• Nghien cu'u - Ky thuat
Xay dyng phirtmg phap dinh Itfong ton dir acid 2,4-dichlorophenoxy acetic trong chi thyc bang sac ky long hieu nang cao
Vii Binh Ducmg '', Pham Van Hien'-^
'Hoc vien Qudn y- Bp Quoc phong 'Dgi hoc Ajou- Han Quoc 'E-mail- [email protected]
Summary
An HPLC method for quantification of 2,4-D residue in Fructus Aurantii immaturus was established:
Colum -C18 (250 x4.6 mm, 5pm); Detector: PDA, scanned at range 210-400 nm; Mobile phase - MeOH and amonium formate, at rate of 50:50 (v/v). The method was validated and proved compatible to the HPLC system, ensured of specificity • repeatability with the recon/ery above 85.0%. The method was of low LOD, proved able of detecting 2,4-D at low dose, and so, practically potential for dertemination of this herbicide residue in Fructus Aurantii immaturus.
Keywords: 2,4-Dichlorophenoxy acetic acid, HPLC, Fructus Aurantii immaturus.
Dat v i n de
Acid 2,4-dichlorophenoxy acetic (2,4-D) la c h i t bao vg thuc vat d u g c sir dung trong ndng nghigp de kich thich s g ra hoa, dau qua. Rigng doi hp cam quyt, su" dyng 2.4-D trong tho'i ki qua non truoc vu thu hoach kich thich tang khoi lugng va kich thuoc cua qua. han che rung qua do iing nuod^'. 2,4-D con (re c h l cac enzym ngi bao. tang cuong dp ben c h i c cua vo qua. cd the bao quan cam quyt den 75 ngay sau thu hogch ma van giu' dugc mau sac, trpng lugng va miii VL Vi thg c h i t nay la ly tuo'ng de tang cudng thoi gian bao quan trong qua trinh van c h u y i n , ton trij tiF ndng trai den khu che x u l t . Tuy nhign.
2,4-D la c h i t cd dgc tinh cao vo'i co quan tieu hda, cac to chu'c duo'i da, mau. gan, than, lach, t u y i n giap, nao cua ngud-i cung nhu dpng vat cd vui'^i. N h i l u bao cao dich t l chi ra rang cd m l i lien quan giu-a ung thu lymphoma non-Hodgkin va s u phoi nhilm 2,4-D'^i. Vi vay. viec ton d u 2,4-D VO'I ham luong cao trong hoa qua va d u g c lieu se anh huong td'i sue khoe con nguoi. 0 Viet Nam, da cd bao cao r i n g cac qua loai cam quyt d Viet Nam cdn ton d u 2,4-D (79-104 pg/kg)"i.
Chi thuc la mpt d u g c li^u hp Cam (Rutaceae) nen nhieu kha nang con ton d u 2,4-D. Chau Au va Nhat Ban da cd quy djnh v l ton d u hda c h i t
, 24 1 \
nay trong cac loai qua hp cam quyt nhung Dugc d i l n Vigt Nam IV chua co chuyen luan rieng ve ton d u 2,4-D trong ch? thuc. Vo'i ly do trgn, chiing tdi thuc hign nghien c u u xiy dung phuong phap dinh lugng ton d u 2,4-D trong d u g c lieu chl thgc b i n g sac ki Idng higu nang cao (HPLC) nhim gdp p h i n kiem soat va dam bao chat lugng dugc ligu Viet Nam.
Doi tu'p'ng va phu'cng phap nghien
CU'U
Ooi tugng va nguyen vat lieu nghien cuu D u g c lieu chi thuc (Fructus Aurantii immaturus) d u g c cung d p boi Cdng ty Dugc lieu Son Lim, Ha Npi dat tigu chuan Dugc dien Vigt Nam IV, kiem tra so bo khong phat hi&n 2,4-D. Chuan 2,4-D d u g c cung d p boi Sigma Aldrich, 99,9%. Methanol (MeOH), acid acetic, acetonitril (ACN), n u o c d t dat tieu chuan dung moi HPLC. Cac hda c h i t dung mdi khac nhu n-hexan, dichloromethan (DCM), aceton, NaCI, HjSO^... dat tigu chuan tinh khiet phan tich (PA).
Cgt c h i l t pha r i n C18-SPE d u g c cung cap bdi Sigma Aldrich. Cac hoa c h i t , dung cy khac dgt tigu c h u i n phan tich.
T A P C H i D i r O C H Q C - 2/201S (SO 466 NAM 55)
Nghien CIFU - Ky thuat
P h u c n g phap nghien CLPU Chuan bi miu
Miu chuin: Chuan 2,4-D d u g c pha trong MeOH thanh dung dich chuan g l c cd ndng dp 1000 pg/ml. Pha loang dung djch chuan g l c de cd cac dung djch c h u i n lam vigc.
Miu tring: Bot chi thuc n g h i i n mjn.
Mau thw. Bpt chi thuc nghien mjn co them cac lugng xac djnh c h u i n 2,4-D.
Chiet xuit
C§n chinh xac khoang 25 g bpt m i u thir va m i u tring vao binh 250 ml va lam am voi 25 ml nudc cat trong 30 phut sau do them 100 ml hon hgp dung moi 5 M HCI - A C N (10'90, v/v). M i u dugc chiet b i n g sieu am. Ly tam thu djch chilt Can duoc chilt t i l p 2 l l n v6i dung mdi tren, Toan bg djch ly tam dugc bdc hoi dung mdi den 20-25 ml va chuyin qua binh chilt cd chua 10 g NaCI Djch chilt dugc k i l m hda bang 100 ml dung dich NaOH 0,6 M va loai tap b i n g n-hexan (50 ml x 3 lln). Pha hexan d u o c bo di. Pha nud-c d u g c acid hda vdi dung djch H^SO^ 6 M d i n pH < 2. Dung djch nay dugc chilt bSng DCM (30 ml x 3). Toan bp djch chilt pha DCM dugc loai nud-c b i n g natri sulfat khan. Djch nay d u g c bdc hoi dung moi b i n g khi N^ tdi c l n ( c l n A). C i n nay co the dugc hda tan trong 2 ml MeOH d l phan tich HPLC hoac t i l p tyc dugc lam giau va loai tap b i n g phuong phap SPE. Vo'i phuong phap 2, d n A duoc tai lgn cgt chilt pha r i n da hoat hoa trudc. Tap dugc loai bd bang l l n lugt rira giai voi dung djch 50% va 20%
aceton trong DCM. T i l p theo 2,4-D duoc rCi-a giai bang 100 ml dung moi 30% MeOH trong DCM.
Djch ripa dugc boc hoi tdi khoang 1 ml dudi ap s u i t giam va sau do d u g c bdc hoi hoan toan td'i can b i n g khi N^. Can nay d u g c hoa tan trong 2 ml MeOH, loc qua mang 0,22 pm va phan tich HPLC.
Lira chgn dieu kien sac ky
Chay sac ky HPLC o cac dieu kien khac nhau.
Trong dd, cgt s i c ky la Gemini 018 (250 x 4,6 mm, 5 pm, Phenomenex, My), detector PDA 996 quet pho lie 210 -400 nm. Dung mdi pha dgng d u g c sir dung voi cac he dung moi khac nhau gdm MeOH:
acid acetic4% (40:60, v/v) va MeOH: amoni format pH 4,5 (50:50, v/v); t h l tich bom m i u la 10 pl. Tdc dp dung moi pha dong la 1 ml/phiit.
Tham djnh phu>o>ng phap phan tich Phuong phap phan tich dugc tham djnh theo yeu cau chung ciia ICH (International Conference on Harmonisation) v l tham dmh phuong phap phan tich ddi vdi cac san pham d u g c sir dung cho nguo'ii^' theo cac chi tieu' Tinh tuong thich he thdng, chgn lgc - dac higu; Khoang ndng dp t u y i n tinh; Do chinh xac; Do dung; Gid'i han phat hien (LOD) va gioi han dinh lugng (LOQ).
X u ly so lieu
Sd hgu duoc phan tich t h i n g kg tren p h i n m i m Microsoft Excel Office 2010 va Origin Pro 8.0.
K^t qua nghien cu'u
K i t qua khao sat li^a chon d i l u kien s i c ky Khao sat dieu kien sac ky vdi 2 he pha dgng nhu tren. Ket qua cho thay, he pha dgng la MeOH : amoni format pH 4,5 (50/50, v/v) cho pic sac net, can doi, thdi gian luu phii hgp. Do vay, dieu kien nay d u g c lua chgn cho nghien cicu tiep theo.
Khi sir dung cpt c h i l t pha r i n SPE, s i c ki dd (SKD) ciia m i u cd sip dung cpt c h i l t pha r i n la it tap hon, pic rd rang so vdi m i u khdng tinh c h l b i n g cgt c h i l t pha r i n (hinh 1). Do vgy, quy trinh XU" ly m i u cd sir dung cdt c h i l t pha r i n d u g c lua chpn cho nghign cipu t i l p .
2.4 D
Hinh 1 : Sic ky dd cua mau thw danh gia Iwa chon phwang phap chiit miu a) Sw dung cot SPE; b) Khdng sO dung cdt SPE
TAP CHi DUOC HQC - 2/2015 (S6 466 N A M 55) 25
Nghien CIFU - Ky thuat
Ket qua tham d m h p h u v n g phap djnh l u g n g 2,4-D
Tfnh tuvyng thich he thdng, chgn lgc - dac hieu
Phan tich s i c ky 5 l l n lign t i l p m i u chuan 2.4-D theo d i l u kien da Iga chgn d u g c d trgn. K i t qua
.._-A,/v.-.-A..
thu d u g c tho'i gian luu va dien tich pic ciia 2,4-D l l n lugt la 7,87 ± 0,07 (x" ± SD, RSD = 0,83%) va 301952,6 + 1467,9 (X ± SD, RSD = 0,49%). Kit qua cho t h i y r i n g RSD cua tho'i gian luu va di?n tich pic 2,4-D deu nhd hon 2%. Vi vay, phuong phap phan tich nay cd tinh tuong thich vdi he thong s i c ky.
A . . ,
.A_1_MA _.___..
Hinh 2: SKD the hien tinh chgn lgc - die hidu cua phuung phip a. SKD miu tring; b) SKD miu chuin; c) SKD miu thw
Phan tich dong tho'i mau chuan 2,4-D, mau t r i n g , va m i u ihir K i t qua cho thay rang, tai vi tri x u l t hign pic ciia 2,4-D trgn s i c ky dd m i u thCp, dong thdi x u l t hien pic tuong irng trgn m i u c h u i n . Tgi vi tri nay khdng x u l t hien pic tuong irng trgn m i u t r i n g . Can cir vao s i c ky d l m i u t h u ta thay pic 2,4-D tach hoan toan khdi pic lap. Pi'c can doi, s i c net. dp rpng chan pic nho.
Do vay, phuong phap nay dam bao dp chgn Ipc
va dgc hieu (hinh 2).
Khoang nong dg tuyen tinh Phan tich sac ky 3 day c h u i n 2,4-D cd ning dp tir 5-160 pg/ml theo d i l u kien da lua chon dugc. K i t qua cho thay, trong khoang khao sat, cd moi lien he tuyen tinh giu'a dien tich pic va nong dp c h i t phan tich. D u o n g h l i quy la duong t h i n g ( y = 16239x-19363) vol he so tuong quan R^gan b i n g 1 (hinh 3).
Hinh 3: £)d thi md ta mdi quan hg tuyen tinh cua dien tich pic vi ndng do 2,4-D trong khoang khao sit
26 TAP CHI DirOC HQC - 2/2015 (SO 466 f^M 55)
• Nghien CIFU - Ky thuat
Dp chinh xac
C h u i n bi c^c m i u chuan LQC (10 pg/ml).
MQC (80 pg/ml) va HQC (120 pg/ml). M l i nong dp chuan bj 5 m i u Phan tich s i c ky theo d i l u kien da lua chgn d u g c trong cung mgt ngay va sau 3 ngay de xac dinh dp chinh xac trong ngay va khac ngay. K i t qua cho t h i y do Igch chuan tuong doi d cac diem khao sat trong ngay va khac ngay deu < 2% (Bang 1). Nhu vay, dd chinh xac cua phuong phap d u g c dam bao.
Bang 1 : Kit qui dinh gli do chinh xic trong ngay va khac ngiy
LQC ll/IQC HQC
• (lOpg/ml) (30pg/ml) (120pg/ml) Trong ngay 9,62±0,ir 79,63 ±1,08* 120,34±1,82*
( X ± SD. MQ/ml) RSD = 1,14% RSD = 1.36% RSD = 1,51%
J<hacngay 9.58±0,16* 78,85±1,51* 119,1712.05*
( X ± SD, Mg/ml) RSD = 1,67% RSD = 1,92% RSD = 1,72%
•; Li ndng dd ngoai suy tw dwang chuin Dg dung
Dp diing cua phuong phap d u g c thuc hign theo phuong phap them c h u i n . Thgm cac lugng chuan xac djnh (10 pg, 200 pg va 300 pg) vao 25,0 g bdt dugc ligu. C h i l t m i u va phan tich sac ky theo quy trinh da lua chpn. Ti le phan tram cua lugng 2,4-D lim t h i y so vdi lugng them vao ban dau dugc xac djnh la do diing ciia phuong phap.
Ket qua chl ra rang, d' 3 diem khao sat. dp thu hoi cua phuong phap deu Id'n hon 85% (bang 2). Do vay, phuong phap djnh lugng nay hoan loan dap ung yeu c l u ve do diing.
Bang 2; Kit qua xic dinh do dCing cua phwang phap
Luvng them vao
ban dau 10 Ui 200 pg 300 ijg
Pi±SD,%) 86,89 ±2,9 6,67*1,8 6,87 + 1,9 Gioi han phat hien va gioi han djnh Iwgng Dga vao dp Igch c h u i n cua dap irng va dud'ng chuan ta tinh d u g c gioi han phat hign va gidi han dinh lugng d u d i . Tigm s i c ky 6 l l n m i u t r i n g , ghi lai dd nhieu dudng nen tai tho'i gian luu tuong ung voi thdi gian luu cua 2,4-D. Phan tich s i c ki mpt day chuan d l xac d|nh diem nong dp tai dd dien tich dap ipng pic cua 2,4-D bang 3 l l n va 10 lan dap irng pic mau trang tai vi tri
tuong Lfng Ket qua cho thay r i n g gidi han phat hien va gidi han dinh lugng d u o i cua 2,4-D lan lugt la 0,05 pg/ml va 0.12 pg/ml.
Ban luan
Dgc tinh cua 2,4-D da dugc ghi nhan ro rang tren co quan ligu hda, cac to chirc d u d i da, mau, gan, than, iach, tuyen giap, nao... C h i t dpc da cam de quoc My dung trong chiln tranh Viet Nam chinh la hdn hop ciia 2,4-D va 2,4,5-T (li le 1:1).
d dpng vat cd vii, 2,4-D tac dyng theo co c h l pha v d qua trinh san sinh nang luong binh thuong cua CO the b i n g each lam thoai b i l n cac phan t u ATpi^^i. N h i l u bao cao djch t l chi ra r i n g cd moi lien quan giiJa ung thu lymphoma non-Hodgkin va s u phoi nhilm 2.4-D!^!. CH mire dd phan t u , 2,4- D lam giam hoat linh ciia cac enzym loai bd goc t g do trong co thei^'; suy giam hormon t y i n giap, cac hormon sinh due nhu lestosteron. estrogen, progesterorf'^i. Nghien ciru nay mang y nghTa thuc tien cao, trgc t i l p phuc vu cdng lac kiem soat va dam bao c h i t luong d u g c lieu, phyc vy cong tac bao ve va cham sdc sire khoe nhan dan.
C h i l t m i u 2,4-D cho phan tich djnh lugng d u g c thuc hien b i n g nhieu phuong phap nhu c h i l t long - long, chilt SPE va chiet sieu loi han.
C h i l t mau idng-ldng mac du don gian, it tdn kem nhung l l n nhilu tap trong m i u phan lich ngn anh hudng den linh dac higu - chpn lgc va lam giam hieu luc cpt s i c kyi"i. Ngoai ra, do han c h l v l dp tan cua 2,4 D trong cac dung mdi nen khd dam bao dp thu hoi cua chat phan lich trong qua trinh c h i l l ' " ' . Chung tdi sir dung cpt SPE de loai bo tgp sau khi da c h i l l x u l t 2,4-D ra khdi nguyen ligu k h i c phuc d u g c cac nhuoc diem ke trgn.
Phuong phap c h i l l sieu tdi han mac dii it tgp chat nhung nd chi phat huy h i t uu d i l m trong trudng hgp loai bd t i n d u hda c h i t bao vg Ihuc vat l u d u g c lieu. Sir dyng de phuc vu chiet x u l t cho qua trinh phan tich gap phai nhuoc d i l m ldn la khdng dam bao dp thu hoi mau do qua trinh thu hdi m i u khd khan vi bj giu lai nhieu trong he t h i n g va l l n tho'i gian'^^i.
Vol c h i l t m i u b i n g SPE, cd nhilu nghign ciru sir dung cac loai col pha ran khac nhau ve ban c h i t c h i t h i p phu nhu C18-SPE, C8-SPE, cgt x6p polymer, opt carbon graphit, cot trao doi anion...I'^i. Cac nghien ciru nay cho thay dp thu hdi cua phuong phap khi s u dyng cpt c h i l t SPE
TAPCHI Di/rfr Hor- jnat^t^n±(y(. viiw « \
Nghien CIFU - Ky thuat d i u > 90% va khdng cd s u khac biet cd y nghTa thong kg vg dp thu hdi giu'a s u dung va khong s u dung cpt chiet SPE de lam giau m i u ' ^ i . Voi phuong phap SLP dung cot chilt SPE de loai tap va lam giau mau phan lich trong nghien cuu ciia chung toi cho t h i y dp thu hoi m i u chilt m i u LOQ la 86,89% ± 2,97% va cac ndng dp khac deu Idn hon 90%. Nhu vay, nghign CLPU ciia chiing tdi thing nhit vdi cac nghien ciru da cdng bo \iuac day.
Djnh lugng 2,4-D trudc day thuong d u g c thgc hign bang phuong phap sac ki khi. Tuy nhign, nhugc diem ciia nd la dp dgc higu va dp nhgy khdng cao. Do do, cac nha nghien ciru lao dan xuat cua 2,4-D v d i mpt chat khac (nhu pentafluorbenzyl bromid, cesium carbonat, trifluor bromid...) thdng qua phan Crng hda hpc de kich thich dd nhay va do dgc hieu ciia phuong phap. Voi each thuc nay 2,4-D cd the dugc phat hien d ndng do t h l p ILF 0,3 ng/ml - 1,0 ng/ml'"'.
Phuong phap cita chiing tdi khdng sir dyng cac phan ipng hda hpc de lao dan x u l t trung gian ngn gioi han phat hign cao hon so vdi cac phuong ph^p khac (50 ng/ml). Tuy nhien, theo quy dinh cua Bg Y t l Nhat Ban, tdn d u 2,4-D la khdng qua 2,0 pg/g'^; Ngoai ra, Gy ban An loan thgc p h i m ChSu Au (European Food Safety Authority) quy dinh ton d u 2,4-D trong cac qua hg cam quyt khdng d u g c qua 1 pg/g'*'. Vo'i phuong phap lam giau m i u b i n g c h i l l SPE va gidi han phat hign trong nghien ciru ciia chiing ldi, hoan loan cd the djnh lugng d u g c ton d u 2.4-D trong chi thuc n l u can cip vao cac gidi han cho phep tren.
Mpt so nghign ciru cung thuc hien dinh lugng 2,4-D b i n g HPLC. Dickovi/ L M. va cpng s u s u dung them mpt c h i t phan Crng vdi 2,4-D de tao thanh mdt d i n xult huynh quang va tin hieu dugc ghi lai bang detector huynh quang. Phuong phap nay cd gidi han phat hien la 62,5 ng/ml'^'. Chi sip dung cpt s i c ki C18 (250 x 4,6 cm; 5 [jm) va detector UV, Zheng Z. M. cung da thuc hign djnh lugng t i n d u 2,4-D trong mpt s6 loai cam, gidi hgn phat hien trong nghien ciru nay la 50 ppb'^l Nghien CU'U ciia chiing tdi cd gioi han phat hien la 50 ng/ml thong n h l l voi cac nghien cuu da cdng bo.
Ket luan
Sa xay dung va t h i m (Snh d u g c phuong phap dinh lugng l6n d u 2,4-D trong dugc ligu bang
phuong phap s i c ky long hieu nang cao. K i t qua chl ra r i n g phuong phap da xay dgng la phii hgp vdi he thdng s i c ky, dam bao do chgn Ipc dgc higu; do dung (> 85%) va dp chinh xac cao.
Tai lieu tham khao I. Bo Y le (2004). "Nghien cuu djnh ti'nh va dmh lugng hoa chit bao VG thgc vat ton du trong mot s6 dugc li^u trong va si> dyng d Viet Nam", Bao cio tdng hop ldt qua de tai cap Bg Y td.
2 A.V Mahk M. N Malik et al (1993), "Control of fruit drop in pine apple sweet orange with growth regulator", Pak. J. Agn. Sa'.. 30(3), 303-306
3. DickowL. M., GerkenD. R, Sams R.A., Ashcraft S. M (2001), "Simultaneous determination of 2,4-D and MORA in canine plasma and urine by HPLC with fluorescence detection using 9-anthryldtazomethane", J. Anal. Toxicol., 25. 35-39.
4 European Food Safety Authority (2011), "Review of the existing maximum residue levels (MRLs) for 2,4-D according to Article 12 of Regulation (EC) No 396/2005", EFSA Journal, 9(11), 2431
5. Fontana A et al (1998), "Incidence rates of lymphomas and environmental measurements of phenoxy herbicides: ecological analysis and case- control study". Arch Environ Health. 53, 384-387.
6. Geng Zhi-ming,Chen Ming et al (2007),
"Determination of 2,4-dichlorophenoxyacetic acid residue in orange using high performance liquid chromatograph", Jiangsu Journal of Agricultural Science, 01.
7. Hiroyuki Nakazawa, Nobuyuki Takahashi et al (2004), "Rapid and simultaneous analysis of dichlorvos, malathion, carbaryl, and 2,4-dichlorophenoxy acetic acid in citrus fruit by flow-injection ion spray ionization tandem mass spectrometry", Talanta, 64, 899-905.
8. ICH (2005), Validation of analytical procedures:
text and methodology, Q2R1.
9. LerdaD., R Rizzi{1991), "Study of reproductive function in persons occupationally exposed to 2,4-D'.
r^utation Research, 262. 47-50.
10. O. P de Amarante et al. (2003), "Determination of 2,4-dichiorophenoxyacetic acid and its major transfomiation product in soil samples by liquidchromatographic analysis", ra/an(a, 60,115-121.
I I . P Sairam, Somsubhra Ghosh et al (2012),
"Supercritical fluid extraction (SFE)-an overview", Asian J. Res Pharm. Sci, Vol. 2, Issue 3, 112-120
12 Palmeira C. M et al (1994), "Interactions of herbicides 2,4-D and dinoseb with liver mitochondrial bioenergetics", Toxicol. Appl. Pharmacol., 127, 50-57, 13,Tibor Cserhati, Esther Forgacs (1998),
"Rhenoxyacetic acids: separation and quantitative detemiination", Joumal of Chromatography B, 717, 157-178.
(Ngiy nhan bai: 12/12/2014 - Ngay duyet ding: 03/02/2015)
hs TAP CHI DUQC HQ*^ - tnnfi (sn dt^ti NAM 5SL^
