Development and validation of a reversed-phase HPLC method for analysis of radiochemical purity in [123I]IBZM

Author’s Accepted Manuscript Development and validation of a reversed-phase HPLC method for analysis of radiochemical purity in [123I]IBZM Shiou-Shiow Farn, Yuen-Han Yeh, Chang-Chin Li, Ching-Shiuann Yang, Jenn-Tzong Chen, ChungShan Yu, Wuu-Jyh Lin www.elsevier.com/locate/apradiso

PII: DOI: Reference:

S0969-8043(16)30880-6 http://dx.doi.org/10.1016/j.apradiso.2017.04.022 ARI7870

To appear in: Applied Radiation and Isotopes Received date: 27 October 2016 Accepted date: 14 April 2017 Cite this article as: Shiou-Shiow Farn, Yuen-Han Yeh, Chang-Chin Li, ChingShiuann Yang, Jenn-Tzong Chen, Chung-Shan Yu and Wuu-Jyh Lin, Development and validation of a reversed-phase HPLC method for analysis of radiochemical purity in [123I]IBZM, Applied Radiation and Isotopes, http://dx.doi.org/10.1016/j.apradiso.2017.04.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Development and validation of a reversed-phase HPLC method for analysis of radiochemical purity in [123I]IBZM Shiou-Shiow Farna,b, Yuen-Han Yeha, Chang-Chin Lia, Ching-Shiuann Yanga, Jenn-Tzong Chena, Chung-Shan Yub, Wuu-Jyh Lina＊

a

Institute of Nuclear Energy Research, Taoyuan 32546, Taiwan

b

Department of Biomedical Engineering and Environmental Sciences, National Tsing-Hua University, Hsinchu 300, Taiwan

*Corresponding author. Wuu-Jyh Lin. Institute of Nuclear Energy Research, Tel.: +886 3 4711400-2750; fax: +886 3 4711416. E-mail addresses: [email protected]

Abstract [123I]IBZM is used widely for in vivo imaging of D2 receptors in human brain and shows relatively fast kinetics and a greater susceptibility to synaptic dopamine release than other single-photon emission computed tomography (SPECT) radioligands. A reliable and reversed-phase HPLC method using UV/VIS and radiometric detectors has been developed for qualitative analysis of BZM and IBZM and radiochemical purity in [123I]IBZM preparations. The method uses gradient elution on a Zorbax XDB C-18 column with a mobile phase that consists of 10 mM 1

3,3-dimethylglutaric acid (DMGA), pH 7.0 and acetonitrile (ACN). The flow rate was 1.0 ml/min with detection at λ = 254 nm. The method was validated for system suitability, precision, accuracy, specificity, linearity, robustness, limit of detection (LOD) and limit of quantification (LOQ), as described in ICH guidelines. The results are described as follows: (1) The system suitability includes the tailing factor, theoretical plate number and resolution, which are 0.962, 10656.11 and 9.367, respectively. (2) For specificity, the BZM and [123I]NH4I did not interfere with the retention time of the [123I]IBZM. (3) The percentage coefficient of variation for analysis of precision, including repeatability and intermediate precision, is less than 2.0%. (4) Accuracy of the method is within the range of 85%–100%. (5) The range of linearity is from 100-70% radiochemical purity (%RCP) of [123I]IBZM, with the correlation coefficient (R) always being above 0.995. (6) The data of method robustness are within acceptance criteria. (7) The LOD and LOQ for impurity (BZM) are 0.145 and 0.50 μg/mL, respectively. All of the analysis results demonstrate that this method is sensitive, specific and suitable for routine analysis of the radiochemical purity in [123I]IBZM preparations.

Keywords: [123I]IBZM, reversed-phase HPLC, radiochemical purity and method validation

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1. Introduction Dopamine receptors are the important targets in the treatment of many disorders such as Parkinson’s disease (PD), Huntington’ s disease and schizophrenia (Seeman and Van Tol, 1994) because the dopamine D2 receptor is involved in the pathogenesis of PD and in neurological or psychiatric disorders. With regard to postsynaptic dopaminergic single-photon emission computed tomography (SPECT) imaging, many radioligands have been proposed and developed for monitoring illness progression and effects of treatment such as [123I]IBZM, [123I]ILIS ( [123I]Iodolisur) , [123I]IBF. (Kung et al., 1988, Brücke et al., 1991, Tatsch et al., 1991,

Chrion et al., 1993,

Ichise et al., 1993,

Laruelle et al., 1997). One of the widely human and translational studies used radiotracers for this receptor is (S)-3-[123I]-iodo-N-[(1-ethyl-2-pyrrolidinyl)]- methyl-2-hydroxy -6-methoxy-benzamide ([123I]IBZM). Because IBZM are subject to competition for in vivo binding to D2 receptors with endogenous dopamine, [123I]IBZM can be used to infer the changes of receptor occupancy by medications to the effect of induced endogenous dopamine (Ronald et al., 2014, Abi-Dargham et al., 2009; Murnane and Howell, 2011, Kugaya et al., 2000) and D2/3 receptor quantification (Tsartsalis et al., 2017) due to high specific dopamine D2 receptor binding (Kd = 0.43 nM, Bmax = 0.48 pmol/mg of protein), relatively fast kinetics and a greater susceptibility to synaptic dopamine release than other SPECT ligands (Kung et al., 1988, Laruelle et al., 1997, Meyer et al., 2008). Generally, [123I]IBZM is prepared by an oxidative electrophillic radioiodination of BZM with [123I]sodium iodide in the presence of peracetic acid (Kung et al., 1991). Several chromatographic methods for analysis of [123I]IBZM and its precursor (BZM) were developed, including thin-layer chromatography (TLC) (Wang et al., 1998) and the

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HPLC systems equipped with radiometric and UV/VIS or Mass Spectrometer (MS) detectors (Kung et al., 1991, Yolanda et al., 1999, Baldwin et al., 2003, Liu et al., 2008). However, the published chromatographic methods investigated were found that there is no suitable resolution between BZM and IBZM or result in asymmetric peak with a large tailing factor. It is unacceptable for analysis of radiochemical purity (%RCP) in [123I]IBZM radiopharmaceuticals as well as chemical purity in IBZM. This article describes the development of a specific HPLC method for analyzing radiochemical purity of in [123I]IBZM preparations which are manufactured at the Radiopharmaceutical Production Center of the Institute of Nuclear Energy Research (Taoyuan County, Taiwan, ROC) using a C-18 column and UV/VIS and radiometric detectors as well as making qualitative analysis of the BZM and IBZM. The method was evaluated for system suitability, specificity, precision, accuracy, linearity, robustness, LOD and LOQ.

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2. Materials and methods 2.1. Chemicals and reagents The reference standard of BZM (≧99.8% purity) was purchased from Advanced Biochemical Compounds (ABX, Radeberg, Germany). The 3,3-dimethylglutaric acid (DMGA) ,3-(N-Morpholino) propanesulfonic Acid (MOPS), tris (Hydroxymethyl) aminomethane (Tris. Base), sodium hydroxide (NaOH), dimethyl sulfoxide (DMSO) and acetonitrile (ACN, HPLC grade) were purchased from Merck (Darmstadt, Germany). Normal saline solution was purchased from TAIWAN BIOTECH (TBC, Taoyuan, R.O.C). IBZM and [123I]IBZM were gifts from Radiopharmaceutical Production Center of the Institute of Nuclear Energy Research, (Taoyuan, ROC). Deionized water of Chromatographic-grade was made up by a Milli-Q system (18.2MΩ, Millipore, Massachusetts, USA).

2.2. Instrumentation Analytical HPLC was carried out with a Perkin Elimer Series HPLC system (Perkin Elimer, Waltham, USA) consisting of a 200 solvent delivery module in a quaternary gradient mode, a 200 autorsampler, a 200 ultraviolet (UV/VIS) detector and a radiometric detector (Packard, Canberra, Australia). Data acquisition was performed by TotalChrom Navigator 200 software (Perkin Elimer, Waltham, USA) operated on an Inter® Pentium 4 microprocessor.

2.3. Analytical procedure A volume of 5.0 μL of each sample was injected on a Zorbax XDB C-18 column of

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150mm×4mm i.d., 5μm particle size, (Agilent Technologies, Santa Clara, USA), at ambient temperature , with detection at λ = 254 nm. Mobile phase A was 7.0 mM DMGA, adjusted to pH 7.0 with NaOH, while mobile phase B was acetonitrile. The separation was obtained at a flow rate of 1.0 mL/min with a gradient program that allowed for 4.5 min at 60% B followed by a 6.5 min step that raised eluent B to 90%. Then washing at 90% B and equilibration at 60% B was performed in a total analysis time of 20 min.

2.4. Stock solutions preparation Stock solution of BZM was made by dissolving accurately weighed 4 mg of the BZM reference standard in 4 mL of DMSO (final concentration, 1 mg/mL). The prepared stock solution was stored at 4 ℃ protected from light. IBZM stock solution was prepared at a final concentration of 1 mg/mL using DMSO and stored at 4℃ protected from light. [123I]IBZM and [123I]NH4I stock solutions were prepared at a final concentration of 37MBq/mL using normal saline solution. All of the stock solutions were freshly prepared during the analysis day.

2.5. Sample preparation For the investigation of specificity, the sample was prepared by mixing 400μl of 1000μg/mL BZM stock solution with 400μL of IBZM stock solution and diluting it with DMSO to final concentration, 400μg/mL, respectively. Solutions for the LOD and LOQ study were prepared by diluting the stock solution of BZM and the volume made up to 1ml with DMSO to obtain linearity range of 1.0, 2.0, 5.0, 10.0 and 20.0 μg/mL. Samples for comparing the retention time between [123I]IBZM and BZM reference

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standard were made by diluting [123I]IBZM solution with IBZM stock solution (1.0 mg/mL) to a final concentration, [123I]IBZM (37MBq/mL) and IBZM (0.4 mg/mL), respectively.

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3. Results and discussion 3.1 Method development To discriminate the BZM from [123I]IBZM during chromatographic process, we used cold IBZM as reference standard and investigated several chromatographic conditions including the type of mobile phase and the type of elution. In preliminary experiments, subsequent assays were performed by isocratic elution and C-8 column using the mobile phase consisting of mixtures of buffer (with 10 mM of MOPS, Tris or DMGA, respectively, pH 7.0) and acetonitrile (30:70, v/v) at a flow rate of 1.0 mL/min. Fig. 1 show the chromatographic results using 10 mM of MOPS, Tris or DMGA as the buffer solution. The results indicate the resolution between BZM and IBZM using DMGA as the buffer is better than the other two buffers. We speculated that the higher the polarity of mobile phase is (Tris ＞ MOPS ＞ DMGA), the stronger the hydrophilic interaction between mobile phase and analytes (BZM and IBZM) is. Therefore, a decrease in hydrophobic interaction between stationary phase and analytes accompanied a decrease in retention of analytes in column and a lower resolution between BZM and IBZM. Due to the silanophilic interaction between stationary phase and analytes, there is an asymmetric peak of IBZM with a greater tailing factor (>1.5) using isocratic elution. The use of a gradient elution resulted in better resolution between BZM and IBZM than isocratic elution. However, we did not observe significant improvement in peak tailing. The stepwise elution is a good approach to minimize this peak tailing effect (table 1). In the chromatographic condition, the tailing factor was within the acceptable range (0.8-1.2) resulting in good peak symmetry and resolution. In addition, changing the column from C-8 bonded phase to C-18 resulted in better resolution between BZM and IBZM and a

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higher number of theoretical plates. Because the hydrogen on the amide may interact with the OH group on the benzene ring, which may strongly change the lipophilicity, the hydrophobic interaction between IBZM and C-18 bonded phase is stronger than with C-8 bonded phase which results in an increase of retention in IBZM within the column. Moreover, the higher number of theoretical plates arose from a decrease of the mass transfer effect (Douglas et al., 1998). The effect is a band broadening process in which analyte molecules at the front of a band are swept ahead before they have time to equilibrate with the stationary phase and thus be retained. Similarly, the equilibrium is not reached at the trailing edge of a band, and molecules are left behind in the stationary phase by the fast-moving mobile phase. Consequently, the effect is decreased, which resulted in sharper peak and higher number of theoretical plates. Fig. 2 illustrates the chromatographic result for [123I]IBZM and IBZM using radiometric and UV/VIS detectors with C-18 column and stepwise elution. The retention times of [123I]IBZM ,IBZM and BZM are 9.417, 8.921 and 5.398 min, respectively. Because the radiometric detector was coupled in series with the UV/VIS detector, the difference of retention time between [123I]IBZM and IBZM reference standard is around 0.5 min.

3.2 Method validation Full method validation for radiochemical purity analysis of [123I]IBZM was performed as described in the relevant ICH guideline (ICH Secretariat, 2005), which includes system suitability, specificity, linearity and range, LOD, LOQ, accuracy, intermediate precision (intra-day) and robustness.

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3.2.1 System suitability In the chromatographic procedure, the system suitability was evaluated by several parameters involving the theoretical plates (N), tailing factor (T), selectivity factor (k’) and resolution (Rs) between the [123I]IBZM and [123I]NH4I. The efficiency of the column as described by the number of theoretical plates for the six replicate injections was 10,656.11±176.16 (mean±S.D), the tailing factor was 0.96±0.03 (mean±S.D), the selectivity factor was 4.89±0.02 (mean±S.D) and resolution was 9.37±0.33 (mean±S.D). These results for analytes are within acceptable criterion (N≧3000, T=0.8-1.3, k’= 2-8, Rs≧1.5), indicating the suitability of the system (Table 1).

3.2.2 Specificity Specificity is the ability to assess unequivocally the analyte in the presence of components that may be expected to be present. The specificity of the analytical method was evaluated by identifying retention times obtained from BZM, IBZM, [123I]IBZM and [123I]NH4I. The retention time of analytes are 5.398, 8.921, 9.556 and 1.621 min. Fig. 2 and Table 1 show that precursor (BZM and [123I]NH4I) of [123I]IBZM did not interfere the retention time of the radiopharmaceutical. The resolution between precursor (BZM) and IBZM was 15.155, which was calculated using the Total Chrom Navigator 200 software. The specificity data indicate the method is specific and peak identification.

3.2.3. Precision Precision of the analysis method was determined based on repeatability (intra-assay)

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and intermediate precision (inter-assay) as described in the ICH guideline (ICH Secretariat, 2005). The repeatability of analytical procedure was determined by performing replicate injections of the [123I]IBZM radiopharmaceutical at the specific activity of 37KBq/μL under the same operating condition over a short period. Intermediate precision was evaluated by comparing within-laboratory variations with six replicate analyses of the analyte on different days and analysts. The calculated results of repeatability and intermediate precision, described as %CV of the peak retention time, was less than 2.0% (Table 2).

3.2.4. Accuracy, as recovery The accuracy of the analysis procedure was studied by comparing the closeness of radiochemical purity (%RCP) of [123I]IBZM between the determined value and an accepted reference value. [123I]IBZM activity sample of 17.575MBq, 16.65MBq, 15.725MBq, 14.8MBq, 12.95MBq were prepared by appropriately diluting the [123I]IBZM stock standard solution with [123I]NH4I solution. (475 μL for [123I]IBZM and 25 μL for [123I]NH4I spiked; 450 μL for [123I]IBZM and 50 μL for [123I]NH4I spiked; 425 μL for [123I]IBZM and 75 μL for [123I]NH4I spiked; 400 μL for [123I]IBZM and 100 μL for [123I]NH4I spiked; 350 μL for [123I]IBZM and 150 μL for [123I]NH4I spiked). [123I]IBZM activity sample of 17.575MBq, 16.65MBq, 15.725MBq, 14.8MBq, 12.95MBq were taken as target samples of radiochemical purity of 95%, 90%, 85%, 80% and 70%, respectively. The experiment was performed using triplicate analyses on three different days for the control sample of target concentration. The accuracy was calculated by use of the

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formula, Recovery (%) = RCPspike / ( RCPoriginal × target percentage)×100%, in which RCPspike and RCPoriginal are the radiochemical purity of spike solution and orignal sample, respectively. Because the activity of [123I]IBZM and [123I]NH4I decay with time, by adding [123I]NH4I in solution, different proportions of radiochemical purity of [123I]IBZM were obtained and then to prove the accuracy (recovery) and reliability of this

method through the recovery analysis of method validation. The considered acceptable recovery criteria is 85%–115% range. All the data of recovery were found within the range of 88%–100%, which is well within the acceptable range.

3.2.5. Linearity The linearity of an analytical procedure is its ability to elicit test results that are proportional to the concentration of analyte in samples within a given range. The calibration curve was evaluated by its correlation coefficient of the radiochemical purity (%RCP) of [123I]IBZM between the determined value and theoretical value. Linearity was constructed with six percentage points, including the range from 70 to 100 %RCP.The six percentage points were assayed in triplicates. The calibration curve was estimated by linear regression analysis, which was calculated on the basis of the least square regression method. The correlation coefficient (R) of all the calibration plots were consistently greater than 0.995. Other linear regression data are listed in Table.3.

3.2.6. Robustness

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The robustness of method is an indication of its reliability during an analytical procedure, which is evaluated by the effect of small but deliberate variation of the chromatographic parameters, including flow rate, concentration of mobile phase, column temperature and the same type of columns of different lots. The data in Tables 4-6 show the robustness of the method.

3.2.6.1. Effect of the same type of columns of different lots. There was no significant change in the retention time of [123I]IBZM using the same type of columns of different lots (Table.4). The %CV of retention time for identifying types of columns with small different usage rates was less than 2.0%. In addition, the variety of columns did not apparently affect the resolution (Rs) between [123I]NH4I and [123I]IBZM. Both resolutions are around 9 which is higher than criteria (Rs.＞1.5).

3.2.6.2. Effect of mobile phase pH. A little difference of the retention time and resolution dependent on the pH of the mobile phase was observed. When the pH of the mobile phase was 6.5, the retention time and resolution significantly became relatively low, increasing the basicity of mobile phase (pH 7.0, 7.5), leading to a better resolution and longer retention time (Table 5). This could be due to amine group of [123I]IBZM in its protonated and unprotonated form. The stronger the hydrophobicity of analyte is in reverse-phase chromatography, the longer the retention of analyte is in column. As a consequence, the charged form of the [123I]IBZM has a much lower retention factor than the uncharged form. Both are in an equilibrium with each other that depends on the pH. Small variations in the pH of the

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mobile phase change the ratio of both forms of the [123I]IBZM ,the retention time and the resolution.

3.2.6.3. Effect of mobile phase flow rate. The faster flow rate of the mobile phase (1.1 mL/min) resulted in a shorter retention time and a better resolution between [123I]NH4I and [123I]IBZM (Table 6). Although the chromatographic resolution is linearly proportional to the difference of retention time among analytes, it is inversely related to the peak width. We speculated that the resolution between different flow rates varies from the longitudinal diffusion effect. The effect in column chromatography is a band broadening process arising from the tendency of molecules to move in directions that tend to parallel the flow. Therefore, the extent of longitudinal broadening is inversely proportional to flow rate. When the flow rate is high, diffusion from the center of the band to the two edges has less time to occur, which results in sharper peak ,narrower peak width and better resolution between [123I]NH4I and [123I]IBZM.

3.2.7. Limit of Detection (LOD) and Limit of Quantitation (LOQ) The detection limit and quantitation limit are analytical parameters for determination of impurities in bulk drug substances. The detection limit is the lowest amount of analyte in a sample that can be detected. In contrast, the quantitation limit is the lowest amount of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. BZM is an impurity and degradation product in [123I]IBZM radiopharmaceuticals.

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BZM samples were injected in triplicates, and the peak area of BZM was estimated. The LOQ and LOD were calculated based on the standard deviation of the response and the slope obtained from the calibration plot of each well-recovered analyte of the standard mixture. The LOQ and LOD were defined as 3.3 α/S and 10 α/S, respectively, where α is the standard deviation of the y-intercept and S is the slope of the regression line. The LOD and LOQ of the method were 0.145 and 0.50 μg/mL, respectively. The calibration curve of LOD consisted of five concentrations, including 1.0, 2.0, 5.0, 10.0 and 20.0μg/mL. The LOQ was evaluated by six replicate analyses of the drug at a concentration of 0.5μg/mL. The precision (%CV) of peak area and retention time for LOQ determination of BZM are within acceptable range (5%). The date of LOD and LOQ indicated that the method can be used for detection and quantification of impurity (BZM) of [123I]IBZM over a very wide range of concentrations (Table 7).

3.3 Limit of BZM According to the previous study, BZM exerted no adverse toxic effects in SD rats at dose levels up to 1,250 μg/kg (Yang et al., 2008), However, a small amount of BZM in the injection that would may influence the result of scintigraphy. By analyzing the BZM content was to understand the actual BZM concentration injected into the human body, D2 receptor may also be competed binding with BZM and [123I]IBZM at the same time that would reduce the result of scintigraphy of [123I]IBZM. Therefore, the recommended limit of BZM concentration is less than 0.4 ug/mL referring to the summary of product characteristics information of [123I]IBZM injection provided by Amersham Health B.V.

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(Amersham Health, 2001). 3.4 Recommended release criteria of quality control for [123I]IBZM Except the BZM impurity and radiochemical purity analysis, other items of quality control are also important, including appearance, pH, bacterial endotoxin, radionuclide purity and identification and sterility test. The recommended release criteria of quality control for [123I]IBZM were described briefly in Table 8.

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4. Conclusions A reliable and reversed-phase HPLC method has been developed in order to analyze [123I]IBZM by a UV/VIS detector coupled with a radiometric detector. This method offers two advantages: First, use of stepwise elution and DMGA/ACN as the mobile phase resulted in good peak symmetry and reasonable resolution. Second, the method was sensitive for the quantitative determination of impurity (BZM). In addition, the validation of the procedure is in compliance with the current ICH requirements, including system suitability, precision, accuracy, specificity, linearity, robustness, LOD and LOQ. All of the validation data were considered within acceptable ranges described by the ICH recommendation, demonstrating that the method is specific, rapid and suitable for analysis of [123I]IBZM. Therefore, this method can be successfully applied in a timely manner for routine analysis of [123I]IBZM radiopharmaceutical and also the release criteria of quality control for [123I]IBZM was recommended.

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Acknowledgments We would like to thank Radiopharmaceutical Production Center of the Institute of Nuclear Energy Research for providing the [123I]IBZM as a gift.

The authors declare that they have no conflict of interest.

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[15] Wang T.S.T., Malaspina D. Van Heerrtum R.L., 1998. A Simple Method of Preparation for[ 123I]-(S)-(-)-IBZM. Appl. Radiat. Isot. 49, 369-372. [16] Zea-Ponce Y. and Laruelle M., 1999. Synthesis of [123I]IBZM: A Reliable Procedure for Routine Clinical Studies. Nucl. Med. Biol. 26, 661–665. [17] Baldwin R.M., Fu X., Kula N.S., Baldessarini R.J., Amici L., Innis R.B. and Tamagnan G.D., 2003. Synthesis and Affinity of a Possible Byproduct of Electrophilic Radiolabeling of [123I]IBZM. Bioorg. Med. Chem. Lett. 13, 4015–4017 [18] Liu K.T., Yang H.H., Hsia Y.C., Yang A.S., Su C.Y., Lin T.S and Shen L.H., 2008. Development and Validation of an HPLC Method for the Purity Assay of BZM, the Precursor of Striatal Dopaminergic D2/D3 Receptor SPECT Imaging Agent [123I]IBZM (Iodobenzamide). J. Food. Drug. Anal. 16, 28-38. [16] Douglas A, Skoog F, Holler J, Nieman TA. Principles of instrumental analysis. 5th ed. Pacific Grove, C.A., USA: Thompson Learning; 1998. [17] ICH Secretariat, 2005. ICH Q2(R1) Validation of Analytical Procedures: Text and Methodology. ICH Secretariat, Geneva. [18] Yang A.S., Chiang T.C., Hsu F.F., Liao M.H., Shen L.H., 2008. Acute intravenous injection toxicity study of IBZM and BZM in rats. Drug Chem Toxicol. 2008;31(4):529-33. doi: 10.1080/01480540802393332. [19] Amersham Health B.V. 2001.“summary of product characteristics of [123I] IBZM

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Fig.1

Chromatograms of IBZM 400 ppm and BZM obtained using isocratic elution with several mobile phases at 1.0 ml/min. (A) 10mM MOPS, pH 7. (B) 10 Mm DMGA, pH 7. (C) 10mM Tris, pH 7.

Fig.2

Chromatograms of BZM, IBZM 400 ppm and [123I]IBZM 0.3 mCi/ml obtained at 1.0 ml/min by (A) a UV/VIS detector and (B) a radioactive detector.

Table 1 System suitability study for the determination of [123I] IBZM (n =6). mobile phase A: 7.0 mM DMGA (pH 7.0), mobile phase B: acetonitrile, flow rate 1.0 mL/min, gradient program, column: Zorbax XDB C-18 column, 150mm length, 4mm inner diameter, radiometric detector at ambient temperature. R.T (min) Mean 9.556 S.D. 0.015 %CV 0.163 Rs: Resolution R.T: retention time

[123I] IBZM Peak Tailing Area Factor 4407092 0.947 29774.38 0.003 0.676 0.377

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Theoretical Plate 10367.75 203.61 1.963

Rs 9.438 0.115 1.222

[123I] NH4I R.T (min) 1.617 0.009 0.581

Table 2 Intra-day and inter-day of precession for the determination of radiochemical of [123I]IBZM (n =6). mobile phase A: 7.0 mM DMGA (pH 7.0), mobile phase B: acetonitrile, flow rate 1.0 mL/min, gradient program, column: Zorbax XDB C-18 column, 150mm length, 4mm inner diameter, radiometric detector at ambient temperature on different days and analysts. PerkinElmer HPLC

Mean Retention Time ± S.D. %CV (n=6) (n=6)

Mean %R.C.P± S.D (n=6)

%CV (n=6)

Day 1

analyst 1

9.556 ± 0.030

0.314

91.463 ± 0.090

0.098

Day 2

analyst 2

9.718 ± 0.021

0.212

98.307 ± 0.084

0.085

Mean retention time ± S.D. (n=12) 9.637 ± 0.088 %RCP: percentage of radiochemical purity

%CV (n=12) 0.915

Table 3 Results of Accuracy (recovery, %) and Linear Regression analysis for the determination radiochemical purity of [123I]IBZM (n =6). mobile phase A: 7.0 mM DMGA (pH 7.0), mobile phase B: acetonitrile, flow rate 1.0 mL/min, gradient program, column: Zorbax XDB C-18 column, 150mm length, 4mm inner diameter, radiometric detector at ambient temperature. 1

No. %RCP

D.V (%)

T.V (%)

100% 94.72 94.72 95% 87.42 89.98 90% 81.67 85.25 85% 74.86 80.51 80% 67.49 75.78 70% 57 66.3 Slope 1.3374 Correlation Coefficient 0.9983 (R) D.V: determined value T.V: theoretical value

2 Recover y (%) 100 97.15 95.8 92.98 89.07 85.97

D.V (%)

T.V (%)

94.83 89.76 85.08 78.12 70.5 59.68

94.83 90.09 85.35 80.61 75.86 66.38 1.2675 0.9975

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3 Recover y (%) 100 99.64 99.69 96.92 92.93 89.91

D.V (%)

T.V (%)

94.92 87.76 84.41 79.43 72.85 60.46

94.92 90.17 85.43 80.68 75.94 66.44 1.1786 0.9963

Recover y (%) 100 97.32 98.81 98.45 95.94 90.99

Table 4 Effect of different Lot number column on chromatographic system for detection of radiochemical purity of [123I]IBZM (n=6). mobile phase A: 7.0 mM DMGA (pH 7.0), mobile phase B: acetonitrile, flow rate 1.0 mL/min, gradient program, column: Zorbax XDB C-18 column, 150mm length, 4mm inner diameter, radiometric detector at ambient temperature. Column Lot #

Mean R.T ± S.D. %CV (n=6) (n=6)

Mean RCP ± S.D. (n=6)

%CV Mean Rs ± S.D. %CV (n=6) (n=6) (n=6)

USKH052556

9.556 ± 0.030

1.222

91.46 ± 0.09

0.10

9.44 ± 0.11

1.22

USKH048004

9.633 ± 0.020

0.209

91.16 ± 0.11

0.13

9.30 ± 0.17

1.83

Summary of Column Lot # [USKH052556 / 048004]

Mean R.T ± S.D. %CV Mean RCP ± S.D. %CV Mean Rs ± S.D. %CV (n=12) (n=12) (n=12) (n=12) (n=12) (n=12) 9.595 ± 0.047

0.494

91.32 ± 0.18

0.20

9.37 ± 0.16

R.T: Retention Time RCP: Raidochemical purity Rs: Resolution

Table 5 Effect of pH of mobile phase for detection of radiochemical purity of [123I]IBZM: (1) pH= 6.5, (2) pH=7.0, (3) pH=7.5 (n=6). mobile phase A: 7.0 mM DMGA (pH 7.0), mobile phase B: acetonitrile, flow rate 1.0 mL/min, gradient program, column: Zorbax XDB C-18 column, 150mm length, 4mm inner diameter, radiometric detector at ambient temperature. pH of Mean R.T ± S.D. Mobile Phase (n=6)

%CV Mean RCP ± S.D. %CV (n=6) (n=6) (n=6)

Mean Rs ± S.D. %CV (n=6) (n=6)

pH 6.5

9.350 ± 0.028

0.302

98.55 ± 0.05

0.05

9.09 ± 0.14

1.53

pH 7.0

9.556 ± 0.030

1.222

91.46 ± 0.09

0.10

9.44 ± 0.11

1.22

pH 7.5

9.801 ± 0.024

0.246

95.04 ± 0.42

0.44

9.74 ± 0.19

1.99

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1.66

Table 6 Effect of flow rate of mobile phase for detection of radiochemical purity of [123I]IBZM: (1) 0.9 mL/min, (2) 1.0 mL/min, (3) 1.1 mL/min (n=6). mobile phase A: 7.0 mM DMGA (pH 7.0), mobile phase B: acetonitrile, flow rate 1.0 mL/min, gradient program, column: Zorbax XDB C-18 column, 150mm length, 4mm inner diameter, radiometric detector at ambient temperature. Flow Rate of Mean R.T ± S.D. Mobile Phase (n=6)

%CV Mean RCP ± S.D. %CV (n=6) (n=6) (n=6)

Mean Rs ± S.D. (n=6)

%CV (n=6)

0.9 mL/min

10.234 ± 0.059

0.302

94.65 ± 0.07

0.08

9.24 ± 0.10

1.03

1.0 mL/min

9.556 ± 0.030

1.222

91.46 ± 0.09

0.10

9.44 ± 0.11

1.22

1.1 mL/min

9.196 ± 0.057

0.618

93.55 ± 0.93

1.00

9.90 ± 0.19

1.93

Table 7 LOD and LOQ for the detection of BZM impurity of [123I]IBZM (n=3). mobile phase A: 7.0 mM DMGA (pH 7.0), mobile phase B: acetonitrile, flow rate 1.0 mL/min, gradient program, column: Zorbax XDB C-18 column, 150mm length, 4mm inner diameter, UV detection: 254 nm at ambient temperature. LOD linear Mean Area ± S.D. (n=3) 1.0 ppm 2246.067 ± 24.836 2.0 ppm 4725.467 ± 29.220 5.0 ppm 12217.467 ± 128.732 10.0 ppm 25550.800 ± 217.714 20.0 ppm 51138.933 ± 593.404 Slope Correlation Coefficient (R2) 2.62E+03 0.9999 2.56E+03 1.000 2.56E+03 0.9999 Mean Slope ± S.D. Mean Correlation Coefficient ± S.D. (n=3) (n=3) 2580.40 ± 32.59 0.9999 ± 0.0001 Mean LOQ ± S.D. Mean Area ± S.D. (n=6) (n=6) 0.56 ± 0.01 (ppm) 1059.12 ± 23.62 LOD: Limit of Detection LOQ: Limit of Quantitation

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%CV 1.106 0.618 1.054 0.852 1.160 Intercept -5.66E+02 -3.83E+02 -3.58E+02 Mean Intercept ± S.D. (n=3) -435.30 ± 113.51 %CV of LOQ (n=6) 1.64

Table 8. Recommended release criteria of quality control for [123I] IBZM Items Acceptance Criteria Appearance Clear, colorless solution, Absent of foreign matter. Product vial is intact. pH 5.0-6.5 Radionuclide must show the 159 KeV gamma spectrum identification Radionuclide purity NLT 99.0% of emission at 159KeV Radiochemical purity NLT 90% Chemical impurity NMT 0.4 ug/mL Radioactivity NLT 5 mCi at calibration time Bacterial endotoxin NMT 25 EU/mL Sterility test No growth observed after 14 days NLT: not less than NMT: not more than

Highlights  [123I]IBZM is used widely for the in vivo imaging of D2 receptors in the brain of human and the validation of the radiochemistry analytical procedure should be performed in compliance with the current ICH requirements.  A reliable and reversed-phase HPLC method has been developed. This method offers two advantages (1) good peak symmetry and reasonable resolution. (2) suitable for the quantitative determination of impurity (BZM).  The method is specific, rapid and suitable for analysis of [123I]IBZM and can be successfully performed in a timely manner for routine analysis for quality control of radiochemistry purity of [123I]IBZM..

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