Preparative HPLC System Research Articles

A practical fully automated radiosynthesis of [18F]Flurpiridaz on the module modular lab-pharmtracer without external purification

Background[18F]Flurpiridaz is a promising novel cardiac PET imaging tracer formed by the radiolabeling of pyridaben derivative with fluorine-18. Clinical studies on [18F]Flurpiridaz are currently at the phase III level for the assessment of MPI. Providing high image quality thanks to its relatively long half-life, F-18 is a high-potential radionuclide for the early detection of CAD. In this study, we aimed to develop a fully automated synthesis of [18F]Flurpiridaz without further preparative HPLC purification.ResultsPrecursor 6 was obtained by multi-step synthesis starting from mucochloric acid (1) as a sole product with 35% yield and identified by spectroscopic measurement. Manually cold labeling experiments were performed using the stable isotope [19F]F, and TBA-HCO3 PTC provided desirable fluorinated compound with high yield. A fully automated [18F]Flurpiridaz synthesis on the ML-PT device provided 55–65% radiochemical yield with more than 98% radiochemical purity. The final product purification method demonstrated that [18F]Flurpiridaz could be obtained without an external preparative HPLC system as a pharmaceutical quality.ConclusionA novel and fascinating strategy was developed for the fully automated synthesis of [18F]Flurpiridaz (7) on ML PT. Organic synthesis of precursor 6 was achieved with a desirable yield and characterized by NMR and HR-MS. A detailed set of cold experiments were completed for optimization conditions before hot trials and TBA-HCO3 increased molar activity with a minimum amount of side products. Radiolabeling showed that our self-designed automated synthesis method enables high radiochemical yield and radiochemical purity for the production of [18F]Flurpiridaz. The desirable radiopharmaceutical quality of the product was obtained without using an additional preparative HPLC system. [18F]Flurpiridaz (7) preserved its stability within 12 h and final specifications were consistent with the acceptance criteria in Ph. Eur. regulations.

Counterfeits of Xanax – combination of designer benzodiazepines clonazolam and flualprazolam

Novel benzodiazepines, emerged on the recreational drug market in past few years, have caused serious problems in many countries, including intoxications and fatalities. Not only, that limited information is still available on their dosage, the situation may be even more complicated if a combination of these designer drugs occurs. For identification of compounds of interest, NMR and HPLC-MS technique were used. NMR spectroscopy was performed on Agilent 400 MR DDR2 (1H NMR 400 MHz, 13C NMR 100 MHz) at room temperature and the spectra were referenced on the residual peak of the solvents (CDCl3). HPLC-MS analyses were performed using the Infinity 1290 LC system coupled with 6460 mass spectrometr (Agilent Technologies, USA). Flash chromatography – Combiflash Rf200 UV/VIS (Teledyne Technologies, California) and preparative HPLC system Autopurification (Waters) were used for isolation and purification of the two benzodiazepines. The obtained benzodiazepines were characterized and used for qualitative analysis of the tablets. Quantitative analysis was performed by LC-MS (see above) in MRM mode using experimental conditions published in the literature. Sample preparation included homogenization of the tablets, dissolution of a tablet equivalent and dilution. Commercial standards were purchased and used as external calibrators. Two independent batches of Xanax counterfeits seized by Customs Service in Prague, Czech Republic were investigated. Each seized batch was about 1000 tablets large; both were of same appearance, very similar to the original product. Both batches had the same dimensions, but they differed in average mass. Using qualitative analysis, we identified clonazolam and flualprazolam in tablets of both seized batches. Furthermore, the tablets contained large amount of caffeine, which was presumably used as excipient. First batch of tablets contained in average 105 μg of clonazolam and 423 μg of flualprazolam per tablet. The second batch contained 81 μg of clonazolam and 385 μg of flualprazolam per tablet. Convincing and remarkably similar to the original product. Both of the tablets batches contained clonazolam and flualprazolam in various concentrations. Based on our analytical results and the literature data survey on the flualprazolam and clonazolam toxicity, these tablets pose a severe health risk for the users as well as for public.

Study on imperatorin extracted from Angelica dahurica and its UV photocatalytic reaction with collagen

Soxhlet extraction method was used to extract imperatorin from Angelica dahurica, and the extraction ratio under different extraction condition was optimized to attain the best condition. Then, XAD-16 macroporous resin was selected as the optimal resin to boost the extraction ratio of imperatorin. Afterwards, the higher purity of imperatorin (96.84±0.2%) was separated by preparative HPLC system. Next, the photocatalytic reaction between the above imperatorin and collagen which the highest levels in skin was investigated using UV-vis spectroscopy, amino acid analysis and HPLC analysis. The results showed that imperatorin reacted with collagen only under ultraviolet light which caused the denaturation of collagen, and three new products were generated. The ultraviolet products were isolated by preparative HPLC system and separately detected by high-resolution mass spectrum. The possible UV photocatalytic reaction mechanism between imperatorin and collagen is that ultraviolet light induces the increase of the activity of the imperatorin to react with the tyrosine in the collagen, resulted in the denaturation of collagen and reestablish of the normal epidermal tissue in skin.

Hyperterpenoids A and B: Two pairs of unprecedented 6/6/4/6/6 polycyclic cyclobutane meroterpenoids with potent neuroprotective and anti-inflammatory activities from Hypericum beanii

Hyperterpenoid A (1) and B (2), two pairs of enantiomers, with an unprecedented 6/6/4/6/6 polycyclic skeleton, along with one known compoud hypermonone A (3) were isolated from Hypericum beanii. The racemate (±)-1 and (±)-2 were successfully separated into the two optically pure enantiomers (ee ≥ 99%) using a preparative HPLC system. Their absolute configurations were elucidated by extensive spectroscopic analyses and single-crystal X-ray diffraction method. The related plausible biogenetic pathways were presented. Compound 1-3 showed significant neuroprotective activity and potential anti-inflammatory activity. The result that (+)-2 and (-)-2 presented different anti-inflammatory properties, may lead us to new discovery of structure activity relationship between racemates, enantiomers, and diastereomers, as well as further research regarding the binding of drugs to target proteins.

Antiplasmodial activity of Heinsia crinita (Rubiaceae) and identification of new iridoids

Ethnopharmacological relevanceHeinsia crinita is used in traditional medicine for the treatment of febrile illness and erectile dysfunction. Its stem bark powder is found in some peripheral markets in the Democratic Republic of the Congo (DRC) as a remedy against malaria. Investigations were conducted on crude extracts of leaves, fruits and stem barks in view to validate their use and to determine which plant part possesses the best antiplasmodial properties. Materials and methodsDifferent plant parts were extracted with methanol, ethanol and dichloromethane. Based on the preliminary assays, the dichloromethane extract of the stem bark was subjected to fractionation using preparative HPLC system and column chromatography. This step led to the isolation of two new iridoids which had their structures elucidated by NMR, UV, MS and FT-IR spectroscopic techniques. Extracts and pure compounds were tested in vitro against the 3D7 strain of Plasmodium falciparum. The inhibition of the parasite growth was evaluated in vitro by colorimetric method (p-LDH assay) and their cytotoxicity evaluated in vitro against the human non-cancer fibroblast cell line (WI38) through WST1 assay.The in vivo antiplasmodial activity was assessed by the inhibition of Plasmodium berghei growth in infected mice treated with the ethanol extract of H. crinita stem bark at the concentrations of 200 and 300mg/Kg/day per os, using a protocol based on the 4-d suppressive test of Peters and compared to a non-treated negative control group of mice (growth =100%). Finally the antioxidant activity of the same extract was evaluated using ABTS, DPPH and cell-based assays. ResultsA moderate in vitro antiplasmodial activity was observed for the dichloromethane extract of the stem bark of H. crinita (IC50 =29.2±1.39µg/mL) and for the two new iridoids, lamalbide 6, 7, 8- triacetate (IC50 =16.39±0.43µg/mL) as well as for its aglycone lamiridosin 6, 7, 8-triacetate (IC50 =0.44.56±1.12µg/mL).The ethanolic stem bark extract (200 and 300mg/kg/day, oral route) showed a moderate in vivo antimalarial activity in Plasmodium berghei-infected mice with 27.84±2.75% and 48.54±3.76% of inhibition of the parasite growth, respectively (p<0.01).). This extract displayed high cellular antioxidant activity using dichlorofluorescein-diacetate (DCFDA) on HL-60 monocytes.These crude extracts and pure compounds tested at the higher concentration of 100µg/mL did not show any cytotoxicity against WI38 cells. ConclusionsThe results showed that H. crinita extracts possess antimalarial activity and contain some unusual iridoids with moderate antiplasmodial activity, therefore justifying to some extent its traditional use by the local population in DRC for this purpose. This is the first report of the isolation and antiplasmodial activity of these two new iridoids.

Antibacterial Activity And Phytochemical Investigation Of Solanum Sp. (Solanaceae)

Solanum (Solanaceae) is a genus present in tropical and subtropical areas with approximately 1500 species mostly distributed in South America [1]. Solanum sp. is a plant species found in Brazilian Cerrado. The EtOH extract of leaves (SEE) of this species showed inhibition against Staphyloccocus aureus and Escherichia coli of 69.15 ± 6.16 and 48.79 ± 5.87%, respectively, and was selected for phytochemical study. The aim of this work was to fractionate the SEE and identify the obtained compounds. Portions of 50 mg of SEE were solubilized in MeOH: H2O (1:1), the suspensions obtained were centrifuged (10.000 rpm, 10 min) and 900µL of the supernatant was injected on to a preparative HPLC system. The elution was carried out using gradient elution of 0.1% formic acid (A) and MeCN with 0.1% formic acid (B), from 5 to 50% of B in 30 min, detection at λ= 210 and 280nm, flow rate of 20 mL/min, generating twelve fractions. TLC analysis of fractions 4, 5 and 6 showed one major spot with fluorescence characteristic of phenolic acids (fraction 4) and flavonoids (fractions 5 and 6). The conjunct analysis of the obtained chromatograms and MS spectra registered in the negative mode allowed the identification of fractions 4 – 6 as chlorogenic acid, rutin and a quercetin glucoside, respectively, by standards co-injection and/or comparison with literature data. Mono and bi-dimensional NMR will be performed to confirm the identification of the isolated compounds. Moreover, the antibacterial and antioxidant activities of the purified compounds will also be evaluated.

Recycling preparative high performance liquid chromatography for the separation of curcumin from curcuminoids incurcuma longaL.

Curcumin is the main bioactive constituent of turmeric rhizome. A wide range of pharmacological activities of curcumin has been reported. There has been growing interest in the development of preparative purification of curcumin. The objective of this study is the isolation of curcumin from other components by using recycling preparative HPLC method from curcuma longa L. The extraction was achieved by using ultrasonic system with three different solvents, including acetone, dichloromethane, and ethanol. After optimization of extraction process, preparative HPLC system was first applied for separation of curcumin. Because of poor resolution of preparative HPLC, recycled preparative HPLC was applied for separation of curcumin with higher purity up to five cycles. Recycled preparative HPLC resulted in improvement in separation factors which led to the increase of curcumin purity up to 99.5%. This method is simple, highly efficient, environmentally friendly, and has been demonstrated to be effective for prepa...

A novel preparative liquid chromatograph for repetitive enrichment and purification of low-abundance compounds

A novel recycle preparative HPLC system was developed to repeatedly enrich and purify low-abundance compounds from complex samples. The recycle separation involves injecting a large volume of the sample through a trap column, releasing the absorbed sample for preparative HPLC separation, and then capturing the target component in the same trap column for the next separation. After several cycles, the final purified target compound absorbed in the trap column is eluted with a small volume of solvent. The capture and recycle procedure was realized by adjusting the strength of the mobile phase using an auxiliary pump and switching the two valves connecting the trap and preparative columns. Compared with standard HPLC or existing recycle chromatography, this system not only reduces the solvent side-effects and sample volume overloading during injection but also avoids peak–broadening and sensitivity loss during the recycle runs. Moreover, the final purified products can be easily concentrated. With the aid of this system, a 10% polyphenol in extracts from Saussurea involucrata cultured cells reached >95% purity with more than 95% recovery after three recycle purifications, and a 1% unstable epimer impurity in the raw material of the drug silybin reached >95% purity with 75% recovery after a six-cycle separation.

Binuclear trans‐Bis(β‐iminoaryloxy)palladium(II) Complexes Doubly Linked with Pentamethylene Spacers: Structure‐Dependent Flapping Motion and Heterochiral Association Behavior of the Clothespin‐Shaped Molecules

The synthesis, structure, and solution-state behavior of clothespin-shaped binuclear trans-bis(β-iminoaryloxy)palladium(II) complexes doubly linked with pentamethylene spacers are described. Achiral syn and racemic anti isomers of complexes 1-3 were prepared by treating Pd(OAc)2 with the corresponding N,N'-bis(β-hydroxyarylmethylene)-1,5-pentanediamine and then subjecting the mixture to chromatographic separation. Optically pure (100 % ee) complexes, (+)-anti-1, (+)-anti-2, and (+)-anti-3, were obtained from the racemic mixture by employing a preparative HPLC system with a chiral column. The trans coordination and clothespin-shaped structures with syn and anti conformations of these complexes have been unequivocally established by X-ray diffraction studies. (1)H NMR analysis showed that (±)-anti-1, (±)-anti-2, syn-2, and (±)-anti-3 display a flapping motion by consecutive stacking association/dissociation between cofacial coordination planes in [D8]toluene, whereas syn-1 and syn-3 are static under the same conditions. The activation parameters for the flapping motion (ΔH(≠) and ΔS(≠)) were determined from variable-temperature NMR analyses as 50.4 kJ mol(-1) and 60.1 J mol(-1) K(-1) for (±)-anti-1, 31.0 kJ mol(-1) and -22.7 J mol(-1) K(-1) for (±)-anti-2, 29.6 kJ mol(-1) and -57.7 J mol(-1) K(-1) for syn-2, and 35.0 kJ mol(-1) and 0.5 J mol(-1) K(-1) for (±)-anti-3, respectively. The molecular structure and kinetic parameters demonstrate that all of the anti complexes flap with a twisting motion in [D8]toluene, although (±)-anti-1 bearing dilated Z-shaped blades moves more dynamically than I-shaped (±)-anti-2 or the smaller (±)-anti-3. Highly symmetrical syn-2 displays a much more static flapping motion, that is, in a see-saw-like manner. In CDCl3, (±)-anti-1 exhibits an extraordinary upfield shift of the (1)H NMR signals with increasing concentration, whereas solutions of (+)-anti-1 and the other syn/anti analogues 2 and 3 exhibit negligible or slight changes in the chemical shifts under the same conditions, which indicates that anti-1 undergoes a specific heterochiral association in the solution state. Equilibrium constants for the dimerizations of (±)- and (+)-anti-1 in CDCl3 at 293 K were estimated by curve-fitting analysis of the (1)H NMR chemical shift dependences on concentration as 26 M(-1) [KD(racemic)] and 3.2 M(-1) [KD(homo)], respectively. The heterochiral association constant [KD(hetero)] was estimated as 98 M(-1), based on the relationship KD(racemic) = 1/2 KD(homo) +1/4 KD(hetero). An inward stacking motif of interpenetrative dimer association is postulated as the mechanistic rationale for this rare case of heterochiral association.

Study of Anticonvulsant Clobazam Drug by Sophisticated Instrumentation

Clobazam (7-Chloro-1-methyl-5-phenyl-1,5-benzodiazepine-2,4(3H)-dione) an anticonvulsant drug used since 1984, with molecular formula C16H13ClN2O2 and molecular mass 300, was characterized with sophisticated instruments like FTIR, UV-NIR, GC-MS, TGA-DTA-DSC and Nano particle size analyzer with zeta potential. The compound was confirmed with the help of GC-MS and the mass of sample was found aggregate with the standard values and mass spectra compare with NIST and WELEY library data. The UV-NIR analysis showed that the λmax was 231 nm with methanol as a diluent. FTIR analysis of the Clobazam gave the characteristics peaks of cyclic amide. HPTLC was used to determine the purity of the drug. Thermal behavior was studied with TGA-DTA-DSC analysis. The particle size of clobazam was measured with the Nano particle size analyzer and Zeta potential of drud found -77.0 mV. The purity of drug check with Preparative HPLC system.

Study of Anticonvulsant Clobazam Drug by Sophisticated Instrumentation

Clobazam (7-Chloro-1-methyl-5-phenyl-1,5-benzodiazepine-2,4(3H)-dione) an anticonvulsant drug used since 1984, with molecular formula C16H13ClN2O2 and molecular mass 300, was characterized with sophisticated instruments like FTIR, UV-NIR, GC-MS, TGA-DTA-DSC and Nano particle size analyzer with zeta potential. The compound was confirmed with the help of GC-MS and the mass of sample was found aggregate with the standard values and mass spectra compare with NIST and WELEY library data. The UV-NIR analysis showed that the λmax was 231 nm with methanol as a diluent. FTIR analysis of the Clobazam gave the characteristics peaks of cyclic amide. HPTLC was used to determine the purity of the drug. Thermal behavior was studied with TGA-DTA-DSC analysis. The particle size of clobazam was measured with the Nano particle size analyzer and Zeta potential of drud found -77.0 mV. The purity of drug check with Preparative HPLC system.

Two-dimensional preparative liquid chromatography system for preparative separation of minor amount components from complicated natural products

An on-line comprehensive two-dimensional preparative liquid chromatography system was developed for preparative separation of minor amount components from complicated natural products. Medium-pressure liquid chromatograph (MPLC) was applied as the first dimension and preparative HPLC as the second one, in conjunction with trapping column and makeup pump. The performance of the trapping column was evaluated, in terms of column size, dilution ratio and diameter-height ratio, as well as system pressure from the view of medium pressure liquid chromatograph. Satisfactory trapping efficiency can be achieved using a commercially available 15mm×30mm i.d. ODS pre-column. The instrument operation and the performance of this MPLC×preparative HPLC system were illustrated by gram-scale isolation of crude macro-porous resin enriched water extract of Rheum hotaoense. Automated multi-step preparative separation of 25 compounds, whose structures were identified by MS, 1H NMR and even by less-sensitive 13C NMR, could be achieved in a short period of time using this system, exhibiting great advantages in analytical efficiency and sample treatment capacity compared with conventional methods.

Isolation of Campesteryl Ferulate and Epi-Campesteryl Ferulate, Two Components of γ-Oryzanol from Rice Bran

Campesteryl ferulate (3a, 24R/α) and epi-campesteryl ferulate (3b, 24S/β), components of rice bran γ-oryzanol, were isolated by the preparative recycle HPLC system using a combination of ODS silica and cholester packed columns at over 99% purity. Their purities and structures of 3a and 3b thus obtained were confirmed by HPLC analysis and physical data (1H- and 13C-NMR, MS spectra, and X-ray crystallography).

New Flavonol Triglycosides from the Leaves of Soybean Cultivars

Soybean (Glycine max) is a major crop in the world. Three new flavonol 3-O-glycosides, kaempferol 3-O-alpha-L-rhamnopyranosyl-(1 --> 4)-[alpha-L-rhamnopyranosyl-(1 --> 6)-beta-D-galactopyranoside] (1), kaempferol 3-O-alpha-L-rhamnopyranosyl-(1 --> 4)-[beta-D-glucopyranosyl-(1 --> 6)-beta-D-galactopyranoside] (4) and quercetin 3-O-beta-D-glucopyranosyl-(1--> 2)-[alpha-L-rhamnopyranosyl-(1 --> 6)-beta-galactopyranoside] (5) were isolated from the leaves of soybean cultivars, together with three known compounds, kaempferol 3-O-beta-D-glucopyranosyl-(1 --> 2)-[alpha-L-rhamnopyranosyl-(1--> 6)-beta-D-galactopyranoside] (2), kaempferol 3-O-beta-D-glucopyranosyl-(1 --> 2)-[alpha-L-rhamnopyranosyl-(1 --> 6)-beta-D-glucopyranoside] (3) and quercetin 3-O-beta-D-glucopyranosyl-(1 --> 2)-[alpha-L-rhamnopyranosyl-(1 --> 6)-beta-D-glucopyranoside] (6), and also common flavonoids. The isolated compounds possess similar structures and high water solubility, and so it was hard to isolate them (in particular 5 and 6) with a normal preparative HPLC system. Their final purification was achieved by a preparative HPLC system equipped with a recycle device.

Development of an Automated Synthesis System for Preparation of Glucuronides Using a Solid-Phase Extraction Column Loaded with Microsomes

An automated synthesis system using a solid-phase extraction (SPE) system and column packed with octadecylsilica (ODS), which was coated with phospholipid and loaded with dog liver microsomes, was developed for synthesis of glucuronides. Preparation of the microsome-immobilized SPE column, glucuronidation of drugs to synthesize the glucuronides and elution of the products were performed by an automated synthesis system. The phospholipid-coated SPE column and then the microsome-immobilized SPE column were readily prepared by allowing a solution containing L-alpha-dipalmitoylphosphatidylcholine to flow through the SPE column, and then by recycling a buffer solution containing dog liver microsomes through the resulting phospholipid-coated SPE column. The microsome-immobilized SPE column exhibiting the uridine diphosphate (UDP)-glucuronosyltransferase activity catalyzed the glucuronidation of mefenamic acid and estradiol to the corresponding glucuronides in the presence of UDP-glucuronic acid, and three glucuronides of mefenamic acid and estradiol were synthesized using the automated synthesis system, by simply recycling a buffer solution containing UDP-glucuronic acid through the microsome-immobilized SPE column loaded with the substrate. We used beta-cyclodextrin as a solubilizing agent for the synthesis of the glucuronides of estradiol that is practically insoluble in aqueous solutions. The productivity of these glucuronides using the microsome-immobilized SPE column was higher than that using the free microsomes (batch method). Furthermore, we developed a fully automated synthesis-isolation system by coupling the automated synthesis system to an automated preparative HPLC system. The automated synthesis system as well as the fully automated synthesis-isolation system should be very useful for synthesizing glucuronides for drug development.

Generation of Formaldehyde by Pharmaceutical Excipients and Its Absorption by Meglumine

Formaldehyde is a well-known air impurity. The possibility was investigated in this study that pharmaceutical excipients commonly used in oral solid dosage forms might also be sources of formaldehyde. The results showed that formaldehyde is generated by the excipients lactose, D-mannitol, microcrystalline cellulose, low-substituted hydroxypropylcellulose, magnesium stearate and light anhydrous silicic acid. Since the quality and safety of pharmaceutical products can be significantly affected by the presence of formaldehyde, various amines were then investigated for their ability to decrease levels of formaldehyde using an aqueous solution system. Of the four amines investigated, only meglumine proved capable of reducing formaldehyde levels. The reaction product between formaldehyde and meglumine was obtained by fractionation using the preparative HPLC system and the structure was clarified by (1)H-, (13)C-NMR, various types of two-dimensional NMR and mass spectroscopy. The reaction product was determined to be a compound with a 1,3-oxazinane skeleton and containing one more carbon than meglumine. It was presumed that formaldehyde reacted with the secondary amino group in meglumine to form the reaction product via an iminium salt intermediate by cyclization. As meglumine is permitted to be used as a pharmaceutical excipient in both oral and parenteral dosage forms by regulations worldwide, the addition of meglumine to pharmaceutical products can be expected to contribute to the stabilization of many drug substances.

Application of conjoint liquid chromatography with monolithic disks for the simultaneous determination of immunoglobulin G and other proteins present in a cell culture medium

The aim of this study was to develop a chromatographic method, as a substitute for enzyme-linked immunosorbent assays, for the rapid and simultaneous detection of IgG, insulin, and transferrin present in a cell culture medium. Conjoint liquid chromatography (conjoint LC) using monolithic disks was applied for this purpose. An anion-exchange disk was combined with a Protein G affinity disk in a preparative HPLC system. IgG bound to the Protein G disk, whereas transferrin and insulin were captured on the quaternary ammonium (QA) disk. Using this method, it was possible to simultaneously determine the concentrations of IgG, transferrin, and insulin in the cell culture medium. Thus, conjoint LC could be used for the rapid and simultaneous detection of different proteins present in a cell culture medium.

A Survey of Composition and Content of Carotenoids, Sugars, and Ascorbic Acid in Watermelons with Various Flesh Colors

About 40 watermelon samples with various flesh colors (red, pink, orange, and yellow) were tested for their carotene, sugar, and ascorbic acid contents. Carotenoids were separated and purified by using a preparative HPLC system and identified by comparing the spectra with standard compounds by using a diode array detector. Sugar and ascorbic acid contents were measured by HPLC methods. Red and pink colored watermelon contained lycopene as the major carotenoid, with a wide range of variation (5 to 51 μg·g-1). Beta-carotene was the second major carotenoid and was less than 6 μg·g-1. There were also lutein and violazanthin in less than 1.5 μg·g-1 range. Yellow and orange flesh watermelons contained a complex mixture of carotenes. Prolycopene, lycopene, or beta-carotene was the major component, depending on the variety, and the contents were less than 24, 3, and 9 μg·g-1, respectively. There were also minor carotenoids, such as violaxanthin, lutein, neurosporene, zea-carotene with a 0 to 3.5 μg·g-1 range. Neurosporene, zea-carotene, and prolycopene were not found in the red watermelons. There was great variation in total sugar content, range being from 22 to 102 mg-1, while the °Brix was from 4.0 to 15.5. Sucrose, glucose, and fructose were the main sugars in the watermelon and their composition were grouped as sucrose-dominant or fructose-dominant groups. Some varieties with very low levels of sucrose were generally low in the total sugar content. Watermelon contained fairly low levels of ascorbic acid, less than 58 μg·g-1 and some varieties had nearly no ascorbic acid. Estimation of total carotenoid in the yellow watermelons by measuring absorbency at 435, 485, or 503 nm was tested and 435 nm showed the highest correlation coefficient (r2 =0.845).

Comparative Enantioseparation of Monoterpenes by HPLC on Three Kinds of Chiral Stationary Phases with an On-Line Optical Rotatory Dispersion under Reverse Phase Mode.

HPLC enantioseparation of chiral monoterpenes was studied using amylose (AD-H), cellulose (OD-H) and β-cyclodextrin (CD-Ph), phenyl carbamate derivatives as chiral stationary phases (CSPs). The contributions of various functional groups of the chiral monoterpenes in capacity factor (k), separation factor (α) and resolution factor (Rs) were investigated. AD-H column clearly showed the chiral recognition in 7 chemicals from a total of 9 analytes and especially for linalool, while the CD-Ph column could achieve efficient enantioseparation on carvone. The enantioseparation mechanism between the analytes and the CSPs is discussed. Chiral HPLC system coupled with ORD detector could be applied to isolate and directly determine the configuration of (3S)-(+)-linalool, which is not commercially available. Moreover, 100% enantiomeric excess of the isolated (3S)-(+)-linalool was observed from the SPME-GCMS result. Additionally, enantioseparation of (3S)-(+)-linalool by preparative HPLC system offered a 500-fold higher sample loading capacity than that of GC.

A Large Scale Separation of Taxanes from the Bark Extract of Taxus yunnanesis and 1H‐ and 13C‐NMR Assignments for 7‐epi‐10‐Deacetyltaxol

AbstractA large‐scale separation of paclitaxel from semi‐purified bark extract of Taxus yunnanesis was investigated. The chromatographic behavior of paclitaxel and two dose editing analogues, cephalomannine and 7‐epi‐10‐deacetyltaxol were systematically studied on a C18 bonded phase column with different mobile phase in reverse phase mode. According to the notably different selectivity of the methanol and acetonitrile with water in the mobile phase and the most important requirement of capacity in preparative chromatography, the optimum suitably mobile phase used in a large‐scale isolation of paclitaxel and 7‐epi‐10‐deacetyltaxol on a preparative C18 column was given. Cephalomannine was eliminated by ozonolysis and after then separated throughout a normal phase silica column. The whole large‐scale process for high purity paclitaxel from the bark extract of Taxus yunnanesis consisted of a preliminary purification with Biotage FLASH 150i system based on a prepacked normal phase silica cartridge followed by using a C18 Nova‐pak™ column in Waters PrepLC™ 4000 preparative HPLC system. The structure of 7‐epi‐10‐deacetyltaxol was elucidated by 2D NMR technologies of TOCSY, DQF‐COSY, HMQC and HMBC, etc.