Development of an Untargeted LC-MS Metabolomics Method with Postcolumn Infusion for Matrix Effect Monitoring in Plasma and Feces.

Untargeted metabolomics based on reverse phase LC-MS (RPLC-MS) plays a crucial role in biomarker discovery across physiological and disease states. Standardizing the development process of untargeted methods requires paying attention to critical factors that are under discussed or easily overlooked, such as injection parameters, performance assessment, and matrix effect evaluation. In this study, we developed an untargeted metabolomics method for plasma and fecal samples with the optimization and evaluation of these factors. Our results showed that optimizing the reconstitution solvent and sample injection amount was critical for achieving the balance between metabolites coverage and signal linearity. Method validation with representative stable isotopically labeled standards (SILs) provided insights into the analytical performance evaluation of our method. To tackle the issue of the matrix effect, we implemented a postcolumn infusion (PCI) approach to monitor the overall absolute matrix effect (AME) and relative matrix effect (RME). The monitoring revealed distinct AME and RME profiles in plasma and feces. Comparing RME data obtained for SILs through postextraction spiking with those monitored using PCI compounds demonstrated the comparability of these two methods for RME assessment. Therefore, we applied the PCI approach to predict the RME of 305 target compounds covered in our in-house library and found that targets detected in the negative polarity were more vulnerable to the RME, regardless of the sample matrix. Given the value of this PCI approach in identifying the strengths and weaknesses of our method in terms of the matrix effect, we recommend implementing a PCI approach during method development and applying it routinely in untargeted metabolomics.

Quantification of 17 Endogenous and Exogenous Steroidal Hormones in Equine and Bovine Blood for Doping Control with UHPLC-MS/MS.

A simple and fast analytical method able to simultaneously identify and quantify 17 endogenous and exogenous steroidal hormones was developed in bovine and equine blood using UHPLC-MS/MS. A total amount of 500 µL of sample was deproteinized with 500 µL of a mixture of methanol and zinc sulfate and evaporated. The mixture was reconstituted with 50 µL of a solution of 25% methanol and injected in the UHPLC-MS/MS triple quadrupole. The correlation coefficients of the calibration curves of the analyzed compounds were in the range of 0.9932-0.9999, and the limits of detection and quantification were in the range of 0.023-1.833 and 0.069-5.5 ppb, respectively. The developed method showed a high sensitivity and qualitative aspects allowing the detection and quantification of all steroids in equine and bovine blood. Moreover, the detection limit of testosterone (50 ppt) is half of the threshold admitted in plasma (100 ppt). Once validated, the method was used to quantify 17 steroid hormones in both bovine and equine blood samples. The primary endogenous compounds detected were corticosterone (range 0.28-0.60 ppb) and cortisol (range 0.44-10.00 ppb), followed by androstenedione, testosterone and 11-deoxycortisol.

Tissue classification by rapid evaporative ionization mass spectrometry (REIMS): comparison between a diathermic knife and CO2 laser sampling on classification performance.

The increasing need for rapid, in situ, and robust tissue profiling approaches in the context of intraoperative diagnostics has led to the development of a large number of ambient ionization-based surface sampling strategies. This paper compares the performances of a diathermic knife and a CO2 laser handpiece, both clinically approved, coupled to a rapid evaporative ionization mass spectrometry (REIMS) source for quasi-instantaneous tissue classification. Several fresh meat samples (muscle, liver, bone, bone marrow, cartilage, skin, fat) were obtained from different animals. Overall, the laser produced cleaner cuts and more reproducible and higher spectral quality signals when compared with the diathermic knife (CV laser = 9-12%, CV diathermic = 14-23%). The molecular profiles were subsequently entered into a database and PCA/LDA classification/prediction models were built to assess if the data generated with one sampling modality can be employed to classify the data generated with the other handpiece. We demonstrate that the correct classification rate of the models increases (+ 25%) with the introduction of a model based on peak lists that are tissue-specific and common to the two handpieces, compared with considering solely the whole molecular profile. This renders it possible to use a unique and universal database for quasi-instantaneous tissue recognition which would provide similar classification results independent of the handpiece used. Furthermore, the laser was able to generate aerosols rich in lipids from hard tissues such as bone, bone marrow, and cartilage. Combined, these results demonstrate that REIMS is a valuable and versatile tool for instantaneous identification/classification of hard tissue and coupling to different aerosol-generating handpieces expands its field of application. Graphical abstract.

MALDI-Mass Spectrometry Imaging to Investigate Lipid and Bile Acid Modifications Caused by Lentil Extract Used as a Potential Hypocholesterolemic Treatment.

This paper reports matrix-assisted laser desorption/ionization mass spectrometry imaging to investigate systematic effects of a lentil extract treatment to lower cholesterol levels. For this purpose, mass spectrometry imaging was used to spatially investigate modifications in the lipid composition and cholesterol levels in the brain, liver, and intestines as well as bile acids in the liver and intestine of rats treated with lentil extract. Neither the lipid composition nor cholesterol levels in the brain samples were found to be significantly different between the treated and not-treated animal groups. The hypercholesterolemic livers showed signs of steatosis (lipid marker PG 36:4), but no modifications in bile acid, cholesterol, and lipid composition. We found significant differences (AUC > 0.75) in the intestines regarding bile acid and lipid composition after treatment with the lentil extract. The treated rats showed a decreased reabsorption (increased excretion) of ursodeoxycholic acid, deoxycholic acid, and chenodeoxycholic acid and an increased deconjugation of taurine-conjugated bile acids (taurochenodeoxycholic acid, taurodeoxycholic acid, taurocholic acid, and 3-keto-taurocholic acid). This indicates that the lentil extract lowers the total cholesterol level in two synergic ways: (i) it increases the excretion of bile acids; hence, new bile acids are produced in the liver from serum cholesterol and (ii) the prebiotic effect leads to free taurine which upregulates the de novo synthesis of bile acid from cholesterol while activating LDL receptors. We demonstrate here that mass spectrometry imaging is a valuable tool for a better understanding of the effects of treatments such as for the synergistic cholesterol-lowering effect of the lentil extract.

Simultaneous quantitation of 9 anabolic and natural steroidal hormones in equine urine by UHPLC-MS/MS triple quadrupole.

A new fast and easy analytical procedure for the simultaneous detection and quantification of 9 anabolic steroids (deslorelin, dexamethasone sodium phosphate, prednisolone, methylprednisolone, stanozolol, boldenone, nandrolone, dexamethasone isonicotinate and altrenogest) in horse urine for doping control have been developed by using the ultra-high-performance liquid chromatography coupled with tandem mass spectrometry technique (UHPLC-MS/MS). A total amount of 400 µl of sample was evaporated, restored and injected in the UHPLC-MS/MS. The proposed method was fully validated showing a recovery higher than 89.12% and a coefficient of variation lower than 6.02%. The correlation coefficients range of the analyzed compound's calibration curves was 0.9955-0.9997, and the limits of detection and quantification were in the range of 0.1 and 0.25 µg/l, respectively.

Detection of endocrine disrupting chemicals and evidence of their effects on the HPG axis of the European anchovy Engraulis encrasicolus.

Natural/synthetic Endocrine Disrupting Chemicals (EDCs) may display estrogenic activity and a lower potency than 17ß-estradiol. Nonetheless, their concentrations and additive effects can affect the endocrine system and reproductive processes related to the Hypothalamic-Pituitary-Gonadal (HPG) axis. Because of their persistence in both the environment and biological systems, they ultimately target multi-level predators, including humans. We detected presence and effects of xenobiotics on wild anchovy Engraulis encrasicolus in the Western Adriatic Sea. Twenty-one PCBs and five organochlorines were detected on the order of ng g-1; vitellogenin, vitellogenin receptor and genes encoding for the zona radiata proteins were evaluated in gonad and/or liver and found transcribed in male specimens; in addition, intersex was histologically identified in the 13% of testis. Our results have developed the understanding of the European anchovy's reproductive toxicological risk and our approach could assist the comprehension of the complex dynamics of commercially relevant Teleost species.

Development and application of a UHPLC-MS/MS method for the simultaneous determination of 17 steroidal hormones in equine serum.

A new, fast and simple analytical method that is able to identify and quantify simultaneously 17 steroid hormones and metabolites (pregnenolone, 17-OH-pregnenolone, progesterone, 17-OH-progesterone, androsterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, cortisol, corticosterone, aldosterone, 11-deoxycortisol, 11-deoxycorticosterone, dihydrotestosterone, estrone and estradiol) has been developed in equine serum using the ultra-high-performance liquid chromatography-tandem mass spectrometry technique. A total of 400 µl of sample was deproteinized with 1000 µl of acetonitrile, evaporated, restored with 50 µl of a solution of 25% methanol and injected in ultra-high-performance liquid chromatography-tandem mass spectrometry triple quadrupole. The recovery percentage obtained by spiking the matrix at two different concentrations with a standard mixture of steroid hormones was in all cases higher than 85.60% and with the percentage of coefficient of variation lower than 8.37%. The range of the correlation coefficients of the calibration curves of the analyzed compounds was 0.9922-0.9986, and the limits of detection and limits of quantification were in the range of 0.002-2 and 0.0055-5.5 ng ml-1 , respectively. The detected limit of quantification for testosterone (i.e. 50 pg ml-1 ) is twofold lower with respect to its threshold admitted in geldings plasma (100 pg ml-1 free testosterone). The high sensitivity and the quantitative aspect of the method permitted to detect most of the steroids in equine serum. Once validated, the method was used to quantify 17 steroid hormones in mare, stallion and gelding serum samples. The main steroids detected were corticosterone (range 37.25-51.26 ng ml-1 ) and cortisol (range 32.57-52.24 ng ml-1 ), followed by 17-OH-pregnenolone, dihydrotestosterone and pregnenolone. Copyright © 2016 John Wiley & Sons, Ltd.