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E L S E V I E RJournal of Pharmaceutical and Biomedical Analysis14 (1996) 1287-129 4

~URNALOFP H A R M A C E U T I C A LA N D B IO M E D I C A L

A N A L Y S I S

High-performance l iquid chromatographic determination oftaurine in human plasma using pre-column extract ion andderivatizationG i l l i a n P . M c M a h o n '~ '*, R i c h a r d O ' K e n n e d y b , M a r y T . K e l l y ~

"Department o f C hemistry, Royal College of Surgeons in Ireland, 123 St. Stephen's Green, Dublin 2, IrelandhSchool of Biological Sciences, Dublin City University, Glasnevin, Du blin 9, IrelandReceived for r eview 13 Septem ber 1995; revised manuscript received 13 N ovem ber 1995

A b s t r a c t

Plasma samples (100 ~1) were t rea ted wi th 150 g l o f ace toni tr i l e and cent r i fuged a t 5800g for 10 min and 50 # l of10 m M bora te buffe r (pH 9 .2) were added to the sup erna tant so lu t ion . This was fo l lowed by the addi t ion o f a 50 p la l iquot of 5 m M fluorescamine in ace toni tr i le and im media te vor tex mixing. A 20 g l sample was in jec ted o n to areversed-phase HPLC sys tem us ing a Bondc lone C-18 l0 pm ana lyt ica l column (300 mm x 3 .9 mm). The mobi lephase was te t ra hyd rofu ran- ace to ni t r i l e - ph osp ha te buffer (15 raM, pH 3 .5) (4 :24:72, v /v /v) . The taur ine de r iva t ivewas de tec ted by measur ing the U V abso rbance of 385 nm. P la te le t -poor p lasma samples were sp iked wi th know namounts of t aur ine and in te r- and in t ra -assay ca l ibra t ion curves were obta ined. The method was appl ied to thede te rm ina t ion o f t aur ine in p la tele t -r ich p lasma .K e y w o r d s : Deriva t iza t ion; F luorescamine ; Human plasma; Reversed-phase chromatography; Taur ine

1 . I n t r o d u c t i o n

T a u r i n e i s a n a t u r a l l y o c c u r i n g f l - s u l p h o n a t e da m i n o a c i d t h a t i s n o t i n c o r p o r a t e d i n t o p r o t e i n s ,b u t f o u n d f r e e o r i n s o m e s i m p l e p e p t i d e s [ 1 ] . I th a s b e e n i m p l i c a t e d i n m a n y p h y s i o l o g i c a l f u n c -t i o n s , p h a r m a c o l o g i c a l a c t i o n s [ 2 ] a n d p a t h o l o g i -c a l c o n d i t i o n s . A m o n g t h e p h y s i o l o g i c a l r o l e s

* C orresponding author.Presented at the Fifth International Symposium on DrugAnalysis, Septem ber 1995, Leuven, Belgium.

a t t r i b u t e d t o t a u r i n e a r e m e m b r a n e s t a b i l i z a t i o n[ 3 ] , a n t i o x i d a t i o n [ 4 , 5 ] , n e u r o m o d u l a t i o n [ 6 ] a n dr e g u l a t i o n o f c a l ci u m h o m e o s t a s i s [7 ]. A l t h o u g hi n t r a c e l l u l a r t a u r i n e c o n c e n t r a t i o n i s s t r i n g e n t l yc o n t r o l l e d [ 8 ] , p l a s m a l e v e l s a r e a l t e r e d d u r i n gt r a u m a [ 9 ] , s e p s i s [ 1 0 ] a n d c a n c e r [ I l l . R e c e n te v i d e n c e s u g g e s t s t h a t t h e l e v el o f t a u r i n e i np l a s m a m a y b e a u s ef u l i n d i c a t o r o f m y o c a r d i a li n f a r c t i o n [ 1 2] . A s t h e r o l e o f t a u r i n e i n v a r i o u sd i s e a s e s t a t e s b e c o m e s m o r e w i d e l y r e c o g n i z e d ,t h e n e e d f o r a s i m p l e , r a p i d a s s a y f o r r o u t i n ep l a s m a t a u r i n e e s t i m a t io n b e c o m e s m o r e i m p o r -t a n t .

0731-7085/96/$15.00 1996 Elsevier Science B.V . All rights reservedS S D I 0731-7085(95)01697-X

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1288 G.P. Mc M aho n e t a l . / J . Pharm. B iomed . A na l 14 (1996) 12 87- 129 4Re c e n t H P LC m e t h o d s i n v o lv i n g d e r iv a t iz a ti o nf o r t h e d e t e r m i n a t i o n o f t a u r i n e h a v e e m p l o y e dortho-phthala ldehyde (OPA) , wh ich requ i res thepresence o f a th io l such as mercap toe than o l to

ensure tha t de r iva t iza t ion occurs in s tan taneous ly .A l t h o u g h t a u r i n e h a s b e e n d e t e r m i n e d in t h is w a ywi th bo th u l t rav io le t (UV) abso rbance [13] andf luo rescence de tec t ion [14] , the re a re s tab il i typ rob lems assoc ia ted wi th th i s reagen t . Dansy lch lo r ide has a l so been used to de te rmine tau r ine[ I 5 ], bu t th is de r iva t iza t ion fo rm s m any s ide p rod-uc ts , r equ i res quench ing and has a long reac t iont ime . Taur ine has been de te rmined by de r iva t iza -t ion w i th dansy l ch lo r ide , fo rmin g a de r iva t ivetha t absorb s in th e v is ib le region [16] , but th ereac t ion requ i res h igh tempera tu res and the p res -ence o f sa l ts ca n have d e t r imen ta l e f fect s on thereact ion yie ld .Othe r ava i lab le de r iva t iz ing agen ts fo r aminoac ids inc lude pheny l i so th iocyana te , bu t the y ie ldcan be adv erse ly a f fected by the p resence o f sa lt s,d iva len t ca t ions and buf fe r s [17 ] . 9 -F luoreny l -me thy l ch lo ro fo rmate fo rms s tab le de r iva t ives ,bu t hydro lys i s p roduc ts o f the reagen t in te r fe reun less removed p r io r to ana lys i s [17 ] .F luorescamine was f i r s t in t roduced fo r the de -t e r m i n a t i o n o f p r i m a r y a m i n e s a n d a m i n o a c id s in1972 [18] and has been used fo r the qu an t i ta t ionof tau r ine (F ig . 1 ) u s ing f luo rescence de tec t ion[19] . In th i s work , UV absorp t ion a t 385 nm waschosen because w hereas the f luo rescence in tens i tym a y d e c r e a s e o v e r a f e w h o u r s , t h e a b s o r b a n c er e m a i n s u n c h a n g e d f o r u p t o 1 w e e k . F l u o -rescamine was se lec ted because i t s r eac t ion wi thp r i m a r y a m i n e s p r o c e e d s i n s ta n t a n e o u s l y a t a m b i -e n t t e m p e r a t u r e i n a l k a l i n e m e d i u m , t h e r e a g e n ta n d i ts m a j o r h y d r o l y s i s p r o d u c t s d o n o t i n t e rf e r ewi th UV de tec t ion and the de r iva t ives a re s tab le .

2 . E x p e r i m e n t a l2.1. Chemicals

Tau r ine (99%) a nd f luo rescamin e (98%) wereo b t a i n e d f r o m t h e A l d r i c h Ch e m i c a l ( G i l l in g h a m ,D o r s e t , U K ) . A l l o t h e r a m i n o a c i d s w e r e o b t a i n e df r o m BD H Ch e m i c a l s ( P o o l e , D o r s e t , U K ) , a s

were pe rch lo r ic ac id (70%) , d i sod ium te t rabora teand po tass ium d ihydrogenphospha te . Bor ic ac idw a s p u r c h a s e d f r o m M e r c k ( D a r m s t a d t , G e r -m a n y ) . A c e t o n i t r i l e , m e t h a n o l a n d t e t r a h y d r o -f u r a n w e r e o f H P LC g r a d e a n d w e r e p u r c h a s e df rom L abscan (D ub l in , I re land) . Super -pur i ty ace -t o n i t r i l e f r o m Ro m i l Ch e m i c a l s ( Lo u g h b o r o u g h ,UK) was used fo r the depro te in iza t ion s tep . Wa-te r was de ion ized us ing an Elgas ta t pu r i f ica t ionsystem.2.2. Materials

Hypersep C-18 ca r t r idges (200 mg , 3 ml) werereceived as a g if t f ro m S ha nd on Scientific (Run -c o r n , U K ) . A n i o n - ( S CX ) a n d c a t i o n - ( P RS ) e x -hange co lumns (200 mg , 3 ml) were k ind lyd o n a t e d b y I S T ( M i d - G l a m o r g a n , U K ) . M i c r o -c o n - 3 c o n c e n t r a t o r s a n d M i c r o p u r e s e p a r a t o r i n -s e r t s w e r e p u r c h a s e d f r o m A m i c o n ( S t o n e h o u s e ,U K ) .2.3, Preparation o f reagents and standa rdsolutions

Ta u r i n e s t a n d a r d s o l u ti o n s w e r e p r e p a re d d a i l yf r o m a s t o c k a q u e o u s s o l u ti o n o f 1 m g m l - ~ t h a twas p repa red o n a w eek ly bas is . F luo rescam ineso lu t ion (5 mM) was p repa red in ace ton i t r i l e andkep t a t room tempera tu re . Such a so lu t ion i ss tab le fo r 12 weeks [20 ] . Bora te bu f fe r was p re -p a r e d b y a d j u s t in g 1 00 m M d i s o d i u m t e t r a b o r a t eso lu t ion to pH 9 .2 wi th 10 m M bor ic acid . Th e 15m M p h o s p h a t e b u f f e r w a s m a d e u p e a c h w e e k b yd i ss o lv i ng p o t a s s i u m d i h y d r o g e n p h o s p h a t e i n w a -te r and ad jus t ing the pH to 3 .5 wi th phosphor ica c i d . Th e m o b i l e p h a s e w a s t e t r a h y d r o f u r a n - a c e -ton i t r i l e - ph osp ha t e bu f fe r (pH 3 .5 ) (4:24 :72 , v /v /v ). A f t e r m i x in g , t h e p H o f t h e m o b i l e p h a s e w a s

s o 3 H c 2 .T s u r i n e F l u o r e s c a m i n e F lu or op ho r

Fig. 1. Derivatization of taurin e with f luorescamine.

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G . P . M cM a h o n et a l . / J . Pha r m . B iomed . An a l . 1 4 ( 1 9 9 6 ) 1 2 8 7 - 1 2 9 4 1 2 8 9

000000

0oo

A q u e o u s T a u ri neSo lut io n/ N00o

000oOO

B l a n k S o l u t i o n

g zs0O 3 0 0 3 6 0 4 2 0( r i m )F i g . 2 . U V absorbancespectrum oft au fin e derivative.

4 8 0~ . ' 1 . ?s 0 0 gO

asse s se d , t h e n i t was f i l t e r e d u n d e r vac u u mt h r o u g h a 0 . 4 5 / z m M i l l i p o r e f i l t e r a n d s o n i c a t e df o r 2 0 m i n .

d e t e c t ion was c ar r i e d ou t a t 385 n m, t h e max i -m u m a b s o r b a n c e w a v e l e n g t h f o r t h e t a u r i n ed e r iva t ive ( F ig . 2 ) .

2.4. HPLC system 2.5. Sample preparat ionT h e h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h

w a s e q u i p p e d w i t h a W a t e r s ( M i l i f o r d , M A ,U S A ) m o d e l 5 1 0 d u a l - p i s t o n p u m p , a W a t e r sM o d e l 4 8 6 t u n e a b l e a b s o r b a n c e d e t e c t o r a n d aW a t e r s m o d e l 7 4 6 d a t a m o d u l e . T h e R h e o d y n ei n j e ct i o n p o r t ( C o t a t i , C A , U S A ) w a s f i tt e d w i t h a2 0 / ~ 1 l o o p . A C - 8 g u a r d c o l u m n w a s f i t t e d p r i o rt o t h e B o n d c l o n e C - 1 8 1 0 / z m s t a i n l e s s - s t e e l a n a -l y ti c al c o l u m n ( 3 0 0 m m x 3 .9 m m i .d . ). T h e f l o wr a te o f t h e e lu e n t w a s 1 m l r a i n - ~ , t h e s y s t e mp r e s s u r e w a s a p p r o x i m a t e l y 1 1 0 0 p s i a n d a l l m e a -s u r e m e n t s w e r e m a d e a t a m b i e n t t e m p e r a t u r e. U V

B l o o d s a m p l e s w e r e t a k e n f r o m f a s t i n g v o l u n -t e e r s in g las s t u b e s c on t a in in g sod iu m c i t r a t e a san t i - c oagu lan t . L ar ge - b or e b u t t e r f ly syr in ge s we r eu s e d s o a s t o m i n i m i z e c e ll d a m a g e , s i n ce t h i s c a nr e su l t in p la t e l e t r u p t ion an d h e n c e g ive r i s e t of a l se ly e l e va t e d l e ve l s o f b asa l t au r in e p r e se n t int h e p l a s m a . P l a t e l e t - r i c h p l a s m a ( P R P ) w a s o b -t a i n e d b y c e n t r i fu g a t i o n f o r 5 m i n a t 170g t r o o mt e m p e r a t u r e . P l a t e l e t - p o o r p l a s m a ( P P P ) w a s o b -t a in e d b y c e n t r i f u gat ion f or 15 r a in a t 1500g a tr o o m t e m p e r a t u r e . C a r e w a s t a k e n d u r i n g p i p e t -t in g so a s n o t t o d i s t u r b t h e b u f f y c oa t l aye r .

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1290 G.P. M cM aho n e t aL / J . Pharm . B iomed . A nal . 14 (1996) 128 7-12 94F o r t h e p r e p a r a t i o n o f s p ik e d p l a t e le t - p o o r

samples , 5 / zl o f the t au r ine s t andard so lu t ionw e r e a d d e d t o 9 5 / ~1 o f p l a s m a t o g iv e s t a n d a r d swi th 0 , 5 , 10, 20 and 30 /Lg m l-~ of taurineadded . Each sample (100 /z l ) was t rea t ed wi th 150/zl o f supe r -pu r i t y ace ton i t r il e , vo r t ex mixed andcen t r i fuged fo r 10 min a t 5800g . Bora t e bu f fe r (50/ tl , 100 mM , p H 9 .2 ) was adde d to ad jus t t hes u p e r n a t a n t t o a p p r o x i m a t e l y p H 9 . T h e n 5 0 / z l o ff luo rescamine in ace ton i t r i l e (5 mM) were addedand the so lu t ion was immed ia t e ly vo r t ex mixed .S a m p l e s w e r e a n a l y s e d o n t h e H P L C s y s t e mwithin 6 h .

3. Resu l ts and d iscuss ion

3.1. Optimization o f protocol3. I .I . Optimization o f sample preparation

In i t i a l exper imen t s cen t red on u l t ra f i l t ra t i on asa m e t h o d o f s a m p l e p r e p a r a t io n . M i c r o c o n - 3 c o n -c e n t r a t o r s w i th a m o l e c u l a r m a s s c u t - o f f o f 3 0 0 0w e r e u s e d i n c o n j u n c t i o n w i t h M i c r o p u r e s e p a r a -to r i n se r t s fo r remo va l o f l a rge molecu les p r io r t ode r iva t i za t i on and ana lys i s . The cen t r i fuga t iont i m e w a s f o u n d t o b e v e r y l o n g ( a p p p r o x i m a t e l y140 min ) and , a l t hough the re was a concen t ra t i nge f fec t , few o f t he l ower molecu la r mass i n t e r fe r -e n t s w e r e r e m o v e d . C l e a n - u p w i t h r e v e r s e d - p h a s eso l id -phase ex t rac t i on ca r t r i dges re su l t ed i n al a rg e d i lu t i o n f a c t o r a n d p o o r c l e a n -u p a n d r e c o v -e ry . Wi th t he an ion - and ca t ion -exchangeco lumn s , t he a im was se l ec t ive ly t o re t a in o rse l ec t ive ly t o e lu t e t he t au r ine . I t was n o t poss ib l et o r e t a i n t h e t a u r i n e o n t h e c o l u m n s u n d e r a n yc i rcumstances and , i n fac t , ex t ra impur i t i e s werein t roduced . Pe rch lo r i c ac id p roved to be ane f f i c i en t dep ro t e in i za t ion agen t , bu t i t was subse -quen t ly d i f fi cu lt t o ra i se t he pH rep rod uc ib ly p r io rto de r iva t i za t i on . Bo i l i ng was found to g ive poorrep roduc ib i l i t y and me thano l was requ i red in a3 :1 ra t i o t o t he sample i n o rde r t o e f fec t com ple t ep rec ip i t a t i on o f p ro t e ins . Depro t e in i z a t ion by ace -ton i t r i l e p roved to be t he mos t fac i l e and rep ro -d u c i b le m e t h o d o f s a m p l e p r e p a r a t i o n a n d w a sthe eas i e s t t o execu te .

3.1.2. Optimization of reaction conditionsThe p H a t wh ich the de r iva t i za ti on o f t au r inet akes p l ace is c ruc i al t o t he reac t ion . T he p H mus t

be t> 8 .5 , and the o p t im um i s 9 . F luo rescam inewas d i s so lved in ace ton i t r i l e because o f i t s com-pa t ib i l i t y w i th t he dep ro t e in i zed superna t an t andi t s su i t ab i l i t y a s a non-hydroxy l i c bu t wa te r -mi s -c ib le so lvent . Immediate mixing i s essent ia l form a x i m u m r e s p o n s e .3.1.3. O ptimization o f mob ile phase

The o r ig ina l mob i l e phase cons i s t ed o f ace ton i -t r i l e and phospha te bu f fe r . Or ig ina l ly , t h i s waso p t i m i z e d w i t h t h e c o m p o s i t i o n a c e t o n i t r i l e -pho sph a te bu f fe r (pH 2 .5 , 15 m M ) (35 :65 , v /v) .A l t h o u g h t h i s g a v e g o o d c h r o m a t o g r a p h y f o ra q u e o u s s t a n d a r d s, i n p l a sm a s a m p l e s t a u ri n e c o -e l u t ed w i t h o th e r c o m p o n e n t s . M e t h a n o l w a s u s e das an o rgan ic mod i f i e r a t va r ious ra t i o s w i th l i t t l esuccess , bu t t e t rahydro fu ran changed the se l ec t i v -i t y , wh ich improved the re so lu t ion be tween t au -r i n e a n d o t h e r e n d o g e n o u s c o m p o u n d s . H e n c e t h ef i n a l c o m p o s i t i o n w a s t e t r a h y d r o f u r a n - a c e t o n i -t r i l e - pho sph a te bu f fe r (15 mM ) (4:24 :72 , v /v /v ) .A va r i e ty o f pH va lues were examined , 1 .6 , 2 .5 ,2 .8 , 3 .5 and 4 .0 , and a t pH 3 .5 taurine wasadequ a te ly sepa ra t ed f rom o the r c lo se ly e lu t ingamino ac id de r iva t ives such as a l an ine , a rg in ine ,asparagine, aspart ic acid , g lu tamic acid , g lu-tamine, g lycine , ser ine , threonine and val ine .3.2. Quantitative analyses o f standards3.2.1. Calibration and calculation

T h e s p i k ed s a m p l e s o f p l a t e le t - p o o r p l a s m awere quan t i f i ed by ex t e rna l s t andard i za t ion . Thes lope and in t e rcep t o f the ca l i b ra t i on g raph s we rede t e rm ined b y unw eigh ted l i nea r reg ress ion o f thet a u r i n e p e a k h e i gh t v e r s u s t h e c o n c e n t r a t i o n o ft au r ine added .3.2.2. PrecisionPrec i s ion was de f ined in t e rms o f t he va r i ab i l i t ybe tween ba t ches ( i n t e r -a ssay ) and wi th in ba t ches( in t ra -assay ) . In t e r -a ssay va r i a t i on was a ssesseds ing ly i n fou r rep l i ca t e runs cover ing the concen-t ra t i on ran ge 5 -3 0 / ~m ml - 1. In t ra -ass ay va r i ab i l -i t y was de t e rmined in quadrup l i ca t e i n t he same

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G.P. McM ahon et a l . / J . Pharm. B iomed. Anal. 14 (1996) 1287-1294 1291Table 1Precision dataAmount added (#g/ml) Mean amou nt found + SD (#g/ml) RSD (%) Difference between added and found (%)lntra-assa y (repeatabili ty)5 (n = 3) 4.56 + 0.41 9.01 -8 .8 710 10.30 + 0.66 6.38 -3 .0 320 20.50 + 0.59 2.90 -2 .5 030 29.63 + 1.17 3.95 + 1.21Mean: 5.56 Mean: 4.38y = 350.28x- 192.77r = 0.9998Inter-assay (reproducibility)

5102030

4.82 _+ 0.26 5.33 + 3.5610.22 +_. 0.58 5.69 -2 .2 120.00 +_ 0.69 3.43 -0 .0 229.95 4- 0.31 1.05 +0.15

Mean: 3. 87 Mean: 2.75y = 347.74 ( + 19.85)x- 179.11 ( + 125.78)r = 0.9995 + 0.0002

c o n c e n t r a t i o n r a n ge . T h e p r e c is i o n o f th e m e t h o dwas desc r ibed by the mean r e l a t ive s t andardd e r i v a t i o n ( R S D ) , a n d t h i s w a s f o u n d f o r t a u r i n ewhen the peak he igh t s were in t e rpo la t ed as un -knowns on the r eg ress ion l i nes . Fo r i n t e r - assayvar i a t ion ( r ep roduc ib i l i t y ) , t he in t e rpo la t ions werebased on the fou r r eg ress ion l i nes genera t ed f romthe fou r r ep l i ca t e r uns , and fo r i n t r a - assay va r i a -t i on ( r epea t ab i l i t y ) , t he in t e rpo la t ions were basedon a s ing le r eg ress ion l i ne genera t ed f rom theq u a d r u p l i c a te r u n . B e c a u s e h u m a n p l a s m a c o n -t a ins a basa l l eve l o f t au r ine , p l a t e l e t -poo r un -sp iked p l asma shows a sma l l peak in t hec h r o m a t o g r a m t h a t w a s a s s u m e d t o c o r r e s p o n d t ot au r ine . An unsp iked p l a t e l e t -poor sample wasrun w i th each ca l ib r a t ion and the he igh t o f t het a u r i n e p e a k w a s s u b t r a c t e d f r o m t h e t a u r i n epeak in each o f t he sp iked samples . P r ec i s ionda ta , ca l cu la t ed on the bas i s o f t he sub t r a c t edresu l t s , a r e p r esen ted in Tab le 1 , and they demon-s t ra t e t h a t t h e r e p ro d u c i b i li t y (m e a n R S D =3 .87%) and r epe a t ab i l i t y (mean R SD = 5 .56%) o fthe me th ods a r e wi th in accep ted va lues fo r c l in i ca lana lyses . These r e su l t s were compared wi th da t af rom unsub t r ac t ed va lues ( i . e . where the peak int h e b l a n k s a m p l e w a s n o t s u b t r a c t e d f r o m t h esp iked s t andards) , and i t was found tha t t he ove r -a l l p r ec i s ion va lues were h igher ( 4 .08% an d 6 .75%

f o r rep ro d u c i b i l i t y a n d rep ea t a b i l i t y , r e s p ec t i v e l y )a n d t h ere w a s g rea t er v a r i a b i l i t y i n p rec i s i o na m o n g s t i n d i v i d u a l v a l u e s .

gk- -

Platei~t-richP l a s m a

P l a t e l e t - p o o rP l a s m aF i g . 3 . Platelet-poor and platelet-rich plasma from one volun-teer. Colu mn, Bondclone C-18, 10 gin, 300 x 3.9 mm i.d.).Mobile phase, tetrahydrofuran-acetonitrile-pho sphate buffer(15 mM, pH 3.5) (4:24:72). Sample preparati on as described intext.

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1292 G.P. Mc Ma hon e t a l. / J . Pharm . B iomed . Ana l . 14 (1996) 1 287 -1294T a b l e 2Co m p ar i so n o f p r ev iou s m eth o d s an d r esu lt sRef . Der ivat izat ing age n t / Method of co l lect ing and t reat ing b lood Tau r ine levels found ( /zg /ml- t )detect ion method[21] OPA/f luorescence H u m a n :

Antecubi tal vein , vacu tainer 7 ml g lass tubeswi th ED TA . PRP: 1000g , 15 rain , 4CPPP: 12 100g, 30 min. 4CH u m a n :Meth o d n o t q u o t edH u m a n :Hepar in ized tubes , 2000g, 10 rainR a t :Wh o le b lo o dR a t :In fer io r vena cava using hepar in ized syr ingesChick:Card iac puncture, 1400g , 10 minFeline: Chilled heparinized syringes, 2677g

[ 2 2 ] O P A / f l u o r e s c e n c e[23] OPA/f luorescence[ 2 4 ] O P A / U V[ 1 3 ] O P A / U V

[25] Dan sy l ch lor ide/fluorescence

P P P : 5 .6 4 -0 .5 P RP : 1 6 -1 9(PC: 200-4 50 000 per ml p lasma)

Quo ted: 7.4 + 1.55.0 + 0.941.9 ___4. 912.6 + 1.613.5 + 0.043.0 _+ 0.3

3.2.3. Linearity and accuracyThe co r re l a t i on coe f f ic i en t o f t he reg ress ion l inefo r t he mean in t ra -assay va lues was 0 .9998 ( sub -t rac t ed da t a ) and 0 .9985 (unsub t rac t ed da t a ) . Ac-cu racy (p resen t ed in Tab le 1 ) , a s de f ined as t hep e r c e n t a g e d i f f e r e n c e b e t w e e n t h e a m o u n t a d d e da n d t h e a m o u n t f o u n d b y b a c k - c a lc u l a ti o n , w a susua l ly l e ss t han 5% wi th mean va lues o f 4 .38%a n d 2 . 7 5 % f o r w i t h i n - b a t c h a n d b e t w e e n - b a t c hanalyses , respect ively .3.2,4. RecoveryR e c o v e r y m a y b e c a l c u l a te d i n a b s o l u t e o r r e l a-t i ve t e rms . In t he ca l cu l a t i on o f abso lu t e recovery ,t h e p e a k r e s p o n s e o f a n e x t r a c te d s t a n d a r d isc o m p a r e d w i t h t h a t o f u n e x t r a c te d s t a n d a r d s t h a ta r e p r e p a r e d t o t h e s a m e t h e o r e t ic a l c o n c e n t r a t i o nas t he ex t rac t s . Re l a t i ve recovery i s ca l cu l a t ed byc o m p a r i n g p e a k r e s p o n s e s o f e x t r a c t e d m a t r i xs t a n d a r d s a g a i n st t h o s e o f e x t r a c te d a q u e o u s s t a n -d a r d s . T h i s p r o c e d u r e w a s u s e d t o a c c o u n t f o r t h ep resence o f ace ton i tr i l e i n t he sam ple t o be i n -j ec t ed . Us ing th i s me thod , t he re l a t i ve recovery o ft a u r i n e f r o m p l a s m a w a s f o u n d t o b e 8 9 . 7 % .3.2.5. Limit of quantitationT h e l im i t o f q u a n t i t a t io n w a s f o u n d t o b e 5 / t gm l - 1 t a u r in e i n p l a s m a s a m p le s .

3.2.6. SelectivityT a u r i n e i s a d e q u a t e l y s e p a r a t e d f r o m e n d o g e -nous p l a sma componen t s , a s can be seen in F ig . 3 .

A num ber o f amino ac ids a re i nhe ren t ly p resen t inbo th p l a t e l e t -poor and p l a t e l e t - r i ch p l a sma andthe t en t hough t mos t p robab le t o i n t e r fe re (a smen t ioned in Sec t ion 3 .1 .3 ) were sub jec t ed to t hesame ex t rac t ion and sepa ra t i on cond i t i ons and , bya d j u s t m e n t o f th e m o b i l e p h a s e p H a n d a q u e o u s -to -o rgan ic ra t i o , i t was poss ib l e t o re so lve t hesecompounds f rom the t au r ine peak . In fac t , i t i se x p e c t e d t h a t t h e m e t h o d d e s c r i b e d c o u l d b e a p -p l i ed to t he s imu l t aneous de t e rm ina t ion o f t au r ineand o the r amino ac ids .3.3. Q uantitative analyses of samples

T h e m a i n d i f f e r e n c e b e t w e e n p l a t e l e t - p o o r a n dp la t e l e t - r i ch p l a sma f rom the same vo lun tee r i st he he igh t o f t he t au r ine p eak (F ig . 3 ) . The l evelo f t a u ri n e i n P R P is u s u a ll y o f t h e o r d e r o f f o u rt imes g rea t e r t han in PPP [21 ] , bu t t h i s dependsn o t o n l y o n t h e n u m b e r o f p l a te l e ts p r e s e n t b u ta l s o o n h o w t h e s a m p l e w a s t a k e n a n d t r e a t e d .The ro l e o f p l a t e l e t s shou ld be cons ide red , s i ncet h e p l a t e l e t c o u n t c a n v a r y f r o m 1 0 0 0 0 0 t o500 000 pe r m l o f p l a sma wi th f ac to rs su ch as age ,sex , hea l th and sample t rea tmen t p l ay ing a pa r t .

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G.P. Mc Ma hon e t a l . / J. Pharm. Biomed. Anal. 14 (1996) 1287-1294 1293In the l i t e ra tu r e , i t i s o f t en u nc lea r w he the r o r no tp l a t e l e t s a r e p r esen t i n p l a sma samples be inga n a l y s e d f o r t a u r i n e b e c a u s e p r o t o c o l s f o r b l o o dc o l le c t io n m a y n o t b e d e s c r i b e d o r m a y o f t e n o m i tce r t a in p rocedura l s t eps (Tab le 2 ) . D i f f e r en t sy -t i nges and co l l ec t ing v i a l s f o r t he b lood samplesa r e used , d i f f e r en t g - fo r ces and t empera tu r es a r ec o m m o n d u r i n g t h e c e n t r if u g a t i o n s t e p a n d p i p e t-t in g o f t h e s u p e m a t a n t c a n c a u s e ce ll r u p tu r e i fsuf f ic ient care i s not taken.I t was found tha t t he p l a t e l e t s cou ld be r e -m o v e d f r o m p l a s m a w i t h m i n i m u m r u p t u r e i f t h esamples a r e co l l ec t ed wi th the l a rge -bore bu t t e r f lysy r inges . Th i s p rocedure ensu res t ha t t au r ine inp la t e l e t -poor p l a sma i s m in imized . However , i tshou ld be no ted tha t t he r e i s a lways a basa l l eve lo f t a u r in e p r e s e n t i n h u m a n p l a s m a . O t h e r w o r k -e r s h a v e c i r c u m v e n t e d t h i s p r o b l e m a n d o b t a i n e dtau r ine - f r ee , p l a t e l e t - f r ee p l a sma by us ing p l asmafrom ki t tens ra ised on a taur ine- f ree d ie t . Fel inep l a s m a n a t u r a l l y c o n t a i n s l o w c o n c e n t r a t i o n s o ft au r ine ( see Tab le 2 ) .S a m p l e s o f P R P f r o m t h r e e d i ff e re n t s o u rc e sw e r e d e t e rm i n e d b y i n t e r p o l a ti o n o f th e p e a khe igh t s a s on a ca l ib r a t ion cu rve (5 - 30 pg ml - 1).Resu l t s f r om the ind iv idua l i n F ig . 3 gave a va lueo f 1 5 .0 p g m l - I , w h il e v a l u es f r o m t w o p o o l e dPRP samples were ca l cu la t ed to be 16 .4 and 18 .5pg ml - 1. Al l three resul ts are wi th in the exp ectedrange acco rd ing to l i t e r a tu r e va lues .

4. ConclusionsF l u o r e s c a m i n e c a n b e u s e d f o r t h e p r e - c o l u m n

der iva t i za t ion o f t au r ine and a l lows the e s t ima t ionof t au r ine l eve l s i n p l a t e l e t - r i ch p l asma . Othe rana lyses have d i f f i cu l t and l abour - in t ens ive sam-p le p r epa ra t ion and some invo lve de r iva t i za t ionp r o c e d u r e s t h a t r e q u i r e q u e n c h i n g a n d / o r t h ep resence o f co - so lven t s in o rde r f o r t he r eac t ion tot ake p l ace . Many a r e p rone to i n t e r f e r ence f romthe de r iva t iz ing agen t i t se l f and the r e a r e d i s -crepanc ies in the s tab i l i ty of der ivat ives. Themethod p r esen ted he r e o f f e r s a s imp le , e f f i c i en ta n d r a p id m e t h o d f o r th e d e t e rm i n a t i o n o f t a u r in ein p l a t e l e t - r i ch p l asma , wi th comparab le sens i t i v -i t y t o f l uo rescence me thods , where a r e su l t i s

f eas ib l e wi th in 1 h o f t ak ing b lo od f ro m a pa t i en t.Wi th e sca l a t ing work in to the quan t i t a t i on o ft au r ine in human p l asma fo r med ica l pu rposes ,the s impl ic i ty of th is assay wi l l be of great benef i tto clinical research.

AcknowledgementsThe au tho r s t hank Dr . Ph i l i p S t ap le ton o f t he

S u r g i c a l R e s e a r c h G r o u p i n B e a u m o n t H o s p i t a l ,Dub l in , f o r dona t ions o f p l a sma . T hey a r e a l sog r a te f u l to A a r o n D e v e n e y o f t h e P h a r m a c o l o g yDepar tmen t a t t he RCSI fo r h i s a ss i s t ance wi tht h e m e t h o d s o f c o ll e c ti o n a n d t r e a t m e n t o f b l o o dsamples .

References[1] R.J. Huxtable, Fed. Proc., Fed. Am. Soc. Exp. Biol. , 39(1979) 2678.[2] R.J . Hux tab le and L.A. Se br ing , Tren ds Pharm . Sci .,(1986) 481.[3] H. Pa san tes-Morales and C . Cruz, J . Neurosci . Res. , 11(1984) 303.[4] P.P. Stapleton and F.J. Bloomfield, J. Biomed. Sci. , 3(1993) 79.[5] J. Milei , R. Ferreira, S. Llesuy, P. Forcada, J. Covarru-

bias and A. Boveris, Am. Heart J. , 123 (1992) 339.[6] K. Kuriyama, Fed. Proc., Fed. Am. Soc. Exp. Biol. , 39(1979) 2680.17] J. Azafi and R.J. Huxtable, Eur. J. Pharm., 61 (1980) 217.[8] N.E. Vin ton , S .A. Laid law, M.E . Am ent and J .D. K op-pie, Am. J. Clin. Nutr., 44 (1986) 398.[9 ] M. Jeev an an d am , D.H. Yo u n g , L . Ram ias an d W.R.Schiller, Am. J. Clin. Nutr., 51 (1990) 1040.

[10] L.I . Woolf , A.C. Grov ers , J .P. Mo ore, J .H. Duff , R.J .F in ley and R .L. Loom er , Surgery , 79 (1979) 283 .[11 ] G .E . G rey , A .M. Lan d e l an d M .M. Meg u id , Nu t r i t io n ,10 (1994) 11-15 .[12] S .K. B hatnagar , J .D. W el ty and A .R. AI Yusef, In t . J .

Card io l . , 27 (1990) 361-366 .[13 ] D .W. P o r t er , M .A. Ban k s , V . Cas t r an o v a a n d W .G.Mart in , J . Chromatogr . , 454 (1988) 311-316 .[14 ] G .M. An d er so n , T .M. Du rk in , M. Ch o k rab o r ty an d D . J .Cohen , J . Chromatogr . , 431 (1988) 400-405 .[15] T.J . Amiss, K.L. Tyc zkowska an d D .P. Auc oin , J . Chro-matogr . , 526 (1990) 375-382 .[16] V. Stocchi, F. P alm a, G. Piccoli , B. Biagiarell i, L. C uc-ch iar in i and M. M agna ni , J . Liq . Chroma togr . , 17 (1994)3 4 7 -3 5 7 .[17 ] C .T . M an t , N .E . Z h o u a n d R .S . Ho d g es , J. Ch ro m ato g r .

7/28/2019 0731-7085-2895-2901697-XHigh-performance liquid chromatographic determination of taurine in human plasma u

8/8

1294 G .P . M c M a h o n e t a l . / J . P h a r m . B i om e d . A n a l 1 4 ( 19 9 6) 1 2 8 7 - 1 2 9 4Libr . , 51B (1992) 75-85 .[18] S . Ude nfr iend , S . S tein , P . Bohlen, W . D airm an, W .Leim grub er and M. W eigele, Science, 178 (1972 ) 871.[19] T. Sakai and T. Nagasawa, J . Chromatogr . , 576 (1992)155-157 .[20] S . De Barnardo , M. Weigele , V. Toome, K. Manhar t , W.Leimgruber , P . B ohlen , S . S tein and S . Ude nfr iend , Arch .Biochem. Biophys., 163 (1974 ) 390.[21] K.H . Ta chik i , H.C. Hendr ie , J . Kel lams and M .H.

Aprison , C l in . Chim. Acta, 75 (1977) 455 .[22] M. Eslami and J .D. S tuar t , J . Liq . Chrom atogr . , 7 (1984)1117-1131.[23] L.L. Hirschberger , J . de La R osa an d M .H. St ipanuk , J .Chrom atogr . , 343 (1985) 303-313 .[24] T. H irai , H. Oyham a and R . K ido , Anal . Biochem., 163(1987) 339-342.[25] T.J . Amiss , K.L. Tyczkow ska and D.P. A ucoin , J . Chro-matogr., 526 (1990) 375-382.