**Nomenclatures**

#### **Abbreviations**

**5.10. Multiclass methods**

354 Honey Analysis

In efforts to increase the cost-effectiveness of antimicrobial residue enforcement programmes, the development of analytical methods able to detect as many contaminant compounds as possible is highly preferred. However, it is well known that one of the difficulties in the development of these procedures is the incompatibility of selective sample treatments with acceptable accuracies for a wide range of analytes. Therefore, only a generic purification protocol such as liquid-liquid extraction or reversed-phase solid-phase extraction is achievable (**Table 11**). Since generally reversed-phase sorbents provide the least selective retention mechanism when compared to normal phase or ion exchange ones, they allow the most universal solid-phase extraction approach retaining most molecules with any hydrophobic character.

There are some considerations to do before to take on multiclass methods for antibiotics: (i) the extraction of nitrofuran metabolites requires acid hydrolysis and derivatization steps that would be destructive to other analytes of interest. Therefore, this class should be extracted apart from a multiclass method to obtain satisfactory recovery and avoid degradation of acidlabile compounds; (ii) as mentioned before, highly polar compounds, such as aminoglycosides, do not perform well in multiclass methods as they are relatively insoluble in organic solvents and exhibit little or no affinity for non-polar stationary phases used in RP-LC.LC. For this reason, in **Table 11** only two papers include aminoglycosides among the determined classes adding an ion-pairing reagent in the mobile phases; (iii) in addition, in honey, the determination of sulphonamides in honey requires a preliminary hydrolysis step to measure residues bound to sugars, and therefore, also in this case, acid-sensitive antibiotics can be destroyed.

In this context, "multiclass" are procedures involving the determination of more than two drug classes. Probably, the first multiclass method in honey has been published in 2004 by Kaufmann et al. [89], reporting the determination of three antibiotic families, including sixteen sulphonamides together with three tetracyclines and flumequine, a quinolone antibiotic for which until now there is no evidence of use in apiculture. In 2008, Hammel et al. [90] developed an LC-MS/MS protocol for 42 substances including five tetracyclines, seven macrolides, three aminoglycosides, eight beta-lactams, two amphenicols and seventeen sulphonamides. Four subsequent liquid-liquid extraction steps were necessary to adequately extract all the analytes. After this paper, many confirmatory multiclass methods have been published mainly applying triple quadrupole platforms [29, 91–98]. This is in accordance with the general trend in analysis of residues in food started in the late 2000s. Although triple quadrupoles have been introduced in the mid-to late-1990s, only in recent years these equipments have improved their electronics enabling the possibility of acquiring

The performances of an analytical method are mainly determined by the applied sample preparation and instrumental technique. The coupling honey-antibiotic (matrix-analyte) can be a "case study" to discuss the general strategies of developing methods for trace

dozens of compounds in the same chromatographic run.

**6. Conclusions**


