DETERMINATION OF ZPT IN SHAMPOOS BY HPLC AND HPLC-MS/MS 267 PREPARATION OF STANDARDS ZPT calibration standards were freshly made up at the start of each batch of analysis. Typically, the standard (20.0 mg) was suspended in 90 ml methanol–acetonitrile (1:9, v/v) and subjected to ultrasonic treatment in the dark. Then the solution was cooled to the room temperature, made up to 100 ml and passed through a 0.22 μm fi lter. A set of standard solutions were produced by diluting the mixture to 1.00(for HPLC-MS/MS method only), 3.20(for HPLC method only), 10, 50, and 100 μg·ml−1. ZPT standards were stored in the refrigerator in darkness when not in use. HPLC ANALYSIS HPLC method was carried out using a Waters 2695 system and Shiseido MG C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase was 0.01 mol·l−1 potassium dihydrogen phosphate, adjusted 0.5 mmol·l−1 EDTA-Na2 at pH 4.0 with phosphoric acid, methanol– acetonitrile gradient of run time of 25 min (Table I). Column temperature was main- tained at 25°C, wavelength was 272 nm, fl ow rate was 1 ml·min−1, injection volume was 10 μl. The retention time of ZPT was 10.5 min. Figure 1 showed the ultraviolet spec- trum of ZPT standard (210–400 nm). HPLC-MS/MS ANALYSIS HPLC-MS/MS method was carried out using a Thermo TSQ Quantum Access system operated under positive polarity. Tandem mass spectrometry was performed under the following conditions: discharge current: 4 μA, vaporizer temperature: 400°C, sheath gas pressure: 40 psi, aux gas pressure: 5 psi, capillary temperature 300°C. Multiple reaction monitoring mode (MRM) was performed using the fragmentation transitions of m/z 317→m/z 173.8, m/z 317→m/z 189.8 (Table II). Gradient separations were performed on a Merck Chromolith Performance RP-18e col- umn (100 mm × 3 mm, 2 μm). The mobile phase was 1mmol·l−1 ammonium acetate– methanol–acetonitrile run over a gradient (Table III). Column temperature was maintained at 30°C, fl ow rate was 0.25 ml·min−1, injection volume was 20 μl. The retention time of ZPT was about 5.0 min. Table I Gradient Elution Program (HPLC) Time (min) Potassium dihydrogen phosphate solution (%) Acetonitrile (%) Methanol (%) 0.0 85 5 10 5.0 85 5 10 8.0 40 50 10 18.0 10 80 10 19.0 10 80 10 20.0 85 5 10 25.0 85 5 10

JOURNAL OF COSMETIC SCIENCE 268 SAMPLE EXTRACTION OF ZPT A 0.500 g amount of sample was accurately weighed into a 50ml-stoppered centrifuge tube, 5–10 ml of water was added. The tube was shaken vigorously, and then centrifuged at 8000 rpm for 5 min. The supernatant was discarded, and the residue was mixed with 40 ml methanol–acetonitrile (1:9, v/v) by a vortex mixer. After ultrasonic extraction for 30 min, the solution was cooled to the room temperature, and diluted to 50 ml with methanol–acetonitrile and centrifuged at 8000 rpm for 5 min. An aliquot of the solution was fi ltered through a 0.22-μm membrane fi lter prior to analysis. The residue was ex- tracted the second time by methanol–acetonitrile in the same way. RESULTS AND DISCUSSION SAMPLE EXTRACTION Although the sample extracted by methanol–acetonitrile could analyze directly, it had much surfactants or impurities that might pollute the chromatography and mass spectrometry system. Since ZPT does not dissolve in water, the research used water to remove surfactant and water-soluble impurities in the sample. After washing the samples with water in the fi rst step, the chromatography of the sample was much cleaner (Figure 2). Table II Mass Parameters of MRM Mode for ZPT Determination Qualitative ion Quantitative ion Collision energy (V) 317 173.8 317 173.8 18 317 189.8 24 Figure 1. UV spectrum of ZPT standard.