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Analysis of Remifentanil with Liquid Chromatography-Tandem Mass Spectrometry and an Extensive Stability Investigation in EDTA Whole Blood and Acidified EDTA Plasma

Koster, Remco A. BSc*; Vereecke, Hugo E. M. MD, PhD; Greijdanus, Ben BSc*; Touw, Daan J. PhD, PharmD*; Struys, Michel M. R. F. MD, PhD; Alffenaar, Jan Willem C. PhD, PharmD*

doi: 10.1213/ANE.0000000000000643
Anesthetic Pharmacology: Research Report

BACKGROUND: Remifentanil is a μ-opioid receptor agonist that was developed as a synthetic opioid for use in anesthesia and intensive care medicine. Remifentanil is rapidly metabolized in both blood and tissues, which results in a very short duration of action. Even after blood sampling, remifentanil is unstable in whole blood and plasma through endogenous esterases and chemical hydrolysis. The instability of remifentanil in these matrices makes sample collection and processing a critical phase in the bioanalysis of remifentanil.

METHODS: We have developed a fast and simple sample preparation method using protein precipitation followed by liquid chromatography-tandem mass spectrometry analysis. To improve the stability of remifentanil, citric acid, ascorbic acid, and formic acid were investigated for acidification of EDTA plasma. The stability of remifentanil was investigated in stock solution, EDTA whole blood, EDTA plasma, and acidified EDTA plasma at ambient temperature, 4°C, 0°C, and at −20°C.

RESULTS: The analytical method was fully validated based on the Food and Drug Administration guidelines for bioanalytical method validation with a large linear range of 0.20 to 250 ng/mL remifentanil in EDTA plasma acidified with formic acid. The stability results of remifentanil in EDTA tubes, containing whole blood placed in ice water, showed a decrease of approximately 2% in 2 hours. EDTA plasma acidified with citric acid, formic acid, and ascorbic acid showed 0.5%, 4.2%, and 7.2% remifentanil degradation, respectively, after 19 hours at ambient temperature. Formic acid was chosen because of its volatility and thus liquid chromatography-tandem mass spectrometry compatibility. The use of formic acid added to EDTA plasma improved the stability of remifentanil, which was stable for 2 days at ambient temperature, 14 days at 4°C, and 103 days at −20°C.

CONCLUSIONS: The analytical method we developed uses a simple protein precipitation and maximal throughput by a 2-point calibration curve and short run times of 2.6 minutes. Best sample stability is obtained by placing tubes containing EDTA whole blood in ice water directly after sampling, followed by centrifugation and transfer of the EDTA plasma to tubes with formic acid. The stability of remifentanil in EDTA plasma was significantly improved by the addition of 1.5 μL formic acid per milliliter of EDTA plasma. This analytical method and sample pretreatment are suitable for remifentanil pharmacokinetic studies.

Published ahead of print February 16, 2015

From the *Department of Clinical Pharmacy and Pharmacology, Laboratory for Clinical and Forensic Toxicology and Drugs Analysis, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; and Department of Anesthesiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

Accepted for publication December 2, 2014.

Published ahead of print February 16, 2015

Funding: None.

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Remco A. Koster, BSc, Department of Clinical Pharmacy and Pharmacology, Laboratory for Clinical and Forensic Toxicology and Drugs Analysis, University of Groningen, University Medical Center Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands. Address e-mail to r.koster@umcg.nl.

Remifentanil is a μ-opioid receptor agonist belonging to the family of phenylpiperidine derivatives and was developed as a synthetic opioid for use in anesthesia and intensive care medicine.1,2 Remifentanil has a unique pharmacokinetic profile characterized by its rapid metabolism in both blood and tissues by endogenous esterases. Unlike the other fentanyl congeners, termination of the therapeutic effect of remifentanil mostly depends on metabolic clearance rather than on redistribution, whereas pharmacodynamically remifentanil is similar to the other fentanyl congeners. The half-life of remifentanil is approximately 3 minutes, independent of the duration of infusion.1 Even after blood sampling, remifentanil is unstable in whole blood and plasma because of the N-substituted methyl propanoate ester group of remifentanil, which is highly susceptible to endogenous esterases and chemical hydrolysis.

The therapeutic analgesic concentration of remifentanil is approximately between 0.5 and 8 ng/mL. In addition to the preferred low sample volume, a sensitive method is mandatory for pharmacokinetic studies evaluating the time course of remifentanil concentrations in plasma.

Degradation of remifentanil is inhibited by acidification of the blood or plasma with citric acid.3–9 Most of the previous publications refer to the article by Selinger et al.3 published in 1994. Here, the acidification of the whole blood with citric acid is described, but the (target) pH of the acidified blood samples was never mentioned. Other publications that described the acidification with citric acid mentioned neither the target pH of the blood nor plasma samples.3–9

Some analytical methods were developed to quantify remifentanil in whole blood instead of plasma to avoid the time necessary for obtaining plasma.3,5,8,10,11 For example, a direct transfer of whole blood to tubes with acetonitrile to overcome the instability of remifentanil has been described.1,11

The applied analytical techniques found in the literature are the following: high-performance liquid chromatography (HPLC) with ultraviolet detection,3,7 gas chromatography,8,11 and HPLC with (tandem) mass spectrometry detection (LC-MS/(MS)).5,9,10 Most of these methods use time-consuming liquid-liquid extraction or solid-phase extraction procedures to process the whole blood or plasma samples.

In addition to citric acid, ascorbic acid and formic acid were investigated for acidification of EDTA plasma and thus the stabilization of remifentanil. During LC-MS/MS analysis, HPLC solvent flow containing the injected sample is vaporized in the heated electrospray ionization source. This requires the use of volatile additives to prevent deterioration of system performance. Because formic acid is volatile, it is considered a more compatible acid for LC-MS/MS analysis than ascorbic acid or citric acid.

The aim of this study was to develop a fast and simple LC-MS/MS method for the analysis of remifentanil in EDTA plasma and to extensively investigate the stability of remifentanil in untreated EDTA whole blood and in EDTA plasma.

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METHODS

Chemicals and Reagents

Remifentanil was purchased from BDG Synthesis Limited (Wellington, New Zealand). The isotopically labeled internal standard [13C6]-remifentanil was purchased from Alsachim (Illkirch Graffenstaden, France). Analytical-grade methanol, formic acid, citric acid monohydrate, and ascorbic acid were purchased from Merck (Darmstadt, Germany). Purified water was prepared by a Milli-Q Integral system (Billerica, MA). Ammonium formate was purchased from Acros (Geel, Belgium). Human EDTA whole blood and plasma were made available according to the guidelines of the University Medical Center Groningen.

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Equipment and Conditions

All experiments were performed on an Agilent 6460A (Santa Clara, CA) triple quadrupole LC-MS/MS system, with a combined Agilent 1200 series LC system. The Agilent 6460A mass selective detector operated in heated electrospray positive ionization mode and performed multiple-reaction monitoring with unit mass resolution. High-purity nitrogen was used for both the source and collision gas flows. In the first quadrupole, single-charged ions were selected for remifentanil and [13C6]-remifentanil. All precursor ions, product ions, optimum fragmentor voltages, and collision energy values were tuned and optimized in the authors’ laboratory. For remifentanil, the precursor ion was set at a mass-to-charge ratio (m/z) of 377.2 and the product ion at an m/z of 317.2. For [13C6]-remifentanil, the precursor ion was set at an m/z of 383.2 and the product ion at an m/z of 323.2. The fragmentor voltage was 115 V and the collision energy was 11 V for both substances. The capillary voltage was set at 4000 V, gas temperature at 300°C, gas flow at 13 L/min, nebulizer gas at 18 psi, sheath gas temperature at 300°C, sheath gas flow at 12 L/min, and the nozzle voltage at 0 V. The Agilent 1290 auto sampler was set at 10°C and the 1260 thermostatted column compartment was set at a temperature of 60°C. The mobile phase consisted of methanol and a 20 mM ammonium formate buffer pH 3.5. Analyses were performed with a 50 × 2.1 mm 3-μm HyPURITY® C18 analytical column from ThermoFisher Scientific (Waltham, MA) equipped with a separate 0.5-μm Varian Frit Filter (Palo Alto, CA). Chromatographic separation was performed by means of a gradient with a flow rate of 0.5 mL/min and a run time of 2.6 minutes with the use of an Agilent 1290 Infinity Binary LC system. The gradient started at 15% methanol and 85% 20 mM ammonium formate buffer pH 3.5 and changed to 30% methanol at 0.11 minutes and increased slowly to 35% methanol in 1.49 minutes. At 1.61 minutes, the methanol increased to 95% and was maintained until 2.10 minutes. From 2.11 to 2.60 minutes, the gradient was kept at 15% methanol to stabilize the column for the next injection. Peak height ratios of the substance and its internal standard were used to calculate concentrations. Agilent Masshunter software for quantitative analysis (version B.04.00) was used for quantification of the analytical results. Regression analysis was performed by applying the software tool Analyse-it, version 2.20 (Analyse-it Software, Ltd., Leeds, UK) in Microsoft Excel (Redmond, WA).

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Method Development

Early method development showed that acidification of the blood caused coagulation during a period of a few hours, resulting in the rejection of this matrix for further method development. To assess the efficacy of varying acids for the acidification of EDTA plasma, citric acid, ascorbic acid, and formic acid were added to the EDTA plasma to decrease the pH of approximately 7.6 to approximately 5.3. When EDTA plasma was acidified to a pH <4.7, coagulation of EDTA plasma was also observed after 24 hours. Acidification of EDTA plasma to a pH of 5.3 showed minimal coagulation, while still stabilizing remifentanil.

The following acidic solutions were added to EDTA plasma to obtain a pH of approximately 5.3. From an almost saturated 1000 g/L citric acid solution, 6 μL was added to 1 mL of EDTA plasma. For ascorbic acid, 37.5 μL of an almost saturated solution of 250 g/L was added to 1 mL of EDTA plasma, and 1.5 μL of a 100% formic acid solution was added to 1 mL of EDTA plasma. Remifentanil was spiked to all 3 acidified EDTA plasmas and to untreated EDTA plasma to obtain concentrations of 50 ng/mL. For stability testing, the spiked EDTA plasma samples were processed according to the sample preparation described further on and analyzed at time zero or stored at ambient temperature and subsequently processed and analyzed at 1.5, 3.0, 19, 26, 44, and 50.5 hours. Only one sample was processed and analyzed at each time point for each solution.

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Sample Preparation

Formic acid was added to EDTA plasma to improve the stability of remifentanil. To each milliliter of EDTA plasma, 1.5 μL formic acid was added.

The sample preparation was performed by means of a protein precipitation. An aliquot of 100 μL EDTA plasma was transferred into a glass 1.5 mL screwneck vial (Fisher Scientific, Breda, The Netherlands), and 400 μL methanol, containing 10 ng/mL [13C6]-remifentanil, was added. After precipitation, the vials were vortex mixed (Multi-Tube Vortexer; Labtek Corporation Ltd., Christchurch, New Zealand) for 1 minute and stored at −20°C for at least 10 minutes. Afterward, the vials were again vortex mixed for 1 minute, centrifuged at 10,000g for 5 minutes, and 5 μL of the clear upper layer of supernatant was injected into the LC-MS/MS.

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Validation

The analytical method validation in acidified EDTA plasma included linearity, accuracy, precision, selectivity, specificity, and stability based on international guidelines.12 Two stock solutions were weighed and dissolved in purified water. One stock solution was used for the preparation of the calibration curve, whereas another stock solution was used for the preparation of the quality control (QC) concentrations. The volume of the spiked stock solution never exceeded 5% of the total EDTA plasma volume used for the preparation of EDTA plasma standards. An 8-point calibration curve was prepared at 0.20, 0.50, 2.0, 5.0, 25, 100, 200, and 250 ng/mL. The following QC concentrations were used for validation: 0.20 ng/mL (lower limit of quantification [LLOQ]), 0.50 ng/mL, 100 ng/mL, 200 ng/mL, and the over the curve concentration of 500 ng/mL. The over the curve concentration was diluted 10 times with blank EDTA plasma before sample processing to validate the dilution. One calibration curve, consisting of 8 calibration points, was analyzed each day to determine linearity on 3 separate days. All 3 calibration curves were assessed with Analyse-it to assess the linear regression of the 8-point calibration curve and to assess whether a 2-point calibration would provide the same linear fit. To maximize sample throughput for routine analysis, the accuracy and precision were calculated for all 3 days using a 2-point calibration curve, consisting of the lowest (0.20 ng/mL) and highest (250 ng/mL) concentrations of the calibration curve. In this way, the 2-point calibration curve was embedded in the validation procedure. The validation was performed with a maximal tolerated bias and coefficient of variation (CV) of 20% for the LLOQ and 15% for all other calibration and QC samples including stability validation. For the determination of accuracy, precision, and over the curve, all QC concentrations were processed and measured 5 times, and the run of 5 was repeated on each of 3 separate days. For each accuracy and precision, concentration bias and CV were calculated per run. Within-run, between-run, and overall CVs were calculated with 1-way analysis of variance.

To test the limit of detection (LOD), a concentration of 0.015 ng/mL was prepared in acidified EDTA plasma and was processed 5 times and analyzed to assess the CV, with a maximal tolerated CV of 20%.

To assess the variation of the acidification procedure between different EDTA plasma lots of different healthy volunteers, 9 lots of EDTA plasma were acidified with 1.5 μL formic acid/milliliter EDTA plasma and followed by pH measurement. These 9 acidified EDTA plasma lots were also used for selectivity and specificity testing because the Food and Drug Administration guideline states the testing of at least 6 sources of the biological matrix. From every batch, an LLOQ was spiked and processed together with a blank sample from each batch. Peaks found in the blank samples should not exceed 20% of the peak height of the LLOQ.

The stability of remifentanil in acidified EDTA plasma was assessed at 0.5 and 200 ng/mL and was processed 5 times and analyzed after 2 days at ambient temperature, 14 days at 4°C, and 14 days as processed sample in the autosampler at 10°C. The long-term stability of remifentanil in EDTA plasma at −20°C was assessed at 103 days, with the use of freshly prepared controls at 0.5 and 200 ng/mL remifentanil. Freeze thaw stability was assessed after 1 and 3 times freezing and thawing and was processed 5 times and analyzed.

Stock stability at 100 μg/mL in purified water was assessed after a 1000 times dilution to 100 ng/mL and followed by triplicate injections after 6 months storage at −20°C and at +4°C.

The stability of remifentanil in EDTA whole blood without acidification was investigated at ambient temperature, 4°C, and 0°C (ice water). EDTA whole blood was spiked at 0.5 and 200 ng/mL and divided over multiple tubes, which were labeled and sealed. At multiple time points during 5 hours, 1 tube of each concentration was centrifuged, and the EDTA plasma was stored at −20°C. The stored samples were prepared in triplicate and analyzed the following day.

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Ion Suppression, Extraction Recovery, Matrix Effect, and Process Efficiency

The presence of ion suppression during an analytical run was tested by infusion of stock solution of remifentanil and [13C6]-remifentanil with the use of a t-piece to combine the flows of the syringe pump with stock solution and the LC pump. The 9 acidified EDTA plasma lots used for selectivity and specificity were processed according to the earlier described sample preparation, without [13C6]-remifentanil. Ion suppression chromatograms were recorded for all 9 lots of processed EDTA plasma.

The extraction recovery, matrix effect, and process efficiency were assessed at 2 concentrations in triplicate. Remifentanil was spiked at 0.5 and 200 ng/mL in EDTA plasma acidified with formic acid (solutions A). For the extraction recovery, acidified blank EDTA plasma samples were spiked at 0.5 and 200 ng/mL after processing (solutions B). For the matrix effect and process efficiency, methanol was spiked at 0.5 and 200 ng/mL (solutions C). The average peak height responses were used to calculate extraction recovery, matrix effect, and process efficiency. The calculations of the extraction recovery, matrix effect, and process efficiency were as follows: extraction recovery = A/B × 100, matrix effect = 100 × (B/C − 1), process efficiency = A/C × 100. Where A, B, and C refer to the prepared solutions mentioned above.

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RESULTS

Method Development

The stability test with unacidified EDTA plasma and EDTA plasma acidified with citric acid, ascorbic acid, and formic acid was performed by single sample analysis for all 7 time points and each matrix. The results of the stability test in EDTA plasma showed that untreated EDTA plasma was very unstable with a decrease in remifentanil concentration of 13% within 3 hours. Citric acid and formic acid showed 0.5% and 4.2% remifentanil degradation, respectively, after 19 hours at ambient temperature, whereas ascorbic acid showed 7.2% degradation (Fig. 1). Formic acid was chosen to acidify EDTA plasma for validation because of the volatility (LC-MS/MS compatibility) and the fact that this acid was also used in the mobile phase.

Figure 1

Figure 1

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Validation Results

The 8-point calibration curve of remifentanil proved to be linear from 0.20 to 250 ng/ml with an R 2 of 0.9995. The linear regression equation with its 95% confidence intervals (CIs) was y = 0.02495 (CI, 0.02471–0.02519) × −0.02342 (CI, −0.05192 to 0.00508) for the 8-point calibration curve. While the regression equation was y = 0.02483 (CI, 0.02400–0.02566) × −0.001312 (CI, −0.14854 to 0.14592) for the 2-point calibration curve. The 95% CI of the intercepts and the slopes of both calibration curves showed overlapping CIs. Therefore, it can be concluded that both calibration curves did not significantly deviate from each other, and the use of a 2-point calibration curve was justified. The accuracy and precision results calculated with a 2-point calibration curve showed the highest overall bias during the validation of −5.0% (CV, 4.3%) for 0.5 ng/mL, whereas the highest overall CV was 5.6% for 0.20 ng/mL. The validation results regarding accuracy, precision, and dilution are shown in Table 1. In Figure 2, chromatograms are shown for a representative blank, LLOQ (0.20 ng/mL), and highest calibration standard (250 ng/mL) for remifentanil and the internal standard [13C6]-remifentanil. The LOD was tested at 0.015 ng/mL and showed a CV of 7.5%.

Table 1

Table 1

Figure 2

Figure 2

The 9 lots of EDTA plasma acidified with formic acid showed reproducible pH values ranging from pH 4.7 to 5.4, with a mean pH of 5.08. Peaks found in the blank samples of the 9 lots of EDTA plasma acidified with formic acid did not exceed 20% of the peak height of the LLOQ.

The results of the stability validation are shown in Table 2. The stock solutions of 100 mg/L proved to be stable at −20°C and at +4°C for 6 months with a maximal bias of 4.3% (CV, 1.3%). The stability of remifentanil in EDTA blood for 2 hours showed an average decrease of approximately 42% at ambient temperature, 12% at +4°C, and <2% decrease at 0°C (ice water) at 0.50 and 200 ng/mL. The results of the stability tests in untreated EDTA whole blood can be seen in Table 3 and Figure 3.

Table 2

Table 2

Table 3

Table 3

Figure 3

Figure 3

At ambient temperature, remifentanil was even more unstable in untreated EDTA whole blood than in EDTA plasma without acidification. After 3 hours at ambient temperature, the remifentanil concentration decreased approximately 54% in untreated EDTA whole blood (Fig. 3) compared with a 13% decrease in untreated EDTA plasma (Fig. 1).

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Ion Suppression, Extraction Recovery, Matrix Effect, and Process Efficiency

The developed analysis method showed extraction recoveries of 99% (CV, 8.2%) and 107% (CV, 1.3%) for 0.5 and 200 ng/mL, respectively. Matrix effects were −8% (CV, 4.1%) for 0.5 ng/mL and −10% (CV, 0.3%) for 200 ng/mL. The total process efficiency was 91% (CV, 8.2%) for 0.5 ng/mL and 96% (CV, 1.3%) for 200 ng/mL. The ion suppression chromatograms showed no ion suppression near the retention time of remifentanil in all 9 lots of acidified EDTA plasma (Fig. 4).

Figure 4

Figure 4

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CONCLUSIONS

We propose LC-MS/MS analysis combined with the use of formic acid to acidify the EDTA plasma sample as a new analytical method to measure remifentanil concentrations in EDTA plasma. We studied the concentration stability over time, at different temperature conditions, and with or without acidification of EDTA plasma to stabilize esterase activity.

During method development, it became clear that acidifying the EDTA whole blood causes coagulation, which impairs the integrity of the sample. For this reason, the stability of remifentanil was also investigated in untreated EDTA whole blood at ambient temperature, 4°C, and 0°C. At ambient temperature, remifentanil was even more unstable in untreated EDTA whole blood than in EDTA plasma without acidification, which is in agreement with the findings of Davis et al.13 These results showed that collected EDTA whole blood should not be stored at ambient temperature. The collected EDTA whole blood tube is best stored temporarily in ice water to cool and should then be centrifuged as soon as possible (preferably before the first hour but not later than 2 hours after the sampling time). To stabilize the esterase activity in EDTA plasma, the resulting EDTA plasma fraction should be acidified with 1.5 μL formic acid per milliliter of EDTA plasma. Subsequently, the sample can be stored in a −20°C freezer.

Compared to earlier used citric acid,3–9 formic acid is volatile, and formic acid was considered a more compatible acid for LC-MS/MS analysis than the nonvolatile ascorbic acid and citric acid.

The pH of acidified EDTA plasma is considered the primary determinant for the stability of remifentanil. We found the pH values in the 9 lots of acidified EDTA plasma within an acceptable range. This indicates that remifentanil concentrations are stable in acidified EDTA plasma of different patient samples. The stability of remifentanil in EDTA plasma acidified with formic acid was extensively investigated and showed good stability results, even at ambient temperature.

Our developed analytical method could be limited by the current LLOQ when bolus injections are used. However, the validated limit of quantitation of 0.20 ng/mL assured that the method was robust and not readily vulnerable to deteriorating sensitivity. The LOD was tested at 0.015 ng/mL and showed a CV of 7.5%. This may indicate that a lower LLOQ could be possible to monitor remifentanil even at the end of the pharmacokinetic curve of bolus injections. In addition, the use of a more sensitive mass spectrometer could also improve the LLOQ.

The application of a 2-point calibration curve, which included the LLOQ and the highest concentration of the linear range, provided excellent accuracy and precision results. The approach of applying a minimal calibration curve already proved to be very efficient for therapeutic drug monitoring.14 The 2-point calibration curve could be impaired when the curve may become nonlinear, possibly due to changing ionization characteristics or overdue maintenance. In our method, an isotopically labeled internal standard is used, which can compensate for changing ionization characteristics. In addition, with the use of QC samples throughout the linear range, linearity issues would result in unacceptable biases for the QCs and run rejection. The 2-point calibration curve was validated during the 3-day validation of linearity, accuracy, and precision, indicating no such linearity problem. QC samples throughout the linear range proved that the 2-point calibration curve was valid. During routine analysis, QC samples are also incorporated in the run, ensuring valid results at all times.

Remifentanil concentrations were frequently reported in whole blood in the past because of the stability issues. Blood was drawn from the patient and directly mixed with acetonitrile to stop the endogenous esterases.1,11 With our procedure, this instability is no longer a problem, and plasma analysis can be easily performed. Due to the instability of remifentanil and the following time-consuming sample processing, the established pharmacokinetic models were based on whole blood. La Colla et al.15 evaluated the predictive performance of the model of Minto et al.16,17 for morbidly obese patients. It should be noted that their analyses were performed in plasma, whereas the model of Minto et al. was developed for whole blood analysis. It is unclear whether La Colla et al.15 acknowledged the blood-to-plasma ratio. As a future perspective, the blood-to-plasma ratio should be investigated before the current pharmacokinetic models are used with plasma analysis.

This developed analytical method uses a simple protein precipitation and maximal throughput by a 2-point calibration curve and short run times of 2.6 minutes. The validation showed excellent accuracy and precision results with a very large linear range of 0.2 to 250 ng/mL. The method showed no ion suppression for all 9 lots of acidified EDTA plasma.

In conclusion, we found that our LC-MS/MS method is suitable for measuring remifentanil concentrations in EDTA plasma for pharmacokinetic trials.

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DISCLOSURES

Name: Remco A. Koster, BSc.

Contribution: This author helped design the study, conduct the study, collect the data, analyze the data, and prepare the manuscript.

Attestation: Remco A. Koster approved the final manuscript, attests to the integrity of the original data and the analysis reported in this manuscript, and is the archival author.

Name: Hugo E. M. Vereecke, MD, PhD.

Contribution: This author helped design the study and prepare the manuscript.

Attestation: Hugo E. M. Vereecke approved the final manuscript, and attests to the integrity of the original data and the analysis reported in this manuscript.

Name: Ben Greijdanus, BSc.

Contribution: This author helped design the study and prepare the manuscript.

Attestation: Ben Greijdanus attests to the integrity of the original data and the analysis reported in this manuscript, and is the archival author.

Name: Daan J. Touw, PhD, PharmD.

Contribution: This author helped prepare the manuscript.

Attestation: Daan J. Touw attests to the integrity of the original data and the analysis reported in this manuscript.

Name: Michel M. R. F. Struys, MD, PhD.

Contribution: This author helped design the study and prepare the manuscript.

Attestation: Michel M. R. F. Struys attests to the integrity of the original data and the analysis reported in this manuscript.

Name: Jan Willem C. Alffenaar, PhD, PharmD.

Contribution: This author helped design the study and prepare the manuscript.

Attestation: Jan Willem C. Alffenaar attests to the integrity of the original data and the analysis reported in this manuscript.

This manuscript was handled by: Tony Gin, MD, FRCA, FANZCA.

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