Identification of a metabolite of propafenone in human urine by means of high-performance liquid chromatography and gas chromatography--mass spectrometry.
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Biotransformation↗
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Analysis of hydroxylated and demethylated metabolites of mephenytoin in man and laboratory animals using gas-liquid chromatography and high-performance liquid chromatography.
Separation of urinary mephenytoin metabolites was evaluated under various gas-liquid chromatographic (GLC) and high-performance liquid chromatographic (HPLC) conditions. A simple and rapid alkylation procedure is described for GLC-using a nitrogen sensitive thermionic detector. The in situ formation of sodium methinylsulfinylmethide is used as base for the perpropylation of hydantoins and their metabolites. Normal-and reversed-phase HPLC of the underivatized compounds was performed using four different types of stationary phases. None of the GLC systems separated all the six hydantoin compounds tested, whereas, normal-and reversed-phase HPLC were able to obtain a complete separation of these compounds. The major metabolites of mephenytoin were 5-phenyl-5-ethylhydantoin, 3-methyl-5-(4-hydroxyphenyl)-5-ethylhydantoin and 5-(4-hydroxyphenyl)-5-ethylhydantoin in man, rat, mouse, rabbit, and guinea pig. 3-Methyl-5-phenyl-5-(2-hydroxyethyl)-hydantoin and 3-methyl-5-(3-hydroxyphenyl)-5-ethylhydantoin are major metabolites in the dog.
Animals↗
Analysis of mesocarb analogues in urine and plasma of rats by high-performance liquid chromatography and thermospray liquid chromatography-mass spectrometry.
The pharmacokinetics and metabolism of synthetic 2-hydroxymesocarb and 4-methyl-2-hydroxymesocarb analyzed by HPLC-DAD and thermospray LC-MS were studied in rats. Multistep liquid-liquid extraction (LLE) was used with diethyl ether at pH 7.0. The major metabolites of 2-hydroxymesocarb in both urine and plasma of the rat were the p-hydroxylated derivative of the phenylcarbamoyl group of the parent drug. The metabolites of 4-methyl-2-hydroxymesocarb in urine of rats may be the oxidized forms at the phenylcarbamoyl group of the parent drug. Absorption (ka) and elimination (ke) rate constants in plasma of 2-hydroxy-mesocarb were 0.0300 and 0.00485 min-1, respectively; those of 4-methyl-2-hydroxymesocarb were 0.0546 and 0.00797 min-1, respectively. The half-lives (t1/2) of 2-hydroxymesocarb and 4-methyl-2-hydroxymesocarb in plasma were 144 and 86 min, respectively.
Animals↗
Quantitative determination of several synthetic corticosteroids by gas chromatography-mass spectrometry after purification by immunoaffinity chromatography.
A study was conducted to test a multiresidue analytical procedure for detecting and quantifying several corticosteroids on which the European Union imposes maximum residue limits (MRLs). Primary extracts from different matrices (liver, milk, urine, faeces) were first purified on C18 cartridges. A new immunoaffinity clean-up step was included. The immunoaffinity gel was used to purify several corticosteroids simultaneously with enrichment of the corresponding fractions. The extracts were treated with an aqueous solution of pyridinium chlorochromate to fully oxidise all corticosteroids and to facilitate their extraction with dichloromethane. After evaporation, the final extract was reconstituted with toluene before injection into the GC-MS apparatus. The analysis was performed in the CI-negative ionisation mode using ammonia as the reactant gas. The estimated detection and quantification limits were, respectively, 0.25 and 0.5 ppb or lower. Overall, the method is reproducible to within 20%. Recovery is between 50 and 80% according to the corticosteroid.
Adrenal Cortex Hormones↗
Combination of high-performance liquid chromatography and thin-layer chromatography separation of five adducted nucleotides isolated from liver resulting from intraperitoneal administration with 7H-dibenzo[c,g]carbazole to mice.
7H-Dibenzo[c,g]carbazole, DBC, is a potent environmental liver carcinogen. Liver DNA from mice treated with DBC exhibited seven distinct DBC-DNA adducts as detected by 32P-postlabeling using multidimensional TLC. To improve quantitation and chemically characterize the adducts, DNA samples were hydrolyzed, 32P-postlabeled and the adducts were separated from the unadducted normal nucleotides on TLC using a D1 solvent, 0.65 M sodium phosphate (pH 6.8). Adducts were eluted from the TLC plates with 4.0 M pyridinium formate, concentrated, resuspended in 50% aqueous methanol and injected onto the HPLC; five individual adduct peaks were resolved and collected by this method. This approach will prove useful to decrease analysis time and improve chemical characterization of tightly clustered DNA adducts generated in vivo.
Animals↗
Characteristics of mass spectrometric analyses coupled to gas chromatography and liquid chromatography for 22-oxacalcitriol, a vitamin D3 analog, and related compounds.
The characteristics of the mass spectra of vitamin D3 related compounds were investigated by GC-MS and LC-MS using 22-oxacalcitriol (OCT), an analog of 1,25-dihydroxyvitamin D3, and related compounds. Fragmentation during GC-MS (electron impact ionization) of TMS-derivatives of OCT and the postulated metabolites gave useful structural information concerning the vitamin D3-skeleton and its side-chain, especially with respect to the oxidation positions of metabolites. In contrast, few fragment ions were observed in LC-MS (atmospheric pressure chemical ionization), showing that LC-MS gave poor structural information, except for molecular mass. However, when comparing the signal-to-noise ratio (S/N) observed during GC-MS and LC-MS analysis for OCT in plasma extracts, the S/N in LC-MS was over ten-times greater than in GC-MS, possibly due to the low recovery on derivatization and thermal-isomerization in GC-MS. Furthermore, both the GC-MS and the LC-MS allowed the analysis of many postulated metabolites in a single injection without any prior isolation of target metabolites from biological fluids by LC. These results suggest that GC-MS and LC-MS analysis for vitamin D3 related compounds such as OCT each have unique and distinct advantages. Therefore, the complementary use of both techniques enables the rapid and detailed characterization of vitamin D3 related compounds.
Animals↗
Biotransformation of 17-alkyl steroids in the equine: high-performance liquid chromatography-mass spectrometric and gas chromatography-mass spectrometric analysis of fluoxymesterone metabolites in urine samples.
In this study the equine metabolism of fluoxymesterone (9alpha-fluoro-11beta-17beta-dihydroxy-17alpha-meth ylandrost-4-ene-3-one) given orally has been investigated. The parent material was not detected, but two major 16-hydroxy metabolites which corresponded to a mono- and a di-hydroxylation product were evident. One of the hydroxylation positions was identified as C-16. Phase II metabolism in the form of glucuronide formation was also common. These steroids will provide target compounds for confirming abuse of this drug in the horse.
Anabolic Agents↗
Liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry of omega- and (omega-1)-hydroxylated metabolites of elaidic and oleic acids in human and rat liver microsomes.
In order to characterize the nature of the active site of cytochrome P450 2E1, the metabolism of various fatty acids with cis/trans geometric configurations has been investigated. A system coupling atmospheric pressure chemical ionization-mass spectrometry detection with HPLC separation was developed as an alternative method for the characterization of hydroxylated metabolites of oleic and elaidic acids in rat and human liver microsomes. Oxidation of oleic and elaidic acids led to the formation of two main metabolites which were identified by LC-MS and GC-MS as omega and (omega-1)-hydroxylated (or 17-OH and 18-OH) fatty acids, on the basis of their pseudo-molecular mass and their fragmentation. The assay was accurate and reproducible, with a detection limit of 25 ng per injection, a linear range from 25 to 1128 ng per injection, no recorded interference, intra-day and inter-day precision with variation coefficients <14%. This LC-MS method was validated with oleic acid by using both radiometric and mass spectrometric detections. A significant correlation was found between the two methods in human (r=0.86 and 0.94 with P<0.05 and 0.01) and rat liver microsomes (r=0.90 and 0.85 with P<0.01 and 0.05) for 17-OH and 18-OH metabolites, respectively. HPLC coupled to mass spectrometry for the analysis of hydroxylated metabolites of elaidic acid offers considerable advantages since the method does not require use of a radioactive molecule, completely separates the two hydroxymetabolites, confirms the identification of each metabolite, and is as sensitive as the radiometric analysis method. This method allowed the comparative study of oleic and elaidic acid hydroxylations by both human and rat liver microsomal preparations.
Animals↗
Qualitative and quantitative determination of sesquiterpenoids in Achillea species by reversed-phase high-performance liquid chromatography, mass-spectrometry and thin-layer chromatography.
A reversed-phase high-performance liquid chromatographic method was developed as a universal analysis system in order to determine and quantify antiphlogistic sesquiterpenoids in different Achillea species. Identification was performed by HPLC and diode array detection as well as by monitoring the HPLC fractions by TLC and MS. Using santonin as internal standard, HPLC separations were achieved with a methanol-water gradient system using RP 8 LiChrospher 100 (5 microm) as stationary phase. For validation, sample analyses were performed, using the two tetraploid species A. collina and A. pratensis. The method allows the identification and quantification of the main compounds achillicin, 8alpha-tigloxy-artabsin, 8alpha-angeloxy-artabsin, arglanin and santamarin with variation coefficients between 3.4 and 4.7% (total content) using santonin as internal standard. For the different compounds recovery was found between 81 and 107% performing multiple analyses of A. collina and A. pratensis.
Calibration↗
Screening of sulphonamides in egg using gas chromatography-mass-selective detection and liquid chromatography-mass spectrometry.
A rapid extraction and clean-up procedure for sulphonamide antibiotics in eggs suitable for both GC-MSD and LC-MS end determinations has been developed. The drugs were extracted using acetonitrile, acidified using acetic acid and cleaned-up using cation- and anion-exchange. For determination by GC-MSD, extracts were derivatised with diazomethane followed by pentafluoropropionic acid anhydride. For LC-MS extracts were taken up in water and used directly. The methodology developed was validated at the 100 and 25 microg kg(-1) levels, equivalent to the MRL and one quarter of the MRL. Results for the GC-MS procedure were quantitated against deuterated sulphadiazine with relative recoveries ranging from 54% for sulphachloropyridazine to 135.5% for sulphamethazine. Recoveries for the LC-MS procedure ranged from 33% for sulphaguanidine to 92% for sulphamethazine and sulphadimethoxine.
Chromatography, Liquid↗
Analysis of cyclophosphamide and five metabolites from human plasma using liquid chromatography-mass spectrometry and gas chromatography-nitrogen-phosphorus detection.
An assay method for the quantification of cyclophosphamide (CY) and five metabolites from human plasma is presented. The procedure is adapted to the chemical properties of the compounds of interest: non-polar compounds are extracted into methylene chloride, concentrated and analyzed by GC-NPD after derivatization, and the remaining aqueous fraction is deproteinated with acetonitrile-methanol prior to separation via reversed-phase HPLC and detection using atmospheric pressure ionization (API)-MS. Standard curves are linear over the required range and reproducible over five months. Plasma concentration-time profiles of CY and metabolites from a patient receiving CY by intravenous infusion (60 mg/kg, once a day for two days) are presented.
Antineoplastic Agents, Alkylating↗
Acidic and neutral drugs screen in blood with quantitation using microbore high-performance liquid chromatography-diode array detection and capillary gas chromatography-flame ionization detection.
Blood previously acidified with aqueous saturated ammonium chloride solution was extracted with ethyl acetate. The dried extract was subjected to acetonitrile-hexane partition. The acetonitrile portion was analysed for the presence of acidic and neutral drugs by HPLC-DAD (200 mm x 2.1 mm I.D. microbore ODS-Hypersil column) and GC-FID (25 m narrow-bore x 0.25 mm I.D. HP-5 column with 0.33 micron film thickness). The protocol was found to be suitable for both clinical toxicology (including emergency toxicology) and postmortem toxicology. At least 66 drugs of interest were unequivocally identified by RRTs (HPLC) and UV spectra (DAD) match while another 12 were unequivocally identified by double RRTs match (HPLC and GC). Quantitation was facilitated by incorporating calibration blood standards in each assay batch. The five drugs most commonly encountered in clinical blood specimens (1150 cases) were: paracetamol (47.4% of the cases); chlormezanone (6.6%), theophylline (1.74%), naproxen (1.65%) and mefenamic acid (1.56%). The following drugs were detected in toxicologically significant quantities in postmortem blood specimens (245 cases): phenobarbitone (1.22% of the cases), naproxen (0.82%), chlormezanone (0.82%), theophylline (0.82%), carbamazepine (0.41%) and paracetamol (0.41%).
Acids↗
Presence of paf-acether in human blood after thin-layer chromatography, but not after high-performance liquid chromatography purification.
After HPLC purification of human blood extracts, paf-acether (paf) was found exclusively as a lipoprotein-bound compound (lipopaf), whereas free-paf was absent. When the same samples (or lipopaf recovered from HPLC) were purified by TLC, both free-paf and lipopaf were detected. The free-paf detected in blood samples could thus result from lipopaf dissociation during TLC purification.
Blood Chemical Analysis↗
7,7,8,8-Tetracyanoquinodimethane as a new derivatization reagent for high-performance liquid chromatography and thin-layer chromatography: rapid screening of plasma for some antidepressants.
Using 7,7,8,8-tetracyanoquinodimethane (TCNQ) as a new derivatization reagent for HPLC and TLC, novel methods are described to detect secondary amine-bearing antidepressants (paroxetine, desipramine, fluoxetine, nortriptyline, maprotiline). The HPLC method is sensitive enough to detect these drugs in plasma at therapeutic levels whereas the latter has potential to detect them in overdose or forensic cases. The methods are based on purple chromogens formed by the displacement reaction of the drugs with TCNQ. The resulting chromogens are directly separated by either reversed-phase HPLC on a C(18) column or TLC on silicagel plates. For HPLC, acetonitrile-water (60:40) was used as mobile phase, with detection at 567 nm and separation in 40 min. For TLC, three developing solvent systems were used. By HPLC, 36 ng ml(-1) spiked plasma concentration of the drugs gave easily detectable signals whereas by TLC, detection limits varied mostly between 240 and 480 ng ml(-1). The HPLC method was applied to real plasma samples. The methods described are simple and very selective; some metabolites of these antidepressants and a vast number of drugs do not interfere with detection.
Antidepressive Agents↗
Determination of 1-phenyl-3-methyl-5-pyrazolone-labeled carbohydrates by liquid chromatography and micellar electrokinetic chromatography.
In this paper, the method for the derivatization of carbohydrates with 1-phenyl-3-methyl-5-pyrazolone (PMP) was simplified. One-third of the derivatization time was saved. Five monosaccharide derivatives have been well separated by MEKC and HPLC under optimized conditions. Good reproducibility could be obtained with relative standard deviation (RSD) values of the migration times within 5.0 and 2.3%, respectively. Furthermore, the developed methods have been successfully applied to the analysis of carbohydrates in Aloe powder and food. These methods are quite useful for routine analysis of monosaccharides and oligosaccharides in real samples.
Antipyrine↗
Chromatographic characteristics of surfactant-mediated separations: micellar liquid chromatography vs ion pair chromatography.
The effects of concentrations of organic solvent and surfactant on elution strength and selectivity in MLC and IPC are studied. It is observed that selectivity between most pairs of solutes used in this study increases in MLC and either decreases or passes through a minimum in IPC, with the volume fraction of organic modifier. In both MLC and IPC, selectivity varies with surfactant concentration; however, the overall variation in selectivity and elution order are more pronounced in MLC. The solvent strength decreases in IPC and increases in MLC as a result of an increase in surfactant concentration. An iterative regression design is used to predict the optimum mobile-phase compositions in terms of solvent strength and selectivity. The correlation between the predicted and measured chromatograms is excellent in MLC and poor in IPC. This is due to a more regular and reproducible retention behavior in MLC which greatly facilitates the development of robust methodologies. For a mixture of amino acids and peptides, a large retention gap between the first and the last eluting solutes is observed in IPC, which makes the use of organic solvent gradient inevitable. However, a better separation for the same mixture of solutes can be achieved in MLC isocratically. Apparently, the general elution problem can be alleviated in MLC by using an optimum eluent composition. It is observed that the efficiencies of MLC and IPC are comparable. The above observations indicate that MLC can be a powerful alternative to IPC in order to achieve optimized separations in shorter analysis time.
1-Propanol↗
Two-dimensional high-performance liquid chromatography using monodisperse polymer beads containing segregated chemistries prepared by pore size specific functionalization. Single-column combinations of size exclusion or ion exchange with reversed-phase chromatography.
Separation media for the complete separation of complex samples that require a combination of size exclusion or ion-exchange with reversed-phase chromatographic modes in a single column have been prepared from size monodisperse 10 microns poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads using a pore size specific functionalization process. To achieve the first combination of chromatographic modes, the large pores of the beads were selectively hydrolyzed to diols using aqueous poly(styrenesulfonic acid), while highly hydrophobic octadecyl groups were introduced into the small pores by reaction of the remaining epoxide groups with octadecylamine. These beads provide excellent protein recoveries and may be used for the direct injection separation of samples containing both hydrophilic proteins and hydrophobic drugs. Beads containing diethylamino groups in the large pores and octadecyl functionalities in the small pores were also prepared by size-selective modification. A plot of log k' against ionic strength of the mobile phase for these beads shows the absence of hydrophobic interactions and documents the clean ion-exchange mechanism of protein separation. Examination of the small pores in both types of separation media confirmed that their hydrophobicity was sufficient to allow the separations of small molecules in reversed-phase mode. Column packed with these dual-chemistry beads exhibited high efficiencies and were used successfully for the separations of proteins and alkylbenzenes or drugs.
Animals↗
Comparison of ion mobility constants of selected drugs after capillary gas chromatography and capillary supercritical fluid chromatography.
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Chromatography↗