化学衍生辅助液相色谱-串联质谱测定枇杷膏中的齐墩果酸和熊果酸

建立了一种液相色谱-串联质谱(LC-MS/MS)同时测定齐墩果酸和熊果酸含量的分析方法,利用衍生化试剂N,N-二甲基乙二胺(DMED)和d4-N,N-二甲基乙二胺(d4-DMED)分别衍生样品和标准工作溶液中的目标分析物,并将标准工作液的衍生产物作为稳定同位素内标。该方法线性关系良好,相关系数均0.99;齐墩果酸和熊果酸的检出限(S/N=3)分别为0.92 ng/L和1.06 ng/L,加标回收率分别为98.7%~102.7%和97.2%~105.0%。该方法简单、快速、准确,可满足枇杷膏中齐墩果酸和熊果酸高灵敏度检测的需求。     

A high-performance polycarbonate electrophoresis microchip with integrated three-electrode system for end-channel amperometric detection

A fully integrated polycarbonate (PC) microchip for CE with end-channel electrochemical detection operated in an amperometric mode (CE-ED) has been developed. The on-chip integrated three-electrode system consisted of a gold working electrode, an Ag/AgCl reference electrode and a platinum counter electrode, which was fabricated by photo-directed electroless plating combined with electroplating. The working electrode was positioned against the separation channel exit to reduce post-channel band broadening. The electrophoresis high-voltage (HV) interference with the amperometric detection was assessed with respect to detection noise and potential shifts at various working-to-reference electrode spacing. It was observed that the electrophoresis HV interference caused by positioning the working electrode against the channel exit could be diminished by using an on-chip integrated reference electrode that was positioned in close proximity (100 microm) to the working electrode. The CE-ED microchip was demonstrated for the separation of model analytes, including dopamine (DA) and catechol (CA). Detection limits of 132 and 164 nM were achieved for DA and CA, respectively, and a theoretical plate number of 2.5x10(4)/m was obtained for DA. Relative standard deviations in peak heights observed for five runs of a standard solution containing the two analytes (0.1 mM for each) were 1.2 and 3.1% for DA and CA, respectively. The chip could be continuously used for more than 8 h without significant deterioration in analytical performance.     

Determination of uric acid in plasma and allantoic fluid of chicken embryos by capillary electrophoresis

Capillary electrophoresis with diode array detection (DAD) was used to determine uric acid (UA) in chicken plasma and the allantoic fluid of chicken embryos. Complete separation of uric and ascorbic acids was attained in less than 10 min in the optimized BGE containing 60 mM MES + 30 mM Tris + 0.001% (w/v) polybrene (pH 6.1). The limit of UA detection (0.2 mg/L) was found to be low enough for sensitive analysis of native plasma and allantoic fluid samples. Range of linearity (1-200 mg/L), repeatability for peak area (CV <4.1%) and migration time (CV < 2.5%), as well as recovery of UA from biological samples (97-100%), were found to be satisfactory. The method was applied to detect the elevated UA concentrations (hyperuricemia) in chicken embryos with induced unilateral renal agenesis. CE/DAD analysis of the chicken plasma can be carried out with a relatively small volume of samples (1 microL).     

Simultaneous voltammetric determination of uric and ascorbic acids in urine

Anodic voltammetric method for simultaneous determination of uric acid (UA) and ascorbic acid (AA) in urine has been developed with the use of a commercial working rotating glassy carbon electrode. UA may be determined in a sample diluted by the buffer supporting electrolyte (HOAc+NH(4)OH; pH 5.1-5.2) approximately 100 times, and AA-in a sample diluted approximately 20 times. Before obtaining the analytical signal the electrode should be maintained in the diluted sample during 3 min at potential 0 V and the working electrode rotating 100 rpm, for achievement of the adsorption equilibrium of inhibitors from the urine matrix. For UA the electron transfer is close to reversible, for AA it is an irreversible one. Optimal voltammetric techniques are the square-wave for UA and the differential pulse for AA. Calibration curves, detection limits and recoveries for both determinations were evaluated as satisfactory.     

Analysis of orotic acid in human urine by on-line combination of capillary isotachophoresis and zone electrophoresis.

The techniques of the on-line combination of capillary isotachophoresis with zone electrophoresis in two coupled capillaries (ITP-CZE) and a single capillary zone electrophoresis (CZE) were used for the sensitive determination of orotic acid (OA) in human urine. The simple CZE system was successfully applied for fast and reliable analyses of urine of healthy adult volunteers (the detection limit 1.7.10(-6) M OA, the total time of analysis 6 min). However, this method failed in analyses of OA in urine of ill children due to more complex matrix of the samples. Here, the ITP preconcentration and preseparation step coupled on-line with CZE proved to serve well with an electrolyte system developed and optimized for this purpose. The maximum selectivity and resolution of OA from other sample constituents in ITP-CZE was achieved by use of an electrolyte system of very low pH 2.15 both for ITP and CZE stage. The sensitivity of detection and simplicity of OA identification were enhanced by use of an external UV scanning detector. High sensitivity of ITP-CZE combination (limit of detection 3.10(-7) M OA), low sample consumption (1 microliter), good reproducibility of migration times (inter-day RSD < 1.86%) and acceptable reproducibility of the determination of OA in urine samples (average RSD = 7.27%) make this technique suitable for routine determination of trace concentration of OA especially in urine of ill children under various pathological conditions and medication.     

Determination of uric acid in human urine by capillary zone electrophoresis with indirect laser-induced fluorescence detection

A CZE with indirect LIF detection method was used for the determination of uric acid (UA) in human urine. UA and its coexisting analytes (i.e. hypoxanthine, xanthine and ascorbic acid) could be well separated within 4.5?min at a voltage of 25?kV with 25°C cartridge temperature in a running buffer composed of 5?mM sodium borate, 10% methanol (v/v) and 0.3?μM fluorescein sodium (apparent pH of the final mixed hydro-organic solution of sodium borate, methanol and fluorescein is 9.5). Under the optimum condition, the method has good linearity relationships (correlation coefficients: 0.9973-0.9987) with ranges of 25-500, 25-350, 25-250 and 25-300?μg/mL for hypoxanthine, ascorbic acid, xanthine and UA, respectively. The detection limits for the analytes were in the range of 0.29-0.90?μg/mL. The intra-day RSD values for migration times and peak areas were less than 0.43 and 3.27%, respectively. This method was applied to the quantitation of UA in human urine with recoveries in the range of 93.1-107.3%.     

Capillary electrophoresis of trace metals in highly saline physiological sample matrices

Trace metal ions in highly saline samples such as urine were determined with capillary electrophoresis (CE) without desalting or off-line preconcentration. By mixing with a dye, 4-(2-pyridylazo) resorcinol (PAR), the metal ions were converted into anionic complexes having strong absorbance near 500 nm. A large volume of the metal-PAR complex sample solution injected into a coated capillary was stacked isotachophoretically and separated under a reverse potential. The salt anion (chloride) and PAR in the sample matrix acted as the leading and terminating electrolytes, respectively. In a sample containing a 250 mM NaCl matrix, more than 400-fold enhancement in the absorbance detector response was realized compared to the normal CE injection mode. Combination of the dye complexation and isotachophoretic stacking provided excellent detection limits (S/N = 3) for three trace metal ions in the low ppb range (Fe(2+), 0.7 ppb, Ni(2+), 0.4 ppb; Zn(2+), 1.2 ppb) with absorbance detection. The migration time reproducibility was excellent (relative standard deviations: standard samples < 1%, urine samples approximately 1%). The proposed method is convenient and fast, and the sample analysis can be completed within 20 min.     

Microfluidic chip capillary electrophoresis coupled with electrochemiluminescence for enantioseparation of racemic drugs using central composite design optimization

Optimization based on central composite design (CCD) for enantioseparation of anisodamine (AN), atenolol (AT), and metoprolol (ME) in human urine was developed using a microfluidic chip‐CE device. Coupling the flexible and wide working range of microfluidic chip‐CE device to CCD for chiral separation of AN, AT, and ME in human urine, a total of 15 experiments is needed for the optimization procedure as compared to 75 experiments using the normal one variable at a time optimization. The optimum conditions obtained are found to be more robust as shown by the curvature effects of the interaction factors. The developed microfluidic chip‐CE‐ECL system with adjustable dilution ratios has been validated by satisfactory recoveries (89.5–99% for six enanotiomers) in urine sample analysis. The working range (0.3–600 μM), repeatability (3.1–4.9% RSD for peak height and 4.0–5.2% RSD for peak area), and detection limit (0.3–0.6 μM) of the method developed are found to meet the requirements for bedside monitoring of AN, AT, and ME in patients under critical conditions. In summary, the hyphenation of CCD with the microfluidic chip‐CE device is shown to offer a rapid means for optimizing the working conditions on simultaneous separation of three racemic drugs using the microfluidic chip‐CE device developed.     

Use of liquid chromatography-atmospheric pressure chemical ionization-ion trap mass spectrometry for identification of oleanolic acid and ursolic acid in Anoectochilus roxburghii (wall.) Lindl

Oleanolic acid (OA) and ursolic acid (UA) are the two important bioactive compounds in Anoectochilus roxburghii (wall) Lindl (A. roxburghii), which has been used as a traditional Chinese medicine. So far, there has been no report to indicate that A. roxburghii contains these two bioactive compounds. It is necessary to develop an effective method to extract and analyze OA and UA in A. roxburghii. In this paper, a quantitative method, consisting of supercritical fluid extraction (SFE) followed by liquid chromatography-atmospheric pressure chemical ionization-ion trap mass spectrometry (LC-APCI-IT-MS) analysis, was developed for identification of OA and UA in A. roxburghii. The extraction was carried out by using CO(2) as the supercritical fluid and ethanol as the modifier before LC separation. The mobile phase used for LC separation consisted of acetic acid (1%, v/v), water (15%, v/v) and methanol (84%, v/v), and the elution was performed at a flow rate of 0.8 ml/min. The mass spectrometer was operated in APCI(+) mode with selected ion monitoring (SIM) to quantify OA and UA at m/z 439.4. Under optimum conditions, the linear responses of OA and UA were obtained in the concentration range of 0.5-80 (r = 0.9992) and 0.5-50 microg/ml (r = 0.9989) with the detection limits of 0.125 and 0.085 microg/ml, respectively. The proposed method has been used for the identification and quantitation of OA and UA in a real A. roxburghii sample.     

A rapid urine creatinine assay by capillary zone electrophoresis.

Using capillary zone electrophoresis, the urine creatinine (uCr) assay was validated in extemporaneous diluted urine, both in healthy subjects and athletes, with the uCr concentration as a reference value to compare excretion rates of other metabolites in the same samples. The electrokinetic sample injection was carried out at 10 kV per 10 s; UV absorbance detection was at 254 nm. Using standard samples, the creatinine migration mean time in 100 mmol/L acetate buffer, pH 4.4, was 3.3+/-0.2 min; the repeatability for absolute migration mean time was 0.6% and peak height repeatability was 2.9%. The correlation coefficient of the standard curve was r = 0.999 and the detection limit was 23.1 micromol/L. Intra- and interassay coefficients of variation (CV) were 3.0 and 3.6%, respectively; recovery was 99+/-3% and linearity was r= 0.98. Normal urine samples were diluted 1:80 in run buffer. The present CE urine creatinine assay showed a good correlation with HPLC and with Jaffe methods (r = 0.98 and r = 0.97, respectively; p < 0.0001). The uCr in the morning urine samples of 34 healthy males (M), 38 healthy females (F), and 83 male athletes (A) was 10.4+/-6.1 mmol/L, 10.8+/-8.1 mmol/L and 13.2+/-6.5 mmol/L, respectively. The uCr difference (p < 0.02) between M and A and a correlation (p < 0.05) with age in A were observed.     

Calibration of migration times of variable salinity samples with internal standards in capillary electrophoresis

A practical approach is presented for identifying the analyte peaks stacked by transient ITP (TITP) in samples of uncontrolled salinity. For TITP with chloride ions acting as the leading electrolyte, the effect of matrix chloride of an unknown concentration was calibrated using multiple internal standards to predict the migration times of weakly acidic anionic analytes behaving as strong electrolytes to an accuracy of over 99.9%. The calibration equations for the migration time of an analyte are given as a function of the migration times of internal standards using the mobilities of the relevant ions as parameters. The effects of matrix chloride and various separation conditions such as the temperature, plug length, ionic strength, and pH of the BGE were completely eliminated from the calibration equations. In addition, the actual mobilities, determined for a standard saline sample under the working conditions, were used, and thus, there was no need to conduct supplementary experiments to determine the absolute mobilities at infinite dilution. The internal standards were dyes, which were easily identified in an auxiliary channel monitoring the absorbance at a longer wavelength. For five standard saline matrices containing 100-300 mM NaCl at intervals of 50 mM, the mean absolute error (MAE) in migration times calibrated with two internal standards was 0.4 s (n=5x13). For an electropherogram of a real standard reference urine sample, peaks of spiked analytes were identified with an MAE of 0.9 s (n=13) without conductivity normalizing or desalting of the sample.     

Development and validation of analytical methodology using capillary electrophoresis for separation and determination of anions in rainwater

A new capillary electrophoresis (CE) procedure was developed for simultaneous determination of both organic and inorganic anions in rain water using a background electrolyte (BGE) containing 5 mM molybdate, 0.15 mM CTAH, 0.01% PVA and 5 mM Tris buffer to adjust pH at 7.9. Under optimised conditions, good repeatability (RSD for sulphate in migration time=0.36% and peak area=4.2%), low detection limit (2 ppb for chloride) and satisfactory working range (50 ppb-20 ppm for hydrodynamic injection, 10 ppb-3 ppm for electrokinetic injection for chloride) were obtained. The reliability of the CE procedure developed was established by satisfactory recovery tests and good agreement of results obtained by both the CE and ion chromatography (IC) methods. The procedure developed had been successfully applied for field monitoring of rainwater showing good repeatability and capability of detecting trace anions at ppb levels beyond the IC working range. Thus, the new CE procedure developed provides a quick, sensitive, economic and reliable method to meet the need for the simultaneous determination of both organic and inorganic anions in the acid rain monitoring programme.     

Determination of oxalate in urine by zone electrophoresis on a chip with conductivity detection

The use of a poly(methylmethacrylate) capillary electrophoresis chip, provided with a high sample load capacity separation system (a 8500 nL separation channel coupled to a 500 nL sample injection channel) and a pair of on-chip conductivity detectors, for zone electrophoresis (ZE) determination of oxalate in urine was studied. Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed and electrophoresis was a dominant transport process in the separations performed on the chip. A low pH of the carrier electrolyte (4.0) provided an adequate selectivity in the separation of oxalate from anionic urine constituents and, at the same time, also a sufficient sensitivity in its conductivity detection. Under our working conditions, this anion could be detected at a 8 x 10(-8) mol/L concentration also in samples containing chloride (a major anionic constituent of urine) at 3.5 x 10(-3) mol/L concentrations. Such a favorable analyte/matrix concentration ratio (in part, attributable to a transient isotachophoresis stacking in the initial phase of the separation) made possible accurate and reproducible (typically, 2-5% relative standard deviation (RSD) values of the peak areas of the analyte in dependence on its concentration in the sample) determination of oxalate in 500 nL volumes of 20-100-fold diluted urine samples. Short analysis times (about 280 s), no sample pretreatment (not considering urine dilution) and reproducible migration times of this analyte (0.5-1.0% RSD values) were characteristic for ZE on the chip. This work indicates general potentialities of the present chip design in rapid ZE analysis of samples containing the analyte(s) at high ionic matrix/analyte concentration ratios.     

Identification and quantification of oleanolic acid and ursolic acid in Chinese herbs by liquid chromatography-ion trap mass spectrometry

A rapid and sensitive method for the identification and quantification of ursolic acid (UA) and oleanolic acid (OA) in Chinese herbs is described. The method combines liquid chromatography (LC) with ion trap‐mass spectrometry (IT‐MS) detection. The UA and OA standard solution were directly infused into IT‐MS for collecting MSⁿ spectra. The major fragment ions of UA and OA were confirmed by MSⁿ at m/z 455, 407, 391, 377 and 363 in negative ion mode, and m/z 457, 439, 411 and 393 in positive mode, respectively. The possible main cleavage pathway of fragment ions was studied. UA and OA provided good signals corresponding to the deprotonated molecular ion [M − H]⁻. The method is reliable and reproducible, and the detection limit is 5 ng/mL. The method was validated in the concentration range of 0.04–40 μg/mL; intra‐ and inter‐day precisions ranged from 0.78 to 2.15%, and the accuracy was 96.5–108.2% for UA and OA. The mean recovery of UA and OA was 97.1–106.2% with RSD less than 1.86%. An LC‐IT‐MS method was successfully applied to determine the UA and OA in nine Chinese herbs. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of the spectrum of low molecular mass organic acids in urine by capillary electrophoresis with contactless conductivity and ultraviolet photometric detection--an efficient tool for monitoring of inborn metabolic disorders

A mixture of 29 organic acids (OAs) occurring in urine was analyzed by capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D) and UV photometric detection. The optimized analytical system involved a 100 cm long polyacrylamide-coated capillary (50 μm i.d.) and the background electrolyte of 20 mM 2-morpholinoethanesulfonic acid (MES)/NaOH + 10% (v/v) methanol, pH 6.0 (pH is related to the 20 mM MES/NaOH buffer in water). The LOD values obtained by C⁴D for the OAs which do not absorb UV radiation range from 0.6 μM (oxalic acid) to 6.8 μM (pyruvic acid); those obtained by UV photometry for the remaining OAs range from 2.9 μM (5-hydroxy-3-indoleacetic acid) to 10.2 μM (uric acid). The repeatability of the procedure developed is characterized by the coefficients of variation, which vary between 0.3% (tartaric acid) and 0.6% (5-hydroxy-3-indoleacetic acid) for the migration time and between 1.3% (tartaric acid) and 3.5% (lactic acid) for the peak area. The procedure permitted quantitation of 20 OAs in a real urine sample and was applied to monitoring of the occurrence of the inborn metabolic fault of methylmalonic aciduria.     

Field-amplified sample injection combined with pressure-assisted capillary electrophoresis UV detection for the simultaneous analysis of allantoin, uric acid, and malondialdehyde in human plasma

The allantoin/uric acid (All/UA) ratio and malondialdehyde (MDA) plasma levels have been proposed as important markers for monitoring oxidation triggered by the action of free radicals (FR). Here, we describe an easy field amplified sample injection capillary electrophoresis method with UV detection for the separation and quantification of All, UA, and free MDA in human plasma. The plasma samples were simply filtered through centrifugation membrane tubes for protein elimination and directly injected on a capillary without complex cleanup and/or sample derivatization procedures. The use of a run buffer composed of 300 mmol/L sodium borate at pH 10 with 50 mmol/L of N-methyl-d-glucamine and an overimposed pressure/voltage of 0.1 psi during the electrophoretic run allows basline resolution of the analytes within 17 min. The electrokinetic injection allows a detection limit of 15 nmol/L for All, 20 nmol/L for UA and 10 nmol/L for MDA in a plasma sample, thus significantly improving the LOD of previous described methods based on capillary electrophoresis. Precision tests indicate a good repeatability of our method both for migration times (CV = 1.85%) and areas (CV = 2.87%). Moreover, a good reproducibility of intra- and inter-assay tests was obtained (CV = 4.63% and CV = 6.59% respectively). The suitability of the method was tested by measuring analyte levels in 40 healthy volunteers.     

Bipolar pulse conductometric detection of enzyme reactions in flow-injection systems Urea in serum and urine

Conductometric monitoring of enzymatic reactions in a flow-injection system is described. Two conductance cells are used and the responses are manipulated automatically to provide a differential signal. The hydrolysis of urea, catalyzed by urease, is used as an example to illustrate the technique. Blood serum and urine control standards are used to assess the precision and accuracy. The conductometric method is shown to be equal to, or better than existing methods for urea with regard to detection limits (0.1 mM in urine, 0.01 mM in serum), working range, precision (3% RSD), accuracy, and sample preparation. The method has a sample throughput of 20 h^?1.     

Electrokinetic supercharging in CE for the separation and preconcentration of barbiturate drugs in urine samples

Three barbiturate drugs, barbital, phenobarbital, and secobarbital were separated and analyzed by electrokinetic supercharging. The influence of different parameters on electrokinetic supercharging performance was evaluated using both univariated and multivariated optimization processes. The parameters studied were sample pH, concentration, and length of the leading and terminating electrolytes, electrokinetic injection of the sample and composition and hydrodynamic injection of the solvent plug. The leading electrolyte (50 mM NaCl) was hydrodynamically injected (50 mbar × 120 s) prior to the sample that was adjusted to pH 9.6 and electrokinetically injected at ?8.5 kV for 300 s. The terminating electrolyte (100 mM of 2‐(cyclohexylamino) ethanesulphonic acid) was then hydrodynamically injected (50 mbar × 140 s). The results showed that this strategy enhanced detection sensitivity around 1050‐fold compared with normal hydrodynamic injection, providing detection limits ranging between 1.5 and 2.1 ng/mL for standard samples with good repeatability in terms of peak area (values of relative standard deviation, %RSD < 3). The applicability of the optimized method was demonstrated by the analysis of human urine samples spiked with the studied compounds at different concentration levels and further liquid–liquid extraction step. The estimated detection limits obtained in the urine samples extract ranged between 8 and 15 ng/mL.     

Application of capillary electrophoresis for organic acid analysis in herbal studies

A capillary electrophoresis (CE) procedure has been developed for the separation of 25 inorganic and organic acid anions using a buffer system consisting of 15 mM tris(hydroxymethyl)aminomethane, 3 mM 1,2,4-benzenetricarboxylic acid, 1.5 mM tetraethylenepentaamine (TEPA) and 20% methanol with pH adjusted to 8.4. A good separation of organic acids extracted from a mixture of Chinese traditional medicine (TCM) containing three herbs, Flos chrysthemi, Spica prunellae, and Folium mori was obtained using the procedure developed with satisfactory working range (0.20-77 mg/g), low detection limit (90-190 microg/g), and good repeatability (relative standard deviation 4.47-6.99%, n = 4). A satisfactory extraction of organic acids was achieved within 20 min using 0.1 M NaOH. The addition of TEPA to provide a reduced electroosmotic flow (EOF) environment was shown to remove interfering organic compounds extracted from TCM. The applicability of using organic acids as markers for determining the mixing ratio of constituent herbs for a TCM mixture was investigated using a three-component mixture with a 1:1:1 mixing ratio. A satisfactory mixing ratio of 1.04:1.09:0.98 was obtained using the methodology developed based on organic acids as markers. The application of our method for determining more complicated TCM mixtures has been discussed.     

Solvent Bar Microextraction with HPLC for Determination and Protein-Binding Characteristics of Oleanolic Acid and Ursolic Acid

An improved solvent bar microextraction (SBME) combined with HPLC was applied to rapidly determine oleanolic acid (OA) and ursolic acid (UA) in Chinese medicinal herbs (CMHs), and to investigate drug–protein binding. Variables influencing SBME were investigated and optimized. Under optimal conditions, the linear ranges of 3.6–1,820 ng mL^?1 for OA and 4.2–2,080 ng mL^?1 for UA were obtained with square correlation coefficients of 0.9996 and 0.9997, respectively. The detection limits were 1.3 ng mL^?1 for OA and 1.5 ng mL^?1 for UA. The relative standard deviations were lower than 9.4 %. The protein-binding rates, the numbers of binding sites, and the binding constants of OA and UA were also obtained via the method. It has been demonstrated that SBME can not only be a simple, rapid and efficient sample preparation method for determination of active components from CMHs but also be a potential research protocol for protein-binding interactions.