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NPH [107-110] reacts with short and longchain FAs, and also straight and branched-chain dicarboxylic
acids in the presence of 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC), as shown
in Fig. 2.23c. The HPLC method with UV detection (230 nm) provides the detection limit of 2-5 pmol
per injection.

NPH Application: HPLC Determination of Straight and Branched-chain Dicarboxylic Acids in
Urine as their 2-Nitrophenylhydrazides [109]

Reagent solutions: an NPH solution (0.02 M) was prepared by dissolving the reagent in ethanol.
Solutions of pyridine (3% v/v) in ethanol and EDC hydrochloric acid (0.25 M) in water were prepared,
and then a working EDC solution was prepared by mixing equal volumes of the pyridine and EDC
hydrochloric acid solutions. A potassium hydroxide solution (15%, w/v) in methanolwater (80:20) was
prepared. Derivatitation procedure: To 50 ml of an aqueous mixture of standard dicarboxylic acids, 100
µl of NPH solution and 200 µl of working EDC solution were added, and the mixture was heated at 60
°C for 20 min. After the addition of 50 µl of potassium hydroxide solution, the mixture was further
heated at 60 °C for 15 min

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