This study used adult male Sprague–Dawley (SD) rats weighing 270–300 g, purchased from Dossy Technology Co., Ltd. (Chengdu, China). Animals were housed under standard laboratory conditions with controlled temperature (22 °C–25 °C) and humidity (50%–60%) under a 12 h light/dark cycle, with free access to food and water. All animal experimental procedures were conducted in accordance with the guidelines for the care and use of laboratory animals and were approved by the Animal Ethics Committee of West China Hospital, Sichuan University (approval number: 20250910005), with all efforts made to minimize animal suffering and reduce the number of animals used.

Anesthesia was induced by placing the rats in an induction chamber with 5% isoflurane delivered in oxygen at a fresh gas flow of 1.0 L/min until loss of the righting reflex was achieved. After induction, anesthesia was maintained with 2% isoflurane via inhalation. Tracheal intubation was then performed, and the rats were connected to a small-animal mechanical ventilator (RWD, Shenzhen, China) for controlled ventilation. A 24-gauge intravenous catheter (Surflo Flash, Terumo, Japan) was inserted into the left femoral artery for serial blood sampling, while a separate 24-gauge catheter was placed in the tail vein for intravenous drug administration. Anesthetic doses were selected to approximate clinically relevant dosing, with ET (1.5 mg/kg), ROC (5 mg/kg), and LID (2 mg/kg) administered sequentially. Blood samples were collected at predefined time points (1, 3, 5, 10, 20, and 30 min) after drug administration for plasma concentration analysis. Given the rapid in vivo metabolism of ET, plasma concentration measurements for ET were performed only up to 20 min post-administration.

The Cell portable MS (C3001-sci, Suzhou Purspec Technology, Suzhou, China) is a tandem mass spectrometry–based analytical platform capable of selective precursor ion fragmentation and multistage mass spectrometric analysis of compounds, including isomers. The system consists of an ion source, ion transmission module, linear ion trap, ion detection unit, and integrated control system. The instrument measures 333 × 235 × 146 mm, weighs less than 8.5 kg, and supports a mass range of 50–1,000 m/z with switchable positive and negative ion modes. Detailed procedures for instrument calibration and quality control are provided in Supplementary Figure S1 and the Supplementary material.

MS detection was performed in positive ion mode. Quantification of ET and LID was conducted using an internal standard (IS) method. For ET, the precursor ion was m/z 245 and the fragment ion was m/z 141, with ET impurity C used as the IS (precursor ion m/z 259, fragment ion m/z 155.1). For LID, the precursor ion was m/z 235.1 and the fragment ion was m/z 86.1, with LID-d10 used as the IS (precursor ion m/z 245.2, fragment ion m/z 96.2). For ROC, the precursor ion was m/z 529.4 and the fragment ion was m/z 487.3. Although verapamil was initially evaluated as an IS, it exhibited pronounced matrix effects in the Cell MS, which could compromise quantitative accuracy; therefore, ROC was quantified using an external standard method. For analysis, A 100 μL of whole blood sample was collected without centrifugation and mixed with 400 μL of acetonitrile. For ET and LID, whole blood samples were mixed with acetonitrile containing the IS. Subsequently, 100 μL of the resulting mixture was transferred into the direct capillary spray reagent kit (Suzhou Purspec Technology, Suzhou, China). Each sample was analyzed using multiple consecutive scans in the Cell portable MS, with a total acquisition time of approximately 4.5 min per sample.

High-performance liquid chromatography with mass spectrometry (HPLC–MS, Agilent 1,260, Japan) was used as the reference method for quantitative analysis. Chromatographic separation was achieved on an Ultimate XB-C4 column using gradient elution with 0.1% formic acid aqueous solution (A) and acetonitrile (B) at a flow rate of 0.3 mL/min. Detection was performed on a triple quadrupole MS equipped with an electrospray ionization source operated in positive ion mode and multiple reaction monitoring (MRM). Plasma samples were processed by protein precipitation with acetonitrile containing the IS, followed by centrifugation and injection of the supernatant. Calibration curves covered ranges of 2–1,000 ng/mL for ET and LID and 10–10,000 ng/mL for ROC. Detailed analytical conditions are provided in the Supplementary material. Representative HPLC–MS chromatograms demonstrating adequate chromatographic separation of ET, ROC, and LID are shown in Supplementary Figure S2. Concentrations determined by HPLC–MS were used as reference values for comparison with Cell portable MS measurements.

Analytical method validation was conducted in accordance with the principles of the International Council for Harmonization (ICH) guidelines, with a focus on key performance characteristics relevant to this preliminary proof-of-concept study. Method specificity, linearity, limit of detection (LOD), and limit of quantification (LOQ) were evaluated to assess the feasibility of the Cell portable MS for rapid quantification of multiple anesthetic drugs.

This study was designed as a preliminary feasibility study; therefore, a small sample size (n = 3 rats) was used. Statistical analysis was performed using GraphPad Prism software (Version 10.4, Boston, USA). Data were presented descriptively, with individual values shown for each animal. The standard curves were fitted by linear regression models. For ET and LID, the theoretical concentrations were used as the independent variable (x), and the IS–corrected peak area ratios were used as the dependent variable (y). For ROC, the theoretical concentration was used as the independent variable (x), and the peak intensity was used as the dependent variable (y). Considering the greater variability in ROC responses at low concentration levels, a weighted least-squares regression with a 1/x weighting factor was applied to improve fitting stability, whereas ordinary least-squares linear regression was used for ET and LID. The goodness of fit was evaluated using the coefficient of determination (R²), with R² ≥ 0.99 indicating good linearity.

Pearson correlation analysis was performed to assess the linear relationship between anesthetic drug concentrations measured by the Cell portable MS and HPLC–MS, with the correlation coefficient (r) and its statistical significance were calculated. An r value > 0.95 was considered to indicate an extremely strong correlation, reflecting high consistency in measurement trends between the two methods (19).

Agreement between the two platforms was further evaluated using Bland–Altman analysis. The percentage difference (%Difference) between measurements obtained by the Cell portable MS and HPLC–MS was plotted on the y-axis against the mean on the x-axis, and the 95% limits of agreement (LoA) were calculated. Agreement was considered acceptable when most data points fell within the 95% LoA and no proportional bias was observed across the concentration range (20).

To evaluate the analytical specificity of the Cell portable MS for ET, ROC, and LID, MS analyses were performed using blank and standard blood samples, and the corresponding full-scan mass spectra are shown in Supplementary material. For ET (Supplementary Figure S3), no interfering signals were observed at m/z 228.0 in blank samples, whereas a distinct ET peak was detected at the same m/z in standard samples. Similarly, no background interference was detected at the characteristic ions of ROC (m/z 487.2; Supplementary Figure S4) or LID (m/z 85.9; Supplementary Figure S5) in blank samples, while clear and well-resolved signals were observed in standard samples. These results demonstrated that the Cell portable MS enabled reliable identification of ET, ROC, and LID based on their characteristic ions and exhibited good analytical specificity in complex blood matrices.

Standard curves were established using a series of standard samples across graded concentration levels. For ET (Figure 1A), calibration was performed over concentrations of 2, 4, 8, 20, 40, 80, 200, and 1,000 ng/mL, yielding a regression equation of y = 0.01496x + 0.3136 with an R² of 0.997. For ROC (Figure 1B), weighted linear regression (1/x) was applied across concentrations of 10, 20, 50, 100, 500, 1,000, 5,000, and 10,000 ng/mL, resulting in the equation y = 1,356.03x + 12,785.42 with an R² value of 0.991. For LID (Figure 1C), calibration over concentrations of 2, 4, 10, 20, 50, 200, 500, and 1,000 ng/mL produced a regression equation of y = 0.01459x + 0.0067 with an R² of 0.999. Detailed regression parameters, including the standard deviation of the slope and intercept and the residual standard deviation, are provided in the Supplementary Table S1. Overall, all three anesthetic drugs demonstrated good linearity within their respective tested concentration ranges.

The LOD and LOQ for the three anesthetic drugs were determined using a signal-to-noise (S/N) approach. For ET, the LOD was 49.35 ng/mL at an S/N ratio of 3, and the LOQ was 213.39 ng/mL at an S/N ratio of 10. Similarly, the LOD and LOQ for ROC were 9.63 ng/mL and 54.10 ng/mL, respectively, while those for LID were 1.36 ng/mL and 5.63 ng/mL. Detailed calculation procedures are provided in the Supplementary material.

Figures 1D–F show the concentration–time curves for the three drugs measured by Cell portable MS and HPLC–MS. The peak concentrations and overall concentration–time trends of ET, ROC, and LID were consistent between the two platforms, in accordance with their expected in vivo pharmacokinetic behavior. Further Pearson correlation analysis (Figures 1G–I) demonstrated strong correlations in the measured concentrations of all three anesthetic drugs between the two analytical platforms, with correlation coefficients close to 1.0 (ET: r = 0.9666; ROC: r = 0.9858; LID: r = 0.9937) and all p values < 0.0001. These results indicated a high degree of consistency in the measurement trends of drug concentration changes between the two instruments.

Bland–Altman analysis was then conducted to evaluate the agreement between the two platforms (Figures 1J–L). The mean biases for ET, ROC, and LID were −9.38%, 6.77%, and −3.44%, respectively, indicating relatively small overall bias. For all three drugs, most points fell within the 95% LoA, and no proportional bias was observed across the tested concentration ranges. These results indicated that the Cell portable MS exhibited good quantitative agreement in the measurement of plasma concentrations of ET, ROC, and LID, with results showing good comparability to those obtained using conventional HPLC–MS.