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The pivaloyloxyethyl ester of methyldopa is an antihypertensive prodrug possessing improved bioavailability properties over methyldopa. A sensitive cation-exchange, high-performance liquid chromatographic assay using electrochemical detection has been developed for the ester in plasma and urine in order to determine the extent of its hydrolysis after oral administration. The chromatographic conditions involve two Altex Partisil 10 SCX columns (25 cm X 4.6 mm) in series; a mobile phase consisting of methanol, potassium phosphate buffer, pH 3.0, and EDTA disodium dihydrate; and an electrochemical detector set at 0.5 V. The pivaloyloxyethyl ester in plasma or urine is extracted into ethyl acetate, back-extracted into 0.1 M sulfuric acid, and analyzed directly by high-performance liquid chromatography. For urine, the ethyl acetate extract is washed with a buffer (pH 8.0) prior to the back-extraction step. The assay gives a linear response over the concentration range of 10-160 ng/ml in plasma and 20-400 ng/ml in urine.

A simple procedure for the simultaneous determination of major metabolites of dopamine and serotonin in plasma, i.e. 3,4-dihydroxyphenylacetic acid, 5-hydroxyindole-3-acetic acid, and 4-hydroxy-3-methoxyphenylacetic acid, was developed. The method is based on rapid isolation of the compounds by one-step clean-up on a small C18 column, followed by high-performance liquid chromatography with dual electrochemical detection. The system is readily used for clinical applications.

Mobile-phase variations were employed to achieve optimal separation by narrow-bore reversed-phase high-performance liquid chormatography of eleven eicosanoids. Separation and quantitation by ultraviolet absorbance at 190 nm using conventional-bore ODS columns were compared. Using the improved sensitivity obtained by means of narrow-bore column, i.e. 250-pg detection limits of a standard solution, analysis of eicosanoids in kidney medulla was achieved. Parallel quantitation by radioactivity, using [1- 14 C]arachidonic acid as substrate, was applied.

A sensitive and selective reversed-phase high-performance liquid chromatographic assay has been developed to determine the concentration of pyrimethamine, sulfadoxine and N4-acetylsulfadoxine in serum and urine after oral administration of the antimalarial remedy Suldox®. Hitherto the literature describes no method being able to quantitate all three compounds in these fluids. The compounds are extracted successively from the same sample and subjected to liquid chromatography followed by ultraviolet detection (280 nm). Calibration curves were linear ( r 2 = 0.999 ; S.E.M. less than 3%; n = 10) in the range 0–300 μg/ml (sulfadoxine) and 0–1000 ng/ml (N4-acetylsulfadoxine and pyrimethamine). The limits of quantitation for the latter compounds were as low as about 5 ng/ml and 1 ng/ml, respectively. At therapeutic serum concentrations of 30 μg/ml (sulfadoxine), 350 ng/ml (N4-acetylsulfadoxine) and 120 ng/ml (pyrimethamine) an interassay reproducibility below 8% (relative standard deviation) was found for all three compounds. The assay was evaluated in a pilot study and proved convenient for pharmacokinetic studies in man following oral co-administration of pyrimethamine and sulfadoxine.

A highly sensitive method for the assay of catechol-O-methyltransferase in erythrocytes is described, which employs high-performance liquid chromatography with fluorescence detection. A newly synthesized catechol compound, 2-(3,4-dihydroxyphenyl)naphtho [1,2-d]thiazole is used as a highly fluorogenic substrate for catechol-O-methyltransferase; the m- and p-methylated products formed enzymatically from the substrate under the optimum conditions, after extraction with n-hexane--chloroform, are separated by normal-phase chromatography on LiChrosorb Si 100. The limits of detection for m- and p-methylated products are 3 pmol per assay tube (60 fmol per injection volume of 20 microliter) in each case. The ratio of m- and p-methylated products was 0.54. This method requires as little as 50 microliter of human erythrocytes.

This paper describes a reversed-phase, high-performance liquid chromatographic (HPLC) method for the isolation, detection, and quantification of TNP-470 (I) and one of its active metabolites, AGM-1883 (II), from plasma. These compounds are initially extracted from plasma with an organic solvent and then separated from one another on a C18 column. Those fractions eluting from the C18 column and containing either I or II are then derivatized through their epoxide moieties with sodium 8-quinolinethiolate (SQT). This derivatization produces fluorescent species that are isolated and quantified by a second reversed-phase HPLC analysis. The assay yields a lower limit of reliable quantification of 2.5 ng/ml and is linear to a concentration at least as high as 160 ng/ml. The inter-assay percent coefficient of variation is less than 18%.

A method for the analysis of epoxy polyunsaturated fatty acids (EpPUFAs) and epoxyhydroxy polyunsaturated fatty acids (EpHPUFAs) in rat tissue homogenate, with homo-γ-linolenic acid (20:3, n − 6), arachidonic acid (20:4, n − 6), eicosapentaenoic acid (20:5, n − 3) or docosahexaenoic acid (22:6, n − 3) as a substrate, has been developed. Extraction with dichloromethane at pH 4–5 and concentration in the presence of pyridine were performed. Spectral analysis of chromatograms obtained with high-performance liquid chromatography—thermospray mass spectrometry showed the presence of EpPUFAs, EpHPUFAs and dihydroxy metabolites (DiHPUFAs) of EpPUFAs corresponding to each precursor fatty acid. On a selected-ion monitoring chromatogram, many EpPUFAs, EpHPUFAs and DiHPUFAs in an extract from an incubation mixture of each precursor fatty acid in aged rat tissue homogenate were detected simultaneously within 70 min. EpPUFAs and DiHPUFAs derived from 20:3 ( n − 6) or 20:5 ( n − 3) were detected in significant amounts. From these results, a highly active cytochrome P450 system or non-enzymic oxidative reactions in aged rat tissue homogenate were suggested.

A sensitive, quantitative reversed-phase high-performance liquid chromatographic method has been established for the simultaneous determination of butorphanol, a synthetic opioid, and its metabolites, hydroxybutorphanol and norbutorphanol, in human urine samples. The method involved extraction of butorphanol, hydroxybutorphanol, and norbutorphanol from urine (1.0 ml), buffered with 0.1 ml of 1.0 M ammonium acetate (pH 6.0), onto 1-ml Cyano Bond Elut columns. The eluent was evaporated under nitrogen and low heat, and reconstituted with the HPLC mobile phase, acetonitrile—methanol—water (20:10:70, v/v/v), containing 10 m M ammonium acetate and 10 m M TMAH (pH 5.0). The samples were chromatographed on a reversed-phase octyl 5-μm column. The analysis was accomplished by detection of the fluorescence of the three analytes, at excitation and emission wavelengths of 200 nm and 325 nm, respectively. The retention times for hydroxybutorphanol, norbutorphanol, the internal standard, and butorphanol were 5.5, 9.0, 13.0, and 23.4 min respectively. The validated quantitation range of the method was 1–100 ng/ml for butorphanol and hydroxybutorphanol, and 2–200 ng/ml for norbutorphanol in urine. The observed recoveries for butorphanol, hydroxybutorphanol, and norbutorphanol were 93%, 72%, and 50%, respectively. Standard curve correlation coefficients of 0.995 or greater were obtained during validation experiments and analysis of study samples. The method was applied on study samples from a clinical study of butorphanol, providing a pharmacokinetic profiling of butorphanol.

[35S]Methionine-labelled envelope polypeptides of herpes simplex virus type 1, strain F, propagated in mammalian cell culture of various origins, were separated by ion-exchange high-performance liquid chromatography on a TSK DEAE-3SW column. Analysis of the fractions by radioimmunoprecipitation followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis of the immunoprecipitates showed similarities as well as distinct differences in the number, migration patterns and molecular mass of the synthesized polypeptides, depending on the host cell. The results show that this method can be used to demonstrate species-specific or organ-specific differences in the processing of virus-specified polypeptides synthesized in host cells.

A reversed-phase high-performance liquid chromatographic method is described to distinguish chlorambucil N-oxide from the parent chlorambucil and quantitate both after separation from biological samples. The influence of solvent pH, alcohol, acid and ion-pairing agent on the separation is described. The stability of chlorambucil and its N-oxide in buffers and alcohols, as well as stability during filtration is discussed with potential application for metabolic studies.

A rapid and sensitive method for the determination of five different alkylphosphocholines including the antineoplastic phospholipid analogues hexadecylphosphocholine and octadecylphosphocholine is presented. The method is based on the separation of the lipids by high-performance liquid chromatography and quantitation by light-scattering mass detection. The lower limit of detection is approximately 50 pmol for each alkyphosphocholine tested. Quantitation is linear over the range 0.05-75 nmol. Hexane-isopropanol extracts of cultured cells can be applied to the column without further cleanup. The high resolution of separation and the sensitivity of detection render this method useful for pharmacokinetic investigations dealing with the uptake of alkylphosphocholines into different types of cells.

The use of a new configuration to control the automated sequential trace enrichment of dialysate (ASTED) system has been used to estimate 1-(β- d -arabinofuranosyl)-5-(1-propynyl)uracil and its metabolite 5-propynyluracil in urine. The system employs “heart-cutting” as a means to improve the efficiency of sample preparation and reduce analysis time. Using this technique the mean within- and between-run imprecision (coefficient of variation) at three different urine analyte concentrations was found to be 1.6 and 3.6% and 1.7 and 3.3% for 5-propynyluracil and 1-(β- d -arabinofuranosyl)-5-(1-propynyl)uracil, respectively.

A high-performance liquid chromatographic method was developed for determining the concentrations of carbenicillin (CBPC) epimers in plasma and urine. Samples were prepared for HPLC analysis by solid-phase extraction and the concentrations of CBPC epimers were determined using reversed-phase HPLC with a mixture of 0.05 M ammonium acetate and methanol as a mobile phase. Baseline separation of the two epimers was observed for both plasma and urine samples with a detection limit of ca. 10 micrograms/ml. No peaks interfering with either of the CBPC epimers were observed on the HPLC chromatograms for blank plasma and urine. The presented method was used to determine the protein binding of CBPC epimers in vitro in human and rabbit plasma. The stereoselectivity of the binding of CBPC appeared to be reversed in human and rabbit plasma.

Caffeine is a popular compound for phenotyping individuals for CYP4501A2, xanthine oxidase (XO) and N-acetyltransferase (NAT) utilising urinary metabolites. The analysis is complex since at least thirteen metabolites are excreted by man. Past methods have been less than satisfactory in that either not all the metabolites have been resolved and/or extractions selective for particular groups of metabolites are required prior to chromatography. We report a method for the rapid analysis of caffeine and metabolites in urine that negates the requirement for an extraction step, and also a method for plasma analysis.

A simple and specific reversed-phase high-performance liquid chromatographic (HPLC) assay for the determination of novobiocin levels in human plasma has been developed. The sample preparation was performed by deproteinization with methanol. Prednisone was used as an internal standard. Both novobiocin and prednisone were separated on a C8 column with a gradient elution of acidic water (pH 3.0)-methanol. The recovery of novobiocin from plasma was nearly complete. The linear range was 5-1000 microM in 0.5 ml of plasma with a minimum limit of determination of 2.25 fmol of novobiocin at 254 nm. The method has been implemented and validated in an ongoing clinical trial.

A high-performance liquid chromatographic method for the sensitive determination of 1-hydroxy-2-(imidazo[1,2-a]pyridin-3-yl)ethane-1,1-bisphosphonic acid monohydrate (YM529) in plasma, urine and bone is described. Plasma obtained in high-dose animal studies is treated by method A, a simple method using 1 ml of plasma, which is based on deproteinization of plasma followed by coprecipitation of the drug with calcium phosphate and dissolution of the precipitate in EDTA. Plasma obtained in low-dose clinical studies is treated by method B, a more sensitive method using 4 ml of plasma, which is based on direct precipitation of the drug prior to the deproteinization in method A. Urine and bone samples are prepared by solid-phase extraction using a Sep-Pak C18 cartridge coupled with method A. The drug is separated with a reversed-phase column using a mobile phase at pH 7, and detected with a fluorescence detector following postcolumn alkalization of the mobile phase to enhance fluorescence intensity. The limit of determination is 0.2 ng/ml for method A and 0.05 ng/ml for method B in plasma, 0.05 ng/ml in urine, and 5 ng/g in bone.

An automated technique involving switching valves and a filter assembly has been developed and evaluated for the on-line precipitation of proteins and peptides from plasma samples. In the set-up, the proteins were precipitated on-line by injecting the plasma sample into a stream of organic precipitating agent. The precipitates so formed are filtered on-line by a set of filter assemblies consisting of ordinary in-line HPLC solvent filters. Evaluation of the technique was performed using ibuprofen and a mixture of three estrogens, estradiol, equilin and estrone, spiked in dog plasma. The coefficients of variation (C.V.) for system suitability parameters were below 10%. Absolute recovery of ibuprofen in plasma ranged from 80% for 100 micrograms/ml to 114% for 5 micrograms/ml spiked concentrations, respectively. Resolution for equilin and estrone, two closely eluting peaks, was 1.79 (C.V. = 5.8%, n = 7). The switching-valve--filter assembly had no significant effect on the efficiency of the HPLC system.

Optimization of the main variables that affect solvent and solid-phase extraction processes, using disposable C18 cartridges and the non-ionic polymeric resin Serdolit AD-2, of human urine containing natural and synthetic corticoids is described. The data were obtained from different HPLC separations of these compounds using calibration graphs obtained before and after extraction of these compounds. The procedures, including sample preconcentration, showed efficiencies over 90%. NaCl was used to avoid emulsion formation in solvent extraction. The results achieved using solvent and solid-phase extraction are discussed.

A gas chromatographic—mass spectrometric (GC—MS) method has been developed, for the determination of trimipramine (TRI), desmethyltrimipramine (DTRI), didesmethyltrimipramine (DDTRI), 2-hydroxytrimipramine (2-OH-TRI) and 2-hydroxydesmethyltrimipramine (2-OH-DTRI). The method includes two derivatization steps with trifluoroacetic acid anhydride and N-methyl-N-( tert. -butyldimethyl silyl)trifluoroacetamide and the use of an SE-54 capillary silica column. The limits of quantitation were found to be 2 ng/ml for DTRI and 4 ng/ml for all other substances. Besides, methods have been optimized for the hydrolysis of the glucuronic acid conjugated metabolites. This specific detection method is useful, as polymedication is a usual practice in clinical situations, and its sensitivity allows its use for single-dose pharmacokinetic studies.

A sensitive and specific method for the determination of the aza alkyl lysophospholipid (AALP) 3-methoxy-2-N,N-methyloctadecylaminopropyloxyphosphorylcholine (I) in rat plasma is described. The target molecule was analyzed by high-performance liquid chromatography (HPLC)-mass spectrometry (MS) after one single liquid-liquid extraction with chloroform-methanol (2:1, v/v). 1,2-Didecanoyl-sn-glycero-3-phosphocholine was used as internal standard. HPLC was carried out using a polymeric reversed-phase column; the coupling to the mass spectrometer was a particle beam (PB) interface, and the ionization method was electron impact (EI). This simple and rugged method permits the measurement of I in rat plasma in the range of 25 ng/ml-5 micrograms/ml with good accuracy and precision and is used in pharmacokinetic studies.

An isocratic liquid chromatographic method for direct sample injection has been developed for the quantitation of felbamate and four metabolites in rat cerebrospinal fluid. The method uses 0.050- or 0.025-ml aliquots of cerebrospinal fluid diluted with equal volumes of internal standard. Chromatography is performed on a 150 mm x 4.6 mm I.D. Spherisorb ODS2, 3-microns HPLC column eluted with a phosphate buffer-acetonitrile-methanol (820:120:60, v/v/v) mobile phase and ultraviolet absorbance detection at 210 nm. The linear quantitation ranges are: felbamate and the 2-hydroxy metabolite 0.195-200 micrograms/ml, the propionic acid metabolite 0.195-50.0 micrograms/ml, the p-hydroxy metabolite 0.781 to 50.0 micrograms/ml, and the monocarbamate metabolite 0.098-50.0 micrograms/ml.

An isocratic liquid chromatographic method employing one extraction step and a 150 mm x 4.6 mm I.D. Spherisorb ODS2, 3-microns HPLC column using UV-absorbance detection at 210 nm has been developed for the quantitation of felbamate and three felbamate metabolites in 0.100-ml aliquots of rat and dog plasmas. The linear quantitation range in rat plasma is 0.195-200 micrograms/ml for felbamate; 1.563-200 micrograms/ml for the p-hydroxy metabolite; 0.391-200 micrograms/ml for the 2-hydroxy metabolite; and 0.098-200 micrograms/ml for the monocarbamate metabolite. The linear quantitation range in dog plasma is 0.195-200 micrograms/ml for felbamate; 0.781-200 micrograms/ml for the p-hydroxy metabolite; 0.195-200 micrograms/ml for the 2-hydroxy metabolite; and 0.098-200 micrograms/ml for the monocarbamate metabolite.

A sensitive high-performance liquid chromatographic assay has been developed to determine the levels of 3'-amino-3'-deoxythymidine (AMT), a cytotoxic metabolite of 3'-azido-3'-deoxy-thymidine (AZT, zidovudine), in human plasma. The sample pretreatment involved solid-phase extraction using cation-exchange extraction columns. Chromatography was carried out on a C8 column, using a mobile phase of methanol-0.01 M ammonium acetate (pH 5)-0.25 M sodium dioctylsulfosuccinate (60:40:4, v/v/v) and ultraviolet detection at 265 nm. The method has been validated, and stability tests under various conditions have been performed. The lower limit of quantitation is 5 ng/ml (using 500-microliters human plasma samples). The bioanalytical assay has been used for the determination of AMT in patients with AIDS who used AZT.

This report describes the analysis of pentamidine by isocratic reversed-phase high-performance liquid chromatography (HPLC) using a commercially available compound (melphalan) as the external standard. Previously described assays use ion-pairing HPLC, an internal standard (hexamidine) that is not readily available, and require a relatively large sample size. In the present assay, pentamidine was extracted from plasma using solid-phase extraction and was analyzed using a C18 column and a mobile phase containing 18% acetonitrile, 2% methanol, 0.2 M ammonium acetate and 0.5% triethylamine. The identity of the eluting peaks was verified using a diode array detector. The extraction yield of pentamidine was 82%. The limit of detection was 8.6 ng/ml with a sample size of 100 μl. The inter-day and intra-day coefficients of variation ranged between 0.3% and 10% with an average of 5%. This method was applied to study the pharmacokinetics of pentamidine in rodents.

1069C85 is a novel tubulin binder developed to circumvent the resistance associated with the Vinca alkaloids. Cytotoxic activity has been demonstrated in vitro against a variety of tumour cell lines, including a variant of the P388 leukaemia with acquired resistance to vincristine. A phase I clinical trial is planned and an assay suitable for preclinical and clinical pharmacokinetics has been developed. A high-performance liquid chromatographic (HPLC) assay is described which allows measurement of 1069C85 in plasma, urine, and tissue samples. The method uses reversed-phase chromatography with isocratic elution and detection by fluorescence at 406 nm following excitation at 340 nm. The assay is specific, sensitive (limit of sensitivity 0.25 ng/ml) and reproducible (coefficient of variation5%). The method has been used to study the pharmacokinetics of 1069C85 in Balb C mice following a single oral dose of 1 mg/kg. The maximum plasma concentration was reached 15 min after administration and subsequent elimination was slow with a half life of 6.5 +/- 2.2 h. The drug remained detectable in plasma, at 1 +/- 0.5 ng/ml, 24 h after this dose. This assay will be used to determine the pharmacokinetic profile of 1069C85 in mice and in a forthcoming phase I clinical trial.

A method is described for the simultaneous determination of vanilmandelic acid, 3-methoxy-4-hydroxyphenylethylene glycol, 5-hydroxyindoleacetic acid, and homovanillic acid in a human plasma sample using reversed-phase high-performance liquid chromatography with column switching and amperometric detection. Two methods of sample preparation were tested. Liquid-liquid extraction yields better recoveries, is more selective and precise than solid-phase extraction and allows a shorter time of chromatographic analysis. Estimated plasma values of the metabolites from healthy controls are in good agreement with previously reported levels. Studies of alcoholics at the beginning of the delirium tremens provided different plasma levels of the metabolites, dependent on the different duration--and hence the severity--of the delirium.

The utility of two deuterium-labelled leukotriene (LT) E4 analogs, e.g. [20,20,20-2H3]LTE4 and [14,15,17,17,18,18-2H6]LTE4, as internal standards for the determination of LTE4 in human urine by gas chromatography-mass spectrometry (GC-MS) was investigated. 2H-Exchange during hydrogenation occurred both in [20,20,20-2H3]LTE4 and [14,15,17,17,18,18-2H6]LTE4 in an extent of 9.4 +/- 0.5% and 67.3 +/- 0.6% (mean +/- S.D., n = 6), respectively. The lower extent of 2H-exchange in [20,20,20-2H3]LTE4 allowed a more accurate quantitation than the use of [14,15,17,17,18,18-2H6]LTE4. Applying [20,20,20-2H3]LTE4 as internal standard the coefficients of variation for the intra- and inter-assay determination of LTE4 in human urine were 5.7% and 6.2% (n = 4), respectively. The inter-assay coefficient of variation for [14,15,17,17,18,18-2H6]LTE4 was 15%. Using [20,20,20-2H3]LTE4 as internal standard and GC-MS, healthy volunteers were found to excrete 17 +/- 10 nmol LTE4 per mol creatinine (mean +/- S.D., n = 11). Similar excretion rates for LTE4 in urine of healthy volunteers were found using GC-tandem MS with [1,1-18O2]LTE4 as internal standard. Our results demonstrate that [20,20,20(-2)H3]LTE4 is a suitable internal standard for the GC-MS determination of urinary LTE4.

An HPLC method for the determination of lisuride hydrogen maleate in plasma is described. After addition of ergotamine tartrate as internal standard, plasma is extracted with diethyl ether. Following evaporation of the solvent and redissolving in methanol the extract is injected on a silica HPLC column and lisuride is monitored by fluorescence detection using an excitation wavelength of 322 nm and an emission wavelength of 405 nm. The method is sufficiently accurate and precise with a detection limit of 20 pg/ml lisuride in plasma. The usefulness of the method is demonstrated by measurements of lisuride levels after oral intake of a 0.6 mg dose of the drug by a healthy male volunteer, showing a peak level of 1266 pg/ml, 45 min after intake.

A sensitive and selective high-performance liquid chromatographic (HPLC) method was developed for the determination of 1,3-di(4-imidazolino-2-methoxyphenoxy)propane (DMP) in rat, dog and human plasma (50-5000 ng/ml) and urine (0.1-10 micrograms/ml). DMP and DMPent (dimethoxyimidizolinopentamidine, the internal standard), are extracted from alkanized plasma with n-butyl chloride-n-butanol (9:1, v/v). The organic phase is dried under nitrogen, reconstituted in mobile phase, and washed with hexane. Separation is achieved by ion-pair chromatography on a Zorbax Rx C8 column with fluorescence detection. The analysis of pooled plasma (80, 400, and 4000 ng/ml) and urine controls (0.3, 1.6, and 8 micrograms/ml) demonstrated excellent precision and accuracy over a three-day period. The recovery of DMP is90% from rat, dog, and human plasma and85% from rat and human urine, and 60-70% from dog urine. The limit of quantitation (LOQ) of the assay is 50 ng/ml in rat, dog and human plasma. Using the high-sensitivity assay, the limit of quantitation was decreased to 5, 2 and 0.6 ng/ml in rat, dog and human plasma, respectively. The LOQ of the assay is 0.1 microgram/ml in rat, dog and human urine. The assay was used to determine plasma and urine concentrations of DMP in pharmacokinetic studies in rat and dog.

An accurate, sensitive, reproducible and selective HPLC assay is presented for the quantitative determination of m-iodobenzylguanidine (MIBG) in human urine. The sample pretreatment involves a solid-phase extraction on Bakerbond SPE cyano columns. The HPLC systems consists of a muBondapak C18 column and 25 mM ammonium phosphate (pH 3.0)-acetonitrile (75:25, v/v) as the mobile phase. Detection is performed by UV absorbance at 254 nm. Log y vs. log x is the response function that yielded the smallest sum of percent relative concentration residuals over the whole concentration range of the assay (0.2-100 micrograms/ml). The excretion profile of MIBG in urine of a three-year old patient is shown.

A high-performance liquid chromatographic method for simultaneous determination of three antiepileptics, phenytoin, phenobarbital, and carbamazepine, in serum for therapeutic drug monitoring is described. The drugs were extracted and injected onto a silica-gel column using a syringe-type minicolumn, Extrashot-Silica, packed with diatomaceous earth granules. We used dichloromethane for extraction-injection and n-hexane containing 0.2% acetic acid, 2% ethanol, and 15% dichloromethane for the mobile phase of a silica-gel HPLC. The eluent was monitored with a UV detector set at 240 nm. Linear relationships between the amount of drug and peak height were confirmed at 1-20 micrograms/ml in serum for carbamazepine and 5-40 micrograms/ml in serum for phenytoin and phenobarbital. When a 5-microliters aliquot of serum was subjected to this method, the observed detection limits of the drugs were far less than therapeutic concentrations. Thus, our method was simple and accurate enough to be used in routine therapeutic drug monitoring and basic pharmacokinetic studies.

This paper describes the application of a combined high-performance liquid chromatography and radioimmunological assay method for the measurement of prostaglandins E1(PGE1) and E2(PGE2). Samples were acidified to pH 3.15, extracted twice with ethyl acetate and further processed through C18 solid-phase extraction cartridges. After HPLC purification, PGE1 and PGE2 were measured by radioimmunological techniques. The limit of detection for PGE1 was 3.9 pg/ml and the intra-assay relative standard deviation was 7.8% for n = 5. The accuracy of the assay procedure was also verified. The method has been applied to the determination of PGE1 and PGE2 in embryo incubates from 10-day pregnant rats.

A coupled TLC-HPLC procedure is proposed for the separation and determination of plasma triglycerides. The method was tested by application to plasma samples corresponding to a normal population of Barcelona (Spain). Eighteen different triglyceride types were identified and their relative proportions were established, in order to give a "normal profile" for men and women. Sex-related differences (p0.05) were only found for dioleostearin and palmitodilinolein + linoleooleopalmitolein (LLP+LOPa). A correlation study showed that palmitodiolein and total cholesterol levels increase with age, whereas LLP-LOPa decreases in men and palmitolinoleoolein + palmitooleopalmitolein in women.

The enantiomers of the racemic analgesic drug etodolac have been resolved by fractional crystallization of the diastereomeric salts with optically active 1-phenylethylamine. A high-performance liquid chromatographic method to determine racemic etodolac (assay I) and its major metabolites (assay II) in urine using a conventional reversed-phase column is described. The determination of the enantiomeric ratios of etodolac and the two metabolites 7-hydroxyetodolac and 8-(1′-hydroxyethyl)etodolac was achieved using different protein-bonded chiral stationary phases. The urinary data for five volunteers are presented and show a marked stereoselectivity of the metabolism of etodolac in humans.

A method for the simultaneous determination of bile acids in rat bile and serum by high-performance liquid chromatography with a post-column enzymic reaction and fluorescence detection has been developed. Without prior fractionation and alkaline hydrolysis, 26 unconjugated, glycine- and taurine-conjugated bile acids were determined. They were separated on a reversed-phase column using a linear gradient solvent system of 200 mM dibasic ammonium phosphate buffer (pH 7.9)—acetonitrile—methanol (73:19:8, v/v/v) and 20 mM dibasic ammonium phosphate buffer (pH 7.9)—acetonitrile—methanol (2:1:2, v/v/v). The limits of detection were 1–5 pmol, and calibration curves were linear for concentrations between 10 and 4000 pmol. This rapid and reliable method is effective for measuring bile acid levels in the bile and serum not only of rats but also of patients with hepatobiliary and other diseases.

A sensitive method suitable for the determination of tricyclic and other antidepressants in postmortem and clinical specimens is presented. The procedure, which utilizes reversed-phase HPLC combined with dual ultraviolet wavelength detection, enables the separation of 17 commonly prescribed antidepressants and some selected metabolites in a single extraction. Peak purity was confirmed using absorbance ratios at 220 nm and 254 nm wavelengths and revealed little interference from other eluting analytes. The blood detection limit for most antidepressants was 50 ng/ml. The most commonly observed antidepressants in 281 forensic cases analysed over a two-year period with the described method were dothiepin, amitriptyline, nortriptyline and doxepin.

Recombinant human extracellular superoxide dismutase produced in chinese hamster ovary cells has been characterized using several chromatographic methods. Peptide mapping confirmed the expected primary structure. The 15 amino acids at the N-terminal end were sequenced and were in accordance with expectations in all positions. The C-terminal amino acids have been confirmed both by amino acid composition studies of a peptide of 42 amino acids and by specific sequential cleavage of the last three C-terminal amino acids with carboxypeptidase A. Both methods demonstrated a full length C-terminus. At physiological ionic strength, the dismutase exists for ca. 25% as octamers or larger polymers, and the amount of polymers increases at lower ionic strength.

A sensitive, stereospecific high-performance liquid chromatographic assay for oxprenolol enantiomers in rat plasma was developed, using a chiral derivatization agent. Racemic oxprenolol and the internal standard (racemic propranolol) are extracted with dichloromethane after alkalinization of the plasma. Quantitation of R (+)- and S (−)-oxprenolol is based on derivatization with the chiral agent S (−)-1-(1-naphthyl)-ethyl isocyanate, followed by chromatographic separation on a C 18 reversed-phase column, with fluorometric detection (excitation at 226 nm, emission at 333 nm). The assay is reproducible as judged by a coefficient of variation of less than 17.5% for both enantiomers at all concentrations used. Preliminary experiments in the rat demonstrate that the method is sufficiently sensitive for pharmacokinetic studies in that species.

An isocratic high-performance liquid chromatographic method has been developed and validated for the quantitative determination of paclitaxel (Taxol), a novel antimitotic, anticancer agent, in human plasma. The analysis required 0.5 ml of plasma, and was accomplished by detection of the UV absorbance of paclitaxel at 227 nm following extraction and concentration. The method involved extraction of paclitaxel from plasma, buffered with 0.5 ml of 0.2 M ammonium acetate (pH 5.0), onto 1-ml cyano Bond Elut columns. The eluent was evaporated under nitrogen and low heat, and reconstituted with the mobile phase, acetonitrile-methanol-water (4:1:5, v/v/v) containing 0.01 M ammonium acetate (pH 5.0). The samples were chromatographed on a reversed-phase octyl 5 microns column. The retention time of paclitaxel was 10 min. The validated quantitation range of the method was 10-1000 ng/ml (0.012-1.17 microM) of paclitaxel in plasma. Standard curve correlation coefficients of 0.995 or greater were obtained during validation experiments and analysis of clinical study samples. The observed recovery for paclitaxel was 83%. Epitaxol, a biologically active stereoisomer, and baccatin III, a degradation product, were also chromatographically separated from taxol by this assay. The method was applied to samples from a clinical study of paclitaxel in cancer patients, providing a pharmacokinetic profiling of paclitaxel.

A method for the derivatization and separation of N-nitroso-N-alkylureas [alkyl = methyl (NMU), ethyl (NEU), and n-butyl (NBU)] has been developed. Fluorescent derivatives were formed with sodium sulphide, taurine and o-phthalaldehyde and separated by reversed-phase high-performance liquid chromatography. The limits of detection of standard NMU, NEU and NBU were 0.25, 0.8 and 1.5 pmol/200 microliters, respectively. The method was applied to the determination of NMU in blood after extraction with acetonitrile in the presence of calcium chloride. NBU was used as the internal standard. The recovery of NMU from blood was ca. 95%, and the limit of detection was 10 pmol/400 microliters blood. NMU levels in rabbit blood following a single oral administration were also measured.

Free phenytoin in human plasma was automatically determined by on-line equilibrium dialysis using the automated sequential trace enrichment of dialysate (ASTED) sample preparation system and HPLC. The dialysis cell was a modification of the cell supplied with the ASTED. Total phenytoin was analysed with the same analytical set-up and plasma protein binding was determined. Free phenytoin was determined in plasma from epileptic patients and the results were compared to those obtained by ultrafiltration. Automated determination of free and total phenytoin in plasma by the ASTED—HPLC combination was shown to be an accurate and reproducible method and the results in free phenytoin analyses were in agreement with those found with ultrafiltration. The sample throughput with the automated on-line combination of dialysis and column-switching HPLC was 75 samples in 24 h when the sample was dialysed at 37°C.

A high-performance liquid chromatographic method for the analysis of hydroxyproline and proline has been developed. The method is based on the derivatization of the secondary amino group with dabsyl-chloride after blocking of the primary amino group with o-phthalaldehyde. Dabsyl-hydroxyproline and dabsyl-proline were separated from other amino acids by high-performance liquid chromatography in the gradient elution mode, and eluted at 10.27 and 16.02 min, respectively. The correlations between the peak areas of dabsyl-hydroxyproline and dabsyl-proline were linear in the range from 20-200 pmol, with equations y = 1.10x - 0.80 (r = 0.999) and y = 1.12x - 0.52 (r = 0.999), respectively. The method was applied to the analysis of rat tail collagen, and the contents of hydroxyproline and proline were 1.55 +/- 0.04 and 2.03 +/- 0.04 nmol/micrograms, respectively.

A highly sensitive method for the measurement of malondialdehyde as thiobarbituric acid-malondialdehyde complex by reversed-phase high-performance liquid chromatography with fluorescence detection in biological samples is described. As samples, 20 microliters of rat plasma or 10% (w/v) liver homogenate mixed with 2.0% thiobarbituric acid in 2 M sodium acetate buffer containing 0.05% butyl hydroxytoluene (pH 3.5) were heated at 95 degrees C for 45 min to give the complex. The complex, extracted with n-butanol, was chromatographed on a system equipped with a reversed-phase column, and the eluted peak was monitored with a fluorescence detector (excitation wavelength 515 nm, emission wavelength 553 nm). The mobile phase was a acetonitrile-water (2:8, v/v) under isocratic conditions at ambient temperature, and a single analysis was done in ca. 4 min. The minimum detection level for malondialdehyde was as low as 0.05 pmol. The n-butanol extract was stable at least for 3 days. The simple mobile phase, the extremely sensitive detection limit, and the stability of the complex make this system applicable to routine clinical analysis with a small amount of tissue or biopsy sample.

A reversed-phase liquid chromatographic method for the separation of 26 phenylthiocarbamyl derivatives of amino acids in human plasma in ca. 35 min. is described. The method used a C18 column (150 x 4.6 mm I.D., 3 micron) thermostatted at 41 degrees C, and a simple multistep linear gradient of two solvents. Solvent A was 0.05 M sodium acetate (pH 5.1)-acetonitrile (98:2, v/v), and solvent B was water-acetonitrile (40:60, v/v). A simple and successful approach to the optimization of the conditions for the separation of the 26 amino acid derivatives was realized. In the initial phase of development, the composition of the gradient, its timings, the column temperature, the flow-rate and the mobile phase compositions were optimized. At the end the influence of pH was studied, and this approach led to a clear resolution of the 26 amino acids. The method was validated by accuracy, precision, and recovery studies, by analyzing patient samples, and by comparing the quality control sample results with the classical ion-exchange method.

Four metabolites of nicotine, including two glucuronides, have been separated by high-performance liquid chromatography. This separation was applied to identification of biliary metabolites of radiolabeled nicotine by radiometric detection. S-(-)-Nicotine N-glucuronide was synthesized and used as a standard in method development.

A high-performance liquid chromatographic method has been developed for the determination of the new podophyllotoxin derivative NK 611 in plasma samples. A solid-liquid extraction procedure with C18 extraction columns was used for extraction of plasma samples containing NK 611. The adsorbed NK 611 was eluted from the extraction columns with methanol-acetonitrile (50:50, v/v). The elution liquid was injected into a reversed-phase system consisting of a Chrompack C18 column. The mobile phase was acetonitrile-20 mM phosphate buffer, pH 7 (30:70, v/v). The UV detection mode allows sensitive determination of NK 611 in plasma within phase I trials. The limit of detection was 10 ng/ml, the limit of quantitation 35 ng/ml (for 1 ml of extracted plasma and 20-microliters injection volume). The calibration curve is linear within the concentration range 100-1000 ng/ml. The recovery of NK 611 from spiked plasma samples was approximately 80%.