Determination of aminoglutethimide enantiomers in pharmaceutical formulations by capillary electrophoresis using methylated-β-cyclodextrin as a chiral selector and computational calculation for their respective inclusion complexes

A capillary electrophoretic method for the separation of the aminoglutethimide (AGT) enantiomers using methylated-β-cyclodextrin (M-β-CD) as chiral selector is described. Several parameters affecting the separation were studied, including the type and concentration of chiral selector, buffer pH, voltage and temperature. Good chiral separation of the racemic mixture was achieved in less than 9 min with resolution factor Rs = 2.1, using a fused-silica capillary and a background electrolyte (BGE) of tris-phosphate buffer solution (50 mmol L⁻¹, pH 3.0) containing 30 mg mL⁻¹ of M-β-CD. The separation was carried out in normal polarity mode at 25 °C, 16 kV and using hydrostatic injection. Acceptable validation criteria for selectivity, linearity, precision, and accuracy/recovery were included. The proposed method was successfully applied to the assay of AGT enantiomers in pharmaceutical formulations. The computational calculations for the inclusion complexes of the R- and S-AGT-M-β-CD rationalized the reasons for the different migration times between the AGT enantiomers.     

Steroselective determination of trihexyphenidyl using carboxylmethyl-β-cyclodextrin by capillary electrophoresis with field-amplified sample stacking

A simple, sensitive and low-cost method using capillary electrophoresis coupled with field-amplified sample stacking (FASS) technique has been developed for enantioselective separation and quantification of trihexyphenidyl (THP) enantiomers in human serum. In this work, three kinds of modified β-cyclodextrin were tested as chiral selectors in CE. Among the CDs studied, THP enantiomers could only be separated by carboxylmethyl-β-cyclodextrin (CM-β-CD). A systematic study of the parameters (CD concentration and pH value in CE buffer, separation voltage and temperature, composition of sample solvent, injection voltage and time) affecting chiral separation and on-line concentration of THP enantiomers were investigated and optimized. The optimum FASS method provided a sensitivity enhancement of about 490-fold compared with usual hydrodynamic injection. Limits of detection for each enantiomer were in the low ng ml^− 1 concentration range (0.92 ng ml^− 1 or 3.06 nM). The quantification of each THP enantiomer in human serum was performed after serum sample extraction. To validate this CE-FASS method, linear regression analysis, intra and inter-day precision and recovery were determined with satisfying results.     

Direct high-performance liquid chromatography enantioseparation of terazosin on an immobilised polysaccharide-based chiral stationary phase under polar organic and reversed-phase conditions

High-performance liquid chromatography (HPLC) enantioseparation of terazosin (TER) was accomplished on the immobilised-type Chiralpak IC chiral stationary phase (CSP) under both polar organic and reversed-phase modes. A simple analytical method was validated using a mixture of methanol–water–DEA 95:5:0.1 (v/v/v) as a mobile phase. Under reversed-phase conditions good linearities were obtained over the concentration range 8.76–26.28 μg mL⁻¹ for both enantiomers. The limits of detection and quantification were 10 and 30 ng mL⁻¹, respectively. The intra- and inter-day assay precision was less than 1.66% (RSD%). The optimised conditions also allowed to resolve chiral and achiral impurities from the enantiomers of TER. The proposed HPLC method supports pharmacological studies on the biological effects of the both forms of TER and analytical investigations of potential drug formulations based on a single enantiomer. At the semipreparative scale, 5.3 mg of racemic sample were resolved with elution times less than 12 min using a mobile phase consisting of methanol–DEA 100:0.1 (v/v) and both enantiomers were isolated with a purity of ≥99% enantiomeric excess (ee). The absolute configuration of TER enantiomers was assigned by comparison of the measured specific rotations with those reported in the literature.     

A novel capillary electrophoresis method for the determination of d-serine in neural samples

A capillary electrophoresis method has been developed for the determination of d-serine in neural samples. d/l-serine was tagged with naphthalene-2,3-dicarboxaldehyde (CBI-d/l-Ser), and the separation of CBI-d/l-Ser enantiomer was achieved by using a dual chiral selector system consisting of β-cyclodextrin (β-CD) and chiral micelles formed by sodium deoxycholate (SDC). No resolution was observed when either β-CD or SDC was used alone. Moreover, the combined use of β-CD with achiral micelles of sodium dodecylsulfate (SDS) exhibited no resolving effect. With laser induced fluorescence detection, the limit of detection was 3.0 × 10⁻⁸ M Ser. Under the separation conditions selected, no other amino acids co-eluted with l-/d-Ser enantiomers. Using the present method, d-Ser level in Aplysia ganglia homogenates was found to vary significantly from animal to animal. Interestingly, d-Ser was not detected in single neurons isolated from Aplysia ganglia.     

Separation of α-cyclohexylmandelic acid enantiomers using biphasic chiral recognition high-speed counter-current chromatography

This work concentrates on a chiral separation technology named biphasic recognition applied to resolution of α-cyclohexylmandelic acid enantiomers by high-speed counter-current chromatography (HSCCC). The biphasic chiral recognition HSCCC was performed by adding lipophilic (−)-2-ethylhexyl tartrate in the organic stationary phase and hydrophilic hydroxypropyl-β-cyclodextrin in the aqueous mobile phase, which preferentially recognized the (−)-enantiomer and (+)-enantiomer, respectively. The two-phase solvent system composed of n-hexane-methyl tert-butyl ether–water (9:1:10, v/v/v) with the above chiral selectors was selected according to the partition coefficient and separation factor of the target enantiomers. Important parameters involved in the chiral separation were investigated, namely the types of the chiral selectors (CS); the concentration of each chiral selector; pH of the mobile phase and the separation temperature. The mechanism involved in this biphasic recognition chiral separation by HSCCC was discussed. Langmuirian isotherm was employed to estimate the loading limits for a given value of chiral selectors. Under optimum separation conditions, 3.5–22.0 mg of α-cyclohexylmandelic acid racemate were separated using the analytical apparatus and 440 mg of racemate was separated using the preparative one. The purities of both of the fractions including (+)-enantiomer and (−)-enantiomer from the preparative CCC separation were over 99.5% determined by HPLC and enantiomeric excess reached 100% for the (±)-enantiomers. Recovery for the target compounds from the CCC fractions reached 85–88% yielding 186 mg of (+)-enantiomer and 190 mg of (−)-enantiomer. The overall experimental results show that the HSCCC separation of enantiomer based on biphasic recognition, in which only if the CSs involved will show affinity for opposite enantiomers of the analyte, is much more efficient than the traditional monophasic recognition chiral separation, since it utilizes the cooperation of both of lipophilic and hydrophilic chiral selectors.     

CE, with Hydroxypropyl-β-Cyclodextrin as Chiral Selector, for Separation and Determination of the Enantiomers of Amlodipine in the Serum of Hypertension Patients

A capillary electrophoretic method for separation of the enantiomers of amlodipine in the serum of hypertension patients has been established and validated. The two enantiomers were separated in a fused-silica capillary with phosphate running buffer (75 mmol L^?1, pH 2.5) containing 15 mmol L^?1 hydroxypropyl-β-cyclodextrin (HP-β-CD). The effects on the separation of buffer pH and concentration, separation potential, and concentration of HP-β-CD were investigated. The range of quantitation for both enantiomers was 2.0–16.0 μg mL^?1. Intra-day and inter-day relative standard deviation (RSD; n = 5) was <10%. The limits of detection (LOD) and quantification (LOQ) of the amlodipine enantiomers, at 214 nm, were approximately 0.5 and 0.7 μg mL^?1, respectively (S/N = 3 and 10, respectively; 5-s injection). Recovery was always >85%. Results from enantiomer separation and quantification showed that concentrations of the enantiomers of amlodipine in serum from an elderly patient were higher than in serum from a young patient administered the same dose. The method was useful for determining the concentration of the enantiomers of amlodipine in hypertension patient serum and for monitoring the transition behavior of the enantiomers in humans. The method proved suitable for application to the separation of the enantiomers of amlodipine and analysis of clinical samples.     

Enantioselective determination of modafinil in pharmaceutical formulations by capillary electrophoresis, and computational calculation of their inclusion complexes

A fast capillary electrophoretic method is described for the separation and determination of the enantiomers of the novel wake-promoting agent, modafinil. Several parameters affecting the separation were studied, including the type and concentration of chiral selector, buffer pH, buffer concentration, voltage and temperature. Good chiral separation of the racemic mixture was achieved in less than 5 min with resolution factor Rs?=?2.51, using a bare fused-silica capillary and a background electrolyte (BGE) of 25 mM H₃PO₄?1 M tris solution; pH 8.0; containing 30 mg mL^?1 of sulfated-β-cyclodextrin (S-β-CD). The separation was carried out in normal polarity mode at 25 ^?C, 18 kV and using hydrostatic injection. Acceptable validation criteria for selectivity, linearity, precision, and accuracy were included. The developed method was successfully applied to the assay of enantiomers of modafinil in pharmaceutical formulations. The computational calculations for the enantiomeric inclusion complexes rationalized the reasons for the different migration times between the modafinil enantiomers.     

Enantiomeric analysis of methotrexate in pharmaceuticals by cyclodextrin-modified capillary electrophoresis

A capillary electrophoresis for the chiral separation of racemic methotrexate (rac-MTX) was developed and validated. The two enantiomers were separated by using fused-silica capillary and a running buffer containing phosphate and hydroxypropyl-β-cyclodextrin (HP-β-CD). Several parameters were studied, including concentration and pH of phosphate buffer, separation voltage, and type and concentration of CD. The quantitative ranges were 12.5-200.0 μM for each enantiomer. The intra- and inter-day relative standard deviations (R.S.D.) and relative errors (R.E.) (n=5) were all <5%. The detection limits were found to be about 4 μM (S/N=3, injection 5 s) at 280 nm. All recoveries were greater than 93%. This method was applied to the assay of l-MTX in pharmaceuticals.     

A novel stacking method of repetitive large volume sample injection and sweeping MEKC for determination of androgenic steroids in urine

In this research, a novel stacking capillary electrophoresis method, repetitive large volume sample injection and sweeping MEKC (rLVSI-sweeping MEKC) were developed to analyze the presence of three androgenic steroids considered as sport doping drugs, testosterone (T), epitestosterone (E) and epitestosterone glucuronide (EG) in urine. This method provides better sensitivity enhancement than the traditional large volume sample stacking-sweeping strategies due to sensitivity enhancement by repetitive injections. This multiple sampling method enhances sensitivity of monitoring of urine samples by UV detection (254 nm). Firstly, the phosphate buffer was filled into an uncoated fused silica capillary and the samples were injected into the capillary at 10 psi for 20 s, and then stacked at −10 kV for 1 min using phosphate buffer containing SDS. The above injecting and stacking steps were repeated five times. Finally, separation was performed at −20 kV, using phosphate buffer containing methanol, SDS and (2-hydroxypropyl)-β-cyclodextrin. Method validation showed that calibration plots were linear (r ≧ 0.997) over a range of 5-200 ng mL⁻¹ for T, 20-200 ng mL⁻¹ for E and 0.5-500 ng mL⁻¹ for EG. The limits of detection were 1.0 ng mL⁻¹ for T, 5.0 ng mL⁻¹ for E and 200.0 pg mL⁻¹ for EG. When evaluating precision and accuracy, values of RSD and RE in intra-day (n = 3) and inter-day (n = 5) analysis were found to be less than 10.0%. Compared with the simple LVSS-sweeping, which is also a stacking strategy, this method further improves sensitivity up to 25 folds (∼2500 folds with MEKC without preconcentration). This method was applied to monitor 10 athletes’ urine, and did not detect any analyte. The novel stacking method was feasible for monitoring of doping by sportsmen.     

Enantioselective analysis of mirtazapine and its two major metabolites in human plasma by liquid chromatography-mass spectrometry after three-phase liquid-phase microextraction

A three-phase liquid-phase microextraction (LPME) method using porous polypropylene hollow fibre membrane with a sealed end was developed for the extraction of mirtazapine (MRT) and its two major metabolites, 8-hydroxymirtazapine (8-OHM) and demethylmirtazapine (DMR), from human plasma. The analytes were extracted from 1.0 mL of plasma, previously diluted and alkalinized with 3.0 mL 0.5 mol L⁻¹ pH 8 phosphate buffer solution and supplemented with 15% sodium chloride (NaCl), using n-hexyl ether as organic solvent and 0.01 moL L⁻¹ acetic acid solution as the acceptor phase. Haloperidol was used as internal standard. The chromatographic analyses were carried out on a chiral column, using acetonitrile-methanol-ethanol (98:1:1, v/v/v) plus 0.2% diethylamine as mobile phase, at a flow rate of 1.0 mL min⁻¹. Multi-reaction monitoring (MRM) detection was performed by mass spectrometry (MS-MS) using a triple-stage quadrupole and electrospray ionization interface operating in the positive ion mode. The mean recoveries were in 18.3-45.5% range with linear responses over the 1.25-125 ng mL⁻¹ concentration range for all enantiomers evaluated. The quantification limit (LOQ) was 1.25 ng mL⁻¹. Within-day and between-day assay precision and accuracy (2.5, 50 and 100 ng mL⁻¹) showed relative standard deviation and the relative error lower than 11.9% for all enantiomers evaluated. Finally, the method was successfully used for the determination of mirtazapine and its metabolite enantiomers in plasma samples obtained after single drug administration of mirtazapine to a healthy volunteer.     

Development and validation of a chiral HPLC method for rapid screening of allylic alcohol asymmetric epoxidation processes

A simple and rapid HPLC method has been developed using a polysaccharide chiral stationary phase (Chiralpak AD-H) for the resolution of glycidyl tosylate enantiomers. These compounds were obtained by asymmetric epoxidation of allyl alcohol with chiral titanium-tartrate complexes as catalyst after in situ derivatization of the intermediate glycidols. Separations were achieved using two types of mobile phase: a normal-phase (n-hexane), and a polar-phase (methanol or acetonitrile). The influence of the type and concentration of organic modifier in the mobile phase (ethanol or 2-propanol), the flow rate and the column temperature was investigated. In normal-phase mode, the optimized conditions were: n-hexane/ethanol 70/30 (v/v) at a flow rate of 1.2 mL min⁻¹ and 40 °C. In polar-phase mode, the optimized conditions were: methanol at a flow rate of 0.8 mL min⁻¹ and 20 °C. In both cases, analysis time was ≤11 min and the chiral resolution was ≥2. Nevertheless, due to the better Rs obtained in normal-phase mode, only this method was validated to avoid peaks overlapping in real samples. This method was found to be linear in the 5-300 μg mL⁻¹ range (R² > 0.999) with an LOD of 1.5 μg mL⁻¹ for both glycidyl tosylate enantiomers. Repeatability and intermediate precision at three different concentrations levels were below 0.5 and 7.2% R.S.D. for retention time and area, respectively. This method was applied successfully for the determination of glycidyl tosylate enantiomers after in situ derivatization of glycidols obtained in allylic alcohol asymmetric epoxidation processes with chiral titanium-tartrate complexes as catalysts.     

Tris(2,2'-bipyridyl)ruthenium(II)-Zirconia-Nafion composite modified electrode applied as solid-state electrochemiluminescence detector on electrophoretic microchip for detection of pharmaceuticals of tramadol, lidocaine and ofloxacin

Tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺)-Zirconia-Nafion composite modified glassy carbon disk electrode as a solid-state electrochemiluminescence (ECL) detector is successfully applied to an electrophoretic microchip system with a wall-jet configuration. Pharmaceuticals such as tramadol, lidocaine and ofloxacin were selected to characterize the performance of this microchip capillary electrophoresis (CE)-ECL detection system. Voltammetric and ECL behaviors of immobilized Ru(bpy)₃²⁺ were investigated in lidocaine system. Influences of the separation electric field to cyclic voltammograms (CVs) of the immobilized Ru(bpy)₃²⁺ were also investigated. Tramadol, lidocaine and ofloxacin can be baseline separated without any additives. The detection limits (S/N = 3) were 2.5 × 10⁻⁵ mol L⁻¹ for tramadol, 5.0 × 10⁻⁶ mol L⁻¹ for lidocaine, 1.0 × 10⁻⁵ mol L⁻¹ for ofloxacin under the sample injection of picoliters, and the linear ranges were from 5.0 × 10⁻⁵ to 2.5 × 10⁻³ mol L⁻¹ for tramadol, 1.0 × 10⁻⁵ to 1.0 × 10⁻³ mol L⁻¹ for lidocaine, and 1.0 × 10⁻⁵ to 2.5 × 10⁻³ mol L⁻¹ for ofloxacin, respectively.     

Method development and validation for the separation and determination of omeprazole enantiomers in pharmaceutical preparations by capillary electrophoresis

A new capillary zone electrophoresis (CZE) method for the separation of omeprazole enantiomers has been developed. Methyl-β-cyclodextrin (methyl-β-CD) was chosen as the chiral selector, and several parameters, such as cyclodextrin structure and concentration, buffer concentration, pH, and capillary temperature were investigated in order to optimize separation and run times. Analysis times, shorter than 8 min were found using a background electrolyte solution consisting of 40 mM phosphate buffer adjusted to pH 2.2, 30 mM β-cyclodextrin and 5 mM sodium disulphide, hydrodynamic injection, and 15 kV separation voltage. Detection limits were evaluated on the basis of baseline noise and were established 0.31 mg/l for the omeprazole enantiomers. The proposed method was applied to five pharmaceutical preparations with recoveries between 84 and 104% of the labeled contents.     

毛细管电泳/非接触式电导法分离检测氧氟沙星对映体

采用毛细管电泳-电容耦合非接触式电导(CE-C4D),以20 mmol/L HAc + 6 mmol/L NaAc+12 mg/L羟丙基甲基纤维素(HPMC)+35 mmol/L羟丙基-β-环糊精(HP-β-CD)为电泳运行液,在熔融石英毛细管柱(45 cm×50 μm i.d.,有效长度 40 cm)中,正高压分离,手性药物氧氟沙星对映体获得良好的基线分离,线性检测范围为0.8～40 mg/L,检出限为0.3 mg/L.考察了电泳运行液组成、二元手性选择剂(HP-β-CD和HPMC)的浓度、进样方式和样品基质等对灵敏度和分离度的影响.本方法应用于市售外消旋和左旋氧氟沙星片剂中对映体的分离测定.     

Enhanced separation of seven quinolones by capillary electrophoresis with silica nanoparticles as additive

This paper describes the enhanced separation of lomefloxacin, sparfloxacin, fleroxacin, norfloxacin, ofloxacin, gatifloxacin and pazufloxacin by capillary zone electrophoresis (CZE) using silica nanoparticles (SiNPs) as running buffer additive. The impact of SiNPs concentration on the resolution and selectivity of separation was investigated and a given value of SiNPs was finally chosen under the optimum conditions. The addition of the SiNPs to the running buffer enabled electroosmotic flow (EOF) decrease and permitted full interaction between SiNPs and analytes. The influence of separation voltage, pH and buffer concentration on the separation in the presence of SiNPs was examined. Interactions between drugs and nanoparticles during the separation are discussed; the determination of interaction constants is also achieved. A good resolution of seven quinolones was obtained within 15 min in a 50 cm effective length fused-silica capillary at a separation voltage of +10 kV in a 12 mM disodium tetraborate-phosphate buffer (pH 9.08) containing 5.2 μg mL⁻¹ SiNPs.     

Stereoselective Determination of Ornidazole Enantiomers in Human Plasma and Urine Samples by Chiral LC: Application to Pharmacokinetic Study

A stereoselective liquid chromatographic method to determine the enantiomers of ornidazole in human plasma and urine has been developed and validated. After addition of the internal standard (naproxen), samples were acidified and extracted with diethyl ether. The separation was performed on a Chiralcel OB-H column, using hexane-ethanol- glacial acetic acid (94:6:0.08, v/v) as the mobile phase. The method was validated for specificity, linearity, sensitivity, precision, accuracy and stability. For each enantiomer of ornidazole, linear calibration curves were obtained over the concentration range of 0.16–20 μg mL^?1 in plasma and 0.32–20 μg mL^?1 in urine. For both enantiomers of ornidazole in plasma and urine, the coefficient of variation for precision were consistently less than 12% and accuracy were within ±14% in terms of relative error. Application of the method to a preliminary pharmacokinetic study showed that this validated method was qualified for the direct determination of ornidazole enantiomers in human plasma and urine.     

Stereoselective quantitation of mecoprop and dichlorprop in natural waters by supramolecular solvent-based microextraction,chiral liquid chromatography and tandem mass spectrometry

Liquid chromatography (LC)/tandem mass spectrometry (MS/MS) after supramolecular solvent-based microextraction (SUSME) was firstly used in this work for the enantioselective determination of chiral pesticides in natural waters. The method developed for the quantitation of the R- and S-enantiomers of mecoprop (MCPP) and dichlorprop (DCPP) involved the extraction of the herbicides in a supramolecular solvent (SUPRAS) made up of reverse aggregates of dodecanoic acid (DoA), analyte re-extraction in acetate buffer (pH = 5.0), separation of the target enantiomers on a chiral column of permethylated α-cyclodextrin under isocratic conditions, and detection of the daughter ions (m/z = 140.9 and 160.6 for MCPP and DCPP, respectively) using a hybrid triple quadrupole mass spectrometer equipped with an electrospray source operating in the negative ion mode. Similar recoveries (ca. 75%) and actual concentration factors (ca. 94) were obtained for both phenoxypropanoic acids (PPAs). The quantitation limits were 1 ng L⁻¹ for R- and S-MCPP, and 4 ng L⁻¹ for R- and S-DCPP, and the precision, expressed as relative standard deviation (n = 6) was in the ranges 2.4–2.7% ([R-MCPP] = [S-MCPP] = 5 ng L⁻¹ and [R-DCPP] = [S-DCPP] = 15 ng L⁻¹) and 1.6–1.8% (100 ng L⁻¹ of each enantiomer). The SUSME-LC–MS/MS method was successfully applied to the determination of the enantiomers of MCPP and DCPP in river and underground waters, fortified at concentrations between 15 and 180 ng L⁻¹ at variable enantiomeric ratios (ER = 1–9).     

Direct chiral resolution and its application to the determination of fungicide benalaxyl in soil and water by high-performance liquid chromatography

A simple chiral high-performance liquid chromatography (HPLC) method with ultraviolet (UV) and circular dichroism (CD) detection was developed and validated for measuring benalaxyl enantiomers using (R,R) Whelk-O 1 column. The effects of mobile phase composition and column temperature on the entioseparation were investigated. A CD detector was used to determine the elution order of the enantiomers. Excellent resolution was easily obtained using n-hexane-polar organic alcohols mobile phase. The chiral recognition mechanism was also discussed. Based on the developed chiral HPLC method, enantioselective analysis methods for this fungicide in environment matrix (soil and water) were developed and validated. Good linearities were obtained over the concentration range of 0.25-25 mg L⁻¹ for both enantiomers. Liquid-liquid extraction and solid phase extraction (SPE) were used for the enrichment and cleanup of soil and water samples. Recoveries for the two enantiomers were 79-91% at 0.02, 0.04 and 0.2 mg kg⁻¹ levels from soil, and 89-101% at 0.0025, 0.01 and 0.05 mg L⁻¹ levels from water. Run-to-run and day-to-day assay precisions were below 10% for both enantiomers at concentrations of 0.5, 1 and 5 mg L⁻¹. Individual detection limits of the two enantiomers were both 2 ng. Limits of detection (LOD) were 0.004 mg kg⁻¹ in soil and 0.001 mg L⁻¹ in water.     

Ultra-high concentration of amylose for chiral separations in capillary electrophoresis

In the present study, a capillary electrophoresis method using high concentration of amylose solutions as separation medium has been developed with the aid of dimethyl sulfoxide (DMSO) as co-solvent. The best buffer conditions for primaquine, trihexyphenidyl (THP), sulconazole and cetirizine enantiomers were optimized as 20 mM sodium phosphate buffer with DMSO/water (40/60, v/v) as solvent at a pH of 3.0, containing 10% (w/v) amylose. Partial-filling and semi-permanent coating techniques were used considering the influences of DMSO on UV detection. High chiral resolution for THP enantiomers was obtained showing good chiral separation capacity of this method. The method showed good linearity (R² > 0.998) over the concentration range of 0.50 and 2.00 mg L⁻¹ for all the enantiomers. The detection limits for the tested enantiomers were in the range from 0.05 to 0.12 mg L⁻¹. The linear calibration models were proven to be adequate for the experimental data by lack-of-fit test. The intra-assay precision, inter-day precision and accuracy were all evaluated to be acceptable. Separation and determination of THP enantiomers in rabbit blood were also carried out.     

Sensitive quantitative determination of oxymatrine and matrine in rat plasma by capillary electrophoresis with stacking induced by moving reaction boundary

A quantitative method of capillary electrophoresis with sample stacking induced by moving reaction boundary (MRB) was developed for sensitive determination of oxymatrine (OMT) and matrine (MT) in rat plasma. The experimental conditions were optimized firstly. Below are the optimized experimental conditions: 20 mM sodium formate solution (HCOONa, adjusted to pH 10.70 by ammonia) as sample solution, 3 min 14 mbar sample injection, 40 mM formic buffer (HCOOH-HCOONa, pH 2.60) as stacking buffer, 7 min 14 mbar injection of stacking buffer, 100 mM HCOOH-HCOONa (pH 4.80) as separation buffer, 73 cm capillary (effective length 64 cm), 21 kV voltage, 210 nm wavelength. Under the optimized conditions, higher than 60-fold sensitivity improvement of the stacking was simply achieved as compared with capillary zone electrophoresis, and the detectable limits obtained for OMT and MT were 0.26 and 0.19 μg mL⁻¹, respectively. Then, numerous demonstrations were carefully performed for the methodological validations of OMT and MT in rate plasma, including high specificity of method, good linearity (r = 0.9993 for OMT, r = 0.9991 for MT), fair wide linear concentration range (1.30-65.00 μg mL⁻¹ for OMT, 0.84-42.00 μg mL⁻¹ for MT), low limit of detection (1.03 μg mL⁻¹ for OMT, 0.38 μg mL⁻¹ for MT), less than 5% intra- and inter-day variance value, and higher than 96% recovery of OMT and MT in plasma. The developed method could be used for the trace analyses of OMT and MT in plasma and was finally used for the investigation on pharmacokinetic study of OMT in rat plasma.