Identification and quantitation of N-acetyl metabolites of biogenic amines in the thoracic nervous system of the locust, Schistocerca gregaria, by gas chromatography-negative-ion chemical ionisation mass spectrometry

The N-acetylated metabolites of p-tyramine, p-octopamine and dopamine were identified unambiguously and quantitatively determined in a single ventral thoracic nerve cord of the locust, Schistocerca gregaria, by gas chromatography-negative-ion chemical ionisation mass spectrometry (GC-NICIMS). Deuterium-labelled analogues of each compound were added to a single ventral thoracic nerve cord in acetonitrile: the tissue was homogenised and the suspension centrifuged. The solvent was removed from the supernatant and the resultant residue was derivatised with trifluoroacetic anhydride. Under negative-ion chemical ionisation conditions, the trifluoroacetyl derivatives produced ions which were sufficiently abundant to be suitable for selected-ion monitoring. This method is highly specific and gave a limit of detection below the picogram levels. N-Acetyl-5-hydroxytryptamine was determined using a previously published GC-NICIMS technique [S.P. Markey, R.W. Colburn and J.N. Johannessen, Biomed. Mass Spectrom., 7 (1981) 301]. The concentrations of N-acetyltyramine, N-acetyloctopamine, N-acetyldopamine and N-acetyl-5-hydroxytryptamine in locust thoracic nerve cords were, respectively, 1.86 +/- 0.71, 1.13 +/- 0.34, 6.77 +/- 8.48 and 0.07 +/- 0.02 ng per tissue.     

Identification and quantitation of phenylalanine, tyrosine and dihydroxyphenylalanine in the thoracic nervous system of the locust, Schistocerca gregaria, by gas chromatography-negative-ion chemical ionisation mass spectrometry

Phenylalanine, tyrosine and dihydroxyphenylalanine (DOPA) were identified unambiguously and quantitatively determined in single ventral thoracic nerve cords from the locust, Schistocerca gregaria, by gas chromatography-negative-ion chemical ionisation mass spectrometry. Deuterium-labelled analogues of each compound were added to a single ventral thoracic nerve cord in hydrochloric acid; the tissue was homogenised and the suspension centrifuged. The remaining hydrochloric acid was eliminated azeotropically by repeated additions of acetonitrile followed by evaporation under a stream of nitrogen and the resultant residue derivatised by reaction with hexafluoroisopropanol and pentafluoropropionic anhydride. Under negative-ion chemical ionisation conditions, the hexafluoroisopropanol-pentafluoropropionyl derivatives produced characteristic ions which were sufficiently abundant to be suitable for selected-ion monitoring. This method is highly specific and gave a limit of detection below the nanogram level. The amounts of phenylalanine, tyrosine and DOPA in a single ventral thoracic nerve cord were, respectively, 194 +/- 81, 347 +/- 88 and 11 +/- 11 ng per tissue.     

Determination of clenbuterol in bovine plasma and tissues by gas chromatography-negative-ion chemical ionization mass spectrometry

A highly sensitive and specific assay was developed for the determination of clenbuterol in bovine plasma and tissues. Clenbuterol and the internal standard [2H9]clenbuterol were measured by gas chromatography-negative-ion chemical ionization mass spectrometry with methane as the reagent gas. Bovine tissues including muscle, liver, heart, kidney, lung, suet, brain, spinal cord and thymus were ground in a buffer of pH 7 and then extracted using ethyl acetate. After two subsequent purification steps, the cleaned-up organic extract was derivatized with pentafluoropropionic anhydride. The mass spectrometer was set to monitor the abundant ions m/z 368 and 377 of the perfluoroacyl derivatives. This assay was performed with 1 ml of plasma or 0.2 g of tissue. The feasibility of this method was demonstrated by the determination of clenbuterol residues as the femtomole level in a variety of tissues.     

Determination of calcium acetylhomotaurinate in human plasma and urine by combined gas chromatography-negative-ion chemical ionization mass spectrometry

A highly sensitive and specific assay has been developed for the determination of calcium acetylhomotaurinate and the internal standard (LM 3041) at the picomole level in human plasma and urine by gas chromatography-mass spectrometry with methane as the reagent gas. After a multiple-step extraction process, the cleaned-up organic extract was derivatized with pentafluorobenzoyl chloride at ambient temperature. Subsequently, chlorination followed by amidation of the sulphonic acid group led to the N-pentafluorobenzoyl di-n-butylamide derivatives. The mass spectrometer was set to monitor the abundant [M - HF]- ions (m/z 424 and 438), which were generated in the ion source switched in the negative-ion chemical ionization mode. This assay required 1 ml of plasma or 50 microliters of urine, and the detection limit was 1 ng/ml. The accuracy of the assay was tested day by day with quality control specimens spiked blind to the analyst. The mean difference between the theoretical and actual values was less than 8%.     

Analysis of diethylstilbestrol, dienestrol and hexestrol in biological samples by immunoaffinity extraction and gas chromatography-negative-ion chemical ionization mass spectrometry

A method has been developed for the detection of diethylstilbestrol, together with dienestrol and hexestrol, using extraction with a single immunoaffinity column containing antibodies raised against diethylstilbestrol, followed by gas chromatography-negative-ion chemical ionization mass spectrometry. Immunoaffinity columns were prepared by coupling immunoglobulin G fractions obtained from rabbit antisera with a Sepharose matrix. The immunizing agent was synthesized by introducing a carboxyl group into the diethylstilbestrol molecule and coupling this product to bovine serum albumin. The columns were used for immunoadsorption of diethylstilbestrol and other estrogens, after dilution of samples with phosphate buffer, and were eluted with acetone-water (95:5 v/v). A derivatization method suitable for gas chromatographic-mass spectrometric analysis of diethylstilbestrol and other estrogens was developed using pentafluorobenzyl bromide and ethanolic potassium hydroxide as reagents. The derivatives obtained were detectable at the sub-picogram level using gas chromatography with negative-ion chemical ionization mass spectrometry. Recoveries of cis- and trans-diethylstilbestrol, dienestrol and hexestrol from the immunoaffinity columns, determined after extraction from urine, plasma and buffer, ranged from 28 to 96%. The sensitivity for diethylstilbestrol in urine samples was ca. 10 ppt. The method was applied to the analysis of urine from calves given a single dose of 10 mg of diethylstilbestrol. Free and glucuronic acid conjugated diethylstilbestrol decreased with time, but their ratio was variable.     

Simultaneous determination of prostanoids in plasma by gas chromatography-negative-ion chemical-ionization mass spectrometry

A method for simultaneous determination of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and thromboxane B2 (TxB2) in plasma was developed. After acidification and addition of 2H- and 3H-labelled internal standards, plasma prostanoids were extracted by reversed-phase cartridges and purified by normal-phase high-performance liquid chromatography. The pentafluorobenzyl, methoxime, trimethylsilyl derivatives were formed. Negative-ion chemical-ionization mass spectra with methane as reagent gas show one intense peak at m/z (M - pentafluorobenzyl). This ion was used for selective-ion monitoring. Prostanoid plasma concentrations (pg/ml) in five healthy volunteers were: PGE2 2.0-10.4, PGF2 alpha 2.2-9.8, 6-keto-PGF1 alpha 0.6-1.8, and TxB2 3.0-45.3. However, there is evidence that the TxB2 values may frequently be falsely high because of ex vivo production during the sampling procedure.     

Trace level determination of phenols as pentafluorobenzyl derivatives by gas chromatography-negative-ion chemical ionization mass spectrometry.

A method for the trace level determination of 11 phenols as pentafluorobenzyl (PFB) derivatives by gas chromatography-mass spectrometry (GC-MS) with negative-ion chemical ionization (NICI) is described. First, the conditions for the PFB derivatisation of phenols were optimized and were found to be reaction temperature 80 degrees C and reaction time 5 h. Second, the detection limits using selected ion monitoring (SIM) were compared between trimethylsilylated (TMS) derivatives in the electron ionization (EI) mode and PFB derivatives in the NICI mode. The responses for the PFB derivatives in the NICI mode were 3.3-61 times higher than those of the TMS derivatives in the EI mode. The instrumental detection limits using NICI-SIM ranged from 2.6 to 290 fg. This method was applied to the analysis of phenols in river water using solid-phase extraction. The recoveries of the phenols from a river water sample spiked with standards at 100 ng l-1 with 2-chlorophenol, 4-chloro-3-methylphenol and pentachlorophenol and at 1000 ng l-1 with phenol, 2,4-dimethylphenol, 2,4-dichlorophenol, 2-nitrophenol, 2,4,6-trichlorophenol and 4-nitrophenol were 81.2-106.3% (RSD 5.1-8.0%), except for 2-methyl-4,6-dinitrophenol and 2,4-dinitrophenol, for which the recoveries were 5.8 and 4.2%, respectively, because water contained in the acetone eluate interfered with the derivatisation of these compounds with two electrophilic nitro groups.     

Analysis of biogenic amines in the brain of the American cockroach (Periplaneta americana) by gas chromatography-negative ion chemical ionisation mass spectrometry

Biogenic amines in the brain of the American cockroach have been identified and quantified by an extraction-derivatisation procedure involving their reaction with ditrifluoromethylbenzoyl chloride (DTFMB) in the aqueous phase followed by extraction into an organic solvent, hydrolysis of phenolic esters and conversion of free hydroxyl groups to trimethylsilyl (TMS) ethers and subsequent analysis by gas chromatography-negative ion chemical ionisation mass spectrometry. The molecular ion of these DTFMB-TMS derivatives carried most of the ion current which made the method highly specific and gave a potential limit of detection below the picogram level. This method establishes unequivocally that the principal amines in cockroach brain are tyramine, p-octopamine, dopamine, 5-hydroxytryptamine and noradrenaline. In contrast to mammalian nervous tissue, the other positional isomers of octopamine, together with the isomeric synephrines, are absent.     

Determination of the lipid peroxidation product trans-4-hydroxy-2-nonenal in biological samples by high-performance liquid chromatography and combined capillary column gas chromatography-negative-ion chemical ionisation mass spectrometry

trans-4-Hydroxy-2-nonenal (HNE) is an aldehyde end-product of lipid peroxidation in biological systems which is capable of producing a range of powerful biological effects. We wish to describe a sensitive and selective strategy for the determination of HNE in biological samples. The method is based on the formation of the O-pentafluorobenzyl (O-PFB) oxime derivatives of HNE and its deuterated internal standard which, after sample clean-up by solid-phase extraction and purification by high-performance liquid chromatography (HPLC), were derivatised further to trimethylsilyl ethers. Subsequent capillary column gas chromatography-negative-ion chemical ionisation mass spectrometry (GC-NICIMS) using selected-ion monitoring allowed quantitation in the low ng/ml range. The use of an internal standard and the O-PFB oxime derivatives circumvented the problems encountered previously by other workers because of the volatility and instability of HNE. The syn-isomer of HNE O-PFB oxime followed the anti-isomer on the HPLC and GC columns used, giving a distinctive pair of peaks of characteristic relative proportion. Moreover, the NICI mass spectra of the geometrical isomers were significantly different, providing further evidence to validate the identity of any endogenous HNE recovered. The method was used to identify and quantify HNE in platelets, monocytes, plasma and oxidised low-density lipoprotein.     

Determination of acetyltrimoprostil and its metabolite trimoprostil in human or dog plasma by gas chromatography-negative-ion chemical ionization mass spectrometry

A sensitive and specific procedure is described for the determination of the antisecretory prostaglandin acetyltrimoprostil and its metabolite trimoprostil in human or dog plasma using gas chromatography--negative-ion chemical ionization mass spectrometry (GC--NICI-MS). Trideuterated analogues of both compounds are added to plasma as the internal standards. The plasma is extracted at pH 7.3 with benzene--dichloromethane (9:1), and the residue of the organic extract is reacted at room temperature with pentafluorobenzyl bromide in the presence of 18-crown-6-ether and potassium acetate. The derivatives are reconstituted in heptane, and appropriate aliquots are analyzed by GC--NICI-MS with selected-ion monitoring of the intense (M--C6F5CH2)- fragment ions of acetyltrimoprostil (m/z 419), trimoprostil (m/z 377), and their respective trideuterated analogues (m/z 422 and m/z 380, respectively). Quantitation of an experimental plasma sample is based on a comparison of the m/z 419 versus m/z 422 and m/z 377 versus m/z 380 ion ratios in each sample to that obtained from the analysis of drug-free plasma fortified with various amounts of both protio compounds, and a fixed amount of each trideuterated internal standard. The limit of quantitation of the assay for human plasma is 0.2 ng ml-1 with mean relative standard deviations at this concentration of 15.5% and 9.7% for acetyltrimoprostil and trimoprostil, respectively.     

Simultaneous determination of clebopride and a major metabolite N-desbenzylclebopride in plasma by capillary gas chromatography-negative-ion chemical ionization mass spectrometry

A procedure for the simultaneous assay of clebopride and its major metabolite N-desbenzylclebopride in plasma has been developed. The method utilizes capillary gas chromatography-negative-ion chemical ionization mass spectrometry with selected-ion monitoring of characteristic ions. Employing 2-ethoxy analogues as internal standards, the benzamides were extracted from basified plasma using dichloromethane. Subsequent reaction with heptafluorobutyric anhydride produced volatile mono- and diheptafluorobutyryl derivatives of clebopride and N-desbenzylclebopride, respectively. The methane negative-ion mass spectra of these derivatives exhibited intense high-mass ions ideal for specific quantitation of low levels in biological fluids. Using this procedure the recovery of the drug and metabolite from human plasma was found to be 84.4 +/- 1.5% (n = 3) and 77.4 +/- 4.7% (n = 3), respectively, at 0.5 ng/ml. Measurement of both compounds down to 0.10 ng/ml with a coefficient of variation of less than 10.5% is described. Plasma levels are reported in four volunteers up to 24 h following oral administration of 1 mg of clebopride malate salt.     

Determination of estrogens in river water by gas chromatography-negative-ion chemical-ionization mass spectrometry.

A method for the determination of estrogens (17alpha-estradiol, 17beta-estradiol, estrone, ethynyl estradiol, and estriol) as pentafluorobenzyl-trimethylsilyl (PFB-TMS) derivatives by gas chromatography-mass spectrometry (GC-MS) with negative-ion chemical-ionization (NICI) is described. The NICI of all the derivatives produced an intense [M-PFB]- ion as the base peak. The reagent gas (methane) flow-rate and the ion source temperature were determined to be 2.0 ml/min and 240 degrees C, respectively, for the optimized NICI-selected ion monitoring (SIM) conditions. The sensitivities of the PFB-TMS derivatives in the NICI mode were 8.0-130 times higher than those of the PFB-TMS derivatives in electron ionization (EI) mode, and 12-25 times higher than those of all the TMS derivatives in the EI mode. This method was applied to the analysis of estrogens in river water using a solid-phase extraction as the sample preparation. The recoveries of the target chemicals from a river-water sample spiked with standards at 2 ng/l level were 85.8-126.5% (RSD, 6.2-13.0%). The methodical detection limits ranged from 0.10 to 0.28 ng/l.     

Determination of benzidine in urine using capillary gas chromatography and negative-ion chemical ionisation mass spectrometry

Summary A highly sensitive and specific gas chromatographic-mass spectrometric (GC-MS) assay for the determination of benzidine in urine is reported. It is based on the solvent extraction of the hydrolysed benzidine conjugates, together with the deuterium-labelled benzidine-d₈ added as an internal standard, and a two-phase derivatisation procedure using pentafluoropropionic anhydride (PFPA) in the presence of pyridine as the phase-transfer catalyst. The reaction is complete within 5 min at room temperature. The pentafluoropropyl derivatives are quantified through capillary column GC-MS using selected ion monitoring (SIM) in the negative-ion chemical ionisation mode (NICI). The lower limit of detection for benzidine was 0.5 g/l and the calibration plot showed linearity between 2 g/l and 200 g/l. The recovery of the analyte added to pooled urine was above 82%. Analysis of 20 urine samples from un-exposed persons and 20 urine specimens of workers employed in a polyurethane-making plant using this procedure showed no substances likely to manifest false positive results in the range of interest.     

High resolution two-dimensional mapping of tissue-specific polypeptides in the desert locust, Schistocerca gregaria.

High resolution two-dimensional gel electrophoresis (2-DE) using immobilized pH gradients was used to map the tissue-specific polypeptides of the desert locust, Schistocerca gregaria. Highly specific comprehensive 2-DE reference maps ("master gels") were developed for the brain, corpus cardiacum, subesophageal ganglion, and hemolymph. The polypeptides were well resolved within the pH 4-7 range in the first dimension and within the 14-94 kDa molecular mass range in the second dimension.     

Quantification of distinct molecular species of the 2-lyso metabolite of platelet-activating factor by gas chromatography-negative-ion chemical ionization mass spectrometry.

The biological activity of platelet-activating factor (PAF) is comprised by a few molecular species of phosphatidylcholine which contain a fatty alcohol connected by an ether linkage to the sn-1 position of the glycerol backbone and an acetate ester at the sn-2 position. The various molecular species of PAF differ in chain length and degree of unsaturation in the fatty alcohol residue side-chain. PAF is rapidly hydrolyzed to lyso-PAF by an acetylhydrolase enzyme which is quite active in a number of cells that synthesize PAF. We describe a method for quantitation of lyso-PAF which involves conversion to its propionate derivative in the presence of an internal standard (deuterium-labelled PAF), digestion to the diglyceride with Bacillus cereus phospholipase C, conversion to the pentafluorobenzoate derivative and capillary column gas chromatographic-negative-ion methane chemical ionization mass spectrometric analysis. Distinct molecular species of lyso-PAF can be individually quantitated at levels of 1 ng or less. These methods are applied to the demonstration of lyso-PAF accumulation in renal tissue from transplanted allografts undergoing acute rejection, in renal tissue from kidneys subjected to cold storage and autotransplantation, and in intestinal mucosa subjected to warm ischemia and reperfusion.