Optimization and validation of a HPLC method for simultaneous determination of aflatoxin B1, B2, G1, G2, ochratoxin A and zearalenone using an experimental design

• Published in: Food additives & contaminants. Part A, Chemistry, analysis, control, exposure & risk assessment Vol. 28; no. 7; pp. 902 - 912
• Main Authors: Rahmani, Anosheh, Selamat, Jinap, Soleimany, Farhang
• Format: Journal Article
• Language: English
• Published: Abingdon Taylor & Francis Group 2011; Taylor & Francis; Taylor & Francis Ltd
• Subjects: Acetic acid; Acetonitrile; Aflatoxin B1; Aflatoxin B1 - analysis; Aflatoxin B1 - toxicity; Aflatoxins; Aflatoxins - analysis; Aflatoxins - toxicity; Biological and medical sciences; Cereal and baking product industries; Cereals; Chromatography, High Pressure Liquid - methods; Data processing; Design; Design of experiments; Edible Grain - chemistry; Edible Grain - microbiology; experimental design; Flow rates; Food additives; Food contamination; Food contamination & poisoning; Food Contamination - analysis; Food industries; Food Microbiology; Food Safety; Food toxicology; Fundamental and applied biological sciences. Psychology; High-performance liquid chromatography; HPLC; Humans; in-house validation; Methanol; Mycotoxins; Ochratoxin A; Ochratoxins - analysis; Ochratoxins - toxicity; Optimization techniques; prediction; Regression analysis; response surface methodology; reversed-phase high performance liquid chromatography; Risk assessment; Solvents; Spectrometry, Fluorescence; Temperature; Temperature effects; Toxins; Zearalenone; Zearalenone - analysis; Zearalenone - toxicity; Validation; Ochratoxin; ochratoxin A; mycotoxins; Mycotoxin; HPLC chromatography; Zearalenone; Method; cereals; Optimization; Toxin; aflatoxins; Experimental design; Aflatoxin B1; Cereal; HPLC; in-house validation; zearalenone
• ISSN: 1944-0057; 1944-0049; 1944-0057
• DOI: 10.1080/19440049.2011.576436

A reversed-phase HPLC optimization strategy is presented for investigating the separation and retention behavior of aflatoxin B₁, B₂, G₁, G₂, ochratoxin A and zearalenone, simultaneously. A fractional factorial design (FFD) was used to screen the significance effect of seven independent variables on chromatographic responses. The independent variables used were: (X1) column oven temperature (20–40°C), (X2) flow rate (0.8–1.2 ml/min), (X3) acid concentration in aqueous phase (0–2%), (X4) organic solvent percentage at the beginning (40–50%), and (X5) at the end (50–60%) of the gradient mobile phase, as well as (X6) ratio of methanol/acetonitrile at the beginning (1–4) and (X7) at the end (0–1) of gradient mobile phase. Responses of chromatographic analysis were resolution of mycotoxin peaks and HPLC run time. A central composite design (CCD) using response surface methodology (RSM) was then carried out for optimization of the most significant factors by multiple regression models for response variables. The proposed optimal method using 40°C oven temperature, 1 ml/min flow rate, 0.1% acetic acid concentration in aqueous phase, 41% organic phase (beginning), 60% organic phase (end), 1.92 ratio of methanol to acetonitrile (beginning) and 0.2 ratio (end) for X1–X7, respectively, showed good prediction ability between the experimental data and predictive values throughout the studied parameter space. Finally, the optimized method was validated by measuring the linearity, sensitivity, accuracy and precision parameters, and has been applied successfully to the analysis of spiked cereal samples.