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A quick and sensitive RP-UHPLC-ESI-MS/MS method for the separation of flavanone, naringenin and hesperetin enantiomers was developed. The separation of analytes was performed using a Chiralpak AD-3R column, and methanol was used as the mobile phase. Detection was carried out using a triple quadrupole tandem mass spectrometer with an electrospray ionisation source. Positive ionisation and multiple reaction monitoring (MRM) were used. The developed method showed satisfactory linearity with determination coefficients greater than 0.996 in the concentration ranges of 2.5-100.0ngmL(-1) for naringenin and flavanone enantiomers and 0.5-100.0ngmL(-1) for hesperetin enantiomers. The limits of quantification varied from 0.1 to 2.0ngmL(-1). The intra-day and inter-day precisions were below 15%, and the accuracy varied from -13.6% to 13.5%. The described method was successfully applied for the chiral separation and determination of flavonoid enantiomers in real samples of spices and herbal root. Due to the occurrence of the natural compounds in the forms of free aglycones and their glycosides, these samples were subjected to hydrolysis in order to obtain free aglycones from the glycosylated forms. Acid and enzymatic hydrolysis techniques were also compared. In the course of this study, the enzymatic hydrolysis technique was selected., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this study, a new analytical method was developed and validated for the simultaneous analysis of β-blockers (metoprolol, propranolol, carvedilol) and their metabolites (5'-hydroxycarvedilol, O-desmethylcarvedilol, α-hydroxymetoprolol, O-desmethylmetoprolol, 5-hydroxypropranolol) in human urine. A salting-out assisted liquid-liquid extraction (SALLE) procedure was used for sample preparation. Several parameters affecting the extraction efficiency and method sensitivity including the type and volume of the extraction solvent, the type and quantity of the inorganic salt, extraction time and sample pH were investigated. Hydrophilic interaction liquid chromatography-ultraviolet detection (HILIC-UV) was used for the determination of all analytes. During method development, the effects of mobile phase components (type, pH, concentration of salt, organic modifier type and content, flow rate, column temperature) on the retention and separation of β-blockers and metabolites on the five different HILIC columns were examined. The method was linear for concentrations ranging from 0.1 to 8.0μg/mL, with determination coefficients higher than 0.993 for all analytes. The limits of quantification were in the range from 0.1 to 0.2μg/mL. Intra- and inter-day precision ranged from 0.1 to 8.9%, and accuracy was within±13% interval for all analytes. Under the optimized conditions, extraction efficiency was greater than 83.4% for determined compounds. The validated method was then applied to the measurement of β-blockers and their metabolites in human urine samples., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A sensitive, stereoselective assay using solid phase extraction and high-performance liquid chromatography (HPLC) with fluorescence detection (FLD) was developed and validated for the analysis of enantiomers of metoprolol and its metabolites (α-hydroxymetoprolol, O-desmethylmetoprolol). Chiral separation was achieved using a CHIRALCEL OD-RH column, packed with cellulose tris-(3,5-dimethylphenyl-carbamate) stationary phase, employing a mobile phase composed by a mixture of 0.2% diethylamine in water and acetonitrile in gradient elution mode. Linear calibration curves were obtained over the range of 0.025-2.0μg/mL (R(2)>0.994) in urine for both enantiomers of metoprolol and its metabolites with quantitation limit of 0.025μg/mL. Intra and inter-day precision and accuracy were below 15% for both metoprolol and metabolites enantiomers. The recovery of enantiomer of metoprolol and its metabolite was greater than 68.0%, utilizing a SPE procedure. The method was tested with urine quality control samples and human urine fractions after administration of 50mg rac-metoprolol., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Given the increasing popularity of aliskiren, particularly in combination with angiotensin converting enzyme inhibitor (e.g. enalapril), it is important to determine whether its use in combination with these agents is associated with potentially life threatening safety events. Analytical methods for the simultaneous determination of both drugs in plasma and urine utilized in clinical studies on efficacy and safety have not been fully described in the literature. In this work, a new, fast and reliable method using a digitally controlled microextraction by packed sorbent (eVol(®)-MEPS) followed by ultra-high performance liquid chromatography (UHPLC) coupled with tandem mass spectrometry (MS/MS) was developed and validated to quantify an aliskiren, enalapril and its active metabolite in both human plasma and urine. Chromatographic separation was accomplished on a Poroshell 120 EC-C18 column with a gradient elution system consisting of 0.1% formic acid in water and acetonitrile (1.5min of total analysis). Detection was performed by multiple reaction monitoring (MRM) mode using electrospray ionization in the positive ion mode. This assay method has been fully validated in terms of selectivity, linearity, accuracy, precision, stability, recovery and matrix effect. The developed method can be applied to the routine determination of selected compounds in human plasma and urine and can be useful to elucidate the mechanisms of the potential risks triggered by the combination of aliskiren and enalapril as well as its active metabolite enalaprilat., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A procedure based on solid-phase extraction (SPE) followed by ultra-high-performance liquid chromatography (UHPLC) with UV detection has been developed for the analysis of multiple drugs in human urine. The compounds evaluated were aliskiren, prasugrel, rivaroxaban, prednisolone, propranolol, ketoprofen, nifedipine, naproxen, terbinafine, ibuprofen, diclofenac, sildenafil and acenocoumarol. Seventeen different solid phase extraction (SPE) cartridges were tested to evaluate their applicability for the isolation of drugs from human urine. Comparison were recovery of different drugs and reproducibility. The samples were analyzed by UHPLC using a Poroshell 120 EC-C18 column and acetonitrile -0.05% TFA in water as the mobile phase under gradient elution conditions. SPE combined with UHPLC-UV allowed the determination of drugs over a linear range of 0.01-30.0μg/mL, with limits of detection at 0.003-0.217μg/mL and precision of 0.8-7.1%. Phenyl (C6H5) sorbent was found to provide the most effective clean-up, removing the greatest amount of interfering substance and simultaneously ensuring analyte recoveries higher than 85.5% with relative standard deviations (RSD) <10%. The method was applied with good accuracy and precision in the determination of drugs in human urine obtained from patients treated with selected drugs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A sensitive and selective ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method was developed for the fast, simultaneous quantification of three novel cardiac drugs (aliskiren, prasugrel and rivaroxaban) in human urine. Sample preparation was performed with microextraction with packed sorbent (MEPS), which is a miniaturization of solid phase extraction. The optimal conditions for MEPS extraction were obtained using C8 sorbent, small sample volumes and a short time period (about 3min for the entire sample preparation step). Chromatographic separation of the selected compounds was achieved in less than 1.5min on a Zorbax Rapid Resolution High Definition SB-C18 column using isocratic elution with 0.1% formic acid and acetonitrile (70:30, v/v) at a flow rate of 0.8mLmin(-1). The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via an electrospray ionization source with positive ionization mode. The method was fully validated according to the latest recommendations of international guidelines. The lower limit of quantification was 5.0pgmL(-1) for aliskiren and rivaroxaban and 0.5pgmL(-1) for prasugrel. The intra- and inter-day precision was within 7.12% and the accuracy ranged from -7.54% to 4.17%. The mean extraction recoveries of the MEPSC8 methodology were found to be 98.3% for aliskiren, 100.3% for rivaroxaban and 99.9% for prasugrel. This MEPSC8-UHPLC-MS/MS method offers a fast, simple and precise way to determine selected novel cardiac drugs in human urine that could be applied to therapeutic drug monitoring and pharmacokinetic studies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A new approach based on microextraction by packed sorbent (MEPS) and a reversed-phase ultra-high pressure liquid chromatography (UHPLC) method was developed and validated for the determination and quantification of nonsteroidal anti-inflammatory drugs (NSAIDs) (acetylsalicylic acid, ketoprofen, diclofenac, naproxen and ibuprofen) in human urine. The important factors that could influence the extraction were previously screened using the Plackett-Burman design approach. The optimal MEPS extraction conditions were obtained using C18 phase as a sorbent, small sample volume (20μL) and a short time period (approximately 5min) for the entire sample preparation step. The analytes were separated on a core-shell column (Poroshell 120 EC-C18; 100mm×3.0mm; 2.7μm) using a binary mobile phase composed of aqueous 0.1% trifluoroacetic acid and acetonitrile in the gradient elution mode (4.5min of analysis time). The analytical method was fully validated based on linearity, limits of detection (LOD), limits of quantification (LOQ), inter- and intra-day precision and accuracy, and extraction yield. Under optimised conditions, excellent linearity (R(2)>0.9991), limits of detection (1.07-16.2ngmL(-1)) and precision (0.503-9.15% RSD) were observed for the target drugs. The average absolute recoveries of the analysed compounds extracted from the urine samples were 89.4-107%. The proposed method was also applied to the analysis of NSAIDs in human urine. The new approach offers an attractive alternative for the analysis of selected drugs from urine samples, providing several advantages including fewer sample preparation steps, faster sample throughput and ease of performance compared to traditional methodologies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

One of the major challenges facing the medicine today is developing new therapies that enhance human health. To help address these challenges the utilization of analytical technologies and high-throughput automated platforms has been employed; in order to perform more experiments in a shorter time frame with increased data quality. In the last decade various analytical strategies have been established to enhance separation speed and efficiency in liquid chromatography applications. Liquid chromatography is an increasingly important tool for monitoring drugs and their metabolites. Furthermore, liquid chromatography has played an important role in pharmacokinetics and metabolism studies at these drug development stages since its introduction. This paper provides an overview of current trends in fast chromatography for the analysis of cardiovascular drugs and their metabolites in clinical applications. Current trends in fast liquid chromatographic separations involve monolith technologies, fused-core columns, high-temperature liquid chromatography (HTLC) and ultra-high performance liquid chromatography (UHPLC). The high specificity in combination with high sensitivity makes it an attractive complementary method to traditional methodology used for routine applications. The practical aspects of, recent developments in and the present status of fast chromatography for the analysis of biological fluids for therapeutic drug and metabolite monitoring, pharmacokinetic studies and bioequivalence studies are presented., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A simple, accurate and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitation of α-ketoglutaric acid (α-KG), L-carnitine (L-CAR) and acetyl-L-carnitine (acetyl-L-CAR) in human urine as potential biomarkers of cardiovascular disease. The separation was performed using an isocratic elution of 0.1% formic acid in water and acetonitrile (97:3, v/v) on an Acclaim 120 C8 column (150 mm × 4.6 mm, 3.0 μm). The flow rate of the mobile phase was 1.2 mL/min and the total assay run time was 3 min. Detection was performed on a triple-quadrupole mass spectrometer in selected reaction monitoring (SRM) mode via an electrospray ionization (ESI) source in positive and negative ion modes. This method covered a linearity range of 0.1-500 ng/mL for L-CAR and acetyl-L-CAR and 1-1000 ng/mL for α-KG with lower limits of quantification (LLOQ) of 0.08 ng/mL for L-CAR, 0.04 ng/mL for acetyl-L-CAR and 0.8 ng/mL for α-KG. The intra-day and inter-day precision and accuracy of the quality control samples exhibited relative standard deviations of less than 5.54% and relative error values from -5.95% to 3.11%. Analyte stability was evaluated under various sample preparation, analysis and storage conditions and varied from -9.89% to -0.47%. A two-step solid-phase extraction (SPE) procedure using silica gel and quaternary amine cartridges was used for urine sample cleanup. The average recoveries for all analyzed compounds were better than 86.64% at three concentrations. The method was successfully applied for the quantitation of α-KG, L-CAR and acetyl-L-CAR in human urine samples., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A sensitive ultra-performance liquid chromatography tandem mass spectrometry method with electrospray ionisation (UHPLC-ESI-MS/MS) was developed for the simultaneous determination of 52 compounds: β-blockers, polyphenols (antioxidants) and their metabolites in mixture of standards and after addition the 52 standard solutions to human urine samples. The analyses of urine samples obtained from patients treated with β-blockers were also carried out. The separation of analytes was performed on a Hypersil GOLD™ column (100 mm × 2.1mm, 1.9 μm) using a gradient elution profile for 10 min and mobile phase consisting of 0.1% formic acid in water and acetonitrile. In these conditions, some of the tested compounds were not separated, but this was compensated by the use of MS/MS detection. The drugs, polyphenols and their metabolites were detected with a tandem mass spectrometer after being ionised positively or negatively (depending on the molecule) using an electrospray ionisation (ESI) source. The MS system was operated in the selected reaction monitoring (SRM) mode, where one quantitation and one confirmation transition was done for each analyte. The quantitative method was validated for selectivity, linearity, low limits of quantitation, accuracy, precision, recovery, matrix effect and analyte stability. The LLOQ varied from 0.01 to 0.40 ng mL(-1) for β-blockers and from 0.05 to 40.0 ng mL(-1) for polyphenols. The linear range was 0.08-1000 ng mL(-1) for the drugs and 0.10-2300 ng mL(-1) for the polyphenols. Intra-day and inter-day precision was less than 8%, and the accuracy ranged from -4.40 to 2.23% for all analytes. The average recoveries for all compounds analysed were better than 90%. The developed method can be successfully used to monitor cardiovascular drugs and their metabolites in urine samples of patients treated with β-blockers and can also be used to study the effect of polyphenols on the metabolism of drugs., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A rapid-resolution ultra high-performance liquid chromatography separation method (UHPLC) for the simultaneous determination of the following β-blockers: milrinone, sotalol, metoprolol, propranolol and carvedilol, and their metabolites: 5'-hydroxylphenyl-carvedilol, O-desmethylcarvedilol, 4-hydroxypropranolol, α-hydroxy-metoprolol, O-desmethyl-metoprolol; the following isoflavones: genistein, daidzein, glycitin, glycitein, puerarin and biochanin A; as well as their metabolites: dihydrogenistein, desmethylglycitein, 8-hydroxygenistein, daidzein-7,4'-diglucoside, 8-hydroxydaidzein, dihydrobiochanin A in human urine was optimized. The analysed compounds were extracted from human urine by means of solid phase extraction (SPE). The effective UHPLC separation of the examined compounds was applied on a Hypersil GOLD™ (50 mm×2.1 mm, 1.9 μm) column with a gradient mobile phase system and a UV detector. The complete separation of all analytes was achieved within 8.0 min. The method was validated for the determination of the aforementioned substances in human urine. The linear ranges, limits of detection (LOD) and limits of quantification (LOQ) for β-blockers, isoflavones and their metabolites were determined. The intra- and inter-day precision (%C.V.) was less than 4.48%, and the intra-day and inter-day accuracy was less than 4.74%. The tested SPE sorbent proved that appropriate absolute recoveries can be obtained for Oasis HLB (Waters). The mean recovery of the analytes, using the new SPE procedure, amounted from 70.14% to 99.85%. The present paper reports, for the first time, the method for the determination of β-blockers, isoflavones and their metabolites in human urine samples. The newly developed method was suitably validated and successfully applied for the analysis of the certain of the aforementioned analytes in human urine samples obtained from the patients suffering cardiovascular disease., (Copyright © 2011 Elsevier B.V. All rights reserved.)