ВЭЖХ анализ пролина и 4-гидроксипролина в биологических жидкостях. HPLC analysis of proline and 4-hydroxyproline in biological fluids

Alexei A. Dutov; Denis A. Nikitin; Maria N. Mishchenko; Anastasia N. Semenova; Anastasia V. Martynova; Anna V. Sverkunova; Ekaterina N. Fedorova; Olga N. Konovalova; Julia L. Lukyanova; Alena V. Yermolina; Joseph S Pinelis

Alexei A. Dutov МD (clinical pharmacology), physician of the highest qualifying category (clinical laboratory diagnostics), Senior Research Fellow Laboratory of Experimental and Clinical Biochemistry and Immunology, Research Institute of Medical Ecology of the Medical Academy, Chita. e-mail: dutovaa@yandex.ru
Denis A. Nikitin head of the laboratory of chemical analysis of the Zabaikalsky State University, Chita, e-mail: nikitinnd@gmail.com
Maria N. Mishchenko cand. sci. med., assistant Department of Dentistry, Medical Academy, Chita
Anastasia N. Semenova post-graduate student Surgical Dentistry 3th year of study Medical Academy, Chita
Anastasia V. Martynova post-graduate student Department of Chemistry 1th-year training Zabaikalsky State University, Chita
Anna V. Sverkunova post-graduate student of chemistry 1th-year learning the Zabaikalsky State University, Chita
Ekaterina N. Fedorova post-graduate student Department of Chemistry, 3th-year training Zabaikalsky State University, Chita
Olga N. Konovalova post-graduate student Department of Chemistry, 3th-year training Zabaikalsky State University, Chita
Julia L. Lukyanova physician ordinator Department of Neurology, Medical Academy, Chita
Alena V. Yermolina physician ophthalmologist Regional Hospital № 2, Chita
Joseph S Pinelis MD, professor, head of dental surgery Medical Academy, Chita

Keywords: HPLC, Fluorimetric detection, proline, 4-hydroxyproline, plasma, saliva, plaintive liquid (tear).

Abstract

An HPLC method for measuring proline and 4-hydroxyproline in human plasma, saliva and plaintive liquid
(or tear) was validated. Biological samples were deproteinized using acetonitrile. Iminoacids derivatization was
achieved with 9-fluorenylmethyl chloroformate (Fmoc-Cl) after blocking primary amino acids with
orthophthaldialdehyde/2-mercaptoethanol. The derivatives were separated by reversed phase (C18) HPLC and
fluorimetric detection at ex260-em330 nm. We are used columns Luna C18(2) (75 ´ 4.6 mm, 3 μm), Gemini C18
(150 ´ 4.6 mm, 5 mm), both from Phenomenex and high-speed columns (100 ´ 4.6 mm) Chromolith Performance
RP-18e from Merck. For screening on hyperprolinemia in newborn proposed separation method for high-speed
columns with monolithic sorbent Chromolith Performance RP-18e. Simplicity, reproducibility and high sensitivity of
a method, allow its use in a clinical practice for an estimation of biochemical changes at eye and stomatologic
diseases and also for screening on congenital hyperprolinemia at newborn

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