Yimin
Wu
a,
Xiaochun
Wang
a,
Qingzheng
Wu
a,
Xiaoping
Wu
*a,
Xucong
Lin
a and
Zenghong
Xie
*ab
aCollege of Chemistry and Chemical Engineering, Fuzhou University, 350108, China. E-mail: wapple@fzu.edu.cn
bXiamen Huaxia Vocational College, Xiamen, 361024, China. E-mail: zhxie@fzu.edu.cn
First published on 15th October 2010
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 = 3) down to 2 nmol L−1. 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.
Accurate measurements of individual BAs eliciting different physiological and pathological responses in vivo are of great importance for clinical and biological research, however, the analysis of BAs in human body fluids is a challenging task. The BAs are usually present in body fluids at micromolar levels and have very poor absorption in UV or visible regions. Moreover, the structural differences between individual BAs based on a steroidal backbone are too small to be separated by conventional separation methods. The major BAs in humans are derivatives of 5β-cholan-24-oic acids. As shown in Fig. 1, the primary BAs cholic acid (CA), CDCA and the secondary BAs deoxycholic acid (DCA), lithocholic acid (LCA) and UDCA only differ in the degree and pattern of hydroxylation, and even contain stereoisomers (diastereomers), i.e. UDCA, DCA, CDCA.7 Chromatographic techniques represent the method of choice for detailed analysis of BAs profiles. Several chromatographic techniques have been employed for BAs determination in biological fluids,7,8 mainly based on thin layer chromatography (TLC),9 gas chromatography (GC),7,10 high performance liquid chromatography (HPLC)8,11–15 and micro column high performance liquid chromatography (μHPLC).16 HPLC is still the most frequently used method for the analysis of BAs due to its outstanding resolving power, even though it usually requires gradient elution and a large volume of sample and solvent. Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC), which combine high efficiency and low sample and solvent consumption, have also been applied for BAs separation in biological fluids.4,7,17 In most cases, cyclodextrins (CD) were needed to improve the resolution of the structurally related BAs, especially for the stereoisomers of UDCA, DCA and CDCA.
Fig. 1 Chemical structure of free bile acids (BAs) included in the study. |
Capillary electrochromatography (CEC), a relatively new miniaturized separation technique that combines the advantages of HPLC and CE,18 has proven to be a powerful tool for analogs separation,19–21 including animal BAs.22 Pressurized CEC (pCEC) is a new CEC mode by applying supplementary pressure on the CEC column to overcome bubbles formation and column drying-out problems,23 which always hinder the repeatability and the extensive application of the CEC technique. In such mode, the mobile phase is driven by both electroosmotic flow (EOF) and pressure, which results in adjustable selectivity, faster elution and less bubble formation than pure EOF-driven CEC. Like other capillary techniques, pCEC is very useful in situations when an efficient separation is needed for the analytes presented in samples with very small amounts, but it also suffers from low concentration sensitivity due to a minute sample volume and limited optical path length when coupled with UV detection. Recently, several selective and sensitive detection methods, such as amperometric detection,24 chemiluminescence detection,25 mass spectrometry,23 laser induced fluorescence detection (LIF),26 nuclear magnetic resonance,27 have been successfully coupled with pCEC for fulfilling the demand of detection sensitivity of various real samples. The hyphenation of pCEC to LIF that fuses the merits of high separation selectivity and high detection sensitivity, have already been developed for the analysis of pollutants,28 pharmaceuticals,29 and amino acids.30 Ultra-trace levels (nmol L−1) of detection limits could be achieved for the analogs with the pCEC-LIF method, which provides a potential powerful separation tool for the analysis of endogenous biological constituents in biological and clinical applications. So far, nearly all CEC/pCEC methods coupled with LIF focus on the analysis of native fluorescence substances or the amino compounds. The analysis of acidic compounds was still rarely performed, which was probably due to the difficulty in effective derivatization. pCEC-LIF will provide a new alternative analytical method for physiologically important carboxylic acids, which are usually non-chromogenic and are present from μmol L−1 to nmol L−1 levels in biofluids.
In this work, an isocratic elution pCEC coupled to a laser diode double pumped solid state (LD-DPSS) LIF method has been developed for the rapid analysis of five structurally related free BAs. To enhance the sensitivity and reduce the interference from endogenous compounds in complex biological samples, an amine-type benzofurazan fluorescence reagent with long excitation and emission wavelengths, 4-nitro-7-N-piperazino-2,1,3-benzoxadiazole (NBD-PZ),31 was first introduced for analysis of free BAs with precolumn derivatization. The reaction was quickly carried out under mild conditions, and produced highly fluorescent adducts that could be selectively separated and sensitively determined by pCEC-LIF on an ODS packed CEC column. The experimental parameters affecting the pCEC analysis were evaluated. Application of the proposed method for simultaneous quantification of free BAs from the human serum is also described.
The commercial packed capillary column (100 μm I.D. × 375 μm O.D.) was obtained from Global Chromatography Ltd. (Su Zhou, China). The total length of the capillary was 45 cm, of which 15 cm was packed with 3 μm octadecyl silica (ODS) particles. A 1∼2 mm detection window was created immediately behind the packed section by removing the polyimide capillary coating with a thermal wire stripper.
BAs stock solutions were prepared as 5 × 10−3 mol L−1 in methanol respectively. The stock solutions were stored in darkness at 4 °C prior to use, and further diluted by ACN as appropriate. 20 mmol L−1 NBD-PZ stock solution was prepared in DMF and diluted to the desired concentration by ACN, then stored in a refrigerator at −20 °C. TPP and DPDS solutions were prepared as 560 mmol L−1 in ACN and also stored in a refrigerator at 4 °C. The CAPS buffer used in this experiment was prepared with CAPS sodium salt and the pH modified with phosphoric acid. The TEAP buffer was prepared with TEA and phosphoric acid, and the phosphate buffer (PBS) was prepared with sodium phosphate monobasic and sodium phosphate dibasic dodecahydrate.
Fig. 2 The reaction scheme for the derivatization of cholic acid (CA) with NBD-PZ. |
Factors affecting the labelling reaction, such as concentration of activation reagents, reaction time, reaction temperature and the molar ratio between NBD-PZ and BAs, were investigated to optimize the labelling efficiency. As shown in Fig. 3a, by comparison of the relative fluorescent intensity of derivatives at different molar ratios between NBD-PZ and CA (concentrations for derivatization were 0.1 mmol L−1), a 100-fold molar excess of NBD-PZ was found to be adequate for achieving a satisfactory reaction yield and a relatively low background. Higher concentrations of NBD-PZ caused no significant enhancement of labelling efficiency. The effect of concentration of the activation reagents (DPDS and TPP) on the yield of the derivatization reaction was investigated (shown in Fig. 3b), and a maximum of labelling efficiency was obtained at a concentration of DPDS and TPP of 400 mmol L−1 respectively. All derivatization reactions were performed at this concentration.
Fig. 3 The influence of (a) molar ratio between NBD-PZ and CA; (b) DPDS and TPP concentration; (c) reaction temperature; (d) reaction time, on the derivatization. Experimental conditions: capillary column: 45 cm (packing length 15 cm) × 100 μm I.D., packed with ODS (3.0 μm), mobile phase: 60% ACN–40% TEAP buffer (10 mmol L−1, pH 7.0); applied voltage 15 kV, supplementary pressure 7.2 MPa, pump flow rate 0.1 mL min−1; wavelength for LIF detection: λex/λem = 473 nm/530 nm. The derivatization conditions were as described as section 2.4. |
In previous reports, the NBD-PZ derivatization was usually performed at room temperature. We found that the derivatization reaction of BAs could be accelerated by increasing the temperature to 32 °C (shown in Fig. 3c). No significant change of the reaction yield could be found with the reaction temperature further increased to 50 °C. Thus, 32 °C is considered as a suitable reaction temperature. The time required for completion of the reaction between NBD-PZ and BAs was also investigated by comparison of the peak area of the derivatives in various time intervals (as shown in Fig. 3d). The derivatization occurred rapidly in the first 1.5 h of the reaction period. With time increasing, the reaction rate decreased and the reaction yield gradually became stable when the reaction time exceeded 2 h. 2 h was used for all of BAs derivatizations in subsequent experiments. Further experiments indicated that the peak area of the derivatives did not decrease within 24 h, which meant that the BAs derivatives were sufficiently stable for quantitative analysis. In order to ensure the reproducibility of this method, all the derivatives were kept at 4 °C in the dark and analyzed within 6 h after completing the reaction.
Five types of buffer solution, PBS, TEAP, CAPS, HEPES and MES, were compared in the same concentration with 60% v/v ACN. It was found that the use of zwitterionic buffers (CAPS, MES, and HEPES) could effectively reduce the analysis time and lower the conductivity. Better peak shape and less interference from the residual NBD-PZ were obtained when using CAPS buffer. Therefore, CAPS buffer was selected as the buffer for the mobile phase in further studies.
Fig. 4 Effect of ACN content on the pCEC separation of five free BAs. Experiment conditions: capillary column: 45 cm (packing length 15 cm) × 100 μm I.D., packed with ODS (3.0 μm), mobile phase: ACN–CAPS buffer (10 mmol L−1, pH 8.0); applied voltage 20 kV, supplementary pressure 10.8 MPa, pump flow rate 0.05 mL min−1, wavelength: λex/λem = 473 nm/530 nm; 4.7 × 10−7 mol L−1 of each analytes. Analytes: 1: NBD-CA; 2: NBD-UDCA; 3: NBD-CDCA; 4: NBD-DCA; 5: NBD-LCA; R: NBD-PZ. |
Fig. 5 Effect of buffer pH on the pCEC separation of five free BAs. Experiment conditions: mobile phase: 60% (v/v) ACN, 40% (v/v) CAPS buffer (10 mmol L−1). All other conditions were the same as in Fig. 4. Analytes: 1: NBD-CA; 2: NBD-UDCA; 3: NBD-CDCA; 4: NBD-DCA; 5: NBD-LCA; R: NBD-PZ. |
Fig. 6 Effect of concentration of CAPS buffer on the pCEC separation of five free BAs. Experiment conditions: mobile phase: 60% (v/v) ACN, 40% (v/v) CAPS buffer (pH 8.0). All other conditions were the same as in Fig. 4. Analytes: 1: NBD-CA; 2: NBD-UDCA; 3: NBD-CDCA; 4: NBD-DCA; 5: NBD-LCA; R: NBD-PZ. |
Fig. 7 Effect of applied voltage on the pCEC separation of five free BAs. Experiment conditions: mobile phase: 60% (v/v) ACN, 40% (v/v) of CAPS buffer (pH 8.0, 10 mmol L−1). All other conditions were the same as in Fig. 4. Analytes: 1: NBD-CA; 2: NBD-UDCA; 3: NBD-CDCA; 4: NBD-DCA; 5: NBD-LCA; R: NBD-PZ. |
In pCEC, the application of supplementary pressure could not only shorten the analysis time, but also assure the reliability and repeatability of the electrochromatographic system, by avoiding the formation of bubbles during the separation. With the pressure increasing from 3.5 to 13.0 MPa by a back-pressure regulator applied to the column inlet, a decrease in retention time of all analytes and a little loss of resolution were observed. A supplementary pressure up to 10.8 MPa could be employed to achieve the compromise between analysis time and resolution.
The optimal pCEC chromatogram of five NBD-PZ labelled free BAs was listed in Fig. 8. All the analytes could be separated within 17 min at the isocratic elution condition, owing to the cooperative action of both pressurized flow and EOF. The separation time of this proposed method could be much shorter than that of the widely used HPLC method (in ca. 1 h).7,8
Fig. 8 The pCEC chromatogram of five free BAs under the optimum derivatization and separation conditions. Experiment conditions: capillary column: 45 cm (packing length 15 cm) × 100 μm I.D., packed with ODS (3.0 μm), mobile phase: 60% (v/v) ACN, 40% (v/v) CAPS buffer (pH 8.0, 10 mmol L−1); applied voltage 20 kV, flow rate 0.05 mL min−1, supplementary pressure 10.8 MPa, wavelength: λex/λem = 473 nm/530 nm; 4.7 × 10−7 mol L−1 of each analytes. Analytes: 1: NBD-CA; 2: NBD-UDCA; 3: NBD-CDCA; 4: NBD-DCA; 5: NBD-LCA; R: NBD-PZ. |
Analytes | Regression equationa | r 2 | Calibration range/mol L−1 | LOD/mol L−1 (S/N = 3) | LOQ/mol L−1 (S/N = 10) |
---|---|---|---|---|---|
a Y: peak area of BAs derivatives (mV s), X: amount concentration (10−8 mol L−1). | |||||
CA | Y = 613.4X + 1794.1 | 0.9983 | 4.0 × 10−8∼5.0 × 10−6 | 2.0 × 10−9 | 4.0 × 10−8 |
UDCA | Y = 769.8X + 1590.1 | 0.9995 | 5.0 × 10−8∼5.0 × 10−6 | 3.0 × 10−9 | 5.0 × 10−8 |
CDCA | Y = 1278.5X + 218.9 | 0.9997 | 5.0 × 10−8∼3.0 × 10−6 | 5.0 × 10−9 | 5.0 × 10−8 |
DCA | Y = 538.7X + 146.3 | 0.9992 | 5.0 × 10−8∼2.0 × 10−6 | 6.0 × 10−9 | 5.0 × 10−8 |
LCA | Y = 335.0X + 750.6 | 0.9999 | 1.0 × 10−7∼4.0 × 10−6 | 9.0 × 10−9 | 1.0 × 10−7 |
The detection limits (LOD, defined as the minimum analyte concentration yielding a signal/noise ratio equal to 3) for the derivatized free BAs were obtained in the range of 2∼9 nmol L−1, and the quantification limits (LOQ, defined as the minimum analyte concentration yielding a signal/noise ratio equal to 10) ranged from 4.0 × 10−8∼1.0 × 10−7 mol L−1. The detection and quantification limits of the proposed method were much lower than the CE4,17 and HPLC12 methods coupled with an UV detector or indirect detection, and has reached the most sensitive level of the previous reports (about nmol L−1∼sub nmol L−1 for LODs) with HPLC-fluorescence14 and HPLC-MS/MS.8,15 The proposed method meets the demand of clinical and biological studies for free BAs in μmol L−1∼nmol L−1 levels. The inter-day and intra-day repeatabilities were also investigated, which includes the repeatability of the whole analytical procedure (both derivatization and instrumental precision). The inter-day RSD (n = 5) of the retention time and peak area of the five analytes were in the range of 0.9%∼2.7% and 1.9%∼5.6% respectively, and intra-day RSD (n = 5) were 2.6%∼6.8% and 5.3%∼9.4% respectively, which indicated acceptable precision of this method for routine analysis. The RSDs of critical resolution were 6.86% for reagent and peak 1, 3.76% for peak 1 and 2, and 2.12% for peak 3 and 4 (intra-day repeatabilities, n = 9). Though the RSD of resolution for reagent and peak 1 was higher than other “critical pairs”, the relative higher resolution (Rs ≥ 3) made it still acceptable for efficient separation and correct quantification.
Analytes | Endogenous value (Mean ± SD)/μmol L−1 | Added value/μmol L−1 | Measured value (Mean ± SD)/μmol L−1 | Recovery (%)a | RSD (%) |
---|---|---|---|---|---|
a Recovery (%) = (Measured value − Endogenous value)/(Added value)] ×100. | |||||
CA | 0.180 ± 0.005 | 0.600 | 0.748 ± 0.035 | 94.8 | 4.6 |
UDCA | 0.102 ± 0.004 | 0.600 | 0.739 ± 0.039 | 106.2 | 5.3 |
CDCA | 0.135 ± 0.008 | 0.600 | 0.790 ± 0.034 | 109.3 | 4.3 |
DCA | 0.130 ± 0.007 | 0.600 | 0.762 ± 0.050 | 105.4 | 6.6 |
LCA | 0.129 ± 0.005 | 0.600 | 0.753 ± 0.028 | 103.9 | 3.7 |
This journal is © The Royal Society of Chemistry 2010 |