Lower Limit Of Sensitivity Research Articles

An HPLC Method for the Determination of Diltiazem and Three of its Metabolites in Serum

Abstract A high performance liquid chromatographic method was developed for the determination of diltiazem and three of its metabolites in serum. Serum samples are extracted with methyl-tert.-butyl ether followed by back-extraction into 0.05N sulfuric acid. Doxepin hydrochloride was used as internal standard. A cyanopropylsilane column in the reverse-phase mode was utilized with a mobile phase of acetonitrile-phosphate buffer-triethylamine (pH 3.5). Using UV detection at 237nm, the lower limit of sensitivity was 1.5–3.0 ng/ml. Recovery and reproducibility results and possible interferences from other compounds are presented and discussed. The assay procedure was applied to a chronic oral dosing study in humans to assess the disposition of diltiazem and its primary metabolites.

Determination of Pineal Melatonin by High‐Performance Liquid Chromatography With Electrochemical Detection: Application for Rhythm Studies and Tissue Explants

High-pressure liquid chromatography with electrochemical detection has been used for the determination of melatonin in rat pineal glands and tissue explants. Rat pineal melatonin content was measured at 2-hr intervals during a 24-hr period by direct injection of supernatants from centrifuged pineal homogenates into a reversed-phase C18 column. The lower limit of sensitivity was 32 pg per gland for a 2:1 signal-to-noise ratio, which allows melatonin measurements during daytime. Both absolute values and circadian rhythmicity obtained with the present method are in agreement with previously validated radioimmunoassays. In addition, isoproterenol-induced release of melatonin from individual rat pineal explants was measured after chloroform extraction of the incubation medium.

Dextran Sulfate Is Poorly Absorbed after Oral Administration

To determine whether dextran sulfate (molecular weight, 7000 to 8000 daltons; 17% to 20% sulfur), a synthetic heparin analogue with anti-human immunodeficiency virus (HIV) activity in vitro, is absorbed after oral administration. Open-label, single-center study in two parts. The first part was a bioavailability study in which six subjects received a single 1800-mg oral dose and a single 225- or 300-mg intravenous dose. The second part was a study of the dose-response relation between dextran sulfate and total plasma lipolytic activity in which twelve subjects were given a single infusion of either 0.05, 0.5, 5, or 50 mg of dextran sulfate. Eighteen healthy volunteers. In the bioavailability study, plasma and urine dextran-sulfate concentrations were measured by a competitive binding assay after each dose. In addition, two bioassays were used to assess plasma concentrations: plasma lipolytic activity and activated partial thromboplastin time (APTT). After the oral dose, plasma concentrations were not measurable with the competitive binding assay (lower limit of sensitivity, 1 microgram/mL); less than 0.5% of the dose was recovered in the urine, the APTT did not increase, and the median increase in the plasma lipolytic activity was only twofold (maximum increase, 11-fold). In contrast, after the intravenous dose of 225 mg, peak plasma concentrations by competitive binding assay were 26 to 35 micrograms/mL (median, 28 micrograms/mL); 25% to 29% (median, 25%) of the dose was recovered in the urine; the APTT increased to 3.5 to 9.2 times the baseline value (median increase, 6.9 times), and the plasma lipolytic activity increased by 185 to 548 times the baseline value (median increase, 438 times). In the dose-response study, intravenous doses as low as 0.5 mg produced significant increases in the plasma lipolytic activity. There was a steep dose-response curve between 0.5 and 50 mg. Dextran sulfate is very poorly absorbed after oral administration.

Exposure to environmental tobacco smoke measured by cotinine 125I-radioimmunoassay.

We describe a polyclonal-antiserum-based 125I-radioimmunoassay for cotinine that is suitable for measuring nonsmokers' passive exposure to tobacco smoke in the environment. The standard curve ranged from 0.25 to 12.0 micrograms/L, with an estimated lower limit of sensitivity of 0.2 microgram/L (95% B/Bo = 0.2 microgram/L; 50% B/Bo = 4.0 micrograms/L). The median within-assay CVs for patients' samples with cotinine values from 0.4 to 1.3, 1.4 to 2.4, 2.5 to 4.6, and 4.7 to 15.6 micrograms/L were 13.9%, 7.2%, 5.1%, and 5.7%, respectively. Between-assay CVs for two quality-control sera with average values of 1.53 and 3.68 micrograms/L were 14.3% and 7.8%, respectively. Analytical recoveries of cotinine from smokers' sera diluted in zero calibrant ranged from 91% to 116%. Cotinine values determined on 79 paired sera and urines from nonsmokers showed significant correlation with self-reported exposure to environmental tobacco smoke (r = 0.49, P less than 0.001 for sera; r = 0.57, P less than 0.001 for urine). The log of the values for serum and urine cotinine were also significantly correlated (r = 0.85, P less than 0.001). Evidently, polyclonal antiserum can be used to develop a cotinine assay for measuring exposure to environmental tobacco smoke that compares well with that described for monoclonal-based assays.

Analysis of histamine and Nτ-methylhistamine in plasma by gas chromatography-negative ion-chemical ionization mass spectrometry

Current methods of quantitation of histamine and its major metabolite N τ-methylhistamine are inaccurate and insensitive to the very low concentrations that exist in plasma samples. Therefore, an accurate and sensitive method for quantification in plasma has been developed using the stable isotope dilution assay with negative ion-chemical ionization mass spectrometry. For histamine, after the addition of [ 2H 4]histamine to 2 ml of plasma, the plasma sample is deproteinized, extracted into butanol, back extracted into HCl, derivatized to the pentafluorobenzyl derivative (CH 2C 6F 5) 3-histamine, purified on silica gel columns, and then quantified with negative ion-chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions m z 430 434 . For N τ-methylhistamine, after the addition of N τ-[ 2H 3]methylhistamine to 2 ml of plasma, the plasma sample is deproteinized, extracted into butanol, back extracted into HCl, derivatized to the heptafluorobutyryl derivative (C 3F 7CO 2) 2- N τ-methylhistamine, purified on silica gel columns, and then quantified with negative ion-chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions m z 497 500 . The precision of the histamine assay is 3.1% and the accuracy is 95.5 ± 2.5% while the precision of the N τ-methylhistamine assay is 1.9% and the accuracy is 106.8 ± 1.9%. The lower limits of sensitivity are 1 pg for histamine and 6 pg for N τ-methylhistamine injected on column. Using the assay in three normal human volunteers, plasma concentrations of histamine were 130, 92, and 85 pg/ml, and of N τ-methylhistamine were 229, 228, and 216 pg/ml. This assay provides a very sensitive and accurate method of quantitation of histamine and N τ-methylhistamine in plasma samples.

Plasma Neuropeptide Y Concentrations in Patients with Neuroendocrine Tumors

In order to develop an immunoradiometric assay for human neuropeptide (hNPY), a recently discovered and potent vasoconstrictor 36-amino-acid peptide, we used hNPY and some of its subpeptides to prepare monoclonal anti-NPY antibodies. Two monoclonal antibodies with high affinity for hNPY, that is affinity constants in the range of 10(10) mol/L-1, which respectively, reacted with the 9-18 portion and the 32-36 portion of hNPY were used in the immunoradiometric system. The assay was highly specific, NPY-related peptides such as pancreatic polypeptide and peptide YY not being detected. The lower limit of sensitivity was 0.5 pmol/L. In 303 normal subjects, plasma NPY concentrations were less than 0.5 pmol/L in 67%, 0.5 to 5.0 pmol/L in 25% and 5.1 to 30 pmol/L in the remaining 8%. A value of 7.5 pmol/L (95th percentile value in the normal group) was considered as the upper limit of normal. Among 111 patients with various neuroendocrine tumors, elevated plasma NPY concentrations were found in patients with pheochromocytomas and neuroblastomas, the highest plasma levels being found in patients with malignant pheochromocytomas. We conclude that patients with neuroendocrine tumors, especially secreting and or malignant tumors of the sympathochromaffin system, often have elevated plasma NPY concentrations.

Measurement of cAMP in Thermomonospora curvata by anion exchange HPLC

A method for quantitation of bacterial cyclic AMP was developed, based on integrator peak height after anion exchange HPLC fractionation. The lower limit of sensitivity and standard deviation with 10 μl samples was 0.4 nmol/ml and ±3.1%, respectively. The presence of a wide variety of other nucleotides did not interfere with the assay. Total culture cyclic AMP production measured by the assay during exponential growth of the filamentous actinomycete, Thermomonospora curvata, was 10–20-fold higher on polymeric substrates such as pectin and cellulose compared to easily utilized sugars (glucose and cellobiose). Enzymatic degradation of cyclic AMP could also be measured by the HPLC method; T. curvata cyclic AMP phosphodiesterase initial reaction rates were linear for about 20 min and proportional to enzyme concentration in mixtures incubated at pH 6.5 and 52°C.

Rapid Confirmation of Enzyme Multiplied Immunoassay Technique (EMIT®) Cocaine Positive Urine Samples by Capillary Gas-Liquid Chromatography/Nitrogen Phosphorus Detection (GLC/NPD)

A rapid gas-liquid chromatographic (GLC) method was developed for the confirmation of benzoylecgonine (BE) positive urine samples screened by the enzyme multiplied immunoassay technique (EMIT) assay. The procedure is performed by solvent extraction of BE from 0.1 or 0.2 mL of urine, followed by an aqueous wash of the solvent and evaporation. The dried residue was derivatized with 50 microL of pentafluoropropionic anhydride and 25 microL of pentafluoropropropanol at 90 degrees C for 15 min. The derivatizing reagents were evaporated to dryness, and the derivatized BE, and cocaine if present, were reconstituted and injected into the gas chromatograph. The column was a 15-m by 0.2-mm fused silica capillary column, coated with 0.25 micron of DB-1, terminating in a nitrogen phosphorus detector (NPD). Cocaine and the pentafluoro BE derivatives retention times were 3.2 and 2.6 min, respectively. Nalorphine was used as reference or internal standard with a retention time of 4.78 min. The complete procedure can be performed in approximately 1.5 h. The EMIT cutoff between positive and negative urine samples is 300 ng/mL of BE. The lower limit of sensitivity of this method is 25 ng of BE extracted from urine. Validation studies resulted in confirmation of 101 out of 121 EMIT cocaine positive urine samples that could not be confirmed by thin-layer chromatography (TLC). This represents 84% confirmation efficiency.

A Rapid HPLC Method for Determination of Imipenem in Plasma

Abstract A high performance liquid chromatographic method was developed for the determination of imipenem concentrations in plasma. This method involves stabilization of plasma with a mixture of 2-(N-morpholine)-ethanesulf onic acid and ethylene glycol (1:1). Samples are ultrafiltered using a membrane separation system and the ultrafiltrate injected directly onto a octyldecyl column. Chromatography is performed in the reverse-phase mode with a mobile phase of acetonitrile-phosphate buffer-triethylamine (pH 7.0). The lower limit of sensitivity was 1mcg/ml using UV detection at 300nm. Recovery and reproducibility results, and interferences from other therapeutic agents are presented and discussed. The assay procedure is applied to estimate pharmacokinetic parameters of imipenem in a thermally injured patient.

Rapid hydrophobic grid membrane filter-enzyme-labeled antibody procedure for identification and enumeration of Escherichia coli O157 in foods.

An O-antigen-specific monoclonal antibody, labeled by horseradish peroxidase-protein A, was used in a hydrophobic grid membrane filter-enzyme-labeled antibody method for rapid detection of Escherichia coli O157 in foods. The method yielded presumptive identification within 24 h and recovered, on average, 95% of E. coli O157:H7 artificially inoculated into comminuted beef, veal, pork, chicken giblets, and chicken carcass washings. In food samples from two outbreaks involving E. coli O157:H7, the organism was isolated at levels of up to 10(3)/g. The lower limit of sensitivity was 10 E. coli O157 per g of meat. Specific typing for E. coli O157:H7 can be achieved through staining with labeled H7 antiserum or tube agglutination.

Simultaneous estimation of nicotine and cotinine levels in biological fluids using high-resolution capillary-column gas chromatography combined with solid phase extraction work-up.

A rapid and sensitive capillary gas-chromatographic method with nitrogen-sensitive detection is reported for the simultaneous analysis of nicotine and cotinine levels occurring in the plasma, saliva, and urine of regular tobacco smokers. The proposed assay has a linear output, has satisfactory accuracy over the range of concentrations of both amines encountered in active smokers, and has also been successful in the analysis of the urine samples of passive smokers. Its lower limit of sensitivity is 0.2 ng of nicotine and 0.5 ng of cotinine per ml of plasma or saliva or per 100 microliters of urine. The beneficial characteristics of the presented method were achieved by the combination of solid phase extraction of 0.1-1.0 ml of fluid specimens, capillary column gas chromatography with splitless injection and nitrogen sensitive detection, and the use of separate, structurally analogous compounds as internal standards for nicotine. The suitability of the assay is shown by plasma concentration-time curves of nicotine and cotinine in a steady smoker during a 24 hours period.

Analyses of drugs by polarographic methods, XXXI. Electroanalysis of brotizolam, 2-bromo-4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-ss]-1,2, 4-triazolo[4,3-alpha]-1,4-diazepine.

AbstractBritton‐Robinson buffers (BRB), pH 4–9, provide for sufficient stability of brotizolam (1) and may be used as supporting electrolytes for its DCt polarography. The polarographic behaviour of 1 was investigated: The i‐E curves are markedly influenced by adsorption processes and cleavage of the C‐Br bond. In spite of these difficulties a precise electrochemical method for the determination of 1 could be developed. A BRB (pH 6.3) is best suited for the assay by DCt and DPP. The lower sensitivity limit is 10−6 mol ± L−1 with DCt polarography and 10−7 mol ±±±± L−1 with DPP. In the analysis of single 0.25 mg tablets the relative standard deviation Srel, is ± 0.76% (DCt) or ± 0.89% (DPP). Therefore, both polarographic methods are most suitable for the content uniformity test (USP XXI).

Stability of serum neuroleptic and prolactin concentrations during short- and long-term treatment of schizophrenic patients.

The potential importance of neuroleptic activity measures in the management of schizophrenia warrants attention to the methods for assessing neuroleptic bioactivity and stability of neuroleptic bioactivity over time. We have carried out measurements of serum neuroleptic and prolactin concentrations in 18 schizophrenic patients treated with haloperidol or thioridazine for up to 1 year. Serum neuroleptic levels were measured by a radioreceptor assay using porcine striatum. The lower limit of sensitivity of the assay was 0.6 ng haloperidol/ml, the intra- and interassay coefficients of variation 3 and 9%, respectively. A linear correlation was observed between haloperidol dose (5-30 mg/d) and serum neuroleptic activity (r = 0.706, P less than 0.001) and a curvilinear relationship between thioridazine dose (50-600 mg/d) and serum neuroleptic activity in schizophrenic outpatients. There was a positive correlation between serum neuroleptic and prolactin concentrations for the patients taking haloperidol (r = 0.620, P less than 0.001) or thioridazine (r = 0.542, P less than 0.001). In patients taking a constant dose of haloperidol or thioridazine for up to 1 year serum neuroleptic activity remained stable, suggesting the absence of metabolic tolerance; the observation of a decrease of 38 +/- 16% (mean +/- SD) in serum prolactin concentrations in patients treated with haloperidol but no prolactin decrease with thioridazine suggests that under certain neuroleptic treatment conditions a functional tolerance develops in the tuberoinfundibular dopamine system.

Determination of aspirin and salicylic acid in uremic patients' plasma using reversed-phase high-performance liquid chromatography.

A simple, rapid, and sensitive method for the determination of aspirin and salicylic acid in plasma of uremic patients is reported. After extraction with hexane, aspirin (ASA) and salicylic acid (SA) were quantified by high-performance liquid chromatography (HPLC) with ultraviolet detection at 229 nm. ASA and SA concentrations and peak-height ratios were linearly related up to 20 micrograms/ml. The lower limit of sensitivity was 0.1 microgram/ml for both compounds. The average recoveries of aspirin and salicylic acid were 27 and 54%, respectively. This procedure offers better performance than do previously described methods. The sample clean-up allows quantitation of a low concentration of aspirin in uremic patients' plasma, commonly rich in interfering endogenous substances.

Mercurated nucleic acid probes, a new principle for non-radioactive in situ hybridization

This report describes the localization of specific nucleic acid sequences in interphase nuclei and metaphase chromosomes by a new hybridocytochemical method based on the use of mercurated nucleic acid probes. After the hybridization a sulfhydryl-hapten compound is reacted with the hybrids formed. A number of such ligands were synthesized and tested. A fluorescyl ligand could be used for the direct visualization of highly repetitive sequences. For indirect immunocytochemical visualization trinitrophenyl ligands were found to be more sensitive than biotinyl analogues. These ligands were applied for the detection of target sequences in metaphase chromosomes and interphase nuclei of somatic cell hybrids, human lymphoid cell lines and blood cell cultures. The sequences were in the range of high to low copy numbers. The lower limit of sensitivity is indicated by the visualization of two human unique DNA fragments (40 and 15.6 kb) in human metaphases. The method is rapid, gives consistent results and can be used for both RNA and DNA probes. Other potentials of the new principle are discussed.

Histamine levels in bronchoalveolar lavage from patients with asthma, sarcoidosis, and idiopathic pulmonary fibrosis

Bronchoalveolar lavage (BAL) has been used extensively as a research tool to elucidate immunologic events occurring in the lower respiratory tract of patients with numerous diseases and, most recently, to study patients with asthma. We assessed mast-basophiloid cell numbers and histamine levels with a sensitive histamine assay, lower limit of sensitivity, 25 pg/ml, in BAL fluid from normal individuals (n = 9) and compared these results to those obtained from patients with sarcoidosis (n = 31), idiopathic pulmonary fibrosis (IPF) (n = 8), and mild asthma (n = 7). Patients with sarcoidosis demonstrated a significant increase in total BAL mast-basophiloid cells, 9.6 ± 4.1 × 10 4, compared to total cells in normal individuals, 0.0, p = 0.03, whereas only patients with IPF had significant elevations in BAL histamine levels. 1315 ± 737 pg/ml, versus levels in normal individuals, 161 ± 54 pg/ml, p = 0.002. A good correlation existed between histamine levels on an aliquot of lysed BAL cells and BAL histamine levels, R = 0.655 and p = 0.02, but not with either the total number or percent mast-basophiloid cells in BAL assessed on Wright's stained cytocentrifuge preparations. Subjects with asthma had both normal numbers of BAL mast-basophiloid cells and histamine levels. These data suggest that (1) BAL histamine levels are easily quantified, (2) the reason(s) for elevations in BAL histamine levels in IPF need further investigation, (3) BAL histamine levels in subjects with asthma are not elevated in those with mild and stable disease, and (4) lumenal mast-basophiloid cells are one major source of BAL histamine.

Studies on immunological assay of vitamin K dependent factors. I. Measurement of factor VII antigen by radioimmunoassay.

A radioimmunoassay (RIA) for factor VII was developed using 125I-factor VII, anti-factor VII rabbit serum and anti-rabbit IgG goat serum. The lower limit of sensitivity in normal reference plasma was 3 X 10(-3) units/ml. Although the level of factor VII antigen (VII:Ag) in normal plasma samples (n = 20) 0.944 +/- 0.176 units/ml, correlated with that of factor VII coagulant activity (r = 0.89), VII:Ag level in paired normal sera showed a higher value (1.469 +/- 0.376 units/ml). The level of antigen according to RIA of factor VIIa activated by factor Xa increased 2.5-fold compared with that of unactivated factor VII. It is suggested that the polyclonal anti-factor VII produced in a rabbit had higher affinity for factor VIIa than for factor VII. In two out of seven patients with congenital factor VII deficiency, VII:Ag was detectable (0.04, 0.31 units/ml, respectively) whereas VII:C was less than 0.01 units/ml. In 12 warfarin-treated individuals, VII:C showed a lower level (0.121 +/- 0.063 units/ml) that that of VII:Ag (0.518 +/- 0.186 units/ml). During 4 weeks observation after stopping warfarin, VII:C and VII:Ag reached normal levels in 1 week. However, VII:C did not reach equivalence with VII:Ag until 4 weeks had elapsed.

Liquid-chromatographic assay of dexamethasone in plasma.

We report a simple, efficient method for removing dexamethasone from plasma by use of solid-phase extraction columns. The dexamethasone is then quantified by "high-performance" liquid chromatography with ultraviolet detection at 254 nm. Dexamethasone concentrations and peak-height ratios were linearly related over the range 5 to 200 micrograms/L. The lower limit of sensitivity was 5 micrograms/L. The average recoveries of dexamethasone and methylprednisolone (internal standard) were 72 and 71%, respectively. This procedure offers improved efficiency over that of previously described methods by decreasing analytical time and improving sample clean-up. Sensitive, specific, and relatively inexpensive, this method is suitable for most clinical applications.

Failure to detect amodiaquine in the blood after oral administration.

Plasma and red blood cell concentrations of amodiaquine (AM) and its metabolite, desethylamodiaquine (DAM), were monitored at intervals up to 21 days after a single oral dose of 600 mg amodiaquine in five normal African subjects. Concentrations of AM and DAM were determined using a high performance liquid chromatography technique with a lower limit of sensitivity of 10 ng ml-1 for both compounds. AM was not detectable in plasma and red blood cell samples withdrawn from any of the subjects. DAM was detectable in both media. It appeared in the blood 0.5-1 h after administering AM, reached a peak in 1.5-2 h and subsequently declined slowly. We conclude that AM is rapidly converted to DAM and its blood concentration, compared with that of DAM, is very low if not negligible.

A rapid method for the determination of blood morphine concentration suitable for use in studies involving acute and chronic pain

This communication provides an improved sample preparation method for blood morphine concentration determination prior to high-performance liquid chromatography analysis with electrochemical detection. The lower limit of sensitivity is 1 ng ml of whole blood, and the standard curve is linear to an upper limit of 500 ng ml . The coefficient of variation of replicate determinations of morphine at concentrations of 10 and 400 ng ml were 4.7 and 2.5%, respectively. This assay procedure is rapid and accurate and allows the large number of samples (usually 30–40 samples) generated by pharmacokinetic studies in acute and chronic pain to be conveniently analyzed in one day.