'High throughput' solid-phase extraction technology and turbo ionspray LC-MS-MS applied to the determination of haloperidol in human plasma.

A quantitative method for the analysis of haloperidol in human plasma is described. Sample clean-up was performed by means of solid-phase extraction using 3M Empore extraction disk plates in the 96-well format, automated with a Canberra Packard pipetting robot. Separation was performed by reversed phase high performance liquid chromatography with turbo ionspray tandem mass spectrometric detection by monitoring the decay of protonated haloperidol of m/z 376 to its fragment at m/z 165, versus the decay of protonated haloperidol-D4 at m/z 380 to its fragment at m/z 169. The validated concentration range was from 0.100 to 50.0 ng ml(-1), with an inaccuracy and overall imprecision below 10% at all concentration levels. Validation results on linearity, specificity, precision, accuracy and stability are shown and are found to be adequate. The average sample preparation time for a batch of 96 samples is approximately 50 min. The chromatographic run time is 3 min. A sample throughput of at least 240 samples per day can be achieved.

Assessment of nitric oxide production by measurement of [15N]citrulline enrichment in human plasma using high-performance liquid chromatography-mass spectrometry.

Nitric oxide (NO) is formed by a class of NO synthases (NOS), which convert arginine into citrulline. A decreased in vivo NO availability can be the result of an increased NO inactivation or a decreased NO production. The latter can be assessed by measurement of isotopic enrichment of plasma citrulline during infusion of isotopically labeled arginine. The potential of high-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) to determine enrichments of [15N2]arginine and [15N]-citrulline in plasma during infusion of [15N2]arginine in humans was investigated. Two types of MS instruments were evaluated: a sector-type mass spectrometer equipped with a frit fast-atom bombardment (FAB) interface and a quadrupole instrument with electrospray ionization (ESI). FAB-MS appeared to be unsuitable for determination of isotope ratios, because background ions influenced the observed isotope ratio in an unpredictable way. In combination with either off- or on-line reversed-phase HPLC, ESI-MS proved to be a more reliable technique. However, the amount of material that is introduced in the mass spectrometer is critical and should be carefully controlled. During infusion of [15N2]arginine in 14 healthy subjects, a mean arginine-to-citrulline conversion rate of 0.22 +/- 0.07 (SD) mumol.kg-1.h-1 was found. In 4 subjects who received an intravenous infusion with the NOS antagonist L-NMMA, the conversion rate decreased from 0.30 +/- 0.14 to 0.10 +/- 0.06 mumol.kg-1.h-1. It is concluded that ESI-MS in combination with HPLC can be successfully applied for determination of arginine and citrulline enrichments in plasma, thus providing a useful tool for assessment of in vivo NO production.

Reversed-phase high-performance liquid chromatographic separation of bovine kappa-casein macropeptide and characterization of isolated fractions.

From complex mixtures of non-glycosylated and differently glycosylated caseinomacropeptides (CMP; kappa-casein fragment 106-169; M(r) approximately 7000) various fractions were isolated and further purified by reversed-phase HPLC. The fractions were characterized by mass determination and composition analysis and also used in gel-permeation chromatography and NMR studies to investigate their molecular size behaviour as a function of pH, ionic strength, peptide concentration and degree of glycosylation. No evidence was found for association of any CMP fraction as a function of the experimental conditions applied, which is in contrast with suggestions made in the literature. The increased molecular size (apparent molecular mass approx. 30-45 kDa) is rather explained by a large voluminosity of the molecular species due to internal electrostatic and steric repulsion. Furthermore, the susceptibility of some non-glycosylated and glycosylated CMP fractions to enzymic attack by the Glu-specific endopeptidase from Staphylococcus aureus V8 was studied. Initial rates of proteolysis by this enzyme were independent of the degree of glycosylation. Only in the case of highly glycosylated CMP was further hydrolysis to smaller fragments inhibited. Hydrolytic products were identified by electrospray ionization and fast-atom bombardment mass spectrometry.

The structure of the lantibiotic lacticin 481 produced by Lactococcus lactis: location of the thioether bridges.

The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis ssp. lactis. This polypeptide contains 27 amino acids, including the unusual residues dehydrobutyrine and the thioether-bridging lanthionine and 3-methyllanthionine. Lacticin 481 belongs to a structurally distinct group of lantibiotics, which also include streptococcin A-FF22, salivaricin A and variacin. Here we report the first complete structure of this type of lantibiotic. The exact location of the thioether bridges in lacticin 481 was determined by a combination of peptide chemistry, mass spectrometry and NMR spectroscopy, showing connections between residues 9 and 14, 11 and 25, and 18 and 26.

Identification of oxidized methionine in peptides.

The positive- and negative-ion collision-induced dissociation spectra of peptides containing methionine, methionine sulphoxide and methionine sulphone have been studied. Characteristic fragmentations were identified and evaluated as possible indicators for the presence of oxidized methionine residues in peptides. It was found that the elimination of CH3SOH (-64 u) from [M + H]+ is unique for peptides that contain methionine sulphoxide. Sequence ions containing the oxidized methionine undergo the same elimination, allowing unambiguous sequence determination. Methionine sulphone exhibits an analogous elimination of CH3SO2H (-80 u) from the protonated molecule, but not from sequence ions.

Identification of a new genetic variant of bovine beta-casein using reversed-phase high-performance liquid chromatography and mass spectrometric analysis.

Various components of the beta-casein fraction from bovine milk were separated by preparative reversed-phase high-performance liquid chromatography (RP-HPLC). They included the genetic variants beta A1, beta A2, beta A3, and an unknown component previously denoted beta X [S. Visser et al., J. Chromatogr. 548 (1991) 361-370]. Tryptic digests of these components were compared by RP-HPLC and most peaks were analysed by mass spectrometry (MS). The tryptic map of beta X was closest to that of beta A1, but with a few mutually different peak components. Electrospray ionisation MS revealed that in the beta X map these components had relative molecular masses of 16 higher than the corresponding ones in the beta A1 map. The main differential peaks represented the 114-169 fragments of beta A1 and beta X, respectively, which were both purified and then cleaved with cyanogen bromide. In the resulting mixtures, each of which contained three fragments, the corresponding peptides representing the 145-156 sequence showed the 16 relative molecular mass difference. In beta X this sequence contained a Leu residue at position 152 instead of the Pro-152 in beta A1, as established by fast-atom bombardment MS-MS. The Leu could be discriminated from an Ile residue by the presence of a side-chain-specific, D-type fragment ion in the MS-MS spectrum of the beta X CNBr peptide. The sequence of the two homologous 145-156 fragments was confirmed by regular amino acid sequence analysis. In accordance with internationally accepted guidelines for the nomenclature of milk proteins, the new genetic variant has been named beta-casein F-5P.