1290 infinity binary lc system

Blood collection took place between 8:00 and 10:00 am, and serum was extracted from the samples. LH was measured with CLIA using the automated platform Advia-Centaur XPi® (Siemens Inc., NY). The lowest level of quantification (LLOQ) was 0.12 U/L, and according to the manufacturer the intra-assay coefficient of variation ranged between 2,3% and 3.0%, and the inter-assay coefficient of variation between 1,5% and 2.9%. Testosterone was also measured with CLIA, using the automated platform Advia-Centaur XPi® (Siemens Inc., NY). The LLOQ was 10 ng/dL, and according to the manufacturer, the intra-assay coefficient of variation ranged between 2.3% and 6.2%, and the inter-assay coefficient of variation between 2.7% and 6.9% (Manufacturer information: 10629910_ES Rev. U, 2014–08, 1–18). Testosterone was also measured with LC MSMS in the validation cohort. An ultra high-pressure liquid chromatography with the 1290 Infinity Binary LC System (Agilent Technologies, Santa, CA) was performed. The system is connected in parallel to a tandem mass spectrometry using the 6430 Series Tripe Quadruple LC/MS System (Agilent Technologies, Santa Clara, CA). The LLOQ was 2 ng/dL and thee intra-assay coefficient of variation ranged from 4% to 5% and the inter-assay coefficient of variation between 7% and 8%, at levels of ST below 50 ng/dL [13 (link)].

For the cytokinin analysis of the fertile or sterile fruit of Ler and ms1 plants, ∼10 mg of fresh-weight material was used per sample (n = 5). Samples were extracted in modified Bieleski buffer (methanol/water/formic acid, 15/4/1 [v/v/v]) with a mixture of stable isotopically labeled internal standards added to each sample for precise quantification (Hoyerová et al., 2006 (link)). The purification of isoprenoid cytokinins (CKs) was carried out according to Dobrev and Kamínek (2002) (link) using the MCX column (30 mg of C18/SCX combined sorbent with cation-exchange properties). Analytes were eluted by two-step elution using a 0.35-M NH₄OH aqueous solution and 0.35-M NH₄OH in a 60% MeOH (v/v) solution. Samples were afterward evaporated to dryness under vacuum at 37°C. Prior to analysis, the samples were dissolved in 40-µL 10% MeOH (v/v). Mass spectrometric analysis and quantification were performed using an ultra-high-performance liquid chromatography–tandem mass spectrometry system consisting of a 1290 Infinity Binary LC System coupled to a 6490 Triple Quad liquid chromatography/mass spectrometry (LC/MS) System with Jet Stream and Dual Ion Funnel technologies (Agilent Technologies, Santa Clara, CA, USA). Ultra-high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry method parameters were adapted from (Svačinová et al., 2012 (link)).

Peptide surrogates of analytical grade (>95% purity) were obtained from JPT (Berlin, Germany) and TAGC (Copenhagen, Denmark). The peptide digests were spiked with stable-isotope labeled (SIL) peptides (Supplementary Table S7). Four μg of peptides from the peptide digests containing the SIL peptide were separated on a C18 reverse-phase column (Acquity UPLC BEH C18 1.7 µm, 2.1 × 100 mm) using a 19 min gradient of water and acetonitrile with 0.1% (v/v) formic acid in an Agilent 1290 Infinity binary LC system. Three mass transitions per peptide were quantified using the scheduled multiple reaction monitoring mode in a QTRAP 6500 mass spectrometer (AB Sciex, Framingham, MA, USA) in electrospray positive mode. Mass transitions are listed in Supplementary Table S7. The calibration curve was linear throughout the calibration range with a correlation coefficient (r) higher than 0.99. Quality controls were run between every eight samples and a maximal divergence of less than 15% from the actual concentration was observed. The lower limit of quantification was 0.1 fmol/μg total protein. Targeted proteomic data was analyzed using Analyst 1.6.2 and MultiQuant 3.0.

Determination of 25‐hydroxycholesterol in cell culture supernatant and BALF was performed based on mass spectrometric methods previously described for different instrumentation (Honda et al, 2009; Huang et al, 2014). 25‐hydroxycholesterol was derivatized, and the product was analyzed using ultra‐high pressure liquid chromatography (UHPLC) coupled with high‐resolution time‐of‐flight mass spectrometry (LC‐HRTOF‐MS). UHPLC separation was performed on a 1290 Infinity Binary LC‐System using an Eclipse C‐18, 1.8 μm, 50 × 2.1 mm I.D. analytical column (both from Agilent Technologies). Mass spectrometric detection was accomplished on a Citius™ High Resolution multi‐reflection time‐of‐flight mass spectrometer (LC‐HRT, Leco).

Leaf samples were extracted, purified, and analyzed according to method described in Šimura et al.^[48] Mass spectrometry analysis of D6‐ABA and ABA was performed by an UHPLC‐ESI‐MS/MS system comprising of a 1290 Infinity Binary LC System coupled to a 6490 Triple Quad LC/MS System with Jet Stream and Dual Ion Funnel technologies (Agilent Technologies, Santa Clara, CA, USA). The quantification was carried out in Agilent MassHunter Workstation Software Quantitative (Agilent Technologies, Santa Clara, CA, USA).