TAP CHI Y D U O t HQC CAN THg - SO 13-14/2018
10. T.N.H. Nguyen, T.L.T. Nguyen, T.TT. Luong, C M T . Nguyen, T.P.P. Nguyen (2016), Synthesis of gold nanorods with a longitudinal surface plasmon resonance peak of around 1250 nm. Adv. Nat. Sci. Nanosci. Nanotechnol. 7, pp. 015006 - 015015.
11. CN. Van, W.S. Chang, J.W. Chen, K.A. Tsai, W.Y. Tzeng, Y.C Lin, et al. (2015), Heteroepitaxial approach to explore charge dynamics across Au/BiVd interface for photoactivity enhcmcementj'Nano Energy. 15, pp. 625-633.
12. C. Nguyen Van, T.H. Do, J.-W. Chen, W.-Y. Tzeng, K.-A. Tsai, H. Song, et al. (2017), WO3 mesocrystal-assistedphotoelectrochemical activity ofBiVOj, NPG Asia Mater. 9, pp 357-362.
13. L.T. Quynh, CN. Van, Y. Bitla, J.W. Chen, T.H. Do, W.Y. Tzeng, et al. (2016)., Self- Assembled BiEe03-E-Fe2O3 Vertical Heteroepitaxy for Visible Light Photoelectiochemistry, Adv. Energy Mater. 6,1600686.
(Ngdynhdnbdi: 18/11/2017 - Ngdy duyjt ddng: 10/01/2018)
NGHIEN ClTU X A Y DlTNG QUY T R I N H D I N H LlTC^fNG D O N G T H 6 I PEPSIN, LIPASE, a-AMYLASE CO T R O N G DICH VI
BANG PHlTONG P H A P LC-MS/MS
Huynh Huynh Anh Thi, Lit Thien Phdc*, Nguyin Phan HaiSam Bd Chdu Minh Vinh Tho, Nguyin Trun^Kiin
Trudng Dgi hgc YDugc Cdn Tha
* Email:[email protected] TOM TAT
Dat van di: Phdn tich su thay ddi ndng do cdc enzym ddnh ddu sinh hgc nhu pepsin, lipase, a-amylase tiong dich vi trong bjnh l^ viem dg ddy Helicobacter pylori Id vdn di dang dugc quan tdm. Vijc xdc dinh hogt do cdc enzym ndy dd dugcthiic hijn bdng mgt sd phuang phdp so mdu, huynh quang, khoi phd Q-TOFchodem thdnh phdn enzym. Mtictieu nghiin cdu: Phdt trien vd thdm dfnh phuang phdp xdc dinh ddng thdi pepsin, lipase, a-amylase tiong dich vf ngudi bang phuang^phdp sdc ky long ghep noi vdi ddu dd khdi phd 3 ldn tu cm: vd ung dyng quy tiinh de xdc dinh ndng dg ciia cdc enzym ndy trong mdu dich vf cua bjnh nhdn tgi bjnh vijn trudng Bgi hoc Y Dugc Cdn Tha. Boi tugng vd phutmg phdp nghiin c^u; 130 mdu djch vidugc cung cdp bdi bjnh vijn Trudng Dgi hgc Y Dugc Cdn Tha. Mdu dich vi dugc tua protein bdng acetonitril Pepsin, lipase, a-amylase thdy phdn cdc ca chdt Tyr-Ala, tiibutyrin vd tinh bgt tgo thdnh tyrosin (TYR), acid butyric (BTR) vd maltose (MAL). Cdc sdn phdm thdy phdn ndy dugc xdc dfnh bdng phuang phdp LC-MSMS, sir dyng chi dg ion hda ducmg (positive) vd dm (negative) vd dugc tdch tien cgt pha ddo hydro-CIS, rira gidi vdi hdn hgp dung mdi nude vd acetonitiil. Kit qud. diiu kijn sac ky thich hgp di tdch tyr, btr vd mai la sii dung cgt synergi hydro rp 80a column (250 x 4.6mm; 4pm) vd hdn hgp ACN.; H2O (58:42). Su ion hda duang (positive) vdi chi dg gidm sdt da phdn ung (MRA^ sii dung di chgn lgc ion me vd mdnh con dgc hiju m/z 181.19-^90.9, m/z 342.10-'203.10, m/z 151.16 -*92.73 tuang dng tyr. mai vd ndi chudn paracetamol (para). Su ion hda dm (negative) vdi chi dg ghi nhdn chgn lgc ion (SIR) m/z 87 cho BTR Phirang phdp dugc thdm dinh dgt yeu cdu theo hudng ddn cua US-FDA vi tinh dgc hiju. dg lgp lgi (USD <15%), tinh tuyin tinh vai hi so tuang quan r^> 0.995. gidi hgn phdt hijn (0.1-100 ppb), ti Ij phuc hdi tiong khodng 90.57j 112.26%. Quy trinh dugc img dung phdn tich nong dg pepsin, lipase, a-amylase ti-ong 130 mdu
TAP CHf Y Dl/QfC HOC CAN THg - SO 13-14/2018
dich vi cua bjnh nhdn. Kit qud thu dugc tdt cd mdu nhiem helicobacler pylori co ndng dg pepsin, lipase cao hem nhiing mdu dich vj bjnh nhdn khdng nhiem helicobacterpylori Kit lugn- phucmg phdp LC-MSMS nhanh vd nhgy de dinh lugng ddng thdi pepsin, lipase, a-amylase trong dich vi ngudi dugc phdt trien vd thdm dinh thanh cong.
TH khda: Pepsin; lipase: a-amylase; djch vi ngudi, khdi pho 3 tii cue ABSTRACT
D E V E L O P M E N T O F LC-MS/MS M E T H O D FOR
SIMULTANEOUSDETERMINATION O F PEPSIN, LIPASE, a- A M Y L A S E IN H U M A N G A S T R I C JUICE
Huynh Huynh Anh Thi, Lu Thien Phuc, Nguyen Phan Hai Sam, Do Chau Minh Vinh Tho, Nguyen Trung Kien Can Tho University of Medicine and Pharmacy Background: Pepsin, lipase, cmd alpha-amylase are crucial digestive enzymes. Analysis of humcm gastric juice biomarker enzymes is important for understanding oral health and disease pathogenesis. However, simultemeous determination of these enzymes by spectrophotometric methods is difficult due to low repeatability. Objectives: development and validation of method for simultaneous determination of pepsin, lipase, cmd alpha-amylase in humcm gastric juice using Acquity LC H-Class tcmdem Xevo TQD mass spectrometer and its application to determine concentration of these bio-makers in gastric juice samples of patients at CTUMP hospital Materials and methods: 130 samples of humcm gastric juice were supplied from CTUMP hospital Firstly, the humcm gastric juice was precipitated protein by acetonitrile, then pepsin, lipase, cmd alpha- amylase in supernatant layer were hydrolyzed into tyrosine, butyric acid, cmd maltose by Tyr-Ala. tributyrin, and starch substrates, respectively. Finally, the hydrolytic products
•were determined by the validated LC-MS/MS, using positive cmd negative mode, separated on a hydro-CIS reversed phase column, cmd eluted with acetonitiile cmd water. Results: The suitable LC- MS/MS conditions for separation of tyrosine, butyric acid cmd maltose were obtained with Synergi Hydro RP 80A column (250 x 4.6mm: 4pm) and mixture of acetonitrile cmd water (58:42) as eluent. ESI ionizer was operated in positive cmd MUM mode using the transition of m/z 181.19~*90.9, m/z 342.10-* 203.10, m/z 151.16-^92.73 to quantify tyrosine, maltose and IS (paracetamol), respectively cmd in negative with selected ion recording mode using m/z 87 for butyric acid. The method was validated cmd shown high selectivity, good reproducibility (RSD <
15%) and linearity (1^> 0.995), low limit of detection (0.1- 50 ppb) and mecm recovery of accuracy in the range of 90.57- 112.26%. The assay was applied for emalyzing gastiic juice sample of 130 patients. The results revealed that all samples of Helicobacter pylori infectious patients had pepsin cmd lipase concentration higher than one in samples without Helicobacter pylori infection. Conclusion: A sensitive cmd rapid iC-AfS/Aff method for the simultaneous quantification of pepsin, lipase, and alpha- amylase in human gastiic juice was successfully developed and validated.
Keywords: Pepsin; lipase: a-amylase; humcm gastric juice, triple quadrupoles.
I. fi^T VAN DE
Su thay doi boat tinh ciia mot hoac m6t vai enzym trong ca the cung la dau hieu dang quan tam ve tinh trang sue khoe. dac bi$t, trong mpt so benh ly su thay doi hoat tinh enzym co y nghia chuan doan, theo doi benh. Tinh trang thay doi boat do cac enzym dich vi trong benh ly viem da day do Helicobacter pylori gay ra, mot trong cac chi tieu danh gia hieu qua dieu tri la theo doi hoat do cac enzym pepsin, lipase va amylase trong dich vi.
viec danh gia doi hoi phai sii dung nhiing phucmg phdp nhay va dac hieu d^ co tbS dua ra
TAP CHfY DlfpC HQC CAN THg - s 6 13-14/2018
k8t qua chinh xac nhat. Hien nay tren the gioi chua co quy trinh nko dinh lupng duac d6ng thai ba enzym tren trong dich vi ciing nhu chua co quy trinh nao dinh luong enzym dich vi bang phuong phap co dp nhay va. chinh xac cao. he thfing phan tich LC-MSMS ra doi la bu0c nhay vpt vk ky thuat phan tich, cho phep dinh lupng dupe nhiing ch4t voi dp nhay (fg/ml), do dung, dac hieu cao. Xuat phat tir tinh hinh thuc ti tren, chiing toi thuc Men ngliien cijru voi muc tieu: Phat trien va thdm dinh phuang phap xac dinh dong thai pepsin, lipase, a-amylase trong dich vi nguoi bang phuang phap s3c ky long ghep n6i voi ddu do kh6i pho 3 lan tu: cue va iing dung quy trinh de xac dinh n6ng dp ciia cac enzym nay trong mdu dich vi cua benh nhan tai benh vien truang Dai hpc Y Dupe Can Tho.
IL DOI TirgfNG VA PHU'ONG PHAP NGHIEN ClTU
2.1. Doi tvong nghien cuu: Chat chudn d6i chieu (san phdm thuy phdn): tyrosin (TYR), acid butyric (BTR), maltose (MAL) va npi chuan phenylalanin (PHE), paracetamol (PARA). Dich vi da loai san phdm thuy phan.Dich vi da loai enzym.
Mau chudn san phdm thuy phan: cac chudn TYR, MAL, BTR dd bi§t nong dO pha trong dich vj trang lo?i san phdm thiiy phan. Mdu thir: dich vi b?nh nhdn c6 va khdng co nhiem Helicobacter pylori.
2.2. Thiet bi nghign cihi: May sdc k^ long ACQUITY UPLC H-CLASS system Waters, Xevo TQD ghep detector khoi pho 3 ldn tijr cue.
2.3. Phirong phdp nghiin cihi: Cdc chdt dir kien ldm n0i chuEin (PHE, PARA) vd TYR, BTR va MAL dupe pha trong hon hpp ACN : nude (1:1, tt/tt) d§n n6ng dp khoang 500 ppb va bam true ti^p vao may khoi ph6 Xevo TQD de xac dinh manh pho, toi uu cac thdng so khoi ph6 nhdm ghi nhdn tin hieu t6t nhdt cho cac chalphdn tich. De khao sat dieu kien sac ky toi uu tach dong thai tyrosin, acid butyric, maltose va n^i chuan trong mau dich vi nguai sac ky pha dao dupe ap dung voi he dimg moi phdn cue bao gom ACN, MeOH, nuac c6 them hoac khong them chdt dieu chinh pH nhu acid formic, trietylamin (<0,1%). Sau khi tim dupe di£u kien kh6i ph6 va sdc ky tiiich hpp sS tiSn hanh tham dinh phuang phap tham khdo cac huong ddn cua AOAC [1], FDA [3].Quy trinh sau khi tham dinh dupe dp dung d6 dinh lupng n6ng dO enzym pepsin, lipase, a- amylase co trong 130 mau dich vi nguoi.
III. KET QUA NGHIEN ClTU
3.L K^t qua xSy dimg phirong phap phan tich: Ket qiid thuc nghidm cho thdy, paracetamol phii hop ban d£ sir dung Iam npi chudn do khong co sdn trong dich vi va dap img tot nhat v^ cuang dp tin hieu cung nhu thoi gian phan tich so v6i phenylalanin. Ket qua cac dieu kien khoi pho toi uu cho cac chat dupe trinh bay trong bang 3.1.
Bang3.1. Cac thong so kh6i ph6 t6i uu — ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ H Thdng so
Th^ mao quan (kV) Thong so
Che dp bat ion J
Manh dinh lirong * The cone (V)
N5ng lugrng bu6ng va dap Thoi gian cho ghi nhan tin higu Positive/negative
J
ES+TYR 4
MAL PARA 1 MRM f 90,96
25 25
203,10 50 22
92,73 30 22
Es- a 3 f i
BTR SIR 87,10 _
30 S
s
0,039 B
(
ES+ ES- •TAP CHf Y Dl/QC HQC CXN THg - SO 13-14/2018
3.1.1. Khao sat dieu kien sac 1^ tach dong Ihoi tyrosin, butyric, maltose va ndi chuan paracetamol
Dieu kien sdc ky thich hop su dung pha tmh la cot Synergi^'^ 4 pm Hydro - RP 80A (250 X 4,6 mm), pha dpng ACN:H20 (58:42) ch^y mdu kidu isocratic, thS tich tiem maulOpL, toe dp d6ng0,6 mL/phut, khong diSu nhiet cot.
L
I
Hinh 3.4. Sac ky d6 tyrosin, maltose, butyric vd npi chudn paracetamol ddieu kien khoi pho va sac kytfiiuu
3.1.2. Xay dmig quy trinh u mau va xu* ly mau
Dieu kien ii mau lua chpn a nhiet dp ca the ngu6i la 37 "C, pH la pH boat dong t6i mi doi voi 2 enzym hpase va a - amylase (pH = 6,8), rieng doi voi pepsin lua chon pH gdn pH toi im do ddc thii phuang phap khoi pho su dung chdt dieu chinh Id acid formic khong th^ dua v6 pH qua thdp (pH = 2,3). Quy trinh u mdu va xu ly mau dupe trinh bay a So do 3.1.
3.2. Ket qua tham dinh phuong phap phan tich 3.2.1. Tinh tirong thich h^ thong
H6n hop chdt chudn dupe pha trong dich vi a n6ng do 400 ppb, n6ng dp npi chudn la IOO ppb, sau do tiem lien ti^p 6 ldn, xac dinh gia tri trung binh, gid tri RSD% ciia thoi gian luu (tp), dien tich pic (Spic), va ti Ie dien tich pic timg chdt chia cho dien tich pic npi chudn (S/Sis) d6u co %RSD <2%. Qui trinh dat tinh tuang thich h?th6ng.
3.2.2. Tinh ddc hi£u, chgn loc
Thuc hign phdn tich tren mdu dimg m6i, mau pha dpng, mau trang gid l^p, mau tidng gia lap them chudn vd mau trdng gid lap them chuan lan 2 cho kSt qua cac pic chdt phan tich tdch hoan todn khoi cac pic khac. Sac ky do so sanh giiia mau trang va mdu trdng them chuan dupe trinh bay d hinh 3.2 vd hinh 3.3.
TAP CHI Y DirpC HQC CAN TH0 - s6 13-14/2018
500 nL Tyr-Ala
500 nL djch vi | | 500 tiL djch vj | « - Nhlet dp 37 °C
20 p h i t ,
500 |JL tinh bpt + TBR Nhi?t dp 37
°C 60 phat 1 mL dich Lay
X
LSy
1600 |iL ACN
LEcxo^y 15 giay Ly t t a 4800 vong/phiit x 10 Dich ly t t a xlO lan voi hon
hopACN-.H20(l:l)(l:l) Them IS
Lpc 0,22 |im
Sa d6 3.1. Quy trnih u mau va xu ly mau dich vi sau li
L
Hinh 3.2. Sic ky d5 mlu trSng va mSu trSng thdm chuan maltose (A) va chuin tyrosin (B)
TAP CHI Y DU'QfC HQC CAN THg - S6 13-14/2018
Hinh 3.3. Sac ky do mau trdng vd mdu ti&ig them chudn butyric (C) va chudn paracetamol (D) Hon niia, voi moi chdt phdn tich deu CO hai ion san phdm dui^ de dinh tinh va dinh lupng do do su CO mat cua TYR, MAL, BTR dupe chdc chdn them nho vao ty Ie cac ion. Theo yeu cdu cua cdc t6 chuc phan ti'ch tren thS gioi nhu AOAC [1], Chdu Au (EC 657^2002) [8], ty 1^ cua ion xac nhan chia cho ion dinh lupng thu dupe trgn mdu khoi^ dupe sai khac qua mpt muc cho phep nhdt dinh. Bdng 3.2 trinh bay ty le ion cua cac chdt phdn ti'ch trong nghien cuu nay.
Bang 3.2. Tl le ion xac nhan va dinh luong cua cac chdt phdn tich trong nghien ciiu Chat phaD tich
TYR MAL
Ion sac nhan 122,93 185,0
lon dinh lugng 90,96 203,10
Ti le ion (%) Thii 1 Chuin 64,6 64,3 27,0 1 21,9
Sai khac
(%)
0,47 23,29 Nhdn xet:ty le sai khdc cua ty le ion TYR vd MAL ldn luprt la 0,47% va 23,29%d^u nho ban quy dinh (<20% d6i voi TYR va <25% d6i voi MAL).
3.2.3. Dirong chuSn, tinh tuyen tfnh, LOD, LOQ
Mau trang gia lap pha dung dich chudn cdc chdt doi chieu vdi nong dp I; 5; 25;
100; 400; 700; 1400; 2000; 2500 va 3000 ng/mL. N6ng dp npi chudn giii c6 dinh la 100 ng/mL. KSt qua thuc nghiem khdo sat gidi han dinh lupng va xdy dung phuang trinh hoi quy khdo sat tinh tuySn tinh dupe trinh bay a Bang 3.3.
Bang 3.3. Gioi ban ^ n h lupng vd duong chudn Chat phan
tich TYR MAL BTR
Phuong trinh hoi quy y = 0,00838157*x -f 0,109349 y = 0,00178535»x + 0,210214 y-0,000I4634»x -0.00055596
R' 0,993558 0,983830 0,999134
LOQ (ng/mL)
0,5 0,5 100
LOD (ng/mL)
0,1 0,13 50
Viing dinh lupng (ng/mL) 5 - 3000 25 - 3000 100-3000 3.2.4. Do flung: Tien hanh phan tich 6 mau dich vi th6m chuto TYR, MAL va BTR.
TigLP CHf Y Dl/QC HQC CAN T H g - SO 13-14/2018
Mdu dich vi tru6c khi them chuan dupe phan tich dS xac dinh n6ng dp TYR va MAL sdn CO. Dp dung dupe danh gid bdng d6 thu h6i cua 3 chdt tren. Kfit qua cho thdy a 3 khodng n^ng dp thdp, trung binh va cao phuang phap co d0 diing cua 3 chdt TYR, BTR va MAL dao dpng tu 90,57% dSn 110,85% (trong khoang 85% d6n 115%).
3.2.5. D? chinh xac trong ngay va Ii£n ngdy: thuc hien quy trinh u mdu tren 6 mdu a n6ng dp enzym PS, LP, AML ldn luot la 50; 5; 5 (|ag/mL). Danh gia k8t qud tren RSD%
cua dien tich pic cac chat TYR, BTR, MAL chia cho dien tich pic IS. Ket qua cho thay do lap lai trong ngdy va lign ngay cua quy trinh u enzym pepsin, a - amylase tao cac san phdm thuy phdn TYR va MAL deu ndm trong khodng duoi 15%, RSD trong ngay thu 3 cua quy trinh u lipase cho BTR dao dpng cao (19,15%).
3.3. iTng dung qui trinh xac dinh pepsin, lipase, a - amylase co trong dfch vi benh nhan nhiSm va kh6ng nhiem Helicobacter pylori
Bang 3.4. Ket qua xdc dinh ndng d6 pepsin, lipase, a - amylase 6 2 nhom benh nhdn Nong dp enzym
(ppm) Pepsin Lipase Amylase
B6nh nhSn nhiSm HP (n=40) 18,65 + 2,38 14,47 + 8,08 18,66±1,12
Benh nhan khong nhiem HP (n=90)
13,2 + 2,4 6,76 + 2,89 41,6±1,91
P
>0,05 V. BAN L U A N
Dieu l d | n khSi pho: Kho khan xdy ra khi khdo sdt khoi ph6 2 Idn cua BTR, cho manh me [BTR-H]" qua buong va dap cac ion t ^ thanh cho tin hieu rat thdp va khong on (finh, khong the sur dung cho dmh lupng. Ly do la vi BTR co phan tur khoi nho, khi va dap de dang tao cac manh khong dac tnmg, tin hieu tao ra khong on dinh. Mpt giai phap cho van de nay la sir dung ch^ d6 bdt ion SIR, ch£ dp SIR chi bdt ion me vd khong cho va dap cr MS2 nM phii hpp de dinh lupng cac chdt co khoi lupng phan tu nho (do khoi lupi^ phdn tu nho it bi triing lap vcfi m6t chdt khac) vd va dap tao cdc mdnh con khdng 6n dinh. Chung toi quyet dinh lua chpn ch6 dp SIR d8 bdt ion [BTR-H]"a ES- voi cdc thong s6 bdt ion dd t6i uu hoa. Che dp bat ion nay ciingdam bao tinh dac hieu do phan tii khoi ciia BTR nho kho trung lap voi mOt cMt khdc trong dich vi a cung fieu kien chpn Ipc khoi.
Lira ch^n noi chuan sv dung: Hai chdt phenylalanin va paracetmol deu phii hap su dung lam n6i chudn, tuy nhien khi dp dung dS phdn tich trgn n^n mdu thuc la dich vi cua benh nhdn viem loet da day ta trang thi trong nhftng mau nay co sdn su hien dien ciia phenylalanin nSn su dung phenylalanin dS lam npi chudn dinh lupng Id khong phu hpp. Viec phenylalanin xudt hien trong mau dich vi co th6 gidi thich Id do thuc an con sot lai cua benh nhdn do ca dja benh viem loet da day ta trang \ i phenylalanin la acid amin cdn thiSt, ca the nguai kh6ng the tu tong hpp dupe. Do do qua viec khdo sat dieu kien khoi pho va ban chdt nen mlu chung toi quyet dinh lira chpn npi chuan sir dungla paraccetamol.
V. K E T L U A N
Phuong phap LC-MSMS nhanh vd nhay de dinh lupng d6ng thaipepsm, lipase, a-amylase trong dich vi nguai dupe phat triSn va thdm dinh dat yeu cdu quy trinh dinh lupng theo huong dan cua AOAC. Phuang phap dupe ung dung thuc xi dh xac dinh ndng d6 cac enzym nay co trong 130 m l u dich vi cua bSnh nhan. K8t qud cho thdy n6ng dO enzym pepsin, lipase tang a nhang nguoi co nhilm Helicobacter pylori.
TAP CHf Y Dl/QC HQC CXN THg - SO 13-14/2018 TAI L I E U T H A M K H A O
1. AOAC (2012). requirements for single laboratoiy validation of chemical methods.
2. Ebbo Schnaith (2009), "Determination of the pepsin activity in human gastric juice, using defined oligopeptides as substrates". Clinical Biochemistry Journal, 22 (2), pp. 91-98.
3. Food and Drug Administration, Centre for Drug Evaluation and Research (2001), Guidance for industry, Bioemalytical Method Validation.
4. Gomes, N., Gonfalves, C , Garcia (2011), "Optimization of a colorimetric assay for yeast lipase activity in complex systems". Analytical Methods, 3(4), pp. 1008-1013.
5. Hasan, F., Shah, A. A., & Hameed, A. (2009), "Methods for detection and characterization of lipases: a comprehensive review". Biotechnology advances, 27(6), pp. 782-798.
6. Rodrigo, Andreia, Daurin F., Louis J., Valderma H. (2003), "A fluorimeti-ic method for the determination ofpepsin activity", ^na/3'«"ca/5/ocftem«/rv, 316 (l),pp.l 1-14.
7. UUa-Maja Bailey,Chamindie P, Justin J., Cooper-White(2012), "Analysis of the extreme diversity of salivary alpha-amylase isoforms generated by physiological proteolysis using liquid chromatography-tandem mass spectrometry". Journal of Chromatography B, 911, pp. 26-29.
8. European Union (2002), Commission decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performcmce of cmalytical methods and the interpretation of results.
(Ngdy nhdn bdi: 19/11/2017 - Ngdy duyjt ddng: 09/01/2018)
N G H I E N C i r u T O N G H O P TAP CHAT LIEN QUAN N - B U T Y R Y L - N - { [ 2 ' - ( 1 H - T E T R A Z 6 L - 5 - Y L ) B I P H E N Y L - 4 - Y L ] M E T H Y L } -
L - V A H N CUA VALSARTAN
Trdn Quoc Phd'" Trmmg Trin Trang+ Nguyin Mgnh Quan^, Nguyen Biic Tuan , Do Chau Minh Vinh Thg
1. Cdng ty cd phdn Dugc Hdu Giang 2. Cdng ty co phdn Dugc phdm COu Long 3. Trudng Dgi hgc YDugc Cdn Tha 4. Truang Dgi hgc YDugc TP HCM
*Email: [email protected] TOM TAT
Bgt vin de: tgp B (N-butyryl-N-{[2'-(lH-tetrazol-5-yl)-biphenyl-4-yl]methyl}-L-valin), phdt sinh trong qud trinh tdng hgp valsartan, cd the dnh hudng den hiju qud dieu tij vd tinh em todn khi sudung valsartan. BP 2014 vd USP 39 yiu cdu phdi kiim tratgp chdt trong nguyen liju valsartcm cUng nhu trong thudc thdnh phdm tuang dng. Tuy nhiin, hijn nay ngudn tgp B chudn khd tdn kem vd chua cd cdng trinh nghien ciru vi tdng hgp tgp B dugc cdng bd. Muc tiiu nghiin ciiu: tdng hgp tgp B cua valsartan vd xdc dinh do tinh khiet bdng phucmg phdp HPLC. Boi tugng vd phuang phdp nghiin cuu: (S)-3-metl^l-2-((2'-(lH-tetiazol-5-yl)-biphenyl-4-yl methyl)- aminoj-butyric acid (DOP) dugc tdng hgp bdng cdch dun hdi luu valsartan trong HCl Them butyryl clorid, pyridin vd acetonitril vdo hdn dich DOP trong acetonitril a -5°C. Them methanol vd nude vdo hdn hgp a nhijt dg phdng. Chinh vi pH = 7,5 bdng Na2COs 10%, cd quay, them
