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HPLC-UV method validation for the identification and quantification of bioactive amines in commercial eggs.

Talanta (2015-05-25)
Tadeu Chaves de Figueiredo, Débora Cristina Sampaio de Assis, Liliane Denize Miranda Menezes, Guilherme Resende da Silva, Isabela Pereira Lanza, Luiz Guilherme Dias Heneine, Silvana de Vasconcelos Cançado
ABSTRACT

A quantitative and confirmatory high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method for the determination of bioactive amines in the albumen and yolk of commercial eggs was developed, optimized and validated by analyte extraction with trichloroacetic acid and pre-column derivatization with dansyl chloride. Phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine standards were used to evaluate the following performance parameters: limit of detection (LoD), limit of quantification (LoQ), selectivity, linearity, precision, recovery and ruggedness. The LoD of the method was defined from 0.2 to 0.3 mg kg(-1) for the yolk matrix and from 0.2 to 0.4 mg kg(-1) for the albumen matrix; the LoQ was from 0.7 to 1.0 mg kg(-1) for the yolk matrix and from 0.7 to 1.1 mg kg(-1) for the albumen matrix. The validated method exhibited excellent selectivity and separation of all amines with coefficients of determination higher than 0.99. The obtained recovery values were from 90.5% to 108.3%, and the relative standard deviation (RSD) was lower than 10% under repeatability conditions for the studied analytes. The performance parameters show the validated method to be adequate for the determination of bioactive amines in egg albumen and yolk.

MATERIALS
Product Number
Brand
Product Description

Supelco
Hydrochloric acid solution, volumetric, 0.1 M HCl (0.1N), endotoxin free
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Hydrochloric acid, 36.5-38.0%, BioReagent, for molecular biology
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Sodium bicarbonate, powder, BioReagent, for molecular biology, suitable for cell culture, suitable for insect cell culture
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Sodium bicarbonate, Hybri-Max, powder, suitable for hybridoma, ≥99.5%
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Trichloroacetic acid, ACS reagent, ≥99.0%
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Trichloroacetic acid, ≥99.0% (titration)
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L-Proline, ReagentPlus®, ≥99% (HPLC)
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Dansyl chloride, BioReagent, suitable for amino acid labeling, powder and chunks, ≥99% (HPLC)
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Putrescine dihydrochloride, powder, BioReagent, suitable for cell culture
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Putrescine dihydrochloride, ≥98% (TLC)
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Spermine tetrahydrochloride, powder or crystals
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Spermine tetrahydrochloride, BioReagent, for molecular biology
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Sodium bicarbonate, BioXtra, 99.5-100.5%
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Putrescine dihydrochloride, γ-irradiated, lyophilized powder, BioXtra, suitable for cell culture
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Acetone, suitable for HPLC, ≥99.9%
SAFC
L-Proline
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Spermine tetrahydrochloride, ≥99.0% (AT), powder or crystals
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Trichloroacetic acid, BioUltra, ≥99.5% (T)
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Trichloroacetic acid, ACS reagent, for the determination of Fe in blood according to Heilmeyer, ≥99.5%
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Hydrochloric acid solution, ~6 M in H2O, for amino acid analysis
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L-Proline, BioUltra, ≥99.5% (NT)
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Dansyl chloride, BioReagent, suitable for fluorescence, ≥99.0% (HPLC)
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Hydrogen chloride solution, 3 M in cyclopentyl methyl ether (CPME)
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Acetonitrile solution, contains 0.05 % (w/v) ammonium formate, 0.1 % (v/v) formic acid, 5 % (v/v) water, suitable for HPLC
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Acetonitrile solution, contains 0.05 % (v/v) trifluoroacetic acid
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Acetonitrile solution, contains 0.1 % (v/v) trifluoroacetic acid, suitable for HPLC
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L-Proline, 99%, FCC, FG
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Spermine tetrahydrochloride, BioUltra, for molecular biology, ≥99.5% (AT)
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Acetone, ≥99%, meets FCC analytical specifications