AGRICULTURAL MATERIALS Determination of Histamine in Tomatoes by Liquid Chromatography/Mass Spectrometry ELEK BOLYGO,PAUL A. COOPER, K. MICHAEL JESSOP, and FRANK MOFFATT 1 Zeneca Agrochemicals, Jealotts Hill Research Station, Bracknell, Berkshire RG42 6ET, UK The histamine level in tomato fruits and pastes was determined by 2 orthogonal techniques as a means of comparing accuracy. Close statistical agreement was found between assays for free histamine by capillary electrophoresis (with UV absorbance detection), and for the dansyl deriva- tive by reversed-phase liquid chromatography (LC). Both techniques have adequate sensitivity for the analysis of endogenous histamine in tomatoes, but LC/electrospray tandem mass spectrometry was more sensitive by at least an order of magnitude, down to a level of 0.05 mg/kg. B iogenic amines such as histamine are natural compo- nents of food and feed that can affect quality and physiology (1). This report provides evidence of the accuracy of a previously reported assay which used capillary electrophoresis (CE)(2) for endogenous histamine in tomatoes by comparing it with an orthogonal liquid chromatographic (LC)-based method. Previous LC methods for histamine in various plant matrixes, used either postcolumn (3) or precol- umn derivatization (4–6) with UV absorbance or fluores- cence detection. Reversed- phase LC requires the use of an ion-pairing agent (3) which would be incompatible with con- ventional electrospray tandem mass spectrometry (MS). The use of such agents can be avoided through precolumn deriva- tization, such as dansylation which is broadly applicable to a wide range of analytes, opening up options of detection by fluorescence (7) and MS on a triple quadrupole instrument. Experimental Reagents and Solvents Histamine dihydrochloride (99% purity) and 1,3- diaminopropane dihydrochloride were used as analytical ref- erence and internal standards, respectively (Sigma-Aldrich Co., Poole, Dorset, UK). L-Proline, 5-[dimethylamino]naph- thalene-1-sulfonyl (dansyl) chloride, and sodium carbonate were of analytical purity grade (Sigma-Aldrich Co.). Buffers were prepared from analytical grade reagents (Merck Ltd. Poole, Dorset, UK). Solvents (acetonitrile, acetone, toluene) were LC grade (Romil, Cambridge, UK). Deionized water was obtained from a Milli Q water purification system (Mil- lipore Ltd., Watford, Hertfordshire, UK). Biological Materials Samples of tomatoes were obtained in-house from Zeneca Plant Sciences or from a retailer (market sample). The green tomatoes were mature but unripe. All samples and extracts were stored at < -18°C when not in use. Instruments LC/tandem mass spectrometry (LC/MS–MS) was per- formed on a Quattro II mass spectrometer (Micromass, Al- tringham, Cheshire, UK), fitted with a Waters Alliance 2690 liquid chromatograph (Waters Ltd., Watford, Hertfordshire, UK). Off-line LC was performed on a standard HP1100 sys- tem fitted with a quaternary pump and UV detector and a Model 1046A fluorescence detector (Hewlett-Packard, Bracknell, Berkshire, UK). Sample Preparation Samples of frozen tomatoes were homogenized with dry ice. A subsample (20 g) was extracted by maceration with aqueous perchloric acid (0.2M), with cooling over an ice bath, at an extraction ratio of 0.33 g tomato fruit/mL extract as described in an earlier publication (2). Samples were for- tified with an internal standard, 1,3 diaminopropane (200 μg), prior to extraction in order to provide an independent means to assess the efficiency of the procedure. An aliquot of the clear extract was dansylated according to the method of Vallee ´ et al. (6), except for an increased reaction time from 5 to 10 min. An aliquot of the extract (100 μL) was treated with saturated sodium carbonate solution (300 μL) in a screw-cap vial (2 mL). Dansyl-chloride solution (400 μL, 7.5 mg/mL in acetone) was added. The mixture was shaken and then left to derivatize (60°C, 10 min) in the dark. After cooling to room temperature the excess reagent was quenched by the addition of L-proline (100 μL, 100 mg/mL in water). After 15 min at room temperature, toluene (500 μL) was added; the mixture was vigorously shaken for 20–30 s, centrifuged at 4000 rpm Received July 27, 1999. Accepted by RN November 30, 1999. 1 Author to whom correspondence should be addressed. BOLYGO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 3, 2000 543 Downloaded from https://academic.oup.com/jaoac/article/83/3/543/5656285 by guest on 03 September 2021