Journal of Chromatography B: Biomedical Sciences and Applications
Quantitative high-performance liquid chromatographic determination of acrolein in plasma after derivatization with Luminarin® 3
Introduction
Oxazaphosphorine drugs are alkylating antineoplastic substances used in various cancer chemotherapy regimens. Cyclophosphamide (CPM, 2) and ifosfamide (IFM, 3) are members of this family and are widely used for the treatment of sarcoma [1], [2]. More particularly, high doses of IFM (up to 9 g/m2) are sometimes administered to children suffering from osteosarcoma and soft tissue sarcoma [3], [4]. Actually, IFM and CPM are non-cytotoxic prodrugs that require a bioactivation step that occurs in the liver via a cytochrome P450-mediated ring oxidation. Indeed, after oxidation at position 4, true alkylating moieties (phosphoramide (4) or isophosphoramide (5) mustards) are spontaneously and concomitantly formed with the release of acrolein (Fig. 1). This latter is responsible for the urotoxic side effects of the therapy [5], [6] and its involvement in CPM-induced cellular toxicity has also recently been proposed by Friedman et al. [7]. Despite the use of large quantities of mesna (sodium mercaptoethanesulfonate) to prevent acrolein toxicity, haemorrhagic cystitis [1], [2], [3], [4] frequently occurs with alarming severity. Since oxazaphosphorine metabolism is sensitive to auto-induction [8], as well as to inter- and intra-individual variations [9], an accurate measurement of acrolein levels in plasma should facilitate individual pharmacokinetic and metabolism studies of these drugs and thus lead to a better understanding of the variation in the metabolism. For these reasons, the monitoring of the acrolein level in plasma is necessary to efficiently adjust the mesna dosage and consequently to optimize IFM and CPM use.
Different methods have been described to achieve the quantitative determination of acrolein in ambient air [10], [11] or in biological samples or urine [12], [13], [14]. Among these methods, the formation of an hydroxyquinoline by condensation of acrolein with 3-aminophenol allowing fluorimetric detection after HPLC has been reported [12], [13]. However, this method, only described for urine or liver microsome extracts analysis, requires drastic conditions (heating of the samples at 100°C) that might modify the real kinetics of the aldehyde formation, by a thermodynamically induced mecanism, in the plasma of patients receiving multitherapy. Another HPLC method including the derivatization of acrolein with 2,4-dinitrophenylhydrazine [14] leading to UV detection has also been proposed but the necessary 254 nm detection dramatically limits its sensitivity and specificity in the case of plasma samples. Facing the crucial need of possessing an accurate method of the quantification of acrolein in plasma, we were eager to develop an HPLC technique with fluorescent detection using only mild conditions and hence suitable for the quantitative determination of acrolein in human plasma.
A few years ago, we demonstrated the favourable fluorescent properties of Luminarin® 3 (1H,5H,11H-[1]benzopyrano[6,7,8-ij]quinolizine-9-acetic acid 2,3,6,7-tetrahydro-11-oxohydrazide) (6) (Fig. 2) derivatives of carbonyl compounds allowing the detection of acrolein at the pico scale [15], [16]. This method being carried out at room temperature, we consequently tried to apply this methodology and have been able to adapt it for the detection of acrolein in plasma samples. In order to prepare a suitable I.S., we studied several other aldehydes and chose the condensation adduct of Luminarin® 3 and acetaldehyde as an I.S. (7) (Fig. 2).
Section snippets
Apparatus
Mass spectrometry (MS) was performed with a Nermag R-1010 instrument. NMR spectra were performed on a Bruker AC 300-P spectrometer.
LC analyses were performed with a Beckman (Gagny, France) 126 binary pump equipped with a Beckman 210A injector, a 20-μl sample loop and a Jasco 820-FP fluorescence LC detector. λexc and λem were set at 407 and 507 nm, respectively (attenuation was 64 and Gain ×1). The LC system was driven by software gold 8.1 Beckman.
RP chromatography was performed on a 150×4.6 mm
Structure of lum-acet
The structure of Lum-acro has already been established in our previous work [13]. The structure of Lum-acet was confirmed by MS, 1H-NMR and 13C-NMR. Its mass spectrum (CI) displayed a quasi-molecular peak at m/z 357 [M+NH4+] and a molecular peak at m/z 340 [MH+]. Unambiguous evidence for the formation and purity of the lum-acet was confirmed by LC, and NMR spectral data (see Experimental). In the 1H-NMR spectrum, the syn/anti forms of Lum-acet were responsible for a 1:1 splitting of H8 and H5′.
Conclusion
Fluorimetric detection is highly sensitive and its use has already permitted the design of numerous methods to specifically quantify metabolites in mixtures. In this paper, we have demonstrated that our previous reported method for the quantification of aldehydes can easily be adapted to allow quantitative determination of acrolein in plasma.
Our method does not require heating of the sample and consequently the risks of modified kinetics, as well as those of denaturation or artefact production
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