Professional Documents
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DSDK KiemNghiemDuocPham
DSDK KiemNghiemDuocPham
V khoa hc v o to
Nh xut bn Y hc
H ni - 2005
Ch bin
PGS.TS. Trn T An
Tham gia bin son
PGS.TS. Trn T An
CN. Trn Tch
DS. Nguyn Vn Tuyn
TS. Chu Th Lc
ThS.Nguyn Th Kiu Anh
Tham gia t chc bn tho
ThS. Ph Vn Thm
Li ni u
Mc lc
Li ni u
3
Chng 1
i cng
Trn Tch
1.1.
1.2.
1.3.
12
14
16
16
18
22
23
26
26
31
34
36
Cu hi t lng gi
36
Chng 2
2.1.
Cc phn ng nh tnh
37
2.2.
48
2.2.1. Mc ch
48
*Phn 2.1 & 2.2. Trn Tch, phn 2.3. & 2.4.: Trn T An
48
50
51
51
52
2.3.3. Xc nh im tng ng
53
54
58
2.4.1. Nguyn tc
58
58
2.4.3. Xc nh im tng ng
59
2.4.4. ng dng
59
2.5.
60
2.6.
61
65
Cu hi t lng gi
66
2.3.
2.4.
Chng 3
3.2.
68
68
79
82
84
84
3.2.2. My HPLC
86
88
94
100
102
104
111
Cu hi t lng gi
111
Chng 4
115
4.1.1. Nguyn tc
115
116
4.1.3. nh gi kt qu
116
4.2.
116
4.3.
117
117
121
4.3.3. Th v trng
124
128
131
137
Cu hi t lng gi
138
4.1.
Chng 5
140
5.2.
145
5.3.
147
5.4.
149
5.5.
153
5.6.
154
5.7.
157
7
5.8.
159
5.9.
160
5.10
166
169
Cu hi t lng gi
169
Chng 6
n nh v tui th ca thuc
Trn T An
6.1.
171
6.2.
i cng v n nh ca thuc
172
6.2.1. nh ngha
172
173
6.2.3. Mc tiu nh gi n nh
174
175
176
ng ho hc dung dch
177
6.3.1. Bc ca phn ng
177
181
Xc nh n nh ca thuc
182
6.4.1. Ly mu
182
183
185
185
Cc dc cht km bn vng
186
188
Cu hi t lng gi
189
6.3.
6.4.
6.5.
Chng 1
i cng
Mc tiu hc tp
Chn on bnh
tra nguyn ph liu, bao b, ng gi, kim tra bn thnh phm, thnh phm
cui cng, nhm sn xut ra c thuc theo d kin v t tiu chun k
thut t ra. Mun vy, phi thc hin c cc yu cu c bn sau:
Trong sut qu trnh sn xut phi c s kim tra, theo di, ghi chp
y chng minh rng tt c cc giai on ca qui trnh sn xut
u c thc hin nghim chnh, c cht lng v s lng sn phm
ph hp vi qui nh.
1.1.1.2. Thc hnh tt phng kim nghim thuc (Good Laboratory Practice)
iu kin v sinh tt
Ly mu, lu mu tt
H s lu tr tt.
Vn ny s c cp k hn phn nghip v ca cng tc kim nghim.
iu kin v sinh tt
Kim tra cht lng thuc hay kim nghim thuc l vic s dng cc
phng php phn tch: l hc, ho hc, ho l, sinh vt, vi sinh vt, qui
nh xc nhn mt thuc hay mt nguyn liu lm thuc c t hay khng
t tiu chun qui nh. Ni mt cch c th l kim tra cht lng thuc
nhm tr li cc cu hi:
Pht hin thuc khng t tiu chun, thuc gi, thuc km phm
cht x l v khng cho php lu hnh trn th trng.
C nhiu yu t, trong c th l:
12
1.1.2.3. Khi nim v thuc t v khng t tiu chun, thuc gi mo, thuc
km phm cht
Thuc t tiu chun (thuc bo m cht lng):
Nh nc giao cho B Y t chu trch nhim qun l ton din cht lng
thuc. V vy, h thng t chc, qun l, kim tra cht lng thuc ca nghnh
y t c chia lm 3 phn: H thng qun l cht lng thuc; H thng kim
tra cht lng thuc; H thng thanh tra Dc.
1.1.3.1. H thng qun l cht lng thuc
Cc qun l Dc Vit Nam:
Ch o, gim st h thng kim tra cht lng thuc trn ton quc.
Phi hp vi thanh tra B Y t thc hin chc nng kim tra, thanh tra
nh nc v cht lng thuc v x l vi phm php lut v cht lng
thuc theo thm quyn.
14
Thc hin chc nng kim tra, thanh tra nh nc v cht lng thuc
v x l vi phm v cht lng thuc trong phm vi a phng.
Sn phm v bn sn phm.
Phng php th: Tiu chun ny bao gi cng phi qui nh km theo
cc ch tiu yu cu k thut. y l phn khng th thiu v n chnh
l qui trnh th nghim hay qui trnh phn tch m t chi tit ton b
qu trnh thc hnh thc hin xem mt ch tiu no ca yu cu
k thut c t hay khng t yu cu t ra.
Tiu chun Vit Nam (tiu chun quc gia, Dc in Vit Nam) TCVN.
18
Hon thnh d tho tiu chun sau khi tin hnh cc vic: Ly kin
gp (cn th t chc hi tho). Thu thp gp , sa cha, hon chnh
h s gi ti c quan qun l v xt duyt.
Cng thc pha ch: Phi trnh by cng thc pha ch ca thuc, ghi r
tn nguyn liu, ph liu vi s lng ghi bng s v bng ch, cng
tiu chun ca chng (v d: t dc t tiu chun Dc dng).
Cht lng thnh phm: Phi nu r v hnh thc cng nh tnh cht
cm quan, mc tinh khit nh: Hnh dng, th cht, mu sc, mi
v, cc c im c bit, bn c hc (vi thuc vin), tan r, sai s
khi lng, sai s th tch.
Yu cu v hm lng.
x (tt) =
x ( tt )
x + x max
2
Trong :
20
nh c; : gi tr thc ).
Mun c gi tr thc so snh, phi c mu chun bit trc
hoc mt mu in hnh c sn xut theo mt phng php m
bo ng nht v i din cho sn phm.
ng ca phng php cng cn c biu th bng t l thu hi
x
100% (: l hm lng cht chun cho vo; x : l hm lng xc
S =
xi x
n 1
hoc RSD % =
(n: s th nghim; xi : kt qu xc nh ln th i;
ca n th nghim).
S
100 %
X
x : kt qu trung bnh
21
di 1ml dung mi
Rt tan :
Tan :
-nt-nt-
1 - 10ml
> 10-30
-ntml
ml
-nt-nt-
Hi tan :
-nt-
> 30-100
Kh tan :
-nt-
> 100-1000ml
-nt-
Rt kh tan :
-nt-
>1000-10.000ml
-nt-
>10.000ml
-nt-
7. V nhit : Dng thang bch phn k hiu oC. Khi khng ghi c
th, qui c :
Nhit chun:
20oC
Nhit thng:
20-30oC
Nc m:
40-50oC
Nc nng:
70-80oC
Nc cch thu:
98-100oC
<-10 oC
Lnh:
2-10 oC
Mt:
10-20 oC
Nhit phng :
20-35 oC
20-25 oC
Nng:
35-40 oC
Rt nng:
> 40 oC
Nung :
400 oC
thm:
600 oC
trng:
900oC
25
10. Khi th tinh khit, nu pht hin thy tp cht l khng ghi trong
chuyn lun th vn phi ghi vo kt qu th.
1.3. Kim nghim thuc theo tiu chun
Nh trn nu, kim nghim thuc l vic tin hnh phn tch mt mu
thuc i din cho l thuc bng cc phng php ho hc, l hc, ho l,
sinh hc c qui nh xem thuc t hay khng t tiu chun t
quyt nh xem c c php lu hnh hoc s dng hay khng. s nh
gi ny chnh xc, i hi phi lm tt 3 vic sau: ly mu kim nghim, thc
hnh phn tch, nh gi kt qu v vit phiu tr li (phiu kim nghim,
phiu phn tch).
1.3.1. Ly mu kim nghim
1.3.1.1. Mt s khi nim
Cc trng hp ly mu:
Trng hp kim tra gim st cht lng hoc thanh tra: u tin ly
mu kim tra v gim st l cc thuc cha bnh, c gi tr kinh t cao,
c cht lng khng n nh v c bit l c nghi ng v hm lng
hoc hiu lc tc dng.
S ly mu:
Vic ly mu phi bo m cho c tnh khch quan, i din cho c
cht lng ca thuc cn ly kim tra. V vy, phi ly theo hng dn ca qui
trnh (hnh 1.1).
L thuc
Mu ring
Mu chung
Mu trung bnh
th nghim
Mu gi
Mu lu ti
c s
Mu lu
thanh tra
Mu lu ti b
Mu lm
kim nghim
28
n = 0,4 N
Trong :
n: s bao gi ly ra
N: s n v bao gi cui cng trong l (th d vi thuc tim: cc ng
ng trong hp giy, cc hp giy ng trong hm th n v bao gi cui cng
l hm).
Ch :
+ Khi tnh theo cng thc trn, nu phn thp phn nh hn 0,5 b qua,
nu ln hn 0,5 th tng thm mt n v.
+ Khi N > 100 lu nmax 30
+ Khi N < 100 c th dng bng:
N
1 -10
11 - 40
41 - 80
81 - 100
n
1
2
3
4
29
32
B Y t
Tn c quan
kim nghim thuc
c lp - T do - Hnh phc
a ch c quan
Kt qu
1.
2.
3.
4.
5.
t (86,5%)
6.
t (98,6%)
t
ng
t (6,75%)
t
Cc nhn vin phi mc trang phc theo qui nh, phi c kim tra
sc kho, khng lm nhim bn vo cc php th v cc dng c, h
thng th nghim...
34
Kim nghim vin tin hnh th nghim phi bo co mt cch chi tit
nhng hin tng gp phi trong qu trnh th nghim vi ph trch.
35
Cu hi t lng gi
1.1. Mc tiu ca cng tc kim tra cht lng thuc? Cc yu cu c
bn t mc tiu trn ?
1.2. Ni dung chnh ca cng tc kim tra cht lng thuc? iu kin
thuc c a vo lu thng, phn phi, s dng? Cc yu t
nh hng n cht lng thuc ?
1.3. Th no l thuc t v khng t tiu chun? Thuc gi mo?
Thuc km phm cht?
1.4. Trnh by h thng t chc kim tra cht lng thuc ca Vit Nam?
1.5. Ni dung chnh ca cng tc tiu chun ho? Ni dung chnh ca
mt tiu chun v thuc?
1.6. Trnh by phng php xy dng tiu chun v yu cu k thut?
v phng php th? Yu cu cht lng i vi mt phng
php th ?
1.7. Ni dung chnh ca cng tc p dng tiu chun trong thc t?
1.8. Trnh by mt s qui nh chung khi s dng Dc in Vit Nam
(dng trong cng tc kim nghim thuc)?
1.9. Cch tin hnh ly mu kim nghim?
1.10. Trnh by cc nhim v ch yu khi tin hnh kim nghim?
1.11. Ni dung chnh ca thc hnh tt phng kim nghim thuc?
36
Chng 2
Mc tiu hc tp
H SO dc
CH 3 COOH + OHC 2 H 5 2 4 CH 3 COOC 2 H 5 + H 2 O
2.1.2. Amoni (mui)
B phn hu khi un nng vi dung dch NaOH, gii phng kh NH3:
+
NH4
OH
NH3
H2O
+ OH
NH4
NH3 + 2K 2 HgI4
NH3
H2O
Hg
+ 3KOH = O
2.1.3. Arseniat
4AsO 34 + 5H 3 PO 2 + 6H +
2.1.4. Arsenit
Phn ng Tile:
38
2.1.5. Bc (mui)
Phn ng vi HCl cho ta trng AgCl, ta khng tan trong HNO3 nhng
tan trong dung dch amoniac:
Ag+ + Cl
AgCl trng
Ag(NH3)2+ + Cl
AgCl + 2NH3
HCHO
Ag(NH3)2+ + Ag + HCOOH
2.1.6. Barbiturat
R1
NH CO
C
O C
NH CO
R2
NH2
O C
NH2
NaOH
+ H2O
to
+ H2O
NH2
O C
COONa
+
NH2
2 NH3
R1
C R2
COONa
CO2
39
Bi
Cl-
BiOCl trng + 2 H+
H2O
Bi
2.1.9. Borat
OC2H5
+
H3BO3
+ 2 C2H5OH
OC2H5
3 H2O
OC2H5
40
Trong mi trng acid, borat chuyn thnh acid boric, acid boric phn ng
vi giy ngh (hoc cn ngh) cho mu nu , sau tm t bng dung
dch kim long (amoniac hoc natrihydroxyd) mu nu chuyn thnh
mu lam hay lc (do s to phc ca cucumin trong ngh vi acid boric).
2.1.10. Bromid
Phn ng vi AgNO3: cho ta vng nht AgBr, ta ny kh tan trong dung
dch amoniac 10M.
Phn ng oxy ho Br- thnh Br2 bng: PbO2 + CH3COOH hoc KMnO4 +
H2SO4. Nhn bit Br2 bng cch chit vo cloroform c mu vng hoc
nu:
Ca
+ (NH4)2C2O4
CaC2O4
trng + 2 NH4
Ca
K4 [(Fe(CN))6] + 2 NH4
= Ca(NH4)2[Fe(CN)6]
trng + 4K
2.1.12. Ch (mui)
Phn ng vi dung dch KI: cho kt ta mu vng, tan trong KI tha:
2+
Pb
PbI 2
2I
2I
PbI 2 vng
2-
PbI4
K2CrO4
PbCrO4 vng
2K+
2.1.14. Clorat
Khng kt ta vi dung dch AgNO3.
un nng vi dung dch HCl, H2SO4 long s b phn hu thnh Cl2 bay ra:
AgCl
+ 2NH3
Ag(NH3)2 Cl
+ HNO3
AgCl
2Cl + I2
2.1.16. ng (mui)
2.1.17. Ethanol
Tc dng vi acid acetic (mi trng H2SO4) to ra ethyl acetat c mi thm:
42
C2H5OH + CH3COOH
C2H5COOCH3 + H2O
2.1.18. Iodid
Phn ng vi Fe3+:
2I + 2Fe3+ = I2 + 2Fe2+
I2 gii phng ra c th chit vo lp cloroform c mu tm .
2 K+
Na3[Co(NO2)6]
2.1.20. Km ( mui )
Mg
2-
+ HPO4
+ NH4
+ OH + 5 H2O =
MgNH4PO4.6H2O trng
43
Na
KH2SbO4
NaH2SbO4 trng
3 (UO2)(CH3COO)2. 2 H2O
+ Zn(CH3COO)2 + CH3COOH + Na
+
H + NaZn(UO2)3(CH3COO)9.6 H2O vng
Phn ng vi nitrobenzen: trong mi trng H2SO4 c to ra mdinitrobenzen, hp cht ny phn ng vi aceton trong mi trng kim
to thnh phc c mu tm (Phn ng Janovsky):
NO2
NO3-
NO2
H2SO4
NO2
H
NO2
CH3COCH3
NaOH
CH2COCH3
NO2
+
Na
NO2
44
NO2
2.1.25. Oxalat
2.1.26. Peroxyd
Phn ng vi K2Cr2O7 trong mi trng acid:
H2O2 + 2I + 2H+ =
I2 + 2H2O
2.1.27. Phosphat
2.1.29. St (II)
Fe 3+ + x(CNS)
= Fe(CNS)x (3x)+ ()
2.1.31. Silicat
Hg2+ + Cu = Hg + Cu2+
Khi lm, trn ming ng s thy vt hn hng sng bng, t nng vt
bng s mt (v Hg bay i).
Vi KI:
2+
Hg
2+
+ 2I-
Hg2 + 2 I-
HgI2
Hg2I2 vng lc
+ 2 I-
+ 2 I-
2-
HgI4
+ Hg en
47
Khi pha dung dch mu phi s dng cn phn tch v dng c th tch
chnh xc.
Hai bnh phn ng so snh phi ging nhau: bng thu tinh khng
mu, c ng knh bng nhau, dy nh nhau
Phi cho cc thuc th vo hai bnh phn ng ging nhau v: thi gian,
s lng v th tch cui.
35,5 0,8238
= 0,0005 g Cl /1ml
58,5 1000
0,0005
= 0,000005g Cl / ml (tng ng 0,005 mg Cl/ ml hay
100
49
0,01
= 20 Ta c
0,0005
Thuc th
Sn phm
Cl-
AgNO3
AgCl
Ta trng
SO42-
BaCl2
BaSO4
Ta trng
NH4+
Nessler
Hg
NH2 I
Hg
Ca2+
(NH4)2C2O4
CaC2O4
Ta trng
Arsen
Zn + HCl
AsH3
PbS
en hoc nu
t vng sang nu
- Na2S (H2S)
Kim loi nng
- thioacetamid
- acid mercaptoSt
acetic
- acid sunfosalicylic
50
- Feri mercaptoacetat
Ferisulfisalicylat
Mu hng
nu hay vng
8-hydroxyquinolin
Nhm
Al/3
Mu vng rm
(tan trong CHCl3)
( pH 6)
(oxyquinolat nhm)
8-hydroxyquinolin
Oxyquinolat Mg
Magnesi
( pH 10)
Mu vng
(tan trong CHCl3)
Phosphat
Sulphomolybdic
(NH4)3H4[P(Mo2O7)6]
Mu vng
Km
K4[Fe(CN)6]
K2Zn3[Fe(CN)6]2
Ta trng
Acid 2 + base 1
V d:
BH+ + S-
B + HS
R NH + CH COOH R N H + CH COO
2
3
3
3
Ngc li nu dung mi c tnh base, n s tng tnh acid ca cht tan HA
A + S+H
HA + S
ki
HA + S
HS+A
solvat ha
HS+ + A
in ly
HS + HS
a H S + . aS = K S
2
V d:
2 CH3COOH
CH3COOH2+ + CH3COO-
KS = [CH3COOH2+ ] [CH3COO-]
2C2H5OH
C2H5OH2+ + C2H5O-
KS = [C2H5OH2+ ] [C2H5O-]
di ca thang pH biu kin tu thuc vo hng s t in ly ca dung
mi. V d:
Methanol: pKS== 17
Ethanol: pKS = 20
2.3.3. Xc nh im tng ng
Cn x l in cc ch th ph hp.
53
Cht phn tch khng ho tan trong nc. Trong kim nghim thuc,
thng gp cc acid v base c khi lng phn t ln t tan trong nc.
Sau y s gii thiu cc loi dung mi, dung dch chun trong nh
lng cc acid v base.
2.3.4.1. nh lng acid
Cc acid carboxylic,
Dung mi
Thng chn dung mi c tnh base tng tnh acid ca cht phn tch
nh: pyridin, dimetylformamid (DMFA). Ngoi ra tert - butanol thng c
dng lm dung mi cho chun acid carboxylic, dn xut ca phenol.
Dung dch chun
Dung dch chun kim loi kim gy sai s base cho in cc thu tinh
khi chun o th.
Dung dch chun R4NOH l base mnh, mnh hn dung dch hydroxyd
kim nh KOH, cho nn c th chun cc acid rt yu. Tuy nhin
cc dung dch ny c 2 nhc im:
+ c do c benzen
+ Pha ch mt nhiu thi gian, kh bo qun (d phn ng vi CO2 ca
khng kh).
54
Cht chun
Kalihydrophtalat, E = M = 204,22.
Phn ng chun
C5H5N + HA
RSAg + C5H5N+HNO3-
tng tnh base ca cht phn tch ngi ta dng cc dung mi acid
nh acid acetic khan.
Acid acetic khan thng c x dng ph bin nht v t c, r tin.
Dng anhydrid acetic cng hay dng v c th nh lng cc base rt yu.
Hn na bc nhy th im tng ng trong anhydrid acetic thng ln
hn trong acid acetic. Tuy nhin anhydrid acetic c nhc im l d acetyl
ha cc amin bc nht v bc hai nht l khi un nng.
(CH3CO)2O + RNH2
CH3CONHR + CH3COOH
Trong trng hp nh lng amin khng dng dung mi ny. Ngoi acid
acetic, acetonitril c dng nh lng cc base vi dung dch chun acid
percloric trong 1,4 dioxan.
Dung dch chun
Dung dch acid percloric trong acid acetic khan thng c dng nhiu
nht. Dung dch ny pha ch t acid percloric thng mi 72% (kl/kl). V vy
khi pha ch phi thm anhydrid acetic loi nc v 48 gi trc khi dng.
Ngoi acid acetic khan, cn dng 1,4 dioxan pha dung dch chun acid
percloric. Dung dch ny km n nh, d chuyn thnh mu nu trong qu
trnh bo qun. V vy ch pha v s dng khi cn thit.
55
Cht chun
Solvat ha
RNH3+CH3COO-
RNH2 + CH3COOH
Trung ho
RNH3+CH3COO- + HClO4
RN+H3Cl-O4 + CH3COOH
2CH3COO-BH+ + HgX2
2CH3COO-BH+ + 2HClO4
2CH3COOH + 2BH+ClO4-
y nh lng mui BH+X- thng qua anion CH3COO- (thay cho anion
X-) tng t Y- trong trng hp trn.
56
BH+ + Cl-
B + HCl
NaCl + H2O
BH+ X- + NaOH
NaX + B + H2O
Mui sulfat
RNH+ClO4- + RNH+HSO4-
2C5H5N.HI + C5H5N.SO3
C5H5N(H)SO4CH3
C5H5N(H)SO4H
R CH (OCH3)2 + H2O
MI2 + H2O
59
I2O7 + 5H2O
IO3- + 3H2O
60
HIO3 + 2 CH3COOH
HIO3 + CH3COOH + CH3CHO
NH2
Trong cc phn ng trn, ngi ta thng nh lng bng cch cho tha
thuc th HIO4 vo mu phn tch. Sau khi phn ng kt thc cho lng
d As2O3 v dung dch KI. Iod gii phng c chun bng dung dch
Na2S2O3. Song song lm mt mu trng. l nguyn tc ca phng
php Fleury. C trng hp ngi ta dng phng php iod xc nh
nng periodat ch vi lng d KI, khng cn dng As2O3.
Cn lu l :
+ S phn t formadehyd to thnh bng s chc alcol bc nht.
+ S phn t acid formic to thnh bng s chc alcol bc 2.
Vi cc aldose ta c th vit:
CH2OH(CHOH)nCHO+(n+1)HIO4
(n+1)HIO3+HCHO + (n+1)HCOOH
q=
za zk
2 k T
e2
Trong :
za v zk l in tch ca anion v cation,
e l in tch ca in t
k l hng s Boltzmann
l hng s in mi ca dung dch,
T l nhit tuyt i (0K)
Khong cch q ph thuc vo in tch z ca ion v hng s : nu in
tch cng ln v cng nh th q cng tng, cp ion to ra cng nhiu.
Vi cht in ly 1 -1 trong dung dch nc 250C th q = 3,6 A0 (1A0 = 10-8
cm). iu c ngha l trong dung dch nc, cht in ly 1-1 ho tan, v d
mui KCl, khi 2 ion K+ v Cl- khong cch q < 3,6 A0 s to thnh cp ion.
Chng chuyn ng t do trong dung dch nh cc ion K+, Cl- v phn t khng
in ly. Cp ion trung ho in tch li do va chm lm gim bt lp v hydrat
ha trong dung dch nc nn d chuyn sang ho tan trong dung mi hu c.
62
R NH2 + H+
R NH 3
R- COOH
+
63
H2 O
Met H+Cl-
Cl- + Met H+
MIn + H+
MInH+
CH2Cl2
Met H+LS-
LS- + Na+
NaLS
MInH+LS-
Chit cc acid hu c
64
Khi xy dng chng trnh HPLC phn tch cc dc cht, thng thng
ngi ta s dng k thut to cp ion. V d, nh lng cc cht hu c
mang in tch dng, ngi ta thng thm vo pha ng cc cht c th to
ra anion to cp nh natri lauryl sulfat, natri heptansulfonat, natri
octansulfonat. Vn ny s c cp c th hn mn hc cc phng
php sc k trong chng trnh cao hc.
65
cu hi t lng gi
2.1. Gii thch phn ng th nh tnh ca:
Phn t: Ethanol
66
CH3 CH CO CH3
NH2
C6H5 CH CH2 CH2OH
OH OH
67
Chng 3
3.1.1.1. hp th
Khi cho bc x n sc i qua mt mi trng c cha cht hp th th
hp th ca bc x t l vi nng ca cht hp th v chiu dy ca mi
trng hp th (dung dch cht hp th). Mi quan h ny tun theo nh
lut Lambert Beer v c biu din bng phng trnh sau:
1
A = lg = lg I = K.C.L
T
I
0
(3.1)
Trong :
T: truyn qua
I0: cng nh sng n sc ti
I: cng nh sng n sc sau khi truyn qua dung dch.
K: l h s hp th ph thuc , thay i theo cch biu th nng
L: l chiu dy ca lp dung dch
C: nng cht tan trong dung dch
68
(3.2)
A =
1 %
1 cm
. L .C
1 %
1 cm
A
L .C
3.3
3.1.1.3. My quang ph
(H0)
404,66 nm
(Hg)
253,70 nm
(Hg)
435,83 nm
(Hg)
287,15 nm
(H0)
486,00 nm
(D)
302,25 nm
(Hg)
486,10 nm
(H)
313,16 nm
(Hg)
536,30 nm
(H0)
334,15
(Hg)
546,07 nm
(Hg)
361,50
(H0)
576,96 nm
(H0)
365,48
(Hg)
579,07 nm
(Hg)
253,65nm
275,28nm
296,73nm
313,16nm
334,15nm
365,02nm
404,66nm
435,83nm
546,97nm
576,96nm
Kim tra hp th
E(1%, 1cm)
235
124,5
122,9 126,2
257
144,0
142,4 145,7
313
48,6
47,6 50,3
350
106,6
104,9 109,2
hp th
235
0,748
257
0,864
313
0,292
350
0,640
Da vo cc i hp th:
Trong dung dch nc
+ Vitamin B12 c 3 cc i hp th cc bc sng:
278 nm + 1 nm
361 nm + 1 nm
548 nm + 2 nm
+ Vitamin B2 c 4 cc i hp th cc bc sng:
223nm
375 nm
267nm
444nm
Mat =
[ x. y ( x. y ]2
( x )2
( y )2
2
x
y
10 3
(3.4)
Trong :
x v y l hp th ca ph th nht v ph th hai cng mt bc sng.
n l s im chn 2 ph.
Cch nh gi Mat:
+ < 900: hai ph khc nhau.
+ 900 - 990: hai ph c nhng im tng ng cn cn nhc khi kt lun.
+ > 990: hai ph tng t nhau.
+ Xp x 1000: hai ph ging nhau hon ton.
nh lng
L = 1cm C =
A
1%
E1cm
(3.5)
AX C X
A
=
C X = CS X
AS C S
AS
(3.6)
AX
CX
A
=
C X = CS ' X
'
AX C S + C X
AX AX
(3.7)
75
AX
CX
thm chun
A th
C dd th
C chun thm vo
76
Trong kim nghim cc dng thuc bo ch, trc tin phi qua cng
on chit hot cht ra khi t dc. Dch chit kh trnh khi mang theo tp
cht. Tp cht ny c th gy sai s cho qu trnh nh lng bng phng
php o quang.
loi tr sai s ny, ngi ta thng s dng k thut o quang vi sai:
Trn ph ca cht nghin cu, chn 2 bc sng 1 v 2, hiu s
hp th A l ln nht .
Amax = A 1 A2
(3.8)
(3.9)
1
2
2
A hh
= E 1 2 C 1 + E 2 2 C
..........
..........
..........
+ ... +
..........
n
A hh
= E 1 n C 1 + E 2 n C
(1 )
E n 2 C n (2 )
+ ... + E n 1 C
..........
......
+ ... + E n n C
(3.10)
(n )
2 1
1 2
A E Ahh
E1
C2 = hh 1
1 2
= E1 E2 E1 2 E21
Phng php ph o hm
(3.11)
(3.12)
(3.13)
Do E l hng s v L khng i nn gi tr ca o hm ca A ch cn
ph thuc tuyn tnh vi nng C ca dung dch.
* Ph o hm ca hn hp nhiu cht :
78
(3.14)
Ti cc im cc tr, o hm bc 1 bng 0.
Ti cc im un, o hm bc 2 bng 0.
Ti gi tr 0 ca ph o hm ca cht ny c th gp gi tr khc 0 ca
ph o hm ca cht kia.
Nu hp th tun theo nh lut Lambert-Beer th o hm bc hai
bc sng bt k no cng c lin h vi nng bi phng trnh sau :
d 2 A d 2E
= 2 C.L
d2
d
(3.16)
79
Nhm chc
S sng (cm-1)
Bc sng m
O-H
3600-3000
2,8-3,3
NH2
Bc hai v bc ba
3600-3100
2,8-3,2
C-H
Thm
3150-3000
3,2-3,3
C-H
Mch thng
3000-2850
3,3-3,5
CN
Nitril
2400-2200
4,2-4,6
CC-
Alkyn
2260-2100
4,4-4,8
COOR
Ester
1750-1700
5,7-5,9
COOH
Acid carboxylic
1740-1670
5,7-6,0
Aldehyd v ceton
1740-1660
5,7-6,0
CONH2
Amid
1720-1640
5,8-6,1
C=C-
Alken
1670-1610
6,0-6,2
Ar-O-R
Thm
1300-1180
7,7-8,5
R-O-R
Mch thng
1160-1060
8,6-9,4
C=O
3.1.2.2. My
81
(+ 0,3 )
1583,1
(+ 0,3 )
2924,0
(+ 2 )
1181,4
(+ 0,3 )
2850,7
(+ 0,3 )
1154,3
(+ 0,3 )
1944,0
(+ 1 )
1069,1
(+ 0,3 )
1871,0
(+ 0,3 )
1028,0
(+ 0,3 )
1801,6
(+ 0,3 )
906,7
(+ 0,3 )
1601,4
(+ 0,3 )
698,9
(+ 0,5 )
(3.17)
y :
K : hng s
I0 : cng ca bc x kch thch
: hiu sut hunh quang
=
S photon pht x
------------------------S photon hp th
(0<<1)
(3.18)
82
3.1.3.3. Chun ho my
C X = CS
I X I OX
I S I OS
(3.19)
y:
CX: nng ca dung dch th
CS : nng ca dung dch chun
IX : tr s o c ca dung dch th
IS : tr s o c ca dung dch chun
I0X v I0S : tr s o c ca cc mu trng tng ng.
Vng trong cng hunh quang t l thun vi nng ca cht
thng rt hp, cho nn t s (IX - I0X)/ (IS - I0S) phi khng c < 0,50 v
khng > 2,0. Nu t s khng nm trong khong trn th phi iu chnh
nng v tin hnh o li.
Trong mt s trng hp, c th s dng cch o so snh vi chun c
nh. Th d: dng knh hunh quang hoc mt cht pht hunh quang khc.
Nu cng hunh quang khng t l nghim ngt vi nng th nn
s dng phng php th chun tng quan gia cng v nng .
nhy ca my c th c kim tra bng mt dung dch bn ca cht
hunh quang khc vi di sng kch thch v pht x tng t nh di ca
cht th, thay th cho cht chun.
V d:
84
k' = K
Vs
Q
t t
t'
= S = R = R o
Vm Qm t o
to
(3.20)
y:
K : h s phn b
Vs : th tch pha tnh
Vm: th tch pha ng
Qs: lng cht trong pha tnh
Qm: lng cht trong pha ng
tR : thi gian lu
tR : thi gian lu hiu chnh
t0: thi gian cht
Cn chn ct, pha ng ... sao cho k nm trong khong ti u : 1 < k< 8.
H s chn lc :
k B'
t
= ' = RB
k A t RA
(3.21)
F=
W
2a
(3.22)
y:
W: Chiu rng pic o 1/20
chiu cao pic,
a: Khong cch t ng vung
gc h t nh pic n mp
ng cong pha trc ti v
tr 1/20 chiu cao pic.
85
S a l thuyt v hiu lc ct N:
Hiu lc ct c o bng thng s: S a l thuyt N ca ct
t R2
t R2
N = 16 2 = 5,54 2
W
W1/ 2
(3.23)
y:
W: Chiu rng o y pic,
W1/2: Chiu rng pic o na chiu cao pic.
phn gii Rs:
RS =
2(t RB t RA ) 1,18(t RB t RA )
=
WB + W A
W1 / 2 B + W1 / 2 A
3.24
Vi:
tRB, tRA: Thi gian lu ca 2 pic lin k nhau (B v A),
WB , WA: rng pic o cc y pic,
W1/2B, W1/2A: rng pic o na chiu cao pic.
Cc gi tr: tRB, tRA, WB, WA , W1/2B , W1/2A phi tnh theo cng mt n v.
Yu cu RS > 1, gi tr ti u RS = 1,5.
3.2.2. My HPLC
86
H bm
H tim mu
Ct
Detector o ch s khc x
C8
Octadecyl
C18
Phenyl
C6 H5
Cyanopropyl
= Si- (CH2)3- CN
CN
NH2
88
pht
Hnh 3.6: Sc tch cc steroid
1- Progesterone
2- Androstendione
5- Andrenosterone
6- Cortisone acetat
3- 4- pregnon-20--ol-one
7- Cortisone
4- 17--hydroxyprogesterone
8- Hydrocortisone
- Pha ng:
- Tc dng:
1,5 ml/pht
- Detector:
UV 254 nm
Pht
2 - Codein
3 - Heroin.
91
X n + Qn+ = [ XQ ]hc
- Ct:
- Pha ng:
- Tc dng:
1 ml/ pht.
- Detector:
UV 280 nm
2
3.272
88
66
4
5.583
3
48
7.059
Mu
100
28
0
0
92
1. Vitamin PP
2. Vitamin B6
3. Vitamin B2
4. Vitamin B1
Hn hp steroid acetat
- Ct:
- Pha ng:
- Tc dng:
- Detector:
254 nm
4. Hydrocortison
5. Prednisolon
3. 9 - Fluorohydrocortison acetat
93
94
95
tan
mu th
Tnh cht
mu th
Hexan
Alcol
K thut SK
Ct
Pha o
C18
- Pha thun
- Hp ph
SiCN
C18 CN
C 8 C3
MW < 2000
Khng
ion ha
Pha o
C18PTH
Amin
C18
Amin
Pha o
cp ion
Nc
C18 - IP
Ion ha
mu
Th
Trao i
ion
SAX SCX
Spherogel
Amin
Pha o
C18 C8
Peptid
Loi c
(ry phn t)
MW > 2000
Tan/
nc
SEC,
pha nc
Khng
tan/nc
SEC,
pha hu c
96
USG SEC
2000
Ct
Pha ng
Detector
Mu th
Acenocumarol
RP - 2
ACN/ H2O/CH3COOH
305
Huyt
tng
Acephyllin
C - 18
280
Huyt
thanh
Acetaminophen
C 18
HCOONH4/ MeOH/H2O
254
Siro ho
254
Vin nn
Guaifenesin
Dextromethorphan
Acetaminophen
C 18
Aspirin
Caffein
Codein
Phenacetin
Salicylamid
Acetaminophen
Phenyl
254
Vin nn
Acetazolamide
C - 18
254
Huyt
tng,
nc tiu
Acetazolamide
Silica
Hexan/CHCl3/MeOH/AcOH
254
Nc tiu
P aminophenol
65 /25 / 10 / 0,25
Acid aminocaproic
C8
345
Huyt
thanh
Acid 5- aminosalicylic
C 18
345
Huyt
tng
Acyclovir
C 18
300; 418
Huyt
tng
Amoxicilin
C 18
395; 485
Nc tiu
225
Huyt
tng
85 / 15 / 0,5
Amoxicilin
C-8
Ampicilin
Nc tiu
Antipyrin
C 18
254
Huyt
tng
Apomorphin
Phenyl
273
Huyt
thanh
Acid ascorbic
C 18
254
Nc tiu
Aspirin
Silica
300
Vin nn
97
Tn thuc
Ct
Pha ng
Detector
Mu th
C- 18
280
Huyt
tng,
nc tiu
Atenolol
Cyano
220
Huyt
tng
Barbiturat
C 18
240
Huyt
thanh
240
Huyt
thanh
254 ; 294
Dung dch
254
Aspirin
Acid salicylic
Acid salicyluric
Acid gentisic
60/ 40
Benzocain
C 18
Benzocain
C 18
Acid benzoic
C 18
Acid salicylic
pH 6,2
Benzoyl peroxyd
C 18
Beta methason
C 18
254
Dung dch
C 18
230
Huyt
tng
Sodium phosphat
Bromazepam
20/ 300/ 70
Caffein
Silica
272
Huyt
thanh
Camphor
Silica
254
Dng phn
liu
Captopril
C 18
340
Huyt
tng
Carbamazepin
C - 18
52/ 48
288
Huyt
tng
280
Huyt
tng
C8
Chlodiazepoxide
C 18
260
Huyt
tng
Chloroquin
C 18
340
Huyt
tng
Cyano
254
Nc tiu
Chlopromazin
Cyano
254
Huyt
tng
98
Tn thuc
Ct
Pha ng
Detector
Mu th
Chlorzoxazon
C 18
254
Huyt
tng
Cimetidin
C 18
180
Huyt
tng
Cimetidin
C 18
Huyt
tng
1000/ 50/ 1
Codein
C 18
254
Dng phn
liu
Cortisone v hydrocortison
C 18
254
Dng phn
liu
Cortisone v hydrocortison
Silica
360
Huyt
thanh
Dexamethason
C 18
254
Dng phn
liu
Dextromethophan
C 18
220
Huyt
tng
35/ 65
Diazepam
C 18
254
Vin nn
Vitamin D3
C 18
H2O/ MeOH 1/ 9
254
Huyt
thanh
Estrogen
C 18
280
Vin nn
Fluorouracil
C 18
270
Huyt
thanh
280
Dng phn
liu
6/ 94
Acid folic
C 18
Furosemid
C 18
254
Vin nn
Gentamicin
C 18
230
Huyt
thanh
80/ 10/ 8/ 4
Griseofulvin
Cyano
254
Vin nn
Heptaminol
C8
338
Huyt
thanh,
nc tiu
Heroin
C 18
135
Nc tiu
350
Huyt
tng
228
Vin nn
254
Huyt
tng
C 18
Hydrochlothiazid
C 18
Hydrocortison
Silica
99
Tn thuc
Ct
Pha ng
Detector
Mu th
Ibuprofen
C 18
230
Huyt
tng
Ketoprofen
C8
255
Huyt
tng
Ketoprofen
Silica
254
Huyt
tng
Lidocain
Phenyl
200
Huyt
tng
Methyldopa
C 18
GCE
Huyt
tng
C 18
+ 0,5 V
313
Huyt
tng
322
Huyt
tng,
nc tiu
(pH 6) 27/ 73
Metronidazol
C 18
Morphine
C 18
254
Dng tim
Acid Nalidixic
C 18
313
Huyt
tng
Naphazolin
C 18
255
Huyt
tng
Tetrahydrozolin
300/ 700
: k .
+ Hiu lc ct (s a l thuyt) : N
+
H s i xng
:F
Chun ho ct sc k pha o
Uracil
0,02 mg/l
Phenol
1,00 mg/l
Anisol
1,50 mg/l
iu kin sc k:
Pha ng
Tc dng
: 1ml/pht
Th tch tim
: 20 L
Detector
: UV 254 nm
2. Phenol
3. Anisol
Tnh ton:
Hiu lc ct (N)
N = 5,54. t2A / W21/2
tA : thi gian lu ca anisol
W1/2 : chiu rng pic o na chiu cao ca pic anisol
101
H s i xng (F)
F = WA / 2a
y:
WA : chiu rng pic anisol o 1/20 chiu cao pic
a: khong cch t ng vung gc h t nh pic n mp ng cong
pha trc ca pic ti 1/20 chiu cao pic.
Chun ho ct sc k pha thun
iu kin sc k :
Pha ng
Tc dng
: 1,0 ml/pht
Th tch tim
: 20 L
Detector
: UV 254 nm
Ct s dng
2,0
< 0,7
1000 a
0,9 2,0
0,9 2, 0
H s dung lng (k )
Hiu lc ct (N)
H s i xng (F)
Ly mu th
Tin hnh sc k
o tn hiu detector
102
Ly mu
Qu trnh chy sc k cn ch :
C th xy ra s phn hu ca cht th trong khi phn tch.
C th xut hin pic l trn sc do dung mi dng ho tan mu th c
cha tp cht. V vy cn phi kim tra bng sc k cc vt tp .
Chun b mu th:
Tn hiu detector:
c X = CS
SX
SS
y:
CX
: nng mu th
CS
+ Xy dng phng trnh hi qui tuyn tnh m t quan h gia din tch
(hoc chiu cao) pic vi nng ca cht cn xc nh.
Y = a + bCx
3.25
Trong :
Y: Din tch pic
a : Giao im ca ng chun vi trc tung.
b : dc ca ng chun.
Cx : Nng ca cht th .
Da vo phng trnh hi quy ny ta tnh c nng cht th .
CX =
Y a
b
3.26
106
FX =
mC S C mC .S IS
:
=
m IS S IS m IS S C
(3.27)
FX =
CC .S IS
C IS S C
(3.28)
y:
- mC , mIS ln lt l khi lng ca cht chun v chun ni.
- CC , CIS ln lt l nng ca chun v chun ni.
- SC , SIS ln lt l din tch pic chun v chun ni.
Cc sai s s c cc tiu ha nu h s FX xp x n v, c ngha l
cht chun v chun ni c cng p ng vi detector. Tuy nhin trong thc t
iu ny thng kh t c hon ho.
* nh lng thnh phn trong mu th.
mT =
ST
m IS FX
S IS
(3.29)
CT =
ST
C IS FX
S IS
(3.30)
SX/ SIS
CX/ CIS
CS/CIS
107
cX = S X
C
S
108
= S2a/S1a ( sc a )
Rb
= S2b /S1b ( sc b )
Rthm = Rb - Ra
Yth
= (Ythm). (Ra)/Rthm
S (H)
dd th
SX
CX
thm chun
C chun thm vo
109
%X =
S .100
.S X .100
= Xn
S X + SY + S Z
Si
i =1
FX =
S S .C X . f S
S X .C S
y:
SS v SX : din tch ca pic chun v pic th
CS v CX : nng ca cht chun v cht th
fS
%X =
S X .FX .100
S X FX + SY FY + S Z FZ
cu hi t lng gi
A257
0,1
0,105
0,2
0,207
0,3
0,318
0,4
0,420
0,5
0,529
tR (pht)
W1/2(pht)
Cht khng lu gi
1,9
10,0
0,76
10,9
0,82
13,4
1,06
Hy tnh :
a/. S a l thuyt trung bnh ca ct ?
b/. Chiu cao ca a ?
c/. phn gii cho:
+ B v A
+ B v C
+ A v C
3.10. Cho ct sc k di 30 cm. Khi chy sc k tch hn hp 2 cht thu c
kt qu:
Cht
tR (pht)
Cht khng lu gi
1,30
16,40
1,11
17,63
1,21
Hy tnh :
phn gii ca ct
S a l thuyt trung bnh ca ct, chiu cao a.
Nu chng ta mun phn gii trong tch 2 cht A v B l RS(m) = 1,5 th:
a/. Yu cu ct phi c s a l thuyt l bao nhiu ?
b/. Nu khng thay i 2 pha, chiu di ct s phi l bao nhiu ?
3.11. Khi chy sc k mt hn hp 2 cht A v B thu c thi gian lu ln
lt l: 10,0 v 10,9 trn ct di 40cm. Mt cht khng lu gi qua ct
1,9 pht.
Chiu rng y pic ca A v B l 1,52pht v 1,64 pht.
Cho bit th tch pha tnh VS = 19,6ml v th tch pha ng VM = 62,6ml.
113
Hy tnh :
a/. H s dung lng k cho mi cht ?
b/. H s phn b cho mi cht ?
c/. H s chn lc cho A v B ?
3.12. Hy so snh sc k lng hiu nng cao vi sc k ct c in ?
3.13. Trnh by v s khc nhau gia sc k trao i ion v sc k loi c (sc
k trn gel) ?
3.14. Hy trnh by v cc loi bm s dng trong HPLC, u, nhc im ca
mi loi ?
3.15. Nguyn tc hot ng ca detector dn nhit v ion ho ngn la ?
3.16. Hy trnh by v phng php s dng chun ni trong nh lng bng
sc k lng hiu nng cao ?
3.17. Hy ni v phng php thm chun trong nh lng bng HPLC ?
114
Chng 4.
Mc tiu hc tp
1. Vit c nhng c im c bn v hnh thi, tnh cht nui cy ca
vi khun, nm mc, nm men p dng trong th nghim vi sinh vt.
2. Bit phng php lm mt mi trng nui cy vi sinh vt v nu
c cc phng php tit trng.
3. Trnh by c mc ch, nguyn tc, phng php th v trng v
m s lng vi sinh vt trong 1 gam (1 ml) dc phm.
Nu c vai tr ca phng php sinh hc trong kim nghim cht
khng sinh, v trnh by c th nghim nh lng cht khng
sinh bng phng php khuch tn.
4.1. M u
Trong ngnh Dc c nhiu phng php khc nhau kim tra cht
lng ca thuc nh phng php ho hc, phng php vt l, phng php
sinh hc.
Phng php ho, l tin hnh nhanh chnh xc nhng ch p dng c
vi cc cht c thnh phn ho hc bit v khng nh gi c bn cht
sinh hc ca mt dc phm. xc nh cc c tnh sinh hc theo yu cu
ca mt s dc phm nh: hot lc tc dng ca cht khng sinh, s v trng,
nhim khun, c tnh bt thng ca mt s loi thuc, hay hiu lc v tnh
an ton ca cc vaccin ... ngi ta phi dng phng php sinh hc.
4.1.1. Nguyn tc
Phng php sinh hc da trn nguyn tc:
So snh hiu lc tc dng hoc cc c tnh ring ca cht th vi cht
chun tng ng trong cng mt iu kin v thi gian th nghim.
Trong lnh vc kim nghim thuc, hai loi th nghim sinh hc c p
dng nhiu nht l:
Cht chun gc: l nhng cht ng nht c tinh khit cao. Cht
chun gc thng c lm cc vin nghin cu quc gia hoc quc t
ring v cht chun sinh hc.
Cht chun th cp: cng l cht c tinh khit cao, c hot tnh sinh
hc c xc nh theo cht chun gc quc t tng ng.
116
Th c tnh bt thng
Th cht h huyt p
Th cht gy st
mt s to bo t. Mi t bo vi khun ch c mt bo t. Mt s vi khun c
kh nng di ng nh s c mt ca mt hoc nhiu lng (flagella).
Phn loi:
Vic phn loi vi khun rt phc tp, phi da vo nhiu c im, hnh
thi, sinh l, sinh ho chia vi khun thnh cc h, chi, loi khc nhau. Vi
mc ch phc v cho cng tc kim nghim thuc, ta khng i su vo nghin
cu phn loi, nhng cn tm hiu vi khun, theo cc nhm da trn hnh th,
tnh cht bt mu thuc nhum Gram v kh nng h hp ca chng.
Sinh sn ca vi khun:
log N
3
4
2
thi gian
118
4.3.1.2. Vi nm (Microfungi)
c im:
Nm mc (Mold):
Nhit :
m:
iu chnh pH:
Lm trong mi trng
ng ng tit trng:
Tia t ngoi (UV) c dng nhiu nht tit trng cc bung pha ch,
t cy vi sinh vt.
n t ngoi phi c chiu trc tip, thng gc vi ni lm th nghim
v liu lng chiu phi vi din tch bung.
Tia UV t c tc dng dit nm, v vy khi kh trng bung pha ch cn
phi hp thm phng php dng ho cht kh nm.
4.3.3. Th v trng
4.3.3.1. Mc ch
Th v trng nhm mc ch pht hin s c mt ca vi khun, vi nm
trong cc ch phm nh dch tim truyn, mt s loi thuc tim, thuc tra
mt v cc dng c y t m theo tiu chun ring cn phi v trng.
4.3.3.2. Nguyn tc
Vi sinh vt c trong ch phm th s pht trin trn cc mi trng dinh
dng thch hp, chng lm c mi trng lng to vng trn b mt hoc
lng cn y ng nghim. Trn mi trng c vi khun, vi nm mc thnh
cc khun lc c trng.
4.3.3.3. Mi trng
Trong nhiu dc in thng dng mi trng Thioglycolat lng pht
hin vi khun hiu kh, k kh v mi trng Casein u tng lng pht
hin vi khun, vi nm. Tuy nhin, c th dng cc mi trng khc cho th
nghim, vi iu kin cc mi trng ny thch hp cho s pht trin ca loi
vi sinh vt cn pht hin. V d c th dng mi trng canh thang cao tht pepton pht hin vi khun hiu kh; mi trng Wilson - Blair pht hin vi
khun k kh; mi trng Sabouraud lng cho s pht hin vi nm.
Kim tra cht lng mi trng:
v trng:
124
Thit b lc thng bng thu tinh, thp khng r hoc nha gm hai b
phn c th tho ri, gia c li mng lc. Mng lc c thnh phn l
nitrat celleulose thng dng lc nc, du, v dung dch alcol yu. Mng
ecetat cellulose lc cc dung dch alcol mnh. L mng lc c nhiu kch
thc khc nhau, trong th v trng thng dng mng c ng knh khong
50mm v ng knh l mng lc 0,45m.
Thit b lc v mng lc phi c tit trng trc khi th nghim .
126
K thut th:
Mu th l dc phm:
Th tch
mi trng (ml)
Ton b mt ng
10
1 ml V< 4 ml
1/2 ng
15
4 ml V < 20ml
2ml
20
5ml
40
10ml
80
Thng 10%
100
- Cht lng:
Th tch V < 1ml
V 100ml
- Cht rn:
Khi lng P< 50mg
Ton b mt n v ng gi
1/2 khi lng ca mt n v
ng gi
20
40
100mg
P 200mg
80
Vi sinh vt ch th:
Staphylococcus aureus (i din vi khun G +).
Escherichia coli (i din vi khun G -).
Candida albicans (i din vi nm).
Nhn xt kt qu:
m s khun lc vi sinh vt trn cc a th nghim.
Gi A l s khun lc a 1
Gi B l s khun lc a 2
Gi C l s khun lc a chng
Nu B A + C mu th khng c cht c ch.
i vi vi khun:
Cho vo mi a petri 1ml cht th nng thch hp sao cho khng qu
300 khun lc trong 1ml. Thm 15 - 20ml mi trng thch casein - u
tng hoc mi trng thch thng ngui di 45oC, xoay nh a
cht th trn u vo mi trng. Nui cy 30 - 350C trong 1 - 2 ngy.
i vi vi nm:
Cho vo mi a petri 1ml cht th nng thch hp sao cho khng
qu 100 khun lc vi nm trong 1ml. Thm 15-20ml thch Sabouraud
+ khng sinh.
Tnh kt qu:
Tng s vi khun hiu kh, vi nm trong 1g (1ml) c tnh theo cng thc:
X=
A1k 1 + A 2 k 2
2
A1
A2
4.3.5.2. Nguyn tc
Hot lc ca mt cht khng sinh c xc nh bng cch so snh kh
nng c ch ca chng vi sinh vt ch th, bi nhng nng bit ca
khng sinh th (cha bit hot lc) v nng bit ca khng sinh chun
( bit r hot lc).
4.3.5.3. Chng ch th
Chng ch th l chng vi sinh vt thun khit, nhy cm i vi mt
cht khng sinh, chng ch th thng c phn lp t cc bo tng ging
Quc gia, c bo qun trong ng ng kh hoc trong mi trng thch hp
4 - 10oC. Tu theo dc in ca mi nc, mi cht khng sinh c th c mt
vi chng ch th khc nhau.
Ch to nh dch vi sinh vt khng c bo t:
Cc chng vi khun:
Staphylococcus aureus
Sarcina lutea
131
Escherichia coli
Micrococcus luteus
Klebsiella faecalis.
Chng vi nm:
Saccharomyces cerevisiae.
Cc chng ch th c bo t:
Bacillus subtilis
Bacillus pumilus
Bacillus cereus
Nu chng ch th c bo t nn ch to dng bo t s dng, v dng
bo t c bo qun di 4oC c th tn ti trong mt thi gian rt di.
Cc chng vi khun trn c cy vo bnh mi trng thch dinh dng
c b mt thch rng (pepton 6g, cao tht 1,5g, cao men 3g, glucose 1g,
K2HPO4 3,68g, KH2PO4 1,32g, thch 15g, nc ct 1000ml)
Mi trng ny c thm MnSO4 vi hm lng 0,001g/lt thc y
s hnh thnh bo t. Nui cy 35 - 37oC trong vng 7 ngy vi B. cereus nui
cy 30oC. Dng nc ct v trng ra lp bo t trn b mt mi trng
to mt nh dch. un cch thy nh dch ny 70oC/ 30 pht, pha long nh
dch n nng 107 - 108 bo t trong 1ml.
4.3.5.4. Cht chun v n v hot lc
Cht chun:
Cht chun l nhng cht c tinh khit cao, c th dng cht chun
gc hoc cht chun th cp c hot lc c xc nh theo mu chun quc t
132
134
S1
T3
T2
S2
S3
T1
4A
R = Anti log 2 +
log I (P = 0,95)
3B
4
A
Hot lc ca mu th =
R hot lc chun
100
135
Ta< ta .V
Tb >> tb .V
Tc < tc .V
Tac < tac .V
V: l tng ca cc hiu gi tr ng knh ln nht v nh nht ca mt
nng .
Ta = (T1 + T2 + T3) (S1 + S2 + S3)
Tb = (T3 + S3) (T1 + S1)
Tab = (S1 + T3 ) (S3 + T1)
Tc = (S1 + S3 + T1 + T3) 2 (S2 + T2)
Tac = (2S2 + T1 + T3) (2T2 + S1 + S3)
ta, tb, tc, tab, tac l cc h s tra bng theo s vng v khun (n) do c ca
mt nng .
Gi tr t:
Gi tr t (P = 0,95)
S vng
v khun (n)
ta
tb
tc
tab
tac
0,80
0,66
1,14
0,66
1,14
0,80
0,66
1,14
0,66
1,14
0,81
0,66
1,14
0,66
1,14
0,82
0,67
1,16
0,67
1,16
0,83
0,68
1,17
0,68
1,17
10
0,84
0,68
1,19
0,68
1,19
136
lch chun:
SM =
M 1, 2 = 0,4013
A
0,301S M
B
Sai s th nghim:
e% =
R1 R
2R
100
Kt qu nh lng c gi tr vi e 5%.
Gi tr Hn
N
10
Hn
1,27
1,03
0,90
0,81
0,75
0,69
0,65
0,65
Cu hi t lng gi
138
139
Chng 5.
Mc tiu hc tp:
1. Trnh by c cc yu cu k thut v phng php th nh gi
cht lng cc dng bo ch: Thuc bt, thuc vin nn, vin nang,
thuc tim, thuc tim truyn, thuc nh mt, thuc m, thuc ung
dng lng, thuc n, thuc trng.
2. nh gi c kt qu kim nghim i vi mt mu kim nghim
thnh phm c th ca cc dng bo ch trn.
Khi tin hnh kim nghim mt mu ch phm thuc mt dng bo ch
c th (thuc bt, vin nn, vin nang, thuc tim, ...) theo chuyn lun ring
ca ch phm trong Dc in, thng thng yu cu ghi phn u ca
chuyn lun l ch phm phi p ng cc yu cu chung ca dng bo ch
v cc yu cu ring ca dc cht, ca ch phm nh tnh cht, nh tnh,
nh lng, tp cht (nu c), ... Cn nu tiu chun kim nghim l tiu
chun ca nh sn xut (TCCS) th trong tiu chun kim nghim c y
yu cu k thut v phng php th c th cho ch phm , mt s tiu ch
c trng cho dng bo ch ca ch phm c tin hnh th theo hng
dn ca Dc in.
Trong ti liu ny, chng ti cp ti yu cu k thut v phng php
th ca cc dng bo ch thng dng nh thuc bt, thuc vin nn, vin
nang, thuc tim, thuc tim truyn, thuc nh mt, thuc m, thuc ung
dng lng, thuc n, thuc trng. Ti liu tham kho chnh c s dng l
Dc in Vit Nam III, ng thi chng ti cng s dng Dc in ca mt
s nc khc nh Dc in Anh, Dc in M, Dc in Trung Quc v
mt s ti liu kim nghim cc dng bo ch khc.
5.1. Kim nghim thuc bt
5.1.1. Yu cu k thut v phng php th
5.1.1.1. Tnh cht
Bt phi kh ti, khng b m, vn, mu sc ng nht. Mi v ty theo
tng loi ch phm ca cc nh sn xut.
Cch th: Tri mt lng bt va thnh mt lp mng trn mt t
giy trng mn. Quan st mu sc bng mt thng, di nh sng t nhin.
140
5.1.1.2. m
Cc thuc bt khng c cha hm lng nc qu 9,0%, tr ch dn khc.
Cch th: theo Xc nh mt khi lng do lm kh - Ph lc 5.16 DVN III.
5.1.1.3. mn
Thuc bt phi t mn quy nh trong chuyn lun ring.
Nu khng c ch dn khc, php th ny dng cho tt c cc thuc bt
kp, cc thuc bt dng p, thuc bt dng pha ch thuc dng cho
mt, tai.
Cc c bt c quy nh da vo cc s ca ry. Ry c li ry v li
ry c th lm bng si kim loi hoc si cc vt liu thch hp khc c dt
thnh nhng mt vung. Li ry dng ry bt thuc c phn loi bng
nhng con s biu th kch thc l ry, quy nh tnh bng m theo bng 5.1.
Cch th: Chn c ry thch hp theo qui nh ca tiu chun. Cn mt
lng thuc bt, em ry qua ry c c qui nh.
i vi bt th hoc na th: ly 25g -100 g bt. Cho vo ry thch hp,
lc ry theo chiu ngang quay trn t nht 20 pht v ry ti khi xong. Cn
chnh xc s lng cn li trn ry v s thu c trong hp hng.
i vi bt na mn, mn hay rt mn: ly khng qu 25 g bt, cho vo ry
thch hp, lc ry theo chiu ngang quay trn t nht 30 pht ri ry ti khi
xong. Cn chnh xc s lng cn li trn ry v s thu c trong hp hng.
i vi cht c du hay bt c xu hng bt mt ry th trong qu trnh
ry thnh thong chi cn thn mt ry, tch ri nhng ng t li khi ry.
Thuc bt t tiu chun v mn nu:
141
C mt ry (mm)
ng knh si ry (mm)
1400
1,400
0,710
710
0,710
0,450
355
0,355
0,224
250
0,250
0,160
180
0,180
0,125
125
0,125
0,090
90
0,090
0,063
Bt th (1400/355)
Bt na th (710/250)
Bt na mn (355/180)
Bt mn (180/125)
Bt rt mn (125/90)
10
Trn 6,00g
Ti 100 mg
15
Trn 100 mg ti 1g
10
Trn 1 g n 5 g
Trn 5 g
Nh hn 300 mg
10
7,5
5.2.1.3. ng u hm lng
Nghin mn bt thuc ring tng vin v tin hnh th v nh gi nh
i vi thuc bt.
5.2.1.4. r
Nu khng c qui nh ring th tin hnh th v nh gi theo "Php
th r vin nn v vin nang" - phn 5.10, trang 154 - 157.
Vin khng cn th r khi php th ha tan c thc hin.
5.2.1.5. ha tan
Trong DVN III, nhiu ch phm thuc vin yu cu nh gi ha
tan. Khi c yu cu s ch dn trong chuyn lun ring.
Cch th: Tin hnh theo Phng php th tc ha tan ca vin nn
v vin nang - phn 5.9, trang 160 - 165.
145
5.2.1.6. nh tnh
Tin hnh nh tnh theo cc phng php c qui nh trong tiu
chun, vin nang phi cho cc phn ng ca cc hot cht c trong ch phm
5.2.1.7. nh lng
Cn thuc trong 20 vin, tnh khi lng trung bnh vin, lm ng nht
bng cch nghin (i vi vin nang cha bt hoc cm) hoc trn u (i vi
nang cha cht lng). Tin hnh nh lng theo cc phng php c qui
nh trong tiu chun, hm lng ca tng hot cht trong ch phm phi
nm trong gii hn cho php theo bng 5.5.
Bng 5.5. Gii hn cho php v hm lng i vi thuc vin nang, vin nn
Lng ghi trn nhn
Ti 50 mg
10
Trn 50 mg ti 100 mg
7,5
Trn 100 mg
146
Ti 80 mg
10
Trn 80 mg n 250 mg
7,5
Trn 250 mg
147
148
Ti 5 ml
+ 15
T 5ml ti 50 ml.
+ 10
Trn 50 ml
+5
152
Dng bt
10
Kt qu:
- Khng c c qu 20 tiu phn c kch thc ln hn 25 m.
- Khng c c qu 2 tiu phn c kch thc ln hn 50 m.
- Khng c c tiu phn no c kch thc ln hn 90 m.
5.5.7. nh tnh
Tin hnh nh tnh theo cc phng php c qui nh trong tiu chun,
thuc nh mt phi cho cc phn ng ca cc hot cht c trong ch phm.
5.5.8. nh lng
Ly thuc ca 5 n v ng gi nh nht bt k, trn u. Tin hnh
nh lng theo cc phng php c qui nh trong tiu chun, hm lng
ca tng hot cht trong ch phm phi nm trong gii hn cho php qui nh
trong chuyn lun.
5.6. Kim nghim thuc ung dng lng
5.6.1. Yu cu k thut v phng php th
5.6.1.1. Tnh cht
Th cht, mu sc, mi v ty theo tng chuyn lun.
Cch th: Bng cm quan.
5.6.1.2. trong
Thuc ung dng dung dch phi t yu cu v trong theo tng
chuyn lun.
Cch xc nh: Ging nh i vi thuc nh mt.
5.6.1.3. Th tch
Cch xc nh: Ging nh i vi thuc nh mt.
Th tch thuc phi nm trong gii hn cho php theo bng 5.9.
5.6.1.4. pH
Mt s ch phm c yu cu th pH phi nm trong gii hn qui nh.
o bng my o pH - Ph lc 5.9 - DVN III.
154
Bng 5.9. Gii hn cho php th tch thuc ung dng lng
Dng thuc
Thuc nc ung
Siro
Ti 20
+ 10
Trn 20 - 50
+8
Trn 50 - 150
+6
Trn 150
+4
Ti 100
+ 10
+8
Trn 250
+6
Bng 5.10. Gii hn cho php v hm lng i vi thuc ung dng lng
Dng thuc
Thuc c A, B
Thuc thng
10
5.6.2.1. Cn thuc
Cn thuc l nhng ch phm lng c iu ch bng cch ngm chit
dc liu thc vt, ng vt hoc bng cch ha tan cao thuc, dc cht theo
t l qui nh trong ethanol cc nng khc nhau.
5.6.2.2. Siro
Siro l dung dch m c (khng di 60%) ca ng trng (Saccharose)
trong nc, c cha cc dc cht hoc cc dch chit t dc liu v cc cht
lm thm.
5.6.2.3. Potio
Potio l dng thuc nc c v ngt, cha mt hay nhiu dc cht, dng
ung tng tha. Potio c th dng hn dch hoc nh dch.
Dung mi ca potio c th l nc, nc thm, nc hm hay nc sc
dc liu. Potio thng cha 20% siro.
5.6.2.4. Dung dch ung (Oral solution)
Dung dch ung l nhng ch phm lng trong sut, cha mt hay nhiu
dc cht ha tan trong mt dung mi hay hn hp nhiu dung mi thch hp
dng bng cch ung.
Dung mi ca dung dch c th l nc, du, ethanol, glycerin...
5.6.2.5. Thuc ung git (Oral drop)
Thuc ung git l thuc ung c dng vi th tch nh bng dng c
phn liu l ng nh git.
5.6.3. Dung dch thuc dng ngoi dng lng
Thuc dng ngoi dng lng l cc ch phm lng ng nht, thng l
dung dch, cn thuc, hn dch hoc nh dch ca mt hay nhiu hot cht
trong cht dn thch hp dng ngoi.
Cc thuc dng ngoi dng lng phi t theo cc yu cu k thut
chung ca thuc ung dng lng v ring tng dng thuc c nhng yu cu
ring. Ring yu cu " nhim khun" khng phi lm.
156
Di 10,0
15
T 10,0 - 20,0
10
Trn 50,0
5.7.1.3. ng nht
Cc tiu phn phi phn tn ng u.
Cch th: Ly 4 n v ng gi, mi n v khong 0,02 -0,03 g, tri ch
phm ln 4 tiu bn, bn trn t mt phin knh. y mi phin knh bng
mt phin knh khc v p mnh cho n khi to thnh mt vt c ng knh
khong 2 cm. Quan st vt thu c bng mt thng, 3 trong 4 tiu bn
khng c nhn thy cc tiu phn. Nu c cc tiu phn nhn thy trong
phn ln s cc vt th phi lm li vi 8 n v ng gi. Trong s cc tiu bn
ny, cc tiu phn cho php nhn thy khng c vt qu 2 tiu bn.
5.7.1.4. nh tnh
Tin hnh nh tnh theo cc phng php c qui nh trong tiu
chun, thuc m phi cho cc phn ng ca cc hot cht c trong ch phm.
5.7.1.5. nh lng
Cn thuc trong 5 n v ng gi nh nht, tnh khi lng trung bnh,
trn ng nht. Cn mt lng ch phm nh ch dn trong chuyn lun,
157
tin hnh nh lng. Hm lng hot cht phi nm trong gii hn qui nh
theo bng 5.12.
Bng 5.12. Gii hn cho php v hm lng i vi thuc m
Nng hm lng ghi trn nhn
Ti 200 mg
15
Trn 200 mg - 1 g
10
Trn 1 - 5 g
7,5
Trn 5 g
5.8.4. nh tnh
Tin hnh nh tnh theo cc phng php c qui nh trong tiu
chun, thuc n v thuc trng phi cho cc phn ng ca cc hot cht c
trong ch phm.
5.8.5. nh lng
Cn thuc trong 20 vin, tnh khi lng trung bnh vin, lm ng nht.
Tin hnh theo phng php qui nh trong tiu chun, hm lng ca tng
hot cht trong ch phm phi nm trong gii hn cho php nh bng 5.5.
5.9. php Th ho tan ca vin nn v vin nang
(Dissolution test for tablets and capsules)
5.9.1. Khi nim
ho tan ca mt ch phm l t l hot cht c gii phng ra khi
dng thuc theo thi gian vi cc iu kin qui nh trong tng chuyn lun.
Vi mi ch phm c cc qui nh c th v thit b th, mi trng ha
tan, thi gian th nghim v phn trm hot cht c gii phng (Ch s Q).
5.9.2. Thit b
Ty thit b c th c mt bnh hoc 6 - 8 bnh th.
5.9.2.1. Thit b kiu gi quay (Basket apparatus)
Thit b bao gm:
1. Mt cc hnh tr C bng thu tinh borosilicat hoc bng cht liu
trong sut thch hp, c y hnh bn cu v c dung tch 1000ml, hnh 5.1 v
hnh 5.2. Ming bnh c vin rng, c y bng np c mt s l nh, trong
c mt l tm ca np.
2. Mt ng c vi b phn iu tc c kh nng duy tr tc quay ca
gi trong phm vi 4% tc t. ng c ny gn vi mt b phn khuy bao
gm trc quay A v gi hnh ng tr B. Tr kim loi phi quay c mt cch
nh nhng, khng b lc l nhiu trong khi quay. B phn gi gm c 2 phn:
160
C.
Hnh 5.1. Thit b th ha tan ca vin nn, vin nang ca Pharma TEST
1. B iu nhit
4. a y
2. Cc ng mi trng ha tan
5. Trc khuy
3. Cnh khuy
6. B phn iu khin
5.9.3. Cch th
5.9.3.1. Chun b mi trng ha tan
Mi trng ho tan c ch dn trong tng chuyn lun ring. Nu l
dung dch m th pH phi iu chnh sai khc khng qu 0,05 n v. Mi
trng ho tan phi loi kh trc khi dng.
Cho mt th tch quy nh mi trng ho tan ui kh vo bnh (th
tch qui nh 1%). Lm m mi trng ho tan n nhit 37 0,5 oC.
5.9.3.2. Cho vin vo thit b th
Nu khng c ch dn g khc th th ng thi trn 6 vin, cho mi vin
vo mt bnh tr hoc mt gi quay.
162
Khi dng thit b gi quay: Cho vin nn hay vin nang vo trong gi
kh. H thp gi vo ng v tr ri cho gi quay.
Khi dng thit b kiu cnh khuy: Cho vin nn hay vin nang chm
xung y bnh trc khi cho cnh khuy quay; c th dng mt dy
xon bng kim loi hay thu tinh gi cho vin thuc nm ngang
di y bnh. Cn loi tr bt khng kh khi b mt vin.
Hoc phn trm dc cht gii phng so vi hm lng ghi trn nhn
c nh gi theo bng 5.13 v bng 5.14 (theo USP 24).
163
Bng 5.13. Gii hn v phn trm dc cht gii phng i vi vin nn,
vin nang nh gi theo tng vin (Theo USP 24 - trang 1943)
Ln
S vin
th
Mi vin phi Q + 5%
12
Yu cu kt qu
(Phn trm hot cht ha tan)
t: Mu th t yu cu v ha tan.
Dng th nghim.
Khng t: Lm ln 2
t: Mu th t yu cu v ha tan.
Dng th nghim.
Khng t: Lm ln 3
t: Mu th t yu cu v ha tan.
Khng t: Mu th khng t yu cu v
ha tan.
Bng 5.14. Gii hn v phn trm dc cht gii phng i vi vin nn, vin nang
nh gi theo cch gp dch lc ca cc vin th (Theo USP 24 - trang 1943)
Ln
S vin
th
Yu cu kt qu
(Phn trm hot cht ha tan)
Trung bnh ca 6 vin phi Q + 10%
t: Mu th t yu cu v
ha tan. Dng th nghim.
Khng t: Lm ln 3
12
t: Mu th t yu cu v
ha tan.
Khng t: Mu th khng t yu
cu v ha tan.
164
Bng 5.15. Gii hn v phn trm dc cht gii phng i vi vin bao, vin nang
tan trong rut (mi trng th l HCl 0,1M) - Theo USP 24 - trang 1947
L
S vin
th
Yu cu kt qu
(Phn trm hot cht ha tan)
12
V d:
Vin nang Cephalexin (DVN III, trang 53 - 54)
Thit b: Gi quay
Mi trng ha tan: 1000 ml nc
Tc : 100 vng/ pht
Thi gian: 45 pht
Ly v x l mu: Lc dung dch sau khi ha tan. Pha long dch lc
bng nc c dung dch cephalexin c nng 25 g/ml.
o hp th ca dung dch thu c bc sng 262 nm vi mu
trng l nc.
Song song lm mt mu chun cephalexin c cng nng .
Yu cu: Khng c t hn 80% lng cephalexin (C16H17N3O4S) ghi trn
nhn c ha tan trong 45 pht.
Vin bao Ibuprofen (DVN III, trang141)
4
2
3
2. B iu nhit
5. ng hnh tr
6. Gi
3. Cc ng mi trng th
7. B phn iu khin
167
5.10.3. Cch th
5.10.3.1. Chun b
Nu c 6 vin u r ht: Mu th t yu cu v r.
Cch pha m phosphat pH 6,8: Trn dung dch acid hydrocloric 0,1N v
dung dch natri phosphat 0,2N theo t l 3: 1. iu chnh pH n 6,8 0,05
bng dung dch acid hydrocloric 2N hoc dung dch natri hydroxyd 2 N.
168
Cu hi t lng gi
5.1. Trnh by tiu chun v k thut kim nghim chung ca thuc bt?
5.2. Trnh by tiu chun v k thut kim nghim chung ca vin nang?
5.3. Phn loi cc loi vin nang? Cho bit nhng yu cu ring cho tng
loi nu c?
5.4. Trnh by tiu chun v k thut kim nghim chung ca thuc vin nn?
5.5. Phn loi cc loi vin nn? Cho bit cch th r i vi tng loi?
169
5.6. Trnh by tiu chun v k thut kim nghim chung ca thuc tim
v thuc tim truyn?
5.7. Trnh by tiu chun v k thut kim nghim chung ca thuc nh mt?
5.8. Trnh by tiu chun v k thut kim nghim chung ca thuc m
v thuc m tra mt?
5.9. Trnh by cch th tc ha tan v nh gi kt qu ca vin nn
paracetamol 500 mg; vin nang cephalexin 500 mg v vin bao
diclofenac 50 mg?
5.10. Anh (ch) hy cho bit cch th r v nh gi kt qu ca vin
nn acid ascorbic 100 mg; vin nang cloxacilin 250 mg v vin bao
cimetidin 400 mg ?
5.11. Anh (ch) hy xut tiu chun v qui trnh kim nghim cho vin
nn A c hm lng hot cht l 1,5 mg/ vin.
5.12. So snh phng php th r ca vin nn ca Dc in Vit
Nam I - tp 1 v Dc in Vit Nam III.
5.13. Ngi ta tin hnh nh lng paracetamol trong vin nn
paracetamol 500 mg nh sau: Cn 20 vin c khi lng 14,6853g.
Nghin mn. Cn 0,2205 g bt vin, ha tan trong va 200 ml
dung mi. Lc u, lc. Pha long 1,00 ml dch lc thnh 100,0 ml
trong bnh nh mc bng dung mi. Mt quang ca dung dch
ny 257 nm l 0,501.
Hi ch phm c t yu cu v hm lng khng? Bit
paracetamol c A (1%, 1cm) bc sng 257 nm l 715.
5.14. Th ha tan ca vin nn ciprofloxacin 250 mg theo DVN III,
sau khi dng khuy, pha long chnh xc mi trng ha tan trong
mi cc th 50 ln. o hp th ca cc dung dch ny theo iu
kin cho c kt qu ln lt tng ng vi cc cc 1; 2; 3; 4; 5 v
6 l 0,435; 0,346; 0,462; 0,378; 0,489 v 0,408. Hi ch phm c t
yu cu v ha tan khng? Bit dung dch ciprofloxacin chun
nng 5,5 g/ ml c hp th l 0,505 o trong cng iu kin.
170
Chng 6.
n nh v tui th ca thuc
Mc tiu hc tp
1. Trnh by c mc tiu v tiu chun nh gi n nh ca thuc.
2. Gii thch c nguyn tc xc nh n nh v cch tnh tui th
ca thuc.
Mt trong nhng yu cu quan trng nht i vi thuc phng v cha
bnh l n nh v cht lng trong sut qu trnh bo qun t khi xut
xng n khi ht hn dng. n nh ca thuc lin quan n qu trnh
sn xut, phn phi v bo qun thuc.
Khi nghin cu trin khai thuc mi hoc hon thin nng cao hiu lc
ca mt thuc c x dng trong lm sng, nh sn xut cn nh gi
n nh ca thuc trong iu kin bo qun xc nh, t tnh ra tui th
ca n. Ngi phn phi, lu gi thuc phi duy tr c iu kin bo qun
thuc qui nh m bo hn dng ca thuc.
Vic nghin cu n nh ca thuc l mt qu trnh hon thin
phng php v cho ti gn y, T chc Y t th gii (WHO) mi c vn bn
chnh thc hng dn nghin cu n nh ca thuc. Dc in ca mt s
nc c chuyn lun v vn ny.
6.1. Qu trnh pht trin nghin cu n nh
Ngi ta c nhng ghi nhn ban u lin quan n bn vng ca
thuc. Mt s ch phm kh bo qun , d phn hy nh aspirin, procain,
mt s vitamin nh A, C. Ngc li, mt s ch phm khc li kh bn vng
nh sulfonamid. T y khi nim v n nh ca thuc dn dn c
hnh thnh.
Nm 1948, tp ch Cng ngh Dc v M phm ca M ng mt kt
qu nghin cu l th v vitamin A: bo qun vitamin A trong 5 tun l
420C s cho mt lng vitamin ny b phn hu tng ng nh khi bo
qun trong hai nm nhit phng. Kt qu nghin cu gi mt cch
nh gi n nh. E.R. Garrett (1955) gi y l phng php th nghim
cp tc. Cng trong thi gian ny ngi ta tin hnh mt php th n
nh ca thuc (gang - testing): Mt nm ngi ta ly mu ngu nhin cc
nh thuc mt ch phm no , tin hnh nh lng hot cht, nh gi s
suy gim ca hm lng. Trn c s kt qu nghin cu, cc nh bo ch M
khuyn co i vi cc ch phm thuc cn:
171
Dng bo ch ca thuc,
Nh vy n nh ca thuc ph thuc ch yu vo c im ca
nguyn liu, qui trnh sn xut v iu kin mi trng. Vic a vo cng
thc bo ch cc cht lm tng n nh ca thuc ch c chp nhn trong
trng hp hu hn v phi c chng minh v mt khoa hc v thc t.
6.2.2. Mt s thut ng lin quan.
Cc ti liu hng dn nghin cu n nh ca thuc cp n nhiu
khi nim v thut ng. y s tm tt mt s thut ng chnh:
Cc php th n nh (stability testings)
l tp hp cc php th c thit k nhm thu c nhng thng tin
v n nh ca ch phm. Trn c s nh ra tui th, hn dng iu
kin ng gi v bo qun xc nh.
Php th n nh di hn (long term (real time) stability testing)
l nhng nghin cu thc nghim nh gi s thay i cc tnh cht
ha l, sinh hc, sinh dc hc .... ca mt ch phm thuc trong qu trnh bo
qun iu kin xc nh. Trn c s kt qu nghin cu ny xc nh hn
dng v khuyn co iu kin bo qun.
Php th n nh cp tc (accelerated stability testing)
y l nghin cu thc nghim c b tr lm tng tc phn hy
ha hc v thay i trng thi vt l ca thuc nh s thay i ca nhit ,
m, chiu sng,... Thng nghin cu c tin hnh nhit cao 35 400C, c khi ti 50 0C, m tng i 80 - 90% hoc hn.
L sn xut (batch N0)
l lng xc nh ca mt sn phm c sn xut theo mt qui trnh
v c coi l ng nht. Trong trng hp sn xut lin tc, l tng ng vi
mt phn nht nh ca qu trnh sn xut v c c trng bi tnh ng
nht ca sn phm.
Hn dng thuc (expiration date)
y l thi im ht hn x dng ca thuc, c ngha l sau thi im
ny ch phm thuc khng cn gi c cc tnh cht nh ng k trong
tiu chun cht lng. Hn dng c xc nh theo l sn xut.
Hn sn xut (manufacture date)
y l thi im kt thc qu trnh sn xut l thuc. Hn ny c ghi
thnh thng v nm cho tng l sn phm. C th tnh hn sn xut l thi
173
Giai on
p dng
TT
Mc tiu
Th nghim cp tc
Xc nh tui th v iu
kin bo qun
Th cp tc v di hn
Th di hn
Lp h s ng k
Thm nh n nh
lin quan n cng thc
v qui trnh sn xut
Th cp tc v di hn
174
Giai on lp h s ng k
Trn c s kt qu thc nghim cp tc v di hn, nh sn xut xc
nh tui th v hn dng ca thuc iu kin bo qun v bao gi thch
hp. Cc thng tin ny c ghi r trn nhn thuc v bao gi. Nh sn
xut trnh vi c quan qun l thuc ton b h s khc xin php lu hnh
thuc trn th trng.
Giai on thuc lu hnh trn th trng
Khi thuc lu hnh trn th trng, nh sn xut tip tc theo di
n nh khng nh tui th ca thuc trong iu kin bo qun xut.
bo m an ton v cht lng thuc lu hnh trn th trng, cc c
quan qun l cng theo di n nh ca thuc thng qua vic thanh tra,
kim tra qui trnh sn xut, ly mu thuc kim nghim.
i vi mt ch phm c cp php lu hnh, nu c s thay i v
cng thc, v qui trnh sn xut hoc qui cch ng gi,... nh sn xut phi
nghin cu b sung v n nh v bo co vi c quan qun l thuc.
6.2.4. Tiu chun nh gi n nh
C 5 tiu chun nh gi n nh ca thuc
n nh ha hc
Cc tnh cht ha hc (thnh phn nh tnh v nh lng) ca (cc)
hot cht c mt trong ch phm nm trong mt gii hn cho php theo tiu
chun cht lng.
n nh vt l
Cc c im vt l ca nguyn liu lm thuc nh: mu sc, trng thi
tinh th, tan, im chy.... khng thay i. Cc c im ca ch phm nh
mu sc, cng, r, ho tan dao ng trong khong gii hn cho php
ca tiu chun cht lng.
n nh vi sinh
v trng hoc gii hn nhim khun ca ch phm phi p ng yu
cu ca tiu chun. Nu ch phm c cha cht khng khun th hm lng
ca n khng vt qu gii hn cho php.
n nh iu tr
Tc dng iu tr ca ch phm khng thay i
n nh c tnh
c tnh ca ch phm khng c tng ln trong sut qu trnh bo
qun v lu hnh trn th trng.
175
Tr s o c ngoi tri
Tr s o c trong kho
Nhit (0C)
m (%)
Nhit (0C)
m (%)
10,9
75
18,7
45
17,0
70
21,1
52
24,4
39
26,0
54
26,5
77
28,4
70
iu kin bo qun
Nhit
NT
m **
m bar *
Nhit
m **
20,0
20,0
42
9,9
21
45
21,5
22,0
52
13,5
25
60
26,4
27,9
35
11,9
30
35
26,7
27,4
76
26,6
30
70
176
Nhiu yu t tc ng ng thi,
Ch phm tn ti nhiu dng khc nhau: dung dch nc, dung dch
du, nh dch, hn dch, bt rn,....
S c mt ca cht xc tc,
Cho mt phn ng:
A+B
(6.1)
v=
dC
= k( m + n ) [ A]m [ B]n
dt
(6.2)
dt
(6.3)
C = C0 k 0 t
(6.4)
hoc C0 (1 a) = k0 ta
(6.5)
C0
2k 0
(6.6)
dC
dt
= k 1C
( 6 .7 )
ln
C
= k 1t
C 0
( 6 .8 )
ln a = k 1 t a
( 6 .9 )
(6.10)
kt0,9 = -0,105
(6.11)
Th nguyn ca hng s k l t -1
178
B
k1
(6.12)
k2
d[ A ]
= k 1[ A ]
dt
( 6 . 13 )
d[ B ]
= k 2 [ B ] + k 1[ A ]
dt
( 6 .14 )
d[C ]
= k 2 [B ]
dt
( 6 . 15 )
(6.16)
(6.17)
k2
k1
[ C ] = A 0 1
exp( k 1 t ) +
exp( k 2 t )
k 2 k1
k 2 k1
( 6 . 18 )
(6.19)
Cefotaxim A (% mol)
B (% mol)
C (hiu s)
0,00
100
0,25
82
10
0,50
70
15
15
0,75
55
18
27
1,00
43
19
38
1,50
28
18
54
2,00
20
15
65
3,00
10
10
80
4,00
90
k1
(6.20)
k2
(6.21)
(6.22)
(6.23)
Lacton
k2
Cefotaxim
k3
sn phm cui
k1
180
sn phm cui
(6. 24)
Tc phn ng
v = d[A]/ dt = k2[A][B]
(6. 25)
(6. 26)
( a b ) k 2 t = ln
(a b)k 2 t = ln
b(a x )
a (b x )
( 6 .27 )
b
ax
+ ln
bx
a
v = k2[A][B] = k2[B]
(6. 28)
(6.29)
181
(6.30)
(6.31)
(6. 32)
y ln A = C1
6.4. Xc nh n nh ca thuc
Khi nh gi n nh cn xem xt n cc yu t:
6.4.1. Ly mu
Vi dc cht tng i n nh, cn ly hai mu hai l sn xut
khc nhau. Nu dc cht km bn hoc c t ti liu cng b, cn ly ba
mu ba l khc nhau. Cc mu ny cn ly i din cho qui trnh sn xut.
i vi nghin cu n nh mt cch lin tc qui m cng nghip
vic ly mu cc l c tin hnh theo chng trnh nh trc, v d:
182
Nhit (0C)
m (%)
II
40 2
75 5
IV
40 2
75 5
183
Hm lng
Thi gian
350C
450C
Thi gian
550C
(thng)
(thng)
0
105
105
105
103
102
102
102
100
99
101
97
97
99
94
94
91
Gii:
V th lnCt t(thng). Tnh hi qui bc nht, ta c hng s tc :
- 350C:
- 450C:
- 550C:
Nhit (0 C)
ln k
35
0,0062
5,083
3,246
45
0,0123
4,398
3,145
55
0,0280
3,576
3,049
184
185
Yu cu ca th nghim.
S liu hin c chng minh rng nhng cng thc tng t c thi hn
bo qun 24 thng hoc hn.
Bo qun di 180C (t ).
186
Tn dc cht
TT
Tn dc cht
50
Hexyl resorcinol
Acid ascorbic
51
Hydralazin, HCl
Acid undecylenic
52
Aminophyllin
53
Hyoscyamin sulfat
Amitryptilin, HCl
54
Lidocain, HCl
Amoni clorid
55
Melarsoprol
Amphotericin
56
Metrifonat
57
Naloxon, HCl
Ampicillin trihydrat
58
10
Antimon tartrat
59
Natri lactat
11
Bc nitrat
60
Natri nitrit
12
Bacitracin
61
Natri p. aminosalicylat
13
62
Natri stibogluconat
14
Benzathin benzylpenicillin
63
Neomycin sulfat
15
64
Nystatin
16
Bephenium hydronaphtoat
65
Orciprenalin sulfat
17
Calci gluconat
66
Oxytetracyclin, HCl
18
Calci p.aminosalicylat
67
Paromomycin sulfat
19
68
Penicillamin
20
Cephalexin
69
Pethidin, HCl
21
Cloral hydrat
70
22
71
23
72
Phentolamin mesilat
24
Clorpromazin, HCl
73
Phenyl butazon
25
Clortetracyclin, HCl
74
Pilocarpin, HCl
26
75
Pilocarpin nitrat
27
Codein phosphat
76
28
Colecalciferol
77
187
TT
Tn dc cht
TT
Tn dc cht
29
Dapson
78
Procain, HCl
30
79
Procarbazin, HCl
31
80
Promazin, HCl
32
Dietyl carbamazin
citrat)
81
Prometazin, HCl
33
Doxycylin hydrat
82
Pyridoxin , HCl
34
Ephedrin
83
Quinin, bisulfat
35
Ephedrin sulfat
84
Quinin, 2HCl
36
Epinephrin
85
Retinol (vitamin A)
37
86
Salbutamol sulfat
38
Ergocalciferol
87
39
Ergometrin hydrogenotartrat
88
40
Ergotamin maleat
89
41
Ergotamin tartrat
90
Tetracain , HCl
42
Etho suximid
91
Tetracyclin, HCl
43
92
Thiamin, HCl
44
Fe(II) sulffat
93
Thiamin mononitrat
45
Fluphenazin decanoat
94
46
Fluphenazin, HCl
95
47
96
Tolbutamid
48
Gentamycin sulfat
97
49
Guanethidin sulfat
(dihydrogeno
cu hi t lng gi
6.1. Ti sao cn t vn nh gi n nh ca thuc.
6.2. Gii thch cc yu t tc ng n n nh ca thuc.
6.3. Phn tch php th n nh di hn v n nh cp tc. Ti sao
cn tin hnh c hai php th ?
6.4. Nhng ch phm no khng thch hp vi php th cp tc? Ti sao?
6.5. Ti sao phi phn thnh 4 vng kh hu khi nghin cu tui th ca
thuc. Theo anh (ch) vng kh hu no nh hng nhiu nht n n
nh ca thuc?
6.6. Gii thch vai tr ca ng ha hc trong nh gi tui th ca thuc.
6.7. Trnh by nguyn tc s dng phng trnh Arrhenius tnh tui
th ca thuc trong phng php th cp tc.
6.8. Nhng phn ng no thng xy ra lm gim n nh ca thuc?
Cho v d.
6.9. Khi xc nh tui th ca thuc da vo s liu hm lng hot cht, cn
thm nh: ng, chnh xc v gii hn nh lng. Gii thch?
6.10. Gii thch l do ti sao khi xc nh tp cht lin quan, tp cht
phn hy cn kim tra tnh c hiu v nhy ca phng php
thc nghim?
6.11. nh gi vai tr ca cc k thut phn tch trong nghin cu n
nh ca thuc.
6.12. Ngi ta nh gi n nh ca mt ch phm vin nn hm
lng 200 mg bng phng php lo ha cp tc trong thi hn 6
thng hai nhit 400C v 600C. S liu thc nghim c trnh
by bng sau:
189
Hm lng (%)
400C
600C
102,1
102,1
101,5
101,6
101,1
101,1
100,6
99,5
100,1
98,9
99,5
98,3
98,8
97,6
190