| Part one: Determination of remifentanil concentration in plasma by HPLC with UV detectionObjective: Remifentanil is a new, ultra-short-actingμ- receptor agonist. It is susceptible to rapid metabolism by esterases in the blood and other tissues for it's unique structure. Remifentanil has a rapid onset and offset of action and rapid blood-brain equilibration properties that facilitate the control of the depth when anesthetizing. In this study, HPLC-UV method was established for determination of remifentanil in plasma. The study will provide academic foundation for the development and clinical use of remifentanil.Methods: The remifentanil in plasma was extracted with 1-butyl chloride, the compound was back extracted into 0.01 mol·L-1 HCl again. The sample was separated on spherisorb- CN(4.6 mm×250 mm, 5μm, Waters) column with a mobile phase of mixture of acetonitrile and phosphate buffer (30: 70) including 0.01% triethylamine at a flow rate of 1.5 mL·min-1, column temperature was room temperature, the detection- wavelength was 210 nm and 200μL of solution was injected into HPLC. To evaluate the methodology by detecting the linear range and limit of test, drawn recovery rate, RSD of inter-day and intra-day, and then to prove whether it could be used for the pharmacokinetic studies for remifentanil or not.We studied six patients of ASAI~II undergoing thoracic surgery, aged 40~65 year and BMI < 30 % of both sexes. Anesthesia was induced with propofol 2.5 mg·kg-1 and remifentanil 4μg·kg-1. When the patients lost conscionsness, rocuronium was given intravenously to facilitate intubation. Anesthesia was maintained with sevoflurane and intermittent intravenously boluses of vecuronium 0.03 mg·kg-1. Venous blood samples were taken before and after remifentanil injection 1, 2, 3, 5 min. For each blood sample, 3 mL venous blood samples were collected into a tube with sodium heparin and centrifugated, then get the supernatant fluid and frozened. The remifentanil blood plasma concentrations were analyzed by HPLC.Results: In this experiment, there existed a district separation between remifentanil in the plasma and the baseline. There was a significant correlation between remifentanil concentrations and area under curve in the scope of 5~300 ng·mL-1 in plasma. Calibration equation of remifentanil in plasma was Y = -1585 +530.54X, r = 0.9996, (n = 7). The limit of determination for remifentanil was 2.5 ng·mL-1 in plasma. Under this condition, the drawn rate of high concentration (200 ng·mL-1), middle concentration (50 ng·mL-1) and low concentration (5 ng·mL-1) were (88.99±2.20)%, (83.72±2.31)% and (83.47±1.7)% respectively. RSD of inter-day were 3.06%, 5.13% and 7.06%, intra-day were 5.33%, 5.69% and 8.91% respectively. The RSD of the stability of zero-temperature and freezing were 5.52% and 5.87% respectively. The remifentanil in the plasma was also shown to be stable after three cycles of freeze (-50oC)-thaw (room temperature).Conclusion: This RP-HPLC method is simple, sensitive and accurate. It is suitable for routine determination of remifentanil levels in human plasma.Part two: Effects of remifentanil on pharmacokinetics of propofolObjective:The interaction between propofol and opioids is complex. It is necessary to define this interaction and provide a rational dosing schemes when such combinations are used for anesthesia. In this study, we aimed to investigate the effect of remifentanil on pharmacokinetics of propofol in humans. Serial blood samples were drawn according to the exact time schedule and concentrations of propofol in patients were determined by RP-HPLC. Our studies provide a good reference for propofol and remifentanil using in combination in pharmacokinetics of human for cancer treatment.Methods:Chromatographic condition:To establish a HPLC method to detect the propofol concentration of plasma. The propofol was separated by Waters Symmetry C18 column (3.9 mm×150 mm, 5.0μm), and the guard column was Symmetry C18, the mobile phase was a mixture of methyl alcohol and water (75: 25, pH = 4 by phosphoric acid ) at a flow rate of 1.5 mL·min-1, column temperature was room temperature. The excitation and emission wavelength were 270 nm and 295 nm respectively. 20μL of supernatant fluid was injected for analysis.Disposal and determination of blood samples:Drug-containing plasma (250μL) and 10μL (10μg·mL-1) internal standard solution were added in a 1.0 mL centrifuge tube for assay. The sample was then extracted with methyl alcohol (750μL) after shaking for 30 s by the vortex mixer. Then the mixture was centrifuged at 10000 r·min-1 for 10 min, and the upper layer was pipetted into another clean centrifuged tube. Filtered it with a 0.45μm filter membrance and 20μL liquid was used by HPLC.Preparation of standard curve:Propofol of various concentration was added to plasma at the final concentration of 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625μg·mL-1. They were processed according to the above procedure. Standard crurve was made with the content of propofol as abscissa and the ratio of peak area as ordinate.Investigation of methodology:Precision, recovery and stability of HPLC method were studied.Administration of drug and collection of blood samples:We studied twenty-one patients of ASAI~II undergoing thoracic surgery, aged 40~65 year and BMI<30%of both sexes. They were assigned to one of three groups (each group has seven patients). In the first group, The patients were given midazolam 0.05 mg·kg-1and atropine 0.5 mg·kg-1 intramuscular before anesthesia. Perioperative monitoring included electrocardiogram and HR, radial mean arterial blood pressure (MAP), SBP, DBP, peripheral oxygen saturation(SpO2), heart rate variability (HRV), bispectral index (BIS), and PETCO2, and 2.5 mg·kg-1 propofol was injected intravenously at the beginning of induction. When the patients lost conscionsness, rocuronium was given intravenously to facilitate intubation. The patients were mechanically ventilated and PETCO2 was maintained between 30~40 mmHg. Anesthesia was maintained with sevoflurane and intermittent intravenously boluses of vecuronium 0.03 mg·kg-1. In the second group, anesthesia was induced with propofol 2.5 mg·kg-1 and remifentanil 4μg·kg-1 but the doses of the other drugs for induction were the same as those in group one. In the last group, anesthesia was induced with propofol 2.5 mg·kg-1 and TCI of remifentanil 6 ng·mL-1 but the doses of the other drugs for induction were the same as those in group one.Propofol was given in bolus through the vein in the forearm slowly, and 2 mL blood sample was taken from the right vein before and after injection at the following time: 2, 4, 6, 8, 10, 15, 30, 45, 60, 90, 120, 180 min. Each sample was thoroughly mixed in tubes containing liquaemin sodium, then centrifuged, the plasma was removed and stored in refrigerator. Statistical analysis:The results of the plasma samples were analyzed with the DAS program to determine the compartment models and the pharmacokinetic parameters,and statistic analyse by SPSS program.Results: Chromatograms of propofol and the specifity: The retention time of propofol is about 6 min and retention time of thymol is about 4 min. Plasma endogenous materials has no interference with propofol.Standard curve and limit of detection:Satisfactory linearity was observed in the range of 0.0625~16μg·mL-1 of the drug. The regression equation was Y = 1.5826X -0.1264 (r = 0.9996, n = 9). In this equation, Y is the peak area comparison, C is the concentration (μg·mL-1) of the drug in plasma and r is the coefficient of correlation. The detectable limit of propofol under these conditions was 0.03125μg·mL-1.Drawn recoveries and intra-day, inter-day precision:Under this condition, draw recoveries of high concentration (8μg·mL-1), middle concentration (1μg·mL-1) and low concentrat- ion (0.125μg·mL-1) were (84.32±1.17)%, (83.21±1.24)% and (79.85±1.50)% respectively. RSD of inter-day and intra-day were both less than 5.90%.Study on Pharmacokinetics:The plasma concentrations of propofol decreased rapidly after intravenous bolus injection in all 3 groups. The present study showed that the sex, age, weight of patients, SBP, DBP and HR have no significant difference between groups during anesthesia (P>0.05). The blood concentration versus time curve for propofol was fitted to a two-compartment modal. There was no significant difference in the pharmacokinetic profile of propofol(T1/2a, T1/2β, AUC, CL, Vc) among the three groups.Conclutions: RP-HPLC method for determinating propofol concentration in plasma is rapid, simple, accurate and sensitive. It is suitable for monitoring propofol and pharmacokinetics study.Induction and TCI of remifentanil have no significant effect on pharmacokinetics of propofol.
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