Identification of Antihistamines in Extracts of Biological Materials Using Thin Layer Chromatography WINSTON W. FlKE and IRVING SUNSHINE Cuyahoga County Coroner’s Office, Cleveland, Ohio
b Thin layer chromatography was applied to the separation and identification of 25 antihistamines. Using Rf values in three chromatographic systems and reactions to two spray reagents, 19 of the 25 drugs can be identified. The other six drugs were separated into three unresolved pairs. The reproducibility of Rf values under various conditions was compared. The method developed was applied to extracts of urine, blood, and liver. The qualitative identification of an antihistamine in biological material can be carried out in 3 hours.
A
reliable method is needed for the detection and identification of antihistamines in samples either of commonly used drugs or of biological fluids obtained from individuals who may have ingested lethal or sublethal doses of these compounds. Cochin and Daly ( 1 ) have previously reported the application of thin layer chromatography to some of these drugs. Using their procedure we were unable to identify satisfactorily a number of drugs. This led to the development of the following procedure for the identification of 25 antihistamines which uses three chromatographic systems and two spray reagents. This study also evaluates some of the parameters related to the reproducibility of Rt values. QUICK
EXPERIMENTAL
Reagents. All chemicals used were reagent grade. Silica gel G was Merck material obtained from Brinkmann Instruments, Inc., Westbury,
N. Y. Chromatographic Systems. Systems used were: I, cyclohexanebenzene-diethylamine (75: 15:10) used with silica gel plates prepared with distilled water; 11, methanol used with silica gel plates prepared with 0.1M sodium hydroxide; 111, methanol used with silica gel plates prepared with 0 . l N potassium bisulfate. Spray Reagents. To prepare Dragendorff’s reagent, solution dissolve 2.2 grams of bismuth subnitrate in a mixture of 25 ml. of glacial acetic acid and 100 ml. of water. Solution U is prepared b y dissolving 50 grams of potassium iodide in 100 ml. of water. To prepare the spray solution, mix 10 ml. each of solutions
.Li a n d B with 20 ml. of glacial acetic acid and 100 ml. of water. Add 1.0 gram of ammonium vanadate to 100 ml. of concentrated sulfuric acid to prepare Mandelin’s reagent; prior to spraying, the mixture should be thoroughly shaken. Drug Standards. Dissolve the commercially available drugs in ethanol to give solutions containing 2 mg./ml. A few drugs require several drops of concentrated hydrochloric acid to ensure complete solution. Apparatus. Standard thin layer chromatography equipment-plates, atomizers, fixed thickness applicator, and development tanks-was obtained from Brinkmann Instruments, Inc. The source of 254-mp ultraviolet light was a Mineralight, Model SL 2637, from Lltraviolet Products, South Pasadena, Calif. Chromatographic Procedure. Five 20- x 20-cm. glass plates were coated with 3 thoroughly stirred mivture of 30 grams of silica gel G a n d 60 ml. of either 0.1M sodium hydroxide, 0 . l X potassium bisulfate, or water. T h e plates were air-dried, then conditioned a t least 16 hours in a room of constant relative humidity and temperature, 52y0 and 22” C., respectively. The developing tanks were stored and used in this same room. L-sing a scriber, columns 1.0 em. wide, parallel to the direction of coating, were drawn on each plate. The two outside columns of each plate were not used. I n each column 2 to 4 pl. of each sample, containing 8 to 10 pg. of drug, were spotted 3.5 cm. from the base of the plates. The plates were then placed in developing tanks to which had been added, a t least 1 hour prior to use, 110 ml. of solvent. The end walls of the tanks were lined with strips of blotting paper freshly saturated with the solvent; the tank lids were sealed m-ith petroleum jelly. Development was allowed to proceed until the solvent front had risen 10 to 12 cm. beyond the original spots. The plates were removed from the tanks and, after marking the solvent front, allowed to air dry. They were then ready for spraying. Standard Rf data for the 25 antihistamines studied were obtained using this procedure for all three system; Chlorcyclizine was included on every plate so the relative R / values could be calculated using it as a standard. For the qualitative identification of an unknou-n antihistamine, a plate was first developed using system 11. ‘C-p to 15 samples and chlorocyclizine were
spotted and developed as described above. The developed plate was then sprayed with Dragendorff’s reagent. Another aliquot of those samples which gave a positive test in system I1 l+as then spotted and developed on a second plate using system I. On this plate up to seven samples were spotted in two series of columns. Chlorcyclizine was spotted in only one column. After development and thorough drying, the two sets of columns were sprayed with Dragendorff’s and Mandelin’s reagents, respectively. During each spraying the other set of columns was covered. The two series of columns sprayed with Dragendorff’s and \landelin’s reagents mere observed and any colored spots noted. Four drugs produce white spots with Mandelin’s reagent which are easily seen against the yellow background. Several drugs give colors immediately, others take up to 60 minutes for full color development depending on the amount present. The plate was then heated a t 85’ C. for 10 to 15 minutes. After being cooled, the plate was observed in daylight and ultraviolet light. If satisfactory identification had not been made using these two plates, another plate was spotted and developed using System 111. The samples were spotted in two series of columns across this plate; those drugs indicated by the results of the first two plates as likely to be present were spotted adjacent to the appropriate sample. -4fter the first development, the silica gel below the starting line of the first series of columns was scraped off and the plate then was developed three additional times with thorough drying between developments. The fully developed plate was then sprayed with Mandelin’s reagent and treated as above. Extraction of Biological Material. I n a glass-stoppered bottle add 10 to 16 volumes of chloroform to one volume of alkalinized urine, blood, or homogenized tissue. Shake gently on a mechanical shaking device for 30 minutes, aspirate the supernatant, and wash the chloroform layer and a n y emulsion twice with a n equal volume of 0.001-11 sodium hydroxide. Extract the washed chloroform with three 20-ml. portions of 0.5.21 sulfuric acid and pool the aqueous acid extracts. Filter the residual chloroform, which contains the neutral fraction, through a dry filter paper and evaporate the filtrate to dryness. Make the pooled aqueous acid extracts strongly alkaline VOL. 37, NO. 1 , JANUARY 1965
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and extract with three 25-ml. portions of chloroform. Filter the pooled chloroform extracts through dry filter paper and evaporate this fraction, which contains the organic bases, to dryness. Take up each residue in 0.5 ml. of ethanol and spot aliquots of both extracth ab described above. RESULTS AND DISCUSSION
The average R, values of 25 antihistamines in three different chromatographic systems are tabulated in Table I. Their R, values relative to chlorcyclizine and their reactions to Mandelin’s reagent are also tabulat’ed in Table
I. Drug Identification. The qualitative procedure described above using three chromatographic systems and two spray reagents will identify all but six of the antihistamines studied. These six drugs consist of three unresolved pairs: buclizine-meclizine, phenirainine-chlorpheniramine, and diphenhydramine - bromodiphenhydramine. However, each of the three pairs is separated from the remaining drugs by its R , values in the three systems. Dragendorff Is reagent was used as a general spray reagent because it gives spots with 3 pg. or more of all 25 antihistamines. Mandelin’s reagent is somewhat less sensitive and requircs 8 to 10 pg. for satisfactory color development. When an R f difference of 0.04 unit and a color distinction with
Table
Drug Antazoline Bromodiphenhydramine Buclizine Carbinoxamine Chlorcyclizine Chlorothen Chlorpheniramine Clemizole Covatin Cyclizine Diphenhydramine Dip henylpyraline Hydroxyzine LIecliziiie Methapheniline Methapyrilene Phenindamine Pheniramine Pheny ltoloxamine Pyrilamine Pyrrobutamine Thenyldiamine Thonzylamine Tripelennamine Tripolidine Plate coated using: Solvent:
I.
Mandelin’s reagent both existed for a pair of drugs--e.g. methapyriline and chlorothen in system I-a mixture of the two drugs was resolved into two distinguishable spots. .A single development in system I11 sufficed to separate some pairs unresolved by the first two systems. Scraping off the silica below the starting line of one set of columns after the first development prevented further development of this set while the multiple development was carried out. The multiple development was mandatory for satisfactory resolution of certain pairs. For example three pairs of drugs which were not separated in one development in system 111 have the folloaing R / values after four developments: chlorothen-pyrilamine, 0.50 and 0.91 0.36; methapheniline-cyclizine, and 0.85; and methapyrilene-tripelennamine, 0.45 and 0.39. In system I l l all nine antihistamines containing a pyridyl group have Rl values less than 0.18; none of the drugs without a pyridyl ring have an R, value below 0.29. Relatively low R j values for antihistamines containing a pyridine ring on both paper and silica gel using acidic systems have been reported by previous workers ( I , 2 ) . This group of drugs fluoresces on the potassium bisulfate plate under the ultraviolet lamp. This latter observation has been used to follow the multiple development of the pyridine-containing drugs in
Thin Layer Chromatographic Data for 25 Antihistamines
System 1Inmc R, Rel. R, 0.11 0.26 0.37 0.84 0.72 1 65 0 21 0 47 0 44 1 00 0 37 0 84 0 19 0 44 0 67 1 53 0 54 1 26 0 46 1 04 0 37 0 85 0 25 0 58 0 59 1 35 0 71 1 62 0 46 1 06 0 36 0 82 0 53 1 21 0 18 0 42 0 51 1 16 0 33 0 76 0 39 0 88 0 32 0 74 0 38 0 86 0 35 0 79 0 40 0 91 0 1M NaOH Methanol
System I I I a R; Rel. tzj 0.61 1.42 0.48 1.05 0.75 1.77 0 05 0 12 0 43 1 00 0 15 0 35 0 08 0 18 0 47 1 09 0 52 1 19 0 41 0 95 0 45 1 04 0 44 1 02 0 56 1 30 0 74 1 72 0 44 1 02 0 14 0 33 0 41 0 95 0 06 0 15 0 42 0 98 0 12 0 29 0 59 I 37 0 12 0 28 0 29 0 68 0 12 0 28 0 17 0 40 0 . lAWKHSOi Methanol
0.08 0.16 0.50 1.02 0.73 1.49 0.29 0.57 0.49 1.00 0 43 0 89 0 38 0 78 0 33 0 96 0 58 1 17 0 55 1 11 0 52 1 06 0 42 0 85 0 08 0 17 0 69 1 43 0 55 1 14 0 47 0 96 0 55 1 12 0 40 0 82 0 46 0 94 0 42 0 84 0 62 1 26 0 47 0 96 0 41 0 83 0 50 1 02 0 41 0 84 Water Cyclohexanebenzenediethylamine (75 15: 10 v /v ) a R, and relative R , values are averages from five different plates. b Similar R, values were obtained using silica gel suspended in 0.1M NaOH. c In several cases the free bases were spotted; identical R; values were obtained
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ANALYTICAL CHEMISTRY
system 111. In some cases less than four developments are necessary to resolve members of a mixture. A number of reports in the literature (1, 4 ) indicate that when a compound has been extracted from a natural product its R j value may be significantly different from that obtained with the pure material. T o check this point samples of urine, liver, and blood were obtained from individuals who were known not to have used antihistamines prior to taking the biological sample. These samples were extracted as previously described. Each of the basic extracts was mixed with a sample of each of the 25 antihistamines and the resulting mixture was spotted and developed as described. R; values of all the drugs in these extracts fell within the range of values noted for the pure compounds. Significantly no fluorescence was observed nor were spots noted with either Dragendorff’s or Mandelin’s reagents in the extracts of normal urines. Each of the antihistamines has been extracted from urine and correctly identified by the procedure outlined here within the limitations noted. Qualitative identification of an antihistamine in biological samples can be carried out in 3 hours. The neutral extract contains bucliaine, covatin, and diphenhydramine, when their concentration in urine was 4 pg./ml. When concentrations of 12 pg./ml. mere used they were found in both the basic and neutral extracts.
Cold Crimson Yellow
Pink
Mandelin’s After hetting at 85 Crimson Brown Red brown Grey brown Purple
uv
fluorescence
Light blue Light blue
White Yellow Yellow Red orange Red White Orange Pink Brown White White
Blue Yellow Yellow Yellow Brown Brown Purple Green Light Brown Orange Purple Yellow Purple Purple Brown
Yellow Light blue
Bright blue
Thus in analyzing samples, both extracts should be spotted. I n the basic extract of urine from two individuals who had each ingested 100 mg. of diphenhydramine and tripelennamine, respectively, a second Dragendorff positive compound, probably a metabolite, was found in addit'ion to the drug. Technique and Reproducibility of R,. Silica gel coated plates have previously been prepared with solutions of sodium hydroxide, oxalic :tcid, and buffers (3). I n this study potassium bisulfate was preferable to the use of other acidic materials either in the adsorbent or in the solvent. The potassium bisulfate coatings gave almost no tailing of spots nor significant flaking of the coating off the plates. The other acidic systems tried--e.g., either acetic or hydrochloric acid in the solvents, or oxalic or phosphoric acid in the coatings-produced either tailing of the spots, a coating which flaked off the 1)lates readily, or both. The developing t,anks cont'aining methanol were used satisfactorily for 25 plates; replenishment of t,he methanol lost during the development was all that was required to attain reproducible R , values. Development of additional plates in the same methanol was not attempted but would appear to be
feasible. Solvent I had to be changed after every five plates since Rj 1-alues were lower on subsequent plates. This change in R, is probably caused by selective evaporation of diethylamine. Because the R j values of many of the antihistamines are similar in each system, the use of an Rj value as a n aid in identification was dependent on its reproducibility. The most crucial factor in attaining reproducibility was securing a constant degree of saturation in the developing tanks prior to inserting the plates. The described procedure has proved satisfactory in this respect. All R j values reported in Table I were obtained on plates conditioned and developed as described in the procedure. Plates oven-dried a t 120" C. for 2 hours and stored over fresh calcium chloride until used have given the same R j values. Plates stored in the laboratory over a relative humidity range of 40 to 55y0 and developed a t temperatures from 22" to 28" C. have not produced Rr values outside of the range found using the more rigid conditions. Plates conditioned a t room temperature and 80% relative humidity did give R j values in both systems I and I1 averaging 0.05 to 0.08 unit higher than those reported in Table I. Ordinary laboratory conditions would thus appear
to suffice for the attainment of reproducible R, values. The ranges of R/ values obtained from the five plates used for the data in Table I were compared with those from five plates developed in unsaturated tanks. I n system I, 22 of the 25 drugs showed ranges of less than 0.07 Rj unit under standard conditions whereas when development was in unsaturated tanks only nine drugs had ranges of less than 0.07 unit. Similar patterns were noted for systems I1 and 111. Relative Rj values partially compensate for this loss of precision but the range distribution indicates that saturated tanks are also desirable when identification is made using relative R j values. LITERATURE CITED
(1) Cochin, J., Daly, J. W., J . Pharm.
Expl. Therap. 139, 160 (1963). (2) Goldbaum, L. K., Kazyak, L., ANAL. CHEM.28, 1289 (1956). ( 3 ) Stahl, E., Arch. Pharm. 292, 411 (1959). ( 4 ) Walker, K. C., Beroza, AI., J . Assoc. O$c. Ogr. Chemists 46, 250 (1963).
RECEIVEDfor review May 3, 1964. Accepted November 19, 1964. Study supported by funds from grant R. G. 9863 of the National Institutes of Health, U. S. Public Health Service.
Experimentu I Reactor Therm a I - Ne utro n Activation Analysis Sensitivities HERBERT P. YULE John Jay Hopkins laboratory for Pure and Applied Science, General Atomic DivisionlGeneral Dynamics Corp.
P . 0. Box 608, San Diego, Calif.
927 72
b Gamma-ray photopeak yields have been experimentally determined for 1 18 reactor thermal-neutron products of all elements from oxygen through lead (except Ne, Kr, and Xe). limits of detection (sensitivities) have been estimated from these data; the median sensitivity is about p.p.m. for a 10-gram sample. The use of reactor thermal neutrons for these measurements has several advantages. Because the yield data have been experimentally determined, they are not subject to inaccuracies found in theoretical estimates caused by poorly known nuclear parameters. The high thermal-neutron flux available in the reactor has made possible the observation of a number of additional activation products not included in previously published lists containing experimentally determined thermalneutron product yields. These additional products offer alternative means of analysis which may b e useful in circumventing matrix activation problems. The present list contains data
on essentially all important thermalneutron products for those elements amenable to neutron activation analysis utilizing gamma-ray counting.
F
of the power of the activation analysis technique is dependent (among other aspects) on a knowledge of the limits of detection for the chemical elements. For most of the elements determinable by neutron activation, the most sensitive method is detection of the radioactivities generated by thermal neutron capture. This paper reports, for many elements, limits of detection by gamma ray counting which have been experimentally determined with reactor thermal neutrons. I n principle, these limits of detection are easily calculable, and a number of tabulations of such sensitivities have been published (3, 7 , 8,10, 11, 13, 1 5 ) . Such calculations depend on experimental parameters of individual nuclides, ULL REALIZATION
and because these pai.ameters are not always accurately known, calculated limits of detection may be in error by more than an order of magnitude as shown by the data in references ( 7 , 15). Experimentally determined limits of detection have also been published ( I , 7 , 9 , f 5 ) ; most of these sensitivities have been determined using thermalized 14-m.e.v. neutrons with fluxes of about lo8 neutrons/sq. cm.-second. I n the present work, the neutron source was the General Atomic Triga Mark I reactor, and the thermal neutron flux was 4.3 X lo1*neutrons/sq. cm.-second. Several advantages are obtained with this much more intense flux: limits of detection are much lower (more sensitive) ; many additional activation products have been observed, including long-lived products not readily available with irradiations with 14-m.e.v. neutron generators, and these additional products offer alternative means of analysis in the event that interferences from activation of the matrix prevent analysis with shorter-lived activities. VOL. 37, NO. 1, JANUARY 1965
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