N g h i e n CLFU - K y t h u g t
/ Xay dung quy trinh san xuat va tieu ciiuan hoa bo kit djnh luong glucose trong huyet thanh
bang phuong phap diing glucose oxidase
Dat vSn d§
Min d l sdn x u i t cdc bO kit thi> dung trong dmh lu-g-ng smh h6a (t cdc nude Id mOt v i n d l da dutyc g\i\ q u y i t khd Idu 6- cdc nud-e trdn t h i gK^i Hidn nay trong nu-b-e ta vi$c nghifin ci>u san x u i t cde bO kit lhi> dung trong xdt nghigm h6a smh a ede p h i n g xdt nghidm eho d i n nay hau nhu ehu'a duoe quan tdm n h i l u , h l u h i t cdc phdng x6t nghidm d i u s u dyng cdc bO kit thu nhdp tu" nud-c ngodi 0 6 Id mOt trong nhOng nguyen nhdn Idm tdng gid thdnh ede xdt nghi$m vd dong thb'i Idm tdng thdm gdnh ndng d i l u tn cho bdnh nhdn. Vide san x u i t trong nub-c nhung bO kit thu cho cdc xdt nghidm h6a smh vdl chat luong I n dmh, gid thdnh re Id m | t d l i hoi c l p thiet Chung t6i chon de tdi ndy nhdm muc dich nghidn cuu vd hud-ng d i n sdn x u i t bO kit thi> dmh luong glucose trong h u y i t thanh d l thay t h i cac san pham ngoai nhdp
D o i t u ' O ' n g v a p h u ' c n g p h a p n g h i e n CLPU
Doi tuccng nghien e u u
Bo kit dmh luong glucose trong h u y i t thanh Nguyen lieu vi hda chat
Glucose oxidase Type-S (Sigma,Anh), peroxidase (Merck, Due), glucose standard solution (My), Glu P-4050 glucose (I S E. Italy)
Trang thiet bi
May do quang pho UV-VIS UVD-3000, Labomed, My
- May do quang pho UV-VIS U2010, Hitachi, Nhat
- May smh hoa tu dong LCD-ECHO Phan mem chuyen dung Design Expert, FormRules, INForm,
Diro'ng T h j T r u e L y ' , Trkn T h a n h Nh«n* ^ ' Trir&ng dtfl hpc Y Dir^ Cm Tha "^
'Dqi hvc Y Dir^c TP Ho Chi Minh P h i r c n g phdp n g h i d n cu'u
XSy dimg cdng thiJiv vi quy trinh di4u ch$
b^ kit, tdi tfu hda c6ng thi>c
BO kit g i m hai toai t h u l c t h u R, & Rj, c6 dup'c tu- trOn thich hep cdc thdnh p h i n vdi n6ng d | thich hep g i m dung d|ch ddm phoshat phenol, glucose oxidase, peroxidase, 4- aminoantipyridin, bdo quan 2°C d i n 8°C.
Quy tnnh d|nh lup-ng glucose: 1 hodc 2ml trdn d i u theo ty Id 1.1 hai Io thu6c thi> R, & R,, cho t h u l c thu R, thdm lOpl huyet thanh. u b nhidt d | 20°C-25°C th&i gian thich h^p, do quang it b\i<K s6ng 505nm'*'- Dimg phin mim chuydn dyng t l i u u h6a e i n g thipc'''
Thim dfnh quy trinh dfnh lu%mg glucose trong huyet thanh
Tinh dde hidu. tinh t u y i n tinh, 30 Idp l?i. 3fi dimg
Thu' d^ dn dfnh b^ kit
ThCf dO 6n djnh theo thO-i gian. sdn xuit (quy m6 p h i n g thi nghidm) 3 16 bO kit thif (dd dirpc t l i uu hda vd t h i m dmh dat cdc tidu ehuin theo ydu e l u ) , O i l u kidn bao quan Iheo d6r nhidt flO 5°C± 3°C. dO I m t u o n g d i i 4 5 % ± 5% theo d O i m l i S , 6. 9, 12thdng'^'.
ThLK dO I n dmh theo p h u c n g phdp Ido h6a cap t i c d i l u kidn bao quan theo dfli" nhidt iSf.
25°C ± 5°C, dfl am t u o n g doi 85% ± 5% theo dOimoi 1,2, 3 , 4 , 5, 6 thdng'^'
Tidu c h u i n ddnh gid b6 kit. nong dfl glucose xdc dmh duoe a thi'i diem ban ddu so sdnh v&\
n i n g dfl sau nhUng khoang thfri gian bao quan, ydu c i u gid trj xdc djnh d u g c a 90% ning flfi th&i d i l m ban d I u - Theo doi hinh thirc cflrti quan thay doi theo thi'i gian so v6\ ih&\ dilm bat dau theo dOi dung dieh trong, khflng mdu. ,
TAP C H I D U O C
• Nghien ctpu - Ky thuat
So sinh bd kit nghien c(>u v&i bp kit i.S.E T i l n hdnh djnh lup-ng glucose trong huyit thanh tren bdnh nhdn tai b$nh vidn qudn 4 vl-i 2 bg kit vd dung so sdnh ddnh gid k i t qud.
K § t q u a
Xay dipng cdng t h i f c vd quy trinh d i l u c h l bo kit
Phan m i m Design Expirt thilt k l cho 16 cflng thirc b | kit theo mfl htnh thye nghidm Taguiehi OA, FormRules, phdn lich lidn quan nhdn qua, anh huong ciia ede thdnh p h i n trong eong thiPC bfl kit eho t h i y ede y i u t l nhu pH, nong dfl phenol, n i n g dfl glucose, n i n g dfl peroxidase efl hdn quan In^rc tiep INForm t l i uu hoa cflng thirc tu 16 cflng thuc ban d i u , xdc djnh duoc thdnh phin cflng thirc toi uu ciia bfl kit.
Cflng thu-c dd duoc i l l uu, bfl kit (thuoc thir R) la gflm 2 loai t h u l c Ihu' R, (ddm phosphai pH
= 8 va phenol 7,51 mmol/l difcfc p h l i hp'p d i n g the lich) vh R; (glucose oxidase 9000 U/l, peroxdase 576,653 U/l, 4-aminoantipyrin 0,4 mmo!/l duoc p h l i hap d i n g t h i tich) trfln VO'i ly le 1 1, dong ehai nhua (5ml/chai/bfl) va bao quan tii mdt {nhiet do tu' 2°- 8°C), trdnh anh sang- Quy trinh dmh luong glucose 1ml trfln d i u theo ty Id 1:1 hai Io thuoc thu' R, & R2, cho t h u l c thir R + lOpI h u y i t thanh, u a nhiet dfl 20''C-25°C trong 20 phut, do quang a buo'c song 505nm
Thdm dinh quy trinh dmh luong glucose trong huyet thanh vfl'i bfl kit nghien ciru
Quy trinh dat cdc yeu cdu tham dinh ve tinh dac hieu; khoang t u y i n tinh tir 40-400 mg/dl vfl'i phuong trinh h l i quy y = 0,0029x + 0,0502. R^ = 0.9956; do lap tai vfl'i RSD = 0,919%, dp dung VO'I ty le phyc hfli trung binh 100,11 %.
T h u do on dmh bo kit Thu'dg dn dmh theo th&i giar^^' Bang 2: K4t qui ndng 66 thay ddi theo theo thai gian (phuung phip dai han)
L6 001 Chu
ky Bat dau 3 thang 6 tliang 9 thang 11 thang
Nong 101,70 100,25 98,10 96.16 94.35
1.6 002 Chu
ky Bat tJau 3 thang 6 thang 9 thang 11 thang
Nong 100,23 99,3 97,75 96,5 95,78
1.6 Chu
ky Bat (Jau 3 thang 6 thang 9 thang 11 thang
003 Nong
do 99,8 98,75 97,55 96,15 95,49
V * i cdc phucng trinh flu'dyng thang h6i quy gi&a nfing do glucose vS thtS-i gian:
L« 001. y = -COOeex + 4, 6263, R2=0.9896 L6 002, y = -C,0042x + 4, 6083, R2=0.9941 L6 003, y = -0,0041 X * 4, 604, R2=0,9968 K 6 I quii t u i i thp bC kit: iO 001 (18 thing), 16 002 (2S thing). IS 003 (25 thSng),
Thu" do 6n (3inli theo phu'ong p h i p iSo h6a c^p l6c.
K^t q u i tu6i tho bO i<it theo pliu-ong p h i p g i i hOa cSp t i c 3 16 theo quy t i c Van't Hoff: i6 001 (18 thing). IS 002 (20 thing). 16 003 (20 thing).
So sinh b^ kit nghiin ciru v&i bd kit I.S.E Bing 3: KSt qui ndng dd glucose tirong img giira bd kit I.S.E vi bd kit theo ddi
Mau b?nh nhSn
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Glucose chuan 1 S E
Bo kit I.S.E [glucose] mg/dl
189,03 270,5 229,9 201,13 150,31 84,97 81,2 100,03 79,32 81,74 137,13 204,36 96,53 91,15 162,95
90.08 188,76 294,17 147,62 93,84 90,08 100.03
Bq kit theo ddi [glucose] mg/dl
185,53 263,51 199,52 193,06 152,46 98,14 86,04 103,52 86.85 86,58 134,45 201,67 94,38 106,48 162.68 89,54 183,38 288,79 178,01 97,07 93,57 104,33 Trie nghiem F so sanh 2 phuong sai vd trdc nghidm t tuong irng tung cdp (T-test- Paired two sample for means) so sanh 2 ket qua cfl duoc tuong irng khdc nhau khflng co y nghla thflng kd
K e t l u a n
Sau thfl'i gian tien hanh d l tai chung tflr Da xay dung cflng thirc vd quy trinh d i l u c h l bo kit dmh lupng glucose theo quy mfl phong thi nghiem voi d i l u kien bao quan bo kit a nhiet do
TAP CHI n [ r n r H n r - 0 2 / 2 0 1 2 f S O 430 NAM 52)
• Nghidn CCPU - Kg thugt
2°C-8°C, trdnh dnh sdng. hgn dCing 18 thdng, xdy dyng vd t h i m djnh quy trinh diing bO kit djnh luqfng glucose trong h u y i t thanh, Dd xdc djnh bfl kit nghidn cii'u c6 gid trj djnh lup-ng glucose trong h u y i t thanh t r l n bdnh nhdn khflng khdc nhau c6 y nghTa t h i n g kd so vO'i bO kit I S E, bfl kil nghiin ciru cO t h i duo'c dCing dinh tu'iyng glucose trong h u y i t thanh b$nh nhdn thay cho bO kit ngoai nhdp
Summary
A kit for serum glucose determination was formulated and the manufacture process was computenzingly optimized, thed in pilot scale, and the kit was investigated in view of stability (expiry date), storage conditions and its creditability by long-term and accelerated tests in comparison with the available Imported kits.
For the proposed kit with reagent R, (buffer solution of phosphate, solution of phenol) and reagent R2 (solution of glucose oxidase.
peroxidase, 4-aminoantipyrin) stored at 2-^C and protected from light, the expiry time v/as 1$
months, but less than 30 days In mixture with R^
and R2.
Key words
Preparation, standardization, long- tenn study, accelerated study.
Tdi li$u tham khdo
1. Odng Vdn Gidp, Thilt k l vd l i t iru h6a cdng IhiKC vd quy trinh, Nhi xuit bin Y hoc, Tp H i CW Minh, (2002), tr.1-97.
2. Odng van H6a, VO Th| Bgch Hud. Cdc phirong phdp tinh tuA. tho thulc. 0^/ hqc Y Difi?c Tp. Hd Cftf fi/linh (200B).
3. Asean, Guideline On Stability Study Of Dnjg Product. (2005) update revision 22.
4 V G.Yuen. J.H mcNeil. Compansion of ttw glucose oxidase method for glucose determination by manual assay and automated analyzer. Joum^ <^
Pharmacological and ToxKOlogical methodes, (2000) vol 44, issue 3, pp. 11-12, 543-546.
Nghien CLFU djnh lu'O'ng... (ndp theo trang si)
cdch ehilt vfli nufl'c bi due do l l n tgp hodc ede htyp chit phenol khflng lan can tri- Idm tdng dfl h i p thu, d i n den tdng ning d l , cfln khi chilt vfl'i dung djch natn cartmnat 20% thi trong s u i t Mdt khde, khi IQC dich chill nufl'c dd phan irng vfl'i thulc tfiu Folin- Ciocalteu thay eo tua mdu xanh duong trdn gily Ipc chirng to cdc hop chit phenol dd bj gii> lai mOt phan ldm cho ty le phyc hoi t h i p (78,5%)
Kdt luan
Phuong phdp quang p h i UV-Vis d l nghi d l dmh luong hop chat phenol todn p h i n trong nhua mu u du pham (C inophyllum L ) cfl dfl dimg 99.3%
va do lap lai la 2,14% dat tidu chuin c h i p nhdn cua AOAC v l dfl dimg (95,0-105%) vd dfl Idp lai (<
3,7%) theo nflng dfl chit can dmh luong Phuong phap sir dung dung dich natn carbonat 20% de chilt va hdm lugng thuoc thir Folin-Csocalteu Id 20% Day Id mot phuang phdp eO d l nhay eao trong dmh luang polyphenol cung nhu protein '^' vd CO the dp dung de danh gid luong hgp chat phenol toan phan irong cac mau nhua du p h i m khde nhau thu duoc trong qua trinh tmh e h l d i u Mu u nhdm khai thac, tan dung
S u m m a r y
An UV-VIS spectrophotometric method for determination of the total phenolic compounds in
resinous residues of poon-oit (Oleum Calophyllae inophyllae - Clusiaceae) obtained m the oil refinement was developed The proposed method was venfied proving to meet AOAC requirements with the accuracy of 99 3% 95- 105%; and precision of 2.14% (<3.7%).
TaJ li^u tham khao 1 Nguyin Khic Quynh Ci>. Trln Thanh Ttigo. VB Thi Bach Hud, Phdn Idp vd xdc djnh d u tnJc ciia calophyllolid ti> hat cdy mu u (Calophyllum inophyllum LJ mpc tet Vi§t Nam, Tap chi Duvc hqc (2002), s6 317/9/02, tr 16-18
2 Celestino Santos-Buelga. Gary Williamson, Methods m Polyphenol Analysis. The Royal Society d Chemistty. UK,(2003) pp 258-260.
3- Arlington, AOAC Peer-Venfied Methods Program, ti^anual on policies andpiocedures. Va., USA (1998).
4, Ikawa M, Schaper TD, Dollard CA, SasnerJJ, Utilization of Folin-Ciocalteu phenol reagent for the detection of certain nitrogen compounds. J. AgiK.
Food C/iem.,(2003) 51 (7), pp 1811-1815 5, Intemationai Standani ISO 14502-1 . Part 1:
Content of total polyphenols in tea-Colorimettic method using Folin-Ciocalteu reagent, ISO, Switzerland, (2005) pp. 1-6.
6- Ralph Nicholson, Wilfred Vermerns, Phenolic Compound Biochemistry, Spnnger. USA, (2008), pp.
152-154,