A rapid and sensitive ultra high performance liquid chromatography with electrospray ionization tandem mass spectrometry method was developed and validated for the simultaneous determination of eight major active components (magnoflorine, menisperine, 20-hydroxyecdysone, cepharanthine, columbamine, jatrorrhizine, columbin, and palmatine) in Radix Tinosporae. The separation was performed on an InterSustainSwift C₁₈ column (1.9 μm, 2.1 id × 100 mm) at 40 °C with a gradient elution. A mixture of acetonitrile and methanol (v/v = 1:1) and ammonium acetate buffer (25 mmol/L ammonium acetate with 0.2% formic acid) were used as mobile phases, and the flow rate was set at 0.4 mL/min. The recovery was tested in real samples and calculated to be 86.97–111.28%, and all the compounds showed good linearity (r > 0.998) in relatively wide concentration ranges. The developed method was applied to the determination of eight active compounds in real herb samples, which were collected from four different places. It has been demonstrated that the proposed method has great potential for the quality control of the traditional Chinese medicine Radix Tinosporae.

In this work, we developed a novel molecularly imprinted solid-phase microextraction with capillary electrophoresis method for the selective extraction and determination of protoberberines in complicated samples. The imprinted monolith was prepared in a micropipette tip-based device by using acrylamide as the functional monomer, ethyleneglyoldimethacrylate as the cross-linker and dimethylsulfoxide as the porogen, and exhibited an imprinting factor of 2.41 to berberine, 2.36 to palmatine and 2.38 to jatrorrhizine. Good capillary electrophoresis separation was achieved by using 20 mM phosphate buffer at pH 7 as running buffer with the addition of organic modifier of 10% methanol. Parameters such as sample pH value, sample flow rate and sample volume were investigated for imprinted monolith-based solid-phase microextraction. An imprinted solid-phase microextraction with capillary electrophoresis method was developed, the method showed a wide linear range (0.3–50 μg/mL), good linearity (R² ≥ 0.9947) and good reproducibility (relative standard deviations ≤ 0.73%), the limit of detection was as low as 0.1 μg/mL, which was lower than some reported methods based on capillary electrophoresis for protoberberines. The method has been applied for determination of three common protoberberines in Cortex Phellodendri Chinensis, by using a molecularly imprinted monolith as the selective sorbent, most of the matrices in the Cortex Phellodendri Chinensis sample were removed and three protoberberines were selectively enriched and well determined.

A capillary-electrophoresis-based method for the screening of tyrosinase inhibitors in traditional Chinese medicines was developed. The method integrated electrophoretically mediated microanalysis with sandwich mode injection, partial filling, and rapid polarity switching techniques, and carried out on-column enzyme reaction and the separation of substrate and product. The conditions were optimized including the background electrolyte, mixing voltage, and the incubation time. Finally, the screening of nine standard natural compounds of traditional Chinese medicines was carried out. The inhibitors can be directly identified from the reduced peak area of the product compared to that obtained without any inhibitor. Chlorogenic acid (100 μM) showed inhibitory activity with the inhibitory percentage of 19.8%, while the other compounds showed no inhibitory activity. This method has great application potential in drug discovery from traditional Chinese medicines.

In this work, a novel magnetic nanomaterial functionalized with a molecularly imprinted polymer was prepared for the extraction of protoberberine alkaloids. Molecularly imprinted polymers were made on the surface of Fe₃O₄ nanoparticles by using berberine as template, acetonitrile/water as porogen, acrylamide as functional monomer and ethylene glycol dimethacrylate as cross-linker. The optimized molar ratio of template/functional monomer was 1:7. The polymeric magnetic nanoparticles were characterized by transmission electron microscopy and Fourier transform infrared spectroscopy. The stability and adsorption capacity of the molecularly imprinted polymers were investigated. The molecularly imprinted polymers were used as a selective sorbent for the magnetic molecularly imprinted solid-phase extraction and determination of jatrorrhizine, palmatine, and berberine. Extraction parameters were studied including loading pH, sample volume, stirring speed, and extraction time. Finally, a magnetic molecularly imprinted solid-phase extraction coupled to high-performance liquid chromatography method was developed. Under the optimized conditions, the method showed good linear range of 0.1–150 ng/mL for berberine and 0.1–100 ng/mL for jatrorrhizine and palmatine. The limit of detection was 0.01 ng/mL for berberine and 0.02 ng/mL for jatrorrhizine and palmatine. The proposed method has been applied to determine protoberberine alkaloids in Cortex phellodendri and rat plasma samples. The recoveries ranged from 87.33–102.43%, with relative standard deviation less than 4.54% in Cortex phellodendri and from 102.22–111.15% with relative standard deviation less than 4.59% in plasma.

The selective extraction of baicalin is important to its quality control especially when the matrices are complicated. In this work, a novel molecularly imprinted polymer was prepared for the selective extraction of baicalin in herbs. The molecularly imprinted polymer was synthesized by the copolymerization of 4-vinyl pyridine and ethylene glycol dimethacrylate in the presence of baicalin by a precipitation polymerization method. After the optimization of parameters for molecularly imprinted polymer preparation, including the functional monomer, porogen, sampling solvent, and washing solvent, good selectivity was obtained, with an imprinting factor of about 4, which is much better than that achieved by the bulk-polymerization method. The performances of the prepared molecularly imprinted polymers were systematically investigated, including adsorption kinetics, isotherm experiment, and Scatchard analysis. On the basis of the good adsorptive capability of the prepared molecularly imprinted polymer, it was also applied for the pretreatment of baicalin in Scutellaria baicalensis Georgi. The result showed that most of the matrices were removed and baicalin was selectively enriched.

Sarcosine, a potential biomarker of prostate cancer, has drawn great attention in recent years. However, controversial research keeps arising about its role as a biomarker that might come from the two isomers (α-alanine and β-alanine) of sarcosine due to their same molecular weight and similar properties, which could interfere with the accurate detection of sarcosine. In this study, a simple and sensitive method was developed for the detection of sarcosine and the two isomers by LC with ion-trap MS through a novel derivatization reagent N,N′-dicyclohexylcarbodiimide. N,N′-Dicyclohexylcarbodiimide is usually considered as a condensation reagent, however, it was directly used as a derivatization reagent through a rearrangement side reaction in this study. The proposed method not only improved the chromatographic retention behavior of sarcosine and its two isomers, which was a benefit to their separation, but also dramatically enhanced the detection sensitivity of sarcosine, which was more favorable for real sample analysis. The factors affecting the productivity of the derivatization reaction, such as reaction time and amount of derivatization reagent, were systematically optimized. The method shows good linearity (R² > 0.99), sensitivity with LODs of sarcosine as low as 1 ng/mL, and repeatability with the RSD < 6.07%. The developed method was applied to the analysis of urine.

Determination of bioactive compounds in traditional Chinese medicines and biological samples is usually interfered with by coexisting components in matrices. In this work, we prepared novel multilayer functional graphene/polydopamine-modified polytetrafluoroethylene microtube for selective solid-phase microextraction of three bioactive compounds in Fructus Psoraleae. Functional graphene/polydopamine-modified polytetrafluoroethylene microtube showed good extraction efficiency toward bavachin, isobavachalcone, and bavachinin; enrichment from 357- to 737-fold was obtained for these compounds. For qualitative analysis, an online solid-phase microextraction with high-performance liquid chromatography method was developed, which showed low limits of detection of 0.02 ng/mL by using UV detection, which is significantly more sensitive than previously reported methods. The proposed method has been used to determine bavachin, isobavachalcone, and bavachinin in Fructus Psoraleae, the contents of three compounds were quantified to be 64.0, 324.0, and 384.5 μg/g; recoveries were 93.4–101.1%. The proposed method has also been applied to determine bavachin, isobavachalcone, and bavachinin in rat plasma samples after oral administration of Fructus Psoraleae.

A nonaqueous CE-IT MS with a nanospray ionization interface method was developed for the identification and quantification of tetrandrine (TET), fangchinoline (FAN), and sinomenine (SIN) using berberine as internal standard. The TET, FAN, and SIN standard solutions were directly infused into IT-MS for collecting MS^1–3 spectra. The major fragment ions of analytes were confirmed and possible main cleavage pathways of fragment ions were studied. A bare fused-silica capillary was used for separation of the analytes. A sheath liquid (50% aqueous methanol containing 0.2% acetic acid) to the capillary effluent with a nanoelectrospray ionization interface was added. Separation buffer comprised 80 mM solution of ammonium acetate, in a mixture of 70% methanol, 20% ACN, and 10% water, which also contained 1% acetic acid. The CE-MS method was validated for linearity, sensitivity, accuracy, and precision, and then used to determine the content of the above components. The detection limits of TET, FAN, and SIN are 0.05, 0.08, and 0.15μg/mL, respectively. The precision was no more than 4.67% and the mean recovery of the analytes were 95.36–99.24%. This method was successfully applied to determine TET, FAN, and SIN in real samples radix Stephaniae tetrandrae and rhizomes of Menispermum dauricum.

The Amaryllidaceae are widely distributed medical plants. Lycorine, lycoramine, lycoremine, and lycobetaine are the major active alkaloids in Amaryllidaceae plants. A nonaqueous CE ESI-IT-MS method for separation, identification, and quantification of the Amaryllidaceae alkaloids has been developed. The MS^1–3 behavior has been studied and the fragmentation pathways of main fragment ions have been proposed. The effects of several factors such as composition and concentration of buffer, applied voltage, composition, and flow rate of the sheath liquid, nebulizing gas pressure, flow rate, and temperature of drying gas were investigated. Under the optimal conditions, the linear concentration range of these compounds was wide with the correlation coefficient (R²) >0.99. RSDs of migration time and peak areas were <10%. The LODs were <240 ng/mL. The proposed method can be successfully applied to the determination of the related alkaloids in the Lycoris radiata roots.

Jasmonic acid (JA) conjugates with amino acids (AAs) are a group of plant hormone in the family of jasmonates. The separation of stereoisomers of JA-AA conjugates is a very challenging work since these stereoisomers have similar chromatographic and electrophoretic behavior. Simultaneous separation of ten (±)-JA conjugates with five AAs including l-Tyr (tyrosine), l-leucine, l-Ile (isoleucine), l-valine, and l-phenylalanine and their stereoisomers has been achieved by MEKC with diode array detector in this work. Optimum separation of the analytes was obtained on a 61.5 cm × 75 μm id capillary using a running buffer containing 80 mM SDS and 50 mM phosphates (pH 7.0) at +18 kV applied voltage and capillary temperature of 35°C. Ten stereoisomers of JA conjugates with five AAs are completely separated in 13 min. The RSDs of the migration times and peak areas of the ten stereoisomers were in the range of 0.48–1.03% and 1.03–2.07%, respectively. In the tested concentration range, good linear relationships (correlation coefficients above 99%) between peak areas and concentrations of the analytes were observed. The proposed method has been successfully applied to the analysis of spiked rice floret sample and original reaction solution of (±)-JA-Ile conjugate and (±)-JA-Tyr conjugate. The recoveries ranged from 91.7 to 107.6% for the rice floret sample and 92.9 to 107.2% for the original reaction solution.

An electrophoretically mediated microanalysis method with partial filling technique was developed for screening aromatase inhibitors in traditional Chinese medicine. The in-capillary enzymatic reaction was performed in 20 mM sodium phosphate buffer (pH 7.4), and sodium phosphate buffer (20 mM, pH 8.0) was used as a background electrolyte. A long plug of coenzyme reduced β-nicotinamide adenine dinucleotide 2′-phosphate hydrate dissolved in the reaction buffer was hydrodynamically injected into a fused silica capillary followed by the injection of reaction buffer, enzyme, and substrate solution. The reaction was initiated with a voltage of 5 kV applied to the capillary for 40 s. The voltage was turned off for 20 min to increase the product amount and again turned on at a constant voltage of 20 kV to separate all the components. Direct detection was performed at 260 nm. The enzyme activity was directly assayed by measuring the peak area of the produced β-nicotinamide adenine dinucleotide phosphate and the decreased peak area indicated the aromatase inhibition. Using the Lineweaver–Burk equation, the Michaelis–Menten constant was calculated to be 50 ± 4.5 nM. The method was applied to the screening of aromatase inhibitors from 15 natural products. Seven compounds were found to have potent AR inhibitory activity.

Selective extraction of bioactive compounds in natural products and traditional medicines is difficult but necessary for drug analysis and new drug development. In this study, baicalin-imprinted polymer particles were prepared for selective recognition and extraction of baicalin from herbs and traditional Chinese medicines. The molecularly imprinted polymer (MIP) was synthesized by co-polymerization of 4-vinylpridine and ethylene glycol dimethacrylate in the presence of baicalin with a ratio of 8 : 40 : 1, where N,N-dimethyl formamide/acetonitrile served as porogen. The prepared MIP particles showed good selectivity by comparing with non-imprinted polymer and possessed an imprinted factor of 2.17; selectivity to other molecules was also tested by extracting an analogue, naringin. The performances of the MIP particles were systematically investigated, including adsorption kinetics, isotherm experiment, and Scatchard analysis. The imprinted particles were also applied to extract baicalin from natural herb Plantago asiatica L, and traditional Chinese medicine Longdanxiegan bolus. The results showed that baicalin in these complex matrixes could be selectively extracted and well isolated with other components. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 1873–1878, 2013

Bavachin and isobavachalcone are the isomeric compounds in the Fructus Psoraleae. The ion trap mass spectrometric fragmentation pathways of the bavachin and isobavachalcone in negative ion mode were elucidated for the identification. A novel method for determination of isomeric bavachin and isobavachalcone has been developed by capillary electrophoresis coupled with mass spectrometric detection. The effects of several factors such as concentration, pH of ammonium acetate buffer, separation voltage, composition and flow rate of the sheath liquid were investigated. Under optimal conditions, the linear concentration range for bavachin and isobavachalcone were 0.8–100 μg/mL with the correlation coefficient of 0.996 and 0.995, respectively. Relative standard deviations of migration time and peak areas were lower than 5%. The limits of detection (signal/noise = 3) were 60 ng/mL. The proposed method can be successfully applied to the determination of bavachin and isobavachalcone in the Fructus Psoraleae and six Fructus Psoraleae-containing preparations.

A simple, sensitive and selective method of high-performance liquid chromatography with electrochemical detection (HPLC-ECD) has been developed for simultaneous determination of bavachin and isobavachalcone in Fructus Psoraleae. At optimized conditions, bavachin and isobavachalcone could be well separated within 15 min at a detection potential of +0.80 V with 0.03 mol/L acetate buffer solution (pH 5.17)/acetonitrile (2:3, v/v) as the mobile phase. The relationships between peak areas and concentrations were linear from 8.26×10⁻⁷ to 1.21×10⁻⁴ mol/L for bavachin, and from 1.01×10⁻⁸ to 1.61×10⁻⁴ mol/L for isobavachalcone, respectively. The method offered excellent linearity with regression coefficient R²>0.995. The method presented detection limits (S/N=3) of 8.81×10⁻⁹ mol/L for bavachin and 1.17×10⁻¹⁰ mol/L for isobavachalcone. It indicates that the sensitivity of electrochemical detection is ten times higher than that of diode array detection (DAD). The mean recoveries around 98% with a relative standard deviation less than 3.1% for the two analytes have been obtained. The proposed separation and detection procedures were successfully applied to the simultaneous determination of bavachin and isobavachalcone in traditional Chinese medicine.

Catharanthus roseus is an important dicotyledonous medicinal plant that contains various anticancer components, such as vinblastine (VLB) and its monomeric precursors (vindoline and catharanthine). A capillary electrophoresis–mass spectrometry (CE-MS) approach for the simultaneous determination of three components was developed in this work. Baseline separation for three components was achieved by using a running buffer consisting of 20 mM ammonium acetate and 1.5% acetic acid in <20 min. Quantification of three components was assigned in positive-ion mode at a protonated molecular ion [M+H]⁺. The CE-MS method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the above components. The detection limits of VLB, catharanthine and vindoline are 0.8, 0.1 and 0.1 μg/mL, respectively. The precision was not more than 4.54% and the mean recovery of the analytes was 95.04–97.04%. The CE-MS method was successfully applied to determine VLB and its monomeric precursors in real sample C. roseus.

The development of new analytical techniques for identifying endogenous peptides in a cell, organ, or organism (the peptidome) is an important work in the fields of peptidomics and proteomics. MS is currently employed as a powerful analytical technique in peptidomics. However, it has a limitation in the identification of peptides with the same amino acid composition but different sequences, as these peptides have the same molecular weights, and generate the same MS spectrum of molecular ions, when MS is used as the detection technique. Therefore, the development of peptide separation techniques like CE and LC coupled with MS is desirable. In this paper, a methodology for the separation of peptides with the same amino acid composition, but different sequences, by CE has been developed using two tripeptides, Gly-Ser-Phe and Gly-Phe-Ser, as a model sample. Excellent separation selectivity was achieved using the separation mode of MEKC. Separation behavior under different conditions and mechanism is discussed.

In order to accomplish the analysis of peptides and proteins by capillary electrophoresis, Lupamin, a high-molecular-weight linear polyvinylamine (PVAm) polymer, was introduced to modify the inner wall of fused-silica capillaries by physical absorption. Thanks to the high density of positively charged amino groups in Lupamin under acidic conditions, not only is a strong reversed electroosmotic flow generated in the coated capillary but the adsorption of analytes on the inner wall of the capillary is also efficiently eliminated. It has been demonstrated that the Lupamin-coated capillary can be used to advantage for the rapid analysis of amino acids, peptides, and proteins with good resolution and peak shape by capillary electrophoresis. In order to evaluate the basic feature of a Lupamin-coated capillary, electroosmotic flows generated by a Lupamin coating layer under different conditions including pH, coating time, concentration, and the composition of electrolytes on Lupamin-coated and uncoated capillaries were investigated. Furthermore, electrospray ionization-mass spectrometry (ESI-MS) detection was carried out for the analysis of amino acids and peptides.