Oasis mcx

Immediately after collection, the anthers were frozen in liquid nitrogen, lyophilized and homogenized while still frozen. Then, 50 mg of pulverized plant material was used for each sample. Auxs were extracted with a mixture of methanol/water/formic acid (15/4/1; v/v/v) according to Dobrev and Kamınek (2002 (link)) with modifications by Stefancic et al. (2007 (link)). Internal isotopic standard mixture consisting of deuterated IAA and KIN labelled with nitrogen ¹⁵N was added to each sample. This extract was fractionated with SPE columns Oasis MCX (Waters). Peak area of each compound was divided by peak area of appropriate internal standard, and thus transformed data were used for calibration table and for quantitation, thereby the efficiency of the process was automatically corrected.

SPE purification of the compounds was achieved using Oasis MCX cation exchange cartridges from Waters (Milford, MA, USA), following the slightly modified protocol of Hartmann et al. [7 (link)]. The cartridges were conditioned with one column volume of methanol and water each, before applying the aqueous sample solution in a concentration that did not exceed 10% of the sorbent mass. After washing the columns with two volumes of water, elution of the MAAs was achieved by flushing with two volumes of 5% acetic acid in water.

To 50 µl cell culture supernatants, 4 nmol N^G-monomethyl-L-arginine (L-NMMA) were added as internal standard, then supplemented with 0.9 ml PBS (pH 6.9) and applied to an Oasis MCX ion exchange column (Waters, Eschborn, Germany). The column was washed with 1 ml each, 0.1 N HCl and methanol, and subsequently cationic amino acids (CAA) were eluted with 1 ml methanol:water: 25% NH₃ (5:4:1), vacuum dried and resuspended in 0.2 ml sodium borate buffer (0.5 mol/l, pH 9.6). L-arginine levels were determined in cell culture supernatants by High Performance Liquid Chromatography (HPLC) using precolumn derivation, exactly as described before (20 (link), 21 (link)).

Frozen samples (ca 10 mg FW) were homogenized and extracted with cold (−20 °C) methanol/water/formic acid (15/4/1, v/v/v) as described in Dobrev and Kaminek [16 (link)] and Dobrev and Vankova [17 (link)]. The following isotope-labelled internal standards (10 pmol/sample) were then added ²H₅-tZ, ²H₅-tZR, ²H₅-tZRMP, ²H₅-tZ7G, ²H₅-tZ9G, ²H₅-tZOG, ²H₅-tZROG, ²H₃-DZ, ²H₃-DZR, ²H₃-DZ9G, ²H₃-DZRMP, ²H₇-DZOG, ²H₆-iP, ²H₆-iPR, ²H₆-iP7G, ²H₆-iP9G, ²H₆-iPRMP (Olchemim, CR, Olomouc, Czech Republic). Phytohormones were separated with a reverse phase-cation exchange SPE column (Oasis-MCX, Waters, Milford, MA, USA) into the acid fraction by elution with methanol and into the basic fraction by elution with 0.35 M NH₄OH in 60% methanol which was used for CK determination. The latter fraction was analyzed using HPLC (Ultimate 3000, Dionex, Sunnyvale, CA, USA) coupled to 3200 Q TRAP hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems, Waltham, MA, USA). Hormone quantification was performed by the isotope dilution method with multilevel calibration curves (r² > 0.99). Data processing was performed with the Analyst 1.5 software package (Applied Biosystems, Waltham, MA, USA).

Chemicals such as acetonitrile, glacial acetic acid, dimethylsulfoxide, formic acid, methanol, ammonium hydroxide, porcine insulin, bovine insulin, and [[13C6] Leu26, 30]-C-peptide (human) were obtained from Sigma (Schnelldorf, Germany). All aqueous buffers and solutions were prepared in purified water (MilliQ quality, Frankfurt, Germany). Recombinant human insulin was obtained from Aventis (Frankfurt, Germany). The labeled insulin (internal) standard [[2H10] LeuB6, B11, B15, B17]-insulin (human) was purchased from PeptaNova (Sandhausen, Germany). The used solid-phase extraction cartridges OASIS MCX (30 mg, 3 mL) were from Waters (Eschborn, Germany), and the synthetic insulin analogs lispro (Humalog), aspart (Novorapid), glulisine (Apidra), and insulin degludec (Tresiba) were supplied by Eli Lilly (Indianapolis, IN, USA), Novo Nordisk (Princeton, NJ, USA), and Aventis (Kansas City, MO, USA), respectively. The glargine metabolite (DesB31-32 glargine) was obtained from IBA (Warsaw, Poland).

Endogenous serum concentrations of ANG I and ANG II were measured by ultra-performance liquid chromatography with tandem mass spectrometry detection, capable of measuring angiotensin peptide levels as low as 10 pg/mL (inVentiv Health Clinique, Quebec City, Quebec, Canada). Following rapid thawing of the serum, samples were stabilized with a combination of aliskiren, pepstatin A, and o-phenanthroline in acidified dimethyl sulfoxide combined with a mixture of EDTA and 4-(hydroxymercury) benzoic acid in phosphate-buffered saline. All samples were spiked with stable-isotope-labeled internal standards for ANG I and ANG II at a concentration of 50 pg/mL. Following protein precipitation using acetonitrile with 1% formic acid and solid-phase extraction (Oasis MCX; Waters Corporation, Milford, MA, USA) of the supernatant, samples underwent liquid chromatography-tandem mass spectrometry analysis using a reverse-phase analytical column (Acquity CSH C18; Waters Corporation) operating in line with an XEVO TQ-S triple quadrupole mass spectrometer (Waters Corporation) in multiple reaction monitoring. The sum of the signal from three different mass transitions per peptide was measured, and angiotensin concentrations were calculated by relating the ratio of peptide signal to internal standard signal.