Preparative isolation and purification of two benzoxazinoid glucosides from Acanthus ilicifolius L. by high-speed counter-current chromatography

The first preparative separation of two benzoxazinoids, (2R)-2-O-beta-d-glucopyranosyl-2H-1,4-benzoxazin-3(4H)-one (HBOA-Glc) and (2R)-2-O-beta-d-glucopyranosyl-4-hydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA-Glc), by means of high-speed counter-current chromatography (HSCCC) from the n-butanol extract of Acanthus ilicifolius L. is presented. The two-phase solvent system containing ethyl acetate-n-butanol-0.5%NH(4)OH (2:3:5, v/v/v, system B) was selected for the one-step HSCCC separation of HBOA-Glc and DIBOA-Glc according to the partition coefficient values (K) for target compounds and the separation factor (alpha) between the two target compounds. In the one-step HSCCC separation using solvent B, from 100mg n-butanol extract of A. ilicifolius, 6.3 mg HBOA-Glc and 6.8 mg DIBOA-Glc were isolated with purities of 90.3% and 80.2%, respectively. In order to obtain the two target compounds with higher purity, a second separation process was developed comprising two steps. In the two-step separation, the sample was first pre-purified by HSCCC using ethyl acetate-n-butanol-water (2:3:5, v/v/v, system A) solvent system and then purified using solvent system B. A 100-mg amount of the n-butanol extracts of A. ilicifolius was separated to yield 5.8 mg of HBOA-Glc and 4.8 mg of DIBOA-Glc with purities of 97.1% and 94.8%, respectively, which were directly used for NMR analyses.     

LC/MS guided isolation of alkaloids from lotus leaves by pH-zone-refining counter-current chromatography

The traditional methods used in natural product separation primarily target the major components and the minor components may thus be lost during the separation procedure. Consequently, it's necessary to develop efficient methods for the preparative separation and purification of relatively minor bioactive components. In this paper, a LC/MS method was applied to guide the separation of crude extract of lotus (Nelumbo nucifera Gaertn.) leaves whereby a minor component was identified in the LC/MS analysis. Afterwards, an optimized pH-zone-refining CCC method was performed to isolate this product, identified as N-demethylarmepavine. The separation procedure was carried out with a biphasic solvent system composed of hexane-ethyl acetate-methyl alcohol-water (1:6:1:6, v/v) with triethylamine (10 mM) added to the upper organic phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase eluent. Two structurally similar compounds--nuciferine and roemerine--were also obtained from the crude lotus leaves extract. In total 500 mg of crude extract furnished 7.4 mg of N-demethylarmepavine, 45.3 mg of nuciferine and 26.6 mg of roemerine with purities of 90%, 92% and 96%, respectively. Their structures were further identified by HPLC/ESI-MSn, FTICR/MS and the comparison with reference compounds.     

An effective method based on medium‐pressure liquid chromatography and recycling high‐speed counter‐current chromatography for enrichment and separation of three minor components with similar polarity from Dracocephalum tanguticum

The separation of minor compounds, especially those with similar polarities from a complex sample, remains challenging. In the proposed study, an effective method based on medium‐pressure liquid chromatography and recycling high‐speed counter‐current chromatography was developed for the enrichment and separation of three minor components from Dracocephalum tanguticum. The crude extract was directly introduced to medium‐pressure liquid chromatography for the enrichment of the three minor components. Based on high‐performance liquid chromatography analysis, the total content of these three compounds increased from 0.48% in the crude extract to 85.3% in the medium‐pressure liquid chromatography fraction. In addition, high‐speed counter‐current chromatography was employed to separate the enriched compounds using the solvent system hexane/ethyl acetate/methanol/water (1.18:8.82:1.18:8.82, v/v/v/v). As a result, compound 3 and a mixture of compounds 1 and 2 were obtained. In order to improve the resolution of compounds 1 and 2 while saving separation time, a recycling and heart‐cut mode was used. Finally, compounds 1 and 2 were obtained after five cycles. These compounds were identified as 3‐phenylethyl β‐d ‐glucopyranoside ( 1 ), tazettoside E ( 2 ), and cirsiliol‐4′‐glucoside ( 3 ). Compounds 1 and 2 were primarily separated from D. tanguticum. Moreover, the developed method provided a reference for the separation of minor components from the complex sample.     

The generally useful estimate of solvent systems method facilitates off-line two-dimensional countercurrent chromatography for isolating compositions from Siraitia grosvenorii roots

Solvent system selection is a crucial and the most time-consuming step for successful countercurrent chromatography separation. A thin-layer chromatography-based generally useful estimate of solvent systems method has been developed to simplify the solvent system selection. We herein utilized the method to select a solvent system for off-line two-dimensional countercurrent chromatography to separate chemical compositions from a complex fraction of the Siraitia grosvenorii root extract. The first-dimensional countercurrent separation using chloroform/methanol/water (10:5.5:4.5, v/v/v) yielded four compounds with high purity and three mixture fractions (Fr I, III, and VII). The second-dimensional countercurrent separation conducted on Fr I, III, and VII using the hexane/ethyl acetate/methanol/water (4:6:6:4, 3:7:3:7, v/v/v) and chloroform/methanol/water (10:9:6, v/v/v) solvent systems, respectively, produced another four compounds. Four triterpenoids and four lignans were finally isolated, including two novel compounds. Hence, the generally useful estimate of solvent systems method is a feasible and efficient approach for selecting an applicable solvent system for separating complex samples. In addition, the off-line two-dimensional countercurrent chromatography method can improve both the peak resolution and the capacity of countercurrent chromatography.     

Separation and purification of intermediates for the preparation of naproxen from synthetic mixtures by countercurrent chromatography

Three key intermediates in the preparation of the nonsteroidal anti‐inflammatory drug naproxen were successfully separated and purified with high purity from synthetic mixtures by countercurrent chromatography with a selected biphasic solvent system. The biphasic solvent system composed of n‐hexane/ethyl acetate/methanol/water (9:1:9:1, v/v/v/v) was selected according to partition performance of the three components using thin‐layer chromatography. Fifty milligrams of the synthetic mixture after the three‐step reaction was injected into a preparative countercurrent chromatography separation column and yielded 3.5, 14.0, and 8.0 mg of three key intermediates with 95.0, 99.0, and 98.0% purity, and the recovery of each component was 65.2, 71.2, and 69.6%, respectively. The results indicated that countercurrent chromatography is an efficient alternative and economical method for the separation and purification of intermediate components from synthetic mixtures.     

Three-phase solvent systems for comprehensive separation of a wide variety of compounds by high-speed counter-current chromatography

Three-phase solvent systems were efficiently utilized for high-speed counter-current chromatography (HSCCC) to separate multiple components with a wide range of hydrophobicity. The compositions of three-phase systems were optimized according to their physical parameters such as volume ratio, viscosity and specific gravity of upper (UP), middle (MP) and lower (LP) phases. The three-phase systems composed of n-hexane-methyl acetate-acetonitrile-water (4:4:3:4, v/v/v/v) was selected for HSCCC separation of a mixture of 15 standard compounds with a wide range in hydrophobicity from beta-carotene to tryptophan. The separation was initiated by filling the column with a mixture of MP and LP both as a stationary phase followed by elution with UP to separate the hydrophobic compounds. Then the mobile phase was switched to MP to elute the moderately hydrophobic compounds, and finally the polar compounds still retained in the column were fractionated by eluting the column with LP. The system successfully resolved all 15 compounds in one-step operation in 70 min.     

Synthesis of indole-based oxadiazoles and their interaction with bacterial peptidoglycan and SARS-CoV-2 main protease: In vitro,molecular docking and in silico ADME/Tox study

In the present study, Indole-based-oxadiazole (1A-17A) compounds were successfully synthesized. The structures of all synthesized compounds were fully characterized by different sophisticated spectroscopic techniques such 1H NMR, 13C NMR, and HREI-MS. Further, the synthesized compounds were explored to investigate their broad-spectrum antibacterial and antibiofilm potential against multidrug resistant Pseudomonas aeruginosa (MDR-PA) and methicillin resistant Staphylococcus aureus (MRSA). The compounds possessed a broad spectrum of antibacterial activity having MIC values of values 1–8 mg/ml against the tested microorganisms. Compound A6 and A7 shows maximum antibacterial activity against MDR-PA, whereas A6, A7 and A11 shows highest activity against MRSA. Furthermore, antibiofilm assay shows that A6, A7 and A11 showed maximum inhibition of biofilm formation and it was found that at 4 mg/ml; A6, A7 and A11 inhibit MRSA biofilm formation by 81.1, 77.5 and 75.9%, respectively; whereas in case of P. aeruginosa; A6 and A7 showed maximum biofilm inhibition and inhibit biofilm formation by 81.5 and 73.7%, respectively. Molecular docking study showed that compounds A6, A7, A8, A10, and A11 had high binding affinity to bacterial peptidoglycan, indicating their potential inhibitory activity against tested bacteria, whereas A6 and A11 were found to be the most effective inhibitors of SARS CoV-2 main protease (3CLpro), with a binding affinity of ? 7.78 kcal/mol. Furthermore, SwissADME and pkCSM-pharmacokinetics online tools was applied to calculate the ADME/Tox profile of the synthesized compounds and the toxicity of these chemicals was found to be low. The Lipinski, Veber, Ghose, and Consensus LogP criteria were also used to predict drug-likeness levels of the compounds. Our findings imply that the synthesized compounds could be a useful for the preventing and treating biofilm-related microbial infection as well as SARS-CoV2 infections.     

Preparative isolation and purification of phillyrin from the medicinal plant Forsythia suspensa by high-speed counter-current chromatography

Forsythia suspensa (Thunb.) Vahl. has been used widely in traditional medicines to treat gonorrhea, erysipelas, inflammation, pyrexia and ulcer. It has also shown antioxidant activity, as well as antibacterial, antiviral, choleretic and antiemetic effects. A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of the bioactive molecule phillyrin from F. suspensa (Thunb.) Vahl. The crude phillyrin was obtained by extraction with 50% ethanol from the dried fruits of F. suspensa (Thunb.) Vahl. under sonication. Preparative HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (1:9:1:9, v/v/v/v) was successfully performed, and the components purified and collected were analyzed by high-performance liquid chromatography. The method yielded 5.6 mg phillyrin at 98.6% purity from 500 mg of the crude extract (1.2% phillyrin) with the recovery of 92% in a one-step separation.     

高速逆流色谱法制备罗汉果根中葫芦素类化合物

葫芦素作为四环三萜类化合物广泛存在于葫芦科植物中,但其含量较低、结构相似,采用常规的柱层析分离法较难得到大量、高纯度的单体化合物,导致其活性的研究与应用受到限制.研究采用高速逆流色谱法(HSCCC),建立了一种从罗汉果根提取物中制备葫芦素类化合物的方法.罗汉果根乙醇提取物经HPD-100大孔树脂、MCI、RP-C18柱...     

Preparative isolation and purification of antioxidative stilbene oligomers from Vitis chunganeniss using high‐speed counter‐current chromatography in stepwise elution mode

Preparative high‐speed counter‐current chromatography (HSCCC) was successfully applied to the isolation and purification of three stilbene oligomers from Vitis chunganeniss using stepwise elution with a pair of two‐phase solvent systems composed of n‐hexane–ethyl acetate–methanol–water at (2:5:2:5, v/v) and (1:2:1:2, v/v). The preparative HSCCC separation was performed on 800 mg of crude sample yielding hopeaphenol (21.1 mg), amurensin G (37.2 mg) and vitisin A (95.6 mg) in a one‐step separation, with purities over 95% as determined by HPLC. The structures of these three compounds were identified by MS, ¹H NMR and ¹³C NMR. In addition, their antioxidant activities were screened by DPPH assay, where vitisin A showed strong antioxidant activity. Further EPR experiments with spin‐trapping technique demonstrated that vitisin A is a potent and selective singlet oxygen quencher, which may be used in singlet oxygen‐mediated diseases as a pharmacological agent.     

高速逆流色谱分离制备椭圆叶花锚中的2种口山酮苷元

椭圆叶花锚的主要活性成分为口山酮类化合物,这类化合物具有利胆、抗炎、抗菌及抗病毒活性。应用高速逆流色谱法建立了2种高纯度口山酮苷元的分离制备方法。对椭圆叶花锚氯仿萃取部位运用高速逆流色谱分离纯化,以正己烷-乙酸乙酯-甲醇-水(5:5:7:5, v/v/v/v)为两相溶剂系统,上相为固定相,下相为流动相。在主机转速800 r/min,流动相流速1.5 mL/min,检测波长254 nm条件下进行分离制备。所得产物经高效液相色谱分析检测,其化学结构由核磁共振氢谱(1H NMR)和核磁共振碳谱(13C NMR)鉴定。在此条件下,从100 mg粗样品中一步分离得到18 mg 1-羟基-2,3,5-三甲氧基口山酮,14 mg 1-羟基-2,3,4,5-四甲氧基口山酮。经高效液相色谱分析,其纯度均达98%以上。该方法简便、快速,所得产物纯度高,适合于椭圆叶花锚口山酮苷元的制备分离。     

Preparative isolation and purification of senkyunolide-I, senkyunolide-H and ferulic acid from Rhizoma Chuanxiong using counter-current chromatography

Three active compounds, senkyunolide-I, senkyunolide-H and ferulic acid (FA), were successfully isolated and purified from the extracts of Rhizoma Chuanxiong by counter-current chromatography (CCC). Based on the principle of the partition coefficient values (k) for target compounds and the separation factor (α) between target compounds, the two-phase solvent system that contains n-hexane-ethyl acetate-methanol-water at an optimized volume ratio of 3:7:4:6 v/v was selected for the CCC separation, and the lower phase was employed as the mobile phase in the head-to-tail elution mode. In a single run, 400 mg of the crude extract yielded pure senkyunolide-I (6.4 mg), senkyunolide-H (1.7 mg) and FA (4.4 mg) with the purities of 98, 93 and 99%, respectively. The CCC fractions were analyzed by high-performance liquid chromatography, and the structures of the three active compounds were identified by MS and (1)H NMR.     

Preparative isolation of diterpenoids from Salvia bowleyana Dunn roots by high-speed countercurrent chromatography combined with high-performance liquid chromatography

The root of Salvia bowleyana Dunn (Lamiaceae) is used as a traditional Chinese medicine that has multiple therapeutic effects. In this study, an efficient strategy was developed to separate diterpenoid compounds, which are the main active ingredients in Salvia bowleyana Dunn roots, from complex crude extracts by high-speed countercurrent chromatography combined with preparative high-performance liquid chromatography. A two-phase solvent system comprising n-hexane–ethyl acetate–methanol–water (7:3:7:3, v/v/v/v) was selected for high-speed countercurrent chromatographic separation. Three major diterpenoids, 6α-hydroxysugiol ( 7 ), sugiol ( 8 ), and 6, 12-dihydroxyabieta-5,8,11,13-tetraen-7-one ( 9 ) were obtained at purities of 98.9, 95.4, and 96.2%, respectively, and minor diterpenoids were enriched via one-step separation. The enriched minor diterpenoids were further purified by continuous preparative high-performance liquid chromatography to yield two new norabietanoids ( 1 , 6 ) and four known compounds ( 2 – 5 ). The structures of these new compounds were determined using NMR spectroscopy, high-resolution electrospray ionization mass spectrometry, and electronic circular dichroism spectroscopy. The results suggest that high-speed countercurrent chromatography combined with preparative high-performance liquid chromatography efficiently isolates diterpenoids, including minor components, from complex natural products.     

Isolation and purification of macrocyclic components from Penicillium fermentation broth by high‐speed counter‐current chromatography

In this paper, high‐speed counter‐current chromatography (HSCCC), assisted with ESI‐MS, was first successfully applied to the preparative separation of three macrolide antibiotics, brefeldin A (12.6 mg, 99.0%), 7′‐O‐formylbrefeldin A (6.5 mg, 95.0%) and 7′‐O‐acetylbrefeldin A (5.0 mg, 92.3%) from the crude extract of the microbe Penicillium SHZK‐15. Considering the chemical nature and partition coefficient (K) values of the three target compounds, a two‐step HSCCC isolation protocol was developed in order to obtain products with high purity. In the two‐step method, the crude ethyl acetate extract was first fractionated and resulted in two peak fractions by HSCCC using solvent system n‐hexane/ethyl acetate/methanol/water (HEMWat) (3:7:5:5 v/v/v/v), then purified using solvent systems HEMWat (3:5:3:5 v/v/v/v) and HEMWat (7:3:5:5 v/v/v/v) for each fraction. The purities and structures of the isolated compounds were determined by HPLC, X‐ray crystallography, ESI‐MS and NMR. The results demonstrated that HSCCC is a fast and efficient technique for systematic isolation of bioactive compounds from the microbes.     

High performance liquid chromatography equipped with a cathodic detector and column-switching device as a high-throughput method for a phosphatase assay with p-nitrophenyl phosphate

A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of coumarin compounds from the Chinese medicinal plant Peucedanum decursivum (Miq.) Maxim (Zihuaqianhu in Chinese) was successfully established by using light petroleum-ethyl acetate-methanol-water (5:5:7:4, v/v) as the two-phase solvent system. The upper phase of light petroleum-ethyl acetate-methanol-water (5:5:7:4, v/v) was used as the stationary phase of HSCCC. Nodakenetin (2.8 mg), 6.1 mg of Pd-C-IV, 7.3 mg of Pd-D-V, 4.7 mg of ostruthin, 7.8 mg of decursidin and 11.2 mg of decursitin C with the purity of 88.3%, 98.0%, 94.2%, 97.1%, 97.8% and 98.4%, respectively, were separated successfully in one-step separation from 150 mg of crude sample from P. decursivum (Miq.) Maxim. After purified by HSCCC again with light petroleum-ethyl acetate-methanol-water (5:5:4:5, v/v) as the two-phase solvent system, the purity of (I) can reach 99.4%. The structures of all the compounds were identified by 1H NMR and 13C NMR.     

Isolation of antioxidants from Psoralea corylifolia fruits using high-speed counter-current chromatography guided by thin layer chromatography-antioxidant autographic assay

A combinative method using high-speed counter-current chromatography (HSCCC) and thin layer chromatography (TLC) as an antioxidant autographic assay was developed to separate antioxidant components from the fruits of Psoralea corylifolia. Under the guidance of TLC bioautography, eight compounds including five flavonoids and three coumarins were successfully separated from the fruits of P. corylifolia by HSCCC with an optimized two-phase solvent system, n-hexane–ethyl acetate–methanol–water (1:1.1:1.3:1, v/v/v/v). The separation produced 5.91 mg psoralen, 6.26 mg isopsoralen, 3.19 mg psoralidin, 0.92 mg corylifol A, and 2.43 mg bavachinin with corresponding purities of 99.5, 99.8, 99.4, 96.4, and 99.0%, as well as three sub-fractions, in a single run from 250 mg ethyl acetate fraction of P. corylifolia extract. Following an additional clean-up step by preparative TLC, 0.4 mg 8-prenyldaidzein (purity 91.7%), 4.18 mg neobavaisoflavone (purity 97.4%) and 4.36 mg isobavachalcone (purity 96.8%) were separated from the three individual sub-fractions. The structures of the isolated compounds were identified by ¹H NMR and ¹³C NMR. The results of antioxidant activity estimation by electron spin resonance (ESR) method showed that psoralidin was the most active antioxidant with an IC₅₀ value of 44.7 μM. This is the first report on simultaneous separation of eight compounds from P. corylifolia by HSCCC.     

Extraction and preparation of 5-lipoxygenase and acetylcholinesterase inhibitors from Astragalus membranaceus stems and leaves

In this study, an efficient method that employs 5-lipoxygenase and acetylcholinesterase as biological target molecules in receptor–ligand affinity ultrafiltration–liquid chromatography was developed for the screening of enzyme inhibitors derived from the Astragalus membranaceus stems and leaves. The effects of the extraction time, number of extraction cycles, ethanol concentration, and liquid–solid ratio on the total yield of the target compounds were investigated using response surface methodology, and the bioactive components were isolated using a combination of semi-preparative high-performance liquid chromatography and high-speed countercurrent chromatography via a two-phase solvent system consisting of n-hexane–ethyl acetate–methanol–water (1:6:2:6, v/v/v/v). Subsequently, 10 naturally-occurring bioactive components in the Astragalus membranaceus stems and leaves, including wogonin, ononin, isoquercitrin, calycosin-7-glucoside, 3-hydroxy-9,10-dimethoxyptercarpan, hyperoside, 7,2′-dihydroxy-3′,4′-dimethoxyisoflavan, baicalein, calycosin, and soyasaponin, were screened using affinity ultrafiltration to determine their potential effects against Alzheimer's disease. Consequently, all target compounds had purities higher than 95.0%, and the potential anti-Alzheimer's disease effect of the obtained bioactive compounds was verified using molecular docking analysis. Based on the results, the back-to-back screening of complex enzyme inhibitors and separation of the target bioactive compounds using complex chromatography could provide a new approach to the discovery and preparation of natural active ingredients.     

Isolation of three sesquiterpene lactones from the roots of Cichorium glandulosum Boiss. et Huet. by high-speed counter-current chromatography

Because of the skeletal complexity and similarity of the polarity, little research was reported on the isolation of sesquiterpene lactones by high-speed counter-current chromatography (HSCCC). Herein, three sesquiterpene lactones were successfully purified from the ethyl acetate extract of the roots of the traditional Uyghur medicinal plant Cichorium glandulosum Boiss. et Huet. by HSCCC. The separation was performed in two steps with two solvent systems: n-hexane-ethyl acetate-methanol-water (1.5:5:2.75:5, v/v/v/v) and ethyl acetate-methanol-water (20:1:20, v/v/v). From 166 mg of the ethyl acetate extract, 19 mg of lactucopicrin was isolated with the first solvent system and 10 mg of 11beta,13-dihydrolactucin and 16 mg of lactucin were obtained with the second solvent system. All purified compounds were over 94% purity as determined by HPLC analysis, and these chemical structures were confirmed by (1)H NMR and (13)C NMR.     

Large-scale preparation of five polar polyphenols including three isomers from Phyllanthus emblica Linn. by preparative high-speed counter-current chromatography

The separation of polar compounds is challenging work due to poor retention and insufficient selectivity. In the present study, an efficient strategy for large-scale preparation of five polar polyphenols including three isomers from Phyllanthus emblica Linn has been established by preparative high-speed counter-current chromatography. Macroporous resin column chromatography was used for the enrichment of the polar polyphenols. However, sugar and other ultra-polar impurities were co-washed out with the targets. Liquid-liquid extraction with ethyl acetate/water (1/1, v/v) solvent system was developed to remove the ultra-polar impurities with a clearance rate of 95%. Finally, the targets were introduced to preparative high-speed counter-current chromatography for separation using ethyl acetate/n-butanol/acetic acid/water (2/7/1/10, v/v/v/v) solvent system. As a result, 191 mg of Mucic acid 1,4-lactone 5-O-gallate, 370 mg of β-Glucogallin, 301 mg of Gallic acid, 195 mg of Mucic acid 1,4-lactone 3-O-gallate and 176 mg of Mucic acid 1,4-lactone 2-O-gallate with purity higher than 98% were obtained from 1.5 g of sample. Mucic acid 1,4-lactone 3-O-gallate, Mucic acid 1,4-lactone 3-O-gallate, and Mucic acid 1,4-lactone 2-O-gallate are isomers. The results showed that high-speed counter-current chromatography could be well developed for the separation of polar compounds from natural products.     

Target-guided separation of Bougainvillea glabra betacyanins by direct coupling of preparative ion-pair high-speed countercurrent chromatography and electrospray ionization mass-spectrometry

In this study, preparative ion-pair high-speed countercurrent chromatography was directly coupled to an electrospray ionization mass-spectrometry device (IP-HSCCC/ESI-MS-MS) for target-guided fractionation of high molecular weight acyl-oligosaccharide linked betacyanins from purple bracts of Bougainvillea glabra (Nyctaginaceae). The direct identification of six principal acyl-oligosaccharide linked betacyanins in the mass range between m/z 859 and m/z 1359 was achieved by positive ESI-MS ionization and gave access to the genuine pigment profile already during the proceeding of the preparative separation. Inclusively, all MS/MS-fragmentation data were provided during the chromatographic run for a complete analysis of substitution pattern. On-line purity evaluation of the recovered fractions is of high value in target-guided screening procedures and for immediate decisions about suitable fractions used for further structural analysis. The applied preparative hyphenation was shown to be a versatile screening method for on-line monitoring of countercurrent chromatographic separations of polar crude pigment extracts and also traced some minor concentrated compounds. For the separation of 760 mg crude pigment extract the biphasic solvent system tert.-butylmethylether/n-butanol/acetonitrile/water 2:2:1:5 (v/v/v/v) was used with addition of ion-pair forming reagent trifluoroacetic acid. The preparative HSCCC-eluate had to be modified by post-column addition of a make-up solvent stream containing formic acid to reduce ion-suppression caused by trifluoroacetic acid and later significantly maximized response of ESI-MS/MS detection of target substances. A variable low-pressure split-unit guided a micro-eluate to the ESI-MS-interface for sensitive and direct on-line detection, and the major volume of the effluent stream was directed to the fraction collector for preparative sample recovery. The applied make-up solvent mixture significantly improved smoothness of the continuously measured IP-HSCCC-ESI-MS base peak ion trace in the experimental range of m/z 50–2200 by masking stationary phase bleeding and generating a stable single solvent phase for ESI-MS/MS detection. Immediate structural data were retrieved throughout the countercurrent chromatography run containing complete MS/MS-fragmentation pattern of the separated acyl-substituted betanidin oligoglycosides. Single ion monitoring indicated clearly the base-line separation of higher concentrated acylated betacyanin components.