Ctc pal autosampler

Laboratory parameters such as number of platelets, erythrocytes, leucocytes, levels of total cholesterol, triglycerides, asparagine aminotransferase (ASAT), alanine aminotransferase (ALAT), creatinine, urea, C-reactive protein (CRP), glucose, and b-natriuretic peptide (BNP), LV-EF and left ventricular end-diastolic diameter (LVEDD) were measured routinely at the Heart and Diabetes Center NRW in Bad Oeynhausen (patients) and the University Hospital Jena (control). Six analytical classes of metabolites (90 GPs, 40 ACs, 21 AAs, 21 BAs, 15 SMs, and 1 sugar) were measured in EDTA plasma samples and quantified with the AbsoluteIDQ^® p180 kit (Biocrates Life Science AG, Innsbruck, Austria) according to the manufacturer’s protocol. An API4000 liquid chromatography–tandem mass spectrometry (LC–MS/MS) system (AB Sciex, Framingham, MA, USA) was used for measurement. The device was additionally equipped with an electrospray ionization source and a CTC PAL autosampler (CTC Analytics AG, Zwingen, Switzerland) and the Analyst 1.6.2. Software (AB Sciex). Calibration curves, quality controls, and samples were evaluated with the MetIQ software package, which is an integrated part of the used kit. Three replicates of a reference sample served for data normalization on the same plate, and the concentrations were exported for the following statistical analysis.

LC-MS/MS was performed using an API 5000 triple quadrupole mass spectrometer (Sciex), coupled to an Agilent 1200 HPLC system (Agilent Technologies). Samples (20 μL) were injected using a CTC PAL Autosampler set at 4°C (CTC Analytics AG, Switzerland). Analytes were resolved through chromatographic separation using a 4-μm Synergi Hydro-RP C18 column (150×2 mm; Phenomenex, Torrance, CA), with column chamber set at 40°C, over a binary gradient with a flow rate of 0.5 mL/min. HPLC gradient conditions were as follows: 0 min, 25:75 A/B; 2.5 min, 20:80 A/B; 7.5 min, 10:90 A/B; 8 min, 0:100 A/B; 10 min, 25:75 A/B; 18 min, 25:75 A/B. Solvent A: 0.1% formic acid in water; Solvent B: methanol. Total run time was 18 min.
LC-MS/MS was performed in positive ion mode, [M + H]⁺, with quantifier and qualifier ion transitions selected for each analyte, at a dwell time of 50 ms. Source parameters were set as follows: positive ion spray voltage, 5000 V; ion source temperature, 500°C; collision gas, 7 psi; curtain gas, 35 psi; nebulizer gas, 25 psi; turbo gas, 45 psi. Transitions were optimized using direct infusion (10 μL/min) with each standard (100 ng/mL). MS/MS parameters are summarized in Table 1. Data were acquired and processed using Analyst^® (Sciex), version 1.6.2.

The extraction of leaf and rhizome VOCs was carried out following the previously reported HS-SPME methods, with some modifications [79 ]. A 50/30 μm DVB/CAR/PDMS preconditioned solid-phase microextraction (SPME) fiber (Supelco, St. Louis, MO, USA) was used in this study.
Sample powders (1.0 g) was placed into the 20 mL headspace GC glass vials (Agilent Technologies Co., Ltd., Waldbronn, Germany). Thereafter, 0.3 g of NaCl (0.3 g/mL), 1.0 mL of 100 mM EDTA (pH 7.5), and 10 μL of an internal standard (IS) of EPA 524.2 fortification solution (20 μg/mL of fluorobenzene, 4-bromofluorobenzene, and 1,2-dichloro-benzene-d4) in methanol were added. Then, the vials were vortexed for 1 min to homogenize the sample powder with the chemicals. VOCs were extracted for 20 min after incubating for 10 min at 80 °C. After the extraction, samples were placed randomly into the CTC PAL autosampler (CTC Analytics AG, Zwingen, Switzerland).

Plasma L-homoarginine was measured from ethylenediaminetetraacetic acid (EDTA) plasma aliquots stored at −80 °C using electrospray ionization-liquid chromatography-mass spectrometry with a high-throughput mass spectrometric assay. Briefly summarized, by adding 100 µL of internal standard (2.5 µmol/L [¹³C₆]-homoarginine) disbanded in methanol to 25 µL of EDTA plasma, proteins were precipitated. The aliquots were centrifuged, vaporized, and afterwards translated to their butyl ester derivatives applying butanolic 1N hydrochloric acid (HCl). After repeated centrifugation, the eluates were dried and again dissolved in 100 µL of methanol:water (25:75) containing 0.1% ammonium format. The plates were positioned in a CTC PAL autosampler (CTC Analytics AG, Zwingen, Switzerland)) and 20 µL samples were injected. Further testing was performed with the mass spectrometer system (Varian 1200 MS, Agilent Technologies, Santa Clara, CA, USA). Lower threshold of quantification for homoarginine was set to be 0.01 µmol/L. Intra- and interassay coefficients of variation were ≤7.5%. Creatinine was determined by routine laboratory method.

The vitamin D profile and kynurenine pathway were measured in serum by the mass spectrometry method. We estimated the concentration of 25(OH)D₃, 24,25(OH)₂D₃, 3-HK, KYN, KYNA, XANA, and PA. The isotope dilution method by liquid chromatography combined with tandem mass spectrometry technique (LC-MS/MS) was applied. All samples were set up and analyzed with the Eksigent ExionLC analytical HPLC system with a CTC PAL autosampler (CTC Analytics AG, Zwingen, Switzerland) coupled with QTRAP^® 4500 MS/MS system (Sciex, Framingham, MA, USA).