Liquid chromatographic determination of enantiomeric purity of [11C]methyl-L-methionine and O-(2-[18F]fluoroethyl)-L-tyrosine by pre-column derivatization with o-phthaldialdehyde and N-isobutyryl-L-cysteine

TL;DR

This paper introduces a faster and more accurate UPLC method to test the purity of two radiopharmaceuticals used in PET scans for brain tumor diagnosis.

Contribution

The study proposes novel UPLC methods with pre-column derivatization for enantiomeric purity analysis of [11C]MET and [18F]FET.

Findings

UPLC methods with OPA and IBLC derivatization effectively separate enantiomers of [11C]MET and [18F]FET within 10–20 minutes.

The methods achieved resolution >1.5, linearity r² >0.997, and recovery between 101.8–105.4%.

The developed protocols meet European Pharmacopoeia and EANM validation requirements for radiopharmaceutical quality control.

Abstract

[11C]methyl-L-methionine ([11C]MET) and O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) are commonly used radiopharmaceuticals in positron emission tomography (PET) for diagnosis of brain tumors. The preparations can be released for human use after the determination of several quality parameters. The enantiomeric purity test is an integral component of the quality control (QC) procedure for these radiopharmaceuticals. In this context, the European Pharmacopoeia monographs recommend thin-layer chromatography (TLC) for the separation of optical isomers of radiolabeled amino acids. To enhance the accuracy and efficiency of the analysis, a liquid chromatographic method should be employed. The aim of this work was to evaluate ultrahigh-performance liquid chromatography (UPLC) for the determination of enantiomeric purity of [11C]MET and [18F]FET, with the goal of reducing analysis time. Additionally, a pre-column derivatization method was applied using o-phthaldialdehyde (OPA) and N‑isobutyryl‑L‑cysteine (IBLC), which are widely used for the separation of optical isomers of amino acids.

In this work, two novel chromatographic methods were proposed for the determination of enantiomeric purity of [11C]MET and [18F]FET. The method development involved studying the effects of the type, composition, and acidity of the mobile phase, as well as the flow rate and column temperature on the separation of DD- and DL-diastereomers obtained from the reaction of amino acids and o-phthaldialdehyde and N-isobutyryl-L-cysteine derivatization reagents. Acquity BEH, CSH, and Kinetex XB stationary phases were tested with particle sizes ranging from 1.7 to 2.8 μm. The finalized method used BEH C18 column (2.1 × 50 mm, 1.7 μm) with mobile phase consisting of 0.1% H3PO4 aqueous solution (A) and 0.1% H3PO4 in acetonitrile (B). In case of [11C]MET, the gradient elution was accomplished by increasing the acetonitrile content from 0 to 5% with 20 min of gradient rate. For [18F]FET, the final acetonitrile ratio reached 40% over 25 min. At a flow rate of 0.6 ml/min the radiolabeled amino acids were separated within 10–20 min with resolution > 1.5. The methods were tested in accordance with EANM guideline on the validation of analytical methods for radiopharmaceuticals. For linearity, r2 > 0.997 was obtained in the concentration range of 8-180 MBq/ml, the repeatability of %Area was < 5% (RSD%), recovery ranged from 101.8 to 105.4%, and the limit of quantitation (LOQ) was between 9 and 13 MBq/ml.

The novel UPLC methods meet the Ph. Eur. monograph specifications and validation requirements. The pre-column derivatization reversed-phase (RP) chromatographic protocols are suitable for determining the enantiomeric purity of [11C]MET and [18F]FET radiopharmaceuticals and can be integrated into the quality control system.

The online version contains supplementary material available at 10.1186/s41181-025-00421-z.

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