Xác định dư lượng một số hóa chất bảo vệ thực vật trong dược liệu khô.pdf

B Y T

TRNG I HC DC H NI

ON HNH DUNG

XC NH D LNG MT S

HA CHT BO V THC VT

TRONG DC LIU KH

KHA LUN TT NGHIP DC S

H NI - 2014

B Y T

TRNG I HC DC H NI

ON HNH DUNG

XC NH D LNG MT S

HA CHT BO V THC VT

TRONG DC LIU KH

KHA LUN TT NGHIP DC S

Ngi hng dn:

1. ThS. ng Th Ngc Lan

2. ThS. Trn Cao Sn

Ni thc hin:

Vin Kim Nghim An Ton V Sinh Thc Phm Quc Gia

H NI - 2014

i

LI CM N

Sau mt thi gian lm vic khn trng c s gip tn tnh c t

gi o, gi n ng bn b, ti on t n K lun tt ng ip Xc nh d

lng mt s ha cht bo v thc vt trong dc liu kh.

Bn kha lun n c thc hin v hon thnh ti Vin kim nghim an ton

v sinh thc phm Quc gi , 48B Tng Bt H - H Ni vi s ng dn ca Th.s

ng Th Ngc Lan v Th.S Tr n C o Sn.

Vi lng knh trng v bit n su sc, em xin trn trng cm n T .s ng Th

Ngc Lan tn tn ng dn v gip em hon thnh bn kha lun tt nghip

ny.

c bit, em xin chn thnh cm n Th.S Tr n C o Sn gi o ti v nhit

tn gip em trong sut qu trnh thc hin kha lun.

Em ng t sc cm n n , nhng ngi lm vic ti Vin kim

nghim an ton v sinh thc phm Quc gia, n it tn gip em trong thi gian

thc hin kha lun

Cui cng, ti xin gi li bit n su s n gi n , c bit l b m v li

cm n n t n n bn b, l ngun ng lc khng th thiu, lun bn cnh gip

ti sut thi gi n i c v trong sut qu trnh thc hin kha lun.

H Ni, ng t ng 5 nm 2014

Sinh vin

on Hnh Dung

ii

MC LC

LI CM N ..................................................................................................................... i

MC LC ......................................................................................................................... ii

DANH MC CC K HIU, CC CH VIT TT ................................................... v

DANH MC CC BNG .............................................................................................. vii

DANH MC CC HNH V, TH ....................................................................... viii

T VN ................................................................................................................... 1

C ng 1: TNG QUAN NI DUNG TI .............................................................. 2

1.1. Gii thiu v ha cht bo v thc vt ................................................................... 2

1.1.1. Khi nim ha cht bo v thc vt ................................................................. 2

1.1.2. Nhm Clo hu ............................................................................................. 3

1.1.3. Nhm ln hu .............................................................................................. 3

1.1.4. Nhm carbamat ................................................................................................ 4

1.1.5. Nhm Pyrethroid. ............................................................................................ 4

1.1.6. Nhm Neonicotinoid ........................................................................................ 4

1.2. M d lng ti trong dc liu...................................................................... 5

1.3. C p ng p p x nh ho cht bo v thc vt ............................................. 6

1.3.1. C p ng p p tru n thng ........................................................................ 6

1.3.2. P ng p p QuEC ERS ................................................................................ 7

1.4. K thut sc k lng khi ph .............................................................................. 10

1.4.1. Nguyn tc chung: .......................................................................................... 11

1.4.2. Sc k lng ..................................................................................................... 12

1.4.4. Khi ph ......................................................................................................... 13

C ng 2: I TNG V PHNG PHP NGHIN CU .................................. 15

2.1. i tng nghin cu ........................................................................................... 16

2.1.1. Ha cht bo v thc vt ................................................................................ 16

iii

2.1.2. i tng phn tch ........................................................................................ 19

2.2. Ni dung nghin cu ............................................................................................. 20

2.2.1. Kho s t p ng p p .................................................................................... 20

2.2.2. Thm n p ng p p ................................................................................ 20

2.2.3. ng dng p ng p p .................................................................................. 20

2.3. P ng p p ng in u ...................................................................................... 20

2.3.1. P ng p p x l mu theo QuEChERS ........................................................ 20

2.3.1.1.Nguyn tc .................................................................................................... 20

2.3.1.2.u im c p ng p p. .......................................................................... 21

2.3.2 P ng p p t m nh................................................................................... 21

2.3.2.1. Tn tng t a h thng .................................................................... 22

2.3.2.2. Gii hn pht hin, gii hn n lng ..................................................... 22

2.3.2.3. Khong tuyn tn v ng chun ............................................................ 23

2.3.2.4. lp li v thu hi. .............................................................................. 23

2.3.3 P ng p p x l s liu ............................................................................... 24

2.4. P ng tin nghin cu ........................................................................................ 24

2.4.1 Thit b, dng c .............................................................................................. 24

2.4.2. Dung mi, ha cht ........................................................................................ 25

C ng 3: KT QU V THO LUN ...................................................................... 27

3.1 Kho st xy dng p ng p p p n t HCBVTV ........................................ 27

3.1.1 Kho s t iu kin phn tch bng LC-MS/MS .............................................. 27

3.1.1.1. Kho s t iu kin khi ph ........................................................................ 27

3.1.1.2. iu kin sc k lng .................................................................................. 29

3.1.2. Kho s t iu kin tch chit HCBVTV trong dc liu kh. ..................... 31

3.1.2.1. Kho st thi gi n ngm nc ................................................................... 33

3.1.2.2. Kho st n ng ca pH thng qua so snh 3 quy trnh ....................... 34

3.1.2.3. Kho st mui chit ..................................................................................... 35

iv

3.1.2.5. n gi v i tr a ni chun trong loi tr n ng nn .................... 36

3.2. Thm n p ng p p ....................................................................................... 37

3.2.1. Gii hn pht hin v gii hn n lng .................................................... 37

3.2.2. Khong tuyn tnh .......................................................................................... 39

3.2.3. lp li v thu hi .................................................................................. 40

3.3. p dng p ng p p x n d lng HCBVTV trn 30 dc liu kh ...... 41

KT LUN V KIN NGH ......................................................................................... 44

TI LIU THAM KHO ............................................................................................... 45

PH LC ........................................................................................................................ 49

Ph l 1: ng chun HCBVTV ............................................................................. 49

Ph lc 2: Mt s s chun ha cht bo v thc vt ........................................... 53

v

DANH MC CC K HIU, CC CH VIT TT

K hiu Ting anh Ting vit

AOAC Association of Official Analytical

Community

Hip i ng ng phn tch

n t

APCI Atmospheric Pressure Chemical

Ionization

C ion p sut k

qu n

CAD Collision Gas Pressure p sut k bn p

CE Collision Energy Nng lng bn p

CUR Curtain Gas Kh mng

CV Coefficient of Variation H s bin thin

CXP Collision Cell Exit Potential T u r

DP Declustering Potential T u vo

d-SPE Dispersive Solid Phase Extraction C it p n t n p rn

ESI Electrospray Ionization Ion p un in t

FAO Food and Agriculture

Organization

T n ng ng ip v lng

t Lin p qu

GCB Graphitized Carbon Black

C rbon ot tn

GC-MS Gas Chromatography Mass

Spectrometry

S k k k i p

GS1 Ion Source Gas 1 Ngun k ion 1

GS2 Ion Source Gas 2 Ngun k ion 2

HCBVTV H t bo v t vt

HPLC High Performance Liquid S k lng iu nng o

vi

Chromatography

IS IonSpray Voltage T ion

LC-MS/MS Liquid Chromatography tandem

Mass Spectrometry

S k lng g p k i p i

l n

LLE Liquid liquid extraction K t ut it lng lng

LOD Limit of Detection Gii n p t in

LOQ Limit of Quantification Gii n n lng

MRL Maximum Residue Limit Gii n tn d ti

MS Mass Spectrometry K i p

PSA Primary Secondary Amine C t p p min b 1, b 2

R(%) Recovery Hiu sut t u i

SD Standard Deviation l un

SPE Solid Phase Extraction C it p rn

TEM Ion source Temperature N it ngun

vii

DANH MC CC BNG

Bng 1.1: Tm tt quy trnh chit xut theo 3 phin bn QuEChERS. ............................ 9

Bng 3.1. C iu kin ti u a MS .......................................................................... 27

Bng 3.2. C iu kin phn tch HCBVTV bng LC-MS/MS ................................... 27

Bng 3.3.Tm tt iu kin gr dient tch hn hp HCBVTV .................................. 30

Bng 3.4: lp li ca h thng v thi gi n lu v din tch peak ............................ 30

Bng 3.5. So s n p ng p p it v pH dch chit o c ............................ 34

Bng 3.6: LOD, LOQ ca cc ha cht bo v thc vt ................................................ 38

Bng 3.7. Kt qu p n t HCBVTV trong dc liu kh ......................................... 41

viii

DANH MC CC HNH V, TH

Hnh 1.1: M hnh h thng LC-MS/MS. ....................................................................... 12

Hnh 1.2: M hnh b phn tch t cc chp ba.............................................................. 15

Hnh 3.1. S HCBVT v chun ni TPP .................................................................. 29

Hn 3.2. S tm tt quy trnh QuEChERS kho st ................................................ 32

Hnh 3.3. n ng thi gian ngm mu trong n n hiu sut chit ................... 33

Hnh 3.4: S lng HCBVTV thu hi cc khong khc nhau .......................... 35

Hnh 3.5: S lng HCBVTV thu hi cc khong khc nhau (>80%; 70-80%

v

1

T VN

Hin nay, vn an ton v sinh thc phm ng l vn b o ng vi ngi

dn v cc cp qun l. Cc v ng c thc phm ng ng gi tng, trong ng

c ho cht bo v thc vt (HCBVTV) cng lm bng ln lo u c ngi tiu dng.

HCBVTV khng ch c s dng trong nng nghip, ng n n n c

dng trong trng v ch bin dc liu. Theo kho st thc t ti mt s vng trng cy

dc liu n t , ngi dn s dng h u ht cc loi ha cht bo v thc vt

ng lu n trn t trng hn ch su bnh, bo v dc liu. Mt ph n khng

nh s dc liu p i k , s ch t n dc liu kh l thnh ph n ch yu

trong cc bi thu ng ng n trong sn phm hin i n t c phm chc

nng.

Hin nay, Vit N m n iu nghin cu v p ng p p x nh

HCBVTV trn dc liu k . Hn n , p ng p p p n t t ng t ng ch

ng n phn tch vi cht hoc mt n m HCBVTV m x n ng thi

nhiu nhm cht trong cng mt l n p n t do t kim so t c hm

lng HCBVTV trong nhng sn phm ny.

Vi l do trn, ng t i la chn ti: Xc nh d lng mt s ha

cht bo v thc vt trong dc liu kh. Thc hin kha lun ny chng ti nhm

ti 3 m tiu n n s u:

1. Kho st p ng p p x n d lng HCBVTV trong dc liu kh bng

LC-MS/MS.

2. Thm n p ng p p x n d lng HCBVTV trong dc liu kh

bng LC-MS/MS.

3. p dng p ng p p o mt s mu dc liu kh ph bin trn th trng

2

Chng 1: TNG QUAN NI DUNG TI

1.1. Gii thiu v ha cht bo v thc vt

1.1.1. Khi nim ha cht bo v thc vt

T chc Nng nghip v Lng t c ca Lin Hip Qu (FAO) r nh

ng k on nh v HCBVTV n s u: HCBVTV l bt k hp cht hay hn

hp c dng vi m ngn nga, tiu dit hoc kim sot cc tc nhn gy hi,

bao gm vt ch trung gian truyn bnh c on ngi ho ng vt, cc b phn

khng mong mun ca thc vt ho ng vt gy hi hoc n ng n cc qu

trnh sn xut, ch bin, bo qun, vn chuyn, mua bn thc phm, nng sn, g v

sn phm t g, th n n nu i, oc hp cht p n t n ln ng vt kim

sot cn trng, nhn i tng khc trong ho trn t chng. HCBVTV

n c dng lm tc nhn iu sin trng thc vt, cht lm rng l, cht lm

k , t n n lm t qu ho ngn n rng qu sm. Cng t dng

HCBVTV cho cy trng tr ng n s u k i t u o bo v sn phm khng

b hng trong qu trnh bo qun v vn chuyn [20].

Vic phn loi ha cht bo v thc vt rt dng, c th c nhng cch phn

loi theo cu to ha hc, theo m s dng (tr su, dit c, dit nm), t eo

c tnh hay theo ngun gc (t nhin hay tng hp). Do ni dung ti l phn tch

d lng thuc bo v thc vt, v vy ha cht bo v thc vt s c phn loi theo

cu to ha hc ca n.

Cc loi ha cht bo v thc vt gm nhiu loi, mt s nhm chnh bao gm:

- HCBVTV n m Clo u

- HCBVTV n m Ln u

- HCBVTV nhm P ret roid tng p

- HCBVTV nhm Carbamat

- HCBVTV nhm Neonicotinoid

3

Ngoi r n mt s n m k n : t tr su v , n m t u tr

su sin

1.1.2. Nhm Clo hu c (organnachlorine OC)

l n ng p t u dng u tin dit su b s u in tr n

t gii t i. HCBVTV lo u l p t u n t n k i t

t ngu n t ydro p n t ydro bon v dn xut bng ngu n t lo.

Trong p n t p t n tn ti vng benzen o d vng (O, N, S),

t ng l dn xut lo mt s p t u n dip en let n, lodien,

benzen, ex n). im qu n trng p t lo l tn o, p

t ng rng n ng km n l v rt bn vng trong m i trng nn t i gi n t

ng lu di (v d n DDT t i gi n b n p n u l 20 nm, ng t b o t i

v t lu vo t sin vt qu ui t n).

i din n m n l DDT s dng p bin trong n ng nm 50 60,

s u n iu t k tng p n lindan, chlordan, thiordan, aldrin,

metoxyclor, c mp lor M d HCBVTV lo t dng dit tr mn i

vi n iu loi su i n ng do vn n im m i trng v d lng HCBVTV

trong n ng sn, s t l v u o i t on ngi v loi ng vt,

m ng n s HCBVTV lo b m s dng [5].

1.1.3. Nhm ln hu c (organophosphorus-OP)

HCBVTV nhm ln hu l ester ca acid phosphoric (H3PO4) v dn

xut. Cc hp cht p osp or c tnh rt cao nn hiu lc dit tr su hi cao v

nhanh chng, ph t ng rng, km bn vng trong m i trng do d b chuyn ha,

phn hy hoc b th p n trong m i trng kim v acid. Hp cht p osphor l

nhng cht c c, va c kh nng t l mnh, v g c cp tnh cho h th n

kinh c on ngi v ng vt. N m n t ng vo th n kinh bng cch c ch

enzym cholinesterase. Enzyme ny c tc dng phn hy acetylcholine. Khi enzyme b

4

c ch lm tng nng acetycholin h th n kin , , dn n t co git, t vong.

Nhm ny bao gm mt s hp cht n p r t ion, m l t ion, di lorvos, lorp rifos,

dimet o t, tri lorfon, edifenp os Hp cht p osphor l n m HCBVTV c

s dng ph bin nht, n ng ng n , do c tnh cao nn rt nhiu cht trong nhm

n b cm hoc hn ch s dng Vit Nam v nhiu quc gia trn th gii [5].

1.1.4. Nhm carbamat (CA)

HCBVTV nhm carbamat l cc ester ca axit carbamic (H2N-COOH) v dn

xut. Cc hp cht carb m t c tnh cao, ph t ng hp n so vi hp cht

lo v p osphor, hiu lc dit tr su hi cao, km bn do d b thy phn trong mi

trng kim v acid. Cc hp cht rb m t ng k nng t l v u c h

th n kinh c on ngi v ng vt n ng c tnh km cc hp cht p osphor.

Khi s dng, ng t ng trc tip vo men cholinesterase ca h th n kinh v c

g c ging n n m ln u . i din o n m n n : rbofur n,

carbaryl, aldicarb, fenobucarb, isopro rb, met om l[5].

1.1.5. Nhm Pyrethroid (PY).

D v kin ng im dn gi n dng bt o ( i Chrysanthenum roseum

v Chrysanthenum cineratiae) dit su b, ngi t x n ot t n

l p ret rin, t dng mn n ng d p n . k p n im ny,

ngi t tng p n iu t u tr tng t gi l p ret roid tng p.

C t n t o gi s v on ngi. N m n t dng lu n gi kn N

m trong mng t bo t n kin g n ng s dn tru n xung t n kin [5].

1.1.6. Nhm Neonicotinoid

Neoni otinoid l n m t u tr su lin qu n n ni otin s dng t

n ng nm 1980. N m t u n tn t p n nhm carbamat v nhm ln

u ; n n tn t p trn ngi. Mt s n n s dng n ng t

5

n m n v mt s bng ng o t ngu g i ng CDD (colony

collapse disorder - ri lon st gim b n) i vi mt ong. Mt s t t u n m

n n imid loprid, et miprid, t i met ox m, dinotefur n .[5].

G n b n chu u qu t n m s dng loi t u tr su

lot i nidin, imid loprid v t i metox m trong mt s trng p n t n 27

n t n vin Lin min u u. Qu t n trn r trn s mt b o

o k o C qu n An ton T p m u u, t eo loi t u tr su

trn t g ngu i o o ong mt - mt t n n t p n t i vi u

t loi t vt trn k p t gii [19].

1.2. Mc d lng ti a trong dc liu

D lng l p n n li ot t, sn p m u n v t n

p n k trong t u , tn ti trn trng, n ng sn, t, n s u mt t i gi n

di t ng sng v iu kin ngoi n . M d lng ti o p p

(MRL) l gii n ti d lng HCBVTV (t ng biu t bng mg/kg), l nng

o n t d lng t u trong mt n v sn p m n ng sn t p m m

t p n n m k ng g i o ngi s dng. MRL tn d

trn tn HCBVTV, d liu v tn d trong sn p m sn xut t eo qu

trn GAP (T n n ng ng ip tt) v lng sn p m m on ngi s dng.

Mi HCBVTV mt gi tr MRL k n u trn tng nn mu. Hn n ,

MRL mi n li k n u do s k n u v s lng sn p m v s

dng tng n . WHO v FAO qu n MRL t eo tiu un Codex, trong

qu n t o mi HCBVTV trong tng nn mu. Tu n in, in n vn n

rt n iu HCBVTV qu n v MRL. gii qu t vn n , mt s qu

gi v t trn t gii n N t Bn, C u u, M s dng gi tr MRL ung

(def ut v lue) o tt HCBVTV l 0,01 mg/kg.

6

i vi d liu v sn p m t d liu trong mt s D in n d

in Vit N m IV, d in M (USP 36) qui n gii n o p p i vi mt s

HCBVTV. Tu n in, vn rt n iu HCBVTV MRL trn d liu v t

p m nng. Do , m 0,01 mg/kg vn xem l MRL ung i

tng n [2].

Ti Vit N m t ng t 46-2007-Q B Y T qui n m gii n ti a

o p p HCBVTV trong sn p m n ng sn: o qu, r u , t t C

tiu un n u d trn tiu un Codex do ng in u Vit N m

v n gi ngu n im HCBVTV. Hin n , m 0,01 mg/kg s

p ng t ng im l n n l m m p ng p p p n t p i t

phn tch HCBVTV [1].

1.3. Cc phng php xc nh ho cht bo v thc vt

x n d lng HCBVTV n p i tri qu qu trn t it l

t n p n t r k i nn mu s u s dng mt k t ut p t in v

n lng.

1.3.1. Cc phng php truyn thng

M p ng p p p n t d lng l p n t ng t i n iu

HCBVTV trong ng mt l n t in, do k t ut it ng ng n it

ng n iu HCBVTV ng tt. C k t ut it t ng t ng n it lng

lng (LLE), it p rn, vi it p rn, it p n t n rn n iu t gi

ng in u v x dng p ng p p p n t HCBVTV.

P ng p p p n t d lng HCBVTV u tin v ni ting n t l

p ng p p Mills p t trin t n ng nm 1960 bi C qun l d v t

p m M (FDA) bi n P.A.Mills. Vo t i gi n , t u tr su n m

lo u k ng p n l n m n p n t . Vi p ng php Mills, cc

thu tr su u v HCBVTV k ng p n u it xut t loi

7

t p m k ng t bo vi etonitril, s u p long vi n , v

t u BVTV p n vo 1 dung mi kh ng p n (petroleum et er) [10].

Nm 1975, Miton Luke v ng s ti p ng t ng im ti Los Angeles,

C liforni t u i t p m v d p m M gii t iu 1 p ng p p mi

p n t d lng HCVTV trong r u qu, ng v n iu mu t p m k .

P ng p p n tr nn rng ri n v bit n vi i tn p ng p p

Luke, o t u i o vi n iu n m HCBVTV ti ng mt t i im (n

n m lo u , p osp or u ). Tu n in p ng p p Luke vn n n iu n

: n s dng dung m i u lo, v u phn tch cc HCBVTV t

p n . Hn n d it ui ng trong dung m i ng dng p n t GC,

n t , p ng p p n k p dng vi mt s HCBVTV k b i o

k ng bn n it o n rb m t, neoni otionid [22].

Mt s ng in u k s dng etonitril o et l et t it HCBVTV

r k i nn mu. D it n v n trong dung m i t p v t

p n t . Tu n in, d it t ng k n iu tp t, do p ng p p

ny thng p i p t m qu trn lm s t ng qu s k t, it p rn

(SPE), s k t m t u gel (GPC)

Nm 2005, ti Vit Nam, T S. Tr n Vit Hng ng t in ng in u x

n HCBVTV trong d liu bng k t ut it nng, it ln o it

S ox let, kt p vi s dng t it p rn (SPE) lm s v lm giu mu v

p n t bng s k k [6].

1.3.2. Phng php QuEChERS

Nm 2003, An st ssi des v ng s [10] gii t iu mt p ng p p mi

p n t d lng HCBVTV, s u n gi l p ng p p QuEC ERS (vit tt

qui k nhanh, easy d, e p r, effe tive iu qu, rugged - n n v s fe

n ton). QuEC ERS l mt p ng p p p n t d lng t u tr su trong

8

n iu loi nn mu k n u, n k ong 70 100% n trong t n p n

(mu k o t m n ). N tn gi n, un b mu t eo QuEC ERS

n iu t un li so vi p ng p p tru n t ng k m vn m bo kt qu

p n t . P ng p p QuEC ERS b n u b o o u tin nm 2002 ti i

ng p n t d lng C u u bi An st ssisdes, Le ot v ng s. Nm 2005,

Le ot v ng s [21] ng in u t m n p ng p p n o t p ng

p p n o kt qu tt vi 207 t trong s 235 t u tr su trn nn mu r u

x l , n o v m; tu n in n ng t n vi pH b n ng r rt. S u

Le ot v ng s t i p ng p p g bng s dng m et t pH 4,8-

5,0 tng t u i t u tr su. P ng p p n s u ng in u

t ng im lin p ng trong 13 p ng t ng im 7 qu gi i vi 30 t u tr su

trn nn mu v tr t n p ng p p n t AOAC 2007.01 [11] vo nm

2007. Cng t i gi n , An st ssi des v ng s Stuttg rt, ng p t trin mt

phng p p QuEC ERS k s dng m itr t pH k ong 5. P ng p p n

t m n lin p ng n iu p ng t ng im v nm 2008 tr t n

p ng p p u u CEN 15662 [17]. S u n , n iu p in bn n p ng

p p QuEC ERS p t trin, gm vi b sung t m C18 vo PSA; o b

sung GCB i vi mu r u x n .

P ng p p QuEC ERS p i t gii n p t in (LOQ) < 0,01 mg/kg

i vi t p n t v k ong tu n tn t n 5 mg/kg t tng t v

t it b s dng. V bn t, t p p PSA t lu gi t u tr su

n m rbox li n 2,4-D v aminozid. Vi mu n iu bo, t u i

n iu t b gim k i m lng bo tng; do o t m t p p C18 v i

tr k ng k i vi mu n iu bo. GCB t dng lm s sterols,

lorop ll, v t u tr p ng. GCB do v ng lu gi t u tr su

u tr p ng (n t i bend zole, terbufos, quitozene, ex lorobenzene). S

9

dng 7,5 mg GCB vi mi ml s o t u i p n n ( ~ 70%) vi t u

tr su dng p ng trong k i loi ~ 90% lorop ll [22].

Bng 1.1: Tm tt quy trnh chit xut theo 3 phin bn QuEChERS

Bc QuEChERS ban

u 2003 [10]

P ng p p m acetat

AOAC 2007.01 [11]

P ng p p m citrat

EN 15662 [17]

Chit - Cn 15g mu

trong ng ly tm

50ml

- Cho 15ml

acetonitril lc

- Thm mui chit:

6g MgSO4+ 1,5g

NaCl. Lc xoy 1

pht

- Ly tm

- Cn 15g mu trong ng

ly tm 50ml

- Cho 15ml acetonitril

1% acid acetic, lc

- Thm mui chit: 6g -

MgSO4 + 1,5g Na

acetate. Lc xoy 1 pht

- Ly tm

- Cn 15g mu trong ng

ly tm 50ml

- Cho 15ml acetonitril

lc

- Thm mui chit: 6g

MgSO4 + 1,5g NaCl.

Lc xoy 1 pht

- Ly tm

Loi tp

bng d-

SPE

- Chuyn 1ml dch

chit vo ng d-

SPE

- d-SPE cha:

150mg MgSO4 +

50mg PSA

- Chuyn 1ml dch chit

vo ng d-SPE

- d-SPE cha: 150mg

MgSO4 + 50mg PSA

- Chuyn 1ml dch chit

vo ng d-SPE

- d-SPE cha: 150mg

MgSO4 + 50mg PSA

Phn tch bng LC-MS/MS hoc GC-MS(/MS)

S k lng k i p 2 l n (LC-MS/MS) v s k k k i p 2 l n (GC-

MS/MS) l n ng p ng tin u iu p i p vi QuEC ERS. Tu n in, vn

on ton t p i p QuEC ERS vi LC, GC s dng dete tor k . N ng

10

do etonitril trong d it, p ng p p n k ng t s dng o GC-NPD

o dete tor b n ng bi lng nit trong p n t. Ngoi r ng t s

dng ethylacetat t v etonitril p p vi t it b n .

Ng n , p ng p p QuEC ERS trin k i trn n iu i tng

khc nhau n , ng ; v ng ng dng p n t p t k ,

n d lng t u t , r l mids, PAHs

Nm 2011 ti Trung Qu tin n p dng QuEC ERS x n

HCBVTV trong d liu k n Trung Qu , G ng Du, Yuelin Song, Yit o Wang

tin n x n d lng 55 HCBVTV trong Traditional Chinese Medicines

(TCM) t m t t (Panax notoginseng), ong k M ng C (Astragalus

mongholicus) v n sm (Salvia miltiorrhiza) bng GC-MS [16].

S u vo nm 2012, ng mt s n kim ng im k tip t tin n

x n d lng 116 HCBVTV trong d liu bng t it b UPLC-MS/MS [14].

Nm 2012, Em d R m d n Att ll v ng s tin n t m n p ng

p p QuEC ERS x n d lng 71 HCBVTV t u n m p osp o u ,

nito u , lo u v p ret roid trong d liu k bng p ng p p GC-NPD

v GC-ECD [12].

1.4. K thut sc k lng khi ph

S k lng k i p l k t ut mi, tu n in n iu t gi ng dng

x n d lng HCBVTV trn n iu i tng mu k n u. l p ng

p p n iu u im n [13].

- C nh o, c bit l sc k lng khi ph hai l n (LC-MS/MS) rt

ph hp p n t d lng.

- C kh nng t t d t eo m/z, do ng vi kh nng t

cht qu HPLC t p ng p p n t phn t ng thi rt nhiu

HCBVTV trong cng mt l n chy.

11

- C tnh chn lc cao, vi kh nng x nh cc cht da vo khi lng v

cu to ca cht nn rt trng o tng cht.

- C kh nng p n t c nhng cht khng th phn tch bng sc k kh,

n t km b i, t khng bn nhit.

Vi n ng u im n v , LC-MS/MS n iu t gi ng in u

p n t HCBVTV bit p p k i p i p vi k t ut QuEC ERS x l

mu. Nm 2003, k i An t ssi des v Le ot l n u tin ng b p ng p p

QuEC ERS o t k nng p i p p ng p p n vi s k lng

k i p v s k k k i p .

Nm 2007, Anastassiades v cng s ng dng k thut QuEChERS tch 80

loi thuc tr su thuc cc nhm ha hc khc nhau t cc sn phm m lng

lipid thp. Hn hp 38 thuc tr su chit t chanh gai, nho kh, bt m v d ut

c phn tch bng GC-MS/MS. Hn hp 42 thuc tr su cn li chit t m, ru

v ng , n o , nho kh v bt m c phn tch bng LC-MS/MS. Cc HCBVTV

la chn cho nghin cu ny bao gm nhng cht t ng xuyn pht hin vt qu

mc gii hn d lng ti (MRL) trong t c phm. Mc thm chun cho cc th

nghim kim tr thu hi l: 0,005; 0,01; 0,02 v 0,2 mg/kg phn tch GC-

MS/MS, v 0,01 v 0,1 mg/kg cho phn tch LC-MS/MS. Kh nng p t in v nh

lng ca cc HCBVTV cao ngay c khi nng thp, t l tn hiu trn nhiu ln,

n x o. thu hi trong khong 70-110% (trung bn trn 98%); lch chun

tng i ni ung di 10% (trung bnh trn 4,3%). Da trn nhng kt qu ny, cc

p ng p p c chng minh l k thut hiu qu v mnh m, ph hp gim

st vic tun th MRL thuc tr su ca mt lot cc sn phm nng nghip [23].

1.4.1. Nguyn tc chung:

V bn, s k lng k i p l p ng p p s k lng s dng b p n

p t in l dete tor k i p :

12

S k lng l qu trn t x r trn t t vi p tn l t rn v p

ng l t lng (s k lng - rn). K i tin n s k, t p n t

p n b lin t gi p ng v p tn . Trong n p t p n t , do

u tr p n t v tn t l o t k n u, nn k nng tng t

ng vi p tn v p ng k n u. Do v , ng di u n vi t k

n u v t r k i n u.

K i p l t it b p n t d trn s x n k i lng p n t

p t bng vi p n t ion p n t t eo t s gi k i lng v

in t (m/z) ng. C ion t to r bng t m bt in t

ng n loi b ele tron, proton ,... C ion to t n n t t eo t s

m/z v p t in, t t o t ng tin v k i lng o u tr p n t

p t.

Mt s tm tt m n t ng LC-MS trn b hnh 1.1.

Hnh 1.1: M hnh h thng LC-MS/MS.

1.4.2. Sc k lng (HPLC

* Pha tnh trong HPLC [3], [4]

Trong HPLC, p tn n l t nhi ct lm nhim v tch hn hp cht

p n t . l n ng cht rn, xp v k t c ht rt nh, t 2 - 5m. Tu theo

bn cht c p tn , trong p ng p p sc k lng pha lin kt t ng chia lm 2

loi: sc k pha thun (NP-HPLC) v s k p o (RP-HPLC)

Sc k lng Ion ha B phn tch

khi

Detector/ Lu

gi s liu

Chn khng

13

- Sc k pha thun: p tn b mt l cc cht phn c ( l sili

tr n ho sili c gn cc nhm alkyl c t cacbon mang cc nhm chc phn

cc: -NH2, -CN...).

- S k p o: p tn t ng l sili c ankyl ho, khng phn

cc, loi thng dng nht l C18H37.

* Pha ng trong HPLC [3], [4]

P ng trong HPLC ng gp mt ph n rt quan trng trong vic tch cc

cht phn tch trong qu trnh sc k nht nh. C th i p ng lm hai loi:

- P ng phn cc: c thnh ph n ch yu l n , tu n in phn tch

cc cht hu , n t m dung m i k gim phn c n met nol,

acetonitril. P ng loi n c dng trong sc k pha lin kt p o.

- P ng khng phn cc: bao gm cc dung mi t phn c n

xyclopentan, n-pentan, n-heptan, n-hexan, 2-chloropropan, cacbondisulfua (CS2),

chlorobutan, CCl4, toluene....

S k p o s dng p bin do tn sn v t g i ti m i

trng so vi s k p t un s dng dung m i k ng p n .

Tu n in p ng mt thnh ph n i k i k ng p ng c kh nng ra

gii, ngi t t ng phi hp 2 3 dung m i dung m i phn cc

t thp n cao ph hp vi php phn tch. S t i thnh ph n p ng theo thi

gian gi l ra gii gradient nng .

P ng trong LC-MS/MS khng ch ng v i tr t t m cn gp

ph n vo kh nng ion o a cht. C ng ng n ng yu c u o n v tinh

khit, m lng cc tp cht. C nhng loi p ng c sn xut ring cho LC-

MS/MS.

1.4.4. Khi ph (Mass Spectrometry - MS)

14

Cu to ca mt thit b khi ph bao gm 3 ph n chnh: ngun ion, thit b

phn tch v b phn pht hin. Trc ht, cc mu c ion ha trong ngun ion, sau

vo b phn phn tch khi tch cc ion theo t s m/z. Cc tn hiu t u c

s chuyn vo m tn x l v lu tr [13].

* Ngun ion: Cht phn tch sau khi ra khi ct tch s c dn ti ngun ion

chuyn thnh dng i v c ion ha nguyn t. H i k t ut ion t ng c s

dng trong sc k lng khi ph l ion p un in t (electrospray ionization

ESI), ion ha ha hc p sut kh quyn (atmospheric-pressure chemical ionization

APCI). Trong nghin cu ny chng ti s dng k thut ion o p un in t (ESI).

K t ut ESI chuyn ha cc ion t dung dch lng thnh cc ion dng kh.

Dung dch mu c dn vo vng trng in t mn c duy tr hiu in th

khong 1 5 kV. Ti , dung dch mu b chuyn thnh cc git nh t in v

t tn in ti li vo ca thit b phn tch khi ph. Cc git nh trc khi

vo thit b phn tch khi ph s c kt hp vi dng k k lm b i dung

mi. C 2 ch bn ph: bn ph vi ch ion dng (ESI+) v ion m (ESI-).

ESI l k t ut ion ha mm, nh o. K t ut ny ng dng phn tch

cc cht khng phn c n : protein, peptid, bon dr t, nu leotid, pol ethilen

glycol,... v cc cht phn cc c khi lng phn t nh.

* B phn phn tch khi: S u k i ion o , ion n b

p n t k i n m loi b n ng ion k ng n t it, l n ion p n t, t

in bn p t m t u ion on. C k t ut p n t k i s dng

p bin l b p n t t , b p n t b ion, b p n t t i gi n b . B

p n t k i b t s dng p bin trong k t ut LC-MS/MS v

n l k t ut ng t i s dng trong ng in u ny.

T u to bi 4 t n in song song to t n mt k ong trng

ion b qu . Mt trng in t to r bng s kt p gi dng mt

iu (DC) v in t t n s r dio (RF). C t ng v i tr n mt b l

15

k i. K i mt trng in t p vo, ion u n ng trong n s d o ng

p t u vo t s gi m/z v trng RF. C n ng ion t s m/z p p mi

t i qu b l n .

B p n t k i b t gm b t g p ni vi n u ( nh 1.2). Trong

, t t n t (Q1) n im v t ion, l n ion m vi m/z n t n

t ngun ion u n n u n n Q2. t t i (Q2) ion p n t b

phn li do va chm vi k tr mt n k N2, Ar, He v mt nng lng bn p

k ong 10 50 eV. B Q2 to r p n l do ion m b p n mn tip t eo to r

ion n n, ion on (d ug ter ions). Q2 k ng ng v i tr l b l ion m n

p n n tt ion do Q1 u n n. S u tt ion on u n qu

b t Q3. B t t b (Q3) u to ging Q1lm n im v t ion

u n t Q2 i ti b p n p t in.

.

Hnh 1.2: M hnh b phn tch t cc chp ba

* B phn pht hin: S u k i i r k i t it b p n t k i, ion

ti p n ui t it b k i p l b p n p t in ion. B p n p t in o

p p k i p to r mt tn iu ion tng ng t ele tron t p

k u i o to r mt dng do in t di u n.

B phn pht hin nhn electron l mt trong nhng detector ph bin nht, c

nhy cao. Cc ion sau khi qua Q3 s p vo b mt diod lm bt ra cc electron.

Cc electron th cp s u c dn ti cc diod tip theo v s to ra electron th

cp nhiu n na, to thnh dng cc electron. C n vy tn hiu c khu i

v c ghi li. Tn hiu ny t l thun vi lng ion do t l vi nng cht

p n t b n u.

Q1

Lc khi

Q2

Bn ph

Q3

Lc khi

Q0

16

Chng 2: I TNG V PHNG PHP NGHIN CU

2.1. i tng nghin cu

2.1.1. Ha cht bo v thc vt

i tng ng in u l 25 HCBVTV v t un ni trip en l p osp t (TPP).

, vi l n HCBVTV trong ng in u n d trn s mt kt qu

k o s t HCBVTV trong d liu ti v t p m nng ngun g d

liu [7], [8]. Kt qu k o s t n o t HCBVTV n xut in trong

mu trn Vit N m.

Bng 2.1. Mt s HCBVTV c nghin cu trong kha lun

STT HCBVTV CTPT; K i lng

p n t (M) (g/mol)

Cu tr Nhm

HCBVTV

1. Acetochlor

C14H20ClNO2

269,76

Chloroaceta-

nilides

2. Aldicarb

C7H14N2O2S

190,26

Carbamat

3. Atrazine

C8H14ClN5

215,68

Clo u

4. Azoxystrobin

C22H17N3O5

403,39

Strobin

5. Abamectin

(Avermectin)

C48H72O14 (B1a)

C47H70O14 (B1b)

Macrocyclic

Lacton

17


p n t (M) (g/mol)

Cu tr Nhm

HCBVTV

6. Acetamiprid C10H11ClN4

Neonicotinoid

7. Carbaryl

(Sevin)

C12H11NO2

201,22

Carbamat

8. Carbofuran

C12H15NO3

221,25

Carbamat

9. Carbendazim

C9H9N3O2

191,19

Benzimidazole

10. Carboxim C12H13NO2S

235,30

Carboxamid

11. Dichlorvos

C4H7Cl2O4P

220,98

Ln u

12. Dimethoate

C5H12NO3PS2

229,26

Ln u

13. Edifenphos C14H15O2PS2

310,36

Ln u

18


p n t (M) (g/mol)

Cu tr Nhm

HCBVTV

14. Emamectin C49H75NO13

886,12

Macrocyclic

Lacton

15. Fenobucarb

C12H17NO2

207,27

Carbamat

16. Imidachlorprid

C9H10ClN5O2

255,661

Neonicotinoid

17. Indoxacarb

C22H17ClF3N3O7

527,83

Carbamat

18. Isoprocarb

C11H15NO2

Carbamat

19. Methiocarb

C11H15NO2S

225,31

Carbamat

20. Methomyl

C5H10N2O2S

162,21

Carbamat

19


p n t (M) (g/mol)

Cu tr Nhm

HCBVTV

21. Profenophos

C11H15BrClO3PS

373.64

Ln u

22. Propoxure

C11H15NO3

209,24

Carbamat

23. Terbuconazole

C16H22ClN3O

307,81

Azole

24. Thiamethoxam

C8H10ClN5O3S

291,71

Neonicotinoid

25. Trichlorfon

C4H8Cl3O4P

257,44

Ln u

26. TPP (IS) C18H15O4P

326,28

Ni un

2.1.2. i tng phn tch

D liu k s dng k o s t v x dng p ng p p l d t

canh v l giang.

ng dng p ng p p p n t HCBVTV trn 30 d liu k l

ti im k n u b o gm:

- C Nin Hip, u n Gi Lm, H Ni (12 mu k iu G01-G12): n ru

ng , ngt, mu, n n tr n, rng , kim tin t o, trin n

hong ung, b ng n , i x , ngi u, o s , t ng k sin .

20

- P Ln ng, qun Hon Kim, H Ni (14 mu k iu L01-L14): n ,

ngt, m , r u m , o n i, k sm, x , xu , b , n ni,

vng, d , si n m, trin n ong cung.

- X Hi L , u n Hi Hu, tn N m n (4 mu k iu H01-H04): dy

t n , in lng, kin gii, dip u.

2.2. Ni dung nghin cu

2.2.1. Kho st phng php

- K o s t iu kin MS

- K o s t iu kin LC

- K o s t iu kin x l mu

2.2.2. Thm nh phng php

- Tn tng t t ng

- Gii n p t in LOD

- Gii n n lng LOQ

- K ong tu n tn

- lp li

- t u i

2.2.3. ng dng phng php

p dng p ng p p mi x dng x n d lng 25 HCBVTV trong

30 d liu k lu n trn t trng.

2.3. Phng php nghin cu

2.3.1. Phng php x l mu theo QuEChERS

2.3.1.1.Nguyn tc

21

P ng p p QuEC ERS d trn it mt l n bng etonitril ( o

etonitril m) v t k i n trong mu bng p n b lng lng n

mui m gie sulf t (MgSO4). Qa trn lm s bng it p n t n p rn (d-SPE)

dng loi cid u , n n d v t n p n k n p i p

t p p min b 1 b 2 (PSA) v MgSO4;( Nu n, C18 v GBC s dng

l n lt loi t bo v olorop ll); d it t bng s k v

p n t bng k t ut k i p .

i tng mu p n t trong lun vn n l d liu k , l mt nn mu

tng i p tp do pH dng, d it n iu lorop l. Do n

p ng p p t it mu t p gim n ng nn mu, tr n lm bn

dete tor, tng k nng p t in. P ng p p QuEC ERS tu mi p t trin

n ng t in n ng u im vt tri so vi p ng p p k c trong phn

t d lng HCBVTV.

2.3.1.2.u im ca phng php [10].

Nhanh (vi 10 mu c th tin hnh x l mu trong 30 pht)

n gin, d thc hin (t tn thit b, dng c n gin, ti thiu c ngun

gy sai s)

t tn km cho qu trnh phn tch (chi ph x l mu khong 100 ng n ng/1

mu).

Tng nhy c p ng p p do dung dch chit khng b pha long nhiu

l n.

n nh

An ton o on ngi v m i trng

Dch chit s u k i c x l bng p ng p p QuEC ERS s i y bng

thit b LC-MS/MS.

2.3.2 Phng php thm nh

22

2.3.2.1. Tnh ph hp ca h thng [2]

C p p t tn p p t ng l p n k ng t t iu mt

p ng p p v dng m bo t ng s k iu nng p p. Hiu

l biu kin, t s p n b k i lng, lu gi t i v s i xng l n ng

t ng s t ng dng n gi iu nng t. C b p n u t n

m p i k nng p ng u u v m k i tin n p p t n

lng.

Nu k ng dn g k trong u n lun t t ng p i t u

u s u :

H s i xng pi n p i trong k ong 0,8 - 1,5, tr k i dn

k trong u n lun. Yu u n p dng rng ri o p p t v n

lng trong u n lun.

l un ti p p o l n tim lp li dung d i iu

kh ng vt qu tr gi qu n trong d in. Yu u n p dng

o p p n lng v k ng p dng o p p t tp t lin qu n.

Tr k i dn ring trong u n lun, l un tng i ti

p p o l n tim lp li l 2,0 %. i vi k i p l un tng i

p n n 5%

2.3.2.2. Gii hn pht hin, gii hn nh lng [9]

Gii hn pht hin LOD: l nng thp nht ca cht phn tch trong mu c

th pht hin n ng t n lng c.

Gii hn nh lng LOQ: l nng ti thiu ca mt cht c trong mu th

m ta c th n lng v cho kt qu chm mong mun.

X nh LOD, LOQ da trn t l tn hiu nhiu ng (S/N): Phn tch mu

thm chun nng thp cn c th xut hin tn hiu ca cht phn tch (n=4). Xc

nh t l tn hiu chia cho nhiu (S/N = Signal to noise ratio).

LOD l nng m ti S/N = 3.

23

LOQ l nng m ti S/N = 10.

trong : S l chiu cao tn hiu ca cht phn tch

N l nhiu ng nn

2.3.2.3. Khong tuyn tnh v ng chun [9]

Khong tuyn tnh ca mt phng php: l khong nng s ph

thuc tuyn tnh gi i lng o v nng cc cht phn tch.

ng chun: l ng biu din s ph thuc tuyn tnh gi i lng c

o v nng cc cht phn tch.

x nh khong tuyn tn ngi ta thc hin o dung dch chun c

nng t i v kho st s ph thuc ca tn hiu vo nng . S u v ng

cong s ph thuc gia din tch peak t u c vo nng , quan st s ph thuc

o n khi khng cn tuyn tnh.

C th xy dng ng chun trn nn mu thc c s dng ni chun, nhm

m loi tr n ng ca nn mu n kt qu phn tch. Ni chun c thm

vo dung dch chun o m , vi nng ph hp v ging nhau (CIS). V ng

cong ph thuc gia t l tn hiu cht ngoi chun v chia cho ni chun (trc tung y)

ph thuc vo nng ngoi chun (trc honh x).

2.3.2.4. lp li v thu hi [9]

lp li ( n gi chm) l m g n nhau ca cc gi tr ring l ca

p p o lp li v c biu din bng lch chun S hay h s bin thin

CV(%):

1

1

2

N

xx

S

N

i

i

Trong :

xi : Nng tn c ca l n th nghim th i

x : Nng trung bn tn c ca N l n th nghim.

N : S l n th nghim.

CV(%) = 100.x

S

24

ng l m g n n u gi tr p n t vi gi tr t o gi tr

p n n. ng l k i nim n tn v biu din n lng di

dng t u i (re over ). thu i ( n gi ng) l t l p n trm gi gi

tr t u so vi gi tr l t u t.

R(%) = C

Cc .100

Trong :

R: t u i (%)

C : Nng t p n t trong mu trng t m un (ng/ml).

Cc : Nng chun thm (l thuyt) (ng/ml).

x n lp li v thu hi c p ng p p p n t , ng t i tin

hnh th nghim thm chun trn nn mu trng 3 mc nng khc nhau (mi mc

lm lp li 6 l n) v tnh ton kt qu theo cng thc trn.

2.3.3 Phng php x l s liu

C kt qu tn to n t ng t eo p n mm p n t t it b (p n

mm An l st 1.5.1, ABS iex). X l kt qu bng p n mm Mi rosotf Ex el.

2.4. Phng tin nghin cu

2.4.1 Thit b, dng c

a. Thit b

H thng sc k lng khi ph khi ph hai l n (LC-MS/MS) gm my sc k lng

LC-20ADXR ca Shimadzu, Nht v my khi ph ba t cc ABI 5500 ca hng

ABSciex, M.

Ct sc k lng XBridge BEH C18 (100 mm x 4,6 mm i.d., c ht 2,5 m) v tin

ct XBridge C18 (20 mm x 3,9 mm i.d. c ht 3,5 m) ca hng Waters, M.

My lc vortex, IKA

25

M ng nht mu HR1843, Phillips

My li tm Z383K, Hermle

Cn phn tch XT22A, Precisa.

Cn k t ut ( chnh xc 0,01g), Precisa.

b. Dng c

Pipet: 1, 2, 5, 10, 20 ml.

Bn nh mc: 5, 10, 50, 100, 250, 1000 ml.

ng li tm 50 ml, 15 ml

Vial loi 1,8 ml.

Micropipet loi 100 l, 200 l, 1000 l, 5000 l v u cn.

2.4.2. Dung mi, ha cht

Cc loi ho cht s dng u thuc loi tinh khit.

o Cc cht chun HCBVTV acetochlor, aldicarb, atrazin, azoxystrobin,

acetamiprid, carbaryl, carbofuran, carbendazim, carboxin, dichlorvos,

dimethoat, edifenphos, emamectin, fenobucarb, imidachlorprid, indoxacarb,

isoprocarb, methiocarb, methomyl, profenophos, propoxure, terbuconazole,

thiamethoxam, trichlorfon ca hng Dr. Ehrenstorfer. Cc cht chun tinh

khit trn 95%.

o Cht ni chun triphenyl phosphat (TPP) ca hng Sigma Aldrich.

o Cc dung mi loi dng cho sc k gm methanol, acetonitril c Mer k, c.

o Cc ha cht CH3COOH, MgSO4 khan, CH3COONa, di-natri citrat sesquihydrat

v tri-natri citrat dihydrat khan ca Merck

o Cht hp ph PSA (primary secondary amines), GCB (graphite carbon black)

c cung cp bi Agilent.

o Nc ct 2 l n t u c t h thng ct nc Halminton.

26

* Chun b dung dch chun:

- Dung dch chun gc 1000 mg/l: Cn chnh xc khong 25 mg cht chun l n

lt ca 25 thuc tr su, ho tan cho vo tng bn nh mc 25ml v nh m n

vch bng acetonitril c cha 1% axit acetic. Chun g c bo qun t 0 5C v

c hn s dng 1 nm k t ngy pha.

- Dung dch chun hn hp 25 cht trung gian 10 mg/l: ly chnh xc 1,00 ml

dung dch chun gc ca mi thuc tr su 1000 mg/l o vo bn nh mc 100 ml

v nh m n vch bng acetonitril c cha 1% axit acetic.

- Dung dch chun hn hp 25 cht lm vic 1 g/l: ly chnh xc 1,00 ml dung

dch chun hn hp trung gi n 10mg/l o vo bn nh mc 10 ml v nh m n

vch bng Acetonitril c cha 1% axit acetic

- Dung dch chun hn hp 3 cht lm vic 100 g/l: ly chnh xc 1,00ml dung

dch chun hn hp lm vic 1 g/l o vo bn nh m 10ml v nh m n vch

bng acetonitril c cha 1% axit acetic

* Chun b dung mi pha ng:

- Kn A: id eti 1% trong nc.

- Knh B: methanol

27

Chng 3: KT QU V THO LUN

3.1 Kho st xy dng phng php phn tch HCBVTV

3.1.1 Kho st iu kin phn tch bng LC-MS/MS

3.1.1.1. Kho st iu kin khi ph (MS)

S dng ngun ion ESI, ion dng, t ion l 5000V. Mi

HCBVTV bn p ion p n t t u i mn ion on, mt dng n lng

v mt dng k ng n . C iu kin ion p n t, ion sn p m v nng lng

bn p ti u x n bng tim n p un HCBVTV nng 100

ng/mL tr tip vo MS v s dng p n mm An l st n gi . C kt qu

tm tt bng 3.1 v bng 3.2.

Bng 3.1. Cc iu kin ti u ca MS

IS (V) 5500

TEM (0C) 450

GS1 (psi) 25

GS2 (psi) 15

CUR (psi) 20

CAD (psi) 7

Bng 3.2. Cc iu kin phn tch HCBVTV bng LC-MS/MS

HCBVTV Ion phn t

(m/z)

Ion nh lng

(nng lng bn

ph)

(m/z(eV))

Ion nh tnh

(nng lng bn

ph

(m/z(eV))

Thi gian

lu

(pht)

Acetochlor 270,0 224 (13) 148 (25) 10,9

Aldicarb 213,0 116 (15) 89 (15) 8,15

Atrazine 216,0 96 (31) 104 (37) 9,67

Azoxystrobin 404,0 372 (19) 344 (31) 9,92

28

HCBVTV Ion phn t

(m/z)

Ion nh lng

(nng lng bn

ph)

(m/z(eV))

Ion nh tnh

(nng lng bn

ph

(m/z(eV))

Thi gian

lu

(pht)

Avemectin 890,5 305 (31) 567,5(17) 14,2

Acetamiprid 223,0 126 (25) 56 (19) 7.03

Carbaryl 202,0 145 (13) 127 (37) 9,14

Carbofuran 222,0 165 (15) 123 (29) 8,86

Carbendazim 192,0 160 (23) 132 (39) 4.05

Carboxim 236,0 143 (19) 87 (29) 9,12

Dichlorvos 221,0 109 (23) 127 (18) 8,83

Dimethoat 230,0 199 (13) 125 (27) 7,12

Edifenphos 311,0 283 (17) 109 (35) 11,1

Emamectin 886,5 158 (39) 302 (35) 10.9

Fenobucarb 208,0 152 (11) 95 (19) 10,2

Imidachlorprid 256,0 209 (21) 175 (27) 6,43

Indoxacarb 528,0 249 (21) 293 (17) 11,3

Isoprocarb 194,0 95 (19) 137 (11) 9,63

Methiocarb 226,0 169 (11) 121 (23) 10,3

Methomyl 163,0 88 (13) 106 (13) 5,05

Profenophos 373,0 303 (23) 345 (17) 12,0

Propoxur 210,0 111 (19) 93 (31) 8,79

Terbuconazole 308,0 125 (39) 151 (31) 11,1

Thiamethoxam 292,0 211 (15) 181 (29) 5,38

Trichlorfon 257,0 109 (23) 221 (15) 7,11

TPP (IS) 327,0 77 (61) 11,1

29

3.1.1.2. iu kin sc k lng (LC)

T eo ng in u tr mt n m t gi ng trn t it b LC-MS/MS ny,

tin n k o s t iu kin LC-MS/MS [7], [8]. Trn s , ng t i tin n

k o s t v t u kt qu n bng 3.3. V n 3.1 l s t p n t

v t un ni TPP p n t r rng iu kin m

Hnh 3.1. Sc HCBVT v chun ni TPP

Methomyl

Thiamethoxam

Carbendazim

Imidaclomirid 1

Trichlofon

Imidaclomirid 2

Dimethoat

e

Aldicarb

Propoxur

Dichlovos

Carbofuran

Carboxim

Carbaryl

Isoprocarb

Atrazine

Azoxytrobin

Methiocarb

Acetochlor

Edifenphos 1

Edifenphos 2

Indoxacarb

TPP

Profenophos

Avermectin

30

Bng 3.3.Tm tt iu kin gradient tch hn hp HCBVTV

Thi gian

(pht)

T dng

(mL/pht)

Knh A:

CH3COOH 0,1 %

Knh B:

Methanol

0,01 0,7 75 25

1,00 0,7 75 25

8,00 0,7 10 90

12,00 0,7 10 90

12,01 0,7 75 25

15,00 0,7 75 25

3.1.1.3. nh gi s ph hp ca h thng

Sau k i k o s t iu kin LC-MS/MS, ng t i ng tin n n gi

lp li t ng HPLC MS/MS. Qu trn tin n bng tim 6

l n mu trng d t n t m un m 0,05 mg/kg v x n l

un din t pe k v t i gi n lu 24 HCBVTV. Kt qu o t l

un v din t peak u n n 2,5%, n l un v t i gi n lu rt t p,

u n n 0,18%. N v kt qu m bo u u v l un v t i gi n

lu p i n n 1% v v din tch peak p i n n 5%.

Bng 3.4: lp li ca h thng v thi gian lu v din tch peak

STT HCBVTV Thi gi n lu Din t pe k

Trung bnh CV% Trung bnh CV%

1. Acetochlor 10,90 0,0 7378333 2,2

2. Aldicarb 8,18 0,1 3885000 2,3

3. Atrazine 9,69 0,11 1810000 2,3

4. Azoxystrobin 9,94 0,15 40600000 2,3

5. Avermectin 14,1 0,0 134167 2,3

31

STT HCBVTV Thi gi n lu Din t pe k

Trung bnh CV% Trung bnh CV%

6. Acetamiprid 7,00 0,17 15566667 2,5

7. Carbaryl 9,16 0,11 10266667 0,80

8. Carbofuran 8,89 0,06 28400000 1,2

9. Carbendazim 7,61 0,25 60833333 1,6

10. Carboxim 9,14 0,10 22733333 1,9

11. Dichlorvos 8,85 0,09 3020000 3,2

12. Dimethoat 7,09 0,15 17216667 1,6

13. Edifenphos 11,1 0,0 19480000 2,0

14. Fenobucarb 10,2 0,0 6921667 3,2

15. Imidachlorprid 6,40 0,18 4425000 1,7

16. Indoxacarb 11,30 0,0 1090167 3,9

17. Isoprocarb 9,65 0,16 15066667 2,0

18. Methiocarb 10,33 0,50 9773333 0,94

19. Methomyl 5,08 0,11 10266667 0,80

20. Profenophos 12,0 0,0 6705000 1,5

21. Propoxure 8,82 0,1 15250000 1,8

22. Terbuconazole 11,15 0,49 326600 4,9

23. Thiamethoxam 5,37 0,3 5681667 1,9

24. Trichlorfon 7,1 0,15 6921667 3,2

3.1.2. Kho st iu kin tch chit HCBVTV trong dc liu kh.

Trn s quy trnh ca AOAC [11], chng ti bt u kho st xy dng p ng

php tch chit o dc liu k n trn n 3.2.

32

Hnh 3.2. S tm tt quy trnh QuEChERS kho st

K i lng mu p n t n ng qu n trng trong p ng p p

QuEChERS. K i lng mu t p t k ng m bo i din o mu, tu n in k i

lng o qu li o n ng nn qu ln, g k k n o vi t it v tn

km dung m i t. C p ng p p QuEC ERS ng dng trn nn mu r u qu

u du tr t l mu:dung m i it l 1:1 (kl/kl). Qu trn it QuEChERS theo

Anasatassiades [10] ng dng trn nn mu r u qu m k ong 80%. Nn mu

d liu k m t p (

33

Trn s qu trn ni trn, ng t i t in mt s k o s t n m ti u

iu sut it i vi nn mu d liu n s u:

- Kho st thi gi n ngm nc mu trc khi chit

- So snh n ng ca pH chit 3 p ng p p QuEC ERS: p ng

p p b n u, p ng p p AOAC (2007), p ng p p EN (2008)

- Thm NaCl vo mui chit p ng p p k o st

- So s n n gi n ng nn

3.1.2.1. Kho st thi gian ngm nc

S dng mu t m un m 500 g/kg. Tin n k o s t t i gi n ngm

mu vi n tr k i it, s u k i t m n mu l k ngm n on

ton, k ong t i gi n 0, 15, 30, 45, 60 p t tr k i it (n = 2) v n

iu kin it k n s n 3.3.

Hnh 3.3. nh hng thi gian ngm mu trong nc n hiu sut chit

N n xt: C kt qu o t ti t i gi n 0 p t, t u nng c

HCBVTV k o s t l o n t, do k ng n t it p i t in ngm mu trong

n tr k i it. Nu ngm mu trong n ng lu, m lng HCBVTV ng

34

gim t do s p n mt s HCBVTV trong n . Kt qu n p p vi

mt ng in u tr trn nn mu t p m nng t n p n t o d

3.1.2.2. Kho st nh hng ca pH thng qua so snh 3 quy trnh

P ng p p QuEC ERS 3 p in bn ng dng trn r u qu, l p ng

p p b n u k ng s dng m t eo An st ssi des [10], p ng p p dng m

acetat theo AOAC 2007.01 [11] v p ng p p dng m t eo EN 15662 [17]. Do

loi d liu pH k n u t n ng n iu sut it nn ng t i

tin n k o s t n ng pH d it n t u i HCBVTV.

- Quy trnh 1: khng s dng m (qu trn QuEC ERS b n u). gi i on

chit pH ph thuc vo pH nn mu.

- Quy trnh 2: S dng m acetate (AOAC 2007.1), pH chit d o ng t 4,8-5.

- Quy trnh 3: S dng h m citrat (Quy trnh EN 15662). pH chit khong 5,0.

S dng 2 nn mu l d t n pH trung tn , v nn mu l gi ng,

pH acid t m un HCBVTV t u nng tng ng 0,1 mg/kg. X

l mu t eo 3 qu trn , lp li 3 l n. pH d h chit trn b bng 3.5

v c kt qu t u i tm tt n 3.4

Bng 3.5. So snh cc phng php chit v pH dch chit o c

P ng p p Mui it Dung m i it pH d it

tha canh l giang

K ng m 6g MgSO4 khan, 1,5g NaCl Acetonitril 5,8 2,0

m et t 6g MgSO4 khan, 1,5g natri

acetat

Acetonitril 1%

acid acetic 5,9 5,2

m itr t

6g MgSO4, 1,5g NaCl, 1,5g

trinatri citrat.2H2O; 0,75g

dinatri citrat 1,5H2O

Acetonitril 4,5 2,7

35

Hnh 3.4: S lng HCBVTV c thu hi cc khong khc nhau (>80%; 70-

80% v

36

Hnh 3.5: S lng HCBVTV c thu hi cc khong khc nhau (>80%; 70-

80% v

37

Hnh 3.6: nh gi vai tr ca ni chun (IS) TPP vi vic loi tr nh hng nn

Kt qu o t , nu k ng s dng ni un n ng nn s t

nm ngoi 20%. K i n lng t eo ni un n ng nn gim r rt, 23/25 p

t n ng nn nm trong k ong 20%, trong k ong 50% s t n

ng nn t p di 5%. Do , vi s dng ni un rt n t it n m loi tr

s i s do nn mu.

3.2. Thm nh phng php

3.2.1. Gii hn pht hin v gii hn nh lng

n gi LOD v LOQ p ng p p, ng t i tin n p n t

mu t m un ti nng 0,005 mg/kg v 0,01 mg/kg (tn trn mu). X n

LOD v LOQ d vo t l tn iu trn n iu (S/N sign l to noise r tio). C kt

qu o t , tt t t n lng ti nng 0,01 mg/kg m k ng

n t m b lm giu mu. Kt qu LOD, LOQ t vi tng HCBVTV

trn b bng 3.6.

38

Bng 3.6: LOD, LOQ ca cc ha cht bo v thc vt

HCBVTV S/N (1

g/l)

S/N (2

g/l)

LOQ

(g/l)

LOQ

(mg/kg)

LOD

(g/l)

LOD

(mg/kg)

Methomyl 10,1 1,0 0,01 0,3 0,0015

Carbendazim 12,8 1,0 0,01 0,3 0,0015

Isoprocarb 24,9 0,5 0,0025 0,15 0,00075

Cabaryl 16,9 0,5 0,0025 0,2 0,001

Fenobucarb 21,9 0,5 0,0025 0,15 0,00075

Imidaclomprid 1,9 10.7 2,0 0,01 0,7 0,0035

Tricloform 2,5 9.4 2,0 0,01 0,7 0,0035

Dimethoate 25,2 0,5 0,0025 0,15 0,00075

Diclovos 7,7 2,0 0,01 0,3 0,0015

Tercobunazole 0,7 13,8 2,0 0,01 0,7 0,0035

Carbofuran 25,8 0,5 0,0025 0,15 0,00075

Aldicarb 17,9 0,5 0,0025 0,2 0,001

Acetamiprid 13,8 1,0 0,005 0,3 0,0015

Carboxim 35,3 0,3 0,0015 0,1 0,0005

Edifenphos 16,5 0,5 0,0025 0,2 0,001

Indoxacarb 6,6 9,8 2,0 0,01 0,7 0,0005

Methiocarb 21,1 0,5 0,0025 0,15 0,00075

Thiamethoxam 14,7 0,75 0,0225 0,25 0,00125

Profenophos 9,7 1,0 0,005 0,3 0,0015

Acetochlo 50,2 0,2 0,001 0,1 0,0005

Propoxur 20,7 0,5 0,0025 0,15 0,00075

Emamectin 124,2 0,1 0,005 0,03 0,00015

Azoxytrobin 57,9 0,2 0,001 0,1 0,0005

Atrazine 21,2 0,5 0,0025 0,15 0,00075

39

3.2.2. Khong tuyn tnh

C ng t i s dng ng un trn nn mu loi tr n ng nn. Mu

trng it t eo qu trn t u l d it, p dung d un trn d

it nng 0; 1; 2; 20; 50; 100 v 200 g/L (tng ng trn nn mu 0; 5;

10; 100; 200; 500; 1000 g/kg). Nng ni un gi n l 100 g/L. X dng

ng p t u gi t l din t peak vi din t peak ni un v nng

tng ng. ng un lp t eo p n mm t it b. Kt qu o o t

trong k ong nng n tn iu HCBVTV t l tu n tn vi nng vi r

> 0,99. ng un mt s HCBVTV trn b n 3.7 v p l 1. Untitled 8 (Imidachlorprid1): "Linear" Regression ("No" weighting): y = 1.01 x + 0.103 (r = 0.9999)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0Analyte Conc. / IS Conc.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0

10.4

An

aly

te A

rea

/ I

S A

rea

Untitled 8 (Fenobucarb1): "Linear" Regression ("No" weighting): y = 2.69 x + 0.317 (r = 0.9995)


0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

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26

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te A

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Untitled 8 (Thiamethoxam1): "Linear" Regression ("No" weighting): y = 1.83 x + 0.112 (r = 0.9999)


0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

12.0

13.0

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Untitled 8 (Methomyl1): "Linear" Regression ("No" weighting): y = 1.32 x + 0.126 (r = 0.9998)


0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

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11.0

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Hn 3.7: ng chun mt s HCBVTV

Imidaclomiprid (r=0.9999) Fenobucarb (r=0.9995)

Thiamethoxam (r=0.9999) Methomyl (r=0.9998)

40

3.2.3. lp li v thu hi

n gi lp li v t u i t ng qu p n t mu t m un 3

m nng 0,01 mg/kg; 0,1 mg/kg v 1 mg/kg, C kt qu s bin t in (CV%)

v t u i (R%) trn b n 3.8 v hnh 3.9.

Hnh 3.8: Kt qu xc nh thu hi ca phng php trn cc nn mu khc

nhau (TC tha canh, TNHC trinh n hong cung, CN c ngt, M m ,

LG l giang) ti cc nng khc nhau (0,01; 0,1 v 1 mg/kg)

41

Hnh 3.9: Kt qu xc nh lp li ca phng php trn cc nn mu khc nhau

(TC tha canh, TNHC trinh n hong cung, CN c ngt, M m , LG l

giang) ti cc nng khc nhau (0,01; 0,1 v 1 mg/kg)

C kt qu o t , s HCBVTV t u i trong k ong t 70-

110%. Trn mt s nn mu bit, ti nng t p 0,01 mg/kg t u i nm

ngoi k ong trn n profenofos (TN 0,01 mg/kg 138%); carbaryl (TN 0,01 mg/kg

65%); carbendazim (TN 0,01 mg/kg 60%); carboxim (TC 0,01 mg/kg 60%). Cc

gi tr t u i t p o o n t loi tr k i s dng p ng p p ni

un.

lp li tn trn mu trng t m un v mu t ( HCBVTV) u

rt tt vi gi tr s bin t in di 10% i vi 94% trng p. K ng

mu no s bin t in vt qu 20% p ng u u v p n t d

lng.

3.3. p dng phng php xc nh d lng HCBVTV trn 30 dc liu kh

C ng t i ng dng p ng p p p n t HCBVTV trong 30 mu d

liu k k n u l ti 3 im. C kt qu trn b trong bng 3.

Bng 3.7. Kt qu phn tch HCBVTV trong dc liu kh

K iu M t mu Kt qu (HCBVTV p t in v nng

- mg/kg)

im l

mu

G01 Ru ng -

X Ninh

Hip, Gi

Lm, H Ni

G02 C ngt -

G03 mu -

G04 N n tr n -

G05 C rng -

G06 Kim tin t o Imidacloprid (0,25)

42


- mg/kg)

im l

mu

G07 Trin n ong

cung

Azoxytrobin (0,15), Carbofuran

(0,015), Fenobucarb (0,010)

G08 B ng n Atrazin (0,020)

G09 Ci x -

G10 Ngi u -

G11 Hoa sa

Tang k sinh -

L01 n -

P Ln ng,

Hon Kim,

H Ni

L02 C ngt -

L03 M

Tercobunazol (0,25), Carbendazim

(12,1), Avermectin (0,30),

Azoxytrobin (1,1)

L04 Rau m -

L05 Hoa nhi Acetamiprid (0,045)

L06 K sm Imidacloprid (0,075)

L07 C x -

L08 Xu -

L09 B -

L10 N ni -

L11 C vng -

L12 Ch dy -

L13 Si n m -

L14 Trin n ong

cung -

43


- mg/kg)

im l

mu

H01 Dy tha canh - X Hi L ,

Hi Hu,

N m n

H02 in lng -

H03 Kin gii -

H04 Dip u Imidacloprid (0,051)

N v , trong s 30 mu d liu p n t 7 mu (k ong 23%)

HCBVTV, trong t n sut xut in ln n t l HCBVTV n m

neoni otinoid vi 4/7 mu.

44

KT LUN V KIN NGH

k o s t x dng v t m n p ng p p QuEC ERS p i p k t ut

LC-MS/MS p n t d lng HCBVTV trong mt s loi d liu k .

- K o s t x dng p ng p p QuEC ERS s i p p vi i tng

d liu k it 25 HCBVTV bng etonitril vi m et te v

loi tp bng d-SPE.

- P ng p p n tt vi LOQ di 0,01 mg/kg, s t

t u i t 70-110% v lp li tt (CV% < 20%) ng m lng t p

0,01 mg/kg.

- p dng p ng p p n gi d lng HCBVTV n trong 30

loi d liu k l ti H Ni v N m n , k ong 23% mu

HCBVTV vi nng trn 0,01 mg/kg.

Trn s n ng kt qu t u , ng t i r mt s kin ng s u:

- Tin n p dng p ng p p t u i vi s bin, sn

xut n iu vi d liu k tr n trng p ng do d lng

HCBVTV.

- Tip t s dng p ng p p k o s t n iu n n i tng mu

d liu k trn t trng, n m t eo di n gi d lng HCBVTV

trong i tng n .

45

TI LIU THAM KHO

Ting Vit

1. B Y t (2007), Quyt nh v vic ban hnh Quy nh gii hn ti a

nhim sinh hc v ha hc trong thc phm, t ng t 46-2007-Q, H Ni.

2. B Y t (2009), Dc in Vit Nam IV, NXB Y hc.

3. B Y t (2011), Kim nghim dc phm, NXB Y hc.

4. Nguyn Min c (2006), Sc k lng hiu nng cao v mt s ng

dng vo nghin cu, kim nghim dc phm, dc liu v hp cht t nhin,

NXB Y hc.

5. Tr n Vn H i (2009) , Gio trnh Ha bo v thc vt ,i hc C n T .

6. Tr n Vit Hng (2005), Kho st v nghin cu kim nghim mt s ha

cht bo v thc vt tn d trong dc liu, Lun n tin s Dc h , i hc

Dc H Ni.

7. Tr n C o Sn, L T Hng Ho, Nguyn Vn Hng, Tr n Th Thy,

Thi Nguyn Hng T u (2013), X n d lng HCBVTV trong thc

phm ch nng t n p n tho dc bng sc k lng hai l n khi ph,

Tp ch phn tch Ho, L v Sinh hc, 18 (3), 69-77.

8. Tr n C o Sn, L T Hng Ho, Hong Th Thu Hng, Thi Nguyn

Hng T u (2013), X n d lng ha cht bo v thc vt trong dc

liu ti bng p ng p p QuEC ERS v sc k lng khi ph, Tp ch dc

hc, 436 (10), 20-25.

9. Vin kim nghim an ton v sinh thc phm quc gia (2010), Thm

nh phng php trong phn tch ha hc & vi sinh vt, NXB Khoa hc v K

thut.

Ting Anh

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10. Anastassiades M., Lehotay S.J., Stajnbaher D., and Schenck F.J. (2003),

Fast and easy multiresidue method employing acetonitrile

extraction/partitioning and dispersive solid-phase extraction for the

determination of pesticide residues in produce, Journal of AOAC international,

86(2), 412431.

11. AOAC Intern tion l (2007), AOAC Offi i l Met od 2007.01 Pesti ide

Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium

Sulfate, AOAC Official method.

12. Attallah E.R., Barakat D.A., Maatook G.R., v Badawy H.A. (2012),

"Validation of a quick and easy (QuEChERS) method for the determination of

pesticides residue in dried herbs", Journal of Food Agriculture Environment, 10,

755762.

13. Ardrey R.E. (2003), Liquid chromatography-mass spectrometry: an

introduction, New York; J. Wiley.

14. Chen L., Song F., Liu Z., Zheng Z., Xing J., Liu S. (2012) , Multi-

residue method for fast determination of pesticide residues in plants used in

traditional chinese medicine by ultra-high-performance liquid chromatography

coupled to tandem mass spectrometr , Journal of Chromatography A, 1225,

132140.

15. Chen G., Cao P., v Liu R. (2011), A multi-residue method for fast

determination of pesticides in tea by ultra performance liquid chromatography

electrospray tandem mass spectrometry combined with modified QuEChERS

sample preparation procedure", Food Chemistry, 125(4), 14061411.

16. Du G., Song Y., v Wang Y. (2011), "Rapid simultaneous determination

of multiple pesticide residues in traditional Chinese medicines using

programmed temperature vaporizer injection-fast gas chromatography coupled

with mass spectrometry", Journal of Separation Science, 34(23), 33723382.

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17. EN 15662 (2009), EN 15662:2009 Foods Of Pl nt Origin -

Determination Of Pesticide Residues Using GC-MS and/or LC-MS/MS) -

Que ers Met od, Europe n St nd rd.

18. Europe n Commission (2002), 2002/657/EC: Commission De ision of

12 August 2002 implementing Council Directive 96/23/EC concerning the

performance of analytical methods and the interpretation of results.

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(EU) No 485/2013 of 24 May 2013 amending Implementing Regulation (EU)

No 540/2011, as regards the conditions of approval of the active substances

clothianidin, thiamethoxam and imidacloprid, and prohibiting the use and sale

of seeds treated with plant protection products containing those active

substances.

20. Food and Agriculture Organization of the United Nations

(2002), International Code of Conduct on the Distribution and Use of Pesticides.

21. Lehotay S. J., de Kok A., Hiemstra M., van Bodegraven P. (2005),

"Validation of a fast and easy method for the determination of residues from

229 pesticides in fruits and vegetables using gas and liquid chromatography and

mass spectrometric detection", Journal of AOAC international, 88(2), 595614.

22. Lehotay S.J. (2011), "QuEChERS sample preparation approach for mass

spectrometric analysis of pestiside residues in foods", Mass Spectrometry in

Food Safety:methods and protocols, 747, 65 - 91.

23. Pay P. , Anastassiades M., Mack D., Sigalova I., Tasdelen B., Oliva J.,

Barba A. (2007), "Analysis of pesticide residues using the Quick Easy Cheap

Effective Rugged and Safe (QuEChERS) pesticide multiresidue method in

combination with gas and liquid chromatography and tandem mass

spectrometric detection", Analytical and Bioanalytical Chemistry, 389(6), 1697-

1714.

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24. Rajski ., Lozano A., Belmonte-Valles N., Ucls A., Ucls S., M.,

Mezcua A., Fernandez-Alba R. (2013), "Comparison of three multiresidue

methods to analyse pesticides in green tea with liquid and gas

rom togr p /t ndem m ss spe trometr , The Analyst, 138(3), 921.

25. Sadowska-Rociek A., Surma M., Cielik E. (2013), "Application of

QuEChERS Method for Simultaneous Determination of Pesticide Residues and

PAHs in Fresh Herbs", Bulletin of Enviromental Contamination Toxicology,

90(4), 508513.

26. Xu R., Wu J., Liu Y., Zhao R., Chen B., Yang M., v Chen J. (2011),

"Analysis of pesticide residues using the Quick Easy Cheap Effective Rugged

and Safe (QuEChERS) pesticide multiresidue method in traditional Chinese

medicine by gas chromatography with electron capture detection",

Chemosphere, 84(7), 908912.

49

PH LC

Ph lc 1: ng chun HCBVTV

Untitled 8 (Carbaryl1): "Linear" Regression ("No" weighting): y = 2.86 x + 0.533 (r = 0.9981)


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Untitled 8 (Carbofuran1): "Linear" Regression ("No" weighting): y = 8.22 x + 2.14 (r = 0.9957)


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Untitled 8 (Edifenphos1): "Linear" Regression ("No" weighting): y = 5.47 x + 0.55 (r = 0.9995)


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Untitled 8 (Tebuconazole1): "Linear" Regression ("No" weighting): y = 0.397 x + -0.00588 (r = 0.9995)


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Untitled 8 (Trichlorfon1): "Linear" Regression ("No" weighting): y = 2.23 x + 0.222 (r = 0.9993)


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Untitled 8 (Dimethoate1): "Linear" Regression ("No" weighting): y = 4.2 x + 0.556 (r = 0.9992)


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Trichlofon (r=0.9993) Dimethoate (r=0.9992)

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Untitled 8 (Aldicarb1): "Linear" Regression ("No" weighting): y = 1.28 x + 0.37 (r = 0.9947)


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Untitled 8 (Dichlorvos1): "Linear" Regression ("No" weighting): y = 1.51 x + 0.375 (r = 0.9971)


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Untitled 8 (Propoxur1): "Linear" Regression ("No" weighting): y = 4.9 x + 1.42 (r = 0.9940)


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Untitled 8 (Acetochlor1): "Linear" Regression ("No" weighting): y = 2.38 x + 0.355 (r = 0.9992)


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Untitled 8 (Isoprocarb1): "Linear" Regression ("No" weighting): y = 6 x + 1.18 (r = 0.9979)


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Untitled 8 (Indoxacarb1): "Linear" Regression ("No" weighting): y = 0.395 x + 0.0244 (r = 0.9997)


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Isoprocarb (r=0.9979) Indoxacarb (r=0.9997)

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Untitled 8 (Atrazine1): "Linear" Regression ("No" weighting): y = 1.62 x + 0.139 (r = 0.9996)


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Untitled 8 (Azoxystrobin1): "Linear" Regression ("No" weighting): y = 10.9 x + 2.73 (r = 0.9967)


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Untitled 8 (Carbendazim1): "Linear" Regression ("No" weighting): y = 14.9 x + 1.79 (r = 0.9995)


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Untitled 8 (Avermectin1): "Linear" Regression ("No" weighting): y = 0.196 x + 0.0203 (r = 0.9976)


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Untitled 8 (Carboxim1): "Linear" Regression ("No" weighting): y = 6.01 x + 1.23 (r = 0.9983)


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Untitled 8 (Methiocarb1): "Linear" Regression ("No" weighting): y = 3.47 x + 0.362 (r = 0.9994)


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Atrazine (r=0.9996) Azoxytrobin (r=0.9967)

Carbendazim (r=0.9995) Avermectin (r=0.9976)

Carboxim (r=0.9983) Methiocarb (r=0.9994)

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Untitled 8 (Edifenphos1): "Linear" Regression ("No" weighting): y = 5.47 x + 0.55 (r = 0.9995)


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Untitled 8 (Profenophos1): "Linear" Regression ("No" weighting): y = 1.38 x + 0.0141 (r = 0.9998)


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