Preparative separation of gambogic acid and its C-2 epimer using recycling high-speed counter-current chromatography

A recycling counter-current chromatographic system was first set up with a high-speed counter-current chromatography instrument coupled with a column switching valve. This system was first successfully applied to the preparative separation of epimers, gambogic acid and epigambogic acid from Garcinia hanburyi using n-hexane-methanol-water (5:4:1, v/v/v) as the two-phase solvent system. As a result, 28.2 mg gambogic acid and 18.4 mg epigambogic acid were separated from 50 mg of mixture. Their purities were both above 97% as determined by HPLC. The chemical structures were then identified by their (1)H NMR and (13)C NMR spectra.     

高速逆流色谱法分离制备中药诃子中的没食子酸

利用高速逆流色谱法从100 mg诃子醇提物中一次性分离制备得到8.6 mg没食子酸。通过分析型高速逆流色谱对5种溶剂系统进行筛选,确定以正己烷-乙酸乙酯-甲醇-水(体积比为1:5:1:5)为两相溶剂体系并放大到制备型上,以上相为固定相,下相为流动相,在主机转速850 r/min、流动相流速2 mL/min、检测波长254 nm的条件下进行分离制备,获得4个分离峰(组分Ⅰ、Ⅱ、Ⅲ、Ⅳ)。经高效液相色谱检测,按照面积归一法计算,其中组分Ⅲ的纯度达96.40%。经电喷雾电离质谱分析,并结合与没食子酸标准品的高效液相色谱测定结果的对比,确定组分Ⅲ为没食子酸。该方法简便、快速、重复性好,适合于诃子中没食子酸的分离制备。     

Peptide nucleic acid and amino acid modified peptide nucleic acid analysis by capillary zone electrophoresis

A rapid, high resolution, and low sample consumption CZE method is developed for peptide nucleic acid (PNA) analysis for the first time. 30% v/v acetonitrile in PNA sample and 20% v/v acetonitrile in 50 mM borax‐boric acid (pH 8.7) as BGE were employed after optimization. The calibration curves were linear for PNA concentration ranging from 1 to 50 μmol/L. LOD and LOQ of PNA were 0.2 and 1.0 μmol/L, respectively. Since the commercially available reagent gives rise to huge PNA peak and an apparent impurity peak, the purity of PNA was evaluated to be about 81.4% by CZE method, obviously lower than the supplier's purity value of 99.9% evaluated by RP–HPLC, and also lower than 94.8% determined with RP–HPLC by our research group. The CZE method takes only 5 min, needs only 90 nL PNA, much less than 20 min and 20 μL PNA in RP–HPLC method. Moreover, the CZE method is applicable for the analysis of glutamic acid modified and lysine modified PNAs, they show different migration time with their corresponding complementary PNAs. Our results show CZE provides a new choice for PNA and modified PNA analysis, also their purity or quality evaluation.     

Isolation of three sesquiterpene lactones from the roots of Cichorium glandulosum Boiss. et Huet. by high-speed counter-current chromatography

Because of the skeletal complexity and similarity of the polarity, little research was reported on the isolation of sesquiterpene lactones by high-speed counter-current chromatography (HSCCC). Herein, three sesquiterpene lactones were successfully purified from the ethyl acetate extract of the roots of the traditional Uyghur medicinal plant Cichorium glandulosum Boiss. et Huet. by HSCCC. The separation was performed in two steps with two solvent systems: n-hexane-ethyl acetate-methanol-water (1.5:5:2.75:5, v/v/v/v) and ethyl acetate-methanol-water (20:1:20, v/v/v). From 166 mg of the ethyl acetate extract, 19 mg of lactucopicrin was isolated with the first solvent system and 10 mg of 11beta,13-dihydrolactucin and 16 mg of lactucin were obtained with the second solvent system. All purified compounds were over 94% purity as determined by HPLC analysis, and these chemical structures were confirmed by (1)H NMR and (13)C NMR.     

HPLC-UV assay for monitoring total and unbound mycophenolic acid concentrations in children

A simple, accurate and sensitive HPLC method was developed for measuring total and unbound mycophenolic acid (MPA) in human plasma. Total MPA was extracted by protein precipitation and ultrafiltration was used to assess unbound MPA concentrations. The supernatant (20 microL) or ultrafiltrate (100 microL) was injected onto a C(18) HPLC column with a mobile phase of 0.05 m sodium phosphate buffer (pH 2.31)-acetonitrile (55:45, v/v for total MPA; 50:50 for unbound MPA) with UV detection at 254 nm. The extraction recovery was over 93% and reproducible. The assay was linear over the concentration range of 0.07-50 mg/L for total MPA and 4-1500 microg/L for unbound MPA. Intra- and inter-day assay reproducibility was less than 10%. Detection limits were 0.04 mg/L and 2 microg/L for total and unbound MPA, respectively. The assay utility was established in samples collected from five paediatric bone marrow transplant recipients who were receiving intravenous doses of mycophenolate mofetil. In these patients MPA concentrations ranged from 0.07 to 7.83 mg/L and unbound drug concentrations ranged from 2.1 to 107.5 microg/L. This method can be effectively applied to MPA pharmacokinetics in paediatric patients.     

Determination of oleanolic acid, ursolic acid and amygdalin in the flower of Eriobotrya japonica Lindl. by HPLC

Simple and accurate HPLC methods were developed for the determination of oleanolic acid (OA), ursolic acid (UA) and amygdalin in loquat (Eriobotrya japonica Lindl.) flower, which is commonly used for the treatment of various diseases as a traditional Chinese medicine. HPLC assay was performed on a reversed-phase C(18) column and all three compounds were detected at 210 nm with a flow rate of 1.0 mL/min. The mobile phase consisted of methanol (A) and 0.03 mol/L phosphate buffer (pH 2.8) (B) with a ratio of 88:12 (A:B, v/v) for simultaneous detection of OA and UA, and 25:75 (A:B, v/v) for detection of amygdalin. The established methods showed good precision and accuracy with overall intra-day and inter-day variation of 0.99-3.55 and 1.05-4.05%, respectively, and overall recoveries of 97.37-99.32% for the three compounds. Application of these methods to determine the OA, UA and amygdalin contents in loquat flower showed that cultivar had a minor effect on the contents of all three compounds, with average amounts of 0.38-0.51 mg OA/g dry weight (DW), 2.15-2.68 mg UA/g DW and 1.23-1.56 mg amygdalin/g DW among five loquat cultivars tested. However, developmental stages and flower tissues showed significant effect on the contents of all three bioactive components.     

Preparative separation of liquiritigenin and glycyrrhetic acid from Glycyrrhiza uralensis Fisch using hydrolytic extraction combined with high-speed countercurrent chromatography

The objective of this paper was to develop a preparative method for the isolation and purification of liquiritigenin and glycyrrhetic acid from Glycyrrhiza uralensis Fisch using hydrolytic extraction combined with high-speed countercurrent chromatography (HSCCC). Liquiritigenin and glycyrrhetic acid were well hydrolyzed from liquiritin and glycyrrhizic acid by hydrochloric acid, respectively. The optimal extraction conditions were obtained by single-factor and orthogonal experiments, which were 100% ethanol, 1.5 mol/L hydrochloric acid, 1:25 ratio of solid to liquid, and extracted 2 h for one time. Using the two-phase solvent system of n-hexane-ethyl acetate-methanol–water (4:5:4:5, v/v), 2.1 mg liquiritigenin (the purity was 96.5% with a recovery of 87.6%) and 12.3 mg glycyrrhetic acid (the purity was 97.1% with a recovery of 74.4%) were obtained from 315-mg crude extraction by HSCCC. The retention ratio of stationary phase was 47.2%. Their structures were identified by HPLC, melting points, UV, Fourier-transform infrared, Electrospray ionization-MS, ¹H nuclear magnetic resonance (NMR), and ¹³C NMR spectra. According to the antioxidant activity assays, liquiritigenin and glycyrrhetic acid had some scavenging abilities on 1,1-diphenyl-2-picrylhydrazyl free radicals; liquiritigenin had stronger scavenging ability on hydroxyl radicals.     

Preparative isolation and purification of rupestonic acid from the Chinese medicinal plant Artemisia rupestris L. by high-speed counter-current chromatography

Rupestonic acid was purified for the first time by high-speed counter-current chromatography from a dichloromethane extract of the traditional Chinese medicinal plant Artemisia rupestris L. The separation was performed in two steps with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (6:4:3.5:6.5, v/v) with 0.5% acetic acid in stationary-phase. From 200 mg of the crude extract, 27.9 mg of rupestonic acid was obtained at over 98% purity as determined by HPLC analysis, and its chemical structure was confirmed by MS, 1H and 13C nuclear magnetic resonance.     

Application of accelerated solvent extraction coupled with high-performance counter-current chromatography to extraction and online isolation of chemical constituents from Hypericum perforatum L

Accelerated solvent extraction (ASE) coupled with high-performance counter-current chromatography (HPCCC) was successfully used for the extraction and online isolation of five chemical constituents from the plant Hypericum perforatum L. The upper phase of the solvent system of ethyl acetate-methanol-water (5:2:5, v:v:v) was used as both the ASE solvent and the HPCCC stationary phase. Two hydrophobic compounds including 28.4 mg of hyperforin with a HPLC purity of 97.28% and 32.7 mg of adhyperforin with a HPLC purity of 97.81% were isolated. The lower phase of ethyl acetate-methanol-n-butanol-water (5:2:2.5:12, v:v:v:v) was used as both the ASE solvent and CCC stationary phase. Three hydrophilic compounds of 12.7 mg of 3,4,5-O-tricaffeoylquinic acid with a HPLC purity of 98.82%, 15.2 mg of 1,3,5-O-tricaffeoylquinic acid with a HPLC purity of 99.46% and 42.5mg of 3-O-caffeoylquinic acid with a HPLC purity of 96.90%, were obtained in a one-step extraction-separation process with less than 3h from 10.02 g of raw material of H. perforatum. The targeted compounds isolated, collected and purified by HPCCC were analyzed by high performance liquid chromatography (HPLC), the chemical structures of all five compounds above mentioned were identified by UV, MS and NMR.     

Investigation of phenolic acids in yacon (Smallanthus sonchifolius) leaves and tubers

Thin-layer chromatographic (TLC) screening of crude extracts of dried leaves and tubers of yacon (Smallanthus sonchifolius, Asteraceae) and products of acid hydrolysis of tubers on the silica gel HPTLC plates using the developing solvents ethyl acetate-formic acid-water (85:10:15, v/v/v) and n-hexane-ethyl acetate-formic acid (20:19:1, v/v/v) proved the presence of chlorogenic, caffeic and ferulic acid. These phenolic acids were isolated from the crude extract of yacon leaves by preparative TLC, and identified after elution by HPLC/MS, as well as by direct injection of the crude extract into the HPLC/MS system. Acid hydrolysis of tubers released the increased amount of phenolic acids (e.g. caffeic acid and ferulic acid), flavonoid quercetin and an unidentified flavonoid, which was detected by TLC analysis. Ferulic acid, isomers of dicaffeoylquinic acid and still an unidentified derivative of chlorogenic acid (Mr = 562) as constituents of yacon leaves and ferulic acid as constituent of yacon tubers are reported here for the first time. These acids gave significant contribution to the radical scavenging activity detected directly on the TLC plate sprayed with 1,1-diphenyl-2-picrylhydrazyl (DPPH).     

高效液相色谱法测定大鼠血浆中的原儿茶酸

建立了大鼠血浆中原儿茶酸含量测定的高效液相色谱方法。采用的色谱柱为DiamondsilTM C18 柱(150 mm×4.6 mm,5 μm);流动相为乙腈-水(体积比为9∶91,用H3PO4 调pH至2.5);流速1.2 mL/min;检测波长260 nm;内标为对羟基苯甲酸。原儿茶酸的线性范围为0.050~3.20 mg/L,线性相关系数为0.9978，最低定量限为0.050 mg/L,日内和日间测定的精密度（以相对标准偏差表示）均低于7.0%,准确度（以相对误差表示）为-1.4%～2.6%；在0.050,0.40,3.20 mg/L低、中、高3个添加浓度水平下，血浆样品的提取回收率分别为83.4%,87.3%,91.1%。该方法简便,灵敏,准确,适用于大鼠体内原儿茶酸的药物动力学研究。     

SEPARATION OF THE MINOR FLAVONOLS FROM FLOS GOSSYPII BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY

An effective high-speed countercurrent chromatography (HSCCC) method was established for further separation and purification of four minor flavonols in addition to five major flavonols which were reported by our previous study from extracts of Flos Gossypii. HSCCC was performed with three two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (7.5:15:6:7, v/v), (2.5:15:2:7, v/v) and (0:1:0:1, v/v). The separation was repeated 3 times, and 3.8 mg of 8-methoxyl-kaempferol-7-O-β-D-rhamnoside (HPLC purity 98.27%), 6.7 mg of astragalin (HPLC purity 94.18%), 3.3 mg of 4'-methoxyl-quercetin-7-O-β-D-glucoside (HPLC purity 94.30%) and 8.2 mg of hyperoside (HPLC purity 93.48%) were separated from 150 mg of the crude sample. The chemical structures of the flavonols were confirmed by MS, (1)H NMR and (13)C NMR. Meanwhile, the results indicated that the target compound with smaller K value (<0.5) can be separated by increasing column length of HSCCC. And four separation rules of flavonols according to the present study and references were summarized, which can be used as a useful guide for separation of flavonols by HSCCC.     

Separation of chlorogenic acid and concentration of trace caffeic acid from natural products by pH‐zone‐refining countercurrent chromatography

Chlorogenic acid and caffeic acid were selected as test samples for separation by the pH‐zone‐refining countercurrent chromatography (CCC). The separation of these test samples was performed with a two‐phase solvent system composed of methyl‐tert‐butyl‐ether/acetonitrile/water at a volume ratio of 4:1:5 v/v/v where trifluoroacetic acid (TFA; 8 mM) was added to the organic stationary phase as a retainer and NH₄OH (10 mM) to the aqueous mobile phase as an eluter. Chlorogenic acid was successfully separated from Flaveria bidentis (L.) Kuntze (F. bidentis) and Lonicerae Flos by pH‐zone‐refining CCC, a slightly polar two‐phase solvent system composed of methyl‐tert‐butyl‐ether/acetonitrile/n‐butanol/water at a volume ratio of 4:1:1:5 v/v/v/v was selected where TFA (3 mM) was added to the organic stationary phase as a retainer and NH₄OH (3 mM) to the aqueous mobile phase as an eluter. A 16.2 mg amount of chlorogenic acid with the purity of 92% from 1.4 g of F. bidentis, and 134 mg of chlorogenic acid at the purity of 99% from 1.3 g of crude extract of Lonicerae Flos have been obtained. These results suggest that pH‐zone‐refining CCC is suitable for the isolation of the chlorogenic acid from the crude extracts of F. bidentis and Lonicerae Flos.     

Determination of phenol, m-, o- and p-cresol, p-aminophenol and p-nitrophenol in urine by high-performance liquid chromatography

A method for the biological monitoring of human exposure to aromatic hydrocarbons, nitrocompounds, amines and phenols has been developed. Phenol, cresols, p-aminophenol, p-nitrophenol and their glucorono- or sulpho-conjugates, were quantified by HPLC; 4-chlorphenol was added as internal standard. After enzymatic hydrolysis, the free compounds were extracted with an organic solvent and analyzed by an isocratic HPLC Perkin Elmer system at ambient temperature and at a flow-rate of 1 ml/min. The column was a reversed-phase Pecosphere 3 x 3 C18 Perkin Elmer; the mobile phase was a 30:70:0.1 (v/v/v) methanol-water-orthophosphoric acid mixture and the chromatogram was monitored at 215 nm. Identification was based on retention time and quantification was performed by automatic peak height determination, corrected for the internal standard. The recovery was ca. 95% for phenol and cresols; 90% for p-nitrophenol; 85% for p-aminophenol; the coefficients of variance were less than 6% within analysis (n = 20) and less than 10% between analysis (n = 20). The detection limits, at a signal/noise ratio of 2, were 0.5 mg/l for phenol and cresols and 1 mg/l for p-aminophenol and p-nitrophenol.     

Supercritical fluid extraction of aurentiamide acetate from Patrinia villosa Juss and subsequent isolation by silica gel and high-speed counter-current chromatography

Supercritical fluid extraction (SFE) of aurentiamide acetate from Patrinia villosa Juss was performed. The optimization of parameters was carried out using an analytical-scale supercritical fluid extraction (SFE) system. Then the extraction was scaled up by 100 times using a preparative SFE system under the optimized conditions of 55 degrees C, 35 MPa and modified CO2 with 10% methanol. Then, the crude extract I obtained by SFE was chromatographed on silica gel and the solvent system composed of petroleum ether-ethyl acetate (5:1, v/v) was used to produce the crude extract II, which was further isolated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:1.2:1.2:1, v/v/v/v). One hundred fifty-five milligrams of aurentiamide acetate was obtained from 400 mg crude extract II (contained 42% target) with a purity of 99.3% determined by HPLC and 92.3% recovery in one-step elution, and identification was performed by UV, MS, 1H NMR and 13C NMR. As far as we know, this is the first report of discovering aurentiamide acetate from the plant of Patrinia genius.     

Validation of the removal of acetylsalicylic acid. Recovery and determination of residues on various surfaces by high performance liquid chromatographic

The validation of a procedure to clean glass, vinyl and stainless steel surfaces that have been exposed to acetylsalicylic acid during its manufacture is described. The cleaning procedure using two cotton swabs moistened with the mobile phase was validated using a wipe-test and a high-performance liquid chromatography (HPLC) method developed to determine low quantities of the acid. The HPLC method involves an octadecylsilane column at 55°C, a mixture of water–acetonitrile–orthophosphoric acid (779:220:1, v/v) as mobile phase and detection at 226 nm. Recoveries of 86%, 90% and 94% were obtained from vinyl, glass and stainless steel plates respectively. The validation gave acceptable levels of sensitivity, recovery, precision and linearity.     

Isolation of fucoxanthin from edible brown algae by microwave-assisted extraction coupled with high-speed countercurrent chromatography

A rapid and efficient method for the separation and purification of fucoxanthin from edible brown algae by microwave-assisted extraction coupled with high-speed countercurrent chromatography was developed. The algae were first extracted using microwave-assisted extraction, then the dried extract was dissolved and directly introduced into the high-speed countercurrent chromatography system with a two-phase solvent system consisting of hexane-ethyl acetate-ethanol-water (5:5:6:4, v/v/v/v). The isolation was done in less than 75 min, and a total of 0.83 mg, 1.09 mg, and 0.20 mg fucoxanthin were obtained from 25.0 g fresh Laminaria japonica Aresch, 1.5 g dry Undaria pinnatifida (Harv) Sur, and 15.0 g dry Sargassum fusiforme (Harv) Setch, respectively. The purity of fucoxanthin determined by HPLC was over 90% and its structure was further identified by LC-ESI-MS and (1) H-NMR.     

Preparative enantioseparation of propafenone by counter‐current chromatography using di‐n‐butyl l‐tartrate combined with boric acid as the chiral selector

This paper extends the research of the utilization of borate coordination complexes in chiral separation by counter‐current chromatography (CCC). Racemic propafenone was successfully enantioseparated by CCC with di‐n‐butyl l ‐tartrate combined with boric acid as the chiral selector. The two‐phase solvent system was composed of chloroform/ 0.05 mol/L acetate buffer pH 3.4 containing 0.10 mol/L boric acid (1:1, v/v), in which 0.10 mol/L di‐n‐butyl l ‐tartrate was added in the organic phase. The influence of factors in the enantioseparation of propafenone were investigated and optimized. A total of 92 mg of racemic propafenone was completely enantioseparated using high‐speed CCC in a single run, yielding 40–42 mg of (R)‐ and (S)‐propafenone enantiomers with an HPLC purity over 90–95%. The recovery for propafenone enantiomers from fractions of CCC was in the range of 85–90%.     

高速逆流色谱法分离制备母丁香和公丁香中的5种非挥发性化合物

应用高速逆流色谱法从母丁香和公丁香中快速分离了3种已知非挥发性化合物,并利用相同方法从公丁香中分离出2种色原酮类化合物。两相溶剂系统A为正己烷-乙酸乙酯-甲醇-水(5:8:6:13, v/v/v/v),系统B为正己烷-乙酸乙酯-甲醇-水(5:8:9:10, v/v/v/v),以系统A的上相为固定相,系统A和B的下相为流动相,利用梯度洗脱方式,在主机转速为880 r/min、流速1.2 mL/min条件下,成功地从70 mg母丁香粗提物中分离得到12.3 mg鞣花酸、9.6 mg鼠李素、17.2 mg槲皮素,从50 mg公丁香粗提物中分离得到5,7-二甲氧基-2-甲基色原酮10.2 mg、5,7-二甲氧基-2,6-二甲基色原酮8.6 mg,纯度均在96%以上。各化合物的结构均由质谱和核磁共振氢谱、碳谱鉴定。利用该方法可以对丁香不同药用部位中的非挥发性化合物进行有效的分离和纯化。     

高速逆流色谱分离纯化蔓荆子中的活性成分

应用高速逆流色谱法(HSCCC)分离纯化蔓荆子中的活性成分。以石油醚-乙酸乙酯-甲醇-水(体积比为3:6:3.6:3)为两相溶剂体系,在转速为800 r/min、流速为1.5 mL/min、检测波长为254 nm的条件下进行分离,所得馏分经高效液相色谱法(HPLC)检测,并经电喷雾电离(ESI)质谱和核磁共振谱(NMR)鉴定化合物的结构。从250 mg蔓荆子粗提物中一次性分离得到4个化合物,分别为23 mg对羟基苯甲酸、15 mg 3,6,7-三甲基槲皮万寿菊素、24 mg蔓荆子黄素和5 mg蒿黄素,其纯度约为93.1%、 97.3%、 98.7%和98.5%。该法具有简便、快速、重复性好的优点,为分离蔓荆子中的活性成分提供了新的方法。