4000 qtrap system

Dried blood spots were used in the assay of metabolomics, which were prepared from capillary whole blood through 8-h fasting. We measured the metabolites by direct infusion mass spectrometry technology equipped with the AB Sciex 4000 QTrap system (AB Sciex, Framingham, MA, USA). High-purity water and acetonitrile were purchased from Thermo Fisher (Waltham, MA, USA), and utilized as diluting agent and mobile phase. 1-Butanol and acetyl chloride were obtained from Sigma-Aldrich (St Louis, MO, USA). Isotope-labeled internal standard samples of amino acids (NSK-A) were purchased from Cambridge Isotope Labo-ratories (Tewksbury, MA, USA), while standard samples of the leucine were purchased from Chrom Systems (Grafelfing, Germany). In brief, 8.5 mL of venous blood was drawn from each participant at 08:00 to 09.30 h in the morning after 8-h fasting. Laboratory tests were carried out at a special diagnostic laboratory. The level of lipid profiles was analyzed by an automatic biochemistry analyzer (Hitachi 7150, Tokyo, Japan). We also assayed the level of HDL-C and LDL-C by the selective solubilization method (12 (link)) (Determiner L-HDL, LDL test kit; Kyowa Medex, Tokyo, Japan).

Details of the metabolomics assessment method were published previously (22 (link)). Briefly, 8 h of fasting blood sample was collected at admission. A total of 23 amino acids, i.e., alanine (Ala), asparagine (Asn), leucine (Leu), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), valine (Val), arginine (Arg), glycine (Gly), proline (Pro), threonine (Thr), citrulline (Cit), glutamine (Gln), histidine (His), lysine (Lys), methionine (Met), serine (Ser), ornithine (Orn), glutamate (Glu), aspartate (Asp), piperamide (Pip), cysteine (Cys), Homocysteine (Hcy) were detected via LC-MS. AB Sciex 4000 QTrap system (AB Sciex, Framingham, MA, USA) was used to conduct direct injection MS metabolomic analysis. Analyst v1.6.0 software (AB Sciex) was used for data collection. ChemoView 2.0.2 (AB Sciex) was used for data preprocessing. Isotope-labeled internal standard samples were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Standard samples of the amino acids were purchased from Chrom Systems (Grafelfing, Germany).

Venous blood samples were collected by finger puncture after 8 h fasting and stored as dried blood spots. Experimental determination was taken at room temperature. Acylcarnitine was extracted from blood samples using the Millipore MultiScreen HV 96-well plate (Millipore, Billerica, MA, USA). Filtrate after extraction and quality control solutions were dried by pure nitrogen gas at 50°CC. Acylcarnitine quality control standards were purchased from Chromsystems (Grafelfing, Germany). Dried samples were dissolved by acetonitrile aqueous solution for final acylcarnitine assays. The assays were performed using an AB Sciex 4000 QTrap system (AB Sciex, Framingham, MA, USA). Acylcarnitine was scanned under positive mode. Analyst v1.6.0 software was applied for the system control and acylcarnitine data collection. Further data preprocessing is carried out through ChemoView 2.0.2 (AB Sciex).

The direct injection MS was used for quantitative metabolomic analysis on an AB Sciex 4000 QTrap system (AB Sciex, Framingham, MA) coupled with an electrospray ionization source, and the MS analysis was conducted under positive mode. A sample volume of 20 μL was injected into the system. The 80% acetonitrile aqueous was used as mobile phase. An initial flow rate was set to be 0.2 mL/min. Flow rate was decreased to 0.01 mL/min within 0.08 min, and remained stable until 1.5 min. Subsequently, the flow rate reverted back to 0.2 mL/min within 0.01 min, and maintained for 0.5 min. MS parameters were set as follows: ion spray voltage 4.5 kV, curtain gas pressure 20 psi, auxiliary gas temperature 350 °C. Sheath and auxiliary gas pressure was set at 35 psi. The scan modes and scan parameters were referred to previous report^{19 (link)}. Analyst 1.6.0 software (AB Sciex) was applied to control system, align spectrum, and collect MS data. ChemoView 2.0.2 (AB Sciex) was used for absolute quantification purposes.

The metabolomics assay method had been described in previous studies (15 (link)). Briefly, dry blood spot samples collected by finger puncture after 8 h fasting were used the metabolomics assay. The metabolomics in dry blood spots were measured using mass spectrometry (MS) technology. The MS metabolomic analysis was conducted using an AB Sciex 4000 QTrap system (AB Sciex, Framingham, MA, USA). Electrospray ionization source was ion source. The ion spray voltage was 4.5 kV. Positive mode was performed to scan analytes. The mobile phase which carried the component to be tested was 80% acetonitrile aqueous solution. Isotope-labeled internal standards of acylcarnitine from Cambridge Isotope Laboratories (Tewksbury, MA, USA) were used for absolute quantification.