Nghién ciru dinh lwong quercetin nguyén liéu bang phuwong phap HPLC Thai Duy Thin, Va Die Loi, Dang Thj Ngoc Thu. at van ad Hién nay, quercetin dang dugc sir dung kha rong rai, ch yéu dudi dang thye pham chic nang va eb mat trén thi trong duoc phdm voi nhing dang bao ché khac nhau nhu: vién nén, vién nang, dang dung dich... véi tae dung chinh la chéng oxy hoa. Viée xéc dinh ham lugng cla quercetin trong nguyén ligu va cc thuc phAm hire nang nay [a rét c&n thiét. Tuy nhign, & nude ta chua cO phyong phap din Iuong quercetin chinh thirc trong cac tiéu chun nganh va quéc gia. Trén thé gidi cé mot s6 phuong phép dinh wong quercetin nhung cing cén hing han ché va chua phil hop voi diéu kién co s6 & Viet Nam®*), Vi vay, ching tdi xin gidi thigu: Phuong phap xéc dinh ham luong quercetin nguyén ligu bang HPLC. Thyc nghiém va két qua Hoa chit, thiét bi Hod chat, thuéc thir ~ Cc héa chat thudc loai HPLC hode PA. ~ Acid phosphoric dc (HsPO,) PA. = Mui kali dihdrophosphat (KHzPO,) PA. = Methanol, acetonitril dung cho HPLC Quercetin: chat di chiéu ( ham long 98%) do Vien Bo luéng chat long cung cép. Thiét bj, dung ow - May sc ky léng higu nang cao: DIONEX- detector UV Diode array- PDA100. ~ May quang phd UV- VIS lamda EZ 210. - May sigu am Sonorex. ~ May do pH Meter 3305 - Jenway. ~ May loc chan kh6ng Sartorius. ~ Cac dung cu thu tinh dat ch&t long ding cho phan tich ‘Déi twgng nghién cou - Cac mu quercetin ca Lién higp khoa hoc san xudt cong nghé méi. ‘TAP CHi DUQC HOC - 7/2010 (SO 411 NAM 50) Tring dai hoc Dure Ha N6i Phuong phap Biéu kign HPLC ~ COt sdc ky Nucleosil C18 ( 250 x 4 mm, 5 um), ~ Detector UV: 371mm. - Pha dong: Methanol - acetonitril - dem phosphat 0,01M pH 3 = 30: 60: 10 = Th tich tiér: 10 yh ~ Tée 46 dong: 0,6 ml phat = Nhi@t 69 phan tich: Nhigt 40 phong thi nghiém Xie ly mau = Mau thi: Can chinh xc khoang 10 mg quercetin nguyén ligu cho vao binh dinh mic 10,0 mi, Héa tan va pha long bang hé dung moi ia pha dong. Lay chinh xac 100 yl dung dich tren cho vao binh dinh mirc 10,0 ml. Thém dung ‘moi vira di dén vach, lac du duoc dung dich cb rnéng dO 10ug/ml, Loc qua mang loc 0,45um, = Mau chuan: Can, pha dung dich quercetin chudn 6 ndng 46 10 ug/ml bang hé dung mdi cia pha déng nhu mau thir. Loc qua mang loc 0.45 um. Két qua va ban luan Séc ky dé cla mau thi va mau chudn ast cence WHET woe an TIPPETT TTT TTT co 100 200 300 «0 sin sm 7m et sos Hinh 4: Sac ky dé cla mau chudnata thy 200 300 «to 00 60) rin 80 a Hinh 2: S&c ky d6 ila mu thir Tién hanh dinh long § mau thir va § mau chudn quercetin theo chong trinh a la chon. Két qua dinh luong duc trinh bay & bang 1 anh gia phwong phap dinh lvgng Ching tdi tién hanh danh gid phuong phap inh lugng quercetin nguyén ligu bang HPLC mdi xay dung voi cdc chi tiéu sau: tinh thich hop ota hé théng s&c ky, tinh chinh xéc, tinh tuyén tinh, tinh ding, tinh dac higu Thir tinh thich hyp cia hé théng sac ky can, pha dung dich quercetin chu&n 06 néng 46 10 jigiml bang hé dung moi cla pha dong nh mau th. Loc qua mang loc 0,45 um, ding dich loc tiem sac ky 5 lan Két qua khdo sat tinh thich hop cia hé théng sdc ky dugc trinh bay & bang 2. Bang 2: Két qua khdo sét tinh thich hop cia he théng'HPLC Pic cia quercetin Bang 1: Két qué dinh Ivgng quercetin grr’ Thbi gan ins Did tich pe yy "4 nguyen ligu mau ‘oni (mAU) mo Miu thi = Mu chun i 422013065 1.75 2 4213 13,198 —_—*1.69 oy én ti ign 3 4.243 13,106 174 “ant Mme ean’ HABE 44207 12919178 (mg) (mAU min) (rng) MN 5 4213 12,905 —_—*41,72 1 102 13,389 10.1 13.216 98.31 SOC) _0,334 0,807. 210.1 13,235 10.2 13,312 98.40 Két qua khdo sat cho thay d6 léch chuan 3402 13708401 13;623 88.62 -—tvong déi (RSD%) cla thoi gian Iuu va dign tich —a 903 13.283 103 13.195 98.36 —_—~ic quercetin trong 5 phép thir dbu nhd hon 2%. S103 13500104 13.556 98.54 —_~DIBU nay ching td he théng HPLC ma chong toi * 56 ligu thing ké cia bing 1 ‘sir dung la phi hop va dm bdo sy én dinh cho easton phép phan tich dinh wong quercetin Sai sé chudn: S, =0,065 Khdo sat, danh gia phuong phép i Tinh chinh xac 6 lech chudn tueng db: RSD = 0.148%; ax S, = 0,18 Khodng tin cay y = 98,43 + 0,18 han xét: Két qua trinh bay & bang 1 cho thay m&u nguyén ligu quercetin 1 dem dinh lang co ham lugng la: 98,43%, Bang 3: Két qué khdo sét tinh chinh xac ac dinh & néng do lam vic ciia quercetin 18 0,01 mg/m Pha 6 mau thi quercetin chuan & ndng 66 0,01 mg/m Két qua khdo sét tinh chinh xae due trinh bay 6 bang 3. str 4 2 3 4 5 6 Dig tich pic(mAU min) 13045 13,055 —«13,045~—=«13,065~—~=«S,085—=«4 3,065 - Sé ligu thong ke: x= 13,055, S=0,0089;__ RSD =0,0684 at qua cho thdy, trong cdc diéu kien sac ky 8 chon, d6 léch chudn tong d6i cla céc két qua [a 0,0684 << 2%. Nhu vay phuong phap la chinh xac. 44 Tinh tuyén tinh Pha 5 mau quercetin o6 ndng 46 lan luot la 0,008; 0,009; 0,010; 0,011; 0,012 mg/ml. Mai néng a6 tién hanh sac ky theo digu kién 48 chon TAP CHi DUQC HOC - 7/2010 (SO 411 NAM 50)3 lan, ly két qua trung binh. Két qua khdo sat khoang tuyén tinh durgc trinh bay & bang 4. Bang 4: Két qué khdo sat tinh tuyén tinh ‘Binh (eZ) Nong a quercetin 0,008 0,009 0,010 0,011 0,012 _{mgimt) Die (eau mn), 10681 11,861 12,960 14,072 15,208 Phuong tinh hei quy: y = 1126,1 x+ 1695, voi R= 0.9999, Sb = 0,0741, 6% d6 tn cay 95% ta cé Khoding tin cay cia y_ intercept 0,0741 4 0,2358 hay -0,1617 < ¥_ intercept < 0,3099 => chira géc toa 46. ‘D6 thi bidu din sy phy thudc tuyén tinh rnéng d6 va dién tich pic cua quercetin duoc ghi ohinh 3 Tinh tuyén tinh Digntich pic(mAU) ° 0.008 0.0101. rnéng a6(mgim) Hinh 3: 86 thi biéu din su phu thudc tuyén tinh gita néng a6 va dién tich pic cua quercetin Nhu vay phyong phap tuyén tinh trong khoang néng do khdo sat tl” 0,008mg/mi - 0,012mgiml hay tuyén tinh trong khoang ndng 6 tir 80% dn 120% néng a6 lam vige. Tinh dang Vi quercetin chua xéc dinh r ham Iuong nén xéc dinh tinh dung phai khdo sat & 3 muc dng dO theo day néng 6 khdo sat la: 80%, 100%, 120% so voi ndng dé lam vigc. Tién hanh + Pha mu thir: Pha ln lyot 3 mic ndng > 0,008 mg/ml, 0,01 mg/ml, 0,12 mg/m! + Pha mau chudn (d6i chiéu): Pha dung dich quercetin chudn néng 49 0,010mg/mi Tién hanh chay sc ky mbi dung dich chay 3 {&n, Tinh ham lugng phan trim cia quercetin cd trong m&u thi dya trén chu&n 6a biét ham wong, tir 46 tinh % tim lai ca mau thi. ét qua dinh gia tinh dung cua phyong phap dugc trinn bay & bang 5, Mau d6i chiéu: S. = 13,189 mAU.min TAP CHI DUQC HQC - 7/2010 (SO 411 NAM 50) Bang 5: Két qué dinh gid tinh dung cua phuong phap Khéi Dien ich wong yng tim ei TB Néng 66 pic cho 7 va MA = Mat wioa (mAUmin) TB ioe (mg) (9) O00emgim! 10.479 80 7045 09,31 0,01 mgiml 13,142 100 98,64 90,64 O.0tamgiml 15,729 120 119,26 99,38 Chudn 13,189 100, “Tye phan tram tim igi = 99/44 %; RSD =0,174 Phuong trinh hdi quy tuyén tinh: 19953 x - 0,075 189 lech chudn cba: Sa = 0,0082 86 lech chudn cia y_intercept: Sy= 0,083 V6 d6 tin cay 95% ta cb Khodng tin cdy cia 46 déc: a # 12,71Sa hay 0,8911 < a < 1,095 => cha 1 nén khong c6 sai sb he théng ty. Khoding tin c€y cia y_intercept: b + 12,71Sb hay - 1,125 chiva gbc toa 49 nén khong c6 sai s6 hang din Vay qua cac két qua trén ta thy phyong phap co dO ding t&t trong khong khdo sat vi kha néing tim lai la 99,44% va RSD = 0,174. Tinh dc hiéw Tién hanh sdc ky dung dich chudn quercetin, rutin chuan, m&u hén hop quercetin va rutin, mau trang va ghi sc ky d6. Két qua ghi & hinh 4;5;6,7. — ean ame {eet —— Teen eae en eT 00 ro 2m 300 4c sm S00 70 om em Hinh 8: Séc ky dé cia mu hén hop quercetin va rutin chudn 45qrcancnn sn ——$—————we] $2 ea 41-3101 Wt an nam si " 4 T 4 Ti Tees ce Te Ta Ty co 10) 200 300 4 sc 600 700 sm 9m Hinh 6: Sc ky dé cua mdu chun rutin Tu cae séc ky d8 cho thay phuong phap dic higu 46i v6i quercetin Két luan 8 xay dung duoc chong trinh dinh lvong quercetin bing phuong phap HPLC don gian, d& thyc hign, véi cdc didu kign sdo ky nhu & trén. BA tién hanh danh gia phurong phap dinh luong va cho thay phuong phap thoa man tinh thich hop cia hé théng sac ky, tinh chinh xc, tinh tuyén tinh, tinh dung, tinh dac higu ‘Summary An HPLC method for determnation of quercetin was proposed. The chromatographic Conditions were: Column - Nucleosil C18 (250 x 4 mm, 5 ym); Detector - UV (A=371nm); Mobile cy on ake seo aon sm Hinh 7: Sac ky dé cila quercetin phase - Methanolacetonitrile-phosphate buffer 0.1M (pH 3) (V/V 30: 60: 10); Injection volume - 10 pi; flow rate - 0.6 mimin, Experimentally, the method was proved time-saving, precise and accurate. Tai ligu tham khao 4. Nguyén Minh Bic, S&c ky lng higu nding cao va mot s6 ting dung ao nghién ciru, kiém nghiém duge phdm, durgc ligu va hgp chat ty nhién, NXB Y hoc, trang 49 -170, (2006) 2. Ping Xiao, Faquiong Zhao, Baizhao Zeng, Voltammetric determination of quercetin at a muli- walled carbon nanotubes paste electrode, Mircochemical Joumal (85), p.244-249, (2007) 3. The United states pharmacopoeia, p.2278- 2282, (2004)