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A rapid micromethod for measuring theophylline in serum by reverse-phase high-performance liquid chromatography
Clin. Biochem 10, (2) 74-77 (1977)
A Rapid Micromethod for Measuring Theophylline in Serum by Reverse-Phase High-Performance Liquid Chromatography S.J. SOLDIN and J.G. HILL Service Division, Department of Biochemistry, The Hospital for Sick Children, and Department of Clinical Biochemistry, University of Toronto, Toronto, Ontario, Canada (Accepted October 25, 1976)
CLBIA, 10, (2) 74-77 (1977) Clin. Biochem. Soldin, S. J., and Hill, J. G.
Service Division, Department o/ Biochemistry, The Hospital for Sick Chil&'en, and Department of Clinical Biochemistry, University of Toronto, Toronto, Ontario, Canada A R A P I D MICROMETHOD FOR M E A S U R I N G T H E O P H Y L L I N E IN SERUM BY R E V E R S E P H A S E H I G H - P E R F O R M A N C E LIQUID CHROMATOGRAPHY We describe an assay system for measuring theophylline in 25 ~l of serum. The procedure involves extraction with a 95:5 mixture of chloroform:isopropanol containing fl-hydroxypropyltheophylline as internal standard, and reverse-phase chromatography on a 4 mm x 30 cm column containing "~t Bondapak C18." Theophylline and fl-hydroxypropyltheophylline are eluted with a 90:10 mixture of sodium acetate buffer (20 mmoles/litre pH 4.0) and acetonitrile a t a flow r a t e of 1.8 ml/min., are detected by their absorbance a t 254 nm, and quantitated by measuring peak areas. Column t e m p e r a t u r e has not been found to be critical in this analysis. Each analysis requires 9 minutes of chromatography time with a total analysis time of 20 minutes. Analytical recoveries were found to be 71 to 75% for theophylline and 94% for ~-hydroxypropyltheophylline. This difference in recovery is corrected when determining the theophylline concentration in unknown samples. The method has good precision (coefficients of variation between 7.0% and 7.9c/e for therapeutic and toxic concentrations). The results obtained with this method comp a r e favourably with results obtained by a published cation-exchange high-performance liquid chromatographic method. None of the metabolites of theophylline, common compounds related to theophylline in structure or drugs tested, have been found to interfere with the analysis described.
t i o n s s i g n i f i c a n t v a r i a t i o n s in i n d i v i d u a l r e s p o n s e s to g i v e n a m o u n t s o f t h e d r u g have been r e c o r d e d , l e a d i n g to s u b o p t i m a l levels a n d t h e r a p e u t i c f a i l u r e s on some occasions, o r excess levels a n d s e r i o u s toxic e f f e c t s on o t h e r s . I n o r d e r to e n s u r e t h e m a x i m u m t h e r a p e u t i c e f f e c t o f t h e o p h y l l i n e , it is d e s i r a b l e to i n d i v i d u a l i z e t h e t r e a t m e n t r e g i m e n f o r each p a t i e n t , a n d t h i s can only be a c h i e v e d b y c a r e f u l a n d f r e q u e n t m o n i t o r i n g o f p l a s m a levels. T h i s is p a r t i c u l a r l y t r u e in m a n a g i n g t i n y i n f a n t s w i t h a p n e a , b u t also a p p l i e s w h e n t h e use o f i n t r a v e n o u s t h e o p h y l l i n e is b e i n g c o n t e m p l a t e d in t r e a t i n g a p a t i e n t in s t a t u s a s t h m a t i c u s . M a n y m e t h o d s h a v e been a d v o c a t e d f o r t h e m e a s u r e m e n t of t h e o p h y l l i n e in s e r u m . M o s t of t h e s e a r e modif i c a t i o n s of t h e u l t r a - v i o l e t m e t h o d of S c h a c k a n d W a x l e r 1, a n d s h a r e to v a r y i n g d e g r e e s t h e d i s a d v a n t a g e s of u s i n g a l a r g e volume of sample, r e q u i r i n g a lengthy analysis time, and lacking specificity. Recently, g a s - l i q u i d c h r o m a t o g r a p h i c ~' "~'4 a n d h i g h - p e r f o r m ance liquid c h r o m a t o g r a p h i c 5' ~"7 p r o c e d u r e s h a v e been described, and these provide many advantages with r e s p e c t to s e n s i t i v i t y a n d s p e c i f i c i t y . H o w e v e r , m o n i t o r i n g s e r u m levels of t h e o p h y l l i n e in p r e m a t u r e inf a n t s o b v i o u s l y r e q u i r e s t h e use o f m i c r o samples, a n d t h e only t r u l y m i c r o m e t h o d p u b l i s h e d to d a t e is t h a t of L e a s t et al~. I n t h a t p r o c e d u r e , t h e o p h y l l i n e is m e a s u r e d in 20 p l of s e r u m b y a g a s - l i q u i d c h r o m a t o g r a p h i c technique employing a nitrogen sensitive detector, and r e q u i r i n g 45 m i n u t e s f o r each a n a l y s i s . T h e h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h i c m e t h od we d e s c r i b e can be p e r f o r m e d on 25 /~l o f s e r u m / p l a s m a , e m p l o y s r e v e r s e - p h a s e c h r o m a t o g r a p h y , h a s an a n a l y s i s t i m e of only 20 m i n u t e s ( i n c l u d i n g n i n e m i n u t e s o f c h r o m a t o g r a p h y t i m e ) , is s p e c i f i c f o r t h e o phylline, a n d h a s p r o v e n r e l i a b l e in a r o u t i n e l a b o r a tory setting.
THEOPHYLLINE HAS BEEN USED in t h e t r e a t m e n t of a s t h m a f o r m a n y y e a r s , a n d m o r e r e c e n t l y in t h e m a n a g e m e n t o f a p n e a o f t h e n e w b o r n . I n b o t h condi-
Apparatus
Correspondence: Dr. S. J. Soldin, Service Division, Dep a r t m e n t of Biochemistry, The Hospital for Sick Children, Toronto, Ontario M5G 1X8
Throughout these studies we used a high-performance liquid chromatograph, Model ALC/GPC-204/6000A, with a Model U6P injection system, and a 4 mm x 30 cm "~ Bondapak C-18" column, all from W a t e r s Associates, Inc., Milford, Mass. 07157. The detector was a W a t e r s Model 440 with 254 nm wavelength kit.
MATERIALS A N D METHODS
ASSAY SYSTEM FOR M E A S U R I N G T H E O P H Y L L I N E IN SERUM
75
RESULTS AND DISCUSSION
Reagents We obtained acetonitrile, methanol, chloroform and dimethylsulfoxide from Burdick and Jackson Laboratories, Inc., 1953 S. Harvey St., Muskegon, MI 449442, U.S.A. Chemicals were also purchased from ICN Pharmaceuticals, Inc., Life Sciences Group, Plainview, N.Y. (hypoxanthine, xanthine, theobromine, 3-methylxanthine and theophylline); from C. H. Boehringer Sohn, Ingelheim im Rhein and distributed by Henley and Co., New York, N.Y. 10004 (/J-hydroxypropyltheophylline); from K & K Laboratories, Inc., Plainview, N.Y. (1-methylxanthine) from Eastman Kodak Co., Rochester, N.Y. 14650 (caffeine); and from Fisher Scientific Company, Chemical Manufact u r i n g Division, F a i r Lawn, N.J. 07410 (uric acid, isopropanol).
Relative Response Factor By i n j e c t i n g k n o w n a m o u n t s of t h e o p h y l l i n e a n d int e r n a l s t a n d a r d , we d e m o n s t r a t e d a l i n e a r r e l a t i o n s h i p between peak a r e a a n d d r u g c o n c e n t r a t i o n over a conc e n t r a t i o n r a n g e f a r in excess of t h a t r e q u i r e d f o r clinical purposes. F i g u r e 1 shows the d a t a obtained. F r o m this graph, a r e l a t i v e response f a c t o r of 1.86 was calculated. F i g u r e 2 shows the type of e l u t i o n p a t t e r n o b t a i n e d for a s e r u m specimen c o n t a i n i n g 15.8 m g t h e o p h y l l i n e per litre.
Procedure
.005 -
Add 250 ill of a 95:5 mixture of chloroform:isopropanol containing 2.2 ~g /#-hydroxypropyltheophylline (as an internal standard) to 25 ~tl of serum or plasma in a 6 x 50 mm glass test tube. Vortex the mixture for 1 minute, then centrifuge for 1 minute. Pipette lower organic phase into a clean test tube and evaporate at 60° under nitrogen (approx. 5 minutes). Dissolve residue in 50 ~l of 20 mmole/litre sodium acetate buffer pH 4.0, vortex for 1 minute, inject 5 ~l of the resulting solution directly into the chromatograph, and elute with the same buffer at a flow rate of 1.8 ml/min. The pressure at the head of the column is approximately 2,000 p.s.i. The detector sensitivity setting is 0.005A Full Scale. Peaks on the chromatogram are identified on the basis of their retention times relative to the internal standard. Theophylline concentration is calculated from the peak areas according to the formula:
O eo~
>.
o. o m
..~
Q.
ob. a. o "0
W
Concentration of theophylline equals A C --X--X-B D
4--
.004 -
,a
.003 -
U
z <
E
F
° c~
where A = peak area for theophylline B = peak area for /~-hydroxypropyltheophylline C = recovery of ~-hydroxypropyltheophylline D--~ recovery of theophylline E : concentration of fl-hydroxypropyltheophylline F : 1/R.R. where R.R. is the relative response (in terms of peak area) of theophylline compared to an equal amount of ~-hydroxypropyltheophylline. The ~t Bondapak columns are washed daily with methanol, and weekly with acetonitrile and dimethylsulfoxide. Under the conditions described, a column may be used for at least 1200 analyses before requiring replacement.
0
t~
<
tO
.002 -
.001
=~
-
Theophylllne 70
° t i50-
o/J~.Hydroxyp¢opyhheophyll~ne
6o
o
40-
lo-
, 0
I 5
I
I 10
I
15
MINUTES
30--
0
I 0
I 10
I 20
I 30
I 40
I 50
'::',i? ! 60
I 70
I 80
I 90
I 100
I 110
u.,,. I 120
ng injecled
Fig. 1 - - Determination o/ the Relative Response Factor. 0 O, Theophylline; @ - - - @ , fl-HydroxypropyltheophyUi~e.
Figure 2 - - The elution pattern obtained using procedure outlined for a ser~m sample containing 15.8 mg th~ophylline/litre.
Precision We assessed the p r e c i s i o n of the method by r e p e a t e d a n a l y s e s of s e r u m s p e c i m e n s c o n t a i n i n g t h e r a p e u t i c a n d toxic c o n c e n t r a t i o n s of theophylline. As s h o w n i n Table 1, the b e t w e e n - d a y p r e c i s i o n (CV) v a r i e d be-
SOLDIN AND H I L L
76
Table 2
Table 1 B E T W E E N - D A Y PRECISION AND RELATIVE RECOVERIES BY REPLICATE ANALYSIS
Number 10 10 10
mg/litre 7.9 15.7 39.3
C.V.
Mean % relative recovery
8.1 7.0 7.9
100.5 98.9 98.7
t w e e n 7.0% a n d 8.1%. T h e s e values a r e a c c e p t a b l e f o r m o s t clinical p u r p o s e s a n d c o m p a r e well w i t h p r e v i o u s l y r e p o r t e d v a l u e s o b t a i n e d w i t h g a s - l i q u i d chromatography ~ and high-performance liquid°chromatog r a p h y 5"'.
RECOVERY
OF
THEOPHYLL1NE AND .J~-HYDROXYPROPYLTHEOPHYLLINE A D D E D
TO i-'LASMA
Plasma Mean level recovery (mg/litre) (%)
Compound Theophylline . . . . . . . . . . . [~-Hydroxypropyltheophylline . . . . . . . . . .
SD of recovery (mg/litre)
7.9 15.7 39.3
75 71 72
0.21 0.65 0.65
75
93
1.25
Table 3 RETENTION
TIME OF THEOPHYLL1NE AND OF RELATED COMPOUNDS
Accuracy W e m e a s u r e d the a b s o l u t e r e c o v e r y f r o m p l a s m a o f t h e o p h y l l i n e a n d t h e i n t e r n a l s t a n d a r d in t h e f o l l o w i n g way. T h e two c o m p o u n d s w e r e a d d e d to d r u g - f r e e plasm a to a c h i e v e t h e c o n c e n t r a t i o n s shown in Table 2. The a n a l y s i s p r o c e d u r e w a s t h e n c a r r i e d out as des c r i b e d e x c e p t t h a t t h e 250 p l of 95:5 solution of chlor o f o r m ' i s o p r o p a n o l a d d e d to t h e 25 /~l a l i q u o t o f p l a s m a c o n t a i n e d no f l - h y d r o x y p r o p y l t h e o p h y l l i n e . C a r e f u l l y m e a s u r e d a l i q u o t s of t h e f i n a l solution w e r e chromatographed and the peak areas determined. Percentage recovery was calculated by comparing these peak areas with the peak areas obtained by the direct i n j e c t i o n o f t h e p u r e compounds. A s shown in T a b l e 2, the absolute % recovery of theophylline at plasma levels o f 7.9, 15.7, a n d 39.3 m g / l i t r e w a s f o u n d to be 75, 71, a n d 72% r e s p e c t i v e l y . T h e a b s o l u t e r e c o v e r y of B - h y d r o x y p r o p y l t h e o p h y l l i n e w a s f o u n d to be 93% a t 75 m g / l i t r e . I n Table 1 we h a v e shown t h a t t h e r e c o v e r y of theop h y l l i n e r e l a t i v e to i n t e r n a l s t a n d a r d v a r i e d b e t w e e n 98.7 a n d 100.5% a t t h e o p h y l l i n e c o n c e n t r a t i o n s bet w e e n 7.9 a n d 39.3 m g / l i t r e . A c c u r a c y w a s f u r t h e r e v a l u a t e d b y a n a l y s i n g a ser i e s of p a t i e n t s ' s p e c i m e n s by a p r e v i o u s l y p u b l i s h e d p r o c e d u r e ( h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y using a strong cation-exchange resin) and the proposed r e v e r s e - p h a s e p r o c e d u r e . A s can be seen in F i g . 3, t h e two p r o c e d u r e s a g r e e d r e m a r k a b l y well, h a v i n g a c o r r e l a t i o n c o e f f i c i e n t of 0.97. Metabolite
and Drug
Interference
In m a n t h e o p h y l l i n e is m e t a b o l i z e d to 1 , 3 - d i m e t h y l u r i c acid, 3 - m e t h y l x a n t h i n e a n d 1 - m e t h y l u r i c a c i d a" 9.10 N o n e o f t h e s e m e t a b o l i t e s , o r a n u m b e r o f closely r e l a t e d compounds, w a s f o u n d to i n t e r f e r e w i t h t h e a n a l y s i s d e s c r i b e d ( T a b l e 3). O u r o r i g i n a l a t t e m p t s to e s t a b l i s h a t h e o p h y l l i n e a s s a y w e r e b a s e d on t h e n o n - e x t r a c t i v e p r o c e d u r e o u t lined by F r a n c o n i et al I1. U n f o r t u n a t e l y , we q u i c k l y f o u n d t h a t two f r e q u e n t l y used a n t i b i o t i c s , a m p i c i l l i n and methicillin, interfered with the analysis by giving r i s e to p e a k s v e r y close to t h e o p h y l l i n e in r e t e n t i o n t i m e . To e l i m i n a t e t h i s p r o b l e m , we m o d i f i e d t h e p r o c e d u r e by i n t r o d u c i n g a p r e l i m i n a r y e x t r a c t i o n s t e p a n d by c h a n g i n g t h e i n t e r n a l s t a n d a r d f r o m 8-chlorot h e o p h y l l i n e to t h e m o r e h y d r o p h o b i c f l - h y d r o x y p r o p y l t h e o p h y l l i n e . T h e m o d i f i e d p r o c e d u r e has now been
Retention time (secs.)
Compound Theophylline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~-hydroxypropyltheophylline . . . . . . . . . . . . . . . Uric acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-methyluric acid . . . . . . . . . . . . . . . . . . . . . . . . . 3-methyluric acid . . . . . . . . . . . . . . . . . . . . . . . . . 1, 3-dimethyluric acid . . . . . . . . . . . . . . . . . . . . . Xanthine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-methylxanthine . . . . . . . . . . . . . . . . . . . . . . . . . 3-methylxanthine . . . . . . . . . . . . . . . . . . . . . . . . . Hypoxanthine . . . . . . . . . . . . . . . . . . . . . . . . . . . . Theobromine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caffeine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
293 534 107 142 120 194 120 176 159 120 219 508
20-
1614-12-10--
I~J
8-
•
ou
"
£iii::
4-
y intercept = --0.28
2C
0 -.
I
I
I
l
l
I
I
I
I
I
2
4
6
8
!0
12
14
16
18
20
REVERSE-PHASE HPLC (mg/iitre)
F i g u r e 3 -~- Comparison of Reverse-Phase H P L C method described w i t h a Cation-Exchange H P L C method.
a p p l i e d to m o r e t h a n two h u n d r e d a n d f i f t y p a t i e n t s ' s p e c i m e n s , a n d no i n t e r f e r e n c e has been o b s e r v e d w i t h ampicillin, methicillin, gentamicin, clindamycin, penicillin G, k a n a m y c i n , c e p h a l o r i d i n e t e t r a c y c l i n e , e r y t h r o m y c i n , o r c i p r e n a l i n e , a n d s a l b u t a m o l . In a d d i t i o n , t h e n o n - i n t e r f e r i n g d r u g s l i s t e d b y F r a n c o n i et aP t m a y r e a s o n a b l y be a d d e d to t h i s l i s t b e c a u s e o f t h e s i m i l a r i t i e s in t h e c h r o m a t o g r a p h i c p h a s e s of t h e t w o procedures.
ASSAY SYSTEM FOR MEASURING THEOPHYLLINE IN SERUM ACKNOWLEDGEMENTS H P L C comparison studies were made possible t h r o u g h the kind cooperation of J e f f r e y R. Koup, School of Pharmacy, State University of New York at Buffalo. Helpful discussions with D. M. Goldberg, and the skilled technical assistance of T. Gero and J. Cook are gratefully acknowledged. REFERENC$S
1. Schack, J. A., and Waxler, S. H. (1949). J. Pharmacol. Exp. Ther. 97, 283-291. 2. Johnson, G. F., DechtiarLtk, W. A., and Solomon, H. M. (1975). Clin. Chem. 21, 144-147. 3. Thompson, R. D., Nagasawa, H. T., and Jenne, J. W. (1974) J. Lab. Clin. Med. 84, 584-593.
77
4. Least, C. J., Johnson, G. F., and Solomon, H. M. (1976). Clin. Chem. 22, 765-768. 5. Sitar, D. S., Piafsky, K. M., Rangno, R. E., and Ogilvie, R. I. (1975). Clin. Chem. 21, 1774-1776. 6. Weinberger, M., and Chidsey, C. (1975). Clin. Chem. 21, 834-837. 7. Evenson, M. A., and Warren, B. L. (1976). Clin. Chem. 22, 851-855. 8. Brodie, B. B., Axelrod, J., and Reichenthal, J. (1952). J. Biol. Chem. 194, 215-222. 9. Johnson, E. A. (1952). Biochem. J. 51, 133-138. 10. Weinfeld, H., and Christman, A. A. (1953). J. Biol. Chem. 200, 345-355. 11. Franconi, L. C., Sandmann, B. J., Hawk, G. L., and Haney, W. G. (1976). Anal. Chem. 48, 372-375.
A Rapid Micromethod for Measuring Theophylline in Serum by Reverse-Phase High-Performance Liquid Chromatography S.J. SOLDIN and J.G. HILL Service Division, Department of Biochemistry, The Hospital for Sick Children, and Department of Clinical Biochemistry, University of Toronto, Toronto, Ontario, Canada (Accepted October 25, 1976)
CLBIA, 10, (2) 74-77 (1977) Clin. Biochem. Soldin, S. J., and Hill, J. G.
Service Division, Department o/ Biochemistry, The Hospital for Sick Chil&'en, and Department of Clinical Biochemistry, University of Toronto, Toronto, Ontario, Canada A R A P I D MICROMETHOD FOR M E A S U R I N G T H E O P H Y L L I N E IN SERUM BY R E V E R S E P H A S E H I G H - P E R F O R M A N C E LIQUID CHROMATOGRAPHY We describe an assay system for measuring theophylline in 25 ~l of serum. The procedure involves extraction with a 95:5 mixture of chloroform:isopropanol containing fl-hydroxypropyltheophylline as internal standard, and reverse-phase chromatography on a 4 mm x 30 cm column containing "~t Bondapak C18." Theophylline and fl-hydroxypropyltheophylline are eluted with a 90:10 mixture of sodium acetate buffer (20 mmoles/litre pH 4.0) and acetonitrile a t a flow r a t e of 1.8 ml/min., are detected by their absorbance a t 254 nm, and quantitated by measuring peak areas. Column t e m p e r a t u r e has not been found to be critical in this analysis. Each analysis requires 9 minutes of chromatography time with a total analysis time of 20 minutes. Analytical recoveries were found to be 71 to 75% for theophylline and 94% for ~-hydroxypropyltheophylline. This difference in recovery is corrected when determining the theophylline concentration in unknown samples. The method has good precision (coefficients of variation between 7.0% and 7.9c/e for therapeutic and toxic concentrations). The results obtained with this method comp a r e favourably with results obtained by a published cation-exchange high-performance liquid chromatographic method. None of the metabolites of theophylline, common compounds related to theophylline in structure or drugs tested, have been found to interfere with the analysis described.
t i o n s s i g n i f i c a n t v a r i a t i o n s in i n d i v i d u a l r e s p o n s e s to g i v e n a m o u n t s o f t h e d r u g have been r e c o r d e d , l e a d i n g to s u b o p t i m a l levels a n d t h e r a p e u t i c f a i l u r e s on some occasions, o r excess levels a n d s e r i o u s toxic e f f e c t s on o t h e r s . I n o r d e r to e n s u r e t h e m a x i m u m t h e r a p e u t i c e f f e c t o f t h e o p h y l l i n e , it is d e s i r a b l e to i n d i v i d u a l i z e t h e t r e a t m e n t r e g i m e n f o r each p a t i e n t , a n d t h i s can only be a c h i e v e d b y c a r e f u l a n d f r e q u e n t m o n i t o r i n g o f p l a s m a levels. T h i s is p a r t i c u l a r l y t r u e in m a n a g i n g t i n y i n f a n t s w i t h a p n e a , b u t also a p p l i e s w h e n t h e use o f i n t r a v e n o u s t h e o p h y l l i n e is b e i n g c o n t e m p l a t e d in t r e a t i n g a p a t i e n t in s t a t u s a s t h m a t i c u s . M a n y m e t h o d s h a v e been a d v o c a t e d f o r t h e m e a s u r e m e n t of t h e o p h y l l i n e in s e r u m . M o s t of t h e s e a r e modif i c a t i o n s of t h e u l t r a - v i o l e t m e t h o d of S c h a c k a n d W a x l e r 1, a n d s h a r e to v a r y i n g d e g r e e s t h e d i s a d v a n t a g e s of u s i n g a l a r g e volume of sample, r e q u i r i n g a lengthy analysis time, and lacking specificity. Recently, g a s - l i q u i d c h r o m a t o g r a p h i c ~' "~'4 a n d h i g h - p e r f o r m ance liquid c h r o m a t o g r a p h i c 5' ~"7 p r o c e d u r e s h a v e been described, and these provide many advantages with r e s p e c t to s e n s i t i v i t y a n d s p e c i f i c i t y . H o w e v e r , m o n i t o r i n g s e r u m levels of t h e o p h y l l i n e in p r e m a t u r e inf a n t s o b v i o u s l y r e q u i r e s t h e use o f m i c r o samples, a n d t h e only t r u l y m i c r o m e t h o d p u b l i s h e d to d a t e is t h a t of L e a s t et al~. I n t h a t p r o c e d u r e , t h e o p h y l l i n e is m e a s u r e d in 20 p l of s e r u m b y a g a s - l i q u i d c h r o m a t o g r a p h i c technique employing a nitrogen sensitive detector, and r e q u i r i n g 45 m i n u t e s f o r each a n a l y s i s . T h e h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h i c m e t h od we d e s c r i b e can be p e r f o r m e d on 25 /~l o f s e r u m / p l a s m a , e m p l o y s r e v e r s e - p h a s e c h r o m a t o g r a p h y , h a s an a n a l y s i s t i m e of only 20 m i n u t e s ( i n c l u d i n g n i n e m i n u t e s o f c h r o m a t o g r a p h y t i m e ) , is s p e c i f i c f o r t h e o phylline, a n d h a s p r o v e n r e l i a b l e in a r o u t i n e l a b o r a tory setting.
THEOPHYLLINE HAS BEEN USED in t h e t r e a t m e n t of a s t h m a f o r m a n y y e a r s , a n d m o r e r e c e n t l y in t h e m a n a g e m e n t o f a p n e a o f t h e n e w b o r n . I n b o t h condi-
Apparatus
Correspondence: Dr. S. J. Soldin, Service Division, Dep a r t m e n t of Biochemistry, The Hospital for Sick Children, Toronto, Ontario M5G 1X8
Throughout these studies we used a high-performance liquid chromatograph, Model ALC/GPC-204/6000A, with a Model U6P injection system, and a 4 mm x 30 cm "~ Bondapak C-18" column, all from W a t e r s Associates, Inc., Milford, Mass. 07157. The detector was a W a t e r s Model 440 with 254 nm wavelength kit.
MATERIALS A N D METHODS
ASSAY SYSTEM FOR M E A S U R I N G T H E O P H Y L L I N E IN SERUM
75
RESULTS AND DISCUSSION
Reagents We obtained acetonitrile, methanol, chloroform and dimethylsulfoxide from Burdick and Jackson Laboratories, Inc., 1953 S. Harvey St., Muskegon, MI 449442, U.S.A. Chemicals were also purchased from ICN Pharmaceuticals, Inc., Life Sciences Group, Plainview, N.Y. (hypoxanthine, xanthine, theobromine, 3-methylxanthine and theophylline); from C. H. Boehringer Sohn, Ingelheim im Rhein and distributed by Henley and Co., New York, N.Y. 10004 (/J-hydroxypropyltheophylline); from K & K Laboratories, Inc., Plainview, N.Y. (1-methylxanthine) from Eastman Kodak Co., Rochester, N.Y. 14650 (caffeine); and from Fisher Scientific Company, Chemical Manufact u r i n g Division, F a i r Lawn, N.J. 07410 (uric acid, isopropanol).
Relative Response Factor By i n j e c t i n g k n o w n a m o u n t s of t h e o p h y l l i n e a n d int e r n a l s t a n d a r d , we d e m o n s t r a t e d a l i n e a r r e l a t i o n s h i p between peak a r e a a n d d r u g c o n c e n t r a t i o n over a conc e n t r a t i o n r a n g e f a r in excess of t h a t r e q u i r e d f o r clinical purposes. F i g u r e 1 shows the d a t a obtained. F r o m this graph, a r e l a t i v e response f a c t o r of 1.86 was calculated. F i g u r e 2 shows the type of e l u t i o n p a t t e r n o b t a i n e d for a s e r u m specimen c o n t a i n i n g 15.8 m g t h e o p h y l l i n e per litre.
Procedure
.005 -
Add 250 ill of a 95:5 mixture of chloroform:isopropanol containing 2.2 ~g /#-hydroxypropyltheophylline (as an internal standard) to 25 ~tl of serum or plasma in a 6 x 50 mm glass test tube. Vortex the mixture for 1 minute, then centrifuge for 1 minute. Pipette lower organic phase into a clean test tube and evaporate at 60° under nitrogen (approx. 5 minutes). Dissolve residue in 50 ~l of 20 mmole/litre sodium acetate buffer pH 4.0, vortex for 1 minute, inject 5 ~l of the resulting solution directly into the chromatograph, and elute with the same buffer at a flow rate of 1.8 ml/min. The pressure at the head of the column is approximately 2,000 p.s.i. The detector sensitivity setting is 0.005A Full Scale. Peaks on the chromatogram are identified on the basis of their retention times relative to the internal standard. Theophylline concentration is calculated from the peak areas according to the formula:
O eo~
>.
o. o m
..~
Q.
ob. a. o "0
W
Concentration of theophylline equals A C --X--X-B D
4--
.004 -
,a
.003 -
U
z <
E
F
° c~
where A = peak area for theophylline B = peak area for /~-hydroxypropyltheophylline C = recovery of ~-hydroxypropyltheophylline D--~ recovery of theophylline E : concentration of fl-hydroxypropyltheophylline F : 1/R.R. where R.R. is the relative response (in terms of peak area) of theophylline compared to an equal amount of ~-hydroxypropyltheophylline. The ~t Bondapak columns are washed daily with methanol, and weekly with acetonitrile and dimethylsulfoxide. Under the conditions described, a column may be used for at least 1200 analyses before requiring replacement.
0
t~
<
tO
.002 -
.001
=~
-
Theophylllne 70
° t i50-
o/J~.Hydroxyp¢opyhheophyll~ne
6o
o
40-
lo-
, 0
I 5
I
I 10
I
15
MINUTES
30--
0
I 0
I 10
I 20
I 30
I 40
I 50
'::',i? ! 60
I 70
I 80
I 90
I 100
I 110
u.,,. I 120
ng injecled
Fig. 1 - - Determination o/ the Relative Response Factor. 0 O, Theophylline; @ - - - @ , fl-HydroxypropyltheophyUi~e.
Figure 2 - - The elution pattern obtained using procedure outlined for a ser~m sample containing 15.8 mg th~ophylline/litre.
Precision We assessed the p r e c i s i o n of the method by r e p e a t e d a n a l y s e s of s e r u m s p e c i m e n s c o n t a i n i n g t h e r a p e u t i c a n d toxic c o n c e n t r a t i o n s of theophylline. As s h o w n i n Table 1, the b e t w e e n - d a y p r e c i s i o n (CV) v a r i e d be-
SOLDIN AND H I L L
76
Table 2
Table 1 B E T W E E N - D A Y PRECISION AND RELATIVE RECOVERIES BY REPLICATE ANALYSIS
Number 10 10 10
mg/litre 7.9 15.7 39.3
C.V.
Mean % relative recovery
8.1 7.0 7.9
100.5 98.9 98.7
t w e e n 7.0% a n d 8.1%. T h e s e values a r e a c c e p t a b l e f o r m o s t clinical p u r p o s e s a n d c o m p a r e well w i t h p r e v i o u s l y r e p o r t e d v a l u e s o b t a i n e d w i t h g a s - l i q u i d chromatography ~ and high-performance liquid°chromatog r a p h y 5"'.
RECOVERY
OF
THEOPHYLL1NE AND .J~-HYDROXYPROPYLTHEOPHYLLINE A D D E D
TO i-'LASMA
Plasma Mean level recovery (mg/litre) (%)
Compound Theophylline . . . . . . . . . . . [~-Hydroxypropyltheophylline . . . . . . . . . .
SD of recovery (mg/litre)
7.9 15.7 39.3
75 71 72
0.21 0.65 0.65
75
93
1.25
Table 3 RETENTION
TIME OF THEOPHYLL1NE AND OF RELATED COMPOUNDS
Accuracy W e m e a s u r e d the a b s o l u t e r e c o v e r y f r o m p l a s m a o f t h e o p h y l l i n e a n d t h e i n t e r n a l s t a n d a r d in t h e f o l l o w i n g way. T h e two c o m p o u n d s w e r e a d d e d to d r u g - f r e e plasm a to a c h i e v e t h e c o n c e n t r a t i o n s shown in Table 2. The a n a l y s i s p r o c e d u r e w a s t h e n c a r r i e d out as des c r i b e d e x c e p t t h a t t h e 250 p l of 95:5 solution of chlor o f o r m ' i s o p r o p a n o l a d d e d to t h e 25 /~l a l i q u o t o f p l a s m a c o n t a i n e d no f l - h y d r o x y p r o p y l t h e o p h y l l i n e . C a r e f u l l y m e a s u r e d a l i q u o t s of t h e f i n a l solution w e r e chromatographed and the peak areas determined. Percentage recovery was calculated by comparing these peak areas with the peak areas obtained by the direct i n j e c t i o n o f t h e p u r e compounds. A s shown in T a b l e 2, the absolute % recovery of theophylline at plasma levels o f 7.9, 15.7, a n d 39.3 m g / l i t r e w a s f o u n d to be 75, 71, a n d 72% r e s p e c t i v e l y . T h e a b s o l u t e r e c o v e r y of B - h y d r o x y p r o p y l t h e o p h y l l i n e w a s f o u n d to be 93% a t 75 m g / l i t r e . I n Table 1 we h a v e shown t h a t t h e r e c o v e r y of theop h y l l i n e r e l a t i v e to i n t e r n a l s t a n d a r d v a r i e d b e t w e e n 98.7 a n d 100.5% a t t h e o p h y l l i n e c o n c e n t r a t i o n s bet w e e n 7.9 a n d 39.3 m g / l i t r e . A c c u r a c y w a s f u r t h e r e v a l u a t e d b y a n a l y s i n g a ser i e s of p a t i e n t s ' s p e c i m e n s by a p r e v i o u s l y p u b l i s h e d p r o c e d u r e ( h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y using a strong cation-exchange resin) and the proposed r e v e r s e - p h a s e p r o c e d u r e . A s can be seen in F i g . 3, t h e two p r o c e d u r e s a g r e e d r e m a r k a b l y well, h a v i n g a c o r r e l a t i o n c o e f f i c i e n t of 0.97. Metabolite
and Drug
Interference
In m a n t h e o p h y l l i n e is m e t a b o l i z e d to 1 , 3 - d i m e t h y l u r i c acid, 3 - m e t h y l x a n t h i n e a n d 1 - m e t h y l u r i c a c i d a" 9.10 N o n e o f t h e s e m e t a b o l i t e s , o r a n u m b e r o f closely r e l a t e d compounds, w a s f o u n d to i n t e r f e r e w i t h t h e a n a l y s i s d e s c r i b e d ( T a b l e 3). O u r o r i g i n a l a t t e m p t s to e s t a b l i s h a t h e o p h y l l i n e a s s a y w e r e b a s e d on t h e n o n - e x t r a c t i v e p r o c e d u r e o u t lined by F r a n c o n i et al I1. U n f o r t u n a t e l y , we q u i c k l y f o u n d t h a t two f r e q u e n t l y used a n t i b i o t i c s , a m p i c i l l i n and methicillin, interfered with the analysis by giving r i s e to p e a k s v e r y close to t h e o p h y l l i n e in r e t e n t i o n t i m e . To e l i m i n a t e t h i s p r o b l e m , we m o d i f i e d t h e p r o c e d u r e by i n t r o d u c i n g a p r e l i m i n a r y e x t r a c t i o n s t e p a n d by c h a n g i n g t h e i n t e r n a l s t a n d a r d f r o m 8-chlorot h e o p h y l l i n e to t h e m o r e h y d r o p h o b i c f l - h y d r o x y p r o p y l t h e o p h y l l i n e . T h e m o d i f i e d p r o c e d u r e has now been
Retention time (secs.)
Compound Theophylline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~-hydroxypropyltheophylline . . . . . . . . . . . . . . . Uric acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-methyluric acid . . . . . . . . . . . . . . . . . . . . . . . . . 3-methyluric acid . . . . . . . . . . . . . . . . . . . . . . . . . 1, 3-dimethyluric acid . . . . . . . . . . . . . . . . . . . . . Xanthine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-methylxanthine . . . . . . . . . . . . . . . . . . . . . . . . . 3-methylxanthine . . . . . . . . . . . . . . . . . . . . . . . . . Hypoxanthine . . . . . . . . . . . . . . . . . . . . . . . . . . . . Theobromine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caffeine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
293 534 107 142 120 194 120 176 159 120 219 508
20-
1614-12-10--
I~J
8-
•
ou
"
£iii::
4-
y intercept = --0.28
2C
0 -.
I
I
I
l
l
I
I
I
I
I
2
4
6
8
!0
12
14
16
18
20
REVERSE-PHASE HPLC (mg/iitre)
F i g u r e 3 -~- Comparison of Reverse-Phase H P L C method described w i t h a Cation-Exchange H P L C method.
a p p l i e d to m o r e t h a n two h u n d r e d a n d f i f t y p a t i e n t s ' s p e c i m e n s , a n d no i n t e r f e r e n c e has been o b s e r v e d w i t h ampicillin, methicillin, gentamicin, clindamycin, penicillin G, k a n a m y c i n , c e p h a l o r i d i n e t e t r a c y c l i n e , e r y t h r o m y c i n , o r c i p r e n a l i n e , a n d s a l b u t a m o l . In a d d i t i o n , t h e n o n - i n t e r f e r i n g d r u g s l i s t e d b y F r a n c o n i et aP t m a y r e a s o n a b l y be a d d e d to t h i s l i s t b e c a u s e o f t h e s i m i l a r i t i e s in t h e c h r o m a t o g r a p h i c p h a s e s of t h e t w o procedures.
ASSAY SYSTEM FOR MEASURING THEOPHYLLINE IN SERUM ACKNOWLEDGEMENTS H P L C comparison studies were made possible t h r o u g h the kind cooperation of J e f f r e y R. Koup, School of Pharmacy, State University of New York at Buffalo. Helpful discussions with D. M. Goldberg, and the skilled technical assistance of T. Gero and J. Cook are gratefully acknowledged. REFERENC$S
1. Schack, J. A., and Waxler, S. H. (1949). J. Pharmacol. Exp. Ther. 97, 283-291. 2. Johnson, G. F., DechtiarLtk, W. A., and Solomon, H. M. (1975). Clin. Chem. 21, 144-147. 3. Thompson, R. D., Nagasawa, H. T., and Jenne, J. W. (1974) J. Lab. Clin. Med. 84, 584-593.
77
4. Least, C. J., Johnson, G. F., and Solomon, H. M. (1976). Clin. Chem. 22, 765-768. 5. Sitar, D. S., Piafsky, K. M., Rangno, R. E., and Ogilvie, R. I. (1975). Clin. Chem. 21, 1774-1776. 6. Weinberger, M., and Chidsey, C. (1975). Clin. Chem. 21, 834-837. 7. Evenson, M. A., and Warren, B. L. (1976). Clin. Chem. 22, 851-855. 8. Brodie, B. B., Axelrod, J., and Reichenthal, J. (1952). J. Biol. Chem. 194, 215-222. 9. Johnson, E. A. (1952). Biochem. J. 51, 133-138. 10. Weinfeld, H., and Christman, A. A. (1953). J. Biol. Chem. 200, 345-355. 11. Franconi, L. C., Sandmann, B. J., Hawk, G. L., and Haney, W. G. (1976). Anal. Chem. 48, 372-375.