Development and validation of a methodology based on Captiva EMR-lipid clean-up and LC-MS/MS analysis for the simultaneous determination of mycotoxins in human plasma.

We report the methodology for the quantification of 19 mycotoxins in human plasma using high performance liquid chromatography-mass spectrometry (triple quadrupole). The studied mycotoxins were: deepoxy-deoxynivalenol, aflatoxins (B1, B2, G1, G2 and M1), T-2 and HT-2, ochratoxins A and B, zearalenone, sterigmatocystin, nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, neosolaniol, diacetoxyscirpenol and fusarenon-X. Sample deproteinization and cleanup were performed in one step using Captiva EMR-lipid (3 mL) cartridges and acetonitrile (with 1% formic acid). The extraction step was simple and fast. Validation was based on the evaluation of limits of detection (LOD) and quantification, linearity, precision, recovery, matrix effect, and stability. LOD values ranged from 0.04 ng/mL for aflatoxin B1 to 2.7 ng/mL for HT-2, except for nivalenol, which was 9.1 ng/mL. Recovery was obtained in intermediate precision conditions and at three concentration levels. Mean values ranged from 68.8% for sterigmatocystin to 97.6% for diacetoxyscirpenol (RDS ≤ 15% for all the mycotoxins). Matrix effects (assessed at three concentration levels and in intermediate conditions) were not significant for most of the mycotoxins and were between 75.4% for sterigmatocystin and 109.3% for ochratoxin B (RDS ≤ 15% for all the mycotoxins). This methodology will be useful in human biomonitoring studies of mycotoxins for its reliability.

Short communication: Analysis of mycotoxins in Spanish milk.

We surveyed the presence of 22 mycotoxins in 191 Spanish cow milk samples. Mycotoxins could be carried over from diet into animal milk and have toxic effects on human and animal health. The interaction of different mycotoxins may be additive or synergetic. Therefore, surveillance of mycotoxin co-occurrence in milk is recommended. Aflatoxins M1, B1, B2, G1, and G2, ochratoxins A and B, nivalenol, deoxynivalenol, deepoxy-deoxynivalenol, 3- and 15-acetyldeoxynivalenol, diacetoxyscirpenol, neosolaniol, fusarenon X, T-2 and HT-2 toxins, fumonisins B1, B2, and B3, sterigmatocystin, and zearalenone were analyzed. Samples were treated by liquid-liquid extraction with acidified acetonitrile, followed by an acetonitrile-water phase separation using sodium acetate. The analysis was carried out by HPLC coupled to a triple quadrupole mass spectrometer. None of the analyzed mycotoxins had a concentration level higher than their detection limit (0.05-10.1 µg/L). The aflatoxin M1 in the samples never exceeded the level established by the European Union.

An LC-MS/MS method for multi-mycotoxin quantification in cow milk.

The simultaneous quantification of 15 mycotoxins in cow milk by liquid chromatography-mass spectrometry, is presented. Extraction was performed with acidified acetonitrile, followed by a cleanup step with sodium acetate. During validation limits of detection (LOD) and quantification, linearity, precision, accuracy, recovery, matrix effect, and stability were studied. LOD values were between 0.02 and 10.14ng/mL for aflatoxins M1, B1, B2, G1, G2, ochratoxins A and B, HT-2 and T-2 toxins, deepoxy-deoxynivalenol, zearalenone, sterigmatocystin and fumonisins B1, B2 and B3. Recovery values were between 82.6 and 94.4% for all the mycotoxins, except for fumonisins. The recovery values for fumonisins were between 42.1% and 64.6%. Matrix effect, between 25.5 and 96.8%, appeared for all of the mycotoxins, especially for deepoxy-deoxynivalenol, zearalenone and sterigmatocystin. The validated method achieves the quantification of those mycotoxins of major concern and mycotoxins that are not frequently studied in milk, such as fumonisins, sterigmatocystin or ochratoxin B.

Development and validation of a high performance liquid chromatographic-mass spectrometry method for the simultaneous quantification of 10 trichothecenes in ultra-high temperature processed cow milk.

An LC-MS/MS (QqQ) method has been developed and validated for simultaneous determination of the following trichothecenes in UHT cow milk: nivalenol (NIV), deoxynivalenol (DON), deepoxy-deoxynivalenol (DOM-1), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), neosolaniol (NEO), diacetoxyscirpenol (DAS), fusarenon X (FUS-X), T-2 and HT-2 toxins. Sample treatment is simple and based on the extraction with acetonitrile (ACN), acidified with 0.2% formic acid, followed by a purification process, adding sodium acetate to the ACN/water extract in order to separate aqueous phase and, consequently, polar components of the milk. Validation of the method for all the 10 mycotoxins was successful; validation parameters taken into account were as follows: limits of detection (LOD) and quantification (LOQ), linearity, precision (within-day and between-day variability), recovery, matrix effect and stability. The LODs were 10.1, 2.5, 1.5, 1.9, 0.1, 0.5, 1.0, 0.08, 0.4 and 0.05ng/mL for NIV, DON, DOM-1, FUS-X, NEO, 3-ADON, 15-ADON, DAS, HT-2 and T-2, respectively. Mean recovery values (obtained in intermediate precision conditions) were between 63.5 and 75.8 (RSDR≤15%) for all the mycotoxins. All the mycotoxins suffered from matrix effects, especially DON.

Role of metal ions on the activity of Mycobacterium tuberculosis pyrazinamidase.

Pyrazinamidase of Mycobacterium tuberculosis catalyzes the conversion of pyrazinamide to the active molecule pyrazinoic acid. Reduction of pyrazinamidase activity results in a level of pyrazinamide resistance. Previous studies have suggested that pyrazinamidase has a metal-binding site and that a divalent metal cofactor is required for activity. To determine the effect of divalent metals on the pyrazinamidase, the recombinant wild-type pyrazinamidase corresponding to the H37Rv pyrazinamide-susceptible reference strain was expressed in Escherichia coli with and without a carboxy terminal. His-tagged pyrazinamidase was inactivated by metal depletion and reactivated by titration with divalent metals. Although Co(2+), Mn(2+), and Zn(2+) restored pyrazinamidase activity, only Co(2+) enhanced the enzymatic activity to levels higher than the wild-type pyrazinamidase. Cu(2+), Fe(2+), Fe(3+), and Mg(2+) did not restore the activity under the conditions tested. Various recombinant mutated pyrazinamidases with appropriate folding but different enzymatic activities showed a differential pattern of recovered activity. X-ray fluorescence and atomic absorbance spectroscopy showed that recombinant wild-type pyrazinamidase expressed in E. coli most likely contained Zn. In conclusion, this study suggests that M. tuberculosis pyrazinamidase is a metalloenzyme that is able to coordinate several ions, but in vivo, it is more likely to coordinate Zn(2+). However, in vitro, the metal-depleted enzyme could be reactivated by several divalent metals with higher efficiency than Zn.

The highly antigenic 53/25 kDa Taenia solium protein fraction with cathepsin-L like activity is present in the oncosphere/cysticercus and induces non-protective IgG antibodies in pigs.

Cathepsin L-like proteases are secreted by several parasites including Taenia solium. The mechanism used by T. solium oncospheres to degrade and penetrate the intestine and infect the host is incompletely understood. It is assumed that intestinal degradation is driven by the proteolytic activity of enzymes secreted by the oncosphere. Blocking the proteolytic activity by an antibody response would prevent the oncosphere penetration and further infection. Serine and cysteine proteases including chymotrypsin, trypsin, elastase, and cathepsin L, are secreted by T. solium and Taenia saginata oncospheres when cultured in vitro, being potential vaccine candidates. However, the purification of a sufficient quantity of proteases secreted by oncospheres to conduct a vaccine trial is costly and lengthy. A 53/25 kDa cathepsin L-like fraction partially purified from T. solium cyst fluid was described previously as an important antigen for immunodiagnostics. In this study we found that this antigen is present in the T. solium oncosphere and is also secreted by the cysticercus. This protein fraction was tested for its ability to protect pigs against an oral challenge with T. solium oncospheres in a vaccine trial. IgG antibodies against the 53/25 kDa cathepsin L-like protein fraction were elicited in the vaccinated animals but did not confer protection.