Establishment And Validation Of A HPLC-MS/MS Method For The Determination Of Tigecycline And Its Application To Population Pharmacokinetics Of Tigecycline

Objective:Tigecycline,the first member of the glycylcycline class of antimicrobial agents,has a wide range of antibacterial activities and is often used to treat severe infections caused by multi-drug resistant bacteria.The recommended dosage of tigecycline is 100mg qd,50mg q12h.However,the condition of critically ill patients is complicated and the diseases develop rapidly.There are large individual differences in the blood concentration of tigecycline,and the dosage in the instructions may not be enough to achieve the expected therapeutic effect.How to use tigecycline optimally has become a clinical issue of increasing concern.Therefore,this study aims to establish and validate a simple,effective,and rapid high-pressure liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method for the quantitative measurement of tigecycline in patients,Then to evaluate the clinical efficacy and microbiological efficacy of high-dose tigecycline in critically ill patients with pulmonary infections.As the same time,to describe the population pharmacokinetic of tigecycline in critically ill patients in China and to optimize the dosing regimens by Monte Carlo simulation.Methods:Tigecycline-d9 was used as an internal standard(IS),and acetonitrile was used as a precipitant to process plasma samples by a protein precipitation method.The analyte and IS were separated on an HSS T3(2.1×100 mm,3.5 μm)chromatographic column using isocratic program with a mobile phase comprising of 80%solvent A(water containing 0.1%formic acid(v/v)with 5 mM ammonium acetate)and 20%solvent B(acetonitrile)with a flow rate of 0.3 mL/min.The sample volume injected by the autosampler was 3 μL.The mass spectrometer,scanning in multi-reaction monitoring(MRM)mode and using an electrospray ion source(ESI),operated in the positive-ion mode.The ion pairs used for quantitative analysis were m/z 586.4→513.3 and m/z 595.5→514.3 for TGC and the IS,respectively.The selectivity,carryover effects,linearity,lower limit of quantification(LLOQ),matrix effect,accuracy and precision of the method,and the stability of plasma samples were investigated.From March 2018 to October 2020,82 patients who were receiving tigecycline treatment for pulmonary infections in the adult ICU of Guangdong Provincial People’s Hospital were enrolled in this study,of them 26 patients had received the treatment of standard-dose tigecycline(loading dose 100mg,maintenance dose 50 mg q12h),while 56 patients had received therapy of high-dose tigecycline(100 mg q12h).Clinical baseline characteristics,including sex,age,BMI,clinical scoring system,diagnoses in ICU admission,underlying diseases,the dosage of tigecycline and course of treatment,efficacy and factors affecting efficacy,as well as prognoses were recorded.Then analyze the factors affecting the clinical efficacy and microbiological efficacy of ICU patients with pulmonary infections through binary logistic regressionThis was a single center,prospective,observational study that was conducted in intensive care units of Guangdong Provincial People’s Hospital.We collected a total of 155 eligible patients’ blood samples,of which 115 were included for establishing the PPK model.Another 40 patients were conducted as a validation group.Tigecycline plasma concentrations were measured by LC-MS/MS that had been established.At the same time,the basic information,including demographic data and laboratory test results was collected.The population pharmacokinetic model of tigecycline was established by NONMEN,and the dosage regimen was optimized by Monte Carlo simulation.Three target exposures,expressed as ratios of the area under the curve to MICs(AUC/MIC),were evaluated(≥17.9 for complicated skin and skin-structure infections,≥6.96 for complicated intra-abdominal infections,≥4.5 for hospitalacquired pneumonia).Results:The standard curve equation of tigecycline y=15.7x+0.0498(r=0.978).The range of the linear calibration curve obtained with this approach was 50～5000 ng/mL.The LLOQ is 50 ng/mL.Intra-and inter-batch precision for TGC quantitation were less than 7.2%.The ISnormalized matrix effect was 87%to 104%.Plasma samples have undergone five repeated freezing and thawing cycles,placed at room temperature and autosampler for 24 hours,and can be stored frozen for at least 130 days at-80℃ with good stability.The rates of clinical efficacy and microbiological eradication in the high-dose tigecycline group were significantly increased compare with the standard-dose tigecycline group after therapy(clinical efficacy rates:62.5%vs 38.5%;microbiological eradication rates:50.8%vs 26.7%),(P<0.05).The independently protective factors of clinical effectiveness were the use of high-dose tigecycline[odds ratio(OR)=3.341,95%confidence interval(95%CI):1.096～10.186,P=0.034]and the duration of tigecycline treatment(OR=1.249,95%CI:1.105～1.411,P=0.0004).A two-compartment model with zero-order absorption and first-order elimination adequately described the data.The estimated values of mean population PK parameters were 16.3 liters/h and 40.7 liters/h for clearance and intercompartmental clearance,respectively,and 145 liters and 345 liters for volume of the central and peripheral compartment,respectively.Creatinine clearance(CrCL)is the only factor affecting clearance,with an adjusting factor of CrCL as 0.0069.The simulation results show that for hospital-acquired pneumonia,only for MIC=0.25 mg/L,the standard dose can reach 100%of the target achievement rate;for MIC≤1 mg/L,the standard dose can achieve a higher target achievement rate(PTA)is only for renal insufficiency patients(CrCL≤60 mL/min);For patients with normal renal function or higher renal clearance(ARC)patients,large doses of tigecycline are needed;for higher MIC,we need higher dose.For complicated intraabdominal infections,the standard dose can achieve higher PTA when MIC=0.25 mg/L and MIC=0.5 mg/L and non-ARC conditions.For complicated skin and skin-structure infections,standard dose can achieve the expected therapeutic effect only for patients with MIC=0.25 mg/L and renal insufficiency patients(CrCL≤60 mL/min).Conclusion:In this study,a fast,sensitive and accurate HPLC-MS/MS method was established to measure the plasma concentration of tigecycline in patients.This approach was effectively applied to study the pharmacokinetics of TGC in critically ill adult patients.For critically ill adult patients with pulmonary infection,high-dose tigecycline and appropriate treatment duration made a significant impact on clinical efficacy and microbial clearance.At the same time,a population pharmacokinetic model of tigecycline in critically ill patients was established,and it was found that creatinine clearance is the only factor affecting clearance.The best dosage regimen was given for different levels of renal function,different MIC concentrations,and different infection types through simulation.