Fluorometric determination of subnanogram levels of nitrite using 5-aminofluorescein

Abstract

Nitrite ion has recently attracted a great deal of attention because of its potential role in producing nitrosoamine, a carcinogenic material, in the human body. The concern stems from the fact that nitrites are commonly added to processed meats.The ability to measure nitrite at extremely low levels is important to those studying nitrite reactions in our food system. A review of various ,techniques has been presented by Streuli and Averell (1). These techniques, in general, lack the sensitivity to reach the low nanogram range. Wiersma ( 2 ) and Dombrowski and Pratt (3) have developed sensitive fluorometric procedures but these are quite complicated relying on solvent extraction.A new procedure, presented here, is sensitive to 50 pg/ml (pg = g) and is also quite simple. Axelrod et al. ( 4 ) previously reported that 5-aminofluorescein is a suitable reagent for the analysis of sulfite. The 5-aminofluorescein is highly fluorescent, and, because it has a primary amino group, it was investigated and found to be useful for the trace determination of nitrite. EXPERIMENTAL Instrumentation. A Perkin-Elmer Model 203 Spectrofluo-rometer equipped with a high-pressure xenon lamp (for continuous spectra) was used in the study. The excitation wavelength was 490 nm and the emission wavelength was 515 nm. All measurements were made in a 22 OC air conditioned room.Reagents. The 5-aminofluorescein (F.W. 347) was obtained from Eastman Kodak Co. (Rochester, N.Y.) and was used as received. All other chemicals were reagent grade; the water used was first passed through a deionizing column and then distilled. Procedure. A 1.5 X 10-3M 5-aminofluorescein (5AF) stock solution was prepared by dissolving the appropriate weight of the dye in 25 ml of absolute methanol in a 100-ml volumetric flask. After the 5AF has dissolved, 4 ml of concentrated HC1 (12.1M) was added and the solution was diluted to volume with distilled water. All further dilutions were made with distilled water.The analysis was performed in the following manner: 7 ml of an aqueous sample was placed in a 25-ml glass stoppered cylinder. Added to this was 1 ml of an appropriate dye concentration followed by 2 ml of 6M HC1. The sample was mixed and allowed to stand for an appropriate time period (up to 60 min-see later discussion). T o this solution, 5 ml of 5.4M NaOH was added. The solution was mixed and allowed to stand for 5 min prior to the fluorescence measurement. The product is stable for measurement up to 24 hr later. Nitrite standards of a suitable concentration range were analyzed in the same manner. In this particular analytical technique, the fluorescence increases with increasing nitrite concentration. Therefore, the standard blank (no nitrite) was used to set the 0 units fluorescence and the highest nitrite standard was used to set the 100 units fluorescence. This procedure was used to measure nitrite concentrations from iO-5M to 5 x I O -~M using final dye concentrations of 10-5M to 3 X 10-8M, respectively. RESULTS AND DISCUSSIONOptimization of Acidity and Basicit...

Analysis of nitrite and nitrate in biological fluids by assays based on the Griess reaction: Appraisal of the Griess reaction in the l-arginine/nitric oxide area of research

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Sensitive determination of nitrite by means of a Landolt-type reaction in fluorescent aggregates of a binaphthyl-based amphiphile

“…All in all, the results in Table 2 indicate the usefulness of the proposed procedure in determining trace amounts of nitrite in water samples down to 5 ng/ml. The sensitivity of the method is similar to those employing fluorescent reagents [6,7] and is higher by about one order of magnitude compared with colorimetric methods [1,2]. The precision of determination (r.s.d.…”

Sequential injection spectrophotometric determination of nanomolar nitrite in seawater by on-line preconcentration with HLB cartridge

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