Simple determination of betaine,l‐carnitine and choline in human urine using self‐packed column and column‐switching ion chromatography with nonsuppressed conductivity detection

A sequential online extraction, clean‐up and separation system for the determination of betaine, l ‐carnitine and choline in human urine using column‐switching ion chromatography with nonsuppressed conductivity detection was developed in this work. A self‐packed pretreatment column (50 × 4.6 mm, i.d.) was used for the extraction and clean‐up of betaine, l ‐carnitine and choline. The separation was achieved using self‐packed cationic exchange column (150 × 4.6 mm, i.d.), followed by nonsuppressed conductivity detection. Under optimized experimental conditions, the developed method presented good analytical performance, with excellent linearity in the range of 0.60–100 μg mL⁻¹ for betaine, 0.75–100 μg mL⁻¹ for l ‐carnitine and 0.50–100 μg mL⁻¹ for choline, with all correlation coefficients (R²) >0.99 in urine. The limits of detection were 0.15 μg mL⁻¹ for betaine, 0.20 μg mL⁻¹ for l ‐carnitine and 0.09 μg mL⁻¹ for choline. The intra‐ and inter‐day accuracy and precision for all quality controls were within ±10.32 and ±9.05%, respectively. Satisfactory recovery was observed between 92.8 and 102.0%. The validated method was successfully applied to the detection of urinary samples from 10 healthy people. The values detected in human urine using the proposed method showed good agreement with the measurement reported previously.     

Semi‐micro reversed‐phase liquid chromatography for the separation of alkyl benzenes and proteins exploiting methacrylate‐ and polystyrene‐based monolithic columns

Monolithic columns were synthesized inside 1.02 mm internal diameter fused‐silica lined stainless‐steel tubing. Styrene and butyl, hexyl, lauryl, and glycidyl methacrylates were the functional monomers. Ethylene glycol dimethacrylate and divinylbenzene were the crosslinkers. The glycidyl methacrylate polymer was modified with gold nanoparticles and dodecanethiol (C₁₂). The separation of alkylbenzenes was investigated by isocratic elution in 60:40 v/v acetonitrile/water. The columns based on polystyrene‐co‐divinylbenzene and poly(glycidyl methacrylate)‐co‐ethylene glycol dimethacrylate modified with dodecanethiol did not provide any separation of alkyl benzenes. The poly(hexyl methacrylate)‐co‐ethylene glycol dimethacrylate and poly(lauryl methacrylate)‐co‐ethylene glycol dimethacrylate columns separated the alkyl benzenes with plate heights between 30 and 60 μm (50 μL min^?1 and 60°C). Similar efficiency was achieved in the poly(butyl methacrylate)‐co‐ethylene glycol dimethacrylate column, but only at 10 μL min^?1 (0.22 mm s^?1). Backpressures varied from 0.38 MPa in the hexyl methacrylate to 13.4 MPa in lauryl methacrylate columns (50 μL min^?1 and 60°C). Separation of proteins was achieved in all columns with different efficiencies. At 100 μL min^?1 and 60°C, the lauryl methacrylate columns provided the best separation, but their low permeability prevented high flow rates. Flow rates up to 500 μL min^?1 were possible in the styrene, butyl and hexyl methacrylate columns.     

Enantioseparation of aromatic amino acids using CEC monolith with novel chiral selector,N‐methacryloyl‐l‐histidine methyl ester

A new type of polymethacrylate‐based monolithic column with chiral stationary phase was prepared for the enantioseparation of aromatic amino acids, namely d ,l ‐phenylalanine, d ,l ‐tyrosine, and d ,l ‐tryptophan by CEC. The monolithic column was prepared by in situ polymerization of butyl methacrylate (BMA), N‐methacryloyl‐l ‐histidine methyl ester (MAH), and ethylene dimethacrylate (EDMA) in the presence of porogens. The porogen mixture included DMF and phosphate buffer. MAH was used as a chiral selector. FTIR spectrum of the polymethacrylate‐based monolith showed that MAH was incorporated into the polymeric structure via in situ polymerization. Some experimental parameters including pH, concentration of the mobile phase, and MAH concentration with regard to the chiral CEC separation were investigated. Single enantiomers and enantiomer mixtures of the amino acids were separately injected into the monolithic column. It was observed that l ‐enantiomers of aromatic amino acids migrated before d ‐enantiomers. The reversal enantiomer migration order for tryptophan was observed upon changing of pH. Using the chiral monolithic column (100 μm id and 375 μm od), the best chiral separation was performed in 35:65% ACN/phosphate buffer (pH 8.0, 10 mM) with an applied voltage of 12 kV in CEC. SEM images showed that the chiral monolithic column has a continuous polymeric skeleton and large through‐pore structure.     

Simultaneous determination of NG‐monomethyl‐l‐arginine,NG,NG‐dimethyl‐l‐arginine,NG,NG′‐dimethyl‐l‐arginine,and l‐arginine using monolithic silica disk‐packed spin columns and a monolithic silica column

We have developed and validated a high‐performance liquid chromatography method that uses monolithic silica disk‐packed spin columns and a monolithic silica column for the simultaneous determination of N^G‐monomethyl‐l ‐arginine, N^G,N^G‐dimethyl‐l ‐arginine, and N^G,N^G′‐dimethyl‐l ‐arginine in human plasma. For solid‐phase extraction, our method employs a centrifugal spin column packed with monolithic silica bonded to propyl benzenesulfonic acid as a cation exchanger. After pretreatment, the methylated arginines are converted to fluorescent derivatives with 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole, and then the derivatives are separated on a monolithic silica column. l ‐Arginine concentration was also determined in diluted samples. Standard calibration curves revealed that the assay was linear in the concentration range 0.2–1.0 μM for methylated arginines and 40–200 μM for l ‐arginine. Linear regression of the calibration curve yielded equations with correlation coefficients of 0.999 (r²). The sensitivity was satisfactory, with a limit of detection ranging from 3.75 to 9.0 fmol for all four compounds. The RSDs were 4.3–4.8% (intraday) and 3.0–6.8% (interday). When this method was applied to samples from six healthy donors, the detected concentrations of N^G‐monomethyl‐l ‐arginine, N^G,N^G‐dimethyl‐l ‐arginine, N^G,N^G′‐dimethyl‐l ‐arginine and l ‐arginine were 0.05 ± 0.01, 0.41 ± 0.07, 0.59 ± 0.11, and 83.8 ± 30.43 μM (n = 6), respectively.     

Determination of l‐tryptophan and l‐kynurenine derivatized with (R)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole by LC‐MS/MS on a triazole‐bonded column and their quantification in human serum

The concentrations of l ‐tryptophan (Trp) and the metabolite l ‐kynurenine (KYN) can be used to evaluate the in‐vivo activity of indoleamine 2,3‐dioxygenase (IDO) and tryptophan 2,3‐dioxygenase (TDO). As such, a novel method involving derivatization of l ‐Trp and l ‐KYN with (R)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐PyNCS) and separation by high‐performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection on a triazole‐bonded column (Cosmosil HILIC®) was developed to determine their concentrations. The optimized mobile phase, CH₃CN/10 mm ammonium formate in H₂O (pH 5.0) (90:10, v/v) eluted isocratically, resulted in satisfactory separation and MS/MS detection of the analytes. The detection limits of l ‐Trp and l ‐KYN were approximately 50 and 4.0 pm , respectively. The column temperature affected the retention behaviour of the Trp and KYN derivatives, with increased column temperatures leading to increased capacity factors; positive enthalpy changes were revealed by van't Hoff plot analyses. Using the proposed LC‐MS/MS method, l ‐Trp and l ‐KYN were successfully determined in 10 μL human serum using 1‐methyl‐l ‐Trp as an internal standard. The precision and recovery of l ‐Trp were in the ranges 2.85–9.29 and 95.8–113%, respectively, while those of l ‐KYN were 2.51–16.0 and 80.8–98.2%, respectively. The proposed LC‐MS/MS method will be useful for evaluating the in vivo activity of IDO or TDO. Copyright © 2016 John Wiley & Sons, Ltd.     

HPLC analysis of synthetic polymers on short monolithic columns

Ultrashort monolithic columns (disks) were thoroughly studied as efficient stationary phases for precipitation–dissolution chromatography of synthetic polymers. Gradient elution mode was applied in all chromatographic runs. The mixtures of different flexible chain homopolymers, such as polystyrenes, poly(methyl methacrylates), and poly(tert‐butylmethacrylates) were separated according to their molecular weights on both commercial poly(styrene‐co‐divinylbenzene) disks (12 id × 3 mm and 5 × 5 mm) and lab‐made monolithic columns (4.6 id × 50 mm) filled with supports of different hydrophobicity. The experimental conditions were optimized to reach fast and highly efficient separation. It was observed that, similar to the separation of monoliths of other classes of (macro)molecules (proteins, DNA, oligonucleotides), the length of column did not affect the peak resolution. A comparison of the retention properties of the poly(styrene‐co‐divinylbenzene) disk‐shaped monoliths with those based on poly(lauryl methacrylate‐co‐ethylene dimethacrylate), poly(butyl methacrylate‐co‐ethylene dimethacrylate), and poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) supports demonstrated the obvious effect of surface chemistry on the resolution factor. Additionally, the results of the discussed chromatographic mode on the fast determination of the molecular weights of homopolymers used in this study were compared to those established by SEC on columns packed with sorbent beads of a similar nature to the monoliths.     

Hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method for the simultaneous determination of l‐valine,l‐leucine,l‐isoleucine,l‐phenylalanine,and l‐tyrosine in human serum

l ‐Valine, l ‐leucine, l ‐isoleucine, l ‐phenylalanine, and l ‐tyrosine are important proposed biomarkers for the early detection and diagnosis of type 2 diabetes. A simple and selective hydrophilic interaction chromatography with tandem mass spectrometry method was developed for the simultaneous determination of these amino acids in human serum, using stable isotope‐labeled amino acids as internal standards. Chromatographic separation was carried out on a Syncronis HILIC column (150 mm × 2.1 mm, 5 μm) with the column temperature of 35°C and a mobile phase consisted of acetonitrile/120 mM ammonium acetate (89:11, v/v), and the run time was 11.0 min. The mass spectrometric analysis was performed using a QTRAP 5500 mass spectrometer coupled with an electrospray ionization source in positive ion mode. As these five amino acids are endogenous compounds in serum, we used the corresponding stable isotope‐labeled amino acids to evaluate the matrix effect and recovery in serum. The matrix effect was 98.7–107.3%, and the recovery was 92.7–102.3%. Calibration curves spiked unlabeled amino acids in water were linear over the range of 0.200–100 μg/mL. The accuracy, inter‐, and intraday precision were below 10.2%. Analytes were stable during the study. This assay method has been validated and applied to the early diagnosis research of type 2 diabetes.     

Direct separation of the enantiomers of ramosetron on a chlorinated cellulose‐based chiral stationary phase in hydrophilic interaction liquid chromatography mode

Ramosetron is an enantiopure active pharmaceutical ingredient marketed in Japan since 1996 and later in a few Southeast Asian countries predominantly as an antiemetic for patients receiving chemotherapy. In this study, a simple and rapid high‐performance liquid chromoatography method for the separation of ramosetron and its related enantiomeric impurity by using hydrophilic interaction liquid chromatography mode is presented. Chiral resolution was performed on an analytical column (100 mm × 4.6 mm id) packed with 3 μm particles of cellulose‐based Chiralpak IC‐3 chiral stationary phase. Using a mobile phase containing acetonitrile–water–diethylamine (100:10:0.1, v/v/v) and setting the column temperature at 35°C, the resolution value was 7.35. At a flow rate of 1 mL/min, the enantioseparation was completed within 5 min. The proposed method was partially validated and it has proven to be sensitive with limit of detection and limit of quantitation of the (S)‐enantiomer impurity of 44.5 and 133.6 ng/mL.     

Developing a novel packed in‐tube solid‐phase extraction method for determination ∆9‐tetrahydrocannabinol in biological samples and cannabis leaves

A novel plate‐like nano‐sorbent based on copper/cobalt/chromium layered double hydroxide was synthesized by a simple coprecipitation method. The synthesized nanoparticels were introduced into a stainless steel cartridge using a dry packing method. Then, the packed cartridge was introduced as a novel on‐line “packed in‐tube” configuration and followed by high performance liquid chromatography for the determination of trace amounts of ^?9‐tetrahydrocannabinol from biological samples and cannabis leaves. The as‐prepared sorbent exhibited long lifetime, good chemical stability, and high anion‐exchange capacity. Several important factors affecting the extraction efficiency, such as extraction and desorption times, pH of the sample solution and flow rates of the sample and eluent solutions, were investigated and optimized. Under optimized conditions, this method showed good linearity for ^?9‐tetrahydrocannabinol in the ranges of 0.09–500, 0.3–500, and 0.4–500 µg/L with coefficients of determination of 0.9999, 0.9991, and 0.9994 in water, serum and plasma samples, respectively. The inter‐ and intra‐assay precisions (n = 3) were respectively in the ranges of 1.8–4.6% and 1.9–4.0% at three concentration levels of 10, 50, and 100 µg/L. The limits of detection were also in the range of 0.02–0.1 µg/L.     

Preparation and evaluation of open‐tubular capillary column combining a metal–organic framework and a brush‐shaped polymer for liquid chromatography

In this work, an open‐tubular capillary liquid‐phase column was prepared by modifying chain polymer on the inner surface of capillary and chemical bonding of metal organic frameworks, NH₂‐UiO‐66, to the brushes of chain polymer (poly(glycidyl methacrylate)). Besides advantages of facial preparation and good permeability, the chain polymer effectively increases the modification amount of NH₂‐UiO‐66 nanoparticles to increase the phase ratio of open‐tubular capillary column and enhance the interactions with analytes. The results of scanning electron microscope energy‐dispersive X‐ray spectra indicated that NH₂‐UiO‐66 nanoparticles were successfully bonded to the chain polymer. Because of the hydrophobic interaction and hydrogen bonding interaction between the analytes and the ligand of NH₂‐UiO‐66, different analytes were well separated on the NH₂‐UiO‐66‐modified poly(glycidyl methacrylate) capillary (1.12 m × 25 μm id × 365 μm od) with the high absolute column efficiency reaching 121 477 plates, benefiting from an open‐tubular column and low mass transfer resistance provided by polymer brush and metal–organic framework crystal. The relative standard deviations of the retention time for run‐to‐run, day‐to‐day, and column‐to‐column (n = 3) runs are below 4.28%, exhibiting good repeatability. Finally, the column was successfully applied to separation of flavonoids in licorice.     

Preparation of metal‐organic framework UiO‐66‐incorporated polymer monolith for the extraction of trace levels of fungicides in environmental water and soil samples

A zirconium terephthalate metal‐organic framework‐incorporated poly(N‐vinylcarbazole‐co‐divinylbenzene) monolith was fabricated in a capillary by a thermal polymerization method. The optimized monolith had a homogeneous structure, good permeability, and stability. The monolith could be used for the effective enrichment of fungicides through π‐π interactions, electrostatic forces, and hydrogen bonds. The potential factors that affect the extraction efficiency, including ionic strength, solution pH, sample volume, and eluent volume, were investigated in detail. The monolith‐based in‐tube solid‐phase microextraction coupled with ultra‐high‐performance liquid chromatography and high‐resolution Orbitrap mass spectrometry was performed for the analysis of five fungicides (pyrimethanil, tebuconazole, hexaconazole, diniconazole, and flutriafol) in environmental samples. Under the optimized conditions, the linear ranges were 0.005–5 ng/mL for pyrimethanil, 0.01–5 ng/mL for flutriafol, and 0.05–5 ng/mL for other fungicides, respectively, with coefficients of determination ≥0.9911. The limits of detection were 1.34–14.8 ng/L. The columns showed good repeatability (relative standard deviations ≤9.3%, n = 5) and desirable column‐to‐column reproducibility (relative standard deviations 5.3–9.4%, n = 5). The proposed method was successfully applied for the simultaneous detection of five fungicides in water and soil samples, with recoveries of 90.4–97.5 and 84.0–95.3%, respectively.     

Simultaneous determination of l‐tetrahydropalmatine and its active metabolites in rat plasma by a sensitive ultra‐high‐performance liquid chromatography with tandem mass spectrometry method and its application in a pharmacokinetic study

A sensitive and reliable ultra‐high‐performance liquid chromatography with tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated for simultaneous determination of l ‐tetrahydropalmatine (l ‐THP) and its active metabolites l ‐isocorypalmine (l ‐ICP) and L ‐corydalmine (l ‐CD) in rat plasma. The analytes were extracted by liquid–liquid extraction and separated on a Bonshell ASB C₁₈ column (2.1 × 100 mm; 2.7 μm; Agela) using acetonitrile–formic acid aqueous as mobile phase at a flow rate of 0.2 mL/min in gradient mode. The method was validated over the concentration range of 4.00–2500 ng/mL for l ‐THP, 0.400–250 ng/mL for l ‐ICP and 1.00–625 ng/mL for l ‐CD. Intra‐ and inter‐day accuracy and precision were within the acceptable limits of <15% at all concentrations. Correlation coefficients (r ) for the calibration curves were >0.99 for all analytes. The quantitative method was successfully applied for simultaneous determination of l ‐THP and its active metabolites in a pharmacokinetic study after oral administration with l ‐THP at a dose of 15 mg/kg to rats.     

Development of a CE‐MS2 method for the enantiomeric separation of L/D‐carnitine: Application to the analysis of infant formulas

A new chiral analytical method based on CE‐MS is proposed for the identification and simultaneous quantification of D /L ‐carnitine in infant formulas. Previous derivatization of carnitine with FMOC enabled the optimization of the chiral separation using CE with UV detection. An optimization of electrospray‐MS parameters using a partial filling of the non‐volatile chiral selector (succinyl‐γ‐CD) was performed. A selective fragmentation using MS² experiments with an ion trap analyser was carried out to confirm the identity of D /L ‐carnitine according to the current legislation. Satisfactory results were obtained in terms of linearity, precision, and accuracy. Interestingly, the CE‐MS² method developed allowed a sensitivity enhancement with respect to UV detection of 100‐fold, obtaining an LOD of 100 ng/g for D ‐carnitine. The determination of L ‐carnitine and its enantiomeric purity in 14 infant formulas supplemented with carnitine was successfully achieved, sample preparation only requiring an ultrafiltration with centrifugal filter devices to retain the components with the highest molecular weights.     

Multilayered particle‐packed column: Evaluation and comparison with monolithic and core–shell particle columns for the determination of red azo dyes in Sequential Injection Chromatography

A recently presented new type of “multilayered” organic–inorganic hybrid silica particle packed column YMC‐Triart C₁₈ (50 mm × 4.6 mm, 5 μm) was used for the development of a sequential injection chromatography method for determination of five azo dyes (Sudan I, Sudan II, Sudan III, Sudan orange G, and para red) in selected food seasonings. The use of a novel sorbent brings attractive features, reduced backpressure, and broader chemical stability together with high separation performance, which are discussed and compared with that of three types of columns typically used in medium‐pressure flow chromatography techniques (classic monolithic, narrow monolithic, and core–shell particle columns). The separation was performed in gradient elution mode created by the zone mixing of two mobile phases (acetonitrile/water 90:10, 1.5 mL + acetonitrile/water 100:0, 2.3 mL) at a flow rate of 0.60 mL/min and time of analysis <9.5 min. The spectrophotometric detection wavelengths were set to 400, 480, and 500 nm. The high performance of the developed method with multilayered particle column was well documented and the results indicate a broad capability of sequential injection chromatography.     

Fast reversed‐phase liquid chromatographic separation of proteins by flow‐through poly(styrene‐co‐divinylbenzene) microspheres

With the explosive growth of the bioscience and biopharmaceuticals, the demand for high efficient analysis and separation of proteins is urgent. High‐performance liquid chromatography is an appropriate technology for this purpose, and the stationary phase is the kernel to the separation efficiency. In this study, flow‐through poly(styrene‐co‐divinylbenzene) microspheres characteristic of the binary pores, i.e. flow‐through pores and mesopores, were synthesized; this special porous structure would benefit the convective mass transfer while guarantee the high specific surface area. Owing to the hydrophobic nature, poly(styrene‐co‐divinylbenzene) microspheres were suitable as the reversed‐phase stationary phase for separation of proteins. For the high permeability of the poly(styrene‐co‐divinylbenzene) microspheres packed column, fast separation of the studied six proteins in ～2 min was achieved. The recoveries of studied proteins were acceptable in the range of 79.0–99.4%. The proposed column had good pH stability of 1–13 and repeatability. Moreover, the column was applied for egg white fast separation, further demonstrating its applicability for complex bio‐sample separation. The flow‐through poly(styrene‐co‐divinylbenzene) microspheres were promising for fast separation of large molecules.     

Poly(glycidyl methacrylate‐co‐N‐methylolacrylamide‐co‐ethylene dimethacrylate) monolith coupled to high‐performance liquid chromatography for the determination of adenosine phosphates in royal jelly

A polymer monolith microextraction method coupled with high‐performance liquid chromatography was developed for the determination of adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate. The monolithic column was synthesized inside fused‐silica capillaries using thermal initiation free‐radical polymerization with glycidyl methacrylate as the monomer, ethylene dimethacrylate as the cross‐linker, cyclohexanol, and 1‐dodecanol as the porogen. N‐Methylolacrylamide, an important hydrophilic monomer, was incorporated into the polymerization mixture to enhance the hydrophilicity of the poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) column. The obtained poly(glycidyl methacrylate‐co‐N‐methylolacrylamide‐co‐ethylene dimethacrylate) monolith was characterized by scanning electron microscopy, Fourier‐transform infrared spectra, and X‐ray photoelectron spectroscopy. Optimum conditions for the preconcentration and separation of the target adenosines were also investigated. Under the optimum conditions, we obtained acceptable linearities, low limits of detection, and good relative standard deviations. The developed polymer monolith microextraction with high‐performance liquid chromatography method exhibited a good performance with recovery values in the range of 76.9?104.7% when applied to the determination of the adenosines in five royal jelly samples.     

Determination of betaine, choline and trimethylamine in feed additive by ion-exchange liquid chromatography/non-suppressed conductivity detection

An ion chromatography method with non-suppressed conductivity detection was developed for the simultaneous determination of betaine, choline and trimethylamine in feed additive. The analytes and the common inorganic cations were well separated by means of cation-exchange chromatography using a 4.5 mmol/L methanesulfonic acid solution containing 10% (v/v) acetonitrile as eluent and an IonPac SCS1 column (250 mm x 4 mm i.d.) as the separation column. The effects of the different chromatographic parameters on the separation were also investigated. Detection limits of betaine, choline and trimethylamine were 0.076, 0.044 and 0.041 mg/L. The relative standard deviations (RSDs) of the retention time and peak area were less than 0.30% and 0.88%, respectively. The recoveries were between 93.2% and 112.6%. The method is suitable for use as a routine method in production quality control of feed additive.     

An optimized mixed‐mode stationary phase based on silica monolith particles for the separation of peptides and proteins in high‐performance liquid chromatography

A phase with both hydrophobic and hydrophilic functionalities has been synthesized by modification of ground silica monolith particles with C18 and 1‐[3‐(trimethoxysilyl)propyl] urea ligands. A series of phases was prepared by changing the ratio of the two ligands to determine the optimal ratio in view of separation efficiency. The resultant optimized stationary phase was packed in narrow‐bore glass‐lined stainless‐steel columns (1 × 300 mm and 2.1 × 100 mm) and used for the separation of synthetic peptides and proteins. The average numbers of theoretical plates (N) of 52 100/column (174 000/m, 5.75 µm plate height) and 35 500/column (118 000/m, 8.47 µm plate height) were achieved with the 300 mm column at a flow rate of 25 µL/min (0.86 mm/s) in 60:40 v/v acetonitrile/30 mM aqueous ammonium formate for the mixture of peptides (Thr‐Tyr‐Ser, Val‐Ala‐Pro‐Gly, angiotensin I, isotocin, and bradykinin) and for the mixture of proteins (myoglobin, human serum albumin, and insulin), respectively. Fast analysis of the peptides and proteins was also carried out at a flow rate of 0.9 mL/min (6.88 mm/s) with the 100 mm column and all the analytes were eluted within 2 min with good separation efficiency.     

Comparative evaluation of a one‐pot strategy for the preparation of β‐cyclodextrin‐functionalized monoliths: Effect of the degree of amino substitution of β‐cyclodextrin on the column performance

To further evaluate the feasibility and applicability of the one‐pot strategy in monolithic column preparation, two novel β‐cyclodextrin‐functionalized organic polymeric monoliths were prepared using two β‐cyclodextrin derivatives, i.e. mono(6‐amino‐6‐deoxy)‐β‐cyclodextrin and heptakis(6‐amino‐6‐deoxy)‐β‐cyclodextrin. In this improved method, mono(6‐amino‐6‐deoxy)‐β‐cyclodextrin or heptakis(6‐amino‐6‐deoxy)‐β‐cyclodextrin reacted with glycidyl methacrylate to generate the corresponding functional monomers and were subsequently copolymerized with ethylene dimethacrylate. The polymerization conditions for both monoliths were carefully optimized to obtain satisfactory column performance with respect to column efficiency, reproducibility, permeability, and stability. The obtained poly(glycidyl methacrylate‐mono(6‐amino‐6‐deoxy)‐β‐cyclodextrin‐co‐ethylene dimethacrylate) and poly(glycidyl methacrylate‐heptakis(6‐amino‐6‐deoxy)‐β‐cyclodextrin‐co‐ethylene dimethacrylate) monoliths exhibited a uniform structure, good permeability, and mechanical stability as indicated by scanning electron microscopy and micro‐high‐performance liquid chromatography experimental results. Because of the probable existence of multi‐glycidyl methacrylate linking spacers on the poly(glycidyl methacrylate‐heptakis(6‐amino‐6‐deoxy)‐β‐cyclodextrin‐co‐ethylene dimethacrylate) monolith, the effect of the ratio of glycidyl methacrylate/heptakis(6‐amino‐6‐deoxy)‐β‐cyclodextrin was especially studied, and satisfactory reproducibility could still be achieved by strictly controlling the composition of the polymerization mixture. To investigate the effect of the degree of amino substitution of β‐cyclodextrin on column performance, a detailed comparison of the two monoliths was also carried out using series of analytes including small peptides and chiral acids. It was found that the β‐cyclodextrin‐functionalized monolith with mono‐glycidyl methacrylate linking spacers demonstrated better chiral separation performance than that with multi‐glycidyl methacrylate linking spacers.     

An efficient strategy based on liquid‐liquid extraction and pH‐zone‐refining counter‐current chromatography for selective enrichment,separation, and purification of alkaloids and organic Acids from natural products

This study presents an efficient strategy based on liquid‐liquid extraction and pH‐zone‐refining counter‐current chromatography for selective enrichment, separation, and purification of alkaloids and organic acids from natural products. First, an acid or base modified two‐phase solvent system with maximum or minimum partition coefficient was developed for the liquid‐liquid extraction of the crude extract. As a result, alkaloids or organic acids could be selectively enriched in the upper or lower phase. Then pH‐zone‐refining counter‐current chromatography was employed to separate and purify the selectively enriched alkaloids or organic acids efficiently. The selective enrichment and separation of five bufadienolide from toad venom of Bufo marinus was used as an example to show the advantage of this strategy. As a result, 759 mg of selectively enriched bufadienolide was obtained from 2 g of crude extract and the total content of five targets was increased from 14.64 to 83%. A total of 31 mg of marinobufagin‐3‐adipoyl‐l ‐arginine, 42 mg of telocinobufagin‐3‐pimeloyl‐l ‐arginine, 51 mg of telocinobufagin‐3‐suberoyl‐l ‐arginine, 132 mg of marinobufagin‐3‐suberoyl‐l ‐arginine, and 57 mg of bufalin‐3‐suberoyl‐l ‐arginine were all simultaneously separated from 500 mg of selectively enriched sample, with the purity of 92.4, 97.5, 90.3, 92.1, and 92.8%, respectively.