125I-Labeled Peptide Mapping and High-Performance Liquid Chromatography I-Peptide Separation of Protein I of Four Strains of Neisseria Gonorrhoeae

Abstract

¹²⁵I-labeled α-chymotryptic peptides of the principal outer membrane proteins (P.Is) of four strains of Neisseria gonorrhoeae were separated and visualized by two-dimensional (2-D) ¹²⁵I-peptide mapping and by high-performance liquid chromatography (HPLC) coupled with a Beckman Biogamma counter. In addition, ¹²⁵I-peptides were recovered from the HPLC separation and re-separated by the 2-D ¹²⁵I-peptide mapping system. The results indicated that the 2-D ¹²⁵I-peptide mapping procedure was best suited for comparative analyses of α-chymotryptic digests whereas the HPLC system, which is able to detect many more peptides than the 2-D system, is ideally suited for preparative separation of the ¹²⁵I-peptides. ¹²⁵I-peptides separated by HPLC could be recovered, rerun on the 2-D system, and the location of each peptide ascertained. The coupling of these two procedures allows for the isolation of specific ¹²⁵I-labeled peptides for further immunological and structural analyses of these outer membrane proteins.     

Labeling of atenolol with radioactive iodine-125 using <Emphasis Type="Italic">N</Emphasis>-bromosuccinimide and hydrogen peroxide as oxidizing agents

An adopted method for the preparation of high radiochemical purity ¹²⁵I-atenolol was investigated. Direct radioiodination of atenolol was carried out using N-bromosuccinamide or hydrogen peroxide as an oxidizing agent. The reaction proceeds well within 30 min at room temperature (25 ± 1 °C) and afforded a radiochemical yield up to 97% as pure as ¹²⁵I-atenolol. Different chromatographic techniques (electrophoresis, TLC and HPLC) were used to determine the radiochemical yield and purity of the labeled product. Biodistribution studies were carried out in normal Albino Swiss mice and the results indicate that ¹²⁵I-atenolol can be used safely as myocardial imaging agent.     

Preparation of <Superscript>125</Superscript>I-celecoxib with high purity as a possible tumor agent

A preparation of ¹²⁵I-celecoxib is carried out via an electrophilic substitution reaction. The reaction parameters studied were celecoxib concentration, reaction temperature, pH of the reaction mixture and kinds of oxidizing agents in order to obtain a high radiochemical yield of the ¹²⁵I-celecoxib. Using 3.7 MBq of Na¹²⁵I, 150 μg (3.9 mM (mmol/L)) of celecoxib, and 1.6 mM (mmol/L) of chloramine-T (CAT) as oxidant at pH 4 and 60 °C for 15 min a maximum radiochemical yield of ¹²⁵I-celecoxib (65%) was obtained. The labeled compound was separated and purified by means of high pressure liquid chromatography (HPLC). The biological distribution in infected mice indicates the suitability of radioiodinated celecoxib as imaging of tumor.     

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.     

Synthesis of18F-2-deoxy-2-fluoro-d-glucose and18F-3-deoxy-3-fluoro-d-glucose with no-carrier-added18F-fluoride

Using a distillation method for the separation of¹⁸F-fluoride from aqueous¹⁸F-solutions obtained after cyclotron irradiation of a water target by means of the¹⁶O(³He, p)¹⁸F reaction, the radiohalogen could be generated as a highly reactive species for nucleophilic substitution reactions. Thus, with the starting compounds 1,3,4,6-tetra-O-acetyl-2-O-trifluoromethanesulfonyl--D-mannopyranose and 1,2:5,6-di-O-isopropylidene-3-O-trifluoromethanesulfonyl--D-allofuranose¹⁸F-2-deoxy-2-fluoro-D-glucose (2-¹⁸FDG) and¹⁸F-3-deoxy-3-fluoro-D-glucose (3-¹⁸FDG) could be synthesized with radiochemical yields of 71.6% and 85.9%, respectively. Including purification by HPLC, the total preparation time was 70 min, yielding the glucose derivatives in a no-carrier-added state. Specific activities coold be calculated to be greater than 10³ Ci/mmol.     

A facile direct nucleophilic synthesis of O-(2-[18F]fluoroethyl)-l-tyrosine ([18F]FET) without HPLC purification

Due to favourable in vivo characteristics, its high specificity and the longer half-life of ¹⁸F (109.8 min) allowing for remote-site delivery, O-(2-[¹⁸F]fluoroethyl)-l-tyrosine ([¹⁸F]FET) has gained increased importance for molecular imaging of cerebral tumors. Consequently, the development of simple and efficient production strategies for [¹⁸F]FET could be an important step to further improve the cost-effective availability of [¹⁸F]FET in the clinical environment. In the present study [¹⁸F]FET was synthesized via direct nucleophilic synthesis using an earlier developed chiral precursor, the Ni^II complex of an alkylated (S)-tyrosine Schiff base, Ni-(S)-BPB-(S)-Tyr-OCH₂CH₂OTs. The purification method has been developed via solid phase extraction thereby omitting cumbersome HPLC purification. The suggested SPE purification using combination of reverse phase and strong cation exchange cartridges provided [¹⁸F]FET in high chemical, radiochemical and enantiomeric purity and 35 % radiochemical yield (decay-corrected, 45 min synthesis time). The method was successfully automated using a commercially available synthesis module, Scintomics Hotbox^one. Based on the current results, the proposed production route appears to be well suited for transfer into an automated cassette-type radiosynthesizers without using HPLC.     

The preparation of the 239Np tracer from 243Am and the purification of the stock solution

The present work describes preparation of ²³⁹Np tracer from ²⁴³Am stock solution and the purification of this solution from ferric cation. The method of the preparation of tracer involves stabilization of Np(IV) by ascorbic acid and ferric nitrate, separation of ²³⁹Np from ²⁴³Am by extraction chromatography and determination of recoveries of ²³⁹Np by means of gamma spectroscopy. We used the commercially available sorbents TEVA^®Resin for the ²³⁹Np preparation and DGA Resin for ²⁴³Am purification. All sorbents were purchased from Eichrom Industries, Inc. The first eluate from the column can be stored for a future preparation of the tracer and fraction with ²³⁹Np will be used to monitor radiochemical yield of ²³⁷Np.     

HPLC determination of domoic acid in shellfish based on magnetic molecularly imprinting polymers

A magnetic molecularly imprinting polymer for domoic acid was fabricated. Synthesis conditions were optimized. The polymer particles have high magnetization for rapid magnetic separation. The apparent maximum absorption amount and dissociation constant of the polymer were 1,600?µg?g^?1 and 20.6?µg?mL^?1, respectively. The polymer retained 90% of adsorption amount after 5 times of repeated use. It was used as an adsorbent for purification and HPLC detection of domoic acid in shellfish with a detection limit of 0.050?µg?g^?1. Thus, the polymer could be applied to the sample pretreatment of aquatic products.     

Radiosynthesis of 10-(2-[18F]fluoroethoxy)-20(S)-camptothecin as a potential positron emission tomography tracer for the imaging of topoisomerase I in cancers

The preparation of 10-(2-[¹⁸F]fluoroethoxy)-20(S)-camptothecin, a potential positron emission tomography tracer for the imaging of topoisomerase I in cancers, is described. 10-(2-[¹⁸F]Fluoroethoxy)-20(S)-camptothecin was synthesized by the [¹⁸F]fluoroalkylation of the corresponding hydroxy precursor molecule with 2-[¹⁸F]fluoroethyl bromide ([¹⁸F]FEtBr) in dimethylsulfoxide (DMSO) at 55 °C for 20 min; this was followed by purification using high performance liquid chromatography (HPLC) with a total preparation time of 60 min. The overall radiochemical yield was approximately 5.4–12 % (uncorrected), and the radiochemical purity was above 96 %.     

Chemical Synthesis of Oligonucleotides Containing N6-Methyladenine Residues in the GATC Site

To investigate by NMR the effects of adenine methylation, a set of oligodeoxynucleotides has been synthesized by phosphotriester or phosphoramidite procedures on silica-gel support. The preparation of fully protected 2′-deoxy-N⁶-methyladenosine 3′-phosphate 4 and (2′-deoxy-N⁶-methyladenosin-3′-O-yl)(methoxy)morpholinophosphine 5 is described. The large-scale chemical synthesis, purification by HPLC, and characterization by MS of these molecules containing N⁶-methyladenine is reported.     

Preparation of iodine-125 seed Part I

We studied the preparation method of ¹²⁵I seeds and found that silver was a good carrier body for ¹²⁵I and the ion-exchange technique was simple and effective for the preparation. Carrier iodine was critical for dose uniformity of the seeds and the minimal pH value for the reaction was 6.5. This paper provided valuable data for the development and preparation of ¹²⁵I seed.     

Evaluation of the radiochemical impurities arising during the competitive fluorination of nosyl group during the synthesis of 3′-deoxy-3′-fluorothymidine, [18F]FLT

The study is aimed at the analysis and identification of radiochemical and chemical impurities present in [¹⁸F]FLT synthesized by a simplified combination-column purification procedure, instead of the currently used HPLC purification. HPLC analysis of the final product showed an anionic radioactive byproduct, which was established as [¹⁸F]4-FBSA. The identity of the product was also confirmed by the radiofluorination of nosyl chloride. Mass spectrum analysis of both a decayed sample of [¹⁸F]FLT and fluorinated nosyl chloride showed a major peak at 242. We have also investigated the possible interference of this byproduct during PET-imaging in rabbits.     

Fully automated and simplified radiosynthesis of [<Superscript>18</Superscript>F]-3′-deoxy-3′-fluorothymidine using anhydro precursor and single neutral alumina column purification

[¹⁸F]-3′-deoxy-3′-fluorothymidine ([¹⁸F]FLT) is an established positron emission tomograph (PET)—radiopharmaceutical to study cell-proliferation rate in tumors. Very low practical yield, uncertain and time-consuming high performance liquid chromatography (HPLC) purification, are the main obstacles for the routine use of [¹⁸F]FLT in clinical PET. To obviate these difficulties, we have developed a fully automated radiosynthesis procedure for [¹⁸F]FLT using 5′-O-(4,4′-dimethoxytriphenylmethyl)-2,3′-anhydro-thymidine (DMTThy) and simplified single neutral alumina column purification. The [¹⁸F]FLT yield was 8.48 ± 0.93% (n = 5) (without radioactive decay correction) in a synthesis time of 68 ± 3 min. The radiochemical purity was greater than 95% as confirmed by analytical HPLC using reference standard FLT and also free of non-radioactive impurity. Soluble aluminum in the final product was much below the permissible limits. Di-methyl sulfoxide (DMSO), the reaction medium, could be detected in the final product in trace amounts, well below the permissible levels. The synthesized [¹⁸F]FLT was sterile and bacterial endotoxin free by appropriate tests. PET imaging study in normal rabbits showed distinct localization of [¹⁸F]FLT in organs having rapid cell division rate like bone marrow, guts and snout and the excretion was through the renal route. There were no significant uptakes in bone and brain. The former finding confirms the in vivo stability of the [¹⁸F]FLT. This simplified radiosynthesis procedure can easily be adapted in any commercial or indigenous [¹⁸F]FDG synthesis module for routine [¹⁸F]FLT synthesis without the need of additional automation for HPLC purification.     

Potential of radioiodinated anti cancer compounds of natural origin for cancer therapy

Plumbagin and Quercetin are naturally occurring compounds which exhibit anti-cancerous activity. To evaluate the effect of radioiodination on cytotoxicity, both Plumbagin and Quercetin were radioiodinated with ¹²⁵I ¹²⁵I-Plumbagin and ¹²⁵I-Quercetin could be prepared in moderate yields and good radiochemical purity and were charactenzed using reverse phase HPLC. In Swiss mice bearing fibrosarcoma, ¹²⁵I-Plumbagin showed a tumor uptake of ∼2.5%ID/g at 3 h p.i. and ∼0.5% ID/g at 24 h p.i. on i.v. injection. When injected intratum orally, greater tumor uptake and retention was observed (∼20%ID/g at 3 h p.i. and ∼14%ID/g at 24 h p.i. respectively).     

Development of the protocol for purification of artemisinin based on combination of commercial and computationally designed adsorbents

A polymeric adsorbent for extraction of the antimalarial drug artemisinin from Artemisia annua L. was computationally designed. This polymer demonstrated a high capacity for artemisinin (120 mg g^?1), quantitative recovery (87%) and was found to be an effective material for purification of artemisinin from complex plant matrix. The artemisinin quantification was conducted using an optimised HPLC‐MS protocol, which was characterised by high precision and linearity in the concentration range between 0.05 and 2 μg mL^?1. Optimisation of the purification protocol also involved screening of commercial adsorbents for the removal of waxes and other interfering natural compounds, which inhibit the crystallisation of artemisinin. As a result of a two step‐purification protocol crystals of artemisinin were obtained, and artemisinin purity was evaluated as 75%. By performing the second stage of purification twice, the purity of artemisinin can be further improved to 99%. The developed protocol produced high‐purity artemisinin using only a few purification steps that makes it suitable for large scale industrial manufacturing process.     

Determination of 1-aminocyclopropane-1-carboxylic Acid in Apple and Peach Extracts by High Performance Liquid Chromatography Coupled to a Fluorescence Detector

A new, sensitive, and simple HPLC method was described for the determination of 1-aminocyclopropane-1-carboxylic acid (ACC) in apple and peach extracts. The method was based on the derivatization of ACC with fluorescamine in borate buffer systems of pH 8.0 to yield a highly fluorescent product. The experimental parameters affecting the derivatization reaction efficiency were optimized by fluorimetric analysis. Under optimum derivatization conditions, the derivative product of ACC in apple and peach extracts without extra purification was successfully chromatographed on a C-18 column by HPLC coupled to fluorescence detection. The derivative product of ACC with fluorescamine could be well separated from other concomitant substances or their derivatives that might interfere with the determination of ACC. The linearity of ACC was measured in the range of 23.82–238.82 µg · L^?1 with a good correlation coefficient of 0.9997. Based on signal-to-noise ratio of 3, a low detection limit of 5.0 µg · L^?1 could be reached. The proposed method was applied successfully to the determination of ACC in the crude apple and peach extracts without extra purification with low RSDs of 0.19–1.9% and good recoveries of 90.89–104.4%. The sensitive HPLC quantitative method is of great significance for the investigations of ACC metabolism in plants.     

Use of RP-HPLC for Purification of a CCK-39 Radio-Ligand and of Its Photoactivable Derivative. Controls of Degradations

Abstract

In order to characterize CCK receptors on guinea-pig pancreatic acini we developped a method of purification and control of degradation of CCK-39 radio-ligands and of their photoactivable derivatives. CCK-39 was radio-labeled with N-hydroxysuccinimide 3-(4-hydroxy 5 [¹²⁵I] phenyl) propionate at pH=8.5. Mono-labeled CCK-39 was separated from di-and tri-labeled CCK-39 on a C-18 column and coupled with N-hydroxysuccinimidyl-4-azido-benzoate. The resulting photoactivable radio-ligand ¹²⁵I-BH-[4-azidobenzoyl]-CCK-39 specifically bound to pancreatic membranes. After photolysis under UV irradiation and SDS-PAGE a major labeled protein of Mr 90 000 could be identified. Furthermore, RP-HPLC permitted the control of degradation of each ligand. Presence of endo-peptidasic and aminopeptidasic activities sensitive to EDTA and bacitracin were demonstrated.     

Preparation of a one-curie <Superscript>171</Superscript>Tm target for the detector for advanced neutron capture experiments (DANCE)

Approximately one curie of ¹⁷¹Tm (T _1/2 = 1.92a) has been produced and purified for the purpose of making a nuclear target for the first measurements of its neutron capture cross section. Target preparation consisted of three key steps: (1) material production; (2) separation and purification; and (3) electrodeposition onto a suitable backing material. Approximately 1.5 mg of the target material (at the time of separation) was produced by irradiating ca. 250 mg of its stable enriched ¹⁷⁰Er lanthanide neighbour with neutrons at the ILL reactor in France. This production method resulted in a “difficult-to-separate” 1:167 mixture of near-neighboring lanthanides, Tm and Er. Separation and purification was accomplished using high-performance liquid chromatography (HPLC), with a proprietary cation-exchange column (Dionex, CS-3) and alpha-hydroxyisobutyric acid (α-HIB) eluent. This technique yielded a final product of ∼95% purity with respect to Tm. A portion (20 μg) of the Tm was electrodeposited onto thin Be foil and delivered to the Los Alamos Neutron Science Center (LANSCE) for preliminary analysis of its neutron capture cross section using the Detector for Advanced Neutron Capture Experiments (DANCE). This paper discusses the major hurdles associated with the separation and purification step, including scale-up issues related to the use of HPLC for material separation and purification of the target material from α-HIB and 4-(2-pyridylazo)resorcinol (PAR) colorant.     

Potential of microbore HPLC within a multiresidue method for the trace analysis of plant-protective substances in water

The performance of microbore HPLC as a “measurement channel” within a true multiclass/multiresidue method for monitoring plant protectants in raw and potable water is demonstrated. The method has a modular design and consists of a non-selective sampling and preparation line generating 250 μL of an “extract” from a 100-mL water sample; this extract can be introduced to up to four measurement channels, as required by the analytical task. The microbore HPLC channel can be used to quantify 34 plant protectants in the 0.1 μg L^–1 concentration range by use of diode-array detection at seven different wavelengths. A solvent change is necessary to link sample preparation to microbore HPLC; this uses 50 μL of the “extract” and is accomplished directly in an autosampler vial. Performance characteristics were evaluated for tap water spiked at 0.2 μg L^–1. Average recoveries were between 65 and 100% and method detection limits were 0.07 μg L^–1 or better. The ability to provide comparable and accurate results was proven by participation in an interlaboratory comparison trial. The procedure for preparing microbore columns from 750 μm i.d. PEEK tubing is described in detail to enable the reader to prepare his own columns. The reproducibility of this preparation procedure was proven by an analysis-of-variance test.     

Preparation of 125I-Lanreotide and its biodistribution in murine model of melanoma

Lanreotide, a somatostatin analogue, was radioiodinated with ¹²⁵I to explore the possibility of using ¹²³I labeled lanreotide as a diagnostic radiopharmaceutical for tumors overexpressing somatostatin (SST) receptors. Radioiodination was carried out with ¹²⁵I using chloramine T as the oxidant. The labeling yield was >90%. Characterization of ¹²⁵I-Lanreotide was carried out by paper electrophoresis as well as HPLC. ¹²⁵I-Lanreotide was purified by chromatography using a C18 Sep-Pak column. Radiochemical purity of the purified ¹²⁵I-Lanreotide thus obtained was >99%. Significant tumor uptake of ¹²⁵I-Lanreotide was observed in C57BL/6 mice bearing melanoma.