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

K hiu/
Ch vit tt
HPLC

US-FDA

3
4
5
6

TSH
SKD
SD
RSD

7
8
9
10

SK
CFC
SSS
WSS

11
12

QC
HQC

13

MQC

14

LQC

15

LLOQ

16

ULOQ

17
18
19

MeCN
MeOH
TB

STT

Ting Anh

Ting Vit

High Performance Liquid

Chromatography
United State-Food Drug
Administration
Bioequivalence (BE)
Bioavailability (BA)
Standard Deviation
Relative
Standard
Deviation
HPLC chromatography
Cefaclor
Stock Standard Solution
Working Standard
Solution
Quality Control Samples
High Quality Control
Samples
Middle Quality Control
Samples
Low Quality Control
Samples
Lower Limit of
Quantification
Upper Limit of
Quantification
Acetonitril
Methanol
Mean

Sc k lng hiu nng cao

Cc qun l thuc v thc
phm Hoa K
Tng ng sinh hc
Sinh kh dng
lch chun
lch chun tng i
Sc k
Chun gc
Chun lm vic
Mu kim tra cht lng
Mu kim tra nng cao
Mu kim tra nng
trung bnh
Mu kim tra nng
thp
Nng di gii hn
nh lng
Nng trn gii hn
nh lng

Trung bnh

MC LC
T VN ..
CHNG I: TNG QUAN...
1.1
Tng quan v cefaclor...
1.1.1
Cng thc ha hc..
1.1.2
Tnh cht Ha L

1
2
2
2
2

1.1.3

Dc l, Dc ng hc 2

1.2
1.2.1
1.2.2
1.2.3
1.2.4

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

C s l thuyt
Mt s i lng c trng trong HPLC
ng dng ca HPLC
ng dng HPLC phn tch cefaclor trong dch sinh hc

1.3

Thm inh phng php php nh lng dc cht 11

trong dch sinh hc
Khi nim 11
Ni dungthm nh phng php 11

1.3.1
1.3.2

4
4
5
7
8

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.

15

2.1.1.2 Dng c, thit b.

2.1.2
Ni dung nghin cu..

16

15
15
15
17

2.2
2.2.1

Phng php nghin cu.. 17

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

2.2.2

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

Xc nh iu kin sc k... 21

2.2.3

Thm nh phng php phn tch. 19

2.2.4.1 Tnh c hiu chn lc 19
2.2.4

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
2.2.4.6
2.3

Hiu sut chit (T l thu hi). 20

n nh ca mu th trong huyt tng 20
Phng php x l kt qu.. 21

CHNG III: THC NGHIM V KT QU

3.1
Xy dng phng php nh lng
3.1.1
Kho st iu kin sc k
3.1.2
Kho st phng php chit tch cefaclor.

22
22
22
25

3.2
3.2.1
3.2.2
3.2.3
3.2.4

Thm inh phng php phn tch.

c hiu chn lc...
ng chun v khong tuyn tnh
Gii hn nh lng di LLOQ
ng, chnh xc trong ngy v khc ngy

27
27
28
30
31

3.2.5
3.2.6

Hiu sut chit. 33

n nh ca mu huyt tng 34

3.3
Bn lun.
CHNG IV: KT LUN V XUT..
4.1
Kt lun..........................
4.2
xut....
PH LC

36
38
38
39

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
Hnh 3.2. SK chun cefaclor 10 g/mL nng m 50 mM trong pha
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

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.

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]-3chloro-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic

[16]

monohydrat

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.

- 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

- 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
H thng cp
dung mi

H thng thu nhn

d liu
(PC/Recorder)

Bm

Detector

B phn tim mu

Ct sc
k
B phn iu nhit

Thi

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

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:
-HCl

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

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
tR: thi gian lu

F: lu lng ca pha ng

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.

= 5.54.

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:

+ 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:

iu kin sc k
Ti
liu

[12]

X l mu
Ct, Detector

Pha ng

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

Ct RP 18
(4x 100mm,
7m )

MeCN:m
phosphate pH 7 =
(10: 90).
Tc dng : 1,0

[12]

[19]

[11]

[8]

mL/pht
To dn xut sau ct
vi Fluorescamin
trong MeCN
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

Chit SPE
Hot ha: 2mL MeOH +
2mL nc ct. Ra gii:
MeOH bc hi dung mi
ha tan cn trong pha
ng.

Ta protein vi axit
Tricloroacetic 6%lc, ly
tm 4000vng/pht x 10pht
Ly dch ni Tim sc
k.

40L mu+ 20 L IS
(chun ni) + 25L pyridine
= 50L 35 mM
CIPIC/800ly tmlc,tim
mu

Ct phn tch MeOH - Nc - Acid Ta protein vi hn hp acid

C18
fomic = (80:20: 1)
fomic: MeOH. Chit SPE
MS/MS

Tc dng : 0,5
mL/pht

Ct Gemini Pha ng:

C18, 5 m; MeOH:

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

Chit SPE

MeCN: Ta protein bng acid

m pecloric, chit loi tp bng

10

250 x 4mm.
phosphate pH3,1
CH2Cl2. Dch thu c tim
Detector : UV (cha TBAH)
sc k
263 nm
t l 10:14:76.
Tc dng: 1,0
mL/pht.
Pha ng: m

[14]

NaH2PO4 0.2 M (pH

Ct : C18 (4,6
3.2 0.05 iu chnh
x 250 mm)
bi axit phosphoric):
Detector: UV
MeOH t l 70:30.
254 nm
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

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.

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 Ni
s dng
xut

Chun

Cefaclor

sn S kim Hm lng
sot
nguyn
m
trng

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

Methanol

HPLC

J.T.Baker, M

Acetonitril

HPLC

J.T.Baker, M

Trichloroacetic acid

P.A

Merck, c

Perchloric acid

P.A

Merck, c

Natri dihydrophosphat

P.A

Merck, c

Tetrabutyl ammonium hydroxyl

sulfat

P.A

Acorss, M

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

13PN07839

Vin huyt hctruyn mu TW

24/06/2014

-400C

13PN07842

Vin huyt hctruyn 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
Metler toledo,
Thy s

Cn phn tch M204

Cn chun

My ly tm
ROTOFIX 32A

Ly tm mu

Hettich, c

My lc xoy

Trn mu

IKA, c

T lnh su - 400C

Bo qun mu

Sanyo DMF-236,
Nht

T lnh

Bo qun mu

Samsung, Hn
Quc

Micopipette

ng chit thy tinh,

dung tch 10 mL

Chun b mu, chiu

mu

Trung Quc

u tp cho
micropipette 0-200 L
v 0-1000 L

Chun b mu, chuyn

mu

Trung Quc

Bnh nh mc,
Pipette chnh xc,
thy tinh Class A

Chun b mu, pha

long mu

Trung Quc

Eppendrof, c

Bng 2.5. Cu hnh h thng HPLC Agilents 1260

17

STT
B phn
1 Bm 4 knh

Model
1260

Ni sn xut
Agilents, c

Autosampler

1260

Agilents, c

Detector UV-Vis-DAD

1260

Agilents, c

Bung n nhit ct

1260

Agilents, c

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
Blank

S1

S2

S3

S4

S5

S6

Nng (g/mL)

0,5

1,0

2,0

5,0

10,0

30,0

VWSS 1 (L)

100

VWSS 2 (L)

100

VWSS 3 (L)

100

VWSS 4 (L)

100

VWSS 5 (L)

100

VWSS 6 (L)

100

VHuyt tng trng (L)

1000

900

900

900

900

900

900

STT

Mu

- 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)

LQC

1,5

50 (WSS3)

950

MQC

10,0

100 (WSS5)

900

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.

Cefaclor

23

Cefaclor

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

ng

Hnh 3.2. SK chun cefaclor 10 g/mL nng m 50 mM trong pha

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

24

Cefaclor

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.

(b)

Cefaclor

Cefaclor

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

ion
(a)

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

tetrabutyl ammonium hydroxgen sulfat; (b) triethanolamin.

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
Din tch
pc

Chiu
cao pc

(mAU*s)

(mAU)

Tetrabutyl
ammonium
hydrogen sulfat

1454,336

113,147

0,64

0,1909

3727

Triethanolamin

880,553

79,530

0,55

0,2056

2253

Cht to cp
ion

H s
rng
S a
bt i pc (pht) l thuyt

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 )

Cefaclor

(a)

(b
)

Cefaclor

27

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 XDBAgilents, 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

28

(b)

Cefaclor

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)

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.

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

1204,621

3745,642

p ng pc
ca
cefaclor 49,503
(mAU.s)
Phng trnh
hi quy
Nng tm
thy (g/mL)

ng(%)

110,636

346,893

624,936

= 125,28 19,893 = 0,9999

0,55

1,04

2,93

5,15

9,77

30,06

110,8

104,2

97,6

102,9

97,7

100,2

p ng pc ca cefaclor

4,000
y = 125,28x - 19,893
r = 0,9999

3,500
3,000
2,500
2,000
1,500
1,000
500
00
0

10
20
30
Nng cefaclor trong huyt tng

40

Hnh 3.8. th ng chun biu din mi tng quan tuyn tnh gia
nng v p ng pc ca 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 LLOQ
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

48,374

0,54

109,0

8,327

45,422

0,52

104,3

46,915

0,53

106,6

44,393

0,51

102,6

6,511

47,117

0,53

107,0

9,632

44,271

0,51

102,4

TB

4,078

46,082

0,53

105,3

2,5

2,5

RSD(%)
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
(%)

1,53

102,1

9,94

99,4

25,32

101,3

1,49

99,5

9,88

98,8

25,66

102,6

1,47

97,9

9,87

98,7

25,29

101,2

1,45

96,3

9,74

97,4

25,11

100,4

1,51

100,9

9,89

98,9

25,55

102,2

1,57

104,7

9,95

99,5

25,26

101,1

TB

100,2

RSD(%)
3,0
(a)
: Tnh t phng trnh hi quy
thc
Kt qu t bng 3.3. cho thy :

98,8
(b)

101,5

0,8
0,8
Phn trm so vi nng

+ 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
LQC (1,5 g/mL)
Ngy/Mu

II

III

MQC (10,0
g/mL)

Nng
ng
(g/mL)
(%)

Nng (a)
(g/mL)

ng(b)
(%)

1,53

102,1

9,94

1,49

99,5

1,47

HQC (25,0 g/mL)

Nng
(g/mL)

ng
(%)

99,4

25,32

101,3

9,88

98,8

25,66

102,6

97,9

9,87

98,7

25,29

101,2

1,45

96,3

9,74

97,4

25,11

100,4

1,51

100,9

9,89

98,9

25,55

102,2

1,57

104,7

9,95

99,5

25,26

101,1

1,59

106,3

9,86

98,6

25,13

100,5

1,48

98,8

10,14

101,4

24,98

99,9

1,52

101,2

9,94

99,4

25,28

101,1

1,46

97,0

9,95

99,5

24,80

99,2

1,47

98,2

9,91

99,1

25,24

101,0

1,41

94,3

9,99

99,9

25,66

102,7

1,47

98,1

9,73

97,3

24,92

99,7

1,48

99,0

9,78

97,8

25,36

101,4

1,47

97,9

9,96

99,6

25,12

100,5

1,48

98,9

9,64

96,4

25,71

102,8

1,42

94,8

9,72

97,2

25,15

100,6

1,44

96,0

9,89

98,9

25,34

101,4

33

TB

99,0

98,8

RSD(%)
3,2
(a)
: Tnh t phng trnh hi quy

101,1

1,2
1,0
(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

Dung

Hiu

Huyt

Dung

Hiu

Huyt

Dung

Hiu

tng

mi

sut
(%)

tng

mi

sut
(%)

tng

mi

sut
(%)

168,93

212,19

79,6

1215,12

1358,70

89,4

3025,57

3523,10

85,9

165,89

205,76

80,6

1198,53

1437,66

83,4

3017,98

3440,07

87,7

171,23

202,44

84,6

1240,33

1409,07

88,0

3125,28

3550,46

88,0

172,71

210,32

82,1

1258,32

1397,25

90,1

2916,06

3498,10

83,4

177,99

205,33

86,7

1279,59

1425,33

89,8

3204,57

3569,55

89,8

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

2,6

1,9

3,1

2,4

1,9

2,9

3,9

1,3

3,2

RSD

(%)

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
Nng (g/mL)
Mu

Mu LQC (1,5 g/mL)

Ban u
Sau 3 chu k
ng-r

Mu HQC (25,0 g/mL)

Ban u
Sau 3 chu k
ng-r

1,53

1,51

25,32

24,85

1,49

1,48

25,66

23,98

1,47

1,46

25,29

24,63

1,45

1,43

25,11

23,47

1,51

1,39

25,55

23,91

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
Nng (g/mL)
Mu

Mu LQC (1,5 g/mL)

X l ngay Sau 4 gi

Mu HQC (25,0 g/mL)

X l ngay
Sau 4 gi

1,57

1,50

25,06

24,74

1,53

1,46

25,36

23,73

1,50

1,48

25,89

24,20

1,45

1,39

24,93

24,98

1,55

1,42

25,16

22,36

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

Nng (g/ml)
Mu

Mu LQC (1,5 g/ml)

Mu HQC (25,0 g/ml)

Ban u

Sau 30 ngy

Ban u

Sau 30 ngy

1,53

1,37

25,32

24,60

1,49

1,46

25,66

23,76

1,47

1,45

25,29

24,22

1,45

1,46

25,11

23,66

1,51

1,39

25,55

24,65

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 XDBAgilents, 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
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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

k'
Symm. Width
Plates Resol Select
RetTime
Area
Height
[min]
[mAU*s]
[mAU]
[min]
ution 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 ***

Data File D:\CEFACLOR\DEF_LC 2014-04-15 18-48-57\003-0301.D

Sample Name: Plasma STD 1.0ug/ml
=====================================================================
Acq. Operator
: CEFACLOR
Location : Vial 2
Acq. Instrument : Instrument 1
Inj :
1
Injection Date : 4/15/2014 7:41:58 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

k'
Symm. Width
Plates Resol Select
RetTime
Area
Height
[min]
[mAU*s]
[mAU]
[min]
ution 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

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

1.07

Data File D:\CEFACLOR\DEF_LC 2014-04-15 18-48-57\004-0401.D

Sample Name: Plasma STD 3.0ug/ml
=====================================================================
Acq. Operator
: CEFACLOR
Location : Vial 2
Acq. Instrument : Instrument 1
Inj :
1
Injection Date : 4/15/2014 8:08:21 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

k'
Symm. Width
Plates Resol Select
RetTime
Area
Height
[min]
[mAU*s]
[mAU]
[min]
ution 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 ***

Data File D:\CEFACLOR\DEF_LC 2014-04-15 18-48-57\005-0501.D

Sample Name: Plasma STD 5.0ug/ml
=====================================================================
Acq. Operator
: CEFACLOR
Location : Vial 2
Acq. Instrument : Instrument 1
Inj :
1
Injection Date : 4/15/2014 8:34:48 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
k'
Area
Height
Symm. Width
Plates Resol Select
[min]
[mAU*s]
[mAU]
[min]
ution 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 ***

Data File D:\CEFACLOR\DEF_LC 2014-04-15 18-48-57\006-0601.D

Sample Name: Plasma STD 10.0ug/ml
=====================================================================
Acq. Operator
: CEFACLOR
Location : Vial 2
Acq. Instrument : Instrument 1
Inj :
1
Injection Date : 4/15/2014 9:01:09 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

k'
Symm. Width
Plates Resol Select
RetTime
Area
Height
[min]
[mAU*s]
[mAU]
[min]
ution 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

7.932

31.83973

8.340

67.77725

0.63

0.1950

3560 1.95

1.13

2.26193

2.83

0.1987

8826 8.91

1.60

2.93632

0.39

0.2778

4998 1.01

1.05

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

Data File D:\CEFACLOR\DEF_LC 2014-04-15 18-48-57\007-0701.D

Sample Name: Plasma STD 30.0ug/ml
=====================================================================
Acq. Operator
: CEFACLOR
Location : Vial 2
Acq. Instrument : Instrument 1
Inj :
1
Injection Date : 4/15/2014 9:27:31 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

k'
Symm. Width
Plates Resol Select
RetTime
Area
Height
[min]
[mAU*s]
[mAU]
[min]
ution 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 ***