Vitamin B12 quantification in human milk - Beyond current limitations using liquid chromatography and inductively coupled plasma - Mass spectrometry.

Vitamin B12 plays a key role in human biological functions and is vital in the neurological development of infants. The assessment of the vitamin B12 intake in exclusively breastfed babies depends on the reliability of its determination in milk. In this report, we present a new accurate and robust method for quantification of vitamin B12 in human milk. A highly specific sample preparation is applied, associated with chromatographic separation and detection by ICP-MS. Excellent sensitivity and accuracy are reported, with recovery values well within acceptability limits (80-120%), within- and between-day variability are lower than 10% and 15% respectively. Strong correlation with a microbiological assay was observed (r2 = 0.9) within the validation range (40-1000 pmol/L, corresponding to 54 to 1355 ng/L). The method can be used to routinely monitor vitamin B12 in clinical or population observational studies, determine infant's intake or assess efficacy of mother's supplementation.

Validation of a method for arsenic speciation in food by ion chromatography-inductively coupled plasma/mass spectrometry after ultrasonic-assisted enzymatic extraction.

A fully validated and rapid quantitative method is presented for determination of inorganic arsenic [arsenite, As(III) and arsenate, As(V)] and organic arsenic species (methylarsonic acid, dimethylarsinic acid, and arsenobetaine) by ion chromatography paired with inductively coupled plasma/MS after ultrasonic-assisted enzymatic extraction (UAEE) in rice- and seafood-based raw materials and finished products. This method gives toxicological meaning to arsenic analysis, since the sum of the toxic chemical forms As(III) and As(V) can be determined. In contrast to classical water-methanol extraction, UAEE enables drastic acceleration of sample extraction (5 min instead of several hours), while total arsenic extraction efficiency is improved without species conversion. Validation was performed to evaluate the method for selectivity, linearity, LOD/LOQ (0.007-0.020 mg/kg), trueness, precision (HorRat values, 0.2-0.6), recovery (93-122%), and uncertainty. The method was also satisfactorily tested using two proficiency tests. Performance characteristics are reported for four certified reference materials, standard reference material (SRM) 1568a (rice flour), Institute for Reference Materials and Measurements 804 (rice flour), SRM 2976 (mussel tissue), certified reference material-627 (tuna fish), and several commercial food samples populating five AOAC triangle food sectors. The results indicated that this speciation method is cost-efficient, time-saving, and accurate, as well as fit-for-purpose, according to International Organization for Standardization/International Electrotechnical Commission 17025:2005 standard, and could be used for routine analysis.

Detection of recombinant bovine somatotropin in milk and effect of industrial processes on its stability.

A LC-MS/MS method has been developed for the direct detection of recombinant bovine somatotropin (rbST) in milk and dairy products. The sample preparation protocol is based on a solid phase extraction step followed by precipitation with cold methanol and enzymatic digestion. The analysis is focused on the tryptic N-terminal peptide, specific of the recombinant form of the hormone and the detection is performed by LC-ESI(+)-MS/MS. This method has been validated according to the European Union criteria described in the Directive 2002/657/EC. Acceptable performances, with a decision limit (CCalpha) of 1.24 ng mL(-1) and detection capability (CCbeta) of 1.92 ng mL(-1) were obtained. Calculation of repeatability and intermediate reproducibility of the signal at 100 ng mL(-1) lead to relative standard deviations lower than 20%, showing the robustness of the method. Samples subjected to various industrial processes namely, heating, freezing, defatting, pasteurization and spray-drying were then analysed in order to determine the consequences of these treatments on the stability of the hormone. Results showed that temperature related processes, such as pasteurization and spray-drying induce a loss of the hormone up to 95%.

Ochratoxin A-mediated DNA and protein damage: roles of nitrosative and oxidative stresses.

Ochratoxin A (OTA) is a mycotoxin occurring in a variety of foods. OTA is nephrotoxic and nephrocarcinogenic in rodents. An OTA-mediated increase of the inducible nitric oxide synthase (iNOS) expression was observed in normal rat kidney renal cell line and in rat hepatocyte cultures, suggesting the induction of nitrosative stress. This was associated with an increased nuclear factor kappa-light chain enhancer of activated B cells activity. The potential consequences of iNOS induction were further investigated. A significant increase in the levels of protein nitrotyrosine residues was observed with OTA. In addition, OTA was found to increase the level of DNA abasic sites in both cell cultures system. This end point was used as an indirect measure of 8-nitroguanine formation. Treatment of the cells with L-N(6)-(1-iminoethyl) lysine, a specific inhibitor of iNOS activity, inhibited the OTA-mediated overnitration of proteins but did not reduce the level of DNA abasic sites. It was found previously that nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) activators were able to restore the cellular defense against oxidative stress and could prevent DNA abasic sites in cell cultures. In the present study, pretreatment of the cells with activators of Nrf2 prevented OTA-mediated increase in lipid peroxidation, confirming the potential of Nrf2 activators to confer protection against OTA-mediated oxidative stress. In addition, it was found that Nrf2 activators could also prevent OTA-induced protein nitration and cytotoxicity. In conclusion, the present data further confirm oxidative stress as a key source of OTA-induced DNA damage and provide additional evidence for a role of this mechanism in OTA carcinogenicity. The exact role of nitrosative stress still remains to be established.

Use of molecularly imprinted solid-phase extraction sorbent for the determination of four 5-nitroimidazoles and three of their metabolites from egg-based samples before tandem LC-ESIMS/MS analysis.

A nitroimidazole, molecularly imprinted polymer (MIP) was tested to extract four 5-nitroimidazoles (i.e., dimetridazole (DMZ), ipronidazole (IPZ), metronidazole (MNZ), and ronidazole (RNZ)) and three of their metabolites (i.e., DMZOH, IPZOH, and MNZOH) from egg powder samples. Various MIP templates were produced, and their selectivity was assessed on nitroimidazole standard solutions using liquid chromatography coupled with ultraviolet detection. The optimal cleanup was then used for the extraction of nitroimidazole in egg powder samples, and their quantification was achieved by isotope dilution LC-ESIMS/MS. The sample preparation entails a solubilization of the samples with water and acetonitrile followed by a MISPE cleanup step before LC-ESIMS/MS analysis. Data acquisition was achieved using selected reaction monitoring, and quantification was done with five deuterated analogues (i.e., DMZ- d(3), RNZ- d(3), IPZ- d(3), DMZOH- d(3), and IPZOH- d(3)). DMZOH- d(3) was used to quantify MNZ and MNZOH since they do not have their corresponding internal standards. The method was validated according to the European Union criteria by spiking experiments at concentration levels of 1, 2, and 3 microg/kg. At these three levels and for compounds having their own internal standards, acceptable performance data were obtained, with internal standard corrected recoveries ranging from 91 to 111%, and decision limits (CCalpha) and detection capabilities (CCbeta) were below 0.34 and 0.39 microg/kg, respectively.

Advantages of molecularly imprinted polymers LC-ESI-MS/MS for the selective extraction and quantification of chloramphenicol in milk-based matrixes. comparison with a classical sample preparation.

A simple and fast selective extraction of the antibiotic chloramphenicol (CAP) from milk (raw milk, skimmed milk, and milk powder) using a molecularly imprinted polymer (MIP) sorbent is described. The method entails a single centrifugation step prior to loading the supernatant onto the MIP cartridge and subsequent elution with a mixture of solvents. CAP was further analyzed by isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) operating in negative ionization acquisition mode. The advantages of the MIP approach were assessed by comparing the data generated from a classical solid-phase and liquid-liquid extractions procedure, previously developed in our laboratory. A better recovery of CAP due to an enhanced selectivity and a faster turnaround time (18 samples processed within 3 h compared to 8 h with the classical approach) were evidenced when using the MIP cleanup. The analysis of CAP in raw milk was further validated according to the 2002/657/EC European Union criteria for the analysis of veterinary drug residues at the minimum required performance limit (MRPL) of 0.3 microg/kg, using CAP-d(5) as internal standard. Non-internal-standard corrected recovery values ranged between 50% and 87% over the range of concentrations considered. The decision limit (CCalpha) and detection capability (CCbeta) were calculated to be 0.06 and 0.10 microg/kg, respectively.

Reduction in antioxidant defenses may contribute to ochratoxin A toxicity and carcinogenicity.

Ochratoxin A (OTA) is a renal carcinogen in rodents. Its human health significance is unclear. It likely depends upon the mechanism of carcinogenesis. In a previous microarray study a reduction in nuclear factor-erythroid 2 p45-related factor 2 (Nrf2)-dependent gene expression was observed in the kidney but not in the liver of rats fed OTA up to 12 months. Nrf2 regulates detoxification and antioxidant gene expression. The present report shows that OTA decreased the protein expression of several markers of the Nrf2-regulated gene battery in kidney in vivo indicating that the effects observed at mRNA level may be of biological significance. The OTA-mediated Nrf2 response could be reproduced in an NRK renal cell line and in primary hepatocyte cultures. In in vitro systems, an OTA-mediated inhibition of Nrf2 activity was demonstrated by electrophoretic mobility shift and Antioxidant Regulatory Element-driven luciferase reporter assays. The reduction of Nrf2-regulated gene expression resulted in oxidative DNA damage as evidenced by formation of abasic sites in vitro and confirmed in kidney in vivo. All OTA-mediated effects observed were prevented by pretreatment of cell cultures with inducers of Nrf2 activity. Our data suggest that reduction of cellular defense against oxidative stress by Nrf2 inhibition may be a plausible mechanism of OTA nephrotoxicity and carcinogenicity.

Quantitative determination of five ergot alkaloids in rye flour by liquid chromatography-electrospray ionisation tandem mass spectrometry.

A confirmatory method for detecting five ergot alkaloids, ergocristine, ergotamine, ergonovine, ergocornine and alpha-ergokryptine, in rye flour is described using high performance liquid chromatography coupled to tandem mass spectrometry detection by monitoring two transition reactions per analyte. The procedure entails a liquid-liquid extraction followed by a clean-up step using a C18 solid-phase extraction (SPE) cartridge. An analogue compound, methysergide hydrogen maleinate, was used to assess both repeatability sample preparation and potential MS response fluctuations. The method was fully validated according to the European Union (EU) criteria. Detection and quantification limits of all analytes were calculated ranging from 7 to 11 microg/kg and from 23 to 37 microg/kg, respectively. Fifteen rye flour samples were investigated with the newly developed method, and none of them were above the current Swiss limits of 200mg/kg for total ergot alkaloids.