Journal of Chromatography B, 791 (2003) 427–430 www.elsevier.com / locate / chromb Short communication Liquid chromatographic determination of hydroxyproline in tissue samples * Paul R. Hutson , Mark E. Crawford, Ronald L. Sorkness UW School of Pharmacy, 777 Highland Avenue, Madison, WI 53705-2222, USA Received 27 September 2002; received in revised form 25 February 2003; accepted 13 March 2003 Abstract We describe a reversed-phase assay of hydroxyproline in rat lung tissue using sarcosine for the internal standard and pre-injection reaction with both o-phthalaldehyde (OPA) and 9-fluorenylmethylchloroformate (FMOC). Intra-assay vari- ability in the concentration range of 25–500 mM hydroxyproline was less than 1%. Normal rat (left) lung was found to have a hydroxyproline content of 1.0860.18 mg / lung. This ability to measure minute amounts of hydroxyproline is being applied to the measure of collagen and pathological fibrosis.  2003 Elsevier Science B.V. All rights reserved. Keywords: Hydroxyproline; Amino acids 1. Introduction with other fluors [1]. 7-Chloro-4-nitrobenzo-2-oxa- 1,3-diazole (NBD-Cl) [2], phenylisothiocyanate This method was developed to satisfy a need to (PITC) [3], and 4-dimethylaminoazobenzene-49-sul- measure small changes in the amount of fibrosis in fonyl chloride (dabsyl chloride, DABS-Cl) [4] have rat lung tissue used as a model for human asthma. all been used in the secondary pre-column deri- Hydroxyproline is one of three predominant amino vatization of hydroxyproline. HPLC has been tried in acids constituting collagen. We sought to develop an the measurement of the hydroxyproline-NBD-Cl assay that could quantify changes in pulmonary derivative but yielded suboptimal sensitivity in the fibrosis by measuring increases in pulmonary hy- aqueous mobile phases [5]. Monboisse et al. have droxyproline content. demonstrated the specificity and sensitivity of an The use of fluorescent derivatives is common for HPLC assay combining sequential precolumn de- the measurement of amino acids. An initial reaction rivatization with OPA and 9-fluorenylmethyl- with o-phthalaldehyde (OPA) is currently used to chloroformate (FMOC) [6]. react with the primary amines, leaving secondary Einarsson and separately Mazzi et al. reported the amines such as proline and hydroxyproline to react application of this OPA/FMOC process to the HPLC analysis of the hydroxyproline in seawater, urine, and serum [7,8]. The FMOC derivative was mea- *Corresponding author. Tel.: 11-608-263-2496; fax: 11-608- sured by fluorescence detection and yielded an 265-5421. E-mail address: prhutson@pharmacy.wisc.edu (P.R. Hutson). extremely sensitive assay for hydroxyproline. Our 1570-0232 / 03 / $ – see front matter  2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S1570-0232(03)00248-4