Luận văn thạc sĩ nghiên cứu định lượng paraquat trong huyết tương người bằng phương pháp điện di mao quản sử dụng detector độ dẫn không tiếp xúc CE c4d

ĐẠI HỌC QUỐC GIA HÀ NỘI TRƯỜNG ĐẠI HỌC KHOA HỌC TỰ NHIÊN ĐỖ THỊ TRANG NGHIÊN CỨU ĐỊNH LƯỢNG PARAQUAT TRONG HUYẾT TƯƠNG NGƯỜI BẰNG PHƯƠNG PHÁP ĐIỆN DI MAO QUẢN SỬ DỤNG DETECTOR ĐỘ DẪN KHÔNG TIẾP XÚC (CE-C4D) LUẬN VĂN THẠC SĨ KHOA HỌC Hà Nội - 2018 ĐẠI HỌC QUỐC GIA HÀ NỘI TRƯỜNG ĐẠI HỌC KHOA HỌC TỰ NHIÊN ĐỖ THỊ TRANG NGHIÊN CỨU ĐỊNH LƯỢNG PARAQUAT TRONG HUYẾT TƯƠNG NGƯỜI BẰNG PHƯƠNG PHÁP ĐIỆN DI MAO QUẢN SỬ DỤNG DETECTOR ĐỘ DẪN KHÔNG TIẾP XÚC (CE-C4D) Chun ngành: Hóa phân tích Mã số: 60440118 LUẬN VĂN THẠC SĨ KHOA HỌC Người hướng dẫn khoa học: PGS.TS Tạ Thị Thảo TS Nguyễn Thị Ánh Hường Hà Nội – 2018 LỜI CẢM ƠN Với lòng biết ơn sâu sắc, em xin chân thành cảm ơn cô giáo PGS.TS Tạ Thị Thảo cô giáo TS Nguyễn Thị Ánh Hƣờng giao đề tài, nhiệt tình hƣớng dẫn tạo điều kiện thuận lợi giúp em thực luận văn Em xin chân thành cảm ơn thầy, mơn Hóa Phân tích nói riêng khoa Hóa học nói chung dạy dỗ, bảo động viên em suốt thời gian em học tập trƣờng Đại học Khoa học Tự nhiên Hà Nội Em xin gửi tới Ban lãnh đạo toàn thể nhân viên trung tâm Chống độc – bệnh viện Bạch Mai, đặc biệt anh chị phòng xét nghiệm độc chất lời cảm ơn sâu sắc nhất, cảm ơn anh chị giúp đỡ, động viên em nhiều trình thực đề tài trung tâm Em xin chân thành cảm ơn Công ty 3SAnalysis (http://www.3sanalysis.vn/) hỗ trợ trang thiết bị nhƣ tƣ vấn kỹ thuật trình thực nghiên cứu Cuối cùng, em xin cảm ơn gia đình, anh chị bạn sinh viên Bộ mơn Hóa phân tích ln động viên tinh thần, tận tình giúp đỡ em thời gian học tập thực luận văn Hà Nội, ngày 09 tháng 02 năm 2018 Học viên Đỗ Thị Trang MỤC LỤC MỞ ĐẦU CHƢƠNG 1: TỔNG QUAN 1.1 Tổng quan paraquat 1.1.1 Cơng thức hóa học, tính chất lý hố học paraquat 1.1.2 Thực trạng sử dụng paraquat 1.1.3 Cơ chế gây độc paraquat 1.1.4 Dƣợc động học paraquat 1.2 Các phƣơng pháp xác định paraquat 1.2.1 Phƣơng pháp quang phổ 1.2.2 Phƣơng pháp sắc ký khí khối phổ (GC-MS) 1.2.3 Phƣơng pháp sắc ký lỏng 1.2.4 Phƣơng pháp điện di mao quản (CE) .12 1.3 Các phƣơng pháp xử lý mẫu sinh học (huyết tƣơng, nƣớc tiểu) nhằm phân tích paraquat 15 1.3.1 Phƣơng pháp chiết lỏng- lỏng 15 1.3.2 Phƣơng pháp chiết pha rắn .17 1.4 Kết luận chung phần tổng quan 20 CHƢƠNG 2: THỰC NGHIỆM 22 2.1 Trang thiết bị, hóa chất 22 2.1.1 Thiết bị, dụng cụ 22 2.1.2 Chất chuẩn 24 2.1.3 Hóa chất, dung mơi 24 2.1.4 Chuẩn bị dung dịch hóa chất 25 2.2 Nội dung đối tƣợng nghiên cứu 26 2.3 Phƣơng pháp nghiên cứu 28 2.4 Các thông số đánh giá độ tin cậy phƣơng pháp phân tích 31 2.5 Ƣớc lƣợng độ không đảm bảo đo 35 2.5.1 Độ không đảm bảo đo đƣờng chuẩn 35 2.5.2 Độ không đảm bảo đo tổng hợp 37 2.6 Phân tích PQ huyết tƣơng bệnh nhân ngộ độc PQ 37 CHƢƠNG 3: KẾT QUẢ VÀ THẢO LUẬN 39 3.1 Khảo sát điều kiện tối ƣu tách paraquat phƣơng pháp điện di mao quản sử dụng detector đo độ dẫn không tiếp xúc (CE – C4D) .39 3.1.1 Khảo sát ảnh hƣởng hệ đệm 39 3.1.2 Khảo sát cation ảnh hƣởng đến trình phân tách PQ 45 3.1.3 Xây dựng đƣờng chuẩn PQ từ dung dịch chuẩn .46 3.1.4 Giới hạn phát (LOD) giới hạn định lƣợng (LOQ) thiết bị (IDL) 48 3.1.5 Đánh giá độ chụm (độ lặp lại) thiết bị 49 3.2 Nghiên cứu quy trình tách chiết PQ mẫu huyết tƣơng phƣơng pháp chiết pha rắn 50 3.2.1 Khảo sát khả loại bỏ cation cột C18 50 3.2.2 Khảo sát khả tách PQ sử dụng không sử dụng EDTA 10-3M 51 3.2.3 Khảo sát nồng độ EDTA bƣớc tiền xử lý mẫu huyết tƣơng 53 3.2.4 Khảo sát pH mẫu 54 3.2.5 Khảo sát loại chất hoạt động bề mặt 55 3.2.6 Khảo sát thành phần, tỉ lệ dung dịch rửa tạp 57 3.2.7 Khảo sát dung dịch rửa giải 59 3.2.8 Khảo sát tỉ lệ axit acetic dung dịch rửa giải 62 3.3 Xác nhận giá trị sử dụng phƣơng pháp 63 3.3.1 Đánh giá độ chọn lọc 63 3.3.2 Xây dựng đƣờng chuẩn PQ mẫu huyết tƣơng 64 3.3.3 Đánh giá phƣơng trình hồi quy đƣờng chuẩn 66 3.3.4 Đánh giá độ xác phƣơng pháp .66 3.3.5 Xác định giới hạn phát phƣơng pháp .68 3.4 Phân tích mẫu thực tế đối chứng kết phân tích phƣơng pháp CE – C4D với phƣơng pháp HPLC 69 3.4.1 Định lƣợng nồng độ PQ mẫu huyết tƣơng bệnh nhân phƣơng pháp CE – C4D .69 3.4.2 Đối chứng kết phân tích với phƣơng pháp HPLC 72 KẾT LUẬN 75 TÀI LIỆU THAM KHẢO 77 DANH MỤC HÌNH Hình 1.1 Cơng thức hóa học paraquat Hình 1.2 Cơng thức hóa học dạng muối paraquat Hình 1.3 Cơ chế gây độc paraquat Hình 2.1 Ảnh chụp hệ thiết bị CE – C4D sử dụng nghiên cứu 23 Hình 3.1 Điện di đồ ảnh hƣởng pH đến phân tách PQ 40 Hình 3.2 Điện di đồ ảnh hƣởng thành phần dung dịch điện di đến khả phân tách PQ 42 Hình 3.3 Điện di đồ ảnh hƣởng nồng độ đệm điện di đến khả phân tách PQ 44 Hình 3.4 Điện di đồ ảnh hƣởng cation đến tín hiệu PQ 45 Hình 3.5 Đƣờng chuẩn PQ dung dịch chuẩn 47 Hình 3.6 Điện di đồ minh chứng loại bỏ cation Na+ 140 mM cột C18 51 Hình 3.7 Điện di đồ minh chứng loại bỏ cation Ca2+ mM cột C18 51 Hình 3.8 Điện di đồ minh chứng hiệu sử dụng EDTA đến phân tách PQ 52 Hình 3.9 Điện di đồ ảnh hƣởng nồng độ EDTA đến phân tách PQ 54 Hình 3.10 Điện di đồ phân tích mẫu SPE điều kiện pH khác .55 Hình 3.11 Điện di đồ ảnh hƣởng chất HĐBM trình SPE đến khả phân tách PQ 56 Hình 3.12 Kết phân tích điện di sau chiết pha rắn sử dụng loại dung dịch rửa tạp khác 57 Hình 3.13 Điện di đồ ảnh hƣởng tỉ lệ dung dịch rửa tạp đến khả phân tách PQ 58 Hình 3.14 Điện di đồ ảnh hƣởng thành phần dung dịch rửa giải đến khả phân tách PQ 60 Hình 3.15 Kết ảnh hƣởng HCl dung dịch rửa giải đến tín hiệu PQ 61 Hình 3.16 Đồ thị thể phụ thuộc diện tích pic PQ vào thể tích HCl thêm vào dung dịch rửa giải .61 Hình 3.17 Điện di đồ thể ảnh hƣởng tỉ lệ axit acetic dung dịch rửa giải đến PQ .62 Hình 3.18 Quy trình chiết dung dịch mẫu cột C18 sử dụng chất tạo cặp ion 63 Hình 3.19 Độ chọn lọc phƣơng pháp .64 Hình 3.20 Đƣờng chuẩn PQ huyết tƣơng .65 Hình 3.21 Điện di đồ thể nồng độ PQ vào viện bệnh nhân ngộ độc PQ 71 Hình 3.22 Điện di đồ thể nồng độ PQ từ lúc vào viện đến sau lọc hấp phụ lần bệnh nhân điển hình .71 Hình 3.23 Đồ thị thể mối quan hệ nồng độ PQ huyết tƣơng phƣơng pháp CE – C4D HPLC .74 DANH MỤC BẢNG Bảng 2.1 Cách pha dung dịch chuẩn gốc 35 Bảng 2.2 Cách pha dung dịch chuẩn trung gian 40 μg/ml 36 Bảng 2.3 Cách pha đƣờng chuẩn độ không đảm bảo đo 36 Bảng 3.1 Kết khảo sát ảnh hƣởng pH đến diện tích pic (Spic) thời gian di chuyển (tdc) PQ chuẩn .40 Bảng 3.2 Kết khảo sát ảnh hƣởng thành phần dung dịch đệm điện di đến diện tích pic (Spic)và thời gian di chuyển (tdc) PQ chuẩn .42 Bảng 3.3 Kết khảo sát phụ thuộc diện tích pic (Spic) thời gian di chuyển (tdc ) PQ vào nồng độ dung dịch đệm điện di .44 Bảng 3.4 Điều kiện tối ƣu cho phân tích PQ phƣơng pháp CE – C4D 46 Bảng 3.5 Sự phụ thuộc diện tích pic vào nồng độ dung dịch PQ chuẩn 47 Bảng 3.6 Giới hạn phát PQ xác định phƣơng pháp điện di mao quản CE – C4D 48 Bảng 3.7 Giới hạn phát (LOD) giới hạn định lƣợng (LOQ) PQ xác định phƣơng pháp điện di mao quản CE – C4D 49 Bảng 3.8 Kết xác định độ chụm thiết bị CE – C4D phân tích định lƣợng PQ 49 Bảng 3.9 Ảnh hƣởng nồng độ EDTA đến hiệu suất thu hồi PQ huyết tƣơng 53 Bảng 3.10 Kết hiệu suất thu hồi PQ pH khác .55 Bảng 3.11 Kết hiệu suất thu hồi PQ sử dụngchất HĐBM khác SPE .56 Bảng 3.12 Kết hiệu suất thu hồi PQ sử dụng loại dung dịch rửa tạp khác .58 Bảng 3.13 Kết hiệu suất thu hồi PQ tỉ lệ dung dịch rửa tạp khác 59 Bảng 3.14 Kết hiệu suất thu hồi PQ sử dụng dung dịch rửa giải khác 60 Bảng 3.15 Sự phụ thuộc diện tích pic vào nồng độ PQ mẫu huyết tƣơng 64 Bảng 16 Kết khảo sát độ phƣơng pháp thêm chuẩn PQ .66 Bảng 3.17 Kết xác định độ lặp lại phƣơng pháp chiết pha rắn định lƣợng PQ huyết tƣơng mẫu thực 67 Bảng 3.18 Kết xác định độ tái lặp phƣơng pháp .68 Bảng 3.19 Kết xác định giới hạn phát phƣơng pháp (MDL) 69 Bảng 3.20 Cách pha dung dịch chuẩn gốc 35 Bảng 3.21 Cách pha dung dịch chuẩn trung gian 40 μg/ml 36 Bảng 3.22 Cách pha đƣờng chuẩn độ không đảm bảo đo 36 Bảng 3.23 Kết định lƣợng PQ 50 bệnh nhân 70 Bảng 3.24 Kết đối chứng nồng độ PQ huyết tƣơng phƣơng pháp HPLC 72 23 Masafumi Tomita Toshiko Okuyama (1992), "Simultaneous Determination of Paraquat and Diquat in Serum Using Capillary Electrophoresis", Biomedical chromatography, 6, 91-94 24 Moreira P N Baltazar M T (2012), "Quantification of paraquat in postmortem samples by gas chromatography-ion trap mass spectrometry and review of the literature", Biomed Chromatography, 26, 338-349 25 Nu´n˜ez O., Moyano E., Puignou L Galceran M.T (2001), "Sample stacking with matrix removal for the determination of paraquat, diquat and difenzoquat in water by capillary electrophoresis", Journal of Chromatography A, 912, 353-361 26 Paixa˜o P., Costa P., Bugalho T., Fidalgo C Pereira L.M (2002), "Simple method for determination of paraquat in plasma and serum of human patients by high-performance liquid chromatography", Journal of Chromatography B, 775, 109-113 27 Proenca P, Vidinha J Teixeira H, "Determination of paraquat in blood and urine by liquid chromatography-electrospray-mass spectrometry" 28 Qingxiang Zhou, Jinli Mao, Junping Xiao Guohong Xie (2010), "Determination of paraquat and diquat preconcentrated with N doped TiO2 nanotubes solid phase extraction cartridge prior to capillary electrophoresis", Analytical Methods, 2, 1063-1968 29 Rafael Lanaro, José Luiz Costa, Luciane C.R Fernandes, Rodrigo Ribeiro Resende Marina F.M Tavares (2011), "Detection of Paraquat in Oral Fluid, Plasma, and Urine by Capillary Electrophoresis for Diagnosis of Acute Poisoning", Journal of Analytical Toxicology, 35, 274-279 30 Rafael Menck de Almeida Mauricio Yonamine (2007), "Gas chromatographic–mass spectrometric method for the determination of the herbicides paraquat and diquat in plasma and urine samples", Journal of Chromatography B, 853, 260-264 31 Rai M.K., Joyce Vanisha Das Gupta V.K (1997), "A sensitive determination of paraquat by spectrophotometry", Talanta, 45, 343-348 32 Richmond R Halliwell B (1982), "Formation of hydroxyl radicals from the paraquat radical cation, demonstrated by a highly specific gas chromatographic technique, the role of superoxide radical anion, hydrogen peroxide, and glutathione reductase", J Inorg Biochem 17, 95-107 33 Seung Kyung Baeck, Young Shin Shin, Hee Sun Chung Myoung Yun Pyo (2007), "Comparison Study of the Extraction Methods of Paraquat in Postmortem Human Blood Samples", Arch Pharm Res, 30, 235-239 34 Watts M (2011), Paraquat, Pesticide action network Asia and the pacific, 10111055 35 Yamamoto HA (2001), "Effects of melatonin on paraquat or ultraviolet light exposure-induced DNA damage", J Pineal Res 31, 308-313 36 Yasaka T (1986), "Further studies of lipid peroxidation in human paraquat poisoning", Arch Intern Med 146, 681-685 37 Yuangao Zou, Yunying Shi, Yangjuan Bai, Jiangtao Tang, Yao Chena Lanlan Wanga (2011), "An improved approach for extraction and high- performance liquid chromatography analysis of paraquat in human plasma", Journal of Chromatography B, 879, 1809-1812 38 ZhaohongWang, ZhipingWang Junbo Xing (2011), "The Quantitative Analysis of Paraquat in Biological Samples by Liquid Chromatography– Electrospray Ionization-Mass Toxicology, 35, 23-27 Spectrometry", Journal of Analytical JournalofChromatograph yB1060(2017)111–117 Contents lists available at ScienceDirect Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb Clinical screening of paraquat in plasma samples using capillary electrophoresis with contactless conductivity detection: Towards rapid diagnosis and therapeutic treatment of acute paraquat poisoning in Vietnam Anh Phuong Vua,b, Thi Ngan Nguyena,b, Thi Trang Doa,b, Thu Ha Doana, Tran Hung Haa, Thi Thao Tab, Hung Long Nguyenc, Peter C Hauserd, Thi Anh Huong Nguyenb,⁎, Thanh Duc Maie,⁎ a Poison Control Center, BACH MAI HOSPITAL, 78 GIAI Phong ROAD, Dong DA, HANOI, Viet NAM DEPARTMENT of ANALYTICAL Chemistry, FACULTY of Chemistry, VNU University of Science, VIETNAM NATIONAL University, HANOI – 19 Le THANH Tong, HANOI, Viet NAM1 c VIETNAM Food ADMINISTRATION, Ministry of HEALTH, 138A GIANG Vo, BA Đinh, HANOI, Viet NAM d University of BASEL, DEPARTMENT of Chemistry, SPITALSTRASSE 51, 4056 BASEL, SWITZERLAND e PNAS, Institut GALIEN de PARIS-SUD, FACULTÉ de PHARMACIE, Université PARIS-SUD, CNRS, rue JB Clément, 92296 CHÂTENAY-MALABRY, FRANCE b ARTICLEINFO Keywords: Purpose-made instrument Capacitively coupled contactless conductivity detection (C4D) Capillary electrophoresis (CE) Paraquat Plasma samples Vietnam ABSTRACT The employment of a purpose-made capillary electrophoresis (CE) instrument with capacitively coupled con- tactless conductivity detection (C4D) as a simple and cost-effective solution for clinical screening of paraquat in plasma samples for early-stage diagnosis of acute herbicide poisoning is reported Paraquat was determined using an electrolyte composed of 10 mM histidine adjusted to pH with acetic acid A detection limit of 0.5 mg/ L was achieved Good agreement between results from CE-C4D and the confirmation method (HPLC-UV) was obtained, with relative errors for the two pairs of data better than 20% for 31 samples taken from paraquat- intoxicated patients The results were used by medical doctors for identification and prognosis of acute paraquat poisoning cases The objective of the work is the deployment of the developed approach in rural areas in Vietnam as a low-cost solution to reduce the mortality rate due to accidental or suicidal ingestion of paraquat Introduction Quaternary ammonium herbicides, with paraquat (PQ) being the most common substance, have been used extensively in the world for controlling the growth of weeds and grasses in order to achieve high agricultural productivity However, these substances have been classi- fied as moderately hazardous compounds by the World Health Organization [1–3] An exposure to paraquat via accidental or suicidal ingestion is extremely toxic to man and frequently fatal, as a result of multi-organ failure and cardiogenic shock [1,2,4,5] After ingestion, the compound primarily accumulates in lungs, resulting in acute pul- monary distress At the same time, it has drastic effects on the gastro- intestinal tract, kidneys, liver and heart Death occurs to the majority of patients within days to weeks whereas survivors suffer severe sequelae [1,4,6] In Vietnam, due to its ubiquitous presence, accidental or sui- cidal ingestion of PQ occurs frequently According to findings at the Poison Control Center – Bach Mai hospital (the largest central hospital in Vietnam), this is the most popularly used compound (even the only herbicide used) by people in rural areas to commit suicide, with more ⁎ than 300 cases reported in 2014 and 391 cases reported in 2015 In most of the cases, the patients, unconscious following acute poisoning, were transported to central hospitals where state-of-the-art equipment is available for clinical diagnosis and treatment Due to the time lost during the transport and the waiting period at central hospitals, which are always overburdened, the majority (80%) of the patients suffered death Also according to the Poison Control Center, this mortality rate would have been significantly reduced if acute poisoning by PQ could be identified immediately at a local hospital followed by urgent treat- ment with blood filtration This however has not been the case due to the lack of inexpensive and easy to use analytical instruments and methods which can be deployed in decentralized hospitals in Vietnam The paraquat concentration in plasma has been used as the most common marker to evaluate the possibility and severity of PQ poi- soning The likelihood of survival is higher for patients whose plasma paraquat levels are less than 2.0, 0.6, 0.3, 0.16, and 0.1 mg/L after 4, 6, 10, 16, and 24 h, respectively [7,8] Plasma levels higher than mg/L are considered to be fatal in most cases [1,9] The identification of PQ poisoning, the prognosis and the efficiency Corresponding authors E-MAIL ADDRESSES: nguyenthianhhuong@hus.edu.vn (T.A.H Nguyen), maithanhduc83@gmail.com (T.D Mai) website: www.CE-Vietnam.com http://dx.doi.org/10.1016/j.jchromb.2017.06.010 Received April 2017; Received in revised form 26 May 2017; Accepted June 2017 1570-0232/©2017PublishedbyElsevierB.V A.P Vu et AL JourNAlofChromAtogrAphyB1060(2017)111–117 of the treatment require first an analysis of PQ in plasma So far, the most cited techniques for analysis of paraquat in biological samples have been LC–MS (see for example [5,10–12]), GC–MS [2,13] and HPLC with ultraviolet detection (UV) [14–18] These instrumental methods however are not affordable for many (local) hospitals or clinical centers in developing countries In Vietnam, paraquat in plasma could to date only be quantified at few central hospitals which are equipped with HPLC-UV, such as Bach Mai hospital There is therefore interest in the development of simpler and less expensive analytical techniques which can be deployed even with modest budget and limited expertise In this context, capillary electrophoresis (CE) can be seen as a more economic alternative Indeed, CE has been employed for determination of paraquat in water, beer, mung bean and biological samples, using either UV or MS as the detection mode (see [19–25] and other precedent publications listed therein) Note that benchtop CE instruments were used in all these cases To the knowledge of the au- thors, the CE approach can be even more cost-effective and better fitted to non-expert use if it is coupled with capacitively coupled contactless conductivity detection (C4D) [26–28] Attractive features of C4D are high versatility, ease in construction and operation, low power con- sumption and the possibility of miniaturization (see more details in the recent reviews [29,30] and references therein) More conveniently, C4D can be used in compact portable CE instruments [31–33] for flexible mobile deployment, both of which can also be built in low cost versions [26–28] Nevertheless, CE-C4D methodology has to the best of our knowledge not been developed for analysis of paraquat in plasma samples Herein a cost-effective and easy to use CE-C4D method for the screening determination of paraquat in plasma samples using a pur- pose-made compact instrument is reported The method developed was applied at the central hospital (Bach Mai hospital) for diagnosis of paraquat acute-poisoning from PQintoxicated patients The results obtained from 31 plasma samples were crossed checked with the well- established HPLC-UV method The collected data were used by the doctors for mortality prognosis and prescription of therapeutic treat- ments to paraquat-intoxicated patients preparation of all solutions and for sample dilution if required pH values of solutions were controlled with an HI 2215 Hanna Instruments pH meter (Woonsocket, RI, USA) Experimental 2.1 CHEMICALS AND MATERIALS All chemicals used in this study were of analytical reagent grade Paraquat in the dichloride salt form purchased from Sigma Aldrich (Hamburg, Germany) was used to prepare stock solutions of paraquat at 200 ppm The structure of paraquat is shown in Fig S1 in the electronic supplementary information (ESI) Its pKa value was estimated to be 9–9.5 [34] L-histidine (His), trichloroacetic acid (TCA), ammonia so- lution (NH3 25%), ascorbic acid (Asc), arginine (Arg), methanol, acetic acid (Ace), sodium hydroxide (NaOH), hydrochloric acid (HCl), phos- phoric acid (H3PO4) 85%, ethylenediaminetetraacetic acid (EDTA), cetyltrimethylammonium bromide (CTAB), sodium 1heptanesulfonate, potassium chloride (KCl) and polyethylenglycol 400 (PEG 400) were purchased either from Merck or Fluka (Germany) Commercial SPE cartridges (Sep-Pak@Vac 3cc), each containing 500 mg of packing material (octadecyl bonded silica particles of 55 μm diameter), were purchased from Waters (Massachusetts, USA) Polyimide coated fused silica capillaries of 75 μm I.D and 365 μm O.D (from Polymicro, Phoenix, AZ, USA) with total (Ltot) and effective (Leff) lengths of 60 cm and 50 cm respectively were used for separa- tions Deionised water purified using a system from Water Pro (Labconco, Kansas City, MO, USA) was used for the 11 8% in methanol through the cartridge at the flowrate of 0.5 mL/min The eluent was dried with nitrogen and the residual was dissolved in mL acetic acid 1–8% in methanol A fold enrichment of PQ was achieved with this SPE procedure The obtained solution was injected into the separation capillary by siphoning at the height of 10 cm for 30 s The capillary before use was preconditioned with M NaOH for 10 and deionised water for 10 prior to flushing with buffer for at least h Electrophoretic separations were then carried out under a voltage of + 20 kV and with the optimized BGE composed of His 10 mM adjusted to pH with acetic acid (refer to Section 3.2 for more details on BGE optimization) Detection with C4D was implemented at an excitation voltage of 500 V, an excitation fre- quency of 600 kHz, a low-pass filter at Hz and with the gain mode within the measurement range of V To minimize the temperature- induced fluctuation of baseline signals during C4D recording, a re- ference signal was subtracted from the signal of the separation capil- lary The reference signal was obtained with a reference capillary which was filled with a solution composed of BGE (90% v/v) and deionized water (10% v/v) For cross check measurements, the HPLC method developed by the Poison Control Centre was used The method was adapted from those reported elsewhere [15,35] Briefly, to mL plasma mL TCA 5% v/v was added and this was then centrifuged at 4000 rpm for 15 The transparent supernatant was filtered through cellulose acetate mem- branes (0.2 μm) prior to injection into the HPLC instrument using a sample loop of 30 μL Chromatographic separation of PQ was carried out at the isocratic mode at the flowrate of 0.5 mL/min using a mobile phase composed of 5% ACN and 95% buffer For preparation of the buffer, 1.1 g heptanesulfonate sodium, g KCl, mL PEG 400 and 200 mL methanol were added into a 1000 mL volumetric flask, which was then filled up close to the graduated level with deionized water The pH of the mobile phase was adjusted to 2.5 with H3PO4 before precisely adjusting the level of the solution to the graduated mark with 2.2 INSTRUMENTATION All CE experiments were performed on a purpose-made portable CE instrument which employs siphoning for sample injection A miniature Spellman unit (UM25*4, 12 V, 200 μA, Pulborough, UK) was used which provides a maximum of 25 kV The high voltage (HV) end of the capillary was isolated in a safety cage equipped with a microswitch to interrupt the HV when the lid was opened Detection was carried out with a commercial C4D (ER815, eDAQ, Australia) As both CE and C4D require a 12 V DC power supply, either mains power or batteries can be used to power the CE-C4D system For cross-check measurements, an HPLC instrument (model: HPLC 1200, Agilent, USA) coupled with a diode-array detector (DAD) was employed These chromatographic separations were carried out with a reversed-phase column (Zorbax C8, 4.6 mm ID × 150 mm, μm packing materials) and a guard column (Zorbax, ZC8-10C5) Buffer capacities of different buffer compositions were estimated with the Phoebus program from Analis (Suarlée, Belgium) 2.3 Procedures The plasma samples from PQ-intoxicated patients were provided by the Poison Control Center (Bach Mai hospital) The procedure for treatment of plasma samples was as follows: mL plasma sample containing PQ after addition of 0.4 mL TCA and 0.6 mL H2O was cen- trifuged at 4000 rpm for 10 0.5 mL NH4OH (25%) and 50 μL EDTA (final concentrations varying from to 10−2 M) were then added to the obtained supernatant after centrifugation The resulting mixture (pH 9) was flushed at mL/min through a C18 cartridge which had been ac- tivated with mL CTAB (1.4 mM) prepared in NH4OH 0.1% and mL sodium 1-heptanesulfonate (100 mM) prepared in NH4OH 0.1% The cartridge that retained PQ was washed with 10 mL H2O and 10 mL methanol at the flowrate of 2.5 mL/min Elution of PQ was then carried out by passing mL acetic acid 11 A.P Vu et AL JourNAlofChromAtogrAphyB1060(2017)111–117 deionized water Detection was implemented at 259 nm, the wave- length of maximum absorption for PQ Fig Schematic drawing of the CE-C4D instrument RC: reference capillary Results and discussion 3.1 INSTRUMENTAL OPTIMIZATION Different purpose-made portable CE-C4D instruments were developed by our groups and transferred to users in various institutes in Vietnam for testing their performances (for details on our recent de- velopments see [26–28,36–39]) Feedbacks from the users were then collected to provide information on the positive features as well as drawbacks of purpose-made CE-C4D setups so that further instrumental improvement and/or methodology adaptation can be realized for more robust operation and better performance The setup that is most pre- ferred by non-expert users is the low-cost and compact version that contains only the essential components required to create electro- phoretic separations, including a miniature high voltage module (up to 25 kV of a predefined polarity), two Platinum electrodes, two vials containing sample or background electrolyte (BGE) solutions, a capillary, a casing with high voltage and ground Manual operation is not an inconvenience, but rather considered a positive (or required) feature for reduction of production cost, power consumption and size and weight of the instrument A good operation with such a CE instrument (e.g no significant fluctuation of migration times, no arcing) can be achieved as long as a working room with an air conditioner is provided to avoid temperature variation and high humidity Good grounding must also be established in order to guarantee safety and a good performance with high voltages To minimize signal drifting when recording with C4D under nonthermostated conditions, the referenced detection mode should be used With this technique, the signal from the separation capillary is corrected to that of the reference capillary by background subtraction [40] This advantageous feature was made possible thanks to simultaneous incorporation of dual capillaries in a single C4D cell As this feature is already available in the recent version of C4D from eDAQ, which is affordable even for a modest budget, the home-made C4D was replaced by this commercial version An arrangement of the home- made CE instrument coupled with a commercial C4D is illustrated in Fig The detector was integrated inside the CE instrument rather than being positioned outside as in our previous CE-C4D instruments Note that the reference capillary is very short (10 cm), and was not immersed in the BGE solutions during CE according to the protocol provided with the commercial detector To our experience with home-made detectors, much better results in terms of background subtraction and signal drift elimination would be achieved if the reference capillary is immersed in BGE solution and undergoes the same CE as the separation capillary Such operation however is rather complicated to (non-expert) users and therefore was not carried out The casing of the home-made CE in- strument was made of mica, which is a type of phyllosilicate, instead of perspex as in previous versions This material, which is locally avail- able, offers a very good electrical isolation between the HV chamber and the other parts of the system The mica sheets (8 mm thickness) used for construction of the casing were fabricated by a local me- chanical workshop Note that in conventional CE setups, the HV elec- trode is opposite to the detector, and injection is normally realized from the HV side of the capillary In our arrangement, the HV chamber is placed close to the detection chamber containing the detector so that the users can carry out siphoning injection from the ground chamber As there are different manual operations when working with our system, manipulation of the solutions and capillary at the ground side helps eliminate any risk associated with high voltage, especially to non- expert users The CE-C4D performance is not affected by this arrange- ment, as long as a good electrical isolation between the detection chamber and HV chamber could be guaranteed 3.2 Development of the CE-C4D methodology for the DETERMINATION of PARAQUAT For separation of PQ by CE, normally acidic conditions were em- ployed [19–25] Conveniently, acidic BGEs are favorable for CE-C4D as they offer better baseline stability and reproducibility of migration times due to suppression of the electro-osmotic flow (EOF) For these reasons, different BGEs, composed of different constituents, namely Arg/Asc, His/Asc, His/Ace, Arg/Ace and Ace alone, were investigated The effect of different buffer compositions under acidic conditions on the separation performance in terms of peak shapes and resolutions is shown in Fig Some inorganic cations, i.e K+ (4 ppm), Ca2+ (4 ppm) and Na+ (3 ppm), were added together with PQ (5 ppm) in the tested standard solutions to mimic the real situations where inorganic cations can still be found in the eluents after SPE pretreatment of plasma samples containing high concentrations of these ions (see Section 3.3) This was for the purpose of referencing the peak position of PQ com- pared to those of the added cations Based on this, BGE optimization can be realized in order to well separate the peak of PQ from those of these interfering cations Note that much higher concentrations of in- organic cations were not used in order to avoid peak overlapping, which in turn renders the peak identification and resolution optimiza- tion difficult As can be seen, the best resolutions were obtained with BGEs containing His/Ace The BGE composed of Ace alone, despite its observed advantages of good baseline stability, reproducibility of mi- gration times and suppression of electroosmotic flow, was not chosen because the migration time of PQ and those of other species were more retarded when using this BGE The peaks of PQ and other cationic species were more broadened (see Fig 2) and the peak resolutions were not improved compared to those from the His/Ace buffer The choice of His/Ace buffer also came from a practical reason: the non-expert users tend to use the same buffer vial repeatedly during their routine analyses, and may want to use the same buffer for different analytes, i.e cations and anions, if possible in order to simplify the operation protocol Then the His/Ace buffer could satisfy these conditions, as it has a better buffer capacity (1050 mmol L−1 pH−1 for 10 mM His adjusted to pH with Ace, calculated with Phoebus) than a solution of Ace alone (5 mmol L−1 pH−1 for 50 mM Ace), and can normally be used for se- parations of both cations and anions [41,42] This BGE was therefore chosen for further optimizations in order to minimize/avoid peak overlapping that may occur with the adjacent major cations in plasma samples BGE solutions at pH from to were also tested by adjusting the Ace concentration in this His/Ace composition It was found that A.P Vu et AL JourNAlofChromAtogrAphyB1060(2017)111–117 50 mV Ca2+ K+ PQNa+ Arg/Asc PQ His/Asc PQ His/Mes PQ His/Ace PQ Ace Migration time (min) Fig Electropherograms for CE- C 4D determination of paraquat with different BGE compositions Electrolyte solutions: His/Asc: 10 mM histidine adjusted to pH 4.0 with ascorbic acid; Arg/Asc: 10 mM arginine adjusted to pH 4.0 with ascorbic acid; Arg/Ace: 10 mM arginine adjusted to pH 4.0 with acetic acid; His/Ace: 10 mM histidine adjusted to pH 4.0 with acetic acid; Ace: 50 mM acetic acid CE conditions: hydrodynamic injection: siphoning at 10 cm high for 30 s; voltage: +20 kV; capillary: fused-silica 75 μm I.D Lt = 60 cm (Leff = 50 cm) Analytes: ppm K+; ppm PQ; ppm Ca2+; and ppm Na+ the separation resolution between PQ and the adjacent cations de- graded at an increase in pH of the BGE (data not shown) At a higher pH, the presence of a stronger EOF in the same direction as the elec- trophoretic migration of cationic species accelerates their arrival at the detector, thus reducing the separation resolution between PQ and the other cations A buffer of 10 mM histidine adjusted to pH with acetic acid was found to offer the best resolution and peak height In our work, instead of using narrow capillaries (10–50 μm i.d.) that were proved to help improve the separation efficiency without having much penalty on detection limit with CE-C4D [43,44], those of 75 μm i.d were em- ployed Indeed, when it comes to real-life practice, when a manual CE system with siphoning injection is carried out by a (non-expert) user, a compromise between the ease of use and the analytical performance has to be made In addition, under the relatively dusty environment in Vietnam, the capillaries of 50 μm or narrower inner diameters were found to be more easily blocked than those of 75 μm i.d For these reasons, the wider capillaries were employed in order to facilitate the (subsequent) operation by non-expert users at local hospitals with CE, matrix removal and PQ enrichment were carried out using liquid extraction with phenol [25], simultaneous electrophoretic con- centration and separation using hydrogels [19], solid phase extraction (SPE) with N-doped TiO2 nanotubes cartridge [24], SPE with silica cartridges [45], sample stacking with an anodic electroosmotic flow [46], field amplified sample injection [21], protein precipitation (for biological samples) [22] and nanoparticle-based extraction [23] In our case, both protein precipitation with TCA and SPE were combined to ensure a good CE-C4D performance (see the procedure in the Section 2.3) The procedure was adapted to the protocols and facilities available at the (local) hospital for the treatment of biological samples Note that offline sample pretreatment was implemented instead of on-line cou- pling of the SPE module to the CE setup By doing so, a high throughput of sample pretreatment could be achieved by using several SPE cartridges at the same time Several pretreated samples thus could be collected simultaneously and made ready for the subsequent CE-C4D analyses The CE-C4D process therefore is not affected or delayed by the long extraction time in the SPE pretreatment step Without the SPE step, there was little chance to see the peak of PQ as those of extremely abundant Na+ and Ca2+ (in plasma samples) overlap completely the adjacent peaks (see Fig S2) With SPE pretreatment, the majority of these interfering cations were removed from the treated samples (i.e the eluents) The efficiencies of interfering cations removal were cal- culated to be more than 98% An electropherogram with CE-C4D of a plasma sample after the sample treatment process is shown in the top trace of Fig As can be seen, major (interfering) cations were still present in the electropherogram even after the sample treatment step Note that C4D is very sensitive to the presence of inorganic cations Three sources of such contamination were identified The major part came from the release of interfering compounds from the C18 cartridges when elution was carried out with acidic conditions (data not shown) A similar situation was encountered by Taguchi et al when using C8/C18 SPE disks to pretreat PQ samples prior to LC–MS analysis [47] This problem became more pronounced when dealing with plasma samples whose inorganic cations were found to be partially retained on the sorbents and then released into the acidic eluent In addition, some contamination with inorganic cations also comes from the dusty and humid air typically found in tropical climates Such contamination becomes more pronounced when realizing siphoning injection where manipulation of the capillary end into and out of the open sample vial is inevitable The unwanted and unavoidable presence of such cation peaks, especially the peak of Ca2+ that follows the PQ peak, whose 0M 50 mV 10-5M PQ 3.3 SAMPLE TREATMENT for MATRIX REMOVAL AND enrichment of PARAQUAT The performance of the CE technique is dependent on the sample matrix injected into the capillary The presence of walladsorbing spe- cies, for example proteins from biological samples, can result in signal disturbance and significant fluctuation of migration times and peak areas A highly conductive sample matrix, caused by high concentra- tions of inorganic ions for example, should also be avoided as this can cause reduction of signal sensitivity When using the hydrodynamic injection mode, preferably the sample conductivity is lower than that of the BGE in order to assure a transient analyte stacking effect As C4D isa bulk detector, which non-selectively records all ionic species in sam- PQ 10-4M PQ 10-3M PQ 10-2M EDTA PQ 3.5 4.0 4.5 5.0 5.5 6.0 ples, a simple sample matrix is preferable for CE-C4D Recourse to sample pre-treatment thus is often needed for removal of interfering compounds in the matrix as well as for analyte enrichment for im- proved detection sensitivity In previous works on PQ determination Migration time (min) Fig Electropherograms for CE-C4D determination of PQ (5 ppm) in plasma samples with different concentrations of EDTA added (0–10−2 M) prior to the SPE procedure Other conditions as for Fig A.P Vu et AL JourNAlofChromAtogrAphyB1060(2017)111–117 achieved for the conditions employed was 0.5 ppm and calibration curves were acquired from 1–20 ppm This is the range of interest which has clinical significance for diagnosis and therapeutic treatment Measurements at higher PQ concentrations were therefore not at- tempted To eliminate any matrix mismatch between the standards used for the preparation of the calibration curve and plasma samples from patients, the standards were prepared by spiking known amounts of PQ into a blank plasma matrix and then passed through the sample treat- ment protocol prior to CE-C4D analyses The repeatabilities for peak areas and migration times were 1.4% and 1.0%, respectively The re- covery after SPE procedure, checked with a solution of ppm PQ pre- pared in blank plasma, was 80% Efforts to increase the recovery were not successful, presumably due to the competitive adsorption of other organic species present in plasma matrices on the C18 cartridges 40 mV PQ 10% 8% 4% 1% 3.4 APPLICATION to PATIENT SAMPLES 0% 4.5 5.0 5.5 6.0 4.0 Migration time (min) Fig Electropherograms for CE-C4D determination of PQ (5 ppm) in plasma samples after elution from the C18 cartridges using CH3COOH (0–10% v/v) in methanol Other conditions as for Fig heights are much more significant than that of PQ, leads to distortion of the PQ peak This phenomenon of CE-C4D has already been described elsewhere [48] To minimize this unwanted phenomenon, EDTA was added to the plasma samples prior to passage through C18 columns The complexation of EDTA with divalent cations, especially Ca2+, helped reduce significantly the saturated zones caused by them in the electropherograms (Fig 3) The minimal distortion effect, reflected by the sharpest PQ peak, was achieved at the EDTA concentration of 10−3 M To elute PQ from the C18 sorbents, an acidic eluent has normally been used [47,49] In our case, acetic acid was used due to its com- patibility with the BGE of the optimized CE-C4D which is composed of histidine and acetic acid Strong inorganic acids, for example HCl or HNO3, were avoided as they can render the solutions too highly con- ducting for injection in CE-C4D The effect of the concentration of acetic acid in the eluent was investigated from to 10% prepared in me- thanol Electropherograms obtained for elution using these concentra- tions of acetic acid are shown in Fig Highest elution efficiency was achieved for acetic acid concentrations equal to or higher than 8% The peak of PQ becomes more broadened at concentration of acetic acid of 10%, which could be explained by a slightly reduced stacking effect due to higher sample conductivity Further study with a similar observation on the effect of the sample matrix’s conductivity on the peaks’ ap- pearance can be found in Fig S3 in the ESI where electropherograms of PQ prepared in Ace up to 60% in methanol (without passing through the SPE step) were shown Note that the conductivity of the sample matrix, which affects the performance of the sample stacking effect, was not drastically augmented with an increase in the concentration of acetic acid in methanol The peak shape and height were therefore not much degraded at a relatively high concentration of acetic acid in the sample matrix Elution was therefore carried out at the optimized acetic acid concentration of 8% in the eluent Note that the presence of an organic solvent (i.e methanol in our case) in the eluent is also needed to achieve efficient elution To further increase the sample enrichment effect, the eluent was evaporated to reduce the sample volume Con- veniently, the use of an eluent composed of volatile compounds (i.e acetic acid and 31 patients with acute PQ poisoning admitted to the Poison Control Center between December 2016 and January 2017 were treated with a methanol) avoids the deposition of residuals in the concentrated samples that may degrade the subsequent CE-C4D per- formance The optimized BGE for determination of paraquat was composed of 10 mM His adjusted to pH with acetic acid The detection limit standardized therapeutic regimen using activated charcoal, immunosuppressive therapy and resin hemoperfusion These operations were performed consecutively until the plasma PQ concentrations be- came undetectable Measurements of the plasma PQ concentrations with CE-C4D were carried out for all patients upon emergency admis- sion at the hospital, and for some patients results obtained following hemoperfusion are also available The concentrations of plasma PQ determined with CE-C4D and for cross-checked by HPLC are shown in Table The relative errors for the two pairs of data are generally very good The highest deviations were found for concentrations close to the lower end of the calibration curves Student’s t-test was also applied using the differences between the data obtained with CE-C4D and those with HPLC The calculated t values for the confidence level of 95% was −0.22, which is smaller than the value referenced in the t table (2.013), meaning that the two sets of data (47 pairs obtained with CEC4D and HPLC) are not significantly different from each other The dynamic monitoring of plasma PQ concentrations allows a guidance of the treatment of patients with acute PQ intoxication The plasma PQ concentrations from the patients treated with resin hemo- perfusion were thus used to evaluate the scavenging effect of hemo- perfusion Electropherograms for determination of plasma PQ from the patient ID22 (see Table 1) before and after hemoperfusion are shown in Fig As can be seen from the data of Table 1, the plasma PQ con- centrations were significantly decreased after the first hemoperfusion, and decreased further after the second hemoperfusion where this was needed Upon conclusion of the third hemoperfusion all plasma PQ tests were negative An exceptional case could be noticed for the patient ID17 This patient suffered from a serious PQ intoxication with an ex- tremely high plasma PQ level of 125 ppm, and did not survive long after hospital admission Hemoperfusion therefore was not carried out for this patient It should be noted that the first diagnostic test of the plasma PQ has to be done as quickly as possible so that the therapeutic treatment can be carried out without delay in order to increase the survivability of the patients In this context, CE-C4D can demonstrate itself as an inexpensive and simple tool to provide fast and efficient information on the PQ poisoning status, which in turns helps medical doctors, especially in decentralized areas, to significantly reduce the fatal rate due to accidental or suicidal PQ ingestion With our present LOD of 0.5 ppm, the developed CE-C4D instrument and methodology are being employed as a screening method for quick identification of PQ intoxication and for quick monitoring of the first and second hemo- perfusion Further treatment after ensuring the survival of the patients would still at this stage require the standard method, i.e HPLC, for monitoring of the plasma PQ levels (down to 0.1 ppm) after the third hemoperfusion Our objective is to further improve the detection limit to better than 0.1 ppm in order to monitor the (second or third) he- moperfusion at the hospital The actual interested calibration curve (5 A.P Vu et AL JourNAlofChromAtogrAphyB1060(2017)111–117 Table Concentrations of paraquat in plasma samples determined with CE-C4D and cross-check results with HPLC-UV After the 2nd hemoperfusion Patient ID Upon emergency admission at the hospital After the 1st hemoperfusion 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 CE (ppm) HPLC (ppm) Deviation% CE (ppm) HPLC (ppm) Deviation% CE (ppm) HPLC (ppm) Deviation % CE (ppm) < 0.5 1.2 81.0 1.1 2.2 69.8 2.7 28.9 0.6 35.8 8.7 5.0 0.5 41.9 2.0 8.3 125.0 1.6 0.9 50.8 0.7 8.0 1.7 9.7 1.9 31.9 17.6 0.5 3.7 0.8 17.5 0.05 1.4 81.0 1.2 2.7 69.0 3.0 24.6 0.6 37.0 7.4 5.9 0.6 41.2 2.5 8.7 124.0 1.8 1.1 48.2 0.7 9.5 1.7 10.7 2.5 32.8 19.2 0.5 4.6 1.1 18.7 – −17.6 −11.9 −19.2 1.2 −10.6 17.3 5.8 −3.2 17.1 −14.8 −17.0 1.8 −20.3 −5.1 1.4 −16.0 −11.9 4.6 1.3 −15.6 −3.2 −9.8 −24.4 −2.8 −8.5 −4.0 −18.4 −21.9 −6.3 < 0.5 < 0.5 – 0.2 – < 0.5 – 0.5 – – 1.2 2.8 – 4.8 1.4 1.3 0.4 – 0.8 1.4 – < 0.5 < 0.5 10.6 < 0.5 0.7 < 0.5 1.7 < 0.5 – 1.0 1.3 – 0.2 0.2 9.8 – – – – – – < 0.5 1.6 – < 0.5 0.6 1.0 – – < 0.5 0.5 – – – – – < 0.5 – – – 0.2 – – 1.0 2.1 < 0.5 4.0 1.0 1.3 < 0.5 – – – – – – −15.4 −23.7 – −16.4 −25.4 −4.7 – – −19.6 8.5 – – – 8.8 – – – 1.7 – 0.3 0.6 1.0 – – 0.4 0.4 – – – – – 0.1 – – < 0.5 0.5 – – – 0.5 – 5.1 – 5.5 – 0.9 – – – – – – – – 1.0 – – – – – – – – – – – – – – −7.7 – – −6.3 −2.0 – – – 11.0 – – – – – – – −6.2 – – – – – – – – – – – – – – < – – < < – – – < – – – – – – – < – – – – – – – 25 mV – 0.9 0.3 1.8 0.2 PQ 0.5 0.5 0.5 0.5 Conclusions PQ B) C) −22.2 – −4.6 – – – – −3.8 – −7.8 0.5 is during the hemoperfusion process that requires quantitative data of plasma PQ concentrations in order for the medical doctors to decide how many cycles of hemoperfusion the patients would need for the treatment A) 3.0 After the 3rd hemoperfusion 3.5 4.0 4.5 5.0 5.5 6.0 Migration time (min) Fig Electropherograms for determination of paraquat in plasma samples from the same patient collected before and after hemoperfusion A) Upon emergency admission at the hospital; B) After the 1st hemoperfusion; C) After the 2nd hemoperfusion Other conditions as in Fig points) is therefore more towards the lower range (1–20 ppm) rather than the higher one (up to 130 ppm with our method) In urgent cases at the hospital, once we observed a high PQ signal out of the upper limit of the calibration range, which indicates A purpose-made portable CE instrument coupled with a miniatur- ized C4D cell was successfully applied to the determination of paraquat in plasma samples collected from poisoned patients in Vietnam Cross- check with the reference method (HPLC-UV) proved the reliability of the results obtained with CE-C4D With the robustness, high portability of the CE-C4D system, simplicity of the operation and the easy avail- ability of the components used in the BGE, the CE-C4D method can be seen as a straightforward and cost-effective option for screening of plasma paraquat concentrations for immediate diagnosis of paraquat poisoning To promote the utilization of the CE-C4D approach at local hospitals, two improvements are still needed: i) further simplification of the sample treatment procedure to more adapt to non-expert users, e.g that the patient is acutely poisoned with PQ, hemoperfusion had to be carried out immediately It technicians and ii) improvement of detection limit In an on-going joint project between the Poison Control Center (Bach Mai hospital) and CE- Vietnam (www.CE-Vietnam.com), we are aiming at further optimiza- tion of the SPE protocol to allow complete removal of the interfering cations and efficient elution of paraquat with a much little elution vo- lume so that the evaporation step can be avoided Upon conclusion of these optimizations, to validate the clinical significance of the devel- oped CE-C4D approach, plasma samples from more than 200 patients will be analyzed with CE-C4D and the results will be used by medical doctors for prescription of therapeutic treatments to these patients Subsequently, deployment of the developed instrumentation and methodology to local hospitals in Vietnam, as well as operation training A.P Vu et AL JourNAlofChromAtogrAphyB1060(2017)111–117 to local staffs, are envisaged Acknowledgements The authors would like to thank 3SAnalysis JSC (www.3SAnalysis vn) for instrumental support References [1] H.-W Gil, M.-S Kang, J.-O Yang, E.-Y Lee, S.-Y Hong, Association between plasma paraquat level and outcome of paraquat poisoning in 375 paraquat poisoning pa- tients, Clin Toxicol 46 (2008) 515–518 [2] R.M de Almeida, M Yonamine, Gas chromatographic-mass spectrometric method for the determination of the herbicides paraquat and diquat in plasma and urine samples, J Chromatogr B 853 (2007) 260–264 [3] C.D.S Tomlin, The Pesticide Manual, 15th ed., British Crop, 2009, p 862 [4] R.J Dinis-Oliveira, J.A Duarte, A Sanchez-Navarro, F Remiao, M.L Bastos, F Carvalho, Paraquat poisonings: mechanisms of lung toxicity, clinical features, and treatment, Crit Rev Toxicol 38 (2008) 13–71 [5] R.D Whitehead, M.A Montesano, N.K Jayatilaka, B Buckley, B Winnik, L.L Needham, D.B Barr, Method for measurement of the quaternary amine com- pounds paraquat and diquat in human urine using high-performance liquid chro- matography-tandem mass spectrometry, J Chromatogr B 878 (2010) 2548–2553 [6] W Klein-Schwartz, G.S Smith, Agricultural and horticultural chemical poisonings: mortality and morbidity in the United States, Ann Emerg Med 29 (1997) 232– 238 [7] A.T Proudfoot, M.S Stewart, T Levitt, B Widdop, Paraquat poisoning – sig- nificance of plasma-paraquat concentrations, Lancet (1979) 330– 332 [8] T.B Hart, A Nevitt, A Whitehead, A new statistical approach to the prognosticsignificance of plasma paraquat concentrations, Lancet (1984) 1222–1223 [9] C Bismuth, R Garnier, S Dally, P.E Fournier, J.M Scherrmann, Prognosis and treatment of paraquat poisoning – a review of 28 cases, J Toxicol Clin Toxicol 19 (1982) 461–474 [10] B Winnik, D.B Barr, M Thiruchelvam, M.A Montesano, E.K Richfield, B Buckley, Quantification of Paraquat, MPTP, and MPP+ in brain tissue using microwave- assisted solvent extraction (MASE) and high-performance liquid chromatography- mass spectrometry, Anal Bioanal Chem 395 (2009) 195– 201 [11] M.M Ariffin, R.A Anderson, LC/MS/MS analysis of quaternary ammonium drugs and herbicides in whole blood, J Chromatogr B 842 (2006) 91–97 [12] X.P Lee, T Kumazawa, M Fujishiro, C Hasegawa, T Arinobu, H Seno, A Ishii, K Sato, Determination of paraquat and diquat in human body fluids by highper- formance liquid chromatography/tandem mass spectrometry, J Mass Spectrom 39 (2004) 1147–1152 [13] N.C Posecion, E.M Ostrea, D.M Bielawski, Quantitative determination of paraquat in meconium by sodium borohdride-nickel chloride chemical reduction and gas chromatography/mass spectrometry (GC/MS), J Chromatogr B 862 (2008) 93–99 [It1o4,]Y.MH ori, M Fujisawa, A Oda, S Katsuyama, Y Hirose, T Yoshioka, Rapid analysis method for paraquat and diquat in the serum using ion-pair highperfor- mance liquid chromatography, Biol Pharm Bull 28 (2005) 725–728 [15] M.R Brunetto, A.R Morales, M Gallignani, J.L Burguera, M Burguera, Determination of paraquat in human blood plasma using reversed-phase ionpair high-performance liquid chromatography with direct sample injection, Talanta 59 (2003) 913–921 [16] P Paixao, P Costa, T Bugalho, C Fidalgo, L.M Pereira, Simple method for de- termination of paraquat in plasma and serum of human patients by high-perfor- mance liquid chromatography, J Chromatogr B 775 (2002) 109–113 [17] P.J Taylor, P Salm, P.I Pillans, A detection scheme for paraquat poisoning: vali- dation and a five-year experience in Australia, J Anal Toxicol 25 (2001) 456–460 [18] K Arys, J Van Bocxlaer, K Clauwaert, W Lambert, M Piette, C Van Peteghem, A De Leenheer, Quantitative determination of paraquat in a fatal intoxication by HPLC-DAD following chemical reduction with sodium borohydride, J Anal Toxicol 24 (2000) 116–121 [19] A Wuethrich, P.R Haddad, J.P Quirino, Simultaneous electrophoretic concentration and separation of herbicides in beer prior to stacking capillary electrophoresis UV and liquid chromatography-mass spectrometry, Electrophoresis 37 (2016) 1122–1128 [20] R Lanaro, J.L Costa, S.O.S Cazenave, L.A Zanolli, M.F.M Tavares, A.A.M Chasin, Determination of herbicides paraquat, glyphosate, and aminomethylphosphonic acid in marijuana samples by capillary electrophoresis, J Forensic Sci 60 (2015) S241–S247 [21] M.M Wang, J Shen, T Song, S.Q Li, H Chen, Rapid, simple and ultrasensitive analysis of paraquat in drinking water by field-amplified sample injection combined with pressure-assisted capillary zone electrophoresis, Chin J Anal Chem 40 (2012) 809–810 [22] R Lanaro, J.L Costa, L.C.R Fernandes, R.R Resende, M.F.M Tavares, Detection of paraquat in oral fluid, plasma, and urine by capillary electrophoresis for diagnosis of acute poisoning, J Anal Toxicol 35 (2011) 274–279 [23] H.P Chu, S.Y Chang, Determination of paraquat and diquat by combination of nanoparticles-Based extraction with capillary electrophoresis, J Chin Chem Soc 58 (2011) 793–797 [24] Q.X Zhou, J.L Mao, J.P Xiao, G.H Xie, Determination of paraquat and diquat preconcentrated with N doped TiO2 nanotubes solid phase extraction cartridge prior to capillary electrophoresis, Anal Methods (2010) 1063–1068 [25] E Vinner, M Stievenart, L Humbert, D Mathieu, M Lhermitte, Separation and quantification of paraquat and diquat in serum and urine by capillary electro- phoresis, Biomed Chromatogr 15 (2001) 342–347 [26] H.A Duong, M.D Le, K.D.M Nguyen, H Peter Hauser, H.V Pham, T.D Mai, In- house-made capillary electrophoresis instruments coupled with contactless con- ductivity detection as a simple and inexpensive solution for water analysis: a case study in Vietnam, Environ Sci Process Impacts 17 (2015) 1941–1951 [27] T.A.H Nguyen, V.R Nguyen, D.D Le, T.T.B Nguyen, V.H Cao, T.K.D Nguyen, J Saiz, P.C Hauser, T.D Mai, Simultaneous determination of rare earth elements in ore and anti-corrosion coating samples using a portable capillary electrophoresis instrument with contactless conductivity detection, J Chromatogr A 1457 (2016) 151–158 [28] T.A.H Nguyen, T.N.M Pham, T.T Ta, X.T Nguyen, T.L Nguyen, T.H.H Le, I.J Koenka, J Saiz, P.C Hauser, T.D Mai, Screening determination of four am- phetamine-type drugs in street-grade illegal tablets and urine samples by portable capillary electrophoresis with contactless conductivity detection, Sci Justice 55 (2015) 481–486 [29] P Kubáň, P.C Hauser, Contactless conductivity detection for analytical techni- ques—developments from 2014 to 2016, Electrophoresis 38 (2017) 95–114 [30] P Kuban, P.C Hauser, Contactless conductivity detection for analytical techniques- developments from 2012 to 2014, Electrophoresis 36 (2015) 195–211 [31] A.V Schepdael, Recent advances in portable analytical electromigration devices, Chromatography (2016) 1–12 [32] M Greguš, F Foret, P Kuban, Portable capillary electrophoresis instrument with contactless conductivity detection for on-site analysis of small volumes of biological fluids, J Chromatogr A 1427 (2016) 177–185 [33] A.P Lewis, A Cranny, N.R Harris, N.G Green, J.A Wharton, R.J.K Wood, K.R Stokes, Review on the development of truly portable and in-situ capillary electrophoresis systems, Meas Sci Technol 24 (2013) 042001 [34] B.L Milman, Cluster ions of diquat and paraquat in electrospray ionization mass spectra and their collision-induced dissociation spectra, Rapid Commun Mass Spectrom 17 (2003) 1344–1349 [35] S Hara, N Sasaki, D Takase, S Shiotsuka, K Ogata, K Futagami, K Tamura, Rapid and sensitive HPLC method for the simultaneous determination of paraquat and diquat in human serum, Anal Sci 23 (2007) 523–526 [36] T.T.T Pham, T.D Mai, T.D Nguyen, J Sáiz, H.V Pham, P.C Hauser, Automated dual capillary electrophoresis system with hydrodynamic injection for the con- current determination of cations and anions and application to the monitoring of biological ammonium removal from contaminated ground water, Anal Chim Acta 841 (2014) 77–83 [37] M.D Le, H.A Duong, M.H Nguyen, J Saiz, H.V Pham, T.D Mai, Screening de- termination of pharmaceutical pollutants in different water matrices using dual- channel capillary electrophoresis coupled with contactless conductivity detection, Talanta 160 (2016) 512–520 [38] T.D Mai, M.D Le, J Saiz, H.A Duong, I.J Koenka, H.V Pham, P.C Hauser, Triple- channel portable capillary electrophoresis instrument with individual background electrolytes for the concurrent separations of anionic and cationic species, Anal Chim Acta 911 (2016) 121–128 [39] T.A.H Nguyen, T.N.M Pham, T.T Doan, T.T Ta, J Sáiz, T.Q.H Nguyen, P.C Hauser, T.D Mai, Simple semi-automated portable capillary electrophoresis instrument with contactless conductivity detection for the determination of beta- agonists in pharmaceutical and pig-feed samples, J Chromatogr A 1360 (2014) 305–311 [40] M Stojkovic, B Schlensky, P.C Hauser, Referenced capacitively coupled con- ductivity detector for capillary electrophoresis, Electroanalysis 25 (2013) 2645–2650 [41] P Kubáň, M Reinhardt, B Müller, P.C Hauser, On-site simultaneous determination of anions and cations in drainage water using a flow injection-capillary electro- phoresis system with contactless conductivity detection, J Environ Monit (2004) 169–174 [42] T.D Mai, S Schmid, B Müller, P.C Hauser, Capillary electrophoresis with con- tactless conductivity detection coupled to a sequential injection analysis manifold for extended automated monitoring applications, Anal Chim Acta 665 (2010) 1–6 [43] T.D Mai, P.C Hauser, Pressure-assisted capillary electrophoresis for cation se- parations using a sequential injection analysis manifold and contactless con- ductivity detection, Talanta 84 (2011) 1228–1233 [44] T.D Mai, P.C Hauser, Anion separations with pressure-assisted capillary electro- phoresis using a sequential injection analysis manifold and contactless conductivity detection, Electrophoresis 32 (2011) 3000–3007 [45] E Mallat, C Barzen, R Abuknesha, G Gauglitz, D Barcelo, Fast determination of paraquat residues in water by an optical immunosensor and validation using ca- pillary electrophoresis-ultraviolet detection, Anal Chim Acta 427 (2001) 165–171 [46] O Nunez, E Moyano, L Puignou, M.T Galceran, Sample stacking with matrix re- moval for the determination of paraquat, diquat and difenzoquat in water by ca- pillary electrophoresis, J Chromatogr A 912 (2001) 353– 361 [47] V.Y Taguchi, S.W.D Jenkins, P.W Crozier, D.T Wang, Determination of diquat and paraquat in water by liquid chromatography-(electrospray ionization) mass spectrometry, J Am Soc Mass Spectrom (1998) 830– 839 [48] P Kubáň, P.C Hauser, Ten years of axial capacitively coupled contactless con- ductivity detection for CZE – a review, Electrophoresis 30 (2009) 176–188 [49] N.J J.T Baker; Phillipsburg, Extraction of Paraquat and Diquat from Pond Water, Bakerbond™, Application Note #EN-020; (1989) ... TRANG NGHIÊN CỨU ĐỊNH LƯỢNG PARAQUAT TRONG HUYẾT TƯƠNG NGƯỜI BẰNG PHƯƠNG PHÁP ĐIỆN DI MAO QUẢN SỬ DỤNG DETECTOR ĐỘ DẪN KHÔNG TIẾP XÚC (CE- C4D) Chun ngành: Hóa phân tích Mã số: 60440118 LUẬN VĂN THẠC... Nguyên tắc định lượng paraquat phương pháp điện di mao quản CE – C4D Phƣơng pháp phân tích phƣơng pháp điện di mao quản sử dụng detector độ dẫn không tiếp xúc kết nối kiểu tụ điện (CE – C4D) Nguyên... nghiên cứu quy trình xử lý mẫu paraquat huyết tƣơng, từ định lƣợng paraquat phƣơng pháp điện dimao quản sử dụng detector đo độ dẫn không tiếp xúc (CE- C4D) CHƢƠNG 1: TỔNG QUAN 1.1 Tổng quan paraquat