Micro-quantitative determination of ciprostene in plasma by gas chromatography—mass spectrometry coupled with an antibody extraction

A simple and highly sensitive method has been developed for the determination in plasma of ciprostene, 9β-methyl-6α-carbaprostaglandin I₂, using gas chromatography—mass spectrometry following solid-phase extraction on an immobilized antibody column. The anti-ciprostene antibody obtained from rabbit serum was coupled to an agarose support matrix, and the immobilized antibody thus prepared was used as an extraction phase for sample clean-up. The extracted drug was treated with pentafluorobenzyl bromide followed by bis(trimethylsilyl)trifluoroacetamide. The derivative was quantitatively analysed by negative-ion chemical ionization gas chromatography—mass spectrometry. The lower limit of quantitation was 50 pg/ml when 1 ml of human plasma was used. The plasma concentration of ciprostene in a dog treated with ciprostene at 2.5 μg/kg was determined successfully by this method.     

Determination of therapeutic and toxic concentrations of doxepin and loxapine using gas—liquid chromatography with a nitrogen-sensitive detector, and gas chromatography—mass spectrometry of loxapine

A gas—liquid chromatographic procedure is presented for the determination of therapeutic and toxic serum levels of doxepin and loxapine, using a nitrogen—phosphorus-sensitive detector. Amitriptyline is used as the internal standard. The method is accurate, sensitive and specific with no derivatization required prior to analysis. An advantage of the procedure is the small serum sample size needed for analysis and the selectivity and sensitivity of the detector, with the limit of detection being 3 and 2 μg/l for doxepin and loxapine, respectively. Nine cases of doxepin and loxapine misuse are presented. Serum doxepin concentrations ranged from 113 to 439 μg/l, with a loxapine concentration of 192 μg/l observed in one patient. The presence of the tricyclics was identified and confirmed by gas chromatography—mass spectrometry and the mass spectrum of loxapine is reported.     

Bioanalysis of erythromycin 2'-ethylsuccinate in plasma using phase-system switching continuous-flow fast atom bombardment liquid chromatography—mass spectrometry

A specific method for the determination of erythromycin 2'-ethylsuccinate (EM-ES) in plasma is described. The method involves a liquid—liquid extraction procedure followed by the analysis of extracts using phase-system switching (PSS) continuous-flow fast atom bombardment (CF-FAB) liquid chromatography—mass spectrometry (LC—MS). In PSS EM-ES is enriched after analytical separation on a short trapping column, from which it is desorbed to the LC—MS interface. In this way, favourable mobile phases can be used for the LC separation and for the MS detection. Using the PSS approach a flow-rate reduction from 1.0 ml/min in the LC system to 15 μl/min going into the mass spectrometer was achieved without splitting. The determination limit for EM-ES was 0.1 μg/ml.     

Thermospray and particle beam liquid chromatographic—mass spectrometric analysis of coumarin anticoagulants

Positive ion mass spectra were obtained from several coumarin oral anticoagulants (phenprocoumon, warfarin, acenocoumarol and dicoumarol) and derivatives by liquid chromatography—thermospray mass spectrometry (LC—TSP-MS) and liquid chromatography—electron impact mass spectrometry (LC—EI-MS) to assess the use of LC—MS methods for the determination of these compounds in biological materials. LC—TSP mass spectra showed a single [M + 1]⁺ ion with no fragmentation; LC—EI mass spectra showed fragment ions which were similar in mass and relative intensities to those obtained by conventional EI-MS. These data should serve as a basis for the development of LC—MS methods for the qualitative and quantitative analysis of coumarin anticoagulants in biological samples. LC—TSP-MS was applied to the determination of phenprocoumon in a plasma extract from an anticoagulated patient.     

Biological monitoring of hexamethylene diisocyanate by determination of 1,6-hexamethylene diamine as the trifluoroethyl chloroformate derivative using capillary gas chromatography with thermoionic and selective-ion monitoring

A GC method using a novel derivatization reagent, 2′,2′,2-trifluoroethyl chloroformate (TFECF), for the derivatization of primary and secondary aliphatic amines with the formation of carbamate esters is presented. The method is based on a derivatization procedure in a two-phase system, where the carbamate ester is formed. The method is applied to the determination of 1,6-hexamethylene diamine (HDA) in aqueous solutions and human urine, using capillary GC. Detection was performed using thermionic specific detection (TSD) and mass spectrometry (MS)—selective-ion monitoring (SIM) using electron-impact (EI) and chemical ionization (CI) with ammonia monitoring both positive (CI)⁺ and negative ions (CI)⁻. Quantitative measurements were made in the chemical ionization mode monitoring both positive and negative ions. Tetra-deuterium-labelled HDA (TDHDA; H₂NC²H₂(CH₂)₄C²H₂NH₂) was used as the internal standard for the GC—MS analysis. In CI⁺ the m/z 386 and the m/z 390 ions corresponding to the [M + 18]⁺ ions (M = molecular ion) of HDA—TFECF and TDHDA—TFECF were measured; in CI⁻ the m/z 267 and the m/z 271 ions corresponding to the [M — 101]⁻ ions. The overall recovery was found to be 97 ± 5% for a HDA concentration of 1000 μg/l in urine. The minimal detectable concentration in urine was found to be less than 20 μg/l using GC—TSD and 0.5 μg/l using GC—SIM. The overall precision for the work-up procedure and GC analysis was ca. 3% (n = 5) for 1000 μg/l HDA-spiked urine, and ca. 4% (n = 5) for 100 μg/l. The precision using GC—SIM for urine samples spiked to a concentration of 5 μg/l was found to be 6.3% (n = 10).     

Simultaneous analysis of phenylglycols and phenylethanols in human urine by gas chromatography—mass spectrometry

A method is described for the determination of the neutral metabolites formed from catecholamines and various other structurally related phenylethylamines by using gas chromatography—chemical ionization—mass spectrometry. These metabolites (phenylglycols and phenylethanols) were extracted from urine specimens and converted to pentafluoropropionyl derivatives which were separated on either 3% OV-1, 3% SP-2250, or 3% QF-1 packed columns. Our results demonstrate the presence in human urine of p-hydroxyphenylglycol, a metabolite of octopamine. One patient excreted 13 and 91 μg/day of free and total (free + conjugated) p-hydroxyphenylglycol, respectively. Treatment with a monoamine oxidase inhibitor reduced the excretion of total p-hydroxyphenylglycol to 30% of baseline level.     

Automated theophylline assay using gas chromatography and a mass-selective detector

An automated gas chromatographic—mass spectrometric assay for theophylline is described. Theophylline is extracted from plasma or urine (50 μl) and transformed into an N-pentyl derivate. The internal standard used for quantitation is [1,3-¹⁵N, 2-¹³C]theophylline. The detection is performed by monitoring the molecular ions 250 for theophylline and 253 for the internal standard with a quadrupole mass specific detector HP 5790 A. The system has been fully automated: injection, calibration, assay, calculation. The method shows excellent analytical parameters: linearity between 2 and 40 μg/ml; day-to-day reproducibility 1.82% for a concentration of 15 μg/ml; repeatability 0.75% (15 μg/ml) and 0.33% (30 μg/ml). Accuracy is also excellent. Due to the use of an internal standard labelled with stable isotopes, the specificity and high analytical quality of the method make it useful as a reference method to compare with routine theophylline assays.     

Atomic energy of Canada Limited

A method is described for determining stable cobalt concentrations in natural lake waters using polarized Zeeman effect graphite furnace atomic absorption spectrometry. The analysis requires small sample volumes, minimal sample pretreatment and preparation and no chelation or solvent extraction procedures. Instrument linearity, per cent cobalt recovery, matrix interferences and the use of the standard additions method are discussed. The increased sensitivity and precision due to Zeeman AA background correction permits precise determination of stable cobalt in the µg.1^–1 range.AECL 8351 reprintAECL 8351 reprint     

Chloride attachment negative-ion mass spectra of sugars by combined liquid chromatography and atmospheric pressure chemical ionization mass spectrometry

A method has been developed for the rapid determination of sugars, including molecular weight measurements, using high-performance liquid chromatography coupled with negative-ion, atmospheric-pressure chemical-ionization mass spectrometry. The chromatography was carried out on a 250 × 4 mm I.D. column packed with 7 μm NH₂-silica. The mobile phase consisted of a high percentage of methanol or acetonitrile with a small amount of chloroform. During the mass spectrometry, a strong base is formed from the polar solvent molecules by corona discharge, followed by ion—molecule reactions in the chemical ionization ion source (e.g. the methoxy anion is formed from methanol). This strong base reacts with the chloroform, generating chloride ions, which in turn react with the neutral sugar molecules as they elute from the chromatograph. The chloride ion and sugar interactions yield chloride-attachment ions, which are further stabilized by successive collisions. In this method, authentic monosaccharides and some oligosaccharides show dominant quasi-molecular ions, [M + Cl]⁻, with little fragmentation, and it is particularly useful for the molecular weight determination of sugars.     

Measurement of plasma uracil using gas chromatography–mass spectrometry in normal individuals and in patients receiving inhibitors of dihydropyrimidine dehydrogenase

A sensitive gas chromatographic–mass spectrometric method is described for reliably measuring endogenous uracil in 100 μl of human plasma. Validation of this assay over a wide concentration range, 0.025 μM to 250 μM (0.0028 μg/ml to 28 μg/ml), allowed for the determination of plasma uracil in patients treated with agents such as eniluracil, an inhibitor of the pyrimidine catabolic enzyme, dihydropyrimidine dehydrogenase. Calibration standards were prepared in human plasma using the stable isotope, [¹⁵N₂]uracil, to avoid interference from endogenous uracil and 10 μM 5-chlorouracil was added as the internal standard.     

Quantitative measurement of a new synthetic hetrazepine derivative, BN50730, in human plasma and urine by combined liquid chromatography—negative chemical ionization mass spectrometry using a particle beam interface

A new simple and sensitive assay has been developed for the quantitative measurement of BN50730 at the picomole level in human plasma and urine. The drug and the internal standard (BN50765) were measured by combined liquid chromatography—negative chemical ionization mass spectrometry with methane as the reagent gas. A simple solid—liquid extraction procedure was used to isolate BN50730 from complex biological matrices. Mild operating conditions were required to assay the parent drug with a particle beam interface from Hewlett-Packard. The mass spectrometer was tuned to monitor the intense ion m/z 333, which was generated in the ion source by a dissociative capture process. This assay was performed with 1 ml of plasma or 0.1 ml of urine, and the quantification limit of the method was statistically calculated as 1 ng ml⁻¹. The very low relative standard deviation and mean percentage of error calculated during the different within-day or between-day repeatability assays clearly demonstrate the ruggedness of the technique for the routine determination of BN50730 in the biological fluids. Some preliminary results on the pharmacokinetics of the drug are presented to illustrate the applicability of this new powerful LC—MS method.     

Determination of l-α-acetylmethadol, l-α-noracetylmethadol and l-α-dinoracetylmethadol in plasma by gas chromatography—mass spectrometry

A method is described for the simultaneous determination of l-α-acetylmethadol (LAAM) and its N-demethylated metabolites, l-α-noracetylmethadol (norLAAM) and l-α-dinoracetylmethadol (dinorLAAM), in plasma by gas chromatography—chemical ionization mass spectrometry. Deuterated internal standards for each analyte serve as carriers and control for recovery during sample purification on a solid-phase extraction column (C₁₈), and subsequent separation and analysis on a DB-17 capillary column. With this method, we have determined levels of LAAM, norLAAM, and dinorLAAM in small volumes of plasma (100 μl). The limit of quantitation for all analytes was approximately 1.0 ng/g plasma and the limit of detection was approximately 0.5 ng/g plasma. An experimental application is also described where these analytes are quantitated in plasma obtained from rats before, during, and after chronic administration of LAAM-HCl. Since this technique affords a selective and sensitive means of detection of LAAM and its active, N-demethylated metabolites in small samples of blood, it may enable patient compliance to be more easily assessed by allowing samples to be collected by a simple finger-prick technique.     

Simultaneous determination of blood concentrations of methohexital and its hydroxy metabolite by gas chromatography and identification of 4′-hydroxymethohexital by combined gas—liquid chromatography—mass spectrometry

A simple, sensitive and selective method is described for the simultaneous determination of low concentrations (less than 50 ng/ml) of underivatized methohexital and its hydroxy metabolite in small (0.1 ml) samples of human and rat plasma or whole blood by gas chromatography with nitrogen-selective detection.Moreover, the main metabolite in rat and man was identified as 4′-hydroxymethohexital by comparison of chromatograms from gas—liquid chromatography (GLC) with data obtained from GLC—mass spectrometry and ¹H-nuclear magnetic resonance spectrometry of this metabolite, produced both by incubating methohexital with isolated rat liver microsomes and by isolating this metabolite from rat urine.     

Nickel toxicity inPseudomonas tabaci: Single cell and bulk sample analysis of bacteria cultured at high cation levels

Summary The growth ofPseudomonas tabaci in nutrient medium is partially inhibited in the presence of 10^–3 M added nickel (threshold toxic concentration), with complete inhibition at 10^–2 M nickel—but no effect at 10^–4 and 10^–5 M. Toxic levels of nickel affect both cell division and cell viability.Spectrophotometric determination of intracellular levels of nickel at different external concentrations showed that the highest internal values occurred with cells cultured in 10^–4M (non-toxic) nickel medium rather than in 10^–3 (toxic) medium—suggesting that nickel toxicity does not primarily relate to internal concentration.X-ray microanalysis, carried out on whole bacterial cells, showed that toxic levels of nickel in the external medium resulted in a range of ionic changes in the cell, including a decrease in the level of K (K efflux) and an increase in the levels of Mn, Fe, Ni, and Cu (transition metal cation influx). Other changes induced by nickel toxicity included an increase in the level of soluble S (with a decrease in insoluble S), an increased cell dry mass, and a conspicuous plasmolysis—which was observed both in whole cells and in ultrathin sections.The results obtained support a primary toxic effect of nickel at the cell surface—possibly directly affecting the transport activity of the plasmalemma. The resulting changes, particularly involving the influx of a range of cations, may lead to secondary toxic activities affecting the whole metabolism, leading to plasmolysis and inhibition of division.     

Rapid,sensitive and specific electron capture—gas chromatographic method for the quantitation of 6-chloro-2-(1-piperazinyl)pyrazine in biological fluids

A highly specific and sensitive gas chromatographic method for the determination of 6-chloro-2-(1-piperazinyl)pyrazine (MK-212), a central serotonin-like agent, in biological fluids is described. MK-212 and a related internal standard are extracted into benzene from an alkaline solution, back-extracted into acid and then re-extracted into benzene at an alkaline pH. The amines are converted to the trifluoroacetyl derivatives (characterized by gas—liquid chromatography—mass spectrometry), chromatographed and detected with a ⁶³Ni electron capture detector. The sensitivity of the method is such that 10 ng of drug can be measured per aliquot of biological fluid. The precision and accuracy of the method are well within acceptable limits. Specificity of analysis was established by gas—liquid chromatography—mass spectrometry techniques.     

On-line measurements of C enrichments in rat breath: Non-invasive method for in vivo study of drug enzymatic induction

A differential kinetic study of ¹³CO₂ enrichment of breath after the intake of specific ¹³C-labelled substrates and co-administration of a drug allows the drug's ability for enzyme induction to be evaluated in vivo. A method and a gas chromatograph—isotope ratio mass spectrometer device for on-line measurements of ¹³CO₂ enrichment in the breath of small animals are described. This system allows on-line breath sample collection from a metabolic cage, purification by gas chromatography, determination of CO₂ by thermal conductivity detection and measurement of ¹³CO₂ enrichment by isotope ratio mass spectrometry. Two protocols for phenobarbital-inducible P450 and 3-methylcholanthrene-inducible P1-450 isoenzymes are described.     

A method for low volume and low Se concentration samples and application to paired cerebrospinal fluid and serum samples

The well-known beneficial health effects of Se have demanded the development of rapid and accurate methods for its analysis. A flow injection (FI) method with inductively coupled plasma mass spectrometry (ICP-MS) as a selenium-selective detector was optimized. Flow injection was carried out using a Knauer 1100 smartline inert series liquid chromatograph coupled with a Perkin Elmer DRC II ICP-mass spectrometer. For sample injection a Perkin Elmer electronic valve equipped with a 25 μL sample loop was employed. Before measurement, standards or samples were administered with 1 μg/L rhodium as internal standard for correction of changes in detector response according to changes in sample electrolyte concentration. The method characterization parameters are: LOD (3σ criterion): 26 ng/L, LOQ (10σ criterion): 86 ng/L, linearity: 0.05–>10 μg/L, r²=0.9999, serial or day-to-day precision at 2 μg/L: 4.48% or 5.6%. Accuracy was determined by (a) recovery experiments (CSF spiked with 2 μg/L Se); (b) comparison of FI-ICP-MS measurement with graphite furnace atomic absorption (GFAAS) measurements of 1:10 diluted serum samples; (c) Se determination in urine and serum control materials. Recovery (a) was 101.4%, measurement comparison with GFAAS (b) showed 98.8% (5 serum samples, 1:10 diluted in the range of 0.5–1.3 μg/L, compared to GFAAS determination, which was set to 100%), and accuracy was 96.8% or 105.6% for the serum or urine control material. Analysis time per sample was short and typically below 2 min for the complete measurement, including sample introduction, sample-line purge and quadruplicate Se determination.This method was used to determine Se in cerebrospinal fluid (CSF) and plasma (here parallel to GFAAS) in 35 paired serum and CSF samples. Se determination gave values in the range of 42–130 μg/L for serum and 1.63–6.66 μg/L for CSF. The median for Se in 35 individual CSF samples was 3.28 μg/L, the mean (±SD) was 3.67 (1.35) μg/L, whilst for individual serum samples the median was 81 μg/L and the mean (±SD) was 85 (26) μg/L. When relating the paired Se concentrations of CSF samples to respective serum samples it turned out that Se-CSF (behind blood brain barrier (BBB)) is independent on Se-serum concentration (before BBB).     

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.     

Improved method for routine determination of nicotine and its main metabolites in biological fluids

Extrelut^R extraction and glass capillary gas chromatography were applied to the routine determination of nicotine and its metabolites cotinine, nicotine-1′-N-oxide and cotinine-1-N-oxide in urine and plasma. After extraction of nicotine and cotinine both N-oxides and phendimetrazine-N-oxide (used as internal standard) were reduced to their bases by SO₂ on-column and eluted by a mixture of diethyl ether and dichloromethane. The minimum detectable concentrations are 0.03 μg/ml for urinary nicotine and cotinine and 0.1 μg/ml for the N-oxides. In plasma samples the corresponding values are 5 ng/ml and 15 ng/ml, respectively, with sample values as small as 2 ml. The advantage of the direct determination of all four compounds of interest in one sample reduced the amount of plasma required. The straightforward and rapid extraction and reduction procedure as well as the long-term stability of the gas chromatographic separation system make the method suitable for routine application.     

Determination of plasma cocaine and ethylcocaine (cocaethylene) in mice using gas chromatography—mass spectrometry and deuterated internal standards

A gas chromatographic—mass spectrometric method is described for the determination of cocaine and ethylcocaine (cocaethylene) from mouse plasma microsamples (50 μl). [²H₃]Cocaine and [²H₅]ethylcocaine served as internal standards, analytical separations were performed on a (5% phenyl)methylpolysiloxane capillary column, and detection was by selected-ion monitoring of electron-impact generated fragment ions [M --- CO₂Ph]. Pilot study plasma concentrations of ethylcocaine following coadministration of cocaine and ethanol were less than 5% of the parent drug.