2 . Experimental
2 .1. Reagents and chemicals
All chemicals used were of analytical grade unless stated otherwise. Acetic acid, acetonitrile, ascorbic acid, citric acid monohydrate, lead(IV)oxide, di-so-dium hydrogen phosphate dihydrate, were purchased from Merck (Darmstadt, Germany). Ammonium hydroxide 25% (m/v), dichloromethane, methanol (HPLC grade), sodium acetate, sodium perchlorate monohydrate, para-toluenesulfonic acid ( p-TSA) were obtained from J.T. Baker (Phillipsburg, NJ, USA). Brilliant green and N,N,N9,N9-tetramethyl-1,4-phenylenediamine dihydrochloride (TMPD) were from Aldrich (Steinheim, Germany), and celite from Acros (Geel, Belgium). N,N-Dimethylformamide
was bought from Rathburn (Walkerburn, UK). Leuco-malachite green and 1-pentanesulfonic acid
(sodium salt) were delivered by Sigma (St. Louis,MO, USA) and malachite green oxalate (Vetrenal
reference standard) by Riedel-de-Hae¨n (Seelze, Ger-many). Aromatic sulfonic acid-bonded SPE columns (3 ml; 500 mg) were purchased from J.T. Baker. Water was of Milli-Q quality (Millipore, Bedford,USA).
2 .2. Apparatus
Screw-capped polypropylene tubes (50 ml and 6 ml) were supplied by Sarstedt (Numbrecht, Germany). Series of SPE columns equipped with 75-ml reservoir adapters were processed simultaneously using a vacuum manifold (J.T. Baker). Samples were dried using an evaporation manifold from Pierce (Rockford, IL, USA).
2 .5. LC–MS–MS analysis
For LC–MS–MS analysis, 10-ml aliquots were injected on an HPLC system consisting of two HPLC pumps (PE200 series, Applied Biosystems), an autowas sampler (PE 200 series, Applied Biosystems) and an API-365 MS detector (Applied Biosystems). The LC–MS–MS was controlled by Analyst software package (version 1.1). The HPLC column was a Phenomenex Luna C (5032 mm, Phenomenex, Torrance, USA) with guard column (SecurityGuard C18 4*2 mm, Phenomenex). The mobile phase was a mixture of 50 mM ammonium acetate, pH 4.4, and acetonitrile (2:3, v/v) flowed at 200 ml /min. Before the eluent was introduced into the ionization chamber for MS detection of the analytes, the effluent was passed through a post-column reactor filled with lead(IV)oxide and celite (see Section 2.4).
The MS was equipped with an ESI interface operating at an ionization voltage of +5500 V and a
source temperature of 400 8C. The entrance, declustering and focusing potentials were set at 29, 40 and 180 V, respectively. Tandem MS analysis was performed using the multi-reaction-monitoring (MRM) mode. Collision energy (CE) was optimized for each product-ion trace measured. The following traces were monitored: m/z 329.3→m/z 165.0 (CE 75 V), m/z 329.3→m/z 208.0 (CE 55 V), m/z 329.3→m/z 313.3 (CE 45 V) and for the internal standard m/z 385.0→m/z 341.0 (CE 50 V) (Fig. 1).