- Email: [email protected]
Chapter 33 Clinical Diagnostics
46 9
Chapter 33 CLINICAL DIAGNOSTICS Various compounds i n p h y s i o l o g i c a l f l u i d s can be analysed u s i n g HPLC, such as p h a r m a c e u t i c a l s , drugs o f abuse, v i t a m i n s , a n t i b i o t i c s and t o x i c a n t s . A p p l i c a t i o n s o f g r a d i e n t e l u t i o n l i q u i d chromatography t o t h e s e p a r a t i o n and determinat i o n o f such compounds and t h e i r m e t a b o l i t e s have been mentioned i n s e v e r a l e a r l i e r c h a p t e r s (Chapters 17-25). Recent medical r e s e a r c h has i n d i c a t e d t h a t hundreds o f t h e m o l e c u l a r c o n s t i t uents o f human body f l u i d s may be r e l a t e d t o b o d i l y f u n c t i o n s and t o v a r i o u s i n borne d e f e c t s and o t h e r diseases. HPLC can be u t i l i z e d f o r automated h i g h - r e s o l u t i o n analyses o f UV-absorbing c o n s t i t u e n t s i n u r i n e , b l o o d serum and o t h e r physi o l o g i c a l f l u i d s . O r i g i n a l l y , anion-exchange chromatography on columns packed w i t h m i c r o p a r t i c u l a t e anion-exchange r e s i n s ( c a . 10
p a r t i c l e d i a m e t e r ) was
proposed and used f o r m o n i t o r i n g compounds i n p h y s i o l o g i c a l f l u i d s . G r a d i e n t e l u t i o n w i t h l i n e a r , convex o r m u l t i - l i n e a r g r a d i e n t s o f sodium a c e t a t e
-
acetic
a c i d b u f f e r c o n c e n t r a t i o n ( f r o m 0.015 t o 6 m o l / l ) a t a c o n s t a n t pH o f 4.4 was employed t o s e p a r a t e a v a r i e t y of a c i d i c , n e u t r a l and b a s i c compounds i n human 1-8 u r i n e . About 100-150 peaks were s e p a r a t e d and some o f them were i d e n t i f i e d
.
The o r i g i n a l t i m e o f a n a l y s i s was more t h a n 40 h w i t h columns 1-3.2 m l o n g , b u t i t was l a t e r decreased t o ca. 20-30 h by u s i n g a simultaneous t e m p e r a t u r e g r a d i e n t
( f r o m 20 t o 60°C)9y10.
W i t h s h o r t e r columns and h i g h e r f l o w - r a t e s t h e s e separa-
t i o n s have been accomplished r e c e n t l y i n ca. 2 h a t t h e expense o f decreased r e s o l u t i o n and peak c a p a c i t y ; however, t h e method m o d i f i e d i n t h i s way has become u s e f u l f o r r o u t i n e c l i n i c a l analyses 11
.
O t h e r g r a d i e n t e l u t i o n systems have been proposed, such as a g r a d i e n t o f i n 12 c r e a s i n g c o n c e n t r a t i o n o f ammonium p e r c h l o r a t e i n w a t e r (0-0.25 m o l / l i n 2 h ) o r a g r a d i e n t o f a c e t o n i t r i l e i n aqueous ammonium c h l o r i d e 1 3 . UV d e t e c t i o n a t v a r i o u s wavelengths (200, 215, 225, 230, 254, 260 and 280 nm) and f l u o r i m e t r i c d e t e c t i o n were used i n t h e s e a p p l i c a t i o n s . C h e m i c a l l y bonded weak a n i o n exchangers w i t h p o l y e t h y l e n i m i n e , d i e t h y l a m i n e o r d i e t h y l a m i n o e t h y l f u n c t i o n a l groups on a c o n t r o l l e d - p o r o s i t y g l a s s s u p p o r t were used f o r s c r e e n i n g UV a b s o r b i n g compounds i n p h y s i o l o g i c a l m a t e r i a l s ( b l o o d serum) u s i n g e l u t i o n w i t h l i n e a r o r m u l t i - l i n e a r g r a d i e n t s o f sodium c h l o r i d e 14 i n 0.05 m o l / l Tris-HC1 b u f f e r
.
S i m i l a r s e p a r a t i o n s t o t h o s e on anion-exchange r e s i n s , b u t c o n s i d e r a b l y f a s t e r , have been achieved u s i n g reversed-phase g r a d i e n t e l u t i o n chromatography. F o r References on p . 470.
470 example, on a s t a n d a r d a n a l y t i c a l column packed w i t h 5 pm o c t a d e c y l s i l i c a m a t e r i a l , i t was p o s s i b l e t o r e s o l v e ca. 120 peaks o f u r i n a r y a c i d s and o t h e r compounds i n
u r i n e samples i n 30 min by e l u t i o n w i t h a g r a d i e n t o f i n c r e a s i n g a c e t o n i t r i l e c o n c e n t r a t i o n i n 0.1 mol/l aqueous phosphate15y16.
An example o f such a separa-
t i o n i s shown on F i g . 4.16. S i m i l a r t e c h n i q u e s have been used f o r t h e a n a l y s i s o f human b l o o d serum 17-22 and o t h e r body f l u i d s 1 9 y 2 2 ( u s u a l l y i n ca. 30-45 m i n ) on columns packed w i t h m i c r o p a r t i c u l a t e o c t a d e c y l s i l i c a m a t e r i a l , u s i n g e l u t i o n 17,18,22 or
w i t h g r a d i e n t s o f methanol i n aqueous potassium d i h y d r o g e n phosphate
sodium a c e t a t e b u f f e r s 19y20 (pH i n t h e range 2.5-5.6). UV d e t e c t i o n a t 254 o r 280 nm i s u s u a l l y employed. Simultaneous UV and f l u o r i m e t r i c d e t e c t i o n i s h e l p f u l i n t h e i d e n t i f i c a t i o n o f sample compounds16 and t h i s may be f u r t h e r improved u s i n g more s e l e c t i v e d e t e c t o r s , such as t h e amperometric type22. The use o f m i c r o b o r e o c t a d e c y l s i l i c a columns (250-1000 mm l o n g , 1 mm I.D.) i n c o n n e c t i o n w i t h g r a d i e n t e l u t i o n seems p r o m i s i n g f o r body f l u i d a n a l y s i s
21
.
33.1, REFERENCES
1 S. K a t z and C.A. B u r t i s , J . Chromatogr., 40 (1969) 270. 2 C.D. S c o t t , J.E. A t t r i l l and N.G. Anderson, Proc. Soc. E p . BioZ. Med., 125 (1967) 181. 3 C.D. S c o t t , CZin. Chem., 14 (1968) 521. P i t t and W.F. Johnson, h e r . J . Clin. Pathol, 4 C.D. S c o t t , R.L. J o l l e y , 53 (1970) 701. 5 E D . S c o t t , F. Wayne and V.E. Walker, Anal. Biochem., 32 (1969) 182. 6 C.A. B u r t i s and K.S. Warren, CZin. Chem., 14 (1968) 29uT x. S c o t t , h e r . J . Clin. PathoZ., 7 C.A. B u r t i s , W.C. B u t t s , W.T. Rainey and C 53 (1970) 739. 8 Katz, J . Chromatogr., 53 (1970) 415. 9 C.A. B u r t i s , J . Chromatogr., 52 (1970) 97. 10 F. Geeraerts, L. S c h i m p f e s s e l a n d R. Crokaert, J . Chromatcgr., 145 (1978) 63. 11 K. Seta, M. Washitake, T. Anmo, N. T a k a i and T. Okuyama, J . Chromatogr., 181 (1980) 311.
m.
12 K. Seta, M. Washitake, I . Tanaka, N. Takai and T. Okuvama, . J . Chromatoqr.. ~. 221 (1980) 215. 13 m i y a g i , 3. Miura, Y. Takata and S. Ganno, CZin. Chem., 25 (1979) 1617. (1976) 321. 14 S. Ho Chang, K.M. Gooding and F.E. Regnier, J . Chrornatogr.,E (19771 391. 15 I. M o l n l r and Cs. H o r v l t h , J . Chromatogr., 16 I. M o l n i r , Cs. H o r v e t h and P. J a t l o w , Chromatographia, 11 (1978) 260. 17 A.M. K r s t u l o v i c , R.A. H a r t w i c k , P.R. Brown and K. L o h s e T J . Chromatogr.,
143
158
(1978) 365.
(1980) 169. 18 S.P. Assenza and P.R. Brown, J . Chromatogr., 19 E. J. Knudzon, Y .C. Lau, H. Veening and D.A. Dayton, CZin. Chem., 24 1978) 686. 20 F.C. S e n f t l e b e r , A.G. H a l l i n e and H. Veening, CZin. Chem., 22 (1976) 1522. (1979) 27. 21 R.P.W. S c o t t and P. Kucera, J . Chromatogr., 22 A.M. K r s t u l o v i c , L. B e r t a n i - D z i e d z i c , S.W. D z i e d z i c and S.E. G i t l o w , J . Chromatogr. , 223 (1981) 305.
185
Chapter 33 CLINICAL DIAGNOSTICS Various compounds i n p h y s i o l o g i c a l f l u i d s can be analysed u s i n g HPLC, such as p h a r m a c e u t i c a l s , drugs o f abuse, v i t a m i n s , a n t i b i o t i c s and t o x i c a n t s . A p p l i c a t i o n s o f g r a d i e n t e l u t i o n l i q u i d chromatography t o t h e s e p a r a t i o n and determinat i o n o f such compounds and t h e i r m e t a b o l i t e s have been mentioned i n s e v e r a l e a r l i e r c h a p t e r s (Chapters 17-25). Recent medical r e s e a r c h has i n d i c a t e d t h a t hundreds o f t h e m o l e c u l a r c o n s t i t uents o f human body f l u i d s may be r e l a t e d t o b o d i l y f u n c t i o n s and t o v a r i o u s i n borne d e f e c t s and o t h e r diseases. HPLC can be u t i l i z e d f o r automated h i g h - r e s o l u t i o n analyses o f UV-absorbing c o n s t i t u e n t s i n u r i n e , b l o o d serum and o t h e r physi o l o g i c a l f l u i d s . O r i g i n a l l y , anion-exchange chromatography on columns packed w i t h m i c r o p a r t i c u l a t e anion-exchange r e s i n s ( c a . 10
p a r t i c l e d i a m e t e r ) was
proposed and used f o r m o n i t o r i n g compounds i n p h y s i o l o g i c a l f l u i d s . G r a d i e n t e l u t i o n w i t h l i n e a r , convex o r m u l t i - l i n e a r g r a d i e n t s o f sodium a c e t a t e
-
acetic
a c i d b u f f e r c o n c e n t r a t i o n ( f r o m 0.015 t o 6 m o l / l ) a t a c o n s t a n t pH o f 4.4 was employed t o s e p a r a t e a v a r i e t y of a c i d i c , n e u t r a l and b a s i c compounds i n human 1-8 u r i n e . About 100-150 peaks were s e p a r a t e d and some o f them were i d e n t i f i e d
.
The o r i g i n a l t i m e o f a n a l y s i s was more t h a n 40 h w i t h columns 1-3.2 m l o n g , b u t i t was l a t e r decreased t o ca. 20-30 h by u s i n g a simultaneous t e m p e r a t u r e g r a d i e n t
( f r o m 20 t o 60°C)9y10.
W i t h s h o r t e r columns and h i g h e r f l o w - r a t e s t h e s e separa-
t i o n s have been accomplished r e c e n t l y i n ca. 2 h a t t h e expense o f decreased r e s o l u t i o n and peak c a p a c i t y ; however, t h e method m o d i f i e d i n t h i s way has become u s e f u l f o r r o u t i n e c l i n i c a l analyses 11
.
O t h e r g r a d i e n t e l u t i o n systems have been proposed, such as a g r a d i e n t o f i n 12 c r e a s i n g c o n c e n t r a t i o n o f ammonium p e r c h l o r a t e i n w a t e r (0-0.25 m o l / l i n 2 h ) o r a g r a d i e n t o f a c e t o n i t r i l e i n aqueous ammonium c h l o r i d e 1 3 . UV d e t e c t i o n a t v a r i o u s wavelengths (200, 215, 225, 230, 254, 260 and 280 nm) and f l u o r i m e t r i c d e t e c t i o n were used i n t h e s e a p p l i c a t i o n s . C h e m i c a l l y bonded weak a n i o n exchangers w i t h p o l y e t h y l e n i m i n e , d i e t h y l a m i n e o r d i e t h y l a m i n o e t h y l f u n c t i o n a l groups on a c o n t r o l l e d - p o r o s i t y g l a s s s u p p o r t were used f o r s c r e e n i n g UV a b s o r b i n g compounds i n p h y s i o l o g i c a l m a t e r i a l s ( b l o o d serum) u s i n g e l u t i o n w i t h l i n e a r o r m u l t i - l i n e a r g r a d i e n t s o f sodium c h l o r i d e 14 i n 0.05 m o l / l Tris-HC1 b u f f e r
.
S i m i l a r s e p a r a t i o n s t o t h o s e on anion-exchange r e s i n s , b u t c o n s i d e r a b l y f a s t e r , have been achieved u s i n g reversed-phase g r a d i e n t e l u t i o n chromatography. F o r References on p . 470.
470 example, on a s t a n d a r d a n a l y t i c a l column packed w i t h 5 pm o c t a d e c y l s i l i c a m a t e r i a l , i t was p o s s i b l e t o r e s o l v e ca. 120 peaks o f u r i n a r y a c i d s and o t h e r compounds i n
u r i n e samples i n 30 min by e l u t i o n w i t h a g r a d i e n t o f i n c r e a s i n g a c e t o n i t r i l e c o n c e n t r a t i o n i n 0.1 mol/l aqueous phosphate15y16.
An example o f such a separa-
t i o n i s shown on F i g . 4.16. S i m i l a r t e c h n i q u e s have been used f o r t h e a n a l y s i s o f human b l o o d serum 17-22 and o t h e r body f l u i d s 1 9 y 2 2 ( u s u a l l y i n ca. 30-45 m i n ) on columns packed w i t h m i c r o p a r t i c u l a t e o c t a d e c y l s i l i c a m a t e r i a l , u s i n g e l u t i o n 17,18,22 or
w i t h g r a d i e n t s o f methanol i n aqueous potassium d i h y d r o g e n phosphate
sodium a c e t a t e b u f f e r s 19y20 (pH i n t h e range 2.5-5.6). UV d e t e c t i o n a t 254 o r 280 nm i s u s u a l l y employed. Simultaneous UV and f l u o r i m e t r i c d e t e c t i o n i s h e l p f u l i n t h e i d e n t i f i c a t i o n o f sample compounds16 and t h i s may be f u r t h e r improved u s i n g more s e l e c t i v e d e t e c t o r s , such as t h e amperometric type22. The use o f m i c r o b o r e o c t a d e c y l s i l i c a columns (250-1000 mm l o n g , 1 mm I.D.) i n c o n n e c t i o n w i t h g r a d i e n t e l u t i o n seems p r o m i s i n g f o r body f l u i d a n a l y s i s
21
.
33.1, REFERENCES
1 S. K a t z and C.A. B u r t i s , J . Chromatogr., 40 (1969) 270. 2 C.D. S c o t t , J.E. A t t r i l l and N.G. Anderson, Proc. Soc. E p . BioZ. Med., 125 (1967) 181. 3 C.D. S c o t t , CZin. Chem., 14 (1968) 521. P i t t and W.F. Johnson, h e r . J . Clin. Pathol, 4 C.D. S c o t t , R.L. J o l l e y , 53 (1970) 701. 5 E D . S c o t t , F. Wayne and V.E. Walker, Anal. Biochem., 32 (1969) 182. 6 C.A. B u r t i s and K.S. Warren, CZin. Chem., 14 (1968) 29uT x. S c o t t , h e r . J . Clin. PathoZ., 7 C.A. B u r t i s , W.C. B u t t s , W.T. Rainey and C 53 (1970) 739. 8 Katz, J . Chromatogr., 53 (1970) 415. 9 C.A. B u r t i s , J . Chromatogr., 52 (1970) 97. 10 F. Geeraerts, L. S c h i m p f e s s e l a n d R. Crokaert, J . Chromatcgr., 145 (1978) 63. 11 K. Seta, M. Washitake, T. Anmo, N. T a k a i and T. Okuyama, J . Chromatogr., 181 (1980) 311.
m.
12 K. Seta, M. Washitake, I . Tanaka, N. Takai and T. Okuvama, . J . Chromatoqr.. ~. 221 (1980) 215. 13 m i y a g i , 3. Miura, Y. Takata and S. Ganno, CZin. Chem., 25 (1979) 1617. (1976) 321. 14 S. Ho Chang, K.M. Gooding and F.E. Regnier, J . Chrornatogr.,E (19771 391. 15 I. M o l n l r and Cs. H o r v l t h , J . Chromatogr., 16 I. M o l n i r , Cs. H o r v e t h and P. J a t l o w , Chromatographia, 11 (1978) 260. 17 A.M. K r s t u l o v i c , R.A. H a r t w i c k , P.R. Brown and K. L o h s e T J . Chromatogr.,
143
158
(1978) 365.
(1980) 169. 18 S.P. Assenza and P.R. Brown, J . Chromatogr., 19 E. J. Knudzon, Y .C. Lau, H. Veening and D.A. Dayton, CZin. Chem., 24 1978) 686. 20 F.C. S e n f t l e b e r , A.G. H a l l i n e and H. Veening, CZin. Chem., 22 (1976) 1522. (1979) 27. 21 R.P.W. S c o t t and P. Kucera, J . Chromatogr., 22 A.M. K r s t u l o v i c , L. B e r t a n i - D z i e d z i c , S.W. D z i e d z i c and S.E. G i t l o w , J . Chromatogr. , 223 (1981) 305.
185