Nghien C I J U - Ky thuat Ket luin
Ham logng ITZ trong m l u pellet vien nang nghidn c d u doge xdc ^ n h b i n g 2 phoang phdp vi sinh va HPLC la khdc nhau khong co y nghTa t h i n g kd. Phoang phdp vl sinh tuy cd d6 ehinh xac thap han so vdi HPLC nhong v i n ddm bao dp dung cda phep djnh logng vdi chi phf t h l p han. Bong thdi, phoang phap vi sinh c6 the cho tjilt hidn trgng hoat lye cua ITZ t r a i g vien nang. Chfnh vi vdy, nen can nhac dng dyng phoang phdp nay trong viee xac djnh ham logng ITZ trong c h i p h i m .
Summaiy
A bioassay for determination of itraconazole in capsules was developed using a specific agar dikjsion tedinique with the strain of Candida albicans ATCC 10231 as test organism. The bioassay was estimated in comparison with the prewously reported HPLC method. Though, higher was tiie precision of the HPLC method, both two methods were comparable in accuracy. Besides, the bioassay enabled evaluation of the potency of itraconazole undemonstrable by HPLC.
Keywords: Determine, itraconazole, HPLC and microbiological assays.
Tai ll#u tham khao 1. Alsarra I. A et al. (2010), "Comparative study of itraconazole-cydodexin inclusion complex and its commercial product". Arch. Pharm. Res.. 33,7, pp.
1009-1017.
2. Dux?c diin Vi$t Nam IV. Phy luc 13, muc 139:
"Xdc anh hogt lye thulc khdng sinh bdng phoong phdp tiid vi sinh vdf, tr. PL-271 vd myc 13.10: "Phdn tich thong kd cac k i t qud thd nghiem sinh hpc", tr.
PL-283.
3. European pharmacopoeia (2005^; Fifth edition, Vol.2, pp. 1852-1853.
4. Hewitt W. (2005), MicmbiokigKal assay for pharmaceutksl analysis. Interpharm / ORG, Boca Raton-London-New York-Washington, D. C. p.97-115..
5. Lima P. M. A. et al. (2009), "Determination of ketoconazole in capsules by high-perfoonance liquid chromatography and mio-obiological assay", J. AOAC (0^,92,4, pp. 1076-1081.
6. Queiroz K. M. et al. (2009), "Comparison of microbiological assay and HPLC-UV for detemiination of fluconazole in capsules", Brazil. J. Pharm. Sci., 45, 4, pp. 693-700.
7. The United States Phanrmxpoem 35. "Aialytical data-interpretation and treatment", pp. 390 - 397.
Nghien cihi dinh lirvng andrographolid trong la cay xuyen tam lien
{Andrographis paniculata (Burm.f.) Nees) bSng phirong phap HPLC
Dat van de
Xuy§n tam lien {Andrographis paniculata (Burm.f,) Nees) Id du'CTc lieu da dug'c s u dyng tCi- lau ix nhieu n u a c l<hdc nhiau tr§n the giai n h u : Trung Quoc, An Dg. Thai Lan, Malaysia.
Viet Nam... de dCing cho mgt s6 t r u a n g hgp n h u : vi§m duo-ng ho hap. vi6m du'd'ng ti&u h6a.
42
'Hoang Vi^t Dung, ^Tru-ong Diic M^nh ' D S Van Binh, 'Ngnyin Thi H6ng Van 'HQC vien Quan y, ^Cuc qudn ly Dupc -BgYte
'Kho 708, Cifc Qudn Y
viem da. cam a i m . s6t, nhi^t miSng... Hien nay, da CO m0t so nghiSn c i i u t i l n hSnh c h i i t xu5t th^nh phlln ho?t chSt ti> xuySn tSm liSn ISm nguyen li^u bao c h i c i c d?ng thuoc nhu' vi6n n^n, vien nang..., t^io thu?ln Ipi cho qu4 trinh si>dgng'^'. Tuy nhlSn, song song v a i vi#c oSi t i l n d?ng bdo che. d n phai I d i m soat d u a c c h i t
T^M" CHi DircJC HQC - 04/2013 (S6 444NAM 53)
• Nghien ci^u - Ky thuat
logng cua d o g e lidu n h i m dam bao chat logng cung n h o higu qud dieu trj eua cdc e h l p h i m . Do vgy, chung tdi tien hdnh nghien c d u xdy dyng phoong phdp djnh l o g n g thdnh phan hogt chat andrographolid trong la edy xuydn tdm lidn n h i m giai q u y l t v i n de dgt ra.
N g u y e n l i d u , t h i e t b i v a p h v a n g p h a p n g h i e n cu>u
Nguyen lieu: Id edy xuydn tdm lien doge thu mua d khu v y c thanh pho Ha Ddng vdo thdng 5/2012.
Hoa chat Andrographolid chuan (Sigma - Aldrieh); Methanol (MeOH), Acetonitril (ACN) (HPLC - Merck).
Thiet bj, dyng cy: hd thong HPLC Waters 2695D; detector 2487 va PDA; cdt RP-C18 phenomenex Genimi 5 pm, 110A", 250x4,6 mm;
mdy lac sieu dm Soniciean 500UT; cdn phdn tfch Metller (AE240 - 0 d c ) dg ehinh xdc 0,1 mg.
Phwong phap nghien cwu Dieu kidn sic k^ '^'^'^^
+ Hd t h i n g HPLC (nho trdn).
+ Detector UV: 230 nm.
+ Too dg ddng: 1 ml/phut.
+ The tieh tiem: 20 pl/phut.
+ Nhidt dg phong thf nghidm.
+ Pha dgng: N o d e - MeOH - A C N ( 45/50/5).
Dung djch tiim sic ky
+ Dung djeh t h d : edn chinh xde khodng 1,0 g Id doge Hdu, ehilt Soxhiet v d i khodng 80 ml MeOH tnang 5 g i d . Djeh ehiet Soxhiet durgic chuyen vdo binh (^nh mde 100 ml, thdm MeOH vda du tdi vaeh. HQt chfnh xdc 1 ml dung djeh tren, cho qua cOt c h i l t pha r i n RP-C18, n>a gidi vdi khoang 15 ml MeOH 75%, djch rda gidi doge chuyin vdo binh (^nh m d e 20 ml, them MeOH 75% v d a du t d i vgch. Loc dung djeh tren qua mdng 0,45 pm.
+ Dung djeh c h u i n : can chfnh xde khoang 50 mg andrographolid ehuan, cho vdo binh djnh mde 50 ml, thdm khodng 40 ml MeOH vao binh, lie sieu dm 15 phut, sau do them MeOH v d a dO tdi vgch. Hut chinh xdc 1 ml dung djch tren eho vdo binh djnh m d c 50 ml, thdm MeOH 75% v d a dd tdi vgch. Lge qua mdng lge 0,45 pm.
K e t q u a v d b d n l u a n T i n h t i p v n g t h i c h h $ t h i n g
De khao sdt tinh thieh hgp cua hd t h i n g , chung tdi tien hdnh tidm lap Igi 6 l l n m | t dung djch chuan vdo hd t h i n g sac k^ v d i d i l u kidn dd ndu tren. K i t qud thu doge nho sau:
Bang 1 : Kit qui khio sit tinh thich hgrp cua hg thing sic k^f
Ndi dung Dien tic^ pfc (pV*s) Thdi gian lu'u (phut) He so bit d i i So dTa \'if thuydt
Kef qua S = 861773; RSD = 0.34%
TR = 6 , 7 9 ; RSD = 0,32%
1,27 3200
Hinh 1 : Sic k^ dd miu tring
Hinh 2 : Sdc ky do miu chuin K i t qua cDa bdng 1 cho thay hg thong sac ky t o a n g thfch v d i mdu phan tfch (RSD cua didn tfch pfc v d thdi gian lou deu <0,5%), tgi thdi diem xuat hign pic trdn sde ky do cCia m l u ehuan khdng xuat hidn pfc tren sde ky d l cua mau trdng.
T i n h dgc h i | u
S d dung detector PDA d l ddnh gid tfnh ddc hidu cua phoo'ng phdp: tgi thdi gian lou eua pie tren sde ky do m l u c h u i n va mdu t h d , xdc dinh pho UV-VIS. Ket qud cho t h i y pho tai ede d i l m tren hai pfc Id g i l n g nhau v l hinh dgng v d s6 dinh hap thy eye dgi chdng t6 phoang phdp ed tfnh dgc hieu eao.
•g^CHt DV(?C HQC - 04/2013 (SO 444 N A M S3)
Nghien CLFU - Ky thuat
Khoang tuyen tinh
De khao sdt khoang tuyln tfnh cua phoang phap, chung toi tiln hdnh pha mgt day dung djch chuan c6 ning do: 4,96; 9,92; 19,84; 29,76;
39,68 pg/ml vd xdc djnh dien tfch pie hogt ehat.
Kdt qud doge trinh bay d bdng sau:
Bang 2: St/" phy thudc cOa diin tich pic vao
nong dd andrographolidN6ng do (tig/ml) 4.96 9.92 19.84 29,76 39,68
Di^n tich (liV*s) 215841 431686 861910 1265625 1726943
Kit qua bang trdn eho thay trong khoang nong dg khao sat, dien tfch pie va nong dd andrographolid eo sy toang quan tuyen tinh chat ehe theo phoang trinh y = 43198x + 50926 vdi R^ = 0,9996.
IJOOOOO
200000
' "'ft'"o'i4fi''^x*
/^
J ^
y ^
j / ^
^ /
^
10 ao 30 «o so
Hinh 3: D6 thi the hiin twrmg quan tuyin tinh
giCra ndng dd androgmpholid vi dien tid\ picD$ Idp lai
D | igp Igi eCia phoang phdp doge ddnh gid dya tren do lap lai cua 6 thi nghiem ridng bidt doge tiln hdnh vdi 6 lln can mau doge ligu khdc nhau vd xd ly theo qui trinh da khao sat.
Kit qud doge thi hign d bdng 3:
Bang 3: Kit qui khao sit dd lap lai __ Lwqmg can „ . , ,.,, Thong ki
TT ^ " Ham Iwgng (%)HLtt>= 3,86%
RSD= 3,80%
1 2 3 4 5
1,0053 1.0016 1.0038 1,0010 1,0034
4,06 3,89 3,75 3,97 3,81
Kit qud cua bang 3 cho thiy phoang phap djnh logng co dp Idp Igi khd cao vdi RSD = 3,80%.
Do dung
Xac djnh dd dOng cua phoang phdp bang phoang phdp them chuan. Xdc djnh gid trj phdn tram tim tai.
- Dung djeh chuin goc: pha dung djch ehuan cd nong do chfnh xac khoang 1000 pg/ml.
- Dung djch A: hiit chfnh xdc 4pil dung djch ehuan glc vao binh djnh mdc 10 ml, thdm MeOH 75% vda du din vgch doge dung djch khodng 400 pg/ml.
- Dung djch thd gde: djch chiet doge lieu bang Soxhiet doge cho vdo binh djnh mdc 100ml, thdm MeOH vda du.
- Dung djeh B: hOt ehinh xdc 5 ml dung djeh thd goc eho vdo binh i^nh mdc 10ml, them dung djeh A vda dO din vgch.
- Dung djeh C: hOt chinh xae 1 ml dung djch B cho qua egt chilt pha ran, rda giai bang MeOH 75% va doge ggp vdo binh djnh mdc 20 ml, them MeOH 75% vda du den vgch. Lge qua mdng 0,45 pm.
Tiln hdnh phan tfch HPLC vdi dilu kien da ndu tren, lap Igi 5 lan thyc nghidm vdi 5 dung djch thd khac nhau. Kit qud doge trinh bay d bang sau:
Bang 4: Kit qui khao sat do dung
TT1 2 3 4 5
Luong them vio (mg) 1,984 1,984 1,984 1,984 1,984 Th6ng ke
Lugmg tim l?i (mg) 1,911 1,899 1,952 2,016 1.941
% tim lai
96,31 95.72 98,37 101,59
97.83
% tim Igi ,b = 97,96 RSD = 2,34 %
Ket qua bang 4 cho thiy phuxyng phap djnh luvng cd dg diing cao vai % tim lai ti> 95,72 - 101,59%, RSD = 2,34%.
Gils'! h^n phat hi^n va gii'i h^n djnh luvng dirdi
Da khio sat vk xic dinh du'g'c:
+ Ndng dg mlu thCf khodng 0,4 jig/mi id gi*l han phat hign, tgi ndng dg ndy d4p ij-ng pic mlu Ihu' c6 di§n tich gip han 3 lln diSn Hch dip ung cua mlu trIng.
T ^ CHi Dtrgrc H Q C - 04/2013 (S6 444 N A M 53)
0 Nghien CLFU - Ky thuat
+ Nong dO m l u t h d khoang 1,2 pg/ml Id gidi hgn djnh l o g n g d o d i , tgi nong dg nay dap dng pic mau t h d co dign tfch g i p han 10 l l n dign tich ddp dng cua m l u trang.
K e t l u a n
Da xdy d y n g 6\j(yc p h o a n g phap ffinh logng andrographolid trong Id cdy xuydn tdm lidn bang phoang phdp HPLC vdi dieu kign sdc ky Id: cot Phenomenex Genimi RP-C18; pha dpng node - MeOH - ACN (45/50/5); detector UV 230nm.
Phoang phap djnh l o g n g ndy ed s y t o a n g quan t u y l n tfnh chat ehe g i d a dign tieh pie va nong d | andrographolid trong khoang n i n g do khao sdt t d 5,0 - 40,0 pg/ml v d i R^ = 0,9996, dg Igp igi (RSD = 3,80%) va dg dung (% tim Igi t d 95,72 - 101,59%, RSD = 2,34%) cao. Thdi gian phdn tich mgt m i u sac ky toang doi ngan (khoang 6,8 phCit).
S u m m a r y
Andrograf^olide in the leaves of Andrographis paniculata (Burm.f.) Nees. was quantitatively detennined by HPLC: Columm: Phenomenex Genimi (RP-C18; 5pm; 100A°; 250x4.6mm);
Mobile phase: Water-Methanol-Acetonitril (45:50:5.0 v/v); Detector: UV 230nm. The method was validated showing good linearity (between
corresponding peak areas and andrographdide concentrations) (1^=0,9996), high repeatibSity (RSD = 3.80%), high accuracy (95.7 - 101.6%;
RSD = 2.34%) and also, time-saving - the time for analyzing a sample was ca. 6.8 minutes.
Key words: Andrographolide, Andrographis paniculata, method of quantification.
Tai lieu tham khao
1. Hoang Wet Dung, Nguyin Minh Chinh (2010)."Nghidn ci>u djnh logng andrographolid trong nguydn lieu chiet xudt doge td la cdy xuyen tam lidn (Andrographis paniculata (Bunn.f.) Nees) bing phoang phdp HPLC, T^p chi Y-Dugc hgc Quin si/, tip 35. so 02/2010.
2. Trln Thj An Tu-ong (1994), "Nghidn cdu dgng bdo c h i thfch hgp tu la cay xuyen tdm lidn", Lugn dn PTS, Hd NOi.
3. Nanthanit pholphana, Nucharnart Rangkadilok et al (2004), "Determination and variation of three active Diterpenoids in Andrographis paniculata (Burm.f.) Nees, Phytochemical Analysis". 15, 365- 371.
4 Qizhen Du, Ceroid Jerz et al (2003), "Separation of Andrographolide and Neoandrographolide from the leaves of Andrographis paniculata using high-speed counter-cun-ent chromatography, J. of Chromatography A , 964. 147-151.
Nghien cihi tac dung khang khuan in vitro va in vivo cua BKOl chi§t tu bo k§t
(Gleditsia australis Hemsl.)
Vu Manh Hung", Phan Van Ki?In^ Khdng Thi Hoa'
^Hoc vi?n Qudn Y 'Vien KH&CN Vi& Nam 'KhoaDu^c, Tmong Bai hoc Y Thdi Binh
Dat van de
Vigc lam dung khSng sinh trong c6ng dong.
si> dgng chua hgp ly trong ngSnh y te cOng vai vi0c k i l m so6t n h i l m khuan chua t i t trong b^nh vi§n d§ ldm cho d l khang khdng sinh cOa vi
k h u i n dang gia tang den mCpc bao dgng b nud'c ta. Co nhieu khang sinh the he III, IV de dieu tr]
nhiem khuan nang do cSc vl khuIn g9y bgnh nguy h l l m cung da x u i t hign vi khuan de khdng.
Vigc si> dgng qua mu'C khang sinh trong chdn