Identification and quantitation of xenobiotics by1H NMR spectroscopy in poisoning cases

M. Imbenottea,*, N. Azaroualb, B. Cartignyc,G. Vermeerschb, M. Lhermittea,c

aLaboratoire de Toxicologie, Faculte des Sciences Pharmaceutiques et Biologiques, BP 83, 59006 Lille, FrancebLaboratoire de Physique, UPRESA CNRS 8009, Laboratoire d’Application RMN, Lille, France

cLaboratoire de Biochimie et Biologie Moleculaire, Hopital Calmette, Lille, France

Received 31 October 2002; received in revised form 18 January 2003; accepted 18 January 2003

Abstract

In order to analyse a wide range of xenobiotics and their metabolites present in biological fluids, NMR spectroscopy can be

used. A large variety of xenobiotics (therapeutic agents, pesticides, solvents, alcohols) can be characterized and quantitated

directly, without sample preparation. NMR investigations were applied to acute poisoning cases, involving drugs such as

salicylates and valproic acid (VPA). In a salicylate poisoning case, the three major metabolites of acetylsalicylic acid have been

detected in crude urine, and rapid identification of lysine revealed the origin of the intoxication, namely lysine acetylsalicylate

(Aspegic1). Valproic acid as its glucuronide was identified in urine samples from two poisoned patients. 1H NMR was also used

to identify and quantitate paraquat (Gramoxone1) in urine owing to its two aromatic signals at 8.49 and 9.02 ppm, in two acutely

poisoned patients (183 and 93 mg/l). An intentional poisoning case with tetrahydrofuran (THF) was also investigated. Serum and

urine samples were collected. THF was characterized by its resonances at 1.90 and 3.76 ppm, and quantified at 813 and 850 mg/l

in the two biological fluids, respectively. Moreover, two other compounds were detected: lactate and g-hydroxybutyric acid

(GHB). 1H NMR spectroscopic analysis of serum samples from three poisoned patients revealed methanol in one case and

ethylene glycol in the two others. Moreover, in the same spectrum, the corresponding metabolites formate and glycolate were

found. Compared with the reference chromatographic or spectrophotometric methods, requiring time-consuming extraction and/

or derivatization steps, NMR spectroscopy allows the determination of many exogenous and endogenous compounds, without

any pre-selection of the analytes.

# 2003 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Biological fluids; 1H NMR spectroscopy; Poisoning; Xenobiotics

1. Introduction

Forensic testing of xenobiotics in biological fluids involves

several analytical methods, the most commonly used requir-

ing chromatographic techniques after extraction hydrolysis of

conjugated forms and sometimes derivatization. NMR spec-

troscopy can be applied to identify compounds in seized drugs

[1], or to precise the mechanisms of postmortem decomposi-

tion [2]. It has been proved to be a very useful complementary

technique to the above-mentioned separative ones, to identify

and quantitate xenobiotics directly invarious biological fluids.

The aim of this paper is to present some benefits linked to

the use of 1H NMR spectroscopy in analytical toxicology. As

no hypothesis has to be made with regards to the compounds

screened for, a wide range of xenobiotics can be analysed,

such as therapeutic agents, pesticides, solvents and alcohols.

2. Material and methods

1H NMRpectra were recorded at 300 MHz on a Bruker

DPX 300 spectrometer using BBI inverse gradient probe.

Forensic Science International 133 (2003) 132–135

* Corresponding author. Tel.: þ33-3-20-964040.

E-mail address: mimbenot@phare.univ-lille2.fr (M. Imbenotte).

0379-0738/03/$ – see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved.

doi:10.1016/S0379-0738(03)00059-8

Prior to Fourier transform, an exponential apodization func-

tion was applied to the signal, corresponding to a line

broadening of 0.3 Hz. A presaturation sequence was used

to suppress the intense water signal. Depending on the

sample concentration, 128–512 transients were collected

into a 16 k data point computer, with a spectral width of

3200 Hz and a 308 pulse.

Chemical standards and b-glucuronidase were obtained

from Sigma–Aldrich (Saint-Quentin Fallavier, France).

Other chemicals were of the highest purity commercially

available.

3. Results

3.1. Salicylate poisoning

To assess acute poisoning by drugs and to check the

applicability of NMR spectroscopy to the analysis of bio-

logical fluids, we first considered a case of known salicylate

poisoning, with a positive colorimetric Trinder test in urine

[3]. In the 1H NMR spectrum, unusual peaks appeared in two

regions (Fig. 1): between 1.4 and 2.0 ppm and in the aro-

matic region (6.5–8.0 ppm).

The first ones were assigned to lysine on the basis of their

chemical shift (d) and coupling constant (J) values. In the

aromatic region, after spectral simplification by two dimen-

sional J-resolved NMR spectroscopy (data not shown), the

three major metabolites of acetylsalicylic acid were identi-

fied. It revealed the origin of the poisoning, namely lysine

acetylsalicylate (commercialized under the trade-name

Aspegic1 in France). Quantitation of each compound was

carried out by integrating the corresponding peaks. Table 1

shows that the sum of the concentration of the three meta-

bolites is nearly equal to the global concentration of sali-

cylates obtained by the classical Trinder method.

3.2. Valproic acid poisoning

Valproic acid (VPA), another therapeutic agent, is widely

used and effective in the treatment of epilepsy. We pre-

viously reported two cases of VPA poisoning [4]. Signals at

0.85, 1.28, and 1.53 ppm could be assigned to H-5, H-4 and

H-3 protons of a valproic acid structure (data not shown).

The fourth signal corresponding to the H-2 protons of

valproic acid, which normally have a chemical shift of

2.23 ppm, was shifted downfield to 2.53 ppm, indicating a

substituted molecule. After an enzymatic hydrolysis with b-

glucuronidase, the 1H NMR spectrum (data not shown) gave

resonance peaks of free VPA, and two free glucuronic acids

with a- and b-anomeric proton chemical shifts at 5.22 and

4.62 ppm, respectively. Two dimensional NMR experiments

and GC/MS analysis confirmed that the major metabolite

was VPA-glucuronide.

3.3. Pesticide poisoning

1H NMR is also very useful in cases of pesticides poisoning

and paraquat (Gramoxone1) can be considered as a high-risk

pesticide since the fatal adult dose ranges from 3 to 6 g when

ingested. Analyses were made on urine samples from two

Fig. 1. 1H NMR spectrum of urine from a patient intoxicated with salicylate. The assignments are as follows: creat, creatinine; Lac, lactic

acid; Gly, glycine; Ci, citrate.; Ha,b,g,d,e, protons of lysine; Ar-protons are those of salicylic, salicyluric and gentisic acids.

Table 1

Quantitation of the three major metabolites of acetylsalicylic acid

Metabolite mmol/l %

Salicylic acid 18.42 75.4

Salicyluric acid 4.94 20.2

Gentisic acid 1.06 4.4

Total 24.42 100

Urine concentration of salicylates determined by Trinder’s method:

22.81 mmol/l.

M. Imbenotte et al. / Forensic Science International 133 (2003) 132–135 133

suicide cases who ingested Gramoxone1 [5]. Two doublets at

8.49 and 9.02 ppm with the same coupling constant of 6.3 Hz,

corresponded to the aromatic paraquat protons. The methyl

resonance appeared as a singlet at 4.48 ppm (Fig. 2). Paraquat

quantitation limit was found to be 25 mmol/l. Comparison of

concentration obtained with spectrophotometric Fuke’s

method gave very similar results: for the first patient, on

admission, parquat concentration was determined at 1081�33 mmol/l by spectrophotometry and 985 � 85 mmol/l by 1H

NMR spectroscopy.

3.4. Solvent poisoning

Tetrahydrofuran (THF) is essentially considered in occu-

pational toxicology, and acute poisoning cases are extremely

rare. Biological fluids from a woman who ingested this

solvent were analysed [6]. In the 1H NMR spectrum of

urine (data not shown), a characteristic signal at 1.90 ppm

could be assigned to the two b-methylene groups of THF.

Moreover, unusual peaks of similar intensities could be

detected: a quintet at 1.77 ppm and two triplets at 2.22

and 3.58 ppm, due to the three methylene groups of 4-amino

butyric acid (GHB). Similar findings were observed on the1H NMR spectrum of the patient’s serum. Quantitative

results for urine and serum are reported in Table 2. The

quantitation limit was found to be 0.1 mmol/l for THF and

0.4 mmol/l for GHB. This example strongly emphasizes the

usefulness of NMR, as GHB was not expected. No other

universal technique can allow the identification and quanti-

tation of GHB in the same analysis as THF. The presence of

THF and GHB was confirmed by GC/MS analyses.

3.5. Alcohol and glycols

The clinical implications of intoxication with methanol

and ethylene glycol are serious and fatality can result because

these xenobiotics as well as their respective metabolites,

formate and glycolate, are toxic. The main biological dis-

turbance is metabolic acidosis with an anionic gap. Analyses

of serum and urine samples from three patients in a metabolic

acidosis state were performed using 1H NMR spectroscopy

and gas chromatography [7]. Characteristic signals of ethy-

lene glycol and glycolic acid could be observed in two cases,

thus confirming the ingestion of ethylene glycol. The third

case was concerned with methanol poisoning: formate serum

concentration was 146 mmol/l at the time of admission

indicating that methanol was largely metabolised.

4. Discussion and conclusion

Other compounds such as analgesics, antiepileptics, anti-

psychotics and antidepressants were previously investigated

by 1H NMR [8]. Some advantages of 1H NMR spectroscopy

can be established: no pretreatment of the sample is needed

and it has reliable specificity based on spectral characteristics.

In spite of its specificity, NMR spectroscopy suffers from a

lack of sensitivity: quantitation may be difficult when very

low concentrations are present or when overlapping signals

are detected. This can occur especially in cases of mixed

Fig. 2. 1H NMR spectrum of urine from a patient intoxicated with paraquat. Assignments are as for Fig. 1 and Hip, hippurate.

Table 2

Concentrations of THF, GHB and lactic acid in patient’s serum and

urine, measured by 1H NMR spectroscopy (mmol/l)

Serum Urine

THF 11.3 11.8

GHB 2.3 28.6

Lactic acid 3.8 25.4

134 M. Imbenotte et al. / Forensic Science International 133 (2003) 132–135

poisoning. Spectral simplification can be achieved using

either multidimensional sequences or 31P NMR spectroscopy,

as in the case of organophosphorus poisonings [9]. As it is a

non-destructive technique, the samples can be later analysed

by complementary methods. Compared with chromato-

graphic or spectrophotometric methods, 1H NMR spectro-

scopy is rapid, only needs a small sample volume (500 ml)

and does not require time-consuming extraction and/or deri-

vatization steps. Moreover, in a single spectrum, three kinds

of information can be collected: the nature of the xenobiotic

compounds, the possible presence of their metabolites, even

of unexpected species such as GHB in the case of THF

poisoning, and finally the consecutive metabolic distur-

bances. NMR spectroscopy can document cases from direct

analysis of biological fluids and when possible, analysis of

the xenobiotic suspected to be at the origin of the poisoning.

In each presented case, quantitative data were validated

and satisfactorily correlated with reference methods, both

for xenobiotic compounds and endogenous abnormalities.

The inherent relatively low sensitivity of 1H NMR spectro-

scopy is counterbalanced by its simplicity and specificity.

This technique is not commonly used in hospital labora-

tories. In spite of this, physicians should be aware of this

laboratory method every time they face an acute poisoning

case in toxicological or forensic science fields.

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