| Polyethylene glycol(PEG)is a kind of synthetic polymer formed by repeating ethylene oxide subunits,and the molecular weight of each subunit is 44 Da.As an essential pharmaceutical excipient,PEG is widely used for its excellent water-solubility,high hygroscopicity,stable chemical property and good safety under normal doses.PEG with low molecular weights mainly act as solvents or surfactants to adjust the solubility and viscosity of drugs.While PEG with high molecular weights is often applied in solid or new formulations,they can also be used as frameworks in combination with other polymers to control the release rate of drugs.The pharmacokinetics of therapeutic agents improves apparently when covalent conjugated with PEG,and this modification technology is called PEGylation.The status of PEG should arouse enough attention after the administration of PEGylated drugs,such as the separated condition of drugs from PEG,the remaining time in the circulation,and the accumulation when taken long-term administration,for it will provide important reference in the research of PEGylated drugs.Therefore,the development of an accurate and effective method for the biological quantitative analysis of PEG with varied chain lengths is of great significance.In recent years,the fast-developing technology of liquid chromatography-tandem mass spectrometry has improved the sensitivity and selectivity of quantitative analysis to a higher level.PEG is a mixture composed of a series of moleculars with similar polymerization degrees,so it has polydisperse molecular weights.In addition,their long chains are prone to be multicharged in the electrospray ionization source,resulting in numerous precursor ions.Because of the defect in the software and hardware design,it is almost impossible for traditional mass analyzer to dissociate all the ions and obtain the data of their fragments sensitively.In this report,we have presented the first attempt Q-TOF mass spectrometry(MS)based on MSAll technique to solve the problem above,and developed the quantitative analyzing method for linear PEG with different polymerization degrees in complicated biological samples.Our strategy was operated as follows:the entire PEG ions passed the first quadrupole directly and entered the collision cell without selecting the parent ions.Then the long chains were dissociated effectively by the high collision energy in the way of collision induced dissociation,generating a few PEG-specific fragments composed by several ethylene oxide subunits,which were common for PEG with different polymerization degrees.These fragments were full scanned by the Time-of-flight analyzer subsequently,and the one with three repeating subunits(theoretical mass-to-ratio was 133.08592)was chosen as the quantitative ion because of its best linearity and reproducibility.Besides,the high-resolution capability of Q-TOF MS could achieve a mass error within 5.41 ppm during our research,leading to a better selectivity.As for the ion chromatogram extraction process,the mass extraction window was optimized to ±5.0 mmu,in order to remove endogenous interference in matrix as much as possible and obtain the best signal-to-noise ration.It was linear over the concentration of 0.05?5.0 ?g/mL with good accuracy and precision.The selectivity and reproducibility could also meet the demand of quantitative bioanalysis.The strategy has been successfully employed in analyzing the concentration of various linear PEG with different molecular weights ranging from 400 to 20000 Da in biological samples.It will provide a promising support to the research of PEG and PEGylated drugs,so as to completely characterize their fate in vivo.It may also bring novel quantitative idea for the method development of PEG with other structures or similar synthetic polymers in further research.The biological behavior in vivo has been researched using the quantitative analyzing method described above.After intravenous administration of PEG with three different molecular weights by the dosage of 3.0 mg/kg,PEG with low and medium molecular weights almost eliminated completely over the initial 12 h and 24 h post-dose,respectively.While the concentration of PEG with high molecular weights decreased gently and a small portion were still remained in plasma 48 h after administration,showing the feature of prolonged circulation.The dynamics process of PEG with different molecular weights fitted two-compartment model,and the t1/2 of PEG prolonged as the molecular weights increased.PEG 4000 distributed widely and rapidly after intravenous administration with Tmax,around 0.25 h,and it could reach the dynamic distribution balance between plasma and tissues at 1.5 to 4 h post-dose.Among these tissues,the concentration of PEG 4000 in spleen,liver,kidney and lung were relatively high,which might be related to their structure and function.First,the blood flow volume of these tissues is large,benefitting substance exchange with plasma.Secondly,there are plenty of reticuloendothelial system in spleen,lung and liver,which can uptake some PEG by macrophages in order to remove these exogenous macromolecules out of body.Moreover,liver can play important roles in metabolism and detoxification,while kidney is the main organ for PEG elimination.As a result,it was not hard to understand that the concentration and amount of PEG 4000 in these tissues was higher than others.There was no PEG 4000 detected in brain,which was probably blocked by the blood barrier.The concentration of PEG 4000 in urine and fence was measured afterwards.The total cumulative excretion of PEG 4000 was 44.0%.The results showed that renal excretion was the dominant pathway for the elimination of PEG 4000.The cumulative urinary excretion of PEG 4000 was 43.4%of the administration dose,which accounted for 98.9%of the total excretion with 92.0%of counts occurring within the initial 8 h.Only 0.56%of the administration dose was excreted in fence.PEG 4000 was almost not broken in vivo and excreted in the form of its original length. |
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