Analysis of monofluoroacetic acid in urine by liquid chromatography-triple quadrupole mass spectrometry and preparation of the positive sample by the bioconversion from monofluoroacetamide to monofluoroacetic acid in vitro
Introduction
Monofluoroacetic acid (FAcOH) is a rodenticide with high acute toxicity and is known as sodium monofluoroacetate (1080) in industry. It is permitted to be used as vertebrate pesticide in New Zealand, Australia, Israel and the USA [1], [2]. FAcOH (as the format of monofluoroacetate) has been found as well in natural plants such as the Fabaceae (Acacia, Gastrolobium), Rubiaceae (Palicourea), Bignoniaceae (Arrabidaea), Malpighiaceae (Amorimia) and Dichapetalaceae (Dichapetalum, Tapura) in the southern continents of Africa, Australia, South America and India [3], [4]. The median lethal dose of mammals (LD50) is less than 1 mg kg−1 in animals and estimated to be 2–5 mg kg−1 in humans within 6–48 h of eating baits [2], [5]. Besides, FAcOH was reported to be the metabolite and biomarker of monofluoroacetamide (FAcNH2, another rodenticide with high acute toxicity) in body [6]. The exposure for both rodenticides to humans or animals includes suicide, ingestion of contaminated food and criminal poisoning [6], [7], [8], [9], [10], [11]. FAcOH and FAcNH2 even can be a powerful tool for terrorist threat due to their colorlessness, tastelessness, easy accessibility and extremely toxicity. The typical case was the criminal threat of FAcOH to adulterate infant formula in New Zealand in 2015 [12].
It is important and imperative to develop a fast and sensitive analytical method of FAcOH in biological matrices to quickly respond to poisoning cases. Gas chromatography–mass spectrometry (GC–MS) was reported to measure FAcOH in biological samples while derivatization techniques were required to fit the polarity of the gas chromatography column [10]. For liquid chromatography measurement of the trace levels of FAcOH in complex matrices, chemical modification was also used to fit the reverse phase separation and produce the fluorescent signal [13]. As a polar and water soluble compound, ion pair reagents were used to improve the retention ability for direct analysis of FAcOH by liquid chromatography-mass spectrometry (LC–MS) [3], [9], [14] or liquid chromatography–triple quadrupole mass spectrometry (LC–MS/MS) [15]. Solid phase extraction (SPE) cartridges with reverse phase function of C18 [9] or HLB [15] were used for clean-up of FAcOH in foods or biological matrices. FAcOH behaves weak retention ability to reverse phase SPE material and only weak polar matrices were removed. In this study, polymeric anion exchange cartridge (PAX) was used to remove both the weak polar and water soluble matrices in urine. Hydrophilic interaction column Amide was planned to directly separate FAcOH under basic conditions without ion pair reagents.
Urine is one of the most typical specimens for forensic diagnosis when poisoning case about FAcOH happens to humans or animals. The positive sample containing FAcOH plays a key role for the development of an accurate and reliable analytical method. Standard spiking can be used to evaluate the method accuracy while the preferable way to obtain a positive sample is the animal experiment. However, the disadvantage of animal experiment is self-evident considering the cost and the animal ethics. As a hot topic for latest study, it is urgent need to find an alternative way to obtain a positive sample without animal experiment. The bioconversion from FAcNH2 to FAcOH (Fig. 1) was found during the stability study of FAcNH2 in urine in vitro. The feasibility for preparation of a positive urine sample containing FAcOH was studied according to this bioconversion. The stopping of the bioconversion, levels of the prepared FAcOH in urine, stability and storage of the positive sample were studied in this study.
Section snippets
Materials
All reagents and solvents were of analytical grade unless specified.
FAcOH (Sodium salt, 98%), FAcNH2 (99%) and isotope labeled internal standard FAcOH-13C2 (Sodium salt, 99%) were provided by Dr. Ehrenstorfer (Augsberg, Germany), Chem Service, Inc (West Chester, PA, USA) and BDG synthesis (Wellington, New Zealand), respectively. HPLC-grade methanol and acetonitrile were supported by Merck (Darmstadt, Germany). Formic acid was obtained from ROE Scientific Inc. (New Castle, DE, USA). Ammonium
LC separation of FAcOH
As a polar compound, FAcOH should be modified with chemical reagent such as 9-chloromethylanthracene [13] or assisted with ion pair reagent such as tributylamine [3], [9], [14] to obtain good retention performance when separated by reverse phase chromatographic columns. Chemical modification steps would result to extra works and labile factors for forensic analysis. Ion pair reagent in the mobile phase would present negative effects to MS measurements. Hydrophilic columns were reported to be
Conclusions
FAcOH is a metabolite and biomarker of FAcNH2 in body. It provided a method to study the possible biomarker in body by bioconversion in vitro and directly obtain a positive urine sample, which was one of the most typical specimens for forensic diagnosis. A reliable experimental model was presented for obtaining a positive urine sample of FAcOH without animal experiment or standard spiking. Besides, the developed method was sensitive and effective for the analysis of FAcOH in urine and was
Acknowledgement
This work was supported by the Science Research Foundation Program of Zhejiang Provincial Medicine and Health (WKJ2013-2-006 and 2015DTA006).
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Analysis of monofluoroacetic acid in human plasma by UFLC–MS/MS and its application in patients with sodium monofluoroacetate or monofluoroacetamide poisoning
2018, Journal of Pharmaceutical and Biomedical AnalysisCitation Excerpt :Liquid chromatography mass spectrometry (LC–MS) was reported to analyze FAcOH (as a high polar compound) with ion pair reagents in the mobile phase to improve the retention ability of FAcOH in reverse phase C18 chromatographic columns [12]. Liquid chromatography-triple quadrupole mass spectrometry (LC–MS/MS) was reported to measure FAcOH in dairy powder [13,14] or urine [15,16] while solid phase extraction cartridges was required to remove the impurities in complex matrices [12–15]. Although sensitive results were obtained from the methods listed above, however the process is complex and not suitable for poisoning monitoring.
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