Journal of Pharmaceutical and Biomedical Analysis
Determination of tetracycline and its major degradation
products by liquid chromatography with fluorescence
A. Pena a,*, A. Carmona b, A. Barbosa b, C. Lino a, I. Silveira a, B. Castillo b
a Laboratory of Bromatology, Faculty of Pharmacy, Uni6ersity of Coimbra, 3000 Coimbra, Portugal
b Laboratory of Instrumentals Techniques, Department of Analytical Chemistry, Faculty of Pharmacy, Complutense Uni6ersity,
Received 15 May 1998; received in revised form 4 September 1998; accepted 13 September 1998
A liquid chromatographic method of tetracycline and its major degradation products on a C -reversed phase
column with acidic mobile phase and fluorescence detection is described. The quantification limit, measured as theamount of sample that gave a signal ten times the peak-to-peak noise of the baseline, was: 0.25 ng for tetracycline(TC) and epitetracycline (ETC), 25 ng for and 4-epianhydrotetracycline (EATC) and 50 ng for anhydrotetracycline(ATC) of injected standard. By means of this liquid chromatography (LC) assay TC, ETC, EATC and ATC as maindegradation products of tetracycline, can be separated and determined with good sensitivity and specificity within 15min. 1998 Elsevier Science B.V. All rights reserved. Keywords: Tetracycline; Epitetracycline; Epianydrotetracycline; Anydrotetracycline; Liquid chromatography; Fluorescence detection
tetracycline (ATC) and 4-epianhydrotetracycline(EATC). Tetracycline used in feed additives are
Tetracycline antibiotic is widely prescribed in
even less pure. The source of the tetracycline used
animal husbandry. It is used for the prevention
in feeds is the residual tetracycline found in the
and control of disease and as feed additives to
dried, ground mycelial powder harvested from
promote weight gain and increase feed conversion
Veterinary pharmaceutical formulations of te-
stored under adverse conditions, e.g. high temper-
tracycline (TC) contain small amounts of impuri-
ature and humidity . It must be realized that
ties namely 4-epitetracycline (ETC), anhydro-
degradation products of tetracyclines can alsooccur in the stomach .
It is important to determine not only the tetra-
* Corresponding author. Fax: + 351-39-27126.
cycline and its major degradation products in
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 6 8 - 4
A. Pena et al. / J. Pharm. Biomed. Anal. 18 (1998) 839 – 845
pharmaceutical formulations but also in biologi-
pseudo-first-order kinetics, leading to ATC at
cal and food samples. Next to a decrease of
very low pH. The epimerization of ATC, and the
potency, degradation can lead to toxic degrada-
dehydration of the ETC lead to the formation of
tion products. This is already proven for EATC
The toxic effects of ATC were attributed to the
Permitted concentrations of these impurities in
relative position of the dimethylamino group (on
pharmaceutical formulations are fixed by the Eu-
Several papers dealing with the liquid chro-
Since the residues in biological and food sam-
matographic determination of tetracyclines and
ples, are products of metabolism they consist of
their degradation products have been published
the parent drug and all compounds derived from
[11,12]. Those reports deal mainly with the deter-
it, such as free metabolites. The maximum residue
mination of tetracyclines in pharmaceutical prepa-
levels (LMR) in foods for TC, established by the
rations, where relatively high concentrations are
European Community (EC)  are for the parent
involved, but it is also important determine their
compound and its epimer. Therefore, it is also
presence in biological and food samples at resid-
very important to develop methods for its deter-
ual levels. Therefore, it was essential to introduce
The stability of TC is poor under strong acidic
Fluorescence detection of tetracyclines is more
and alkaline conditions with reversible formation
specific and also in many cases more sensitive
than UV detection normally used , and its a
(ETC) in weak acid (pH 3) and to anhydro-TC
important tool in the analysis of tetracycline
under strong acidic (below pH 2) conditions .
residues in biological and food samples.
Epimerization on carbon-4 in tetracycline,
In this paper a LC method with fluorescence
which is a reversible first-order process, occurs
detection, according to the Haagsma method 
most rapidly between pH 3 and 5. The presence of
validated in our laboratory for the residue analy-
a hydroxyl group at C favors dehydration and
sis of oxytetracycline (OTC), tetracycline (TC)
aromatization of the C-ring of tetracycline follow
and chlortetracycline (CTC), was tested for the
Fig. 1. Structures of tetracycline and its major related substances. A. Pena et al. / J. Pharm. Biomed. Anal. 18 (1998) 839 – 845
analysis of TC, ETC, EATC and ATC. The pre-
phases in well-ventilated chemical fume hood.
sented method utilizes the fluorescence produced
when tetracyclines reacted with magnesium ions.
1. Water was purified by distillation and passage
Most tetracyclines fluorescence in the presence
through Milli-Q system (Millipore). The water
of magnesium ions, and this fluorescence is in-
was filtered through a 0.45 mm filter under
tensified by the addition of a base such as
vacuum and degassed by ultrasonication.
2. All the solvents were LC grade and were pur-
Tetracycline was found to fluorescence more if
chased from Carlo Erba (Italy). Oxalic acid,
heated to produce anhydro-salts before complex-
magnesium acetate, boric acid, potassium hy-
ation . During dehydration, two double
droxide and sodium hydroxide were analytical
bonds are added to the tetracycline nucleus,
reagent grade chemical obtained from Merck
producing stable anhydro forms and increasing
fluorescence. Therefore, this property can be
3. Tetracycline (TC) was obtained from Sigma
used for a more sensitive detection of the anhy-
dro forms, measured as anhydro-TC – magne-
purchased from European Pharmacopoeia. In-
dividual stock standard solutions of TC, ETC,
which gives fast separation and determination
with good sensitivity and selectivity, of TC,
into a volumetric flask, and was stored at
− 20°C in brown glass vials for a maximum
tetracycline, suitable for their precise routine
period of one month. Tetracycline was cor-
analysis in biological and food samples.
The working solutions were a mixture of the
four compounds prepared by a serial dilution of
the stocks and were stored in brown glass vials at4°C. These solutions were prepared immediately
The mobile phases used for analysis containing
model 7125 loop injector (Rheodyne, Cotati,
aqueous oxalic acid solution (pH 2.0; 0.01 M) and
CA) and a Perkin Elmer LS-3B fluorescence de-
20 – 40% of acetonitrile. The mobile phases were
tector operated at an excitation wavelength of
filtered through a 0.45 mm filter under vacuum,
385 nm and an emission wavelength of 500 nm
were used. The spectral band width was 10 nm
The reagent post-column was prepared daily,
for both excitation and emission. Mobile phase
dissolving 6.0 g of magnesium acetate, 6.1 g boric
flow 0.8 ml min−1. The derivatization reagent
acid and 2.5 g of potassium hydroxide in 950 ml
was delivered at a flow rate of 0.45 ml min−1.
of water, adjusted with 1 M sodium hydroxide
The results were recorded on a 3390A chro-
and make up to 1 l. Its important to follow the
order of addition of these reagents, because pre-
column used was a Chromspher C , 100-3 mm
cipitations may occur. This solution was filtered
through a 0.45 mm filter under vacuum and de-
gassed by ultrasonication, was held in a brownglass bottle.
All glassware was cleaned with Extran MA 03,
Merck, Germany; 10% v/v, rinsed in concentrated
Caution: tetracyclines are irritants and tetracy-
acid-dichromate solution, washed thoroughly with
cline itself is a possible teratogen. Handle tetra-
tap water, rinsed with deionized water and dried
cyclines standards with care. Prepare mobile
A. Pena et al. / J. Pharm. Biomed. Anal. 18 (1998) 839 – 845
Table 1Linear regression of the assay response for TC, ETC, EATC and ATC
these solutions: they were always kept at 4°C andprotected from light, immediately after the injec-
The standard curves were tested for linearity in
tion in the chromatographic system, they were not
the range of 2.5 – 25 ng for ETC and TC, and
allowed to stand in the laboratory at room tem-
50 – 250 ng for EATC and ATC, of injected
perature. Under these conditions no appreciable
decomposition was observed in these solutions for
The linear regressions of the assay response for
approximately 1 working day (8 – 12 h).
TC, ETC, EATC and ATC are shown in Table 1.
The good repeatability mentioned in the cali-
The correlation coefficients for the regression
bration is an indication for the good stability of
this compounds during the chromatographic
greater than 0.993, showing the standard curve to
be linear within the range of standards used.
The quantification limits, measured as that
amount of sample that gave a signal ten times the
peak-to-peak noise of the baseline, were: 0.25 ngfor TC and ETC, 25 ng for EATC and 50 ng for
The quantification limits achieved by the
present method are in agreement with the MRL
The quantification of small amounts of impuri-
established by the European Comminity for TC
ties can be realized owing to the good separation
and its epimer in foods and in the same chromato-
graphic run we can also averiguate the presence of
the toxic EATC and ATC. Since the anhydro-
analysing five standard solutions at two levels of
TC – magnesium complexes are highly fluorescent
concentration (ETC and TC at 25 and 2.5 ng and
the method is more sensitive for these compounds
EATC and ATC at 250 and 50 ng), whilst be-
permitting the separation of very small amounts
tween-day precision were determined by analysing
the same standard solutions on 5 successive days.
can be separated from TC, according to the Eu-
We obtained coefficients of variation between 2.4
ropean Pharmacopoeia permitted concentrations
and 3.5% and 9.0 and 4.1% respectively, showing
Chromatography of anhydrotetracyclines is
The individual stock standard solutions of TC,
some what more difficult to achieve than that the
ETC, EATC and ATC were prepared in methanol
parent compounds because of the lower polarity
and stored at − 20°C in brown glass vials. We did
not observe significant alterations over a maxi-
A study on reversed-phase LC of TC and its
mum period of 1 month. The working solutions
degradation products using acid mobile phases
were a mixture of the four compounds prepared
was first published by Knox et al. .
by a serial dilutions of the stocks with methanol,
The method presented utilizes a C Chromo-
in brown glass vials, and were prepared daily,
spher column at room temperature with acetoni-
immediately before use. Some care was taken with
trile – 0.01 M oxalate buffer (pH 2) as the mobile
A. Pena et al. / J. Pharm. Biomed. Anal. 18 (1998) 839 – 845
phase. Tetracyclines form chelate complexes with
and ATC altering the concentration of organic
ions at i-diketones (C –C ) and carboxyamide
(C )  and bind with silanol groups in the
We have tried several modifications of the per-
stationary phase . In an acidic medium (pH
centage of acetonitrile in the mobile phase, so
1 – 2.5) the tetracycline molecule is fully proto-
those anhydrotetracyclines eluted after the tetra-
nated and exists in its cationic form [20,21] and
cycline with good resolution, permitting also the
can be paired with a suitable anion such as ox-
separation of tetracycline and his epimer.
alate . On the other hand, all tetracyclines had
The isocratic analysis using acetonitrile – oxalic
the best asymmetric values at pH 2 .
acid solution (pH 2.0; 0.01 M) (30:70, v/v) as
Following elution, tetracyclines are derivatized
mobile phase, allows the separation of the four
with Mg2+ ions at room temperature to produce
a highly fluorescent derivative. The fluorescence
With 20% of acetonitrile the resolution between
detector is set with an excitation wavelength of
385 nm and an emission wavelength of 500 nm.
eluted very late with bad resolution.
The development of the fluorimetric method
An increase of the acetonitrile concentration to
was based upon experience obtained with the
40% finally enable the elution of EATC and ATC
analysis of OTC, TC and CTC. The fluorescence
but with this mobile phase ETC overlapped with
response is dependent upon pH. For a maximum
fluorescence a pH greater than 8 is essential, and
Several variations of the ratio of acetonitrile in
is associated with the ionized form of the pheno-
the acid mobile phases did not yield substantially
lic-i-diketone site of these molecules. The pH is
better separations, and some improvement was
adjusted with the addition of the post-column
achieved by the use of gradient elution as we can
reagent, prepared in alkaline solution at pH 9.
observe in Fig. 2, showing greater resolution of
The fluorescence response is also dependent
these compounds. The gradient was chosen based
upon the flow rate of derivatization reagent. In
in this work experience, to allow for optimum
our study the fluorescence response reached a
separation. We have applied the follow gradient:
maximum at 0.45 ml min−1. Post-column deriva-
from 0 to 5 min 20% of acetonitrile and 80% of
tization does not directly affect the chromato-
oxalic acid (pH 2.0; 0.01 M), from 5 to 16 min
graphic properties of the tetracyclines, however
40% of acetonitrile and 60% of oxalic acid (pH
the reaction chemistry must be rapid on the chro-
2.0; 0.01 M) and at 17 min 30% of acetonitrile
matographic time scale in order to preserve the
and 70% of oxalic acid (pH 2.0; 0.01 M), at a
Post-column derivatization has also the advan-
tage that a separate sample treatment step is notrequired and the analytes are better separated
from interferences prior to derivatization.
Preliminar work was carried out on isocratic
Methanol was chosen as the universal solvent
analysis, making several variations of the ratio of
for the tetracyclines, because of its ability to
organic modifier and aqueous oxalic acid in the
dissolve the tetracyclines and its miscibility with
aqueous and organic solvents. Methanol was also
The composition of acid mobile phases must be
chosen because aqueous solvents tend to acceler-
established very well, because complete separation
ate degradation of tetracyclines compounds.
of TC and ETC is obtained only with mobile
Because many reversed-phase materials are un-
phases which are too weak to eluted the more
stable at pH lower than 2 – 3 it was necessary to
strongly retained EATC and ATC within a rea-
flush the columns with a neutral solvent (e.g.
sonable time. The retention decrease directly with
water – acetonitrile 50:50) for 1 h at the end of
concentration of acetonitrile in the mobile phase.
each working day . This practice contributed
We can obtain shorter retention times of EATC
markedly to the prolonging of the column life. A. Pena et al. / J. Pharm. Biomed. Anal. 18 (1998) 839 – 845
The epimer of TC is always eluted first, while
suitable for analysing the impurities of TC, show-
its anhydro-forms are most retained in agreement
ing better resolution (R ) values, we obtained
with the polarity of the tetracycline compounds.
good symmetrical peaks using a Chromspher C
Accordingly to polarity of the chromatographic
column. Our results show that even though a C
column was used, we obtained good performance.
eluted with good resolution only with a high
This method enables good separations of TC,
percentage of acetonitrile in the mobile phase.
With this LC method we can differentiate be-
The methodology reported herein use a simple
tween the tetracycline and their major degrada-tion products, at residue level within 15 min. Thus
solvent system containing a low concentration of
it is possible to ascertain whether the tetracyclines
buffer, avoiding the drawbacks related with its use
determined in biological and food samples are the
for the chromatographic system. Its also allows
intact molecule originally applied or some fluores-
analysis of the tetracyclines and its degradation
cent degradation and food products. The high
products with the same chromatographic column.
degree of selectivity achieved in using a post-
Although the analysis was performed at pH 2,
column reaction and fluorescence detection, that
this system was proven not to cause any epimer-
is less prone to interference from other com-
ization of TC. Formation of EATC and ATC due
pounds in the sample matrix, has considerable
to partial degradation of ETC and TC in the
potential as a basis for the development of a
strong acidic mobile phase also was not observed.
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