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 NaC2HsOn3H20
ANALYTICAL CHEMISTRY, VOL. 42, NO. 12, OCTOBER 1970
1435
in 500 ml of distilled water and add 107 ml of glacial acetic acid. Dilute to 1 liter. FLUORIDE REAGENT.To 300 ml of reagent grade acetone, add 63 ml of the acetate buffer and shake. Follow this with 9.10 ml of AC solution and then 9.10 ml of cerium nitrate solution. Dilute to 500 ml with fluoride-free distilled water and shake well. The reagent is stable for at least five days at room temperature. STANDARD FLUORIDE SOLUTION (Primary). Dissolve 1.103 grams of anhydrous NaF in 1 liter of distilled water. The preparation of a secondary standard by further dilution will be necessary. The primary standard solution is stable for months. Procedure. Add 10 ml of seawater to 30 ml of fluoridefree distilled water (dilution is necessary to come within the concentration range where Beer’s law is followed). Follow this by accurately adding 20 ml of fluoride reagent. After at least 20 and not more than 60 minutes, read the absorbance of the samples and of a distilled water blank against distilled water at 625 nip, in a 2.5-cm cell (0.12-mm slit width). Determine the concentrations by reading from a distilled water (40-ml samples) curve plotted from sample values within the range of 600-1800 pg of F-/liter (these values are on the basis of 10 ml before dilution to 40 ml, as would be the case with 10-ml seawater samples before dilution). For the distilled water standard curve, add 20 ml of fluoride reagent to the 40-ml samples and allow to react for not less than 20 and not more than 60 minutes. The absorbancies of samples containing 600, 1200, and 1800 pg of F-/liter are approximately 0.13, 0.30, and 0.47, respectively (after subtraction of the large blank value of approximately 0.388). The distilled water standard curve is nonlinear below 80 pg of F-/ liter causing the intercept to be to the right of zero. Therefore, it is desirable to read the unknown values directly from the standard curve.
natural seawater. The artificial seawater contained 23.3 grams of NaCl, 10.63 grams of MgClz.6H20, 3.9 grams of Na2S04,1.11 grams of CaCL, 0.725 grams ofKC1,0.20 grams of NaHCO3,0.044 grams of H3B04, 0.0014 grams of LiC1, and 0.097 gram of KBr made up to 1 liter with distilled water. By effectively increasing the salinity to 58%, (salt content equal to that of a 10-ml seawater sample of salinity %go;,,after diluting to 40 ml as in the procedure), the slope remained identical to the natural seawater value, thus showing any salt effect to be quite small. pH and Time. The optimum pH for seawater analysis was found to be 3.85 to 4.40. A value of 4.35 is suggested to allow the use of the same reagent for seawater and distilled water (optimum pH range is 4.25-4.55) use. The optimum time for development of color in seawater samples, read against distilled water blanks, was 10-60 minutes, while distilled water blanks containing the reagent required 20-60 minutes. The time of 20-60 minutes is recommended in the procedure to allow for the slightly longer time required for stabilization of the distilled water reagent blank. DATA FROM THE COLUMBIA RIVER PLUME
The analysis of 196 samples of chlorinity range 17.99-18.99 from the Columbia River Plume region produced 27 samples in the fluoride/chlorinity range of 6.05-6.14 ( X 61 of the 5.95-6.04 (x lO-5), 31 of the 5.85-5.94 ( X lO-5), 46 of the 5.75-5.84 ( X lO-b), and 31 of the 5.65-5.74 ( X 10-5) range. ACKNOWLEDGMENT
DISCUSSION
The authors wish to gratefully acknowledge the technical assistance, helpful conversations, and critical evaluation of this work by Fred E. Palmer, Meredythe Meller, William W. Broenkow, Ralph W. Riley, Joel D. Cline, John C. Jorgenson, Robert V. Thurston, and Clifford A. Barnes.
Standard Curves and Salt Effect. The slope of the linear portion of the standard curve (80-2640 pg of F-/liter) is fairly constant from run to run (absorbance/F- in pg liter N 0.001066), being identical to that run in artificial seawater, and within 1.45% at the 95% confidence level to that run in
RECEIVED for review April 5, 1968, resubmitted December 5 , 1969. Accepted July 2, 1970. Work supported by the U S . Atomic Energy Commission, Grant AT(45-1)-1725 (ref. RLO1725-114). Contribution 453 from the Department of Oceanography, University of Washington, Seattle, Wash.
e
Detection of Sugars and Sugar Derivatives by Perchloric Acid on Cellulose Thin-Layers Kinzo Nagasawa, Akira Ogamo, Harue Harada, and Kazuko Kumagai Faculty of Pharmaceutical Sciences, Kitasato University, 5-9-1, Shirokane, Minato-ku, Tokyo, Japan
DURINGTHE DETECTION of sugar phosphate esters on paper chromatogram by the Hanes and Isherwood reagent ( I ) , we found that 2-deoxyribose phosphate showed a specific and sensitive coloration different from other sugar phosphates. Later, it was found that aqueous perchloric acid alone, one of the components of the reagent, could cause this color reaction. In general, detection of sugars on chromatograms is based
on the reaction whereby furfural-type compounds resulting from sugars in a strong acidic condition reacted with aromatic amines, phenols, or aldehydes to form colored substances. A mixture of aqueous perchloric acid and vanillin was first reported as a specific spray reagent for sugar alcohols and ketose by Godin (2). A more detailed study revealed that deoxysugars besides sugar alcohols and ketoses were also
(1) C. S. Hanes and F. A. Isherwood, Nature, 164, 1107 (1949).
(2) P. Godin, Nature, 174, 134 (1954).
1436
ANALYTICAL CHEMISTRY, VOL. 42, NO. 12, OCTOBER 1970
