An ELISA for quantification of murine IgG in rat plasma: application to the pharmacokinetic characterization of AP-3, a murine anti-glycoprotein IIIa monoclonal antibody, in the rat

An enzyme-linked immunosorbent assay (ELISA) has been developed to determine concentrations of murine IgG in rat plasma. Specifically, the assay was developed to measure a murine anti-glycoprotein IIIa antibody (AP-3) in rat plasma to facilitate future investigations of AP-3 pharmacokinetics and pharmacodynamics in the rat. The working range of the assay is 15-100 ng ml(-1), corresponding to a limit of quantification of 1.5 microg ml(-1) in rat plasma. The assay was validated with respect to accuracy, precision, and cross-reactivity with both pooled rat and mouse IgG. Intra-assay recoveries of AP-3 in rat plasma ranged from 93 to 103%, with CV% values ranging from 5.2 to 8.5%. Inter-assay recoveries of the plasma AP-3 samples ranged from 107 to 119% with CV% values ranging from 17.7 to 25.1%. The assay has no appreciable cross reactivity with pooled rat IgG and full cross reactivity with pooled mouse IgG, making this an ideal assay to determine plasma pharmacokinetics of mouse antibodies in the rat. The assay was used to determine the pharmacokinetics of AP-3 in a Sprague-Dawley rat.     

Bromperidol radioimmunoassay: human plasma levels

A sensitive radioimmunoassay (RIA) procedure was developed to assay for bromperidol levels in human plasma after therapeutic drug administration. The antisera used in the RIA procedure was generated in rabbits against a haloperidol-bovine serum albumin conjugate. Tritiated haloperidol was used as the radioligand in the assay. A single ether extraction of alkalinized plasma was used to separate bromperidol from its more polar metabolites and to reduce assay variability encountered with a direct plasma assay. The lower limit of detection was approximately 0.5 ng/mL of parent drug in plasma. The assay exhibited within- and between-assay variabilities of approximately 9 and 14%, respectively. A 103-106% recovery of bromperidol from quality control plasma samples was observed over the concentration range of 1-150 ng/mL. A correlation coefficient of 0.9999 with respect to measured versus expected bromperidol content in the quality control plasma samples was exhibited. Cross-reactivity characteristics of the antisera indicated that dehydrobromperidol could significantly interfere (approximately 25% cross-reactivity) with the RIA procedure. However, biotransformation studies have not suggested this compound as a metabolite of bromperidol. Predose ( Cmin ) plasma levels of bromperidol in schizophrenic patients maintained on drug therapy are also reported.     

Improved radioreceptor assay for the determination of plasma levels of dihydropyridine calcium channel blockers in humans

Ways of improving sensitivity of the radioreceptor assay to determine the plasma levels of dihydropyridine calcium antagonists were investigated. Extraction of the drug from plasma with organic solvent was found to enhance the sensitivity of the assay (method 1). Alternatively, the inhibitory effect observed when plasma is added directly to the binding assay can be counteracted by increasing the amount of membranes in the assay (method 2). Plasma levels after single oral doses of nitrendipine and nicardipine were followed with method 1. Plasma levels of isradipine were measured with methods 1 and 2 and by mass fragmentography. The data confirm that nitrendipine plasma level kinetics vary widely from patient to patient, whereas for nicardipine the drug level profile is more homogeneous. The similarity of the data obtained from the radioreceptor assay and from mass fragmentography suggests the absence of any active metabolite of isradipine.     

A sensitive assay of 5-fluorouracil in plasma by gas chromatography-mass spectrometry.

1 A gas chromatographic-mass spectrometric method was developed for determining 5-fluorouracil in plasma, using methylated thymine as an internal standard. 2 5-fluorouracil was extracted from plasma by a novel procedure which removed plasma components interferring with the sensitivity of the assay. The method included heating the plasma, washing with ether and extracting the drug under optimum conditions. 3 The sensitivity of the assay was 10 ng/ml plasma, sufficient to determine the low concentrations of 5-fluorouracil found in plasma during continuous infusion of the drug in patients receiving chemotherapy for cancer.     

Determination of cyclonite (RDX) in human plasma by high-performance liquid chromatography

A simple and sensitive HPLC method has been developed for the assay of cyclonite (RDX) in human plasma. The assay involves solid-phase extraction on Tox-clean RC SPE cartridges and isocratic reversed-phase chromatography with diode-array detection. The assay was linear over the concentration range of 0.01-2.0 microg/ml for plasma with a lower limit of detection of 0.005 microg/ml. Both the within-day and day-to-day reproducibilities and accuracies were less than 10.15 and 1.9%, respectively. The method was applied to evaluate RDX concentration in plasma samples obtained from soldiers exposed RDX.     

Statistical analysis of performance of a liquid chromatographic assay of anticonvulsants involving solvent-demixing extraction and reciprocal internal standardization

Analysis of variance both factorial and nested was used to validate a HPLC method intended for routine clinical assay of ethosuximide, phenobarbital, phenytoin and carbamazepine. Drugs were salted out, together with the solvent, from 0.5 ml acetonitrile-deproteinized plasma samples with 80-90% recovery. The acetonitrile extraction solution contained a known amount of all four drugs. This added amount of any drug was used when absent from plasma as an internal standard for those present and when present as a calibrator. Results showed that assay precision was acceptable (CV 6%) over and above the therapeutic range when additions did not exceed the lower therapeutic plasma level and if as many replications were made as there were drugs to assay. In return for some loss of sensitivity, reciprocal internal standardization provides increased assay reliability owing to the usual availability of more than one internal standard and to easier identification of interfering chromatographic peaks.     

Phenytoin and phenobarbital assayed by the ACCULEVEL method compared with EMIT in an outpatient clinic setting

The ACCULEVEL (Syntex) therapeutic drug assay technique was evaluated for phenytoin and phenobarbital in 30 patients with epilepsy who attended a neurology outpatient clinic. This finger-prick whole blood method is calibrated by the manufacturer to give assay results equivalent to plasma drug concentration. The results were compared with EMIT (Syva) technique measurements on the plasma from venous blood drawn simultaneously. The results presented show regression lines of y = 0.91x + 10.8, (r2 = 0.92) and y = 0.97x + 5.01, (r2 = 0.77) for phenytoin and phenobarbital (microM), respectively, when the ACCULEVEL finger-prick blood assay was compared with the EMIT venous plasma assay. Acceptable precision and accuracy data are presented for replicated ACCULEVEL assays. The ACCULEVEL method was found to be reliable when performed by a laboratory technician and provides a very convenient quantitative drug assay that could easily be performed by a variety of individuals at a site remote from laboratory facilities.     

Brotizolam radioimmunoassay: development, evaluation, and application to human plasma samples

A radioimmunoassay for the determination of the hypnotic agent brotizolam (11) was developed. With this procedure, an antiserum was used which was obtained from rabbits immunized with the hapten 10 (We 934) covalently bound to bovine serum albumin and tritium-labeled brotizolam as the radioligand. Compound 10 represents a structural analogue of brotizolam: the bromine was replaced by a carboxyethyl group. By such manipulation high assay specificity against the primary human metabolites was achieved. The sensitivity limit of the assay was about 100 pg of brotizolam per mL of plasma when 0.1-mL samples were used. The assay showed good accuracy and high precision. Repeated assays after keeping plasma samples frozen for various periods again indicated high precision as well as the stability of the brotizolam molecule under these conditions. Application of the assay to plasma samples of eight subjects who received single oral 0.25-mg doses of brotizolam showed a mean maximum plasma concentration of 4.6 ng of unchanged drug per mL at 0.9 h after administration. The brotizolam plasma concentration declined with a mean elimination half-life of 5.1 h. The pharmacokinetic parameters estimated by RIA agree well with those obtained with other specific brotizolam determination procedures.     

Subnanogram quantitation of chlorpheniramine in plasma by a new radioimmunoassay and comparison with a liquid chromatographic method

A new radioimmunoassay (RIA) procedure for the quantitation of chlorpheniramine in plasma is described. The assay allows the determination of chlorpheniramine levels up to 96 h after oral administration of a single 4-mg tablet to healthy volunteers. This procedure was sensitive to a 156-pg/mL plasma concentration when a 100-microL plasma sample was used. The mean coefficient of variation over the linear range of the assay from 0.156 to 20 ng/mL was 3.79%. The specificity of the assay was investigated, and the antisera showed 7% cross-reactivity with the N,N-didemethyl analogue and 17% cross-reactivity with the N-demethyl analogue. This high degree of specificity was also evident from the findings that the plasma concentrations determined by this newly described RIA procedure in samples of two healthy male volunteers who were administered 4 mg of chlorpheniramine maleate orally gave a strong correlation (r2 = 0.88) with values obtained by an HPLC-UV procedure. The antiserum cross-reacted 100% with brompheniramine and, thus, can be used for its analysis in plasma. The described RIA procedure is precise, simple, and capable of handling a large number of plasma samples with a minimal turnaround time.     

Plasma levels of fluphenazine during fluphenazine decanoate treatment in schizophrenia

The authors measured plasma fluphenazine levels in 20 schizophrenic patients receiving 25 or 50 mg fluphenazine decanoate (FPZ-D) by IM injection every 2 weeks. The plasma levels were determined by a sensitive gas-liquid chromatographic (GLC) assay with a nitrogen detector device developed in their laboratory. Using this chemical assay method, they replicated the finding of a sharp initial plasma peak within 24 h after the injection followed by a low but rather stable plasma level as previously reported by nonchemical assay methods. The interval plasma levels (averages of day 4–10 after injection) ranged from 0.17–0.61 ng/ml in 10 patients who received 25 mg; and 0.20–0.93 ng/ml in 7 patients who received 50 mg FPZ-D every 2 weeks. This four-fold variation in plasma levels during FPZ-D injection was smaller than previously reported levels achieved with oral antipsychotic drug treatment. Based on the study of plasma levels achieved with FPZ-D injection and oral FPZ-H (fluphenazine HCl) in 6 patients, the dosage requirement of FPZ-D appeared to be difficult to predict from the oral dosage of FPZ-H in the same patient. Two weeks past injection, fluphenazine was undetectable in approximately half the samples with the GLC method. Thus, radioimmunoassay or radioreceptor assay, which also measures metabolites, might be more suitable for the study of plasma levels in patients receiving FPZ-D injection.     

Specific assay for the quantitation of indocyanine green in rat plasma using high-performance liquid chromatography with fluorescence detection

A rapid and sensitive reversed-phase HPLC assay employing fluorescence detection was developed for quantitating indocyanine green (ICG) in rat plasma. Sample preparation entailed precipitation of plasma proteins with acetonitrile prior to injection on the column. The assay was linear from 0.4 to 200 micrograms/mL, with a detection limit of 3 ng on column. The plasma concentration-time profile of ICG was characterized by this HPLC method and compared with the traditional spectrophotometric assay following iv bolus and iv infusion administration of 5 mg/kg of ICG to rats. Concentrations of ICG obtained using the spectrophotometric assay were consistently higher than those determined by HPLC. In animals receiving ICG by infusion, the maximum difference between the two assays was observed 1 min post-infusion and became negligible by 5 min post-infusion. The calculated pharmacokinetic parameters for ICG, systemic clearance and apparent volume of distribution, were higher using the HPLC assay as compared with the spectrophotometric procedure. The data suggest that a biotransformation or degradation product of ICG is formed in the rat and interferes with the determination of ICG by the spectrophotometric assay. Since the HPLC assay is specific for the parent dye, it is suggested that this assay method be used when determining pharmacokinetic parameters of ICG in rats.     

ELISA on a microchip with a photodiode for detection of amphetamine in plasma and urine

A rapid and sensitive assay was developed for the detection of amphetamine in plasma and urine. The method relies on the principle of competitive ELISA (enzyme-linked immunosorbent assay). A flow microchip with a total volume of 7 microL was used for the development of a chemiluminescent ELISA technique. Solutions, samples, and the chemiluminescence substrate were injected by a flow system, and a photodiode detector was used to measure the light intensity. The incubation time of the competitor (competition phase) was reduced to 10 min. Calibration curves corresponding to analyte concentrations ranging from 40 to 1,000 microg/L in urine samples and from 6 to 96 microg/L in plasma samples were obtained. The detection limits were in the region of 20 and 6 microg/L in urine and plasma, respectively. The main focus of the work was on speed, reliability, reproducibility, and operational stability of the assay. This method was proven readily adaptable to automation and provided reproducible results.     

Competitive protein binding assay for piritrexim

A competitive protein binding assay for piritrexim (PTX, 1) that makes use of a commercially available radioassay kit for methotrexate has been developed. After it is selectively extracted from plasma, PTX competes with [125l]methotrexate for binding to dihydrofolate reductase isolated from Lactobacillus casei. Free drug is separated from bound drug by adsorption to dextran-coated charcoal. Piritrexim is measurable over a range of 0.01 to 10.0 micrograms/mL in plasma with a coefficient of variation less than 15%. The limit of sensitivity of the assay is approximately 2 ng/mL. An excellent correlation between this assay and a previously published HPLC method was found. published HPLC method was found.     

Simultaneous determination of BNP7787 and its metabolite mesna in plasma and tissue by micro-HPLC with a dual electrochemical detector

Sensitive, accurate, and precise assays are described to determine BNP7787 (disodium 2,2'-dithio-bis-ethane sulfonate) and its metabolite mesna (sodium 2-mercaptoethane sulfonate) simultaneously in plasma and tissue by micro-high-performance liquid chromatography (HPLC) with dual electrochemical detection. After separation of BNP7787 and mesna by micro-HPLC, the disulfide BNP7787 was reduced to mesna by a reactor cell with a glassy carbon working electrode (-1.6 V versus Hy-REF). At the second electrode, which consisted of a gold wall-jet electrode, the mesna generated from BNP7787 and the mesna already present in the samples were detected (+0.85 V versus Ag/AgCl). The lower limit of quantification (LLQ) of both compounds was 3 microM in plasma and 20 nmol/g in tissue. The dynamic range of the assay in plasma was 3-120 microM for mesna and 15-1200 microM for BNP7787. In tissue, the dynamic range was 20-2000 nmol/g for both compounds. The recovery of mesna from plasma and tissue ranged from 61.4 to 90.5% and 82.7 to 90.2%, respectively, and seemed to be concentration dependent. The recovery of BNP7787 from plasma and tissue was complete (i.e., 101.5 and 96.4%, respectively). The within- and between-day accuracy and precision for the plasma and tissue assay were within 14 and 7%, respectively. The utility of the assay was shown by determination of the stability of mesna and BNP7787 in a kidney sample of a rat and by analysis of plasma samples obtained from a patient receiving 18.4 g/m(2) BNP7787 as a 15-min intravenous infusion.     

Plasma pentazocine radioimmunoassay.

A sensitive and specific radioimmunoassay of dog and human plasma pentazocine is described. Rabbit antiserum and the second antibody method separated bound from free pentazocine. The radioimmunoassay employed an 125I-labeled radioligand and required extraction from the sample prior to quantitation. The method had a detection limit of approximately 200 pg/assay tube (1 ng/ml). The assay was used successfully to measure pentazocine in the plasma of beagle hounds given 0.3 mg of pentazocine/kg iv. The decline in plasma levels fitted a two-compartment body model with a 100-min mean overall half-life and a 3.2-liters/hr mean plasma clearance rate.     

Determination of nitroimidazoles in biological fluids by differential pulse polarography.

A sensitive differential pulse polarographic assay was developed for the determination of metronidazole, ornidazole, and 2-nitro-1H-imidazole-1-(3-methoxy-2-propanol) in plasma. The compounds are selectively extracted into ethyl acetate from a protein-free filtrate of plasma, buffered to pH 7.0 +/- 0.2. The residue of the ethyl acetate extract is dissolved in 0.1 N NaOH and analyzed by differential pulse polarography for the reduction of the nitro group at approximately -0.600 v versus the saturated calomel electrode. The overall recovery from plasma was about 55 +/- 3.0% (SD) for the three compounds investigated. A TLC step after the ethyl acetate extraction may also be included to ensure specificity. This step reduced the overall recovery to approximately 45%. The sensitivity limit of detection from plasma using a 2-ml sample is 0.1 mug/ml. The assay may also be employed for the analysis of urine. The urine is adjusted to pH 7.0 +/- 0.2 and extracted with ethyl acetate, and the residue is analyzed as described for plasma. The assay was applied to the determination of ornidazole in blood and urine in the dog following 10 mg/kg po.     

Advanced method for determination of omeprazole in plasma by HPLC

An advanced and sensitive high-performance liquid chromatographic (HPLC) method for determination of omeprazole in human plasma has been developed. After omeprazole was extracted from plasma with diethylether, the organic phase was transferred to another tube and trapped back with 0.1 N NaOH solution. The alkaline aqueous layer was injected into a reversed-phase C8 column. Lansoprazole was used as an internal standard. The mobile phase consisted of 30% of acetonitrile and 70% of 0.2 M KH2P04, pH 7.0. Recoveries of the analytes and internal standard were >75.48%. The coefficients of variation of intra- and inter-day assay were <5.78 and 4.59% for plasma samples. The detection limit in plasma was 2 ng/ml. The clinical applicability of this assay method was evaluated by determining plasma concentration-time courses of the respective analytes in 24 healthy volunteers after oral administration 40 mg of omeprazole. The present assay is considered to be simple, accurate, economical and suitable for the study of the kinetic disposition of omeprazole in the body.     

Determination of LY295501 in human plasma by reverse phase HPLC with UV detection

A high performance liquid chromatographic (HPLC) assay was developed and validated for the quantitative determination of LY295501 in human plasma. A structural analog, LY186641, was selected as the internal standard. The samples were processed by protein precipitation with acetonitrile followed by concentration of the supernatants and reconstitution. Chromatographic resolution of LY295501 from endogenous plasma components was accomplished with a Waters Novapak C18 HPLC column (3.9 x 150 mm, d(p) 4 mm). Detection was by absorbance at 260 nm. The linear dynamic range was from 5 to 400 microg ml(-1) of human plasma using a 0.25 ml aliquot. The inter-day precision (%RSD) and accuracy (%RE) in plasma ranged from 2.4 to 4.7, and -4.9 to 1.4, respectively. This assay is both simple and rapid, and has been used to successfully analyze over 1500 samples from human clinical trials.     

Comparison of sandwich enzyme-linked immunoadsorbent assay and radioimmunoassay for determination of exogenous glucagon-like peptide-1(7–36)amide in plasma

A sensitive sandwich enzyme-linked immunoadsorbent assay (ELISA) for determination of exogenous glucagon-like peptide-1(7–36)amide (GLP-1(7–36)amide) in plasma samples from pharmacokinetic studies is described. The assay employs an N-terminally directed antibody and a C-terminally directed antibody. The ELISA has a working range from 10 to 500 pmol 1⁻¹, and can be applied to plasma samples from humans, dogs, pigs, minipigs, cats, rabbits, and rats. The assay was compared to a validated radioimmunoassay (RIA), employing an antibody directed against the mid-region of GLP-1. After s.c. administration of GLP-1(7–36)amide, the plasma immunoreactivity of GLP-1 (P-GLP-1-IR) measured by ELISA was markedly lower than P-GLP-1-IR measured by RIA. After HPLC fractionation of plasma samples with subsequent RIA and ELISA analyses of the fractions, this difference was shown to be due to cross reaction with biologically inactive fragments of GLP-1(7–36)amide in the RIA but not in the ELISA.     

Analysis and pharmacokinetics of olanzapine (LY170053) and two metabolites in rat plasma using reversed-phase HPLC with electrochemical detection

A sensitive HPLC assay for measurement of the antipsychotic drug, olanzapine, in plasma has been developed. The assay has a limit of quantitation of 1 ng ml⁻¹ in plasma and utilizes solid-phase extraction and electrochemical detection. The method provides a linear response for olanzapine over a concentration range of 1–100 ng ml⁻¹ with coefficients of determination greater than 0.9912. The inter-assay precision was 15.9% at the limit of detection and ranged from 7.33% to 8.47% over the range of 5–100 ng ml⁻¹. The intra-assay precision was in the range 0.97%–26.0%. The inter-assay accuracy ranged from 98.9 to 118% and the intra-assay accuracy ranged from 92.5% to 125% of the theoretical value. In addition, the assay was extended to measure the plasma levels of two metabolites of olanzapine, namely the N-desmethyl- and the 2-hydroxymethyl analogs. The utility of the assay was demonstrated following the administration of a single oral dose of ¹⁴C-olanzapine to rats where, at several time-points after dosing, the plasma was assayed for total radioactivity, levels of olanzapine, and the two metabolites. Olanzapine and two of its metabolites accounted for less than 50% of the total plasma radiocarbon; olanzapine accounting for approximately 39% at the C_max, N-desmethyl for 5% and 2-hydroxymethyl for 8% respectively. The plasma elimination half-times for olanzapine and the two metabolites were approximately the same, ranging from 3.3 to 4.4 h.