Application of high-speed counter-current chromatography coupled with high-performance liquid chromatography-diode array detection for the preparative isolation and purification of hyperoside from Hypericum perforatum with online purity monitoring

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 3.5 h, and a total of 83.0-mg hyperoside at over 99.0% purity was yielded from 300 mg of the partially purified extract. This new strategy possesses general utility in the preparation of bioactive compounds from traditional Chinese medicine (TCM).     

2-chloro-(4R,5R)-bis[(1R,2S,5R)-menth-1-yloxycarbonyl)]-1,3,2-dioxaphospholane: a practical chiral pool-derived reagent for determining enantiomeric purity of alcohols

2-Chloro-(4 R,5 R)-bis[(1 R,2 S,5 R)-menth-1-yloxycarbonyl)]-1,3,2-dioxaphospholane is a practical reagent for reliably determining enantiomeric purity of chiral alcohols via (31)P NMR spectroscopy. The compound is available as a crystalline solid on a 20 g scale from PCl 3 and bis[(1 R,2 S,5 R)-menth-1-yl] tartrate. It is comparatively inert toward spontaneous hydrolysis under conventional laboratory conditions but undergoes quantitative substitution of alkoxide for chloride if treated with a chiral alcohol. Nonequivalent (31)P NMR signals of diastereomeric 2-alkoxy-1,3,2-dioxophospholanes were dispersed by approximately 1.4-0.1 ppm. The associated integral ratios reflected enantiomeric purities of preweighted samples of ( R)- and ( S)-1-phenylethanol, (+)- and (-)-menthol, and a set of primary, secondary, and tertiary alcohols with a precision of +/-0.4-1.0%.     

Dual detection approach to a more accurate measure of relative purity in high-throughput characterization of compound collections

The accurate determination of compound purity is crucial for characterizing library purity, monitoring the stability of storage compounds, and obtaining meaningful high-throughput screening results. However, current high-throughput techniques for the determination of compound purity are inadequate. We evaluated on-line chromatography detectors, including UV(TWC), UV(214), and ELSD detectors, in a series of studies of 233 compound mixtures prepared with known compositions. Results indicate that both UV(TWC) and UV(214) overestimate the minor component in a mixture whereas ELSD underestimates the minor component. An average of UV(TWC) and ELSD purities gives a more accurate measure of the relative purity for a wide range of compounds in various purity ranges. This technique was applied to 959 compounds from our compound collection to more accurately determine their relative purity.     

Separation and purification of two minor typical diarrhetic shellfish poisoning toxins from harmful marine microalgae via combined liquid chromatography with mass spectrometric detection

A novel method was developed for the purification of two typical diarrhetic shellfish poisoning toxins from toxin‐producing marine microalgae using macroporous resin, high‐speed countercurrent chromatography–mass spectrometry, and semipreparative high‐performance liquid chromatography–mass spectrometry. Analytical high‐performance liquid chromatography–mass spectrometry was used for identification and purity analysis of okadaic acid and dinophysistoxin‐1 because they exhibit no visible or ultraviolet absorption. First, four kinds of macroporous resins were investigated, and HP‐20 macroporous resin was selected for the preenrichment and cleanup of the two target toxins. Second, the resin‐purified sample was further purified using high‐speed countercurrent chromatography coupled with a mass spectrometer. The purities of the obtained okadaic acid and dinophysistoxin‐1 were 89.0 and 83.0%, respectively, as determined through analytical high‐performance liquid chromatography–mass spectrometry. Finally, further purification was carried out using semipreparative high‐performance liquid chromatography with mass spectrometry, and the purities of the final okadaic acid and dinophysistoxin‐1 products were both over 98.0% based on the analytical high‐performance liquid chromatography–mass spectrometry chromatograms and fraction spectra. This work demonstrates that the proposed purification process is a powerful method for the preparation of high‐purity okadaic acid and dinophysistoxin‐1 from toxin‐producing marine microalgae. Moreover, it is particularly important for the purification and preparation of minor toxins that exhibit no visible or ultraviolet absorption from harmful marine algae.     

Preparation and biodistribution of [111In]-rHuEpo for erythropoietin receptor imaging

Human recombinant erythropoietin (EPO) was successively labeled with [¹¹¹In]-indium chloride after conjugation with freshly prepared cyclic DTPA-dianhydride (ccDTPA). The best results of the conjugation were obtained by the addition of 100 i.u. of an EPO pharmaceutical solution (in phosphate buffer, pH 7.5) to a glass tube pre-coated with DTPA-dianhydride (0.01 mg) at 25 °C with continuous mild stirring for 30 minutes. Radio thin layer chromatography (RTLC), instant thin layer chromatography (ITLC) and high performance liquid chromatography (HPLC) showed overall radiochemical purity of higher than 95% at optimized conditions (specific activity = 1.2–1.5 GBq/mg, labeling efficiency 80%). Preliminary in vivo studies in normal rat model was performed to determine the biodistribution of the radiotracer up to 1 hour using scarification. The high kidney uptake of the tracer was consistent with the reported EPO receptor distribution.     

Coulometric assay of the primary standards potassium dichromate and ammonium hexanitratocerate

The high-precision coulometric titration apparatus of Eckfeldt and Shaffer (Leeds & Northrup Company) has been used for the precision assay of two primary standard materials for oxidation-reduction work. The purity found for NBS 136b Potassium Dichromate. 99.975 %, standard deviation 0.002 %, checks the earlier value of Marinenko and Taylor. Ammonium hexanitratocerate, if low in thorium, is also an excellent primary standard, two recent commercial preparations having purities of 99.972 and 99.984%, a higher standard deviation, 0.005 %, reflecting a minor difficulty in end-point detection caused by platinum oxide formation on the indicating electrodes.     

Semipreparative enantiomeric separation of omeprazole by supercritical fluid chromatography

Omeprazole, a widely used antiulcer drug, has been enantiomerically separated at semipreparative scale on a polysaccharide based chiral stationary phase by supercritical fluid chromatography (SFC). For this work, a modular supercritical fluid chromatograph was adapted to operate at semipreparative scale and a Chiralpak AD (250 mm x 10 mm) column was used. The effect of two organic modifiers (ethanol and 2-propanol) was studied, and different injection volumes and concentrations of the omeprazole racemic mixture were evaluated in order to obtain high enantiomeric purities and production rates. Better results were achieved using concentration overloading instead of volume overloading. The recoveries decreased when the requirements of enantiomeric purity or the load increased, but it was possible to recover 100% of both enantiomers at an enantiomeric purity higher than 99.9% under some loading conditions, like injecting 1 and 2 ml of a solution of 3g/l. As far as production rates are concerned, the best result for S-(-)-omeprazole at that purity (27.2mg/h) was achieved with sample concentrations of 10 g/l and the injection of 2 ml, while a volume of 4 ml was better in the case of R-(+)-omeprazole (20.5mg/h).     

Use of semipreparative supercritical fluid chromatography to obtain small quantities of the albendazole sulfoxide enantiomers

The semipreparative separation of the albendazole sulfoxide enantiomers using chiral supercritical fluid chromatography is presented in this work. For this purpose, a modular SFC chromatograph was adapted to work at semipreparative scale and a Chiralpak AD (250 x 10 mm) column was used. Different injection volumes were evaluated in order to obtain high purities and throughputs. Using the maximum load, it was possible to obtain 37 mg/h of the first eluted enantiomer with a purity of 99.9%, and 36.5 mg/h of the second eluted enantiomer with a purity of 95%.     

A new traceability scheme for the development of international system-traceable persistent organic pollutant reference materials by quantitative nuclear magnetic resonance

Quantitative nuclear magnetic resonance (qNMR) was used for the purity determination of neat compounds of persistent organic pollutants (POPs). qNMR is a unique quantitative method that is not only traceable to the International System of Units (SI), but it also does not require a standard of its own. The purities of the POP compounds determined in this work were traceable to a single certified reference material (CRM), which is extremely attractive for reference material producers. The purities observed by qNMR were equivalent to those observed by gas chromatography with flame ionization detection (GC/FID) or a differential scanning calorimetry (DSC) combined with a thermogravimetric analyzer (TGA). The uncertainties obtained by the qNMR method were comparable to being slightly larger than those observed by DSC.     

Comparison of high-speed counter-current chromatography instruments for the separation of the extracts of the seeds of Oroxylum indicum

Analytical Milli high-speed counter-current chromatography (HSCCC) was used for the selection and optimization of the two-phase solvent system to separate flavonoids from the extracts of the seeds of Oroxylum indicum. The optimum solvent system obtained from Milli-CCC was also the best solvent system for preparative HSCCC and led to the successful separation of two crude flavonoids from the seeds of O. indicum by Lab/Prep (laboratory preparative) HSCCC using different sized coils. Four flavonoids were isolated by preparative HSCCC: baicalein-7-O-diglucoside (25.0 mg, 92% purity), baicalein-7-o-glucoside (50.4 mg; 95% purity), baicalein (75 mg; purity 98%) and chrysin (100 mg; purity 98%).     

Partial-discard strategy for obtaining high purity products using simulated moving bed chromatography

A new "partial-discard" operation strategy was developed to improve the performance, especially purity, achievable in simulated moving bed (SMB) chromatography. This strategy was applied to the four-zone SMB process with two columns per zone for binary separation within the linear range. The "partial-discard" strategy significantly enhanced the purity or enrichment when the discard time and discard length were controlled. In addition, the "partial-feed with partial-discard" strategy improved remarkably the extract and raffinate purities at an intermediate feed time compared with the "partial-feed" operation. Adjustments of the discard length and discard time played key roles in achieving the desired product purity in SMB chromatographic performance.     

Some considerations governing the choice of method for purity determinations by cryoscopy

Some theories of freezing and melting are considered and as a result it is concluded that the stationary temperatures observed in dynamic systems in one laboratory may not be suitable for use for the determination of purities in another. Factors which give rise to a slow attainment of equilibrium between solid and liquid are considered. The use of a comparative technique for the determination of purities by freezing point measurements is advocated and a new method for the determination of purities by melting curves is described. This method allows the purities of certain materials to be ascertained when equilibrium between solid and liquid is reached rather slowly. Finally an outline is given of the theory of a method suitable for the intercomparison of the purity of several samples simultaneously.     

高速逆流色谱法分离制备鱼藤根中的2种鱼藤酮类化合物

建立了分离制备鱼藤根中2种鱼藤酮类化合物的高速逆流色谱法。以正己烷-乙酸乙酯-甲醇-水(体积比为7:0.25:5:3)为两相溶剂系统,上相为固定相,下相为流动相,在主机转速850 r/min、流速2.0 mL/min、检测波长254 nm条件下进行分离制备,从50 mg鱼藤根粗提物中得到了2种鱼藤酮类化合物,分别为6.4 mg纯度为96.60%的鱼藤酮和23.4 mg纯度为97.87%的鱼藤素。该方法为鱼藤酮类化合物的深入研究提供了物质基础。     

The high-mass component (>m/z 10 000) of coal tar pitch by matrix-assisted laser desorption/ionisation mass spectrometry and size-exclusion chromatography

The size-exclusion chromatography (SEC) of acetone-soluble, pyridine-soluble and pyridine-insoluble fractions of a coal tar pitch indicates a bimodal distribution in each fraction. The proportion of high-mass material excluded from the SEC column porosity increases with solvent polarity. The polymer calibration of SEC shows the mass range of the small molecules to be from approximately 100 u to approximately 6000 u, with the mass range of the large excluded molecules above 200 000 u and up to several million u. In contrast, matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) shows a similar low-mass range of ion abundances (< m/z 6000), but with a smaller range of high-mass ion abundances, from approximately m/z 10 000 to 100 000. The large molecules may have three-dimensional structures to allow molecules of relatively low mass to behave as if they are of large size in SEC. Laser desorption mass spectrometry of the acetone- and pyridine-soluble fractions produced molecular ions of polycyclic aromatics that can be related to the known compositions from gas chromatography (GC) mass spectrometry. The experimental conditions used to generate the bimodal distribution by MALDI-MS involve reducing the ion signal intensities to avoid overload of the detector and enable detection of the high-mass ions, by reducing the high-mass detector voltage (i.e. sensitivity) and increasing the laser power.     

Semi-preparative gas chromatographic separation of all-trans-perhydrotriphenylene enantiomers on a chiral cyclodextrin stationary phase

Enantiomers of all-trans-perhydrotriphenylene (PHTP) were separated by gas chromatography using heptakis(6-O-tert.-butyldimethylsilyl-2,3-di-O-methyl)-beta-cyclodextrin (TBDMS-beta-CD) as the chiral selector. Conditions for semi-preparative separations were established using a 2 m x 2 mm I.D. packed column and subsequently extended to a 1.8 m x 4 mm I.D. column which enabled separations on a mg scale. The column packing was TBDMS-beta-CD dissolved in SE-54 coated on Chromosorb P AW-DMCS 80-100 mesh. Optimization of the chromatographic conditions (oven temperature, carrier gas flow, and column load) with respect to better efficiency and peak retention resulted in a system capable of separating up to 10 mg of the racemate per day. Purities of separated enantiomers were determined by capillary gas chromatography. Yields and purities of the fractions obtained by single- and double-step separations are compared. Highly enriched enantiomers with purities of up to 99.6% (99.2% ee) were obtained by a single separation step.     

Determination of optical purity in partially racemized samples of l-aspartic acid by means of liquid crystals

A method for the determination of the optical purity of partially racemized samples of l-aspartic acid, based on the measurement of the pitch and handedness of cholesteric mesophases induced in MBBA (p-methoxybenzylidene-p'-n-butylaniline), is described. The minimum quantity of aspartic acid required varies with the optical purity of the sample (from 50 mug for an enantiomeric purity of 10-20% down to 5 mug for purities around 90%).     

Evaluation of field desorption mass spectrometry for the analysis of polyethylene

Field desorption mass spectrometry (FD-MS) has been evaluated for the analysis of low molecular weight polyethylene by using samples in the molecular weight range 600–2000 u as determined by gel permeation chromatography. The repeat units and end groups were characterized by FD-MS, but it was demonstrated that accurate molecular weight distribution data cannot be obtained for polyethylene by FD-MS because there is mass discrimination against the higher molecular weight polymers.     

Continuous chromatographic separation of a baclofen precursor (N-Boc-4-[p-chloro-phenyl]-2-pyrrolidone) in a simulated moving bed using a polysaccharide carbamate as chiral stationary phase

Liquid chromatography is known as one of the most flexible, efficient and cost-effective methods to resolve racemic mixture in order to attend the growing demand of the pharmaceutical industry for pure enantiomeric compounds. Cellulose tris(3,5-dimethylphenylcarbamate) is frequently used as a stationary phase for enantiomeric separations because of its attractive properties, including high enantioselectivity, high loading capacity and good mechanical stability. In this study, we investigated the usefulness of cellulose tris(3,5-dimethylphenylcarbamate) as the stationary phase and of ethanol and hexane mixtures as the mobile phases for the chromatographic separation of potential pharmaceutical intermediates. Using adsorption equilibrium data, we determined the optimal operational conditions for the separation of the N-Boc-4-[p-chloro-phenyl]-2-pyrrolidone enantiomers - a baclofen precursor - in a semi-preparative scale simulated moving bed unit. This unit was used to obtain high purity enantiomers on a scale of 1g/day. The outlet streams were analyzed by an on-line system that consisted of a UV-vis spectrophotometric unit, a polarimeter, and HPLC. Enantiomeric purities of up to 97% were obtained for the raffinate stream and up to 90% for the extract stream.     

Influence of Storage Conditions on the Stability of Lactide

The objective following of the present study was to investigate the effect of different storage time and conditions on the stability of lactide. In this study the samples of lactide with different purities were stored under three various storage conditions and the compound integration was monitored for a period of 2 weeks. The storage conditions selected were (1) in the desiccator, (2) in the glove box under argon and (3) under ambient atmosphere. The purity of samples was performed on HPLC and Karl Fischer titration during all period of storage. It was shown that the initial purity of the lactide has a significant bearing on the lactide degradation.     

Production of malachite green oxalate and leucomalachite green reference materials certified for purity

Malachite green oxalate (MG oxalate) and leucomalachite green (LMG) have been prepared and certified as pure reference materials. The purities of MG oxalate and LMG were assessed by high-performance liquid chromatography–diode array detection (HPLC–DAD), nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry (DSC), Karl Fischer titration, ashing and thermogravimetric analysis (TGA). MG oxalate was purified by supercritical fluid extraction (SFE). Prior to purification, commercial MG oxalate purity was estimated to be about 90%. The main impurities present in SFE-purified MG oxalate were identified and quantified using HPLC–DAD. The main impurities were found to be monode-MG (monodemethylated MG oxalate synthesis impurity), 4-(dimethylamino)benzophenone (4-DMABP), MG-carbinol and LMG. The homogeneity of both reference materials was also determined. Issues associated with the stability of LMG and MG oxalate in solution forced an extensive study investigating different parameters i.e. solvent, acid, analyte concentration and temperature. MG oxalate (100 μg/mL) was found to be stable in acetonitrile containing 1% v/v glacial acetic acid for at least 155 days and LMG (100 μg/mL) was stable in acetonitrile for at least 133 days. The final purity value for MG oxalate was 94.3 ± 1.4% m/m at the 95% confidence interval (or 67% m/m if MG cation is reported). For LMG, the certified purity was found to be 98.8 ± 0.8% m/m at the 95% confidence interval. Figure Calibration reference materials for malachite green and leucomalachite green, certified for purity, are essential in characterising these key analytes in a fish matrix reference material