Analysis of alkaloids in Coptis chinensis Franch by accelerated solvent extraction combined with ultra performance liquid chromatographic analysis with photodiode array and tandem mass spectrometry detections

A new method based on accelerated solvent extraction (ASE) followed by ultra performance liquid chromatography (UPLC) analysis has been developed for the identification and quantification of major alkaloids in extracts of Coptis chinensis Franch. The UPLC system consisted of a dual detection system of photodiode array detector (PDA) and positive ion electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in sequential configuration. The operational parameters of ASE including extraction solvent, extraction temperature, static extraction time and extraction cycles were optimized. UPLC analysis was performed on an ACQUITY UPLC BEH C₁₈ column eluted by a mobile phase of acetonitrile spiked with a buffer solution consisting of 0.50% acetic acid and 20 mmol L⁻¹ ammonium acetate. A tandem quadrupole spectrometer operating in either full scan mode or in MS/MS mode for multiple reaction monitoring (MRM) was used for the identification and quantitative analysis of eight major alkaloids in C. chinensis Franch extracts. The samples were also analyzed on a high-performance liquid chromatography-electrospray ionization-time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) system to confirm the identification results. Three of the eight major alkaloids, berberine, palmatine and jatrorrhizine were quantified by UPLC-PDA and UPLC-MS/MS. The results indicated that both UPLC-PDA and UPLC-MS/MS methods were simple, sensitive and reliable for the determination of alkaloids in C. chinensis Franch. Seven Huanglian samples from different locations were analyzed using the established methods. UPLC fingerprints based on the distribution of the eight major alkaloids can serve as a rapid and reliable method for the authentication and quality evaluation of traditional Chinese medicine (TCM) herbs.     

Quality evaluation and species differentiation of Rhizoma coptidis by using proton nuclear magnetic resonance spectroscopy

Rhizoma coptidis, a broadly used traditional Chinese medicine, derives from the dried rhizomes of Coptis chinensis Franch, Coptis deltoidea C.Y. Cheng et Hsiao and Coptis teeta Wall. Quantitative determination of protoberberine alkaloids in R. coptidis is critical for controlling its quality. In this study, a rapid, simple and accurate quantitative ¹H NMR (qNMR) method was developed for simultaneous determination of berberine, jatrorrhizine, epiberberine, coptisine, palmatine and columbamine in R. coptidis from the three species. Method validation was performed in terms of selectivity, precision, repeatability, stability, accuracy, robustness and linearity. The average recoveries obtained were in the range of 96.9–102.4% for all the six alkaloids. In addition, the qNMR data were analyzed with analysis of variance (ANOVA), hierarchical clustering analysis (HCA) and principal component analysis (PCA), and the results showed that the contents of the active alkaloids have significant difference among the three species. Compared with the conventional HPLC approach, the proposed qNMR method was demonstrated to be a powerful tool for quantifying the six alkaloids due to its unique advantages of high robustness, rapid analysis time and no need of standard compounds for calibration curves preparation. These findings indicate that this method has potential as a reliable method for quality evaluation of herb medicines, especially for protoberberine alkaloid-containing ones.     

Optical sensor for berberine utilizing its intrinsic fluorescence enhanced by the formation of inclusion complex with butylated-β-cyclodextrin

An optical sensor for berberine, the basic ingredient of the widely used traditional Chinese medicine Coptis Chinensis, based on its intrinsic fluorescence enhanced by butylated-β-cyclodextrin (HDB-β-CD) immobilized in plasticized poly(vinyl chloride) (PVC) membrane has been developed. The drastic enhancement of fluorescence intensity of berberine was attributed to the formation of an inclusion complex between HDB-β-CD and berberine, which has been utilized as the basis of the fabrication of a berberine-sensitive fluorescence sensor. The proposed sensor was quite distinct from those fluorescent sensors for berberine reported so far which relied upon quenching the fluorescence of the sensing reagent immobilized on membrane by berberine. The response mechanism of optode membrane was discussed in detail from the view of molecular dynamics and the optimum steric configuration of the inclusion complex was presented by molecular dynamics simulation. The analytical performance characteristics of the proposed berberine-sensitive sensor were investigated. The sensor can be applied to the quantification of berberine with a linear range covering from 4.0×10⁻⁷ to 2.0×10⁻⁵ mol l⁻¹ with a detection limit of 8.0×10⁻⁸ mol l⁻¹. The sensor exhibits excellent reproducibility, reversibility and selectivity. The recommended method was successfully used for the determination of berberine in pharmaceutical preparations.     

Determination of ethylenediaminetetraacetic acid, ethylenediaminedisuccinic acid and iminodisuccinic acid in cosmetic products by capillary electrophoresis and high performance liquid chromatography

A capillary electrophoresis (CE) and a high performance liquid chromatography (HPLC) method are described for the simultaneous determination of ethylenediaminetetraacetic acid (EDTA), S,S′-ethylenediaminedisuccinic acid (EDDS) and R,S-iminodisuccinic acid (IDS) complexing agents as their Fe(III) complexes in cosmetics like shower cream and foam bath. The non-biodegradable EDTA is used in combination with biodegradable analogues like EDDS and IDS in many commercial products. The HPLC method involves separation by reversed-phase ion pair chromatography on a C₁₈ column using methanol-formate buffer (20 mM tetrabutylammonium hydrogen sulfate, 15 mM sodium formate adjusted to pH 4.0 with formic acid) (10:90, v/v) as mobile solvent at a flow rate of 0.8 mL min⁻¹ at 24 °C using UV detection at 240 nm. The CE separation was performed in a fused silica capillary of 50 μm i.d. with the total length of 50 cm with a 10 mM MES and MOPSO (pH 5.5) at an applied voltage of −25 kV. The samples were introduced by applying a 50 mbar pressure for 2 s. Absorbances at 215 and 225 nm were monitored for the detection of the complexes. The methodology performance of the two methods was evaluated in terms of linearity, limit of detection (LOD), limit of quantitation (LOQ) and reproducibility. The LOD values obtained from HPLC are low when compared with CE. The applicability of both the methods was demonstrated for the analysis of cosmetic products such as shower cream and foam bath. The results obtained by both CE and HPLC were found to be comparable and in good agreement.     

Determination of quinolizidine alkaloids in Sophora medicinal plants by capillary electrophoresis

A new capillary electrophoresis (CE) method for the determination of quinolizidine alkaloids in Sophora medicinal plants was developed. A total of seven alkaloid components (cytisine, sophocarpine, matrine, lehmannine, sophoranol, oxymatrine and oxysophocarpine) were separated within 15 min. The running buffer was a 50 mM phosphate buffer containing 1%HP-β-CD and 3.3% isopropanol. The linear calibration ranges were 5.50-88.0 μg ml⁻¹ for cytisine and lehmannine, 5.00-88.0 μg ml⁻¹ for sophocarpine and sophoranol, 5.60-89.6 μg ml⁻¹ for matrine and oxysophocarpine, and 24.0-384 μg ml⁻¹ for oxymatrine. The recoveries of the seven alkaloids were 96.0-102.9% with relative standard deviations from 1.50 to 3.00% (n = 5). The method was successfully applied to different Sophora medicinal plants including Sophora flavescens, Sophora tonkinensis and Sophora alopecuroides.     

Quantification of isoorientin and total flavonoids in Passiflora edulis fruit pulp by HPLC-UV/DAD

A method is reported for the quantification of isoorientin (using a standard addition method) and total flavonoids (expressed as rutin, using the external standard method) in passion fruit pulp (Passiflora edulis Sims f. flavicarpa Degener, Passifloraceae). Extraction of flavonoids was optimized by experimental design methodology, and quantitative analysis was performed by high-performance liquid chromatography with photo-diode array detection (HPLC-UV/DAD). The method was developed and validated according to ICH requirements for specificity, linearity, accuracy, precision (repeatability and intermediate precision), LOD and LOQ. Rutin was chosen as standard for the quantification of total flavonoids in order to propose a HPLC method feasible for routine analysis of the flavonoids in the passion fruit pulp. The passion fruit pulp contained 16.226 ± 0.050 mg L^− 1 of isoorientin and 158.037 ± 0.602 mg L^− 1 of total flavonoid, suggesting that P. edulis fruits may be comparable with other flavonoid food sources such as orange juice or sugarcane juice.     

Comparison of laser ablation-inductively coupled plasma-mass spectrometry and micro-X-ray fluorescence spectrometry for elemental imaging in Daphnia magna

Visualization of elemental distributions in thin sections of biological tissue is gaining importance in many disciplines of biological and medical research. Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and scanning micro-X-ray fluorescence spectrometry (micro-XRF) are two widely used microanalytical techniques for elemental mapping. This article compares the capabilities of the two techniques for imaging the distribution of selected elements in the model organism Daphnia magna in terms of detection power and spatial resolution. Sections with a thickness of 10 and 20 μm of the fresh water crustacean Daphnia magna were subjected to LA-ICP-MS and micro-XRF analysis. The elemental distributions obtained for Ca, P, S and Zn allow element-to-tissue correlation. LA-ICP-MS and micro-XRF offer similar limits of detection for the elements Ca and P and thus, allow a cross-validation of the imaging results. LA-ICP-MS was particularly sensitive for determining Zn (LOD 20 μg g⁻¹, 15 μm spot size) in Daphnia magna, while the detection power of micro-XRF was insufficient in this context. However, LA-ICP-MS was inadequate for the measurement of the S distributions, which could be better visualized with micro-XRF (LOD 160 μg g⁻¹, 5 s live time). Both techniques are thus complementary in providing an exhaustive chemical profiling of tissue samples.     

Simple hollow fiber renewal liquid membrane extraction method for pre-concentration of Cd(II) in environmental samples and detection by Flame Atomic Absorption Spectrometry

A hollow fiber renewal liquid membrane (HFRLM) extraction method to determine cadmium (II) in water samples using Flame Atomic Absorption Spectrometry (FAAS) was developed. Ammonium O,O-diethyl dithiophosphate (DDTP) was used to complex cadmium (II) in an acid medium to obtain a neutral hydrophobic complex (ML₂). The organic solvent introduced to the sample extracts this complex from the aqueous solution and carries it over the poly(dimethylsiloxane) (PDMS) membrane, that had their walls previously filled with the same organic solvent. The organic solvent is solubilized inside the PDMS membrane, leading to a homogeneous phase. The complex strips the lumen of the membrane where, at higher pH, the complex Cd-DDTP is broken down and cadmium (II) is released into the stripping phase. EDTA was used to complex the cadmium (II), helping to trap the analyte in the stripping phase. A multivariate procedure was used to optimize the studied variables. The optimized variables were: sample (donor phase) pH 3.25, DDTP concentration 0.05% (m/v), stripping (acceptor phase) pH 8.75, EDTA concentration 1.5 × 10⁻² mol L⁻¹, extraction temperature 40 °C, extraction time 40 min, a solvent mixture N-butyl acetate and hexane (60/40%, v/v) with a volume of 100 μL, and addition of ammonium sulfate to saturate the sample. The sample volume used was 20 mL and the stripping volume was 165 μL. The analyte enrichment factor was 120, limit of detection (LOD) 1.3 μg L⁻¹, relative standard deviation (RSD) 5.5% and the working linear range 2-30 μg L⁻¹.     

Copper nanoparticles entrapped in SWCNT-PPy nanocomposite on Pt electrode as NOx electrochemical sensor

A highly sensitive NO_x sensor was designed and developed by electrochemical incorporation of copper nanoparticles (CuNP) on single-walled carbon nanotubes (SWCNT)-polypyrrole (PPy) nanocomposite modified Pt electrode. The modified electrodes were characterized by scanning electron microscopy and energy dispersive X-ray analysis. Further, the electrochemical behavior of the CuNP-SWCNT-PPy-Pt electrode was investigated by cyclic voltammetry. It exhibited the characteristic CuNP reversible redox peaks at −0.15 V and −0.3 V vs. Ag/AgCl respectively. The electrocatalytic activity of the CuNP-SWCNT-PPy-Pt electrode towards NO_x is four-fold than the CuNP-PPy-Pt electrode. These results clearly revealed that the SWCNT-PPy nanocomposite facilitated the electron transfer from CuNP to Pt electrode and provided an electrochemical approach for the determination of NO_x. A linear dependence (r² = 0.9946) on the NO_x concentrations ranging from 0.7 to 2000 μM, with a sensitivity of 0.22 ± 0.002 μA μM⁻¹ cm⁻² and detection limit of 0.7 μM was observed for the CuNP-SWCNT-PPy-Pt electrode. In addition, the sensor exhibited good reproducibility and retained stability over a period of one month.     

Zwitterionic hydrophilic interaction chromatography coupled with post-column derivatization for the analysis of glutathione in wine samples

In this study the development, validation and application of a new chromatographic method for the determination of glutathione (GSH) in wine samples is presented. The separation of the GSH was carried out using a sulfobetaine-based hydrophilic interaction chromatography (HILIC) analytical column whereas its detection was carried out spectrofluorimetrically (λ_ext/λ_em = 340/455 nm) after post-column derivatization with o-phthalaldehyde. GSH was separated efficiently from matrix endogenous compounds of wines by using a mobile phase of 15 mmol L⁻¹ CH₃COONH₄ (pH = 2.5)/CH₃CN, 35/65% (v/v). The parameters of the post-column reaction (pH, amount concentration of the reagent and buffer solution, flow rate, length of the reaction coil) were investigated. The linear determination range for GSH was 0.25–5.0 μmol L⁻¹ and the LOD was 19 nmol L⁻¹. No matrix effect was observed, while the accuracy was evaluated with recovery experiments and was ranged between 89% and 108%.     

A environmentally friendly reversed-phase liquid chromatography method for phthalates determination in nail cosmetics

A simple and rapid analytical method was developed for the determination of phthalates, usually employed in nail cosmetic products. The method is based on an ultrasonic extraction of the sample with ethanol-water (90:10, v/v) followed by HPLC separation and quantitation. HPLC was carried out using a C18 column and spectrophotometric detection at 254 nm. A linear gradient elution was performed with ethanol-water starting from 50 to 95% ethanol in 30 min. Standard calibration curves were linear for all the analytes over the concentration range 5-200 μg ml⁻¹ with LOD values of about 0.5 μg ml⁻¹.The proposed green analytical method has been successfully applied for the analysis of commercial samples in order to check the presence of phthalates and to determine their concentration.     

A simplified approach to the determination of N-nitroso glyphosate in technical glyphosate using HPLC with post-derivatization and colorimetric detection

A simple and sensitive HPLC post-derivatization method with colorimetric detection has been developed for the determination of N-nitroso glyphosate in samples of technical glyphosate. Separation of the analyte was accomplished using an anionic exchange resin (2.50 mm × 4.00 mm i.d., 15 μm particle size, functional group: quaternary ammonium salt) with Na₂SO₄ 0.0075 M (pH 11.5) (flow rate: 1.0 mL min⁻¹) as mobile phase. After separation, the eluate was derivatized with a colorimetric reagent containing sulfanilamide 0.3% (w/v), [N-(1-naphtil)ethilendiamine] 0.03% (w/v) and HCl 4.5 M in a thermostatized bath at 95 °C. Detection was performed at 546 nm. All stages of the analytical procedure were optimized taking into account the concept of analytical minimalism: less operation times and costs; lower sample, reagents and energy consumption and minimal waste. The limit of detection (k = 3) calculated for 10 blank replicates was 0.04 mg L⁻¹ (0.8 mg kg⁻¹) in the solid sample which is lower than the maximum tolerable accepted by the Food and Agriculture Organization of the United Nations.     

Dual-wavelength β-correction spectrophotometric determination of trace concentrations of cyanide ions based on the nucleophilic addition of cyanide to imine group of the new reagent 4-hydroxy-3-(2-oxoindolin-3-ylideneamino)-2-thioxo-2H-1,3-thiazin-6(3H)-one

A simple, fast, low cost and sensitive direct β-correction spectrophotometric assay of cyanide ions based on its reaction with the reagent 4-hydroxy-3-(2-oxoindolin-3-ylideneamino)-2-thioxo-2H-1,3-thiazin-6(3H)-one, abbreviated as HOTT in aqueous media of pH 7-10 is described. The electronic spectrum of the produced brown-red colored species showed well defined and sharp peak at λ_max = 466 nm. The effective molar absorptivity for the produced cyano compound was 2.5 × 10⁴ L mol⁻¹ cm⁻¹. Beer's law and Ringbom's plots were obeyed in the concentration range 0.05-2.0 and 0.30-1.5 μg mL⁻¹ cyanide ions, respectively. The proposed method offers 16.0 and 50.3 μg L⁻¹ lower limits of detection (LOD) and quantification (LOQ) of the cyanide ion, respectively. The analytical utility of the method for the analysis of cyanide ions in tap and drinking water samples was demonstrated and the results were compared successfully with the conventional cyanide ion selective electrode. The short time response and the detection by the naked eye make the method available for the detection and quantitative determination of cyanide in a variety of samples e.g. fresh and drinking water. Moreover, the structure of the produced colored species was determined with the aid of spectroscopic measurements (UV-Vis, IR, ¹H and ¹³C NMR) and elemental analysis.     

An effective method for fast determination of artemisinin in Artemisia annua L. by high performance liquid chromatography with evaporative light scattering detection

Artemisinin isolated from the aerial parts of Artemisia annua L., is a promising and potent antimalarial drug, which meets the dual challenge posed by drug-resistant parasites and rapid progression of malarial illness. The aim of the current study was to develop a reliable and fast analytical procedure for the determination of artemisinin in A. annua using high performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD) in couple with microwave-assisted extraction (MAE) as an efficient sample preparation technique. The HPLC conditions were Agilent C18 column using water:acetonitrile (40:60 v/v) mixture as mobile phase at a flow rate of 1 mL min⁻¹. ELSD conditions were optimized at nebulizer-gas flow rate of 2.0 L min⁻¹ and drift tube temperature of 70 °C under the impactor off-mode, and the gain was set at 2. Afterwards, method validation system for HPLC-ELSD analysis was developed. Calibration range was 0.2-1.0 mg mL⁻¹ and correlation coefficient r was above 0.9990. Precision experiments showed relative standard deviation (R.S.D.) of retention time was less than 0.5% and R.S.D. of peak area was less than 1.30%. Inter-day and intra-day variabilities showed that R.S.D. was ranged from 1.01% to 4.66%. Limit of detection was less than 40 μg mL⁻¹ and limit of quantification was less than 100 μg mL⁻¹. Accuracy validation showed that average recovery was between 98.23% and 104.97%. The developed analytical procedure was successfully applied to determine the contents of artemisinin in the different parts of A. annua plants.     

p-Aminobenzoate ion determination in pharmaceutical formulations by using a potentiometric sensor immobilized in a graphite matrix

The characteristics, performance, and application of an electrode, namely, Pt|Hg|Hg₂(PABzt)₂| graphite, where PABzt stands for p-aminobenzoate ion, are described. This electrode responds to PABzt with sensivity of (58.1±1.0) mV per decade over the range 1.0×10⁻⁴ to 1.0×10⁻¹ mol l⁻¹ at pH 6.5-8.0 and a detection limit of 3.2×10⁻⁵ mol l⁻¹. The electrode shows easy construction, fast response time (within 10-30 s), low-cost, and excellent response stability (lifetime greater than 6 months, in continuous use). The proposed sensor displayed good selectivity for p-aminobenzoate in the presence of several substances, especially, concerning carboxylate and inorganic anions. It was used to determine p-aminobenzoate in pharmaceutical formulations by means of the standard additions method. The results obtained by using this electrode compared very favorably with those given by an HPLC procedure.     

Pretreatment of plasma samples by a novel hollow fiber centrifugal ultrafiltrate device for the determination of cefaclor concentrations in human plasma

A simple sample preparation method was developed by using a centrifugal ultrafiltration (CF-UF) device with hollow fiber (HF) for the determination of cefaclor in plasma by HPLC. Samples were placed into a homemade device, which was consisted of a glass tube and a U-shaped hollow fiber. The filtrate was withdrawn from the hollow fiber into a syringe after centrifugation and 20 μL was directly injected into the HPLC for analysis. The HPLC method had a linear calibration curve in the concentration range of 6.00 × 10⁻²–30.7 μg mL⁻¹(r = 0.9996). The limit of detection (LOD) and limit of quantitation (LOQ) were 0.02 and 0.06 μg mL⁻¹, respectively. The intra and inter-day precisions (RSD) were 1.7%, 1.2%, 1.0% and 3.6%, 2.5%, 1.9%, respectively, for three concentrations. Assay accuracy was higher than 99.2% and the absolute recovery was 86.8–92.5%. It is feasible to use this novel and low cost device for sample pretreatment for the analysis of cefaclor in plasma.     

Catalytic adsorptive stripping voltammetry versus electrothermal atomic absorption spectrometry in the determination of trace cobalt and chromium in human urine

Two methods of the determination of cobalt and chromium in human urine of non-occupationally exposed populations—highly sensitive catalytic adsorptive stripping voltammetry (CAdSV) and electrothermal atomic absorption spectrometry (ET-AAS)—are evaluated and compared. The CAdSV methods are based on adsorptive accumulation of a cobalt-nioxime (1,2-cyclohexanedione dioxime) or a chromium-DTPA (diethylenetriammine-N,N,N′,N″,N″-pentaacetic acid) complexes on a hanging mercury drop electrode, followed by a stripping voltammetric measurement of the catalytic reduction current of the adsorbed complex in the presence of sodium nitrite in case of cobalt or in the presence of sodium nitrate in case of chromium determination. In the CAdSV procedure UV-photolysis was used for the sample pre-treatment; the ET-AAS determination did not require any separate preliminary decomposition of the analyte urine samples. The accuracy of the procedures was checked by the analysis of commercially available quality control urine samples. The detection limits (3σ) were 0.13 μg l⁻¹ for Co and 0.18 μg l⁻¹ for Cr in ET-AAS determination and 0.007 μg l⁻¹ for Co and 0.002 μg l⁻¹ for Cr in CAdSV measurements. Precision (R.S.D.) was less than 5% for both methods. The study has shown that the CAdSV is a more reliable and sensitive technique for the determination of very low cobalt and chromium contents in urine, the detection of which is not possible when using the AAS technique.     

A highly sensitive amperometric sensor for oxygen based on iron(II) tetrasulfonated phthalocyanine and iron(III) tetra-(N-methyl-pyridyl)-porphyrin multilayers

The development of a highly sensitive sensor for oxygen is proposed using a glassy carbon (GC) electrode modified with alternated layers of iron(II) tetrasulfonated phthalocyanine (FeTsPc) and iron(III) tetra-(N-methyl-pyridyl)-porphyrin (FeT4MPyP). The modified electrode showed excellent catalytic activity for the oxygen reduction. The reduction potential of the oxygen was shifted about 330 mV toward less negative values with this modified electrode, presenting a peak current much higher than those observed on a bare GC electrode. Cyclic voltammetry and rotating disk electrode (RDE) experiments indicated that the oxygen reduction reaction involves 4 electrons with a heterogenous rate constant (k_obs) of 3 × 10⁵ mol⁻¹ L s⁻¹. A linear response range from 0.2 up to 6.4 mg L⁻¹, with a sensitivity of 4.12 μA L mg⁻¹ (or 20.65 μA cm⁻² L mg⁻¹) and a detection limit of 0.06 mg L⁻¹ were obtained with this sensor. The repeatability of the proposed sensor, evaluated in terms of relative standard deviation (R.S.D.) was 2.0% for 10 measurements of a solution of 6.4 mg L⁻¹ oxygen. The sensor was applied to determine oxygen in pond and tap water samples showing to be a promising tool for this purpose.     

A colloidal suspension of nanostructured poly(N-butyl benzimidazole)-graphene sheets with high oxidase yield for analytical glucose and choline detections

A colloidal suspension of nanostructured poly(N-butyl benzimidazole)-graphene sheets (PBBIns-Gs) was used to modify a gold electrode to form a three-dimensional PBBIns-Gs/Au electrode that was sensitive to hydrogen peroxide (H₂O₂) in the presence of acetic acid (AcOH). The positively charged nanostructured poly(N-butyl benzimidazole) (PBBIns) separated the graphene sheets (Gs) and kept them suspended in an aqueous solution. Additionally, graphene sheets (Gs) formed “diaphragms” that intercalated Gs, which separated PBBIns to prevent tight packing and enhanced the surface area. The PBBIns-Gs/Au electrode exhibited superior sensitivity toward H₂O₂ relative to the PBBIns-modified Au (PBBIns/Au) electrode. Furthermore, a high yield of glucose oxidase (GOD) on the PBBIns-Gs of 52.3 mg GOD per 1 mg PBBIns-Gs was obtained from the electrostatic attraction between the positively charged PBBIns-Gs and negatively charged GOD. The non-destructive immobilization of GOD on the surface of the PBBIns-Gs (GOD-PBBIns-Gs) retained 91.5% and 39.2% of bioactivity, respectively, relative to free GOD for the colloidal suspension of the GOD-PBBIns-Gs and its modified Au (GOD-PBBIns-Gs/Au) electrode. Based on advantages including a negative working potential, high sensitivity toward H₂O₂, and non-destructive immobilization, the proposed glucose biosensor based on an GOD-PBBIns-Gs/Au electrode exhibited a fast response time (5.6 s), broad detection range (10 μM to 10 mM), high sensitivity (143.5 μA mM⁻¹ cm⁻²) and selectivity, and excellent stability. Finally, a choline biosensor was developed by dipping a PBBIns-Gs/Au electrode into a choline oxidase (ChOx) solution for enzyme loading. The choline biosensor had a linear range of 0.1 μM to 0.83 mM, sensitivity of 494.9 μA mM⁻¹ cm⁻², and detection limit of 0.02 μM. The results of glucose and choline measurement indicate that the PBBIns-Gs/Au electrode provides a useful platform for the development of oxidase-based biosensors.     

Determination of four major saponins in the seeds of Aesculus chinensis Bunge using accelerated solvent extraction followed by high-performance liquid chromatography and electrospray-time of flight mass spectrometry

A new method based on accelerated solvent extraction (ASE) followed by a reliable high-performance liquid chromatography-diode array detector (HPLC-DAD) and positive ion electrospray-time of flight mass spectrometry (ESI-TOF/MS) analysis has been developed for the characterization and quantification of four major saponins in extracts of the seeds of Aesculus chinensis Bunge (Semen Aesculi). The saponins escin Ia, escin Ib, isoescin Ia and isoescin Ib were extracted from seeds of A. chinesis Bunge via ASE, and the operational parameters of ASE were optimized, such as extraction solvent, extraction temperature, static extraction time and extraction cycles. The optimized procedure employed 70% MeOH as extraction solvent, 120 °C of extraction temperature, 7 min of static extraction time, 60% flush volume and the extraction recoveries of the four compounds were nearly to 100% for two cycles. The HPLC conditions are as follows: SinoChrom ODS BP C₁₈ (4.6 mm × 200 mm, 5 μm) column, acetonitrile and 0.10% phosphoric acid solution as mobile phase, flow rate is 1.0 mL min⁻¹, detection length of UV is 203 nm, injection volume is 10 μL. The results indicated that the developed HPLC method is simple, sensitive and reliable for the determination of four major saponins in seeds of A. chinesis Bunge with a good linearity (r² > 0.9994), precision (relative standard deviation (R.S.D.) <1.5%) and the recovery ranges of 95.2-97.3%. The limits of detection (LOD) of the four compounds were in the range of 0.40-0.75 μg mL⁻¹. This assay can be readily utilized as a quality control method for Semen Aesculi and other related medicinal plants.