Separation and determination of structurally related free bile acids by pressurized capillary electrochromatography coupled to laser induced fluorescence detection

Abstract

A sensitive pressurized capillary electrochromatography-laser induced fluorescence detection (pCEC-LIF) method has been developed for the simultaneous analysis of five structurally related free bile acids including cholic acid (CA) and lithocholic acid (LCA), as well as the stereoisomers of chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA). 4-nitro-7-N-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was used for the precolumn derivatization of non-chromogenic free bile acids to form strongly fluorescent adducts. Efficient separation of these derivatization products was performed within 17 min by isocratic elution pCEC on an octadecyl silica (ODS) packed capillary column, using a mobile phase consisting of acetonitrile and 10 mM CAPS buffer (pH 8.0) (60 : 40 v/v), 20 kV of applied voltage and 10.8 MPa of supplementary pressure. The derivatized bile acids could be determined by a LIF detector at an excitation wavelength of 473 nm and an emission wavelength of 530 nm, with detection limits (signal/noise ¼ 3) down to 2 nmol L À1 . Applicability of this pCEC-LIF method to the analysis of human serum samples was demonstrated. Accepted recoveries ranging between 94.8% and 109.7% for all analytes in spiked human serum samples were achieved. This method has potential for the trace analysis of physiologically important acidic analogs.

Analysis of bile acids in human biological samples by microcolumn separation techniques: A review

“…In addition, Wu et al. [87] developed a method for the simultaneous analysis of five free serum BAs by pressurized capillary electrochromatography coupled to LIF. The carboxyl group of BAs was derivatized with 4‐nitro‐7‐ N ‐piperazino‐2,1,3‐benzoxadiazole prior to analysis.…”

“…The analysis of 15 BAs was performed under 12 min, which is comparable or faster than the previously mentioned HPLC-MS methods (see Table 1). In addition, Wu et al [87] developed a method for the simultaneous analysis of five free serum BAs by pressurized capillary electrochromatography coupled to LIF. The carboxyl group of BAs was derivatized with 4-nitro-7-N-piperazino-2,1,3-benzoxadiazole prior to analysis.…”

An underestimated technique. Does pressurized and pressure-assisted capillary electrochromatography have potential in drug and pharmacological-active compounds analysis?

“…Another product designed for pCEC is the 7100 CE System from Agilent. This also offers the advantage of several operation modes using one instrument [16].…”

Recent advances in development and application of derivatization reagents having a benzofurazan structure: a brief overview

“…After trypsin digestion, the generated DAABD-peptides were analyzed by HPLC-FL and LC-tandem mass spectrometry (LC/MS/MS; Koshiyama and Imai, 2010). As the benzofurazan structure is moderately hydrophobic and DAABD-F has a dimethylamino group as an ionization moiety, DAABD-labeled compounds were suitable for analysis by electrospray ionization-MS. NBD-PZ (4-nitro-7-piperazino-2,1,3-benzoxadiazole) was used for the analysis of carboxylic acids such as bile acids (Wu et al, 2010) and D-lactate (Lin et al, 2011). DBD-ED [4-(N,Ndimethylaminosulfonyl)-7-(2-aminoethylamino)-2,1,3-benzoxadiazole] was used for the analysis of N-acetylaspartic acid (Song et al, 2012b), N-acetylneuraminic acid (NANA), and 4-(acetylamino)-2,4-dideoxy-D-glycero-D-galacto-octonic acid (ADOA) (Ota et al, 2013).…”