Soman A, Qiu Y, Chan Li Q
Analytical Sciences Department, Research and Development Center, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT 06877, USA.
J Chromatogr Sci. 2008 Jul;46(6):461-5. doi: 10.1093/chromsci/46.6.461.
A reversed-phase high-performance liquid chromatographic method (HPLC) with diode-array detection is developed and validated for the quantitative determination of formaldehyde in a drug substance. Formaldehyde (HCHO) is reacted with 2,4-dinitrophenylhydrazine (DNPH) to form a Schiff base (HCHO-DNPH derivatization product), which has an absorbing maximum (lambda max) at 360 nm. The HPLC method employs a C8, 3-microm particle size analytical column (150 mm x 4.6 mm), 15-microL injection volume, column temperature controlled at 30 degrees C, detection at 360 nm, and a water-acetonitrile (55:45, v/v) mobile phase at a flow rate of 1 mL/min. These conditions resolve the HCHO-DNPH product from unreacted DNPH, the drug substance and related impurities, as well as diluent peaks within 20 min. The retention time of the HCHO-DNPH product is approximately 6.4 min. The method is linear, accurate in the specified range (0.33-333 ppm), and robust based on analyte (HCHO-DNPH derivatization product) stability in standard and sample. Detection limit is 0.03 ng (0.1 ppm).
开发并验证了一种采用二极管阵列检测的反相高效液相色谱法（HPLC），用于定量测定原料药中的甲醛。甲醛（HCHO）与2,4-二硝基苯肼（DNPH）反应形成席夫碱（HCHO-DNPH衍生化产物），该产物在360nm处有最大吸收峰（λmax）。HPLC方法采用C8、3μm粒径的分析柱（150mm×4.6mm），进样体积15μL，柱温控制在30℃，检测波长为360nm，流动相为水-乙腈（55:45，v/v），流速为1mL/min。这些条件可在20分钟内将HCHO-DNPH产物与未反应的DNPH、原料药及相关杂质以及稀释剂峰分离。HCHO-DNPH产物的保留时间约为6.4分钟。该方法在规定范围（0.33 - 333ppm）内呈线性、准确，且基于标准品和样品中分析物（HCHO-DNPH衍生化产物）的稳定性具有稳健性。检测限为0.03ng（0.1ppm）。
