Gradient HPLC System Research Articles

Liquid Chromatography−Mass Spectrometry of cis- and all-trans-Lycopene in Human Serum and Prostate Tissue after Dietary Supplementation with Tomato Sauce

Several epidemiological studies suggest a lower incidence of prostate cancer in men who routinely consume tomato products. Tomatoes are the primary dietary source of lycopene, which is among the most potent antioxidants of the carotenoids. Men with clinical stage T1 or T2 prostate adenocarcinoma were recruited (n = 32) and consumed tomato sauce based pasta dishes for 3 weeks (equivalent to 30 mg of lycopene per day) before radical prostectomy. Prostate tissue from needle biopsy just before intervention and prostectomy after supplementation from a subset of 11 subjects was evaluated for both total lycopene and lycopene geometrical isomer ratios. A gradient HPLC system using a C(18) column with UV-vis absorbance detection was used to measure total lycopene. Because the absorbance detector was insufficiently sensitive, HPLC with a C(30) column and positive ion atmospheric pressure chemical ionization mass spectrometric (LC-MS) detection was developed as a new assay to measure the ratio of lycopene cis/trans isomers in these samples. The limit of detection of the LC-MS method was determined to be 0.93 pmol of lycopene on-column, and a linear response was obtained over 3 orders of magnitude. Total lycopene in serum increased 2.0-fold from 35.6 to 69.9 microg/dL (from 0.664 to 1.30 microM) as a result of dietary supplementation with tomato sauce, whereas total lycopene in prostate tissue increased 3.0-fold from 0.196 to 0.582 ng/mg of tissue (from 0.365 to 1.09 pmol/mg). all-trans-Lycopene and at least 14 cis-isomer peaks were detected in prostate tissue and serum. The mean proportion of all-trans-lycopene in prostate tissue was approximately 12.4% of total lycopene before supplementation but increased to 22.7% after dietary intervention with tomato sauce. In serum there was only a 2.8% but statistically significant increase in the proportion of all-trans-lycopene after intervention. These results indicate that short-term supplementation with tomato sauce containing primarily all-trans-lycopene (83% of total lycopene) results in substantial increases in total lycopene in serum and prostate and a substantial increase in all-trans-lycopene in prostate but relatively less in serum.

Relationship of serum α-tocopherol, carotenoids and retinol with the risk of breast cancer

The relationship of serum antioxidant vitamin concentration and the risk of breast cancer was investigated in a case-control study in Korea. This study was carried out with 389 subjects consisting of 160 breast cancer patients and 229 control subjects from June 1994 to July 1995. The serum concentrations of α-tocopherol, carotenoids and retinol were measured simultaneously by a reverse phase, gradient HPLC system. Average serum concentrations of α-tocopherol, β-carotene and retinol were 7.63 ± 0.42 ug/ml, 40.5 ± 2.05 ug/dl, and 49.0 ± 4.10 ug/dl for cases and 11.0 ± 0.73 ug/ml, 48.3 ± 1.59 ug/dl and 64.6 ± 1.95 ug/dl for controls, respectively. Serum levels of α-tocopherol, β-carotene, cryptoxanthin, lutein+zeaxanthin, lycopene and retinol of breast cancer patients were significantly lower than those of controls. Odds ratios (ORs) adjusted for age, body mass index, smoking habit and alcohol drinking revealed a significant gradient of decreasing risk of breast cancer with increasing serum antioxidant vitamins. There were significant interactions between menopausal status and serum levels of antioxidant vitamins. The adjusted odds ratios for highest quintile compared with lowest quintile were 0.41 (95% CI, 0.18, 0.93) for α-tocopherol, 0.33 (95% CI, 0.15, 0.73) for β-carotene, 0.13 (95% CI, 0.06, 0.31) for zeaxanthin+lutein in premenopausal women; 0.13 (95% CI, 0.03, 0.66) for α-tocopherol, 0.28 (95% CI, 0.07, 1.12) for β-carotene, 0.12 (95% CI, 0.03, 0.58) for zeaxanthin+lutein in postmenopausal women. This results support inverse association between serum antioxidant vitamins and the risk of breast cancer in Korea and indicate that antioxidant vitamins may protect against breast cancer.

A HPLC–Fluorescence Detection Method for Determination of Cardiac Phospholipase D Activity in Vitro

A nonradioactive assay for the investigation of phospholipase D (PLD) activity in cardiac membranes has been developed. A fluorescent derivative of phosphatidylcholine [2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3proprionyl)amino) undecyl) sn-glycero-3-phosphocholine] was utilized as substrate in an in vitro PLD-catalyzed transphosphatidylation reaction utilizing ethanol as second substrate. Unreacted phosphatidylcholine and the products of phospholipase activity (PEtOH, phosphatidylethanol; PA, phosphatidic acid; DAG, diacylglycerol) were separated by a binary gradient HPLC system and detected by fluorometry. The detection limit of this assay is approximately 0.6 pmol PEtOH. The reaction proceeded at a linear rate for up to 45 min and increased linearly with increasing amounts of rat cardiac membrane protein in a range of 0.625 μg up to at least 25 μg. In the presence of potassium fluoride, formation of fluorescent PA increased at the expense of DAG generation, demonstrating the presence of PA phosphohydrolase activity in rat cardiac membranes. PEtOH formation was unchanged in the presence of the PA phosphohydrolase inhibitor, indicating that the phosphatidylalcohol is not subject to further metabolism by this enzyme. Activation of protein kinase C by phorbol ester significantly increased PLD activity in cardiac membranes. This assay proved to be sensitive for accurate and rapid assessment of PLD activity in cardiac membranes permitting further characterization of the regulation of PLD signal transduction in the heart.

Substrate specificities of 3-oxoacyl-CoA thiolase A and sterol carrier protein 2/3-oxoacyl-CoA thiolase purified from normal rat liver peroxisomes. Sterol carrier protein 2/3-oxoacyl-CoA thiolase is involved in the metabolism of 2-methyl-branched fatty acids and bile acid intermediates.

The two main thiolase activities present in isolated peroxisomes from normal rat liver were purified to near homogeneity. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the first enzyme preparation displayed a single band of 41 kDa that was identified as 3-oxoacyl-CoA thiolase A (thiolase A) by N-terminal amino acid sequencing. The second enzyme preparation consisted of a 58- and a 46-kDa band. The 58-kDa polypeptide reacted with antibodies raised against either sterol carrier protein 2 or the thiolase domain of sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCP-2/thiolase), formerly also called sterol carrier protein X, whereas the 46-kDa polypeptide reacted only with the antibodies raised against the thiolase domain. Internal peptide sequencing confirmed that the 58-kDa polypeptide is SCP-2/thiolase and that the 46-kDa polypeptide is the thiolase domain of SCP-2/thiolase. Thiolase A catalyzed the cleavage of short, medium, and long straight chain 3-oxoacyl-CoAs, medium chain 3-oxoacyl-CoAs being the best substrates. The enzyme was inactive with the 2-methyl-branched 3-oxo-2-methylpalmitoyl-CoA and with the bile acid intermediate 24-oxo-trihydroxycoprostanoyl-CoA. SCP-2/thiolase was active with medium and long straight chain 3-oxoacyl-CoAs but also with the 2-methyl-branched 3-oxoacyl-CoA and the bile acid intermediate. In peroxisomal extracts, more than 90% of the thiolase activity toward straight chain 3-oxoacyl-CoAs was associated with thiolase A. Kinetic parameters (Km and Vmax) were determined for each enzyme with the different substrates. Our results indicate the following: 1) the two (main) thiolases present in peroxisomes from normal rat liver are thiolase A and SCP-2/thiolase; 2) thiolase A is responsible for the thiolytic cleavage of straight chain 3-oxoacyl-CoAs; and 3) SCP-2/thiolase is responsible for the thiolytic cleavage of the 3-oxoacyl-CoA derivatives of 2-methyl-branched fatty acids and the side chain of cholesterol.

Analysis of Corticosteroids in Biofluids by Capillary Electrochromatography with Gradient Elution

Capillary electrochromatography (CEC) with gradient elution was used to separate mixtures of corticosteroids (adrenosterone, hydrocortisone, dexamethasone, fluocortolone) in extracts of equine urine and plasma. Urine samples were first purified using solid phase extraction. Two purification steps were necessary to prevent contamination of the CEC column. Plasma was purified using automated dialysis. A laboratory-built CEC interface, connected to a gradient HPLC system, delivered samples and mobile phase to the CEC column. CEC was performed in fused-silica capillaries of 50 μm i.d., 24 cm total length, and 16 cm effective length packed with Apex ODS, 3 μm particle size. The mobile phase was ammonium acetate (5 mM) in water/acetonitrile. Acetonitrile in the mobile phase was varied from 9 to 80% (v/v) using the gradient HPLC system. Detection was by UV absorbance at 240 nm. Samples, 10-250 μL, were injected into the mobile phase stream and loaded onto the CEC column under an applied field of 1.04 kV cm(-1) and a CEC column head pressure of 12 bar. Mobile phase flow rate through the sampling interface was 100 μL min(-1). The system was reproducible and could be left in unattended operation for long periods. After injection of 200 urine extracts, a broadening of peaks was observed but the CEC column was still serviceable.

Determination of adapalene (CD271/differin®) and retinol in plasma and tissue by on-line solid-phase extraction and HPLC analysis

Adapalene, the active constituent of Differin®, is a novel potent retinoid (vitamin A analogue) for the topical treatment of acne vulgaris. The clinical usefulness of retinoids is limited by a number of side effects, such as teratogenicity and skin irritation. A method has been developed for simultaneous determination of adapalene and retinol in plasma and tissue in in vivo and in vitro studies for the determination of the pharmacokinetic profile and the influence of adapalene on the endogenous retinol level. The new method was developed by coupling an autosampler to an automated solid-phase extraction unit on-line with a gradient HPLC system using UV and fluorescence detection. The low detection limit (0.25 ng mL−1 for adapalene), the small sample weight (50 mg) and the high degree of automation make this method convenient for analysis of biological samples in animal and human studies.

Metallothionein isoform analysis by liquid chromatography-electrospray ionisation mass spectrometry.

Metallothionein (MT) is a class of small metal-binding proteins [ l ] whose mass can vary from under 6OOO to over 7000 daltons depending on a) variation in non-conserved amino acid regions b) the degree of metal saturation and c) the species of metal bound. Many mammalian species have several functioning genes of MT and accurate mass values of the corresponding protein isoforms can be deduced from published gene sequences. However, the translated product of each gene has yet to be confirmed in purified MT protein samples because the isoforms can be difficult to separate by conventional chromatography or electrophoresis. In the case of sheep which have at least 4 functioning MT genes [2], the deduced N-terminally acetylated apoprotein masses are 5993 (MT-la), 6023 (MT-lb), 6027 (MT-lc) and 6070 (MT-2). The objective of this work was to study the proteins by high accuracy, high resolution electrospray mass spectrometry and to confirm their predicted mass values. Metallothionein was purified from the liver of a gray-faced sheep which had been injected with Zn. For LC-MS studies, an LDC 4100 MS gradient HPLC system with 2 x 125 mm C,, column and UV detector was coupled to a MAT 900 mass spectrometer fitted with a PATRIC detector and using an electrospray ionisation source (Finnigan Mat GmbH). Separations were performed using a 2-step linear gradient of 030% acetonimle with 0.1% TFA over 5 min. followed by 30-40% acetonitrile with 0.1% TFA during the remaining 40 min. Flow rate was 300 pl/min. and sample injection was 20 p1 at a concentration of 1 pg/pL. Separated components were detected by UV monitoring at 214 nm prior to electrospray at 3 kV (1 pA) with a capillary temperature of 270'C. Mass spectra were collected over the range lo00 2300 amu using a resolution of 1400 at 5 sec/decade. For flow injection, the same MS parameters were used and samples were dissolved in either water/acetonitrile (1/1) or water/acetonitrile (4/6) + 0.1% TFA. They were then analysed using a 5 FL loop injection with a flow rate of 30 pUmin. Analysis of sheep MT-I proteins by on-line HPLCESI-MS revealed components with distinct mass profiles whose chromatograms can be seen in Fig. 1. Mass spectra and derived deconvoluted spectra of the components resolved by chromatography confirmed the presence of proteins whose masses were within a fraction of predicted values for MT-la, -1b and -1c (Fig. 1, inset table). Indeed, the relative abundance of these isoforms was similar to that reported for the corresponding MT mRNA in sheep liver [3] and the translation of all 3 MT-1 proteins is consistent with the evidence for isoform heterogeneity obtained using capillary electrophoresis techniques [4]. A further minor isoform of mass 5951 was detected in MT-1 (Fig. 1). which is consistent with the presence of a form of deacetylated MT la. This may have physiological relevance since the rate of protein degradation may be influenced by the degree of deacetylation. The study of MT-1 by flow injection ESI-MS at acid and neutral pH gave rise to complex spectra (Fig. 2). A repetitive pattern with a repeat interval of 63-64 amu can be seen, with components at neutral pH being of higher mass than those at low pH. This pattern was caused by a varying degree of Zn or Cu dissociation from MT isoforms depending on the combined effect of pH and electrospray conditions.

HPLC for Windows: A Computer Simulation of High-Performance Liquid Chromatography

HPLC for Windows is a new and updated version of a program previously published for DOS (). It includes a number of new features and has all the advantages of a Windows program. Like the earlier HPLC, it combines emulation of a typical modern binary gradient HPLC system with sophisticated modeling of reversed-phase column behavior. The modeling, based primarily on the work of L. R. Snyder(), provides realistic chromatographic output for separations implemented over a broad range of operating conditions. The program is intended as a pedagogic aid for people who want to learn HPLC theory and practice. As such, it provides easy, inexpensive access to many of the features of a real instrument without risk of damage, consumption of supplies, and the need for constant supervision. It can be used effectively in instrumentation courses as a training exercise prior to hands-on work with the real instrument.

Estimation of ranolazine and eleven Phase I metabolites in human plasma by liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry with selected-ion monitoring

The estimation of ranolazine, a novel piperazine derivative, and eleven of its Phase I metabolites has been undertaken by liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry (LC-APCI-MS). Plasma samples, taken on day 5 of a multiple-dose study, were extracted by solid-phase extraction (SPE) and analysed, using a gradient HPLC system coupled to the APCI source of a Finnigan MAT TSQ 700 mass spectrometer. Metabolites were analysed in selected-ion monitoring (SIM) mode, using an instrument control language (ICL) procedure. The LC-MS combination allowed resolution of all eleven metabolites, including four hydroxylated metabolites and five unresolved components. The results from the linear regression showed good correlation ( r 2>0.980) for all the metabolites. Plasma concentrations indicated that three metabolites were present at levels higher than 10% of the parent compound.

Simultaneous fluometuron and norflurazon analysis in soil extracts and leachates

Abstract Rapid, methanol‐extraction techniques for fluometuron (N, N‐dimethyl‐N'‐[3‐(trifluoromethyl) phenyl] urea) and norflurazon (4‐chloro‐5‐(methylamino)‐2‐(3‐(trifluoromethyl)phenyl)‐3(2(H)‐pyridazinone) from fortified soils have been reported to attain >90% recoveries. Analytical methods involving chromatographic separation coupled with fluorescence detection have also been described. The objectives of this study were to describe an analytical method for the simultaneous detection of fluometuron and norflurazon using ultraviolet spectro‐scopy in soil leachates and extracts and to examine the influence of residence time on herbicide recovery from fortified soil. The analytical method requires a gradient HPLC system, a reverse‐phase C‐18 column, and ultraviolet spectroscopy at a wavelength of 240 nm. The method is characterized by high reproducibility (spike recovery and diluted sample results are generally within 10% of the expected herbicide concentrations), low limits of detection (less than 1 (μg/...

Simultaneous HPLC gradient analysis of 15 benzodiazepines and selected metabolites in postmortem blood.

The simultaneous identification and quantitation of 15 benzodiazepines and selected metabolites in postmortem blood, serum, or liver homogenate is described. The assay involves extraction with diethylether, followed by an acid clean-up step of the ether. Chromatographic separation was achieved on a Nova-Pak phenyl 18 column using ultraviolet detection at 240 nm. A gradient HPLC system was developed to improve separation of nitro-reduction metabolites from the solvent front and endogenous peaks. The mobile phases consisted of a gradient from 15 to 28% acetonitrile in 40 mM potassium phosphate buffer. Within-run and day-to-day precision were generally 10-15%. The method described is sensitive and reproducible for the analysis of benzodiazepine concentrations in postmortem tissues.

HPLC: A computer simulation of high-performance liquid chromatography

This software program combines emulation of a typical modern binary gradient HPLC system with sophisticated molding of reversed-phase column behavior.

Scale-up from analytical to preparative reversed-phase high-performance liquid chromatography of soybean glycinin

Analytical (50 μg) reversed-phase high-performance liquid chromatography (RP-HPLC) of fractions from soy protein purifications has been scaled up to injections in the preparative (1–200 mg) range to obtain sufficient quantities of glycinin subunits for sodium dodecyl sulfate-polyacrylamide gel electrophoresis characterization. We describe here the glycinin separations obtained by using analytical, semi-preparative, and preparative columns with an analytical gradient HPLC system. Parameters such as injection volume, flow-rate, and detector sensitivity are noted. Our results suggest that analytical RP-HPLC separationis of proteins can be conveniently scaled up in most HPLC systems.

High-performance liquid chromatography of water-soluble choline metabolites

We have developed a new method for the separation of [ 3H]choline metabolites by high-performance liquid chromatography. Using this method it is possible to separate, in one step, all of the known major water-soluble choline metabolites present in crude acid extracts of cells that have been incubated with [ 3H]choline, with baseline or near-baseline resolution. We use a gradient HPLC system with a normal-phase silica column as the stationary phase, and a linear gradient of increasing polarity and ionic strength as the mobile phase. The mobile phase is composed of two buffers: Buffer A, containing acetonitrile/water/ethyl alcohol/acetic acid/0.83 m sodium acetate (800/127/68/2/3), and buffer B (400/400/68/53/79), pH 3.6. A linear gradient from 0 to 100% buffer B, with a slope of 5%/min, is started 15 min after injection. At a flow rate of 2.7 ml/min and column temperature of 45°C, typical retention times for the following compounds are (in min): betaine, 10; acetylcholine, 18; choline, 22; glycerophosphocholine, 26; CDP-choline, 31; and phosphorylcholine, 40. This procedure has been applied in tracer studies of choline metabolism utilizing the neuronal NG 108-15 cell line and rat hippocampal slices as model systems. While the compounds labeled in the NG108-15 cells were primarily phosphorylcholine and glycerophosphocholine, reflecting high rates of phospholipid turnover, in the hippocampal slices choline and acetylcholine were the major labeled species. Identification of individual peaks was confirmed by comparing the elution profiles of untreated cell extracts with extracts that had been treated with hydrolyzing enzymes of differing specificities. This HPLC method may be useful in studies of acetylcholine and phosphatidylcholine metabolism, and of the possible interrelationships of these compounds in cholinergic cells.