Journal of Chromatography B, 799 (2004) 311–321 Reversed-phase liquid chromatography method to determine COL-3, a matrix metalloproteinase inhibitor, in biological samples Jing Li a , Hung Huynh b , Eli Chan a,∗ a Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore b Laboratory of Molecular Endocrinology, National Cancer Center, Singapore 169610, Singapore Received 11 June 2003; received in revised form 22 October 2003; accepted 28 October 2003 Abstract A reversed-phase high-performance liquid chromatographic (HPLC) method with ultraviolet (UV) detection was developed and vali- dated for the quantification of 6-deoxy-6-demethyl-4-dedimethylamino-tetracycline (COL-3), a matrix metalloproteinase (MMPs) inhibitor, in rat serum. This simple, sensitive, rapid and reproducible assay involved a preliminary serum deproteinization by adding a mixture of acetonitrile–methanol–0.5 M oxalic acid (70:20:10 (v/v)), as the combined precipitant and metal blocking agent, into serum samples (2:1 (v/v)). An aliquot (20 l) of the supernatant was injected into the HPLC system linked to a Waters XTerra TM RP 18 column (150 mm × 4.6 mm i.d., particle size 5 m). The compound was eluted by a mixture of acetonitrile–methanol–0.01 M oxalic acid (40:10:50 (v/v), pH 2.00), as the mobile phase, and detected at the wavelength of 350 nm. The total running time was 10 min. The low and high concentration calibration curves were linear in the range of 50–1200 ng/ml and 1200–12,000 ng/ml, respectively. The intra- and inter-day coefficients of variation at three quality control concentrations of 100, 1200, and 12,000 ng/ml were all less than 6%, while the percent error ranged from -2.5 to 6.6%. The limit of quantitation (LOQ) for COL-3 in serum was 50 ng/ml. This assay was successfully employed to study the serum concentration–time profiles of COL-3 after its intravenous and oral administration in rats. The method with some minor modifications in sample pretreatment was also applicable to the determination of the concentrations of COL-3 in rat bile, urine and feces. © 2003 Elsevier B.V. All rights reserved. Keywords: COL-3; Metalloproteinase inhibitor 1. Introduction 6 - Deoxy - 6- demethyl- 4- dedimethylamino - tetracycline (COL-3), also named as Metastat ® or CMT-3 (Fig. 1), is a new synthetic non-antimicrobial tetracycline analogue. It has been developed as a potential anti-tumor agent based on its inhibition of matrix metalloproteinases (MMPs), which belong to a family of Zn 2+ -dependent proteinases that proteolytically degrade the extracellular matrix [1,2]. An imbalance between active MMPs and MMP inhibitors causes degradation of the basement membrane to allow angiogenesis, tumor growth, and metastasis [3–5]. COL-3 directly inhibits MT1-MMP (membrane-type-1 MMP) expression and activity and pro-MMP-2 expression in osteosarcoma cells [1]. In addition, COL-3 inhibits cell ∗ Corresponding author. Tel.: +65-68742932; fax: +65-67791554. E-mail address: phaelic@nus.edu.sg (E. Chan). proliferation and induces apoptosis in a number of tu- mor cells [3,6]. In vivo studies have demonstrated that COL-3 has both anti-tumor and anti-metastasis activities [7,8]. COL-3 has undergone the Phase I clinical trial in the pa- tients with refractory metastatic cancer [9] and AIDS-related Kaposi’s sarcoma [10]. A reversed-phase high-performance liquid chromatography (HPLC) with atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) de- tection has been developed for quantification of COL-3 in human plasma [11]. COL-3 is separated on a Waters Symmetry ® C 18 column with oxalic acid (0.01 M, pH 2.2)–acetonitrile (55:45 (v/v)) mobile phase. This method is specific and sensitive, with the limit of quantitation (LOQ) at 30 ng/ml for a 25 l injection of the reconstituted drug solution [11]. However, this assay method is hampered by the relatively large inter- and intra-day errors (mean, <18.3%) and coefficients of variation (mean, <14.9%) for all of the three quality control concentrations tested (50, 1570-0232/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2003.10.066