separation-Omeprazole

Figure 4: Separation of Omeprazole (tR value=2.06) from Tinidazole (tR value=1.36) from Clarithromycin (tR value=5.44) upon using acetonitrile, methanol and buffer pH 3.5 (33: 17 : 50, v/v/v).

Peak purity was confirmed for the HPLC peaks of OMP, TND and CLR by a pilot run using a photodiode array detector. Calibration graph was obtained by plotting the relative peak area ratios against concentrations. Linearity range was found to be 0.2-250 μg mL-1 for OMP, 0.5-250 μg mL-1 TND and 75-2000 μg mL-1 CLR. The regression equation for OMP: A=0.1832C+0.1946 (r=0.9999), for TND: A=0.0241C+0.0513 (r=0.9999) and for CLR: A=0.0021C+0.0280 (r=0.9999) where A is the relative peak area ratio, C is the concentration in μg mL-1 and r is the correlation coefficient. The mean percentage recovery was found to be 100.08 ± 0.454 for OMP, 100.40 ± 0.535 for TND and 100.65 ± 0.862 for CLR (Tables 1 and 2).

Parameter OMP TND CLR
Range (µg mL-1) 0.2-250 0.5-250 2000-75
Slope 0.183 0.024 0.002
Intercept 0.195 0.051 0.028
Variance 0.206 0.286 0.743
Coefficient of variation 0.454 0.535 0.862
Correlation coefficient (r) 1 1 1
Accuracy
mean
RSD%
100.08
0.454
100.40
0.535
100.65
0.862
Precision (RSD%
Repeatability
Intermediate precision
0.201
0.332
  0.184
0.409
  0.237
0.294
Specificity
mean
RSD%
99.96
0.407
100.14
0.332
  99.97
0.216

Table 1: Validation and regression parameters for the determination of OMP, TND & CLR by the proposed HPLC method.

  OMP TND CLR
OMP :TND: CLR Taken
ug mL-1
Found
ug mL-1
R (%)   Taken
ug mL-1
Found
ug mL-1
R (%)   Taken
ug mL-1
Found
ug mL-1
R (%)  
3  : 4  : 90 30 29.75 99.17 40 40.15 100.38 900 900.14 100.02
0.5 :  1 : 144 2.5 2.503 100.12 5 4.99 9980 720 721.33 100.18
1 :  0.2  : 26 50 49.92 99.84 10 10.02 100.20 1300 1299.05 99.93
7  : 4  :  130 7 6.98 99.71 4 3.98 99.50 130 130.1 100.08
0.5  :  50 : 336 0.5 0.502 100.40 50 50.11 100.22 336 334.82 99.65
30  :  12.5 : 168 60 60.23 100.38 25 25.09 100.36 336 336.71 100.21
6  : 2.5 : 124 30 29.96 99.87 12.5 12.52 100.16 620 620.14 100.02
2 : 0.2 :  23 20 20.04 100.20 2 2.01 100.50 230 229.19 99.65
Mean ± SD 99.96± 0.407 100.14± 0.332 99.968  ± 0.216

Table 2: Determination of OMP, TND and CLR in laboratory prepared mixtures containing different ratios (3: 4: 90, 0.5: 1: 144, 1: 0.2: 26, 7: 4: 130, 0.5: 50: 336, 30: 12.5: 168, 6: 2.5: 124, 2:0.2:23 ) of OMP, TND and CLR, respectively by the proposed method.

Analysis of laboratory prepared mixtures containing different ratios of OMP, TND and CLR

The suggested HPLC method was successfully applied for the determination of the studied drugs in their laboratory prepared mixtures. The precision of the proposed method was checked by the analysis of different concentrations (Table 2).

The mean percentage recovery was found to be:

99.96 ± 0.407 for OMP

100.14 ± 0.332 for TND

99.97 ± 0.216 for CLR.

Analysis of dosage form (Heli-cure tablets)

The suggested HPLC method was successfully applied for the determination of the studied drugs in their pharmaceutical formulation which is Heli-cure tablets. The precision of the proposed method was checked by the analysis of different concentrations (Table 3).The mean percentage recovery was found to be:

OMP TND CLR
Taken
ug mL-1
Found
ug mL-1
R (%) Taken
ug mL-1
Found
ug mL-1
R (%)   Taken
ug mL-1  
Found
ug mL-1
R (%)  
10 10.03 100.30 4 4.01 100.25 125 124.67 99.74
15 14.99 99.93 5 4.98 99.60 250 250.71 100.28
20 20.09 100.45 30 30.05 100.17 500 501.33 100.27
100.227± 0.268 100.01± 0.354 100.097  ± 0.216

Table 3: Determination of OMP, TND and CLR in Helicure® tablets by the proposed method

100.23 ± 0.268 for OMP

100.01 ± 0.354 for TND

100.097 ± 0.216 for CLR.

Conclusion

Validation of the accuracy of the proposed HPLC method was confirmed using standard addition technique (Table 4). Statistical comparison with the official and reported methods showed that the proposed HPLC is sensitive and precise (Table 5 and 6). Application of the proposed methods to the analysis of OMP, TND and CLR in their pharmaceutical formulation (Table 3) shows that excipients do not interfere with the determination. The system suitability parameters of the proposed HPLC method (Table 7). The proposed method has advantage of being sensitive and applicable over wide range. The proposed method can be used for routine analysis of omeprazole, tinidazole and clarithromycin in quality control laboratories.

OMP
Recovery (%) Authentic found (ug mL-1) Authentic added (ug mL-1) Claimed amount taken (ug mL-1)
00.55 20.11 20.00 40.00
00.28 40.11 40.00
00.23 60.14 60
00.35±0.172 Mean ± SD
TND

Recovery (%)

Authentic found (ug mL-1) Authentic added (ug mL-1) Claimed amount taken (ug mL-1)
00.40 0.04 0.00 25.00
99.76 24.94 25.00
99.90 49.95 50.00
00.02±0.336 Mean ± SD
CLR

Recovery (%)

Authentic found (ug mL-1) Authentic added (ug mL-1) Claimed amount taken (ug mL-1)
99.38 987.62 2000.00 000.00
99.95 499.23 500.00
99.97 3998.60 4000.00
99.78±0.335 Mean ± SD

Table 4: Application of the standard addition technique to the proposed HPLC method of OMP, TND & CLR in their pharmaceutical formulation.

Parameters HPLC B.P official  method2 OMP B.P official method2 TND B.P official method2 CLR
OMP TND CLR
Mean± S.D 100.076 100.403 100.647 100.30 100.13 100.16
0.454 0.535 0.862 0.401 0.722 0.531
Variance 0.206 0.286 0.743 0.161 0.521 0.282
F-test 1.28 (4.95)a 1.82 (4.95)a 2.63 (5.05)a      
Student,s t-test 0.723 (2.201)a 0.763 (2.201)a 1.176 (2.23)a      
N 7 7 6 6 6 6

Table 5: Statistical comparison for the results obtained by the proposed method and the official method for analysis of OMP, TND and CLR.

Parameters   HPLC   Reported  method48 OMP Reported  method48 TND Reported  method48 CLR
OMP TND CLR
Mean ± S.D 100.227 100.01 100.097 100.20 100.12 100.12
0.268 0.354 0.216 0.283 0.167 0.281
Variance 0.072 0.125 0.047 0.080 0.028 0.079
F-test 1.14 (5.79)a 4.46 (5.79)a 1.68 (5.79)a      
Student,s t-test 0.13 (2.57)a 0.497 (2.57)a 0.123 (2.57)a      
N 3 3 3 4 4 4

Table 6: Statistical comparison for the results obtained by the proposed HPLC method and the reported TLC method for analysis of OMP, TND and CLR in dosage form.

Parameter OMP TND CLR Limit (49,50)
Retention time (tR) 2.063 1.353 5.443  
Resolution (RS) 3.95 R S> 2
Tailing factor (T) 0.80 0.88 0.40 T = 1 for a typical symmetric peak
Capacity factor  (K’) 1.58 1.48 5.06 1 -10 acceptable
Selectivity factor (α) 3.27 α> 1
Column efficiency (N) 9811 6991 2451 N > 2000
Height equivalent to theoretical plate (HETP) 0.003 0.004 0.010 The smaller the value, the higher the column efficiency

Table 7: System suitability parameters of the proposed HPLC method.

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