离子色谱-三重四极杆质谱法同时测定血浆和尿液中百草枯和敌草快

Simultaneous determination of paraquat and diquat in plasma and urine by ion chromatography-triple quadrupole mass spectrometry

百草枯和敌草快是广泛使用的非选择性触杀型除草剂,中毒后会造成急性肺损伤,病死率高,同时监测血浆和尿液中百草枯和敌草快的浓度,可以为临床早期诊断和预后提供有价值的信息。血浆和尿液中百草枯和敌草快的主要检测方法为液相色谱-质谱法。百草枯和敌草快为强极性水溶性化合物,在反相色谱柱上难以保留,多采用离子对色谱法或亲水色谱法进行分离。采用离子对色谱法时,加入的离子对试剂有离子抑制作用,降低了质谱检测的灵敏度,还给质谱系统增加了额外的污染;亲水色谱法易受基质成分影响,保留时间不稳定。考虑到百草枯和敌草快在水溶液中以双电荷联吡啶离子状态存在,更适合采用阳离子交换色谱法,建立了离子色谱-三重四极杆质谱测定血浆和尿液中百草枯和敌草快的检测方法。血浆和尿液样品经水稀释后,直接过混合型聚合物反相吸附和弱阳离子交换固相萃取柱(Oasis WCX)净化,经IonPac CS 18型阳离子色谱柱(250 mm×2.0 mm,6.0μm)分离,以自动在线产生的甲磺酸进行梯度洗脱,色谱柱流出液经阳离子抑制器抑制后进入质谱系统,在ESI+、多反应监测(MRM)模式下检测,稳定同位素内标法定量。百草枯和敌草快分别在1.0~150μg/L和0.5~75μg/L范围内线性关系良好,血浆中的平均基质效应分别为84.2%~89.3%和84.7%~91.1%,尿液中平均基质效应分别为50.3%~58.4%和51.9%~59.4%;血浆中百草枯和敌草快的平均加标回收率分别为93.5%~117%和91.7%~112%,相对标准偏差(RSD)分别为3.4%~16.7%和2.8%~13.2%;尿液中百草枯和敌草快的平均加标回收率分别为90.0%~118%和99.2%~116%,RSD分别为5.6%~14.9%和2.4%~17.3%(n=6);血浆和尿液中百草枯和敌草快的检出限分别0.3μg/L和0.2μg/L,定量限分别为1.0μg/L和0.5μg/L。该法灵敏度高,准确性好,可用于血浆和尿液中百草枯和敌草快的中毒检测。

Paraquat(PQ)and diquat(DQ)are widely used as non-selective contact herbicides.Several cases involving accidents,suicide,and homicide by PQ or DQ poisoning have been reported.Poising by PQ,which is mainly concentrated in the lungs,causes acute respiratory distress syndrome and leads to multiple organ toxicity.The toxic effects of DQ are similar to those of PQ but relatively less intense.The mortality rates in PQ and DQ poisoning are high.Simultaneous monitoring of the PQ and DQ concentrations in plasma and urine can provide valuable information for early clinical diagnosis and prognosis.High performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)is the main analytical method used to detect PQ and DQ in plasma and urine.As both these compounds are highly polar and water soluble,they cannot be retained effectively on a reversed-phase column with conventional mobile phases.The separation of PQ and DQ by ion-pair chromatography or hydrophilic chromatography has been reported.The use of an ion-pairing reagent helps in improving the retention capabilities of PQ and DQ.However,the sensitivity of MS detection is noticeably decreased because of ion suppression caused by the ion-pairing reagent in the mobile phase;furthermore,ion-pairing reagents may contaminate the MS system.The separation of PQ and DQ by hydrophilic chromatography is easily affected by matrix components in the sample,and their retention times are not stable.Considering PQ and DQ are bicharged cation species in solution,they are more suitable for separation by cation-exchange chromatography.A method based on ion chromatography-triple quadrupole mass spectrometry was established for the determination of PQ and DQ in plasma and urine.The plasma and urine samples were diluted with water,and then purified on a solid-phase extraction column containing a polymer-reversed phase and weak ion-exchange mixed-mode adsorbent(Oasis WCX).PQ and DQ were separated on an IonPac CS 18 analytical column(250 mm×2.0 mm,6.0μm)with gradient elution using a methylsulfonic acid solution electrolytically generated from an on-line eluent generation cartridge.An in-line suppressor was used to remove methylsulfonate and other anions from the eluent before the eluent entered the mass spectrometer.Between the suppressor and the ion source in MS,the addition of 3%(v/v)formic acid in acetonitrile as an organic modifier(using an auxiliary pump and a T-piece)aided desolvation in the ion source,resulted in a one-or two-fold improvement of the response,and eliminated the residual effects of the adsorption of PQ and DQ caused by ion source.The analytes were detected by triple quadrupole tandem mass spectrometry using positive electrospray ionization in the multiple reaction monitoring(MRM)mode.PQ-d8 and DQ-d4 were used as internal standards.The calibration curves for PQ and DQ showed good linear relationships in the ranges of 1.0-150μg/L and 0.5-75μg/L,respectively,and the correlation coefficients were>0.999.The average matrix effects of PQ and DQ in plasma were 84.2%-89.3%and 84.7%-91.1%,while the average matrix effects of PQ and DQ in urine were 50.3%-58.4%and 51.9%-59.4%.The average recoveries of PQ and DQ in plasma were 93.5%-117%and 91.7%-112%,respectively,with relative standard deviations(RSDs)of 3.4-16.7%and 2.8%-13.2%,and that in urine were 90.0%-118%and 99.2%-116%,with relative standard deviations of 5.6%-14.9%and 2.4%-17.3%(n=6).The limits of detection of PQ and DQ in plasma and urine were 0.3μg/L and 0.2μg/L,respectively,with the corresponding limits of quantification being 1.0μg/L and 0.5μg/L.This method is sensitive and accurate,and it can be used to determine PQ and DQ for clinical diagnosis and prognosis in patients.