Vancomycin was purchased from Sigma-Aldrich, and atenolol (ATEN) as an internal standard (99.9%) was provided by the Institute of Drug Quality Control Ho Chi Minh City in Vietnam.

Acetonitrile, methanol, and water were of LC-MS grade and supplied by Merck (Darmstadt, Germany). All chemicals or solvents used in sample preparation met analytical standards.

Six batches of human plasma were provided by the Can Tho Hematology Blood Transfusion Hospital in Can Tho (Vietnam) and were kept at -20 °C. The protocol of this study was approved by the Human Investigation Ethics Committee in Biomedical Research of the Can Tho University of Medicine and Pharmacy, Vietnam, code 22.152.SV/PCT-HDDD. The authors hereby declare that all the procedures and experiments used in this study conformed with the ethical standards stipulated in the Helsinki Declaration of 1975, as revised in 2013, as well as national laws (World Medical Association 2013).

The experiments were performed on the ACQUITY UPLC H-Class PLUS System (Waters, Milford, MA, United States) coupled to Xevo TQD Triple Quadrupole Mass Spectrometry (Waters, Milford, MA, United States) equipped with electrospray ionization (ESI). The data were analyzed by the MasslynxTM version 4.2 software (Waters Corporation, Milford, MA, USA).

Chromatographic separation was performed on the Poroshell 120 Phenyl Hexyl Column (4.6 × 150 mm, 2.7 μm) at 25 °C. The mobile phase consisted of methanol and 0.1% formic acid (40:60, v/v) with a constant flow rate of 0.5 mL.min-1. The injection volume was 10 μL, and the total run time was 3.5 min. After the injection of each sample, the needle was rinsed alternately with methanol and water (80:20, v/v).

The ion source was set as ESI in positive mode with multiple reaction monitoring (MRM) modes. Each analyte was monitored in two different transitions, as presented in Table 1. Nitrogen was used as the desolvation gas, under the following conditions: capillary voltage: 4 kV; desolvation temperature: 400 °C, desolvation gas flow: 950 L.h-1. The collision gas was argon. Cone voltages and collision energies were optimized for each analyte individually, as presented in Table 1. The dwell time was 1.8 s.

Stock solutions of VAN and internal standard (IS) were prepared in methanol at 1000 μg/mL and 500 μg/mL, respectively. Working standard solutions had a concentration of 100 μg/mL for VAN and 500 ng/mL for IS by diluting the stock solutions in the same solvent.

Calibration standards and quality control (QC) samples were obtained by spiking human blank plasma with the working standard solutions. Calibration standards were prepared at concentrations corresponding to 0.1, 0.15, 0.3, 9, 18, 32, 40, 50, 60, and 75 μg/mL. QC samples were prepared at five concentration levels, including lower limit (LLOQ), low (LQC), medium (MQC), high (HQC), and ultra limit (ULOQ) concentrations of 0.1 μg/mL, 0.3 μg/mL, 18 μg/mL, 40 μg/mL, and 75 μg/mL, respectively. Internal standards were present in all of the calibration standards and QC samples at a constant concentration.

After thawing, a 300 µL aliquot of plasma was transferred into a microcentrifuge tube, followed by the addition of 150 µL of the IS working solution. To precipitate the protein, add 500 µL acetonitrile, 450 µL methanol, and 100 µL 0.1% formic acid. It underwent vortex mixing for 1 min. After centrifugation (at 10,000 rpm for 7 min), 500 µL of the supernatant phase was transferred to another tube with the addition of 500 µL formic acid 0.1% and then filtered via a 0.22 µm membrane before being injected into the UPLC-MS/MS system. The flowchart of the extraction procedure is provided in Fig. 1.

Figure 1. 

Process of plasma sample treatment.

The approach was validated in accordance with the US Food and Drug Administration (FDA) bioanalytical method guidelines, encompassing: system suitability, specificity, lower limit of quantification, linearity and calibration curve, inter- and intra-day accuracy, precision, extraction recoveries, stability experiments, matrix effect, carryover, and dilution integrity (FDA 2018).

In this study, method development was initiated by meticulously optimizing various critical aspects, including ionization, fragmentation conditions, chromatographic separation conditions, and sample preparation conditions.

Ionization and fragmentation conditions for vancomycin and atenolol were fine-tuned using the Masslynx™ version 4.2 software’s auto-tune function (Waters Corporation, Milford, MA, USA). Subsequently, parameters were carefully confirmed and manually adjusted within the software interface to ensure optimized analytical settings for precise and reliable mass spectrometry analysis. Positive-mode electrospray ionization (ESI) was chosen for its greater sensitivity in detecting both vancomycin and atenolol. To enhance specificity in identifying compound fragments, the MRM mode was employed. This combination ensures heightened sensitivity and improved specificity in detecting and characterizing target compound fragments. Vancomycin’s precursor ion appeared at an m/z ratio of 725.66, representing a doubly charged [M + 2H]2+ ion. The product ion of vancomycin at the m/z ratio of 144.16 results from the separation of the two parts of the glycoside group. Atenolol has been used as an internal standard in several studies (Zhang et al. 2007; Shou et al. 2014). Atenol served as an internal standard to enhance the accuracy, precision, and robustness of quantitation. Table 1 displays optimal mass spectrometry parameters, and Figs 2, 3 depict the mass spectrum plot and fragmentation mechanism for VAN and IS.

Figure 2. 

MS/MS spectra of VAN (A) and IS (B).

Figure 3. 

Fragmentation mechanism for VAN and IS.

Due to vancomycin’s polarity (XLogP3-AA = -2.6), reversed-phase liquid chromatography was employed to achieve separation and reduce retention time (Cheng et al. 2010; König et al. 2013; Lu et al. 2022). The Poroshell 120 Phenyl Hexyl Column (4.6 × 150 mm, 2.7 μm) was chosen for its unique selectivity for aromatic compounds like vancomycin, a characteristic not commonly found in other reversed-phase packings such as C18 and C8. Formic acid served as a pH adjuster, with this study exploring concentrations (0.05%, 0.1%, and 0.2%) as additives to the mobile phases, using methanol and acetonitrile as organic mobile phases (Zhang et al. 2007; Liu et al. 2018; Xiao et al. 2021). The final chromatographic conditions were established with a mobile phase of methanol and 0.1% formic acid in a 40:60 (v/v) ratio, maintaining a constant flow rate of 0.5 mL/min. The injection volume was 10 μL, and retention times for vancomycin and atenolol were 2.58 min and 2.82 min, respectively, within a total run time of 3.5 min. This brief analysis duration is crucial for ensuring efficient application in TDM processes and contributes to cost-saving measures.

The study aimed to devise a straightforward and time-efficient sample processing approach for vancomycin analysis using UPLC-MS/MS in human plasma. While solid-phase extraction (SPE) has proven effective in handling complex biological sample matrices, it often involves multiple steps and increases costs (Javorska et al. 2017). Our sample preparation method prioritizes simplicity, involving protein precipitation, centrifugation, and filtration. Acetonitrile, methanol, and 0.1% formic acid were employed for protein precipitation. Organic solvent precipitants enhance molecule attraction, reduce the dielectric constant of plasma protein solutions, and promote electrostatic protein interactions. Additionally, organic solvents displace organized water molecules around hydrophobic areas on protein surfaces (Polson et al. 2003). After centrifugation, formic acid is added to the supernatant to alter the medium’s pH, influencing the sample’s polarity and separation (Thao Nguyen Ngoc Nha et al. 2020). Adjusting the pH enhances peak shape, ensuring precise and high-quality analytical results. The method demonstrates high recovery rates.