ated Dru G.J
Pupariello,
S. C.S l ~ c k ,OR^ W . J.
Mader
icsenrch Depurtmenf? CiBP P!I,-rniaceufical Products, Inc., Summif, h'. j.
cwtain organizational ri.vieir-. I t 1s Iiop(~1 t t>rgxriiz;ition is T I K I ~ . O I ~ \ . ~ J I I I : I I ~to iis(., III c*cxrc;iins h o w v;ii(w a c!i;.niic:il subttit!,(l
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ALs Ilot 1, I)? uctiiv~!s iiud>
i,i\isiOli
ALKALOIDS
Isoquirioline Graup. !i r g a n i c hawaiIcti as cotieiri,., nnr? amiclopyrine form C * I I I I ~ ; ! ~ V:vitli S mtlium tetraphenj-li ~ ~ ~ ~ :,i lik.!:, ) r i on i,b:ic.tion with meriirii,cli!c~i.itic~,!ii)erato hydrogitn chloride (270). 'ri'liis I.Kll:tim povides a of thew chroniochromatograplilc rrwthocis fcbr thc detect>ion 01 :i:ir~.xit,uhL~ i n n!criphine ! ~ n diiarcotine in codeine (371:. .I method for : i i c ciu:tntitative separatioii of papavc111ic f r c , i i L nnrcotine by forming t,he i1iso;iible papaverine reiuwkatc has been >. Lee and others sugd tpectrophotometry ioiiowed by LL nonaq ii(~oustitration for tieterniiiiing narcwtinc in papaverine rriistiirrs (2821. .I method which deptm& n the fluorescent:! produced by re:lc:tion with concentrated wlfuric acid folio\retl ~.iyireatnient with ammonia is prescvited for determining small xniumts of morphine (338). h simiiar fluorometric: method along with a hriinet,ric method for the det,ermi:iaon of pap:tverinc, has been reported (.?3lj. Van der Pol has developed a inethod of differential spectrophotom(:try which is used in the detcrmination of morphine in thi: presence of t>he s
3tIit.r opium :tikaloida (51.g). It hm been found that the infrared spectruni of the carhon tetr:icliloride solution of the dried d h o f o r n i extract from an acetic acids-ater extract of opium shows a quantit.ati7.e relationship bet'wern the absorbrtiii't'd a t !TGi, 1602, arid 1160 em.-' :ind the concentration of narcotine, thebaiix, arid papaverine ( 2 2 ) . The per c e n t concentrations are caiculated with three scp:tr:ite equations in which corrections for ttie other t\vo alkaloids are i!i:idc. Thosc, alkaloids containing a ~ I t w t i i o sgroup ~~ c i n lie quantily determined by liberation of ldeh\.tic, on hydrolysis and detcrriiining fo!maidcliydc~ by developilli'llt of a yioiet color on reactioii with c~lironiotropi;~ acid in sulfuric acid (279). I. 1 l i o dcgrec of drgradation of papaverine injcrtion \v;iS studied by polaro~rapliic dc~temiinxtion of papai-cr:ildii:e (971j . i'al,u.erinc :tiid pal,averinol did not iiitcdcw \vith the tn o-elertrcin \vave corr.i,sponding to the reduction of the carbony! group uf papavi,ralc!irie. Puiarog~ i l ~ l iisy also iiseti by llili'hers in the e p i n a t i o n of niorphifie in blood 7 ) . d s iittk as (0 ug. uf morphine 112 10 nil. of blood can he dcterniined by this methoci. .4 iicphelon,etric deinination of mwphint~ in poppy ~apsu!es has been L i e \ t,l,l;~ed (286). All the alkaioiJs arc prchcipitatd by animonium molybdate esvept for morphine which, after filtration, i q transIcmned into n i l irisoluble compound by the d d i t i u n of amiiioniuni w i l d a t e . Yarious pztper cliromntogra~;hi!,methods lor the separatioii of op' mixtures have been pi 353, 482). Elwtrophoresis has also btwi applied to this separat,ivri problem (p959). A method for the spectrophotoZietric det'ermination of papaverine iiydrochloride is dcscribd by determining the absorpt'ion maxima a t 240 rnM and an Ai:;, of 1500 (203). 13iglino hrts used countcrvurrent dist,rib!ltion to separate hJdrastine, canadine! and bcrherine in artificial mixtures, as n-ell :is in tiiicturcs (Jf Flydrastis cannclcnsis ( 6 6 ) . .I nicthod for determilling the total aikaloids and for the separate dctermi:intion of cnictine and cephaeline r c metric measurement of intriisity of a yellow color arising by the action of iu1 iodine solution in the presence of an aqueous sodium acetate solution on the ipecac alkaloida (I 06).
Purine Group. Sorlaqueous titrirnetry has b e t ~ i suggested ?q many authors as a means of determining the purine nucleus xlkaloitls, such as caffpine, theobroniine, and thcophylline 1116, 212, 248, 394, 414, 4.15, ,503). T'arious solveiit mi.;turcs, techniques, a d indicators are described i i i these publications. Ogaira amperoinetrically titrated caffeine, cinrhonine, and amidopyrine in a hydroc.hlori~~ acid solution using tungstosilicic wid :is titrant (562). Kassau found that sublimed a-ystnls of caffeine, thcobroniiiie, and t!ieophyl.linc appear :IS 1600-micron pr!sms, 3-micron crj,stals of indefinite Cimnj and 30-iiiicron crystals of varied &~apc.s,respectively ( 2 5 2 ) . Thus. this suthor iras able to determinr !is little :IS 1 pg. uf alkaloiii h) subliniiiig the imre material: its salts, or crude drugs containing them anti viewing the sub!imate with a microscope. Caffeine in a mixture containing theobromine and theophylline can be deterniined photometrically according t'o ll-achsmuth ami \.an Koeckhown ( ~ 5 3 2 ) . h photonephelometric method for the determination of caffeine, baaed on the use of tungstophosphoric acid, has also beta presented (49). Caffeine in the presence of antipyrine i i ~ dphenacetii! can he determined by an ion exchange method (455). Yoshino reports that xanthine basths can be separated tiy ion exchange chromat,ography nsiiig a xeak acid type cation exchanger and gradient elution with hydrochloric acid solutions of increasing acidity as the desorption agents ( b 6 6 ; . Tropane Group. Several d o r i metric riiethods h ~ v ebeen suggested for the drtermination of the solanaceous alkaloids. Amopine and related alkaloids in pharmaceutical preparations can be tiett.rmined colorinietricaliy by nitration f ( J l h ? d by reduction and then coupling after diazotizat,ion with .Y-(l-naphthyl)ethylenediamine (288, ,347). Atropine, hyoscyamine, and hyoscine react with p-aininol)enzaldehyde in concentrated sulfuric acid to give a color ( 4 ) . Paper chromatography with both ascending and descending development has been used successfully for the separation of mixtures of tropane nucleus alkaloids (i+4,119,134! 623). Indole Group. Haycock and others described a spectrophotofluorometric procedure for the determination of VOL. 33, NO. 5, APRIL 1901
113 R
rewrpiiie in the presence of rescinnariiincs hascd on the natural fluoresccnce of rcueriiine a t 360 mp when Iiiasimally t,scited a t 280 nip (1.97). The excitation : i d fluorescence maxima in niillimicrons a n d the relative fluorescent sensitivity of other Rauivolfia alkaloids and related compounds w r e also deterniincd by thrse workers. -1 reIatcd method for determining reserpine a t t h e O.OOO27, concentration level generally found in feeds was also developed a t this laboratory (196). Maynard has published an infrared spcctrophotometric method for the detcrmination of reserpine in tablets and eapsu1i.s with a procedure for dt+rmining 3Yc or more of the common impiiritirs deserpidinr and rcscinnamine (314‘1. -1new niethod for the assaying of the total alkaloids of Rnirccoijia scrpentina has been presented ( 6 4 ) . Protoveratrine arid reserpine in tablets can be determined by the method o; Gyenes mid others (191). In this method, the tablrts are cstractcd with chloroform or carbon tetrachloride containing 2 to 3% of phenol and then the bases are immediately titrated with p-toluenesulfonic acid. i\ general review of th(1 p a p u chromatographic, colorimetric. and fluorometric methods used in determining resperine in pharmaceutical preparations has been presented ( I f 5 ) . I3ayer describes a niethod for the determination of the total ergot alkaloidal content and a separation proccdure so that the ergotoxine content, the ergotamine content, and the water-soluble ergot alkaloidal content can be determined (39, @). This method of analysis depends on the lysergic acid part of the ergot alkaloids which has an absorption niasimum in the ultraviolet region of the spectrum. Several colorimetric methods for the determination of ergotanline have also bren drveloped (556). Paper chromatographic separat’ioris were performed on ergot alkaloidal systenis (5’82. 460). hsensitive and selective polarographic and oscillopolarographic determination of brucine in the presence of stryclinitie and other alkaloids is made possible by quantitatively converting brucine into ‘Lbruciquinonc” in nitric acid (158). “Bruciquinone” is then reduced polarographically. Ijriner gives a method for determining strychnine in n u s vomica with the aid of paper chromatography (69). Dusinsky and TyllovL also utilized paper chromatography in the separation of the t\vo major n u x vomica alkaloids. strychninc, arid brurinc, ( 1 3 9 ) . .\ method has been proposed for the determination of stryeh~iine n liich is bawd on the prcci1Tit:ition of the alkaloidal salts with potassium tctraiodomcrcuratc and subsequent titration of the libcrated iiicxrrriric ion ivitli ELlT.1
(558). Phenylalkylamine Group.
1 14 R
I.:plicd-
ANALYTICAL CHEMISTRY
rine in the p r t w n c e of ephedrone may be determined l7y the production of a color with tlie u s e of ninhytlrin a t specified pH valuvs (f6Sj. 1IethJ-lamine, liberated ti!. alkaline hydrolysis of ephedrine, can be deterniinctl on the semimicro scale with the use of the Kjeldahl nicthod (209). Feigl and Silva deriwd :ispot test for the detection of v f ) h d r i n e in the prestwc of amphctariiiiie ( I 5 4 ) . Quinoline Group. nietbod for determining quinine by preeipitah ing it with potassium cadmium iodide and then titrating the excess cadmium ion with a standard solution of disodium cliliydrogen(ethylenedinitri1o)tetraacetate has been developed (98). Petkovii. has v-orkedout a rapid proredure for the differentiation of quininr from quinidine (369). Column chromatograph5 follon.ed by ultravio1r.t sl)ectrol,hotometry is used by Smolonshi and others in the deterniination of colchicine ( 4 5 7 ) . Lupinane Group. h column chromatogral)hic proccdure followed by :I color tlevclopmciit a n d photometric measurement pmiiits the separation and detcirniination of the principal lupinane nuclcus alkaloids (550). Sparteine arid lupiiiinc can br quantitativelj- detormind 13y a conductometric! tit,ration niethocl (555). Miscellaneous. The alkaloidal aglycones from Soianz~m have been sep:irat,cd 11y p:iper chromatography (60,C), and I)!- two-dimensional paper chromatography atid elwtrophorcsis conibincd ($14,. Conditions for the qualitatirt. :innlnis of certain alkaloid extracts and tinctures by nirans of filtration chromatoqrapliy have lieen invcstigated (523). The alkaloid bands on thcl strii) paper chromatograms can be detected by means of a special micarovolt Sb )‘KC1 0.1S Hg2CI2 KC’1 0.1S Hg cell corinectetl to a suitalik potentiomctc,r (540). On moving the terminals every 3 mni. along the treated strip, the potential changes, the distances are recorded, and the position of tlie alkaloitls is dctrrmined. \Yachsmuth ant1 van K o c ~ k hoven suggest a coloririietric~ drtrmiin:rtiori of yarious alkaloids by forming the alkaloidal hydrosaniatt~s \vhich react wit,h ferric2 chlorid(, anti produce a violrt color ( 5 3 4 ) . -1 paper chromatogrnpliic nwthod suit:iI~li~for forensic purposw has brcn rlcsc.ribed by Vorrl (.%?S). This niitlior also lists tlie R, w l u r s of 36 alkaloids and related conipoutitls. Smdri drscribes the use of bromoauric acid (421 anti bronioIilatiriic acid (.$22)as rc,:tge’nts in niicrochemical detwtion of alkaloids and organic basw. A\lkaloidal analysis by use of the rragent sodium tctraphenylboron has lieen \vel1 developed (340, 636). r\ brief rc,vitn- of coloririictry in has bcvn prescntcd
+
by Arzamastsev (16). -1 table oi the characteristic fluorescenw of 131 powdered vegctsblc drugs trEtc.1. treztment with various wagciits h:is been developed (261). ANTIBIOTICS
Penic’illin T’ a d p1icnosy:icetic acid arc’ detc~riiiin4in ferment:itiori liquors h y rcaction with nitric :inti sulfuric. acids (57). l‘hc. phenolic derivative formed i j yt,llorv in alkaline solution. Magor and Papay (SO?) st:ibilize t,he comples formed in the assay of penicilliri via the irori(II1) 112-drosamate method by extracting with ioobutanol. The color is measured at 4 T O to 500 mi*. Factors affecting the official iodometric assay of penicillin are discussed by Weiss (544). The use of Biccillzc~ megatheri.iim in the cup-plate assay of penicillin has been reportcd 1363). Penicillin may be determined spectrophotometrically after degradation to peniciilinic acid by heating \!-ith acetate buffer (20s). 1 new fluorometric mrthod for the determination of eh1ortetr:wycline has been described (500). The stabilitjof aqueous tetracycline solutions bj- the spectrophotometric method has been investigated (379). The colorimetric method of Saknguchi for the titterminntion of chlortetracycline x i t h boric acid in concentrated suifuri(’ avid is applied to the ass:iy of tetracycline ($.C2). Inoue, Ito, and 0gan.a i218) describe mathematical formulas for
of other subjtances. A colorimetric mcthod for t,etracyclines using a thorium reagent is reported i.5 1 3 ) . Hoc’lininiin and l3ayc,r (206) cliscauss tile liigh-frequency titrimetric. d(~terniiiintioi1 of The nonaqueous titraline antibiotics in commercial prrparatioiir ha> bc,-ii suggested (56‘2). .I method for the dt:tc.rmination of tetracycline :!!drhlortetracycline in Inistures 11 respectiw stability i n is rrported (266). The sy>c’trophotometric method for tetra chlortetrwyiline is applied of pharniac.eutica1 prepirat T h e bromatonietric and titai,oiiicdric assay of chloranipheniroi is dcscribctl ( 1b). Estimation of clilorampliciiicol by di:tzotization is reiiortcd ( 5 ~ $ 1 ) . Thc sl)cctlophotnIiietrie control of chloraniplic~nicolin comples prc,parations 111. spe~troi)hotomctryhas been suggestc.d by Ihnchi and Loniuto (26, 2 6 ) . Salvesen reports the analytical control oi chloramphenicol and c~lilorampheriicol palmitate by nonaqueous titration (416). Chloramphenicol and benzocairie are detrrmincd in aural !)reparstions by ultraviolet si)ectrophotonit.try (563).
,
;\,I i
o\.uliiucin is b:~scduiion
’ of aiiinionia from the car-
1)anioyl group by alkaline hydrolysis (?OO,.Xovobiorin in the presence of isonovobiorin is determined by the r1cxv:igc of the novobiocins with trifluuru:icc’tic acid ( 161). The priodate i,oii-unil)tion of the product is a measure of tliv novobiocin concentration. . \ i i autoniated system for the chcmi1.31di~tc~rniination of streptomyvin and pcwicillin in fermentation media lias bren dcwribcd (157). Ikeda and Ikeda (216) determine dihydrodeosystreptoniyciii by measuring the ultraviolet :tbsorption of the acid-hydrolyzed produvt. -1gasonietric method for the assa>- of strt~ptomycinand dehydrostreptomycin in complex preparations has I ) I ~ C I I suggcsted (519). Electrophoresii is usid for the identification and estimation of streptidine in streptomycin :iddihydrostreptomycin (372). ‘I’he furfural and anthrone methods for the assay of kanamycin are discussed :iiid iwriiixirisons to the biologiral asS ~ J .n l c made (264). hlie\. (6) suggests a new qualitative rcwtion for syntoniycin and levomyvcxtiii. .c;l)ectrol)hotonietry is correlated with lo-< of biological activity to predicit tlw stxbility of pharmaceutical preparation> of strcptovaricin ( 1 7 2 ) . Tlic ecy~ration and purification of :ic4tioniycin K by chromatography has II(YW iqiorted ( 1 S 3 ) . .I c,nloriiiietric method for the dcti>riiiiti:ttion of cyclohesimide based iipon tlic, forniation of a ferric hydros:tnintc h:is hwii suggested (160). Ilasic aiitibiotics may be determined 11). liigh-frcquency titrations (350). l’wic, ~ircviirsorsof antibiotics cannot I w rirtc~niiined. JIicrobiological assay of niitiliiotic solutions using sodium wwzuriii lias brcn proposed (123). ‘i‘lio w l o r test for arginine with 1naphthol and hypobromite has been xpplicd to the assay of Sakaguchiantibiotics (485). Electrohas been suggested for the tl(tcction of a number of antibiotics in iiiistiircs (8.;).The solubilities of antiliiotic,s in a number of solvents are i,c-l)ortrtl ( 1 I , 5 4 5 ) . CARBOHYDRATES AND GLYCOSIDES
Carbohydrates. Sucrose in U.S.P. m a y be determined by an c~nzyii1:ttivspectrophotomet,ric method (ZO.?). Siigxrs may bc identified by the, I Y J ! ~ ) ~ Iiroduced lvhen they are trc,:itcd \\ ith 3,4-dinitrobenzoic acid rvugtBiit ( . $ I , ‘I‘he influence of the cmnlio*itiiJii of reactants on the deterniinat k J l l of glucose by the Gabrien I3rrti,;Liid method is discussed (15). A riiic,rnclc:tcrniiiiation of glucose is carried o u t by titrating with zinc sulfate solutivii :ifter oridation (376). ,4 com-
>ii,iiI)s
parison of a glucosc~olidase nicthod lvitli a n official mc,thod for the deterniination of glucose is discussed (3Ssj. The green color produced when fructose is heated with phenol and concentJrated sulfuric arid is the basis of an assay (292). p-hnisyl blue tetrazolium chloride has been proposed for the ultramicrodeterniination of fructose (891). .\ comparison of titrimetric and rolorimetric methods for the determination of fructose in the presence of glucose has been made by Matsuo and S a m b a (312). Factors affecting the colorimetric method of determining reducing sugars with dinitrosalicylic acid are thoroughly discussed (65, 26‘8, 325). The interference of citric acid in the estimation of reducing sugars with alkaline copper reagents is reported (365). Carbon-l$labeled cyanide has been used in thc determination of reducing sugars and reducing end groups in polysaccharides (336). Bevenue and IVilliams (55’) discuss the use of 4,5-dinitroveratrole for the ultramicrodetermination of reducing sugars on paper chromatograms by a reflectance method. A two-stage method of chromophoric developmcnt is described for the spectrophotometric procctlure for chromatographic analysis of sugars 1380). Ai review of color reactions of the deosy sugars is supplemented by a number of color reactions for digitosose (408). Two new specific color reactions are proposed for the qualitative and quantitative determination of tetroses (132). The hydroxamic acid reaction has been applied t o the determination of reducing sugars and sugar acids (206). The separation and deterniination of methyl mannosides is accomplished in a n automatically controlled cellulose partition column (335). The Molisch reaction as applied to cyclic keto1 groups is discussed (507). The differentiation of y- and &lactones of aldonic acids by an infrared technique is proposed (31). A nepheloco!orinietric assay for lactose has been proposed (179). The zinc complex of toluene-3,4-dithiol has been suggested as a reagent for ketose sugars (99). X new indirect method for the microtitration of certain reducing mono- and dis:rr.c:harirlrs uses sodium cuprosulfosalicglatc (392). Aldosaccharides may IIC determined colorimetrically using nodiuni 4-aniinosalicy!ate (14.5). llatfiiiosc~t ~ n d melibiose are assaycd qu:intitnti\.r.l!. by paper chroniatogral~hy (521. Glycosides. The wpur;ition of thcx on d ai,\- ,:l!~c~oriclcs of Digitalis purpurea leaf by chromatographing on :iliiniiniini oxidc columns and subsrquent d(,tc,rniination of the glycosidrs liy tlic 3.5dinitrobenmica acid reaction arc reported ( . W 5 ) . The significanw of tlic
infrared absorjitioii s;jectra of some digitalis glycosides and aglycons in their identification and differentiation has been discussed by Bell (45). The constituents of Digitalis purpurea ha1.e been separated by countercurrent distribution (194). hkatsuka proposes the colorimetric determination of cardiotonic glycosides n i t h 3,S-dinitrobenzcnesulfonic acid ( 2 ) . The rrsults of four methods for the determination of cardiac glycosides in Digitalis purpurea and Digitalis lanata are summarized and compared to a biological method (267). Tattje has revien-ed the various chemical methods for the assay of digitalis leaves and has recommended a procedure (@9). colorinietric assay for digitalis glycosides with 2,4-dinitrophenyl sulfone has been described (4SS). n’achsmuth and van Koeckhoven discuss the reactions of deoses and their application to the determination of glycosides (533). The use of papcr chromatography continues to receive wide attention in the separation and identification of the cardiac glycosides. S e w systems for separation and reagents for identification have been proposed (162, 167, 381, 4S6, 509). The colorimetric deterinination of rutin by its reaction ivith diazotized p-aminobenzoic acid has been reported (117). nicthod for thc assay of rutin has bem developed based upon its reaction with aqueous solutions of uranyl acetate t o produce an intense stable yellow color which is measured photometrically ( 7 2 ) . A \
STEROIDS AND HORMONES
Colorimetric analysis of steroid- has bren given considerable attention in the past few years. Wagner and others used a modified tetrazolium reaction t o determine photonietrically the prednisolone content of compressed tablets (.539). A colorimetric procedure v hich enables one to determine simultaneously the cortisone and hydrocortisone content of a pharmaceutical preparation has been presented (13). This method uses 2,6-di-terl-butyI-p-cresol as a reagent. Bianchi has used this reagent for the analysis of A4-3-ososteroids in the presence of the corresponding dehydro derivatives ( 5 5 ) . A method has been presented for the determination of A5-3-P-hydro.;y steroids in biological fluids based upon the yellow color formed by reaction with sulfuric acid-ethyl alcohol reagent (351). The coupling of estrone and a-estradiol nith diazobenzene-p-sulfonic acid hydrochloride is the basis for the colorimetric determination of these compounds (410). Toluene-p-sulfonic acid is the reagent introduced by Turner and Eales for the colorimetric deterniination of cholesterol in seruni (506). Testosterone VOt. 33, NO. 5, APRIL 1961
115 R
has been determind phototnetricalljurine (87, 141, 313, 466, 5 5 4 ) . Antias its green indonnilinc derivativc mony chloride was used to give a color (307). Steroidal colorimetric annlysis reaction suitable for the detection and by use of a Ecrric chlnritle--sulfuric estimation of steroids after paper chrowid rcagrnt \vas further studied, matographic separation (341, 479). motlifictl, and ap~ilicd(58, 400. $ 4 1 ) . ~‘iSmith and Focll used isonicotinic acid Ecrrous ammonium sulfate-sulfuric acid hydrazide reagent for the differcntial rcagcnt \\as used in a one-stcp procedetection of A4-3-ketostcroids and A1~4durc to d&rniinr c.stradiol, rstrone: %ketosteroids on paper chromatograms :in11wtriol i\l$9), (455). Various other chromatographic &b:wik:ili:iii and othcrs prcsent ail procedures for a nuniher of different infrarid method iyliich makes it possteroid systcms h a w been dewlopcd in sible t o dc~tcrniiriethc Ei~drosylcquivthe past few years (109, 131, 2.59, 294, :ilrnts in strroirls (243). Thc in300, 456,. Seher has matic a eomprefrmvl slitlctra of a number of ling-TI hensive rcviev of thr chromatographic steroid lactoncs w r e examined and methods uscd for stwolq steroids, found to have three absorption freand rrlated compounds (344). Cox, clucni.ics cliaractcristic of such moleHigh, and .Jonc.s suggcst that strroid c,iilcs ( io?). Jleda has t a b u l a t d the sidl: chains can be investigated by fission frcqucncic~i;of thc infrarcd abcnrption of tho strroid under standard conditions b:iiitls of ketostcroids in the c>arhnyi and then an rsamination of the resultarid double-bond region, arid the, Ereing products by gas-liquid chroniiatogc p w y shifts on halogenation (.315). raphy (10,T). Iiiirnrctl qiectroscopy has also heen Prcdnisonc in tablrt formulations uwi.1 for tlic quantitative analysis of map be determined hy use of polarography (125’). A fluorometric mt~thodfor the difticosteroids n.ith v r y conrcntrate(1 frrcritial estimation of metanephrine and phosphoric acid or sulfuric acid has riornietanephrine has been published been further studicd as a mc~tliotl of (51). The reaction of epinephrine or mulysis for thtsc steroids (182, 2j.7’. norcpincphrinc with ethylcriedianiine 4.51‘). The fluorescence of certain sterproduces complex n.istures which can oids i n sulfuric acid has been found to be scparated by paprr chromatographic incrimt, considerably upon the addition methods ( S S 7 ) . Epinephrine is quanof :illout 30% of phosphorus osychlotitatively osidized with potassium ferriride (501). EStrogenic hormones found cyanide t o adrenochrome, nnd the rein tlic. urii!~.arc’ fluorometricnlly or i d o r sulting ferrocyanide is determined by a inictricn1I:- i i ~ ~ t i v m i n iby d us? of a hydead-stop end point titration ivith zinc droquiiioiie -suliuric. a d reagent folsulfate solution as the titrant (378). li~n-ctiby an t ’ \ traction procedur -1 sensitive differential fluorometric .‘Yo p-nitropli~iiolin vhlorioforni method for determining adrenaline and (L’licn h3s reported that salicyloyl noradrenaline is based upon the differential transformation of the oxidized forms, adrenochrome and noradrenochrome, to the fluorescent forms (409). rcaction can be i1si.d for cal determination of these VITAMINS
tlctwiiincd hy means of ultraviolet ?i’ectrni)liotonic,try, hut this method is dcpc~ntic~nt on tlie difference in rates of thioseniicnrbazotie formation ( I 5 9 ) . Klrin and others have developed an ii1travioli.t sl,cctrophotonietric assay for combinations of ethynylestradiol :ind nieth?-ltestostr,rone (260). Prednisolone in prednisolone and neomy(,in ointmimt has bcen determinrd by use of ultrairiolet siicctrophotomctry ( 2 7 ? ) . Phenolic stixroids were dvtrrm i n d s ~ ~ e c ~ t r o ~ j l i i ~ t o m e t rin i ~ a the lly presence of :in (’si liy e1imin:ition of alisorption by rc,tluction with potassium l ~ o r o l i ~ ~ i l r i(ai‘.$). ile
JIvistrr and othi’rs use paprr chroniatography as n nirans of prednisolone in tahlc9t formulations (.?I 6 ). C’hroiii:itogr:il)liic proc*edures huv(’ h t X ndc~\c~!o])ctl to drtrrmine the amount of c,rrtain .steroids fonnd in
116 R
ANALYTICAL CHEMISTRY
-1new s1)ectrophotometric method for the determination oi vitamin X has been devised (2%). This method is based on the formation of the red color which devdops, aftcr a transicnt bliie, when a solution of tungstophosphoric acid is added to a chloroform solution of vitamin A in the prescncc of acetic anhydridr. A ijhotoneljhelomctric determination of vitamin I%, has been prcwntcd by Bernshtcin (50). An investigation of the method for dctermiiiing tliianiinc by incasurenicnt of the color produced with tliazotizcd 6-aminothyniol has shoivn that t1i:izotization is riot ncwssarily involved in the rcactiun arid that a similar color is p r o d u d by dirvct troritnirnt with an alkalinc solution of 6-aminothymol (199). 150th nic,otiiric ac~idand nicotinamide in a mixture Jr-ith cyunogc~nbromide and barbituric acid give extinction rn:irinia a t 400 nip,
but nicotinamide gives a second masimum at 513 mp, t>huspcrniitting simultaneous determination of nicotinamide and nicotinic acid (298). The use of t,ungstosilicic acid as a precipitating agent before the colorimetric. detcirniination of nicotinamide with cyanogen bromide in multivitamin preparations removes the interfering thiamine and stabilizes the color development (445). MaeArthur and Lehmann have developed a procedure for separating pyridosine, pyridosal, and pyridoxaminc in aqueous solution with a cation exchange resin (296). A nevi color reaction for pyridoxal based on its interaction with thiophene has been discovrred (287). Pyridoxal, but not pyridoxine, pyridosamine, or pyridosic acid, reacts with thiophene t o form a jade-green color. Pyridoxine reacts with diethyl-p-phenylenediamine in the presence of oxidants to form a color (212). This reaction can be used for the quantitative determination of pyridoxine in the presence of most other vitamins and compounds with the esception of ascorbic acid, salicylic acid, nnd phenol. Webb and others present a method for the determination of folic acid in pharmaceutical preparat’ions ($33). I n this method the folic acid is determined by digesting in alkaline buffered solution, splitting with potassium permanganate. diazotizing the resulting 4-aminobenzoic acid, and mcasuring the color produced by coupling n-ith -Y- (1-naphthyl) -ethylenediamine dihydrochloride. Another colorimetric method for determining folic acid is based on the color which develops when nitric acid solutions of folic acid are builed s n d then t r e a t d with ammonium hydroxide (517 ) . Cords and Katym describe ci method for the determination of ryanocobalamin in solid and liquid Litamin BI2 preparations (105). ‘The total E,,(‘ontent is measured by the light absorption a t 550 mp of a i,yaniiie-treated sample and the cj-ariocc)balaniin/U12 ratio is determined by quantitative paper chromatograph!,. method has been developed for the dttcrmination of cyanocobn1:min in :tr:iourits of 5 t o 50 pg. (.S,W). In this mcthod the cyanocobalamin is irradiatrd with a mercury vapor lamp. and thc liberated HCT is distillrd and dctcmiiicd photometrically folloir-ing t h t :ddition of a guaiac-copper sulfate rciigent. The separation and determination of mixturcs of vitamin and adi~nosine5monopliosiihoric acid (3543 and vitamin B, (4.93) ha1.c bccn treatcd. I’oteiitionictric and nmpcronietric methods :ire dcccribrtl for thc, titration of ascorbic. wid with potassium perniangnn:itc in tlir prcwncc’ of potassium iodide and sulfuric acid ( 2 2 6 ) . ,\ dead-stop end point method u-i:ig Imtansiurn fcrric~yanitlc (37‘7) and :iii
amperometric method using bromate as titrant (425) have also been presented. Sant reports a ferricyanometric titration of ascorbic acid in the presence of zinc acetate with iodine-starch used as a n internal indicator (424). Mitsuta and Shikanai used a n indophenol-butanol solution in a colorimetric deternlination of ascorbic acid (329). A procedure has been described by Schmall and others for the relatively rapid photometric determination of vitanlin D in the presence of vitamin A in simple mixtures of dry A and D powders and some multivitamin preparations by using chromatography on Florex XXS (437). Using this method, the assay results compare favorably with those from bioassays. Sternberg and others have proposed the use of the least squares matrix method as a n analytical curve-fitting technique to provide analysis of the complex irradiation mixtures of ergosterol using ultraviolet spectrophotometric data (473). The components in the mixture studied were ergosterol, lumisterol2, tachystcrolp, precalcifero12, and calciferol2. Vendt and Drokova made a study of the specificity and sensitivity of various colorimetric methods used in the determination of vitamin E ( 5 1 5 ) . The determination of a-tocopherol, the biologically most active form of vitamin E, and of p-, y- and &tocopherols, important antioxidants in oils, is based on the ability of p-, y-, and &tocopherols t o give a yellow color reaction with sodium nitrite in contrast t o a-tocopherol ( 1 2 7 ) . Green and Rlarcinkiewicz investigated various coupling reagents in an effort to obtain a differential coupling reaction for the complete analysis of tocopherol mixtures (1P7). I t has been reported that 5-imino-3thiono-1,2,4-dithiazolidineis a promising reagent for the colorimetric determination of vitamin K (436). A spectrophotometric method based on the formation of isosbestic points at various pH's has been developed for mixtures of vitamins B1, BPI Bs, and nicotinamide (82). Other methods for determining the vitamin content in certain multivitamin preparations have also been presented (18, 518). ACIDS AND ACID DERIVATIVES
A new colorimetric determination of the higher fatty acids is based on the fact that the copper salt of a fatty acid dissolved in chloroform in the presence of triethanolamine shows a stable blue color (224). This can be used for a simple and rapid determination of saturated and unsaturated fatty acids from C,, to C Z 2 . Carboxylic acid derivatives (180) and dicarboxylic acid derivatives (168) react with hydroxylamine to form hydroxamic acids which, in turn, react with ferric ion to give a
measurable colored product. The determination of both esters and amides is possible by this method. This method has been applied t o the determination of total esterified fatty acids in plasma (334) and other ester determinations (374). A new spectrophotometric method for the determination of lactic acid has been presented (130). I n this method, the lactic acid is converted t o iodoform which is dissolved in chloroform and measured spectrophotometrically a t 347 mp. A spectrophotometric determination of small amounts of malonic acid, which is based on its reaction with acetic anhydride t o give a colored polymer, has been described (230). Pinchas and Avinur have reported a spectrophotometric method for determining mixtures of the three isomeric toluenesulfonic acids (375). Phenylacetic acid in fermentation broth can be colorimetrically determined by nitration according to Yoshii (564). A spectrophotometric method of determining aspirin, phenacetin, and caffeine in prescription mixtures and tablets has been described (75). Sodium p-aminosalicylate has been determined colorimetrically after forming a complex with uranyl ions (21). The separation and colorimetric determination of p-aminobenzoic acid in procaine have been described (97). Hydroxybenzoic acids have also been determined by use of a colorimetric procedure (538). Papariello and others have devised a chromatographic procedure for the analysis of the pharmaceutically important esters of fatty acids and polyethylene glycols, glycerol, and ethylene glycol (357). Partition chromatography followed b y spectrophotometric measurements of the separated components was used by Heuermann and Levine in the total analysis of mixtures of acetylsalicylic acid, acetophenetidin, and caffeine with codeine, barbiturates, or antihistamines (204). Gas-liquid chromatography has been used in the determination of a series of malonic esters (4Q6) and in the determination of a system of methyl esters of highly unsaturated fatty acids (475). Nicotinuric acid ana nicotinamide mixtures are analyzed by paper chromatographic separation followed by ultraviolet absorption measurements (239). Descending papcr chromatography was used in the separation of Ce to CI1 fatty acids (404). Higher aliphatic carboxylic acids from Clo to CZ2were also separated by means of paper chromatography (41). An anion exchange resin was used for the separatory determination of glucuronic acid and various glucuronides (219). Malmstadt and Vassallo state that the nonaqueous titration of carboxylic acids, sulfonamides, imides, mercaptans, phenols, and enols can be performed a i t h a
single titrant-solvent combination and a n automatic derivative potentiometric or spectrophotometric end point determination (303). These acids in 90% acetone solution are titrated with tri-n-butylmethylammonium hydroxide in 80% benzene-20yo 2-propanol solvent. A platinum (loyo rhodium)graphite electrode pair connected directly to the input of a Sargent-Rlalmstadt titrator was used in the potentiometric procedure. Many indicators which are suitable for spectrophotometric procedures were tabulated. By using tetraalkylammonium hydroxides in benzenemethanol as titrants a wide variety of organic acids have been titrated in nonaqueous solvents (559). Milligram quantities of components in mixtures containing hydrochloric acid, acylhalides, anhydrides, organic acids, and alkyl halides n ere determined by titration in nonaqueous solutions with the bases, sodium methylate, Triton 13, or tributylamine (361). Dean and Hackman describe a nonaqueous titration method for the assay of fatty acids in the presence of their zinc salts in antifungal preparations (118). The drug Oxeladin can be titrated with perchloric acid using acetic acid as the solvent and determining the end point electrometrically (83). A number of generalizations which permit the prediction of the titration behavior of organic acids in pyridine have been derived (478). Icha has reported a polarographic method of analysis for orotic acid (214). A polarographic as J5ell as gravimetric method of assay of derivatives of xanthen-9-carboxylic acidnamely, methanthelinium bromide and propantheline bromide-has been described (269). An assay has been suggested for tartaric acid in which the acid is converted by a photochernical reaction in the presence of uranyl acetate into glyoxal which is then polarographically determined (70). Methyl salicylate (54) and phenyl salicylate (133) have also been assayed by polarography. Van Meurs and Dahmen have conductometrically titrated carboxylic and phenolic acids in nonaqueous solutions (321-323). Gaslini and Nahum have conductometrically titrated many weak acids with aqueous lithium hydroxide in aqueous ammonia or triethylamine with better than 1% accuracy (173). Shain and Svoboda have applied constant-current potentiometry to nonaqueous titrations of weak acids (4 46). 0- and m-Hydroxybenzoic acids in mixtures can be determined fluorometrically by varying the p H of the solution (497). Salicylic acid in biologic tissues has been determined by a fluorometric procedure (96). Fluorescence methods have been suggested for the analysis of gibberellic and gibberellenic acids in fermentation prodVOL. 33, NO. 5, APRIL 1961
* 117R
ucts (253), of cholic acid in acid (551), and of gallic acid Quantitative ,analysis of mono- and polybasic benzcne carboxylic acids in potassium bromide disks by infrared spectroscopy is discussed by Sicholson (34.5). Tritclbauni has used infrared spectroscopy along n-ith various srparation methods to stiidy the constitution of essrntial oils ($94). Jordan and Dumbaugh rnude a stud!. of the thcrniomctric titration of \veal; monoprotic acids (24.0). An inversib isotope dilution analysis of salic!-lic, acid has bcen presriited (483). .In improi-cd mcthod for the dpterminatioii of a,+unsaturated acids has bcen presentrd liy Critchfield (If I j . This abthor has found that the convercion of the unsatur:itcd a d s to their corrcsponding sodium or potassium salts will pmiiit a rapid and m o o t h addition of brominc. 1Iethanesulfonic acid tlrrivativcs, such as dipyronc and sulfaniipj-rinc, have bcen dctcrmined by nil iodomctric method (439). INORGANICS
h simple! direct, and rapid gas-liquitl chromatographic method for the CIPtermination of w t e r in pharmaceutic:il preparations has been rejiortc,d ( 1 4 7 ) . Small aniounts of water in fluids art’ deterniiii,:tl by an automstically olicrating, coiiloiiictrir titration method (30;. Formaniitli. 1 1 : ~bcen suggested as :HI estractiori solvcxnt in the Karl Fischcr niethotl for ~lc~tc~rniiniiig water in lactosc and m:iltoso i 298). Meyer and Iloycl describe. :I iiit’thod for determining \\-atc>r by titr:itiori I\ ith coulonietrically generatc.rl Karl Fischcr rragent (3+?$, 1fic.rotlcterIiiiiiutioii of water with Karl Fischcr rt~igcnthas been reported (34). Existing nic’tliotls for t h e determination of \vatc,r are surveyed (49s). LOK concentratioiis of frec chlorincb Iia\-e bccn tletermincd by a spri,trophotoiiittric- procetlurc (463). Prcc.ision null-point pottxntioiiietry has bcrn used for lo\\. cwncrntlntion chloride dctr~rnii1i:itions (30.4). Lcffert,s re,ports the iiiicrotletc,rmiiiation of chloride by nonaqueous potentiometric titration (283). Chloroiotloquinolinol is assayed for halogen conteiit by combustion, distillation, and aniperometric titration of the halides (464). Cuprous copper has been uscd for the coulometric titration of bromidc ( I $ ) . Potentiometric titration of small amounts of iodide in the presence' of chloride has bcen proposcd (476). Iiolthoff and Sambucetti rcport the' tlitc~rniination of fluoridc at a rotatcd aliiininuni clectrode (26‘3). Cornbudion hy the Schoniger mcxthotl is used in preparing the sample in a rapid colorimetric method for organicallj- bound fluorine (443). A new spot test, for fluoride ion utilizes ccrium(II1)alizarin c,otnplr~one(44). 1 18 R
ANALYTICAL CHEMISTRY
Kress advocatw the use of sodium (1iethyldithioc:trbamate in pharmacopeia tests for heavy metals (272). Contributions t o the mc%hods for determining PbA2,CiiA2,and Fef2 have h e m mad(. by Khokhlovn (266). Sodium rhodizonate has bcrn proiioscd as n spt,cific rcagrnt for ltwl (90). XyIcnol Orange is used in the complrsometric drtermination of k x r l i n p11:~rrnaw i i t icnl pre para t ions (ii68) .
trophotometry zinc1 its appliration ts the determination of c llcium. A direct method for calciiini using a single-cell flame photometer has heen discussed (391). Phospliomolybtlt~iiumblue, coinplrscs anti thvir importance in the :iss.iy of phosph:ite are rtyorted (438). Rirdc~l proposcs thc indirect drtermination of phosphate 1l.it h (cthj.lcncdi ni trilo) tetraacetate (,$Of). 1Irthods for the cictwtion :incl de. d spot ttsst for nitr:ittl ion is h a w i trrmination of pot:tssium ivith sodium upon its roai,tioii \vitli (lipli ,n!-l:iininc~ tctraphenylboratc hnvt, kwcn p i ~ ~ p o s e d (100). h vo1uiri;trit~ nicthoc! csniiilo!.(,q, 364). Precipitation \sit11 sodium ing st~and:ircl so(lium nitritcs lixs 1 ) o ( ~ i tctraphcny1bor:itc is r c ~ c o r n n i c d t d in proposed for the, tlctt~rmination of nitkterniining small quantities of Iiotastratt, and nitritc’ (I((’,$). yium (i?f5! 343). F h m e photometry is n.idrly iiscd in the r1rtcmnin:ition of NITROGEN-CONTAINING COMPOUNDS 130th sodium and pot:wiuiii (100.$17). Potas3ium is titratcd 1)otcntioinctricaily Amines. Thirtos a n d I3utrin rci i i aqucxous solutions using :intimonyport thc usc of qiiin:izolinium salts wlmiiim and aritimony-snt~ur~tt~d cdoin the co1oi:iiiicti~ic~tlctrrmination of niel clcctrotlrs (23’). 5-Xitrobarbituprimary amines (33). The optimum i i c acid Ixis bcen suggested fortlit’ coloriconditions for the rractiori of arid dyes nictric tlttcmnination of potassium with organic basm have been tliscussetl (3.iG). Aniline tartrate has bcrn used for thi> r:~pid gravimetric clcterminawith a(-etous p(~chloric:arid in chlorotion of potassium in some pharmaceutibcnztne with a glass r h t r o i l c and :i cal preparations (447). slecve-type c:ilonirl chtrocle (368). A simplc prclcise method for selenium Sitromothane, because of its iveakly cl(>pendsupon the color produced with acidic* naturc, reatlily lends itself to ,‘3,3-diaminobenzidinc (481). The forpotcntionietric titrations of organic bases iiintion of a rolortd conip1c.s nith iodine (477). Primary aliphatic amines may is thc basis of a colorimetric assay for be dcterniined colorimetrically in microvlcnium proposed by Ray (3%). X gram qu:intitics using 2-(3-oso-l-inn c ~ wcolor test for selenium is suggrstcd danylit~enr)-ind:inc.-l.3-diorie (462). ,i hy Snwicki (430). color reaction of primary :Imines with Small qu:intitir,s of fcrrica ion in solus:ilicylal~lehydc~ ha.; becri suggested (328). tion can bc titrated with ethylcneGuanidinium con~pountlsexhibit a green cli~mitic~di(o-hy~~rosyl,hc.n!-lacc.ti~ acid) fluorescence n-hen treated n i t h ninhy(;:If), Iron ?:in lie dctcrmined spt’ctrodrin (103). 1,ocal :inrjthctirs with .z iJliotonic,tric:LlIy a t 2riO nip with EDTA primary aniirio group iiixy bc dctcr(.?If?), A cwlorirnctric detc~rrnin:itionof mined with 1,2-riaphtlioc~iiiiione-4-suliron based upon the reaction of Fe(1IT) fonate (155). Substitution of nitro:ind aniliiic ncxtivated by 2,2’-bip!-ridinc? syl broniidc for nitroiiq :ic*iclin the van h:ic h e m dtwribcd ( 1 ) . The direct Slyke dct(,rniination of 1iriiii:iry amino c,oinlilesometric determination of iron groups avoicls high rvsiilts (244). A \sith Xj-lcnol Ornnge as the indic:Ltor method for the tlt,tc,riiiin:itioii of‘ priis :it,c.iiratc,and sc~lt~ctivc (530). mary, sccond:irj-, and tortiary :imino .i]iplic,ations of t hc t.oin1)It~somttric groujis a.lic~iprcscvit togt>tlicrhas been reported (f 71j . Salting-out chromat1ttc~riiiin:rtion of bismuth to tlic, control xrc tlisc*ussctl by Ddgn of nicdic~:iiiic~nts tography has ber’n :ipplit~d to the :iiitl Storc-k (124). I ~ y c r s wports :I separation of niilligrum amounts of ~ l ~ c ~ i f rc:ic.tion ic, for bismuth tlqicntiing primary, serondary. or tprtiary aliphatic* or aromatic :iininr< (427). on :I ycllon bithiourca c~omplcs(18). Sccond:irj- aliph:itic amines can be ‘I‘hv analytical applit*ations of Xy~,\-tinc,tionof their Ic,nol Orange in t,hr dctwmination of nitrosnmincss (33 tr:icv of zirconium h i v e heen tlisc,usscd by Chcng (B3: 04). Thiocliglyiiic~tliotl~ for the estimation of tcrti:iry :imines have been collic acid has bccn proposrd as a rcagcnt proposed. I n o i i ~ ,the iwc,tion of the for zirconium (426). Sano suggests the amine with ac.ouitic> :inhytlritle is used use of 1~2-nitrophenylfliiororlc for the and in the other, the, rc’nctioii with chlors1)cc.trol)hotonictrir drtcrmination of zirconium 1423). Frtst Gray RA has ani1 (428). Lon. coni4eiitr:itions of procaine may be tl(~tc~rmint~cl i ) c ~ t ~ rused i as a colorimctric reagcwt in caolorimetrizii~i~oiiir~ drtcrminations m (254). cally with sodium I .~-n:i]~~ithoquinone4-sulfonate (2.9), Spcat.tro! ihotometric Tlit’ (slimination of anionic intertitration of proc8:iinc n n t l other local forcwc in the flamc photomcxtric deanesthetics with so(Iiiini nitrite has tcBrmination of calcium has b w n discussed (561). Rcil1c.y ant1 Rildc~brand been suggrstcd (388): 1’ri)c:iine may be ) rvport, a mcthod of indircsct ~ j w ( ’ - sep:irateci from ~ ~ - : i i i i i i i i J 1 ~ c , i i ~ oacid ic
by electrophoresis a t p H 5 to 9 (250). The zones are tl&ctcd by Dragcndorff’s reagent. Thc principles correlating absorption characteristics with solution pH for proposycaine in the presence of procaine HC1 have been discussed (156). The formation of a cobalt complex is usc.d in thc colorimetric assay of amylocaine solutions (185). Diphcriylaniine is uscd as an internal indicator in thtx titration of aromatic amines with nitrite (136). The aut.0matic titration of aromatic amines with nitrous :wid has becn described (77). T‘anillin rcxcts with primary aromatic nniincs a t pH 0.5 to give a stable yellow color (121). A g:is-licluid chromatogr:iphic prorcdurc for the analysis of c,oniplex m i x t u r c ~ of aromatic amines has betw rcported (238). Kaatz and O’Rcilly disruss the infrared spectra of substitutctl anilines and give proce(lures for distinguishing between the various siii)Stitution patterns (241). 1Iisturc.s of ~tnilineand -V-cthylaniline w i i h :iii:iIyzed with a standard deviat,iori of :it)oiit It 1% using the N-H overtour : t i i d cwnbination bands in the ncsr infr:\rcatl (548). Weak bases such :IS anilinc, c:in be titrated potcntioiiir:tric:illy i n strong aqueous solutions i J f ncritr:il salts (112). Indicators may : i l x hi) uw(I. I-Xnphthylamine and 2iiapht 1iyl:iniinc may be determined co1orinic~tric:ilIyby their reaction with furfurxl t 360). C~itcc,lit,l:iriiiiit,smay be distinguished from corresponding Y-methylated deriixtivcs by reaction with ferric chloride2,2’-tlipyridyl reagent (406). Amino Acids. hrginine-containing licytidcs c a n be separated on .\riiherlite IRC-50 ion exchange resin v i t h a tot:rl Tecovcry of peptides of 997, (10). C’ystcline is determined colorimetric:~lly after the hydrolysis of the Sriitroso derivative as an azo dye (289). Ainp(~rnmctric titration of cysteine hrtncen two polarized electrodes is (wried out with a precision within 1 in millimole quantities (537). (ilycinc is separated from a mixture of riinino acids by precipitation with C X ~ C S S potassium triosalatochromate (459). After reaction with copper phosphate, the copper is determined by icdornetric titration. The microdetermination of hydroxyp r i i i i r c with chloramine-?‘ and pri~iiic~thylaminobenzaldehyde is reported A highly sensitive, specific trace for use in paper chromatograbeen proposed (469). ivan and Mijal report a n improved crilorimetric method for lanthionine (480). A new colorimetric procedure for lysine and its detcrmination after separation from othvr basic amino acids has been described (4f8,419). Ribonucleic acid is determined in
microgram quantities by a photo graphic-photometric procedure in monochromatic ultraviolet light (143). Coult discusses the use of cathoderay polarography in thc determination of gamma quantities of amino acids (107). A colorimetric mcthod for amino acids depends upon thc rcd color produced with p-b(~nzoquinonc under appropriate conditions (543). Mixtures of aniino acids (mi lie separated by gas partition chromatography after conversion t o the methyl esters (38). Borchers reports that the sensitivity of the alkaline copper salt method for amino acids is increased by the use of cuprizone (63). Improved procedures for the chromatographic determination of amino acids on sulfonated polystyrene resins has been rcportcd (332). Factors affecting t.he ultraviolet densitometry of amino acid chromatograms have been discussed in detail (516). The elimination of the interaction of amino acids during their chromatography by the use of their copper complexes is suggestcd ( 4 2 ) . Barbiturates. T h e use of x-ray diffraction methods for t h e idcntification of substituted barbituric acid derivatives has been reported (365, 553). Chatten and Levi have made an infrared study of the reaction of barbiturates with p-nitrobenzyl chloride ( 8 9 ) . The analysis of barbiturates by the pressed bromide sampling method and infrared spectrophotometry has been suggested (306). A colorimetric procedure for determining barbituric acid derivatives as mercury complexes has been reported (59). Barbituric and 2-thiobarbituric acids were detrrmined colorimetrically as the sodium or potassium violurate and 2-thioviolurate in a method suggested by Drozdov and Krylov (185). A number of authors report the determination of barbiturates by titration in nonaqueous solvents (175, 181, 280, 5Z4). The technique of alternating current polarography has been applied to phenobarbital (295). Distinctive analytically useful waves wcre obtained in p H 8 borate buffer containing 1;lI potassium nitrate. Phenobarbital lvas determined in both liquid and solid dosage forms with an accuracy of 27,. The separation of barbituric acid derivatives has been accomplishcd by countercurrent distribution (68). Barbiturates of pharmaceutical interest may be separated by means of clcctrophorcsis (81). The degradation products of phenobarbital sodium are separated by paper chromatography (2n‘f). Miscellaneous Nitrogen Compounds. Chlorpromazine may be separated from compounds of similar chemical constitution by elcctrophoresis and may be determined colorimetrically by its reaction with phosphoric and iodic acids (79, 80).
Polarographic oxidation stutlicBs of fourteen phenothiazine tranquilizcrs :it :I rotating gold wire electrode have bwn rcportcd (242). X-ra!. t1iffr:iction sl)c’c‘tra have heeii obtninid for t\vc,lvt* phenothiazinc tlcrivativcs ( I O ] ) . Son:iqueous titration nivtlioil~ 1i:ivc b t ~ ~ 1 i described for lironiazinc~. c~lilorl)rnmazine, and thcir salts (326). I1y:in describes a method for the colorinic~tric(ICtormination of unositlizc3cl pIit~notlii:~zine derivatives (.$12). d colorimotric mcthod for thc, ass 1y of prmi~iziri(~ :itit1 its separation from chlor~,rom:iziiit~ and proniethaziiie 111s lx~c~nrqiortcd (S6). Phenothiazine dcrivntivcs h:ivc bcen precipitated from solution by c x ( ~ s scadmium iodide ant1 detcrniincd complexomctrically nith Xylcnol Orange as indicaatnr (*?go). A nwthod for the assay of chlorproniaziiic in tablcts and injcctions is based upon the pink color developed with t u n p t o phosphoric acid (f5f). Freed and Salmre rcport that intlolc df>rivatices indistinguishable by tlir’ir fluorescence may be easily tliffcrcntiatcd by ~)hosphorescenccspectra ( 1 6 4 ) . Paper chromatography has bcen cstensivcilj- used in the separation of a large number of indolc conipoui~cls (373, 359, 585). The oxidation titration of isoninzid methanesulfonatc has bren esamincd with reference to the effect of starch, lactose, and carboxyniethylcellulose (487). 4 procedure to climinatc these interfering substances is proposed. A spectrophotometric nivthod for cycloserine and isoniazitl based on thc. differences in absorption :it 219 :ind 272 m p has been reported (557). Iodine monochloridc has brrn used for the quantitative ampcronictric titrstion of antipyrine in aqueous solution (176). Rlilligram amounts of antipyrine may be determincxl coulometrically (484). The method is rt~conimended for routine usc. A new procedure For the tl(tcmiination of aminopyrine is prcwntc~l,lx~scd on the osidation to diliy[lroxy:iiiiiiiopyrine and cleavage to dinictliylnmiiic which is titrated (62). Current methods for determining methenamine have been studied and recommendations made (128). The use of Kesslcr’s reagent for thc assay of methenamine and methenamine mandelate has bcen proposed (189). The gas-liquid chromatography of pyridines usiug a new solid support has been reported (120). A method for pyridine anti its dcrivatiws which utilizes the color change \\.hen pyridines are trcntcd with cyanogen cliloriclc is suggested (346). A solution-tcnil)crnture mcthod is suggcstrd for the. determination of small amounts of apicoline in pyridine (I). 2-Hydrox~~in~omcthyl -LY-nictl~ylpyridinium mcthanesulfoii:tte, an :tntitlote VOL. 33, NO. 5, APRIL 1961
F19R
to organophosphorous poisoning, may be estimated in aqueous solution and in biological materials by its ultraviolet absorption in alkalinc solution (110). Hoycis discusses the analytical characteristics of p-chlorobenzhydrylpiperazine derivatives and proposes methods for their extraction and determination (210). A colorimetric method for piperazine citrate has been proposed (317). p-Saphthoquinone-4-sulfonate is used for the colorimetric determination of azacyclonol (246). An accurate and sensitive photometric method for the determination of hydrazinophthalazines based on the formation of a yellow azine by the action of acid solution is described (546). Chen (92) reports the fluorescence characteristics of some salicyloyl hydrazones. Ultraviolet absorption spectra are used for the identification and analysis of certain quaternary ammonium compounds (367). A turbidimetric assay for benzalkonium chloride has been suggested (385). Titrimetric and gravimetric methods for the assay of benzethonium chloride using sodium tetraphenylborate are reported (362). Mercuric acetate has been used as an analytical agent in a nonaqueous titrimetric method for the determination of quaternary ammonium halides (274). Schill and Danielsson (434, 435) report the use of hexanitrodiphenylamine in the photometric determination of certain quaternary ammonium compounds. Titration of a number of quaternary ammonium salts nith sodium tetraphenylborate has been suggested (257, 51 2 ) . Nonaqueous titrimetry has been applied to the determination of benziminazoles (201). Porter reports the use of chromic acid as an oxidizing agent in the colorimetric determination of benzimidazolone and some derivatives (384). A procedure for the separation of naphazoline from its hydrolytic degradation products and a discussion of the kinetics of the base-catalyzed hydrolysis of naphazoline have been reported (471, 472). Wimer has reported data on the potentiometric titration of about forty amides using perchloric acid as the titrant (555). Acetophenetidin may be determined in tablet mixtures by a potentiometric method (24). Mixed formulations of phenacetin are assayed colorimetrically by the formation of indophenol (221). The infrared spectra of a n homologous series of monosubstituted amides is reported (43). Two tests for the detection of nitriles and amides designed to supplement the hydroxamic acid test have been proposed (502). Bovalini and Piazzi report the potentiometric and heterometric titrations of solutions of thioacetamide with silver nitrate (66). 120 R
ANALYTICAL CHEMISTRY
A fluorometric procedure is described for the determination of glutethimide in tablet formulations which is based on the fluorogen resulting from the reaction of glutethimide with concentrated sulfuric acid containing formald(4iyde (198). Alkaline bromine has applications as a volumetric reagent in the estimation of urea and thiourea (190). The coulometric determination of thiourea using a mercury anodc and a platinum cathode is reportcd (256). The paper chromatographic separation of thiourea and thiouracil is describrd ( inietric assay of sulfathiourea is b a s d upon its diazotization and coupling with thymol (667). Franchi dctcmiincs 1butyl-3. sulfanylurea and l-butyl-3-(ptolylsulfony1)urea by titration in nonaqueous solvents (162, 163). lleprobamate reacts with furfural in acid medium to produce a blue color which is measured a t 550 mp ( 2 7 7 ) . Hoffman and Ludwig have reported a colorimetric method for nieprobamatc using dimethylaminobenzaldehyde and antimony trichloride in acetic anhydridc (207). The color is proportional to concent,ration from 0.5 to 10 pg. 3,j-Dinitrobenzoic acid and its steroid esters may be determined by a polarographic method in lithium chloride (47). The colorimetric ferrous sulfate method for determining organic nitrate has been modified to include aliphatic and cyclic nitramine compounds (275). The color produced is stable for 2 hours. A polarographic method has been reported for the assaj- of 2-acetamido-5nitrothiazole in pharmaceutical compounds (306). Bandelin and Pankratz have described a colorimetric procedure for the determination of organic nitro compounds used as vasodilators (27). An analytical study of nitrofuramine has been reported (308). PHENOLS, ALCOHOLS, A N D CARBONYL C O M POUNDS
Phenols. A spectrophotometric method for determining the resorcinol content of an ointment is based on the color reaction of resorcinol with 9hydroxyxanthene (660). The determination of the capsaicin content of capsicum and its preparations is made by spectrophotometric methods (9). Phenolic compounds found in pharmaceutical preparations can be determined by forming a color with 4-aminophenazone (235). Huckel and Wehrung report that many phenols of pharmaceutical interest form stable complexes with ferric chloride which can be used for quantitative photocolorimetric determinations of these phenols (213). Duerr and Pappas applied this ferric chloride reagent to the determination of diethylstilbestrol (137). Banes developed a colorinietric procedure for the
determination of dic~tli~~lstill~c~strol n-liic,Ii overcomes the difficulty due to thc prosence of phenolic contaminants (281, A measurable orange-yc,llow color i h produced by rracting :inthralin \\-itti sodium nitrite (35s’). I’ol!~hytlros>~ phenols can bc dctermincd colorinicxtrically after reacting with p-tlinic~tli\~laminob~~nz:iltl~~h~ctc (262). H e r d m :inti R o g m PuggCst tho de)terminxtion of 1- and 2-naphthol 1))measurcincmt of tliv fluorcwc~iiw i i i alkali solution a t 480 and 426 nip, rvspectively (902). Sewr:il spot tests ivhich can be used to int1ic~:itc~ tlic, l)r(’>encc of phenolic compounds l i : i \ - o I X Y , I I reportcd ( I 53, :?I 7’. 220). JanBk and his coivorlic~rsli:i\-c. c,:irri(,ci on a rather i,stcnsivc, stiitiJ- o n tliv gas-liquid chroniatograpliic~:Lii:il>.sis of mono- and dih>-dric plic~nol- ( 22:229). These author? 1i.d the, -;lic~.ific* elution volumes for v:irious Iihcviols of pharmaceutical intercst . Othvr :iutliors who have carried on gas-liquitl rlironiatographic studies of ph(,nol systc’nis suggest that the ph(,nols be, cmnvc~rtc~cl to thcir triniethylsilj~l othc>rs (2;S) or their methyl or ethyl othc>rsids) l)t.forcs analysis. Papcr c1iroiii:itogr:il)lii~~ methods have been uscd for tlic tion of phenol and isorncric (330), the determination of c:it(~.IioIs (566), and the sc3pnration of :ilkylsubstituted phenols (169). ‘1‘lic R,i values of 37 phenols and phenolic ctlicrs for five solvents have becn tabu1:itcul along with the characteristic rolors :mi required reaction timrs i v i t h ncntr:d silver nitrate in aqueous ncctonc ( 7 6 ) . Xitrophenol systems have hcc~ rated by use of paper clectrol (520, 521). Phenolic hydroxyl can bv dctcrmincd by a nonaqueous titration in pyridinc ~ i t han antimony indicating clrctrotic~ and platinum reference rlwt rot let :iccording to Greenhow and Pniitli (188). A photometric titration p r o c ~ ~ l u rthat c~ can be uscd for the t1rtc~riiiiii:itioriof phenols a t concentrations of 10-R.ll or greater has been dcscrihcti (290). Phenols have been drtcrmined as weak acids using a high-frequency conductometric titration procedure (226). Phenols and cresols can bc dctermined by an electronietric method based on the electrolytic gencration of bromine and its subseqncnt rwction with phenols (f14>2SS). Alcohols. A spcc t i o phot o mc,t I,ic: method for determining hut>-lalcohols is presented (440). In this mc~tiiod.the, butyl alcohols are convertcti into tlvir nitrites and thcir absorption spr(1tr:L in the ultraviolet region of tliv spcct~ruin are observed. The r:idi(xl \vIii(,li is bound to the 0 N : O group cscrt- :L measurable influcmcc on tlic posit ii)n of the band and its m:isimuni a11sorlltion. This spectropliotomc’tric‘ nicthocl is furthrr applied by this author to thi.
mobile phase. lfolar absorptivitirs for a numbcr of altlchyrlc~santl kctows are listcd. Shcrmn and R i ~ m n nmade a study of thc dution of kctoncs t,hrough ion exchange rrsins with aqucous solutions of acctic acid or the lon-er alcohols as clucnts (448). Carbonyl compounds have bcen dctermincd bj- osimation in methanolisopropanol and titration of the rsccss hydrosylnminc with pc~rcxhloric acid to a potentionic,tric or visual cnd point with IIartius ycllow as indicator (166). Altlehyctcs r a n be detcrmined by titrating the hydroc.hloric acid produced in t,he osimation rcaction with sodium hydroside solution in the prescncc of methyl orangc (370). Polarographic tcnis of pharniaceutical intcrcst have been made. Camphor tlc~rivntivcs ( I ? ) , %-phcnyl-I,3indandione (I@), santonin (4.99),and formaldehyde antl acetaldehyde misturcs (420) h a w all been detcrmined by polarographic techniques. Vanillin can be determined gravimetrically as its semicarbazone as described by Kaistha (245). Sobotka and Trutnovsky suggest the use of sodium borohydride as a reducing agent for carbonyl compounds in a microdetermination procedure (458). The hydrogen gas which is liberated from the excess reagent on addition of acid is measured in this procedure. Several color tests that can be used in detecting the presence of carbonyl compounds hare been described (12, 431, 432, 464, 465). SULFUR-CONTAINING COMPOUNDS
Sulfonamides. -\ eolorinictric method for the determination of p-aminomethylbenzenesulfonamide is described, based on its reaction n i t h lactose in alkaline solution (85). Sulfanilamide is detected in the presence of other sulfonamides by a colorimetric reaction n i t h cupric acetate (186). Sulfanilamide derivatives are detected by diazotization and coupling with resorcinol (148). Matsuno and Mishimura propose a spectrophotometric method for the determination of mised sulfa drugs (311). Sulfonamides may be titrated by anhydrous perchloric acid by Fritz's method (320). An acetylation method is described for the assay of sulfonamides in powders, tablets, and injections (170). Vanillin in sulfuric acid is used for the idcntification of a number of sulfonamides (467). Some sulfa compounds have been identified by the fluoromicroscopy of their Schiff's bases (504). Goudsivaard lists four reagents suitable for differentiating commonly used sulfonamides (186). The spectrophotometry, chromatography, and electrophoresis of chlorothiazide are discussed by Ruggieri (411). Nonaqueous titration for the
determination of cliiorothiazitic has been proposed using 0 . 2 s potassium hydroside in isopropyl alcwhol (88). Rehm and Smith (3-98) r c y ~ fphotometric methods suitable for the routine determination of hydrochlorothiazide and its hydrolysis product, 4-aniino-6chloro - m benzenedisulfonaniide. The riisulfonamide resulting from the hydrolysis of chlorothiazide is dcterminctl by reaction with nitrous acid and coupling of t,he diazonium compound nith chromotropic acid. The rcsulting rcd color is mcasured a t 500 mp. Thiols and Miscellaneous Compounds. A scheme for the colorimetric determination of microgram amounts of thiols is based on their convvrsion t o S-nitroso derivati\,es which yield nitric acid on hydrolysis (429). The nitric acid is used in the formation of a brilliant azo dye from sulfnnilamick. Colorimetric microdetermination of thiols in alcoholic medium is carried out by reacting with X-ethylmaleimide (71). A spectrophotometric method utilizing S-ethylmalcimide in phosphate buffer has been reported (5). .4 colorimetric mcthod for mercaptans based on their reaction with di-(p-nitropheny1)disulfide has been published (246). A titrimetric method for determining alkyl mercaptan, dialkyl sulfide, and alkyl mercaptan-alkyl disulfide mistures has been proposed (231). Thiocarbon>-l groups have been tietected by infrared spectroscopy (461). Thiopental sodium has been detcrmined coulometrically by means of bromine generated electrolytically (249).
-
METHODS AND TECHNIQUES
This section is not intended to be a complete review of all the general methods and techniques which have been presented recently but merely a summation of those articles that have been found applicable t o industrial pharmaceutical analysis. In the last few years there have been many publications which facilitate the use of gas-liqufd chromatography, a tool TI hich has already found many applications in the pharmaceutical industry. Tenney has tested 18 different substrates as to their selectivity towards types of hydrocarbons and oxygenated compounds (495). Bayer has also investigated the selectivity of the internal phase and the choice of a support (36). Raupp investigated the use of such internal phases as didecyl phthalate, bis(ethylhexy1) sebacate, silicone oil DC 200, tetraethylene glycol dimethyl ether, fluorinepicric acid, and polyethylene glycol (393). High-temperature gas-liquid chromatography of aromatic hydrocarbons has been studied by Baxter and Keen (35). Ogilvie and VOL. 33, NO. 5, APRIL 1961
121 R
ot'hers also considered gas-liquid chromatography systems at high temperatures (353). Gas-liquid chromatography of many natural products and tht4r derivatives ( I & ) , as well as synthetic arid natural aroma materials ( S T ) , has been discussed. Bennett and others report on the use of gas-liquid chromatography for trace analysis (46). A procedure has been presented for estimating the smallest amount of sample in gas-liquid chromatograpliy t h a t can be determined reliably by R detector (236). Thc effect of thcx carriw gas on the sensitivity of :t thermal conductivity detector has been considercd (396). 1Iessni.r and others have reported on the rorrelation that cssists betn.etw the rclativc thc~rmaldetwtor response and the molecular weight within a structurally similar homologous series (319). This enables one to prcdict responses values for given compounds whrw rc1:itcXtl inforniation i1 eswllent rcviews of thc, broad field of gas-liquid cliromatography have been publislied rccimtly ( 1 93, 195,405). Infrared spectrophotometry is :mother method which is used widel!. in pharmaceutical analj.sis. The use of infrared spectrophotometry as a tool for quantitative analysis has becn tliscussed by Fabbri (150) and by Cupples (113). Kard and Philpotts have m:tdc a study of the errors due t o solute-solvent interactions in quantitative analysis by infrared methods (542). Refinements in the use of the potassium bromide disk technique havc. been presented (234, 402,' 440). The infrared analysis of been reviewed by D Rogers has studied the influence of spectral slit width on the absorption of visible or ultraviolct light by pharmaceutical substances (403). Drug identification using ultraviolet spectrophotometry has been reviewed by \Villiams ( 5 5 2 ) . Tunnicliff presents mathcmatiral relationships which show the effcct of light absorption by the solvent on absorption measurements (,505). Methods for obtaining correction factors for fluorescence spectra as tlctcrmined with the Aminco-Bowman Speetrophotofluorometer have been discussed (549). Adaptions of the Beckman l\lodrl B (237) and D U (297) spectrophotometers for use in t h r measuremcnt of dilute fluorescent solutions have been presented. A review of spectrofluorometry and its application to chrmical analysis has been published (32). Stock and Purdy have reviewed the potentiometric electrode systems used in nonaqueous titrimetry (4'74). The selertion of bimetallic electrodes for potentiometric titrations is discussed by Ueno and Tachikana (510). A review
122 R
ANALYTICAL CHEMISTRY
of titrations in nonaqueous solvents has been presented (587). A method for the quantitative determination of drugs by means of ion exchange and spectrophotometry has been described (192). The development of analytical methods for pharmaceuticals and drugs has been discussed by Stcphen (470). Vlict has suggested an in vitro procedure for measuring the rat'e of drug release from timed release tablets and capsules (526). A general method of analysis of organic materials based on the kinetics of the reactions involved has been described by Blaedel and Petitjean (60). This method is applicable t o integral-order or complex reactions. An in vitro colorimetric method for determining the effect of the hydrophil-lipophil balance on drug rdease from ointment bases has been reported (466). Since the analytical chemist in the pharmaceutical field is intirnatcly involved in the stability testing of drugs, a mention here of the recent publications on the general methods involved seems appropriate. Lachman has outlined the methods involved in the prediction of the shelf life of parenteral solutions from accelerated stability studies (276). AlcLeod and others present the method for the prediction of expiration dates for multivitamin preparations by accelerated storage tests (301). A review of the stability testing of pharmaceuticals has been presented by Cooper and Lachman il04). LITERATURE CITED
(1) Ade:, I, A,, Proctor, Ii. A , , -4,ialysl 84, 461-3 (1959). 1148) En, II., Ostronski, W., Kralvczyk, A , , Zesryty Problemowe :Tankc' Polskiej 9 , 8 1 ( 1 956).
(143) Edstrom, J. E., J . .Yeurocheru. 3, 10@-6 (1958).
CIIE:>I, 31, ltl!) -10 (1950). ( 103) Conn, K. B., J r . , Davis, R. E., \-/tiitre 183, lfK5.3-5 (1959).
1144) Eelinton. G.. Hamilton. It. J:. Hedges, K.,'Iisphilel, Ii. A,, 'Chem. R. Ind. ( L o n d o n ) 1959, 955-7. (145) Ek, J., Haltman, E., Scand. J . Clzn. Lab. I n ?est. 9, 915-16 (1957).
1042-4 (1959). (161) Forist, A. A , , Theal, S,,Struck, \V, .I., Ibid., 31, 100-2 (1959). (102) Franchi, G., Atti accad. Jsi'ocrit. S i e m 4 , 56-60 (1956-57). (163) lbid., 61-4. (164) Freed, S.,Salmre, IV., Science 128, 1341-2 (1958). (165) Friedman, P. J., Cooper, J. R . , .%S.iL. CHEM. 30, 1674-6 (1958). (166) Fritz, J, S., Yaniamiira, S. S., Bradford, E. C., I b i d . , 31, 260-3 (1050). (167) Fuchs, I,,, M'irhtl, )I., Jnrhs, H., rlrck. Pharni. 291, 103-208 (1058). (168) Fujimoto, R., Use, S., J . Pharni. Soc. Japan 79, 371-4 (1959). (16!lj Fiijiwara, F., dugimoto, C., R a g a k n l o K6yy6 32, 265-8
(l!)58).
( 1 70) Gaind, I., Rezinger, S . S . , Zhttr. .4nal. Khim. 13, 003-7 (1058). (172) Garrett, 1:. I
