Fluorescence confirmatory

Certain salts of divalent metals (e.g., lead and copper formate, calcium acetate) are exceptional in giving bright green fluorescences. In each case confirmatory tests must always be employed. 

A method should be able both to quantify the amount of marker drug residue present in the sample and to identify the compound unambiguously. Historically, this required two distinct procedures a determinative procedure used to quantify the analyte, and a confirmatory procedure used to unequivocally identify the analyte. The need for two procedures was driven by the limitations of available technology. Most determinative methods over the last two decades have been based on liquid chromatography, usually with ultraviolet (UV)/visible or fluorescence defection. Limitations of cost. 

Applications With the current use of soft ionisation techniques in LC-MS, i.e. ESI and APCI, the application of MS/MS is almost obligatory for confirmatory purposes. However, an alternative mass-spectrometric strategy may be based on the use of oaToF-MS, which enables accurate mass determination at 5 ppm. This allows calculation of the elemental composition of an unknown analyte. In combination with retention time data, UV spectra and the isotope pattern in the mass spectrum, this should permit straightforward identification of unknown analytes. Hogenboom et al. [132] used such an approach for identification and confirmation of analytes by means of on-line SPE-LC-ESI-oaToFMS. Off-line SPE-LC-APCI-MS has been used to determine fluorescence whitening agents (FWAs) in surface waters of a Catalan industrialised area [138]. Similarly, Alonso et al. [139] used off-line SPE-LC-DAD-ISP-MS for the analysis of industrial textile waters. SPE functions here mainly as a preconcentration device. 

Because the confirmatory study also represents "worst-case" conditions, there should be few chances that an unstable product is not discovered. Examples of sources currently used by the pharmaceutical industry are xenon- and metal halide lamps (Option 1), artificial- and full spectrum daylight fluorescent tubes (Option 1), white fluorescent- and near UV-fluorescent tubes (Option 2). In (Northern) Europe (e.g., Scandinavia, England, and Germany), it seems that Option 1 with the xenon lamp is the preferred source. 

Confirmatory methods should preferably be based on MS, providing direct information on the molecular structure of the analyte(s), especially for residues of prohibited substances or substances for which no MRLs are set. However, validated chromatographic methods with specific detectors (e.g., photodiode array, fluorescence) or using two or more different chromatographic separation systems may be used, particularly for contaminants for which MRLs are specified. 

The problem with the simple TLC analysis of aflatoxins is that many natural products fluoresce blue or green under UV and many of these compounds have an Rp value close to, or even identical with, that of one of the four common aflatoxins (Figure 1). The early literature contains a number of false-positive reports of the presence of aflatoxins, and even their production by species of mold other than Aspergillus flavus, A. parasiticus, and A. nomius, so it is essential that a confirmatory test be carried out such as derivatization coupled with two-dimensional TLC. In the case of the aflatoxins, a useful confirmatory test is to derivatize as the hemiacetals with trifluoroacetic acid by superimposing 2 pi of a 50% solution of the reagent in chloroform onto the spot of both the unknown and a standard marker before chromatography. Aflatoxins Bi and Gi, but not B2 and G2, form hemiacetals that have the same fluorescence as the parent compound but run significantly more slowly... 

Because of its higher separation power, higher sensitivity, and accuracy, and the possibility of automating the instrumental analysis, HPLC is now the most commonly used technique in analytical laboratories. HPLC using fluorescence detection has already become the most accepted chromatographic method for the determination of aflatoxins. For its specificity in the case of molecules that exhibit fluorescence. Commission Decision 2002/657/EC, concerning the performance of analytical methods, considers the HPLC technique coupled with fluorescence detector a suitable confirmatory method for aflatoxin identification. 

Muscarella, M., Lo Magro, S., Palermo, C., and Centonze, D. 2007. Validation according to European Commission Decision 2002/657/EC of a confirmatory method for aflatoxin Ml in milk based on immunoaffinity columns and high performance liquid chromatography with fluorescence detection. Anal. Chim. Acta 594 257-264. 

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