Journal of Pharmaceutical and Biomedical Analysis
Tetracycline, oxytetracycline and chlortetracycline
determination by flow injection potentiometry
Cristina M.C.M. Couto, Jose´ L.F.C. Lima, M. Conceic¸a˜o,
CEQUP/ Departamento de Quı´mica Fı´sica-Faculdade de Farma´cia do Porto, Rua Anı´bal Cunha, 164-4050 Porto, Portugal
Received 15 May 1998; received in revised form 21 July 1998; accepted 15 August 1998
Abstract
This paper describes tetracycline (TCH), oxytetracycline (OTCH) and chlortetracycline (CTCH) determination by
flow injection potentiometry. In the flow system proposed TC samples are inserted in a carrier solution and convergedwith a Cu(II) solution of known concentration; the Cu(II) decrease due to its complexation with tetracyclines (TC)was monitored. The detector used was a homogeneous crystalline CuS/Ag S double membrane tubular electrode with
increased sensitivity. The present system allows tetracyclines determinations within a 48.1 – 4.8 × 103 ppm for TCH,49.1 – 4.9 × 103 ppm for OTCH and 51.5 – 5.1 × 103 ppm for CTCH and a precision better than 0.4% for the three TCspecies. This procedure accomplishes 150 – 200 samples h−1 with a Cu(II) consumption of about 13 mgdetermination−1. 1998 Elsevier Science B.V. All rights reserved. Keywords: Tetracyclines; Cu(II) tubular electrode; Increased sensitivity; FIA
1. Introduction
logical family is used in human pathologies aswell as in veterinary medicine, animal nutrition
The fast advances in pharmaceutical industry
and feed additives for cattle breeding.
impose the development of more rigorous analyti-
The biological method of evaluation of micro-
cal methods, particularly faster and inexpensive,
biological potency is commonly recognised as offi-
cial method for its determination in pharma-
ceutical preparations [2,3]. However, the fact that
Tetracyclines are amongst the most essential
it is time-consuming and of difficult operation as
antibiotic families characterized by their wide
well as subject to random error due to the inher-
range of antibacterial effect [1]. This pharmaco-
ent variability of biological responses, led to thedevelopment of other methodologies, particularlybased on batch procedures with spectrophotomet-
* Corresponding author. E-mail: mcbranco@ff.up.pt.
0731-7085/98/$ - see front matter 1998 Elsevier Science B.V. All rights reserved.
PII: S 0 7 3 1 - 7 0 8 5 ( 9 8 ) 0 0 2 0 7 - 6
C.M.C.M. Couto et al. / J. Pharm. Biomed. Anal. 18 (1998) 527 – 533
The requirements demanded by pharmaceutical
improved operational characteristics. In order to
industry concerning automation and higher sam-
determine tetracyclines in pharmaceutical prod-
pling rate led to the promotion of flow injection
ucts by using an accurate, fast, simple and inex-
systems for TC determination. Therefore flow sys-
pensive method, a flow system incorporating a
tems with spectrophotometric [7 – 11], amperomet-
Cu(II) tubular detector with improved character-
istics was used to monitor the complexing reac-
potentiometric [18] detection were developed. The
tion between TC and copper(II) cation.
automated flow injection analysis systems withspectrophotometric detection referred in literaturefor TC analysis were based on their indirect deter-
2. Experimental section
minations by monitorization of the coloured com-plexes formed by tetracyclines with Fe(III) [7,8],
WO2− [9], 4-aminophenazone and hexacyanofer-
rate (III) [10] and Cu(II) [11]. These systems
All solutions were prepared with bideionized
present as main disadvantages their application to
water (specific conductivity less than 0.1 ms cm−1)
a very limited concentration range, the need of a
and with reagents of analytical-reagent grade
compensation procedure for the measurements
due to the samples intrinsic colour, and also the
The stock solution of tetracyclines (Sigma), as
fact that, when referred [7,10] they accomplish
the hydrochlorides, was daily obtained by care-
very low sampling rates, of about 17 [7] and 70
fully weighing the solid and was kept in a refriger-
samples h−1 [10]. Similar sampling throughputs
ator before and after use. The TC standard
are also referred for the amperometric FIA sys-
solutions were daily prepared from the previous
tems described [12 – 14], despite their high analyti-
cal sensitivity. Chemiluminescence flow systems
Copper nitrate stock solution was obtained by
[15 – 17] using hydrogen peroxide/potassium per-
weighing the solid and standardised by potentio-
sulfate pair [15], bromine [16] or N-bromosuccin-
metric titration against a standard EDTA solu-
imide [17] as oxidising agents are proposed as a
tion (Titriplex III, 0.1 M Merck 109992).
good alternative. Nevertheless, these methodolo-gies are very expensive and the oxidising reagents
Potentiometric detection FIA systems have
been widely used in the analysis of pharmaceutical
2002 potentiometer (sensitivity of 90.1 mV) con-
preparations due to their low cost, easy operation
nected to a Kipp & Zonen BD 111 recorder.
and automation [18]. TC-sensitive conventional
electrodes for direct determinations have been
Minipuls 2 peristaltic pump and samples were
previously considered by some authors [19] as an
inserted through a Rheodyne 5020 injection valve.
alternative to microbiological procedure although
The components of the FIA system were con-
the results showed poor reproducibility.
nected with PTFE tubes (0.8 mm i.d.). Auxiliary
As organic molecules containing N or O atoms
laboratory-made devices, namely joints, ground-
can actuate as electron donors, are able to form
ing electrode, tubular and reference electrode sup-
complexes with metal ions, which are electron
ports, were used and constructed as previously
acceptors, if the equilibrium constant is suffi-
ciently high [20], these compounds can be deter-
The homogeneous crystalline Cu(II) double-
mined by titration with metal salts followed by
membrane tubular electrode construction and
the appropriate metal ion-selective electrode [18].
evaluation are described in [21]. An ORION 90-
The development of tubular electrodes of in-
00-02 AgCl/Ag double junction electrode, with a
creased sensitivity [21] presented both the capacity
10% KNO solution in the outer compartment,
of being steadily adapted to flow systems and
C.M.C.M. Couto et al. / J. Pharm. Biomed. Anal. 18 (1998) 527 – 533
Fig. 1. FIA manifold for the evaluation of TC in pharmaceutical preparations. PB, peristaltic pump; V , injection valve; L1, reaction
coil (35 cm); S, sample; C, carrier solution; CR, complexing Cu(II) solution; X, confluence; GE, ground electrode; TD, tubulardetector; RE, reference electrode; SD, summing device; mV, voltimeter; Rec, recorder; W, waste.
The summing of the potentials of the mem-
The quality of results obtained by FIA was also
branes comprised in the tubular potentiometric
assessed by comparison with those given by the
detector was carried out by means of a labora-
corresponding procedure described in Pharmaco-
tory-made summing device similar to that de-
poeia Helvetica [4]. According to Pharmacopoeia
Helvetica samples were diluted in a 1 × 10−2 N
A Pye-Unicam SP6-500 UV spectrophotometer
HCl solution and their absorbance measured by
spectrophotometry at 245 nm (TCH), 254 nm(OTCH) and 280 nm (CTCH).
2.3. Sample preparations for TC determination
Potentiometric determinations were carried out
3. Results and discussion
in different pharmaceutical formulations of TCavailable in Portugal. For solid samples (coated
Considering the good working characteristics of
tablets and capsules) powdering and homogenisa-
CuS/Ag S double membrane tubular electrodes,
tion were performed after determination of the
namely increased sensitivity, calibration slope of
average weight. For ophthalmic ointment a quan-
62 mV dec−1, lower limit of linear response of
tity equivalent to 50 mg TC was added to 1 ×
5 × 10−5 M and wide pH operational range (3.0 –
10−2 M HCl solution which was then kept for a
12.0), a flow system for TC determination in
few minutes in a 37°C ultra-sonic bath to allow
pharmaceutical products was attempted.
dispersion and dissolution of the dosage form
Therefore, a FIA manifold with potentiometric
contents in the aqueous solution. The veterinary
detection was established and then optimised (Fig.
powder and ophthalmic solution were analysed
1). The sample (200 ml) was inserted in a carrier
after homogenisation of the packet contents. Af-
terwards, the different samples were diluted in
with a Cu(NO ) complexing solution. The varia-
bideionised H O to obtain solutions of about
tion of Cu(II) concentration, caused by the com-
7 × 10−4 – 1 × 10−3 M, within linear concentra-
plexing reaction of TC with Cu(II) solution, was
tion range of the Cu tubular electrode.
monitored downstream by the potentiometric
Recoveries of potentiometric measurements
double membrane tubular detector sensitive to
were obtained by using the specified methodology,
after addition of 0.3 – 0.5 ml of a 1 × 10−2 M TC
The manifold was optimised regarding the infl-
solution to 25 or 50 ml sample solutions, in order
uence of hydrodynamic and physic-chemical
to achieve a TC concentration of about 7 ×
parameters to facilitate TC determinations within
a wide concentration range and with the highest
C.M.C.M. Couto et al. / J. Pharm. Biomed. Anal. 18 (1998) 527 – 533
Fig. 2. Variation of the FIA system analytical sensitivity in relation to Cu(II) concentration (A) and pH (B) variation for each ofthe TC evaluated and (", OTCH; a, TCH; , CTCH).
possible sensitivity (mV dec−1 of activity or con-
The pH influence on the development of the
centration) and sampling rate. Hence, consecutive
complexation reaction was also evaluated, being
calibrations were performed to obtain a linear
studied within an interval of 1.0 – 9.7 pH units
relationship between peak height and logarithm of
(Fig. 2B). A 0.1 M KNO solution (C) was used
copper (II) concentration, for average TC concen-
as carrier for ionic strength adjustment and differ-
trations of the different pharmaceutical prepara-
ent pH values were established by adding HNO3
or NaOH. The results obtained presented an in-
The selection of copper cation concentration in
crease of signal sensitivity as pH diminished what
the complexing solution affected the potential dif-
corresponded to a major extent of the complexa-
ference required for the tubular detector to per-
tion reaction in acidic medium that may be re-
form the measurements (Fig. 2A), and restrained
lated to copper precipitation occurring as oxides
the TC concentration levels to be determined so,
and hydroxides, at pH levels higher than 5/6. The
the effect of different Cu(NO ) concentrations
low sensitivity obtained at pH 1 might be ex-
(CR), ranging from 5 × 10−5 to 0.1 M, was stud-
plained by the fact that Cu(II)-TC complexes’
ied. Copper concentration levels higher than 5 ×
studies demonstrated that complexation majority
10−3 M affected the measurements on samples
occur for pH values higher than 2. The highest
with TC concentrations inferior to 7.5 × 10−4 M,
analytical signal corresponding to a greater extent
originating a very low peak height of the analyti-
of the complexation reaction was found at a pH
cal signal. For copper concentrations lower than
value of 2 for the three TC species studied.
5 × 10−5 M the potential variation of the most
After selection of pH and copper concentra-
concentrated solutions was insignificant due to
tions in the complexing solutions, other parame-
insufficient titrant solution to promote the reac-
ters such as injection volume, reactor length and
tion. Besides the baseline potentials were unstable
and baseline returning was very slow. The optimi-
The influence of the injected volume (V ) was
sation of this parameter was therefore restrained
assessed for volumes from 100 to 1000 ml (Fig.
by the TC concentration levels in the prepared
3A). Volumes higher than 500 ml demands higher
samples and hence, a 5 × 10−5 M Cu(II) concen-
concentrations for the Cu(II) titrant solution,
tration was selected for TC determination in the
huge coil lengths (L1) and consequently low sam-
50 to 500 ppm range and a concentration of
pling rates. With an injection volume of 100 ml
5 × 10−4 M for higher TC concentrations (350 to
low peak height signal was attained, what com-
5 × 103 ppm). In both cases a high sensitivity
promised the determination of analyte concentra-
(mV dec−1) was obtained (Fig. 2A).
tions lower than 5 × 10−4 M. Therefore, an
C.M.C.M. Couto et al. / J. Pharm. Biomed. Anal. 18 (1998) 527 – 533
Fig. 3. Variation of the FIA system analytical sensitivity in relation to the injection volume (A) and reactor length (L1) (B) variationfor each of the TC evaluated (", OTCH; a, TCH; , CTCH).
injection volume of 200 ml was selected since it
OTCH and 51.5 – 5.1 × 103 ppm, for CTCH (Fig.
allowed the attainment of a good sensitivity and
reproducibility without prejudice of the sampling
Reproducibility of the system was assessed
rate and avoiding great sample consumption.
throughout a working day by performing six cali-
For the fixed injection volume (200 ml) different
brations for about 8 h work. The potential varia-
reactor lengths (35 to 300 cm) from the confluenceto the detector (L1) were also studied (Fig. 3B). An optimum value was chosen so that the sam-pling rate would not be compromised and thetotal mixing of sample and complexing solutionwould be assured. A decrease of sensitivity andsampling rate was observed with the increase ofthe sample dispersion for the three TC species. Areactor of 35 cm, which was the shortest possiblelength required for the connections between thedifferent components of the FIA system, wasenough to promote a complete complexing reac-tion without compromising the analytical signalsensitivity (peak height), besides providing a goodreproducibility with high sampling rates.
The effect of the flow rate was evaluated keep-
ing the same value for both channels (C and CR). The total flow rate was varied from 2.7 to 8.0 mlmin−1. No significant analytical signal variationwas observed for the interval studied. Therefore, avalue of 8.0 ml min−1 allowed analytical calibra-tions with good sensitivity as well as high sam-pling
described above enabled a maximum sampling
Fig. 4. Recorder output corresponding to the tracing of a
rate of 200 samples h−1 and a minimum of 150
calibration curve for OTCH determination and respective
samples h−1, for TC concentrations of 48.1 –
calibration equation. OTCH standard solutions injected: A,
4.8 × 103 ppm for TCH, 49.1 – 4.9 × 103 ppm for
6 × 10−4; B, 7 × 10−4; C, 1.05 × 10−3 and D, 2 × 10−3 M. C.M.C.M. Couto et al. / J. Pharm. Biomed. Anal. 18 (1998) 527 – 533
Table 1Results obtained from TCH, CTCH and OTCH determination in commercial pharmaceutical preparations by the proposed methodand reference procedure
a Mean and standard deviation of four determinations for the same sample. Results expressed as mg g−1. b Recovery values of TC obtained by using the specified methodology, after addition of 0.3–0.5 ml of a 1×10−2 M TC solution
tion for TCH, OTCH and CTCH was less than
less than the fixed value (2.365) for a reliable
92.6, 1.1 and 1.4 mV day−1, respectively.
The within-run precision of FIA methodology
was assessed by calculating the relative standarddeviation after performing 12 replicate injections
The usefulness of the proposed method for the
of each of the three samples (one of each type of
assay of commercial TC formulations was as-
TC) of 318.0 mg g−1 (OTCH), 761.3 mg g−1
sessed by studying the effect of some common
(TCH) and 19.8 mg g−1 (CTCH) concentrations.
excipients used in pharmaceutical preparations.
The values obtained showed a good precision with
The influence of some inorganic and organic com-
a relative standard error less than 0.4%.
pounds, namely glucose, sucrose, lactose, starch,urea, polyethylene glycol, PEG 4000, Na SO and
NaCl on the FIA system were evaluated. No
4. Conclusions
interference was observed from any of the excipi-ents tested other than NaCl.
The determination of tetracyclines by using a
The present FIA manifold was therefore ap-
flow injection system with potentiometric detec-
plied to the determination of TC in different
tion proved to be an advantageous method re-
pharmaceutical formulations available in Portu-
gal. The results obtained are presented in Table 1.
since determinations within a wide concentration
The quality of the results obtained with the FIA
range, regardless of the samples colour and tur-
system (C ) was assessed by comparison with the
bidity, could be accomplished. The potentiometric
results provided by the reference procedure (C )
detection system of increased sensitivity provides
described in Pharmacopoeia Helvetica. A linear
improved precision, high sampling rates and bet-
ter reproducibility than the previously reported
showing a good agreement between methods. Re-
This system facilitated the determination of
covery assays performed for all the pharmaceuti-
these compounds with high sampling rates, from
cal samples, presented values close to 100%.
150 to 200 samples h−1, and a low consumption
of reagent, about 13 mg Cu(II) determination−1.
Student t-test was also carried out and a theo-
The results obtained in this work enable to
retical value of 0.696 was obtained for the deter-
conclude that this methodology can be applied to
mination of TC by the proposed method, being
the analysis of other antibiotics chemically similar
C.M.C.M. Couto et al. / J. Pharm. Biomed. Anal. 18 (1998) 527 – 533
to TC, only requiring the adjustment of the com-
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