Cisplatin is the most widely used anticancer drug in the world. Mono-chloro and none-chloro complexes of cisplatin may be believed to be the activated compounds. The separation of these compounds using octa decyl silyl column or aminopropylsilyl silica gel column is difficult because of high-reactivity and structural similarity. In this study, cisplatin, hydroxo complexes, and OH-dimer were determined by HPLC using a naphthylethyl group bonded with silica gel (πNAP) column. The analytical conditions of HPLC were as follows: analytical column, πNAP column; wave length, 225 nm; column temperature, 40°C; mobile phase, 0.1 M sodium perchlorate, acetonitrile, and perchloric acid (290 : 10 : 3), flow rate, 1.0 mL/min. Sample (20 µL) was injected onto the HPLC system. Retention time of cisplatin, mono-chloride, OH-dimer, and none-chloride was 3.2, 3.4, 3.6, and, 4.3-6.6 min, respectively. Measurable ranges with this method were 1 10 5 to 4 10 3 M for cisplatin. Correlation coefficient of the calibration curves of cisplatin was 0.999 (p<0.01). The within-and between-day variations of coefficient of variation (CV) were 5% or lower. In this study, injectable formulations in physiological saline solution, water for injection, 5% glucose solution, and 7% sodium bicarbonate precisely were measured the stability and compositional changes upon mixing by πNAP column rather than C18 column. We successfully determined cisplatin, hydroxo complexes, and OH-dimer by HPLC using a πNAP column. Thus the measurement of cisplatin (cis-diamminedichloro-platinum(II), cis-[PtCl 2 (NH 3 ) 2 ]) (CDDP) should be done using a πNAP column rather than a C18 column or aminopropylsilyl silica gel column.Key words cisplatin; mono-chloro; none-chloro; OH-dimer; naphthylethyl group bonded silica gel column; aminopropylsilyl silica gel columnis an injectable anticancer agent used frequently in clinical practice.1-3) The chlorine (Cl) in CDDP is replaced with a water molecule in cells to form a hydrated complex and react with DNA.4) In addition, CDDP has been reported to change into various forms such as a hydrated complex and hydroxo-bridged complex (OH-dimer) 5) (Fig. 1). For the measurement of platinum agents including CDDP, the HPLC method as well as the atomic absorption spectrometry method and inductively coupled plasma-mass spectrometry method are employed. 6,7) For the former method, derivatization by an octadecyl chemically bonded silica (C18, ODS) column such as a 5C18-MS-II, NH 2 -bonded column, and cholester column is performed. However, this could not separate the mono-chloro, none-chloro, and dimer body of detached chlorine from CDDP (di-chloro). Therefore, CDDP derivatized by sodium diethyldithiocarbamate (NaDDTC) is used for the measurement.8-11) Furthermore, the identification tests by HPLC using aminopropylsilyl silica gel column (normal phase chromatography) is described in the Japanese Pharmacopoeia, Seventeenth Edition (JP17). For the latter method, the total content of platinum itself is being measured. To date, no studies have sep...