Simultaneous quantification of soman and VX adducts to butyrylcholinesterase, their aged methylphosphonic acid adduct and butyrylcholinesterase in plasma using an off-column procainamide-gel separation method combined with UHPLC–MS/MS

Liu, Chang-Cai; Huang, Guilan; Xi, Hailing; Shilei, Liu; Liu, Jingquan; Yu, Huilan; Zhou, Shi-Kun; Liang, Long-Hui; Yuan, Liang

doi:10.1016/j.jchromb.2016.09.044

Cited by 17 publications

(11 citation statements)

References 32 publications

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“…Hence, it is essential to extract HuBuChE from highly concentrated proteins in plasma before digestion and analysis by LC/MS to avoid ion suppression. Current purification methods are based on HuBuChE extraction from plasma by affinity chromatography on procainamide gels [4,8,21,22] or by antibodies immobilized on magnetic beads activated with streptavidin or protein G [23][24][25][26][27]. Procainamide gels are relatively cheap but not selective enough, implying the need of additional purification steps, which extends the extraction time and reduces the total recovery of the method [4,28].…”

Section: Introductionmentioning

confidence: 99%

Development of immunosorbents coupled on-line to immobilized pepsin reactor and micro liquid chromatography–tandem mass spectrometry for analysis of butyrylcholinesterase in human plasma

Bonichon

Combès

Desoubries³

et al. 2017

Journal of Chromatography A

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Human butyrylcholinesterase (HuBuChE) has been widely used as a biomarker of exposure to organophosphorus (OPs) warfare agents. Indeed, intoxication by OPs can be proven by LC-MS/MS analysis of a specific HuBuChE nonapeptide on which OPs covalently bind. Therefore, we developed a fast, selective and sensitive on-line set-up for the analysis of HuBuChE from plasma that combines immunoextraction by anti-HuBuChE antibodies, pepsin digestion on Immobilized Enzyme Reactors (IMER) and microLC-MS/MS analysis of the target nonapeptide, FGESAGAAS. Two pepsin-based IMERs were prepared and characterized in terms of grafting and digestion yields and were coupled on-line to microLC-MS/MS analysis. In addition, immunosorbents were prepared by covalent grafting of three anti-HuBuChE antibodies on CNBr-sepharose and epoxy-polymethacrylate supports and packed in precolumns. The best antibody grafting yields were obtained with sepharose-based supports, with grafting yields up to 98%. B2 18-5 monoclonal antibody grafted on sepharose led to the best immunosorbent, with HuBuChE recovery close to 100%. The immunosorbent was introduced upstream of the on-line digestion set-up and immunoextraction of HuBuChE was achieved in 14min while digestion was performed in 20min, allowing detection of the target nonapeptide in less than 1h. The global recovery of the nonapeptide was higher than 42% using the best immunosorbent with a RSD value lower than 7% (n=3). Finally, the limit of quantification evaluated in plasma sample was 2fmol of nonapeptide. This value, corresponding to 0.5fmol of HuBuChE tetramer, is well below the average amount of HuBuChE tetramer in 50μL of plasma (590fmol).

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Section: Introductionmentioning

confidence: 99%

Development of immunosorbents coupled on-line to immobilized pepsin reactor and micro liquid chromatography–tandem mass spectrometry for analysis of butyrylcholinesterase in human plasma

Bonichon

Combès

Desoubries³

et al. 2017

Journal of Chromatography A

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“…Procainamide-sepharose gel from GE Healthcare (2.5 mL) was preconditioned with 15 mL of buffer A (15 mmol.L -1 Na 2 HPO 4 , 5 mmol.L -1 NaH 2 PO 4 , pH = 6.7) and 100 µL of plasma was added and gently mixed with the gel, following Liu et al protocol [27]. The mixture was incubated for 1 hour at room temperature and shaken at a speed of 60 rpm.…”

Section: Procainamide Gelmentioning

confidence: 99%

“…The efficiency of immunosorbents to selectively extract HuBuChE from plasma was compared to the most frequently used or efficient approaches found in literature to purify HuBuChE. Procainamide gel, has been widely used to extract HuBuChE, combined or not with additional purification steps [25][26][27][29][30][31]49]. The method depicted by Liu et al [27] put forward that more than 90% HuBuChE could be extracted with only one purification step with off-column procainamide gel.…”

Section: Comparison Of the On-line Set-up With The Procainamide And Hmentioning

confidence: 99%

“…Procainamide gel, has been widely used to extract HuBuChE, combined or not with additional purification steps [25][26][27][29][30][31]49]. The method depicted by Liu et al [27] put forward that more than 90% HuBuChE could be extracted with only one purification step with off-column procainamide gel. Consequently, this method was chosen and reproduced in this work closely as possible to compare HuBuChE recovery obtained after purification by procainamide to that obtained after on-line immunoextraction.…”

Section: Comparison Of the On-line Set-up With The Procainamide And Hmentioning

confidence: 99%

“…On-column procainamide gel extractions have been frequently used to purify HuBuChE from plasma [25][26][27][28][29][30][31][32][33][34]. However, this method is considered as poorly sensitive, time-consuming and requires large volume of sample or additional purification steps.…”

Section: Introductionmentioning

confidence: 99%

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Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of sarin and soman-butyrylcholinesterase adducts in human plasma

et al. 2017

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Organophosphorus nerve agent (OPNA) adducts formed with human butyrylcholinesterase (HuBuChE) can be used as biomarker of OPNA exposure. Indeed, intoxication by OPNAs can be confirmed by the LC/MS analysis of a specific HuBuChE nonapeptide on which OPNAs covalently bind. A fast, selective, and highly sensitive online method was developed to detect sarin and soman adducts in plasma, including immunoextraction by anti-HuBuChE antibodies, pepsin digestion on immobilized enzyme reactors (IMER), and microLC/MS analysis of the OPNA adducts. The potential of three different monoclonal antibodies, covalently grafted on sepharose, was compared for the extraction of HuBuChE. The online method developed with the most promising antibodies allowed the extraction of up to 100% of HuBuChE contained in plasma and the digestion of 45% of it in less than 40 min. Moreover, OPNA-HuBuChE adducts, aged OPNA adducts, and unadducted HuBuChE could be detected (with S/N > 2000), even in plasma spiked with a low concentration of OPNA (10 ng mL). Finally, the potential of this method was compared to approaches involving other affinity sorbents, already described for HuBuChE extraction. Graphical abstract Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of organophosphorous nerve agent adducts formed with human butyrylcholinesterase.

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Untargeted and targeted analysis of sarin poisoning biomarkers in rat urine by liquid chromatography and tandem mass spectrometry

et al. 2021

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Simultaneous quantification of soman and VX adducts to butyrylcholinesterase, their aged methylphosphonic acid adduct and butyrylcholinesterase in plasma using an off-column procainamide-gel separation method combined with UHPLC–MS/MS

Cited by 17 publications

References 32 publications

Development of immunosorbents coupled on-line to immobilized pepsin reactor and micro liquid chromatography–tandem mass spectrometry for analysis of butyrylcholinesterase in human plasma

Development of immunosorbents coupled on-line to immobilized pepsin reactor and micro liquid chromatography–tandem mass spectrometry for analysis of butyrylcholinesterase in human plasma

Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of sarin and soman-butyrylcholinesterase adducts in human plasma

Untargeted and targeted analysis of sarin poisoning biomarkers in rat urine by liquid chromatography and tandem mass spectrometry

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