Thermospray liquid chromatography-mass spectrometry of nucleosides and of enzymatic hydrolysates of nucleic acids.

Nucleosides dissolved in aqueous buffered solutions undergo ionization during direct introduction of the solution into a mass spectrometer using a thermospray interface. The principal ions formed represent the protonated molecule, the corresponding protonated free base, and sugar. In addition to potential utility for characterization of new nucleosides, the technique can be used to monitor nucleosides separated from enzymatic hydrolysates by liquid chromatography. The selectivity of chromatographic detection is significantly greater than with UV absorbance alone so that independent detection of components of unresolved chromatographic peaks is usually possible. Detection limits, with signal/noise greater than 10 for most nucleosides, are approximately 0.1-1 ng per component for selected ion monitoring and 10-50 ng for full-scan mass spectra. Examples are given from the detection of modified nucleosides in enzymatic hydrolysates of 0.05 A260 units (2.5 micrograms) of rabbit liver tRNAVal and of unfractionated H. volcanii tRNA.     

Comparative studies of HPLC-fluorometry and LC/MS method for the determination of N-acetylneuraminic acid as a marker of deteriorated ophthalmic solutions

Methods for determining the deterioration of ophthalmic solutions using both high-performance liquid chromatography (HPLC) with fluorescence detection and liquid chromatography coupled with selected ion monitoring mass spectrometry (LC/MS) are described. The methods are based on the determination of N-acetylneuraminic acid (NeuAc) released by the hydrolysis of foreign bodies that contaminate eye drops during use. The released NeuAc was either labeled with 1,2-diamino-4,5-methylenedioxybenzene (DMB) for fluorometric detection or detected without derivatization by mass spectrometry. The calibration curves for NeuAc showed good linearity between 1.2 ng/mL and 39 ng/mL for fluorometric HPLC and 5.0 ng/mL and 100 ng/mL for LC/MS, respectively. Detection limits for fluorometric HPLC and LC/MS were 0.20 ng/mL and 0.88 ng/mL, respectively. The NeuAc content of some model glycoproteins determined by LC/MS method were 62-78% of those determined by fluorometry. The differences are attributed to matrix effects but the LC/MS method afforded sufficiently high sensitivity that NeuAc in the foreign bodies could be determined in eight of nine test samples.     

Analysis of oxidative warfarin metabolites by thermospray high-performance liquid chromatography/mass spectrometry

Oxidative metabolites of the anticoagulant, warfarin [4-hydroxy-3-(3-oxo-1-phenylbutyl)-2H-1-benzopyran-2-one], produced by the actions of cytochromes P450 were analyzed by thermospray high-performance liquid chromatography/mass spectrometry. Warfarin, dehydrowarfarin, and the 6-, 7-, 8-, and 4'-hydroxy derivatives of warfarin were found to ionize well by the thermospray process in the presence of ammonium acetate. Thermospray mass spectra of these compounds were generally dominated by the protonated molecule, (M + H)+, and ions formed by the loss of water from the protonated molecule, (M + H - H2O)+. Fragment ions arising from the hydroxycoumarin, benzylhydroxycoumarin, and phenylbutanone portions of the molecules were observed, and the relative intensity of these fragment ions was greatly increased with filament ionization and application of a high repeller potential (100-130 V). Selected-ion monitoring of the (M + H)+ and (M + H - H2O)+ ions provided sensitivities for these compounds in the 2 to 10 ng range. A method employing thermospray HPLC/MS with selected-ion monitoring and internal standard quantitation for the analysis of the oxidative metabolites of warfarin is described.     

Analysis of underivatized glycosphingolipids by high-performance liquid chromatography/atmospheric pressure ionization mass spectrometry

Analytical conditions for underivatized glycosphingolipids by using high-performance liquid chromatography atmospheric pressure ionization mass spectrometry (HPLC/API-MS) were investigated. The analysis was performed by using an ordinary reversed-phase column (4.6 X 150 or 4.6 X 250 mm) at a flow rate of 1 ml/min. The glycosphingolipids could be characterized from the HPLC/API-MS in terms of molecular weight, ceramide composition, and partial oligosaccharide sequence. In order to obtain an adequate spectrum the amount of material needed is in the range of a few micrograms of lipid. By selected ion monitoring the sensitivity of the method allowed characterization of only 60 ng of glycosphingolipid. The method will be very useful in the characterization of small quantities of glycosphingolipids from biological samples.     

Partial isotope fractionation during high-performance liquid chromatography of deuterium-labelled internal standards in plant hormone analysis: A cautionary note

Deuterium-labelled indole-3-acetic acid, abscisic acid and phthalimido-1-aminocyclopropane-1-carboxylic acid were found to separate from the unlabelled compounds on reverse-phase high-performance liquid chromatography (HPLC). A similar separation was found for the methyl esters of these compounds on normal-phase HPLC. Such separations may lead to substantial errors when these compounds are used as internal standards for quantitation by gas chromatography-mass spectrometry/selective ion detection, unless the complete chromatographic peaks are collected.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - IAA indole-3-acetic acid - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - Pht-ACC phthalimido-ACC - SIM selected ion monitoring     

The use of negative ion thermospray liquid chromatography/tandem mass spectrometry for the determination of bile acids and their glycine conjugates.

A study was carried out on the negative ion thermospray liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry of a group of bile acids and their glycine conjugates. The liquid chromatographic/tandem mass spectrometric experiments were performed using low-energy collisions on a triple-quadrupole mass spectrometer. It was found that relatively high collision energies and collision gas pressures were required to produce fragmentation and that some unusual fragments were produced.     

Generation of hydroxyeicosatetraenoic acids by human inflammatory cells: analysis by thermospray liquid chromatography-mass spectrometry

Arachidonic acid was converted to a series of hydroxyeicosatetraenoic acids (HETEs) by mixed human inflammatory cells following stimulation with the calcium ionophore A23187. HETEs were purified by a simple one-step extraction procedure followed by HPLC. The HPLC was coupled to a Finnigan quadrupole mass spectrometer using the now commercially available thermospray liquid chromatography-mass spectrometry interface. The HPLC eluant was monitored 'on line' by the mass spectrometer. Soft ionisation occurs, generating intense molecular ion species in the negative ion mode (M - H-:m/z 319) for each of the isomeric HETEs. The (M + H+ - H2O) ion at m/z 303 is the major species in the positive ion spectra of HETEs. Mass spectra were obtained on-line post-HPLC for HETEs formed by the human cells, and the HPLC-MS profile compared with that obtained from standards; species corresponding to the 11-, 9- and 5-HETEs were observed.     

Determination of trimethoprim in bovine serum by high-performance liquid chromatography with confirmation by thermospray liquid chromatography—mass spectrometry

A high-performance liquid chromatographic (HPLC) method with a detection limit of 5 ng/ml was developed for the analysis of trimethoprim in bovine serum. Trimethoprim and the internal standard, ormetoprim, under alkaline conditions, were first extracted into dichloromethane and then back-extracted into dilute sulphuric acid (0.15 M) and cleaned-up on a C₁₈ cartridge. Trimethoprim was quantified on a C₁₈ column using a triethylammonium acetate—acetonitrile—methanol (16:3:1, v/v/v) mobile phase at a flow-rate of 1.5 ml/min, with ultraviolet detection at 225 nm. This method was used to verify the accuracy of test responses obtained with the Brilliant Black Reduction test, a rapid screening method, for trimethoprim levels in the serum of steers treated with Trivetrin. Confirmation of the presence of trimethoprim in the sample extract was obtained by thermospray HPLC—mass spectrometry.     

Determination of morphine and morphine glucuronides in human plasma by 96-well plate solid-phase extraction and liquid chromatography-electrospray ionization mass spectrometry

There is considerable interest in quantifying morphine and its major metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). Available assays use gas chromatography-mass spectrometry or high-performance liquid chromatography (HPLC) with single or tandem mass spectrometry, ultraviolet, electrochemical, or fluorimetric detection. Nevertheless, few methods provide adequate sensitivity for all analytes, in a single injection, with the desired rate of sample throughput. A rapid and sensitive method for quantification of morphine, M3G and M6G from human plasma using HPLC with electrospray ionization mass spectrometry was developed using a Waters Oasis MCX 96-well plate for extracting both lipophilic morphine and its hydrophilic glucuronides, C18 separation using an isocratic mobile phase (methanol, acetonitrile and formic acid), and selected ion monitoring. Recoveries of morphine, M3G and M6G, respectively, were 81, 90 and 82% at the low (2, 25 and 2 ng/ml), 80, 77 and 75% at the medium (10, 250 and 10 ng/ml), and 74, 62 and 72% at the high (100, 1000 and 100 ng/ml) quality control samples. The limit of quantitation was 0.5 ng/ml morphine and M6G, and 5 ng/ml M3G. Analytes were validated over a linear range of 0.5-200 ng/ml morphine and M6G, and 5-2000 ng/ml M3G. This assay represents an improvement over existing methods through solid phase extraction with increased sample throughput (96-well plates), use of small samples (0.5 ml), and sub-nanogram detection.     

Optimization of immobilized enzyme hydrolysis combined with high-performance liquid chromatography/thermospray mass spectrometry for the determination of neuropeptides

Peptidases, including chymotrypsin, thermolysin, trypsin, V8 protease, and carboxypeptidases A, B, and Y, were immobilized for use in conjunction with HPLC/thermospray MS for the analysis of neuropeptides. The optimal operating conditions for each immobilized enzyme bioreactor were determined. Optimal hydrolysis usually occurred at the highest percentage of aqueous solution in the mobile phase at pH 7-8 and 40-50 degrees C. Often post-HPLC column addition of aqueous solutions before the bioreactor could improve activity and thermospray sensitivity without changing the HPLC separation. Enzymatic hydrolysis requirements were compatible under conditions for HPLC separation and thermospray MS detection of the selected neuropeptides. Synthetic alpha-, beta-, and gamma-endorphins were the primary neuropeptides used to evaluate on-line immobilized enzyme bioreactor/MS. HPLC followed by peptidase hydrolysis produced characteristic hydrolysis products for confirming the peptides' identity using thermospray MS detection. Furthermore, the peptide formed from enzymatic hydrolysis resulted in a MS ion current 10-40 times higher than that of the [M + 2H]2+ ion for unhydrolyzed beta-endorphin. The increased sensitivity achieved for detecting the hydrolysis products permits detection and quantitation of synthetic peptides down to 800 fmol.     

Determination of bile acids in biological fluids by liquid chromatography-electrospray tandem mass spectrometry

A simple, sensitive, and specific liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the determination of bile acids in human bile has been developed. The bile acids were extracted with a C(18) (octadecyl) reversed-phase column and identified and quantified by simultaneous monitoring of their parent and daughter ions, using the multiple reaction monitoring mode. Identification and quantification of conjugated bile acids in bile was achieved in 5 min. The detection limit was 1 ng, and the determination was linear for concentrations up to 100 ng. The percent recovery of standards made of single conjugated (glycine and taurine) bile acid or of mixture of glycine- or taurine-conjugated cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, and lithocholic acid averaged 71.73% to 95.92%. The percent recovery of the same standard bile acids was also determined by gas chromatography-mass spectrometry (GC-MS), using the selected ion monitoring mode, and averaged 66% to 96%. A biliary bile acid profile of human gallbladder bile was obtained by LC-MS/MS and GC-MS.The results showed a good correlation between the two techniques and no significant differences between the two methods were observed. The LC-MS/MS method was also used for the analysis of serum, urine, and fecal bile acids. In conclusion, LC-MS/MS is a simple, sensitive, and rapid technique for the analysis of conjugated bile acids in bile and other biological samples. - Perwaiz, S., B. Tuchweber, D. Mignault, T. Gilat, and I. M. Yousef. Determination of bile acids in biological fluids by liquid chromatography-electrospray tandem mass spectrometry. J. Lipid Res. 2001. 42: 114;-119.     

Determination of lipid ester ozonides and core aldehydes by high-performance liquid chromatography with on-line mass spectrometry

Unsaturated triacylglycerols (TG) and choline (PC) and ethanolamine (PE) phosphatides of known structure were subjected to ozonization and reduction with triphenylphosphine to yield the corresponding lipid ester core aldehydes. Mono- and di-C₉ aldehyde palmitoylglycerols were prepared from oleoyldipalmitoyl and oleoyllinoleoylpalmitoyl glycerols, respectively, while egg yolk PC and PE provided the mono-C₅ and mono-C₉ aldehydes of palmitoyl-and stearoyl glycerophospholipids. The aldehydes were isolated in the free form and as the dinitrophenylhydrazone (DNPH) derivatives by thin-layer chromatography (TLC). The intermediate ozonides, free aldehydes and hydrazones were identified by reversed phase high performance liquid chromatography (HPLC) with on-line negative ion thermospray and normal phase HPLC with on-line positive ion electrospray mass spectrometry (LC-MS). The synthetic aldehydes were used as carriers during isolation from natural sources and as reference compounds in quantitative analyses     

Development of a coupled-column liquid chromatographic–tandem mass spectrometric method for the direct determination of betamethasone in urine

Different hyphenated liquid chromatographic (LC) and mass spectrometric (MS) techniques were investigated in order to set-up a method for the fast, direct analysis of betamethasone in hydrolysed and non-hydrolysed urine using large-volume sample injection. After the optimisation of the LC parameters using a traditional UV detector and of the thermospray and mass spectrometric parameters by flow injection, urine samples (0.5 ml) were submitted to analysis by either LC combined with tandem mass spectrometry (MS–MS), coupled-column LC (LC–LC) combined with single quadrupole MS, and LC–LC–MS–MS. Both the three-step configurations (LC–MS–MS and LC–LC–MS) did not provide satisfactory results: loss of sensitivity was noted in the case of LC–MS–MS (likely due to reduced efficiency in the ionisation of betamethasone in the thermospray owing to the presence of large amounts of matrix interference), while in the case of LC–LC–MS a high chemical noise resulting in insufficient selectivity of detection was observed. On the contrary, LC–LC–MS–MS analysis proved to meet the demand of high speed of analysis (sample throughput, 4.5 h⁻¹), selectivity, and sensitivity (LOQ, 1 ng/ml; LOD, 0.2 ng/ml). Notwithstanding the complex analytical system adopted, the developed procedure was manageable and very robust, provided that at the beginning of each analytical session the performance of the system was controlled by checking the retention time of the analytes on the first analytical column with UV detection and by optimising vaporiser temperature of the thermospray by flow injection.     

Development and validation of a liquid chromatography-mass spectrometry method for the quantitation of naltrexone and 6beta-naltrexol in guinea pig plasma

A quantitative liquid chromatographic-electrospray ionization mass spectrometry method for the determination of naltrexone and 6beta-naltrexol in guinea pig plasma has been developed and validated using naloxone as an internal standard. A single step precipitation-extraction technique was carried out to extract the plasma samples using acetonitrile:ethyl acetate (1:1, v/v). The chromatographic separation was performed on a C(18) column using a mobile phase consisting of 35:65 (v/v) acetonitrile:2 mM ammonium acetate with 0.01 mM ammonium citrate at a flow rate of 0.25 mL/min. The analyte was detected after positive electrospray ionization using selected ion monitoring (SIM) mode. The mean recoveries for naltrexone, naltrexol, and naloxone were 91.7, 89.3, and 99.0%, respectively. The lower limit of quantification (LLOQ) for naltrexone and 6beta-naltrexol was 1.25 ng/mL, and the limit of detection (LOD) was 0.75 ng/mL. The method was applied to a pharmacokinetic study in order to assess the drug disposition of naltrexone in guinea pigs.     

Highly sensitive determination and characterization of intact cellular ester-linked phospholipids using liquid chromatography-plasma spray mass spectrometry

Liquid chromatographic class separations of common cellular phospholipids combined with plasma spray ionization of the effluents were investigated. Comparison with true thermospray ionization involving ammonium acetate buffering revealed a gain in total ionization in the plasma spray of a factor of approximately 10 using a cation-exchange column and a solvent mixture consisting of acetonitrile-methanol-water (400:100:15, v/v). Plasma spray ionization studies of bovine brain polyphosphoinositides interrelated by the phosphate content in the inositol moeity showed almost identical monoglyceride and diglyceride ion clusters, indicating possibilities of studying the biochemical turnover of such phospholipids. Plasma spray ionization liquid chromatography-mass spectrometry of bacterial membrane phospholipids (Pseudomonas fluorescens) revealed possibilities of obtaining indications of individual fatty acid compositions from the spectra of the phosphatidylinositol and phosphatidylethanolamine fractions present. Conventional gas chromatographic fatty acid analysis agreed with the direct mass spectrometric structure elucidations. Interestingly, the two phospholipid classes had different relative fatty acid compositions with a significantly higher degree of cyclic fatty acids in the phosphatidyl ethanolamines. Plasma spray ionization yielded linear dose-response curves for both the monoglyceride and diglyceride fragment signals in the selected-ion monitoring mode. The detection limit for the monoglyceride and diglyceride species of phosphatidylcholine under the chromatographic and mass spectrometric conditions used was found to be in the picogram range.     

Application of liquid chromatography—thermospray mass spectrometry in the analysis of glycerophospholipid molecular species

We report the application of high-performance liquid chromatographic (HPLC) separation with ultraviolet detection and direct, on-line, structural analyses by mass spectrometry of glycerobenzoate derivatives from complex mixtures of phospholipid molecular species. Individual phospholipids were resolved from total lipid extracts by thin-layer chromatography (TLC). Diradylglycerols were released from phospholipids by phospholipase-C treatment, converted to diradyl glycerobenzoates and subsequently separated by TLC into subclasses (alk-1-enylacyl, alkylacyl and diacyl types). The molecular species within each subclass were resolved by HPLC with an octadecyl reversed-phase column in acetonitrile—isopropanol (80:20, v/v). Individual peaks were quantitated at the picomole level by measuring absorbance at 230 nm. After post-column addition of methanol—0.2 M ammonium acetate (50:50, v/v), peaks were introduced through the thermospray interface into a VG Masslab 30–250 quadrupole mass spectrometer. Molecular species showed as base peaks the salt adducts of the molecular ion which permitted easy deduction of the overall fatty acyl composition. In addition, the diglyceride fragment of each species was found at [MH — 122]⁺ and two fragments formed by the loss of the fatty acyl groups (R) in the sn-1 or sn-2 position were found at [M — R₁]⁺ and [M — R₂]⁺, respectively. Since preferential release of either fatty acyl group was observed in positional isomers, the ratio of the intensity of these fragments gave information on the position of the fatty acyl groups in the individual HPLC peaks. We show that the use of on-line mass spectrometry, however, provides easy identification of all molecular species present in a complex phospholipid mixture, even when more than one molecular species is contained in an HPLC peak.     

Simultaneous determination of selegiline-N-oxide,a new indicator for selegiline administration,and other metabolites in urine by high-performance liquid chromatography–electrospray ionization mass spectrometry

In order to discriminate selegiline (SG) use from methamphetamine (MA) use, the urinary metabolites of SG users have been investigated using high-performance liquid chromatography (HPLC)–electrospray ionization mass spectrometry (HPLC–ESI–MS). Selegiline-N-oxide (SGO), a specific metabolite of SG, was for the first time detected in the urine, in addition to other metabolites MA, amphetamine (AP) and desmethylselegiline (DM-SG). A combination of a Sep-pak C₁₈ cartridge for the solid-phase extraction, a semi-micro SCX column (1.5 mm I.D.×150 mm) for HPLC separation and ESI–MS for detection provided a simple and sensitive procedure for the simultaneous determination of these analytes. Acetonitrile–10 mM ammonium formate buffer adjusted to pH 3.0 (70:30, v/v) at a flow-rate of 0.1 ml/min was found to be the most effective mobile phase. Linear calibration curves were obtained over the concentration range from 0.5 to 100 ng/ml for all the analytes by monitoring each protonated molecular ion in the selected ion monitoring (SIM) mode. The detection limits ranged from 0.1 to 0.5 ng/ml. Upon applying the scan mode, 10–20 ng/ml were the detection limits. Quantitative investigation utilizing this revealed that SGO was about three times more abundant (47 ng/ml, 79 ng/ml) than DM-SG in two SG users’ urine samples tested here. This newly-detected, specific metabolite SGO was found to be an effective indicator for SG administration.     

Determination of deuterium-labeled tryptophan in proteins by means of high-performance liquid chromatography and thermospray mass spectrometry

In order to develop direct methods for determining the extent of metabolic incorporation of isotopically labeled amino acids into a protein, the determination of deuterated tryptophan in [2H5]tryptophan-bacteriorhodopsin was investigated. The isotopically modified protein was subjected to alkaline hydrolysis. After phenyl isothiocyanate derivatization of the hydrolysate, the mixture was separated by reversed-phase liquid chromatography. Field desorption mass spectrometry and thermospray mass spectrometry were investigated for their ability to determine the ratio between [2H5]tryptophan and total tryptophan in the collected fractions. In order to check the procedure a set of known tryptophan/[2H5]tryptophan mixtures were passed through the same derivatization, HPLC separation, and lyophilization procedure as used for the biological samples.     

Quantification of roxatidine in human plasma by liquid chromatography electrospray ionization tandem mass spectrometry: application to a bioequivalence study

A sensitive and specific method using a one-step liquid-liquid extraction (LLE) with ethyl acetate followed by high-performance liquid chromatography (HPLC) coupled with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed and validated for the determination of roxatidine in human plasma using famotidine as an internal standard (IS). Data acquisition was carried out in multiple reaction monitoring (MRM) mode, by monitoring the transitions m/z 307.3-->107.1 for roxatidine and m/z 338.4-->189.1 for famotidine. Chromatographic separation was performed on a reverse phase Hydrosphere C(18) column at 0.2 mL min(-1) using a mixture of methanol-ammonium formate buffer as mobile phase (20:80, v/v; adjusted to pH 3.9 with formic acid). The achieved lower limit of quantification (LLOQ) was 1.0 ng mL(-1) and the standard calibration curve for roxatidine was linear (r(2)=0.998) over the studied range (1-1000 ng mL(-1)) with acceptable accuracy and precision. Roxatidine was found to be stable in human plasma samples under short-, long-term storage and processing conditions. The developed method was validated and successfully applied to the bioequivalence study of roxatidine administrated as a single oral dose (75 mg as roxatidine acetate hydrochloride) to healthy female Korean volunteers.     

Selective determination of sultopride in human plasma using high-performance liquid chromatography with ultraviolet detection and particle beam mass spectrometry

We developed a sensitive and selective method for determining levels of sultopride, a neuroleptic drug of the substituted benzamide, in human plasma using high-performance liquid chromatography (HPLC) combined with UV detection and particle beam mass spectrometry (PBMS). Sutopride was extracted with tert.-butylmethyl ether using a salting-out technique. Tiapride served as an internal standard (I.S.). Sutopride and I.S. were separated by HPLC on a silica column with a mobile phase of acetonitrile-0.1 M ammonium acetate (94:6, v/v). The calibration curves were linear over the concentration range from 5 to 1000 ng/ml by HPLC with UV detection and from 10 to 1000 ng/ml with PBMS detection. The limit of quantitation was 5 ng/ml with UV detection and 10 ng/ml with PBMS detection. The absolute recovery was 92% and the within-day coefficients of variation were 2.9–7.1% at plasma concentrations from 50 to 500 ng/ml, determined by HPLC with UV detection. Using this method, we measured the plasma concentrations of sultopride with replicate analyses in four hospitalized patients and steady-state plasma levels were determined to be 161.6±30.8, 321.1±93.7, 726.5±143.1 and 1273.6±211.2 ng/ml, respectively.