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Determination of a new angiotensin-converting enzyme inhibitor (CS-622) and its active metabolite in plasma and urine by gas chromatography-mass spectrometry using negative ion chemical ionization
129
Journal of Chromatography, 496 (1989) 129-135 BromedLcal Applrcatrons Elsevler Science Publishers B V , Amsterdam - Pnnted m The Netherlands
CHROMBIO
4943
DETERMINATION OF A NEW ANGIOTENSIN-CONVERTING ENZYME INHIBITOR (CS-622) AND ITS ACTIVE METABOLITE IN PLASMA AND URINE BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY USING NEGATIVE ION CHEMICAL IONIZATION
HIROAKI SHIOYA*, MASAKO SHIMOJO and YUKINORI KAWAHARA Product Development Laboratortes, Shanagawa-ku, Tokyo 140 (Japan)
Sankyo Co, Ltd ,2-58, HtromachL 1 -chome,
(First received May 3rd, 1989, revised manuscript received July 6th, 1989)
SUMMARY A sensltlve and speclflc gas chromatographlc-mass spectrometrlc method for the simultaneous determmatlon of aneotensm-convertmg enzyme mhlbltor (I, CS-622) and lte active desethyl metabohte (II, RS-5139) m plasma and urine was developed Compound D5-RS-5139 was used as an internal standard and measurements were made by electron-capture negative Ion chemical lomzatlon Extraction from plasma and urme was carried out usmg Sep-Pak Cl8 and silica cart&es The extract of plasma or urme was treated with dlazomethane followed by trlfluoroacetlc anhydride to convert I and II mto their methyl ester trlfluoroacetyl derlvatlves The detectlon hmlt of I and II was 0 5 ng/ml m plasma and 5 ng/ml m urme The proposed method was satlsfactory for the determmatlon of I and II m plasma and urme with respect to accuracy and preclslon Thus it 1s suitable for measurement of bloavallabdlty and pharmacokmetlcs of I and II m body flu&
INTRODUCTION
In recent years, inhibitors of angiotensin-convertmg enzyme (ACE; EC 3.4 15.1) have offered a new approach to the treatment of hypertension and congestive heart failure. a-{ (2S,6R)-6- [ (lS)-l-Ethoxycarbonyl-3-phenylpropyllammo-5-oxo-2(2-thienyl)perhydro-1,4-thiazepin-4-yl) acetic acid hydrochloride (I, CS-622) is a new ACE inhibitor, and it is a prodrug that is deesterified m vivo by esterase to release an active metabolite RS-5139 (II, Fig 1). Compound I has a very high potency, so it IS therapeutrcally active at 0378-4347/89/$03
50
0 1989 Elsevler Science Pubhshers B V
D Fig 1 Structures of I, II and the internal standard (IS )
low concentration in plasma. Thus it is very important to determine its concentration in plasma and urine in order to study the pharmacokmetics and pharmacodynamlcs Analytical methods m use at present for the measurement of I and II are inhibitor binding assay (IBA) [l], radioimmunoassay [2] and liquid or gas chromatography [ 3,4]. IBA and radioimmunoassay are very sensitive, but it is difficult to distinguish the prodrug and its active metabohte at the same time. Column liquid and gas chromatography are specific but less sensitive than other methods. This paper describes a gas chromatographic-mass spectrometrlc (GC-MS) method that uses negative ion chemical lomzatlon (NICI ). The fused-silica capillary GC column and the selected-Ion monitoring (SIM) technique make the method very specific and highly sensitive EXPERIMENTAL
Mater&s and reagents Compounds I and II and the internal standard (IS, Fig. 1) were synthesized by Yanaglsawa et al. [5]. All reagents were of analytical grade Sep-Pak C,, and silica cartridges were products of Waters Assoc. (Milford, MA, US A.) Trifluoroacetic anhydride (TFAA) was purchased from Tokyo Kasei (Tokyo, Japan) and used without further purification. N-Methyl-N-mtroso-p-toluenesulphonamide (Dmzald), for diazomethane generation, was supplied by Aldrich (Milwaukee, WI, U.S.A. ) Gas chromatography-muss spectrometry A Hewlett-Packard 5988A GC-MS system equipped with a Hewlett-Packard 5890 gas chromatograph was used. The gas chromatograph was equipped
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Fig 2 Mass spectra of derwatwes of I (A), II (B) and IS (C)
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, 600
133
ACE inhibitors, such as enalapril and ramiprd. So this method is applicable to the measurement of the other non-sulphydryl-containing ACE inhibitors in the body fluids. The SIM chromatograms of spiked plasma are shown in Fig. 3 and no interfering peaks were observed The retention times of I, II and IS were 5.2, 5.06 and 5.04 min, respectively. (I: The standard curves of I and II showed good linearity Ion 2
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Fig 3 SIM chromatograms of the extract from a l-ml plasma sample, spiked with 156 ng of I (m/z 302 ) and II (m/z 288 ) , and 200 ng of IS (m/t 293 ) TABLE I ACCURACY AND PRECISION OF THE PROPOSED METHOD FOR THE DETERMINATION OF I AND II IN HUMAN PLASMA AND URINE Compound
Plasma I II
Urrne I II
Concentration added
Concentration found (mean+S D )
(ng/ml 1
(ng/ml )
Coefficient of vanatlon (o/o)
5 15 20 60
65*02 163&07 193&07 586?13
49 46 38 25
50 150 200 600
506?22 X15%-42 200 1+38 5788+76
30 28 19 13
134
_
200
z m 150
t
0’ ; E
100
42
G 8F
50
0 0
12
24
36
4%
Time (h)
Fig 4 Plasma concentrations (mean t S D n = 6) of I ( 0 ) and II (0 ) after oral admmlstratlon of5mgofI
I
6
12
18
Y 24
Time (h)
Fig 5 Cumulatwe urinary excretion ratios (mean k S D , n =6) of I (0 ) and II ( l ) after oral admmlstratlon of 5 mg of I
y=7 6*10W3x+ 1.0*10-2,r2=0.999, II y=5.2~10-3x+0.8~10-2,r2=0 999) over the concentration range 156-100 ng/ml for a l-ml sample volume. It is evident from the data m Table I that the proposed method is satisfactory in both accuracy and precision. The detection limrt is 0 5 ng/ml m plasma and 5 ng/ml in urine at a signal-to-noise ratio of 3. The method was apphed to the determinatron of I and II m the plasma and urme of healthy volunteers orally dosed with 5 mg of I Plasma concentrations of I and II are shown in Fig 4. The cumulative urmary excretions of I and II are shown m Fig 5 It is apparent that I 1s rapidly absorbed and metabohzed to the pharmacologically active II
135
In conclusion, this GC-MS method can be applied to the determination of I and II m plasma and urine at the same time and 1s suitable for studies of the pharmacokmetlcs and pharmacodynamlcs of I and II ACKNOWLEDGEMENTS
The authors thank Dr H Yanaglsawa and his co-workers for the synthesis of the pentadeuterated compound D5-RS-5139. REFERENCES 1 D J Tocco, F A de Luna, A E W Duncan, T C Vassal and E H Ulm, Drug Metab D~spos ,10 (1982) 15 2 M Hlkens, E L Hand and W S Mulcahy, Llgand Q ,4 (1981) 43 3 A Rakhlt andV Tlpms, Clm Chem ,30 (1984) 1237 4 GV Tlpms and A Rakhlt, J Chromatogr ,345 (1985) 396 5 H Yanaglsawa, S Ishn, A Ando, T Kanazakl, S Mlyamoto, H Kolke, Y Iqlma, K Olzuml, Y Matsushita and T Hata, J Med Chem ,30 (1987) 1984 6 P HaJdu, D Schmlt, M Bomm, L Hack, and A Keller, Arznelm -Forsch ,34 (1984) 1431
Journal of Chromatography, 496 (1989) 129-135 BromedLcal Applrcatrons Elsevler Science Publishers B V , Amsterdam - Pnnted m The Netherlands
CHROMBIO
4943
DETERMINATION OF A NEW ANGIOTENSIN-CONVERTING ENZYME INHIBITOR (CS-622) AND ITS ACTIVE METABOLITE IN PLASMA AND URINE BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY USING NEGATIVE ION CHEMICAL IONIZATION
HIROAKI SHIOYA*, MASAKO SHIMOJO and YUKINORI KAWAHARA Product Development Laboratortes, Shanagawa-ku, Tokyo 140 (Japan)
Sankyo Co, Ltd ,2-58, HtromachL 1 -chome,
(First received May 3rd, 1989, revised manuscript received July 6th, 1989)
SUMMARY A sensltlve and speclflc gas chromatographlc-mass spectrometrlc method for the simultaneous determmatlon of aneotensm-convertmg enzyme mhlbltor (I, CS-622) and lte active desethyl metabohte (II, RS-5139) m plasma and urine was developed Compound D5-RS-5139 was used as an internal standard and measurements were made by electron-capture negative Ion chemical lomzatlon Extraction from plasma and urme was carried out usmg Sep-Pak Cl8 and silica cart&es The extract of plasma or urme was treated with dlazomethane followed by trlfluoroacetlc anhydride to convert I and II mto their methyl ester trlfluoroacetyl derlvatlves The detectlon hmlt of I and II was 0 5 ng/ml m plasma and 5 ng/ml m urme The proposed method was satlsfactory for the determmatlon of I and II m plasma and urme with respect to accuracy and preclslon Thus it 1s suitable for measurement of bloavallabdlty and pharmacokmetlcs of I and II m body flu&
INTRODUCTION
In recent years, inhibitors of angiotensin-convertmg enzyme (ACE; EC 3.4 15.1) have offered a new approach to the treatment of hypertension and congestive heart failure. a-{ (2S,6R)-6- [ (lS)-l-Ethoxycarbonyl-3-phenylpropyllammo-5-oxo-2(2-thienyl)perhydro-1,4-thiazepin-4-yl) acetic acid hydrochloride (I, CS-622) is a new ACE inhibitor, and it is a prodrug that is deesterified m vivo by esterase to release an active metabolite RS-5139 (II, Fig 1). Compound I has a very high potency, so it IS therapeutrcally active at 0378-4347/89/$03
50
0 1989 Elsevler Science Pubhshers B V
D Fig 1 Structures of I, II and the internal standard (IS )
low concentration in plasma. Thus it is very important to determine its concentration in plasma and urine in order to study the pharmacokmetics and pharmacodynamlcs Analytical methods m use at present for the measurement of I and II are inhibitor binding assay (IBA) [l], radioimmunoassay [2] and liquid or gas chromatography [ 3,4]. IBA and radioimmunoassay are very sensitive, but it is difficult to distinguish the prodrug and its active metabohte at the same time. Column liquid and gas chromatography are specific but less sensitive than other methods. This paper describes a gas chromatographic-mass spectrometrlc (GC-MS) method that uses negative ion chemical lomzatlon (NICI ). The fused-silica capillary GC column and the selected-Ion monitoring (SIM) technique make the method very specific and highly sensitive EXPERIMENTAL
Mater&s and reagents Compounds I and II and the internal standard (IS, Fig. 1) were synthesized by Yanaglsawa et al. [5]. All reagents were of analytical grade Sep-Pak C,, and silica cartridges were products of Waters Assoc. (Milford, MA, US A.) Trifluoroacetic anhydride (TFAA) was purchased from Tokyo Kasei (Tokyo, Japan) and used without further purification. N-Methyl-N-mtroso-p-toluenesulphonamide (Dmzald), for diazomethane generation, was supplied by Aldrich (Milwaukee, WI, U.S.A. ) Gas chromatography-muss spectrometry A Hewlett-Packard 5988A GC-MS system equipped with a Hewlett-Packard 5890 gas chromatograph was used. The gas chromatograph was equipped
132 s b 9
P
302
283
20:
\ 10
/ 138
I I!
0
584
1,” 5
‘3 ’
’
i’a, ! I /
300 Mass/Charge
200 lel0
70
,
/ ,i ,
5ti0
S&I0
! I! 400
L
B
SO a0
.’
288
i @-y-$Q”
35
1
CO&H,
CF3
II
R = CH,
40 287 30
\
20 570 /
t-lass/Charge
IS
20
R = CH3
2?3
J, 200
300 Mass/Charge
400
Fig 2 Mass spectra of derwatwes of I (A), II (B) and IS (C)
504 /
,/
500
575 /
,,I,
i.
, 600
133
ACE inhibitors, such as enalapril and ramiprd. So this method is applicable to the measurement of the other non-sulphydryl-containing ACE inhibitors in the body fluids. The SIM chromatograms of spiked plasma are shown in Fig. 3 and no interfering peaks were observed The retention times of I, II and IS were 5.2, 5.06 and 5.04 min, respectively. (I: The standard curves of I and II showed good linearity Ion 2
268
00
Ion
amu
302.00
amu
2E4
2.0E4 3.0E4
7.0E5 6.0ES i1.4E4
:
:1.2E4 D
: -o
s
: 10000
:
4.0ES
2
i
6000
: BE5
: ,2
BE4
: P
3.0E5
6000 4000
0 4 0 Tlms
(mfn
0 4.0 Time
6.0 >
0L 6 (mln
0 )
6 Ttn”,
(mfn
0 I’
Fig 3 SIM chromatograms of the extract from a l-ml plasma sample, spiked with 156 ng of I (m/z 302 ) and II (m/z 288 ) , and 200 ng of IS (m/t 293 ) TABLE I ACCURACY AND PRECISION OF THE PROPOSED METHOD FOR THE DETERMINATION OF I AND II IN HUMAN PLASMA AND URINE Compound
Plasma I II
Urrne I II
Concentration added
Concentration found (mean+S D )
(ng/ml 1
(ng/ml )
Coefficient of vanatlon (o/o)
5 15 20 60
65*02 163&07 193&07 586?13
49 46 38 25
50 150 200 600
506?22 X15%-42 200 1+38 5788+76
30 28 19 13
134
_
200
z m 150
t
0’ ; E
100
42
G 8F
50
0 0
12
24
36
4%
Time (h)
Fig 4 Plasma concentrations (mean t S D n = 6) of I ( 0 ) and II (0 ) after oral admmlstratlon of5mgofI
I
6
12
18
Y 24
Time (h)
Fig 5 Cumulatwe urinary excretion ratios (mean k S D , n =6) of I (0 ) and II ( l ) after oral admmlstratlon of 5 mg of I
y=7 6*10W3x+ 1.0*10-2,r2=0.999, II y=5.2~10-3x+0.8~10-2,r2=0 999) over the concentration range 156-100 ng/ml for a l-ml sample volume. It is evident from the data m Table I that the proposed method is satisfactory in both accuracy and precision. The detection limrt is 0 5 ng/ml m plasma and 5 ng/ml in urine at a signal-to-noise ratio of 3. The method was apphed to the determinatron of I and II m the plasma and urme of healthy volunteers orally dosed with 5 mg of I Plasma concentrations of I and II are shown in Fig 4. The cumulative urmary excretions of I and II are shown m Fig 5 It is apparent that I 1s rapidly absorbed and metabohzed to the pharmacologically active II
135
In conclusion, this GC-MS method can be applied to the determination of I and II m plasma and urine at the same time and 1s suitable for studies of the pharmacokmetlcs and pharmacodynamlcs of I and II ACKNOWLEDGEMENTS
The authors thank Dr H Yanaglsawa and his co-workers for the synthesis of the pentadeuterated compound D5-RS-5139. REFERENCES 1 D J Tocco, F A de Luna, A E W Duncan, T C Vassal and E H Ulm, Drug Metab D~spos ,10 (1982) 15 2 M Hlkens, E L Hand and W S Mulcahy, Llgand Q ,4 (1981) 43 3 A Rakhlt andV Tlpms, Clm Chem ,30 (1984) 1237 4 GV Tlpms and A Rakhlt, J Chromatogr ,345 (1985) 396 5 H Yanaglsawa, S Ishn, A Ando, T Kanazakl, S Mlyamoto, H Kolke, Y Iqlma, K Olzuml, Y Matsushita and T Hata, J Med Chem ,30 (1987) 1984 6 P HaJdu, D Schmlt, M Bomm, L Hack, and A Keller, Arznelm -Forsch ,34 (1984) 1431