Extracts Of Human Urine Research Articles

Complete separation of ephedrines by graphite-layer open-tubular (GLOT) column

The preparation of graphite-layer capillary column is described together with their application to the analysis of aliphatic and aromatic amines. Attention is mainly focused on the separation of ephedrines which, being sympathomimetic amines, are often present in pharmaceutical preparations, but are also illegally used by athletes as stimulants. Complete separation of these compounds, using direct GC-NPD analysis of human urine extract, without derivatisation, has been obtained.

Quantitating Staphylococcal Enterotoxin B in Diverse Media Using a Portable Fiber-Optic Biosensor

A new, portable fiber-optic biosensor has been used to detect staphylococcal enterotoxin B, a causative agent of food poisoning, at levels as low as 0.5 ng/ml in buffer. The toxin (SEB) can also be detected and quantitated in other relevant media: human serum, urine, and aqueous extract of ham. The level of toxin, from 5 to 200 ng/ml, can be accurately predicted in these media by calibrating each fiber and by comparing results to a single standard curve based on toxin in buffer. The quantitative fluorescent sandwich immunoassay provides results in 45 min; qualitative results are provided in 15–20 min. Using a blender and a benchtop centrifuge, fast, simple aqueous extracts of contaminated ham samples were prepared and tested. Ham spiked with 5 or 40 μg SEB per 100 g food resulted in biosensor readings indicative of 11 or 69% recovery of the toxin, respectively. Finally, the SEB assay is highly specific; SEA and SED give only 2–3% of the signal at 5000 ng/ml as SEB gives at 1000 ng/ml. This specific, sensitive assay for SEB on the portable fiber-optic biosensor permits easy monitoring of clinical samples or on-site analysis of suspect food samples.

Identification of non-steroidal anti-inflammatory drugs and their metabolites in solid phase extracts of human urine using capillary electrophoresis–mass spectrometry

Download options Please wait... Article information DOI https://doi.org/10.1039/AI9953200459 Article type Paper Download Citation Anal. Proc., 1995,32, 459-462 BibTex EndNote MEDLINE ProCite ReferenceManager RefWorks RIS Permissions Request permissions Identification of non-steroidal anti-inflammatory drugs and their metabolites in solid phase extracts of human urine using capillary electrophoresis–mass spectrometry A. E. Ashcroft, H. J. Major, S. Lowes and I. D. Wilson, Anal. Proc., 1995, 32, 459 DOI: 10.1039/AI9953200459 To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page. If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given. If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page. Read more about how to correctly acknowledge RSC content. Search articles by author Alison E. Ashcroft Hilary J. Major Stephen Lowes Ian D. Wilson

Blunted natriuretic response to human urine extracts with Na +, K +-ATPase inhibiting activity in experimental cirrhosis

Human urine and plasma extracts contain a material that inhibits the enzyme Na+,K(+)-ATPase (the endogenous sodium pump) and produces natriuresis in the bioassay animal. This endogenous sodium pump inhibitor(s), also known as digitalis-like factor, is thought to be involved in sodium and extracellular fluid volume homeostasis. Increased urine and plasma sodium pump inhibiting activity have been reported in patients with cirrhosis and sodium retention. The aim of the study was to assess the renal response to i.v. administration (0.2 ml/min per kg bw for 10 min) of a human urine extract containing sodium pump inhibiting activity (28.5 nmol equivalent ouabain/ml) in eight conscious rats with cirrhosis and ascites and eight control rats. Baseline urinary excretion of Na+,K(+)-ATPase inhibiting activity was significantly higher in cirrhotic rats with ascites than in control rats (235 +/- 40 vs 91 +/- 16; p < 0.01). Human urine extract induced a significant (p < 0.05) increase in glomerular filtration rate in control (3.2 +/- 0.4 to 4.2 +/- 0.5 ml/min) and cirrhotic rats (3.0 +/- 0.3 to 4.0 +/- 0.5 ml/min). In control rats it also increased urinary sodium excretion (1.47 +/- 0.22 to 2.43 +/- 0.5 microEq/min, p < 0.01) and fractional sodium excretion (0.29 +/- 0.01 to 0.43 +/- 0.04%, p < 0.025). In contrast, in cirrhotic rats with ascites neither sodium excretion nor fractional sodium excretion was significantly affected. No changes were observed in plasma aldosterone and atrial natriuretic peptide concentrations in either group. These data suggest that in cirrhosis there is a renal resistance to the natriuretic effect of endogenous sodium pump inhibitor(s).

Tandem mass spectrometric approaches for the analysis of alkylguanines in human urine

AbstractHuman exposure to carcinogenic alkylating agents can lead to the formation of covalently bound adducts in DNA, some of which are excreted in urine as alkylated purines following DNA degradation and repair. Tandem mass spectrometric methods have been developed for the qualitative and quantitative determination of such alkylpurines in human urine. Short‐chain alkyl‐ and hydroxyalkylguanines have been synthesized with the substituents at the N‐7‐, O6‐ and N2‐positions of guanine. Examination of the product ion scans of their molecular ions (electron impact (EI) ionization) revealed that the ion at m/z 151, [guanine]+, was common to all of the alkylguanines studied, with the exception of the methylated analogues. Precursor ion scans of this ion on partially purified human urine extracts showed the presence of several ions (e.g. m/z 179, 195) which were consistent with molecular ions for alkylguanines. The presence of these and other constituents was confirmed by product ion spectra of molecular ions (EI and fast atom bombardment), and by high‐performance liquid chromatographic separation prior to tandem mass spectrometry (MS/MS). Evidence was obtained for the presence of N‐7‐methyl‐, N2‐dimethyl‐, N2‐dimethyl‐, N2‐ethyl‐ and N‐7‐(2‐hydroxyethyl)guanine. Quantitative methods were established for these five alkyl guanines using gas chromatography mass spectrometry (GC/MS) and GC/MS/MS. Deuterated internal standards were synthesized and added to the urine prior to extraction of alkylpurines by Sep‐Pak cartridge chromatography. The products were converted into their tert‐butyldimethylsilyl derivatives and analysed by selected ion monitoring (SIM) of [M – 57]+ or by multiple reaction monitoring (MRM) of the fragmentation M+˙ → [M – 57]+. The MRM method yielded values for N‐7‐methylguanine of 2.57 ± S.D. 1.32 mg day−1 (n = 6), N2‐methylguanine of 0.31 ± 0.10 mg day−1 (n = 10) and N2‐dimethylguanine of 0.21 ± 0.23 mg day−1 (n = 10). N2‐Ethyl‐ and N‐7‐(2‐hydroxyethyl)guanine could only be detected by SIM at levels of ∼0.5 and 2 μg day−1, respectively. The MRM analyses, although inherently less sensitive than the SIM analyses, exhibit greater selectivity and consequently fewer contaminant ions.

Quantitative determination of N-acetyl(-L-)cysteine derivatives in human urine by tandem mass spectrometry.

Collision-induced dissociation (CID) methods are described for the quantification of nanogram per millilitre (ppb) concentrations of 2-acetamido-3-(3'-hydroxypropylthio)-propanoic acid (I) and 2-acetamido-3-phenylthiopropanoic acid (II) in human urine extracts. I and II are potential detoxification products of acrolein and benzene in conjugation with N-acetyl(-L-)cysteine derived from glutathione. We have studied the potential of tandem mass spectrometry (MS/MS) under electron impact (EI) and chemical ionization (CI) conditions as a confirmatory screening technique for these compounds. Our main goals were high selectivity and low detection limits along with little or no sample clean-up. The effects of the mode of ionization and of collision conditions on the CID spectra have been investigated. Direct insertion probe without any derivatization or short-column gas chromatographic separation techniques are used. Total instrument and data analysis time is about 15 min for direct insertion probe MS/MS and about 30 min for short-column gas chromatography (GC)/MS/MS. Detection limits are: direct insertion probe MS/MS (EI mode), 50 ppb (100 pg) for compound I; short-column GC/MS/MS (EI mode), 1.5 ppb (5 pg) for compound II; and short-column GC/MS/MS (CI mode), 0.6 ppb (2 pg) for the methyl ester of compound II. Results are compared with non-mass spectrometric methods. The MS/MS methods were applied for the determination of I (EI mode) and II (CI mode) in urinary samples of a smoker and eight non-smokers. After smoking, the urinary levels of I and II were elevated, whereas no increase was observed after experimental passive smoking.

Substances in human urine that strongly inhibit bacterial mutagenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and related heterocyclic amines.

Extracts of human urine were shown to contain substances that strongly inhibited the liver S9-mediated mutagenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in Salmonella typhimurium TA98 strain in a liquid incubation assay. The inhibitory effect was unrelated to cytotoxicity and was similar with urine extracts from smokers and non-smokers. Under similar assay conditions, the mutagenicity of the related amino-imidazoazaarenes, 2-amino-3-methyl-imidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]-quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline was also found to be strongly inhibited by urine extracts. Decreased or enhanced mutagenicity was seen with 2-acetyl-aminofluorene and 2-aminoanthracene depending on the type of assay, and the time of incubation in liquid medium. A weak inhibition of the mutagenicity of 2-nitrofluorene, a direct-acting mutagen, was observed only after a short incubation time. Mutagenicity of 4-nitroquinoline N-oxide was not altered by the presence of urine extracts at concentrations shown to be inhibitory for the mutagenicity of heterocyclic aromatic amines. Our data suggest that the inhibitory substances in urine act through their capacity to non covalently bind the parent heterocyclic and aromatic amines, thus affecting their availability in aqueous medium for diffusion into liver microsomes where metabolic activation takes place.

Identification of caffeine and its metabolites in human urine extracts by electron impact ionization tandem mass spectrometry

AbstractElectron impact ionized purine bases such as caffeine and its metabolites (theopbylline, theobromine, paraxanthine, 1‐methylxanthine, 3‐metnybumthine and 7‐methyIxanthine) were characterized by tandem mass spectrometry. The direct analysts of human urinary extracts by tandem mass spectrometry allows the unambiguous identification of caffeine and its metabolites present simultaneously in the urine.

MICs of ciprofloxacin and trimethoprim for Escherichia coli: Influence of pH, inoculum size and various body fluids

The influence of pH, inoculum size, human urine and prostatic extract on the MICs of ciprofloxacin and trimethoprim for Escherichia coli was investigated. There was no influence by the bacterial inoculum size within wide ranges on either drug. An increase in pH had a variable influence on the MICs of trimethoprim for E. coli but lowered those of ciprofloxacin considerably. Human prostatic extract increased the trimethoprim MIC for E. coli but lowered those of ciprofloxacin as compared to Mueller Hinton broth. Human urine increased the MICs of both drugs for E. coli.

骨肉腫に対するＡｎｔｉｎｅｏｐｌａｓｔｏｎ Ａ１０の抗腫よう効果について

Antineoplaston A 10 (3-phenylacetylamino-2, 6-piperidinedione) is a modified dipeptide analogue originally isolated from extracts of human urine. Antineoplastic effect of antineoplaston A 10 was tested in tissue culture of osteosarcoma. Antineoplaston A 10 produced a cytostatic effect at 16mg/ml and a cytocidal effect at 32mg/ml. Antineoplaston A 10 is virtually devoid of toxicity even at very high dosages and does not appear to attack normal cells. It is very attractive as an new agent for chemotherapy of osteosarcoma.

Endothelin-1-Like Immunoreactivity in Human Urine

By using a radioimmunoassay specific for endothelin-1 (ET-1), we studied whether ET-1-like immunoreactivity (LI) is present in human urine. Significant amounts of ET-1-LI were present in human urine, and the daily urinary excretion of ET-1-LI in 30 normal subjects was 67.6 +/- 35.5 ng/day. The mean urinary excretion of ET-1-LI determined in spot urine was 82.8 +/- 38.2 pg/mg creatinine (n = 30), while the mean plasma concentrations of ET-1-LI in the same individuals were 1.1 +/- 0.5 pg/ml. There was no significant correlation between urinary ET-1-LI excretion and its plasma levels. Reverse-phase high-pressure liquid chromatography of human urine extract revealed a major ET-1-LI component coeluting with standard ET-1. The present study demonstrates the presence of ET-1-LI excreted in human urine, although its exact source and physiological significance in renal tissues remain to be determined.

Analysis of methylated and oxidized purines in urine by capillary gas chromatography-mass spectrometry

Methylated purine bases from urine can be quantitated by capillary gas chromatography-mass spectrometry with selected ion monitoring. The method is based on a separation of the purines in urine using XAD-2 column chromatography and bonded-phase extraction methods. The extracted samples are derivatized with N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide and analyzed by capillary gas chromatography-mass spectrometry. The tert-butyldimethylsilyl derivatives of the purines showed excellent GC and GC/MS properties suitable for low-level detection in extracts of human urine as well as in urine extracts from animals treated with the methylating carcinogen N-nitrosodimethylamine. The oxidized purine bases 7-methyl-8-hydroxyguanine, 8-hydroxyadenine, and 8-hydroxyguanine were also detected in urine. To our knowledge, the latter two modified bases have not been previously reported in normal urine.

Induction of SOS response in Escherichia coli strain PQ37 by 16 chemical compounds and human urine extracts

The SOS Chromotest on Escherichia coli strain PQ37 was used to detect DNA damage induced by 16 chemical compounds and urine samples from smokers and a non-smoking psoriatic patient treated with mineral coal tar. The results confirmed the strong SOS inducing activity of 2-aminoanthracene and benzo[a]pyrene with metabolic activation and N-methyl-N'-nitro-N-nitrosoguanidine, mitomycin C and 4-nitroquinoline-N-oxide without metabolic activation. A weaker response in the absence of microsomal enzymes was observed with hydroxyurea (only at high doses) and the soluble Cr(VI) compounds potassium chromate and potassium dichromate. No effect was observed with ampicillin, cadmium chloride, cyclophosphamide, griseofulvin, the insoluble Cr(VI) compound lead chromate, the soluble Cr(III) compounds chromium nitrate, chromium chloride, chromium potassium sulphate, and the chelating agent sodium nitrilotriacetate. Among the Cr(III) compounds only chromium acetate produced a low but significant increase of SOS inducing activity. Solubilization by nitrilotriacetate of genotoxic Cr(VI) from insoluble lead chromate was observed, whereas no interaction occurred between nitrilotriacetate and the soluble Cr(VI) and Cr(III) compounds. Using urinary XAD-2 extracts, we found the SOS Chromotest poorly sensitive to the mutagens present in urine from tobacco smokers which, on the other hand, were detected by the gene mutation assay in Salmonella typhimurium (Ames test). A urine sample obtained from a psoriatic patient, therapeutically treated with mineral coal tar, had a significant SOS inducing activity with and even without metabolic activation, whereas in the Ames test it was active only in the presence of metabolic activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Liquid chromatography—mass spectrometry in bioanalysis: Some applications and a new approach for non-volatile buffer systems

Combined liquid chromatography mass spectrometry (LC/MS) is still a rapidly developing area. The advantages and limitations of the most important interfaces, i.e. the moving belt interface, the thermospray interface, and continuous-flow fast atom bombardment, are discussed from a bioanalytical point of view. The discussion is underlined by two bioanalytical applications of LC/MS using a moving belt interface. The first example is the identification of unknown compounds in alkaline extracts of post-mortem human urine and plasma. The second example is actually concerned with a new approach in LC/MS: phase-system switching using a short column between the LC chromatography and the moving belt interface. The perspectives of the latter are outlined.

Characterization of a high-molecular-weight form of epidermal growth factor in an extract of human urine

We purified from a side fraction of the commercial preparation of urokinase from large volumes of human urine a high-molecular-weight (HMW) form of human epidermal growth factor (hEGF). Sequence analysis of the amino terminus of the intact molecule and of two tryptic fragments and carboxypeptidase Y analysis revealed the molecule to correspond to residues 828–1023 of the hEGF precursor predicted by the nucleotide sequence of human renal hEGF mRNA, with hEGF forming its carboxyl terminus. HMW hEGF bound poorly to concanavalin A-agarose, quite avidly to wheat germ lectinagarose, and completely to phenyl boronate-agarose, suggesting that it was O-glyco-sylated. Sephacryl S-200 chromatography of freshly-voided urine revealed mostly hEGF, with smaller amounts of a much higher molecular weight hEGF, but little material that was the size of the HMW hEGF we characterized. The large fragment we characterized presumably is cleaved from the larger form by enzyme(s) present in urine during the collection, storage, and processing of urine. We have confirmed that hEGF is synthesized as a large precursor molecule, as predicted by the nucleotide sequence of hEGF mRNA.

Characterization of digoxin and related cardiac glycosides by fast atom bombardment mass spectrometry

The potential of using fast atom bombardment mass spectrometry for the detection and the characterization of digoxin and a series of related cardenolide analogues was investigated. The spectra were dependent upon the type of support-matrix in which they were recorded; thioglycerol proved to be satisfactory for the characterization of digoxin and allowed for its detection in human urine extract spiked with ca 11 ng ml −1.

Inhibition of follicle-stimulating hormone/diethylstilbestrol-stimulated ovarian growth by extracts of pregnancy urine.

We devised an in vivo biological assay for ovarian growth inhibiting activity to examine extracts of human pregnancy urine for the presence of ovarian growth inhibiting factor. Diethylstilbestrol (DES) capsules were implanted sc in immature hypophysectomized female rats; FSH was injected sc with or without test substance for 5 days. Rats with unstimulated ovaries were implanted with blank capsules and given the vehicle without FSH. Twenty four hours after the last injection, the ovaries were removed and weighed. The ovarian growth inhibition of the ovarian weight gain achieved in rats treated with DES and FSH. Crude commercial human CG (hCG) preparations, extracted from pregnancy urine, were chromatographed on Sephadex G-100, and the fractions were tested for ovarian growth inhibiting activity. The peak of ovarian growth inhibiting activity was found in fractions eluting from the column in the mol wt range of 12,000-20,000. Ovarian growth inhibiting activity was heat sensitive, not extracted by ether, and precipitated by acetone. Ovarian growth inhibiting activity was stable in acid at pH 2, but was inactivated by digestion with trypsin. The ovarian growth inhibiting activity was purified by chromatography on Concanavalin A-Sepharose and diethylaminoethyl-Sephacel. The active material contained hCG alpha, hCG beta, and beta-carboxyterminal peptide-immunoreactivity and its inhibiting activity could be removed from solution by immunoadsorption with antisera specific for hCG beta. The ovarian growth inhibiting activity was further purified on an anti-hCG alpha-immunoglobulin G affinity column. The activity was eluted from the affinity column at low pH, and eluted material contained all of the immunodeterminants of hCG. Virtually identical dose-response curves of ovarian inhibition were obtained using equivalent doses of beta-carboxyterminal peptide immunoreactivity of purified inhibitor and purified hCG (CR123). The inhibiting activity reached plateau of 80-90% at doses of 50-100 ng hCG/rat. Upon rechromatography on Sephadex G-100, the ovarian growth activity that was pooled from fractions corresponding to the 12,000-20,000 mol wt range was recovered in fractions corresponding to the elution position of hCG. We conclude that the low mol wt inhibiting activity observed in the crude pregnancy extracts is due to hCG and that hCG is a very potent inhibitor of FSH/DES stimulation of ovarian growth.

Partial purification and characterization of epidermal growth factor in human breast milk.

Human epidermal growth factor (hEGF), a potent growth stimulator of many tissues in culture, has been isolated from human urine and subsequently identified in many human biological fluids including breast milk. In this study, partial purification and characterization of hEGF-like substance(s) in human milk were performed using homologous hEGF radioimmunoassay (RIA) and radioreceptor assay (RRA). hEGF-like material(s) was extracted from pooled human milk by ethanol precipitation, followed by adsorption to cation- and anion-exchange resin. DEAE-Sephadex G-25 ion-exchange chromatography of human milk extracts revealed three major components with hEGF activity (peak I, II, III) eluted with a linear gradient by ammonium acetate. The competitive binding curves for these components were parallel to those for standard hEGF in both RIA and RRA. The apparent molecular weight of peak I was approximately 6,500 and that of peak II and III was approximately 7,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The pI value for peak I was approximately 4.5 and that for peaks II and III was approximately 5.0 by isoelectric focusing. These data are comparable to the size and charge heterogeneity of hEGF in human urine extracts. In conclusion, the major components of hEGF in human milk appear to be physicochemically, immunologically and biologically (receptor binding activity) indistinguishable from hEGF of urinary origin.

Identification of conjugated metabolites of antipyrine in urine by pyrolysis-electron-impact mass spectrometry

Pyrolysis-electron-impact mass spectrometry can be employed as a fast and sensitive method for the identification of unconjugated and conjugated metabolites of antipyrine at different stages of the work-up procedure. The method avoids derivatization of the highly polar conjugates, generally a basic requirement for electron-impact investigation. Using crude extracts of human and rat urine, endogeneous products of the metabolic process did not interfere with the studied metabolite. It is demonstrated that all phase-I metabolites so far described in the literature can be thermally liberated from their corresponding conjugates and subsequently identified by electron-impact mass spectrometry. The isomeric main metabolites 3-hydroxymethylantipyrine and 4-hydroxyantipyrine can be distinguished by specific pyrolytic reactions. The different temperatures of decomposition of the conjugated sulphates and glucuronides give additional information on the nature of the conjugates. Thus, the characteristic difference in the metabolic patterns in man and rat is reflected in the mass thermograms of the aglycones obtained by thermal decomposition of the conjugate mixtures.

Trace analysis of doxylamine succinate in animal feed, human, urine, and wastewater by GC using a rubidium-sensitized nitrogen detector.

Doxylamine succinate, a drug used as a sleep-inducing agent, an antihistamine, and in a therapeutic formulation taken by pregnant women as an antinauseant, was scheduled for toxicological evaluation as part of a structure activity relationship study, with rats and mice, because a deficiency of such data exists with regard to many antihistamines. Analytical chemical procedures that ensure proper concentration, homogeneity, and stability of the drug in dosed feed, as well as the safety of personnel and the environment, were prerequisites for the toxicological tests. GC methods using a rubidium-sensitized nitrogen detector were developed for analysis of doxylamine succinate in animal feed, human urine, and wastewater at levels as low as 1 ppm, 100 ppb, and 100 ppb, respectively. Sample extracts were cleaned up by liquid-liquid partitioning, followed by additional cleanup on a column of silica gel. Data are presented concerning the stability of the drug in animal feed, extraction efficiencies, and the use of the silica gel cleanup column to separate the caffeine interference from doxylamine in extracts of human urine. Partition values and ancillary data concerning analysis of the drug in feed, by HPLC at levels as low as 10 ppm, are also reported.