Determination of cyanide in whole blood by capillary gas chromatography with cryogenic oven trapping

Cyanide, one of the most important toxic substances, has been found measurable with high sensitivity by capillary gas chromatography (GC) with cryogenic oven trapping upon injection of headspace (HS) vapor samples. The entire amount of cyanide in the HS sample could be cryogenically trapped prior to on-line GC analysis. A 0.5-mL volume of blood in the presence or absence of cyanide and propionitrile (internal standard, IS) was added to a vial containing 0.25 mL of distilled water, 0.3 g of Na2-SO4, 0.2 mL of 50% H3PO4, and 0.1 g of ascorbic acid (when needed), and the mixture was heated at 70 degrees C for 15 min. A 5-mL volume of the HS vapor was introduced into a GC capillary column in the splitless mode at -30 degrees C oven temperature that was programmed up to 160 degrees C for GC analysis with nitrogen-phosphorus detection. A sharp peak was obtained for cyanide under the present conditions, and backgrounds were very clean. The extraction efficiencies of cyanide and IS were 2.89-3.22 (100 or 500 ng/mL) and 2.42%, respectively. The calibration curve showed good linearity in the range of 25-1000 ng/mL and the detection limit was approximately 2 ng/mL. The coefficients of intraday and interday variations were 2.9 and 11.8%, respectively. The mean blood cyanide level measured for actual fire victims was 687 +/- 597 ng/mL (mean +/- SD, n = 9). Endogenous blood cyanide concentration for healthy subjects was 8.41 +/- 3.09 ng/mL (mean +/- SD, n = 6).     

Quantitative analysis of melphalan and its major hydrolysate in patients and animals by reversed-phase high-performance liquid chromatography

The detection of 4-bis-(2-hydroxyethyl)amino-1-phenylalanine (L-DOH) in blood samples taken from patients after treatment with melphalan [4-bis-(2-chloroethyl)amino-1-phenylalanine, L-PAM] suggests that the quantification of this major hydrolysate of L-PAM can be of considerable importance in L-PAM chemotherapy. A reversed-phase high-performance liquid chromatographic procedure has been developed for the quantitative analysis of both L-PAM and L-DOH in biological samples, with a detection sensitivity of 0.1 ppm. This method provides a distinct separation of L-PAM (retention time 12 min) and L-DOH (retention time 6.5 min), with no interference from the biological background (retention time 1.4--3 min).     

Quantitative determination of pentaerythrityl tetranitrate and its metabolites in human plasma by gas chromatography/mass spectrometry

Levels of anticeramide antibodies and S-100 antigen in leprosy patients with and without reaction are compared in this study. The increase in levels of IgM anti ceramide antibody in the tuberculoid group of patients with reaction, when compared to those without reaction, is significant (P < 0.05). Similarly, significant increase (P < 0.01) was observed in the borderline group with reaction. No significant change in anti ceramide antibody level was observed in the lepromatous group of patients with and without reaction. Mean levels of S-100 were slightly lower in all three groups of patients with reaction, but the differences were not statistically significant.     

Determination of propofol in rat whole blood and plasma by high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatographic method was developed for the determination of propofol, an intravenous anaesthetic agent, in rat whole blood or plasma samples. The method is based on precipitation of the protein in the biological fluid sample and direct injection of the supernatant into an HPLC system involving a C18 reversed-phase column using a methanol-water (70:30) mobile phase delivered at 1 ml/min. Propofol and the internal standard (4-tert.-octylphenol) were quantified using a fluorescence detector set at 276 nm (excitation) and 310 nm (emission). The analyte and internal standard had retention times of 6.3 and 10.5 min, respectively. The limit of quantification for propofol was 50 ng/ml using 100 microl of whole blood or plasma sample. Calibration curves were linear (r2=0.99) over a 1-10 microg/ml concentration range and intra- and inter-day precision were between 4-11%. The assay was applied to the determination of propofol whole blood pharmacokinetics and propofol whole blood to plasma distribution ratios in rats.     

Quantitative analysis of plasma acylcarnitines using gas chromatography chemical ionization mass fragmentography

A stable isotope dilution gas chromatography chemical ionization mass spectrometry (GC-CI-MS) method was developed for the quantitative profiling of plasma acylcarnitines. The clean-up procedure was comprised of a solid-phase cation exchange extraction using PRS-columns from which the acylcarnitines were eluted with a barium chloride solution. Isolated acylcarnitines were transformed into acyloxylactones and analyzed by positive GC-CI-MS using isobutane as reactant gas. The selected monitoring of a common ion at m/z [85]+ and the protonated molecular ion enabled a selective and sensitive detection of all C2-C18 acylcarnitines. An accurate quantitation was achieved by the use of stable isotope-labeled internal standards (C2-C18) and acylcarnitines could be analyzed in the sub-nanomolar range. Control values for C2-C18 acylcarnitines in plasma were established. Concentrations ranged from 0.02 micromol/L for C14-acylcarnitine to 4.90 micromol/L for C2-acylcarnitine. The diagnostic suitability of the method was demonstrated for patients with medium-chain acyl-CoA dehydrogenase deficiency and very long-chain acyl-CoA dehydrogenase deficiency.     

Determination of mitoxantrone in mouse whole blood and different tissues by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method was developed for the specific determination of mitoxantrone (MTO) in whole blood and different tissues of mice (liver, heart, spleen, kidneys). MTO was extracted into dichloromethane with ametantrone (AMT) as internal standard. The different tissues were homogenised in citrate buffer (pH 3.0) containing 20% ascorbic acid. Separation of MTO and AMT was carried out using a Nucleosil C18 column. The mobile phase consisted of acetonitrile (33%) and 0.16 M ammonium formate buffer, pH 2.7. UV detection was used at 658 nm. Baseline separation of AMT and MTO was achieved in all matrices. The calibration curves were linear in all matrices (r > 0.999) in the concentration range of 2-200 micrograms/l for whole blood and 2-700 micrograms/l for tissue homogenates, respectively. The within-day and between-day precision studies showed good reproducibility with coefficients of variation below 4.5% for whole blood and below 10% for tissue homogenates, respectively. The extraction efficiencies of MTO are 60% in whole blood and 38% in tissue homogenates. The method described is suitable for pharmacokinetic studies on the distribution of MTO in different tissues of mice.     

Peroxynitrite augments fMLP-stimulated chemiluminescence by neutrophils in human whole blood

The neutrophil respiratory burst was examined by the technique of luminol-dependent chemiluminescence (LDCL) triggered by submaximal concentrations of N-formyl-methionyl-leucyl-phenylalanine (fMLP) in diluted whole blood. We sought to identify the chemical species responsible for LDCL in whole blood, to examine the role of leukotriene B4 (LTB4) and other arachidonic acid metabolites as mediators of the fMLP signaling pathway, and to investigate the effect of peroxynitrite on this response. Both sodium azide and taurine significantly inhibited LDCL (93% inhibition with 100 microM azide, 52% inhibition with 10 mM taurine). More modest inhibition was seen with superoxide dismutase (SOD), catalase, the nitric oxide synthase inhibitor monomethyl-L-arginine (L-NMMA), and with inhibitors of the cyclooxygenase (indomethacin), lipoxygenase (AA-861; no effect), and cytochrome P-450 (SKF 525-A) pathways of arachidonic acid metabolism. The nitric oxide donor SIN-1 (1-100 microM) and peroxynitrite (10-300 microM) also augmented fMLP-induced LDCL. The augmentation seen with peroxynitrite and SIN-1 was attenuated by SOD. Despite the increase in LDCL, peroxynitrite caused a dose-related inhibition of fMLP-stimulated LTB4 release. In summary, our results indicate that (1) LDCL elicited by fMLP in diluted whole blood appears primarily mediated by hypochlorous acid derived from myeloperoxidase; (2) pretreatment with the nitric oxide donor SIN-1 or with peroxynitrite augments LDCL; and (3) LTB4 release does not contribute to fMLP-stimulated LDCL or in the modulation of LDCL by SIN-1 or peroxynitrite.     

Quantitative determination of constituents in various licorice roots by means of high performance liquid chromatography

During the course of our studies on the chemical constituents of botanically identified Chinese licorice roots of various origins, we have identified 15 kinds of saponins and 49 kinds of flavonoids (including flavonoid glycosides). With these chemical constituents at hand, we have carried out the chemical evaluation studies of licorice roots available in Japan by means of high performance liquid chromatography (HPLC). In the HPLC analysis using an ODS reversed-phase column, a combination of 1% aqueous acetic acid and acetonitrile was used as the mobile phase in a gradient solvent system, and the eluate was monitored with UV absorption at 254 nm (for saponins with a diene or a conjugated carbonyl moiety, liquiritin, and liquiritin apioside) or at 350 nm (for other flavonoid constituents). By our analytical method described here, quantitative analyses of saponin and flavonoid constituents were effected simultaneously. We have found an interesting piece of information concerning the origin of parent plants and their characteristic constituents such as saponins and flavonoids.     

Determination of acrolein in human urine by headspace gas chromatography and mass spectrometry

The aim of the present study is to examine whether noradrenergic neurons of the locus coeruleus (LC) of the rat contain monoamine oxidase (MAO) activity. Sections were processed initially for MAO enzyme histochemistry using tyramine as a substrate, followed by fluorescence immunohistochemistry for tyrosine hydroxylase (TH). In the LC, virtually all TH-immunoreactive neurons (i.e., noradrenergic neurons) were also positive for MAO. No MAO activity was found in any TH-negative neurons. Neurons in the LC have previously been shown to form dopamine during noradrenaline biosynthesis and to produce serotonin from exogenously administered l-5-hydroxytryptophan. Moreover, dopamine- and serotonin-degrading MAO activity has also been found in LC neurons. Therefore, our results indicate that MAO activity is localized within noradrenergic neurons in the LC and is likely involved in the degradation of dopamine that is endogenously synthesized, and also in the elimination of serotonin that is produced from exogenous precursors.     

Quantitative analysis of synthetic dyes in lipstick by micellar electrokinetic capillary chromatography

The separation of synthetic dyes, used as color additives in cosmetics, by micellar electrokinetic capillary chromatography (MEKC) is described in this study. The separation of seven dyes, namely eosine, erythrosine, cyanosine, rhodamine B, orange II, chromotrope FB and tartrazine has been achieved in about 3 min in an untreated fused silica capillary containing as background electrolyte a 25 mM tetraborate/phosphate buffer, pH 8.0, and 30 mM sodium dodecyl sulfate. The electrophoretic method exhibits precision and relatively high sensitivity. A detection limit (LOD, signal/noise = 3) in the range of 5-7.5 X 10(-7) M of standard compounds was recorded. Intra-day repeatability of all the studied dye determinations (8 runs) gave the following results (limit values), % standard deviation: 0.24-1.54% for migration time, 0.99-1.24% for corrected peak areas, 0.99-1.24% for corrected peak area ratio (analyte/internal standard) and 1.56-2.74% for peak areas. The optimized method was successfully applied to the analysis of a lipstick sample where eosine and cyanosine were present.     

Quantitative analysis of ceramide molecular species by high performance liquid chromatography

BACKGROUND: Hepatocyte growth factor/scatter factor (HGF/SF) is a potent mitogen for various neoplastic cells, including neoplastic bronchial epithelia. METHODS: Immunoreactive hepatocyte growth factor/scatter factor (HGF/SF) was measured in extracts prepared from 129 nonsmall cell lung carcinoma (NSCLC) specimens, using an enzyme-linked immunosorbent assay. These specimens represented 5 cases of solitary/localized bronchioloalveolar cell carcinoma (BAC), 4 cases of diffuse/infiltrative BAC, 90 cases of non-BAC adenocarcinoma, 25 cases of squamous cell carcinoma, and 5 cases of large cell carcinoma. RESULTS: The mean concentration of immunoreactive HGF/SF was more than 19-fold higher in tissue extracts from diffuse-type BAG (265.0 +/- 110.2 ng/100 mg protein) than in those from solitary-type BAC (13.9 +/- 15.9, P < 0.005), non-BAC adenocarcinoma (13.8 +/- 14.9, P < 0.001), squamous cell carcinoma (13.2 +/- 14.4, P < 0.001), or large cell carcinoma (11.2 +/- 6.5, P < 0.005). When immunohistochemical staining for HGF/SF was performed, intense HGF/SF staining was uniformly observed in diffuse-type BAC tumor cells, but not in solitary-type BAC. CONCLUSIONS: Although BAC is included as a subtype of adenocarcinoma in the World Health Organization classification, diffuse-type BAC should be considered a distinct biologic entity, at least in terms of HGF/SF expression, from solitary-type BAC or non-BAC adenocarcinoma. In addition, the solitary and diffuse forms of BAC are known to be associated with different prognoses; for the latter, the prognosis is much poorer than for the former. The results of this study may at least partly explain this difference in prognosis.     

Quantitative determination of propranolol, propranolol glycol and N-desisopropylpropranolol in brain tissue by electron capture gas chromatography

A method for the quantitative determination of propranolol and two of its active metabolites, 3-(alpha-naphthoxy)-1,2-propanediol (propranolol glycol) and N-des-isopropylpropranolol, in brain tissue of mice is described. Tissues are homogenized in perchloric acid-acetonitrile. Propranolol and its metabolites are isolated from the supernatant by solvent extraction and separated and detected as their trifluoroacetyl derivatives by electron capture gas chromatography. Chemical structures of the derivatives were confirmed by gas chromatography-mass spectrometry. The electron capture detector response of all three compounds is high, 0.7-2.0 X 10(-16) moles/sec. Brain levels of 10-250 ng/g can be detected of all three compounds with high specificity and good precision.     

Processing of adenosine receptor agonists in rat and human whole blood

A stability study of adenosine receptor agonists in rat and human whole blood was performed. The compounds were incubated at 37 degrees in fresh blood, and aliquots of the incubation mixture were hemolyzed at regular time intervals and analyzed with HPLC. N6-cyclopentyladenosine (CPA) and N6-cyclobutyladenosine (CBA) were degraded, whereas N6-cyclohexyladenosine, N6-cycloheptyladenosine and N6-sulfophenyladenosine were not. 2-Chloroadenosine had a half-life very similar to that of CPA. However, the 2'-, 3'-, and 5'-deoxyribose derivatives of CPA remained intact. The nucleoside transport inhibitor nitrobenzylthioinosine attenuated CBA and CPA metabolism in rat blood as did the inhibitor of adenosine deaminase erythro-9-(2-hydroxy-3-nonyl)adenine, albeit at relatively high concentrations. Complete blockade of CBA and CPA degradation was achieved by a preincubation of rat and human blood with the adenosine kinase (AK) inhibitor 5'-amino-5'-deoxyadenosine. We conclude that the two adenosine analogues are metabolized by AK both in rat and in human whole blood.     

Rapid fluorometric analysis of unbound salicylate in whole blood

A method has been developed for the analysis of unbound salicylate by membrane cone ultrafiltration of either whole blood or plasma with quantitation by spectrofluorometry. The method was used to study the distribution of salicylate in whole blood over a wide range of concentrations. It was found that at a concentration of 50 ug/ml in whole blood only 9% of the salicylate is present as free drug in the plasma water, while at a concentration of 500 ug/ml, 23% is free. At concentrations exceeding 500 ug/ml the plasma protein binding sites appear to be fully satrated since the free drug concentration increases linearly with respect to the whole blood concentration. Changes in hematocrit have a relatively minor effect on the ultrafiltrate salicylate concentration. The blood/plasma salicylate concentration ratio was found to vary widely over the blood salicylate range studied, 50-1000 ug/ml.     

Contrast-agent gas bodies enhance hemolysis induced by lithotripter shock waves and high-intensity focused ultrasound in whole blood

Fresh canine whole blood was exposed in 1.3-mL disposable pipette bulbs with or without added Albunex contrast agent to lithotripter shock waves (rate: 2 Hz; mean pressure amplitude: 14.8 MPa) or 1.28-MHz high-intensity focused ultrasound up to a mean pressure amplitude of 17.8 MPa. Significant shock-wave-induced hemolysis up to about 10% was detected after 500 or more shock waves without added agent, after 200 with 1% added agent and after 100 with 10% added contrast agent. Hemolysis was increased somewhat with added contrast agent, but this enhancement was not statistically significant. For ultrasound exposure, significant hemolysis approaching 100% was detected for 100-ms continuous exposure at 17.8 MPa without added gas bodies. Addition of 1% contrast agent enhanced the hemolysis by reducing the apparent threshold to 10 MPa. Burst mode exposure with a 100-ms total on-time (20 microseconds, 100 microseconds and 1-ms bursts) also yielded hemolysis enhancement with added contrast agent, with results similar to the 100-ms continuous exposure. The added gas bodies appear to lower cavitation thresholds by serving as cavitation nuclei in the blood.     

Lipopolysaccharide-induced interleukin 8 production by human whole blood is enhanced by epinephrine and inhibited by hydrocortisone

To determine the effect of epinephrine and hydrocortisone on lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) production, human whole blood was stimulated with LPS in the presence or absence of these stress hormones. Epinephrine caused a dose-dependent increase in LPS-induced IL-8 production, which was mediated exclusively via beta-adrenergic receptors, as reflected by the facts that beta (but not alpha) receptor blockade reversed the epinephrine effect and beta (but not alpha) receptor stimulation reproduced the epinephrine effect. Further, elevating cellular cyclic AMP (cAMP) concentrations, a known effect of beta-adrenergic stimulation, by addition of dibutyryl cAMP also enhanced LPS-induced IL-8 production. Epinephrine-induced upregulation of IL-10 production masked an even more pronounced stimulating effect of this hormone on IL-8 synthesis, as indicated by the finding that the extent of IL-8 upregulation was greater in the presence of anti-IL-10 than in the absence of anti-IL-10. Hydrocortisone dose-dependently inhibited LPS-induced IL-8 production and reversed epinephrine-induced enhancement of IL-8 production. Epinephrine and hydrocortisone have opposite effects on IL-8 production, which may be relevant for the understanding of endogenous and therapeutic stress hormone influences on IL-8 mediated inflammation.     

A method for the estimation of 2-phenylethylamine in human urine by gas chromatography

The fine structure of the retinal epithelium has been studied by electron microscopy in the opossum (Didelphis virginiana). The retinal epithelium, over most of the retina, is typical of that in other vertebrates and consists of a single layer of heavily pigmented, cuboidal cells. These cells display extensive basal (scleral) infoldings and numerous apical (vitreal) processes which enclose photoreceptor outer segments. A semicircular area of the retinal epithelium in the superior fundus is further specialized as a tapetum lucidum. The reflecting material consists of a large quantity of lipoidal spheres scattered throughout the epithelial cells. Centrally in the tapetal area very few or no melanosomes are found, indicating a non-occlusible tapetum. Peripherally in be tapetum, the epithelial cells contain both reflecting material and melanosomes. As in the non-tapetal area, the epithelial cells of the tapetum display large amounts of smooth endoplasmic reticulum and numerous mitochondria. Bruch's membrane everywhere displays the usual pentalaminate structure described for most vertebrates. The choriocapillaris is also typical, in that numerous fenestrations are present in the endothelium bordering Bruch's membrane.