Development and Validation of a Novel GC–MS Method for the Determination of Flavonoids in mallorcan Carob (Ceratonia siliqua) Honey

Carob (Ceratonia siliqua) honey from the Balearic Islands is a unique monofloral product recognized for its sensory properties and rich content in bioactive compounds. Despite its growing economic and nutraceutical importance, the compositional profile of this honey—especially its flavonoid content—remains largely unexplored. In the present study, we report the development and validation of a novel gas chromatography–mass spectrometry (GC–MS) method specifically optimized for the sensitive and selective quantification of flavonoid compounds in Mallorcan carob honey.

The analytical workflow includes liquid–liquid extraction with ethyl acetate, derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in the presence of pyridine as a catalyst, and subsequent chromatographic separation coupled with mass spectrometric detection. Derivatization conditions—particularly the volume ratio of BSTFA to pyridine, reaction temperature, and time—were optimized using a multivariate experimental design to maximize the yield and stability of trimethylsilyl derivatives. This chemometric approach enabled efficient silylation of structurally diverse flavonoids while minimizing thermal degradation and byproduct formation.

A total of 22 analytes, including quercetin, kaempferol, luteolin, apigenin, and pinocembrin, were successfully separated within a 17-minute analytical window. Method validation demonstrated excellent linearity (R² > 0.995), low detection limits (LODs: 5–30 ng/mL), and high repeatability (RSD < 5%).

The optimized GC–MS protocol was complemented by total polyphenol content (TPC) and total flavonoid content (TFC) spectrophotometric assays, alongside three antioxidant activity assays—FRAP, CUPRAC, and ABTS—providing a comprehensive evaluation of the honey’s polyphenolic and antioxidant profile. This is the first comprehensive application of GC–MS for flavonoid profiling in Mallorcan carob honey, offering a strong analytical basis for compositional authentication and valorization of this regional product.