Simultaneous Quantification of Propofol, Ketamine and Rocuronium in Just 10 Μl Plasma Using Liquid Chromatography Coupled With
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Received: 1 November 2018 Revised: 19 February 2019 Accepted: 21 March 2019 DOI: 10.1002/bmc.4540 RESEARCH ARTICLE Simultaneous quantification of propofol, ketamine and rocuronium in just 10 μL plasma using liquid chromatography coupled with quadrupole mass spectrometry and its pilot application to a pharmacokinetic study in rats Teodora Shopova1 | Daniel Kiefer1 | Beate Wolf1 | Felix Maurer1 | Daniel I. Sessler2 | Thomas Volk1 | Tobias Fink1 | Sascha Kreuer1 1 CBR‐ Center of Breath Research, Department of Anaesthesiology, Intensive Care and Pain Abstract Therapy, Saarland University Medical Center The combination of propofol, ketamine and rocuronium can be used for anesthesia of and Saarland University Faculty of Medicine, Homburg/Saar, Germany ventilated rats. However, reliable pharmacokinetic models of these drugs have yet to 2 Michael Cudahy Professor & Chair, be developed in rats, and consequently optimal infusion strategies are also unknown. Department of Outcomes Research, Development of pharmacokinetic models requires repeated measurements of drug Anesthesiology Institute, Cleveland Clinic, Cleveland, Ohio, USA concentrations. In small animals, samples must be tiny to avoid excessing blood extraction. We therefore developed a drug assay system using high‐performance liq- Correspondence Teodora Shopova, CBR ‐ Center of Breath uid chromatography coupled with quadrupole mass spectrometry that simultaneously Research, Department of Anaesthesiology, determines the concentration of all three drugs in just 10 μL rat plasma. We Intensive Care and Pain Therapy, Saarland University Medical Center and Saarland established a plasma extraction protocol, using acetonitrile as the precipitating University Faculty of Medicine, Building 57, reagent. Calibration curves were linear with R2 = 0.99 for each drug. Mean recovery 66421 Homburg, Germany. Email: [email protected] from plasma was 91–93% for propofol, 89–93% for ketamine and 90–92% for rocuronium. The assay proved to be accurate for propofol 4.1–8.3%, ketamine 1.9– 7.8% and rocuronium −3.6–4.7% relative error. The assay was also precise; the intra‐day precisions were propofol 2.0–4.0%, ketamine 2.7–2.9% and rocuronium 2.9–3.3% relative standard deviation. Finally, the method was successfully applied to measurement the three drugs in rat plasma samples. Mean plasma concentrations with standard deviations were propofol 2.0 μg/mL ±0.5%, ketamine 3.9 μg/mL ±1.0% and rocuronium 3.2 μg/mL ±0.8% during ventilation. KEYWORDS anesthesia, ketamine, propofol, quadrupole mass spectrometer, rocuronium 1 | INTRODUCTION humans (Solima, Mofeed, & Momenah, 2017; Stevic et al., 2017; Wixson, White, Hughes, Lang, & Marshall, 1988). However, a useful Rats are commonly used in medical and other types of research; intravenous approach is to combine propofol (a GABAA‐receptor similar‐sized animals such as ferrets are also used, although much less agonist), ketamine (an NMDA‐receptor antagonist) and rocuronium commonly. For some studies, general anesthesia is required. There are (a nondepolarizing nicotinic receptor antagonist). The drugs are com- various anesthetic approaches for small animals, just as there are in bined to provide hypnosis, analgesia and relaxation during anesthesia. Biomedical Chromatography. 2019;33:e4540. wileyonlinelibrary.com/journal/bmc © 2019 John Wiley & Sons, Ltd. 1of8 https://doi.org/10.1002/bmc.4540 2of8 SHOPOVA ET AL. For this purpose, adequate blood levels must be achieved for each of buffer pH 4.9 in water (A) and 4 mM ammonium formate buffer the drugs. Insufficient doses can result in animals being conscious and pH 4.9 in 90% acetonitrile (B) and was applied for the separation of stressed during painful procedures; excessive doses can cause cardio- ketamine and rocuronium. The pH value of 4.9 was adjusted with vascular collapse or delayed recovery. 0.1% formic acid. A linear gradient with time program of the A:B vol- Even in humans, there is no method of assessing blood concentra- ume ratio was used as follows: 0 min (0:100) → 4 min tion of propofol, ketamine and rocuronium in real time. Clinicians thus (40:60) → 8 min (40:60) → 9 min (0:100) → 14 min (0:100). depend on complex multicompartment nonlinear pharmacokinetic A second gradient of 0.025% ammonium hydroxide in 70% aceto- models to guide drug administration. In practice, drugs such as nitrile was used to separate propofol under isocratic conditions for propofol are often given by target‐controlled computer systems that 4 min. The flow rate was set at 0.4 mL/min for both gradients. The col- use model parameters to continuously adjust infusion rates to target umn temperature was constant at 35°C for rocuronium and ketamine, a desired plasma concentration (Hu, Ph, Horstman, & Shafer, 2005). and 40°C for propofol. An Agilent 6130B quadrupole mass spectrom- The development of accurate models requires dozens of blood eter was used for the detection and quantification of the three com- samples over time in each animal, including the “tail” periods when pounds. Propofol was acquired in negative single ion mode using a drug concentrations are extremely low. However, measurements of 3 μL injection volume, and a second injection with the same volume drug concentrations in blood require a certain volume of blood. This was used for the detection of ketamine and rocuronium in positive can be a problem especially in small animals because of their very lim- single ion mode by electrospray ionization (Table 1). ited blood volume if numerous blood samples are taken within a short The following parameters were set for the spray chamber: capillary time (Johnson et al., 2003; Rönn, Lendemans, De Groot, & Petrat, voltage −3 kV for propofol and + 3 kV for ketamine and rocuronium. 2011). This can negatively affect the health of the test animal and thus The drying gas flow was set at 12 L/min and 350°C for the three falsify the study data. drugs. The data acquisition was performed using OpenLab CDS We therefore developed a new method for plasma extraction and C.01.05 Agilent. measurement of drug concentrations in just 10 μL plasma based on liquid chromatography (LC) coupled with a mass‐selective detector 2.3 | Standard solutions, calibration standards and (MS). Our goal was to develop and validate a system that simulta- quality control samples neously and accurately assays propofol, ketamine and rocuronium in an extremely small volume that is not critical even with frequent blood The propofol standard solution was obtained as a certificated sub- withdrawals in small animals. We initially tested the new quantifica- stance at a concentration of 1 mg/mL in methanol and was diluted at tion approach under laboratory experimental conditions, and thereaf- 0.25, 0.5, 1, 2, 3, 4 and 5 μg/mL. The ketamine standard solution ter in rats. contained 1 mg/mL ketamine in water and was diluted at 1, 2, 3, 4, 5, 6, 7, and 8 μg/mL. Standard solution of rocuronium was prepared 2 | EXPERIMENTAL in 10 mM ammonium acetate buffer pH 4.0 at a concentration of 1 mg/mL and calibration standards of 1, 2, 3, 4, 5, 6, 7 and 8 μg/mL were used. Stock solutions of propofol and ketamine were stored at 2.1 | Chemicals and reagents −20°C; the stock solution for rocuronium was stored at 8°C. Quality control solutions were prepared at 1 and 4 μg/mL for propofol, and Propofol was provided by Sigma Aldrich (Munich, Germany), Esmeron at 2 and 6 μg/mL for ketamine and rocuronium, all of which were (rocuronium bromide) was provided by Essex Pharma (Munich, Ger- stored at −20°C. All calibration standards and quality controls (QCs) many), and Ketamine O.K. (ketamine hydrochloride) was provided by were diluted in drug‐free rat plasma. Rotexmedica (Trittau, Germany). Acetonitrile and water were pur- chased from VWR (Darmstadt, Germany). Ammonium formate (purity >99%), formic acid (purity >99%), ammonium acetate (purity >99%) 2.4 | Sample preparation and ammonium hydroxide (25%) were obtained from Sigma‐Aldrich (Munich, Germany). All chemicals and reagents used in the study were Protein precipitation was used to prepare the plasma samples. A 10 μL MS analytical grade. plasma sample was added to a tube containing 390 μL acetonitrile TABLE 1 Mass spectrometry parameters for the detection of 2.2 | Instrumentation and LC–MS conditions propofol, ketamine and rocuronium. Dwell time was always set to 100 ms Chromatographic separation was performed on an Agilent (Santa Single ion mode Fragmentor Nebulizer Clara, CA, USA) 1260 Infinity Liquid Chromatography system Analytes m/z (V) (psi) equipped with an XSelect CSH C column (3.5 μm, 2.1 × 100 mm, 18 Propofol 177 70 35 Waters, USA) for propofol and an XBridge BEH Amide XP HILIC col- Ketamine 238 70 35 umn (2.5 μm, 3.0 × 150 mm, Waters, USA) for ketamine and Rocuronium 529 70 35 rocuronium. The mobile phase contained 4 mM ammonium formate SHOPOVA ET AL. 3of8 which was then vortexed for 10 s. After centrifugation at 10 000 rpm for 2.5.5 | Recovery and matrix effects 10 min at 4°C, 150 μL supernatant was transferred to a vial. The extraction recoveries for propofol, ketamine and rocuronium 2.5 | Validation were determined from spiked plasma concentrations. The recoveries were calculated by comparing the concentration of quality control The method was validated for linearity, limit of quantification (LOQ), samples with the concentration of identical standards. Identical stan- lower limit of quantification (LLOQ), extraction recovery, matrix dards were prepared from 20 μL drug‐free plasma precipitated with effects, precision and stability. The acceptance of all validation tests 780 μL of acetonitrile. After centrifugation, 390 μL supernatant was was selected at ±15%. taken out and spiked again with 10 μL 1 or 4 μg/mL propofol in ace- tonitrile, 2 or 6 μg/mL ketamine in acetonitrile, and 2 or 6 μg/mL 2.5.1 | Selectivity rocuronium in acetonitrile.