Simple,sensitive and rapid HPLC–MS/MS method for the determination of cepharanthine in human plasma

A rapid, sensitive and specific method for the determination of cepharanthine in human plasma using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) was described. Cepharanthine and the internal standard (I.S.), telmisartan, were extracted from human plasma by methanol to precipitate the protein. A centrifuged upper layer was then evaporated and reconstituted with 100 μL methanol. Chromatographic separation was performed on an AGILENT XDB-C₈ column (150 mm × 2.1 mm, 5.0 μm, Agilent, USA) using a gradient mobile phase with 1 mmol/L ammonium acetate in water with 0.05% formic acid and methanol. Detection and quantitation was performed by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the positive ion mode. The most intense [M+H]⁺ MRM transition of cepharanthine at m/z 607.3 → 365.3 was used for quantitation and the transition at m/z 515.5 → 276.4 was used to monitor telmisartan. The calibration curve was linear within the concentration range of 0.5–200.0 ng/mL (r = 0.9994). The limit of quantification (LOQ) was 0.5 ng/mL. The extraction recovery was above 81.1%. The accuracy was higher than 92.3%. The intra- and inter-day precisions were less than 9.66%. The method was accurate, sensitive and simple and was successfully applied to a pharmacokinetic study after single intravenous administration of 50 mg cepharanthine in 12 healthy Chinese volunteers.     

A simple and sensitive HPLC–ESI-MS/MS method for the determination of andrographolide in human plasma

A liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS) method for the determination of andrographolide in human plasma was established. Dehydroandrographolide was used as the internal standard (I.S.). The plasma samples were deproteinized with methanol and separated on a Hanbon C₁₈ column with a mobile phase of methanol–water (70:30, v/v). HPLC–ESI-MS/MS was performed in the selected ion monitoring (SIM) mode using target ions at [M?H₂O–H]^?, m/z 331.1 for andrographolide and [M?H]^?, m/z 331.1 for the I.S. Calibration curve was linear over the range of 1.0–150.0 ng/mL. The chromatographic separation was achieved in less than 6.5 min. The lower limits of quantification (LLOQ) was 1.0 ng/mL. The intra and inter-run precisions were less than 6.95 and 7.22%, respectively. The method was successfully applied to determine the plasma concentrations of andrographolide in Chinese volunteers.     

A rapid gas chromatographic method for the determination of poly-β-hydroxybutyric acid in microbial biomass

Summary The gas chromatographic method for the determination of poly--hydroxybutyric acid (PHB) consists of a mild acid or alkaline methanolysis of poly--hydroxybutyric acid directly without previous extraction of PHB from the cells; this is followed by gas chromatography of the 3-hydroxybutyric acid methylester. The method is characterized by high accuracy and excellent reproducibility, permitting determinations as low as 10^–5 g/l. Only 4 h is required from sampling from the fermenter till completion of the PHB determination.     

Effect of colloidal β-cyclodextrins-Fe(3) O(4) magnetic nanoparticles on the chemiluminescence enhancement of luminol-Ag(III) complex for rapid and sensitive determination of cysteine in human serum

Colloidals solution of Fe₃O₄ magnetic nanoparticles (MNPs), capped with β‐cyclodextrins (β‐CD) as inclusion complexes, were found to enhance the chemiluminescence (CL) intensity of the luminol–diperiodatoargentate(III) (DPA) system. On injection of cysteine into the luminol–DPA–β‐CD–Fe₃O₄ MNPs inclusion complexes system, the CL intensity is strongly enhanced. The enhanced CL signal is ascribed to the catalytic effect of Fe₃O₄ MNPs capped with β‐CD, which is assumed to stabilize the CL intermediate. Based on these findings, a rapid and sensitive assay was developed for the determination of cysteine in human serum. The effects of analytical variables on the CL signal were studied and optimized. Under the optimum conditions, the CL intensity was directly proportional to the concentration of cysteine in the range 8.0 × 10^–9–1.0 × 10^–6 mol/L. The detection limit was 2.8 × 10^–9 mol/L (3 S_b/m) and the relative standard deviation (RSD) for 10 replicate determinations of 1.0 × 10^–7 mol/L cysteine was 3.5%. The proposed method was applied to the sensitive determination of cysteine in human serum samples, and compared with the Ellman method with satisfactory results. Copyright © 2011 John Wiley & Sons, Ltd.     

Effect of K+ channels in the apical plasma membrane on epithelial secretion based on secondary active Cl− transport

Summary Models of epithelial salt secretion, involving secondary active transport of Cl^– [9], locate the K⁺ conductance of the plasma membrane exclusively in the basolateral membrane, although there is considerable experimental evidence to show that many secretory epithelia do have a significant apical K⁺ conductance. We have used an equivalent circuit model to examine the effect of an apical K⁺ conductance on the composition and flow rate of the fluid secreted by an epithelium in which secretion is driven by the secondary active transport of Cl^–. The parameters of the model were chosen to be similar to those measured in the dog tracheal mucosa when stimulated with adrenaline to secrete. We find that placing a K⁺ conductance in the apical membrane can actually enhance secretion provided that proportion of the total cell K⁺ conductance in the apical membrane is not greater than about 60%, the enabling effect on secretion being maximal when the proportion is around 10–20%. We also find that even when the entire cell K⁺ conductance is located in the apical membrane, the secreted fluid remains relatively Na⁺ rich. Analysis of the sensitivity of model behavior to the choice of values for the parameters shows that the effects of an apical K⁺ conductance are enhanced by increasing the ratio of the paracellular resistance to the transcellular resistance.     

Differential Effect of High Extracellular Ca2+ on K+ and Cl− Conductances in Murine Osteoclasts

Effects of the extracellular Ca²⁺ concentration ([Ca²⁺] _o ) on whole cell membrane currents were examined in mouse osteoclastic cells generated from bone marrow/stromal cell coculture. The major resting conductance in the presence of 1 mm Ca²⁺ was mediated by a Ba²⁺-sensitive, inwardly rectifying K⁺ (IR_K) current. A rise in [Ca²⁺] _o (5–40 mm) inhibited the IR_K current and activated an 4,4′-diisothiocyano-2,2′-stilbenedisulfonate (DIDS)-sensitive, outwardly rectifying Cl⁻ (OR_Cl) current. The activation of the OR_Cl current developed slowly and needed higher [Ca²⁺] _o than that required to inhibit the IR_K current. The inhibition of the IR_K current consisted of two components, initial and subsequent late phases. The initial inhibition was not affected by intracellular application of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) or guanosine 5′-O-(2-thiodiphosphate) (GDPβS). The late inhibition, however, was enhanced by GTPγS and attenuated by GDPβS, suggesting that GTP-binding proteins mediate this inhibition. The activation of the OR_Cl current was suppressed by pretreatment with pertussis toxin, but not potentiated by GTPγS. An increase in intracellular Ca²⁺ level neither reduced the IR_K current nor activated the OR_Cl current. Staurosporine, an inhibitor for protein kinase C, did not modulate the [Ca²⁺] _o -induced changes in the IR_K and OR_Cl conductances. These results suggest that high [Ca²⁺] _o had a dual action on the membrane conductance of osteoclasts, an inhibition of an IR_K conductance and an activation of an OR_Cl conductance. The two conductances modulated by [Ca²⁺] _o may be involved in different phases of bone resorption because they differed in Ca²⁺ sensitivity, temporal patterns of changes and regulatory mechanisms. Received: 28 May 1996/Revised: 28 January 1997     

New method for the determination of doxorubicin, 4′-epidoxorubicin and all known metabolites in cardiac tissue

4′-Epidoxorubicin, doxorubicin (internal standard) and eight metabolites were extracted from heart tissue homogenate by a mixture of tetrahydrofuran-water (1:2, v/v) and purified by C₁₈ Sep-Pak cartridges. The buffer used to prepare the homogenate contained glucaric acid-1,4-lactone and glucose, to prevent decomposition of the 4′ -epidoxorubicin glucuronides. Anthracyclines were separated by high-performance liquid chromatography within 14 min and detected by fluorescence. Recoveries ranged from 49 to 75%. The detection limits of the individual anthracyclines ranged from 0.5·10⁻¹¹ to 2.5·10⁻¹¹ mol/g wet weight. The peak-height ratios of the fluorescence intensities of the anthracyclines versus doxorubicin were linear from 2.5·10⁻¹¹ to 250·10⁻¹¹ mol/g wet weight. Within- and between-day precisions of the assay varied between the anthracyclines and were in the ranges 3–12% (n=6) and 2–11% (n=6), respectively.     

Rapid,highly sensitive method for the determination of morphine and its metabolites in body fluids by liquid chromatography–mass spectrometry

A rapid, highly sensitive method for the determination of morphine and its metabolites morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G) and normorphine has been developed using high-performance liquid chromatography–electrospray mass spectrometry, with the deuterated analogues as internal standards. The analytes were extracted automatically using end-capped C₂ solid-phase extraction cartridges. Baseline separation of morphine, M3G and M6G was achieved on a LiChrospher 100 RP-18 end-capped analytical column (125×3 mm I.D., 5 μm particle size) with water–acetonitrile–tetrahydrofuran–formic acid (100:1:1:0.1, v/v) as the mobile phase. Morphine and normorphine coeluate and were separated mass spectrometrically. The mass spectrometer was operated in the selected-ion monitoring mode using m/z 272 for normorphine, m/z 286 for morphine, m/z 462 for morphine-6-glucuronide. Due to an interfering peak, M3G was measured by tandem mass spectrometry in the daughter-ion mode. The limits of quantitation achieved with this method were 1.3 pmol/ml for morphine, 1.5 pmol/ml for normorphine, 1.0 pmol/ml for M6G and 5.4 pmol/ml for M3G in serum or cerebrospinal fluid. The limits of quantitation achieved in urine were 10 pmol/ml for morphine, 20 pmol/ml for normorphine and M6G and 50 pmol/ml for M3G using a sample size of 100 μl. The method described was successfully applied to the determination of morphine and its metabolites in human serum, cerebrospinal fluid and urine in pharmacokinetic and drug interaction studies.     

Development of a sensitive and selective LC-MS/MS method for the determination of α-fluoro-β-alanine, 5-fluorouracil and capecitabine in human plasma

A sensitive and selective quantitative method to determine α-fluoro-β-alanine (FBAL), 5-fluorouracil (5-FU), and capecitabine (Cape) from a single human plasma aliquot (50 μL) has been developed and validated. First, 5-FU and Cape were extracted by liquid–liquid extraction (LLE) using a mixture of acetonitrile and ethyl acetate. This was followed by derivatization with dansyl chloride. The dansyl-derivatives from 5-FU and Cape were further purified using LLE with methyl tertiary-butyl ether (MTBE) and analyzed using a reversed-phase analytical column “Primesep D” (2.1 mm × 50 mm; 5 μm) with embedded basic ion-pairing groups. The remaining aqueous phase containing FBAL was treated with dansyl chloride and the dansyl-FBAL was purified by solid phase extraction. Ultra high pressure liquid chromatography (UPLC) technology on a BEH C18 stationary phase column with 1.7 μm particle size was used for analysis of dansyl-FBAL. The method was validated over the concentration ranges of 10–10,000, 5–5000, and 1–1000 ng/mL for FBAL, 5-FU, and Cape, respectively. The results from assay validation show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies where approximately 300 samples can be extracted and analyzed in 1 day.     

The effect of air containing O2 −,O2 +, CO2 − and CO2 + on the growth of seedlings ofHordeum Vulgaris

Equipment was devised which permitted the addition of specific gaseous ions to the atmosphere of plastic chambers in which seedlings of HORDEUM VULGARIS were grown in sand culture supplied with chemically defined nutrient solutions. Moderate densities of O₂ ^– or O₂ ⁺ ions (1.8×10⁴/cm³)in air containing an added 8% of O₂ accelerated the growth rate. A like number of CO₂ ^– or CO₂ ⁺ ions in air containing 8% of CO₂ inhibited growth, impeded the production of chlorophyll and devitalized the young seedlings. Evidence is presented that O₂ ^– and O₂ ⁺ stimulate the production of cytochromes and other Fe-containing enzymes through their action on the plant regulatory system responsible for the control of Fe metabolism. The toxic effect of CO₂ ^– and CO₂ ⁺ cannot be explained as yet.

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Zusammenfassung Eine Apparatur wurde entwickelt, die die Zufuhr von ionisiertem Gas der AtmosphÄre in Kammern gestattet. Darin wurden Keimlinge von HORDEUM VULGARIS in Sand mit chemisch definierten NÄhrlösungen gezüchtet. Konzentrationen von 1,8×10⁴/cm³ O₂ ^– und O₂ ⁺ in Luft mit zusÄtzlich 8% O₂ beschleunigten die Wachstumsrate. Die gleiche Menge CO₂ ^– und CO₂ ⁺ in Luft mit zusÄtzlich 8% CO₂ hemmte die Wachstumsrate, die Bildung von Chlorophyll und entkrÄftigte die Keimlinge. Es wird gezeigt,dass O₂ ^– und O₂ ⁺ die Bildung von Cytochrom und anderen eisenhaltigen Enzymen anregen durcn ihre Wirkung auf das den Fe-Stoffwechsel regulierende System der Pflanze. Die toxische Wirkung von CO₂ ^– und CO₂ ⁺ lÄsst sich noch nicht erklÄren.

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Resume On a construit un appareil permettant d'introduire dans 1'atmosphères des ions de gaz déterminés. On a alors effectué de telles adjonctions à l'air contenu dans des cellules de plastique dans lesquelles on cultivait HORDEUM VULGARIS sur du sable et dans une solution nutritive chimiquement définie. Des densités modérées d'ions O₂ ^– ou O₂ ⁺ (1,8×10⁴/cm³) dans de l'air additionné de 8% d'O₂ accélèrent la croissance. La meme concentration de CO₂ ^– et CO₂ ⁺ additionnée de 8% de CO₂ a ralenti la croissance et la formation de chlorophylle et a diminué la vitalite des plantes nouvellement germées. On démontre que O₂ ^– et O₂ ⁺ active la formation de cytochrome et d'autres enzymes ferreuses par suite de l'action de ces ions sur le système régularisant le métabolisme du fer dans la plante. L'effet toxique du CO₂ ^– et CO₂ ⁺ reste encore inexpliqué.

     

Fluorescence ‘on-off-on’ chemosensor for sequential recognition of Fe3+ and Hg2+ in water based on tetraphenylethylene motif

A novel selective and sensitive fluorescence ‘on-off-on’ probe based on tetraphenylethylene (TPE) motif for sequential recognition of Fe³⁺ and Hg²⁺ in water has been developed. Especially the complex 6-Fe³⁺ could behave as a ‘turn on’ fluorescent sensor over a wide-range pH value for detection of Hg²⁺. The selectivity of this complex for Hg²⁺ over other heavy and transition metal ions is excellent, and its sensitivity for Hg²⁺ is at 2 ppb in water.     

C3−C4 Intermediate species in the genus Flaveria: leaf anatomy,ultrastructure, and the effect of O2 on the CO2 compensation concentration

Leaf anatomical, ultrastructural, and CO₂-exchange analyses of three closely related species of Flaveria indicate that they are C₃–C₄ intermediate plants. The leaf mesophyll of F. floridana J.R. Johnston, F. linearis Lag., and F. chloraefolia A. Gray is typical of that in dicotyledonous C₃ plants, but the bundle sheath cells contain granal, starch-containing chloroplasts. In F. floridana and F. chloraefolia, the chloroplasts and numerous associated mitochondria are arranged largely centripetally, as in the closely related C₄ species, F. brownii A.M. Powell. In F. linearis, fewer mitochondria are present and the chloroplasts are more evenly distributed throughout the bundle sheath cytosol. There is no correlation between the bundle sheath ultrastructure and CO₂ compensation concentration. () values of these C₃–C₄ intermediate Flaveria species. At 21% O₂ and 25°C, for F. chloraefolia, F. linearis, and F. floridana is 23–26, 14–19, and 8–10 l CO₂ l^-1, respectively. The O₂ dependence of is the greatest for F. chloraefolia and F. linearis (similar to that for C₃–C₄ intermediate Panicum and Moricandia species) and the least for F. floridana, whose O₂ response is identical to that for F. brownii from 1.5 to 21% O₂, but greater at higher pO₂. The variation in leaf anatomy, bundle sheath ultrastructure, and O₂ dependence of among these Flaveria species may indicate an active evolution in the pathway of photosynthetic carbon metabolism within this genus.Abbreviations CO₂ compensation concentration - IRGA infrared gas analysis Published as Paper No. 7068, Journal Series, Nebraska Agricultural Experiment Station     

A novel spectrofluorimetric method based on a reaction with an azoisoxazoles–benzenesulfonamide derivative for determination of uranium (VI) ions in water samples

Selective fluorometric detection and determination of uranium ions is provided here using a novel fluorescent reagent, namely (E)-4-([4-hydroxynaphthalen-1-yl]diazenyl)-N-(5-methyleisoxazol-3-yl) benzenesulfonamide (U^VI reagent). The U^VI reagent offers a selective fluorescence enhancement behaviour at emission wavelength = 557 nm. The parameters affecting fluorometric detection of uranium ions, such as the pH, solvent type, ligand concentration, interaction time, and interfering ions, were investigated and adjusted. The proposed U^VI reagent can detect and determine uranium ions even at low concentrations, for which the obtained limit of detection was 0.1 ppm. Additionally, this proposed determination protocol was successfully used to detect, monitor, and determine uranium ions in actual water samples.     

Development and validation of a sensitive and rapid non-aqueous LC–ESI-MS/MS method for measurement of diosgenin in the plasma of normal and hyperlipidemic rats: A comparative study

A sensitive and specific electrospray ionization liquid chromatography–tandem mass spectrometry method was developed to detect diosgenin in the plasma of normal and hyperlipidemic rats. Diosgenin was extracted with n-hexane–ethyl acetate (9:1, v/v) using sarsasapogenin as an internal standard. With multiple reaction monitoring modes, linear calibration curves were obtained in the range 10–1500 ng/mL (r ≥ 0.9979) and the limit of quantification was 10 ng/mL. Intra- and inter-assay variabilities were within 7.74%, and accuracies were between ?5.33% and 1.50%. The assay was successfully applied to study pharmacokinetics in rats after oral administration of diosgenin. Significantly different pharmacokinetics between normal and hyperlipidemic rats were observed, which would be beneficial for the clinical use of diosgenin.     

Development and validation of a sensitive gas chromatography–ammonia chemical ionization mass spectrometry method for the determination of tabun enantiomers in hemolysed blood and plasma of different species

The aim of this study was to develop and validate a fast, sensitive and easily applicable GC–MS assay for the chiral quantification of the highly toxic organophosphorus compound tabun (O-ethyl-N,N-dimethylphosphoramidocyanidate, GA) in hemolysed swine blood for further use in toxicokinetic and toxicodynamic studies. These requirements were fulfilled best by a GC–MS assay with positive chemical ionization with ammonia (GC–PCI-MS). Separation was carried out on a β-cyclodextrin capillary column (Supelco BetaDex^® 225) after reversed phase (C18) solid-phase extraction. The limit of detection was 1 pg/ml for each enantiomer (approximately 500 fg on column) and the limit of quantification 5 pg/ml. The GC–PCI-MS method was applied for the quantification of tabun enantiomers in spiked swine blood after hemolysis and in spiked plasma of different species including humans.     

Characterisation of CO2 and HCO3 − uptake in the cyanobacterium Synechocystis sp. PCC6803

The availability of a complete genome database for the cyanobacterium Synechocystissp. PCC6803 (glucose-tolerant strain) has raised expectations that this organism would become a reference strain for work aimed at understanding the CO₂-concentrating mechanism (CCM) in cyanobacteria. However, the amount of physiological data available has been relatively limited. In this report we provide data on the relative contributions of net HCO₃ ⁻ uptake and CO₂ uptake under steady state photosynthetic conditions. Cells were compared after growth at high CO₂ (2% v/v in air) or limiting CO₂ conditions (20 ppm CO₂). Synechocystishas a very high dependence on net HCO₃ ⁻ uptake at low to medium concentrations of inorganic carbon (Ci). At high Ci concentrations net CO₂ uptake became more important but did not contribute more than 40% to the rate of photosynthetic O₂ evolution. The data also confirm that high Ci cells of Synechocystissp. PCC6803 possess a strong capacity for net HCO₃ ⁻ uptake under steady state photosynthetic conditions. Time course experiments show that induction of maximal Ci uptake capacity on a shift from high CO₂ to low CO₂ conditions was near completion by four hours. By contrast, relaxation of the induced state on return of cells to high CO₂, takes in excess of 230 h. Experiments were conducted to determine if Synechocystissp. PCC6803 is able to exhibit a `fast induction' response under severe Ci limitation and whether glucose was capable of causing a rapid inactivation in Ci uptake capacity. Clear evidence for either response was not found. This revised version was published online in August 2006 with corrections to the Cover Date.     

A sensitive and specific liquid chromatography–tandem mass spectrometric method for determination of belinostat in plasma from liver cancer patients

A novel, sensitive and reliable liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and validated for the determination of belinostat (PXD101) in human plasma. Oxamflatin was used as the internal standard. Liquid–liquid extraction of the plasma sample was performed using tert-butyl methyl ether as the organic solvent. Chromatographic separation was achieved on a BDS Hypersil C18 column (2.1 mm × 100 mm, 5 μm) using gradient elution mode using 0.05% formic acid in water and 0.05% formic acid in acetonitrile as solvents A and B, respectively, 60/40. The run time was 6 min. The mass spectrometer was operated under a positive electrospray ionization condition and a multiple reaction monitoring mode. An excellent linear calibration was achieved in the range of 0.5–1000 ng/mL. An average recovery of belinostat for four quality controls was 72.6% and the recovery of the internal standard at 1000 ng/mL was 67.8%. The intra-day and inter-day precisions for belinostat were ≤8.0 and ≤10.3%, respectively, and their accuracy ranged from 100.2 to 106.7%. No significant matrix effect was identified. In analysis of patient samples, belinostat glucuronide was identified and baseline separated from belinostat. This well-validated assay has been applied for quantification of belinostat in plasma samples within 24 h after the start of infusion for Asian hepatocellular carcinoma patients in a dose escalation study.     

A method for the determination of confidence limits for the P/2e − ratio for chosen values of Y ATP max from the results of continuous culture experiments

A model is described, which allows the determination of 95% confidence limits for the maintenance coefficient and the efficiency of oxidative phosphorylation for chosen values of the growth yield for ATP corrected for energy maintenance (Y _ATP ^max ). As experimental data the specific rates of substrate consumption, product formation and oxygen uptake in chemostat cultures at various growth rates are used.     

UHPLC method for the simultaneous determination of β-blockers, isoflavones and their metabolites in human urine

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.