An improved radioenzymatic assay for plasma norepinephrine using purified phenylethanolamine N-methyltransferase

Radioenzymatic assays have been developed for norepinephrine (NE) using either catechol O-methyltransferase (COMT) or phenylethanolamine N-methyltransferase (PNMT). Assays using PNMT are specific for NE but have been considered less sensitive than the more complex assay procedures employing COMT. An improved purification procedure for bovine PNMT has permitted development of a NE assay with substantially improved sensitivity (less than 0.5 pg), reproducibility, and decreased manipulative effort. PNMT was purified by sequential pH 5.0 treatment and dialysis and by column chromatographic procedures using DEAE-Sephacel, Sephacryl S-200 and Phenyl Boronate-agarose. Recovery of PNMT activity through the purification scheme was 50% while blank recovery was less than 0.001%. Norepinephrine can be directly quantified in 25 microliters of human plasma and a seventy-tube assay can be routinely completed within 4 h. The capillary to venous plasma NE gradient was examined in eight normotensive male subjects. Capillary plasma NE (211 +/- 21.7 pg/ml) was significantly lower than venous plasma NE (367 +/- 32.7 pg/ml) in all subjects (p less than 0.005). This difference suggests the concentration of NE in capillary blood may be a unique indicator of sympathetic nervous system activity in vivo.     

High-performance liquid chromatographic determination of a potent AII receptor antagonist (DMP 811) in plasma

An HPLC assay for DMP 811, 4-ethyl-2-propyl-1-[(2′-(1H-tetrazol-5-yl)biphenyl-4-yl)-methyl]imidazole-5-carboxylic acid (I) in rat and dog plasma has been developed. Compound I was isolated from plasma using a liquid—liquid back extraction procedure. The extraction recovery was greater than 81%. Separation of I from endogenous components in plasma was achieved on an E. Merck C₈ column using a mobile phase of 0.05 M ammonium acetate, brought to pH 3.75 with acetic acid, and acetonitrile (78:22, v/v). The eluent was monitored by fluorescence with excitation and emission set at 235 and 370 nm, respectively. The assay was linear from 2 to 2000 ng/ml. Inter- and intra-day coefficients of variation for the rat-plasma assay ranged from 0.9 to 5.2% (5–2000 ng/ml) and 2.7 to 16.5% (2–2000 ng/ml), respectively. The respective coefficients of variation for the dog-plasma assay were 1.9 to 5.6% and 1.2 to 14.0%. The percent differences from the accuracy results were 12% or less. Using 0.5 ml of plasma for extraction, the minimum quantifiable limit was 2 ng/ml. This method has been used to quantify plasma levels of I in rats or dogs following 3–10 mg/kg i.v. or p.o. doses.     

II. Validity and reliability of liquid chromatography with electrochemical detection for measuring plasma levels of norepinephrine and epinephrine in man

Liquid chromatography with electrochemical detection (LCEC) provides a rapid, sensitive, and specific technique for measuring human plasma norepinephrine (NE) and epinephrine (E) levels. We tested the reliability and validity of this technique against that of the catechol-O-methyl-transferase radioenzymatic (COMT-RE) assay. In healthy, resting humans, mean NE and E values were similar using the LCEC and COMT-RE techniques (311 vs. 300 pg/ml for NE; 57 vs. 52 pg/ml for E). In a series of 25 plasma samples obtained from a variety of sources, the correlation between the two methods was 0.99 for both NE and E. Coefficients of variation were similar for catecholamine levels above 100 pg/ml, but below this, the COMT-RE technique appeared to be more reliable. The advantages of the LCEC method are its speed, simplicity of sample preparation, low cost per assay, lack of use of radionuclides, and ease in trouble-shooting. The COMT-RE technique is preferable for small sample sizes or large numbers of samples. LCEC offers a reasonable alternative to the COMT-RE technique for measuring plasma norepineprhine and epinephrine.     

Validation of a simplified method for determination of cimetidine in human plasma and urine by liquid chromatography with ultraviolet detection

A HPLC method was developed for determination of cimetidine in human plasma and urine. Plasma samples were alkalinized followed by liquid extraction with water-saturated ethyl acetate then evaporated under nitrogen. The extracts were reconstituted in mobile phase and injected onto a C(18) reversed-phase column; UV detection was set at 228 nm. Urine samples were diluted with an internal standard/mobile phase mixture (1:9) prior to injection. The lower limit of quantification in plasma and urine were 100 ng/ml and 10 microg/ml, respectively; intra- and inter-day coefficients of variation were 相似文献   

Determination of grepafloxacin in plasma and urine by a simple and rapid high-performance liquid chromatographic method

A rapid, specific, sensitive and economical method has been developed and validated for the determination of grepafloxacin in human plasma and urine. The assay consisted of reversed-phase HPLC with UV detection. Plasma proteins were removed by a fast and efficient procedure that has eliminated the need for costly extraction and evaporation. For the urine samples, the only required sample preparation was dilution. Separation was achieved on a reversed-phase TSK gel column with an isocratic mobile system. The method had a quantification limit of 0.05 μg/ml in plasma and 0.5 μg/ml in urine. The coefficients of variation (C.V.) were less than 4% for within- and between-day analyses. The method was successfully applied to a pharmacokinetic study, and was proved to be simple, fast and reproducible.     

Simple high-performance liquid chromatographic determination of the protease inhibitor indinavir in human plasma

Indinavir is a member of a class of protease inhibitors that actively prevent the acquired immunodeficiency syndrome virion from maturing. A high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of indinavir in human plasma. Indinavir and the internal standard were isolated from the plasma by ether extraction. The residue after evaporation of ether was reconstituted with buffer and injected onto a C₄ reversed-phase column eluted isocratically with a mobile phase consisting of 35:65 (v/v) of acetonitrile and buffer. A wavelength of 210 nm was found to be optimum for detection. The calibration range of this assay was from 10 to 5000 ng/ml and coefficients of variation for the assay ranged from 4.6% to 11.0% for three different drug concentrations and the limit of quantitation was 10 ng/ml. During the validation, short-term stability of the drug in plasma, stability during heat deactivation and on repeated freezing and thawing of plasma was evaluated. The overall recovery of indinavir by the ether extraction method was 91.4%. This HPLC assay was found to be a simple and reproducible method for monitoring indinavir levels in human plasma obtained during clinical trials of the drug.     

Liquid chromatography-tandem mass spectroscopy assay for quercetin and conjugated quercetin metabolites in human plasma and urine

A sensitive and specific method was developed and validated for the quantitation of quercetin in human plasma and urine. The application of liquid chromatography-tandem mass spectrometry (LC/MS/MS) with a TurboIonspray (TIS) interface in negative mode under multiple reactions monitoring was investigated. Chromatographic separation was achieved on a C12 column using a mobile phase of acetonitrile/water with 0.2% formic acid (pH 2.4) (40/60, v/v). The detection limit was 100 pg/ml and the lower limit of quantification was 500 pg/ml for plasma samples; the detection limit was 500 pg/ml and the lower limit of quantification was 1 ng/ml for urine samples. The calibration curve was linear from 1 to 800 ng/ml for plasma samples and was linear from 1 to 200 and 50 to 2000 ng/ml for urine samples. All the intra- and inter-day coefficients of variation were less than 11% and intra- and inter-day accuracies were within +/-15% of the known concentrations. This represents a LC/MS/MS assay with the sensitivity and specificity necessary to determine quercetin in human plasma and urine. This assay was used to determine both parent quercetin and the quercetin after enzymatic hydrolysis with beta-glucuronidase/sulfatase in human plasma and urine samples following the ingestion of quercetin 500 mg capsules.     

A radioimmunoassay for epinephrine and norepinephrine in tissues and plasma

An I¹²⁵ radioimmunoassay (RIA) has been developed for the measurement of plasma and tissue epinephrine (E) and norepinephrine (NE). The assay utilizes an antibody which specifically binds metanephrine. E and NE are detected by conversion to metanephrine with the enzymes catechol-0-methyltransferase and phenylethanol-amine-N-methyltransferase. The assay is very specific and will allow the measurement of E and NE in less than 500 μl of normal human plasma. E and NE concentrations were determined by both the RIA and a radioenzymatic assay in canine, human and rat biologic samples. The correlation coefficients between the two assays were .962 for E and .956 for NE. The RIA is sensitive, specific, precise and significantly less costly and time consuming than present radioenzymatic methods.     

High-performance liquid chromatography method with ultraviolet detection for the quantification of the BCR-ABL inhibitor nilotinib (AMN107) in plasma, urine, culture medium and cell preparations

An isocratic and sensitive HPLC assay was developed allowing the determination of the new anticancer drug nilotinib (AMN107) in human plasma, urine, culture medium and cell samples. After protein precipitation with perchloric acid, AMN107 underwent an online enrichment using a Zirchrom-PBD precolumn, was separated on a Macherey-Nagel C18-HD column and finally quantified by UV-detection at 258 nm. The total run time is 25 min. The assay demonstrates linearity within a concentration range of 0.005-5.0 microg/ml in plasma (r(2)=0.9998) and 0.1-10.0 microg/ml in urine (r(2)=0.9913). The intra-day precision expressed as coefficients of variation ranged depending on the spiked concentration between 1.27-9.23% in plasma and 1.77-3.29% in urine, respectively. The coefficients of variation of inter-day precision was lower than 10%. Limit of detection was 0.002 microg/ml in plasma and 0.01 microg/ml in urine. The described method is stable, simple, economic and is routinely used for in vivo and in vitro pharmacokinetic studies of AMN107.     

High-performance liquid chromatographic assay for the quantitation of irbesartan (SR 47436/BMS-186295) in human plasma and urine

A selective, accurate, precise and reproducible high-performance liquid chromatographic assay was developed for the quantitation of irbesartan, an angiotensin II antagonist, in human plasma and urine samples. The method involved solid-phase extraction of irbesartan and internal standard (I.S.) using a 100-mg Isolute CN cartridge. A portion of the eluate was injected onto an ODS analytical column connected to a fluorescence detector that was set at an excitation wavelength of 250 nm and an emission wavelength of 371 nm. The mobile phase consisted of 50% acetonitrile and a 50% weak phosphate-triethylamine solution, pH 3.5, at a flow-rate of 0.8 ml/min. The assay was linear from 1 to 1000 ng/ml with both plasma and urine. In either matrix, the lower limit of quantitation was 1 ng/ml. The analyses of quality control samples indicated that the nominal values could be predicted with an accuracy >95%. The inter- and intra-day coefficients of variation for the analyses in both matrices were <8%. Irbesartan was stable in both human plasma and urine for at least seven months at −20°C. The application of the assay to a pharmacokinetic study is described.     

Simultaneous quantification of beclomethasone dipropionate and its metabolite, beclomethasone 17-monopropionate in rat and human plasma and different rat tissues by liquid chromatography-positive electrospray ionization tandem mass spectrometry

A sensitive, rapid and selective liquid chromatography-positive electrospray ionization tandem mass spectrometry (LC-(ESI+)-MS-MS) method has been developed and validated for the simultaneous quantification of beclomethasone dipropionate (BDP) and its active metabolite, beclomethasone 17-monopropionate (17-BMP) in rat plasma and different tissues using fluticasone propionate (FP) as the internal standard. The method was validated over a linear range from 0.05 to 5 ng/ml for both analytes. A solid-phase extraction procedure was used for plasma samples and a liquid-liquid extraction procedure for tissues samples (lung, liver and kidney). The between-day and within-day coefficients of variation for all compounds were 相似文献   

Determination of plasma propofol levels using gas chromatography—mass spectrometry with selected-ion monitoring

Propofol (2,6-diisopropylphenol, I.C.I. 35 868) is a rapid-acting, intravenous anesthetic agent recently introduced for the induction and maintenance of general anesthesia. This paper describes a gas chromatographic—mass spectrometric procedure using selected-ion monitoring for the determination of plasma propofol levels. The drug and the internal standard (thymol) were extracted from plasma into diethyl ether—pentane, and derivatized to their trimethylsilyl derivatives before analysis. The reproducibility of the daily standard curves had coefficients of variation ranging from 2.7% to 10.2%. The precision of the assay yielded a coefficient of variation ranging from 4.5% to 5.6%, and the concentration means for the seeded control samples were found to be within −1.6% to +0.6% of the theoretical values for propofol. No interfering peaks have been observed in application of this procedure to either normal volunteer or patient samples. The minimum detectable level under the conditions described was 0.20 ng propofol/ml plasma. This assay and a high-performance liquid chromatographic assay with fluorescence detection were both used to measure plasma propofol concentrations in 89 human plasma samples, and the correlation between the two methods was excellent.     

Quantitation of the antitumor agent N-(Phosphonacetyl)-l-aspartic acid in human plasma and urine by gas chromatography—mass spectrometry—selected ion monitoring

N-(Phosphonacetyl)-l-aspartic acid (PALA) is an antitumor agent which is currently under clinical study. A gas chromatography—mass spectrometry—selected ion monitoring assay procedure using [¹³C]PALA as the internal standard has been developed for the quantitation of PALA in biological samples. Standard curves which related ion intensity peak height ratios (m/e 220/221) to PALA concentrations in plasma and urine were described by a non-linear least square analysis with correlation coefficients of R² > 0.995 and > 0.996, respectively. Over concentration ranges for PALA of 1–60 μg/ml of plasma and 1–160 μg/ml of urine the coefficient of variation from the fitted curve was 4–18%. This methodology has been used to quantitate PALA in human plasma samples in a study on the clinical pharmacology of the drug.     

Quantitative determination of 4-hydroxy-4-androstene-3,17-dione (4-OHA), a potent aromatase inhibitor, in human plasma, using isotope dilution mass spectrometry

An original method is described for the determination in human plasma of 4-hydroxy-4-androstene-3,17-dione (4-OHA), a potent aromatase inhibitor, by isotope dilution mass-spectrometry using 7,7-[2H2]-4-OHA as internal standard. This compound was synthesized starting from 7,7-[2H2]-4-androstene-3,17-dione. The procedure includes an extraction step using an Extrelut 1 column and a derivatization with N,o-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The minimum detection level of the method is 0.650 pg and the coefficients of variation for the 0.5 ng/ml (plasma) and 5 ng/ml (plasma) concentrations are 3.2% (within assay) and 6.7% (between assay) and 1.86% (within assay) and 2.3% (between assay) respectively.     

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 bumetamide 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.     

Determination of dihydroergocryptine in human plasma and urine samples using on-line sample extraction-column-switching reversed-phase liquid chromatography-mass spectrometry

A rapid and sensitive assay for the determination of dihydroergocryptine (DHEC) in human plasma and urine samples with dihydroergotamine (DHET) as the internal standard was developed. The procedure employs on-line sample preparation using an extraction pre-column and an octadecylsilylsilica (ODS) analytical column. After centrifugation human plasma or urine were injected onto the pre-column, concentrated and extracted, back-flushed onto the analytical column and eluted with a binary methanol--aqueous formic acid gradient. Either determination of DHEC as well of its mono- and dihydroxy-metabolites was performed by measurement of the signal responses from MS detection in the selected reaction monitoring (SRM) mode using the transition of the respective parent ions to the common daughter ion at m/z=270.2 amu. The limit of quantitation (LOQ) for determinations of DHEC in both plasma and urine were 25 pg/ml for injected sample volumes of 400 microl. Proportionality of signal responses versus concentration was accomplished within the range of 25-1000 pg/ml. Recovery of target analyte from plasma was 99%. Mean values of the coefficients of variation (CV) for the target analyte in plasma ranged from 1.7 to 13.8% (within-day) and 5.0 to 9.1% (between-day) and accuracy from 91.7 to 102.6% for the within-day and from 95.8 to 98.8% for the between-day measurements. The corresponding values for determinations in urine were 1.7-14.5% (within-day) and 5.3-11.8% (between-day) for CV and 95.8-110.7% (within-day) and 100.1-104.6% (between-day) for accuracy.     

Simultaneous determination of nefiracetam and its metabolites by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic assay was developed to simultaneously quantitate nefiracetam (NEF), a novel nootropic agent, and its three known oxidized metabolites (N-[(2,6-dimethylphenylcarbamoyl)methyl]succinamic acid (5-COOH-NEF), 4-hydroxy-NEF and 5-hydroxy-NEF) in human serum and urine. The quantitative procedure was based on solid-phase extraction with Sep-Pak C₁₈ and ultraviolet detection at 210 nm. The calibration curves of NEF and the metabolites were linear over a wide range of concentrations (0.5–21.5 nmol/ml for NEF and 0.4–9.5 nmol/ml for metabolites in serum and 4–86 nmol/ml for NEF and 8–190 nmol/ml for metabolites in urine). Intra- and inter-day assay coefficients of variation for the compounds were less than 10%. The limit of detection was 0.1 nmol/ml for NEF, 5-COOH-NEF and 4-hydroxy-NEF, and 0.2 nmol/ml for 5-hydroxy-NEF in both serum and urine. This method is applicable for the determination of NEF and its metabolites in human serum and urine with satisfactory accuracy and precision.     

Determination of phenethyl isothiocyanate in human plasma and urine by ammonia derivatization and liquid chromatography-tandem mass spectrometry

Phenethyl isothiocyanate (PEITC) is a dietary compound present in cruciferous vegetables that has cancer-preventive properties. Our objective was to develop and validate a novel liquid chromatography-tandem mass spectrometry procedure to analyze PEITC concentrations in human plasma and urine. Following hexane extraction, ammonia was added to samples to derivatize PEITC to phenethylthiourea. Chromatographic separation was achieved on a C(18) column with acetonitrile/5 mM formic acid (60:40, v/v) as the mobile phase followed by tandem mass spectrometry detection in multiple reaction monitoring mode. Deuterium-labeled PEITC was used as the internal standard. The detection limit was 2 nM and calibration curves were linear from 7.8 to 2000 nM. The intra- and inter-day coefficients of variation were less than 5 and 10%, respectively. The intra- and inter-day accuracies ranged from 101.0 to 104.2% and from 102.8 to 118.6%, respectively. The recovery from spiked human plasma and urine ranged from 100.3 to 113.5% and from 98.3 to 103.9%, respectively. The assay was used to measure PEITC in plasma and urine samples obtained from subjects after consumption of 100g of watercress. This novel assay represents the first analytical method with the sensitivity and specificity to determine plasma and urine concentrations of PEITC.     

Radioenzymatic assay of sulfate conjugates of catecholamines and DOPA in plasma

The addition of a commercially available sulfatase to the incubation mixture for the single isotope radioenzymatic assay of the catecholamines permits the simultaneous assay of the catecholamine sulfates in plasma. The sulfatase is compatible with the catechol-0-methyl transferase of the radioenzymatic assay and cleaves the sulfate to form the free catechol. With the multiple enzyme action it is possible to assay total (sulfated plus free) catecholamines and DOPA. A second series of assays performed in the absence of the sulfatase provides results for each of the free catecholamines and DOPA. In a study with ten normotensive male subjects supine free NE was 19% (range of 10–24%) of the total, E was 12% (range 6–16%), DA was 1% (range 0.5–2%) and DOPA was 45% (range 14–71%) of the total. Upon standing, free NE increased an average of 71% from 196±53 pg/ml (supine) to 336±69 pg/ml. Free NE (30%) and free E (18%) comprised a larger proportion of the total plasma NE and E in standing subjects than in the plasma of the supine subjects. Changes in the free levels and in the ratio of free to total levels of DA and DOPA were less than those noted for NE and E. This assay methodology may be useful in demonstrating individual variations in the ability to sulfate endogenous catechols.     

Determination of indinavir in human cerebrospinal fluid and plasma by solid-phase extraction and high-performance liquid chromatography with column switching

A rapid, sensitive and robust sample preparation procedure for the quantitative determination of indinavir in human cerebrospinal fluid (CSF) and plasma is described. Indinavir and the internal standard were isolated from CSF or plasma samples by cation-exchange solid-phase extraction with SCX cartridges, while the chromatographic separation was adopted from a previous method, using a cyano column connected by a switching valve to a C₁₈ column. UV detection was set at 210 nm. The standard curve was linear over the concentration range of 2 to 2000 ng/ml in CSF and 5 to 2000 ng/ml in plasma. The intra-day coefficients of variation at all concentration levels were ≤5.9%. The inter-day consistency was assessed by running QC samples during each daily run. The coefficients of variation for quality control samples in both matrixes were ≤6.1%. The method has been utilized to support clinical pharmacokinetic studies.