Haarhoff Zuzana, Wagner Andrew, Picard Pierre, Drexler Dieter M, Zvyaga Tatyana, Shou Wilson
Bristol-Myers Squibb, Wallingford, CT, USA
Bristol-Myers Squibb, Wallingford, CT, USA.
J Biomol Screen. 2016 Feb;21(2):165-75. doi: 10.1177/1087057115607184. Epub 2015 Sep 29.
The move toward label-free screening in drug discovery has increased the demand for mass spectrometry (MS)-based analysis. Here we investigated the approach of coupling acoustic sample deposition (ASD) with laser diode thermal desorption (LDTD)-tandem mass spectrometry (MS/MS). We assessed its use in a cytochrome P450 (CYP) inhibition assay, where a decrease in metabolite formation signifies CYP inhibition. Metabolite levels for 3 CYP isoforms were measured as CYP3A4-1'-OH-midazolam, CYP2D6-dextrorphan, and CYP2C9-4'-OH-diclofenac. After incubation, samples (100 nL) were acoustically deposited onto a stainless steel 384-LazWell plate, then desorbed by an infrared laser directly from the plate surface into the gas phase, ionized by atmospheric pressure chemical ionization (APCI), and analyzed by MS/MS. Using this method, we achieved a sample analysis speed of 2.14 s/well, with bioanalytical performance comparable to the current online solid-phase extraction (SPE)-based MS method. An even faster readout speed was achieved when postreaction sample multiplexing was applied, where three reaction samples, one for each CYP, were transferred into the same well of the LazWell plate. In summary, LDTD coupled with acoustic sample deposition and multiplexing significantly decreased analysis time to 0.7 s/sample, making this MS-based approach feasible to support high-throughput screening (HTS) assays.
药物研发中向无标记筛选的转变增加了对基于质谱（MS）分析的需求。在此，我们研究了将声学样品沉积（ASD）与激光二极管热解吸（LDTD）-串联质谱（MS/MS）相结合的方法。我们评估了其在细胞色素P450（CYP）抑制试验中的应用，其中代谢物形成的减少表明CYP受到抑制。测量了3种CYP同工型的代谢物水平，分别为CYP3A4-1'-羟基咪达唑仑、CYP2D6-右啡烷和CYP2C9-4'-羟基双氯芬酸。孵育后，将样品（100 nL）声学沉积到不锈钢384孔LazWell板上，然后通过红外激光直接从板表面解吸到气相中，通过大气压化学电离（APCI）进行离子化，并通过MS/MS进行分析。使用该方法，我们实现了2.14秒/孔的样品分析速度，其生物分析性能与当前基于在线固相萃取（SPE）的质谱方法相当。当应用反应后样品多路复用时，实现了更快的读出速度，即将三个反应样品（每个CYP一个）转移到LazWell板的同一孔中。总之，LDTD与声学样品沉积和多路复用相结合显著将分析时间缩短至0.7秒/样品，使得这种基于质谱的方法能够支持高通量筛选（HTS）试验。
