Rapid and sensitive tapered-capillary microextraction combined to on-line sample stacking-capillary electrophoresis for extraction and quantification of two beta-blockers in human urine.

A tapered-capillary microextraction (tCap-µEx) combining with field-amplified stacking (FASI) method for CE analysis was developed. The tCap-µEx method is based on the construction of a micro solid phase extraction (SPE) column by narrowing the end of a silica capillary from 530µm (inner diameter) to 20µm, enabling the packing of 45µm sorbent particles without a frit. Various parameters that may affect the microextraction and FASI-CE analysis have been investigated and optimized. This study shows that microextraction exhibits advantages of small sample and sorbent volumes (less than 200µL sample and 2µL sorbent) and fast extraction time of 6min. The method was successfully applied for efficient determination of atenolol and metoprolol in human urine samples, with recovery of 93.7-105.5% and RSD (n=3) lower than 8.5%. Twenty-one-fold and nineteen-fold average enhancement of detection sensitivity was achieved for atenolol and metoprolol, respectively, versus the CE method without tCap-µEx and FASI. The method is environmentally friendly and allows reuse of the sorbent at least 8 times without an obvious loss in performance. The results indicate that the proposed method could be potentially applied in a wide range of doping control, clinical, forensic toxicology, food analysis and environmental analyses.

Spectrofluorimetric determination of atenolol from human urine using high-affinity molecularly imprinted solid-phase extraction sorbent.

This study presents a novel, sensitive and selective molecularly imprinted solid-phase extraction (MISPE)-spectrofluorimetric method for the removal and determination of atenolol from human urine. Molecularly imprinted and non-imprinted polymers were synthesized thermally using a radical chain polymerization technique and used as solid-phase extraction sorbents. Acrylic acid ethylene glycol dimethacrylate, dibenzoyl peroxide and dichloroethane were used as a functional monomer, cross-linker, initiator and porogen, respectively. The calibration curve was in the range of 0.10-2.0 µg/ml for the developed method. Limit of detection and limit of quantification values were 0.032 and 0.099 µg/ml, respectively. Owing to the selectivity of the MISPE technique and the sensitivity of spectrofluorimetry, trace levels of atenolol have been successfully determined from both organic and aqueous media. Relatively high imprinting factor (4.18) and recovery results (74.5-75.3%) were obtained. In addition, intra- and interday precision values were 0.38-1.03% and 0.47-2.05%, respectively, proving the precision of the proposed method. Thus, a selective, sensitive and simple MISPE-spectrofluorimetric method has been developed and applied to the direct determination of atenolol from human urine.

On-line double focusing of atenolol and metoprolol in human urine using capillary electrophoresis with the aid of ß-cyclodextrin.

A maneuverable and sensitive on-line double focusing technique combined field amplified sample stacking (FASS) with micelle to solvent stacking (MSS) with the aid of ß-cyelodextrin (ß-CD) is developed to detect the contents of AT and ME in human urine by capillary electrophoresis (CE) with UV detector. Small amount of ß-CD not only increase the critical micelle concentration (CMC) of SDS, but also strengthen the interaction between SDS and the aimed compound by forming inclusion complexes. The result indicates that the addition of ß-CD affords 5.5- and 3.5-fold improvements for atenolol (AT) and metoprolol (ME) in sensitivity than that of in the absence of ß-CD in the double focusing system, respectively. Under the optimal conditions, about 200-fold improvement in sensitivity for analytes is achieved compared with conventional CE method. The limits of detection (LODs) at a signal-to-noise of 3 (S/N = 3) of the two ß-blockers can be reached 3.3 and 3.7 ng mL-1 respectively, which are lower than minimum required performance levels (MRPLs) set by the World Anti Doping Agency. The relative standard deviations (RSDs) of peak areas of intra-day and inter-day are 3.51-3.38% and 2.34-4.28% (n = 6), respectively. AT and ME in urine without special pretreatment and additional instrument are analyzed. The recoveries are 82-98% with RSDs less than 2.0%.

Cyclodextrin Micellar LC for Direct Selective Analysis of Combined Dosage Drugs in Urine.

Two cyclodextrin micellar liquid chromatographic methods were developed and applied to the simultaneous determination of bisoprolol/hydrochlorothiazide and atenolol/chlorthalidone combinations in urine matrices without sample pretreatment. These combined ß-blockers and diuretics chemotherapies are commonly used in the treatment of hypertension and cardiovascular diseases. Hybrid isocratic mobile phases containing hydroxypropyl-ß-cyclodextrin, sodium dodecyl sulfate, phosphate buffer and methanol on a Luna C18 column with 0.5 mL min(-1) flow rate and 25.0°C column temperature were used. The methods were sensitive enough for the determination of analytes at the therapeutic urine levels with limits of detections down to 1.0 µg mL(-1); relative standard deviations and recoveries were ranged between 1.5-4.4% and 98.00-109.52%, respectively. Urinary excretion studies showed that the detection of drugs is possible up to 24 h after their ingestion. The selective proposed separations with less consumption of organic solvents over the hitherto ones could be attributed to the four point competitive interactions among analysts, pseudostationary phases and a real stationary phase.

A novel solid-phase extraction-spectrofluorimetric method for the direct determination of atenolol in human urine.

A novel, simple, sensitive and selective solid-phase extraction (SPE)-spectrofluorimetric method has been developed for the determination of atenolol (ATE) in human urine. Because an extraction procedure is required to isolate ATE or eliminate the interfering molecules present in complex human urine for the direct spectrofluorimetric determination, a pH-sensitive poly(acrylic acid-ethylene glycol dimethacrylate) [poly(AA-EGDMA)] hydrogel was developed and used as a SPE adsorbent. Some factors affecting the ATE extraction efficiency, such as washing solvent type and volume, and the volume of elution solvent were optimized. Eluates from SPE cartridges were analyzed using a spectrofluorimeter (λ(ex) = 277 nm and λ(em) = 300 nm). The calibration graph was linear over the concentration range 0.15-4.0 µg/mL. Limit of detection (LOD) and limit of quantification (LOQ) values were found to be 0.03 and 0.10 µg/mL, respectively. Relatively high intraday [2.06%, mean relative standard deviation (RSD)] and interday (2.6%, mean RSD) precisions were achieved. High mean recovery (95.4%) and low RSD values (3.8%) were obtained for spiked ATE in human urine. The spectrofluorimetric method presented here can be easily applied to assay trace amounts of ATE in pharmaceuticals and biological samples.

Microfluidic chip capillary electrophoresis coupled with electrochemiluminescence for enantioseparation of racemic drugs using central composite design optimization.

Optimization based on central composite design (CCD) for enantioseparation of anisodamine (AN), atenolol (AT), and metoprolol (ME) in human urine was developed using a microfluidic chip-CE device. Coupling the flexible and wide working range of microfluidic chip-CE device to CCD for chiral separation of AN, AT, and ME in human urine, a total of 15 experiments is needed for the optimization procedure as compared to 75 experiments using the normal one variable at a time optimization. The optimum conditions obtained are found to be more robust as shown by the curvature effects of the interaction factors. The developed microfluidic chip-CE-ECL system with adjustable dilution ratios has been validated by satisfactory recoveries (89.5-99% for six enanotiomers) in urine sample analysis. The working range (0.3-600 µM), repeatability (3.1-4.9% RSD for peak height and 4.0-5.2% RSD for peak area), and detection limit (0.3-0.6 µM) of the method developed are found to meet the requirements for bedside monitoring of AN, AT, and ME in patients under critical conditions. In summary, the hyphenation of CCD with the microfluidic chip-CE device is shown to offer a rapid means for optimizing the working conditions on simultaneous separation of three racemic drugs using the microfluidic chip-CE device developed.

Determination of atenolol in human urine by emission-excitation fluorescence matrices and unfolded partial least-squares with residual bilinearization.

A second-order multivariate calibration method based on a combination of unfolded partial least-squares (U-PLS) with residual bilinearization (RBL) has been applied to second-order data obtained from excitation-emission fluorescence matrices for determining atenolol in human urine, even in the presence of background interactions and fluorescence inner filter effects, which are both sample dependent. Atenolol is a cardioselective beta-blocker, which is considered a doping agent in shoot practice, so that its determination in urine can be required for monitoring the drug. Loss of trilinearity due to analyte-background interactions which may vary between samples, as well as inner filter effects, precludes the use of methods like parallel factor analysis (PARAFAC) that cannot handle trilinearity deviations, and justifies the employment of U-PLS. Successful analysis required to include the background in the calibration set. Unexpected components appear in new urine samples, different from those used in calibration set, requiring the second-order advantage which is obtained from a separate procedure known as residual bilinearization (RBL). Satisfactory results were obtained for artificially spiked urines, and also for real urine samples. They were statistically compared with those obtained applying a reference method based on high-performance liquid chromatography (HPLC).

Determination of atenolol in human urine by gas chromatography-mass spectrometry method.

A sensitive and efficient method was developed for the determination of atenolol in human urine by gas chromatography-mass spectrometry (GC-MS). Atenolol and metoprolol (internal standard, IS) were extracted from human urine with a mixture of chloroform and butanol at basic pH with liquid-liquid extraction. The extracts were derivatized with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and analyzed by GC-MS using a capillary column. The standard curve was linear (r = 0.99) over the concentration range of 50-750 ng/mL. Intra- and inter-day precision, expressed as the relative standard deviation were less than 5.0%, and accuracy (relative error) was better than 7.0%. The analytical recovery of atenolol from human urine has averaged 91%. The limit of quantification was 50 ng/mL. Also, the method was successfully applied to a patient with hypertension who had been given an oral tablet of 50 mg atenolol.

Determination of enalapril maleate and atenolol in their pharmaceutical products and in biological fluids by flow-injection chemiluminescence.

A chemiluminescent method using flow injection (FI) was investigated for rapid and sensitive determination of enalapril maleate and atenolol, which are used in the treatment of hypertension. The method is based on the sensitizing effect of these drugs on the Ce(IV)-sulfite reaction. The different experimental parameters affecting the chemiluminescence (CL) intensity were carefully studied and incorporated into the procedure. The method permitted the determination of 0.01-3.0 microg mL(-1) of enalapril maleate in bulk form with correlation coefficient r = 0.99993, lower limit of detection (LOD) 0.0025 microg mL(-1) (S/N = 2) and lower limit of quantitation (LOQ) 0.01 microg mL(-1). The linearity range of atenolol in bulk form was 0.01-2.0 microg mL(-1) (r = 0.99989) with LOD of 0.0003 microg mL(-1) (S/N = 2) and LOQ of 0.01 microg mL(-1). In biological fluids the linearity range of enalapril maleate was 0.1-2.0 microg mL(-1) in both urine and serum, and for atenolol the linearity range was 0.1-1.0 microg mL(-1) in both urine and serum. The method was also applied to the determination of the drugs in their pharmaceutical preparations.

Use of teicoplanin stationary phase for the enantiomeric resolution of atenolol in human urine by nano-liquid chromatography-mass spectrometry.

Nano-liquid chromatography (nano-LC) was used for the enantiomeric resolution of atenolol employing a teicoplanin modified silica stationary phase prepared in our laboratory. Experiments were carried out in a fused silica capillary of 75 microm i.d. packed with chiral modified silica particles of 5 microm diameter. Separated enantiomers were revealed by on-line UV detector at 205 nm or electrospray-ion-trap mass spectrometer (ESI-MS). Atenolol enantiomers were eluted utilizing a mobile phase with the following composition: 500 mM ammonium acetate pH 4.5/methanol/acetonitrile 1:60:39 (v/v/v) allowing to achieve good enantioresolution in a reasonable analysis time (about 8 min) with a flow rate of about 900 nL/min. After comparing the sensitivity of the nano-LC method using a conventional UV detector for capillary electrophoresis, a zeta cell (3 nL volume) employed in nano-LC and the ion-trap MS the method was validated with the MS detector offering the highest sensitivity (limit of detection (LOD)=50 ng/mL; limit of quantification (LOQ)=400 ng/mL for each atenolol enantiomer). (-)-Psi-Nor-ephedrine was used as the internal standard. The method was successfully applied to the analysis of atenolol enantiomers present in human urine samples of a patient under atenolol therapy.

Gas chromatographic-mass spectrometric differentiation of atenolol, metoprolol, propranolol, and an interfering metabolite product of metoprolol.

Over a 10-year period, 1993-2002, Federal Aviation Administration identified 50 pilot fatalities involving atenolol, metoprolol, and propranolol, which is consistent with the fact that these drugs have been in the lists of the top 200 drugs prescribed in the U.S. In a few of the 50 pilot fatality cases, initial analysis suggested the presence of atenolol and metoprolol. However, there was no medical history with these cases supporting the use of both drugs. Therefore, atenolol, metoprolol, and/or propranolol, with their possible metabolite(s), were re-extracted from the selected case specimens, derivatized with pentafluoropropionic anhydride (PFPA), and analyzed by gas chromatography-mass spectrometry (GC-MS). The MS spectra of these three antihypertensives and a metoprolol metabolite are nearly identical. All of the PFPA derivatives had baseline GC separation, with the exception of a metoprolol metabolite product, which co-eluted with atenolol. There were four primary mass fragments (m/z 408, 366, 202, and 176) found with all of the PFPA-beta-blockers and with the interfering metabolite product. However, atenolol has three unique fragments (m/z 244, 172, and 132), metoprolol has two unique fragments (m/z 559 and 107), propranolol has four unique fragments (m/z 551, 183, 144, and 127), and the metoprolol metabolite product has two unique fragments (m/z 557 and 149). These distinctive fragments were further validated by using a computer program that predicts logical mass fragments and performing GC-MS of deuterated PFPA-atenolol and PFPA-propranolol and of the PFPA-alpha-hydroxy metabolite of metoprolol. By using the unique mass fragments, none of the pilot fatality cases were found to contain more than one beta-blocker. Therefore, these mass ions can be used for differentiating and simultaneously analyzing these structurally similar beta-blockers in biological samples.

Effects of orange juice on the pharmacokinetics of atenolol.

OBJECTIVE: Fruit juices can significantly change the pharmacokinetics of several drugs. Our objective was to investigate the effect of orange juice on the pharmacokinetics of the beta-blocking agent atenolol. METHODS: In a randomized cross-over study with two phases and a washout of 2 weeks, ten healthy volunteers took either 200 ml orange juice or water thrice daily for 3 days and twice on the fourth day. On the morning of day 3, each subject ingested 50 mg atenolol with an additional amount of either 200 ml orange juice or water. The plasma concentrations of atenolol and the cumulative excretion of atenolol into urine were measured up to 33 h after its dosing. Systolic and diastolic blood pressures and heart rate were recorded in a sitting position before the intake of atenolol and 2, 4, 6, and 10 h after. RESULTS: Orange juice decreased the mean peak plasma concentration (C(max)) of atenolol by 49% (range 16-59%, P<0.01), and the mean area under the plasma atenolol concentration-time curve (AUC(0-33 h)) by 40% (range 25-55%, P<0.01). The time of the peak concentration (t(max)) and the elimination half-life (t(1/2)) of atenolol remained unchanged by orange juice. The amount of atenolol excreted into urine was decreased by 38% (range 17-60%, P<0.01), but the renal clearance remained unaltered. The average heart rate was slightly higher during the orange juice+atenolol phase than during the water+atenolol phase. CONCLUSIONS: Orange juice moderately interferes with the gastrointestinal absorption of atenolol. This food-drug interaction can be of clinical significance.

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 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.

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.

Micellar liquid chromatography: a worthy technique for the determination of beta-antagonists in urine samples.

Several beta-antagonists (acebutolol, atenolol, celiprolol, labetalol, metoprolol, nadolol, propranolol) were determined in urine samples with fluorometric detection after direct injection, in less than 15 min, with a micellar mobile phase of 0.1 M sodium dodecyl sulfate (SDS), 15% propanol, and 1% triethylamine at pH 3. The limits of detection (38 criterion) were usually between 3 and 30 ng/mL. The addition of propanol and triethylamine and the reduction of the pH of the mobile phase improved the efficiency of the chromatographic peaks that was rather low in pure micellar eluents. The selection of the composition of the mobile phase was easily performed through the use of an interpretive procedure which considered the retention times and peak shapes of the beta-antagonists in six chromatograms, obtained at varying concentrations of SDS (0.05-0.15 M) and propanol (5-15% v/v). The chromatograms of urine samples from healthy volunteers, which were administered atenolol, metoprolol, and propranolol, showed only one peak for the former drug and several peaks for the other two. These peaks corresponded to the parent drug and metabolites, which indicated the partial and the extensive degradation of metoprolol and propranolol, respectively.

Simultaneous determination of the beta-blocker atenolol and several complementary antihypertensive agents in pharmaceutical formulations and urine by capillary zone electrophoresis.

A simple capillary zone electrophoresis method is developed for the quantitation of the beta-blocker atenolol and the complementary antihypertensive agents bendroflumethiazide, amiloride, and hydrochlorothiazide in human urine samples. The electrophoretic separation is performed using a 78-cm x 75-micron-i.d. (70-cm effective length) fused-silica capillary. A borate buffer (pH 9) is used as running electrolyte. The sample is hydrostatically introduced for 20 s, and the running voltage is 25 kV at the injector end of the capillary. The analysis of urine samples requires the optimization of solid-phase extraction methods, achieving recoveries > or = 61% for all the drugs and good separation from the urine matrix. The method is successfully applied to the determination of these compounds in pharmaceutical formulations and in urine samples collected after the intake of Neatenol Diu (100 mg atenolol-5 mg bendroflumethiazide) and Kalten (50 mg atenolol-25 mg hydrochlorothiazide-2.5 mg amiloride). The method is validated in terms of reproducibility, linearity, and accuracy.

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.

The influence of gastrointestinal transit on drug absorption in healthy volunteers.

1. The effect of variability of gastric emptying and oro-caecal transit on the absorption of a multicomponent solution of frusemide, atenolol, hydrochlorthiazide and salicylic acid has been studied in six healthy subjects. Each subject was studied on five separate occasions: three times under basal conditions, once following metoclopramide and once following codeine pretreatment in an attempt to speed and slow transit respectively. 2. Inter-subject variability of gastric emptying, oro-caecal transit and the rate and extent of drug absorption was considerable. 3. The absorption of salicylic acid appeared rate-limited by gastric emptying but the rate and extent of frusemide, atenolol and hydrochlorthiazide absorption were unrelated to measures of gastric emptying or oro-caecal transit. 4. Codeine phosphate caused a two-fold delay in oro-caecal transit but did not influence gastric emptying while metoclopramide had no significant effect on either function. 5. Metoclopramide and codeine had no significant effect on the rate or extent of absorption of any of the study drugs. 6. Within the limits of this experiment, oro-caecal transit time did not appear to be an important determinant of frusemide, atenolol, hydrochlorothiazide or salicylic acid absorption. Other factors must account for the observed variability in drug absorption.

Effects of a non-absorbable osmotic load on drug absorption in healthy volunteers.

1. We have studied the effects of a non-absorbable osmotic load on the absorption of a multicomponent solution of frusemide, atenolol, hydrochlorothiazide and salicylic acid in six healthy volunteers. 2. Each subject was studied on up to four separate occasions. The drugs were administered in one of four solutions: a) a mannitol/electrolyte solution, b) a double-strength mannitol/electrolyte solution, c) a glucose/electrolyte solution and d) water. Lactulose or sulphasalazine were added as oro-caecal transit markers. Lactulose was included in the mannitol- and glucose-based solutions, adding a further non-absorbable osmotic load, and sulphasalazine was added to the water, adding little osmotic load. 3. The absorption of atenolol and hydrochlorothiazide was two- to three-times less from all lactulose-containing solutions than from the sulphasalazine-containing solution. The absorption of frusemide and salicylic acid was similar from all four solutions. 4. The largest non-absorbable osmotic load impaired the absorption of atenolol and hydrochlorothiazide most and the incorporation of glucose only partly restored absorption. 5. These results suggest that transmucosal water movement is an important determinant of atenolol and hydrochlorothiazide absorption but is less relevant for the absorption of frusemide and salicylic acid. Furthermore, these data demonstrate a previously unrecognised interaction between a commonly prescribed laxative--lactulose, and atenolol and hydrochlorothiazide.