Determination of fluoroacetic acid in water and

Anal Bioanal Chem DOI 10.1007/s00216-006-0713-x

ORIGINAL PAPER

Determination of fluoroacetic acid in water and biological samples by GC-FID and GC-MS in combination with solid-phase microextraction Nadezhda L. Koryagina & Elena I. Savelieva & Natalia S. Khlebnikova & Nikolay V. Goncharov & Richard O. Jenkins & Andrey S. Radilov

Received: 15 May 2006 / Revised: 20 July 2006 / Accepted: 27 July 2006 # Springer-Verlag 2006

Abstract A novel procedure has been developed for determination of fluoroacetic acid (FAA) in water and biological samples. It involves ethylation of FAA with ethanol in the presence of sulfuric acid, solid-phase microextraction of the ethyl fluoroacetate formed, and subsequent analysis by GC-FID or by GC-MS in selected-ion-monitoring mode. The detection limits for FAA in water, blood plasma, and organ homogenates are 0.001 μg mLj1, 0.01 μg mLj1, and 0.01 μg gj1, respectively. The determination error at concentrations close to the detection limit was less than 50%. For analysis of biological samples, the approach has the advantages of overcoming the matrix effect and protecting the GC and GC-MS systems from contamination. Application of the approach to determination of FAA in blood plasma and organ tissues of animals poisoned with sodium fluoroacetate reveals substantial differences between the dynamics of FAA accumulation and clearance in rabbits and rats. Keywords SPME . GC-MS . Fluoroacetate . Fluoroacetic acid . Ethylation . Fluorocitrate

Introduction Salts of fluoroacetic acid (FAA), primarily sodium fluoroacetate (SFA), are strong metabolic poisons that cause N. L. Koryagina : E. I. Savelieva : N. S. Khlebnikova : N. V. Goncharov : A. S. Radilov Research Institute of Hygiene, Occupational Pathology and Human Ecology, Saint-Petersburg, Russian Federation R. O. Jenkins (*) School of Allied Health Sciences, De Montfort University, Leicester LE1 9BH, UK e-mail: [email protected]

death because they are converted into fluorocitrate, which blocks aconitase in the Krebs cycle. Their action characteristically involves a latent period. SFA (also known as compound 1080) is readily soluble in water, has neither taste nor odor, and is used in Australia and New Zealand to control the population of some vertebrate species [1]. The compound_s acute sub-lethal and target organ toxicity and its non-target effects and general toxicity have been widely studied [1, 2]. Many compounds are metabolized to FAA as an intermediate product: these are the anticancer drugs 5fluorouracil and fluoroethyl nitrosourea, N-(2-fluoroethyl) derivatives of the narcotic analgesics normeperidin and normetazocin, the pesticides fluoroacetamide and 1,3difluoro-2-propanol, and the industrial chemicals fluoroethanol and 1-(di)halo-2-fluoroethanes [3–8]. Cases of intentional and unintentional misuse of SFA or other precursors of FAA have been described in the literature [9–16]. Probably many more cases are not described because of uncertainty in genuine causes of intoxication and poor understanding of specific signs of FAA poisoning. For this reason analysis of biological samples for FAA is often the only tool for clinical and forensic expertise. Forensic analysis in cases of poisoning with unknown poisons, and scientific research on the toxicokinetics of FAA salts, necessitate development of methods for determination of FAA in biological fluids and tissues. Chemical analysis of FAA and its salts is a challenging problem because of the high polarity of the fluorine-carbon bond. The first gas chromatographic (GC) analysis of FAA was reported in 1965 by Gershon and Renwick [17] who attempted to separate low-molecular-mass fluorinated carboxylic acids on a short copper column. Stevens et al. were the first to report analysis of FAA in biological samples [18], using a Porapak Q glass column and flame-ionization detector, but separation of FAA from some biological components was not entirely resolved. The first attempts

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to analyze FAA by gas chromatography-mass spectrometry (GC-MS) [19, 20] were also unsuccessful, because of absorption of the analyte on the hot metallic surfaces of the GC-MS interface. Because FAA is a nonvolatile substance, it has most commonly been analyzed by GC as the methyl [18, 21], ethyl or n-propyl [19], and pentafluorobenzyl esters [22–24]. Derivatization with 2,4-dichloroaniline in the presence of N, N-dicyclohexylcarbodiimide has been used for GC determination of SFA in water [25] and blood serum [26]. Liquid chromatography has also been used for the determination of FAA in various media [27–29]. Most recently, Minnaar et al. [30] described the analysis of FAA in plants and stomach contents by HPLC with UV detection. Ozawa and Tsukioka [31] used the method for determination of SFA after its extraction from biological samples and soil using a strongly basic anion exchanger. Procedures developed by Ozawa and Tsukioka [31] have been extensively modified by Eason et al. [32, 33], and low detection limits for FAA have been reported: 0.01 μg gj1 in plasma and urine and 0.002 μg gj1 in the tissue and feces of sheep and goats. GC with electron-capture detection at this level of sensitivity is, however, traditionally regarded as less reliable for unambiguous identification of analyte. The procedure, which includes two successive steps of deproteinization and two successive stages of solidphase extraction, is, moreover, labor and time-consuming. Analysis of foreign compounds or their metabolites in biological matrices is accomplished by injection of either unpurified or purified extracts containing target analytes into a chromatographic system. The first approach is associated with contamination of the chromatographic system with matrix components whose concentrations in unpurified extracts are usually an order of magnitude higher than the concentrations of the target analytes. Purification of extracts most commonly involves preparative adsorption and/or ion-exchange chromatography. Such approach enables parallel processing which greatly increases the velocity and throughput of analysis. It is, however, inevitably associated with analyte losses, even if standard cartridges are used, mainly because of its non-specific and irreversible sorption on the cartridges. According to our experience with FAA, these losses vary over a wide range even if cartridges from the same company are used. The efficiency of retention and elution of analytes is highly dependent on the qualitative and quantitative composition of macro components of the sample. Variation of this composition in biological samples precludes development of a universal procedure for analysis of FAA. Although a head-space analysis approach circumvents most of the above-mentioned drawbacks, it is insufficiently sensitive. Solid-phase microextraction (SPME) from an equilibrium vapor phase has all the advantages of head-space analysis, but is a

much more sensitive technique. Advantages of SPME in headspace mode for analysis of biological samples were recently demonstrated by Domeno et al. [34]. We report here a novel procedure for determination of FAA in water and biological samples, involving ethylation of FAA with ethanol in the presence of sulfuric acid, SPME of the ethyl fluoroacetate formed from the equilibrium vapor phase, and subsequent analysis by GC-MS. The developed SPME-GCMS method is principally different from that of Sporkert et al. [35] - which involves derivation with pyrenyldiazomethane on the microfiber - in that derivation of the analyte is performed directly in the sample, producing more reproducible results.

Experimental Chemicals and materials Ethanol, toluene, carbon tetrachloride, acetonitrile, and sulfuric acid of at least chemical grade purity were used (Ekros, Russia). Sodium fluoroacetate was synthesized as reported elsewhere [36] at the Research Institute of Hygiene, Occupational Pathology, and Human Ecology (St Petersburg, Russia). According to GC-MS and elemental analysis data the purity of the synthesized sample was no less than 98%. All SPME fibers, holders, and capillary columns used in this research (manual SPME sampling stand; CarboxenPDMS 75-μm microfiber; polyacrylate coating 85-μm microfiber; Carboxen-PDMS StableFlex 85-μm microfiber; and SPB-5 capillary columns (30 m0.2 mm0.2 μm) were obtained from Supelco (Shneedorf, Germany)). Instrumentation A Hewlett-Packard HP-5890 gas chromatograph (USA) fitted with a narrow-bore (0.75 mm i.d.) injector liner (Supelco) and flame-ionization detection (FID) was used for tap-water analysis. A Shimadzu (Japan) GC 17 Gas chromatograph linked to a QP5000 quadrupole MS instrument fitted with a narrow bore (0.75 mm i.d.) injector liner (Supelco) was used for analysis of biological samples. Sample preparation and solid-phase microextraction (SPME) Blood plasma and brain, liver, kidney, and heart homogenates obtained from Chinchilla rabbits and Wistar rats exposed to SFA at a dose of 12LD50 (0.12 mg kgj1 for rabbits and 0.8 mg kgj1 for rats) were analyzed. Tissue homogenates were prepared by grinding liquid nitrogen-

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frozen biological material with an equal weight of distilled water; samples were stored frozen for no more that seven days. Control samples were taken from animals not exposed to SFA. Tap water spiked with SFA was also analyzed. Blood plasma (1 mL) was diluted with 3 mL acetonitrile and the mixture was thoroughly shaken and centrifuged for 10 min at 7000 rpm. The supernatant was transferred to a 4-mL screw-top vial with a phenolic cap and a PTFEsilicone rubber septum. The precipitate was shaken with 3:1 acetonitrile-water (1 mL) and centrifuged under the same conditions. the combined supernatants were evaporated to dryness. Tissue homogenates were defrosted and mixed with acetonitrile (4 mL per 1 g organ homogenate) and the mixture was centrifuged for 15 min at 7000 rpm. The supernatant was transferred to a 4-mL screw-cap headspace vial with a phenolic cap and a PTFE-silicone rubber septum. the precipitate was shaken with 3:1 acetonitrilewater (21 mL) and centrifuged under the same conditions. The combined supernatant was evaporated to dryness. FAA in the dry residue (from blood plasma or organ homogenate samples) was ethylated by adding concentrated (96%) sulfuric acid (30 μL) and ethanol (70 μL) to the dry residue in the same vial; the ethanol was spiked with toluene or carbon tetrachloride (2–20 μg mLj1) as internal standard (i.s.). For SPME of ethyl fluoroacetate in the equilibrium vapor phase the sample mixture was heated at 55 -C for 15 min, with continuous stirring, in a tightly sealed 4-mL vial mounted in a manual SPME sampling stand, after which a Carboxen-PDMS StableFlex 85-μm microfiber was inserted through the seal into the vapor phase over the reaction mixture and exposed for 10 min. Thermal desorption of the analytes from the microfiber in the GC injector was at 280 -C. Ethylation of FAA in 1-mL spiked water samples and subsequent SPME of the formed ethyl fluoroacetate was also performed as described above. Chromatographic analysis GC-MS was performed by an internal calibration technique (internal standard toluene or carbon tetrachloride) with a Shimadzu QP5000 GC-MS system fitted with a Supelco SPB-5 capillary column (30 m0.2 mm0.2 μm). Splitless injection was performed with a purge-off time of 0.3 min. The column oven temperature was maintained at 40 -C for 1 min then programmed to 200 -C at 5- minj1; the injector and detector temperatures were 280 -C. The mass spectrometer was operated in SIM mode (Table 1). GC-FID was performed with a Hewlett-Packard HP5890 chromatograph under conditions identical with those used in GC-MS analysis.

Table 1 GC-MS parameters for determination of FAA in biological samples by the internal calibration technique Compound

m/z qual.a

m/z quant.a

Retention time (min)

Carbon tetrachloride (i.s.) Ethyl fluoroacetate

117, 119

117

5.1

61, 78, 91 91, 92

61

5.7

91

7.4

Toluene (i.s.) a

m/z qual. and m/z quant. are mass numbers used for identification and quantification, respectively, of the analyte

Results and discussion Mori et al. [37] described a simple procedure for analysis of SFA in water that involved static head-space analysis of SFA as ethyl fluoroacetate, using FID and a DB-5 capillary column. In this procedure, aqueous solutions of SFA were evaporated to dryness and the dry residue was ethylated with ethanol in the presence of sulfuric acid, with subsequent vapor phase analysis by GC using toluene as internal standard. The reported linear range for SFA in water was 5–200 μg mLj1 and the detection limit was 0.5 μg mLj1. There are only two conceivable ways of increasing the sensitivity of such a determination - increasing the volume of either the aqueous or vapor sample. The first entails a much longer analysis time, because any attempt to intensify the evaporation procedure results in enhanced analyte losses. Increasing the vapor sample to Q1 mL requires use of special facilities, which makes the procedure more expensive and intricate while not resulting in a substantial gain in sensitivity. The sensitivity of the analysis could be much improved (by approximately two orders of magnitude) by use of SPME. SPME analysis of ethyl fluoroacetate has not yet been described in the literature. In SPMEGC analysis of nonvolatile compounds, the latter can be derivatized directly in the sample, on the microfiber, or in a GC injector. For FAA analysis one should chose between the first two approaches. Spokert et al. [35] reported a procedure for analysis of FAA in biological samples involving derivatization of the analyte with 1-pyrenyldiazomethane on a StableFlex DVBCarboxen microfiber. The linear range in the analysis of FAA in blood serum was 0.02–0.5 μg mLj1. On attempted reproduction of this procedure we failed to obtain reproducible results at the reported sensitivity level. We therefore investigated direct in-sample derivatization of FAA as a more rational approach. For successful SPME, correct choice of microfiber and conditions for adsorption (temperature, time, stirring speed, and the ionic strength of the solution) and desorption (temperature and sampling time) are imperative. In a search

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for optimum SPME conditions we initially chose conditions used by Sarrion et al. [38] for analysis of chloroacetic and bromoacetic acids in water. The same authors [39] subsequently reported a more “sparing” procedure for alkylation of these acids with diethyl or dimethyl sulfide. The reaction was performed in water in the presence of a phase-transfer catalyst. The procedure [39] has the advantages that it does not involve evaporation of the aqueous sample to dryness and exposure of the microfiber to aggressive sulfuric acid vapor is avoided. In our work, however, we failed to adapt the procedure to FAA analysis at a sensitivity higher than 0.1 μg mLj1. Thus use of SPME under these conditions had no advantages over the headspace analysis procedure described by More et al. [37]. When ethylating FAA with ethanol in the presence of sulfuric acid there is a potential problem of short life-time of the SPME fiber, because of the need to take samples in the presence of sulfuric acid fumes. For extraction of highly polar compounds an 85-μm layer of polyacrylate on a quartz fiber is commonly used. Indeed, application of an 85-μm polyacrylate microfiber resulted in much higher sensitivity than use of 75-μm Carboxen-PDMS or 85-μm Carboxen-PDMS StableFlex. We could not use this microfiber, however, because of its short life-time in the aggressive vapor phase in which ethylation occurred. Polyacrylates are, moreover, soluble in aromatic hydrocarbons such as toluene (used as internal standard). The sensitivity was sharply reduced after a single extraction only. The 75-μm Carbogen-PDMS microfiber was also found to be insufficiently resistant. Polydimethylsyloxane (PDMS) decomposed into monomers in the aggressive medium; some of the degradation products co-eluted with the analyte and the internal standard, affecting the analysis and interfering with identification. The sensitivity of the 75-μm Carbogen-PDMS microfiber decreased markedly after only four or five extractions. The 85-μm StableFlexmodified Carbogen-PDMS Microfiber was more resistant to aggressive media and proved to be relatively stable in the vapor phase of the ethylation mixture. Reduction of the adsorption time to 10 min enables use of this microfiber for 10–12 extractions without significant loss of sensitivity. On the basis of the dependence of peak area of ethyl fluoroacetate on reaction time during head space analysis, equilibrium in the vapor phase was achieved after mixing the reactants for 25 min (data not shown). The microfiber was therefore placed in the vapor phase 15 min after mixing the reagents and was kept there for 10 min (an optimum time for adsorption taking into account maximum life-time of the microfiber). Sarrion et al. [38] reported that the most significant drawback of their procedure for ethylation of chloroacetic and bromoacetic acids in the presence of sulfuric acid was the short life-time of the microfiber. To minimize this

problem we used a more stable PDMS-Carboxen StableFlex microfiber (75 μm) and a shorter adsorption time. The GC-FID, combined with SPME under the optimum conditions, enabled reliable determination of FAA in water in the concentration range 0.001–10 μg mLj1. When analyzing the tap water samples, satisfactory results were obtained if GC combined with a non-selective detector (FID) was used. The absence of components coeluting with the analyte or the internal standard was justification for conducting the procedure with a single internal standard (toluene). The most important distinguishing feature of biological matrices is that they contain many organic compounds, including those co-eluting with FAA derivatives. In this regard ethyl fluoroacetate is very difficult to detect in biological samples with a universal (non-selective) GC detector. In analysis of animal blood plasma and tissue homogenates for FAA we therefore made use of GC-MS in the SIM mode. Because of a probability of partial overlapping of the peak of the internal standard with components of the sample matrix, quantification was performed with use of two internal standards-carbon tetrachloride (Rt 5.1 min) and toluene (Rt 7.4 min). Calibration studies were also performed with the two standards. The chromatographic purity of the peaks of the internal standards was studied in parallel with analysis of the samples; if the chromatographic purity of one of the peaks was insufficient the other standard was used. Blood plasma samples from control animals (in this work, rabbits) spiked with SFA were used for calibration. The calibration plots for the determination of SFA in biological samples were linear in the SFA concentration range 0.01–5.0 μg mLj1 for both internal standards. For blood plasma a linear relationship was observed between peak area (S) and concentration in the range 0.01 to 5.0 μg mLj1 (r=0.95). With toluene as internal standard the linear regression equation was Y=0.014X (where Y was the ratio S (ethylFA)/S(toluene) and X was the concentration of SFA, μg mLj1). The RSD for fluoroacetate quantification at 0.1 μg mLj1 was 12% (n=5). For plasma with carbon tetrachloride as internal standard a linear relationship was observed in the range 0.01 to 5.0 μg mLj1 (r=0.98). The linear regression equation was Y=0.1656X (where Y was the ratio S(ethylFA)/S(CCl4) and X was the concentration of fluoroacetate, μg mLj1). The RSD for fluoroacetate quantification at 0.1 μg mLj1 was 6% (n=5).The detection limit was 0.01 μg mLj1 (S/N=3). The same calibration studies were performed for rat liver, kidney, brain, and heart homogenates; the calibration data obtained were identical with those for plasma. A mass chromatogram obtained from blood plasma containing a quantity of FAA close to the detection limit is given in Fig. 1; the corresponding data are listed in Table 1.

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In tap water analysis a linear relationship was observed in the range 0.1 to 50.0 μg mLj1 (r=0.98). The linear regression equation was Y=0.0128 X (where Y was the ratio S(ethylFA)/S(toluene) and X was the concentration of fluoroacetate, μg mLj1). The RSD for fluoroacetate quantification at 0.1 μg mLj1 was 14% (n=5); at 1 μg mLj1 it was 6% (n=5) and at 20 μg mLj1 it was 6% (n=5). Results from determination, by the developed procedure, of FAA (calculated as SFA) in rat and rabbit organs and plasma in the first 24 h after poisoning, were averaged over 4–6 runs with, predominantly, pooled samples from 2–3 animals. For rabbits poisoned with 0.12 mg kgj1 SFA, FAA levels in plasma were highest 1 h after exposure (0.5T0.07 μg mLj1) then decreased in 3 h to 0.2T 0.05 μg mLj1 and had fallen to below the detection limit within six hours of exposure. FAA was also detected in kidney tissue 3 h after poisoning (0.03T0.005 μg gj1); FAA was not detected in other rabbit organs at any stage after poisoning with SFA. Variation in the concentration of FAA in rat plasma and organs after poisoning with 0.8 mg kgj1 SFA is depicted in Fig. 2. Plasma levels of FAA were highest 3 h after exposure and dropped to undetectable levels within 24 h after poisoning. The highest concentrations of FAA in kidney and heart were also observed 3 h after poisoning. For brain tissue, however, levels of FAA were highest after 6 and 24 h. The liver was the only organ in which the highest concentration of FAA was detected 1 h after poisoning. The absence of FAA from all control samples was confirmed by GC-MS. These data demonstrate for the first time that retention of SFA is longest in the brain tissue of rats; this has an affect on development of encephalopathy in rats. 1/2LD50 doses of SFA were different for rats and rabbits - 0.8 and 0.12 mg kgj1, respectively. This difference should account for different kinetic behavior and dynamic effects of FAA in these species. These data on the specific features of the toxicokinetics and toxicody-

Fig. 1 SPME-GC-MS determination of FAA as ethyl fluoroacetate in blood plasma. Peak number and analyte: 1, carbon tetrachloride (internal standard), m/z 117; 2, ethyl fluoroacetate, m/z 61; 3, toluene (internal standard), m/z 91. The mass chromatogram shows FAA at a quantity close to the detection limit

Fig. 2 Data from determination of FAA (calculated as SFA) in rat tissue homogenates and blood plasma 1 h (hatched), 3 h (dotted), 6 h (white), and 24 h (black) after poisoning with SFA at a peroral dose of 1/2LD50 (0.8 mg kgj1). Asterisks indicate not detected (