Normal Phase HPLC Column Research Articles

Application of Normal-Phase Silica Column in Hydrophilic Interaction Liquid Chromatography Mode for Simultaneous Determination of Underivatized Amino Acids from Human Serum Samples via Liquid Chromatography-Tandem Mass Spectrometry.

In neonatal screening, amino acids have a significant diagnostic role. Determination of their values may identify abnormal conditions. Early diagnosis and continuous monitoring of amino acid disorders results in a better disease outcome. An easy and simple LC-MS/MS method was developed for the quantitation of underivatized amino acids. Amino acids were separated using a normal-phase HPLC column having a totally porous silica stationary phase and using classical reversed-phase eluents. Mass spectrometry in multiple reaction monitoring mode was used for the analysis, providing high selectivity and sensitivity. A standard addition calibration model was applied for quantitation using only one isotope-labeled internal standard for all amino acids. Five calibration points were used for quantitation, and the method was successfully validated. The slopes of the calibration curves of the individual amino acids in parallel measurements were found to be similar. Since the measured slopes were reproducible, one serum sample could represent every series of serum samples of a given day. The method was tested on human serum samples and adequate results were obtained. This new method can be easily applied in clinical laboratories.

Determination of Lutein and Zeaxanthin Esters and Their Geometric Isomers in Carotenoid Ester Concentrates Used as Ingredients in Nutritional Supplements: Validation of a Combined Spectrophotometric-HPLC Method.

A combined spectrophotometric-LC method is described for the determination of total lutein and zeaxanthin ester content in carotenoid ester concentrates, including their main geometrical isomers. The concept of composite-specific absorbance is introduced for this purpose. The method is applicable to carotenoid ester concentrates used as ingredients in oil suspensions and dosage forms. The sample is dissolved in a hexane-2-propanol mixture (95 + 5, v/v) for spectrophotometric measurement at a maximum absorption of ~445 nm. Subsequently, in parallel, a sample is saponified and chromatographed on a normal-phase HPLC column to determine the relative percentage profile of the main geometrical isomers of both carotenoid esters. This, in turn, is used to calculate the composite-specific absorbance of the sample for the final calculation of results. The method, which solely uses reference standards to validate chromatographic conditions, avoids the common error of applying the specific absorbance of only the trans isomer for the calculation of total carotenoid content when cis isomers are present.

Argentation high performance liquid chromatography on-line coupled to gas chromatography for the analysis of monounsaturated polyolefin oligomers in packaging materials and foods

Multidimensional chromatography based on two-dimensional high performance liquid chromatography on-line coupled to gas chromatography (on-line HPLC–HPLC–GC) enables the separate analysis of saturated, monounsaturated and aromatic hydrocarbons in packaging materials like polyolefins or paperboard and their migrates into foods. Since normal-phase HPLC on silica gel did not preseparate saturated from monounsaturated hydrocarbons, a separation step on a normal-phase HPLC column treated in the laboratory with an optimized amount of silver nitrate was added. The preparation of this HPLC column and the instrumental set-up are described, followed by examples showing the potential of the method. In a preliminary investigation of 11 polyolefin granulates for food contact up to 40% monounsaturated hydrocarbons among the oligomers C16–35 were determined.

Bloodspot Assay Using HPLC–Tandem Mass Spectrometry for Detection of Barth Syndrome

Barth syndrome (BTHS) is a serious X-linked, metabolic, multisystem disorder characterized by cardiomyopathy, neutropenia, myopathy, and growth delay. Because early diagnosis and appropriate treatment are of key importance for the survival of affected boys, we developed a biochemical BTHS screening method based on analysis of the monolysocardiolipin:cardiolipin ratio in bloodspots. We performed chloroform/methanol extraction on quarter-inch punches of dried bloodspots on Guthrie cards from BTHS patients and controls. Extracts were dried (60 degrees C, N(2)) and reconstituted in CHCl(3)/methanol/H(2)O [50:45:5 vol/vol/vol, 0.1% NH(3) (25%)]. HPLC-tandem mass spectrometry analysis was performed with a normal-phase HPLC column and multiple reaction monitoring transitions for monolysocardiolipin (MLCL) and cardiolipin (CL) with a total run time of 10 min. The ratio of MLCL and CL was used as screening parameter. All BTHS patients (n = 31) had monolysocardiolipin:cardiolipin ratios >0.40 and all controls (n = 215) had monolysocardiolipin:cardiolipin ratios <0.23. Using a cutoff point of 0.30, a blind test of 206 samples (199 controls, 7 BTHS) had sensitivity and specificity of 100%. Bloodspots could be stored at 4 degrees C or room temperature for >1 year without affecting the test outcome. Three neonatal Guthrie cards of BTHS patients taken 3.6 to 5.8 years previously were correctly identified as positive for BTHS. HPLC-tandem mass spectrometry analysis of dried bloodspots is an unambiguous screening test for BTHS with potential for rapid screening of neonates suspected of having BTHS, making remote and retrospective diagnosis accessible for a disease that is almost certainly underdiagnosed.

Purification, Characterization, and Cloning of a Spodoptera frugiperda Sf9 β-N-Acetylhexosaminidase That Hydrolyzes Terminal N-Acetylglucosamine on the N-Glycan Core

Paucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a beta-N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been identified. We report the purification of a beta-N-acetylhexosaminidase from the culture medium of Spodoptera frugiperda Sf9 cells (Sfhex). The purified Sfhex protein showed 10 times higher activity for a terminal N-acetylglucosamine on the N-glycan core compared with tri-N-acetylchitotriose. Sfhex was found to be a homodimer of 110 kDa in solution, with a pH optimum of 5.5. With a biantennary N-glycan substrate, it exhibited a 5-fold preference for removal of the beta(1,2)-linked N-acetylglucosamine from the Man alpha(1,3) branch compared with the Man alpha(1,6) branch. We isolated two corresponding cDNA clones for Sfhex that encode proteins with >99% amino acid identity. A phylogenetic analysis suggested that Sfhex is an ortholog of mammalian lysosomal beta-N-acetylhexosaminidases. Recombinant Sfhex expressed in Sf9 cells exhibited the same substrate specificity and pH optimum as the purified enzyme. Although a larger amount of newly synthesized Sfhex was secreted into the culture medium by Sf9 cells, a significant amount of Sfhex was also found to be intracellular. Under a confocal microscope, cellular Sfhex exhibited punctate staining throughout the cytoplasm, but did not colocalize with a Golgi marker. Because secretory glycoproteins and Sfhex are cotransported through the same secretory pathway and because Sfhex is active at the pH of the secretory compartments, this study suggests that Sfhex may play a role as a processing beta-N-acetylhexosaminidase acting on N-glycans from Sf9 cells.

Lecithin:Retinol Acyltransferase Is Responsible for Amidation of Retinylamine, a Potent Inhibitor of the Retinoid Cycle

Lecithin:retinol acyltransferase (LRAT) catalyzes the transfer of an acyl group from the sn-1 position of phosphatidylcholine to all-trans-retinol (vitamin A) and plays an essential role in the regeneration of visual chromophore as well as in the metabolism of vitamin A. Here we demonstrate that retinylamine (Ret-NH2), a potent and selective inhibitor of 11-cis-retinal biosynthesis (Golczak, M., Kuksa, V., Maeda, T., Moise, A. R., and Palczewski, K. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 8162-8167), is a substrate for LRAT. LRAT catalyzes the transfer of the acyl group onto Ret-NH2 leading to the formation of N-retinylpalmitamide, N-retinylstearamide, and N-retinylmyristamide with a ratio of 15:6:2, respectively. The presence of N-retinylamides was detected in vivo in mice supplemented with Ret-NH2. N-Retinylamides are thus the main metabolites of Ret-NH2 in the liver and the eye and can be mobilized by hydrolysis/deamidation back to Ret-NH2. Using two-photon microscopy and the intrinsic fluorescence of N-retinylamides, we showed that newly formed amides colocalize with the retinyl ester storage particles (retinosomes) in the retinal pigment epithelium. These observations provide new information concerning the substrate specificity of LRAT and explain the prolonged effect of Ret-NH2 on the rate of 11-cis-retinal recovery in vivo.

Liquid chromatographic determination of geometrical retinol isomers and carotene in enteral feeding formulas

The objective of the present study was to evaluate a practical method for separation and determination of geometrical retinol isomers and carotene in enteral feeding formulas and to analyze 17 samples of commercial formulas. By using a normal-phase HPLC column and a mobile phase consisting of 1-octanol in n-hexane, seven isomers of retinol were separated and identified from the standard solution after photolysis. For evaluation of Vitamin A activity in these formulas, simultaneous determination of total carotene was performed. The data about linearity, recovery, accuracy and precision showed the reliability of analytical procedures. In the unsaponifiable portion of samples of commercial formulas, six retinol isomers were identified: ( E)-retinol; ( Z)-13; ( Z)-9; ( Z, Z)-9,13; ( Z, Z)-11,13 and ( Z)-7-retinol. ( Z)-13/( E)-retinol ratios ranged between 3 and 37%. The range of total Z/( E)-retinol isomers fell between 5 and 42%. Despite the high concentration of Z-isomers observed in various commercial enteral feeding formulas, none of the samples presented Vitamin A activity below 90% of that specified on the label.

Determination of Bisphenol A diglycidyl ether (BADGE) and its hydrolysis products in canned oily foods from the Austrian market

Bisphenol A diglycidyl ether (BADGE) is determined in canned oily foods from Austria using a new simplified HPLC method. Samples are extracted with pentane, back extracted with methanol, and finally dissolved in the mobile phase (cyclohexane/tert–butyl methyl ether). Separation is performed on a normal-phase HPLC column using fluorescence detection. Verification of the BADGE-containing peak is carried out by using off-line GC-MS. Additionally, the synthesis and determination of BADGE hydrolysis products, Bisphenol A bis(2,3-dihydroxypropyl) ether (BADGE.2H2O) and Bisphenol A glycidyl (2,3-dihydroxypropyl) ether (BADGE.H2O) are presented. From 67 analyzed cans, containing various fatty meat or fish products, 16% were above the maximum quantity of 1 mg/kg tolerated by the European Community, 45% were in the range between 0.1–1 mg/kg, 24% between 0.02 and 0.1 mg/kg, and in 15% the BADGE concentrations were below the detection limit of 0.02 mg/kg. The hydrolysis product BADGE.H2O was not detected in any sample, whereas BADGE.2H2O was found in some samples up to a concentration of 0.5 mg/kg.

Methodology for Bioassay-Directed Fractionation Studies of Air Particulate Material and Other Complex Environmental Matrices

A normal phase HPLC methodology using a semi-preparative polyaminocyano column in conjunction with a selection of short-term genotoxicity assays has been developed for bioassay-directed fractionation studies of complex environmental mixtures. To illustrate the effectiveness of this methodology, an organic extract prepared from respirable air particulate samples collected in Hamilton, Canada was separated into a non-polar aromatic fraction and a polar aromatic fraction using a combination of alumina and Sephadex LH20 chromatography. These fractions were evaluated for their genotoxic potential using the Salmonella/microsome (Ames) assay with six different strains of Salmonella. The non-polar aromatic fraction was analyzed by normal phase HPLC and the eluent was collected in one-minute subfractions ; these subfractions were bioassayed in three different Salmonella strains (YG1021 -S9. YG1024 -S9 and YG1029 +S9) to afford three different mutation profiles of this sample. Some subfractions which exhibited high mutagenic responses were subjected to further chemical analyses using GC/MS in order to identify those compounds responsible for the genotoxic responses. The nitroarene compounds 2-nitrofluoranthene, 1-nitropyrene and 2-nitropyrene and higher molecular weight polycyclic aromatic hydrocarbons such as benzo[a]pyrene and indeno[1,2,3-cd]pyrene were identified and quantified in some of the biologically active subfractions. The normal phase gradient conditions afforded very reproducible retention times for a series of polycyclic aromatic standards with a broad range of compound polarities. In addition, polycyclic aromatic hydrocarbons (PAH) were observed to elute from the normal phase HPLC column in a series of peaks ; successive peaks contained PAH of increasing molecular weight while any individual peak was shown to contain PAH of the same molecular weight.

Protocol for the study of the metabolism of retinyl esters in plasma lipoproteins during postprandial lipemia.

An efficient protocol is described for the study of the kinetics of retinyl esters in whole plasma and several lipoprotein fractions following the consumption of an oral fat load containing vitamin A (retinol). To allow for a more complete characterization of the kinetics of retinyl esters in different lipoprotein fractions, a simplified two-step ultracentrifugation procedure is reported for the efficient and reproducible isolation of triglyceride-rich chylomicrons from nonfasting subjects, VLDL-sized lipoprotein particles, and the triglyceride-poor lipoprotein fraction. The present method for the determination of retinyl esters is based on the direct application of the lipid fraction onto a normal phase HPLC column without requiring the lipid extract to be desiccated and resolubilized. All of the commonly occurring esters of retinol elute as a single peak with a retention time of 1.6-1.8 min followed by retinyl acetate (serving as the internal standard) and retinol with retention times of 2-2.5 min and 5-5.5 min, respectively. With this system, a new sample can be processed every 10 min and a complete set of 60 samples from a typical oral fat load can analyzed in one working day with minimal technical interaction. By normalizing to the area under the internal standard to correct for variability in the injected volume, the coefficient of variability for the concentration retinyl esters within a single run is less than 5% and less than 10% between runs.

Chemistry of Succinimido Esters. XXI

A sensitive determination of sphingolipid bases (1) was made using synthetic Cn-dihyrosphingosine and naturally occurring C18- (1) aromatized with N-succinimidyl arylacetate (2a). The amino group in (1) easily reacted with (2a) to give the corresponding N-arylacetyl derivative (3a). The isomers of (1) were separated on a normal phase HPLC column in the form of (3a) which was detectable by the absorption in UV region. The isomer content of (1) as determined by the present method using (3a) having a different substituent showed agreement with that determined by GC method in the form of N-acetyl-O, O-bis (TMS) derivatives of (1) and the HPLC method in the form of N-arylcarbonyl derivative of (1), (3b).

Chemistry of Succinimido Esters. XVII.*1

The sensitive determination of erythro/threo ratios of sphingolipid bases without trimethylsilylation was made using synthetic Cn-dihydrosphingosines (1) labelled by N-succinimidyl benzoates (2). Compound (1) reacted with (2) in a refluxing acetonitrile to give the corresponding N-arylcarbonyl derivative, (3). The erythro and threo isomers of (1) could be separated on a normal phase HPLC column in the form of (3) which was detectable at 254nm. The erythro content of (1) as determined by the present method using (2) with a different substituent showed good agreement and was essentially the same as that by the GLC method using O, O-di-TMS derivative of (3).

Selective Separation of Polar Compounds Using an Electric Field Coupled Normal Phase HPLC Column

Abstract An electric field coupled method for separation of polar compounds using normal phase high performance liquid chromatography is described. The method, which is not based on electrophoresis or electrochromatography, selectively changes the column capacity for polar analytes. The use of different columns showed improved column efficiency and up to a 62% increase in the number of theoretical plates. The theory for the improvement is presented. The significance and utility of the method for selective analysis of polar fractions in environmental samples is discussed.

The erythro/threo Separation of Non-trimethylsilylated Sphingolipid Bases by Normal Phase HPLC and GLC

The facile determination of erythro/threo ratios of sphingolipid bases without trimethylsilyl groups was made using synthetic Cn-dihydrosphingosines (1) (n=12, 14 and 16) as standard substances. The erythro/threo isomers of (1) could be separated on a normal phase HPLC column using N, O, O-triacetyl and N-acetyl derivatives of (1) [(2) and (3)], and on a GLC column (polyethylene glycol 20 M) using (2). The erythro content of (1) determined by the present methods showed good agreement, and with that by previously reported GLC methods using the trimethylsilyl derivatives of (1) and (3). Deuterium content of synthetic 2-d-Cn dihydrosphingosines can be determined using GC-MS based on the present GLC method.