• Nghien ciru - ^
Ky thuat
Tong hop va xac dinh do tinh khiet captopril disulfid
Lfr Thien PhiicS Nguyen T3n DatS D6 Chau Minh VTnh Thg' Nguyen Due TuSn^*, Dang Van Tinh^
' Khoa Duac. TnrangDai hoc Y Dupe Cdn Tiia '-Khoa Duac. Dai hoc YDuac TP. H6 Chi Minh ' E-mail: [email protected]
Summary
Captopril disulfide, a closely related compound of captopril, was successfully synthesized by oxidation reaction of captopril with hydrogen peroxide. The process was optimized by the JMP 5.1 software. The synthesis achieved captopril disulfide in the purity above 99% in the yield of approximately 63%. Moreover, an HPLC method for quantitative determination of captopril disulfide was developed and validated according to International Conference on Harmonization (ICH) guidelines. The method showed desirable selectivity, wide linearity range, and high precision.
Keywords: Captopril disulfide, synthesis, hydrogen peroxide, quantitative determination.
EC131001, ham lupng 100,65% tinh tren c h i pham nguyen trang, han dung 29/09/2016.
Chat chuan doi chieu: Captopril do Vi^n K i i m nghiem thudc TP Ho Chi Minh cung d p , so kiem soat QT 146050614, ham lupng 99,09 % tinh tren c h i pham nguyen trang.
Hoa chit va dung moi. Acid hydroclone, methanol (Trung Qudc) va nude oxy gia 30%
(Viet Nam) dat tieu c h u i n tinh khiit hoa hpc.
Acetonitril (ACN) dat tieu chuan dung cho s i c ky Idng (Merck). Benzen, ethyl acetat (EtOAc), acid formic, cloroform, methanol (MeOH), n-butanol (Bu-OH) va acid phosphoric dat tieu ehuIn phan tieh (Merck) Ban mdng silica gel GF^gj (Merck),
Thiet bi
He t h i n g s i c ky long hieu nang cao (HPLC) Hitachi 2000 d i u do PDA. May quang pho IR Bruker alpha-T May LC-MS Agilent 1200 nguon ESI. May eong hudng tLF hat nhan Bruker Advance ll.
P h i n m i m JMP 5 1 cd chue nang thiet k i thi nghiem, tdi uu hoa theo md hinh thiit k i be mat dap ung Box-Behnken cCia SAS Campus.
PhLPO-ng phap nghien CCPU Tong hgp CPDS
CPDS dupe t i n g hpp l u captopnl vdi t^c nhan oxy hoa la nude oxy gia Phan ung tong hpp nhu sau:
Dat van de
Captopril disulfid (CPDS) la tap ehat lien quan cCia eaptoprii, phat sinh do phan ung oxy hoa giu'a captopril va oxy khdng khi '^'^i trong qua trinh san xult, b i o quan va luu thdng phan phoi nen gay cinh hudng khdng nhd d i n hieu qua dieu tri lam s^ng va dae tinh an toan cua captopril doi vdi benh nhSn ''•^•''1. Do dd, trong chuyen luan captopril cua Dupe d i i n Vipt Nam va Dupe dien mgt so nude nhu BP 2013. USP 36 d i u qui dinh phai k i i m tra gidi hgn ham lupng CPDS ca trong nguyen lieu v^ thanh pham c a p t o p n l ' " ' . Trong khi do. c h u i n tgp captopril disulfid dang dupe ban vdi gia r l t d I t v^ phai mua tiF nude ngoai nen gay khdng it khd khan eho cdng tae kiem nghiem tap c h i t nay trong nguyen lieu va thanh p h l m captopril. Cho d i n nay, chua cd nghien cuu nao trong nude cdng bd qui trinh tong hpp captopnl. T u nhu cau thyc t i tr«n, nghien euu tong hop CPDS ti> captopril va hydrogen peroxid ( H ^ ^ ) ^^ '^"^b'C thue hien, dong thdi vi^c xay dyng quy trinh xac djnh do tinh khiit cOa s i n p h l m tong hpp b i n g ky thuat HPLC cung dupe t i i n h^nh n h I m hudng d i n viec thiit lap chit d6i chiiu CPDS.
Nguyen li$u va phu'o'ng phap
Nguyen li^u
Nguy6n llfu captopril: Nha san x u l t Changzhou Pharma (Trung Quoc). So Id
T/fcPCHf n n n r HOC -10/2014 (SO 462 > A M 54)
• Nghien CIFU - Ky thuat
2 HSCH, C = 0 , C - 0 up _ X = ° 9 H , 0=<^
Captopril Thu' tinh khiet CPDS tong hgp San p h l m sau khi tong hop dupe t h i i tinh k h i i t b i n g s l e ky Idp mdng va do nhiet dp ndng ehay.
sic ky lap mong: Ban mong silica gel GF^^^
vdi 3 he dung mdi cd do phan eye khac nhau:
CgHg-EtOAc-HCOOH (30.70.10), CHClj-MeOH- HCOOH (85:15:10), B u O H - H p - H C O O H (40:60:10), Mau thu, 1 mg/ml MeOH.
Nhiet do nong chay: T i i n hanh do nhiet do ndng chay 3 l l n , l l y gia tri trung binh.
Xac djnh cau true
San pham tong hpp d u p e xac dinh c l u true b i n g cac phuang phap pho nghiem IR, MS va NMR.
Xac djnh do tinh khiet CPDS bang HPLC-PDA Dp tinh k h i i t eua CPDS dupe xac djnh b i n g phuang phap HPLC qui v i 100% dien tieh pic, D i i u kien s l e ky: cot Phenomenex Gemini NX C I S (150 X 4,6 mm, 5 pm), pha dpng A C N - nude:acid phosphoric pH 2 (30:70), t i c do ddng 1 ml/phut, dau dd PDA vdi b u d c sdng phat hien 214 nm, the tich tiem mau 10 pi, nhiet do cot SO^'C. Mau thLF cd nong do 500 pg/ml MeOH. M l u ehuIn captopril ed ndng do 500 pg/ml MeOH.
Kdt qua va banluan
Khao sat so" bp phan u'ng tong hp'p CPDS CPDS dupe tong hpp b i n g each hda tan khoang 5 g eaptoprii vao 100 ml nude c i t , sieu am 5 phut d 30''C, Igc qua g i l y loe, sau dd eho 5 ml dung dich H p ^ 30% vao dung dieh captopnl, khuay d i u , de yen 12 gid, euoi cung n i a tua vdi 20 ml nude lanh va s l y khd. San p h l m s l y khd dupe k i t tinh trong MeOH, thu dupe 1,54 g va cd nhiet dp ndng chay khoang 234°C '^i t u o n g d u o n g vdi nhiet dp ndng chay e(Ja CPDS K i t qua thi> tinh k h i i t b i n g sac ky Idp mdng vdi
Bang 1: Ma hoa cac yeu tS khao sat va bd tri thi
Captopril disulfid
3 he dung moi eho t h I y san pham tong hpp cd mpt v i t ehinh duy n h l t . Hieu s u i t phan ung tong h p p va tinh che khoang 3 1 % .
Khao sat cac yeu t d anh hyo'ng den hieu suat p h a n u'ng
Khao sat ty le H^OJcaptopril (ml/g) HjO/captopril da d u p e khao sat d cae ty le 0,4:1; 1:1; 2 : 1 ; 3 : 1 ; 4 : 1 ; 5 : 1 ; 6 : 1 ; 8:1; 10:1; 12:l";
14:1. Trong khi dd mot sd d i i u kien phan ung khae d u p e cd djnh nhLP: thdi gian phan u'ngt= 12 gid, dung mdi 100 mi nude e l t . K i t qua eho thay ty le the tieh H^O^ 30% va khdi lupng eaptoprii cd anh hudng den hieu s u i t phan ung va ty le 2:1 cho hieu s u i t phan ung eao nhat.
Khao sat pH phan O'ng
T i i n hanh khao sat d cac pH 0,5; 1; 1,2; 1,9;
2,2; 2,5; 3,0; 4,0; 5,0; 7,0; 8,0; 9,0; 10,5 vdi mpt s i d i i u kien phan ung khac d u p e co djnh nhu:
thdi gian phan ung t = 12 gid, ty le Hp^/captopril 2:1 (ml/g). K i t qua eho t h I y pH ed anh hudng den hieu s u i t phan ung va pH = 2,2 eho higu s u i t phan ung eao n h l t .
Khao sat th&i gian phan u'ng T i i n hanh khao sat d eae khoang thdi gian 3, 6, 9, 12, 24, 36 va 48 gid vdi mdt sd dieu kign phan ung khac diji'ac eo dinh n h u : ty le i^pJ captopril 2:1 (ml/g), pH = 2,2 K i t qua cho thIy thai gian phan iFng ed anh hudng d i n higu s u i t phan ung va thdi gian phan ung 9 gid cho higu s u i t phan ung cao n h l t .
Toi y u hoa qui trinh tong hp<p CPDS bing p h i n m i m JMP 5.1 vo'i mo hinh Box-Behnken Dua vao k i t qua khao sat eae y i u to I n h h u d n g d i n hieu s u i t phan LFng, t i i n hanh ma hoa eae yeu td khao sat va bo tri eac thi nghiem theo bang 1.
nghiem Yeu to
X, (ml/g) Ty le H^oycaptopnl Aftre duvri -1 X, (gid)' Thdi gian phan LFng
pH phan ung
T A P C H I D U O C H O C - 10/2014 (SO 462 NAM 54)
• Nghien cipu - Ky thuat
Ma tran thue nghiem: chpn md hinh Box- Behnken de thue hien t i l uu hda T i i n hanh eae thi nghiem nhu bang 1, mdi phan ung thuc hien 3 Ian, l l y ket qua trung binh, sau do nhap d u lieu
thu d u p e vao p h i n m i m JMP 5.1. Hinh 1 minh hoa m i i tuang tac giua cac y i u t i khao sat len hieu s u i t phan ung.
Hinh 1 : Tu^ng tac giwa cac yeu to khao sat len hieu suit phan ung Phuong trinh be mat dap ung vdi hieu s u i t
cfiFoe thiet lap nhu sau:
Y = 4 9 , 1 5 -I- 1,44X2 "^ 4,34X3 "^ 3.26X,X2 -r 2,19X^X3 - 6,22X,^ - 4 , 7 6 X / + 11.76X3^
Dl,]' doan d i i u kien phan ung t i i u u ' p h i n mem JMP 5.1 vdi mo hinh Box-Behnken eho k i t
qua d u doan hieu s u i t tdi uu cua qua trinh tong hap CPDS la 66,04% vdi d i i u kien X, = 2,13/1, X^ = 10,28; X3 = 3,4 De k i i m chung qui trinh tong hap CPDS toi u u , t i i n hanh thuc nghiem 3 l l n vdi d i i u kien d u doan. Bang 2 trinh bay hieu s u i t phan ung tong hpp CPDS.
Bang 2: Hieu suit phan ung tdng hap CPDS theo dieu kien du doan n Ty le H^O./captopril
1 2,13 (ml/g) 2 2,13 (ml/g) 3 2,13 (ml/g)
Trung binh
Th&i gian pH
lOgio 30 phut
Hieu suat 60,52%
65,05%
63,37%
62,98%
Khoi Iwang san pham (g) 3,03 3,35 3,17 3,18 Thu' tlnh khiet san pham tong hpp
San p h l m tong hpp d u p e do nhiet dp ndng chay 3 Ian, cho k i t qua 234 ± VC, tuong duong vdi nhiet dp ndng ehay cua CPDS. K i t qua
khai trien sac ky Idp mdng vdi 3 he dung moi cd do phan cue khac nhau cho thay san p h l m t i n g hpp chi cho mpt vet duy nhlt.
Xac dmh c l u true
Hinh 2: Pho IR (KBr) cua san pham tong hap Pho ESI-MS (-) cua san p h l m tong hpp cd m/z = 431,13 [M-H]-; M phu hpp vdi cong thue piiSn t u cua CPDS- CjgH^gNPgS^ Dua vao
Biing 3: DO' li$u phd cua san phim tong hap
Hinh 3: Phd MS cua san pham tong hap 6iy lieu pho cua bang 3 cho k i t luan san p h l m tong hgp chinh la captopnl disulfid (CPDS).
JR (KBr): v_.Jcm') . 1746,9 (HO-C=0)
'H-NMR (500 MHz. CDCI J: 8^ (ppm) '^C-NMR (125 MHz, CDCI J: S, (ppm) 4.22 (dd, IH, CH;pyrolidm) 58,31, 58,53 (C-2) 1601,4 (-N-C=0) 3,58 (t, J = 6,5 Hz; CH^, pyrolidm) 46,46, 46,02 (C-5)
H P C H f R I J O C H O C - 10/2014 (SO 462 NAM 54)
• Nghien ciru - Ky thuat
490 (-S-S-) 2,60 - 2.70 (brd; 2H: CH,) 2,87-3,00(brd;1H;CH) 1,81 - 2 , 1 8 (m; 2H; CH,; pyrolidin) 1,92 (m; J = 6,5 Hz; CH,; pyrolidin) 1,09(s;3H, J=6,5Hz;CH.)
41,21; 40,64 (C-3') 36,89 (C-2') 28,64; 30,85 (C-3) 24,34; 22,00 (C-4) 16,46 (CH,-C-2') 172,21; 172,69; 173,25; 173,68 (C=0)
Cong thu'c cau tap cua captopril disulfid Xac djnh do tinh khiet CPDS b i n g HPLC VO'I 6ku do PDA
Qui trinh xac dinh do tinh khiet CPDS b i n g HPLC-PDA da d i i g c tham dinh theo huang d i n
Bang 4: Kit qua khao sat tlnh phCi hap cua he thdng cua phLK/ng phap (n
ciia iCH w, bao gom khao sat tfnh phu h^p cOa he th6ng, tinh chon ioc, khoang tuyen tfnh, do chinh xac. Ket qua tham djnh du'p'c the hien o bang 4, hinh 4 va bang 5.
Gia tri thong ke Trung binh
RSD
Thm gian Iwu (phut) 3.575 0,10%
Dien tich pic (pVx giay) He sd doi xu-ng 46829226 1,1
1.07% 0.94%
So dia lir thuyet 30515 1.63%
Nhan xet: RSD eua eae thdng sd sac ky deu
< 2% va he sd ddi xung cua pie CPDS n l m trong
khoang 0,8 - 1,5. N h u vay qui trinh xac djnh dp tinh k h i i t CPDS dat tinh phu hpp eua he t h i n g .
Hinh 4 : Sic ky dd khao sat K i t qua khao sat tinh chpn lpc eho t h I y trong cung d i i u kien phan tich: s i c ky do m l u chuan captopril va m l u thiF CPDS x u l t hien pic chinh cd thai gian luu khae nhau hoan toan. S l e ky dd pha dpng va dung mdi pha mau khdng x u l t hien pie cd thdi gian luu tuong d u a n g vdi thai gian luu eua eaptoprii va CPDS trong s l e ky do m l u chuan va m l u thu. SLF dung ehuc nang kiem tra dp tinh k h i i t pic cho t h I y pic captopril va CPDS tinh k h i i t Vay qui trinh ed tfnh chgn Ipc.
Bang 5: Kit qua khao sat khoang tuyin tinh va dd chinh xac
Phwong trinh hoi qui Khoang tuyen tinh (ppm) He so tuveng quan (R) Do chinh xac (RSD, n=6) Do tinh khiet CPDS'
y = 73823 X + 7482096 200 - 800
0.9998 1.23%
99.45%
'Dupe xac dinh tir ket qua khao sat do chinh xac K i t qua thong ke cho t h I y qui trinh xac djnh dp tinh khiet CPDS ed khoang t u y i n tinh rpng
tinh chon Ioc cua phuang phap
vdi gia tri cua he sd t u a n g quan cao, dat yeu eau v i do ehinh xac. K i t qua xae dinh dp tinh khilt CPDS theo p h u a n g phap qui ve 100% dipn tieh pic la 99,45% tinh tren e h i pham nguyen trgng.
K§t tuan
Captopril disulfid da d u p e tong hpp thanh cdng tLF captopril b i n g phuang phap oxy hoa vdi H p ^ . <^?t ^0 tinh k h i i t tren 99% tinh tren che p h l m nguyen trang. Qui trinh tong hap da dupe tdi u u hda bang p h i n mem JMP 5.1 vdi md hinh Box-Behnken eho hieu s u i t eao n h l t khoang 63% (3,18 gam). San p h l m tong hpp du d i l u kien de t h i i t lap c h i t chuan doi chieu,
Tai lieu tham l<hao
1, Julia Aparecida L, Souza (2012), "Quantification of captopril disulfide as a degradation product in captopnl tablets using near infrared spectroscopy and chemometiics". Vibrational Spectroscopy, 62, pp. 35-41.
70 TAPCHI DUOC HOC - 10/2014 (SO 462 NAM 54)
