Simultaneous determination of barbiturates in human biological fluids by direct immersion solid-phase microextraction and gas chromatography-mass spectrometry

Simultaneous determination of seven barbiturates in human whole blood and urine by combining direct immersion solid-phase microextraction (DI-SPME) with gas chromatography-mass spectrometry (GC-MS) is presented. The main parameters affecting the DI-SPME process, such as SPME fibers, salt additives, pHs, extraction temperatures and immersion times were optimized for simultaneous determination of the drugs. The extraction efficiencies were 0.0180-0.988 and 0.0156-2.76% for whole blood and urine, respectively. The regression equations of the drugs showed excellent linearity for both samples; the correlation coefficients (r(2)) were 0.994-0.999. The detection limits for whole blood were 0.05-1 microg x ml(-1), and those for urine 0.01-0.6 microg x ml(-1). Actual quantitation could be made for pentobarbital in whole blood and urine obtained from volunteers, who had been orally administered a therapeutic dose of the drug. The DI-SPME/GC-MS procedure for barbiturates established in this study is simple and sensitive enough to be adopted in the fields of clinical and forensic toxicology.     

Determination of cadmium in blood, plasma, and urine by electrothermal atomic absorption spectrophotometry after isolation by anion-exchange chromatography

The determination of cadmium in whole blood, urine, or plasma by atomic absorption using electrothermal atomization is described. In preparation for atomic absorption analysis, cadmium was concentrated on an anion-exchange column, significantly lowering the limit of detection and allowing for the first time the accurate and precise determination of plasma cadmium concentrations in persons/animals with low-level cadmium exposures. Recovery of 109Cd from spiked whole blood, plasma, and urine into supernatants of nitric acid-deproteinated samples averaged 99, 100, and 95%, respectively. Anion-exchange isolation of the anionic chlorocadmium complex removed 99.8% of the major elements associated with a deproteinated whole blood sample. The recovery of 109Cd from the anion-exchange column was 92.2 +/- 0.9% (mean +/- SE, N = 35). The separation of cadmium from constituents in blood, urine, or plasma in this manner allowed comparison of unknown samples to aqueous standards with a defined acid matrix using commercially available acids. The mean intra-assay coefficient of variation (CV) was 12 +/- 3% (mean +/- SE, N = 6) for blood, plasma, and urine samples having cadmium concentrations of 0.1-0.8 microgram/liter. The interassay CV was 13% (N = 7) for a blood sample containing 0.6 microgram Cd/liter. The recovery of known amounts of cadmium added to blood, plasma, and urine in the range of 0.2 to 5.0 micrograms Cd/liter was 97 +/- 6% (mean +/- SE, N = 4).     

Analysis of methanol or formic acid in body fluids by headspace solid-phase microextraction and capillary gas chromatography

Methanol and its metabolite formic acid have been found extractable from human whole blood and urine by headspace solid-phase microextraction (SPME) with a Carboxen/polydimethylsiloxane fiber. The headspace SPME for formic acid was carried out after derivatization to methyl formate under acidic conditions. The determinations of both compounds were made by using acetonitrile as internal standard (IS) and capillary gas chromatography (GC) with flame ionization detection. The headspace SPME–GC gave sharp peaks for methanol, methyl formate and I.S.; and low background noises for whole blood and urine samples. Extraction efficiencies were 0.25–1.05% of methanol and 0.38–0.84% formic acid for whole blood and urine. The calibration curves for methanol and formic acid showed excellent linearity in the range of 1.56 to 800 and 1.56 to 500 μg/0.5 ml of whole blood or urine, respectively. The detection limits were 0.1–0.5 μg/0.5 ml for methanol and 0.6 μg/0.5 ml for formic acid for both body fluids. The within-day relative standard deviations in terms of extraction efficiency for both compounds in whole blood and urine samples were not greater than 9.8%. By using the established SPME method, methanol and formic acid were successfully separated and determined in rat blood after oral administration of methanol.     

Sensitive determination of alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine using gas chromatography-mass spectrometry

We devised a sensitive and simple method to determine alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine, using gas chromatography-mass spectrometry (GC-MS). AMT and 5MeO-DIPT were extracted using an Extrelut column with an internal standard, bupivacaine, followed by derivatization with acetic anhydride. The derivatized extract was used for GC-MS analysis of EI-SIM mode. The calibration curves of AMT and 5MeO-DIPT were linear in the concentration range from 10 to 750 ng/ml in both blood and urine samples. The method detection limit (MDL) of AMT and 5MeO-DIPT were 1 ng/ml each in whole blood and 5 ng/ml each in urine. This method should be most useful to accurately determine the presence of these drugs in blood and urine in clinical and forensic cases.     

The in vitro loss of penicillamine in plasma,albumin solutions,and whole blood: Implications for pharmacokinetic studies of penicillamine

The recent development of a high performance liquid chromatography assay method for the analysis of penicillamine in biological samples such as plasma, whole blood, and urine has provided a specific and sensitive assay method to aid in the study of penicillamine pharmacokinetics. Several investigators have reported measuring the plasma concentration of penicillamine. Some of these investigators have indicated that the plasma must be assayed immediately. However, such restrictions can limit the feasibility of a pharmacokinetic study. The results of this paper demonstrate the instability of penicillamine in plasma, albumin solutions, and whole blood. The rate of loss of penicillamine was shown to be influenced by the concentration of albumin. As a result of the significant loss of penicillamine over a short period of time, plasma or whole blood samples must be deproteinated immediately upon collection to avoid the loss of reduced penicillamine. Methods are presented for the preparation of biological samples so that the oxidation of penicillamine is prevented and the samples can be held for several days prior to analysis.     

Indicator ability of biosubstances in monitoring the moderate occupational exposure to toxic metals

In order to improve the monitoring system, watching influence of toxic metals on human health in industrial plants, indicator properties of different biosubstances were compared. Four types of samples (whole blood, plasma, urine, and hair) from 263 workers of the “Khimprom” chemical plant (Novocheboksarsk, Russia) were subjected to multielement analysis by ICP-AES/ICP-MS. 19–25 chemical elements, including main toxic metals (Cd, Hg, Pb, etc.) were determined. The results were calculated with regard to workers’ individual data on occupational exposure to chemical elements. Hair was found to be the most sensitive to toxic and conditionally toxic trace metals: Pb, Mn, Cr, Be, Ni, while occupational contact with macro elements (Na, P), trace metalloids (Si, B) and some other metals (Zn) was not reflected in hair. Whole blood relatively weakly indicated a moderate occupational level of metals except Pb and Mn, but effectively reflected deficiencies of essential elements: I, Cr, and shifts in K/Na ratio, which are likely to be secondary effects of harmful occupational factors. Blood plasma reflected only contact with Be, P; urine – only with Ni. In both whole blood and plasma the changes for the absolute majority of elements were similar. Thus, hair analysis is useful for monitoring the occupational exposure to toxic and conditionally toxic chemical elements, while a general estimation of occupational harmful influence on mineral metabolism requires simultaneous investigation of two biosubstances: hair and whole blood, or hair and blood plasma, with whole blood being more preferable. Analysis of urine is appropriate for monitoring particular chemical elements, e.g. nickel.     

High-performance liquid chromatographic method for determination of amodiaquine,chloroquine and their monodesethyl metabolites in biological samples

A high-performance liquid chromatographic method for determination of amodiaquine (AQ), desethylamodiaquine (DAQ), chloroquine (CQ) and desethylchloroquine (DCQ) in human whole blood, plasma and urine is reported. 4-(4-Dimethylamino-1-methylbutylamino)-7-chloroquinoline was used as internal standard. The drugs and the internal standard were extracted into di-isopropyl ether as bases and then re-extracted into an acidic aqueous phase with 0.1 M phosphate buffer at pH 4.0 for AQ samples and at pH 2.5 for CQ filter paper samples. A C(18) column was used and the mobile phase consisted of methanol-phosphate buffer (0.1 M, pH 3)-perchloric acid (250: 747.5:2.5, v/v). The absorbance of the drugs was monitored at 333 nm and no endogenous compound interfered at this wavelength. The limit of quantification in whole blood, plasma and urine was 100 nM for AQ and DAQ (sample size 100 microliter) as well as for CQ and DCQ in blood samples dried on filter paper. For 1000 microliter AQ and DAQ samples, the limit of quantification was 10 nM in all three biological fluids. The within-assay and between-assay coefficients of variations were always <10% at the limits of quantification. Plasma should be preferred for the determination of AQ and DAQ since use of whole blood may be associated with stability problems.     

Automated high-performance liquid chromatographic method for the determination of nedocromil sodium in human urine using bimodal column switching

An automated HPLC method is described for the determination of nedocromil sodium in human urine. An HPLC autosampler is used to inject urine samples onto a short reversed-phase column. This column acts as a concentration column and performs a preliminary extraction. The concentration column is automatically backflushed onto an ion-exchange column where final separation of nedocromil sodium from urine constituents occurs. Recovery, accuracy, precision, sensitivity and specificity were investigated. The method has been applied to urine samples from clinical studies, and the results were compared to those obtained using a radioimmunoassay developed previously.     

Determination of melarsoprol in biological fluids by high-performance liquid chromatography and characterisation of two stereoisomers by nuclear magnetic resonance spectroscopy

The analysis of melarsoprol in whole blood, plasma, urine and cerebrospinal fluid is described. Extraction was made with a mixture of chloroform and acetonitrile followed by back-extraction into phosphoric acid. A reversed-phase liquid chromatography system with ultraviolet detection was used. The relative standard deviation was 1% at concentrations around 10 μmol/l and 3–6% at the lower limit of determination (9 nmol/l in plasma, 93 nmol/l in whole blood, 45 nmol/l in urine and 10 nmol/l in cerebrospinal fluid). Melarsoprol is not a stable compound and samples to be stored for longer periods of time should be kept at −70°C. Plasma samples can be stored at −20°C for upt to 2 months. Chromatography showed that melarsoprol contains two components. Using nuclear magnetic resonance spectroscopy the two components were shown to be diastereomers which slowly equilibrate by inversion of the configuration at the As atom.     

Towards a rapid molecular diagnostic for melioidosis: Comparison of DNA extraction methods from clinical specimens

Optimising DNA extraction from clinical samples for Burkholderia pseudomallei Type III secretion system real-time PCR in suspected melioidosis patients confirmed that urine and sputum are useful diagnostic samples. Direct testing on blood remains problematic; testing DNA extracted from plasma was superior to DNA from whole blood or buffy coat.     

A rapid,sensitive method for the determination of 3-methylhistidine levels in urine and plasma using high-pressure liquid chromatography

A method is presented for the precolumn derivatization and subsequent high-pressure liquid chromatographic separation of 3-methylhistidine from urine and plasma. The solvent system is 10 mm sodium phosphate (pH 7.5) and acetonitrile. The elution can be performed isocratically and requires less than 10 min. Both fluorescent and ultraviolet detection may be utilized. This method is at least 10³ times more sensitive than conventional ion-exchange chromatography using ninhydrin. 3-Methylhistidine determinations performed on plasma and urine samples from normal volunteers correlated well with published literature values.     

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.     

A practical and reliable method for determination of urinary 3-methylhistidine

A practical and reliable semiautomated method for analysis of urinary 3-methylhistidine (3-MH) was designed combining the isolation of 3-MH by ion-exchange chromatography with the color reaction given by ninhydrin-orthopthalaldehyde (ninhydrin-OPT) reagent after alkalinization. 2 ml of urine were passed through disposable columns packed with an ion-exchange resin (Dowex 50-X8, 200–400 mesh) and the acidic and neutral amino acids were eluted with 10 ml of 0.2 M pyridine solution. Then, the 3-MH was quantitatively eluted and separated from histidine with a volume of 9 ml of a 1.5 M pyridine solution. Standard Autoanalyzer equipment was used for the automation of spectrophotometry. The method permits the analysis of 40 samples in duplicate per day. The 3-MH color reaction was linear for concentrations from 0.015 to 0.24 μ mol/ml. The mean recoveries of 3-MH from standards and urine were 98.6 ± 1.3 and 99.0 ± 1.3%, respectively. Duplicate determinations of urine samples showed a variation coefficient of 1.88%. An excellent agreement was obtained between urine samples analyzed by the present method and by an amino acid analyzer. The need for the elimanation of the interfering amino acids was clearly demonstrated.     

Determination of tryptophan and several of its metabolites in physiological samples by reversed-phase liquid chromatography with electrochemical detection

A new method for the concurrent assay of three tryptophan metabolites at the picomole level is described. The method has been developed for blood, urine, cerebrospinal fluid, and tissue samples such as whole brain, brain parts, and endocrine glands. Tryptophan itself, serotonin, and 5-hydroxyindoleacetic acid are isolated initially on extraction columns, eluted with a suitable solvent, and injected onto a liquid chromatograph with an amperometric detector. This general approach may be applicable to a variety of other tryptophan metabolites and should be useful in both research and clinical investigations.     

Measurement of trimethylselenonium ion in human urine

A comparison is made between two methods (ion-exchange chromatography vs a difference method) for the quantitative measurement of trimethylselenonium ion (TMSe) in human urine. It is shown that the difference method yields reliable data only if TMSe constitutes a relatively large fraction of urine selenium. Under normal conditions of selenium intake in man, accurate measurement of this important metabolite can, at present, be carried out only with the ion-exchange chromatographic procedure. Preliminary data from a human subject employing stable isotope tracer methodology are given to show that the fraction of urine selenium present as TMSe varies with the level of intake as well as other factors.     

Hypoxanthine and xanthine levels determined by high-performance liquid chromatography in plasma, erythrocyte, and urine samples from healthy subjects: the problem of hypoxanthine level evolution as a function of time

The levels of hypoxanthine and xanthine are determined in plasma, erythrocyte, and urine samples by a reverse-phase high-performance liquid chromatographic (HPLC) method. The hypoxanthine concentration increases in erythrocyte and plasma samples when whole blood is stored at room temperature between sampling and centrifugation. Furthermore, the hypoxanthine concentration increases in erythrocyte samples when they are kept apart at room temperature before analysis, whereas the plasma hypoxanthine level remains constant. This result proves an endogenous formation of hypoxanthine in erythrocytes with time, at room temperature. These studies show the necessity of rigorous conditions for the collection, transport, and treatment of blood samples. In order to achieve accurate results, the blood must be centrifuged immediately after collection. The erythrocyte and plasma samples must be stored frozen until deproteinization and HPLC analysis. Under these conditions, the concentrations of hypoxanthine and xanthine in plasma are 2.5 +/- 1 and 1.4 +/- 0.7 microM, respectively. In erythrocyte samples, hypoxanthine concentration reaches 8.0 +/- 6.2 microM.     

Polyamines-an improved automated ion-exchange method

An accurate, precise, and improved automated cation-exchange chromatographic method with ninhydrin detection for the analysis of di- and polyamines (putrescine, cadaverine, spermidine, and spermine) has been developed. We have shown that different types of biological fluids such as urine, blood plasma, blood sera, tissue extracts, and cancer cell culture media can be analyzed under identical chromatographic conditions. The simplicity and precision of the method was achieved by eliminating the sample pre-separation and using an internal standard technique. Thus, not only has the sample preparation been simplified, but the accuracy and precision and sensitivity of the method have been greatly improved. Twenty-four unattended analyses were performed each day. With minor modifications of the instrument a two-fold analytical output can be achieved with analysis time cut to 30 min. The ruggedness and applicability of the method has been demonstrated in our laboratory during the past six months. More than two thousand urine and hundreds of other physiological samples have been analyzed by this method with a relative standard deviation from 3.3 to 7.8%, and recoveries of 94 to 97%.This automated ion-exchange chromatographic method for the polyamines will be useful to researchers in biological markers programs for monitoring the course of cancer and effectiveness of chemotherapy.     

Simultaneous determination of selegiline and desmethylselegiline in human body fluids by headspace solid-phase microextraction and gas chromatography-mass spectrometry

A method for the simultaneous determination of selegiline and its metabolite, desmethylselegiline, in human whole blood and urine is presented. The method, which combines a fiber-based headspace solid-phase microextraction (SPME) technique with gas chromatography-mass spectrometry (GC-MS), required optimization of various parameters (e.g., salt additives, extraction temperatures, extraction times and the extraction properties of the SPME fiber coatings). Pargyline was used as the internal standard. Extraction efficiencies for both selegiline and desmethylselegiline were 2.0-3.4% for whole blood, and 8.0-13.2% for urine. The regression equations for selegiline and desmethylselegiline extracted from whole blood were linear (r(2)=0.996 and 0.995) within the concentration ranges 0.1-10 and 0.2-20 ng/ml, respectively. For urine, the regression equations for selegiline and desmethylselegiline were linear (r(2)=0.999 and 0.998) within the concentration ranges 0.05-5.0 and 0.1-10 ng/ml, respectively. The limit of detection for selegiline and desmethylselegiline was 0.01-0.05 ng/ml for both samples. The lower and upper limits of quantification for each compound were 0.05-0.2 and 5-20 ng/ml, respectively. Intra- and inter-day coefficients of variation for selegiline and desmethylselegiline in both samples were not greater than 8.7 and 11.7%, respectively. The determination of selegiline and desmethylselegiline concentrations in Parkinson's disease patients undergoing continuous selegiline treatment is presented and is shown to validate the present methodology.     

The variability of arsenic in blood and urine of humans

BackgroundHumans are exposed to inorganic and organic arsenic. The total arsenic (As) concentration in urine is a commonly used biomarker of exposure. However, little is known about variability of As in biological fluids and the diurnal variation of As excretion.ObjectivesMain objectives were to assess the variability of As in urine, plasma (P-As), whole blood (B-As), and the blood cell fraction (C-As), and to assess diurnal variation of As excretion.MethodsSix urine samples were collected at fixed times during 24 h on two different days around one week apart among 29 men and 31 women. Blood samples were collected when the morning urine samples were delivered. The intra-class correlation coefficient (ICC) was calculated as the ratio of the between-individuals variance to the total observed variance.ResultsGeometric mean (GM) 24 h urinary excretions of As (U-As_24 h) were 41 and 39 µg/24 h on the two days of sampling. Concentrations of B-As, P-As and C-As were highly correlated with U-As_24 h and As in first void morning urine. No statistically significant differences were observed for the urinary As excretion rate between the different sampling times. A high ICC was observed for As in the cellular blood fraction (0.803), while ICC for first morning urine corrected for creatine was low (0.316).ConclusionsThe study suggests that C-As is the most reliable biomarker for use in exposure assessment of individual exposure. Morning urine samples have low reliability for such use. No apparent diurnal variation was observed in the urinary As excretion rate.     

A rapid gas-liquid chromatographic method for the quantitation of urinary estrone, estradiol-17 , estriol, and pregnanediol throughout pregnancy

A rapid, sensitive and inexpensive method is presented for the determination of urinary estrone (E₁), estradiol-17β (E₂), estriol (E₃), and pregnanediol (P₂) in pregnancy urine. The assay involves enzymatic hydrolysis, ion-exchange chromstography, and final quantitation of the steroids as heptafluorobutyric anhydride derivatives in a gas Chromatograph. Twenty to 30 samples may be prepared for gas-liquid chromatography in one working day. For six samples, the results including gas-liquid chromatography and calculation can be obtained eight hours after receiving the urine specimens thus competing in speed with radioimmunoassays. The method is sensitive enough to allow urines from less than six weeks after the last menstrual period to be quantitated.