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Article Cite This: ACS Omega 2017, 2, 7120-7126
http://pubs.acs.org/journal/acsodf
Development and Validation of a High-Pressure Liquid Chromatography Method for the Determination of Chemical Purity and Radiochemical Purity of a [68Ga]-Labeled Glu-Urea-Lys(Ahx)HBED-CC (Positron Emission Tomography) Tracer Silvia Migliari,*,† Antonino Sammartano,† Maura Scarlattei,† Giulio Serreli,‡ Caterina Ghetti,‡ Carla Cidda,† Giorgio Baldari,† Ornella Ortenzia,‡ and Livia Ruffini† †
Nuclear Medicine and Molecular Imaging Department and ‡Medical Physics Unit, University Hospital of Parma, via Gramsci 14, 43126 Parma, Italy ABSTRACT: Background: Prostate-specific membrane antigen (PSMA) has gained high attention as a useful biomarker in the imaging evaluation of prostate cancer with positron emission tomography (PET) during recent years. [68Ga]labeled Glu-urea-Lys(Ahx)-HBED-CC ([68Ga]-PSMA-HBEDCC) is a novel PSMA inhibitor radiotracer which has demonstrated its suitability in detecting prostate cancer. Preparation conditions may influence the quality and in vivo behavior of this tracer, and no standard procedure for the quality control (QC) is available. The aim of this study was to develop a new rapid and simple high-pressure liquid chromatography method of analysis for the routine QCs of [68Ga]-PSMA-HBED-CC to guarantee the high quality of the radiopharmaceutical product before release. Methods: A stepwise approach was used based on the quality by design concept of the International Conference of Harmonisation Q2 (R1) and Q8 (Pharmaceutical Development) guidelines in accordance with the regulations and requirements of European Association of Nuclear Medicine, Society of Nuclear Medicine, International Atomic Energy Agency, World Health Organization, and Italian Association of Nuclear Medicine and Molecular Imaging. The developed analytical test method was validated because a specific monograph in the pharmacopoeia is not available for [68Ga]-PSMA-HBED-CC. Results: The purity and quality of the radiopharmaceutical obtained according to the proposed method resulted high enough to safely administrate it to patients. An excellent linearity was found between 0.8 and 5 μg/mL, with a detection limit of 0.2 μg/mL. Assay imprecision (% CV) was 95%. 5.5.5. Limit of Quantitation. Experimental LOQ has been determined by analyzing a series of diluted solutions of GaPSMA-HBED-CC and standard PSMA-11, until a concentration level quantified with a precision >95% is reached. The experimental value determined as above described the need to be confirmed through a precision analysis, using a sample at a concentration corresponding to the found LOQ. Acceptance criterion is CV % < 5%. 5.6. Validation of the HPLC Method To Determine Radiochemical Purity. Validation of the analytical method for the determination of radiochemical purity is presented here. In Table 3, the validation parameters and their acceptance criteria are summarized:
urea-Lys(Ahx)-HBED-CC (PSMA-11) were purchased from ABX Radeberg (Germany). The γ-ray spectrometry tests included the identification of principal γ-photon (499−521 keV peak) and 68Ge content (decay of 499−521 keV peak ≥ 48 h) using a large volume counter linked to a multichannel analyzer system (HPGe detector ORTEC GEM 30P4-76). The half-life of 68Ga was calculated after measuring the radioactivity of a sample in the dose calibrator at four consecutive intervals (5, 10, 15, and 20 min) and then using the equation t1/2 = ln(1/2)/λ, where λ = decay constant. 5.4. Standard Solution. Stock solutions (5 μg/mL) and appropriate dilutions of Glu-urea-Lys(Ahx)-HBED-CC (PSMA-11) and natGa-labeled reference Glu-urea-Lys(Ahx)[Ga(HBED-CC)] (DKFZ-GaPSMA-11) were prepared in CH3CN/H2O (1:1) and stored at −20 °C. 5.5. Validation of the HPLC Method To Determine Chemical Purity. Validation of the analytical method for the determination of chemical purity of [68Ga]-PSMA-HBED-CC was carried out according to ICH Q2 (R1) guidelines. The parameters assessed for the validation were specificity, linearity, precision (repeatability), accuracy, and LOQ.15 The acceptance criteria for each parameter are listed in Table 2. Table 2. Test and Acceptance Criteria in Determining Chemical Purity Using HPLC test
acceptance criteria
specificity linearity repeatability LOQ accuracy
≥2.5 R2 ≥ 0.99 CV % < 2% CV % < 5% bias % > 95%
Table 3. Test and Acceptance Criteria in Determining Radiochemical Purity Using HPLC
5.5.1. Specificity. Specificity determination is performed by analyzing the mixture containing critical components that might be present in the finished product [68Ga]-PSMA-HBEDCC solution and by demonstrating that the method is capable to distinguish the various components present at the limited concentration for the considered standards. The preparation method of the development of [68Ga]-PSMA-HBED-CC was not considered for chemical impurities, except for free gallium68. Thus, analyses were performed using a series of solutions containing [68Ga]-PSMA-HBED-CC and Ga-68. 5.5.2. Linearity. Determination of linearity was done on sets of standard solutions with different concentrations for each of the analytes of interest (Ga-PSMA-HBED-CC and PSMA-11). Such solutions are usually prepared by serial dilution starting from a “mother” solution with the highest concentration (5, 4, 3.125, 1.25, and 0.8 μg/mL). The statistical function used is linear regression with least squares. The curve equation, the correlation coefficient, and the determination coefficient (r2) are calculated through the equation: y = ax + b, where y is the peak area, a is the slope, x is the analyte concentration, and b is the intercept. 5.5.3. Precision. Precision may be considered at different levels as a measure of repeatability or intermediate precision. 5.5.3.1. Repeatability. Repeatability may be calculated based on the content of standard Ga-PSMA-HBED-CC and PSMA11. The statistical parameter of concern is the CV % or RSD, which is determined using the equation: CV % = s/m × 100, where s is the standard deviation of the peak areas and m is the average of the peak areas.
test
acceptance criteria
specificity linearity repeatability LOQ accuracy
not applicable R2 ≥ 0.99 CV % < 2% not applicable not applicable
In the validation of the methods for radioactive compounds, some of the ICH guidelines of validation parameters may not be of concern and do not apply. 5.6.1. Linearity. Considering the radioactive nature and the short half-life of 68Ga, the typical experimental approach based on the preparation of a series of solutions with different concentrations does not apply. On the contrary, in this case, one sample solution only, with a suitable radioactive concentration, is analyzed five times, at defined time intervals (15 min). Indeed, the radioactivity being the physical parameter of concern for radiochemical detectors, the radionuclide decay itself provides the necessary linear series of values. R2 may be extrapolated from the calibration curve by analyzing five different radioactive concentrations of [68Ga]-PSMA-HBEDCC. 5.6.2. Precision. Precision may be considered at different levels as a measure of repeatability or intermediate precision. 5.6.2.1. Repeatability. Here, also the same considerations apply as that described for linearity, that is, the decay of the radionuclide 68Ga inevitably leads to a decrease over time of the radioactivity. However, repeatability may be evaluated by analyzing a series of HPLC runs obtained with repetitive injections of a single [68Ga]-PSMA-HBED-CC sample and by recalculating the obtained peak area values with the decay equation: ln A0 = ln A + λt, where λ = 0,693/t1/2, A0 = corrected peak area, A = measured peak area, t = time interval between 7125
DOI: 10.1021/acsomega.7b00677 ACS Omega 2017, 2, 7120−7126
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Article
the considered injection and the first one. t1/2 = half-life (68Ga = 67.63 min). The peak area values normalized for decay may then be compared and yield a consistent statistical analysis. Average, standard deviation, and CV % are then calculated. Repeatability has to be determined in three different days, to verify the instrument outcome during the course of time.
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(8) Kitajima, K.; Murphy, R. C.; Nathan, M. A. Choline PET/CT for imaging prostate cancer: an update. Ann. Nucl. Med. 2013, 27, 581− 591. (9) Lütje, S.; Heskamp, S.; Cornelissen, A. S. PSMA Ligands for Radionuclide Imaging and Therapy of Prostate Cancer: Clinical Status. Theranostics 2015, 5, 1388−1401. (10) Jadvar, H. PSMA PET in prostate cancer. J. Nucl. Med. 2015, 56, 1131−1132. (11) Eder, M.; Eisenhut, M.; Babich, J.; Haberkorn, U. PSMA as a target for radiolabelled small molecules. Eur. J. Nucl. Med. Mol. Imaging 2013, 40, 819−823. (12) Eiber, M.; Maurer, T.; Souvatzoglou, M.; Beer, A. J.; Ruffani, A.; Haller, B.; Graner, F.-P.; Kübler, H.; Haberhorn, U.; Eisenhut, M.; Wester, H.-J.; Gschwend, J. E.; Schwaiger, M. Evaluation of Hybrid 68 Ga-PSMA Ligand PET/CT in 248 Patients with Biochemical Recurrence After Radical Prostatectomy. J. Nucl. Med. 2015, 56, 668−674. (13) Eder, M.; Neels, O.; Müller, M.; et al. Novel preclinical and radiopharmaceutical aspects of [68Ga]Ga-PSMA-HBED-CC: A New PET tracer for imaging of prostate cancer. Pharmaceuticals 2014, 7, 779−796. (14) Eder, M.; Schäfer, M.; Bauder-Wüst, U.; et al. 68Ga-Complex Lipophilicity and the Targeting Property of a Urea-Based PSMA Inhibitor for PET Imaging. Bioconjugate Chem. 2012, 23, 688−697. (15) Zarghi, A.; Foroutan, S.; Shafaati, A.; Khoddam, A. A rapid HPLC method for the determination of losartan in human plasma using a monolithic column. Arzneimittelforschung 2011, 55, 569−572.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: 0039 3335939138 (S.M.). ORCID
Antonino Sammartano: 0000-0002-1709-4758 Author Contributions
S.M., A.S., and L.R. have contributed to the organization of the content for this manuscript. S.M. and A.S. collected relevant information and prepared the draft. L.R. drafted and revised the manuscript. All authors read and approved the final manuscript. All authors have read and approved the paper for publication. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge Claudia Silva and Lucia Battistini, University of Parma, for their feedback on the text during its composition and revision stages.
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ABBREVIATIONS CT, computed tomography; QC, quality control; CV %, coefficient of variation; GMP, good manufacturing practice; GRP, good radiopharmaceutical practices; GC, gas chromatography; LAL, Limulus amebocyte lysate; HPGe, high-purity germanium; HPLC, high-pressure liquid chromatography; ICH, International Conference of Harmonisation; MR, magnetic resonance; NBP-MN, Norme di Buona Preparazione in Nuclear Medicine; SPECT, single photon emission computerized tomography; TLC, thin layer chromatography; TFA, trifluoroacetic acid; RSD, relative standard deviation; PCa, prostate cancer; PSA, prostate-specific antigen; PSMA, prostate-specific membrane antigen; PET, positron emission tomography; US, ultrasound
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REFERENCES
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DOI: 10.1021/acsomega.7b00677 ACS Omega 2017, 2, 7120−7126
