Xây dựng phương pháp định lượng Cefaclor trong huyết tương bằng HPLC.pdf

B Y T TRNG I HC DC H NI

TRNG I

XY DNG

PHNG PHP NH LNG

CEFACLOR TRONG HUYT TNG

BNG HPLC

H NI - 2014

B Y T TRNG I HC DC H NI

TRNG I

XY DNG

PHNG PHP NH LNG

CEFACLOR TRONG HUYT TNG

BNG HPLC

Ngi hng dn:

1. TS. L nh Chi.

2. TS. o Danh Sn.

Ni thc hin:

1. B mn Ha phn tch v c cht.

2. Cng ty c phn tp on Merap.

H NI - 2014

LI CM N

***

Trc ht, ti xin c by t lng bit n chn thnh v su sc ti TS. L nh

Chi, ng cm n TS. o Danh Sn, tn tnh hng dn v gip ti trong sut qu

trnh thc hin ti ny.

ng thi, ti cng chn trng cm n cc thy c gio, cc anh ch k thut vin

trong b mn Ha phn tch v c cht - Trng i hc Dc H Ni; Cng ty c

phn tp on Merap tn tnh gip v to nhng iu kin thun li nht ti

hon thnh kha lun tt nghip ny.

Cui cng, ti xin php c gi nhng tnh cm su sc v lng bit n v hn

ti gia nh, ngi thn v bn b, nhng ngi lun gip , ng vin ti trong sut

qu trnh hc tp v hon thnh kha lun tt nghip.

Ti xin chn thnh cm n.

H Ni, ngy 12 thng 5 nm 2014

Trng i

DANH MC K HIU, CH VIT TT

STT K hiu/

Ch vit tt Ting Anh Ting Vit

1 HPLC High Performance Liquid

Chromatography

Sc k lng hiu nng cao

2 US-FDA United State-Food Drug

Administration

Cc qun l thuc v thc

phm Hoa K

3 TSH Bioequivalence (BE) Tng ng sinh hc

4 SKD Bioavailability (BA) Sinh kh dng

5 SD Standard Deviation lch chun

6 RSD Relative Standard

Deviation

lch chun tng i

7 SK HPLC chromatography Sc k

8 CFC Cefaclor

9 SSS Stock Standard Solution Chun gc

10 WSS Working Standard

Solution

Chun lm vic

11 QC Quality Control Samples Mu kim tra cht lng

12 HQC High Quality Control

Samples

Mu kim tra nng cao

13 MQC Middle Quality Control

Samples

Mu kim tra nng

trung bnh

14 LQC Low Quality Control

Samples

Mu kim tra nng

thp

15 LLOQ Lower Limit of

Quantification

Nng di gii hn

nh lng

16 ULOQ Upper Limit of

Quantification

Nng trn gii hn

nh lng

17 MeCN Acetonitril

18 MeOH Methanol

19 TB Mean Trung bnh

MC LC

T VN .. 1

CHNG I: TNG QUAN... 2

1.1 Tng quan v cefaclor... 2

1.1.1 Cng thc ha hc.. 2

1.1.2 Tnh cht Ha L 2

1.1.3 Dc l, Dc ng hc 2

1.2 Tng quan v sc k lng hiu nng cao (HPLC).. 4

1.2.1 C s l thuyt 4

1.2.2 Mt s i lng c trng trong HPLC 5

1.2.3 ng dng ca HPLC 7

1.2.4 ng dng HPLC phn tch cefaclor trong dch sinh hc 8

1.3 Thm inh phng php php nh lng dc cht

trong dch sinh hc

11

1.3.1 Khi nim 11

1.3.2 Ni dungthm nh phng php 11

CHNG II: I TNG NGHIN CU V PHNG

PHP NGHIN CU.

15

2.1 iu kin thc nghim v ni dung nghin cu. 15

2.1.1 iu kin thc nghim 15

2.1.1.1 Nguyn liu, ha cht, dung mi. 15

2.1.1.2 Dng c, thit b. 16

2.1.2 Ni dung nghin cu.. 17

2.2 Phng php nghin cu.. 17

2.2.1 Chun b mu huyt tng t to cha chun cefaclor.. 17

2.2.2 Xy dng quy trnh chit cefaclor trong huyt tng.. 19

2.2.3 Xc nh iu kin sc k... 21

2.2.4 Thm nh phng php phn tch. 19

2.2.4.1 Tnh c hiu chn lc 19

2.2.4.2 ng chun v khong tuyn tnh 19

2.2.4.3 Gii hn nh lng di (LLOQ).. 20

2.2.4.4 ng, lp li. 20

2.2.4.5 Hiu sut chit (T l thu hi). 20

2.2.4.6 n nh ca mu th trong huyt tng 20

2.3 Phng php x l kt qu.. 21

CHNG III: THC NGHIM V KT QU 22

3.1 Xy dng phng php nh lng 22

3.1.1 Kho st iu kin sc k 22

3.1.2 Kho st phng php chit tch cefaclor. 25

3.2 Thm inh phng php phn tch. 27

3.2.1 c hiu chn lc... 27

3.2.2 ng chun v khong tuyn tnh 28

3.2.3 Gii hn nh lng di LLOQ 30

3.2.4 ng, chnh xc trong ngy v khc ngy 31

3.2.5 Hiu sut chit. 33

3.2.6 n nh ca mu huyt tng 34

3.3 Bn lun. 36

CHNG IV: KT LUN V XUT.. 38

4.1 Kt lun.......................... 38

4.2 xut.... 39

PH LC

DANH MC CC BNG TRONG KHA LUN

Bng 1.1 Mt s quy trnh nh lng cefaclor (trong dch sinh hc) bng

HPLC

Bng 2.1. Cc cht chun s dng trong nghin cu

Bng 2.2. Cc thuc th v dung mi s dng trong nghin cu

Bng 2.3. Cc mu huyt tng trng s dng trong nghin cu

Bng 2.4. Cc thit b s dng trong nghin cu

Bng 2.5. Cu hnh h thng HPLC Agilents 1260

Bng 2.6. Cch chun b ng chun mu huyt tng t to

Bng 2.7. Cch chun b mu QC

Bng 3.1. Bng so snh cc thng s ca pc cefaclor trong dung dch 10

g/mL vi cc cht to cp ion khc nhau

Bng 3.2. S tng quan gia nng v p ng pc ca cefaclor trong

huyt tng

Bng 3.3. Kt qu xc nh gii hn di LLOQ

Bng 3.4. Kt qu kho st ng, lp li trong ngy

Bng 3.5. Kt qu kho st ng, lp li khc ngy

Bng 3.6.Kt qu kho st hiu sut chit cefaclor

Bng 3.7. Kt qu nghin cu n nh ca mu huyt tng sau 3 chu k

ng - r

Bng 3.8. Kt qu nghin cu n nh mu huyt tng thi gian ngn

Bng 3.9. Kt qu n nh ca mu huyt tng thi gian di

DANH MC CC HNH TRONG KHA LUN

Hnh 1. 1 .S khi ca mt my sc k lng hiu nng cao.

Hnh 3.1. SK chun cefaclor 10 g/mL nng m 10 mM trong pha

ng


ng

Hnh 3.3. SK chun cefaclor nng 10 g/mL khng c cht to cp ion

Hnh 3.4. SK chun cefaclor nng 10 g/mL c cht to cp ion: (a)

tetrabutyl ammonium hydroxgen sulfat; (b) triethanolamin

Hinh 3.5 Sc k mu huyt tng ta protein bng MeCN

Hnh 3.6.a. SK mu huyt tng ta protein bng axit pechloric.

Hnh 3.6.b. SK mu huyt tng ta protein bng axit tricloroacetic.

Hnh 3.7. Sc k mu (a) huyt tng trng v (b) cefaclor 10,0 g/mL

trong huyt tng trng

Hnh 3.8. th ng chun biu din mi tng quan tuyn tnh gia nng

v p ng pc ca cefaclor trong huyt tng

1

T VN

Cefaclor l khng sinh cephalosporin th h th hai, c nhiu u im

ni bt nh tc dng mnh trn vi khun Gram (-) v bn vi cc men -

lactamase ca vi khun, do vy c th trnh c s khng

thuc...Cefaclor c s dng trong cc trng hp nhim vi khun nhy cm

vi thuc, iu tr vim nhim trng ng h hp trn, nhim khun tai mi

hng, c bit l vim tai gia, vim xoang [5]. Ngy nay cefaclor c

sn xut trong nc di dng bo ch vin nang nh vin nang cefaclor

Stada 250 mg ca cng ty STADA Vit Nam, vin nang Mekocefaclor 250

cng ty C phn Ha-Dc Mekophar....Cefaclor cng l thuc c tn trong

Danh mc cc dc cht yu cu bo co s liu th tng ng sinh hc

khi ng k thuc do B y t ban hnh [6].

Ti Vit Nam cng b quy trnh x l mu v nh lng cefaclor

trong huyt tng ngi tnh nguyn bng HPLC [8]. Tuy nhin, vic p

dng ti quy m phng th nghim va v nh, c s vt cht cn thiu thn

ti cc Trung tm Kim nghim Dc M phm tuyn tnh v cc cng ty sn

xut thuc trong nc l cn kh khn. Hng ti mc tiu nng cao cht

lng v hiu qu iu tr ca thuc cefaclor sn xut trong nc th cc n

v kim nghim u mun c ch ng phn tch, gim st nng

cefaclor trong huyt tng ngi.

Xut pht t yu cu thc t , chng ti tin hnh thc hin ti:

Xy dng phng php nh lng cefaclor trong huyt tng bng sc

k lng hiu nng cao vi 2 mc tiu chnh:

1. Xy dng phng php x l mu, kho st iu kin sc k v nh lng

cefaclor trong huyt tng bng sc k lng hiu nng cao.

2. Thm nh phng php xy dng.

2

CHNG I: TNG QUAN

1.1. Tng quan v Cefaclor

1.1.1. Cng thc ha hc

- Cng thc phn t: C15H14ClN3O4S.H2O [9],[11]

- Khi lng phn t: 367,8g/mol [ 9]

- Cng thc cu to:[9],[13]

- Tn khoa hc: (6R,7R)-7-[[(2R)-2-amino-2-phenylacetyl]amino]-3-

chloro-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic monohydrat

[16]

1.1.2. Tnh cht ha l

- Bt trng hoc vng nh.

- Kh tan trong nc (10g/L), thc t khng tan trong methanol v

methylene chlorid, chloroform, benzene (0,5g/L).

- Nhit nng chy dng khan: 2200 C

- Hng s phn ly: pKa1= 1,5; pKa2 = 7,17;

- H s phn b du nc logP (octanol/nc) = 0,35 [12] nn vic chit

cefaclor trong huyt tng bng phng php chit lng - lng l kh khn.

- Cefaclor hp thu bc sng UV c max = 263 nm, do vy c th nh

lng cefaclor bng HPLC vi detector t ngoi [3],[12],[20].

- n nh pH 2,5 - 4,5 [7],[9],[12],[16].

1.1.3. Dc l, dc ng hc

*Dc ng hc:

Cefaclor c hp thu tt khi ung lc i. Vi liu 250 mg v 500 mg

ung lc i, Cmax t 13 g/mL sau 60 pht.

3

- Thi gian bn thi ca cefaclor trong huyt tng t 30-60 pht. Chc

nng thn mt hon ton th thi gin bn thi ko di thm 2, 3 n 22, 8 gi.

- Cefaclor phn b rng ri khp c th, i qua nhau thai v bi tit trong

sa m nng thp.

- Cefaclor thi tr nhanh qua thn, 85% liu s dng c thi tr qua

nc tiu dng khng i trong vng 8gi, phn ln thi tr trong 2 gi u.

- Tng tc thuc: Probenecid lm chm bi tit cefaclor, mt s t

cefaclor c o thi qua thm tch mu [4], [5], [12].

*Ch nh

- iu tr cc nhim khun ng h hp do cc vi khun nhy cm, c

bit sau khi dng cc khng sinh thng thng m tht bi.

- Vim tai gia cp, vim xoang cpm vim hng vim amidan ti pht.

- Vim ph vim ph qun mn trong t din bin.

- Nhim khun ng tit niu di khng bin chng (vim bng

quang).

- Nhim khun da v phn mm do Staphylococus aureus nhy cm v

Streptococus pyogenes [4], [5].

*Chng ch nh

- Ngi c tin s d ng vi khng sinh nhm cephalosporin [4], [5].

*Thn trng

- Bnh nhn c tin s d ng vi penicillin.

- Thn trng khi ch nh cho bnh nhn c tin s mn cm vi

cephalosporin, c bit l vi cefaclor v penecilin.

- Ngi bnh c tin s ng tiu ha, c bit l vim i trng.

- Dng cefaclor di ngy c th gy vim i trng gi mc [4].

*Liu iu tr v cc dng

- Dng ng ung, vo lc i

+ Ngi ln:

- Vim hng, vim amidan, vim ph qun, nhim khun da v m mm,

nhim khun ng tit niu di: 500-1000mg/ln x ngy 2 ln hoc

500mg/ln/ngy 3 ln

4

- Nhim khun nng: 1g/ln/ngy 3 ln

- Liu gii hn k n: 4g/ngy [4], [5].

+Tr em:

- Liu thng dng : 20-40mg/kg cn nng/2-3 ln/ngy

- Vim tai gia: 40mg/kg/cn nng/2-3 ln/ngy, liu tng cng trong

ngy khng qu 1g.

- Liu t a: 1,5g/ngy [4], [5].

1.2. Tng quan v sc k lng hiu nng cao (HPLC) [1]

1.2.1. C s l thuyt [1]

HPLC cn gi l sc k lng hiu nng cao, 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.

Tc di chuyn khc nhau lin quan n i lc tng i ca cc cht

ny vi pha tnh v pha ng. Th t ra gii cc cht ra khi ct v vy ph

thuc vo cc yu t nh bn cht ca pha tnh, phn cc ca pha ng,

pH, nhit

Cc cht sau khi ra khi ct s c pht hin bi Detector v s c

chuyn sang b phn x l kt qu [1]

* Nguyn tc cu to chung ca mt my sc k lng hiu nng cao

Hnh 1. 1 .S khi ca mt my sc k lng hiu nng cao.

H thng cp

dung mi

Bm B phn tim mu

B phn iu nhit

Ct sc

k Detector

H thng thu nhn

d liu

(PC/Recorder)

Thi

5

Loi pha tnh ph bin nht c ch to t silica. Nhm - OH trn b

mt silica phn ng vi dn cht clorosilan to ra dn cht siloxan:

R l mch thng c 8 hoc 18 carbon hoc cc nhm hu c khc nh

amin mch thng, nitril, hydrocarbon thm. Tnh phn cc ca pha tnh thay

i ty theo gc R [1]

Ph thuc vo phn cc tng i ca pha ng v pha tnh m sc

k c phn thnh sc k pha thun v sc k pha o:

- Sc k pha thun: Pha tnh phn cc, cn pha ng l dung mi t phn

cc nh: hexan, iso propyl etherCht t phn cc nht c ra gii u

tin, khi tng phn cc ca pha ng th thi gian lu gim dn.

- Sc k pha o: Pha tnh khng phn cc, cn pha ng l dung mi

phn cc hn nh : acetonitrile, methanol,Cht phn cc nht c ra gii

u tin, khi tng phn cc ca pha ng th thi gian lu tng dn [1]

1.2.2. Mt s i lng c trng trong HPLC

*H s phn b (partition coefficient):

L t s gia nng cht tan trong pha tnh vi nng ca n trong

pha ng:

=

Trong : K: h s phn b.

Cs: nng cht tan trong pha tnh

Cm: nng cht tan trong pha ng

H s phn b khc nhau cng nhiu, kh nng tch 2 cht cng ln.

*Thi gian lu (retention time, tR):

Thi gian lu ca mt cht tan l thi gian t lc tim mu th ti khi

xut hin nh ca cht tan trn sc k , l thng tin v mt nh tnh. Trong

-HCl

6

mt iu kin sc k nht nh, thi gian lu l mt hng s i vi mt cht

tan. Nu tR nh qu th s tch km, nu tR qu ln th pc b long, thi gian

phn tch ko di [1].

*Th tch lu (retention volum, VR):

VR ca mt cht tan l th tch ca lng pha ng b y ra khi ct t

lc bt u tim mu th n khi xut hin nh ca cht tan trn sc k .

VR= tR x F

Trong : VR: th tch lu F: lu lng ca pha ng

tR: thi gian lu

Trn thc t, th tch cht l lng tng cng pha ng tnh t lc tim

mu n khi ra khi h thng.

*H s dung lng (capacity factor, k):

L t s gia lng cht tan trong pha tnh v lng cht tan trong pha

ng:

=

H s dung lng l mt thng s khng ph thuc vo lu lng pha

ng F v chiu di ca ct m ch ph thuc vo bn cht cht cn phn tch

v c tnh ca pha tnh v pha ng.

Tr s ti u 1 k 5; nu k> 5 thi gian phn tch s ko di, nh b

dn rng v nhy tn hiu thp.

* chn lc (selectivity,):

L i lng c trng cho tc di chuyn t i ca hai cht A, B.

=

=

=

Theo quy c B l cht b lu gi mnh hn A nn >1

tch ring 2 cht thng chn trong khong 1,05 - 2,0.

Nu qu ln thi gian phn tch s di [1], [2]

*S a l thuyt N:

Hiu lc ct c o bng s a l thuyt N ca ct

N = 16.

2 = 5.54.

/2

7

Trong : W: chiu rng y pc

W1/2: chiu rng pic o na chiu cao ca pic

S a l thuyt N cng ln th hiu lc ct cng cao (nh nhn, hp)

* phn gii (resolution - Rs):

L i lng biu th tch ca cc cht ra khi nhau trn cng mt

iu kin sc k, c tnh theo cng thc

Rs = ()

Trong : tR1, tR2: thi gian lu ca 2 nh 1 v 2

W1: chiu rng ca nh 1

W2: chiu rng ca nh 2

phn gii Rs th hin 2 yu t nh nhn v khong cch gia cc

nh pic, thng yu cu Rs 5.

1.2.3. ng dng ca HPLC:

*ng dng trong phn tch nh tnh:

Sc k cho ta thi gian lu hoc v tr trn pha tnh ca cht phn

tch; cng vi cc iu kin sc k l nhng thng tin gip ngi phn tch

khng nh s c mt ca cht phn tch trong mu.

nh tnh bng sc k phi kt hp vi quang ph hng ngoi hoc kt

hp vi khi ph cho kt qu chc chn hn. Ph sc k nhn bit s vng

mt ca mt cht no trong hn hp; do ng dng kim tra tp cht

treong mu. Trn sc k khng c pic ti thi gian lu ca tp cht (dng

cht i chiu) khi chy sc k trong cng iu kin.

*ng dng trong phn tch nh lng:

y l ng dng quan trng nht ca HPLC gip nh lng nhiu loi

hp cht khc nhau. C th xc nh nng cha bit ca mt cht trong

dung dch bit trc nng , hay xc nh cu trc ca hot cht khi ghp

ni vi khi ph MS/MS..Ngy nay HPLC cn cho php nh lng cc

thnh phn c hm lng thp, thm ch cc thnh phn di dng vt trong

hn hp phc tp.

Cc phng php nh lng gm c:

8

+ Phng php ng chun (ngoi chun)

+ Phng php thm ng chun

+ Phng php ni chun

+ Phng php chun ha din tch.

1.2.4. ng dng HPLC phn tch cefaclor trong dch sinh hc

Nhng nm va qua c mt s nghin cu c tin hnh nhm

mc ch phn tch cefaclor trong dch sinh hc, da trn k thut HPLC.

Di y l mt vi phng php x l mu tiu biu v iu kin sc k

km theo ca cc tc gi m chng ti tham kho, lit k:

9

Ti

liu

iu kin sc k X l mu

Ct, Detector Pha ng

[12]

Supelcosil LC

-18 (4,6 x 250

mm; 5 m).

Detector UV

265 nm

MeOH : THF : m

(1g

Na.heptansulfonat +

5ml T.E.A/1L nc,

chnh pH 2,3) =

(16:4:80).

Tc dng: 1,4

mL/pht

Chit SPE

Hot ha: 2mL MeOH +

2mL nc ct. Ra gii:

MeOH bc hi dung mi

ha tan cn trong pha

ng.

[12]

Ct RP 18

(4x 100mm,

7m )

MeCN:m

phosphate pH 7 =

(10: 90).

Tc dng : 1,0

mL/pht

To dn xut sau ct

vi Fluorescamin

trong MeCN

Ta protein vi axit

Tricloroacetic 6%lc, ly

tm 4000vng/pht x 10pht

Ly dch ni Tim sc

k.

[19]

Ct ODS Rp

(150 x 4,6

mm, 5m)

To dn xut

trc ct.

A.CN: 0.1M

Triethylamin (pH

8.5) = ( 35:65).

Tc dng: 0,6

mL/pht

40L mu+ 20 L IS

(chun ni) + 25L pyridine

= 50L 35 mM

CIPIC/800ly tmlc,tim

mu

[11]

Ct phn tch

C18

MS/MS

MeOH - Nc - Acid

fomic = (80:20: 1)

Tc dng : 0,5

mL/pht

Ta protein vi hn hp acid

fomic: MeOH. Chit SPE

0,5mL plasma + 100 L IS...

Chit SPE

[8] Ct Gemini

C18, 5 m;

Pha ng: MeCN:

MeOH: m

Ta protein bng acid

pecloric, chit loi tp bng

10

Bng 1.1: Mt s quy trnh nh lng cefaclor trong dch sinh hc bng

HPLC

Nhn xt:

- iu kin sc k:

a s cc ti liu tham kho u s dng ct sc k pha o nh C8,

C18 vi h dung mi pha ng cha MeOH hoc MeCN phi hp vi h m

phosphate, phn tch trn my HPLC - Detector UV. Vi iu kin sc k nh

trn c th d dng p dng quy m phng th nghim kim nghim va v

nh ca cc nh my x nghip sn xut cng nh trung tm kim nghim

dc m phm tuyn tnh.

- Phng php x l mu:

Do cefaclor c h s cn bng du nc bng 0,35 nn kh dng

phng php chit cefaclor trong huyt tng bng dung mi hu c v vy

cc ti liu tham kho trn u x l mu vi k thut c bn l loi protein

trong huyt tng bng cc tc nhn to ta nh MeOH, hn hp acid fomic:

MeOH, acid pecloric, hay axit tricloroacetic.

250 x 4mm.

Detector : UV

263 nm

phosphate pH3,1

(cha TBAH)

t l 10:14:76.

Tc dng: 1,0

mL/pht.

CH2Cl2. Dch thu c tim

sc k

[14]

Ct : C18 (4,6

x 250 mm)

Detector: UV

254 nm

Pha ng: m

NaH2PO4 0.2 M (pH

3.2 0.05 iu chnh

bi axit phosphoric):

MeOH t l 70:30.

Tc dng: 1,0

mL/pht

Ta protein. 500 l huyt

tng +, 100 l IS (cefaclor)

nng (5 g/mL) + 400 L

MeOH ly tm, ly dch lc

tim sc k

11

i vi quy trnh x l mu bng chit SPE, hoc to dn xut trc v

sau ct, s dng detector quang ha tng i phc tp v chi ph cao.

Trong iu kin phng th nghim nc ta vic p dng phng php ny

thc t cn gp nhiu kh khn do t phng th nghim c kinh ph mua

ct, trang thit b chit pha rn v kinh nghim s dng phng php chit ct

pha rn cn hn ch.

Trn c s tham kho cc phng php nh lng c cng b,

chng ti d kin xy dng phng php nh lng cef trong huyt tng

ngi chn quy trnh x l mu l ta protein v s dng my HPLC detector

UV c ng, chnh xc...ph hp vi nghin cu.

1.3. Thm nh phng php phn tch trong dch sinh hc [7], [8], [19]

1.3.1. Khi nim:

Thm nh mt phng php phn tch dc cht sinh hc l qu trnh

xc nh bng nghin cu trong phng th nghim nhng c im c trng

ca phng php m bo phng php t yu cu vi cc ng dng

phn tch thc t.

Do qu trnh phn tch mu sinh hc b nh hng bi nhiu yu t nh

lng mu t, nng thp, ln nhiu tp cht l cc cht ni sinh (cc mui

v c, lipid, protein v cht chuyn ha) v s khc nhau gia cc c th, nn

phng php phn tch phi c thit lp v thm nh m bo tin

cy. [7]

1.3.2. Ni dung thm nh phng php

*Tnh chn lc - c hiu

Tnh chn lc hay tnh c hiu l kh nng nh gi mt cch r rng cht

phn tch khi c mt cc thnh phn khc, c th l tp cht, hoc cc thnh

phn cn tr khc. Trong HPLC, tnh chn lc - c hiu c th hin khi

kt quthu c trn sc k (t mu trng, mu th v mu chun pha

trong mu trng) cho thy pc ca cht cn phn tch c phn tch hon

ton vi cc pc tp [8], [19]

12

*ng chun v khong tuyn tnh

ng chun l ng biu din mi quan h gia p ng ca pc v

nng cht phn tch trong dch sinh hc. Mi quan h ny thng c

nh gi bng phng php hi huy, thu c t phng php hi quy tuyn

tnh. Khong tuyn tnh l khong nng t thp nht n cao nht trong

mt ng chun c p ng tuyn tnh. [8], [19]

ng chun phi c t nht 6 nng ca cht chun pha trong cng

mt mu dch sinh hc.

Khong tuyn tnh phi bao gm ton b khong nng ca cc mu

cn phn tch. Trong khong nng cn kho st, ng chun phi tuyn

tnh, c h s tng quan r 0,98 v phi p ng cc iu kin sau:

- lch so vi gi tr thc ca cc nng phi 15%, tr im gn

LOQ c chp nhn 20%.

- C t nht 4/6 im trong dy chun t tiu chun trn, bao gm c LOQ

v im c nng cao nht.

*Gii hn nh lng di LLOQ

Gii hn nh lng di th hin nhy, chnh xc, ng ca

phng php.

Gi tr LLOQ thng nm trong khong 1/30-1/10 Cmax

Nng chun thp nht trn ng chun c chp nh LLOQ khi tha

mn cc iu kin sau:

- p ng ca mu LLOQ phi gp t nht 5 ln p ng ca mu trng

ti cng thi gian lu

- ng phi bng 80% - 120% so vi nng thc.

- chnh xc th hin bng gi tr RSD % khi tin hnh phn tch trn

t nht 5 mu LLOQ c lp phi nh hn hoc bng 20% [8], [19]

* ng- chnh xc:

ng ca phng php: l gi tr phn nh gn st ca kt qu phn

tch thu c bng phng php vi gi tr ca cht phn tch ny.Gi tr ny

nm trong khong 85% - 115%, ring ti cc im gn gii hn di c th

trong khong 80%-120%.

13

chnh xc ca phng php: l mc thng nht gia cc kt qu

ring bit khi lp li quy trnh phn tch nhiu ln trn cng mu th ng

nht. chnh xc c biu th bng lch chun (SD) hoc lch chun

tng i (RSD). [8], [9].

Yu cu:

- i vi chnh xc trong ngy th gi tr RSD % phi 15%.

- i vi chnh xc gia cc ngy th gi tr RSD % phi 15%.

*Hiu sut chit:

Hiu sut chit hay t l thu hi hot cht l t s gia p ng/nng

hot cht c trong mu c chit tch theo quy trnh chn so vi mu c

cng nng khng c x l qua chit tch.

Yu cu:

- Phng php chit tch, x l mu l thch hp khi hiu sut chit

khng qu 11% v khng thp h 30%.

- Gi tr RSD % gia cc p ng ca cht phn tch trong cc mu kim

tra (QC) chit tch mi nng phi nh hn hoc bng 15%.

- T l thu hi trung bnh ti cc nng khng khc nhau qu 15%.

* n nh

nh gi n nh ca cht phn tch trong dch sinh hc cn c nh

gi trong qu trnh ly mu, x l v bo qun mu. nh gi n nh,

ngi ta thng so snh kt qu phn tch mu bo qun vi cc mu mi

chun b. Kt qu so snh c nh gi theo thng k da trn tin cy p

dng trong nh gi sinh hc. Trong thm nh phng php cn xc nh cc

loi n nh sau y:

- n nh ca dung dch chun gc bao gm: n nh nhit

phng, n nh khi bo qun di ngy, n nh khi pha long.

- n nh trong mu huyt tng bao gm

+ n nh sau 3 chu k ng - r ng: Nng thuc trong mu

sau 3 chu k ng - r phi sai khc vi nng ban u khng qu 15%

RSD gia cc kt qu nh lng mi nng phi nh hn hoc bng 15%

14

+ n nh thi gian ngn nhit phng i vi dung dch mu

th sau khi chit tch: Nng n thuc trong mu c x l sau khi c

bo quan mt thi gian nht nh nhit phng phi sai khc vi nng

x l ngay khng qu 15% v gi tr RSD % gia cc kt qu nh lng

mi nng phi nh hn hoc bng 15%.

+ n nh thi gian di ng lnh ca mu th trong thi gian d

nh: Nng cefaclor trong mu sau khi bo qun mt thi gian nht nh

lng mi nng phi nh hn hoc bng 15%.

+ n inh trong qu trnh tim mu [8], [19].

15

CHNG II: I TNG NGHIN CU V PHNG

PHP NGHIN CU

2.1. iu kin thc nghim v ni dung nghin cu

2.1.1. iu kin thc nghim

2.1.1.1. Nguyn liu, ha cht, dung mi

Nguyn vt liu, dung mi, ha cht v thuc th dng trong nghin

cu c trnh by cc bng 2.1, 2.2 v 2.3

Bng 2.1. Cc cht chun s dng trong nghin cu

STT Tn Mc ch

s dng

Ni sn

xut

S kim

sot

Hm lng

nguyn

trng

m

1 Cefaclor Chun VKNT TW 0309042 94,71% 5,06%

Bng 2.2. Cc thuc th v dung mi s dng trong nghin cu

STT Tn tinh

khit Ni sn xut

1 Methanol HPLC J.T.Baker, M

2 Acetonitril HPLC J.T.Baker, M

3 Trichloroacetic acid P.A Merck, c

4 Perchloric acid P.A Merck, c

5 Natri dihydrophosphat P.A Merck, c

6 Tetrabutyl ammonium hydroxyl

sulfat P.A Acorss, M

7 Triethanol amin P.A Merck, c

16

Bng 2.3. Cc mu huyt tng trng s dng trong nghin cu

STT L mu Ni cung cp Hn dng Bo qun

1 13PN07839 Vin huyt hc-

truyn mu TW 24/06/2014 -400C

2 13PN07842 Vin huyt hc-

truyn mu TW 30/08/2014 -400C

2.1.1.2. Dng c, thit b

Cc thit b chnh v cu hnh h thng HPLC-Agilents dng trong nghin

cu c trnh by cc bng 2.4 v 2.5

Bng 2.4. Cc thit b s dng trong nghin

STT Tn thit b Mc ch s dng Ni sn xut

1 Cn phn tch M204 Cn chun Metler toledo,

Thy s

2 My ly tm

ROTOFIX 32A Ly tm mu Hettich, c

3 My lc xoy Trn mu IKA, c

4 T lnh su - 400C Bo qun mu Sanyo DMF-236,

Nht

5 T lnh Bo qun mu Samsung, Hn

Quc

6 Micopipette Eppendrof, c

7 ng chit thy tinh,

dung tch 10 mL

Chun b mu, chiu

mu Trung Quc

8

u tp cho

micropipette 0-200 L

v 0-1000 L

Chun b mu, chuyn

mu Trung Quc

9

Bnh nh mc,

Pipette chnh xc,

thy tinh Class A

Chun b mu, pha

long mu Trung Quc

Bng 2.5. Cu hnh h thng HPLC Agilents 1260

17

STT B phn Model Ni sn xut

1 Bm 4 knh 1260 Agilents, c

2 Autosampler 1260 Agilents, c

3 Detector UV-Vis-DAD 1260 Agilents, c

4 Bung n nhit ct 1260 Agilents, c

5 Phn mm iu khin&x l kt

qu 1260 Agilents, c

2.1.2. Ni dung nghin cu

* Xy dng phng php nh lng cefaclor trong huyt tng bng phng

php HPLC:

- La chn iu kin chit tch cefaclor: tin hnh kho st cc cch x

l mu nh: ta protein trn cc mu huyt tng t to la chn phng

php x l mu thch hp da trn cc nguyn l chit mu.

- La chn iu kin sc k, xy dng phng php phn tch m bo

phn tch cefaclor ra khi cc thnh phn tp trong huyt tng, cho php

nh lng cefaclor c trong mu vi nng chnh xc thch hp.

* Thm nh phng php phn tch v cc ch tiu:

Tnh c hiu, chn lc, tuyn tnh, ng, chnh xc, gii hn

nh lng, hiu sut chit, n nh ca cefaclor trong huyt tng trong

cc iu kin (r ng, cch ngy).

2.2. Phng php nghin cu

2.2.1. Chun b mu huyt tng t to cha chun cefaclor

- Mu huyt tng trng khng cha cefaclor.

- Mu huyt tng t to cha chun cefaclor: Pha dung dch chun

gc cefaclor nng chnh xc khong 1000 g/mL trong MeOH. T dung

dch chun gc chun b 6 dung dch chun lm vic WSS1, WSS2, ...WSS6

trong MeOH 50% c nng chnh xc khong 5, 10, 30, 50, 100 v 300

g/mL.

18

Pha dy ng chun gm 1 mu huyt tng trng (blank plasma) v

6 mu huyt tng c pha chun cefaclor v chun ni cephalexin theo bng

2.6.

Bng 2.6. Cch chun b ng chun mu huyt tng t to

STT Mu Blank S1 S2 S3 S4 S5 S6

Nng (g/mL) 0 0,5 1,0 2,0 5,0 10,0 30,0

1 VWSS 1 (L) - 100 - - - - -

2 VWSS 2 (L) - - 100 - - - -

3 VWSS 3 (L) - - - 100 - - -

4 VWSS 4 (L) - - - - 100 - -

5 VWSS 5 (L) - - - - - 100 -

6 VWSS 6 (L) - - - - - - 100

7 VHuyt tng trng (L) 1000 900 900 900 900 900 900

- Mu kim chng (QC) gm 3 loi:

+ LQC: Nng gp 3 ln nng LLOQ

+ MQC: Nng bng 50-80% Cmax

+ HQC: Nng bng 75-90% nng ULOQ

Cc mu LQC, MQC, HQC c nng ln lt l: 1,5; 10,0; 25,0

g/mL c chun b tng t nh cc mu huyt tng t to cha chun

cefaclor. Cch chun b cc mu QC c trnh by bng 2.7.

Bng 2.7. Cch chun b mu QC

STT QC Nng (g/mL) VWSS (L) VHuyt tng trng (L)

1 LQC 1,5 50 (WSS3) 950

2 MQC 10,0 100 (WSS5) 900

3 HQC 25,0 500 (WSS4) 500

- Chun b mu gii hn nh lng di LLOQ 0,5 g/mL nh sau:

Dng micropippette ly chnh xc 100 L dung dch chun lm vic WSS1,

pha long bng huyt tng trng va 1 mL.

19

2.2.2. Xy dng quy trnh chit cefaclor trong huyt tng

Mt iu quan trng trong vic phn tch dc cht trong mu mu

huyt tng l loi b cc protein tan trong nc. Cc phng php thng

dng kt ta protein; chit lng-lng v chit pha rn. Da trn tnh cht ha

l ca cefaclor l mt cht rt phn cc v c tnh cht lng tnh nn rt kh

chit khi huyt tng bng phng php chit lng-lng. Do vy chng ti

tin hnh kho st chit tch cefaclor trong huyt tng bng phng php

ta protein vi cc tc nhn gy ta nh MeOH, MeCN, axit Trichloroacetic,

axit perchloric

2.2.3. Xc nh iu kin sc k

Kho st phng php phn tch trn h thng sc k Agilents s dng

ct sc k pha o C18 (4,6 x 250 mm, 5 m, Zorbax Eclipse XDB-Agilents,

USA) vi cc pha ng c thnh phn t l khc nhau. T kt qu kho st

la chn nhng iu kin sc k ph hp c th tin hnh nh lng

cefaclor trong huyt tng bng HPLC vi detector UV-Vis.

2.2.4. Thm nh phng php phn tch

Thm nh phng php phn tch theo hng dn ca US-FDA, tin

hnh trn cc mu: Mu trng, mu chun v mu QC; nh gi cc ch tiu:

2.2.4.1. Tnh c hiu chn lc

X l mu theo quy trnh la chn, tin hnh chy sc k theo iu

kin xc nh ca cc mu: Mu trng, mu chun, mu th. Ghi li cc

sc k v p ng pc ti cc v tr tng ng vi thi gian lu ca hot

cht.

2.2.4.2. ng chun v khong tuyn tnh

Chun b mt dy nng chun ca cefaclor trong huyt tng trng

vi cc nng trong khong 0,5 30 g/mL. Tin hnh phn tch cc mu.

T p ng pc ca cefaclor ti cc nng tng ng, xc nh s tng

quan tuyn tnh gia p ng pc v cc nng pha.

Xy dng phng trnh hi quy v xc nh h s tng quan tuyn

tnh r. Tnh li nng theo phng trnh hi quy xy dng, xc nh

ng vi tng nng .

20

2.2.4.3. Gii hn nh lng di (LLOQ)

Tin hnh tim sc k 6 mu trng v 6 mu chun c nng trong

khong 0,5 g/ml. Ghi li p ng pc ca mu trng v mu chun. Tnh li

nng ca cc mu kho st da vo ng chun xy dng trong cng

iu kin. Tnh % lch gia cc kt qu ca 6 ln phn tch.

2.2.4.4. ng, lp li

a. ng

Tin hnh chy sc k cc l mu bao gm LQC (1,5 g/mL), MQC

(10,0 g/mL), HQC (25,0 g/mL). Mi l mu gm t nht 5 mu c lp c

cng nng . Xc nh kt qu nh lng cc mu QC theo ng chun

xy dng trong cng iu kin.

So snh gi tr trung bnh cc ln nh lng ti mi nng vi gi tr thc.

b. lp li

- lp li trong ngy:

Da theo ng chun pha trong huyt tng trng tin hnh trong

cng iu kin, tnh li nng ca cc mu QC. Xc nh lp li trong

ngy bng cch tnh ton RSD gia cc ln nh lng ca mi nng c

phn tch trong cng mt ngy.

- lp li khc ngy:

Tin hnh tng t nh xc nh lp li trong ngy ca cc mu QC.

Tnh gi tr RSD ca kt qu nh lng cho mi nng cho mi nng

trong t nht 3 ngy phn tch khc nhau.

2.2.4.5. Hiu sut chit (T l thu hi)

Tin hnh sc k cc l mu QC bao gm LQC, MQC, HQC theo quy

trnh xy dng, mi l mu gm t nht 5 mu c lp c cng nng .

Song song tin hnh nh lng cc mu chun pha trong dung mi khng

qua giai on chit tch, c nng tung ng vi cc mu QC.

So snh kt qu p ng ca cefaclor trong mu QC pha trong huyt tng

(c qua chit tch) vi p ng ca cefaclor trong mu pha trong dung mi

(khng qua chit tch)

2.2.4.6. n nh ca mu th trong huyt tng

21

a. n nh sau 3 chu k ng r

Kho st n nh sau 3 chu k ng-r thc hin trn 2 nng

LQC v HQC. Bo qun mu nhit -400C v r ng nhit

phng. Sau 3 chu k ng-r, tin hnh phn tch xc nh nng cefaclor

c trong mu.

So snh vi kt qu xc nh nng cefaclor c trong cc mu tin

hnh phn tch ngay sau khi pha (nng ban u).

b. n nh ca mu huyt tng nhit phng trong thi gian ngn

Phn tch mu huyt tng 2 nng LQC v HQC sau khi r

ng v phng trong mt thi gian nht nh (6 gi), so snh vi nng

mu c x l ngay sau khi r ng.

c. n nh ca mu huyt tng thi gian di

Khong thi gian th nghim n nh di ngy ti thiu phi tin

hnh ly mu mu v phn tch ht s mu huyt tng ca ngi tnh

nguyn. Bo qun mu huyt tng 2 nng LQC v HQC nhit -

400C, phn tch mu ti thi im ban u v sau tng khong thi gian bo

qun nht nh (7, 14, 30 ngy.).

So snh nng ca mu huyt tng sau khi bo qun vi nng

ca mu ti thi im ban u.

2.3. Phng php x l kt qu

- Gi tr trung bnh, lch chun tng i, phng trnh hi quy, h

s tng quan hi quy c xc nh bng phn mm Microsoft Excel 2010.

- Nng cefaclor trong mu chun c xy dng trong mi ngy

phn tch.

22

CHNG III: THC NGHIM V KT QU

3.1. Kt qu xy dng phng php nh lng

3.1.1. Kho st iu kin sc k

Da vo nhng c tnh ha-l ca cefaclor v cc cng trnh nghin cu

c cng b [3],[8],[15],[13],[18], chng ti tin hnh nghin cu

phng php nh lng cefaclor trn h thng sc k HPLC-Agilents vi

iu kin sc k thay i nh sau:

- Ct RP C18 (4,6 x 250 mm, 5 m, ZORBAX Eclipse XDB-Agilents,

USA).

- Pha ng cha 10 - 100 mM Natridihydro phosphat (NaH2PO4) pH 3,1

v MeOH hay MeCN vi cc t l khc nhau.

- Tc dng khong 1,0 mL/pht.

- Detector: UV-Vis vi bc sng ghi sc k 263 nm gn vi bc

sng max ca cefaclor [20].

- Th tch tim: 100 L.

la chn c iu kin sc k ph hp, chng ti tin hnh kho st

bng cch thay i cc iu kin nh: nng NaH2PO4 trong pha ng, cht

to cp ion, t l cc thnh phn trong pha ng.

a. Kho st nng dung dch NaH2PO4 trong pha ng:

a s cc ti liu c cng b u s dng h m cha mui phosphate

[8], [12], [14], [15], do m phosphat cng c la chn trong phng

php ny. Nng m trong pha ng l nng NaH2PO4 trong nc.

Thay i nng NaH2PO4 trong dung dch t 10, 30, 50, 100 mM. Sc k

i din ca cc mu chun cefaclor vi nng m 10 mM v 50 mM

trong pha ng c th hin qua cc hnh 3.1 v 3.2.

23


ng


ng

Nhn xt:

Kt qu kho st trn cc mu cefaclor chun pha trong pha ng tng

ng cho thy: c 4 nng , thi gian lu v cc thng s khc (chiu cao,

din tch) ca pc khng khc nhau nhiu. C th la chn nng ca h

m trong pha ng l 10 mM.

Tuy nhin, khi kho st nh lng cefaclor trong huyt tng vi cc

nng NaH2PO4 10 mM; 30 mM; 50 mM v 100 mM th ch c nng 50

mM v 100 mM cho thi gian lu n nh. V vy, nng NaH2PO4 50 mM

c la chn s dng cho kho st bc tip theo.

b.La chn cht to cp ion:

Theo phng php nh lng cefaclor trong USP 32, pha ng la chn

hai cht to cp ion l 1-pentansulfonat natri v triethylamin. Trong s cc ti

liu tham kho cng b [8], [19], [12], [15] pha ng u b sung cht to cp

ion. Tuy nhin la chn cht no ph hp nht cng nh nng ca cht to

cp ion t c cp. V vy, chng ti tin hnh kho st hai cht to cp

Ce

faclo

r

Ce

faclo

r

24

ion l tetrabutyl ammonium hydroxyl sulfat v triethanolamin, la chn nng

ph hp.

Thay i cc cht to cp ion trong thnh phn pha ng: tetrabutyl

ammonium hydroxyl sulfat, triethanol amin vi nng l 10 mM. Kt qu

SK ca cefaclor chun nng 10 g/mL khng c v c cht to cp ion

th hin qua hnh 3.3 v 3.4.

Hnh 3.3. SK chun cefaclor nng 10 g/mL khng c cht to cp

ion

(a)

(b)

Hnh 3.4. SK chun cefaclor nng 10 g/mL c cht to cp ion: (a)

tetrabutyl ammonium hydroxgen sulfat; (b) triethanolamin.

Ce

faclo

r

Ce

faclo

r C

efa

clo

r

25

Bng 3.1. Bng so snh cc thng s ca pc cefaclor trong dung dch 10

g/mL vi cc cht to cp ion khc nhau

Cht to cp

ion

Din tch

pc

(mAU*s)

Chiu

cao pc

(mAU)

H s

bt i

rng

pc (pht)

S a

l thuyt

Tetrabutyl

ammonium

hydrogen sulfat

1454,336 113,147 0,64 0,1909 3727

Triethanolamin 880,553 79,530 0,55 0,2056 2253

Nhn xt:

Qua hnh 3.3 cho thy trn SK pc ca cefaclor b b vai khi khng c

cht to cp ion. iu ny c khc phc khi c thm cht to cp ion trong

pha ng. Cefaclor p ng pc tt hn khi thm cht to cp ion tetrabutyl

ammonium hydroxgen sulfat so vi triethanol amin trn SK (hnh 3.4, bng

3.1). V vy, chng ti la chn tetrabutyl ammonium hydrogen sulfat l cht

to cp ion cho bc pht trin phng php tip theo.

c. T l cc thnh phn trong pha ng:

Cc ti liu nghin cu c cng b u ghi nhn thnh phn pha ng

gm h m v dung mi MeOH hoc MeCN. Tuy nhin, trong qu trnh

kho st chng ti nhn thy cn c s phi hp ng thi hai dung mi

MeOH v MeCN trong thnh phn pha ng p ng cc yu cu phn

tch dc cht trong huyt tng. Kt qu ny cng ph hp vi cc nghin

cu c cng b [8], [16]. Thay i t l cc thnh phn chng ti la

chn t l thnh phn nh sau: m phosphate-MeOH-MeCN (75:16:9, v/v/v).

T l pha ng ny c s dng cho bc kho st phng php tip theo.

3.1.2. Kho st phng php chit tch cefaclor

Vi h pha ng c kho st trn, chng ti tin hnh chun b mu

vi hai nhm tc nhn to ta l dung mi MeCN v cc axt nh perchloric,

hoc trichloroacetic.

26

X l mu:

1 mL huyt tng + 2 mL MeCN/ 250 L axt perchloric 20% hoc axt

trichloroacetic 6% lc trn u 30s, ly tm 3500 vng/pht ly phn dch

trong, tim sc k.

Nhn xt:

i vi tc nhn gy ta l dung mi MeCN: mu huyt tng sau khi x

l em i phn tch sc k th nhn thy ng nn nhiu v pc cefaclor

trng vi pc ca tp cht (hnh 3.5). Mt khc th tch dung mi em x l

mu gp hai ln th tch mu, mu b pha long sau qu trnh chit tch dn

ti gim gi tr pht hin v nh lng ca phng php. Do vy, chng ti

khng la chn ta protein bng dung mi hu c.

Hnh 3.5. SK ca mu huyt tng trng loi protein bng MeCN

Tc nhn gy ta l axt (axt perchloric 20%, trichloroacetic 6%): i vi

tc nhn ta l axt perchloric 20% th trn sc k pc ca cefaclor dnh vi

pc tp c trong nn mu (hnh 3.6a). Do axt trichloroacetic 6% c la

chn loi protein trong mu (hnh 3.6b )

27

(a) (b

)

Hnh 3.6. SK ca mu huyt tng trng ta protein bng

(a) axit pechrolic 20%; (b) trichloroacetic 6%.

Nhn xt v la chn iu kin:

T cc kt qu thc nghim v kho st cc iu kin sc k v x l mu

trn, chng ti bc u xy dng c phng php nh lng cefaclor

trong huyt tng vi cc iu kin sau:

* iu kin sc k:

- Ct RP C18 (4,6 x 250 mm, 5 m, ZORBAX Eclipse XDB-

Agilents, USA). Tin ct RP C18 (4,6 x 12,5 mm, 5 m, XDB-Agilents,

USA).

- Nhit ct 400C.

- Pha ng cha 50 mM Natridihydro phosphat + 10 mM

tetrabutyl ammonium hydrogen sulfat pH 3,1 : MeOH : MeCN vi t l

75 : 16 : 9 (v/v/v)

- Tc dng: 1,0 mL/min.

- Detector: 263 nm ;

- Th tch tim:100 L

* Quy trnh x l mu:

1 mL huyt tng + 250 L trichloroactic 6%, lc xoy 30s. Ly tm 3500

vng/pht trong 10 pht. Ly dch ni, lc qua mng lc 0,2 m, tim sc k.

3.2. Thm nh phng php phn tch

3.2.1. c hiu chn lc

Tin hnh phn tch cc mu trng, mu chun (nng 10,0 g/mL

cefaclor trong huyt tng) theo phng php c xy dng. Trn SK

Ce

faclo

r

Ce

faclo

r

28

ca chun cefaclor trong huyt tng th pc cefaclor c tch ring ra khi

cc pc khc ca nn huyt tng (Hnh 3.7). Trong SK ca huyt tng

trng khng xut hin pc no ti thi im thi gian lu ca cefaclor. Chng

ti tnh ton LLOQ (xem bng 3.2) v so snh p ng pc cefaclor ti

nng ny vi p ng ti cng thi gian trn SK ca mu huyt tng

trng.

(a)

(b)

Hnh 3.7. SK mu (a) huyt tng trng v (b) cefaclor 10,0 g/mL

trong huyt tng trng.

Kt qu phn tch cho thy: p ng pc ca mu trng nh hn 20% p

ng pc trn mu LLOQ (xem bng 3.3.), trn sc k pc cefaclor tch khi

cc thnh phn tp; ti v tr pc ca cefaclor khng ghi nhn s xut hin ca

pc no ti v tr . Do phng php c tnh c hiu-chn lc vi

cefaclor.

3.2.2. ng chun v khong tuyn tnh

Tin hnh phn tch cc mu chun cha cefaclor c nng t 0,5 - 30

g/mL theo phng php c xy dng. Thit lp mi quan h tng

quan gia nng cefaclor vi p ng pc, xc nh phng trnh hi quy,

h s tng quan r (Hnh 3.8). Tnh li cc nng theo ng chun xy

dng c xc nh ng ca nng tm li. Kt qu phn tch c

trnh by trong bng 3.2.

Ce

faclo

r

29

Bng 3.2. S tng quan gia nng v p ng pc ca cefaclor trong

huyt tng

Mu S1 S2 S3 S4 S5 S6

Nng

(g/mL) 0,5 1,0 3,0 5,0 10,0 30,0

p ng pc

ca cefaclor

(mAU.s)

49,503 110,636 346,893 624,936 1204,621 3745,642

Phng trnh

hi quy

= 125,28 19,893 = 0,9999

Nng tm

thy (g/mL) 0,55 1,04 2,93 5,15 9,77 30,06

ng(%) 110,8 104,2 97,6 102,9 97,7 100,2

Hnh 3.8. th ng chun biu din mi tng quan tuyn tnh gia

nng v p ng pc ca cefaclor trong huyt tng.

y = 125,28x - 19,893r = 0,9999

00

500

1,000

1,500

2,000

2,500

3,000

3,500

4,000

0 10 20 30 40

p

ng p

c c

a

cefa

clo

r

Nng cefaclor trong huyt tng

30

Nhn xt :

Trong khong nng t 0,5 - 30 g/mL, c s tng quan tuyn tnh

gia nng hot cht v din tch pc (r=0,9999). ng ca cc nng

tm li t 97,6% n 110,8% p ng yu cu trong khong 85 - 115%. Nh

vy, ng chun v khong tuyn tnh thch hp nh lng cefaclor

trong mu huyt tng.

3.2.3. Gii hn nh lng di LLO Q

Phn tch cc mu trng v mu chun c nng thp nht trong khong

tuyn tnh (nng cefaclor khong 0,5 g/mL). Tnh ton nng ca cc

mu chun theo phng trnh hi quy xy dng c trn. Kt qu c

trnh by trong bng 3.3.

Bng 3.3. Kt qu xc nh gii hn di LLOQ

STT Mu trng Mu chun (0,5 g/mL)

p ng

(mAU.s)

p ng trong

mu chun

cefaclor

(mAU.s)

Nng

tm thy

(g/ml)

ng

(%)

t/Khng

t

1 - 48,374 0,54 109,0 t

2 8,327 45,422 0,52 104,3 t

3 - 46,915 0,53 106,6 t

4 - 44,393 0,51 102,6 t

5 6,511 47,117 0,53 107,0 t

6 9,632 44,271 0,51 102,4 t

TB 4,078 46,082 0,53 105,3

t RSD(%) 2,5 2,5

p ng trung bnh ca mu

LLOQ/mu trng 11,3 ln

Nhn xt:

p ng ca mu trng ti cc thi im trng vi thi gian lu ca

cefaclor nh hn 20% p ng ca cc pc ny trong mu nng 0,5 g/mL

31

v ng khi tnh li theo ng chun xy dng theo mc 3.2.2 t

102,4% n 109,0%. Cc gi tr v ng u nm trong khong 80% - 120%

vi RSD nh hn 20% (RSD=2,5%). Do vy gii hn nh lng di

(LLOQ) i vi phng php l 0,5 g/mL.

3.2.4. ng, chnh xc trong ngy v khc ngy

*Kho st ng, chnh xc trong ngy

Phn tch cc mu LQC, MQC, HQC theo phng php c xc nh.

Ghi li kt qu p ng ti mi nng (mi mu QC tin hnh o 6 ln

xc nh ng v lp li). T p ng pc ca cefaclor xc nh nng

thc ca cefaclor theo ng chun xy dng trn. T nng c xc

nh ng, lp li trong ngy c trnh by trong bng 3.4.

Bng 3.4. Kt qu kho st ng, lp li trong ngy

Mu LQC (1,5 g/mL) MQC (10,0 g/mL) HQC (25,0 g/mL)

Nng

(a)

(g/mL)

ng(b)

(%)

Nng

(g/mL)

ng

(%)

Nng

(g/mL)

ng

(%)

1 1,53 102,1 9,94 99,4 25,32 101,3

2 1,49 99,5 9,88 98,8 25,66 102,6

3 1,47 97,9 9,87 98,7 25,29 101,2

4 1,45 96,3 9,74 97,4 25,11 100,4

5 1,51 100,9 9,89 98,9 25,55 102,2

6 1,57 104,7 9,95 99,5 25,26 101,1

TB 100,2 98,8 101,5

RSD(%) 3,0 0,8 0,8 (a) : Tnh t phng trnh hi quy (b) Phn trm so vi nng

thc

Kt qu t bng 3.3. cho thy :

+ Mu LQC: c ng t 96,3% - 104,7% vi RSD = 3,0%

+ Mu MQC: c ng t 97,4% - 99,5% vi RSD = 0,8%

+ Mu HQC: c ng t 100,4% - 102,6% vi RSD = 0,8%

32

Nh vy cc mu QC u c ng nm trong khong 85-115%, vi

lch chun tng i RSD nh hn 15%

* Kho st ng, chnh xc khc ngy

Tip tc kho st ng v lp li khc ngy qua vic kho st cc

mu QC, mi mu tin hnh trong 3 ngy-mi ngy o 6 mu tng ng vi

mi mu QC. Kt qu xc nh c trnh by bng 3.5.

Bng 3.5. Kt qu kho st ng, lp li khc ngy

Ngy/Mu

LQC (1,5 g/mL) MQC (10,0

g/mL) HQC (25,0 g/mL)

Nng (a)

(g/mL)

ng(b)

(%)

Nng

(g/mL)

ng

(%)

Nng

(g/mL)

ng

(%)

I

1 1,53 102,1 9,94 99,4 25,32 101,3

2 1,49 99,5 9,88 98,8 25,66 102,6

3 1,47 97,9 9,87 98,7 25,29 101,2

4 1,45 96,3 9,74 97,4 25,11 100,4

5 1,51 100,9 9,89 98,9 25,55 102,2

6 1,57 104,7 9,95 99,5 25,26 101,1

II

1 1,59 106,3 9,86 98,6 25,13 100,5

2 1,48 98,8 10,14 101,4 24,98 99,9

3 1,52 101,2 9,94 99,4 25,28 101,1

4 1,46 97,0 9,95 99,5 24,80 99,2

5 1,47 98,2 9,91 99,1 25,24 101,0

6 1,41 94,3 9,99 99,9 25,66 102,7

III

1 1,47 98,1 9,73 97,3 24,92 99,7

2 1,48 99,0 9,78 97,8 25,36 101,4

3 1,47 97,9 9,96 99,6 25,12 100,5

4 1,48 98,9 9,64 96,4 25,71 102,8

5 1,42 94,8 9,72 97,2 25,15 100,6

6 1,44 96,0 9,89 98,9 25,34 101,4

33

TB 99,0 98,8 101,1

RSD(%) 3,2 1,2 1,0 (a) : Tnh t phng trnh hi quy (b) : Phn trm so vi nng thc

Nhn xt :

Kt qu t bng 3.4 cho thy, ng khc ngy ca cc mu QC nm

trong khong 85-115% vi RSD nh hn 15%. Nh vy phng php nh

lng cefaclor t v ch tiu ng, lp li trong ngy v khc ngy.

3.2.5. Hiu sut chit

Tin hnh o ti 3 nng ca 3 mu QC. Chun b cc l mu LQC,

MQC, HQC mi l mu gm 5 mu c lp v cc mu chun pha trong pha

ng c nng tng ng. Tin hnh nh lng trong cng iu kin sc

k. So snh din tch pc ca cefaclor thu c t cc mu QC qua x l

chit tch vi din tch pc thu c t cc mu tng ng pha trong pha

ng khng qua chit tch.

Kt qu xc nh hiu sut c trnh by trong bng 3.6.

Bng 3.6. Kt qu kho st hiu sut chit cefaclor

ST

T

p ng pc cefaclor

LQC (1,5 g/mL)

p ng pc cefaclor

MQC (10,0 g/mL)

p ng pc cefaclor

HQC (25,0 g/mL)

Huyt

tng

Dung

mi

Hiu

sut

(%)

Huyt

tng

Dung

mi

Hiu

sut

(%)

Huyt

tng

Dung

mi

Hiu

sut

(%)

1 168,93 212,19 79,6 1215,12 1358,70 89,4 3025,57 3523,10 85,9

2 165,89 205,76 80,6 1198,53 1437,66 83,4 3017,98 3440,07 87,7

3 171,23 202,44 84,6 1240,33 1409,07 88,0 3125,28 3550,46 88,0

4 172,71 210,32 82,1 1258,32 1397,25 90,1 2916,06 3498,10 83,4

5 177,99 205,33 86,7 1279,59 1425,33 89,8 3204,57 3569,55 89,8

6 175,70 211,34 83,1 1253,33 1405,73 89,2 2897,34 3505,82 82,6

TB 172,07 207,90 82,8 1240,87 1405,62 88,3 3031,13 3514,52 86,2

RSD

(%) 2,6 1,9 3,1 2,4 1,9 2,9 3,9 1,3 3,2

Nhn xt: Kt qu cho thy c 3 nng , phng php cho hiu sut

chit t yu cu nm trong khong 30 -110%, hiu sut chit trung bnh ti

34

mi nng c RSD nh hn 15%. Nh vy phng php x l mu ph hp

chit tch cefaclor t huyt tng.

3.2.6. n nh ca mu huyt tng

* n nh sau 3 chu k ng-r

Phn tch cc l mu LQC, HQC, mi l gm 6 mu. Phn tch cc mu

theo phng php c xy dng. Xc nh nng cefaclor trong cc

mu ban u (phn tch ngay sau khi pha mu) v cc mu qua 3 chu k

ng-r theo ng chun c xy dng. Kt qu c trnh by bng

3.7

Bng 3.7. Kt qu nghin cu n nh ca mu huyt tng sau 3 chu

k ng - r

Mu

Nng (g/mL)

Mu LQC (1,5 g/mL) Mu HQC (25,0 g/mL)

Ban u Sau 3 chu k

ng-r

Ban u Sau 3 chu k

ng-r

1 1,53 1,51 25,32 24,85

2 1,49 1,48 25,66 23,98

3 1,47 1,46 25,29 24,63

4 1,45 1,43 25,11 23,47

5 1,51 1,39 25,55 23,91

6 1,57 1,34 25,26 22,99

TB 1,50 1,43 25,37 23,97

RSD(%) 3,0 4,5 0,8 2,9

lch tng

i(%) -4,6 -5,6

Nhn xt : Kt qu c 2 khong nng thp v cao cho thy: RSD ca

mi mu u nh hn 15%, lch tng i nng cefaclor mu tri qua

3 chu k ng-r v mu ban u chnh lch khng qu 15% so vi mu ban

u. Mu t tiu chun v n nh sau 3 chu k ng-r.

* n nh ca mu huyt tng nhit phng trong thi gian ngn

35

Phn tch mu huyt tng 2 l mu LQC v HQC, mi l 6 mu sau khi

r ng v nhit phng mt thi gian nht nh (4 gi), so snh vi

nng mu c x l ngay sau khi r ng. Kt qu c trnh by trong

bng 3.8

Bng 3.8. Kt qu nghin cu n nh mu huyt tng thi gian ngn

Mu

Nng (g/mL)

Mu LQC (1,5 g/mL) Mu HQC (25,0 g/mL)

X l ngay Sau 4 gi X l ngay Sau 4 gi

1 1,57 1,50 25,06 24,74

2 1,53 1,46 25,36 23,73

3 1,50 1,48 25,89 24,20

4 1,45 1,39 24,93 24,98

5 1,55 1,42 25,16 22,36

6 1,46 1,43 25,73 25,14

TB 1,51 1,45 25,35 24,19

RSD(%) 3,3 2,8 1,5 4,3

lch tng

i(%) -3,7 -4,7

Nhn xt : Kt qu c 2 khong nng thp v cao cho thy: RSD ca

mi mu u nh hn 15% ; lch tng i nng cefaclor mu khi

r ng v nhit phng sau 4 gi chnh lch khng qu 15% so vi

mu c x l ngay. Mu t tiu chun v n nh trong thi gian ngn.

* n nh ca mu huyt tng trong thi gian di

Tin hnh th n nh ca mu sau mt thng. Kt qu c trnh by

bng 3.9

36

Bng 3.9. Kt qu n nh ca mu huyt tng thi gian di

Mu

Nng (g/ml)

Mu LQC (1,5 g/ml) Mu HQC (25,0 g/ml)

Ban u Sau 30 ngy Ban u Sau 30 ngy

1 1,53 1,37 25,32 24,60

2 1,49 1,46 25,66 23,76

3 1,47 1,45 25,29 24,22

4 1,45 1,46 25,11 23,66

5 1,51 1,39 25,55 24,65

6 1,57 1,35 25,26 21,76

TB 1,50 1,41 25,37 23,78

RSD(%) 3,0 3,4 0,8 4,5

lch (%) -5,9 -6,4

Nhn xt: Kt qu 2 khong nng thp v cao cho thy: cc mu c

RSD nh hn 15%, lch ca mu sau 30 ngy so vi mu u tin nh hn

15% nn mu t yu cu v ch tiu n nh di ngy.

3.3. Bn lun

V phng php x l mu :

Cefaclor l hot cht c h s phn b du nc kh thp, kh tan trong

mt s dung mi hu c nn vic chit cefaclor trong huyt tng bng

phng php chit lng - lng l kh khn. Sau khi tham kho cc ti liu v

kho st vi cc tc nhn gy ta protein, kha lun xy dng c

phng php x l mu nh lng cefaclor trong huyt tng c tnh kh

thi cao.

Phng php x l mu c la chn l ta protein bng axt

chloroacetic. Phng php ny c u im n gin, d dng thc hin, t c

hi v c bit tnh kinh t cao v khng phi dng dung mi, ha cht thit b

v cc vt t tiu hao t tin nh cc phng php khc.

37

V phng php nh lng bng HPLC

Vi thit b phn tch hin c l h thng HPLC, detector UV-Vis. Chng

ti tin hnh kho st v a ra iu kin sc k ph hp, la chn pha

ng thng dng c b sung thm cht to cp ion tetrabutyl ammonium

hydrogen sulfat v iu chnh pH bng Natridihydro phosphat. Vi iu kin

sc k khng qu phc tp, cefaclor tch hon ton khi cc tp cht c

trong huyt tng vi thi gian trin khai sc k khong 5pht. V vy c th

p dng nh lng c nhiu mu trong mt ngy, tit kim chi ph v nhn

lc.

Phng php nh lng cefaclor trong huyt tng trong kha lun c

thm nh theo cc ch tiu ca US-FDA gm: tnh chn lc-c hiu, khong

tuyn tnh-ng chun, ng, chnh xc, gii hn nh lng di

LLOQ, mt s ch tiu v n nh trong huyt tng.

Vi nhu cu ca Doanh nghip l kho st s b kt qu nghin cu tng

ng sinh hc lm c s cho vic nh gi chnh thc th phng php ny

gip cc doanh nghip dc trong nc ch ng kim sot v nng cao cht

lng thuc sn xut trong tng lai, hng ti hi nhp ha, quc t ha.

38

CHNG IV: KT LUN V XUT

4.1. Kt lun :

Sau mt thi hon thnh kha lun ny, chng ti t c cc kt qu

nh sau :

1. Xy dng c phng php nh lng cefaclor trong huyt tng vi

cc iu kin sc k v x l mu tng i n gin nh sau :

*Quy trnh x l mu: ly 1 mL huyt tng cho vo ng ly tm. Thm

250 L trichloroactic 6%. Lc xoy 30s sau ly tm 3500 vng trn pht

trong vng 10 pht. Ly lp dch ni, lc qua mng lc 0,2 m v tim sc k.

*iu kin sc k:

- Ct RP C18 (4,6 x 250 mm, 5 m, ZORBAX Eclipse XDB-

Agilents, USA). Tin ct RP C18 (4,6 x 12,5 mm, 5 m, XDB-Agilents,

USA).

- Pha ng : 50 mM Natridihydro phosphat + 10 mM tetrabutyl

ammonium hydrogen sulfat pH 3,1 : MeOH : MeCN vi t l 75 : 16 : 9

(v/v/v)

- Tc dng 1,0 mL/min.

- Nhit ct 400C.

- Detector DAD: 263 nm.

- Th tch tim 100 L

2. Tin hnh thm nh phng php HPLC nh lng cefaclor trong huyt

tng theo hng dn ca US-FDA vi cc kt qu nh sau:

- Phng php c chn lc cao vi cefaclor

- Khong tuyn tnh : 0,5 - 30,0 g/ mL

- ng, lp li trong ngy : 96,3% - 104,7% vi gi tr RSD

nh 0,8-3,2%

- T l thu hi hot cht l :79,6 - 90,1%

- n nh huyt tng trong thi gian ngn: 4 gi nhit

phng.

- n nh huyt tng di ngy: 30 ngy/-400 C.

39

- n nh huyt tng 3 chu k: ng - r.

Kt qa thm nh cho php kt lun phng php nh lng cefaclor

trong huyt tng bng HPLC m chng ti xy dng trn c bn p

ng cc yu cu ca phng php phn tch thuc trong dch sinh hc theo

quy nh ca t chc FDA - Hoa K. Nh vy, kt qu p ng c mc

tiu ra ca kha lun.

4.2. xut:

Trong khun kh ca mt kha lun tt nghip, chng ti mi ch xy

dng phng php vi chun cefaclor trong nn huyt tng trng. Mc d

phng php p ng c y cc ch tiu v phng php nh lng,

nhng nh gi phng php c tnh n nh cao cn c kho st trn

cc nn mu th khc nhau, chy mu vi thi gian di, lin tcNhm loi

tr ti a cc sai st k thut khi trin khai thc t.

TI LIU THAM KHO

Ting Vit

1. Trn T An (2007), Ha phn tch II NXB Y hc, trang 125-142, 168-189.

2. Trn T An (2007), Kim nghim dc phm, NXB Y hc, trang 101 - 110.

3. L Th Vn Anh (2012), Xy dng phng php nh lng cefaclor trong

ch phm bng HPLC, kha lun tt nghip Dc s, Trng i hc Dc H

Ni.

4. B Y t, (2007), Dc l hc 2, NXB Y hc, H ni, trang 132-133, 149-151

5. B Y t (2007), Dc th quc gia Vit Nam, Ti bn trn b 600 chuyn lun,

NXB Y hc, H ni, trang 272-275.

6. B Y t, thng t s 08/2010/TT-BYT, Hng dn bo co s liu nghin cu

sinh kh dng, tng ng sinh hc trong ng k thuc.

7. Hi ng dc in Vit Nam (2010), Dc in Vit Nam IV.

8. T Mnh Hng (2006), Nghin cu nh gi tng ng sinh hc vin nang

Cefaclor Stada 250 mg sn xut ti Vit Nam, lun vn Thc s Dc hc,

trng i hc Dc H Ni, trang 11- 23, 27-50.

9. Thi Duy Thn (2007), Ha Dc tp 2, NXB Y hc trang 123-127.

10. Vin kim nghim thuc Trung ng (2012), K yu cc cng trnh nghin

cu khoa hc 2007-2011.

Ti liu ting anh

.

11. Chen X.Y, Zhong D.F (2003), Determination of cefaclor in human plasma

by a sensitive and specific liquid chromatographic- tandem mass spectrometric

method", Journal of Chromatograp B, Volume 784, Issue 1, p.17-24.

12. Clarker's Analysis of Drugs and Poisons. Third edition, edited by Snthony C

Moffat, M David Oselton and Brian Widdop p.762 -763

14. Kai M., Kimoshitaa H., Ohtaa K., et al (2003), "Sensitive determination of a

beta lactam antibiotic, cefaclor by liquid chromatography with

chemiluminesence detection", Journal of Pharmaceutical and Biomedical

Analysis, Volum 30, Issue 6, p. 1775-1771

15. Lunn G., Schumuff N.R (1997), HPLC Method for Pharmacetutical analysis,

John Wiley & Sons, Inc., p.257-263.

13. Golam Mortuza Shahed et al (2011), A Simple RPHPLC Method for the

Determination of Cefdinir in Human Serum: Validation and Application in a

Pharmacokinetic Study with Healthy Bangladeshi Male Volunteers, Dhaka

Univ. J. Pharm. Sci. 10(2): 109-116.

17. The United States Pharmacopeia XXVI (2003), p. 350-352, 2340 - 2345 and

2350-2352

18. Rao Basaw M.V., Prasanthi V., et.al (2011) Development and validation of

New RP-HPLC method for the determination of cefaclor in pharmaceutical

dosage forms in human plasma, Research Journal of Chemical Sciences Vol.

1(5), 36-39.

16. L. Manna, L. Valvo, et al. (2004) Development and Validation of a Fast

Reversed-Phase Ion-Pairing Liquid Chromatographic Method for Simultaneous

Determination of Eight Cephalosporin Antibiotics in Pharmaceutical

Formulations, Chromatographia Vol 60(11-12), 645-649

19. U.S Department of Health and Human Services, Food and Drug

Administrion, Center for Drug evaluation and Research (2001): Guidance for

industry- Bioanalytical Method Validation

20. UV and IR Spectra Pharmaceutical Subtances (UV and IR) and

Pharmaceutical and Cosmetic Excipients (IR); 2002 ECV Editio contor verlag.

Cc trang web s dng trong ti:

http://en.wikipedia.org/wiki/Cefaclor

PH LC

MT S SC K THU C TRONG QU TRNH THC NGHIM

Data File D:\CEFACLOR\DEF_LC 2014-04-15 18-48-57\002-0201.D Sample Name: Plasma STD 0.5ug/ml

===================================================================== Acq. Operator : CEFACLOR Location : Vial 2 Acq. Instrument : Instrument 1 Inj : 1 Injection Date : 4/15/2014 7:15:35 PM Inj Volume : 100.0 l Last changed : 4/15/2014 6:23:04 PM by CEFACLOR Analysis Method: C:\CHEM32\1\METHODS\CEFACLOR PLASMA.M

===================================================================== Area Percent Report with Performance

=====================================================================

Multiplier: : 1.0000 Dilution: : 1.0000 Use Multiplier & Dilution Factor with ISTDs

Signal: Sig=263,4 Ref=360,100

RetTime [min]

k' Area [mAU*s]

Height [mAU]

Symm. Width [min]

Plates Resol ution

Select ivity

-------|------|----------|----------|-----|-------|-------|-----|------ 2.208 - 374.60794 78.35150 1.45 0.0748 4820 - -

2.516 - 35.04243 2.72541 0.84 0.2132 770 1.26 1.14 3.065 - 7211.79053 1341.34424 1.60 0.0661 11920 2.31 1.22 3.750 - 438.78043 48.56980 1.01 0.1534 3303 3.67 1.22

3.928 - 772.22473 63.11826 0.60 0.1845 2511 0.62 1.05 4.417 - 1029.40442 85.97210 0.83 0.1800 3331 1.57 1.12 4.895 - 30.63786 2.73370 0.57 0.1762 4274 1.58 1.11 5.398 - 25.40115 2.07225 0.83 0.3339 1449 1.16 1.10 7.592 - 44.34795 2.19983 0.33 0.3233 3056 3.92 1.41

===================================================================== *** End of Report ***




=====================================================================



RetTime [min]

k' Area [mAU*s]

Height [mAU]

Symm. Width [min]

Plates Resol ution

Select ivity

-------|------|----------|----------|-----|-------|-------|-----|------ 1.984 - 6103.01611 404.70901 0.81 0.2717

2.503 - 36.98906 2.87527 0.72 - 3.066 - 7237.89258 1416.69714 1.57 0.0609 3.810 - 308.35361 36.48083 2.42 0.1342 4474 4.48 1.24

4.008 - 788.50256 59.40178 1.08 0.2446 1486 0.61 1.05 4.417 - 982.97321 77.32500 0.95 0.1933 2896 1.10 1.10 4.912 - 63.94221 5.27695 0.70 0.1933 3578 1.50 1.11 5.325 - 13.26284 1.16884 0.49 0.2029 3820 1.23 1.08 7.111 - 231.31358 3.55900 3.20 1.2530 179 1.44 1.34

7.636 - 107.15672 4.33793 0.44 0.4067 1952 0.37 1.07

===================================================================== *** End of Report ***




=====================================================================



RetTime [min]

k' Area [mAU*s]

Height [mAU]

Symm. Width [min]

Plates Resol ution

Select ivity

-------|------|----------|----------|-----|-------|-------|-----|------ 1.769 - 3.47501e4 2684.72852 0.35 0.1900 479 - -

2.809 - 409.12155 52.90077 2.53 0.1143 3341 4.01 1.59 3.036 - 7015.67139 1235.46594 0.98 0.0758 8873 1.41 1.08 4.035 - 1100.55371 60.17056 1.44 0.2417 1543 3.70 1.33

4.406 - 1047.04224 76.33371 0.76 0.2111 2414 0.96 1.09 4.938 - 427.45520 24.16481 0.66 0.2600 2000 1.33 1.12 7.743 - 17.57202 1.59301 0.68 0.1683 11714 7.70 1.57

===================================================================== *** End of Report ***




=====================================================================



RetTime [min]

k' Area [mAU*s]

Height [mAU]

Symm. Width [min]

Plates Resol ution

Select ivity

-------|------|----------|----------|-----|-------|-------|-----|------ 1.684 - 6.70043e4 3442.68164 0.32 0.2911 183 - -

3.018 - 8693.20313 1258.21875 1.30 0.0744 9122 4.29 1.79 4.063 - 998.69391 59.32290 1.70 0.2067 2143 4.37 1.35 4.401 - 1017.47827 77.92806 0.86 0.1944 2835 0.99 1.08

4.952 - 752.57727 49.13673 0.66 0.2222 2751 1.55 1.13 7.796 - 32.74215 1.79181 0.68 0.2933 3916 6.48 1.57 8.323 - 51.61734 1.60471 0.22 0.4200 2177 0.87 1.07

===================================================================== *** End of Report ***




=====================================================================



RetTime [min]

k' Area [mAU*s]

Height [mAU]

Symm. Width [min]

Plates Resol ution

Select ivity

-------|------|----------|----------|-----|-------|-------|-----|------ 1.637 - 4.33298e4 3441.54663 0.70 0.2040 356 - -

1.801 - 3.50066e4 3351.18311 0.27 0.1386 937 0.56 1.10 2.567 - 166.77661 15.99122 1.13 0.2240 729 2.48 1.43 2.967 - 3138.21802 916.05005 1.29 0.0600 13570 1.66 1.16

3.034 - 3653.64282 996.69366 0.68 0.0621 13235 0.64 1.02 3.667 - 63.34018 10.14215 0.79 0.1025 7101 4.52 1.21 3.981 - 426.53995 48.33870 0.76 0.1350 4823 1.55 1.09 4.176 - 38.62766 8.83680 0.80 0.1050 8771 0.96 1.05 4.386 - 673.38666 72.06301 0.66 0.1422 5262 0.99 1.05

4.946 - 1476.63354 111.36246 0.63 0.1950 3560 1.95 1.13

7.932 - 31.83973 2.26193 2.83 0.1987 8826 8.91 1.60

8.340 - 67.77725 2.93632 0.39 0.2778 4998 1.01 1.05

===================================================================== *** End of Report ***




=====================================================================



RetTime [min]

k' Area [mAU*s]

Height [mAU]

Symm. Width [min]

Plates Resol ution

Select ivity

-------|------|----------|----------|-----|-------|-------|-----|------ 1.624 - 4.38988e4 3449.21460 0.85 0.2060 344 - -

1.803 - 3.87327e4 3404.58887 0.48 0.1760 582 0.55 1.11 2.531 - 192.38908 19.32635 0.71 0.2008 879 2.27 1.40 2.952 - 2205.79272 510.85840 1.42 0.0699 9869 1.83 1.17

3.037 - 4486.14355 1156.58044 0.83 0.0575 15456 0.79 1.03 3.657 - 57.95285 8.35603 0.65 0.1219 4986 4.06 1.20 3.981 - 435.39230 40.10500 0.70 0.2081 2031 1.15 1.09 4.194 - 238.21764 31.84163 1.40 - - - 1.05 4.385 - 965.89355 79.92015 0.79 0.1911 2915 - 1.05

4.953 - 4450.80078 308.86703 0.64 0.2178 2863 1.63 1.13

7.978 - 25.55027 1.68783 2.47 0.1960 9177 8.59 1.61

8.324 - 47.31979 1.66135 0.22 0.3000 4265 0.82 1.04

===================================================================== *** End of Report ***

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