Journal of Chromatography, 57 i (t 99 I) 283-290 Biomedical Applications Elsevier Science Publisher~; B.V., A m s t e r d a m CHROM BIO. 6018
Short Communication High-performance liquid chromatographic determination of lansoprazole and its metabolites in human serum and urine 1SAMU AOKI*
Takeda Atlal)'tical Research Laboratories. Lt~L. dusa-Hmmauchi. Ymtogan'a-ktt, Oset~'a 532 (J~. ~an) MINORU OKUMURA
Pkatvnacetttical Product/ott Research Laboratories. Takeda Chemh'al Dtdustri~.s. LttL. Jttso-Honmachi, Yodogau'a-ku. Osaka 532 (Japan) and TAKATSUKA YASHIKI
Analytical Laboratories. Takeda Ckemk'al huhtstries. Ltd.. Jtt.vo-Honmttchi. Yoth~gawa-ktt. Osaka 532 (Japan) (First received January 16th, 1991: revised manuscript received May ! 5th, 1991 ) ABSTRACT A simple and sensitive high-performance liquid c h r o m a t o g r a p h i c m e t h o d with uitraviole! detection is described for the simultaneous determination o f tansoprazole and its metabolitcs in hum:m serum and urine. T h e analytes in serum or urine were extracted with diethyl cther-diclalorometlaane (7:3, v/v) followed by e v a p o r a t i o n , dissolution and injection into ,'1 reversed-phase column. T h e reco,eries o f aufl~entic analyres added to serum at 0.05-2 ltg/ml o r to urine at 1-20 l~g/ml werc greater than 88"/,,, with the coellicients o f variation less than 7 . I % . T h e mininaum determinable concentrations o f all analytes were 5 ngtml in serum and 50 ng/mi in urine. T h e rnethod was successfully applied to a pharmacokinctic study o f lansoprazole in human.
INTRODUCTION
Lansoprazole, (4-)-2-[[[3-methyl-4-(2,2,2_trifluoroethoxy)_2_pyridyl]naethyl]_ sulphinyl]benzimidazole (AG-1749), is a new substituted benzimidazole compound, which markedly inhibits the secretion of basal and stimulated gastric acid and is.useful in the treatment of duodenal ulcers, gastric ulcers and reflux esophagitis [I-3]. The metabolism of kmsoprazole was investigated with animals (rats, mice and dogs), and the main metabolites were identified [4,5] as shown in Fig. i. The metabolites o f lansoprazole inclttde not only its sulphide (tX~-l) and sulphone (M-VII) but also 5-hydroxy metabolites (M-V!. -IV and -IX) of these. 0378-4347/91/S03.50
O
1991 Elsevier Science Publishers B.V. All rights reserved
SHORT COMMUNICATIONS
284 l ~ .
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~../g~ao--cu,...~n..
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1: fl llsO.S~
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"~ OCII=CF=
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M-I
M-1V
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H OCII~CF~ M--- ~ql
OC
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M-IX
Fig. I. Structurcs o f hmsoprazole :rod its possible metabolites in humans.
In the present paper, a simple and sensitive high-performance liquid c h r o m a ' tographic (HPLC) method for the simultaneous determination of lansoprazole and its main metabolites in human serum and urine is decribed. EXPERIMENTAL
Reageots and materials Test samples and reagents. Lansoprazoie and its metabolites were synthesized in the Research and Development Division of Takeda Chemical Industries. T y p e VII fl-glucuronidase was purchased from Sigma (St. Louis, MO, USA). Dichloromethane, acetonitrile and ethanol were of H P L C grade. All other reagents were of analytical-reagent grade. Mobile phase 1 (MP-I) was water--acetonitrile-n-octylamine (620:380:1, v/v), adjusted to pH 7 with 85°/; phosphoric acid. Mobile phase II (MP-II) was 0.07 M phosphate buffer (pH 7.2)-ethanol-acetonitrile (20:10:3, v/v). The propylene glycol (PG) solution, propylene glycol-diethyl ether-dichloromethane (1:140:60, v/ v), was used t o depress the evaporation of the analytes. Internal standard solutions 1 (IS-I) and I1 (IS-II) consisted of 25 pg/ml isobutyl p-hydoxybenzoate in dichloromethane and 6 pg/ml isopropyl p-aminobenzoate in MP-II, respectively. Serum and urine samples were collected from ten healthy male volunteers participating in the Phase I study, and the control samples were collected before administration of the test compound. Standard samples. A serum standard sample was prepared to contain lan-
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s o p r a z o l e , M - I , M - I V , M ' V I , M - V I I a n d M - I X ( e a c h 0.5 llg/ml) in a c o n t r o l serum, a n d a urine s t a n d a r d s a m p l e was p r e p a r e d to c o n t a i n M - I V , M - V I a n d M - I X (each 5 .'.tg/ml) in a m i x t u r e o f c o n t r o l urine a n d 0.47 M s o d i u m b i c a r b o ' nate solution (I: 1, v/v).
Extraction o f the analytes.fi'om serum and to'hw samples A 0. l-ml p o r t i o n o f IS-I w a s a d d e d to 0.5 ml o f a s e r u m s a m p l e a n d a s e r u m s t a n d a r d sample, a n d the a n a l y t e s were e x t r a c t e d twice with 3 ml o f diethyl e t h e r - d i c h l o r o m e t h a n e (7:3, v/v). A f t e r c e n t r i f u g a t i o n , 0.5 ml o f the p r o p y l e n e glycol ( P G ) solution was a d d e d to the s u p e r n a t a n t , a n d the solvent w a s e v a p o r a t ed u n d e r a n i t r o g e n s t r e a m . T h e residue was dissolved in 0.5 ml o f M P - I , a n d a 100-1d p o r t i o n o f the s o l u t i o n w a s subjected to H P L C . T o 0.5 ml o f a urine s a m p l e a n d a urine s t a n d a r d s a m p l e w a s a d d e d 0.5 ml o f fl-glucuronidase solution (1000 U / m l in 0.07 M p h o s p h a t e buffer o f p H 7), a n d the m i x t u r e was i n c u b a t e d at 37"C for 60 min. T h e a n a l y t e s were extracted twice with 3 ml o f diethyl e t h e r - d i c h i o r o m e t h a n e (7:3, v/v). A f t e r c e n t r i f u g a t i o n , 0.5 ml o f the P G solution w a s a d d e d to the s u p e r n a t a n t , a n d the solvent w a s e v a p o r a t e d u n d e r a n i t r o g e n s t r e a m . T h e residue was dissolved in 0.4 ml o f IS-II, a n d a 20-1d p o r t i o n o f the solution was subjected to H P L C .
htstrttments and conditions./br determhlation T h e H P L C system consisted o f a S h i m a d z u L,~--,A ~ ,t o r L C - 6 A 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 e q u i p p e d with a n a u t o s a m p l e r with cooling device (lshido, C h i b a , J a p a n ) . A r e v e r s e d - p h a s e c o l u m n o f T S K gel O D S - I 2 0 T (particle size 5 ltm; 250 m m x 4.6 m m I . D . , T o s o h , T o k y o , J a p a n ) was used. T h e c o l u m n t e m p e r a t u r e a n d the flow-rate were 40°C a n d 1.0 m l / m i n , respectively. M P - I a n d M P - i I were used for the d e t e r m i n a t i o n o f a n a l y t e s 1,'_ s e r u m a n d urine, respectively. L a n s o p r a z o l e , M - I , M - I V , M - V I I a n d internal s t a n d a r d in s e r u m were detected by U V a b s o r p t i o n at 285 n m , a n d M - V ! a n d M - I X were detected at 303 nm; M - I V , M - V I a n d M - I X in urine were detected at:303 rim.
Admhlistration o f lansoprazole and sample collection A capsule c o n t a i n i n g 30 m g o f l a n s o p r a z o l e as enteric-coated g r a n u l e s (lans o p r a z o l e capsule, 30 mg) w a s orally a d m i n i s t e r e d to each o f ten fasted h e a l t h y male volunteers. T h e s e r u m samples were collected before a d m i n i s t r a t i o n a n d 0.5, 1, 2, 3, 4, 6, 8, 12 a n d 24 h a f t e r a d m i n i s t r a t i o n . T h e samples were preserved at 20"C until analysis. T h e urine samples were collected before a d m i n i s t r a t i o n a n d at the intervals o f 0 - 6 , 6 - 1 2 a n d 12-24 h a f t e r administratior~. T o stabilize the analytes, the s a m e v o l u m e o f 0.47 M ~ndlum b i c a r b o n a t e solution was a d d e d i m m e d i a t e l y a f t e r the collection o f a urine s a m p l e a n d the m i x t u r e w a s stored at - 2 0 ° C until analysis. -
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DISCUSSION
F r o m the results of metabolism of lansoprazole in animals, the sulphide (M-I) and sulphone (M-VII) metabolites a nd three corresponding 5-hydroxy metabolites (M-VI, M-IV, M-IX) were expected as metabolites in h u m a n s . Thus, we investigated a s i m u l t a n o u s determination m e t h o d for lansoprazole and its five possible metabolites to develop a reversed-phase H P L C technique with UV detection. Lansoprazole and its metabolites were unstable in an acid solution, as is omeprazole [6], a lthough they were rather stable in an alkaline solution; the half-lives o f lansoprazole at 25"C were ca. 30 min at p H 5, c a . 18 h at pH 7 and c a . 37 h at pH 8. Accordingly, the analytes were stable in a serum sample (pl-I c a . 8), but unstable in a urine sample depending on the p H . Therefore, we stabilized the analytes by a d d i n g an equal volume of 0.47 M s o d i u m bicarb~T~ate solution to each urine sample to keep the pH c a . 9. The c h r o m a t o g r a m s of serum samples are shown in Fig. 2. The calibration curves of the peak-height ratios (analyte to
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Fig. 2. C h r o m a t o g r a m s o f e x t r a c t s f r o m ( A ) a n a l y t e - f r e c s e r u m , (B) s e r u m s p i k e d w i t h a n a l y t e s ( e a c h c o n c e n t r a t i o n c a . 0. i gtg,q~l) a n d (C) the s e r u m s a m p l e o f a v o l u n t e e r ( M - V I , 162 n g / m l : l a n s o p r a z o l e , 1748 n g / m l ; M - V I I , 268 n g / m l ) . P e a k s : I = M - V I ; 2 --- M - I X ; 3 = M - I V : 4 = l a n s o p r a z o l e ; 5 = M - V I I : 6 = internal standard: 7 = M-I.
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internal standard) v e r s u s the concentration of the analytes show straight lines passing through the origin (correlation coefficient >0.999). The average recoveries of the analytes in the concentration range 0.05-2 lzg/ml were more than 88%, with coefficients of variation (C.V) of less t h a n 7.1% (Table I). The minimum determinable concentrations (signal-to-noise ratio of 3) for lansoprazole and its metabolites were all 5 ng/ml when 0.5 ml of a serum sample was used. Preliminary investigation on the metabolites of lansoprazole in human urine revealed that only 5-hydroxy metabolites (M-IV, M-VI, M-IX) were excreted, mainly as the glucuronides. As the authentic samples of the glucuronides were not available'," the cenditions for the hydrolysis of glucuronides were investigated using fl-glucuronidase. As a result, incubation at 37°C for 1 h was found enough for the hydrolysis. The chromatograms of urine samples (after hydrolysis) are shown in Fig. 3. The calibration curves of the peak-height ratios (analyte to internal s t a n d a r d ) v e r s u s the concentration of the analytes show straight lines passing through the
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Fig. 3. C h r o m a t o g r a m s o f extracts f r o m (A) analyte-free urine (after hydrolysis) a n d (B) the urine sample o f a volunteer (after hydrolysis: M - I X , 5.75 /tg/ml; M - V I , 4.35 pg/ml; M - I V , 17.09 lig/ml); detection wavelength, 303 nm. Peaks: 1 = M - I X ; 2 = M - V I ; 3 = internal standard; 4 -- M - I V .
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Concentration o f analytes in urine (/ig/ml) I (n =
5)
2 (n -- 5) 4 (n -- 5)
10 ( n = 5 ) 20 (n = 5) Overall (n = 25) Correlation coefficient (r)
289
REPRODUCIBII URINE
ITY
FOR
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METABOLITES
ADDED
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Recovery (mean 4- R.S.D.) (%) M-IX
M-V!
M-IV
92.9 96.1 95.8 96.5 96.9 95.7
89.6 96.5 93,1 94.6 94.1 93.6
90.2 94.8 94.5 96.9 97.4 94.7
(4-0.8) (4-0.7) (4- 1.3) (4-0.6) (4-0.9) ( 4- 1.8)
0.99995
(4- 1.8) (4- !.5) (4- 1.0) (4-0.9) (4- 1.0) ( 4- 2.8)
0.9999g
(4- !.3) (=t=0.8) (4- 1.2) (4-0,8) (4- 1.0) ( 4- 2.9)
0.99997
origin (correlation coefficient > 0.9999). T h e average recoveries o f the analytes in the c o n c e n t r a t i o n range 1-20 # g / m l were m o r e t h a n 9 0 % , with coefficients o f v a r i a t i o n less t h a n 2 . 9 % (Table II). T h e m i n i m u m d e t e r m i n a b l e c o n c e n t r a t i o n s
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Fig. 4. Serum levels o f lansoprazole and its metabolites. Data show the mean values and the standard deviations obtained from ten healthy volunteers (single oral dose of" 30-rag lansoprazole •capsule in the •fasted state). ( O ) Lansoprazole; ( I ) M-VI; (r-l) M.VII. • . . . . . .
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(signal-to-noise ratio of 3) for lanaoprazole a nd its metaboiites were all 50 ng/ml when 0.5 ml o f a urine samples was used. By using the established method, the determination of lansoprazole and its m a i n metabolites in serum and urine was performed after oral a d m i n i s t r a t i o n of the lansoprazole capsule (30 mg) to ten fasted, healthy male volunteers. Th e average time-courses of the serum concentrations are shown in Fig. 4. In serum, lansoprazole showed a higher concentration than M-V1 or M-VII. The serum concentration of lansoprazoie reached the m a x i m u m 2-3 h after a d m i n i s t r a t i o n and disappeared after 24 h. M-IV, M-VI and M - I X were excreted m a i n l y as glucuronides in the urine samples, and the total excretion ratios of the metabolites (glucuronides plus free forms) in 24 h were 8.55, 3.90 and 1.54%, respectively. ACKNOWLEDGEMENTS
The authors t h a n k Mr. T. Shima of T a k e d a Service Co. Ltd. a nd Mr. T. A m a n o o f T a k e d a Analytical Research Laboratories for their cooperation. REFERENCES 1 H, Satoh, N. hmtomi, H. Nagaya, !. lnada, A. N o h a r a and Y. Maki, Jpn. J. Pha~vracol., 40 (Suppl.) (I 986) 226. 2 H. Satoh, N. Inatomi, H. Nagaya, I. Inada, A. N o h a r a , N. N a k a m u r a and Y. Maki, J. PharmacoL Exp. Ther., 248 (1989) 806. 3 H. Nagaya, H, Satoh, K. K u b o :rod Y. Maki, J. PharmtwoL Exp. Ther., 248 (1989) 799. 4 K. Miwa, M. Mitani, T. T s u k a m o t o , K. Yoshida, T. Kobayashi, T. Kimura, H. S h i m o m u r a and S. T a n a y a m a , Jim. PharmacoL Ther., 18 (1990) 3413. 5 T. Kobayashi. M. Mitani, K. Miwa, K. Yoshida, I. Naeshiro, M. Ikeda, T, Kimura, H. S h i m o m u r a and S. T a n a y a m a , Jl m, Pharmacol. Ther., 18 (1990) 3437. 6 A. Pilbrant and C. Cederberg, Scand. J. Gastroenterol., 20 (Suppl.) (I985) ! 13.
Short Communication High-performance liquid chromatographic determination of lansoprazole and its metabolites in human serum and urine 1SAMU AOKI*
Takeda Atlal)'tical Research Laboratories. Lt~L. dusa-Hmmauchi. Ymtogan'a-ktt, Oset~'a 532 (J~. ~an) MINORU OKUMURA
Pkatvnacetttical Product/ott Research Laboratories. Takeda Chemh'al Dtdustri~.s. LttL. Jttso-Honmachi, Yodogau'a-ku. Osaka 532 (Japan) and TAKATSUKA YASHIKI
Analytical Laboratories. Takeda Ckemk'al huhtstries. Ltd.. Jtt.vo-Honmttchi. Yoth~gawa-ktt. Osaka 532 (Japan) (First received January 16th, 1991: revised manuscript received May ! 5th, 1991 ) ABSTRACT A simple and sensitive high-performance liquid c h r o m a t o g r a p h i c m e t h o d with uitraviole! detection is described for the simultaneous determination o f tansoprazole and its metabolitcs in hum:m serum and urine. T h e analytes in serum or urine were extracted with diethyl cther-diclalorometlaane (7:3, v/v) followed by e v a p o r a t i o n , dissolution and injection into ,'1 reversed-phase column. T h e reco,eries o f aufl~entic analyres added to serum at 0.05-2 ltg/ml o r to urine at 1-20 l~g/ml werc greater than 88"/,,, with the coellicients o f variation less than 7 . I % . T h e mininaum determinable concentrations o f all analytes were 5 ngtml in serum and 50 ng/mi in urine. T h e rnethod was successfully applied to a pharmacokinctic study o f lansoprazole in human.
INTRODUCTION
Lansoprazole, (4-)-2-[[[3-methyl-4-(2,2,2_trifluoroethoxy)_2_pyridyl]naethyl]_ sulphinyl]benzimidazole (AG-1749), is a new substituted benzimidazole compound, which markedly inhibits the secretion of basal and stimulated gastric acid and is.useful in the treatment of duodenal ulcers, gastric ulcers and reflux esophagitis [I-3]. The metabolism of kmsoprazole was investigated with animals (rats, mice and dogs), and the main metabolites were identified [4,5] as shown in Fig. i. The metabolites o f lansoprazole inclttde not only its sulphide (tX~-l) and sulphone (M-VII) but also 5-hydroxy metabolites (M-V!. -IV and -IX) of these. 0378-4347/91/S03.50
O
1991 Elsevier Science Publishers B.V. All rights reserved
SHORT COMMUNICATIONS
284 l ~ .
rl
.m
I N ' ~ " 8° - -
~../g~ao--cu,...~n..
Cl lr"-,y,j~ N'---
r
tt
,
!i0 ~-"t'~.,,, ' v ~
1: fl llsO.S~
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OC|[a(2F3
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M
H 8 - - CH=
lllC~
rN [
-
VI
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"~ OCII=CF=
OCI|=CF,~
M-I
M-1V
11
H OCII~CF~ M--- ~ql
OC
H=CF
M-IX
Fig. I. Structurcs o f hmsoprazole :rod its possible metabolites in humans.
In the present paper, a simple and sensitive high-performance liquid c h r o m a ' tographic (HPLC) method for the simultaneous determination of lansoprazole and its main metabolites in human serum and urine is decribed. EXPERIMENTAL
Reageots and materials Test samples and reagents. Lansoprazoie and its metabolites were synthesized in the Research and Development Division of Takeda Chemical Industries. T y p e VII fl-glucuronidase was purchased from Sigma (St. Louis, MO, USA). Dichloromethane, acetonitrile and ethanol were of H P L C grade. All other reagents were of analytical-reagent grade. Mobile phase 1 (MP-I) was water--acetonitrile-n-octylamine (620:380:1, v/v), adjusted to pH 7 with 85°/; phosphoric acid. Mobile phase II (MP-II) was 0.07 M phosphate buffer (pH 7.2)-ethanol-acetonitrile (20:10:3, v/v). The propylene glycol (PG) solution, propylene glycol-diethyl ether-dichloromethane (1:140:60, v/ v), was used t o depress the evaporation of the analytes. Internal standard solutions 1 (IS-I) and I1 (IS-II) consisted of 25 pg/ml isobutyl p-hydoxybenzoate in dichloromethane and 6 pg/ml isopropyl p-aminobenzoate in MP-II, respectively. Serum and urine samples were collected from ten healthy male volunteers participating in the Phase I study, and the control samples were collected before administration of the test compound. Standard samples. A serum standard sample was prepared to contain lan-
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285
s o p r a z o l e , M - I , M - I V , M ' V I , M - V I I a n d M - I X ( e a c h 0.5 llg/ml) in a c o n t r o l serum, a n d a urine s t a n d a r d s a m p l e was p r e p a r e d to c o n t a i n M - I V , M - V I a n d M - I X (each 5 .'.tg/ml) in a m i x t u r e o f c o n t r o l urine a n d 0.47 M s o d i u m b i c a r b o ' nate solution (I: 1, v/v).
Extraction o f the analytes.fi'om serum and to'hw samples A 0. l-ml p o r t i o n o f IS-I w a s a d d e d to 0.5 ml o f a s e r u m s a m p l e a n d a s e r u m s t a n d a r d sample, a n d the a n a l y t e s were e x t r a c t e d twice with 3 ml o f diethyl e t h e r - d i c h l o r o m e t h a n e (7:3, v/v). A f t e r c e n t r i f u g a t i o n , 0.5 ml o f the p r o p y l e n e glycol ( P G ) solution was a d d e d to the s u p e r n a t a n t , a n d the solvent w a s e v a p o r a t ed u n d e r a n i t r o g e n s t r e a m . T h e residue was dissolved in 0.5 ml o f M P - I , a n d a 100-1d p o r t i o n o f the s o l u t i o n w a s subjected to H P L C . T o 0.5 ml o f a urine s a m p l e a n d a urine s t a n d a r d s a m p l e w a s a d d e d 0.5 ml o f fl-glucuronidase solution (1000 U / m l in 0.07 M p h o s p h a t e buffer o f p H 7), a n d the m i x t u r e was i n c u b a t e d at 37"C for 60 min. T h e a n a l y t e s were extracted twice with 3 ml o f diethyl e t h e r - d i c h i o r o m e t h a n e (7:3, v/v). A f t e r c e n t r i f u g a t i o n , 0.5 ml o f the P G solution w a s a d d e d to the s u p e r n a t a n t , a n d the solvent w a s e v a p o r a t e d u n d e r a n i t r o g e n s t r e a m . T h e residue was dissolved in 0.4 ml o f IS-II, a n d a 20-1d p o r t i o n o f the solution was subjected to H P L C .
htstrttments and conditions./br determhlation T h e H P L C system consisted o f a S h i m a d z u L,~--,A ~ ,t o r L C - 6 A 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 e q u i p p e d with a n a u t o s a m p l e r with cooling device (lshido, C h i b a , J a p a n ) . A r e v e r s e d - p h a s e c o l u m n o f T S K gel O D S - I 2 0 T (particle size 5 ltm; 250 m m x 4.6 m m I . D . , T o s o h , T o k y o , J a p a n ) was used. T h e c o l u m n t e m p e r a t u r e a n d the flow-rate were 40°C a n d 1.0 m l / m i n , respectively. M P - I a n d M P - i I were used for the d e t e r m i n a t i o n o f a n a l y t e s 1,'_ s e r u m a n d urine, respectively. L a n s o p r a z o l e , M - I , M - I V , M - V I I a n d internal s t a n d a r d in s e r u m were detected by U V a b s o r p t i o n at 285 n m , a n d M - V ! a n d M - I X were detected at 303 nm; M - I V , M - V I a n d M - I X in urine were detected at:303 rim.
Admhlistration o f lansoprazole and sample collection A capsule c o n t a i n i n g 30 m g o f l a n s o p r a z o l e as enteric-coated g r a n u l e s (lans o p r a z o l e capsule, 30 mg) w a s orally a d m i n i s t e r e d to each o f ten fasted h e a l t h y male volunteers. T h e s e r u m samples were collected before a d m i n i s t r a t i o n a n d 0.5, 1, 2, 3, 4, 6, 8, 12 a n d 24 h a f t e r a d m i n i s t r a t i o n . T h e samples were preserved at 20"C until analysis. T h e urine samples were collected before a d m i n i s t r a t i o n a n d at the intervals o f 0 - 6 , 6 - 1 2 a n d 12-24 h a f t e r administratior~. T o stabilize the analytes, the s a m e v o l u m e o f 0.47 M ~ndlum b i c a r b o n a t e solution was a d d e d i m m e d i a t e l y a f t e r the collection o f a urine s a m p l e a n d the m i x t u r e w a s stored at - 2 0 ° C until analysis. -
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286
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DISCUSSION
F r o m the results of metabolism of lansoprazole in animals, the sulphide (M-I) and sulphone (M-VII) metabolites a nd three corresponding 5-hydroxy metabolites (M-VI, M-IV, M-IX) were expected as metabolites in h u m a n s . Thus, we investigated a s i m u l t a n o u s determination m e t h o d for lansoprazole and its five possible metabolites to develop a reversed-phase H P L C technique with UV detection. Lansoprazole and its metabolites were unstable in an acid solution, as is omeprazole [6], a lthough they were rather stable in an alkaline solution; the half-lives o f lansoprazole at 25"C were ca. 30 min at p H 5, c a . 18 h at pH 7 and c a . 37 h at pH 8. Accordingly, the analytes were stable in a serum sample (pl-I c a . 8), but unstable in a urine sample depending on the p H . Therefore, we stabilized the analytes by a d d i n g an equal volume of 0.47 M s o d i u m bicarb~T~ate solution to each urine sample to keep the pH c a . 9. The c h r o m a t o g r a m s of serum samples are shown in Fig. 2. The calibration curves of the peak-height ratios (analyte to
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Fig. 2. C h r o m a t o g r a m s o f e x t r a c t s f r o m ( A ) a n a l y t e - f r e c s e r u m , (B) s e r u m s p i k e d w i t h a n a l y t e s ( e a c h c o n c e n t r a t i o n c a . 0. i gtg,q~l) a n d (C) the s e r u m s a m p l e o f a v o l u n t e e r ( M - V I , 162 n g / m l : l a n s o p r a z o l e , 1748 n g / m l ; M - V I I , 268 n g / m l ) . P e a k s : I = M - V I ; 2 --- M - I X ; 3 = M - I V : 4 = l a n s o p r a z o l e ; 5 = M - V I I : 6 = internal standard: 7 = M-I.
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internal standard) v e r s u s the concentration of the analytes show straight lines passing through the origin (correlation coefficient >0.999). The average recoveries of the analytes in the concentration range 0.05-2 lzg/ml were more than 88%, with coefficients of variation (C.V) of less t h a n 7.1% (Table I). The minimum determinable concentrations (signal-to-noise ratio of 3) for lansoprazole and its metabolites were all 5 ng/ml when 0.5 ml of a serum sample was used. Preliminary investigation on the metabolites of lansoprazole in human urine revealed that only 5-hydroxy metabolites (M-IV, M-VI, M-IX) were excreted, mainly as the glucuronides. As the authentic samples of the glucuronides were not available'," the cenditions for the hydrolysis of glucuronides were investigated using fl-glucuronidase. As a result, incubation at 37°C for 1 h was found enough for the hydrolysis. The chromatograms of urine samples (after hydrolysis) are shown in Fig. 3. The calibration curves of the peak-height ratios (analyte to internal s t a n d a r d ) v e r s u s the concentration of the analytes show straight lines passing through the
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Fig. 3. C h r o m a t o g r a m s o f extracts f r o m (A) analyte-free urine (after hydrolysis) a n d (B) the urine sample o f a volunteer (after hydrolysis: M - I X , 5.75 /tg/ml; M - V I , 4.35 pg/ml; M - I V , 17.09 lig/ml); detection wavelength, 303 nm. Peaks: 1 = M - I X ; 2 = M - V I ; 3 = internal standard; 4 -- M - I V .
SHORT C O M M U N I C A T I O N S
288
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TABLE
II
RECOVERY CONTROL
AND HUMAN
Concentration o f analytes in urine (/ig/ml) I (n =
5)
2 (n -- 5) 4 (n -- 5)
10 ( n = 5 ) 20 (n = 5) Overall (n = 25) Correlation coefficient (r)
289
REPRODUCIBII URINE
ITY
FOR
LANSOPRAZOLE
METABOLITES
ADDED
TO
Recovery (mean 4- R.S.D.) (%) M-IX
M-V!
M-IV
92.9 96.1 95.8 96.5 96.9 95.7
89.6 96.5 93,1 94.6 94.1 93.6
90.2 94.8 94.5 96.9 97.4 94.7
(4-0.8) (4-0.7) (4- 1.3) (4-0.6) (4-0.9) ( 4- 1.8)
0.99995
(4- 1.8) (4- !.5) (4- 1.0) (4-0.9) (4- 1.0) ( 4- 2.8)
0.9999g
(4- !.3) (=t=0.8) (4- 1.2) (4-0,8) (4- 1.0) ( 4- 2.9)
0.99997
origin (correlation coefficient > 0.9999). T h e average recoveries o f the analytes in the c o n c e n t r a t i o n range 1-20 # g / m l were m o r e t h a n 9 0 % , with coefficients o f v a r i a t i o n less t h a n 2 . 9 % (Table II). T h e m i n i m u m d e t e r m i n a b l e c o n c e n t r a t i o n s
,-.
t000-
i
see.
"
\ T
9
' O"
24 •
Time(b)
Fig. 4. Serum levels o f lansoprazole and its metabolites. Data show the mean values and the standard deviations obtained from ten healthy volunteers (single oral dose of" 30-rag lansoprazole •capsule in the •fasted state). ( O ) Lansoprazole; ( I ) M-VI; (r-l) M.VII. • . . . . . .
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(signal-to-noise ratio of 3) for lanaoprazole a nd its metaboiites were all 50 ng/ml when 0.5 ml o f a urine samples was used. By using the established method, the determination of lansoprazole and its m a i n metabolites in serum and urine was performed after oral a d m i n i s t r a t i o n of the lansoprazole capsule (30 mg) to ten fasted, healthy male volunteers. Th e average time-courses of the serum concentrations are shown in Fig. 4. In serum, lansoprazole showed a higher concentration than M-V1 or M-VII. The serum concentration of lansoprazoie reached the m a x i m u m 2-3 h after a d m i n i s t r a t i o n and disappeared after 24 h. M-IV, M-VI and M - I X were excreted m a i n l y as glucuronides in the urine samples, and the total excretion ratios of the metabolites (glucuronides plus free forms) in 24 h were 8.55, 3.90 and 1.54%, respectively. ACKNOWLEDGEMENTS
The authors t h a n k Mr. T. Shima of T a k e d a Service Co. Ltd. a nd Mr. T. A m a n o o f T a k e d a Analytical Research Laboratories for their cooperation. REFERENCES 1 H, Satoh, N. hmtomi, H. Nagaya, !. lnada, A. N o h a r a and Y. Maki, Jpn. J. Pha~vracol., 40 (Suppl.) (I 986) 226. 2 H. Satoh, N. Inatomi, H. Nagaya, I. Inada, A. N o h a r a , N. N a k a m u r a and Y. Maki, J. PharmacoL Exp. Ther., 248 (1989) 806. 3 H. Nagaya, H, Satoh, K. K u b o :rod Y. Maki, J. PharmtwoL Exp. Ther., 248 (1989) 799. 4 K. Miwa, M. Mitani, T. T s u k a m o t o , K. Yoshida, T. Kobayashi, T. Kimura, H. S h i m o m u r a and S. T a n a y a m a , Jim. PharmacoL Ther., 18 (1990) 3413. 5 T. Kobayashi. M. Mitani, K. Miwa, K. Yoshida, I. Naeshiro, M. Ikeda, T, Kimura, H. S h i m o m u r a and S. T a n a y a m a , Jl m, Pharmacol. Ther., 18 (1990) 3437. 6 A. Pilbrant and C. Cederberg, Scand. J. Gastroenterol., 20 (Suppl.) (I985) ! 13.
