Optimization of an analytical method for detecting paraphenylenediamine (PPD) by GC/MS-iontrap in biological liquids

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Abstract

A method for qualitative and quantitative determination of paraphenylenediamine in biological fluids (blood, urine and gastric content) was developed. This analytical procedure involves deproteneisation or hydrolysis, liquid–liquid extraction, derivatization with TFA followed by GC–MS iontrap analysis. Benzidine was the internal standard used for quantification and the extraction recovery test was about 85%. The detection limit of paraphenylenediamine was determined at 0.1 pg (S/N = 10).

Introduction

The p-phenylenediamine (PPD) is used for manufacturing azo dye intermediate, dyeing furs, photochemical measurements, photographic developing agent, intermediate in manufacture of antioxidants and accelerators for rubbers. In Morocco, this product is mainly used for dyeing furs of animals and traditionally for the coloration of hairs, alone or mixed with Henna (Lawsonia alba), as a substitute for galls of Tamarix orientalis (plant of south of Morocco, containing gallotannins responsible of dyeing). Reputed for its high toxicity (lethal dose in rats is 80 mg/kg orally), PPD has been forbidden for sale to the public, but continues to be procured by illegal process from the herbalist.

The statistical data of the Centre Anti-Poison du Maroc (CAPM) report that the PPD is responsible of 25% of poisonings assigned to products of traditional pharmacopeia. The young women are the most touched (75.4%), especially relating to abortion or suicide attempts; the death occurs in more than 30% of cases. Otherwise, a retrospective study [1] between October 1999 and May 2000 of 20 cases of death owing to this product shows a predominance of deliberate ingestion to suicide.

If the symptoms of poisonings to PPD are well known and described in the literature [2], [3], [4], [5], a small work relative to its screening in the biological liquids is available. Lately, a study [6] was undertaken for determination of crude PPD in post-mortem blood and gastric contents from a poisoned case by ingesting this product. PPD was determined using thin-layer chromatography, ultraviolet spectrometry, GC/MS and was quantified by HPLC/UV. Great quantity of biological fluids (126 g of gastric contents for example) was used for extraction, and no derivatization was required to detect the PPD by GC/MS.

When only low sample amounts of biological fluids are available, we need to display an analytical method with lower detection limits of PPD. To contribute for developing analytical studies, this work proposes an optimization of a method for extraction, screening and quantification of PPD in small quantities of biological liquids (2 ml). This was enabled by reducing the affinity of PPD to usual stationary phases, by trifluoroacetylation. To quantify the analyte, we use external standardisation for high PPD concentrations and internal standardisation for detecting PPD traces. Finally, these methods were validated on poisoning cases with PPD.

Section snippets

Chemicals

All solvents were chemical grade from Merck, Iodomethane from Panreac 99% trifluoroacetic anhydride from Merck >99%, potassium carbonate from ACROS >99%. Internal standard, benzidine, was obtained from Sigma at 95%, and PPD standard was purchased from Merck >98%.

The biological liquids were provided from the Centre Anti-Poison du Maroc (CAPM) or during criminal investigation cases of Gendarmerie Royale.

Extraction

For urine samples, 2 ml are hydrolysed [7] in 6 ml, 6N HCl for 6 h at 100 °C and filtered. The

Derivatization

The derivatization of PPD with iodomethane (Fig. 1 ) produces three kinds of products, resulting from di (N,N′-dimethyl), tri (N,N,N′ trimethyl) and tetramethylation (N,N,N′,N′-tetramethyl) of PPD, characterized on GC/MS by their ion parents (136, 150 and 164 amu, respectively). On the other hand, the derivatization of PPD with trifluoroacetic anhydride (Fig. 2) produces only one compound due to its N,N′-trifluoroacetylation identified on GC/MS by its ion parent (300 amu). These results show that

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