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Utility of -Acceptors in Alkaloid Assay.

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310177
rr-Acceptors in Alkaloid Assay
485
Arch. Pharm. (Weinheim) 310,485-494 (1977)
Aly Taha* and Gerhard Riicker
Utility of rr-Acceptors in Alkaloid Assay
Institut fur Pharmazeutische Chemie der Westfilischen-Wilhelms-Universitat,Miinster
(Eingegangen am 26. Juli 1976)
The interaction of alkaloids with selected polyhaloquinone and polycyanoquinone n-acceptors was
found to yield intensely coloured radical ions. This finding was developed into a sensitive spectrophotometric assay for alkaloids of general applicability and with adequate accuracy and precision. 7,7,8,8-Tetracyanoquinodimethaneis used as the reagent.
Anwendung von rr-Acceptoren zur quantitativen Analyse von Alkaloiden
Akaloide geben mit rr-Acceptoren vom Typ der Polyhalogenchinone und Polycyanochinone intensiv gefirbte Radikal-Anionen. Auf Grund dieser Reaktion wurde eine allgemein anwendbare,
empfindliche photometrische Bestimmungsmethode fur Alkaloide entwickelt, die auf der Umsetberuht.
zung mit 7,7,8,8-Tetracyanochinondimethan
n-Acceptors, such as tetracyanoethylene (TCNE), 7,7,8,8-tetracyanoquinodimethane
(TCNQ), chloranil and 2,3-dichloro-5,6-dicyanoquinone
(DDQ) are known to yield
charge-transfer complexes and radical ions with a variety of electron donors which
include amines’ - ’). This donor-acceptor interaction has not been investigated hitherto for alkaloids as a group of n-electron donors which were recently demonstrated to
participate in charge-transfer complexation with iodine4- ‘). Furthermore, relatively
few previous publications concerned the analytical applications of this interaction,
and were mainly centered on indoles and aromatic amines with TCNE’-’’).
* On leave of absence from the University of Assiut, Assiut, Egypt. This research was supported
by a grant from the Alexander von Humboldt Foundation.
1 L. R. Melby in “The Chemistry of the Cyano Group”, pp. 639-670, S. Patai, Editor, Interscience, New York 1970.
2 R. Foster “Organic Chage-Transfer Complexes”, Academic Press, London 1969.
3 C. N. R. Rao, S. N. Bhat and P. C. Dwivedi, in “Applied Spectroscopy Reviews”, Vol. 5
pp. 1-1 70, E. G. Brame, Editor, Dekker, New York 1972.
4 A. M. Taha, A. K. S. Ahmad, C. S. Gomaa and H. El-Fatatry, J. Pharm. Sci. 63, 1853 (1974).
5 C. Gomaa and A. Taha, J. Pharm. Sci. 64, 1398 (1975).
6 A. M. Taha and C. S. Gomaa, J. Pharm. Sci. 65,986,(1976).
7 D.N.Dhar, Chem. Rev. 67,611 (1967).
8 G. H.Schenk, P. Warner and W. Bazzelle, Anal. Chem. 38, 907 (1966).
9 G. H. Schenk, M. Santiago and P. Wines, Anal. Chem. 35,167 (1963).
10 R. A. Heacock, J. E. Forrest and 0. Hutzinger, J. Chromatogr. 72, 343 (1972).
Overlag Chemie, GmbH, Weinheim 1977
486
Taha und Rucker
Arch. Pharm.
In acetonitrile solution alkaloids were found to yield intense colours causing characteristic long wavelength absorption bands, frequently with numerous vibrational
maxima, in the electronic spectrum (Table 1, Fig. 1). Data of Table 1 indicate that
the predominent chrornogen with TCNQ is the blue coloured radical-anion, TCNQ?
(l),which probably resulted through the dissociation of an original donor-acceptor
(DA) complex with alkaloids (Equation I). The dissociation of the DA-complex was
~).
suppromoted by the high ionizing power of the solvent a ~ e t o n i t r i l e ~ ' " ~ 'Further
port to this assignment was provided by the identity of the absorption bands (Table
1) with those of TCNQ? anion radical produced by the iodide reduction method in
acet onitri1el3).
DA-ComDlex
NC A
0 CN
TCNQ
TCNQ?
1
The resulting bands of the alkaloids with DDQ, fluoranil and chloranil (Table 1)
are similar to the maxima of radical-ions of these acceptors obtained by the reduction
ith alkarnethcd'41 and coincide with the values reported in the literature ' , I 5 - l 7 ) . W'
loids arid TCNE, however, the characteristic "cocksornb" shaped absorption band
of TCNE? radical-ion ( 2 ) with reported18) maxima in acetonitrile centered at 432 nm,
was not formed. Instead, doublet at 393 and 412 nm in acetonitrile (at 398 and 419
I1 R. Foster and J. W. Morris, Red. Trav. Chim. Pays-Bas 89, 636 (1970).
12 W. Liptay, G. Briegleb and K. Schindler, 2. Elektrochem. 66, 331 (1962).
13 L. R. Melby, R. J. Harder, W. R. Hertler, W. Mahler, R. E. Benson and W. E. Mochel, J. Am.
Chem. SOC.84, 3374 (1962).
14 H. A. Terrey and W. H. Hunter, J. Am. Chem. SOC.34, 702 (1912).
15 A. Yamagishi, Bull. Chem. SOC.Jpn. 48, 2440 (1975).
16 N. Skai, I. Shirotani and S. Minomura, Bull. Chem. SOC.Jpn., 44,675 (1971).
17 A. Fulton, Aust. J. Chem. 21, 2847 (1968).
18 0. W. Webster, W. Mahler and R. E. Benson, J. Am. Chem. SOC.84, 3678 (1962).
310177
n-Acceptors in Alkaloid Assay
487
in 1,2-dichloroethane) was formed which corresponds to the 1 ,I ,2,3,3,-pentacyanopropenide anion (PCNPF) (3). PCNP? was reported to be produced by the action of
bases TCNE in presence of waterlg). Therefore it may be assumed that there was
enough water to cause the basic hydrolysis of TCNE directly to PCNPQ’9-2’) at the
low concentrations of reactants employed in the analytical procedure (ca. 3 x lo-’ M).
This finding is in agreement with previous reports on analogous ~ y s t e r n s ~ ’ - ~ ~ ) .
From the quantitative point of view, PCNPO is preferable to TCNE?.on grounds
of its higher em, value (22 600)19)as compared to TCNE? (7 10O)l8) giving some
3-fold increase in sensitivity.
CN
NC,
OC-C;
/
NC/
CN
yN
/CN
C=C-CO
NC’
CN
NC,
TCNE?
PCNF
2
3
The failure of papaverine to interact with chloranil and fluoranil (Table l),
is certainly related to its weak basicity (pKa 6.4 at 25”) which is insufficient to induce the ionization of these relatively weaker n-acceptors which possess lower electron affinities than DDQ, TCNQ and TCNE29z4).With reserpine and ephedrine additional different maxima were obtained with some acceptors (Table 1) due to side
reactions. Thus reserpine was probably oxidized by DDQ and fluoranil, on the basis
of the known susceptibility of this alkaloid to o x i d a t i ~ n ~ particulary
~ - ~ ~ ) with
these quinone acceptors which are characterized by strong dehydrogenating powerz8).
The prominent peaks at 387 nm (DDQ and fluoranil) and at 390 nm (chloranil) are
probably due to dehydr~reserpines~~~~~).
On the other hand, ephedrine, being a secondary aliphatic amine, probably entered into a displacement reaction with TCNE
giving an N-tricyanovinyl derivative A,,
330 (Table 1). This suggestion is compatible with analogous reactions of TCNE with primary and secondary aminesz9).
1 9 W. J. Middleton, E. L. Little, D. D. Coffman and V. A. Engelhardt, J. Am. Chem. SOC.80,
2795 (1958).
20 J. Bolard, J. Chim. Phys. 66, 221 (1969).
21 H. J. Shine and R. D. Goodwin, J. Org. Chem. 35, 949 (1970).
2 2 K. Wallenfels, Angew. Chem. 76. 275 (1964).
23 P. G. Farrell and K. K. Wojtowski, J. Chem. SOC.C, 1970, 1394.
24 G. Briegelb, Angew. Chem. 76, 326 (1964).
25 R. E. Schirmer in “Analytical Profiles of Drug Substances”, K. Florey, Editor, Vol. 4, p. 401,
Academic Press, New York 1975.
26 D. Banes, J. Wolef, H. 0. Fallscheer and J. Carol, J. Amer. Pharm. Assoc. Sci. Ed. 45, 710
(1956).
27 G. E. Wright and T. Y. Tang, J. Pharm. Sci. 61, 299 (1972).
28 H.-D. Becker in “The Chemistry of the Quinoid Compounds” part. 1, pp. 335-416, S.
Patai (Editor), Wiley, New York 1974.
29 B. C. Mckusick, R. E. Heckert, T. L. Cairns,D. D. Coffmann and H. R. Mower, J. Am. Chem.
SOC.80, 2806 (1958).
488
Taha und Riicker
Arch. Pharm.
The relative sensitivity of the five acceptors in analytical work may be compared by
E-values of the chromogens (Table 2) using atropine as a reference example.
TCNQ, exhibiting the highest intense band, was selected for all further quantitative work. In all cases studied, Beer’s law plots were linear, with 0 or very small intercepts, in the general concentration range of 3 x 10-6 to 4 x 1OP5 M alkaloid base
(ca. 1-12 pg base/ml for alkaloid of average molecular weight of 300).
The sensitivity of the assay, expressed as €-values, varied with different alkaloids
in a regular pattern which was found dependent upon the pKa values of alkaloids
(Table 3, Fig. 2).
Regression analysis of the above correlation by the method of least squares3’)
afforded: correlation coefficient r=O. 966 (theoretical for 10 degrees of freedom at
p = 0.01 is 0.70830));slope ( p ) 0.135; relative standard deviation (Ro) of 0.081
(Fig. 2). From molar absorptivity of 43 30013)for TCNQ? the extent of ionization
of TCNQ or in effect the yield of TCNQ? produced by each alkaloid was calculated
(Table 3). Here again the dependence on pKa is evident. The deviations from linearity are to be expected on the basis of differences between the proton-transfer ionization of alkaloids in water expressed by pKa values, and the extent of the electrontransfer reaction in the aprotic solvent acetonitrile, as expressed in terms of equation
1. The deviations are towards higher E-values exhibited by alkaloids rich in methoxyl
groups. It is possible that these groups may induce further formation of TCNQ? by
an electron donating mechanism analogous to N-atom donation.
As an assay solvent, acetonitrile afforded maximum sensitivity (Table 2) owing to
its high dielectric constant which promotes maximum yield of TCNQ?. In addition,
it has good solvent power for the reagent (up to 0.01 5 M) and for all alkaloids examined. However, its water-miscibility necessitates an extra step during the assay of
alkaloidal salt in that a preliminary extraction with a water-immiscible solvent in
alkaline pH (ca. 9.5) is required. 1,2-Dichloroethane may be used directly both as an
extraction and assay solvent at the expense of decreased sensitivity (Table 2).
Of the other solvents examined, only methylene chloride is a possible substitute. Benzene
was found unsuitable because of itr low solvent capacity for the reagent and some alkaloids and
its low ionizing power giving poor yields of TCNQ?, in addition to high background blank-reading
due to DA-complex formation with TCNQ. In spite of its good solvent qualities, chloroform was
rejected as an assay solvent due to its interaction with alkaloids by hydrogen bonding, similar to
’~)
its behaviour with other corn pound^^^'^^), as well as by charge-transfere c ~ m p l e x a t i o n ~resulting in competition with TCNQ and low yields of TCNQ? particular1 at low alkaloid concentra&
tion. In tetrahydrofuran, instead of the characteristic bands of TCNQ another orange coloured
a ,
30 A. N. Martin, J. Swarbrick and A. Cammarata ‘‘ Physical Pharmacy” 2 nd. ed., p. 24, Lea and
Febiger, Philadelphia 1969.
3 1 J. Lascombe, J. Devaure and M.-L. Josien, J. Chim. Phys. 61, 1271 (1 964).
32 M. D. Johnston, F. P. Gasparro and I. D. Kuntz, J. Am. Chem. SOC.91, 5715 (1969).
310177
489
n-Acceptors in Alkaloid Assay
product ( L a x 483 nm) was obtained upon addition of alkaloids. It is likely that the formation
of this product also involves interaction with solvent molecules”).
The n-acceptor spectrophotometric method was applied to assay a number of
commercial preparations containing an alkaloid as the sole or major ingredient (Tabli
4). Common ingredients of formulations such as tablet excipients were found not to
interfere in the method when following the proper extraction procedure, as outlined
in the experimental part.
Potential interference in the assay by other ingredients of some formulations was
eliminated by adopting a suitable extraction method. Thus in tablets of total belladonna alkaloids with phenobarbitone, the single partition at the alkaline pH of the
Table 1: Electronic spectra of Alkaloids with rr-Acceptors in Acetonitrile
TCNQ~)
Lax
TCNE
[nm] with:
DDQ
Atropinea)
Codeinea)
Quininea)
Papaverine
Reserpine
845, 752,685
845,752,687
845,752,687
845, 752, 685
845, 754, 687
412, 393
412,393
412,393
412, 393
412, 393
Ephedrine
845, 752,684
458,415
396,330
576,545
576,545
578,546
576, 545
580, 555,
387
592,550,
353
Alkaloid
Fluoranil
Chloranil
538, 383
540,355
540,381
no reaction
410, 387
442,420
442,418
442,420
no reaction
442,390
358
378
a) These represent typical spectra which were also obtained with scopolamine, strychnine,
brucine, cocaine, ergometrine, morphine, nicotine, pilocarpine and veratrine.
b, Minor vibrational peaks were at 830, 768 and 630 nm.
Table 2: Molar Absorptivities of Chromogens with Atropine
Reagenta)
Chloranil
Fluoranil
DDQ
TCNE
TCNQ
TCNQC)
fmaxb) ( L a x ) in
Acetonitrile
1,2-Dichloroethane
83 (442)
722 (538)
10350 (576)
17100 (393)
38600 (845)
45100 (845)
no reaction
no reaction
7580 (460)
7225 (398)
29500 (854)
37000 (854)
a) 0.2 mg/ml unless indicated b) Practical f. Average value of 3 determinations, based on the
molecular weight of alkaloid, c) 0.4 mg/ml.
33 J. Diekmann and C. J. Pedersen, J. Org. Chem. 28, 2874 (1963).
490
Taha und Rucker
Arch. Pharm.
476nm 1
/-
1000 nm
L__
[nml
pr7g?'
-
Fig. I : - Spectrum of the chromogen resulting from interacting quinine with TCNQ in acetonitrile.
40-
p i 0 13'7
= 0966
30-
20-
16-
Eh NI:
I
I
I
pKa
uf
I
I
25'
Fig. 2: - Correlation of pKa of alkaloid and band intensity of TCNQ?. anion radical at 845 nm
in acetonitrile: See Table 3 for abbreviation symbols for individual alkaloids.
procedure was sufficient to separate the barbiturate from alkaloids. In tablets of papaverine hydrochloride with penicillin and theophylline, all of the penicillin and most
of the purine base remained in the aqueous phase. Residual theophylline in the final
assay solution was found not to interfere. This was confirmed by experiments on the
purine bases, caffeine, theophylline and theobromine, which revealed no radical ion
310177
49 1
n-Acceptors in Alkaloid Assay
Table 3: Correlation of molar Absorptivity and pKa values for Alkaloids with TCNQ
Alkaloid
Papaverine
Reserpine
Pilocarpine
Nicotine
Morphine
Codeine
Strychnine
Brucine
Cocaine
Quinine
1-Ephedrine
Atropine
Tropine
pKa
6.4
6.6
6.87
8.02
8.20
8.21
8.26
8.28
8.41
8.52
9.36
9.65
10.43
E~~~
20.0
26.0
21.5
29.6
32.0
35.0
32.4
38.0
34.4
35.6
41.2
45.1
48.0
. 10K3
yield of TCNQ?
Symbol in Fig. 2
[%I
57.81
76.02
62.34
70.27
81.99
90.84
87.60
92.82
85.14
82.12
93,54
101.23
96.55
pv
Rs
P1
Nc
MI
Cd
St
Bc
cc
Qn
EP
At
Tr
a) at 25', from Reference3')
oY
ALKALOID 10
,pg per ml
15
Fig. 3: - Calibration curves of various alkaloids based on the anion-radical band of TCNQT. at
845 nm in acetonitrile.
492
Arch. Pharm.
Taha und Riicker
Table 4: Assay of Commercial Dosage Forms
Preparationa)
Alkaloidal Salt content, mg/unit
Label
Claim
Ephedrine HCI- Tablets
50.0
Foundb) Added
50.25
50.0
Recovered
total
S. D.
+%
100.8
1.43
Total Belladonna
Alkaloids-Tablets
0.25
0.249
0.5
0.752
2.76
Bellad. Alkaloids
with Phenobarbitone
0.25
0.253
0.5
0.756
2.05
Reserpine Tablets
0.25
0.243
0.5
0.745
1.68
Papaverine HCl Tabl.
with Penicillin
40.0
40.4
40
81.5
1.47
Morphine HCl Amp.
20.0c)
20.0
10
29.6
2.1 1
Atropine Sulfate
Eye Drops
50d)
48.5
25
74.0
1.83
Scopolamine Eye
Drops
207e)
209.1
100.0
310.0
1,55
Hyoscine n-butylbromide Ampoule
20.0c)
19.6
20
40.2
0.96
a) Name of the drug and detailed composition in Experimental section;
b, Average value of 6 determinations, c, per 1 ml ampoule; dl per 10 ml solution;
calculated as base per 100 g solution
formation with TCNQ in acetonitrile. This finding was expected because these compounds actually behave as weak acids3’).
The quaternized salts of some alkaloids, particularly those of the tropane group
such as atropine methylnitrate, homatropine methylbromide and hyoscine n-butylbromide were assayed by little modification of the same procedure for alkaloids. They
were converted to the respective iodide salts by addition of excess iodide ion. This
conversion facilitated the complete extraction of the salts into a halogenated solvent
and hence quantitative recovery63Mi3) and made possible the facile reduction of
, , ,A
TCNQ with the iodide ion’3) and the release of TCNQ‘?, absorbing at the same
equivalent to the quaternary salt originally present. In popular mixtures of papaverine
with tropane quaternary salts, an extraction step with pH 9.5 buffer gave the tertiary
base, while a second extraction with excess iodide ion in neutral or faintly acidic pH
34 E. Jungermann, “Cationic Surfactants” pp. 420-422, Dekker, New York 1970.
35 R. Reiss, Arzneim.-Forsch. 6, 77 (1956).
n-Acceptors in Alkaloid Assay
310177
493
yielded the quaternary salt. This procedure was applied to synthetic mixtures with
good recoveries (98.43-100.31 %) and precision (SD*1.28 to f 2.63 %).
The results of Table 4 confirm the suitability of the n-acceptor spectrophotometric method for analysis of alkaloids in the micro range. The method is particulary
attractive in the assay of originaly weak absorbing alkaloids such as the tropane group,
ephedrine and codeine. The strongly red shifted band combined with the intensity
of absorption and very low reagent background obviously recommend this method
for routine alkaloid analysis with minimum of interference.
Experimental
Material: Pharmaceutical grade alkaloidal bases and salts were used as working standards. All
other chemicals used were reagent grade or purified according to literature methods. Solvents
used were spectrograde.
Dosage Forms: The following commercial preparations were analyzed
a) Ephetonin@tablets (Merck): contain DL-ephedrine HC1 50 mg per tablet.
b) Bellefolin@ tablets (Sandoz): contain total alkaloids of belladonna as malates 0.25 mg per
tablet.
c) Belladenal@ tablets (Sandoz): contain total alkaloids of belladonna 0.25 mg and phenbarbitone 50 g, per tablet.
d) Serpasil tablets CIBA): contain reserpine 0.25 mg per tablet.
8
d
e) Broncho-binotal tablets (Bayer): contain Da-aminobenzyl penicillin trihydrate (577 mg),
theophylline (100 mg), papaverine hydrochloride (40mg) and guaiacol glycerol ether
(100mg) per tablet.
f) Amphiole morphinum hydrochloricum@(Merck): contains 20 mg morphine hydrochloride
per ml of injection.
g) Atropine-POS Augentropfen@(Bernhard Buxman & Co): contain atropine sulfate, 50 mg,
per 1 0 ml b ffer
. ..
h) Boroscopal (Dr. Winzer) contains Scopolamine boric. 0.25 %, equivalent to 207 mg base
per 100 g uffer
.:
i) Buscopan Injection (Boehringer Ingelheim): contains hyoscine n-butylbromide, 20 mg, per
rnl -P injection.
6
&
Reagents:
a) n-acceptors: 1mgper ml of reagent in acetonitrile or 1,2-dichloroethane were used. For assay
2 mg TCNQ per ml in acetmitrile was used (stable for at least 1 weak at 4O).
b) Buffer: adjust 0.2 M K2HF04 to pH 9.5.
c) KI-Solution: 0.2 M in water
Standard So1utions:Dissolve calculated amount of alkaloid base in acetonitrile and dilute quantitatively to 0.1 mg base/ml. For alkaloid salts, dissolve calculated amount in water and dilute
to equivalent of 1.0 mg baselml. Pipet 1 ml into 30-ml separator. Add 5 ml buffer and extract
with three 5 ml portions of chloroform, passing separated organic layers through 2 g anhydrous
Na2S04 supported in a small funnel. Remove chloroform in a stream of nitrogen. Dissolve residue in acetonitrile and dilute t o 10.0 ml with same solvent in a volumetric flask.
494
Taha und Riicker
Arch. Pharm.
Sample Solutions a) Liquid Preparations: Transfer aliquot equiv. of 1 mg alkaloid salt, or measured contents of singe1 dose container of injection, into 5 ml buffer in 30 ml separator and continue as described above for standard solutions.
b) Tablets of Alkaloidal salts: Place equiv. of 1tablet (containing at least 1 mg alkaloid) from
composite of 10 powdered tablets in 5 ml buffer in 30 ml separator and continue as described
above.
c) Reserpine Tablets: Transfer equiv. of 4 tablets from composite of 10 powdered tablets in 30
ml beaker. Extract with warm (ca. 40') chloroform. Filter the extract and remove solvent with
N2-stream. Dissolve residue in acetonitrile and complete to 10.0 ml with same solvent in a volumetric flask.
d) Quaternary salt Preparations: Follow procedure as for alkaloidal salts substituting 5 mlO,2 M
KI for the buffer.
General Procedure
Pipet 1 ml acetonitrile solution of the preparation or standard into dry 5 ml volumetric flask.
Add 1 ml TCNQ reagent (2 mg/ml) and dilute to volume with acetonitrile. Read absorbance of
solution at 845 nm against reagent blank treated similarly.
Calculate amount of alkaloid in preparation by reference to corresponding calibration curves
(Fig. 3) and using equation
Y = mx + b (Y = absorbance of solution)
x = concentration of alkaloid base, mg/ml of final dilution. The values of m (slope) and b (intercept) were calculated by the method of least squares**:
m
b
Ephedrine
Atropine
Hyoscine n-butylbromide
Morphine
Scopolamine
Papaverine
Reserpine
4.2485
6.4995
7.6100
8.8174
11.8370
17.0530
23.8701
-0.0 29 85
-0.03152
+0.06724
+0.04599
-0.1 5324
+0.01426
-0.1004
Specrrophotomerer: Carl Zeiss model DMR 21 equiped with M 4 QIII monochromator.
Anschrift: Prof. Dr. A. Taha, Hittorfstr. 58-C., 44 Miinsterh'estf.
[Ph 7431
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