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Anileridine and pethidine were established by gas and thin layer chromatography and mass spectroscopy. In the mass spectrum the main peak of anileridine is found at m/e 246 and that of pethidine at m/e 71. The determination was made by gas chromatography from the blood, urine, liver, muscle and stomach contents.
We compared doxorubicin and metabolite pharmacokinetic data obtained from thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) assay of plasma samples from six patients who had been treated with doxorubicin. Duplicate 1-ml samples were extracted with chloroform: isopropanol (1:1) and assayed using a sensitive HPLC system incorporating a dual pump gradient with tetrahydrofuran as the mobile phase and fluorescence detection. Duplicate 1-ml samples from the same specimens were assayed using a modification of a previously described TLC assay. Areas under the curve for doxorubicin by HPLC (3.36 +/- 2.30 microM X h) and TLC (4.16 +/- 2.50 microM X h) were not significantly different (P = 0.5). Terminal half-life of doxorubicin by HPLC (28.0 +/- 6.98 h) and TLC (23.2 +/- 7.8) (P = 0.29) and the calculated total-body clearances by HPLC (0.55 +/- 0.29 l/min) and TLC (0.45 +/- 0.23) (P = 0.55) were not significantly different. Areas under the curve for doxorubicinol by HPLC (2.75 +/- 1.4 microM X h) and TLC (2.53 +/- 7.1 microM X h) (P = 0.73) showed no significant differences. HPLC detected a mixed 7-deoxydoxorubicinol aglycone-doxorubicin aglycone peak, 7-deoxydoxorubicin aglycone, and two nonpolar, unidentified metabolites. TLC detected the following aglycone metabolites: doxorubicin aglycone, doxorubicinol aglycone, 7-deoxydoxorubicinol aglycone, an unidentified polar metabolite, and several unidentified nonpolar metabolites. From these data we conclude that HPLC and TLC detect concentrations of doxorubicin and doxorubicinol from human plasma equally well to concentrations of 7.0 nM (4 pmol injected doxorubicin). Aglycones do circulate in human plasma at concentrations above the detection limits of both assays. Doxorubicinol aglycone, which is detected by TLC but not by HPLC, may be formed from artifactual breakdown of doxorubicinol during TLC development. Unidentified nonpolar compounds seen on HPLC and TLC may represent further doxorubicin metabolism than previously described.
Antiserum to nortriptyline was generated in male New Zealand rabbits innoculated with n-succinylnortriptyline-bovine serum albumin conjugates. The antiserum was used at a final dilution of 1:4000 and tritiated imipramine was used as the label antigen. An accurate, sensitive, and specific radioimmunoassay of depressed patients' plasma or serum nortriptyline is described. The accuracy was good with a recovery range of 90-100% with a mean of 94%. The method can be used to measure nortriptyline concentration in the range of 0.1 microgram/liter to 100 micrograms/liter without prior extraction and purification of plasma or serum. Results of this method correlate well with those obtained by high-pressure liquid chromatography (r = 0.979) and by gas-liquid chromatography (r = 0.98). The specificity of the antiserum was examined by studying the cross-reactivity of 20 different psychopharmacological compounds, including nortriptyline's metabolites.
Methods for the screening of fungal cultures for toxic secondary metabolites are reviewed. Thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) are good general analytical methods for secondary metabolites in unpurified extracts. The combination of normal phase TLC using different chemical spray reagents with reversed phase HPLC, using alkylphenone retention indices and diode array detection, is a powerful technique for identifying the individual mycotoxins detected. The results of the screening methods are very dependent of the growth media and incubation conditions and a general method for the detection of Penicillium, Aspergillus, Fusarium, Alternaria and Cladosporium toxins is suggested.
Thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and mass spectrometry (MS) methods have been developed for the analysis of the antibiotic nybomycin, its derivatives deoxynybomycin and nybomycin acetate, during the fermentation and isolation of nybomycin. Using a quantitative HPLC based assay, the time course of nybomycin production (nybomycin titers) in 1000 liter fermentations was determined. Desorption chemical ionization mass spectrometry (DCI/MS) of standard nybomycin samples, fermentation broth samples and purified fractions suggested the co-production of deoxynybomycin which was not reported previously from this organism. TLC and HPLC were used to confirm the presence of deoxynybomycin in the crude extracts of fermentation broths.
The detection of 4-hydroxycoumarin rodenticides in poisoned domestic animals requires a highly sensitive method as tissue and serum levels of anticoagulants may be very low owing to rapid elimination, metabolism or post-mortem degradation. Thin-layer chromatography (TLC) and reversed-phase high-performance liquid chromatography (RP-HPLC) with fluorescence detection were used to identify the anticoagulants in spiked tissues and in suspicious samples. The analysis of ten suspicious samples highlighted the limitations of both methods. Only the three samples of baits were found positive by TLC whereas one of the five anticoagulants was detected in eight samples by RP-HPLC with fluorescence detection. Therefore, RP-HPLC with fluorescence detection proved to be the more sensitive method for detecting low levels of 4-hydroxycoumarins in blood serum, liver and ingesta, whereas TLC is usually sufficient for analysing baits.
The urinary metabolites of metoclopramide (4-amino-5-chloro-N-[2-diethylaminoethyl]-2-methoxybenzamide) were identified in cows. The drug was administered intravenously, voided urine was collected, and individual urine extracts were analysed by gas chromatography-mass spectrometry and high-performance liquid chromatography-photodiode array detection. The parent compound and one major metabolite (4-amino-5-chloro-N-[2-(ethylamino)ethyl]-2-methoxybenzamide) were common to all individuals. In addition to the parent and major metabolite, a second, minor metabolite was identified in two cows as 4-amino-5-chloro-N-[2-(diethylamino)ethyl]-2-hydroxybenzamide. The identity of the minor metabolite was confirmed by comparison with a standard synthesized by a new method. Metabolite identification and characterization in food animal species allows the design of safety and environmental impact studies and relative metabolite ratios between dose treatment groups.
Human milk triacylglycerols were separated by high-performance liquid chromatography. A 5-mu Supelcosil LC-18 column (Supelco, Inc., Bellefonte, PA) was used with acetone/acetonitrile (64:36, vol/vol) as mobile phase. Triacylglycerols were tentatively identified based on theoretical carbon number and relative retention time. Despite changes resulting from dietary fat variation, the major component triacylglycerols were those composed of palmitic, oleic and linoleic acids. Triacylglycerols with palmitic, stearic and oleic acids were present as minor components. Fatty acids were quantified by gas chromatography relative to an internal standard. Ratios of n-6/n-3 fatty acids were found to be higher than previously reported.
High-temperature gas chromatography and gas chromatography-mass spectrometry for the analyses of oligosaccharides derived from glycoproteins or glycosphingolipids has been developed. Permethylated oligosaccharides with up to about 12 sugar residues and masses up to 2500 Daltons can be analyzed. This approach is discussed and exemplified.
An investigation of the liquid chromatography of the minor capsaicinoids in a commercial capsaicinoid mixture is reported. Twelve stationary phases including C8, C18, C30, phenyl, and cation-exchange chemistries were examined in combination with isocratic aqueous methanol and aqueous acetonitrile mobile phases. A phenyl stationary phase and aqueous acetonitrile mobile phase baseline-resolved 7 of 11 capsaicinoids, and selected ion chromatograms (LC-ESI-MS) demonstrated this was the most effective reversed-phase separation. Argentation chromatography with an alkyl or phenyl column and aqueous silver nitrate-methanol mobile phase revealed the presence of the 6-ene-8-methyl and 6-ene-9-methyl homocapsaicin isomers and the absence of 7-ene-9-methyl homocapsaicin. A mixed phenyl-cation-exchange stationary phase (charged with silver ion) enabled unique and useful separations of the capsaicinoids.
n-Butyl benzyl phthalate (BBP) is an endocrine-disrupting chemical. A bacterium species capable of using BBP as the sole source of carbon and energy was isolated from mangrove sediment. Effects of BBP concentration, pH, temperature, and salinity on BBP biodegradation were studied. The optimum pH, temperature, and salinity for the BBP biodegradation were 7.0, 37 degrees C, and 15 g L(-1), respectively. BBP was completely degraded within 6 days under optimum conditions, and the biodegradation of BBP could be fitted to a first-order kinetic model. The major metabolites of BBP biodegradation were identified as mono-butyl phthalate, mono-benzyl phthalate, phthalic acid, and benzoic acid by using high-performance liquid chromatography and gas chromatography-mass spectrometry. A preliminary metabolic pathway was proposed for the biodegradation of BBP.
Commercial cheese products were analyzed for their composition and content of conjugated linoleic acid (CLA) isomers. The total lipids were extracted from cheese using petroleum ether/diethyl ether and methylated using NaOCH3. The fatty acid methyl esters (FAME) were separated by gas chromatography (GC), using a 100-m polar capillary column, into nine minor peaks besides that of the major rumenic acid, 9c,11t-octadecadienoic acid (18:2), and were attributed to 19 CLA isomers. By using silver ion-high performance liquid chromatography (Ag+ -HPLC), CLA isomers were resolved into seven trans,trans (5-9%), three cis/trans (10-13%), and five cis,cis (<1%) peaks, totaling 15, in addition to that of the 9c,11t-18:2 (78-84%). The FAME of total cheese lipids were fractionated by semipreparative Ag+ -HPLC and converted to their 4,4-dimethyloxazoline derivatives after hydrolysis to free fatty acids. The geometrical configuration of the CLA isomers was confirmed by GC-direct deposition-Fourier transform infrared, and their double bond positions were established by GC-electron ionization mass spectrometry. Reconstructed mass spectral ion profiles of the m + 2 allylic ion and the m + 3 ion (where m is the position of the second double bond in the parent conjugated fatty acid) were used to identify the minor CLA isomers in cheese. Cheese contained 7t,9c-18:2 and the previously unreported 11t,13c-18:2 and 12c,14t-18:2, and their trans,trans and cis,cis geometric isomers. Minor amounts of 8,10-, and 10,12-18:2 were also found. The predicted elution orders of the different CLA isomers on long polar capillary GC and Ag+ -HPLC columns are also presented.
Peroxidation of membrane lipids has been implicated in the toxicity of reactive oxygen intermediates and of several hepatotoxins, but the specific products of this peroxidation in vivo have not been chemically identified. A method for the isolation, identification, and quantitation of specific lipid hydroperoxy and hydroxy acids formed in vivo has been developed. Hydroxylated derivatives of linoleic, arachidonic, and docosahexaenoic acids formed in mouse liver phosphatidylcholines following carbon tetrachloride administration were isolated by high-pressure liquid chromatography and identified as the trimethylsilyl ether methyl ester derivatives by gas chromatography-mass spectrometry. This methodology should be important for the investigation of the role of lipid peroxidation in a variety of normal physiologic and pathologic processes.
The retention (k') for various biomolecules was studied as a function of the mobile phase composition in reversed-phase chromatography (RPC) and ion-exchange chromatography (IEC). The "elution window" (EW) for a molecule is defined as the mobile phase composition in which 1 less than k' less than 10. The following relations were verified: (i) EW(RPC) less than EW(IEC), and (ii) EW(large Mr) less than EW(small Mr). The results support the theory that in both RPC and IEC larger molecules interact with the stationary phase by multiple-site binding. The results are used to explain why, in both RPC and IEC, larger molecules are best separated on short columns using gradient elution while smaller molecules often require longer columns and isocratic conditions.
Analytical methods, using capillary gas chromatography and normal-phase high-performance liquid chromatography, were developed for the analysis of the neurotoxic chemicals n-hexane, 2-hexanone, and 2,5-hexanedione and their suspected metabolites. Two gas chromatographic methods, using a 50-m glass capillary OV 101 column and cyclohexane as an internal standard, were employed. In both methods, the injector and detector temperatures were 220 and 280 degrees C, respectively. In method I the following temperature program was used: isothermic at 50 degrees C for 30 min, followed by a temperature increase of 10 degrees C/min to a final temperature of 180 degrees C, which was then maintained for 7 min. This method was used to analyze the following compounds: n-hexane, 2,5-dimethylfuran, 2-hexanone, 3-hexanone, hexanal, 1-hexanol, 2-hexanol, 3-hexanol, 5-hydroxy-2-hexanone, gamma-valerolactone, 2,5-hexanedione, and 2,5-hexanediol. Method II, which was developed for n-hexane and eight of its more common metabolites, used the following temperature program: isothermic at 70 degrees C for 15 min, followed by a temperature increase of 40 degrees C/min to a final temperature of 220 degrees C, which was maintained for 5 min. A linear relationship between peak area and amount injected was observed over a 100-fold range. The minimum detectable amounts ranged from 0.05 to 1 microgram, depending on the compound. Normal-phase HPLC, using a 5-micron silica cartridge fitted into an RCM-100 radial-compression separation system, was utilized to analyze 2-hexanone and its metabolites 2,5-dimethylfuran, gamma-valerolactone, 5-hydroxy-2-hexanone, and 2,5-hexanedione.(ABSTRACT TRUNCATED AT 250 WORDS)
A method of analysis of 3-indolylacetic acid (IAA) and abscisic acid (ABA), allowing the simultaneous extraction of both regulators from plant material, has been developed. The method involves extraction with methanol, isolation of the acid fraction, diazomethane methylation, separation of the hormones through reverse-phase preparative high-performance liquid chromatography, and quantification of both compounds by gas-liquid chromatography. The recovery percentage at each step was monitored with radioactive compounds added at the beginning of the process. The final recovery was 70% for IAA and 96% for ABA. The method was applied to the analysis of the IAA and ABA content of stems of hazel (Corylus avellana L.).
A reversed-phase high-performance liquid chromatography method, with L-proline and copper as chiral mobile phase, is described for the enantiomeric resolution of various alpha-substituted ornithine and lysine analogs. Although ornithine gives no separation with the chiral eluant used, excellent resolutions are obtained for various alpha-alkyl-, alpha-halogenomethyl-, alpha-vinyl-, and alpha-ethynyl-substituted ornithines. Similar separations are also observed for the dehydroornithine and lysine analogs. Gas chromatography on a chiral stationary phase, Chirasil-Val, allows the resolution of the ornithine and lysine analogs after derivatization into the monofluoroacyl derivatives of their corresponding lactams. No resolution or only a poor resolution is obtained by GC on Chirasil-Val for the dehydroornithine analogs as their di-N-perfluoroacyl alkyl esters. The chiral eluant HPLC procedure is easily scaled up for the semipreparative resolution of several ornithine analogs, i.e., alpha-fluoromethylornithine, alpha-difluoromethylornithine, alpha-chlorofluoromethylornithine, and alpha-fluoromethyldehydroornithine, which are known as potent ornithine decarboxylase inhibitors in vitro and in vivo.
We have developed a reproducible and sensitive procedure for the isolation and measurement of choline, phosphocholine, glycerophosphocholine, phosphatidylcholine, lysophosphatidylcholine and acetylcholine in a single 100-mg sample of biological tissue. Tissues were spiked with 14C-methyl- and 2H-methyl- or 15N-choline labeled internal standards for each compound. They were extracted with chloroform/methanol/water and the aqueous and organic phases were dried. The organic phase was resuspended in chloroform/methanol (1/1, v/v) and an aliquot was applied to a silica-gel thin-layer chromatography plate. The plate was developed in chloroform/methanol/water (65/30/4, v/v). Segments which cochromatographed with external standards of phosphatidylcholine and lysophosphatidylcholine were stained, scraped, and hydrolyzed in 6 M methanolic-HCl at 80 degrees C for 60 min, liberating free choline. The aqueous phase was resuspended in methanol/water and injected onto a silica HPLC column. Choline and its metabolites were eluted using a binary nonlinear gradient of acetonitrile/ethanol/acetic acid/1 M ammonium acetate/water/0.1 M sodium phosphate (800/68/2/3/127/10, v/v changing to 400/68/44/88/400/10, v/v). Peaks were detected with an on-line radiometric detector, collected, and dried under vacuum. Each choline ester was digested in 6 M HCl at 80 degrees C to form choline. Choline was then converted to the propionyl ester and demethylated with sodium benzenethiolate. This volatile derivative was then isolated using gas chromatography and measured with a mass selective detector. Deuterated internal standards were used to correct for variations in recovery. Choline, glycerophosphocholine, phosphocholine, phosphatidylcholine, lysophosphatidylcholine, and acetylcholine were measured in rat liver, heart, muscle, kidney, plasma, red blood cells, and brain and in human plasma.(ABSTRACT TRUNCATED AT 250 WORDS)