2.4. Amino Acid and Carbohydrate Analysis

Amino acids and carbohydrates were analyzed using high performance liquid chromatography (HPLC) from Agilent Technologies 1260 Series provided with an Agilent 1260 Infinity Quaternary Pump (G1311C, Agilent Technologies, Böblingen, Germany), an Agilent 1260 Infinity Standard Autosampler (G1329B), and an Agilent 1260 Infinity Thermostatted Column Compartment (G1316A), to maintain the temperature for amino acids at 40 °C and for carbohydrates at 30 °C.

Amino acids were separated on a Zorbax Extend-C18 column (3.0 × 150 mm, 3.5 µm, Agilent Technologies, Böblingen, Germany), preceded by a guard column Zorbax Extend-C18 (2.1 × 12.5 mm, 5 µm, Agilent Technologies, Böblingen, Germany), and were detected by an Agilent 1260 Infinity System Diode Array Detector (DAD, G4212B) with a flow rate of 1 mL min⁻¹. Prior to injection, amino acids were derivatized with either ortho-phthalaldehyde (OPA, Agilent Technologies, Böblingen, Germany, for primary amino acids: alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tyrosine, and valine) or 9-fluorenylmethyl chloroformate (FMOC, Agilent Technologies, Böblingen, Germany, for proline) [54,55,56]. Amino acids were separated by a solvent gradient with a buffer (1 L ultra-pure water, 10 mM Na₂HPO₄, 10 mM Na₂B₄O₇, 0.5 mM NaN₃, pH 8.2) used as polar phase and acetonitrile-methanol-water (45%:45%:10% (v/v), all CHROMASOLV^®, Sigma-Aldrich Chemie GmbH, Munich, Germany) used as non-polar phase [54,55]. We started with a 2%:98% non-polar to polar phase, then gradually changed the ratio to 57%:43% for 13 min, until finally increasing the non-polar phase to 100% for a period of 2 min, followed by a re-equilibration to 2%:98% non-polar to polar phase for about 9 min [54]. Solvent flow rate was 0.750 mL min⁻¹ [54].

Carbohydrates were separated on a NH2 column (Zorbax: 4.6 × 250 mm, 5 µm, Agilent Technologies) preceded by a NH2 guard column (Zorbax: 4.6 × 12.5 mm, 5 µm, Agilent Technologies) under isocratic conditions using an elution buffer with 78%:22% (v/v) acetonitrile and ultra-pure water and a flow rate of 1.5 mL min⁻¹. Carbohydrates were detected by a refractive index detector (RID, Agilent 1260 Infinity, G1362 A) [57].

Four different concentrations of a standard comprising 17 amino acids (Amino Acid Standard solution, Sigma-Aldrich Laborchemikalien GmbH, Hannover, Germany) or three carbohydrates (sucrose, fructose, and glucose, HPLC grade, Sigma-Aldrich Laborchemikalien GmbH, Hannover, Germany) were run every five samples as an external reference. All amino acids and carbohydrates in nectar samples were identified based on standard reference compounds. HPLC control and compound quantification was carried out with Agilent ChemStation for LC 3D systems (Agilent Technologies, Böblingen, Germany).