Liquid chromatographic determination of triethylenetetramine in human and rabbit sera based on intramolecular excimer-forming fluorescence derivatization
Introduction
Wilson’s disease is a genetic disorder characterized by the accumulation of excessive amounts of copper in some organs, such as liver, brain, cornea and kidney, and its major clinical manifestations are liver cirrhosis, advanced extrapyramidal symptom and Kayser-Fleisher rings, which subsequently lead to death [1], [2], [3]. Wilson’s disease has been treated with oral administration of triethylenetetramine dihydrochloride (N,N′-bis(2-aminoethyl)-1,2-ethanediamine 2HCl, TETA·2HCl) which chelates copper ions to increase their urinary excretion of the ion. Even though TETA is relatively safe and effective among the chelating agents for Wilson’s disease [4], [5], it still causes mild anemia in some patients [6], probably depending on individual differences in disposition of TETA. Therefore, drug monitoring in each patient is necessary for good therapy.
Several liquid chromatographic (LC) methods coupled with fluorescence [7], [8], [9], [10] or conductometric [11] detection have been developed for the determination of TETA in human serum. The fluorometric method by precolumn derivatization using fluorescamine is highly sensitive and selective, but it requires a rather complicated clean-up procedure using solid-phase extraction on a cation-exchange cartridge [7]. On the other hand, the postcolumn fluorescence method using o-phthalaldehyde requires only simple deproteinization as a sample pretreatment, but the resulting chromatograms are rather complicated due to the presence of many endogenous amino compounds [8]. The conductometric method does not have enough sensitivity to be applied to drug monitoring in serum.
The aim of the present study was to develop a highly sensitive, selective and simple method for the determination of TETA in serum. Recently, we have developed a highly selective fluorescence derivatization technique for endogenous polyamines (putrescine, cadaverine, spermidine, and spermine) and basic amino acids (lysine and ornithine), in which the polyamino compounds were converted to the corresponding polypyrene-labeled derivatives by reaction with 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE) [12], [13]. The derivatives can emit intramolecular excimer fluorescence (450–550 nm), which can clearly be discriminated from the normal fluorescence of pyrene (370–420 nm). Since the chemical structures of TETA and the endogenous polyamines resemble each other closely, we applied the excimer-forming derivatization technique to the determination of TETA by optimizing derivatization and LC separation conditions. In the established method, TETA was converted to the polypyrene-labeled derivative by reaction (90 °C, 30 min) with PSE and the derivative afforded the intramolecular excimer-fluorescence in the LC mobile phase. The method was successfully applied to the determination of TETA in human and rabbit sera with a simple pretreatment, deproteinization with organic solvents (acetonitrile and tetrahydrofuran, (THF)). 1,6-Hexanediamine (HDA) was used as an internal standard (I.S.).
Section snippets
Chemicals and solutions
All chemicals and solvents were of the highest purity available and were used as received. Deionized and distilled water was used for all aqueous solutions. TETA·2HCl was purchased from Sigma (St. Louis, MO, USA), and a therapeutic capsule containing 250 mg TETA·2HCl (metalite capsule, Tsumura, Tokyo, Japan) was used for the drug monitoring studies. HDA was obtained from Kanto Chemical (Tokyo, Japan). PSE was purchased from Molecular Probes (Eugene, OR, USA) and used without further
Excimer fluorescence from TETA derivative
Fig. 1 shows normalized fluorescence emission spectra of the column eluate for TETA peak and PSE dissolved in the LC mobile phase. PSE gave only normal (monomer) fluorescence (370–420 nm), whereas the pyrene derivative of TETA in the peak eluate emitted excimer fluorescence (450–550 nm). The shape of the spectrum for TETA did not changed when the solution was diluted 10 times with the mobile phase. These observations imply that the produced TETA derivative under the present conditions is a
Conclusions
The present excimer fluorometric LC method utilizing PSE only needs a one-step deproteination procedure for pretreatment of the serum sample. Moreover, it offers higher sensitivity to permit the quantification of TETA in 25 μl of human serum after oral administration of the drug. The method can thus be useful in the therapeutic and pharmaceutical investigation of TETA.
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