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8Research Results in Pharmacology
Research Results in Pharmacology 9(2): 55-59
UDC: 615.074 DOI 10.18413/rrpharmacology.9.10027
Development and validation of a quantitative HPLC/MS/MS method for the determination of piperacillin in blood plasma
Vladimir I. Petrov1-2, Ivan S. Anikeev1-2, Tatyana E. Zayachnikova3, Andrey V. Strygin1-2-4, Anna M. Dotsenko1-2-4
1 Volgograd State Medical University of the Ministry of Health of the Russian Federation, 1 Pavshikh Bortsov Sq., Volgograd 400131 Russia
2 Center for Innovative Medicines with Pilot Production of Volgograd State Medical University of the Ministry ofHealth of the Russian Federation, 39Novorossiyskaya St., Volgograd 400087Russia
3 Institute of Continuing Medical and Pharmaceutical Education of Volgograd State Medical University of the Ministry of Health of the Russian Federation, 1 Pavshikh Bortsov Sq., Volgograd 400131 Russia
4 State budgetary institution Volgograd Medical Research Center, 1 Pavshikh Bortsov Sq., Volgograd 400131 Russia
Corresponding author: Ivan S. Anikeev (anikeivan(ô)yandex.ru)
Academic editor: Tatyana Avtina ♦ Received 11 February 2023 ♦ Accepted 21 April 2023 ♦ Published 01 June 2023
Citation: Petrov VI, Anikeev IS, Zayachnikova TE, Strygin AV, Dotsenko AM (2023) Development and validation of a quantitative HPLC/MS/MS method for the determination of piperacillin in blood plasma. Research Results in Pharmacology 9(2): 55-59. https://10.18413/rrpharmacology.9.10027
Introduction: Reduced mortality in patients with sepsis taking piperacillin is possible when they receive a long-term infusion, which improves the effect of antimicrobials. However, such piperacillin therapy requires therapeutic drug monitoring, the use of the latest analytical equipment and developed methods for the quantitative determination of piperacillin.
Materials and Methods: Dry samples of the appropriate certified piperacillin standards were used to prepare stock and standard solutions of piperacillin. Separation of the components was performed using an Agilent 1260 HPLC system with a binary pump and a temperature controlled autosampler. Analyses were detected using a Sciex QTRAP 5500 hybrid mass spectrometric system. Validation of the developed method was carried out in accordance with the rules for conducting bioequivalence studies of drugs within the framework of the Eurasian Economic Union; 2016, in Astana.
Results and Discussion: Piperacillin ions-"precursors" corresponded to particles m/z 518.2. The most intense ions-"products" registered during the fragmentation of protonated molecules in the collision cell were particles m/z 143.1 and m/z 115.0. During the validation of the developed method, the main validation parameters were established: linearity, accuracy, accuracy, and sensitivity (lower limit of quantitation).
Conclusion: The validated analytical range of the method was 0.5-100 ng/mL in plasma. The resulting analytical range makes it possible to apply the developed method for conducting the analytical part of studies of the pharmacokinetics of piperacillin.
Abstract
Copyright Petrov VI et a I. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Graphical abstract:
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Keywords
HPLC/MS, validation, quantitation, piperacillin, bioanalytics
Introduction
A reduction in mortality in patients with sepsis taking piperacillin is possible when they receive a long-term infusion, which improves the effect of antimicrobials. However, such piperacillin therapy requires therapeutic drug monitoring (TDM), which is a promising strategy for individualization and increasing the effectiveness of antibiotic therapy. Therapeutic drug monitoring with dosing adapted to the altered pharmacokinetics of the individual patient will avoid underdose or overdose. (Barco et al. 2015; Schmitt et al. 2017; Verhoven et al. 2018; Capiauet al. 2019).
However, for the correct application of therapeutic drug monitoring and the study of a sufficiently large number of samples, it is necessary to use the latest analytical equipment and developed methods for the quantitative determination of piperacillin (Cohen-Wolkowiez et al. 2014; Berm et al. 2016; Anikeev et al. 2018; Hagel et al. 2019).
Therefore, our goal was to develop and validate a highly sensitive and selective HPLC-MS/MS method for the determination of piperacillin in human plasma.
Aim of the study. Finding the optimal conditions for the quantitative determination of piperacillin in blood plasma using HPLC-MS/MS and validation of the method.
Materials and Methods
Chemicals and reagents
For the preparation of stock and standard solutions of piperacillin and prazosin as an internal standard, dry
weights of the corresponding certified standard of piperacillin (Sigma Aldrich, Milan, Italy), special highly purified water (Milli-Q), formic acid for HPLC-MS, acetonitrile, (Scharlab, Spain) were used. The matrix for the preparation of calibration standards and quality control samples was human blood plasma obtained from healthy volunteers.
Equipment
Dry substances were weighed using an Ohaus Explorer EX225/AD semi-microanalytical balance (Ohaus, USA). Separation of the components was performed using an Agilent 1260 HPLC system with a binary pump and a temperature controlled autosampler (USA). Analyzed substances were detected using a hybrid mass spectrometric system Sciex QTRAP 5500 (Singapore), Shaker (Biosan, Latvia), Special analytical software Analyst 1.6.
Preparation of stock and standard solutions
To prepare a standard solution of piperacillin and an internal standard with a concentration of 1.00 mg/mL, dry samples were weighed and then placed in a 25-mL volumetric flask and made up to the mark with ultrapure water. The final concentration of working solutions was: 5; 10; 25; 50; 100; 200. and 1000 mcg/mL.
Preparation of calibration and quality control samples
Using a dispenser, 100 jil of plasma were transferred into 1.5 ml microtubes, 10 jil of a working solution of the appropriate concentration was added with a dispenser until calibration solutions of 0.5; 1; 2.5; 5; 10; 20 and 100 |ug/ml for piperacillin.
Quality control (QC) samples were prepared at the following four concentration levels: 0.5 |g/mL (LQQ), 1.5 |g/mL (low QC), 50 |g/mL (medium QC), and 75 |g/mL (high QC) for piperacillin.
Chromatographic and Mass Spectrometric Conditions
Chromatographic separation of the components was carried out on a Poroshell 120 C18 column (4.6x50 mm, 2.7 |m). When developing the conditions for the mass spectrometric detection of the desired substances by the method of multiple reaction monitoring (MRM), ions-" precursors" and the corresponding ions - "products" were determined. Piperacillin "precursor" ions corresponded to particles m/z 518.2. The most intense ions-"products" registered during the fragmentation of protonated molecules in the collision cell were particles m/z 143.1, m/z 115.0 (Li et al. 2012, 2013; Kegeli et al. 2014; Barco et al. 2015; D'Cunha et al. 2018).
Validation
Validation of the developed method was carried out in accordance with the rules for conducting bioequivalence studies of drugs within the framework of the Eurasian Economic Union; 2016, in Astana (Council of the Eurasian Economic Commission 2016). During the validation of the developed method, the main validation parameters will be established: linearity, accuracy, correctness, sensitivity (lower limit of quantitation), and linearity.
To validate the linearity, the calibration samples were examined, which corresponded to the concentrations: 1; 2; 5; 10; 20; 50 and 100 |g/mL for piperacillin. Calibration curves were constructed by plotting peak area ratios versus plasma concentrations using a weighted 1/ xA2 linear regression model. The lower limit of quantitation was defined as the lowest concentration on the calibration curve, which was 1 |g/mL.
Statistical analysis
For the detection of analytes, a Sciex QTRAP 5500 hybrid mass spectrometric system was used. Chromatography-mass spectra were recorded using the Analyst 1.6 software. Peak integration, calculation of the quantitative content of the studied compounds, and statistical data processing were carried out using the MultiQuant 2.4 program.
For quantitative determination, a calibration curve method with a weighting factor of 1/x2 was used. The ratio of the peak areas of the analyte and the internal standard was taken as a parameter (Timmerman et al. 2011; Jager et al. 2014; Fan et al. 2019; Li et al. 2013; Mu 2019).
Results and Discussion
Piperacillin ions-"precursors" corresponded to particles m/z 518.2. The most intense ions - "products" registered during the fragmentation of protonated molecules in the collision cell were particles m/z 143.1, m/z 115.0 (Fig. 1). In the course of optimizing the conditions for chromatographic separation, an isocratic elution mode was chosen.
The mobile phase was 80/20 acetonitrile/water at a flow rate of 0.3 mL/min. The mobile phase modifier was 0.1% formic acid, which was added to both the
aqueous and organic components of the mobile phase. Under these conditions, the retention time of piperacillin was 1.86 min (Fig. 2).
Figure 1. Mass spectrum of piperacillin in blood plasma: along the abscissa axis - the ratio of mass to charge m/z (Da), along the ordinate axis - the signal intensity.
Figure 2. Chromatogram of piperacillin in blood plasma: along the abscissa axis - time (min); along the ordinate axis - signal intensity.
During the validation of the developed method, the main validation parameters were established: linearity, precision, correctness, sensitivity (lower limit of quantitation), stability, and matrix effect. The developed method confirmed its linearity in the concentration range from 0.5 to 100 ^g/mL using a weighted coefficient 1/ xA2, with rA2 > 0.99. The coefficient of variation (%) calculated when determining the inter- and intraday accuracy did not exceed 15% for the main range of concentrations (Table 1).
Successful TDM requires the use of specific validated methods, knowledge of new physicochemical approaches to sample preparation of biosamples, and the availability of modern equipment.
We have developed and validated methods for the quantitative HPLC-MS/MS determination of: piperacillin in blood plasma using standard sample preparation methods (protein precipitation, etc.).
When developing the conditions for the mass spectrometric detection of the desired substances by the method of multiple reaction monitoring (MRM), ions-" precursors" and the corresponding ions-"products" were determined. The "precursor" ion of piperacillin was the ion m/z 518.2. The most intense ions-"products" registered during the fragmentation of protonated molecules in the collision cell were particles m/z 143.1 and m/z 115.0.
The developed methods confirmed their linearity in the selected concentration ranges using the weighted
Table 1. Table of validation parameters for the quantitative determination of piperacillin for the method using protein precipitation as a sample preparation
Parameter Value
LLOQ (0.5 ^g/mL) LQC (1.5 ^g/mL) MQC (50 ^g/mL) HQC (75 ^g/mL)
Within run Precision (CV %) Between cycles 4.1 14.8 11.2 14.2 3.70 7.1 5.5 6.7
Within the cycle 113.5 98.8 92.4 102.1
Correctness (%)
Between cycles 92.1 95.8 97.5 105.2
Stability (%) Selectivity (%) Correlation coefficient
5.1
96.8
0.99
107.9
Note: LLOQ - lower limit of quantification (lower limit of quantification); LQC - Low level (lower concentration level); MQC - Medium level (medium concentration level); HQC - High level (upper concentration level).
coefficient 1/xA2, with rA2>0.99. The coefficient of variation (%) calculated in determining the inter- and intraday accuracy did not exceed 15% for the main range of concentrations for all drugs.
The lower limit of quantification of the procedure was determined based on linearity, precision, and precision data.
This analytical method for the determination of piperacillin in blood plasma using HPLC-MS/MS will make it possible to use it in a wide routine practice, especially for the correct use of therapeutic drug monitoring and the study of a sufficiently large number of samples in the determination of piperacillin.
Conclusion
In this study, the optimal conditions for the quantitative determination of piperacillin in human plasma were established using chromatographic and mass spectrometric equipment.
The method has been validated according to all the requirements of the Eurasian Economic Union and the European Medicines Agency and meets all the requirements for bioanalytical methods. The validated analytical range of the method was 0.5-100 ^g/mL in plasma. The resulting analytical range makes it possible to apply the developed method for conducting the analytical part of studies of the pharmacokinetics of piperacillin.
Conflict of Interests
The authors declare no conflict of interests.
Funding
The study was supported by the Russian Science Foundation (Project no. 22-25-20207).
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Author Contributions
■ Vladimir I. Petrov, Doctor of Sciences (Medicine), Professor, Member of the Russian Academy of Sciences; Head of the Department of Clinical Pharmacology and Intensive Care, Volgograd State Medical University; Director of the Scientific Centre of Innovative Medicines with Pilot Production, Volgograd State Medical University; Chief freelance specialist - clinical pharmacologist of the Ministry of Healthare the Russian Federation; Honored Scientist of the Russian Federation; Honored Doctor of the Russian Federation; e-mail: brain@sprintnet.ru; ORCID ID https://orcid.org/0000-0002-0258-4092. The author's contribution: development of the design of the study, editing and final approval of the article.
■ Ivan S. Anikeev, Head of the Laboratory of Pharmacokinetics, Scientific Center of Innovative Medicines with Pilot Production; Assistant of the Department of Fundamental Medicine and Biology, Volgograd State Medical University; e-mail: anikeivan@yandex.ru; ORCID ID https://orcid.org/0000-0002-9384-4338. The author's contribution: conception, planning of the article, review of literary sources, collection of materials, writing and editing the article.
■ Tatyana E. Zayachnikova, Candidate of Sciences (Medicine), Associate Professor, Professor of the Department of Pediatrics and Neonatology, Institute of Continuous Medical and Pharmaceutical Education, Volgograd State Medical University; e-mail: guz5deti@mail.ru; ORCID ID https://orcid.org/0000-0001-6758-4686. The author's contribution: conception, planning of the article, review of literary sources, collection of materials, writing and editing the article.
Andrey V. Strygin, Candidate of Sciences (Medicine), Associate Professor, Head of the Department of Fundamental Medicine and Biology; Deputy Director of Scientific Center of Innovative Medicines with Pilot Production; Head of the Laboratory of Genomic and Proteomic Research, Volgograd Medical Research Center; email: drumsav@mail.ru; ORCID ID https://orcid.org/0000-0002-6997-1601. The author's contribution: development of the design of the study, editing and final approval of the article.
■ Anna M. Dotsenko, Assistant of the Department of Fundamental Medicine and Biology, Volgograd State Medical University; Junior Researcher, Laboratory of Genomic and Proteomic Research, Volgograd Medical Research Center; e-mail: ev8278@mail.ru; ORCID ID https://orcid.org/0000-0003-3324-3351. The author's contribution: conception, planning of the article, review of literary sources, collection of materials, writing and editing the article.
