Gas chromatography in anaesthesia: I. A brief review of analytical methods and gas chromatographic detector and column systems

Practical applications and relevant studies involving the anaesthetic gases, have been extensively described in the literature. Many eminent analytical methods have already been developed for medical practice where routine analysis of anaesthetics is frequently needed, particularly during anaesthesia, and in related and respiratory research programmes. The determination of halothane, isoflurane, enflurane and nitrous oxide concentrations from vaporizers, in exhaled and inhaled gas mixtures, in body fluids and tissues is necessary to control anaesthetic concentrations, and thus, the relevant and adverse effects successfully. Therefore, a literature review, with particular emphasis on gas chromatography would provide important information for investigators in the search for a suitable analytical method for the analysis of multi-component mixtures of anaesthetic gases.     

Endogenous lithium and boron red cell-plasma ratios: normal subjects versus bipolar patients not on lithium therapy

This study was undertaken to compare endogenous lithium concentrations in human blood and its components from normal donors versus bipolar patients. The patients were not on lithium therapy at the time that the blood samples were donated and had not received any lithium therapy for at least 2 yr. Blood components were separated by centrifugation. The analytical method for lithium as developed in this laboratory consists of thermal-neutron activation of freeze-dried samples. 3H is produced via the reaction 6Li + n = 3H + 4He, and high-sensitivity rare gas mass spectrometry is used to measure 3He formed from beta-decay of 3H. Boron measurements are made concurrently using 4He from the reaction 10B + n = 4He + 7Li. Seven normal donors and seven patients with a diagnosis of bipolar disorder participated in this study. Measurements of lithium and boron were made in whole blood, plasma, and red cells. Red cell-plasma ratios R(Li) and R(B) were calculated after corrections were made for trapped plasma in the red cells. The results show that bipolar patients may have higher concentrations of lithium in blood, plasma, and red cells (p = 0.08, 0.02, and 0.02, respectively) and may have higher R(Li) values than normal donors (p = 0.01). No evidence was found for bipolar-normal differences in these four parameters for boron. Although our sample size is admittedly very small, the results clearly show that the endogenous red cell ratio R(Li) and plasma or red cell lithium concentrations may become useful diagnostic indicators for bipolar illness if the analytical methods are further developed.     

Determination of chlorpromazine and its major metabolites by gas chromatography/mass spectrometry: application to biological fluids

A method for the quantitative determination of chlorpromazine and five of its major metabolites in a single sample of biological fluid in the ng/ml range has been developed utilizing gas chromatography/mass spectrometry with selected ion recording. The assay is highly specific and quantification is accomplished by an inverse stable isotope dilution technique, using deuterium-labeled variants of the compounds as internal standards. In this way the concentrations of chlorpromazine and five of its major metabolites (the sulfoxide, the N-oxide, the monodemethylated, the didemethylated, and the 7-hydroxylated compounds) can be determined in biological fluids. Levels in humans have been measured both in plasma and in red blood cells and are compared to those found in related in vitro studies.     

Dissolved atmospheric gas in xylem sap measured with membrane inlet mass spectrometry

A new method is described for measuring dissolved gas concentrations in small volumes of xylem sap using membrane inlet mass spectrometry. The technique can be used to determine concentrations of atmospheric gases, such as argon, as reported here, or for any dissolved gases and their isotopes for a variety of applications, such as rapid detection of trace gases from groundwater only hours after they were taken up by trees and rooting depth estimation. Atmospheric gas content in xylem sap directly affects the conditions and mechanisms that allow for gas removal from xylem embolisms, because gas can dissolve into saturated or supersaturated sap only under gas pressure that is above atmospheric pressure. The method was tested for red trumpet vine, Distictis buccinatoria (Bignoniaceae), by measuring atmospheric gas concentrations in sap collected at times of minimum and maximum daily temperature and during temperature increase and decline. Mean argon concentration in xylem sap did not differ significantly from saturation levels for the temperature and pressure conditions at any time of collection, but more than 40% of all samples were supersaturated, especially during the warm parts of day. There was no significant diurnal pattern, due to high variability between samples.     

Methods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids

In human organism, the gaseous radical molecule nitric oxide (NO) is produced in various cells from L-arginine by the catalytic action of NO synthases (NOS). The metabolic fate of NO includes oxidation to nitrate by oxyhaemoglobin in red blood cells and autoxidation in haemoglobin-free media to nitrite. Nitrate and nitrite circulate in blood and are excreted in urine. The concentration of these NO metabolites in the circulation and in the urine can be used to measure NO synthesis in vivo under standardized low-nitrate diet. Circulating nitrite reflects constitutive endothelial NOS activity, whereas excretory nitrate indicates systemic NO production. Today, nitrite and nitrate can be measured in plasma, serum and urine of humans by various analytical methods based on different analytical principles, such as colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis and mass spectrometry. The aim of the present article is to give an overview of the most significant currently used quantitative methods of analysis of nitrite and nitrate in human biological fluids, namely plasma and urine. With minor exception, measurement of nitrite and nitrate by these methods requires method-dependent chemical conversion of these anions. Therefore, the underlying mechanisms and principles of these methods are also discussed. Despite the chemical simplicity of nitrite and nitrate, accurate and interference-free quantification of nitrite and nitrate in biological fluids as indicators of NO synthesis may be difficult. Thus, problems associated with dietary and laboratory ubiquity of these anions and other preanalytical and analytical factors are addressed. Eventually, the important issue of quality control, the use of commercially available assay kits, and the value of the mass spectrometry methodology in this area are outlined.     

Effects of asphyxia on arterial blood pressure, formation of nitric oxide in medulla and blood parameters in the cat

The rostral ventrolateral medulla (RVLM) plays an important role in the integration of cardiovascular functions. We examined the effect of asphyxia on cardiovascular responses, on sympathetic vertebral nerve activity (VNA) and nitric oxide (NO) formation in the RVLM, on hemodynamics, and on plasma concentrations of catecholamines, blood gas partial pressures and carbohydrate metabolites. Using 16 anesthetized cats we found that the systemic arterial pressure (SAP), VNA, NO formation and the release of plasma catecholamine components of norepinephrine and epinephrine were increased during asphyxia. The onset of NO production was significantly earlier than that of SAP and VNA. The venous partial pressure of O2 decreased, while the partial pressure of CO2 increased. Furthermore, metabolism of glucose and lactate increased, as did the blood concentrations of white and red blood cells, hemoglobin and platelets. Thus, asphyxia increased SAP, VNA and NO formation. It increased the plasma catecholamines, blood gases, carbohydrate metabolites and blood cells.     

Quantitative gas chromatographic determination of two oxidized metabolites of the diuretic mefruside in human urine,plasma and red blood cells

A gas chromatographic method is reported for the quantitative analysis of two metabolites of mefruside, viz., 5-oxo-mefruside (mefruside lactone) and its hydroxycarboxylic acid analogue in human body fluids. Use was made of extractive methylation as the derivatization technique, and quantitation was achieved, with a suitable internal standard, by means of a nitrogen-sensitive detector.Because the two metabolites are linked chemically through a lactone—open acid equilibrium, interconversion prior to their separation had to be avoided. A pH partitioning study was performed to find optimal separation conditions. The lactone could be extracted quantitatively at pH 7.4, without any trace of co-extracted hydroxy acid. The latter was extracted either at pH 2 directly (in the case of plasma and urine), or after conversion to the lactone at pH 7.4 (in the case of red cells or whole blood). Concentrations down to 25 ng per sample of both compounds could be analysed with a standard deviation of 5%.The two metabolites of mefruside equilibrated instantaneously between red cells and plasma in vitro. At 37°, the red cell/plasma concentration ratio was 20 for the lactone, but only 0.1 for the open acid compound. 5-Oxo-mefruside was able to displace mefruside from its red blood cell binding sites in vitro.     

Sex steroid regulation of urinary excretion of carnitine in rats

The concentration of acid soluble carnitine was determined in several body tissues and fluids in rats under various conditions of sex steroid regulation. Intact female rats had significantly greater liver carnitine concentrations and urinary excretion rates, and lower blood plasma and heart carnitine concentrations than intact male rats. Ovariectomy increased blood plasma carnitine concentrations (P < 0.01) and the excretion of carnitine in the urine (P < 0.05). The administration of either estradiol or testosterone to ovariectomized rats did not alter blood plasma concentrations or urinary excretion of carnitine. Orchidectomized rats had similar blood plasma carnitine concentrations when compared to intact males but excreted significantly (P < 0.01) greater quantities of carnitine in their urine. Administration of testosterone to orchidectomized rats reduced (P < 0.01), whereas estradiol stimulated (P < 0.05) the excretion rate of carnitine in the urine; however, blood plasma carnitine concentrations were not affected by these hormones. These data suggest that a major site for modulation of body carnitine concentration in the male resides in the control of kidney excretion by androgens. Liver, heart and skeletal muscle carnitine concentrations were not altered by the administration of either estradiol or testosterone to orchidectomized or ovariectomized rats.     

Microvascular experimental evidence on the relative significance of restoring oxygen carrying capacity vs. blood viscosity in shock resuscitation

The development of volume replacement fluids for resuscitation in hemorrhagic shock comprises oxygen carrying and non carrying fluids. Non oxygen carrying fluids or plasma expanders are used up to the transfusion trigger, and upon reaching this landmark either blood, and possibly in the near future oxygen carrying blood substitutes, are used. An experimental program in hemorrhagic shock using the hamster chamber window model allowed to compare the relative performance of most fluids proposed for shock resuscitation. This model allows investigating simultaneously the microcirculation and systemic reactions, in the awake condition, in a tissue isolated from the environment. Results from this program show that in general plasma expanders such as Ringer's lactate and dextran 70 kDa do not sufficiently restore blood viscosity upon reaching the transfusion trigger, causing microvascular collapse. This is in part restored by a blood transfusion, independently of the oxygen carrying capacity of red blood cells. These results lead to the proposal that effective blood substitutes must be designed to prevent microvascular collapse, manifested in the decrease of functional capillary density. Achievement of this goal, in combination with the increase of oxygen affinity, significantly postpones the need for a blood transfusion, and lowers the total requirement of restoration of intrinsic oxygen carrying capacity.     

High-performance liquid chromatographic assay of 5-fluorouracil in human erythrocytes,plasma and whole blood

A HPLC assay method was modified and validated for the determination of 5-fluorouracil in human red blood cells, plasma and whole blood with a two-fold increased sensitivity (detection limit=10 ng/ml). The assay was linear from 25 to 1500 ng/ml and the accuracy ranged from 96.7 to 103.2% at 25 ng/ml, 94.8 to 99.4% at 500 ng/ml, and 98.9 to 99.5% at 1500 ng/ml. Intra-assay and inter-assay coefficients of variation were less than 8% over the range of concentrations and less than 8% over 10 days of analysis. After intravenous bolus and infusion of 5-fluorouracil in patients with colorectal cancer, the concentrations of 5-fluorouracil in whole blood were 108–111% of plasma concentrations, while packed red blood cells levels were 8–15% of plasma concentrations in the five patients studied. By utilising basic analytical hardware, this represents an accurate, precise, reproducible and affordable method for 5-fluorouracil pharmacokinetics investigation and therapeutic drug monitoring.     

Haemolysis during sample preparation alters microRNA content of plasma

The presence of cell-free microRNAs (miRNAs) has been detected in a range of body fluids. The miRNA content of plasma/serum in particular has been proposed as a potential source of novel biomarkers for a number of diseases. Nevertheless, the quantification of miRNAs from plasma or serum is made difficult due to inefficient isolation and lack of consensus regarding the optimal reference miRNA. The effect of haemolysis on the quantification and normalisation of miRNAs in plasma has not been investigated in great detail. We found that levels of miR-16, a commonly used reference gene, showed little variation when measured in plasma samples from healthy volunteers or patients with malignant mesothelioma or coronary artery disease. Including samples with evidence of haemolysis led to variation in miR-16 levels and consequently decreased its ability to serve as a reference. The levels of miR-16 and miR-451, both present in significant levels in red blood cells, were proportional to the degree of haemolysis. Measurements of the level of these miRNAs in whole blood, plasma, red blood cells and peripheral blood mononuclear cells revealed that the miRNA content of red blood cells represents the major source of variation in miR-16 and miR-451 levels measured in plasma. Adding lysed red blood cells to non-haemolysed plasma allowed a cut-off level of free haemoglobin to be determined, below which miR-16 and miR-451 levels displayed little variation between individuals. In conclusion, increases in plasma miR-16 and miR-451 are caused by haemolysis. In the absence of haemolysis the levels of both miR-16 and miR-451 are sufficiently constant to serve as normalisers.     

Determination of citrulline in human plasma, red blood cells and urine by electron impact (EI) ionization gas chromatography-mass spectrometry

A method was developed by using gas chromatography-mass spectrometry in the electron impact ionization mode to quantify citrulline in plasma, red blood cells (RBC) and urine. For all three fluids, citrulline was extracted on ion exchange resins, before derivatization to its propyl-heptaflorobutyryl-ester. Assay precision (coefficient of variation, CV) was <5%, recovery% was >90% and the within- and between-day CV were <10% on 200 microL of plasma and RBC, and 400 microL of urine. The current method allows for the detection of 20 pmol of natural citrulline in aqueous standards, and small volumes (<100 microL) of biological fluids.     

Streamer breakdown of long gas gaps

Results obtained from numerical simulations of the streamer breakdown of long (longer than 10 cm) gas gaps at atmospheric pressure are reviewed. Most attention is focused on air under normal conditions and at elevated temperatures characteristic of the rebreakdown in the postspark channel that is cooled after the primary spark discharge has come to an end. The main stages of the evolution of a streamer discharge into an arc are considered, and the features of this phenomenon are analyzed as functions of the initial conditions. The main macroscopic processes that govern the composition and dynamics of the streamer plasma in different discharge stages are revealed. The experimental data and the results of computer simulations provide evidence for a nonthermal mechanism for streamer breakdown in noble gases.     

Simultaneous determination of superoxide dismutase and catalase in biological materials by polarography.

The polarographic method of catalytic currents applied to a wave of oxygen permits the simultaneous assay of superoxide dismutase and catalase in biological materials with high speed and reproducibility and minimal manipulation of tissues. Washed red blood cells and tissue homogenates give rise to a strong polarographic maximum, apparently due to heme proteins, which interferes with the measurement. This maximum is suppressed by addition of approximately 0.2% plasma. Therefore, the determination of the two enzymes in red blood cells can be carried out by direct addition of whole blood to the polarographic solution. Thirty microliters of blood are enough for optimal determination of both enzymes. The method can determine superoxide dismutase and catalase at concentrations as low as 2 × 10⁻¹¹m and 5 × 10⁻¹⁰m, respectively, and shows a linear correlation between measured activity and enzyme levels. The average values of the two enzymes in human red blood cells was found by this method to be 2.6 × 10⁻⁶m for catalase and 1.8 × 10⁻⁶m for superoxide dismutase, which agree with previously reported values.     

Antioxidant capacity of human blood plasma and human urine: simultaneous evaluation of the ORAC index and ascorbic acid concentration employing pyrogallol red as probe

The oxygen radical absorbance capacity (ORAC) methodology has been employed to estimate the antioxidant capacity of human blood plasma and human urine using pyrogallol red (ORAC-PGR) as target molecule. Uric acid, reduced glutathione, human serum albumin, and ascorbic acid (ASC) inhibited the consumption of pyrogallol red, but only ASC generated an induction time. Human blood plasma and human urine protected efficiently pyrogallol red. In these assays, both biological fluids generated neat induction times that were removed by ascorbate oxidase. From these results, ORAC-PGR method could be proposed as a simple alternative to evaluate an ORAC index and, simultaneously, to estimate the concentration of ascorbic acid in human blood plasma or human urine.     

ReviewMethods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids

In human organism, the gaseous radical molecule nitric oxide (NO) is produced in various cells from l-arginine by the catalytic action of NO synthases (NOS). The metabolic fate of NO includes oxidation to nitrate by oxyhaemoglobin in red blood cells and autoxidation in haemoglobin-free media to nitrite. Nitrate and nitrite circulate in blood and are excreted in urine. The concentration of these NO metabolites in the circulation and in the urine can be used to measure NO synthesis in vivo under standardized low-nitrate diet. Circulating nitrite reflects consitutive endothelial NOS activity, whereas excretory nitrate indicates systemic NO production. Today, nitrite and nitrate can be measured in plasma, serum and urine of humans by various analytical methods based on different analytical principles, such as colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis and mass spectrometry. The aim of the present article is to give an overview of the most significant currently used quantitative methods of analysis of nitrite and nitrate in human biological fluids, namely plasma and urine. With minor exception, measurement of nitrite and nitrate by these methods requires method-dependent chemical conversion of these anions. Therefore, the underlying mechanisms and principles of these methods are also discussed. Despite the chemical simplicity of nitrite and nitrate, accurate and interference-free quantification of nitrite and nitrate in biological fluids as indicators of NO synthesis may be difficult. Thus, problems associated with dietary and laboratory ubiquity of these anions and other preanalytical and analytical factors are addressed. Eventually, the important issue of quality control, the use of commercially available assay kits, and the value of the mass spectrometry methodology in this area are outlined.     

Analysis of creatine, creatinine, creatine-d3 and creatinine-d3 in urine, plasma, and red blood cells by HPLC and GC-MS to follow the fate of ingested creatine-d3

Creatine, which is increasingly being used as an oral supplement, is naturally present in the body. Studies on the fate of a particular dose of creatine require that the creatine be labeled, and for studies in humans the use of a stable isotopic label is desirable. The concentrations of total creatine and total creatinine were determined using HPLC. Creatine and creatinine were then separated using cation exchange chromatography and each fraction was derivatized with trifluoroacetic anhydride and the ratio of the deuterated:undeuterated species determined using GC-MS. Ratios of creatine:creatine-d(3), and creatinine:creatinine-d(3), and the concentrations of each of these species, were able to be determined in urine, plasma and red blood cells. Thus, the uptake of labeled creatine into plasma and red blood cells and its excretion in urine could be followed for a subject who ingested creatine-d(3). Creatine-d(3) was found in the plasma and red blood cells 10 min after ingestion, while creatine-d(3) and creatinine-d(3) were found in the urine collected after the first hour.     

Metabolic stratification driven by surface and subsurface interactions in a terrestrial mud volcano

Terrestrial mud volcanism represents the prominent surface geological feature, where fluids and hydrocarbons are discharged along deeply rooted structures in tectonically active regimes. Terrestrial mud volcanoes (MVs) directly emit the major gas phase, methane, into the atmosphere, making them important sources of greenhouse gases over geological time. Quantification of methane emission would require detailed insights into the capacity and efficiency of microbial metabolisms either consuming or producing methane in the subsurface, and establishment of the linkage between these methane-related metabolisms and other microbial or abiotic processes. Here we conducted geochemical, microbiological and genetic analyses of sediments, gases, and pore and surface fluids to characterize fluid processes, community assemblages, functions and activities in a methane-emitting MV of southwestern Taiwan. Multiple lines of evidence suggest that aerobic/anaerobic methane oxidation, sulfate reduction and methanogenesis are active and compartmentalized into discrete, stratified niches, resembling those in marine settings. Surface evaporation and oxidation of sulfide minerals are required to account for the enhanced levels of sulfate that fuels subsurface sulfate reduction and anaerobic methanotrophy. Methane flux generated by in situ methanogenesis appears to alter the isotopic compositions and abundances of thermogenic methane migrating from deep sources, and to exceed the capacity of microbial consumption. This metabolic stratification is sustained by chemical disequilibria induced by the mixing between upward, anoxic, methane-rich fluids and downward, oxic, sulfate-rich fluids.     

Purine and pyridine nucleotides in rabbit red blood cells of different maturity

Using reversed-phase high-performance liquid chromatography purine nucleotides, nucleosides and nucleobases as well as pyridine nucleotides were determined in extracts of reticulocytes and mature red blood cells of rabbits. The concentrations of almost all compounds measured decrease during the last phase of red blood cell maturation. These changes were interpreted with respect to the loss of mitochondria, accompanied by shifting the energy production from the preferentially oxidative mode to the exclusively glycolytic one and variations in the concentrations of purine compounds in blood plasma during reticulocytosis.