NGHigN COTU DjNH Ll/CfNG CIPROFLOXACIN TRONG HUY^TTl/dNG NGl/^J BANG P H I / O N G PHAP HPLC
LE THI LA. NGUYEN THI CHAM, TA MANHHl/NG, DOAN CAO SON Vi$n Kiem nghiem thudc Trung mmg
1. Dat van de
Ciprofloxacin (Ci^HjgFNjOj; KLPT: 331,36) la thudc khang sinh bdn tdng hgp nhom fiuoroquinolon, cd tdc dvng tdt vdi cdc vi khudn da khang lai khdng sinh thudc nhdm khdc (aminoglycosid, cephalosporin..,).
Ciprofloxacin hap thu tdt qua dudng tieu hda, ndng dp dinh trong mau khodng 2,4 pg/ml dat dupe sau khi udng lidu 500 mg tii 1 gid den 2 gid [1]. Dd tridn khai ddnh gia tuong duong sinh hoc cac che phdm chira hoat chat Ciprofloxacin san xuat va luu hanh trdn thi trudng Viet Nam, chiing tdi da tien hanh nghidn cuu dinh luong Ciprofloxacin trong huydt tuong ngudi bdng phuang phap sdc ky long hidu nang cao vdi detector PDA.
2. Thurc ngbif m 2.1. Nguyen vgt li^u
- Chdt chudn: Ciprofloxacin hydroclorid: Chuan DDVN, SKS: 0212029.02, ham luong: 93,47%
C, ^H, gFNjOj -HCl (tinh theo nguy dn tr^g).
Cefiuoxim Nafri (CEFU): chudn DDVN, SKS:
0212175.02,ham luang: 906,96 pgCigHigN^OgS/mg tinh theo nguyen trang. Cefiiroxim Natri dugc dimg Idm chudn noi trong phuong phap phdn tich.
- Dung moi, hoa chdt: Acetonitnl, Methanol, Acid phosphoric 85%, Dicloromethan dat tidu chuan tinh khidt dung cho phdn tich hodc tinh khidt sdc ky.
- Huyit tuang trdng: Khdng cd Ciprofloxacin (CIPRO) ciia Vi?n Huyet hgc vd Tmyen mau Trung uang.
2.2. Thiit bfvd dftng cfi phdn tich
- Thiet bi: May sac ky Idng hidu ndng cao Shimadzu LC20AT; can phan tich Sartorius CP224S, dp chinh xdc 0,1 mg; tii lanh sdu -35''C ± -5°C, may ly tdm lanh, mdy Igc nuac... Cdc thiet bi dugc quan ly vd hieu chudn theo cac quy dinh cua iSO/IEC 17025 vd GLR
- Dung c^: Binh dinh miic, pipet loai A, dng ly tdm.,.
2.3. Bdi tirgng, npi dung vdphuffngphdp nghiin cuu - Ddi tucfng nghien cuu: Cac mlu huyet tuong (HT) trdng khdng chura CIPRO va cdc mau HT tvr tao chua CIPRO dupe chudn bi bdng each hda tan chat chudn CIPRO vdo cac mlu HT trdng vai cdc ndng dp khac nhau tii 0,1 pg/ml den 10,0 ng/ml.
- NQI dung vd phuang phdp nghiin ciru: Dua vao cac ddc tinh ly hda ciia hoat chat can phdn tich, ddc diem ciia ddi tuong nghidn cuu vd tham khdo mpt sd tai h?u [3],[4],[5] chiing tdi khao sat l\ra chpn phuong phap ket hgp tiia protein bang acetonitnl vd chiet loai tap bdng dicloromethan de chidt tdch CIPRO khdi nen mdu HT sau do xdc dinh ndng dp CIPRO trong mdu bang phuang phdp sac ky long vai detector PDA. Dieu kien sac ky vd xu: ly mlu huyet tuong dugc trinh bdy d phdn dudi. Tidn hanh thdm dinh do ddc hi^u/chpn Ipc, gidi han dinh lugng dudi, dudng chudn va khoang myen tinh, do diing, do chinh xdc, dO dn dinh... ctta phuang phdp theo cac hudng ddn tham dinh phuang phdp dinh lugng thudc trong dich sinh hpc [2],[6].
2.3.1. Dieu kien sdc ky
- cot sdc ky: C18; 250 x 4,6 mm; 5 pm. Nhidt dg cpt40'>C.
- Pha ddng; Acetonitnl (MeCN) - Methanol - dung dich H3PO4 0,025 M pH 3,0 (ty 1? thich hpp),
-Tdcdp ddng: I,4ml/phut.
- Detector: PDA, 280 nm - Thd tich tidm: 50 ^il.
2.3.2. Xu ly mdu HT, chiet tdch CIPRO
Them 100 \il dung dich chudn ndi (ndng dp chinh xac khoang 50 |J.g/ml) vdo I ml mdu HT da rd ddng d nhiet dp phong. Them 3 ml MeCN, lac, ly tdm, ldy Idp dich them dicloromethan, Idc, ly tam, hiit lop dung djch phia tren, tiem sdc ky.
T^p chi KIEM NGHI$M THUOC - S6 3.2014; T|p 12.(45)
2.3.3 Phuang phdp tinh ftc/ qud
Xdc dinh ndng dO CIPRO cd hong cdc mlu thit (chua biet nong do) dua vao fy le di^n tich pic CIPRO/CEFU thu dugc tir sdc ky dd ciia mlu thur vd duong chudn bidu thi mdi tuong quan giiia ndng dp CIPRO cd trong cdc mlu chudn vdi ty 1? di$n rich pic CIPRO/CEFU ciia mdu chudn.
3. Kk qua vd b ^ ludn
3.1. Dp 0c hi$u - chgn ipc cua phuang phdp Tien hdnh phdn rich cac mau HT trdng, mdu HT tu tao chua n^i chudn vd mdu HT tu tao chiia CIPRO nong dp khoang 0,1 pg/ml (ndng dp gicri hgn djnh lugng dudi) theo phuong phap dd xay dung vd ghi Igi sdc ky dd (Hinh 1,2 va 3).
D
.—i L
i
i 1
Hinhl. SM) mdu HT trang Htnh 2. SKD mdu HT trdng co chudn noi cefiiroxim
Wndnlll
\.
\ ' i . , i . II
Hinh 3. SKD mdu HT trdng co pha chudn CIPRO a nong dp LLOQ (0.1 fig/ml)
Tren SKD ciia mlu HT trdng (Hinh 1), tai cdc khodng thdi gian triing vdi thdd gian luu ciia CIPRO vd CEFU trong SKD ciia mdu HT trdng thdm chudn va chudn ndi khdng thdy xudt hien cdc pic tap. Do vay, phuang phdp phdn tich cd dp dac hi^u/chgn Ipc vdi CIPRO theo cac quy djnh ciia phuang phap phdn rich thudc trong dich sinh hpc.
T?p chi K I £ M N G H I E M THUOC - S6 3.2014; Tfip 12.(45)
3.2. Duang chudn vd khodng tuyin tinh Phdn tich cdc mlu HT chiia chudn CIPRO cd ndng dd khoang 0,1 pg/ml ddn 10,0 pg/ml theo quy trinh da xdy dymg. Xdc dinh s^ tuong quan gifta ndng do CIPRO trong huydt tuong vd ti Id didn tich pic CIPRO/CEFU thu dupe ti-en SKD bdng phuong phdp
hdi qui tuyen tinh, su dung he sd ty trpng l/(n6ng dd)^. Tir phuong trinh hdi qui tuyen tinh va ndng dp ly thuydt ciia dudng chudn xdc dinh gid tri dp diing.
Tidn hanh hen 5 dudng chudn.
Ket qua xac dinh moi tuong quan nay dupe trinh bay 6 Bdng 1.
Bdng 1. Ket qud thdm dinh khodng tuyen tinh cua phuang phdp Budng chuan 1
Nong d$ (fig/nil) Ti If CIPRO/CEFU
D$ dUng (%) 0,1 0,025 100,3
0,2 0,050 101,5
0.5 0,115
95,7 1,0 0,241 100,8
2,0 0,448
93,8 5,0 1,152 96,6
7,0 1,686 101,0
10,0 2,633 110,4 Phinmg trinh hoi qui y = 0,236 x + 0,001; r = 0,9983; Weighting: 1/x^
Birdfng chu&n 2 N^ng d$ (t^ml)
Ti 16 CIPRO/CEFU D$ dung (%)
0,1 0,026 100,8
0,2 0,050 100,6
0,5 1 1,0 1 2,0 0,115 j 0,238 1 0,445 95,6 1 99,9 1 93,7
5,0 1,149 97,3
7,0 1,682 101,8
10,0 2,600 110,2 Phinrag trinh hdi qui y = 0,233 x + 0,003; r = 0,9983; Weighting: 1/x^
Dudng chuan 3 Ndng dfi Oig/ml)
Ti If CIPRO/CEFU D$ dung (%)
0,1 0,024
99,4
0,2 1 0,5 0,049 101,4
0,119 98,3
1,0 0,252 104,6
2,0 1 5,0 0,469 1 1,164 97,5 1 96,8
7,0 1,634 97,1
10,0 2,520 104,9 Phuong trinh hdi qui y = 0,237 s +0,001; r = 0,9993; Weighting: 1/x*
Bit&ng chu&n 4 Nong d$(^g/nil)
Ti If CIPRO/CEFU DO diing (%)
0,1 0,027 103,5
0,2 0,049
96,1 0,5 0,112
90,3 1,0 0.256 104,1
2,0 1 5,0 0,485 1 1,203 99,0 1 98,4
7,0 1,765 103,2
10,0 2,581 105,6
^ Phinmg trinh hdi qui y = 0,241 x + 0,002; r = 0,9983; Weighting: 1/x^
Birdng chuan 5 Ndng d$ (»ig/nil)
Ti If CIPRO/CEFU Do dung (%)
0,1 0,027 103,4
0,2 0,049
96,4 0,5 0,112
90,2 1,0 0,255 103,9
2,0 0,485
99,0 5,0 1,200 98,2
7,0 1,762 103,1
10,0 2,582 105,8 Phuong trinh hdi qui y = 0,241 x + 0,002; r = 0,9983; Weighting: 1/x^
(y Id ti 14 dien tich pic CIPRO/CEFU; x Id nong do lig/ml Ciprofloxacin trong huyet tuang)
Kdt qua thdm dinh cho thdy trong khodng ndng do tir 0,1 pg/ml ddn 10 pg/ml cd svr tuong quan mydn tinh giiia ndng dO CIPRO cd trong mlu va ty le didn tich pic CIPRO/CEFU tren sdc ky dd tiiu dugc vdi he sd tuong quan xdp xi bdng 1. Ndng dp CIPRO xdc dinh tir dudng chudn so vdi gia tri ly thuyet deu dat
dp diing tir 90,2% den 110,4% va nam trong gidi han cho phep theo quy dinh ciia phuong phap phdn tich thudc trong dich sinh hgc.
3.3. Gidi hgn ^nh luang dudi cuaphuctngphdp Phdn rich cac mlu HT trdng va mlu HT chijra CIPRO ndng dp chinh xac khoang 0,1 |ig/ml (mlu Tap chi KIEM N G H I E M T H U 6 C - S6 3.2014; T^p 12.(45)
LLOQ). Xac dinh ndng dp CIPRO cd trong cac mlu trong cung dieu kien. Kdt qua tiidm dinh gidi ban djnh LLOQ tu cac dudng chudn tidn hanh Idm song song lupng dudi ciia phuang phdp dugc trinh bdy d Bdng 2.
Bdng 2. Kit qud xdc dinh gid tri LLOQ cda phuang phdp
STT
1 2 3 4 5 6 TB
lHSu trSng Difn tich pic
0 0 0 0 0 0 0
Mdu chuan (9,1 pg/mt) Difn tich pic CIPRO
10636 11630 11245 12501 11931 10592 11423
Difn tich pic IS 321322 318582 322012 320765 316606 311759 318508 CV(%)
Dip Irng trung binh cua man trSng/mau LLOQ
TilfCIPRO/IS 0,033 0,037 0,035 0,039 0,038 0,034 0.036 6 3
NSng ^ CIPRO em thiy (pg/ml)
0.101 0,110 0,106 0,117 0,113 0,104 0,109 5,5
B^dung(%) 100,8 109,8 105,6 116,4 113,0 103,1 108,1 5,5 0
Kdt qua thdm dinh cho thdy ty le giifa dien rich 3.4. Xdc ^nh do dung, m^pi^ cuaphuffngphdp pic CIPRO trong cac mlu LLOQ ddu Idn hon 5 Idn Tien hdnh thdm dinh do dimg, dp Idp lai tren 3 Id so vdi dien ti'ch pic t?p cd cung thdi gian luu trong mdu LQC, MQC, HQC chiia CIPRO cd ndng dp tirong cdc mlu trang. Ty le gifta ndng dd CIPRO xdc dinh ling la 0,3 pg/ml; 4,0 pg/ml; 8,0 pg/ml. Xac dinh ham tu dudng chudn so vdi ndng do ly thuyet cd trong cdc lugng CIPRO cd trong cac mau bdng dudng chudn mlu LLOQ ndm trong khodng 100,8% - 113,0%i va phdn rich trong cung didu ki?n. Dp diing ciia phuong gia tri CV(%) < 6%, ddp ling yeu cdu gidi han dinh phdp Id ty 1^ % giiia ndng do xdc dinh dugc so vdi ndng lugng duoi cua phuong phdp phan rich thudc trong dg ly thuydt. Do ISq) lai cua phuong p h ^ dugc bidu thi dich sinh hpc. bdng gia tri CV%. Kdt qud xac dmh dO dung, dp %) lai
cua phuang phap dugc trinh bdy d Bang 3.
Bdng 3. Kit qud thdm dinh dp dung, dg lgp Igi trong ngdy vd khdc ngdy
D$ dAng, d$ l$p lai
TVong ngiy (n=6) Kh^c ngay (n=3)
LQC (^,3 ftg/ml) Do dung (%)
94,1 95,5
CV(%) 3,7 5,6
MQCM.Ofig/Ml) Dpdung(%)
103,5 101,1
CV(%) 3,0 4,1
HQC^,0/igdnl) Do diing (%)
103,0 101,2
cviy^
3,1 5,7
Kdt qud thdm djiih cho thdy d cdc ndng do thdp, trung binh vd cao, phuong phdp cd dd diing trong ngay va khdc ngay trong khodng 94,1% -103,5%); dp lap lai tix)ng ngdy va khdc ngay vdi gid hi CV dao dpng tir 3,0%) - 5,7%, ddp ung yeu cdu ciia phuong phdp phan rich thudc trong dich sinh hpc.
3,5. Nghien cuu ^ on ^h cua hogt chdt trong hiQ/it tuang
Tidn hdnh nghidn ciiu dp dn dinh cua CIPRO trong HT ti-dn cdc Id mdu LQC va HQC. Danh gia dp dn dinh cua CIPRO trong HT bdng each so sanh ndng dp CIPRO co trong cdc mlu dugc bdo qudn vd cdc mlu cd ndng dg tuong img dugc phdn tich ngay sau khi hda tan chudn CIPRO vdo HT trdng. Kdt qud nghien cuu dd dn djnh CIPRO dugc trinh bdy trong Bang 4.
Tap chi KIEM NGHI$M THUOC - S6 3.2014; T§p 12.(45)
Bdng 4, Kit qud nghien cuu dQ on dfnh cua CIPRO trong HT
B^ on djnh
3 chu ki dOng - ra
D$ 6ii djnh dai ng&y (-35"C±-5»C,57ngJy)
DQ on djnh thoi gian ngSn (3 gid, nhiet d$ ph6ng)
e $ 6n dmh trong autosampier (20 giirM^C) Miu
LQC HQC LQC HQC LQC HQC LQC HQC
Nong dp ly thuyit TB±SD (Pg/ml; n=6) 0,276 ±0,010 8,480 i 0,381 0,276 ±0.010 8,480 ±0,381 0,276 ±0,010 8,480 ±0,381 0,304*0,023 7,884±0,412
Ndng d$ phSn tich TB±SD (pg/ml; n=6)
0,288 ± 0,007 7,808 ± 0.304 0,304 ±0,023 7,884*0,412 0,300 ±0,007 9,095 ± 0,096 0,295*0,011 7,647 ±0,197
%Sai khdc
4,5%
-7,9%
10,1%
-7,0%
8,9%
7,3%
-2,8%
-3,0%
4.Kltlu|n
Nghien cuu dd xdy dung dugc phuong phap djnh lugng Ciprofloxacin trong huydt tuong ngudi bdng phuong phap HPLC vdi detector PDA. Ket qua thdm dinh cho thay, phuong phap c6 gid trj gidi han dinh lugng dudi nhd (0,1 pg/ml); khodng tuydn tinh rgng (0,1 pg/ml ddn 10,0 pg/ml); dO diing cao
(94,1% - 103,5%); dp Idp l^i vdi gid tii CV% nhd (3,0% - 5,7%) ddp ung yeu cau doi vdi phuong phdp phdn rich thudc trong dich sinh hpc cua US-FDA.
Phuang phdp da xdy dipig cd thd umg dung trong cdc nghidn curu ddnh gid tuong duong sinh hpc d6i vdi chd phdm chlia ho^t chit Ciprofloxacin.
Tdi lif u tham khdo
1. Bp Y te (2009), Dui^c thu qudc gia Vi^t Nam, Idn xudt bdn thii 2, nha xudt ban Y hoc.
2. T? M?nh Hiing, Le Thj Thu Huygn, Trin Hodng, Nguyin Thi Thanh Thao, Phung Thj Vinh (2013), "Huong dan thdm dinh phirong phap sic k^ long hi|u ndng cao 6k phdn tich thuoe trong huyet tuong ngudi tinh nguyen tham gia cac nghiSn ciiu tirorng duong sinh hpc", Tgp chi kiim nghi$m thudc, tap 11, s6 40, trang 1 - 4.
3. T? Manh Hiing, Phiing Thi Vmh, Trinh Vdn Qui* (2004), "Xdy d\mg phirong phap dinh lupng norfloxacin trong huy^t tucmg bang HPLC", Tgp chi kiim nghiem thudc, s6 4/2004, trang 12-16.
4. Imre S, Dogaru MT, Van CE, Muntean T, Kelemen L (2003), "Validation of an HPLC method for the determination of ciprofloxacin in human plasma", JPharm BiomedAnal. 2003 Voi 33(1), pp. 125-30.
5. Maya MT, Gon9alves NJ, Silva NB, Morais JA (2001), "Simple high-performance liquid chromatographic assay for the determination of ciprofloxacin in human plasma with ultraviolet detection", J Chromatogr B BiomedSci Appl. Voi.
755(1-2), pp. 305-9.
6. U.S Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Reseach (2003): (juidance for IjuJustry - Bioanalytical Method Validation.
SUMMARY
A simple, specific and sensitive HPLC method has been developed for the determination of ciprofloxacin in human plasma. Separation of ciprofloxacin and cefuroxime sodium (internal standard) was achieved on a CIS column (5 pm;
250 x4-6 mm) using PDA detection with wavelength 280 nm. The mobile phase consists of acetonitrile. methanol andpH 3.0 phosphoric acid solution with suitable ratio. The assay was linear over the concentration range of 0.1-10.0 pg/mL The LLOQ was O.I pg/ml. The intra-day and inter-day accuracy were within 94.1% - 103.5%. This method can be used for BE studies of ciprofloxacin preparations.
T9P chi KIEM NGHIEM THU6c - S6 3.2014; T^p 12.(45)
