Purification and Characterization of hydroxytyrosol

Hydroxytyrosol is a phenolic compound with multiple biological activities, commonly found in olive oil and olive leaves. HPLC-MS (high-performance liquid chromatography-mass spectrometry) and nuclear magnetic resonance (NMR) are important methods for identifying the purity and structure of hydroxytyrosol. The following is a detailed introduction:

I. HPLC-MS Identification Method

1. Sample Preparation

The hydroxytyrosol sample to be purified is dissolved in a suitable solvent (such as methanol or acetonitrile) to prepare a solution of appropriate concentration, typically in the range of 1–10 mg/mL, to ensure suitable peak area and signal intensity in HPLC detection.

2. HPLC Analysis

Chromatographic Column Selection: Reverse-phase columns such as C18 columns are usually selected, which have good separation performance and can effectively separate hydroxytyrosol and its possible impurities.

Mobile Phase Preparation: Common mobile phase systems consist of water and organic solvents (such as acetonitrile or methanol). An appropriate amount of acid (such as formic acid or acetic acid) can be added to improve peak shape and separation efficiency. For example, mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is 0.1% formic acid acetonitrile solution. The gradient elution program can be optimized according to the sample.

Detection Conditions: Set suitable flow rate (generally 0.8–1.2 mL/min), column temperature (usually 25–35°C), and detection wavelength (hydroxytyrosol has a characteristic absorption at about 280 nm, which can be selected for detection).

Separation and Purity Judgment: Hydroxytyrosol chromatographic peaks are obtained through HPLC separation. The purity of the sample is judged based on the purity and symmetry of the peaks. If the peaks are sharp and symmetrical with no obvious impurity peaks, the sample purity is high. Meanwhile, the retention time is recorded as a reference for subsequent mass spectrometry analysis.

3. MS Analysis

Ion Source Selection: Common ion sources include electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). ESI is more commonly used and suitable for analyzing polar and thermally unstable compounds. Hydroxytyrosol has good polarity and is suitable for ESI ion sources.

Mass Spectrometry Parameter Setting: Set appropriate parameters such as capillary voltage, cone voltage, and ion source temperature. For example, the capillary voltage is generally 3–5 kV, the cone voltage is 20–50 V, and the ion source temperature is 100–150°C.

Molecular Weight Determination: The molecular weight of hydroxytyrosol is determined through the mass spectrum. In positive ion mode, hydroxytyrosol produces a [M + H]⁺ peak, and the molecular weight is calculated based on the mass-to-charge ratio (m/z) of this peak. The theoretical molecular weight is 154, and if the detected [M + H]⁺ peak has an m/z of 155, it matches the molecular weight characteristics of hydroxytyrosol.

Fragment Ion Analysis: Further analysis of fragment ion information in the mass spectrum helps determine the structure of hydroxytyrosol. By analyzing the m/z and relative abundance of fragment ions, the breaking mode of chemical bonds and the presence of functional groups in the molecule can be inferred.

II. Nuclear Magnetic Resonance (NMR) Identification Method

1. Sample Preparation

The purified hydroxytyrosol sample is dissolved in a suitable deuterated solvent, such as deuterated chloroform (CDCl₃) or deuterated methanol (CD₃OD). The sample concentration is generally 10–50 mg/mL to ensure clear signals in the NMR spectrum.

2. Proton Nuclear Magnetic Resonance (¹H-NMR) Analysis

Instrument Parameter Setting: Set appropriate parameters such as pulse sequence, number of scans (generally 16–64 times), and sampling time.

Chemical Shift Analysis: Observe the chemical shift values in the ¹H-NMR spectrum. Hydrogen atoms at different positions in the hydroxytyrosol molecule have different chemical shifts. For example, the chemical shift of hydrogen atoms on phenolic hydroxyl groups is usually between 4–12 ppm, and that of methyl hydrogen atoms is generally between 0.8–2.5 ppm. The structure of hydroxytyrosol is preliminarily judged by comparing with the chemical shift data of known compounds.

Peak Splitting and Coupling Constants: Analyze the splitting pattern and coupling constants of peaks. The coupling effect between different types of hydrogen atoms leads to peak splitting, and the number and spatial relationship of adjacent hydrogen atoms can be inferred from the splitting pattern and coupling constants. For example, a doublet with a coupling constant of approximately 7 Hz usually indicates two hydrogen atoms on an adjacent carbon atom.

3. Carbon Nuclear Magnetic Resonance (¹³C-NMR) Analysis

Instrument Parameter Setting: Similarly, set appropriate parameters such as pulse sequence and number of scans.

Chemical Shift Analysis: Observe the chemical shift values in the ¹³C-NMR spectrum. Carbon atoms at different positions in the hydroxytyrosol molecule have different chemical shifts. For example, the chemical shift of carbonyl carbon atoms is generally between 150–200 ppm, and that of methyl carbon atoms is between 0–50 ppm. The structure of hydroxytyrosol is further confirmed by comparing with the chemical shift data of known compounds.

DEPT Experiment: The DEPT (distortionless enhancement by polarization transfer) experiment can be performed to distinguish different types of carbon atoms (such as CH₃, CH₂, CH, C). The connection mode and chemical environment of carbon atoms in the molecule can be more accurately determined through the DEPT spectrum.

4. Two-Dimensional Nuclear Magnetic Resonance (²D-NMR) Analysis

COSY (Correlation Spectroscopy): The COSY spectrum shows the spin-spin coupling relationship between hydrogen atoms in the molecule, helping to determine the connection mode of adjacent hydrogen atoms. The coupling network of hydrogen atoms in the molecule can be inferred by analyzing the cross-peaks in the COSY spectrum.

HSQC (Heteronuclear Single Quantum Coherence): The HSQC spectrum combines information from ¹H-NMR and ¹³C-NMR to determine the corresponding relationship between hydrogen atoms and adjacent carbon atoms. The HSQC spectrum can accurately correlate the hydrogen atom signals in the ¹H-NMR spectrum with the carbon atom signals in the ¹³C-NMR spectrum.

HMBC (Heteronuclear Multiple-Bond Correlation): The HMBC spectrum detects the coupling relationship between hydrogen atoms and carbon atoms separated by two or three bonds, helping to determine the long-range structure of the molecule and the connection mode of functional groups. The HMBC spectrum can further confirm the positions and connection order of various functional groups in the hydroxytyrosol molecule.