A Selective LC Method for the Determination of Reboxetine in Human Plasma with Fluorescence Detection

A rapid, simple, accurate, sensitive and reproducible high performance liquid chromatographic method for the quantitation of reboxetine (REB) in human plasma using fluvoxamine as an internal standard (IS) has been developed and validated. The method is based on derivatization with 7-chloro-4-nitrobenzofurazan (NBD-Cl). The NBD-derivatives in plasma were extracted by liquid–liquid extraction and chromatographed on a reversed phase C₁₈ column with isocratic elution using acetonitrile and aqueous nitric acid (pH 3) solution. Calibration curve was linear over the range 2.0–200.0 ng mL⁻¹ with inter- and intra-assay precision (RSD%) of less than 4%. The mean recovery was about 94% for REB. The applicability of the method to the plasma was also studied.

     

Quantitative and Qualitative Analysis of CKD-501, Lobeglitazone,in Human Plasma and Urine Using LC–MS/MS and Its Application to a Pharmacokinetic Study

A simple, rapid and sensitive LC–MS/MS method in positive ion mode was developed and validated to determine CKD-501, lobeglitazone, in human plasma and urine using glipizide as an internal standard (IS). Lobeglitazone is a novel thiazolidinedione (TZDs)-based peroxisome proliferator-activated receptor (PPAR) agonist, used for the management of type-2 diabetes. After mixing the IS, dissolved in acetonitrile, with a plasma or urine sample containing lobeglitazone, 10 μL of supernatant was injected into the LC–MS/MS system. Quantification was performed in the multiple reaction monitoring (MRM) mode using transition of 481.5 → 152.2 (m/z) for lobeglitazone and 446.1 → 321.2 (m/z) for the IS. The method showed good linearity over concentration ranges of 0.5–1,000 ng mL⁻¹ for plasma and 0.2–250 ng mL⁻¹ for urine (r ² ≥ 0.9996). The mean percent extraction recovery of lobeglitazone was 90.8 % for plasma and 87.3 % for urine, while the recoveries of the IS were greater than 86.4 % for both. The intra-day and inter-day precision of plasma ranged from 1.1 to 3.7 and 2.5 to 3.3 % (RSD), respectively, and the intra- and inter-day precision of urine ranged from 1.5 to 2.7 and 3.2 to 3.5 %, respectively. This method is simple, sensitive, and applicable for the pharmacokinetic study of lobeglitazone in human plasma. Most of the urine concentrations of lobeglitazone were below the LLOQ because the lobeglitazone is extensively metabolized.

     

A Simultaneous Determination of Trazodone and Its Metabolite 1-m-Chlorophenylpiperazine in Plasma by Liquid Chromatography with Electrochemical Detection

Abstract

A liquid chromatographic method coupled with electrochemical detection was developed to measure plasma trazodone and its metabolite 1-m-chlorophenylpiperazine (m-CPP). Following extraction from 1 ml of alkaline plasma with methyl-t-butyl ether, the extracts were chromatographed on a reversed phase trimethylsilyl bonded column using a 0.05 M phosphate buffer and acetonitrile (90:10) with n-nonylamine and sodium heptane sulfonate added to the mobile phase. The compounds were detected via a thin layer electrochemical transducer with glassy carbon electrodes at a potential of + 1.15V vs Ag/AgCl reference electrode. The recovery of trazodone ranged from 91–97% and the coefficient of variation was less than 5% for between run and within-run analyses. The recovery of m-CPP ranged from 82–86% and the coefficient of variation was less than 8% for between run and within-run analysis. Steady state plasma concentration data are presented from several patients.     

An HPLC Assay for a Prostacyclin Analogue,Ciprostene Calcium,in Human Plasma

Abstract

A sensitive assay has been developed for the quantification of the prostacyclin analogue, ciprostene calcium, in human plasma. The method involves solid phase extraction of ciprostene calcium and internal standard, carbacyclin, from a small volume of human plasma. The extract is derivatized with 4-bramamethyl-7-acetoxycoumarin, and the derivatized product extracted with a polar solid phase cartridge and concentrated by evaporation. The final extract is separated by reversed phase HPIC and measured by a fluorimetric detector following post-column alkaline hydrolysis. The overall extraction efficiency is better than 75%, and the assay is linear over the concentration range studied (5–20 ng/ml). The limit of quantification was approximately 5 ng/ml. Ultimate sensitivity was limited by interfering peaks endogenous to the biological matrix. Coefficients of variation at mid-range concentrations are less than 10%.     

A new HPLC method for the simultaneous determination of ascorbic acid and aminothiols in human plasma and erythrocytes using electrochemical detection

A new, simple, economical and validated high-performance liquid chromatography linked with electrochemical detector (HPLC-ECD) method has been developed and optimized for different experimental parameters to analyze the most common monothiols and disulfide (cystine, cysteine, homocysteine, methionine, reduced (GSH) and oxidized glutathione (GSSG)) and ascorbic acid present in human plasma and erythrocytes using dopamine as internal standard (IS). Complete separation of all the targets analytes and IS at 35 °C on Discovery HS C18 RP column (250 mm × 4.6 mm, 5 μm) was achieved using 0.05% TFA:methanol (97:3, v/v) as a mobile phase pumped at the rate of 0.6 ml min⁻¹ using electrochemical detector in DC mode at the detector potential of 900 mV. The limits of detection (3 S/N) and limits of quantification (10 S/N) of the studied compounds were evaluated using dilution method. The proposed method was validated according to standard guidelines and optimization of various experimental parameters and chromatographic conditions was carried out. The optimized and validated HPLC-ECD method was successfully applied for the determination of the abovementioned compounds in human plasma and erythrocytes. The method will be quite suitable for the determination of plasma and erythrocyte profile of ascorbic acid and aminothiols in oxidative stress and other basic research studies.     

Determination of Cefaclor in Human Plasma by Reversed-Phase High-Performance Liquid Chromatography with UV Detection and Its Application to the Bioequivalence Studies

Abstract

A selective and sensitive reversed-phase high-performance liquid chromatography method was developed and validated for quantitation of cefaclor in human plasma using cefradine as an internal standard. Calibration curve was linear over range of 0.1–20 mg · L^?1. The intra- and inter-run relative standard deviations of the assay were less than 7%. The mean absolute recoveries determined at the concentrations of 0.3, 3.0, 8.0, and 15.0 mg · L^?1 were 69.9%, 69.9%, 77.1% and 72.0%, respectively. The analytical method established was proved to be specific, precise, sensitive, and suitable for applying in the pharmacokinetic and bioequivalence studies of cefaclor in human.     

Combining Low Pressure Liquid Affinity Chromatography and Flow Injection Analysis for the Quantitation of Immunoglobulins

Abstract

A simple, accurate method combining low pressure liquid affinity chromatography and flow injection analysis is described for the quantitation of immunoglobulins in biologicals fluids. The affinity matrix consists of Protein A covalently immobilized to a 2-fluoro-1 methylpyridinium salt activated Fractogel support. Utilizing a 1 cm³ affinity column, optimized binding and eluting buffer flow rates of 0.7 and 1.5 mL min^?1, respectively, and a sample loop size of 100 μL, IgG's can be eluted from the affinity column in about 9 min. Linear standard curves (r > 0.99) were obtained at concentrations up to at least 4.0 and 8.0 mg mL^?1 respectively for human and bovine IgG. Recovery yields are good ranging from 96–100%. The within day CV for human and bovine IgG was found to be less than 3.0% whereas the day to day CV was less than 3.4% (n=10). IgG concentrations of spiked and unspiked bovine plasma samples obtained by the low pressure affinity/flow injection method when compared to those obtained by the radial immunodiffusion agreed to within 4%.     

Electrochemical Behavior of Dopamine at a Mercury Electrode in the Presence of Citrate: Analytical Applications

Abstract

Dopamine can be determined by voltammetric methods using a mercury electrode, previously oxidized at +0.30 V. The oxidation product formed is stabilized in the presence of citrate and undergoes reduction at ?0.31 V. This work describes the electrochemical behavior of dopamine at a mercury electrode in the presence of citrate and its application in the development of a square‐wave voltammetric method for the dopamine determination in pharmaceutical formulations. The method was in‐house validated for determination of dopamine in injectable formulations. The detectability of the method was 0.02 µg ml^?1.     

Simultaneous LC Determination of Quercetin, Kaempferol and Isorhamnetin in Rabbits after Intragastric Administration of an Ethanol Extract from Pollen Typhae

A simple and rapid reversed-phase LC method was developed and validated for simultaneous determination of three flavonoids, quercetin (QU), kaempferol (KA) and isorhamnetin (IS), in rabbit blood plasma. The plasma was deproteinized using 10% trichloroacetic acid and extracted by n-butanol–acetoacetate solvent prior to LC analysis. The analyte was separated on a reversed-phase column with acetonitrile and 0.1% phosphoric acid in water (27:73, v/v) as mobile phase at a flow-rate of 0.8 mL min^?1, and UV detection wavelength at 369 nm. By this developed method, the concentrations of QU, KA and IS were linearly related to their responses in the range of 0.05–2.5 μg mL^?1. The precision and accuracy for QU, KA and IS in plasma were within ±15% except for the limit of quantitation (LOQ), where they were within ±20%. The validated method has been successfully applied in the pharmacokinetic study of QU, KA and IS in rabbits after intragastric administration of an ethanol extract from traditional Chinese medicine Pollen Typhae.     

Analysis of Biogenic Amines and Their Acidic Metabolites by Reversed-Phase Liquid Chromatography with Electrochemical Detection

Abstract

An improved reversed-phase liquid chromatography analysis with an electrochemical detector was developed to determine concurrently concentrations of the basic neurotransmitters, serotonin, dopamine, norepinephrine and epinephrine and their acidic metabolites. A combination of extraction procedures and a novel method of peak identification using applied potentials from 0.35 to 0.95 volts are described.     

Ion-Pair Liquid Chromatographic Analysis of Phenylpropanolamine in Plasma and Urine by Post-Column Derivatization with O-Phthalaldehyde

Abstract

A high pressure Liquid chromatographic analysis of phenylpropano Lamine in plasma and urine by post-column derivatzaition with o-phthalaldehyde is described. Plasma samples are extracted with methylene chloride under alkaline conditions. Urine is diluted with mobile phase without extraction. Using fluorescence detection, the method is sufficiently sensitive (2 ng/ml in 0.5 ml of plasma and 0.5 mcg/ml in 0.2 ml of urine) so that phenylpropano lamine concentrations in plasma or urine may be measured for up to 24 hours following a 75 mg oral dose. Coefficients of variation for inter-day and intra-day precision are less than 10%.     

Metabolomics analysis of human plasma metabolites reveals the age- and sex-specific associations

Abstract

Objectives: The objective of this study was the investigation of age- and sex-associations in a set of blood plasma metabolites in healthy male and female subjects.

Methods: A comparison study design with male and female subjects of various ages was used. Metabolic profiling was performed using electrospray ionization tandem mass spectrometry that yielded 186 metabolite concentrations for each study participant. The key age-related metabolites were identified using an integrative analysis of absolute concentrations, metabolite ratios and the differential correlation of pairwise metabolite concentrations. All of the age-associated metabolites were adjusted prior to the analysis to account for differences in Body Mass Index (BMI).

Results: A total of 236 plasma samples from 140 female and 96 male subjects aged 20 to 82 years-old were collected and analyzed in the study. 13 and 14 age-associated metabolites (|r|?>?0.33 and p < 6.6×10^?5), 438 and 337 age-associated metabolite ratios (|r|?>?0.37 and p < 3.5×10^?6), and 5 and 10 core metabolites were discovered in the female and male groups, respectively. 80% of the metabolites displaying associations with age belonged to sphingolipids and phosphatidylcholines, and the two sexes shared less than 50% of the age-associated metabolites.

Conclusion: The study found that changes in metabolite concentrations, metabolite ratios and differential correlations were age and sex-specific.     

Quantitation of plasma and biliary cefpiramide concentrations in human samples using high-performance liquid chromatography

Cefpiramide is frequently used to treat biliary infections. However, no bioanalytical method has been validated to quantitate cefpiramide in human samples, particularly in bile. Therefore, this study was conducted to develop a simple, selective and validated high-performance liquid chromatographic method to determine cefpiramide in human plasma and bile. A protein precipitation procedure was used to extract cefpiramide and cefoperazone (internal standard, IS) from 200 μl of plasma and bile. Utilizing a Capcell Pak C₁₈ column (4.6 × 250 mm), cefpiramide and IS were separated using the timed-gradient mobile phase consisting of 0.1 m sodium acetate (pH 5.2) and acetonitrile at a flow rate of 1 ml/min with photodiode array detector (wavelength set at 273 nm). The calibration curves showed linearity at concentrations ranging from 1 to 150 μg/ml in both plasma and bile (r² > 0.999). The within- and between-run coefficients of variation (CVs) for plasma samples were 0.570–4.43 and 1.10–2.76%, respectively; for bile samples, the within- and between-day precision (CV) was 0.814–6.34 and 2.05–4.00%, respectively. Our newly developed bioanalytical method was successfully employed to quantify cefpiramide concentrations in both plasma and bile at multiple time points in patients with acute cholangitis.     

Determination of Pyridostigmine in Human Plasma by High-Performance Liquid Chromatography

Abstract

An easy to perform, specific, reproducible and sensitive high performance liquid chromatographic (HPLC) method to measure pyridostigmine concentration in human plasma was developed and validated. Sample clean-up consists of ion-pair extraction into dichloromethane in the presence of neostigmine as internal standard, followed by back extraction into an aqueous phase. Mean recovery of 110% (with a standard deviation of 10%) was determined for concentrations of 5 – 100 ng/ml. Chromatography on a 125·4 mm CN-propyl column using a mobile phase composed of 10% acetonitrile in 3.5×10^?4M NaH₂PO₄ and UV detection at 270 nm, yields clean chromatograms without any interferences from endogenous plasma components. Using 1 ml plasma samples the method has a limit of detection (LD) of 3 ng/ml, with %CV (precision) and bias (accuracy) ≥ 10% for concentrations in the range of 0–100 ng/ml. The method is being used in human pharmacokinetic studies of oral dosage forms of pyridostigmine.     

Determination of Dirithromycin,LY281389 and Other Macrolide Antibiotics by HPLC with Electrochemical Detection

Abstract

Sensitive and rapid assays have been developed for the determination of the macrolide antibotics dirithromycin, erythromycylamine, and LY281389 in plasma. The methods utilize dichloromethane extraction of alkalinized plasma and isocratic reverse phase HPLC with electrochemical detection. The lower limit of detection is 10 ng/ml. Calibration curves are linear and highly reproducible over the range of 20–500 ng/ml. Precision of the calibration curves is very good having relative standard deviations of 5% or less over the dynamic range. These methods can be used for other macrolide antibiotics with minor modifications to the mobile phase. The electrochemical response of various macrolides was found to be dependent upon the functionality at C-9 of the macrolide ring.     

Quantification of Quetiapine in Human Plasma by LC–MS–MS

A rapid liquid chromatographic method with electrospray ionization tandem mass spectrometric detection has been developed and validated for quantification of quetiapine in heparinized human plasma. Plasma samples, without a drying and reconstitution step, were extracted by solid-phase extraction and eluted with acetonitrile. The analyte and zolpidem tartrate (internal standard, IS) were chromatographed on a C₁₈ column; the mobile phase was 85:15 (v/v) acetonitrile–5 mM ammonium formate, pH adjusted to 4.5 with formic acid, at a flow rate of 0.5 mL min⁻¹. The retention times of quetiapine and the IS were 1.25 and 1.05 min, respectively, and the run time was 1.8 min per sample. Selected reaction monitoring of MH⁺ at m/z 384.12 and 308.11 resulted in stable fragment ions with m/z 253.02 and 235.09 for quetiapine and the IS, respectively. Response was a linear function of concentration in the range 1.0–240.0 ng mL⁻¹, with r ≥ 0.9994. Recovery of quetiapine and the IS ranged from 74.82 to 85.57%. The assay has excellent characteristics and has been successfully used for analysis of quetiapine in healthy human subjects in a bioequivalence study.

     

Quantitative and Qualitative Analysis of CKD-501, Lobeglitazone, in Human Plasma and Urine Using LC–MS/MS and Its Application to a Pharmacokinetic Study

A simple, rapid and sensitive LC–MS/MS method in positive ion mode was developed and validated to determine CKD-501, lobeglitazone, in human plasma and urine using glipizide as an internal standard (IS). Lobeglitazone is a novel thiazolidinedione (TZDs)-based peroxisome proliferator-activated receptor (PPAR) agonist, used for the management of type-2 diabetes. After mixing the IS, dissolved in acetonitrile, with a plasma or urine sample containing lobeglitazone, 10?μL of supernatant was injected into the LC–MS/MS system. Quantification was performed in the multiple reaction monitoring (MRM) mode using transition of 481.5?→?152.2 (m/z) for lobeglitazone and 446.1?→?321.2 (m/z) for the IS. The method showed good linearity over concentration ranges of 0.5–1,000?ng?mL^?1 for plasma and 0.2–250?ng?mL^?1 for urine (r ²?≥?0.9996). The mean percent extraction recovery of lobeglitazone was 90.8?% for plasma and 87.3?% for urine, while the recoveries of the IS were greater than 86.4?% for both. The intra-day and inter-day precision of plasma ranged from 1.1 to 3.7 and 2.5 to 3.3?% (RSD), respectively, and the intra- and inter-day precision of urine ranged from 1.5 to 2.7 and 3.2 to 3.5?%, respectively. This method is simple, sensitive, and applicable for the pharmacokinetic study of lobeglitazone in human plasma. Most of the urine concentrations of lobeglitazone were below the LLOQ because the lobeglitazone is extensively metabolized.     

High-performance liquid chromatographic analysis of amphotericin B in rat plasma using α-naphthol as an internal standard

A simple, sensitive and accurate reverse phase high-performance liquid chromatographic (RP-HPLC) method with photo-diode array detector (PDA) was developed and validated for the determination of amphotericin B (AMB) in the rat plasma using a new internal standard (IS) α-naphthol. The plasma samples were subjected to protein precipitation with methanol prior to a HPLC analysis. Chromatographic separations were achieved on a Nucleosil^® 100-5C18 (150 mm × 4.6 mm) column. The mobile phase consisted of acetonitrile and sodium acetate buffer (pH 4; 10 mM) in a gradient mode. Detection was carried out at a wavelength of 407 and 294 nm for AMB and IS, respectively. The retention times of AMB and IS were about 6.8 and 7.8 min, respectively. The calibration curve was linear in the range of 10-2000 ng mL⁻¹ for AMB (r² > 0.998). No significant matrix effect was observed on quantification of AMB or IS. At three quality control concentrations of 20, 500, and 2000 ng mL⁻¹, the intra-day and inter-day relative standard deviation ranged from 1.13% to 4.91%. The limit of detection (LOD) was 5 ng mL⁻¹ and the limit of quantification (LOQ) was 10 ng mL⁻¹ for AMB in rat plasma. This method is simple, sensitive, rapid and does not require any extensive sample purification before injecting into HPLC.     

LC–ESI–MS Determination of Lovastatin in Human Plasma

A convenient, selective and sensitive liquid chromatographic-electrospary ionization mass spectrometry (LC–ESI–MS) method was developed and validated to determine lovastatin in human plasma. The analyte was extracted from human plasma samples by typical liquid–liquid extraction, separated on a C₁₈ column by using the mobile phase consisting of water–methanol (13:87, v/v). Simvastatin was used as the internal standard (IS). The method was linear within the range of 0.1–20 ng mL⁻¹. The lower limit of quantification (LLOQ) was 0.1 ng mL⁻¹. The intra- and inter-run precision, calculated from quality control (QC) samples was less than 10.2%. The accuracy as determined from QC samples was in the range of 99.3–102.9% for the analyte. The mean recoveries for lovastatin and IS were 84.8 and 88.0%, respectively. The method was successfully applied for evaluation of the pharmacokinetic of lovastatin in healthy volunteers.     

High Performance Liquid Chromatographic Assay of Methoclopramide in Plasma Using a Silica Gel Column and An Aqueous Meobile Phase

Abstract

A high performance liquid chromatographic method for quantitating matoclopramide in plasma is presented. Proteins ara precipitated from the plasma sample with acetonitri la containing the internal standard, procainamida. The treated samples ara than analyzad using an Ultrasphere Si column, an aqueous solution at pH 7 of 65% CH³CN and 5.0 mM (NH₄)₂HPO₄ as a mobile phase, and a fluorescence detector. The retention times for drug and intarna1 standard ara 11.2 and 13.2 min, respectively. The caibration curve is Linear from 0.89 to 44.5 ng/ml. The detection limit is 0.89 ng/ml [signaL/hoisa = 31] for 0.2 ml plasma samples Pracision is measured by intraday and intarday coefficients o f variation, which are less than 10%. This method is currently being used for pharmacokinetic studies of methoclopramide.