Liquid chromatographic-electrospray tandem mass spectrometric determination of bisoprolol in human plasma

An analytical method for the determination of bisoprolol in human plasma has been developed based on liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analyte and internal standard (IS) diphenhydramine were cleaned up by protein precipitation with acetonitrile, reconstituted in mobile phase and separated by reversed-phase high-performance liquid chromatography (HPLC) using methanol:10 mm ammonium acetate:formic acid (70:30:0.1 v/v/v) as mobile phase. Detection was carried out by multiple reaction monitoring (MRM) on an LC-MS/MS system and was completed within 2.5 min. The assay was linear over the range 0.5-100 ng/mL with a limit of quantitation (LOQ) of 0.5 ng/mL. The intra- and inter-day precision levels were within 5.54 and 9.95%, respectively, while the accuracy was in the range 89.4-113%. This method has been utilized in a pharmacokinetic study, where healthy volunteers were treated with an oral dose of 5 mg bisoprolol.     

An HPLC method for the determination of bisoprolol in human plasma and its application to a pharmacokinetic study

A new high-performance liquid chromatographic method is described for the determination of bisoprolol in human plasma. The proposed method was based on the derivatization of bisoprolol with 4-chloro-7-nitro-2,1,3-benzoxadiazole in borate buffer at pH 9.5 to yield a fluorescent product. Chromatographic separation of bisoprolol was achieved by using isocratic elution at a flow rate of 1.2 mL/min on a C18 reversed-phase column (Inertsil, 4 μm, 150 4.6 mm) at 40°C. The mobile phase used for the analysis was methanol-water (70:30, % v/v). Fluorescence detector was used at the excitation and emission wavelengths of 458 and 525 nm, respectively. The method was validated for linearity, limit of detection, limit of quantification, precision, accuracy, recovery and system suitability. The assay was linear over the concentration range of 10-2000 ng/mL. This method was applied in pharmacokinetic studies of bisoprolol preparations in healthy volunteers.     

Rapid determination of gemcitabine in plasma and serum using reversed-phase HPLC

Gemcitabine (2'2'-difluorodeoxycytidine) is a pyrimidine analog used in the treatment of a variety of solid tumors. After intravenous (i.v.) administration, it is rapidly inactivated to 2'-deoxy-2',2'-difluorouridine (dFdU). A sensitive analytical method for the quantitation of gemcitabine is required for the assessment of alternative dosage and treatment schemes. A rapid and robust RP-HPLC assay for analysis of gemcitabine in human and animal plasma and serum was developed and validated using 2'-deoxyuridine (dU) and 5-fluoro-2'-deoxyuridine (5FdU) as internal standards. It is based on protein precipitation, the use of an Atlantis dC18 column of 100 mm length (inner diameter, 4.6 mm; particle size, 3 microm) and isocratic elution using a 10 mM phosphate buffer, pH 3.0, followed by isocratic elution with the same buffer containing 3% of ACN. For gemcitabine, RSD values for intraday and interday precision were < 4.4 and 5.3%, respectively, the LOQ was 20 ng/mL, and the assay was linear in the range of 0.020-20 microg/mL with an accuracy of > or =89%. The recovery for gemcitabine, dU and 5FdU was 86-98%. The assay was applied to determine gemcitabine levels in plasma samples of patients collected during and shortly after conventional infusion of 25-30 mg/kg body mass (levels: 2.0-18.9 microg/mL) and rats that received lower doses (1.5 mg/kg) via i.v., subcutaneous and oral drug administration (levels: 0.20-2.60 microg/mL). It could also be applied to estimate dFdU levels in human plasma.     

Improved HPLC determination of fluoxetine and norfluoxetine in human plasma

Summary An HPLC method with fluorescence detection has been developed for the determination of fluoxetine and its main metabolite norfluoxetine in human plasma. Pretreatment of the biological samples by liquid-liquid extraction was used to improve the sensitivity of a previously published SPE procedure. The method uses 200 μL plasma and recovery is good for both analytes. On a C₈ column with a mixture of perchlorate buffer and acetonitrile as mobile phase fluoxetine, norfluoxetine and the internal standard (paroxetine) were eluted in less than 9 min, without interference from the biological matrix. Response for both analytes was linearly dependent on concentration over the range 2.5–500 ng mL⁻¹, and repeatability (RSD%) was <4%. The limit of detection was 1 ng mL⁻¹ for both fluoxetines. Application to plasma samples from depressed patients treated with fluoxetine gave good results. There was no interference from other common CNS drugs. This method seems to be a useful tool for clinical monitoring, because it requires small plasma samples and is highly sensitive and highly selective.     

Validated HPLC method for the determination of fluconazole in human plasma

A high-performance liquid chromatographic assay with UV detection was developed for the determination of fluconazole in human plasma. The method utilized solid-phase extraction for sample clean-up. The separation was performed on a C18 column by isocratic elution with a mobile phase of 10 mM acetate buffer at pH 5.0 and methanol and UV detection at 210 nm. Validation was performed according to the current recommendations of the USFDA bioanalytical method validation guidance. The method proved to be specific, accurate, precise and linear between 200 and 10,000 ng/mL with correlation coefficients greater than 0.999. The coefficient of variation was within 11% and relative deviation was less than 10%.     

Liquid chromatography tandem mass spectrometry method for determination of bisoprolol in human plasma using d5‐bisoprolol as the internal standard

A simple, reliable and sensitive liquid chromatography tandem mass spectrometry (LC‐MS/MS) protocol was developed and validated for quantification of bisoprolol in human plasma. The sample was pretreated with a simple procedure of protein precipitation and an isotope‐labeled d5‐bisoprolol was used as internal standard. The chromatographic separation was performed on a Capcell Pak C₁₈ MG III column (100 mm × 2.0 mm, 5 µm). The protonated ion of the analyte was detected in positive ionization by multiple reaction monitoring mode. The mass transition pairs of m/z 326.3 → 116.3 and m/z 331.3 → 121.3 were used to detect bisoprolol and the internal standard, respectively. Linearity, accuracy, precision, recovery, matrix effect, dilution test and stability were evaluated during method validation over the range of 0.5–100 ng/mL. The validated method was successfully applied to analyze human plasma samples in a bisoprolol bioavailability study. Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid determination of haloperidol and its metabolites in human plasma by HPLC using monolithic silica column and solid-phase extraction

A rapid, sensitive and reproducible HPLC method was developed and validated for the analysis of haloperidol and its three main metabolites in human plasma. The analysis was carried out on a monolithic silica column (Chromolith Performance RP-18e, 100 x 4.6 mm). The mobile phase consisted of sodium phosphate (0.1 m, pH 3.5)-acetonitrile (80:20, v/v) at a flow rate of 2.0 mL/min. UV detection at 230 nm was used, with the detection limits of these compounds ranging from 2 to 5 ng. The separation factors of all studied compounds were in the range 2.30-16.32, while the resolution factors were from 1.00 to 5.37.     

Rapid determination of quinidine in human plasma by high-performance liquid chromatography

A sensitive and specific method for the determination of quinidine in plasma is described. Quinidine is extracted with chloroform, the extract is evaporated and reconstituted with methanol and the methanol extract is analyzed by high-performance liquid chromatography using a silica gel column. The recovery of quinidine from plasma varied between 93.0 to 100%. Quinidine levels as low as 12 ng/ml can be detected by this method.     

Rapid determination of ranitidine in human plasma by high-performance liquid chromatography

Summary An HPLC method has been developed for the quantification of rantidine in plasma for pharmacokinetic studies. Metoclopramide was used as internal standard. The method uses a simple and rapid sample clean-up procedure involving single-step extraction with organic solvent to extract ranitidine from plasma. After evaporation and reconstitution the samples are chromatographed on a 250 mm×4 mm base-stable reversed-phase column with 0.05 M ammonium acetate-acetonitrile, 75∶25 (v/v) as mobile phase and UV detection at 313 nm. The calibration graph was linear for quantities of ranitidine between 10 and 2000 ng mL⁻¹. Intra- and inter-dayCV did not exceed 11.64%. The quantitation limit was 10 ng mL⁻¹ for human plasma. The applicability of this method for pharmacokinetic studies of ranitidine after oral administration are described. Approximately 90 samples can be processed in 24 h.     

HPLC determination of plasma taurine

Summary The taurine content in plasma was determined by high performance liquid chromatographic analysis of its dansyl derivative with fluorometric detection. After the reaction with dansyl chloride, the derivative was extracted from an aqueous mixture by using tetrabutylammonium as a counter-ion. The influences of different tetrabutylammonium salts and of the eluent mixture composition were studied. Omotaurine, added as the internal standard to the plasma samples, assured good reproducibility. The procedure was applied to the determination of taurine in specimens from different mammalian species.     

The issue of HPLC determination of endogenous lipoic acid in human plasma

Lipoic acid (LA) is used extensively as a therapeutic agent for the treatment of various diseases. Many methods have been reported for the determination of LA plasma levels and its metabolites after its supplementation, but available information concerning endogenous plasma levels is still scarce. Studies which directly focused on determining the endogenous plasma levels provided highly controversial results, <4.9 nmol/L or 143.7–197.0 nmol/L. The main aim of this study was to verify the levels of free LA in the plasma of 40 individuals (17 women, 23 men). This group was nonsupplemented with LA and met the conditions for incorporation into the blood donors register. We measured the levels of LA using an HPLC method with very sensitive coulometric detection after previous sample preparation including deproteination and solid‐phase extraction with a Phenyl cartridge. Our limit of detection was 1.85 nmol/L and was better than the values reported in studies that directly focused on determining the endogenous plasma levels of LA: 2.4 and 4.9 nmol/L respectively. However, the levels of free LA in the plasma of nonsupplemented voluntary blood donors were not detectable in all cases. The presented results of our study show that endogenous concentrations of LA are <1.85 nmol/L.     

A validated HPLC determination of the flavone aglycone diosmetin in human plasma

Diosmetin, 3',5,7-trihydroxy-4'-methoxy flavone, is the aglycone of the flavonoid glycoside diosmin that occurs naturally in foods of plant origin. Diosmin exhibits antioxidant and anti-inflammatory activities, improves venous tone and it is used for the treatment of chronic venous insufficiency. Diosmin is hydrolyzed by enzymes of intestinal micro flora before absorption of its aglycone diosmetin. A specific, sensitive, precise, accurate and robust HPLC assay for the determination of diosmetin in human plasma was developed and validated. Diosmetin and the internal standard 7-ethoxycoumarin were isolated from plasma by liquid-liquid extraction and separated on a C8 reversed-phase column with methanol-water-acetic acid (55:43:2, v/v/v) as the mobile phase at 43 degrees C. Peaks were monitored at 344 nm. The method was linear in the 10-300 ng/mL concentration range (r > 0.999). Recovery for diosmetin and internal standard was greater than 89.7 and 86.8%, respectively. Intra-day and inter-day precision for diosmetin ranged from 1.6 to 4.6 and from 2.2 to 5.3%, respectively, and accuracy was better than 97.9%.     

Simultaneous HPLC determination of 14 tricyclic antidepressants and metabolites in human plasma

A HPLC method has been developed for the simultaneous determination of seven tricyclic antidepressants (TCAs) and seven metabolites in human plasma. The analyte separation was obtained using a C8 reversed phase column and a mobile phase composed of 68% aqueous phosphate buffer at pH 3.0 and 32% ACN. The UV detector was set at 220 nm and loxapine was used as the internal standard. A careful pre‐treatment procedure for plasma samples was developed, using SPE on C2 cartridges, which gives satisfactory extraction yields (>80%) and good sample purification. The LOQs were always lower than 9.1 ng/mL and the LODs always lower than 3.1 ng/mL for all analytes. The method was successfully applied to plasma samples from depressed patients undergoing therapy with one or more TCA drugs. Precision data (RSD <8.1%), as well as accuracy results (recovery >80%), were satisfactory and no interference from other drugs was found. Hence the method seems to be suitable for the therapeutic drug monitoring of patients treated with TCAs under monotherapy or polypharmacy regimens.     

Rapid HPLC analysis of the antiepileptic lamotrigine and its metabolites in human plasma

A liquid chromatographic method with diode array detection (DAD) has been developed for the analysis of the antiepileptic agent lamotrigine (LTG) and its metabolites, lamotrigine 2-N-glucuronide and 2-N-methylated in plasma samples. The analytes were separated on a C8 RP column, using a mobile phase composed of methanol and a 0.45 mM, pH 3.5 phosphate buffer containing 0.17% triethylamine (24:76 v/v). Melatonin was used as the internal standard (IS). The DAD detector was set at 220 nm for the detection of all the analytes. A simple protein precipitation with methanol guaranteed high extraction yield values (>90%) and good purification from matrix interference. Good linearity was obtained in the 0.1-15.0 microg/mL range for LTG and lamotrigine 2-N-glucuronide and in the 0.1-2.0 microg/mL range for lamotrigine 2-N-methylated. The analytical method was validated in terms of precision, extraction yield, and accuracy. These assays gave RSD% values for precision always lower than 4.3% and mean accuracy higher than 80%. The method seems to be suitable for the analysis of plasma samples from patients treated with Lamictal.     

反相高效液相色谱法测定兔血浆中的灯盏乙素

建立一种测定兔血浆中灯盏乙素浓度的高效液相色谱法.以芦丁为内标,采用甲醇沉淀蛋白法对血浆样品进行预处理.HPLC色谱柱为Kromasil C18柱(4.6 mm×250 mm,5 μn),流动相为水(用醋酸调pH 3.0)-乙腈(7822),检测波长335 nm,流速1.0 mL/min,柱温为室温;本法的线性范围为0.025～4μg/mL,最低定量浓度为0.025 μg/mL;方法回收率为95.9%～104.8%,日内RSD为1.9%～7.3%,日间RSD为1.9%～8.9%.方法学评价结果表明本方法灵敏、准确,操作简便,可用于深入研究灯盏乙素在动物体内的药代动力学过程.     

Enantioanalysis of bisoprolol in human plasma with a macrocyclic antibiotic HPLC chiral column using fluorescence detection and solid phase extraction

A sensitive, enantioselective, high-performance liquid chromatographic (HPLC) method was developed and validated to determine S-(-)- and R-(+)-bisoprolol in human plasma. Baseline resolution was achieved using the teicoplanin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic T with a polar ionic mobile phase (PIM) consisting of methanol-glacial acetic acid-triethylamine (100 : 0.02 : 0.025, v/v/v) at a flow rate of 1.5 ml/min and fluorescence detection set at 275 nm for excitation and 305 nm for emission. All analyses with S-(-)-atenolol as the internal standard were conducted at ambient temperature. The assay involved the use of a solid-phase extraction procedure for human plasma samples prior to HPLC analysis. The C18 cartridge gave good recovery rates for both enantiomers without any interference. The method was validated over the range of 20-200 ng/ml for each enantiomer concentration. Recovery rates for S-(-)- and R-(+)-bisoprolol enantiomers were in the range of 95-102%. The method proved to be precise (within-run precision expressed as % RSD ranged from 1.0-6.2% and between-run precision ranged from 0.9-6.7%) and accurate (within-run accuracies expressed as percentage error ranged from 0.2-4.8% and between-run accuracies ranged from 0.3-1.7%). The limit of quantitation and limit of detection for each enantiomer in human plasma were 20 and 5 ng/ml, respectively.     

HPLC determination of irbesartan in human plasma: its application to pharmacokinetic studies

A simple and rapid HPLC method using fluorescence detection was developed for determination of irbesartan in human plasma. Sample preparation was accomplished through a simple deproteinization procedure with 0.4 mL of acetonitrile containing 800 ng/mL of losartan (internal standard), and to a 0.1 mL plasma sample. Chromatographic separation was performed on a Zorbax Xclipse XDB C₁₈ column (150 × 4.6 mm, i.d., 5 µm) at 40°C. An isocratic mobile phase, acetonitrile:0.1% formic acid (37:63, v/v), was run at a flow‐rate of 1.0 mL/min, and the column eluent was monitored using a fluorescence detector set at excitation and emission wavelengths of 250 and 370 nm, respectively. The retention times of irbesartan and losartan were 4.4 and 5.9 min, respectively. This assay was linear over a concentration range of 10–5000 ng/mL with a lower limit of quantification of 10 ng/mL. The coefficient of variation for this assay precision was less than 8.48%, and the accuracy exceeded 94.4%. The mean relative recoveries of irbesartan and losartan were 98.4 and 99.1%, respectively. This method was successfully applied for pharmacokinetic study after oral administration of irbesartan (300 mg) to 23 Korean healthy male volunteers. Copyright © 2009 John Wiley & Sons, Ltd.     

HPLC determination of diltiazem in human plasma and its application to pharmacokinetics in humans

A simple and sensitive reversed-phase high performance liquid chromatographic method (HPLC) has been developed and validated for the routine analysis of diltiazem in human plasma and the study of the pharmacokinetics of the drug in the human body. Diltiazem and diazepa (internal standard) were extracted with a mixed organic solution of hexane, chloroform and isopropanol (60:40:5, v/v/v), and then HPLC separation of the drugs was performed on an Spherisorb C(18) column and detected by ultraviolet absorbance at 239 nm. The use of methanol-water solution (containing 2.8 mm triethylamine, 80:20, v/v) as the mobile phase at a fl ow-rate of 1.2 mL/min enables the baseline separation of the drugs free from interferences with isocratic elution. The method was linear in the clinical range 0-300 ng/mL and the lower limit of detection of diltiazem in plasma was 3 ng/mL. The range of percentage of relative standard deviation (%RSD) was from 3.5 to 6.8% for within-day analyses and from 6.2 to 8.4% for between-day analyses, respectively. The extraction recoveries of diltiazem from spiked human plasma (n = 5) at three concentrations were 91.4-104.0%. The method has been used to determine diltiazem in human plasma samples from eight volunteers who had taken diltiazem hydrochloride slow release tables and the data obtained was fitted with a program on computer to study the pharmacokinetics. The results showed that the peak level in plasma approximately averaged 118.5 +/- 14.3 ng/mL at 3.1 +/- 0.4 h, and the areas under the drug concentration curves (AUC) was 793.1 +/- 83.1 ng.h/mL.