Atenolol In Plasma Research Articles

Simple and rapid HPLC method for simultaneous determination of atenolol and chlorthalidone in spiked human plasma

A simple, sensitive and rapid chromatographic method was developed and validated for the simultaneous quantification of atenolol and chlorthalidone in human plasma using hydrochlorothiazide as internal standard (IS). The method utilized proteins precipitation with acetonitril as the only sample preparation involved prior to reverse phase-HPLC. The analytes were chromatographed on Shim-pack cyanopropyl column with isocratic elution with 10 mM KH 2PO 4 (pH 6.0) – methanol (70:30, v/v) at ambient temperature with flow rate of 1 mL min −1 and UV detection at 225 nm. The chromatographic run time was less than 10 min for the mixture. The calibration curves were linear over the range of 0.1–10 μg mL −1. The method was validated in terms of accuracy, precision, absolute recovery, freeze–thaw stability, bench-top stability and re-injection reproducibility. The within- and between-day accuracy and precision were found to be within acceptable limits <15%. The analytes were stable after three freeze–thaw cycles (deviation <15%). The proposed method was specific for the simultaneous determination of atenolol and chlorthalidone in human plasma where there was no interference from endogenous biological substances.

GC–MS Determination of Atenolol Plasma Concentration after Derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide

An analytical procedure was developed and validated for the determination of atenolol in human plasma. Atenolol and metoprolol (internal standard) were extracted from human plasma with a mixture of chloroform and butanol at basic pH. The extracts were derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide and analyzed by GC–MS. Calibration curves were linear over the concentration range 15–250 ng mL−1. Intra- and inter-day precision values for atenolol in human plasma were less than 7.4, and accuracy (relative error) was better than 6.4%. Recovery of atenolol from human plasma averaged 90.46%. The limits of detection (LOD) and quantitation (LOQ) of atenolol were 5.0 and 15 ng mL−1. This method was successfully applied to six patients with hypertension who had been given an oral tablet of 50 mg atenolol.

Reversed‐phase liquid chromatographic determination of enantiomers of atenolol in rat plasma using derivatization with Marfey's reagent

An HPLC method was established for enantioseparation of (R,S)-atenolol (ATE) and determination of enantiomers in rat plasma. Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide, FDNP-L-Ala-NH(2), MR) was used as chiral derivatizing reagent with detection of diastereomers at 340 nm. It was shown that the R-isomer eluted before the S-isomer. The method was validated for linearity, repeatability, limits of detection and limit of quantification (LOQ). Recovery of ATE at LOQ was 92.8% for (R)-ATE and 92.6% for (S)-ATE.

Simultaneous Determination of Atenolol, Rosuvastatin, Spironolactone, Glibenclamide and Naproxen Sodium in Pharmaceutical Formulations and Human Plasma by RP‐HPLC

AbstractA rapid and sensitive high‐performance liquid chromatographic method was developed and validated for the simultaneous determination and quantification of atenolol, rosuvastatin, spirnolactone, glibenclamide and naproxen sodium in bulk drugs, pharmaceutical formulations and in human plasma in the presence of internal standard (flurbiprofen). Chromatograms were developed with methanol and water (80:20, v/v) solvent system on a Purospher start, C18 (5 μm, 250 × 4.6 mm) column and pH was adjusted to 3.40 with ortho‐phosphoric acid. Mobile phase was pumped with a flow rate of 0.90 mL/minute with 235 nm UV detection. Standard curves were linear over the concentration range 0.25‐30 μg/mL−1. The coefficients of variation (C.V.%), were &lt; 3% and LOD and LOQ were &lt;0.0154 &amp; 0.06 for inter‐ and intra‐day, respectively. The method was applied to drug interaction studies of atenolol with rosuvastatin, spironolactone, glibenclamide and naproxen to illustrate the scope and application of the methods to manage four different therapeutic classes of drugs, as they are co‐administered.

Simultaneous Determination of Atenolol and Chlorthalidone by LC–MS–MS in Human Plasma

A simple, sensitive, selective and rapid liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous separation and quantitation of atenolol and chlorthalidone in human plasma using metoprolol and hydrochlorothiazide as internal standard. Following solid phase extraction, the analytes were separated by an isocratic mobile phase on a reversed-phase C18 column and analyzed by MS in the multiple reaction-monitoring mode (atenolol in positive and chlorthalidone in the negative ion mode). The limit of quantitation for this method was 10 and 15 ng mL−1 and the linear dynamic range was generally 10–2,050 ng mL−1 and 15–3,035 ng mL−1 for atenolol and chlorthalidone, respectively.

Determination of Atenolol in Human Plasma by Pseudo Reversed Phase Liquid Chromatography-Tandem Mass Spectrometry

Previous HPLC determination of atenolol on reversed-phase packings has often required a mobile phase containing three components: organic modifier, buffer and ion-pairing reagent or organic amine. In addition to the complexity of the eluents employed, alkyl sulphonates and organic amines in the mobile phase can reduce the life of silica-based bonded columns. A new simple, rapid and sensitive method—pseudo reversed-phase liquid chromatography/tandem mass spectrometry has been developed for the analysis of atenolol in human plasma using bare silica as the stationary phase coupled with a simple mobile phase consisted of 5% acetonitrile and 95% formate buffer. The optimization of separation is fast and easy. The assay was validated for the concentration range 1–100 ng mL−1 with a detection limit of 1 ng mL−1. Moreover, the silica column was durable with the mainly aqueous eluents. No obvious loss in performance was observed for 30,000 column volumes of eluent.

A Micromethod for the Quantification of Atenolol in Plasma Using High-Performance Liquid Chromatography With Fluorescence Detection

A simple, rapid, selective, and sensitive analytical method was developed for the quantification of atenolol in small volumes of plasma, by high-performance liquid chromatography with fluorescence detection. Only 200 microL of plasma was used for chromatographic analysis. Separation was performed on a C18 reverse-phase column (4 microm) using a binary mobile phase consisting of 0.05 M of phosphate buffer, pH 5.5, and methanol (80:20, vol/vol) at a flow rate of 0.7 mL/minute. The retention times of atenolol and of the internal standard (sotalol) were 12.7 and 10.4 minutes, respectively. Validation of this analytical method showed a good linear correlation (8-2000 ng/mL), high sensitivity (quantification limit: 8 ng/ml and detection limit: 4 ng/mL), accuracy of 99.3%, and intraday and interday precision of 5.3% and 6.9%, respectively. Absolute recovery was 93.7%. The method was found to be robust, with acceptable stability. The analytical method was validated by the quantification of atenolol in plasma obtained from 2 patients with unstable angina, scheduled for myocardium revascularization surgery, who were chronically treated with 50 mg of atenolol administered per os once a day. The method developed was found to be adequate for use in pharmacokinetic studies and in adjusted dose pharmacotherapy.

Screening for library-assisted identification and fully validated quantification of 22 beta-blockers in blood plasma by liquid chromatography–mass spectrometry with atmospheric pressure chemical ionization

A liquid chromatographic-mass spectrometric assay with atmospheric pressure chemical ionization (LC-APCI-MS) is presented for screening for, library-assisted identification (both in scan mode) and quantification (selected-ion mode) of the beta-blockers acebutolol, diacetolol, alprenolol, atenolol, betaxolol, bisoprolol, bupranolol, carazolol, carteolol, carvedilol, celiprolol, esmolol, labetalol, metoprolol, nadolol, nebivolol, oxprenolol, penbutolol, propranolol, sotalol, talinolol and timolol in blood plasma after mixed-mode (HCX) solid-phase extraction (SPE) and separation by reverse-phase liquid chromatography with gradient elution. The validation data were within the required limits. The assay was successfully applied to authentic plasma samples allowing confirmation of diagnosis of overdose situations as well as monitoring of patients' compliance.

Screening for library-assisted identification and fully validated quantification of 22 beta-blockers in blood plasma by liquid chromatography–mass spectrometry with atmospheric pressure chemical ionization

A liquid chromatographic–mass spectrometric assay with atmospheric pressure chemical ionization (LC–APCI–MS) is presented for screening for, library-assisted identification (both in scan mode) and quantification (selected-ion mode) of the beta-blockers acebutolol, diacetolol, alprenolol, atenolol, betaxolol, bisoprolol, bupranolol, carazolol, carteolol, carvedilol, celiprolol, esmolol, labetalol, metoprolol, nadolol, nebivolol, oxprenolol, penbutolol, propranolol, sotalol, talinolol and timolol in blood plasma after mixed-mode (HCX) solid-phase extraction (SPE) and separation by reverse-phase liquid chromatography with gradient elution. The validation data were within the required limits. The assay was successfully applied to authentic plasma samples allowing confirmation of diagnosis of overdose situations as well as monitoring of patients’ compliance.

Enantioselective analysis of atenolol in biologic fluids: comparison of liquid–liquid and solid-phase extraction methods

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 C 8 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 10–5000 ng/ml for both methods. These methods can be used in pharmacokinetic studies.

Determination of the β-blocker atenolol in plasma by capillary zone electrophoresis

A capillary zone electrophoretic method was optimised for the determination of the β-blocker atenolol in plasma. Separation was performed in an uncoated silica capillary of 58.5 cm (effective length 50 cm)×75 μm I.D., and detection was at 194 nm. The effects of the buffer (concentration and pH), the injection time, the voltage applied and the plasma clean-up procedure were studied. The determination of atenolol was achieved in less than 3 min, using an electrolyte of 50 m M H 3BO 3–50 m M Na 2B 4O 7 (50:50, v/v) pH 9, injected hydrodynamically for 4 s at 50 mbar and applying a voltage of +25 kV. This method was applied to the determination of atenolol in plasma of nine hypertensive patients (male and female, aged from 39 to 73 years). Atenolol concentrations found vary from 30 to 585 ng/ml.

Enantioselective analysis of ( R)- and ( S)-atenolol in urine samples by a high-performance liquid chromatography column-switching setup

An HPLC column-switching method for the enantioselective determination of ( R, S)-atenolol in human urine was developed and validated. Diluted urine samples were injected onto a LiChrospher ADS restricted access column and atenolol was separated from most of the matrix components using 0.01 M Tris buffer. The atenolol peak was sharpened by a step gradient of 30% acetonitrile and the atenolol-containing fraction was switched onto an enantioselective column. Separation of the atenolol enantiomers was carried out on a Chirobiotic T (Teicoplanin) column using acetonitrile–methanol–acetic acid–triethylamine (55:45:0.3:0.2, v/v/v/v) as eluent. Detection of the effluent was performed by fluorescence measurement. Several experiments were carried out to suppress the high blank reading, which was efficiently achieved using Tris buffer in the first dimension. For the enantioselective analysis of ( R)- and ( S)-atenolol in plasma under the same conditions the sample capacity of the ADS column is considerably lower.

Two reproducible and sensitive liquid chromatographic methods to quantify atenolol and propranolol in human plasma and determination of their associated analytical error functions

Two liquid chromatography (LC) methods with fluorimetric detection have been developed to measure atenolol and propranolol in human plasma. The same 5 μm Nucleosil RP-18 column, extraction procedure and mobile phase (containing acetonitrile, water, triethylamine and phosphoric acid, pH 3) were used. The linearity ranges were 25–800 ng/ml for atenolol and 3.13–100 ng/ml for propranolol. The coefficients of variation for validation assays were lower than 15% at the concentration assayed. The functions of the analytical error were linear: SD (ng/ml)=7.698+0.037C for atenolol and SD (ng/ml)=0.126+0.036C for propranolol.

Automated liquid chromatographic determination of atenolol in plasma using dialysis and trace enrichment on a cation-exchange precolumn for sample handling

A fully automated method involving dialysis combined with trace enrichment was developed for the liquid chromatographic (LC) determination of atenolol, a hydrophilic β-blocking agent, in human plasma. The plasma samples were dialysed on a cellulose acetate membrane and the dialysate was reconcentrated on a short trace enrichment column (TEC) packed with a strong cation-exchange material. All sample handling operations can be executed automatically by a sample processor (ASTED system). After TEC conditioning, the plasma sample, to which the internal standard (sotalol, another hydrophilic β-blocker) was automatically added, was introduced in the donor channel and dialysed in the static/pulsed mode. The dialysis liquid consisted of 4.3 m M phosphoric acid. When the dialysis process was discontinued, the analytes were eluted from the TEC in the back-flush mode by the LC mobile phase and transferred to the analytical column, packed with octyl silica. The LC mobile phase consisted of phosphate buffer, pH 7.0–methanol (81:19; v/v) with 1-octanesulfonate. Atenolol and the internal standard were monitored photometrically at 225 nm. The different parameters influencing the dialysis and trace enrichment processes were optimised with respect to analyte recovery. The influence of two different kinds of cation-exchange material on analyte recovery and peak efficiency was also studied. The method was then validated in the concentration range 25–1000 ng/ml. The mean recovery for atenolol was 65% and the limit of quantitation was 25 ng/ml.

Validation of an automated method for the liquid chromatographic determination of atenolol in plasma: application of a new validation protocol

In order to test the applicability of a new strategy by a Commission of the Société Française des Sciences et Techniques Pharmaceutiques (SFSTP) for the validation of bioanalytical methods, an automated method was developed for the determination of atenolol in human plasma. This method was based on the use of dialysis as sample purification step, followed by enrichment of the dialysate on a precolumn and liquid chromatographic analysis. All sample handling operations were carried out automatically by means of an ASTED system. Atenolol and its internal standard (sotalol) were separated on a C 8 column with a mixture of pH 7.0 phosphate buffer containing 1-octanesulphonate and methanol (81/19; v:v) as mobile phase and monitored photometrically at 225 nm. The validation strategy comprises two steps. The experiments performed during the first step, the so called pre-validation step, have permitted the selection of the most appropriate model for the calibration curve by means of a decision tree, i.e. a least squares regression model obtained after transformation of data (square root in this case), the estimation of the limit of quantitation at 25 ng/ml by means of an accuracy profile, the determination of the calibration range (from 25 to 1000 ng/ml), the estimation of the limit of detection at 9 ng/ml and the calculation of the mean extraction efficiency (about 65%). The second step is the validation itself, comprising the evaluation of method selectivity towards endogenous components, the confirmation of the limit of quantitation, the verification of linearity and the assessment of method precision (repeatability and intermediate precision) as well as accuracy using quality control samples at different concentration levels over the range investigated. The relative standard deviation values for repeatability and intermediate precision were between 2.7% and 9.0%. Moreover, the method was found to be accurate. Indeed, the 95% one-sided confidence limits of the mean recovery did not exceed the acceptance limits of 80% and 120%.

Simultaneous determination of atenolol and chlorthalidone in plasma by high-performance liquid chromatography application to pharmacokinetic studies in man

An HPLC method developed to detect in a single run both atenolol and chlorthalidone, extracted from plasma, using two detectors (UV for chlorthalidone and fluorometric for atenolol) connected in series, is described. The drugs were separated on an ODS column at room temperature using a 0.05 M sodium dodecyl sulphate in phosphate buffer (pH 5.8)- n-propanol (95:5, v/v) solution, delivered at a flow-rate of 1.3 ml/min. Having ascertained the sensitivity (10 ng/ml of both drugs) and the intra-day reproducibility (pre-study validation), the reliability of the method was verified by inter-day assays (within-study validation) carried out during the analysis of plasma samples collected from healthy volunteers after single-dose treatment with atenolol+chlorthalidone tablets (pharmaceutical preparations containing 100+25 mg and 50+12.5 mg of the two drugs, respectively).

Efficient assay for the determination of atenolol in human plasma and urine by high-performance liquid chromatography with fluorescence detection.

An efficient method for the determination of atenolol in human plasma and urine was developed and validated. alpha-Hydroxymetoprolol, a compound with a similar polarity to atenolol, was used as the internal standard in the present high-performance liquid chromatographic analysis with fluorescence detection. The assay was validated for the concentration range of 2 to 5000 ng/ml in plasma and 1 to 20 microg/ml in urine. For both plasma and urine, the lower limit of detection was 1 ng/ml. The intra-day and inter-day variabilities for plasma samples at 40 and 900 ng/ml, and urine samples at 9.5 microg/ml were <3% (n=5).

Direct enantiospecific HPLC bioanalysis of (R,S)-atenolol on a chiral stationary phase.

The simultaneous determination of the enantiomers of the beta1-selective adrenergic antagonist atenolol in human plasma and urine is described. After an alkaline pre-extraction atenolol is extracted from biological material at pH 12.3 using dichloromethane/propan-2-ol. The separation of the underivatized enantiomers is achieved by high-performance liquid chromatography on a chiral stationary phase (Chiralcel OD, cellulose tris-3,5-dimethylphenylcarbamate, coated on silica gel) with fluorimetric detection. (--)-)S)-Pindolol is used as an internal standard. The detection limits of 5 ng/ml enantiomer in plasma and 50 ng/ml enantiomer in urine are sufficient for pharmacokinetic studies after therapeutic doses.

A Liquid Chromatographic Method for the Determination of Atenolol in Human Plasma

Abstract A reliable, highly reproducible, accurate and time-efficient high performance liquid chromatographic (HPLC) method to measure atenolol concentration in human plasma was developed and validated. Sample clean-up consists of simple and efficient liquid-liquid extraction (mean recovery 103%) which allows a high sample throughput. Chromatography on a CN-propyl column yields symmetrical and well resolved peaks for atenolol and for the internal standard (metoprolol) without any interference from endogenous plasma components. Using 1 ml plasma samples the method has a limit of detection of 12.6 ng/ml (calculated at a 99.9% confidence level) with %CV (precision) ≥ 8.8% and bias (accuracy) ≥ 3.8% for concentrations in the range of 10 – 1000 ng/ml. We now routinely use this method in human pharmacokinetic studies of atenolol dosage forms.

Improved High-Performance Liquid Chromatography Assay for Atenolol in Plasma and Urine Using Fluorescence Detection

An improved assay for racemic atenolol (AT) concentrations in human plasma and urine is described using a high-performance liquid chromatographic method with fluorescence detection. The method has a sensitivity limit of 0.5 micrograms/L in plasma with acceptable within- and between-run reproducibilities, and demonstrated linearity at concentrations up to 2,000 micrograms/L. A pilot clinical evaluation of the assay was undertaken on 56 trough plasma specimens from 36 outpatients on established AT therapy. Atenolol concentrations in these patients showed large variations at all prescribed doses, including undetectable levels in four patients (revealing unsuspected noncompliance). Because of its sensitivity and applicability to urinary analysis, the method can be used for pharmacokinetic studies and, under certain circumstances, may be valuable in clinical therapeutic drug monitoring.