Resolution of the stereoisomers of leucovorin and 5-methyltetrahydrofolate by chiral high-performance liquid chromatography

Leucovorin (5-formyltetrahydrofolate, LV) is a reduced folate that has been in clinical use for many years as a rescue agent following methotrexate (MTX) therapy. Commercially available LV is a 1:1 mixture of [6R]-and [6S]-isomers. Due to the lack of a specific method for directly separating and quantitating the stereoisomers of LV, it has been difficult to precisely define the pharmacokinetic and biological characteristics of each stereoisomer. We have now developed a novel HPLC method to completely separate [6S]-LV and [6S]-5-methyltetrahydrofolate (MeTHF) from their respective [6R]-isomers using bovine serum albumin (BSA)-bonded silica as the chiral stationary phase. Baseline separation was achieved using 5 and 25 mM sodium phosphate buffers (pH 7.4) as the mobile phase with resolution factors of 1.65 for LV and 2.31 for MeTHF, respectively. The purity of each isomer prepared by this HPLC method is greater than 99%. The stereoisomers were identified by examining their ability to protect CEM cells from MTX (0.04 microM)-induced inhibition of growth. In the LV chromatogram, the first eluted peak provided complete protection from MTX growth inhibition when LV concentrations of 0.1 microM and above were used, whereas the last eluted peak failed to reverse MTX toxicity at concentrations up to 1.0 microM. Chemically pure synthetic [6R]-and [6S]-LV standards confirmed that the first eluted, biologically active peak is the [6S]-isomer. For MeTHF, only the last eluted peak effectively protects cells from MTX growth inhibition and is therefore believed to be the [6S]-isomer. This new HPLC method will serve as a useful tool to elucidate the clinical and cellular pharmacology of the stereoisomers of LV and MeTHF.     

Evaluation of the stereoselective metabolism of the chiral analgesic drug etodolac by high-performance liquid chromatography

The enantiomers of the racemic analgesic drug etodolac have been resolved by fractional crystallization of the diastereomeric salts with optically active 1-phenylethylamine. A high-performance liquid chromatographic method to determine racemic etodolac (assay I) and its major metabolites (assay II) in urine using a conventional reversed-phase column is described. The determination of the enantiomeric ratios of etodolac and the two metabolites 7-hydroxyetodolac and 8-(1′-hydroxyethyl)etodolac was achieved using different protein-bonded chiral stationary phases. The urinary data for five volunteers are presented and show a marked stereoselectivity of the metabolism of etodolac in humans.     

Separation of phosphohydroxyamino acids by high-performance liquid chromatography

A high-performance liquid chromatography system has been developed which resolves O-phosphoserine, O-phosphothreonine, and O⁴-phosphotyrosine. Separation is performed on an anion-exchange resin eluted with phosphate buffer of low pH. Detection of the amino acid derivatives is accomplished using O-phthalaldehyde in an in-stream fluorometric system. This highly sensitive method has been applied to the detection of phosphohydroxyamino acids in bovine myelin basic protein phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase and in whole bovine myelin phosphorylated by endogenous kinases.     

Separation of neutral oligosaccharides by high-performance liquid chromatography

We have developed a method for separating reduced, neutral oligosaccharides by high-performance liquid chromatography on columns of MicroPak AX-5 (Varian Associates) with a mobile phase consisting of acetonitrile:H₂O. Individual glucose oligomers containing from 1 to 20 glucose moieties can be separated in a single 1-h analysis with a solvent program of decreasing acetonitrile concentration. We have applied this method to both the analysis and preparative isolation of glycoprotein-derived oligosaccharides obtained by enzymatic release with endoglycosidases or chemical release by hydrazinolysis. Introduction of ³H by reduction with NaB³H₄ permits the detection of subnanomole quantities of oligosaccharides. This method offers previously unattainable rapidity and resolution for the analysis of oligosaccharides.     

Separation of anionic oligosaccharides by high-performance liquid chromatography

We have developed methods for rapid fractionation of anionic oligosaccharides containing sulfate and/or sialic acid moieties by high-performance liquid chromatography (HPLC). Ion-exchange HPLC on amine-bearing columns (Micropak AX-10 and AX-5) at pH 4.0 is utilized to separate anionic oligosaccharides bearing zero, one, two, three, or four charges, independent of the identity of the amnionic moieties (sulfate and/or sialic acid). Ion-exchange HPLC at pH 1.7 allows separation of neutral, mono-, di-, and tetrasialylated, monosulfated, and disulfated oligosaccharides. Oligosaccharides containing three sialic acid residues and those bearing one each of sulfate and sialic acid, however, coelute at pH 1.7. Since the latter two oligosaccharide species separate at pH 4.0, analysis at pH 4.0 followed by analysis at pH 1.7 can be utilized to completely fractionate complex mixtures of sulfated and sialylated oligosaccharides. Ion-suppression amine adsorption HPLC has previously been shown to separate anionic oligosaccharides on the basis of net carbohydrate content (size). In this study we demonstrate the utility of ion-suppression amine adsorption HPLC for resolving sialylated oligosaccharide isomers which differ only in the linkages of sialic acid residues (alpha 2.3 vs alpha 2.6) and/or location of alpha 2,3- and alpha 2,6-linked sialic acid moieties on the peripheral branches of oligosaccharides. These two methods can be used in tandem to separate oligosaccharides, both analytically and preparatively, based on their number, types, and linkages of anionic moieties.     

Simultaneous determination of the stereoisomers of guggulsterone in serum by high-performance liquid chromatography

Simultaneous separation of E- and Z-guggulsterone, which is the main ingredient of ‘Guggulip', an ayurvedic drug, was accomplished by HPLC on a C₁₈ column using methanol, acetonitrile, buffer and tetrahydrofuran as a mobile phase. The compounds were monitored at 248 nm on a photodiode array detector. The assay method was used for the simultaneous determination of stereoisomers (E and Z) of guggulsterone in spiked serum and dosed (50 mg/kg, p.o.) rats. The recoveries of E- and Z-isomers from serum samples were always greater than 90%. The calibration graph was linear over the range of 25–2500 ng/ml for Z- and E-isomers. Lowest quantitation limit of Z- and E-guggulsterones was 25 ng/ml.     

Simultaneous analysis of several non-steroidal anti-inflammatory drugs in human urine by high-performance liquid chromatography with normal solid-phase extraction

A practical and reproducible high-performance liquid chromatographic method using normal solid-phase extraction has been developed for the simultaneous analysis of twelve non-steroidal anti-inflammatory drugs (NSAIDs) in human urine. A urine specimen mixed with acetate buffer pH 5.0 was purified by solid-phase extraction on a Sep-Pak Silica cartridge. The analyte was chromatographed by a reversed-phase Inertsil ODS-2 column using a phosphate buffer-acetonitrile at pH 5.0 as the mobile phase, and the effluent from the column was monitored at 230 or 320 nm. Absolute recoveries were greater than 73% for all of the twelve NSAIDs. The present method enabled simple manipulation and isocratic HPLC with UV analysis as well as high sensivity of 0.005 μg/ml for naproxen, and 0.05 μg/ml for sulindac, piroxicam, loxoprofen, ketoprofen, felbinac, fenbufen, flurbiprofen, diclofenac, ibuprofen and mefanamic acid as the quantitation limit in human urine using indomethacin as an internal standard.     

Separation of porphyrin isomers by high-performance liquid chromatography.

A reversed-phase gradient elution system is described for the simultaneous separation of the type I and type III isomers of 8-, 7-, 6-, 5- and 4-carboxylated porphyrins and isocoproporphyrins. The method, adaptable for isocratic and stepwise separation of individual groups of isomers, is also suitable for preparative isolation of pure porphyrins. The analyses of porphyrin isomers in the urine and faeces of porphyric patients are examples of applications.     

Separation of galactolipid molecular species by high-performance liquid chromatography

A technique for the separation, detection, and quantification of molecular species of monogalactosyldiglyceride and digalactosyldiglyceride is described. Use of the technique to analyze the molecular species composition of the galactolipids isolated from Dunaliella salina chloroplasts is presented. The results indicate that the respective compositions of the two lipids are quite different. This suggests that the enzymes involved in galactolipid metabolism are very specific with respect to acyl chain composition and pairing, or that extensive retailoring of constituent acyl chains occurs following formation of digalactosyldiglyceride from monogalactosyldiglyceride.     

Separation of hemoglobin types by cation-exchange high-performance liquid chromatography

The use of a recently developed cation-exchange HPLC packing material for the separation of hemoglobin types in human blood has been investigated. Adult and newborn hemolysates from normal individuals and from subjects with hemoglobin disorders were analyzed using a weak cation carboxymethyl-bonded phase on 5-micron-particle-size silica. Elution was accomplished using a Bistris (2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1, 3-propanediol) gradient. Seven well-resolved HbA1 fractions eluted before the major HbA peak. Hbs A1a, A1b, A1c and an HbA1 fraction that increased with aging of the hemolysates were separately eluted. HbF when present or when added to the hemolysates eluted as a distinct peak. HbA was followed by Hbs A2, S, and C when present. An early-eluting peak corresponding to Hb Bart's was identified in newborn hemolysates. It is concluded that cation-exchange HPLC provides a new tool for the reliable separation of minor hemoglobin components.     

Separation of drug enantiomers by liquid chromatography and capillary electrophoresis, using immobilized proteins as chiral selectors

Proteins display interesting chiral discrimination properties owing to multiple possibilities of intermolecular interactions with chiral compounds. This review deals with proteins which have been used as immobilized chiral selectors for the enantioseparation of drugs in liquid chromatography and capillary electrophoresis. The main procedures allowing the immobilization of proteins onto matrices, such as silica and zirconia particles, membranes and capillaries are first presented. Then the factors affecting the enantioseparation of drugs in liquid chromatography, using various protein-based chiral stationary phases (CSPs), are reviewed and discussed. Last, chiral separations already achieved using immobilized protein selectors in affinity capillary electrochromatography (ACEC) are presented and compared in terms of efficiency, stability and reproducibility.     

Separation of the intermediates of leucine catabolism by high-performance liquid chromatography

A method has been developed for the separation of leucine, 2-ketoisocaproic acid, isovaleryl CoA, 3-methylcrotonyl CoA, 3-hydroxy-3-methylglutaryl CoA, 3-methylglutaconyl CoA, acetyl CoA, and acetoacetic acid by ion-exchange high-performance liquid chromatography. The analysis requires 180 min. Use of this method to assess the catabolism of radiolabeled leucine in normal cultured human skin fibroblasts shows that these cells do not accumulate CoA esters, but convert leucine mainly to 2-ketoisocaproic acid, glutamate, and hydroxyisovalerate. In the fibroblasts of a patient with maple syrup urine disease, only 2-ketoiscaproic acid is produced from leucine.     

Separation of the major adrenal steroids by reversed-phase high-performance liquid chromatography

The major adrenal steroids were separated by multistep gradient elution with a reversed-phase high-performance liquid chromatography system, employing water and 1-propanol as solvents. With this solvent system, a wide range of 21 5-ene-3 beta-ol and 4-ene-3-one steroids can be resolved in a single chromatogram, which was not possible with previously published gradient solvent systems. In particular, intermediate steroids of the biosynthetic pathway, 17 alpha-hydroxyprogesterone, 17 alpha-hydroxypregnenolone, and dehydroepiandrosterone, were separated with baseline or sufficient resolution to allow accurate quantitation. Using the 1-propanol-water gradient, the separations of 5-ene and 4-ene steroids were compared on different octadecylsilyl packings. Optimum resolution was obtained with a fully covered, spherical particle. The 1-propanol-water gradient was compared to a previously published methanol-water gradient in the analysis of steroidogenesis by adrenocortical cell cultures. HPLC analysis of the steroid production was quantitatively the same with both gradient solvent systems. However, qualitatively, the methanol-water gradient system did not resolve the above-mentioned intermediate steroids.     

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.     

Separation and quantitation of leukotrienes by reversed-phase high-performance liquid chromatography

A rapid and sensitive reversed-phase HPLC procedure is reported which allows the simultaneous separation and quantitation of LTC₄, 11t-LTC₄, LTD₄, LTB₄, 12epi,6t,8c-LTB₄, 6t-LTB₄ + 12epi,6t-LTB₄, two trihydroxy-eicosatetraenoic acids tentatively identified as 20-OH-LTB₄ and 20-OH,12epi,6t,8c-LTB₄ and several not yet identified 15-series leukotrienes produced by the cytosol of porcine polymorphonuclear leukocytes.     

Separation of brain monosialoganglioside molecular species by high-performance liquid chromatography

A method for the separation of molecular species of brain monosialogangliosides by high-performance liquid chromatography is described. GM4, GM3, GM2, and GM1 were purified from human brain and their individual molecular species were separated on a C18 reversed-phase column. Peaks were identified by mass spectrometry of the intact ganglioside, by gas-liquid chromatography of the fatty acids, and by high-performance liquid chromatography of the long chain bases. A characteristic elution sequence of molecular species permitted their identification based upon their retention times on the reversed-phase column.     

Separation and quantitation of dolichyl esters by high-performance liquid chromatography

An HPLC procedure for the isolation and quantitation of total and individual dolichyl esters in tissues has been developed. The purified lipid extracts are subjected to sequential reversed-phase, straight-phase, and reversed-phase HPLC, which yield complete resolution and high recovery of the individual dolichyl esters. The isoprenoid distribution in the esterified fraction was similar to that of the free alcohol fraction in liver, kidney, and spleen. All fatty acids present in the total fraction were also recovered in all the individual polyisoprenoids. Dolichyl esters thus appear to differ from other lipid esters in tissues in containing a broader range of fatty acids.