Isolation and identification of S-adenosylmethionine from human urine

Procedures are described for the isolation and identification of adenosylmethionine from human urine. Previously described preliminary separative procedures using anion and cation exchange columns and an XAD-4 resin column have been extended to permit the separation of adenosylmethionine. The adenosylmethionine has been identified by conversion to methylthioadenosine followed by rechromatography of the latter compound with three different types of columns and elution systems. Mean adenosylmethionine values for urine were as follows: adults, 0.26; children, 0.36 nmole/mumole creatinine. Recovery of adenosylmethionine added to urine and determined by this separative procedure was 52%.     

Improved HPLC method for the simultaneous determination of allantoin, uric acid and creatinine in cattle urine

An HPLC procedure developed for the rapid and simultaneous determination of purine derivatives (PD) in ruminants' urine was investigated, since the adoption of a single method for the simultaneous detection of PD and creatinine was not carried out due to elution of polar co-extractives and also due to overlapping of the peaks of allantoin and creatinine. The experimental conditions chosen in the present study avoid the presence of chemically competitive compounds and afford a good separation of the peaks of allantoin and creatinine. The recoveries of the standard compounds added to urine samples were 94-104%. This method can be proposed as a possible reference method for the estimation of allantoin, uric acid and creatinine in cattle urine.     

Determination of dialysate creatinine by micellar electrokinetic chromatography

Micellar electrokinetic chromatography with UV absorbance detection has been applied for fast and selective determination of creatinine in samples of postdialysate fluid. Optimization of the method was performed, with the best results being obtained using a 30 mM borate-100 mM sodium dodecyl sulphate background electrolyte, pH 9, with the detector set at 235 nm and an applied voltage of 17 kV across a fused-silica capillary of 67 cm/75 micro m I.D. The linear range of the technique was over 2 orders of magnitude (5-1000 micro M). The developed analytical procedure is useful for the monitoring of clinical hemodialysis treatment, because creatinine levels in real undiluted samples of postdialysate range from 80 to 350 micro M. The separation system allows the analysis of about six to seven samples of spent dialysate per hour in almost real time. The determinations are not influenced by other components of dialysate fluid nor by other surrogates extracted from patient blood. The results of analysis using the developed procedure and the kinetic spectrophotometric Jaffe method conventionally used in clinical settings for creatinine determination are fully comparable. Successful clinical evaluation of the analytical system was performed. The developed system is useful for bloodless estimation of bioanalytical parameters of hemodialysis sessions such as creatinine-time profiles and total creatinine removal. Both these parameters are important in clinical models of hemodialysis therapy.     

Detection and quantification of α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid: a metabolite of asymmetric dimethylarginine

Nitric oxide is an ubiquitary cell signaling substance. Its enzymatic production rate by nitric oxide synthase is regulated by the concentrations of the substrate L-arginine and the competitive inhibitor asymmetric dimethylarginine (ADMA). A newly recognized elimination pathway for ADMA is the transamination to α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid (DMGV) by the enzyme alanine-glyoxylate aminotransferase 2 (AGXT2). This pathway has been proven to be relevant for nitric oxide regulation, but up to now no method exists for the determination of DMGV in biological fluids. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of DMGV. D(6)-DMGV was used as internal standard. Samples were purified online by column switching, and separation was achieved on a porous graphitic carbon column. The calibration was linear over ranges of 10 to 200 nmol/L for plasma and 0.1 to 20 μmol/L for urine. The intra- and interday accuracies and precisions in plasma and urine were better than 10%. In plasma samples, DMGV was present in concentrations between 19.1 and 77.5 nmol/L. In urine samples, concentrations between 0.0114 and 1.03 μmol/mmol creatinine were found. This method can be used as a tool for the scientific investigation of the ADMA conversion to DMGV via the enzyme AGXT2.     

Gas—liquid chromatography of isobutyl ester,N(O)-heptafluorobutyrate derivatives of amino acids on a glass capillary column for quantitative separation in clinical biology

A gas chromatographic method adapted to routine analysis has been developed for quantitative separation on glass capillary columns for free proteic and other known amino acids normally or abnormally found in physiological fluids. The procedure involves ion-exchange chromatography and isobutyl ester, N(O)-heptafluorobutyrate derivatization of free plasma and urine amino acid samples. Derivatized components were ascertained by combined gas chromatography—mass spectrometry. The use of glass for the capillary column is mandatory to achieve qualitative and quantitative analysis of the known occurring amino acids in urine and small plasma samples. Quantitative analysis of several types of human amino acid disorders are presented.     

The amperometric titration of plasma chloride and urine chloride

It has been shown that the method of Laitinen, Jennings, and Parks for the amperometric titration of chloride can be easily adapted to analyses of plasma (serum) and urine chlorides. For the procedure 1.0-ml. samples are diluted so that the mixture being titrated is 50% in acetone and 0.8 N in nitric acid. Silver nitrate, 0.0100 N, is used as the titrant, and the rotating platinum electrode is used as the indicator electrode. The method is simple, rapid, and as accurate as existing methods.When 0.1-ml. samples are analyzed, the accuracy is decreased a little over that obtained when 1.0-ml. samples are used.The method is not satisfactory for the analysis of chloride in the presence of whole blood or muscle tissue.     

Determination of free and total S-phenylmercapturic acid by HPLC/MS/MS in the biological monitoring of benzene exposure

Urinary S-phenylmercapturic acid (SPMA) is a biomarker suggested by the American Conference of Governmental Industrial Hygienists (ACGIH) for assessing occupational exposure to benzene. A possible cause of the miscorrelation between environmental monitoring and biological monitoring for benzene exposure, which many authors complain about, is the existence of a urinary metabolite that turns into SPMA by acid hydrolysis. Forty urine samples were tested to determine which concentration value would correspond to the ACGIH Biological Exposure Index (BEI) of 25 µg g^-1 creatinine if exposure assessment was based on the determination of SPMA after quantitative hydrolysis of its precursor. An aliquot of each sample was hydrolysed with 9 M H₂SO₄, a second one was brought to pH 2 and a third one was used as it was (free SPMA). SPMA was determined by high-performance liquid chromatography/tandem mass spectrometric technique (HPLC/MS/MS) using an internal standard. The analytical method was validated in the range 0.5-50 µg l^-1. The average SPMA in pH 2 samples is 45-60% of the total, while free SPMA varies from 1% to 66%. The hydrolysis of pre-SPMA reduces the likelihood of variability in the results by reducing pH differences in urine samples and increasing the amount of measured SPMA. The BEI limit value would be about 50 µg g^-1 creatinine.     

Detection and quantification of α-keto-δ-(N,N-dimethylguanidino)valeric acid: A metabolite of asymmetric dimethylarginine

Nitric oxide is an ubiquitary cell signaling substance. Its enzymatic production rate by nitric oxide synthase is regulated by the concentrations of the substrate l-arginine and the competitive inhibitor asymmetric dimethylarginine (ADMA). A newly recognized elimination pathway for ADMA is the transamination to α-keto-δ-(N^G,N^G-dimethylguanidino)valeric acid (DMGV) by the enzyme alanine-glyoxylate aminotransferase 2 (AGXT2). This pathway has been proven to be relevant for nitric oxide regulation, but up to now no method exists for the determination of DMGV in biological fluids. We have developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantification of DMGV. D₆-DMGV was used as internal standard. Samples were purified online by column switching, and separation was achieved on a porous graphitic carbon column. The calibration was linear over ranges of 10 to 200 nmol/L for plasma and 0.1 to 20 μmol/L for urine. The intra- and interday accuracies and precisions in plasma and urine were better than 10%. In plasma samples, DMGV was present in concentrations between 19.1 and 77.5 nmol/L. In urine samples, concentrations between 0.0114 and 1.03 μmol/mmol creatinine were found. This method can be used as a tool for the scientific investigation of the ADMA conversion to DMGV via the enzyme AGXT2.     

Analysis of 8-hydroxy-2'-deoxyguanosine in urine using high-performance liquid chromatography-electrospray tandem mass spectrometry

8-hydroxy-2'-deoxyguanosine (8-OHdG) is a widely used biomarker of oxidative stress in research related to DNA, protein damage as well as lipid peroxidation. HPLC-MS/MS with electrospray ionization (ESI) and the use of isotopically labelled 8-OHdG as an internal standard allows a simple quantification of 8-OHdG in urine samples. HPLC separation utilized the peak cutting technique and a 1.5 mmx120 mm analytical anion exchange column. Novel method entails only minimal sample handling including the addition of a buffer and an internal standard followed by centrifugation before the samples are ready for analysis. The levels of 8-OHdG in human urine samples (n=246) varied from 0.16 to 16.48 microg/L and the corresponding creatinine-normalized values were ranged from 0.49 to 14.27 microg of 8-OHdG/g creatinine. The correlation between the developed HPLC-MS/MS method and the existing HPLC-EC method was good with an R2 value of 0.8707.     

Analysis of theaflavins in biological fluids using liquid chromatography–electrospray mass spectrometry

A HPLC–MS procedure for the sensitive and specific analysis of the black tea flavonoid theaflavin in human plasma and urine was developed. Levels were measured after enzymatic deconjugation, extraction into ethyl acetate, and separation by HPLC, using tandem mass spectrometry as a detecting system. Two healthy volunteers consumed 700 mg theaflavins, equivalent to about 30 cups of black tea. The maximum concentration detected in blood plasma was 1.0 μg l⁻¹ in a sample collected after 2 h. The concentration in urine also peaked after 2 h at 4.2 μg l⁻¹. Hence, only minute amounts of theaflavins can be detected in plasma and urine samples of healthy volunteers after ingestion.     

Automated determination of orotic acid, uracil and pseudouridine in urine by high-performance liquid chromatography with column switching

A column-switching high-performance liquid chromatographic method, requiring no sample preparation apart from filtration, is described for quantification of urinary orotic acid, uracil and pseudouridine. The analyses were carried out using a reversed-phase octadecylsilane-bonded column for sample clean-up and a cation-exchange column for separation; 5–20 ]sml samples of urine were directly analysed, and more than 100 samples could be analysed consecutively. Each sample required only 30 min. Detection limits of these compounds were 5 pmol. Creatinine-related urinary uracil excretion was lowest in the newborn period (17.3 ± 14.4 μmol/g of creatinine). A patient with partial ornithine transcarbamylase deficiency and his mother usually excreted a high level of uracil during the period of normal orotic acid excretion and normal serum ammonia level.     

Determination of free and total S-phenylmercapturic acid by HPLC/MS/MS in the biological monitoring of benzene exposure

Abstract

Urinary S-phenylmercapturic acid (SPMA) is a biomarker suggested by the American Conference of Governmental Industrial Hygienists (ACGIH) for assessing occupational exposure to benzene. A possible cause of the miscorrelation between environmental monitoring and biological monitoring for benzene exposure, which many authors complain about, is the existence of a urinary metabolite that turns into SPMA by acid hydrolysis. Forty urine samples were tested to determine which concentration value would correspond to the ACGIH Biological Exposure Index (BEI) of 25 µg g^?1 creatinine if exposure assessment was based on the determination of SPMA after quantitative hydrolysis of its precursor. An aliquot of each sample was hydrolysed with 9 M H₂SO₄, a second one was brought to pH 2 and a third one was used as it was (free SPMA). SPMA was determined by high-performance liquid chromatography/tandem mass spectrometric technique (HPLC/MS/MS) using an internal standard. The analytical method was validated in the range 0.5–50 µg l^?1. The average SPMA in pH 2 samples is 45–60% of the total, while free SPMA varies from 1% to 66%. The hydrolysis of pre-SPMA reduces the likelihood of variability in the results by reducing pH differences in urine samples and increasing the amount of measured SPMA. The BEI limit value would be about 50 µg g^?1 creatinine.     

Determination of dichloroanilines in human urine by GC–MS, GC–MS–MS, and GC–ECD as markers of low-level pesticide exposure

Methods for the determination of 3,4-dichloroaniline (3,4-DCA) and 3,5-dichloroaniline (3,5-DCA) as common markers of eight non-persistent pesticides in human urine are presented. 3,5-DCA is a marker for the exposure to the fungicides vinclozolin, procymidone, iprodione, and chlozolinate. Furthermore the herbicides diuron, linuron, neburon, and propanil are covered using their common marker 3,4-DCA. The urine samples were treated by basic hydrolysis to degrade all pesticides, metabolites, and their conjugates containing the intact moieties completely to the corresponding dichloroanilines. After addition of the internal standard 4-chloro-2-methylaniline, simultaneous steam distillation extraction (SDE) followed by liquid–liquid extraction (LLE) was carried out to produce, concentrate and purify the dichloroaniline moieties. Gas chromatography (GC) with mass spectrometric (MS) and tandem mass spectrometric (MS–MS) detection and also detection with an electron-capture detector (ECD) after derivatisation with heptafluorobutyric anhydride (HFBA) were employed for separation, detection, and identification. Limit of detection of the GC–MS–MS and the GC–ECD methods was 0.03 and 0.05 μg/l, respectively. Absolute recoveries obtained from a urine sample spiked with the internal standard, 3,5-, and 3,4-DCA, ranged from 93 to 103% with 9–18% coefficient of variation. The three detection techniques were compared concerning their performance, expenditure and suitability for their application in human biomonitoring studies. The described procedure has been successfully applied for the determination of 3,4- and 3,5-DCA in the urine of non-occupationally exposed volunteers. The 3,4-DCA levels in these urine samples ranged between 0.13 and 0.34 μg/g creatinine or 0.11 and 0.56 μg/l, while those for 3,5-DCA were between 0.39 and 3.33 μg/g creatinine or 0.17 and 1.17 μg/l.     

Speciation of organotin compounds in urine by GC–MIP-AED and GC–MS after ethylation and liquid–liquid extraction

A method for the determination of organotin compounds in urine samples based on liquid–liquid extraction (LLE) in hexane and gas chromatographic separation was developed and optimized. Seven organotin species, namely monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT), tetrabutyltin (TeBT), monophenyltin (MPhT), diphenyltin (DPhT) and triphenyltin (TPhT), were in situ derivatized by sodium tetraethylborate (NaBEt₄) to form ethylated less polar derivatives directly in the urine matrix. The critical parameters which have a significant effect on the yield of the successive liquid–liquid extraction procedure were examined, by using standard solutions of tetrabutyltin in hexane. The method was optimized for use in direct analysis of undiluted human urine samples and ways to overcome practical problems such as foam formation during extraction, due to various constituents of urine are discussed. After thorough optimization of the extraction procedure, all examined species could be determined after 3 min of simultaneous derivatization and extraction at room temperature and 5 min phase separation by centrifugation. Gas chromatography with a microwave-induced plasma atomic emission detector (MIP-AED) as element specific detector was employed for quantitative measurements, while a quadrupole mass spectrometric detector (MS) was used as molecular specific detector. The detection limits were between 0.42 and 0.67 μg L^?1 (as Sn) for the quantitative LLE–GC–MIP-AED method and the precision between 4.2% and 11.7%, respectively.     

Enantioselective analysis of levetiracetam and its enantiomer R-α-ethyl-2-oxo-pyrrolidine acetamide using gas chromatography and ion trap mass spectrometric detection

A gas chromatographic–mass spectrometric method was developed for the enantioselective analysis of levetiracetam and its enantiomer (R)-α-ethyl-2-oxo-pyrrolidine acetamide in dog plasma and urine. A solid-phase extraction procedure was followed by gas chromatographic separation of the enantiomers on a chiral cyclodextrin capillary column and detection using ion trap mass spectrometry. The fragmentation pattern of the enantiomers was further investigated using tandem mass spectrometry. For quantitative analysis three single ions were selected from the enantiomers, enabling selected ion monitoring in detection. The calibration curves were linear from 1 μM to 2 mM for plasma samples and from 0.5 mM to 38 mM for urine samples. In plasma and urine samples the inter-day precision, expressed as relative standard deviation was around 10% in all concentrations. Selected ion monitoring mass spectrometry is suitable for quantitative analysis of a wide concentration range of levetiracetam and its enantiomer in biological samples. The method was successfully applied to a pharmacokinetic study of levetiracetam and (R)-α-ethyl-2-oxo-pyrrolidine acetamide in a dog.     

One-chip biosensor for simultaneous disease marker/calibration substance measurement in human urine by electrochemical surface plasmon resonance method

We have developed a miniaturized electrochemical surface plasmon resonance biosensor for measuring two biomolecules that have very different molecular sizes, one is transferrin (MW=75 kDa) as a disease marker protein, the other is creatinine (MW=113) as a calibration marker for the accurate measurement of human urinary samples. The sensor has a PDMS based microchannel that is 2 mm wide and 20 μm deep. Two gold films were integrated in the microchannel; one was modified with anti-transferrin antibody for immuno-reaction, and the other was modified with osmium-poly-vinylpyridine wired horseradish peroxidase (Os-gel-HRP). We further immobilized a tri-enzyme layer of creatininase, creatinase and sarcosine oxidase in order to measure creatinine by converting it to hydrogen peroxide in the upstream channel. We measured the transferrin concentration from the refractive index change involved in an immuno-complex formation, and we were simultaneously able to measure creatinine by employing the refractive index change in the Os-gel-HRP caused by oxidation with the hydrogen peroxide produced from creatinine by the tri-enzyme. The effects of ascorbic acid and uric acid in urine samples were sufficiently eliminated by adding ascorbate oxidase and uricase to the urine samples during sampling. We were able to measure two analyte concentrations within 15 min by one simple injection of 50 μL of diluted human urine into our sensor. The detectable transferrin and creatinine ranges were 20 ng/mL to 10 μg/mL, and 10 μM to 10 mM, respectively, which are sufficient levels for clinical tests. Finally, we compared the results obtained using our sensor with those obtained with a conventional immunoassay and the Jaffe method. We obtained a similar trend that can reduce the fluctuation in the urinary transferrin concentration from three different samples by calibrating the creatinine concentration.     

Lipid quantitation in formalin-fixed liver sections

We describe a simple, sensitive, and quantitative procedure for measurement of triglycerides and protein contents in formalin-fixed liver sections. The method can detect as little as 0.27 microgram of triglycerides per mg of protein. It is based on selective binding of Sudan IV and Fast Green FCF to fat and total proteins, respectively, and their sequential elution with solvents. Sudan IV is eluted readily with acetone and Fast Green with NaOH-methanol, and the absorbances obtained at 500 and 610 nm can be used to determine the amount of triglycerides and total protein. The color equivalence for Fast Green was obtained after destaining the sections and measuring the protein contents by micro-Kjeldahl analysis. The color equivalence of Sudan IV was estimated by determining the triglyceride content in liver homogenates by an enzymatic procedure and then measuring the amount of dye bound to multiple fixed sections. There was a strong linear correlation between the triglyceride content as determined chemically and that obtained using the equivalence colors (r2 = 0.98). This method is useful to measure fat content in tissue samples and could be applied to evaluate the progression of liver disease.     

Determination of phenethyl isothiocyanate in human plasma and urine by ammonia derivatization and liquid chromatography-tandem mass spectrometry

Phenethyl isothiocyanate (PEITC) is a dietary compound present in cruciferous vegetables that has cancer-preventive properties. Our objective was to develop and validate a novel liquid chromatography-tandem mass spectrometry procedure to analyze PEITC concentrations in human plasma and urine. Following hexane extraction, ammonia was added to samples to derivatize PEITC to phenethylthiourea. Chromatographic separation was achieved on a C(18) column with acetonitrile/5 mM formic acid (60:40, v/v) as the mobile phase followed by tandem mass spectrometry detection in multiple reaction monitoring mode. Deuterium-labeled PEITC was used as the internal standard. The detection limit was 2 nM and calibration curves were linear from 7.8 to 2000 nM. The intra- and inter-day coefficients of variation were less than 5 and 10%, respectively. The intra- and inter-day accuracies ranged from 101.0 to 104.2% and from 102.8 to 118.6%, respectively. The recovery from spiked human plasma and urine ranged from 100.3 to 113.5% and from 98.3 to 103.9%, respectively. The assay was used to measure PEITC in plasma and urine samples obtained from subjects after consumption of 100g of watercress. This novel assay represents the first analytical method with the sensitivity and specificity to determine plasma and urine concentrations of PEITC.     

Micromethod for the quantitative determination of leucine from biological experimental material using a dansylation technic]

A method is described allowing quantitative determination of leucine from biological material up to 1 - 10(-11) M by means of dansylation of amino acids and their thin-layer chromatographic separation on polyamide microfoils. By selecting [14C]-U-L-leucine as an 'internal' standard, which is added to the tissue during the homogenization procedure, it is possible to correct for data scattering related to variations of the conversion rate or the yield of the dansylation reaction, and to losses in the different steps of thin-layer chromatography that are difficult to standardize.