High-Speed Counter-Current Chromatography Combined with Column Chromatography for Isolation of Methyllycaconitine from Delphinium pseudocyanthum

Preparative high-speed counter-current chromatography (HSCCC) combined with conventional column chromatography (CC) has been used for isolation and purification of methyllycaconitine from Delphinium pseudocyanthum. n-Hexane-ethyl acetate-methanol-water, 1:1:1:2 (v/v), was used as the solvent system for HSCCC. Separation of methyllycaconitine from an HSCCC fraction was successfully achieved by CC on silica gel using chloroform-methanol, 7:1 (v/v), as mobile phase. A total of 113.45 mg methyllycaconitine of purity >95% was obtained from 1.044 g extract of D. pseudocyanthum. Its structure was identified by MS and NMR.

     

Preparative Isolation of Imperatorin, Oxypeucedanin and Isoimperatorin from a Traditional Chinese Herb Using a HSCCC Strategy Based on Optimization of Rapid Flow Rate

Preparative high-speed counter-current chromatography has been used successfully for the isolation and purification of imperatorin, oxypeucedanin and isoimperatorin from traditional Chinese herb “bai zhi”—Angelica dahurica (Fisch. ex Hoffm) Benth. et Hook using high-speed counter-current chromatography (HSCCC). This was achieved in two stages. The first stage used a high flow HSCCC protocol with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (HEMW) with volume ratios of 5:5:5:5, v/v which isolated isoimperatorin but co-eluted imperatorin and oxypeucedanin. The second stage used HEMW 5:5:4:6, v/v at low flow rate to resolve the co-eluted components from the first stage. The flow rate was optimized by preparative HSCCC. 300 mg of the crude extract was separated, yielding 18.5 mg of imperatorin, 8.3 mg of oxypeucedanin and 9.8 mg of isoimperatorin all at a high purity of over 98%.     

Simultaneous Isolation and Purification of Mollugin and Two Anthraquinones from Rubia cordifolia by HSCCC

A preparative high-speed countercurrent chromatography (HSCCC) method for isolation and purification of three bioactive components, tectoquinone; 1-hydroxy-2-methylanthraquinone and mollugin from the Chinese medicinal plant Rubia cordifolia was successfully established. With a two-phase solvent system composed of light petroleum/ethanol/diethyl ether/water (5:4:3:1, v/v), 10 mg tectoquinone, 19 mg 1-hydroxy-2-methylanthraquinone and 16 mg mollugin were obtained from 100 mg of the crude petroleum extract in a one-step separation at a purity of 98.8, 95.8 and 98.3%, respectively. The structures of mollugin and anthraquinones were identified by ¹H NMR and ¹³C NMR.     

Efficient Protocol for Large-Scale Purification of Naringin with High Recovery from Fructus aurantii by Macroporous Resin Column Chromatography and HSCCC

Several kinds of resins were investigated in the first step and D101 macroporous resin was selected for cleaning-up naringin (NAR), a major flavonoid glycoside from Fructus aurantii. In the subsequent column chromatography, 10% aqueous ethanol was first used to elute the column to remove the undesired constituents and 70% aqueous ethanol was used to elute the target. The content of NAR was 57.1% with 95.7% recovery in this process. In the second step, the obtained crude sample was directly isolated by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of ethyl acetate–n-butanol–water at a volume ratio of 2: 0.8: 3.2 (v/v/v), and 331 mg NAR with 98.3% purity was obtained from 600 mg crude extract in only one run. The recovery of the compound in this step was 95.0%. Thus, the total recovery of NAR was 90.9% after the two step purification. The established protocol for large-scale isolation and separation of NAR with high purity and recovery from F. aurantii was simple, efficient, and suitable for pharmace- utical and commercial use.     

Preparative Isolation and Purification of Two Phenylpropanoids from Daphne giraldii Nitsche by HSCCC

Preparative high-speed counter-current chromatography (HSCCC) was successfully applied to purify phenylpropanoids from the stem and root bark of Daphne giraldii Nitsche, a traditional Chinese medicine. Their structures were identified on the basis of ¹H NMR and ¹³C NMR technology. The two-phase solvent system composed of n -hexane–ethyl acetate–methanol–water (2: 3: 0.5: 4, v/v/v/v) was selected for HSCCC. A total of 8.0 mg woonenoside XI (1) and 18.0 mg daphnetin (2) were obtained in one-step separation from 200 mg of the crude extract with purity of 96.0 and 99.1%, respectively, as determined by LC. And the major compound (2) showed antithrombotic activity in vitro.     

Separation and Purification of Three Flavonoids from Helichrysum arenarium (L.) Moench by HSCCC

Following an initial clean-up step on a Sephadex LH-20 column, high-speed countercurrent chromatography was successfully applied to the isolation and purification of three flavonoids from a crude sample of Helichrysum arenarium (L.) Moench. HSCCC was performed with a two-phase solvent system composed of ethyl acetate–water (1:1, v/v). Naringenin-7-O-β-d-glycoside (2.3 mg), isoquercitrin (3.5 mg), and astragalin (6.7 mg), with purities of 96.05%, 93.63%, 95.23%, respectively, were separated from 160 mg of crude sample in a one-step separation. The structure identification was by ¹H NMR and ¹³C NMR.     

Preparative Isolation and Purification of Flavonoids and Protocatechuic Acid from Sea Buckthorn Juice Concentrate (Hippophaë rhamnoides L. ssp. rhamnoides) by High-Speed Counter-Current Chromatography

High-speed counter-current chromatography (HSCCC)—a support free all liquid–liquid chromatography technique—has been successfully used for the preparative isolation of isorhamnetin 3-O-β-d-glucoside, isorhamnetin 3-O-β-rutinoside, quercetin 3-O-β-d-glucoside, syringetin 3-O-β-d-glucoside and protocatechuic acid from sea buckthorn juice concentrate (Hippophaë rhamnoides L. ssp. rhamnoides, Elaeagnaceae). The preparative HSCCC instrument was a multilayer coil planet centrifuge equipped with three preparative coils. Separation was performed with a two phase solvent system (n-hexane–n-butanol–water, 1:1:2 v/v/v) in ‘head-to-tail’ mode. Each injection of 4.1 g crude ethyl acetate extract yielded isorhamnetin 3-O-β-d-glucoside (95 mg), isorhamnetin 3-O-β-rutinoside (10 mg), quercetin 3-O-β-d-glucoside (5 mg), and protocatechuic acid (34 mg) with purities >98%. The flavonoid syringetin 3-O-β-d-glucoside (2 mg) was a novel compound for H. rhamnoides. Chemical structures of all compounds were determined by HPLC–ESI–MS–MS, 1D-NMR (¹H, ¹³C, DEPT 135) spectroscopy and for elucidation of glycosidic linkages 2D-NMR (HMBC) spectroscopy was used.     

Combined Ultrahigh Pressure Extraction and High-Speed Counter-Current Chromatography for Separation and Purification of Three Glycoside Compounds from Dendrobium officinale Protocorm

As an alternative to Dendrobium candidum, protocorm-like bodies (PLBs) of Dendrobium candidum are of great value due to their high yield and low cost. In this work, three glycoside compounds, β-D-glucopyranose 1-[(E)-3-(4-hydroxyphenyl)-2-propenoat] (I), β-D-glucopyranose 1-[(E)-3-(3, 4-dihydroxyphenyl)-2-propenoat] (II), and 1-O-sinapoyl glucopyranoside (III), were extracted and isolated by ultrahigh pressure extraction (UPE) coupled with high-speed counter-current chromatography (HSCCC) from PLBs of D. officinale. First, the target compounds were optimized and prepared with 50% ethanol solution at a 1:30 (g/mL) solid/liquid ratio in 2 min under 300 MPa by UPE. Then, the crude extract was chromatographed with a silica gel column, and primary separation products were obtained. In addition, the products (150 mg) were separated by HSCCC under the solvent system of MTBE-n-butyl alcohol-acetonitrile-water (5:1:2:6, v/v/v/v), yielding 31.43 mg of compound I, 10.21 mg of compound II, and 24.75 mg of compound III. Their structures were further identified by ESI-MS, ¹H NMR, and ¹³C NMR. The antioxidant results showed that the three compounds expressed moderate effects on the DPPH· scavenging effect. Compound II had the best antioxidant capacity and its IC₅₀ value was 0.0497 mg/mL.     

Ion Chromatography in Combination with ESI Ion Trap MS for the Analysis of Diphacinone in Animal Livers

Analysis of diphacinone is often performed on C18 column by reserved phase liquid chromatographic methods coupled with ultraviolet detection or by ion-pair chromatography coupled with mass spectrometry. In this work, a sensitive and selective method using eluent generator reagent free ion chromatography coupled with electrospray ionization ion trap mass spectrometric detection for the quantification of diphacinone in animal livers has been developed. Samples were extracted with 10% (v/v) methanol in acetonitrile after spiking with warfarin (internal standard, IS) and cleaned by solid-phase extraction. The analytes were separated on an IonPac AS11 analytical column (250 mm × 4.0 mm) using 30.0 mmol L^?1 KOH (achieved by a KOH eluent generator) containing 10% (v/v) methanol as organic modifier at a flow rate of 1.0 mL min^?1. Detection was performed by negative electrospray ionization and ion trap tandem mass spectrometry in multiple reaction monitoring mode with a transition of m/z 339 → 167 for diphacinone, and 307 → 161 for IS, respectively. The calibration curve was linear (r ² = 0.993) in the concentration range of 1.0–200.0 ng mL^?1 with a lower limit of quantification of 0.3 ng g^?1 in animal livers. Intra- and inter-day relative standard deviations (RSDs) were less than 8.7 and 12.5%, respectively. Recoveries of diphacinone ranged from 81.2 to 89.5%. It was confirmed that this method could be used in clinical diagnosis or forensic toxicology.     

Isolation of Terpenoids from Trichilia quadrijuga (Meliaceae) by Droplet Counter-Current Chromatography

The Trichilia genus (Meliaceae) consists of about 230 species distributed throughout tropical America and its phytochemical profile is rich in terpenic metabolites. Droplet counter-current chromatography (DCCC) was used for the isolation and purification of secondary metabolites obtained from a dichloromethane fraction (2.9 g) of Trichilia quadrijuga stems. A hexane–ethyl acetate–methanol–water (1:2:1.75:1, v/v/v/v) solvent system was chosen to isolate 2β,3β,4β-trihydroxypregnan-16-one (1, 24.5 mg, 0.8%), kudtdiol (2, 45.0 mg, 1.6%) and 3-O-β-d-glucopyranosylsitosterol (3, 6.0 mg, 0.2%). The results showed that DCCC was a very effective tool for the isolation of terpenes from T. quadrijuga.     

Direct Injection of Plasma Samples and Micellar Chromatography of Procainamide and Its Metabolite N-Acetylprocainamide

Micellar liquid chromatography was employed for the monitoring of procainamide and its metabolite N-acetylprocainamide using a C18 column. Sodium dodecyl sulphate (SDS) and modifier concentrations were optimised in order to obtain minimum analysis time, maximum sensitivity and good resolution. The optimum chromatographic conditions were as follows: flow rate 1 mL min^?1, injection volume 20 μL, temperature at 25 °C, respectively. The mobile phase consisted of 0.05 M SDS-1% (v/v) butanol–phosphate buffer (10 mM, pH 7, 0.9%, w/v) NaCl using a detection wavelength at 280 nm. Validation studies were carried out according to the ICH guideline and included the determination of calibration curves (r ² > 0.999), intra- and inter-day precisions (CV < 3.9%), robustness and interference studies, respectively. The recoveries in spiked serum samples were adjusted. Finally, the optimized method was applied to serum samples of patients treated with antiarrhythmics, and the results were compared with those given by a reference method where a good correlation was obtained.     

Enantioselective Analysis of Fluoxetine and Norfluoxetine by LC in Culture Medium for Application in Biotransformation Studies Employing Fungi

A liquid chromatography method is described for the analysis of fluoxetine and norfluoxetine enantiomers in fungi cultures. The analytes were separated simultaneously by LC employing a serial system. The resolution was performed using a mobile phase of ethanol: 15 mM ammonium acetate buffer solution, pH 5.9: acetonitrile (77.5:17.5:5, v/v/v). UV detection was at 227 nm. Hexane: isoamyl alcohol (98:2, v/v) was used as extractor solvent. The calibration curves were linear over the concentration range of 12.5–3,750 ng mL^?1 (r ≥ 0.996). The values for intra- and inter-day precision and accuracy were ≤10% for all analytes. The validated method was used to evaluate fluoxetine biotransformation to its mammalian metabolite, norfluoxetine, by selected endophytic fungi. Although the desired biotransformation was not observed in the conditions used here, the method could be used to evaluate the biotransformation of fluoxetine by other fungi or to be extended to other matrices with adequate procedures for sample preparation.     

Separation of abietane-type diterpenoids from Clerodendrum kaichianum Hsu by high-speed counter-current chromatography using stepwise elution

High-speed counter-current chromatography (HSCCC) was successfully used for the separation of abietane-type diterpenoids from the medicinal plant C. kaichianum, which were not separated in our previous study using preparative HPLC. The HSCCC separation employed the lower phases of n-hexane–ethyl acetate–methanol–water (HEMW) 4:5:4:5 and HEMW 4:5:5:4 as the mobile phase for stepwise elution while the upper phase of HEMW 4:5:4:5 was used as the stationary phase. HSCCC separation yielded 90.5 mg of compound 1(kaichianone A), 137.7 mg of compound 2 (kaichianone B), 125.0 mg of compound 3 (teuvincenone E), and 227.6 mg of compound 4 (taxusabietane A) with purities of 95.3%, 97.2%, 97.8%, and 98.6%, respectively, as determined by HPLC. Compounds 1–2 are two new abietane-type diterpenoids while Compounds 3–4 are known abietane-type diterpenoids, analyzed by ESIMS and NMR data. The results demonstrated that HSCCC can be an excellent alternative for other separation methods. The two new compounds showed significant cytotoxicity against ileocecal carcinoma HCT-8 and breast adenocarcinoma MCF-7 cells.     

An Economical Method for Isolation of Dioscin from Dioscorea nipponica Makino by HSCCC Coupled with ELSD, and a Computer-Aided UNIFAC Mathematical Model

An economical method for isolation of dioscin from Dioscorea nipponica Makino by high-speed counter-current chromatography (HSCCC) was successfully developed by using a UNIFAC mathematical model coupled with computer-aided counter-current chromatography solvent-selection software (CCC-SSS) for separate preparation of the components of the solvent system (i.e., the stationary and mobile phases). The solvent system n-hexane–ethyl acetate–ethanol–water 2:5:2:5 (v/v) was selected to demonstrate the feasibility of the approach. A comparative study was also carried out on different methods for preparation of the solvent system, namely conventional preparation of the mobile and stationary phases together in the same vessel and the method developed for separate preparation of the phases. The results indicated that purity and recovery of dioscin were no different when solvent systems prepared by the different methods were used for HSCCC separation. Much less n-hexane, ethyl acetate, and ethanol was used when the mobile and stationary phases were prepared separately, however. This was not only environmentally sensible, but also enabled conservation of resources. Use of the UNIFAC mathematical model combined with the CCC-SSS technique for separate preparation of the components of the solvent system in HSCCC is reported and explained. It is a simple and economical means of isolating pure dioscin from Dioscorea nipponica Makino.     

Separation and Identification of Ergosta-4,6,8(14),22-tetraen-3-one from Ganoderma atrum by High-Speed Counter-Current Chromatography and Spectroscopic Methods

High speed counter-current chromatography in semi-preparative scale was used to separate and purify ergosta-4,6,8(14),22-tetraen-3-one from Ganoderma atrum, a famous traditional Chinese medicine. A two-phase solvent system composed of a mixture of n-hexane–ethanol–water (6: 5: 1, v/v/v) was used and the separation conditions were optimized. In a typical run in less than 400 min, 100 mg of samples can be separated to yield 14 mg of ergosta-4,6,8(14),22-tetraen-3-one with 99.1% purity. The structure of this compound was elucidated by UV, EI-MS, ¹H NMR and ¹³C NMR.     

Spectrometric and Chromatographic Study of Reactive Oxidants Hypochlorous and Hypobromous Acids and Their Interactions with Taurine

In this study, we focused on the studying of taurine complexes with phenol and sodium hypochlorite, and of taurine with sodium hypobromite by spectrometry, reverse phase chromatography and ion-exchange chromatography. The formed complexes were studied under various conditions such as temperature (10, 20, 30, 40, 50 and 60 °C), and/or time of interaction (0, 5, 10, 15, 20, 25 and 30 min). In addition, we optimized high performance liquid chromatography coupled with UV detector for detection of taurine and its complexes with the acids. Taurine–phenol–hypochlorite complex was effectively separated under isocratic elution, mobile phase water:methanol 30:70 %, v:v, flow rate 1 mL min^?1 and 55 °C. Taurine-bromamine complex was isolated under the following optimized conditions as isocratic elution, mobile phase water:methanol 85:15 % v:v, flow rate 1 mL min^?1 and 55 °C. The limits of detection (3 S/N) were estimated as 1 μM for both types of complexes, i.e. for taurine. Further, we estimated recovery in one sample of urine (male 25 years), commercially achieved energy drink and tea leaves and varied from 79 to 86 %. Further, we aimed our attention at investigating the ability of the above characterized taurine and taurine complexes to scavenge reactive oxygen species. For this purpose, an ion-exchange liquid chromatography with post-column derivatization with ninhydrin and VIS detector was used. It clearly follows from the results obtained that taurine itself reacts with peroxide more intensely than in a bound form, which can be associated with the highest signal decrease. Complexes stabilized structure taurine against peroxide radicals, resulting in slower decreasing of peak heights. The most stable was taurine complexes with phenol and hypobromite.     

SPE and LC–ESI–MS for Quantitative Analysis of Mitiglinide in Human Plasma in a Bioequivalence Study

Liquid chromatography–electrospray ionization mass spectrometry has been used for rapid, selective, and sensitive quantitative analysis of mitiglinide in human plasma. Sample pretreatment involved solid-phase extraction from plasma with gliclazide as internal standard. Separation was performed on a C₁₈ column (150 × 2.0 mm) with 71:29 (v/v) acetonitrile–water (containing 0.1% formic acid and 0.2 mmol L^?1 ammonium acetate) as mobile phase at a flow rate of 0.2 mL min^?1. The method was validated then successfully applied to a clinical bioequivalence study of mitiglinide in 20 healthy volunteers after oral administration.     

Tetrazole-Functionalized Silica for Hydrophilic Interaction Chromatography of Polar Solutes

In this work, tetrazole-functionalized stationary phase was prepared with nitrile-modified silica by an ammonium-catalyzed (3 + 2) azide-nitrile cycloaddition reaction. The prepared stationary phase was used for separation of nucleobases and nucleosides by hydrophilic interaction chromatography (HILIC) mode. A typical HILIC mechanism was observed at higher content of acetonitrile (>85%, v/v) in the mobile phase. The retention mechanism of the column was investigated by the models used for describing partitioning and surface adsorption through adjustment ratio of water in the mobile phase, and by the influence of salt concentration, buffer pH, and temperature on the retention of solutes. The results illustrated that the surface adsorption through hydrogen bonding dominated the retention behavior of nucleobases/nucleosides under HILIC mode. From the separation ability, the tetrazole-functionalized stationary phase could become a valuable alternative for the separation of the compounds concerned.     

Sequential Injection Magneto Chromatography Determination of Non-Steroidal Anti-Inflammatory Drugs in Pharmaceutical Formulations

A magnetic separation method based on the use of magnetic silica as the stationary phase in sequential injection chromatography was used for simultaneous determination of nonsteroidal anti-inflammatory drugs (acetaminophen, naproxen, diclofenac, and ibuprofen) in tablets. The method is based on a thin layer paramagnetic stationary phase retained on the inner wall of a mini-column through the action of an external magnetic field. The influence of the variables involved was evaluated and the optimal conditions were found to be: a methyl-silica magnetic adsorbent was used as the stationary phase, the mobile phase was methanol-water (60:40, v/v), pH 2.5 adjusted with 98% phosphoric acid, a flow rate 0.60 ml min^?1, and UV detection at 225 nm. Under these conditions, the linear range of the calibration curve ranged from 3–6 mg L^?1 to 100 mg L^?1 with limits of detection ranging between 1 to 2 mg L^?1. The proposed method was validated by comparing the results obtained against those provided by high performance liquid chromatography; no significant differences were seen.     

Single-Step Preparative Extraction of Artemisinin from Artemisia annua by Charcoal Column Chromatography

Activated charcoal column chromatography was successfully applied for preparative extraction of artemisinin with high-purity from Artemisia annua L. in one single run for the first time in this study. The adsorption kinetics study showed that powdery activated charcoal had high adsorption speed for artemisinin. The experimental adsorption data fitted well using the Langmuir adsorption model. The optimal parameters for the extraction of artemisinin were obtained using a column packed with activated charcoal: the loading amount, 1 g extract/5 g activated charcoal; the elution mode, CH₂Cl₂?CMeOH (2:5, v/v) for 5 BV (bed volume) after the removal of some impurities with CH₂Cl₂?CMeOH (1:10, v/v). With this optimized condition, artemisinin was finally obtained with purity at 95.2% and the recovery at 72.3%. The developed technique would provide a feasible large-scale method for the extraction of artemisinin in pharmaceutical industry.