Separation and purification of sphingomyelin diastereomers by high-performance liquid chromatography

All naturally occurring sphingomyelins have the d-erythro-(2S,3R) configuration of the sphingoid base. We have developed a normal-phase HPLC method for the separation of this natural stereoisomer from the l-threo-sphingomyelin, which is the other stereoisomer commonly present in semisynthetic preparations of acyl-chain defined sphingomyelins. The chromatographic method was developed by modification of a previously reported method for phospholipid separation on a normal-phase diol column. The separation was accomplished by a binary gradient of solvent mixtures (A) hexane:isopropanol:acetic acid (82:17:1.0 by vol) and (B) isopropanol:water:acetic acid (85:14:1.0 by vol) with 0.08 vol% triethylamine added to both solvent mixtures. The program of gradient elution was optimized for maximal separation of sphingomyelin diastereomers. For detection of the lipids, a light-scattering detector was used. This analytical scale HPLC method was also used for purification of the stereoisomers (up to 0.5 mg of N-oleoyl-sphingomyelin in a single injection). The purified stereoisomers were at least 99% pure according to high-performance thin-layer chromatography and analytical HPLC.     

Preparative separation of four isomers of synthetic anisodamine by HPLC and diastereomer crystallization

Anisodamine (654‐1), a well‐known cholinergic antagonist, is marketed as synthetic anisodamine (mixture of four isomers, 654‐2) in China. To preparative resolution and comparison of the bioactivities of the four isomers of synthetic anisodamine, current work explores an economic and effective separation method by using preparative high performance liquid chromatography (HPLC) and diastereomer crystallization. Their absolute configurations were established by single‐crystal X‐ray diffraction and circular dichroism method. The purities of each isomer were more than 95%. Among them, 654‐2‐A2 (6R, 2′S configuration) exhibited better effect on cabachol preconditioned small intestine tension more than 654‐2 and other isomers. The direct separation method without using HPLC was tried as well, which was still on progress. This is the first report of the method for preparative separation of four isomers of synthetic anisodamine which could be used for large‐scale production in industry.     

Enantioseparation of citalopram enantiomers by capillary electrophoresis: Method development through experimental design and computational modeling

Citalopram (CIT) is a frequently used modern antidepressant that inhibits selectively serotonin reuptake in the brain. It has a chiral center in its structure and is used in therapy as both racemic mixture and pure enantiomer as its pharmacological effect is almost entirely associated with S-CIT. The aim of this study was the development of a simple and rapid capillary electrophoresis (CE) method for the separation and quantification of CIT enantiomers. To establish the optimum chiral selector, several native and derivatized, neutral, and ionized cyclodextrins (CDs) were examined at different pH levels. An experimental design strategy was adopted for method optimization; a fractional factorial design was applied for screening purposes to identify significant experimental factors followed by a face-centered central composite design used for optimization purposes. Computational modeling was used to obtain information on the interaction energy and the geometry of the complexes to aid in the understanding of chiral separation mechanism. The best results were obtained when using a 25-mM phosphate buffer at pH 7.0, 3-mM CM-β-CD as chiral selector, 17.5°C temperature, 15-kV voltage, and 50 mbar/s hydrodynamic injection. The separation time was fast, below 3 min, and the migration order was S-CIT followed by R-CIT. The analytical performance of the method was verified in terms of precision, linearity, accuracy, sensibility, and robustness, and the method was applied for the determination of CIT enantiomers from pharmaceutical preparations.     

Separation and quantitative analysis of alkyl sulfobetaine-type detergents by high-performance liquid chromatography and light-scattering detection

An improved high-performance liquid chromatographic (HPLC) method for the separation of zwitterionic detergents is described. It is based on a reversed-phase liquid chromatography with evaporative light-scattering detection (ELSD). The method was shown to be highly specific, allowing the separation of three detergents of the alkyl sulfobetaine family: 3-(N-dodecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate (SB12), 3-(N-tetradecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate (SB14) and 3-(N-hexadecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate (SB16). It was further used to develop a quantitation method for SB14, which was validated for linearity, precision, robustness, limits of detection and quantitation, specificity and accuracy. Linearity was found in the range of 50-500 microg/ml with a correlation coefficient of 0.9938+/-0.0029. The mean value of slope and intercept were 1.567+/-0.06 and 0.1541+/-0.0271, respectively. The limits of detection (LOD) and quantitation (LOQ) were 2 and 10 microg/ml, respectively. The validated method was used to determine the concentration of SB14 in different biological samples, specially in bulks of a recombinant membrane protein, the Klebsiella pneumoniae outer membrane protein A, which is produced at the pilot scale for human clinical studies.     

Analysis of oligosaccharides by on-line high-performance liquid chromatography and ion-spray mass spectrometry.

Oligosaccharides were analyzed by a combination of high-performance liquid chromatography (HPLC) and mass spectrometry (MS). First, oligosaccharides labeled with 2-aminopyridine were studied to see if they could be analyzed by MS under the conditions used for separation by HPLC. Pyridylamino (PA)-oligosaccharides could be analyzed under these conditions, although the mass spectra were affected. Then, liquid chromatography-mass spectrometry was used to analyze a PA-oligosaccharide mixture derived from human immunoglobulin G. The PA-oligosaccharides were separated on a reversed-phase column and mass-analyzed directly. The observed molecular weights were close to or identical to those expected from the structures, which were estimated from the elution position on HPLC. This method is rapid and simple, as the mass spectrometer can give the accurate molecular weight of each PA-oligosaccharide in one chromatography run, even if the HPLC separation is incomplete. This method can be used to extend the so-called two-dimensional mapping of PA-oligosaccharides. The structure can be studied in greater detail by tandem MS.     

Chiral separation of salbutamol and bupivacaine by capillary electrophoresis using dual neutral cyclodextrins as selectors and its application to pharmaceutical preparations and rat blood samples assay

An attempt for the simultaneous separation of salbutamol (Sal) and bupivacaine (Bup) enantiomers was performed by capillary elecytrophoresis with a dual mixture of neutral cyclodextrins as chiral selector. The influence on the separation of several parameters such as buffer composition, pH, the concentration ratio of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) to dimethyl-beta-cyclodextrin (DM-beta-CD) was investigated. A better separation was obtained for Sal and Bup with the CD mixtures compared to the use of HP-beta-CD or DM-beta-CD alone. The best simultaneous separation of Sal and Bup enantiomers was achieved with a 20 mM HP-beta-CD and 20 mM DM-beta-CD at pH 2.5 in a triethanolamine (TEA)/phosphate buffer. This method-utilized chlorphenamine (Chl) as an internal standard was found to be linear in the range 0.5-100 microg/mL and 0.5-150 microg/mL for Sal and Bup enantiomers, respectively. The limits of detection for both enantiomers of Sal and Bup were 0.18 and 0.24 microg/mL, respectively. The proposed method was applied to monitor Sal and Bup enantiomers concentration change in rat blood samples obtained from a male rat after celiac doses administration 0-30 min of Sal and Bup racemate. The method could also be used to determine Sal enantiomers in a pharmaceutical aerosol.     

Simple and efficient method for enantioselective determination of omeprazole in human plasma

A practical and selective HPLC method for the separation and quantification of omeprazole enantiomers in human plasma is presented. C18 solid phase extraction (SPE) cartridges were used to extract the enantiomers from plasma samples and the chiral separation was carried out on a Chiralpak AD column protected with a CN guard column, using ethanol:hexane (70:30) as the mobile phase, at a flow rate of 0.5 ml/min. The detection was carried out at 302 nm. The method proved to be linear in the range of 10-1000 ng/ml for each enantiomer, with a quantification limit of 5 ng/ml. Precision and accuracy, demonstrated by within-day and between-day assays, were lower than 10%.     

Simultaneous determination of 8 HIV protease inhibitors in human plasma by isocratic high-performance liquid chromatography with combined use of UV and fluorescence detection: amprenavir, indinavir, atazanavir, ritonavir, lopinavir, saquinavir, nelfinavir and M8-nelfinavir metabolite

A simple, accurate and fast method was developed for determination of the commonly used HIV protease inhibitors (PIs) amprenavir, indinavir, atazanavir, ritonavir, lopinavir, nelfinavir, M8-nelfinavir metabolite and saquinavir in human plasma. Liquid-liquid extraction was used with hexane/ethylacetate from buffered plasma samples with a borate buffer pH 9.0. Isocratic chromatographic separation of all components was performed on an Allsphere hexyl HPLC column with combined UV and fluorescence detection. Calibration curves were constructed in the range of 0.025-10 mg/l. Accuracy and precision of the standards were all below 15% and the lowest limit of quantitation was 0.025 mg/l. Stability of quality control samples at different temperature conditions was found to be below 20% of nominal values. The advantages of this method are: (1) inclusion and determination of the newly approved atazanavir, (2) simultaneous isocratic HPLC separation of all compounds and (3) increased specificity and sensitivity for amprenavir by using fluorescence detection. This method can be used for therapeutic drug monitoring of all PIs currently commercialised and is now part of current clinical practice.     

Fractional factorial design optimization of the separation of pilocarpine and its degradation products by capillary electrophoresis

The separation of pilocarpine and its degradation products by micellar electrokinetic capillary chromatography (MECC) has been optimized by using fractional factorial design of the experiments. Critical parameters were identified in a screening design, and an optimization design was used to optimize the separation. The optimal separation method was based on a borate buffer with sodium dodecyl sulfate (SDS). It is concluded that by using fractional factorial design it is possible to improve the separation of pilocarpine, it trans epimer, isopilocarpine and their hydrolysis products, pilocarpic acid and isopilocarpic acid.     

Chiral separation and quantification of R/S-amphetamine, R/S-methamphetamine, R/S-MDA, R/S-MDMA, and R/S-MDEA in whole blood by GC-EI-MS

The enantioselective composition of the amphetamines is of interest, as the enantiomers show differences in their pharmacological effects and several methods for chiral separation of amphetamines have been described. Only a few methods have used whole blood as matrix and none of these separates both classic amphetamines (amphetamine and methamphetamine) and designer amphetamines (MDA, MDMA and MDEA). The aim of this study was, therefore, to develop a method for enantioselective analysis of AM, MA, MDA, MDMA, and MDEA in whole blood. The amphetamines were extracted from 0.5 g of whole blood by liquid-liquid extraction. After derivatization with R-MTPCl, the resulting diastereomers were separated by GC on a HP-5MS column and detected by SIM-MS. R-MTPCl was used as derivatization reagent because of the stability of this reagent and good separation of these analytes. Through the method, development time and temperature of the derivatization were optimized, and by admixture of 0.02% triethylamine it became possible to detect the amphetamines in adequately low concentrations as more analytes were derivatized. The method was validated and it was linear from 0.004 to 3 microg/g per enantiomer. The accuracy was within 91-115%, while the repeatability and reproducibility were < or =15% R.S.D. A method suitable for enantioselective separation and analysis of the amphetamines has been achieved, and the method was applied to analysis of whole blood samples originating from traffic and criminal cases and post mortem cases.     

Measurement of various polyaromatic hydrocarbons in the urban area of Naples]

In order to investigate the organic compound fraction of the Naples aerosol a chromatographic method was used for the separation and analysis of the polycyclic aromatic (PAH). As a first step a suitable one-step thin-layer chromatography (TLC) separation of the cyclohexane extractable material from airborne particulate was sought. After the TLC separation the concentrated samples were analyzed by reverse-phase liquid chromatography with fluorescence detection. We obtained chromatographic separation of five PAH on the EPA Priority Pollutant List and we determined the concentration of these PAHs present in atmospheric matter.     

Capillary electrophoresis enantioselective separation of vigabatrin enantiomers by precolumn derivatization with dehydroabietylisothiocyante and UV-vis detection

A simple and reliable capillary electrophoresis (CE) method with UV-vis detection is presented for the enantioselective separation and determination of vigabatrin enantiomers. Dehydroabietylisothiocyante (DHAIC), a novel chiral derivatizing reagent, was used for precolumn derivatization of vigabatrin enantiomers. Optimal separation was obtained with a running buffer consisting of 50 mM Na2HPO4 (pH 9.0), 17 mM sodium dodecyl sulfate (SDS) and 25% acetonitrile. The enantiomeric separation of vigabatrin derivatives was achieved within 25 min, and the resolution was found to be 2.1. Detection was followed by direct UV absorptiometric measurements at 202 nm. A calibration curve ranging from 0.3 to 6.0 microg/ml was shown to be linear, and the limit of detection was 0.15 microg/ml. The developed method has been applied to the determination of vigabatrin enantiomers spiked in human plasma, no interferences were found from endogenous amino acids.     

Method for rapid separation of 3,5,3'-triiodothyroacetic acid in human serum by fast protein liquid chromatography

The 3,5,3'-triiodothyroacetic acid (TRIAC) has been approved as a valuable agent in the management of hyperthyroidism secondary to inappropriate secretion of thyrotropin. We have developed a fast protein liquid chromatography (FPLC) method for separation and quantification of TRIAC. Serum samples charged with TRIAC were extracted with methanol/ammonium acetate, the supernatants were evaporated to dryness, reconstituted in NaOH and injected on a reversed phase column for chromatography. For separation an isocratic elution method (methanol water; 0.1% trifluoroacetic acid) was used. The area under the curve (ml%) was compared with those of the calibration curves. Recoveries were 70 +/- 10.8%. TRIAC was eluted in 2.33 ml. Conclusively, the present method shows that TRIAC can be measured by FPLC and may be applied to the measurement of TRIAC in pharmacological studies.     

Separation of nucleoside phosphoramidate diastereoisomers by high performance liquid chromatography and capillary electrophoresis

Separations of five diastereoisomers of nucleoside phosphoramidate derivatives (pronucleotides) were performed by both HPLC method using derivatized cellulose and amylose chiral stationary phases and CE method using anionic cyclodextrins added in the background electrolyte (BGE). An optimal baseline separation (Rs > 1.5) was readily obtained with all silica-based celluloses and amyloses using in a normal-phase methodology. Capillary electrophoresis was used as an alternative technique to HPLC for the separation of pronucleotides. The diastereoisomers were fully resolved with sulfated cyclodextrins at both BGE pH (2.5 and 6.2). Limits of detection and limits of quantification, calculated for both methods, are up to 200 times higher in CE separations than in HPLC separations. The analytical HPLC method was then applied in a preliminary study for the pronucleotide 1 quantification in cellular extract.     

Separation of human from mouse and monkey adenosine deaminase by ion-exchange chromatography following retroviral-mediated gene transfer

A method for the chromatographic separation of human adenosine deaminase (ADA) from murine and monkey ADA is described. This procedure was developed in order to detect the expression of low or moderate levels of human ADA following retroviral-mediated gene transfer of cloned human ADA gene sequences into both mouse and monkey cells. Protein separation was achieved on a Mono Q (HR 5/5) anion-exchange column using the Pharmacia fast protein liquid chromatography system and was found to be a highly reproducible method yielding enzymatically active protein. An increasing linear gradient extending from 0.05 to 0.5 M potassium chloride (pH 7.5) was used to elute the enzyme. Under these conditions, most human ADA does not bind to the column and elutes in the low-salt buffer (0.05 M KCl), while murine ADA elutes at 0.12 M KCl and monkey ADA at 0.15 M KCl. The column fractions were assayed for ADA activity, and the characteristic isozyme banding patterns for human, mouse, and monkey ADA were confirmed by starch gel electrophoresis. This procedure allows the rapid and reproducible separation of human ADA from that of other species and yields partially purified enzymatically active protein.     

Serum LDL- and HDL-cholesterol determined by ultracentrifugation and HPLC

Simple and precise methods for LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C) measurements are essential for assessment of cardiovascular disease (CVD) risks and for lipid and lipoprotein studies. We report here an ultracentrifugation (UC) and HPLC method that requires substantially less specimen volume and provides the necessary reliability and throughput required by large-volume, high-quality research and clinical studies. 2-Mercaptoethanol (ME) was used to dissociate serum lipoprotein [a] (Lp[a]) into apolipoprotein [a] and Lp[a] remnant (Lp[a-]) and eliminated the contamination of Lp[a] in HDL separated by UC. Serum aliquots were centrifuged at a density of 1.006 kg/l for the separation of HDL plus LDL, and in the presence of ME at a density of 1.063 kg/l for the separation of HDL. Cholesterol concentrations of the bottom fractions were analyzed by HPLC. LDL-C and HDL-C determined using this method were equivalent to those with β-quantification and the designated comparison method of the Centers for Disease Control. The total coefficient of variations for LDL-C and HDL-C were 0.65-1.12% and 0.96-2.07%, respectively. This method requires a small amount of specimen and is easy to operate. This method may be used in research or in clinical laboratories where precise and specific lipoprotein cholesterol analysis is needed.     

Sugaring out: A new method for removal of acetonitrile from preparative RP-HPLC eluent for protein purification

Reverse phase-high pressure liquid chromatography (RP-HPLC) with an acetonitrile–water mixture as the eluent is widely used for purification of proteins. The separation of acetonitrile (ACN) in RP-HPLC eluent is important for protein recovery. Cooling below subzero temperature and salting out have been used to remove ACN, each with its limitations. In this work we have explored the use of sugaring-out, a new phase separation method developed at University of Illinois for the separation of ACN from a simulated preparative RP-HPLC effluent. The effect of glucose concentration, temperature, and initial amount of ACN in the effluent on phase separation was investigated. Results showed that a good phase separation can be achieved at near room temperature (18 °C). With the optimized conditions, we found that more than 60% (w/w) of ACN was removed and more than 95% (w/w) of water-soluble proteins (bovine serum albumin, trypsin, and pepsin) were recovered.     

Determination of octopamine in human plasma by capillary electrophoresis with optical fiber light-emitting diode-induced fluorescence detection

A new analytical method based on capillary electrophoresis (CE) separation and optical fiber light-emitting diode (LED)-induced fluorescence detection has been developed for the determination of octopamine. Naphthalene-2,3-dicarboxaldehyde (NDA) was used for precolumn derivatization of octopamine. The separation and determination of the derivative was performed using a laboratory-built CE system with an optical fiber LED-induced fluorescence detector. Optimal separation was obtained at 20 kV using a background electrolyte solution consisting of 25 mM sodium borate (pH 9.2). High sensitivity detection was achieved by the optical fiber LED-induced fluorescence detection using a purple LED as the excitation source. The limit of detection (signal/noise=3) for octopamine was 5.0 x 10(-9)M. A calibration curve ranging from 1.0 x 10(-8) to 5.0 x 10(-7)M was shown to be linear. Using this method, the levels of octopamine in human plasma from healthy donors were determined.     

High-performance liquid chromatography of pyridylamino derivatives of unsaturated disaccharides produced from chondroitin sulfate isomers by chondroitinases

A sensitive method was developed for the separation and quantitation of four unsaturated disaccharides (delta Di-0S, delta Di-4S, delta Di-6S, and delta Di-diS) by high performance liquid chromatography. The unsaturated disaccharides were coupled with a fluorescent compound, 2-aminopyridine. Complete separation of the resulting pyridylamino derivatives was achieved on a column of muBondapak-C18 with 8 mM KH2PO4-Na2HPO4 (pH 6.0)/methanol (30/l, by volume) as a mobile phase. There was a linear relationship between the fluorescence emission (peak height), and the amount of each authentic disaccharide used for the coupling reaction. This method was applied to analyze commercially available chondroitin sulfates A and C, dermatan sulfate, and urinary glycosaminoglycans obtained from patients with mucopolysaccharidosis after digestion with chondroitinases. The data indicated that the present method is useful for the separation and quantitation of nmol-pmol levels of the unsaturated disaccharides produced from chondroitin sulfate isomers by chondroitinases and can be used for their structural characterization.     

Improved separation and characterization of lipopolysaccharide related compounds by reverse phase ion pairing-HPLC/electrospray ionization-quadrupole-mass spectrometry (RPIP-HPLC/ESI-Q-MS)

A new approach for the separation and inline characterization of lipopolysaccharide (LPS) related compounds has been developed. The separation was based on the difference in the number of charged phosphate and ethanolamine groups, as non-stoichiometric substituents, on the polysaccharide backbone, and was achieved with reverse phase ion-paring chromatography (RPIP-HPLC). Tributylamine was used as an ion-pair reagent. In the conditions used in this study, tributylammonium then binds to the LPS related compounds through the negatively charged phosphate groups. This changes the hydrophobicity of the analytes at different positions and allows for separation based on both the number and position of the substituents on the analyte. The RPIP-HPLC was found to be effective for the separation of the O,N-deacylated derivative (deON) and polysaccharide portion (PS) from the LPS of Escherichia coli C strain. Post-column fluorescence derivatization (FLD), using sodium periodate and taurine, was used to detect the separated LPS related species. On the other hand, the separated species were also detected by direct infusion into the ESI-Q-MS using a volatile ammonium acetate buffer rather than the more traditional potassium phosphate buffer. The signal to noise ratio (S/N ratio) was low for the total ion chromatogram, however, high S/N ratios as well as good resolution were attained by selected ion monitoring (SIM) using m/z numbers corresponding to species with different numbers of non-stoichiometric substituents. Five species for deON and ten species for PS were clearly identified on the SIM chromatogram on the RPIP-HPLC/ESI-Q-MS. Accordingly, the present method allows for the effective separation and inline identification of the species corresponding to the diverse non-stoichiometric substitutions in LPS related compounds.