Because weak binding for Type 2 sites implies
7’1’’
>
Tz”,the normalized equation for testing is
Y2n
[l
+ T34’Y)
(A-28a)
This equation without stepwise character may apply to glasses with higher alumina contents typical of commercial compositions. If all cations are mobile, the usual Equation 36 is found. Thus Case I1 is intermediate between Case I and the conventional equation. Mean Ion Activities in the Glass Surface. Simultaneous ion exchange on two types of sites has been studied previously (61, 62) and non-ideal solution theory applied to two sites considered as a mixture by Doremus (63). Reworking his results for different standard states gives mean activities (61) J. P. Cornaz and H. Deuel, H e h . Chim. Acta, 39, 1220, 1227
(1956), (62) R . M. Barrer and W. M. Meier, Trans. Faraday SOC., 55, 130 (1959). (63) R. H. Doremus, J , Phys. Chem., 72, 2665 (1968).
and equilibrium constant [K2(therm.)IC2/Cfor the reaction UM
+ Rmean
L
[Kl(therm.)]C1’C aH
+
a,,,,
x
(A-30)
The effect of these mean values is to replace [UH’ + KzUM’] in Equations 28 and 29 with [UH’ + KlUM’lC1 x [UH’ + KZQM’]C~/C. This new term does not fit the data used by us, but may be appropriate for other glassy systems. The means used in Equation 20ab are possibly satisfactory for two-phase patchwork surfaces while A-29ab applies to one phase solution-like surfaces.
ACKNOWLEDGMENT The author thanks Ron Ellis, Joseph Edwards, Robin Porter, and John Boles for aid in computations and accumulating data for Figure 11. Received for review June 9, 1972. Accepted November 20, 1972. This paper was presented a t the 163rd National Meeting, ACS, Boston, Mass., in the Analytical Chemistry Division, No. 59, April 14, 1972. The author recognizes support by the Materials Research Center (University of North Carolina) under Contract DAHC-15-67-(2-0223 with the Advanced Research Projects Agency and National Science Foundation Grant GP20524.
Determination of an Indolyl-l,4-benzodiazepine in Blood by Spectrophotofluorometry J. Arthur F. de Silva, Nancy Munno, and Norman Strojny Department of Biochemistry and Drug Metabolism, Hoffmann-La Roche lnc., Nutley, N.J. 071 70
A sensitive fluorometric assay for 5-(3-lndolyi)-2,3-dihydro-1 H-l,4-benzodiazepine (I) was developed based on selective extraction and hydrolysis in 2 N H2S04, for quantitative conversion to the ppenylquinolone (ll), which fluoresces strongly in concentrated H2S04, with activation at 325 nm and emission at 390 nm. The overall recovery of I from blood is 86 f 5.0% and the sensitivity limit of the assay is 0.02 to 0.03 pg/ml of blood or plasma. The method was applied to the determination of blood level fall off curves in a dog given a single 70-mg dose by oral and intravenous routes and in a human subject volunteer following single oral doses of 100 mg and 150 rng given nine days apart. Urinary excretion data in the patient were determined using a simpler UV spectrophotometric method. A tentative characterization of the “free” or unconjugated urinary metabolites in man and in the dog is presented.
5-(3-Indolyl)-2,3-dihydro-lH-l,4-benzodiazepine (I), was synthesized ( I , 2) and was clinically evaluated as a psychostimulant. A sensitive spectrofluorometric assay was developed based on the selective extraction of the compound into diethyl ether from blood or plasma made alkaline with NaOH. The drug is back extracted into 2N HzS04, and is hydrolyzed at 100 “C to convert I to the phenylquinolone (11) (Figure l ) , which fluoresces strongly in concentrated H2S04, with activation a t 325 nm and emission a t 390 nm. Prolonged hydrolysis of I leads to the formation of the indoloquinoline (111) which is also highly fluorescent but at different wavelengths of activation (350
E. E. Garcia, J . G. Riley, and R. I. Fryer, J. Org. Chem, 33, 2868 (1968). (2) R. I. Fryerand L. H. Sternbach, U.S.P. No. 3.391, 158 (1968) (1)
ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1973
0
665
1
i: 2N H2S0,
A
IOO'C-2
Hrr
I
I-
UNSTABLE
r
TERMEDIATE
In w
e
N
0
V
(r W
W
n
0
z
0
a
f&--%~. - C H ~ - N H ~
H30@
*
CONDENSATION CI H 2 - C H Z - N H 2
\
[I111
[I11
F L U O R E S C E S IN E T H A N O L ACT 350 / E m 4 5 0 I m
F L U O R E S C E S I N C O N C H2S04 A C T 3 2 5 / E m 390 n m
Figure 1. Chemical reactions of 5-(3-indolyl)-1,4-benzodiazepine ( I )
nm) and emission (450nm). The mechanisms of these hydrolytic rearrangement reactions have been described (3). 5-(3-Indolyl)-2,3-dihydro-1H-1,4-benzodiazepine (I), mol wt 261.31, mp 215 to 223 "C; 1-(2-aminoethyl)-3-(2-aminophenyl)-4(1H)-quinolone (11), mol wt 279.0, m p 162.5 to 163.5 "C; and 5-(2-aminoethyl)-5H-indolo-[3,2-c]-quinoline (111), mol wt 261.31, mp 175 "C were synthesized by Garcia et al. ( I ) , for use in this study as reference standards. The overall recovery of I from blood is 85 5.0% and the sensitivity limit of the assay is 0.02 to 0.03 pg/ml of blood or plasma. The method was applied to the determination of blood levels of I in a dog given a single 70-mg dose by oral and intravenous routes and in a female patient following single oral doses of 100 mg and 150 mg given nine days apart. The urinary excretion data were determined in the dog and in man using a simpler UV spectrophotometric method. A tentative characterization of the major urinary metabolites in man and in the dog is presented.
*
EXPERIMENTAL Apparatus. A Farrand Spectrofluorometer (equipped with a Xenon arc energy source and an RCA IP-28 photomultiplier) was used for all fluorescence measurements. The monochromators were corrected for the fundamental Hg lines, but the fluorescence measurements were not corrected for energy and other instrumental artifacts. The instrument sensitivity was adjusted for constant energy for each day of operation using a reference Pyrex rod and a standard solution of the compound to be measured. The 10-nm slit arrangement was used a t all four positions. Reagents. All reagents used were of analytical grade purity (>99%) and were used without further purification. All aqueous reagents were made up in double distilled water and included 2.0.11 HzS04, 2.ON NaOH, 36N HzS04 (reagent grade), 27N HzS04, made by mixing 25 ml of 2N HzS04 with 75 ml of concd 13) R. I. Fryer.J. HeterocycL Chem., 9, 747 (1972)
666
0
ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1073
(36N) HzSOl to make up 100 ml of reagent, and diethyl ether (absolute anhydrous) Mallinckrodt. Standards. Weigh out 10.00 mg of I into a 10-ml volumetric flask. Dissolve and make up to volume in ethanol. This stock solution (A) contains l mg/ml. Make two 1 : l O serial dilutions of A in ethanol to give a working solution (B) containing 100 ng 1/10 PI.
Prepare respective standard solutions of I1 in a similar manner to yield a working solution (B') containing 100 ng 11/10 pl. Procedure. I. Spectrofluorometric assay in blood. Into a 50-ml glass-stoppered centrifuge tube, add 4 ml of blood, 4 ml of 2 . O N NaOH, and 15 ml of diethyl ether. Extract by shaking on a reciprocating shaker for 15 minutes and centrifuge for 10 minutes a t 2000 rpm, preferably a t 0 to 4 "C in a refrigerated centrifuge. Along with each set of unknowns run a 4-ml specimen of control blood and duplicate 4-ml specimens of control blood to which 500 ng of I have been added [50 g1 of solution B evaporated to dryness under nitrogen] as internal standards. Transfer a 10-ml aliquot of the ether phase into a fresh 5 m l centrifuge tube containing 1 ml of 2.ON H2S04. Extract, shaking for 10 minutes, centrifuge for 5 minutes, and then aspirate off the ether. While this step is in progress, re-extract the remainder of the original sample with another 5-ml aliquot of ether. Shake for 15 minutes and centrifuge for 10 minutes a t 2000 rpm. Transfer the ether layer quantitatively into the tube containing the HzS04. Shake for 10 minutes and centrifuge for 5 minutes a t 2000 rpm. Aspirate the ether and discard. Wash the extract with 2 X 5-ml aliquots of ether for 60 seconds on a Vortex Super-Mixer (Lab-Line), centrifuge, and aspirate off the ether. The H2S04 extract should be clear (transparent) when viewed against the light. Place the sample tubes in a boiling water bath (100 "C) for 5 minutes to equilibrate and to expel any residual ether. Stopper the tubes (sealing with a drop of distilled water) and hydrolyze for 1 hour a t 100 "C. While the hydrolysis step is in progress, prepare external standards of I1 as follows: Transfer two 50-pl aliquots of standard solution B' into separate 15-ml centrifuge tubes and evaporate to dryness under nitrogen. Add 4 ml of 27N HzS04 to each tube and mix well for 60 seconds on a Vortex Super-Mixer. These samples contain 500 ng of I1/4 ml of 27N and are used as the refer-
I
I
250
I
I
I
300
I
I
I
I
I
I
I
350
I
I I
1
400
430
WAVELENGTH I N NANOMETERS
Figure 2. Absorption spectra of I in 1.ON HCI before a n d after extraction from blood (Ext std = authentic I and rec std = I recovered from blood
ence external standards. Use 4 ml of 27N HzS04 as a reagent blank. After hydrolysis, cool the sample tubes in ice and carefully add 3 ml of concd HzS04 (36N) dropwise to the 1-ml solution of 2N HzS04. Mix well on a Super-Mixer for 60 seconds and allow the samples to warm up to room temperature for about 10 minutes. Read the fluorescence of all the hydrolyzed samples and the external standard solutions of I1 in a 1-cm path cell a t 390 nm, activating a t 326 nm. Calculations. The fluorescence readings, (Transmittance [TI X Meter Multiplier [MI = [ T M ] )of the external standards of II are corrected for the reagent blank reading, and the fluorescence ( T M ) of the blood specimens are corrected for the control blood reading. A samp1e:blank fluorescence ratio of 2 : l is used as the limit of detectability.
Determination o j Unknowns.
(TM) U n k n o w n (TM) Int Std
X
C o n c n o f Int S t d (0.50 p g )
4 ml of b l o o d s a m p l e = p g I / m l of blood
Determination of 70Recouery ( T M / p g / m l ) Int Std (TM/,ug/ml) Ext Std
X
0.94
X
100 = % R e c o v e r y
where 0.94 is the Molar Conversion factor of m o l w t of I 261.31 = - = 0.94 mol w t of I1 279.00 Per cent recovery is determined routinely as a check on analytical precision and reproducibility. II. Spectrophotometric (UV) assay for I and Its Major Metabolites in Urine. Into a 50-ml glass stoppered centrifuge tube, add 5 ml of urine, 5 ml of 1.ON NaOH and 15 ml of diethyl ether. With each series of unknowns, run a 5-ml specimen of control urine (taken from the patient prior to medication) and duplicate 5-ml specimen of control urine added to 10 pg of I (0.10 ml of standard solution A evaporated to dryness under nitrogen a t 35-40 “ C ) . Stopper the tubes, and shake for 10 minutes on a reciprocating shaker. Centrifuge the samples for 5 minutes a t 2000 rpm and transfer the ether supernatant into a 50-ml centrifuge tube without transferring any of the aqueous phase. Re-extract the specimen with another 10-ml portion of ether, centrifuge, and combine the ether extracts.
Evaporate the ether extracts to dryness, wash down the walls with 1 ml of ether to collect the entire residue a t the tip of the tube and evaporate to dryness. Dissolve the residue in 100 p1 of ethanol, and transfer quantitatively onto a 20 X 20-cm Brinkmann F254 thin layer chromatoplate coated with silica gel G/F, rinsing the centrifuge tube with 50 pl of ethanol to assure quantitative transfer. Develop the plate in a vapor saturated chamber using acetone:ammonia 100:2 (v/v) until the solvent front has ascended a distance of 12 cm. The parent drug (I) is located by comparison with an authentic standard (Hf0.60) developed alongside the sample extracts, as a yellow colored spot under visible light or a dark spot under short wave UV. Scrape off the silica gel covering the entire area of I. Also, process a separate clean area from the plate as a control reagent blank. Transfer the silica gel into clean 15-ml centrifuge tubes, add 4 ml of 1N HC1 and slurry the samples on a Vortex Action Super Mixer for 60 seconds. Compound I is eluted into the HC1 solution as its hydrochloride salt. Wash the HC1-silica gel slurry with 2 X 5-ml portions of ether on the Super Mixer for 60 seconds each. After each wash, centrifuge and remove the supernatant ether. This clean-up step is required to remove certain hydrocarbon impurities “octoils” which otherwise contaminate the sample. The ether washed HC1 solution should be clear (transparent) when viewed against the light. Transfer a 3-ml aliquot of the HC1 solution into a 1-cm path cell using a capillary Pasteur pipet taking care not to disturb the silica gel a t the bottom of the tube, and read the absorbance of I a t 371 nm in a suitable spectrophotometer. Calculations. All sample absorbance readings are corrected for control blood blank reading. Determination of Unknowns
AsT1( U n k n o w n ) A3il ( I n t S t d )
X
p g of I n t Std (10 p g ) m l of s a m p l e
= p g I / m l of u r i n e
Determination of 70 Recovery The per cent recovery is determined by direct comparison of the absorbance per pg per ml of the recovered internal standard to that of an external standard of 10 pg of I (0.10 ml of solution A evaporated to dryness under nitrogen) dissolved in 4 ml of 1.ON HC1 and read a t 371 nm in a 1.0-cm path cell against 4.0 ml of 1 . O N HC1 as the reagent blank, thus; ( A l p g l m l ) R e c o v e r e d Int S t d ( A i p g i m l ) E x t e r n a l Std
X
100 = % R e c o v e r y
ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1973
0
667
Table I. Urinary Excretion Data of I A. In the dog Following Oral Administration of a 70-mg dose ( 5 mgjkg)
Excretion period, hours
pg/ml
Total, pg
Cumulative Total, pg
0-7.5 7.5-24 24-48
4.95 12.50 1.18
361 1875 443
36 1 2236u 267gb
Excretion rate, psi hour 48.2 113.6 18.4
6 .In man (1) Following a 100-mg oral dose. Patient C.S. ( f ) , age 33, weight 111 kg 0-4 4-8 8-1 2 12-24 24-48
1.59 1.29
0.50 0.19 0.16
215 529 308 222 341
215 744 1052 1274 1615"
53.81 132.3 77.0 18.5 14.2
1034 1594 21 8 0 321 5 3401* (
258.5 140.0 146.5 86.3 7.8
(2) Following a 150-rng oral dose 01
IO
10 0
1000
CONCENTRATION PQ [llj/4ml H ~ S O (I2 7 N ) L1 CONCENTRATION pg [ I ] A O D E O ml OF BLOOD A
Figure 3 A Linear dynamic range of fluorescence vs. concentration of I1 in 27N HzS04, and B the recovery of I added to blood, determined by spectrofluorometry
The A3n/pg:rnl = 0.059 (Figure 2 ) . The per cent recovery of added I should be calculated routinely as a check on analytical precision and reproducibility RESULTS AND DISCUSSION The recovery of microgram amounts of I added to blood and urine was determined using the spectrophotometric (UV) method. Recovery of added I was quantitative (87% f 4.0) in the concentration range of 2.0-60 &ml of blood or urine. The A371/pg/ml of compound was 0.059 giving a sensitivity limit of 0.5-1.0 pg/ml of blood or urine using a 2 : l sample to blank absorbance ratio as the limit of sensitivity and 4 ml of blood or 5 ml of urine per assay. Thin layer chromatography in acetone: NH4OH (concd) 100:2 (v/v) gives good separation of I ( R , 0.60) from other ether extractable components and metabolites present in urine thus ensuring specificity of the assay. Although the compound I possesses adequate UV absorbance for quantitation in the microgram range, it did not exhibit any intrinsic fluorescence in acidic, neutral, or basic media for quantitation in the submicrogram range. The investigation of chemical derivatives of I that may be suitable for fluorometric or GLC analysis indicated that acid hydrolysis in 2N H z S 0 4 a t 100 "C for 1 hour resulted in the benzodiazepine ring opening a t the >C5 = N4-position followed by rearrangement with a quantitative yield of compound 11. Figure 1. This derivative (11) fluoresces strongly in concentrated ( 2 7 N ) H2SO4 with activation a t 325 nm and emission at 390 nm. The fluorescence yield is linear with concentration in the range of 0.10 to 10.0 pg II/4 ml, Figure 3.4. The blood recovery curve of I was also linear within the concentration range of 0.10 to 10.0 pg of I/ml of blood (Figure 3B). The overall recovery from blood was 85.6% f 5.0. The sensitivity limit of the assay is of the order of 0.02 to 0.03 pg of I per ml of blood using a 4-ml sample per assay and a samp1e:blank fluorescence T M ratio of 2 : 1as the limit of detectability. Prolonged hydrolysis of I in 2 N H2S04 for 32 hours resulted in 11, while prolonged hydrolysis of I in 6 N HC1 for 9 days or of I1 in 2 N HzSO4 acid for 66 hours resulted in a condensation reaction between the anilino amine and the 668
ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1973
0-4 4-8 8-12 12-24 24-48
1.23 1.40 1.15 2.10 c.11
1034
560 586 1035
186
O3.19% of dose recovered in 24 hours. b3.83% of dose recovered in 48 hours. '1.62% of dose recovered in 48 hours. d2.27% of dose recovered in 48 hours.
quinolone carbonyl groups to yield the indoloquinoline (111) in good yield (I). This compound 111 is even more intensely fluorescent than is I1 with significantly different wavelengths of activation (350 nm) and emission (450 nm), (Figure l), and is potentially more sensitive than the method reported. However, the long reaction times required (>24 hours) for the quantitative conversion of I or I1 to I11 even in the submicrogram range of concentration, rendered this avenue impractical as an analytical procedure. The synthesis of authentic reference standards of I1 and III was achieved by hydrolysis of I in strong acids (1). Application of the Method to Biological Specimen. Blood Levels and C r i n a o Excretion of I in the Dog. A male beagle (14.2 kg) was administered a 5-mg/kg (70-mg total) dose of I by intravenous and oral routes 11 days apart, and blood specimens were collected prior to medication (control) and thereafter a t appropriate time points over a 48-hour experimental period. Urine specimens were collected prior to medication (control) and thereafter as pooled specimens during the 0-24, and 24-48 hour periods after oral dosing. A semilogarithmic plot of the blood levels us. time are shown in Figure 4. The blood level fall-off curve following I.V. administration shows the typical biphasic pattern with a rapid initial distribution phase followed by the slower elimination phase with an apparent half-life of 2.83 hours. The oral dose blood level curve indicated a slow and prolonged absorption phase of about 3 hours followed by the elimination phase with an apparent half-life of 2 hours. The urinary excretion data (Table I, A ) indicated that only 3.19% of the 70-mg oral dose was recovered in 24 hours and 3.83% recovered in 48 hours as the intact drug. Blood Levels and Urinary Excretion of I in M a n . A female patient (C. S.) (Age 33. Wt 111.34 kg) was administered single oral 100-mg and 150-mg doses of I given 9 days apart and blood specimens were collected over a 12hour period as shown in Figure 5 . Urine specimens were collected prior to dosing (control), followed by collections during 0-4, 4-8. 8-12, 12-24 and 24-48 hour periods post dosing.
0-0 = 7 0 m g
I V
DOSE
D-0 i 70 rng O R A L DOSE
nm
I
L A ; 7I 5
0 01
3HOURS A F T E R
2
DOSING
'+-?+-74
6
Figure 4. Blood level curves of I in a dog following t h e administration of a single 5 mg/ kg dose by intravenous and oral routes Patient
I
Wt
C.S.( f 1 33
Age :
HI 34
kg = 150 rng dose
020r
0 01
& A
I
I
,
I
2
3
, 4
5
1
6
7
HOURS A F T E R
Figure 5.
= i O O m g oose
8
9
\
10
II
I2
13
DOSING
Blood level curves of I in man following the administration of single oral doses of 100 mg and 150 mg given nine days apart
A semilogarithmic plot of the blood levels us. time (Figure 5 ) indicated a prolonged absorption phase of about 3 hours and an apparent half-life of elimination of 4.42 hours following the 100-mg dose and 4.14 hours after the 150-mg dose. The urinary excretion data (Table I, B) show that following the 100-mg oral dose only 1.62% of the dose was recovered as the intact drug in 48 hours, and that 2.27% of the 150-mg dose was recovered in 48 hours, indicating extensive biotransformation and distribution and/or an alternate route of elimination. Specificity of the Assay. Thin layer chromatographic analysis of' the blood ether extracts in the dog (oral and I.V.) and in the human (oral) experiments using Brinkmann (F254) silica gel G chromatoplates and acetone:
"*OH = 100:2 (v/v) as the developing solution indicated that intact I was the only drug related component present following single doses. However, chronic administration of I may reveal metabolites which would then necessitate the use of the above TLC separatior, step prior to spectrofluorometric determination. The material isolated as the intact drug from blood was characterized as authentic I by its UV spectrum and fluorescence spectrum after acid hydrolysis, and by its K f on TLC analysis. Since metabolites are absent in the blood ether extract after single oral doses, the assay is deemed to be specific for the intact drug. Thin layer chromatography of the diethyl ether extracts of urine in the dog and in man, Figure 6, shows a more complex situation. Again in the dog (oral and I.V.) and in ANALYTICAL CHEMISTRY, VOL. 45, NO. 4 , APRIL 1973
669
A.
DOG
URINE ( 4 m l ) D O S E : 7 0 m g ‘1.V and ORAL
I
B.
HUMAN
URINE ( 5 m l ) DOSE: 3 0 m q ORAL
SOLVENT SYSTEM
:
ACETONE . NHqOH ( c o n c ) 100:2 ( V / V )
Figure 6. Thin layer chromatograms of diethyl ether extracts of urine from a dog and a human subject following the administration of I
6A) whereas following I.V. dosing only the intact drug (I)
@d x
>c=o
>CHOH X I
>
or
X=
c=o
m.w..
259
or >CHOH
M.W..236
m/e * 219
was seen in a 0- to 6-hour urine collection. Following oral administration in the human, the only metabolite seen in the urine corresponded to M 1in the dog (Figure 6B). The parent drug and the three metabolites observed were isolated from the TLC plate by elution with ethanol and characterized by high resolution mass spectrometry using a Consolidated Electrodynamics Corp. Model 21-110 spectrometer. Characterization of Urinary Metabolites by Mass Spectrometry. The spectrum of I isolated from urine was identical with that of an authentic standard both of which showed a molecular ion ( M + ) at m l e 261 and major fragmentation peaks at mle 233 (M-28) due to the loss of (-CHzN), and m / e 204 (M-57) due to the loss of (-C2HSN2), with weaker peaks at m / e 130, 116 (indole), and 102, respectively. Characterization of the metabolites was complicated due to the presence of hydrocarbons (“octoils”) co-extracted from the silica gel and also by the absence of a halogen group which is present in all the reported benzodiazepines ( 4 - 8 ) . The presence of a halogen serves as a useful “handle” to differentiate isotopic fragments due to the compound of interest from extraneous peaks due to co-extracted impurities. The spectra due to M1 and M2 showed peaks above background at m l e 279, 259, 258, 245, 244, 236, and 235 for M1 and at m l e 245, 244. 236, 233, 219, and 205 for M2, respectively. The major peaks in M1 a t m l e 259, 258, 245, and 244 suggest that the aromatic phenyl and indole rings are intact and that the major changes have occurred in the benzodiazepine ring. Based
Figure 7. Postulated structures for the metabolites of I isolated from dog urine determined by high resolution mass spectrometry
the human (oral), I was the major. drug related component. Following oral administration in the dog, the chromatoplate showed the presence of three yellow colored metabolites MI, ( R , 0.40), M2, ( R f 0.70), M3, (Kf0.80) (Figure 670
0
ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1973
(4) M. A. Schwartz, P. Bommer, and F. M. Vane, Arch. Biochern. Biophys., 121, 508 (1967). (5) M. A. Schwartz, F. M. Vane, and E . Postma. Biochern. Pharmacoi., 17, 965 (1968). (6) M. A. Schwartz, F. M. Vane, and E. Postma, J. Med. Chern., 11, 770 (1968). (7) W. Sadee and E. Van der Kleijn, J. Pharrn. Sci., 60, 135 (1971). (8) A. Forgione, P. Martelli, F. Marcucci, R. Fanelli, E. Mussini. and G. C. Jommi. J. Chrornatogr., 59, 163 (1971).
on data published on other benzodiazepines these changes could be due to the addition of oxygen (16 amu) a t either C2 and/or C3 in the benzodiazepine ring (4-6). Because of the significant difference in R, values of the two compounds in the TLC system used, M2 is also less polar than MI. The postulated structures of M1 and M2 are shown in Figure 7. The spectrum of M3 showed weak peaks a t m / e 260, 259, 258 [possibly related to the parent compound (I)], and strong peaks at mle 236 and 216 (compatible with indolophenone). In addition, some weaker peaks were seen at mle 235, 220, 206, and 188. The major peaks a t mle 236 and 216 indicate that M3 is 2-amino-indolophenone (M3-B), however, the weak peaks a t mle 260, 259, and 258 (due to loss of H from I) suggest a structure compatible with M3-A in Figure 7. Thus M3 appears to be mainly composed of structure B with some possible contamination with A. The major biotransformation reactions of I are apparently oxidation in the 2- and 3-C-positions of the benzodiazepine ring of the molecule. Oxidation at the 2-C-position (MI) can yield the secondary alcohol as an intermediate leading to the 1,4-benzodiazepin-2-one, while oxidation at the 3-C-position (M2) usually leads to the formation of the secondary alcohol in preference to a ketone. The formation of the 2-amino-indolophenone (M3) may be explained by the hydrolytic cleavage of either a 1,2-dehydro-1.4-benzodiazepine (M3-A), formed by dehydration of a 2-hydroxy-1,4-benzodiazepineor from a 1,4-benzodiazepin-2-one as a precursor. Similar metabolic pathways were observed in the biotransformation of medazepam (a 5-phenyl-N-l-methyl-7-chloro-1,4-benzodiazepine) in the dog and rat (9) and in man (20). The metabolites isolated in these studies represent only the unconjugated diethyl ether extractable components in (9) M. A . Schwartz and S. Koiis, J. Pharm. Expt. Therap., 180, 180 (1972). (10) M. A . Schwartz and J. J. Carbone, Biochem. Pharmacol., 19, 343 (1970).
urine. The conjugated (glucuronide/sulfate) fraction which could contain the major percentage of the dose excreted may also contain phenolic metabolites in addition to those isolated so far (1 I ) .
CONCLUSIONS
A highly sensitive and specific spectrofluorometric assay was developed for a 5-indolyl-1,4-benzodiazepine using a novel rearrangement process which the compound undergoes in concentrated (27N) HzS04, a t elevated temperatures. Unlike other 5-phenyl-1,4-benzodiazepinessuch as medazepam (12, 13) or 5-phenyl-3-hydroxy-l,4benzodiazepin-2-ones such as oxazepam (14) which fluoresce in concentrated H2SO4 or perchloric acid at room temperature [probably because of the formation of the quinazoline-carboxaldehyde derivative (7, 8) by a dehydration and rearrangement mechanism], this indolyl-1.4-benzodiazepine required more drastic chemical conditions to produce a fluorescent derivative. The process was successfully scaled down to be reproducible a t the submicrogram level and be of analytical utility.
ACKNOWLEDGMENT The authors are indebted to E. E. Garcia and R. I. Fryer for the synthesis of authentic standards of compounds I1 and 111, to J. Carbone for conducting the dog experiments, to A. S. Leon for conducting the clinical study a t the Newark Beth Israel Medical Center, Newark, N.J., to F. Vane, Department of Physical Chemistry for the mass spectral work, and to T. Daniels and R. McGlynn for the drawings of the figures presented. Received for review September 25, 1972. Accepted November 20,1972. (11) M. A. Schwartz in "Benzodiazepines," S. Garattini and L . 0. Randall, Ed.. Raven Press, New York, N.Y., 1972, in press. (12) J. A. F. deSilvaandC. V . Puglisi,Ana/. Chem., 42, 1725 (1970). (13) S. Lauffer and E. Schmid, Arzneim.-Forsch., 19, 740 (1969). (14) S. S. Walkenstein, R. Wiser, C. H. Gudrnundsen, H. 6. Kimmei, and R. A. Corradino, J. Pharm. Sci., 53, 1181 (1964).
Trace Element Determination with Semiconductor Detector X-Ray Spectrometers Robert D. Giauque, F r e d S. Goulding, Joseph M. Jaklevic, and Richard H. P e h l Lawrence Berkeley Laboratory, University of California, Berkeley, Calif. 94720
A method of obtaining high sensitivity and accuracy in X-ray fluorescence analysis using semiconductor detector spectrometers is discussed. Mono-energetic exciting radiation is employed to generate characteristic X-rays from trace elements in thin, uniform specimens. Corrections for absorption effects are determined; enhancement effects are omitted as they are negligible for many thin specimens. A single element thin-film standard is used to calibrate for the X-ray geometry, and theoretical cross sections and fluorescent yield data are employed to relate the X-ray yields for a wide range of elements to the thin-film standard. Various corrections which affect the accuracy of the method are discussed including the
method for determining X-ray spectral background. Results obtained in the analyses of biological and geological specimens, and of air particulate filters are reported. Using a single excitation energy, the concentrations of more than fifteen trace elements may be simultaneously determined during a fifteen-minute interval for concentrations of 1 ppm or less. This corresponds to less than 10 ng/cm' on air particulate filters.
The analytical technique of X-ray emission spectroscopy depends upon the ability to excite and accurately measure characteristic K and L X-rays emanating from the ANALYTICAL CHEMISTRY, VOL. 45,
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APRIL 1973
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