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PubMed2003

A novel sensitive high-performance liquid chromatography/electrochemical procedure for measuring formaldehyde produced from oxidative deamination of methylamine and in biological samples.

Formaldehyde is a well-known environmental toxic hazard. It is also a product of oxidative deamination of methylamine catalyzed by semicarbazide-sensitive amine oxidase (SSAO). Increased SSAO-mediated deamination has been implicated in some pathophysiological conditions, such as diabetic complications. The measurement of formaldehyde in the enzymatic reactions and in vivo production using conventional methods was not straightforward due to limitations of selectivity and sensitivity. A novel high-performance liquid chromatography (HPLC)/electrochemical procedure for the measurement of formaldehyde has been developed. The measurement is based on the formation of adducts between formaldehyde and dopamine. These adducts can be selectively purified and concentrated using a batch method of alumina absorption, separated by HPLC, and electrochemically quantified. The method is highly selective and substantially more sensitive, i.e., detection of picomole levels of formaldehyde, than the conventional methods. The procedure not only facilitates the assessment of SSAO activity in vitro but also is useful for assessing formaldehyde in tissues and biological fluids.

Animals↗
PubMed1989

Chromosome aberrations in peripheral blood lymphocytes of welders and characterization of their exposure by biological samples analysis.

Chromosomal aberrations in cultured lymphocytes obtained from 55 welders and 55 matched controls were analyzed. Depending on the welding techniques and the nature of the consumables and metals welded, three separate groups of welders were examined. Chromium, nickel, and manganese levels in serum and urine were measured to assess the exposure to welding fumes. A statistically significant increase of chromosomal aberrations was found in one of the three analyzed groups of welders. This group used the semi-automatic metal active gas welding process with cored wire containing nickel for welding mild steel. These welders had significantly higher concentrations of serum and urine manganese and, unlike the other welders, significantly elevated concentrations of nickel, both in serum and urine. However, no significant correlations between nickel or manganese levels and the frequency of chromosomal aberrations were found. There was a significant correlation between the length of welding employment of these welders and the frequency of chromosomal breaks, although there was no significant correlation between age and the frequency of chromosomal aberrations. The other two groups of welders, for which the analyses of biologic fluids proved chromium and manganese exposure, had no statistically significant higher frequency of chromosomal aberrations. One of these groups used the manual metal arc welding process with coated electrodes for welding mainly mild steel and the other group used the tungsten inert gas welding process for welding stainless steel. A significant correlation between the daily amount of cigarettes smoked and the frequency of chromosomal breakages, in controls as in welders, was observed. The present data indicate that certain welding processes may generate fumes that seem to have a clastogenic activity.

Adult↗
PubMed1993

Levels of selected elements (Fe, As, Cd, Pb, Zn, Mn) in biological samples from ferrochromium workers.

A long-term exposure to metals poses the question of whether the impaired health condition is caused exclusively by the quantitatively dominant exposure metal. In 67 workers exposed to ferrochromium alloys on average 12.5 +/- 1.09 years (mean +/- SE), the subgroups of ferrochromium workers and operators of dust removing equipment, average Cr values in urine were 0.233 +/- 0.032 mumol.l-1 (mean +/- SE). In the control group (K) the average Cr values were 0.034 +/- 0.004 mumol.l-1 of urine (mean +/- SE). The levels of plasmatic Fe were assessed photometrically; the levels of Fe, As, Cd, Pb, Mn and Zn in hair, urine and blood were assessed by atom-absorption spectrophotometry (Varian). The results were compared with the control group not significantly different in age (t-test). All the values were correlated with age, years of exposure and the number of cigarettes smoked during life. The values of the single selected metals in furnace workers and operators of dust removing devices were not higher than the limits set for the groups of workers exposed to metals. But compared to the original biological background, there was observed a significant Cd increase (hair, blood, urine) year by year. Average values of As, Pb, Fe, and Mn in hair of the exposed workers were also higher compared to the group K. As and Mn values in hair were increasing up with the years of exposure (r = 0.465, p < 0.05; r = 0.580, p < 0.001). No expected higher values of plasmatic Fe were found. Zn values in hair were falling down with the years of exposure as well as with age, in all groups including the group K. The authors discuss the possible causes of the quantitative changes in the selected metals in connection with professional exposure and non-professional effects (especially smoking). Higher cancer incidence of the organs affected has not been observed so far in the study plant. (Tab. 2, Fig. 8, Ref. 41.)

Adult↗
PubMed1986

Determination of ciprofloxacin in biological samples by reversed-phase high performance liquid chromatography.

Previously reported high performance liquid chromatography (HPLC) assays for ciprofloxacin have used cumbersome fluorescence detection. UV absorbance is more commonly used for assay of antibiotics. Separation of ciprofloxacin and nalidixic acid (internal standard) was achieved using UV absorption at 313 nm, and a reversed phase C-18 Nova-Pak column. The mobile phase consisted of 35% phosphate buffers adjusted to pH 7.4, 65% methanol, and 5.5 mM hexadecyltrimethylammonium bromide. Retention times were 4.3 and 7.3 min, respectively, for ciprofloxacin and nalidixic acid. Serum sample preparation involved protein precipitation with acetonitrile (1:2), followed by methylene chloride and 2-propanol extraction (90:10). After evaporation, reconstitution with a minimal volume of mobile phase allowed for 5X concentration of the sample. The sensitivity limit of the assay was 0.06 microgram/ml. The response was linear from 0.125 to 10.0 micrograms/ml (r greater than 0.999). The coefficient of variation for day-to-day analysis was less than 5.3%, and the recovery was 55%. When compared with microbiological assay in serum, the correlation coefficient was 0.922 (n = 58). This HPLC method using UV detection provided comparable results to those obtained by fluorimetry. Data from three pharmacokinetic studies showed this method to be reliable and accurate.

Biological Assay↗
PubMed2004

Synergistic use of synchrotron radiation techniques for biological samples in solution: a case study on protein-ligand recognition by the peroxisomal import receptor Pex5p.

Circular dichroism spectropolarimetry and X-ray scattering data, obtained using synchrotron radiation, can yield information about the secondary and tertiary structure of proteins in solution. These techniques have been used to analyse the architecture and shape of a complex of two proteins in solution. The crystal structures of two separate proteins, the C-terminal domain of Pex5p and SCP2, are available but their complex has not previously been structurally characterized. Circular dichroism spectropolarimetry indicated that complex formation requires little secondary structure rearrangement. X-ray scattering data fit an elongated irregular 'shoe'-shaped particle of the complex of the two proteins, with dimensions of the order of 30 A x 40 A x 90 A. Comparison with the known crystal structures suggests that this 'shoe' shape requires a conformational change of the C-terminus of SCP2 to appropriately locate its peroxisomal targeting signal type-1 recognition motif into the binding pocket of the Pex5p receptor. Implications of the combined use of synchrotron-based circular dichroism spectropolarimetry and X-ray scattering in structural biology and proteomics are discussed.

Binding Sites↗
PubMed2004

Single-drop microextraction combined with low-temperature electrothermal vaporization ICPMS for the determination of trace Be, Co, Pd, and Cd in biological samples.

A new method of single-drop microextraction combined with low-temperature electrothermal vaporization (LTETV)-ICPMS was proposed for the determination of trace Be, Co, Pd, and Cd with benzoylacetone (BZA) as both extractant and chemical modifier. Several factors that influence the microextraction efficiency, such as sample flow rate, microdrop volume, and extraction time, were investigated and the optimized microextraction conditions were established. Be, Co, Pd, and Cd in the postextraction organic phase were directly determined by LTETV-ICPMS with the use of BZA as chemical modifier. The chemical modification of BZA in LTETV-ICPMS was studied, and the factors affecting the formation of chelates and vaporization/transportation of chelates were investigated. Under the optimized conditions, the detection limits of the method were 0.12, 0.99, 1.5, and 0.27 pg/mL for Be, Co, Pd, and Cd, and the relative standard deviations for 0.1 ng/mL Be, Co, Pd, and Cd were 16, 14, 14, and 11%, respectively. After 10 min of extraction, the enrichment factors were 160 (Be), 125 (Co), 40 (Pd), and 180 (Cd). The proposed method was applied to the determination of trace Be, Co, Pd, and Cd in biological reference materials, and the determined values were in good agreement with the certified values.

Beryllium↗
PubMed2002

Comparison of electrospray, atmospheric pressure chemical ionization, and atmospheric pressure photoionization in the identification of apomorphine, dobutamine, and entacapone phase II metabolites in biological samples.

The applicability of different ionization techniques, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and a novel atmospheric pressure photoionization (APPI), were tested for the identification of the phase II metabolites of apomorphine, dobutamine, and entacapone in rat urine and in vitro incubation mixtures (rat hepatocytes and human liver microsomes). ESI proved to be the most suitable ionization method; it enabled detection of 22 conjugates, whereas APCI and APPI showed only 12 and 14 conjugates, respectively. Methyl conjugates were detected with all ionization methods. Glucuronide conjugates were ionized most efficiently with ESI. Only some of the glucuronides detected with ESI were detected with APCI and APPI. Sulfate conjugates were detected only with ESI. MS/MS experiments showed that the site of glucuronidation or sulfation could not be determined, since the primary cleavage was a loss of the conjugate group (glucuronic acid or SO3), and no site-characteristic product ions were formed. However, it may be possible to determine the site of methylation, since methylated products are more stable than glucuronides or sulfates. Furthermore, the loss of CH3 is not necessarily the primary cleavage, and site characteristic products may be formed. Identification and comparison of conjugates formed from the current model drugs were successfully analyzed in different biological specimens of common interest to biomedical research. A fairly good relation was obtained between the data from in vivo and in vitro models of drug metabolism.

Animals↗
PubMed1994

Simultaneous determination of oxidized and reduced coenzyme Q and alpha-tocopherol in biological samples by high performance liquid chromatography with platinum catalyst reduction and electrochemical detection.

A convenient and sensitive high performance liquid chromatographic method with post-column platinum catalyst reduction and electrochemical detection was applied to the simultaneous determination of oxidized and reduced coenzyme Q (CoQ and CoQH2) and alpha-tocopherol (alpha-TP) in human and rat samples. After the separation of oxidized and reduced CoQ homologues (CoQ8-CoQ10 and CoQ8H2-CoQ10H2) and alpha-TP on a reversed-phase column, oxidized CoQ homologues were reduced on a platinum catalyst reduction column, and then all the reduced forms were quantified with an electrochemical detector operated in the oxidation mode (+ 0.6 V vs. Ag/AgCl). The order of elution was alpha-TP, CoQ8H2, CoQ9H2, CoQ8, CoQ10H2, CoQ9, and CoQ10 and all were well separated within 13 min. The detection limits at a signal-to-noise ratio of 3 were 20 pg for alpha-TP, 70 pg for CoQ8 and CoQ8H2, and 100 pg for CoQ9, CoQ9H2, CoQ10 and CoQ10H2. Quantitative recoveries (93-102%) from serum and tissues were obtained for all the analytes studied. This method was applied satisfactorily to the simultaneous determination of CoQ, CoQH2 and alpha-TP in biological materials.

Adult↗
PubMed2005

Critical review of the application of liquid chromatography/mass spectrometry to the determination of pesticide residues in biological samples.

A critical review is made on the use of hyphenated liquid chromatography/mass spectrometry (LC-MS) for the identification and quantification of pesticides and their metabolites in human biosamples (whole blood, plasma, serum and urine). The first applications of LC-MS in this field began in the early 1990s. Since then, increasing interest has been shown in applying this powerful technique, with most applications dealing with the determination of a variety of chemically diverse metabolites in urine. The use of different LC-MS interfaces and mass spectral detection modes are discussed. Special attention is given to tandem mass spectrometry (MS/MS) due to its inherent advantages of increased sensitivity and selectivity, as well as its advantages for identification and confirmation of analytes in samples. Quantification can be severely affected by matrix effects, the most common being inhibition of the ionisation of analytes in the mass spectrometer, which leads to unacceptable errors if no correction is made. Different approaches can be employed to minimise this undesirable matrix effect, the preferred being the use of labelled internal standards (when available) in isotope dilution methods or the application of an efficient clean-up, performed off-line or automated on-line. Adequate criteria for confirming the identities of residues detected are required in order to avoid false positives. The criterion most commonly used with a triple quadrupole instrument is the monitoring of two MS/MS transitions together with the ion abundance ratio. TOF mass analysers are seldom used in pesticide residue analysis despite their improved resolution and mass accuracy characteristics, which makes them very suitable for confirmation purposes. The main reasons for the relative unpopularity of TOF MS in residue analysis are its limited sensitivity and its high acquisition cost. In this paper, we present a critical assessment on current techniques, trends and future developments, and give illustrative examples to point out the main characteristics of LC-MS for pesticide residue analysis in biological fluids.

Animals↗
PubMed2006

Sol-gel zirconia coating capillary microextraction on-line hyphenated with inductively coupled plasma mass spectrometry for the determination of Cr, Cu, Cd and Pb in biological samples.

A sol-gel zirconia coating was developed for the preconcentration/separation of trace Cr, Cu, Cd and Pb by capillary microextraction, and the adsorbed analytes were on-line eluted for detection using inductively coupled plasma mass spectrometry (ICP-MS). By immobilizing sol-gel zirconia on the inner surface of a fused-silica capillary, the sol-gel zirconia coating was simply prepared. Its adsorption properties, stability and the factors affecting the adsorption behaviors of Cr, Cu, Cd and Pb were investigated in detail. In the pH range from 7.8 to 10, the zirconia-coated capillary (35 cm x 0.15 mm) is selective towards Cr, Cu, Cd and Pb, and the analyzed ions could be desorbed quantitatively with 0.2 mL of 0.5 mol/L HNO(3) at a rate of 0.2 mL/min. With a consumption of 1.25 mL sample solution, an enrichment factor of 6.25, and detection limits (3sigma) of 9.9 pg/mL Cr, 17.9 pg/mL Cu, 4.5 pg/mL Cd and 3.7 pg/mL Pb were obtained. The precisions for nine replicate measurements of 1 ng/mL Cr, Cu, Cd and Pb were 4.9% Cr, 2.2% Cu, 2.0% Cd and 3.2% Pb (RSD), respectively. The proposed procedure has been applied to the determination of Cr, Cu, Cd and Pb in human urine, which was subjected to microwave-assisted digestion prior to analysis, and the recoveries for these elements were 89.2-101.8%. In order to validate the developed procedure, a NIES No.10-a Rice Flour-Unpolished certified reference material and a BCR No. 184 Bovine Muscle certified reference material were analyzed, and the results are in good agreement with the certified values.

Biological Assay↗
PubMed2006

High-resolution solid modeling of biological samples imaged with 3D fluorescence microscopy.

Optical-sectioning, digital fluorescence microscopy provides images representing temporally- and spatially-resolved molecular-scale details of the substructures of living cells. To render such images into solid models for further computational analyses, we have developed an integrated system of image acquisition, processing, and rendering, which includes a new empirical technique to correct for axial distortions inherent in fluorescence microscopy due to refractive index mismatches between microscope objective immersion medium, coverslip glass, and water. This system takes advantage of the capabilities of ultra-high numerical aperture objectives (e.g. total internal reflection fluorescence microscopy) and enables faithful three-dimensional rendering of living cells into solid models amenable to further computational analysis. An example of solid modeling of bovine aortic endothelial cells and their nuclei is presented. Since many cellular level events are temporally and spatially confined, such integrated image acquisition, processing, rendering, and computational analysis, will enable, in silico, the generation of new computational models for cell mechanics and signaling.

Animals↗
PubMed2003

Trypsin activity assay in substrate-specific one- and two-dimensional gels: a powerful method to separate and characterize novel proteases in active form in biological samples.

To separate and identify the proteases, a substrate-specific, sensitive assay in sodium dodecyl sulfate (SDS)-polyacrylamide gels after two-dimensional (2-D) electrophoresis has been developed. This method allows simultaneous determination of protease cleavage specificity, molecular weight, isoelectric point, and if necessary, amino acid sequencing. After isoelectric focusing in immobilized pH gradient (IPG) strips (pH 6-11) (first dimension), trypsin was electrophoresed in 12% SDS polyacrylamide gels (second dimension) copolymerized with Boc-Gln-Ala-Arg-MCA (4-methyl-coumaryl-7-amide). The gels were washed in cold 2.5% Triton X-100 and water, and incubated in assay buffer (6.3 mM Bicine, 100 mM NaCl). Trypsin cleavage of the peptide-MCA generated fluorescent 7-amino-4-methyl-coumarin. In 1-D gels, as low as 500 pg trypsin could be detected and trypsin band volumes correlated linearly with the amounts of trypsin (R(2) = 0.999). In 2-D gels, the lowest amount of trypsin detected was 1 ng. The linear regression of spot volume and loading amount was still good (R(2) = 0.974). To optimize renaturation conditions, 5x5 min washes with 2.5% Triton X-100 and water, respectively, gave the strongest band volume. For fluorescence development, an assay buffer at pH 9 was the best; incubation at 37 degrees C for 30 min was sufficient. The method has application for identifying novel proteases as it does not rely on antibodies.

Biological Assay↗
PubMed2002

Arachidonate geometrical isomers generated by thiyl radicals: the relationship with trans lipids detected in biological samples.

The presence of trans fatty acids in mammalians is attributed to exogenous sources; nevertheless, trans isomers could be easily formed by free radical-catalyzed isomerization processes in vivo. The isomerization of methyl arachidonate (all-cis isomer) catalyzed by thiyl radical is proposed as a methodology applicable in biochemical laboratories, which produces mono- and di-trans isomers. Carbon-13 nuclear magnetic resonance spectroscopy shows that the carbon atom in position 15 is characteristic for each mono- and di-trans isomer. Antioxidants, such as alpha-tocopherol and all-trans-retinol acetate, inhibited the isomerization process. Trans phospholipids are formed in erythrocyte membranes by exposing blood to gamma-irradiation in the presence of thiols, which is in contradiction with the known role of these compounds as radioprotectors. Trans isomers are also analyzed in tissues harvested from breast cancer patients and compared to the adipose breast tissue taken a few centimeters from the edge of the tumor from the same patient. This work is generally aimed at contributing to the debate on trans fatty acids and stimulating a reconsideration of the current view on the exclusive presence of cis double bonds in cell membranes by studying radical processes that could affect or protect this natural configuration.

Adipose Tissue↗
PubMedDate not supplied

Electrochemiluminescence from tunicate, tunichrome-metal complexes and other biological samples.

Low level intrinsic electrochemiluminescence (ECL) was induced from body fluids and homogenized tissues of oysters and several species of tunicates. No significant ECL was detected in human blood cell lysates, or bovine haematin, but minor ECL was observed in avian blood cell lysates. Both terrestrial grass and seagrass exhibited ECL, which is probably attributable to chlorophyll, since dead (brown) grass did not demonstrate ECL. It was postulated that organic-metal complexes in marine invertebrates were, at least in part, responsible for the intrinsic ECL, since such animals are known to be rich in organically bound metals. However, alternative biochemical mechanisms for the observed ECL, which do not involve metal chelates, are possible. Various metal ions were added to the invertebrate preparations to determine whether exogenous metals could enhance or inhibit the ECL reactions. Strongly oxidizing metal ions such as Ag+, Au+, Cu2+, Hg2+ and Sb2+ at > or = 100 ppm severely inhibited the intrinsic ECL response. No statistically significant ECL enhancement due to addition of metal ions was noted. ECL "profiles' were generated which demonstrated differences in the ECL responses of individual tunicate preparations to the presence of various exogenous metal ions. Differences in ECL profiles may represent differences in types or levels of endogenous metal chelates or other biochemical constituents. In addition, synthetic tunichromes (tunicate pigments) were analysed for ECL in the presence and absence of various added metal ions. One synthetic tunichrome isomer demonstrated a specific ECL interaction with Hg2+, while the other demonstrated broader ECL activity with several metal ions.

Animals↗
PubMed1998

A flow immunoassay for studies of human exposure and toxicity in biological samples.

This paper describes a heterogeneous competitive flow immunoassay with a high sample throughput which can be used for the screening of smaller analytes in various samples. The method is based on off-line incubation of the analyte (Ag), a fluorescent labelled tracer (Ag*) and the corresponding antibody (Ab). The separation of bound (Ab-Ag*) and free tracer (Ag*) is based on a size exclusion and reversed phase mechanism utilizing a restricted access (RA) column. The column traps the free unbound tracer (Ag*) in its hydrophobic (C18) inner cavity but excludes the large Ab-Ag* complex, which is passed on and measured by the fluorescence detector. The flow immunoassay was developed using the triazine herbicide atrazine as a model compound owing to its human toxicity and widespread use. A sample throughput of 80 samples per hour and a detection limit of 300 pg ml-1 in water were obtained. Urine samples were successfully applied for direct injections into the flow system, while for human plasma samples an additional clean-up step using solid phase extraction was efficiently included where pure extract is obtained with the highly stable and biocompatible extracting column material. The resulting detection limits for atrazine in plasma and water samples using this clean-up and trace enrichment procedure were found to be 2 ng ml-1 and 20 pg ml-1 respectively.

Animals↗
PubMed1999

Iodine speciation in biological samples by capillary electrophoresis-inductively coupled plasma mass spectrometry.

A hyphenation of capillary electrophoresis (CE) to inductively coupled plasma mass spectrometry (ICP-MS) was employed for the speciation of iodine. The separation method used a buffer sandwich of phosphate (pH 2.3), NaOH, sodium dodecyl sulfate (SDS) and borate buffer (pH 8.3) for stacking, aiming at sufficient separation of iodide, iodate, thyroxine (T4) and triiodothyronine (T3). These four iodine species were separated within 15 min and subsequently detected during a pressure-driven detection step (baseline-separated) at 19.5, 29.1, 36.6 and 42.2 s. The detection limits were determined at 0.08 microg I/L (iodide), 0.3 microg I/L (iodate), 3.5 microg I/L (thyroxine) and 2.5 microg I/L (triiodothyronine). This method was applied on iodine speciation in human serum ("healthy" and after thyroid gland operation) and urine. The serum from the healthy person contained iodide (13 microg I/L), T4 (61 microg I/L) and T3 (7.5 microg I/L), whereas the serum from the thyroid-operated person lacked T3. As no "free" I-hormones are known in serum, the role of the thyroid hormone binding globulin (TBG) was investigated. We found that spiked T4 or T3 immediately bound to TBG. Investigations on human urine showed only a peak for iodide.

Electrophoresis, Capillary↗
PubMed2000

The need for chromatographic and mass resolution in liquid chromatography/tandem mass spectrometric methods used for quantitation of lactones and corresponding hydroxy acids in biological samples.

Because of the potential in-source conversion between a lactone and the corresponding hydroxy acid, it has been recognized that a liquid chromatography/tandem mass spectrometric (LC/MS/MS) method developed for quantitation of a lactone drug in the presence of its hydroxy acid metabolite (or vice versa) must incorporate chromatographic separation between the two compounds, unless in-source conversion between the two compounds has been eliminated by the appropriate selection of the LC/MS/MS parameters. We now report that chromatographic separation between a lactone and its hydroxy acid will be required under certain LC/MS/MS conditions used even in the absence of in-source conversion. This is due to the fact that the 18-mass-unit difference between a lactone and its hydroxy acid is, by coincidence, different by only one mass unit from the 17-mass-unit difference between the [M + H](+) and [M + NH(4)](+) ions of the lactone or the hydroxy acid. Thus, the [M + H](+) ion of a hydroxy acid is higher than the [M + NH(4)](+) ion of its lactone by only one mass unit. Therefore, in a method developed for quantitation of a hydroxy acid drug utilizing a selected-ion-monitoring (SRM) scheme that incorporates its [M + H](+) ion as the precursor ion, the quantitation would be inaccurate due to the interference by the contribution of the A + 1 isotope response from the [M + NH(4)](+) ion of the lactone metabolite present in the sample, unless there is a chromatographic separation between the two compounds. This is true even if Q1 is operated under a unit-mass resolution. The implication of this type of interference, arising from the presence of both the [M + H](+) and [M + NH(4)](+) ions of a drug and its metabolite, to the selection of LC and MS conditions (including mass resolution) will be discussed using the data obtained with a model lactone drug and its hydroxy acid metabolite.

Chromatography, High Pressure Liquid↗
PubMed2000

On-line coupling of solid-phase extraction with mass spectrometry for the analysis of biological samples. II. Determination of clenbuterol in urine using multiple-stage mass spectrometry in an ion-trap mass spectrometer.

Solid-phase extraction (SPE) was coupled to ion-trap mass spectrometry to determine clenbuterol in urine. For SPE a cartridge exchanger was used and, after extraction, the eluate was directly introduced into the mass spectrometer. For two types of cartridges, i.e. C18 and polydivinylbenzene (PDVB), the total SPE procedure (including injection of 1 mL urine, washing, and desorption) has been optimised. The total analysis, including SPE, elution, and detection, took 8.5 min with PDVB cartridges, while an analysis time of 11.5 min was obtained with C18 cartridges. A considerable amount of matrix was present after extraction of urine over C18 cartridges, resulting in significant ion suppression. With PDVB cartridges, the matrix was less prominent, and less ion suppression was observed. For single MS, a detection limit (LOD) of about 25 ng/mL was found with PDVB cartridges. With C18 cartridges an LOD of only about 50 ng/mL could be obtained. Applying tandem mass spectrometry (MS/MS) did not lead to an improved LOD due to an interfering compound. However, a considerable improvement in the LOD was obtained with MS3. The selectivity and sensitivity were increased by the combination of efficient fragmentation of clenbuterol and reduction of the noise. Detection limits of 2 and 0.5 ng/mL were obtained with C18 and PDVB cartridges, respectively. The ion suppression was 4 to 45% (concentration range: 250 to 1.0 ng/mL) after extraction of urine using PDVB cartridges, and up to 70% ion suppression was observed using C18 cartridges. With MS4, no further improvement in selectivity and sensitivity was achieved, due to inefficient fragmentation of clenbuterol and no further reduction of noise.

Amino Acid Sequence↗