Zhang Jun, Gage Eric M, Ji Qin C, El-Shourbagy Tawakol A
Department of Drug Analysis, Abbott Laboratories, Abbott Park, IL 60064, USA.
Rapid Commun Mass Spectrom. 2007;21(14):2169-76. doi: 10.1002/rcm.3046.
Levosimendan (Simdax) is an approved drug in approximately 40 countries and currently in phase III clinical studies in the USA and Europe. An accurate, high-throughput and rugged assay is critical to support these clinical trials. Due to the mechanism of drug metabolism, the drug and its active metabolites often have significant differences in their chemical properties. In order to achieve high assay throughput and low sample volumes, a single bioanalytical assay for the drug and its metabolites is preferred. However, this need may prevent the optimization of both high-performance liquid chromatography (HPLC) and mass spectrometric ionization conditions. The chemical properties of levosimendan are significantly different from those of its two active metabolites, OR-1855 and OR-1896. Here, we present a novel strategy for high-throughput analysis of levosimendan and its metabolites. A 96-well liquid/liquid extraction procedure was developed for sample preparation. A single liquid chromatography/tandem mass spectrometry (LC/MS/MS) system with two separate mobile phases, shared backwash solvent and conditioning solvent, was developed to perform sequential LC separation for levosimendan and the metabolites. Levosimendan was eluted by 5 mM ammonium acetate in 33.3% acetonitrile and detected using negative ionization mode MS/MS monitoring. The metabolites were eluted by 5 mM ammonium acetate and 0.2% acetic acid in 20% acetonitrile and detected with positive ionization mode MS/MS monitoring. The method has been demonstrated to have excellent precision and accuracy, with high assay ruggedness during method validation and clinical sample analysis. The linear dynamic ranges were approximately 200-50,000 pg/mL for levosimendan and approximately 500-130,000 pg/mL for both metabolites. The coefficient of determination (r2) for all analytes was greater than 0.9985. The intra-assay %CVs for QC samples were from 0.9% to 2.0% for levosimendan, 0.9% to 3.2% for OR-1855, and 0.4% to 4.9% for OR-1896. The inter-assay %CVs for QC samples were from 1.2% to 1.8% for levosimendan, 1.3% to 2.7% for OR-1855, and 1.4% to 3.4% for OR-1896. The mean % biases for QC samples were from 1.5% to 5.5% for levosimendan, -1.4% to 2.6% for OR-1855, and -0.3% to 4.5% for OR-1896. By using a single extraction approach coupled with sequential LC/MS/MS analysis for levosimendan and its metabolites, the assay maintained high throughput and low sample volume usage.
左西孟旦（Simdax）在约40个国家已获批准，目前正在美国和欧洲进行III期临床试验。一种准确、高通量且耐用的分析方法对于支持这些临床试验至关重要。由于药物代谢机制，药物及其活性代谢物在化学性质上往往存在显著差异。为了实现高通量分析和低样本量，针对药物及其代谢物采用单一生物分析方法较为可取。然而，这一需求可能会妨碍高效液相色谱（HPLC）和质谱电离条件的优化。左西孟旦的化学性质与其两种活性代谢物OR - 1855和OR - 1896有显著不同。在此，我们提出一种用于左西孟旦及其代谢物高通量分析的新策略。开发了一种96孔液 - 液萃取程序用于样品制备。开发了一种配备两种独立流动相、共享反冲洗溶剂和调节溶剂的单液相色谱/串联质谱（LC/MS/MS）系统，用于对左西孟旦及其代谢物进行顺序LC分离。左西孟旦用含5 mM醋酸铵的33.3%乙腈溶液洗脱，并采用负离子模式MS/MS监测进行检测。代谢物用含5 mM醋酸铵和0.2%乙酸的20%乙腈溶液洗脱，并采用正离子模式MS/MS监测进行检测。该方法在方法验证和临床样品分析过程中已证明具有出色的精密度和准确性以及高耐用性。左西孟旦的线性动态范围约为200 - 50,000 pg/mL，两种代谢物的线性动态范围约为500 - 130,000 pg/mL。所有分析物的决定系数（r2）均大于0.9985。质量控制（QC）样品的批内变异系数（%CV）对于左西孟旦为0.9%至2.0%，对于OR - 1855为0.9%至3.2%，对于OR - 1896为0.4%至4.9%。QC样品的批间变异系数（%CV）对于左西孟旦为1.2%至1.8%，对于OR - 1855为1.3%至2.7%，对于OR - 1896为1.4%至3.4%。QC样品的平均偏差百分比对于左西孟旦为1.5%至5.5%，对于OR - 1855为 - 1.4%至2.6%，对于OR - 1896为 - 0.3%至4.5%。通过对左西孟旦及其代谢物采用单一萃取方法结合顺序LC/MS/MS分析，该分析方法保持了高通量和低样本量使用。
