Optimization of Capillary Isotachophoretic Method for Histidine Determination in Protein Matrices

Abstract

The capillary isotachophoretic method was optimized and used for histidine determination in food samples. The optimum conditions for histidine separation and determination were found on the experimental conditions such as: selectivity, separation speed, pH, concentration of the leading and terminating electrolytes, and electroosmotic flow additives. The optimum electrolytes composition [leading electrolyte: 7 mM NH₄OH + 15 mM 2-(N-morpholino)ethanesulfonic acid + 1% hydroxyethylcellulose; pH = 6.10 and terminating electrolyte: 15 mM aminocaproic acid +5 mM acetic acid +40% methanol; pH = 5.10] and conditions of analysis were adopted for histidine determination in food samples (meat and fish products). The proposed electrolyte system was characterized by linearity (10–100 and 100–430 mg · L⁻¹ with R² = 0.9976 and 0.9991), accuracy (99.5% and 98%), intra-assay of the relative step height (1.40% for standard and 3.20% for food samples analysis), inter-assay of the relative step height (3.65% and 6.30%) and satisfactory quantification and detection limits. The obtained results were compared to a chromatographic method (reversed-phase (RP)-HPLC) for determination of histidine. The average concentrations of histidine in the samples assayed by both methods were statistically comparable. It should be noted that the proposed histidine determination method can be considered as a contribution to Green Analytical Chemistry.

Keywords:

• Food samples
• Histidine
• Optimization of isotachophoretic condition
• Optimization of sample preparation

Acknowledgments

The author wishes to thank Polish Ministry of Science and Higher Education for the financial support: grant No. NN 312 465640 (4656/B/P01/2011/40).

Notes

Note: EOF, electroosmotic flow; MES, 2-(N-morpholino)ethanesulfonic acid; HEC, hydroxyethylcellulose; PVA, polyvinyl alcohol; EACA, aminocaproic acid.

Note: S, standard deviation; R², coefficient of determination; DL, Limit of Detection (y + 3 · s_y/x)/b, where s_y/x is the standard deviation in the y axis; QL, Limit of Quantification (10 · s_y/x/b).

RSH, relative step height; , within-day analyses; , between- days analyses.

Note: X, average value (mg · 100 g⁻¹ fresh mass); μ, confidence limit (μ = (t_n-1 · s/n^1/2); p = 95%); CV – coefficient of variation (%).