Omeprazole assay

Assay preparation Transfer about 100 mg of Omeprazole, accurately weighed, to a 50-ml volumetric flask, dissolve in and dilute with Diluent to volume, and mix. Transfer 5 ml of this solution to a 50-ml volumetric flask, dilute with Diluent to volume, and mix. 

Procedure Separately inject equal volumes (about 20 pT) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C17H19N3O3S in the portion of omeprazole taken by the formula ... 

Procedure After 2 h, remove each sample from the basket, and quantitatively transfer into separate volumetric flasks to obtain a solution having a final concentration of about 0.2 mg/ml. Proceed as directed for the Assay preparation in the Assay, starting with "Add about 50 ml of Diluent. Calculate the quantity, in mg, of omeprazole (C17H19N3O3S) dissolved in the Medium by the formula ... 

Assay preparation Weigh and mix the contents of not fewer than 20 Capsules. Transfer an accurately weighed portion of the mixture, equivalent to about 20 mg of omeprazole, to a 100-ml volumetric flask, add about 50 ml of Diluent, and sonicate for 15 min. Cool, dilute with Diluent to volume, mix, and pass through a membrane filter having 0.45 /im or finer porosity. [Note Bubbles may form just before bringing the solution to volume. Add a few drops of dehydrated alcohol to dissipate the bubbles if they persist for more than a few minutes]. 

Dhumal et al. [26] described an individual UV spectrophotometric assay method for the analysis of omeprazole from separate pharmaceutical dosage forms. Powdered tablets, equivalent to 50 mg of the drug, were sonicated with 35 ml of 0.1 M sodium hydroxide for 5 min and diluted to 50 ml with 0.1 M sodium hydroxide. The solution was filtered and a 2-ml portion of the filtrate was diluted to 200 ml with 0.1 M sodium hydroxide before the absorbance of the solution was measured at 305 nm versus 0.1 M sodium hydroxide. Beer s law was obeyed for 6-25 /[Pg.205]

Sastry et al. [27] described four simple and sensitive spectrophotometric methods for the assay of omeprazole in pure and in dosage forms based on the formation of chloroform soluble ion-associated under specified experimental conditions. Four acidic dyes Suprachen Violet 3B (SV 3B, method A), Tropaeolin 000 (TP 000, method B), Boromocresol Green (BCG, method C), and Azocarmine G (AG, method D) are utilized. 

El-Kousy and Bebawy [31] described two stability-indicating spectro-photometric methods for the determination of omeprazole in the presence of its photodegradation products. In the first method, omeprazole from capsules or vials were dissolved in acetonitrile/water (1 1) and UV-VIS spectrophotometry used to determine the first-, second-, and third-derivative absorption curves between 200 and 400 nm. The level of omeprazole was assayed from the values of ordinates of the three curves at 290.4,... 

Wahbi et al. [32] used a spectrophotometric method for the determination of omeprazole in pharmaceutical formulations. The compensation method and other chemometric methods (derivative, orthogonal function, and difference spectrophotometry) have been applied to the direct determination of omeprazole in its pharmaceutical preparations. The method has been validated the limits of detection was 3.3 x 10 2 /ig/ml. The repeatability of the method was found to be 0.3-0.5%. The linearity range is 0.5-3.5 /ig/ml. The method has been applied to the determination of omeprazole in its gastro-resistant formulation. The difference spectrophotometric (AA) method is unaffected by the presence of acid induced degradation products, and can be used as a stability-indicating assay method. 

Radi [41] used an anodic voltammetric assay method for the analysis of omeprazole and lansoprazole on a carbon paste electrode. The electrochemical oxidations of the drugs have been studied at a carbon paste electrode by cyclic and differential-pulse voltammetry in Britton-Robin-son buffer solutions (0.04 M, pH 6-10). The drug produced a single oxidation step. By differential-pulse voltammetry, a linear response was obtained in Britton-Robinson buffer pH 6 in a concentration range from 2 x 10-7to 5 x 10 5 M for lansoprazole or omeprazole. The detection limits were 1 x 10 8 and 2.5 x 10 8 M for lansoprazole and omeprazole, respectively. The method was applied for the analysis of omeprazole in capsules. The results were comparable to those obtained by spectrophotometry. 

Motevalian et al. [62] developed a rapid, simple, and sensitive HPLC assay method for the simultaneous determination of omeprazole and its major metabolites in human plasma using a solid-phase extraction procedure. Eluent (50 /d) was injected on a /rBondapak Ci8 reversed-phase column (4.6 mm x 250 mm, 10 /un). The mobile phase consisted of 0.05 M phosphate buffer (pH 7.5) and acetonitrile (75 25) at a flow-rate of 0.8 ml/min. UV detection was at 302 nm. Mean recovery was greater than 96% and the analytical responses were linear over the omeprazole concentration range of 50-2000 ng/ml. The minimum detection limits were 10, 10, and 15 ng/ml for omeprazole, omeprazole sulfone, and hydroxyomeprazole, respectively. The method was used to determine the plasma concentration of the respective analytes in four healthy volunteers after an oral dose of 40 mg of omeprazole. 

Orlando and Bonato [73] presented a practical and selective HPLC method for the separation and quantification of omeprazole enantiomers in human plasma. Ci8 solid-phase extraction cartridges were used to extract the enantiomers from plasma samples and the chiral separation was carried out on a Chiralpak AD column protected with a CN guard column, using ethanol-hexane (70 30) as the mobile phase, at a flow-rate of 0.5 ml/min. The detection was carried out at 302 nm. The method is linear in the range of 10-1000 ng/ml for each enantiomer, with a quantification limit of 5 ng/ml. Precision and accuracy, demonstrated by within-day and between-day assays, were lower than 10%. 

Rezk et al. [74] developed and validated a reversed-phase HPLC assay method for the simultaneous quantitative determination of omeprazole and its three metabolites in human plasma. The method provides excellent chromatographic resolution and peak shape for the four components and the internal standard within a 17-min run time. The simple extraction method results in a clean baseline and relatively high extraction efficiency. The method was validated over the range of 2-2000 ng/ml. The resolution and analysis for the four analytes omeprazole, hydroxyome-prazole, omeprazole sulfone, and omeprazole sulfide and the internal standard utilized a Zorbax C18 (15 cm x 3 mm, 5 /im) with a Zorbax C18 (12.5 cm x 4.6 mm) guard column. The mobile phase consisted of two components. Mobile phase A was 22 mM phosphate monobasic, adjusted to a pH of 6 with diluted sodium hydroxide. This solution was filtered through a 0.45-/im membrane filter, then mixed as 900 ml buffer to 100 ml methanol. Mobile phase B was composed of 100 ml of the phosphate buffer as mobile phase A, mixed with 800 ml of acetonitrile, 100 ml of methanol, and 100 /A of trifluoroacetic acid with an initial flow-rate of 0.55 ml/min and detection at 302 nm. 

Zarghi et al. [76] developed an HPLC method, using a monolithic column, for quantification of omeprazole in plasma. The method is specific and sensitive with a quantification limit of 10 ng/ml. Sample preparation involves simple, one-step extraction procedure, and analytical recovery was complete. The separation was carried out in reversed-phase conditions using a Chromolith Performance (RP-18e, 100 x 4.6 mm) column with an isocratic mobile phase consisting of 0.01 mol/1 disodium hydrogen phosphate buffer-acetonitrile (73 27) adjusted to pH 7.1. The wavelength was set at 302 nm. The calibration curve was linear over the concentration range 20-1500 ng/ml. The coefficients of variation for intra- and interday assay were found to be less than 7%. 

Sultana et al. [88] developed a reversed-phase HPLC method for the simultaneous determination of omeprazole in Risek capsules. Omeprazole and the internal standard, diazepam, were separated by Shim-pack CLC-ODS (0.4 x 25 cm, 5 m) column. The mobile phase was methanol-water (80 20), pumped isocratically at ambient temperature. Analysis was run at a flow-rate of 1 ml/min at a detection wavelength of 302 nm. The method was specific and sensitive with a detection limit of 3.5 ng/ml at a signal-to-noise ratio of 4 1. The limit of quantification was set at 6.25 ng/ml. The calibration curve was linear over a concentration range of 6.25—1280 ng/ml. Precision and accuracy, demonstrated by within-day, between-day assay, and interoperator assays were lower than 10%. 

Stenhoff et al. [117] determined enantiomers of omeprazole in blood plasma by normal-phase liquid chromatography and detection by atmospheric-pressure ionization tandem mass spectrometry. The enantioselec-tive assay of omeprazole is using normal-phase liquid chromatography on a Chiralpak AD column and detection by mass spectrometry. Omeprazole is extracted by a mixture of dichloromethane and hexane and, after evaporation, redissolution and injection, separated into its enantiomers on the chiral stationary phase. Detection is made by a triple quadrupole mass spectrometer, using deuterated analogs and internal standards. The method enables determination in plasma down to 10 nmol/1 and shows excellent consistency suited for pharmacokinetic studies in man. 

Bonato and Paias [136] developed two sensitive and simple assay procedures based on HPLC and capillary electrophoresis for the enantio-selective analysis of omeprazole in pharmaceutical formulations. Racemic omeprazole and (S)-omeprazole were extracted from commercially available tablets using methanol-sodium hydroxide 2.5 mol/1 (90 10). Chiral HPLC separation of omeprazole was obtained on a ChiralPak AD column using hexane-ethanol (40 60) as the mobile phase and detection at 302 nm. The resolution of omeprazole enantiomers by capillary electrophoresis was carried out using 3% sulfated /1-cyclodextrin in 20 mmol/1 phosphate buffer, pH 4 and detection at 202 nm. 

Lin and Wu [137] established a simple capillary zone electrophoresis method for the simultaneous analysis of omeprazole and lansoprazole. Untreated fused-silica capillary was operated using a phosphate buffer (50 mM, pH 9) under 20 kV and detection at 200 nm. Baseline separation was attained within 6 min. In the method validation, calibration curves were linear over a concentration range of 5-100 /iM, with correlation coefficients 0.9990. RSD and relative error were all less than 5% for the intra- and interday analysis, and all recoveries were greater than 95%. The limits of detection for omeprazole and lansoprazole were 2 fiM (S/N = 3, hydroxynamic injection 5 s). The method was applied to determine the quality of commercial capsules. Assay result fell within 94—106%. 

Naesdal et al. [144] studied the pharmacokinetics of 14C-omeprazole and its metabolites after single intravenous and oral doses of 20 0 mg, respectively, to 12 patients with chronic renal insufficiency. Blood samples for determination of total radioactivity, omeprazole, hydroxyomeprazole, sulfone, and sulfide were taken for 24 h. Urine was collected over 96 h for determination of total radioactivity and during the first 24 h for additional assay of omeprazole and metabolites. The mean systemic availability was 70% and the mean plasma t1/z of omeprazole was 0.6 h. 

Zhao and Lou [164] studied the metabolism of omeprazole to its two major metabolites, hydroxyomeprazole and omeprazole sulfone, in rat liver microsomes by a reversed-phase HPLC assay. The formation of metabolites of omeprazole depended on incubation time, substrate concentration, microsomal protein concentration, and was found to be optimal at pH 7.4. The Pmax and Km of omeprazole hydroxylation in the rat liver microsomal preparation were 2033 nmol /(min mg protein), and 46.8 ymol/l, respectively. The effects of seven drugs on omeprazole metabolism were tested. Mephenytoin, five benzodiazepines and pava-verine caused inhibition of omeprazole metabolism. 

Quercia et al. [171] studied the stability of omeprazole 2 mg/ml in an extemporaneously prepared oral liquid. The contents of five 20-mg omeprazole capsules were mixed with 50 ml of 8.4% sodium bicarbonate solution in a Luer-Lok syringe. Three vials of this liquid were prepared for storage at 24,5, and — 20 °C. A 3-ml sample of each was taken initially and on days 1, 2, 3, 4, 6, 8, 10, 12, 14, 18, 22, 26, and 30 and assayed by HPLC. The liquids stored at 5 and — 20 °C did not change color during the study period, but the color of the liquid stored at 24 °C changed from white to brown. 

The assay of omeprazole and its metabolite is usually performed using HPLC and UV detection (Lager-strom 1984 Ieri 1996 Yim 2001 Tybring 1997) orLC-MS/MS assay (Kanazawa 2002). 

Yim DS, Jeong JR, Park JY (2001) Assay of omeprazole and omeprazole sulfone by semi-microcolumn liquid chromatography with mixed-function precolumn. J Chromatogr B 754 487-193... 

The effect of gastric HVK -ATPase inhibitors on enzyme activity (ATP cleavage) can be studied in vitro with partly purified HVK -ATPase preparations [27]. This assay has been used more effectively to study the mechanism of action of H /K -ATPase inhibitors in detail than to study the structure-activity relationship of such inhibitors [28]. Since HVK -ATPase inhibitors of the omeprazole-type need acid activation and the enzyme assay should be performed at neutral pH values, a pre-incubation period at the lowest possible pH of about 6 was used to initiate the acidic conversion of the test compound into its active principle. This reflects more the chemical instability of the test compound at neutral pH values than its effect during conditions of much higher acidity within the secretory cannaliculus of the parietal cell during acid secretion. Many chemically labile inhibitors are therefore very active in this test system. However, they do not cause an inhibition in more complex test systems and, therefore, are without any practical usefulness [28]. 

The structure-activity relationship of HVK -ATPase inhibitors of the omeprazole type is based on the balance between chemical stability at neutral pH values and acid-induced conversion into the active sulphenamide. Derivatives, which are too unstable at neutral pH, are very active in the test assay of partly purified HVK" -ATPase. This assay has been performed at pH 7.4 after preincubation at pH 6 of the enzyme protein with the derivative to be tested. The high activity was therefore the result of the conversion of the derivative in solutions of neutral pH values and this does not reflect the situation of high acidity within the secretory compartment of the parietal cell [28]. The derivatives which are very unstable at neutral pH do not inhibit gastric acid secretion in vivo because their transformation had already occurred prior to the active principle reaching the target enzyme. Chemically very stable derivatives do not show any inhibitory effect either in vitro or in vivo. 

Table 3.5 and Fig. 3.9). Where possible, their potency relative to omeprazole, measured under the same assay conditions, is indicated. However, notwithstanding differences in the stimulant used, comparison of their in vivo... 

Andersson, T. Lagerstrom, P.-O. Miners, J.O. Veronese, M.E. Weidolf, L. Birkett, D.J. High-performance liquid chromatographic assay for human liver microsomal omeprazole metabolism. J.Chromatogr., 1993, 619, 291-297... 

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