Semi-industrial isolation of salicin and amygdalin from plant extracts using slow rotary counter-current chromatography

Salicin in the bark extract of Salix alba and amygdalin in the fruit extract of Semen armeniacae were each separated by slow rotary counter-current chromatography (SRCCC). The apparatus was equipped with a 40-L column made of 17 mm i.d. convoluted Teflon tubing. A 500g amount of crude extract containing salicin at 13.5% was separated yielding 63.5 g of salicin at 95.3% purity in 20h using methyl tert-butyl ether-l-butanol (1:3) saturated by methanol-water (1:5) as a stationary phase and methanol-water (1:5) saturated by methyl tert-butyl ether-1-butanol (1:3) as a mobile phase. A 400g amount of crude extract containing amygdalin at 55.3% was isolated to yield 221.2g of amygdalin at 94.1% purity in 19h using ethyl acetate-1-butanol (1:2) saturated by water as a stationary phase and water saturated by ethyl acetate-1-butanol (1:2) as a mobile phase. The flow rate of the mobile phase was 50 ml/min. The results show that industrial SRCCC separation of salicin and amygdalin is feasible using a larger column at a higher flow rate of the mobile phase.     

Determination of the epimerization rate constant of amygdalin by microemulsion electrokinetic chromatography

A new method for separation and determination of amygdalin and its epimer (neoamygdalin) in the epimerization of amygdalin by MEEKC is proposed. For the chiral separation of amygdalin and neoamygdalin, a running buffer composed of 80 mM sodium cholate, 5.0% v/v butan‐1‐ol, 0.5% v/v heptane and 94.5% v/v 30 mM Na₂B₄O₇ buffer (pH 9.00) is proposed. Under optimum conditions, the basic separation of amygdalin and neoamygdalin can be achieved within 7 min. The calibration curve for amygdalin showed excellent linearity in the concentration range of 20–1000 μg/mL with a detection limit of 5.0 μg/mL (S/N=3). The epimerization rate constant of amygdalin in basic microemulsion was first determined by monitoring the concentration changes of amygdalin, and the epimerization rate constant of amygdalin was found to be 2×10^?3 min^?1 at 25°C under the above optimum microemulsion conditions.     

高效液相色谱法测定山楂中的苦杏仁甙

 建立了从山楂中提取苦杏仁甙的方法,样品先用石油醚脱脂，然后用甲醇进行索氏提取。用高效液相色谱法定量测定了山楂中的苦杏仁甙,色谱条件如下：反相C18柱,流动相为15%的甲醇水溶液,检测波长为215 nm。测定了含不同比例山楂籽的山楂样品,结果表明含山楂籽比例高的山楂样品中苦杏仁甙的含量高,且山楂粗粒样品中苦杏仁甙的提取量比粉末样品的提取量高。     

Densitometric thin-layer chromatographic determination of artemisinin and its lipophilic derivatives,artemether and arteether

A thin-layer chromatograpy (TLC) method is developed to analyze artemisinin (AT) and its derivatives, artemether (AM) and arteether (AE), using a silica-gel plate with a mobile phase containing pure chloroform. After development, all products are visualized after dipping in a 4-methoxybenzaldehyde dipping reagent of 1% (v/v) in an acidic solution of sulphuric acid (98%, v/v) and acetic acid (96-98%, v/v) (respectively, 2% and 10%, v/v in alcohol-water, 60:30, v/v), presenting a purple color against a slightly colored background. This TLC system is quantitatively evaluated in terms of stability of the color, precision, accuracy, and calibration. Activation is performed at 110 degrees C. Stability of the color of both analytes is reached after 12 min. Precision, less than 5%, is obtained at two levels. Good linearity is obtained in the range of 0.5-8 micro g for all analytes. Some applications show its utility in the quality control of capsules. The prederivatization technique, applying the described dipping reagent before development, reveals the presence of various reaction products, possibly isomers. These results prove that TLC can be a cheap and easy alternative for the analysis of AT and its lipophilic derivatives, AM and AE, as pure powder and in pharmaceutical-dosage forms.     

Determination of oleanolic acid, ursolic acid and amygdalin in the flower of Eriobotrya japonica Lindl. by HPLC

Simple and accurate HPLC methods were developed for the determination of oleanolic acid (OA), ursolic acid (UA) and amygdalin in loquat (Eriobotrya japonica Lindl.) flower, which is commonly used for the treatment of various diseases as a traditional Chinese medicine. HPLC assay was performed on a reversed-phase C(18) column and all three compounds were detected at 210 nm with a flow rate of 1.0 mL/min. The mobile phase consisted of methanol (A) and 0.03 mol/L phosphate buffer (pH 2.8) (B) with a ratio of 88:12 (A:B, v/v) for simultaneous detection of OA and UA, and 25:75 (A:B, v/v) for detection of amygdalin. The established methods showed good precision and accuracy with overall intra-day and inter-day variation of 0.99-3.55 and 1.05-4.05%, respectively, and overall recoveries of 97.37-99.32% for the three compounds. Application of these methods to determine the OA, UA and amygdalin contents in loquat flower showed that cultivar had a minor effect on the contents of all three compounds, with average amounts of 0.38-0.51 mg OA/g dry weight (DW), 2.15-2.68 mg UA/g DW and 1.23-1.56 mg amygdalin/g DW among five loquat cultivars tested. However, developmental stages and flower tissues showed significant effect on the contents of all three bioactive components.     

Preparative separation and purification of two highly polar alkaloids derived from Semen Strychni extracted with dichloromethane by high‐speed countercurrent chromatography

Brucine chloromethochloride and strychnine chloromethochloride, the two chloromethochloride derivatives formed during the extraction of Semen Strychni in which dichloromethane was used as the extracting solvent, were isolated and purified by high‐speed countercurrent chromatography for the first time. The two‐phase solvent system composed of chloroform/methanol/0.3 mol/L hydrochloric acid (4:3:2, v/v/v) was selected for separation. From 300 mg of the crude extracts, 56.2 mg of brucine chloromethochloride and 60.2 mg of strychnine chloromethochloride were obtained with the purity of 99.78 and 96.99%, respectively, and the structures were confirmed by mass spectrometry, ¹H, ¹³C, and two‐dimensional NMR spectroscopy. The results indicated that the present method is a powerful technology for large‐scale isolation of alkaloids from Semen Strychni.     

超高效液相色谱-串联四极杆飞行时间质谱和超高效液相色谱-串联三重四极杆质谱用于血浆中苦杏仁苷及其代谢产物野黑樱苷的定性和定量分析

建立了大鼠灌胃麻杏石甘汤后血浆中苦杏仁苷、野黑樱苷的定性及定量方法。样品经液液萃取净化处理,定性采用超高效液相色谱-串联四极杆飞行时间质谱仪（UPLC-QTOF-MS/MS）,经Shim-pack XR-ODS Ⅲ色谱柱（75 mm×2.0 mm,1.6 μm）分离,定量采用超高效液相色谱-串联三重四极杆质谱仪（UPLC-Q-TRAP-MS）,经Agilent C₁₈色谱柱（50 mm×2.1 mm,1.7 μm）分离,电喷雾负离子化（ESI）及MRM模式测定,流动相均为乙腈-0.1%（v/v）甲酸水溶液。结果显示苦杏仁苷、野黑樱苷在相应浓度范围内线性关系良好（相关系数分别为0.9990、0.9970）,精密度（RSD）小于9.20%,回收率为82.33%~95.25%,检出限（LOD）约为0.50 ng/mL。本方法快速简便,为血浆样品中苦杏仁苷、野黑樱苷的定性和定量分析提供良好参考。     

Simultaneous assay of olanzapine and fluoxetine in tablets by column high-performance liquid chromatography and high-performance thin-layer chromatography

This paper describes validated high-performance liquid chromatographic (LC) and high-performance thin-layer chromatographic (TLC) methods for the simultaneous estimation of olanzapine and fluoxetine in pure powder and tablet formulations. The LC separation was achieved on a Lichrospher 100 RP-180, C18 column (250 mm, 4.0 mm id, 5 microm) using 0.05 M potassium dihydrogen phosphate buffer (pH 5.6 adjusted with o-phosphoric acid)-acetonitrile (50 + 50, v/v) as the mobile phase at a flow rate of 1 mL/min and ambient temperature. The TLC separation was achieved on aluminum sheets coated with silica gel 60F254 using methanol-toluene (40 + 20, v/v) as the mobile phase. Quantitation was achieved by measuring ultraviolet absorption at 233 nm over the concentration range of 10-70 and 40-280 microg/mL with mean recovery of 99.54 +/- 0.89 and 99.73 +/- 0.58% for olanzapine and fluoxetine, respectively, by the LC method. Quantitation was achieved by measuring ultraviolet absorption at 233 nm over the concentration range of 100-800 and 400-3200 ng/spot with mean recovery of 101.53 +/- 0.06 and 101.45 +/- 0.35% for olanzapine and fluoxetine, respectively, by the TLC method with densitometry. These methods are simple, precise, and sensitive, and they are applicable for simultaneous determination of olanzapine and fluoxetine in tablet formulations.     

Development and Validation of HPLC,TLC and Derivative Spectrophotometric Methods for the Analysis of Ezetimibe in the Presence of Alkaline Induced Degradation Products

Reversed phase‐high performance liquid chromatography (RP‐HPLC), thin layer chromatography (TLC) densitometry and first derivative spectrophotometry (1D) techniques are developed and validated as a stability‐indicating assay of ezetimibe in the presence of alkaline induced degradation products. RP‐HPLC method involves an isocratic elution on a Phenomenex Luna 5μ C₁₈ column using acetonitrile: water: glacial acetic acid (50:50:0.1 v/v/v) as a mobile phase at a flow rate of 1.5 mL/min. and a UV detector at 235 nm. TLC densitometric method is based on the difference in Rf‐values between the intact drug and its degradation products on aluminum‐packed silica gel 60 F₂₅₄ TLC plates as stationary phase with isopropanol: ammonia 33% (9:1 v/v) as a developing mobile phase. On the fluorescent plates, the spots were located by fluorescence quenching and the densitometric analysis was carried out at 250 nm. Derivative spectrophotometry, the zero‐crossing method, ezetimibe was determined using first derivative at 261 nm in the presence of its degradation products. Calibration graphs of the three suggested methods are linear in the concentration ranges 1–10 mcg/mL, 0.1–1 mg/mL and 1–16 mcg/mL with a mean percentage accuracy of 99.05 ± 0.54%, 99.46 ± 0.63% and 99.24 ± 0.82% of bulk powder, respectively. The three proposed methods were successfully applied for the determination of ezetimibe in raw material and pharmaceutical dosage form; the results were statistically analyzed and compared with those obtained by the reported method. Validation parameters were determined for linearity, accuracy and precision; selectivity and robustness and were assessed by applying the standard addition technique.     

HPLC, TLC, and first-derivative spectrophotometry stability-indicating methods for the determination of tropisetron in the presence of its acid degradates

Three stability-indicating assay methods were developed for the determination of tropisetron in a pharmaceutical dosage form in the presence of its degradation products. The proposed techniques are HPLC, TLC, and first-derivative spectrophotometry (1D). Acid degradation was carried out, and the degradation products were separated by TLC and identified by IR, NMR, and MS techniques. The HPLC method was based on determination of tropisetron in the presence of its acid-induced degradation product on an RP Nucleosil C18 column using methanol-water-acetonitrile-trimethylamine (65 + 20 + 15 + 0.2, v/v/v/v) mobile phase and UV detection at 285 nm. The TLC method was based on the separation of tropisetron and its acid-induced degradation products, followed by densitometric measurement of the intact spot at 285 nm. The separation was carried out on silica gel 60 F254 aluminum sheets using methanol-glacial acetic acid (22 + 3, v/v) mobile phase. The 1D method was based on the measurement of first-derivative amplitudes of tropisetron in H2O at the zero-crossing point of its acid-induced degradation product at 271.9 nm. Linearity, accuracy, and precision were found to be acceptable over concentration ranges of 40-240 microg/mL, 1-10 microg/spot, and 6-36 micro/mL for the HPLC, TLC, and 1D methods, respectively. The suggested methods were successfully applied for the determination of the drug in bulk powder, laboratory-prepared mixtures, and a commercial sample.     

Selective separation and determination of isoproterenol on thin layers of bismuth silicate ion-exchanger

A simple and sensitive method for the separation and determination of isoproterenol from other doping drugs has been developed on thin layers of bismuth silicate, a synthetic inorganic ion exchanger as adsorbent in thin layer chromatography (TLC). A mixture of methanol and 0.1 mol/L formic acid (3:7, v/v) was employed as the mobile phase. The development time was 32 min. The quantitative measurement were performed with a Camag TLC Scanner-3 at wavelength (λ) of 410 nm. The isoproterenol recovery in this procedure was 98.9%. The linear correlation coefficient was greater than 0.9871 and the relative standard deviation (RSD) was less than 0.94. The limit of detection (LOD) and limit of quantification (LOQ) were 7.7×10^-7mol/L and 3.85 ×10^-6mol/L, respectively. This method has been applied in the determination of isoproterenol in dosage forms and in biological fluids.     

HPLC with Column Switching Coupled to APCI–MS for Pharmacokinetic Study of Amygdalin in Rabbit Plasma

A fast and validated assay was established for the pharmacokinetic study of amygdalin in Armeniacae Semen in rabbit. The method involved column switching (CS) enrichment, separation, post-column derivatization, and atmospheric pressure chemical ionization (APCI) mass spectrometric detection. Plasma sample was enriched by CS using a MAYI-ODS as the first column. Analytes of interest were isolated and analyzed on a second column of Zorbax SB-C₁₈. To detect amygdalin in plasma samples, a T-piece was connected between the HPLC outlet and the APCI source to add a mixture of dichloromethane and methanol to the eluent by an isocratic pump. Calibration graphs showed good linearity over a range of 1.0–1,280 ng mL⁻¹. The detection limit was 0.2 ng mL⁻¹. The intra- and inter-day accuracies were within 3.9%. The method was successfully applied to a study of the pharmacokinetics of amygdalin after an intravenous injection of amygdalin extracts to rabbits with a dose of 400 mg kg⁻¹. The results indicate that amygdalin is a one-compartment open model with a first order absorption phase.     

Isolation and determination of p-hydroxybenzoylcholine in traditional Chinese medicine Semen sinapis Albae

A special component is isolated from Semen sinapis Albae (white mustard seed), a traditional Chinese medicine. According to the physical and chemical investigation and spectroscopic identification, this component can be known as p-hydroxybenzoylcholine bisulfate, a choline base. This component in the drug is also determined by RP-HPLC. A reversed-phase C₁₈ column is used to separate the p-hydroxybenzoylcholine with an eluent of methanol–0.05 mol/L monopotassium phosphate solution (30:70) (adjusted by phosphoric acid to pH 3.6) at the flow rate of 0.5 mL/min. Detection is carried out with a UV detector operated at 285 nm, and the column temperature is 25 °C. It reveals that there is 0.021% (w/w) of p-hydroxybenzoylcholine bisulfate in Semen sinapis Albae and 0.037% (w/w) in stir-baked Semen sinapis Albae.     

Development of a reversed-phase thin-layer chromatographic method for artemisinin and its derivatives

In this study a clear separation between seven analogues of artemisinin on thin-layer chromatography (TLC) is presented. The developed TLC method is carried out on a RP-C18 thin-layer plate using acetonitrile-water (50:25 v/v) as the mobile phase. Spots are visualized by derivatization with an acidified 4-methoxybenzaldehyde reagent in methanol-water. This method allows the separation of a diverse group of compounds that have versatile hydrophilic/lipophilic characteristics; namely artemisinin, artesunate (AS), artelinic acid (AL), arteether (AE), both isomers of artemether (AM) (alpha and beta), dihydroartemisinin, and desoxyartemisinin. Separation of some degradation products and impurities, down to 2%, allows quality control and stability investigation of all actives in raw material and pharmaceutical formulations. The method is further developed via densitometric measurement for quantitative determination purposes for AL and AS. The derivatization technique is evaluated, showing good stability and reproducibility of the coloring process. Percent relative standard deviation values are less than 5% for replicates, and linearity is obtained in the range of 0.5 to 8 microg. A comparative study with high-performance liquid chromatography (HPLC) on a C18 column, applying the same mobile phase, proves the suitability of the TLC method, in which almost all presented analytes are separated from each other. In contrast, HPLC requires at least a 20-min analysis to chromatograph all of the compounds and only betaAM and AE are clearly separated from each other and from the other compounds.     

Image analysis of phenylisothiocyanate derivatised and charge-couple device-detected glyphosate and glufosinate in food samples separated by thin-layer chromatography

The paper presents the application of pre-chromatographic derivatisation reaction of aminophosphonic acids (glyphosate and glufosinate) with phenylisothiocyanate in thin-layer chromatography (TLC). Silica gel as stationary phase and a mixture of methanol–water–diethyl ether (2:1:1, v/v/v) and ethanol–water–diethyl ether (4:1:2, v/v/v) were used as the mobile phase, respectively. Detection was performed by spraying TLC plates with a freshly prepared mixture of sodium azide (1%), starch solution (1% for glyphosate and 2% for glufosinate), and potassium iodide (1.0 × 10^–2 mol L^?1) adjusted to pH 6.0 and exposed to iodine vapour for 15 s. Both glyphosate and glufosinate as phenylthiocarbamates (PTC-derivatives) were visible as white spots against a violet background which were converted into chromatograms using TLSee software. The calibration curves for glyphosate and glufosinate were within the ranges of 8.45–84.5 ng and 1.98–79.2 ng per spot, respectively. The limits of detection and quantification for glyphosate were at a level of 4 and 8.45 ng per spot, and for glufosinate were 0.99 and 1.78 ng per spot, respectively. The proposed method was successfully used in the determination of aminophosphonic acids in spiked plants samples.     

An Improved Method to Resolve Plant Saponins and Sugars by TLC

Thin-layer chromatography (TLC) plays an important role in the initial selection of mutants having a unique seed saponin composition from the germplasm collections of the subgenus Soja. In the conventional TLC procedure, the dehydrated free sugars are retained just below the major saponins and interrupt the identification of some minor saponin constituents. To resolve this problem, we developed an efficient and reliable method to move sugars from the saponin area on TLC. A developing chamber was saturated with the lower phase of chloroform:methanol:water (65:35:10, v/v) for 2?h and the TLC plates were developed in it for 50?min. Plates were then dried at 100?°C for 10?min to evaporate the excess mobile phase and developed again with 10?% H₂SO₄ for 15?min. While sulfuric acid migrates over the surface of SiO₂, sugar molecules are dehydrated and hydrophilic interactions between free sugars and SiO₂ are strongly reduced. Thus, the positions of dehydrated sugars were shifted to above the saponin area on the TLC plate. This resulted in easy recognition of the saponin composition without any discrimination. This amended protocol would be applicable to all TLC analyses in which the target components should be separate from the interrupting sugar molecules.     

Improved thin-layer chromatographic method for the separation of cholesterol,egg phosphatidylcholine,and their degradation products

Degradation products of egg phosphatidylcholine (EPC) and cholesterol were analyzed with different normal- and reversed-phase thin-layer chromatography (TLC) systems. The best separation, in terms of the highest number of degradation products from both analytes, was obtained with a reversed-phase system, using butanol-methanol-water-96-98% (v/v) acetic acid (40 + 40 + 20 + 4, v/v/v/v) as the mobile phase after overnight saturation at 25 degrees C. A special development technique was used. After a first development, the plate was dried and a second development was performed in the same direction. This method enabled us to separate lysophosphatidylcholine, several free fatty acids and hydroperoxides, and several undefined degradation products of EPC and cholesterol. All products were visualized after the plate was dipped in a 1% (v/v) solution of 4-methoxybenzaldehyde in 98% sulfuric acid-96-98% (v/v) acetic acid-ethanol-water (2 + 10 + 60 + 30), presenting a blue color or a white spot against a colored background. After activation at 110 degrees C, a stable color for both analytes was reached after 12 min. Precision of <5% was obtained at 2 levels of analysis. Good linearity was obtained in the range of 5-30 microg for EPC (r = 0.991) and 5-40 microg for cholesterol (r = 0.991). These results show that TLC can be an inexpensive and easy alternative for the analysis of EPC and cholesterol.     

Development and validation of high-performance thin-layer chromatographic method for determination of amygdalin

ABSTRACT

A new method for the extraction and quantitative determination of amygdalin has been proposed. Accelerated solvent extraction was applied for the extraction, and reversed-phase high-performance thin-layer chromatography method was developed, validated, and applied for the determination of amygdalin in the extracts of apricot, plum, almond, and peach kernels. The chromatographic system used was RP-18 silica, as stationary phase and acetonitrile/water (50:50, v/v), as mobile phase. Densitometric scanning was performed at 210 nm. The method was validated with respect to specificity, linearity, precision, and accuracy. The results showed that the peak area responses were linear within the concentration range of 2.5–50.0 µg/spot (R² = 0.9984). The limit of quantification was 4.28 µg/spot, and the detection limit 1.28 µg/spot. The intra-day and inter-day reproducibility, in terms of %RSD, were in the range of 0.81–1.15 and 1.32–1.89, respectively. The accuracy data were in the range from 99.98 to 100.56%. The method is linear, quantitative and reproducible, and could be used as an efficient and economical green chromatographic procedure for the determination of amygdalin in the fruit kernel.     

A comparative study of HPLC and TLC separation of amino acids using Cu(II) ion

Separation of amino acids using Cu(II) by RP-HPLC and impregnated TLC is reported. For HPLC the sample mixture of amino acids containing copper (II) was injected into the column while for TLC the silica gel plates were impregnated with Cu(II). The mobile phase used in HPLC was acetate buffer (0.3 M , pH 6.0)-acetonitrile (9:1, v/v), and that in TLC was acetate buffer (0.3 M , pH 6.0):acetonitrile-n-butanol (12:5:10, by vol.). The results have been compared and discussed.