HPLC Measurement of Cyclosporine in Blood Plasma and Urine and Simultaneous Measurement of its Four Metabolites in Blood

Abstract

A high performance liquid chromatographic assay has been developed for the estimation of cyclosporine and its four major metabolites in blood and for cyclosporine alone in plasma and urine samples. This assay employs a rapid and very reproducible solid-liquid extraction system. Isocratic chromatographic conditions allow the simultaneous measurement of cyclosporine and its four major metabolites in blood. The method is linear up to 2500 ng/ml and the minimum quantifiable limit for cyclosporine is 30 ng/ml, when 1 ml of sample is analyzed.     

Straight-Phase Ion-Pair Chromatography of Zimelidine and Similar Divalent Amines Part I Bioanalysis

Abstract

Methods are presented for the quantitative determination of ZIMELIDINE, a new antidepressant drug, and its active metabolite norZIMELIDINE in biological material (whole blood, plasma, urine and rat brain). The extraction is optimized regarding recoveries and blank chromatograms and the compounds are separated by high performance ion-pair liquid chromagraphy with perchlorate as counter ion in the stationary phase. Internal standards are chlorpheniramine and the geometrical isomer to norzimelidine. The precision for determinations in plasma ranges 2 - 7% (CV) for the concentrations 100 - 5 ng/ml, and the detection limits are 150 pg/ml but can be lowered about five times by using larger sample volumes. The selectivity against metabolites is investigated and the use of the method in routine is discussed. The isolation and identification of the primary amine metabolite by collecting the peak for subsequent GC-MS-analysis is demonstrated.     

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.     

Microanalysis of dialkylphosphorus metabolites of organophosphorus pesticides in human blood by capillary gas chromatography and by phosphorus-selective and ion trap detection

A procedure for the determination of dialkyphosphorus metabolites of organophosphorus pesticides in human blood has been worked out. Dimethyl and diethyl phosphates, phosphorothioates and phosphorodithioates, extracted with diethyl ether from plasma acidified with hydrochloric acid, were methylated with diazomethane and analysed by capillary gas chromatography with an alkali flame ionization detector and an ion trap detector. The extraction of metabolites was preceded by n-hexane extraction of parent organophosphorus pesticides without a negative effect on the efficiency of metabolite extraction. If plasma samples, containing 2 μ/ml of each metabolite, were not saturated with sodium chloride before extraction, only dialkyl phosphorothioates were recovered by more than 80%. The recoveries of other metabolites were less than 25%. The extraction of plasma samples saturated with sodium chloride resulted in higher recoveries of all metabolites. At concentrations ranging from 0.2 to 2.8 μg/ml the accumulation effeciencies (%±S.D.) of dimethyl and diethyl phosphorothioates were 92±20 and 97±11, and those of corresponding phosphorodithiotes 79±7 and 71±4. A significantly lower recovery (36±12%) was determined for dimethyl phosphate at concentrations in plasma below 2 μg/ml. The recovery of diethyl phosphate was dependent on the initial metabolite concentration in plasma being 31±5% at concentrations lower than 0.5 μg/ml, 51±12% at concentrations ranging from 0.7 to 1.7 μg/ml and 97±3% at concentrations at or above 2 μg/ml. Detection limits of metabolites in plasma using the phosphorus selective detector were 150 ng/ml for dimethyl phosphate and 50 ng/ml for other metabolites. Those values were for dialkyl phosphates and phophorothioates three to five times lower and for diakyl phosphorodithiotes even 30 times lower than detection limits achieved by the use of ion trap detector. The procedure was applied for the evidence and confirmation of human poisoning with organophosphorus pesticides.     

Determination of Doxorubicin,Daunorubicin and some of their Metabolites in Mouse Plasma by High-Performance Reversed-Phase Liquid Chromatography with Amperometric Detection

Abstract

A selective and sensitive reversed-phase liquid chromatographic method with electrochemical detection for the analysis of doxorubicin, daunorubicin and some of their metabolites in plasma is reported. A mobile phase consisting of acetonitrile-phosphate buffer solution-tetrahydrofuran (25–71,5–3,5) flowing at 1 ml/min through a Lichrocart RP 18 column was employed. The influence of various parameters on the separation (solvent composition, pH, tetrahydrofuran content) has been examined. An extraction of anthracyclines from plasma was performed using chloroform-ethanol mixture (4: 1) with high extraction efficiency; reproducible results were attained by working with a 1 M phosphate buffer which ensured a real buffering of the plasma samples. The sensitivity of amperometric detection makes this method suitable for analyzing small amounts of the parent drugs and their metabolites. The precision was better than 4% in the range 0.2 to 5 μg/ml plasma.     

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.     

Determination of Adriamycin and Aclacinomycin a in Plasma by High Pressure Liquid Chromatography and Spectrophot of Luorometry

Abstract

Procedures for the determination of adriamycin and aclacinomycin A in plasma using high pressure liquid chromatography for resolution and fluorescence for detection are described. Separations of these anthracyclines from some of their metabolites—adriamycinol and 4?-hydroxy aclacinomycin A isomers—are also illustrated.     

Specific HPLC Method for the Separation of Verapamil and Four Major Metabolites After Oral Dosing

Abstract

We have developed a high performance liquid chromatographic (HPLC) method which resolves verapamil, norverapamil, D620, D617 and what we believe to be another verapamil metabolite which has been previously unreported. An alkyl-phenyl column is used with a mobile phase of 0.005% sulfuric acid in methanol. The extraction recoveries of verapamil, norverapamil and the internal standard (imipramine) from plasma ranged between 98% and 104%. The day-to-day, and within-day coefficients of variations for verapamil and norverapamil at plasma concentrations of 7.3 and 233 ng/ml ranged between 1.7 and 6.1%. The limit of sensitivity was slightly less than 1 ng for both verapamil and norverapamil. Chromatograms of extracts of serum and urine obtained from five normal subjects who took single oral verapamil doses, indicated the presence of verapamil, norverapamil, and two other known metabolites. Chromatograms of serum extracts also indicated an additional peak which is probably another verapamil metabolite.     

Rapid Determination of Propranolol and 4-Hydroxypropranolol in Plasma by High Pressure Liquid Chromatography

Abstract

The resolving power of high pressure liquid chromatography has been combined with the sensitivity of fluorescence detection to develop a method for the determination of propranolol and 4-hydroxypropranolol in plasma. A single selective extraction step precedes chromatography on a high efficiency bonded phase column. The limits of quantitation are approximately 2 ng/ml of plasma for each drug. The analysis of a number of clinical samples has demonstrated the application of the method in pharmacokinetic studies, but the possible interference of other drugs and their metabolites is still under investigation.     

The Determination By Means of Capillary Gas Chromatography of the Benzodiazepines-Clobazam,Clonazepam, Diazepam,and Nitrazepam-and Their Principal Metabolites Currently Used In the Treatment of Epilepsy

Abstract

The authors developed a gas chromatographic method allowing the quantification of those benzodiazepines in current use in the treatment of epilepsy, name ly clobazam, clonazepam, diazepam and nitrazepam. This method involved a butyl acetate extraction from plasma, and an analysis on a CP-Sil 5 WSCOT capillary column with electron-capture detection. Intra-day precision and accuracy were better than 10% for each of the compounds and their main metabolites, N-desmethyldiazepam, oxazepam, N-desraethylclobazam. the quantification limit was about 0.5 to 1 ng/ml for each compound. Linearity proved satisfactory between 1 and 4000 ng/ml. Adequate han previously. Besides, its sensitivity is sufficient for kinetic studies following a single dose administration.     

High Performance Liquid Chromatoraphic Determination of R-831, A New Antiallergic Agent,in Dogs and Humans

Abstract

A simple, selective and sensitive HPLC method has been developed to measure R-831 levels in dogs and humans. It is an internal standard technique with a single step extraction and one wash. Samples are chromatographed on a reversed-phase system with ultraviolet detection. The lowest detectable concentration for plasma is 25 ng R-831/ml with a 1 ml sample and the linear range is 25–8000 ng R-831/ml. The lowest detectable concentration for urine is 250 ng R-831/ml with a 0.1 ml sample and the linear range is 250–8000 ng R-831/ml. This method has been used to quantitate levels of R-831 in bioavailability and toxicity studies in dogs, and in pharmacokinetic and efficacy studies in humans.     

Gas Chromatographic Analysis of Cyclophosphamide in Plasma and Tissues Using Nitrogen-Phosphorus Detection

Abstract

A gas chromatographic method for the analysis of cyclophosphamide in plasma, blood, and organ tissues is described. This method involves extraction of aliquots of plasma or tissue homo-genate in alkaline condition with ether. The extracted drug is derivatized with heptafluorobutyric anhydride followed by gas chromatographic separation via a glass column of 183 cm × 2 mm i. d. packed with 3% SE-30 on chromosorb W-HP. The derivatized cyclophosphamide and isophosphamide, an added internal standard, are detected by a nitrogen-phosphorus detector. The sensitivity limit of this method is 10 ng per gm or ml of sample and gives linearity over 100-fold of concentration range.     

Size Exclusion Chromatographic Determination of PEG 3350 in Human Plasma and Urine

Abstract

A size exclusion chromatographic method is reported that quantitated PEG 3350 in human plasma and urine to a concentration of 10 μg/ml in plasma, or urine. The chromatographic system consisted of a 500 A gel permeation analytical column, a differential refractometer detector, and chloroform as the eluting solvent. Organic extraction was used as an initial separation technique. PEG 400 was used as the internal standard for PEG 3350, and showed similar extraction properties. Spiked plasma standards yielded standard calibration curves with correlation coefficients of greater than 0.99, and relative standard deviations (n=3) of 2.19% (500 μg/ml) and 6.97% (20 μg/ml). The analytical technique was used to estimate the elimination rate constant of PEG 3350 from 5 normal human subjects after oral administration of 240 grams of PEG 3350. K_E was found to be 0.1079 hr^?1 0.03781^?1 hr (mean s.d.) using the Sigma-minus data analysis method on total urines collected from the 5 subjects at varying intervals.     

Comparison of Solid-Phase Extraction and Liquid-Liquid Extraction Methods for Liquid Chromatographic Determination of Diltiazem and its Metabolites in Plasma

Abstract

A rapid solid-phase extraction method for the determination of diltiazem and its metabolites from plasma was compared to a conventional liquid-liquid extraction procedure we have described previously. Analytical recovery for all compounds was greater than 90 % for solid-phase extraction whereas for liquid-liquid extraction, mean recovery ranged from 67 to 82 %. The increase of extraction efficiency was closely related to an improvement of the detection limit for the metabolites. Solid phase extraction procedure was found to be more convenient, rapid and sensitive than liquid-liquid extraction and represents a useful analytical tool for the monitoring of diltiazem and its metabolites in clinical investigations.     

High Performance Liquid Chromatographic Analysis of Tolmetin,Indomethacin and Sulindac in Plasma

Abstract

Isocratic and gradient reversed phase high-performance liquid chromatographic (HPLC) methods for the quantitation of tolmetin, indomethacin, and sulindac and their respective metabolites in plasma were developed. Only the determination of the parent drugs was possible using the isocratic technique. Specific, simultaneous determination of each drug and its respective metabolites was achieved using the gradient technique. The effect of pH and ionic concentration of the mobile phase on retention time was studied. Statistical analysis demonstrated excellent precision and linearity over the following ranges: 1–40, 0.1–3, and 0.1–3 ug/ml plasma for tolmetin, indomethacin, and sulindac respectively. Both methods have been applied to the analysis of patient samples.     

Determination of Trace Amounts of Chlorogenic Acid and Three of Its Metabolites Using Time-Resolved LPME and LC-UV Detection in Biological Specimens

Chlorogenic acid (CGA) is an effective antitumor, anti-inflammatory and antimicrobial agent. Since the absorption and metabolism of CGA remains controversial, time-resolved binary-solvent synergy liquid-phase microextraction (TRBSS-LPME) using hollow fiber was developed for the extraction of CGA and its metabolites: caffeic acid, p-hydroxycinnamic acid and ferulic acid, from biological specimens. In this technique, the target drugs were extracted into a binary-solvent immobilized in the wall pores of hollow fiber. The extraction occurred due to a pH gradient between the two sides of the fiber. After extraction, an aliquot was analyzed by LC. Under the optimal conditions, the CGA, caffeic acid, p-hydroxycinnamic acid and ferulic acid had good correlation of determination values (R > 0.97) and the detection limits (LODs) were 1.0, 1.0, 2.0, and 5.0 ng mL^?1 in plasma; and 1.0, 50, 10, and 50 ng mL^?1 in urine. The mean recoveries in plasma were 90.8–119.8% for CGA and its metabolites: caffeic acid, p-hydroxycinnamic acid and ferulic acid evaluated and the mean recoveries of caffeic acid and p-hydroxycinnamic acid in urine were 81.6–111.6%. Finally, TRBSS-LPME was successfully used for the determination of target drugs in biological specimens. It not only extended the linear range of CGA determination in biological samples and improved the sensitivity, but also eliminated interferences from complex constituents in the biological specimens and reduced the LOD.     

Quantitation of Cyproheptadine in Plasma and Urine by HPLC

Abstract

A sensitive, reliable and specific high performance liquid chromatographic procedure has been developed for the quantitation of cyproheptadine in plasma or urine. After extraction of the drug with ethyl acetate from alkalinized samples, the organic extract was evaporated to dryness, reconstituted with acetonitrile and chromatographed using a C₈ reversed-phase analytical column with UV detection at 254 nm. The average recoveries of cyproheptadine from spiked plasma and urine samples in the concentrations ranging from 0.2 – 3 mcg/ml were 95.7 and 100.3%, respectively and their respective CV was 4.1 and 3.9%. Regression analyses for the calibration plots for plasma and urine standards obtained on three different days for the drug concentrations between 0.2 – 3 mcg/ml indicated excellent linearity (r > 0.999) and reproducibility (CV < 2.0%, p > 0.01). The limit of sensitivity was 50 ng/ml for both plasma and urine samples. The method was applied to monitor the plasma concentration versus time profile of cyproheptadine following a single bolus IV dose of 1 mg/kg in a dog.

Urine samples taken from a human subject for the duration of 24 hours following a single oral dose of 8 mg showed that the cumulative amount excreted in urine as cyproheptadine was approximately 1% of the dose.     

Quantitation of Benzodiazepine Hydrolysis Products in Urine Using Solid-Phase Extraction and High Performance Liquid Chromatography

Abstract

Solid-phase extraction and high performance liquid chromatography (HPLC) were used to indirectly (via products of acid hydrolysis) confirm the presence of benzodiazepine metabolites in the urine of patients who had received overdoses of these compounds. The use of solid-phase extraction method for quantitation of benzodiazepine hydrolysis products in urine offers numerous advantages in comparison to extraction with chloroform. The chromatograms of urine extracts were free of interferences. The recoveries of the benzodiazepine hydrolysis products and the internal standard were greater than 96%, which makes this method highly reliable for quantitative analytical purposes.     

An HPLC Method for the Determination of Theophylline and Its Metabolites in Serum and Urine

Abstract

A urine and a serum assay have been developed to quantitate theophylline and its major metabolites:1,3-dimethyluric acid, 3-methylxanthine and 1-methyluric acid. Reverse phase chromatography follows a serum acetone extraction procedure and a urine anion exchange clean-up procedure. Lower limits of sensitivity are 0.04 μg/ml for serum metabolites and 1 μg/ml for urine metabolites. Both assays are free of interference from endogenous substances. These assays have been tested successfully in pharmacokinetic and metabolic studies of theophylline.     

Ion-pair extraction and high-performance liquid chromatographic determination of tianeptine and its metabolites in human plasma, urine and tissues

An isocratic reversed-phase ion-pair liquid chromatographic method for the determination of tianeptine and its two main metabolites in plasma, urine and tissues, using an internal standard, is reported. The influence of two stationary phases on the retention of the drugs was studied. The drugs were extracted as ion pairs, using a heptane-octanol-tetraheptylammonium bromide mixture (98:2:0.5, v/v/w) as extraction solvent. This extraction procedure yielded plasma drug recoveries of greater than 60% and allowed UV detection at 220 nm without interference from endogenous components of plasma, urine or tissues. Linear standard curves up to 1.00 micrograms/ml and drug determination down to 0.01 microgram/ml were observed. This method has been successfully applied to the analysis of human plasma and urine samples and of encephales from tianeptine-dosed rats.