(T1030-01-02) Evaluation of LC-MS Coupled with a Single Quadrupole Mass Spectrometer for Drug Concentration Analysis in Plasma Samples

Purpose: A Waters ARC HPLC system coupled with a Waters SQD2 single quadrupole mass spectrometer was used. Chromatographic separation was achieved using a Waters XBridge C18 column (4.6 × 50 mm, 3.5 μm particle size; Waters, USA). Detection was performed in selective ion monitoring mode, targeting OL at m/z 447.4 and its internal standard (IS) sorafenib at m/z 465.3, MET at 455.1 and its IS tinidene at m/z 248, and SIM at m/z 441.3 and its IS sorafenib at m/z 465.3. LC methods including mobile phases, flow rates and injection volumes were optimized for each drug. LC-MS Method validation for plasma sample measurement followed FDA guidelines, evaluating specificity, linearity, accuracy, precision, and stability in the autosampler.

Methods: A Waters ARC HPLC system coupled with a Waters SQD2 single quadrupole mass spectrometer was used. Chromatographic separation was achieved using a Waters XBridge C18 column (4.6 × 50 mm, 3.5 μm particle size; Waters, USA). Detection was performed in selective ion monitoring mode, targeting OL at m/z 447.4 and its internal standard (IS) sorafenib at m/z 465.3, MET at 455.1 and its IS tinidene at m/z 248, and SIM at m/z 441.3 and its IS sorafenib at m/z 465.3. LC methods including mobile phases, flow rates and injection volumes were optimized for each drug. LC-MS Method validation for plasma sample measurement followed FDA guidelines, evaluating specificity, linearity, accuracy, precision, and stability in the autosampler.

Results: LC methods for three drugs are summarized in Table 1. After optimization of mobile phases, all drugs were fully separated from interferences in plasma. Protein precipitation was used for sample preparation, with direct injection of the extracted samples into LC without drying. By optimizing LC conditions and MS parameters specifically for precursor ions (e.g., minimizing precursor ion collisions), the sensitivity of the single quadrupole MS was enhanced for drug quantification. This LC-MS method offers rapid sample preparation, a short run time ( < 5 minutes), and a low injection volume ( < 20 µL). As shown in Table 2, sensitivity, accuracy, and precision of each drug achieved the criteria of the FDA guidance and were comparable to those of triple quadrupole MS methods previously reported in literature. Injection samples were stable in the autosampler over 10 hours at 4°C.

Conclusion: By using three different drugs, we demonstrated that LC-MS coupled with a single quadrupole MS can quantitatively analyze OL, MET and SIM in plasma samples, offering an alternative to traditional triple quadrupole MS. These validated single quadrupole LC-MS methods offer reliable, rapid, and economical alternatives for drug quantitation in plasma. The methods delivered performance comparable to that of triple quadrupole systems while simplifying sample preparation and reducing analysis time. These findings highlight the potential of single quadrupole LC-MS systems for routine bioanalytical applications, particularly in early-stage research and resource-limited settings. This also provides the opportunity for laboratory with budget constrictions. These LC-MS methods are well-suited for quantitative analysis and can be effectively applied to measurements in preclinical studies for OL, MET and SIM.

References: 1. G. Loos, A. Van Schepdael, D. Cabooter, Quantitative mass spectrometry methods for pharmaceutical analysis, Philos Trans A Math Phys Eng Sci, 374 (2016).
2. X. Wang, D.I. Vernikovskaya, T.N. Nanovskaya, E. Rytting, G.D. Hankins, M.S. Ahmed, A liquid chromatography method with single quadrupole mass spectrometry for quantitative determination of indomethacin in maternal plasma and urine of pregnant patients, J Pharm Biomed Anal, 78-79 (2013) 123-128.

Acknowledgements: This work was supported by the National Institutes of General Medical Sciences [1R35GM138225] to Dong, X.

Table 1. LC methods for olmesartan, methotrexate and simvastatin.

Table 2. Precision, accuracy, LOQ and LOD of LC-MS methods for olmesartan, methotrexate and simvastatin. CV: coefficient variation. LOQ: limit of quantification. LOD: limit of detection.