High-performance liquid chromatography determination of pipecolic acid after precolumn ninhydrin derivatization using domestic microwave

• Published in: Analytical biochemistry Vol. 308; no. 2; pp. 320 - 327
• Main Authors: Moulin, Michaël, Deleu, Carole, Larher, François Robert, Bouchereau, Alain
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-09-2002
• Subjects: Amino acids; Brassica napus - chemistry; Brassica napus - drug effects; Brevibacterium - chemistry; Brevibacterium - drug effects; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Humans; Indicators and Reagents - chemistry; Microwaves; Ninhydrin - chemistry; Nitrous acid; Nitrous Acid - pharmacology; Osmotic stress; Pipecolic Acids - analysis; Plant Leaves - chemistry; Plant Leaves - drug effects; Secondary amines; Seeds - chemistry; Seeds - drug effects; α-Imino acids; α-Imino acids; Amino acids; Secondary amines; Nitrous acid; Osmotic stress
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/S0003-2697(02)00202-6

A novel procedure to specifically quantify low amounts of pipecolic acid and structurally related compounds in several types of biological materials has been characterized. From crude extracts of various types of biological material, the first step was to clear all low-molecular-weight compounds containing primary amino groups by a treatment of nitrous acid. Using a microwave-assisted reaction, the remaining substances containing secondary amino groups were then derivatized with ninhydrin and made soluble in glacial acetic acid. The derivatives produced were resolved by reverse-phase HPLC and detected by spectrophotometry at 570
nm. This procedure allowed more rapid determination of pipecolic acid since microwave heating shortened the time needed for derivatization compared with heating at 95
°C in a water bath. The complete analysis of the chromogens for pipecolic acid and related substances was achieved in 20
min. Under such conditions, the detection threshold for pipecolic acid was about 20
pmol. The suitability of the technique was assessed in various biological matrices known to contain significant amounts of this amino acid. The data obtained are in accordance with those available in the literature. To our knowledge, this is the first method using the ninhydrin reaction in a precolumn, microwave-assisted derivatization procedure for detection and determination of heterocyclic α-amino acids.