Mass spectrometric quantification of urinary 6-sulfatoxymelatonin: age-dependent excretion and biological variation

ObjectivesRegulators of circadian rhythm, including melatonin, influence fundamental biological processes. Measuring the melatonin metabolite 6-sulfatoxymelatonin in urine can estimate melatonin production. 6-sulfatoxymelatonin is mainly analyzed by immunoassays, but these methods are hampered by cross-reactivity and poor reproducibility when used to analyze small molecules. Therefore, we validated a high-throughput liquid chromatography with tandem mass spectrometry (LC–MS/MS) method to quantify 6-sulfatoxymelatonin in urine. We evaluated age-dependent 24-h excretion of 6-sulfatoxymelatonin into urine and the biological variation of urinary excretion in healthy individuals.MethodsThe online solid phase extraction method combined with LC–MS/MS was validated according to international guidelines, and used to measure the excretion of 6-sulfatoxymelatonin into urine of 240 healthy individuals. Biological variation of 6-sulfatoxymelatonin excretion was examined in 10 healthy individuals.ResultsUrinary 6-sulfatoxymelatonin results were well within the validation criteria (interassay coefficient of variation: <5.4%, quantification limit: 0.2 nmol/L). There was an age-related decrease in 6-sulfatoxymelatonin excretion into 24-h urine [F(5, 234)=13.9; p<0.001]. Within-subject variation of 6-sulfatoxymelatonin was 39.2% in day urine, 15.1% in night urine, and 12.2% in 24-h urine. Between-subject variation was 39.1% in day urine, 37.9% in night urine, and 36.8% in 24-h urine.ConclusionsThis MS-based method enables straightforward, reproducible, and sensitive quantification of 6-sulfatoxymelatonin in urine. Urinary 6-sulfatoxymelatonin levels decreased with age. Biological variation of 6-sulfatoxymelatonin excretion into urine was high between subjects and lower within subjects, indicating that repeated measurements of 6-sulfatoxymelatonin in 24-h urine are needed in future studies.

Enzyme-Linked Immunosorbent Assay for the Determination of Five Organophosphorus Pesticides in Camellia Oil

A matrix solid-phase dispersion and direct competitive enzyme-linked immunosorbent assay (MSPD-ELISA) was developed for five organophosphorus pesticides (OPs) in camellia oil. Seven haptens with different substituents in the aromatic ring were used to prepare different competitors; the ELISA showed highest sensitivity and specificity to OPs when the competitor had moderate heterology to the immunizing hapten. Several assay conditions were optimized to increase the ELISA sensitivity. The optimized ELISA for five OPs had 50% inhibitory concentrations of 6.3 ng/ml (parathion), 18.9 ng/ml (methyl parathion), 120.7 ng/ml (fenitrothion), 110.4 ng/ml (fenthion), and 20.7 ng/ml (phoxim). The average recoveries of five OPs in camellia oil ranged from 75.7 to 105.3%, with the interassay coefficient of variations ranging from 6.0 to 13.4%. Compared with the results previously reported, the ELISA that was developed in the present study showed a much higher sensitivity. Additionally, MSPD was used in the sample preparation to minimize the matrix effect. Recoveries from the method developed here were in agreement with those obtained by gas chromatography, which indicated that the detection performance of the MSPD-ELISA could meet the regulatory requirements of different governments and international organizations.

Quantitative analysis of chloramphenicol residues in shrimp muscle tissues by Chemiluminescent enzyme immunoassay

A competitive indirect chemiluminescent enzyme immunoassay (ic-CLEIA) has been developed for the determination of chloramphenicol (CAP) residues in shrimp. After the optimisation of four physico-chemical parameters, i.e. incubation time, concentration of Tween-20, concentration of PBS and its pH, the method developed gave a limit of detection of 0.01 ng/ml and a detection range from 0.03 ng/ml to 23.7 ng/ml, with an ED₅₀ of 0.47 ng/ml. The developed method has been validated on spiked shrimp samples in terms of precision (intra- and interassay coefficient variations of less than 10% and 15%, respectively), and of accuracy (mean recovery from 95% to 123%). All these parameters being better than those of the ELISA method which is widely used to detect chloramphenicol, it may be suggested that the CLEIA method can be used to detect aquatic samples instead of ELISA. &nbsp;

Radioreceptor assay of an endothelin A receptor antagonist in plasma and urine

Orally active nonpeptide antagonists of endothelin (ET) receptors may prove beneficial in the treatment of cardiovascular and renal disease. The pharmacodynamics and pharmacokinetics of these drugs are not sufficiently known, and practical methods for their analysis have not been developed. We describe a simple, sensitive, and reproducible radioreceptor assay (RRA) for LU135252, a selective antagonist of the ETA receptor, using porcine aortic smooth muscle membranes as the acceptor and 125I-endothelin-1 as the ligand. With methanol extraction of plasma and urine samples, recovery of LU135252 ranged from 79% to 91% at 60–1000 nmol/L. The logit-log transformed calibration curves constructed with LU135252 added to plasma or to urine were linear (r = 0.993 ± 0.005, n = 11) in the range from 18.7 to 2400 nmol/L. The detection limit with plasma- and urine-based calibration curves was 19 nmol/L. The interassay coefficient of variation was 12.6% at 70 nmol/L (n = 9) and 6.5% at 590 nmol/L (n = 9). Endothelin-1 did not interfere in the RRA at pathophysiologically and clinically relevant concentrations [up to 15 pmol/L (40 pg/mL)]. When LU135252 was added to plasma, the signal was completely stable after storage for 1 week at 4 °C, although there was a modest loss of the signal after 24 h at room temperature. The practical performance of this RRA was then tested in plasma samples obtained from (a) rats after a single oral administration of LU135252, (b) from coronary-ligated rats chronically treated with LU135252, and (c) in plasma and urine samples obtained from dogs during intrarenal infusion of LU135252.

Radioimmunoassay of inhibin in the serum of male monkeys

ABSTRACT A heterologous inhibin radioimmunoassay method to measure inhibin in serum of male cynomolgus (Macaca fascicularis) and rhesus (Macaca mulatta) monkeys has been validated using a specific antibody against bovine 31 kDa inhibin and 125I-labelled 31 kDa inhibin as tracer. A serum pool from male monkeys was used as standard. Serial dilutions of normal monkey serum showed parallel logit–log dose–response curves to purified porcine and bovine inhibin as well as to a female human serum pool. The intra-assay coefficient of variation was 4·2% (n=10) and the interassay coefficient of variation 5·1% (n=10). No loss of inhibin immunoactivity was noted after storage at 23 °C for 5 days or repeated thawing and freezing of the serum samples. Serum from castrated monkeys showed undetectable levels of inhibin. Treatment with a gonadotrophin-releasing hormone agonist for 15 weeks led to a marked suppression of peripheral serum inhibin to concentrations similar to those after hypophysectomy or pituitary stalk section. Journal of Endocrinology (1989) 122, 477–483

A two-site immunoenzymometric assay of 52-kDa pro-cathepsin D, and its use in human breast diseases.

After isolating monoclonal antibodies specific for the 52-kDa precursor of cathepsin D (cath-D), which is secreted in excess in both hormone-dependent and hormone-independent breast cancer, we developed a two-step double-determinant immunoenzymometric assay that is specific for this pro-enzyme. The assay combines the use of a monoclonal antibody specific for the precursor and bound to microtiter plates, and a second antibody directed against a smaller processed form of the mature enzyme, coupled to alkaline phosphatase. The specificity of the assay relies on separate and sequential additions of the antigen and the conjugated second antibody. It allows rapid measurement of the analyte in plasma and cytosols of normal and neoplastic mammary tissues, with a detection limit of 5 fmol and a maximal interassay coefficient of variation of 9%. This assay is particularly useful for tissue cytosol samples where the pro-enzyme form co-exists with large quantities of the mature processed forms of the enzyme. Comparative assays of 52-kDa pro-cath-D and total cath-D in cytosols of breast cancers and benign mastopathies indicate that the present assay better discriminates between benign and cancerous mammary tumors.

Nonisotopic "sandwich" immunoassay of thyroglobulin in serum by the biotin-streptavidin technique: evaluation and comparison with an immunoradiometric assay.

In this sequential assay, the thyroglobulin in serum binds to polystyrene beads coated with two mouse monoclonal antibodies. These beads then react with a rabbit polyclonal antibody, biotinylated sheep anti-rabbit IgG, streptavidin-horseradish peroxidase, and a peroxidase substrate to yield a colored product that is measured spectrophotometrically at 492 nm. The range of the standard curve is 1.6 to 100 micrograms/L. The detection limit of the assay is 1.2 micrograms/L. The interassay coefficient of variation is 14.0% at 6.2 micrograms/L, 5.7% at 32.7 micrograms/L; the intra-assay CVs range from 7.8% to 14.8%. The reference intervals are 2.7 to 42.1 micrograms/L for euthyroid persons and less than or equal to 5 micrograms/L for athyreotic patients not on thyroxin replacement therapy. Some autoantibodies to thyroglobulin cause thyroglobulin values to be falsely low. The concentration of autoantibodies is not correlated with the analytical recovery of human thyroglobulin. The coefficient of correlation between the measurement of thyroglobulin by this assay and by an immunoradiometric assay was 0.969 for 186 autoantibody-negative samples, 0.963 for 37 autoantibody-positive samples.

Time-resolved immunofluorometric assay of sex-hormone binding globulin.

A time-resolved immunofluorometric assay (trlFMA) for human sex-hormone binding globulin (SHBG) is described in which antibody-coated tubes or microliter strip-wells and a europium (Eu) chelate-labeled monoclonal antibody are used. The trlFMA sensitivity is similar to that of other SHBG immunoassays, and other analytical variables compare favorably with an SHBG immunoradiometric assay (IRMA) kit and a steroid binding capacity assay: the interassay coefficient of variation (CV) is less than 8% and the intra-assay CV is less than 6% for concentrations between 6 and 200 nmol/L. The reference intervals (means +/- SD) for SHBG concentrations (nmol/L) in serum from 10 men, 10 women, and 10 pregnant women were 23 +/- 12, 65 +/- 39, and 439 +/- 122, respectively. In 14 hirsute women the mean +/- SD serum SHBG concentration (37 +/- 21 nmol/L) was significantly lower (P less than 0.01) than the mean for an age-matched, nonhirsute female comparison group. The trlFMA is technically simple, requires no centrifugation or separation reagent, and takes a counting time of only 1 s. In addition, the Eu-label is nontoxic, presents no waste-disposal problems, and has a long shelf life.

Improved Enzyme-Linked Immunosorbent Assay for Aflatoxin Bj in Agricultural Commodities

An improved enzyme-linked immunosorbent assay (ELISA) for aflatoxin B, in cornmeal and peanut butter was developed. Aflatoxin B, in cornmeal and peanut butter samples was extracted with 70% methanol in water containing 1% dimethylformamide diluted with assay buffer to a final concentration of 7.0% methanol, and directly subjected to an ELISA procedure that took less than 1 h for quantitative analysis and less than 30 min for screening tests. Analytical recoveries for 5-100 ppb B, added to the cornmeal and peanut butter were 91 and 95.4%, respectively. The interwell and interassay coefficient of variation was 10% or less at the 20 ppb level and above. Agreement for B, levels in more than 30 naturally contaminated corn, mixed feed, and peanut butter samples was excellent between the ELISA data and the data obtained from different independent laboratories using TLC or other analytical methods

Development and characterization of a radioimmunoassay to measure human tissue kallikrein in biological fluids

ABSTRACT A direct radioimmunoassay has been developed to measure tissue kallikrein in human biological fluids. These fluids include serum, plasma, urine, pancreatic juice and saliva. Purified kallikreins from human urine and human saliva were used to raise rabbit antibody and each was labelled with Na125I for use in the radioimmunoassay. A comparison of the different antigen-antibody systems was then made. Bound and free enzyme were separated by a double-antibody technique. The usable range of the standard curve was from 2·5 to 100 μg kallikrein/l. The intra-assay coefficient of variation was 4·7%, the interassay coefficient of variation 8·9% and the recoveries of purified kallikrein added to the samples were 99·3, 96·0, 110·8 and 81·2% for urine, saliva, serum and plasma respectively. Parallel dilution curves were obtained for serum and plasma, as well as for urine, saliva and pancreatic juice. However, plasma anticoagulated with EDTA or heparin gave consistently lower values than serum, when measured in the radioimmunoassay. From eight different subjects plasma (EDTA) values were on average 50% lower than those of serum. Experiments designed to determine the cause of this difference revealed that treatment of blood with some anticoagulants, in particular heparin and EDTA, resulted in a marked reduction in the amount of measurable tissue kallikrein. J. Endocr. (1984) 101, 173–179