Visualization and Quantification of Radiochemical Purity by Cerenkov

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Visualization and quantification of radiochemical purity by Cerenkov luminescence imaging Yeong su Ha, Woong Hee Lee, Jung-Min Jung, Nisarg Soni, Darpan N Pandya, Gwang Il An, Swarbhanu Sarkar, Won Kee Lee, and Jeongsoo Yoo Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01098 • Publication Date (Web): 11 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

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Analytical Chemistry

Visualization and quantification of radiochemical purity by Cerenkov luminescence imaging

Yeong Su Ha1, Woonghee Lee1, Jung-Min Jung1, Nisarg Soni1, Darpan N. Pandya1, Gwang Il An2, Swarbhanu Sarkar1, Won Kee Lee3, Jeongsoo Yoo1,*

1

Department of Molecular Medicine, BK21 Plus KNU Biomedical Convergence

Program, School of Medicine, Kyungpook National University, Daegu, Korea; 2

Molecular Imaging Research Center, Korea Institute of Radiological and Medical

Sciences, Seoul, Korea; 3Medical Research Collabration Center in KNUH & Kyungpook National University School of Medicine, Daegu, Korea

Corresponding Author Jeongsoo Yoo, Department of Molecular Medicine, School of Medicine, Kyungpook National University, Daegu 41944, Korea. E-mail: [email protected]. Phone: +82-53-420-4947. Fax: +82-53-426-4944

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ABSTRACT

Determination of radiochemical purity is essential for characterization of all radioactive compounds, including clinical radiopharmaceuticals. Radio-thin layer chromatography (radio-TLC) has been used as the gold standard for measurement of radiochemical purity; however, this method has several limitations in terms of sensitivity, spatial resolution, two-dimensional scanning, and quantification accuracy. Here, we report a new analytical technique for determination of radiochemical purity based on Cerenkov luminescence imaging (CLI), whereby entire TLC plates are visualized by detection of Cerenkov radiation. Sixteen routinely used TLC plates were tested in combination with three different radioisotopes (131I,

124

I, and

32

P). All TLC

plates doped with a fluorescent indicator showed excellent detection sensitivity with scanning times of less than 1 min. The new CLI method was superior to the traditional radio-TLC scanning method in terms of sensitivity, scanning time, spatial resolution, and two-dimensional scanning. The CLI method also showed better quantification features across a wider range of radioactivity values compared with radio-TLC and classical zonal analysis, especially for b--emitters such as 131I and 32P.

Key Words: radiochemical purity; Quantification; radio-TLC, Cerenkov luminescence imaging


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Determination of radiochemical purity is an essential step during the development of radioactive compounds and the routine daily production of radiopharmaceuticals in the clinic.1,2 Recently, application of molecular imaging technologies based on nuclear imaging has expanded rapidly, and the markets for both therapeutic radiopharmaceuticals and diagnostic radiopharmaceuticals, especially for positron emission tomography (PET) imaging in the latter case, are growing quickly.3-5 The method that is typically used for determination of radiochemical purity is radio-thin layer chromatography (radio-TLC), whereby a TLC plate is developed and then scanned using a radio-detector; the results of radio-TLC analysis are displayed in chromatograms.6,7 Because radioactive compounds are handled in extremely low quantities (typically < 10-9 mol), radioactive spots on TLC plates cannot be visualized using standard UV lamps or any staining methods used in the laboratory. High performance liquid chromatography (HPLC) instruments equipped with radioactivity detectors can also be used to quantify radiochemical purity6,8; however, because of the high instrumentation costs, necessity of time-consuming analysis, and requirement for professional knowledge in HPLC operation, this method is not preferred unless high-resolution separation of the relevant compounds is needed. Zonal analysis,6,9 a labor-intensive method whereby a TLC plate is cut into multiple pieces and the activity of each piece is measured using a gamma counter or liquid scintillation counter (LSC) depending on the decay mode of the radioisotope, is possible as a last resort when other methods are not available; however, this method provides only a rough assessment of radiochemical purity and also requires specialized radio-detectors for quantification of radioactivity. Cerenkov radiation refers to the dim light emitted when charged particles, such as beta particles (electrons and positrons) or alpha particles, move faster than light



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through a particular medium.10,11 The photon intensity of Cerenkov radiation is higher at wavelengths in the ultraviolet light region and is proportional to 1/λ2.12-14 The intensity of Cerenkov radiation also depends strongly on the refractive index of the medium, increasing with the refractive index of the medium.15,16 Very recently, a new molecular imaging technique based on Cerenkov radiation has been developed, known as Cerenkov luminescence imaging (CLI); this technique involves imaging of Cerenkov light originating from radioisotopes injected into living organisms using the highly sensitive charge-coupled device (CCD) cameras of optical imaging systems. 17-20

In 2010, we also reported that luminescence imaging based on Cerenkov radiation holds promise as a new optical imaging modality, demonstrating that Cerenkov luminescence imaging (CLI) offers high spatial resolution and tissue penetration compared with other conventional optical imaging modalities.17 In a proof-of-concept demonstration, a radio-TLC plate spotted with an

131

I-labeled

compound was imaged by CLI and also scanned using a radio-TLC scanner, and the results of these analyses were shown to be comparable.18 However, further in-depth studies of the practical application have not yet been pursued. Recently Spinelli et al. devised a simple imaging system composed of a CCD coupled with lens mounted on a black light-tight box for the detection of different type of radiations including Cerenkov luminescence light.21 In the current study, we performed a comprehensive screening experiment to determine whether CLI could be used as a universal analytical method for the determination of radiochemical purity. This new method enabled visualization of radio-TLC plates in a similar manner to conventional TLC plates under a UV lamp and accurate quantification of radiochemical purity. The accuracy of the new method


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was directly compared with that of conventional radio-TLC and zonal analysis methods.

EXPERIMENTAL SECTION Radioisotopes and Materials. [32P]Phosphoric acid and [methyl-3H]thymidine were purchased from Perkin Elmer (Waltham, USA), and [131I]NaI was purchased from the Korea Atomic Energy Research Institute (KAERI, Daejeon, Korea). [124I]NaI was produced at the Korea Institute of Radiological and Medical Sciences (KIRAMS, Seoul, Korea) using an MC50 Cyclotron (Scanditronix, Uppsala, Sweden). In total, 16 types of TLC plates were tested in the current study. TLC silica gel 60 F254, TLC aluminum oxide 60 F254 neutral, TLC silica gel 60 RP-18 F254s, HPTLC silica gel 60 F254, TLC cellulose F, TLC silica gel 60 F254 (backing: plastic), TLC silica gel 60 F254 (backing: glass), PLC silica gel 60 F254 0.5 mm, PLC silica gel 60 F254 1 mm, PLC silica gel 60 F254 2 mm, and TLC silica gel 60 (without a fluorescent indicator) were purchased from Merck (Darmstadt, Germany). ITLC-SG (instant thin layer chromatography medium) was purchased from Agilent Technologies (Santa Clara, USA), and Grade 1 Chr, Grade 17 Chr, and Grade 31ET Chr chromatography paper was purchased from Whatman (Maidstone, UK). The features of the 16 TLC plates and the notation used in this study are summarized in Table 2. Fluorescence indicator green 254 nm (manganese-doped zinc silicate, Zn2SiO4) was purchased from Sigma Aldrich (St. Louis, USA). The liquid scintillation cocktail Insta-Gel Plus was purchased from Perkin Elmer (Waltham, USA). A transparent glass plate with a refractive index of 1.5 and thickness of 1.2 mm was used to cover the TLC plates for amplification of the luminescence signal. A glass plate with a refractive index of 1.6



and thickness of 1 mm was purchased from Hongsung Optical (Bucheon, Korea). Refractive indices were measured using a spectroscopic ellipsometer (SOPRA, Courbevoie, France).

Spotting of radioactive samples on TLC plates. Three-fold serial dilutions of each radioisotope solution were prepared to give activities of 37, 12.3, 4.11, 1.37, and 0.46 kBq/µL. The resulting solutions were spotted on the top portion of each TLC plate at 1 cm intervals, and three of the solutions (37, 12.3, and 4.11 kBq/µL) were also spotted on the lower portion of the plate at 5 mm intervals (Figure 1A).

Cerenkov luminescence imaging (CLI) studies. All CLI studies were performed using IVIS Spectrum or Lumina II instruments together with Living Image software (Perkin Elmer, Waltham, USA). The Cerenkov luminescence signal was acquired using a bioluminescence channel with no excitation light or emission filter. All Cerenkov luminescence images were obtained using a fixed scan time of 1 min. A threshold was applied for the luminescence images to maximize visualization of the regions of interest (ROIs) and to minimize background luminescence. The imaging parameters were as follows: IVIS Spectrum - block excitation; open emission filter; field of view (FOV) = D (22.5 × 22.5 cm); Bin = 8; f = 1; IVIS Lumina II - block excitation; open emission filter; FOV = D (12.5 × 12.5 cm); Bin = 4; f = 1. Luminescence was quantified in each image by measurement of photon radiance (photons/second/cm2/steradian) by ROI analysis. Quantitative analysis was conducted using Living Image software.


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Fluorescence spectroscopy. Fluorescence measurements were recorded on a F-7000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) at the Korea Basic Science Institute (KBSI, Daegu, Korea). Fluorescence emission spectra were recorded using an excitation wavelength of 250 nm and slit widths of 5.0 nm for both excitation and emission. The adsorbent on plate 5 (Si(0.2)_F_Al) was scraped off, weighed, and placed in a quartz cuvette with a path length of 1 cm, and the fluorescence emission spectrum of the adsorbent was obtained using a fluorescence spectrophotometer. The powdered form of the fluorescent indicator, in the same amount (100 mg) as was obtained by scraping the adsorbent from plate 5, was weighed and placed in a quartz cuvette, and the fluorescence emission spectrum of this sample was measured using the parameters described above. To obtain the luminescence spectrum of the fluorescent indicator in 131I solution, the same amount of the fluorescent indicator was prepared in a quartz cuvette, and

131

I solution (37 KBq), diluted to 500 µL in water,

was added. The resulting mixture was vortexed vigorously for 1 min before luminescence measurement. The sample was excited with Cerenkov radiation emitted from

131

I, with no additional excitation light, and the luminescence spectrum was

collected from 300 nm to 700 nm using a fluorescence spectrophotometer with a scan speed of 2400 nm/min. Luminescence spectra of F254 (100 mg) and liquid scintillator (30 µL) in 131I solution were measured in the similar way as above.

Effect of covering TLC plates with glass on luminescence intensity. TLC plate 11 (iTLC-Si) was spotted with the radioisotope samples, air-dried at room temperature for 5 min, and then imaged for 1 min by Cerenkov luminescence imaging using an IVIS Lumina II system. The scan was immediately repeated after covering the TLC plate with a glass plate with a refractive index of 1.5 or 1.6.



Effect of spraying scintillation cocktail on the luminescence intensity of TLC plates. We selected four TLC plates (plate 5, Si(0.2)_F_Al; plate 9, Si(0.2)_Al; plate 11, iTLC-Si; plate 13, Paper(0.18)) to examine the effect of scintillation cocktail solution on the non-fluorescent TLC plates. The TLC plates were spotted with the radioisotope samples, air-dried at room temperature for 5 min, and then imaged for 1 min by Cerenkov luminescence imaging using an IVIS Lumina II system. Insta-Gel Plus scintillation cocktail solution was then sprayed onto the TLC plates, using a small sprayer to wet the plates evenly. Luminescence images of the wet TLC plates were obtained again for 1 min using the IVIS Lumina II system. To quantify the luminescence signal intensity, photon radiance (photons s-1.cm-2.sr-1) was measured by ROI analysis.

Two-dimensional (2D) images. 2D radio-TLC analysis was performed immediately after Cerenkov luminescence imaging using an AR-2000 radio-TLC scanner together with WinScan 2D software (Eckert & Ziegler Radiopharma, Hopkinton, USA) to obtain sequential 1D images. The radioactivity on the TLC plates was scanned at 1 cm intervals between 0 and 12 cm, and then represented in 2D images.

Comparison of CLI, radio-TLC, and zonal analysis methods for quantification accuracy. All 16 TLC plates were imaged simultaneously in the same field of view (22.5 × 22.5 cm) on the IVIS Spectrum system, then scanned each TLC plate individually using the radio-TLC scanner for 1 min. For zonal analysis, all TLC plates except the glass-backed TLC plates (plates 5-16) were cut into 1-cm-wide


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pieces using scissors. To quantify the activity of each spot on the four glass-backed TLC plates (plates 1-4), 1-cm-wide segments of the silica gel containing the radioactive samples were scraped. The activities of

131

I and

124

I were measured for 1

min using a Wallac Wizard 1480 gamma counter (Perkin Elmer, Waltham, USA), while the activity of

32

P was measured for 1 min using a Tri-Carb 2910TR liquid

scintillation counter (LSC) (Perkin Elmer, Waltham, USA) in Cerenkov counting mode, using pure water as the solvent. To compare quantification accuracy, the activities of the five upper spots on each TLC plate were measured using each method (Figure 1A). For each TLC plate, the activities of the four lower spots were divided by the activity of the uppermost spot with the highest activity, and the four ratios were plotted separately for each TLC plate (Figure 6). The ten TLC plates doped with fluorescent indicator (plates 1-8, 10, and 12) were used for comparative analysis.

RESULTS AND DISCUSSION Firstly, we assessed the general applicability of this new imaging method via analysis of three different radioisotopes with different decay modes (32P, 131I, and 124I) using 16 different types of TLC plates, a total of 48 different conditions. 32P is a 100% beta minus (b-) emitter, 131I is a 100% β- emitter that also emits gamma radiation, and 124

I decays via beta plus (b+) emission (23%) (Table 1)22. We also carefully selected

16 different commonly used TLC plates, which differed in terms of the TLC adsorbent (silica gel, aluminum oxide, glass microfiber, cellulose paper), the thickness of the adsorbent (0.1-2 mm), the backing material (glass, aluminum, plastic), and the presence of a fluorescent indicator (Table 2).



In order to assess the sensitivity and spatial resolution of CLI, samples with different amounts of activity were spotted on TLC plates at intervals of 1 cm or 0.5 cm (Figure 1A). Three-fold serial dilutions of each radioisotope (37, 12.3, 4.1, 1.4, and 0.46 kBq, 1 µL each) were spotted on TLC plates at 1 cm intervals from the top of the plate, and three of these samples (37, 12.3, 4.1 kBq) were also spotted at 0.5 cm intervals on the lower part of the plate. Sixteen sets of TLC plates spotted with [131I]NaI, [124I]NaI, or [32P]phosphoric acid were scanned simultaneously for 1 min using an IVIS Spectrum imaging system (Perkin Elmer, Waltham, USA) with no excitation light and no emission filters.

Effect of fluorescent indicator doped on TLC plates on luminescence intensity. Luminescence images of the sixteen

131

I-spotted TLC plates are shown in

Figure 1B. At first glance, at least three of the spots with the highest activity were clearly visible on 10 of these plates (plates 1-8, 10, and 12). However, no significant luminescence signal was detected on the other six plates, except for a barely visible spot for the highest activity sample on plate 15. The common feature of the 10 plates for which luminescence signals were clearly visible was the presence of a fluorescent indicator (F254). Regardless of their packing or backing materials, the other six plates did not show strong luminescence signals at the positions of any radioactive spots. Even though some differences in overall intensity were observed for the other isotopes (124I and

32

P), the same absolute dependence of signal sensitivity on the

presence of the fluorescent indicator was observed (Supporting Information Figure S1 & S2). This phenomenon can be explained by considering the relationship between the wavelength dependence of Cerenkov radiation, the optical properties of the fluorescent indicator, and the detection sensitivity of the luminescence imager.


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Cerenkov radiation exhibits a continuous spectrum, and the intensity of Cerenkov radiation is inversely proportional to its wavelength, which means that Cerenkov radiation is strongest in the UV region.23 The detection sensitivity of a typical luminescence detector is poor in this region, but much greater in the visible and near-infrared regions (400-800 nm).24 The fluorescent indicator most commonly used in TLC is manganese-activated zinc silicate (Zn2SiO4:Mn, commonly denoted F254), which displays maximum absorption between 220 and 320 nm and maximum emission between 520 and 540 nm.25 The fluorescent indicator doped on TLC plates seems to absorb strong Cerenkov radiation in the UV region, resulting in strong emission in the visible spectrum, which can be sensitively detected using the CCD detector of an optical imager. Consistent with this hypothesis, the adsorbent of plate 5 (silica gel doped with the fluorescent indicator F254), which was scraped off and analyzed by fluorescence spectrometry, showed a fluorescence emission spectrum almost identical to that of manganese-activated zinc silicate (Supporting Information Figure S3.A, B). Furthermore, the luminescence spectrum of manganese-activated zinc silicate in a solution containing

131

I resembled the emission spectrum of

manganese-activated zinc silicate obtained following excitation at 250 nm (Supporting Information Figure S3.C). When F254 was mixed with

131

I solution

(Cerenkov luminescence excitation), the intensity of the original Cerenkov luminescence at a lower wavelength (Figure S3D) diminished significantly, and new strong light at 531 nm was emitted (Figure S3F). These data strongly imply that the fluorescent indicator (F254) absorbs most of the strong Cerenkov radiation generated by

131

I in the UV region (Figure S3D) before emitting intense fluorescence around

530 nm, which is much greater in intensity than the unabsorbed Cerenkov radiation itself and is ultimately detected efficiently by CCD detector. Liquid scintillator also



absorbs most Cerenkov luminescence generated by

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131

I solution and emits a strong

fluorescence at 431 nm (Figure S3H). In a recently developed “Cerenkov radiation energy transfer” technique, Cerenkov radiation in the UV region are absorbed by fluorescent dye or quantum dot and reemitted as a strong fluorescence light in longer wavelength for the better tissue penetration,10,26,27 which is basically the same phenomenon observed in current study.

Effects of the adsorbent and backing materials of TLC plates on luminescence intensity. The relative luminescence intensities of each plate were accurately compared by quantification of the photon radiance of the illuminating spots. Circular ROIs of the same size were drawn on the uppermost spot of each TLC plate, and the photon radiance associated with each spot was measured (Figure 2). First, we compared three TLC plates (plates 1, 5, and 10) with the same adsorbent (silica gel of a similar thickness) but different backing materials (glass, aluminum, and plastic, respectively). The luminescence signals associated with each plate were comparable, indicating that the effect of the backing material on luminescence intensity is minimal. However, comparison of plates 5 and 12, which have different adsorbents (silica gel and aluminum oxide, respectively) of the same thickness (0.2 mm), showed that the luminescence intensity increased by 162% when aluminum oxide was used as the adsorbent rather than silica gel (1.722 ± 0.017 ´ 106 photons s-1cm-2sr-1 vs. 1.065 ± 0.114 ´ 106 photons s-1cm-2sr-1). This observation can be explained by considering the different refractive indices of the adsorbents. Cerenkov radiation is strongly dependent on the refractive index of the medium through which the relevant charged particles pass. The energy threshold for the emission of Cerenkov radiation is


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correlated with the refractive index of the medium according to the following equation28:

𝑇 keV = 511

1 1−1

+

𝑛*

*

−1

where T is the threshold for the emission of Cerenkov radiation, and n is the refractive index of the medium. As the refractive index of the medium increases, the threshold energy decreases, resulting in Cerenkov radiation of greater intensity. Because the refractive indices of aluminum oxide and silica gel are 1.7682 and 1.4585, respectively29, all beta particles with an energy of > 109 keV emitted during 131I decay can emit Cerenkov radiation in an aluminum oxide medium, while only beta particles with an energy of > 191 keV can contribute to Cerenkov radiation in a silica gel medium. All five spots were detected on the aluminum oxide plate (plate 12), but only four spots were visible on the silica gel plate (plate 5). Plate 9, a silica gel plate identical to plate 5 except that it does not contain a fluorescent indicator, exhibited a luminescence intensity 133-fold lower than that of plate 5 (0.008 ± 0.001 ´ 106 photons s-1cm-2sr-1) (Figure 2). The other two iTLC-SG and cellulose plates that did not contain a fluorescent indicator also showed negligible photon values.

Effect of the thickness of the adsorbent of TLC plates on luminescence intensity. The second dominant factor affecting luminescence intensity was found to be the thickness of the adsorbent of the TLC plates (Figure 3). As the thickness of the adsorbent (silica gel) was increased from 0.25 mm to 0.5, 1, and 2 mm in TLC plates



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1, 2, 3, and 4, respectively, the photon radiance of the highest activity spot (37 kBq) on each plate increased from 1.11 to 1.48, 2.57, and 3.33 ´ 106 photons s-1cm-2sr-1, respectively. Even though the overall luminescence intensities of the paper TLC plates without fluorescent indicators were much lower than those of plates doped with a fluorescent indicator, the same dependence of luminescence intensity on the thickness of the TLC plate was clearly observed; cellulose paper of 0.92 mm thickness (plate 15) showed 156% and 346% increases in Cerenkov luminescence compared with similar cellulose paper plates with 0.5 mm thickness (plate 14) and 0.18 mm thickness (plate 13), respectively (Figure 3, right). As the thickness of an adsorbent with a higher refractive index than air is increased, conversion of the kinetic energy of ejected beta particles to Cerenkov radiation is also increased.15,30 The refractive indices of silica gel and cellulose are 1.4585 and 1.4613, respectively, while that of air is only 1.0003.29

Auxiliary strategies to increase luminescence intensity. Using the same strategy, specifically, by increasing the thickness of the medium that the charged particles (b- particles in the case of 131I) pass through, we found that the luminescence signal could be amplified simply by covering the TLC plates with a glass plate (Figure 4). Instant TLC plates (iTLC, plate 11) are commonly used in radiochemistry laboratories31 because they can be developed relatively quickly; however, the Cerenkov radiation observed using this plate was very weak because of the lack of a fluorescent dopant (Figure 2). However, when the iTLC plate was covered with a 1.2-mm-thick glass plate with a refractive index of 1.5, amplification of the luminescence signal was observed for all radioisotopes (Figure 4A); for

131

I and

124

I,

the spots with the highest activity (37 kBq), which were previously invisible, became


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clearly visible, and for

32

P, the luminescence intensity of the formerly faint spot

increased significantly. The photon radiance associated with the original spots increased 4.22-, 5.34- and 7.55-fold for

131

I,

124

I, and

32

P, respectively, after the plates were covered with glass

(Figure 4B). These differences in the relative increase in luminescence intensity can be explained by consideration of the mean energies of the ejected beta particles and the branching ratios for each radioisotope (Table 1). The mean energies of beta particles emitted by 32P and 124I (Eb- mean 695 keV, Eb+ mean 819 keV, respectively) are much higher than the mean energy of beta particles emitted by 131I (Eb- mean 182 keV). Even though the mean energy of 124I-derived beta particles is slightly higher than that of 32P-derived beta particles (819 vs. 695 keV), the branching ratio of b- decay for 32P is four times higher than that of b+ decay for

124

I (100 vs. 23%), clearly explaining

why the increase in luminescence intensity was greatest for 32P, followed by

124

I and

131 22

I.

When the TLC plates were covered with glass plates with a higher refractive index (1.6), further amplification of the luminescence signal was achieved (Figure 4A). Even though the glass cover with the higher refractive index was slightly less thick than the glass cover with the lower refractive index (1 vs. 1.2 mm), the photon radiance increased 4.23-, 6.18- and 7.67-fold for

131

I,

124

I, and

32

P, respectively

(Figure 4B). The luminescence intensities of TLC plates without fluorescent indicators could also be increased significantly by application of a liquid scintillation cocktail. As discussed above, the fluorescent indicator doped on TLC plates plays a critical role in amplifying Cerenkov radiation to enable detection using common CCD detectors; TLC plates without a fluorescent indicator showed very weak luminescence signals.



However, application of solid fluorescent indicators to TLC plates is not straightforward. Instead, we sprayed the TLC plates with a liquid scintillator, which is a cocktail solution commonly used for liquid scintillation counting. The luminescence intensities of three TLC plates having no fluorescent indicator (plate 9, Si(0.2)_Al; plate 11, iTLC-Si; plate 13, Paper(0.18)) increased dramatically after application of the cocktail solution (Figure 4C, D). Quantitatively, the luminescence signals were amplified more than 2,000-fold (4,684 vs. 10,643,000 photons s-1cm-2sr-1), 200-fold (58,582 vs. 13,565,000 photons s-1cm-2sr-1), and 150-fold (37,064 vs. 5,972,800 photons s-1cm-2sr-1) for the aluminum, glass fiber, and paper plates, respectively, after application of the liquid scintillator. The use of TLC plates sprayed with liquid scintillator as opposed to plates containing a fluorescent indicator generally reduced the spatial resolution, presumably because of spreading of the spots. However, plates containing aluminum as the backing material (for example, plate 9) that were treated with liquid scintillator yielded spatial resolution comparable to analogous plates that contained a fluorescent indicator (plate 5 in Figure 1). On the other hand, the use of glass microfiber (plate 11) and paper (plate 13) TLC plates yielded lower spatial resolution upon application of liquid scintillator (Figure 4D). This signal amplification technique could also be applied to 3H, which emits b– particles too low in energy (Eβ- mean 6 keV) to generate Cerenkov radiation (Table 1). Even though the amplification of the luminescence signal was lower for 3H than for the other radioisotopes, up to three spots with the highest activity could be visualized by luminescence imaging of plates 5 and 9 (Supporting Information Figure S4). The effect of liquid scintillator application on signal amplification for 3H was higher for plate 9 (Si(0.2)_Al, 120-fold, 1,475 vs. 177,430 photons s-1cm-2sr-1) and plate 11


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(iTLC-Si, 120-fold, 812 vs. 99,334 photons s-1cm-2sr-1) than for plate 5 (Si(0.2)_F_Al, 12-fold, 15,897 vs. 194,800 photons s-1cm-2sr-1) and plate 13 (Paper(0.18), 20-fold, 448 vs. 8,738 photons s-1cm-2sr-1).

Two-dimensional (2D) scanning. The advantages of the new Cerenkov luminescence imaging technique over traditional radio-TLC methods are more evident when the application of each technique to two-dimensional (2D) scanning is considered. 2D images of four TLC plates (plates 1-4) obtained using a radio-TLC scanner and luminescence imager are displayed in Figure 5. First, while the luminescence images of the four TLC plates could be obtained in less than 1 min, recording of the whole 2D radio-TLC image required more than 10 min when a distance interval of 1 cm was used. Increasing the size of the imaging area would further increase the difference in imaging time, because luminescence imaging captures the entire imaging area simultaneously, while the radio-TLC scanner reconstructs 2D images by assembling a series of 1D scanned images. In addition to imaging speed, a further advantage of the new luminescence-based method over the traditional radio-TLC method is greater spatial resolution. Spots of different activity, even at 5 mm intervals, were clearly distinguishable on TLC plates by luminescence imaging. However, in the 2D image produced by the radio-TLC scanner, the spots appeared blurred and, more problematically, were distorted in the horizontal direction, resulting in oval-shaped rather than circular spots. CLI is also advantageous for activity quantification; the photon radiance of each spot could be accurately quantified simply by selecting the relevant ROI. This new technique would be able to be applied efficiently to analysis of proteins, DNA, RNA, and metabolites separated in gels and blots.32-36



Scanning time, neighboring radiation and spatial resolution issues. Compared with conventional radio-TLC methods, the new Cerenkov luminescence imaging technique has several advantages for measuring radiochemical purity. First, the new imaging technique enables high-throughput screening. As indicated in Figure 1, high quality luminescence images of a total of 16 TLC plates were obtained within 1 min. The field of view in a modern luminescence imager is typically larger than 20 ´ 20 cm. Radio-TLC, which necessitates scanning of each plate individually, requires at least 16 min of scanning time alone; data acquisition for a real experiment, which requires additional steps such as positioning each TLC plate and adjusting the acquisition settings for each plate, would take much longer. The time required to determine the radiochemical purity of radiopharmaceuticals before they are released to the clinic is a critical factor in radiopharmacy, especially when handing short-lived radioisotopes such as 11C (t1/2 20 min), 13N (t1/2 10 min), and 15O (t1/2 2 min).37 Another advantage of the CLI technique is that the luminescence signal of each spot is highly independent of neighboring activity, regardless of its intensity. Because CLI detects only luminescence generated as a result of Cerenkov radiation rather than the radiation itself, each spot can be imaged and quantified accurately without interference from radiation derived from neighboring spots with high activity. Most of the kinetic energy of the beta particles (positrons or electrons) is converted to Cerenkov radiation as the particles travel through few millimeters of medium. Highly penetrating gamma rays do not interfere with CCD detector in optical imagers. As shown in Supporting Information Figure S5, nine TLC plates consisting of three different radioisotopes (131I,

124

I, and

32

P) in the same field of view could be

visualized with appropriate signal intensities without any interference between


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radioisotopes, giving identical results to plates containing a single radioisotope (Figures 1, S1, and S2). When luminescence images and radio-TLC chromatograms derived from the same TLC plate (plate 3) are compared, the higher spatial resolution of the CLI technique is clearly evident (Supporting Information Figure S6). In the case of the radio-TLC chromatogram, the chromatographic peaks were well separated using an interval of 1 cm between spots. However, when the distance between adjacent spots was decreased to 5 mm, the peaks overlapped not to identify each peaks, especially in the cases of 124I and 32P. In contrast, luminescence imaging enabled clear visualization of individual spots even at 5 mm intervals. Luminescence imaging also offers a major advantage in post-imaging manipulation, in that the color scale representing the signal intensity of luminescence images can be adjusted to maximize sensitivity and spatial resolution. Decreasing the overall signal intensity of the luminescence images resulted in clearer separation of the spots separated by 5 mm, with no overlap between spots (data not shown).

Quantification comparison studies. The accuracy of the new Cerenkov luminescence method for radiochemical purity was compared with the accuracy of traditional radio-TLC and zonal analysis methods. For each TLC plate, the activities of the second, third, fourth, and fifth spots from the top of the plate were divided by the activity of the first spot with the highest activity (37 kBq); these four values were then plotted versus the theoretical values, and the least squares line was computed for each plate (Figure 6). In comparison with the radio-TLC method, which is currently the gold standard, the new CLI method yielded more accurate quantification of radioactivity (Figure 6).



For all three radioisotopes, most of the least squares lines obtained using the CLI method were in close agreement with the theoretical red least square lines (Figure 6A, D, G). In contrast, many of the least squares lines derived from radio-TLC analysis deviated from the theoretical least squares lines, especially in the cases of 131I and 32P (Figure 6B, H), which indicates that quantification based on radio-TLC analysis does not accurately reflect the actual activity on the TLC plates. Analysis of individual ratios categorized by radioisotope and quantification method clearly shows that the new CLI method enables more accurate quantification of radioactivity (Supporting Information Figure S7). For all three radioisotopes, using the CLI method, the observed values of the activity ratios were more closely clustered around the calculated values, while using the radio-TLC method, the ratio values were scattered across a wider range, especially for S7A-D, I-L). In the case of

124

131

I and

32

P (Supporting Information Figure

I, comparable degrees of dispersion were achieved

using radio-TLC and CLI; however, the median values of the observed ratios obtained using the radio-TLC method deviated further from the theoretical ratios (red dotted line) than those obtained using the CLI method, especially in the case of the fourth ratio (Supporting Information Figure S7H). Zonal analysis showed better accuracy compared to radio-TLC analysis for radioactivity quantification, even though the former method is much more time-consuming and cumbersome. However, although the degree of dispersion of the ratios obtained by zonal analysis was comparable with that obtained using CLI, the median values of the categorized ratio values obtained by zonal analysis showed greater deviations from the expected values than those obtained by CLI, especially for 124

I (Supporting Information Figure S7H) and

32

P (Supporting Information Figure

S7J-L).


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CONCLUSIONS A new optical imaging technique for the efficient measurement of radiochemical yield and purity was developed based on Cerenkov radiation, rather than classical gamma radiation, emitted by radioisotopes. Three radioisotopes with different decay modes (131I,

124

I, and

32

P) spotted on TLC plates could be clearly visualized and

accurately quantified using this new Cerenkov luminescence imaging (CLI) technique. Using this method, all radio-TLC plates containing a fluorescent indicator, regardless of the adsorbent or backing materials, could be visualized and quantified within one minute with excellent sensitivity. A facile method for boosting the luminescence signals of TLC plates without a fluorescent indicator was also developed. CLI was far superior to radio-TLC with respect to scanning time and spatial resolution, especially for two-dimensional scans. In terms of radioactivity quantification, the new CLI method showed better accuracy across a wider range of activities compared with the traditional radio-TLC method and zonal analysis. Future development of luminescence imagers dedicated to radio-TLC scanning, which could enable automatic quantification of the activities of all spots on a TLC plate, will have wide applications in the accelerated development of radioactive compounds and in radiopharmacy.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:



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Cerenkov luminescence image of TLC plates spotted with I-124 and P-32. fluorescence emission spectra, amplification of luminescence signal intensity of TLC plates, Cerenkov luminescence images of TLC plates spotted with different radioisotopes, comparative images of Cerenkov luminescence signals and radio-TLC chromatograms, and statistical analysis of three quantification methods (PDF)

AUTHOR INFORMATION Corresponding Author *J.Y.: phone, +82-53-420-4947; e-mail, [email protected]. ORCID Jeongsoo Yoo: 0000-0002-0752-5745 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by an R&D program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (No. 2016R1A2B4011546,

2013R1A4A1069507,

2016H1D3A1907667,

and

2017M2A2A6A02018506) and supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI17C0221).


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REFERENCES (1) Saha, G. B. In Fundamentals of Nuclear Pharmacy. Saha G. B., Ed.; Springer: New York, 2010; pp 153-174. (2) Vallabhajosula, S.; Killeen, R. P.; Osborne, J. R. Semin. Nucl. Med. 2010, 40, 220-241. (3) Rudin, M.; Weissleder, R. Nat. Rev. Drug Discovery. 2003, 2, 123-131. (4) Volkert, W. A.; Hoffman, T. J. Chem. Rev. 1999, 99, 2269-2292. (5) Preshlock, S.; Tredwell, M.; Gouverneur, V. Chem. Rev. 2016, 116, 719-766. (6) Roberts, T. R. Radiochromatography: the chromatography and electrophoresis of radiolabelled compounds; Elsevier: Amsterdam, 1978. (7) Sherma, J.; DeGrandchamp, D. J. Liq. Chromatogr. Relat. Technol. 2015, 38, 381-389. (8) Veltkamp, A. C. J. Chromatogr. B: Biomed. Sci. Appl. 1990, 531, 101-129. (9) Sherma, J.; Fried, B. Handbook of thin-layer chromatography, 2nd ed.; Marcel Dekker: New York, 1996. (10) Thorek, D. L. J.; Ogirala, A.; Beattie, B.J.; Grimm, J. Nat. Med. 2013, 19, 1345-1350. (11) Kotagiri, N.; Niedzwiedzki, D. M.; Ohara, K.; Achilefu, S. Angew. Chem. Int. Ed. 2013, 52, 7756-7760. (12) Shaffer, T. M.; Pratt, E. C.; Grimm, J. Nat. Nanotechnol. 2017, 12, 106-117. (13) Thorek, D. L. J.; Riedl, C. C.; Grimm, J. J. Nucl. Med. 2014, 55, 95-98. (14) Robertson, R.; Germanos, M. S.; Li, C.; Mitchell, G. S.; Cherry, S. R.; Silva, M. D. Phys. Med. Biol. 2009, 54, N355-N365. (15) Mitchell, G. S.; Gill, R. K.; Boucher, D. L.; Li, C.; Cherry, S. R. Philos. Trans. R. Soc. A. 2011, 369, 4605-4619. (16) Ruby, K. G.; Gregory, S. M.; Cherry, R. C. Phys. Med. Biol. 2015, 60, 4263-4280. (17) Park, J. C.; Yu, M. K.; An, G. I.; Park, S. -I.; Oh, J.; Kim, H. J.; Kim, J. -H.; Wang, E. K.; Hong, I. -H.; Ha, Y. S.; Choi, T. H.; Jeong, K. -S.; Chang, Y.; Welch, M. J.; Jon, S.; Yoo, J. Small. 2010, 6, 2863-2868. (18) Park, J. C.; An, G. I.; Park, S. -I.; Oh, J.; Kim, H. J.; Ha, Y. S.; Wang, E. K.; Kim, K. M.; Kim, J. Y.; Lee, J.; Welch, M. J.; Yoo, J. Nucl. Med. Biol. 2011, 38, 321-329. (19) Liu, H.; Ren, G.; Miao, Z.; Zhang, X.; Tang, X.; Han, P.; Gambhir, S. S.; Cheng, Z. PLoS One. 2010, 5, e9470. (20) Ruggiero, A.; Holland, J. P.; Lewis J. S.; Grimm, J. J. Nucl. Med. 2010, 51, 1123-1130. (21) Spinelli, A. E.; Gigliotti, C. R.; Boschi, F. Biomed. Opt. Express. 2015, 6, 2168-2180. (22) Eckerman, K.F.; Endo, A. MIRD: Radionuclide Data and Decay Schemes; Society of Nuclear Medicine: Reston, 1989. (23) Fülöp, L.; Biró, T. Int. J. Theor. Phys. 1992, 31, 61-74. (24) Janesick, J. R.; Elliott, T.; Collins, S.; Blouke, M. M.; Freeman, J. Opt. Eng. 1987, 26, 692-714. (25) Jagnnathan, R.; Lakshminarayanan, R.; Rajaram, N.; Venkatesan, V. K. Bull. Electrochem. 1987, 3, 677-679. (26) Bernhard, Y.; Collin, B.; Decréau, R. A. Sci. Rep. 2017, 7, 45063. (27) Sun, X.; Huang, X.; Guo, J.; Zhu, W.; Ding, Y.; Niu, G.; Wang, A.; Kiesewetter, D. O.; Wang, Z. L.; Sun, S.; Chen, X. J. Am. Chem. Soc. 2014, 136, 1706-1709.



(28) Rengan, K. J. Chem. Educ. 1983, 60, 682-684. (29) Lide, D. R. CRC handbook of chemistry an physics, 85th ed.; CRC press LLC: Boca Raton, 2004. (30) Cho, J. S.; Taschereau, R.; Olma, S.; Liu, K.; Chen, Y. -C.; Shen, C. K. -F.; Dam, R. M. V.; Chatziioannou, A. F. Phys. Med. Biol. 2009, 54, 6757-6771. (31) Amin, K.; Patel, S.; Doke, A.; Saha, G. B. J. Nucl. Med. Technol. 2011, 39, 51-54. (32) Jiang, D.; Jia, Y.; Zhou, Y.; Jarrett, H. W. J. Proteome. Res. 2009, 8, 3693-3701. (33) Gerner, C.; Vejda, S.; Gelbmann, D.; Bayer, E.; Gotzmann, J.; Schulte-Hermann, R.; Mikulits, W. Mol. Cell. Proteomics. 2002, 1, 528-537. (34) Malloff, C. A.; Fernandez, R. C.; Lam, W. L. J. Mol. Biol. 2001, 312, 1-5. (35) Rio, D. C. Cold Spring Harb. Protoc. 2014, 2014, 793-797. (36) Voytas, D.; Ke, N. In Current Protocols in Immunology; John Wiley & Sons, Inc.: Hoboken, 2001; pp A.3J.1-A.3J.10. (37) Stoll, H. -P.; Hutchins, G. D.; Winkle, W. L.; Nguyen, A. T.; Appledorn, C. R.; Janzen, I.; Seifert, H.; Rübe, C.; Schieffer, H.; March, K. L. J. Nucl. Med. 2001, 42, 1375-1383.


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TABLES Table 1



Table 2


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FIGURES Figure 1



Figure 2


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Figure 3



Figure 4


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Figure 5



Figure 6


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FIGURE LEGENDS

Table 1. Summary of the physical properties of selected radioisotopes. Table 2. Composition of the 16 TLC plates tested in this study. In the notation for each plate, Si, Alu, Cel, F, Gl, Al, and Pl denote silica, aluminum, cellulose, fluorescent indicator, glass, aluminum, and plastic, respectively. Figure 1. Cerenkov luminescence imaging of 16 TLC plates spotted with

131

I. (A)

Spotting pattern of radioactive samples on each TLC plate. (B) Cerenkov luminescence signals of 131I on various TLC plates imaged simultaneously in the same field of view. 1: Si(0.25)_F_Gl, 2: Si(0.5)_F_Gl, 3: Si(1)_F_Gl, 4: Si(2)_F_Gl, 5: Si(0.2)_F_Al, 6: Si(0.25)_F_Al, 7: HP-Si(0.2)_F_Al, 8: RP-18(0.2)_F_Al, 9: Si(0.2)_Al, 10: Si(0.2)_F_Pl, 11: iTLC-Si, 12: Alu(0.2)_F_Al, 13: Paper(0.18), 14: Paper(0.5), 15: Paper(0.92), 16: Cel(0.1)_Al.

Figure 2. Dependence of luminescence signal intensity on the existence of a fluorescent indicator integrated into the adsorbent of the TLC plate. (A) Luminescence images of TLC plates containing (left) or not containing (right) a fluorescent indicator F254. (B) Quantitative analysis of the luminescence signal of the uppermost spot on each TLC plate (n = 3). The dotted circle indicates the region of interest for which the photon radiance was measured.

Figure 3. Dependence of luminescence signal intensity on the thickness of the adsorbent. (A) Luminescence images of silica (left) and paper (right) TLC plates of varying thickness. (B) Quantitative analysis of the luminescence signal of the



Page 34 of 36

uppermost spot on each TLC plate (n = 3). The black circle indicates the region of interest for which the photon radiance was measured.

Figure 4. Amplification of luminescence signal using auxiliary techniques. (A-B) Dependence of luminescence signal intensity on the refractive index of the glass plate covering the TLC plate. (B) Quantitative analysis of the luminescence intensity. (C-D) Luminescence signal intensity of various TLC plates spotted with

131

I (C) before and

(D) after application of liquid scintillation cocktail solution. Plate 5: Si(0.2)_F_Al; plate 9: Si(0.2)_Al; plate 11: iTLC-Si; plate 13: Paper(0.18).

Figure 5. Comparison of two-dimensional (2D) images obtained by radio-TLC and CLI. (A) 2D chromatogram obtained using a radio-TLC scanner. (B) Cerenkov luminescence image. Plate 1: Si(0.25)_F_Gl; plate 2: Si(0.5)_F_Gl; plate 3: Si(1)_F_Gl; plate 4: Si(2)_F_Gl.

Figure 6. Comparison of the accuracy of radioactivity quantification between the CLI, radio-TLC, and zonal analysis methods. For each radioisotope and each quantification method, the plot shows the observed values versus the calculated values of the activity ratios between the serially 1/3-diluted samples for 10 TLC plates. The radioactivity of 131

I and 124I was assessed using CLI (A, D), a radio-TLC scanner (B, E), and a gamma

counter (C, F, respectively). The radioactivity of 32P was measured using CLI (G), a radio-TLC scanner (H), and a liquid scintillation counter (I). The activity ratios derived from different TLC plates are denoted as follows: ◆ indicates trend line of measured value by Si(0.25)_F_Gl TLC plate, ■ Si(0.5)_F_Gl, ▲ Si(1)_F_Gl, × Si(2)_F_Gl, Ж Si(0.2)_F_Al, ● Si(0.25)_F_Al, + HP-Si(0.2)_F_Al, - RP-18(0.2)_F_Al, ̶


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Si(0.2)_F_Pl, ◆ Alu(0.2)_F_Al. The four black dots (●) fitted with the red line (R2 = 1) indicate the calculated values (1/3, (1/3)2, (1/3)3, (1/3)4) of the activity ratios.



for TOC only


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Visualization and Quantification of Radiochemical Purity by Cerenkov

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