Use Of Sep-Pak C18 Cartridges Research Articles

Some Methodic Aspects in Optimizing the Radioimmunoassay of β-Endorphin

A specific double antibody radioimmunoassay (RIA) for human beta-endorphin (beta-EP) using an antibody to synthetic beta h-endrophin was established. This antibody cross-reacted with beta-Lipotropin (beta-LPH) at 42 per cent on molar basis and allowed a usable range of 20 pg to 4 ng of beta-endorphin per ml in the assay. Comparing the efficiency of the conventional extraction procedures under various conditions using Corning glass, Vycor glass, QUSO G-32, silicic acid and controlled pore glass 75, the optimal result was obtained by Corning glass, with a recovery rate of more than 80 per cent. The most simple and rapid method with an extraction efficiency of more than 90 per cent was found to be the extraction by use of Sep-Pak C18 cartridges. The separation of beta-endorphin from beta-LPH was studied using Sephadex G-50, G-75, G-100 and Bio-Gel P-60 columns and different elution media. The use of a Sephadex G-50 column (0.9 X 55 cm) and elution with 0.1 N acetic acid-0.05 per cent HSA gave the best result. The reliability of the radioimmunoassay was shown under physiological and pathophysiological conditions.

Substrate reduction reduces gangliosides in postnatal cerebrum-brainstem and cerebellum in GM1 gangliosidosis mice

II3NeuAc-GgOse4Cer (GM1) gangliosidosis is an incurable lysosomal storage disease caused by a deficiency in acid beta-galactosidase (beta-gal), resulting in the accumulation of ganglioside GM1 and its asialo derivative GgOse4Cer (GA1) in the central nervous system, primarily in the brain. In this study, we investigated the effects of N-butyldeoxygalacto-nojirimycin (N B-DGJ), an imino sugar that inhibits ganglioside biosynthesis, in normal C57BL/6J mice and in beta-gal knockout (beta-gal-/-) mice from postnatal day 9 (p-9) to p-15. This is a period of active cerebellar development and central nervous system (CNS) myelinogenesis in the mouse and would be comparable to late-stage embryonic and early neonatal development in humans. N B-DGJ significantly reduced total ganglioside and GM1 content in cerebrum-brainstem (C-BS) and in cerebellum of normal and beta-gal-/- mice. N B-DGJ had no adverse effects on body weight or C-BS/cerebellar weight, water content, or thickness of the external cerebellar granule cell layer. Sphingomyelin was increased in C-BS and cerebellum, but no changes were found for cerebroside (a myelin-enriched glycosphingolipid), neutral phospholipids, or GA1 in the treated mice. Our findings indicate that the effects of N B-DGJ in the postnatal CNS are largely specific to gangliosides and suggest that N B-DGJ may be an effective early intervention therapy for GM1 gangliosidosis and other ganglioside storage disorders.

Determination of local anaesthetics in body fluids by gas chromatography with surface ionization detection

Ten local anaesthetics were tested for their detection by gas chromatography (GC)-surface ionization detection (SID). Lidocaine, mepivacaine and bupivacaine were detected with the highest sensitivity; their detection limit was 5-10 pg in an injected volume. The sensitivity of other drugs, such as procaine, dibucaine tetracaine and oxybuprocaine, was an order of magnitude lower than that of the above three local anaesthetics. A detailed procedure for isolation of local anaesthetics from human whole blood and cerebrospinal fluid (CSF) by the use of Sep-Pak C18 cartridges, before the GC-SID, is also presented. The recovery of lidocaine, mepivacaine and bupivacaine, which had been added to 1 ml of whole blood or CSF, was close to 100%.

Rapid isolation with Sep-Pak C18 cartridges and wide-bore capillary gas chromatography of some barbiturates.

A simple and rapid method for isolation of nine barbiturates with Sep-Pak C18 cartridges from human urine, plasma and whole blood, is presented; the detection of drugs was made by wide-bore capillary gas chromatography (GC) with flame ionization. The drug-containing samples, after mixing with dilute acid solution, were directly applied to the cartridges and eluted with either chloroform/methanol (9:1) or acetonitrile. Separation of the nine drugs was satisfactory with use of an intermediately polar HP-17 capillary column. Recoveries of most compounds were excellent for both chloroform/methanol and acetonitrile as elution solvents. However, backgrounds were cleaner and evaporation time was much shorter for the chloroform/methanol system. The present isolation method with use of Sep-Pak C18 cartridges and wide-bore capillary GC seem very useful in the fields of forensic chemistry, clinical toxicology and clinical pharmacology.

Improvements in the simultaneous determination of calcidiol and calcitriol in human serum or plasma.

Technical improvements have been applied to reduce the time required for the determination of calcidiol and calcitriol in serum or plasma. The modifications include the use of Sep-Pak C18 cartridges for the extraction of calciol metabolites from serum instead of a classical liquid/liquid extraction, a considerable shortening of the HPLC purification time compared with our previously described method, and the application of HPLC with UV detection at 254 nm of calcidiol as an alternative to the usual competitive protein binding methods. In none of the 199 samples where calcidiol was determined by HPLC did we observe a detectable peak of ercalcidiol. Quantitation of calcidiol by HPLC and competitive protein binding was compared in 5 series of assays. The correlation between the two methods was 0.99. The average slope of the linear regression line when the HPLC values were plotted versus the competitive protein binding values was 1.14. The average intercept was 0.19 nmol/l. The mean within-run coefficient of variation for calcidiol in these series was 5% for the competitive protein binding method, and 4% for HPLC method. Between-run coefficients of variation were 6% and 12% for the competitive protein binding and for the HPLC method, respectively. Within-run and between-run coefficients of variation for calcitriol were 6% and 15%, respectively.

‘Dynorphin’ in plasma: enzymatic artifact and authentic immunoreactivity

The potent opioid peptide dynorphin (DYN) is found in posterior pituitary vasopressinergic neurones and in adrenal medullary cells suggesting that secretion into plasma is likely. We have developed a sensitive radioimmunoassay in order to study plasma DYN in man. It transpired that extraction prior to assay was essential since unextracted plasma caused gross and non-parallel inhibition of binding of tracer. Plasma extracted using leached silica glass (Vycor) caused inhibition of tracer binding which diluted in parallel to synthetic DYN suggesting the presence of substantial amounts of DYN-like immunoreactivity (irDYN) in plasma. Further investigation however demonstrated that this irDYN was artifactual and caused by enzymatic degradation of tracer. Although use of Seppak C18 cartridges resulted in reliable extraction of synthetic procine DYN from acidified plasma, we have not detected irDYN in any plasma so far studied using this technique. However, extraction of non-acidified plasma using our antibody coupled to Sepharose CNBr-activated 4B followed by gel filtration chromatography demonstrated a single peak of irDYN of molecular size similar to DYN. These data suggested that a small amount of a DYN-like peptide does circulate in human plasma although this is not identical to porcine DYN(1–17). The implication of our results for the measurement of other similar peptides in plasma is discussed.

An evaluation of the use of sep-pak C18 cartridges for the extraction of vitamin D 3 and some of its metabolites from plasma and urine

The use of Sep-Pak C18 cartridges for the extraction of vitamin D and some of its metabolites from plasma and urine has been evaluated by studying the recovery of added tritiated secosteroids. The preparation of the cartridges, recoveries, extraction and elution with a number of solvents, effect of varying flow rates for application and elution, and the effect of increasing volumes of plasma and urine have been investigated. Two methods for the application of secosteroids present in plasma to Sep-Pak C18 cartridges have been examined, using methyl cyanide extracts removing precipitated protein by centrifugation, and using acidified methanolic plasma. Methyl cyanide extracts applied to Sep-Pak C18 cartridges and eluted with methanol or methyl cyanide gave the cleanest extracts suitable for direct HPLC. Acidified methanolic plasma, applied to Sep-Pak C18 cartridges and eluted with methanol or methyl cyanide gave extracts which could not be applied directly to an HPLC-further fractionation using Sep-Pak SIL cartridges was necessary. Recoveries of added tritiated secosteroids using both methods were greater than 80% with the exception of vitamin D itself which was poorly recovered-methyl cyanide extraction giving only 30% recovery and use of acidified methanolic plasma giving 66% recovery.

SEP-PAK Preparative Chromatography: Use in Radiopharmaceutical Synthesis

Abstract The use of SEP-PAKR 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.

The use of Sep-Pak C18 cartridges during the isolation of gangliosides.

The use of Sep-Pak C18 reverse-phase cartridges to adsorb gangliosides from aqueous solutions was studied. When upper phases formed from chloroform-methanol tissue extracts or aqueous salt solutions containing gangliosides are rapidly passed through the cartridges, the lipids are retained and the non-lipid components can be washed through. Gangliosides and other retained lipids can subsequently be eluted with methanol or chloroform-methanol