Toward a Harmonized and Standardized Protocol for the Determination of Total Hydroxytyrosol and Tyrosol Content in Virgin Olive Oil (VOO). The Pros of a Fit for the Purpose Ultra High Performance Liquid Chromatography (UHPLC) Procedure

Abstract

Τoward a harmonized and standardized procedure for the determination of total hydroxytyrosol and tyrosol content in virgin olive oil (VOO), the pros of a recently published in house validated ultra high performance liquid chromatography (UHPLC) protocol are discussed comparatively with those of other procedures that determine directly or indirectly the compounds hosted under the health claim on "olive oil polyphenols" (EC regulation 432/2012). Authentic VOOs were analyzed with five different liquid chromatographic separation protocols and ¹H-NMR one in five different laboratories with expertise in VOO phenol analysis within three months. Data comparison indicated differences in absolute values. Method comparison using appropriate tools (Passing-Bablok regression and Bland Altman analyses) for all protocols vs. the UHPLC one indicated slight or statistically significant differences. The results were also discussed in terms of cost effectiveness, detection means, standard requirements and ways to calculate the total hydroxytyrosol and tyrosol content. Findings point out that the in-house validated fit for the purpose UHPLC protocol presents certain pros that should be exploited by the interested parties. These are the simplicity of sample preparation, fast elution time that increase the number of samples analyzed per day and integration of well-resolved peaks with the aid of only two commercially available external standards. Importance of correction factors in the calculations is stressed.

Keywords: 1H-NMR spectroscopy; European Commission Regulation 432/2012; HPLC-DAD; LC-HRMS; UHPLC-DAD; health claim; hydroxytyrosol; standardization; tyrosol; virgin olive oil.

Figure 1

Profile at 280 nm of S-26 VOO polar fraction (PF) prior (a,c) and after (b,d) acidic hydrolysis using protocol A (a,b) and protocol B (c,d).

Figure 2

Profile at 280 nm of S-26 VOO PF using UHPLC-diode array detection (DAD; protocol C; 1: Htyr, 2: Tyr, 3: Vanillic acid, 4: Caffeic acid, 5: Syringic acid (IS), 6: Vanillin, 7: p-coumaric acid, 9: Ferulic acid, 11a: Decarboxymethyl oleuropein aglycone, oxidized dialdehyde form, 12: Decarboxymethyl oleuropein aglycone, dialdehyde form, 14: Oleuropein aglycone, dialdehyde form, 16a: Decarboxymethyl ligstroside aglycone, oxidized dialdehyde form 17: Decarboxymethyl ligstroside aglycone, dialdehyde form, 18: Pinoresinol, 1-acetoxy-pinoresinol; 20: Ligstroside aglycone, dialdehyde form, Lu: Luteolin, 23: Oleuropein aglycone, aldehyde and hydroxylic form, Api: Apigenin, 27: Ligstroside aglycone, aldehyde and hydroxylic form).

Figure 3

Profile at 278 nm of S-26 VOO PF using HPLC-DAD (protocol D) (peak numbers: 1: Htyr, 2: Tyr, 3: Decarboxymethyl oleuropein aglycone, dialdehyde form, 3′: Other structure of decarboxymethyl oleuropein aglycone, dialdehyde form, 4: Decarboxymethyl ligstroside aglycone, dialdehyde form, 4′: Other structure decarboxymethyl ligstroside aglycone, dialdehyde form, 5: Oleuropein aglycone, 6: Ligstroside aglycone).

Figure 4

Extracted ion chromatograms in negative ionization mode (protocol E) for molecular ions of a: Htyr, b: Decarboxymethyl oleuropein aglycone oxidized dialdehyde form, c: Decarboxymethyl oleuropein aglycone, d: Oleuropein aglycone dialdehyde form (sum of isomers), e: Oleuropein aglycone aldehyde and hydroxylic form (sum of isomers), f: Decarboxymethyl ligstroside aglycone, g: Ligstroside aglycone dialdehyde form (sum of isomers), h: Ligstroside aglycone aldehyde and hydroxylic form (sum of isomers), i: Oleuropein aglycone oxidized aldehyde and hydroxylic form (sum of isomers) in S-26 VOO PF by liquid chromatography-heated electrospray ionization-high resolution mass spectrometry (LC-HESI-HRMS).

Figure 5

¹H-NMR spectra (400 MHz, CD₃CN, aldehyde region) from top to bottom: Decarboxymethyl oleuropein aglycone dialdehyde form (oleacein), decarboxymethyl ligstroside aglycone dialdehyde form (oleocanthal), sample S-26.