Sensitive Determination of Piritramide in Human Plasma by High-Performance Liquid Chromatography

Abstract

A selective and sensitive method for the determination of piritramide in human plasma is described. After addition of 50 μl of 2 M ammonia and 20 μl of aqueous promethazine solution (100 ng/10 μ1) as an internal standard, 1 ml of plasma was extracted with 5 ml of toluene (extraction efficiency: 93.9 × 2.6%; mean × S. D.; n = 5). HPLC was performed with a phenyl hypersil NC-04 column, particle size 5 μm, 250 × 4 mm I. D.; mobile phase: 8 parts of acetonitrile and 2 parts of 10 mM potassium phosphate buffer (pH 3. 3). The flow rate was set to 2 ml/min and the column temperature was 22°C. The assay was linear in a concentration range of 3.75 ? 3000 ng/ml (r = 0.999), with a lower limit of detection of 3 ng/ml. The precision was determined using spiked plasma samples (15 ng/ml; 300 ng/ml), with coefficients of variation of 6.1 and 5.9% (intraday; n = 5) and 6.5 and 0.2% (interday; n = 3). In the range of 5.6 ? 1500 ng/ml, the accuracy of the assay was 2.82%. The method was used for the determination of piritramide plasma concentrations in patients receiving intra- or postoperative analgesia.     

Determination by High Performance Liquid Chromatography of Stability of Tetrahydro-β-carbolines at Different Ambient Temperatures

Abstract

To determine the stability of tetrahydro-β-carboline compounds over time and at different temperatures, a reversed-phase high pressure liquid chromatography system with electrochemical detection was utilized. Noreleagnine (1,2,3,4-tetrahydro-β-carboline) and tetrahydroharman (1-methyl-1,2,3,4-tetrahydro-β-carboline) were dissolved in water or ascorbate (0.1 mg/ml) vehicle and stored at ?20°C, 22°C, or 37°C for one, seven or 12 days. After each solution was injected in the column in a concentration of 400–600 ng/10 μl, peak height values were obtained for the compound under each condition. Analysis of percent recovery showed that the two β-carbolines were relatively stable with a maximal degradation of 14% occurring only at the 12-day assay interval. These results suggest that this class of compound can be used in pharmacological studies in which they can be dispensed from a mini-pump implanted in tissue. Further, an HPLC system with electrochemical detector provides a valid and reliable procedure for quantification of indoleamine-aldehyde condensation products.     

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.     

A quantitative gas-liquid chromatographic method for the determination of neostigmine and pyridostigmine in human plasma

A sensitive and selective analytical method was used to measure the concentration of neostigmine and pyridostigmine in human plasma. The procedure involved preliminary ion-pair extraction of the drugs into dichloromethane, followed by concentration and analysis of the ion-pair complex using a gas-liquid chromatographic system fitted with a nitrogen detector. Using the peak area ratio technique, pyri-dostigmine bromide was used as the internal standard for the quantitation of neo-stigmine in plasma; neostigmine bromide was the internal marker for the determination of pyridostigmine. The method depends on the thermal dequaternisation of the quaternary amines, and can be used to detect 5 ng/ml in a 3-ml plasma sample. Accurate measurement can be made at levels of 50–1000 ng/ml. This assay procedure has been applied to the separate determination of the plasma concentration of neostigmine and pyridostigmine after single administration of intravenous doses in aneasthetised patients.     

Simultaneous quantification of mycophenolic acid and its glucuronide metabolites in human plasma by an UPLC–MS/MS assay

The aim of this study was to develop a reliable UPLC–MS/MS assay for accurate quantification of mycophenolic acid (MPA) and its glucuronide conjugates in human plasma. Plasma proteins were precipitated with acetonitrile and the chromatographic separation was achieved on a C₁₈ column with a gradient elution. The detection was performed by a triple quadrupole mass spectrometer in the positive electrospray ionization and multiple reaction monitoring mode. Linearity of the assay was demonstrated over the range of 20–10,000 ng/mL for MPA and MPA glucuronide (MPAG), and 2–1000 ng/mL for acyl MPA glucuronide in human plasma. The assay was precise and accurate with coefficient of variation and bias <15%. MPA and MPAG were stable at 25 °C up to 1 day in both heparin‐ and EDTA‐treated blood. In heparin‐ and EDTA‐plasma, MPA and MPAG were stable for at least 1 week at 25 and 4 °C, and 1 month at ?20 °C. However, 99% acyl MPA glucuronide degraded in both heparin‐ and EDTA‐blood as well as plasma when stored at room temperature for 1 day. All the analytes remained stable for at least 3 months in acidified EDTA‐plasma at ?80 °C. The assay was successfully applied on patients post hematopoietic stem cell transplantation. Copyright © 2016 John Wiley & Sons, Ltd.     

Simultaneous Analysis of Cimetidine and Ranitidine in Human Plasma by HPLC

Abstract

A rapid method for the simultaneous quantitation of the H₂-receptor antagonist drugs cimetidine and ranitidine in human plasma by isocratic ion-pair reverse-phase HPLC is described. The method involves a simple organic extraction step of the alkalinized plasma containing added internal standard followed by back extraction of the extract with dilute acetic acid and subsequent analysis of the aqueous acidic phase on a reverse-phase (C18) column. The eluting solvent was acetonitrile-water (20:80 v/v) containing 0.005 mole/litre octanesulphonic acid and was monitored at 229 nm. The run time for the assay was 12.5 minutes, with a detection limit for cimetidine of 50 ng/m1/(0.2 μmole/1) and that for ranitidine was 20 ng/ml (0.06 umole/1).     

Combined High-Performance Liquid Chromatography and Radioimmunoassay for A Novel Retinobenzoic Acid Derivative, 4-[(5,6,7,8-Tetrahydro-5,5,8,8-Tetramethyl-2-Naphthyl)-Carbamoyl] Benzoic Acid (AM-80), in Human Plasma

Abstract

4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-carbamoyl] benzoic acid (Am-80) is a novel retinobenzoic acid derivative possessing retinoid activity. Plasma concentration of this drug in clinical use is very low, less than 1 ng/ml, and could not be measured with direct immunoassays. A combination of high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA) was developed for this drug in human plasma. Am-80 in 0.5 ml of human plasma was extracted by solid-phase extraction, and the extract was purified by reversed-phase HPLC. The immunoreactivity in the fraction of the eluate was measured by competitive RIA. The within- and between-assay variances were 4.1 to 15.5% and 4.1 to 7.0%, respectively. The limit of quantification was 0.04 ng/ml in human plasma, which was much lower than that of direct RIA, 0.6 ng/ml, previously reported. This assay system could be used to observe the pharmacokinetics of Am-80 in human even after dermal application at very low dose.     

Assessment of a two-step high-performance liquid chromatographic assay using dual-wavelength ultraviolet monitoring for 25-hydroxyergocalciferol and 25-hydroxycholecalciferol in human serum or plasma

The technique of dual-wavelength monitoring was used to verify the purity of high-performance liquid chromatographic (HPLC) peaks quantified as 25-hydroxyergocalciferol and 25-hydroxycholecalciferol. The data obtained show the need for a second HPLC step prior to quantitation. Potential inaccuracy arising from inadvertent collection of radio-labelled decomposition products was assessed. Between-day coefficients of variation were 7.3, 5.0 and 3.6%, respectively for 11.3 (n = 12), 17.1 (n = 14), and 32.9 (n = 8) ng/ml of 25-hydroxycholecalciferol. For 25-hydroxyergocalciferol, these values were 6.4 and 3.8% for 11.1 (n = 12) and 20.1 (n = 8) ng/ml concentrations, respectively. Comparison of total 25-hydroxycalciferol with a competitive protein binding assay was made. The comparison produced a correlation coefficient (r) of 0.94 and a relationship of y = 1.03x + 3.3. Four of the samples contained more than 10 ng/ml of 25-hydroxyergocalciferol and the results are consistent with the reported 100% cross-reactivity of the competitive binding protein method for 25-hydroxyergocalciferol and 25-hydroxycholecalciferol. A simple regeneration procedure is also described which enables Sep-Pak C18 cartridges to be reused up to eighteen times. Samples may be stored at -18 degrees C for upto several months before assay and either serum or plasma may be used.     

Sensitive HPLC Assay for Ketoprofen in Human Plasma and its Application to Pharmacokinetic Study

Abstract

A selective and sensitive HPLC method was developed for the analysis of ketoprofen in human plasma. The assay involves an extraction of the drug and the internal standard (piroxicam) into diethyl ether from acidified plasma and then back-extracted into a small volume of alkaline aqueous solution before injection onto the HPLC column. A microbore column (2 mm I.D. × 10 cm) packed with a C18 reversed-phase material (5 pm ODS Hypersil) was used. The chromatographic separation was accomplished with a mobile phase comprising a mixture of acetonitrile-methanol-water (15 :20 : 65, v/v) containing 10 mM Na2HP04 buffer, pH 4. The mobile phase was pumped at a flow rate of 0.5 dmin. The eluant was monitored at 258 nm. With this procedure coefficients of variation were less than 10%. The detectionlimit was 0.05 μg/ml (i.e., 50 ng/ml) of plasma. The highly sensitive nature of this method was applied successfully to the dewmination of ketoprofen in human plasma for phmacokinetic studies.     

Reversed Phase High - Performance Liquid Chromatographic Analysis of Tricyclic Antidepressants in Plasma

Abstract

A reversed phase HPLC procedure is described for measuring the plasma concentration of four commonly used tricyclic anti-depressants (TCA): amitriptyline, desipramine, imipramine and nortriptyline in the range of 25 to 800ng per ml. The procedure involves rapid extraction, and HPLC analysis using a μBondapak C-18 column at 50°C, and a 254nm detector. Coefficients of variation are less than 5% for within run, and 7% for day-to-day experiments. Detection limits are: desipramine -0.5ng, nortriptyline or imipramine -0.6ng, and amitriptyline -0.7ng. Propoxyphene interferes with amitriptyline while chlorpromazine interferes with clomipramine. The procedure is easily adapted for clinical drug monitoring of TCA.     

High Performance Liquid Chromatographic Analysis of Desipramine in Human Plasma Using a Microbore Column Technique

Abstract

A reversed-phase, isocratic HPLC method has been developed for the quantitation of desipramine in human plasma. the method involved the use of cloimpramine as an internal standard. the chromatographic separation was accomplished with a mobile phase comprising acetonitrile-aqueous solution (60:40. v/v) containing 10 mM disodium hydrogenphosphate and 80 mM sodium dodecyl sulfate adjusted to pH 2. the mobile phase was pumped at a flow rate of 0.5 ml/min. the column used was a microbore column (2 mm I.D. × 100 mm) packed with a C18 reversed-phase material (5μm ODS Hypersil). Plasma samples were extracted at basic pH with diethyl ether followed by back-extraction into 0.1 N sulfuric acid. Using UV detection at 250 nm, the lower limit of sensitivity was 10 ng/ml. the inter- and intra-assay coefficients of variation were found to be less than 10%. the assay procedure was applied to a long term oral dosing study in patients to monitor the plasma concentration of desipramine.     

A Direct and Sensitive Method for the Determination of Salicylamide in Microplasma Samples by High-Performance Liquid Chromatography Using Fluorescence Detection

Abstract

A quantitative analysis of salicylamide in microplasma volumes by high-performance 1iquid chromatography using fluorescence detection is reported. The procedure is extremely simple and very rapid, involving the direct introduction of the plasma sample on the HPLC column. The assay procedure is linear over the concentration range studied, 0–100 ng/ml with correlation coefficient for the linear regression, r = 0.998. This assay procedure enables the detection of salicylamide as low as 5.0 ng/ml in plasma, using sample volume of 100 μl.     

Letter to the Editor

Abstract

The experiments show that Mo(VI) has very strong catalytic effect on the slow redox reaction between methyl orange and hydrazine sulfate in sulphuric acid medium at 100°C. And methyl orange exhibits a sensitive second derivative wave at ?0.72 V vs. SCE in the supporting electrolyte of NaOH. This provides a novel catalytic method for the determination of trace amounts of molybdenum using linear scan voltammetry as detection technique. A Calibration graph from 1 to 80 ng/ml and detection limit of 0.3 ng/ml Mo were obtained by the fixed-reaction time procedure. This new catalytic method has been applied to determining molybdenum in ore and standard steel samples, with excellent results.     

Determination of Chlordiazepoxide and Its Metabolites in Plasma by High Pressure Liquid Chromatography

Abstract

A rapid, sensitive and specific high pressure liquid chromatographic (HPLC) assay was developed for the determination of chlordiazepoxide and its metabolites from plasma. The assay involves extraction of chlordiazepoxide and its metabolites into diethyl ether from plasma buffered to pH 9. The overall recovery of chlordiazepoxide is 80 ± 5.0% (S.D.) and the sensitivity limit of detection is 50 to 100 ng/ml of plasma, using a 1 ml specimen. The assay was used in the determination of plasma levels of chlordiazepoxide and its metabolites in man following oral administration of chlordiazepoxide. HCl.

The chromatographic behavior of other clinically important benzodiazepines and their major metabolites is also reported.     

Reverse Phase HPLC Determination of Azq in Biological Fluids

Abstract

A sensitive and specific reverse phase HPLC method employing a simple sample preparation procedure and utilizing an internal standard was developed to measure the new antitumor agent AZQ in biological fluids. A single chloroform extraction gave drug recoveries of greater than 88% from plasma, urine and CSF in the range of expected physiological concentrations (20–800 ng/ml). Isocratic reverse phase HPLC with UV detection at 340 nm resulted in a limit of quantisation of 5 ng/ml although smaller amounts of the drug could be detected. This assay was successfully applied to determine the single dose plasma pharmacokinetics of AZQ in rats. The potential of this method for determining AZQ disposition and pharmacokinetics in human subjects was demonstrated by analysis of patient CSF.     

A Stability-Indicating Liquid Chromatographic Method for the Analysis of Erythromycin in Stored Biological Fluids Using Amperometric Detection

Abstract

A simple, sensitive and reliable high-performance liquid chromatographic procedure has been developed for the determination of erythromycin in human serum and urine using amperometric detection. A solid-phase extraction procedure was used followed by chromatography on a reverse-phase column. The mean recovery of erythromycin from serum and urine was 80%. Calibration plots for erythromycin base in serum and urine were linear over the ranges 0.25–5.0 μg/ml and 1.25–25.0 μg/ml respectively, with a sensitivity limit of 0.1 μg/ml.

This method allows both erythromycin and its principle degradation product, anhydroerythromycin, to be determined during a period of sample storage at 4°C and ?15°C. The method is sufficiently sensitive and precise and is thus highly suited for use in both pharmacokinetic and stability studies.     

Sensitive and precise HPLC method with back‐extraction clean‐up step for the determination of sildenafil in rat plasma and its application to a pharmacokinetic study

A sensitive HPLC method was developed and validated for the determination of sildenafil concentrations in rat plasma (200 μL) using a liquid–liquid extraction procedure and paroxetine as an internal standard. In order to eliminate interferences and improve the peak shape, a back‐extraction into an acidic solution was utilized. Chromatographic separation was achieved on a cyanopropyl bonded‐phase column with a mobile phase composed of 50 m m potassium dihydrogen phosphate buffer (pH 4.5) and acetonitrile (75:25, v/v), pumped at the flow rate of 1 mL/min. A UV detector was set at 230 nm. A calibration curve was constructed within a concentration range from 10 to 1500 ng/mL. The limit of detection was 5 ng/mL. The inter‐ and intra‐day precisions of the assay were in the ranges 2.91–7.33 and 2.61–6.18%, respectively, and the accuracies for inter‐ and intra‐day runs were within 0.14–3.92 and 0.44–2.96%, respectively. The recovery of sildenafil was 85.22 ± 4.54%. Tests confirmed the stability of sildenafil in plasma during three freeze–thaw cycles and during long‐term storage at ?20 and ?80°C for up to 2 months. The proposed method was successfully applied to a pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Liquid Chromatographic Method for Determination of Free and Niosome‐Entrapped Nimodipine in Mouse Plasma and Different Tissues

Abstract

A rapid HPLC method for the quantification of nimodipine in mouse plasma and tissues has been developed in this study, with simple procedure of sample preparation by one‐step protein precipitation. The results of HPLC analysis indicated that linear calibration curves were obtained over the concentration range 0.10–10.00 µg/ml for plasma, 0.10–20.00 µg/g for heart, liver, spleen, kidneys, and brain, and 1.00–200.00 µg/g for lung, respectively. The desirable precision and accuracy were achieved, both intraday and interday for plasma and tissue homogenates. Thus, this newly developed procedure was successfully applicable for determination of nimodipine in mouse plasma and tissues following intravenous administration of free and novel niosome‐entrapped nimodipine.     

A High-Performance Liquid Chromatographic Method for the Determination of Doxefazepam in Human Plasma Using a Solid-Phase Extraction Column

Abstract

A procedure for the rapid, quantitative isolation of doxefazepam from plasma with Supelclean LC-18 cartridges is described together with a sensitive HPLC assay for the quantitative determination of the drug. The recovery of doxefazepam was greater than 80 % over an investigated range of 0.1–2.0 μg/ml of plasma. The column extraction of doxefazepam coupled with the versatility of HPLC make this procedure well suited for detailed pharmacokinetic studies and as well as routine plasma analysis of doxefazepam.     

A Sensitive High Performance Liquid Chromatographic Determination of Clopamide in Human Plasma

Abstract

A simple and sensitive high-performance liquid chromatographic method for quantitation of clopamide in human plasma has been developed. the assay uses a reversed-phase C18 microbore column (2 mm I.D. × 100 mm) packed with 5 μm ODS Hypersil. the chromatographic separation was achieved by using an isocratic mobile phase comprising acetonitrile-10 mM phosphate buffer pH 4 (17:83, v/v) at a flow rate of 0.5 ml/min. the eluant was monitored by a UV detector operating at 241 nm. the assay was based on an organic extraction before chromatographic separation. to 1 ml plasma sample, 100 μl of the internal standard, methylparaben (300 ng/ml), and 8 ml of diethyl ether were added. the samples were shaken and centrifuged, the organic layer was then transferred to a tapered centrifuge tube and evaporated to dryness. the residue was reconstituted and injected onto the HPLC column. the inter-and intra-assay coefficients of variation were found to be less than 10%. the lowest limit of detection for clopamide in plasma was 5 ng/ml. the method is sensitive, specific and allows for routine analysis in the pharmacokinetic studies.