Automated Workflow for High-Throughput LC-MS/MS-Based Therapeutic Monitoring of Cannabidiol and 7-Hydroxy-cannabidiol in Patients with Epilepsy

Abstract

This study describes the development and validation of a fully automated workflow for serum sample preparation, enabling the quantitative determination of cannabidiol (CBD) and its active metabolite, 7-hydroxy-CBD, via liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. Implemented on an automated platform, the workflow performs key steps such as solvent dispensing, mixing, centrifugation, filtration, and supernatant transfer, producing 96-well plates ready for analysis. Human serum samples were obtained from patients with epilepsy treated with CBD. All samples were processed using both manual and automated methods to evaluate method agreement. Quantification was performed by LC-MS/MS with CBD-d₃ as the internal standard (IS). Method validation was conducted in accordance with European Medicine Agency (EMA) guidelines, confirming that the automated protocol meets the recommended acceptance criteria for both intraday and interday precision and accuracy. Calibration curves demonstrated excellent linearity across the concentration ranges. Comparative analysis using Passing-Bablok regression and Bland-Altman plots demonstrated strong agreement between the methods. These findings support the clinical applicability of the automated method for the therapeutic drug monitoring (TDM) of CBD and 7-hydroxy-CBD, and its robust performance and scalability provide a solid foundation for the development of an expanded analytical panel covering a broader range of antiseizure medications (ASMs), enabling more standardized TDM protocols in clinical practice.

Keywords: 7-hydroxy-CBD; CBD; LC-MS/MS; TDM; automation; epilepsy.

Figure 3

Representative chromatogram obtained from a serum sample processed using the manual preparation method, showing CBD and 7-hydroxy-CBD at concentrations of 49 ng/mL and 81 ng/mL, respectively. CBD (red line); 7-hydroxy-CBD (grey line); CBD-d₃ (light blue line).

Figure 4

Representative chromatogram obtained from a serum sample processed using the automated preparation method, with measured concentrations of CBD and 7-hydroxy-CBD at 49 ng/mL and 80 ng/mL, respectively. CBD (red line); 7-hydroxy-CBD (grey line); CBD-d₃ (light blue line).

Figure 1

Representative curves of CBD (a) and 7-hydroxy-CBD (b) in serum (manual sample preparation).

Figure 2

Representative curves of CBD (a) and 7-hydroxy-CBD (b) in serum (automated sample preparation).

Figure 5

Passing–Bablok regression plots showing the correlations of CBD (a) and 7-hydroxy-CBD (b) concentrations, reported in ng/mL, between the manual and automated methods (n = 70 pairs), with dotted lines representing the 95% CIs.

Figure 6

Bland–Altman plots comparing CBD (a) and 7-hydroxy-CBD (b) concentrations obtained from serum samples prepared using manual versus automated methods (n = 70 pairs). The x-axis represents the mean concentration of each pair, while the y-axis indicates the difference between the two preparation methods. For CBD, the mean bias was 7.1316 ng/mL (95% CI: −57.3373 to 71.6004). The lower limit of agreement (LoA) (95% CI) was −0.7113 ng/mL (−70.9216 to −43.7530), and the upper LoA (95% CI) was 14.9745 ng/mL (58.0162 to 85.1847). For 7-hydroxy-CBD, the mean bias was −6.9539 ng/mL (95% CI: −50.0299 to 36.1221), with a lower LoA (95% CI) of −12.1942 ng/mL (95% CI: −59.1064 to −40.9533) and an upper LoA (95% CI) of −1.7135 ng/mL (95% CI: 27.0456 to 45.1987). The solid line denotes the mean bias, while the dotted lines represent the LoA. A fitted regression line with its 95% CI is also included.

Figure 7

Schematic representation of the high-throughput automated sample preparation platform: (1) Biomek i7 hybrid workstation; (2) positive pressure unit; (3) microplate centrifuge; (4) robotic arm; (5) ambient storage unit.

Figure 8

Schematic layout of the Biomek i7 deck configuration: (1) tip racks; (2) source labware; (3) 96-well elution plate; (4) solvent reservoir and centrifuge balance; (5) 96-well filtration plate positioned for integration with the positive pressure unit; (6) Biomek i7 servo shuttle system for plate positioning; and (7) moving tray enabling automated labware transfer to the positive pressure unit module.