A gas chromatography-mass spectrometry (GC-MS) method for the determination of propofol in human plasma has been developed and validated. Propofol was extracted from human plasma by using mixed-mode cation exchange/reversed-phase (MCX) cartridges. As propofol easily volatilizes during concentration, 100% methanol was injected directly into GC-MS to elute propofol. Despite avoiding concentration process of the eluted solution, lower limit of quantization (LLOQ) of propofol was 25 ng/mL. The validated method exhibited good linearity (
Propofol (2,6-diisopropylphenol) is an intravenously administered hypnotic/amnestic agent for induction and maintenance of anesthesia. A therapeutic dose of intravenous propofol produces hypnosis rapidly and smoothly within forty seconds [
However, high lipophilicity of propofol can cause hypertriglyceridemia and bradycardia, subsequent hypotension, and transient apnea at high dose infusion [
Many studies for propofol determination were conducted using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Propofol and 3 main metabolites (propofol-glucuronide, 1-quinol-glucuronide, and 4-quinol-glucuronide) in human plasma were extracted by using the solid phase extraction and validated with the LC-MS/MS [
In the propofol analysis that used LC-MS, derivatization or restricted mass condition for propofol determination should be embraced because of the low ionization efficiency of propofol in LC-MS/MS. The absence of ionization group and nonpolarity of propofol cause such low ionization efficiency. To overcome this hindrance, N-methylpyridinium ether derivatization and dansyl chloride derivatization were applied to the propofol analysis [
The therapeutic dose of propofol is quite high (6–10 mg/L for induction of anesthesia and 2–4 mg/L for maintenance of anesthesia) [
In this experiment, mixed-mode cation exchanger with two binding sites, sulfate ion site for ionic interaction and benzene ring site for reversed-phase interaction, was applied to extract propofol from human plasma. Also, propofol volatility was evaluated using two concentrators: nitrogen gas and centrifugal vacuum. This is a simple, rapid, and accurate quantification method to determine the propofol in human plasma, without sample concentration. It could be a new guideline for propofol analysis in pharmaceutical and/or in forensic fields.
Cannabinol (CBN), used as internal standard, and propofol were purchased from Sigma (St. Louis, MO, USA). Methanol was supplied by Burdick and Jackson (Muskegon, MI, USA). Water was purified using a Millipore (Chem-science, USA) purification system. Oasis MCX® (3 mL, 60 mg) cartridges were purchased from Waters (Milford, MA, USA).
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The SPE cartridges (Oasis MCX) were preconditioned with 2 mL methanol and 2 mL distilled water, sequentially. After being preconditioned, the centrifuged plasma sample was then loaded into the cartridges, previously washed with 2 mL distilled water and 2 mL cyclohexane, and dried under reduced pressure for 5 min. Elution of analytes was carried out with 2 mL of methanol. 100
The GC-MS analysis was performed with a 7890A gas chromatography instrument, combined with 5975C mass spectrometer equipped with electron ionization (EI) and quadrupole analyzer (Agilent technologies, Palo Alto, CA, USA).
Propofol was separated using a 100% dimethyl polysiloxane fused-silica capillary column (HP-5MS 30 m × 250
The analytical method was validated in terms of the limit of detection, the limit of quantification, linearity, intra- and interday precision and accuracy, recovery, and the matrix effect. Propofol free plasma samples from five different volunteers were used as blanks, and various concentrations of propofol standards were spiked to the samples. To evaluate the selectivity of the method, six different sources of blank plasma samples were extracted and analyzed to check interfering peak on propofol and the internal standard. Recovery was assessed to compare the concentration of spiking propofol, at three different concentrations, between, before, and after extraction. Propofol spiked plasma from six different sources was compared with propofol calibrator spiked phosphate buffer saline to achieve the matrix effect.
The linearity of calibrator was calculated by analyzing five calibration curves, ranging from 25 to 5,000 ng/mL. The limit of detection (LOD) and the lower limit of quantification (LLOQ) were assessed, using signal to noise ratio. The signal to noise ratios of LOD and LLOQ were 3 and 10, respectively. Accuracy and precision were achieved by analyzing propofol spiked plasma at three different concentrations (25, 500, and 5,000 ng/mL) and were replicated five times. In order to examine interday precision and accuracy, five sets of each sample were analyzed on three different days.
Two blood samples were obtained from the police for forensic analysis. These samples were stored at 4°C in refrigerator and were used for this (validated) method. Propofol concentration was achieved using peak area and calibration curve.
The nitrogen gas evaporation was performed at an EvaT-0200 total concentration system (Goojung, Seoul, South Korea), with the temperature of nitrogen gas concentrator set at 45°C and the pressure of nitrogen gas at 20 psi. 100
Centrifugal vacuum concentration system consisted of a UNIVAPO 100H vacuum concentrator centrifuge and a UNIJET II refrigerated aspirator (UNIEQUIP, Munich, Germany). The temperatures of the vacuum concentrator centrifuge and the refrigerated aspirator were set at 40 and 7°C, respectively. The pressure of the refrigerated aspirator was 145 psi.
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The MCX cartridge has two binding sites. The first is the benzene ring site binding to the benzene ring of the target analyte with
While propofol was loaded to the mixed-mode cation cartridge with phosphate buffer (pH 6.8), propofol was in neutral form and not in ionic condition because p
After the washing steps, some chemicals like propofol bound to the benzene ring of cartridge sorbent with reversed-phase retention. Other analytes with cation group binding to the negatively charged site of sorbent with ionic binding were not eluted through water and cyclohexane washing. Among the combined materials, only chemicals including propofol bound to the cartridge, with the reversed-phase retention being extracted at the methanol elution step (Figure
The extracted ion chromatograms of blank sample (a) and propofol spiked plasma sample (b).
The propofol standards at seven concentration levels, 25, 50, 250, 500, 1,000, 2,500, and 5,000 ng/mL, were used for the method validation. The linearity of 5 calibration curves was drawn, and their correlation coefficients (
Intra- and interday precision and accuracy of propofol.
Concentration (ng/mL) | Intra-assay | Interassay | ||
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Precision (CV%) | Accuracy (bias, %) | Precision (CV%) | Accuracy (bias, %) | |
25 | 5.4 | 5.3 | 11.6 | 11.7 |
500 | 4.0 | −4.2 | 5.4 | −5.8 |
5,000 | 3.7 | −0.1 | 4.1 | −1.2 |
Recovery of propofol in human plasma (
Concentration |
Recovery |
CV value of recovery |
---|---|---|
25 | 96.6 ± 3.3 | 9.1 |
500 | 98.8 ± 2.2 | 5.3 |
5,000 | 99.4 ± 0.9 | 2.3 |
All the concentrations in mg/L of plasma; mean values ± standard error.
Matrix effect of propofol in human plasma (
Concentration |
Matrix effect |
CV value of matrix effect |
---|---|---|
25 | 95.3 ± 5.9 | 13.7 |
500 | 100.2 ± 2.5 | 6.2 |
5,000 | 101.4 ± 0.3 | 0.1 |
All the concentrations in mg/L of plasma; mean values ± standard error.
Plasma samples of 2 patients were extracted according to this developed method. The propofol concentrations of heart blood and peripheral blood were determined and calculated. Their representative chromatograms are shown in Figure
Propofol concentration of authentic human heart blood and peripheral blood.
Concentration |
Case |
Case |
---|---|---|
Heart blood | 0.238 | 0.454 |
Peripheral blood | 0.988 | 0.997 |
The representative extracted ion chromatograms of authentic heart plasma sample (a) and peripheral plasma sample (b).
GC-MS analysis was used to evaluate the loss of propofol by nitrogen gas concentration and centrifugal vacuum evaporation. Various volumes of sample but with equal quantity of propofol were concentrated with nitrogen gas evaporator and centrifugal vacuum evaporator and quantified with GC-MS. However, regardless of concentration methods, all the results showed loss of propofol, and the inconsistent quantification results were turned up (Table
The quantitation results of concentrated propofol standards with two different kinds of evaporation.
Added methanol volume ( |
0 | 100 | 400 | 900 | 1900 |
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Recovery (%) of nitrogen gas evaporation |
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Recovery (%) of centrifugal vacuum evaporation |
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Recovery was calculated by dividing the area of 0
All the recoveries are displayed with mean values ± standard error.
The nitrogen gas concentration and centrifugal evaporation tests suggested propofol to be a very volatile compound, indicating that the general propofol quantification methods with nitrogen gas concentration result in inaccurate quantification value. However, in this study, propofol was extracted from human plasma with mixed-mode cartridge, stably without any loss due to concentration. Also, this simple and accurate method was validated and the results were satisfactory. Furthermore, authentic human plasma samples were successfully applied to the method. Thus, this extraction method could be a new guideline for other researchers and can be employed in forensic and other analytical fields.
The author declares that they have no competing interests.
This research was supported by Kyungsung University Research Grants in 2016.