Microchip isotachophoresis for green and sustainable pharmaceutical quality control: Method validation and application to complex pharmaceutical formulations.

Universal microchip isotachophoresis (µITP) methods were developed for the determination of cationic and anionic macrocomponents (active pharmaceutical ingredients and counterions) in cardiovascular drugs marketed in salt form, amlodipine besylate and perindopril erbumine. The developed methods are characterized by low reagent and sample consumption, waste production and energy consumption, require only minimal sample preparation and provide fast analysis. The greenness of the proposed methods was assessed using AGREE. An internal standard addition was used to improve the quantitative parameters of µITP. The proposed methods were validated according to the ICH guideline. Linearity, precision, accuracy and specificity were evaluated for each of the studied analytes and all set validation criteria were met. Good linearity was observed in the presence of matrix and in the absence of matrix, with a correlation coefficient of at least 0.9993. The developed methods allowed precise and accurate determination of the studied analytes, the RSD of the quantitative and qualitative parameters were less than 1.5% and the recoveries ranged from 98 to 102%. The developed µITP methods were successfully applied to the determination of cationic and anionic macrocomponents in six commercially available pharmaceutical formulations.

Separation, identification and cardiovascular activities of phospholipid classes from the head of Penaeus vannamei by lipidomics and zebrafish models.

Phospholipids not only have high nutritional value, but also have a positive effect on cardiovascular disease, cancer and nervous system diseases. However, the activity of individual phospholipid classes of shrimp phospholipids is rarely studied. This paper researched phospholipids in the by-products of Penaeus vannamei processing. The phospholipid classes of the head from P. vannamei (PV) were separated by column chromatography, analyzed with UHPLC-Q-Exactive HF/MS, and quantified using ammonium ferrothiocyarate spectrophometry. In addition, their cardiovascular activities in zebrafish models were evaluated. A total of 5 phospholipid classes were obtained, including PV-PC, PV-PE, PV-PI, PV-PS and PV-SM, and identified as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and sphingomyelin (SM), respectively. In the phospholipid profiling analysis, PV-PC (308 molecules) had the highest proportion with 85.24%, followed by PV-PE (139 types) with 9.32%, PV-SM (41 structures) with 4.75%, PV-PS (24 types) with 0.16%, and PV-PI (6 molecules) with 0.03%. In the quantitative analysis, the content of PV was 45.7%, and the purity of phospholipid classes was 75.5-88.1%. In the cardiovascular activity assays, the effects of different phospholipid classes were different. For example, PV-PC groups had strong angiogenesis activity, but PV-PE groups showed the opposite property. Our comprehensive profiling analysis and in vivo bioactivity evaluation of phospholipids from the head of P. vannamei can provide evidence for their targeted applications in the future.

Determination of drugs in exhumed liver and brain tissue after over 9 years of burial by liquid chromatography-tandem mass spectrometry-Part 1: Cardiovascular drugs.

This paper should serve as support for future exhumations in which an analysis of cardiovascular drugs is issued after over 9 years of burial. Amiodarone, amlodipine, atropine, bisoprolol, cafedrine, clonidine, esmolol, furosemide, hydrochlorothiazide, lisinopril, nifedipine, nitrendipine, phenprocoumon, torsemide verapamil, and xipamide were determined in liver and brain tissue of over 100 cases in which exhumation was performed after over 9 years of burial. Diagrams, showing the detectability depending on postmortem period as well as condition of tissues, are presented for furosemide.

Fatal intoxication with ivabradine: First case report.

Ivabradine is a bradycardic drug used worldwide in the treatment of chronic stable angina and chronic heart failure. We presented here a case of a 61-year-old woman who was admitted to emergency department for overdose. She presented with drowsiness, bradycardia (45bpm) and a low blood pressure (116/21mmHg). She died ten hours after admission from multiple organ failure. Ivabradine was quantified in different matrices sampled during autopsy using a method on LC-MS/MS (TSQ Vantage Thermo Fisher Scientific®), after a double liquid-liquid extraction with a mixture of hexane/ethyl acetate (1/1; v/v) and then chloroform/isopropanol (80/20; v/v). Chromatographic separation was achieved using a Hypersyl gold PFP column (200×2.1mm, 1.9µm) and an acetonitrile/formiate 2mM, 0.1% formic acid buffer gradient. Method was fully validated on whole blood. The mean overall recovery was 90%. Linearity was validated in the 5-500ng/mL range, with intra and inter-day precision lower than 14.3%. The ivabradine concentration found in patient post-mortem blood was 1210ng/mL. Ivabradine was also quantified in different viscera like lung (2910ng/g), kidney (1510ng/g), liver (1050ng/g), heart (900ng/g), and brain (110ng/g). The vitreous humor concentration was 760ng/mL. Pregabalin and zopiclone were also found in blood at 50µg/mL and 206ng/mL, respectively. This case seems to be the first report of a fatal intoxication involving ivabradine and the first published concentrations in organs.

Understanding the multi-herbal composition of Buyang Huanwu Decoction: A review for better clinical use.

ETHNOPHARMACOLOGICAL RELEVANCE: Buyang Huanwu Decoction (BHD) is a multi-herbal composition commonly prescribed in the treatment of cerebrovascular diseases such as stroke. Although studies have been conducted at the cellular (in vitro), animal and human (in vivo) level, there was no detailed analysis on how the composition and proportion of BHD is modified according to target diseases. AIM OF STUDY: The purpose of this study is to investigate the composition and proportion of each herb in BHD to summarize how the original BHD was modified according to the target disease. MATERIALS AND METHODS: Electronic literature searches were performed in three databases, collecting sixty-eight studies for the final analysis. The studies were divided into three types: cell studies, animal experiments and clinical trial. In the analysis, the decoction formula including the composition and the weight proportion of the herbs in BHD used in the studies and the target diseases were examined. RESULTS: The result showed that in cell studies, the targets were mostly cell differentiation, cell injury and immune activation. In animal studies, cerebrovascular diseases such as cerebral ischemia were the most identified target diseases followed by nervous system and cardiovascular diseases. While the proportions of the herbs in BHD used in these studies were in general similar to the original formula, some studies reduced the amount of Astragali Radix to half of the original amount. Modified BHDs were used in four studies for cerebrovascular and peripheral nerve diseases. However, no significant correlation has been observed between the target diseases and the change of the proportion of the herbs in BHD. CONCLUSIONS: The most commonly used formula was the original composition of BHD, and modified BHDs were reported to be used to treat cerebrovascular and nervous diseases. Further studies about the effects of BHD by composition and proportion of herbs are needed in the future.

Screening and identification of potential active components in Ophiopogonis Radix against atherosclerosis by biospecific cell extraction.

Atherosclerosis is a chronic disease and an important pathological process associated with cardiovascular disease. Endothelial dysfunction, vascular smooth muscle cells (VSMCs) proliferation and neutrophil activation are involved in the development of atherosclerosis. Ophiopogonis Radix is a common traditional Chinese medicine use to treat cardiovascular diseases, however, its active constituents remain to be elucidated. In this study, primary vascular endothelial cells, primary VSMCs and neutrophils were prepared, and extract of Ophiopogonis Radix (EOR) was investigated to ameliorate H2O2 induced reactive oxygen species (ROS) and nitric oxide (NO) production. The results showed that EOR decreased levels of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, its protective effects against oxidative damage of endothelia and endothelial dysfunction. Additionally, EOR treatment inhibited oxidized low-density lipoprotein-induced VSMC proliferation, phorbol-12-myristate-13-acetate-mediated ROS production and neutrophil activation, malondialdehyde production, and decreased superoxide dismutase activity and myeloperoxidase release. By HPLC-Q-TOF-MS/MS analysis, 51 compounds in EOR were identified including 22 saponins and 24 homoisoflavonoids. Then biospecific cell extraction and LC-MS technique were employed to screening the antiatherosclerosis active components in Ophiopogonis Radix. After co-cultured with EOR, the multi-effective active constituents including four saponins and two homoisoflavonoids were acquired and subsequently verified to restore properties including endothelial injury, VSMC proliferation and neutrophil activation, indicating that these compounds may be multi-effective active constituents that were responsible for atherosclerosis and the cardiovascular protection of Ophiopogonis Radix.

Insights on the Multifunctional Activities of Magnolol.

Over years, various biological constituents are isolated from Traditional Chinese Medicine and confirmed to show multifunctional activities. Magnolol, a hydroxylated biphenyl natural compound isolated from Magnolia officinalis, has been extensively documented and shows a range of biological activities. Many signaling pathways include, but are not limited to, NF-κB/MAPK, Nrf2/HO-1, and PI3K/Akt pathways, which are implicated in the biological functions mediated by magnolol. Thus, magnolol is considered as a promising therapeutic agent for clinic research. However, the low water solubility, the low bioavailability, and the rapid metabolism of magnolol dramatically limit its clinical application. In this review, we will comprehensively discuss the last five-year progress of the biological activities of magnolol, including anti-inflammatory, antimicroorganism, antioxidative, anticancer, neuroprotective, cardiovascular protection, metabolism regulation, and ion-mediating activity.

Determination of cardiovascular drugs in sewage sludge by matrix solid-phase dispersion and ultra-performance liquid chromatography tandem mass spectrometry.

The current study presents a single step sample preparation procedure for the simultaneous determination of five antihypertensive (propranolol, losartan, irbesartan, telmisartan, and valsartan), three antiarrhythmic drugs (flecainide, dronedarone, and amiodarone), and one of their metabolites (N-desethylamiodarone) in sludge from municipal sewage treatment plants (STPs). Matrix solid-phase dispersion (MSPD) and ultra-performance liquid chromatography (UPLC) with tandem mass spectrometry (MS/MS) detection were selected as sample preparation and determination techniques, respectively. Under optimal conditions, MSPD extractions were carried out with freeze-dried samples (0.5 g) dispersed on 2 g of C18. Exhaustive extraction of target compounds was achieved with 10 mL of a methanol/acetonitrile/formic acid (30:69:1) solution. The obtained extract was ready for UPLC-MS/MS analysis without any further treatment, except filtration. The overall recoveries of the method (calculated against solvent-based standards) varied from 82 to 124%, with standard deviations in the range from 2 to 16%. Thus, the method was free of matrix effects during electrospray ionization. The achieved limits of quantification stayed between 2 and 10 ng g-1, and the linear response range extended to 5000 ng g-1. The occurrence of target compounds was investigated in sludge from 14 different STPs. High detection frequencies were observed for all compounds, with average concentrations above 100 ng g-1 for six cardiovascular drugs.

Electrochemical simulation of three novel cardiovascular drugs phase I metabolism and development of a new method for determination of them by liquid chromatography coupled with tandem mass spectrometry.

In this study electrochemistry (EC) coupled with electrospray ionization mass spectrometry (ESI-MS) was used to study the metabolic fate of three novel cardiovascular drugs: rivaroxaban (RIV), aliskiren (ALS), and prasugrel (PRS). Mimicry of the oxidative phase I metabolism was achieved in a simple amperometric thin-layer cell equipped with a boron-doped diamond (MD) working electrode. Structures of the electrochemically-generated metabolites were elucidated from MS/MS experiments. Additionally, a sensitive, specific, and rapid ultra-high performance liquid chromatography-tandem mass spectrometer (UHPLC-MS/MS) method has been developed and validated for the selected drugs in human urine samples. Three different sample preparation methods were compared and finally, sample preparation was accomplished through an ultrasound-assisted emulsification microextraction process (USAEME). The drugs were detected using a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via an electrospray ionization source with positive ionization mode (ESI(+)). The results obtained by EC-MS were compared with conventional in vivo studies by analyzing urine samples from patients. Data from in vivo experiments showed good agreement with the data from electrochemical oxidation. Thus, EC-MS is very well-suited for the simulation of the oxidative metabolism of rivaroxaban, aliskiren, and prasugrel as well. Moreover, electrochemical conversion of target compounds appears to be a new in vitro technology for the prediction of potential metabolites.

Fighting fake medicines: First quality evaluation of cardiac drugs in Africa.

BACKGROUND: The growing menace of poor quality and falsified drugs constitutes a major hazard, compromising healthcare and patient outcomes. Efforts to assess drug standards worldwide have almost exclusively focused on anti-microbial drugs; with no study to date on cardiovascular drugs. Our study aims to assess quality of seven routinely used cardiovascular medications (anticoagulants, antihypertensives and statins) in ten Sub-Saharan African countries. METHODS: Drugs were prospectively collected using standardized methods between 2012 and 2014 from licensed (random pharmacies) and unlicensed (street-markets) places of sale in Africa. We developed a validated reversed-phase liquid chromatography with tandem mass spectrometry method to accurately quantify the active ingredient in a certified public laboratory. Three quality categories were defined based on the ratio of the measured to the expected dosage of the active ingredient: A (good quality): 95% to 105%, B (low quality): 85 to 94.99% or 105.01 to 115%, C (very low quality): <85% or >115%. RESULTS: All expected medicines (n=3468 samples) were collected in Benin, Burkina-Faso, Congo-Brazzaville, the Democratic Republic of Congo, Guinea, Côte d'Ivoire, Mauritania, Niger, Togo and Senegal. Out of the 1530 samples randomly tested, poor quality (types B and C) was identified in 249 (16.3%) samples. The prevalence of poor quality was significantly increased in certain specific drugs (amlodipine 29% and captopril 26%), in generic versions (23%) and in drugs produced in Asia (35%). The proportion of poor quality reached 50% when drugs produced in Asia were sold in street-markets. CONCLUSION: In this first study assessing the quality of cardiovascular drugs in Africa, we found a significant proportion of poor quality drugs. This requires continued monitoring strategies.

Carboxymethyl ß-cyclodextrin as chiral selector in capillary electrophoresis: Enantioseparation of 16 basic chiral drugs and its chiral recognition mechanism associated with drugs' structural features.

Herein we present the enantioseparation of 10 cardiovascular agents and six bronchiectasis drugs including propranolol, carteolol, metoprolol, atenolol, pindolol, esmolol, bisoprolol, bevantolol, arotinolol, sotalol, clenbuterol, procaterol, bambuterol, tranterol, salbutamol and terbutaline sulfate using carboxymethyl-ß-cyclodextrin (CM-ß-CD) as chiral selector. To our knowledge, there is no literature about using CM-ß-CD for separating carteolol, esmolol, bisoprolol, bevantolol, arotinolol, procaterol, bambuterol and tranterol. During the course of work, changes in pH, CM-ß-CD concentration, buffer type and concentration were studied in relation to chiral resolution. Excellent enantiomeric separations were obtained for all 16 compounds, especially for procaterol. An impressive resolution value, up to 17.10, was obtained. In particular, most of them achieved rapid separations within 20 min. Given the fact that enantioseparation results rely on analytes' structural characters, the possible separation mechanisms were discussed. In addition, in order to obtain faster separation for propranolol enantiomers in practical application, the effective length of capillary was innovatively shortened from 45 to 30 cm. After the validation, the method was successfully applied to the enantiomeric purity determination of propranolol in the formulation of drug substances.

Occurrence of cardiovascular drugs in the sewage-impacted Vistula River and in tap water in the Warsaw region (Poland).

In recent years, cardiovascular diseases were the second most common cause of death worldwide. Therefore, the consumption of drugs used to treat cardiovascular diseases is high. So far, there were no such comprehensive reports regarding the presence of cardiovascular drugs in surface and tap waters, particularly in Central and Eastern Europe. The aim of our study was to determine the presence of 30 pharmaceutically active compounds and some of their metabolites, at specific points of the Vistula River and in tap water samples in the Warsaw region. The analysis was performed using the liquid chromatography-electrospray ionization-tandem mass spectrometry method, coupled to solid-phase extraction. To the best of the authors' knowledge, this is the first time where the presence of ciprofibrate in the environment was investigated. Cardiovascular drugs found at the highest concentrations (reaching 1 µg/L or higher) in surface water were beta-blockers, sartans and diuretics. In tap water samples, trace amounts of pharmaceuticals were detected, for almost all target compounds. This highlights their inadequate elimination by the treatment facility used in the Warsaw region. The presence of cardiovascular compounds in the aquatic environment could have a long-term effect even at a low exposure level, since synergy effects amongst pharmaceuticals may occur.

Determination of selected cardiovascular active compounds in environmental aquatic samples--Methods and results, a review of global publications from the last 10 years.

In recent years cardiovascular diseases were the second most common cause of death worldwide. Therefore, the consumption of cardiovascular drugs is high, which might result in an increase of them in the environment. The major source of aquatic environmental contamination is still effluents of wastewater treatment plants (WWTPs). Unfortunately removal of cardiovascular active compounds and/or their metabolites in WWTP is still unsatisfactory. Among microbial and abiotic degradation of these compounds during wastewater processes, photolysis and photodegradation of cardiovascular drugs also play an important role. New formed compounds may be more toxic or retain the properties of parent compounds. Thus the main goal of this paper was to provide a detailed and comprehensive review of used analytical methods, coupled to liquid chromatography-tandem mass spectrometry, to determine the presence of cardiovascular compounds in surface waters as well as WTTPs effluents and influents. Exhaustive preparation for mass spectrometry detection and quantitation including samples pre-treatment, and the common problem of the matrix effect are thoroughly explored in this paper. Additionally, the article provides some hints in respect of recently noted problematic issue related to the availability of specific standards for the analysis of drug's metabolites. Furthermore, information concerning the metabolism of cardiovascular active compounds including differences in metabolism within enantiomers is described. This article also touches on the problems associated with environmental risk assessment due to the presence of cardiovasculars in the environment. The paper also tries to explain differences in concentrations among cardiovascular compounds between countries worldwide.

Determination of the cardiac drug amiodarone and its N-desethyl metabolite in sludge samples.

For the first time, a procedure for the simultaneous determination of the iodinated drug amiodarone and its major metabolite, N-desethylamiodarone, in sludge from urban sewage treatment plants (STPs) is proposed. Matrix solid-phase dispersion (MSPD) followed by on-line cationic exchange clean-up, in modular configuration, was used as sample preparation technique. Liquid chromatography with tandem mass spectrometry (LC-MS/MS), based on a hybrid quadrupole time-of-flight (QTOF) system, was employed for the selective determination of target compounds. The optimized procedure provided exhaustive recoveries with little effect of the sample matrix in the efficiency of electrospray ionization (ESI). The overall recoveries of the method ranged between 95 and 111%, for samples spiked at different concentration levels. The achieved limits of quantification (LOQs) remained below 10ngg(-1) for both compounds, and the linear response range extended up to 2500ngg(-1). Amiodarone and N-desethylamiodarone were ubiquitous in sludge samples, from different STPs located in the Northwest of Spain, with maximum concentrations above 300ngg(-1) referred to the freeze-dried matrix. They were also present in stabilized sludge (mixed with lime and thermally dehydrated), which is mostly disposed in agriculture fields as fertilizer. Furthermore, mono-iodinated analogues of amiodarone and N-desethylamiodarone were also tentatively identified in some samples from their accurate MS and MS/MS spectra.

Simultaneous quantitation of aspirin, amlodipine and simvastatin in a fixed dose combination of encapsulated tablet formulation by HPLC-UV method.

A high-pressure liquid chromatography (HPLC-UV) based simple and specific method for simultaneous quantitative determination of aspirin, amlodipine besylate and simvastatin in a capsule formulation has been developed and validated according to ICH guidelines. Chromatographic separation of the three drugs was carried out by aSpherisorbODS2 reverse phase column (4.6 x 250 mm; 5 µm) using amobile phase, which consisted of 70: 30 (v/v) mixture of acetonitrile and triethylamine phosphate buffer (pH 3; 0.015 M) with final pH adjusted to 2.5 using dilute ortho-phosphoric acid, at a flow rate of 1mL/min. The eluents were detected at UV wavelength of 237 nm and the retention times for aspirin, amlodipine besylate and simvastatin were ~2.7 mins, ~6.1 mins and ~10.5mins, respectively. This method is suitable and specific for the three drugs and was found to be linear (R2>0.995), accurate, specific, reproducible and robust in the concentration range of 375 to 1125mcg/ml for aspirin, 25 to 75mcg/ml for amlodipine besylate and 50 to 150mcg/ml for simvastatin. This simple and convenient method could be easily utilized for the characterization and quantitation of the three drugs in a single formulation for combination therapy of cardiovascular diseases.

Cardiovascular drugs in environmental waters and wastewaters: method optimization and real sample analysis.

A sensitive method for determination of the eight most prescribed drugs used in combined cardiovascular therapy in Serbia was developed and optimized. The method was based on SPE followed by LC/ion trap-MS/MS with positive electrospray ionization. Parameters that affect the SPE were optimized, such as the eluent, sample pH, and sample volume. Good recoveries from groundwater (87.6-120.9%) as well as wastewater (84.5-106.6%) were achieved with this method, except in the case of atorvastatin (26.1 and 45.2%, respectively). The method was applied in the analysis of four river water samples collected in Serbia, as well as nine corresponding groundwater samples. Residues of the P-blockers metoprolol and bisoprolol as well as the anticoagulant clopidogrel were detected for the first time in river water. Groundwater samples did not contain drug residues. Influents and effluents of two wastewater treatment plants showed the predominant presence of metoprolol and enalapril. The removal rate of metoprolol was generally low, whereas enalapril was eliminated with the highest efficiency. Atorvastatin was detected in influents and completely removed in the treatment.

Current approaches to glycoprotein analysis.

Glycoproteins are becoming increasingly relevant in therapeutics, including tissue plasminogen activator for the treatment of myocardial infarction and strokes, erythropoietin for anemia and various monoclonal antibody-based treatments for cancer. Protein N- and O-glycosylation is perhaps the most crucial and immensely complex posttranslational modification that proteins undergo, and its characterization presents a major challenge. This review will discuss current techniques for the characterization of glycoproteins, with a focus on therapeutic glycoproteins where available. The crucial analytical techniques, such as high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), and mass spectrometry (MS) will be described, alongside the necessary chemical labeling methods for sensitive detection. The well-established chemical and enzymatic methods for oligosaccharide release from proteins will be discussed, as will more modern methods based on exhaustive protein hydrolysis with non-specific proteases.

Removal and seasonal variability of selected analgesics/anti-inflammatory, anti-hypertensive/cardiovascular pharmaceuticals and UV filters in wastewater treatment plant.

Seasonal removal efficiency of 16 pharmaceuticals and personal care products was monitored in a wastewater treatment plant in Ceské Budejovice, Czech Republic, over a period of 1 year (total amount of samples, n = 272). The studied compounds included four UV filters, three analgesics/anti-inflammatory drugs and nine anti-hypertensive/cardiovascular drugs. In most cases, elimination of the substances was incomplete, and overall removal rates varied strongly from -38 to 100%. Therefore, it was difficult to establish a general trend for each therapeutic group. Based on the removal efficiencies (REs) over the year, three groups of target compounds were observed. A few compounds (benzophenon-1, valsartan, isradipine and furosemide) were not fully removed, but their REs were greater than 50%. The second group of analytes, consisting of 2-phenylbenzimidazole-5-sulfonic acid, tramadol, sotalol, metoprolol, atenolol and diclofenac, showed a very low RE (lower than 50%). The third group of compounds showed extremely variable RE (benzophenon-3 and benzophenon-4, codeine, verapamil, diltiazem and bisoprolol). There were significant seasonal trends in the observed REs, with reduced efficiencies in colder months.

Improving sensitivity in microchip electrophoresis coupled to ESI-MS/MS on the example of a cardiac drug mixture.

A comprehensive study for a sensitivity optimization in MCE with mass spectrometric detection is presented. As a text mixture, we chose a mixture of the cardiac drugs propranolol, bisoprolol, lidocaine, procaine and studied the effect of different chip layouts and experimental parameters with the aim of achieving both high sensitivity in MS detection and adequate chip electrophoretic separation. An important aspect was a comparison of microfluidic layouts containing various sheath-flow channels with that avoiding sheath-flow junctions on-chip. We utilized glass chips with monolithically integrated nanospray emitter tips coupled dead volume-free to an IT mass spectrometer running in fragmentation mode (MS(n) ). With this setup, detection limits down to 0.6 ng/mL for the model compound propranolol were achieved.