Quantitative analysis of amino acids and acylcarnitines combined with untargeted metabolomics using ultra-high performance liquid chromatography and quadrupole time-of-flight mass spectrometry.

Metabolomics is an "omic" technique being increasingly used in epidemiological and clinical studies. We developed a method combining untargeted metabolomics with the quantitative determination of eight amino acids (AA) and eight acylcarnitines (AC) in plasma using ultra-high pressure liquid chromatography (UHPLC), electrospray ionization (ESI) and quadrupole time-of-flight mass spectrometry (QTOFMS). Separation of metabolites is performed by ion-pair reverse phase UHPLC using a HSS T3 column (2.1×100mm, 100Å, 1.8μm particle size) and formic acid-ammonium acetate-heptafluorobutyric acid in water and formic acid-ammonium acetate in methanol as mobile phases. Metabolite identification and quantification are achieved using a QTOFMS operating in ESI-positive and full-scan mode along with MS(E) acquisition of fragmentation patterns. Targeted metabolites are quantified using the appropriate labeled standards and include branched-chain AA (leucine, isoleucine, valine), aromatic AA (phenylalanine, tyrosine) as well as acetylcarnitine and propionylcarnitine, which have been identified as biomarkers of future cardiometabolic disease risk. The inter-day precision (relative standard deviation) for the targeted method was <15% for all but one metabolite and accuracy (bias) of amino acids ranged from 0.5% to 13.9% using SRM 1950 as the external standard. Untargeted metabolomics in 30 plasma samples from the general Canadian population revealed 5018 features, of which 48 metabolites were identified using the MZmine 2.19 software including 23 by our in-house library that comprises 671 annotated metabolites. SRM 1950 analysis revealed 11,684 features, among which 154 metabolites were identified. Our method is currently applied in several epidemiological studies to better characterize cardiometabolic diseases and identify new biomarkers for disease prevention.

Roy C ，Tremblay PY ，Bienvenu JF ，Ayotte P ... -  《-》

Pseudotargeted metabolomics method and its application in serum biomarker discovery for hepatocellular carcinoma based on ultra high-performance liquid chromatography/triple quadrupole mass spectrometry.

Chen S ，Kong H ，Lu X ，Li Y ，Yin P ，Zeng Z ，Xu G ... -  《-》

A quantitative method for acylcarnitines and amino acids using high resolution chromatography and tandem mass spectrometry in newborn screening dried blood spot analysis.

We have developed a high resolution liquid chromatographic separation with electrospray ionization (ESI) mass spectrometry detection for the combined analysis of twelve acylcarnitines and seven amino acids commonly measured in newborn screening heritable metabolic disorders. Samples were prepared by punching 3.2 mm disks out of dried blood spots and extracting with a mixture of methanol and 0.1% formic acid containing stable isotopically labeled internal standards. Analysis was performed on an UHPLC system using a HILIC amide, 2.1 mm × 50 mm, 1.7 μm column. A normal phase gradient, employing 10mM ammonium acetate in 90:10 acetonitrile/water for mobile phase B and 0.1% formic acid in water for mobile phase A, was used. Optimized multiple reaction monitoring (MRM) was used for detection of amino acids and acylcarnitines on a Waters Premier mass spectrometer. Quantification of analytes was performed using internal calibration by fortification of sodium heparin whole blood with analytes at appropriate levels to encompass the range around the reported cut-off values. The method was fully validated with respect to precision, accuracy, recovery, linearity, matrix suppression and extraction robustness. Precision and accuracy were evaluated over 3 days and determined to be acceptable with an overall precision within 10% and accuracy within 15% of theoretical for all analytes except for acetylcarnitne at one fortified level, which quantitated 21.8% lower than the expected value. This method is suitable as a second-tier test for newborn screening of specific disorders associated with abnormal levels of acylcarnitines and amino acids, potentially reducing false positive cases and shortening the time to diagnosis.

Miller JH 4th ，Poston PA ，Karnes HT 《-》

An improved pseudotargeted metabolomics approach using multiple ion monitoring with time-staggered ion lists based on ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Pseudotargeted metabolomics is a novel strategy integrating the advantages of both untargeted and targeted methods. The conventional pseudotargeted metabolomics required two MS instruments, i.e., ultra-high performance liquid chromatography/quadrupole-time- of-flight mass spectrometry (UHPLC/Q-TOF MS) and UHPLC/triple quadrupole mass spectrometry (UHPLC/QQQ-MS), which makes method transformation inevitable. Furthermore, the picking of ion pairs from thousands of candidates and the swapping of the data between two instruments are the most labor-intensive steps, which greatly limit its application in metabolomic analysis. In the present study, we proposed an improved pseudotargeted metabolomics method that could be achieved on an UHPLC/Q-TOF/MS instrument operated in the multiple ion monitoring (MIM) mode with time-staggered ion lists (tsMIM). Full scan-based untargeted analysis was applied to extract the target ions. After peak alignment and ion fusion, a stepwise ion picking procedure was used to generate the ion lists for subsequent single MIM and tsMIM. The UHPLC/Q-TOF tsMIM MS-based pseudotargeted approach exhibited better repeatability and a wider linear range than the UHPLC/Q-TOF MS-based untargeted metabolomics method. Compared to the single MIM mode, the tsMIM significantly increased the coverage of the metabolites detected. The newly developed method was successfully applied to discover plasma biomarkers for alcohol-induced liver injury in mice, which indicated its practicability and great potential in future metabolomics studies.

Wang Y ，Liu F ，Li P ，He C ，Wang R ，Su H ，Wan JB ... -  《-》

Determination of carnitine and acylcarnitines in plasma by high-performance liquid chromatography/electrospray ionization ion trap tandem mass spectrometry.

A high-performance liquid chromatography/mass spectrometry method was developed for the determination of carnitine, its biosynthetic precursor butyrobetaine, and eight acylcarnitines in plasma. The procedure includes a solid-phase extraction for carnitine and short- and medium-chain acylcarnitines, and a liquid-liquid extraction for protein-bound long-chain acylcarnitines, followed by separation on a reversed-phase column in the presence of a volatile ion-pairing reagent. Detection was achieved using an ion-trap mass spectrometer run in the tandem mass spectrometry (MS/MS) mode. The choice of the matrix for calibrators, used for quantification of these endogenous compounds, was also investigated. Validation was performed for standard quality controls diluted with 4% bovine serum albumin solution and for spiked plasma quality control samples at concentrations between 0.5 and 80 micromol/L, depending on the compound. Intra- and inter-day precisions for the determination of carnitine were below 3.4% and accuracies were between 95.2 and 109.0%. Application of the method to the diagnosis of pathological acylcarnitine profiles of metabolic disorders in a patient suffering from methylmalonic aciduria is presented. The method allows quantification of carnitine, butyrobetaine, acetylcarnitine and propionylcarnitine, and semiquantitative analysis of medium- and long-chain acylcarnitines. In contrast with other methods, no derivatization step is needed.

Vernez L ，Wenk M ，Krähenbühl S 《-》