TAP CHi KHOA HOC - BAI HOC BONG NAI, SO 03 - 2016 ISSN 2354-1482_
P H A T T R I £ N PHU'OfNG P H A P SAC K Y L O N G S I E U H I E U N A N G D A U D O D I O D A R R A Y TU" N G O A J D E X A C D I N H C E F A L E X I N
T R O N G N i r 6 c T H A I
TkS. Nguyin Huy Hodi \ PGS. TS. Nguyin Van Hgp ' TS. Trdn Vinh Tkien TOMTAT
Phuffng phdp sac ky long nhanh siiu hiiu ndng (UPLC), su dung ddu do diod array tu ngoai (UV-PDA) da duac pkdt tiien vd ddnh gid dexdc dinh cefalexin tiong nuffc thdi. Qua trinh phdn tich duac thifC hiin tiin he thong ACQUITY UPLC vai cot Cortecs-C18, hi pha dong Methanol Acetonitiil kali dihydrogen orthophosphate 13.6 g/L - nuorc cdt (2:5:10:83). toe do ddng 0,3 ml/phut vai ddu dd UV-PDA. Ket qud ddnh gid cho thdy phuang phdp dat cdc yiu cdu ve tinh dqc hieu, do lap lqi, dp nhgy, tinh tuyen tinh vd dQ diing.
Tir kkoa: HPLC. UPLC, MS. FDA, Cefalexin
t T»-* >• j i Theo Duac Difin Anh (British
l . D a t v a n d e ^ Su Men di?n ngay cang nhieu
cua duoc ch4t (API) trong moi truang da tra thanh moi quan tam do kha nang gay tac dong sinh thai khong mong muon [I], la mot trong nhung nguy ca gay ra nhung benh nhu ung thu, roi loan sinh truang. Dac biet la cac chSt khang sinh ton tai trong moi truang kich thich su phat trien cua cac vi sinh v^t khang thuoc. Khi cac vi sinh vat khang thuoc lay nhilm sang nguoi lam tang nguy ca tu vong vi chung lam giam hieu luc ciia thuoc khang sinh (Antibiotics) [2].
Trong cac thuoc khang sinh, cefalexin la mgt trong nhung thuoc su dung kha pho bien, duac chi dmh trong dieu tri nhiem khuan duang ho hap, nhiem trung duong tieu, nhilm khuan da [3]... Hien nay, rat nhilu che pham chiia cefalexin da dugc Cue Quan ly Dugc - Bg Y t l Viet Nam c4p giay phep san xuat va luu hanh.
Pharmacopoeia 2015), Dugc Dien My (United States Pharmacopeia 36) tlu cefalexm dugc dinh lugng trong cac dang chl ph4m bSng phuang phap sSc ky long hieu nang cao (HPLC) dau dd tu ngoai kha kiln (UV-VIS) [4,5]. Tuy nhien, khi phan tich chat a nong do rat thap "vit" thi phuong phap HPLC/UV- VIS thucmg khong phii hgp do kem nhay. Hien nay voi su phat trien ky thuat sac ky long sieu hieu nang (UPLC) voi dau do UV-PDA, nhugc diem neu tren da dugc khSc phuc [6].
Vi vay muc dich ciia d l tai la cai tien va chuan hoa phuang phap UPLC/UV-PDA dua tren phuang phap HPLC sao cho hgi tu cac yeu t6: phan tich nhanh, it tieu ton dung moi, du nhay d l xac dinh vet cefalexin trong nu6c thai.
'Cong ty C6 phk PYMEPHARCO Phu Yen
^ Truong D?i hgc Khoa hgc Hue Trucmg Dai hpc Phii Yen
TAP CHl KHOA HOC - DAI HOC BONG NAI, SO 03 - 2016 ISSN 2354-1482
H N ^ v s .
Hinh 1. Cong thurc ciu tao cefalexm 2. V | t lifu va phuong ph^p 2.7. Vat lieu
MSU nudc thai dugc thu gom tu nha mdy san xuat cefalexin. Cac hoa chdt dugc sur dung bao gom: Kali dihydrogen orthophosphate (tinh kiet phan tich). Methanol HPLC (tinh khilt sSc ky), Acetonitril HPLC (tmh khilt sac ky), nudc cat 2 \hn. Chdt chudn:
Cefalexin c6 thong tm nhu a bang 1
B^ng 1. Thong tin chat chuan Chuan
Cefalexin
Nguon g6c
Vien Idem nghiem TP.H6
Chi Mmh
S6ld
QT006 130615
Ham luong
(%)
99,27
Ham am
6,8
Cac trang thiet hi dugc su dung gdm: He thing sac ky long sieu hieu nang ACQUITY UPLC-My voi d4u do 2.2. Pkirffng pkdp pkan tick Dilu kien s^c ky
Dua tren dilu kien sac ky do HPLC da dugc d l cap trong Dugc diln [4], nhom nghien cuu da thay doi thong s6 t6c do dong, cot, buac song de phii hgp vai he thing UPLC voi dilu kien sac ky nhu sau:
- Cot: Cortecs; C18 (2,7, 4,6 mm X 100 mm)
Detecter UV (PDA): buoc s6ng 264 nm
- Thi tich tiem mlu: 10 pi . T6c dp dong: 0,3 ml/phiit.
(detector) PDA UV-VIS, phan mem xii ly pho Empower 3
Pha dgng: Methanol Acetonitril kali dihydrogen orthophosphate 13,6 g/L - nuac cat (2:5:10:83), Igc qua mang lgc 0,45 pm va duoi khi.
Chuan bi
Day dung dich cefalexin chuan:
Hoa tan chat chuan cefalexin trong nuac cdt va pha loang vai nuac cat de dugc day dung dich chuan khoang 3 jig/L- den 60 pg/L.
Dung dich thu: Lay 50 ml nudc thai, ly tam d toe do 2000 vong/phiit trong 10 phlit, thu lay ldp dimg dich trong, lgc qua mang lgc 0,45 fun.
Tinh to^n ket qua
TAP CHt KHOA HOC - DAI HOC DONG NAI, S6 03 - ISSN 2354-1482 Tri6n khai sac ky do day dung
dich chudn, dung dich thir, xac dinh lugng cefalexin trong mlu nudc thai bang phuang phap dudng chuan dua tren dien tich dinh chinh tren sac ky do ciia dung dich thii va dudng tuyen tinh mo ta sy ph\i thuoc giiia di?n tich dinh/peak va ning dd cefalexin chuan.
2.3. Ddnk gid chdt lu^tg pkuang phdp [7]
Chdt lugng ciia phuang phap phan tich dugc danh gid bing tinh dac hieu, do lap lai, do diing (bdng each phan tich mau them chuan - spiked sample), tinh tuyen tinh, gidi han phat hien va gidi han dinh lugng. Cac s6 lieu thi nghifm dugc x\i ly bdng phdn m i m Excel 2010 d l xac djnh gia tri trung binh, do lech, thiet l^p dudng tuyin tinh, hf s6 tuong quan.
3. K i t qua v^ thao lu3n 3.1. Tink dqc hi$u
Tfnh dac hi?u ciia phuang phap phan tich dugc thuc hien bdng each frien khai sdc k^ d l bdng ddu do diod array tu ngoai 3 chilu (3D) ddng thdi 3 thdng sd; thdi gian luu dinh (RT), budc sdng tCr 200-400 nm d l danh gia s\r anh hudng dilu kien sdc kj' va dinh phu len chat cdn phan tich-cefalexin.
Dua tren sdc ky dd cua mau trdng, m l u thir (nudc thai) va chudn cefalexin d hinh 2. nhdn thdy; RT (thdi gian luu dinh) m l u trdng khong tning vdi RT thit (khoang 2,353 phut) va chudn (khoang 2,350 phlit), RT thir tuang duang vdi RT cefalexin chudn. Hon nua, khi phan tich pho UV cua m l u trang (hinh 3) khdng c6 dinh hap thu trung vdi cue dai hSp thu mau thir (hinh 4) va cue dai hap th\i mau thu phu hgp vdi cue d 0 hSp th\i chuan (264nm) (hinh 5). Nhu v§y, phuang phap phan tich dat yeu cau ve tinh dac hieu.
144
TAP CHl KHOA HQC - BAI HOC BONG NAI, S6 03 - 2016 ISSN 2354-1482
Hinh 2. Sac ky do mau nudc thai (a), chuan cefalexin (b), mdu trang (c)
" " I '" ISM I
m mo mi m m ma ma xia ma xm sua xsi MU ma
a) b) Hinh 3. Sac ky do 3 chieu (a) va pho UV (b) cua mau trdng
, " " 1 V
a) b) Hinh 4. Sic ky d6 3 chiSu (a) va ph6 UV (b) cua mlu thir
AI
c " I
a) b) Hinh 5. sac ky d6 3 chieu (a) va pho UV (b) cua chuan cefalexin
TAP CHI KHOA HQC - Dy»I HQC DONG NAI, S6 03 - 2016 ISSN 2354-1482 3.2. Do lap Iffi
De xac djnh do lap lai cua phuang phap phan tich, tien hanh phan tich lap lai 6 lan mlu thu - nudc thai roi tinh dp lech chudn tuang d l i (RSD) :
SOxlOO RSD(%) =
^ i b (1)
Trong do, SD la do lech chuan cua cdc ket qua phan tich xi, X2, ..xe (n
= 6); Xtb la tnmg binh sd hgc ciia cac kSt qua phan tich (n = 6).
Tiln hdnh phan tich lap lai 6 ldn mdu nudc thai co chua cefalexin, ket qud xdc dinh dd lap lai dugc neu d bang 3.
Bang 3. Ket qua xac dinh dp lap lai cua phuang phap phan tich Lan do
1 2 3 4 5 6 Xtb SD RSD (%) '/jRSDH (%)
Nong do cefalexin (tig/L) 5,2 6,1 5,5 5,0 5,3 5,8 5,5 0,4 7,0 17,5 Theo Horwitz [8], sai sd gjiia
cac phdng thi nghiem (danh gia qua do lech chuan tuang doi - RSD) khi phan tich nong do C neu dat dugc RSD nhd han R S D H (do lech chuan tuang ddi, tinh theo phuang trinh Horwitz (2) la chap nhan dugc.
RSD(%) = 2'""'"^^ (2) Trong do, C la ndng do chdt
phan tich dugc bieu diln bang phan sd;
Vdi C = 5,6 X 10^ -*
R S D H = 35 %
Khi xac dinh nong do C trong ngi bg mgt phong thi nghiem, thi RSD nhd han y2RSDH xac dinh theo phuang trinh (2) la chap nhan dugc. Nhu vay,
theo ket qua xdc dinh va tinh todn d bang 3 cho thay, phuang phap phan tich da dat dugc do lap lai theo yeu cau quoc tl.
3.3. Dd dUng
Do dung cua phuang phap phan tich dugc danh gia qua do thu h6i (Recovery/Rev), bang each phan tich mlu them chuan:
Rev(%) xlOO
(3) Trong do, Xo la ndng dp chdt phan tich trong mlu; Xi la ndng dg chdt chuan them vdo mau; Xi la ndng dg xac dinh dugc trong mau da them chudn.
Ket qua xac dinh dp dung cua phuang
TAP CHi KHOA HQC - BAI HQC DONG NAI, S6 03 - 2016 ISSN 2354-1482 phdp phan tich cefalexin dugc n8u d bang 4.
Bang 4. Kit qua xac dinh dp diing cua phuang phdp phan tich vit cefalexin
Lan do 1 2 3 Xtb
x„'-'
5,4 5,4 5,4
X, 5,0 5,0 5,0
X2 9,4 9,9 9,8
Rev (%) 79,8 89,8 87,2 85,6 Theo quy dinh sd 2002/657/EC
Clia Uy ban Chau Au (EU) [9], khi phan tich nhung nong dp trong mau cd I ppm - 10 ppm, neu dat dugc dp thu h l i tir 70 - 110% la dat yeu cdu. Nhu vay, cd the cho rang, phuang phdp phan tich dat dugc dp diing tdt khi phan tich cefalexin.
3.4. Tink tuyin tinh Pha cac dung djch cefalexin cd ndng dp Xi trong khoang 3 pg/L- din 60 pg/L. Do tin hieu dien tich dinh/peak bdng phan tich sdc ky thu dugc cdc gia tri yi tuong ling.
Bang 5. Dien tich dinh (peak) sac ky tuang ung vdi cdc nong dp cefalexin STT
I 2 3 4 5
Nong do cefalexin (^g/L ) 3,0 6,0 10,0 30,0 60,0
Dign tfch dinh (^Au.min) 10097 20551 29561 85990 172250 Xay dung phucmg ttinh hoi quy
tuygn tinh y = ax + b sir phu thuOc gifla dien tich dinh va nfing do cua cefalexin bang each xac dinh he s6 goc (a), hang s6 (b), he s6 tuoiig quan (R^), kft qua duac trinh bay a bang 5 va hinh 6. Ket
qua cho thay quy trinh phan tich dat dp tuyen tinh tot vai phuang trinh hoi quy y = 2876,5x co R^= 0,9992 > 0,99, dat yeu cau theo huang dan cua Hiep hoi cdc nha khoa hoc phan tich My (AOAC) [9]
147
TAP CHl KHOA HOC - BAI HOC B 6 N G NAI, SO 03 - 2016 ISSN 2354-1482 2 0 0 0 0 0 -
18000G - 16OO0O - 1 4 0 0 0 0 -
^ 1 2 0 0 0 0 -
"ra lOOOOO -
^ 8 0 0 0 0 - c 6 0 0 0 0 - Cl 4 0 0 0 0 - 20O0O - 0 . 0
y = 2S76.5X
R^ = 0 . 9 9 9 2 ^ ^
2 0 . 0 4 0 . 0 6 0 . 0 N o n g (Jo Cefalexin {[ig/L)
S C O
Hinh 6. Phuang trinh dudng chuan cua cefalexin 3.S. Gidi han phdt kiin
(LOQ) va gidi han dink lirffng (LOD) Dua tren dii liSu cua phep thu tinh tuyin tinh, xac dinh LOD va LOQ theo cong thuc:
L O D = i ^ ( 4 ) L O Q = ' 5 i l (5)
Trong do: s la s6 du dp lech chuan, a la he sd gdc cua dudng hoi quy tuyen tinh (y= ax + b) giiia nong do va dien tich dinh.
Vdi qua thu dugc: LOQcefeiexin = 1,3 pg/L, LOQcefalexm= 4,2 pg/L.
Bang 6. So sdnh cdc phuang phdp sac
Phuang phap la du nhay de kiem vet cefalexin trong nudc thai [10].
3.6. So sank giira pkirffng pkdp UPLC/UV'FDA vd HPLC~UV
Su kit hgp cua cot kich ttiudc nhd-rdng (Cortecs) vdi ky thuat sac ky ldng sieu hieu nang dau do PDA de phan tich cefalexm trong nudc thai cho thdy phuang phdp UPLC/UV-FDA cd nhieu uu diem nhu: dp nhay cao, giam thdi gian phan tich mau va co thi thu nhan dugc tin hieu da budc song..., Ket qua so sdnh dugc md ta d bang 6.
ky phan tich cefalexin trong nudc thai Thong so
Cpt sac ky Ap suat (psi)
Thai gian phan tich (phut) Lupng pha dpng tieu tdn (inl)/lan phan tich Tin hieu thu nhan LOD(ng/L)
HPLC-UV (*) Xterra;C18(5,0(im,4,6x
150 mm) 2000
5,6 500 sac ky d6 dan bucfc song
20
UPLC/UV-PDA Cortecs; C18 (2,7|im, 4,6x100 mm)
6000 2,3 100 sac ky do da buac s6ng
va ca ph6 UV-VIS 1,3
TAP CHi KHOA HOC - BAI HQC BONO NAI, SO 03 - 2016 ISSN 2354-1482
•Ghi chu: K^t qua nghien cim chi ' ' ' " S H dinh danh va dinh luang du tiet cua phuang phap phan Uch HPLC- ' " " " S cefalexm ttong nuac thai. Ch^t UV da dupc trinh bay ft nghiSn cuu '"""S ™^ P ^ ^ S P'''P,<'» ''•"'"^ ^ ^ khac [111 gia dat y6u cau qu6c te ve tinh dac hieu, 4. Ket luan ^^ ^^P ^^' ^ °hiiy, tttih tuyin tinh va Phuang phap UPLC dju do U V- * ' '•™8-
PDA da dupc phat tri8n va chuan h6a
TAI LIEU THAM KHAO
1. Irena Branawska (201U Using HPLC Method with DAD Detection for the Simultaneous Determination of 15 Drugs in Suiface Water and Wastewater,
Polish Joumal of Environmental Studies; Vol. 20 Issue 1, Pages 21
2. Jhon F. NARVAEZ V (2012), Pharmaceutical Products in the Environment: Sources, Effects And Risks, Vol 19, no.l
3. Dupc Thu Qu6c Gia (2012) 4. Dupc Dien My 36 (2014) 5. Dupc Dien Anh (2015)
6. Angela L. Batt (2008), Analysis of Ecologically Relevant Pharmaceuticals in Wastewater and Surface Water Using Selective Solid-Phase Extraction and UPLC-MS/MS, Analytical Chemistty 80 (13), pp 5021-5030
7. J.C. Miller and J.N. Miller (1988), Statistics for Analytical Chemistry, John Wiley & Sons, 2nd Edition
8. AOAC Guidelines for Single Laboratory Validation of Chemical Methods for Dietary Supplements and Botanicals (December 12, 2002)
9. Jared Anderson (2015), Analytical Separation Science, Wiley -VCH, December
10. Johan Bengtsson-Palme, Concentrations of antibiotics predicted to select for resistant bacteria: Proposed limits for environmental regulation. Environment
International, Volume 86, January 2016, Pages 140-149
11. Nguyin Huy Hoai (2012), Xdc dinh du lucmg khang sinh nhom Beta- lactam trong trong nhd mdy sdn xudt duac phdm (de tai ca so), Cong ty c6 phan PYMEPHARCO
149
TAP c m KHOA HOC - BAI HQC BONG NAI, S6 03 - 2016 ISSN 2354-1482_
DEVELOPIVIENT AND VALIDATION OF THE ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY METHOD WITH PDA DETECTOR FOR DETERMINATION OF CEFALEXIN IN WASTE WATER
ABSTRACT
A rapid Ultra Performance Liquid Chromatography method has been developed and validated for the determination of cefalexin in waste water.
Chromatography was performed by ACQUITY UPLC with Cortecs-C18 column using a mobile phase comprising methanol R, acetonitrile R, a 13.6 g/l solution of potassium dihydrogen phosphate R. water R (2:5:10:83 V/V/V/V). at a flow rate 0,3 ml/min and a UV-PDA detector. Method validation investigated parameters such as the speciftcity. sensitivity, linearity, precision, accuracy, giving results within the acceptable range.
Keywords: HPLC, UPLC, MS, FDA, Cefalexin
