The compound was eluted with 85%acetonitrile/0.1%% formic acid from Eclipse Plus C18 column (2.1 mm × 100 mm, 3.5 μm, Agilent, USA) at a flow rate of 0.4 ml/min.
The compound was eluted off the resin with 0.5 n aqueous ammonia, and the product-containing fractions (detected with ninhydrin stain on a TLC plate) were combined.
BA identity was further confirmed by MS analysis; BA reference and isolated compound was eluted at the same retention time (10.56 min), and the mass spectral isotopic pattern of isolated BA matches that of reference BA (455.35, 456.35 and 457,35 m/z).
As shown in Fig. 6, the signal for the internal standard is constant up to approximately 4 min of runtime; afterwards it decreases to about 50%% of the initial value at 10-min runtime, when the last mercury compound is eluted.
The compounds were eluted with a gradient elution of mobile phase A (100% acetronitrile) and B (1% acetic acid in deionized water) where A increased from 3%to8%8% in 5 minutes, to 10% by 25 minutes and was maintained at 10% for 20 minutes, then returned to initial condition (3%) in 10 minutes and remained for 5 minutes before next injection.
The extraction solution was pumped into the sample loop and then introduced into the CCC column; the target compounds were eluted with the lower aqueous phase of the two-phase solvent system.
Finally, the upper phase of the solvent system of n-hexane n-butanol methanol water (4.2:1.0:1.2:4.5, v:v:v:v) was used as both the PLE solven-hexane n-butanol methanol watertracted at 125 °C), an-hexane n-butanol methanol watered with the corresponding lower phase of the solvent system to separate the low polar compounds.
At the second stage, the upper phase of the solvent system of ethyl acetate n-butanol methanol water (3.1:0.6:1.0:2.6, v:v:v:v) wacetate n-butanol methanol waterand HSCCC stationary phase (extracted at 110 °C), and the target compounds were eluted with the corresponding lower phase of the solvent system to separate the moderate polar compounds.
The volatile compounds are eluted first under isobaric conditions (1500 psi) in less than a minute followed by a linear gradient of the column back pressure (from 1500 to 3500 psi in 5 min) for the elution of the non-volatile compounds up to C40.
At the first stage, the upper phase of the solvent system of ethyl acetate n-butanol water (1.1:1.0:2.1, v:v:v) wacetate n-butanol water solvent and HSCCC stationary phase, and the target compounds were eluted with the corresponding lower phase of the solvent system to separate the polar common compounds.
Subsequently, the retained compounds were eluted and interrogated using experimental conditions designed to maximize mass spectrometric information content; these conditions included optimized solvent systems, optimized flow rates, chemical manipulation of the sample, extended acquisition time, and other appropriate mass spectral techniques.
