Analysis of Oil-Based Pharmaceuticals1
`EDWARD SMITH, Division of Drug Chemistry, Office of Pharmaceutical Research
`and Testing, Bureau of Drugs, Food and Drug Administration, Washington, DC. 20204
`
`ABSTRACT
`
`Oils of animal, mineral and vegetable origin are
`used in the formulation of pharmaceuticals. In most
`cases the analysis of these pharmaceuticals requires
`the separation of the drug substance from the oil
`components prior to its quantitation,
`the required
`degree of separation depending on the specificity of
`the quantitation method. When the physical proper-
`ties of the drug substance are quite similar to those of
`components of the oil,
`the separation of the drug
`becomes complex. Column partition chromatography
`provides an excellent means of separating steroids
`from the vegetable oil vehicles used in injectables.
`Both reverse phase and direct phase techniques are
`applied to the separation of the steroids from the
`glyceride, sterol and triterpenoid fractions of the oil.
`Illustrations are provided to demonstrate the appli-
`cation of thin layer chromatography, paper chroma-
`tography,
`gas
`chromatography,
`gel permeation
`chromatography and adsorption chromatography in
`the analysis of oil solutions, oil-based dermatological
`preparations and suppositories.
`
`Oils and related substances are used in pharmaceutical
`preparations because of their solvent properties and their
`ability to serve as an effective means of administration of
`the drug for
`a specified activity in the body. Such
`pharmaceuticals include depot-injections of steroids, derma-
`tological preparations and suppositories. In addition, vola-
`tile oils such as peppermint oil are used as flavors and as
`active ingredients of pharmaceuticals. These are beyond the
`scope of this presentation.
`Oils of animal, mineral and vegetable origin and products
`derived from them, e.g., ethyl oleate, glyceryl monostearate
`and hydrogenated glycerides, are used in the formulation of
`pharmaceuticals. The analysis of these pharmaceuticals
`requires,
`in most cases, separation of the drug substance
`from the oil components prior to its quantitation. The
`degree of separation needed will depend on the specificity
`of the quantitation method. When the physical properties
`of the drug substance are similar to those of components of
`the oil, the separation of the drug becomes quite involved.
`The FDA, as a regulatory agency, must devise meaning—
`ful assay procedures suitable for regulatory control. The
`procedures should include the following: (a) separation of
`the drug from any substance which interferes in the
`quantitation step; (b) a suitable specific identification or
`differentiation test, or both; and (c) a suitable test for
`synthesis precursors or degradation products.
`The analysis of steroids in oil solution has been a
`problem for many years. The vehicles commonly used in
`these preparations
`are castor, corn, cottonseed, olive,
`peanut and sesame oils, together with adjuvants such as
`benzyl benzoate and benzyl alcohol. These oils consist, of
`course, essentially of the glycerides of fatty acids, with
`minor amounts of sterols and triterpenoids.
`A biological assay was specified in the USP XIV (1)
`Testosterone Propionate Injection monograph because of
`the lack of a suitable chemical analysis. The assay in USP
`XV-XVII
`(2-4) was a gravimetric measurement of the
`
`semicarbazone derivative, based on the procedure of Madi-
`gan et a1. (5). For samples containing less than 10 mg/ml of
`oil,
`a preliminary partition between 90% alcohol and
`petroleum ether
`is used to separate the testosterone
`propionate from the bulk of the oil prior to conversion to
`the semicarbazone (5). Dry heat sterilization is usually
`employed in the preparation of these oil injections. During
`an investigation of this procedure it was found that heated
`samples yielded higher values due to increased coprecipi-
`tation of unidentified material (6). The melting point of the
`semicarbazone is used as the criterion of identification in
`this monograph;
`this does not differentiate between the
`semicarbazane of
`testosterone propionate and that of
`unesteritied testosterone.
`Quantitative procedures for progesterone and testoster-
`one propionate have been described (7-9) based on colori-
`metric determination of the 2,4-dinitrophenylhydrazone
`prepared from the ketosteroid after preliminary liquid-
`liquid extraction using 90% alcohol and n-heptane. Um-
`berger used an adsorbent magnesium silicate (Florisil) for
`the chromatographic separation of testosterone propionate
`and progesterone from their oil solution prior to colori—
`metric
`determination of
`their
`isonicotinylhydrazones.
`Tappi and coworkers (1 1) also used the adsorbent magne~
`sium silicate to separate steroids from olive oil.
`The application of partition chromatography to the
`separation of drugs from oil dosage forms was investigated
`in our laboratories. Our initial work considered only those
`dosage forms in vegetable oils other than castor oil, because
`the latter, which consists primarily of the glycerides of
`hydroxy fatty acids, has properties quite different from
`those of other vegetable oils.
`Jones and Stitch (12)
`previously reported separation of mixtures of steroids in
`biological material by partition chromatography, using the
`polar organic solvent nitromethane as immobile phase on a
`silicic acid support and 3% CHCl3 in petroleum ether as the
`mobile phase. Wolff
`(13)
`in our
`laboratories isolated
`progesterone from its oil injection with nitromethane as the
`immobile phase, using Celite 545 as support and n-heptane
`as the mobile phase. The glycerides and sterols emerged
`virtually with the solvent front, and the progesterone eluted
`
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`1One of five papers in the symposium “Fats and Oils in
`Cosmetics and Pharmaceuticals,” presented at the AOCS Meeting,
`Atlantic City, October 1971.
`
`FIG. 1. Separation of chlorotrianisene in corn oil. Column 25 x
`300 mm. 7.0 g Celite 545 A.W., 8.0 ml nitromethane; mobile phase
`n-heptane. (a) Sterol and glyceride fractions; (b) chlorotrianisene.
`
`409
`
`InnoPharma Exhibit 1071.0001
`
`

`

`4] 0
`
`JOURNAL OF THE AMERICAN OIL CHEMISTS” SOCIETY
`
`VOL. 49
`
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`FIG. 3. Separation of testosterone cypionate from sesame oil.
`Column 25 x 300 mm.
`(1) 15.0g Celite 545 A.W., 17 m1
`nitromethane; (2) 15.0g Celite 545 A.W., l7 ml nitromethane—90%
`methanol 1:1, mobile phase n-heptane. (a) Sterol and glyceride
`fractions; (b) testosterone cypionate; (c) sesamin.
`
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`FIG. 2. Separation of testosterone propionate from sesame oil.
`Column 25 x 300 mm. 8.0 g Celite 545 A.W., 9.0 ml nitromethane;
`mobile phase n~heptane.
`(a) Sterol
`fractions;
`(b)
`testosterone
`propionate; (c) sesamin.
`
`after a significant interval. He found that the background
`UV absorbance of
`the progesterone fraction from the
`sterols in the oil was reduced to negligible proportions. This
`procedure was modified in our laboratory and applied to
`the assay of chlorotrianisene, a nonsteroidal estrogen, in oil
`capsules (E. Smith, unpublished data); the modified proce~
`dure was adopted as official
`in NF XII (14).
`In these
`separations there is an interval of at least 40 ml between the
`complete removal of the glyceride and sterol fractions of
`the oil and the emergence of the desired substance, as
`shown in Figure
`1. Because of
`lesser differences in
`polarities, this prooedure cannot be adapted directly to the
`determination of testosterone propionate in oil injectables.
`Instead of having the large interval,
`the testosterone
`propionate follows the elution of the sterol fraction so
`closely that sharp separation may not be achieved.
`In
`chromatographing injections at
`lower concentrations of
`testosterone propionate, the large amount of glycerides in
`the sample required by the assay tends to tail and affect the
`separation.
`Testosterone propionate is effectively separated from
`the bulk of the oil in these preparations by reverse phase
`partition chromatography (6). In this system the support,
`which is rendered hydrophobic by silanization, retains the
`nonpolar solvent as the immobile phase. A polar solvent,
`90% alcohol, may be used as
`the mobile phase. The
`glycerides, which constitute the major portion of the oil,
`are retained in the immobile phase, while the eluate
`contains the sterols and triterpenoid fractions of the oil,
`together with the testosterone propionate. Any free testo-
`sterone which may be present
`in the sample would also
`appear inuthis eluate. The final separation of the testosten
`one propionate from the sterols and triterpenoids of the oil,
`as well as free testosterone,
`is achieved with the Celite~
`nitromethane column, using n-heptane as mobile phase.
`Figure 2 illustrates this separation. The sterols are removed
`completely by the first 15 ml of n-heptane. A distinct
`interval of ca. 30 m1 then follows before the appearance of
`testosterone propionate in the eluate. The triterpenoids,
`such as sesamin in sesame oil, are not eluted with the
`volume of n-heptane specified, but are retained on the
`Celite-nitromethane column. These polar compounds would
`emerge only after 400 ml of eluant. Unesterified testoster—
`one is also retained on this column.
`The reverse phase chromatographic separation has been
`incorporated in the USP XVIII (15) assays of androgenic
`steroids in oil injections. The steroid fractions are quanti-
`tated colorimetrically as their isonicotinylhydrazones. Since
`they do not interfere with the quantitation, no separation
`
`of the sterol and triterpenoid fractions nor adjuvants is
`necessary.
`Testosterone cypionate and testosterone enanthate are
`not amenable to separation from oil by the Celite-nitro-
`methane column procedure. From their structure one could
`predict that they would be relatively more nonpolar than
`testosterone propionate and close in polarity to the sterols.
`They are eluted together with the bulk of the oil from a
`Celite~nitromethane column. By changing the stationary
`phase to a mixture of nitromethane and 90% methanol 1:1
`and employing a longer Celite column (15 g), these esters of
`testosterone are completely separated from all fractions of
`the oil (Fig. 3). Because these two esters are administered in
`relatively high doses (50-200 mg/ml), an analytical sample
`will contain only a small amount of oil;
`therefore the
`preliminary reverse phase clean-up is not required.
`The esters of estradiol which are administered as oil
`injections have the l7—hydroxy or the 3-phenolic hydroxy,
`or both, esterified. Only the benzoate and propionate
`phenol esters are marketed,
`the latter being the diester.
`The estradiol esters are relatively more polar than the
`androgenic and progestrogenic esters. The effect on the
`polarity of the compound by the change in the identity of
`the fatty acid moiety of the l7-hydroxy ester is predict-
`able;
`the longer the chain the greater the decrease in
`polarity with resultant increased rate of elution from a
`nitromethane column. The optimum separation of the
`estrogenic esters
`from their oil solutions by partition
`chromatography is illustrated in Figures 47. As with the
`androgen esters, a stationary phase of nitromethane-90%
`methanol is required for the least polar esters (E. Smith,
`unpublished data). Compounds of like polarity, i.e., estra-
`diol Valerate and estradiol isovalerate, and, as previously
`mentioned, testosterone cypionate and testosterone enan-
`thate, will show no difference in their elution behavior.
`The estrogenic steroid esters are administered at much
`lower levels than the androgens, usually at levels of 0.140
`mg/ml, compared to 10-200 mg/ml
`for
`the androgens
`(although one product, estradiol valerate, is available at the
`20 and 40 mg/ml levels). Therefore most analytical meth-
`ods for these incorporate colorimetry, fluorimetry or gas
`chromatography for the determinative step, rather than a
`UV quantitation.
`assay for Estradiol Valerate
`(16)
`The USP XVIII
`Injection does not incorporate a separation step. It applies
`differential UV directly to the sample solution. The
`quantitation depends on the difference in absorbence of the
`phenolate and the free phenol forms of the compound. The
`NF XIII (17) method for Estradiol Cypionate Injection
`utilizes a shakeout procedure with 85% ethanol and hexane
`to separate the steroid from the bulk of the oil prior to
`colorimetric determination of the steroid with modified
`Kober
`reagent.
`In the assay of Estradiol Dipropionate
`
`InnoPharma Exhibit 1071.0002
`
`

`

`JULY, 1972
`
`/////
`
`SMITH: OIL-BASED PHARMACEUTICAL ANALYSIS
`
`4] 1
`
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`
`FIG.4. Separation of estradiol benzoate from sesame oil. Col-
`umn 25 x 300 mm. 8.0g Celite 545 A.W., 9.0 ml nitromethane,
`mobile phase n—heptane.
`(a) Sterol and glyceride fractions; (b)
`estradiol benzoate.
`
`Injection (18), the sample is treated with base to hydrolyze
`the phenolic ester prior to colorimetric measurement. In
`the Estradiol Benzoate Injection monograph (19)
`the
`steroid is separated from the vehicle by adsorption chroma-
`tography on dry Silica Gel G with benzene—ethyl acetate as
`the mobile solvent, prior to hydrolysis. Banes (20) sepa-
`rated diethyl stilbestrol, a synthetic estrogen, from its oil
`solutions
`as
`its phenolate by partitioning it between
`isooctane and 1N NaOH, prior
`to quantitation as
`its
`photochemical isomerization product.
`The NF XIII procedure for Progesterone Injection (21)
`is simply a gravimetric determination of the progesterone
`dinitrophenylhydrazone formed without prior separation
`from the oil. The USP XVIII procedure for Hydroxypro«
`gesterone Caproate Injection (22), and the NF XIII
`monographs for Nandrolone Deconate (23) and Nandrolone
`Phenpropionate (24) Injections are based on the color-
`imetric measurement of the isonicotinylhydrazone, again
`formed without prior separation from the oil.
`Hydroxyprogesterone caproate and estradiol valerate
`injections in which castor oil
`is
`the vehicle are also
`marketed. Castor oil, comprised primarily of the glycerides
`of hydroxy fatty acids, has quite different properties from
`those of other vegetable oils commonly used as the vehicle.
`Consequently it is not amenable to the partition chromato~
`graphic procedures described. Because castor oil is soluble
`in methanol, the isonicotinylhydrazone and differential UV
`procedures in the official compendia can be applied directly
`to solutions of the injections.
`Paper chromatography has been applied to the separa-
`tion of steroids from oil solutions. Roberts and coworkers
`(25) utilized this technique to isolate estradiol valerate
`from castor oil;
`the isolated steroid was determined
`spectrophotofluorimetrically. Hydroxyprogesterone
`cap-
`roate in castor oil and testosterone enanthate in sesame oil
`were colorimetrically quantitated as
`their
`isonicotinyl»
`hydrazones after isolation from their chromatogram (26).
`Paper chromatography has been applied to the separation
`of nandrolone decanoate (27) from its oil solutions prior to
`colorimetric determination as its isonicotinylhydrazone.
`Talmadge and coworkers (28) used paper chromatography
`to separate quingesterone from its decomposition products,
`progesterone and 601- and 6B-hydroxyprogesterone, in its oil
`solutions. In all of these paper chromatographic methods,
`the separate spots were cut out and the steroid was
`extracted for quantitation.
`Bican—Fister (29) and Cavina and Moretti (30) utilized
`
`
`
`50
`
`lOO ml
`
`15C
`
`206
`
`FIG. 5. Separation of estradiol valerate from sesame oil. Column
`25 x 300 mm. 10.0 g Celite 545 A.W. 11.0 ml nitromethane, mobile
`phase n-heptane. (a) Sterol and glyceride fractions;
`(b) estradiol
`valerate.
`
`thin layer chromatography (TLC) to separate steroids from
`their oil solutions. Bican-Fister (29) colorimetrically deter-
`mined progesterone and testosterone propionate after
`extracting the isolated zones from the TLC plate. For
`testosterone propionate, progesterone, l9 norOtestosterone
`propionate and estradiol cypionate at levels of 10 mg/ml or
`greater, Cavina and Moretti (30) subjected the sample to
`continuous ascending chromatography for up to 8 hr. The
`area of the separated steroid is extracted with chloroform
`and then quantitated by UV or colorimetrically as the
`isonicotinylhydrazone. For
`lower
`level preparations of
`estradiol dipropionate and estrone—3-benzoate, the isolated
`spots
`are saponified without prior
`removal
`from the
`adsorbent (31). The steroid is then extracted and deter-
`mined colorimetrically or by gas liquid chromatography
`(GLC)
`as
`its trimethylsilyl ether (31). The same TLC
`procedure was also applied by these workers (32) to two
`monoesters of estradiol, estradiol-3-benzoate and cstradiol»
`l7 B—cypionate, at levels of 2 mg/ml of oil. In a recent paper
`(33) they omitted the thin layer chromatographic separao
`tion for samples at
`levels of 10 mg/ml or greater; they
`separated the steroids by partition between 80% alcohol
`and hexane and applied their GLC procedure directly to the
`extract. In a later paper these workers (34) utilized gradient
`elution on a silicic acid column to separate the steroids
`from their oil solutions. This was followed by a UV or GLC
`quantitation. They observed effects of the ester structure
`change on the order of elution similar to those we observed
`in our partition procedures.
`Talmadge and coworkers (35) quantitated ethinyl estra‘
`diol by GLC after extracting it from its oil solution by first
`partitioning it between heptane and NaOH solution and
`then back extracting it with chloroform from the acidified
`extract.
`
`Penner et a1. (36) recently reported a procedure which
`provides selective adsorption of l7oL-ethynyl steroids on
`silver nitrate-impregtated Florisil to separate it from its oil
`vehicle. After displacement with ethanolic ammonium
`chloride the I7a-ethynyl steroid, quingestanol acetate, was
`determined spectrophotometrically.
`The vehicles employed for the formulation of ointments,
`creams and similar preparations are usually vegetable and
`mineral oils, petrolatum, lanolin and oil~in—water or water-
`in-oil emulsions. The method of analysis of these dosage
`
`InnoPharma Exhibit 1071.0003
`
`

`

`412
`
`JOURNAL OF THE AMERICAN OIL CHEMISTS’ SOCIETY
`
`VOL. 49
`
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`FIG. 6. Separation of estradiol cypionate from sesame oil.
`Column 25 x 300 mm.
`(1) 8.0g Celite 545 A.W., 9.0 ml
`nitromethane; (2) 10.0g Celite 545 A.W., 12.0 ml nitromethane-
`90% methanol 1:1, mobile phase n-heptane. (a) Sterol and glyceride
`fractions; (b) estradiol cypionate.
`
`FIG.7. SeparatiOn of estradiol dipropionate from sesame oil.
`Column 25 x 300 mm.
`(1) 8.0g Celite 545 A.W., 9.0 ml
`nitromethane; (2) 15.0g Celite 545 A.W., 17.0 ml nitromethane—
`90% methanol 1:1, mobile phase n-heptane. (a) Sterol and glyceride
`fractions; (b) estradiol dipropionate.
`
`the vehicle
`is predicated upon the nature of
`forms
`(hydrophilic or hydrophobic) and upon the chemical nature
`of the drug substance. As would be expected, the separa-
`tion of drugs with either basic or acidic characteristics from
`the vehicle is relatively straightforward. Basic drugs can be
`separated from 1yophilic vehicles by partitioning between
`aqueous acid and an immiscible solvent. The drug is
`extracted as its salt into the aqueous acid and the oil base
`into the solvent layer. This mode of separation is employed
`in ,the NF XIII assays for such products as benzocaine
`ointment, dibucaine cream and tetracaine ophthalmic oint-
`ment
`(37). In the case of a hydrophilic ointment
`the
`process is reversed:
`the basic drug is extracted as the free
`base by an immiscible organic solvent while the vehicle
`remains in the aqueous phase. This process is the basis of
`the USP XVIII assay of lidocaine ointment and lidocaine
`jelly (38).
`More complex separation is required for drug combina-
`tions. The AOAC procedure for benzoic and salicylic acid
`ointment (39) utilizes a two column partition chromato-
`graphic system in which ferric chloride-urea and sodium
`bicarbonate solutions are used as the stationary phases. A
`CHC13 solution of
`the ointment
`is passed over
`these
`columns;
`salicylic acid is
`retained as
`its
`ferric-phenol
`complex on the first column and benzoic acid is retained as
`its sodium salt on the second column, while the ointment
`base passes through. The salicylic and benzoic acids are
`recovered from the individual columns after acidification of
`the stationary phases in situ with a solution of acetic acid in
`chloroform.
`Two general analytical schemes are used for the analysis
`of antibiotics
`in oil bases.
`If
`the antibiotic is
`in a
`hydrophilic vehicle it is blended with a buffer or aqueous
`acid with the acid of polysorbate 80. If it is in a 1yophilic
`vehicle a preliminary liquid-liquid extraction step is used
`whereby the ointment or oil vehicle is retained in the
`organic phase and the antibiotic is extracted into the
`appropriate buffer or acidic aqueous phase. The official
`procedures apply a microbiological assay to the extracts
`(40). Van Giessen and Tsuji (41) recently reported a GLC
`method for neomycin in petrolatum-based ointments. The
`ointment base is dissolved in chloroform and the neomycin
`is removed by centrifugation.
`As in the case of oil injectables of neutral compounds,
`separation procedures
`for corticosteroids in ointments,
`creams and related preparations are more complex. As with
`injectables the determinative step may be applied without
`any prior separation of the drug from the vehicle. In the
`
`monographs for Hydrocortisone and Hydrocortisone Ace—
`tate (42) the ointment or cream is heated with alcohol to
`dissolve the steroid and part of the base. The solution is
`cooled, which congeals the base, and the alcohol solution is
`decanted. The steroid in the alcohol solution is measured
`colorimetrically with blue tetrazolium. This procedure
`cannot be applied to mastitis preparations because they
`usually contain other drugs,
`including antibiotics and
`sulfonamides, together with the corticosteroid. Bracey et al.
`(43) applied partition chromatography to isolate hydro-
`cortisone and hydrocortisone acetate from these prepara-
`tions. The sample mixed with dry Celite is packed on a
`column employing a methanol-water stationary phase over
`a sodium bicarbonate solution trap layer. The interfering oil
`fractions are eluted first with methylene chloride-isooctane
`(1-9). The steroid is then eluted with methylene chloride,
`leaving trapped on the sodium bicarbonate the other
`substances which would cause interference in the blue
`tetrazolium quantitation step.
`Adsorption chromatography is used in the monograph
`flurandrenolide
`cream and
`ointment
`(44). The
`for
`ointment
`is extracted with hot alcohol as above. The
`alcohol is diluted with water and the steroid is extracted
`with chloroform. The cream is simply dissolved in chloro-
`form and filtered over sodium sulfate. The respective
`chloroform solutions are then passed over a chromato-
`graphic magnesium silicate column which retains the steroid
`while the vehicle is eluted. Finally the steroid is eluted with
`a 1:19 solution of alcohol in chloroform.
`Another technique, gel permeation chromatography, was
`applied by Cosi and Bichi (45) to separate fluocinolone
`acetonide from its ointment base. They utilized a Sephadex
`LH 20 column with chloroform as the solvent. Levorato
`(46) separated corticosteroids from their vehicles in creams,
`ointments and lotions by thin layer chromatography on
`Silica Gel GF prior to UV or colorimetric determination.
`Most drugs formulated in oil base suppositories are
`either acidic or basic in nature; therefore the problems of
`isolation are identical with those in 1yophilic ointments.
`Monographs for amines such as aminophylline (47), chlor-
`promazine (48), bisacodyl (49) and prochlorperazine (50)
`utilize the same extraction procedure which is applied to
`ointments, wherein the drug is separated from the vehicle
`by partition between ether and aqueous acid. The phenolic
`compound diethylstilbestrol is separated from oleaginous
`suppositories with NaOH as its phenolate from an isooctane
`solution of the suppository (51). In the assay of aspirin
`suppositories (52), chloroform solution of the suppository
`
`InnoPharma Exhibit 1071.0004
`
`

`

`JULY, 1972
`
`SMITH: OIL~BASED PHARMACEUTICAL ANALYSIS
`
`413
`
`is passed over a partition chromatographic column con-
`taining sodium bicarbonate solution as the stationary phase.
`The vehicle is eluted, while aspirin is trapped as the sodium
`salt on the column. The aspirin is
`then eluted after
`acidification of the column in situ with a solution of acetic
`acid in chloroform.
`Again, just as with the ointments, the separation of drug
`combinations in suppositories requires more complex oper-
`ations. The procedure for
`the analysis of ergotamine
`tartrate and caffeine suppositories (53) applies partition
`between ether and a tartaric acid solution to separate the
`alkaloids from the suppository vehicle. The alkaloids are
`then extracted with chloroform and subjected to partition
`chromatography over
`a column containing citric acid
`solution on Celite. The caffeine is eluted with chloroform
`while the ergotamine is retained on the column. The latter
`is extracted with chloroform as the free base from the
`extruded Celite mass.
`Cometti and coworkers (54) recently reported on gas
`chromatographic analysis of multicomponent suppositories.
`Depending on the composition of the vehicle, absolute
`ethanol, chloroform or
`a mixture of chloroform and
`alcohol-containing internal standards is added to the melted
`suppository to dissolve the sample. The hot solution is
`injected onto the gas chromatograph.
`AC KNOWLEDGMENTS
`
`L. Wong, C.J. Reamer, M. Sharkey and B. Ross helped in the
`study of the analysis of steroids in oil injectables; and J. Levine
`contributed valuable suggestions. All are members of the Division of
`Drug Chemistry.
`
`REFERENCES
`
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`2. Ibid., 15th Revision, Mack Publishing Co., Easton, Pa., 1955, p.
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`3. Ibid., 16th Revision, Mack Publishing Co., Easton, Pa., 1960, p.
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`4. Ibid., 17th Revision, Mack Publishing Co., Easton, Pa., 1965, p.
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`14. “The National Formulary,” 12th Edition, Mack Publishing Co.,
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`15. “United States Pharmacopeia,” lsth Revision, Mack Publishing
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`16. Ibid., 18th Revision, Mack Publishing Co., Easton, Pa., 1970, p.
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`
`17.
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`“The National Formulary,” 13th Edition, Mack Publishing Co.,
`Easton, Pa., 1970, p. 284.
`Ibid., 13th Edition, Mack Publishing Co., Easton, Pa., 1970, p.
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`Ibid., 13th Edition, Mack Publishing Co., Easton, Pa., 1970, p.
`281.
`Banes, 1)., J. Ass. Offic. Anal. Chem. 43:249 (1960).
`“The National Formulary,” 13th Edition, Mack Publishing Co.,
`Easton, Pa., 1970, p. 599.
`“United States Pharmacopeia,” 18th Revision, Mack Publishing
`Co., Easton, Pa., 1970, p. 320.
`“The National Formulary,” 13th Edition, Mack Publishing Co.,
`Easton, Pa., 1970, p. 68.
`Ibid.. 13th Edition, Mack Publishing (30., Easton, Pa., 1970, p.
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`Roberts, H.R., and M.R. Siino, J. Pharm. Sci. 522370 (1963).
`Roberts, H.R., and K. Florey, Ibid. 51 :794 (1962).
`Drug Standards Laboratory, Ibid. 53:98 {1964).
`Talmadge, J.M., M.H. Penner and M. Geller, Ibid. 53:76 (1964).
`Bican~Fister, T., J. Chromatogr. 22:465 (1966).
`Cavina, G., and G. Moretti, Ibid. 22:41 (1966).
`Cavina, G., G. Moretti and J. Sardi de Valverde, Ann. Inst.
`Super. Sanita 4:75 (1968).
`Moretti, G., G. Cavina and J. Sardi de Valverde, J. Chromatogr.
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`Cavina, G., G. Moretti and P. Siniscalchi, Ibid. 47: 186 (1970).
`Cavina, G., G. Moretti, A. Mollica and R. Antonini, Farmaco
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`Talmadge, 1.M., M.H. Penner and M. Geller, J. Pharm. Sci.
`54:1194 (1965).
`Penner, M.H., D.C. Tsilifonsis and L. Chafetz, Ibid. 6021388
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`“The National Formulary,” 13th Edition, Mack Publishing Co.,
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`“United States Pharmacopeia,” 18th Revision, Mack Publishing
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`Weber, 1.1)., J. Ass. Offic. Anal. Chem. 48:1151 (1965).
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`Van Giessen, 13., and K. Tsuji, J. Pharm. Sci. 60:1068 (1971).
`“United States Pharmacopeia,” 18th Revision, Mack Publishing
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`Bracey, A., L. Garrett and PJ. Weiss, J. Pharm. Sci. 55:1113
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`“The National Formulary,” 1 3th Edition, Mack Publishing Co.,
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`Cosi, G.,and G.L. Bichi, Farmaco Ed. Prat. 25:248 (1970).
`Levorato, C., Ibid. 24:227 (1969).
`“United States Pharmacopeia,” 18th Revision, Mack Publishing
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`Ibid., 18th Revision, Mack Publishing Co., Easton, Pa., 1970, p.
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`“The National Formulary,” 13th Edition, Mack Publishing Co.,
`Easton, Pa., 1970, p. 99.
`Ibid., 13th Edition, Mack Publishing Co., Easton, Pa., 1970, p.
`597.
`“United States Pharmacopeia,” 18th Revision, Mack Publishing
`Co., Easton, Pa.,1970, p. 189.
`Ibid., 18th Revision, Mack Publishing Co., Easton, Pa., 1970, p.
`54.
`“The National Formulary,” 13th Edition, Mack Publishing Co.,
`Easton, Pa., 1970, p. 268.
`Cometti, A., G. Bagnasco and N. Maggi, J. Pharm. Sci. 60:1074
`(1971).
`
`{Received December 11, 1971]
`
`InnoPharma Exhibit 1071.0005
`
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