Prostate Cancer Patients-Negative Biopsy Controls Discrimination by Untargeted Metabolomics Analysis of Urine by LC-QTOF: Upstream Information on Other Omics.

The existing clinical biomarkers for prostate cancer (PCa) diagnosis are far from ideal (e.g., the prostate specific antigen (PSA) serum level suffers from lack of specificity, providing frequent false positives leading to over-diagnosis). A key step in the search for minimum invasive tests to complement or replace PSA should be supported on the changes experienced by the biochemical pathways in PCa patients as compared to negative biopsy control individuals. In this research a comprehensive global analysis by LC-QTOF was applied to urine from 62 patients with a clinically significant PCa and 42 healthy individuals, both groups confirmed by biopsy. An unpaired t-test (p-value < 0.05) provided 28 significant metabolites tentatively identified in urine, used to develop a partial least squares discriminant analysis (PLS-DA) model characterized by 88.4 and 92.9% of sensitivity and specificity, respectively. Among the 28 significant metabolites 27 were present at lower concentrations in PCa patients than in control individuals, while only one reported higher concentrations in PCa patients. The connection among the biochemical pathways in which they are involved (DNA methylation, epigenetic marks on histones and RNA cap methylation) could explain the concentration changes with PCa and supports, once again, the role of metabolomics in upstream processes.

Study of exhaled breath condensate sample preparation for metabolomics analysis by LC-MS/MS in high resolution mode.

Metabolomic analysis of exhaled breath condensate (EBC) requires an unavoidable sample preparation step because of the low concentration of its components, and potential cleanup for possible interferents. Sample preparation based on protein precipitation (PP), solid-phase extraction (SPE) by hydrophilic and lipophilic sorbents or lyophilization has demonstrated that the analytical sample from the last is largely the best because lyophilization allows reconstitution in a volume as small as required (preconcentration factors up to 80-times with respect to the original sample), thus doubling the number of detected compounds as compared with the other alternatives (47 versus 25). In addition, PP and/or SPE cleanup are unnecessary as no effect from the EBC components removed by these steps appears in the chromatograms. The total 49 EBC compounds tentatively identified and confirmed by MS/MS in this research include amino acids, fatty acids, fatty amides, fatty aldehydes, sphingoid bases, oxoanionic compounds, imidazoles, hydroxy acids and aliphatic acyclic acids.

Stable isotopic internal standard correction for quantitative analysis of hydroxyeicosatetraenoic acids (HETEs) in serum by on-line SPE-LC-MS/MS in selected reaction monitoring mode.

The influence of the inclusion of a stable isotopic labeled internal standard (SIL-IS) on the quantitative analysis of hydroxyeicosatetranoic acids (HETEs) in human serum is evaluated in this research. A solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) platform, one of the preferred approaches for targeted analysis of biofluids through the selected reaction monitoring (SRM) operational mode, was used to determine HETEs. These compounds were chosen as targeted metabolites because of their involvement in cardiovascular disease, cancer and osteoporosis. 15HETE-d8 was chosen as internal standard to evaluate matrix effects. Thus, the physico-chemical properties of the SIL-IS were the basis to evaluate the analytical features of the method for each metabolite through four calibration models. Two of the models were built with standard solutions at different concentration levels, but one of the calibration sets was spiked with an internal standard (IS). The other two models were built with the serum pool from osteoporotic patients, which was spiked at different concentrations with the target analytes. In this case, one of the serum calibration sets was also spiked with the IS. The study shows that the IS allowed noticeable correction of matrix effects for some HETE isomers at certain concentration levels, while accuracy was decreased at low concentration (15ng/mL) of them. Therefore, characterization of the method has been properly completed at different concentration levels.

LC-MS/MS quantitative analysis of paclitaxel and its major metabolites in serum, plasma and tissue from women with ovarian cancer after intraperitoneal chemotherapy.

A method for determination of the antineoplastic drug paclitaxel and its main metabolites (viz. 6α-hydroxypaclitaxel and p-3'-hydroxypaclitaxel) at the sub-ng/ml level is here presented. Sample preparation consisted of a liquid-liquid extraction step for cleanup and preconcentration of the target analytes prior to chromatographic analysis by tandem mass spectrometry detection (LC-ESI-MS/MS). The determination step was optimized by selected reaction monitoring (SRM) mode for highly selective identification and sensitive quantitation of paclitaxel and its metabolites in human serum, plasma and tissue. The detection limits were in the range 0.03-0.15ng/ml for serum and 0.07-0.62ng/g for tissue, with intra-day variability range from 0.5 to 2.7%, expressed as relative standard deviation. The method was applied to determine paclitaxel and its metabolites in serum and tissue from 13 women suffering from ovarian peritoneal carcinomatosis, after hyperthermic intraperitoneal intraoperative chemotherapy (HIPEC) treatment. The method reported here can be considered a suited tool to monitor the concentration of this drug in patients subjected to HIPEC as strategy to evaluate the toxicity and efficiency of this treatment.