High-performance Liquid Chromatography-electrospray Mass Research Articles

Enantioselective determination of terazosin in human plasma by normal phase high-performance liquid chromatography–electrospray mass spectrometry

A sensitive and selective analytical method for the enantioselective determination of terazosin in human plasma has been developed. The chromatography is based on the normal-phase chiral separation utilizing the analogue prazosin as the internal standard. The detection involves the direct introduction of the normal-phase eluent into an electrospray source followed by mass-selective detection. No pre-column derivatization was required prior to analysis. The chiral stationary phase used was Chiralpak AD 100 mm×2.1 mm I.D. (10 μm particle size). The method was utilized to determine the concentrations of terazosin enantiomers in human subjects following a 5 mg single oral dose. Results were compared with those from an enantioselective HPLC–fluorescence method from the same subject plasma samples. The LC–MS results confirm with confidence that the two terazosin enantiomers have different elimination profiles.

Separation and characterization of the tryptic peptide mapping of recombinant bovine growth hormone by reversed-phase high-performance liquid chromatography electrospray mass spectrometry.

Reversed phase high-performance liquid chromatography (RPHPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) was used to separate and characterize the peptides resulting from the tryptic cleavage of somidobove, a recombinant bovine growth hormone. The tryptic digestion of somidobove was carried out at room temperature for 15 hours. The tryptic peptides were separated on a Zorbax SB300 C8 column with trifluoroacetic acid--acetonitrile gradient elution and characterized by LC/ESI-MS. Resulting single and combined peptide fragments were characterized by comparing the observed molecular mass with the calculated mass for these fragments.

Rapid pharmacokinetic screening of salbutamol in plasma samples by column-switching high-performance liquid chromatography–electrospray mass spectrometry

In order to obtain pharmacokinetic data from studies in humans, a sensitive and selective assay for the quantification of salbutamol in human plasma samples was required. This report describes an automated high-performance liquid chromatography–mass spectrometry assay with pre-column enrichment using internal standard calibration for the quantification of salbutamol and the validation of the assay. The lower limit of quantitation is 0.2 ng/ml with an accuracy and imprecision of less than 7%. The analysis time is 8 min per sample.

A general method for the dereplication of flavonoid glycosides utilizing high performance liquid chromatography/mass spectrometric analysis

Using high performance liquid chromatography–electrospray mass spectrometry a method was developed to separate flavonoids and flavonoid glycosides and to obtain molecular weight information. Collision-induced dissociation was then used to obtain the molecular masses of the flavonoid aglycones. Thus, the mass of a flavonoid glycoside and its aglycone can now be determined in a mixture, and it is not necessary to isolate the components. An aqueous extract of Eugenia jambos L. was used to demonstrate the utility of the technique in analysing extracts for flavonoid glycosides. © 1997 John Wiley & Sons, Ltd.

Application of micropellicular poly-styrene/divinylbenzene stationary phases for high-performance reversed-phase liquid chromatography electrospray-mass spectrometry of proteins and peptides

Short columns packed with highly crosslinked 2.3 μm poly-styrene/divinylbenzene (PS/DVB) particles were used for rapid and efficient separation of proteins and peptides by reversed-phase high-performance liquid chromatography at elevated temperatures. Enhancement of the diffusivities of the sample components at elevated temperatures together with the short diffusion pathlength with the micropellicular polymeric stationary phases were responsible for high efficiency, high speed of analysis, and short column regeneration times. Underivatized PS/DVB beads as well as PS/DVB microspheres which have been modified with polyvinylalcohol or octadecyl chains on the surface were synthesized, employed, and compared to HY-TACH-C18, a commercially available micropellicular octadecyl-silica stationary phase, for the separation of proteins, octapeptides and tryptic protein digests. Highest performance was obtained with the silica- and PS/DVB-based octadecyl stationary phases, which exhibited similar column efficiencies but different selectivities for proteins and peptides. The minimum detectability at 214 nm and the maximum loading capacity for ribonuclease A using analytical 30×4.6 mm I.D. columns were 10 ng (0.6 pmol) and 1 μg, respectively. Finally, reversed-phase HPLC with a 60×2 mm I.D. narrow-bore column packed with micropellicular octadecyl PS/DVB was coupled successfully to electrospray mass spectrometry at a flow-rate of 0.15 mL min−1 and on-line full-scan mass spectra for molecular mass determination and identification of proteins in the lower picomol range were obtained.

Rapid Quantification of Biotin in Human Skin Extracts After Dermal Application Using High-performance Liquid Chromatography–Electrospray Mass Spectrometry

A method for the determination of the vitamin biotin in extracts from different layers of human skin is presented, which has a detection limit of 800 pg ml–1 without further sample preparation. The quantification was performed using an HPLC procedure on a reversed phase column, which takes only 4 min, coupled to a single quadrupole mass spectrometer via an electrospray ionization interface. The detection was performed in the positive selected ion monitoring mode. Additional MS–MS and MSn experiments with biotin using an ion trap mass spectrometer are described.

Isolation and characterization of recombinant human apolipoprotein C-II expressed in Escherichia coli.

A full-length recombinant human apolipoprotein C-II (ApoC-II) has been successfully expressed in Escherichia coli using the T7 expression system. The recombinant ApoC-II. which was expressed intracellularly in the inclusion bodies, was solubilized with 8 M urea and purified using Sephadex G-75 gel permeation chromatography. Four liters of the bacterial culture yielded 16-20 mg of purified recombinant ApoC-II. Sequencing and mass spectrometric analyses indicated that the isolated recombinant ApoC-II contained predominantly (64%) the native form with threonine as the N-terminus, but also contained a minor (36%) molecular form of ApoC-II with an additional methionine at the N-terminus (Met-ApoC-II). Analysis of the recombinant ApoC-II by tryptic digestion and high performance liquid chromatography-electrospray mass spectrometry provides additional conclusive evidence that, with the exception of the N-terminus of Met-ApoC-II, the expressed ApoC-II has the expected peptide sequence. However, this extra N-terminal methionine residue can be excised by further in vitro treatment with methionine aminopeptidase. The purified recombinant ApoC-II was found to be competent in the activation of bovine milk lipoprotein lipase. Thus, the recombinant ApoC-II prepared from E. coli may have a pharmacological application for the treatment of patients with genetic hypertriglyceridemia caused by ApoC-II deficiency.

High performance liquid chromatography —Electrospray tandem mass spectrometry (HPLC-ESI-MS-MS) for the analysis of heterocyclic aromatic amines (HAA)

Sensitive and selective detection of the sixteen most abundant heterocyclic aromatic amines (HAA) has been achieved by application of high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS-MS) in combination with selected reaction monitoring (SRM). Detection limits between 0.1 and 50 ng mL−1 were established by use of HAA model solutions.

Investigation of crudes of synthesis of neuropeptide Y by high-performance liquid chromatography-electrospray mass spectrometry

Neuropeptide Y (NPY) and its modified form, [Leu 31, Pro 34]NPY, are both thirty six amino acids long and they have relative molecular masses of 4250 and 4220. Solid-phase synthesis of both peptides resulted in complex crudes of reaction, which were investigated by means of combines high-performance liquid chromatography-electrospray mass spectrometry (HPLC-ES-MS). The combination of these two powerful analytical techniques allowed rapid and reliable identification of the target peptides and furnished comprehensive information on other reaction products, which were mainly peptidic chains containing a smaller number of amino acids compared to those present in the intact peptides. The possible origin of such side-products and the eventual purification and unambiguous identification of both peptides are discussed.

High-performance liquid chromatographic-electrospray mass spectrometric determination of morphine and its 3- and 6-glucuronides: application to pharmacokinetic studies.

A rapid and selective assay of morphine and its 3- and 6-glucuronides in serum, based on high-performance liquid chromatography-electrospray mass spectrometry has been developed. The analytes and the internal standard, codeine or naltrexone, were subjected to solid-phase extraction, using ethyl solid-phase extraction columns, prior to chromatography. A reversed-phase column and a gradient mobile phase consisting of water and methanol were used. The mass spectrometer was operated in the selected-ion monitoring mode. The following ions were used: m/z 286 for morphine, m/z 300 for codeine, m/z 342 for naltrexone, and m/z 462 for morphine 3- and 6-glucuronides. The limit of quantitation observed with this method was 10 ng/ml morphine, 50 ng/ml morphine-6-glucuronide and 100 ng/ml morphine-3-glucuronide. The present method proved useful for the determination of serum levels of the parent drug and its metabolites in pain patients, heroin addicts and in morphine-treated mice.

Analysis of oligonucleotides using coupled high performance liquid chromatography-electrospray mass spectrometry

Improved HPLC and ESMS conditions have been established, allowing the separation and analysis of oligodesoxyribonucleotides by coupled HPLC-ESMS.

Determination of penicillin G, ampicillin, amoxicillin, cloxacillin and cephapirin by high-performance liquid chromatography—electrospray mass spectrometry

This report contributes to a preliminary investigation of high-performance liquid chromatographic (HPLC)—mass spectrometric (MS) methods for confirming β-lactam antibiotic residues in bovine milk. Initial work for each antibiotic evaluated the collisional activated dissociation (CAD) spectra that could be generated between the capillary and skimmer in the electrospray (ESP) interface. The drugs show various characteristic fragmentation, mostly within the β-lactam ring and the amide group. Response for a particular compound in a given solvent can vary drastically. Usually, the more organic component in the solvent, the higher the ESP response. In many cases use of acetonitrile also results in slightly better ion currents than for methanol when comparing equal percentages of either organic solvent in water. The ESP response of most of the tested antibiotics can be enhanced by the addition of formic acid or acetic acid to the mobile phase methanol—water (1:1). In general, the negative ion spectra are lower in intensity, exhibiting an [M  H] − ion and producing less fragmentation at higher CAD voltages as compared to positive ion spectra. An isocratic reversed-phase HPLC method for the separation of a mixture of five common β-lactam antibiotics was developed using acetic acid as a mobile phase additive and optimized for detection with a new ESP HPLC-MS interface. A post-column split ratio of 70:1 for the eluent from a 150 × 2 mm I.D. column was chosen to provide the required lower flow-rate (approximately 4 μ1/min). The limit of detection for the simultaneous determination of these antibiotics was estimated to be 100 ppb. Electrospray HPLC-MS could be used to confirm these antibiotics for quantities down to about 100 pg entering the mass spectrometer. Multiresidue analysis with microbore HPLC-ESP-MS has the advantage that no post-column splitting of the eluent is required and all of the analyte (on-column injected) will be transferred into the ESP interface. Preliminary work showed good mass spectrometric sensitivity down to the level of regulatory interest, but chromatographic separation efficiency must be improved.