tractant of barbituric acids than chloroform, as illustrated in Table 111. The solvent-aqueous partition coefficients of most compounds are considerably higher with chloroform than with butyl ether, but the butyl ether-aqueous and CHC18-aqueous partition coefficients for a given barbituric acid are nearly the same. Acids stronger than the barbituric acids, such as salicylic acid, are exceptions, but usually only a low proportion of such a one present in blood is extracted into butyl ether, and all of this is removed from the ether by Borax 1. Many partition methods utilizing the partition coefficients of the barbiturates for their identification were tested. Some of these were superior t o the method described for differentiation of certain barbiturates but were not as generally applicable. The use of the simple 240-mp absorbances for Borax 1 and 2 is not as accurate as the use of the Goldbaum absorbance difference-type procedure, since substances other than barbiturates absorb a t 240 mp. Addition of 8N sodium hydroxide to these and use of their 260-mp absorbance differences have been tried, but are usuaily not required. The use of Equation 4 for determination of phenobarbital is nearly as accurate as the total barbiturate procedure for clinically significant concentrations of 2 mg. % or more and has the great advantage that it can be used in the presence of mixtures of the other common three, since the contributions of the latter t o the absorbance of Boras 1 and 2 are nearly the same. Equation 5 is less satisfactory but it need not be used, since Equation 4 can be used for compounds more polar than Nostal and Equation 1 for com-
pounds less polar than aprobarbital. If Equation 4 indicates no more than 0.2 mg. % and there is no peak at 240 mp, this may be due to blank or interfering substance. If a single barbiturate is present, the total barbiturate procedure can be used t o check the results of application of Equations 1, 4, or 5 . If this is done, the value of the expression 100 (mg. % barbiturate from Equation l)/(mg. % ' barbiturate from Equation 6) will equal the per cent in 1N NaOH in Table I and' will be more accurate for identification of the nonpolar barbiturates than the use of Ratio 1; however, this requires an additional extraction. Mixtures of barbiturates appearing close to each other in Table I are not readily identified as mixtures. It is for this reason, since mixtures are often found, that it is felt that this partition procedure alone cannot give positive identification. Likervise, it does not appear that additional partition procedures to sharpen differences between the barbiturates are worthwhile, since most values might be simulated by a mixture. Accordingly, the final goal of the procedure is the partial resolution of mixtures of barbiturates into fractions whose constituent barbiturates can be positively identified by other procedures to be described in a system for identification in a later. paper. The most significant difference between this and earlier methods for quantitative determination of barbiturate is the separate determination of classes of barbiturates of different potencies. This has proved in cases hospitalized for barbiturate poiscning to be a satisfactory substitute for identification following determination of total
barbiturate, though totai barbiturate alone is satisfactory when the identity of the barbiturate is known with certainty. Nevertheless, in some cases positive identification is desirable. It is anticipated that in forensic practice also this partition procedure will usually be sufficient without need of positive identification. ACKNOWLEDGMENT
The author thanks Frank McKee, Director of the Clinical Laboratories, UCLA Medical Center, for supplying blood samples, and Raymond Abernethy, Head Toxicologist, Los Angeles County Coroner's Office, for his kind cooperation. LITERATURE CITED
(1) Dybing, F., Scand. J . Clin. & Lab. InUeSt. 7. SUDD1. 20. 114 (1955). (2) Go1dbaum;'L. R., A&L. CHEM.24, 1604 (1952). (3) Helldorff, I., Scand. J . Clin. h Lab. Invest. 7, Suppl. 20, 127 (1955). (4) Leyda, J. P., Lamb, D. J., Harris, L. E.. J . Am. Pharni. Assoc.. Sn'. Ed. 49, 581 (1960). (5) Locket, S., Proc. Roy. SOC. Med. 49, 585 (1956). (6) Stevenson, G. R., ANAL.CHEM.32, 1522 (1960). (7) Stevenson, G. W., University of California, Los Angeles, Calif., unpublished d a h ..
(8) Sunshine, I., Hackett, E., Clilz. Chem. 3, 125 (1957).
RECEIVEDfor review May 8, 1958. Resubpitted March 2, 1961. Accepted April 26, 1961. Investigation supported by research grant B-1106 from National Institute of Xeurological Diseases and Blindness, National Institutes of Health, U. S. Public Health Service. California Association of Crirrinalists, Los Angeles, Calif., April 1958. An-erican Academy of Forensic Sciences, Chicago, Ill., February 1959.
Separation and Identification of Barbiturates by Gas Chromatography KENNETH D. PARKER and PAUL L. KIRK School of Criminology, University o f California, Berkeley, Calif.
b Separation and identification of 23 barbituric acid derivatives, in the form of free acids, are described. Microgram samples, in organic solvent, are subjected to separation in the argon gas chromatograph with an SE-30 stationary phase. Preparation of the sample, as well as class identification, involves a double basic-acidic extraction from blood, coordinated with examination by ultraviolet spectrophotometry. All of the barbiturates tested produced single peaks whose 1378
*
ANALYTICAL CHEMISTRY
retention times could be used for identification. With proper calibration, the areas may be used for quantitative estimation.
N
of distinguishing among the barbiturates have been employed in toxicology. However, the low specificity of all chemical methods and the small structural differences that exist among many of the barbiturates have made such identiUMEROUS METHODS
fications difficult unless each compound is purified carefully. Ultraviolet absorption (4, 5 ) distinguishes the common barbiturates and the thiobarbiturates as groups: the absorptions being influenced by the pH. Differential hydrolysis (9) and differential partition between solvents (1, 10) have been successful with single compounds, but are not satisfactory for mixtures. Melting points, x-ray diffraction, and infrared absorption are suitable with pure solid material, in milligram quanti-
ties. Color and crystal tests are relatively nonspecific, and of limited utility. Paper chromatography has been the preferred method for small samples of limited purity (3,9). The conditions with respect to pH, solvent system, and temperature require careful control t o secure reproducible R, values. As regards the time required for development, often 8 to 20 hours are needed for sufficient separation of barbiturates. The longer the chromatographic run, the more diffused the spot becomes, which demands more sensitive methods of detection. To obviate repeated runs, the practice of spotting three different concentrations of the unknown extract is often used. This in turn decreases the practical sensitivity of the method and raises the minimum size of sample to 30 pg. or more. Although 5 t o 7 barbiturates are very common, there are some 37 barbiturates, of the malonylurea type alone, in use under some 162 different trade and pharmaceutical names. Pharmaceutical firms tend more and more to blend two or more different barbiturates in tablets or capsules, often with the incorporation of other dtugs. Examples of such formulations are,Tuinal, Nidar, and Banesin Forte tablets. Speedier and more reliable methods for the separation and identification of barbiturates are among the more important immediate needs of the toxicologist. The seriousness of the problem is intensified by the large proportion of fatal Foisonings that occur from overdosage with barbiturates, singly and in combination (9, 11). There is also a need to interpret tissue levels, since barbiturates generally differ in potency in reverse order of the duration of their action. In this paper a gas chromatographic method is described by which barbiturates, singly and in combination, may be detected, separated, and identified in microgram quantities rather quickly. REAGENTS AND APPARATUS
The Pye argon chromatograph (W. G. Pye and Co., Ltd., Granta Works, Cambridge, England) equipped with an ionization ,%ray (strontium-90) detector, and a l-mv. Minneapolis-Honeywell-Brown recorder with Disc integrator Model K 1-1, was employed. To inject samples readily into the heated portion of the column, a shorter than normal column was used and equipped with a diaphragm injection port made of borosilicate glass, as shown in Figure 1. The sample was injected with a Hamilton microsyringe of either 1.0or 10.0-pl. capacity, with a 7-cm. needle which penetrated to the heated portion of the column. The chromatographic column was a borosilicate glass tube of Smm. internal diameter, 4 feet in length. It was packed with 100- to 120-mesh acid-
Figure 1. A. 8. C.
.
Sampling attachment
Diaphragm injection port Gas supply Top of column
washed firebrick coated with SE-30 (6) 5y0 by weight. The temperature was 180' f 0.5" C. The flow rate of argon was kept at 28.6 cc. per minute under an inlet pressure of 105.0 cm. of Hg. The column was preconditioned for 24 hours a t 280" C. A variety of other chromatographic conditions was investigated to find a compromise set of conditions which would give sufficient separation and reasonable peak sharpness without too great a sacrifice of time. Various combinations of conditions were studied, with alterations of liquid phases, solid supports, temperatures, and flow rates. Higher temperatures and more rapid flow rates caused sharper peaks for many of the barbiturates, but a t the sacrifice of separation. Initially, some barbiturate salts and the corresponding free acids were investigated. The salts generally did not offer evidence of emergence a t temperatures between 180" and 220" C. All df the free acids, however, proved satisfactory for chromatographic analysis, giving single-peak responses. The barbiturates (some in the form of salts, and some as free acids) were supplied by pharmaceutical supply houses for this study. Basic chloroform extractions of the salts provided the free acids, which were recrystallized from alcohol and water and desiccated over activated alumina. The melting point of each preparation was determined and found to correspond with literature values (7,8). Appropriate weighed quantities of the free acids were dissolved in acetone to give standard solutions containing 10 pg./pl. PROCEDURE
To determine the applicability of the method to physiological materials tests were made by extracting samples of blood from coroners' death cases. Previous investigation had shown that each of these samples contained a barbiturate or a mixture of barbiturates; in some instances the particular barbiturates had been identified.
A 3- to 5-ml. blood sample was estracted with 45 ml. of chloroform, the extract being filtered through Whatman No, 1 paper. A 35-ml. portion of this filtrate was extracted with 5 ml. of 0.45N NaOH. At this point one would normally investigate the ultraviolet absorption of the solution and perhaps quantitate total barbiturates. This basic extract was brought to p H 4 with hydrochloric acid, re-extracted with three 15ml. portions of chloroform, and filtered as before. The extract was concentrated by evaporation in a beaker on a water bath. The resulting residue was desiccated in vacuo over activated alumina to remove any water or HC1 which might have been carried over with the chloroform. The dry extract was clean, containing only slight traces of blood extractives which survived the double extraction. The desiccated extract was washed into a 15-ml. centrifuge cone with small portions of warm acetone. The solution was reduced in volume on the water bath, using a 50-pl. micropipet (Kirk type) to wash the walls of the cone with the concentrating acetone solution. By alternate evaporating and taking up into the micropipet, the entire residue was finally contained in the 50rl. volume and could then be transferred to a very small glass-stoppered container to await analysis. From 1 to 5 pl. of this solution was injected into the apparatus. In this kind of investigation the gas Chromatograph does not replace the spectrophotometer, but is a valuable adjunct to it. Materials derived from blood by double extraction are first examined spectrophotometrically to determine whether or not the blood sample contains barbiturate. If so, the barbiturate may be re-extracted from the cuvette sample, as the acid, in chloroform. Following the procedure already described, the extract may then be injected into the gas chromatograph, which will identify the partirular barbiturate or barbiturates that are present. At only one third of the recorder's maximum sensitivity, 1-to 5pg. quantities caused half- to full-scale response. The method has the added advantage of speed. The preparatory phases, exclusive of desiccation, require about 20 minutes for a single sample; five may be concurrently prepared in an hour. Desiccation is not essential to the procedure. An additional half hour. should be allowed for the gas chromatographic identification, in consideration of the longest retention time observed. Acidic compounds other than barbituric acid derivatives no doubt exist which would survive the double extraction outlined here. Some of these might be of toxicological importance and could, conceivably, show retention times under a single set of conditions that would confuse them with barbiturates. For this reason preliminary ultraviolet class identification as barbiturate is recommended, as are also other independent methods for a more rigorous identification of the individual barbiturates. Graphs were made from the gas VOL. 33, NO. 10, SEPTEMBER 1961
1379
Table 1.
Barbituric Acid Derivation
Official Name (Chemical groups on barb.) Metharbital (NNR) (5,Fdiethyl-1-methyl) Barbital (USP) (5,bdiethyl) Probarbital (NNR) ( 5-ethyl-5-isopropyl) Diallyl barbituric acid (NN (5,5-diallyl) Allyl barbituric acid (NNR (5-allyl-5-isobiityl) Aprobarbital (NNR) ( 5-allyl-5-isopropyl) Butethal (NNR) (5-ethyl-5-n-bn tyl) Butabarbital INNR)
[5-allyl-5-(I-methylpropyl)] Amobarbital (USP) (5-ethvl-5-isoamvl) Pentobarbital f TisPi IB-ethvl-.i Secobarbital (NI , [f+allyl-5-( 1-met hylbutyl)] Thiopental (USP) 15-ethyl-54 1-m ethylbuty1)-2thio] Hexobarbital (NNR) [3,5-djmethyl-5-(1-cyclohexen-1-yl)] Cyclopal [5-allyl-5-(2-cyclopenten-l-yl)] Mephobarbital (NNR) I
( l-methyl-5-ethyl-5-phenyl)
Hexethal (NNR) (5-ethyl-5-n-hexyl) Thiamylal (NNR) [5-allyl-5-(l-methylbutyl)-2-thio] Phenobarbital (USP) (5-ethyl-5- henyl) Cyclobarbitar(N&R) [ 5-ethyl-5-( cyclohexen-1-yl)] Alphenal ( 5-allyl-5-phenyl)
Area/
Mol. Wt.
M. P.
R. T. (minutes)
198.22
151-155
3.2
970
184.19
188-192
4.2
868
198.21
202-203
4.7
381
208.21
171-173
5.5
62 1
224.25
138-139
6.2
286
210.23
140-142
6.5
316
212.24
124-127
6.8
357
212.24
165-168
7.2
793
226.29
160-1 61
7.8
370
B5.24
...
8.0
363
226.27
156-158
8.4
363
225.26
158
i.8
310
224.24
161-163
9.3
212
238.27
96-100
9.7
133
rg.
242.33
159
10.0
365
236.26
145-147
12.3
248
234.25
139-140
13.6
30
246.26
176
14.0
200
240.29
125-126
14.3
254
254.34
132-133
14.5
130
232.23
174-178
20.1
180
236.26
171-1 74
21.3
209
244.24
156-157
24.8
85
Chromatography conditions: Column: 4-foot packed with SE30 5% on firebrick 100/120. Temp. 180’ C. Flow: (argon) 28.6 cc. per min. Detector voltage: 1750 volts. Sensitivity: ~ 3 .
chromatograms of five barbiturates, plotting the integrated area under the curve against the weight of the sample. The samples ranged in weight from 0.5 to 20 pg. In each case a straight-line graph resulted. This linear response of the detector under given conditions should be of valuo in quantitation. It was possible, in cases where mixed barbiturates caused overlapping peaks, to estimate the relative concentration of the barbiturates present, duplicate the mixture on this basis, and come close to duplicating the overlapping peaks of the unknown. To determine possible interference by materials ex%racted from physiological fluids and tissues, blank extractions were run with blood, liver tissue, and urine (20 mi., 50 grams, and 50 ml., respectively) subjected to the same preliminary treatment as described. The specimens were run in the same
1380
0
ANALYTICAL CHEMISTRY
manner as the samples known to contain barbiturates. RESULTS
The behavior of standard solutions of pure barbiturates was first tested under the conditions described above. It was early noted that the retention time showed an effect due to variations in sample size. Slightly shorter retention times were observed with smaller microgram quantities. This effect could be avoided when the sample size was chosen to give a peak height of approximately one half the chart width. With a little experience, the shift in retention time could be estimated rather well from the peak height. When utilized solely for identification of individual barbiturates, it was found desirable
to choose a standard set of conditions, as given in Table I, which shows the retention times and integrated areas of peaks, with molecular weights and melting points of 23 barbiturates. The basis of choice of this set of conditions was, in part, the emergence of alphenal a t 25 minutes, which was the slowest barbiturate to appear. Extracts of biological materials, as listed, gave no specific responses in the gas chromatograph, nor did they interfere with other materials that gave such responses. Some effect was observed in the form of a 3-minute tailing of the acetone peak and a slight but definite rise in the base line. The injected quantities of biological samples represented extracts from blood, 2 ml., liver tissue, 5 grams, and urine, 5 ml. These quantities represented more than four times the amount of material that would be injected into the chromatograph in the described procedure for barbiturate extraction from blood. This suggests the possibility that less pure extractions would be satisfactory and, further, seriously opens the possibility of preparative gas chromatography in this area. The procedure was tested with 16 blood samples from coroners’ cases, in which death had occurred from barbiturate poisoning. The samples contained from 30 to 90 pg. per ml. Twelve contained but one barbiturate. In seven instances this was pentobarbital, in three it was phenobarbital; one contained secobarbital, and one, amobarbital. Two samples contained two barbiturates in misture, pentobarbital with phenobarbital, and secobarbital with amobarbital, respectively. One sample contained butabarbital, amobarbital, and barbital, while another single sample had four compounds: barbital, butabarbital, amobarbital, and phenobarbital. All coroners’ samples were resolved rapidly and easily into their components and identified by retention time as compared with standards. The data of Table I were used as a guide in preliminary identification. Inasmuch as only 1- to 5-pg. samples were required, only a small portion of the prepared extract was used for each determination, leaving sufficient for rechecking with different size of sample when this was found desirable. Recent investigation in this laboratory has also demonstrated the utility of the flame ionization detector in the chromatographic identification of the barbiturates. LITERATURE CITED
(1) Rrackett, J. W., California Association of Criminalists,Sprinq Meeting, 1959. ( 2 ) Broughton, P. hZ. O., Biochem. J . 63, 207 (1956). (3) Curry, A. S., Nature 183, 1052 (1959).
( 4 ) Goldhaurn, L. R., ANAL. CHEM.24,
1604 (1952). ( 5 ) Gould, T. D., Hine, C. H., J . Lab. Clin. M e d . 34, 1402 (1949). (6) Lloyd, H. A., Fales, H. M., Highet, P. F., VanderHeuvel, W. J. A., Wildman, W. C., J . Am. Chem. Soc. 82, 3481, 3791 (1960). (7) Merck Index, 7th ed. Merck & Co., Inc., U. S. A. (1960).
(8) Royal Canadian Mounted Police Seminar No. 1, Feb. 1, 1954, Headquarters, Ottawa. (9) Smith, Ivor, ed., "Chromatographic and Electrophoretic Techni ues," vol. 1, p. 381, Interscience, J e w York, 1960. (10) Stevenson, G. W., California Associstion of Criminalists, Spring Meeting, 1958.
(11) Stewart, C. P., Stolman, A., 'Toxicology," vol. 1, p. 71, Academic Press, New York, 1960. RECEIVEDfor review May 18, 1961. Accepted July 12, 1901. Work supported by grants from the National Institutes of Health, U. S. Puhlic Health Service (RG-4372 and RG-5802), and from the Research Committee, University of California.
Naphthyl Azoxine S as a Complexometric Indicator JAMES S. FRITZ, JANET E. ABBINK, and MARILEE A. PAYNE Institute for Atomic Research and Departmenf of Chemistry, Iowa State University, Ames, Iowa
b Naphthyl Azoxine SI or NAS, is an excellent indicator for the complexometric titration of metal ions. This indicator can be used for titrations in either acidic or basic solution. Using NASI methods for the titration of 25 elements with EDTA are described. Various masking agents improve the selectivity of EDTA titrations with NAS indicator.
T
HE 7-arylazo derivatives of 8-hy-
droxyquinoline-5-sulfonic acid are excellent metal ion indicators for complexometric titrations (1). Of these, the 7-(l-naphthylazo)-8-hydroxyquinoline-5-sulfonic acid, which has been termed Naphthyl Azoxine, is the best. Unlike Eriochrome Black T, Naphthyl Azoxine can be used in acidic solution and is not blocked by metal ions such as copper(II), cobalt(II), and nickel(I1). 1-(2-Pyridylazo)3-naphthol (PAN) indicator is similar to Naphthyl Azoxine in its application, but in aqueous solution a t room temperature the color change of PAN at the end point of a titration is much slower than with Naphthyl Azosine. Guerrin, Sheldon, and Reilley (8) introduced the indicator, 7-(4-sulfo-lnaphthylazo) - 8 - hydroxyquinoline - 5sulfonic acid, which they called SNAZOXS. This indicator differs from Kaphthyl Azoxine only by a sulfonic acid group in the naphthalene part of the molecule. SNAZOXS retains all of the advantages of Naphthyl Azoxine, but can be used as a metal ion indicator in both acidic and basic solution. Naphthyl Azosine is a satisfactory indicator only in solutions more acidic than about p H 7. When the paper by Guerrin, Sheldon, and Reilley appeared, we had synthesized both the 4sulfo-1-naphthylazo and 6sulfo-2-naphthylazo derivatives of 7-aryl-8-hydroxyquinoline-5-sulfonic acid. However, we had chosen 7 - ( 6 sulfo - 2 naphthylazo) - 8 - hydroxy-
-
quinoline-5-sulfonic acid, which we named Naphthyl Azoxine S or NAS, for development as a metal ion indicator for titrations with EDTA UT other complexing titrants. Actually NAS and SNAZOXS are very similar in properties and application. It is very probable that the two indicators can be used interchangeably for the analyses reported below. One purpose of this paper is to indicate the metal ions that can be titrated with EDTA using NAS indicator. The successful titration of several elements not previously titrated with SNAZOXS or Naphthyl Azoxine is reported. Another purpose is to show how masking agents can be used in conjunction with NAS indicator to increase the selectivity of EDTA titrations. PREPARATION AND PURIFICATION OF INDICATORS
Prepare the indicators by diazotizing either Camino-1-naphthalenesulfonic acid sodium salt or 6-amino-2-naphthalenesulfonic acid sodium salt and coupling with 8-hydroxy-5-quinolinesulfonic acid. Dissolve 10.16 grams (0.021tl) of the amine in 20 ml. of water with gentle heating in a 100-ml. beaker. Add 20 ml. of concentrated HC1 and stir the pasty mixture while cooling to 0" to 5" C. in an ice bath. Dissolve 2.76 grams (0.02M) of sodium nitrite in 8 ml. of water and cool to 0" to 5" C. Slowly add the sodium nitrite to the amine solution through a funnel with its tip extending below the surface of the amine solution. Continue stirring and test for excess nitrous acid with starchiodide paper. Dissolve 9.00 grams (0.02Al) of finely ground 8-hydrosyquinoline-5-sulfonic acid in 40 ml. of water in a 400-ml. beaker. Cool the solution to 0" to 5" C. Slowly add the diazonium salt alternately with a 1.2N solution of NaOH cooled to 0" to 5" C., keeping the p H between 5 and 8. Stir for 10 to 30 minutes. Add NaCl to precipitate the
dye; collect the dye by suction filtration. To purify the dyes, dissolve in a minimum amount of water a t 70" C. Filter to remove any insoluble residue. Add dioxane a t 70" C. equal to four to five times the volume of water and cool. Filter by suction and dry the dye a t 100" C. overnight in an oven. Determine the molecular weight and the per cent of purity of the dyes by a potentiometric titration with 0.02M tetrabutylammonium hydroxide. Use a sleeve calomel with KC1-saturated methanol and glass electrode system. Standardize the tbutylammonium hydroside against benzoic acid. Dissolve approximately 70 mg. of the dye in 7 ml. of water and 25 ml. of acetone. Titrate potentiometrically with 0.02M tetrabutylammonium hydroxide. REAGENTS AND SOLUTIONS
NAS. Prepare a 0.5% aqueous solution of 7-(6-sulfo-2-naphtl1ylazo)-8-hydroxyquinoline-5-sulfonic acid, disodium salt. Metal Solutions. Prepare 0.05M aqueous solutions of the metal nitrates or perchlorates unless otherwise indicated. 0.05M S C + ~ Dissolve . 0.3455 gram of S C Z Oin~ concentrated HC1 and dilute to 100 ml. 0.05M TiC4. Dissolve 0.05111 Tic14 in concentrated HzSOd and evaporate to fumes of HzS04. Dilute back to volume. 0.05M VO+2. Prepare a 0.05M aaueous solution of vanadvl sulfate. 'Citrate. Prepare a io% aqueous solution of ammonium citrate. Tartrate. Prepare a 10% aqueous solution of ammonium tartrate. 2,CPentanedione. Prepare a 10% alcoholic solution. Fluoride. Prepare a 0.1M aqueous solution of sodium fluoride. 0.05M EDTA. Prepare an aqueous solution from the reagent grade disodium salt of (ethylenedinitri1o)tetraacetic acid. Standardize by titrating zinc nitrate (pure zinc metal as primary standard) a t p H 8 to 10 with Friochrome Black T indicator. VOL. 33, NO. 10, SEPTEMBER 1961
1381
