Automated radiochemical synthesis of pharmaceutical grade [18 F]FLT using 3-N-Boc-5'-O-dimethoxytrityl-3'-O-nosyl-thymidine precursor and its Sep-Pak® purification employing selective elution from reversed phase.

Pharmaceutical grade 3'-deoxy-3'-[18 F]fluorothymidine [18 F]FLT was synthesized using 3-N-Boc-5'-O-dimethoxytrityl-3'-O-nosyl-thymidine (BOC-Nosyl) precursor, in the general purpose TRACERlab FX modules. Purification of [18 F]FLT, via solid phase extraction (SPE) after radiosynthesis, using a combination of different SPE cartridges, yielded satisfactory results, with radiochemical and chemical purity >99%. While the non-decay corrected radiochemical yield (RCY) with 20 mg (24 µmole) of BOC-Nosyl precursor was found to be 6.80 ± 0.16%, the decay corrected radiochemical yield (RCY) was 9.95 ± 0.24%. Residual acetone, acetonitrile, and ethanol levels were found to be 22.97 ± 0.76, 109.08 ± 0.93, and 7,666.45 ± 3.7 ppm, respectively. A simplified method for solid-phase purification of [18 F]FLT was developed, circumventing the need for HPLC purification. Biodistribution in C57BL/6 mice with B16F10 cell line-induced melanoma showed tumor to blood ratio of ~3.8 at 90 min. PET/CT imaging of normal rabbit injected with [18 F]FLT shows selective uptake in the bone marrow and small intestine. [18 F]FLT was found to be excreted through the kidneys and get collected in the urinary bladder, 120 min post injection. PET/CT imaging performed in rabbit model at 30, 60, 90, and 120 min post [18 F]FLT injections showed concordance with tissue distribution kinetics of mice tumor model.

Automated Synthesis of Fluorine-18 Labeled CXCR4 Ligand via the Conjugation with Nicotinic Acid N-Hydroxysuccinimide Ester (6-[18F]SFPy).

The C-X-C motif chemokine receptor 4 (CXCR4) is a seven-transmembrane G protein-coupled receptor that is overexpressed in numerous diseases, particularly in various cancers and is a powerful chemokine, attracting cells to the bone marrow niche. Therefore, CXCR4 is an attractive target for imaging and therapeutic purposes. The goal of this study is to develop an efficient, reproducible, and straightforward method to prepare a fluorine-18 labeled CXCR4 ligand. 6-[18F]Fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester (6-[18F]FPy-TFP) and nicotinic acid N-hydroxysuccinimide ester (6-[18F]SFPy) have been prepared using 'fluorination on the Sep-Pak' method. Conjugation of 6-[18F]SFPy or 6-[18F]FPy-TFP with the alpha-amino group at the N terminus of the protected T140 precursor followed by deprotection, yielded the final product 6-[18F]FPy-T140. The overall radiochemical yields were 6-17% (n = 15, decay-corrected) in a 90-min radiolabeling time with a radiochemical purity >99%. 6-[18F]FPy-T140 exhibited high specific binding and nanomolar affinity for CXCR4 in vitro, indicating that the biological activity of the peptide was preserved. For the first time, [18F]SFPy has been prepared using 'fluorination on the Sep-Pak' method that allows rapid automated synthesis of 6-[18F]FPy-T140. In addition to increased synthetic efficiency, this construct binds with CXCR4 in high affinity and may have potential as an in vivo positron emission tomography (PET) imaging agent. This radiosynthesis method should encourage wider use of this PET agent to quantify CXCR4 in both research and clinical settings.

Fluorine-18 Labeled Fluorofuranylnorprogesterone ([18F]FFNP) and Dihydrotestosterone ([18F]FDHT) Prepared by "Fluorination on Sep-Pak" Method.

To further explore the scope of our recently developed "fluorination on Sep-Pak" method, we prepared two well-known positron emission tomography (PET) tracers 21-[18F]fluoro-16α,17α-[(R)-(1'-α-furylmethylidene)dioxy]-19-norpregn-4-ene-3,20-dione furanyl norprogesterone ([18F]FFNP) and 16ß-[18F]fluoro-5α-dihydrotestosterone ([18F]FDHT). Following the "fluorination on Sep-Pak" method, over 70% elution efficiency was observed with 3 mg of triflate precursor of [18F]FFNP. The overall yield of [18F]FFNP was 64-72% (decay corrected) in 40 min synthesis time with a molar activity of 37-81 GBq/µmol (1000-2200 Ci/mmol). Slightly lower elution efficiency (~55%) was observed with the triflate precursor of [18F]FDHT. Fluorine-18 labeling, reduction, and deprotection to prepare [18F]FDHT were performed on Sep-Pak cartridges (PS-HCO3 and Sep-Pak plus C-18). The overall yield of [18F]FDHT was 25-32% (decay corrected) in 70 min. The molar activity determined by using mass spectrometry was 63-148 GBq/µmol (1700-4000 Ci/mmol). Applying this quantitative measure of molar activity to in vitro assays [18F]FDHT exhibited high-affinity binding to androgen receptors (Kd~2.5 nM) providing biological validation of this method.

Rapid synthesis of maleimide functionalized fluorine-18 labeled prosthetic group using "radio-fluorination on the Sep-Pak" method.

Following our recently published fluorine-18 labeling method, "Radio-fluorination on the Sep-Pak", we have successfully synthesized 6-[18 F]fluoronicotinaldehyde by passing a solution (1:4 acetonitrile: t-butanol) of its quaternary ammonium salt precursor, 6-(N,N,N-trimethylamino)nicotinaldehyde trifluoromethanesulfonate (2), through a fluorine-18 containing anion exchange cartridge (PS-HCO3 ). Over 80% radiochemical conversion was observed using 10 mg of precursor within 1 minute. The [18 F]fluoronicotinaldehyde ([18 F]5) was then conjugated with 1-(6-(aminooxy)hexyl)-1H-pyrrole-2,5-dione to prepare the fluorine-18 labeled maleimide functionalized prosthetic group, 6-[18 F]fluoronicotinaldehyde O-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl) oxime, 6-[18 F]FPyMHO ([18 F]6). The current Sep-Pak method not only improves the overall radiochemical yield (50 ± 9%, decay-corrected, n = 9) but also significantly reduces the synthesis time (from 60-90 minutes to 30 minutes) when compared with literature methods for the synthesis of similar prosthetic groups.

Bioactivity assays and phytochemical analysis upon Alcea glabrata; focusing on xanthine oxidase inhibitory and antimalarial properties.

Alcea glabrata from the family Malvaceae, was selected for evaluating its xanthine oxidase inhibitory, anti-malarial, and antioxidant activities. In addition, some phytochemical analysis upon different extracts of A. glabrata were performed. Aerial parts of the collected A. glabrata plant material were dried and solvent extracted via soxhlet apparatus using different solvents. Various chromatographic techniques were used for extra fractionation of the achieved extracts. Xanthine oxidase (XO) inhibitory, antimalarial and antioxidant activity assays upon different A. glabrata extracts and fractions were carried out and reported in terms of IC50s. Total phenolic and flavonoid contents of the A. glabrata methanol extract (MeOH) were determined using the 2,2-Di Phenyl-1-Picryl Hydrazyl (DPPH) assay, aluminum chloride colorimetric, and Folin-Ciocalteu reagents, respectively. In addition, A. glabrata essential oil was obtained through hydrodistillation by a Clevenger apparatus. Analysis and identification of essential oil compounds were carried out through gas chromatography mass spectrometry (GC-MS) analysis. MeOH extract showed the highest XO inhibitory activity with the IC50 of 0.37 ± 0.12 mg/mL antioxidant activity with the RC50 of 0.24 ± 0.06 mg/mL. While, chloroform extract revealed the strongest antimalarial activity with the IC50 of 0.4 ± 0.05 mg/mL. The total flavonoid and phenolic contents of the A. glabrata methanol extract were 39.8 mg quercetin equivalent and 6.1 g gallic acid equivalent per 100 g of dry plant material, respectively. GC-MS analysis showed that the monoterpenes were prevailing in A. glabrata essential oil where the major constituents: octacosane (30.7%), eugenol (12.3%), and anethole (12.0%). Concerning the results of this study, A. glabrata extracts and its ingredients could be considered as a novel promising herbal medicine in the design and also treatment of new drugs for the relief of gout and malaria diseases.

An investigation of aspects of radiochemical purity of 99mTc-labelled human serum albumin nanocolloid.

BACKGROUND: Nanocolloidal human serum albumin radiolabelled with 99mTc provides a diagnostic radiopharmaceutical for sentinel node lymphoscintigraphy. NanoHSA (Nanotop), a commercially available kit, enables the simple preparation of this radiopharmaceutical via reconstitution with pertechnetate eluted from a generator. Thin-layer chromatography is widely used for determining radiochemical purity in clinical nuclear medicine. Quality control methods recommended by the manufacturer were sometimes reported to yield variable results. Therefore, we proposed and evaluated three alternative thin-layer chromatography methods for the quality control of [99mTc]Tc-NanoHSA from a commercially available kit. RESULTS: The radiochemical purity of [99mTc]Tc-NanoHSA determined with all methods was reproducible and met the requirements of the SPC and the European Pharmacopoeia (≥ 95%). Our quality control using iTLC-SG chromatographic paper in methyl ethyl ketone mobile phase identified only free pertechnetate as impurity, resulting in > 99% RCP. The quality control using iTLC-SG in 85% methanol or iTLC-SA in 0.9% NaCl identified an additional small fraction of a hydrophilic impurity, resulting in 95-97% RCP. Glucose was identified as a potential 99mTc-carrying hydrophilic species contributing to hydrophilic impurities. CONCLUSION: Our quality control of [99mTc]Tc-NanoHSA with non-polar mobile phase tended to underestimate the amount of hydrophilic impurities, although without compromising the final quality of the radiopharmaceutical. Alternative TLC methods using aqueous mobile phases enabled a more accurate determination of hydrophilic impurities.

Development of a solid-phase extraction method with simple MEKC-UV analysis for simultaneous detection of indole metabolites in human urine after administration of indole dietary supplement.

This work presents the development of a solid phase extraction method with simple MEKC-UV analysis for the simultaneous determination of indole-3-carbinol (I3C) and its metabolites (3, 3'-diindolylmethane (DIM), indole-3-carboxaldehyde (I3CAL), indole-3-acetonitrile (I3A)) in human urine after oral administration of an indole dietary supplement. Solid phase extraction (SPE) method was applied for the first time for simultaneous analysis of these indole metabolites. The MEKC separation method was developed in a previous work. Three commercial SPE cartridges, each with different sorbent materials, were investigated: Sep-Pak® C18, Oasis® HLB and Oasis® WCX. The Sep-Pak® C18 material provided the highest extraction recovery of 88-113% (n = 9), for the four target indole metabolites (I3C, DIM, I3CAL and I3A). The optimal washing and elution solutions were 40% methanol/water (v/v) and 100% methanol, respectively, and optimal elution volume was 2.0mL. The specificity of the proposed SPE method was evaluated with negative control urine samples (n = 10) from healthy volunteers who had not taken the dietary supplement or vegetables known to contain indole compounds. Linear calibration curves were in the range of 0.2-25µgmL-1 (r2 > 0.998) using diphenylamine (DPA) as the internal standard. Intra-day and inter-day precisions were 3.5-12.3%RSD and 2.7-14.1%RSD, respectively. Limits of detection and quantification were 0.05-0.10µgmL-1 and 0.10-0.50µgmL-1, respectively. The four target indole compounds were separated within only 5min by MEKC-UV analysis. Urine from 5 subjects who had taken a dietary supplement containing I3C and DIM were found to contain only the DIM metabolite at concentrations ranging from 0.10 to 0.35µgmL-1. Accuracy of the proposed method based on the percentage recovery of spiked urine samples were 70-108%, 82-116%, 82-132% and 80-100% for I3C, I3CAL, I3A and DIM, respectively. The Sep-Pak®C18 cartridge was highly effective in extraction and sample cleanup for the downstream simultaneous detection of urinary indole metabolites by MEKC-UV method.

Analysis of organophosphorus pesticides in whole blood by GC-MS-µECD with forensic purposes.

In the present work, two multi-residue methods for the determination of ten organophosphorus pesticides (OPs), namely chlorfenvinphos, chlorpyrifos, diazinon, dimethoate, fenthion, malathion, parathion, phosalone, pirimiphos-methyl and quinalphos, in post-mortem whole blood samples are presented. The adopted procedure uses GC-MS for screening and quantitation, and GC-µECD (electron capture detector) for compound confirmation. Three different Solid Phase Extraction (SPE) procedures for OPs with Oasis(®) hydrophilic lipophilic balanced (HLB) and Sep-Pak(®) C18 cartridges were tested, and followed by GC-µECD and GC-MS analysis. The Sep-Pak(®) C18 cartridges extraction procedure was selected since it generated analytical signals 5 times higher than those obtained with the two different Oasis(®) HLB cartridges extraction procedures. The method has shown to be selective for the isolation of selected OPs as well as to the chosen internal standard (ethion) in postmortem blood samples. Calibration curves between 50 and 5000 ng/mL were prepared using weighted linear regression models (1/x(2)). It was not possible to establish a working range for fenthion by GC-µECD due to the lower sensitivity of the detector to this compound, whereas for pirimiphos-methyl it was set between 500 and 5000 ng/mL. The limit of quantitation was established at 50 ng/mL for all analytes, except for pirimiphos-methyl by GC-µECD analysis (500 ng/mL). The average extraction efficiency ranged from 72 to 102%. The developed methods were considered robust and fit for the purpose, and had already been adopted in the laboratory routine analysis.

Rapid Preparation and Quality Control of 99mTc-ECD, MAG3 and MIBI using Microwave Heating and Sep-Pak Cartridges / 대한핵의학회잡지

PURPOSE: We evaluated a rapid preparation procedures for the labeling and quality control of 99mTc-ECD, MAG3, and MIBI using microwave heating and Sep-Pak cartridges. MATERIALS AND METHODS: 99mTc labeling of ECD, MAG3, and MIBI kit preparation was performed according to the package inserts with microwave heating modification. Heating time was 10-15 sec, and heating was performed with 3 mm plastic bottle with screw cap to prevent radiation contamination. Labeling efficiency was obtained with C18 or Alumina N Sep-Pak cartridges. RESULTS: The radiochemical purity of 93~96% for 99mTc-ECD and 95~99% for 99mTc-MIBI was obtained using Alumina N Sep-Pak cartridge. The optimum irradiation time of microwave method for 3 ml 99mTc-labeled radiopharmaceutical solution was 10 sec for 99mTc-ECD and 99mTc-MIBI, and 15 sec for 99mTc-MAG3. The RESULTS of quality control data with Sep-Pak cartridges were well correlated with TLC method. The total preparation time of these radiopharmcaeuticals was 5~6 min including quality control procedure. CONCLUSION: This study demonstrates that radiopharmaceuticals preparation by microwave heating and quality control by Sep-Pak cartridges can be efficiently utilized as an alternative to the recommended method by manufacturer's manual.