Validation of a new planar chromatographic method for quantification of the heterocyclic aromatic amines most frequently found in meat

摘要

A new HPTLC method, in which all the HPTLC steps are performed automatically, has been established for quantification of the five most frequently found heterocyclic aromatic amines (HAA: PhIP, MeIQx, 4,8-DiMeIQx, norharmane, and harmane). The method was used for trace analysis (low μg kg?1 range) of HAA in meat samples and found to be a cost-effective alternative to HPLC, because it enables simultaneous chromatography of up to 20 samples within 30 min. After preconditioning of the HPTLC silica gel layer with ammonia vapour the plate was developed with methanol–chloroform, 1:9 (v/v). At least 4ó separations were obtained and selectivity for the meat matrix was achieved. In repeatability tests relative standard deviations (RSD, n = 14, peak height) were less than or equal to ±3.3%. In tests of intermediate precision (mean value for 14 tracks on six different plates on six different days) RSDs were better than ±2% (peak height). Measurement of the reproducibility of migration distances on six different plates resulted in RSDs less than or equal to ±1.3%. Limits of detection and quantification (S/N 3 and 10, respectively) for the five HAA ranged between 0.4 and 5 ng per band and between 0.8 and 14 ng per band, respectively. With paraffin–n-hexane, 3:7 (v/v), as fluorescence enhancer more sensitive determinations (up to 8.5-fold greater signal intensities) were achieved for PhIP, norharmane, and harmane. In the working range (1:10) polynomial regressions for PhIP, MeIQx, and 4,8-DiMeIQx and linear regressions for norharmane and harmane resulted in RSDs between ±1.9 and 3.6%. To confirm the absence of potentially coeluting minor HAA, rarely formed in meat, mass spectra were occasionally recorded by online ESI–MS. Robustness tests showed that preconditioning with ammonia was essential for successful separation and that relative humidity had only a minor effect on the chromatography.