Qualitative Identification of Local Anesthetics in Their Dosage Forms HENRY M. KOEHLER and EDWARD G. FELDMANNl Division of Chemistry, American Dental Association, Chicago 11 , 111.
b Methods are described for the identification of local anesthetics in dosage forms, including tablets, ointments, jellies, and solutions. Ultraviolet analysis may b e used for quantitative determinations in selected cases. The preparation of solid picrate and tetraphenylborate derivatives is described and the crystalline materials are characterized. Separation of combinations of anesthetics by descending paper chromatography provides a relatively quick way to determine the active ingredients of these mixtures. Vasoconstrictors, salts, and excipients found in dosage forms do not interfere with the methods as described. By combining several of the techniques, the local anesthetic or anesthetics involved may b e unequivocally identified.
T
HE qualitative identification of local anesthetics in various dosage forms, such as jellies, ointments, topical solutions, and injectable solutions is of interest to governmental and university laboratories as well as clinicians, hospitals, and pharmaceutical manufacturers. Identification of these compounds has proved difficult when physical properties alone or classical chemical procedures are considered, because of the close chemical relationships n-hich many of these drugs display. This study has been generally limited to those local anesthetic agents n-hich are of primary interest in dentistry. While some of the constants detailed herein have been previously listed in scattered literature reports, this paper represents a first attempt to compile and complete these data and to develop experimental procedures which provide a satisfactory identification scheme for all such agents in any dosage form encountered. A large group of local anesthetics have been characterized using three clearly defined techniques. Formation of derivatives, the usual approach, presents several problems. Foremost of these is the low concentration in which
1 Present address, American Pharmaceutical Sssociation, 2215 Constitution Ave., N.W., Washington 7, D. C.
28
ANALYTICAL CHEMISTRY
the drugs are usually employed. Most dosage forms of local anesthetics contain about 2% of the active ingredient; few contain 4y0 or more. The most frequently used form is the cartridge which contains the drugs in aqueous solution for injection, often accompanied by a vasoconstrictor in very low concentration. Therefore, derivatives were chosen which could be prepared directly from dilute aqueous solutions. Preparation of picrates of some local anesthetics has been described by Steiger and Kuhni, among others (22). Picrate formation is well suited for the detection of local anesthetic compounds, because most of these drugs are substituted amines. This method of identificiation has produced characteristic derivatives for each of the drugs tested in any of their dosage forms, with a minimum of difficulty in extraction from ointments and jellies and n i t h no interference from any of the vasoconstrictors encountered in injectable solutions. Recently, sodium tetraphenylborate has been used for the formation of derivatives of amines by Crane (S), Gautier and coworkers ( I 5 ) , Schultz and XIayer ( d l ) , and Fischer and Karan-ia ( I d ) , who have reported the preparation of a few local anesthetic tetraphenylborate derivatives. Flaschka, Holasek, and Amin (14) have applied the reaction of mercuric chloride with the tetraphenylborate ion to the quantitative determination of several drugs, among them procaine. More recently, Chatten, Pernarowski, and Levi (2) have extended the use of the tetraphenylborate derivatives to the quantitative determination of certain official (USP, K.F.) local anesthetics and have reported extensive ultraviolet and infrared spectral data for these drugs and their derivatives. As the tetraphenylborate derivatives are also easily prepared from dilute aqueous solutions, they are liken ise well suited for characterization of these agents. With the increasing use of local anesthetics in dental practice, a number of local anesthetic mixtures have appeared, such as combinations of butethamine and procaine hydrochlorides (Graham Chemical Gorp.) or tetracaine
and procaine hydrochlorides (CookWaite Laboratories). Before the components of these mixtures can be identified, the active ingredients must be separated. I n this laboratory such separation has been attempted by paper electrophoresis, column chromatography with various ion exchange resins, and buffered paper chromatography according to the method of Schmall, Wollish, and Shafer (19). These methods met with only limited success, probabIy because the components of the mixtures, such as procaine hydrochloride and butethamine hydrochloride, are chemically nearly identical and have almost identical p K values. Clear-cut separations of various mixtures were, hon ever, achieved by descending paper chromatography, with solvent systems as described by Wagner (24) and by Castagnola and coworkers (1). Paper chromatography was applied to dosage forms containing only one anesthetic compound as well as to local anesthetic mixtures. One of the most useful and most widely applied methods for identifying local anesthetic compounds employs ultraviolet spectrophotometry. -41though highly suited for quantitative as well as qualitative analysis of selected drugs of this class, the structural similarities of many compounds of the group are such that the spectra of the drugs are identical or nearly so. This analytical method is further complicated in the case of samples containing mixtures of anesthetics. However, in selected cases quantitative analysis by ultraviolet procedures can follow unambiguous identification by the methods outlined in the experimental section. K i t h these methods, a number of physical constants can be obtained which unequivocally identify the local anesthetic. An analyst seeking to identify an unknom-n or questioned dosage form need only determine several of the physical constants to identify the drug with certainty. By judicious application of a number of the procedures employed and the use of known reference standards n hen tentative identification has been made, all the other possibilities can be eliminated. -4s new local anesthetics are developed, suitable reference parameters can be
determined, and the list of constants which serve to identify the drugs will be lengthened. I n the event that subsequent quantitative analysis is required, spectrophotometric analysis, or especially in the case of mixtures, more specific methods of analysis such as those previously reported (6, IO, l l ) ,can be used. EXPERIMENTAL
Local Anesthetics. T h e generic, trade, and chemical names of t h e local anesthetics studied are listed in Table I. T h e same numbers apply in subsequent tables. Extraction of Dosage Forms. Except for aqueous solutions, dissolve or disperse a n accurately weighed 2- t o 4-gram sample in 10 ml. of 1% hydrochloric acid. Filter the solution or suspension through qualitative grade filter paper a n d collect t h e filtrate in a small separatory funnel. Introduce exactly 10 ml. of reagent grade chloroform and about 3 ml. of 10% ammonium hydroxide solution; shake the funnel vigorously for 1 minute and allow the phases t o separate. Remove the lower (chloroform) layer and evaporate a 5 d . aliquot of this solution to near dryness. Then add about 5 ml. of 1% hydrochloric acid solution and heat until the odor of chloroform is absent. Quantitatively transfer the aqueous acid solution t o a IO-ml. volumetric flask and fill to the mark with water. Portions of this solution may be used to prepare picrate and tetraphenylborate derivatives as described below, for paper chromatographic analysis, and, upon further appropriate dilution, a n aliquot may be employed for ultraviolet analysis.
local anesthetics can be used directly for t h e formation of crystalline picrate or tetraphenylborate derivatives. Where t h e solution contains more than one local anesthetic, identification via formation of solid derivatives is not possible unless further analytical procedures are used.
PICRATES.Add 1 ml. of water to 2 ml. of the aqueous solution of the local anesthetic or t o 2 ml. of the solution resulting from extraction of other dosage forms, and heat to boiling. Introduce 1 ml. of a saturated solution of picric acid in 2001, ethyl alcohol; allow the solution t o cool slowly. If crystallization does not occur, chill the liquid further in a n ice bath. (In the case of metabutoxycaine picrate, prolonged standing in a refrigerator may be necessary for the formation of a crystalline solid.) Filter the crystalline
KO. 1
2 3 4 5 6
7 8
9
10 11
12 13 14
15
16
Formation of Solid Derivatives. I n general, injectable solutions of
Table I. Local Anesthetics Trade Same Chemical Kame Butyn 3’-Dibutylaminopropyl 4-aminobenzoate Monocaine 2-Isobutylaminoethyl 4-aminobenzoate Versacaine 2’-Diethylaminoethyl Zchloro-4-aminobenzoate Dyclonine Dyclone 4’-Butoxy-3-N-piperidinopropiophenone Ethyl aminobenzoate Benzocaine Ethyl 4-aminobenzoate Yylocaine a-Diethylamino-2,6-acetoxylidide Lidocaine Oracaine 2-Methyl-2-propylaminopropyl benzoate Meprylcaine Metabutethamine Unacaine 2’-Isobutylaminoethyl 3-aminobenzoate 3fetabutoxycaine Primacaine 2‘-Diethylaminoethyl 2-butoxy-3-aminobenzoate Amylsine 2’-hmylaminoethyl 4aminobenzoate Kaepaine Parethoxycaine Intracaine 2’-Diethylaminoethyl 4-ethoxybenzoate Piperocaine ilfetycaine 3-(2’-Pvlethylpiperidino)propyl benzoate Pl’ovocaine 2’-Diethylaminoethy14-aminobenzoate Procaine Proparacaine Ophthaine 2’-Diethylaminoethyl 3-amino-4-propoxybenzoate Propoxycaine Ravocaine 2’-Diethylaminoethyl 2-propoxy-4aminobenzoate Pontocaine 2’-Dimethylaminoethyl 4-butylaminobenTetracaine zoate Generic Name Butacaine Butethamine Chloroprocaine
Table II.
Local Anesthetic Derivatives
Picrates Melting Point, ’ C. Found Lit. value 92.5-94 167-1 7 1
Calcd. 13.07 15.04
Found 12.89 14.21
14.01 10.99 14.46 15.11 12.06 15.04 13.02 14.60 10.88 11.42
13.82 10.37 14.72 14.55 11.72
11 12
147-151 116-118 128-130.5 230-234 200-202 147-149 58.5-61 101-104 112-1 14 123-125
12.65 14.72 10.73 11.72
13
135-136.5
15.04
15.08
XO.
1
2
3 4 5 6
7
8
9 10
14
147-152(4) 131 ( 1 6 ) 231-232 ( 2 3 ) 196-200 (’71 ’ 147-149 (6j
131.5-133.5 (18)
precipitate with suction and dry the solid material in a vacuum desiccator over magnesium perchlorate. If necessary, recrystallize the picrates from aqueous ethyl alcohol in the conventional manner. The melting points of crystalline picrates, all of n-hich were found by nitrogen analysis to be monopicrates, are tabulated in Table 11. They were determined on a Fisher-Johns melting point apparatus n-hich had been calibrated with USP melting point reference standards. TETRAPHEKYLBORATES. TO 4 ml. Of the aqueous acid solution prepared by extraction or to 4 ml. of the injectable solution, add 1 to 2 ml. of 6 N hydrochloric acid. I n certain instances i t may be necessary to raise the pH of the local anesthetic solution by the addition of portions of 114’ sodium
%N
Tetraphenylboratee Melting Point, C. %r\J Found Lit. value Calcd. 97-100 4.47 112-115” 91.5-108 ( 2 ) 5.04 (90-98)b 143-145 4.74 115-1 17.5 2.29 O
74 -~ 28 __
Found 4.37 4.60 4.66 2.12
c
154-1 58 141-143 130-132 106-109 72-76 131-131 127-130“ (90-102)b 168-170d (:120-140)b
143-147 122-124 13.33 12.86 127-131 15 130-132 130-132 (9) 13.35 12.93 135- 137 16 118-120 14.18 13.86 Recrystallized from hot methanol and water. Melting point of unrecrystallized derivative. c Ethyl aminobenzoate does not form a solid precipitate with sodium tetraphenylborate. d Recrystallized from 1 to 1 acetone-dioxane and water.
113-116 ( 2 )
5.06 2.52 5.04 4.45 4.91 2.31 2.43
4.27 4.65 2 42 2.33
147-151(12)
5.04
4.68
4.54 4.56
4.63 4 05 4 87
106-109 (8) 110.5-112.5 ( 2 )
141-143 ( 2 ) 119-120 ( 2 )
4.81
1.71 2 11 1 77
5
VOL. 32, NO. 1, JANUARY 1960
29
hydroxide. However, the solution must, in all cases, be acid. Add, with stirring] 6% aqueous sodium tetraphenylborate solution until no evidence of further precipitate formation, as shown by increased cloudiness, occurs. Allow the white or cream-colored precipitate to settle or agglutinate and collect on a sintered glass funnel of medium porosity, under vacuum. Dry the collected solid overnight in a vacuum desiccator over magnesium perchlorate.
If the precipitate has retained moisture after this treatment, drying may be continued under vacuum a t 60" C. for 8 to 24 hours. Crane (3) reports that the tetraphenylborates need not be recrystallized; where doubt of purity existed, some of these derivatives have been recrystallized from hot acetone-dioxane in 1 to 1 mixtures by the addition of O . l ~ c acetic acid until the first sign of cloudiness. Addition of excess aqueous acetic acid will result in the formation of oils. Recrystallization in good yield c r n also be achieved by taking up the crude tetraphenylborates in minimum volumes of hot methanol. Addition of small amounts of water followed by careful cooling will produce a good yield of pure derivative. Melting points of solid tetraphenylborates, all of which were found by nitrogen analysis to be monotetraphenylborates, are similarly tabulated in Table 11. Chromatographic Analysis. Two solvent systems are used for clear-cut separation and identification of the local anesthetic drugs. HCl SOLVENT SYSTEM. Combine 30 ml. of redistilled analytical grade butyl alcohol, 5 ml. of 37.5% hydrochloric acid, and 37.5 ml. of water in aseparatory funnel. Shake for 1 minute and allow the layers to separate; then withdraw each layer individually and save both layers. The R j values which result from chromatograms prepared n ith this solvent system are strongly dependent on the hydrochloric acid concentration of the organic phase. The concentration of hydrochloric acid in the organic layer must therefore be carefully established and must be kept constant in subsequent experiments for purposes of comparison. Place 1.5 ml. of the aqueous layer in a Petri dish on the bottom of a chromatography jar. Assemble the chromatography apparatus, fitted with paper strips which have been spotted with 0.01 ml. of the anesthetic solution, and seal the jar. Allow the paper strips to equilibrate with vapors of the aqueous layer. After at least a 3-hour equilibration period, add 8 ml. of the organic layer to the trough a t the top of the jar and allow solvent migration to 30
ANALYTICAL CHEMISTRY
Table 111.
Chromatographic Results
Rt HCl system 0.82 0.36 0.36 0.82 1.0 0.72 0.74 0.59 0.68 0.67 0.72
NO.
1
2 3 4 5 6
7 8 9 10 11
12 13 14 15
HOAc system 0.86 0.77 0.78 0.87 0.64 0.71
0.87 0.79 0.83 0.85 0.86
16
Table IV.
Ultraviolet Absorption Characteristics
hbsorw tivity, I%, 1 Cm. 499 631 506 492 810 12.5 422
Wave Length, M p Alas. hIin. 288 241 1 2 290 241 3 29 1 240 4 282 238 5b 283 24 1 263 275 6b 7 232 257 310 273 8 90 9 67 313 272 lob 625 287 238 2'27 560 260 11 12 388 230 208 13 660 290 240 14 735 231 252 270 268 290 147 310 15 462 303 288 278 252 418 251 16 770 311 a Aqueous solutions prepared from mineral acid salts of respective anesthetics. Free base made up in sufficient hydrochloric acid t o dissolve and diluted t o volume with mater. X0.a
~~
to 135 ml. with water. Prepare the spray solution fresh daily. T o prepare another suitable spray solution, dissolve 1.5 grams of potassium permanganate in 1 ml. of 614' hydrochloric acid and dilute to 100 ml. with water. HOAc SOLVEKT STBTEY. Combine 40 ml. of butyl alcohol, 10 ml. of glacial acetic acid, and 50 ml. of n-ater in a sepnrstory funnel. Shake for 1 minute and allow the layers to separate; save both 1a):ers. Prepare chromatograms in the same manner as with the hydrochloric acid system, but allow the equilibration to proceed overnight a,nd solvent migration to continue for a masimum of 8 hours. Use strips such as Whatman No. 1 paper, 1 inch wide and long enough to permit the solvent front to move at least 16 inches for both solvent. systems. Chromatographic constants pertaining to both solvent systems are shown in Table 111. Routinely, 0.01 ml. of an aqueous 1 to 2% solution of the local anesthetic hydrochloride is used. Eon-ever, highcLr concentrations up t.o 4yc have be:,n successfully chromatographed without' significant streak formation resulting. The use of a n ultraviolet lamp for detection of the spots requires at least 0.01 ml. of a n 0.2% solution; spraying with potassium permanganate 1%-ill allon. detection of certain anesthetics in lower concentrations. The concentrations of the solutions prepared for derivative formation as outlined a b o w are well within these limits. Ultraviolet Analysis. Preparation of the solution for ultraiiolet analysis depends on the material undergoing analysis. I n the case of an aqueous solution, simple dilution will usually suffice; for other dosage forms, estraction is used as described previously. Table Is' lists the v a v e lengths of maxinium and minimum absorption of t h e local anesthetics, with their approximate absorpt'ivities a t the masims. DISCUSSION
proceed for 8 to 18 hours. The solution volumes suggested here and for the other system are for chromatography jars 6 inches in diameter and 18 inches high. The volumes should be adjusted for jars of other sizes. Air-dry the strips for a t least 4 hours and then locate the spots by any of several methods. View the strips in a dark room with a n ultraviolet lamp for quickest identification of those compounds, which strongly absorb in the ultraviolet range. Alternatively spray the strips Iyith a modified Dragendorff reagent. Prepare this reagent as follows: Dissolve 7 grams of potassium iodide and 1.6 grams of bismuth subnitrate in a solution of 20 ml. of water and 1 ml. of concentrated hydrochloric acid. Cool the solution, add 4.5 grams of iodine, and dilute the solution to twice the original volume with water. Keep in a glass container. T o use as a spray, dilute 2 ml. of this stock solution and 2 ml. of concentrated hydrochloric acid
-1 revieTv of the literature s h o w various values for the melting points of a number of crystalline local anesthetic picrates (4; 6, 7, 9, 22, 2 3 ) . Steiger and Kuhrii (22) report, with values for several nionopicrates, the formation of a procaine dipicrate, melting point 183" to 190' C. In this stud!- only nionopicrates \\-ere prepared. Elemental analysis of the derivatives listed in Table I1 indicates purity of the samples and correct assignnient of molecular ratios of format ion, The formation of n.ater-insoluble tetraphenylborate derivatives of local anesthetics was first reported b y Fischer and Iiarawia ( I @ , n-ho applied the reagent to procaine. Schultz and Mayer (21) reported that the reagent would give a quantitative precipitate with procaine, benzocaine, and tetracaine. Procaine and t'etracaine, among
other drugs, were prepared as tetraphenylborates b y Gautier and conorkers (15). Flaschka (14) devised a nonaqueous titration procedure for the quantitative determination of tetraphenylborate derivatives. In this laboratory the method was applied to solid tetraphenylborate derivatives of metabutethamine, metabutoxycaine, and meprylcaine, n itli quantitative results. Chatten ( 2 ) , employing a somem hat similar procedure for the preparation of tetraphenylborate derivatives, reported quantitative precipitation of selected anesthetic derivatives. While complete recoveries n ere not generally achieved 17 ith the prwent procedure, high yields of derivatives were regularly obtained, which had melting points substantially higher, in most cases, than those reported by Chatten. Differences in the method of preparation of the solid tetraphenylborate derivatires may lead to polymorphism, which may explain the differences in the melting points obtained by Chatten et al. and those reported here. Benzocaine n a s the only drug of this series which could not be identified in this study by a tetraphenylborate derivative. Every attempt to prepare the derivative resulted in the formation of a gum n-hich could not be crystallized, regardless of the p H (1 to 6 range) of the aqueous benzocaine solution. Benzocaine, because of its neak basic properties, might be dissolved in some other solvent to enhance solid precipitate formation. The techniques of paper chroniatography have been applied to local anesthetics by Wagner (Pi), n h o used the ascending technique, and by Reichelt ( I t ? ) , !Tho used descending solvent flon. Fischer and Otterbeck ( I S ) have identified many local anesthetics with their test-tube chromatography method, which requires relatively short time but also necessitates the use of four solvent systems for unequivocal itlmtification. The quantitative aspects of chromatographj have also been studied (PO, 2 4 ) . Quantitative elution of the separated coiistituents of mixed anesthetics n ithin reasonable limits was not achieved in this laboratory, although ethyl alcohol, methanol, and n a t e r have all been employed as eluents. Reproduction of R J values as listed in Table IV requires carc,ful duplication of the solvent systems. As Wagner has pointed out, the concentration of hydrochloric acid in the hydrochloric acid s j stem is critical anti must be kept constant if sets of data are to be compared. I n the acetic acid system the concentration of acetic acid, though perhaps not as critical, is important; large deviations from the system lead to
R , values that are different from those in the table. Other solvent systems have been used \$ith some success (13). Kagner and Zinimer (25) discuss ionophoretic separation of local anesthetics from their degradation products, especially p-aminobenzoic acid. They also describe a more complicated system of buffered paper chromatography. The ultraviolet absorption spectra of most compounds testcd show distinct maxima and minima; several of the drugs have more than one peak. K i t h suitable standard solutions this fact can be utilized for determination of concentration in dosage forms containing only one local anesthetic, or, in special cases, the absorption data can be used in the determination of each of t n o local anesthetics (11). I n some instances the spectra depend on the p H of the solvent solution. Ultraviolet spectra of the local anesthetic bases in various solvents usually differ considerably from those of the local anesthetic mineral acid salts in aqueous acid solution. Preparation of the solution for ultraviolet analysis depends on the material undergoing analysis. The presence of vasoconstrictor agents, preservatives, and salts in the original local anesthetic solution nil1 not interfere with the analysis because of the high dilution required. For example, procaine hydrochloride is often marketed as a solution containing 2y0 procaine hydrochloride and epinephrine (adrenaline), 1 to 50,000. For ultraviolet analysis this solution must be diluted to 1 to 2000, n hich reduces the epinephrine level to 1 to lo&,a concentration nhich does not interfere Kith the ultraviolet analysis of procaine hydrochloride. The ultraviolet spectra of procaine and propoxycaine have been studied intensively (11). Kalow (17) has described the absorption spectra of procaine, butacaine, tetracaine, and piperocaine. The wive lengths of maximum absorption which he reports agree closely Fi-ith those listed in Table ITT. Another set of absorption data recently reported ( 2 ) presents slightly different values. This may be attributed, a t least in part, to differences in the solvents employed. ACKNOWLEDGMENT
The authors express their appreciation to J. Roy Doty for his helpful comments and to Helen Jones for invaluable technical assistance. Samples of the local anesthetics employed were generously supplied by their manufacturers: tetracaine hydrochloride and propoxycaine hydrochloride by Cook-Waite Laboratories, Inc.,
subsidiary of Sterling Drug; butethamine hydrochloride, metabutethamine hydrochloride, metabutoxycaine hydrochloride, and naepaine hydrochloride by Sovocol Chemical Manufacturing Co., Inc.; butacaine sulfate, ethyl aminobenzoate, and procaine hydrochloride by Abbott Laboratories; lidocaine by Astra Pharmaceutical Products, Inc.; meprylcaine hydrochloride by Oradent Chemical Co., Inc.; piperocaine hydrochloride by Eli Lilly and Co.; parethoxycaine hydrochloride and proparacaine hydrochloride by E. R. Squibb & Sons, Division of Olin Mathieson Chemical Corp.; dyclonine hydrochloride by Pitman-Noore Co.; and chloroprocaine hydrochloride by Lederle Laboratories Division, American Cyanamid co. LITERATURE CITED
(1) Castagnola, Luigi, Marangoni, Attilio, Massarotti, Aldo, Riva, Aldo, Schwezz. Monatsschr. Zahnheilk. 67, 621-38
(1957). (2) Chatten, L. G., Pernarowski, M., Levi. L.. J . Am. Pharm. Assoc.. Sci. Ed. 48, 276-83 (1959). (3) Crane, F. E., Jr., ANAL. CHEM.28, 1794 (1986). (4) Drug Standards 27, 30-2 (1959). (5) Feldmann, E. G., J . Am. Pharm. Assoc., Sci. Ed. 48, 197-201 (1959). (6) Feldmann, E. G., Koehler, H. >I., Drug Standards 2 5 , 128-30 (1957). ( 7 ) Zbid., pp. 130-2. ( 8 ) Ibid., 26, 147-8 (1958). (9) Ibid., pp. 148-50. (10) Feldmann, E. G., Koehler, H. &I., J . A m . Pharm. Assoc.. Sci. Ed. 48. 54952 (1959). (11) Feldmann, E. G., Mahler, W., Koehler, H. XI.,Ibid.,47, 676-80 (1958). (12) Fischer, R., Karan-ia, >I. S., Mzkrochim. Acta 1953, 366. (13) Fischer, R., Otterbeck, N., Scz. Pharm. 2 6 , 76-8 (1958). (14) Flaschka, H., Holasek, A., Amin, A. XI., Artneimittel-Forsch. 4, 38-40 (1954). (15) Gautier, J. A , , Renault, J., Pellerin, F., Ann. pharm. franG. 14, 377 (1956). (1:) Heilbron, I., Bunbury, H. &I.,eds., Dictionary of Organic Compounds,” 2nd ed., Vol. I, p. 254, Oxford Univ. Press, New York, 1953. (17) Kalorr, \T7., J . Phaimacol. Ezptl. Therap. 104, 122-34 (1952). (18) Reichelt, J., Ceskoslov. farm. 4 , 297-301 (1955). (19) Schmall, >Wollish, I., E. G., Shafer, E. G. E., A S A L . CHEM. 28, 1373-6 (1926). (20) schriftman, H., J . S m . Pharm. ASSOC., Sci. Ed. 48, 111-13 (1959). (21) Schultz, 0. E., Mayer, G., Deut. Apotheker 2.92, 388 (1952). (22) Steiger, K., Kuhni, E., dcta Pharm. Intern. 2 , 1-17 (1951). (23) “Tests and Standards for New and Konofficial Remedies,” p. 148, J. B. Lippincott Co., Philadelphia 1953. (24) Wagner, G., Arch. Pharm. 286, 23241 (1953). (25) Wagner, G., Zimmer, U., Pharm. Acta Helv. 30, 385-407 (1955). RECEIVEDfor review June 17, 1959. Bccepted October 23, 1959. Fourth in a series on “Analysis of Local Anesthetics.” For preceding paper see ( 6 ) .
VOL. 32, NO. 1, JANUARY 1960
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31
