Determination of Boronophenylalanine in Biological Samples Using Precolumn o-Phthalaldehyde Derivatization and Reversed-Phase High-Performance Liquid Chromatography

Abstract

A reversed-phase high-performance liquid chromatographic method for the detection of boronophenylalanine is described. Determination was obtained by precolumn reaction of o-phthalaldehyde with a mixture of standard amino acids containing boronophenylalanine and separating the corresponding o-phthalaldehyde derivatives, using a Kromasil C-18, 250 ؋ 4.6 mm, 5-m particle size column, a step gradient with two buffers, a flow rate of 1.2 ml/min, a column temperature of 23°C, and fluorimetric detection (excitation and emission wavelengths of 330 and 430 nm, respectively). The use of such a method for assaying boronophenylalanine in biological samples was tested in neutralized perchloric acid blood and cerebral tissue extracts of rats treated with intracarotid administration of 300 mg/kg of body weight boronophenylalanine. Results of these experiments showed that the present HPLC method represents a valid alternative to currently available analytical techniques for assaying boronophenylalanine based on boron determination in terms of reproducibility, recovery, or sensitivity. Therefore, it is suggested that the present method may routinely be used in all preclinical and clinical studies in which quantification of circulating and tissue concentrations of boronophenylalanine is critical for the application of boron neutron capture therapy.

L-DOPA Preloading Increases the Uptake of Borophenylalanine in C6 Glioma Rat Model: A New Strategy to Improve BNCT Efficacy

“…In Experiment 2, BPA quantification in animal tissues was performed using high performance liquid chromatography (HPLC). As previously shown, this technique is a reliable method for analysis of BPA incorporation in several biological tissues, including cerebral samples (35).…”

“…Tumor tissue, normal brain (obtained from both hemispheres, ipsi-and contra-lateral to the tumor implantation), and blood samples were collected for HPLC analysis. HPLC procedure for BPA quantification was performed as previously described (35). Briefly, the reversed-phase HPLC method exploits BPA derivatization with OPA and a spectrofluorimetric detector.…”

Boronophenylalanine, a boron delivery agent for boron neutron capture therapy, is transported by ATB0,+, LAT1 and LAT2

“…On a methodological note, HPLC was extensively used throughout this study to detect and quantify BPA, instead of direct‐current plasma atomic emission spectrometry (DCP‐AES). Our HPLC method derivatizes BPA with NBD‐F and detects NBD‐BPA with a detection limit of 0.1 pmol per injection, whereas DCP‐AES requires a minimum of 16 nmol BPA/sample for detection . Di Pierro et al .…”

“…Our HPLC method derivatizes BPA with NBD-F and detects NBD-BPA with a detection limit of 0.1 pmol per injection, whereas DCP-AES requires a minimum of 16 nmol BPA ⁄ sample for detection. (36) Di Pierro et al (36) reported an HPLC application with o-phthalaldehyde derivatization and a similar detection limit. Such derivatization methods are applicable to compounds with amino groups with which the derivatizing reagents react.…”

Fast Analysis of Free Amino Acids in Tobacco by HPLC with Fluorescence Detection and Automated Derivatization

“…6 More recently, interest has been focused on precolumn derivatization followed by reversed-phase HPLC which constitutes a much faster and more efficient and sensitive alternative to former amino acid analyzers. [7][8][9] Reagents employed for this purpose are o-phtalaldehyde (OPA), 10 dansyl chloride (DANS-Cl), 11 N-benzoyl chloride (Ben-Cl), 12 phenylisothiocyanate (PTIC) 13-14 or 9-fluorenylmethyl chloroformate (FMOC-Cl). 15 DANS-Cl reacts both with primary and secondary amines, but high temperature and a long time are required.…”