Colorimetric Determination of Organic Nitro Compounds Used as Vasodilators F. J. BANDELIN and
R. E. PANKRATZ’
Analytical Control laboratory, Flint, Eaton and Co., Decafur, 111. ,The colorimetric ferrous sulfate method for nitrates has been modified for the determination of various organic nitro compounds used as vasodilators in the treatment of hypertension. This reaction appears to be group; the specific for the -ON02 resultant color obeys Beer’s law. Procedures and data are given for determination of the nitro compounds in their usual carbohydrate diluents and in certain pharmaceutical dosage forms. The method is rapid and accurate within the usual limits of colorimetric analysis.
T
nitro esters of organic nlcoliols are extensively used in niedicinc in the treatment of hypertension. Pharmacologically they produce vasodilation, thus reducing the blood pressure. Their assay as raw material and in pharmaceutical preparations is a problem in the pharmaceutical control laboratory. 91though several methods have been published for one or more of these compounds, no single comprehensive method for this class has appeared nor has any systematic investigation of any mctl~od lwen published. The USP X I V method for mannitol hrsanitrate tablets ( 7 ) ,Ivhich consists of extracting the mannitol heyanitrate from the powdered tablets Fvith ether, rvsporating the ether, and weighing the residue, is not specific and cannot be used in the presence of other ether-soluHe materials. Cannon and Heurman ( 2 ) published a procedure for mannitol hexanitrate, utilizing Devarda’s alloy in alcoholic solution to reduce the nitro groups to ammonia, with subsequent distillation and titration. This is the official L S P method for the determination of nitroglycerin in nitroglycerin tablets. The colorinietric method for pentaerythritol tetranitrate of the British Pharmacopoeia ( 1 ) n-as applird by S:moff for the assay of mannitol hewnitrate (6). This procedure, using phenoldisulfonic acid, although sensitive, develops a yellolv color with a maximum absorbance a t 408 mp, a region in t h r near ultraviolet where a great number of interferences occur. HE
Turthcr111~c~. thc cwlor is ilclicn(lcnt upon the purity of the rc:igcnt, a s prwc’ncr of the in0110 iionin. r a u ~ c s:I ( l ~ v i x tion in w ~ v length c a1)Yorption. Rcceiitly Sn ann and ,itl:ims (6)proposed a rolorimetric nicthotl for nitrocellulose t m e d on the classic ljrown ring test ( 3 ) . A reagent composed of 0.5yo ferrous sulfate in 75% sulfuric acid reacts n i t h the nitro groups to produce a stable purple color which, according to Feigl ( d ) , has the structure FcSOJO. The reaction, with crrtain modificntions, is applicable to the colorimetric tlcterniinntion of the nitro compounds. generally used as vasodilators, in various pliarniaceutical dosage forms as w l l :I? in ndmisturc with lactose or mnnnitol. The nitro compound? to which the method is applied in this study arc pentaerythritol tetranitrate, mannitol hexanitrate, glyceryl trinitrate, inositol hexanitrate, and amyl nitrite. (Thew compounds, because of their explosive nature, arc usually handled and compounded in pharmaceutical preparationq diluted with lactose or mannitol.) I n adapting the original method of Swann and ddams ( 6 ) to the organic nitro compounds, certain changes in time, temperature, volumes. acid convmtration, n ave length for dcterniining nhorptivity, and method of evtraction ncre found nccessary. Use of sodium sulfite as an accelerator specded up the rmrtion time. PREPARATION OF STANDARD CURVE
Because of its purity, stability, and
Tn o grams of pota5sium nitrate (nnalytical reagent grade), accurately weighed, wAre dissolved in distilled water to make euactly 500 ml. Aliquots of 2, 4, 6,8, and 10 nil. of this solution 11-ere pipetted into each of five 100-ml. volumetric flasks and evaporated to drynes3 under reduced pressure n ith gentle m r m i n g . To each flask was added 2 nil. of glacial acetic acid to take up t h e r e d u e , the solution was diluted to volume with ferrous sulfate-sulfuric acid reagent, and finally 5 mg. of sodium sulfite was added. The ferrous sulfatesulfuric acid reagent \\-as prepared b j dissolving 0.5 gram of ferrous sulfate hcptahydrate (analytical reagent grade) in 2.50 ml. of water, cautiously mixing with i 5 0 ml. of concentrated sulfuric acid (nitrogen-free), and cooling to room temperature before using. The flasks w r e tightly stoppered and in\ erted yeveral times and the niilture wns &red n itli a magnetic stirrcr. The color devcloped rapidly and the ahsorbance over the range of from 400 to 600 i n p was plotted (Figure 1). The most desirable point in this range for determination is a t 510 nip. Spcctrophotometric readings of the dilutions obtained a t this wave length were plotted as the standard curve (Figure 2 ) . The absorptivity (extinction corfficient), E:,,?, for potassium nitratc nt 510 mp is 24.07. Considering that potassium nitrate contains 13.867, nitrogen. the absorptivity for the nitrogen available for color by this reaction is 173.6. This theoretical value is plotted
0.7
0 6.-
0 3;-
- ~ --- ~~
~~
400
Present address, Fort Dodge Laborstories, Inc., Fort Dodge, Iowa.
ncccs4ldity, potns-iuni nitrntc \\:is i e Iwtt~tlas :I prini:iiv itnnilnrrl f r r the. nitro cdor.
450
+:AJE
1
Figure 1 .
~~
500 LENGTH
550
_____-
600
n_u
Absorption spectra of nitro color VOL. 30, NO. 8, AUGUST 1958
1435
i n Figure 2. Standard curves for all tlw organic> nitro conipound~could bc prepared eaily, ubing iiliquots of 40 to 200 nig. per 100 cc. after extraction as outlined. APPLICATION OF ORGANIC COMPOUNDS
0 6.
I 0 5-
NITRO
-411 the organic nitro compounds under consideration, with the exception of amyl nitrite, are solids and all are eyplosire. They are therefore available commrrcially only as mi.cturt.7 with desensitizing 511gars or ninnn 1t 01, I n each deterinination a quantity of the sugar-organic nitro compound equivalent to approximately 100 mg. of the nitro compound was accurately weighed and placed in a suitable glassstoppered flask. To this was added 25 ml. of chloroform (pentaerythritol required the use of acetone instead of chloroform), the flask was stoppered, and the mixture was stirred with a magnetic stirrer for 1 hour. The mixture mas then filtered into a 100-ml. volumetric flask and the residue on the filter FT ashed with t n o additional 10-ml. portions of chloroform. The combined chloroform filtrates ere evaporated to dryness on a steam bath under reduced pressure until all the solvent had evaporated. To the flask was then added 2 nil. of glacial acetic acid, which mas swirled about the inner surface of the flask to take up the nitro compound, then diluted to volume with ferrous sulfate-sulfuric acid reagent, and 5 mg. of sodium sulfite was added. The flask was stoppered and agitated with a magnetic stirrer. If a minute quantity oi white precipitate developed during color formation, it was removed by centrifuging. The absorbance of the solution n a s then determined with a Beckman DU spectrophotometer a t .510 mp.
0 2
Figure 2. 1.
2. 3.
4.
Inositol hexanitrate Nitroglycerin Mannitol hexanitrote
5. 6.
obeyed oyer the range used and the color was stable in all C R S W for a t least 24 hours. The volatility of amyl nitrite made impossible the extraction and evaporation technique used above. I n this case the compound was added directly to the glacial acetic acid with a micropipet and this solution \vas transferred to the 100-ml. volumetric flask for color development with the aid of the reagent and finally ma& u p to volume with thr, rragent . EFFECT
Standard curves for these compounds were plotted using aliquots of 4, 8, 12, 16, and 20 mg. per 100 cc. for color development (Figure 2). Beer's law was
Table 1.
Standard curves for various nitro compounds and calculated curve for nitro nitrogen
OF
VARIABLES ON COLOR DEVELOPMENT
Optimum concentrations of sulfuric acid and ferrous sulfate were determined by varying the concentration of these
Determination of Nitro Compounds
Nitro Compound, 70 Detd. by by Dumas eutn. anti wight Colorimetric
% Kitrogen Calcd. from yo?j by Dumas
i.12570 mannitol hexanitrate in lactose 20% pentaerythritol tetranitrate in lactose 7.134 inositol hexanitrate in mannitol 1077. nitroglycerin i n plactose
1.30
6.993
3.29
18.57
1.35
7 .'LBO:
1.78
10.10
Table II.
1436
e
ANALYTICAL CHEMISTRY
Yfi
K i n Compd. 18 59 18 58 17 7% 18 50 11 96
7.138
19.81
19.75
7 285
7.221
9.90
10.00
two compouiids over a wide range. Xaximum color development occurred with the recommended concentrations. CORRELATION
OF
RESULTS
Xitrogen determinations on the sugarnitro compoiind mixtures were carried out by the micro-Dumas method. From tlic. value thiis obtained tlw amount of nitro compound present in the sugar mixture n-as determined. Another corroborative method vias extraction of the nitro compound from the excipient with an appropriate organic solrent, and evaporation of the solvent a t less than 35" C. The residual compounds were dried in a vacuum desiccator and weighed. The results of these two methods were compared with those obtained with the colorimetric method (Tahle I). If the absorptivity of potassium nitrate (24.07 a t 510 mp) is divided by the grams per cent of nitrogen in potassium nitrate, a factor of 1.72 is derived. The grams per cent of nitrogen in any of the nitro compounds multiplied by this factor gives the absorptivity of that compound. C a l d n t e d valurs were in good agreement n it11 actual values obtained from sprctropliotomcter rcadings (Tablc 11). DISCUSSION
Absorptivity
Theorrt.
bhnnitol hexanitrate Inositol hexanitrate Pentaerythritol tetranitra te Nitroglycerin ilmyl nitrite
7.140
Pentaerythritol tetranitrate Potassium nitrate Amyl nitrate
E'I
n
____ C:ilcti. from c L S ~
32,3 32.3 30 8
32 1 20 8
L",
.
-.
-~
Found :3 1 :i 52 4 30 4
3% 1 20 5
The optiinuin conditions for the coloriinetric deterinination of this group of organic nitro compounds n rre identical from the standpoint of concentration of the reagent components and maximum color development. After extraction with solvent, considerable caution must
I ) ( % ot)avrvctl i n c.r:cporation of the aolr e n t and drying, The temperaturo should not exceed 35" C., as thc compounds decompose a t higher temperatures or on prolonged exposure to air. This is especially true of nitroglycerin and pentaeryt'hritol tetranitrate. Amyl nitrite is volatile and care must be taken to avoid loss on prolonged heating or exposure to air. Addition of glac-ial acetic acid to the nitro compound residue after extraction seenis to stabilize the compound and solubilizes it for inow rapid color development. Attempts to confirm the totnl nitrogcn contcnt directly on rxtractcd material by the micro-Dumas nicthod were unsuccessful, yielding low nitrogen valLICS which indicated that considerable nitrogen was lost betir-een the time it. was extracted and the time the Dumas dcterminations were made. All microDumas determinations were run on the sugar-nitro compound mixtures. This reaction seems to be specific for the pharmacologically active -OXO, group, since nitromethane, nitrobenzene, nitrotoluene, and nitrourea gave no color. The reproducibility of the method with innnnitol hexaiiitrate in lactose alone and in cornprcasc3d tahlcts in cornbination with rauwolfia, rutin, and bes-
Iciiiovod froiri the sugar Iiy solrrmt e l Table 111. Reproducibility of Colorimetric Determination o f Mannitol Hexanitrate
M g . er Tablet
rc in
Bound (Calcd. 16 M g . pcr Tablet)
1,actose 7 09 7.12 7 11 7 08
16 1
7.11 7.14 7.10
ktction before color drvclopment. In spite of thc preliminary solvent estrartion necessary in this method, it is considerably less time-consuming than the degradation-distillation methods. Rrsults are rrproducible and well within the usual limitations of colorimetric methods. For these reasons the method lends itself we11 t o pharmaceutical control laboratory work.
16.1
15.8
15 9 16.2 lt5 8
7.10 7.09 7.12 Average 7 . I O St,d.dev. 0 . 0 178
lG.O
I d , 97 0.141
LITERATURE CITED
(1) British
Pharmacopoeia, 1932, 7th Addendum, p. 46, Constable &! Co., 10 Orange St., Lricester Square, 1,ondon \VCL%, 1943. (2) Cannon, J. H., Heurman, R. F., J . ilssoc. O j i c . 4 g r . Chemists 34, 717 11951). (3) Desbassins de Richemont, Chem. \
peridin is shown in Talile 111. Siinilar results were obtained with the other nitro conipounds. A number of medicinal agents commonly used in conjunction with these nitro compounds in therapy apparently do not interfere in determinations n i t h this method. These include rauwolfia, reserpine, phenobarbital, sodium pentobarbital, rutin, hesperidin, and theopliyllin. In a11 c a s ~ sthe nitro compound was
,
Zenlr. 1835, 782. (4) Fcligl, Fritz, "Spot Tests," Vol. I, 4th ed., I).299, Elsevier, New York, 1954. (5) Sarnoff, Evelyn, J . Sssoc. Ofic. Agr. Chemzsts 38, 637 (1955); 39, 63 (1956). ( 6 ) Swann, M. H., Adams, M. L.. ANAL. CHEM.28, 1630 (1956). (7) U. S. Pharmacopoeia, 14th Revision, p. 331, Mack Publishing Co., Easton, Pa., 1950.
REFLIVED for review August 29, 1957. .icceytcd February 21, 1958
An a lysis of Am ino- Fo rma Ide hy de Resins JOSEPH
C. MORATH and JOHN T. WOODS
Organic Chemicals Division, American Cyanamid Co., Bound Brook, N. J. F M e t h o d s f o r analysis o f textile resins of the amino-formaldehyde t y p e a r e reviewed and summarized and, in some instances, a r e given in detail. Methods a r e included for the amino portion of the resin and the various forms o f formaldehyde and/or methanol. The chromotroDic acid method for total formaldehyde should b e of particular interest because of its speed and simplicity.
0
0
H&C?jHCH,OH l\lononiethylol urea
Dimethylo1 urpa
T
varii4y of rel-iiii of tlic uiiiiiio-foriiialtIfli?-dc type wet1 iii tlw tcstilc intlustrj. are constantly groning. Those most, romnionly used are formed b y the condensation of formaldehyde with melamine, urea, ethylene urea, or thiourea. They may be sirnplc condensations or cocondensstions of tn-o or more amino-type resins. liesins m a j ~also he rnetliylatcd or utimetliylated. The following structures illustrate a fen of the forms in which uiea-formaltleli!-de resins may exist.
Ii
I
I1
111,;S ~ M I ~ L I: i{i i t l
0 11
SH-C-S-CH~OH I
I
CHZ I
CH2
€I()CII:---S---C---s
I1
~i
0
Dimethylol urea (ring dimer)
HC)CHJTHCNCH,OH CH,NHCSI CHzOH 'I 0 Ilimethylol urea (chain dimer)
As the ~ i u m b e r and complexity of these resins grow! the need for analytical methods grows ako, to gire information on simple resins as \yell :ih niisturcs of rwirih.. This papvr w i i i i i a r i ~ r bthv i i u n i e r o i ~ ~ rnetliod~,iciittc,recl throughout the litcrature. for the determination of the aniino or formaldehyde portions of the resins, and in some cases proposes modifications. The following resin components are discussed. Amine Cres
1:thylenrI U I N llelnmine Thiourea
Formaldehyde Methanol Total Total I'rcle Free Methylol XIethylated Methylenic
VOL. 30, NO. 8, AUGUST 1958
1437
