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By the action of UVA light, fluphenazine reacted with nucleophiles through a mechanism involving defluorination of its trifluoromethyl group, giving rise to carboxylic acid derivatives that were easily detected by electrospray mass spectrometry. This photoreaction took place with alcohols, sulphydryls, and amines. When irradiation of fluphenazine was carried out in the presence of an amino acid at pH 7.4, the alpha-amino group was covalently bound to the drug. With amino acids possessing a further nucleophilic residue on the side chain, such as lysine, tyrosine, and cysteine--but not serine--both groups reacted, resulting in a fluphenazine-amino acid-fluphenazine diadduct. The same occurred with the physiological peptide glutathione (gamma-glutamylcysteinylglycine). By means of MALDI mass spectrometry, it was shown that fluphenazine also covalently bound to peptides and proteins such as calmodulin. This binding may result in the formation of antibodies, ultimately leading to the destruction of the granulocytes and thus suggesting that photoactivation of this drug may play a role in its clinical side effects, such as agranulocytosis.

Antisera have been raised in rabbits to the immogen 2-trifluoromethylphenothiazine-10-beta-propionate bovine serum albumin. An [125I]-labelled tyrosine methyl ester derivative of the immunogen precursor has been synthesised and used with the antisera to develop a simple, precise and sensitive radioimmunoassay for phenothiazines and thioxanthenes bearing 2-trifluoromethyl substituents. The assay can detect 0.4 ng/ml of fluphenazine, trifluorperazine or (Z)-flupenthixol in 100 microliter of human serum without interference from their sulphoxide or 7-hydroxylated metabolites. An acceptable correlation between this assay and an established fluphenazine radioimmunoassay using commercial [3H]fluphenazine has been obtained for plasma samples.