| Objectives: To establish a HPLC method for determination of norvancomycin in cerebrospinal fluid?CSF?;To investigate the drug concentration in CSF of patients who is administrated norvancomycin by intravenous infusion or intrathecal injection for the treatment of intracranial infection after neurosurgical operation;To summarize the results and give the conclusion about clinical significance.Methods:1 Chromatographic parameters are listed as follows: Column: Diamonsil C18 column?150 mm×4.6 mm,5?m?;Mobile phase: methanol: monopotassium phosphate?0.025 mol/L,pH 3.0?=18:82?v/v?;flow rate: 1.0 mL·min-1;Column temperature: 30?;Detection wavelength: UV 236 nm;Injection size: 20 ?L;Internal standard: vancomycin.2 CSF sample processing: 400 ?l of CSF sample is added to a centrifuge tube,10% perchloric acid is added and mixed thoroughly,centrifuge for 5 min at 10000r·min-1,20 ?l of the supernatant is injected into the chromatographic system.3 Linearity and Range: a series of norvancomycin CSF reference standard solution with concentrations of 50,20,10,5,2,1,0.5,0.2 and 0.1?g·mL-1 are prepared and processed as described above,20 ?l of each supernatant is injected into the chromatographic system.Calibration curve is drawn by regression analysis based on the peak area ratio of norvancomycin to internal standard?Y?versus the norvancomycin concentration in CSF?X?,the linearity and range is obtained.4 Recovery: norvancomycin CSF reference standard solution with concentration of 0.2,2,and 40?g·mL-1 are prepared and injected into the chromatographic systems.The absolute recovery of norvancomycin is obtained by comparing the peak area of norvancomycin in CSF reference standard solution with that of reference standard solution;also,the absolute recovery of internal standard is obtained by comparing the peak area of internal standard in CSF reference standard solution with that of reference standard solution.The relative recovery is obtained by comparing the concentration of norvancomycin calculated according to calibration curve with that of theory.5 Precision: norvancomycin CSF reference standard solution with concentration of 0.2,2,and 40?g·mL-1 are prepared and injected into the chromatographic systems,5 determinations are performed for each concentration in different times within a day to obtain intra-day precision;or prepared and determined in 3 days to obtain inter-day precision.6 Stability: norvancomycin CSF reference standard solution with concentration of 0.2,2,and 40?g·mL-1 are determined at 0,6,12 h to investigate the stability of sample stored at room temperature;norvancomycin CSF reference standard solution with concentration of 0.2,2,and 40?g·mL-1 are determined immediately or after freeze-thaw for thrice to investigate the stability of samples after repeated freeze-thaw;norvancomycin CSF reference standard solution with concentration of 0.2,2,and 40?g·mL-1 are determined under stored at-40? for 30 day,240 day to investigated the stability of samples stored in low temperature for a long time;processed norvancomycin CSF reference standard solution with concentration of 0.2,2,and 40?g·mL-1 are determined at 0,2,4h to investigate the stability of processed samples.Norvancomycin reference standard solution with concentration of 0.2,2,and 40?g·mL-1 are determined at 0,4,12 h to investigate the stability of standard solution stored at room temperature;norvancomycin reference standard solution with concentration of 0.2,2,and 40?g·mL-1 are determined at 0,2,5 day to investigate the stability of standard solution stored in refrigerator.7 collection of samples: to adult patients of intracranial infection after neurosurgical operation with lumbar cistern tube,norvancomycin is administrated by intravenous infusion,2 ml of CSF sample is collected at 0 h,2 h,4 h,6 h,8 h and 12 h;10 mg of norvancomycin is administrated by intrathecal injection,2ml of CSF sample is collected at 2 h,4 h,8 h,12 h and 24 h.Centrifuge,the supernatant is stored at-40? for further analysis.8 Pharmacokinetic analysis: the norvancomycin concentrations in CSF are determined by HPLC,the datum of two groups are processed using SPSS statistic software.Results:1 The retention times of norvancomycin and internal standard are 5.8 min and 7.4 min,respectively.The endogenous substance in CSF don't affect the determination of norvancomycin and internal standard.2 Calibration curve: Y=0.679X+0.021?R2=0.994?,and the linear range is 0.150?g·mL-1.3 Recovery: the absolute recoveries of norvancomycin at concentrations of 0.2,2,and 40?g·mL-1 are?77.54±1.68?%,?79.46±2.05?% and?88.08± 5.04?%,respectively;the absolute recovery of internal standard is?80.11±4.84?%;the relative recoveries of norvancomycin at concentrations of 0.2,2,and 40?g·mL-1 are?96.90±4.84?%,?96.26±5.13?% and?102.36±3.09?%,respectively.4 Precision: the values of RSD of intra-day precision of norvancomycin CSF reference standard solution at concentrations of 0.2,2,and 40?g·mL-1 are 5.00%,5.33% and 3.03%,respectively;the values of RSD of inter-day precision of norvancomycin CSF reference standard solution at concentrations of 0.2,2,and 40?g·mL-1 are 6.43%,3.86% and 8.78%,respectively.5 Stability: the norvancomycin CSF reference standard solution are stable when stored at room temperature for 12h;the norvancomycin CSF reference standard solution are stable when freeze-thaw for thrice;the norvancomycin CSF reference standard solution are stable when stored at-40? for 240 day;the processed norvancomycin CSF reference standard solution are stable when stored at room temperature for 4h;the norvancomycin reference standard solution are stable when stored at room temperature for 12h;the norvancomycin reference standard solution are stable when stored at 4? for 5days.6 During the trial,19 CSF samples of intravenous infusion group are collected,of which,6 patients receive intrathecal injection when the treatment is not effective.CSF sample is collected at 0,2,4,6,8,12 h for intravenous infusion group and at 2,4,8,12,24 h for intrathecal injection group,the average concentrations at each sampling time for intravenous infusion group and intrathecal injection group are 2.810±1.907,2.278±1.487,1.313±0.812,1.005±0.668,0.716±0.559,0.503±0.465 and 150.72±34.29,75.52±13.02,28.81±8.39,17.11±7.46,4.66±1.71,respectively.For the intravenous infusion group,dose is 0.8g and interval is 12 h.The results show that: the norvancomycin concentration in CSF is higher than the MIC of common pathogens?P<0.05?at 0h;it is about equal to the MIC of common pathogens?P<0.05?at 2h;it is lower than the MIC of common pathogens?P<0.01?at 4h.For the intrathecal injection group,dose is 10 mg and interval is 24 h.The results show that: the trough concentration is higher than the MIC of common pathogens?P<0.05?,however,it is still lower than the effective treatment concentration of 10?g·mL-1?P < 0.01?;It reaches the effective treatment concentration of 10?g·mL-1 at 12 h.The results indicate that the trough concentration is likely to reach effective treatment concentration when the dosing interval is 12 h.Conclusion:The HPLC method established for determination of norvancomycin concentration in CSF is simple,accurate,sensitive and reproducible,and it is suitable for its intended use.When norvancomycin is administrated by intravenous infusion for treatment of intracranial infection after neurosurgical operation,although the drug concentration in CSF could reach the effective treatment concentration,the duration is too short,and it varied in different patients,as a result,the effectiveness couldn't be ensured;when intrathecal injection is adopted,the drug concentration in CSF is high and could meet the requirement of treatment.As a conclusion,intrathecal injection is a good choice when intravenous infusion is not effective. |
PDF Full Text Request