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by ion-exclusion chromatography, pre-column derivatization by reaction with FDNB, and determined
by RP-paired-ion HPLC with UV detection at 405 nm. The detection limit corresponded to 0.1 nmol
trimethyllisine per injection [15].

FDNB can modify the side chain of lysine, tyrosine, histidine, and cysteine residue in proteins, and the
N-terminal amino group. Thus, FDNB is suitable for chromophoric labeling for selective mapping [16].
In addition, the DNP group can be removed from tyrosine, histidine, and cysteine residues by thiolysis
with 2-mercaptoethanol [16].

FDNB Application: Determination of Amikacin in Serum by HPLC [10]

Remove most of the stem of a Pasteur pipette and plug the remainder of the stem with glass wool.
Incubate CM-Sephadex C-25 cation exchanger in a 0.2 M sodium phosphate solution in water at room
temperature for at least 24 h. Fill the Pasteur pipette with sufficient of the Sephadex slurry to obtain a
column height of 1.5 cm (5.4 mm I.D.). Pipette 200 µ1 of the serum sample into a centrifuge tube and
add 20 µ1 of a solution containing 250 mg/1 kanamycin sulfate in water (IS), Vortex and dispense the
contents of the centrifuge tube on top of the column. Elute the column with 2 ml of a solution
containing 1 mM HCl and 0.2 M sodium sulfate (initial eluent). Discard the eluate (dead volume of the
column). Elute the column with 1 ml of 50 mM NaOH and collect this eluate in a 5-ml ampoule. Add
2.5 ml of a solution of FDNB in methanol (30 g/1). The formation of a precipitate is observed, which
redissolves upon mixing. Heat-seal the ampoule and place in boiling water for 5 min. After cooling, a
150-µ1 portion of the final reaction mixture is applied onto RP-HPLC with UV detection at 365 nm.
The sensitivity is 1 mg/1 for amikacin with samples of 200 µ1. Precision, expressed R. S. D., is about
3%.

FDNB Application: Derivatization Used in Peptide Mapping [16]

For peptide mapping, each protein (<5 mg) was first modified with an equal weight of maleic anhydride
at pH 8.5, using a pH stat. Urea (1 g/ml of sample volume) and NaHCO (0.1 g/ml) were dissolved in
3
the sample, and 50% FDNB in CH CN (50 µ1) was added. The mixtures were stirred in the dark at
3
room temperature overnight, then dialyzed extensively against water, then 0.2 M NH CO , using
4
3
dialysis tubing with a 3500 molecular weight cut-off (Spectra-Por3). The modified protein was digested
with thermolysin (1:25 enzyme: substrate ratio) at 37 °C for 8 h, and freeze-dried. The modified
peptides were separated by RP-HPLC (monitored at 270 and 320 nm). The collected peptide was
hydrolyzed in 6 N HCl at 110 °C for 22 h for amino acid analysis.

2.2.1.2—
4-Fluoro-3-nitrotrifluoromethylbenzene [17] (4-Fluoro-3-nitrobenzotrifluoride) (FNBT)

The reaction of FNBT with primary amines is shown in Fig. 2.2. FNBT reacts with polyamine and
produces the N-2'-nitro-4-trifluoromethylphenyl polyamine (NTP-polyamine) (Fig. 2.2).
NTPpolyamines could be extracted by organic solvents. 2-methylbutane was selected because it
extracted less by-products of the reaction and could easily be removed by evaporation. FNBT does not
react with secondary amines. The reaction products of polar compounds such as amino acids with
FNBT were not extracted by 2-methylbutane. Thus histidine was used to scavenge excess FNBT.
NTPpolyamines have absorption maxima at 242 and 410 nm.

FNBT Application: Derivatization of Polyamines [17]

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