Design of Experimental Approach for Development of Rapid High Performance Liquid Chromatographic Process for Simultaneous Estimation of Metoprolol, Telmisartan, and Amlodipine from Formulation: Greenness and Whiteness Evaluation.

The design of an experimental approach, the Box-Behnken design, was implemented to optimize the chromatographic condition to develop a rapid HPLC procedure for quantification of a ternary mixture of metoprolol (MET), telmisartan (TEL), and amlodipine (AML) from the formulation. The perturbation plots, contour, and 3D response surface pictures were developed to study the impact of each variable on the analytes' retention time and the probable interaction between the parameters with fewer chromatographic runs. The optimized HPLC method separated the three analytes within 5 min with excellent selectivity and peak shape on a Zorbax C18 HPLC column using acetonitrile and phosphate buffer (20 mM, pH 5.8) with isocratic elution at a 1.1 mL/min flowrate. A wavelength 230 nm was utilized to monitor the elute. The validation of proposed method demonstrated a wide linearity range of 10-200 µg/mL for MET and TEL and 5-50 µg/mL for AML along with an excellent correlation coefficient. The correctness of the HPLC approach was further confirmed by excellent recovery of the added amount of analytes utilizing the standard addition technique. The recommended HPLC approach was employed safely for quality assurance of the formulation, because the evaluation of the method's greenness and whiteness confirmed the environmentally friendly nature of the approach.

Metoprolol and Its Degradation and Transformation Products Using AOPs-Assessment of Aquatic Ecotoxicity Using QSAR.

Pharmaceuticals are found in waterbodies worldwide. Conventional sewage treatment plants are often not able to eliminate these micropollutants. Hence, Advanced Oxidation Processes (AOPs) have been heavily investigated. Here, metoprolol is exposed to UV irradiation, hydrogen peroxide, and ozonation. Degradation was analyzed using chemical kinetics both for initial and secondary products. Photo-induced irradiation enhanced by hydrogen peroxide addition accelerated degradation more than ozonation, leading to complete elimination. Degradation and transformation products were identified by high-performance liquid-chromatography coupled to high-resolution higher-order mass spectrometry. The proposed structures allowed to apply Quantitative Structure-Activity Relationship (QSAR) analysis to predict ecotoxicity. Degradation products were generally associated with a lower ecotoxicological hazard to the aquatic environment according to OECD QSAR toolbox and VEGA. Comparison of potential structural isomers suggested forecasts may become more reliable with larger databases in the future.

Enantioseparation of propranolol, amlodipine and metoprolol by electrochromatography using an open tubular capillary modified with ß-cyclodextrin and poly(glycidyl methacrylate) nanoparticles.

The inner wall of a capillary was coated with glycidyl methacrylate (GMA) to form tentacle-type coating, and poly(glycidyl methacrylate) nanoparticles (PGMA NPs) were then immobilized on the film. Ethanediamine-ß-cyclodextrin as chiral selector was covalently bonded into the PGMA NPs through the ring-open reaction. The materials were characterized by SEM, TEM and FT-IR. The modified column was applied to the enantioseparation of the racemates of propranolol, amlodipine and metoprolol. Compared to a capillary with a single layer of CD-PGMA (without GMA coating) and to a CD-GMA system (without PGMA nanoparticles), the performance of the capillary is strongly improved. The effects of buffer pH value and applied voltage were optimized. Best resolutions (propranolol: 1.27, metoprolol: 1.01 and amlodipine: 2.93) were obtained when using the PGMA-coated capillary system. The run-to-run, day-to-day and column-to-column reproducibility were tested and found to be highly attractive. The new stationary phase is likely to have a large potential and scope in that it may also be applied to chiral separations of other enantiomers, such as amino acids and biogenic amines. Graphical abstract Schematic presentation of the preparation of a capillary column with glycidyl methacrylate (GMA) coating which was then immobilized with poly(glycidyl methacrylate) nanoparticles and ethanediamine-ß-cyclodextrin. This novel open tubular column was applied to construct capillary electrochromatography system for separation of basic racemic drugs.

Simultaneous determination of amlodipine and metoprolol in their combined dosage form using a synchronous fluorescence spectrofluorimetric method.

Highly sensitive, rapid, accurate and precise synchronous fluorescence spectrofluorimetric method has been developed for simultaneous analysis of a mixture of amlodipine (AMD) and metoprolol (MET). The method relies on measuring the relative synchronous fluorescence intensity of both drugs at Δλ of 90 nm in acetate buffer solution at pH 5. The experimental parameters influencing the developed method were investigated and optimized. The method was linear over the ranges 0.2-2 µg/ml and 0.5-10 µg/ml for AMD and MET, respectively. The limits of detection were 50 ng/ml for AMD and 130 ng/ml for MET while the limits of quantitation were 150 ng/ml for AMD and 390 ng/ml for MET. The developed method was applied successfully for the determination of the two drugs in their co-formulated tablet. The mean percent recoveries were found to be 100.51 and 99.57 for AMD and MET, respectively.

Detection of Metoprolol in Human Biofluids and Pharmaceuticals via Ion-Transfer Voltammetry at the Nanoscopic Liquid/Liquid Interface Array.

Metoprolol (MTP) is one of the most widely used antihypertensive drugs yet banned to use in sport competition. Therefore, there has been an increasing demand for developing simple, rapid, and sensitive methods suited to the identification and quantification of MTP in human biofluids. In this work, ultrathin silica nanochannel membrane (SNM) with perforated channels was employed to support nanoscale liquid/liquid interface (nano-ITIES) array for investigation of the ion-transfer voltammetric behavior of MTP and for its detection in multiple human biofluids and pharmaceutical formulation. Several potential interfering substances, including small molecules, d-glucose, urea, ascorbic acid, glycine, magnesium chloride, sodium sulfate and large molecules, bovine serum albumin (BSA), were chosen as models of biological interferences to examine their influence on the ion-transfer current signal of MTP. The results confirmed that the steady-state current wave barely changed in the presence of small molecules. Although BSA displayed an apparent blockade on the transfer of MTP, the accurate determination of MTP in multiple human biofluids (i.e., urine, serum and whole blood) and pharmaceutical formulation were still feasible, thanks to the molecular sieving and antifouling abilities of SNM. A limit of detection (LOD) within the physiological level of MTP during therapy could be achieved for all cases, i.e., 0.5 and 1.1 µM for 100 times diluted urine and serum, respectively, and 2.2 µM for 1000 times diluted blood samples. These results demonstrated that the nano-ITIES array behaved as a simplified and integrated detection platform for ionizable drug analysis in complex media.

Measured and predicted environmental concentrations of carbamazepine, diclofenac, and metoprolol in small and medium rivers in northern Germany.

This study evaluated the impact of secondary municipal effluent discharge on carbamazepine, diclofenac, and metoprolol concentrations in small and medium rivers in northern Germany and compared the measured environmental concentrations (MECs) to the predicted environmental concentrations (PECs) calculated with four well-established models. During a 1-year sampling period, secondary effluent grab samples were collected at four wastewater treatment plants (WWTPs) together with grab samples from the receiving waters upstream and downstream from the wastewater discharge points. The carbamazepine, diclofenac, and metoprolol concentrations were analyzed with high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS-MS) after solid phase extraction. In the secondary effluents, 84-790 ng/L carbamazepine, 395-2100 ng/L diclofenac, and 745-5000 ng/L metoprolol were detected. The carbamazepine, diclofenac, and metoprolol concentrations analyzed in the rivers downstream from the secondary effluent discharge sites ranged from <5 to 68, 370, and 520 ng/L, respectively. Most of the downstream pharmaceutical concentrations were markedly higher than the corresponding upstream concentrations. The impact of wastewater discharge on the MECs in rivers downstream from the WWTPs was clearly demonstrated, but the correlations of the MECs with dilution factors were poor. The smallest rivers exhibited the largest maximum MECs and the widest ranges of MECs downstream from the wastewater discharge point. Three of the four tested models were conservative, as they showed higher PECs than the MECs in the rivers downstream from the WWTPs. However, the most detailed model underestimated the diclofenac concentrations.

Preliminary study of an offline simultaneous determination of metoprolol tartrate and hydrochlorothiazide in powders and tablets by reflectance near-infrared spectroscopy.

A preliminary study of the feasibility of using near-infrared spectroscopy (NIRS) for the offline simultaneous determination of metoprolol tartrate (MTP) and hydrochlorothiazide (HTZ) in powders and tablets has been carried out. An industrial tableting process was simulated using an instrumented tablet press replicator - Presster™. Conventional reference analytics were replaced with gravimetric analysis. The NIRS models for powder and tablet analysis were developed using 55 samples, and tested on 80 independent samples. Powder mixture components were weighed in glass vials to collect reference values, mixed and manually transferred to a tablet press replicator and compacted to form tablets. NIRS calibration models were developed using spectral and gravimetric reference data. The two model drugs were simultaneously quantified exhibiting root mean-squared error of prediction (RMSEP) of 1.69 and 1.31 mg for HTZ powder and tablet samples, respectively, and RMSEP of 3.15 and 3.00 mg for MTP powder and tablet samples, respectively. NIRS analysis of MTP and HTZ in powder and tablet form has not been reported elsewhere.

A quantitative HPLC-MS/MS method for studying internal concentrations and toxicokinetics of 34 polar analytes in zebrafish (Danio rerio) embryos.

An analytical method using high-performance liquid chromatography-tandem mass spectrometry was developed to determine internal concentrations of 34 test compounds such as pharmaceuticals and pesticides in zebrafish embryos (ZFE), among them, cimetidine, 2,4-dichlorophenoxyacetic acid, metoprolol, atropine and phenytoin. For qualification and quantification, multiple reaction monitoring mode was used. The linear range extends from 0.075 ng/mL for thiacloprid and metazachlor and 7.5 ng/mL for coniine and clofibrate to 250 ng/mL for many of the test compounds. Matrix effects were strongest for nicotine, but never exceeded ±20 % for any of the developmental stages of the ZFE. Method recoveries ranged from 90 to 110 % from an analysis of nine pooled ZFE. These findings together with the simple sample preparation mean this approach is suitable for the determination of internal concentrations from only nine individual ZFE in all life stages up to 96 h post-fertilization. Exemplarily, the time course of the internal concentrations of clofibric acid, metribuzin and benzocaine in ZFE was studied over 96 h, and three different patterns were distinguished, on the basis of the speed and extent of uptake and whether or not a steady state was reached. Decreasing internal concentrations may be due to metabolism in the ZFE.

[Intentional poisoning of two wives by their husband?]. / Vorsätzliche Vergiftung zweier Frauen durch ihren Ehemann?

The authors report on the death of two women who were married to the same man one after the other. Exhumation and toxicological investigation of the first wife, who had died 7 years before, did not produce any conclusive evidence of homicide. With regard to the circumstances of death of the second wife the husband made different statements. According to the result of the chemical and toxicological investigations death was caused by acute intoxication with the beta-blocker metoprolol. The man was found guilty of killing on request (which is a criminal offence in Germany) by administering the beta-blocker metoprolol through a transnasal gastric tube.

Development of a RP-HPLC method for simultaneous determination of atenolol, metoprolol tartrate and phenol red for in-situ rat intestinal perfusion studies.

Single-pass intestinal perfusion (SPIP) method is a widely used experimental model to determine the intestinal permeability of drugs. These studies are performed in the presence of a reference standard (metoprolol, MT) and a zero permeability marker (phenol red, PR). Therefore, it is important to develop a validated method for simultaneous determination of the investigated compound along with MT and PR. The aim of this study was to develop a reversed phase high-performance liquid chromatography (RP-HPLC) method with UV-detection for the simultaneous determination of atenolol (ATN), MT, and PR in the perfusion medium used in SPIP experiments. Separation of compounds were performed using an InertSustain C18 (250 × 4.6 mm, 5 µm) HPLC column at 35 °C. The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 7.0, 12.5 mM) in gradient elution, and was delivered at a flow rate of 1 mL/min. The acetonitrile ratio of the mobile phase increased linearly from 10 to 35 % over 15 min. The injection volume was 20 µL, and ATN, MT and PR were detected at 224 nm. The retention times under optimum HPLC conditions were 5.028 min, 12.401 min, and 13.507 min for ATN, MT and PR, respectively. The developed RP-HPLC method was validated for selectivity, specificity, calibration curve and range, accuracy and precision, carry-over effect, stability, reinjection reproducibility, recovery and robustness. The method was linear for ATN (0.76-50 µg/mL), MT (1.14-50 µg/mL), and PR (0.47-20 µg/mL) with determination coefficients of 0.9999, 0.9994 and 0.9998, respectively. The results obtained for all validation parameters of the developed RP-HPLC method met the required limits of the ICH M10 Guideline.

Potentiometric propranolol-selective sensor based on molecularly imprinted polymer.

A novel potentiometric sensor based on molecularly imprinted polymer (MIP) for propranolol, an adrenergic-blocking drug, was designed. The influence of molecularly imprinted polymer particle content and sodium tetraphenylborate additives in polyvinylchloride membrane was shown. The electrodes show near-Nernstian responses down to 10(-4)-10(-5) M propranolol concentration. The potentiometric response of MIP-based sensor for propranolol in mixed nonaqueous medium was shown at first. Sensor selectivity relative to various inorganic cations, atenolol and metoprolol, was reported. Direct potentiometry was used to determine propranolol in aqueous modeling solutions and pharmaceutical preparations with good results.

Multi-residue enantiomeric analysis of pharmaceuticals and their active metabolites in the Guadalquivir River basin (South Spain) by chiral liquid chromatography coupled with tandem mass spectrometry.

This paper describes the development and application of a multi-residue chiral liquid chromatography coupled with tandem mass spectrometry method for simultaneous enantiomeric profiling of 18 chiral pharmaceuticals and their active metabolites (belonging to several therapeutic classes including analgesics, psychiatric drugs, antibiotics, cardiovascular drugs and ß-agonists) in surface water and wastewater. To the authors' knowledge, this is the first time an enantiomeric method including such a high number of pharmaceuticals and their metabolites has been reported. Some of the pharmaceuticals have never been studied before in environmental matrices. Among them are timolol, betaxolol, carazolol and clenbuterol. A monitoring programme of the Guadalquivir River basin (South Spain), including 24 sampling sites and five wastewater treatment plants along the basin, revealed that enantiomeric composition of studied pharmaceuticals is dependent on compound and sampling site. Several compounds such as ibuprofen, atenolol, sotalol and metoprolol were frequently found as racemic mixtures. On the other hand, fluoxetine, propranolol and albuterol were found to be enriched with one enantiomer. Such an outcome might be of significant environmental relevance as two enantiomers of the same chiral compound might reveal different ecotoxicity. For example, propranolol was enriched with S(-)-enantiomer, which is known to be more toxic to Pimephales promelas than R(+)-propranolol. Fluoxetine was found to be enriched with S(+)-enantiomer, which is more toxic to P. promelas than R(-)-fluoxetine.

Simultaneous enantiomeric determination of propranolol, metoprolol, pindolol, and atenolol in natural waters by HPLC on new polysaccharide-based stationary phase using a highly selective molecularly imprinted polymer extraction.

A simple high performance liquid chromatography method HPLC-UV for simultaneous enantiomeric determination of propranolol, metoprolol, pindolol, and atenolol in natural water samples was developed and validated, using a molecularly imprinted polymer solid-phase extraction. To achieve this purpose, Lux(®) Cellulose-1/Sepapak-1 (cellulose tris-(3,5-dymethylphenylcarbamate)) (Phenomenex, Madrid, Spain) chiral stationary phase was used in gradient elution and normal phase mode at ambient temperature. The gradient elution program optimized consisted of a progressive change of the mobile phase polarity from n-hex/EtOH/DEA 90/10/0.5 (v/v/v) to 60/40/0.5 (v/v/v) in 13 min, delivered at a flow rate of 1.3 ml/min and a sudden change of flow rate to 2.3 ml/min in 1 min. Critical steps in any molecularly imprinted polymer extraction protocol such as the flow rate to load the water sample in the cartridges and the breakthrough volume were optimized to obtain the higher extraction recoveries for all compounds. In optimal conditions (100 ml breakthrough volume loaded at 2.0 ml/min), extraction recoveries for the four pairs of ß-blockers were near 100%. The MIP-SPE-HPLC-UV method developed demonstrates good linearity (R(2) ≥ 0.99), precision, selectivity, and sensitivity. Method limit detection was 3.0 µg/l for propranolol and pindolol enantiomers and 20.0 and 22.0 µg/l for metoprolol and atenolol enantiomers, respectively. The proposed methodology should be suitable for routine control of these emerging pollutants in natural waters for a better understanding of the environmental impact and fate.

Identification and characterization of stressed degradation products of metoprolol using LC/Q-TOF-ESI-MS/MS and MS(n) experiments.

A rapid, specific and reliable isocratic high-performance liquid chromatography combined with quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) method has been developed and validated for the identification and characterization of stressed degradation products of metoprolol. Metoprolol, an anti-hypertensive drug, was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per ICH-specified conditions. The drug showed extensive degradation under oxidative and hydrolysis (acid and base) stress conditions. However, it was stable to thermal, neutral and photolysis stress conditions. A total of 14 degradation products were observed and the chromatographic separation of the drug and its degradation products was achieved on a C(18) column (4.6 × 250 mm, 5 µm). To characterize degradation products, initially the mass spectral fragmentation pathway of the drug was established with the help of MS/MS, MS(n) and accurate mass measurements. Similarly, fragmentation pattern and accurate masses of the degradation products were established by subjecting them to LC-MS/QTOF analysis. Structure elucidation of degradation products was achieved by comparing their fragmentation pattern with that of the drug. The degradation products DP(2) (m/z 153) and DP(14) (m/z 236) were matched with impurity B, listed in European Pharmacopoeia and British Pharmacopoeia, and impurity I, respectively. The LC-MS method was validated with respect to specificity, linearity, accuracy and precision.

[Methods for the protection against counterfeit medications. Part 2. The assessment of interlot dispersion of the metoprolol succinate tablets fabricated by different manufacturers].

This paper reports the results of analysis of the metoprolol succinate tablets fabricated by two different manufacturers, Akrikhin (Russia) and AstraZeneka (Sweden) by near-IR spectroscopy in the combination with the chemometric processing of the data obtained (discriminative analysis). It is concluded that this method is applicable for the assessment of interlot dispersion of the metoprolol succinate tablets.

[The methods for the protection against counterfeit medications. Part 1. The estimation of the quality of metoprolol succinate substance and tablets from different manufacturers in terms of "identity" and "active ingredient content"].

This paper was designed to report the results of analysis of metoprolol succinate substance and tablets obtained from two manufacturers, Akrikhin (Russia) and AstraZeneca (Sweden). The analysis was performed by spectroscopy in the near IR region and followed by the chemometric treatment of the data obtained. The method was used to confirm the "identity" of metoprolol succinate tablets. The approach to distinguishing the differences between pharmaceutical dosage forms produced by different manufacturers is proposed. Also, the method for the qualitative determination of metoprolol succinate in the pharmaceutical formulations has been developed.

Utility of synchronous fluorimetry for the concurrent quantitation of metoprolol and ivabradine.

Metoprolol combined with ivabradine have been determined up to nanogram level simultaneously relied on the synchronous fluorescence spectra. First derivative amplitudes of the synchronous spectrofluorimetric spectra are recorded at Δλ = 40 nm using ethanol as diluting solvent. Metoprolol can be measured at 286 nm which is the zero-crossing point of ivabradine, and the later can be measured at 296 nm. The calibration plots were found to be linear over the ranges of concentrations: 100.0-1000.0 ng/mL and 10.0-200.0 ng/mL for metoprolol and ivabradine, respectively. Validation of the procedure was performed using the International Council of Harmonization guidelines. Values of LODs were found to be 28.89, 2.80 ng/mL and LOQs were 87.56, 8.49 ng/mL for metoprolol and ivabradine, respectively. As the two drugs are co-administered safely and effectively to reduce heart rate, angina attacks, the current methodology is utilized for the concurrent analysis of them in their single ingredient pharmaceutical preparations, synthetic mixtures, and biological fluids. The designed method, being cost-effective and simple procedure, is the first method for metoprolol and ivabradine simultaneous analysis. The results agreed statistically with the comparison methods.

Vortex assisted liquid-liquid microextraction based on in situ formation of a natural deep eutectic solvent by microwave irradiation for the determination of beta-blockers in water samples.

In this study, a simple, green, and reliable method combining vortex-assisted liquid-liquid microextraction based on in situ formation of a novel hydrophobic natural deep eutectic solvent (NADES-VA-LLME) and high-performance liquid chromatography (HPLC) was developed for the determination of metoprolol and propranolol in water samples. The novel NADES was synthesized in situ within only 20 s by subjecting the water sample containing azelaic acid and thymol to microwave irradiation at 50 ˚C. Initial studies indicated that a 17:1 ratio of thymol to azelaic acid yielded the highest response for analytes. The influence of 7 parameters, including NADES volume, salt amount, sample pH, vortex time, centrifugation time, microwave time, and temperature, were screened using a 27-3 fractional factorial design. The obtained significant parameters were optimized by response surface methodology employing a Box-Behnken design. The method displayed satisfactory linearity (r=0.9996) for metoprolol and propranolol with limits of detection of 0.2 and 0.1 µg/L, respectively. The relative standard deviation at 2.5, 40, and 80 µg/L levels was lower than 6%, with accuracy in the range of 90.8-100.2%. Enrichment factors were 147.0 and 144.4 for metoprolol and propranolol, respectively. This study demonstrates that the developed in situ NADES-VA-LLME-HPLC technique can be considered as a fast and environmentally friendly alternative for isolation/preconcentration of ß-blockers from water samples.

Optimization and establishment of a validated stability-indicating HPLC method for study of the stress degradation behavior of metoprolol succinate.

A stability-indicating HPLC method has been established for analysis of metoprolol succinate in the presence of products generated in a stress degradation study. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis, and thermal decomposition. Extensive degradation was found to occur in an alkaline medium and under thermal stress. Minimum degradation was observed in an acidic medium and under photolytic and oxidative stress. Successful separation of the drug from its degradation products formed under stress conditions was achieved on a C18 column using sodium dihydrogen phosphate buffer-acetonitrile (70 + 30) mobile phase. The flow rate was 1 mL/min, and the detection wavelength was 274 nm. The method was validated for linearity, range, precision, accuracy, LOQ, and LOD. Because the method effectively separates the drugs from their degradation products, it can be used as a stability-indicating method.

Simultaneous time-course measurements of metoprolol and α-hydroxyl metoprolol in fingermarks after oral administration by liquid chromatography tandem mass spectrometry.

The aim of this study was to evaluate whether fingerprints are suitable to be applied as the biometric identification samples by testing the orally administered drugs needs to be taken daily. The dosage of BETALOC® was administered to subjects following single and multiple doses and its active ingredient metoprolol and its main metabolite α-hydroxyl metoprolol were selected as the analytes. The subjects washed their hands and pressed fingertips onto glass slides at fixed sampling points (from 1 h to 7 days), and the analytes were extracted using cotton swabs 30 times followed by ultrasonic assistance in 30℃ methanol solution for 5 min with working power of 2000 W after optimization. The drugs in blood were taken from their elbow vein and deproteinized before analysis. Analysis were performed using liquid chromatography-tandem mass spectrometry (LC/MS/MS), and their concentration time course in fingerprints and blood were evaluated and compared. Results showed that metoprolol was detected 1 h after ingestion both in fingerprints and blood, while α-hydroxyl metoprolol was detected from sampling points of 2 h in fingerprint and 3 h in blood, respectively. Drugs could be detected for longer periods in blood than in fingerprint in single dose administration. However, in multiple doses, they could reach a steady detectable state in fingerprints from the fifth day after oral administration, and could serve as a more rapid and simpler alternative for drug analysis. We demonstrate that fingerprints could be applied as the biometric identification samples for orally administered drugs in multiple-doses, and could be applied to drug testing in criminal investigations.