Microliter-scale reaction arrays for economical high-throughput experimentation in radiochemistry.

The increasing number of positron-emission tomography (PET) tracers being developed to aid drug development and create new diagnostics has led to an increased need for radiosynthesis development and optimization. Current radiosynthesis instruments are designed to produce large-scale clinical batches and are often limited to performing a single synthesis before they must be decontaminated by waiting for radionuclide decay, followed by thorough cleaning or disposal of synthesizer components. Though with some radiosynthesizers it is possible to perform a few sequential radiosyntheses in a day, none allow for parallel radiosyntheses. Throughput of one or a few experiments per day is not well suited for rapid optimization experiments. To combat these limitations, we leverage the advantages of droplet-radiochemistry to create a new platform for high-throughput experimentation in radiochemistry. This system contains an array of 4 heaters, each used to heat a set of 16 reactions on a small chip, enabling 64 parallel reactions for the rapid optimization of conditions in any stage of a multi-step radiosynthesis process. As examples, we study the syntheses of several 18F-labeled radiopharmaceuticals ([18F]Flumazenil, [18F]PBR06, [18F]Fallypride, and [18F]FEPPA), performing > 800 experiments to explore the influence of parameters including base type, base amount, precursor amount, solvent, reaction temperature, and reaction time. The experiments were carried out within only 15 experiment days, and the small volume (~ 10 µL compared to the ~ 1 mL scale of conventional instruments) consumed ~ 100 × less precursor per datapoint. This new method paves the way for more comprehensive optimization studies in radiochemistry and substantially shortening PET tracer development timelines.

Automated processing of solid target 86Y using enriched SrO powder.

86Y (t1/2 = 14.74 h, 32% ß+) has significant potential in theranostic applications as a simultaneous PET imaging partner to 90Y-labelled antibody therapy. However, the complex and costly nature of producing 86Y has led to this radiometal being difficult for hospitals and researchers to obtain. The aim of this work was to develop a simple and cost-efficient method for safely producing 86Y. Our approach was twofold: to develop a method of target preparation that would significantly increase the cost efficiency of producing 86Y, and to design and construct an automated purification system that would eliminate manual radiation handling risks and exposure. Multiple automated productions of high radionuclidic purity (99.45%) 86Y were performed resulting in saturation yields of between 518 MBq/µA and 1332 MBq/µA, dependent on target thickness.

Economical droplet-based microfluidic production of [18F]FET and [18F]Florbetaben suitable for human use.

Current equipment and methods for preparation of radiopharmaceuticals for positron emission tomography (PET) are expensive and best suited for large-scale multi-doses batches. Microfluidic radiosynthesizers have been shown to provide an economic approach to synthesize these compounds in smaller quantities, but can also be scaled to clinically-relevant levels. Batch microfluidic approaches, in particular, offer significant reduction in system size and reagent consumption. Here we show a simple and rapid technique to concentrate the radioisotope, prior to synthesis in a droplet-based radiosynthesizer, enabling production of clinically-relevant batches of [18F]FET and [18F]FBB. The synthesis was carried out with an automated synthesizer platform based on a disposable Teflon-silicon surface-tension trap chip. Up to 0.1 mL (4 GBq) of radioactivity was used per synthesis by drying cyclotron-produced aqueous [18F]fluoride in small increments directly inside the reaction site. Precursor solution (10 µL) was added to the dried [18F]fluoride, the reaction chip was heated for 5 min to perform radiofluorination, and then a deprotection step was performed with addition of acid solution and heating. The product was recovered in 80 µL volume and transferred to analytical HPLC for purification. Purified product was formulated via evaporation and resuspension or a micro-SPE formulation system. Quality control testing was performed on 3 sequential batches of each tracer. The method afforded production of up to 0.8 GBq of [18F]FET and [18F]FBB. Each production was completed within an hour. All batches passed quality control testing, confirming suitability for human use. In summary, we present a simple and efficient synthesis of clinically-relevant batches of [18F]FET and [18F]FBB using a microfluidic radiosynthesizer. This work demonstrates that the droplet-based micro-radiosynthesizer has a potential for batch-on-demand synthesis of 18F-labeled radiopharmaceuticals for human use.

Radiochemical separation by liquid-liquid extraction for the determination of selenium in Mentha pulegium L.: Toxicity monitoring and health study.

In Algeria, Data and studies on the non-metal trace element selenium (Se) are presently lacking, therefore, the aim of this investigation is to provide new data on (Se) element via its determination for the first time from Mentha pulegium L. plant. The plant samples were collected in summer of 2012 from Ain-Oussera region, Djelfa province, Algeria; they were dried and powdered. After the neutron irradiation, the samples were digested using high oxidative reagents including H2SO4, HNO3, H2O2 and HCl. The end of this process gave two phases, organic and aqueous discard phase. By using a separating funnel, the organic phase was transferred into a vial in order to measure their induce radionuclide 75Se using gamma-ray spectrometer. A non-chromatographic and sensitive analytical technique RNAA (Radiochemical Neutron Activation Analysis), was applied in this investigation due to its great significant minor systematic error. Results were determined using two distinguish calculation methods, relative-RNAA and k0-RNAA, the findings were quite significant, whereas, the average separation yield was about 85% for both calculation methodologies. Moreover, (Se) concentration obtained from M. pulegium L., is close to the minimal FAO (Food and Agriculture Organization of the United Nations) recommended consumption.

Transforming an Academic Radiochemistry Facility for Positron Emission Tomography Drug cGMP Compliance.

In light of the United States Food and Drug Administration (FDA) requirement of 21 CFR 212 current Good Manufacturing Practice (cGMP) for FDA-approved position emission tomography (PET) drugs, the University of California Los Angeles (UCLA) Biomedical Cyclotron (BMC) transformed from a pre-cGMP era academic cyclotron and radiochemistry facility to a current cGMP-compliant PET drug manufacturer. In this article, we share the financial and regulatory compliance aspects of the "transformation" required to develop a sustainable quality system to support the production of two PET drugs under Abbreviated New Drug Applications (ANDAs).

Quality of radiochemical purity in multiple samples of various fractionated cold kits: Testing a cost & time effective technique.

OBJECTIVE: This study was aimed to assess technical aspects of fractionation of commonly used cold kits in Nuclear Medicine. MATERIALS AND METHODS: A total of 90 samples (30 samples each) of technetium-99m methylene diphosphonate (99mTc-MDP), 99mTc-diethylenetriaminepentaacetic acid (DTPA) and 99mTc-dimercaptosuccinic acid (DMSA III) were taken on various days. The radiochemical purity was calculated of each fraction of these cold kits by using paper chromatography. RESULTS: The mean value of radiochemical purity of 99mTc -MDP, 99mTc -DTPA and 99mTc-DMSA(III) were calculated as ~ 95.12%, 91.43% and 95.68% and standard deviation (SD) were ~ 5.43, 8.36 and 3.88, respectively. Maximum time in which fractionation procedure completed i.e. time required for preparing the fraction or thawing was 10 minutes. All fractionated aliquots were between 1 and 15 days. Radiopharmaceutical bio-distribution was found to be appropriate during imaging in all samples. CONCLUSION: Fractionation of cold kits using standardised technique is a time and cost-effective method and does not deteriorate the quality of labelling in commonly used pharmaceuticals in our study. We have used fractionated aliquots up to 3 days of preparation in patients with clinically usable radiochemical purity. Deep frozen fractions can be used up to 15 days in our experience.

"In-loop" 18 F-fluorination: A proof-of-concept study.

There is a great demand to develop more cost-efficient and robust manufacturing processes for fluorine-18 (18 F) labelled compounds and radiopharmaceuticals. Herein, we present to our knowledge the first radiofluorination "in-loop," where [18 F]triflyl fluoride was used as the labelling agent. Initial development of the "in-loop" [18 F]fluorination method was optimized by reacting [18 F]triflyl fluoride with 1,4-dinitrobenzene to form [18 F]1-fluoro-4-nitrobenzene. This methodology was then applied for the syntheses of two well-known radiopharmaceuticals, namely, [18 F]T807 for imaging of tau protein and [18 F]FEPPA for imaging the translocator protein 18 KDa. Both radiotracers were synthesized and formulated using an automated radiosynthesis module with nondecay corrected radiochemical yields of 27% and 29% (relative [18 F]F- ), respectively. The overall syntheses times for [18 F]T807 and [18 F]FEPPA were 65 and 55 minutes, respectively. In these cases, our "in-loop" radiofluorination methodology enabled us to obtain equal or superior yields compared with conventional reactions in a vial. The radiochemical purities were more than 99%, and the molar activities were more than 350 GBq/µmol at the end-of-synthesis for both radiotracers. This novel method is simple, efficient, and allows for a reliable production of radiofluorinated compounds and radiopharmaceuticals.

Internal radiation dose assessment of radiopharmaceuticals prepared with cyclotron-produced 99m Tc.

PURPOSE: Technetium-99m (99m Tc) is the radioisotope most widely used in diagnostic nuclear medicine. It is readily available from 99 Mo/99m Tc generators as the ß- decay product of the 99 Mo (T½  = 66 h) parent nuclide. This latter is obtained as a fission product in nuclear reactors by neutron-induced reactions on highly enriched uranium. Alternative production routes, such as direct reactions using proton beams on specific target materials [100 Mo(p,2n)99m Tc], have the potential to be both reliable and relatively cost-effective. However, results showed that the 99m Tc extracted from proton-bombarded 100 Mo-enriched targets contains small quantities of several Tc radioisotopes (93m Tc, 93 Tc, 94 Tc, 94m Tc, 95 Tc, 95m Tc, 96 Tc, and 97m Tc). The aim of this work was to estimate the dose increase (DI) due to the contribution of Tc radioisotopes generated as impurities, after the intravenous injection of four radiopharmaceuticals prepared with cyclotron-produced 99m Tc (CP-99m Tc) using 99.05% 100 Mo-enriched metallic targets. METHODS: Four 99m Tc radiopharmaceuticals (pertechnetate, sestamibi (MIBI), hexamethylpropylene-amine oxime (HMPAO) and disodium etidronate (HEDP)) were considered in this study. The biokinetic models reported by the International Commission on Radiological Protection (ICRP) for each radiopharmaceutical were used to define the main source organs and to calculate the number of disintegrations per MBq that occurred in each source organ (Nsource ) for each Tc radioisotope present in the CP-99m Tc solution. Then, target organ equivalent doses and effective dose were calculated for each Tc radioisotope with the OLINDA/EXM software versions 1.1 and 2.0, using the calculated Nsource values and the adult male phantom as program inputs. Total effective dose produced by all Tc isotopes impurities present in the CP-99m Tc solution was calculated using the fraction of total activity corresponding to each radioisotope and compared with the effective dose delivered by the generator-produced 99m Tc. RESULTS: In all cases, the total effective DI of CP-99m Tc radiopharmaceuticals calculated with either versions of the OLINDA software was less than 10% from 6 up to 12 h after EOB. 94m Tc and 93m Tc are the Tc radioisotopes with the highest concentration in the CP-99m Tc solution at EOB. However, their contribution to DI 6 h after EOB is minimal, due to their short half-lives. The radioisotopes with the largest contribution to the effective DI are 96 Tc, followed by 95 Tc and 94 Tc. This is due to the types of their emissions and relatively long half-lives, although their concentration in the CP-99m Tc solution is five times lower than that of 94m Tc and 93m Tc at the EOB. CONCLUSIONS: The increase in the radiation dose caused by other Tc radioisotopes contained in CP-99m Tc produced as described here is quite low. Even though the concentrations of the 94 Tc and 95 Tc radioisotopes in the CP-99m Tc solution exceed the limits established by the European Pharmacopoeia, CP-99m Tc radiopharmaceuticals could be used in routine nuclear medicine diagnostic studies if administered from 6 to 12 h after the EOB, thus maintaining the effective DI within the 10% limit.

Radiochemical Evaluation and In Vitro Assessment of the Targeting Ability of a Novel 99mTc-HYNIC-RGD for U87MG Human Brain Cancer Cells.

BACKGROUND: Labeled RGD peptide that specifically targets ανß3 integrin has great potential for the early diagnosis of malignant tumors.αvß3 integrin receptors appear specifically more on the surface of glioblastoma (malignant glioma) cells rather than normal cells. OBJECTIVE: The aim of this study was to identify a novel RGD that can be radiolabeled with99mTc with in vitro assessment of its targeting ability for U87MG human brain cancer cells. METHOD: Novel RGD was designed by Amino Acid retro-inversion technique. The peptide HYNIC conjugate was radiolabeled with 99mTc at 95°C for 10 min and radiochemical analysis was performed using ITLC and HPLC methods. The stability of the radiopeptide was checked in the presence of human serum at 37°C up to 24 h. Binding properties and internalization were studied with U87MG cells. RESULTS: Novel HYNIC-RGD has shown high radiochemical purity over 98%. Radioconjugate binding and internalization in U87MG cells were high and specific (13.96% and 12.38% at 4 h respectively). The radiolabeled peptide revealed good affinity for glioblastoma cells (Kd =1.46 ±0.26nM). CONCLUSION: The in vitro study demonstrated the targeting ability of novel 99mTc-HYNIC-RGD for glioblastoma cells. Therefore, more in vivo studies are required.

Untangling the web of European regulations for the preparation of unlicensed radiopharmaceuticals: a concise overview and practical guidance for a risk-based approach.

Radiopharmaceuticals are highly regulated, because they are controlled both as regular medicinal products and as radioactive substances. This can pose a hurdle for their development and clinical use. Radiopharmaceuticals are fundamentally different from other medicinal products and these regulations are not always adequate for their production. Strict compliance may have a huge resource impact, without further improving product quality. In this paper we give an overview of the applicable legislation and guidelines and propose a risk-based approach for their implementation. We focus on a few controversial Good Manufacturing Practice topics: cleanroom classification, air pressure regime, cleanroom qualification and microbiological monitoring. We have developed an algorithm to assess the combined risk of microbiological contamination of a radiopharmaceutical preparation process and propose corresponding Good Manufacturing Practice classification levels. In our opinion, the risk of carry-over of radiopharmaceuticals by individuals cannot be contained by pressure differences, and complicated regimes with underpressured rooms are not necessary in most situations. We propose a sterility assurance level of 10 for radiopharmaceuticals that are administered within a working day, irrespective of their use. We suggest the adoption of limits for environmental monitoring of microbial contamination, as proposed by Bruel and colleagues, on behalf of the French Society of Radiopharmacy. Recently launched regulatory documents seem to breathe a more liberal spirit than current legislation and recognize the need for the use of risk assessment. We argue that future legislation be further harmonized and state risk assessment as the gold standard for implementation of drug quality regulations for the preparation of unlicensed radiopharmaceuticals.

EURADOS intercomparison on emergency radiobioassay.

Nine laboratories participated in an intercomparison exercise organised by the European Radiation Dosimetry Group (EURADOS) for emergency radiobioassay involving four high-risk radionuclides ((239)Pu, (241)Am, (90)Sr and (226)Ra). Diverse methods of analysis were used by the participating laboratories for the in vitro determination of each of the four radionuclides in urine samples. Almost all the methods used are sensitive enough to meet the requirements for emergency radiobioassay derived for this project in reference to the Clinical Decision Guide introduced by the NCRP. Results from most of the methods meet the requirements of ISO 28218 on accuracy in terms of relative bias and relative precision. However, some technical gaps have been identified. For example, some laboratories do not have the ability to assay samples containing (226)Ra, and sample turnaround time would be expected to be much shorter than that reported by many laboratories, as timely results for internal contamination and early decisions on medical intervention are highly desired. Participating laboratories are expected to learn from each other on the methods used to improve the interoperability among these laboratories.

Radiosynthesis of 68Ga-labelled DOTA-biocytin (68Ga-r-BHD) and assessment of its pharmaceutical quality for clinical use.

OBJECTIVES: Biocytin analogues labelled with indium-111, yttrium-90 and lutetium-177 have shown their effectiveness in the imaging of infections/inflammation in patients with osteomyelitis and function as efficient tools in pretargeted antibody-guided radioimmunotherapy. In this study, the labelling of a biocytin analogue coupled with DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), namely, r-BHD, with gallium-68 (68Ga) was optimized, and the quality and stability of the preparations were assessed for clinical use. MATERIALS AND METHODS: Synthesis of 68Ga-r-BHD was carried out by heating a fraction of the 68Ge/68Ga eluate in a reactor containing the biocytin analogue with the appropriate buffer. The influence of the precursor amount (from 2.5 to 140 nmol), the pH of the reaction (from 2 to 5.5) and the buffer species (1.5 mol/l sodium acetate, 1.5 mol/l sodium formate, 4.5 mol/l HEPES) on radiochemical yield and radiochemical purity was assessed. Studies on stability and binding to avidin (Av) were also conducted in different media. RESULTS: Under the best labelling condition (56 nmol of precursor, 3.8 pH, sodium formate buffer) synthesis of 68Ga-r-BHD resulted in a yield of 64 ± 3% (not decay corrected). Radiochemical purity was around 95% because a 68Ga-coordinated sulfoxide form of the ligand was detected as a by-product of the reaction (68Ga-r-SBHD). The by-product was identified and characterized by liquid chromatography-electrospray ionization tandem mass spectrometry. At the natural 1 : 4 Av/68Ga-r-BHD molar ratio, affinity results were 62 ± 2 and 80 ± 2% in saline and human serum, respectively. Stability of 68Ga-r-BHD and of the radiotracer/Av complex remains almost constant over 180 min. 68Ga-r-BHD appears to be a good candidate for clinical applications.

Emergency radiobioassay preparedness exercises through the NIST radiochemistry intercomparison program.

The present challenge for the international emergency radiobioassay community is to analyze contaminated samples rapidly while maintaining high quality results. The National Institute of Standards and Technology (NIST) runs a radiobioassay measurement traceability testing program to evaluate the radioanalytical capabilities of participating laboratories. The NIST Radiochemistry Intercomparison Program (NRIP) started more than 10 years ago, and emergency performance testing was added to the program seven years ago. Radiobioassay turnaround times under the NRIP program for routine production and under emergency response scenarios are 60 d and 8 h, respectively. Because measurement accuracy and sample turnaround time are very critical in a radiological emergency, response laboratories' analytical systems are best evaluated and improved through traceable Performance Testing (PT) programs. The NRIP provides participant laboratories with metrology tools to evaluate their performance and to improve it. The program motivates the laboratories to optimize their methodologies and minimize the turnaround time of their results. Likewise, NIST has to make adjustments and periodical changes in the bioassay test samples in order to challenge the participating laboratories continually. With practice, radioanalytical measurements turnaround time can be reduced to 3-4 h.

Assessment of radon equilibrium factor from distribution parameters of simultaneous radon and radon progeny measurements.

In Canada, a radon and radon progeny survey was carried out in the 1970s in 19 cities. To the authors' knowledge, this is the only large survey of simultaneous radon and radon progeny measurements up to the present time. However, the survey was carried out for the purpose of establishing geographic variation of radon and radon progeny; therefore, radon equilibrium factors, F, were not assessed at that time. From the summary results of this large simultaneous radon and radon progeny survey, the characteristics of radon equilibrium factor were assessed. The average F factor assessed from this survey in 12,576 houses is 0.54. The current assessment may indicate that the typical F value of 0.4 recommended by the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR), and the International Commission on Radiological Protection (ICRP) could lead to a downward bias in the estimation of radon doses to the lung.

A radiochemical technique with potential for revealing novel fungal metabolites according to expression of specific biosynthetic activities.

Fungal secondary metabolites are mostly derived from a few key intermediates in primary metabolism, such as acetate and some amino acids. Classical screens for novel fungal compounds of possible industrial interest have used chromatographic displays of extract components, as was the practice for plant natural products, followed by structural determination and pharmacological study. In contrast, modern robotic screens usually focus initially on specific bioassay applied to fermentation products and crude extracts. Both strategies are expensive in terms of human resources and/or in sophisticated equipment. A relatively inexpensive technique, exploiting biosynthetic principles through use of 14C-labelled probes to recognise particular structural features by autoradiography of tlc plates is described and discussed. Application to 80 isolates of filamentous fungi from Caribbean and European/Mediterranean environments enabled recognition of arrays of metabolites according to their biosynthetic origin, showing the potential of the technique in novel product discovery in unsophisticated laboratory facilities, as exemplified by reference to subsequent discovery of novel metabolites produced by Amorosia littoralis.

Assessment of the global fallout of plutonium isotopes and americium-241 in the soil of the central region of Saudi Arabia.

A radiochemical technique for determination of plutonium isotopes and 241Am in soil samples is tested against IAEA-standard reference materials to determine its accuracy and precision for reliable results. The technique is then used in the investigation of topsoil samples, collected from the natural environment of the central region of Saudi Arabia, to assess the effect of fallout accumulation of these radionuclides in the region. Plutonium and americium were sequentially separated from all other components of the sample by anion-exchange chromatography and co-precipitated with Nd3+ as fluorides. The precipitates were mounted on membrane filters and measured using a high-resolution alpha-spectrometer. The results of the analysis of the reference materials showed satisfactory sensitivity and precision of the technique. The results of the analyzed soil samples show activity levels ranging from < LLD to 0.089 and from

The radiochemist's role in the quality evaluation and assessment of radiological data in environmental decision making.

The quality evaluation and assessment of radiological data is the final step in the overall environmental data decisionprocess. This quality evaluation and assessment process is performed outside of the laboratory, and generally the radiochemist is not involved. However, with the laboratory quality management systems in place today, the data packages of radiochemical analyses are frequently much more complex than the project/program manager can effectively handle and additionally, with little involvement from radiochemists in this process, the potential for misinterpretation of radiological data is increasing. The quality evaluation and assessment of radiochemistry data consists of making three decisions for each sample and result, remembering that the laboratory reports all the data for each analyses as well as the uncertainty in each of these analyses. Therefore, at the data evaluation and assessment stage, the decisions are: (1) is the radionuclide of concern detected (each data point always has a number associated with it?); (2) is the uncertainty associated with the result greater than would normally be expected; and (3) if the laboratory rejected the analyses is there serious consequences to other samples in the same group? The need for the radiochemist's expertise for this process is clear. Quality evaluation and assessment requires the input of the radiochemist particularly in radiochemistry because of the lack of redundancy in the analytical data. This paper describes the role of the radiochemist in the quality assessment of radiochemical data for environmental decision making.

Monte Carlo analysis of multi-layer targets for production of 15O.

A Monte Carlo simulation code was written to analyze multi-layer targets for production of 15O. The code models beam-particle transport through the target and production and transport of generated radionuclides; it can be used to assess the effects on radionuclide production of several beam and target design variables. A pathlength correction feature is included that relaxes restrictions inherent in previous ion transport codes and a variance reduction procedure is implemented that significantly improves code efficiency.

The Monte Carlo simulation of the biological effect of the 10B(n, alpha)7Li reaction in cells and tissue and its implication for boron neutron capture therapy.

The energy deposition in the nucleus of cells exposed to the 10B(n, alpha)7Li neutron capture reaction has been calculated and compared to the measured biological effect of this reaction. It was found that a considerable distribution of hit sizes to the nucleus occurs. The comparison of hit size frequency with the observed survival indicates that not every hit, independent of its size, can lead to cell death. This implies the existence of a hit size effectiveness function. The analysis shows that the location of boron relative to the radiation-sensitive volume of the cell is of great importance and that average dose values alone are of limited use for predicting the biological effect of this reaction. Boron accumulating in the cell nucleus is much more efficient in cell killing than the same amount of boron uniformly distributed; its presence in one cell, however, has little effect on its neighboring cells in a tissue. When boron is present on the cell surface of a tissue (as presumably delivered by antibodies), its cell-killing effect is greatly reduced compared to that in uniform distribution. However, in this case much of the dose to one cell comes from neutron capture reactions occurring on the surface of its neighbor cells. These data have implications for the choice of boron carries in neutron capture therapy. The mathematical analysis carried out here is similar to that proposed recently for low-level exposure effects of radiation, taking mutation and/or carcinogenesis as biological effects. The results here show that high-level exposure to high-LET particles (resulting in cell killing) should be treated in an analogous manner.