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Abstract: Integration of supercritical fluid/mass spectrometry (SFC/MS) and reversed phase liquid (HPLC/MS) chromatographic screening techniques into a single chromatographic system and utilized in “walk up” mode, enabled us to produce an orthogonal data set for selecting purification conditions for medicinal chemistry compounds. To streamline the overall workflow, we also demonstrate the use of automated batch data processing of individual data files to identify suitable separation conditions without user intervention. We have addressed the chromatographic challenges that hinder the identification of the intended target and thus the selection of ideal purification conditions. For instance, multiple component-of-interest (COI) peaks, co-elution of impurities with the COI, and chromatographic suitability factors such as retention times and peak shapes are all important considerations when selecting appropriate methods for purification and, therefore, are bottlenecks to an automated approach. Since SFC and HPLC data were collected in parallel from separate instruments in our workflow, the time required for the separation scientist to analyze acquired data from both systems was a time-limiting factor. To reduce data processing time and accelerate or “FastTrack” samples to purification, two unique and automated solutions were introduced. We describe the implementation of an integrated, multi-column, walk-up HPLC/SFC/MS system, and the implementation of an intelligent, automated method selection application which uses advanced data evaluation criteria to selectively score and identify the most practical separation conditions for SFC/MS and HPLC/MS methodologies. [Copyright &y& Elsevier]

Abstract: This paper demonstrates a combined approach for separating an active pharmaceutical ingredient (API) from a heavily contaminated waste stream. The approach uses organic solvent nanofiltration (OSN) to improve the application of counter-current chromatography (CCC) in an industrial process. OSN provides an efficient route for exchanging solutes from the process solvent into the desired mobile phase for CCC, generating a CCC feed containing less than 0.01% (area % by GC) of the original process solvents. The high solvent burden of CCC was additionally reduced through recovery of mobile phase using OSN, with the recovered solvent containing less than 1% (area % by HPLC) impurities. The recovered solvent was then successfully recycled into a subsequent CCC run with no indication of impurity build-up. Coupling OSN with CCC improved the mass-intensity of the CCC process, reducing the solvent use by 56%. OSN can be a useful tool in facilitating the application of CCC to pharmaceutical process streams. [Copyright &y& Elsevier]

Abstract: The accumulation of saxitoxins (STXs) in fish from freshwater aquaculture was investigated for the first time in the present study. Cyanotoxins have been monitored in liver and muscle samples of Oreochromis niloticus by chromatographic methods, both before and after the depuration process. The results show that tilapia can accumulate STXs. Our findings suggest that depuration with clean water is an alternative process to eliminate STXs from fish and, therefore, improve the safety of tilapia for consumers. [Copyright &y& Elsevier]

Abstract: High-speed counter-current chromatography (HSCCC) and preparative high-performance liquid chromatography (prep-HPLC) were successively used for the separation of epigallocatechin and flavonoids from Hypericum perforatum L. The two-phase solvent system composed of ethyl acetate–methanol–water (10:1:10, v/v) was used for HSCCC. About 900mg of the crude extract was separated by HSCCC, yielding 7.8mg of quercitrin at a purity of over 97%, 12.6mg of quercetin at a purity of over 93%, and 38.9mg of a mixture of hyperoside, isoquercitrin and miquelianin constituting over 97% of the fraction. A mixture of epigallocatechin and avicularin pooled from three HSCCC runs, a total amount of 54.3mg, was further separated by prep-HPLC yielding 23.4mg of epigallocatechin and 15.3mg of avicularin each at a purity of over 97%. [Copyright &y& Elsevier]

Abstract: The proanthocyanidin extract from tea (Camellia sinensis) leaves was purified for the further study of the biological role of proanthocyanidins in blister blight leaf disease of tea, which is caused by the fungus Exobasidium vexans. An aqueous acetone extract of proanthocyanidins prepared from healthy tea leaves was partially purified using Sephadex LH-20 chromatography. The crude proanthocyanidin extract obtained was fractionated with high-speed counter-current chromatography (HSCCC) using the solvent system n-hexane–EtOAc–MeOH–water (1:5:1:5). The purity of the each isolated fraction after a single HSCCC run was evaluated by high-performance liquid chromatography (HPLC). Seven fractions of high purity were isolated. The identity of the compound present in each fraction isolated was established using electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. Five proanthocyanidins and two flavanol digallates, (−)-epigallocatechin digallate (EGCDG) and (−)-epicatechin digallate (ECDG) were isolated. Comparison of spectral data of the proanthocyanidins isolated with those previously reported indicated that all five were known B-type proanthocyanidins with 2,3-cis stereochemistry in both the upper (u-unit) and the terminal (t-unit) units, and 4R configuration of the C-ring in the u-unit. The proanthocyanidins were established to be dimers composed of (−)-epigallocatechin gallate (EGCG), (−)-epicatechin gallate (ECG) and (−)-epiafzelechin gallate (EAG) units with the following structures: EGCG-(4β→6)-EGCG, ECG-(4β→6)-EGCG, EGCG-(4β→6)-ECG, EAG-(4β→6)-EGCG, ECG-(4β→6)-ECG by analysis of spectral data. Therefore HSCCC offers a powerful method for the separation of a group of closely related naturally occurring compounds. [Copyright &y& Elsevier]

Abstract: Apple pomace, a by-product in the processing of apple juice, was investigated as a potential source of polyphenols. Two methods of separation and purification of polyphenols from apple pomace extract were established by combination of gel chromatography with high-speed counter-current chromatography (HSCCC) and solvent extraction with HSCCC, respectively. The optimal separation was performed on a Sephadex LH-20 column using gradient aqueous ethanol as eluting solvent from 0% to 100% in increments of 10%. HPLC analysis indicated that main polyphenols existed in fractions eluted between 40% and 50% aqueous ethanol. The fractions of interest from column were separated by HSCCC with the solvent system hexane–ethyl acetate–1% aqueous acetic acid (0.5:9.5:10, v/v/v). Ethyl acetate fractionation of the apple pomace extract followed by direct HSCCC separation by the same solvent system in the volume ratio of 1:9:10 also produced a good separation of the main polyphenols of interest. Six high-purity polyphenols were achieved tentatively and identified by HPLC/MS: chlorogenic acid (1, m/z 354), quercetin-3-glucoside/quercetin-3-glacaside (2, m/z 464), quercetin-3-xyloside (3, m/z 434), phloridzin (4, m/z 436), quercetin-3-arabinoside (5, m/z 434), and quercetin-3-rhamnoside (6, m/z 448). These results provided a preliminary foundation for further development and exploration of apple pomace. [Copyright &y& Elsevier]

Abstract: Experiments were performed to evaluate whether counter-current chromatography (CCC) could function as an alternative purification method to reversed-phase high-performance liquid chromatography (RP-HPLC) and normal-phase supercritical fluid chromatography (SFC). RP-HPLC and SFC are the routine methods currently used in our high-throughput purification (HTP) facility for the purification of high-throughput organic synthesis (HTOS) libraries and medicinal chemistry reaction mixtures. Pre-equilibration of the solvent mixture layers was not mandatory for effective chromatography when hexanes–ethyl acetate–methanol–water (HEMW) solvent mixtures were used. Key to the use of CCC for high-throughput applications is the ability to effectively select a solvent system appropriate to each library member. Pilot-scale CCC elution time was used to estimate a starting solvent ratio and RP-HPLC retention time was then used to adjust solvent ratios within a particular library. It was also found that dimethyl sulfoxide (DMSO) and DMSO–methanol were suitable as sample injection solvents when using the HEMW solvent systems. [Copyright &y& Elsevier]

Abstract: In this paper, a rapid separation approach has been developed using high-capacity high-speed counter-current chromatography (high-capacity HSCCC) to isolate and purify honokiol and magnolol, which are the main bioactive constituents from Houpu. The optimization of the solvent selection process, sample loading volume and flow rate is systematically studied using analytical high-capacity HSCCC. The optimized parameters obtained rapidly at analytical scale were used for a 1000× scale-up preparative run using pilot scale high-capacity HSCCC in a MAXI-DE centrifuge. A crude sample of 43g was successfully separated and the fractions were analysed by high-performance liquid chromatography (HPLC). This large scale preparative single step run yielded 16.9 and 19.4g of honokiol and magnolol with purities of 98.6 and 99.9%, in only 20min. This is the first time that high-performance counter-current chromatography has been used to purify multiple gram grade bioactive compounds in less than 1h and at such high concentrations of final products (10.8g/l for magnolol and 7.0g/l for honokiol). [Copyright &y& Elsevier]

Abstract: Following preparative isolation and purification by high-speed counter-current chromatography (HSCCC), the collected fractions were generally analyzed by high-performance liquid chromatography (HPLC) to determine the relative purities of each fraction. Our paper reports for the first time a preparative isolation-purity detection hyphenated system: online coupling of HSCCC with high-performance liquid chromatography–diode array detection (HSCCC–HPLC–DAD). The introduction of online purity analysis in HSCCC has dramatically improved the efficiency of this technique by overcoming the drawbacks of post analysis in HSCCC isolation. The effluent from the outlet of HSCCC was splitted into two parts: one was collected, while the other was introduced directly into an HPLC–DAD system for purity analysis through a switch valve. Therefore, the purities of the obtained fractions from HSCCC were monitored, and fractions with high purities were collected. This strategy has been successfully demonstrated with the preparative isolation and purification of hyperoside from Hypericum perforatum (St. Jone''s Wort); a model of TBE-300A HSCCC was used to isolate and separate hyperoside from H. perforatum with a two-phase solvent system composed of ethyl acetate–ethanol–water at the volume ratio of 5:1:5 (v/v) using online detection technique. The isolation was done in less than 3.5h, and a total of 83.0-mg hyperoside at over 99.0% purity was yielded from 300mg of the partially purified extract. This new strategy possesses general utility in the preparation of bioactive compounds from traditional Chinese medicine (TCM). [Copyright &y& Elsevier]