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In this work, an efficient method termed as continuous flow membrane microextraction coupled with high performance liquid chromatography is introduced for a highly selective determination of metoprolol and propranolol in the biological samples. According to this method, an aqueous source phase of the analytes (donor phase, 10 mL) is circulated into an extraction cell, which is separated from an aqueous acceptor phase (100 μL) by a small piece of polypropylene membrane sheet whose pores are impregnated by an organic solvent (1-octanol, 15 μL). The analytes are extracted from the donor phase into the organic solvent. They are subsequently selectively back-extracted into the acceptor solution due to the pH gradient. The proposed method is very convenient and has the capability of being fully automated. It provides a good preconcentration and an excellent repeatability. The extractant is an aqueous phase, and by prevention of the extraction of macromolecules through the membrane, the developed method provides a high sample clean-up. In order to maximize the extraction efficiency, the influential parameters including the type of mediator solvent, pH values for the donor and acceptor solutions, extraction time, ionic strength, stirring rate, and volume of the acceptor solution are optimized. The calibration curves were obtained with a reasonable linearity (r 2  = 0.999) in the range of 3-1000 ng mL - 1 . The limits of detection were 0.5 and 1.0 ng mL -1 , and excellent relative standard deviations were obtained (between 3.2% and 4.0%). Finally, the reliability of the procedure is evaluated by determination of metoprolol and propranolol in the human urine and plasma samples, which indicates the suitability, sensitivity, and high sample clean-up of the proposed method., (Copyright © 2019 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.)

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.)

In this work we have compared three analytical techniques (ELISA, GC-MS, and LC-MS) for the analysis of 16 beta-blockers: acebutolol, alprenolol, atenolol, betaxolol, bisoprolol, carteolol, labetalol, metipranolol, metoprolol, nadolol, oxprenolol, pindolol, propranolol, sotalol, timolol, and bupranolol. Several sample-preparation methods were optimized for each technique and enabled compounds of interest to be extracted from small urine samples (1-2.5 mL). The results enabled us to assess the possibilities and the sensitivity of each technique for application to doping tests. ELISA, whose selectivity is very poor and sensitivity the lowest one, is, nevertheless, useful as a rapid screening method. GC/MS and LC/MS provide confirmation procedures with the identification and quantification of the beta-blockers with good sensitivity, accuracy, precision. The LC-MS analytical procedure allows the determination of the target analytes in the lower ng/mL range (0.53-2.23 ng/mL). The methodology was applied to the analysis of beta-blockers in different urines.

Described herein are two general screening procedures for the simultaneous determination of 49 exogenous compounds (21 diuretics, 19 beta-blockers, eight stimulants and two steroidal drugs) in human urine by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Urine samples were extracted using a simple and robust solid phase extraction (SPE) method. Samples were injected onto reversed phase HPLC and UPLC columns connected to tandem mass spectrometers capable of scan-to-scan polarity switching. The methods were validated according to the ISO 17025 international standard for the validation of a qualitative method. Sixty urine samples submitted for routine analysis were tested using both methods, the results of which correlated with results obtained from previously validated procedures. Both methods proved to be useful for routine urine analysis; most notably, the use of UPLC-MS/MS demonstrated that samples can be reliably screened with significantly reduced analysis times.

A novel high-performance liquid chromatography (HPLC) assay for the estimation of formoterol in urine samples was developed and validated. A solid phase extraction (SPE) using Oasis HLB was optimised to isolate formoterol from a urine matrix followed by HPLC with UV detection. This extraction procedure concentrated the final analyte forty times so that UV detection can be used to determine even a low concentration of formoterol in urine samples. The urinary assay was performed in accordance with FDA and ICH regulations for the validation of bioanalytical samples. The samples were injected onto a C18 Spherisorb (250 mm x 4.6 mm x 5 microm) analytical column maintained at 30 degrees C. The mobile phase consisted of 5 mM of potassium dihydrogen orthophosphate buffer (adjusted to pH 3 with ortho phosphoric acid):acetonitrile (ACN) (70:30, v/v), and the formoterol peak was detected at wavelength 214 nm. The extraction recovery of formoterol from the urine sample was >95%. The calibration curve was linear (r2=0.99) over formoterol concentrations ranging from 1.5 to 25 ng/mL (n=6). The method had an accuracy of >92% and intra and inter-day precision CV% of <3.9% and <2.2%, respectively, at three different concentrations low, medium and high (10, 15, 20 ng/mL). The limit of quantification (LOQ) for formoterol was found to be 1.50 ng/mL. The accuracy and precision at the LOQ level were 95% and %CV <3.7% (n=10), respectively. The method reported is simple, reliable, precise, and accurate and has the capacity to be used for determination of formoterol in urine samples.

Enantioselective separations on chiral stationary phases with or without derivatization were developed and compared for the HPLC analysis of (+)-(R)- and (-)-(S)-metoprolol acidic metabolite in human plasma and urine. The enantiomers were analysed in plasma and urine without derivatization on a Chiralcel OD-R column, and in urine after derivatization using methanol in acidic medium on a Chiralcel OD-H column. The quantitation limits were 17 ng of each enantiomer/ml plasma and 0.5 microgram of each enantiomer/ml urine using both methods. The confident limits show that the methods are compatible with pharmacokinetic investigations of the enantioselective metabolism of metoprolol. The methods were employed in a metabolism study of racemic metoprolol administered to a patient phenotyped as an extensive metabolizer of debrisoquine. The enantiomeric ratio (+)-(R)/(-)-(S)-acid metabolite was 1.1 for plasma and 1.2 for urine. Clearances were 0.41 and 0.25 l/h/kg, respectively, for the (+)-(R)- and (-)-(S)-enantiomers. The correlation coefficients between the urine concentrations of the acid metabolite enantiomers obtained by the two methods were >0.99. The two methods demonstrated interchangeable application to pharmacokinetics.

A rapid chromatographic procedure with a C18 column, a mobile phase of 0.15 M sodium dodecyl sulfate (SDS)-10% (v/v) 1-propanol at pH 3 (0.01 M phosphate buffer), and fluorimetric detection, is reported for the control of propranolol (PPL) intake in urine samples, which are injected directly without any other treatment than filtration. The peak of PPL was only observed in samples taken a few hours after ingestion of the drug due to its extensive conjugation and metabolisation. The detection of several unconjugated PPL metabolites was therefore considered: desisopropylpropranolol (DIP), propranolol glycol (PPG), alpha-naphthoxylactic acid (NLT) and alpha-naphthoxyacetic acid (NAC). NLT showed the best characteristics: it eluted at a much shorter retention time than PPL, its concentration in urine samples was greater and it did not present any interference from endogeneous compounds in urine, common drugs or drugs administered in combination with PPL. The limit of quantification, measured as the concentration of analyte providing a relative standard deviation of 20%, was 24 ng/ml, and the day-to-day imprecision was below 4% for concentrations above 200 ng/ml. The procedure allows the routine control of PPL at therapeutic urine levels. Urinary excretion studies showed that the detection of NLT is possible at least up to 20-30 h after oral administration.

In this study we evaluated a liquid-liquid extraction procedure and a solid-phase extraction procedure for sample preparation for the enantioselective analysis of atenolol in plasma and urine by high-performance liquid chromatography. A Chiralcel OD-H column was used for the resolution of atenolol enantiomers with hexane-ethanol (85:15, v/v) plus 0.1% diethylamine as the mobile phase. In the liquid-liquid extraction procedure, atenolol was extracted from alkalinized body fluids with 5 ml chloroform-2-propanol (4:1, v/v). In the solid-phase extraction procedure, atenolol was isolated from plasma using a C8 column and methanol. Both extraction procedures were efficient in recovering atenolol and removing endogenous interferents. The RSDs and deviation from nominal values were lower than 10% for both within-day and between-day assays. The results show that there were no statistically significant differences in between-day variation. The t-test showed that there were no significant differences between the real concentrations and the determined concentrations. The limit of quantitation was 10 ng/ml and the linear range was 10-5,000 ng/ml for both methods. These methods can be used in pharmacokinetic studies.