Chromabond sb

Two hundred microliters of plasma (baseline and standing samples) were diluted with double-distilled water (1:2, vol/vol) and loaded on pre-conditioned anion exchange columns (Chromabond SB, Macherey-Nagel, Düren, Germany). After a washing step with double-distilled water nitrite and nitrate were eluted with 1 mL 0.5 mol/L sodium chloride. In the eluates, nitrite and nitrate were determined simultaneously by means of HPLC analysis according to a previously published method (Romitelli et al., 2007 (link)) but with some modifications. Briefly, the HPLC consisted of a L-2200 autosampler, two L-2130 HTA pumps, and a L-2450 diode array detector (all: VWR Hitachi, VWR, Vienna, Austria). Separation was performed on a Hypersil ODS column (5 μm; 250 × 4 mm I.D.) with 10.0 min isocratic elution (buffer A: 0.1 mol/L NaH₂PO₄, pH = 5.5, containing 5.9 mmol/L tetrabutylammonium hydrogensulphate) followed by a linear gradient to 20% buffer B (buffer B: 0.1 mol/L NaH₂PO4, pH = 5.5, containing 5.9 mmol/L tetrabutylammonium hydrogensulphate/acetonitrile, 3:1, vol/vol) within another 10 min. The injection volume of standard and sample solutions was 40 μL. The absorbance at 205 nm was recorded. Data acquisition and subsequent analysis was done with the EZchrom Elite (VWR) program. Retention time was ~7.80 min for nitrite and ~14.5 min for nitrate.

Sample purification was performed by solid phase extraction (SPE) according to the method presented by [35 (link)]. Chromabond SB (Macherey-Nagel, Düren, Germany) polypropylene columns with a bed weight of 500 mg and a capacity of 6 mL were used. The dry extracts were dissolved in 2 mL methanol. Then, 0.5 mL was taken and evaporated to dryness under a stream of nitrogen and next redissolved in 2 mL of methanol, further mixed with 8 mL of deionized water to give a solution of the sample in 20% methanol. The samples were applied to SPE columns and washed with 5 mL of deionized water under normal pressure. Residual water was removed under reduced pressure. Aglycones were eluted from the columns into reaction vessels with 5 mL methanol and evaporated to dryness under a stream of nitrogen.