Monitoring And Prediction Of Unbound Concentration Of High Protein Bound Antibiotics In Children

BackgroundAfter the drug enters the bloodstream,part of it binds to plasma proteins,and part of it remains free.The free drugs are responsible for the pharmacokinetic and pharmacodynamic activity,for only the unbound part is considered pharmacologically active and can be eliminated.Various factors may affect the protein binding,such as age,disease,physiological state and endogenous&exogenous substance replacement,etc.Children are in a stage of rapid growth and development,their pharmacokinetic characteristics,including protein binding,will be influenced by organ development,enzyme development,transporter activity,etc.There are still significant technique barriers in applying TDM of unbound drugs in pediatric clinical practice,mainly due to the restricted vascular access,which limits the volume and number of blood samples we can obtain.Moreover,compared with the total concentration,the measurement of unbound concentration requires a larger sample volume,the extra time and additional cost to perform.Ceftriaxone and cefoperazone,high protein bound antimicrobial agents,are commonly used in pediatrics.Previous studies have shown that the unbound fractions of high protein bound drugs in children(especially newborns and infants)are significantly different from adults,and there are also large individual differences.Extreme free concentrations may lead to treatment failure or toxic reactions,which may produce adverse clinical outcomes.Therefore,the unbound fraction and its influencing factors can provide valuable information for children's drug treatment,and achieve the purpose of improving the effectiveness of treatment and reducing adverse reactions.PurposeThe study aims to develop and validate sensitive method to quantify the unbound ceftriaxone and cefoperazone concentrations in children,providing an effective tool for therapeutic drug monitoring and pharmacokinetic studies.Furtherly,we aim to study the factors which influencing the unbound fraction of ceftriaxone and cefoperazone in children,and simple mathematical formulas was used to predict the free concentration.When it is inconvenient to measure directly,the free concentrations of ceftriaxone and cefoperazone in children can be easily and quickly obtained.MethodsUsing high-performance liquid chromatography combined with UV detector to develop and validate the analysis methods for the quantification of total and free ceftriaxone.Blood samples of pediatric patients treated with ceftriaxone were collected,and the total and free concentrations of the samples were obtained using the established analytical methods.Combined with the clinical information,a multiple linear regression model was established using SPSS.The influence on the protein binding of total concentration,albumin,age and other factors were evaluated.The prediction performance of the empirical in vivo equation,disease adapted equation and multiple linear regression equation was assessed by MAPE,MPE and other indicators.Using high-performance liquid chromatography combined with UV detector to develop and validate an analysis method for the quantification of total cefoperazone.An LC-MS/MS method for the analysis of free cefoperazone in plasma has been developed and validated.Blood samples of pediatric patients treated with cefoperazone were collected,and the total and free concentrations of the samples were obtained using the established analytical methods.Combined with the clinical information,a multiple linear regression model was established using SPSS.ResultsThe quantitative ranges of bioanalysis methods were 2-600 ?g/mL and 0.5-150?g/mL for total and unbound ceftriaxone,respectively.A total of 92 infants and young children were included in the study and 95 plasma samples were collected.The average total and unbound ceftriaxone concentrations were 126.18± 81.46 ?g/mL and 18.82 ± 21.75 ?g/mL,and the unbound fraction varied greatly from 4.8%to 40.0%,showing concentration-dependent protein binding and large individual variability.The results of multiple linear regression have shown that total concentration,albumin,bile acid and prealbumin were significant factors which influence the free concentration.Total concentration and albumin were strong influencing factors.Compared with the other two equations,the disease adapted equation showed the best predictive performance.The quantitative ranges of bioanalysis methods were 0.05-100 ?g/mL for total cefoperazone.The accuracy was in the range of 90.5%-95.5%,and the intra-day and inter-day precision were less than 8.3%.The quantitative ranges of bioanalysis methods were 5-5000 ng/mL for unbound cefoperazone.After validation,there was no obvious matrix effect and the recovery is stable;the accuracy is between 91.5%-105.0%,and the precision is less than 8.1%.A total of 17 neonatal patients were included in the study,and 17 blood samples were collected at the valley concentration.The total and unbound concentrations of the patients were in the range of 1.1-45.6?g/mL and 0.4-11.2?g/mL,respectively.The unbound fractions of cefoperazone are in the range of 11.2-48.0%,which is significantly higher than the reported ratio in the adult.After multiple linear regression analysis,the total cefoperazone was a significant factor influencing the free concentration.Albumin had a negative correlation with the unbound fraction.The multiple linear regression equation predicts the free concentration of cefoperazone well.ConclusionIn this study,we developed and validated fast and reliable HPLC-UV analysis methods for the quantification of total and free ceftriaxone in plasma,and we clarified the influencing factors of ceftriaxone and plasma protein binding in children.The estimation of free ceftriaxone can promote the clinical study of free concentration of ceftriaxone in children.In addition,we developed and validated a sensitive,fast,and accurate LC-MS/MS method for the determination of free cefoperazone in small volume of plasma firstly,which can be used to conduct therapeutic drug monitoring in children(neonatal).At the same time,we found that the unbound fraction of cefoperazone in the neonatal population was significantly higher than that in adults.The study will provide guidance information for the treatment of cefoperazone in neonates.