Gas chromatographic—mass spectrometric determination of ethambutol in human plasma

A quantitative gas chromatographic—mass spectrometric assay has been developed for the determination of ethambutol (EMB) in human plasma. Plasma samples were taken from a patient after oral administration of EMB (with proven tuberculosis infection). Deuterated EMB and a non-deuterated analogue of EMB were synthesized and used as internal standards in this procedure; both gave excellent agreement in the analysis. The derivatizing agent used was trifluoroacetic anhydride (TFAA) and quantitative derivatization was complete in one hour, forming EMB-(TFA). Selective ion monitoring was utilized to monitor the gas chromatographic effluent. Ions were generated by electron impact at 70 eV. The limit of detection was 36 ng EMB per ml plasma. This method is compared with the electron-capture gas chromatographic procedure of Lee and Benet.     

Determination of phenelzine in human plasma with gas chromatography—mass spectrometry using an isotope labeled internal standard

A quantitative gas chromatographic—mass spectrometric assay was developed for the determination of phenelzine in human plasma. Phenelzine, in aqueous solution or in plasma reacts at room temperature with pentafluorobenzaldehyde to form quantitatively a hydrazone derivative. The derivative has good gas chromatographic characteristics. The assay utilizes selected ion monitoring in a gas chromatographic effluent, the molecular ion generated by electron impact ionization of phenelzine derivative. Phenelzine-d₇ was synthesized and used as an internal standard. The assay can measure 2 ng/ml of the drug with about 10% precision.The method was used for the determination of steady state levels of phenelzine in the plasma of patients taking a therapeutic dose of the drug.     

Development of a gas chromatographic selected ion monitoring assay for thyroxine (T4) in human serum

The components of a gas chromatographic mass spectrometric-selected ion monitoring (SIM) assay for thyroxine (T4) in human serum are described. The internal standard for the assay was synthesized from deuterium-labelled 3,5-diiodotyrosine and 3,5-diiodo-4-hydroxyphenylpyruvic acid. A novel method was developed for isolating the products of the coupling reaction. The results obtained by gas chromatography mass spectrometry SIM were compared with those of radioimmunoassay. The gas chromatographic mass spectrometric SIM assay would form the basis of a reference assay for T4.     

Measurement of arachidonic acid release from human polymorphonuclear neutrophils and platelets: comparison between gas chromatographic and radiometric assays

a simple gas chromatographic method for the assay of phospholipase A2 (PLA2) has been described in which arachidonic acid released from endogenous phospholipid pools is measured following its extraction and derivatization to pentafluorobenzyl esters. Using this assay, PLA2 activities in control and calcium ionophore-stimulated human neutrophils, as well as in control, thrombin, and calcium ionophore stimulated human platelets, have been measured. These values are compared with those obtained by monitoring the release of radioactivity from [3H]- or [14C]arachidonic acid prelabeled cells. While the radiometric assay measures only the release of exogenously incorporated radioactive arachidonic acid, the gas chromatographic assay measures arachidonic acid released from all the endogenous pools. Thus, the apparent increase in PLA2 activity in stimulated cells measured by the gas chromatographic assay is four- to fivefold higher than that by the radiometric assay. Inclusion of fatty acid free bovine serum albumin in the reaction buffer significantly increases the amount of arachidonic acid that is measured by gas chromatography. The gas chromatographic method has also been successfully utilized for measuring PLA2 activity in cell-free preparations derived from physically disrupted human neutrophils.     

Small-molecule inhibition of APE1 induces apoptosis,pyroptosis, and necroptosis in non-small cell lung cancer

Apurinic/apyrimidinic endonuclease 1 (APE1) plays a critical role in the base excision repair (BER) pathway, which is responsible for the excision of apurinic sites (AP sites). In non-small cell lung cancer (NSCLC), APE1 is highly expressed and associated with poor patient prognosis. The suppression of APE1 could lead to the accumulation of unrepaired DNA damage in cells. Therefore, APE1 is viewed as an important marker of malignant tumors and could serve as a potent target for the development of antitumor drugs. In this study, we performed a high-throughput virtual screening of a small-molecule library using the three-dimensional structure of APE1 protein. Using the AP site cleavage assay and a cell survival assay, we identified a small molecular compound, NO.0449-0145, to act as an APE1 inhibitor. Treatment with NO.0449-0145 induced DNA damage, apoptosis, pyroptosis, and necroptosis in the NSCLC cell lines A549 and NCI-H460. This inhibitor was also able to impede cancer progression in an NCI-H460 mouse model. Moreover, NO.0449-0145 overcame both cisplatin- and erlotinib-resistance in NSCLC cell lines. These findings underscore the importance of APE1 as a therapeutic target in NSCLC and offer a paradigm for the development of small-molecule drugs that target key DNA repair proteins for the treatment of NSCLC and other cancers.Subject terms: Non-small-cell lung cancer, Apoptosis     

Simultaneous quantitative analysis of methyl salicylate,ethyl salicylate and salicylic acid from biological fluids using gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric (GC–MS) assay was developed for the quantitative analysis of methyl salicylate (MeS), ethyl salicylate (ES) and salicylic acid (SA) from biological fluids. The method was validated from 100-μl rat liver homogenate preparations (5 mg/ml protein) in 70 mM KH₂PO₄ (pH 7.4) buffer and from 100 μl rat plasma. The samples were extracted with chloroform, derivatized with BSTFA and quantitated by GC–MS in the SIM mode. The standard curves ranged from 31 ng/ml to 800 or 1250 ng/ml. Relative standard deviations and bias were less than 11% in plasma and homogenate for all compounds except SA which evidenced greater variability. The assay was used in preliminary experiments to characterize the pharmacokinetics of MeS in rats.     

Comparison of conventional and fast gas chromatography in human plasma fatty acid determination

A fast gas chromatographic technique for the determination of fatty acids in human plasma was developed. Its validation and comparison to a conventional method are here reported. The fast method significantly reduced the time required for analysis by a factor of 5 (total time of 3.2 min) while maintaining a similar resolution. Reproducibility of qualitative and quantitative data was measured in both applications. The results demonstrated that the applied fast gas chromatography (GC) conditions do not affect the analytical quality of the assays, allowing a short analysis time.     

Determination of MK-287, a new platelet-activating factor antagonist, in plasma and serum by gas chromatography chemical ionization mass spectrometry

MK-287 is a novel platelet-activating factor antagonist. A sensitive and specific gas chromatographic/mass spectrometric assay has been developed for the determination of the drug in serum and plasma. The assay utilizes an extraction with methyl-t-butyl ether and subsequent trimethylsilylation of the hydroxyl function. The gas chromatographic/mass spectrometric determinations are carried out with temperature-programmed capillary gas chromatography and ammonia negative chemical ionization mass spectrometry. The method has sufficient sensitivity, precision, accuracy and selectivity for the analysis of drug concentrations in clinical samples.     

Determination of urinary 18β-glycyrrhetinic acid by gas chromatography and its clinical application in man

A sensitive and quantitative gas chromatographic assay for the determination of 18β-glycyrrhetinic acid (18β-GA), the main metabolite of glycyrrhizin after oral licorice consumption in human urine, has been developed and validated. For the extraction of 18β-GA from urine two Sep-Pak C₁₈ extractions, hydrolysis with Helix pomatia and three liquid–liquid extractions were performed, using 18α-glycyrrhetinic acid (18α-GA) as internal standard. Both 18β-GA and internal standard were converted into their pentafluorobenzyl-ester/trimethylsilyl-ether derivatives and detected by flame ionization detection using a WCOT-fused-silica capillary column. Good quality control data were obtained in precision and accuracy tests. The detection limit of the gas chromatographic method was 10 μg/l with a urine volume of 10 ml. A detection limit of 3 μg/l was obtained by performing GC–MS. The GC method was used to monitor the urinary excretion of 18β-GA after licorice consumption by two healthy volunteers and a patient suspected of licorice abuse. Furthermore, it was shown that this GC assay enables to detect other metabolites related to licorice consumption.     

Gas-liquid chromatography for the determination of acetylcholinesterase activity

Method has been modified and used for quantitative gas chromatographic determination of microtraces in a large volume of test mixture under study. The method can be applied for the differentiated determination of genuine and false blood cholinesterase by final products of specific substrate hydrolysis.     

Activation of muscarinic K<Superscript>+</Superscript> channels by arecaidine propargyl ester in isolated guinea-pig atrial myocytes

Summary Arecaidine propargyl ester (APE) was developed as a potential candidate compound for the treatment of Alzheimer’s disease. APE has been shown to have cardiovascular effects. APE produces negative chronotropic and inotropic effects in isolated atria. However, the ionic mechanisms underlying the cardiovascular effects of APE in guinea-pig atria are unclear. The aims of this study were: (1) to examine the shortening effect of APE on action potential duration (APD) and to compare the difference in potency between APE and muscarine in isolated single guinea-pig atrial myocytes by using the current clamp method, (2) to examine by using patch clamp techniques the ionic mechanisms underlying the cardiac effects of APE, and (3) to determine whether the cardiac effects caused by APE affect the usefulness of APE as a potential candidate for the treatment of Alzheimer’s disease. The APE significantly reduced the APD in guinea-pig atria and produced no direct effect on ventricular myocytes. APE is approximately 20 times as potent as muscarine in shortening the APD. Attenuation of the APD was consistently accompanied by a hyperpolarization of the resting membrane potential in a concentration-dependent manner. The APE activated muscarinic K⁺ channels and increased potassium conductance in guinea-pig atrial myocytes. In the cell-attached configuration, the APE contained in the pipette increased the channel-opening probability and decreased the closed-state time interval. The proposal that APE can be used as a potential remedy for the treatment of Alzheimer’s disease should be taken into consideration the undesirable cardiovascular side effects that APE causes at lower concentrations.     

Comparison of the limulus amebocyte lysate test and gas chromatography-mass spectrometry for measuring lipopolysaccharides (endotoxins) in airborne dust from poultry-processing industries.

The lipopolysaccharide (endotoxin) content in airborne dust samples from three different poultry slaughterhouses was determined with both the chromogenic Limulus amebocyte lysate assay and gas chromatography-mass spectrometry analysis of lipopolysaccharide-derived 3-hydroxy fatty acids. Gram-negative cell walls were also measured by using two-dimensional gas chromatography/electron-capture analysis of diaminopimelic acid originating from the peptidoglycan. The correlation between the results of the Limulus assay and those of gas chromatography-mass spectrometry for determination of the lipopolysaccharide content in the dust samples was poor, whereas a good correlation was obtained between lipopolysaccharide and diaminopimelic acid concentrations with the gas chromatographic methods. The results suggest that it is predominantly cell-wall-dissociated lipopolysaccharides that are measured with the Limulus assay, whereas the gas chromatographic methods allow determination of total concentrations of lipopolysaccharide, including Limulus-inactive lipopolysaccharide, gram-negative cells, and cellular debris.     

A nonradioactive assay method for determination of enzymatic activity of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)

A sensitive and nonradioactive assay method for activity determination of Rubisco is described. The method is based on thin-layer chromatographic separation of 3-phosphoglycerate (3-PGA) and D-ribulose-1,5-bisphosphate (RuBP). This assay method allows the quantitative determination of Rubisco activity. Rates of carbon dioxide fixation on RuBP determined by this method were comparable to those obtained independently by other methods. This assay method is reproducible and relatively free from interference.     

Comparison of the limulus amebocyte lysate test and gas chromatography-mass spectrometry for measuring lipopolysaccharides (endotoxins) in airborne dust from poultry-processing industries

The lipopolysaccharide (endotoxin) content in airborne dust samples from three different poultry slaughterhouses was determined with both the chromogenic Limulus amebocyte lysate assay and gas chromatography-mass spectrometry analysis of lipopolysaccharide-derived 3-hydroxy fatty acids. Gram-negative cell walls were also measured by using two-dimensional gas chromatography/electron-capture analysis of diaminopimelic acid originating from the peptidoglycan. The correlation between the results of the Limulus assay and those of gas chromatography-mass spectrometry for determination of the lipopolysaccharide content in the dust samples was poor, whereas a good correlation was obtained between lipopolysaccharide and diaminopimelic acid concentrations with the gas chromatographic methods. The results suggest that it is predominantly cell-wall-dissociated lipopolysaccharides that are measured with the Limulus assay, whereas the gas chromatographic methods allow determination of total concentrations of lipopolysaccharide, including Limulus-inactive lipopolysaccharide, gram-negative cells, and cellular debris.     

Determination of methionine sulfoxide in proteins: comparison of a gas-chromatographic and electrophoretic method

Two methods for the determination of methionine in proteins have been used to estimate the extent of methionine sulfoxide obtained upon exposure of proteins to oxidizing agents. Both methods are based on prior treatment with cyanogen bromide, which attacks methionines (but not the sulfoxide derivative) with the resultant formation of methyl thiocyanate and peptides. The amount of methyl thiocyanate is determined quantitatively by gas chromatography, while the number of peptides is ascertained by SDS-polyacrylamide gel electrophoresis. The gas chromatographic estimate of CH3SCN offers an accurate and precise method (down to nanogram values) for the quantitative determination of methionine sulfoxide in proteins. Due to its simplicity and the use of low-cost equipment, the electrophoretic method appears to be a valuable complement to the gas chromatographic method, and the two methods in conjunction provide novel results.     

Comparison of a commercial ELISA assay for indole-3-acetic Acid at several stages of purification and analysis by gas chromatography-selected ion monitoring-mass spectrometry using a c(6)-labeled internal standard

Quantitative analysis of indole-3-acetic acid (IAA) using selected ion monitoring gas chromatography-mass spectrometry (GC-MS) with ¹³C₆[benzene ring]-IAA as the internal standard was used to compare the quantitative accuracy of commercial enzyme-linked immunoabsorbent assay (ELISA) kits. Plant materials differed in the amount of purification required prior to use of ELISA for reliable estimates to be made. Purification similar to that obtained by at least one high performance liquid chromatographic (HPLC) step was generally necessary prior to ELISA analysis of plant materials. Additional levels of purification appeared to be required for some plant materials prior to HPLC in order to obtain an accurate estimate by ELISA techniques. In no case was it possible to obtain reasonable estimates of IAA from crude extracts or even from acidic fractions of extracts of plant tissues. GC-MS techniques provide a rapid and simple method for checking the validity of ELISA techniques. Quantitative GC-MS, or a similar technique that provides an independent quantitative validation, should, whenever possible, be applied to each new plant material under study if use of the ELISA is planned.     

Quantitative measurement of sulforaphane, iberin and their mercapturic acid pathway metabolites in human plasma and urine using liquid chromatography-tandem electrospray ionisation mass spectrometry

A quantitative liquid chromatography positive ion electrospray tandem mass spectrometric method for the simultaneous determination of sulforaphane, iberin and their metabolites in human urine and plasma is described. The stability of the metabolites was determined in aqueous solution and in human plasma. Gradient liquid chromatographic separation was performed on a Zorbax SB-Aq 3.5 microm (100 x 2.1mm) column, using a mobile phase (flow rate 0.25 mL/min) consisting of ammonium acetate buffer at pH 4 and acetonitrile. Butyl thiocarbamoyl l-cysteine was used as internal standard. The assay was linear (r(2)>0.99) over the range of 0.03-300 microM in urine and 0.03-15 microM in plasma with intra- and inter-day assay precision (<10% CV) and accuracy (<20%). The lower limits of quantitation were in the range of 10-150 nmol/L. The method has been used to report, for the first time, individual quantitative measurement of each of the mercapturic acid pathway metabolites of sulforaphane and iberin in both human plasma and urine following a dietary study of broccoli consumption.     

Analytical methods for the quantitative determination of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors in biological samples

Published analytical methods for the quantitative determinations of presently available five 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors ("statins"), lovastatin, simvastatin, pravastatin, fluvastatin and atorvastatin, are reviewed for therapeutic drug monitoring purpose in patients. Almost all assay reviewed are based on high-performance liquid chromatography or gas chromatography. Some purification steps (liquid-liquid extraction, solid-phase extraction, etc.) have been used before they are submitted to separation by chromatographic procedures and they are detected by various detection methods like UV, fluorescence and mass spectrometry. This review shows that most method may be used quantitative determination of statins in plasma and they are suitable for therapeutic drug monitoring purpose of these drugs.     

Gas chromatographic analysis of ketamine and norketamine in plasma and urine: Nitrogen-sensitive detection

A sensitive gas chromatographic method for quantitative analysis of ketamine and norketamine in human and animal biological fluids is described. The nitrogen-sensitive detection procedure used is more stable than electron-capture detection and reduced analysis time. The method used bromo-ketamine as an internal standard for quantitation and is linear from 10–25,000 ng/ml. No interferences were shown with drugs commonly associated with cardiac surgery with cardiopulmonary by-pass. This assay is sensitive, specific, using either native or derivatized drugs and can be used for routine analysis of ketamine and norketamine in plasma or urine.     

Valproic acid analysis in saliva and serum using selected ion monitoring (electron ionization) of the tert.-butyldimethylsilyl derivatives

A highly sensitive ion monitoring method for the determination of valproic acid in saliva and in serum has been developed based on the gas chromatographic—mass spectrometric analysis of the tert.-butyldimethylsilyl derivatives. Extraction methods are simple and the techniques for derivatization are rapid and convenient. Selected ion monitoring was carried out using electron ionization conditions and a common ion m/z 201 (M⁺ − 57) present in valproic acid and the internal standard octanoic acid. The lower limit of sensitivity that has acceptable precision for assay purposes is 0.1 mg/l based on a 200-μl sample size. The ion monitoring method (derivatized) was compared to a gas chromatographic method (underivatized) for serum valproate assays and found to be essentially identical.The assay methodology was used in a kinetic study of valproic acid in two normal subjects. Saliva levels of drug were found to give reasonably good correlations with serum total and with serum free concentrations of drug in both individuals.