Liquid Chromatography-mass Spectrometric Analysis Research Articles

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.

High performance liquid chromatography–mass spectrometric analysis of sulphonated dyes and intermediates

This paper reports our results in the analysis of polysulphonated anionic dyes and their intermediates using high-performance liquid chromatography–mass spectrometry (HPLC–MS). Negative-ion electrospray ionization is the most suitable ionization technique for the molecular mass determination of polysulphonated dyes or other dyes carrying a negative charge. From the series of [M– xH] x− ions and their sodiated adducts [M–( x+ y)H+ yNa] x− , the molecular mass and the number of sulphonic and carboxylic groups can be determined. The mobile phase should be compatible with the mass spectrometric detection, which rules out non-volatile tetraalkylammonium salts usually used as ion-pair mobile phase additives for the HPLC of sulphonated compounds. Some mono- and disulphonated dyes and intermediates can be separated with aqueous–organic mobile phases containing 5 m M ammonium acetate, which is the most suitable additive as far as compatibility with MS detection is concerned. However, the retention of compounds with two or more sulphonic groups is too low for a successful separation both with this mobile phase additive and with ion-pair additives with short alkyl chains. The dihexylammonium acetate ion-pairing reagent offers a reasonable compromise in terms of sufficient volatility and adequate retention and separation selectivity for the HPLC–MS analysis of polysulphonated dyes.

IDENTIFICATION OF Hb VILLEJUIF [β123(H1)Thr→Ile] IN SOUTHERN ITALY

Hb Villejuif [β123(H1)Thr→Ile] is a silent and asymptomatic variant described in 1989 in an 87-year-old woman of French origin suffering from coincidental polycythemia vera. This paper reports the second observation of Hb Villejuif in three related subjects from Montesarchio, Southern Italy. All routine techniques for hemoglobin analysis yielded normal results with the exception of a slight increase in the Hb A2 value. The occurrence of a variant β-globin was rapidly assessed by liquid chromatography mass spectrometric analysis and the abnormal chain purified by high performance liquid chromatography. The amino acid replacement Thr→Ile at β123 was determined by tandem electrospray mass spectrometric analysis of the tryptic digest of the variant β chain. The corresponding DNA mutation was established as C→T at the second position of codon 123 (ACC → ATC) by polymerase chain reaction amplification techniques.

Comparison of different liquid chromatography methods for the determination of corticosteroids in biological matrices

Various extraction techniques can be combined with column liquid chromatography (LC) and ultraviolet (UV) or mass spectrometric (MS) detection for the determination of synthetic corticosteroids in biological matrices. Target analysis of low concentrations of 25 μg/kg of dexamethasone in feed can be performed by combining immunoaffinity chromatography (IAC) and LC with UV detection. A straightforward multi-analyte procedure is obtained by tandem solid-phase extraction (SPE) and subsequent LC–UV. However, the limits of detection for feed samples are then relatively poor, viz. 100 μg/kg. A multi-analyte method which meets modern demands of about 5 μg/kg detection limit requires one-step SPE combined with LC–MS analysis. As regards urine corticosteroids can be determined down to a level of 0.5 μg/l by either SPE–LC–MS–MS or SPE(IAC)–LC–MS.

High-performance liquid chromatography–mass spectrometric analysis of furosemide in plasma and its use in pharmacokinetic studies

This study presents a rapid, specific and sensitive high-performance liquid chromatography–mass spectrometric (LC–MS) assay for the determination of furosemide in human plasma using diclofenac as an internal standard (IS). Both compounds were extracted from human plasma with ethyl acetate at pH 1 and were chromatographed using Shim-Pack GLC-CN column and a mobile phase consisting of acetonitrile and 20 mM ammonium acetate buffer solution pH 7, 4:1 (v/v) at a flow rate 1 ml min −1. Furosemide and IS were detected by mass spectrometer operated in the negative single ion monitoring mode using APCI as an ionization process at m/ z 329.2 and 294.1, respectively. The assay linearity of furosemide was confirmed over the range 50–2000 ng ml −1. Detection limit for furosemide in plasma was 10 ng ml −1. The selected concentration range corresponds well with the plasma concentrations of furosemide for pharmacokinetic study. Intraday and interday relative standard deviations were 1.3–4.7 and 2.7–11.5%, respectively. The extraction recovery percentages of furosemide and IS from plasma were in the range 89.3–97.1%. The developed LC–MS procedure was applied for the determination of the pharmacokinetic parameters of furosemide after an oral administration of tablet formulation (40 mg) to two healthy male volunteers. The calculated parameters were in good agreement with the reported values.

High-performance liquid chromatographic and tandem mass spectrometric quantitation ofN7-methyldeoxyguanosine in methylated calf thymus DNA

Quantitation ofN7-methyldeoxyguanosine (N7-MedG) produced in thein vitro N-methyl-N-introsourea (NMU) action on calf thymus DNA has been achieved by enzymatic degradation, liquid chromatographic separation and desorption chemical ionization tandem mass spectrometry. In conjunction with the resolving power of HPLC in the separation of isomers, desorption chemical ionization tandem mass spectrometry has been utilized in determining modified nucleosides at low levels using a stable-isotope labeled compound as an established by an independent HPLC analysis of methylated calf thymus DNA. A sensitive and specific methodology for the quantitation ofN7-MedG at the picomole level using HPLC combined with tandem mass spectrometry without radioisotope labeling process is presented. The potential of the liquid chromatographic tandem mass spectrometric analysis shows the detection ofN7-MedG as a possible marker for human exposure to methylating agentsin vitro.

Liquid chromatography mass spectrometric analysis of ouabainlike factor in biological fluid.

Ouabainlike factor (OLF), assayed as ouabainlike immunoreactivity (OLI), is a probable endogenous digitalislike factor (EDLF). Liquid chromatography/mass spectrometry (LC/MS) is one of the most highly sensitive tools for obtaining structural information regarding low-molecular weight materials in a target compound, and to measure the concentrations of these materials. We have previously reported that OLI can be isolated from the culture supernatant of the rat pheochromocytoma cell line, PC12, by several reverse-phase chromatography and LC/MS techniques. The present study was performed to characterize OLF from biological fluids such as plasma and culture supernatant of PC12 cells by LC/MS. The previous applications of LC/MS to OLI in plasma have been limited to structural identification at the final stages of isolation, in which the starting volume of plasma has been over 10 I. In the present study, we tried to minimize the volume of plasma, and to develop a new preclearing step to gain adequate LC/MS characterization using MS/MS analysis. The plasma was acidified, and OLI was purified by ODS column chromatography. OLI in chromatographic fractions from plasma was assayed by a sensitive enzyme-linked immunosorbent assay for ouabain. After Sep-Pak treatment and two rounds of ODS column chromatography, OLI was identified from 80 ml of plasma. The structure of the purified OLI was identical to authentic ouabain and digoxin, as assessed by LC/MS. In conclusion, we identified the chemically or structurally clarified ouabain and digoxin as the circulating form in plasma by LC/MS.

Oxidation of glycerophospholipids from biological membranes by reactive oxygen species: liquid chromatographic–mass spectrometric analysis of eicosanoid products

Peroxidation of glycerophospholipids present in cellular membranes results in the formation of a complex mixture, with many products derived from the oxidation of esterified arachidonic acid. Techniques of chromatography and mass spectrometry have facilitated the elucidation of the structure of individual components present as intact glycerophospholipids as well as the oxidized fatty acyl groups liberated from the glycerol backbone by saponification. Previously reported studies are summarized in this overview concerning those oxidized products of arachidonic acid derived from the red blood cell membrane, studied by techniques of electrospray tandem mass spectrometry developed to analyze eicosanoid products.

Liquid chromatography–mass spectrometric analysis of phenylurea herbicides and their degradation products using an atmospheric pressure ionization interface

A method was developed for the determination of two phenylurea herbicides and their degradation products using liquid chromatography–atmospheric pressure chemical ionization (APCI)-mass spectrometry without any additives in the mobile phase. The APCI detection sensitivity of these compounds was maximized when the vaporizer temperature was set at 250 °C and a positive-ion detection mode was chosen. Under these conditions, the detection limits (S/N = 3) of these compounds in total-ion and selected-ion monitoring were 2.5–3.0 and 0.12–0.26 ng, respectively. In a biodegradability test on diuron, small quantities of degradation products could be determined by the proposed method.

Metabolism of Leukotriene C4 in γ-Glutamyl Transpeptidase-deficient Mice

We have investigated the metabolism of leukotriene C4 (LTC4) in gamma-glutamyl transpeptidase (GGT)-deficient mice (Lieberman, M. W., Wiseman, A. L., Shi, Z-Z., Carter, B. Z., Barrios, R., Ou, C-N., Chevez-Barrios, P., Wang, Y., Habib, G. M., Goodman, J. C., Huang, S. L., Lebovitz, R. M., and Matzuk, M. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7923-7926) and have found substantial conversion of LTC4 to leukotriene D4 by high performance liquid chromatography and continuous flow fast atom bombardment-tandem mass spectrometric analyses. LTC4-converting activity has a tissue distribution different from GGT with highest activity in spleen followed by small intestine, kidney, and pancreas and lower activity in liver and lung. The activity is membrane-bound and is inhibited by acivicin, a known inhibitor of GGT. The enzyme was partially purified from the small intestine of GGT-deficient mice by papain treatment and gel filtration chromatography. The partially purified fragment released by papain has an apparent molecular mass of 65-70 kDa and the same substrate specificity as the tissue homogenate. In addition to LTC4, S-decyl-GSH is also cleaved. GSH itself, oxidized GSH, and the synthetic substrates used to analyze GGT activity (gamma-glutamyl-p-nitroanilide and gamma-glutamyl-4-methoxy-2-naphthylamide) are not substrates for this newly discovered enzyme. These data demonstrate that in addition to GGT at least one other enzyme cleaves LTC4 in mice. To reflect this enzyme's preferred substrate, we suggest that it be named gamma-glutamyl leukotrienase.

Studies on the dynamics of the production of destruxins by Metarhizium anisopliae Direct high-performance liquid Chromatographic and fast atom bombardment mass spectrometric analysis correlated with biological activity tests

In this work, the new strain Ma23 of the fungus Metarhizium anisopliae is demonstrated to be an effective source of destruxins, known insecticidal cyclodepsipeptides, under optimized culture conditions. The dynamics of the destruxin production is analysed by fast atom bombardment MS and HPLC, using wide-pore reversed-phase packings without any pretreatment of the culture media samples. By this approach, we show that twelve different destruxins are produced by M. anisopliae. The most abundant toxins have been unequivocally identified and HPLC quantitation shows that E destruxin, the most toxic compound of the series, accounts for ~35–40% of the whole toxin production from Ma23. A good correlation between the pathogenic effect of the fungal samples and amounts of destruxins produced is also demonstrated with an optimum of toxicity after four days.

Identification, characterization and pharmacological profile of three metabolites of (R)-(+)-1,2,3,6-tetrahydro-4-phenyl-1-[(3-phenylcyclohexen-1- yl)methyl]pyridine (CI-1007), a dopamine autoreceptor agonist and potential antipsychotic agent.

Liquid chromatographic-mass spectrometric (LC-MS) analysis of plasma taken from cynomolgus monkeys dosed orally with (R)-(+)-1,2,3,6-tetrahydro-4-phenyl-1-[(3-phenylcyclohexen-1- yl)methyl]pyridine (1), a dopamine (DA) autoreceptor agonist and potential antipsychotic agent, revealed several metabolites. The molecular masses of three major metabolites suggested that they were mono- and dihydroxylated derivatives of 1. We synthesized compounds 2 and 3, the two possible mono-p-hydroxyphenyl derivatives of 1, along with the bis-p-hydroxyphenyl derivative 4. These compounds coeluted by HPLC with the three hydroxylated metabolites of 1. Compounds 2-4 all had high affinities for DA D2 and D3 receptors and moderate affinities for D4 receptors. Like 1, compound 2 decreased DA synthesis and neuronal firing in rat brain, indicative of DA autoreceptor activation. Compound 2 inhibited exploratory locomotor activity in rodents and was active in the Sidman avoidance test in squirrel monkeys, predictive of antipsychotic activity in humans. Compounds 3 and 4 showed weak activity in all these tests. After squirrel monkeys were dosed with 1 orally at the ED100 dose of the Sidman avoidance test, the plasma concentration of 2 was below the limit of quantitation. Therefore, these metabolites are unlikely to contribute greatly to the potent activity seen with 1 in the Sidman avoidance test.

Polar organic pollutants in the Elbe river Liquid chromatographic-mass spectrometric and flow-injection analysis-mass spectrometric analyses demonstrating changes in quality and concentration during the unification process in Germany

During the unification process of the two German states from 1989 to 1994, water extracts from the Elbe river were analyzed by gas chromatography-mass spectrometry (GC-MS) and by high-performance liquid chromatography (HPLC) combined with ultraviolet (UV) and/or mass spectrometric detection (MS) in order to monitor the pollutants in one of the most contaminated European rivers. After column chromatography (LC) or flow-injection analysis (FIA) bypassing the analytical column, ionization was performed by a thermospray interface (TSP). Semiquantitative estimations of the pollution of the Elbe were made form the total-ion current traces (TIC) of the extracts. Determination of the total dissolved organic carbon (DOC) indicated a reduction of more than 55% of pollutants in the water phase. The pollutants were identified by tandem mass spectrometry (MS-MS), generating daughter-ion spectra by collision-induced dissociation (CID) using either column chromatography or direct mixture analysis. Compound-specific analyses indicated a reduction in the amount of pollutants as well as a change in their composition. This could be explained by reduced industrial production and by intensified construction of biological sewage treatment plants.

Quantitative liquid chromatographic thermospray-tandem mass spectrometric analysis of some analgesics and tranquilizers of the methadone, butyrophenone, or diphenylbutylpiperidine groups in whole blood.

The results of a liquid chromatographic-mass spectrometric method for the quantitative determination of some medicaments of the methadone, butyrophenone, or diphenylbutylpiperidine groups in whole blood are presented. The method includes an extraction procedure with Bond Elut columns. The liquid chromatograph is connected to a mass spectrometer by a thermospray interface, and to obtain as high a sensitivity and selectivity as possible, a selected reaction monitoring mass spectrometric technique in the daughter ion mode is applied.

Analysis of isomers of cholesteryl acetate hydroperoxides by HPLC and GC-ITDMS

Gas chromatographic, high performance liquid chromatographic and GC-ion trap detector mass spectrometric analyses of the hydroperoxides formed during thermal peroxidation of cholesteryl acetate, are reported and discussed. The hydroperoxides were separated from the thermal-oxidised mixture by solid-phase extraction, fractionated and individually collected by HPLC. Each hydroperoxide was then chemically reduced with NaBH4 to a hydroxy-derivative. The GC and MS characteristics of hydroxy- and TMS-derivatives were compared against available standards. Only 7β-hydroperoxide and 7α-hydro-peroxide of cholesteryl acetate were found, as reported in the literature for cholesterol.

Determination of chloramphenicol in muscle using a particle beam interface for combining liquid chromatography with negative-ion chemical ionization mass spectrometry

A simple and rapid liquid chromatographic—mass spectrometric analysis of chloramphenicol in calf muscle is presented. A particle beam interface was used, with negative-ion chemical ionization mass spectrometry using methane as the reagent gas. The method specificity was tested for three related compounds, dehydrochloramphenicol, nitrosochloramphenicol and nitrophenylaminopropanediol. The extraction procedures require 5 g of muscle, and the quantification limit is 2 μg/kg for chloramphenicol. Residues were detected in calf muscle 48 h after intravenous administration of chloramphenicol (25 mg/kg body weight).

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 ([ 15N 2, 2H 2]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.

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 precipitations is a complex procedure and the recovery is limited.

Identification of chlorophyll transformation products in a lake sediment by combined liquid chromatography-mass spectrometry

Negative-ion liquid chromatographic-mass spectrometric analysis of the pigment composition of the bottom sediment (15–20 cm) of a eutrophic lake revealed the presence of a novel series of chlorophyll transformation products, in which a series of C 27C 30 sterols and stanols are esterified to a pyrophaeophorbide a nucleus of algal origin. The major components are algal-derived chlorophyll a and b degradation products, and the presence of a bacteriochlorophyll-related phaeophytin indicates the presence of anoxic conditions extending into the photic zone when the sediment was laid down.

Liquid chromatographic—mass spectrometric analysis for screening of patients with cystinuria, and identification of cystine stone

Analyses of amino acids in the urine of a normal human and of patients with heterozygous and homozygous cystinuria have been carried out, using liquid chromatography—mass spectrometry with an atmospheric pressure ionization interface system. A kidney cystine stone was also analysed by this system. Very intense quasi-molecular ions ([M + H] +) of standard cystine, arginine, lysine and ornithine were observed on mass chromatograms as base peaks. Mass chromatograms of the urine samples from a normal human and from patients with heterozygous and homozygous cystinuria were easily distinguishable. The retention times in the mass chromatogram and mass spectrum of kidney stone cystine was almost the same as that of authentic cystine.