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Letters |
1
Lab. de Biochim.
2
Service de Réanimation, Hôpital Calmette, CHR et U de Lille, Bd. du Pr. J.Leclerc, 59037 Lille Cedex, France,
3
Lab. de Phys., URA CNRS 351, Lab. d'Application RMN de L'Université de Lille II, BP 83, 59006 Lille Cedex
4
Lab. de Chim. des Biomolécules, URA CNRS 1309, Institut Pasteur de Lille, 1 rue du Pr. Calmette, BP 245, 59019 Lille Cedex
5
Lab. de Toxicol., Faculté des Sci. Pharm. et Biol. de Lille, BP 83, 59006 Lille Cedex
a Author for correspondence.
To the Editor:
High-resolution NMR spectroscopy is increasingly being used to analyze a variety of physiological fluids (1)(2) and may constitute a new tool in clinical diagnosis. Many important low-molecular-mass metabolites can be readily detected and quantified by 1H-NMR. New sequences, such as 1H–1H J-resolved map, have proved very useful (3). We have used these methods to investigate a urine sample obtained in a case of acute chloroquine poisoning.
A 41-year-old man was admitted to the hospital after attempting suicide. He was conscious, without neurological deficit; the electrocardiogram showed an increased QT duration, and his blood potassium concentration was 3 mmol/L. A toxicological screen was requested and reported a plasma chloroquine concentration of 890 µg/L.
For NMR measurements, we used a urine sample collected at admission. Spectra were recorded at 300 MHz with a Bruker (Wissembourg, France) AC300 spectrometer. One-dimensional spectra of the crude urine were obtained by operating in the pulsed Fourier-transform mode with quadrature detection; there was a selective reduction in the T2 of the water protons by chemical exchange with NH4Cl (0.8 mol/L) and a saturation of the water signal. Two-dimensional J-resolved maps of the same urine sample were realized in the same conditions.
We also used a Bruker DMX600 spectrometer operating at 600 MHz with a 5-mm triple-nucleus inverse- geometry self-shielded gradient probe. Two-dimensional total correlation spectroscopy (TOCSY) spectra of urine were obtained with water peak suppression.
For 1H-NMR analysis, we added 20 µL of deuterium oxide, containing 3-(trimethylsilyl)-2,2',3,3'-tetradeuteropropionic acid (TMSP-d4) used as internal chemical shift reference, to 500 µL of the crude urine. The sample was adjusted to pH 7.0 and introduced into a 5-mm-diameter NMR tube; the spectra were run at room temperature.
Compared with that for a normal subject, the 1H-NMR
spectrum for this patient's urine shows intense peaks between 1.2 and
1.5 ppm and near 8.3 ppm (Fig. 1A
). The J-resolved
pulse sequence was used to identify unusual peaks. Expansions of the
1H–1H J-resolved map of the sample are shown
in Figs. 1B
(aliphatic region) and 1C (aromatic region). The spectrum
of chloroquine in pure form, run under the same conditions, allowed the
assignment of H-2, H-3, H-5, H-12, and H-17 chloroquine protons. The
spectrum showed also signals for major endogenous metabolites (e.g.,
lactate, alanine, 3-D-hydroxybutyrate, hippurate) as well
as ethanol.
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At 600 MHz, no addition of NH4Cl (0.8 mol/L) was needed and the water signal was suppressed by a field gradient. The resolution at 600 MHz is much improved over that at 300 MHz, and the one-dimensional spectrum allows us to trace and remove less-intense unusual signals that partly overlap the signals of chloroquine H-2, H-3, H-5, and H-12 protons, perhaps corresponding to monodesethylchloroquine resonances. The spectrum of monodesethylchloroquine in pure form was run under the same conditions. TOCSY two-dimensional mapping was performed and showed the cross peaks of all the aliphatic and aromatic chloroquine protons. This also showed that the weak unusual peaks near the chloroquine signals on the one-pulse spectrum can be assigned to monodesethylchloroquine protons.
In conclusion, the results of high-field 1H-NMR spectroscopy at different frequencies (300 and 600 MHz) were coherent and led to the identification of chloroquine in the urine of the intoxicated patient. The major metabolite, monodesethylchloroquine, could be characterized only at the higher field (600 MHz). Because only 500 µL of biological sample is required, NMR spectroscopy has the potential for use in rapid toxicological screening in routine clinical diagnosis. For this purpose, we will have to characterize by NMR spectroscopy the major xenobiotics involved in poisonings and generate a database of structures and 1H-NMR characteristics.
References
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