Purification of Membrane-Bound Catechol-O-Methyltransferase by Arginine-Affinity Chromatography

Affinity chromatography strategies using amino acids as immobilize d ligands have been successfully applied for the purification of different biomolecules from complex mixtures. Therefore, in this work, several supports with immobilized amino acids were applied for the purification of membrane-bound catechol-O-methyltransferase (MBCOMT) from Pichia pastoris lysates and it was verified that l-arginine provided the required selectivity for MBCOMT isolation. The optimization of the binding and elution buffers composition allowed the recovery of purified MBCOMT in a biological and immunologically active state from the arginine support. Additional optimization experiments varying the mobile phase pH, temperature and the concentration of the injected sample were carried out and an improvement of MBCOMT adsorption and purity was observed. Indeed, the optimized conditions for MBCOMT isolation and purification consisted in: loading of 4 mg of total protein onto the column previously equilibrated at 20 °C where the target enzyme was recovered in a purified fraction using 500 mM NaCl, 10 mM DTT and 0.5 % (v/v) Triton X-100 in 10 mM Tris buffer (pH 7) with a total bioactivity recovery of 24 ± 2.2 % and a purification fold of 4.95 ± 0.23, a value that is consistent with the best values ever reported for MBCOMT. Moreover, the l-arginine support demonstrated the ability to bind the target protein in a wide range of pH values (above and below the pI of the target protein) and the MBCOMT elution occurs in a single peak pattern. Finally, the strategy here reported can aid in the implementation of crystallization studies with MBCOMT in complex with clinically relevant inhibitors since it is obtained in a purified form with biological activity. In conclusion, a novel affinity chromatography strategy was developed and implemented for recombinant MBCOMT purification in a highly immunological and biologically active state.

     

Semi‐preparative high‐speed counter‐current chromatography separation of alkaloids from embryo of the seed of Nelumbo nucifera Gaertn by pH‐gradient elution

A new high‐speed counter‐current chromatography method for semi‐preparative separation and purification of alkaloids from embryo of the seed of Nelumbo nucifera Gaertn was developed by using pH‐gradient elution mode. Diethyl ether was used as the stationary phase of the two‐phase solvent system and Na₂HPO₄/NaH₂PO₄ buffer solution with pH values of 7.5 and 7.2 in gradient mode as the mobile phase. Consequently, 33 mg of liensinine, 42 mg of isoliensinine, and 67 mg of neferine were obtained from 200 mg of crude extracts. The purities of them were all over 98% as determined by HPLC area normalization method, and the structures were identified by ¹H‐NMR and ¹³C‐NMR.     

Octyl 1‐(5‐tert‐butyl‐1H‐pyrazol‐3‐yl)‐2‐(4‐chlorophenyl)‐1H‐benzimidazole‐5‐carboxylate: complex sheets built from N—H...N,C—H...N and C—H...O hydrogen bonds

In the title compound, C₂₉H₃₅ClN₄O₂, the bond lengths provide evidence for aromatic delocalization in the pyrazole ring but bond fixation in the fused imidazole ring, and the octyl chain is folded, rather than adopting an all‐trans chain‐extended conformation. A combination of N—H...N, C—H...N and C—H...O hydrogen bonds links the molecules into sheets, in which the hydrogen bonds occupy the central layer with the tert‐butyl and octyl groups arranged on either side, such that the closest contacts between adjacent sheets involve only the octyl groups. Comparisons are made with the supramolecular assembly in some simpler analogues.     

Simultaneous determination of seven phthalic acid esters in beverages using ultrasound and vortex‐assisted dispersive liquid–liquid microextraction followed by high‐performance liquid chromatography

A sensitive, rapid, and simple high‐performance liquid chromatography with UV detection method was developed for the simultaneous determination of seven phthalic acid esters (dimethyl phthalate, dipropyl phthalate, di‐n‐butyl phthalate, benzyl butyl phthalate, dicyclohexyl phthalate, di‐(2‐ethylhexyl) phthalate, and di‐n‐octyl phthalate) in several kinds of beverage samples. Ultrasound and vortex‐assisted dispersive liquid–liquid microextraction method was used. The separation was performed using an Intersil ODS‐3 column (C₁₈, 250 × 4.6 mm, 5.0 μm) and a gradient elution with a mobile phase consisting of MeOH/ACN (50:50) and 0.2 M KH₂PO₄ buffer. Analytes were detected by a UV detector at 230 nm. The developed method was validated in terms of linearity, limit of detection, limit of quantification, repeatability, accuracy, and recovery. Calibration equations and correlation coefficients (> 0.99) were calculated by least squares method with weighting factor. The limit of detection and quantification were in the range of 0.019–0.208 and 0.072–0.483 μg/L. The repeatability and intermediate precision were determined in terms of relative standard deviation to be within 0.03–3.93 and 0.02–4.74%, respectively. The accuracy was found to be in the range of –14.55 to 15.57% in terms of relative error. Seventeen different beverage samples in plastic bottles were successfully analyzed, and ten of them were found to be contaminated by different phthalic acid esters.     

High‐performance anion‐exchange chromatography coupled with diode array detection for the determination of dencichine in Panax notoginseng and related species

A high‐performance anion‐exchange chromatography coupled with diode array detection method was developed for the determination of dencichine in Panax notoginseng and related species. The analysis was performed on an Eprogen Synchropak WAX column (4.6 × 250 mm, 6 μm) with 50 mM NaH₂PO₄ aqueous solution isocratic elution. The method was validated in terms of linearity, sensitivity, precision, stability, and accuracy. It was found that the calibration curve for dencichine showed good linearity (R² = 0.9999) within the test range. The LOD and LOQ were 0.77 and 3.06 ng, respectively. The RSD for intra‐ and interday repeatability was 0.2 and 0.5%, respectively. The test solution of dencichine is stable at least for three days at room temperature and for seven days at 4°C. The mean recovery of dencichine was 102.0%. The established method was successfully applied to determine dencichine in the raw root of P. nogoginseng, P. ginseng, and P. quinquefolium as well as the steamed root of P. notoginseng. Compared with previous reports, this method is sensitive, selective, and accurate, which is helpful to evaluate the quality of P. notoginseng and related species.     

ATNRC and SET‐NRC synthesis of PtBA‐g‐PEO well‐defined amphiphilic graft copolymers

A series of new well‐defined amphiphilic graft copolymers containing hydrophobic poly(tert‐butyl acrylate) backbone and hydrophilic poly(ethylene oxide) side chains were reported. Reversible addition‐fragmentation chain transfer homopolymerization of tert‐butyl 2‐((2‐bromopropanoyloxy)methyl)acrylate was first performed to afford a well‐defined backbone with a narrow molecular weight distribution (M_w/M_n = 1.07). The target poly(tert‐butyl acrylate)‐g‐poly(ethylene oxide) (PtBA‐g‐PEO) graft copolymers with low polydispersities (M_w/M_n = 1.18–1.26) were then synthesized by atom transfer nitroxide radical coupling or single electron transfer‐nitroxide radical coupling reaction using CuBr(Cu)/PMDETA as catalytic system. Fluorescence probe technique was employed to determine the critical micelle concentrations (cmc) of the obtained amphiphilic graft copolymers in aqueous media. Furthermore, PAA‐g‐PEO graft copolymers were obtained by selective acidic hydrolysis of hydrophobic PtBA backbone while PEO side chains kept inert. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Extraction and isolation of lithospermic acid B from Salvia miltiorrhiza Bunge using aqueous two‐phase extraction followed by high‐performance liquid chromatography

A rapid and effective method integrating separation and purification of lithospermic acid B from Salvia miltiorrhiza Bunge was developed by combining an aqueous two‐phase system extraction with preparative chromatography. An aqueous two‐phase system of n‐butyl alcohol/KH₂PO₄ was chosen from seven systems. The influence of parameters including concentration of KH₂PO₄, n‐butyl alcohol concentration, pH, and the ratio of an aqueous two‐phase system to crude extract were investigated using a single factor design. Response surface methodology was subsequently used to find the optimal compositions of an aqueous two‐phase system. Keeping a solvent‐to‐solid ratio of 10, the final optimized composition of an aqueous two‐phase system was 39.1% w/w n‐butyl alcohol and 22.6% w/w KH₂PO₄. Under these conditions a recovery yield of 99.8% and a high partition coefficient of 310.4 were obtained. In a pilot‐scale experiment using optimized conditions, 18.79 g of lithospermic acid B with a purity of 70.5% and in a yield of 99.8% was separated from 0.5 kg of crude extract. Subsequently, 9.94 g lithospermic acid B with a purity of 99.3% and recovery yield of 70.3% was obtained with a preparative chromatographic process, and the two‐step total recovery was 70.1%.     

Separation of Anthraquinones by Capillary Electrophoresis and High‐Performance Liquid Chromatography

The separation and determination of twelve anthraquinones, viz. anthraquinone 1, chrysphanol 2, aloe‐emodin 3, alizarin 4, anthraquinone‐2‐carboxylic acid 5, purpurin 6, sennoside B 7, sennoside A 8, emodin 9, quinalizarin 10, rhein 11, and anthraflavic acid 12, were achieved by capillary electrophoresis (CE) and high‐performance liquid chromatography (HPLC). Detection at 260 nm with a buffer solution containing 30 mM sodium borate (adjusted to pH = 10.56 with 0.05N NaOH) and acetonitrile (9 : 1) in CE or with a linear gradient elution containing 20 mM KH₂PO₄ with 0.05% phosphoric acid (pH = 2.91) and methanol in HPLC was found to be the most suitable approach for this separation. Contents of six components (2, 3, 7, 8, 9, 11) in crude Rhei Rhizoma extract could easily be determined within 39 min by CE or 63 min by HPLC. The effects of buffers on this separation and the validation of the two methods were studied.     

Sodium desoxycholate‐assisted capillary electrochromatography with methacrylate ester‐based monolithic column on fast separation and determination of coumarin analogs in Angelica dahurica extract

A rapid and sensitive CEC method with methacrylate ester‐based monolithic column has been developed for separation and determination of five coumarins (byakangelicin, oxypeucedanin hydrate, xanthotoxol, 5‐hydroxy‐8‐methoxypsoralen and bergapten) in Angelica dahurica extract. Surfactant sodium desoxycholate (SDC) was introduced into the mobile phase as the pseudostationary to dynamically increase the selectivity of analytes instead of increasing the hydrophobicity of stationary phase. In addition, other factors, pH of phosphate buffer, ACN content and applied voltage, for instance, have also an obvious effect on the resolution but little on the retention time. Satisfactory separation of these five coumarins was achieved within 6 min under a 30:70 v/v ACN–buffer containing 20 mM sodium dihydrogen phosphate (NaH₂PO₄) and 0.25 mM SDC at pH 2.51. The RSDs of intraday and interday for relative peak areas were less than 3.0% and 4.7%, respectively; and the recoveries were between 87.5% and 95.0%. The LODs were lower than 0.15 μg/mL and the LOQs were lower than 0.30 μg/mL, respectively, while calibration curves showed a good linearity (r² > 0.9979). Finally, five target coumarins from the crude extracts of A. dahurica were separated, purified, and concentrated by D‐101 macroporous resin, and were successfully separated and quantitatively determined within 6 min.     

tert‐Butyl (6S)‐4‐hydroxy‐6‐isobutyl‐2‐oxo‐1,2,5,6‐tetra­hydropyridine‐1‐carboxyl­ate and tert‐butyl (4R,6S)‐4‐hydroxy‐6‐iso­butyl‐2‐oxopiperidine‐1‐carboxyl­ate

X‐ray studies reveal that tert‐butyl (6S)‐6‐iso­butyl‐2,4‐dioxo­piperidine‐1‐carboxyl­ate occurs in the 4‐enol form, viz. tert‐butyl (6S)‐4‐hydroxy‐6‐iso­butyl‐2‐oxo‐1,2,5,6‐tetra­hydropyri­dine‐1‐carboxyl­ate, C₁₄H₂₃NO₄, when crystals are grown from a mixture of di­chloro­methane and pentane, and has an axial orientation of the iso­butyl side chain at the 6‐position of the piperidine ring. Reduction of the keto functionality leads predominantly to the corresponding β‐hydroxy­lated δ‐lactam, tert‐butyl (4R,6S)‐4‐hydroxy‐6‐iso­butyl‐2‐oxo­piperidine‐1‐car­boxyl­ate, C₁₄H₂₅NO₄, with a cis configuration of the 4‐hydroxy and 6‐iso­butyl groups. The two compounds show similar molecular packing driven by strong O—H⋯O=C hydrogen bonds, leading to infinite chains in the crystal structure.     

5,5′‐Di‐tert‐butyl‐2,2′‐di­hydroxy‐3,3′‐methyl­enedibenz­aldehyde and 6,6′‐di‐tert‐butyl‐8,8′‐methyl­ene­bis­(spiro­[4H‐1,3‐benzodioxin‐2,1′‐cyclo­hexane])

Two related compounds containing p‐tert‐butyl‐o‐methyl­ene‐linked phenol or phenol‐derived subunits are described, namely 5,5′‐di‐tert‐butyl‐2,2′‐di­hydroxy‐3,3′‐methyl­ene­di­benz­aldehyde, C₂₃H₂₈O₄, (I), and 6,6′‐di‐tert‐butyl‐8,8′‐methyl­ene­bis­(spiro­[4H‐1,3‐benzo­di­oxin‐2,1′‐cyclo­hexane]), C₃₅H₄₈O₄, (II). Both compounds adopt a `butterfly' shape, with the two phenol or phenol‐derived O atoms in distal positions. Phenol and aldehyde groups in (I) are involved in intramolecular hydrogen bonds and the two dioxin rings in (II) are in distorted half‐chair conformations.     

Homooxacalix[3]arene Derivatives as Ionophores for Serotonin‐Selective Membrane Electrodes

Homooxacalix[3]arene derivatives are effective ionophores for constructing serotonin‐selective membrane electrodes. An electrode based on one of the derivatives, tris(methoxyphenylpropyloxy)hexahomooxacalix[3]arene‐triethyl ether, with potassium tetrakis(p‐chlorophenyl)borate (20 mol% relative to the ionophore) as an ionic additive and bis(2‐ethylhexyl) sebacate as a solvent mediator in a poly(vinyl chloride) membrane matrix, displayed much better selectivity for serotonin than for various organic ammonium ions and inorganic cations. The electrode exhibited a near‐Nernstian response to serotonin in the concentration range of 2×10^?4 to 1×10^?2 M with a slope of 56.4 mV per concentration decade in physiological saline containing 150 mM NaCl and 10 mM Na₂HPO₄/NaH₂PO₄ (pH 7.4). The limit of the detection was 8×10^?5 M. The selectivity pattern of this electrode was quite different from that of an electrode using calix[6]arene‐hexaacetic acid hexaethyl ester, a well‐known ionophore for primary organic ammonium ions, which did not induce an enhanced response to serotonin. The developed electrode was used for the active loading of serotonin in liposomes induced by transmembrane pH gradients.     

Reaction of Ketene Dithioacetals with Pyrazolylcarbohydrazide： Synthesis and Biological Activities of Ethyl 5-Amino-1-（5＇-methyl-1 ＇-t-butyl-4＇-pyrazolyl）carbonyl-3-methylthio-1 H-pyrazole-4-carboxylate

The title compound, C16H23N5O3S, ethyl 5-amino-1-(5‘-methyl-1‘-t-butyl-4‘-pyrazolyl)carbonyl-3-methylthio-1H-pyrazole-4-carboxylate (5) has been synthesized by the treatment of ethyl 2-cyano-3,3-dimethylthioacrylate with 1-t-butyl-5-methyl-4-hydrazinocarbonylpyrazole (4) in refluxed ethanol. The possible mechanism of the above reaction was also discussed. The results of biological test show that the title compound has fungicidal and plant growth regulation activities.     

四（对叔丁基）硫杂杯[4]芳烃选择性包合四氟硼酸分子：晶体结构和理论研究

X－射线晶体衍射测定了4－(对叔丁基)硫杂杯[4]芳烃选择性地包合四氟硼酸分子。晶体学数据为：C₄₀H₄₉O₄S₄BF₄，M_r=808.88， 四方锥， 空间群 P4/nmm，a=1.5887(2), b=1.5887(2), c=0.8428(0) nm, V=2.127(2) nm³, Z=2, D_c=1.263 g·cm^-3, R₁=0.0405, _WR[I>2σ(I)]=0.1218. ¹⁹F NMR谱中，在-151.4 ppm处出现的峰，证实了四氟硼酸的存在。用Bader的分子中的原子理论方法计算了分子结构中的非共价键相互作用。结果显示，在四氟硼酸包合物中，除了F…H－C氢键作用和阳离子－阴离子的静电作用外，F^δ--C^δ+静电作用的存在也对4－(对叔丁基)硫杂杯[4]芳烃憎水空腔包合氟硼酸分子起到了稳定作用。     

Synthesis and characterization of block copolymers by metal‐ and solvent‐free ring‐opening polymerization of cyclic carbonates initiated from PEG‐based surfactants

Ring‐opening polymerizations of trimethylene carbonate (TC) and 2,2‐dimethyltrimethylene carbonate (DTC) are initiated from hydroxyl‐terminated polyethylene glycol (PEG) and PEG‐based surfactants (Triton X‐100 or Triton X‐405) in the absence of any catalysts. The metal‐ and solvent‐free polymerizations proceed under melt at 150 °C, affording Triton X‐100‐block‐poly(TC) with M_n of 1400–5200 and Triton X‐100‐block‐poly(DTC) with M_n of 1800–7100 in excellent yields. The molecular weights and the comonomer composition of the resulting copolymers are controlled by the feed ratios of the monomers to the initiators, confirmed by gel permeation chromatography and ¹H NMR spectroscopy. The solubilities of the block copolymers composed of hydrophilic PEG segment and hydrophobic poly(TC) or poly(DTC) segment depend on both the compositions and the components. For example, Triton X‐100‐block‐poly (TC) (TC/EG = 9.5/9.5) and Triton X‐405‐block‐poly(TC) (TC/EG = 28/40, 46/40) milky suspend in water, while Triton X‐405‐block‐poly(TC) (TC/EG = 9.7/40) dissolves in water. A dynamic light scattering study reveals that the particle distribution of a copolymer, Triton X‐405‐block‐poly(TC) (TC/EG = 9.7/40) in water, has a monodisperse unimodal pattern ranging from 92 to 368 nm. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 1985–1996, 2006     

A Fluorescent Chemosensor for Dihydrogen Phosphate Ion Based on 2‐[2‐Hydroxy‐4‐(diethylamino) phenyl]‐1H‐imidazo[4,5‐b]phenazine‐Fe3+ Ensemble

A long wavelength emission fluorescent (612 nm) chemosensor with high selectivity for H₂PO₄^? ions was designed and synthesized according to the excited state intramolecular proton transfer (ESIPT). The sensor can exist in two tautomeric forms ('keto' and 'enol') in the presence of Fe³⁺ ion, Fe³⁺ may bind with the 'keto' form of the sensor. Furthermore, the in situ generated GY‐Fe³⁺ ensemble could recover the quenched fluorescence upon the addition of H₂PO₄^? anion resulting in an off‐on‐type sensing with a detection limit of micromolar range in the same medium, and other anions, including F^?, Cl^?, Br^?, I^?, AcO^?, HSO₄^?, ClO₄^? and CN^? had nearly no influence on the probing behavior. The test strips based on 2‐[2‐hydroxy‐4‐(diethylamino) phenyl]‐1H‐imidazo[4,5‐b]phenazine and Fe³⁺ metal complex ( GY‐Fe³⁺ ) were fabricated, which could act as convenient and efficient H₂PO₄^? test kits.     

Versatile online SPE–HPLC method for the analysis of Irinotecan and its clinically relevant metabolites in biomaterials

Monitoring levels of Irinotecan and its metabolites during cancer therapy could help link broad interpatient variations in antitumor activity and toxicity to the patient's metabolic status. We have developed and validated a versatile and highly sensitive method for the simultaneous determination of Irinotecan and its clinically relevant metabolites 7‐ethyl‐10‐hydroxy‐camptothecin (SN‐38) and SN‐38 glucuronide. Sample clean‐up involves precipitation by acetone/methanol/0.5 M trichloroacetic acid at 4:4:2 v/v followed by extraction of the metabolites on an SPE column by 20% methanol in 25 mM KH₂PO₄ pH 2.9. Online transfer to an analytical μBondapak C18 column, elution with 24% acetonitrile (ACN) in 0.1 M KH₂PO₄ pH 2.9 and fluorescence detection with excitation at 375 nm and emission at 430 nm for SN‐38 glucuronide and Irinotecan or 540 nm for SN‐38 results in high sensitivity (1–2 pg) and short (～10 min) run times. The method was used to determine the degree of SN‐38 glucuronidation in mice after Irinotecan administration and in cultured cancer cells exposed to SN‐38. The method may be used to better understand Irinotecan metabolism, personalize therapy, and develop Irinotecan‐based tumor targeting therapies.     

Synthesis of hydroxyl‐terminated poly(ether ether ketone) with pendent tert‐butyl groups and its use as a toughener for epoxy resins

Hydroxyl‐terminated poly(ether ether ketone) with pendent tert‐butyl groups (PEEKTOH) was synthesized by the nucleophilic substitution reaction of 4,4′‐difluorobenzophenone with tert‐butyl hydroquinone with potassium carbonate as a catalyst and N‐methyl‐2‐pyrrolidone as a solvent. Diglycidyl ether of bisphenol A epoxy resin was toughened with PEEKTOHs having different molecular weights. The melt‐mixed binary blends were homogeneous and showed a single composition‐dependent glass‐transition temperature (T_g). Kelley–Bueche and Gordon–Taylor equations gave good correlation with the experimental T_g. Scanning electron microscopy studies of the cured blends revealed a two‐phase morphology. A sea‐island morphology in which the thermoplastic was dispersed in a continuous matrix of epoxy resin was observed. Phase separation occurred by a nucleation and growth mechanism. The dynamic mechanical spectrum of the blends gave two peaks corresponding to epoxy‐rich and thermoplastic‐rich phases. The T_g of the epoxy‐rich phase was lower than that of the unmodified epoxy resin, indicating the presence of dissolved PEEKTOH in the epoxy matrix. There was an increase in the tensile strength with the addition of PEEKTOH. The fracture toughness increased by 135% with the addition of high‐molecular‐weight PEEKTOH. The improvement in the fracture toughness was dependent on the molecular weight and concentration of the oligomers present in the blend. Fracture mechanisms such as crack path deflection, ductile tearing of the thermoplastic, and local plastic deformation of the matrix occurred in the blends. The thermal stability of the blends was not affected by blending with PEEKTOH. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 541–556, 2006     

Order in amorphous di‐n‐alkyl itaconate polymers,copolymers, and blends

The presence of a main‐chain correlation distance (d_II) in the poly(di‐n‐alkyl itaconate)s was confirmed with small‐angle X‐ray scattering/wide‐angle X‐ray scattering measurements taken over the temperature range of 293–478 K. Data for a series of alkyl acrylate polymers were also obtained for comparison. The intensity of the itaconate d_II peak was significant and indicated a greater level of nanophase formation than in analogous systems. In the lower members of the series, nanophase formation appeared to be further enhanced in the temperature range above the glass‐transition temperature (T_g). This was ascribed to the rapidly increasing main‐chain mobility in this region. Macroscopically phase‐separated itaconate blends displayed the individual d_II nanospacings of each homopolymer component. Copolymers, on the other hand, showed more interesting behavior. Poly(methyl‐co‐di‐n‐butyl itaconate) followed an average behavior in which the d_II spacing and T_g changed progressively with the comonomer content. In contrast, the side‐chain pairing in poly(methyl‐co‐di‐n‐octyl itaconate) generated d_II spacings characteristic of separate methyl and octyl nanodomains. The observation of the dioctyl nanodomains, along with the dioctyl side‐chain lower T_g relaxation event, confirmed the concept of independent side‐chain‐domain relaxation in these polymers. The temperature behavior of the poly(methyl‐co‐di‐n‐octyl itaconate) small‐angle X‐ray scattering profiles and scattering correlation lengths indicated that the two nanodomains were not completely structurally independent. © 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 4000–4016, 2004     

Determination of arsenic species in solid matrices utilizing supercritical fluid extraction coupled with gas chromatography after derivatization with thioglycolic acid n‐butyl ester

A method using derivatization and supercritical fluid extraction coupled with gas chromatography was developed for the analysis of dimethylarsinate, monomethylarsonate and inorganic arsenic simultaneously in solid matrices. Thioglycolic acid n‐butyl ester was used as a novel derivatizing reagent. A systematic discussion was made to investigate the effects of pressure, temperature, flow rate of the supercritical CO₂, extraction time, concentration of the modifier, and microemulsion on extraction efficiency. The application for real environmental samples was also studied. Results showed that thioglycolic acid n‐butyl ester was an effective derivatizing reagent that could be applied for arsenic speciation. Using methanol as modifier of the supercritical CO₂ can raise the extraction efficiency, which can be further enhanced by adding a microemulsion that contains Triton X‐405. The optimum extraction conditions were: 25 MPa, 90°C, static extraction for 10 min, dynamic extraction for 25 min with a flow rate of 2.0 mL/min of supercritical CO₂ modified by 5% v/v methanol and microemulsion. The detection limits of dimethylarsinate, monomethylarsonate, and inorganic arsenic in solid matrices were 0.12, 0.26, and 1.1 mg/kg, respectively. The optimized method was sensitive, convenient, and reliable for the extraction and analysis of different arsenic species in solid samples.