Talanla, Vol. 35, No.5, pp. 375-378, 1988
Printed in Great Britain. All rights reserved
0039-9140/88$3.00+ 0.00
Copyright ((;) 1988 Pergamon Presspic
COMPLEXOMETRIC-SPECTROPHOTOMETRIC ASSAY
OF TETRACYCLINES IN DRUG FORMULATIONS
SALAH M. SULTAN, IBRAHIM Z. ALZAMIL and NAWAL A. ALARFAJ
Chemistry Department, College of Science,King Saud University, Riyadh 11451,P.O. Box 2455,
Saudi Arabia
(Received3 October 1986. Revised15 April 1987.Accepted23 November 1987)
,
Summary-An accurate,rapid and very simple spectrophotometric method for the assayof tetracyclines
(tetracycline.HCI, chlorotetracycline.HCI, demeclocycline, oxytetracycline.HCI and doxycycline) has
been developed. The method is based on the complexation of iron(lII) with tetracyclines in O.OOIM
sulphuric acid. It has beensuccessfullyapplied to the assayof tetracyclinesin drug formulations, and the
interferencesof excipients have been examined. The results have been statistically compared with those
obtained by two standard methods and found to be very satisfactory.
..,
..
..,
The tetracyclines and their derivatives (tetracycline,
oxytetracycline,
chlorotetracycline,
demeclocycline
and doxycycline) are extensively employed as bacte.
.
"b.."
.
nostatI~ a~tl 10tlC drugs. Va~ous ~ethods for ~h.elr
determInatIon have been revIewed. In the Bntlsh
Pharmacopoeia2 a biological assay for tetracycline is
given but it is an elaborate method not suitable for
routine analysis. Iron(lII)
was earl recommended
.
. 3
y..
m the U.S. PharmacopeIa for the determInation of
oxytetracycline. A simple spectrophotometric method
Ferric ammonium sulphate solution (I mg/ml) was prepared by dissolving.abo~t I g, accurately weighed,in a litre
of O.OOIM~ulphunc.acid.
Tetracycline solution (I mg/ml) was freshly prepared by
dissolving the required amount in O.OOIMsulphuric acid by
warming, then cooling and making up to volume in a
standard flask. For analysis of capsules,the contents of 10
were mixed and weighed and a quantity of the powder
equivalent ~o250 ~g of tetracycline was accurate~yw~ighed
out, and stirred with 200 ml of O.OOIMsulphunc acid for
10min, with warming, then the solution was filtered (Whatman No. 41 filter-paper), the paper was washed with hot
O.OOIMsulphuric acid and the filtrate and washings were
diluted to volume with ~.OOIM sulphuric acid in a 250-ml
standard flask after cooling to room temperature.
for the assayof tetracycline was recently describedI
but it is not suitable for the assay of other derivatives.
T
t
h t
t .
th d ..
th
f
wo spec rop 0 orne nc me 0 s lor
e assay 0
some tetracycline derivatives have been described
based on oxidation with ammonium vanadate4 and
sodium cobaltinitrite 5 but neither is specific.
The
.
+
J.,
present
work
'.
IS based
on
the
. "
abIlity
of
Generalprocedure
Place 10ml of ferric ammonium sulphate solution and an
appropriate amou!1t of tetracycline sol~tion in a 50-.ml
standard
flask. Swirl and leave for 20 mm for tetracycline
hydrochloride
but only 5 min for the other derivatives,
then
tet-
racyclines to form metal-ion complexes.6,7
Chelation
diluteto themarkwith O.OOIM sulphuric acid. Measurethe
with cations such as iron(III), aluminium, copper(II),
nickel, cobalt(II), zinc, vanadium(III), thorium, lan-
absorbanceat t~e.appropriate wavelengthagainst a reagent
blank treated similarly.
thanum, magnesium and calcium is well established.s-is It has been reported that iron(lII)
has
RESULTSAND DISCUSSION
higher affinity than other cations for chelation with
tetracyclines in vitro.6.8-14,19
I h
h d
. .
t e met 0 presented here, tetracycline IS added
to Iron(lII) ~nd the absorbance of the brown complex formed IS measured.
Mechanism
...
The tetracyclines were added to Iron(lII) solutIon
in sulphuric acid of different concentrations. A brown
soluble compound was always obtained, with each
derivative giving a characteristic wavelength of max-
~
EXPERIMENTAL
Apparat~
A Vanan.Model DMS 100Spectrophotomet~rconnected
to a Vanan Model DS 15 Data Station and a
Hewlett-Packard Model 82905 B Printer was used for all
absorbance measurements.Matched sets of 10-mm cells
were used throughout.
R
eagents
.
Hlgh-punty
imum absorption, at 423 nm for tetracycline
nm for the four derivatives examined. The
can be attributed to complex formation of
with the tetracyclines. When iron(lI) was
.
colour change was observed. The tetracycline structure contains numerous sites at which chelation with
metal cations might occur, the most important of
these being in the portion
...
contaIns
distilled
water
was
used
throughout.
Stock
and 435
maxima
iron(lII)
used no
.
.
6,2~22
t
he
two
eno
of the molecule which
I.Ize d I , 3- d.I k etone
..
group-
solutions were prepared from analytical or pharmaceutical mgs.
~uch ~nol gr~ups r~adlly form SIXgrade chemicals,and working solutions were prepared from membered nngs with metal Ions, with the two oxygen
these by appropriate dilution.
atoms as donors.21,22Job's method23,24showed that
375
376
SALAHM. SULTANet al.
the ratio of iron(lII) to tetracycline is I: 2. This ratio
1.~
hasalsobeenreportedfor combinationof copper(II),
\
nickel and zinc with tetracycline}OAccordingly, it is
suggestedthat iron(lII) chelateswith tetracycline as
follows:
W=
CH
3"
H3
2
~
OH
~
OH
0
OH
\
1:2 I
\
9
CH
I
OH
C-NH'
If
2
0
+ Fe3+
I
I
~o.
~
3
OH
I
7
/'
1
-
/
~
I
\
~ o.
\
\
/"~1
2
\
0
'\..-/
o.
CH3,
/
;.
CH3
,~
'N
~7
~
I
II
HN-C
2
HO=
0
HO
HO
~
OH
0
U
+ 2H+
H3C
660
750
Wavelength(nm)
molarities:
'Y
:
1-0.0001';
2-0.0005;
3-0.001;
4-0.003;
5-0.005; 6-0.01; 7-0.05; 8-0.10; 9-2.
~
.-.:;
HO
570
Fig. I. Absorption spectra for reactants [3 mg of tetracycline + 6 mg of iron(IIl») in 50 ml of solution, measured
against a reagent blank at different sulphuric acid
]$~~~~
I
I
/N"
3
OH I C-NH2
~
0" ,,0
Fe3
,..
0
0
0.0
300
OH
i:;~$[H3 OH
"
0
~'\
Applications
CH
The method was applied to the assay of tetracyclines in drug formulations (commercial products
3
CH3
randomly collected from local pharmacies).Typical
All the iron(lII) complexesthus formed are stable
for 24 hr. Figure 1 shows that the absorbanceof the
tetracycline-iron(lII) complex at 423 nm decreasesas
the sulphuric acid concentration is increased, becoming minimal at 0.1M acidity. In the sulphuric acid
range 0.OOO5-0.002Mthe absorbanceis almost constant and O.OOIMacidity is optimal. At sulphuric acid
concentrations> O.IM protonation of the tetracycline molecule is possible and an absorbancepeak
for the protonated form appears at 441 nm as in
curve 9. At acidities lower than 0.0005M, hydrolysis
of iron(lII) is expectedand the maximum is not as
well defined (curve I). For this reason, O.OOIM
sulphuric acid was selectedas optimum.
Spectral data
resultsare given in Table 2 and show good agreement
with those obtained by other methods. The sodium
molybdate method was usedfor tetracycline, and the
sodium cobaltinitrite methodS for the other four
compounds. The t-values (Table 3) showed no
significant difference between the means obtained
(95% confidencelimit).
Interferences
The results for analysis of the compounds in drug
formulations with the constituents listed in Table 2
indicate that the excipients usually presentin dosage
forms, such as starch, lactose and glucose, did not
interfere. Troleandomycin, glucosamine.HCl and
vitamin K3 did not interfere with the determination
of tetracycline. Thiamine, pyridoxine and folic acid
did not interfere with the determination of oxy-
Beer'slaw was found to hold over the range 10-200
tetracycline. Vitamins B1, B2, B6, B12,nicotinamide
J1gjml for all five compounds examined. Table I gives
the molar absorptivities at the wavelength of maximum absorption.
and calcium pantothenate did not interfere with the
determination of tetracycline or oxytetracycline. Sulphamethizole and phenazopyridine.HCl,
however,
Table I. Analytical appraisal for compounds (in pure form) investigated
Generic name
Supplier
Am...
nm
i,
I. mole-I. cm-1
Tetracycline.HCl
Demethylchlorotetracycline
Chlorotetracycline.HCl
Oxytetracycline.HCl
Doxycycline
Lederle
Lederle
Lederle
Pfizer
Pfizer
423
435
435
435
435
4646
5190
5533
5240
4922
~
i
:,.;
Assay of tetracyclines
377
Table 2. Resultsfor all drugs investigatedby the proposedmethod and/or the sodium molybdate8and sodium cobaltinitriteb
methods
Proposed method
Drug proprietary name,
and supplier
.
.,
Nominal
composition, mg
Found per
capsule,mg-
Error,
%t
Standard
method,
found, mg
250 tetracycline.HCI
125 tetracycline.HCl
250 sulphamethizole
50 phenazopyridine.HCI
250 tetracycline.HCI
250 tetracycline.HCI
167tetracycline.HCI
83 troleandomycin
250 tetracycline.HCI
250 tetracycline.HCI
250 glucosamine.HCI
262
429
+4.9
+243
2628
3028
257
251
155
+2.6
+0.5
-7.0
2588
2538
1558
249
149
-0.3
-40
249'
2638
102
478
+ 1.5
+282
102b
330b
173
-31
381b
260b
Generic name
Tetrerba, Carlo Erba
Uropol, Bristol-Myers
Tetracycline
Tetracycline
Dumocycline, Dumex
Tetrambezim, Lepetit
Sigmamycin, Pfizer
Tetracycline
Tetracycline
Tetracycline
Achromycin, Lederle,
Latycin, Biochemie
Tetracycline
Tetracycline
2.5 Vito B1
2.5 Vito B2
2.5 Vito B6
~
2.5VitoB'2
~,
Vibramycin, Pfizer,
Urobiotic, Pfizer
Doxycyclin
Oxytetracycline.HCI
Terramycin S.F., Pfizer
Oxytetracycline.HCI
Terramycin, Pfizer
Oxytetracycline.HCI
75 Vito C
0.5 Vito KJ
25 nicotinamide
5 calcium pantothenate
100 doxycycline
125 oxytetracycline.HCI
250 sulphamethiazole
50 phenazopyridine.HCI
250 oxytetracycline.HCI
2.5 thiamine
2.5 riboflavin
25 niacinamide
5 calcium pantothenate
0.5 pyridoxine
0.375 folic acid
I Vito B'2
75 ascorbic acid
250 oxytetracycline
259
Demethylchloro-
pure analytical reagent
-
+4.0
Demeclocycline,
(another batch)
pure analytical
-
-0.5
Lederle
Chlorotetracycline
tetracycline
Chlorotetracycline.HCI
- 1.4
reagent (another batch)
-Mean of 7 determinations.
tDifference from nominal content.
~
;
Table 3. Statistical comparison of the results obtained (7 replicates) by the proposed method
with results obtained by the sodium molybdate' and/or sodium cobaltinitriteS method
4
~~
~
-
Recovery:t standarddeviation,%
Drug proprietary name
Method-
104.8:t 0.9
102.6:t0.6
105.0:t 0.2
103.2:t0.2
Tetrambezim
Sigmamycin
Vibramycin
Terramycin
Demeclocycline
Chlorotetracycline.HCI
lOO.5:t0.7
93.0:t 0.2
101.6:t0.4
103.6:t0.6
99.1:t0.2
95.3:t 0.6
101.2:t0.4
93.0:t 0.8
-
AchromycinV
99.8:t0.7
-Proposedmethod.
tSodiummolybdatemethod.
§Sodium cobaltinitrite method.
~Theoretical value = 2.45 (P = 0.05).
c"
Methodt
Tetrerba
Dumocycline
99.5:t0.4
Method§
-
102.0:t0.5
104.1:t 0.8
99.5:t0.2
94.9:t 0.5
t Calculated~
0.38
1.71
0.81
1.75
0.51
0.68
0.56
1.10
0.43
-
378
SALAH
M. SULTAN
et al.
gave a large positive error in the determination of
both tetracycline and oxytetracycline.
Vitamin C also interfered with the determination of
d
I. b
.h
t.
I.
tetracyc me an oxytetracyc me, ut WIt a nega Ive
systematicerror. This may be due to decreasein the
iron(III) concentration by reduction with ascorbic
acid, but addition of extra iron(III) failed to eliminate
the interference.
6. P. J. Neuvonen,Drugs,1976,II, 45.
7. J. J. ~tezowski,Jerusalem
Symp.QuantumChern.Biochem.,1977,9,375.
8. A. Albert, Nature,1953,172,201.
9. A. Albert and C. W. Rees,ibid., 1956,177,433.
10. Idem,ibid., 1963,172,201.
II. P. J. Neuvonen,G. Gothoni, R. Hackmanand K.
Bjorksten,Brit. Med.J., 1970,4, 532.
12. P. J. ~euvonen,P. J. Pentikainen
andG. Gothoni,Brit.
J. Clm. Pharmacol.,1975,2, 94.
Conclusion
13. T. F. Chin andJ. L. Lach,Am. J. Hosp.Pharm.,1975,
32,625.
This method is accurate,simple, rapid and suitable 14. T. Sakaguchi,M. Toma,T. Yoshidaand H. Takasu,
for routine analysis of all commonly used tetraPharm.Bull., 1958,78,177.
cyclines. There is no interference from most com- 15. M. Saiki and F. W. Lima, J. Radioanal.Chem.,1977,
pounds added to drug lormu
so
Iatlons
.
contammg
.,
tet ra36,435.
16 D E W' II '
L
I.
. . . I lamson and G . W . Everett, Jr., J . Am. C,.em.
cyc meso
Soc.,1975,97,2397.
Acknowledgements-The
authorsthan~LederleLaborato- 17. f!..A. Hochstein,C. R. Stephens
and L. H. Conover,
ries and Pfizer Corporation,for supplyingthe standard
IbId.,1953,75, 5455:
samples.
Mrs NawalA. Alarfaj thanksKing SaudUniver- 18. H: Ibsen,M. R. Unst and S. Ugeno,Proc.Soc.Eptl.
.t so
19 J J S' I
dH M D . R P
Q .
197214
!Ii
Sl
Y
lor
0
.
enng
h
a
c
t
ance
d
0
rea
so
th
lor
M
e
S
.
d
c.
BIoi.
egree.
REFERENCES
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5. M. S. Mahrousand M. M. Abdel-Khalek,ibid., 1984,
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~~"-~..."'~~".
'..":I_;~l:;!~~~!!~;:'
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,
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",
t
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t~~~
~'*~
~
_1