WGHliN C&UKHOA HQC
DjNH Ll/dNG NGUYEN U£U AMIKACIN SULFAT
BANG PHi/dNG PHAP HPLC V 6 I DETECTOR DI£N H6A
1. Bat van dk
Amikacin sulfat (C22H43N50,3.2H2S04) Id mpt khdng sinh nhom aminoglycosid, duac dimg dilu tr|
nhiem khudn nang do nhftng chung nhay cdm trong nhilm khudn huylt (cd d tie so sinh), nhigm khudn ndng dudng hd hap, xuong, khdp, tilt ni$u, he thdn kinh trung uang (cd viem mang nao), da mo mim, nhilm khuan tiong 6 byng (viem phiic mac), nhilm khuan do bdng, nhilm triing h|u phau [1].
Hipn nay tiong cdc Dupe diln, cdc khang sinh thupc nhdm Aminoglycosid dang dupe chuyin tft dinh lupng bdng phuong phap vi sinh sang phuang phdp HPLC. Dupe diln Anh, Dupe diln Viet Nam IV dang dp dyng dinh lupng Amikacin sulfat bdng phuong phdp sdc ky Idng pha dao ddng cdt C18, boat chdt dupe tao ddn xudt trudc khi di vao cpt va sau dd dupe phdt hien bang detector UV. Day la phuang phap CO khdu chudn bi mau tuang doi phuc t?Lp, nhieu cdng doan vd dieu kien tien hanh phdn ftng nghiem ngdt.
Tft Dupe dien My 29, chuyen Iuan Amikacin sulfat da dp dyng dinh lupng bdng phuong phdp HPLC sft dyng detector difn h6a vdi uu diem phuong phap phat hi$n rdt chpn Ipc, co dp nhay va tinh dac hieu cao, kh6ng can tao dan xuat de phat hi§n ndn tiet kiSm thdi gian [3],[4].
Tren the gidi eung eo nhieu nghien ciiu ve phuang phdp HPLC vdi detector di$n h6a, tuy nhi8n d Viet Nam detector di8n hda van chua dupe ftng dyng nhi6u vi vay can phdi xem xet viee dp dyng detector di?n hoa cho phft hpp vdi dilu ki?n phong thf nghiem tiong nude. Vi ly do tien, chftng toi tien hdnh kilm tia su phu hpp cua phuang phap HPLC vdi detector dien hda dinh lupng nguySn lieu Amikacin sulfat, ding thdi gidi thifu phuong phdp dinh lupng Amikacin sulfat theo Dupe dien My su dyng detector dien hoa vdi quy trinh thuc nghiem don gian hem d6 ban dpc tham khdo vd dp dyng d ph6ng tiii nghiem hien tai.
2. Thirc nghif m
MAI THANH HA. LE THI THU. TRAN VIET HUNG Vien Kiem nghiem thudc Trung irffng 2.1. Thiet bf, dung cu, hoa chdt, chdt chuin 2.1.1. Thiet bi, dung cu
Cdc thilt bi, dyng cu ciia \^?n Kiem nghiem thuoe Trung uang (da dupe hieu chuan theo ISO/IEC 17025 vd GLP) gom:
- Mdy sac ky Idng hieu nang eao - Infinity 1260 (detector ECD);
- C6t L47 phenomenex: 250 x 4,6 mm, 5 (xm;
- Can phdn tich Mettler toledo MS 105: dp chinh xdc 0,01 mg;
- Mdy do pH Metiohm;
- Cac dyng cy thuy tinh can thiet cho qua trinh thyc nghiem.
2.1.2. Hoa chdt, dung moi, chdt chudn
Chdt chudn Amikacin sulfat USPRS Lot. K0I162: 0,987 mg Amikacin/mg (khan), ham lupng nude; 8,36% (xdc dinh bdng phuang phdp KF trudc khi sft dyng).
- Chdt chudn Kanamyein sulfat (chudn Asean), Lot:T185031 (812 lU/mg nguyen tiang)
- Natri hydroxyd logi tinh kMet phan tich.
- Nude loai dung cho sac ky.
2.2. Doi tu^ft^ vaphmmgphdp nghien cmt 2.2.1. Doi tuang nghien dm
Nguyen lieu Amikacin sulfat (Mexico), Lot. AMKS-40011104
2.2.2. Phuong phdp nghien cim 2.2.2.1. Dilu kien sdc ky [3],[4],[5]
Theo chuyen Man nguydn lieu Amikacm sulfat ciia USP. 36, chftng tdi dp dung dilu kifn sdc ky nhu sau:
- Pha dpng: Dung dich Natri hydroxyd 0,115N.
- Dung dich kiem tia su phii hpp ciia h$ thing:
0,02 mg Amikacin sulfat chudn vd 0,008 mg Kanamyein sulfat chuan/ml nude.
- Dung dich chudn: Hda tan 20,0 mg Amikacin sulfat chuan tiong nude, th6m ciing dung mdi vira dft T^p chi KIEM NGHIEM THUOC - So 3.2014; T^p 12.(45)
50,0 ml. Hut ehinh xdc 1,0 ml dung dich, them nude vua dli 20,0 ml (ning dp 0,02 mg/ml).
- Dung dich thft: Hda tan 20,0 mg nguyen li?u Amikacin sulfat tiong nude, them cung dung m6i vua du 50,0 ml. Hiit ehinh xdc 1,0 ml dung dich, them nude vua du 20,0 ml.
-Dieukien sac ky:
+ Cpt: L47 Phenomenex 250 x 4,6 mm, 5 jim.
+ Detector ECD vdi di$n eye 1dm viec Au vd dien eye so sdnh pH Ag-AgCl.
+ T6c dO d6ng: 0,5 ml/phiit.
+ ThI tich tigm: 20^1.
+ Chuang trinh di?n hoa:
E | = + 0,1V t , = 200 ms
£3 = +0,86V t2=I90ms
£3 =-0,74V t3= 190 ms
2.2.2.2. Danh gia sy phu hpp cua phuong phdp dinh lupng [2]
Ddnh gia vdi cac chi tieu: Dp dac hieu, tinh tMch
h p p cua hf tllUilg oav. ti-y, uu l u j ^ u u u u , uy uuug, UO ehinh xac, dp chinh xdc tiimg gian, khoang xae dinh.
3.KltquSvdb^nlu§n 3.1. Bp ^c hi^u
Chiing toi tien hanh tiem cdc mdu sau vao hS thing sic ky da lya chpn:
- Mdu tiing: nude.
- Dung dich chudn, thu, dung dich kilm tra su phfi hpp cua h$ thing sic ky: xem myc 2.2.2.1.
Kit qud phdn tich cho thiy:
- Tren sic k^ dl cCia dung dich thft cho pic co flioi gian luu tuong ling vdi thdi gian lini cua pic Amikacin sulfat thu dupe tii sac 1^ dl ciia dung dich chudn (t^ khodng 8,3 phiit).
- Tren sdc k^ do ciia mlu tring khdng xuat hien pie d tiong khodng thdi gian luu tuang ftng voi thoi gian luu cua pic Amikacin sulfat.
- Hai pic ciia Amikacin va tgp D (Kanamyein) tach rd rang vdi R = 4,4.
Ket qua dupe the hien d Hinh 1, 2,3 vd 4.
Hinh 1. Sac ky do cm mdu trang
"*"
.^
« 0 . .
«».-
A D C 1 A . A D C 1 CHANNEL A (AM lACI NTD\AM 1KACINTHOQ
L A
^r
7'fl N VH rm
Hinh 3. Sac Icy do ciia dung dich tiiir
s e o o o -
ADC1 A . A D C 1 CHANNEL A (AMIKACINnnAMDMCINTDW
—1
Hinh 2. Sac icy do ciia dung dich chudn
Hinh 4. Sdc Ic^ do dung dich kiim tra sifphit ht?p cua he. ',l|
thong sdc ity ft
T?p chi KlfeM NGHI$M THU6C - S6 3.2014; Tap 12.(45)
3.2. Tinh thich hpp cAa h$ thong sac k^
Tidn hdnh tiSm mlu trdng, dung dich chudn vd dung djch kiem tra su phu hpp ciia hp thong sac ky (xem myc 2.2.2.1) vao he thdng sdc ky, kit qua the hifndBdng 1,2.
Bdng 1. Tinh thich hop cua he thong sac ky
Bdng 2. Cdc thong so cua sdc ky
STT I 2 3 4 5 6 Trung binh
RSD (%)
ThMgian liru -1,^ (pititt) 8,293 8,303 8,287 8,315 8,330 8,353 8,313 0,31
Di^n tich pic (mAV.S) 80713,6 79977,1 79607,0 80881,0 80966,7 80848,0 80498,9 0,70
Thdng s6 Thdi gian luu tuang ddi (Rg) ciia Kanamyein
D9 phan giai (R) giiia Kanamyein va Amikacin
SSdIi.lythuyh(N„,,„„) H | s 6 k 6 o a u S i ( T , „ „ „ )
Yen cht 0,8
>3,0
>1500
<2,0 Kit qud
0,8 4,4 3400
1,1 Nhu vay, he thfing sdc ky cd dp Idp lai tot vdi RSD cua ttidi gian luu Id 0,31% vd diSn tich pic Id 0,70%
(< 1,0%), cac thong so sdc ky diu dat y6u clu (R>3,0;
N>150O; T<2,0).
3.3. Bp tuyin tinh, khodng xac ^h
Pha mdt day cac dung dich chuan tiong nude khft ion e6 ning dp Amikacin sulfet tft 1 0 - 3 0 ^ig/ml, ti6n hanh sdc ky theo dieu kipn da chpn. Ket qud thS hien d Bang 3 vaHinhS.
Bdng 3. Kit qud khdo sdt do tuyen tinh cua Amikacin sulfat Dung dich
Nong dp (pg/ml) Dien tich pic (mAU.s)
/
0,010 37433,7
2 0,016 59836,1
3 0,020 73611,3
4 0,024 89653,3
5 0,030 111407,0 Phinmg trinh hM quy: y = 3702594x + 336; H$ s6 tiromg quan r ^ 0,9999; % HS s6 chSn t^i nong a$ 20 fig/ml = 0,46%
Birfrng chuin Amikacin
Hinh 5. Do thi bieu dien moi tuong quan tuyen tinh giiia ndng dp vd dien tich pic Amikacin sulfat
Nhu vay, co sy tuong quan tuyen tinh chat che gifta nong dp va dien rich pic Amikacin sulfat tiong khoang nong do khdo sdt vdi h% si tuang quan r ~ 1,000 va % hp so chan tai ndng dp dinh lupng
= 0,46% (< 2,0%).
3.4. Dp dung
Ddnh gia dp diing cua phuong phdp bdng each them chudn. Them mpt lupng chinh xac chat chuan Amikacin sulfat vdo mdi mlu thft lln lupt khodng 2,4 mg, 4,8 mg vd 7,2 mg, tien hanh dinh lupng vd xdc dinh lupng chdt chudn tim lai dupe so vdi lupng them vao. Kit qua khao sat dO diing cfta phuang phdp dupe trinh bdy d Bdng 4.
Bdng 4. Kit qua ddnh gid dQ dung cua phuang phdp STT
1 2 3 4 5 6
LupitgcSnm^ dta (mgi 21,52 21,04 21,37 21,52 21,04 21,37
Litpr^dudnthem
2,42 2,42 2,42 4,84 4,84 4,84
Dipt tich pic (mAU.s) 77815,5 77435,7 77895,8 87392,6 8681S,1 86955,6
Lupng chuan
23,63 23,51 23,65 26,53 26,36 26,40
Tylf aiuhSi(%)
98,7 i 100,21 99,41 100,64 101,85 100,72
TnmgHaUt
99,40%
101,07%
T;p chi KIEM NGHIEM THUOC - S6 3.2014; T|p 12.(45)
7 8 9 TB
21,52 21,04 21,37
7,26 7,26 7,26
95562,1 94495,5 95516.8
28,69 29,00
101,38 101,29 100,56% ; n = 9; RSD = 0,98%
101,16%
-
Tft kit qud khdo sdt cho thay phuang phdp co t j lp ttiu hli cao tu 98,71% - 101,85%. Ty le thu hIi tiimg binh la 100,56%. Vdy phuong phap d£it yeu edu v l dO dung.
3.5. Dp chinh xdc 3.5.1. Dp lgp Igi
Tiln hdnh djnh lupng 6 mdu thft ddc lap theo dilu ki^n sic ky dd lya chpn. Xac dinh ham lupng nguySn lipu Amikacin sulfat theo chuan Amikacin sulfat
USPRS Lot.K0I163. Dp Idp lai cfta phucfng phap dupe ddnh gid dua tr8n dp Ipch chuan tuang dli cua kit qua dinh lupng 06 mdu thu (% RSD). Ket qua thi hi8ndBdng5.
3.5.2. Dp chinh xdc trung gian
Dp ehinh xae trung gian dupe danh gia dua fcrSn dp lech chudn tuong ddi ciia 12 mlu thu duac phan tich rieng bi?t tien cung thilt bj d 2 ngay khdc nhau vd 2 kilm nghiem vien khdc nhau. Kit qua dupe thi hien d Bdng 5.
Bdng 5. Ket qud ddnh gid dp chinh xdc cua phuang phdp Mitt thu
1 2 3 4 5 6 HLTB (%)
KNVl Lir^g cSn
('"g>
20,92 21,22 20,91 20,24 20,47 20,01
Di^n tich pic (mAU.s) 64413,4 66335,2 65366,1 62320,5 64131,0 61561,8
Hdim luffng
87,45 88,78 88,78 87,45 88,98 87,38 88,14%; n - 6; RSD = 0,89%
KNVl Lirfng c&n
20,90 21,32 20,51 20,80 21,82 21,80
Di^n tich pic (mAU.s) 60885,3 62455,1 59213,4 60484.3 63885.7 63605,1
f/am lirfmg
88,45 88,94 87,65 88,29 88,89 88,58 88,47%; 11-6; RSD - 0,53%
TB = 88^0% ; n = 12 ; RSD - 0,72%
Dupe dien My 36 da dupe kilm tra sy phu hop cua phuang phdp vl tinh ddc hi$u, dp chinh xac, d6 tuy^
tinh, khoang xdc dinh, va dp dung. Phuong phap.
da dupe ddnh gia hodn toan phu hpp dl djnh luong Amikacin sulfat tiong nguySn li8u.
Ket qua cho thdy, phuang phdp cd dp chinh xac cao, dp lap lai tiong ngdy la 0,53% vd 0,89% (vdi n = 6) va khdc ngdy Id 0,72% (vdi n = 12).
4. Kit Iu§n
Phuang phdp dinh lupng Amikacin sulfat theo Tai lieu tham khao 1. BoYtl(2OO2),Di«7'c//iM-0wo'cgiaf7e(iVo7W,NXBYhpc,HaNoi,tr. 130-132
2. Ta Manh Hung, Doan Cao Son (2012), "Hirdng din chung thim dinh quy trinh phan tich bing phuong phdp HPLC", Tgp chi Kiem nghiem thuoe, 10(4), Vc. 1-8.
3. The Bristish Pharmacopoeia (2012).
4. The United States Pharmacopoeia 36 (2013), 5. Electrochemical Detector user manual (Antec).
SUMMARY i A HPLC method with electrochemical detector referred jrom USP 36 was introduced to determine Amilatcin sulfc^
Amikacin sulfate is dissolved in water and diluted to have final concentration about 0.02 mg/ml, then determined!^,
HPLC with electrochemical detector j
6 Tap chi KIEM NGHlfM THU6C - Sh 3^014; Tdp 12.(45}
