Table I. Comparison of Phosphate Buffer and Sodium Chloride as Eluents for Chromatography of Barbiturates and Metabolites Retention volume, ml Compound KHQPOI" NaClb Phenobarbital 5.9 11.o Hydroxyphenobarbital 10.0 16.5 5.4 10.3 Pentobarbital Hydroxypentobarbital 1.1 4.7 Amobarbital 6.1 10.6 Hydroxyamobarbital 1.5 4.3 Hexobarbital 1.4 10.8 Ketohexobarbital ... 1.3 a Conditions: 2.0 mM KHQPO~, pH 5 . 5 , 80 "C, flow rate 32-36 ml/hr. * Conditions: Low concentrate chamber, O.lmM NaCl; high concentrate chamber, 1.OM NaC1; flow of high concentrate into gradient chamber, 12 ml/hr; column flow, 24 ml/hr; initial volume, 50 ml; temperature, 80 " C ; inlet pressures, 700-900 psig.
increased as the pH became less acidic. The effect of column temperature on the peak shape was marked and operation at higher temperatures resulted in sharper peaks (Figure 2). The finding of improved peak shape at higher temperatures is similar to the results obtained by Horvath and Lipsky (2) who observed that high column temperatures produced favorable conditions for the separation of purine and pyrimidine bases. The separation of diphenylhydantoin and hydroxydiphenylhydantoin was insensitive to changes in molarity, pH, and temperature over the ranges given above. The same conditions that proved to be optimum for the chromatography of diphenylhydantoin and its hydroxylated metabolite were also suitable for the separation of phenobarbital and hydroxyphenobarbital. Analysis of barbiturates and their metabolites, other than phenobarbital, using phosphate buffers as eluents in gradient or nongradient modes of operation was unsatisfactory inasmuch as poor peak shape was obtained. Further studies showed, however, that the use of a linear sodium chloride gradient gave satisfactory results and both barbiturates and
their metabolites eluted as sharp symmetrical peaks (Figure 3). In addition, metabolites and parent compounds were well separated. As shown in Table I, alcoholic or ketonic metabolites consistently showed retention volumes less than that of the parent molecule. On the other hand, the phenolic metabolite of phenobarbital was retained longer than the parent compound. Separation efficiency was substantially greater when a linear sodium chloride gradient was employed than during nongradient phosphate buffer operation. As expected, operation of the column with neutral sodium chloride solutions resulted in an increased sensitivity as compared to the use of acidic phosphate buffers. This is attributable to the increased absorptivity of barbiturates at more alkaline pH values. In contrast to the results obtained with barbiturates and their metabolites, diphenylhydantoin and hydroxydiphenylhydantoin were not eluted from the column by a sodium chloride gradient. In conclusion, high-speed ion exchange chromatography has been shown to be a useful method for the analysis of barbiturates, diphenylhydantoin and metabolites of these compounds. Nongradient elution with phosphate buffers proved effective for the separation of phenobarbital, diphenylhydantoin, and their phenolic metabolites. Optimum conditions for the above analysis were found to be 20.0mM phosphate buffer, pH 3.5,and a column temperature of 80 "C. Separation of barbiturates and metabolites were successfully carried out with a linear sodium chloride gradient at 80 "C. Studies are in progress to extend these techniques to the analysis of drugs in biological fluids. ACKNOWLEDGMENT
The authors thank Mehroo J. Cooper for the preparation of ketohexobarbital. Tom Flanagan, Smith Kline and French Laboratories, supplied the hydroxyamobarbital used in these experiments, and A. J. Glazko, Parke, Davis and Company, supplied hydroxydiphenylhydantoin. RECEIVED for review April 20, 1970. Accepted July 15, 1970. This research was supported by U.S.P.H.S. grants G M 17511 and G M 15477.
Identification of Barbiturates by Chemical Ionization Mass spectrometry' H. M. Fales, G . W. A. Milne, and T. Axenrod2 Laboratory of Chemistry, National Heart and Lung Institute, National Institutes of Health, Bethesda, Md., 20014 THEUSE OF BARBITURATES as sedatives is so widespread that accidental and intentional overdoses are daily occurrences. This problem is further compounded by the fact that a wide variety of substituted barbituric acids with considerably different biological activities is in common use. They are obtainable, moreover, in many different formulations and mixtures. Therefore, when an overdose has been taken, it 1 This is Part V of the series on Chemical Ionization Mass Spectrometry of Complex Molecules. NIH Special Fellow, 1969. O n leave from the City College of the City University of New York.
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is difficult to determine from an examination of the patient the identity of the drug or drugs responsible for his condition. This information is essential to a correct choice of treatment and a reliable method for the identification of specific barbiturates in stomach washings, blood, or urine would be of considerable value. A wide variety of chemical and colorimetric methods is available ( I ) for the identification of barbiturates as a class (1) See for example, D. M. Baer, Amer. J. Clin. Puthol., 44, 114 (1965) and references cited therein.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 12, OCTOBER 1970
T
FFFCWFBITAL,
M = 210,
- €.I.
Figure 1. Electron impact mass spectra of seconal and aprobarbital but since these methods, without exception, identify the barbituric acid nucleus, they are unable to distinguish between the various members of the class. On the other hand, the quantitative data so obtained are vital to treatment once the drug is identified. Gas chromatography has been effectively utilized ( 2 ) to differentiate between the various derivatives and is a most promising method. However, many other substances may be present in biological samples and these may give misleading peaks even in a purified sample that contains mainly the acidic barbiturates. Mass spectrometry coupled with gas chromatography can often provide positive identification of such peaks and while the apparatus is relatively complex and expensive at present, there is every indication that this will not be so in the future. Behavior of the barbiturates on electron impact (EI) has been investigated by a number of groups (3-5) all of whom note the disturbing fact that the odd-electron molecular ions are very unstable and often cannot be detected. Fragmentation usually occurs by the loss of CO and one of the two side chains. However, many barbiturates possess one side chain in common so that loss of the other substituent may cause confusion. Thus loss of the isopentyl side chain from the molecular ion of seconal [l, R = CH3CH2CH2CH(CH3)-] gives the same fragment ion 2 as is formed by the loss of the corresponding side chain of aprobarbital [Alurate, 1, R = (CH&CH-]. These are very important processes. As a re(2) B. J. Gudzinowicz and S. J. Clark, J . Gas Chromatogr., 3 , 147 (1965). (3) A. Costapanagiotis and H. Budzkiewicz, Momtsh. Chem., 91, 1800 (1965). (4) W. F. Griitzmacher and W. Arnold, Tetrahedron Lett., 1365 (1966). ( 5 ) R. T. Coutts and R. A. Locock, J . Pliarm. Sci.,57, 2096 (1968).
sult, both spectra are virtually identical, as is shown in Figure 1. The molecular ions of these compounds would have different masses but they are of such low abundance as to be of little value in identification of the compounds in a biological matrix.
1
2
We demonstrate in this work that chemical ionization (CI) mass spectrometry with methane provides intense quasimolecular (QM+) ions at m/e ( M 1)+ in all eight representative barbiturates investigated. Isomeric barbiturates such as amytal and pentobarbital, cannot be distinguished in this way but can be identified from their E1 mass spectra which can be measured immediately upon closure of the methane admittance valve.
+
EXPERIMENTAL
Equipment. All mass spectra, both E1 and CI were measured at low resolving power on an MS-902 double focusing mass spectrometer equipped with a Scientific Research Industries dual EIjCI source described previously (6). In all cases, the reactant gas for CI was methane, at a pressure of about 1 torr. The samples were admitted to the source cia a direct insertion probe and the temperature of the source was kept between 100 and 150 "C, at which temperatures the free barbituric acids are volatile. In all the CI mass spectra (6) H. M. Fales, G. W. A. Milne, and M. L. Vestal, J. Amer. Cliem. Soc., 91, 3682 (1969).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 12, OCTOBER 1970
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271
103 T
E W
=
"I
20
100
W
80
140
a W
2 b-
40
U
d a:
20
Figure 2. Electron impact and chemical ionization mass spectra of ortal 7
PENTOBARBITAL
M.226
r * i i ~ l l l u i L w i L11~U,
I
I
140
150
I
I
I
1
l
l
l
l
d i $ & L d -
/
l
'
160 170 180 190 200 210220230240250 227 239
Figure 3. Chemical ionization mass spectrum of extract of gastric contents reported, the ions derived from methane alone are subtracted from the spectra, The intensities of the remaining ions are reported as a percentage of the total ion current represented by all such ions.
3, R = n-C6Hn];phenobarbital (3, R = C6H5);amobarbitaand cyclobarbital (Phanl [Amytal, 3, R = (CH3)2CHCH2CH2], odorn, 3, R = cyclohex-1-enyl). r!
"&
O W N V O
RESULTS AND DISCUSSION
The E1 and CI mass spectra of the following eight derivatives of barbituric acid were studied: Seconal [l,R = CHICH2CH2CH(CH3)-]; butalbital [l, R = (CH3)tCHCHt-]; aprobarbital [Alurate, 1, R = CHI]; pentobarbital [Nembutal, 3, R = CH3CH2CH2CH(CH3)-];hexethal [Ortal, 1434
C*
t!JH
R
O
3
In CI mass sDectrometry (7) the molecule under investiga(7) F. H. Field, Accourits Chem. Res., 1, 42 (19h8).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 12, OCTOBER 1970
Table I. Chemical Ionization Mass Spectra of Barbituric Acids QM+ ion (mle); relative Compound (MW) abundance, % I / Z I Pentobarbital (226) 227; 63% Hexethal(240) 241; 55% 233; 60% Phenobarbital (232) Amobarbital (226) 227; 81% Cyclobarbital (236) 231; 33% 239; 41% Seconal (238) Butalbital(224) 225; 13% Aprobarbital(210) 211; 46%
tion is protonated by collision with either of the Bronsted acids CH5+ or C2Hf which are well-known (8) as the major species in the mass spectrum of methane at 1 torr. The amount of available energy in such collision-induced ionizations is only -230 kcals and the even electron QM+ ions so derived should be very stable relative to the odd-electron ions formed by EI. Our experience with alkaloids (9), diketones (IO), and amino acids (11)has shown this to be true and Figure 2 shows that such reasoning also holds in the case of barbituric acid derivatives such as hexethal (Ortal, 1, R = n-CeH11). In the E1 mass spectrum, the molecular ion (m/e 240) is of very low abundance (-2 % of the base peak) and among the various fragmentation processes may be observed loss of CzHs(giving the ion of m/e 211) and loss of C6HlZ (giving the ion at m/e 156). Such processes are much less important in the CI mass spectrum where the most abundant ion is the (8) . , F. H. Field and M. S. B. Munson, J. Amer. Chem. SOC..87, 2289 (1965). (9) H. M. Fales, H. A. Lloyd, and G. W. A. Milne, ibid., 92, 1590 (1970). (10) H. M. Fales, H. Ziffer, G. W. A. Milne, and F. H. Field, ibid., 92, 1597 (1970). (11) Part IV, H. M. Fales, G. W. A. Milne, and T. Axenrod, J. Amer. Chem. SOC.,92,5170 (1970).
QM+ ion at m/e 241 whose intensity constitutes some ,55 of the total ion current. Very similar results are obtained from all the other barbiturates studied. In every case the QM+ ion is the base peak of the CI mass spectrum. In Table I is given the relative intensity (expressed as a percentage of the total ion current) of the QM+ ion of each individual barbiturate (12). The practical value of this technique is illustrated by the following example. The acidic organic material, containing any barbituric acids present, was extracted with chloroform from the acidified stomach washings obtained from a comatose patient who was suspected to be suffering from an overdose of a sedative drug. Removal of the solvent left an oily residue which was submitted directly to CI mass spectrometry. The CI mass spectrum obtained, Figure 3, shows many materials to be present, but from the overall complex spectrum, it may be seen that ions at m/e 233 and 239 are prominent. These correspond to the QM+ ions of phenobarbital and seconal, respectively, and thus a tentative identification of these two drugs was made. Institution of the appropriate treatment resulted in this case in the recovery of the patient who was able to confirm this identification (13). CONCLUSIONS Mainly because of the very large cross-section for proton capture possessed by the barbituric acid nucleus, QM+ ions derived from barbiturates in CI mass spectrometry are very intense and can be used to help identify the specific barbiturates. The value of this method in actual cases of drug identification has been demonstrated and the application of the technique to other dangerous drugs outside the barbiturate family is under study in this laboratory. RECEIVED for review May 8, 1970. Accepted July 15, 1970. (12) The 13Csatellite of the QM+ ion is ignored in the calculation
of these figures. (13) Sample submitted by Mr. N. Law, Chemist at Suburban Hospital, Old Georgetown Road, Bethesda, Md.
Spectrophotometric Determination of Fluoride in Seawater Richard A. Kletschl and Francis A. Richards Department of Oceanography, University of Washington, Seattle, Wash. 98105 THE FRESH WATER spectrophotometric fluoride method of Yamamura et al. ( I ) , has been modified for the determination of fluoride in seawater [using a cerium-alizarin complexone chelate in an acetate buffered 2 0 Z (v/v) acetone solution]. The seawater reaction requiring only 10 ml of seawater, is complete in approximately 20 minutes. An average standard deviation of 10 pg of fluoride at the 1080-pg F-/liter level, was found using 60 duplicate samples. Present address, Chemistry Department, Centralis College, Centralia, Wash. 98531. (1) S. S. Yamamura, M. A. Wade, and J. H. Sikes, ANAL.CHEM., 34, 1308 (1962).
EXPERIMENTAL
Apparatus. A Beckman Model DU spectrophotometer and 2.5-cm borosilicate glass cells were used for absorbance measurements. Distilled water or reagent blanks were used in the reference cell. Reagents. CERIUMNITRATE(0.0167M). Dissolve 3.626 grams of Ce(N0&.6 H 2 0 in distilled water and dilute to 500 ml. ALIZARIN COMPLEXONE (AC). Suspend 0.643 gram of AC in 50 ml of distilled water and dissolve by adding 0.25 ml of concentrated N H 4 0 H . Add O.Z5 ml of concentrated acetic acid and dilute to 100 ml. This solution is stable for at least two weeks if refrigerated. The AC was obtained from Jackson and Burdick Laboratories, Muskegon, Mich. ACETATE BUFFER. Dissolve 60 grams of NaC2HsOn 3H20
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