Identification of the in vivo metabolites of the antimalarial arteether by thermospray high-performance liquid chromatography/mass spectrometry.

The thermospray mass spectra of arteether and 16 of its potential metabolites all showed strong [M + NH4]+ ions and with only a few exceptions these compounds also showed spectral peaks corresponding to [M + NH4 - HOR]+ and [M + H - HOR]+, where OR represents the alkoxy or hydroxy group at the 12-position. A method for quantifying the metabolites was developed in which the plasma was spiked with an internal standard (the propyl ether analog of arteether), extracted using a C-18 solid-phase cartridge, then subjected to thermospray high-performance liquid chromatographic/mass spectrometric analysis using selected ion monitoring and a C-18 reversed-phase analytical column. Following the intravenous administration of arteether (11.6 mg kg-1), the plasma was found to contain 12 metabolites of arteether in the 10-1000 ng ml-1 range 15 min post-injection, and within 60 min two of these metabolites attained higher concentrations than that of the parent compound, while several other of the metabolites attained concentrations similar to the parent compound. The pseudo-first-order half-life of arteether was found to be 10.0 +/- 0.6 min, while the apparent half-lives of most of the metabolites were in the 15-30 min range. Nine of these metabolites were identified by comparison to authentic reference standards and the structures of three remaining metabolites were tentatively assigned from their spectral and chromatographic properties. The metabolic pathways leading to these 12 metabolites was a rather complex, multiple-step process, but most of the metabolites arose from an enzymatic oxidation at one of three sites; 3 alpha, 9 alpha, or the CH2 of the side-chain. Conversion of the endoperoxide group to an cyclic ether was not a major pathway. The in vitro antimalarial activity of reference standards of several of the metabolites was determined and all of those tested were found to be active in the low nanogram per milliliter range.     

Analysis of phenylthiohydantoin amino acid mixtures for sequencing by thermospray liquid chromatography/mass spectrometry

Phenylthiohydantoin (PTH) amino acids, the derivatives of amino acids liberated in the course of automated N-terminal sequence analysis of peptides and proteins, are most commonly identified by high-performance liquid chromatography. This communication describes an extension to the methodology for PTH amino acid identification which exploits thermospray liquid chromatography/mass spectrometry for use in the confirmation of PTH amino acid identifications previously made solely on the basis of retention times. Thermospray mass spectra of the 19 synthetic PTH amino acids corresponding to the residues commonly observed during N-terminal sequencing have been acquired. These spectra show strong signals for the protonated molecular ion, accompanied in several cases by ions produced by limited fragmentation of the amino acid side chain and/or the PTH ring system. A reverse-phase separation protocol has been adapted for use with thermospray. The method permits recognition of the protonated molecular ions of all the standard PTH amino acids at the 150-pmol level on the basis of signal-to-noise ratios of 10:1 or better with full scanning. The method has been tested on the N-terminal amino acid sequence analysis of 200 pmol of the standard protein beta-lactoglobulin A, and has been found useful in the study of selected side-products of the sequencing chemistry.     

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.     

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.     

A comparison of thermospray and direct liquid introduction high-performance liquid chromatography/mass spectrometry for the analysis of candidate antimalarials

The analysis of antimalarials by high-performance liquid chromatography (HPLC)/mass spectrometry demonstrates a new dimension in specificity along with increased sensitivity compared to conventional HPLC detection methods. Both direct liquid introduction and thermospray HPLC/mass spectrometry interfaces provided molecular weight information as well as characteristic fragment ions for antimalarials not normally amenable to direct probe or gas chromatographic/mass spectrometric techniques. The direct liquid introduction interface, which incorporated a 1/100 split, showed a detection limit of 30 ng using selected ion monitoring. The thermospray technique showed less than 1 ng detection limits using selected ion monitoring.     

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.     

Analysis of plant galactolipids by reversed-phase high-performance liquid chromatography/mass spectrometry with accurate mass measurement

The composition of plant membrane lipids was investigated by reversed-phase high performance liquid chromatography mass spectrometry with accurate mass measurement. The data dependent methods for the analysis of monogalactosyldiacylglycerols (MGDGs) and digalactosyldiacylglycerols (DGDGs) have been developed. The optimised chromatographic systems were based on a 2.0mm i.d. Nucleosil C18 column with methanol/water (MGDGs) or acetonitrile/methanol/water (DGDGs) gradients. The galactolipids were ionised by electrospray operated in the positive ion mode and identified based on their MS/MS spectra. High resolution spectra with accurate masses were found to be essential for correct interpretation of the MS data. The elution order of non-oxidised MGDGs and DGDGs followed the equivalent carbon numbers. The methods were applied for detailed characterisation of the MGDGs and DGDGs in the leaves of Arabidopsis thaliana and Melissa officinalis.     

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.     

Analysis of oligosaccharides by on-line high-performance liquid chromatography and ion-spray mass spectrometry.

Oligosaccharides were analyzed by a combination of high-performance liquid chromatography (HPLC) and mass spectrometry (MS). First, oligosaccharides labeled with 2-aminopyridine were studied to see if they could be analyzed by MS under the conditions used for separation by HPLC. Pyridylamino (PA)-oligosaccharides could be analyzed under these conditions, although the mass spectra were affected. Then, liquid chromatography-mass spectrometry was used to analyze a PA-oligosaccharide mixture derived from human immunoglobulin G. The PA-oligosaccharides were separated on a reversed-phase column and mass-analyzed directly. The observed molecular weights were close to or identical to those expected from the structures, which were estimated from the elution position on HPLC. This method is rapid and simple, as the mass spectrometer can give the accurate molecular weight of each PA-oligosaccharide in one chromatography run, even if the HPLC separation is incomplete. This method can be used to extend the so-called two-dimensional mapping of PA-oligosaccharides. The structure can be studied in greater detail by tandem MS.     

Determination of andrographolide in human plasma by high-performance liquid chromatography/mass spectrometry

In this paper, a rapid method based on high-performance liquid chromatography/electrospray-mass spectrometry (HPLC/ESI-MS) method for the quantitative determination of andrographolide (AND) in human plasma has been developed and validated. A liquid-liquid extraction (LLE) procedure was selected to isolate AND from biological matrixes. Isosorbide-5-mononitrate (IS-5-MN) was selected as the internal standard (IS). The correlation coefficient of the calibration curve was 0.998, in the range of 9.9-320.0 ng/mL. The validated method may be used to assess the bioavailability and pharmacokinetics of the drug.     

Analysis of N-glycans from recombinant immunoglobulin G by on-line reversed-phase high-performance liquid chromatography/mass spectrometry

An on-line reversed-phase (RP) high-performance liquid chromatography/mass spectrometry (MS) method has been developed for profiling and characterizing N-glycans from recombinant immunoglobulin G antibodies. In this method, released N-glycans are derivatized at their reducing end with 2-aminobenzamide (2AB) and separated on a RP column with on-line fluorescence and MS detection. The method achieves good resolution of all major glycans and segregates glycan types (high-mannose, hybrid, and complex) to different regions of the chromatogram, thus allowing accurate quantification of N-glycans from the fluorescent signal alone. Moreover, the mobile phase used allows high quality on-line MS detection. The 2AB-labeled N-glycans demonstrate good ionization efficiency in electrospray and generate primarily doubly charged [M+2H](2+) ions. The mass and structural information can be readily obtained from the on-line MS and tandem MS data. As little as 70 fmol glycan species can be detected and identified.     

High-throughput high-performance liquid chromatography/mass spectrometry for modern drug discovery

High-throughput high-performance liquid chromatography/mass spectrometry can be used in the analysis of high-throughput organic synthesis products, bioanalytical target analysis for preclinical and clinical studies, and early absorption, distribution, metabolism and excretion (ADME) screening. New techniques are emerging, including system automation, faster analysis, programmed multiple extraction and analysis columns, multiple electrospray ionization channels, and automated 96-well sample preparation.     

Quantification of malonyl-coenzyme A in tissue specimens by high-performance liquid chromatography/mass spectrometry

We present a validated high-performance liquid chromatography/mass spectrometry (HPLC/MS) method for the quantification of malonyl-coenzyme A (CoA) in tissues. The assay consists of extraction of malonyl-CoA from tissue using 10% trichloroacetic acid, isolation using a reversed-phase solid-phase extraction column, HPLC separation, and detection using electrospray MS. Quantification was performed using an internal standard ([(13)C(3)]malonyl-CoA) and multiple-point standard curves from 50 to 1000pmol. The procedure was validated by performing recovery, accuracy, and precision studies. Recoveries of malonyl-CoA were determined to be 28.8+/-0.9, 48.5+/-1.8, and 44.7+/-4.4% (averages+/-SD, n=5) for liver, heart, and skeletal muscle, respectively. Accuracy was demonstrated by the addition of known amounts of malonyl-CoA to tissue samples. The malonyl-CoA detected was compared with the malonyl-CoA added, and the resulting relationships were linear with slopes and regression coefficients equal to 1. Precision was demonstrated by repetitive analysis of identical samples. These showed a within-run variation between 5 and 11%, and the interbatch repeatability was essentially the same. This procedure was then applied to rat liver, heart, and skeletal muscle, where the malonyl-CoA contents were found to be 1.9+/-0.6, 1.3+/-0.4, and 0.7+/-0.2nmol/g wet weight, respectively, for these tissues. This analytical approach can be extended to the quantification of other acyl-CoA species with no significant modification.     

Quantitative liquid chromatography/mass spectrometry/mass spectrometry warfarin assay for in vitro cytochrome P450 studies.

A sensitive assay using high-performance liquid chromatography tandem mass spectrometry (MS/MS) has been established for the quantitative analysis of cytochrome P450 form-specific activities using warfarin as a probe substrate. Four metabolites, 6-, 7-, 8-, and 10-hydroxywarfarin, were chromatographically resolved within 10 min using gradient mobile phases. The mass spectrometry was operated under negative ionization mode. The MS/MS product ion spectra of warfarin and the metabolites were generated using collision-activated dissociation and interpreted. The abundant product ions of the metabolites were selected for quantification applying multiple reaction monitoring. Quantification was based on a quadratic or power curve of the peak area ratio of the metabolite over the internal standard against the respective concentration of the metabolite. This assay has been validated from 2 to 1000 nM for 10-hydroxywarfarin and from 2 to 5000 nM for 6-, 7-, and 8-hydroxywarfarin and successfully applied to evaluate cytochrome P450-mediated drug-drug interactions in vitro using human hepatocytes and liver microsomal preparations.     

Analytical method for keratan sulfates by high-performance liquid chromatography/turbo-ionspray tandem mass spectrometry

We established a highly sensitive LC/MS/MS method for the analysis of the disaccharides produced from keratan sulfates (KS). It was revealed that the disaccharides produced by keratanase II enzymatic digestion of KS could be determined with high sensitivity by the negative-ion mode of multiple reaction monitoring. Furthermore, monosulfated and disulfated disaccharides can be separated using a short column of Capcell Pak NH2 UG80 (35 mm x 2 mm i.d.). The complete analysis of one sample can be performed within 5 min. The assay method was validated and showed satisfactory sensitivity, precision, and accuracy, which enabled quantitation at subpicomole levels. From the results of analyses of KS obtained from cornea, nasal cartilage, and brain, it was found that the degree of sulfation at the C-6 position of the galactose residues differed among those samples in the following order: nasal cartilage > cornea > brain. Our analytical method is very useful for the analyses of KS in various biological materials and for comparison of the degree of sulfation of KS from various biological samples.     

Simultaneous glycosylation analysis of human serum glycoproteins by high-performance liquid chromatography/tandem mass spectrometry

Changes in the glycosylation of some serum proteins are associated with certain diseases. In this study, we performed simultaneous site-specific glycosylation analysis of abundant serum glycoproteins by LC/Qq-TOF MS of human serum tryptic digest, the albumin of which was depleted. The glycopeptide peaks on the chromatogram were basically assigned by database searching with modified peak-list text files of MS/MS spectra and then based on mass differences of glycan units from characterized glycopeptides. Glycopeptide of IgG, haptoglobin and ceruloplasmin were confirmed by means of a comparison of their retention times and m/z values with those obtained by LC/MS of commercially available glycoproteins. Mass spectrometric carbohydrate heterogeneity in the assigned glycopeptides was analyzed by an additional LC/MS. We successfully demonstrated site-specific glycosylation of 23 sites in abundant serum glycoproteins.     

Assessment of [18F]fluoroethylflumazenil metabolites using high-performance liquid chromatography and tandem mass spectrometry

A simple procedure using HPLC and tandem mass spectrometry has been developed for the determination of fluoroethylflumazenil metabolites. Samples were precipitated with acetonitrile, evaporated to dryness followed by reconstitution with methanol. As mobile phase, 50 mM ammonium formate–methanol (58:42, v/v) was used. The method is valid both for cold and radiolabelled metabolites. Various cold metabolites (hydroxylated and/or dealkylated) were identified in rat and human microsome preparations. Radiolabelled metabolites arise from two or more transformations including hydroxylation. The methodology developed can be applied for further characterisation of metabolites, and for the determination of non metabolised [¹⁸F]fluoroethylflumazenil in routine clinical analysis.     

Analysis of tetracyclines in raw urine by column-switching high-performance liquid chromatography and tandem mass spectrometry

The aim of the project was to develop a fast and reliable method for the quantification of the three tetracyclines: tetracycline, oxytetracycline and chlortetracycline in urine. The method is based on column-switching high-performance liquid chromatography with detection by MS–MS. Buffer is added to the sample before it is injected into the chromatographic system, and the first column which is an internal surface reversed-phase column separates the tetracyclines from the bulk of other compounds in urine. The tetracyclines are collected and concentrated on the analytical column before they are separated and eluted into the mass spectrometer in which the tetracycline are detected. The mass spectrometer is a triple quadrupole instrument and is equipped with an electrospray ion source. The MH⁺ ions are selected in the first quadrupole and collisionally activated in the collision cell. Upon collision, activation all three tetracyclines form fragment ions which could be assigned as: [M+H–H₂O–NH₃]⁺ which are selected in the sond mass filter. The detection limits for all three tetracyclines are about 10 ppb, and the calibration curves are linear from 10 to 1000 ppb.     

Investigation of piceid metabolites in rat by liquid chromatography tandem mass spectrometry

There is considerable evidence that stilbenes provide health benefits. Trans-piceid is one of the major stilbenoid compounds in red wine and other plants. The purpose of this study is to investigate the metabolism of piceid in rats, including its conversion product by intestinal microflora in vitro and urinary metabolites. A HPLC-MS/MS method with electrospray ionization (ESI), negative ion mode and collision induced dissociation (CID), was used to elucidate the structures of the major metabolites of piceid. Three metabolites resveratrol, dihydropiceid and dihydroresveratrol were detected after incubating with gut microbiota for 5h. Four urinary metabolites of piceid were identified as resveratrol, dihydroresveratrol monosulfate, piceid monosulfate and piceid monoglucuronide.     

Analysis of secondary metabolites from eschscholtzia californica by high-performance liquid chromatography

A rapid and precise analytical HPLC method has been developed for screening the major benzophenanthridine alkaloids produced by cell cultures of Eschscholtzia califomica, namely, sanguinarine, chelirubine, macarpine, chelerythrine and chelilutine. Separation was achieved on a C18, reversed-phase column with gradient elution using acetonitrile and 50 mM phosphoric acid. Detection was performed by both fluorescence (lambda(ex) 330 nm, lambda(em) 570 nm) and photodiode array, leading to good selectivity and precision in determining peak purity. A simple and quick sample preparation protocol was elaborated involving a methanolic extraction for the measurement of intracellular concentrations of the alkaloids and a solid phase extraction for their quantification in culture medium. Owing to the non-availability of commercially standards, a method for the purification of chelirubine, macar pine and chelilutine by semi-preparative HPLC was developed. Coupled together, the isolation method and the analytical method were highly reliable for screening the alkaloids of interest produced by E. califomica.