Solid Extraction of Steroid Conjugates from Plasma and Milk

Abstract

Extraction of steroids and steroid conjugates with Amberlite XAD-2 and Sep-Pak C₁₈ has been studied. Amberlite XAD-2 has ionic sites with high affinity for conjugated steroids, particularly steroid disulphates. Nearly quantitative recoveries of steroid conjugates are obtained when the resin is washed with an aqueous solution of a sulphate prior to elution with methanol. Such treatment is not required using Sep-Pak C₁₈ cartridges.

The adsorbents were used for extraction of steroids and their conjugates from plasma and milk. Steroid-protein and steroid-lipid interactions were minimized by diluting the fluid twice with 0.5 M aqueous triethylamine sulphate and passing the solution through the adsorbent a t 64°C. Steroids were eluted with methanol and methanol/chloroform at room temperature. Essentially quantitative recoveries were obtained with both adsorbents.     

A Novel Method for the Quantitation of Warfarin and its Metabolites in Plasma

Abstract

A procedure for the rapid, quantitative recovery of warfarin and its metabolites (diastereoisomeric alcohol, 4′-, 6-, 7-, 8-benzylic-hydroxywarfarin and dehydrowarfarin) from plasma with Sep-Pak C₁₈ cartridges has been developed. A solution of warfarin and its metabolites in plasma was acidified with NH₄OAc buffer (pH 4.85), adsorbed on the Sep-Pak C₁₈ resin, washed free of polar constituents and eluted with methanol. Dilution of the eluate with buffer followed by gradient high performance liquid chromatography permitted accurate quantitation of the desired compounds when detected at 313 nm. The recovery of warfarin and each metabolite was greater than 95% over an investigated range of 0.5–10.0 μg/ml of plasma and the limit of quantitation by the assay was 0.1 μg/ml of plasma. For more rapid quantitation of warfarin, without simultaneous analysis of metabolites, the chromatographic parameters were modified so that elution of warfarin occurred within 13 minutes, and the sensitivity of the assay increased to 0.03 μg of warfarin/ml of plasma. The quantitative recovery of warfarin and its metabolites coupled with the chromatographic versatility of the method make it ideally suited for either detailed pharmacokinetic studies or routine plasma analysis of warfarin.     

Reliable System for the Analysis of Riboflavin in Foods by High Performance Liquid Chromatography and UV Detection

Abstract

A high performance liquid chromatographic (HPLC) method has been developed to determine riboflavin in food samples. A reverse phase C₁₈ column with ultraviolet detection was employed. Sample preparation included acid and enzimatic hydrolysis, followed by purification on Florisil and Sep-Pak cartridges. Recoveries of 98% were obtained. A detection limit of 0.4 ng/injection has been achieved.     

Improved Extraction Method and Chromatographic Separation of Ginsenosides from Ginseng Roots,Leaves and Ginseng Drug Preparations

A new extraction method for ginsenosides from ginseng roots, ginseng leaves and ginseng drug preparations by Sep-Pak C₁₈ cartridges has been studied. Ginsenoside extraction by Sep-Pak cartridges is a rapid, efficient, reproducible method. In addition, the extracts were analyzed by high performance thin layer chromatography (HPTLC) and reverse phase high performance liquid chromatography (HPLC). The major components of ginseng saponins were effectively separated using an ODS-120T column.     

Determination of Organophosphorus Pesticides in Water Using C18 or Florisil Sep-Pak Cartridge and Gas Chromatography with Flame-Photometric Detection

Simple methods for the concentration and clean-up of fifteen organophosphorus pesticides in water using a C₁₈ Sep-Pak cartridge or a Florisil Sep-Pak cartridge and subsequently determining the pesticides by gas chromatography with flame photometric detection have been developed. The conditions for stepwise or simultaneous desorption of these pesticides from the Sep-Pak cartridges are given.     

Purification of Radioiodinated Peptides with PRP-1 Polystyrene Cartridges and HPLC: Application to Atrial Natriuretic Factor and Vasopressin

Abstract

A simple and rapid cleanup procedure is described for the purification of iodinated peptides using PRP-1 polystyrene cartridges following the radioiodination process. The method is validated using different volumes and solvent systems and compared to the standard Sep-Pak C₁₈ procedure. In this study, the method is used to prepare ¹²⁵I-labeled atrial natriuretic factor and arginine-vasopressin which are further purified by reverse phase HPLC giving maximally obtainable specific activity required for the radioimmunoassays of these peptides.     

A Comparison of Sample Preparation Techniques for the Determination of an Organo-phosphorous Pesticide from Water Using Reverse-Phase HPLC

Abstract

A methylene chloride liquid/liquid extraction and Sep-Pak C₁₈ cartridge adsorption techniques were used to quantify the pesticide, Dursban, in contaminated environmental water samples. Results showed a large disparity between Dursban levels using these two techniques, due primarily to the presence of a large adsorbed fraction of pesticide. A Sample Clarification Kit was used to isolate the particulate fraction, which can subsequently be stripped of its adsorbed pesticide compliment by means of a methanol rinse. Lastly, the filtrate from the Sample Clarification Kit may be trace enriched on a Sep-Pak C₁₈ cartridge to isolate the dissolved fraction of pesticide.     

Fast solid phase extraction of polychlorobiphenyls and chlorinated pesticide residues from mussels using sep-pak cartridges

Summary A rapid method for the determination of chlorinated pesticides and polychlorinated biphenyls in mussels (Mytilus sp.) is reported. The mussel sample is homogenized and extracted with acetonitrile. The organic solution is concentrated and successively diluted with distilled water solution (12 g L⁻¹ NaCl). The organic compounds from water solution are adsorbed onto a NH₂ Sep-Pak cartridge. The clean-up step, in which the polychlorobiphenyls and chiorinated pesticides are separated in different eluates, is achieved by passing 25 mL of a 40% methanol aqueous solution through the NH₂ Sep-Pak and the C₁₈ Sep-Pak cartridges connected in series. The polychloroblphenyls are desorbed from the NH₂ Sep-Pak cartridge whilst the chlorinated peslicides are recovered from the C₁₈ Sep-Pak cartridge. In the separation of polychlorobiphenyls from the chlorinated pesticides tested in this work, only aldrin, hepatachlor and 4,4′-DDD are partially adsorbed with the polychlorobiphenyls onto the NH₂ Sep-Pak cartridge. The average recovery is ≥95.0% with a relative standard deviation ≤5.0%. The limits of detection for different pesticides and polychlorobiphenyl congeners are 0.01 and 0.008 μg Kg⁻¹. The final determination is carried out by capillary gas chromatography with ECD.     

Determination of Chlorinated Hydrocarbons Using Sep-Pak Cartridges and Gas Chromatography

Methods are described for the determination of chlorinated hydrocarbons at levels of parts per billion in water and nonaqueous environmental samples by gas chromatography with electron-capture detection. C₁₈, Florisil, alumina N and silica Sep-Pak cartridges were compared to evaluate their cleanup ability. The accuracy of the results of this analytical technique was proved by the analysis of a certified reference material (lake sediment EC-2).     

Rapid Quantitative Method for the Determination of Dextrose,Mannitol, and Sorbitol in Meat Products by Liquid Chromatography

Abstract

A liquid chromatographic (LC) method for the determination of dextrose, mannitol, and sorbitol in meat products was developed. Dextrose, mannitol, and sorbitol were extracted from comminuted meat products with 52% ethanol. After filtration, the extracts were purified by passing through a C₁₈ Sep-Pak cartridge and two ion exchange resin Econo-Columns in series. After concentration and filtration, extracts were analyzed by liquid chromatography using a cation exchange analytical column and a differential refractometer detector. Homogeneously ground samples of cooked and fresh sausages and ground beef were fortified with dextrose, mannitol, and sorbitol at four different concentrations. Average overall recovery for all three compounds at all four levels of fortification was greater than 80% with coefficients of variation less than 10%.     

Preconcentration of Volatile Hydrocarbons as Air Pollutants by C-18 Bonded Silica used for Solid Phase Extraction

ABSTRACT

C₁₈-Silica used for Solid-Phase Extraction exhibits the same degree of adsorption of volatile hydrocarbons as compared to conventional Tenax adsorbent. The vapor pressure of the hydrocarbons and the velocity of the air sample through the sorbent are dominants of the preconcentration. Recovery of 80% for n-hexane and 98% for p-xylene at a concentration of 10 mg.m^?3 was obtained at 10 ml.g^?.min^? velocity.

C₁₈Bond Elut cartridges have been successfully used for quantitative determination of hydrocarbons as air pollutants by gas chromatography. The detection limit for p-xylene using preconcentration from 1 L air sample and a S/N ratio of 5 was 0.1 mg.m^?3. After regeneration of C₁₈Bond Elut cartridges by washing with acetonitrile and diethyl ether and drying at 85°C/15 min, their preconcentration potential remain acceptable upon reusing at least three times.     

Detection of organic toxic pollutants in water and waste-water by liquid chromatography and in vitro cytotoxicity tests

In vitro toxicity tests with fish cell lines have proved to be correlated with in vivo toxicity tets on fish. A group of toxicity tests on RTG-2 cell line (a fibroblastic line derived from rainbow trout) has been standardized in order to enhance reproducibility and sensitivity. The liquid chromatographic (LC) separation of organic chemicals from industrial effluents and polluted waters and the use of in vitro toxicity tests on RTG-2 as a biological detector of toxicity in the eluted peaks are reported. Effluents and polluted waters were concentrated, if required, using Sep-Pak C₁₈ cartridges, and analysed by reversed-phase LC using a 30-cm C₁₈ column with an acetonitrile gradient from 10 to 100% in water in 60 min at a flow-rate of 1 ml min^?1 and UV detection at 254 and 280 nm. The cytotoxicity test was adapted to use 20-μl fractions of acetonitrile-water mixtures, allowing toxicity detection every 12 s with eight replicates per sample (or every 5 s with four replicates).     

SEP-PAK Preparative Chromatography: Use in Radiopharmaceutical Synthesis

Abstract

The use of SEP-PAK^R C₁₈ cartridges for the isolation and purification of radiopharmaceuticals, labeled with the 20.4 minute half-life radionuclide carbon-11, is reported. Synthesis and SEP-PAK preparative chrotmatographic purification of [1-¹¹C]palmitic acid, [¹¹C-methyl]benzyl methyl ether, [1-¹¹C]butan-1-ol, and [1-¹¹C]pyruvic acid are described. The use of SEP-PAK C₁₈ cartridges has allowed development of rapid and remote methods for handling of high amounts (> 100 mCi) of radioactive products.     

A Rapid Method for Extraction and Estimation of Abscisic Acid from Plant Tissue Using High Performance Liquid Chromatography

Abstract

A simple, rapid and economical method for the determination of the abscisic acid content in different plant organs was described. Silica Sep-Pak prepacked cartridges were used for prepurification of plant extracts. The abscisic acid content in the extract was determined by HPLC.     

A Fractional Factorial Design Applied to the Optimization of Microwave- and Ultrasound-Assisted Acid Leaching Methods for Heavy Metals Determination in Sludges by Flame Atomic Absorption Spectrometry

Abstract

The sampling performance of C₁₈ cartridges coated with DNPH has been studied for twenty four C₁-C₉ carbonyls in experiments involving sampling of parts per billion levels of carbonyls in urban air. indoor air and laboratory experiments. The cartridge background carbonyl content in thirty six batches of cartridges averaged 85, 137 and 155 nanogram/cartridge for formaldehyde, acetaldehyde and acetone, respectively, and was below analytical detection for all other carbonyls. Carbonyl-DNPH derivative recovery from the cartridge was complete in the first elution with 2 mL acetonitrile, and this for twenty four carbonyls at concentrations of 0.02–73 μg carbonyl/cartridge. Studies carried out using two cartridges in series showed no breakthrough, for the sixteen carbonyls tested, at concentrations of 0.10–49 μg carbonyl/cartridge and volumes of air sampled = 6–370 L. Average relative standard deviations (RSD) for replicate analyses were 0.20–13.2% for twenty one carbonyls. Average RSD for co-located samples were 0.9–16.2% for eighteen carbonyls. Comparison of RSD for replicates and RSD for co-located samples for thirteen carbonyls indicated that the overall method precision was limited by sampling precision rather than by analytical precision.     

High-Performance Liquid Chromatographic Determination of Trace Quantities of Azinphos Methyl and Azinphos Methyl Oxon in Various Water Sources by Direct Injection and Trace Enrichment

Abstract

A high-performance liquid chromatographic (HPLC) method is described for the determination of trace amounts of the organophosphate insecticide, azinphos methyl and its degradation product, azinphos methyl oxon, by direct injection and by trace enrichment. The compounds were analyzed on a uBondapak C₁₈ column with UY detection at 224 nm. The mobile phase for the analysis was acetonitrile-water (50:50) at a flow rate of 1.3 ml/min. Ten minutes were required for the chromatographic analysis. Water from three sources, public water supply, stream, and ocean was analyzed for azinphos methyl and azinphos methyl oxon at concentrations as low as 11.9 and 11.3 ppb, respectively, without a clean-up, concentration or derivatization step. Azinphos methyl was quantitated at 0.29 ppb and azinphos methly oxon at 0.29 ppb by employing a concentration step involving a C₁₈ Sep-Pak cartridge. The coefficients of variation for all determinations ranged from 0.77 to 9.06%. Peak heights were used for quanti-tation. Several other pesticides have been shown not to interfere with the analysis of either compound.     

The Semi-Preparative Separation of Peptides on Reversed Phase Silica Packed into Radially Compressed Flexible-Walled Columns

Abstract

The semi-preparative separation of underivatised peptide mixtures from the tryptic digestion of thyroid and pituitary proteins has been accomplished on a 10dp spherical octadecyl-silica stationary phase, Radial Pak A/C₁₈, packed into flexible-walled polyethylene cartridges (10 × 0.8cm). With volatile ionic modifiers, such as ammonium bicarbonate, excellent resolution and peptide recoveries were obtained with this support     

An Improved Technique for Extraction,Identification, and Quantification of Leukotrienes

Abstract

An improved method for extraction, separation and quantification of leukotrienes is described. Critical steps include extraction in thoroughly cleaned Sep-Pak C₁₈ mini columns, elution with 90 percent methanol in water, addition of 0.25 percent Na₄EDTA to the solvent system of methanol/water 60/40, pH 7.4, injection of the samples in 1 ml of the same system after lowering the pH to 3.0 with glacial acetic acid and utilization of a Z-module containing a 5 μm C₁₈ reverse phase cartridge. Recovery of the leukotrienes was 94 pm 8% mean pm SD for LTC₄, 89 pm 5% for LTD₄, 89 pm 3% for LTB₄ and 84 pm 6% for LTE₄. We had no problems with precipitation of Na₄EDTA and occlusion of the HPLC fittings. The method is simple, reproducible and easily adaptable to any research laboratory.     

Enzymatic Fluorimetric Determination of Ursodeoxycholic Acid in Urine Using Clostridium Absonum 7β-Hydroxysteroid Dehydrogenase

Abstract

A simple and rapid enzymatic fluorimetric method for the determination of ursodeoxycholic acid (UDCA) and its glycine (GUDCA) and taurine (TUDCA) conjugates in urine has been developed. Octadecylsilane-bonded silica cartridges (Sep-Pak C₁₈) are used for the solid-phase extraction of bile acids (BA) from urine samples. the method is based on the fluorimetric monitoring of NADPH formed via the reaction of 7β hydroxylated BA (7β-BA) with β-nicotinamide adenine dinucleotide phosphate (β-NADP⁺) catalysed by 7β hydroxysteroid dehydrogenase (7β-HSD). the 7β-HSD, which is not yet commercially available, was isolated from Clostridium absonum cultures (ATCC # 27555) and purified by affinity chromatography.

The method has a limit of detection of 2 μmol/L (initial sample concentration), within-run precision varied from 8.3% to 5.3% and between-run precision varied from 12% to 1.8% for low and high concentrations respectively. the recovery of ursodeoxycholic acid added to urine samples was about 98% (range 88–110%). the method was successfully applied for UDCA determination in urine samples from patients subjected to UDCA therapy. Randomly collected urine samples from patients and controls were used and the results were expressed as ratio of [UDCA]/[creatinine] to correct for variation in urine flow.     

Rapid sample preparation for the gas-chromatographic determination of methohexital in serum using reversed phase cartridges

Conclusion The proposed sample preparation for the gaschromatographic determination of methohexital in serum by using SEP-PAK C₁₈ cartridges is superior to the usual extraction by organic solvents because of its good reliability and practicability. Its main advantages are: 1) constantly high recovery; 2) complete removal of endogenous substances that might interfere with the determination resulting in a rather specific method; 3) ease of handling.

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Schnelle Probenvorbereitung für die gas-chromatographische Bestimmung von Methohexital im Serum unter Verwendung von Reversed-Phase-Kartuschen