Ba d^sig ssmk kgc vd cdc chai ca fiasc tmh stri'" Age
NGHliN ClJU ©jNH UfOHQ HgP CHAT SARCOPHINE TCT CAO CHi^T LOAI SAN H 6 MiM SARCOPHYTON EHRENBERGI B A N O
PHirONG PHAP HPLC-DAD
Vu Anh Td', Chdu Ng^c Bifp, Ninh Thj Ng^c', Cao Ng^c Anh^, Dodn Cao Son', Nguyin Vfin Thanh'*, Nguyin Xudo Cuong', Nguyen Hodi Nam', Chau VSn Minh'
' Vign Hoa sinh biin (IMBC). Vign Hdn lam Khoa hpc va Cong ngM Vi$t Nam (VAST)
^Vign Kiim nghigm thuoc Trung uang Abstract
High perforaiance liquid chromatography '.vith diode array defector (HPLC-DAD) was developed for the quantitative determination of sarcophir-e (1) in methanol extract of the soft coral Sarcophyton ehrenbergi. The separations were effected by using a mobile phase of acetonitrile and water. The gradient elution of the mobile phase was 50-95%
ACN in 0-25 min, 95% ACN in 25-35 rain. The flow rate was 0.8 ml/min aid the detector wavelength was set at 210 nm.
This method was validated m terms of compatibility, specificity, linearity, piscision, accuracy, limit of detection and limit of quantitation.
Keywords. Sarcophine, Sarcophyton ehrenbergi, HPLC, validation.
I.DATVAND6
Cembranoid la nhiing h<jp chat diterpene v6ng 14 rit pho bien ij cic lo^ san ho mem Trong so d6 sarcophine U m$t h(;rp chdt kb& n6i ti^ng voi nhilu ho^t tfnh sinh hoc khic nhau mi no! b^t nhat la kh^
n ^ g ch6ng ung thu cua no [1] [2]. Nhung dieu tra vl h6a hgc cho thay h(?p chat nay co m5t trong nhieu loii san h6 chi Sarcophyton nhu S. glaucum, [3] [4]
[2], S. mililatensis [5], Sarcophyton trocheliopkorum [6], Sarcophyton ehrenbergi [7]
[81. G ^ day, trong chuong trinh nghiSn cihi tim kilm cic ho9t chit sinh hijc tu .an h3 mSm ving bien Song b3c bo, chung toi aa p h ^ I|p duQ'c sarcophme tir cic loii san ho mera Lobophytum sp.
va Sarcophyton ehrenbergi. Trong bai bao niy chung toi bao cio phucmg p h ^ dinh lugmg sarcophme tii cao chiSt MeOH mau san h6 mem Sarcophyton ehrenbergi bing HPLC-DAD.
Hinh 1: Cau true hpp chat sarcophine 2.THUCNGHI$M
2.1. Thiet bi vk hda chit
H§ th6ng sSc ky I6ng hi?u nSng cao (HPLC) Agilent 1200,4 k6nh dung moi, bom mau tyr dong, detector DAD, c$t s5c ky Zorbax Eclipse XDB-C18 (4.6 X 150 mm, 5 pm).
can phSn tich Ohaus-USA dp chhih xic 0,0001 g, ntay si@u im, cac dyng ci^ thdy tinh, blnh d)nh rm^c, micropipette c6 d$ chinh xac phii hgp.
Dung moi Methanol (MeOH), Acetonitril (ACN)_ lo^i dung cho HPLC (Merck).
Sic ky jpt (CC) sii d\mg silica gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, Merck) vi YMC RP-18 (30-50 pm. Fuji Silysia Chemiral Ltd.). sic ky lap mong (TLC) sii dpng bin tring sin sihca gel 60 F254 (1.05554.0001, Merck) vi RP-18 F254S plates (l.!5685.0001, Merck). V§t ch^t dupe phit hi?n bing each phun dlu thuoc thi axit H2SO4 1 10% vi ho nong tir tir din khi hifn mau. j 2.2. Nguyen lifU
Mau san ho ralm Sarcophyton ehrenbergi (v.
Marenzeller, 1886) dupc thu th$p t^i khu v\ic Di Den, Quing Ninh, vio thing 3 nim 2015 vi dupe!
PGS. TS. S3 C6ng Thung vi?n Tii nguydn Moi ttiiCfii^ biln dinh l:;ii. MIU tieu bin dupe Imi gift tgi Vi?n Hoa sinh biln vi Vi#n Tii nguyen va Moi truong ^)iln.
23. Phan l^p hfp ch4t (1) I Miu san ho mem Sarcophyton ehrenbergil
dupe sly kho. xay nhd (2.0 kg), sau d6 chiet sidu im [ trong MeOH ba lin ^ nhi§t dp ph6ng. Dich chilt MeOH sau khi c4t Iogi dung m6i 6 ip suit giim (250
Hgi thdo khoa hgc ky mem 40 ndm ihdnk Igp lien Ildp? ldm KH&CN ViSiNam
g) lan lupt dupe chiet phan doan vol cac dimg moi n-hexan va CHzCb.
C§n chilt n-hexan (80 g) dupe phan tich bing h? thdng s5c k;? long trung ip tren silica gel pha thucmg, rua giii gradient bing hon hpp n- hexan/acetone (50/1 - 1/1) thu dupe cac phan dogn.
Phin dogn chlia hpp chat (1) tilp tyc dupe chgy sic ky cOt pha dio vai h? dung m6i acetone/HzO (2/1) thu dupe 15 mg chit (1). Hpp chit niy dupe tmh chl them bing b? thdng HPLC dieu chl, nia giai bing hon hpp ACN/HiO thu dirpc 12 mg sarcophme tinh khiet, sii dyng lam chit chuan IS dinh lupng,
c i u true hpp chit (1) dupe xic djnh bing c. -\
so sanh dCc lieu ph6 c§ng hudng tir hat nhan (W,IR) vi cic hing -o vgt 1^ v6i cic tai lieu di cong b6 ;"9].
2.4. Chuir- b( man phSn tich
Can khoing 100 mg cao chilt MeOH miu san h6 mem Sarcophyton ehrenbergi, chuyin vao binh d^nh raijc 10 inl, thfim MeOH tdi vgch miic va lie deu. Dung djch thu dupe dupe Ipc qua mang Ipc 0.45 pm tnnfrc khi phan tich biug HPLC.
2.5. Chuin bj mSu chuin
Cic dung djch chuin dupe pha ttr sarcophine tinh khi^t trong MeOH 6 cac ning d0 10, 20, 50,
100, 150 pg/ml va dirpc bao quan trinh anh sing a 4''C cho den khi phan tfch.
2.6. Bieu Iden sfic ky (*)
Cpt sic ky Zorbax Echpse XDB-Ci8 (4.6 s 150 mm, 5 pm), detector DAD, buoc song phit hifn 210 nm, toe dp dong 0.8 ml/ph, the tich tiem miu 20 p!, pha dong ACN - H2O vdi chuong trinh dung ra6i: 50-95% ACN tir 0-25 phut, 95% ACN Iii 25- 35 phut.
3. KET QUA VA THAO LU^N 3.1. Xay dung phinmg phap phin tfch
Qua tien hanh khio sit budc song cho tip hip thy eye dai, ty I? thanh phin pha dpng, thl tich ti6m mau, toe dp dong, chung toi da xa - dyng dupe didu ki$n sic ky thich hpp de djnh lupng hpp chit (1) li dieu ki^n (*). Vdi dieu ki^n sic ky nay chat (1) cd thdi gian luu li 10.21 phiit va dupe tach khoi cac pic khac b^ng mSu phan tich (hinh 2). Him lupng chit phin tich dirpc xic dinh dua vao di^n tich pic thu dupe va dudng chain.
1W
•Mi
nwUJ
1
S 10 15 so
(1)
I
^ " M J
A S IS Vl ii f 1
t 1. • • • 1. • • • • i . • • •
(a)
(b)
^ " r-
(c) !
h 3i wk
Hinh 2: SSc kjr do miu t i ^ g (a), chit chuin ndng dp 20 pg/ml (b) va dfch chilt MeOH cua san hd mim Sarcophyton ehrenbergi nong dO 10 mg/ml (c)
B(s dgsig si^h h^c vd cdc chde cd hoat Sinh sink hgc
3.2. Tham Spih phiFffng phap
Thim dinh phuang phap phan tich dupe tie,.i hSnh theo cic chi tieu: dp tuang tiiich » ^ h? thing sic ky, dp chpn Ipc - dgc hi?u, khoing ndng dp tuyin tioh, dO chinh xac, dp diing, gidi hgn phit hipn va gidi hgn djnh lupng [10] [11].
3.2.L Do tuong thich ciia h4 thong sdc ^ Chgy sic ky 6 lin n^u chuan ndng dp 20 pg/ml theo dilu ki?n sic ky C^ chpn. Ket qui "ho thay RSD ciia thai gian luu vi dien tich pic deu nho ban 2% (bing 1) chfaig td he thing sic k^ tuong thich cho qui trinh phin tich n^u.
Bdng I: Kit qua Ithio sat tmh tuong thich ciia h?
Sdng 2: Kit qui khao sat khoang ndng dp myln tinh
th6ng TT
1 2 3 4 5 6 RSD (%)
Thfri gian lini (phit) 10.251 10.259 10.250 10.251 10.247 10.249 0.04
Difn tich pic (1) (mAu) 996.88 993.01 995.71 991.03 991.67 990.54 0.26 3.2.2. DQ chQn IQC — Bdc hi^u
T\ka hinh phin tich mau tring, miu chuin vi miu phin tich theo phuang phip da xiy dyng. Kit qua cho thay (hinh 2) tren sfic IP? d6 cua miu bring tai vi bi thdi gian lim cua (1) Ididng xuat hifn pic nhu d miu chuin vd nmu phan tich. Pic cua (1) trong miu phin tich dupe tach Ididi cac pic khic. Nhu vgy phuang phip cd tinh chpn Ipc tdt a dO d^c hifu cao.
3.2.3. Khodngnongdp tuyen tiii
Tien hanh sac ky cic mau chuan co nong d$ tir 10-150 pg/ml thi^o phuong ph^p di chpn vi thilt l§p in6i quan h? giiia di?n tich pie -, di n6ng dO chit chuin. Ket qui cho thiy trong khoang nong dp tren cd sy tuong quan tuyin tinh giira difn tich pic vi ndng dp (1) vdi hf sd tirong quan r = 0.99940.
TT 1 2 3 4 5
&
Nong d$ ch^t chuan (^g/ml)
0 10 20 50 100 150
Dien tich pic (j) (mAU)
0 510.21 993.0!
2792.87 5138.57 8045.06 Phuang trliih h6i quy: y = 53.25x - '. 5.30 ^
H? s6 tucmg quan: r = 0.99940 -^/ea-
3000- 4000- 2000 T
-''^
3 ^ , ^ - - ^
Xi-^"'^
" 0 100 Amountfmco/mll
,-'^
Hinh 3: Dudng chuin bilu dien mil tuo^g quan gifta di?n tich pic vdi ndng dO (1)
3.2.4. Dp chinh xdc
Bang 3: Kit ,^ui khio sat dp chinh xic cua phuong phip
TT 1 2 3 4 5 6
Nong ad mau phan tich (mg/ml)
10 10.2
10 9.8 10.4 9.6
Difn tich pic (l)(mAU)
3901.62 3734.10 3669.85 3638.80 3815.61 3554.51 Trung binh
- 3 ( % )
H^m lugng
(%)
0.694 0.690 0.692 0.700 0.692 0.698 0.69 0.57 Tiln hanh dinh lupng 6 miu phan tich khic nJiau (dupe chuan bi theo quy trmh bong myc 2.4) vdi dieu ki?n sic ky di lya chpn. Ket qua (bing 3) xic dinh dirpc him lupng (1) trong cao chilt miu san hd mim Sarcophyton ehrenbergi la 0.69%, vdi dp Ifcb chuin tuang d6i li 0.57%. Nhu vgy phuang pbip cd dp chinh xic tot, dip iing ydu ciu phsLn tich.
Hgi thdo khoa hgc ky nipn 40 ndm thdnh l§p Vien Mdn ldm KH&CN f-lftNam
3.2.5. Dp dung
ThSm chinh xic dung dich chuin vio cac mau phan tich <^y 1? 1:1 vl thl tich) dl thu dirpc cae dung dich cd ndng dp (1) hiong iing khoang 80%, 100%
va 120% so vdi ndng dp dinh lupng, Tiln hanh sic Bdng 4: Kft qua khio sat dp dung ciia phuong phap
ky theo phiroTig phap da xay dyng df xac djnh lupng ch^t chuin thu hdi. Kit qua cho thiy (b^g 4) phuang phap co dp diing dap iing dupe ySu ciu ciia phuang phap phan tich HPLC vdi t^ If thu hoi trung binh la 100.27% va RSD = 0.86%.
TT 1 2 3 4 5 6 7 8 9
Nong d§
man (mg/ml)
9.8 10 10.2 9.8
10 10.2 9.8
10 10.2
N^ng df ChSt chuan thim v£ko (llg/ml)
50 50 50 70 70 70 100 100 100
Difn tich pic (l)(mAXq
3154.17 3159.77 3207.04 3644.81 3675.85 3732.15 4465.2!
4527.32 4554.62
LuoTig chat chuan tim 1^1
(J'g'ml) 50.42 50.05 50.62 68.85 VJ.43 70.34 99.66 101.41 101.15
Ty If thu hSi (%) 100.84 100.09
!01.23 98.35 99,19 100.48 99.66 101.41 101.15 Trung Mnh
Trung hinh 100.72
99.34
100.74 100.27
RSD (%)
0.57
1.08
0.94 0.86 3.2.6. Gi&i hqn phdt hi$n (LOD) vd gidi hcfn dinh
lu(mg (LOQ)
Tien hinh chgy sac 1^ mau chuan theo dieu ' kifn (*) (l$p Igi 4 lan) d ndng d$ thap sao cho chieu cao pic chat chuin gip 3 lln d$ nhieu dudng nen.
; Kit qui cho thiy gidi han phit hifn cua (1) la 0.06 pg/ml vi gidi hgn d;nh lupng li 0 " • Z'TII.
4. K £ T LD^
Da xiy dyng dupe phuang phap djnh lupng sarcophine trong cao chiet mau san hd mem
^Sarcophyton ehrenbergi bing phucmg phip HPLC, 'detector DAD vdi cic dilu kifn sic k;? nhu tren. Ket
•qui thim djnh cho thiy phuong phap thda man dp hrong thich ciia hf th6ng s^c k^, d§ chpn Ipc - dgc , h i ^ khoing nong dp tuyin Unh, dp chinh xac, dp Jhkg, gidi hgn phit hifn va gidi han dinh lupng.
LM cim on: Cong trinh ndy dupe hodn thanh vdi 3lf tdi trp kinh phi cua Vi$n Hdn ldm Khoa hpc vd Vdng ngh$ Vi0t Ham, di tdi ma so:
YAST.TB.DAB.02/I3-I5.
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Lien hf: NguySn VSn Thanh
Vifn Hda sinh biln, Vifn Hin lim Khoa hpc vi Cdng nghf Vift Nam So 18, Hoing Quoc Vift, Ciu Giiy, Ha Npi. Vift Nam Email: thanh cm gutoiyahoo. com
