0 Nghien CIFU - Ky thuat
Xay dyng quy trinii dinh lyang dong thoi chat bao quan natri benzoat va kali sorbat
r •? y
trong mot so thtfc pham bang HPLC
Le Thj Kim Clii\ Tr^n Hiru Dung^' 'Trung tdm KiSm nghiem Thuoc, My phdm, Thtfcphdm Thira Thien - Hue
^Khoa Dugc, Truang Dgi hgc YDugc, Dgi hgc Hue
*E-mail: huudung76@,gmail.com
SummaryA reverse phase HPLC procedure for simultaneous analysis of sodium benzoate and potassium sorbate in food products was introduced. Injection solutions were obtained by treating the analyzed samples with acidic or basic solutions and discarding the residues in potassium fericyanid and zinc acetate solutions. The method was validated for system suitability, proving a good repeatability (RSD
= 1.35 -1.90%), suitable accuracy (recovery ratios: 93.10 -103.80 %); low detection limit (0.69 - 0.76 ppm), with a good linear concentration range. By experimental application to some kinds of marketed food products with sodium benzoate and potassium sorbate as preservative additives, the method proved really reliable.
Keywords: Reverse, HPLC, natri benzoate, kali sorbate, food.
Dat van de
Hign nay, hon 2500 phy gia da dyge sd dyng trong c6ng nghg thyc pham vdi nhieu mye dfch khdc nhau, trong dd, viec sd dgng hai logi phy gia chdng vi sinh vgt va nam mde Id natri benzoat vd kali sorbat kha phd biln. Mgt trong nhyng nguyen nhdn cCia viec gia tang sd dyng hai loai phu gia ndy id do sy thay ddi trong sdn xuit vd tidu thu cdc thye pham ... '^' ^'. Cae phu gia khi dyoe sd dung qua iiiu lygng trong mpt thdi gian ddi se gay ra cac ngp dge nhat djnh nhat Id ddi vdi tre em, hoge nhgy cdm cua mdt sd ngydi ngay cd khi sd dung d mde an toan'^.
Be djnh lyong hai hoat chat tren theo Thydng qui ky thuat (4021/2003/QO-BYT) vd Qui chuan Vigt Nam (QCVN 4-12: 2010/BYT) eua Bg Y te ehi xac djnh trong nguyen iieu hda chat bang phyong phdp chuan dp trong m6i trydng khan, con trong nen mau phdc tgp cCia thyc pham phyong phdp nay khdng the dp dyng dygc.
Trong nghien cdu ndy, chung t5i gidi thieu quy trinh xac djnh ddng thdi natri benzoat va kali sorbat tnDng mau thyc pham bang phyong phdp HPLC.
Nguyen v f t iieu va p h u a n g phap
Chuan bj cdc dung djch
Dung djeh ehuan gdc 1000 ppm natri benzoat vd kali sorbat: cdn 0,1 g ehuan mdi logi, them 2 - 3 ml methanol, lie cho tan hoan todn, them nyde cat vda du 100 ml. Td dung djeh ehuan gdc, pha ra cdc chuan Idm vige eo ndng dd thufic khodng 1 - 200 ppm.
- Dung djch K3Fe(CN)6: hod tan 10,6 g K3Fe(CN)6 trong 50 ml nydc, lac cho tan hodn todn, them nyde vda du 100 ml.
- Dung djch K2HPO4 0,02 M: hda tan 3,484 g K2HPO4 trong 100 ml nydc.
Phyo>ng phap nghien cdoj
Phwong phip xv ly mau thiPC pham d^ng long
Hut chinh xdc 20 ml mau cho vdo cdc cd md
250 ml, thdm 25 mi dung djch NaOH IN, dun
ndng tren bep cdch thuy d nhigt dg 70°C trong
30 phut. Ldm ngudi v l nhigt dg phdng, ehuyin
vdo binh djnh mde 100 ml, trung hda mlu bing
dung djch H2SO4 10%. Them Iln lygt 2 ml dung
djch fericyanid vd 2 ml dung djch kem acetat, ldc
T^P CHI DlTt^C HQC-08/2013 (S6 448 N A M 53)
• Nghien CIJU - Ky thuat
deu gay tCia d l loai tgp. Them 10 ml dung djeh K2HP04 0,2 M, Ide ky, vd dinh m d e methanol v d a du 100 ml. Lpc qua mang Ipe 0,45 pm t r y d e khi bom vao may HPLC .
Ket qua tinh theo cdng thdc:
C(mg/I) = Cx X n/R
Trong do: - R: He so thu hdi sau qua chidt tach miu; - C^: sd pg natri benzoat vd kali sorbat trong 1 ml dung djch chidt; - n: he sd pha loang.
Phuvng phap xiP ly mau tiiirc phim la dang ian
Can ehinh xae khoang 5 g mau da d y g c ddng nhat va xay nhd cho vao cdc cd md 250 mi, thdm 25 mi dung djch NaOH 0,1 N, dun nong tren bdp each thuy d nhiet dp 70°C trong 30 phut. Ldm ngudi ve nhiet dp phdng, chuyin vdo binh djnh mde 100 mi, tmng hoa mau bang dung djch H2SO4 10%. Them khoang 2 ml dung djeh fericyanid, iac, thdm 2 mi dung djeh kem aeetat de tgo tua iogi tgp. Them 10 ml dung djeh K3HPO4 0,2 M, iac ky, va djnh mde methanol vda dCi 100 ml.
Loc qua mang 0,45 pm, bom vao may HPLC.
K i t qua d y g c tinh theo cfing thdc:
C ( m g / 1 0 0 g ) = ( C , 1 0 ) / ( P x R)
Trong do: P(g): sd gam mdu dem phan tieh.
D i l u kidn sac ky
Trien khai tren may sac ky long hieu nang eao Series 20A (Shimadzu), cfit pha dao RPI 8 (150 X 4,6 mm; 3 pm) d nhiet dd phfing vdi pha dpng la dikali hydrophosphat: methanol [70:30], Ipe qua mang 0,45 pm, iac sidu dm dudi bpt khi.
The tfch tidm mdu id 20 pl vdi tde do ddng 1,5 ml/phOt vd phdt hien tgi byde song A = 230 nm.
K e t q u a v a b a n l u a n Xdc djnh tfnh tu'ong thich he thdng Tfnh t y o n g thfeh he thdng eua phyong phdp d y g e ddnh gid tren mdu dung djeh ehuan hdn hgp ehuan natri benzoat vd kali sorbat, mdu
d y g e tiem iap lai 5 i l n vdi d i l u kien sde ky nhy da neu, ket qua d y g c ghi d bang 1.
Bang 1 : Tinh tuung thich cCia h$ thdng sic ky dum xay dimg
STT Thong so Th&i gian \vfu IR 1 (philt) RSD
H& s6 dot xifng RSD Dien tich pic
•* RSD So Sia ly thuyet
RSD DO phan giai
RSD
Natri Ixnzoat 6,134 0,046 0,738 0,102 2963546
0,097 5032 0,216
Kali sorbat 8,438 0,030 0,837 0,065 3284176
0,036 7048 0,514 5,691 0,054 Cde th6ng sd sac k^ nhy sd dTa ly thuylt, he sd bat ddi (he sd keo dudi) vd he sd phdn giai d y g c tfnh toan d y a tren cae gid tri thdi gian l y u va dp rpng ede pfe thanh p h i n thu d y g c t d hai pfc natri benzoat vd kali sorbat T d kdt qua thu d y g e eho thay, dieu kien phdn tfch HPLC d y g c thda man khi tat ca cac thfing sd sde ky deu eho g i a t r i R S D < 2 % t ^ ' ^ l
Xac djnh tinh dgo hieu
Tinh dge hieu dygc nghien cdu tren tren sac ky dd cua m i u thd, mau chuan va mdu thd them ehuan (hinh 1) cho thay tat ca eac pic cau t d deu sde nhpn. Thdi gian iyu eua eac pic natri benzoat (6,134 phut) va kali sorbat (8,438 phut) tren mau thd tyong t y vdi thdi gian lyu cua eae thanh phan dd tren sdc ky 36 cCia mdu chuan va mau thd thdm ehuan. Ngodi ra, t d phd dd eua natri benzoat va kali sorbat cua m i u thd cho thay hinh dang tyong t y vdi mau chuan khi phan tich bdng detector PDA (hinh 2). Nhy vay, phyong phdp ed tfnh chpn ipc tdt, kh6ng cd thanh phdn hay c h i t phy gia nao cua mau trong qua trinh nj-a gidi gay dnh hudng den phep xdc djnh cac thanh phdn phdn tieh'''' ^.
Hinh 1 : SSc ky do cOa mau thi> (a), m§u thu them chuan (b) va m§u chuan (c) thu duvfc dime vol didu kien sic ky duxyc chgn li/a
TAP CHf DirOC HOC - 08/2013 (SO 448 NAM S31
Hinh 2: Phd dd cOa natri benzoat (a), kali sorbat (b)
tren mau thi> thu du^v vdi didu kien chay thich hgp sCr dung detector PDA , thu dygc cho thay cd mdi tyong quan tuyen tinh cao eua di^n ttch pie tyong dng vdi ndng dp cua natri bonzoat vd kali sortaat trong khodng ndng dp [0,25 - • 100 pg/ml] khi cde hg sd tyong quan diu Idn hon 0,999 (hinh 3) f"'^'.
Xac djnh mdi tuvng quan tuyen tinh Tfnh tuyen tinh eua phyong phdp dygc xdc djnh bang each tiem eac dung djch ehuan natri benzoat va kali sorbat d 6 mde tyong dng td 10 - 300% cua ndng do dung djch mau thy. Kit qua
Duimg chuin natiiienzoat Dirdng d i t i i n kaliiortat
7000 6000 sooo .i!«00
1000
y = 65 632X-0 2313 F ? = 1 ^ , ^
n6ngd9 n£nga6
Hinh 3: Dd thj bieu dien khoang tuydn tinh cua ndng dd natri benzoat (a), kali sorbat (b) theo dd hip thu tuxmg uvg tren HPLC v6i bifoc sdng phat hien tai 230 nm Xac djnh d$ lap lai phuxmg phap
flfi chinh xdc cCia phyong phdp dyge thyc hien bdng cdch phdn tfch 5 mlu thd theo dilu kien sdc Wj/ da xdy dyng. Hdm lygng trung binh
Bang 2: NghiSn cm do chinh xac cua phuang phap
cita natri benzoat vd kali sorisat lan lygt Id 251,0%
± 1,00 vd 206,4 % + 0,92 vdi cdc gid tri RSD <
2% (jbang 2). Oieu ndy da ehdng tfi dO lap lai cua hg thdng HPLC rat tdt'"' ^.
STT Lwqmg mau thip (ml) Him Iwgng natii benzoat (iig/ml) Ham lipqmg kali sorbat (iig/mt)
2 3 4 5
50 60 50 50
255,5 246,5 250,5 252,8 249,5
205,5 210,2 200,5 209,8 206,0 RSD (%)
251,0 ±3,4 1,35
206,4 ± 3,9 1,90 Xac djnh do dung cua phuvng phap
06 dung cua phu'ang phap du-Q-c xac dinh bing Phu'O'ng phap them chuan, tii'c th§m chmh xac cac luvng da biet ciJa natri benzoat va kali sortrat vao mau thi> vdi ty le them 50. 100 v^ 150
|jg/mi, sau db xac cHnh luong chat chuan tim lai dU'O'C so vd'i ban dau. Tien hanh tren 5 miu thO
12
vol dieu ki$n XCK ly va quy trinh phSn tich nhu trgn. ty le thu h6i cua natri benzoat vd kali sorbat lan lugt id 96.7 ± 6,1 va 96,8 ± 2.9 (bang 3).
Vol sy xic djnh cac ham lu'p'ng thip trong mlu (pg/mi) lufin tgo ra cac sai so trong thao t i c thg'C hi$n ndn dO diing cua phuong phdp HPLC ducc x§y dyng \k chap nhan dyp'c ''*' ^.
TAP CHi Dl/qC HQC - 08/2013 (S6 448 NAM 53)
• Nghien CIFU - Ky thuat
Bang 3: Kit qui l<hio sat dq dung trin miu thir thSm chuan (n= 5) Hoftchit Mau
1 Natri benzoat 2
3
Lirpng c6 san trong Luvng them vao Lu-ong tim thay Ty /# thu h6i — dung dich (pg/mO (pglml) (pg/mlj (%) ^ <"'''>
50 100 150 50 100
78.25 124.64 187.24 88.52 141.24
93.42 93.10 103.80 94.80 100.12
184,37
95,50 Xdc d j n h g i d i han phat hi$n va gloN handjnh lu*gng
Gidi hgn phdt hign (LOD) vd gidi han djnh lygng (LOQ) d y g c t h y c hidn theo "qui tde 3a"
b i n g edeh pha loang dung djch c h u i n trong phdp t h d tinh tuyen tinh cua natri benzoat vd kail sorbat d l phdn tfch trdn hg thdng HPLC d y g c xdy dyng, LOD cua natri benzoat va kali sorbat d y g c xdc djnh b i n g t^ Id tin higu/nhidu (signal-to-noise) a 3, I l n l y g t Id 0,76 va 0,69
pg/ml. LOQ cua natri benzoat vd kali sorbat d y g c xdc djnh bdng ty le tfn hieu/nhidu > 10 vd nam trong khoang t u y l n tinh t y o n g dng Id 2,28 - 3,04 va 2,07 - 2,76 pg/ml '''• ^'
A p d u n g phuYing phap phan t i c h tren thiPc te
Dya tren phyong phap HPLC dyge xay dyng vdi cdc ttifing sd ky thuat da tdi y u hod, nhom nghien edu ap dung d l phdn tieh 65 mau thyc pham tren thj trydng Thda Thien-Hue (hinh 4).
(0 AU
a.
1
hteiHensty 4 , 0 0 0 ^IA
Hinh 4: Sic ky dd cua mau nu^ nggt (a), mau torn chua (b) thu dirgic vin dieu kien sic ky duofc chon lim
K i t qud phdn tfch hdm i y g n g chat bao thudc, my p h i m , t h y c p h i m T h d a Thien - Hue quan trdn cdc mau t h y e pham efing bd chat deu nam d y d i m d e qui djnh eua Bp Y te (bang l y g n g d y g c gdi den Trung tdm Kiem nghiem 4 p ' .
Bang 4: Ket quit khao sat ham lux^ng chat bao quan tren mot sd mau thiic pham tai Trung tam Kidm nghi$m ThOa Thidn-Hue
STT 1 2 3 4 5 6
Ten miu Nydc ngpt cam Nudc ngpt chanh
Nudc mang c l u Nydc ngpt coca Nydc ngpt lang lyc
Mlu tdm chua
MOv chit lugmg yeu ciu
< 600 mg/1 (dung mgt minh hay kit hgp ca 2 loai)
< 600 mg/1 (diing mgt minh hay ket hgp ca 2 loai)
< 600 mg/1 (dung mgt minh hay k i t hgp ca 2 loai)
< 600 mgfl (dung mgt minh hay k i t hgp ca 2 loai)
< 600 mg/1 (dung mpt minh hay ket hgp ca 2 logi)
< 1000 mg/1 (dung mOt minh hay ket hgp ca 2 loai)
Ham luvng (mg/1) Natri benzoat
182,10 168,98 177,06 202,55 150,81
Kali sorbat
274,12 K e t l u $ n
P h y o n g phdp djnh l y g n g ddng thdi hai hoat c h i t natri benzoat vd kali sorbat trong mdt sd
thye pham ddng bao bi bdng phyong phdp HPLC da d y g e xay d y n g vd tham djnh. Cde k i t qud thye nghigm thu d y g c cho thay phyong TAP CHI DirOC HOC - 08/2013 (S6 448 NAM 531
• Nghien CIFU - Ky thuat
phap ed tfnh t y o n g thieh he thdng cao, eho dfi chfnh xdc va dp dung khd tdt vdi khoang gidi hgn djnh i y g n g rfing. Quy trinh da d y g c ap dung de kiem tra hon 65 mau t h y c p h i m cdng bd chdt i y g n g d y g e gdi d i n Trung tam K i l m nghiem thudc, my pham, t h y e pham T h d a Thien H u l . Qua k i t qua k i l m tra tat ed deu dgt gidi han chat bao quan natri benzoat va kali sorbat cho phep cua Bfi Y t l .
T a i l i e u t h a m k l i a o 1. 66 Y ti, CtJC an toan v$ sinh (/7uc pham (2005),
"Cac van ban quy pham phap luSt v l v§ sinh an toan
thyc pham", Nha xuat ban Hd Ngi, tdp II, tr. 186-192.
2. Bd Yti, QCVN 4-2010/BYT. "Qui chuan cdc Ity thugt qudc gia ve phu gia thyc phim - Chit bao quan", Nhd xuit ban Ha Noi, phy luc 2,5.
3. Bp y ti (1998), "Danh myc cdc tieu chuan ve smh ddi vdi lyong thyc thyc pham" QD s6 867/1998/Qe-BYT cCia Bg trydng Bg Y t l ng^y 04 thdng 04 nam 1998, tr. 4-7.
4. Snyder L. R., Wrkland J. J., Glajch J. L. (1997), Practical HPLC Method Development, 2nd ed., John Wiley & Son: NewYork '
5 AOAC (2005), Official IWethods of Analysis of AOAC International,
r r '?
Phan lap va thiet lap chat chuan majonosid - R2 tir sam Viet Nam {Panax vietnamensis Ha et Grushv.)
Le Thi H6ng VSn\ L§ Thi Mai Sirffng^ Nguyen Ngoc Kh8i\
Nguyen Ddc Tuan', Dirong H^ng T 6 Quy&n\
H^ Difu Ly^, Nguyin Minh Dwc'*
'Khoa Dugrc, Dgi hgc Y-Duffc Thdnh pho Ho Chi Minh
^Vien Kiem Nghiem Thudc TP. Ho Chi Minh E-mail: [email protected] S u m m a r y
Saponin f^Aajonosid - R2 (M-R2) cf ocotillol type, known as an important active and characteristt substance of Vietnamese Ginseng (Panax vietnamensis Ha et Gmshv.), was isolated and purified by means of Diaion HP-20, vacuum chromatography, and preparative HPLC. The isolated fi/IR-2 was preliminarily checked by TLC, HPLC-PDA and further simdurally confinned by IR, MS, and NMR. The HPLC quariMfve analysis of MR-2 was validated for system suitability, selectivity, linearity range and precisbn by two independent GLP laboratories. A reference standard of MR-2 was established with the purity of 98.86% and showed applicable to quality control of the Vietnamese Ginseng and preparations made Uiereof.
Keywords: Panax vietnamensis Ha et Grushv., primary standard reference, majonosid - R2.
trong ede edy sdm Panax cd gid tri khde nhy;
nhdn sdm, tam that vd sdm Hoa ky. M-R2 ehilm gan 50% saponin todn phan v d co nhieu tde dung d y g c ly quan trgng'^^... Do dd, M-R2 la mpt trong nhdng hogt e h i t quan trpng v d can thiet trong kiem tra, ddnh gia chat l y g n g sam Viet Nam vd ede ehe pham, n h i t Id trong tlnh hinh sdm Vidt Nam l y u hdnh tren thj t r y d n g b'i gia mgo rat n h i l u do gid trj cao vd ngudn cung D a t v a n d e
Sdm Vidt Nam tde sdm Ngpc Linh, ten khoa hpe Panax vietnamensis Ha et Grushv.
(Araliaceae), id mot ioai sdm dac hdu cua Viet Nam. Cac eong trinh nghidn c d u khoa hpc da efing bd cho thay sam Vidt Nam la mpt ioai sdm quy'^'^l Trong thanh phan saponin, majonosid - R2 (M-R2) Id saponin chinh thufic nhdm ocotillol, dge tryng eho sdm Viet Nam, khfing cd