Determination of urinary trans,trans-muconic acid by gas chromatography-mass spectrometry

A sensitive and specific method for the determination of trans,trans-muconic acid (t,t-MA) in urine is described. After clean-up on an anion-exchange cartridge, t,t-MA was derivatized with BF₃-methanol to the dimethyl ester and analyzed by gas chromatography-mass spectrometry (GC-MS), with 2-bromohexanoic acid as an internal standard. The limit of detection was 0.01 mg/l, the coefficient of variation for duplicate analysis in a series of urine samples (n = 50) was 2.6% and the recovery rate ranged from 93.3 to 106.3%. The between-day and within-day precision for the analysis were 7.4 and 14.6%, respectively. The method was applied to the determination of t,t-MA in urine samples from smokers and non-smokers. The mean concentration of t,t-MA in urine of 10 smokers was 0.09 ± 0.04 mg/g creatinine and was significantly (p = 0.012) higher than that found in urine of 10 non-smokers (0.05 ± 0.02 mg/g creatinine). In contrast to the results obtained with the commonly used high-performance liquid chromatographic ultraviolet detection (HPLC-UV) methods, no interference between t,t-MA and other urinary compounds was found. This GC-MS method is both specific and sensitive for biomonitoring of low environmental benzene exposure.     

A note on urinary trans,trans-muconic acid level among Thai press workers

Benzene is a common toxic volatile substance associated with many industrial processes. Benzene exposure is of particular concern because recent research indicates that it can result in chronic toxicity and thousands of workers in industrial plants experience ongoing exposure. Therefore, the determination and control of benzene exposure among at-risk workers is very important. Urinary trans,trans-muconic acid (ttMA) determination is a helpful test for monitoring groups of at-risk workers for exposure to benzene. In this study, 103 urine samples were obtained from 60 controls and 43 occupational exposed press workers in a press factory in Bangkok. All samples were analysed for ttMA using a previously reported method. The average urinary ttMA levels for the control and exposed groups were 0.08±0.03 mg g^-1 creatinine and 0.56±0.65 mg g^-1 creatinine, respectively. Significantly higher urinary ttMA levels were observed among the press workers (p=0.03). The introduction of public health policies concerning the prevention of exposure to benzene among at-risk workers is recommended, and more widespread use of biological monitoring for the assessment and control of occupational exposure to industrial chemicals is encouraged.     

A note on urinary trans,trans-muconic acid level among Thai press workers

Benzene is a common toxic volatile substance associated with many industrial processes. Benzene exposure is of particular concern because recent research indicates that it can result in chronic toxicity and thousands of workers in industrial plants experience ongoing exposure. Therefore, the determination and control of benzene exposure among at-risk workers is very important. Urinary trans,trans-muconic acid (ttMA) determination is a helpful test for monitoring groups of at-risk workers for exposure to benzene. In this study, 103 urine samples were obtained from 60 controls and 43 occupational exposed press workers in a press factory in Bangkok. All samples were analysed for ttMA using a previously reported method. The average urinary ttMA levels for the control and exposed groups were 0.08±0.03 mg g^?1 creatinine and 0.56±0.65 mg g^?1 creatinine, respectively. Significantly higher urinary ttMA levels were observed among the press workers (p=0.03). The introduction of public health policies concerning the prevention of exposure to benzene among at-risk workers is recommended, and more widespread use of biological monitoring for the assessment and control of occupational exposure to industrial chemicals is encouraged.     

A note on urinary trans,trans-muconic acid level among Thai press workers.

Benzene is a common toxic volatile substance associated with many industrial processes. Benzene exposure is of particular concern because recent research indicates that it can result in chronic toxicity and thousands of workers in industrial plants experience ongoing exposure. Therefore, the determination and control of benzene exposure among at-risk workers is very important. Urinary trans,trans-muconic acid (ttMA) determination is a helpful test for monitoring groups of at-risk workers for exposure to benzene. In this study, 103 urine samples were obtained from 60 controls and 43 occupational exposed press workers in a press factory in Bangkok. All samples were analysed for ttMA using a previously reported method. The average urinary ttMA levels for the control and exposed groups were 0.08+/-0.03 mg g(-1) creatinine and 0.56+/-0.65 mg g(-1) creatinine, respectively. Significantly higher urinary ttMA levels were observed among the press workers (p=0.03). The introduction of public health policies concerning the prevention of exposure to benzene among at-risk workers is recommended, and more widespread use of biological monitoring for the assessment and control of occupational exposure to industrial chemicals is encouraged.     

A sensitive spectrophotometric method for determination of Tris

The paper describes a sensitive spectrophotometric method for qualitative and quantitative determination of Tris, in the 0.1–1.0 μmole range. The procedure is almost identical with that used for histidine. The green-colored reaction product has two absorption peaks with maxima at 422 nm and 640 nm, in contrast to the single peak (540 nm) in the case of histidine. Absorbance and Tris concentration are mutually linear at both wavelengths, but reading conditions are more favorable at 640 nm.     

A sensitive gas chromatographic method for determination of protein-associated sulfur

A sensitive method for the determination of elemental sulfur bound to serum albumin and other proteins has been devised. The sample is treated with a hexane solution of triphenylphosphine, and the triphenylphosphine sulfide formed is determined by gas chromatography with a flame photometric sulfur detector. The detection limit of the method is 0.3 microM albumin-associated sulfur. Protein-associated sulfur was not detected in plasma from rats or normal human beings, findings that do not support an earlier suggested transport function of serum albumin for sulfur. Significant amounts of protein-associated sulfur, however, were found in certain rat tissues.     

Fast liquid chromatographic determination of urinary trans,trans-muconic acid

trans,trans-Muconic acid (1,3-butadiene-1,4-dicarboxylic acid, MA), a minor urinary metabolite of benzene exposure, was determined, after clean-up by solid-phase anion-exchange chromatography, by reversed-phase HPLC on a C₁₈ column (5 × 0.46 cm I.D., 3 μm particle size), using formic acid-tetrahydrofuran-water (14:17:969) as mobile phase and UV detection at 263 nm. The recovery of MA from spiked urine was > 95% in the 50–500 μg/l range; the quantification limit was 6 μg/l; day-to-day precision, at 300 μg/l, C.V. = 9.2%; the run time was less than 10 min. Urinary MA excretion was measured in two spot urine samples of 131 benzene environmentally exposed subjects: midday values obtained in non-smokers (mean±S.D.=77±54 μg/l, N = 82) were statistically different from those of smoerks (169±85 μg/l, N = 30) (P<0.0001); each group showed a statistically significant increase between MA excretion in midday over morning samples. Moreover, in subjects grouped according to tobacco-smoke exposure level, median values of MA were positively associated with and increased with daily smoking habits.     

Personal exposure to different levels of benzene and its relationships to the urinary metabolites S-phenylmercapturic acid and trans,trans-muconic acid

This report is part of an extensive study to verify the validity, specificity, and sensitivity of biomarkers of benzene at low exposures and assess their relationships with personal exposure and genetic damage. The study population was selected from benzene-exposed workers in Tianjin, China, based on historical exposure data. The recruitment of 130 exposed workers from glue-making or shoe-making plants and 51 unexposed subjects from nearby food factories was based on personal exposure measurements conducted for 3-4 weeks prior to collection of biological samples. In this report we investigated correlation of urinary benzene metabolites, S-phenylmercapturic acid (S-PMA) and trans,trans-muconic acid (t,t-MA) with personal exposure levels on the day of urine collection and studied the effect of dose on the biotransformation of benzene to these key metabolites. Urinary S-PMA and t,t-MA were determined simultaneously by liquid chromatography-tandem mass spectrometry analyses. Both S-PMA and t,t-MA, but specifically the former, correlated well with personal benzene exposure over a broad range of exposure (0.06-122 ppm). There was good correlation in the subgroup that had been exposed to <1 ppm benzene with both metabolites (P-trend <0.0001 for S-PMA and 0.006 for t,t-MA). Furthermore, the levels of S-PMA were significantly higher in the subgroup exposed to <0.25 ppm than that in unexposed subjects (n=17; P=0.001). There is inter-individual variation in the rate of conversion of benzene into urinary metabolites. The percentage of biotransformation of benzene to urinary S-PMA ranged from 0.005 to 0.3% and that to urinary t,t-MA ranged from 0.6 to approximately 20%. The percentage of benzene biotransformed into S-PMA and t,t-MA decreased with increasing concentration of benzene, especially conversion of benzene into t,t-MA. It appears that women excreted more metabolites than men for the same levels of benzene exposures. Our data suggest that S-PMA is superior to t,t-MA as a biomarker for low levels of benzene exposure.     

Gas chromatographic method for the determination of urinary acetylpolyamines

A gas chromatographic method was developed for the determination of monoacetylputrescine, monoacetylcadaverine, N¹-acetylspermidine and N⁵-acetylspermidine in human urine. The amines were isolated from urine by silica gel column chromatography. 1, 10-Diaminodecane was used as internal standard. The amines were reacted with ethyl chloroformate in aqueous medium to four ethyloxycarbonyl derivatives prior to application to gas chromatography using a flame ionization detector. Separation and determination of the derivatives were carried out on a Uniport HP column (1.0 m) impregnated with 0.5% SP-1000 under temperature-programmed conditions. The monoacetylpolyamines could be measured accurately at the nanomole level. The method was used for the determination of the monoacetylpolyamines in urine of healthy volunteers. The values obtained were in the range of the published data.     

High-performance liquid chromatographic method for sensitive determination of the alkylating agent CB1954 in human plasma

A high-performance liquid chromatography (HPLC) method is described for the measurement of the weak alkylating agent CB1954 in human plasma. CB1954 can be used as an innocuous prodrug designed for activation by bacterial nitroreductases in strategies of gene-directed enzyme–prodrug therapy, and becomes activated to a potent bifunctional alkylating agent. The HPLC method involves precipitation and solvent extraction and uses Mitomycin C (MMC) as an internal standard, with a retention time for MMC of 5.85±0.015 min, and for CB1954 of 10.72±0.063 min. The limit of detection for CB1954 is 2.9 ng/ml, and this compares favourably with systems involving direct analysis of plasma (limit of detection 600 ng/ml, approximately). The method is now being used for pharmacokinetic measurements in plasma samples from cancer patients entering phase I clinical trials of CB1954. Results using serial plasma samples from one patient are presented. The patient was treated intravenously with CB1954 (6 mg/m²), and plasma clearance of the drug showed biphasic kinetics with α half-life 14.6 min, and β half-life 170.5 min.     

A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts

Superoxide dismutase (EC 1.15.1.1) has been assayed by a spectrophotometric method based on the inhibition of a superoxide-driven NADH oxidation. The assay consists of a purely chemical reaction sequence which involves EDTA, Mn(II), mercaptoethanol, and molecular oxygen, requiring neither auxiliary enzymes nor sophisticated equipment. The method is very flexible and rapid and is applicable with high sensitivity to the determination of both pure and crude superoxide dismutase preparations. The decrease of the rate of NADH oxidation is a function of enzyme concentration, and saturation levels are attainable. Fifty percent inhibition, corresponding to one unit of the enzyme, is produced by approximately 15 ng of pure superoxide dismutase. Experiments on rat liver cytosol have shown the specificity of the method for superoxide dismutase. Moreover, common cellular components do not interfere with the measurement, except for hemoglobin when present at relatively high concentrations. The assay is performed at physiological pH and is unaffected by catalase.     

Simple and sensitive high-performance liquid chromatographic method for the determination of an everninomycin,SCH 27899, in rat plasma

A simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of SCH 27899, an everninomycin antibiotic, in rat plasma. The method involved plasma protein precipation with acetonitrile, followed by reversed-phase HPLC analysis using a polymeric column and a mobile phase containing acetonitrile and ammonium phosphate, pH 7.8. The linear relationship between detector response and concentration was demonstrated with a correlation coefficient of larger than 0.996 at concentrations ranging from 0.2 to 100 μg/ml. The results showed that the HPLC method was accurate (bias ≤6%) and precise (coefficient of variation, C.V.≤6%). The limit of quantitation was 0.2 μg/ml with a C.V. of 2.6% and bias of 5%. SCH 27899 was stable in rat plasma at −20°C for at least 40 days. The HPLC method has been utilized for the determination of SCH 27899 in plasma samples from rats following single intravenous administration (3 mg/kg).     

A high-performance liquid chromatographic method for the determination of polyphosphoinositides in brain

A simple high-performance liquid chromatographic method for the determination of endogenous phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in brain has been developed. PIP and PIP2 were derivatized with 9-anthryldiazomethane to yield (9-anthryl)PIP and di(9-anthryl)-PIP2. The derivatives were separated on a reversed-phase column using isocratic elution and detected with a uv detector. The detection limits of PIP and PIP2 were 0.25 micrograms. The method with uv detection was sufficiently sensitive to measure the concentrations of PIP and PIP2 in rat brain. The levels of PIP and PIP2 were increased in developing rat brain and were decreased after 10 min of ischemia.     

A new high-performance liquid chromatographic method for determination of warfarin enantiomers

Warfarin is the most common agent used for control and prevention of venous as well as arterial thromboembolism. Although warfarin is administered as a racemic mixture of two stereoisomers (S and R), the S-form is mainly responsible for the anticoagulant effect. The anticoagulant effect of the drug is monitored by analysis of prothrombin complex (International Normalised Ratio,INR). In some cases, however, the measurements of plasma warfarin concentration are needed. Here, we present a new, rapid, sensitive and cost-effective HPLC-method for the determination of warfarin enantiomers in plasma. The chromatographic system consisted of Waters 616 gradient pump, Waters 996 photo diode array detector, Gilson 230 autoinjector and Pirkle (R,R) Whelk-O1 column (25 cmx4.6 mm I.D., 5 microm). An isocratic mobile phase of methanol/acetonitrile/water (50/10/40, v/v) with 0.1% glacial acetic acid was used. The follow rate was 1 mL/min. Data analysis was carried out with Waters Millennium32. The absorbance at 305 nm was measured with a total run-time of 15 min. Method linearity was studied by establishing regression data containing eight points over the range 0.08-10 microg/mL. In this range, warfarin showed to be linear (r2=0.9997 for S-warfarin and r2=0.9998 for R-warfarin). The limit of detection in plasma was 16 ng/mL for S-warfarin and 18 ng/mL for R-warfarin. Limit of quatitation was defined as 10xLOD. The extraction recovery was approximately 80%. Also the relation between INR and warfarin concentration was investigated. As expected, there was a low correlation between these two variables (r=0.23, y=0.3044x+0.9712). This method offers a rapid and cost-effective determination of warfarin enantiomers in human plasma.     

Simple high-performance liquid chromatographic method to verify the direct barbituric acid assay for urinary cotinine

An isocratic HPLC method is described to determine urinary concentrations of nicotine and cotinine after derivatization with cyanogen chloride and barbituric acid. This method has been used to assess the reliability of the direct barbituric acid assay to determine smoking status. It is concluded that the direct barbituric acid assay is a very reliable indicator of smoking status, provided that urine blank samples are prepared to correct for background absorbance. If the direct barbituric acid assay is in disagreement with self-reported smoking status, this HPLC procedure is a useful method to resolve the discrepancy.     

Rapid, sensitive high-performance liquid chromatographic method for the determination of cyclosporin A and its metabolites M1, M17 and M21

Cyclosporin A (CyA) and its metabolites seem to have nephro-, hepato- and neurotoxic side effects. Immunosuppressive therapy is a narrow path between the risk of rejection by inderimmunosuppression and toxic organ damage by overdosage. Thus CyA dosage must be calculated to avoid the risks of organ rejection through underdosage and toxic organ damage through overdosage or accumulation of metabolites. In routine monitoring of CyA therapy, it can be important to measure not only the patent drug but also the metabolites. We describe a rapid and isocratic high-performance liquid chromatographic method for measurement of CyA and its metabolites M1, M17 and M21 in whole blood. CyA was detected by ultraviolet absorption at 212 nm with a CN analytical column maintained at 50°C and recycling of hexane-isopropanol as mobile phase for improved long-term column stability and efficiency. The minimum detectable concentration of CyA and the three metabolites was 10 ng/ml blood. Our modified HPLC method for the determination of CyA and its metabolites is a simple (isocratic), rapid (the retention times were 7.1 min for CYD, internal standard, 8.9 min for CyA, 11.0 min for M21, 12.9 min for M17 and 16.3 min for M1) and economical method suitable for measuring the concentration of the major metabolite, M17, and for routine monitoring of CyA-treated patients.     

A simple and sensitive spectrophotometric method for the quantitative determination of solid supported amino groups

A simple and sensitive method for the quantitative determination of free amino groups on solid support is described. This approach is a modification of Ngo's [(1986) J. Biochem. Biophys. Methods 12, 349-354] method reported earlier. The method is based on the reaction of the solid support with an excess of 5'-O-(4,4'-dimethoxytrityl)-thymidine-3'-O-(2,4-dinitrophenyl) succinate (DTDS) in the presence of a catalytic amount of 4-dimethylaminopyridine. After removing the excess reagent, solid support is treated with perchloric acid to release 4,4'-dimethoxytrityl cation into the solution. The released 4,4'-dimethoxytrityl cation, which has a strong absorption at 498 nm (epsilon 498 = 70,000), is then determined spectrophotometrically. A comparative study of DTDS, N-succinimidyl-3-(2-pyridyldithio)propionate and 4,4-dimethoxytrityl chloride is also included. The method was found to be very useful to determine those amino groups which are available for functionalization of solid supports, especially, monitoring the functionalization of solid supports for affinity chromatography and synthesis of biopolymers.