789
Anal. Chem. 1981, 5 3 , 789-793
and no reaction occurred. These model compound studies support the analyses performed on the asphalt fractions and further justify the use of T P T H as a convenient reagent for quantitatively determining the carboxylic acids, carboxylate salts, and anhydrides in petroleum asphalts and similar bituminous materials.
LITERATURE CITED (1) Petersen, J . C. Anal. Chem. 1975, 47, II;!. (2) Barbour, R. V.; Petersen, J . C. Anal. Chem. 1974, 46, 273. (3) Plancher, H.; Dorrence, S. M.; Petersen, J . C. Asphalt Paving Techno/. 1977, 46, 151. (4) Jones, R. N.; Ramsey, D. A.; Keir, D. S.; Dubrinev, K. J. Am. Chem. SOC. 1952, 74, 80.
(5)
Dorrence, S. M.; Barbour, F. A.; Petersen, J . C. Anal.
Chem. 1974,
46, 2242.
(6) (7)
Petersen, J. C.; Barbour, F. A.; Dorrence, S. M. Anal. Chem. 1975, 47, 107. Poller, R. C. "The Chemistry of Organotin Compounds"; Logos: London, 1970; Chapter 14.
(8) Sawyer, A. K. "Organotin Compounds"; Marcel Dekker: New York, 1972; Voi. 3, Chapter 12. (9) Mesubi, M. Adediran Spectrochim. Acta, Part A 1976. 32A, 1327.
RECEIVED for review November 24,1980. Accepted February 2, 1981. Mention of specific brand names or models of equipment is made for information only and does not imply endorsement by the Department of Energy.
Determination of pK, Values and Total Proton Distribution Pattern of Spermidine by Carbon-13 Nuclear Magnetic Resonance Titrations Mary M. Kimberly and J. H. Goldstein" Department of Chemistry, Emory University, Atlanta, Georgia 30322
The 13C resonance assignments of the carbons of spermidine have been made over the pH range 5-13 for a series of concentrations. Analysls of the data has provided the three thermodynamic pK, values as well as the total proton dlstrlbution pattern,. The charge distributlon deduced from the data appears to be reasonable, reflecting the asymmetry of the molecule and the expected effects of charge repulsion.
Polyamines are found in bacteria, bacteriophages, and plant and animal tissues (1). They appear to have a variety of biological roles ranging from acting as growth factors in some microorganisms to exerting stabilizing effects on nucleic acids (I). They have been found as degradation products and/or precursors of some alkaloids, for example, lunarine and palustrin (2,3) and as inhibitors of enzymes such as adenylate cyclase ( 4 ) . Wright et al. postulate that polyamines are modulators of cell membrane function ( 4 ) . Elevated levels of the polyamine, spermidine, and an increased spermidine/spermine ratio have been found in the blood of patients with cystic fibrosis (5). It is believed that, spermidine and its metabolic degradation products may play important roles in the pathogenesis of membrane dysfunctions in this disease (6). (For more detailed discussions of the functions of polyamines, see reviews by Tabor and Tabor (1) and Theoharides (7) and references therein.) At physiological pH ranges, these molecules are polycations and, therefore, have a high affinity for cellular polyanions such as fatty acids, phospholipids, nucleotides, and nucleic acids (8-16). One of the main functions of the polyamines seems to be that of acting as protective or stabilizing agents by being involved in interactions with these anions. Some of the most interesting of the interactions are those involving the various forms of nucleic acids (including DNA, rRNA, and tRNA) (8, 9,11,12,14-16). At sufficiently high concentrations, the polyamines have the ability to precipitate these macromolecules. The complexes are formed by ionic 0003-2700/8 1/0353-0789$0 1.25/0
interactions between the cationic amine groups of the polyamine and the anionic phosphate groups of the nucleic acid. This report describes an investigation of the protonation of spermidine
NH,CH,CH,CH,NHCH,CH,CH,CH,NH, a
1
2
3
b
4
5
6
7
c
utilizing 13C NMR spectrometry at natural abundance. The NMR titration technique provides pK, values and, in this case, information concerning the total proton distribution pattern (17-20). The NMR parameters obtained can provide a basis for further studies of spermidine interactions in other environments. To date, only symmetrical polyamines have been studied by NMR titrations. The unsymmetrical nature of spermidine should be reflected in its proton distribution pattern and in the various interactions mentioned above.
EXPERIMENTAL SECTION Sample Preparation. Spermidine was purchased as the trihydrochloride (Calbiochem,La Jolla, CA) and was used without further purification. First, 1.5 mL of a stock solution was titrated
potentiometrically with a known concentration of KOH. The data from this titration were plotted as pH vs. mL of KOH. From this graph, estimates of volumes of KOH needed for a range of pH values were made. NMR samples were prepared by adding the estimated volumes of KOH from a buret to individual 25-mL volumetric flasks containing 1.5 mL of the stock solution. The volume was brought to 25 mL with deionized water. The concentrations used in the NMR studies were 0.337,0.051,0.078, and 0.160 M spermidine. NMR and pH Measurements. Natural abundance 13CNMR spectra were obtained on a Bruker WH-90 apectrometer. All resonances were referred to external dioxane (67.4 ppm downfield from Me4Si). External D20was used as the lock material. The 0.160 M samples required a sweep width of 1400 Hz and 50 scans in order to obtain spectra. The 0.078 and 0.051 M samples required a sweep width of 3000 Hz and 2000 scans. The 0.037 M samples required a 3000 Hz sweep width and 5000 scans. pH measurements were made on a Corning 109 pH meter equipped with a microelectrode. For the NMR titrations, spectra 0 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 6, MAY 1981
790
'
I - -
'
7 -
t
t
'I
J./ ii
-. 1 -.
/
E/
x;; a
4 4
5000
4000
3000
2000
ppm
6.
7.
e.
9.
10.
11.
12.
13.
PH
Flgure 1. Chemical shift vs. pH data and best fit line calculated from nonlinear parameters for C5 of spermidine at 0.051 M.
were obtained at each concentration between pH 5 and 12.5. pH measurements and NMR spectra were obtained at 30.7 "C. Data Analysis. The deprotonation equilibria of spermidine (SP) proceed as follows: H3SP3++ HzSP2++ H+
(1)
HzSP2++ HSP+ + H+
(2)
HSP+ SPo+ H+ (3) with the corresponding equilibrium constants, K1, KP,and K3, respectively. The observed chemical shift is defined by the following equation:
[SPO] [HSP+] [H3SP3+] + 62+ 63- [HzSP2+] + 64- [SPI (4) [SPI [SPI [SPI where 61, S2, a3, and b4 are the chemical shifts of the non-, mono-, di-, and triprotonated species, respectively. Since it is not possible to measure the concentrations of the individual species, do^ needs to be defined in terms of measurable quantities. After expressions for the concentrations of the species are substituted, the final equation is KiKzK361 + K1K26z[Ht] + K163[HtI2 + 64[H+I3 (5) 6obd = KiK2K3 + K1Kz[H+] + K1[H+I2+ ["I3 6obad
= 61-
This equation defines a titration curve. The chemical shifts, Bow, are obtained from the NMR spectra, and [H'] is calculated from pH = -log [H']. The data obtained in these studies were analyzed by nonlinear regression using eq 5. The parameters obtained from the analysis are the equilibrium constanta and the chemical shifts. Since there are seven carbons in spermidine, seven sets of data were analyzed for each concentration. For each concentration study, the initial estimates for the equilibrium constants were the same for all carbons but the shifts were different for each carbon. These initial parameters were estimated graphically from plots of pH vs. chemical shift for each nucleus in the molecule. The pK, values from all the graphs for one concentration were averaged and used in the nonlinear program. After convergence, the data were plotted and the best fit curve was drawn through the points using the final parameter estimates given by the nonlinear regression program. The data and best fit line for C6are shown in Figure 1.
For each carbon, the pK, values were graphed as a function of concentration and the best fit line drawn from a linear
Flgure 2. Series of '3CNMR spectra for spermidine at various pH values: (a) pH 8.88, (b) pH 7.90, (c) pH 7.54, and (d) pH 3.98. Scale
is ppm downfield from Me,Si.
least-squares analysis for each pK,. The y intercept (infinite dilution) was taken as the thermodynamic pK, value, Le., pK,". Expressions for the equilibrium constank can be used to derive equations that describe a distribution curve for the species during the deprotonation process (21). The fractions of each species at any hydrogen ion concentration are expressed as [H3SP3+]/[SP] = [H'] /KIF
(6)
[H&P2+]/[SP] = 1/F
(7)
[HSP+]/ [SP] = K2/ [H+]F
(8)
[SPO]/ [SP] = K&/
(9)
[H']'F
where F = [H+]/Kl + 1 + K2/[H+]
+ K2K3/[H'l2
(10)
For the determination of the proton distribution pattern, equations of the following general form were used (22): N
A6,C =
Cc,f,
(11)
,=1
- 6prot) of the where A6,C is the calculated protonation shift ith methylene carbon resonance, chIis the protonaton shift constant of the ith resonance for total protonation of the jth basic site, and f, is the average fraction of time the jth site is protonated. Values for A6,C were obtained from the results of the nonlinear analysis as protonation shifts between the non- and triprotonated, non- and diprotonated, and non- and monoprotonated species. Values for cIl were calculated from the protonation shift parameters in Tables VI and VI1 of Sarneski et al. (23). These authors did not have parameters for all of the substituents needed to accurately calculate values of c,, for spermidine. However, the substituent most closely resembling the actual substituent was used. Values for f, were then determined by solving simultaneous equations.
RESULTS AND DISCUSSION The spermidine spectrum is shown in Figure 2 for several values of pH. The four downfield peaks correspond to the a-carbons and the three upfield peaks correspond to the pcarbons. Table I lists chemical shifts calculated from additivity constants for each of the carbon nuclei (23). During the course of the NMR titration, the molecule is deprotonated and all the carbon resonances shift downfield. At the lower end of the pH range, the resonances from Cz and C6 appear to merge and then move away slightly. The shift
53, NO. 6,MAY 1981
ANALYTICAL CHEMISTRY, VOL. I
Table I. Calculated Chemical Shifts for Nonprotonated Spermidine carbon
shift oalcd,a ppm
carbon
shift calcd,bl ppm
4 3 7 1
50.23 48.59 43.88 42.45
2 6 5
32.18 30.36 28.03
I
I
I
I
I
c
I
4
791
I
. t
i;
' Calculated with respect to an external reference of Me,Si. Table 11. pKa0 Results for Spermidine carbon no. PKl PK, PK3 4 7.99 f 0.18 9.12f 0.12 10.80 f 0.17 8.83 f 0.18 10.70 f 0.12 3 7.98i 0.21 9.53 f 0.33 11.11 f 0.34 7 8.13 * 0.26 9.13 f 0.36 10.81 i 0.10 1 8.13 f 0.12 9.71 f 0.09 10.88f 0.15 6 8.19* 0.09 2 8.31 f 0.03 9.71 f 0.08 10.91 f 0.15 8.65 f 0.41 10.81 f 0.22 5 8.16 f 0.14 av 8.25 f 0.09' 9.71 f 0.12' 10.90 f 0.21' Alternative Assignments (Noncrossing Peaks) 2 8.38 f 0.04 9.82 f 0.06 10.95 f 0.16 9.69 f 0.09 10.85 f 0.18 6 8.09 f 0.09 av 8.24 f 0.09' 9.76 f O.lla 10.90 f 0.24' a Average pK,' values for carbons 6 and 2. O
O
O
assignments for these carbon nuclei will obviously depend on whether their peaks do or do not cross. It was found that predictions based on protonation substituent effects for monoamines as reported by Sarneski et al. could not resolve the alternative assignments (23). Although this feature has not been clarified from the NMR data, the shift difference is so slight (approximately 0.1 ppm) that the derived pKa values and proton distribution pattern are not significantly affected. The results and error limits of the pKao values obtained from this study are shown in Table 11. The results from both alternative assignments for C2 and c6 are reported. The errors of the pKao values are the standard deviations of the y intercept obtained from the linear least-squares analysis. The errors reported for the final pKao values were calculated from [ z\( A)'] I/'. After the pK," values were obtained, they were used in eq 6 through 9 to calculate distribution curves at infiiite dilution. These curves are shown in Figure 3. The plot gives the fractions of each species present at any pH over the range 5.5-12.4. The results obtained from the nonlinear analysis for the values of al, 6 , and are shown in Table 111. There were
s
5.
7.
g.
11.
la.
*.
Flgure 3. Species dlstribution curves for spermidine: trlprotonated, 0;diprotonated, A; monoprotonated, *; nonprotonated,
no chemical shift changes due to changes in concentration of spermidine. The errors are those obtained from the regression analysis. Table I11 also records the protonation shifts for the 13C nuclei. Figure 4 shows the titration curves for all the carbon atoms at a concentration of 0.051 M. According to these curves and Table 111, the largest protonation shifts are exhibited by the &carbons, c 2 , c6, and Cg. c2and c6 are the most sensitive of the &carbons and provide the smallest errors in pK,. Accordingly, the three final pKao values shown in Table I1 are the averages obtained from C2 and Cg. These values are pK1 = 8.25 f 0.09, pK2 = 9.71 f 0.12, and pK3 = 10.90 f 0.21. It would be expected that if the assignments of resonances to C2 and at low pH made any difference, there would be significant differences between the two alternatives in the errors of the final results. However, as Table I1 shows, this is not the case. Palmer and Powell have reported pKa values for the protonation of spermidine obtained from potentiometric measurements in sodium chloride solutions, using a method described by Hedwig and Powell (24, 25). Their values for deprotonation are pK1 = 8.34 f 0.03, pK2 = 9.81 f 0.02, and pK3 = 10.89 f 0.05 (at 25 "C and ionic strength I = 0.1 M).
Table 111. Chemical Shift" Results and Protonation Shifts of 0.051 M Spermidine carbon
a
no.
61
4 3 7 1 6 2 5
49.36 f 0.04 47.08 I 0.03 41.54 f 0.08 39.75 f 0.04 30.55 f 0.06 32.56 f 0.09 26.84 f 0.04
6,
15
PH
6 3
48.67 f 0.05 48.45 f 0.05 46.63 i- 0.04 46.40 f 0.04 40.38 f 0.06 41.17 f 0.07 39.38f 0.04 38.87 f 0.03 28.69 f 0.08 25.76 f 0.06 27.85 * 0.09 30.40 f 0.11 25.67 f 0.05 25.26 f 0.06 Alternative Assignments (Noncrossing Peaks) 2 32.57 f 0.10 30.51 f 0.13 27.94 f 0.10 6 30.55 f 0.07 28.52 f 0.10 25.63 f 0.06 All chemical shifts are reported as ppm downfield from Me4Si. A6 = 6 - S .,.
64
AS6
48.10 f 0.04 45.56 f 0.03 39.96 f 0.06 37.71 f 0.02 24.90 * 0.06 24.78f 0.08 23.65 f 0.04
1.26 1.52 1.58 2.04 5.65 7.78 3.19
24.85 f 0.08 24.83 f 0.07
7.72 5.72
ANALYTICAL CHEMISTRY, VOL. 53, NO. 6,MAY 1981
792
r
I
I
I
I
I
I
I
I
Table V. pKa Results from Nonlinear Analysis of Potentiometric Data
-1
I
I
6.
I
I
I
I
E.
10.
I
I
I
I2
I 14.
PH
Figure 4. Titration curves for ail carbons in spermidine. Assignments are indicated to right of curve. Table Iv. Values of f a , f b , and f , for 0.037 M Spermidine at the Various Stages of Protonationa stage of protonation fa fb fc total tri 0.94 0.95 0.92 2.81 di 0.67 0.42 0.86 1.95 mono 0.32 0.32 0.43 1.07 pH 7.4 0.92 0.86 1.00 Alternative Assignments for C, and C, 0.93 0.94 0.94 2.81 di 0.63 0.42 0.86 1.91 mono 0.29 0.32 0.43 1.04 pH 7.4 0.90 0.86 1.02 tri
The values for cu used in eq 8 to calculate f a , f b , and f c were the following: c% = 5.56,C2b = 2.66,c 2 , = 0.00, C5a 0.00, C5b = 2.66,c5c = 0.70, C6a 0.00,c 6 b = 0.52, and C e C
5.56.
These are not thermodynamic values, however. Anichini et al. reported thermodynamic results (-AGO) obtained from calorimetric measurements (26). By use of their results, a value for the thermodynamic pK, can be determined from AGO = -RT In K O . Calculated results at the temperature of these studies, 30.7 OC, for deprotonation using their data are pK1 = 8.18,pK, = 9.62,and pK3 = 10.67. It is interesting to note that the values for AGO reported by Anichini et al. predict the same pK, values as those obtained by Palmer and Powell, at 25 "C, using potentiometric titration data: pKl = 8.34,pK2 = 9.81,pK3 = 10.88. The calculated values of fa,fb, and f, for a concentration of 0.037M are listed in Table N. The values calculated from both alternative assignments for C2and c6 are reported. (The values calculated for the other concentrations were not significantly different from those reported.) When 1 equiv of acid has been added, spermidine is monoprotonated. At this pH, which is approximately midway between pK3 and pK,, N, and Nb are protonated 32% of the time while N, is protonated 43% of the time.
concn,M 0.03 0.051 0.078 0.160
pK,
8.56 f 0.13 8.57 i: 0.11 8.67 f 0.09 9.20f 0.42
PK,
10.04 i: 0.11 10.06 i: 0.10 10.13 f 0.08 10.42f 0.33
PK, 11.10 f 0.11 10.84 f 0.10 10.99 f 0.08 11.69 f 0.44
When the molecule is diprotonated, N, and N, are protonated 67% and 86% of the time, respectively, while Nb is protonated 42% of the time. With this distribution of protonation, the molecule presumably adopts the energetically most favorable situation by minimizing the electrostatic repulsion (22,27,28). In the triprotonated form, N, is protonated 94% of the time, Nb is protonated 95% of the time, and N, is protonated 92% of the time. These results show that the molecule is fully protonated; within experimental error, the values for fa,fb, and f, are essentially the same. It is interesting to determine the distribution of protonation a t the physiological pH, 7.4. To do this, we determined the chemical shifts of C2, C5, and (26 for this pH by using eq 5. Then the protonation shifts were calculated and used in the equations derived from eq 11. The results of these calculations are included in Table IV. Table IV also shows that the values of fa,fb, and f, for the two alternative assignments are very nearly the same within experimental error. The differences are too small to influence the assignment of resonances to C2 and c6 at low pH. At each stage of titration fa + fb + f, should ideally equal the number of equivalents of acid added. As seen in Table IV, there is a small discrepancy in these totals. This is not surprising since the substituent parameters employed were obtained from data on monoamines and, hence, cannot account for the effects due to multiple charge sites. Nevertheless, the general character of the values in Talbe IV appears to be reasonable. For example, the charge distribution in the triprotonated species is approximately uniform, as is to be expected. Also, the asymmetries of the charges in the di- and monoprotonated structures reflect the expected effects of charge repulsion (23). A more detailed study of an appropriate series of polyamine compounds would help clarify these features. As a check of the pK, values obtained from the 13Ctitration study, the preliminary potentiometric data were also fit to equations obtained from Hamann (29). The nonlinear regression analysis was used, and the results are recorded in Table V. A linear least-squares analysis gave the following values: pK, = 8.30 f 0.06,pK, = 9.90f 0.02,and pK3 = 10.66 f 0.21.
ACKNOWLEDGMENT The helpful assistance and discussions of Robert C. Long, Jr., are gratefully acknowledged.
LITERATURE CITED (1) Tabor, H.; Tabor, C. W. Pharmacol. Rev. 1084, 16, 245-300. (2) Badawi, M. M.; Bernauer, K.; van den Broek, P.; Groger, D.; Guggisberg, A.; Johne, S.; Kompis, I.; Schneider, F.; Veith, H.J.; Hesse, M.; Schmid. H. Pure Appl. Chem. 1973, 33, 81-108. (3) Potier, P.; LeMen, J.; Janot, M. M.; Bladon, P. Tetrahedron Leff.1980, 16, 36-38. (4) Wright, R. K.; Buehler, B. A.; Schott, S. N.; Rennert, 0. M. Pediafr. Res. 1078, 12, 830-833. (5) Rennert, 0. M.; Frias, J.; Shukla, J. B. rex. Rep. Biol. Med. 1976, 34, 187-197. (6) Arvanitakis. S.; Mangos, J.; McSherry, N. R.; Rennert, 0. rex. Rep. BIOI. Med. 1078, 34, 175-186. (7) Theoharides. T. C. Me Sci. 1980, 27, 703-713. (8) Bolton, P. H.; Kearns, D. R. Biochemistry 1077, 76, 5729-5741. (9) Cohen, S. S. Nature (London) 1978, 274, 209-210. (10) Bunce, S.; Kong, S. W. Biophys. Chem. 1978, 8 , 357-368. (11) Kalser, D.; Tabor, H.; Tabor, C. W. J . Mol. Biol. 1963, 6 , 141-147.
Anal. Chem. 1981, 53, 793-797 (12) Kimes, B. W.; Morris, D. R. Biochemistry 1973, 12, 442-449. (13) Long, K. R,; Long, R. C.; Goldstein, J. H. J. Magn. Reson. 1972. 8, 207-210. (14) Tabor, H. Biochemistry 1962, 1 , 496-501. (15) Welss, R. L.; Morris, D. R. Slochemistry 1973, 12, 435-441. (10) Weser, U.; Strobel, G.J.; Rupp, H.; Voeiter, W. Eur. J. Biochem. 1974, 50, 91-99. (17) Sarneski, J. E.; Reilley, C. N. Essays Anal. Chem. 1977, 35-49. (18) Sarneski, J. E.; Surprenant, H. L.;Reiiley. C. N. Spectrosc. Len. 1978, 9, 805-894. (19) van de Weijer, P.; Thijsse, H.; van der Meer, D. Org. M a p . Reson. 1976, 8, 187-191. (20) van de Weijer, P.; van den Ham, D. M. W.; van der Meer. D. Org. Magn. Reson. 1977, 8 , 281-284. (21) Olson, A. R.; Koch, C, W.; Pimentel, G. C. “Introductory Quantitative Chemistry”; W. H. Freeman: San Franclsco, CA, 1950; Chapter 12.
793
(22) Sudmeier, J. L.; Reiiley. C. N. Anal. Chem. 1984, 36, 1098-1700. (23) Sarneski, J. E.; Surprenant, H. L.; Molen, F. K.; Reiiley, C. N. Anal. Chem. 1975, 47, 2116-2124. (24) Palmer, 8. N.; Powell, H. K. J. J . Chem. Soc., Dalton Trans. 1974, 2080-2088. (25) Hedwlg, G. R.; Powell, H. K. J. Anal. Chem. 1971, 43, 1206-1212. (20) Anichlnl, A.; Fabbrizzl, L.; Barbucci, R.; Mastroianni, A. J. Chem. Soc., Dalton Trans. 1977, 2224-2228. (27) Prue, J. E.; Schwarzenbach, G. HeEv. Chim. Acta 1050, 33,985-995. (28) Schwarzenbach, G. Helv. Chlm Acta 1950. 33, 974-985. (29) Hamann, S. D. Aust. J. Chem. 1970, 23, 1749-1705.
RECEIVED for review November 13,1980. Accepted January 23,1981. This research was supported in part by a grant (GM 10848) from the National Institutes of Health.
Determination of Flurazepam in Human Plasma by Gas Chromatography-Electron Capture Negative Chemical Ionization Mass Spectrometry B. J. Miwa” and W. A. Garland Department of Biochemistry and Drug Metabollsm, Hoffmann-La Roche Inc., Nutley, New Jersey 071 10
P. Blumenthal Department of Clinical Pharmacology, Hoffmann-La Roche Inc., Nutley, New Jersey 071 10
A specific and extraordlnarily sensitive GC/MS assay has been developed to quantitate flurazepam, a commonly prescribed hypnotic, In human plasma. The assay requires 2 mL of plasma and features the use of electron capture negative chemical ionization. A decadeuterated analogue of flurazepam is used as Internal standard. A 4 ft X 1 mm mlcropacked column contalning OV-17 on pPartkorb Is used for the GC analysis. A quadrupole mass spectrometer Is used to monitor in the GC effluent the M-• Ions of flurazepam and Its Internal standard. Methane is used as both GC carrier gas and negative chemical ionization reagent gas. The sendtlvlty of the assay Is 12 pg mL-‘ and at a concentration of 135 pg mL-’ the assay precision is 4 %. Three subjects who received a 30-mg oral dose of flurazepam showed peak flurazepam concentrations of 3.9, 2.3, and 0.4 ng mL-’ at 0.5, 1, and 1 h postdoslng, respectively. The ellminatlon half-llves for the three subjects were 1.9, 3.0, and 2.0 h, respectively.
Flurazepam (Dalmane, I, Table I) was introduced into general medical practice in 1970 as an orally active agent for the treatment of insomnia. Presently, flurazepam is the most widely prescribed drug for insomnia ( I ) and is the thirteenth most prescribed drug in the United States (2). Studies on the biotransformation and elimination of flurazepam in humans have shown that the compound is extensively metabolized and has a large apparent volume of distribution (3). Peak plasma concentrations of the intact drug rarely exceed several nanograms per milliter following the single or multiple dose administration of therapeutic amounts of flurazepam to humans (4,5).While de Silva and co-workers have reported spectrophotometric and EC/GC procedures which can sometimes measure the peak concentration of flurazepam following dosing (4,5), only the recently published RIA procedure of Glover et al. (6) can measure flurazepam 0003-2700/81/0353-0793$01.25/0
Table I. Chemical Structures and Their Designations
designation
over a sufficient period of time to determine the drug’s pharmacokinetic profile. In this paper, we report a sensitive and specific method for the quantitation of flurazepam which is the first reported “chemical” assay for flurazepam with sufficient sensitivity to measure useful concentrations of this widely used hypnotic in plasma. The assay uses electron capture negative chemical ionization mass spectrometry (EC/NCIMS) as the method of detection. NCIMS has been successfully used to analyze trace amounts of other medically used 1,4-benzodiazepin-2ones, i.e., desmethyldiazepam (7) and clonazepam (8),in biological matrices. The sensitivity of EC/NCI depends on the ability of the molecule in question to capture a thermal electron and form a stable molecular anion. In this regard, electron capture by the 1,4-benzodiazepin-2-onesis aided by the presence of two aromatic electron systems which can resonance stabilize the resulting anion (9). The highly adsorptive nature of flurazepam makes it a particularly difficult compound to quantitate. In addition to a deuterated internal standard (11), we also add a structural analogue, compound 111,to the plasma to increase extraction efficiencies and reduce adsorptive losses (IO). Another 0 1981 American Chemlcal Society
