VIETNAM MEDICAL JOURNAL N"! - JAHUARY • 2017 of the WHO Intemational Agency for Research on
Cancer Monograph Worldng Group. Special Report:
Policy. A review of human cardnogens - Part B:
biological agents. Lancet. 2009; 10: 321-322.
6. Parkin DM. The global health burden of infection-
associated cancers in the year 2002. Internatio/im Journal of Cancerl^ae; 118(12):3030-3044. ; f 7. Schiffman M, Castle PE, Jeronimo 3, R o d n g ^
AC, Wacholder S. Human papillomavirus aM cervical cancer. Lancet2007; 370(9590):890-90f
NGHIEN CU'U XAY DU'NG QUY TRINH DINH LU-gNG DONG T H a i PARACETAMOL, CAFEIN VA PHENYLEPHRIN TRONG CHE PHAM
DA T H A N H PHAN BANG PHU'aNG P H A P H P L C - D A D
Nguyen Thi Ngoc Van*, HuJ'nh Thanh Giang*
T O M TAT
Muc tieu: Xac djnh dieu kien sac ky de djnh lutfng dong "thdi paracetamol, cafein va phenylephrin bang phu'dng phap HPLC-DAD; Tham dmh quy trinh djnh lutJng dong thcfi paracetamol, cafein va phenylephrin bSng phu'dng phap HPLC-DAD. Ket qua: da )rac dinh du'dc dieu kien sac ky thich hdp: cot GL Sciences Ca (250mm X 4,6mm, Spm); toe do dong: 0,8 mj/phut;
buSc song phat hien 215 nm. Pha ddng la hon hdp ACN: I^ieOH: HjO (10:30:60); nhiet do c6t: rihiet do phong; ttie tich tiem lOpI; quy trinh dinh lu'dng da du'dc tham dinh theo hu'dng dan cua ICH vdi cac chi tieu do chinh xac va do dung cd RSD < 2%; khoang tuyen tfnh ciia ba chat paracetamol tff 50 - SOOpg/ml/
cafein tff 10 -lOOpg/ml, phenylephrin tff 5 - 50pg/ml va cd r^ > 0,99. Ket lu^n: Quy trinh co tinh chon Ipc, dp chinh xac va dp diing cao co the ffng dung de djnh lubng dSng thdi 3 thanh phan tren trong cac che pham tffdng tff tr§n thi trffdng.
S U M M A R Y
SIMULTANOUS D E T E R M I N A T I O N OF PARACETAMOL, CAFFEINE AND PHENYLEPHRINE I N TABLET BY HPLC-DAD
Objective: A reversed-phase high performance liquid chromatography (HPLC) method for simultaneous determination of paracetamol, caffeine, and phenylephrine in tablet was developed and validated. Results: These active ingredients were completely separated on a GL Sciences RP - Ca column (250 X 4.6 mm, 5 pm) with mobile phase consisting of acetonitrile - methanol - water (10: 30: 60) with DAD detector highly selective to HPLC system. The flow rate was 0.8 ml min"^ and the photodiod - array detection wavelength was 215 nm. All calibration cun^es showed good linear regression (r >0.99) within
* lOioa Dddc, Tnidng Dai hge YDddc Can Ihd Chju trach nhiem chinh: Nguyin Thj Ngpc Van Email: [email protected]
Ngay nhan bai: 21.10.2016 Ngay phan bien khoa hoc: 2.12.2016 Ngay duyet bai: 8.12.2016
the concentration ranges of 50 - 800 pg/ml for paracetamol, 10 -lOOpg/ml for caffeine, 5 - 5 0 pg/ml for phenylephrine. The accuracies for four compon,eiits were in the range of 98-102% of the expected valups with RSD < 2%; the inter-day and intra-day relative standard deviations were below 2%. Conclusion: The proposed method can be effectively applied for tiie simultaneous determination of three compounds In bulk and in combined dosage form.
Td khoa: HPLC-DAD, paracetamol, cafein, phenylephrin
I.
OAT VANo l ^ 3
Tren thi trffdng, cac che pham da thanh phan ngay cang pho bien, dac biet la hon hdp cac thuoc ha nhiet, giam dau, so mui, khang histamin... vdi nhieu biet dffdc. Ba hoat chat thffdng thay trong thanh phan cac biet dffde nay la paracetamol (PAR), cafein (CAF), . va phenylephrin (PHE) vdi ham Iffdng chenh lenh nhau kha ldn trong che pham (5 -500mg). :De djnh Iffdng nhu'ng hoat chat nay thffdng sff dung hai quy trinh rieng biet, dieu nay thiic day vi^c xay dffng mgt fluy trinh phu hdp de djnh lffdng dong thdi nhieu hoat chat trong clie pham.
Trong cae ehuyen luan cua DDVN IV [1] va Dffdc dien cac nffdc [2] hau het chi co chuyen Iuan eho cae che pham co chffa mgt thanh phan,;
trong USP 36 tuy co de cap m6t so chuyen luan' ve cac che pham chffa ba hoac bon hoat chat nhffng viec phan tich cac chat dffdc tien hanh, rieng biet tren nhChig dieu kien phan tich khac nhau, gay ton kem ve dung m5i, hoa chat, thdi gian. Vi vay, de dap ffng nhu cau kiem soat chat Iffdng, chung toi tien hanh nghien cffu xay dffng quy trinh djnh Iffdng dong thdi paracetamol, cafein va phenylephrin trong che pham da thanii phan bang phffdng phap sac ky long hieu nang cao, vdf 2 mue tieu; (1) Xac djnh dieu kien sac ky de dinh Iffdng dong thdi PAR, CAF, v^ PHE bang phffdng phap HPLC-DAD; (2) Tham djnh
62
TAP CHi Y HOC VlgT NAM TAP 450 - THANG 1 - gO 1 - 2017
quy trinh djnh Iffdng dong thdi PAR, CAF, va PHE
bang phutfng phap HPLC-DAD.
n. DOI TUONG VA PHLTONG PHAP NGHllN COU 2.1. Odi tu'dng nghien cu'u
Boi tu'dng: Vien nen bao phim Panadol cam cum chfe 500mg paracetamol, 25mg csfein, 5mg phenylephrin hydrododrid dffdc san xuat bdi cong ty Co phan Dffdc pham Sanofi - Synthelabo Viet Nam, so dang ky: VD-16582-12.
Dung moif hda chat: Cae chat chuan:
paracetamol (ham Iffdng 100,10%), cafein (ham Iffdng 99,83%), phenylephrin hydrododrid (ham Iffdng 100,02%) cua Vien kiem nghiem thuoc TP.HCM. Acetonitril, mettianol tinh kliiet phan tfeh eua Merck (£>ffc) dung eho HPLC
Trang thiet bi: He thong HPLC Hitachi L- 2000 dau do DAD L-2455 (Nhat Ban).
2.2. Phu'dng jihap nghiSn cu'u Bieu luin sac kjy: Khao sat ve thanh phan pha dgng, t^ le dung moi, toe do ddng, chung toi xay dffng dffdc chffdng trinh sac ky nhff sau: cot GL Sciences Q (250mm x 4,6mm, 5pm); toe do ddng: 0,8 ml/phut; bffdc song phat hien 215 nm.
Pha dong la hon hdp ACN: MeOH: H2O (10:30:60); nhiet dp cot: nhiet do phong; the tfeh flem lOpl.
Dung didi ciiuan: Can chinh xac khoang 100 mg ehuan doi chieu paracetamol, hut 25ml eafiein (20Dpg/ml) va 5ml phenylephrin (2QD|jg/ml) vao binh djnh mffe 200 ml hoa tan va pha bang den vach bang pha dgng, Igc qua mang Igc 0,45 pm. Hon hdp mau cliuan eo nong do: paracetamol 500pg/ml, cafein 25pg/ml, phenylephrin 5pg/ml.
Dung didi mau thut: Can 20 vien Panadol cam cum, xae djnh khoi Iffdng trung binh vien.
cho vao coi nghien thanh bgt mjn, dong nhat.
Can chfnh xac mgt Iffdng bgt thuoc tffdng ffng 1 vien cho vao binh djnh mffc 100 ml; them khoang 30 ml methanol roi sieu am eho den khi tan hoan toan, de nguoi ve nhiet do phong, bo sung methanol vffa du ioo ml, Igc bo 10 ml djch Igc dau. Hut chfnh xac 10,0 ml djch Igc; pha loang bang pha dgng vij^ dii eho vao binh djnh mffe 100 ml; Igc qua mahg Igc 0,45 pm trffdc khi tien hanh sac ky.
Tham dinh guy trinh: Xac djnh tfnh tffdng thieh he thong, tfnh dac hieu cua phffdng phap, tfnh tuyen tinh, do clifnh xac va do diing eua phffdng phap khi tiiem 80-120% mau chuan vao mau thff theo hffcfng dan cua ICH Q2A [3].
III. KFT QUA
3.1. Khao sat va lu^ chpn dfeu Iden sac k^
Quet pho hap thy UV trong khoang 200-400 nm tai ttidi gian Iffu cua cac pic PAR, CAF va PHE tren sac ky do nham xac djnh bffdc song hap thu cdc dai tffng chat tff do chgn bffdc song thieh hdp ciio quy trinh djnh Iffdng. Tai bffdc song 215 nm, ca ba chat cung cho tfn hieu hap thu manh, phu hdp, pie PAR, CAF va PHE dat yeu cau ve hinh dang pic (0,8<As<l,5).
TTiffc hien sac ky hon hdp 3 chat vdi he pha dong gom 2 thanh phan MeOH va nffdc vdi t^ le thay doi cua MeOH tff 60- 40% (AC = 10%), toe do dong 0,8 ml/phut. Ket qua cho thay khi giam ty le dung moi hffu cd, thdi gian rffa giai cac pic tang len dan den pic PAR va CAF tach nhau, nhffng hinh dang pic CAF khong can doi (As = 1,92). Nhff vay, he pha dgng MeOH va nffdc khong phiJ hdp de t^ch CAF (xem hinh 1).
-
^ E ^ " "• fAI
-
^ C " " " ' " fRl
=
Hinh 1. Sac k^ do hon hdp chuan vdi he pha dong MeOH:H20 (A) va he pha dong ACN:H20 (B)
Tiep tuc khao sat he pha dpng gom 2 thanh phan ACN va nffdc vdi t^ le thay doi cua ACN tff 60- 40% (AC = 10%), toe do dong 0,8 ml/phut.
Ket qua eho thay khi giam ty le dung moi hffu ed, pic PAR va CAF tach nhau va hinh dang pic CAF eai thien ro vdi he so bat doi dat yeu cau
(As =1,82). Tuy nhien vdi he pha dgng nay pic
PHE khong Iffu giff tren cot (tR = tO) va tfn hieu
gan bang tfn hieu dffdng nen. Tff ket qua tren
eho thay, he pha dgng ACN va nffdc khong phu
hdp de tach PHE (xem hinh 1).
VIETNAM MEDICAL JOURHAL N°1 - JANUARY - 2017
Khi ket hdp ACN: MeOH: HjO (10: 30: 60) can doi, ehan pic hep va deu dat cac quy CRnhpk phat hien dffdc dong thdi tfnh hieu cua ea ba thong so trong sac ky. Do do day la he pha dgr^
hoat chat, thdi gian Iffu cua PHE khoang 3 phut. 6ddc chgn de phan tfeh d5ng thdi PAR, CAF va P H ^ Ca ba pie tach rdi nhau hoan toan, hinh dang pic
Hinh 2. Sac ky d& hdn Nhff vay da lffa chgn dffdc dieu kien sac ky thfch hdp djnii Iffdng dong thdi phenylephrin, paracetamol va cafein trong che pham bang phffdng phap HPLC/DAD nhff da trinh bay trong phan phffdng phap nghien cffu (hinh 2). Vdi dieu kien nay thdi gian Iffu cua phenylephrin la 2,67 ph6t, paracetamol la 5,39 phut, cafein la 6,42 phut, phii hdp cho quy trinh phan tfch thffdng quy (< lOphiit). Tham khao mgt so cong trinh nghien cffu [4,5,6,7] de rffa giai dffdc tot eac chat nay thffdng dung pha dgng la cac muoi vo cd nhff: kali dihydrophosphat, natri heptan sulphonie... ma theo xu hffdng nghien cffu ngay nay eae muoi vo cd thffdng khong dffdc khuyen Bang 1. Ket qua khao sat tfnh phu hcfp he thonq
T a n hnaf- f^hTaf -H. nhii-l- fn — ^ ^ Plii^n f i ,
hdp dung djch chuan
khfch sff dung do hien tffdng dong muoi he thong sac k^ khi khdng rffa cot dung each. Vdi dieu ki$n sac ky vij^ khao sat co he pha dpng ddn gian khong sff dung dem muoi gay an mon he thong, phu hdp cfe trien khai dinh Iffcfng • thffdng quy.
3.2. Tham dinh phu'dng phap
3.2.1. Tinh iddng tJiidie^a he tJi&ng sac kj \ Tfnh tffdng thfch cua he tJiong sac ky dffdc xac dinh bang each tien hanh tieni lap Iai 6 I3n dung djch chuan co nong dp paracetamol khoang | 500pg/ml, cafein khoang 25pg/ml v a | phenyleptirin khoang 5pg/ml. Ket qua duWc trinh '
bay trong bang 1. \ Ten hoat chat
Phenylephrin Paracetamol Cafein
tR phut (n=5) 2,571±1,18 5,398±0,20 5,422±0,19
Dien tich pic 212.276±1,23 36.626.667±0,63
7.765.821±0,70
Bo phan qiai 7,76 -
3,99
He so doi XL^nq 1,36 1,09 1,D8 3.2.2, Tinh dac hiiu
Tien hanh sac ky mau trang, pha dpng, dung djch chuan, dung djch thff va dung dich thff tliSm chuan. Ket qua cho thay dung dich thff eho cae pic khong trung vdi cac pie eua mlu trang va pha dpng; mau thff them chuan cd sff tang Ien ve chieu cao va di^n tfch dinh tai eac pic co thdi
gian lffu tffdng ffn^ vdi mau chuan. Kiem tra SLT tffdng dong ve pho UV-Vis eua hai pie chiian, thD"
CO thdi gian Iffu va pho UV-Vis ti/dng ffng vdi cac pic phenylephrin, paracetamol va cafein (Hinh 2)^ Kiem tra dp tinh khiet ciia ba pic chfnh trong mau thff deu tren 99%. Ket qua tren khang djnh quy trinh co tfnh dac hieu vdi 3 hoat chat.
Hinh 2. Tffdng dong"pho~UV giffa chuatTva thff ciia (1) phenylephrin, (2) paracetamol, (3) cafein.
tai thdi gian-Iffu tffdng ffng tren sac ky do.
Ti°»P CHi Y HOC VIET NAM TAP 450 • THANG 1 - SO 1 - 2017
3.2.3. Tinh tuyen tinh
Khao sat sff phu thupe ciia dien tfch pie vao nong do cac chat can djnh Iffdng bang each pha mpt day dung djch ehuan phenylephrin, paracetamol va cafein. Ti€n hanh sac ky theo dieu kien sac k^ da Iffa chpn. Ket qua khao sat khoang tuyen tfnh dffdc trinh bay trong bang 2.
Bang 2. K^ qua khao sat khoang tuyen tfnh cua phenylephrine para(xtamof va cafefn Ten hoat chSit
Phenylephrin Paracetamol Cafein
NBng dd (pg/ml) 2-50 50 - 800 10 - 100
Phu'dng trinh hoi quy y =34.529x+ 36,047
y =71.063x+2.187 y =200,053x+2.751.194
i l l sff tu'tfng quan (r^) 0,9977 0,9996 0,9992 Nhan xet: Ket qua khao sat cho thay trong khoang nong dp khao sat, cac chat cd sff tffdng quan tuyen tfnh chat che giffa nong dp va dien tfch pic dap ffng vdi he s6 tffdng quan xap xT bang 1 nen co the ap dung cac phffdng trinh nay vao dinh Iffdng.
Ducmg cbuaii cafeia (-275U94
Hinh 3. Bu'dng bieu dien tuyen ffnh cua phenylephrine paracetamol vi cafein 3.2.4. Dp diinh xac
Danh gia dp chfnh xac cua phffdng phSp du^ vao kha sat do lap lai (dd chfnh xac trong ngay) va do chfnh xac trung gian (d6 chfnh xac liSn ngay) tren chS pham vien nen Panadol cam eOm. Ket qua khao sat dp chfnh xac the hien trong bang 3 va bang 4.
Bang 3. Ket qua khao sat dp l§p lai Ham iLTdngCmg)
Phenylephrin Paracetamol Cafein
1 4,927 489,01 24,676
2 5,031 502,41 24,676
3 5,025 510,32 24,676
4 5,03 495,33 24,676
5 4,934 498,17 24,676
6 5,145 486,83 24,676
TB 5,015 497,01 24,676
RSD{%) 1 1,58 1,75 1,59 Ket qua cho thay, do l§p Iai ham Iffdng ciia 3 boat chat trong 6 mau thff trong cung ngay va trong 3 ngay lien tiep co RSD < 2% nhff vay phffdng phap dat do chfnh xae.
Bang 4. Ket qua khlfo sat do chfnh xac trung gian tIam lu'tfng(mg)
Phenylephrin Paracetamol Cafein
1 5,016 497,015
24,506 2 5,052 499,245
24,562 3 5,229 505,482
24,945 TB 5,099 500,581
24,67
RSD (°/o) 1,82 0,71 0,79 Nhan xet: Vdi chffdng trinh sSc ky da chgn, phffdng phap djnh Iffdng ed dp lap lai cao vdi RSD deu nho hdn 2% (trong.cCing ngay va khac ngay).
3,2.5. Bg dUng
Dp dung ciia phffdng phap dffdc xac djnh bang phffdng phap them chuan, xac djnh gia tri phan
tram phuc hoi. Thffc hien d ba mffe nong dp khac nhau, moi nong dp dffdc thffe hien 3 Ian. Ket qua
du'dc trinh bay d bang 5.
VIETNAM MEDICAL JOURNAL N°1 - JANUARY - 2017 Bang 5. Ket qua khao sat dp dung ciia phffdng phap
Mu'c nong do
Phenylephrin Ty le phuc
hoi (%)
RSD (n=3) f % )
T y l e phuc I RSD(n=3)
hoi (%) I (%)
Cafein T y le phuc
hoi f % )
RSD (n=3)
(%)
100,08 101,43 1,48
100% 99,42 1,84 99,80 0,79
120% 3,97 1,54 102,00 0,19 99,02
0,57Trung binh
99,49 1,85 101,08 99,66 1,55
Nhin xdt: Jy le phuc hoi ciia mau thff them chuan d tiTng mffe them ehuan 8 0 % , 100%, 120%' eua ca'ba heatxhat phenylephrin, paracetamol va cafein deu dat trong khoang 98 - 1 0 2 % . RSD eua tffng mffc them, chuan d ea ba hoat chat deu dat, nho hdn 2 % . Vay quy trinh djnh Iffdng dong thdi paracetamol, cafein va phenylephrin dat do dung theo hffdng dan eiia ICH [3]
IV.
BAN LU^NNghien cffu cua Kotaiah nam 2013 [4] sff dung pha dgng ed he dem phosphat, he dem vo ed nay neu sff dung thffdng xuyen va khong rffa he thong sac k^ dung quy each se gay hien tffdng ddng muoi va an mon he thong, d§c biet la he thong bdm, dan den he thong sac lc/ ro n va lam ket qua sai lech. Dieu kien sac ky theo nghien eiru ciia Salshi Sawant [6] khong sff dung dem phosphat ma sff dung he pha dpng kha phffc tap bao gom hon hdp acid 1-oetan sulfonic dieu chinh den pH 2,8 bang acid orthophosphoric - acetonitril vdi chffdng trinh gradient phffc tap. Khi sff dung tac nhan tao cap ion la acid l-octan sulfonic acid se lam he thong ap suat cao hdn do acid l-octan sulfonic la chat CO do nhdt cao. Trong khi do, nghien effu cua ehiing toi sff dung he pha dgng ddn gian vdi dung moi thffdng quy trong phong thi nghiem.
Quy trinh djnh Iffdng dong thdi 3 hoat chat paracetamol, cafein va phenylephrin bang phffdng phap HPLC/DAD dffdc cong bo Ian dau tien. Nghien effg sff dung pha tinh la ept Cs va pha dpng la hon hdp ACN: MeOH: H2O (10:30:60). Day la cac dieu kien phan tfch sac ky long cd ban va co the de dang ap dung. Dieu kifn sac ky vffa khao sat tach dffdc dong thdi 3 hoat chat co ham Iffdng chenh lech nhau IQO lan (paracetamol 500 mg va phenylephrin 5 mg). Ket qua xSy dutig phffdng phap eho thay, cac chi tieu tham djnh dap iffig tieu chuan ciia ICH vdi thdi gian phan h'ch ngan (7 phiit) do do ed the sff dung quy trinh nay cho phan tfch thffdng quy nham kiem tra chat Iffdng dffdc pham chffa 3 hoat chat nay tren thj trffdng.
V. KET L U A N
Quy trinh djnh Iffdng dong thdi paracetamol, cafein va phenylephrin da dffdc xay dffng thanh
cong bang phffdng phap HPLC vdi dau do DAD,, sff dung pha dpng khong eo dung djch dem. Quy trinh cd tfnh chpn Ipc, do chfnh xac va dp dungvj eao CO the irng dung de djnh Iffdng dong thdi 3 thanh phan tren trong cac che pham tffdng tff tren thi trffdng.
TAI UEU THAIVI KHAO
1 . Hoi dong Du'dc dien ( 2 0 0 9 ) , Dudc dieh Wit Nam W, Nha xuat ban Y hoc. IT. 841-842.
2. British Pharmacopoeia (2013), p. 3128-3135, 4548-4552.
3. ICH (2005), Validation of analytical procedurd:^
Text and Methodology Q2(R1). ''-' 4. Kotaiah, P. and Kamarapu. SK, (2013),
"Method development and validation of RP-HPLC method for simultaneous estimation of dextromethorphan hydrobromide, phenylephrine hydrochloride and trlprolidine hydrochloride in bulk and combined tablets dosage fomis", Intemational:
Joumai of Pharmacy and Biological Sdenixsj
volume 3, Issue 3, pp.172-179. „ ^ 5. LoharV.R,Mane Aruna, Chavan jaylcar, 1 ^
Palled M.S, BhatA.R ( 2 0 1 1 ) , "Simultaneous Estimation ^of Paracetamol, Caffeine, Pseudoephedrine Hydrochloride, Dexbomethorpiian,g Hydrobromide and Loratadine In Tablet Dosage form by Fy^-HPLC", Asian Joumal of Re^ardi in Chemistry,fi
Vol 4, Issue 6, pp. 1141-1147. "^
6. Sakshi Sawant and Nitin Borkar (2014),
"Method development of a simultaneous determination of common cough and cold ingredients by high performance liquid chromatography (HLPC) in multi component cough and cold oral drug product"^ International joumal of pharmaceutical, chemical and biological sdences, 4(4), pp. 1029-1037.
7. Vijay Kumar Rekuiapally and Vinay U. Rao ( 2 0 1 5 ) , "A novel stability indicating RP-HPLC method development and validation for simultaneous estimation of phenylephrine, acetaminophen, guaifenesin and dextromethorphan in tablet dosage form", Der Pharmada Letire, 7 (7), pp. 329-339.
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