GC Labs

Sung-Eun Cho, M.D., Ph.D.

Endocrine Substance Analysis Center

The Necessity of Introducing Automated Pretreatment Method for Quantitative Mass Spectrometry Analysis

With the advancement of liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods, renowned for their high accuracy and precision, the complexity of sample pretreatment methods has also significantly increased. The sample pretreatment step is intricate, labor-intensive, and time-consuming, making it a critical determinant of the turnaround time (TAT) for LC-MS/MS testing. It is also one of the most challenging steps in adopting and implementing LC-MS/MS methods.

The sample pretreatment step, often referred to as sample cleanup or sample preparation, primarily involves separating and concentrating target analytes from the sample matrix while removing or minimizing complex matrix interferences to refine the sample for LC-MS/MS analysis. This process enhances chromatographic performance and ensures more reliable analytical results. A variety of techniques are employed for pretreatment method in quantitative mass spectrometry. However, most methods remain manual or partially automated. Representative pretreatment methods are summarized in Table 1.

Table 1. MS/MS sample preparation methods

Recently, efforts have been made to automate various stages of LC-MS/MS, including the sample pretreatement step. Automation allows laboratories to manage staff more efficiently, reduce TAT, enhance the reliability of test results, and increase sample throughput. 

At our institution, the demand for LC-MS/MS tests for metanephrine and normetanephrine has surged, creating an urgent need for the adoption of automated pretreatment methods. In response, the Endocrine Substance Analysis Center (ESAC) implemented Janus G3 Automated Workstations (Revvity, Turku, Finland) to develop an automated pretreatment method for plasma metanephrine and normetanephrine quantification. This method was established as a laboratory-developed test (LDT). The clinical utility of this automated method is compared to manual methods and introduced in this study. A front view of the automated workstation is provided in Figure 1.

Fig. 1. Picture of the Janus G3 Automated Workstations (Revvity) in GCLabs ESAC

Development Process of Automated Pretreatment Method for Quantitative Mass Spectrometry

This automated pretreatment method is not only the first successfully developed in Korea but also the first case of its practical application in actual operations. Based on solid-phase extraction (SPE), this automated method represents an innovative approach in which all steps, from the initial sample barcode reading after sample loading to dispensing the sample into insert vials for mass spectrometry injection, are fully automated. The program for executing the automated pretreatment method was developed and configured according to each sequence, and the execution sequence of the automated pretreatment method was set up as follows: 

The automated pretreatment equipment is set up with barcode tube racks for samples, vial racks, 1000 µL pipette tip racks, MDT pipette tip racks, reagent reservoirs (troughs), shaking modules, Oasis WCX plates for SPE, positive pressure manifolds, and reaction plates (Fig. 2).

Fig. 2. There are barcode tube racks for sample, vial racks, 1000 µL pipette tip racks, Modular dispense technology (MDT) pipette tip racks, reagent reservoir (trough), shaking module, solid phase extraction (SPE) module, positive pressure manifold, reaction plate, elution plate, and incubator module in this Revvity Janus G3 Automated Workstations (Revvity, Turku, Finland).

The troughs are filled with 0.1% formic acid in ACN, 2% formic acid in ACN, IS, 100% MeOH, and DW, respectively. The barcode labels attached to the sample tubes are scanned automatically to identify double blank samples, blank samples, calibrators, quality control materials (QC materials), and sample information. Then, 150 µL of IS (2 ng/mL in water) is automatically dispensed into each well of the 96-well reaction plate, followed by the automatic addition of 150 µL of double blanks, blanks, calibrators, QC materials, and samples into each well. All processes, from barcode scanning to sample dispensing, are fully automated. The steps of the automated pretreatment method are summarized in Table 2.

Table 2. Serial steps of automated sample pretreatment method using Janus G3 Automated Workstations (Revvity)

Clinical Utility of the Automated Pretreatment Method for Quantitative Mass Spectrometry

Upon completing the setup of the method, all performance evaluations demonstrated excellent results. Compared to the manual pretreatment method, the automated method showed the following for plasma metanephrine: a correlation coefficient of 0.9958, a slope of 1.008 (0.994–1.022), and an intercept of -0.002 (-0.007–0.003). For plasma normetanephrine, the correlation coefficient was 0.9976, the slope was 0.993 (0.982–1.004), and the intercept was 0.005 (-0.008–0.018), indicating strong correlations for both analytes.

In terms of time efficiency, the manual pretreatment method required a total of 81 minutes when performed by skilled personnel and 184 minutes by unskilled personnel. In contrast, the automated system’s intrinsic processing time was 49 minutes, significantly shorter than the manual method. Including the manual tasks preceding automation (sample centrifugation and barcode labeling), the total processing time for the automated pretreatment method was 90 minutes, compared to 121 minutes (skilled personnel) and 234 minutes (unskilled personnel) for the manual method.

After the adoption of the automated pretreatment method, the foundation successfully reduced TAT, alleviated examiner workloads, and improved job satisfaction among examiners. Furthermore, the pretreatment step was carried out stably and efficiently, regardless of the examiners’ skill levels, enhancing the overall quality of the final test results. This improvement is expected to lead to increased test requests in the future. Despite a rise in test volumes, it is anticipated that all requested tests can be successfully completed without fluctuations in TAT.

Future Prospects of the Automated Pretreatment Method for Quantitative Mass Spectrometry

The automated pretreatment method requires highly specialized personnel for the complex LDT setup process, and the initial implementation cost is substantial. These factors pose challenges to its widespread adoption in most mass spectrometry laboratories. However, in laboratories with mass spectrometry experts where a high workload from manual pretreatment methods causes examiner burdens and increased TAT, the adoption of this automated method may offer significant advantages over maintaining manual processes.

The foundation plans to continue developing and implementing automated pretreatment methods for various mass spectrometry applications. The Janus G3 Automated Workstations (Revvity) used for this process provide extensive scalability. Future adaptations will include supported liquid extraction (SLE) pretreatment methods, incubation processes, and nitrogen drying processes, among others. These newly developed automated methods will also be applied to mass spectrometry-based assays, such as renin activity measurement and aldosterone quantification.

Ultimately, the goal is to establish a fully automated total laboratory automation (TLA) system encompassing all phases, from pre-analytical stage to post-analytical stages, promoting the activation and efficiency of mass spectrometry testing.

References

01.    Grebe SKG, Singh RJ. LC-MS/MS in the Clinical Laboratory - Where to From Here?. Clin Biochem Rev. 2011;32:5-31.

02.    Chae H, Kim H, Kim JE, Lee JJ, Cho SE, Kim S, et al. Evaluation of a Robotic Liquid Handler, Andrew+, for Sample Preparation for LCMS/MS-Based Quantitation of Immunosuppressants in Whole Blood. Lab Med Online. 2023;13: 199-204.

03.    Vogester M and Kirchhoff F. Progress in automation of LC-MS in laboratory medicine. Clin Biochem 2011;44:4-13.

04.    Salvagno GL, Danese E, Lippi G. Mass spectrometry and total laboratory automation: opportunities and drawbacks. Clin Chem Lab Med. 2020;58:994-1001 

05.    Yu S, Zhou W, Yu J, Li M, Zhang S, Jian X, et al. An automated magnetic bead extraction method for measuring plasma metanephrines and 3-methoxytyramine using liquid chromatography tandem mass spectrometry. Anal Bioanal Chem. 2022;414:3541-9.

06.   Stone JA, van Staveren DR, Fitzerald RL. Automated Sample Preparation Enables LC-MS/MS as a Routine Diagnostic Analysis for Serum Testosterone. J Appl Lab Med. 2017;2:33-46.