Influence of photoperiod on seed development in the genetic line of peas G2 and its relation to changes in endogenous gibberellins measured by combined gas chromatography — Mass spectrometry

When apical senescence in the genetic line of peas G2 was prevented by short days fruit development was also found to be retarded. The levels of GA₂₀ and GA₂₉ in cotyledons and pods grown under long or short days were measured by gas chromatography — mass spectrometry multiple ion monitoring using extracts derivatised with deuterated trimethylsilyl groups as internal standards. The levels of GA₂₀ but not GA₂₉, were increased by short days. Conventional gas chromatography — mass spectrometry showed that relative to GA₂₉ the levels of GA₁₉, the other GA identified in G2 cotyledons, were also increased in short days. The levels of GA₂₀ in the pods were highest during the main phase of pod growth early in fruit development.Abbreviations GA_n gibberellin A_n - GC/MS gas chromatography — mass spectrometry - MIM multiple ion monitoring - Me methyl ester - SIM single ion monitoring - TIC total ion current - TMS trimethylsilyl ether - TLC thin layer chromatography - TTLC instant thin layer chromatography     

Determination of 6-keto-PGF1α, 2,3-dinor-6-keto-PGF1α, thromboxane B2, 2,3-dinor-thromboxane B2, PGE2, PGD2 and PGF2α in human urine by gas chromatography—negative ion chemical ionization mass spectrometry

A method for quantification of 6-keto-PGF_1α, 2,3-dinor-6-keto-PGF_1α, TXB₂, 2,3-dinor TXB₂, PGE₂, PGD₂ and PGF_2α in human urine samples, using gas chromatography—negative ion chemical ionization mass spectrometry, is described. Deuterated analogues were used as internal standards. Methoximation was carried out in urine samples which were subsequently applied to phenylboronic acid cartridges, reversed-phase cartridges and thin-layer chromatography. The eluents were further derivatized to pentafluorobenzyl ester trimethylsilyl ethers for final quantification by gas chromatography—mass spectrometry. The overall recovery was 77% for tritiated 6-keto-PGF_1α and 55% for tritiated TXB₂. Urinary levels of prostanoids were determined in a group of six volunteers before and after intake of the thromboxane synthase inhibitor Ridogrel, and related to creatinine clearance.     

Improved screening method for beta-blockers in urine using solid-phase extraction and capillary gas chromatography—mass spectrometry

An improved screening method for beta-blockers in urine is proposed, involving enzymatic hydrolysis, solid-phase extraction and capillary gas chromatography—mass spectrometry. Several extraction methods for beta-blockers, such as conventional liquid—liquid and solid-phase extraction procedures, have been evaluated for at least eight beta-blockers. Additionally, the gas chromatographic properties and mass fragmentation of the trimethylsilyl—trifluoroacetyl, trifluoroacetyl and cyclic n-butylboronate derivatives of beta-blockers have been compared and evaluated with respect to their efficiency for screening urine. The resulting screening method proved to be a specific and sensitive procedure, enabling these analytes to be detected and identified up to 48 h after the administration of a dosage, usually encountered in doping cases.     

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 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.     

Gas chromatographic—mass spectrometric screening procedure for the identification of formaldehyde-derived tetrahydroisoquinolines in human urine

A gas chromatographic—mass spectrometric method has been developed for the identification of 1,2,3,4-tetrahydroisoquinoline and six metabolites extracted from urine in the picogram range. The derivatization procedure for the substances, formed by reaction of formaldehyde with biogenic amines, employs propionic anhydride and can take place in aqueous medium. In this way artificial formation of these compounds via condensation of biogenic amines with aldehydes or α-keto acids during the work-up procedure is eliminated. The procedure results in hydrophobic compounds, which are quantitatively extractable by liquid—liquid extraction with organic solvents. Further clean-up was performed by solid-phase extraction on C₁₈ sample preparation columns.     

Derivatization of thiol-containing compounds

The determination of thiol-containing compounds in biological fluids is important in biochemistry and clinical chemistry. In this paper, derivatization reagents for thiols are reviewed with respect to their reactivity, selectivity, spectroscopic characteristics and their applicability especially to high-performance liquid chromatography. Derivatization used in ultraviolet and electrochemical detection. The derivatization reagents contain a functional group, e.g. an N-substituted maleimide, active halogen or aziridine, which react with the thiol group. Derivatization for use in flow injection analysis, thin-layer chromatography or gas chromatography—mass spectrometry is also described.     

Determination of clenbuterol in urine as its cyclic boronate derivative by gas chromatography—mass spectrometry

A rapid and reliable gas chromatographic—mass spectrometric method for the determination of clenbuterol in urine is described. Penbutolol was used as internal standard. Four derivatization procedures have been tested, of which 1-butaneboronic acid gave the best results. The method includes extraction of the alkalinized urine (3 ml) with tert.-butyl methyl ether—n-butanol (9:1), derivatization with 1-butaneboronic acid (15 min at room temperature), and analysis in the selected-ion monitoring mode of the derivatives of clenbuterol at m/z 243, 327 and 342 and of penbutolol at m/z 342 and 357. The detection limit is 0.5 ng/ml and the recovery better than 90%.     

Determination of free salsolinol concentrations in human urine using gas chromatography—mass spectrometry

The urine concentrations of free salsolinol were determined in six healthy volunteers, using a gas chromatographic—mass spectrometric method with electron-capture negative-ion chemical ionization after derivatization with pentafluoropropionyl anhydride. The sensitivity of this method allows the quantification of salsolinol concentrations of 0.55 pmol/ml. The synthesis of [²H₄]salsolinol from dopamine and [²H₄]acetaldehyde via a Pictet—Spengler condensation is described; [²H₄]salsolinol was used as the internal standard for salsolinol quantification. The urine concentrations of free salsolinol ranged from ca. 1 to 6 pmol/ml.     

Determination of ephedrines in urine by gas chromatography–mass spectrometry

A selective gas–liquid chromatographic method with mass spectrometry (GC–MS) for the simultaneous confirmation and quantification of ephedrine, pseudo-ephedrine, nor-ephedrine, nor-pseudoephedrine, which are pairs of diastereoisomeric sympathomimetic amines, and methyl-ephedrine was developed for doping control analysis in urine samples. O-Trimethylsilylated and N-mono-trifluoroacetylated derivatives of ephedrines — one derivative was formed for each ephedrine — were prepared and analyzed by GC–MS, after alkaline extraction of urine and evaporation of the organic phase, using d₃-ephedrine as internal standard. Calibration curves, with r²>0.98, ranged from 3.0 to 50 μg/ml depending on the analyte. Validation data (specificity, % RSD, accuracy, and recovery) are also presented.     

Determination of lorazepam in plasma and urine as trimethylsilyl derivative using gas chromatography–tandem mass spectrometry

A procedure based on gas chromatography–tandem mass spectrometry for identification and quantitation of lorazepam in plasma and urine is presented. The analyte was extracted from biological fluids under alkaline conditions using solid-phase extraction with an Extrelut-1 column in the presence of oxazepam-d₅ as the internal standard. Both compounds were then converted to their trimethylsilyl derivatives and the reaction products were identified and quantitated by gas chromatography–tandem mass spectrometry using the product ions of the two compounds (m/z 341, 306 and 267 for lorazepam derivative and m/z 346, 309 and 271 for oxazepam-d₅ derivative) formed from the parent ions by collision-induced dissociation in the ion trap spectrometer. Limit of quantitation was 0.1 ng/ml. This method was validated for urine and plasma samples of individuals in treatment with the drug.     

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).     

The reaction of oxalyl thiolesters with nucleophiles, especially thiols

Since there is evidence that oxalyl thiolesters (RSCOCOO⁻) are present in animal cells, and possibly may participate in the control of metabolism, the present study was undertaken to characterize their reactivity with nucleophiles so that one could gain a better understanding of how they might be affecting the activities of enzymes. At 25°C and neutral pH, N-acetyl-S-oxalyl-2-aminoethanethiol (NAC-S-Ox) reacts rapidly with cysteamine (2-aminoethanethiol) to give N-acetylcysteamine and N-oxalylcysteamine. Under similar conditions, other aminothiols, such as cysteine, homocysteine, penicillamine, and cysteine ethyl ester, also react rapidly with NAC-S-Ox, but non-thiol-containing amines, such as alanine, alanine ethyl ester, glycine, and S-methylcysteine, react more than four orders of magnitude less rapidly. The aminothiol reactions apparently proceed by rate-determining oxalyl transfer to the thiol followed by a rapid intramolecular S- to N-oxalyl migration. The reactions follow second-order kinetics with the thiolate anion being the reactive nucleophile. At 25°C and ionic strength 1.0 , k_N, defined in the equation, rate = k_N[RS⁻][NAC-S-Ox], has the following values ( ⁻¹ s⁻¹) for the anion of the reacting thiol: cysteamine, 170; cysteine, 260; cysteine ethyl ester, 76; homocysteine, 380. Rate data for the reaction of NAC-S-Ox with hydroxylamine, imidazole, hydroperoxide, and hydroxide were also obtained. The reaction of S-oxalyl-p-thiocresol with thiol anions under the same conditions gives the following values for k_N ( ⁻¹ s⁻¹ × 10⁻³): glutathione, 5.6; N-acetylcysteamine, 3.7; pantetheine, 4.8; 8-mercaptooctanoic acid, 4.5; 6-mercaptooctanoic acid, 1.0; dihydrolipoic acid, 8.2. These results indicate that oxalyl transfers from oxalyl thiolesters to thiol anions occur more than two orders of magnitude more rapidly than corresponding acetyl transfers, and that under physiological conditions any in vivo oxalyl thiolester would equilibrate within minutes with virtually every thiol in the cell, including those attached to enzymes. Consequently, it is proposed that one mechanism by which oxalyl thiolesters may function in vivo to alter the catalytic activities of enzymes is to covalently modify enzymic thiols by acylation with an oxalyl group.     

Determination of isoprostaglandin F2α type III in human urine by gas chromatography–electronic impact mass spectrometry. Comparison with enzyme immunoassay

F₂-Isoprostanes are stable lipid peroxidation products of arachidonic acid, the quantification of which provides an index of oxidative stress in vivo. We describe a method for analysing isoprostaglandin F_2α type III (15-F_2t-IsoP) in biological fluids. The method involves solid-phase extraction on octadecyl endcapped and aminopropyl cartridges. After conversion to trimethylsilyl ester trimethylsilyl ether derivatives, isoprostaglandin F_2α type III is analysed by mass spectrometry, operated in electronic impact selected ion monitoring mode. We have compared enzyme immunoassay (EIA; Cayman, Ann Arbor, MI, USA) to this method with 30 human urine aliquots following the same extraction procedure in order to determine the agreement between both methods. Isoprostaglandin F_2α type III concentrations determined with gas chromatography–mass spectrometry (GC–MS) did not agree with those determined with EIA. Our results suggest that GC–MS and EIA do not measure the same compounds. As a consequence, comparison of clinical results using GC–MS and EIA should be avoided.     

Determination of urinary 5-hydroxytryptophol by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method for the routine determination of elevated urinary levels of the serotonin metabolite 5-hydroxytryptophol (5-HTOL) is described. Urine samples were treated with β-glucuronidase, and 5-HTOL was isolated by solid-phase extraction on a small Sephadex G-10 column prior to injection onto an isocratically eluted C₁₈ reversed-phase column. Detection of 5-HTOL was performed electrochemically at +0.60 V vs. Ag/AgCl. The limit of detection was ca. 0.05 μM, and the intra-assay coefficients of variation were below 6% with urine samples containing 0.2 and 2.1 μM 5-HTOL and a standard solution of 2.0 μM (n = 5). The recovery of 5-HTOL after the sample clean-up procedure was close to 100%. A good correlation (r² = 0.97; n = 12) was obtained between the present method and a sensitive and specific gas chromatographic—mass spectrometric method. The total (free plus conjugated) 5-HTOL levels in urine were normally below 0.2 μM, but after an acute dose of alcohol they increased to 0.5–15 μM.     

Sample purification using a C18-bonded reversed-phase cartridge for the quantitative analysis of corticosteroids in adrenal cell cultures by high-performance liquid chromatography or gas chromatography—mass spectrometry

Quantitative extraction and subsequent purification of small biological samples often involve cumbersome procedures. We have devised a short and efficient method for the quantitative extraction of the corticosteroid and the 20α reduced steroid series from culture medium containing 20% sera in a single, pure fraction with separation from cholesterol. Passage through a C₁₈-bonded reversed-phase Sep-Pak^® cartridge of the acidified culture medium and subsequent extraction of the steroid fraction with methanol yields a single fraction containing all steroids in 90% recovery and reduced quantities of cholesterol down to 30%. The extract can then be used without further purification for quantitative analysis by high-performance liquid chromatography or derivatized and analyzed by gas chromatography and gas chromatography—mass spectrometry.     

Reversible reaction via a carbanion intermediate in the elimination of ammonia from l-histidine catalysed by histidine ammonia-lyase

l-[5′-²H₂]Histidine was used as a substrate to investigate the enzymatic reaction mechanism with histidine ammonia-lyase from Pseudomonas fluorescens. The study was performed to determine the exchange rate of deuterium at C-5′ of the imidazole ring with solvent hydrogen relative to the net urocanic acid production. The extent of hydrogen exchange at C-5′ of histidine or urocanic acid was measured by gas chromatography—mass spectrometry—selected ion monitoring, monitoring the molecular ion intensities of the respective gas chromatographic derivatives, at m/z 380 and 379 for histidine and at m/z 267 and 266 for urocanic acid. The observed hydrogen exchange at C-5′ suggested a reversible mechanism via a carbanion intermediate in the reaction with histidine ammonia-lyase.     

Application of negative-ion chemical ionization isotope dilution gas chromatography—mass spectrometry to single-dose bioavailability studies of mefloquine

An electron-capture negative-ion chemical ionization gas chromatographic—mass spectrometric assay for mefloquine, an antimalarial drug used in the treatment of drug-resistant Plasmodium falciparum malaria, is described. The method, developed in support of bioavailability studies involving the co-administration of different tableted formulations of the drug and an aqueous solution of its ¹³C₃-labeled analog, enables quantification of both dosage forms. Quantitative analysis of extracted plasma samples was performed on the O-tert.-butyldimethylsilyl (t-BDMS) derivative of the drug by selected-ion monitoring, using a VG Trio 2000 quadrupole mass spectrometer and monitoring the [M — t-BDMSOH]^−√ ions of the analytes. The method, incorporating [²H₆]mefloquine as an internal standard, demonstrated good accuracy and precision over the 1–200 ng ml⁻¹ range, with correlation coefficients greater than 0.990 for all standard curves and a detection level of 50 fg on-column. Replicate analysis of plasma samples over a 90-day period exhibited a mean intra-day and inter-day variation of less than 4.5% and 5.5%, respectively. The high stability and sensitivity of the assay, combined with the inherent selectivity of mass spectrometric detection, make the method well-suited for such studies.     

Solid-phase extraction and reversed-phase high-performance liquid chromatographic determination of sulphur mustard in blood

A reversed-phase high-performance liquid chromatographic method is reported for the analysis of sulphur mustard in blood with the aid of solid-phase extraction sample preparation. Sulphur mustard is extracted from blood samples (both in vitro and in vivo) of rats with a solution of 0.05 M sodium dodecyl sulphate and pre-concentrated over Sep-Pak C₁₈cartridges pre-coated with Tween-20. A Polygosil C₁₈ column is used with acetonitrile—water (52:48, v/v) as mobile phase for separation and sulphur mustard was detected at 200 nm.     

Derivatization and gas chromatographic—mass spectrometric detection of anabolic steroid residues isolated from edible muscle tissues

A method was developed for the detection of anabolic steroid residues in edible muscle tissues. After enzymic digestion of the tissue and purification on disposable C₁₈ solid-phase extraction columns, the extract was injected onto a C₁₈ reversed-phase high-performance liquid chromatographic column. Three fractions or windows were collected, each containing specific analytes. After evaporation to dryness, the residues were subjected to a derivatization procedure which yielded suitable derivatives. After gas chromatographic—mass spectrometric analysis, both gas chromatographic retention data and mass spectral data were used for the detection and identification of nortestosterone, testosterone, estradiol, ethinylestradiol, trenbolone, methyltestosterone, chlormadinone acetate, medroxyprogesterone acetate and megestrol acetate.