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LC-MS analysis of cyanuric acid in soil extracts

Cyanuric acid (2, 4, 6-trihydroxy-1, 3, 5-triazine, CA), a suspected gastrointestinal or liver toxicant, can be regarded as a marker of symmetric triazine herbicide's degradation in water and soil environment. It is formed by chemical oxidation, photooxidation and microbial degradation of parent compounds. CA is also used to stabilize the chlorine disinfectant in swimming pools because it is able to form N-chlorinated isocyanurates and to prevent the rapid photolytic degradation of residual chlorine. CA is a highly polar, hydrophilic compound, fairly stable under many environmental conditions. In solution it occurs as a mixture of keto (isocyanuric acid) and enol tautomers, the latter form being stable under alkaline conditions (pH>7.2). Analytical methods for CA analysis mostly employ reverse-phase high-performance liquid chromatography (HPLC) using modified silica columns, phosphate buffer eluents, and UV detection at 213 nm [1, 2]. However, due to the coeluting interferences absorbing UV light below 220 nm, UV detection of CA is often not sufficiently selective. Non-volatile eluent components used for HPLC-UV analysis of CA are not compatible with mass spectrometric (MS) detection systems. Mass spectrometric methods developed so far for determination of CA are based either on stable association complex electrospray mass spectrometry using a solid probe technique [3] or on capillary gas chromatography coupled with mass selective detection of methylated CA [4]. In this study we compared the performance of two HPLC columns of different polarity: Hypersil ODS (octadecyl-modified silica) and Lichrospher 100 CN (cyano-modified silica) for determination of CA in standard solutions prepared in LC-grade water and in phosphate buffer (pH 7.0). Both columns were 250 mm x 4.6 mm i.d. with a 5-micrometer particle size. The isocratic elution was carried out with following mobile phases: acetonitrile/water (5/95 and 50/50, v/v) and acetonitrile/phosphate buffer, pH 7.0 (5/95, v/v). The volume injected was 100 microlitre and the flow-rate 1 ml/min. The UV diode-array detector (DAD) was set at 213 nm. The results indicated the comparable efficiency (number of theoretical plates) of both columns. The advantage of octadecyl column over the cyano column was for 1 minute longer CA retention time under the same chromatographic conditions allowing a better resolution from chromatographic void volume interferences. However, CA dissolved in LC-grade water could be detected by UV(DAD) as a peak of good shape only if analysed on cyano column. For analysis on octadecyl column CA has to be dissolved in phosphate buffer in order to avoid the peak splitting caused by keto-enol tautomerism. The UV(DAD) response was linear for both columns at CA mass concentrations ranging from 0.2 microgram/ ml to 4.5 microgram/ml (i.e. from 20 ng to 450 ng of injected CA). The LiChrospher 100 CN column with acetonitrile/water (50/50, v/v) as the mobile phase was employed for LC-MS analysis of CA dissolved in water. The MS detector was a quadrupole-time of flight mass spectrometer (Q-TOF MS) using the electrospray ionisation in negative mode. CA was determined by selective monitoring of deprotonated molecule (m/z 128). The identity of the analyte was additionally confirmed by accurate mass measurements of deprotonated molecule using the Q-TOF MS equipped with lock-spray option. CA retention time (approx. 3 min) in the applied LC-MS system was nearly the same as in the HPLC-UV(DAD) system but the chromatographic peak shape was highly dependent on the sample volume injected. Acceptable injection volume, resulting in symmetric and sharp peaks, was up to 20 microlitres. The MS detector response was linear at CA mass concentrations ranging from 0.2 microgram/ml to 3.0 microgram/ml (i.e. from 4 ng to 60 ng of injected CA). The applicability of the LC-MS method for determination of CA in soil was tested by analysis of blank soil extracts spiked with CA at mass concentrations ranging from 0.25 microgram/ml to 2.5 microgram/ml. The blank soil extracts were prepared by ultrasonic extraction of four soils, differing in organic matter content and other pedological properties, with methanol or with 2:1 acetone:n-hexane mixture. The greatest influence of soil matrix in both extraction solvents on MS response was observed at the lowest CA mass concentration level. Compared to the analysis of standards in LC-grade water the CA peak area in soil extracts was reduced for 20 % to 35 %. At higher concentrations the difference in the chromatographic response to CA in water standards and extracts was less than 10 %. To determine the extraction recovery of CA from soil, an acidic (pH 3.95) forest soil with 4.6 % of organic matter was spiked with CA at 15 mg kg− 1 level and ultrasonically extracted with methanol and 2:1 acetone:n-hexane mixture. The extracts were evaporated and the dry residue was redissolved in water for LC-MS analysis. Interference-free chromatograms were obtained due to the high ion selectivity of Q-TOF MS system. CA extraction recoveries achieved with two different extraction solvents were comparable: 67 % (RSD 4 %) with methanol and 76 % (RSD 7 %) aceton:n-hexane mixture. Any attempt to determine CA extracted from soil by HPLC-DAD without additional extract purification failed because of coeluting interferences originating from soil matrix. [1] R. Cantú, O. Evans, F. K. Kawahara, J. A. Shoemaker, A. P. Dufour, Anal. Chem. 72 (2000) 5820-5828. [2] R. Cantú, O. Evans, F. K. Kawahara, L. J. Wymer, A. P. Dufour, Anal. Chem. 73 (2001) 3358-3364. [3] M. L: Magnuson, C.A. Kelty, R. Cantú, J. Am. Soc. Mass Spectrom. 12 (2001) 1085-1091. [4] Y. C. Fiamegos, C. N. Konidari, C. D. Stalikas, Anal. Chem. 75 (2003) 4034-4042.

cyanuric acid; soil; HPLC analysis; electrospray ionisation; Q-TOF MS

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