Three‐phase hollow fiber liquid‐phase microextraction combined with HPLC for determination of three trace acidic plant growth regulators in Anoectochilus roxburghii (Wall.) Lindl

The analysis of plant growth regulators presents a challenge due to their trace quantities and complex matrices. A novel, simple, and effective analytical method for the determination of three trace acidic plant growth regulators in Anoectochilus roxburghii (Wall.) Lindl was developed to address this issue. Three‐phase hollow fiber liquid‐phase microextraction combined with high‐performance liquid chromatography was applied for the enrichment, purification, and determination of three acidic plant growth regulators, namely, indole‐3‐acetic‐acid, indole‐3‐butyric‐acid, and (+)‐abscisic acid. The factors affecting extraction performance, including extractant species, pH of donor and acceptor phases, salt addition dosage, extraction time, temperature, and stirring rate, were investigated and optimized. Under optimum conditions, the proposed method provided good linearity (R², 0.9994–0.9999), low limit of detection (0.038–0.12 ng/mL), and acceptable relative recoveries (56.7–117.6%). The enrichment factors were between 153 and 328. The developed method was successfully applied to the enrichment and determination of plant growth regulators in Anoectochilus roxburghii (Wall.) Lindl and exhibited increased purification capacity, higher sensitivity, and decreased organic solvent consumption compared with conventional sample preparation methods. This method may provide a testing platform for the monitoring of plant growth regulator residues, ensuring the safe and effective use of traditional Chinese medicine.     

Determination of plant growth regulators in pears by microwave‐assisted extraction and liquid chromatography with electrospray ionization mass spectrometry

A new method for the determination of six plant growth regulators, 3‐indolylacetic acid, 3‐indolepropionic acid, 2‐naphthoxyacetic acid, 2,4‐dicholrophenoxyacetic acid, 1‐naphthlcetic acid, and methyl naphthalene‐1‐acetate, in pears was established by liquid chromatography with electrospray ionization mass spectrometry. In this study, a microwave‐assisted extraction technique was first applied for the determination of plant growth regulators in fruit and three cleanup techniques were, respectively, investigated for the purification of pear samples. The chromatographic separation was performed on a Diamonsil C₁₈ column by using 0.01 mol/L formic acid/ammonium formate buffer solution (pH 3.5)/methanol (35:65, v/v) as the mobile phase with a flow rate of 0.7 mL/min in 1:1 split mode. The LODs ranged from 0.3 to 1.9 μg/kg. Under optimized conditions, the average recoveries (five replicates) for six plant growth regulators (spiked at 0.01, 0.05, and 0.5 mg/kg) ranged from 78.9 to 118.0%, and the RSDs were 1.4–10.3%.     

Simultaneous determination of multiclass plant growth regulators in fruits using the quick,easy, cheap,effective, rugged,and safe method and ultra‐high performance liquid chromatography‐tandem mass spectrometry

In this study, a modified quick, easy, cheap, effective, rugged, and safe method combined with ultra‐high performance liquid chromatography and tandem mass spectrometry was developed for the multiclass determination of 28 plant growth regulators in various fruits. Different extraction solvents and adsorbents, including primary secondary amine, octadecylsilyl, graphitized carbon black, and zirconia‐based sorbent, were investigated. Internal calibration and isotope internal standards, chlormequat chloride‐d₄, mepiquat chloride‐d₆, indole‐3‐acetic acid‐d₂, and forchlorfenuron‐d₅ were used to improve accuracy. For method validation, good linearity, low limits of detection and quantification were obtained. At three spiked concentrations (10, 50, and 100 µg/kg), satisfactory recoveries with relative standard deviations of 2.4–17.5% were obtained for strawberries (75.2–119.8%), grapes (70.5–114.0%), tangerines (71.7–115.4%), apples (72.7–115.4%), and kiwi fruits (71.7–119.2%). Samples analysis revealed that 15.6% of the samples (n = 96) were contaminated with one or two kinds of plant growth regulators, including chlormequat chloride, forchlorfenuron, paclobutrazol, 2,4‐dichlorophenoxyacetic acid, 2‐diethylaminoethyl hexanoate, and mepiquat chloride. Similar results were obtained by ultra‐high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry, indicating the robustness, effectiveness, and suitability of the developed method for routine monitoring of plant growth regulator residues in fruits.     

Determination of multiple phytohormones in fruits by high‐performance liquid chromatography with fluorescence detection using dispersive liquid–liquid microextraction followed by precolumn fluorescent labeling

Plant hormone determination in food matrices has attracted more and more attention because of their potential risks to human health. However, analytical methods for the analysis of multiple plant hormones remain poorly investigated. In the present study, a convenient, selective, and ultrasensitive high‐performance liquid chromatography method for the simultaneous determination of multiple classes of plant hormones has been developed successfully using dispersive liquid–liquid microextraction followed by precolumn fluorescent labeling. Eight plant hormones in fruits including jasmonic acid, 12‐oxo‐phytodienoic acid, indole‐3‐acetic acid, 3‐indolybutyric acid, 3‐indolepropionic acid, gibberellin A₃, 1‐naphthylacetic acid, and 2‐naphthaleneacetic acid were analyzed by this method. The conditions employed for dispersive liquid–liquid microextraction were optimized systematically. The linearity for all plant hormones was found to be >0.9993 (R² values). This method offered low detection limits of 0.19–0.44 ng/mL (at a signal‐to‐noise ratio of 3), and method accuracies were in the range of 92.32–103.10%. The proposed method was applied to determine plant hormones in five kinds of food samples, and this method can achieve a short analysis time, low threshold levels of detection, and a high specificity for the analysis of targeted plant hormones present at trace level concentrations in complex matrices.     

高效液相色谱-串联质谱法测定果蔬中7种植物生长促进剂残留

建立了果蔬样品中对氯苯氧乙酸、赤霉酸(GA3)、2,4-二氯苯氧乙酸、α-萘乙酸、吲哚丁酸、6-苄氨基嘌呤、氯吡脲残留的高效液相色谱-串联质谱(HPLC-MS/MS)分析方法。样品用甲醇匀质提取2次,经Waters C18固相萃取小柱净化后,在高效液相色谱-串联质谱仪(HPLC-MS/MS)选择反应监测(SRM)模式下测定。采用质谱定性,外标法定量。色谱柱为Hypersil GOLD aQ(150 mm×2.1 mm,3μm)柱,以甲醇和水为流动相,梯度洗脱。果蔬样品在低、中、高3个加标水平下的平均回收率为79%～97%,相对标准偏差均不高于7.6%。7种植物生长促进剂在果蔬样品中的方法检出限为0.40～20.0μg/kg。该方法灵敏度高、操作简单,可作为大批量果蔬中植物生长促进剂残留的检测方法。     

Comparison of extraction solvents and sorbents in the quick,easy, cheap,effective, rugged,and safe method for the determination of pesticide multiresidue in fruits by ultra high performance liquid chromatography with tandem mass spectrometry

An improved analytical method was developed for the simultaneous quantification of several plant growth regulators and fungicides (carbendazim, pyrimethanil, metalaxyl, triadimefon, paclobutrazol, thiophanate, prochloraz, dimethomorph, difenoconazole, (4‐chlorophenoxy)‐acetic acid, (2,4‐dichlorophenoxy)‐acetic acid, thiadiazuron, forchlorfenuron and gibberellins) in fruits followed by ultra high performance liquid chromatography with tandem mass spectrometry. Samples were extracted and purified using a modified QuEChERS method. Different extraction solvents and sorbents in the QuEChERS method were compared. Optimum results were followed by the addition of 1% acetic acid in acetonitrile; C₁₈ sorbent was added due to the acidic nature of several pesticides. The recoveries of the pesticides were in the range 73.7–118.4%, with relative standard deviations lower than 16.63%. Limits of detection ranged from 0.1–1.0 μg/kg. The method presented here is simple, rapid, sensitive and can be applied to large‐scale monitoring programs to screen the presences of pesticides in fruits.     

DES-Fe3O4 composite for rapid extraction of residual plant growth regulators in edible vegetable oil

DES-Fe₃O₄ composite achieved a good performance on rapid extraction of residual plant growth regulators in edible vegetable oil with the help of external magnetic field.     

Analysis of diphenylarsinic acid in human and environmental samples by HPLC–ICP‐MS

A simple, rapid and robust analytical method for determining diphenylarsinic acid in human and environmental samples was developed based on a combination of hydrophilic polymer‐based gel‐permeation high‐performance liquid chromatography (HPLC) and inductively coupled plasma mass spectrometry (ICP‐MS). Hair and nail samples were digested with alkali, and liberated diphenylarsinic acid (derivative) was extracted with diethyl ether, redissolved in water and injected for HPLC–ICP‐MS analysis. Human urine, groundwater and water extracts from soils were injected for HPLC–ICP‐MS directly after filtration. Using the method, diphenylarsinic acid in a solution was quantified in 7 min duration for an analysis with a detection limit of sub‐nanograms per milliliter. The method has been applied to groundwater arsenic pollution recently uncovered in Japan. Copyright © 2005 John Wiley & Sons, Ltd.     

Solid‐phase extraction of phenoxyacetic acid herbicides in complex samples with a zirconium(IV)‐based metal–organic framework

A zirconium(IV)‐based metal–organic framework material (MOF‐808) has been synthesized in a simple way and used for the extraction of phenoxyacetic acids in complex samples. The material has good thermal and chemical stability, large specific surface area (905.36 m²/g), and high pore size (22.18 Å). Besides, it contains a large amount of Zr‐O groups, easy‐to‐form Zr‐O‐H bond with carboxyl groups of phenoxyacetic acids, and possesses biphenyl skeleton structure, easy to interact with compounds through π‐π and hydrophobic interactions. These characteristics make the material very suitable for the extraction of certain compounds with a high extraction efficiency and excellent selectivity. The extraction conditions were optimized, and then an analytical method was successfully established and applied for analysis of actual samples. The solid‐phase extraction method based on prepared material had a wide linear range of 0.2–250 μg/L and a low detection limit of 0.1–0.5 μg/L for four phenoxyacetic acid compounds including 2,4‐dichlorophenoxyacetic acid, 2‐(2,4‐dichlorophenoxy) propionic acid, 4‐chlorophenoxyacetic acid, and dicamba. The relative standard deviations of intra‐ and interday precision were 1.8–3.8 and 4.3–6.9%, and the recoveries after spiking were between 77.1 and 109.3%. The results showed that the material is a desired substituent for the extraction of compounds with benzene ring structure containing carboxyl groups.     

Simultaneous determination and tissue distribution studies of four phenolic acids in rat tissue by UFLC–MS/MS after intravenous administration of salvianolic acid for injection

A rapid, simple and sensitive ultra‐fast liquid chromatography tandem mass spectrometric method was developed and validated for simultaneous determination and tissue distribution studies of rosmarinic acid, salvianolic acid D, lithospermic acid and salvianolic acid B in rats after intravenous administration of salvianolic acid for injection. The tissue homogenate samples were pretreated by protein precipitation with pre‐cooled acetonitrile. Chromatographic separation was achieved on a Waters Cortecs UPLC C₁₈ column (1.6 μm, 2.1 × 100 mm) with a mobile phase composed of 0.1% formic acid–water and 0.1% formic acid–acetonitrile. Analytes were detected by electrospray ionization mass spectrometry and quantitated using multiple reaction monitoring. The method was fully validated. The calibration curves for the four phenolic acids were linear in the given concentration ranges. The precisions (relative standard deviation) in the measurement of quality control samples were <10% and the accuracies (relative error) were in the range of 0.28–11.22%. The reliable method was successfully applied to the tissue distribution studies of the four phenolic acids. The results showed that rosmarinic acid, salvianolic acid D, lithospermic acid and salvianolic acid B were rapidly distributed in tissues with the major amount found in kidney, and little crossed the blood–brain barrier. The developed method and the results provide a basis for further studies.     

Comparative pharmacokinetic study of four major bioactive components after oral administration of Zhi‐Zi‐Hou‐Po decoction in normal and corticosterone‐induced depressive rats

A highly selective and efficient LC–MS/MS method was developed to determine the plasma concentration of magnolol, hesperidin, neohesperidin and geniposide following oral administration of Zhi‐Zi‐Hou‐Po decoction in normal and depressed rats. Plasma samples were pretreated by protein precipitation with methanol. Chromatographic separation was performed on an XTerra^® MS C₁₈ column using a gradient elution with a mobile phase composed of acetonitrile–0.1% aqueous formic acid. The proposed method was validated to be specific, accurate and precise for the analytes determination in plasma samples. The calibration curves displayed good linearity over definite concentration ranges for the analytes. The intra‐ and inter‐day precision of the proposed method at three different levels were all within <11.13% and the relative errors ranged from ?8.46 to 8.93%. The recovery of the four compounds ranged from 82.72 to 89.08% and no apparent matrix effect was observed during sample analysis. After full validation, the established method was successfully applied for comparing the pharmacokinetics of four components between normal and depressed rats. The results showed that the AUC and C_max of four analytes in depressed rats were significantly different from those in normal rats and might provide helpful information to guide the clinical use of Zhi‐Zi‐Hou‐Po to treat depression.     

Development of air‐assisted dispersive micro‐solid‐phase extraction‐based supramolecular solvent‐mediated Fe3O4@Cu–Fe–LDH for the determination of tramadol in biological samples

A simple method, air‐assisted dispersive micro‐solid‐phase extraction‐based supramolecular solvent was developed for the preconcentration of tramadol in biological samples prior to gas chromatography–flame ionization detection. A new type of carrier liquid, supramolecular solvent based on a mixture of 1‐dodecanol and tetrahydrofuran was combined with layered double hydroxide coated on a magnetic nanoparticle (Fe₃O₄@SiO₂@Cu–Fe–LDH). The supramolecular solvent was injected into the solution containing Fe₃O₄@SiO₂@Cu–Fe–LDH in order to provide high stability and dispersion of the sorbent without any stabilizer agent. Air assisted was applied to enhance the dispersion of the sorbent and solvent. A number of analytical techniques such as Fourier transform‐infrared spectrometry, field emission scanning electron microscope, energy‐dispersive X‐ray spectroscopy and X‐ray diffraction measurements were applied to assess the surface chemical characteristics of Fe₃O₄@SiO₂@Cu–Fe–LDH nanoparticles. The effects of important parameters on the extraction recovery were also investigated. Under optimized conditions, the limits of detection and quantification were obtained in the range of 0.9–2.4 and 2.7–7.5 μg L^?1 with preconcentration factors in the range of 450–472 in biological samples. This method was used for the determination of tramadol in biological samples (plasma, urine and saliva samples) with good recoveries.     

Preparation of doxycycline‐imprinted magnetic microspheres by inverse‐emulsion suspension polymerization for magnetic dispersion extraction of tetracyclines from milk samples

A magnetic dispersion extraction method was developed based on a molecularly imprinted magnetic microsphere (MIMM) for the selective clean‐up and enrichment of tetracycline antibiotics from milk samples. The MIMMs were prepared by inverse‐emulsion suspension polymerization, using doxycycline, trimethylolpropane trimethacrylate, acrylamide, methacrylic acid, and surface‐modified Fe₃O₄ as a template molecule, crosslinker, functional monomer, and magnetic component, respectively. Synthesis and extraction conditions were optimized for obtaining excellent affinity and high selectivity. The magnetism, covering amount, and selectivity of the magnetic microspheres were characterized by a vibrating sample magnetometer, thermogravimetric analysis, and a competitive recognition experiment. The MIMMs were applied to separate tetracycline antibiotics from milk samples by magnetic dispersion extraction, and an enrichment factor of 9.28 and a good sample clean‐up were obtained. The average recoveries of four tetracycline antibiotics were obtained in the range of 74.5–93.8% with a precision of 1.2–5.2%. The LODs and LOQs of the proposed method were in the range of 7.4–19.4 and 24.7–64.7 μg/kg, respectively. The results indicated that magnetic dispersion extraction using MIMMs is a powerful tool for food‐sample pretreatment with high selectivity and a simplified procedure.     

Biosurfactant trehalose lipid‐enhanced ultrasound‐assisted micellar extraction and determination of the main antioxidant compounds from functional plant tea

Hydrosoluble trehalose lipid (a biosurfactant) was employed for the first time as a green extraction solution to extract the main antioxidant compounds (geniposidic acid, chlorogenic acid, caffeic acid, and rutin) from functional plant tea (Eucommia ulmoides leaves). Single‐factor tests and response surface methodology were employed to optimize the extraction conditions for ultrasound‐assisted micellar extraction combined with ultra‐high‐performance liquid chromatography in succession. A Box‐Behnken design (three‐level, three‐factorial) was used to determine the effects of extraction solvent concentration (1–5 mg/mL), extraction solvent volume (5–15 mL), and extraction time (20–40 min) at a uniform ultrasonic power and temperature. In consequence, the best analyte extraction yields could be attained when the trehalose lipid solution concentration was prepared at 3 mg/mL, the trehalose lipid solution volume was 10 mL and the extraction time was set to 35 min. In addition, the recoveries of the antioxidants from Eucommia ulmoides leaves analyzed by this analytical method ranged from 98.2 to 102%. These results indicated that biosurfactant‐enhanced ultrasound‐assisted micellar extraction coupled with a simple ultra‐high‐performance liquid chromatography method could be effectively applied in the extraction and analysis of antioxidants from Eucommia ulmoides leaf samples.     

Preparation and application of molecularly imprinted polymer solid‐phase microextraction fiber for the selective analysis of auxins in tobacco

As signal molecules, auxins play an important role in mediating plant growth. Due to serious interfering substances in plants, it is difficult to accurately detect auxins with traditional solid‐phase extraction methods. To improve the selectivity of sample pretreatment, a novel molecularly imprinted polymer ‐coated solid‐phase microextraction fiber, which could be coupled directly to high‐performance liquid chromatography, was prepared with indole acetic acid as template molecule for the selective extraction of auxins. The factors influencing the polymer formation, such as polymerization solvent, cross‐linker, and polymerization time, were investigated in detail to enhance the performance of indole acetic acid‐molecularly imprinted polymer coating. The morphological and chemical stability of this molecularly imprinted polymer‐coated fiber was characterized by scanning electron microscopy, infrared spectrometry, and thermal analysis. The extraction capacity of the molecularly imprinted polymer‐coated solid‐phase microextraction fiber was evaluated for the selective extraction of indole acetic acid and indole‐3‐pyruvic acid followed by high‐performance liquid chromatography analysis. The linear range for indole acetic acid and indole‐3‐pyruvic acid was 1–100 µg/L and their detection limit was 0.5 µg/L. The method was applied to the simultaneous determination of two auxins in two kinds of tobacco (Nicotiana tabacum L and Nicotiana rustica L) samples, with recoveries range from 82.1 to 120.6%.     

Application of a dispersive solid‐phase extraction method using an amino‐based silica‐coated nanomagnetic sorbent for the trace quantification of chlorophenoxyacetic acids in water samples

Herein, an amino‐based silica‐coated nanomagnetic sorbent was applied for the effective extraction of two chlorophenoxyacetic acids (2‐methyl‐4‐chlorophenoxyacetic acid and 2,4‐dichlorophenoxyacetic acid) from various water samples. The sorbent was successfully synthesized and subsequently characterized by scanning electron microscopy, X‐ray diffraction, and Fourier‐transform infrared spectroscopy. The analytes were extracted by the sorbent mainly through ionic interactions. Once the extraction of analytes was completed, they were desorbed from the sorbent and detected by high‐performance liquid chromatography with ultraviolet detection. A number of factors affecting the extraction and desorption of the analytes were investigated in detail and the optimum conditions were established. Under the optimum conditions, the calibration curves were linear over the concentration range of 1–250, and based on a signal‐to‐noise ratio of 3, the method detection limits were determined to be 0.5 μg/L for both analytes. Additionally, a preconcentration factor of 314 was achieved for the analytes. The average relative recoveries obtained from the fortified water samples varied in the range of 91–108% with relative standard deviations of 2.9–8.3%. Finally, the method was determined to be robust and effective for environmental water analysis.     

A hybrid triazine‐imine core‐shell magnetic covalent organic polymer for analysis of pesticides in fruit samples by ultra high performance liquid chromatography with tandem mass spectrometry

A novel hybrid triazine‐imine core‐shell magnetic covalent organic polymer with high adsorption capacity and excellent stability was fabricated by surface‐assisted in situ growth technique. The composite possesses porous and extended π‐conjugated system, and was applied as the magnetic sorbent for efficient enrichment and rapid separation of pesticides. A new analytical method for simultaneous determination of eight pesticides in fruit samples was developed by magnetic solid phase extraction combined with ultra high performance liquid chromatography and tandem mass spectrometry. The effect of extraction time, desorption time, and the type of desorption solvent on the extraction efficiency were evaluated. The established method shows good repeatability and high sensitivity. The repeatability of this method was estimated with relative standard deviations in the range of 0.7–7.0% (n = 5) for the same batch, and 1.7–10% (n = 3) for batch to batch. Good linearity for eight pesticides was obtained with coefficient of determination in the range of 0.9942–0.9990. Limit of detections ranged from 0.4 to 1.2 ng/L. Real sample determination showed that four and two pesticides were detected in strawberry and grape, respectively. The results demonstrated that the established method was efficient, sensitive, and convenient for trace determination of pesticides in fruit samples.     

Odour‐causing compounds in air samples: Gas–liquid partition coefficients and determination using solid‐phase microextraction and GC with mass spectrometric detection

A quantification method based on solid‐phase microextraction followed by GC coupled to MS was developed for the determination of gas–liquid partition coefficients and for the air monitoring of a group of odour‐causing compounds that had previously been found in wastewater samples including dimethyl disulphide, phenol, indole, skatole, octanal, nonanal, benzothiazole and some terpenes. Using a divinylbenzene/carboxen/polydimethylsiloxane fibre, adsorption kinetics have been studied to define an extraction time that would avoid coating saturation. It was found that for an extraction time of 10 min, external calibration could be performed in the range of 0.4–100 μg/m³, with detection limits between 0.1 and 20 μg/m³. Inter‐day precision of the developed method was evaluated (n = 5) and RSD values between 12 and 24% were obtained for all compounds. The proposed method has been applied to the analysis of air samples surrounding a wastewater treatment plant in Catalonia (Spain). In all air samples evaluated, dimethyl disulphide, limonene and phenol were detected, and the first two were the compounds that showed the highest partition coefficients.     

Molecularly imprinted solid‐phase extraction method for the gas chromatographic analysis of organochlorine fungicides in ginseng

A highly selective molecularly imprinted solid‐phase extraction coupled with gas chromatography method was developed for the simultaneous isolation and determination of four organochlorine fungicides (pentachloronitrobenzene, pentachloroaniline, methylpentachlorophenyl sulfide, and hexachlorobenzene) in ginseng samples. A novel molecularly imprinted polymer with pentachloronitrobenzene as template was synthesized by precipitation polymerization employing butanone/n‐heptane (6.5:3.5, v/v) solution as porogen. The limit of detection of the method was 0.001 mg/kg, and the limit of quantification was 0.002 mg/kg. The different spiked levels of ginseng samples were 0.05, 0.5, 2.0 for pentachloronitrobenzene and pentachloroaniline, and 0.01, 0.1, 1.0 for methylpentachlorophenyl sulfide and hexachlorobenzene. The average recoveries of four organochlorine fungicides were 87.6–92.3% of pentachloronitrobenzene, 79.3–95.2% of pentachloroaniline, 80.3–90.4% of methylpentachlorophenyl sulfide, and 83.5–91.7% of hexachlorobenzene, respectively. This new method could be applied to direct determination of four organochlorine fungicides in ginseng samples.     

Solid‐phase extraction based on amino‐functionalized graphene oxide nanocomposites for analysis of short‐chain perfluorinated alkyl acids in human serum by ion chromatography mass spectrometry

A highly sensitive method was developed for the analysis of short‐chain perfluorinated alkyl acids (PFAAs) in serum samples using solid‐phase extraction (SPE) coupled with ion chromatography–electrospray ionization–mass spectrometry. The synthesized amino‐functionalized graphene oxide nanocomposites were used as an SPE sorbent for the enrichment of trace analytes and purification of samples. They exhibited high selectivity to polar compounds. The suppressor was employed to remove counterions and reduce background signals of mobile phase. These two crucial steps could effectively eliminate matrix effects and enhance analytical sensitivity. The lowest limits of quantification were 2.0 μg L⁻¹ for perfluorobutanoic acid and perfluorovaleric acid, 1.0 μg L⁻¹ for perfluorocaproic acid and 0.50 μg L⁻¹ for perfluorobutane sulfonic acid, respectively. The procedure was successfully applied for determination of trace PFAAs in 25 serum samples. Mean recoveries ranged from 86.3 to 101.4% with relative standard deviations of 1.6–6.8%. The method allowed an excellent separation and quantification of short‐chain PFAAs that were difficult to analyze by conventional chromatography.