Imprinted ß-ketoenamine-linked covalent organic frameworks as dispersive sorbents for the fluorometric determination of timolol.

Timolol accompanied the formation of fluorescent ß-ketoenamine-linked covalent organic frameworks (COFs) via the Sc(Tof)3-catalyzed condensation of derivated carbaldehyde and hydrazide in a 1,4-dioxane/mesitylene porogen to construct timolol-imprinted COFs (TICOFs). With high imprinting factors, the synthesis-optimized TICOFs were characterized by fluorescence, UV-Vis spectrometry, X-ray diffraction, N2 adsorption/desorption analyses, scanning electron microscopy, and FTIR spectrometry. The TICOF fluorescence measured at 390 nm/510 nm is dynamically quenched by timolol and was thus utilized to quantify timolol in a linear range of 25-500 nM with a LOD of 8 nM. The TICOF recovered 99.4% of 0.5% timolol maleate in a commercial eye drop (RSD = 1.1%, n = 5). In addition, TICOF was used as a dispersive sorbent to recover 95% of 2.0 nM timolol from 20 mg of TICOF in 25 mL phosphate buffer. Dilution factors of 25 and 75 were the maximum tolerated proportions of the urine and serum matrix spiked with 2.0 nM timolol to reach recoveries of 92.4% and 90.3%, respectively.

A versatile HPLC method with an isocratic single mobile phase system for simultaneous determination of anti-glaucoma formulations containing timolol.

Timolol is a non-cardioselective beta blocker and has different combined ophthalmic dosage forms for treatment of glaucoma. This research introduce an HPLC method for the separation of three drugs used in combination with timolol simultaneously by applying isocratic mobile phase system in a single run and the same detection wavelength with short time. The drugs included in the separation procedures are; dorzolamide, brinzolamide, and brimonidine. The HPLC method was carried out through a single mobile phase system, which contains acetonitrile: 0.05M phosphate buffer at the ratio of 30:70, respectively at pH 3.5 and wavelength of 220nm. The method, regarding its simplicity allows determination of the studied drugs simultaneously using single run in about 8minutes. The method was rectilinear in the ranges of concentration: 1.25-25µg/mL for timolol, 4-80µg/mL for dorzolamide, 5-50µg/mL for brinzolamide and 2-20µg/mL for brimonidine. Different factors affecting the separation are thoroughly studied. The developed method was validated based on the official guidelines and the results were compared statistically with previously published methods and showed non-significant difference.

Deformability properties of timolol-loaded transfersomes based on the extrusion mechanism. Statistical optimization of the process.

The purpose of this work was to analyze the deformability properties of different timolol maleate (TM)-loaded transfersomes by extrusion. This was performed because elastic liposomes may contribute to the elevation of amount and rate of drug permeation through the corneal membrane. This paper describes the optimization of a transfersome formulation by use of Taguchi orthogonal experimental design and two different statistical analysis approaches were utilized. The amount of cholesterol (F1), the amount of edge-activator (F2), the distribution of the drug into the vesicle (F3), the addition of stearylamine (F4) and the type of edge-activator (F5) were selected as causal factors. The deformability index, the phosphorous recovery, the vesicle size, the polydispersity index, the zeta potential and percentage of drug entrapped were fixed as the dependent variables and these responses were evaluated for each formulation. Two different statistical analysis approaches were applied. The better statistical approach was determined by comparing their prediction errors, where regression analysis provided better optimized responses than marginal means. From the study, an optimized formulation of TM-loaded transfersomes was prepared and obtained for the proposed ophthalmic delivery for the treatment of open angle glaucoma. It was found that the lipid to surfactant ratio and type of surfactant are the main key factors for determining the flexibility of the bilayer of transfersomes. From in vitro permeation studies, we can conclude that TM-loaded transfersomes may enhance the corneal transmittance and improve the bioavailability of conventional TM delivery.

[Determination of enantiomeric impurity of timolol maleate in bulk substances and eye drops].

OBJECTIVE: To determine the enantiomeric impurity contents of domestic timolol maleate in bulk drugs and eye drops. METHODS: Enantiomer impurity of timolol was assayed by chiral high performance liquid chromatography. The chromatographic conditions were as follows:chiralcel OD chiral column (4.6 mm ×150 mm, 5µm), detection wavelength:297 nm, mobile phase:hexane-isopropanol-diethylamine (480:20:1), column temperature:25 ℃, flow rate:1.0 ml/min, sample injection volume:5 µl. RESULTS: The resolution between R- and S-timolol was more than 4. The enantiomeric impurity contents were less than 0.67% on average in two batches of timolol maleate bulk drugs, and 0.31% on average in three batches of timolol maleate eye drops. CONCLUSION: Enantiomeric impurity contents in each batch of products all meet European Pharmacopoeia criteria, which can be used as references in Chinese Pharmacopoeia criteria.

Colorimetric Concurrent Determination of Ultra-Trace Amounts of Pilocarpine and Timolol as Anti-Glaucoma Drugs in Binary Mixtures Using a Multivariate Calibration Approach Based on the Aggregation of Gold Nanoparticles.

BACKGROUND: To study the ultra-trace simultaneous determination of drugs, the colorimetric method in combination with chemometrics can be used. OBJECTIVE: In this study, a simple, rapid, and sensitive UV-Vis spectrophotometric method using gold nanoparticles (AuNPs) was introduced for the simultaneous determination of ultra-trace amounts of pilocarpine (PIL) and timolol (TIM) in binary mixtures and biological samples. METHODS: AuNPs interacted with components and the aggregation mode of NPs occurred, and, finally, the color change of the solution (red to gray) was observed with the naked eye without the most modern and expensive instruments. The characterization of AuNPs was evaluated by transmission electron microscopy (TEM) and dynamic light scattering (DLS). RESULTS: The validation of the colorimetric way was studied in the concentration range of 100-800 and 100-600 µg/L with good linearity equal to 0.9772 and 0.9891 for PIL and TIM, respectively. The limit of detection (LOD) was found to be 165.00 and 92.40 µg/L, where the limit of quantitation (LOQ) was 500.00 and 280.00 µg/L for PIL and TIM, respectively. The effect of some factors such as interaction time, the concentration of components, and the volume of buffer on absorbance was investigated. Partial least squares (PLS) as an efficient multivariate calibration method was combined with colorimetry for the simultaneous determination of PIL and TIM in binary mixtures. The optimum number of latent variables was selected by k-fold cross-validation based on minimum mean square error prediction (MSEP), and the number of components equal to 1 with MSEP of 1.085 and 0.763 was considered for PIL and TIM, respectively. The mean recovery was obtained at 100.20 and 101.55% for PIL and TIM, respectively. CONCLUSIONS: The colorimetric method can be introduced as a proper option for the simultaneous determination of components in pharmaceutical formulations and other samples. HIGHLIGHTS: A colorimetric method using AuNPs was proposed. The PLS method was coupled with a colorimetric method for the ultra-trace simultaneous estimation of PIL and TIM in binary mixtures. Ultra-trace amounts of PIL and TIM were also determined in biological samples. The proposed method is simple, fast, and less expensive than chromatography methods.

Development and Validation of Facile RP-HPLC Method for Simultaneous Determination of Timolol Maleate, Moxifloxacin Hydrochloride, Diclofenac Sodium and Dexamethasone in Plasma, Aqueous Humor and Pharmaceutical Products.

The present study aimed to develop a validated RP-HPLC method for the simultaneous determination of timolol maleate (TM), moxifloxacin hydrochloride (MOXI), diclofenac sodium (DS) and dexamethasone (DEXA) in human plasma, bovine aqueous humor and pharmaceutical preparations. The chromatographic separation was studied using the C18 column. The chromatographic conditions, such as composition, pH, the flow rate of mobile phase, the temperature of column, wavelength of absorption and injection volume of the sample, were studied. The method was validated to confirm specificity, linearity and accuracy in accordance with an International Conference on Harmonization guidelines. The optimum conditions for separation included mobile phase 0.05 M monobasic phosphate buffer: acetonitrile (65:35 v/v), pH of buffer adjusted to 6.2 and the flow rate of 1 mL/minute. The optimum temperature of the column was found to be 35°C, absorption wavelength 265 nm and injection volume 50 µL. The baseline separation of all four drugs with good sensitivity, resolution, and a less than 15 min run time was achieved. The retention time of TM, MOXI, DS and DEXA were 4.3,5.7,9.9 and 13.5 minutes respectively. The limit of detection (LOD) values were 6.2, 4.8, 0.8 and 1.2 ng/mL for TM, MOXI, DS and DEXA, respectively, whereas their respective limit of quantification (LOQ) values was: were 42.6, 26.8, 5.6 and 6.2 ng/mL. The coefficient of variation for intra-day and inter-day were in the range of 0.32-1.57 and 1.29-3.07%, respectively. The method was found to be sensitive, precise and accurate in human plasma and bovine aqueous humor and can be applied for the quantification of these compounds in plasma, aqueous humor and pharmaceuticals.

[Causes of gelatinous material on ocular surface of a patient using timolol maleate long-acting eye drops].

PURPOSE: To ascertain the causes of the formation of gelatinous material observed on the ocular surface of a patient using a betamethasone sodium phosphate ophthalmic solution containing fradiomycin sulfate (Rinderon-A) together with a timolol maleate long-acting ophthalmic gel-forming solution (Timoptol-XE). METHODS: The gellan gum in the Timoptol-XE was suspect as it might have been gelatinized by the fradiomycin sulfate in the Rinderon-A. Mixtures of the chemical compounds such as fradiomycin sulfate with the Timoptol-XE was tested in vitro to find out whether any resulted in gelation. RESULTS: It was confirmed that Timoptol-XE was gelatinized by the aminoglycoside drugs in vitro. The density of the aminoglycoside drugs needed for the gelation was only about 0.2 mM on average. Gelation was also observed with benzalkonium chloride and vancomycin. CONCLUSION: The results suggest that the gellan gum in the Timoptol-XE gelatinized by the fradiomycin sulfate in the Rinderon-A and that aminoglycocide compounds act strongly on gellan gum as polycation.

Full Factorial Design for Development and Validation of a Stability-Indicating RP-HPLC Method for the Estimation of Timolol Maleate in Surfactant-Based Elastic Nano-Vesicular Systems.

A novel isocratic stability-indicating chromatographic method was developed, optimized and validated using Design-Expert® following ICH guidelines for the quantification of Timolol maleate (TM). The intrinsic stability of TM was assessed by force degradation studies, which concluded no extensive degradation except under alkaline and oxidative conditions. TM was quantified accurately in the surfactant-based elastic vesicular system by separating it on Hypersil BDS C8 column using triethylamine in H2O (0.15%v/v; pH 3.0) and acetonitrile (ACN; 65:35%v/v). The influence of variable factors like mobile phase pH, injection volume (µL), flow rate (mL/min) and ACN content (%) on method responses were assessed using a full factorial design. The method was linear between 0.05 and 10 µg/mL with an R2 value of 0.9993. Limit of detection and limit of quantification were found to be 0.90 and 27.2 ng/mL. The method was specific, with recovery in plain drug solution of 89-92% and elastic nanovesicles of 90-93%. The experimental model was significant (P < 0.0001) as indicated by deliberate changes in the method analyzed through analysis of variance. The total drug content in elastic nanovesicles was estimated to be 9.53 ± 0.01 mg/20-mL dispersion and entrapment efficiency was 44.52 ± 0.73%. The developed method was rapid, economic and precise for the quantification of TM in bulk and vesicular system.

1H and 13C NMR characteristics of ß-blockers.

The ß-blockers are important drugs and decades of clinical experience proved their high medical status. However, to the best of our knowledge, there is no complete assignment of (1)H and (13)C NMR resonances of popular representatives: acebutolol, alpenolol, pindolol, timolol and propranolol and the published NMR data on carvedilol and atenolol are incorrect. Therefore, (1)H and (13)C NMR spectroscopy was applied for the characterization of a series of ß-adrenolytics: carvedilol (1), pindolol (2), alprenolol (3), acebutolol (4), atenolol (5), propranolol (6) and timolol (7). Two-dimensional NMR experiments (COSY, HMQC, HMBC, NOESY) allowed the unequivocal assignment of (1)H and (13)C spectra for solution (DMSO-d(6) ). Salts and bases can be easily distinguished based on (13)C chemical shifts which are within 65.0-65.5 ppm (OC2) and 46.9-47.0 (NC3) for hydrochlorides and larger, ca. 68.4 ppm (OC2) and 50.3-52.6 (NC3) for bases. NMR data of 1-7 should be included in pharmacopoeias.

Simultaneous spectrofluorometric analysis of tablets containing hydrochlorothiazide combined with timolol maleate or amiloride hydrochloride.

Green and sensitive spectrofluorometric methods have been developed and validated for the determination of timolol maleate (TML)/hydrochlorothiazide (HCT) and amiloride hydrochloride (AMH)/hydrochlorothiazide in tablets. The proposed spectrofluorometric procedures were found to be linear in the range of 4-12, 5-35 and 0.025-0.2 mg L-1 for HCT, TML and AMH, resp. The excitation and emission wavelengths for HCT, TML and AMH at room temperature were 270 and 375, 295 and 435, 330 and 415 nm, resp. The methods were validated with respect to ICH guidelines. The AMH showed higher sensitivity with lower values of LOD and LOQ values compared to HCT and TML. The proposed methods were applied to two pharmaceutical formulations; the method for HCT and AMH has proven as reliable assaying method, whereas the method for TML, when combined with HCT, is applicable to screening semi-quantitative analyses.

Analytical quality by design development of an ecologically acceptable enantioselective HPLC method for timolol maleate enantiomeric purity testing on ovomucoid chiral stationary phase.

Official method in Ph. Eur. for evaluation of timolol enantiomeric purity is normal-phase high performance liquid chromatography (NP-HPLC) method. Compared to other HPLC modes, NP is depicted as quite expensive with high consumption of organic solvents which leads to chronic exposure of analysts to toxic and carcinogenic effects. In order to overcome above-mentioned drawbacks, the aim of this study was to develop new method with better eco-friendly features. This was enabled by using protein type Chiral Stationary Phase (CSP) in reversed-phase mode that required up to 10 % (v/v) of organic solvent. Therefore, an enantioselective HPLC method was developed and validated for quantification of (S)-timolol and its chiral impurity, (R)-isomer. Optimized separation conditions on ovomucoid column were set using Analytical Quality by Design (AQbD) approach in method development. Optimization step was performed following the Box-Behnken experimental plan and the influence of three critical method parameters (CMPs) towards enantioseparation of the above-mentioned peak pair was examined. CMPs included variation of acetonitrile content in the mobile phase (5-10 %, v/v), pH value of the aqueous phase (6.0-7.0) and ammonium chloride concentration in the aqueous part of the mobile phase (10-30 mmol L-1). The most relevant critical method attributes (CMAs) in this case were the separation criterion between studied critical pair and retention factor of the second eluting peak, (S)-timolol. Qualitative Design Space (DS) was defined by Monte Carlo simulations providing adequate assurance of method's qualitative robustness (π = 95 %). The selected working point situated in the middle of the DS was characterized by following combination of CMPs: acetonitrile content in the mobile phase 7 % (v/v), pH value of the aqueous phase 6.8 and concentration of ammonium chloride in aqueous phase 14 mmol L-1. In the next step, the quantitative robustness was tested by Plackett-Burman experimental design. The validation studies confirmed adequacy of the proposed method for its intended purpose. Finally, Analytical Eco-Scale metric tool was applied to confirm that developed method represents excellent green analytical method compared to the official one.

Assessment of ion transfer amperometry at liquid-liquid interfaces for detection in CE.

In this research, ion transfer across the interface between two immiscible electrolyte solutions (ITIES) was used as a method of detection in a CE separation system. This method allows for the electrochemical detection of ionic analytes that cannot be easily oxidized or reduced. Method development revealed that the optimal separation conditions for three model ions (tetraethylammonium, tetrabutylammonium, and benzensulfonate) were found to be 5 mM sodium tetraborate buffer pH 9.2 with a separation voltage of 20 kV using a 40 cm, 50 microm id fused silica capillary. Constant potential amperometry and pulsed amperometric detection were applied at the ITIES in which the organic phase was gelled. A miniaturized ITIES within a pipette tip was investigated, which resulted in improved separation efficiency and LOD. To demonstrate the ability of the system to detect substances of bioanalytical interest, the beta-adrenergic receptor blockers timolol and propranolol were detected. The simplicity of the detection platform means that it may be useful for analytical situations not requiring trace or ultratrace detection capabilities.

Electrochemical determination of antihypertensive drugs by employing costless and portable unmodified screen-printed electrodes.

Hypertension increases the risk of heart disease and stroke, is commonly known as a silent killer disease and considered as one of the key risk factor for premature death and disability over the world. Herein, we report for the first time a sensitive, costless and reproducible voltammetric method for individual determination of five antihypertensive drugs namely, propranolol (PRO), timolol (TIM), amlodipine (AML), amiloride (AMI) and triamterene (TRI) using differential pulse voltammetry at bare/unmodified screen-printed carbon electrodes (SPEs) in presence of sodium dodecyl sulfate (SDS). Each drug exhibits an electrochemical signal in aqueous media which is significantly enhanced in presence of optimized concentration of SDS due to accumulation of the protonated drug molecules and electrostatically interaction with negatively charged micellar structures. As a result, the spherical micellar orientation of SDS onto the graphitic surface of SPEs offered the analytically sensitive determination of the target drugs over a wide linear concentration range with nano-molar detection limits possible negating the need for any complicated surface modifications. Finally, the proposed voltammetric method was successfully utilized in the individual determination of the target antihypertensive drugs in pharmaceutical formulations and human urine samples.

The application of artificial neural networks and support vector regression for simultaneous spectrophotometric determination of commercial eye drop contents.

In the present study, artificial neural networks (ANNs) and support vector regression (SVR) as intelligent methods coupled with UV spectroscopy for simultaneous quantitative determination of Dorzolamide (DOR) and Timolol (TIM) in eye drop. Several synthetic mixtures were analyzed for validating the proposed methods. At first, neural network time series, which one type of network from the artificial neural network was employed and its efficiency was evaluated. Afterwards, the radial basis network was applied as another neural network. Results showed that the performance of this method is suitable for predicting. Finally, support vector regression was proposed to construct the Zilomole prediction model. Also, root mean square error (RMSE) and mean recovery (%) were calculated for SVR method. Moreover, the proposed methods were compared to the high-performance liquid chromatography (HPLC) as a reference method. One way analysis of variance (ANOVA) test at the 95% confidence level applied to the comparison results of suggested and reference methods that there were no significant differences between them. Also, the effect of interferences was investigated in spike solutions.

Validated HPLC-DAD Method for the Simultaneous Determination of Six Selected Drugs Used in the Treatment of Glaucoma.

This work presents a simple, sensitive, and generic HPLC-diode-array detection method for the simultaneous determination of six drugs prescribed for the treatment of open-angle glaucoma and ocular hypertension. The investigated drugs include brimonidine tartarate (BMN), acetazolamide (AZA), brinzomaide (BZA), dorzolamide HCl (DZA), levobunolol HCl (LVB), and timolol maleate (TIM). Efficient chromatographic separation was achieved using a Thermo Hypersil BDS C18 column (4.6 × 250 mm, 5 µm) with a mobile phase consisting of phosphate buffer pH 5 and acetonitrile in a ratio of 78 + 22. The flow rate was 1 mL/min, and quantification was based on measuring peak areas at 298 nm for TIM and 254 nm for the other drugs. Peaks were perfectly resolved, with retention times at 3.06, 3.87, 4.53, 5.78, 7.31, and 10.78 min for BMN, AZA, DZA, TIM, LVB, and BZA respectively. The developed method was validated according to International Conference on Harmonization guidelines with respect to system suitability, linearity, ranges, accuracy, precision, robustness, and LODs and LOQs. The proposed method showed good linearity in the ranges of 2-80, 2.5-100, 2.5-100, 5-200, 3.75-150, and 1.75-70 µg/mL for BMN, AZA, DZA, TIM, LVB, and BZA, respectively. LODs were 0.20-1.01 µg/mL for the analyzed compounds. Applicability of the proposed method to real-life situations was assessed through the analysis of five different pharmaceutical formulations, and satisfactory results were obtained.

Ketopinic acid and diisoproylideneketogulonic acid as chiral ion-pair selectors in capillary electrophoresis. Enantiomeric impurity analysis of S-timolol and 1R,2S-ephedrine.

1S,4R-(+)-ketopinic acid [(+)-KPA] has been introduced as a chiral selector for the separation of pharmacologically active amines by non-aqueous capillary electrophoresis (NACE). (+)-KPA gave enantioresolution for most of the compounds previously separated by 2R,3S,4R,5S-(-)-2,3:4,6-di-O-isopropylidene-2-keto-L-gulonic acid [(-)-DIKGA], but with a reversed migration order. A complete enantioresolution (Rs=4.2) was obtained for timolol, a compound that could not be resolved using (-)-DIKGA as the selector. Thus, (+)-KPA was evaluated for the enantiomeric purity determination of S-timolol. A method based on pre-concentration by transient isotachophoresis (tITP) provided a limit of detection (LOD) of 0.2% R-timolol in S-timolol samples. Because of the lack of enantioresolution of ephedrine when (+)-KPA was used as the selector, a method with (-)-DIKGA has been developed and validated for determination of the enantiomeric purity of the 1R,2S enantiomer. The method gave good precision and accuracy with an LOD (S/N=3) of 0.033% for the enantiomeric impurity 1S,2R-ephedrine.

Robustness testing of a chiral NACE method for R-timolol determination in S-timolol maleate and uncertainty assessment from quantitative data.

A robustness test of a capillary electrophoresis method for the chiral separation of timolol in nonaqueous acidified media was performed. A two-level Plackett-Burman design was applied in which one qualitative and six quantitative factors were examined. Resolution, migration times and relative migration times to pyridoxine (selected as internal standard) were examined as qualitative responses to evaluate electrophoretic performance. A quantitative response, the content of R-timolol in S-timolol maleate sample, was also considered. Even though some significant factor effects were observed on the qualitative responses, it was still possible to quantify the R-timolol in the S-timolol maleate samples properly. The quantitative response was not significantly affected by the selected factors, demonstrating the robustness of the procedure. However, the use of different HDMS-beta-CD batches seemed to affect both types of responses necessitating to introduce a warning in the procedure. Since the experiments of the Plackett-Burman design can be assimilated to laboratories in an interlaboratory study, uncertainty can be evaluated using the robustness test data. The robustness test was set-up in such a way that the required variances could be estimated.

NMR analysis of ion pair formation between timolol and sorbic acid in ophthalmic preparations.

Ion pair formation between timolol and sorbic acid was investigated using NMR spectroscopy in order to clarify their interactions within ophthalmic preparation. (13)C and (1)H NMR spectra of timolol, sorbic acid, and a mixture of the two were obtained, and the signal changes induced by pairing were observed. The carbon signals of the butylaminopropanol moiety of timolol were markedly shifted in the mixture, as were the carboxyl and conjugated carbons assigned to sorbic acid. The localizations of the changes in each molecule revealed the binding sites. The profiles of butylaminopropanol carbon chemical shifts plotted against a molar ratio of sorbate were synchronized, which suggested a single type of interaction with sorbic acid. The Job plot showed a typical pattern with a single-maximum at a mole function of 0.5, indicating the presence of a 1:1 complex of timolol and sorbic acid. The stability constants (K) of the timolol-sorbate and timolol-maleate pairs were 1.9x10(1) and 2.2x10(2)M(-1), respectively. The higher K value of the timolol-maleate interaction suggested that it was dominant to the timolol-sorbate interaction when maleate and sorbate coexisted within a timolol solution. Here, we demonstrated evidence of an interaction between timolol and sorbic acid using simple NMR measurements, which suggested the existence of ion pair formation derived from charge neutralization. Our analysis using NMR spectroscopy should advance the understanding and optimization of formulations that are based on ion pair.

A New UPLC Method with Chemometric Design-Optimization Approach for the Simultaneous Quantitation of Brimonidine Tartrate and Timolol Maleate in an Eye Drop Preparation.

A new ultra-performance liquid chromatography (UPLC) with photodiode array was proposed for the quantitation of Brimonidine Tartrate (BRI) and Timolol Maleate (TIM) in eye drop using experimental design and optimization methodology. A 33 full factorial design was applied to uncover the effects of the selected factors and their interactions on the chromatographic response function for the optimization of experimental conditions in the development of a new UPLC method. As a result, the optimal chromatographic conditions giving a better separation and short analysis time were found to be 49.2°C for column temperature; 0.38 mL/min for flow rate and 56.7 % (v/v) for 0.1 M CH3COOH used in mobile phase. The elution of BRI and TIM was reported as 0.508 and 0.652 min within a short runtime of 1.5 min, respectively. Calibration graphs for BRI and TIM were obtained by the regression of the concentration on the peak area, which was detected at 246 and 298 nm, respectively. The method validation was performed by the analysis of the synthetic mixtures, intra-day and inter-day samples and standard addition samples. This study shows that the optimized and validated UPLC method is very promising and available for the quantification of BRI and TIM in an eye drop formulation.

A simple spectrophotometric method for the determination of beta-blockers in dosage forms.

A simple, extraction-free spectrophotometric method is proposed for the analysis of some beta-blockers, namely atenolol, timolol and nadolol. The method is based on the interaction of the drugs in chloroform with 0.1% chloroformic solutions of acidic sulphophthalein dyes to form stable, yellow-coloured, ion-pair complexes peaking at 415 nm. The dyes used were bromophenol blue (BPB), bromothymol blue (BTB) and bromocresol purple (BCP). Under the optimum conditions, the three drugs could be assayed in the concentration range 1-10 microg ml(-1) with correlation coefficient (n = 5) more than 0.999 in all cases. The stoichiometry of the reaction was found to be 1:1 in all cases and the conditional stability constant (K(F)) of the complexes have been calculated. The free energy changes (DeltaG) were determined for all complexes formed. The interference likely to be introduced from co-formulated drugs was studied and their tolerance limits were determined. The proposed method was then applied to dosage-forms the percentage recoveries ranges from 99.12-100.95, and the results obtained were compared favorably with those given with the official methods.