Solid-phase extraction-field-amplified sample injection coupled with CE-ESI-MS for online pre-concentration and quantitative analysis of brain-gut peptides.

In order to improve the sensitivity of CE-ESI-MS for the analysis of brain-gut peptides, a solid-phase extraction combined with field-amplified sample injection was used for the pre-concentration of the brain-gut peptides. Compared with the conventional pressure injection method, the sensitivity in the detection of brain-gut peptides was improved more than 100-fold. The possible factors affecting sample stacking, such as the sample matrix, the composition and the length of the water column, the types and the volumes of eluent, have been investigated in detail. Under the optimum conditions, the detectable concentration of brain-gut peptides was found to be as low as 0.02 µM. A linear response concentration for the detection was developed in the range of 0.08-25 µmol/L. A real sample of human cerebrospinal fluids, which was spiked with brain-gut peptides, was also examined in order to evaluate the reliability of the proposed approach. The recovery of the method was in a range from 69.2 to 85.4%. The method was found to be reliable, accurate and potentially applicable for clinical drug analysis.

Analysis of methylene blue and its metabolites in blood by capillary electrophoresis/electrospray ionization mass spectrometry.

A method for the determination of methylene blue (MB) and its metabolites (azure A, azure B and azure C) in rat blood by CE-electrospray ionization mass spectrometry (CE-ESI-MS) was developed in this paper. Different analytical parameters were investigated in detail such as pH and concentration of separation buffer, and ESI-MS instrumental parameters. Under the optimum conditions, MB and its metabolites were separated and detected in 27.3 min. LODs (defined as S/N=3) of this method were 0.22, 0.25, 0.10 and 0.30 µg/mL for MB, azure A, azure B and azure C, respectively. To get a satisfactory extraction efficiency of MB and its metabolites in rat blood, different extraction solutions were studied. By using this method, MB and its metabolites (azure A, azure B and azure C) were successfully analyzed in rat blood samples.

CE-ESI-MS coupled with dynamic pH junction online concentration for analysis of peptides in human urine samples.

In this article, an approach has been developed for the analysis of some small peptides with similar pI values by CE-ESI-MS based on the online concentration strategy of dynamic pH junction. The factors affected on the separation, detection and online enrichment, such as BGE, injection pressure, sheath flow liquid and separation voltage have been investigated in detail. Under the optimum conditions, i.e. using 0.5 mol/L formic acid (pH 2.15) as the BGE, preparing the sample in 50 mM ammonium acetate solution (pH 7.5), 50 mbar of injection pressure for 300 s, using 7.5 mM of acetic acid in methanol-water (80% v/v) solution as the sheath flow liquid and 20 kV as the separation voltage, four peptides with similar pI values, such as L-Ala-L-Ala (pI = 5.57), L-Leu-D-Leu (pI = 5.52), Gly-D-Phe (pI = 5.52) and Gly-Gly-L-Leu (pI = 5.52) achieved baseline separation within 18.3 min with detection limits in the range of 0.2-2.0 nmol/L. RSDs of peak migration time and peak area were in the range of 1.45-3.57 and 4.93-6.32%, respectively. This method has been applied to the analysis of the four peptides in the spiked urine sample with satisfactory results.

Enantiomeric separation of chiral dipeptides by CE-ESI-MS employing a partial filling technique with chiral crown ether.

Enantiomer of chiral dipeptides were separated by CE-ESI-MS in a bare fused-silica capillary using (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18C6H4) as the chiral selector. As 18C6H4 is a kind of nonvolatile chiral selector, in order to prevent from 18C6H4 into the ion-source of CE-ESI-MS, a partial filling technique was employed in this study. Some dipeptides with one chiral center or two chiral centers, such as DL-Leu-DL-Leu, D-Ala-D-Ala and L-Ala-L-Ala, Gly-D-Phe and Gly-L-Phe were used to evaluate this CE-ESI-MS system. Optimized conditions were achivevd with 2.0 mol/L acetic acid (pH 2.15) as the running electrolyte, 5 mM 18C6H4 in 3.0 mol/L acetic acid (pH 2.00) was injected hydrodynamically (50 mbar for 960 s) before sample injection. In total 7.5 mM acetic acid in 80% v/v methanol-water was used as the sheath liquid, and 20 kV applied voltage was used. Under the optimum conditions, these dipeptides were separated and detected. LODs (defined as S/N=3) of this method were 0.20, 0.10, 0.05 and 0.10 micromol/L for D-Ala-D-Ala, L-Ala-L-Ala, DL-Leu-DL-Leu, Gly-L-Phe and Gly-D-Phe, respectively. The RSDs (n=7) of the method were 0.68-2.08% for migration times and 2.32-5.24% for peak areas. The proposed method was also successfully applied to the enantioselective analysis of these dipeptides in the spiked serum samples with satisfactory results.

On-line preconcentration and quantitative analysis of peptide hormone of brain and intestine using on-column transient isotachophoresis coupled with capillary electrophoresis/electrospray ionization mass spectrometry.

A new approach was described to achieve very sensitive analysis of peptide hormone of brain and intestine by capillary electrophoresis coupled with a transient isotachophoresis (tITP) preconcentration method. The system used was electrospray ionization mass spectrometry (ESI-MS) as detector and equipped with a sheath flow configuration. The effects of sample matrix, pH and concentration of leading electrolyte (LE), sample injection time, and ESI-MS instrumental parameters on the efficiency of the sample stacking were investigated in detail. Under the optimized conditions, lower than micromole (0.01 microM) concentrations of the peptides were easily detected. Compared to traditional hydrodynamic injection methods, about 40-230-fold increase in detection sensitivity was obtained by this technique. A distinguishing feature of the described approach is that the background electrolyte can serve as terminating electrolyte (TE), which simplifies the process of the experiments. The method was further evaluated by the analysis of low concentration active peptide mixtures spiked in hypothalamus tissue of the rat.

Rapid separation and sensitive detection method for beta-blockers by pressure-assisted capillary electrochromatography-electrospray ionization mass spectrometry.

A new method for rapid separation and sensitive detection of beta-blockers by pressure-assisted capillary electrochromatography (pCEC) with electrospray ionization mass spectrometry (ESI-MS) using silica-based monolithic column was studied in this paper. The proposed method has been confirmed to be very powerful since the fast mass transfer property and good permeability of silica monolithic column was used in this pCEC-ESI-MS system. In this work, a silica monolithic column was prepared with sol-gel method for simultaneous fast separation of beta-blockers. Furthermore, in order to obtain the highly selective and sensitive result of pCEC-ESI-MS, both the CEC separation and MS detection parameters were optimized in detail. Under the optimized conditions, namely 80% acetonitrile and 20% 20 mmol/L ammonium acetate (pH 6.0) as the mobile phase, 20 kV and 8 bar as the separation voltage and the assisted pressure, isopropanol/water (1:1, v/v) containing 7.5 mmol/L acetic acid as the sheath liquid, and 3 microL/min as the flow rate of sheath liquid, seven beta-blockers were well separated within 11 min with detection limits in the range of 0.15-0.80 ng/mL (defined as S/N=3). The recoveries of spiked urine samples of these beta-blockers were between 86.3 and 103% with the RSDs lower than 8.0%. The real samples from some male volunteers were successfully analyzed and confirmed with the proposed method. Comparing with GC-MS or LC-MS, the new method has some superiority (such as fast analysis capacity and simple pretreatment) in clinical practice and doping control.

Pressure-assisted capillary electrochromatography with electrospray ionization-mass spectrometry based on silica-based monolithic column for rapid analysis of narcotics.

A pressure-assisted CEC (pCEC) with ESI-MS based on silica-based monolithic column was developed for rapid analysis of narcotics. Combining the extremely high permeability and separation efficiency of silica-based monolithic column with the high selectivity and sensitivity of pCEC-ESI-MS, the developed system exhibited its prominent advantages in separation and detection. A systematic investigation of the pCEC separation and ESI-MS detection parameters was performed. Experiment results showed that the optimized separation efficiency could be obtained at 8 bar assisted pressure with 25 kV separation voltage, using the solution containing 65% ACN v/v and 20 mmol/L ammonium acetate with pH 6.0 as running buffer. 3 microL/min of sheath liquid was considered as the optimized flow rate since it could provide the maximum signal intensity. Under the optimum conditions, the tested five narcotics could be completely separated within 10 min with the detection limit in the range of 2.0-80 nmol/L. The proposed method has been successfully used for detection of narcotics in real urine samples.

Determination of peptide hormones of brain and intestine by CE with ESI-MS detection.

A new method for the determination of the peptide hormones of brain and intestine based on CE coupling with a DAD and ESI-MS was established. Several electrophoretic and ESI-MS parameters were investigated in detail, such as electrolyte nature and concentration, organic solvent and sheath liquid compositions, nebulization gas pressure and the ESI capillary voltage. Optimized conditions were achieved with 25 mM formic acid-ammonium formate (pH 2.9) as the optimal electrolyte, 2 mM formic acid in 80% methanol in water as the sheath liquid, and 20 kV applied voltage. Under the optimized conditions, four protonated peptides were separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath flow ESI interface. LODs for the four peptides (neurotensin hexapeptide, neurotensin, cholecystokinin tetrapeptide, and pentagastrin) were in the range of 0.10-0.60 micromol/L at an S/N of 3. The RSDs (n = 8) of the method were 0.70-1.5% for migration times and 1.6-6.1% for peak areas. This method is simple, rapid, and selective compared with RIA and ELISA techniques, and has been applied to the analysis of rat hypothalamus tissue.

A new method for screening and determination of diuretics by on-line CE-ESI-MS.

A rapid, high-resolution and effective new method for analyzing 12 diuretics by CE-ESI-MS was established in this paper. Ten diuretics (except two neutral compounds) could be fast separated by CE with a DAD at 214 nm with a 20 kV voltage within 6 min, using a 50 microm id and 48.5 cm effective length uncoated fused-silica capillary in a 40 mM ammonium formate buffer (pH 9.40). CE was coupled to the mass spectrometer applying an orthogonal electrospray interface with a triple-tube sheath liquid arrangement. The sheath liquid was composed of isopropanol-water (1:1 v/v) containing 30 mM acetic acid with a flow rate of 4 microL/min. Mass spectrum was employed in the positive mode and both full scan mode and SIM scan mode were utilized. All 12 diuretics could be detected and confirmed by MS in a single analysis. Under optimized conditions, LODs for the 12 diuretics were in the range of 0.13-2.7 micromol/L at an S/N of 3, and the correlation coefficients R(2 )were between 0.9921 and 0.9978. The RDSs (n = 5) of the method was 0.24-0.94 % for migration times and 1.6-8.8 % for peak areas. The recoveries of spiked samples of 12 diuretics were between 72.4% and 118%. The real urine samples were injected directly for analysis, with only simple filtration through a 0.22 microm membrane filter in order to remove solid particles, which may cause capillary blockage. Based on the migration times and characteristic ions, the diuretics in urine samples were detected successfully. This CE-ESI-MS method for analyzing diuretics will hopefully be applied to doping control.