10.10. nh lng vitamin A

 

Hot lc ca vitamin A c tnh theo n v quc t (k hiu IU). 1 IU vitamin A tng ng vi 0,300 mg retinol; 0,344 mg retinyl acetat; 0,359 mg retinyl propionat hoc 0,550 mg retinyl palmitat.

Vitamin A rt d b oxy ho nn iu kin tin hnh chung l phi nhanh chng, trnh nh sng v s dng km theo cht chng oxy ho.

Ty theo c im ca vitamin A trong cc thuc v nguyn liu lm thuc m p dng 1 trong 5 phng php di y nh lng.

Phng php 1

(Phng php o quang trc tip):

C th p dng i vi cc nguyn liu vitamin A ester tng hp (retinyl acetat, retinyl propionat hoc retinyl palmitat ) v cc du hoc nang mm cha vitamin A ester vi hm lng ln (5000 IU/g).

Cn chnh xc khong t 25 mg n 100 mg ch phm v em ho tan trong 5 ml pentan (TT) ri pha long trong isopropanol (TT1) c dung dch cha chnh xc khong 10 n 15 IU vitamin A trong 1 ml. Xc nh bc sng c hp th cc i v hp th ca dung dch ti cc bc sng 300 nm, 326 nm, 350 nm v 370 nm trong cc o dy 1 cm, dng isopropanol (TT1) lm mu trng. Tnh t l hp th ti cc bc sng 300 nm, 350 nm v 370 nm so vi hp th ti bc sng 326 nm (Al/A326).

Nu cc i hp th nm trong di sng t 325 nm n 327 nm v t l Al/A326 khng ln hn cc gi tr ghi di y:

0,593 렠 l = 300 nm.

0,537 렠 l = 350 nm.

0,142 렠 l = 370 nm.

th tnh kt qu hm lng vitamin A trong mu th (IU/g) theo cng thc:

Trong :

A326 l hp th quang ti bc sng 326nm.

m l lng ch phm em th (g).

V l lng th tch dung dch thu c sau khi pha long em o (ml).

Nu cc i hp th nm ngoi di sng t 325 nm n 327 nm hoc c mt gi tr Al/A326 ln hn gi tr quy nh th kt qu khng c gi tr, tin hnh nh lng ch phm ny theo phng php 2 hoc 3 hoc 4.

Phng php 2

(Phng php o quang sau khi chit tch vitamin A):

C th p dng cho cc nguyn liu vitamin A c ngun gc t nhin v a s cc dng thuc cha vitamin A: Vin nang, vin nn, thuc bt, thuc m, du gan c...

Ly chnh xc 1 lng ch phm khng t hn 500 IU vitamin A v khng nhiu hn 1 gam cht bo cho vo bnh nt mi, thm 30 ml ethanol (TT), 3 ml dung dch kali hydroxyd bo ha (TT), vi vin mm si, un si 30 pht trn cch thu c lp ng sinh hn hi lu, di dng kh nitrogen khng c oxygen. Lm ngui nhanh ri dng 30 ml nc ct chuyn ht hn hp sang bnh gn, thm 4 gam natri sulfat (TT) nghin mn. Chit vitamin A vi 150 ml ether (TT) v lc 2 pht (nu to thnh nh tng th chit thm 3 ln na, mi ln vi 25 ml ether). Tp trung dch chit li ri ra dch chit 4 ln, mi ln 50 ml nc ct, ch lc rt nh nhng 2 ln u trnh to thnh nh tng. Lm bay hi dch chit ether trn cch thu di dng kh nitrogen khng c oxygen hoc ct quay chn khng nhit khng qu 30 0C n ht dung mi. Ho tan cn trong mt lng isopropanol (TT) va thu c dung dch cha 10 - 15 IU vitamin A trong 1 ml.

o hp th ca dung dch ny cc bc sng 300 nm, 310 nm, 325 nm, v 334 nm trong cc dy 1cm vi mu trng l isopropanol (TT), sau xc nh bc sng c hp th cc i.

Nu cc i hp th nm trong di sng t 323 nm n 327 nm v t l A300/A325 0,73 th kt qu c tnh nh sau:

Tnh hp th hiu chnh A325(K):

A325(K) = 6,815A325 - 2,555A310 - 4,260A334

NuA325(K)/A325 0,970 th tnh kt qu hm lng vitamin A trong mu th (IU/g) theo cng thc sau:

(IU/g)

Trong :

A325 l hp th quang ti bc sng 325nm.

m l lng ch phm em th (g).

V l lng th tch dung dch thu c sau khi ho tan em o (ml).

Nu A325(K)/A325 < 0,970 th kt qu vn tnh theo cng thc trn nhng thay A325 bng A325(K).

Nu cc i hp th nm ngoi khong 323 - 327 nm

hoc t l A300/A325 > 0,73 th phi tin hnh theo phng php 5.

Ch :

Trong cch tin hnh phng php 2 nu ch phm dng vin nang, vin nn hoc cht rn khc nhau sau khi b v, nghin thnh bt m vn khng x phng ho c (do dng vi nang) th phi x l trc bng cch un nng vi 10 ml nc ct trn cch thy 5 pht, dng a thy tinh nghin nh cc cht rn cn li v gi nng 5 pht na.

Khi tin hnh x phng ha, nu khng c kh nitrogen c th thay th bng cch cho cht chng oxy ha vo bnh phn ng nh 3 ml dung dch hydroquinon 1% trong ethanol, hoc 30 mg BHT (butyl hydroxytoluen).

chit vitamin A c th thay ether bng ether du ha hoc n- hexan.

Phng php 3

(Phng php sc k lng trc tip)

C th p dng i vi cc nguyn liu vitamin A tng hp, cc thnh phm cha vitamin A tng hp m trong thnh phn ch cha vitamin A mt dng ester ng nht hoc dng retinol.

Tin hnh theo phng php sc k lng (Ph lc 5.3).

iu kin sc k:

Pha ng: Methanol - ethyl acetat - nc (90 : 7 : 3).

Dung dch th:

Ly chnh xc mt lng ch phm c cha khong 4000 IU vitamin A cho vo bnh nh mc dung tch 100 ml, thm ethanol (TT), lc k ri thm ethanol (TT) n nh mc, lc u, lc.

Dung dch chun:

Pha cht chun vitamin A (dng ging vi dung dch th: Retinol, Retinyl acetat, Retinyl palmitat ) trong ethanol (TT) thu c dung dch chun c nng vitamin A chnh xc khong 40 IU/ml.

Ct thp khng g (25 cm 4 mm) c nhi Octadecylsilyl Silica gel (5 mm hoc 10mm).

Detector quang ph t ngoi t bc sng 325 nm.

Tc dng: 1,5 - 2,0 ml/pht.

Th tch tim: 20 ml.

Cch tin hnh:

Kim tra kh nng thch hp ca h thng sc k: Tin hnh sc k vi dung dch chun, lch chun tng i ca din tch pic thu c trn sc k ca 6 ln tim lp li mu chun (RSD) khng c ln hn 2%.

Tin hnh sc k ln lt vi dung dch chun v dung dch th . Cch tnh kt qu:

Hm lng vitamin A trong mu th (IU/g) c tnh theo cng thc:

ST C 100

SC mT

Trong :

ST v SC l din tch ca pic vitamin A trn sc ca th v chun.

mT l lng cn mu th (g).

C l nng dung dch chun em o sc k (IU/ml).

Ch :

Trong vic chun b dung dch th, nu ch phm dng vin nang, vin nn hoc cht rn khc nhau, sau khi b v, nghin thnh bt, m vitamin A vn khng ho tan c trong ethanol (do dng vi nang) th phi x l trc bng cch un nng vi 10 ml nc ct trn cch thu 5 pht, dng a thu tinh nghin nh cc khi rn cn li v gi nng thm 5 pht na ri mi cho ethanol vo ho tan.

Nu mu th c nhiu t dc dng du th nn thm 2 ml ethyl acetat(TT) hoc 2 ml n-hexan (TT) v lc k trc khi cho ethanol vo ha tan.

Nu trn sc thu c ngoi pic ca dng vitamin A cn o cn c pic ca dng vitamin A khc th phi tin hnh theo phng php 2 hoc 5.

 

Phng php 4

(Phng php sc k lng sau khi thu phn)

C th p dng i vi nguyn liu vitamin A tng hp dng bt, dng du v dng nh tng.

Tin hnh theo phng php sc k lng (Ph lc5.3).

Pha ng: Hn hp Methanol - nc (95 : 5).

Dung dch th:

Dung dch th A:

i vi nguyn liu dng bt: Ly chnh xc mt lng ch phm c cha khong 120000 IU vitamin A cho vo bnh nh mc dung tch 50 ml. Thm khong 20 mg n 30 mg bromelains (TT), 20 mg n 30 mg BHT (TT), 2,0 ml nc v 0,15 ml isopropanol (TT). Lm nng trong cch thu 60 oC 65 oC trong thi gian 2 n 5 pht. Lm lnh ti 30 0C ri thm 20 ml dung dch tetrabutylamoni hydroxyd 0,1 M trong isopropanol. Lc siu m 5 pht. Thm isopropanol (TT) n vch, lc u (lc xoay trn, trnh to bt kh).

i vi nguyn liu dng du hoc nh tng: Ly chnh xc mt lng ch phm c cha khong 120000 IU vitamin A cho vo bnh nh mc dung tch 50 ml, ha tan ngay trong 5 ml pentan (TT). Thm khong 20 mg n 30 mg BHT (TT) v 20 ml dung dch tetrabutylamoni hydroxyd 0,1 M trong isopropanol. Lc siu m 5 pht. Thm isopropanol (TT) n vch, lc u (lc xoay trn, trnh to bt kh).

Dung dch th B:

Cho khong 20 mg n 30 mg BHT (TT) vo bnh nh mc dung tch 50 ml, thm 5 ml isopropanol (TT), lc cho tan, thm 5,0 ml dung dch th A, thm isopropanol (TT) ti vch, lc u, lc.

Dung dch chun:

Dung dch chun A:

Cn chnh xc mt lng cht chun Retinyl acetat tng ng vi 120000 IU vitamin A cho vo bnh nh mc dung tch 50 ml. Thm ngay 5 ml pentan (TT), thm khong 20 mg n 30 mg BHT (TT) v 20 ml dung dch tetrabutylamoni hydroxyd 0,1 M trong isopropanol. Lc siu m 5 pht. Thm isopropanol (TT) n vch, lc u (lc xoay trn, trnh to bt).

Dung dch chun B:

Cho khong 20 mg n 30 mg BHT (TT) vo bnh nh mc dung tch 50 ml, thm 5 ml isopropanol, lc cho tan, thm 5,0 ml dung dch chun A ri thm isopropanol (TT) n vch, lc u.

iu kin sc k:

Ct thp khng g (25 cm 4 mm) c nhi Octadecylsilyl Silica gel (5 mm hoc 10mm).

Detector quang ph t ngoi t bc sng 325 nm.

Tc dng: 1 ml/pht.

Th tch tim: 20 ml.

 

Cch tin hnh:

Ging nh phng php 3 vi dung dch chun B v dung dch th B.

Cch tnh kt qu:

Hm lng vitamin A trong mu th (IU/g) c tnh theo cng thc:

ST C mC

 


SC mT

Trong :

ST, SC l din tch pic ca vitamin A trn sc ca th v chun.

mC, mT l lng cn mu chun, th (g).

C l hm lng cht chun vitamin A (IU/g).

Kt qu c chp nhn nu:

lch chun tng i ca din tch pic thu c trn sc k ca 6 ln tim lp li mu chun (RSD) khng c ln hn 2%.

Trn sc ca dung dch chun ngoi pic ca Retinol khng c pic ca Retinyl acetat cha thy phn.

Phng php 5:

(Phng php sc k lng sau khi chit tch vitamin A)

C th p dng i vi cc ch phm phc tp v c cha vitamin A vi hm lng thp khng thc hin c 1 trong 4 phng php trn.

Tin hnh bng phng php sc k lng (Ph lc 5.3).

Pha ng: Hn hp Methanol - nc (97 : 3).

iu kin sc k:

Ct thp khng g (25 cm 4 mm) c nhi Octadecylsilyl Silica gel (5 mm hoc 10 mm).

Tc dng: 1 ml/pht.

Detector quang ph t ngoi t bc sng 325 nm.

Th tch tim: 10 ml.

Dung dch th:

Ly chnh xc mt lng ch phm c cha khong 2000 IU vitamin A em x phng ha, chit v bay hi dch chit n cn ging nh phng php 2. Ho tan cn thu c v chuyn hn hp sang bnh nh mc 50 ml vi isopropanol (TT).

Dung dch chun m:

Ho tan cht chun Retinyl acetat trong isopropanol (TT1) thu c dung dch c nng chnh xc khong 1000 IU vitamin A trong 1 ml. Xc nh nng chnh xc ca dung dch chun m bng cch pha long trong isopropanol (TT1) ti nng 10 - 15 IU trong 1 ml ri o hp th bc sng 326 nm trong cc o dy 1 cm, dng isopropanol (TT1) lm mu trng.

 

Tnh nng vitamin A trong dung dch chun m (IU/ml) theo cng thc:

A326 1900 V2

(IU/ml)

100 x V1

Trong :

A326 l hp th quang ca dung dch 326 nm.

V1 l th tch dung dch chun m em pha long (ml).

V2 l th tch dung dch long pha (ml).

Dung dch chun:

Ly chnh xc 2 ml dung dch chun m em x l hon ton nh mu th. Xc nh nng chnh xc ca dung dch chun bng cch pha long trong isopropanol (TT1) ti nng 10 - 15 IU trong 1 ml ri o hp th bc sng 325 nm trong cc o dy 1 cm, dng isopropanol (TT1) lm mu trng.

Tnh nng vitamin A trong dung dch chun (IU/ml) theo cng thc:

A325 1830 V4

(IU/ml)

100 V3

Trong :

A325 l hp th ca dung dch 325 nm.

V3 l th tch dung dch 3 em pha long (ml).

V4 l th tch dung dch long pha (ml).

Cch tin hnh :

Ging nh phng php 3.

Kt qu c chp nhn nu p ng 3 yu cu sau:

Pic Retinol thu c t dung dch th c thi gian lu ging vi pic thu c t dung dch chun.

Khi dng phng php thm chun vo dung dch th th kh nng tm li phi ln hn 95%.

Nng dung dch chun xc nh bng phng php o quang phi ln hn 95% so vi nng tnh t dung dch chun m.

Tnh kt qu:

 

Hm lng vitamin A trong mu th (IU/g) c tnh theo cng thc:

A1 C V

A2 m

Trong :

A1 l din tch pic ca retinol trn sc ca dung dch th.

A2 l din tch pic ca retinol trn sc ca dung dch chun.

C l nng dung dch chun gc pha (IU/ml).

m l lng ch phm em x l thnh dung dch th (g).

V l th tch dung dch chun gc em x l (ml).