Determination of metoprolol in rabbit blood using capillary electrophoresis with laser-induced fluorescence detection

This work described a sensitive method for determination of metoprolol in rabbit plasma.The method involved purification by ultrafiltration,derivatization with fluorescein isothiocyanate,determination by capillary electrophoresis(CE) coupled with laser-induced fluorescence(LIF) detector.Other components in plasma including a variety of amino acids and proteins did not interfere with the determination of metoprolol in experimental condition.The assay had a wide range(2.0-500 ng/mL) of linearity and a detection limit of 0.8 ng/mL.The intra- and inter-day precisions were satisfactory with relative standard deviation(RSD) less than 10.0%and accuracy within 10.0%.This method was successfully applied to pharmacokinetic study of metoprolol in rabbit blood.     

Determination and Pharmacokinetics of Mexiletine in Rabbit Plasma by Gas Chromatography with Mass Spectrometry

This paper describes a gas chromatography/mass spectrophotometry method for determination of mexiletine in rabbit plasma. Mexiletine and internal standard metoprolol were extracted from rabbit plasma with a mixture of ethylacetate and diethylether at basic pH with liquid-liquid extraction. Calibration curves were linear over the concentration range 45–2000 ng/mL. Intra- and inter-day precision (relative standard deviation) for mexiletine in rabbit plasma were less than 6.9%, and accuracy (relative error) was better than 6.8%. Recovery of mexiletine from rabbit plasma averaged 92.6%. This method was successfully applied to six rabbits which had given an oral capsule of 200 mg mexiletine.     

Development,validation and analytical error function of two chromatographic methods with fluorimetric detection for the determination of bisoprolol and metoprolol in human plasma

This work describes two high-performance liquid chromatographic methods for the individual determination of bisoprolol and metoprolol in human plasma. Analytical methods involve two different liquid-liquid extractions of human plasma, with diethyl ether for bisoprolol and with dichloromethane for metoprolol, coupled with a similar Nucleosil C(18) reversed-phase HPLC column. Fluorimetric detection was used to identify both beta-blockers. Retention times for bisoprolol and metoprolol were 8.7 and 3.2 min, respectively. Linear regressions for the calibration curves were linear at a concentration range of 6.25-200 ng/mL. Intra- and inter-day precision coefficients of variations and accuracy bias were acceptable (within 15%) over the entire range for both drugs. Average recovery was 89% for metoprolol and 98% for bisoprolol. Once the methods had been validated, analytical error functions were established as standard deviation (SD; ng/mL) = 2.216 + 3.608 x 10(-4)C(2) (C = theoretical concentration value) and SD-(ng/mL) = 0.408 + 0.378 x 10(-1)C for bisoprolol and metoprolol, respectively. The methods developed and their associated analytical error functions will be suitable for pharmacokinetic studies and for determination of plasma concentration if posology individualization of these drugs is needed.     

Determination of metoprolol in human plasma and urine by high‐performance liquid chromatography with fluorescence detection

A simple, specific and sensitive HPLC method has been developed for the determination of metoprolol in human plasma and urine. Separation of metoprolol and atenolol (internal standard) was achieved on an Ace C₁₈ column (5 μm, 250 mm×4.6 mm id) using fluorescence detection with λ_ex=276 nm and λ_em=296 nm. The mobile phase consists of methanol–water (50:50, v/v) containing 0.1% TFA. The analysis was performed in less than 10 min with a flow rate of 1 mL/min. The assay was linear over the concentration range of 3 – 200 and 5 – 300 ng/mL for plasma and urine, respectively. The LOD were 1.0 and 1.5 ng/mL for plasma and urine, respectively. The LOQ were 3.0 and 5.0 ng/mL for plasma and urine, respectively. The extraction recoveries were found to be 95.6 ± 1.53 and 96.4 ± 1.75% for plasma and urine, respectively. Also, the method was successfully applied to three patients with hypertension who had been given an oral tablet of 100 mg metoprolol.     

HPLC quantification of metoprolol with solid-phase extraction for the drug monitoring of pediatric patients

Although the analytical literature seems abundant for the determination of metoprolol in human plasma, a method using standard equipment providing a sensitive and simple high-performance liquid chromatographic (HPLC) method for limited blood volume, e.g. where 1 mL of blood in a 1 kg infant equals 70 mL of adult blood volume, has rarely been addressed. Therefore, in 500 microL of plasma, metoprolol was extracted using an internal standard and solid-phase extraction columns. Chromatographic analysis was performed on a Spherisorb C(6) column (5 microm particle size) at ambient temperature and fluorimetric detection with an excitation wavelength of 225 nm, and emission wavelength of 310 nm. The mobile phase [30% acetonitrile and 70% 0.25 m potassium acetate buffer (pH 4)] was pumped with 1 mL/min. Metoprolol recovery was determined at 73.0 +/- 20.5%, and the limit of quantitation was 2.4 ng/mL. Precision values of intra- and inter-assay were below 15.5% and those for accuracy were between 90 and 110%. This method was developed for monitoring and determination of pharmacokinetic parameters of metoprolol in pediatric patients and therefore metoprolol plasma concentrations in a 2-year-old child with ventricular tachycardia are reported. .     

Determination of azasetron hydrochloride in rabbit plasma by liquid chromatography tandem mass spectrometry and its application

A sensitive and selective liquid chromatography tandem mass spectrometry method for determination of azasetron hydrochloride in rabbit plasma was developed. After addition of doxapram hydrochloride as internal standard (IS), protein precipitation by 10% trichloroacetic acid was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB-C(18) (2.1 × 50 mm, 3.5 μm) column with acetonitrile-water as mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring mode was used to quantification using target fragment ions m/z 349.9 → 223.5 for azasetron hydrochloride and m/z 378.9 → 291.8 for the IS. Calibration plots were linear over the range of 6-1000 ng/mL for azasetron hydrochloride in plasma. The lower limit of quantitation for azasetron hydrochloride was 6 ng/mL. The mean recovery of azasetron hydrochloride from plasma was in the range 85.6-92.7%. The RSDs of intra-day and inter-day precision were both less than 12%. This method is simple and sensitive enough to be used in pharmacokinetic research for determination of azasetron hydrochloride in rabbit plasma.     

Determination of metoprolol enantiomers in human plasma and saliva samples utilizing microextraction by packed sorbent and liquid chromatography–tandem mass spectrometry

A sensitive, accurate and reliable bioanalytical method for the enantioselective determination of metoprolol in plasma and saliva samples utilizing liquid chromatography–electrospray ionization tandem mass spectrometry was developed and validated. Human plasma and saliva samples were pretreated by microextraction by packed sorbent (MEPS) prior to analysis. A new MEPS syringe form with two inputs was used. Metoprolol enantiomers and internal standard pentycaine (IS) were eluted from MEPS sorbent using isopropanol after removal of matrix interferences using aliquots of 5% methanol in water. Complete separation of metoprolol enantiomers was achieved on a Cellulose‐SB column (150 × 4.6 mm, 5 μm) using isocratic elution with mobile phase 0.1% ammonium hydroxide in hexane–isopropanol (80:20, v/v) with a flow rate of 0.8 mL/min. A post‐column solvent‐assisted ionization was applied to enhance metoprolol ionization signal in positive mode monitoring (+ES) using 0.5% formic acid in isopropanol at a flow rate of 0.2 mL/min. The total chromatographic run time was 10 min for each injection. The detection of metoprolol in plasma and saliva samples was performed using triple quadrupole tandem mass spectrometer in +ES under the following mass transitions: m/z 268.08 → 72.09 for metoprolol and m/z 303.3 → 154.3 for IS. The linearity range was 2.5–500 ng/mL for both R‐ and S‐metoprolol in plasma and saliva. The limits of detection and quantitation for both enantiomers were 0.5 and 2.5 ng/mL respectively, in both matrices (plasma and saliva). The intra‐ and inter‐day precisions were presented in terms of RSD values for replicate analysis of quality control samples and were <5%; the accuracy of determinations varied from 96 to 99%. The method was able to determine the therapeutic levels of metoprolol enantiomers in both human plasma and saliva samples successfully, which can aid in therapeutic drug monitoring in clinical laboratories. Copyright © 2016 John Wiley & Sons, Ltd.     

Development of a method for the determination of ibafloxacin in plasma by HPLC with flourescence detection and its application to a pharmacokinetic study

A simple, rapid, and sensitive high-performance liquid chromatographic method is developed for the determination of ibafloxacin in rabbit plasma. Plasma proteins are precipitated with acetonitrile, and after extraction with methylene chloride followed by desecation, ibafloxacin is determined by reversed-phase chromatography with fluorescence detection exciting at 330 nm and emission at 368 nm. Peaks corresponding to ibafloxacin and the internal standard (salycilic acid) are obtained at 9.8 and 5.2 min, respectively. The method is validated for a limit of quantitation of 10 ng/mL. The intraday relative standard deviation ranges from 4.78-7.15%, and the interday precision ranges from 1.32-4.03%. The method shows linearity for the two calibration curves used (10-100 ng/mL and 100-2000 ng/mL). The procedure described is applied successfully to a pharmacokinetics study of ibafloxacin in rabbits.     

Hyphenation of field‐amplified sample injection and transient isotachophoresis in CE for the determination of sotalol and metoprolol in human urine samples

A sensitive approach of capillary electrophoresis coupled with field‐amplified sample injection and transient isotachophoresis was developed for the simultaneous determination of two β‐blockers: sotalol and metoprolol. In this dual focusing technique, the samples were prepared via only dissolution in ultrapure water and then injected electrokinetically. Phosphate acted as both the background electrolyte and the leading electrolyte. Its optimized concentration was 80 mM. A total of 25 mM of glycine was used as the terminating electrolyte. Under optimum conditions, good separation of sotalol and metoprolol was achieved within 10 min. In comparison with the conventional method, the sensitivity enhancement factors were up to 1031 and 919 for sotalol and metoprolol, respectively. The proposed method was employed in the determination of sotalol and metoprolol in spiked human urine samples. The limits of detection and limits of quantitation obtained via ultraviolet detection were 5 and 12 ng/mL, respectively, for sotalol, and 10 and 25 ng/mL, respectively, for metoprolol. The intraday repeatability values were lower than 2.7 and 1.7% for peak area and migration time, respectively. The assay is a simple and efficient strategy with potential for application in clinical and biochemical laboratories for monitoring sotalol and metoprolol.     

Gas chromatography-mass spectrometry method for determination of metoprolol in the patients with hypertension

A sensitive and efficient method was developed for determination of metoprolol in human plasma by gas chromatography-mass spectrometry (GC-MS). Metoprolol and atenolol (internal standard, IS) were extracted from human plasma with a mixture of ethylacetate and diethylether at basic pH with liquid-liquid extraction. Calibration curves were linear over the concentration range 15-500 ng/ml. Intra- and inter-day precision values for metoprolol in human plasma were less than 6.4, and accuracy (relative error) was better than 8.8%. The analytical recovery of metoprolol from human plasma averaged 91.20%. The limits of detection (LOD) and quantification (LOQ) of metoprolol were 5.0 and 15 ng/ml, respectively. Also the developed and validated GC-MS method was successfully applied to three patients with hypertension who had been given an oral tablet of 100 mg metoprolol.     

Determination of metoprolol and its alpha-hydroxide metabolite in serum by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the simultaneous quantitation of metoprolol and alpha-hydroxymetoprolol concentrations in serum is developed. Analysis is performed on a C18 column using a 70:30 mixture of two solutions, A and B respectively. Solution A is 1 L of a 1% acetic acid solution with 25 mL of 0.005 M 1-heptanesulfonic acid. Solution B is 1 L of acetonitrile with 25 mL of 0.005 M 1-heptanesulfonic acid. Column effluent is monitored by fluorescence detection at an excitation wavelength of 225 nm. A 320-nm band pass filter is employed. The limit of sensitivity is approximately 2 ng/mL for both compounds. No potential sources of interference are identified. A coefficient of variation of less than 10% is observed on replicate determinations at 10 ng/mL for both compounds and at 300 and 180 ng/mL for metoprolol and alpha-hydroxymetoprolol, respectively. The method has the advantages of complete resolution of the metabolite of metoprolol, a high degree of reproducibility, adequate sensitivity, and an easily accessible internal standard.     

Determination of curdione in rabbit plasma by liquid chromatography mass spectrometry

A sensitive and selective liquid chromatography mass spectrometry method for determination of curdione in rabbit plasma was developed. After addition of tramadol as internal standard (IS), protein precipitation by acetonitrile was used for sample preparation. Chromatographic separation was achieved on a Zorbax SB‐C₁₈ (2.1 × 50 mm, 3.5 µm) column with acetonitrile–0.1% formic acid as mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive‐ion mode; selective ion monitoring was used for quantification using target fragment ions m/z 237 for curdione and m/z 264 for the IS. Calibration plots were linear over the range of 20–4000 ng/mL for curdione in plasma. The lower limit of quantification for curdione was 20 ng/mL. Mean recovery of curdione from plasma was in the range 94.3–98.4%. The RSD of intra‐day and inter‐day precision were both less than 9%. This method is simple and sensitive enough to be used in pharmacokinetic research for the determination of curdione in rabbit plasma. Copyright © 2011 John Wiley & Sons, Ltd.     

High performance liquid chromatography determination of doxorubicin and daunorubicin in plasma using UV detection and column switching

A method for the determination of doxorubicin and daunorubicin in plasma is described. The plasma is injected directly into a loop column and then washed with water. After switching the injection valve, the sample is separated on a phenyl column using detection at 254 nm. The detection limit is 10 ng/mL, the coefficient of variation is 7% for 100 ng/mL of doxorubicin and 4% for 200 ng/mL of daunorubicin.     

Simultaneous determination of niflumic acid and its prodrug, talniflumate in human plasma by high performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of niflumic acid and its prodrug, talniflumate, in human plasma. Niflumic acid and talniflumate were eluted isocratically with methanol-water (73:27, v/v, adjusted to pH 3.5 by acetic acid) at a fl ow rate of 1 mL/min. Indomethacin was used as an internal standard. Signals were monitored by an UV detector at 288 nm. Retention times of indomethacin, niflumic acid and talniflumate were 5.9, 7.2 and 13.5 min, respectively. Calibration plots were linear over the range 50-5000 ng/mL for niflumic acid and 100-5000 ng/mL for talniflumate. The limits of quantitation were 50 ng/mL for niflumic acid and 100 ng/mL for talniflumate. The intra- and inter-day relative standard deviations (RSD) of niflumic acid and talniflumate were less than 10% and the accuracies were higher than 90%. This method is rapid, sensitive and reproducible for the determination of niflumic acid and talniflumate in human plasma.     

Use of 18O3-clodronate as an internal standard for the quantitative analysis of clodronate in human plasma by gas chromatography/electron ionisation mass spectrometry

A sensitive and specific method for the quantitative determination of clodronate in human plasma is presented. The drug was extracted from plasma by anion-exchange chromatography and derivatised to the tetra-tert-butyldimethylsilyl derivative. 18O3-Clodronate was prepared from unlabeled clodronate and used as an internal standard. Gas chromatography/mass spectrometry (GC/MS) under electron ionisation conditions was used for quantitative measurement of the drug, using m/z 643.16 and 651.17 for target and internal standard, respectively. Calibration graphs were linear within the range of 10-1280 ng/mL plasma. Intra-day precision was 1.8% (10 ng/mL), 0.5% (40 ng/mL), 1.0% (120 ng/mL), 0.5% (200 ng/mL), 0.5% (400 ng/mL) and 2.7% (800 ng/mL) and inter-day variability was found to be 0.7% (10 ng/mL), 1.6% (40 ng/mL), 1.3% (120 ng/mL), 2.3% (200 ng/mL), 2.5% (400 ng/mL) and 1.2% (800 ng/mL). Intra-day accuracy showed deviations of 0.8% (10 ng/mL), 0.8% (40 ng/mL), 0.9% (120 ng/mL), 0.9% (200 ng/mL), 1.9% (400 ng/mL) and 0.3% (800 ng/mL) and intra-day accuracy was of -1.4% (10 ng/mL), 0.0% (40 ng/mL), -0.7% (120 ng/mL), -0.4% (200 ng/mL), -1.2% (400 ng/mL) and -3.3% (800 ng/mL). The stable isotope labeled standard was found to be stable under analysis conditions.     

Determination of orbifloxacin in rabbit plasma by high-performance liquid chromatography with fluorescence detection.

A simple and sensitive high-performance liquid chromatography method is developed for the determination of orbifloxacin (ORB) in rabbit plasma. Sample preparations are carried out by adding phosphate buffer (pH 7.4, 0.1 M) and extracting with trichloromethane. ORB and the internal standard, norfloxacin (NOR), are separated on a reversed-phase column using an aqueous phosphate buffer-acetonitrile (80:20, v/v) mobile phase. The concentrations of ORB and NOR eluting from the column with retention times of 2.16 and 3.09 min, respectively, are monitored by fluorescence detection at 338 (excitation) and 425 nm (emission). The method is shown to be linear from 4 to 1500 ng/mL (regression coefficient r2 = 0.999). The quantitation and detection limits are 4 and 9 ng/mL, respectively. Mean recovery is determined as 92% by the analysis of plasma standards containing 150, 750, and 1500 ng/mL. Inter- and intra-assay precisions were 4 and 3%, respectively.     

Quantitative determination of pravastatin and its metabolite 3α‐hydroxy pravastatin in plasma and urine of pregnant patients by LC‐MS/MS

This report describes the development and validation of a chromatography/tandem mass spectrometry method for the quantitative determination of pravastatin and its metabolite (3α‐hydroxy pravastatin) in plasma and urine of pregnant patients under treatment with pravastatin, as part of a clinical trial. The method includes a one‐step sample preparation by liquid–liquid extraction. The extraction recovery of the analytes ranged between 93.8 and 99.5% in plasma. The lower limits of quantitation of the analytes in plasma samples were 0.106 ng/mL for pravastatin and 0.105 ng/mL for 3α‐hydroxy pravastatin, while in urine samples they were 19.7 ng/mL for pravastatin and 2.00 ng/mL for 3α‐hydroxy pravastatin. The relative deviation of this method was <10% for intra‐ and interday assays in plasma and urine samples, and the accuracy ranged between 97.2 and 106% in plasma, and between 98.2 and 105% in urine. The method described in this report was successfully utilized for determining the pharmacokinetics of pravastatin in pregnant patients enrolled in a pilot clinical trial for prevention of preeclampsia. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous determination of beta-blockers in human plasma using liquid chromatography-tandem mass spectrometry

A detailed procedure for the analysis of four beta-blockers, acebutolol, labetalol, metoprolol and propranolol, in human plasma by high-performance liquid chromatography (LC)-tandem mass spectrometry (MS-MS) using an MSpak GF column, which enables direct injection of crude plasma samples, is presented. Protein and/or macromolecule matrix compounds were eluted first from the column, while the drugs were retained on the polymer stationary phase of the MSpak GF column. The analytes retained on the column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. All drugs showed base peak ions due to [M + H]+ ions by LC-MS with positive ion electrospray ionization, and the product ions were produced from each [M + H]+ ion by LC-MS-MS. Quantification was performed by selected reaction monitoring. The recoveries of the four beta-blockers spiked into plasma were 73.5-89.9%. The regression equations for all compounds showed excellent linearity in the range 10-1000 ng/mL of plasma, with the exception of propranolol (10-800 ng/mL). The limits of detection and quantification for each drug were 1-3 and 10 ng/mL, respectively, of plasma. The intra- and inter-day coefficients of variation for all drugs in plasma were not greater than 10.9%.     

Determination of metoprolol and its alpha-hydroxylated metabolite in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed for the simultaneous determination of metoprolol and its alpha-hydroxylated metabolite in plasma, Metoprolol, alpha-hydroxymetoprolol and alprenolol (internal standard) are extracted from plasma at alkaline pH with diethyl ether-dichloromethane (4:1, v/v) and back-extracted with 0.01 N sulfuric acid. A 100-microliter volume of the acidic extract is injected into the chromatographic system. The compounds are eluted in about 12 min with acetonitrile-acetate buffer (75:25, v/v) on a LiChrosorb RP-8 (5 micron) column. The quantitative determinations are made fluorometrically. Concentrations down to 35 nmol/1 (10 ng/ml) of metoprolol base and 30 nmol/1 (8 ng/ml) of alpha-hydroxymetoprolol base in plasma can be determined with good precision and accuracy.     

Determination of artemether in plasma and whole blood using HPLC with flow-through polarographic detection

An HPLC method with polarographic detection for the trace determination of artemether in plasma and whole blood was developed and applied to pharmacokinetic and clinical pharmacological studies. The method showed high sensitivity and selectivity because of the easy reduction of the peroxide linkage of artemether at the mercury drop electrode. The detection limit was 10 ng and the detector response was linear over the range of 10 ng to 1 microgram artemether injected onto the column. The largest relative standard deviation of 10 replicate measurements of standard solutions (concentrations of 10 ng/mL-1 microgram/mL) was 8%. The recovery from whole blood and plasma of added drug (concentrations of 15-480 ng/mL) was 71-100%.