Meropenem Pharmacokinetic Study Of CRRT Patients And Non-CRRT Patients By LC-MS/MS Method

Meropenem is second generation of carbapenem antibiotics with a wide spectrum of activity.It shows bacterial killing of Gram-positive and Gram-negative pathogens.Kidney plays a pivotal role in meropenem elimination.Meropenem is usually used to treat pneumonia,gynaecology infection,urinary tract infection,meningitis and sepsis.Acute Kidney Injury occurs usually in patient with severe infection or sepsis.CRRT displays good biocompatibility,slow and continuous elimination,so CRRT is the best therapy to choose in treatment of AKI.Due to the influence of CRRT in medicine pharmacokinetics,researches on meropenem pharmacokinetics have important clinical significance.Objective:1 To establish LC-MS/MS method,the method will be applied in determination of meropenem concentration in human plasma.2 To discuss the effect of different CRRT dose on meropenem pharmacokinetics with same drug dose.3 To discuss the difference of meropenem pharmacokinetics between CRRT patients and non-CRRT patients when patients’ creatinine clearance rate are lower than 50ml/min.4 To discuss influence of renal function on meropenem pharmacokinetics without CRRT.Methods:1 Determination of meropenem in human plasma using LC-MS/MS.1.1 Chromatographic conditionschromatographic column: Kinetex HILIC column(100×2.1 mm,2.6um)(Phenomenex,USA);Mobile phase: A: aqueous solution of 0.2% Formic acid and 8mM ammonium acetate,B: ACN;flow rate: 0.4ml/min;injectionvolume: 5μl;Column temperature: 40℃;Gradient elution.1.2 Mass spectrometry conditionsUsing electrospray ion source,Positive ion mode,MRM.Mass spectrum parameters: IS:5500V;Ion source temperature:500℃;GS1:50 psi;GS2:50 psi;CUR:25psi;Meropenem: m/z 384.2→m/z 141.1,DP:65V,CE:25 V;prazosin: m/z384.1→m/z 247.0,DP:100V,CE:45 V.1.3 plasma sample preparationAdding 10μl prazosin solution and 800μl ACN to centrifuge tube with100μl plasma,vortex 3min,and centrifuge(15000g,10min),separate 500μl supernatant,centrifuge(15000g,3min),then determine supernatant.2 Meropenem pharmacokinetics of CRRT patients and non-CRRT patients.2.1 Patients and meropenem dosePatients who received meropenem treatment in ICU of Fourth Hospital of Hebei Medical University were involved.16 patients received CRRT and meropenem of 1g,q12 h.20 patients received meropenem of 1g,q8 h or 1g,q12 h.2.2 Samples collection and conservation2.2.1 Samples collection and conservation of CRRT patientsAfter CRRT patients was given meropenem more than once,collected2 ml blood,time of collection were 0 h,0.5 h,1h,1.5 h,2 h,4 h,6 h,8 h,10 h,12 h after meropenem intravenous drip,then centrifuge and conserved plasma by microtubes in refrigerator(-70℃).2.2.2 Samples collection and conservation of non-CRRT patientsAfter meropenem distribution reached steady state in non-CRRT patients,collect 2ml blood,time of collection were 0 h,1h,2 h,4 h,6 h after meropenem intravenous drip,then centrifuge and conserved plasma by microtubes in refrigerator(-70℃).2.3 Data processingProcess meropenem plasma concentration by WinNonlin6.4 and SPSS21.0,blood samples which collected before filter were convert to plasmaconcentration by coefficient.Data were calculated by non-compartment model and linear trapezoidal linear interpolation in WinNonlin6.4.SPSS 21.0 express data using Mean±SD,t-test was used in two groups comparison.Multiple groups were compared after homogeneity test of variances,one-way analysis of variance was used in multiple groups comparison,Dunnett-t test was used in multiple groups comparison if difference existed.Results:1 Determination of meropenem in human plasma using LC-MS/MS.Specificities of meropenem and prazosin in plasma were good in this method.Endogenous substances in plasma have no interference in determination.Liner of meropenem was good in 0.5μg/ml-50μg/ml,the low limit of quantification was 0.5μg/ml.The extraction recoveries of low concentration(1μg/ml),middle concentration(8μg/ml),high concentration(40μg/ml)were in the range of 99%-106%,intra-day RSD% and inter-day RSD% were lower than 8%,RSD% of low limit of quantification was lower than 20%.Meropenem stabilities of freeze thawing,storage in-70℃ for 60 days,placement in automatic sampler for 12 hours,placement in room temperature for 4 hours,placement in 4℃ for 4 hours satisfied validation guidelines of biological sample quantitative analysis.The matrix effect and stability of dilution meet validation guidelines too.2 Meropenem pharmacokinetics of CRRT patients and non-CRRT patients2.1 Meropenem pharmacokinetics of 1g,q12 h and different dose of CRRTHalf life of meropenem in patients receiving 25mL/kg/h and 35mL/kg/h of CRRT were(3.31±0.63)h and(2.63±0.38)h,Vd were(23.17±6.16)L and(16.82±3.68)L,comparisons of half life and Vd had statistical differences,comparisons of Cmax,AUC,CL had no statistical difference in two groups.2.2 Meropenem pharmacokinetics of CRRT patients and non-CRRT patients when patients’ creatinine clearance rate are lower than 50ml/min.Meropenem pharmacokinetics of patients receiving CRRT of 25mL/kg/h were compared with non-CRRT patients,the Cmax,Vd and AUC were lower than non-CRRT patients,CL was higher than non-CRRT patients.Comparisonof Cmax,,AUC,Vd and CL had statistical differences,Meropenem pharmacokinetics of patients receiving CRRT of 35mL/kg/h were compared with non-CRRT patients,half life,AUC and Vd were lower than non-CRRT patients,CL was higher than non-CRRT patients,these parameters had statistical differences.2.3 Meropenem pharmacokinetics of non-CRRT patients with different renal functionMeropenem pharmacokinetics of patients with creatinine clearance in a range of 60-90ml/min and 50-60ml/min were compared with patients with creatinine clearance > 90ml/min,half life prolonged,AUC and Vd were higher than patients with creatinine clearance >90ml/min,CL was lower than patients with creatinine clearance > 90ml/min.These comparisons had statistical differences.Conclusion:1 The study establishes LC-MS/MS method to determine meropenem concentration in human plasma.This method is accurate,convenient and has good specificity and it can be used in meropenem pharmacokinetics.2 When patients are given meropenem at a dose of 1g,q12 h,half life and Vd are lower of meropenem in patients receiving CRRT dose of 35mL/kg/h.And these comparisons have statistical differences(P<0.05).Comparisons of AUC and CL have no statistical differences.So,CRRT dose of 35mL/kg/h can accelerate metabolism of meropenem.3 When patients receive meropenem of 1g,q12 h and creatinine clearance<50ml/min,CRRT have a significant impact on meropenem pharmacokinetics when compared with non-CRRT patients.CRRT accelerate metabolism and CL of meropenem as a way of blood purification.4 When patients receive meropenem of 1g,q8 h,meropenem have a lower metabolism and CL in patients with poor renal function,medication of meropenem should regulate according to renal function to prevent adverse events.