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.     

Thermospray liquid chromatographic-mass spectrometric analysis of mutagenic substances present in tryptophan pyrolysates

During protein pyrolysis, as can occur when broiling meat or fish, mutagenic substances are formed, as shown by in vitro mutagenicity assays. Some of the most active compounds have been shown to originate from tryptophan (Trp). Hundreds of grams of Trp had to be used previously to study the formation of these compounds by classical separation and detection methods. Studies have been made of the formation of two active heterocyclic amines, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), by heating Trp at different temperatures and for different periods at time. Advantage was taken of the high selectivity and sensitivity of thermospray liquid chromatography-mass spectrometry coupling, which permitted the use of much smaller amounts (10 g) of starting material. These conditions permit a more accurate control of the pyrolysis temperature and the method of extraction can be shortened and simplified. The results show that Trp-P-1 and Trp-P-2 were already formed at 250 degrees C. These substances were detectable in the low ppb range, i.e., less than the threshold levels necessary to elicit a positive response in the Ames test under standard conditions.     

Gas chromatographic-mass spectrometric method for analysis of a pyrimido-s-triazine and some of its metabolites in dog urine.

3-Phenylpyrimido-[3,4-a]-s-triazines exhibit antiparasitic, antibacterial and antifungal activity. In order to study the metabolism of these heterocycles, 9,9-diethyl-3-phenyl-6,8-dioxo-2,3,4,5,6,7,8,9-octahydropyrimido[3 ,4-a]-s- triazine (TZ) was administered to dogs. Three potential metabolites were synthesized, and these models were identified and quantified with gas chromatography-mass spectrometry. The heterobicyclic compounds, TZ and its hydroxy derivative, underwent thermal degradation under chromatographic conditions. Dog urine spiked with the model metabolites was extracted, and the substances were quantified. The urine of dogs treated with TZ was studied, and two of the potential metabolites were recovered, identified and quantified.     

Strategies for the liquid chromatographic-mass spectrometric analysis of non-polar compounds

Electrospray ionization and atmospheric pressure chemical ionization (APCI) have evolved recently as very useful tools for the liquid chromatographic–mass spectrometric (LC–MS) analysis of polar substances. Non-polar compounds, however, are difficult to analyze with these atmospheric pressure ionization techniques due to their soft ionization mechanism. Recently, new approaches have been introduced which are likely to overcome this obstacle, at least partly. On-line electrochemical conversion of the analytes to more polar reaction products, atmospheric pressure photoionization, atmospheric pressure electron capture negative ion-MS and coordination ionspray-MS are four techniques which are presented in detail, compared and discussed critically with respect to their current status and future perspectives. Particular focus is directed from a chemical viewpoint on the substance groups which are accessible by each of the new approaches.     

Atmospheric pressure photoionization liquid chromatographic-mass spectrometric determination of idoxifene and its metabolites in human plasma

This paper describes a comparison between atmospheric pressure chemical ionization (APCI) and the recently introduced atmospheric pressure photoionization (APPI) interface for the LC–MS determination of idoxifene and its major metabolite, SB245419 (SB19), in human plasma. The results indicate that analyte response in APPI is highly dependent on the solvent composition, especially to water in the mobile phase. Other parameters investigated are the mobile phase flow-rate, the chemical noise, and signal suppression by matrix interferences. APPI appears to be six to eight times more sensitive than APCI for idoxifene and its SB245419 metabolite; the response for the SB245420 metabolite is considerably better than for APCI conditions, but still not sufficient for trace level pharmacokinetic determinations in human plasma. The LOQ for the parent drug and its major metabolite were 10 and 25 ng/ml, respectively, in human plasma. From post-column infusion experiments we conclude that there is little difference in matrix suppression between APCI and APPI. From these studies we suggest APPI may be an additional tool in pharmaceutical LC–MS applications.     

Validation of high-performance liquid chromatographic-mass spectrometric method for the analysis of lidocaine in human plasma

A sensitive and simple liquid chromatography-tandem mass spectrometry method is developed and validated for the determination of lidocaine in human plasma. Bupivacaine is used as an internal standard, and the plasma extraction is performed by a simple liquid-liquid extraction. The limit of quantitation (LOQ) is 0.5 ng/mL with a signal-to-noise ratio greater than 5. The calibration curve is linear from 0.5 to 250 ng/mL with an r2 greater than 0.99. The coefficients of variation for within- and between-assay imprecision, including LOQ, are < or = 13% and < or = 8%, respectively. The percentage of inaccuracy for within- and between-assay, including LOQ, are < or = 9% and < or = 5%, respectively. The absolute recovery of lidocaine and bupivacaine are greater than 84% and 82%, respectively. The higher sensitivity and accuracy of this method allow the measurement of low concentrations of lidocaine in plasma from a clinical study of topically applied lidocaine in healthy subjects.     

Thermospray liquid chromatographic-mass spectrometric analysis of anti-AIDS nucleosides: quantification of 2',3'-dideoxycytidine in plasma samples.

Thermospray liquid chromatography-mass spectrometry was investigated as a method for quantification of 2',3'-dideoxycytidine (DDC) from human plasma. A stable isotope analog of DDC ([15N2,2H2]DDC) was used as an internal standard. Selected ion monitoring of the protonated molecular ions for DDC and the internal standard was used to record mass chromatograms. The areas of the peaks in the mass chromatograms were used for quantification. The detection limit of DDC in this assay was 50 pg on-column. The calibration curve was linear over the desired range, 0.25-20 ng/ml. The major advantages of this assay over others are: no derivatization, high sensitivity, high specificity and short assay time.     

Liquid chromatographic-mass spectrometric analysis of glucuronide-conjugated anabolic steroid metabolites: method validation and interlaboratory comparison

Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for simultaneous and direct detection of 12 glucuronide-conjugated anabolic androgenic steroid (AAS) metabolites in human urine is described. The compounds selected were the main metabolites detected in human urine after dosing of the most widely abused AAS in sports, e.g. methandienone, methenolone, methyltestosterone, nandrolone and testosterone, and certain deuterium-labeled analogs of these metabolites. Sample preparation and the LC-ESI-MS/MS method were optimized, validated, and the overall process was implemented and the results between seven laboratories were compared. All the metabolites were extracted simultaneously by solid-phase extraction (SPE) and analyzed by LC-ESI-MS/MS with positive ionization mode and multiple reaction monitoring (MRM). Recovery of the SPE for the AAS glucuronides was 89-100% and ten out of twelve compounds had detection limits in the range of 1-10 ng/ml in urine. The results for inter/intraday repeatability were satisfactory and the interlaboratory comparison with authentic urine samples demonstrated the ease of method transfer from one instrument setup to another. When equivalent triple quadrupole analyzers were employed the overall performance was independent from instrument manufacturer, electrospray ionisation (ESI) or atmospheric pressure chemical ionization (APCI) and liquid chromatohraphic (LC) column, whereas major differences were encountered when changing from one analyzer type to another, especially in the analysis of those AAS glucuronides ionized mainly as adducts.     

Thermospray high-performance liquid chromatographic-mass spectrometric characterization of biological macromolecules. I. Analysis of acid hydrolysate of peptides

A method for the routine analysis of phenylthiocarbamyl (PTC) amino acids by thermospray high-performance liquid chromatography-mass spectrometry (TSP LC-MS) is described. Data were acquired on a small dedicated TSP LC-MS system in which the temperature of the vaporizer and ion source block were optimized. PTC-amino acids exhibited unique TSP mass spectra containing sufficient fragment ions to determine structural data. Therefore, using this method the amino acids contained in the acid hydrolysates of unique and modified peptides were able to be positively identified. Additionally, the amino acid composition of peptides as determined by TSP LC-MS in the selected ion monitoring mode corresponded well with the theoretical value. The detection limits for the PTC-amino acids were at the low picomole level.     

Thermospray high performance liquid chromatographic/mass spectrometric analysis of some fusarium mycotoxins

Thermospray high performance liquid chromatography/mass spectrometry (TSP HPLC/MS) was used to analyze five Fusarium mycotoxins in porcine plasma and urine. Four cytotoxic trichothecene mycotoxins, T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), T2 tetraol, and the fungal estrogen zearalenone (F-2 toxin) were analyzed. The thermospray mass spectrum contained molecular weight information with few, if any, fragment signals. Detection limits ranging from 1 to 10 ng of mycotoxin injected onto the HPLC column were obtained using selected ion monitoring (SIM) HPLC/MS. Neither the plasma nor the urine matrix interfered with TSP HPLC/MS analysis of these mycotoxins and no sample derivatization was necessary for the analysis. The TSP HPLC/MS technique appears to be ideal for very sensitive analysis of mycotoxins in biological samples.     

Gas chromatographic-mass spectrometric method for polycyclic aromatic hydrocarbon analysis in plant biota

Using gas chromatography-mass spectrometry, a new method was developed for the identification and the quantification of polycyclic aromatic hydrocarbons (PAHs) in plants. This method was particularly optimised for PAH analyses in marine plants such as the halophytic species, Salicornia fragilis Ball et Tutin. The saponification of samples and their clean up by Florisil solid-phase extraction succeeded in eliminating pigments and natural compounds, which may interfere with GC-MS analysis. Moreover, a good recovery of the PAHs studied was obtained with percentages ranging from 88 to 116%. Application to the determination of PAH in a wide range of coastal halophytic plants is presented and validated the efficiency, the accuracy and the reproducibility of this method.     

Simultaneous determination of tropatepine and its major metabolites by high-performance liquid chromatographic-mass spectrometric identification. Application to metabolic and kinetic studies

Tropatepine is used to combat against extrapyramidal syndrome induced by neuroleptic drugs. A high-performance liquid chromatographic method was proposed for the simultaneous determination of tropatepine and its potential metabolites in biological fluids. After double extraction of compounds in hexane and back-extraction in hydrochloric acid, the chromatographic separation was performed on a reversed-phase column with an acetonitrile--perchlorate buffer mixture as mobile phase. Compounds were detected at 229 nm and the detection limit was about 15 ng/ml. The method was applied to bile and urine samples collected in rats, after a single high oral dose of 100 mg/kg of tropatepine hydrochloride. Gas chromatography-mass spectrometry was used for identification of the potential metabolites. Nortropatepine and tropatepine S-oxide were identified in this way, and it seemed that tropatepine was subjected to a large and intense metabolic process. The analytical procedure and the results of the metabolic investigation were applied to a preliminary pharmacokinetic study in patients undergoing long-term oral therapy with tropatepine.     

Capillary gas chromatographic-mass spectrometric method for the identification and quantification of some benzodiazepines and their unconjugated metabolites in plasma

A gas chromatographic-mass spectrometric method for the identification and/or quantification of diazepam, clobazam, flunitrazepam, triazolam, midazolam, oxazepam and lorazepam and some of their desmethylated and hydroxylated metabolites in plasma is described. Benzodiazepines were extracted from plasma with butyl acetate at pH 9; the hydroxylated compounds were then silylated with N,O-bis (trimethylsilyltrifluoroacetamide). Analysis was performed using a compact mass-selective detector operating in the electron-impact mode. Depending on the concentration, identification was performed either by direct comparison of the observed mass spectra with reference spectra or by the relative intensities of the most intense and characteristic ions in the selected-ion monitoring (SIM) mode. Quantification was performed in the SIM mode using the most intense ion. The intra-assay precision and accuracy were better than 5-6%; linearity was satisfactory up to 1-2 micrograms/ml. The detection limit was 1-5 ng/ml for most of the benzodiazepines. This method can be easily used in clinical situations when a safe and rapid response is essential for patient treatment.     

A gas chromatographic-mass spectrometric method for analysis of the contents of aerosol irritant projectors

A convenient method for the sampling and total analysis of the propellents and active agents contained in aerosol irritant projectors has been developed. A gas chromatograph equipped with a column packed with 6% Carbowax 20M on Chromosorb G and coupled to a mass spectrometer provides both quantitative and qualitative analysis for the components of a typical irritant projector. The main lachrymator, bromoacetone, represented 1.5% and the propellents dichlorodifluoromethane and trichlorofluoromethane, comprised 97.7% of the aerosol mixture.     

Pre-column derivatization of free bile acids for high-performance liquid chromatographic and gas chromatographic-mass spectrometric analysis

Pentachlorophenyl (PCP) esters of five free bile acids (FBA) were obtained by reacting the FBA and Kovacs' complex (KC) in a 1:8 molar ratio in acetone at 65 degrees C, and were purified by column chromatography on silica gel. The esters were crystallized from benzene-hexane, derivatized as trimethylsilyl ethers for gas chromatography on a DB-1 capillary column and for gas chromatography-mass spectrometry with a DB-5 column, and mass spectrometry (MS) in the electron-impact (EI) positive-ion mode at 70 eV. The reaction is specific for FBA even in the presence of glycine and taurine conjugates of bile acids. The PCP esters were treated with benzylamine in chloroform or methanol to produce N-benzyl derivatives of FBA. The N-benzylamides were separated by high-performance liquid chromatography (HPLC) on a 4-microns Nova-Pak C18 column, studied by thermospray-LC-MS, and in the direct insertion probe-EI positive-ion mode.     

Thermospray high-performance liquid chromatography/mass spectrometric determination of cyclosporins.

The cyclic undecapeptides cyclosporin (Csp) A, CspD and dihydro CspC (HCspC) were analyzed by high-performance liquid chromatography/thermospray-mass spectrometry (HPLC/TSP-MS) on line with a UV detector. Positive ion partial (1000-1300 u) mass spectra of these compounds could be obtained with 1-2 pmol injected on column. Mass spectra were characterized by signals corresponding to the [M+H] ions as well as fragment ions derived from the loss of 112 (CspA and CspD) or 44, 114 and 114 + 44 (HCspC) mass units from the parent ion. The same qualitative profile was observed for negative-ion acquisition where the ions were formed by proton abstraction. The application of the technique to the characterization of CspA and its major hydroxylated and dealkylated metabolites in human blood samples is presented.