Determination of Caffeine in Honey by HPLC

• Analyte: Caffeine
• System: EClassical 3200 HPLC System with UV Detector
• Column: SinoChrom ODS-BP (5 μm, 4.6 × 250 mm)
• Highlight: Reliable HPLC-UV method for caffeine analysis in honey

Honey is a natural health food rich in carbohydrates and various bioactive compounds. Caffeine, a central nervous system stimulant, is sometimes found in honey and is regulated as a doping substance by the International Olympic Committee. Therefore, monitoring caffeine levels in honey is important for quality and safety. 

Standards: Caffeine reference standard (≥99%)

Reagents:Acetonitrile (HPLC grade), glacial acetic acid (analytical grade), sodium hydroxide solution (analytical grade), chloroform (analytical grade), deionized water (18.2 MΩ·cm). 

Stock solution (1 mg/mL):Accurately weigh 0.1 g (to 0.0001 g) of caffeine standard, dissolve in water, and dilute to 100 mL in a volumetric flask.

Working standards: Dilute the stock solution with water to appropriate concentrations as needed for calibration (e.g., 0.5, 1.0, 2.0, 5.0, 10.0 μg/mL). Prepare fresh before use.

1. Accurately weigh 20 g (to 0.01 g) of honey sample into a 50 mL centrifuge tube.
2. Add 10 mL of sodium hydroxide solution and mix well.
3. Add exactly 20.0 mL of chloroform, cap the tube, and vortex mix for 5 min.
4. Centrifuge at 2000 r/min for 5 min. Discard the upper aqueous layer.
5. Wash the organic phase twice with 5 mL of sodium hydroxide solution, discarding the aqueous layer each time.
6. Mix the organic phase and filter through dry filter paper, discarding the first 3 mL of filtrate.
7. Collect 10.0 mL of the filtrate and evaporate to near dryness under a stream of air at 45°C.
8. Dissolve the residue in exactly 2.0 mL of water and filter through a 0.45 μm membrane filter before HPLC analysis..

HPLC System: EClassical 3200 equipped with P3200 high-pressure pump, D3210 UV-Vis detector, S3210 autosampler, O3220 column oven, T3200 solvent bottle tray, Kromstation CDS.

Pretreatment equipment: Centrifuge, rotary evaporator, nitrogen evaporator, vortex mixer, analytical balance, filtration apparatus, etc.

Column:              SinoChrom ODS-BP column (5 μm, 4.6 × 250 mm)

Mobile Phase:     Acetonitrile / Water / Acetic acid = 16 / 83 / 1 (v/v/v)

Flow Rate:          1.0 mL/min

Detection:           UV at 273 nm

Injection Vol.:     20 μL

Column Temp.:   40°C

A typical chromatogram of a honey sample containing caffeine is shown in Figure 1. The peak shape and retention time are suitable for quantification.

Fig. 1. chromatogram of a honey sample