Determination of osteocalcin in meat and bone meal of bovine and porcine origin using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry and high-resolution hybrid mass spectrometry.

A method has been developed for determining the origin of meat and bone meal (MBM) by detecting species-specific osteocalcin (OC) using matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) and high-resolution hybrid mass spectrometry (HR-Q/TOF MS). The analysis is based on the detection of typical species-specific OC and its tryptic peptide fragments which differ in mass due to differences in the amino-acid sequences between species. After dissolving the MBM samples in EDTA buffer, purification after ultrafiltration was performed using two methods: solid-phase extraction using Zip-Tip C(18) or size exclusion coupled with reverse-phase chromatography. Fractions containing partially purified intact OC were analyzed using LC-Q/TOF and MALDI/TOF mass spectrometry. Species-specific OC was detected at the typical protonated and doubly protonated molecular ions. Furthermore, typical porcine- and bovine-derived tryptic fragments from MBM were detected after enzymatic digestion. In order to determine the underlying amino-acid sequences and to confirm the assignment to OC-derived peptides, MS/MS analysis was carried out. In conclusion, we were able to detect OC in bovine and porcine MBM with high sensitivity and the MS-based method described here by which total OC mass and marker peptides of digested OC are recorded can be used as an alternative approach to detect genus-specific differences in MBM and can be applied as a confirmatory method to mainly immunological osteocalcin screening methods.

Permeation of topically applied caffeine through human skin--a comparison of in vivo and in vitro data.

AIMS: Due to ethical reasons, in vivo penetration studies are not applicable at all stages of development of new substances. Therefore, the development of appropriate in vitro methods is essential, as well as the comparison of the obtained in vivo and in vitro data, in order to identify their transferability. The aim of the present study was to investigate the follicular penetration of caffeine in vitro and to compare the data with the in vivo results determined previously under similar conditions. METHODS: The Follicular Closing Technique (FCT) represents a method to investigate the follicular penetration selectively. In the present study, FCT was combined with the Franz diffusion cell in order to differentiate between follicular and intercellular penetration of caffeine into the receptor medium in vitro. Subsequently, the results were compared with the data obtained in an earlier study investigating follicular and intercellular penetration of caffeine in vivo. RESULTS: The comparison of the data revealed that the in vitro experiments were valuable for the investigation of the follicular penetration pathway, which contributed in vivo as well as in vitro to approximately 50% of the total penetration, whereas the kinetics of caffeine penetration were shown to be significantly different. CONCLUSIONS: The combination of FCT with the Franz diffusion cell represents a valuable method to investigate follicular penetration in vitro. Nevertheless, in vivo experiments should not be abandoned as in vitro, structural changes of skin occur and blood flow and metabolism are absent, probably accounting for reduced penetration rates in vitro.

Development of a validated liquid chromatography/tandem mass spectrometry method for the distinction of thyronine and thyronamine constitutional isomers and for the identification of new deiodinase substrates.

Thyronines (THs) and thyronamines (TAMs) are two groups of endogenous iodine-containing signaling molecules whose representatives differ from each other only regarding the number and/or the position of the iodine atoms. Both groups of compounds are substrates of three deiodinase isozymes, which catalyze the sequential reductive removal of iodine from the respective precursor molecule. In this study, a novel analytical method applying liquid chromatography/tandem mass spectrometry (LC-MS/MS) was developed. This method permitted the unequivocal, simultaneous identification and quantification of all THs and TAMs in the same biological sample. Furthermore, a liquid-liquid extraction procedure permitting the concurrent isolation of all THs and TAMs from biological matrices, namely deiodinase (Dio) reaction mixtures, was established. Method validation experiments with extracted TH and TAM analytes demonstrated that the method was selective, devoid of matrix effects, sensitive, linear over a wide range of analyte concentrations and robust in terms of reproducible recoveries, process efficiencies as well as intra-assay and inter-assay stability parameters. The method was applied to study the deiodination reactions of iodinated THs catalyzed by the three deiodinase isozymes. With the HPLC protocol developed herein, sufficient chromatographic separation of all constitutional TH and TAM isomers was achieved. Accordingly, the position of each iodine atom removed from a TH substrate in a Dio-catalyzed reaction was backtracked unequivocally. While several established deiodination reactions were verified, two as yet unknown reactions, namely the phenolic ring deiodination of 3',5'-diiodothyronine (3',5'-T2) by Dio2 and the tyrosyl ring deiodination of 3-monoiodothyronine (3-T1) by Dio3, were newly identified.

Investigation of the feeding effect on the 13C/12C isotope ratio of the hormones in bovine urine using gas chromatography/combustion isotope ratio mass spectrometry.

The effect of the feeding on the 13C/12C isotope ratio of four endogenous steroid hormones testosterone (T), epi-testosterone (epi-T), dehydroepiandrosterone (DHEA) and etiocholanolone (ETIO) in bovine urine was investigated. An analytical method to determine the accurate isotope ratio was developed including an extensive clean up followed by enrichment of the analytes in two steps of HPLC fractionation. Feeding experiments with four young animals were performed using C3 and C4 plants (grass, maize silage, hay, etc.) over a time period of about 280 days. One cattle was used as a control animal with no change of its diet over the full period. The detection of the 13C/12C isotope ratio of the acetylated extracts was performed by gas chromatography/combustion isotope ratio mass spectrometry. After the first change of the feeding from C4 to C3 plants significant changes of the delta 13C % values were observed from the -19 to -23% level to the -24 to -32% level for etiocholanolone and epi-testosterone in urine of three animals, whereas the DHEA values remained under the level of the two metabolites. Testosterone could not be detected with GC-C-IRMS due to its low concentration in young animals. After the second change of the diet from C3 to C4 plants (after 222 days), the measured delta 13C % values have been stabilised at the original level. The results show that in case of the feeding with only C3 plants the endogenous delta values of -32% can be reached. In this case the contribution of exogenous material with a delta value of -32% could not be detected independently of the concentration. If the diet contains C4 plants the difference or the ratio of the delta 13C % values becomes the determinant in the discriminatory power. For validation of the method a human and a cattle were treated with testosterone and the delta 13C % values were measured in incurred human and cattle urine.