B Y T

TRNG I HC DC H NI

NGUYN TH LAN ANH

XY DNG PHNG PHP NH LNG

CEFIXIM TRONG NC THI BNG

SC K LNG HIU NNG CAO

KHA LUN TT NGHIP DC S

H NI 2013

B Y T

TRNG I HC DC H NI

NGUYN TH LAN ANH

XY DNG PHNG PHP NH LNG

CEFIXIM TRONG NC THI BNG

SC K LNG HIU NNG CAO

KHA LUN TT NGHIP DC S

Ngi hng dn:

1.PGS.TS. Nguyn Th Kiu Anh

2.DS. Trn Th Thanh Hu

Ni thc hin:

1. B mn Ha Phn Tch

2. Phng Dc l Vin KN thuc TW

H NI 2013

LI CM N

Vi lng knh trng v bit n su sc, ti xin chn thnh cm n PGS.TS

Nguyn Th Kiu Anh, ngi thy trc tip hng dn ti trong sut qu trnh

nghin cu v hon thnh kha lun tt nghip.

Ti cng xin gi li cm n n TS. Nguyn Th Kim Hng Trng khoa

Dc l Vin kim nghim thuc Trung ng cng tp th cn b trong khoa

nhit tnh hng dn v to iu kin cho ti trong qu trnh thc hin kha lun.

c bit, ti xin chn thnh cm n DS. Trn Th Thanh Hu, ngi gip

rt tn tnh v cho ti nhng gp chn thnh trong sut thi gian ti thc hin

kha lun.

Nhn dp ny ti cng xin gi li cm n n Ban gim hiu cng ton th cc

thy c gio trng i hc Dc H Ni dy d v to mi iu kin thun li

cho ti trong thi gian ti hc tp ti trng v thc hin kha lun.

Cui cng, ti xin cm n gia nh v bn b, nhng ngi lun ng vin,

khch l, to ng lc cho ti trong sut qu trnh hc tp v thc hin kha lun

ny.

H Ni, ngy 8 thng 5 nm 2013

Sinh vin

Nguyn Th Lan Anh

MC LC

MC LC

DANH MC CC K HIU, CH VIT TT

DANH MC CC BNG

DANH MC CC HNH V, TH

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

CHNG 1: TNG QUAN ..................................................................................... 3

1.1 TNG QUAN V CEPHALOSPORIN ............................................................ 3 1.1.1 Lch s ra i, ngun gc........................................................................... 3 1.1.2 Cng thc cu to, phn loi v ph tc dng ........................................... 3 1.1.3 c tnh ca cc cephalosporin nghin cu ............................................... 5

1.2 TNG QUAN V CEFIXIM ............................................................................ 6 1.2.1 Cng thc cu to ...................................................................................... 6 1.2.2 Tnh cht l ha........................................................................................... 6 1.2.5 Ch nh ...................................................................................................... 7 1.2.6 Mt s phng php nghin cu nh lng cefixim ................................ 7

1.2.6.1 Nghin cu nh lng cefixim bng phng php o quang ............ 7 1.2.6.2 Nghin cu nh lng cefixim bng phng php HPLC ................ 7

1.4 SC K LNG HIU NNG CAO ................................................................ 10 1.4.1 Khi nim ................................................................................................. 10 1.4.2 Nguyn tc ............................................................................................... 10 1.4.3 Phn loi ................................................................................................... 10 1.4.4 Cc thng s c trng ............................................................................. 10 1.4.5 ng dng ca HPLC ................................................................................ 12

CHNG 2: I TNG V PHNG PHP NGHIN CU ................... 15

2.1. I TNG NGHIN CU ........................................................................ 15 2.2. PHNG TIN NGHIN CU.................................................................... 15

2.2.1 Ha cht thuc th - cht chun ............................................................ 15 2.2.2. Thit b - dng c .................................................................................... 16

2.3. PHNG PHP NGHIN CU .................................................................. 17 2.3.1 Kho st phng php x l mu ............................................................. 17 2.3.2. Xy dng iu kin sc k ....................................................................... 17 2.3.3. nh gi phng php ............................................................................ 17 2.3.4 Phng php x l s liu ........................................................................ 18

CHNG 3 : KT QU NGHIN CU............................................................. 19

3.1 XY DNG QUY TRNH PHN TCH ....................................................... 19 3.1.1 Kho st phng php x l mu ............................................................. 19

3.1.1.1 La chn loi dung mi x l mu .................................................... 19 3.1.1.2. La chn t l dung mi x l mu ................................................... 21

3.1.2 Kho st quy trnh phn tch .................................................................... 23 3.1.2.1 Kho st la chn ct sc k ............................................................. 24 3.1.2.2 Kho st thnh phn pha ng v tc dng ................................ 25 3.1.2.3. Kho st th tch tim mu ............................................................... 26

3.1.3 Quy trnh phn tch................................................................................... 27 3.2.NH GI PHNG PHP......................................................................... 29

3.2.1. thch hp ca h thng ....................................................................... 29 3.2.2 c hiu .............................................................................................. 30 3.2.3 tuyn tnh ............................................................................................ 32 3.2.4. ng ................................................................................................... 34 3.2.5 Kho st chnh xc .............................................................................. 36

3.2.5.1 lp li ........................................................................................... 36 3.2.5.2 chnh xc trung gian .................................................................... 36

3.2.6.Gii hn pht hin (LOD) v gii hn nh lng (LOQ) ....................... 38 3.2.6.1.Gii hn pht hin ............................................................................ 38 3.2.5.2 Gii hn nh lng .......................................................................... 39

3.3.BN LUN .................................................................................................... 39

CHNG 4: KT LUN V XUT ............................................................. 42

4.1. KT LUN .................................................................................................... 42 4.2. XUT ....................................................................................................... 43

TI LIU THAM KHO

DANH MC CC K HIU, CH VIT TT ACN Acetonitril

BP 2010 The Bristist Pharmacopoeia (Dc in Anh 2010)

C18 Octadecyl

DAD Diode array derector (derector chui diod)

HPLC High perform liquid chromatography (sc k lng hiu nng cao)

HPLC-MS High perform liquid chromatography Mas Spectrometry (sc k

lng khi ph)

MeOH Methanol

mg miligram

ml mililit

PA Pro analysis (tinh khit phn tch)

SK S ng k

USP 32 The United States Pharmacopoeia (Dc in M 34)

UV - VIS Ultraviolet and visiable absorption Spectrophotometry (ph hp

th t ngoi kh kin)

DANH MC CC BNG STT Tn bng Trang Bng 1.1. Ph tc dng ca cc cephalosporin ........................................................... 4

Bng 1.2. c tnh ca cc cephalosporin nghin cu trong ti ............................ 5

Bng 3.1. Kho st la chn dung mi x l mu .................................................... 20

Bng 3.2. Kt qu kho st th tch tim mu .......................................................... 27

Bng 3.3. Kt qu thch hp ca h thng ........................................................... 29

Bng 3.4. Kt qu kho st tuyn tnh ................................................................. 33

Bng 3.5. Kt qu kho st ng ......................................................................... 35

Bng 3.6. Kt qu lp li v chnh xc trung gian .......................................... 37

Bng 3.7. Kt qu kho st LOD .............................................................................. 38

DANH MC CC HNH V, TH STT Tn hnh v, th Trang Hnh 1.1. Cng thc chung ca cephalosporin .......................................................... 3

Hnh 1.2. Cng thc cu to ca cefixim ................................................................... 6

Hnh 2.1 Ha cht,thuc th, cht chun, thit b, dng c ..................................... 16

Hnh 3.1. Sc k kho st la chn dung mi x l mu...................................... 20

Hnh 3.2. Sc k kho st la chn lng dung mi x l mu ........................... 22

Hnh 3.3. Kho st la chn ct sc k ..................................................................... 25

Hnh 3.4. Sc k kho st thnh phn pha ng v tc dng .......................... 26

Hnh 3.5. Mi lin quan gia th tch tim mu v din tch pic ............................. 27

Hnh 3.6. Sc k kho st c hiu: mu chun (a), mu trng (b), mu th (c) .............................................................................................................................. 31

Hnh 3.7. Ph cefixim trong dung dch th v trong dung dch chun .................... 31

Hnh 3.8. Sc k kho st c hiu khi phn tch cefixim v 6 cephalosporin khc ........................................................................................................................... 32

Hnh 3.9.a. th biu din s ph thuc gia nng (ng/ml) v din tch pic (mAu.s) ca cefixim .................................................................................................. 33

Hnh 3.9.b. Sc k kho st tuyn tnh ............................................................ 34

Hnh 3.10. Sc k st LOD................................................................................... 39

1

1 T VN Nm 1929, Alexander Fleming pht hin ra kh nng khng khun ca nm

Penicillium notatum, m u cho nghin cu v s dng khng sinh. T ti nay

c rt nhiu dng khng sinh mnh, ph rng nh cephalosporin, macrolid,

quinolon, cloramphenicol c ra i. S ra i ca cc loi thuc khng khun

v khng sinh l thnh tu v i ca y hc. Cc bc s c c nhng loi thuc

k diu gi r v hiu qu trong iu tr nhim khun. Khng sinh cng vi cc bin

php v sinh, tim phng gp phn gia tng tui th ca con ngi trong nhiu nm,

thm ch nhiu thp k. Mi ln pht hin mt loi vi khun khng thuc, cc phng

th nghim li a ra mt loi thuc khng sinh mi[7], [25].

Tuy nhin theo cnh bo ca WHO, nhng loi khng sinh cng hiu nht

trong iu tr bnh ang dn b v hiu ha. Trong cuc chin gia vi khun v con

ngi, phn thng ang nghing v pha cc loi vi khun khng thuc. y gn

nh l du chm ht cho thi k hong kim ca thuc khng sinh [26]. T nm 1987

khng h c mt loi khng sinh no c pht hin cn vi khun li vn ang

khng ngng bin i [25], [26]. Do , gim t l khng thuc ca vi khun l

cng vic cp bch hn bao gi ht. Mt trong nhng nguyn nhn quan trng dn

n vi khun khng thuc l do khng sinh tn d trong nc thi c bit l trong

nc thi nh my sn xut khng sinh. Vic loi b khng hon ton khng sinh

trong nc thi ti nh my dn n vic x nc vo mi trng tim nng nh

hng n sc khe con ngi v tc ng n h sinh thi, to iu kin cho vi

khun khng thuc ngay c nng du vt [24]. Do vy hng nghin cu v d

lng khng sinh, cht chuyn ha ca chng v nh hng ca chng ln con

ngi v mi trng ang rt c quan tm ch c bit l cc nc pht trin

[24], [17] c nhiu nghin cu nh lng khng sinh trong nc thi v nh

hng ca n ln ng thc vt v sc khe con ngi [19].

Ti Vit Nam vn d lng khng sinh trong nc thi cng ang c

quan tm nghin cu nh nh lng khng sinh trong nc thi bnh vin [3].

Cng nghip dc ang pht trin mnh vi gia tng s lng cc nh my sn xut

2

dc phm cng vi lng khng sinh thi ra mi trng ngy cng nhiu. Tuy

nhin li cha c nghin cu v d lng khng sinh trong nc thi nh my sn

xut dc phm.

Cefixim l mt khng sinh cephalosporin th h 3 c ph rng, c hiu qu

trn nhiu vi khun khng nhiu loi khng sinh nh cephalosporin th h 1,

cephalosporin th h 2, penicillin, c bit cefixim dng theo ng ung mt

ng dng n gin v d s dng hn so vi nhiu cephalosporin khc. V vy

m cefixim ngy cng c s dng nhiu. gp phn kim sot d lng khng

sinh, c th l khng sinh cefixim trong nh my sn xut dc phm chng ti thc

hin ti:

Xy dng phng php nh lng cefixim trong nc thi bng sc

k lng hiu nng cao

Vi mc tiu:

- Xy dng phng php x l mu v iu kin phn tch nh lng

cefixim trong nc thi nh my sn xut dc phm.

- Thm nh phng php nh lng cefixim trong mu nc ny.

3

CHNG 1: TNG QUAN 1.1 TNG QUAN V CEPHALOSPORIN

1.1.1 Lch s ra i, ngun gc

Nm 1945, Giuseppe Brotzu t ngun nc bin ti Cagliari (Italia) phn

lp c chng nm Cephalosporium acremonium. Dch lc mi trng nui cy

nm ny c tc dng c ch s pht trin ca t cu vng Staphylococcus aureus v

c tc dng iu tr bnh nhim khun do t cu vng v bnh thng hn ngi.

T nm 1948 n nm 1956, Abraham v Neuton (Anh) t mi trng nui cy ny

(nay c gi l Acremonium chrysogenum) chit c mt hn hp khng sinh

cephalosporin P1, cephalosporin C v penicillin N. Nm 1971, t chng Norcardia,

Nagarazan chit xut c cephamycin cng c cu trc gn vi cephalosporin C.

C nhiu cephalosporin t nhin c sinh tng hp t cc chng nm mc

khc nhau nhng chng hu nh khng c ngha trong tr liu. V vy ngi ta

tm cch to ra cc cephalosporin bn tng hp trn c s cu trc vng -lactam.

u th ca cc cephalosporin l c tc dng khng khun i vi c cc vi khun c

hot tnh -lactamase, nhiu vi khun Gram (-) v c tnh thp. V vy chng

nhanh chng chim v tr quan trng trong tr liu [8].

1.1.2 Cng thc cu to, phn loi v ph tc dng

* Cng thc chung ca cephalosporin:

S

N

NHR1

O

R2 H

O

OOH

HH

R3

Hnh 1.1: Cng thc chung ca cephalosporin

Cephalosporin l dn cht acyl ha ca acid 7-aminocephalosporanic

(A7AC), mang cu trc 3-cephem, 3 mi lin hp in t vi vng lactam

hp cht c tc dng sinh hc.

Trong A7AC, cc carbon bt i C(6) v C(7) u c cu hnh R, v hu nh

l nguyn nhn lm cho cc cephalosporin c tnh hu truyn kh mnh [7].

4

* Phn loi v ph tc dng

Hin nay, cephalosporin c phn loi thnh 4 th h da trn ph tc

dng. Cc cephalosporin th h trc tc dng trn vi khun Gram (+) mnh hn,

nhng trn Gram (-) yu hn th h sau v ngc li. Ph chung ca cc th h

cephalosporin c gii thiu ti bng 1.1 [9].

Bng 1.1 Ph tc dng ca cc cephalosporin

Tn

nhm

Tn i

din Ph tc dng

Th h I Cephalexin

Cephalothin

Cephazolin

- Ph tc dng trung bnh, tc dng trn cc vi khun

Gram (+) nh t cu, lin cu, ph cu (tr lin cu

khng methicillin).

- Thuc cng c tc dng trn mt s vi khun Gram

(-) nh E.coli, Kebsiella pneumoniae, Proteus

mirabilis v Shigella.

Th h II Cephaclor

Cephuroxim

Cefotetan

- Ph tc dng tng t nh th h 1. Tuy nhin tc

dng trn vi khun Gram (+) yu hn cn tc dng

trn vi khun Gram (-) mnh hn.

Th h III Cefixim

Cefoperazon

Cefotaxim...

- Tc dng tt trn vi khun Gram (-), bn vng vi -

lactamase, tc dng trn c P. aeruginosa.

- Trn vi khun Gram (+), tc dng km th h 1.

Th h IV Cefdinir

Cefepim

- Ph tc dng tng t nhng mnh hn th h 3.

Thuc tc dng tt vi cc vi khun

Enterobacteriaceae, Haemophilus, Pseudomonas,

Streptococcus, lu cu, no m cu.

- Bn vng vi -lactamase.

5

1.1.3 c tnh ca cc cephalosporin nghin cu

Trong khun kh ca ti chng ti xin gii thiu c tnh ca mt s

cephalosporin c s dng nghin cu trong ti ti bng 1.2 [6], [7], [9], [13],

[18], [22].

Bng 1.2 c tnh ca cc cephalosporin nghin cu trong ti

Tn cht Th h Cng thc phn t Tnh cht vt l

1. Cephadroxil monohydrat

I C16H17N3O5S.H2O - Bt mu trng hoc gn nh trng. - Kh tan trong nc, rt kh tan trong ethanol 96%.

2.Cefuroxim II C20H22N4O10S - Bt trng hoc hi vng. - t tan trong nc, tan trong aceton, ethyl acetat v methanol, t tan trong ethanol.

3.Cefdinir III C14H13N5O5S2 - Bt trng hoc hi vng. - Tan trong dung dch m phosphat 0,1M (pH = 7), t tan trong nc, trong alcol v diethyl ether.

4.Ceftazidim III C22H22N6O7S2.5H2O - Bt trng hoc hi vng. - Tan trong dung dch kim, v trong dimethyl sulfoxid, t tan trong dimethylformamid, methanol, nc, khng tan trong aceton, ethanol, cloroform, dioxan, ether, ethyl acetat, toluen.

5.Cefotaxim natri

III C16H16N5NaO7S2 - Bt trng hoc hi vng. - D tan trong nc, hi tan trong methanol, thc t khng tan trong ether.

6.Cefoperazon III C25H26N9NaO8S2 - Bt trng hoc hi vng. - D tan trong nc, tan trong methanol, hi tan trong ethanol 96%.

6

1.2 TNG QUAN V CEFIXIM

1.2.1 Cng thc cu to

Hnh 1.2: Cng thc cu to ca cefixim

+ Cng thc phn t:C16H15N5O7S2.3H2O

+ Khi lng phn t: 507,5

+Tn khoa hc: (6R, 7R)- 7- [[(Z)- 2- (2- aminothiazol- 4- yl)- 2- [(carboxy

methoxy) imino] acetyl] amino]- 3- ethenyl- 8- oxo- 5- thia- 1-azabicyclo[4.2.0] oct-

2- en- 2- carboxylic trihydrat [6], [13], [22].

1.2.2 Tnh cht l ha

Bt trng hoc gn nh trng, hi ht m. t tan trong nc, aceton, glycerin,

tan trong methanol, propylen glycol, hi tan trong ethanol, thc t khng tan trong

ether, ethyl acetat v hexan, hu nh khng tan trong dung dch sorbitol 70% v

octanol. Dung dch cha 0,7 mg cefixim trong 1 ml nc c pH t 2,6 n 4,1 [6],

[13], [22].

1.2.4 Dc l

Cefixim l mt khng sinh cephalosporin th h 3, c dng theo ng

ung.

Cefixim c bn vng cao vi s thy phn ca beta-lactamase m ha bi

gen nm trn plasmid v chromosom. Cefixim c tc dng c invitro v trn lm

sng vi hu ht cc chng ca cc vi khun sau y:

- Vi khun Gram (+): Streptococcus pneumonia, Streptococcus pyogenes.

.3H2O

7

- Vi khun Gram (-): Haemophilus influenzae (tit hoc khng tit beta

lactamase), Moraxella catarrhalis (a s tit beta lactamase), Escherichia coli,

Proteus mirabilis, Neisseria gonorrhoeae (tit hoc khng tit penicillinase).

Cefixim khng c hot tnh i vi Enterococcus, Staphylococcus,

Pseudomonas aeruginosa v hu ht cc chng Bacteroides v Clostridia [5], [9],

[11], [12], [18].

1.2.5 Ch nh

- Nhim khun ng h hp trn v di, vim tai gia cp tnh.

- Nhim khun ng niu khng bin chng.

- Vim niu o do lu cu.

- iu tr cc nhim khun nng do cc vi khun khng cephalosporin th

h I v th h II: Vim mng no cp, p xe no, nhim khun ng huyt, vim

mng trong tim, nhim khun ng h hp nng, nhim khun tiu ha, nhim

khun ng mt, nhim khun tit niu v sinh dc [5], [18].

1.2.6 Mt s phng php nghin cu nh lng cefixim

Nghin cu nh lng cefixim rt phong ph vi nhiu loi nn mu khc

nhau. Chng ti xin gii thiu mt s nghin cu nh lng cefixim.

1.2.6.1 Nghin cu nh lng cefixim bng phng php o quang

- nh lng cefixim trong vin nn khi nh gi ha tan ti = 288 nm,

mu trng l dung dch m phosphat 0,05 M pH = 7,2 (6,8 g KH2PO4, iu chnh

pH bng dung dch NaOH 1M) [6], [22].

1.2.6.2 Nghin cu nh lng cefixim bng phng php HPLC

Mu: nguyn liu, vin nn, hn dch - Ct: C18 (0,125 m x 4 mm, 5m)

- Pha ng: ACN : dung dch tetrabutylamoni hydroxyd 0,4 M pH = 6,5 (chnh

pH bng dung dch H3PO4 1,5 M) (1:3)

- Detector: UV = 254 nm

- Tc dng: iu chnh cho thi gian lu ca cefixim khong 10 pht [6],

[13], [22].

8

Mu dch sinh hc Phm Thu Trang: mu huyt tng

- Ta protein bng dung dch acid HClO4 20%

- iu kin phn tch:

o Ct Rp 18e (250 x 4,6 mm, 5m) c bo v ct, nhit ct 40 o Pha ng: MeOH : m phosphat pH = 2,1 (NaH2PO4 12,5 mM, KH2PO4 5

mM; TBAH 0,06 mM) = 35 : 65, tc dng 1,5 ml/pht

o Detector: DAD =285nm [10]. Abbas Khan v cng s: mu huyt tng

- Ta protein bng acid tricloroacetic

- iu kin phn tch:

o Ct Supelco Discovery HS C18 (150 mm x 4,6 mm, 5 m), nhit ct 50 o Pha ng: ACN : methanol (50:50): dung dch trifloroacetic(19:81), tc

dng 2 ml/pht

o Detector: UV = 285 nm [16]. Joy A. McAteer v cng s: mu huyt tng

- Ta protein bng ACN

- iu kin sc k:

o Ct Altex Ultrasphere C8 (150 x 4,6 mm, 5 l) o Pha ng: MeOH : dung dch m phosphat pH 2,6 = 20:80, tc dng 2

ml/pht

o Detector: UV = 254 nm [19]. Emirhan Nemutlu v cng s: mu huyt tng v nc i

- Chit pha rn ct polymeric sorbent (Strata X)

- iu kin sc k:

o Ct Xterra C18 (250 x 4,6 mm, 5 l), nhit ct 32 o Pha ng: dung dch m phosphat 40mM pH = 3,2 v MeOH, gradient, tc

dng 0,85 ml/pht

o Detector: DAD = 285 nm [20].

9

Mu: nc b mt, nc ngm, nc ung Nghin cu xc nh cefixim trong mu nc b mt, nc ngm, nc ung

c cng b

- Chit pha rn ct SPE: Isolute EVN 100 mg

- iu kin sc k:

o Ct Luna C18 (250 x 2 mm, 5 m) o Pha ng: Acid formic 0,1% : ACN : MeOH, gradient thnh phn pha ng, tc dng 0.2 ml/pht

o Detector: MS-MS [23]. Nhn xt

Cefixim l mt khng sinh cephalosporin th h 3 c s dng trong nhiu

trng hp.Vy nn cefixim l mt trong nhng khng sinh c quan tm nghin

cu nhiu. Cc nghin cu v cefixim rt phong ph a dng t nh lng cefixim

trong ch phm, huyt tng ti nh lng trong nc thi.

Cc dc in cc nc u c chuyn lun ring v cefixim [6], [13], [22].

Nghin cu nh lng cefixim trong huyt tng cng rt c quan tm

ch . Trn th gii c nhiu nghin cu nh lng ring cefixim hay nh

lng cefixim ng thi vi cc cht khc [16], [19], [20]. Ti Vit Nam, cng

c nhng nghin cu nh lng cefixim trong huyt tng [10].

Tuy nhin nhng nghin cu v cefixim trong nc thi nh my cn kh t.

Cc quy trnh nh lng cefixim trong ch phm v huyt tng khng th p dng

cho nh lng cefixim trong nc thi nh my. Chng ti mi ch tm thy mt

nghin cu nh lng cefixim trong nc thi nh my [17]. Nghin cu ny s

dng phng php phn tch bng LC-MS/MS, x l mu bng chit pha rn.

Phng php ny cho c hiu cao, gii hn pht hin thp. Tuy nhin, h thng

LC-MS/MS v chit pha rn gi thnh cao, hin nay cha ph bin ti Vit Nam.

Trong khi , h thng HPLC UV-DAD v chit lng - lng kh thng dng, d

kim, d p dng ti Vit Nam. V vy chng ti xy dng quy trnh phn tch

cefixim trong nc thi nh my vi h thng HPLC UV-DAD v chit lng - lng.

10

1.4 SC K LNG HIU NNG CAO

1.4.1 Khi nim

HPLC l k thut phn tch da trn c s ca s phn tch cc cht trn mt

pha tnh cha trong ct, nh dng di chuyn ca pha ng lng di p sut cao.

Sc k lng da trn c ch hp th, phn b, trao i ion hay loi c ty thuc vo

loi pha tnh s dng [1].

1.4.2 Nguyn tc

Mu phn tch c ha tan trong pha ng. Pha ny l mt cht lng c

cho qua ct cha cc ht pha tnh mt cch lin tc. Cc cht tan l thnh phn ca

mu s di chuyn qua ct theo pha ng vi tc khc nhau ty thuc vo h s

phn b gia cht tan vi pha tnh v pha ng. Nh tc di chuyn khc nhau

cc thnh phn ca mu s tch ring bit thnh di, lm c s cho phn tch nh

tnh v nh lng [1], [2].

1.4.3 Phn loi

Cc k thut sc k lng: sc k phn b, sc k hp ph, sc k trao i ion,

sc k loi c, sc k i lc, sc k cc ng phn quang hc.

Trong khun kh ca ti ti xin trnh by chi tit v sc k phn b.

Sc k phn b l k thut c s dng rng ri nht hin nay. C th phn

thnh 2 dng ty thuc vo pha tnh:

- Sc k lng lng: Pha tnh l lp cht lng bao quanh cc ht cht mang

rn, l cht nhi ct. Qu trnh lu gi cht phn tch lin quan n s phn b

gia 2 pha lng.

- Sc k pha lin kt: pha tnh c lin kt ha hc vi b mt cht mang rn v

d nh silica, alumina... [1], [2].

1.4.4 Cc thng s c trng

* H s phn b K

Trong CS

11

CS : nng mol ca cht tan trong pha tnh.

CM: nng mol ca cht tan trong pha ng.

K cng ln, s di chuyn ca cc cht ca cht tan qua pha tnh cng chm.

Nu cc cht trong hn hp c hng s K khc nhau cng nhiu th kh nng tch

din ra cng d dng hn [1].

* Thi gian lu tR

Thi gian lu tR l khong thi gian t lc tim mu vo ct n khi cht

phn tch n derector. Trn cng mt iu kin HPLC chn, thi gian lu ca

mi cht l hng nh. V vy c th dng thi gian lu pht hin nh tnh cc

cht [1].

Thi gian lu ph thuc vo cc yu t:

- Bn cht ca pha tnh

- Bn cht, thnh phn, tc ca pha ng

- Cu to v bn cht phn t ca cht tan

- Trong mt s trng hp cn ph thuc vo pH pha ng.

* H s dung lng k

k = = K.

Trong :

K: h s phn b

VS: th tch pha tnh

VM: th tch pha ng

Cn chn ct, pha ng... sao cho k nm trong khong ti u [1].

* H s i xng ca pic AF

AF =

y:

W: Chiu rng pic o 1/20 chiu cao pic,

: Khong cch t ng vung gc h t nh pic n mp ng cong pha

trc ti v tr 1/20 chiu cao pic

12

Trong php nh lng yu cu 0,8 AF 2 [1].

* S a l thuyt v hiu lc ct N:

Hiu lc ct c o bng thng s: S a l thuyt N ca ct. 2

2/1

2

54,516

=

=

Wt

WtN RR

Trong : W1/2 l chiu rng pic o na chiu cao ca nh [1].

* phn gii

- phn gii nh gi kh nng tch hai cht trn sc k cho 2 pic lin k.

AB

RARB

AB

RARBS WW

ttWWttR

2/12/1

)(18,1)(2+

=+

=

- Trong : tRA, tRB : Thi gian lu ca 2 pic lin k

WA, WB : rng ca pic o cc y pic.

W1/2A, W1/2B : rng ca pic o na chiu cao cc pic.

Cc gi tr tRA, tRB ,WA, WB , W1/2A, W1/2B phi tnh theo cng n v [1].

- Yu cu RS > 1, gi tr ti u RS = 1,5.

1.4.5 ng dng ca HPLC

- nh tnh :

- S ging nhau v thi gian lu trn sc k ca chun v th l c s ca

php nh tnh. Trn cc my HPLC hin i vi detector mng diod ta c th qut

ph UV-VIS ca cht phn tch trn sc k mu chun v mu th ri chng ph

vi nhau xc nh s ging nhau v dng ph thng qua h s Match (SI-

similarity index).

- H s Match xp x 1,000 ch ra mt ph nh tnh ging nhau hon ton.

- H s Match cng gn 1,000 th s tng t ca ph cng cao.

- H s Match 0,990 : hai ph tng t nhau.

- H s 0,900 Match 0,990 : hai ph c nhng im tng ng.

- H s Match 0,900 : hai ph khc nhau.

- nh lng :

13

Vic so snh ln tn hiu thu c t cht phn tch trong dung dch (din

tch pic hoc chiu cao pic) trong cng mt iu kin sc k l c s ca php nh

lng

Cc phng php nh lng c th p dng l :

- Phng php dng chun ngoi.

- Phng php dng chun ni.

- Phng php thm cht chun.

- Phng php chun ha din tch.

Trong phng php dng chun ngoi c s dng nhiu. y cng l

phng php s dng trong nghin cu ny. V vy sau y chng ti xin trnh by

chi tit v phng php chun ngoi dng trong nh lng bng HPLC.

* Phng php dng chun ngoi

Phng php dng chun ngoi l phng php nh lng c bn. Trong

c mu chun v mu th u c tin hnh sc k trong cng mt iu kin.

Nguyn tc : so snh din tch pic (hoc chiu cao pic) ca mu th vi din

tch pic (hoc chiu cao pic) ca mu chun tnh nng cc cht trong mu th.

C th s dng chun ha 1 im hoc chun ha nhiu im.

Chun ha 1 im : Chn nng ca mu chun xp x vi nng ca mu th.

Tnh nng mu th theo cng thc :

CX = CS . SX/SS Trong :

CX : nng cht phn tch trong mu th

CS : nng cht phn tch trong mu chun

SX (HX) : din tch (chiu cao) ca pic cht phn tch trong mu th

SS (HS) : din tch (chiu cao) ca pic cht phn tch trong mu chun

Chun ha nhiu im :Tin hnh qua cc bc nh sau : Chun b mt dy chun vi cc nng tng dn ri tin hnh sc k. Cc

p ng thu c l din tch pic hoc chiu cao pic ca mi dung dch chun.

14

tnh ton nng ca cht phn tch c th s dng 1 trong 2 cch sau :

- V th ng chun biu din s tng quan gia din tch pic S (hoc

chiu cao H) ca pic vi nng ca cht chun (C). T din tch (hoc chiu cao)

pic ca cht th v ng chun xy dng s suy ra c nng ca cht th.

- Xy dng phng trnh hi quy tuyn tnh m t quan h gia din tch

(hoc chiu cao) pic vi nng cht cn xc nh.

Y = a. CX + b

Trong :

Y : Din tch pic

b : Giao im ca ng chun vi trc tung

a : dc ca ng chun

CX : Nng cht th

Da vo phng trnh hi quy tnh nng cht th :

CX = (Y-b)/a

Lu : ln ca din tch (hoc chiu cao) pic ca mu th phi nm trong

on tuyn tnh ca ng chun [1], [2].

15

CHNG 2: I TNG V PHNG PHP NGHIN

CU 2.1. I TNG NGHIN CU

S dng nc thi nh my dc phm ca cng ty Mediplantex mt trong

nhng cng ty c dy chuyn sn xut cc cephalosporin trong c cefixim lm

nn mu trong nghin cu ny.

Tin hnh ly mu nc thi ca cng ty Mediplantex vo ngy khng sn

xut cefixim, thm cefixim chun t xy dng phng php nh lng

cefixim trong nc thi nh my.

2.2. PHNG TIN NGHIN CU

2.2.1 Ha cht thuc th - cht chun

Cc ha cht, thuc th, cht chun s dng trong nghin cu :

Cc cht chun :

- Cefixim : Chun quc gia, SKS : 0105192, hm lng 98,88%, m

10,57%, ni sn xut VKNTTW.

- Cefuroxim natri, cephadroxil, cefdinir, ceftazidim, cefotaxim, cefoperazon :

chun lm vic.

Thuc th, dung mi s dng trong nghin cu nh sau :

- ACN, MeOH loi tinh khit dng cho HPLC, ni sn xut Merck, c.

- Dicloromethan, cloroform, diethyl ether, natri dihydrophosphat, acid

phosphoric loi PA, ni sn xut Merck, c.

- Giy th pH, ni sn xut VKNTTW

16

a.H thng HPLC Merck - Hitachi

b.Mt s cht chun ha cht

c.Mu nc thi ca nh my Mediplantex

Hnh 2.1. Ha cht,thuc th, cht chun, thit b, dng c

2.2.2. Thit b - dng c

Cc thit b, dng c ti Vin Kim Nghim Thuc Trung ng (c chun

theo ISO/IEC 17025 v GLP ) gm c :

- My lc siu m LK 106 (c)

- Cn phn tch Metler Toledo (d=0,01 mg, e = 0,1 mg) (Thy S)

- My li tm Hettich zentrifugen (c)

- Thit b sc k lng hiu nng cao (HPLC) Merck Hitachi vi detector

DAD, phn mm Multi-system manager, Interface D700, Nht Bn

- Dng c thy tinh cc loi

17

2.3. PHNG PHP NGHIN CU

2.3.1 Kho st phng php x l mu

- Mc ch: tm c loi dung mi, t l dung mi x l mu c hiu sut cao,

loi c nhiu tp cht.

- Tin hnh x l mu th cha cefixim bng cc cch khc nhau. nh lng

hot cht theo quy trnh la chn. nh gi v tm ra cch x l ph hp.

2.3.2. Xy dng iu kin sc k

Nghin cu ti liu tham kho, tin hnh kho st cc iu kin sc k v ct

sc k, thnh phn v tc pha ng, th tch tim mu trn thit b sc k lng

hiu nng cao vi detector DAD nhm la chn c iu kin sc k ph hp nht

nh lng cefixim.

2.3.3. nh gi phng php

X l cc mu nghin cu theo phng php x l xy dng, tin hnh sc

k cc dung dch thu c theo iu kin kho st, thm nh phng php da

trn cc ch tiu: ph hp ca h thng, c hiu, tuyn tnh, ng v

chnh xc, gii hn pht hin v gii hn nh lng.

thch hp ca h thng: nh gi n nh ca h thng v thi gian lu v din tch pic khi tim lp li 1 mu chun 6 ln lin tip.

Yu cu: RSD% v thi gian lu v din tch pic < 2% [15].

c hiu :Trn sc k , mu trng phi khng c pic c thi gian lu tng ng vi thi gian lu ca mu chun [15].

tuyn tnh: Kt qu phn tch thu c t l vi nng (trong khong nht nh) ca cht phn tch trong mu th.

Xy dng ng chun 6 im (X1 n X6) ca dung dch c cha cht chun

cefixim trong khong nng ph hp.

Tin hnh sc k theo iu kin chn. Tim 3 ln mi mu. Tnh gi tr

trung bnh.

Xc nh phng trnh hi quy tuyn tnh v h s tng quan r ca cefixim.

Yu cu: r > 0,997 [15].

18

ng: s ng nht gia gi tr tm thy vi gi tr thc hoc gi tr i chiu c chp nhn.

ng ca phng php c tin hnh theo quy trnh phn tch trn mu t

to cha lng hot cht bit trc hm lng. Tin hnh trn 3 im nng : 2

- 3 ln LLOQ, 40 - 60% ULOQ, 70 90% ULOQ. Tnh t l chun thu hi li c

v lng chun thm vo c c ng ca phng php.

Yu cu:

- Nng t 100 1000 ng/ml: thu hi t 80 110 % [21].

- Nng t 10 100 ng/ml: thu hi t 60 115 % % [21].

chnh xc: s thng nht (mc phn tn) kt qu gia mt lot php o t nhiu ln ly mu trn cng mt mu th ng nht di nhng iu kin m

t.

chnh xc c th chia lm 3 cp : lp li, chnh xc trung gian v

ti lp. Chng ti tin hnh kho st lp li v chnh xc trung gian.

- lp li: tin hnh cng iu kin th nghim trong khong thi gian ngn.

Yu cu: Nng t 100 1000 ng/ml: RSD% t 11 15% [21].

- chnh xc trung gian: thc hin trong cng mt phng th nghim, thc

hin hai ngy khc nhau.

Yu cu: Nng t 100 1000 ng/ml: RSD% t 11 15% [21].

- Gp kt qu 2 ngy: FTN < FLT

Vi FTN = (FTN > 1)

FLT (0,05,5,5) Kho st gii hn pht hin (LOD) v gii hn nh lng (LOQ)

- Gii hn pht hin (LOD) ca cefixim c xc nh da vo h s gc ca

ng chun v lch chun ca p ng.

- Gii hn nh lng (LOQ) c tnh bng 3,3 ln gii hn pht hin [15].

2.3.4 Phng php x l s liu

Cc kt qu thng k c tnh da vo cc hm trong Microsoft Excel.

19

CHNG 3 : KT QU NGHIN CU 3.1 XY DNG QUY TRNH PHN TCH

3.1.1 Kho st phng php x l mu

Qua tham kho ti liu chng ti tin hnh x l mu bng cch kt ta protein

v loi tp bng dung mi trc khi phn tch HPLC v phng php ny n gin,

kinh t, tit kim thi gian. Chng ti kho st la chn dung mi x l mu v

lng dung mi tm ra quy trnh x l mu sch nht, hiu sut cao, d thc hin.

Kt qu kho st c nh gi qua hiu sut x l mu : lng cefixim tnh c

trong mu sau x l so snh vi lng cefixim thm vo mu nc thi.

3.1.1.1 La chn loi dung mi x l mu

Cefixim c gi tr log Poctanol/nc = -0,64 nn vic chuyn cefixim t pha nc

(nc thi) sang pha dung mi l kh kh khn. Chng ti tin hnh x l mu vi

mc ch loi tp cht tan trong dung mi hu c, cefixim vn trong pha nc.

Do cn la chn dung mi t ha tan cefixim nht. Chng ti kho st x l mu

nc thi cha cefixim vi 3 dung mi : dicloromethan, diethyl ether, cloroform vi

cng t l mu th : ACN : dung mi = (5 : 1 : 3)

Chun b cc mu th v mu chun :

- Mu chun gc (cefixim 50 g/ml trong nc ct): Cn chnh xc khong 25

mg cefixim ha tan trong va 500 ml nc ct.

- Mu th: pha long chnh xc 2 ml mu chun gc trong va 200 ml mu

nc thi trng.

- Mu chun: pha long mu chun gc trong nc ct c nng

cefixim 500 ng/ml.

Tin hnh: - Ln lt x l mu th vi tng dung mi trong 3 dung mi dicloromethan,

cloroform, diethyl ether.

- Dung dch th: mu th c acid ha vi HCl 1N n pH khong bng 3,

th bng giy ch th mu pH. Ly chnh xc 5 ml dung dch th vo cc ng

nghim, thm 1ml ACN, lc xoy trong 30s, em ly tm tc 3000 vng/pht

20

trong 3 pht, ly dch trong ni pha trn. Thm 3 ml dung mi chit lc trong 5

pht, cho tch lp. Ly lp nc (i vi dicloromethan, cloroform ly dch pha

trn, i vi diethyl ether ly dch pha di) ly tm nu cn, lc qua mng lc 0,45

m.

- Dung dch chun: mu chun c lc qua mng lc 0,45 m .

- Tin hnh sc k theo iu kin sc k chn mc 3.1.3.

Kt qu thu c nh hnh 3.1 v bng 3.1.

Bng 3.1.Kho st la chn dung mi x l mu

Chun

Chit vi dicloromethan

Chit vi cloroform

Chit vi diethyl ether

Din tch pic(mAu.s) 153672 124109 75993 64927 Hiu sut chit (%) - 80,76% 49,45% 42,25%

Hnh 3.1.Sc k kho st la chn dung mi x l mu

b.X l mu vi cloroform

c.X l mu vi diethyl ether

a.X l mu vi dicloromethan

21

Nhn xt :

- C 3 dung mi u d tch lp, khng b nh ha trong qu trnh x l mu.

Qu trnh x l mu tng i d dng v tn t thi gian.

- Mu x l vi dicloromethan cho hiu sut cao nht.

- Mu x l vi dicloromethan v diethyl ether tng i sch, trong khi

mu chit vi cloroform vn cn nhiu tp cht.

Do da trn tiu ch v thi gian x l mu, hiu sut, sch tp cht ca

mu, n gin chng ti chn dicloromethan l dung mi x l mu.

3.1.1.2. La chn t l dung mi x l mu

Chng ti tin hnh kho st t l th tch ca mu th, ACN, dicloromethan

la chn t l dung mi hp l nht.

Chun b cc dung dch th v chun nh phn 3.1.1.1

Tin hnh : - X l mu th vi t l dung mi dicloromethan khc nhau. Chng ti tin

hnh kho st 3 t l v th tch ca mu th, MeCN, dicloromethan nh sau :

+ Mu th : ACN : dicloromethan = (5 : 1 :2)

+ Mu th : ACN : dicloromethan = (5 : 1 :3)

+ Mu th : ACN : dicloromethan = (5 : 1 :4)

- Cc bc tin hnh nh mc 3.1.1.1

- Kt qu thu c nh hnh 3.2.

22

Hnh 3.2 Sc k kho st la chn lng dung mi x l mu

Nhn xt:

- Vi c 3 t l dung mi x l mu khc nhau, cc mu u khng b nh ha,

d dng tch lp.

- Vi t l mu th : ACN : dicloromethan = (5:1:2), nn mu vn cn nhiu

tp cht, hiu sut thp.

- Vi t l mu th : ACN : dicloromethan = (5:1:4), nn mu sch tuy nhin

hiu sut thp.

- Vi t l mu th : ACN : dicloromethan = (5:1:3), nn mu sch, hiu sut

tng i cao 80,76%.

a.Mu th : ACN : dicloromethan = (5 : 1 :2)

c. Mu th : ACN : dicloromethan = (5 : 1 :4)

b. Mu th : ACN : dicloromethan = (5 : 1 :3)

23

Do da trn tiu ch v sch tp cht trong mu, hiu sut cefixim sau

x l mu chng ti la chn t l mu th : ACN : dicloromethan = (5: 1 :3) tin

hnh x l mu trc khi phn tch.

Nh vy, qua qu trnh kho st la chn dung mi, lng mi dung x l

chng ti la chn quy trnh x l mu c th nh sau:

Acid ha mu th bng HCl 1N n pH khong bng 3, th bng giy ch th

mu pH. Ly chnh xc 5 ml mu th v 1ml ACN cho vo ng nghim, lc xoy

30s, ly tm 3000 vng trong 3 pht, ly dch ni pha trn. Thm 5 ml

dicloromethan, lc trong 5 pht, yn cho tch lp. Ly lp nc pha trn (ly tm

nu cn), lc qua mng lc 0,45 m.

3.1.2 Kho st quy trnh phn tch

Mt nh my sn xut cephalosporin thng sn xut ng thi nhiu

cephalosporin, do vy iu kin sc k i hi phi nh lng c cefixim khi c

mt cc cephalosporin khc trong nn mu. Chng ti tin hnh kho st thay i

iu kin sc k v ct sc k, thnh phn v tc pha ng, th tch tim mu

nhm tm ra iu kin sc k ti u nht c kh nng tch c cefixim trong hn

hp gm 6 cephalosporin khc: cephadroxil, cefdinir, cefuroxim, ceftazidim,

cefotaxim, cefoperazon.

Tin hnh: pha mt hn hp gm cefixim v 6 cephalosporin k trn c nng

500 ng/ml trong nc ct, lc qua mng lc 0,45 m v tin hnh sc k.

Qua qu trnh kho st chng ti la chn c iu kin sc k nh sau:

Ct: Ct Luna C18 (250 x 4,6 mm; 5 m)

Detector DAD: = 285 nm

Th tch tim mu: 200 l

Nhit ct: 32C

Pha ng: methanol dung dch m phosphat pH = 3,2 (NaH2PO4 40 mM,

chnh pH bng H3PO4)

Pha ng v tc dng: gradient

24

Thi gian (pht) MeOH (%) m phosphat (%) Tc d dng (ml/pht)

0 18 82 1,0

10 45 55 1,0

16 55 45 0,85

18 18 82 0,85

la chn c iu kin sc k nh trn chng ti tin hnh kho st

chn ct sc k, thnh phn v tc pha ng, th tch tim mu, qut ph dung

dch chun tm max nh sau:

3.1.2.1 Kho st la chn ct sc k

- Chng ti tin hnh kho st trn 3 loi ct Hypersil C18 BDS (250 x

4.6mm, 5m), ct Zorbax SB (300 x 4.6mm, 5m), ct Luna C18 (250 x 4.6mm,

5m) vi cc iu kin khc nh mc 3.1.3.

Kt qu nh hnh 3.3

25

Hnh 3.3 Kho st la chn ct sc k

Nhn xt:

- Sc k tin hnh trn ct Hypersil C18 BDS v ct Zorbax SB18 pic ca

cefixim khng tch khi pic ca cc cephalosporin khc v khng cn xng.

- Sc k tin hnh trn ct Luna C18, pic cefixim tch khi pic ca cc

cephaloporin khc, pic cn xng. Th t ra gii cc cephalosporin ln lt nh sau

cephadroxil, ceftazidim, cefdinir, cefixim, cefuroxim, cefotaxim, cefoperazon.

Do chng ti la chn ct Luna C18 tip tc nghin cu.

3.1.2.2 Kho st thnh phn pha ng v tc dng

Chng ti tin hnh kho st 2 chng trnh pha ng v tc dng. Cc

iu kin sc k khc nh mc 3.1.3. Chng trnh pha ng v tc dng kho

st nh sau:

a. Ct Hypersil C18 BDS b. Ct Zorbax SB18

c. Ct Luna C18

26

- Chng trnh 1: chng trnh ng dng pha ng

+ Pha ng: hn hp ACN : MeOH (50:50) : dung dch acid

trifluoroacetic(19:81)

+ Tc dng: 2ml/pht

+ Nhit ct: 50 C

+ Cc iu kin khc nh mc 3.1.3.

- Chng trnh 2: chng trnh gradient pha ng

Cc iu kin sc k ca chng trnh gradient pha ng nh mc 3.1.3.

Kt qu thu c nh hnh 3.4

Hnh 3.4 Sc k kho st thnh phn pha ng v tc dng

Nhn xt:

- Sc k kho st chng trnh ng dng pic cefixim khng tch khi cc

cephalosporin khc v pic khng cn xng.

- Vi sc k kho st chng trnh gradient pha ng pic cefixim tch khi

cc cephalosporin khc, pic cn xng.

V vy chng ti s dng chng trnh gradient tin hnh cc nghin cu

tip theo.

3.1.2.3. Kho st th tch tim mu

Chng ti tin hnh kho st cc th tch tim mu: 20 l, 50 l, 200 l.

Cc iu kin sc k khc nh mc 3.1.3.

a.Chng trnh ng dng pha ng

b.Chng trnh gradient pha ng

27

Kt qu thu c nh sau:

- V kh nng tch cefixim khi cc cephalosporin khc: vi c 3 th tch tim

mu, pic cefixim u tch khi pic ca cc cephalosporin khc, pic cn xng.

- V din tch pic ca cefixim kt qu c ghi li trong bng 3.2

Bng 3.2 Kt qu kho st th tch tim mu

Th tch tim mu (l) 20 50 200

Din tch pic (mAu.s) 32501 66860 148302

Hnh 3.5 Mi lin quan gia th tch tim mu v din tch pic

Vi th tch tim mu 200 l, pic cefixim vn tch khi pic cc cephalosporin

khc, pic cn xng, tng p ng din tch pic. Do vy, chng ti chn th tch tim

mu 200 l tin hnh nghin cu tip.

Tin hnh sc k theo iu kin mc 3.1.3 dung dch chun pha nh mc

3.1.1.1 t = 220 400 nm thu c max = 285 nm. V vy, chn bc sng pht

hin ti = 285 nm.

3.1.3 Quy trnh phn tch

T nhng kt qu kho st trn, chng ti a ra quy trnh nh lng

cefixim trong nc thi c th nh sau:

Chun b dung dch th v dung dch chun:

28

- Mu chun gc (cefixim 50 g/ml): Cn chnh xc khong 25 mg cefixim

ha tan trong va 500 ml nc ct.

- Mu th (cefixim 500ng/ml): pha long chnh xc 2 ml mu chun gc trong

va 200 ml mu trng.

- Mu chun (cefixim 500ng/ml) trong nc ct : pha long mu chun gc

trong nc ct c nng 500ng/ml.

- Dung dch th: Acid ha mu th bng HCl 1N n pH khong bng 3, th

bng giy ch th mu pH. Ly chnh xc 5 ml mu th v 1 ml ACN cho vo ng

nghim, lc xoy 30s, ly tm 3000 vng trong 3 pht, ly dch ni pha trn. Thm

3 ml dicloromethan lc trong 5 pht. Ly lp nc pha trn (ly tm nu cn), lc

qua mng lc 0,45 m.

- Dung dch chun: mu chun (cefixim 500 ng/ml) lc qua mng lc 0,45 m.

iu kin sc k: - Ct: Ct Luna C18 (250 x 4,6 mm; 5 m)

- Detector DAD: = 285 nm

- Th tch tim mu: 200 l

- Nhit ct: 32

- Pha ng: methanol dung dch m phosphat pH = 3,2 ( NaH2PO4 40 mM,

chnh pH bng H3PO4)

- Pha ng v tc dng: gradient

Thi gian (pht) MeOH (%) m phosphat (%) Tc d dng (ml/pht)

0 18 82 1,0

10 45 55 1,0

16 55 45 0,85

18 18 82 0,85

Tin hnh: Tin hnh sc k dung dch th v dung dch chun, ghi thi gian lu v din tch

pic cefixim trn sc k dung dch th v dung dch chun.

29

Tnh ton kt qu: - nh tnh: nh tnh cefixim c th c trong mu th da vo so snh thi

gian lu v ph UV ca pic xut hin trong sc k ca mu th vi thi gian lu

ca cefixim trong mu chun.

- nh lng: Nng cefixim trong 100 ml mu th (X%)

X (g /100ml) = X C x 100

Trong :

- AT v AS ln lt l din tch pic ca cefixim trong sc k ca mu th v mu chun.

- C: nng cefixim trong dung dch chun tnh theo g/ml. 3.2.NH GI PHNG PHP

3.2.1. thch hp ca h thng

Pha long mu chun gc chun b nh mc 3.1.3 bng nc ct c mu

cefixim chun nng 600 ng/ml, lc qua mng lc 0,45 m. Tim 6 ln dung dch

chun cefixim 600 ng/ml. Tin hnh sc k theo iu kin chn nh mc 3.1.3

chng ti thu c kt qu nh sau:

Bng 3.3 Kt qu thch hp ca h thng

Th t Thi gian lu (pht) Din tch pic (mAu.s) 1 9,68 189691 2 9,76 189182 3 9,76 192665 4 9,76 191288 5 9,76 193989 6 9,68 188626

Trung bnh 9,73 190906,8 SD 0,0413 2116,21

RSD% 0,424 1,11 Nhn xt:

30

Kt qu cho thy RSD ca thi gian lu RSD% = 0,424 1% v RSD ca

din tch pic RSD% = 1,11% 2%, t yu cu cho php phn tch nh lng

cefixim.

3.2.2 c hiu

c hiu c nh gi qua phn tch cc dung dch th, dung dch chun,

dung dch mu trng.

Chun b cc dung dch chun, dung dch th, dung dch mu trng nh sau:

- Dung dch th, dung dch chun: chun b nh mc 3.1.3.

- Dung dch mu trng chun b nh dung dch th nhng khng cho cefixim

vo mu trng.

Tin hnh: Tim cc dung dch chun, dung dch th, dung dch mu trng vo my sc k hiu nng cao v tin hnh sc k theo iu kin chn nh phn

3.1.3.

Kt qu thu c nh hnh 3.3

a. Mu chun

c.Mu th

b.Mu trng

31

Hnh 3.6 Sc k kho st c hiu: mu chun (a), mu trng (b), mu th (c)

Ly ph DAD cefixim ti thi gian lu tng ng trong dung dch th v

dung dch chun, tin hnh so ph. Kt qu thu c nh hnh 3.7

Hnh 3.7.Ph cefixim trong dung dch th v trong dung dch chun

Ngoi ra chng ti cn kho st c hiu khi phn tch ng thi cefixim

v 6 cephalosporin khc gm cephadroxil, cefdinir, cefuroxim, ceftazidim,

cefotaxim, cefoperazon. Chng ti tin hnh pha mt hn hp gm 6 cephalosporin

ny v cefixim, mt hn hp khc ch gm 6 cephalosporin ny vi nng 500

ng/ml mi cht trong nc ct tin hnh sc k theo iu kin chn nh mc

3.1.3. Kt qu c ghi li trong hnh 3.8

a.Hn hp 6

cephalosporin v cefixim b. Hn hp gm 6 cephalosporin

khng c cefixim

32

Hnh 3.8 Sc k kho st c hiu khi phn tch cefixim v 6 cephalosporin khc

Nhn xt:

- Trn sc k ca dung dch mu trng khng xut hin pic no ti thi gian

lu tng ng ca cefixim trong sc k dung dch chun.

- Trn sc k ca dung dch th xut hin pic tng ng vi thi gian lu

ca cefixim trong sc k dung dch chun.

- Bc sng hp th cc i ca chun l = 285 nm, ca th l = 285 nm,

so ph DAD cht ny cho h s SI = 0,9902 0,990.

Do c th kt lun phng php ny c c hiu cao.

Khi phn tch hn hp 6 cephalosporin khc v cefixim:

- Trn sc k phn tch 6 cephalosporin khc khng cha cefixim khng

xut hin pic no ti thi gian lu tng ng ca cefixim trong sc k dung dch

chun.

- Trn sc k phn tch 6 cephalosporin v cefixim xut hin pic ti thi

gian lu tng ng ca cefixim trong sc k dung dch chun.

Nh vy, phng php ny c hiu cao ngay c khi phn tch cefixim trong

dung dch th c cha mt hay nhiu loi trong 6 cephalosporin k trn.

3.2.3 tuyn tnh

Chng ti tin hnh kho st tuyn tnh gia din tch sc k v nng

cc cht phn tch, c th nh sau:

T mu chun gc chun b nh phn 3.1.3 chng ti chun b cc mu chun

lm vic: pha long mu chun gc vi nc ct c cc dung dch c nng

t 30 ng/ml n 1200 ng/ml, lc qua mng lc 0,45 m.

Tin hnh sc k theo iu kin chn nh mc 3.1.3, chng ti thu c kt

qu nh sau:

33

Bng 3.4 Kt qu kho st tuyn tnh

Dung dch Nng thc

(ng/ml)

Din tch pic

(mAu.s)

1 30,46 10007

2 60,91 16931

3 121,82 36312

4 609,10 188626

5 1218,20 385332

Phng trnh y = 316,70x - 1838

r = 0,99995 H s tng quan

Hnh 3.9.a th biu din s ph thuc gia nng (ng/ml) v din tch pic

(mAu.s) ca cefixim

34

Hnh 3.9.b.Sc k kho st tuyn tnh

Nhn xt: Kt qu kho st tnh tuyn tnh cho thy trong khong nng t

30 ng/ml n 1200 ng/ml c s tng quan cht ch gia nng v din tch pic

(h s tng quan r = 0,99995 0,997).

3.2.4. ng

ng ca phng php c tin hnh theo quy trnh phn tch trn mu t

to cha lng hot cht bit. T mu chun gc 50 g/ml chun b nh mc

3.1.3, chng ti pha long 5 ln c mu chun nng 10 g/ml.

Ly mu chun nng 10 g/ml thm vo mu trng c cc mu th c

nng ln lt l 60 ng/ml, 500ng/ml, 800ng/ml. Mi nng 3 mu. Lc qua

mng lc 0,45 m.

Tin hnh phn tch theo quy trnh la chn nh phn 3.1.3

Tnh lng chun thu hi c theo phng trnh hi quy xy dng v so

snh vi lng chun thm vo. Kt qu thu c nh bng 3.5.

35

Bng 3.5 Kt qu kho st ng

Th t

mu th

Nng

(ng/ml)

Lng

chun

thm vo

(g/100ml)

Din tch

pic

(mAu.s)

Lng

chun tm

li

(g/100ml)

Kh

nng

thu hi

(%)

Chun 162893

1.1 60 6,24 13873 4,96 79,49

1.2 60 6,24 12791 4,62 74,04

1.3 60 6,24 11985 4,36 69,87

Trung bnh 74,47

RSD (%) 6,48

2.1 500 51,99 132012 42,25 81,27

2.2 500 51,99 135313 43,29 83,27

2.3 500 51,99 125926 40,33 77,57

Trung bnh 80,70

RSD (%) 3,58

3.1 800 83,18 223377 71,09 85,47

3.2 800 83,18 215313 68,55 82,41

3.3 800 83,18 209546 66,72 80,21

Trung bnh 82,70

RSD (%) 3,19

Nhn xt:

- Ti nng 60 ng/ml, thu hi l 74,47 % (nm trong khong t 60

115%), RSD% = 6,48 (nm trong khong 21%).

- Ti nng 500 ng/ml, thu hi l 80,70 % (nm trong khong t 80

110%), RSD% = 3,58 (nm trong khong 15 %).

36

- Ti nng 800 ng/ml, thu hi l 82,70 % (nm trong khong t 80

110%), RSD% = 3,19 (nm trong khong 15 %).

Kt qu cho thy phng php c ng t yu cu.

3.2.5 Kho st chnh xc

3.2.5.1 lp li

lp li ca phng php c tnh trn kt qu phn tch cc mu c lp

trong cng mt iu kin phn tch. Chng ti s dng 6 mu th chun b nh

phn 3.1.3.

Tin hnh nh lng c lp mu th theo quy trnh la chn mc 3.1.3.

Tnh hm lng trung bnh, tnh RSD ca hm lng thu c kt qu nh

bng 3.7.

Nhn xt:

Kt qu th lp li ca phng php cho thy vi cc iu kin phn tch

chn, lch chun tng i thu c khong 4,88% < 15%. Nh vy phng

php c lp li p ng c yu cu.

3.2.5.2 chnh xc trung gian

- Chng ti kho st chnh xc trung gian ca phng php trn c s phn

tch cc mu c lp trong cng mt iu kin phn tch. Chng ti s dng 6 mu

th c chun b nh phn 3.1.3

- nh lng 6 ln trn mu th theo quy trnh chn phn 3.1.3, nhng

tin hnh vo ngy phn tch khc.

- Tnh kt qu hm lng cc cht trong mu th, lch chun, thu c kt

qu nh bng 3.6.

37

Bng 3.6 Kt qu lp li v chnh xc trung gian

Din tch pic (mAu.s)

Nng (g/100ml)

Thng s thng k

lp li

Chun 150252 48,72

CTB = 39,78 (g/100ml) SA = 1,93

RSD (%) = 4,88

Ln 1 116667 37,41 Ln 2 127300 40,76 Ln 3 118239 37,90 Ln 4 129555 41,58 Ln 5 130141 41,56 Ln 6 119321 38,25

chnh xc trung

gian

Chun 154624 49,31

CTB = 39,25 (g/100ml) SB = 3,00

RSD (%) = 7,65

Ln 1 126353 40,47 Ln 2 119206 38,21 Ln 3 129887 41,58 Ln 4 123073 39,43 Ln 5 105313 33,82 Ln 6 131169 41,99

Nhn xt:

Kt qu chnh xc khc ngy c lch chun tng i 7,65 % < 15%.

nh gi kt qu phn tch c tng t nhau khi phn tch cng mt mu

th vo ngy khc, chng ti so snh vi kt qu ca phn lp li ca phng

php.

So snh kt qu th lp li gia hai ln th nghim s dng test F:

FTN =

Kt qu : FTN = = 2,42

FTN < 5,05

(FLT (0,05,5,5) =5,05) Vy phng php c chnh xc t yu cu

38

3.2.6.Gii hn pht hin (LOD) v gii hn nh lng (LOQ)

3.2.6.1.Gii hn pht hin

Chng ti xc nh LOD ca phng php da vo ng tuyn tnh v

lch chun ca p ng.

- Dung dch th: T dung dch chun 500 ng/ml (chun b nh phn 3.1.3),

pha long vo mu trng c cc nng cefixim chnh xc khong 20 ng/ml.

Chun b 6 mu.

- Tin hnh phn tch theo quy trnh xy dng 6 dung dch, ghi li p ng

din tch pic.

- Tnh lch chun ca din tch pic cefixim.

- Gii hn pht hin di c tnh theo cng thc:

LOD =

Trong :

S: lch chun ca p ng

a: dc ng chun

Kt qu thu c bng 3.7 v hnh 3.10

Bng 3.7 Kt qu kho st LOD

Th t Din tch pic (mAu.s) 1 9912 2 9372 3 9382 4 7890 5 7818 6 9612

Gi tr trung bnh 8997,7 lch chun (S) 907,721

H s gc (a) 316,70 LOD (ng/ml) 9,46

39

Hnh 3.10 Sc k st LOD

Nhn xt:

Nh vy, gii hn pht hin ca cefixim l 9,46 ng/ml.

3.2.5.2 Gii hn nh lng

Gii hn nh lng l lng nh nht ca cht phn tch trong mu th c th

nh lng c vi ng v chnh xc thch hp.

Cch xc nh:

LOQ = 3,3 LOD = 3,3 x 9,46 = 28,37 ng/ml.

Nh vy, gii hn nh lng ca phng php l 28,37 ng/ml.

3.3.BN LUN

Nh vy qua nghin cu, chng ti xy dng c quy trnh nh lng

cefixim trong nc thi nh my vi s c mt ng thi ca 6 cephalosporin khc.

Quy trnh phn tch bao gm giai on x l mu v giai on phn tch bng h

thng HPLC UV-DAD.

Giai on x l mu bao gm kt ta protein v loi tp bng dung mi loi

b c nhiu tp cht trong mu nc thi, nn mu tng i sch, hiu sut kh

cao trn 80%. Quy trnh x l mu tng i n gin, d thc hin, chi ph thp, c

th p dng rng ri. Tuy nhin khi so snh vi SPE th quy trnh x l ny c hiu

sut sau x l thp hn v c bit cha lm giu c mu trc khi phn tch.

Phn tch bng h thng HPLC UV-DAD c th p dng rng ri v h thng

HPLC UV-DAD kh ph bin ti cc phng th nghim. Trong khi , h thng

40

LC-MS hoc LC-MS/MS t tin v cha ph bin ti Vit Nam. Tuy nhin, h

thng LC-MS hoc LC-MS/MS li cho php phn tch mu nc c nng

cefixim rt nh thch hp cho phn tch d lng khng sinh trong nc thi. V

khng sinh c th gy khng thuc vi khun ngay nng du vt.

Nng cefixim trong mu nc thi dao ng ln do vy chng ti kho

st tuyn tnh trong khong rng t 30 ng/ml n 1200 ng/ml.

Phng php chng ti xy dng c kh nng nh lng cefixim trong

mu nc thi c cha 6 cephalosporin khc cephadroxil, cefdinir, cefuroxim,

ceftazidim, cefotaxim, cefoperazon. iu ny l ph hp vi thc t mi dy

chuyn sn xut cephalosporin khng ch sn xut n l mt loi cephalosporin m

sn xut ng thi nhiu cephalosporin cng lc. 6 cephalosporin v cefixim cng

l nhng loi khng sinh c s dng v sn xut nhiu ti Vit Nam, do vy

phng php c th p dng cho mu nc thi ca nhiu nh my khc nhau.

Hin nay, vn d lng khng sinh trong nc thi cha c quan tm

ng mc. Ngy 28/12/2011, B Ti Nguyn v Mi Trng ban hnh thng t s

47/2011/TT-BTNMT quy nh quy chun quc gia v mi trng. Ban hnh km

theo thng t ny l quy chun k thut quc gia v nc thi cng nghip. Trong

c quy nh gi tr ti a cho php ca cc thng s nhim trong nc thi

cng nghip khi x ra ngun tip nhn nc thi. Cc thng s ny quy nh cc ch

tiu v pH, mu, nhit , oxy trong nc, d lng cc kim loi nng nh ch, thy

ngn, ng, km,, mt s cht hu c ni chung nh tng lng cyanua, tng

lng du m khong, cht bo v thc vt, cht phng x[4]. D lng khng

sinh trong nc thi cha c quy nh c th. Trong khi , vi ngnh cng

nghip dc ang pht trin mnh m nhiu nh my sn xut khng sinh c ra

i. Nhiu nh my ch ngh n li ch trc mt, thi trc tip nc thi cha qua

x l vo mi trng. Ngun nc ny c d lng khng sinh cao. Tn d khng

sinh trong nc thi s dn n chng c mt trong nc b mt, nc ngm v

cui cng l nc ung. Cefixim cng nh nhiu khng sinh khc c pht trin

41

da trn tc dng ca n trn sc khe con ngi, chng c tc dng ln h thng

sinh hc ngay nng du vt.

Nguy c ln nht khi cefixim tn d trong nc thi l c th lm cho vi sinh

vt thch ng nhiu hn v nhiu chng khng thuc pht trin trong vng nc t

nhin. Tuy nhin li cha c mt nghin cu no a ra mc nng c th ca

cefixim trong nc thi c th gy khng thuc trn vi khun. Jerker Fick v cng

s nghin cu nng cefixim trong nc thi nh hng n c, t xut

vi nng cefixim trong nc thi cao hn mc 6,2 g/ml c th gy nh hng

tiu cc ln mi trng v sc khe con ngi [22]. Vi mc nng ny, phng

php nh lng chng ti nghin cu hon ton c th nh lng c.

42

CHNG 4: KT LUN V XUT 4.1. KT LUN

Qua qu trnh thc hin ti, chng ti t c cc kt qu nh sau:

xy dng phng php x l mu v iu kin phn tch nh lng cefixim trong nc thi nh my sn xut dc phm.

Quy trnh x l mu Acid ha mu th bng HCl 1N n pH khong bng 3, th bng giy ch th

mu pH. Ly chnh xc 5 ml mu th v 1 ml ACN cho vo ng nghim, lc xoy

30s, ly tm 3000 vng trong 3 pht, ly dch ni pha trn. Thm 3 ml diclorometan

lc trong 5 pht. Ly lp nc pha trn (ly tm nu cn), lc qua mng lc 0,45

m.

iu kin sc k - Ct: Ct Luna C18 (250 x 4,6 mm; 5 m)

- Derector DAD: = 285 nm

- Th tch tim mu: 200 l

- Nhit ct: 32

- Pha ng: methanol dung dch m phosphat pH = 3,2 ( NaH2PO4 40 mM,

chnh pH bng H3PO4)

- Pha ng v tc dng: gradient

Thi gian (pht) MeOH (%) m phosphat (%) Tc d dng (ml/pht) 0 18 82 1,0

10 45 55 1,0 16 55 45 0,85 18 18 82 0,85

tin hnh thm nh phng php vi cc ch tiu: thch hp ca h thng: RSD% ca thi gian lu 0,43; RSD% ca

din tch pic 1.1085.

43

Phng php c hiu khi nh lng cefixim trong nc thi v nh lng cefixim khi c mt ng thi c 6 cephalosporin khc trong nn mu:

cephadroxil, cefdinir, cefuroxim, ceftazidim, cefotaxim, cefoperazon.

Khong tuyn tnh: 30 1200 ng/ml. ng:

Nng (ng/ml) thu hi (%) RSD% 60 74,47 6,48

500 80,70 3,58 800 82,70 3,19

chnh xc: - lp li ca phng php RSD% = 4,88%.

- chnh xc trung gian

+RSD% = 7,65 %.

+ Test F: < FTN = 0,41 < 5,05

Gii hn pht hin l 9,46 ng/ml, gii hn nh lng l 28,37 ng/ml. 4.2. XUT

- Nghin cu n nh ca cefixim trong mu nc thi.

- p dng phng php nh lng cefixim trong nc thi, pht trin

phng php xc nh thm 6 cefalosporin bao gm cephadroxil, cefdinir,

cefuroxim, ceftazidim, cefotaxim, cefoperazon trong nc thi v hon thin thm

nh.

TI LIU THAM KHO Ting Vit

1. Trn T An (2007), Kim nghim dc phm, Nh xut bn y hc, tr 84-110.

2. Trn T An, Thi Nguyn Hng Thu (2006), Ha phn tch II, Nh xut bn

y hc, tr 123.

3. Dng Hng Anh (2006), Phn tch nh gi s c mt ca cc khng sinh

h floquinilon trong nc thi bnh vin, ti nghin cu khoa hc ca

Trng i hc Quc Gia H Ni.

4. B ti nguyn v mi trng (2011), Thng t quy nh quy chun k thut

quc gia v mi trng.

5. B y t (2007), Dc th quc gia Vit Nam, Nh xut bn Y hc.

6. B y t (2009), Dc in Vit Nam IV, Nh xut bn Y hc.

7. Trn c Hu (2007), Ha dc tp 2, Nh xut bn y hc, trang 118-119.

8. T Minh Kong (2007), K thut sn xut dc phm tp 2: K thut sn

xut thuc bng phng php sinh tng hp, Nh xut bn y hc, trang 113-

115.

9. Mai Tt T, V Th Trm (2007), Dc l hc tp 2, Nh xut bn y hc,

trang 150-152.

10. Phm Thu Trang (2011), Xy dng phng php nh lng cefixime trong

huyt tng bng sc k lng hiu nng cao, kha lun tt nghip dc s, tr

31- 48.

Ti liu ting Anh

11. AHFS drugs information 2008.

12. Bristish national formulary 61, p342.

13. British Pharmacopoeia 2010.

14. Fick Jerker , Richard H. Lindberg et al (2010),Predicted critical

environmental concentrations for 500 pharmaceuticals, Regulatory

Toxicology and Pharmacology 58 516523.

15. ICH, Validation of analytical procedures (2005): Text and Methodolody

Q2(R1).

16. Khan Abbas, Zafar Iqbal et al (2011),Simultaneous determination of

cefdinir and cexime in human plasma by RP-HPLC/UV detection

method:Method development, optimization, validation, and its application to

a pharmacokinetic study, Journal of Chromatography B, 879 p24232429.

17. Lindberga Richard, Per-Ake Jarnheimer et al (2004),Determination of

antibiotic substances in hospital sewage water using solid phase extraction

and liquid chromatography/mass spectrometry and group analogue internal

standards, Chemosphere 57 14791488.

18. Martidale thirty-six edition, p 224.

19. McAteer Joy A., Mark F. Hlltke et al (1987), Liquid-Chromatographic

Determination of Five Orally Active Cephatosporins-Cefixime, Cefaclor,

Cefadroxil, Cephalexin, and Cephradine-in Human Serum, CLIN. CHEM.

33/1 0, 1788-1 790.

20. Nemutlua Emirhan, Sedef Kra et al (2009), Simultaneous multiresponse

optimization of an HPLC method to separate seven cephalosporins in plasma

and amniotic uid: Application to validation and quantication of cefepime,

cexime and cefoperazone, Talanta 80 p117126.

21. The scientific asociation dedicated to analytical excellence (2012), Standard

Format and Guidance for AOAC Standard Method Performance

Requirement (SMPR) Documents.

22. The United States Pharmacopoeia 32 (2009).

23. Water analysis & research center of K water (2008), Development of multi

residual analytical method for pharmaceuticals, perfluorinated compounds,

mitrosamines, hormones, persistent organic pollutants, in water volume 1,

Reports on International Collaborative Research, p 151-169.

24. Xu Haomin (2010), Photochemical Fate of Pharmaceuticals in Natural

Waters, Dissertation submitted in partial satisfaction of the requirements for

the degree of doctor of philosophy in Engineering with a Concentration in

Environmental Engineering, University of California, Irvine, p16 17.

Trang web

25. Lm An (2013), Ngy tn th ca thuc khng sinh, th gii v Vit Nam,

a ch truy cp:

http://www.tgvn.com.vn/Item/VN/KhoaHoc/2013/3/C2CAAF59F6DA8B88/

26. Hng Nhung (2012), Con ngi trc thm ha thuc khng sinh v tc

dng, Ngi a tin, a ch truy cp:

http://www.nguoiduatin.vn/con-nguoi-truoc-tham-hoa-thuoc-khang-sinh-vo-

tac-dung-a60726.html

LI CM NMC LCDANH MC CC K HIU, CH VIT TTDANH MC CC BNGDANH MC CC HNH V, TH1 T VN CHNG 1: TNG QUAN1.1 TNG QUAN V CEPHALOSPORIN1.1.1 Lch s ra i, ngun gc1.1.2 Cng thc cu to, phn loi v ph tc dng1.1.3 c tnh ca cc cephalosporin nghin cu

1.2 TNG QUAN V CEFIXIM1.2.6 Mt s phng php nghin cu nh lng cefixim1.2.6.1 Nghin cu nh lng cefixim bng phng php o quang1.2.6.2 Nghin cu nh lng cefixim bng phng php HPLC

1.4 SC K LNG HIU NNG CAO1.4.1 Khi nim1.4.2 Nguyn tc1.4.4 Cc thng s c trng1.4.5 ng dng ca HPLC

CHNG 2: I TNG V PHNG PHP NGHIN CU2.1. I TNG NGHIN CU2.2. PHNG TIN NGHIN CU2.2.1 Ha cht thuc th - cht chun2.2.2. Thit b - dng c

2.3. PHNG PHP NGHIN CU2.3.1 Kho st phng php x l mu2.3.2. Xy dng iu kin sc k2.3.3. nh gi phng php2.3.4 Phng php x l s liu

CHNG 3 : KT QU NGHIN CU3.1 XY DNG QUY TRNH PHN TCH3.1.1 Kho st phng php x l mu3.1.1.1 La chn loi dung mi x l mu3.1.1.2. La chn t l dung mi x l mu

3.1.2 Kho st quy trnh phn tch3.1.2.1 Kho st la chn ct sc k3.1.2.2 Kho st thnh phn pha ng v tc dng3.1.2.3. Kho st th tch tim mu

3.1.3 Quy trnh phn tch

3.2.NH GI PHNG PHP3.2.1. thch hp ca h thng3.2.2 c hiu3.2.3 tuyn tnh3.2.4. ng3.2.5 Kho st chnh xc3.2.5.1 lp li3.2.5.2 chnh xc trung gian

3.2.6.Gii hn pht hin (LOD) v gii hn nh lng (LOQ)3.2.6.1.Gii hn pht hin3.2.5.2 Gii hn nh lng

3.3.BN LUN

CHNG 4: KT LUN V XUT4.1. KT LUN4.2. XUT

TI LIU THAM KHO