High-performance purification of quaternary alkaloids from Corydalis yanhusuo W. T. Wang using a new polar-copolymerized stationary phase

An efficient method for purification of alkaloids from Corydalis yanhusuo W. T. Wang using HPLC was developed, featuring a polar-copolymerized stationary phase named C18HCE. As ionizable solutes, the crude alkaloid sample often suffered from serious peak tailing problem on conventional RP-LC columns, and the separation would rapidly became destroyed with the increasing of load amount. However, on the new stationary phase, good peak shapes (asymmetry factor <1.5) as well as good loadability were easily obtained in a commonly used acidic mobile phase condition. The loading amount could reach 10 mg per injection on an analytical C18HCE column for laboratory-scale purification. About 6.8 mg of palmatine (HPLC purity >98%) and 44.4 mg of dehydrocorydaline (HPLC purity >98%) were rapidly derived from 200 mg of the crude alkaloid sample, and the recoveries of these two compounds were 76.5 and 81.7%, respectively. The purified alkaloids were characterized by comparing retention times with standard compounds as well as (1)H-NMR data. The new method is simple and high yielding, and it may provide a promising tool for purification of alkaloids as well as other alkaline compounds.     

Two-dimensional RPLC-RPLC system with different pH in two dimensions for separation of alkaloids from Corydalis yanhusuo W. T. Wang

An effective method utilizing the same RP chromatographic column with different pH in first and second LC dimensions has been developed for separation of the basic compounds from traditional Chinese medicines (TCMs). In this work, the alkaloids in Corydalis yanhusuo which is an important TCM were selected as a model to develop the method. The additives and pH values of the mobile phase were optimized in this work. To investigate the feasibility of this method, off-line mode separation was performed in the experiments. According to the UV-absorption intensity, there were eight fractions collected in acidic conditions. All the fractions were analyzed in basic conditions. The results showed that the chromatographic selectivities were significantly different in the separations performed with acidic and alkaline elution systems. Complementary separation was achieved in this work. It is demonstrated that this method would be an effective tool for alkaloids research. Based on the different pH of the mobile phase in this method, it could also be suitable to analyze compounds which were sensible to the pH of the solution.     

A new protoberberine alkaloid from Corydalis yanhusuo W.T.Wang

A new protoberberine alkaloid,named 5,6-dihydro-10-hydroxy-2,3,9-trimethoxy-13-methyldibenzo[a,g]quinolizinium(1) was isolated from the 60%ethanol extract of the tubers of Corydalis yanhusuo W.T.Wang,together with a new natural product,13- methylpalmatrubine(2).Their structures were established by spectroscopic methods.     

Preparative separation of quaternary ammonium alkaloids from Corydalis yanhusuo W. T. Wang by pH-zone-refining counter-current chromatography

The optimal extraction condition for extracting quaternary ammonium alkaloid dehydrocorydaline from Corydalis yanhusuo W. T. Wang was investigated using orthogonal experimental design. pH‐zone‐refining counter‐current chromatography (CCC) with normal phase elution was successfully applied to preparative separation of alkaloids from the crude extract of Corydalis yanhusuo. The separation was performed with a biphasic solvent system composed of chloroform (CHCl₃)–methanol (MeOH)–water (2:1:1, v/v), in which the lower organic phase containing 10 mM of triethylamine was used as the mobile phase, while the upper aqueous phase containing 10 mM of hydrochloric acid was used as the stationary phase. The separation mechanism of quaternary ammonium alkaloids using pH‐zone‐refining CCC was discussed in comparison with standard high‐speed CCC. In the present study, the separation of 1.200 g of crude sample yielded 129 mg of dehydrocorydaline and 12 mg of palmatine at a high purity of 94 and 92%, respectively. Recovery for dehydrocorydaline and palmatine was 85 and 86%, respectively.     

Purification of flavonoids from licorice using an off‐line preparative two‐dimensional normal‐phase liquid chromatography/reversed‐phase liquid chromatography method

An orthogonal (71.9%) off‐line preparative two‐dimensional normal‐phase liquid chromatography/reversed‐phase liquid chromatography method coupled with effective sample pretreatment was developed for separation and purification of flavonoids from licorice. Most of the nonflavonoids were firstly removed using a self‐made Click TE‐Cys (60 μm) solid‐phase extraction. In the first dimension, an industrial grade preparative chromatography was employed to purify the crude flavonoids. Click TE‐Cys (10 μm) was selected as the stationary phase that provided an excellent separation with high reproducibility. Ethyl acetate/ethanol was selected as the mobile phase owing to their excellent solubility for flavonoids. Flavonoids co‐eluted in the first dimension were selected for further purification using reversed‐phase liquid chromatography. Multiple compounds could be isolated from one normal‐phase fraction and some compounds with bad resolution in one‐dimensional liquid chromatography could be prepared in this two‐dimensional system owing to the orthogonal separation. Moreover, this two‐dimensional liquid chromatography method was beneficial for the preparation of relatively trace flavonoid compounds, which were enriched in the first dimension and further purified in the second dimension. Totally, 24 flavonoid compounds with high purity were obtained. The results demonstrated that the off‐line two‐dimensional liquid chromatography method was effective for the preparative separation and purification of flavonoids from licorice.     

Systematic screening and characterization of tertiary and quaternary alkaloids from corydalis yanhusuoW.T. Wang using ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry

Ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) is an effective technique for analysis of complex samples with offering rapid, efficient separation in combination with accurate mass measurement and tandem mass spectrometry (MS/MS). This paper exploits this technique to identify the alkaloids in corydalis yanhusuo, an important antalgic Traditional Chinese Medicine (TCM). The mass spectral fragmentation behavior of one tertiary alkaloid and two quaternary alkaloids was studied in detail. Low-abundance product ions of tertiary and quaternary alkaloids were investigated and compared between each other. Sixteen alkaloids were screened out by using a systematic screening method developed in our laboratory; structures of eight therein were identified by characteristic UV absorption spectrum and positive ion mode of Q-TOF-MS/MS; and two of them were discovered for the first time in corydalis yanhusuo to our knowledge. This research demonstrates the potential of UPLC-Q-TOF-MS in structural characterization and identification of components in traditional Chinese herbal medicines.     

Purification and preparation of compounds from an extract of Scutellaria barbata D. Don using preparative parallel high performance liquid chromatography

Preparative parallel high performance liquid chromatography combined with solvent partition and other pretreatments were adopted to separate and purify compounds from an extract of Scutellaria barbata D. Don. Mass-triggered fraction collection allowed the rapid and precise isolation of target compounds. Twelve compounds were isolated from the extract of S. barbata D. Don, their purity in area percent was determined by HPLC analysis, and the structures of seven compounds were further identified with HPLC/ESI-MS, (1)H NMR, and( 13)C NMR, among which 4-(3,4-dihydroxy-phenyl)-but-3-en-2-one, acacetin-7-diglucuronide, and luteolin-7-diglucuronide were the first to be identified from this plant. The results demonstrated that multi-channel parallel preparative HPLC/UV/MS is an efficient method for isolation and purification of compounds from natural products.     

Preparative isolation of alkaloids from Corydalis bungeana Turcz. by high-speed counter-current chromatography using stepwise elution

High‐speed counter‐current chromatography (HSCCC) was successfully applied for the preparative separation and purification of alkaloids from Corydalis bungeana Turcz. (Kudiding in Chinese) for the first time. After the measurement of partition coefficient of seven target alkaloids in the nine two‐phase solvent systems composed of CHCl₃–MeOH–(0.1 M; 0.2 M; 0.3 M) HCl (4:1.5:2; 4:2:2; 4:3:2, v/v), CHCl₃–MeOH–0.2 M HCl (4:2:2, v/v) and CHCl₃–MeOH–0.3 M HCl (4:3:2, v/v) were finally selected for the HSCCC separation using the first upper phase as the stationary phase and the stepwise elution of the two lower mobile phases. Consequently, sanguinarine (10 mg), corynoline (25 mg), protopine (20 mg), corynoloxine (18 mg), and 12‐hydroxycorynoline (8 mg) were obtained from 200 mg of crude alkaloid extracts with purities of 94–99% as determined by HPLC. Their chemical structures were characterized on the basis of ¹H‐NMR, ¹³C‐NMR, and LC‐ESI‐Q‐TOF‐MS/MS analyses.     

A new denudatine type C20-diterpenoid alkaloid from Aconitum sinchiangense W. T. Wang

A new denudatine-type C₂₀-diterpenoid alkaloid, designated as sinchianine (1), together with eight known diterpenoid alkaloids, 12-acetyl-12-epi-napelline (2), 12-epi-napelline (3), neoline (4), talatisamine (5), 14-O-acetylsenbusine A (6) and benzoylaconine (7), songorine (8) and aconitine (9), were isolated from the whole herb of Aconitum sinchiangense W. T. Wang. Their structures were elucidated on the basis of extensive spectroscopic analyses (NMR and HR-ESI-MS) and comparison with data reported in the literature.     

Preparative separation of isoquinoline alkaloids from Corydalis impatiens using a middle‐pressure chromatogram isolated gel column coupled with two‐dimensional liquid chromatography

We established a two‐dimensional strong cation exchange/reversed‐phase liquid chromatography protocol to isolate and purify isoquinoline alkaloids from Corydalis impatiens. Isoquinoline alkaloids were first enriched from a C. impatiens extract in which liposoluble components were removed using a medium‐pressure chromatographic tower containing middle chromatogram isolated gel. A strong cation exchange column was employed to separate and obtain 30 fractions. We chose fractions 22–29 for reversed‐phase liquid chromatography purification using characteristic isoquinoline alkaloid ultraviolet absorption spectra. Several isoquinoline alkaloid fractions (22–29) were further separated, and those of low resolution were isolated via two‐dimensional liquid chromatography in the orthogonal plane. A total of eight novel isoquinoline alkaloids with characteristic ultraviolet spectra were obtained from C. impatiens. We thus demonstrate the benefits of off‐line two‐dimensional strong cation exchange/reversed‐phase liquid chromatography to isolate isoquinoline alkaloids from C. impatiens.     

延胡索及水煎液中14种无机元素的测定

为了探索延胡索中无机元素与其活血，理气，止痛的药效关系，用等离子体发射光谱法测定了延胡索及水煎液中１４种元素的含量，其含量较高的有Ｆｅ，Ｍｇ，Ｚｎ，Ｃａ，Ｍｎ。     

Two‐dimensional hydrophilic interaction chromatography × reversed‐phase liquid chromatography for the preparative isolation of potential anti‐hepatitis phenylpropanoids from Salvia prattii

Traditional Tibetan medicine is important for discovery of drug precursors. However, knowledge of the chemical composition of traditional Tibetan medicines is very limited due to the lack of appropriate chromatographic purification methods. In the present work, Salvia prattii was taken as an example, and an off‐line hydrophilic interaction liquid chromatography/reversed‐phase liquid chromatography preparative method was developed for the purification of phenylpropanoids with high purity from a crude sample of Salvia prattii. Based on the separation results of four different chromatographic stationary phases, the first‐dimensional preparation was performed on an XAmide preparative column with the crude sample concentration of 62.0 mg/mL, and five main fractions were obtained from the 12.4 g crude sample with a recovery of 54.8%. An XCharge C₁₈ preparative column was applied in the second‐dimensional preparation to further isolate the phenylpropanoids from the redissolved first‐dimensional fractions with concentration of approximately 50.0 mg/mL. The purities of the phenylpropanoids isolated from the crude sample of Salvia prattii were higher than 98%, indicating that the method was efficient for the purification of phenylpropanoids with high purity from Salvia prattii. Additionally, this method showed great potential in the preparation of phenylpropanoids and can serve as a good example for the purification of phenylpropanoids from other plant materials.     

Preparative HPLC. Part 2. Purification of β-lactam derivatives using laboratory-assembled equipment

The capabilities of a preparative chromatograph assembled at moderate cost from commercially available components are examined in relation to the purification of β-lactam derivatives. It is shown that high efficiency separations can be achieved rapidly, economically, and on a scale sufficient to provide adequate quantities of material for spectroscopic identification and biological testing. In the synthesis of a large batch of aminopropyl silica for preparative HPLC purification of an impure sample of cephacetrile, three different grades of silica were investigated. The material giving the best performance was prepared on a 100 g scale and baseline separations for 0.5 g loadings of the impure cephacetrile were obtained on a 30 cm × 2.2 cm i.d. column. The pure cephalosporin was recovered in high yield by freeze drying of the eluate.     

Efficient purification of high‐purity compounds from the stem of Lonicera japonica Thunb using two‐dimensional preparative chromatography

Purification of high‐purity compounds from traditional Chinese medicines (TCMs) plays an important role in investigating their bioactivity. Nevertheless, it is often quite difficult to isolate compounds with high purity because of the complexity of TCMs in chemical composition. In this work, a two‐dimensional preparation method was successfully developed for the preparation of high‐purity compounds from the stem of Lonicera japonica Thunb, based on two novel polar copolymerized RP stationary phases, XAqua C3 and XAqua C18. An XAqua C3 prep column was used to separate the sample in the first‐dimensional preparation, and 14 g of sample was fractionated into eight fractions with a recovery of 82%. An XAqua C18 prep column was selected to prepare high‐purity compounds in the second‐dimensional preparation for its good orthogonality with the XAqua C3 stationary phase. As a result, major compounds in the sample were isolated with more than 99% purity. This method is a potent method to realize the efficient purification of compounds with high purity from the stem of L. japonica Thunb and it shows great potential in the separation of high‐purity compounds from complex samples.     

Large‐scale separation of antipsychotic alkaloids from Rauwolfia tetraphylla L. by pH‐zone‐refining fast centrifugal partition chromatography

pH‐zone‐refining centrifugal partition chromatography was successively applied in the large‐scale separation of close R_f antipsychotic indole alkaloids directly from CHCl₃ fraction of Rauwolfia tetraphylla leaves. Two experiments with increasing mass from 500 mg to 3 g of crude alkaloid extracts ( 1 C) of R. tetraphylla were carried out in normal‐displacement mode using a two‐phase solvent system composed of methyl tert‐butyl ether/ACN/water (4:1:5, v/v/v) where HCl (12 mM) was added to the lower aqueous stationary phase as a retainer and triethylamine (5 mM) to the organic mobile phase as an eluter. The two centrifugal partition chromatography separations afforded a total of 162.6 mg of 10‐methoxytetrahydroalstonine ( 1 ) and 296.5 mg of isoreserpiline ( 2 ) in 97% and 95.5% purity, respectively, along with a 400.9 mg mixture of α‐yohimbine and reserpiline ( 3 and 4 ). Further, this mixture was resolved over medium pressure LC using TLC grade silica gel H (average particle size 10 μm), which afforded 160.4 mg of α‐yohimbine ( 3) and 150.2 mg of reserpiline ( 4) in >95% purities. The purity of the isolated antipsychotic alkaloids was analyzed by high‐performance LC and their structures were characterized on the basis of their 1D, 2D NMR and electrospray ionization‐mass spectroscopic data.     

Analytical and semi‐preparative enantioresolution of (RS)‐ketorolac from pharmaceutical formulation and in human plasma by HPLC

An efficient, simple, validated, analytical and semi‐preparative HPLC method has been developed for direct enantioresolution of (RS)‐Ketorolac (Ket) using monochloro‐methylated derivatives of cellulose and amylose, i.e. cellulose (tris‐3‐chloro‐4‐methylphenylcarbamate) and amylose (tris‐5‐chloro‐2‐methylphenylcarbamate) as chiral stationary phases (CSPs) with photo diode array detection at 320 nm. Enantioresolution was carried out in samples of human plasma spiked with (RS)‐Ket under normal and reversed‐phase elution modes with suitable mobile phase compositions. The effect of nature of alcohols (MeOH, EtOH, PrOH and n‐BuOH) and other solvents (MeCN and MeOH) as organic modifiers in the mobile phase was investigated on the separation performance of two CSPs in terms of retention and separation of enantiomers. The best resolution was observed on cellulose‐based CSP using EtOH, while using 2‐PrOH (15%) and amylose‐based CSP obtained the highest retention. Under reversed‐phase elution mode the best enantioseparation was observed using 30% MeCN with ammonium formate buffer. The elution order of enantiomers was ascertained by determining specific rotations. The limit of detection and quantitation values were 5 and 15.5 ng/mL for each enantiomer of (RS)‐Ket, respectively. Copyright © 2016 John Wiley & Sons, Ltd.     

Preparative isolation and purification of 12 main antioxidants from the roots of Polygonum multiflorum Thunb. using high‐speed countercurrent chromatography and preparative HPLC guided by 1,1′‐diphenyl‐2‐picrylhydrazyl‐HPLC

A hyphenated strategy by off‐line coupling of 1,1′‐diphenyl‐2‐picrylhydrazyl‐high‐performance liquid chromatography, high‐speed countercurrent chromatography, and preparative high‐performance liquid chromatography was established to screen and separate antioxidants from ethyl acetate fraction of the roots of Polygonum multiflorum. Under the targeted guidance of 1,1′‐diphenyl‐2‐picrylhydrazyl‐high‐performance liquid chromatography experiment, 12 compounds were identified as potential antioxidants and readily isolated by high‐speed counter‐current chromatography and preparative high‐performance liquid chromatography. Ultraviolet spectroscopy, mass spectrometry, and ¹H NMR spectroscopy were employed to identify their structures, which were assigned as gallic acid ( 1 , 6.2 mg, 98.28%), catechin ( 2 , 8.8 mg, 90.69%), epicatechin ( 3 , 4.1 mg, 96.71%), polydatin ( 4 , 5.3 mg, 94.91%), 2,3,5,4′‐tetrahydroxy stilbene‐2‐Ο‐β‐D‐glucoside ( 5 , 20.2 mg, 95.23%), piceatannol ( 6 , 5.3 mg, 96.85%), rutin ( 7 , 5.4 mg, 97.92%), resveratrol ( 8 , 5.2 mg, 96.94%), isorhapontigenin ( 9 , 11.4 mg, 94.81%), hyperoside ( 10 , 9.7 mg, 98.52%), rhein ( 11 , 4.9 mg, 97.46%), and emodin ( 12 , 8.2 mg, 95.74%). Notably, compounds 6 and 9 were isolated from Polygonum multiflorum for the first time. In addition, antioxidant activity of compounds 1–12 were evaluated, and compounds 1–8 and 10 exhibited stronger antioxidant activity than ascorbic acid (positive control). These results indicated that the proposed method is a highly efficient strategy to screen and isolate antioxidants from complex natural products.     

Enantioselective isolation of methyl jasmonate using permethyl‐β‐cyclodextrin HPLC

A method based on the use of HPLC for the enantioselective resolution of the four stereoisomers of methyl jasmonate (MJ) with no need for the previous formation of the diastereoisomers is developed. To that end, a Nucleodex‐β‐PM column as well as an optimization process considering different flow rates and mobile phase compositions were required. As a result, 0.8 mL/min and 55:45 methanol/water composition were the conditions selected to carry out the separation of the stereoisomers. Isolation of pure (–)‐ and (+)‐MJ was accomplished by collecting the HPLC fractions corresponding to their elution time. SPE was subsequently used to concentrate and change the solvent of the HPLC fractions collected. Chiral GC and polarimetry were additionally employed to evaluate the purity and optical rotation, respectively, of the enantiomers separated. The results found in this study are particularly relevant considering that MJ stereoisomers are not commercially available.     

Application of an efficient strategy based on liquid–liquid extraction,high‐speed counter‐current chromatography,and preparative HPLC for the rapid enrichment,separation, and purification of four anthraquinones from Rheum tanguticum

This study presents an efficient strategy based on liquid–liquid extraction, high‐speed counter‐current chromatography, and preparative HPLC for the rapid enrichment, separation, and purification of four anthraquinones from Rheum tanguticum. A new solvent system composed of petroleum ether/ethyl acetate/water (4:2:1, v/v/v) was developed for the liquid–liquid extraction of the crude extract from R. tanguticum. As a result, emodin, aloe‐emodin, physcion, and chrysophanol were greatly enriched in the organic layer. In addition, an efficient method was successfully established to separate and purify the above anthraquinones by high‐speed counter‐current chromatography and preparative HPLC. This study supplies a new alternative method for the rapid enrichment, separation, and purification of emodin, aloe‐emodin, physcione, and chrysophanol.