Transfer Of China-made Remifentanil Across The Single Cotyledon Of Term Human Placenta

Objective: To establish the dual perfused of single cotyledon humanplacental models for the further study of remifentanil across the term humanplacenta. To establish a HPLC/MS/MS method for the determination ofremifentanil and meet the demand of clinic applications.Methods:10placentas were obtained from healthy full term parturientswithin10min after cesarean section delivery. The placentas were perfusedwith heparinized Krebs Ringer buffer (KRB)(pH=7.4,4℃).Perfused by aumbilical artery and squirt to the surface of the placenta, to maintains thefunction of placenta and prevent the blood coagulation. Afer visual inspectionfor tears, an artery and vein supplying a well-defined cotyledon werecannulated with infant feeding tubes and go on to perfused with KRB. Ifarterial and venous flow rates were equal, the placenta preparation was placedin a chmber with the maternal side facing upward. After transferring theplacental chamber to the perfusion cabinet, the maternal intervillous spacewas perfused by inserting three blunt-tipped19-gauge needleds2-3mm belowthe maternal plate. At the beginning of each experiment,the fetal flow rate(1-3ml/min) were adjusted to provide perfusate pressure of60-75mm Hg.Maternal flow were maintained at (10-12ml/min), to provide perfusatepressures of35-45mm Hg. The pH of both perfusates was held constant at7.4±0.05by manipulating the amount of carbon dioxide in the gas mixture. At1-min intervals, pressure and pH were recorded. To test the integrity andviability, glucose consumption and lactate production were measured at theend of the experiments.The dual perfused of single cotyledon human placenta models were made.The closed (recirculating) models or the open (single-pass or nonrecirculation)models of placental perfusion was used to investigate the placental passage of remifentanil. The closed method: the group of maternal to fetal (MTF, n=3)and the group of fetal to maternal (FTM, n=3). Remifentanil (10ng/ml) andantipyrine (1μg/ml) wer added to either the maternal or fetal reservoir afer a30-min equilibration period.1ml perfusate samples were collected from bothfetal an maternal reservoirs at15,30,60,90and120min after the addition oftest drugs. Then determinate the concentrations of remifentanil, antipyrine,glucose and lactate. Glucose consumption rate, lactate generation rate and therelative and absolute transfer ratio of reminfentanil were calculated. Theclosed models use fixed volumes for donor (250ml) and recipient (100ml)circuits. In all6closed experiments performed, the human fresh frozen plasma(FFP) as perfusate in the maternal circuit and KRB with4g/100ml albumin inthe fetal circuit.The open models:(MTF, n=4) Both the maternal an fetal circuit use KRBas perfusate.The open models use large maternal (3000ml) and fetal (1000ml)reservoirs from which the perfusates are pumped through the placenta and outto two waste reservoirs. Progressively increasing maternal perfusateconcentrations of remifentanil (10，20，50and100ng/ml) were perfusated tothe placenta. During each of these2-h perfusions,2-ml samples were takensimultaneously from the maternal reservoir and the fetal venous outflow at15-min intervals. Then determinate the concentrations of remifentanil and thespeed of the fetal venous outflow. Then the transfer of speed werecalculated.Establish a HPLC/MS/MS method for the determination of remifentanil,use fentanyl cirate as an internal standard. The sample were separated over anPhenomenex SB-C18（2.1×30mm，1.8um）and the mobile phase consisted ofa mixture of A: methanol,B:water with0.1%formic acid. The triplequadrupole mass spectrometer with the turbo was set at100℃. The pressureof atomization gas was25.0psi. The multi-reaction monitoring (MRM)mode was employed to scan the ions. The selected ions (two product-ions)were (317.1，228.2) for remifentanil, fentanyl were (105.1，188.2) and (56.1，76.9) for antipyrine. Results: Remifentanil crossed the placenta rapidly in both (Mâ†'F andFâ†'M) directions. Mâ†'F(0.66±0.04) and Fâ†'M (0.76±0.03)remifentanil/antipyrine transfer ratios were not significationly different in theexperiment（sn=6，P＞0.05）.A linear increase in the remifentanil transfer ratewas observed with increasing maternal circuit remifentanil concentration （1,10,20,50and100ng/ml）.Conclusion: Absolute transfer ratio of remifentanil increased with theperfusion time. Remifentanil appears to be bound by the placental tissues, butonly less. This may be due to that the distribution coefficient of alcohol/waterof remifentanil is very low. The method of HPLV/MS/MS,is rapid,accurateand sensitive, it can meet the demand of remifentanil determination in clinicapplication.