On-line SPE-HPLC Method Research Articles

Extraction and determination of terpenoids from Zexie Decoction based on a porous organic cage-doped monolithic cartridge

A monolithic solid-phase extraction (SPE) cartridge packed with a composite adsorbent was fabricated via polymerization using dodecene as the monomer with the porous organic cage (POC) material doped, combing with an analytical column through a high-performance liquid chromatography (HPLC) instrument, which was used for the online extraction and separation of 23-acetyl alismol C, atractylodes lactone II and atractylodes lactone III from Zexie Decoction. The POC-doped adsorbent shows porous structure with a relatively high specific surface area of 85.50 m2/g, which was obtained from the characterizations of a scanning electron microscope and an automatic surface area and porosity analyser. Efficient extraction and separation of three target terpenoids was achieved by an online SPE-HPLC method based on the POC-doped cartridge, which exhibits strong matrix-removal ability and good terpenoids-retention ability with a high adsorption capacity, due to the interactions of hydrogen bond and hydrophobicity between the terpenoids and the POC-doped adsorbent. Method validation shows good linearity (r ≥ 0.9998) of the regression equation, and high accuracy with the spiked recovery in the range of 99.2 %-100.8 % of the proposed method. Compared to the generally disposable adsorbent, this work fabricated a reusable monolithic cartridge, which can be used for at least 100 times, with the RSD based on the peak area of the three terpenoids less than 6.6 %.

A simple and efficient method for the extraction and purification of tuberostemonine from Stemonae Radix using an amide group-based monolithic cartridge

Stemonae Radix shows multiple pharmacological effects, mainly due to the alkaloid of tuberostemonine contained in it. An online SPE-HPLC method was fabricated by combining the solid-phase extraction (SPE) cartridge with a C18 analytical column through high-performance liquid chromatography (HPLC) system, which was used for the quantitative determination of tuberostemonine from Stemonae Radix extract and plasma. The SPE cartridge shows excellent matrix removal ability and specific selectivity for the tuberostemonine. The methodology validation confirms its good accuracy with the spiked recovery of 100.6%∼101.7%, a good repeatability with the relative standard deviations of 0.3%∼0.7% (tR) and 5.8%∼7.7% (A). Furthermore, the proposed monolithic SPE cartridge was used for the preparative separation and purification of tuberostemonine from Stemonae Radix, which was carried out on a semi-preparative HPLC instrument, with a high yield of 1.08 mg g−1 obtained. These results show that the present monolithic cartridge is an outstanding SPE adsorbent for the extraction, separation, enrichment and purification of tuberostemonine from Stemonae Radix, which can be extended to the analysis of similar alkaloids from complex samples.

Extraction and Determination of Evodiamine from Euodia Fructus with SPE-HPLC Based on a Homemade Phenyl-Based Monolithic Cartridge.

A phenyl-based monolithic adsorbent was prepared in a 50-mm-long stainless steel tube, which was initiated by the redox system, using ethylene glycol phenyl ether acrylate as the monomer and ethylene glycol dimethacrylate as the crosslinker. The effects of monomer/crosslinker ratio and the porogens on the permeability and morphology of the resulting adsorbents were investigated, and the optimal adsorbent shows relatively uniform pore structure according to the characterizations of scanning electron microscopy and nitrogen adsorption-desorption method. The column that filled with the adsorbent was used as the solid-phase extraction (SPE) cartridge, exhibiting unique selectivity for the extraction of evodiamine from Euodia fructus (the fruits of Euodia rutaecarpa (Juss.)Benth.), which attributes to the interactions of π-π and hydrogen bonding between the adsorbent and evodiamine. Combined with a C18 analytical column via high-performance liquid chromatography (HPLC) system, an online SPE-HPLC method was established for extraction, enrichment and determination of evodiamine from Euodia fructus. Method validation demonstrates that the relative standard deviation of the precision is less than 0.66%, and the spiked recovery is in the range of 93.11-98.06%. Furthermore, it is worth noting that the prepared SPE cartridge can be reused for no less than 100 times. These results show that the developed method is simple and efficient for online extraction and enrichment of evodiamine from Euodia fructus.

Amino-modified Scholl-coupling mesoporous polymer for online solid-phase extraction of plant growth regulators from bean sprouts

A new amino-modified Scholl-coupling mesoporous polymer (NH2@SMPA)-online solid-phase extraction method, coupled with high-performance liquid chromatography (online SPE-HPLC) was established for the analysis of six plant growth regulators (PGRs) in bean sprouts. NH2@SMPA was synthesized by acid-catalyzed deacetylation of acetylamino-Scholl-coupling mesoporous polymer (SMPA). The diversity of functional groups, such as aromatic, acetylamino, and NH2, was conducive to multiple binding interactions between NH2@SMPA and PGRs. NH2@SMPA exhibited superior extraction capability for PGRs, compared with SMPA and commercial adsorbents. The extraction conditions, including loading solvent, pH of loading solution, eluting solvent, and flow rates of loading and elution, were optimized. Under the optimized conditions, wide linear ranges (0.01–500 μg kg−1) and low detection limits (2.34–20.2 ng kg−1) were obtained. The recoveries were satisfactory, i.e., 86.0% to 109% with relative standard deviations ≤9.8% (n = 3). Finally, the online SPE-HPLC method was successfully used for determination of PGRs in bean sprouts.

Hydroxyapatite-embedded monolithic column for selective on-line solid-phase extraction of adenosine triphosphate and its phosphorylated metabolites

A novel hydroxyapatite-embedded monolithic column has been facilely prepared in a stainless-steel column with inner diameter of 2.1 mm by the strong adhesion of urea-formaldehyde (UF) resin and exploited as a sorbent for selective on-line solid-phase extraction (on-line SPE) of adenosine triphosphate and its phosphorylated metabolites. The composition for this preparation, including the amount of hydroxyapatite nanopowders and the porogen were investigated to obtain a suitable monolith with large surface area and satisfactory permeability. Owing to anion exchange interaction of hydroxyapatite and hydrophilic interaction of UF monolithic matrix, the prepared monolith showed good extraction efficiency and selectivity towards these phosphorylated analytes. Several parameters for on-line SPE, including ACN percentage in the sampling solution, collection time span, salt concentration of the eluent, sampling and elution flow rate, were optimized with respect to the extraction efficiencies of the target compounds. Under the optimized conditions, the LODs of the analytes were in the range of 0.01–0.04 μg/g, the recoveries in the spiked samples ranged from 78.3%–92.5% with RSDs <4.7%. Due to the excellent extraction ability towards phosphorylated compounds in practical samples, a simple on-line SPE-HPLC method using hydroxyapatite-embedded monolith as sorbent has been proposed for monitoring freshness of grass carp.

Novel nanofibrous sorbents for the extraction and determination of resveratrol in wine

On-line SPE HPLC method using nanofibrous sorbents for the extraction and determination of resveratrol in wine was developed and validated. Different types of nanofibrous and microfibrous polymers were tested and compared with commercial monolithic C18 sorbent. Polyamide and polyacrylonitrile nanofibers and composite materials composed of respective polycaprolactone and poly(vinylidene difluoride) nanofibers at microfibrous scaffold were included among tested materials. Two different polycaprolactone-based materials were prepared and their effect on the extraction properties studied. Alternatively, dopamine-coated polycaprolactone fibers were also used. Poly(vinylidene difluoride) nanofibers/polycaprolactone microfibers composite was found as the most effective sorbent and utilized for the method validation. Resveratrol in red wine was determined using our validated on-line SPE HPLC method.

On-line coupling of molecularly imprinted solid phase extraction with liquid chromatography for the fast determination of coumarins from complex samples

In this work, an on-line SPE-HPLC method with spectrophotometric detection was developed for the determination of coumarins in complex samples. For the on-line cleanup of samples, a molecularly imprinted polymer was packed into the column cartridge and coupled directly with HPLC (MISPE-HPLC) using a column switching system. The separation of coumarins was performed on a C18 core-shell column (100×4.6mm, 5μm) with a mobile phase consisting of 0.3% acetic acid/acetonitrile with gradient elution at a flow-rate of 1mLmin−1. The total time of the whole analytical run, including the extraction step, was 13.25min. The on-line MISPE-HPLC method was optimized and validated. The results showed good linearity (0.10–100μgmL−1) with correlation coefficients higher than 0.99. The LOD values were from 0.03 to 0.15μgmL−1. The proposed method was successfully applied for analysis of real samples (Cassia cinnamon, chamomile tea, and Tokaj specialty wines) and obtained recoveries varied from 78.7% to 112.2% with an RSD less than 9%.

Simultaneous determination of cordycepin and 2'-deoxyadenosine in Cordyceps genus by online SPE-HPLC

An online SPE-HPLC method for simultaneous determination of cordycepin (3'-deoxyadenosine) and 2'-deoxyadenosine in Cordyceps genus (C. sinensis,C. militaris,Hirsutella sinensis and C. sobolifera) was developed. The samples were enriched on a ZORBAX SB-AQ (4.6 mm×12.5 mm,5 μm) column with isocratic elution by 9% methanol solution. The separation of analytes was performed on a ZORBAX SB-AQ (4.6 mm×150 mm,5 μm) column with gradient elution by 0.1% formic acid solution and methanol (91∶9). The flow rate was 1.0 mL•min⁻¹. Column temperature was 40 ℃ and detection wavelength was 260 nm. This method has been applied for analysis of different Cordyceps genus. The 2'-deoxyadenosine was detected in C. sinensis,Hirsutella sinensis and C. sobolifera. The cordycepin was detected in C. militaris. In summary,the cordycepin chromatographic peak from C. sinensis in some past reports may be the 2'-deoxyadenosine chromatographic peak or the mixture peak of 2'-deoxyadenosine and cordycepin in which 2'-deoxyadenosine content was higher than cordycepin. The developed method is suitable for analysis of cordycepin and 2'-deoxyadenosine in Cordyceps genus.

Pharmacokinetics of calycopterin and xanthmicrol, two polymethoxylated hydroxyflavones with anti-angiogenic activities from Dracocephalum kotschyi Bioss.

BackgroundRecently flavonoids have attracted the attention of researchers in the fight against cancer. Calycopterin and xanthomicrol, are two polymethoxylated flavonoids found in the aerial parts of Dracocephalum kotschyi Bioss.. We have recently shown that these compounds possess antiangiogenic activity and may be of value as potential anticancer agents. In order to demonstrate putative in vivo antitumor effect of these compounds we needed preliminary information on both pharmacokinetics and toxicological properties of these two agents.MethodA new online SPE HPLC method for measurement of calycopterin and xanthomicrol in rat plasma was developed. Pharmacokinetic parameters of calycopterin and xanthomicrol, after i.v. administration in rats, were determined.ResultsThe plasma half-life for both agents was around 4 h, however, the volume of distribution of calycopterin appeared to be about 8 times greater than xanthomicrol. This was probably due the greater hydrophobicity of the former which had other consequences such as much smaller maximum plasma concentration of calycopterin compared to its less methoxylated congener. Preliminary toxicological study of xanthomicrol failed to show any behavioral, histological and biochemical adverse effects after repeated administrations of high doses.Graphical Pharmacokinetics of xanthomicrol in rats.

An on-line SPE–HPLC method for effective sample preconcentration and determination of fenoxycarb and cis, trans-permethrin in surface waters

A new on-line SPE–HPLC method using fused-core columns for on-line solid phase extraction and large volume sample injection for increasing the sensitivity of detection was developed for the determination of insecticides fenoxycarb and cis-, trans-permethrin in surface waters. The separation was carried out on fused-core column Phenyl–Hexyl (100×4.6mm), particle size 2.7µm with mobile phase acetonitrile:water in gradient mode at flow rate 1.0mLmin−1, column temperature 45°C. Large volume sample injection (1500µL) to the extraction dimension using short precolumn Ascentis Express RP C-18 (5×4.6mm); fused-core particle size 2.7µm allowed effective sample preconcentration and efficient ballast sample matrix removal. The washing mobile phase consisting of a mixture of acetonitrile:water; 30:70, (v/v) was pumped at flow rate of 0.5mLmin−1 through the extraction precolumn to the waste. Time of the valve switch for transferring the preconcentrated sample zone from the extraction to the separation column was set at 3rd min. Elution of preconcentrated insecticides from the extraction precolumn and separation on the analytical column was performed in gradient mode. Linear gradient elution started from 40% of acetonitrile at time of valve switch from SPE column (3rd min) to 95% of acetonitrile at 7th min. Synthetic dye sudan I was chosen as an internal standard. UV detection at wavelength 225nm was used and the method reached the limits of detection (LOD) at ngmL−1 levels for both insecticides. The method showing on-line sample pretreatment and preconcentration with highly sensitive determination of insecticides was applied for monitoring of fenoxycarb and both permethrin isomers in different surface water samples in Czech Republic. The time of whole analysis including on-line extraction, interferences removal, chromatography separation and system equilibration was less than 8min.

Investigation on Simultaneous Determination of Deltamethrin, Carbaryl, Microcystin-LR and Benzo[a]pyrene by Online SPE-HPLC Method

In this paper, simultaneous analysis method for deltamethrin, carbaryl, microcystin-LR and benzo[a]pyrene in aqueous solution was established by online solid phase extraction (SPE)-HPLC technology. The target pollutants were enriched in SPE column, eluted by switching the six-way valve, separated by analytical column, and finally measured by UV-VIS Detector (UVD) and Fluorescence Detector (FLD). The results showed that online SPE-HPLC method was suitable for determination of these four organic pollutants. The correlation coefficient of the linear curve was all larger than 0.9990, and the detection limit and the limit of quantitation could also meet the Chinese standard method for drinking water (GB/T 5750-2006 and GB/T 3838-2006). This method achieved simultaneous online enrichment and efficient removal of substrate; avoided tedious manual pre-treatment process, saved the sample volume and processing time, and also had high recovery rate and sensitivity.

Simultaneous determination of five quinoxaline-1,4-dioxides and two major metabolites in surface water by on-line solid phase extraction coupled to high-performance liquid chromatography

Veterinary antibiotics are environmental contaminants of recent concern, so this study was designed to develop an analytical method of simultaneous determination for quinoxaline-1,4-dioxides (QdNOs) and their metabolites in the aqueous environment at trace levels. The new method is based on on-line solid phase extraction (SPE) using cigarette filter (CGFR) as the sorbent coupled to high-performance liquid chromatography (HPLC). Five QdNOs (carbadox, olaquindox, cyadox, mequindox, quinocetone) and two major metabolites (quinoxaline-2-carboxylic acid and methyl-3-quinoxaline-2-carboxylic acid) were detected in the surface water. The cleaned CGFR precolumn selectively retained target analytes when untreated water samples were upload by a flow-inject pump. Then the enriched extracts were subsequently eluted by HPLC mobile phase to the analytical column for chromatographic analysis. The on-line setup was uncomplicated and automated. The on-line SPE conditions were optimized in detail including sample pH, sample loading flow rate, sample volume, eluent, elution time and sorbents. Under the optimal experimental conditions, the enrichment factors were 33.3–630.0 by preconcentrating 25.0 mL of water samples. Limits of detection (S/N = 3) ranged from 1.6 to 24.3 ng L−1. Satisfactory recoveries were obtained and ranged from 87.1 to 107.5% at two spiked levels in real water samples with high precision (RSD, 1.3–3.8%). The on-line SPE-HPLC method is simple, rapid, reliable, sensitive and could be applied for multiresidue determination of the QdNOs and their metabolites in water samples.

On-line SPE-HPLC Method using Alumina Filtering to Selectively Extract Phenolic Compounds from Environmental Water

A on-line SPE (solid phase extraction)-HPLC preconcentration method was developed for the determination of phenolic compounds at trace levels in environmental water sample. XAD-4 and Dowex 1-X8 were used as sorbent in the on-line SPE-HPLC method for the selective enrichment of nine phenolic compounds, which are included in the priority pollutants list of the US EPA. Also alumina prefiltering considerably reduced the amount of interfering peaks due to humic substances that could accumulated due to the preconcentration step and prevent quantification of polar phenolic compounds in environmental water samples. This method was used to determine the phenolic compounds in tap and river water and superiority to the US EPA 625 method in its enrichment factor, pretreatment time, recoveries, and detection limit. The limits of detection were in the range of <TEX>$0.3-0.9\;{\mu}g/L$</TEX> in tap water sample.

Abstract B69: Potential cancer chemopreventive compounds from Oplopanax horridus, a native North American plant

Abstract Distributed throughout the west of North America, the plant Oplopanax horridus, or devil's club, belongs to the genus Oplopanax, which consists of three species (O. elatus, O. japonicus and O. horridus). Although many studies on the other two species have been performed, phytochemical and pharmacological investigations on O. horridus have been limited, especially cancer chemopreventive studies. After comparing differences of antiproliferative potentials among different plant parts of O. horridus, the root bark was selected to perform further phytochemical isolation to prepare active anticancer fractionations and compounds. The hydrophobic fractions showed potent antiproliferative effects on human HCT-116 and SW-480 colorectal cancer cells. Subsequent isolation of the hydrophobic fractions gave six polyynes and one polyene; in which two novel compounds, Oplopantriol A and Oplopantriol B, were obtained and their absolute structures were elucidated by detailed spectroscopic analyses, including 1D and 2D NMR techniques and chemical methods. The content of these seven compounds in the O. horridus root bark was determined using an on-line SPE-HPLC method. Pharmacological studies showed that two polyynes, including a novel compound (Oplopantriol A), possessed strong cancer cell inhibitory activities (P &amp;lt; 0.001). The IC50 of these two polyynes were observed at 1-5 µM on the colorectal cancer cells. Treatment with active fraction and compounds noticeably induced apoptosis, and distinctly induced the G2/M phase arrest of the cell cycle in a time- and concentration-dependent manner. The trend of increasing cyclin A and cyclin B1 were similar to the increase of G2/M phase cells in all treated groups. These results suggested that active O. horridus fraction and selected polyynes had potential antiproliferative activities on human colorectal cancer cells. The observed cancer chemopreventive effects could be related to the induction of apoptosis and regulation of cell cycle transition. (This work was supported in part by the NIH/NCCAM grants AT003255, AT004418 and AT005362). Citation Information: Cancer Prev Res 2010;3(12 Suppl):B69.

Determination of Aniline Derivatives in Water Samples by High Performance Liquid Chromatography Coupled with On-Line Flow Injection Preconcentration

An on-line SPE-HPLC method using a cigarette filter as the sorbent was developed and applied to analyze the aniline derivatives in water samples. A preconcentration column packed with cigarette filters was used to replace a conventional sample loop on the injector valve of an HPLC for on-line SPE. With the consumption of a 7.5 mL sample solution, the enhancement factors ranged from 287 to 349 in comparison with direct injections of 20 μL sample solution. The detection limits (S/N = 3) for preconcentration of 7.5 mL of sample solution ranged from 0.27 to 5.46 μg/L at a sample throughput of 6 samples per hour.

Quantitative analysis of six polyynes and one polyene in Oplopanax horridus and Oplopanax elatus by pressurized liquid extraction and on-line SPE–HPLC

A pressurized liquid extraction and on-line SPE–HPLC method was developed for simultaneous determination of six polyynes, including falcarindiol, oplopandiol, (11 S,16 S,9 Z)-9,17-octadecadiene-12,14-diyne-1,11,16-triol,1-acetate, oplopandiol acetate, oplopantriol A, oplopantriol B, and one polyene, ( S, E)-nerolidol, in Oplopanax horridus and Oplpanax elatus. The analysis was conducted on a Grace Prevail C 18 column (3 μm, 7 mm × 33 mm) with gradient elution of acetonitrile and water after the sample loaded and washed with 42%ACN in 0.3 min on a phenomenex Strata-X on-line Extraction Cartridge SPE column (2.5 μm, 2.0 × 20 mm). All calibration curves of seven analytes showed good linearity within the test ranges. The validated method was successfully applied to quantify six polyynes and one polyene in two species of Oplopanax, O. horridus and O. elatus.

Extraction and Determination of Cefazolin Sodium and Cefotaxime Sodium in Human Urine with a Weak Ion Exchange Monolithic Column

An online solid phase extraction (SPE) method was developed for the simultaneous screening of cefazolin sodium and cefotaxime sodium in human urine. A short weak anion exchange monolithic column was prepared and used as the selective extraction sorbent. The column exhibited good permeability and biocompatibility. During the online SPE process, most matrices in the urine samples were removed rapidly, while cefazolin sodium and cefotaxime sodium were effectively trapped in this monolithic column. Reversed phase high performance liquid chromatography (HPLC) was performed on a C18 column with ultraviolet detection at 254 nm. The mobile phase consisted of methanol-0.005 mol/L phosphate (21/79, v/v, pH = 5.0) and the flow rate was set to 0.7 mL/min. A good linear relationship was demonstrated at analyte concentrations ranging from 1 to 100 µg/mL. The absolute recovery ranged from 79.4% to 81.5% and the inter- and intra-day relative standard deviations were <5.0%. These results suggest that the online SPE-HPLC method is suitable for the simultaneous monitoring of cefazolin sodium and cefotaxime sodium in human urine samples because it avoids tedious pretreatments and provides a fast, economical, and reproducible method for assaying trace drugs in biological samples.

Determination of nicardipine and amlodipine in human plasma using on-line solid-phase extraction with a monolithic weak cation-exchange column

An on-line solid-phase extraction (SPE)-HPLC method was developed for simultaneous screening of nicardipine and amlodipine in human plasma. A short monolithic poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) [p(GMA-EDMA)]-based weak cation-exchange (WCX) column was prepared and employed as the selective extraction sorbent, which exhibited good permeability and biocompatibility. During the on-line SPE protocol, high-abundance proteins (human serum albumin, immunoglobulin G, immunoglobulin A and transferrin) and most matrixes in plasma were fast removed while nicardipine and amlodipine were effectively trapped on this monolithic column. Furthermore, the monolithic WCX sorbent could be continuously reused more than 300 times without obvious changes in analytes extraction and proteins cleanup. The proposed method was linear over a range of 0.5–50.0 ng mL −1 for both analytes with a linear regression coefficient greater than 0.998, and the limit of detection (LOD) for each analyte was 0.2 ng mL −1. Validation assays also demonstrated acceptable precision and adequate recovery for simultaneous quantitative screening of nicardipine and amlodipine in human plasma. Real plasma samples from hypertensive patients receiving a dosing of 5 mg antagonists were examined by using the proposed method. Results indicated that the on-line SPE-HPLC method could be applied for simultaneously monitoring of nicardipine and amlodipine in clinical plasma samples.

On‐line solid‐phase extraction with a monolithic weak cation‐exchange column and simultaneous screening of α1‐adrenergic receptor antagonists in human plasma

An on-line SPE-HPLC method, using a monolithic poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (poly(GMA-EDMA)) based weak cation-exchange (WCX) column, was developed for simultaneous determination of alpha1-adrenergic receptor antagonists in human plasma. The monolithic WCX column was prepared by an in-situ polymerization protocol and modified stepwise with ethylenediamine and chloroacetic acid. On connecting this column to an injection valve, an on-line SPE protocol could be established for removal of matrices (mainly proteins and lipids) and preconcentration of four alpha1-adrenergic receptor antagonists in human plasma. This method was validated and then used for determination of terazosin, alfuzosin, prazosin, and doxazosin in clinical plasma samples. For all analytes, each calibration curve was found to be linear over a range of 0.005-5 microg/mL (R>0.997), and the limit of detection for each analyte was 0.5 ng/mL. Recovery (>80%) and precision (RSD<15%) for inter- and intra-day assay were tested at three concentration levels of each analyte and showed acceptable results for quantitative assay. Real samples from hypertensive patients were monitored and results were in agreement with those of the conventional liquid-liquid extraction-HPLC method. Furthermore, due to its good permeability and biocompatibility, the monolithic WCX sorbent could be reused more than 300 times. The proposed method was especially appropriate for multi-analyte monitoring in plasma samples.