TP CH NGHIN CU Y HC

NH LNG ACID AMIN T DO TRONG DCH SINH HC

BNG PHNG PHP SC K LNG SIU HIU NNG
L Duy Cng1, Trn Th Chi Mai2
1
Bnh vin a khoa Quc t Vinmec, 2Trng i hc Y H Ni

Nghin cu ny nhm thm nh quy trnh k thut ci tin nh lng acid amin t do trong dch sinh
hc bng sc k lng siu hiu nng (UPLC). Cc acid amin trong mu th c dn xut bng AQC
(6 - aminoquinolyl - N - hydroxysuccinimidyl carbamate) chuyn thnh cc cht c th pht hin c bng
u d cc tm (UV), phn tch cc dn xut AQC ca acid amin bng phng php UPLC, nh lng bng
u d TUV. Gii hn nh lng ca phng php l 5 mol/L. Khong tuyn tnh ca phng php l t 5
n 1000 mol/L. chnh xc ngn hn CV 2,4%, chnh xc di hn CV 3%. Qui trnh k thut ci
tin nh lng acid amin t do trong dch sinh hc bng phng php UPLC chnh xc v xc thc, c th
s dng phc v chn on v theo di iu tr cc ri lon chuyn ho bm sinh.

T kho: acid amin, dch sinh hc, sc k lng siu hiu nng, ri lon chuyn ho bm sinh

I. T VN
nh lng acid amin t do trong mu v thn kinh, bnh tim mch, khng insulin,
nc tiu l mt cng c quan trng trong nghin cu sinh l, dc l, nui cy t bo
chn on v theo di cc ri lon chuyn ha v phn tch gi tr dinh dng thc phm
bm sinh. S dng phn tch acid amin t do [3; 4; 5].
trong chng trnh sng lc s sinh cho
Theo cc bo co t Khoa Ni tit v Ri
php chn on nhanh mt s ri lon chuyn
lon chuyn ha Bnh vin Nhi Trung ng
ha bm sinh nh bnh phenylceton niu,
trong vi nm gn y, s tr mc cc bnh
MSUD (maple syrup urine disease), mt s ri
ri lon chuyn ha bm sinh vi t l 1/5000
lon trong chu trnh ur, bnh homocystine
tr sinh ra, trong t l t vong ln n
niu [1; 2]. Vic theo di cn thn v duy tr
48%. ng thi vic xt nghim chn on
nng acid amin trong mu mc ti u
rt kh khn, cc mu bnh phm nh lng
nhng bnh nhn ny s mang li nhng li
acid amin t do phi gi ra nc ngoi dn
ch lu di trong iu tr. Bn cnh ng dng
n kt qu xt nghim rt chm. Vit
trong nh gi tnh trng dinh dng bnh
Nam, c nhiu bnh nhn mc ri lon
nhn mc ri lon chuyn ha bm sinh, nh
chuyn ha bm sinh nhng khng c
lng acid amin t do trong cc dch sinh vt
chn on xc nh sm dn n nhng
khc cn c p dng trong mt s nghin
di chng nng n [1]. Trn Th gii, nh
cu nh: tnh trng suy t bo gan, bnh l
lng acid amin t do trong mu v nc
ung th, chuyn ha c, ri lon dn truyn
tiu c tin hnh t nhng nm u
thp nin 60 ca th k XX [6; 7]. Phng
a ch lin h: L Duy Cng, Bnh vin a khoa Quc
php ph bin nht c s dng l sc k
t Vinmec
trao i ion vi dn xut hu ct bng
Email: v.cuongdl@vinmec.com
Ngy nhn: 25/7/2016 ninhydrin. y l phng php c u im r
Ngy c chp thun: 18/12/2016 tin, d s dng. Tuy nhin, sc k trao i

62 TCNCYH 102 (4) - 2016


ion c nhc im l thi gian phn tch di
(120 pht/mu) v i hi v bo tr phc tp
nn k thut ny tr nn khng ph hp vi
cc phng xt nghim [2,8,9]
Trong nhng nm gn y, mt phng
php mi c ng dng thay th cho
phng php sc k trao i ion: sc k lng
siu hiu nng (Ultraperformance
chromatography - UPLC). So vi sc k trao
i ion, phng php UPLC c u th vt
tri hiu nng v rt ngn c thi gian
phn tch [8; 9; 10]. Khoa Ha sinh, Bnh vin
Nhi Trung ng c trang b h thng
UPLC nhm phn tch acid amin t do trong
cc dch sinh vt, phc v cho vic chn on,
sng lc, theo di iu tr mt s ri lon
chuyn ha bm sinh. c th p dng thit
b UPLC vo phn tch acid amin t do, cn
phi nh gi phng php trc khi a vo
s dng. Do , ti c tin hnh vi
mc tiu: Thm nh quy trnh nh lng acid
amin t do trong mu v nc tiu bng
phng php UPLC.
II. I TNG V PHNG PHP
1. Trang thit b v cht liu nghin cu
H thng sc k lng siu hiu nng
(UPLC) Acquity ca Waters; Ct sc k
MassTrak AAA Column, 1,7 m 150 x 2,1 mm;
Chun acid amin ca Sigma-Aldrich; Thuc
th MassTrak AAA to dn xut acid amin ca
Waters. Pha ng A: MassTrak AAA Eluent A
Concentrate, pha long 1:10 (8 - 10%
acetonitrile, 4 - 6% formic acid, 84 - 88%
ammonium acetate/H2O). Pha
MassTrak AAA Eluent B m c ( 95%
acetonitrile, 5% acetic acid).
2. K thut nh lng acid amin bng
UPLC
TCNCYH 102 (4) - 2016
TP CH NGHIN CU Y HC
Mu th c kh protein bng dung dch
acid sulfosalicylic (SSA) 10% cha ni chun
norvaline 250 M vi th tch tng ng, ly
tm dung dch trong thi gian 5 pht tc
13000 vng/pht, thu dch ni v tin hnh to
dn xut theo quy trnh ca nh sn xut.
Phng php phn tch acid amin bao
liquid gm cc giai on sau:
(1) Kim ho mu chun, mu th, mu
trng (blank) c kh tp bi SSA bng
dung dch NaOH/ borat.
(2) Phn ng to dn xut: Dn xut ho
cc acid amin trong mu th bng AQC (6 -
aminoquinolyl - N - hydroxysuccinimidyl
carbamate) chuyn cc acid amin bc mt
v bc hai thnh cc cht c th pht hin
c bng u d cc tm (UV).
(3) Phn tch cc dn xut AQC ca acid
amin bng phng php UPLC, nh lng
bng u d TUV.
3. Thm nh phng php nh lng
acid amin t do trong dch sinh hc bng
UPLC
- Kho st tuyn tnh ca phng php:
Nc ct tinh khit v chun nng cao
gn vi gi tr trn d on ca khong phn
tch. Trn hai mu chun trn vi cc t l th
tch nh sau: 1/0, 4/1, 3/2, 2/3, 1/4 v 0/1.
Tin hnh phn tch nh lng cc acid amin
trong 6 mu trn. Mi mu lm lp li 03 ln.
Tnh gi tr trung bnh thu c ca mi mu.
V th nh gi tuyn tnh bng phn
mm c tch hp trong h thng UPLC vi:
ng B:
trc honh X l nng pha long (0%, 20%,
40%, 60%, 80%, 100%), trc tung Y l nng
trung bnh o c mi nng qua 3 ln.
Thit lp phng trnh Y = aX + b. Tnh h s
tng quan R, nu R2 0,99 th chp nhn
63
TP CH NGHIN CU Y HC

di gi tr ny, nu khng t th phi thu hp bnh thng c phn tch trong mt ln

2
di nng cho n khi t c R 0,99. chy, o lp li 10 ln (n = 10), thu thp d
- Kho st gii hn nh lng (LOQ) liu v nh gi chnh ngn hn.

LOQ c xc nh bng phn tch cc + Xc nh chnh xc di hn ( ti

dung dch acid amin chun cc mc lp): mu QC c chy trong 20 m khc
khc nhau trong mt ln chy. Pha long dung nhau (n = 20) trong vng 3 thng, trong mi
dch chun c nng cao nht thu c m, mu QC c o lp li 3 ln, thu thp
cc dung dch c t l pha long 2, 4, 8, 16, d liu v nh gi chnh xc di hn.
32, 64 ln. Cc chun pha long c chy + Tnh gi tr trung bnh, lch chun
lp li 3 ln trong mt m phn tch. Tnh SD (SD) v h s bin thin (CV) nh gi
v CV, LOQ l gi tr nng thp nht m ti chnh xc ngn hn v di hn. Tiu
phng php vn cn tuyn tnh cht ch chun chp nhn: theo AOAC, chnh chp
vi R > 0,99, CV 20%.
nhn c khi phn tch nng 10-6 - 10-4
- chnh xc ca phng php mol/L l CV < 11% i vi chnh xc di
+ Xc nh chnh xc ngn hn ( lp hn, v CV < 5,3% i vi chnh xc ngn
li): mu QC c to t huyt tng ca tr hn. [3]

III. KT QU

Hnh 1. Kt qu phn tch nhit 430 C

nhit c khuyn co bi Waters trong quy trnh chun, hiu nng phn tch km
cc cp His + PEA, EA + Asp, Lys + Tyr, Arg + Gly. Cc cp His + PEA v Lys + Tyr b ln vo
nhau.

Hnh 2. Kt qu phn tch nhit 41,20 C

64 TCNCYH 102 (4) - 2016

 TP CH NGHIN CU Y HC

C s ci thin r tt c cc cp peak kh phn tch: His + PEA, Arg + Gly, EA + Asp, Lys +
Tyr.

Hnh 3. S ci thin hiu nng phn tch khi thay i gradient pha ng

Kho st nhit 41,20 C, chng ti thu c s ci thin r rt ti cc thi gian lu xut

hin cc cp peak kh phn tch: His/PEA, Arg/Gly, EA/Asp, Lys/Tyr.

Bng 1. So snh gradient ca hai phng php

Gradient tiu chun (Waters) Gradient ci tin

Thi gian (pht) mL/pht %A %B Curve Thi gian (pht) %A %B Curve

4,5 0,400 98,5 1,5 6 4,5 98,1 1,9 6

7,0 0,400 98,0 2,0 6 7,0 97,5 2,5 5
8,0 0,400 98,0 2,0 6 8,0 97,9 2,1 5
20,0 0,400 88,0 12,0 6 20,0 87,0 13,0 6
21,0 0,400 88,0 12,0 6 21,0 88,0 12,0 6

Thay i t l % A/B c ngha trong vic thay i nh mc pH ti mi thi im mong mun,

ti pht 4,5 (His + PEA), 7 (Arg), 8 (Gly) v 20 (Lys + Tyr) gim % A v tng % B lm tng nh pH
ca hn hp pha di ng. Pht 21 t li t l ban u nhm thit lp pH nh trong trong
phng php tiu chun. Kt qu c ci thin sau khi thay i t l % gia A v B.

Bng 2. Kho st tuyn tnh trong khong 5 - 1000 mol/L

TT Acid amin R R2 TT Acid amin R R2

1 Ala 0,999810 0,999620 14 Thr 0,999972 0,999944
2 His 0,998439 0,996881 15 Cyst 0,999885 0,999769
3 PEA 0,987842 0,975832 16 Phe 0,999079 0,998160
4 Asn 0,999897 0,999793 17 Orn 0,999777 0,999554
5 Ser 0,999508 0,999016 18 Lys 0,999516 0,999032

TCNCYH 102 (4) - 2016 65

TP CH NGHIN CU Y HC

TT Acid amin R R2 TT Acid amin R R2

6 Gln 0,999973 0,999947 19 Tyr 0,999225 0,998451
7 Carn 0,999709 0,999418 20 Tryp 0,998685 0,997372
8 Arg 0,999697 0,999394 21 Met 0,999664 0,999329
9 Gly 0,999800 0,999600 22 Val 0,999994 0,999988
10 EA 0,999218 0,999177 23 Ile 0,999983 0,999966
11 Asp 0,997961 0,995925 24 allo 0,999948 0,999896
12 Glu 0,998928 0,997858 25 Leu 0,999968 0,999936
13 Cit 0,999953 0,999907 26 HCys 0,999813 0,999627

Tt c cc acid amin in hnh u c h s tng quan R > 0,99 trong khong nng 5 -
1000 mol/L.
Gii hn nh lng (LOQ): Trong nghin cu ny, LOQ ca cc acid amin l 5 mol/L.
Bng 3. Minh ho gii hn nh lng ca Valine

66 TCNCYH 102 (4) - 2016

 TP CH NGHIN CU Y HC

(STD5, STD10 cc mc nng 5, 10 mol/L. % Deviation: phn trm lch.

Trong gii hn t 5 - 1000 mol/L, R v R2 ca valine u tha mn u tha mn iu kin
> 0,99, ng thi lch 8,15%. Nng kho st cng mc thp th lch cng tng.

Bng 4. chnh xc di hn ( lp li)

Acid
Ala His PEA Asn Ser Gln Carn Arg Gly EA Asp Glu Cit Thr
amin

Mean
216,0 210,5 217,0 220,1 221,9 222,1 223,2 221,8 216,6 215,4 216,9 215,8 221,3 221,6
(mol/L)

SD
2,71 2,13 5,49 3,19 3,19 2,99 3,67 4,16 2,20 3,07 3,01 2,92 2,84 2,80
(mol/L)

CV (%) 1,3 1,0 2,5 1,5 1,4 1,3 1,6 1,9 1,0 1,4 1,4 1,4 1,3 1,3

Acid
Cyst Phe Orn Lys Tyr Tryp Met Val lle Allo Leu HCys
amin

Mean 226, 220, 218, 214, 219, 221, 219, 217,

229,8 222,0 218,7 225,3
(mol/L) 7 8 9 5 9 0 7 9

SD
3,36 3,10 3,64 3,66 4,63 3,55 5,02 2,81 3,31 2,93 2,76 4,03
(mol/L)

CV (%) 1,5 1,4 1,7 1,7 2,0 1,6 2,3 1,3 1,5 1,3 1,3 1,8

chnh xc di hn ca tt c cc acid amin u c CV < 3,0%.

Bng 5. chnh xc ngn hn ( lp li)

Acid
Ala PEA Asn Ser Gln Carn Gly EA Asp Glu Cit Thr
amin

Mean
344,2 265,6 56,1 124,6 371,39 8,6 182,9 10,8 12,8 193,3 24,8 98,8
(mol/L)

SD 0,08 0,09 0,06 0,07 0,12 0,07 0,51 0,07 0,03 0,06 0,01 0,01

CV (%) 0,02 0,04 0,122 0,06 0,03 0,87 0,28 0,69 0,20 0,03 0,05 0,01

Acid amin Cyst Phe Orn Lys Tyr Met Val lle Leu Tryp

Mean (mol/L) 6,8 48,6 68,5 188,4 52,2 18,2 208,3 71,3 113,4 41,3

SD 0,04 0,03 0,04 0,15 0,01 0,06 0,02 0,39 0,13 0,37

CV (%) 0,67 0,13 0,07 0,08 0,22 0,36 0,01 0,55 0,11 0,91

chnh xc ngn hn ca cc acid amin < 1%.

TCNCYH 102 (4) - 2016 67

TP CH NGHIN CU Y HC

IV. BN LUN 5), Arg/Gly(pht 7 - 7,5), EA/Asp (pht 7,5 -

8,0), Lys/Tyr (pht 19 - 21). Da theo im
Khi p dng phng php UPLC tiu
ng in (pI- isoelectric point) ca tng acid
chun ca hng Waters vo iu kin thc t,
amin: His (7,59), Arg (10,76), Gly (5,97), Lys
mt s cc cp peak b chng nh His/PEA
(9,74), Tyr (5,66) [5], chng ti thay i t
(pht 4,4 - 5), Arg/Gly (pht 7 - 7,5), EA/Asp
l % dung dch A v B ti cc thi gian lu
(pht 7,5 - 8,0), Lys/Tyr (pht 19 - 21). V vy
tng ng vi tng cp acid amin nu trn
khng th nh lng chnh xc nng ca
nhm nng cao hiu nng phn tch. Ti pht
cc acid amin ny trong mu phn tch. Do
4,5, chng ti gim t l % A/B t 98,5/1,5 v
chng ti quyt nh ci tin phng php
98,1/1,9 dn n lm tng nh pH do acid
UPLC tiu chun nng cao hiu sut phn
formic trong dung dch A mnh hn acid ace-
tch. Cc thng s c kho st bao gm:
tic trong dung dch B. S thay i ny gip
nhit ct, t l phn trm ca pha di ng A
tng pH ca hn hp pha di ng ph hp vi
v B. Kt qu thu c cho thy, nhit
pI xu hng kim ca His. Kt qu thu c
cng cch xa 430C th hiu nng phn tch
l s ci thin hiu qu phn tch ca cp
ca cp Lys/Tyr cng tng. Cng gim nhit
His/PEA. Ti pht th 7, t l % A/B t 98/2
th hiu nng phn tch ca cp EA/Asp
v 97,5/2,5, gy tng nh pH ph hp vi pI
cng tng, trong khi hiu nng phn tch ca
10,76 ca Arg, v kt qu thu c l ci
cp Arg/Gly cng gim. Cp His/PEA l tr
thin ng k kh nng phn tch cp Arg/
ngi ln nht trong qu trnh kho st do d
Gly. Ti pht 8, pI ca Asp nm trong vng pH
tng hay gim nhit u phn tch c
thp nn chng ti khi phc li t l %
so vi 430C nhng hiu nng phn tch l
A/ B gn ging vi t l chun ban u ca
khng cao, cha th phn tch hon ton.
phng php Waters tiu chun - nhm h
Nhng kh khn ny cng c cng b khi
nh pH. Kt qu l chng ti thnh cng
tin hnh nghin cu trn mu huyt tng
trong phn tch cp EA/Asp. Ti pht 20, pI
[4]. Tuy nhin, nghin cu khc th hiu
ca Lys nm trong vng pH kim, nn chng
nng phn tch l ti u khi tin hnh phng
ti nng pH ca dung dch ra gii bng
php UPLC tiu chun (430C) [2]. iu ny
cch thay i t l % A/B t 88/12 v 87/13,
cho thy, khng phi cng mt phng php
ti pht 21, pI ca tyrosin nm trong vng pH
tiu chun ca nh sn xut khi p dng
acid nn chng ti khi phc li t l % A/B
nhng phng xt nghim khc nhau th cng
ban u: 88/12. S thay i ny ci thin
cho ra mt kt qu ti u ging nhau, c bit
r rng hiu nng phn tch ca cp Lys/Tyr.
l vi phng php sc k. Kt qu kho st
cho thy 41,20C, hiu nng phn tch l ti Trong nghin cu v acid amin c
u nht. Nhit ny cng gn ging vi cng b, gradient c gi nguyn nh gra-
nhit 410C trong mt nghin cu khc [4]. dient tiu chun ca Waters, nhng pH c
Sau khi thu c nhit ti u, chng ti thay i trong ton b qu trnh phn tch do
tin hnh kho st nh hng ca t l % ca dung dch B c thay i t acid acetic thnh
pha ng A v B ln hiu nng phn tch. acid formic v kt qu thu c l s ci thin
Kho st ny s tp trung ti cc cp peak c hiu nng tch ca cp Lys/ Tyr v lm gim
hiu nng phn tch thp: His/PEA (pht 4,4 - lch qua cc ln chy lp li [4].

68 TCNCYH 102 (4) - 2016

 TP CH NGHIN CU Y HC

Sau khi thay i phng php so vi tuyn tnh khi s dng phng php UPLC
phng php tiu chun, cc thc nghim - MS/MS l rt cao [6].
nh gi c tin hnh bao gm: khong Trong nghin cu ca chng ti, gii hn
tuyn tnh, gii hn nh lng, chnh xc. nh lng (LOQ) l 5 mol/L, LOQ cc
Trong di 1 - 1000 mol/L chng ti cha thu nghin cu c cng b trc y u bng
c h s R2 > 0,99 tt c cc acid amin 2 mol/L [2; 4], so vi cc nghin cu ny th
nh mong mun. Tin hnh thu hp di phn LOQ trong nghin cu ny cao hn. Nng
tch v 5 - 1000 mol/L, chng ti thu c acid amin mu v nc tiu trong di tuyn
2 tnh ca chng ti bao gm cc gi tr c
h s R > 0,99 hu ht cc acid amin,
ngoi tr phosphoethanolamine (PEA) Vi R2 tnh quyt nh lm sng, do gii hn tuyn
> 0,97- c xem l c s tng quan tuyn tnh ny vn m bo pht hin ng cc
tnh rt cht ch, v vi R > 0,99, c xem ri lon chuyn ha bm sinh. chnh xc
nh tim cn gi tr 1. Do c th ni ca phng php c th hin qua lch
tuyn tnh trong phng php UPLC l rt chun v h s bin thin (CV). Kt qu
cao, y cng l u im chung cho cc nghin cu ny tha mn tiu chun ca
phng php sc k hin i. So snh gi tr AOAC vi CV chnh xc ngn hn < 5,3%
R trong nghin cu ca chng ti vi cc tc v CV chnh xc di hn < 11% mc
gi khc c th thy rng, trong cng thit k nng 10-6 - 10-4 mol/L [3; 7]. H s bin
nghin cu nh lng acid amin bng UPLC thin trong nghin cu ca chng ti cng
- TUV, cc tc gi khc cng b h s R tng ng vi cc nghin cu khc ca cc
trong khong t 0,8658 - 0,9932 vi nhit tc gi: CV < 5% [4], CV 0,9 - 2,9% [8], CV 1,8
v gradient chun [2]. Trong nghin cu ca - 4,9% [9]) CV 6% [6], CV < 4% [10].
tc gi khc R nm trong khong 0,85 - 0,99 V. KT LUN
(ngoi tr vi methionine, R = 1). Trong ,
Phng php ci tin nh lng acid
0,85 < R < 0,9 i vi cc acid amin: Gluta-
mate, proline, citrulline, valine) [4]. H s R amin trong dch sinh hc bng UPLC c:

trong nghin cu ny ln hn, c s khc bit Khong tuyn tnh ca cc acid amin l

ny c th do cch ly gi tr khong tuyn 5 - 1000 mol/L. chnh xc ngn hn CV <

tnh. Nghin cu ny gii hn tuyn tnh l 5- 1%, chnh xc di hn CV < 3%.

1000 mol/L, trong khi nghin cu ca cc tc
gi khc l 2 - 3500 mol/L v 2 - 2000 mol/L
[2; 4]. Khong tuyn tnh cng rng th lch
chun cng cao, iu ny gii thch ti sao R
li cng gim. Cng vi , dung dch chun
vi nng cng thp th nn mu (matrix)
cng b thay i so vi dung dch chun gc
(stock) do b pha long vi t l ln. Nghin
cu nh lng acid amin trong dch c th
ca nm (2009) vi phng php UPLC - MS/
MS, R2 > 0,99 (R > 0,999) i vi tt c cc
acid amin, vi LOQ l 10 mol/L. Nh vy,
Li cm n
Nhm nghin cu xin by t li cm n
su sc ti Ban gim c Bnh vin Nhi Trung
ng h tr kinh ph cho vic thc hin
nghin cu ny.
TI LIU THAM KHO
1. Hong Hnh Phc (2008). Chn on
bnh ri lon chuyn ha bm sinh bng k
thut sc k kh khi ph. Tp ch nghin cu
y hc, 57(4), 1 - 5.

TCNCYH 102 (4) - 2016 69

TP CH NGHIN CU Y HC

2. Narayan S. B (2011). Measurement of 7. Vin Kim Nghim An Ton V Sinh

plasma amino acids by Ultraperformance(R)
Liquid Chromatography. Clin Chem Lab Med,
49(7),1177 - 1185.
3. AOAC International methods commit-
tee (2012). Guidelines for Standard Method
Performance Requirements. J AOAC Int, Ap-
pendix F, 9.
4. Peake, R.W (2013). Improved separa-
tion and analysis of plasma amino acids by
modification of the MassTrak AAA Solution
Ultraperformance(R) liquid chromatography
method. Clin Chim Acta, 423, 75 - 82.
5. Chauhan, B.S (2008). Principles of Bio-
chemistry and Biophysics. Laxmi Publications
Pvt Limited, 150 - 151.
6. Waterval W. A (2009). Quantitative
UPLC-MS/MS analysis of underivatised amino
acids in body fluids is a reliable tool for the
diagnosis and follow-up of patients with inborn
errors of metabolism. Clin Chim Acta, 407(1-
2), 36 - 42.
Thc Phm Quc Gia (2010). Thm nh
phng php trong phn tch ha hc v vi
sinh vt. Nh xut bn khoa hc v k thut
H Ni, 11 - 47.
8. Fiechter, G. and H.K. Mayer (2011).
Characterization of amino acid profiles of cul-
ture media via pre-column 6-aminoquinolyl-N-
hydroxysuccinimidyl carbamate derivatization
and ultra performance liquid chromatography.
J Chromatogr B Analyt Technol Biomed Life
Sci, 879(17-18), 1353 - 1360.
9. Terrlink, T., P.A. van Leeuwen, and A.
Houdijk (1994). Plasma amino acids deter-
mined by liquid chromatography within 17 min-
utes. Clin Chem, 40(2), 245 - 249.
10. Fekkes, D (1995). Validation of the
determination of amino acids in plasma by
high-performance liquid chromatography using
automated pre-column derivatization with
o - phthaldialdehyde. J Chromatogr B Biomed
Appl, 669(2), 177 - 186.

Summarry
MEASUREMENT OF AMINO ACIDS IN BIOLOGICAL SAMPLES
BY ULTRAPERFORMANCE CHROMATOGRAPHY (UPLC)
The aim of this study was to validate the modified method for quantitation of free amino acids
by UPLC. The amino acids in samples were derivatized by AQC (6 - aminoquinolyl - N-
hydroxysuccinimidyl carbamate), separated by UPLC method and quantified by TUV detector. The
limit of quantitation was 5 mol/L for all amino acids. The linearity range was 5 to 1000 mol/L.
The short - term precision was 2.4%, the long-term precision was 3%. The proposed method for
quantitation of free amino acids in biological fluid using UPLC was precise and accurate. This method
could be applied to the routine diagnosis and treatment monitoring of inborn error of metabolism.

Key words: amino acids, physiolosical samples, UPLC, inborn error of metabolism

70 TCNCYH 102 (4) - 2016