Accurate Determination of the Degree of Deacetylation of Chitosan Using UPLC-MS/MS

Abstract

The mole fraction of deacetylated monomeric units in chitosan (CS) molecules is referred to as CS's degree of deacetylation (DD). In this study, 35 characteristic ions of CS were detected using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS). The relative response intensity of 35 characteristic ion pairs using a single charge in nine CS samples with varying DDs was analyzed using 30 analytical methods. There was a good linear relationship between the relative response intensity of the characteristic ion pairs determined using ultrahigh performance (UP) LC-MS/MS and the DD of CS. The UPLC-MS/MS method for determining the DD of CS was unaffected by the sample concentration. The detection instrument has a wide range of application parameters with different voltages, high temperatures, and gas flow conditions. This study established a detection method for the DD of CS with high sensitivity, fast analysis, accuracy, stability, and durability.

Keywords: UPLC–MS/MS; chitosan; degree of deacetylation determination; relative response intensity of the characteristic peak.

Figure 1

Time-of-flight (TOF) mass spectral feature map. (a) Blank, (b) CS500-70, (c) CS500-78, and (d) CS600-90.

Figure 2

(a–h) Relationship among the ion intensity, degree of deacetylation (DD), and molecular weight (Mw) of chitosan (CS). (i) Ion response intensity and relative abundance of each polysome in different CSs. (j) Polysome combination by D and A; the relative abundance between the polysomes of the degree of polymerization (DP) 1–7 tended to be constant, i.e., DP 1:DP 2:DP 3:DP 4:DP 5:DP 6:DP 7 were relatively stable, with possible forms of D and A; D₂A₀, D₁A₁, and D₀A₂; D₃A₀, D₂A₁, D₁A₂, and D₀A₃; D₄A₀, D₃A₁, D₂A₂, D₁A₃, and D₀A₄; D₅A₀, D₄A₁, D₃A₂, D₂A₃, D₁A₄, and D₀A₅; D₆A₀, D₅A₁, D₄A₂, D₃A₃, D₂A₄, D₁A₅, and D₀A₆; and D₇A₀, D₆A₁, D₅A₂, D₄A₃, D₃A₄, D₂A₅, D₁A₆, and D₀A₇, respectively. The overall response of each DP is the addition of the response intensities of all possible monomeric unit forms of the DP. (k,l) Response intensity and precision results of nine CSs in detecting 35 characteristic ion pairs.

Figure 3

Correlation between the degree of deacetylation (DD) and relative response intensity (RRI). (a) DP 1, (b) 2, (c) 3, (d) 4, (e) 5, (f) 6, and (g) 7. (h) The relative errors (RE) between the degree of deacetylation (DD) of chitosan calculated using the established standard curve and that measured using ¹H NMR. The area between the grey dashed lines illustrates RE ± 2.5%. (i) RRI precision of the characteristic ion pairs of chitosan.

Figure 4

Relative response intensity (RRI) of CS 500-78, CS 200-87, and CS 300-95 with different concentrations in the range of 500–4000 ng/mL.

Figure 5

(a–c). Response intensity of 27 of the 35 characteristic ion pairs detected of CS200-87 under different mass spectrometry parameter conditions. (d) Relationship between Relative response intensity (RRI) and each capillary voltage from 200 to 5000 V; (e) desolvation temperature, 300–400 °C; (f) desolvation gas flow, 600–700 L/h; cone gas flow, 150–250 L/h, and nebulizer gas flow, 5.5–6.5 bar.