Simultaneous determination of biogenic amines and morphine in discrete rat brain regions by high-performance liquid chromatography with electrochemical detection

A simple and sensitive method has been developed for the simultaneous determination of norepinephrine, epinephrine, dopamine, 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, and morphine in discrete rat brain regions by reversed-phase high-performance liquid chromatography with electrochemical detection. Perchloric acid extracts of the tissue were directly injected into the chromatographic system. Each of these compounds gave a linear response over the range of 20-160 ng/ml cerebellar homogenate (0.4-3.2 ng on column). Recoveries of these compounds, added to the homogenates, were complete when compared with standards dissolved in perchloric acid. The average between-run coefficients of variation for all these compounds were lower than 7.4% over the range of 20-160 ng/ml, and the within-run coefficients of variation at 20 ng/ml were lower than 8.7%. The present method has been applied to a study of the effects of intraperitoneal administration of morphine on biogenic amines in several discrete rat brain regions.     

Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition.

A high-performance liquid chromatographic method for the measurement of bumetanide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C18 column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C18 Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol-water-glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5-2000 ng/ml.     

Capillary column gas chromatographic method using electron-capture detection for the simultaneous determination of nicardipine and its pyridine metabolite II in plasma

A rapid, specific and direct method based on capillary column gas chromatography with electron-capture detection is described for the simultaneous determination of nicardipine, a new calcium antagonist, and its pyridine metabolite II in human plasma. In this method, the nicardipine, its pyridine metabolite II and internal standard are extracted from the plasma and then partially purified by acid-base partitioning prior to the final injection onto the capillary column gas chromatograph for quantification by means of an electron-capture detector. The quantification limit of the method is 1 ng/ml of plasma for both nicardipine and its pyridine metabolite II. The coefficients of variation for nicardipine and the pyridine metabolite II at concentrations of 1-50 ng/ml are less than 7% and less than 9% (n = 4), respectively. The method has been validated against a previously developed high-performance liquid chromatographic method (sensitivity 5 ng/ml).     

New, high-sensitivity high-performance liquid chromatographic method for the determination of acyclovir in human plasma, using fluorometric detection.

A high-performance liquid chromatographic method for the determination of acyclovir in human plasma has been developed. It is the first published chromatographic method capable of determining acyclovir in plasma with sufficient sensitivity and for sufficiently long periods of time following oral administration of a standard dose of acyclovir during pharmacokinetic investigations. Following precipitations of the proteins with perchloric acid, the sample is chromatographed with a strongly acidic mobile phase on a reversed-phase column, and is then subjected to fluorometric detection (excitation 260 nm, emission 375 nm). The determination limit is 6-10 ng/ml human plasma. The calibration is linear in the range 10-12,400 ng/ml plasma, with the coefficients of variation less than 8%. The absolute recovery rate is between 102 and 113%. This method has already been used to analyse several thousand plasma samples.     

Determination and identification of amiloride in human urine by high-performance liquid chromatography and gas chromatography-mass spectrometry.

A simple and sensitive high-performance liquid chromatographic method was developed to screen and determine amiloride (I) in human urine. The detection limit of the method is 0.12 micrograms/ml and the recovery of amiloride from urine was 80.4-85.5% at different concentrations. The coefficients of variation were less than 2.8 and 4.4% for intra- and inter-assays, respectively. Total urinary excretion of I in 24 h after oral administration of 5 mg or 15 mg of I ranged from 22.0 to 33.3% of the total dose for three different subjects. I could be detected in urine up to at least 44 h after a 5-mg dose and 72 h after a 15-mg dose. A gas chromatographic-mass spectrometric (GC-MS) confirmatory method was established based on the methanolysis of I to methyl 3,5-diamino-6-chloropyrazine-carboxylate (II). The di-N-trimethylsilyl derivative of II showed very good GC-MS properties and provided reliable structure information for confirmation analysis of I. This is the first time that a reliable GC-MS method has been reported for the detection of urinary I.     

High-performance liquid chromatographic method for the quantitation of bupivacaine, 2,6-pipecoloxylidide and 4'-hydroxybupivacaine in plasma and urine.

A high-performance liquid chromatographic method with ultraviolet detection at 210 nm for quantitation of bupivacaine and two of its metabolites from plasma and urine is described. The compounds are extracted into n-hexane-isopropanol (5:1), evaporated and the reconstituted residue injected onto a reversed phase C18 column. Standard curves for all compounds were linear (r2 greater than 0.999) in the range 20-2000 ng/ml, with a limit of detection of 10 ng/ml. The inter-day coefficients of variation ranged between 2.7 and 12.2%. The method was applied to analyse bupivacaine and metabolite concentrations in patients on long-term epidural bupivacaine-fentanyl infusions.     

Quantitation of a new cholecystokinin and gastrin receptor antagonist (L-365,260) in dog and rat plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) procedure has been developed for the quantification of L-365,260 (I), a cholecystokinin and gastrin receptor antagonist, in dog and rat plasma. The method involves liquid-liquid extraction and HPLC with ultraviolet detection. Standard curves were linear over the range 7.5-2000 ng/ml for rat and dog plasma. The method is reproducible and reliable with a detection limit of 7.5 ng/ml in biological fluids. The mean coefficients of variation for concentrations within the range of the standard curve range were 3.84 and 2.56%, respectively, for intra-day analysis and 4.48 and 4.26%, respectively, for inter-day analysis. Application of the development was successfully demonstrated by quantifying the concentration of I in both dog and rat plasma samples following an intravenous or oral dose of 5 mg/kg I.     

Automated high-performance liquid chromatographic method for the simultaneous determination of cefotiam and delta 3-cefotiam in human plasma using column switching.

An automated high-performance liquid chromatographic method using column switching was established for the simultaneous determination of cefotiam (I) and delta 3-cefotiam (II) in human plasma after oral administration of cefotiam hexetil dihydrochloride. The method allowed the determination of analytes in plasma by the direct injection of diluted specimen with phosphate buffer. The analytes were enriched onto the C18 short pretreatment column by 0.05 M phosphate buffer (pH 7.7), while proteins and endogenous hydrophilic substances in plasma were washed off to waste. The enriched analytes were then back-flushed onto the analytical C18 column, separated by a mixture of 0.05 M phosphate buffer (pH 7.7)-acetonitrile (88:12, v/v) and detected by the ultraviolet absorbance at 254 nm. Recoveries from spiked plasma were quantitative, and the coefficients of variation were below 4%. The lower detection limits in plasma were 10 ng/ml for both I and II. Concentrations of I and II in plasma determined by the present method were in good agreement with those obtained by the conventional deproteinization method.     

Determination of nalbuphine by high-performance liquid chromatography with electrochemical detection: application to clinical samples from postoperative patients

A rapid, selective and reproducible high-performance liquid chromatographic assay with electrochemical detection was developed for the determination of nalbuphine in human plasma. The method involves extraction with chloroform-isopropanol at pH 9.4, back-extraction into dilute phosphoric acid and reversed-phase chromatography on a microBondapak phenyl column. The recovery of nalbuphine and naltrexone (internal standard) was greater than 90%. Calibration curves were linear over a concentration range of 3-36 ng/ml with coefficients of variation, within-day or between-day, not exceeding 8% at any level. Although the limit of detection was 0.3 ng/ml based on a signal-to-noise ratio of 3, the reliable limit of quantitation was 1 ng/ml (coefficient of variation 12%) using 1 ml of plasma. The dual-electrode detector was operated in the screening mode of oxidation (electrode 1, 0.3 V and electrode 2, 0.6 V), providing a greater specificity and reducing background noise. This procedure was applied to a large number of clinical samples in an intravenous dose-range pharmacokinetic study in patients.     

Determination of sialic acids in human serum by reversed-phase liquid chromatography with fluorimetric detection.

A simple, rapid and highly sensitive reversed-phase liquid chromatographic method has been developed for the determination of sialic acids in human serum. The sialic acids, released by hydrolysis of serum, are converted in borate buffer with malononitrile to highly fluorescent compounds. The reaction mixture is separated isocratically within 5 min using an octadecyl-bonded silica column and a mobile phase of methanol and ammonium acetate buffer (15:85, v/v; pH 5.5). Measurement of the fluorescence intensity of the reaction mixture at 434 nm with irradiation at 357 nm allowed determination of 30-1000 ng/ml of sialic acids with high reproducibility. The limit of detection was 2 ng/ml. Intra-day and inter-day coefficients of variation for assaying 300 ng/ml N-acetylneuraminic acid (NANA) were 1.5% (n = 9) and 2.6% (n = 7), respectively. The recoveries of NANA were 98.5-101.1% for serum. The method has been used for clinical determinations.     

High-performance liquid chromatographic determination of lansoprazole and its metabolites in human serum and urine.

A simple and sensitive high-performance liquid chromatographic method with ultraviolet detection is described for the simultaneous determination of lansoprazole and its metabolites in human serum and urine. The analytes in serum or urine were extracted with diethyl ether-dichloromethane (7:3, v/v) followed by evaporation, dissolution and injection into a reversed-phase column. The recoveries of authentic analytes added to serum at 0.05-2 micrograms/ml or to urine at 1-20 micrograms/ml were greater than 88%, with the coefficients of variation less than 7.1%. The minimum determinable concentrations of all analytes were 5 ng/ml in serum and 50 ng/ml in urine. The method was successfully applied to a pharmacokinetic study of lansoprazole in human.     

Rapid high-performance liquid chromatographic method for the determination of propranolol levels in canine and feline plasma.

A sensitive high-performance liquid chromatographic method that does not require organic extraction has been developed for the determination of propranolol levels in canine and feline plasma. Equal volumes of plasma and a mixture of methanol-acetonitrile-0.1 M sodium hydroxide (3:3:4, v/v/v) were added to a microseparation unit with a 10,000 molecular mass cut-off filter. The ultrafiltrate was analyzed by reversed-phase liquid chromatography with fluorimetric detection. The consistency of the recoveries obtained eliminated the need for an internal standard (coefficients of variation less than 4%). Linear regressions for the standard curves (2.5-100 ng/ml) gave correlation coefficients above 0.9955. The detection limit was 1 ng/ml. The assay retains high sensitivity while eliminating laborious sample preparation.     

High-performance liquid chromatographic determination of 1,2-diethyl-3-hydroxypyridin-4-one and its 2-(1-hydroxyethyl) metabolite in rat blood.

A sensitive and selective high-performance liquid chromatographic (HPLC) method for the analysis of 1,2-diethyl-3-hydroxypyridin-4-one (CP94, I) and its 2-(1-hydroxyethyl) metabolite (II) in rat blood is described. I, II and the internal standard, 1-propyl-2-ethyl-3-hydroxypyridin-4-one (CP95, III) were extracted into dichloromethane (3 x 5 ml, with the addition of 1 g of sodium chloride) from blood (0.25 ml plus 0.75 ml of pH 7.0 morpholinopropanesulphonic acid buffer). Extractability approached 100% for I and III, and approximately 65% for II under these conditions. Chromatographic analysis was carried out using a Hypercarb porous graphitised carbon HPLC column (10 cm x 0.46 cm). The mobile phase was 14:86 (v/v) acetonitrile-NaH2PO4 buffer (10 mM, containing 2 mM EDTA, pH adjusted to 3 with phosphoric acid) and detection was by ultraviolet at 280 nm. Calibration curves were linear (correlation coefficient greater than 0.99) and reproducible over the concentration range 0-80 micrograms/ml and the coefficient of variation was less than 16% even at low (1 microgram/ml) concentrations. The minimum quantifiable level was 0.5 microgram/ml for both I and II.     

Simultaneous determination of the camptothecin analogue CPT-11 and its active metabolite SN-38 by high-performance liquid chromatography: application to plasma pharmacokinetic studies in cancer patients.

CPT-11 (I; 7-ethyl-10-[4-(1-piperidino)-1- piperidino]carbonyloxycamptothecin) is a new anticancer agent currently under clinical development. A sensitive high-performance liquid chromatographic assay suitable for the simultaneous determination of I and its active metabolite SN-38 (II) in human plasma, and their preliminary clinical pharmacokinetics, are described. Plasma samples were processed using a solid-phase (C18) extraction step allowing mean recoveries of I, II and the internal standard camptothecin (III) of 84, 99 and 72%, respectively. The extracts were chromatographed on a C18 reversed-phase column with a mobile phase composed of acetonitrile, phosphate buffer and heptanesulphonic acid, with fluorescence detection. The calibration graphs were linear over a wide range of concentrations (1 ng/ml-10 micrograms/ml), and the lower limit of determination was 1 ng/ml for both I and II. The method showed good precision: the within-day relative standard deviation (R.S.D.) (5-1000 ng/ml) was 13.0% (range 4.9-19.4%) for I and 12.8% (6.7-19.1%) for II; the between-day R.S.D. (5-10,000 ng/ml was 7.9% (5.4-17.5%) for I and 9.7% (3.5-15.1%) for II. Using this assay, plasma pharmacokinetics of both I and II were simultaneously determined in three patients receiving 100 mg/m2 I as a 30-min intravenous infusion. The mean peak plasma concentration of I at the end of the intravenous infusion was 2400 +/- 285 ng/ml (mean +/- standard error of the mean). Plasma decay was triphasic with half-lives alpha, beta and gamma of 5.4 +/- 1.8 min, 2.5 +/- 0.5 h and 20.2 +/- 4.6 h, respectively. The volume of distribution at steady state was 105 +/- 15 l/m2, and the total body clearance was 12.5 +/- 1.9 l/h.m2. The maximum concentrations of the active metabolite II reached 36 +/- 11 ng/ml.     

Simple high-performance liquid chromatographic method for the determination of captopril in biological fluids.

A rapid, simple and sensitive column-switching high-performance liquid chromatographic procedure for the determination of captopril in plasma and urine had been developed. p-Bromophenacyl bromide was used as a derivatizing reagent to react with captopril to form a product that showed ultraviolet-absorbing properties. For plasma samples the protein was removed with 6% perchloric acid before injection. The urine samples were directly injected into the chromatograph. The column-switching system was equipped with a pre-column (5 cm x 0.5 cm I.D.) packed with muBondapak C18 (37-50 microns) and an analytical column (15 cm x 0.5 cm I.D.) packed with YWG-C18, 10 microns. Impurities were washed from the pre-column with 0.2% acetic acid and the retained substances were eluted into the analytical column with acetonitrile-water-acetic acid (35:65:0.4, v/v). Captopril was detected at 260 nm. The calibration curve was linear in the range 20-1000 ng/ml for plasma and 10-200 micrograms/ml for urine. The recoveries averaged 103.2 and 99.5% for plasma and urine, respectively. The coefficients of variation were all less than 10%.     

Determination of fluorescent trypanocidal diamidines by quantitative thin-layer chromatography

The fluorescent trypanocidal diamidines 2-(4-amidinophenyl)indole-6-carboxamidine dihydrochloride (I, DAPI), 2-(4-amidinophenyl)benzo[b]thiophene-6-carboxamidine dihydrochloride (II) and 2-(4-amidinophenyl)-1-benzofurane-5-carboxamidine dihydrochloride (III) were determined in plasma, urine, faeces and tissues of experimental animals using quantitative thin-layer chromatography. Samples were extracted with n-octanol after addition of sodium hydroxide and subsequently re-extracted into 0.1 M hydrochloric acid. Chromatography was performed on silica gel plates under nitrogen with n-butanol saturated with 2 M hydrochloric acid. Quantitation was performed by measuring native fluorescence using a fluorodensitometer. The respective diamidines were used as internal standards for each other to ensure precision (coefficient of variation less than 7%) and accuracy of the assay. Calibration curves were linear up to 150 ng/ml of sample solution with detection limits of 10 ng/ml of sample solution for I and III and 50 ng/ml for II. The described method has been successfully used for pharmacokinetic studies in experimental animals.     

Quantification of amitriptyline, nortriptyline, and 10-hydroxy metabolite isomers in plasma by capillary gas chromatography with nitrogen-sensitive detection

A selective, sensitive method for the determination of amitriptyline and its metabolites is described. This method involves liquid-liquid extraction and capillary gas chromatography with nitrogen-sensitive detection. The detection limits of amitriptyline, nortriptyline, 10-hydroxy(E)amitriptyline, 10-hydroxy(E)nortriptyline, and 10-hydroxy(Z)nortriptyline were slightly less than 0.5 ng/ml in 1.0-ml plasma samples. The coefficients of variation for within-run and between-run analyses of samples containing 100 ng/ml were less than 12% and 9%, respectively. The method offers rapid analysis of individual isomers, increased sensitivity over high-performance liquid chromatographic methodology and the conveniences of the gas chromatographic technique.     

Determination of clozapine and its major metabolites in human serum using automated solid-phase extraction and subsequent isocratic high-performance liquid chromatography with ultraviolet detection.

An isocratic high-performance liquid chromatographic (HPLC) method with ultraviolet detection is described for the quantification of the atypical neuroleptic clozapine and its major metabolites, N-desmethylclozapine and clozapine N-oxide, in human serum or plasma. The method included automated solid-phase extraction on C18 reversed-phase material. Clozapine and its metabolites were separated by HPLC on a C18 ODS Hypersil analytical column (5 microns particle size; 250 mm x 4.6 mm I.D.) using an acetonitrile-water (40:60, v/v) eluent buffered with 0.4% (v/v) N,N,N',N'-tetramethylethylenediamine and acetic acid to pH 6.5. Imipramine served as internal standard. After extraction of 1 ml of serum or plasma, as little as 5 ng/ml of clozapine and 10 or 20 ng/ml of the metabolites were detectable. Linearity was found for drug concentrations between 5 and 2000 ng/ml as indicated by correlation coefficients of 0.998 to 0.985. The intra- and inter-assay coefficients of variation ranged between 1 and 20%. Interferences with other psychotropic drugs such as benzodiazepines, antidepressants or neuroleptics were negligible. In all samples, collected from schizophrenic patients who had been treated with daily oral doses of 75-400 mg of clozapine, the drug and its major metabolite, N-desmethylclozapine, could be detected, while the concentrations of clozapine N-oxide were below 20 ng/ml in three of sixteen patients. Using the method described here, data regarding relations between therapeutic or toxic effects and drug blood levels or metabolism may be collected in clinical practice to improve the therapeutic efficacy of clozapine drug treatment.     

A generic method for the determination of acrylamide in thermally processed foods

A generic sample preparation method for the determination of acrylamide in foods was developed. The method entails extraction with methanol, purification with Carrez I and II solutions, evaporation and solvent change to water, and cleanup with Oasis HLB solid-phase extraction (SPE) cartridge. The final extract was analyzed by liquid chromatography-mass spectrometry (LC-MS) for quantitation. The chromatographic separation was performed on ODS-3 column using the isocratic mixture of 0.01 mM acetic acid in 0.2% aqueous solution of formic acid at a flow rate of 0.6 ml/min at 25 degrees C. The recoveries of acrylamide from potato chips, biscuits and coffee ranged between 92.8 and 101.5% with relative standard deviations of 4.1% or less. The limit of detection (LOD) and the limit of quantitation (LOQ) were 2 ng/g and 6 ng/g in the basis of signal to noise ratios of 3:1 and 9:1, respectively.     

Determination of HP 749, a potential therapeutic agent for Alzheimer's disease, in plasma by high-performance liquid chromatography.

N-(n-Propyl)-N-(4-pyridinyl)-1H-indol-1-amine hydrochloride (HP 749, I), a non-receptor-dependent cholinomimetic agent with noradrenergic activity, is a potential agent for the treatment of Alzheimer's disease. Pharmacokinetic studies in animals and humans showed that I was well absorbed and metabolized primarily to the N-despropyl metabolite (P7480, II) after oral administration. To facilitate the kinetic studies, a sensitive and selective high-performance chromatographic assay was developed. I and II are extracted from plasma by a mixture of cyclohexane-ethyl acetate and chromatographed on an isocratic reversed-phase high-performance liquid chromatographic system employing an analytical phenyl column with acetonitrile-ammonium formate as mobile phase. The concentrations of these two compounds, quantitated by internal standardization, are monitored by ultraviolet detection. The method is linear in the plasma assay over a concentration range of 0.5-500 ng/ml for both compounds with a quantitation limit of 0.5 ng/ml. The precision and accuracy of the calibration curves and/or method are less than 10%. The recovery of I and II from plasma is 63-74 and 63-68%, respectively, over a concentration range of 0.5-500 ng/ml.