Nghien
CLFU- Ky thuat
Xay dyng quy trinh xac dinh du* luong hoa chat bao ve thuc vat dimethoat va
r• • •
fenitrothion tren dira can bang phuong phap
r
sac ky long hieu nang cao
Dat van de
To chirc Y t i t h i gidi WHO da cd canh bao ve
" Nguyen lieu lam thudc co ngudn gdc thwc vat co thi bi anh hwdng boi dw Iwgng cua hoa chit bao ve thwc vat (HCBVTV) tich tu trong qua trinh canh tac va cung c6 thi bi nhiim chit khang nim trong qua trinh bao quan" '^'. Oe dam bao tinh an toan trong str dung dygc lieu, dygc dien mdt so nyde (USP, BP, EP ..) cd qui dinh gidi han cho phep doi vdi dy lygng HCBVTV. Trong khi dd, Dygc dien Viet Nam xuit ban l i n thir ba (DOVN III, 2002) thi chya ed qui djnh nay va cung chya cd phyong phap kiem tra. De gdp phin kiem tra dp an toan cua dygc lieu dung lam thuoc chung tdi xay dirng qui trinh xac dinh dy lygng HCBVTV dimethoat va fenitrothion tren dira can tyoi va khd bing phyong phap s i c ky Idng hieu nang cao.
Ddi turnig va phirong phap nghien cuu 0 6 i tu'O'ng nghien cii'u
Dira can (tyoi (dp am ~ 85%) va khd (dd am ~ 5%)) dygc phun cac thudc trir sau sau: Bian 40 EC chira dimethoat 40% (kl/kl) eua cdng ty CP BVTV An Giang, Visumit 50 ND chira fenitrothion 50% (kl/tt) cua cdng ty Thuoc sat triing Viet Nam (VIPESCO).
Dira can dygc trdng tai i p Ninh Lgi, xa Ninh Thanh, th| xa Tay Ninh, tinh Tay Ninh.
Chit c h u i n , trang thiit bj, hoa chit, dung moi Chatchuan
Dimethoat (95,7%) va fenitrothion (96%):
nguon goc tir edng ty VIPESCO.
Thiet bi
He thing HPLC HP 1100 - Hewlett Packard (My), d i u dd PDA. Cdt s i c ky Luna C18 100A (250
Le Thi Chau Pha', VTnh Djnh^
'Cong ty Co phdn Dirac phdm OPC 'Khoa Dirac, trirdng dgi hoc Y dirac TP. HCM x 4,6 mm, 5 pm), cdt SPE Chromabond C18ec (500 mg/ 6 ml), cdt SPE florixil tu- nhdi (1,5 g/10 ml).
Hoa chat, dung moi
Chloroform, diethyl ether, n-hexan dat tieu chuan phan tieh. Acetonitril, methanol dat tieu chuan dung trong s i c ky Idng cao ap.
Phu'O'ng phap nghien ciru Chuin bi mau
Dira can dygc thu l i y toan bd p h i n eay tren mat dat, tiiy vao ban c h i t d y g c lieu ed each xu- ly m i u kh^c nhau, d y g c lieu tyoi d y g c c i t nhd va bam nhuyin, dygc lieu khd xay thanh bdt thd bing may xay dygc lieu.
Xac dmh khoi Imyng miu phan tich
Bang 1: Cach tinh khdi Iwgng dwgc lieu di phan tich
DiTcyc lieu
Tryccc khi thu hoach Sau khi thu hoach
Ham am (%)
a
b
Khoi lu'O'ng mau can (g)
X
.x.(lOO-a) y = —^^
100-6
x>
X (khdi lygng dygc lieu tyoi trydc khi thu hai):
V.C
MRL
{V: the tich cuoi cung ciia dich thir dem phan tich sic ky (ml): MRL:mg/kg(/jg/g): c:LOQ ciia phwong phap phan tich (/jg/ml)).
Xay dirng qui trinh phan tich va khao sat hieu suit thu hoi cua qui trinh chiet
- Tao m i u nhiim HCBVTV: 30 g dira can tyoi (4,8 g dira can khd), them chinh xac 1ml dung djch ehit ehuan 15 ppm dimethoat va 15 ppm fenitrothion pha trong methanol, lie d i u , de yen qua dem.
TAP CHl DUQC HOC - 8/2009 (SO 400 NAM 49)
Nghien CLFU - Ky thuat
- Chiet xuit: 80 ml chloroform / m i u dira can tyoi (40 ml/ dira can khd), lic/10 phiit, sieu am/30 phiit. Lpc/2 ldp giiy lpc. 40 ml chloroform/
ba dira can tyoi (20 ml/ ba dira can khd), lic/5 phiit, lpc/ 2 Idp giiy lpc. Gdp t&t ca djch Ipc, cd quay chan khdng/40 °C d i n c i n khd. Hda cin/10 ml ether, Ipc/giiy Ipc, hda c i n cdn lai nhy tren vdi 10 ml ether. Gdp eac dich lpc, bay hoi d i n c i n khd (Cin A).
- Tinh c h i :
+ Qua SPE florixil: Hda tan c i n A/5 ml ether, them 5 ml n-hexan, l i c d i u , lpc/ bdng. Cho dieh Ipc qua cdt SPE florixil da hoat hda. Thu l i y dich thu- qua cdt va dich rCra giai b i n g 5 ml ether (Dung dich B). T i i p tuc cho 5 ml ether qua cdt, loai bd phin nay. Cho t i i p 10 ml ether-methanol ( 9 : 1 ) qua cdt, thu l i y dich rCra giai nay (Dung dich C). Bay hoi dung dich B, dung djch C ty nhien trong tu hot thu d y g c e i n khd B va C.
Bang 2; Diiu kien tiin hanh sic ky gradient (ti le
+ Qua SPE C18: Hda tan c i n B/ C b i n g 5 ml methanol, lpe qua bdng. Hiit chinh xae 1 ml dich lpc vao edc ed chira s i n 9 ml nyde, l i e d i u , de yen 15 phiit, cho qua cdt SPE C18 da hoat hda.
Loai tap bing 10 ml nyde, hiit khd cdt dydi chan khdng. Rya giai b i n g 5 ml ether-methanol (95:5)/
c i n B (5 ml ether-methanol (90:10)/ c i n C). Thu hdi dich ru-a giai, bay hoi tu- nhien trong tu hdt d i n e i n khd ( c i n B', e i n C ) . Hda tan cac c i n B', C vdi 1 ml methanol, lpe/ 0,45 |.im, dygc cac dich s i c ky Bsk' va Csk'. (' Bsk: phan tich fenitrothion: Csk: phan tich dimethoat)
- Phan tich djnh lygng dung djch Bsk va Csk (theo d i i u kien d bang 2)
Dung dich tring: Chiit theo qui trinh dung dich thy nhyng khdng n h i i m thuoc trir sau vao dygc lieu.
Dung dich ddi chiiu 1: 3 ppm dimethoat chuin trong methanol.
Dung dich ddi chiiu 2: 3 ppm fenitrothion chuan trong methanol.
% cac dung moi theo thoi gian)
Tdc do dong: 1 Thoi gian (phiit)
Acetonitril MeOH
Nyo-c
0-10 10 10 80
Dimethoat
ml/phut: nhiet do budng cot:
15-25 30 10 60
30 10 10 80
30-35 10 10 80
3^C:
0-6 30 10 60
Fenitrothion the tich bam:
6,01 40
0 60
SOful 15-25
70 0 30
30-35 30 10 60 Thim dinh qui trinh xac dinh dw Iwo'ng
dimethoat va fenitrothion da phun len dira can bang phuxyng phap HPLC
Dung dich thir 1: 30 g dira can tuoi (4,8 g dira can khd), them chinh xac 1ml dung dich Bian 40 EC da pha loang trong methanol (15 ppm dimethoat/ 1 ml). L i e d i u , de yen qua dem, tiin hanh chiiL
Dung dich thir 2: 30 g diJ-a can tyoi (4,8 g dira can khd), them chinh xac 1ml dung djch Visumit 50 ND pha loang trong methanol (15 ppm fenitrothion/ 1 ml). L i e d i u , de yen qua dem, tiin hanh chiit.
Khao sat dw luxyng dimethoat, fenitrothion moi ngay sau khi phun thuoc trir sau len dira
Bang 3: Thudc trw sau sir dung va ndng do phun
can, thu hai va phcfi kho dwai dieu kien tw nhien
Dira can trdng tren 3 ludng, mdi ludng 12 m^, cac ludng each nhau 1 m. Trydc khi thu hoaeh 3 gid phun eae thuoe trir sau theo bang 3. Thu hoach toan cay bo re, phoi d y d i d i i u kien tu- nhien tren 3 khu vyc tach biet. L i y m i u kiem mdi ngay, b i t d i u tir ngay thu hoach, cho d i n khi khdng cdn thiy dy lygng thudc BVTV. L i y m i u ; dygc lieu tren mdi khu vu-c d y g c l i y 5 m i u theo dydng cheo gdc, mdi m i u it n h i t 3 cay dygc lieu nguyen. Trdn 5 m i u vdi nhau. Xac djnh ham am, tinh khdi lygng m i u se can. T i i n hanh chiit, diing mdt lygng dung mdi chiit du ngap m i u va chiit lai ba vdi Vz the tich dung l i n mdt.
len cac ludng dwgc lieu Dwov lieu
Lu6ng 1 Lu6ng 2 Lu6ng 3
Dien tich
12 m^
12 m^
12 m^
Thuoc trif sau
BIAN 40 EC VISUMIT 50 ND
Ty le pha thuoc trong nw&c 2,4 m l / 5 0 0 ml 2,4 m l / 5 0 0 ml Khong phun thu6c trir sau
Ham Iwong thuoc trir sau 80 mg/m^
100 mg/m^
10 TAP CHI DUOC HOC - 8/2009 (SO 400 NAM 49)
Nghien
CLFU- Ky thuat
K§t qua va ban luan
Hieu suit thu hdi cua qui trinh chiit
Bang 4: Hieu suit thu hdi cita dimethoat va fenitrothion tren miu nhiem Ca m i u = 3
Hieu suit RSD
Dimethoat
Dira can twai Dira can kho 112,64% 119,48%
4,53% 3,15%
Fenitrothion
DCra can twai DCra can kho 83,21 yo 94,76%)
5,76% 5,90%
« u - |
^.
...
,«.
»
.,.. , -.„ . , ,u.- „.... ,.,. .... ; . . r.,.i .^ . .
1
Q
1
^,.^fU'j\'-- ..^.:. / '
io i? 11 10 IB :-j
Hinh 1: Lin Iwgt sic ky dd phan tich dimethoat, fenitrothion trong miu thir bang HPLC. Tinh twong thich he thdng tren may HPLC
Bang 5; Kit qua 6 lin bom lap lai ciia cac dung dich thir
H
a
N afDimethioat TB 18,400 213,481 23,318 1,23 1,01 106486 1,13 RSD 0,43% 1,85% 1,97% 1,51% 1,05% 1,67% 1,30%
fenitrothion TB 18,975 242,511 23,507 1,51 1,02 116789 0,82 RSD 0,83% 1,93% 1,59% 0,50% 1,01% 1,73% 1,20%
Thim dinh phwang phap
Bang 6: Kit qua thim dinh phwong phap phan tich bing HPLC
Chi tieu Dimethoat Fenitrothion
Tinh tuyen tinh He sd twong quan R^
Miin gii tri (ppm)
YA==83,451X +0,7816 0,9997
0,03-17,66
YA = 94,878X-0,5592 0,9995
0,04- 17,89
Gioi han phat hien 0,017 ppm 0,008 ppm
Gidl han dinh Iwgng 0,07 ppm 0,04 ppm
Do chlnh xac (RSD), n = 6 4,2% - 6,2% 5,1%-7,3%
Do diing (R%,),n = 9 89,2%-112,2% 88,7%- 110,7%
Khao sat dp chinh xae eua dimethoat va fenitrithion deu dat theo BP2007 (18,75% vdi nong dp 0,5 ppm). Dd diing so vdi tieu ehuin BP2007 (70-110%): dimethoat vygt qua gidi han tren 2,2%; fenitrothion qua gidi han tren 0,7%.
K i t qua du' lu'O'ng dimethoat, fenitrothion mdi ngay sau khi phun thudc trip sau len diFa can, thu hai va phai kho du'di diiu kien tip nhien
Diiu kien phoi tw nhien: Trong khoang thdi gian phoi dira can, trdi cd liic ning to, thmh
thoang cd mya (dira can d y g c dya vao chd rao), da sd cac ngay it ning.
Dy lygng dimethoat r i t cao vao ngay dau tien khi phun thude, giam r i t nhanh sau khi phoi khd mdt ngay va dydi mirc 0,5 ppm tir ngay thir 9 sau khi phun thudc, khdng phat hien vao ngay thir 16. Dy lygng fenitrothion r i t cao vao ngay d i u tien khi phun thudc, giam r i t nhanh sau khi phoi khd mdt ngay va d y d i mirc 0,5 ppm tir ngay thir 14 sau khi phun thudc; khdng phat hien vao ngay thir 18.
TAP CHI DUQC HOC - 8/2009 (SO 400 NAM 49) 11
Nghien CLFU - Ky thuat
Khong co pic dimethoat
Hinh 2: Lin Iwgt sSc ky dd dich chiit dimethoat sau khi thu hai 3 ngay va 16 ngay
Khong co pic fenitrothion
.P
Hinh 3: Lin Iwgt sic ky dd dich chiit fenitrothion sau khi thu hai 9 ngay va 18 ngay
dimethoat va fenitrothion trong dCra can sau khi phun, thu hai va phoi khd.
S u m m a r y
A reserve-phase HPLC method was developed for determination of two pesticides of different solubilities - dimethoat and fenitrothion - in fresh and dried Catharanthus roseus.
Dimethoat and fenitrothion were simultaneously extracted with CHCI3. The extracts were purified by SPE florixil (1.5g/10ml) and SPE C18 (500mg/6ml), respectively, for sample cleanup, then concentrated to get 2 separate fractions, one contained dimethoat and the other fenitrothion. A simultaneous and complete separation of dimethoat and fenitrothion from unknown adjacent peaks was achieved by using a Phenomenex Luna C18 column (250 x 4,6 mm;
5 ^m) with a mixture of MeOH - acetonitril - water as eluents in gradient mode, flow rate of 1 ml/min, column temperature at 3^C and detection at 205 nm. The method was validated to have adequate reliability for determination of residual dimethoat and fenitrothion in Catharanthus roseus extracts. By the built analysis methods, the residue of these pesticides on Catharanthus roseus was followed: Dimethoat (Bian 40 EC) was less than 0.5 ppm after the 9'"
day, then undetectable on the '/6"' day;
fenitrothion (Visumit 50 ND) was less than 0.5 ppm after the 14"^ day, and undetectable on the
18'^ day. The blanks were affirmed with samples of Catharanthus roseus uncontaminated with dimethoat and fenitrothion by the other reference methods as GC-FID, HPLC-MS.
Ket luan
o i tai nghien eiru da xay dirng dygc mdt phyong phap chiit xuit, lam sach, lam giau m i u cung liic cua dimethoat va fenitrothion (cd dd tan khae nhau: dimethoat hoi tan trong nyde, fenitrothion khdng tan trong nyde) trong dygc lieu dira can tyoi va khd vdi hieu s u i t phue hdi >
70%. Oa xay dyng dygc qui trinh djnh lygng dy lygng dimethoat, fenitrothion trong dira can khd va tygi b i n g phyong phap HPLC-PDA. l i n g dung qui trinh da xay dirng de khao sat dy lygng
Tai lieu tham khao
1. Plant Protection Department of Vietnam, Asean Secrectarial, Croplife Asia, Asean Argochemical Residue workshop, 10/2002; pp.
17, 21, 24, 128-129.
2. The British Pharmacopoeia (2005), CD ROM, Volume I V - A 2 6 3 .
3. The European Pharmacopoeia IV (2002), pp. 184-187.
4. The United States Pharmacopoeia XXIX (2006), pp. 2600-2603.
