Reverse-FIA Technique for the Determination of Omeprazole Using Chemiluminescence Detection Rebwar O. Hassan Chemistry Department-College of science – University of Salahaddin-Erbil-Iraq Email:
[email protected]
Abstract Simple, accurate, sensitive and reliable reverse flow injection analysis (rFIA) technique with chemiluminescence detection was applied for the determination of omeprazole in pharmaceutical products. This rFIA system was based on the inhibition effect of omeprazole on the generated chemiluminescence light from the in situ peroxynitrite (NO2 and H2O2 in acidic medium) and luminol system, when the latter was injected as a reagent. All analytical parameters participated in the determination procedure were evaluated statistically to assess the applications of the methods. Calibration curve was constructed between different omeprazole concentrations (µg/mL) versus the differences in CL-intensity in the form of the peak height (ΔCL = CL1 (Blank) - CL2 (omeprazole)). The calibration curve was linear in concentration range of 3.0-15.0 µg/mL (R2= 0.9987 and DL= 1.30 µg/mL). The method shows good selectivity as no serious effects of interferences were observed. Applications of the present method for the determination of omeprazole in pharmaceutical products were performed and the results show good agreement with that obtained from HPLC technique. Keywords: Omeprazole determination, Chemiluminescence, Reverse Flow injection, Pharmaceutical products, Peroxynitrite.
1
1. Introduction
Omeprazole
(OMP),
chemically
(5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-
yl)methylsulfinyl)-1H-benzimidazole (Table 1), is one of the most world wide consumed proton pump inhibitor employed as an antiulcer drug and against other acid related diseases (Bosch 2007, p. 831). The drug was used in conditions where the inhibition of gastric acid secretion may be beneficial, including aspiration syndromes, dyspepsia, gastro-esophageal reflux disease, peptic ulcer disease, and the Zollinger–Ellison syndrome (Brittain 2010, p.153). A detailed survey in literature reveals a variety of analytical methods used for determination of OMP in pharmaceutical formulations and body fluids include spectrophotometry (Salama et al. 2003, p. 411 & Sharma and Sharma 2012), electrophoresis (Nevado et al. 2014, p. 211), Liquid chromatography–mass spectroscopy (LC-MS) (Zhang et al. 2010, p. 1169 & Boix et al. 2014, p. 706), electrochemical (Cavalcanti 2013, p. 1803), polarography (Qaisi, Tutunji and Tutunji 2006, p. 384 & ELEnay, Belal and Rizk 2008, p.889 ), HPLC (Rezk, Brown and Kashuba 2006, p. 314 & Nataraj et al. 2012, p. 366) and FIA-CL (Shu-hua 2007, p.51 & Yun and Yin-huan, 2010, p. 378). A reverse flow injection analysis (rFIA) technique was adopted successfully to eliminate background absorption from sample matrix as well as to simplify the flow system (Liu and Feng 1998, p.47 &Jing-fu and Gui-bin 2001, p.329). In case of continuous signal forming, rFIA approach was employed in order to avoid their effect on the baseline noise (Hassan and Faizullah 2011,p. 373). The aim of the present work was to develop a simple, accurate and fast rFIA method with chemiluminescence detection used for the determination of OMP in different pharmaceutical formulations. In this method, a decrease in the chemiluminescence light intensity generated from nitrous acid, H2O2 and luminol in basic medium in the presence of OMP was used as a base for the determination of OMP.
2
2. Experimental 2.1. Apparatus The schematic design of rFIA-system used in this work was illustrated in Fig.1. A peristaltic pump (DESAGA Heidelberg-England, with 6-channels) provided with silicon pump tubes (0.8 mm i.d.) used to deliver four flow streams. The reagent was injected into the flowing stream via a six-way injection valve (Rheodyne-USA) supplied with variable loops volumes. A PTFE tubes with homemade T- pieces and mixing coils (60 cm, 1.0 mm i.d.). A home-made flow cell used in the present study was made by winding a length of a glass tubing (0.8mm i.d.) to form a coil with 60 μL volume, in a way that the reagent and sample are mixed exactly at the entrance of the cell. The detection house of a spectrophotometer was completely light tight during the measurement. The flow cell placed in the front of the optical window of the detector (spectrophotometer, Cecil CE303) the light source of which was blocked. 2.2. Reagents and samples All chemicals used of analytical grade reagent unless otherwise stated. Reagent Luminol stock solution 5.0×10-3 M solution was prepared by dissolving 0.886 g of the luminol (Surechem-LTD) in 1.0 L of 0.12 M sodium carbonate ((Fluka)) solution. Other solutions were prepared by serial dilutions of the stock solution with 0.12 M sodium carbonate solution. Hydrogen peroxide stock solution (GCC, 3.0 % (v/v)) 0.1 M was freshly prepared by diluting 10.2 mL of H2O2 to 100 mL in a volumetric flask. A 0.225 M HCl stock solution was prepared by appropriate dilution of concentrated acid (BDH, 37 % (w/w)). Sodium nitrite solution (0.1 M) was prepared by dissolving 0.69 g in 100 mL distilled water. Standard stock Solution The standard stock solution (1000 µg/mL) of OMP, provided from Samarra Drug Industries-Iraq (SDI), was prepared by dissolving 0.1 g standard OMP in 10.0 mL ethanol 3
and diluted to 100 mL with distilled water. Other solutions were prepared by serial dilutions of the stock solution with distilled water. Sample solutions A contains of ten capsules were poured and homogeneously mixed. Accurately a portion equal to 10 mg of OMP was weighed and dissolved in 10.0 mL ethanol then stirred by using an ultrasonic bath until the material is either dissolved completely in the solution or uniformly dispersed. This solution was filtered and transferred to a 100 mL volumetric flask. 3. Results and Discussions 3.1. Mixing order and manifold design optimization The CL light generated by two steps: 1- acidic nitrite ion react with H2O2 to generate peroxynitrouse acid, which frequently change to peroxynitrite (a powerful oxidizing agent); 2- product of the step-1, reacts with luminol in basic medium to emit CL light (Bhattacharyya and Veeraraghavan 1977, p.629; Merenyi, Lind and Eriksen 1990, p.53; Radi et al. 1993,p.51 & Sahaet al. 1998, p.653).The overall chemiluminescence reaction can be explained as follow: First step: HNO2 + H2O2
HOONO + H2O –
ONOO– + H2O
HOONO + OH Second step:
ONOO– + Lu–
Lu· – + O2·– + NO–
Lu·– + O2·–
light + aminophthalate
While reaction of OMP with nitrite ion in acidic medium is a nitrosation reaction as follows (Baraka et al 2014, p.54): H N H3CO
O S
N H 3C
N CH 3 OCH 3
NaNO2 HCl
ON
H N
H3CO
N NO
O S
H 3C
N CH 3 OCH 3
4
Determination of OMP was performed by indirect method, because the CL light signals (as peak height) decreased in the presence OMP owing to its reaction with nitrous acid. In the present study, the rFIA technique was selected to eliminate background absorption and generated great noise from continuous CL light formed, as well as to simplify the flow system. The suggested reagent injection system in this case, was either by injection of nitrite ion into HCl stream or by injection of luminol into H2O2 stream. Thus, unreproducible signal and un-stability of base line eliminate the former suggestion. Consequently, manifold design based on the injection of luminol to H2O2 stream selected. 3.1. Optimization of reagent concentrations Formation of nitrous acid likewise peroxynitrous acids depends mainly on the acids type and concentration. In this study, 0.1 M of different types of acids: HCl, HNO3, H2SO4 and CH3COOH were tested. The best result was obtained when HCl was used due to its role in the peroxynitrous acid formation (Lu 2002, p.107). The effect of various concentration of HCl on CL intensity was successively tested and shown in Fig. 2. The emitted light increased with increasing HCl concentration up to 0.225 M, and then the signal began to decrease. The higher acidity tends to decrease the CL-light generated from luminol system since the latter requires an alkaline medium. Subsequently, 0.225 M HCl was chosen as the optimum concentration. Other chemical optimizations including sodium nitrite, hydrogen peroxide and luminol concentrations were made and the results are tabulated in Table 2. Sodium carbonate concentration was found to have great effect on the CL determination method. This may attribute to the fact that the CL light intensity from luminol oxidation increased in a basic carbonate medium (Radi et al.1993, p.51 & Lu et al. 2004, p.29). Besides the stability of nitrosation products, reaction speed was also increased in basic medium. Figure 3 illustrates such effect and 0.12 M sodium carbonate was found to be optimum as it causes a significant change in the CL signals. 3.2. Effect of system flow rates Optimum system flow rate was selected in such away, that the complete CL reaction could be recorded before the excited product leaves the flow cell, thus, a rapid mixing of 5
the reactants in the CL flow cell was necessary. As shown in Fig. 4, maximum CL intensity was obtained using a 3.0 mL min−1. 3.3. Effect of reaction coil length According to the developed rFIA-CL system in this investigation, and under optimum regnants concentrations and flow rate, three reacting coils were tested. Coil 1, for nitrous acid formation by mixing HCl stream with that of NaNO2; Coil 2, for the complete reacting of OMP sample with in situ generated nitrous acid. Coil 3, for mixing injected luminol reagent with H2O2 carrier stream. Optimum coil lengths were chosen in a way to obtain highest CL signals. Fig. 5 shows that the best coil lengths in the proposed method were 20, 80 and zero cm for coil 1, 2, and 3, respectively. 3.4. Effect of injected reagent volume The variation of CL emission with the injected volume (50–200 µL) of the reagent was studied. The signal increased upto 100 µL; hence, this volume was selected for further experiments. Larger sample volumes cause insufficient mixing and dispersion. 4. Validity of the analysis method 4.1. Calibration curve Under optimum reaction conditions in Table 3, the calibration graph was constructed by plotting concentration in μg/mL of the OMP against the differences in CL-intensity as a peak height (ΔCL), when ΔCL= CLblank – CLOMP). It was found that the calibration curve was linear in the concentration range of 3.0-15.0 μg/mL (Fig. 6), and the regression coefficient calculated by least squares produced for the calibration equation are recorded in Table 4 with other statistical values. 4.2.
Accuracy and precision
Accuracy of the work was measured by the standard addition method. Solution of preanalyzed drug sample contain 5.0 µg/mL of OMP were spiked with three different concentrations (3.0, 5.0, and 8.0 µg/mL) of standard OMP and the mixtures were 6
analyzed by the developed method. The experiment was performed in triplicate. The Recovery % and standard error (SE) were calculated for each concentration (Table 5). Precision study was achieved by measuring three replicate analyses (n=3) at two concentrations level (5.0 and 10.0 μg/mL) for intra-day (repeatability) and inter-day (intermediate precision) RSD% and accuracy (E%). The results in Table 6 predicted that the present study was more précises. 4.3.
Robustness
To predicate the capacity and efficiency of the present method to remain unaffected by small, but deliberate, variations in method parameters and good indication of the procedure’s reliability during normal usage. Number of method parameters, such as flow rate, reagent injection volume, and coil length are varied within a realistic range, and the quantitative influence of the variables are determined for the pre-analyzed sample solution, contains 5.0 μg/mL OMP. The results obtained, as shown in Table 7, illustrate that the proposed method is robust since the results are not very sensitive to variations in the experimental conditions. 5. Interferences study The applicability of the present rFIA-CL method for determination of OMP in different pharmaceutical products was investigated. Most of the formulations contain excepients, which are added along with the active drug constituents. These substances may cause some interference during estimation of the active drug constituents, so the recovery study was employed. This study was performed by comparing the signal obtained from (10.0 µg/mL) OMP present alone and in the presence of different concentrations of the excipients commonly present in those products. A substance was not considered to be an interference if the variation of the CL intensity was < ±5 %. The results obtained and tabulated in Table 8, indicate that no serious interferences were observed.
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6. Application of the method Under optimum experimental conditions, the proposed method was applied for the determination of OMP in pharmaceutical samples commonly consumed in Erbil city (Table 8). The results obtained, together with the results from HPLC method described by Nataraj et al. (2012, p.366), are given in Table 10. The analysis results showed no significant difference compared with those obtained by the published work (F-test=0.982 and t-test=1.658). 7. Conclusion The developed rFIA method with CL detection applied for the determination of OMP is simple, accurate, fast, specific (as no interfering effects were observed) and inexpensive. Furthermore, the method was specific enough to differentiate the drug from the soluble excipients, impurities and degradation products. Literature surveys predicate that only two FIA with CL detection methods were applied for omeprazole determination (Shu-hua 2007, p.51 & Yun and Yin-huan, 2010, p. 378), Table below demonstrate the differences between this developed method and that published previously. The only apparent difference in favor of other methods is the detection limit value, and this simply attributed to our devices response in comparison to others. In contrast, there are many advantage of reverse method on normal one. In the rFIA, it is the reagent that is injected into the flowing system which is made up of the sample and some other solutions. This technique was also successfully adopted to eliminate background absorption from sample matrix. One of the strength points in this research was the selectivity achieved by combination of three fast reactions a) Nitrosation reaction (HCl+NaNO2+OMP) b) Peroxynitrous formation (HCl+NaNO2+H2O2) c) CL-light generation (Peroxynitrous acid (HCl+NaNO2+H2O2) + Luminol (Luminol + Base)). As well-known one of the poor point in any CL method is selectivity. In other CL-system we have a lack in the selectivity in comparison with our method. Additionally, the methods depend on oxidation and reduction reaction. While in the present study the main 8
determination reaction was nitrosation reaction, and as a limited number of compounds undergo this reaction this gives a great strength to our methods. On the other side, no sophisticated instrument designs are required like capillary electrophoresis. They also offer several advantages over the use of conventional methods such as HPLC, in a way that they do not require the use of relatively large amounts of potentially hazardous and expensive organic solvents. Moreover, the present method was applied successfully for determination of OMP in pharmaceutical products and the results obtained were in good agreement with those obtained using published methods for comparison. 8. Acknowledgements The author would like to thanks all stuff members of Department of Chemistry - College of Science - Salahaddin University -Erbil for their helping during research interval, which in so many ways were involved in the present work.
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9. References 1. Baraka, M. M., Esadek, M. E., Abdelaziz, L. M. & Elbermawi, S. S. (2014) Spectrophotometric Determination of Omeprazole Via Nitrosation Reaction, International Journal of Current Pharmaceutical Research, 6 (3), pp. 54-57. 2. Bhattacharyya, P. K. & Veeraraghavan, R. (1977) Reaction Between Nitrous Acid and Hydrogen Peroxide in Perchloric Acid Medium, International Journal Of Chemical Kinetics, 9, pp. 629-640. 3. Boix, C., Ibáñez, M., Zamora, T., Sancho, J. V., Niessen, W. M. A. & Hernández, F. (2014) Identification of New Omeprazole Metabolites in Wastewaters and Surface Waters, Science of the Total Environment, 468-469, pp. 706-714. 4. Bosch, M. E., Sànchez, A. J. R., Rojas, F. S. & Ojeda, C. B. (2007) Analytical Methodologies for the Determination of Omeprazole: An Overview, Journal of Pharmaceutical and Biomedical Analysis, 44, pp. 831-844. 5. Brittain, H. G. (2010) Profiles of Drug Substances, Excipients, and Related Methodology, Volume 35; Chapter 4, Omeprazole, Elsevier, USA, pp. 153. 6. Cavalcanti, E. B., Segura, S. G., Centellas, F. & Brillas, E. (2013) Electrochemical Incineration of Omeprazole in Neutral Aqueous Medium Using a Platinum or BoronDoped Diamond Anode: Degradation Kinetics And Oxidation Products; Water Research, 47(5), pp.1803-1815. 7. EL-Enany, N., Belal, F. & Rizk, M. (2008) The Alternating Current Polarographic Behavior and Determination of Lansoprazole and Omeprazole In Dosage Forms and Biological Fluids, Journal of Biochemical and Biophysical Methods, 70(6), pp. 889896. 8. Hassan, R.O. and Faizullah, A. T. (2011) Reverse-Flow-Injection Analysis (FIA) for The
Determination
of
Vitamin
C
in
Pharmaceutical
Formulation
with
Chemiluminescence Detection, African Journal of Pure and Applied Chemistry, 5(11), pp. 373-382. 9. Jing-fu, L. & Gui-bin, J. (2001) Evaluation of a Reagent-Injection Flow Injection Technique for Sample Background Absorption Elimination: Determination of Chloride in Cigarettes, Talanta, 54, pp. 329– 332.
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10. Liu, J. & Feng, Y. (1998) Determination of Nicotine by Reagent-Injection Flow Injection Photometric Method, Talanta, 47, pp. 833-840. 11. Lu, C., Lin, J., Huie, C. W. & Yamada, M. (2004) Chemiluminescence Study of Carbonate and Peroxynitrous Acid and Its Application to the Direct Determination of Nitrite Based on Solid Surface Enhancement, Analytica Chimica Acta, 510, pp. 29– 34. 12. Lu, C., Qu, F., Lin, J. & Yamada, M. (2002) Flow-Injection Chemiluminescent Determination of Nitrite in Water Based on the Formation of Peroxynitrite From the Reaction of Nitrite and Hydrogen Peroxide, Analytica Chimica Acta, 474, pp. 107114. 13. Merenyi, G., Lind, J. & Eriksen, T. E. (1990) Luminol Chemiluminescence; Chemistry, Excitation, Emitter, Journal of Bioluminescence and Chemiluminescence, 5, pp. 53-56. 14. Nataraj, K. S., Duza, M. B., Pragallapati, K. & Kumar, D. K. (2012) Development and Validation of RP-HPLC Method for the Estimation of Omeprazole in Bulk and Capsule Dosage Forms, International Current Pharmaceutical Journal, 1(11), pp. 366-369. 15. Nevado, J. J. B., Peñalvo, G. C., Dorado, R. M. R. & Robledo, V. R. (2014) Simultaneous Determination of Omeprazole and Their Main Metabolites in Human Urine Samples by Capillary Electrophoresis Using Electrospray Ionization-Mass Spectrometry Detection, Journal of Pharmaceutical and Biomedical Analysis, 92, pp. 211-219. 16. Qaisi, A. M., Tutunji, M. F. & Tutunji, L. F. (2006), Acid Decomposition of Omeprazole in the Absence of Thiol: A Differential Pulse Polarographic Study at the Static Mercury Drop Electrode (SMDE), Journal of Pharmaceutical Sciences, 95(2), pp. 384-391. 17. Radi, R., Cosgrove, T. P., Beckman, J. S. & Freeman, B. A. (1993) Peroxynitriteinduced luminol chemiluminescence, Biochemistry Journal, 290, pp. 51-57. 18. Rezk, N. L., Brown, K. C. & Kashuba, A. D. M. (2006) A Simple and Sensitive Bioanalytical Assay for Simultaneous Determination of Omeprazole and Its Three
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Major Metabolites In Human Blood Plasma Using RP-HPLC After a Simple Liquid– Liquid Extraction Procedure, Journal of Chromatography B, 844(2), pp. 314-321. 19. Saha, A., Goldstein, S., Cabelli, D. & Czapski, G. (1998) Determination of Optimal Conditions for Synthesis of Peroxynitrite by Mixing Acidified Hydrogen Peroxide With Nitrite, Free Radical Biology and Medicine, 24(4), pp. 653-659. 20. Salama, F., El-Abasawy, N., Abdel-Razeq, S. A., Ismail, M. M. F., & Fouad, M. M.(2003) Validation Of The Spectrophotometric Determination of Omeprazole and Pantoprazole Sodium Via Their Metal Chelates, Journal of Pharmaceutical and Biomedical Analysis, 33(3), pp. 411-421. 21. Sharma, S. & Sharma, M. C. (2012) Development and Validation of New Analytical Methods for Simultaneous Estimation of Drotaverine Hydrochloride in Combination With Omeprazole in a Pharmaceutical Dosage Form, Arabian Journal of Chemistry, (article under press). 22. Shu-hua, H. (2007) Determination of Omeprazole by Flow Injection Combined with Chemiluminescence, Journal of Analytical Science, 23, pp. 51-53. 23. Yun, Y. & Yin-huan, L. (2010) Determination Of Omeprazole With a New Flow Injection Chemiluminescence Method, Journal of Xi’an Jiaotong University (Medical Sciences), 390(3), pp. 378-381. 24. Zhang, W., Han, F., Guo, P., Zhao, H., Lin, Z., Huang, M., Bertelsen, K., & Weng, N.
(2010) Simultaneous Determination of Tolbutamide, Omeprazole, Midazolam and Dextromethorphan in Human Plasma By LC-MS/MS-A High Throughput Approach To Evaluate Drug-Drug Interactions, Journal of Chromatography B, 878, pp. 11691177.
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Waste
Fig. 1: suggested rFIA system for the determination of OMP; R 1= 0.08 M H2O2, OMP (10.0 µg/mL), L = 5.0×10-4 M luminol in 0.1 M Na2CO3 (injected regent) , R2= 0.225 M HCl , R3= 0.1 M NaNO2, Co= reaction coil and D= Detector.
Fig. 2: Effect of HCl concentration (M)
Fig. 3: Effect of Na2CO3 concentration (M)
13
Fig. 4: Effect of system flow rate (mL min-1)
Fig. 5: Effect of coils length (cm)
Fig. 6: Calibration Curve for the determination of OMP
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Table 1: Chemical structure of OMP Mol. Formula C17H19N3O3S
M. wt
Chemical Structure
345.42 (anhyd.)
Table 2: Chemicals optimization range Chemicals NaNO2 H2O2 Luminol
Studying concentration range (mol L-1) 0.04-0.16 0.02-0.14 0.1×10-3 – 1.2×10-3
Optimum concentration (mol L-1) 0.12 0.08 0.5×10-3
Table 3: Optimum experimental parameters of the standard calibration Parameter Hydrochloric acid (HCl) Sodium nitrite (NaNO2) Hydrogen peroxide (H2O2) Luminol Sodium carbonate (Na2CO3) Flow rate Coil length Coil 1 Coil 2 Injected volume
Unit M M M M M mL/min
Value 0.225 0.1 0.08 5.0 ×10-4 0.12 3.0
Cm Cm µL
20 80 100
Table 4: The statistical parameters of calibration results Parameters Analyte Blank signal (mV) Linear range (µg/mL) Slope Intercept Coefficient of determination (R2) Detection limit (µg/mL)
Value Omeprazole (OMP) 450 3.0-15.0 29.43 5.7 0.9987 1.3
15
Table 5: Accuracy measurement of the present method OMP (µg/mL) Recovery % E% Found X ± SE; SD 3.0 8.04 ± 0.066; 0.112 101.40 1.40 5.0 5.0 9.89 ± 0.118; 0.205 97.76 -2.24 8.0 12.93 ± 0.079; 0.137 99.18 -0.82 X=Mean of three replicate (n=3), SE=Standard error, SD=Standard Deviation, E% =Relative error. Sample solution
Added
Table 6: Results Analysis of precision tests Analyte
Taken (μg/mL)
Intra-day Found (μg/mL) E% X ± SE, SD
RSD%
Inter-day Found (μg/mL) E% X±SE, SD
RSD%
5.00 4.89 ± 0.061; 0.106 -2.28 2.17 4.87 ± 0.079; 0.136 2.64 2.80 10.00 9.86 ± 0.073; 0.127 -1.4 1.29 9.77 ± 0.090; 0.157 2.34 1.60 X=Mean of three replicate (n=3), SE=Standard error, SD= Standard deviation, RSD %= Relative standard deviation, E %=Relative error.
OMP Standard
Table 7: Robustness results of the method OMP (µg/mL) Parameters
2.8 Flow rate (mL/min) 3.2 Reagent injection volume (µL)
90 110
Sample
Added
5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
4.00 8.00 4.00 8.00 4.00 8.00 4.00 8.00
Found
Recovery %
RSD%
(X ± SE, SD) 8.98 ± 0.127; 0.220 12.78 ± 0.087 ; 0.150 9.11 ± 0.139 ; 0.240 13.01 ± 0.211 ; 0.367 9.04 ± 0.149 ; 0.258 12.86 ± 0.120 ; 0.208 9.17 ± 0.084 ; 0.146 12.65 ± 0.234; 0.405
99.60 97.20 102.65 100.18 100.95 98.30 104.25 95.65
2.45 1.17 2.64 2.82 2.85 1.62 1.59 3.20
Coil length (cm) 10 5.0 4.00 8.95 ± 0.109 ; 0.188 98.80 2.10 30 5.0 8.00 13.11 ± 0.222 ; 0.385 101.33 2.94 70 5.0 4.00 8.73 ± 0.069 ; 0.120 93.30 1.38 Coil 2 90 5.0 8.00 12.75 ± 0.118 ; 0.205 96.88 1.61 X=Mean of three replicate (n=3), SE=Standard error, SD= Standard deviation, RSD %= Relative standard deviation. Coil 1
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Table 8: Effect of common excipients on the determination of OMP by the present rFIA-CL
Glucose Fructose
60 60
10 10
OMP (µg/mL) Found (X ± SE, SD) 10.20 ± 0.079; 0.136 10.37 ± 0.130; 0.225
Sucrose
60
10
10.41± 0.138; 0.239
104.12
2.29
6
Talc
60
10
10.16 ± 0.210; 0.364
101.62
3.59
6
Starch
30
10
9.54 ± 0.179; 0.310
95.40
3.25
3
Na+
100
10
9.97 ± 0.106; 0.184
99.74
1.84
10
PO43-
60
10
9.98 ± 0.224; 0.388
99.78
3.89
6
Substances
MAC (µg/mL)
Added
% Recovery
RSD%
TCR
102.04 103.66
1.33 2.17
6 6
X=Mean of three replicate (n=3), SE=Standard error, SD= Standard deviation, RSD%= Relative standard deviation, MAC= Maximum Allowable Concentrations, TCR= Tolerable Concentration Ratio with no interferences = (Interferent (µg/mL) / Omeprazole (µg/mL)).
Table 9: Pharmaceutical products analyzed by the present method Sample form
Capsule products
Name
Product manufacture and country
Gasec Aprazole Omiz Pepzol Gastrimut Risek Ome TAD Oprazole Epirazol
Mepha – Portugal Ajanta –India Tabuk -Saudi Arabia Hikma Pharma S.A.E.- Egypt Lab. Normon S.A –Spain Julphar –UAE TAD Pharma-Germany Hikma pharmaceuticals –Jordan Eipico – Egypt
OMP content (mg/capsule) 20 mg 40 mg 20 mg 40 mg 40 mg 20 mg 20 mg 20 mg 20 mg
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Table 10: Application of the present rFIA-CL method for the determination of OMP in pharmaceutical products Sample product
Labeled amount (mg/capsule)
OMP found (mg) ± SE; SD/ Capsule Published method
Proposed method
RSD %E F-value t-value 19.73 ± 0.157 ; 1.78 1.11 Gasec 20 19.52 ± 0.374 ; 0.837 0.352 19.48 ± 0.079 ; 0.90 1.48 Omiz 20 19.20 ±0.293 ; 0.656 0.176 19.67 ± 0.093 ; 1.06 8.90 Risek 20 18.06 ± 0.448; 1.002 0.208 19.51 ± 0.078 ; 0.90 -0.10 Ome TAD 20 19.53 ± 0.225; 0.503 0.175 19.69 ± 0.151 ; 1.72 1.86 Oprazole 20 19.33 ± 0.258 ; 0.577 0.982 1.658 0.338 18.44 ± 0.094 ; 1.14 0.42 Epirazol 20 18.37 ± 0.144 ; 0.321 0.210 39.17 ± 0.050 ; 0.29 0.94 Pepzol 40 38.81 ± 0.316; 0.707 0.114 39.51 ± 0.023 ; 0.13 -1.02 Gastrimut 40 39.92 ± 0.278 ; 0.621 0.052 39.63 ± 0.061 ; 0.34 1.36 Aprazole 40 39.09 ± 0.157 ; 0.352 0.137 X: mean of five replicate (n=5); SD: standard deviation; RSD: relative standard deviation; tabulate students t-test and variances F-test at 95% confidence limit (n = 5) were 2.228 and 2.98, respectively.
Table 11: Comparison between the present method with that published by Shu-hua and Yun. Variables
Present method
Shu-hua Method
Yun Method
Indirect
Direct
Direct
CL-Regent
HNO2+H2O2+Luminol
Na2S2O4+Ce(IV)+ HNO3
KMnO4+NaOH +Luminol
FIA-System
Reverse
Normal
Normal
Liner range
3.0-15.0 µg/mL
0.02-10.0 µg/mL
0.3 -10 µg/mL
Detection limit
1.3 µg/mL
0.003 µg/mL
0.16µg/mL
mixing tubing
20 and 80 cm
-
150cm
Analysis method
18
