Residual detection method of hydroxytyrosol

Hydroxytyrosol, a functional component in plant-derived foods like olive oil, its residue detection holds significant importance in food quality control and biomedical research. LC-MS/MS (liquid chromatography-tandem mass spectrometry), with its high sensitivity and specificity, has become the core technology for quantifying hydroxytyrosol in foods and plasma. The detection method system is as follows:

I. Detection Principle and Technical Advantages

LC-MS/MS achieves precise analysis of hydroxytyrosol through "liquid chromatography separation + tandem mass spectrometry qualitative and quantitative analysis":

Chromatographic separation: Utilizing the polar characteristics of hydroxytyrosol, reverse-phase chromatographic columns (such as C18, C8) are employed for gradient elution in an acidic mobile phase (e.g., 0.1% formic acid aqueous solution-acetonitrile system) to separate the target substance from matrix interference components.

Mass spectrometry detection: In the electrospray ionization (ESI) mode, hydroxytyrosol easily forms [M-H]- negative ions. Tandem mass spectrometry is used to select parent ions (m/z 153) and characteristic fragment ions (such as m/z 135, m/z 93) for multiple reaction monitoring (MRM), and quantitative analysis is realized by combining retention time and ion pair intensity.

Technical advantages: Compared with the traditional HPLC-UV method, the detection limit of LC-MS/MS can be reduced to the ng/mL level, and it can exclude the interference of structural analogs (such as tyrosol, flavonoids) in the matrix, which is especially suitable for the detection of trace hydroxytyrosol in complex biological matrices such as plasma.

II. LC-MS/MS Quantitative Process of Hydroxytyrosol in Foods

1. Sample Pretreatment: From Matrix Separation to Target Enrichment

Oily foods such as olive oil:

Liquid-liquid extraction (LLE) or solid-phase extraction (SPE) is used to remove oil interference: for example, after degreasing with n-hexane, hydroxytyrosol is extracted by ultrasonic treatment with methanol-water (5:1, v/v), purified by a C18 SPE column (eluent is 50% methanol aqueous solution), dried under nitrogen, and redissolved with the mobile phase.

Polar matrices such as fruit juices and dairy products:

Macromolecular impurities are directly removed by centrifugation or ultrafiltration, extracted with ethyl acetate under acidic conditions (pH 3-4), or the target substance is adsorbed by a solid-phase extraction column (such as Oasis HLB), eluted with methanol-water (containing 0.1% formic acid), and concentrated.

Key control points: For oily samples, attention should be paid to the degreasing efficiency to avoid the inhibition of mass spectrometry ionization by residual lipids; acidic conditions can prevent the dissociation of phenolic hydroxyl groups of hydroxytyrosol and improve the extraction recovery rate (usually, the recovery rate needs to be verified to be in the range of 80%-120%).

2. Optimization of Chromatographic and Mass Spectrometry Conditions

Chromatographic conditions:

Column temperature 30-40℃, flow rate 0.3-0.5 mL/min, mobile phase gradient such as acetonitrile increasing from 5% to 25% in 0-5 min, and increasing to 40% in 5-10 min, ensuring that the retention time of hydroxytyrosol is about 8-10 min (effectively separated from impurities).

Mass spectrometry parameters:

ESI negative ion mode, spray voltage -3.5 kV, drying gas temperature 350℃, sheath gas pressure 35 arb, MRM ion pairs select m/z 153→135 (quantitative ion) and m/z 153→93 (qualitative ion), and the collision energies are set to 10 eV and 20 eV respectively.

3. Quantitative Methods and Quality Control

The isotope internal standard method (such as deuterated hydroxytyrosol - d3) is used to correct the matrix effect. The standard curve range is usually 0.1-100 ng/mL, and the linear correlation coefficient R²>0.995.

Each batch of samples should include blank matrix, standard solution, and spiked samples (low, medium, and high concentrations) to verify the precision (RSD<10%) and accuracy (recovery rate 85%-115%) of the method.

III. LC-MS/MS Detection Strategy of Hydroxytyrosol in Plasma

1. Biological Matrix Pretreatment: Anti-Interference and Stability Control

Sample collection and preservation: Plasma samples need to add anticoagulants (such as sodium heparin), immediately centrifuge after collection (4℃, 3000 g, 10 min), and store the supernatant at -80℃ to avoid oxidation of hydroxytyrosol caused by repeated freezing and thawing.

Pretreatment methods:

Protein precipitation method: Add 3 times the volume of acetonitrile (containing 0.1% formic acid) to the plasma, vortex, centrifuge (14000 g, 15 min), take the supernatant, dry under nitrogen, redissolve with the mobile phase, and filter through a 0.22 μm membrane.

Solid-phase extraction method: For low-concentration samples, a weak anion exchange SPE column (such as Oasis WAX) is used for enrichment, loaded under acidic conditions, eluted with ammoniated methanol, and the detection limit can be reduced to below 0.5 ng/mL.

Antioxidant measures: 0.1% ascorbic acid or BHT is added during the pretreatment process to prevent the degradation of hydroxytyrosol under oxidative conditions.

2. Mass Spectrometry Detection and Quantitative Optimization

Endogenous substances (such as amino acids, fatty acids) in the plasma matrix easily interfere with the detection. It is necessary to optimize the chromatographic separation conditions (such as extending the gradient elution time to 15-20 min) and improve the specificity of mass spectrometry scanning (such as increasing the collision energy to screen characteristic fragments).

Quantification uses the standard addition method or isotope internal standard method. The standard curve range is usually 1-50 ng/mL, which is suitable for the pharmacokinetic study of hydroxytyrosol in plasma after oral administration of olive oil (such as the determination of peak concentration Cmax and elimination half-life t1/2).

IV. Method Applications and Challenges

Applications:

Food field: LC-MS/MS can be used for the authenticity identification of olive oil (detecting the content of hydroxytyrosol to evaluate the quality of extra virgin olive oil) and the stability monitoring of hydroxytyrosol in functional foods (such as the degradation law after adding to beverages and health products).

Medical research: Through the analysis of plasma hydroxytyrosol concentration, the bioavailability after dietary intake can be evaluated, and the association between its physiological activities such as antioxidant and anti-inflammatory and disease prevention (such as epidemiological studies of cardiovascular diseases and cancer) can be explored.

Existing challenges:

The concentration of hydroxytyrosol in plasma is extremely low (usually <10 ng/mL), and it is necessary to further optimize the pretreatment efficiency to reduce the matrix effect.

The removal of interfering substances in complex food matrices (such as fermented foods and deep-processed products) is still a difficulty in method development. In the future, two-dimensional liquid chromatography (2D-LC) or ultra-high performance liquid chromatography (UPLC) can be combined to improve the separation efficiency.