**4. Methods for the determination of drug residues in honey: analytical techniques**

Until early 2000s, LC-UV-Vis and LC-FLD were the most used equipments to detect residues in food. UV-Vis detectors measure solute analytes by their absorbance in the ultraviolet or visible region. A UV detector employs a deuterium discharge lamp (D2 lamp) as a light source, with the wavelength of its light ranging from 190 to 380 nm. If substances are to be detected at longer wavelengths, that is, in the visible region (380–700 nm), a UV-VIS detector is used with an additional tungsten lamp (W lamp). Nowadays, photodiode arrays and DAD (semiconductor devices) have replaced UV-Vis detectors, and its use is mandatory to definitively confirm the presence of residues of permitted veterinary drugs in food [6]. A DAD detects the absorption in UV to VIS region. While a UV-VIS detector has only one sample-side light-receiving section, a DAD allows the acquisition of full wavelength spectrum at one time thanks to multiple photodiode arrays. Spectra are measured at regular intervals (one second or less) during the LC separation with continuous eluate delivery. Therefore, to identify a compound, in addition to the retention time, DAD enables the comparison between the spectrum of the authentic standard and of the analyte. It is important to underline that according to Commission Decision 2002/657/EC, only the coupling between LC and DAD (not between LC and UV-Vis) allows the definitive confirmation of residues of permitted substances in food.

Fluorescence detectors have greater sensitivity and selectivity over the UV-Vis ones. This is an advantage for the measurement of specific fluorescent species in samples; however, only about 15% of all compounds have a natural fluorescence. Compounds having specific functional groups are excited by shorter wavelength energy and emit higher wavelength radiation. This phenomenon is called fluorescence. Generally, the presence of aromatic conjugated pi-electrons produces the most intense fluorescent signal. Most unsubstituted aromatic hydrocarbons fluoresce with quantum yield increasing with the number of rings, their degree of condensation and their structural rigidity. In addition, aliphatic and alicyclic compounds with carbonyl groups and substances with highly conjugated double bonds fluoresce, but usually to a lesser extent. Among veterinary drugs, quinolones possess native fluorescence; some other antibiotic classes can be efficiently derivatized to give fluorescent compounds (e.g. sulphonamides and aminoglycosides).

For the analysis of residues in food, nowadays, LC-MS is the standard internationally accepted technology already available in most laboratories that is capable of providing structural information about the analytes. Different mass spectrometer platforms have been successfully employed for the analysis of veterinary drugs in honey [14]. Since early 2000s, triple quadrupole mass spectrometer (LC-MS/MS) platform has been introduced in routine worldwide laboratories, and at present, this MS technology is the gold standard for routine analysis of complex sample extracts. The LC-MS/MS, also known as LC-QqQ, is a tandem MS technique in which the first and third quadrupoles act as mass filters and the second, a radiofrequency-only quadrupole, fragments the analyte through interaction with a collision gas. The most used acquisition mode is multiple reaction monitoring (MRM). Increased selectivity, improved signal-to-noise ratio (S/N), lower limits of quantitation, wider linear range and improved accuracy are some of the benefits of this technique. LC-MS/MS instrumentation tends to give better quantitative precision and improved sensitivity than alternative configurations, making it a superior choice for routine analysis of specific targeted contaminants.

An alternative to LC-MS/MS system is the coupling of liquid chromatography with high-resolution mass spectrometry (LC-HRMS). At the beginning, these analysers were mainly used for research purposes, but after 2007 they started to be applied in routine analysis, too. With HRMS analysers, full-scan spectra are continuously obtained throughout the analytical run allowing for exhaustive qualitative post-acquisition analysis. There are two technologies of high-resolution mass spectrometry: time-of-flight (TOF) and orbitrap. However, single-stage high-resolution mass spectrometry demonstrated to not be suitable for the confirmation of residues at very low concentrations in highly complex matrices such as honey. More recently, hybrid platforms have been available at the bench level such as Q-TOF and Q-Orbitrap combining a quadrupole with an accurate mass analyser. These configurations provide exceptional selectivity and sensitivity over single-stage equipment, and they are increasingly applied in residues analysis of food.

With regard to the chromatographic separation, although the coupling between gas chromatography and mass spectrometry (GC-MS) has been realized before LC-MS, gas chromatography is rarely used for the determination of antibiotics, due to their polar nature, low volatility and thermal instability. Therefore, high-performance liquid chromatography (HPLC) is the technique of choice for antibiotic analysis. Since its introduction in 1970s, HPLC progressively improved mainly thanks to the evolution of packing materials used to carry out the separation. Columns packed with 10 and 5 μm fully porous particles dominated the field for nearly thirty years (1975– 2000). In 2004, a great advance in instrumentation and column technology was made achieving very significant increases in resolution, speed and sensitivity. Columns with smaller particles (sub 2-μm) and instrumentation able to deliver mobile phase at 15,000 psi (1000 bar) allowed the achievement of a new level of performance. This new step of HPLC is known as UHPLC technology. In 2007, LC columns with core-shell (superficially porous) particles were introduced. This new generation of microspheres provides the same high efficiency of sub 2-μm UHPLC totally porous particles, but with lower backpressures. The first commercially available coreshell sorbent was the Halo® from Advanced Material Technologies. Currently, the most applied core-shell columns are Kinetex® (Phenomenex), Poroshell® (Agilent), Accucore® (Thermo Fisher Scientific), Ascentis Express® (Supelco), Cortecs® (Waters) and Nucleoshell® (Macherey Nagel). Many of these have been used to determine residues in honey (see **Tables 3**–**11**).
