Clin. Biochem. 11 (4) 172-174 (1978)
A Sensitive Method for the Determination of Hydrochlorothiazide in Serum by High Pressure Liquid Chromatography W.T. ROBINSON* and L. COSYNS Department of Biochemistry, Ayerst Research Laboratories, P.O. Box 6115, Montreal, Quebec (Accepted January 23, 1978)
CL'BIA, 11, (4) 172-174 (1978) Clin. B~ochem. Robinson, W.T., and Cosyns, L.
Department of Biochemistry, Ayerst Research Laboratories, P.O. Box 6115, Montreal, Quebec A S E N S I T I V E METHOD FOR T H E D E T E R M I N A T I O N OF H Y D R O C H L O R O T H I A Z I D E IN SERUM BY H I G H P R E S S U R E L I Q U I D CHROMATOGRAPHY A high pressure liquid chromatographic method for the determination of hydrochlorothiazide in serum has been elaborated and verified in a blood level study in normal individuals given a single oral dose of 50 mg hydrochlorothiazide. Based on 5 ml of serum, the limit of detection of the method is 2 ng/ml.
PUBLISHED COLORIMETRIC METHODS FOR THE DETERMINATION of t h e d i u r e t i c h y d r o c h l o r o t h i a z i d e in b l o o d ~1°~ l a c k b o t h s p e c i f i c i t y a n d s e n s i t i v i t y . M e t h ods b a s e d on g a s l i q u i d c h r o m a t o g r a p h y r e q u i r e i n v o l v e d s a m p l e c l e a n i n g up a s w e l l a s d e r i v a t i z i n g procedureg3'4~; a l i m i t o f d e t e c t i o n of 2-3 n g / m l h a s b e e n c l a i m e d c4~. A m e t h o d b a s e d on h i g h p r e s s u r e liquid c h r o m a t o g r a p h y ( H P L C ) as r e p o r t e d b y Cooper et al c5~ ( s e n s i t i v i t y : 50 n g / m l ) in o u r h a n d s , f a i l e d due to c o n t a m i n a t i o n p r o b l e m s when e f f o r t s w e r e m a d e to i n c r e a s e i t s s e n s i t i v i t y . M o r e r e c e n t l y a n o t h e r H P L C m e t h o d h a s been r e p o r t e d , b u t a g a i n t h e l i m i t of d e t e c t i o n w a s 50. n g / m l ce~. I n view o f o u r i n t e r e s t in a m e t h o d s u f f i c i e n t l y s e n s i t i v e to m e a s u r e , in man, t h e blood level p r o f i l e a f t e r a s i n g l e o r a l dose o f 50 m g h y d r o c h l o r o t h i a z i d e , we h a v e e l a b o r a t e d a n d r e p o r t h e r e w i t h a m e t h o d , b a s e d on H P L C , w i t h a l i m i t o f d e t e c t i o n of 2 n g / m l serum. MATERIALS AND METHOD
Apparatus A high pressure liquid chromatograph (Spectra Physics, Model 3500) equipped with an oven, a Valco loop injection valve and a speetrophotometric detector (PerkinElmer, model LC-55) was used. Chromatography was *Present address: Squibb Inc., Route 10, E a s t Hanover, N.J., 07936. Correspondence: Dr. L. Cosyns A y e r s t Research Laboratories P.O. Box 6115 Montreal, Quebec Canada H3C 3J1
accomplished on a column of 5 micron Lichrosorb (4.4 x 500 ram) packed a t 10,000 psi using an acetone s l u r r y technique and a pneumatic pump (Haskel Engineering and Supply Co., System N. 29426-100). Alternatively, two columns of 5 micron Spherisorb (3 x 250 mm; SpectraPhysics) connected in series m a y be used.
Reagents The reagents used in the extraction procedure were: potassium phosphate solution (0.05 M, p H 6.0; Baker Analyzed R e a g e n t ) ; methyl isobutyl ketone (MIK, all glass distilled reagent; American Chemicals Ltd., Montr e a l ) ; 1.0 N HC1; 0.1 N N a O H ; sodium bicarbonate (reagent grade, M c A r t h u r Chemical Ltd.) and ethyl acetate (reagent grade; American Chemicals Ltd.). Hexane (reagent grade; American Chemicals Ltd.) and ethanol (denatured with 10% methanol; Canadian International P a p e r ) were used for chromatography.
Procedure Into a 40 ml glass stoppered centrifuge tube pipette 5.0 ml of 0.05 M potassium phosphate solution, 5.0 ml Iserum and 12 ml of MIK, shake (80 rain) and centrifuge. Place a 10 ml aliquot of the organic phase in a 20 ml glass stoppered round bottom centrifuge tube containing 5.0 ml of 1.0 N HC1, shake for 5 rain and centrifuge. T r a n s f e r 9.0 ml of the organic phase to another 20 ml centrifuge tube containing 5 ml 0.1 N NaOH, shake (15 rain), centrifuge, then a s p i r a t e and discard the organic phase. Finally, t r a n s f e r 4.0 ml of the aqueous phase to another 20 ml centrifuge tube, add 0.4 ml 1.0 N HC1, N 500 mg NaHC03 and 10 ml ethyl acetate; shake (15 min) and centrifuge. Pipette 9.0 ml of the organic layer into a disposable test tube and evaporate the ethyl acetate under nitrogen; reconstitute with 0.5 ml of 30% ethanol in hexane and mix (Vortex) before direct injection onto the c h r o m a t o g r a p h y column. Chromatography was performed on a "micro-pack" silica column (4.4 x 500 ram) maintained at 50"C and eluted a t a rate of 2.0 m l / m i n with 45% ethanol in hexane. Quantitation was based on the optical density measured a t 271 nm as peak height at 0.02 absorbanee full scale deflection (A.F.S.D.) Standardization was performed by c a r r y i n g 750 ng of hydrochlorothiazide through the method. The amount of hydroehlorothiazide in the sample was calculated by direct comparison of response to the standards. TABLE 1 EFFECT OF SERUM ALIQUOT VOLUME ON THE RECOVERY OF HYDROCHLOROTHIAZIDE*
Serum aliquot (ml) 0.5 2.0 3.0 5.0 *250 ng added
Percent Recovered 86.8 86.8 85.2 86.4
173
D E T E R M I N A T I O N OF H Y D R O C H L O R O T H I A Z I D E IN SERUM BY HPLC A
B
C
T
i
±
.¢
f
f
::=:: r l D,q~lb
M~
Fig. I -- Chromatograms of A ) a hydrochlorothiazide standard, B) a serum blank (5 ml) taken through the method and C) a serum sample (5 ml) containing 10 ng/ ml hydroehlorothiazide taken through the method.
Amaunb
Fig. 2 - - Effect of concentration on the recovery chlorothiazide.
a) Chromatography
TABLE
of
hydro-
H y d r o c h l o r o t h i a z i d e can be d e t e c t e d b y i t s abs o r p t i o n a t 226 n m (E ---- 3.04 x 104 ) o r 271 nm ( E = 1.70 x 104). Occasionally, s e r u m b l a n k s d i s p l a y e d i n t e r f e r i n g p e a k s a t 226 n m b u t n o t a t 271 nm. T h e r e f o r e , d e s p i t e a t w o f o l d loss in sensit i v i t y , t h e m e a s u r e m e n t w a s done a t 271 nm.
:RESULTS AND DISCUSSION
The determination of hydrochlorothiazide by HPLC,5, e) h a s been done b y r e v e r s e p h a s e liquidliquid p a r t i t i o n c h r o m a t o g r a p h y . Since we have observed increased efficiency using absorption c h r o m a t o g r a p h y we chose to c h r o m a t o g r a p h h y d r o c h l o r o t h i a z i d e on silica gel. U s i n g e t h a n o l : h e x a n e a s t h e mobile phase, good e f f i c i e n c y ( t y p i c a l l y 6000-9000 t h e o r e t i c a l p l a t e s / 5 0 cm, F i g u r e 1A) was achieved. Problems with final sample prepar a t i o n , e.g. s o l u b i l i t y a n d a b s o r p t i o n to glass, w e r e a v o i d e d b y u s i n g 30% e t h a n o l in h e x a n e to dissolve t h e s a m p l e b e f o r e i n j e c t i o n onto t h e column. Upon i n j e c t i o n , t h e s a m p l e c o n c e n t r a t e s on t h e top of t h e column a n d only elutes w i t h 45% e t h a n o l h e x a n e ; t h e r e f o r e , volumes, of up to 0.15 ml could be i n j e c t e d w i t h o u t loss in a p p a r e n t column e f f i ciency. W i t h 45% e t h a n o l in hexane, a column h e i g h t of 50 cm w a s a d e q u a t e to s e p a r a t e h y d r o chlorothiazide from extractable endogeneous material. A column t e m p e r a t u r e o f 50°C r e d u c e d t h e p r e s s u r e to 1000-3000 p.s.i, a n d s h o r t e n e d t h e c h r o m a t o g r a p h y t i m e to 6 min.
r'nq/ml']
fqddmd
b) Extraction In our hands, t h e use of ethyl a c e t a t e f o r t h e e x t r a c t i o n of h y d r o c h l o r o t h i a z i d e f r o m s e r u m (1, 2, 3, 5) r e s u l t e d in emulsions. No e m u l s i o n s and good r e c o v e r i e s w e r e o b t a i n e d w i t h n - p r o p y l n i t r i l e , t - b u t y l n i t r i l e and MIK'"); b a s e d on e x t r a c t i o n a n d c h r o m a t o g r a p h y of s e r u m blanks, t h e l a t t e r w a s selected a s t h e m o r e selective solvent ( F i g . 1). T h e e x t r a c t i o n w i t h M I K w a s p H d e p e n d e n t and o p t i m a l b e t w e e n p H 5.0 a n d 6.5. A c i d " w a s h i n g " of t h e M I K e x t r a c t a n d back e x t r a c t i o n into dilute sodium hydroxide separated hydrochlorothiazide f r o m i n t e r f e r i n g m a t e r i a l . I t is i m p o r t a n t to acidi f y t h e e x t r a c t w i t h o u t d e l a y so as to avoid possible d e c o m p o s i t i o n of h y d r o c h l o r o t h i a z i d e . T h e p H is t h e n a d j u s t e d w i t h s o d i u m b i c a r b o n a t e and t h e solution e x t r a c t e d w i t h ethyl a c e t a t e ; a s a t u r a t e d bicarbonate solution was required for maximal r e c o v e r y (~).
2
RECOVERY OF HYDROCHLOROTHIAZIDEFROMSERUM
Percent Recovery
Amount Added (ng),
Mean b
75 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1500 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
89.6 86.3 84.3 84.1 85.0
Average . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
85.8
1500 (day 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1500 (day 3) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
87.0 89.0
,Total added to 3 ml serum bn=4
Range 80.6 85.1 81.8 83.3 83.3
------
98.6 87.4 86.2 86.0 85.6
S.E. on Mean
% C.V.
3.7 0.7 i.i 0.6 0.6
7.1 1.3 2.3 1.3 1.1
--
0.85
4.3
85.9 - - 88.6 86.3 - - 91.4
0.6 0.1
1.1 2.1
174
ROBINSON AND COSYNS
n~
Eu,
Fig. 3 - - Serum concentrations of hydroehloro~hiazide in male volunteers given 50 ~ng hydroehlorothiazide.
%
m=lm
Z in Hn, Iw ¢
I.Jm Z,m
lg= Wm LXMIT
m
a'.no
s'.oo
e'.on
~F
~ETECT~BN
I~,.oo
z~.oo
,koo
HBUR~ RF'TER 1 1 B S I N G
c) Recovery and Sensitivity The recovery of added hydrochlorothiazide from pooled human serum was independent of concentration or serum volume (0.5-5 ml; Table 1). While the recovery was reproducible from day to day (Table 2), we prefer to determine the recovery for each set of analysis. The response of standards carried through the method was linear at concentrations up to 500 ng/ml (Table 11, Figure 1). A typical serum blank is shown in Figure lb. There are no interfering peaks near that of hydrochlorothiazide. It is important to use a good grade or redistilled MIK since, upon chromatography, some commercially available MIK produced a solvent front moving close to the hydrochlorothiazide peak. With poorer g r a d e s , addition of 20% isooctane to MIK before extraction removes the interference, albeit at a 10% loss in overall recovery. Based on the response obtained with 5.0 ml serum containing 10 n g / m l of hydrochlorothiazide (Fig. lc) the detection limit of the method was assessed as 2 n g / m l (7 mm peak; approximately 4x above baseline noise).
d) Precision and Selectivity An indication of the precision of the method is provided in recovery studies (Table 2). The coefficient of variation is typically 1-2.%. At lower concentrations slight errors due to alkaline decomposition, glass absorption, and loss t h r o u g h evaporation begin to show. Therefore with 75 ng (25 n g / m l for 3 ml serum or 15 n g / m l for 5 ml of serum) the coefficient of variation is approximately 7%.
gi.or,
a'q.oo
The method by virtue of the extraction procedure and the chromatography should be very selective. Commonly used compounds such as phenyl-butazone, salicyclic acid, nicotinic acid, clofibric acid, propoxyphene, phenobarbital, diphenylhydantoin, dicoumarol, diazepam, thiosulfil, and hydroflumethiazide do not interfere.
e) Application of the Method The usefulness of the method was assessed by measuring ~ the concentration of hydrochlorothiazide in the serum of five normal male volunteers receiving hydrochlorothiazide (HydroDiuril; Merck Sharp and Dohme). As illustrated in Fig. 3, the method was sufficiently sensitive to determine the blood level profile of hydrochlorothiazide produced by a single oral dose of 50 mg HydroDiuril. ACKNOWLEDGMENTS
We thank Dr. D. Dvornik for advice in the preparation of the manuscript and Mr. M. Caron and Dr. R. Moisan (University of Montreal) for conducting the blood level study in man. REFERENCES
1. Baer, J. E., Leidy, H. L., Brooks, A. V. and Beyer, K. H. (1959). J. Pharm. Exp. Therapeutics, 125, 295302. 2. Sheppard, H., Mowles, T. F. and Plummer, A. J. (1960). J. Am. Pharm. Assoc. 49, 722-723. 3. Vandenheuvel, W. J. A., Gruber, V. F., Walker, R. W. and Wolf, F. J. (1975). J. Pharm. Sci. 64, 1309-12. 4..LindstrSm, B., Molander, M. and Groschinsky, M. (1975). J. Chromatography, 114, 459-62. 5. Cooper, M. J., Sinaiko, A. R., Auders, M. W. and Mirkin, B. L. (1976). Anal. Chem., 48, 1110-1111. 6. Christophersen, A. S., Rasmussen, K. E. and Salvesen, B. (1977). J. Chromatography, 132, 91-97.
A Sensitive Method for the Determination of Hydrochlorothiazide in Serum by High Pressure Liquid Chromatography W.T. ROBINSON* and L. COSYNS Department of Biochemistry, Ayerst Research Laboratories, P.O. Box 6115, Montreal, Quebec (Accepted January 23, 1978)
CL'BIA, 11, (4) 172-174 (1978) Clin. B~ochem. Robinson, W.T., and Cosyns, L.
Department of Biochemistry, Ayerst Research Laboratories, P.O. Box 6115, Montreal, Quebec A S E N S I T I V E METHOD FOR T H E D E T E R M I N A T I O N OF H Y D R O C H L O R O T H I A Z I D E IN SERUM BY H I G H P R E S S U R E L I Q U I D CHROMATOGRAPHY A high pressure liquid chromatographic method for the determination of hydrochlorothiazide in serum has been elaborated and verified in a blood level study in normal individuals given a single oral dose of 50 mg hydrochlorothiazide. Based on 5 ml of serum, the limit of detection of the method is 2 ng/ml.
PUBLISHED COLORIMETRIC METHODS FOR THE DETERMINATION of t h e d i u r e t i c h y d r o c h l o r o t h i a z i d e in b l o o d ~1°~ l a c k b o t h s p e c i f i c i t y a n d s e n s i t i v i t y . M e t h ods b a s e d on g a s l i q u i d c h r o m a t o g r a p h y r e q u i r e i n v o l v e d s a m p l e c l e a n i n g up a s w e l l a s d e r i v a t i z i n g procedureg3'4~; a l i m i t o f d e t e c t i o n of 2-3 n g / m l h a s b e e n c l a i m e d c4~. A m e t h o d b a s e d on h i g h p r e s s u r e liquid c h r o m a t o g r a p h y ( H P L C ) as r e p o r t e d b y Cooper et al c5~ ( s e n s i t i v i t y : 50 n g / m l ) in o u r h a n d s , f a i l e d due to c o n t a m i n a t i o n p r o b l e m s when e f f o r t s w e r e m a d e to i n c r e a s e i t s s e n s i t i v i t y . M o r e r e c e n t l y a n o t h e r H P L C m e t h o d h a s been r e p o r t e d , b u t a g a i n t h e l i m i t of d e t e c t i o n w a s 50. n g / m l ce~. I n view o f o u r i n t e r e s t in a m e t h o d s u f f i c i e n t l y s e n s i t i v e to m e a s u r e , in man, t h e blood level p r o f i l e a f t e r a s i n g l e o r a l dose o f 50 m g h y d r o c h l o r o t h i a z i d e , we h a v e e l a b o r a t e d a n d r e p o r t h e r e w i t h a m e t h o d , b a s e d on H P L C , w i t h a l i m i t o f d e t e c t i o n of 2 n g / m l serum. MATERIALS AND METHOD
Apparatus A high pressure liquid chromatograph (Spectra Physics, Model 3500) equipped with an oven, a Valco loop injection valve and a speetrophotometric detector (PerkinElmer, model LC-55) was used. Chromatography was *Present address: Squibb Inc., Route 10, E a s t Hanover, N.J., 07936. Correspondence: Dr. L. Cosyns A y e r s t Research Laboratories P.O. Box 6115 Montreal, Quebec Canada H3C 3J1
accomplished on a column of 5 micron Lichrosorb (4.4 x 500 ram) packed a t 10,000 psi using an acetone s l u r r y technique and a pneumatic pump (Haskel Engineering and Supply Co., System N. 29426-100). Alternatively, two columns of 5 micron Spherisorb (3 x 250 mm; SpectraPhysics) connected in series m a y be used.
Reagents The reagents used in the extraction procedure were: potassium phosphate solution (0.05 M, p H 6.0; Baker Analyzed R e a g e n t ) ; methyl isobutyl ketone (MIK, all glass distilled reagent; American Chemicals Ltd., Montr e a l ) ; 1.0 N HC1; 0.1 N N a O H ; sodium bicarbonate (reagent grade, M c A r t h u r Chemical Ltd.) and ethyl acetate (reagent grade; American Chemicals Ltd.). Hexane (reagent grade; American Chemicals Ltd.) and ethanol (denatured with 10% methanol; Canadian International P a p e r ) were used for chromatography.
Procedure Into a 40 ml glass stoppered centrifuge tube pipette 5.0 ml of 0.05 M potassium phosphate solution, 5.0 ml Iserum and 12 ml of MIK, shake (80 rain) and centrifuge. Place a 10 ml aliquot of the organic phase in a 20 ml glass stoppered round bottom centrifuge tube containing 5.0 ml of 1.0 N HC1, shake for 5 rain and centrifuge. T r a n s f e r 9.0 ml of the organic phase to another 20 ml centrifuge tube containing 5 ml 0.1 N NaOH, shake (15 rain), centrifuge, then a s p i r a t e and discard the organic phase. Finally, t r a n s f e r 4.0 ml of the aqueous phase to another 20 ml centrifuge tube, add 0.4 ml 1.0 N HC1, N 500 mg NaHC03 and 10 ml ethyl acetate; shake (15 min) and centrifuge. Pipette 9.0 ml of the organic layer into a disposable test tube and evaporate the ethyl acetate under nitrogen; reconstitute with 0.5 ml of 30% ethanol in hexane and mix (Vortex) before direct injection onto the c h r o m a t o g r a p h y column. Chromatography was performed on a "micro-pack" silica column (4.4 x 500 ram) maintained at 50"C and eluted a t a rate of 2.0 m l / m i n with 45% ethanol in hexane. Quantitation was based on the optical density measured a t 271 nm as peak height at 0.02 absorbanee full scale deflection (A.F.S.D.) Standardization was performed by c a r r y i n g 750 ng of hydrochlorothiazide through the method. The amount of hydroehlorothiazide in the sample was calculated by direct comparison of response to the standards. TABLE 1 EFFECT OF SERUM ALIQUOT VOLUME ON THE RECOVERY OF HYDROCHLOROTHIAZIDE*
Serum aliquot (ml) 0.5 2.0 3.0 5.0 *250 ng added
Percent Recovered 86.8 86.8 85.2 86.4
173
D E T E R M I N A T I O N OF H Y D R O C H L O R O T H I A Z I D E IN SERUM BY HPLC A
B
C
T
i
±
.¢
f
f
::=:: r l D,q~lb
M~
Fig. I -- Chromatograms of A ) a hydrochlorothiazide standard, B) a serum blank (5 ml) taken through the method and C) a serum sample (5 ml) containing 10 ng/ ml hydroehlorothiazide taken through the method.
Amaunb
Fig. 2 - - Effect of concentration on the recovery chlorothiazide.
a) Chromatography
TABLE
of
hydro-
H y d r o c h l o r o t h i a z i d e can be d e t e c t e d b y i t s abs o r p t i o n a t 226 n m (E ---- 3.04 x 104 ) o r 271 nm ( E = 1.70 x 104). Occasionally, s e r u m b l a n k s d i s p l a y e d i n t e r f e r i n g p e a k s a t 226 n m b u t n o t a t 271 nm. T h e r e f o r e , d e s p i t e a t w o f o l d loss in sensit i v i t y , t h e m e a s u r e m e n t w a s done a t 271 nm.
:RESULTS AND DISCUSSION
The determination of hydrochlorothiazide by HPLC,5, e) h a s been done b y r e v e r s e p h a s e liquidliquid p a r t i t i o n c h r o m a t o g r a p h y . Since we have observed increased efficiency using absorption c h r o m a t o g r a p h y we chose to c h r o m a t o g r a p h h y d r o c h l o r o t h i a z i d e on silica gel. U s i n g e t h a n o l : h e x a n e a s t h e mobile phase, good e f f i c i e n c y ( t y p i c a l l y 6000-9000 t h e o r e t i c a l p l a t e s / 5 0 cm, F i g u r e 1A) was achieved. Problems with final sample prepar a t i o n , e.g. s o l u b i l i t y a n d a b s o r p t i o n to glass, w e r e a v o i d e d b y u s i n g 30% e t h a n o l in h e x a n e to dissolve t h e s a m p l e b e f o r e i n j e c t i o n onto t h e column. Upon i n j e c t i o n , t h e s a m p l e c o n c e n t r a t e s on t h e top of t h e column a n d only elutes w i t h 45% e t h a n o l h e x a n e ; t h e r e f o r e , volumes, of up to 0.15 ml could be i n j e c t e d w i t h o u t loss in a p p a r e n t column e f f i ciency. W i t h 45% e t h a n o l in hexane, a column h e i g h t of 50 cm w a s a d e q u a t e to s e p a r a t e h y d r o chlorothiazide from extractable endogeneous material. A column t e m p e r a t u r e o f 50°C r e d u c e d t h e p r e s s u r e to 1000-3000 p.s.i, a n d s h o r t e n e d t h e c h r o m a t o g r a p h y t i m e to 6 min.
r'nq/ml']
fqddmd
b) Extraction In our hands, t h e use of ethyl a c e t a t e f o r t h e e x t r a c t i o n of h y d r o c h l o r o t h i a z i d e f r o m s e r u m (1, 2, 3, 5) r e s u l t e d in emulsions. No e m u l s i o n s and good r e c o v e r i e s w e r e o b t a i n e d w i t h n - p r o p y l n i t r i l e , t - b u t y l n i t r i l e and MIK'"); b a s e d on e x t r a c t i o n a n d c h r o m a t o g r a p h y of s e r u m blanks, t h e l a t t e r w a s selected a s t h e m o r e selective solvent ( F i g . 1). T h e e x t r a c t i o n w i t h M I K w a s p H d e p e n d e n t and o p t i m a l b e t w e e n p H 5.0 a n d 6.5. A c i d " w a s h i n g " of t h e M I K e x t r a c t a n d back e x t r a c t i o n into dilute sodium hydroxide separated hydrochlorothiazide f r o m i n t e r f e r i n g m a t e r i a l . I t is i m p o r t a n t to acidi f y t h e e x t r a c t w i t h o u t d e l a y so as to avoid possible d e c o m p o s i t i o n of h y d r o c h l o r o t h i a z i d e . T h e p H is t h e n a d j u s t e d w i t h s o d i u m b i c a r b o n a t e and t h e solution e x t r a c t e d w i t h ethyl a c e t a t e ; a s a t u r a t e d bicarbonate solution was required for maximal r e c o v e r y (~).
2
RECOVERY OF HYDROCHLOROTHIAZIDEFROMSERUM
Percent Recovery
Amount Added (ng),
Mean b
75 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1500 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
89.6 86.3 84.3 84.1 85.0
Average . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
85.8
1500 (day 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1500 (day 3) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
87.0 89.0
,Total added to 3 ml serum bn=4
Range 80.6 85.1 81.8 83.3 83.3
------
98.6 87.4 86.2 86.0 85.6
S.E. on Mean
% C.V.
3.7 0.7 i.i 0.6 0.6
7.1 1.3 2.3 1.3 1.1
--
0.85
4.3
85.9 - - 88.6 86.3 - - 91.4
0.6 0.1
1.1 2.1
174
ROBINSON AND COSYNS
n~
Eu,
Fig. 3 - - Serum concentrations of hydroehloro~hiazide in male volunteers given 50 ~ng hydroehlorothiazide.
%
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lg= Wm LXMIT
m
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~ETECT~BN
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HBUR~ RF'TER 1 1 B S I N G
c) Recovery and Sensitivity The recovery of added hydrochlorothiazide from pooled human serum was independent of concentration or serum volume (0.5-5 ml; Table 1). While the recovery was reproducible from day to day (Table 2), we prefer to determine the recovery for each set of analysis. The response of standards carried through the method was linear at concentrations up to 500 ng/ml (Table 11, Figure 1). A typical serum blank is shown in Figure lb. There are no interfering peaks near that of hydrochlorothiazide. It is important to use a good grade or redistilled MIK since, upon chromatography, some commercially available MIK produced a solvent front moving close to the hydrochlorothiazide peak. With poorer g r a d e s , addition of 20% isooctane to MIK before extraction removes the interference, albeit at a 10% loss in overall recovery. Based on the response obtained with 5.0 ml serum containing 10 n g / m l of hydrochlorothiazide (Fig. lc) the detection limit of the method was assessed as 2 n g / m l (7 mm peak; approximately 4x above baseline noise).
d) Precision and Selectivity An indication of the precision of the method is provided in recovery studies (Table 2). The coefficient of variation is typically 1-2.%. At lower concentrations slight errors due to alkaline decomposition, glass absorption, and loss t h r o u g h evaporation begin to show. Therefore with 75 ng (25 n g / m l for 3 ml serum or 15 n g / m l for 5 ml of serum) the coefficient of variation is approximately 7%.
gi.or,
a'q.oo
The method by virtue of the extraction procedure and the chromatography should be very selective. Commonly used compounds such as phenyl-butazone, salicyclic acid, nicotinic acid, clofibric acid, propoxyphene, phenobarbital, diphenylhydantoin, dicoumarol, diazepam, thiosulfil, and hydroflumethiazide do not interfere.
e) Application of the Method The usefulness of the method was assessed by measuring ~ the concentration of hydrochlorothiazide in the serum of five normal male volunteers receiving hydrochlorothiazide (HydroDiuril; Merck Sharp and Dohme). As illustrated in Fig. 3, the method was sufficiently sensitive to determine the blood level profile of hydrochlorothiazide produced by a single oral dose of 50 mg HydroDiuril. ACKNOWLEDGMENTS
We thank Dr. D. Dvornik for advice in the preparation of the manuscript and Mr. M. Caron and Dr. R. Moisan (University of Montreal) for conducting the blood level study in man. REFERENCES
1. Baer, J. E., Leidy, H. L., Brooks, A. V. and Beyer, K. H. (1959). J. Pharm. Exp. Therapeutics, 125, 295302. 2. Sheppard, H., Mowles, T. F. and Plummer, A. J. (1960). J. Am. Pharm. Assoc. 49, 722-723. 3. Vandenheuvel, W. J. A., Gruber, V. F., Walker, R. W. and Wolf, F. J. (1975). J. Pharm. Sci. 64, 1309-12. 4..LindstrSm, B., Molander, M. and Groschinsky, M. (1975). J. Chromatography, 114, 459-62. 5. Cooper, M. J., Sinaiko, A. R., Auders, M. W. and Mirkin, B. L. (1976). Anal. Chem., 48, 1110-1111. 6. Christophersen, A. S., Rasmussen, K. E. and Salvesen, B. (1977). J. Chromatography, 132, 91-97.
