Full-scan accurate mass selectivity of ultra-performance liquid chromatography combined with time-of-flight and orbitrap mass spectrometry in hormone and veterinary drug residue analysis.

The applicability of ultra-performance liquid chromatography (UPLC) combined with full-scan accurate mass time-of-flight (TOF) and Orbitrap mass spectrometry (MS) to the analysis of hormone and veterinary drug residues was evaluated. Extracts from blank bovine hair were fortified with 14 steroid esters. UPLC-Orbitrap MS performed at a resolving power of 60,000 (FWHM) enabled the detection and accurate mass measurement (<3 ppm error) of all 14 steroid esters at low ng/g concentration level, despite the complex matrix background. A 5 ppm mass tolerance window proved to be essential to generate highly selective reconstructed ion chromatograms (RICs) having reduced background from the hair matrix. UPLC-Orbitrap MS at a lower resolving power of 7500 and UPLC-TOFMS at mass resolving power 10,000 failed both to detect all of the steroid esters in hair extracts owing to the inability to mass resolve analyte ions from co-eluting isobaric matrix compounds. In a second application, animal feed extracts were fortified with coccidiostats drugs at levels ranging from 240 to 1900 ng/g. UPLC-Orbitrap MS conducted at a resolving power of 7500 and 60,000 and UPLC-TOFMS detected all of the analytes at the lowest investigated level. Thanks to the higher analyte-to-matrix background ratio, the utilization of very narrow mass tolerance windows in the RIC was not required. This study demonstrates that even when the targeted sample preparation from conventional LC-MS/MS is applied to UPLC with full-scan accurate mass MS, false compliant (false negative) results can be obtained when the mass resolving power of the MS is insufficient to separate analyte ions from isobaric co-eluting sample matrix ions. The current trend towards more generic and less selective sample preparation is expected to aggravate this issue further.

Comparison of various liquid chromatographic methods involving UV and atmospheric pressure chemical ionization mass spectrometric detection for the efficient trace analysis of phenylurea herbicides in various types of water samples.

The performance of mass spectrometric (MS) detection and UV detection in combination with reversed-phase liquid chromatography without and with the use of coupled column RPLC (LC-LC) has been compared for the trace analysis of phenylurea herbicides in environmental waters. The selected samples of this comparative study originated from an inter-laboratory study. For both detection modes, a 50 mm x 4.6 mm I.D. column and a 100 mm x 4.6 mm I.D. column packed with 3 microm C18 were used as the first (C-1) and second (C-2) column, respectively. Atmospheric pressure chemical ionization mass spectrometry was performed on a magnetic sector instrument. The LC-LC-MS analysis was carried out on-line by means of direct large volume (11.7 ml) injection (LVI). The performance of both on-line (LVI, 4 ml of sample) and off-line LC-LC-UV (244 nm) analysis was investigated. The latter procedure consisted of a solid-phase extraction (SPE) of 250 ml of water sample on a 500 mg C18 cartridge. The comparative study showed that LC-LC-MS is more selective then LC-LC-UV and, in most cases, more sensitive. The LVI-LC-LC-MS approach combines direct quantification and confirmation of most of the analytes down to a level of 0.01 microg/l in water samples in less then 30 min. As regards LC-LC-UV, the off-line method appeared to be a more viable approach in comparison with the on-line procedure. This method allows the screening of phenylurea's in various types of water samples down to a level of at least 0.05 microg/l. On-line analysis with LVI provided marginal sensitivity (limits of detection of about 0.1 microg/l) and selectivity was sometimes less in case of surface water samples. Both the on-line LVI-LC-LC-MS method and the LC-LC-UV method using off-line SPE were validated by analysing a series of real-life reference samples. These samples were part of an inter-laboratory test and contained residues of herbicides ranging from 0.02 to 0.8 microg/l. Beside good correlation between the methods the data agreed very well with the true values of the samples.

A single naturally processed measles virus peptide fully dominates the HLA-A*0201-associated peptide display and is mutated at its anchor position in persistent viral strains.

We studied the natural MHC class I display of measles virus (MV) epitopes. Peptide ligands associated with HLA-A*0201 were purified from a B lymphoblastoid cell line prior to and after infection with MV. Infection-induced peptides were revealed using microcapillary reversed phase high performance liquid chromatography electrospray ionization/mass spectrometry (microLC-ESI/MS) by subtraction of the "infected" and "uninfected" ion traces. Three naturally processed viral epitopes derived from different MV proteins were identified through tandem MS sequencing. These peptides were expressed at widely divergent levels of HLA-peptide complexes, but had similar binding capacities to HLA-A*0201. The most abundant viral peptide species, identified as residues 84-92 (KLWESPQEI) of the MV nonstructural C protein, was expressed at an unprecedented high density (> 10(5) copies per cell) and was immunogenic in HLA-A2/Kb-transgenic mice. Furthermore, natural mutants of this epitope, occurring in persistent lethal MV strains, were shown to have lost their HLA-A*0201 binding capacity. Thus, here we report for the first time the direct discovery through microLC-ESI/MS of a uniquely dominant viral HLA class I ligand, KLWESPQEI, with features eligible for immune selection pressure.

A microcapillary column switching HPLC-electrospray ionization MS system for the direct identification of peptides presented by major histocompatibility complex class I molecules.

A microcapillary column switching high-performance liquid chromatography (HPLC) system was developed for the separation of major histocompatibility complex (MHC) class I associated peptides. Combination of the column switching system with electrospray ionization mass spectrometry (ESIMS) enabled the detection and identification of the peptides at low-femtomole levels. Sample volumes of 30-50 microL were injected and concentrated onto a short, 100-micron-i.d. precolumn. The precolumn was coupled to a 100-micron-i.d. reversed-phase analytical HPLC column via a six-port valve. Peptides were separated on the analytical column using an ESI-compatible mobile phase at a flow rate of 0.5 microL/min. Peptides were eluted directly into the ESI source of either a magnetic sector MS or an ion trap MS. Peptides associated with human leukocyte antigen A*0201 molecules were determined in immunoaffinity-purified extracts from either measles virus infected cells or uninfected cells by microcapillary column switching HPLC-ESIMS. The approach toward detection of virus-specific peptides we used was based on the comparison of ion chromatograms obtained from the LC-MS analysis of extracts from virally infected cells and their uninfected counterparts. In this way, the molecular mass of peptides unique to virus infected cells was obtained. The utility of the system is demonstrated by the identification of a candidate epitope. Microcapillary column switching HPLC was used along with ESI ion trap tandem MS to identify the naturally processed viral peptide KLWESPQEI. This peptide was found to derive from the measles virus nonstructural protein C. The approach described here provides a versatile and sensitive method for the direct identification of viral peptides associated with MHC class I molecules.

On-line derivatization of peptides for improved sequence analysis by micro-column liquid chromatography coupled with electrospray ionization-tandem mass spectrometry.

An on-line method has been developed for the derivatization and coupled liquid chromatography (LC)/electrospray ionization (ESI) MS analysis of peptides at the femtomol level. Peptides are reacted with N-succinimidyl-2(3-pyridyl)acetate (SPA) in buffered aqueous medium at pH7 following loading on a precolumn (PC) in a microcolumn switching system. The fast-hydrolysing reagent is dissolved in dry methanol and mixed, in a 3 vol% ratio, with a buffer just before reaching the sample on the PC. Following the reaction and wash, the N-pyridylacetyl (PA) derivatives formed are transferred to the analytical micro-high-performance (HP) LC column for separation and subsequent ESI tandem MS analysis. The reaction is nearly quantitative and takes place selectively at the N-terminal and lysine amino functions, the latter providing a chemical means to distinguish between isobaric residues lysine and glutamine. In some cases, a minor reaction was observed with the tyrosine hydroxyl group. The N-terminal PA group was able to alter the collision-induced fragmentation pathway of peptides in favour of the formation of abundant b-type ions, a helpful feature for sequence elucidation of unknown peptides, particularly with quadrupole ion trap instruments.

Conformation and quantification of chloramphenicol in cow's urine, muscle and eggs by electron capture negative ion chemical ionization gas chromatography/mass spectrometry.

A method is described for the determination of residues of the antibiotic chloramphenicol in biological samples. The method is based on gas chromatography/negative ion chemical ionization mass spectrometry and uses (37Cl2)chloramphenicol as internal standard. Selective ion monitoring of four analyte-specific ions enables the determination of chloramphenicol levels in urine of 3 micrograms l-1 with a coefficient of variation of 8%. The limit of detection of the method is 0.1 p.p.b. for urine, muscle and egg.