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Anal. Chem. 1980, 52, 1086-1089
Gas Chromatography/Selected Ion Monitoring Mass Spectrometric Determination of Captopril in Human Blood Phillip T. Funke, Eugene Ivashkiv, Mary F. Malley, and Allen I. Cohen" Department of Analytical Research, The Squibb Institute for Medical Research, Princeton, New Jersey 08540
A gas-liquid chromatography/selected ion monitoring mass spectrometric method has been developed to measure Captopril (SQ14,225), a new angiotensin-converting enzyme inhibitor, in human blood. Oxidative degradation of the thiol function in whole blood was prevented by quantitative reaction with N-ethylmaleimide immediately after sampling. The separation procedure includes quantitative adsorption of the N-ethylmaleimide derivative onto XAD-2 resin. The limit of detection and the practical detection limit with a 9 0 % confidence limit are 5.5 and 16.5 ng per mL of blood, respectively.
acid, acetone, Sylon-CT (Supelco), "V,O-bis(trirnethylsilyl)trifluoroacetamide (BSTFA, Pierce Chemical Co.), metaphosphoric acid, sodium phosphate dibasic (Na2HP0,.7H20, instant methanolic hydrochloride kit (Applied Science Lab.), 80-200 mesh Brockman Activity 1 neutral alumina, and sodium chloride were used without further purification. Purified XAD-2 resin (Applied Science Lab.) was conditioned by transfer of 100 g of resin to a column, washed successively with 500 mL of methanol, 500 mL of acetone, and 1000 mL of water. The resin was stored as a suspension in water. Ethyl acetate was purified immediately before use by passing 600 mL through a neutral alumina 2.5 X 40 ern column to remove impurities. Glassware. Columns with Teflon stopcocks, 250 X 10.5 mm with 100 mm X 50 mm cylindrical reservoirs, 5-mL Reacti-Vials (Pierce Chemical Co.), 1-mL GLC vials (Hewlett-Packard) with sealable Teflon-lined caps and 150-mm unscratched screw-cap culture tubes were used. Solutions. Phosphate buffer, p H 7.0, was prepared by dissolving 40 g of NazHPO4.7H20 in 1000 mL of distilled H,O, adjusting the p H to 7.0 with phosphoric acid and then diluting to 2000 mL with HzO. Metaphosphoric Acid Solution, Ten percent solutions were prepared daily with 25 g of metaphosphoric acid dissolved in approxiniately 225 mL of distilled H 2 0 , sonicated in a beaker in an ultrasonic bath, and diluted to 250 mL. Methanolic Hydrochloric Acid. A 0.25-mL portion of chilled redistilled acetyl chloride was added to 5 mL of LIPOPURE methanol in a clean, dry 150-mm test tube, cooled for 5 min in an ice bath. The solution was mixed and transferred to a 5-mL Reacti-Vial, which was capped and sealed. S t a n d a r d Solutions. Twenty-five mg of internal reference standard, 2,SO.O mg of 1 and 25.0 mg of disulfide, 3, were weighed separately. The internal reference standard, 2, was transferred to a 100-mL volumetric flask as were 1 and 3 to another 100-mL volumetric flask, each flask containing 250 mg of NEM and 10 mL of p H 7 phosphate buffer. After standing for 15 min, the dissolved samples were diluted to mark with acetone and filtered through sintered glass of fine porosity. The concentration of the stock solution of internal reference standard was 250 pg/mL and that of the stock solution of captopril (1) and disulfide (3) was 500 and 250 pg/mL, respectively. Working solutions were prepared from the two stock solutions by diluting one to ten with acetone. The concentrations of 1 and 3 in the working solution were 50 and 25 pg/mL, respectively, and that of 2 was 25 pg/mL. When kept in a tightly-sealed brown container and stored in a freezer, the concentrated and working solutions were stable for at least 3 months. P r e p a r a t i o n of Control Blood Samples a n d Construction of S t a n d a r d Curve. T o 5 mL of citrated blood in 150-mm screw-cap culture tubes, each containing 2,5 mg of N E M , a n appropriate amount of the standard solution containing 1 and 3 was added to yield blood concentrations from 0 to 2000 ng/mI, of 1 and half as much as 3. To each 5-mL sample. 0.1 mL of the diluted internal reference standard solution (2.5 p g of 2) was added. These control blood samples were processed as described below, The ratios were determined and a straight line plot constructed of the ratio of the peak height intensities (I,,,, l,i,,/ I,,,, 248)r R , vs. the n g ratio of 1 t o 2, R'. The intercept, I , is the ratio for the blank blood extract ( R ' = 0 ) . The line was fitted by linear regression t o Equation 1
A hypothetical model of t h e active site of t h e angiotensin-converting system has led t o the design of a new class of specific inhibitors of t h e enzyme (1-3). One of these com-L-proline, pounds, 1-[2(S)-3-mercapto-2-methyl-l-oxopropyl] captopril (1, CAS Registry 62571-86-2) has been shown to be a n orally active angiotensin converting-enzyme inhibitor in man ( 4 ) , as demonstrated in clinical studies ( 5 ) . An analytical method was needed t o measure blood levels of 1. Because of extreme reactivity of thiols, such as 1, t h e success of t h e gas-liquid chromatography (GLC) selected ion monitoring mass spectrometry method depends upon preventing oxidative degradation of captopril during storage of the blood samples prior t o processing. T h e thiol alkylating agent N-ethylmaleimide (NEM) (6-8) was chosen to eliminate this problem because N E M immediately reacts with captopril i n blood to form a stable thioalkyl derivative, l A , as shown by disposition studies in mouse (9) and human (10) blood after oral administration of %-captopril. This paper describes t h e selective extraction and purification procedures a n d the GLC/selected ion monitoring measurement of from 10- to 2000 ng of 1 p e r m L of blood (Figure 1).
EXPERIMENTAL I n s t r u m e n t a t i o n . The modified Electronic Associates, Inc. QUAD 300 quadrupole mass spectrometer, previously described ( I I ) , is now under the control of a Teknivent 69 KD data acquisition system operating in the selected ion mode. Peak height measurements and ratio calculations were performed with programs provided with the computer system. The ionization voltage was maintained at 50 eV with a total emission current of 2 mA. The other ion source parameters were adjusted for sensitivity and resolution. The analysis was carried out on a 70 cm x 2 mm i.d. silanized glass column packed with 3% OV-101 on 80/100 mesh Supelcoport, which was connected by a glass-lined stainless steel transfer line to the source of the spectrometer (11). All glass surfaces were previously silanized with Sylon-CT reagent before assembly. The injector temperature was maintained a t 280 "C, while the transfer line was kept a t 240 "C. The column temperature was programmed from 200 to 280 "C a t a rate of 20 "C/min, commencing from the time of injection. Dried helium gas (11) was regulated at 6 mL/min. R e a g e n t s . c a p t op r i 1 ( 1) , 1- [ ( s ) ,4S] - 4- f 1uo ro - 1- ( D- 3 mercapto-2-methyl-l-oxopropyl)-l-proline (2), and captopril disulfide (3) were pharmaceutical grade materials (E. R. Squibb & Sons) used without additonal purification. Methanol, A'ethylmaleimide, hydrochloric acid, sodium bicarbonate. phosphoric 0003-2700/80/0352-1086$0 1 O O / O
R=l+kR' t(i C
ebtahlish the slope. k , and the intercept. I (Figure 2).
1980 American Chemical Society
(1)
ANALYTICAL CHEMISTRY, VOL. 52, NO. 7, JUNE 1980 % *
i O
-1 CAPTOPRIL -2 4-Fluoro-1
H F
R
H H
1A
217 235
-3 35
2A 16 28
H F H F
H
H CH3 CH3
342 360 356 374
-_ kJ Rv
MW
CWg
432 460
Figure 1. Structures of captopril, related compounds, and their derivatives
0
1
2
Nanogram Ratio 1/2,
3 RI
Figure 2. Calibration curves, ratio of intensities of captopril to 4fluorocaptopril Blood Collection. From 12 to 15 mL of blood were drawn by Vacutainer (Becton-Dickinson). Each blood sample was immediately poured into a prepared tube containing 75 mg of NEM. The tube was capped, sealed, and the contents mixed vigorously on a Vortex mixer. The coded samples were placed in a dry ice chest and frozen. Citrated blood used for calibration and other experiments was stored under the same conditions, but without the addition of NEM. E x t r a c t i o n Procedure. Measured volumes, preferably 5 mL, of thawed blood samples were transferred to 150-mm screw-cap culture tubes. Then 0.1 mL of the diluted internal reference solution (2.5 pg of 2) was added to each of these blood samples. One 5-mL control blood sample containing 5000 ng of 1, derivatized with NEM, and 2500 ng of 3, was prepared as described for the standard curve. To each tube, a 10% metaphosphoric acid solution was added dropwise from a buret, with mixing, until 8 m L had been added and then a t a faster rate until a total of 25 m L had been added to each sample. After standing for 30 min, the solutions were centrifuged a t about 2000 rpm for 20 min. The supernatant was filtered through a Nalgene 0.45-wm disposable membrane and the filtrate transferred to a disposable 150-mm test tube. The excess moisture from the XAD-2 resin was removed by vacuum drying on a glass filter, just prior to use, and 1-g portions were transferred to 150-mm screw-cap culture tubes, followed by 20 mL of the sample filtrates. The capped culture tubes were shaken mechanically for 60 min. The contents of the culture tubes were transferred to a chromatographic column containing a small plug of silanized glass wool. The plastic cap was washed twice with 5-mL portions of 0.1 N HC1. All remaining solids in the culture tube were transferred to the column, by carefully injecting 10-mL portions of 0.1 N HC1 from a syringe
1087
equipped with a long canule into the inverted culture tube held over the column. The column was then washed twice with 5-mL portions of acid and finally with about 10 mL of acid under the slight vacuum generated by an aspirator until the excess 0.1 N HCl was removed from the resin. T o the dried resin, 10 mL of ethyl acetate were added and the column eluted a t a rate of 1.5 mL/min. The eluate was collected in a 150-mm screw-cap culture tube containing 3 mL of 5% NaHC03 solution. A second 10-mL portion of ethyl acetate was added to the column and collected in the culture tube. The remaining solvent was forced from the resin into the culture tube by air pressure from a hand bulb on the head of the column. The tubes were capped, shaken for 5 min, centrifuged a t about 2000 rpm for 5 min, unsealed, and the upper ethyl acetate layer was removed by aspiration and discarded. T o each aqueous solution, 1 mL of 6 N phosphoric acid was added and mixed. Then 3 g of sodium chloride were added, and the tube was capped and placed on a shaker for 5 min. To each tube, 5 mL of purified ethyl acetate were added; the tube was sealed and shaken for 5 min. T h e tube was then centrifuged for 5 min a t about 3000 rpm. The centrifugate was decanted into a disposable 13 X 100 mm test tube. Using a 5-mL serological pipet, 4.5 ml, of the ethyl acetate layer was transferred to a 5-mL Reacti-Vial and the solvent was reduced to approximately 0.1 mL under a nitrogen stream on a 50 "C water bath. By means of an automatic pipet, 0.2 mL of ethyl acetate was added to the residue and mixed on a Vortex mixer. The solution was transferred with a disposable Pasteur pipet to a 1-mL GLC serum vial. The Reacti-Vial was rinsed with an additonal 0.2 mL of ethyl acetate. The rinse was transferred to the smaller vial. The solvent was evaporated with a Mini-Vap under a stream of nitrogen and then dried at room temperature in a vacuum oven for 30 min. Methylation. To the dried residue, 0.1mL of methanolic HCl solution was added, the vial was immediately capped and sealed with a Teflon-lined serum septum and the solution was mixed on a Vortex mixer. The sealed vial was then heated on a dry block heater, containing glass powder, a t 60 "C for 10 min, cooled a t room temperature for an additional 10 min, the caps were removed, and the sample was dried in a vacuum desiccator for 1 h. The vials were recapped with a Teflon-lined serum septum and stored in a dark refrigerator. For injection onto the GLC column, the sample was reconstituted by adding 20 pL of acetone through the septum and mixing thoroughly on a Vortex mixer. Properly extracted solutions yielded a light salmon colored solution. Selected Ion Monitoring. A 5-pL syringe was washed with six 5-pL aliquots of acetone. BSTFA, 0.5 pL, was drawn up into the syringe, followed by 1pL of reconstitued sample solution. The solution plus BSTFA was injected onto the column. BSTFA was used to prolong the life of the column. The peak height intensity data of the m / z 230 and 248 ions was collected by selected ion monitoring. The concentration of captopril was calculated from the equation C = R'(F/M) (2) where C is the concentration of 1, in ng/mL blood, F is the ng quantity of internal reference standard, 2, added to M mL of blood and R' is the ng ratio of 1 to 2 calculated from Equation 1.
RESULTS AND DISCUSSION Initial a t t e m p t s a t measuring 1 in blood without t h e immediate derivitization were unsuccessful since significant quantitites of oxidation products were formed. However, t h e addition of N E M t o whole blood produced a stabilized blood sample. At least 90% of t h e N E M derivative, l A , remained in t h e blood samples after storage in t h e freezer for t h r e e months. Blood proteins are precipitated prior to adsorption of 1A a n d 2A o n t o XAD-2 resin. T h e precipitating agents tried included 5% trichloroacetic acid, 0.5 N HC104 and 10% metaphosphoric acid. T h e latter produced t h e cleanest solutions at t h e optimum pH for adsorption onto XAD-2 resin (Table I). T h e use of the XAD-2 resin as %on-polar" ext r a c t a n t is very convenient. Radioactivity studies show that t h e NEM derivative, lA, can be completely adsorbed onto one gram of resin from aqueous solutions at p H 1 t o 2.5. It c a n
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 7 , JUNE 1980 ~
'B
Table I. Adsorption of "'C-1A on XAD-2 Resin as a Function of Solution pH solution pH
% adsorption
t
100.2 99.6
1.5 1.7
2 .o
.
m/z 248
5
I-
100.6
m/z 230 .
-
rn
100.8
2.5
,
'8
1B
z -
NON-OXIDIZED 100
70
m/z 230
80
28
12E
0
m/z 248
'
60
0 40
T
2
l
l
l
'
4 6 TIME (minutes)
l
a
Figure 4. GLClselected ions m l z 230 and 248 Oxldized extract (upper trace), non-oxidized extract (lower trace) 230 1c I
1
1, Lu
2
356 M ;
B.
88
100
I-
Id€
Q, Lu
cz:
'?
rn/z 230
3B
80
'? 60
40
248
0
20
0
It
50
2
4
6
8
TIME (minutes)
I
,I 100
150
200
250
300
350
MASS CHARGE (m/z)
Figure 3. (A) Mass spectrum of GLC derivative 1B. (8)Mass spectrum of GLC derivative 2B
be quantitatively eluted with dioxane, methanol, ethyl acetate, acetonitrile, acetone, isopropanol, methyl ethyl ketone, and tetrahydrofuran. Diethyl ether and halogenated solvents, such as methylene chloride a n d chloroform, were found t o be unsuitable. While more polar solvents completely displaced 1A from t h e resin, they also eluted impurities which interfered with t h e GLC measurements. T h e reference compound, 2, was chosen because it forms a n NEM derivative which can be extracted along with 1A and forms a methyl ester under identical conditions as IA. T h e retention times of 1B a n d 2B were essentially the same and their major fragment ions differ by 18 amu (compare Figure 3A with 3R). T h e mlz 230 a n d 248 ions were used in the quantitative measurements because they showed the fewest interferences from other extracted blood components. During the method development, a shoulder appeared on the sides of GLC peaks of 1B and 2B (Figure 4). Its intensity varied from 0.1 to equal to that of the authentic peaks. Control samples that exhibited this behavior nevertheless gave measured ratios within 3% of those controls having only single peaks.
Figure 5. GLClselected ion m l r 2 3 0 of 1C (upper trace). GLClselected ions m l z 230 and 248 of mixture containing l B , 2 8 , and 1C (lower trace)
An investigation of the extraction procedure showed t h a t when t h e alumina purified ethyl acetate was not freshly prepared, peroxides were formed which oxidized the NEM derivatives, 1A and 2A, to the respective sulfoxides. T h e GLC of authentic 1C had the identical retention time as the second peak (Figure 5 ) . T h e presence of small amounts of 1B a n d the disulfide 3B are observed at their expected retention times. When 1C was mixed with control samples containing 1B a n d 2B, the selected ion monitoring indicated a mlz 230 ion with a shoulder but no shoulder on the mlz 248 ion profile (Figure 5). T h e NEM sulfoxides undergo thermolysis to dehydrated products, presumably 1D and 2D, with respective M+ of m l z 354 a n d 3 7 2 , a n d t h e same m / z 230 a n d 248 ions as 1B a n d 2B (Figure 6). T h e reliability of the assay was enhanced by establishing the calibration curve under similar conditions as the samples. Addition of one control blood sample with each day's set of extracted blood samples served to check t h e extraction procedures as well as t h e sensitivity and resolution of t h e instrument and the responseslope of the calibration curve. This ratio was maintained by suitable adjustment of the quadrupole mass resolution instrument parameters (focus, ion energy). When this ratio changed significantly, a new calibration curve was determined.
ANALYTICAL CHEMISTRY, VOL. 52, NO. 7, JUNE 1980
1089
~
Table 11. Comparison of Duplicate Blood Extract Results Concentration of 1, in ng/mL Blood mean deviation, extract I extract I1 m e a n , X A IOO(A/~) 210 242 339 337 343 395 396 44 5 435 447 47 4 454 507 50 1 555 509 577 585 589 612 60 2 613 666 654 67 6 67 6 6Y2 685 719 750 756 697 806 776 811
790 869 79 3 85 1 902 913 936 998 1055 1087 1144 1174 1236
221 267 345 347 39 6 397 428 47 5 493 5 21 512 543 568 5 89 559 638 594 598 595 626 6 37 632 675 69 6 684 714 699 717 771 763 787 871 824 858 8 27 887 87 0 956 900 914 944 945 1016 1124 1136 1161 1265 1263
215 254 342 342 3 69 396 412 460 464 484 493 498 5 37 545 557 573 585 591 592 619 619 622 67 0 675 6 80 695 695 7 01 7 45 7 56 771 784 815 817 819 8 38 8 69 87 4 87 5 908 9 28 9 40 1007 1089 1111
1152 1219 1249
5 12 3 5 27 1
2.3 4.7 0.9 1.5 7.3 0.2 3.9 3.3 6.3 7.6 3.9 9.0 5.8 8.1 0.4
16 15 29 37 i9 45 31 44 2 65 9
11.3 1.5
7 3
1.2 0.5
7 18 10 5 21 4 19 4 16 26 7 16 87
2.9 1.6 0.7 3.1 0.6 2.7 0.6 2.3 3.5 0.9 2.1
9
41 8 49 1
82 25 6 16
4 9 35 25 9 46 14
1.1
11.1 1.1
5.0 1.0 5.8 0.1 9.4 2.8 0.7 1.7 0.5 0.9 3.2 2.2 0.7 3.7 1.1
T h e mean ratio, R , for the control sample containing 1000 ng of 1 and 500 ng of 2 / m L blood for 115 control samples was 4.245 (s = 0.226). T h e precision was kept well within the 5% level established for all controls through t h e periodic reevaluation of t h e calibration curve (Figure 2). Detection limits were determined from t h e replication of the zero concentration values of the calibration, prepared from extracts of blood. The standard deviation (s) of the calibration blank was 5.5 ng 1 per m L of blood. For a single measurement, t h e practical detection limit with a 90% confidence limit is three s or 16.5 n g / m L (12). In 42 of 55 blood samples taken immediately before the oral administration of captopril, the concentration of 1 was determined t o be zero while the 13 other samples had t h e following concentrations (and frequency): 2(5), 3(2), 4(2), 3(1), 6(2), a n d 9(1). For purposes of t h e statistical evaluation of the bioavailability, the actual a m o u n t determined was tabulated, even though as a n indi-
R = F \--..
m
2
248
1D M t m l z 354 M:m/z 372
-
Figure 6. Proposed thermolysis of 1C and 2C to 1D and 2 0
vidual value the confidence level of the measurement was low. Duplicate extracts were measured of blood samples taken 4 h after administration of 1. Mean values of from 5 t o 50 ng of 1 per m L were determined with a n s = 7 ng/mL, which compares favorably with the 5.5 ng/mL determined from the measurement of t h e s t a n d a r d blanks. Likewise, duplicate extracts of 1-h blood samples with levels of from 200 t o 1250 n g / m L were measured (Table 11). Significant improvement in t h e precision was achieved by t h e replacement of the older computer system ( 1 I ) and the elimination of the oxidation of t h e N E M derivatives. More t h a n 1000 human blood samples have been processed with this method. Correlation studies after administration of 14Ccaptopril are being completed a n d will be reported elsewhere (13). Preliminary results indicate that the duplicate analyses are in good agreement.
ACKNOWLEDGMENT T h e authors thank K. J. Kripalani, B. H. Migdalof, a n d S. M. Singhvi, Drug Safety Evaluation, for their technical advice and the TLC-radioactivity correlation results. T h e discussions a n d encouragement provided by G. Brewer, J. Dunham, a n d K. Florey, Analytical Research, are also gratefully acknowledged.
LITERATURE C I T E D (1) Cushman. D. W.; Cheung, H. S.; Sabo, E. F.; Ondetti, M. A. Prog. Cardiovasc. Dis. 1978, 21. 176-182. (2) Ondetti, M. A.; Rubin, B.; Cushman, D. W. Science. 1977, 796, 441-444. (3) Cushman, D. W.; Cheung, H. S.; Sabo, E. F.; Ondetti, M. A . Biochem. 1977, 1 6 , 5484-5491. (4) Gavras, H.; Brunner, H. R.; Turini, G. A,; Kershaw, G. R.; Tifft, C. P.; Cuttelod. S.;Gavras, I.; Vukovich, R. A,; McKinstry, D. N. N . Engl. J . Med. 1978, 298, 991-995. (5) Case, D. B.; Atlas, S. A,; Laragh, J. H.; Sealey, J. E,; Sullivan, P. A,; McKinstry, D. N. Prog. Cardiovasc. Dis. 1978. 27, 195-206. (6) Fontana, A.; Toniolo, C. "The Chemistry of the Thiol Group", Patai, S., Ed.; John Wiley and Sons: New York, 1974;Chapter 5, pp 294-6. (7) Gregory, J. D. J . A m . Chem. SOC. 1955, 77, 3922-3923. (8) Portanova, J. P.; Shrift. A. J . Chromatogr. 1977, 739, 391-394. (9) Kripaiani, K, J.; Singhvi, S. M.; Shaw, J. M.; Ross, J. J., Jr.: Meeker, F. S., Jr.; Migdalof, B. H. Pharmacologist 1978. 20, 213. (IO) Singhvi, S. M.; Kripalani, K. J.; McKinstry, D. N.; Shaw, J. M.; Willard, D. A.; Migdalof, B. H.; Vukovich, R. A. Pharmacologist 1978, 20, 214. (11) Funke, P. T.; Malley, M. F.; Ivashkiv, E.;Cohen, A. I.J . Pharm. Sci. 1978, 67,653-657. ( 1 2) Winefordner, J. D. "Trace Analysis:Spectroscopic Methods for Elements" Winefordner, J. D., Ed.; John Wiley and Sons: New York, 1976;Chapter 1. (13) Singhvi, S. M., Squibb Inistitute for Medical Research, New Brunswick, N.J., 1978,unpublished work, May 1979.
RECEIVED for review August 2,1979. Accepted March 3, 1980.
