Gel Chromatography Column Scanning (GCS) Method of Choice for Quality Control of 99mTc-Plasmin Preparations

Abstract

Gel chromatography column scanning (GCS) is useful for testing compounds labelled with gamma-emitting radionuclides. The elution volume used in this technique is so small that no components of the sample are eluted from the column. In the radioactivity distribution of the sealed column various species are recorded in characteristic zones of the column. Plasmin labelled with ^99mTc is used for scintigraphic detection of deep vein thrombosis. The quality of the ^99mTc-plasmin preparation has been tested by various methods. The GCS method employing small columns offers a very fast testing procedure and adequate resolution for quality control in routine radiopharmaceutical work.     

PREPARATION OF L-CYSTEINE-35S HYDROCHLORIDE BY REDUCTION OF L-CYSTINE-35S

Abstract

The method for the synthesis of L-cysteine hydrochloride labelled with ³⁵ is described. L-Cystine- ³⁵ S, obtained from baker's yeast, was reduced with tin in hydrochloric acid and radiochemical pure L-cysteine- ³⁵-hydrochloride was isolated by ion-exchange chromatography on a column.     

α-Melanotropin Labelled at its Tyrosine2 Residue: Synthesis and Biological Activities of 3′-Iodotyrosine2-,3′-125Iodotyrosine2-,3′,5′-Diiodotyrosine2-, and (3′,5′-3H2)tyrosine2-α-Melanotropin,and of Related Peptides

α-MSH was labelled at its tyrosine² residue with tritium and iodine. Several synthetic routes were investigated by preparing 13 precursor or mode compounds and 4 different labelled products (via about 40 intermediates). Their melanotropic activity was determined with an in vitro frog skin assay and, for some of the compounds, with a tyrosinase assay. The tritiation was performed on [Tyr(I₂)²]α-MSH by catalytic halogen/tritium exchange, yielding α-MSH of high specific radioactivity (34 Ci/mmol) and full biological activity. Iodination was studied in detail using five different techniques. An equimolar chloramine T procedure proved to be the most convenient and reproducible method, resulting in monoiodinated α-MSH containing 99% of the label in position 2. The biological activity was 50% that of α-MSH; the specific radioactivity, determined in a competitive binding assay with a highly specific α-MSH antiserum and [Tyr(I)²]α-MSH as competitor, was 1530 Ci/mmol. The labelling techniques and the bioligical results are discussed.     

Tritium labelling of hexamethyldisiloxane for 3H NMR referencing

Tritium labelled hexamethyldisiloxane is a suitable material for the internal referencing of ³H NMR spectra. Such a referencing procedure is necessary where the instrument is not capable of scanning ¹H resonance under identical experimental conditions. Hexamethyldisiloxane may be tritiated to high activity (typically 1 Ci ml^?1) by Raney nickel catalysed exchange with elemental tritium in a simple one-step procedure. A variety of heterogeneous catalysts are shown to be inactive in the common labelling method involving exchange with tritiated water.     

Tritium nuclear magnetic resonance spectroscopy. 14—analysis of tritiated methyl groups

The complete analysis of the tritium distribution in tritiated methyl groups in labelled compounds is achieved by a direct method for the first time, through ³H NMR spectroscopy aided by resolution enhancement.     

Study of kinetics,equilibrium and isotope exchange in ion exchanger systems

An automatic, rapid combustion method has been developed for the determination of tritium and¹⁴C in singly or doubly labelled organic materials by liquid scintillation counting. The sample is burned in a stream of oxygen. The water formed and its tritium content are retained from the gas stream in an absorber containing a small amount of diethyleneglycol monoethyl ether. Radioactive carbon dioxide, if included in the combustion products, is transferred into 3-methoxypropylamine. The final solutions ready for counting are obtained in less than three minutes. Quantitative collection recoveries for both tritium and¹⁴C are achieved and no cross-contamination occurs.     

A semi-automatic combustion method for the simultaneous determination of3H and35S in doubly labelled organic materials by liquid scintillation counting

A sample preparation method developed for the simultaneous liquid scintillation assay of tritium and³⁵S in doubly labelled organic materials is described. The sample is burnt in a stream of oxygen and the radioactive isotope carriers formed are collected separately for individual counting.³⁵S content of the sample is measured as a dilute sulfuric acid solution, while the tritium is counted as water. The procedure is free of cross-contamination and memory effect, provides quantitative analytical recovery, and the final solutions ready for counting are obtained in twelve minutes.     

Purification of Radio-Iodinated Cholecystokinin Peptides by Reverse Phase HPLC

Abstract

The purification of the iodinated tracers of CCK peptides using Sphadex G5O chromatography does not allow for a good separation between non-modified peptides and labelled peptides. We present, in this paper a simple and rapid purification method using reverse phase HPLC with a C-18 column for four of these tracers. The biological characteristics of the molecules obtained demonstrate their strong specific radioactivity and their high degree of purity.     

Preparation of substituted cinnamic acids labelled with deuterium and tritium at the ring positions

A series of substituted cinnamic acids labelled at the ring positions with deuterium or tritium has been easily obtained via the Doebner modification of Knoevenagel condensation reaction between labelled benzaldehyde derivatives and malonic acid in pyridine. The substituted benzaldehyde precursors were synthesized by isotope exchange method in deuterated or tritiated water at 80 °C in acidic medium. Ring-tritiated substituted cinnamic acids with specific activity ranging from 207 GBq /5.6 Ci/ mol^–1 to 4366 GBq /118 Ci/ mol^–1 was obtained using ca. 37 GBq /1 Ci/ of HTO.     

High Performance Liquid Chromatographic Determination of Pirenzepine Dihydrochloride in Its Pharmaceutical Formulation

Abstract

A high-performance liquid chromatographic procedure for the determination of pirenzepine dihydrochloride as a bulk material and in its tablet dosage form (Gastrozepin^R) is presented. Normal phase liquid chromatography has been performed on a Micro-pack Si-10 column using ammonium hydroxide (28–30% NH₃) in methanol (0.75: 99.25% v/v) as mobile phase at a flow rate of 2 ml/min. Clobazam has been used as internal standard with retention times of 1.9 and 2.8 minutes for clobazam and pirenzepine dihydrochloride, respectively at 254 nm. Analytical calibration yields a linear relationship between 5 and 25 μg/ml, with correlation coefficient of 0.999. Tablets each labelled to contain 25 mg pirenzepine dihydrochloride give mean percentage found of 99.98 ± 0.4. A plot of logarithm of concentration against time for a solution in 6 N hydrochloric acid gives a straight line with a slope of - 0.197 day^?1. The proposed method is, therefore, a stability indicating method.     

Trace amine receptor ligands: tritiation at high specific activity

d-amphetamine and tyramine are both amines which are active at the recently discovered trace amine-associated receptor 1 (TAAR1) and analogues of these compounds labelled with tritium would be useful reagents for neurochemistry investigators. This paper describes the tritiation and characterization of these substances. d-amphetamine was radiolabelled by a direct tritium catalytic method and characterized by proton decoupled tritium NMR. For tyramine, a bromination??catalytic tritium debromination approach worked best to afford a tritiated analogue at high specific activity.     

Screening of Benzoylecgonine in Urine by Cyclobond Solid Phase Extraction and High Performance TLC

Abstract

A method is described for screening of the cocaine metabolite benzoylecgonine in urine at 0.2 μ/ml using a Cyclobond cyclodex^?trin solid phase extraction cartridge and high performance thin layer chromatography. Results are compared with those obtained using liquid-liquid and diatomaceous earth column extraction and C-18 solid phase extraction.     

Two Approaches for Sequential Extraction of Radionuclides in Soils: Batch and Column Methods

Abstract

A three-step sequential extraction protocol designed by Community Bureau of Reference (BCR) is applied to two types of soil (sandy and sandy-loam) which had been previously contaminated with a radionuclide aerosol containing ¹³⁴Cs, ⁸⁵Sr and ^110mAg. This scheme is applied using both batch and column methods. The radionuclide distribution obtained with this scheme depends both on the method and on soil type. Compared with the batch method, column extraction is an inadvisable method. Kinetic aspects seem to be important, especially in the first and third fractions. The radionuclide distribution shows that radiostrontium has high mobility, radiocaesium is highly retained by clay minerals whereas Fe/Mn oxides and organic matter have an important role in radiosilver retention.     

Separation of Testosterone and Dihydrotestosterone in Semen by High Performance Liquid Chromatography

Abstract

We have established a method for the separation of testosterone and dihydrotestosterone in seminal plasma by high performance liquid chromatography. The separation column is a reversed phase column (ODS). The mobile phase is methanol-water (8:2) and the flowrate is 1.5 m1/min. It takes 12–14 min to separate a sample. The recoveries are 96.4% and 96.5% respectively by radioactivety labelled steroids. We have separated 600 seminal plasma samples with this method. The separated testosterone and dihydrotestosteroneare determined by radioimmunoassay. The result have shown that the method is satisfactory.     

High Performance Liquid Chromatographic Method for Prostaglandin E2 Determination in Human Gastric Juice Without Derivatization

Abstract

A rapid and practical method for the separation and quantitation of PGE² in human gastric juice by high performance liquid chromatography is described. Separation on a reversed-phase column and UV detection allows quantities as little as 20 nanograms of PGE² to be detected. Specificity, sensitivity, high yield and reproducibility make this method particularly suitable for prostaglandin determination in human gastric juice.     

Synthesis of radiolabelled compounds

Compounds labelled with radioisotopes such as¹⁴C,³H,³²P,³⁵S and¹²⁵I, are essential tools for research and especially in the life sciences. Syntheses can be directed to isotopic labelling or to non-isotopic labelling. In isotopic labelling a compound is labelled with an isotope of an element already present in the compound whereas non-isotopic labelling is achieved with an isotope is foreign to the material or compound being labelled. This paper reviews the properties of the more important and commonly used radioisotopes in research and methods currently employed for the synthesis of radiolabelled compounds. The relative ease of measurement of radioactivity in large numbers of samples, when compared for example with the measurement of mass, and the great sensitivity with which very small quantities of radioactive compounds can be accurately measured, has resulted in the widespread need and use of radiolebelled compounds. Methods for the practicable labelling of compounds for use as radiotracers all into three main categories, namely, chemical systhesis, biochemical methods and isotope exchange reactions. Examples are chosen to illustrate these methods for the labelling of amino acids, peptides, proteins, carbohydrates, lipids, neurochemicals, nucleic acids, steroids and miscellaneous compounds of interest in biological research. Most methods of labelling employed lead to specific labelling, that is, to modecules in which the position(s) of the radioactive atom(s) is known with certainty. However, isotope exchange reactions which are especially useful for labelling molecules with tritium are often non-specific. The routine use of tritium nuclear magnetic resonance spectroscopy has resolved any uncertainties in the specificity of labelling with tritium. Examples are given illustrating the effectiveness of this valuable analytical technique. A review of the synthesis of radiolabelled compounds would not be complete without reference to the special properties of the labelled compounds themselves which affect their useful shelf-life, self-decomposition, purification and analysis; factors which all need to be clearly understood in order to use radiolabelled compounds with confidence in scientific research.     

Determination of five nucleosides by LC‐MS/MS and the application of the method to quantify N6‐methyladenosine level in liver messenger ribonucleic acid of an acetaminophen‐induced hepatotoxicity mouse model

Ribonucleic acid N⁶‐methyladenosine methylation plays an important role in a variety of biological processes and diseases. Acetaminophen‐induced hepatotoxicity is one of the major challenges faced by clinicians. To date, the link between N⁶‐methyladenosine and acetaminophen‐induced hepatotoxicity has not been studied. In this study, a simple ultra high performance liquid chromatography with tandem mass spectrometry method was developed for the simultaneous determination of five nucleosides (adenosine, uridine, cytidine, guanosine, and N⁶‐methyladenosine) in messenger ribonucleic acid. After enzymatic digestion of messenger ribonucleic acid, the nucleosides sample was separated on an Acquity UPLC column with gradient elution using methanol and 0.02% formic acid water, and detected by a Qtrap 4500 mass spectrometer with an electrospray ionization mode. The method was validated over the concentration ranges of 4–800 ng/mL for adenosine, uridine, cytidine, and guanosine and 0.1–20 ng/mL for N⁶‐methyladenosine. It was successfully applied to the determination of N⁶‐methyladenosine levels in liver messenger ribonucleic acid in an acetaminophen‐induced hepatotoxicity mouse model and a control group. This study offers a method for the determination of nucleoside contents in epigenetic studies and constitutes the first step toward the investigation of ribonucleic acid methylation in acetaminophen‐induced hepatotoxicity, which will facilitate the elucidation of its mechanism.     

Studies in Pheromone Biosynthesis: Preparation of 3H Labelled Precursors of Drosophila Pheromones

Two synthetic schemes were designed giving access to tritium labelled potential precursors of Drosophila pheromones. An intermediate in the first scheme allowed the preparation of [³H]-labelled vaccenyl acetate.     

Iodination of Peptide Hormones and Purification of Iodinated Peptides by HPLC

Abstract

Hexagastrin, Arg⁸-vasopressin, oxytocin, Tyr¹-somatostatin, and ACTH 1-39 were iodinated in order to yield precursors for tritium labelling or radioiodinated tracers for radioimmunoassay, respectively. The heterogeneous mixture of iodination products was purified via reversed-phase high-performance liquid chromatography. Iodination of peptides resulted in a marked increase in retention time on the reversed-phase adsorbent. A simple and quick method was applied for purification of radioiodinated peptides on a Sep-pak® C-18 cartridge for rapid sample preparation.     

Comparison Of Highly Sensitive Sandwich Enzyme Immunoassay And Radioimmunoassay For Human Ferritin

Abstract

A highly sensitive sandwich enzyme immunoassay (EIA) for human ferritin was developed using rabbit anti-ferritin IgG-coated polystyrene balls and affinity-purified rabbit anti-ferritin Fab' labelled with β-D-galactosidase from Escherichia coli and compared with the corresponding sandwich radioimmuno assay (RIA). The specific and nonspecific binding of labelled anti-ferritin to the polystyrene balls were examined in relation to the amount of labelled anti-ferritin used per tube, and the highest sensitivity of each immunoassay (0.2 amol/tube in EIA and 2.5 amol/tube in RIA) was obtained by using the minimal amount of the corresponding labelled anti-ferritin (0.71 fmol in EIA and 4.5 fmol=4436 cpm in RIA) which gave a reliable calibration curve. The sandwich RIA was less sensitive, largely because the specific radioactivity of ¹²⁵I-labelled anti-ferritin used was not sufficiently high.