Nghien CCFU - Ky thuat
Dinh lirong calci atorvastatin va simvastatin
trong mot so che pham bang sac ky long hieu nang cao
Dang Thj Ngoc Lan*, Pham Le Minh, Vu Thi Quynh, Thai Nguyin Hung Thu
Trudng Dai hpc Duac Hd Npi 'E-mail:[email protected]
Summary
A reverse-phase HPLC pnDcedure was developed for analysis of statin derivatives (atorvastatin, simvastatin) as cholesterol lowering agents: Column - C,g (250 x4,6 mm, 5 pm); Detector -UV(238nm for simvastatin, and 246 nm for calcium atorvastatin); Mobile phase - acetonitrile-phosphate buffer pH 2.5 (70:30 v/v); Flow rate -1.2 ml/min; Injection volume -10 pi. The method was validated for the specificity (RSD of peak area < 0.20%; RSD oft^ < 0.25%); linearity (R^ = 0.9998), and accuracy (recovery: 98.40'% -100.91% for calcium atorvastatin and 98.94% - 100.37% for simvastatin). The method was applied to quantitative detenvination of the mentioned actives in some pharmaceutical preparations currently available in local market. For the tested samples, the content of calcium atorvastatin and simvastatin was 98.00%-99.39% and 98.05%-99.51%, respectively
Keywords: HPLC, calci atorvastatin, simvastatin.
Dat van de
O nudc ta, benh vtVa xa ddng mach vdi eae bilu hien lam sang nhu suy maeh vanh, ddt tip, nhdi mau co tim, nhdi mau nac... dang cd xu hudng tang nhanh theo nhjp dp phat trien eiia xa hdi. Nguyen nhan ehii ylu la do cac rdi loan lipid mau. Atorvastatin va simvastatin la nhu'ng thudc ha lipid mau duge sir dung nhieu nhit hien nay. Tuy nhien, treng cac tai lieu trong nudc va nudc ngoai ehiing toi tham khao duge ehua cd nhiiu phuang phap d i djnh lugng hai chit nay Vi vay chiing tdi tiin hanh nghien ciru nay nhim bd sung them mdt phuang phap cd the djnh lugng ealei atcrvastatin va simvastatin trong mdt sd c h i phim ed tren thj tapdng bang sic ki Idng hieu nang cao (HPLC).
Doi tu'O'ng va phu'ong phap nghien cu'u Hoa chit, thilt bj
Hoa chat
- Aeetonitril, methanol tinh khiit loal diing che HPLC (Merck).
Chit chuin: Simvastatin (99,14%) va atorvastatin calcium (98,77%) ciia Vien Kilm nghiem thudc TW.
- Natri dihydrophesphatdihydrat (NaH2P0^.2H20) (Merck).
-Acid ortho-phosphoric 85% (Merck).
- Nude cit 2 lin diing cho HPLC.
Thief bj, dung cu
- May sic k^^ Idng hieu nang cao Agilent 1100.
Cdt sac ky Apollo C18 5u ( 4,6 x 250 mm, 5 pm).
Can phan tieh Sartorius TE214S cd dp chinh xac ± 0,1 mg. May khuiy tip WiseStir MSH-20A (Han Qudc). May de pH510 Euteeh. May sieu am Ultrasonic LC 60 H. May 11 tam Harmonic Series.
May hut chan khdng. Mang loc 0,2 pm. Autopipet 100-1000 pi, 1000-5000 pi (Nhat).
Phu'O'ng phap nghien cu'u
Xay dgng mdt phuang phap cd t h i djnh lugng dugc ca ealei atorvastatin va simvastatin trong ehl phim bing phugng phap HPLC dugc tiin hanh qua cac budc:
- Lga chpn diiu kien sac ki thich hgp: cdt, pha ddng, tie dp ddng, the tich tiem mau, chuin ndi va quy trinh XLP ly miu.
- Danh gia chuang trinh s i c ki da xay dgng ve: dp thich hgp eua he thing sac ki, dp dac hieu, kheang tuyin tinh, dp lap lal va dp dung.
ling dung_ phuang phap da xay dgng d l djnh lugng cae c h i pham da chpn.
Doi tu>gng nghien ciru
Cac che pham co chu'a simvastatin 10 mg Vien nen Simvastatin 10 mg (STADA-VN), NSX: 02-07-2014, HSD: 10-07-2016.
- Vien nen bao film Winthrop (Sanofi Aventis), NSX: 09-05-2014, HSD: 09-05-2016.
Cac che phim co chira calci atorvastatin 10 mg - Vien nen Lipitad (STADA- Viet Nam), NSX:
03-05-2014, HSD: 07-05-2016.
- Vien nen Atostin (Cdng ty DP Trudng Tho), NSX: 17-02-2014, HSD: 17-02-2017.
Vien nen Hacortin (COHAVINA-XNDP150), NSX: 30-11-2014, HSD: 30-11-2017.
Ket qua va ban luan
Xay dgng phu'ong phap phan tich Quy trinh xii' ly mau
Can 10 vien, tinh khdi lugng tmng binh. Nghiin mjn thude thanh bdt, can lugng bdt tuong irng vdi 1,5 mg dugc chit cho vae binh djnh miPC 10 ml.
38 TAP CHI DlTOC '
Nghien CLFU - Ky thuat
them methanol tdi 2/3 binh, dem sieu am 5 phiit, de yen 15 phut, them methanol tdi vach. Ly tam 3000 vdng/phiit trong 10 phut. Hut djch dem Ipe qua rriang 0,2 pm, bd 2 - 3 ml djch lpc diu, djeh Ipc dem sac ki.
Quy frinh phan tich
Bieu kien sic ki: trien khai thir nghiem tren ca sd tham khap tai lieu ^ ^i, chpn duge diiu kien sae ki nhu sau:
- Cot C18 (250 mm x 4,6 mm, 5 pm).
- Buac sdng dinh lugng: simvastatin 238 nm, atorvastatin 246 nm.
- Pha ddng: aeetonitril: dem phosphat pH 2,5 (70:30).
- Tdc dp dong: 1,2 ml/phiit.
- Thi tieh tiem miu: 10 pi.
Tiem rieng biet dung djeh ehuan va dung djeh thip vao he thdng HPLC vdi cac diiu kien sic ki nhu tren. Ghi lal sac ki dd.
Tham djnh phu'O'ng phap
Danh gia finh chgn Igc cua phirong phap De khang djnh sg ed mat ciia ta duge va dung mdi pha mau khdng anh hudng tdi qua trinh djnh lugng cac dugc chat, chiing tdi tiin hanh phan tich cac miu:
- Miu tring: la methanol, lap lai 6 lin.
- Miu thir: ehl pham dugc xip li nhu muc Quy trinh xir ly miu de cd dugc ndng dp dugc chat nghien ciru khoang 0,15 mg/ml.
- Miu them chuin phu miu thip tren duge bd sung them chuan cdn lai.
- Miu chuin kep: Cd ea 2 ehit chuan d ndng dp khoang 0,15 mg/ml pha trong pha ddng.
SP sanh sac ki dd cua miu chuin kep vdi miu trang, mau thir va miu them chuin phu d tren d i danh gia tinh chpn Ipc.
Hinh 2: Sic ki dd eiia cac miu them chuin phu Miu thif calci atorvastatin them chuin simvastatin
(a) va miu thii'simvastatin them chuin calci atorvastatin (b)
Hinh 3: Sae ki dd mau ehuan kep Kit qua: Miu tring khdng cd tin bleu phan tich.
Cae mau thir diu eho met pie duy nhit tuong irng vdl dugc chit cd trong miu. Cac miu thir them chuin phu va miu ehuan kep diu eho cac pic tren sac ki dd tuang irng vdi calci atorvastatin (kheang 3,7 phut) va simvastatin (khoang 11,3 phiit).
Cac kit qua tren eho thiy phuong phap cd dp dae hieu d l xac dinh ealei atcrvastatin va simvastatin.
Khao sat finh tirong thich he thong Tiem mdi miu 6 lin, vdi ndng dp khpang 0,15 mg/ml. Tlln hanh sic ki vdi diiu kien da chpn.
Bang 1: Kit qua khao sat tinh tuang thieh he thing
Hinh 1: Sae ki dd cua mau trang (a), miu thir chua ealei aton/astatiri (b) va miu thir chira simvastatin (c)
Lan1 Lan 2 Lan 3 Lan 4 Lan 5 Lan 6 Trung binh
SD RSD (%)
Simsvastatin 0,15 mg/ml Thai gian
Im (phiit) 11,278 11,280 11,260 11,271 11,232 11,222 11,257 0,025
0,22
Dien tich pic ImAOs]
3866,1665 3875,4370 3870,7727 3873,4868 3867,4412 3877,3342 3871,7731 4,4382
0,11
Caici ator/astatin 0,15 mg/ml Thiri gian Dien tich pic luu (phut) (mAU.s)
3,678 2606,1321 3,680 2603,6951 3,678 2609.2781 3,681 2612,8919 3,681 2606,5449 3,677 2618,0386 3,679 2609,4301 0,002 5,2575 0,05 0,20
TAP CHI DLTOC HOC - 6/2016 (SO 482 NAM 56) 39
Nghien CLFU - Ky thuat
K i t qua che t h i y eae gia trj dp lech c h u i n t u o n g ddi thu d u g e d i u r i t nhd. RSD (%) cua dien tich pie khdng qua 0,20% va RSD (%) eiia thdi gian luu gitra cac l i n tiem khdng qua 0,25%.
Khio sat khoang tuyen finh giira nong dp va dien tich pic
Be xac djnh khoang t u y i n tinh, pha 1 day dung djeh c h u i n cd ndng dp n h u trong bang va cd them chuan ndi trong mdi m i u (ealei atorvastatin la chuan ndi ciia simvastatin va nguge lai), djnh mirc bang dung mdi pha ddng. T i i n hanh s i e ki n h u d i i u kien d muc quy trinh phan tich. K i t qua eho t h i y treng kheang ndng dp khao sat ed s g t u a n g quan t u y i n tinh g'liea dien tieh pie va ndng dp.
Bang 2: Kit qua khao sat khoang tuyin tinh eiia calci atorvastatin
Nong do (mg/ml) 0,05 0,07 0,12 0,15 0,28 Tyle dien tieh pie (mAU.s) 34,34 38,32 46,83 52,72 76,65 Phuong trinh duong ehuan S = 183,53.C + 25,18 vgi R^ = 0,9998
Bang 3: Kit qua khao sat khoang tuyin tinh ciia simvastatin
Nong do C (mg/ml) 0,05 0,10 0,15 0,22 0,25 Tyle dien tieh pic S(mAU.s) 39,90 49,82 59,74 72,21 77,55 Phuong trinh duong chuan S = 187,84.C 130,92 voi R' = 0,9993
Dp lap lai - Dp dung
Dd diing d u g c tinh bang each them met lugng c h i t ehuan xae djnh vac m i u thir, lugng chuan them vao bang 10%, 20%, 3 0 % lugng d u g e c h i t ed treng m i u thir. T i i n hanh s i e ki m i u thip khdng them chuan va cac m i u thir cd them c h u i n , moi m i u lam 4 l i n . Tinh ty le thu hdi tip chenh lech ndng dp d u g e c h i t xae djnh d u g e trcng cae m i u them chuan vdi ndng dp trung binh cd trcng m i u thip ban d i u va lugng chuan da them vac m i u . Dp diing ddi vdi calci atorvastatin d u g e t l l n hanh tren m i u Lipitad va ddi vdi simvastatin d u g e t l l n hanh tren m i u STADA. Ndng dp trung binh m i u thir Lipitad khdng them c h u i n la 0,1487 mg/ml va vdi m i u STADA la 0,1496 mg/ml. K i t qua d u g c trinh bay d bang 4.
Bang 4: Kit qua khao sat dd dung ddi vai calci atorvastatin va simvastatin
TT
1 2 3 4 5
Nong do ealei atorvastatin (mg/ml)
Them vao 0,015 0,015 0,015 0,015 0,030
Xac dinh duoe 0,1644 0,1645 0,1645 0,1644 0,1795
% thu hoi 98.40 99,25 99,27 98,59 99,63
Nong dd simvastatin (mg/ml)
Them vao 0,015 0,015 0,015 0,015 0,030
Xac dinh dugc 0,1635 0,1636 0,1637 0,1635 0,1786
% thu hoi 98.94 99,54 100,01
99,01 99,80
6 7 8 9 10 11 12
0,030 0,030 0,030 0,045 0,045 0,045 0,045
0,1798 0,1795 0,1795 0,1945 0,1947 0,1950 0,1949 Trung binh
RSDt
'%)
100,64 99,64 99,67 99,76 100,24 100.91 100,65 99,72 0,76
0,030 0,030 0,030 0,045 0,045 0,045 0,045
0,1786 0,1788 0,1786 0,1938 0,1933 0,1938 0,1937
99,74 100,33 99,73 100,27 99,10 100,37 100,05 99,74 0,49 Dp lap lai ciia p h u g n g phap d u g c danh gia qua k i t qua ham l u g n g ealei atorvastatin heac simvastatin trong mdt m i u e h l pham d u g c xae djnh qua 6 l i n dinh l u g n g khac nhau vdi 2 mau Lipitad va Simvastatin STADA. K i t qua d u g c trinh bay d bang 5.
Bang 5: K i t qua khao sat do lap lai ciia phuang phap ddi vdi ealei atorvastatin va simvastatin
Nliu Litipad _ . (ealei atorvastatin)
Luung bdt vien thir (mg) 1 44,8 2 45,1 3 44,8 4 45,3 5 44,0 6 45,5
Trung binh RSD(%)
Ham luong so vol nhan (%j
98,89 99,09 98,75 9941 98,20 99,00 98,89 0,41
Mau Simvastatin STADA (simvastatin) Luung bdt vien
thif (mq) 45,8 45,9 45,7 46,7 46,1 45,7
Ham luung so voinhan(%j
99,29 99,50 99,51 99,61 99,27 99,87 99,51 0,22 Ket qua thge nghiem che thay p h u a n g phap cd dp lap lal tdt qua 6 l i n phan tich ddc lap ( d i u cd gia tri RSD t h i p , 0,41 % vdl calci atorvastatin va 0,22%
vdi simvastatin). Ty le thu hdi eae: tip 98,40% d i n 100,91% vdi calci atorvastatin va tip 98,94% d i n 100,37% vdi simvastatin. N h u vay, p h u a n g phap phan tich xay d g n g da t h i hien dp lap lai, dp diing tdt, phil hgp vdi muc dich nghien ciru.
Ap dung p h y a n g phap de djnh lu'gng vai mgt so c h i pham
Ap dung p h u a n g phap vipa xay d g n g de djnh lugng calci atorvastatin va simvastatin treng mpt sd c h i p h i m mua tren thj t r u d n g . K i t qua thu d u g c n h u d bang 6 va cac e h l p h i m d i u cd ham lugng boat c h i t nam trong khoang 98,0% d i n 9 9 , 5 1 % so vdi nhan.
Bang 6: Kit qua dinh lugng mot sd ehi phim cd calci atorvastatin va simvastatin
40
Ham luong Che pham Hoat chat ghi tren nhan
(mg/vien)
Atostin 10 Hacortin . ''^^, 10
Lipitad 10 Ham lirong dinh luong (mg/vien)
9,80 9,84 9,89
Ham lirong so vainhan
(%)
98,00 99,39 98,89
TAP CHI DLTOC " < " " " z:/-.-.,^.
Nghien CLFU - Ky thuat
Winthrop
Simvastatin Simvastatin STADA
9,81 98,05 3,95 99,51
Ket luan
Bang ky thuat HPLC, ehiing tdi da xay dgng d u g c mdt quy trinh cd the dinh lugng hai hoat c h i t calci atorvastatin va simvastatin. Phuang phap phan tieh cho k i t qua dac hieu, diing va ehinh xac cao, cd the ap dung d i djnh lugng eae c h i pham chira
cac e h i t nay dang luu hanh tren thi trudng Viet Nam.
Tai lieu tham khao
1. Chu'cng Ngpe Nai, NguySn Ngoc Vinh, Nguyen Birc Tuan (2009), "Djnh lu'gng dong thai atorvastatin, lovastatln va simvastatin bing ky thuat s i c ky mixen dien dpng", Tap chi Dugc hoc, so 9 -2009, trang 45-50.
2. Lucie Novakova, Dalibor Satinsky, Petr Sollch (2008), "HPLC methods for the detemnination of simvastatin and atorvastatin", Trends in Analytical Chemistry, Vol. 27, No. 4, 2008, p. 352-367.
{Ngay nhan bai: 12/11/2015 - Ngay duyet dang: 02/06/2016)
T o n g h o p m o t s o . . . (TUP theo trang 33)
( I H , s, -NH-); 8,20 ( I H , s, -NH^); 8,04 ( I H , s, -NH^); 8,02 ( I H , s, -CH=N-); 7,83 (2H, d, J = 8,5 Hz, -CH=); 7,45 (2H, d, J = 8,5 Hz, -CH=). Phd MS (m/z) C3H3CIN3S [M-i-Hr d g k i l n : 214,0127; [M+H]*
tim t h i y : 214,0168; [M-H]- d g k i l n : 212,0127; [M- H]-tim t h i y : 212,0075.
2-(3-Nltrobenzyllden)hydrazln-
carbothloamld (21): Tinh the mau vang, hieu s u i t 78%, nhiet dp nong chay: 224-225 °C, R,= 0,78 (EtOAc:PE 3:1). Phd IR (KBr, c m ' ) : 3352 (N-H); 1618 (C=N); 1533 (=CH tham); 1508 (N-O);
1265 (C=S). Phd 'H-NMR (500 MHz, DMSO-d^, 5 ppm): 11,37 ( I H , s, -NH-); 8,46 ( I H , t, J = 1,5 Hz, -CH=); 8,21-8,25 ( I H , m, -CH=); 8,23 ( I H , s, -NHj); 8,14 ( I H , s, -CH=N-); 8,16-8,19 ( I H , m, -CH=); 8,05 ( I H , s, -NH^); 7,75 ( I H , t, J = 8,5 Hz, -CH=). Phd MS (m/z) CjHgN.O.S [M+H]* d g k i i n : 225,0211; [M-t-H]* tim t h i y : 225,0192; [M-H]- d u k i l n : 223,0211; [M-H]- tim t h i y : 223,0198.
K i t luan
Mdt sd dan c h i t semicarbazon va thiosemicarbazon t i i m nang d u g c tdng hgp thanh cdng b i n g phuong phap sir dung phan irng ngung tu giO'a aldehyd thom va semiearbazid heac thiosemicarbazid. Dieu kien tdi u u d u g c xac djnh n h u sau: ti le moi semiearbazid hydroclorid/
thiosemicartDazid:CH3COONa la 1:1; nhiet dp 80 °C (tdng hgp semicarbazon) va 100 °C (tdng hgp thiesemiearbazon). Hieu s u i t phan irng cao eiing vdi thdi gian phan irng ngan va each tinh e h l don gian la nhij-ng u u diem ndi trdi eiia phuang phap tdng hgp nay. Cac dan c h i t semicarbazon va thiosemicarbazon dang d u g e thir nghiem heat tinh khang khuan, khang n i m va khang ung thLP tren
mdt sd md hinh te bao. Ngoai ra, cac d i n c h i t nay eung d u g e sir dung lam nguyen lieu tong hgp met sd dan c h i t oxadiazol va thladiazel ed t i l m nang khang ung t h u se d u g e edng bd trong nghien ciru t i l p theo.
Tai lieu tham khao
1. Agganwal N., Mishra P. (2004), "Synthesis and evaluation of 4-substituted semicarbazone of levulinic acid for anticonvulsant activity", Joumal of Zhejiang University SCIENCE B, 6, 617-621.
2. Afraslabi Z., Shin E., Lin Wma Y, Campana C , Padhye S (2005), "Nickel (II) complexes of naphthaquinone thiosemicarbazone and semicarbazone: synthesis, structure, spectroscopy and biological activity", Joumal of Inorganic Biochemistry, 99, 1526-1531.
3. Vernna K., Pandeya S. N., Singh U. K., Gupta S., Prashant P., Anurag, Gautam B. (2009), "Synthesis and pharmacological activity of some substituted menthone semicarijazone and thiosemicarbazone derivatives"
Intemational Journal of Pharmaceutical Sciences and Nanotechnology, 1,357-362.
4. Sathisha M. R, Revankar V. K., Pai K. S .R. (2008),
"Synthesis, stmcture, electrochemistry, and spectral characterization of bis-lsatin-thiocarbohydrazone metal complexes and their antitumor activity against ehriich ascites carcinoma in Swiss albino mice", tVlet-Based Drugs, 1, 1-11.
5. Cerchiaro G., Fen-eira A. M. (2006), "Oxindoles and copper complexes with oxindole-derivatives as potential phannacological agents", Joumal Brazilian Chemical Society, 17, 1473-1485.
6. Negi Parul, Nandy Subhangkar, Mahato Arun (2012), "Antimicrobial activity of different thiosemicarbazone compounds against microbial pathogens", international Research Joumal of Pharmacy, 3 (5), ISSN 2230-8407.
{Ngay nhan bai: 30/11/2015 - Ngay duyet dang: 02/06/2016) TAP Cffl DirgfC HOC - 6/2016 (SO 482 NAM 56)
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