Separation of bisbenzylisoquinoline alkaloids by micellar electrokinetic chromatography

The micellar electrokinetic chromatographic (MEKC) separation of seven bisbenzylisoquinoline alkaloids has been developed. The effects of various separating factors were studied. Optimum separation was achieved using a buffer (pH 9.2) of 20 mM sodium borate and 20 mM sodium dihydrogen phosphate buffer containing 55 mM sodium cholate; the optimum voltage and injection time were 21 kV and 0.05 min, respectively. Highest peak efficiency was obtained when the analytes were dissolved in 10 mM sodium dodecyl sulphate as sample matrix for injection. The elution order of the bisbenzylisoquinoline alkaloids was related to their lipophilicity. The resolution, run time and detection limits of the MEKC method were compared with those of an HPLC method developed previously.     

Determination of active components in rhubarb and study of their hydrophobicity by micellar electrokinetic chromatography

A micellar electrokinetic chromatographic (MEKC) method has been developed for the determination of five anthraquinones and one distyrene derivative in rhubarb. The separation conditions were optimized and two kinds of rhubarb plants and rhubarb-containing medicines were analyzed. The negatively charged solutes migrated toward the anode and were retarded by their interaction with the micelle. Hydrophobicity of the solutes was studied by both MEKC with SDS and SDS-free capillary zone electrophoresis in the buffer of 15 mmol/L NaH(2)PO(4)+ 20 mmol/L borax and 15% ethanol (v/v). Linear correlation between log k' and log P(OW) was obtained for the five anthraquinones in SDS micelle system. The capacity factor, k', and free energy differences delta(deltaG) derived from this method provided fundamental information on the interaction between the solutes and the micelle.     

The effect of tyrosine conjugation on the critical micellar concentration of free and glycine-conjugated bile salts

We investigated the effect of conjugation with the aromatic amino acid tyrosine on the critical micellar concentration (CMC) of bile salts. The CMC values were determined by surface tension and by dye solubilization. The surface tension measurement employed the Du Nouy ring detachment method and the dye solubilization measurement utilized a water-insoluble dye, 1-O-tolylazo-2-naphthol. We compared the CMC values of the sodium salts of cholyltyrosine (cholylTyr), deoxycholyltyrosine (deoxycholylTyr), deoxycholylglycyltyrosine (deoxycholylGlyTyr) chenodeoxycholyltyrosine (chenodeoxycholylTyr), chenodeoxycholylglycyltyrosine (chenodeoxycholylGlyTyr), cholyldiglycyltyrosine (cholylGlyGlyTyr) and cholylglycyltyrosine (cholylGlyTyr) with their respective glycine conjugated bile salts. Both techniques of CMC determination indicated that tyrosine conjugation to free and glycine-conjugated bile salts reduced their CMC significantly.     

Spectrophotometric determination of the critical micellar concentration of bile salts using bilirubin monoglucuronide as a micellar probe. Utility of derivative spectroscopy.

We have developed a simple biologically non-invasive method for determining the critical micellar concentration (CMC) of bile salts using pure naturally occurring bilirubin IX alpha monoglucuronide (BMG), an important bile pigment present in virtually all mammalian biles. This methodology employs visible absorbance spectroscopy of BMG in bile salts over a range of bile salt concentrations that include the reported CMC. Using 100 microM-BMG in 0.4 M-imidazole buffer at pH 7.8, we calculated that the CMC for sodium taurochenodeoxycholate is between 2.5 and 3.0 mM based on: (1) an abrupt change in lambda max. in this concentration range, (2) a precipitous decrease in the amplitude of the absorbance shoulder at 450 nm, (3) a sudden decrease in the second derivative absorbance of BMG at 400 nm and an increase in absorbance at 470 nm, (4) a sharp change in the 4th derivative absorbance at 375 and 395 nm. In contrast, sodium taurocholate, a bile salt that reportedly does not have a CMC but continuously self-associates over a wide concentration range, exhibited none of these changes. The use of derivative spectroscopy enhances the ability to detect the CMC changes and also indicates the number of BMG species in solution and their relative energy states.     

Analysis of synthetic derivatives of peptide hormones by capillary zone electrophoresis and micellar electrokinetic chromatography with ultraviolet-absorption and laser-induced fluorescence detection

Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were used for the analysis of new synthetic derivatives of hypophysis neurohormones--vasopressin and oxytocin, and pancreatic hormone--human insulin (HI) and its octapeptide fragment, derivatized by fluorescent probe, 4-chloro-7-nitrobenzo[1,2,5]oxadiazol (NBD). The suitable composition of background electrolytes (BGEs) was selected on the basis of calculated pH dependence of effective charge of analyzed peptides. Basic ionogenic peptides were analyzed by CZE in the acidic BGE composed of 100 mM H3PO4, 50 mM Tris, pH 2.25. The ionogenic peptides with fluorescent label, NBD, were analyzed in 0.5 M acetic acid, pH 2.5. The best MEKC separation of non-ionogenic peptides was achieved in alkaline BGE, 20 mM Tris, 5 mM H3PO4, with micellar pseudophase formed by 50 mM sodium dodecylsulfate (SDS), pH 8.8. Selected characteristics (noise, detectability of substance, sensitivity of detector) of the UV-absorption detectors (single wavelength detector, multiple-wavelength photodiode array detector (PDA), both of them operating at constant wavelength 206 nm) and laser-induced fluorescence (LIF) detector (excitation/emission wavelength 488/520 nm) were determined. The detectability of peptides in the single wavelength detector was 1.3-6.0 micromol dm(-3) and in the PDA detector 1.6-3.1 micromol dm(-3). The LIF detection was more sensitive, the applied concentration of NBD derivative of insulin fragment in CZE analysis with LIF detection was three orders lower than in CZE with UV-absorption detector, and the detectability of this peptide was improved to 15.8 nmol dm(-3).     

Evaluation of the efficiency of extraction of ultraviolet-absorbing pollen allergens and organic pollutants from airborne dust samples by capillary electromigration methods

Capillary electromigration methods, zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC), have been used for evaluation of the efficiency of different extraction agents applied to the extraction of pollen allergens and organic pollutants from dust samples collected during different periods (before, during and after pollen seasons) and in different locations in air-filtration devices (car-traffic tunnel in Prague and a metro station in Paris). Water and acetic acid extracts were analyzed by CZE using acetic acid as background electrolyte (BGE). Water and alkaline water-SDS-buffer extracts were analyzed by MEKC in Tris-phosphate BGE with anionic detergent sodium dodecylsulfate (SDS) micellar pseudophase. More material was extracted and more components were found in the water-buffer extracts than in the water extracts, and better resolution of the components was achieved by MEKC than by CZE. Significant differences have been found in the analyses of dust extracts of different origin. More material and more components have been found in the extracts of the dust collected in the pollen-rich period (March, April) than in the pollen-free period (December, January).     

An Edge Selection Mechanism for Chirally Selective Solubilization of Binaphthyl Atropisomeric Guests by Cholate and Deoxycholate Micelles

Combining micellar electrokinetic capillary chromatography (MEKC) and nuclear magnetic resonance (NMR) experimentation, we shed light on the structural basis for the chirally selective solubilization of atropisomeric binaphthyl compounds by bile salt micelles comprised of cholate (NaC) or deoxycholate (NaDC). The model binaphthyl analyte R,S‐BNDHP exhibits chirally selective interactions with primary micellar aggregates of cholate and deoxycholate, as does the closely related analyte binaphthol (R,S‐BN). Chiral selectivity was localized, by NMR chemical shift analysis, to the proton at the C12 position of these bile acids. Correspondingly, MEKC results show that the 12α‐OH group of either NaC or NaDC is necessary for chirally selective resolution of these model binaphthyl analytes by bile micelles, and the S isomer is more highly retained by the micelles. With NMR, the chemical shift of 12β‐H was perturbed more strongly in the presence of S‐BNDHP than R‐BNDHP. Intermolecular NOEs demonstrate that R,S‐BNDHP and R,S‐BN interact with a similar hydrophobic planar pocket lined with the methyl groups of the bile salts, and are best explained by the existence of an antiparallel dimeric unit of bile salts. Finally, chemical shift data and intermolecular NOEs support different interactions of the enantiomers with the edges of dimeric bile units, indicating that R,S‐BNDHP enantiomers sample the same binding site preferentially from opposite edges of the dimeric bile unit. Chirality 28:525–533, 2016. © 2016 Wiley Periodicals, Inc.     

Determination of diterpenoid triepoxides in Tripterygium wilfordii by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatographic (MEKC) method has been established for the identification and determination of diterpenoid triepoxides in the Chinese herb Tripterygium wilfordii Hook F. and its preparations. Studies of the influence of boric acid and borax buffer concentration and pH, and of sodium dodecylsulphate (SDS) concentration have been carried out, and the optimum separation for the triepoxides was achieved using 20 mM boric acid and 10 mM borax with 20 mM SDS as the running buffer. MEKC was found to exhibit good accuracy, precision and repeatability. The sensitivity of the assay was sufficient to monitor the three active components in T. wilfordii and its preparations.     

Mono-N-acyl-2,6-diaminopimelic acid derivatives: Analysis by electromigration and spectroscopic methods and examination of enzyme inhibitory activity

Thirteen mono-N-acyl derivatives of 2,6-diaminopimelic acid (DAP)—new potential inhibitors of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18)—were analyzed and characterized by infrared (IR) and nuclear magnetic resonance (NMR) spectroscopies and two capillary electromigration methods: capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Structural features of DAP derivatives were characterized by IR and NMR spectroscopies, whereas CZE and MEKC were applied to evaluate their purity and to investigate their electromigration properties. Effective electrophoretic mobilities of these compounds were determined by CZE in acidic and alkaline background electrolytes (BGEs) and by MEKC in acidic and alkaline BGEs containing a pseudostationary phase of anionic detergent sodium dodecyl sulfate (SDS) or cationic detergent cetyltrimethylammonium bromide (CTAB). The best separation of DAP derivatives, including diastereomers of some of them, was achieved by MEKC in an acidic BGE (500 mM acetic acid [pH 2.54] and 60 mM SDS). All DAP derivatives were examined for their ability to inhibit catalytic activity of DapE from Haemophilus influenzae (HiDapE) and ArgE from Escherichia coli (EcArgE). None of these DAP derivatives worked as an effective inhibitor of HiDapE, but one derivative—N-fumaryl, Me-ester-DAP—was found to be a moderate inhibitor of EcArgE, thereby providing a promising lead structure for further studies on ArgE inhibitors.     

Micellar electrokinetic chromatography-electrospray ionization mass spectrometry for the identification of drug impurities

Previously, we have presented a system hyphenating continuous micellar electrokinetic chromatography (MEKC) with electrospray ionization mass spectrometry (ESI-MS). Here we evaluate this technique for its applicability in impurity profiling of drugs using galantamine and ipratropium as test samples. A background electrolyte (BGE) of 10mM sodium phosphate (pH 7.5), 12.5-15% acetonitrile and 20mM sodium dodecylsulfate (SDS) was used for the MEKC-MS analysis of a galantamine sample containing a number of related impurities, and a heat-treated solution of ipratropium containing a number of unknown degradation products. MEKC provided efficient separation of all sample constituents. Despite the presence of non-volatile BGEs, all impurities in the galantamine sample could be detected by ESI-MS in their respective extracted ion traces (XICs) with a detection sensitivity in the sub-microg/ml range (full-scan mode). MS/MS detection provided useful product spectra allowing the structural characterization of the respective galantamine impurities. With the MEKC-MS/MS system, two degradation products could be revealed and identified in the heat-stressed ipratropium sample. The presented method shows good potential for the detection and structure elucidation of minor impurities in drug substances.     

Stability of mixed micellar bile models supersaturated with cholesterol.

The maximal equilibrium solubility of cholesterol in mixtures of phosphatidylcholine (PC)1 and bile salts depends on the cholesterol/PC ratio (Rc) and on the effective ratio (Re) between nonmonomeric bile salts and the sum (CT) of PC and cholesterol concentrations (Carey and Small, 1978; Lichtenberg et al., 1984). By contrast, the concentration of bile salts required for solubilization of liposomes made of PC and cholesterol does not depend on Rc (Lichtenberg et al., 1984 and 1988). Thus, for Rc greater than 0.4, solubilization of the PC-cholesterol liposomes yields PC-cholesterol-bile salts mixed micellar systems which are supersaturated with cholesterol. In these metastable systems, the mixed micelles spontaneously undergo partial revesiculation followed by crystallization of cholesterol. The rate of the latter processes depends upon Rc, Re, and CT. For any given Rc and Re, the rate of revesiculation increases dramatically with increasing the lipid concentration CT, reflecting the involvement of many mixed micelles in the formation of each vesicle. The rate also increases, for any given CT and Re, upon increasing the cholesterol to PC ratio, Rc, probably due to the increasing degree of supersaturation. Increasing the cholate to lipid effective ratio, Re, by elevation of cholate concentration at constant Rc and CT has a complex effect on the rate of the revesiculation process. As expected, cholate concentration higher than that required for complete solubilization at equilibrium yields stable mixed micellar systems which do not undergo revesiculation, but for lower cholate concentrations decreasing the degree of supersaturation (by increasing [cholate]) results in faster revesiculation. We interpret these results in terms of the structure of the mixed micelles; micelles with two or more PC molecules per one molecule of cholesterol are relatively stable but increasing the bile salt concentration may cause dissociation of such 1:2 cholesterol:PC complexes, hence reducing the stability of the mixed micellar dispersions. The instability of PC-cholesterol-cholate mixed systems with intermediary range of cholate to lipids ratio may be significant to gallbladder stone formation as: (a) biliary bile contains PC-cholesterol vesicles which may be, at least partially, solubilized by bile salts during the process of bile concentration in the gallbladder, resulting in mixtures similar to our model systems; and (b) the bile composition of cholesterol gallstone patients is within an intermediary range of bile salts to lipids ratio.     

Effect of free and conjugated bile salts on alpha-amylase activity.

The effect of individual bile salts on alpha-amylase hydrolysis of Cibachron Blue starch was studied at pH 6.0. With sodium cholate, taurocholate and taurodeoxycholate, enzyme activity was increased to 150-160 percent of the control value, at a concentration of similar to 1 mmol/l bile salt. The increased activity extended up to 4 mmol/l. The bile salts sodium deoxycholate and taurochenodeoxycholate exerted activation and inhibition depending on the concentration. With deoxycholate (0.75 mmol/l), activation (150 percent) was evident, while inhibition was apparent above 2.5 mmol/l. With taurochenodeoxycholate maximum activity (135 percent) was observed at 0.25 mmol/l, while inhibition was evident above 1.5 mmol/l. Chenodeoxycholate and lithocholate exerted marked inhibition at concentrations as low as 0.5 mmol/l. Inhibition of alpha-amylase by chenodeoxycholate was competitive with both soluble and insoluble starch substrates. Since the pH of the jejunum is in the region of 6.0 the phenomenon of activation and inhibition of alpha-amylase by bile salts at this pH could be of physiological significance.     

Development and Validation of a Micellar Electrokinetic Chromatography Method for Quantitative Determination of Butenolides in Piper malacophyllum (C. Presl) C. DC.

Introduction – A large number of natural and synthetic compounds having butenolides as a core unit have been described and many of them display a wide range of biological activities. Butenolides from P. malacophyllum have presented potential antifungal activities but no specific, fast, and precise method has been developed for their determination. Objective – To develop a methodology based on micellar electrokinetic chromatography to determine butenolides in Piper species. Methodology – The extracts were analysed in an uncoated fused‐silica capillaries and for the micellar system 20 mmol/L SDS, 20% (v/v) acetonitrile (ACN) and 10 mmol/L STB aqueous buffer at pH 9.2 were used. The method was validated for precision, linearity, limit of detection (LOD) and limit of quantitation (LOQ) and the standard deviations were determined from the standard errors estimated by the regression line. Results – A micellar electrokinetic chromatography (MEKC) method for determination of butenolides in extracts gave full resolution for 1 and 2 . The analytical curve in the range 10.0–50.0 µg/mL (r² = 0.999) provided LOD and LOQ for 1 and 2 of 2.1/6.3 and 1.1/3.5 µg/mL, respectively. The RSD for migration times were 0.12 and 1.0% for peak area ratios with 100.0 ± 1.4% of recovery. Conclusions – A novel high‐performance MEKC method developed for the analysis of butenolides 1 and 2 in leaf extracts of P. malacophyllum allowed their quantitative determined within an analysis time shorter than 5 min and the results indicated CE to be a feasible analytical technique for the quantitative determination of butenolides in Piper extracts. Copyright © 2010 John Wiley & Sons, Ltd.     

Head group-independent interaction of phospholipids with bile salts. A fluorescence and EPR study

Bile salts are essential for phospholipid secretion into the bile. To study the relevance of the structure of phospholipids for their interaction with bile salts, we used spin-labeled or fluorescent phospholipid analogues of different head groups and acyl chain length. Those analogues form micelles in aqueous suspension. Their solubilization by bile salts resulting in the formation of mixed micelles was followed by the decrease of spin-spin interaction of spin-labeled analogues or by the relief of fluorescence self-quenching of (7-nitro-2-1,3-benzooxadiazol (NBD))-labeled analogues. Solubilization of analogue micelles occurred at and above the critical micellar concentration (CMC) of the bile salts. As revealed by stopped-flow technique, solubilization of NBD-analogues was very rapid with half times as low as 0.1 sec above the CMC of taurocholate. Both kinetics and extent of solubilization were independent of the phospholipid head group, but were significantly affected by the fatty acid chain length. Furthermore, using vesicles with varying phospholipid composition and different types of analogues in self-quenching concentrations, we could show that bile salt-mediated vesicle solubilization depended on the fatty acid chain length of phospholipids. In contrast, neither for phospholipids nor for analogues could an influence of the lipid head group on the solubilization process be observed. These findings support a head group-independent mechanism of bile salt-mediated enrichment of specific phospholipids in the bile fluid.     

Neutral amino acids monitoring in phenylketonuric plasma microdialysates using micellar electrokinetic chromatography and laser-induced fluorescence detection

Neutral and non-polar amino acids such as phenylalanine (Phe), valine (Val), tyrosine (Tyr), threonine (Thre) and GABA are hard to resolve by capillary zone electrophoresis (CZE). Their separation is possible by adding a surfactant to the mobile phase. This method is called micellar electrokinetic chromatography (MEKC). We used MEKC with laser-induced fluorescence detection (LIFD) to separate and quantitate these amino acids in plasma microdialysates of patients with phenylketonuria (PKU). This disease is an inborn enzymatic defect with decreased conversion of Phe to Tyr that causes severe neurological damage and mental deterioration, which is diagnosed by measuring plasma Phe and Phe/Tyr ratio. The amino acids tested had linear concentration–signal relation. PKU patients had significantly higher Phe, lower Tyr, 21 times higher Phe/Tyr ratio and decreased values of Val and Thre than controls. These results show that microdialysis of biological fluids coupled with MEKC–LIFD is a convenient technique to measure neutral amino acids in clinical disorders such as PKU.     

Quantitative relationships between structure and antimicrobial activity of new “Soft” bisquaternary ammonium salts

New surface-active bisquaternary ammonium salts derived from bis-(2-dimethylaminoethyl) ester of glutaric acid are highly effective against representatives of Gram-positive, Gram-negative bacteria and yeasts. Relationships between structure, lipophilicity and antimicrobial effectiveness were demonstrated by quantitative structure-activity methodology. The nonlinear dependence of biological activity on the structure as well as lipophilicity (expressed as critical micelle concentration—CMC) was shown using Kubinyi’s bilinear model. The most effective compounds were those with the alkyl chain of 11–12 carbon atoms and with the CMC values around 0.7−1.0 mmol/L. These derivatives possessed higher antimicrobial activity particularly to Gram-negative bacteria. Part 45 of the seriesQuaternary Ammonium Salts; part 44: Brádlerová A.et al., Pharmazie, in press.     

Surface behaviour of bile salts and tetrahydrolipstatin at air/water and oil/water interfaces.

The surface behaviour of two bile salts, sodium deoxycholate (NaDC) and sodium taurodeoxycholate (NaTDC), as well as that of tetrahydrolipstatin (THL), a potent gastrointestinal lipase inhibitor, was studied at air/water and oil/water interfaces, using interfacial tensiometry methods. The surface behaviour of NaDC and NaTDC was comparable at both oil/water and air/water interfaces. A fairly compact interfacial monolayer of bile salts is formed well below the critical micellar concentration (CMC) and can help to explain the well-known effects of bile salts on the kinetic behaviour of pancreatic lipases. Using the Wilhelmy plate technique, the surface pressure-molecular area curves recorded with THL at the air/water interface showed a collapse point at a surface pressure of 24.5 mN.m(-1), corresponding to a molecular area of 70 A(2). Surprisingly, using the oil drop method, a limiting molecular area of 160 A(2) was found to exist at the oil/water interface. On the basis of the above data, space-filling models were proposed for bile salts and THL at air/water and oil/water interfaces.     

Quantification of meropenem in plasma and cerebrospinal fluid by micellar electrokinetic capillary chromatography and application in bacterial meningitis patients

A high-performance micellar electrokinetic capillary chromatography (MEKC) has been demonstrated for the determination of meropenem in human plasma and in cerebrospinal fluid (CSF) and application in meningitis patients after intravenous (IV) administration. Plasma sample was pretreated by means of solid-phase extraction (SPE) on C(18) cartridge and CSF sample was by direct injection without sample pretreatment, with subsequent quantitation by MEKC. The separation of meropenem was carried out in an untreated fused-silica capillary (40.2 cm x 50 microm I.D., effective length 30 cm) and was performed at 25 degrees C using a background electrolyte consisting of Tris buffer (40 mM, pH 8.0) solution with sodium dodecyl sulfate (SDS) as the running buffer and on-column detection at 300 nm. Several parameters affecting the separation and sensitivity of the drug were studied, including pH, the concentrations of Tris buffer and surfactant. Using cefotaxime as an internal standard (IS), the linear ranges of the method for the determination of meropenem in plasma and in CSF were all over 0.5-50 microg/mL; the detection limits (signal-to-noise ratio=3) of meropenem in plasma and in CSF were 0.2 microg/mL and 0.3 microg/mL, respectively.     

胶束动电毛细管色谱法测定功能运动饮料中的水溶性维生素

目前,在功能运动饮料中测定水溶性维生素含量的研究相对较少,为此,本研究开发了一种通过胶束动电毛细管色谱法(MEKC)测定几种能量饮料和运动饮料中水溶性维生素的方法,并根据背景电解质组成(硼酸盐含量, p H值,表面活性剂类型等)和其他MEKC参数研究维生素的分离,以及对可能干扰维生素测定的干扰化合物进行了研究。研究显示,背景电解质为pH值8.5,50 mmol/L硼酸盐,50 mmol/L SDS和5%MeOH时,可有效地检测出能量和运动饮料中的水溶性维生素含量。该方法可成功地检测各种能量和运动饮料中的水溶性维生素,并可用于软饮料行业的质量控制。     

Inhibition of human pancreatic lipase by tetrahydrolipstatin: Further kinetic studies showing its reversibility

Tetrahydrolipstatin (THL, Orlistat) is a potent inhibitor of gastrointestinal lipases. Using the pH-stat technique we report that, in the absence of substrate, THL (at a molar excess of 100) inhibits rapidly (after few minutes of incubation) human pancreatic lipase (HPL). Bile salts over their critical micellar concentration (CMC) were found to accelerate the inhibition process.At variance with the generally accepted model of a covalent and quasi-irreversible acyl-lipase complex, we showed here that the inhibition of HPL could be rapidly and partially reversed in the presence of an emulsion of short- or long-chain triacylglycerols, as indicated by a kinetic reactivation process. The presence of bile salts in the incubation medium, containing THL and HPL, was found to stabilise the covalent complex as reflected by a decrease in the reactivation rate. Paradoxically, the presence of bile salts in the lipase assay enhanced this reactivation process probably by forming mixed micelles between bile salts and THL, which accelerates the deacylation phenomenon.On the basis of this kinetic study, a general model is proposed to describe the inhibition of lipases by THL in the aqueous phase as well as its partial reactivation process at the lipid–water interface.