Neuroendocrine tumors (NETs) are a heterogeneous group of neoplasms that arise from cells of the endocrine and nervous systems. These tumors can originate from various areas of the body but are most commonly in the gastrointestinal or bronchopulmonary system.
Many NETs may be characterized by a spectrum of overexpression of somatostatin receptors (SSTRs) on the cell surface.
Radiolabeling of DOTA-conjugate peptides with the positron emission tomography (PET) tracer Ga-68 allows to detect SSTRs overexpressed on NET cells providing in vivo visualization of primary tumor and metastatic lesions.
Recent availability of good manufacturing practice Good manufacturing practices (GMP)-grade generators for the production of the positron emission tomography (PET) tracer Ga-68 and publication of European Pharmacopoeia (Ph. Eur.) monograph (#2464) on Ga-68-chloride from generators have changed the imaging scenario. Finally, various new diagnostic agents based on Ga-68 labeling have been added to the clinical activity due to the use of automated synthesis systems.[3,4]
The continuing development of new tools for molecular imaging is not accompanied by a coherent effort in the development, standardization, and validation of quality control (QC) methods to guarantee high-quality radiopharmaceutical production, especially in the routine clinical setting.
Analytical methods play an essential role in the final product quality. However, the quality can only be reached if the analytical method undergoes an appropriate validation process. Analytical validation comprises a formal, systematic, and documented tool that measures the ability of an analytical method to provide reliable, accurate, and reproducible results.[5,6]
In this paper, we describe the validation process of a new selective and sensitive high-performance liquid chromatography (HPLC) method developed for determining chemical and radiochemical purity of Ga-68-DOTATATE (PET) tracer.
Materials and Methods
All reagents were high-purity pharmaceutical grade.
The reagents used for high-pressure liquid chromatography (HPLC) and instant thin-layer chromatography (ITLC) phase (trifluoroacetic acid [TFA], acetonitrile, and ammonium acetate) were ultrapure or trace metal free and were purchased from Sigma Aldrich (Saint Louis, Missouri, USA).
The GMP-grade peptide DOTATATE, or DOTA-Tyr3-Octreotate (TATE), was obtained from ABX pharmaceuticals (ABX, Advanced Biochemical Compounds, Radeberg, Germany) as a 1 mg powder which is dissolved in 1 ml of water and used in 10 μl aliquots.
Ga-68 was obtained from a GMP-compliant 68Ge/68Ga generator (GalliaPharm® Eckert and Ziegler, E and Z, Berlin, Germany) eluted with 0.1N HCl (Rotem GmbH, Germany).
Synthesis and Quality control analysis of [68Ga] Ga-DOTATATE
The synthesis of 68Ga-DOTATATE was performed using a fully automated module (Scintomics GRP®, Fürstenfeldbruck, Germany) specifically designed for the synthesis of radiopharmaceuticals.
The radiolabeling procedure and QC tests for Ga-68-DOTATATE [radiochemical purity (HPLC), radiochemical impurity 68Ga3+ (HPLC and ITLC), chemical purity (HPLC and GC), pH (pH-strips), radionuclidic purity (principal γ-photon), 68Ge-content, Ga-68 half-life (γ-ray spectrometry), and sterility/endotoxin assay] were described previously. The results of QC tests for Ga-68-DOTATATE are illustrated in Table 1.
Chromatographic conditions for analytical method development and validation
HPLC analyses were performed on a Dionex UltiMate 3000 HPLC system (Thermo Fisher Scientific) equipped with an Acclaim™ 120 C18 column 3 μm 120Š (3.0 mm × 150 mm) and a UV and a γ-detector (Berthold Technologies, Milan, Italy). The flow rate of mobile phase was pumped at a rate of 0.600 mL/min with a total run of 16 min. The detection wavelength for chemical impurities was 220 nm. T Column temperature was kept at 30°C, and sample injection was performed manually using an inert injector with a multiport valve and a 20 μL calibrated loop.
The software system Chromeleon 7 (Thermo Scientific™ Dionex™) was used to put together the information.
The mobile phase was as follows:
- -phase A: ACN: TFA in the ratio (100:0,1), (v/v);
- -phase B: Water: TFA in the ratio (100:0,1), (v/v).
The gradient as shown in Table 2 was used in the HPLC analysis.
Standard stock solution preparation
The stock standard solution of DOTATATE was prepared in water (TraceSelectUltra; Fluka, Switzerland) at a concentration of 3 μg/mL and stored in a refrigerator at −20°C.
Different working standard solutions of DOTATATE (0.5–3 μg/ml) were prepared by diluting the above-mentioned stock solution in water and were stored at −20°C.
Method of validation
Validation of HPLC method to determine the chemical purity
The HPLC method to determine the chemical purity of Ga-68-DOTATATE was validated in term of specificity, linearity, precision (repeatability), accuracy, and limit of quantification (LOQ) according to the International Conference on Harmonization (ICH) requirements and EDQM guidelines.[6,7]
The acceptance criteria for each parameter are listed in Table 3.[8,9]
The specificity of the HPLC method was evaluated to ensure that there was no interference from the excipients present in the formulations. The specificity was studied by analyzing mixture containing critical components that might be present in the finished product Ga-68-DOTATATE solution and demonstrating that the method is capable to distinguish the various components present at the limit of concentration for the considered standards. The specificity of the method was evaluated by injecting solutions of standard, blank, 68Ga-DOTATATE, and Ga-68.
To evaluate linearity and range of the method, different standard solutions were prepared by diluting the standard stock solution with water in different concentrations: 3.00; 2.00; 1.5; 1.00; 0.8; and 0.5 μg/mL, which cover 100%, 67%, 50%, 33%, 27%, and 17% of the target concentration, respectively.
The statistical function used to assess linearity was the linear regression with least squares. The curve equation, the correlation coefficient, and the determination coefficient (r2) were calculated through the equation y = ax + b, where y is peak area, a the slope, x the analyte concentration, and b the intercept.
Limit of detection and limit of quantification
Limit of detection (LOD) and LOQ are the lowest concentration in a sample that can be detected but not necessarily quantified under the stated experimental conditions.
LOQ is the lowest concentration of analyte that is determined by analyzing a series of diluted solutions of standard DOTATATE until a concentration level quantified with acceptable accuracy and precision >95% is reached.
The experimental value determined as described above needs to be confirmed through a precision analysis, using a sample at the concentration corresponding to the found LOQ. Acceptance criteria are CV% <5%.
Moreover, to determine the detection limit (DL) and the quantification limit (QL), we considered the standard deviation of the y-axis intercept of calibration curve (S intercept) and the average of its angular coefficient (slope of the line).
The DL and QL values were expressed in concentration and were based on the ratio of five times the baseline noise for DL, and ten times for QL, as shown in the following equations (1 and 2):
Precision and accuracy
Precision may be considered, at different levels, as a measure of repeatability or intermediate precision.
Repeatability may be calculated based on the content of standard DOTATATE, and the statistical parameter of concern is the variation coefficient (CV%) or relative standard deviation (RSD), which is determined using the equation: CV% = s/m × 100, where m is the average of the concentrations and s is the standard deviation.
Accuracy is a measure of the degree of conformity of a value generated by a specific procedure to the assumed or accepted true value, and it is expressed through bias% value.
Bias% value measures the difference between the experimental value (calculated from replicate measurements) and the nominal (reference) values. Acceptance criteria are bias% >95%.
The data were obtained by replicate analysis of samples containing known amounts of analyte at three different concentrations, chosen so that they are representative of the validation interval. Five replicates were performed for each concentration for 3 consecutive days.
Intraday and interday precision are expressed through the coefficient of variation, which must be ≤2% for all concentrations; the intraday and interday accuracy are expressed through the bias%, which must not deviate more than 5% for all concentrations.
Validation of HPLC method to determine the radiochemical purity
Some of the ICH guidelines validation parameters may not be of concern and do not apply for radioactive compounds.
The validation of the analytical method for the determination of the radiochemical purity is presented here.
The validation parameters and their acceptance criteria are summarized in Table 4.
Considering the radioactive nature and the short half-life of Ga-68, 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 5 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 value.
r2 may be extrapolated from the calibration curve by analyzing five different radioactive concentrations of Ga-68-DOTATATE.
Here also apply the same considerations described for linearity, i.e., the decay of the radionuclide Ga-68 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 Ga-68-DOTATATE sample and recalculating the obtained peak area values with the decay equation: lnA0 = lnA + λt, where λ = 0,693/t1/2, A0 = corrected peak area, A = measured peak area, t = time interval between 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 3 different days to verify the instrument outcome during the time course.
Under the chromatographic conditions described in the materials and methods section, DOTATATE and the internal standard peaks were well resolved.
The blank chromatogram in Figure 1, the DOTATATE in Figure 2, the Ga-68-DOTATATE in Figure 3, and gallium-68 chromatograms in Figure 4 were used for the validation of the analytical HPLC method.
The average retention time of Ga-68-DOTATATE was 5.565 min ± 0.016.
The determination of linearity of DATATATE was done on 6 solution sets in the concentration range 0.5–3 μg/mL.
From the evaluated chromatograms, we determined the area's peaks and height and plotted graphically their values against concentrations determining the correlation coefficient (R2). From the regression analysis, a linear equation was obtained: y = 1.0298X − 0.4624. The correlation coefficient value was 0.9998, in accordance to the acceptance criteria [Table 3], indicating a linear relationship between the concentration of analyte and area under the peak. Linearity was statistically confirmed for each calibration curve [Figure 5].
For each point of calibration standard, the concentrations of DOTATATE were recalculated from the equation of linear regression curve. The coefficients of variation (CV%) were <2%, while the bias values% does not deviate more than 5% for all concentrations [https://links.lww.com/IJNM/A20].
For Ga-68-DOTATATE, linearity was determined analyzing one sample solution with a suitable radioactive concentration [Table 5]. As shown in Figure 6, the calibration curve was linear and the linearity of this method was confirmed from the regression analysis, Y = 0.1712X + 9.9952. The r2 was 0.9935 and the average coefficient of variation (CV%) was <2% which is in accordance to acceptance criteria [Table 4].
Limit of detection and limit of quantification
The LOD and limit of quantitation (LOQ) values were determined by the analysis of samples with known concentrations of analytes in which the analyte can be reliably detected
LOD and LOQ were calculated as 0.5 and 0.1 μg/mL, respectively [Table 6].
Accuracy and precision of HPLC analytical method: validation intraday and interday
Tables 7 and 8 show the average values of the QC concentrations recalculated on the basis of the calibration line, the coefficient of variation%, as index of the precision of the collected data, and the accuracy, expressed as bias% ([average concentration observed/nominal concentration × 100] − 100).
The intra and interday precision were expressed as coefficient of variation% (CV%) which must be b2% for all the concentrations. The method was considered precise obtaining coefficients of variation between 0.22% and 0.52% for intraday and 0.20% and 0.61% for the interday precision. The intraday and interday accuracy is expressed through the bias% which must not deviate more than 5% for all concentrations. The bias% did not deviate more than 5% for all the concentrations.
Transition of radiopharmaceuticals to routine clinical use needs standardized procedures for assessing drug quality, to guarantee safety and efficacy of the final product and to comply with good manufacturing practice. Recently, guidance on the validation of radio-analytical methods for radiopharmaceuticals has been published by EANM, recommending the general approach with two practical examples.
During recent years, attention to QC methods has grown up in the development of radiopharmaceuticals, especially in establishing clinical grade compounds. So that, HPLC method has become crucial for identification/characterization of the final product, demanding higher resolution than standard TLC method.
Our group is expending many efforts in developing and validating radiolabeled probes for molecular imaging.[8,9,10,11,12,13,14,15,16,17] However, the expanding field of molecular imaging drives a growing development of new radiopharmaceuticals targeting a specific biologic process.
The lack of generic and peer-reviewed QC guidelines and recommendations for their application in human patients is a major concern.
Therefore, it is necessary to establish acceptance criteria for the selected quality parameters and validate them to transfer radiopharmaceutical production in the clinical/research activity.
The analytical method conditions and the mobile phase solvents used in the validation process provided good resolution for DOTATATE. In addition, the main features of the developed method are the short run time and retention time around 5.5 min.
Results evidenced that the proposed HPLC method is suitable for chemical and radiochemical purity evaluation of Ga-68-DOTATATE considering operational conditions of our laboratory, owing to its adequate specificity, linearity, LOD, LOQ, accuracy, and precision. The method was validated in accordance with ICH guidelines.
Consent for publication
All of the authors have read and approved the paper for publication.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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