Chromafil xtra pet 45 25

According to Analytica—EBC 7.7 method [47 ] HPLC was employed to determine the α- and β-acids in hop extracts. Extracts were filtered through a disposable syringe filter, Chromafil Xtra PET-45/25 (Macherey-Nagel, Dueren, Germany) and a 10 µL injection loop on the HPLC injector was used. The separation was achieved on the Nucleodur 5–100 C18, 125 × 4 mm HPLC analysis column (Macherey-Nagel, Dueren, Germany). The isocratic mobile phase consisting of distilled water, methanol (J.T.Baker, USA) and 85% aqueous solution of ortophosphoric acid (MERCK, Germany, Taufkirchen) in a ratio of 775/210/9 (v/v/v) was used, and the detection was carried out with a Diode array detector (DAD) set at 314 nm for α-and β-acids and 370 nm for detection of xantohumole, respectively. The quantification was performed by the external standard ICE4 (NATECO2, Wolnzach, Mainburg, Germany) for α-and β-acids and by XH 90% (Steiner Hopfen GmBH, Germany). All solvents were of analytical grade purity.

Bones, rind, subcutaneous fat, and innards were removed from the meat samples. Following mixing, the samples were freeze-dried and ground in a cryomill (SamplePrep6870 Freezer Mill, C3 Process and Analysis Technology GmbH, Haar, Germany). The ground, dry samples were stored in a freezer (−20°C) until use. The meat powder (500 mg) was extracted with 6 mL of water. After the samples were mixed on a test tube shaker (Multi Reax, Heidolph, Schwabach, Germany) for 10 min, samples were centrifuged at 3,000 rpm [relative centrifugal force (RCF), 1,690 x g] for 15 min. The aqueous supernatant was passed through a syringe filter (Chromafil Xtra PET −45/25, Macherey-Nagel, Düren, Germany) into a centrifuge tube. The 3 kDa ultrafiltration filters (Vivaspin^®, Sartorius, Goettingen, Germany) were rinsed three times with 2 mL of water each to remove glycerol [10 min at 3,000 rpm, relative centrifugal force (RCF), 1,690 × g]. After the cleaning step, 800 μL of the meat extract was transferred to the 3 kDa ultrafiltration unit, and samples were centrifuged at 3,000 rpm [relative centrifugal force (RCF), 1690 × g] for 1.5 h. An aliquot of the obtained filtrate (500 μL) was mixed with 250 μL of 3 M sodium dihydrogen phosphate buffer (pH 6) and 75 μL of TSP (dissolved in D₂O). A 600 μL-aliquot of this mixture was transferred to a 5-mm Boro 300-5-8 (Deutero, Bad Kreuznach, Germany) NMR tube.

High-performance liquid chromatography (HPLC) was used to determine α- and β-acids and xanthohumol in the hop according to the Analytica-EBC 7.7 method [54 ]. The separation was achieved on a Nucleodur 5–100 C18, 125 × 4 mm HPLC analysis column (Macherey-Nagel, Düren, Germany) while a 10 µL injection loop on an HPLC injector was used. The isocratic mobile phase constituted distilled water, methanol (Sigma-Aldrich, Taufkirchen, Germany), and 85% aqueous solution of ortophosphoric acid (Sigma-Aldrich, Germany) in a ratio of 775/210/9 (v/v/v). The detection was carried out with a diode array detector (DAD) set at 314 nm for α- and β-acids (Figure 1) and 370 nm for xanthohumol (Figure 2). The quantification was performed according to the external standard ICE4 (Labor Veritas, Zürich, Switzerland). All solvents were of analytical grade purity.
Just before the analysis, extraction solutions were volumetrically diluted by a factor of 10 with a mobile phase and filtered through disposable syringe filters, Chromafil Xtra PET-45/25 (Macherey-Nagel, Germany).