Optimisation of acid hydrolysis conditions of choline esters and mass spectrometric determination of total choline in various foods – PubMed Black Hawk Supplements

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Determining the content of the nutrient choline in foods and obtaining the required amount from the diet are crucial. One way to measure choline in foods is by converting choline esters to free choline via acid hydrolysis, followed by quantifying the total choline, as adopted by the AOAC method (AOAC-Choline); however, certain choline esters are difficult to hydrolyse. Here, we investigated various acid hydrolysis conditions to establish a reliable method for determining the total choline in…
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Optimisation of acid hydrolysis conditions of choline esters and mass spectrometric determination of total choline in various foods - PubMed

Optimisation of acid hydrolysis conditions of choline esters and mass spectrometric determination of total choline in various foods

Yoshinari Hirakawa et al. Sci Rep. .

Abstract

Determining the content of the nutrient choline in foods and obtaining the required amount from the diet are crucial. One way to measure choline in foods is by converting choline esters to free choline via acid hydrolysis, followed by quantifying the total choline, as adopted by the AOAC method (AOAC-Choline); however, certain choline esters are difficult to hydrolyse. Here, we investigated various acid hydrolysis conditions to establish a reliable method for determining the total choline in foods by detecting free choline using highly sensitive and selective mass spectrometry. Hydrolysis in 0.055 mol/L HCl for 8 h in an autoclave (121 °C) was found to be optimal for the hydrolysis of choline esters in various foods. Twenty-four foods, including grains, seed, vegetables, fruits, mushroom, algae, fish, meats, beverage, processed foods, and egg, were measured. The trends in the total choline content were consistent with previous reports; however, the choline content was 10-20% higher than that measured using AOAC-Choline. Therefore, re-evaluation of the total choline content in foods using our constructed method is recommended. This reassessment will allow for a more reliable determination of choline intake for maintaining health.

Keywords: Acid hydrolysis; Choline; Choline esters; Liquid chromatograph-tandem mass spectrometry; Total choline; Various foods.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1

(a) Structure of choline esters and their acid hydrolysis under three conditions. (b) Preliminary studies on conditions for hydrolysis of choline esters. Each test solution ((A) 50.00 mg/100 mL phosphocholine, (B) 90.60 mg/100 mL glycerophosphorylcholine, (C) 1985 mg/100 mL phosphatidylcholine, or (D) 356.2 mg/100 mL sphingomyelin) was hydrolysed under three different conditions. After hydrolysis, the solution was analysed by LC–MS/MS. Detailed conditions are described in the Materials and methods. The data represent the mean ± SD (n = 3). Significant differences from Condition 3 were analysed using one-way ANOVA followed by Dunnett’s test (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 2
Figure 2

Optimisation of hydrolysis conditions using test solutions. Each test solution ((A) 50.00 mg/100 mL phosphocholine, (B) 90.60 mg/100 mL glyceryl phosphorylcholine, (C) 1985 mg/100 mL phosphatidylcholine, or (D) 356.2 mg/100 mL sphingomyelin) was hydrolysed under Condition 3 with varying HCl concentrations and hydrolysis times. After hydrolysis, the solution was analysed by LC–MS/MS. Detailed conditions are described in the Materials and methods. The data represent the mean ± SD (n = 3). Significant differences from 16 h were analysed using one-way ANOVA followed by Dunnett’s test (*P < 0.05, ***P < 0.001).

Figure 3
Figure 3

Optimisation of hydrolysis conditions using food samples. Each food sample (egg yolk, raw chicken liver, soybean powder, and pistachio nuts) was hydrolysed under Condition 3 with varying hydrolysis times. After hydrolysis, the solution was analysed by LC–MS/MS. Detailed conditions are described in the Materials and methods. The data represent the mean ± SD (n = 3). Significant differences from 16 h were analysed using one-way ANOVA followed by Dunnett’s test (*P < 0.05, **P < 0.01).

Figure 4
Figure 4

LC–MS/MS calibration curve and SRM chromatogram for food samples hydrolysed under optimal conditions. (A) The choline stock solution was diluted to 0.005, 0.010, 0.020, 0.040, 0.080, 0.120, and 0.160 μg/mL with 0.1% formic acid, after which IS was added at 0.040 μg/mL and analysed by LC–MS/MS. (B) The hydrolysed solution of each food sample (egg yolk, NIST 1869, pistachio nuts, and raw carrot) was injected into LC–MS/MS to analyses free choline. Detailed LC–MS/MS conditions are described in the Materials and methods. The intensity (%) in the graph for each food is based on choline SRM as 100%.

Figure 5
Figure 5

SIM chromatogram of food samples hydrolysed under optimal conditions and correlation coefficient for average values from LC–MS/MS and LC–MS. (A) The hydrolysed solution of each food sample (egg yolk, NIST 1869, pistachio nuts, and raw carrot) was injected into LC–MS to analyse free choline. Detailed LC–MS conditions are described in the Materials and methods. The intensity (%) in the graph for each food is based on choline SIM as 100%. (B) The 24 foods were measured 10 times using both LC–MS/MS and LC–MS, and the average value was calculated. The average value for yogurt determined using LC–MS was calculated from six analyses. The average value from LC–MS/MS is plotted on the Y axis of the graph, and the average value from LC–MS is plotted on the X axis of the graph; the correlation coefficient was determined.

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Optimisation of acid hydrolysis conditions of choline esters and mass spectrometric determination of total choline in various foods – PubMed