TECHNICAL NOTE Cryogenic grinding pre-treatment improves extraction efficiency of fluoroquinolones for HPLC-MS/MS determination in animal tissue Manuel Lolo & Sandra Pedreira & Beatriz I. Vázquez & Carlos M. Franco & Alberto Cepeda & Cristina A. Fente Received: 6 October 2006 / Revised: 5 December 2006 / Accepted: 11 December 2006 / Published online: 3 February 2007 # Springer-Verlag 2007 Abstract An efficiency extraction of fluoroquinolones in chicken muscle was achieved by pulverizing it in a freezer mill before treatment with NaOH (10 mM)/MeCN (1:1). The improvement of cryogenic grinding in the extraction was demonstrated for the same piece (whole leg) of four chickens treated with enrofloxacin in equal doses. A confirmatory method based on high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/ MS) was used to analyze the extracts. The chromatographic separation was achieved in 5 min with a Synergi Fusion-RP 80A (50 x 2 mm, 4 μm) column filled with a hybrid polymer. The HPLC was coupled with a detector based in a quadrupole–linear ion trap Q-TRAP that allows a confir- matory detection according to the European legislation. The specificity of the method was assessed by testing a number of representative blank muscle samples (n = 10) to verify the absence of potential interfering compounds. The limits of detection and quantitation were 2 and 5 ng g -1 of quinolones in muscle samples, respectively. The chromato- graphic method was demonstrated to be linear for the range studied (5–500 ng g -1 ) with the P value for lack-of-fit in the ANOVA table greater or equal to 0.10 (calibration coefficient 0.9998 and 0.9996 for ciprofloxacin and enro- floxacin, respectively). The mean intra-day relative stan- dard deviation (RSD) (n = 6, c = 50ng g -1 ) was 6%; inter-day assay gave a RSD of 12%. The extraction and clean-up were carried out in one step with very satisfactory recovery data (between 65 and 101%). Keywords Cryogenic grinding . HPLC-MS/MS . Chicken . Food residues Introduction Quinolone and fluoroquinolone antimicrobial agents are highly effective in treating gram-positive, gram-negative, and micoplasma infections [1]. Quinolones have been used extensively in animal production, but the presence of residues of these compounds in meat, milk, or other animal foods could present a risk for human health. The European Union has established maximum residue limits (MRLs) in chicken muscle of 100 μg kg -1 for the sum of enrofloxacin and its principal metabolite ciprofloxacin [2]. Reviewing the recent methods proposed for the analysis of quinolones in foods [3–9], LC-MS is the technique most used. It must be noticed that most methods involve a preliminary extraction step followed by a later clean-up step. In our previous work we have developed methods for the analysis of enrofloxacin residues, in eggs with a diphasic-dialysis extraction and purification in one step [10], and in chicken muscle, raw and cooked, by means of a liquid extraction with acetonitrile [11]. Nevertheless, when calculating the effectiveness of the extraction procedures for residues in complex matrices like the muscle, the blank samples are overloaded with the analyte. However, it is impossible in real samples to ensure that all the analyte in contact with the matrix is removed because they have been incorporated “while alive” during the metabolization of drugs. For this reason we have decided to use a type of mill that pulverizes the samples, frozen with liquid nitrogen, so that complete rupture of the cells is obtained. Thus, in this paper we describe an extraction method for determination of Anal Bioanal Chem (2007) 387:1933–1937 DOI 10.1007/s00216-006-1090-1 M. Lolo : S. Pedreira : B. I. Vázquez : C. M. Franco : A. Cepeda : C. A. Fente (*) Laboratorio de Higiene, Inspección y Control de Alimentos, Área de Nutrición y Bromatología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain e-mail: cfente@lugo.usc.es