lau mieng bang khan sach, mem (nhiing nudc da dun sdi de ngudi). Vdi tte ldn, cho siic mieng nude mudi (pha loang ed dp man nhu nudc mat). Nhd mit, nhd mui thude khing sinh.
- Cho an nhe, dii chat; cho udng nhieu nude (dung dieh oresol, nuac qui tuai) khi tte sdt eao, tieu chay. Vdi tte dang bii, tiep tuc cho bii me. Tre dang an bd sung, ngoai sua me can uu tien tre khiu phan du chat dinh duong nhit la nhiing thue phim giiu protid va earoten.
- Tre sdt eao lau khan am, cho udng paracetamol, thudc an than. Cho udng thudc giam ho.
- Khdng dirag thudc khang sinh cho tte bj sdi vdi muc dieh du phdng bien chiing vi de gay lo£in khuan va di ling. Chi dimg thudc khang sinh khi cd bang chiing bdi nhiSm vi khuan: hi viem tai gi&a, viem thanh - khi - phS quan, viem phdi... va ehi diing khi ed chi dinh ciia thay thudc.
- Bk de phdng khd mat do thieu vitamin A, ed thi cho tte udng vitamin A 100.000 don vi ttong hai ngay diu. Sau khi sdi bay cho udng them mdt liiu nhu the.
- Chii y theo ddi than nhiet hing ngay, nhit li khi sdi bay cd the xay ra bien chiing. Khi sdi bay ma tte vin sdt can phai nghi din biin chiing va dua tte di benh vien ngay.
4. Bien phip phdng b^nh
NguySn nhan chinh din den dich sdi quay ttd
l^i chinh la vi8c ttong thdi gian qua do xiy ra nhiiu su viec lien quan din vie xin nen nhiiu phu buynh khdng dua tte di tiem vie xin phdng benh sdi hoSc tiem ehua dii sd miii.
Tre sinh ra tir nhiing ngudi me da bi b$nh sdi trudc diy sg dupe miln dich thu dpng do me tmyin cho ttong vdng 6 - 9 thing.
De chil ddng phdng ttinh benh sdi, eieh tdt nhit li cho tre tiem phdng vac xin sdi diy du, diing lich; ti6m mui thii 1 khi tte dii 9 thing tudi va tiem mui thii 2 khi tte dupe 18 thing tudi d l dim bao dii liiu lupng, nong dp khing thi giiip tte mien djch tdt vdi loai virus sdi.
ViSc tiem miii thii 2 cd thi tao mien dich tdi 99%. Tre da dupe tiem phdng vin ed mdt ti le nhd bi mic bgnh nhung thudng d the nhe, it gay biin ehiing.
Vi-rut sdi chi tdn tai ngoii khdng khi 4 tiing ddng hd, nen dg phdng ttanh benh sdi ngudi din co thg lau nha, cac be mat tigp xiic bang cac dung dich sit khuan thdng thudng; nia cac dung eu hpc tap, dac biet la dd choi cua tte em bang nudc javen. Dgc biet, thuc hanh nia tay thudng xuygn bing xa phdng vk nude sach, trudc va sau khi di ve sinh, trudc va sau khi cham sdc con tt8 va ngudi benh, se giiip giim din 50%
nguy ca mie eie benh truyen nhiem ttong dd cd sdi.
Dung khan hoae tay ehe mieng khi ho, hit hai.
Ngoai ra, cic bac eha me can ting siic dfi khing cho tte bing each bd sung day du cic ehSt dinh dudng vi khi eo thi khde manh thi sieu vi se rit khd tan cdng.
NGHIEN CUV KHOA HQC
•J
NGHIEN Cl/UOINH Ll/pNG ATENOLOL TRONG HUY^T TUONG NGllPl B A N G PHUONG PHAP sAc KY LONG K H 6 | P H 6
1. Bat van d€
Atenolol {Ci^^-,2^p^.^ M = 266,3 g/mol) la din chit eua benzenaeetamid, dupe dung ttong dilu tri tang huylt ip. Thude cd tic dung lie ehl chpn Ipc ttgn thu die p,- adrenergic vi it bi chuyin hda lin diu qua gan [1]. Ndng dp dinh ttong huylt tuong khoang I pg/ml, dat dupe sau khi udng liiu don 100 mg atenolol [4], Hien nay, mdt sd doanh nghiep dupe ttong nudc da va dang nghien ciiu sin xuit cac ehl phim chiia atenolol nen nhu cau nghign ciru v l sinh khi dung va tuang
TA M4NHHUNG, PHAN THI NGHIA, TRJNH VAN LAU Vien Kiim nghiem thudc Trung irffng duong sinh hpc l i dilu cin thilt va xuit phat tii diuc te. Dvra tren dilu kien ttang thilt bi hien ed vi tham khio tai lipu [2],[5] chiing tdi da tiln hinh nghiSn eiiu xiy dung phuang phap sic ky Idng ghep ndi vdi detector khdi phd kilu tu cue chip ba (LC-MS/MS) co du dp nhay, die hieu, chinh xac, dinh lupng dupe ndng do atenolol ttong cie miu huylt tuong ngudi. Phuong phip phan tich sg dupe ip dung ttong cac nghien ciiu sinh kha dung vi tuong duong sinh hoc chl phim chiia atenolol.
Tap chi KIEM NGHIEM THU6C - So 2.2014; Tip 12.(44)
2. Thuc nghiem
2.1. Thiet bj, dung C(i, hda chdt vd chdt chudn 2.1.1. Thiel bi vd diing cu phan lich
Tat ca eie thiet bi v i dung cu phan tieh da dupe hieu ehuin theo qui dinh ciia ISO/IEC vi GLP. Tiln hinh phin tich tten may sic ky long khdi phd TSQ Quantum Vantage (Thermo Scientific).
2.1.2. Dungmoi, hda chdt vd chdl chudn - Chit chuin:
+Atenolol - Chuin Qudc gia, SKS: 0102093, him lupng 100,53%; dd im 0,11%.
+ Diltiazem hydroclorid - Chuan chinh, SKS:
WS 0108218, ham lupng 100,85%; dp im 0,30%.
dupe su dung lim chit npi chuan ttong phuong phip phan tich.
- Dung mdi, hda chit: Dat tieu chuan tinh khilt diing cho HPLC vi LC/MS.
- Huyet tuong tting khdng cd atenolol: Vien Huylt hpc va Truyen miu Trung uong cung cap.
2.2. Bdi tuang vd phuongphdp nghien ciru 2 2.1. Doi lirang nghien cuu
Miu huylt tuong (HT) tu tao chira atenolol (ATN) dupe ehuin bi bang cich hda tan chuan ATN
vio cac mau huyet tuong ttang vdi cic ndng dp khic nhau.
2.2.2. Phuongphdp nghien ciru
- Khao sat cae dieu kien khdi phd (kieu ion hda, dien the, nhiet dp ngudn ion hda, nang lupng phin manh ATN...); dilu kien sac ky long (cpt, pha dpng, tde dp ddng...) va phuong phap xir ly-chiet tieh boat chit cd ttong miu de dinh lupng ATN ttong cac mau HT.
- Tien hanh tham dinh dp die hieu-ehpn Ipe, gidi ban dinh lupng dudi, dp dung, dp chinh xac,... ciia phuong phip theo cac hudng dan tham dinh phuong phap dinh lupng thudc trong dich sinh hpc cua US-FDA viEMEA [31,[6].
3. Ket qua va ban luan
3.1. Xay difng phuang phdp phan tich
Sau qui trinh khao sit, xay dung dupe phuong phap LC/MS-MS dinh lupng ATN trong HT ngudi nhu sau:
- Diiu kien khoi pho:
+ Kieu phd khoi: MS/MS, ngudn ion hda ESI (+);
+ Cac thdng sd cua thiet bi khdi phd de phit hign ATN vi diltiazem (DIL) nhu sau:
Hoal chdl Thdng so ' -— ______
0i^n thi nguon phun (Spray voltage) Nhi?t do nguon phun (Spray temperature) Ap suat khi mang (Sheath gas pressure) Ap suat Idii bo tra (Auxilary gas pressure) Ap su3t khi quel ion (Ion sweep gas pressure) Nhi?t dp mao quan (Capillary temperature) Dien the bd ph^n hpi tu (Tube lens offset) Ion ban dau (Parent ion)
The phan manh ion (Collision energy) Ion tgo thanh (Product ion)
Atenolol 3200 V
50°C 20psi 5psi 1 psi seo-c 80 V m/z = 267,0
I7V m/z= 190,0
Diltiazem 3200V
SO^C 20 psi 5 psi 1 psi SfiO'-C 80 V m/z = 415,l
22 V m/z= 178,1 - Diiu kiin sdc ky:
-I-Cpt RP 18e: 50 X 2 mm; 1.8 pm. Nhiet dp cdt 40 "C.
+ Pha ddng: Aeetonittil - Amoni acetat 2 mM, ty le phu hpp. Tdc dO ddng 0,2 ml/phut.
+ The tich ti8m miu: 5 pi.
- Phuongphdp xu ly mdu: Mau HT de ra ddng d nhiet dp phdng. Liy 1 ml HT, them 50 ml dung dich IS sau dd chiet vdi 5 ml dung mdi eloroform. Lie, ly tim 3000 vdng/phiit x 5 phiit. Liy ldp dung mdi, cd dudi ddng khi nita din ein. Hda tan can ttong 1 ml pha ddng, tiem sac ky.
3.2. Thdm dinh phuong phdp phan tich 3.2.1. Do ddc hiiu — chon loc cua phuong phdp
Phan tieh cic man HT ttang va eae mau HT tu tao chiia chuan ATN vdi ndng dp khoang 5 ng/ml (bing khoang 1 /60 C^^ khi udng lieu don 50 mg ATN) ed chiia ndi chuan DIL theo phuang phap da dupe xiy dung.
Tren sac ky do man HT tring (Hinh !) tai cac thdi diem 1,4 va 1,8 phiit (ttiing vdi thdi gian luu cua ATN va DIL ttong miu chuin - Hinh 2), khdng xuat hien eie pic ed cic minh phd khdi m/z = 267 —* 190 (ATN) va m/z = 415-^178(DIL).
Tap chi KIEM NGHIEM THU6C - So 2.2014; Tap 12.(44)
Hinh 1. Sdc ky do mdu huyet tinmg trang Hinh 2. Sdc ky dd mdu huyit luong tu tao chua chudn ATN (5 ng/ml) vd ngi chudn (DIL) Chil giai hinh: Tr6n- S5c ky d6 t6ng ion (Total Ion Chromalograms - TIQ
Giiia' sac ky d6 chon Ipc ion ciia ATN (SRM, m/z: 267 ^ 190) Duoi: Sackyd6chonlgcioncuan9ichuinDlL(SRM, m/z, 415 -> 178)
Kit qua phan tich cung eho thiy, dip iing cua miu 3.2.2. Dudng chudn vd khodng tuyen tinh tting t?i thdi diim triing vdi thdi gian luu cua ATN xiln hanh pha eac miu HT chiia ehuan ATN cd ndng (1,4 phiit) nhd hon 2% dip iing ciia pic ATN d ndng dp khoang tir 5 ng/ml din 1000 ng/ml. Phan tieh theo dd 5 ng/ml va dap iing cua miu tting tai thdi dilm quy ttinh da xay dung. Xac dinh sir hrang quan giGa ttimg vai thdi gian luu cua DIL (1,8 phut) khdng vupt no"g ^d ATN (x) cd ttong miu v i ty le d i ^ tich pic , ° , ,- , . rMT r^ - u u- ATN/DIL (y) bang phuong phap hoi quy ttiyen ttnh, su qua 1% dip ung cua pie DIL. Do vay, phuang phap y' , , , i *^2^ TZ-* - i^. A;^h „iAi
^ f & f .J f D r r j ^ ^ g j^g g^ ^ trotig (1/nong do-^). Ket qua xae dinh moi die hieu va chpn loc ddi vdi ATN. ^^^ ^ ^ ^i^ ^^ ^^^^ ^ ^ ^ ^^^ ^^^^ Bing 1.
Bdng 1. Do dung cua cdc mdu thudc cdc duong chudn
Mau Si S2 S3 S4 S5 S6 S7 S8
Nong d^ (ng/ml) 5,2 10.4 20,7 51,8 103,6 310,7 621,4 1035,6 Phinmg trinh hoi quy
Do mng (%) Bir&ng chuan 1
99,4 97,6 104,9 105,2 101,2 101,7 98,9 91,1 a = 0,0022 b = 0,0008 r-0,9986
OKWJig chuan 2 i06,4
91,8 92,1 97,1 95,4 101,6 109,5 106,0 a = 0,0019 b = 0,0018 r = 0,9969
Dtrefng chuan 3 105,0
93,4 93,1 100,6 97,6 102,8 106,1 101,4 a-0,0020 b - 0 , 0 0 2 2 r - 0 , 9 9 8 4
Bir&ng chu&n 4 98,3 99,0 108,5 100,7 i00,6 101.7 99,3 92,0 a = 0,0022 b = 0,0023 r = 0,9986
Btcdng chuan 5 100,2 96,5 105,8 100,1 i00,7 103,9 100,8 92,0 a-0,0023 b-0,0025 r =0,9988
Tap chi KIEM NGHIEM THUOC - S6 2.2014; Tjp 12.(44)
Ket qui tham dinh cho thiy ttong khoing ndng dp tir 5 ng/ml den 1000 ng/ml cd su tuong quan tuyen tinh giiia ndng dp ATN v i ty le dien ticb pic giiia pic ciia ATN/DIL vdi be sd tuong quan xip xi bang 1.
Ndng dp ATN xae dinh tii dudng chuan so vdi gia tri ly thuyet deu dat xip xi 100% va nam ttong gidi ban cho phep theo qui dinh ciia phuang phap phan tich thudc ttong dich siiih hoe.
3.2.3. Giai- han dinh luong dual (LLOQ) cua phuong phdp Phin tich cie miu huylt tuong ttang va miu huylt tuong ehiia ATN ndng dp 5 ng/ml (mau LLOQ). Xic dinh dien ticb pic ATN va DIL ciia cac mau LLOQ vi xie dinh ndng dp ATN ed ttong cie miu tir dudng ehuan tien hanh lam song song ttong cung dieu kien.
Ket qui tham dinh gidi ban dinh lupng dudi cua phuong phip dupe trinh biy d Bing 2.
Bdng 2. Ket qud xdc dinh gid tri LLOQ Sa
1 2 3 4 5 6
Ty le dipt tich pic ATN/DIL 0,010 0,010 0,010 O.Oii 0,01i O.OiO
Nong dp xac djnh tit dtrc/ng chuan (ng/ml)
4,2 4,3 4,4 4,8 4,9 4,5 Trung binh (%)
CV(%)
Bp dung so v&l nong dpthttc(%)
95,0 83.3 84.2 92,3 8i,4 86,1 87,1 6,2
Gid tri S/N ciia pic ATN S/N > 10 S/N> 10 S/N > 10 S/N > 10 S/N>iO S/N > 10
Ket qua tham dinh cho thiy gia tri S/N ciia pic ATN ttong eie miu LLOQ (ndng dp 5 ng/ml) diu
> 10; ndng dp ATN xie dinh tii dudng chuin so vdi ndng dp thuc cd ttong miu nim ttong khoang tu 80 - 120% v i CV < 20% dap iing yeu ciu gidi ban dinh lupng dudi cua phuong phip phan tich thudc ttong dich sinh hpc.
3.2.4. Do dung, do lap lai cua phuong phdp Tien hanh thim dinh dp diing, dp lap lai tten 4 Id miu thir LLOQ, LQC, MQC va HQC chira ATN cd ndng dp tuong irng la 5; 15; 400 va 800 ng/ml. Xac dinh ndng dp ATN ed ttong man bang phuong phip dudng ehuin. Ket qua dp diing, dp lap lai ciia phuong phip dupe ttinh bay d Bang 3.
Bdng 3. Kel qud thdm dinh do dung, do lap lai trong ngay vd khac ngay
ffp dung, Bg lap lfi
Trong ngay (n = 6) Khac ngay
( n - 5 )
Mau LLOQ (5 ng/ml) Do dung
88,5
94,6
Dp lap lai (CV%)
6,2
11,8
Mdu LQC (15 ng/ml) Dg diing
94,4
94,8
Dp lap tgi (CV%)
4,1
7,5
MSu MQC (400 ng/ml) Do dimg
(%)
97,4
98,9
Dp lap lai (CV%)
5,0
5,5
Mau HQC (800 ng/ml) Dp dung
(%)
99,9
102,2 Do lap lai
(CV%)
2,3
6,3
Kit qua tham dinh cho thiy d cae khoang ndng dp thip; trung binh va cao, phuong phip cd dp diing ttong ngay, khic ngay xip xi 100%; dp lip lai ttong ngiy, khic ngay vdi gii tri CV < 15,0%; dap iing eie yeu ciu v& dp diing, dp lap lai cua phuong phap phin tich thudc ttong dich sinh hpc theo hudng din eua
US-FDA vaEMEA.
3.2.5. Do on dinh cua hogl chdt trong huyet tirong Ket qui nghien eiiu dp dn dinh eua ATN ttong huylt tuong d ea hai khoang ndng dp thap v i eao dupe trinh bay ttong Bang 4.
Tap chi KIEM NGHIEM THUOC - S6 2.2014; Tap 12.(44)
Bdng 4. Kit qud nghien cim do on dinh cua ATN trong huyit tuang
Dp on dinh
3 chu ky dong - ra dong {Three freeze-ihaw Cycles)
Do on dinh dai ngay (-40 "C, 52 ngay)
Do on dinh thai gian ngan (5 h; nhiet dg phong)
Do ba dinh trong autosampler (20''C;18h)
Lofi mau
LQC HQC LQC HQC LQC HQC LQC HQC
N&ng dp ly thuyet (ng/ml; n=5)
16,1 925,7 16,1 925,7 12,1 758,0 13,5 831,3
Nong 3^ trung bmh xic dinh' (ng/ml; n=5)
14,1 919,2 14,9 842,2 13,4 779,3
15,1 901,8
CV(%)
1,9 1,9 1,5 1,2 4,6 2,0 7,2 4,3
%
Saikhac -12,1 -0,7 -7,1 -9,0 10,8 2,8 11,3 8,5 (*): X a c dinh tir d u d n g c h u i n p h a n tieh ttong cimg dieu kien
4. K i t l u i n (5 ng/ml) x i p xi b i n g 1/60 gia tti C^^^, khoang tuyen N g h i e n c i u da x a y d u n g d u a c p h u o n g phap s i c « » ! ' ••*"8 «> ^ d i n 1000 n g / m l , v a thoi gian p h t o ticl ky long g h e p n6i v o l detector kh6i p h 6 kiSu t u c u e " g S " P . " P t " ' ^lio ™ . m a u ) , p h u o n g p h a p phan tich chap ba dinh l u o n g atenolol trong cac m l u huySt '^° "-6 ™ g d v n g t r o n g cac n g h t e n e u u sinh kha dtjng t u o n g n g u d i . V d i gia tri gidi han dinh l u p n g d u d i va t u o n g d u o n g sinh h p c c a c e h e p h i m ehiia atenolol.
T i i li^u t h a m k h i o
1. Bo Y t l (2009), Duac thu qudc gia Viet Nam, lan xuat ban thu 2, nha xuat ban Y hoc - Ha Ndi.
2. D. Sridharan, A. Thenmozhi, S. Sundaranandavalli (2010), "Bioanalytical method development and validation of atenolol in human plasma by LC-MS", Asian Journal of Pharmacettca! and Clinical Reseach, Volume 3, Issue 2, pp. 92-94
3. European Medicines Agency, Coramittee for Medicinal Products for Human Use (2011), Guideline on Validation of Bioanalytical Methods.
4. Fe-lin Lui Wu, Po-fen Chen (2003), Comparative pharmacokinetics of two atenolol products. Journal of Food and Drug Analysis, Vol.11, N o . l , 2003, page 4-7.
5. L. R, P. Abreu, S. A. C. Castro (2003), "Atenolol Quantification in Human Plasma by High-Performance Liquid Chromatography: Application to Bioquivalence Study", AAPS Pharmacetical Sciences, Vol 5(2), Article 21, 6. U.S Department of Health and Human Services, Food and D m g Administtation, Center for Dmg Evaluation and Reseach (2001), Guidance for Industry - Bioanalytical Method Validation.
SUMMARY
A high throughput and specific method using high performance liquid chromatography tandem mass spectrometry WAS developed for determination of atenolol in human plasma. The atenolol and internal standard were extracted from human plasma with chloroform. Analytes were chromatographedon a CIS column (50x2 0 mm. i d, 1.8 pm) with isocratic elution at a flow rate of 0.2 ml/min using 2 mM ammonium acetate buffer and acetonitrile with suitable ratio. The Thermo TSQ LC-MS/MS was operated under the selected reaction-monitoring mode using the electrospray ionization technique. The method was validated over the concentration range 5 lo 1000 tr^/.r! for atenolol. The intra-day and inter-day precision and accuracy were within 85% -115.0%. This method can be usi.;, or BA-BE studies of atenolol preparations
T a p chi K I E M N G H I E M T H U O C - S6 2.2014; T a p 12.(44)
