Simultaneous Quantification of Propofol, Ketamine and Rocuronium in Just 10 Μl Plasma Using Liquid Chromatography Coupled With

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

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.

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 acetonitrile, 2 or 6 μg/mL ketamine in acetonitrile, and 2 or 6 μg/mL 2.5.1 | Selectivity rocuronium in acetonitrile. The recovery was calculated as:

Selectivity was evaluated by comparing the chromatograms of blank peak area of quality control Recovery ¼ × 100% rat plasma (n = 6) with the corresponding plasma samples spiked with peak area of identical standard propofol, ketamine and rocuronium.

2.5.2 | Linearity and lower limit of quantification Plasma matrix effects were evaluated to identify any suppression or enhancement of signal from an interfering substance around the Calibration curves were constructed for all calibration standards at retention time of propofol, ketamine and rocuronium using the known concentration against the measured peak area according to matrix factor (ME, %): Section 2.3. The linearity of the response was investigated by plotting ‐ the calibration curves (regression lines using the least squares method) peak area of spiked standard in plasma 2 ME ¼ 1 − × 100% and calculating the correlation coefficient (R ). At least 75% of the peak area of standard in acetonitrile back‐calculated concentrations of the calibration standards should be with ±15% of the nominal value. The LLOQ was defined as the lowest where ME = 0 indicates the absence of matrix affect, otherwise concentration on the calibration curve. The precision and accuracy for denoting ionization suppression (ME <0) or enhancement (ME >0). LLOQ samples were accepted within 20%.

2.5.3 | Limit of quantification 2.5.6 | Stability and carryover

Limits of quantification were determined from a specific calibration The stability of propofol, ketamine and rocuronium in plasma was curve with calibration standards (250, 125, 62.5, 31.2, 15.6 and evaluated on QC samples at low and high concentration levels. The 7.8 ng/mL) measured in triplicate for each drug. The standard devia- QCs were analyzed after the applied storage condition: 60 days at tion of the lowest calibration standard (σ) and the slope of the regres- −20°C, or 24 h at 8°C. The concentrations of all samples were investi- sion line (S) were used for the calculation. gated against calibration curve, obtained from freshly spiked calibration standards. Carryover was investigated on the peak area of blank 10*σ plasma samples on the expected retention time for propofol, ketamine LOQ ¼ S and rocuronium after application of high‐concentration standards at 5 or 8 μg/mL. 2.5.4 | Accuracy and precision 2.6 | Animals Accuracy and precision were evaluated using QC samples prepared in ‐ six replicates at low and high concentrations levels. The intra and With approval by the Institutional Animal Care and Use Committee ‐ inter day precisions (RSD, %) and the accuracy (RE, %) were measured (no. 45/2016 Landesamt für Soziales, Saarland, Saarbrücken, using four runs analyzed on the same day and on three separate days. Germany), 20 male Sprague–Dawley rats (200–350 g) were obtained RSD and RE values were determinated as: from Charles River Laboratories International (Sulzfeld, Germany) for the determination of the plasma concentrations of the three drugs. A standard deviation of mean measured concentration continuous infusion of 30 mg/kg/h of propofol and the same dose of RSD ¼ × 100% mean measured concentration ketamine was given through a jugular venous catheter. We also gave the rats a single bolus injection of 10 mg/kg rocuronium which was followed by an infusion at 12.5 mg/kg/h. Blood samples (30 μL, ðÞmeasured concentration − nominal concentration sufficient to produce 10 μL plasma) were collected via a catheter RE ¼ × 100% nominal concentration from the carotid artery three times for each rat. 4of8 SHOPOVA ET AL.

3 | RESULTS AND DISCUSSION were ±15% of the nominal value. The correlation coefficients R2 obtained were >0.9850 for all curves validated as critical validation 3.1 | Method validation parameters. The mean regression equations and correlation coefficients are shown in Table 2. 3.1.1 | Selectivity 3.1.3 | Limit of quantification The mean column retention times of propofol, ketamine and rocuronium were 1.72, 3.34 and 6.19 min, respectively (Figure 1). The limit of quantification was 10.1 ng/mL for propofol, 2.4 ng/mL for Blank plasma from six rats was tested and no interfering endogenous ketamine and 7.6 ng/mL for rocuronium, and was defined as the low- peaks were observed on the relevant retention times, which indicated est concentration of the standard curve that can be quantitated with that the assay was selective. statistical assurance.

3.1.2 | Linearity and lower limit of quantification 3.1.4 | Accuracy and precision

The method was linear across the concentration ranges tested for all The assessment of accuracy and precision were conducted by analyz- three drugs. All measured concentrations of the calibration standards ing six replicates of QC samples through four validation days. The

FIGURE 1 Typical chromatograms for (a1) blank rat plasma, (b1) blank rat plasma spiked with propofol and (c1) a rat plasma sample after venous administration of propofol, (a2) blank rat plasma, (b2) blank rat plasma spiked with ketamine and rocuronium and (c2) a rat plasma sample after venous administration of ketamine and rocuronium SHOPOVA ET AL. 5of8

TABLE 2 Precision (%, RSD), accuracy (%, RE), regression equation, correlation coefficient (R2) and lower limits of quantification (LLOQ) for propofol, ketamine and rocuronium of calibration standard samples in drug‐free plasma

Nominal Inter‐day (n =4) concentration Regression equation LLOQ Analytes (μg/mL) Measured (μg/mL) RSD (%) RE (%) y = R2 (μg/mL)

Propofol 5 4.95 1.2 −0.6 9389x + 1251 0.999 0.25 4 3.94 0.5 −1.6 3 3.03 2.2 1.0 2 2.08 1.6 4.1 1 0.95 0.8 −5.0 0.5 0.48 5.1 −4.0 0.25 0.24 7.8 −3.1 Ketamine 8 7.74 1.0 −3.2 365,962x + 289,626 0.994 1 7 7.03 0.3 0.5 6 5.99 1.2 −0.2 5 5.35 0.5 7.1 4 4.11 0.4 2.7 3 3.02 1.2 0.8 2 1.88 2.0 −5.8 1 0.87 1.6 −13.4 Rocuronium 8 7.88 0.4 −1.5 92,510x + 36,410 0.996 1 7 7.03 0.8 0.5 6 5.84 0.8 −2.6 5 5.18 1.2 3.6 4 4.19 2.6 4.6 3 3.11 2.1 3.8 2 1.9 4.5 −4.8 1 0.89 1.4 −14.2

intra‐ and inter‐day accuracy from plasma was between −4.7 and 8.3% et al., 2012; Lian et al., 2012; Probst, Blobner, Luppa, & Neumeier, with the RSD ˂ 5.2% at all QC levels (Table 3). The intra‐ and inter‐day 1997; Radford et al., 2017; Seno, He, Tashiro, Ueyama, & Mashimo, accuracy and precision met the acceptance limits (±15%). This indi- 2002; Seplúveda et al., 2011; Thieme, Sachs, Schelling, & Hornuss, cated that the developed LC–MS was precise and accurate for the 2009; Veilleux‐Lemieux, Beaudry, Hélie, & Vachon, 2012). quantitative analysis of propofol, ketamine and rocuronium from 10 μL of plasma over the established concentration range. Our 3.1.5 | Recovery and matrix effects method distinctly improves on previously reported methods which separately evaluate the three drugs, and require more than of 50 μL The extraction recoveries and matrix effects of propofol, ketamine and of plasma or whole blood for each analysis. The accuracy and precision rocuronium are shown in Table 4. The extraction recoveries ≥89% of our method was generally better than those of previous ones. In the were consistent at different concentration levels and no significant previous publications, the accuracy and precision were 5–11% and matrix effect was detected in plasma. In our case matrix effects were 2.5–8.1%, respectively (Cirimele, Villain, Pépin, Ludes, & Kintz, 2003; between 3.2 and 8.0%, well under 15% as a critical validation param- Giroux, Santamaria, Hélie, & Burns, 2016; Knibbe et al., 1998; Li eter. Our one‐step extraction protocol can be useful for laboratories

TABLE 3 Precision (%, RSD) and accuracy (%, RE) for propofol, ketamine and rocuronium of QC samples in plasma

Intra‐day (n = 6) Inter‐day (n = 18) Nominal Analytes concentration (μg/mL) Measured (μg/mL) RSD (%) RE (%) Measured (μg/mL) RSD (%) RE (%)

Propofol 1 1.08 4.0 8.3 1.03 4.9 2.5 4 4.16 2.0 4.1 4.02 2.7 0.5 ketamine 2 2.16 2.7 7.8 2.04 4.1 1.8 6 6.11 2.9 1.9 6.10 5.2 1.6 Rocuronium 2 1.93 3.3 −3.6 1.95 3.2 −2.3 6 5.72 2.9 −4.7 5.79 2.0 −3.4 6of8 SHOPOVA ET AL.

TABLE 4 Recovery (%) and matrix effects (%, ME) for propofol, the MS signal (Bolze & Boulieu, 1998; El Hamd et al., 2015; Farenc ketamine and rocuronium of QC samples in plasma (n =6) et al., 2001; Lian et al., 2012). However, plasma preparation protocols ‐ Nominal Recovery Matrix based on re extraction steps are time intensive (extra preparation concentration (mean ± SD, %) effect (%) steps) and device‐dependent (vaporization of the organic phase after Analytes (μg/mL) extraction). Propofol 1 93 ± 2.2 7.0 4 91 ± 1.8 8.0 3.1.6 | Stability and carryover Ketamine 2 89 ± 5.0 5.3 6 93 ± 5.2 4.8 No quantifiable carryover effects were observed when injecting drug‐ Rocuronium 2 90 ± 2.4 −3.2 free rat plasma immediately after the highest calibration standards 6 92 ± 2.5 −4.0 (Figure 2). Stability was investigated by analyzing QCs under different storage conditions in six replicates. The results for all samples indi- without complex equipment and with a high number of samples for cated that propofol, ketamine and rocuronium were stable under the determination per day. Re‐extraction steps with different organic sol- storage conditions listed in Table 5. The precision (RSD) was ≤6.8% vents after protein precipitation can be added if matrix effects are and accuracy (RE) was ≤7.3%. The method was indicated to be appli- suspected because they could suppress or enhance the response of cable for routine analysis.

FIGURE 2 Carryover for (a1) blank rat plasma, (b1) blank rat plasma spiked with propofol at 5 μg/mL, (c1) blank rat plasma sample measured after the injection of 5 μg/mL of propofol, (a2) blank rat plasma, (b2) blank rat plasma spiked with ketamine and rocuronium at 8 μg/mL and (c2) blank rat plasma sample measured after the injection of 8 μg/mL of ketamine and rocuronium SHOPOVA ET AL. 7of8

TABLE 5 Stability, precision (%, RSD) and accuracy (%, RE) of propofol, ketamine and rocuronium under different storage conditions of QC samples in plasma (n =6)

8°C in autosampler for 24 h −20°C in freezer for 60 days Nominal Analytes concentration (μg/mL) Measured (μg/mL) RSD (%) RE (%) Measured (μg/mL) RSD (%) RE (%)

Propofol 1 0.97 5.9 −3.9 1.07 4.9 7.3 4 3.88 3.2 −3.2 4.11 1.4 2.8 Ketamine 2 1.96 6.8 −2.1 1.91 5.2 −4.3 6 5.94 3.4 −1.0 6.08 7.3 1.2 Rocuronium 2 1.89 2.3 −5.3 1.98 3.1 −0.8 6 5.75 1.5 −4.9 5.87 1.1 −2.2

3.2 | Plasma concentrations concentrations in blood plasma for several hours of ventilation, without clinical signs of awareness. Sixty blood samples were investigated. The mean concentration of propofol with standard deviation was 2.0 μg/mL ± 0.5% with a mini- 4 | CONCLUSIONS mum of 0.9 μg/mL and maximum of 2.9 μg/mL. The mean concentration of ketamine with standard deviation was 3.9 μg/mL ± 1.0% with a Our novel LC–MS method permits accurate and precise simultaneous minimum of 2.1 μg/mL and maximum of 6.5 μg/mL. The mean concen- determination of propofol, ketamine and rocuronium concentrations tration of rocuronium with standard deviation was 3.2 μg/mL ± 0.8% in only 10 μL plasma. with the minimum of 1.6 μg/mL and a maximum of 5.1 μg/mL (Figure 3). CONFLICT OF INTEREST During the observation period, there were no significant differ- None of the authors has a financial interest in this report. ences between the plasma concentrations of propofol and ketamine. The rocuronium plasma decreased after 7 h and was significantly dif- DISCLOSURE OF FUNDING ferent compared with the beginning of the bolus administration. The established infusion protocol of propofol and ketamine led to a reliable This work was supported by internal funds only. B. Braun Melsungen anesthesia with stable vital parameters and constant drug (Melsungen, Germany) loaned the devices that were used in this analysis.

ORCID

Teodora Shopova https://orcid.org/0000-0002-0537-5683

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