Automated analysis of the pyridinium crosslinks of collagen in tissue and urine using solid-phase extraction and reversed-phase high-performance liquid chromatography.

A fully automated method for assaying the collagen crosslinking amino acids, pyridinoline and deoxypyridinoline, in human urine samples or tissue hydrolysates is described. Samples were processed using a Gilson ASPEC system with solid-phase extraction of the crosslinks on columns containing 100 mg of microgranular cellulose. Introduction of an additional solvent step during sample preparation allowed direct analysis by reversed-phase HPLC and elimination of the drying step used previously in a manual method. Use of a synthetic pyridinoline derivative as internal standard enabled accurate quantification of the crosslinks by correcting for recoveries through the whole assay. Samples were analyzed in sequential mode with a total assay time of 30 min. The automated assay showed close correlation with the manual method for both free and total crosslink determinations in human urine (r > 0.97). Reproducibility was improved, as seen from replicate analyses of human urine (CV < 3% for automated pyridinoline measurement compared with 8-12% previously observed for the manual method). Crosslink excretion is the most useful marker of collagen degradation in metabolic bone diseases and arthritic disorders. The automated assay which has been developed is rapid, convenient, and reliable and will greatly facilitate the monitoring of urinary collagen crosslinks and their tissue levels in clinical investigations.     

A computational method for prediction of excretory proteins and application to identification of gastric cancer markers in urine

A novel computational method for prediction of proteins excreted into urine is presented. The method is based on the identification of a list of distinguishing features between proteins found in the urine of healthy people and proteins deemed not to be urine excretory. These features are used to train a classifier to distinguish the two classes of proteins. When used in conjunction with information of which proteins are differentially expressed in diseased tissues of a specific type versus control tissues, this method can be used to predict potential urine markers for the disease. Here we report the detailed algorithm of this method and an application to identification of urine markers for gastric cancer. The performance of the trained classifier on 163 proteins was experimentally validated using antibody arrays, achieving >80% true positive rate. By applying the classifier on differentially expressed genes in gastric cancer vs normal gastric tissues, it was found that endothelial lipase (EL) was substantially suppressed in the urine samples of 21 gastric cancer patients versus 21 healthy individuals. Overall, we have demonstrated that our predictor for urine excretory proteins is highly effective and could potentially serve as a powerful tool in searches for disease biomarkers in urine in general.     

Identification of pyridoxine 3-sulfate, pyridoxal 3-sulfate, and N-methylpyridoxine as major urinary metabolites of vitamin B6 in domestic cats

Preliminary studies of vitamin B6 metabolism in three adult domestic cats detected very little pyridoxic acid in the urine. At oral doses of 49 to 490 mumol of [14C]pyridoxine hydrochloride, 50% of the excreted dose occurred as pyridoxine 3-sulfate and 25% as N-methylpyridoxine. The identity of these two metabolites was confirmed by isolation from urine and comparison with known compounds. A third compound was identified as pyridoxal 3-sulfate on the basis of chromatographic behavior and fluorescent properties before and after hydrolysis. At pyridoxine intakes of 0.97 mumol/day, the concentration of pyridoxal 3-sulfate in the urine sometimes exceeded the concentration of pyridoxine 3-sulfate. Pyridoxic acid remained a minor urinary metabolite at pyridoxine intakes ranging from 0.97 to 490 mumol/day. Although sulfation of phenol groups and methylation of the ring nitrogen are well-known detoxication reactions, this appears to be the first time such reactions have been observed in normal metabolism of vitamin B6. These observations provide further evidence of the diversity of vitamin B6 metabolism between species. While such diversity complicates the extrapolation of data from animal studies to humans, it does provide a variety of models for examining the influences of various factors on vitamin B6 metabolism.     

Steroid metabolism in the rabbit. Biliary and urinary excretion of metabolites of [4-14C]cortisone

1. [4-(14)C]Cortisone was administered to anaesthetized male and female New Zealand White rabbits as a single injection or as a 45-60min infusion. 2. The method of administration of the steroid did not significantly affect the total excretion of radioactivity in bile and urine [83.8+/-10.8%(s.d.)]. 3. The mean ratio of metabolites in urine to those in bile was 0.97+/-0.23% (range 0.64-1.3). 4. When bile and urine samples were hydrolysed successively by beta-glucuronidase, cold acid and hot acid, neutral metabolites extracted by ethyl acetate-ether were found mainly after hydrolysis by beta-glucuronidase. 5. An approximately equal proportion of the dose was converted into substances not extractable from alkaline aqueous solution after hydrolysis.     

Excretion of trimethylselenonium ion in human urine

A method to permit isolation and measurement of trimethylselenonium ion [TMSe, (CH3)3Se+] from 1 liter of human urine was developed. The method was based on precipitation of TMSe with ammonium reineckate, preseparation with anion-exchange resin, and final thermal decomposition and collection of the product in HNO3. It was tested for recovery and separation from other selenium moieties present in urine using both in vivo-labeled rat urine and human urine spiked with unlabeled TMSe. Recoveries from the former were in the range 76.8-87.0% (mean +/- SD: 81.8 +/- 3.7%, n = 5), while for the latter they were in the range 72.0-93.0% (mean +/- SD for three occasions (%): 80.9 +/- 5.5, 81.4 +/- 7.8, and 78.9 +/- 1.0). The reliability of the method was tested against an HPLC procedure using in vivo-labeled rat's urine. The mean (+/- SD) percentage of urine radioactivity appearing as TMSe was 36.0 +/- 5.7% for the present and 36.2 +/- 6.6% for the HPLC method. The mean of deviations, as percentage of the HPLC method, was -0.03 +/- 8.8%. The linear regression equation for the two methods was y = -0.805 + 1.029x (r2 = 0.81). Excretion of TMSe was measured in urine samples from several persons (range: 0.18-0.37 micrograms Se/liter; mean +/- SD: 0.26 +/- 0.07, n = 9). One subject consumed three separate doses of unlabeled selenite on alternate days (Day 1, 197 micrograms Se; Day 3, 395; and Day 5, 592). For the first 24 h of each period, TMSe excretions (micrograms Se/24 h) were 0.24, 0.53, and 0.97, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stabilization and determination of a PPAR agonist in human urine using automated 96-well liquid-liquid extraction and liquid chromatography/tandem mass spectrometry

Two stability challenges were encountered during development of an urine assay for a proliferator-activated receptor (PPAR) agonist, I (2-{[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy}-2-methyl propionic acid), indicated for the treatment of Type II diabetes. First, the analyte was lost in urine samples due to adsorption on container surface which is a common problem during clinical sample handling. Secondly, the acylglucuronide metabolite (III), a major metabolite of I, displayed limited stability and effected the quantitation of parent drug due to the release of I through hydrolysis. Therefore, a clinical collection procedure was carefully established to stabilize I and its acylglucuronide metabolite, III, in human urine. The metabolite was not quantitated with this method. The urine samples are treated with bovine serum albumin (BSA) equal to 1.75% of the urine volume and formic acid equal to 1% of urine volume. Compound (I) and internal standard (II) were extracted from urine with 1 mL ethyl acetate using a fully automated liquid-liquid extraction in 96-well plate format. The analytes are separated by reverse phase high-performance liquid chromatography (HPLC) with tandem mass spectrometry in multiple-reaction-monitoring (MRM) mode used for detection. The urine method has a lower limit of quantitation (LLOQ) of 0.05 ng/mL with a linearity range of 0.05-20 ng/mL using 0.05 mL of urine. The method was validated and used to assay urine clinical samples.     

Automated column-switching high-performance liquid chromatography for the determination of aflatoxin M1

An extractionless method for determining aflatoxin M1 (AFM1), a major metabolite of aflatoxin B1 (AFB1), in human urine was developed. The biological fluid is injected directly into the chromatographic system after simple dilution and centrifugation. A pre-column, packed with a cation-exchange phase and coupled on-line to a column-switching liquid chromatography (LC) system, is used for sample pre-treatment and concentration. The analytes are non-selectively desorbed with the LC eluent and cleaned by means of a column-switching procedure. Pre-treatment and analysis were performed within 40 min. Average AFM1 recovery reached 97% in the 10–100 ng/l range of urine. The detection limit of AFM1 in urine and milk was 2.5 ng/l for 1 ml of injected sample. A comparison with an immunoaffinity column clean-up and LC method was performed. The method was applied to determine AFM1 in the urine of AFB1 gavaged rats, and in the urine of both potentially exposed and supposedly unexposed workers. The method was also extended to milk.     

Pregnanetriolone, a normal steroid metabolite: its excretion by normal, Cushing's syndrome and congenital adrenal hyperplasia subjects.

Synthesis of 3H-pregnanetriolone permitted the estimation of pregnanetriolone in urine with a sensitivity in excess of most previous claims. A good correlation (r = +0.97) was obtained between the values from gas liquid chromatography and those of a double isotope derivative method. In contrast to previous reports, these methods indicated that pregnanetriolone is excreted by normal adults. Urinary pregnanetriolone levels were 18-59 mug/24hr for normal subjects, 35-290mug/24hr in Cushing's syndrome and 250-7000 mug/24hr with congenital adrenal hyperplasia. It is concluded that pregnanetriolone is a normal steroid metabolite and its occurrence in Cushing's syndrome does not necessary indicate an abnormal steroid biosynthetic pathway.     

Time-resolved fluoroimmunoassay for equol in plasma and urine

We present a method for the determination of the isoflavan equol in plasma and urine. This estrogenic isoflavan, which is formed by the action of the intestinal microflora, may have higher biological activity than its precursor daidzein. High urinary excretion of equol has been suggested to be associated with a reduction in breast cancer risk. The method is based on time-resolved fluoroimmunoassay, using a europium chelate as a label. After synthesis of 4′-O-carboxymethylequol the compound is coupled to bovine serum albumin (BSA), then used as antigen to immunize rabbits. The tracer with the europium chelate is synthesized using the same 4′-O-derivative of equol. After enzymatic hydrolysis (urine) or enzymatic hydrolysis and ether extraction (plasma) the immunoassay is carried out. The antiserum cross-reacted to variable extent with some isoflavonoids. For the plasma method the cross-reactivity does not seem to influence the results, which were highly specific. The overestimation of the values using the urine method (164%) compared to the results obtained by a gas chromatography–mass spectrometry (GC–MS) method is probably due to some influence of the matrix on the signal, and interference of structurally related compounds. It is suggested that plasma assays are used but if urine samples are measured a formula has to be used to correct the values making them comparable to the GC–MS results. The correlation coefficients between the time-resolved fluoroimmunoassay (TR-FIA) methods and GC–MS methods were high; r-values for the plasma and urine method, were 0.98 and 0.91, respectively. The intra-assay coefficient of variation (CV%) for the TR-FIA plasma and urine results at three different concentrations vary between 5.5–6.5 and 3.4–6.9, respectively. The inter-assay CV% varies between 5.4–9.7 and 7.4–7.7, respectively. The working ranges of the plasma and urine assay are 1.27–512 and 1.9–512 nmol/l, respectively.     

Simultaneous detection of methylphenidate and its main metabolite, ritalinic acid, in doping control

Two analytical methods for the simultaneous detection in urine of methylphenidate and its main metabolite, ritalinic acid, are described. Both procedures are based on solid-phase extraction of urine samples on Bond Elut Certify columns, and capillary gas chromatographic—mass spectrometric detection of O-trimethylsilyl, N-trifluoroacetyl derivatives. The former method is used as a general screening procedure for the detection of basic polar nitrogen-containing compounds in urine such as stimulants, narcotic and adrenergic drugs. The latter procedure is proposed as a specific method to confirm methylphenidate ingestion. The two methods are sensitive enough to detect methylphenidate and ritalinic acid in urine at least for 24 h after administration of a therapeutic dose (20 mg oral dose) of methylphenidate.     

Urinary Scandium as Predictor of Exposure: Effects of Scandium Chloride Hexahydrate on Renal Function in Rats

Some of the rare earth elements such as Sc are believed to be non-toxic and, at present, are widely utilized for the replacement of toxic heavy metals in technological applications, but they are not entirely free of toxicity, with hidden potential health risks. In this animal experiment, we report the urinary scandium (Sc) excretion rate and nephrotoxiciy in male Wistar rats. For this purpose, the rats were given a single dose of a solution of scandium chloride by intraperitoneal injection. The Sc excretion (U-Sc) was determined in 24-h urine samples by inductively coupled plasma–argon emission spectrometry along with the Sc nephrotoxicity, urine volume (UV), creatinine (Crt), β-2-microglobulin (β2-MG) and N-acetyl-β-d-glucosaminidase (NAG). A dose-dependent Sc excretion of 0.0063% (r = 0.97) via 24-h urine was confirmed. The administration of Sc induced a significant decrease of UV and Crt and a significant increase of NAG and β2-MG. These results suggest that U-Sc can be a useful tool for monitoring Sc exposure. The formation of Sc colloidal conjugates that deposit in glomeruli may be the cause of a reduction of the glomerular filtration rate. We propose that the analytical method and results described in this study will be of great importance for future toxicological studies on Sc exposure.     

Combined gas chromatographicmass spectrometric procedure for the measurement of captopril and sulfur-conjugated metabolites of captopril in plasma and urine

A gas chromatographicmass spectrometric method is described for the simultaneous measurement of the novel anti-hypertensive drug captopril, and the following metabolites: captopril disulfide dimer, S-methyl captopril, and S-methyl captopril sulfone. With this method all derivatives can be chromatographed using conventional gas chromatography of hexafluoroisopropyl esters in one temperature-programmed run and these can then be quantitated using selected-ion monitoring techniques.Using urine or plasma, captopril, S-methyl captopril and the disulfide dimer of captopril in concentrations as low as 1 ng/ml, 10 ng/ml and 25 ng/ml, respectively can be detected. The reproducibility of the method is satisfactory both within-assay and inter-assay.This technique has demonstrated that the pattern of urinary excretion of these compounds in both man and rat was similar. Excretion of unchanged captopril, S-methyl captopril and the disulfide dimer over 6 h in man given captopril (50 or 100 mg) chronically was 18.3%, 0.97% and 3.06%, respectively. Corresponding excretion of these three compounds in the rat following a single 10 mg/kg dose was 18.3%, 2.69% and 1.8%, respectively. A possible sulfone oxidation product of S-methyl captopril was not detected in the urine of either man or rat.     

High-performance liquid chromatography of the renal blood flow marker p-aminohippuric acid (PAH) and its metabolite N-acetyl PAH improves PAH clearance measurements

PAH (N-(4-aminobenzoyl)glycin) clearance measurements have been used for 50 years in clinical research for the determination of renal plasma flow. The quantitation of PAH in plasma or urine is generally performed by colorimetric method after diazotation reaction but the measurements must be corrected for the unspecific residual response observed in blank plasma. We have developed a HPLC method to specifically determine PAH and its metabolite NAc-PAH using a gradient elution ion-pair reversed-phase chromatography with UV detection at 273 and 265 nm, respectively. The separations were performed at room temperature on a ChromCart^® (125 mm×4 mm I.D.) Nucleosil 100-5 μm C₁₈ AB cartridge column, using a gradient elution of MeOH–buffer pH 3.9 1:99→15:85 over 15 min. The pH 3.9 buffered aqueous solution consisted in a mixture of 375 ml sodium citrate–citric acid solution (21.01 g citric acid and 8.0 g NaOH per liter), added up with 2.7 ml H₃PO₄ 85%, 1.0 g of sodium heptanesulfonate and completed ad 1000 ml with ultrapure water. The N-acetyltransferase activity does not seem to notably affect PAH clearances, although NAc-PAH represents 10.2±2.7% of PAH excreted unchanged in 12 healthy subjects. The performance of the HPLC and the colorimetric method have been compared using urine and plasma samples collected from healthy volunteers. Good correlations (r=0.94 and 0.97, for plasma and urine, respectively) are found between the results obtained with both techniques. However, the colorimetric method gives higher concentrations of PAH in urine and lower concentrations in plasma than those determined by HPLC. Hence, both renal (Cl_R) and systemic (Cl_S) clearances are systematically higher (35.1 and 17.8%, respectively) with the colorimetric method. The fraction of PAH excreted by the kidney Cl_R/Cl_S calculated from HPLC data (n=143) is, as expected, always <1 (mean=0.73±0.11), whereas the colorimetric method gives a mean extraction ratio of 0.87±0.13 implying some unphysiological values (>1). In conclusion, HPLC not only enables the simultaneous quantitation of PAH and NAc-PAH, but may also provide more accurate and precise PAH clearance measurements.     

Sensitivity of methods for calculating energy expenditure by use of doubly labeled water

Attempts to estimate human energy expenditure by use of doubly labeled water have produced three methods currently used for calculating carbon dioxide production from isotope disappearance data: 1) the two-point method, 2) the regression method, and 3) the integration method. An ideal data set was used to determine the error produced in the calculated energy expenditure for each method when specific variables were perturbed. The analysis indicates that some of the calculation methods are more susceptible to perturbations in certain variables than others. Results from an experiment on one adult human subject are used to illustrate the potential for error in actual data. Samples of second void urine, 24-h urine, and breath collected every other day for 21 days are used to calculate the average daily energy expenditure by three calculation methods. The difference between calculated energy expenditure and metabolizable energy on a weight-maintenance diet is used to estimate the error associated with the doubly labeled water method.     

A simple rapid method for the detection of rat urinary proteins by agarose electrophoresis and nigrosine staining

The method described is sufficiently sensitive to detect major changes in the protein excretion patterns of rat urine, and the short time required for technical procedures makes the method suitable for screening large numbers of rat urine samples. The patterns observed for normal adult male rats are similar to previously published data, and the method may also be used to identify pseudoproteinuria.     

Determination of the naturally occurring monoacetyl derivatives of di- and polyamines

A method is described for the determination of pmol quantities of monoacetylputrescine, N¹-acetylspermidine, N⁸-acetylspermidine and related compounds. The method is based on the derivatization of these compounds with 5-dimethylaminonaphthalene-1-sulphonyl-chloride, followed by thin-layer chromatographic separation. Cleanup steps allow the application of the method to urine analyses. From the repeated determination of acetylated polyamines in the urine of healthy individuals it can be concluded that these conjugates are the major excretory form of di- and polyamines.The cleanup steps used in this procedure and the method described for the stabilization of 5-dimethylaminonaphthalene-1-sulphonyl derivatives on thin-layer plates are advantageous also for the analyses of total polyamines in urine hydrolysates, and in related applications of the dansylation method.     

Sensitive method for the quantification of urinary pyrimidine metabolites in healthy adults by gas chromatography-tandem mass spectrometry

Enzyme deficiencies in pyrimidine metabolism are associated with a risk for severe toxicity against the antineoplastic agent 5-fluorouracil. To assess whether urinary levels of pyrimidines and their metabolites can be used for predicting patients' individual phenotype, a new gas chromatographic-tandem mass spectrometric method was developed which allows the simultaneous determination of uracil and thymine and their metabolites dihydrouracil, dihydrothymine, beta-ureidopropionic acid, beta-ureidoisobutyric acid, and the amino acids beta-alanine and beta-aminoisobutyric acid in human urine. Small aliquots (2-20 microl) of the urine samples were evaporated and derivatized to the tert.-butyldimethylsilyl derivatives before quantification, using the respective stable isotope-labelled analogues as internal standards. Analytical variation was acceptable with an intra-day imprecision (RSD) below 10%, for beta-ureidoisobutyric acid below 15%. The method was used for investigating the stability of urine samples and the influence of urine collection at different times.     

Determination of urinary triclosan by stir bar sorptive extraction and thermal desorption-gas chromatography-mass spectrometry

We have developed an analytical method for the determination of urinary 5-chloro-2-(2,4-dichlorophenoxy)phenol (triclosan), which utilizes stir bar sorptive extraction (SBSE) and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS). Human urine sample is de-conjugated by treatment with beta-glucuronidase and sulfatase. A stir bar coated with polydimethylsiloxane (PDMS) is added to the urine sample in a vial and the sample is stirred for 60 min at room temperature (25 degrees C). Then, the PDMS stir bar is subjected to TD-GC-MS. The detection limit of triclosan is 0.05 ng mL(-1). The method shows linearity over the calibration range (0.1-10 ng mL(-1)) and the correlation coefficient (r) is higher than 0.993 for triclosan standard solution. The average recoveries of triclosan in human urine sample are 102.8-113.1% (RSD: 2.4-6.7%). This simple, sensitive, and selective analytical method may be used in the determination of trace amounts of triclosan in human urine samples.     

An approach for the development and selection of chromatographic methods for high-throughput metabolomic screening of urine by ultra pressure LC-ESI-ToF-MS

Since the components of a sample for open metabolomic analysis are unknown a priori a pragmatic approach to method development has been taken in order to develop and select a chromatographic method suitable for high-throughput open metabolomic screening of urine by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS). A total of 848 injections of diluted rat urine were made onto a UPLC-ESI-ToF-MS system using several different gradient profiles and run times to determine a suitable method for analysis of urine from male and female rats. Peak integral and multivariate data analysis were performed to investigate the quality of separation and information obtained from these multiple analyses. A suitable 8 min method was selected and is now used routinely for open profiling metabolomic analyses of urine. The use of a sample-relevant QC mix is also discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Determination of urinary ortho- and meta-cresol in humans by headspace SPME gas chromatography/mass spectrometry

ortho-Cresol (o-C) and meta-cresol (m-C) are minor urinary metabolites of toluene, a widely used chemical with neurotoxicological properties. A new assay for their determination in human urine is here proposed. Urinary cresol sulphates and glucuronates are submitted to acid hydrolysis, urine is neutralized, added with o-cresols-d8, and analytes are sampled in the headspace of urine by SPME using a polydimethylsiloxane fiber. Analysis is performed by GC/MS using, for separation, either a SupelcoWax10 (for o-C) or a chiral CP Cresol (for o-C and m-C) column. The method is very specific, with a range of linearity 0-5.0 mg/l, within- and between-run precision, as coefficient of variation, <15% and <19%, limit of detection of 0.006 mg/l for o-C and 0.007 mg/l for m-C. The procedure is applied to the quantification of cresols in urine from workers exposed to toluene and from subjects belonging to the general population.