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.     

Thermospray liquid chromatographic-mass spectrometric method for the analysis of metribuzin and its metabolites

A thermospray liquid chromatographic-mass spectrometric (TSP LC-MS) method has been developed for the analysis of the herbicide metribuzin and its three major metabolites in plant tissue. Metribuzin and its metabolites exhibited widely varying sensitivities in positive-ion TSP, with metribuzin being the most sensitive and deaminated diketo metribuzin being the least sensitive. All four compounds of interest were detected in an extract of a soybean plant which had been treated with metribuzin.     

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.     

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.     

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.     

Liquid chromatographic-mass spectrometric determination of phenolic compounds using a capillary-scale particle beam interface.

A capillary-scale particle beam interface was used to detect 18 phenolic compounds in red wine samples. This technique allows reproducible, library searchable electron ionization spectra at only 1 microliter/min mobile phase flow-rate for a sensitive detection of the analytes in complex matrices. The method makes use of a narrow bore, reversed-phase packed capillary column for sample separation. Detection limits were in the low picogram range for most compounds. Sensitivity and response linearity were evaluated for eight phenolic acids, which are often encountered in red wines. The phenolic compound composition was outlined in two red wines obtained using different aging processes.     

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.     

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.     

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.     

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.     

Gas chromatographic-mass spectrometric analysis of volatile constituents in saliva

Present methods for the development of metabolic profiles are limited to the use of headspace techniques and solvent extraction methods. A new method for the development of saliva profiles which provides information complementary to existing analyses has been developed. The results of the developed methodology provide a reliable, reproducible method for metabolic profiling. Gas chromatographic-mass spectrometric analysis of the volatile constituents provided positive identification of 39 compounds. Application of the developed protocol toward the investigation of saliva as a vehicle for the non-invasive detection of certain pathological states, specifically diabetes mellitus and liver disorders, may be possible.     

High-performance liquid chromatographic-mass spectrometric assay of busulfan in serum and cerebrospinal fluid.

A liquid chromatographic-mass spectrometric method has been developed to determine busulfan concentrations in the cerebrospinal fluid and serum of some children undergoing bone marrow autotransplantation. After two liquid-liquid extraction steps with dichloromethane on a biological matrix, the separation of busulfan was carried out by isocratic reversed-phase chromatography. The mass spectrometric system was operated in electron-impact mode. Principal ions at m/z 175, 111 and 79 were observed for busulfan, but only m/z 175 was chosen for the quantification of the analyte. The retention time of busulfan was 2.5 min. The detection limit of 100 ng/ml allowed the determination of cerebrospinal fluid and serum busulfan concentrations during the four days of high-dose (1 mg/kg) treatment prior to autotransplantation in five child patients.     

Rapid liquid chromatographic-mass spectrometric analysis of withanolides in crude plant extracts by use of a monolithic column

Summary A rapid analytical method has been developed for the mutual resolution of three steroidal compounds, withaferin A, iochromolide, and withacnistin. Liquid chromatography was performed on a Chromolith analytical column (4.6 mm i.d.×50 mm), made from a cylindrical silica rod, operated at a flow rate of 4 mL min⁻¹ with a simple linear gradient prepared from 0.1% aqueous formic acid and 0.1% formic acid in acetonitrile. Under optimum conditions simultaneous separation of the compounds was achieved in less than 7 min, one eighth the time required for conventional LC separation. The overall analysis time was reduced without sacrificing chromatographic performance—essential for the resolution of positional isomers such as iochromolide and withacnistin. The column was coupled to a single-quadrupole mass spectrometer and the method was characterized by good performance in terms of repeatability, selectivity, linearity, and sensitivity. Detection limits in the single-ion-monitoring mode were 0.15 μg mL⁻¹ or below. Finally, the developed method was successfully applied to the determination of withanolides in extracts fromlochroma gesnerioides obtained by three different processes—traditional Soxhlet extraction and two faster methods, microwave-assisted extraction and pressurized solvent extraction.     

Liquid chromatographic-mass spectrometric analysis of supercritical-fluid extracts of rosemary plants

A two-step supercritical fluid extraction process of rosemary leaves, on a pilot plant scale, is proposed to divide the oleoresin into two fractions with different antioxidant activities and essential oil composition. Rosemary leaves were extracted by using different conditions of pressure and temperature as well as different conditions for fractionation of the extracts. Conditions can be tuned to selectively extract one antioxidant fraction with almost no residual aroma. In the present investigation, the antioxidant fraction was exhaustively studied in terms of antioxidant activity measurements as well as of chemical composition. An LC–MS method was adapted to perform the analysis and identification of the compounds responsible for the antioxidant activity of the extracts. Different extraction and fractionation conditions were studied in order to correlate the process conditions with the antioxidant activities obtained.     

Matrix effect and correction by standard addition in quantitative liquid chromatographic-mass spectrometric analysis of diarrhetic shellfish poisoning toxins

An evaluation of the feasibility of liquid chromatography-mass spectrometry (LC-MS) with atmospheric pressure ionization was made for quantitation of four diarrhetic shellfish poisoning toxins, okadaic acid, dinophysistoxin-1, pectenotoxin-6 and yessotoxin in scallops. When LC-MS was applied to the analysis of scallop extracts, large signal suppressions were observed due to coeluting substances from the column. To compensate for these matrix signal suppressions, the standard addition method was applied. First, the sample was analyzed and then the sample involving the addition of calibration standards is analyzed. Although this method requires two LC-MS runs per analysis, effective correction of quantitative errors was found.     

Solid-phase microextraction-gas chromatographic-mass spectrometric analysis of garlic oil obtained by hydrodistillation

Garlic (Allium sativum L.) is highly consumed worldwide. This crop is mainly known for its flavor and odor, although the many medicinal properties that are attributed to it, including anticarcinogenic, antiatherosclerotic, and antithrombotic potential, among several others, have called the attention of scientists since very early times. It is known that sulfur-containing volatiles are the principal compounds responsible for such properties. The aims of this work were to develop a solventless extraction method for sulfur-containing volatiles from garlic, as well as their chemical characterization. Since garlic volatiles are rather thermolabile, low-pressure hydrodistillation was chosen as the extracting method. The analysis of all compounds was performed on an HP-FFAP chromatographic column mounted in a GC-MS system. For volatile transfer and injection method, solid-phase microextraction was selected, with the use of eight different fibers. The most abundant volatile compound was diallyl disulfide, followed by diallyl trisulfide. Among the 47 totally identified compounds, 18 were linear sulfur-containing volatile compounds, 6 were of non-sulfur nature, and the other 23 were cyclic compounds. However, linear sulfur volatiles accounted for 94% of the total amount.     

Gas chromatographic-mass spectrometric analysis of methylphenidate and p-hydroxymethylphenidate using deuterated internal standards

A gas chromatography-mass spectrometry assay is described for the simultaneous determination of threo-dl-methylphenidate and threo-dl-p-hydroxymethylphenidate in plasma and urine using selected ion monitoring of electron impact generated fragments of their pentafluoropropionyl derivatives. The use of recently available deuterated analogues as internal standards improves overall performance relative to previous methods. The practical limit of quantifiable detection of the assay is 0.5 ng/ml for both methylphenidate and p-hydroxymethylphenidate. p-Hydroxymethylphenidate appears to be a significant urinary metabolite of methylphenidate in rats but not in humans.     

High-performance liquid chromatographic-mass spectrometric analysis of oligosaccharides from enzymatic digestion of glycosaminoglycans. Application to human samples.

Glycosaminoglycan contents were evaluated in plasma and urine samples from volunteers treated intravenously with a mixture of dermatan sulphate and heparin, combining a novel liquid chromatographic-mass spectrometric technique for the determination of oligosaccharides from glycosaminoglycans with a classical technique for the extraction of glycosaminoglycans from biological samples (precipitation with cetylpyridinium chloride). In plasma samples dermatan sulphate and heparin can be measured for 2 h after treatment; urine excretion was detectable for 24 h. These results suggest that this novel approach is promising for future studies on the pharmacokinetics of glycosaminoglycans, although some technical aspects need further improvement, mainly regarding the procedures for sample clean-up; cetylpiridinium precipitation is a complex procedure and the recovery is limited.