MSACL 2017 US Abstract

‘Chromatogra-Free’: Ultra-Rapid LC-MS/MS Analysis Demonstrated using Clozapine and Norclozapine

Lewis Couchman (Presenter)
Viapath, King's College Hospital

Bio: Lewis is a clinical scientist at Viapath Analytics, London. He is extensively involved in the development, evaluation and implementation of clinical mass spectrometry methods in clinical chemistry, in therapeutic drug monitoring, and in toxicology. He has a particular interest in novel sample preparation methods and high-throughput analysis using LC-MS/MS.

Authorship: Lewis Couchman, Danielle Fisher, Krithika Subramaniam, Simon Handley, Robert Flanagan
Viapath Analytics, Toxicology Unit, Department of Clinical Biochemistry, King’s College Hospital, London, UK.

Short Abstract Typically, targetted quantitative LC-MS/MS analyses using gradient elution are carried out at the rate of a few minutes per injection. In this paper, an approach for ultra-rapid LC-MS/MS analysis will be described in which the complete injection-to-injection cycle-time is just 36 seconds, or one 96-well plate per hour. This cycle time is more akin to that of direct injection methods such as flow-injection analysis, but includes efficient chromatographic separation of target analytes and matrix components. Practical considerations and requirements for this approach will be demonstrated using two target analytes, clozapine and norclozapine, following automated extraction from plasma samples.

Long Abstract

Typically, targetted quantitative LC-MS/MS analyses using gradient elution are carried out at the rate of a few minutes per injection. In this paper, an approach for ultra-rapid LC-MS/MS analysis will be described in which the complete injection-to-injection cycle-time is just 36 seconds, equating to one 96-well plate per hour. This cycle time is more akin to that of direct injection methods such as flow-injection analysis, but includes efficient chromatographic separation of target analytes and matrix components. Practical considerations and requirements for this approach will be demonstrated using two target analytes, clozapine and norclozapine, following automated extraction from plasma samples.

Using a fully-automated and selective sample preparation method, we have previously demonstrated that direct injection of sample extracts is suitable for the analysis of these compounds. However, the use of direct injection is not widely applicable, and throughput is limited by (i) MS/MS data-file processing time and (ii) the autosampler cycle time. The latter of these is especially important to consider for assays prone to carry-over, in which extensive needle-washing is required. The total analysis time (injection-to-injection) for our previously validated direct injection method for clozapine and norclozapine was 60 s [Couchman et al., 2016]. In this paper, we describe the implementation and validation of a novel method for the analysis of clozapine and norclozapine using the same fully-automated sample preparation method, but with extracts analysed by ultra-rapid LC-MS/MS. Despite the inclusion of a chromatographic step, the total analysis time was much less than that described in our previously validated direct injection approach.

Typical retention times were 11.4 s and 12.4 s for norclozapine and clozapine, respectively. The internal standards co-eluted (± 0.3 s) with each analyte, and the peak width (at base) was < 1 s. MRM product ion ratio (% peak area of quantifier ion transition) acceptance criteria were 50 ± 5 % for clozapine and 60 ± 5 % for norclozapine. The isotopic purity for each of the ISs was >97 %. Intra-batch and inter-batch precision (% RSD) and accuracy (% nominal concentrations) were < 8 % and 95-110 %, respectively for both analytes. Carryover was less than 0.3 % for both analytes.

For the analysis of clozapine and norclozapine, this ultra-rapid LC-MS/MS approach has allowed a reduced total analysis time compared to our previously validated direct injection method, with the added analytical robustness of LC-MS/MS. As we previously concluded, direct analyis of sample extracts is not applicable to analyses that require LC to resolve isobaric species/metabolites, or those where matrix effects are not compensated for by suitable internal standards. This novel approach could therefore have much wider application in targetted analyses in analytical toxicology and in other areas.

References & Acknowledgements:

Couchman L et al. Automated analysis of clozapine and norclozapine in human plasma using novel extraction plate technology and flow-injection tandem mass spectrometry. Ther Drug Monit 2016; 38: 42-49.

Thanks to Robert Boughtflower (GSK, Ware, UK) for valuable input, and Christopher Benton (Agilent Technologies) for support.

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