Studies On The Determination Of The Concentration Of Ethacridine Lactate In Amniotic Fluid Of Pregnant Women Terminated Pregnancy In Second Trimester

Ethacridine lactate is commonly used as a drug for second trimester termination of pregnancy by injecting into the amniotic cavity in China, which proved to be associated with the lowest rate of complications. And the intra-amniotic administration could avoid the shortcomings, such as the risk of systemic absorption of ethacridine lactate and the risk of infection. Now in China, intra-amniotic administration of ethacridine lactate is widely and legally used as an abortion route in government agency or public hospitals. However, due to the traditional Chinese view that sons are better than daughters and the one-child policy in China, ethacridine lactate has been abused illegally in recent years to induce termination of second trimester pregnancies and expulsion of a female fetus shown by B ultrasound scanning, and thus to acquire a second pregnancy opportunity for a male fetus. This kind of gender-selection behavior has led to a serious imbalance of the sex ratio at birth in China, reported that since 1980s the sex ratio at birth has been high and even continued to increase. It is 111:100 in the fourth population census of 1990,and 117:100 in the fifth population census of 2000,which deviates the normal range seriously, and has a direct impact on economic development, society stablilization, and the quality of people's lives. To avoid the worsening of this imbalance, the development of a sensitive and validated method for the qualitative and quantitative determination of ethacridine lactate in biological samples to detect the illegal abuse of ethacridine lactate is becoming more important. Ethacridine lactate, 2-ethoxy-6,9-diaminoacridine lactate hydrate, is employed as a potent antimicrobial agent. It could cause the contraction of uterus in vivo and vitro. After ethacridine lactate has been injected into the amniotic cavity, the frequency and extent of uterus muscle contraction would increase and finally expulse the fetus and placenta. Success rates of the abortifacient are above 95% for clinical use, which is widely used as a technique to terminate the second trimester pregnancy. There is a little research about the determination method of ethacridine lactate, especially in biological samples. Therefore, the samples have been collected and the concentration of ethacridine lactate has been determined. In this work, a simple, sensitive and accurate method to detect ethacridine lactate in amniotic fluid has been developed.The main contents are divided into some sections as follows:1. Determination of the concentration of ethacridine lactate in amniotic fluid by spectrophotometryThe concentration of ethacridine lactate in amniotic fluid had been detected by UV-vis. The blank amniotic fluid sample had absorption on the corresponding wavelengths. It was hard to find the suitable absorption wavelength in ultraviolet spectra of the low concentration of ethacridine lactate to do quantitative determination.The concentration of ethacridine lactate in amniotic fluid had been detected by spectrofluorometry (SPF). Ethacridine lactate had been extracted one time by using n-butanol. And the excitation and emission waves had been scaned in the range of 220 nm~760 nm on WFY-28 spectrofluorophotometer. The excitation and emission wavelength were 370 nm and 495 nm in this experiment. The recovery was 93.5% and relative standard deviation (RSD) was 3.0%. This method was convenient, rapid and had good sensitivity. However, the blank amniotic fluid had some emission in the wavelength. When the concentration of ethacridine lactate in the amniotic fluid was too low, it was hard to determine whether there was ethacridine lactate in the amniotic fluid. Therefore, this method had some certain limitations.2. Determination of the concentration of ethacridine lactate in amniotic fluid by high performance liquid chromatography (HPLC)The HPLC method was used to determine the content of ethacridine lactate in amniotic fluid. The analysis of ethacridine lactate was carried out on Waters Spherisorb C18 column(4.6 mm×250 mm×5μm). The mobile phase was MeOH–ACN-0.2 mol/L NH4AC(60:37:3,v/v/v). The flow-rate was 1.0 ml/min. The detection excitation and emission wavelength were 366 nm and 500 nm. And the column temperature was 40℃. External reference method was applied to quantitate the ethacridine lactate in amniotic fluid. Retention time (RT) of ethacridine lactate was about 7 min in this experiment. The entire sampling time was 10~15 min. The liner range of ethacridine lactate was 0.005~1.00μg/ml, and the correlation coefficient (r) was 0.9984. The limit of quantitation was 0.0007μg/ml. The recoveries were 92.1% and 98.3%. The intra-day RSDs were 1.5% and 1.4%. The inter-day RSDs were 6.5% and 2.2%. The methodology validation showed that this method conformed to the Chinese Pharmacopoeia published in 2005 completely, and had been applicable to determine the concentration of ethacridine lactate in amniotic fluid for clinical use.