Determination of enantiomeric excess of ethyl 3,5-dihydroxy-6-benzyloxy hexanoate by chiral reverse phase high performance liquid chromatography

A simple and reliable chiral HPLC method was developed for the determination of enantiomeric excess of a chiral dihydroxy intermediate for the chemoenzymatic synthesis of side chain of statin drugs. After evaluating different columns and conditions, the four stereoisomers of ethyl 3,5-dihydroxy-6-benzyloxy hexanoate were well resolved by a simple gradient elution on OD-RH column, and the enantiomeric excess of the desired 3R,5S-enantiomer was accurately measured. This study provides a simple, rapid, accurate, and reliable method to assess the enantiomeric quality of such important intermediates.     

Determination of the molecular species composition of diacylglycerols in human adipose tissue by solid-phase extraction and gas chromatography on a polar phase

The free diacylglycerols (DAGs) in adipose tissue are involved in the metabolism of stored lipids and hence are related to the supply of fatty acids for other tissues. This paper describes a simple, fast, and reproducible method for the identification and quantification of different molecular species of DAGs in human adipose tissue. The method comprised solid-phase extraction on a diol-bonded phase column combined with capillary GC analysis of silylated DAG derivatives on a polar phase (65% phenylmethylsilicone). Separation of the DAGs was achieved based on chain length, isomeric structure (1,2- and 1,3-DAGs), and degree of unsaturation. The main DAGs were 1,2-OO, 1,2-OP, 1,2-LO and 1,2-LP. The composition was corroborated by analysis of the component fatty acids of the DAGs, 18:1(n-9), 16:0, and 18:2(n-6) being the three major fatty acids obtained.     

Analysis of aminophospholipid molecular species by high performance liquid chromatography

A new method is described for the separation of individual molecular species of the aminophospholipids, phosphatidylethanolamine and phosphatidylserine. Trinitrobenzene-sulfonic acid was used to derivatize both aminophospholipids and the derivatives were purified by thin-layer chromatography. A reversed-phase high performance liquid chromatography technique was developed to separate and quantify individual molecular species based upon ultraviolet detection of the attached chromophore. The retention times of the molecular species on the C18 reversed-phase column were longer with increasing carbon chain length and decreasing degree of unsaturation of fatty acyl chain. The overall procedure allowed a quantitative recovery of the aminophospholipid species. The lower limit of detection was about 10 pmol and a linear response was observed in the range of 0.1-10 nmol of phospholipid. Using this method, we were able to separate and quantify trinitrophenyl-phosphatidylethanolamine molecular species of both subclasses (diacyl and alkenyl) from human red blood cells and rat brains. Separation of species was confirmed by gas-liquid chromatographic analysis of the fatty acid content of each peak and by thermospray liquid chromatography-mass spectrometry. This new method provides a convenient and sensitive technique for studies of aminophospholipid molecular species composition. Furthermore, it appears to be a useful tool for the analysis of asymmetric distribution of these species in biological membranes.     

Separation of derivatized glycosphingolipids into individual molecular species by high performance liquid chromatography

The high performance liquid chromatography separation of the perbenzoyl derivatives of the neutral glycosphingolipids (GlcCer, LacCer, GbOse3Cer, GbOse4Cer, and GgOse3Cer) and the p-bromophenacyl and 2,4-dinitrophenyl hydrazide derivatives of the gangliosides (GM4, GM3, GM2, GM1, GD1a) into individual molecular species on a C18 reversed-phase column is described. Peaks were identified by comparing their relative retention times to the relative retention time of the corresponding glycosphingolipid of known molecular species composition. As little as 5 to 10 pmol of each molecular species of neutral glycosphingolipids and 3 to 5 pmol of the gangliosides can be detected. The effects of changes in the proportion of acetonitrile, methanol, and water in the mobile phase and of column temperature on the molecular species separation are described. A procedure for the tentative identification of glycosphingolipid molecular species based on their relative retention times is presented.     

Determination of enantiomeric impurity in besifloxacin hydrochloride by chiral high-performance liquid chromatography with precolumn derivatization

Besifloxacin hydrochloride is a novel chiral broad-spectrum fluoroquinolone developed for the treatment of bacterial conjunctivitis. R-besifloxacin hydrochloride is used in clinics as a consequence of its higher antibacterial activity. To establish an enantiomeric impurity determination method, some chiral stationary phases (CSPs) were screened. Besifloxacin enantiomers can be separated to a certain extent on Chiral CD-Ph (Shiseido Co., Ltd., Japan), Chiral AGP, and Crownpak CR (+) (Daicel Chemical IND., Ltd., Japan). However, the selectivity and sensitivity were both unsatisfactory on these three CSPs. Therefore, Chiral AGP, Chiral CD-Ph, and Crownpak CR (+) were not used in the enantiomeric impurity determination of besifloxacin hydrochloride. The separation of enantiomers of besifloxacin was further performed using a precolumn derivatization chiral high-performance liquid chromatography method. 2,3,4,6-Tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate was used as the derivatization reagent. Besifloxacin enantiomer derivates were well separated on a C(18) column (250 × 4.6 mm, 5 μm) with a mobile phase that consisted of methanol-KH(2)PO(4) buffer solution (20 mM; pH 3.0) (50:50, v/v). Selectivity, sensitivity, linearity, accuracy, precision, stability, and robustness of this method were all satisfied with the method validation requirement. The method was suitable for the quality control of enantiomeric impurity in besifloxacin hydrochloride.     

Direct enantiomeric resolutions of chiral triazole pesticides by high-performance liquid chromatography

Cellulose-tris (3,5-dimethylphenylcarbamate; CDMPC) was synthesized and coated on aminopropylsilica to prepare chiral stationary phase (CSP). Normal-phase high-performance liquid chromatography (HPLC) methods for the resolutions of five chiral triazole pesticides, diniconazole, tebuconazole, hexaconazole, triadimefon and flutriafol, on the CSP were developed. Several operating parameters such as mobile phase composition, modifier and column temperature were studied for the optimization of the resolutions. Better separations were achieved using 2% iso-butanol for diniconazole, 2% ethanol for tebuconazole, 2% iso-propanol for hexaconazole, 1% n-butanol for triadimefon and 2% n-propanol for flutriafol as modifiers in n-hexane at 0 degrees C with the resolution factors (Rs) of 1.62, 1.66, 2.46, 1.68 and 1.98, respectively. Low temperature was better for the resolutions. Validation of the methods included linearity and precision.     

Immobilized vancomycin as chiral stationary phase in packed capillary liquid chromatography

Fused silica-packed capillary columns containing vancomycin immobilized by reductive amination on an aldehyde-silica were used to separate enantiomers of some non-steroidal anti-inflammatory drugs. Attempts have been made to qualitatively explain the influence of various mobile phase compositions on the enantioselective retention. The effects of mobile phase pH, buffer, and organic modifier concentrations were investigated as well as the influence of salts of hydrophobic ions added to the mobile phase to induce ion pair retention. Chirality 10:273–280, 1998. © 1998 Wiley-Liss, Inc.     

Determination of methylputrescine oxidase by high performance liquid chromatography

N-Methyl-Δ¹-pyrrolinium chloride, the product of the title enzyme, was synthesized by methylation of aminobutyraldehyde diethylacetal followed by acidic cleavage. After purification to homogeneity, it was characterized by NMR and UV spectroscopy. The compound had an absorption maximum at 210 nm; previous data indicating a maximum at 267 nm were shown to arise from an impurity. An HPLC method for the assay of N-methylputrescine oxidase from plant material was developed based on the separation of N-methyl-Δ¹-pyrrolinium chloride on a cation exchange column and direct detection at 210 nm. The enzyme activity was measured in the protein fraction extracted from plant roots and treated by gel filtration on disposable PD 10 columns. A K_m value of 1.9 mM was determined for methylputrescine and the enzyme from tobacco roots. The enzyme activities from N. tabacum and Datura stramonium were compared.     

Analysis of molecular species of plant polar lipids by high-performance and gas liquid chromatography

Total lipid extracts from potato tubers and tobacco leaves are separated into lipid classes by two step HPLC using a silicic column. Elution is first performed for 20 min with a programmed linear gradient of two mixed solvents running from 100% of solution A (isopropanol-hexane, 4:3) to 100% of solution B (isopropanol-hexane-water, 8:6:1.5); the column is then eluted with pure solution B in an isocratic mode for 20 min more. The main polar lipids (MGDG, DGDG, PC, PE, PG) from both plant tissues can be collected and further separated into component molecular species on a simplified HPLC system with a C₁₈ column eluted in an isocratic mode with a polar solvent. Molecular species separations are achieved within 35 min; quantifications are made through GLC analysis of attached fatty acids. Three to five main molecular species are thus clearly identified in each lipid class. In potato tuber, phospholipids (PC, PE) 18:2/18:2 species are predominant. In tobacco leaf, six double bond species (18:3/18:3 and 16:3/18:3) are predominant in galactolipids, whereas PC contains a greater number of molecular species varying by their degree of unsaturation (from 18:3/18:3 to 16:0/18:2). Only certain molecular species of PG contain Δ³-trans-hexadecenoic acid.     

15种獐牙菜属植物中主要药用成分的高效液相色谱测定

对青藏高原和云贵高原的15种獐牙菜属植物进行了3种苦味苷,即獐牙菜苦苷(swertiamarin)、龙胆苦苷(gentiopicroside)、苦龙苷(amarogentin)、一种黄酮苷-当药黄素(swertisin)、及5种口山酮苷-芒果苷(mangiferin)、当药醇苷(swertianolin)、7-O-[a-L-吡喃鼠李糖-(1→2)-β-D-吡喃木糖]-1,8-二羟基-3-甲氧基口山酮(7-O-[a-L-rhamnopyranosyl-(1-2)-β-D-xylopyranosyl]-1,8-dihydroxy-3-methoxyxanthone)、7-O-β-D-吡喃木糖-1,8-二羟基-3-甲氧基口山酮(7-O-β-D-xylopyranosyl-1,8-dihydroxy-3-methoxyxanthone)、3-O-β-D-吡喃葡萄糖-1,8-二羟基-5-甲氧基口山酮(3-O-β-D-glucopyranosyl-1,8-dihydroxy-5-methoxyxanth-one)等9种主要药效成分同时进行了高效液相色谱的含量测定(Kromasil C18柱,甲醇一水梯度洗脱,二级管阵列检测);并对其主要药效成分的分布进行了比较。     

Determination of stereo- and positional isomers of oxygenated triterpenoids by reversed phase high performance liquid chromatography

Twenty four oxygenated triterpenoids, including eight pairs of stereoisomers and five pairs of positional isomers, could be separated by reversed phase HPLC. The capacity factors obtained in methanol-water and acetonitrile-water solvent systems made it possible to correlate the molecular polarities due to the presence of multiple oxygenated functional groups in these compounds. It was found that the number and position of functional groups as well as the stereochemistry of these functional groups played important roles in governing the polarity of these lanostanoid acids. The polarity weighting factors were in the following order: 3 beta-OH greater than 3 alpha-OH greater than 3 alpha-OAc greater than 3 beta-OAc. The contribution to polarity due to 15 alpha-OAc and 22 beta-OAc was probably very similar. The unique stereochemical character and eluting sequences of the lanostanoid acids provide information to generate empirical rules for predicting the role of individual polar functional groups in the chromatographic behavior in reversed phase HPLC.     

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.     

高效液相法测定蜂蜜中甘油的含量

建立了以混合溶剂直接提取测定蜂蜜中甘油含量的方法.采用ZORBAX Carbohydrate a-nalysis(4.6 mm×250 mm 5-Micro)色谱柱,以乙腈/水(80:20,v/v)为流动相,示差检测器,使用乙腈/甲醇/水混合作为溶剂快速检测甘油.结果表明,应用此法的甘油浓度在10.0 mg/L～250...     

Separation of molecular species of glucosylceramide by high performance liquid chromatography of their benzoyl derivatives.

The method of separation of glucosylceramide by HPLC was reported. Glucosylceramide was perbenzoylated and separated on a packed muBondapack C18 column, using methanol as eluting solvent. The pattern obtained by HPLC closely resembled that obtained by GLC of the TMS-glucosylceramide, and reflected the molecular species of fatty acid components. This method is reproducible, and sensitive as GLC. This method also can be used for analysis of higher glycolipids.     

HPLC法测定牙膏中柚皮苷的含量

建立了高效液相色谱测定牙膏中柚皮苷含量的方法。采用的色谱条件:Hypersil BDS C18色谱柱(250 mm×4.6 mm,5μm);柱温为40℃;以水(A相)和乙腈(B相),梯度洗脱程序为:0～15 min,10%～100%B;流速为1.0 mL/min;检测波长为283 nm;进样量为20μL。结果表明,柚皮苷的质量浓度在14.55～116.40μg/mL范围内与峰面积呈良好的线性关系(r=0.9999),平均加标回收率为97.56%。该方法稳定、准确,重现性好,可作为牙膏中柚皮苷的含量测定和质量控制方法。     

Enantioseparation of 2-aryl-1,3-dicarbonyl analogues by high performance liquid chromatography using polysaccharide type chiral stationary phase

The HPLC chiral separation of 21 kinds of 2-aryl-1,3-dicarbonyl analogues was investigated in normal phase mode with amylose tris(3,5-dimethylphenylcarbamate), amylose tris((S)-1-phenylethylcarbamate), cellulose tris(3,5-dimethylphenylcarbamate), and cellulose tris(4-methylbenzoate) chiral stationary phases, respectively. The whole set of 2-aryl-1,3-dicarbonyl analogues shows better enantioselectivity and enantioseparation on amylose tris(3,5-dimethylphenyl carbamate) (Chiralpak AD-H). The temperature dependence of enantioselectivity was studied to improve the enantioseparation. In addition, efforts are made to relate analyte structure with the quality of the achieved chiral separation.     

Direct measurement of enantiomeric ratios of enzymatic resolution by chiral high-performance liquid chromatography

(R)- and (S)-Methyl 2-(phenoxy)propionate and their acids could be separated simultaneously by a Chiralcel OD or OK column, while (R)- and (S)-methyl 2-(4-chlorophenoxy)propionate and their acids were separated concurrently only by an OK column. This is a novel and facile way to measure the enantiomeric excesses of the remaining substrate and product in the reaction of enzymatic resolution; enantiomeric ratios could then be calculated.     

Separation and quantification of alkylphosphocholines by reversed phase high performance liquid chromatography

Alkylphosphocholines represent a new class of drugs with remarkable antineoplastic and antiprotozoal activity. For instance, hexadecylphosphocholine has been approved for the topical treatment of skin metastasis. In addition, it was successfully studied in India for the treatment of leishmaniasis. Different phase-I and phase-II-trials resulted in cure rates of more than 97%. To optimize antitumor or antiprotozoal activity, we have prepared alkylphosphocholines differing in chain length and unsaturation. For the qualitative and quantitative analysis of these longer chain analogues, we have used isocratic high performance liquid chromatography. The separation of the alkylphosphocholines with different chain lengths in this reversed phase HPLC system was achieved on a YMC-TMS column with a mobile phase consisting of methanol-water (85:15; v/v) at a flow rate of 1.0 ml/min. Furthermore the cis-/trans-isomers such as oleylphosphocholine and elaidylphosphocholine were clearly separated on a YMC-C8 column with a methanol-water mixture (80:20; v/v) as mobile phase. In the described reversed phase HPLC systems simple refractive index detection and UV detection allow the sensitive and quantitative determination of alkylphosphocholines. These methods are very important for reproducible identification and quantitative determination of saturated and mono-unsaturated alkylphosphocholines with alkyl residues containing up to 25 carbon atoms.     

Rapid separation of anionic oligosaccharide species by high performance liquid chromatography

We have developed a method for the rapid separation of anionic oligosaccharide species by high-performance liquid chromatography utilizing a MicroPak AX-10 ion-exchange column (Varian Associates) with the mobile phase consisting of 25–500 mm KH₂PO₄, pH4.0. Separation of oligosaccharides bearing zero, one, two, three or four sialic acid residues requires less than 45 min. Oligosaccharides containing mannose-6-PO₄ moieties in monoester or diester linkage can also be analyzed in this system. Preparative separations of as much as 20 mg of oligosaccharide can be accomplished in a single chromatographic analysis with quantitative yields of oligosaccharide. This method should prove useful for the rapid isolation and characterization of anionic oligosaccharide species.