Fluorometric detector, HPLC

Levels of IAA were analyzed by HPLC with a fluorometric detector and indole-propionic acid as an internal standard. Levels of ABA were analyzed by GLC with an ECD and [14C]ABA as an internal standard. Means of results of 3 experiments and standard errors are given (n=3). Reproduced and revised with permission from ref. 8. 

Determination of human plasma levels of aloe-emodin and rhein using HPLC with a fluorometric detector has been described (Krumbiegel and Schulz, 1993). 

The dansyl derivatives, which have a napthtalene structure, are excellent derivatives for primary amines (Mietz and Karmas, 1978). They are easily formed and detected by a uv (HPLC) detector in amounts as little as 10 ng, therefore the method does not require high sensitivity or fluorometric detectors. Gradient elution improves the separation, allowing for a broad range of derivatives to be separated in a relatively short time (40 min). Use of reverse-phase microparticular (5-10 /u,m) columns can further improve the HPLC separation (Gouygou et al, 1992). Desiderio et al (1987) described the use of a reversed-phase HPLC method for the quantification of putrescine, cadaverine, spermidine, and spermine from brain extracts as the dansyl derivatives. 

Various analytical techniques such as spectrophotometry/colorimetry, FL and infrared spectrometry, voltammetry, thin-layer chromatography (TLC), gas chromatography (GC), and HPLC based on ultraviolet, diode array, or fluorometric detectors have been reported in the literature for analysis of vitamin E. Various critical and comprehensive reviews are available on vitamin E quantification in food and clinical samples (Ball, 1988, 1998 Lumley, 1993). The AOAC International Official Methods of Analysis (1995) provides several methods based on older, chemical approaches. The applications of these analytical techniques are briefly summarized below. 

Figure 10.4 Assay of indoleamities such as S-HIAA and tryptophan in urine by HPLC with natural fluorescence detection. The chromatographic conditions were column, 100 mm X 4.6 mm (5 pm) ODS Hypersil eluent, 50 mM ammonium acetate (pH 5.25)-methanol (250 30, v/v) flow rate, 1ml mininjection, 20 pi temperature, ambient detector, fluorometric (Jasco with xenon lamp 280 nm 335 nm).

Fig. 3 Mild acid hydrolysis-fluorometric HPLC analysis. Mini Q anion exchange chromatography of a2— 8-linked di/oligo/polyNeu5Ac-DMB. 2— 8-Linked oligo/polyNeuSAc was labeled with DMB and applied to a mini Q HR5/5 anion exchange column (1 mL, Cl -form). The column was eluted with 5 mM Tris-HCl (pH 8.0) with a gradient from 0 to 0.3 M NaCI for 75 min and 0.3 M NaCl to 0.4 M NaCl for 120 min after 15 min wash. The elution was monitored by a fluorescence detector (set at wavelength of 373 nm excitation and 448 mn emission). Each peak is assigned from the order of elution, based on DP...

The enzymatic method described above has two disadvantages (1) trapping of CO2 is a cumbersome procedure, and (2) the use of a radioactive substrate requires special precautions for use and disposal of reagents. Measurement of the primary amine formed by decarboxylation of the amino acid can also be exploited to monitor the PLP-dependent, enzyme-catalyzed reaction. This principle has been applied by Allenmark et al. (106), who used L-3,4-dihydroxyphenyl-alanine (L-DOPA) as substrate for tyrosine decarboxylase the dopamine produced by the decarboxylation reaction was determined by HPLC followed by amperometric detection. Both Hamfelt (107) and Lequeu et al. (108) utilized apo-tyrosine decarboxylase with tyrosine as substrate. The tyramine produced by the decarboxylation reaction was separated from the substrate (tyrosine) by HPLC and quantitated by either amperometric (108) or fluorometric (107) detection. The procedures discussed above are still subject to the main disadvantage of enzymatic methods possible interference by other materials present in the PLP containing extract which could either inhibit reconstitution of the holoenzyme or alter the reaction rate of enzyme catalysis. Moreover, HPLC with amperometric detection can hardly be described as less cumbersome than CO2 trapping difficulties in baseline-stabilization encountered with these detectors are well known. 

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