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. 2025 Oct;39(10):e70214.
doi: 10.1002/bmc.70214.

A Method for Analysis of Free and Total Ropivacaine in Dog Plasma Using UHPLC-MS/MS

Natali Verdier  1 , Karin Hummel  2 , Ebrahim Razzazi-Fazeli  2
Affiliations

A Method for Analysis of Free and Total Ropivacaine in Dog Plasma Using UHPLC-MS/MS

Natali Verdier et al. Biomed Chromatogr. 2025 Oct.

Abstract

Ropivacaine is a local anesthetic commonly used in veterinary anesthesia. A liquid chromatography-mass spectrometry (LC-MS) method was developed to quantify free and total ropivacaine in dog plasma, which included rapid equilibrium dialysis. The method was validated for selectivity, specificity, matrix effect, calibration curve and range, accuracy and precision, carry-over, stability, and reinjection reproducibility according to the International Conference on Harmonization M10 guidelines. After ultra-high performance liquid chromatographic (UHPLC) separation, detection and quantification of ropivacaine was performed using a triple quadrupole tandem mass spectrometer with electrospray ionization. LC-MS method validation was carried out in a range of 0.05-1000 ng/mL ropivacaine in dog plasma in two dilutions (1:1 and 1:4). The precision and accuracy of the method were determined at four concentration levels and ranged from 0.40% to 5.30% and 85.50% to 113.30%, respectively. The lower limit of quantification was as low as 0.30 and 0.05 ng/mL, for the quantitation of protein-bound (1:4) and free (1:1) ropivacaine, respectively. All validation parameters met acceptance criteria. This UHPLC-MS/MS method was successfully applied in a clinical study that involved the intraperitoneal instillation of ropivacaine to anesthetized dogs and can be used to quantify free and total ropivacaine in dog plasma.

Keywords: liquid chromatography; mass spectrometry; method validation; rapid equilibrium dialysis; ropivacaine.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Sample preparation scheme including rapid equilibrium dialysis.
FIGURE 2
FIGURE 2
Chromatograms of ropivacaine standard (XIC m/z 275.1 ➔ 84.0) and the internal standard D7‐ropivacaine (XIC m/z 282.1 ➔ 85.0) comparing the Kinetex C18 and the Kinetex F5 UHPLC column using gradient elution with two different flow rates: Kinetex C18 (a) 0.4 mL/min and (c) 0.3 mL/min, Kinetex F5 (b) 0.4 mL/min and (d) 0.3 mL/min.
FIGURE 3
FIGURE 3
Overlaid blank chromatograms of six dogs: (a) quantifier ropivacaine 1:1 dilution, (b) quantifier IS in 1:1 dilution, (c) quantifier ropivacaine in 1:4 dilution, (d) quantifier IS in 1:4 dilution, (e) qualifier ropivacaine in 1:1 dilution, (f) qualifier IS in 1:1 dilution, (g) qualifier ropivacaine in 1:4 dilution, and (h) qualifier IS in 1:4 dilution. The lack of peaks at 2.4 min demonstrates the absence of any significant interfering components.
FIGURE 4
FIGURE 4
Standard curve in (a) 1:1 dilution (used for buffer fraction) and (b) 1:4 dilution (used for plasma fraction).
FIGURE 5
FIGURE 5
Mean concentrations of ropivacaine and their standard deviation over time analyzed in (a) buffer and (b) plasma fractions of study samples after intraperitoneal instillation of 1 mg/kg ropivacaine to anesthetized dogs (R1, n = 4), and mean concentrations of ropivacaine and their standard deviation over time in (c) buffer and (d) plasma fractions of study samples after intraperitoneal instillation of 3 mg/kg ropivacaine to anesthetized dogs (R3, n = 4).
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References

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Web References

    1. European Medicines Agency . 2022. “ICH Guideline M10 on Bioanalytical Method Validation and Study Sample Analysis, Step 5.” Accessed February 20, 2025. https://www.ema.europa.eu/en/documents/scientific‐guideline/ich‐guidelin....

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