Talanta 166 (2017) 223227

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Talanta
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A feasible method for indirect quantication of L-T4 in drugs by iodine MARK

determination

Marcia Foster Mesko , Alessandra Cortes Teotonio, Dirce Taina Teixeira Oliveira, Diogo La Rosa
Novo, Vanize Caldeira Costa
Centro de Cincias Qumicas, Farmacuticas e de Alimentos, Universidade Federal de Pelotas, Campus Capo do Leo, 96160 000, Capo do Leo, Brazil

A R T I C L E I N F O A BS T RAC T

Keywords: In this work, a method combining microwave-induced combustion (MIC) for sample preparation of commercial
Sodium levothyroxine determination levothyroxine sodium (L-T4) drugs (L-T4: 25200 g/tablet), and potentiometry with ion selective electrode
Iodine determination (ISE) for iodine determination and subsequent indirect quantication of L-T4 was proposed. The type and
Drug analysis concentration of the absorbing solution were evaluated to select the most suitable conditions for this study.
Microwave-induced combustion
Using the MIC method, it was possible to use solutions as diluted as 150 mmol L1 (NH4)2CO3 (for samples
Sample preparation
containing 25200 g of L-T4/tablet) for I absorption. In these conditions, recoveries for L-T4 were between
Ion selective electrode
99% and 101%, and relative standard deviations were lower than 10%. The limit of detection for L-T4 was
11.2 g/tablet, which is almost two times lower than the minimum concentration of L-T4 in commercially
available drugs. Thus, the MIC was suitable for the digestion of several L-T4 drugs for subsequent I
determination by ISE and indirect quantication of L-T4. Furthermore, the proposed method presents high
throughput with low reagent consumption and consequently lower waste generation, making it suitable for
routine determination of L-T4 in drugs. From the obtained results, it was possible to observe that one of the
analyzed samples is not in agreement with the limits established by the United States Pharmacopeia, indicating
the importance of the drug quality control. The United States Pharmacopeia establishes that each tablet must
contain between 90% and 110% of the amount of active substance declared by the manufacturer.

1. Introduction Methods for analysis of the activity pharmaceutical ingredient and/

The thyroxine (T4) and triiodothyronine (T3) hormones secreted
from the thyroid gland present an important biological role in the
development of the central nervous system in infants, the skeletal
growth and for the normal function of multiple organ system in adults
[1]. The deciency of these hormones in the organism causes
hypothyroidism, which is a disorder with numerous symptoms, such
as myxedema with struma and debility [2]. For the treatment of the
hypothyroidism, pharmaceutical preparations containing the thyroid
hormones are used. The levothyroxine sodium (L-T4) - a sodium salt of
levo-isomer of T4 - is the most common drug prescribed to treat this
disease [1,2]. The L-T4 has a narrow therapeutic index; thus, doses
outside of the therapeutic window may cause the side eects of
hyperthyroidism or hypothyroidism in patients, such as adverse cardiac
and/or metabolic eects [3,4]. In this way, the determination of L-T4 in
commercially available drugs using a sensitive method is important to
ensure that the dose present in the medicament corresponds to the
informed.
or tablets containing L-T4 are described in ocial compendiums. These
methods usually involve the use of the high-performance liquid
chromatography (HPLC) for the determination of L-T4 [5,6], which
presents as disadvantages the time consuming for analysis, interfer-
ences from matrix compounds, among others. Moreover, it is impor-
tant to highlight that, in general, the interferences in L-T4 assay are
related to the matrix composition and solubility of the excipients, and it
can cause high variability in the results [7]. Another method involves a
sample preparation using the Schniger ask that generally allows the
digestion of a small sample mass and low throughput [8,9]. In this
method, the determination of L-T4 is performed indirectly by the
titration of the iodine present in the sample. Nevertheless, the titration
is a technique with low sensibility and contributes to the obtainment of
high limits of detection (LODs), making dicult the determination of
L-T4 in low concentrations [10].
In this context, it is also important to mention that there are
relatively few studies in literature reporting methods for the determi-
nation of L-T4 in drugs [13,1119]. In most of the studies, the

Corresponding author.
E-mail address: marcia.mesko@pq.cnpq.br (M.F. Mesko).

http://dx.doi.org/10.1016/j.talanta.2017.01.039
Received 12 December 2016; Received in revised form 11 January 2017; Accepted 12 January 2017
Available online 13 January 2017
0039-9140/ 2017 Elsevier B.V. All rights reserved.
M.F. Mesko et al.
commercial L-T4 drugs were dissolved in a suitable medium and
analyzed by HPLC [13,12]. However, depending on the concentration
of L-T4 in pharmaceutical preparations, the sensitivity of HPLC with
ultraviolet detection can be not suitable, especially at low concentra-
tions, for detection and quantication of subtle but signicant dier-
ences in L-T4 concentrations [3,4]. Other methods used for L-T4
determination in commercial drugs involve the use of immunoassays,
capillary electrophoresis, and electroanalytical and spectrometric tech-
niques [13,1519]. In spite of some electroanalytical techniques had
been used for the determination of L-T4 and the potentiometry with ion
selective electrode (ISE) to be a simple and an inexpensive device, it is
important to highlight that this technique still was not evaluated for the
determination of I and the indirect quantication of L-T4 in drugs.
Considering the advantages of the potentiometry with ISE, such as
relatively good sensitivity and selectivity and fast response time, it is
possible to consider the use of this technique as an alternative for the
indirect quantication of L-T4 in routine analyses. However, the
potentiometry with ISE is extremely prone to interferences caused by
ions with hydration energy and ionic radius similar to the analyte or
that form complexes with the specie to be determined [20,21]. Thus, it
is necessary to use an appropriate sample preparation method to
ensure the release of I from the sample matrix and the accuracy of L-T4
determination by potentiometry with ISE in commercial drugs. In this
sense, considering the major organic composition of L-T4 drugs and the
unsuitability of wet digestion for the subsequent determination of I,
combustion methods can be considered as an alternative for the
digestion of drug samples and the subsequent determination of I by
ISE for the indirect quantication of L-T4 [22].
The microwave-induced combustion (MIC) method allows the
conversion of an organic matrix to the respective oxidation products,
and the high temperature reached during combustion enables quanti-
tative halogen release from the sample [8,23]. In the MIC, it is possible
to digest sample masses higher than those commonly digested using
Schniger ask (sample preparation method recommended in ocial
compendium for indirect determination of L-T4) due to the possibility
of pressurization of the system with oxygen. Moreover, the MIC has a
high sample throughput and, as the Schniger ask, allows the use of
reagents suitable to the analytes and the determination technique [8].
For digestion of organic samples by the MIC, samples are usually
prepared as pellets and positioned on a quartz holder. The combustion
occurs in a closed quartz vessel pressurized with oxygen, and the
ignition step is performed using microwave radiation and an ammo-
nium nitrate solution. After combustion, the analytes are absorbed in a
suitable solution, which can be compatible with the analytes and the
determination technique [22,24].
Recently, the MIC has been successfully applied to the digestion of
organic samples for further determination of halogens, such as foods,
polymers, and drugs [23,2531]. In addition, the MIC is recommended
in Brazilian Pharmacopeia for digestion of organic samples that may
not be eciently digested even using wet digestion in closed system,
and it is reported as a suitable sample preparation method for
subsequent halogens determination in drugs. However, this method
still was not recommended for the digestion of L-T4 drugs and the
subsequent determination of I [32].
Thus, considering the importance of the determination of L-T4 in
commercial drugs, in the present work, a method using the MIC is
proposed for drug digestion and the subsequent determination of I by
ISE for the indirect quantication of L-T4. For both, a systematic study
was performed paying special attention to the sample preparation step.
The accuracy of the proposed method was evaluated by the comparison
of the obtained results by ISE to those determined in digests by IC and
by recovery tests using an L-T4 standard solution.
224
Talanta 166 (2017) 223227
Table 1
Operational parameters for iodine determination by IC.
Parameter Operation conditions
Mobile phase 3.2 mmol L1 Na2CO3/1.0 mmol L1 NaHCO3 (pH 10.5)
Flow rate 0.7 mL min1
Sample loop 20 L
Column Anion-exchange, Metrosep A Supp 5 (polyvinylalcohol with
quaternary ammonium groups, 250 mm x4 mm i.d.)
Guard column Metrosep A Supp 4/5 Guard (polyvinylalcohol with quaternary
ammonium groups, 5 mm x4 mm i.d.)
Suppressor Chemical type
Detection Conductivity
2. Experimental section
2.1. Instrumentation
The MIC method was performed in a microwave oven (Multiwave
3000 microwave sample preparation system, Anton Paar, Austria)
equipped with eight high-pressure quartz vessels with an internal
volume of 80 mL (maximum pressure and temperature of 80 bar and
280 C, respectively). Quartz holders were inserted inside the vessels as
support for the samples during the combustion process.
Iodine determination was carried out using a potentiometer (HI
3221 pH/ORP/ISE meter, HANNA Instruments, USA) equipped with
an ISE of iodide (HI 4111, HANNA Instruments, USA). A hot plate
with agitation (RH Basic, IKA, USA) was used for the homogenization
of the samples during I determination by ISE. Additionally, for
comparison of results, the determination of I was performed using an
ion chromatograph (IC 2.861.0020, Metrohm, Switzerland), and the
operating conditions are shown in Table 1.
2.2. Reagents and samples
All solutions were prepared using ultrapure water (18 M cm)
obtained from a purication system (MegaUp, MegaPurity, South
Korea), and the reagents used were of analytical grade or higher purity.
Water and ammonium carbonate solutions (50150 mol L1) were
used as absorbing solution in the MIC. Solutions of (NH4)2CO3 and a
solution of ammonium nitrate (6 mol L1), used as the igniter of the
combustion, were prepared by the dissolution of solid reagents (Merck,
Germany) in water.
Small discs (12 mg, 15 mm of diameter) of lter paper (0.5% ash
content, Qualy, J Prolab, Brazil) were used as a combustion aid, and
polyethylene (PE) lms were used to wrap the samples for digestion by
the MIC. Before combustion, the discs of lter paper and PE lms were
cleaned by immersion in 10% (v v1) HNO3 (Vetec, Brazil) for 20 min
in an ultrasonic bath (USC-1800 A, Unique, 40 kHz, 155 W, Brazil),
subsequently rinsed with ethanol (Synth, Brazil) and ultrapure water,
and dried in a class-100 laminar bench (CSLH-12, Veco, Brazil).
Oxygen (99.6%, White Martins, Brazil) was used for the vessels
pressurization for the MIC method.
Vessels and holders were cleaned with 6 mL of 14.4 mol L1 HNO3
(Vetec, Brazil) using a microwave heating program set at 1000 W for
10 min and 0 W for 20 min (cooling step). After, this procedure was
repeated using 6 mL of water instead of HNO3.
The standard solutions used for the determination of I by IC and
ISE were prepared by the dilution of a stock standard solution
(0.1 mol L1, HI 4011, HANNA Instruments, USA) in a suitable
medium. Moreover, for I determination by ISE, 2 mL of ionic strength
adjuster (HI 4000-00, HANNA Instruments, USA) were used per
100 mL of sample or standard solution. The mobile phase
(3.2 mmol L1 sodium carbonate and 1.0 mmol L1 sodium bicarbo-
nate) used for the determination of I by IC was prepared by the
dissolution of the respective salts (Merck, Germany) in water.
M.F. Mesko et al. Talanta 166 (2017) 223227

Sodium levothyroxine (high purity L-T4 from Pharmanostra was possible to digest up to 800 mg of the L-T4 drug.
Company, Brazil) was diluted in 500 mmol L1 NH4OH to obtain the However, taking into account that the samples contained a sig-
reference solution (L-T4: 1875 mg L1) used for L-T4 recovery tests. nicant iodine concentration and the sample mass commonly used in
The solubilization of L-T4 in an alkaline solution was necessary, the MIC method [8,34], the subsequent studies were performed using
because it has low solubility in water. 400 mg of sample. In this context, it is important to mention that this
Commercial L-T4 drugs of the same brand (labeled as brand A) sample mass was also selected to avoid excessive dilutions, considering
and containing 25, 50, 100, 150 or 200 g of L-T4 per tablet (produced the content of I in some therapeutic doses, and for safety aspects.
in Germany or Mexico) were purchased in local drugstores. Drugs However, it is important to mention that depending on I content in
containing 100 or 200 g of L-T4 per tablet were chosen for initial drug higher sample mass can be used.
experiments because these samples had an intermediary and high Therefore, 400 mg of the L-T4 drug were digest by the MIC using a
concentration of L-T4, respectively, when compared to the other drugs. PE lm for sample wrapping and a pressure of O2 of 20 bar. Moreover,
After, optimized conditions were used for digestion of the samples in these conditions, the maximum pressure achieved was lower than
containing 25, 50 or 150 of L-T4 per tablet. 30 bar, which corresponds only about 35% of the maximum operating
In addition, the proposed method was applied to L-T4 determina- pressure of the microwave system (80 bar).
tion in drug samples of two other brands (produced in Germany,
Mexico or Puerto Rico, and labeled as brands B and C) containing
3.2. Inuence of absorbing solution on I recoveries after the digestion
low (25 g/tablet) or high (200 g/tablet) concentrations of L-T4.
of commercial L-T4 drugs by the MIC
Before digestion by the MIC, the samples were ground using a
porcelain mortar and pestle to homogenize the samples and reduce
To select the most suitable solution for the absorption of I after the
the particle size, and dried at 60 C for 3 h.
digestion of drug samples by the MIC, water or (NH4)2CO3 solutions
(50150 mmol L1) were evaluated. These solutions were selected
2.3. Sample preparation by MIC
according to previous works that aimed the determination of halogens
in a variety of matrices [29,3436]. Experiments were carried out by
Commercial L-T4 drugs (400 mg) were weighed and wrapped with a
adding of 100 L of the L-T4 reference solution (1875 mg L1) on drug
PE lm (88 cm). After closing the PE lm as a small wrap, the lm
samples containing 100 or 200 g of L-T4/tablet prior to digestion by
was sealed by heating, and its excess was removed (PE lm mass used
the MIC. The drugs containing 100 or 200 g of L-T4/tablet were
in the MIC was approximately 30 mg). The wrap of PE containing the
selected for initial studies, because these samples had an intermediary
sample was placed with a small disc of lter paper moistened with an
and high concentration of L-T4, respectively, contributing to the
NH4NO3 solution (50 L of 6 mol L1) on the base of a quartz holder.
evaluation of the proposed method in a broad manner. Results are
The quartz holder was then introduced into the quartz vessel contain-
shown in Fig. 1.
ing 6 mL of absorbing solution (water or 50, 100 or 150 mmol L1
As shown in Fig. 1, when water was used as an absorbing solution,
(NH4)2CO3). Vessels were closed, positioned in the rotor, and pressur-
recoveries for L-T4 were lower than 40%. Probably, this behavior is
ized with 20 bar of oxygen. The rotor with the vessels was placed inside
related to the low potential hydrogenionic of the solutions obtained
of the microwave oven, and the heating program (1400 W for 5 min,
after digestion of both samples by the MIC (pH < 3.0). In this
and 0 W for 20 min for cooling) was started [24,29]. After combustion,
condition, the conversion of I to volatile species, such as HI and I2,
the pressure of each vessel was released and resultant solutions were
is favored, and so the analyte may be lost by volatilization, even using
transferred to volumetric asks and diluted with water up to 25 mL for
closed vessels. Thereby, the water is not a suitable solution for the
subsequent I determination by ISE and IC. The indirect quantication
absorption of I during digestion of commercial L-T4 drugs by the MIC.
of L-T4 in the analyzed samples was carried out by calculations that
Although good recoveries (97101%) had been obtained for L-T4
related to the I concentration in the samples with I content in the drug
using (NH4)2CO3 solutions (50, 100 or 150 mmol L1) during digestion
molecule, as well as with the molecular mass of L-T4.
of the sample containing 100 g of L-T4/tablet, the same behavior was
The accuracy of the proposed method was evaluated by analyte
not observed for the drug containing 200 g of L-T4/tablet. For the
recovery tests. For this, a reference solution containing 1875 mg L1 L-
sample containing the higher dosage of L-T4, quantitative recoveries
T4 (100 L) was added on the sample (400 mg) before digestion by the
only were obtained (99 9%) when 150 mmol L1(NH4)2CO3 was used
MIC for the subsequent I determination by ISE and the indirect
as an absorbing solution. This behavior can be associated with the
quantication of L-T4.
increase of iodine content, which requires the use of more concentrated
All statistical calculations were performed using the software
solutions for its absorption.
GraphPadInStat (GraphPadInStat Software, Inc., version 3.00, 1997),
and a condence level of 95% was used.

3. Results and discussion

3.1. Commercial L-T4 drugs digestion by the MIC preliminary

studies

For the digestion of the L-T4 drugs by the MIC, initial experiments
were carried out to evaluate a suitable way to insert samples into the
combustion system. Initially, the digestion of a commercial tablet was
evaluated, and 100 mg of sample (medium mass per commercial
tablet) was successfully burned. However, in order to digest a higher
sample mass, the samples were comminuted and wrapped with PE
lms before being inserted in the combustion system. Polyethylene
lms were used as described in a previous work [30]. Moreover, in Fig. 1. Recoveries for L-T4 after the digestion of commercial drugs containing 100 g of
these experiments, 50 L of 6 mol L1 NH4NO3 solution were used as L-T4/tablet or 200 g of L-T4/tablet by MIC using water or 50, 100 or
igniter, the initial pressure of O2 was 20 bar, and 6 mL of water were 150 mmol L1 (NH4)2CO3 as an absorbing solution. Determination by ISE (sample mass:
used as the absorbing solution [26,33]. By using these conditions, it 400 mg, n=3).

225
M.F. Mesko et al.
Table 2
Iodine and L-T4 concentrations in L-T4 commercial drugs after MIC digestion using
150 mmol L1 (NH4)2CO3 as absorbing solution, and I- determination by ISE (mean
standard deviation, n=3).
Brand Informed L-T4 I (g/tableta) Calculated L-T4
concentration (g/tableta) concentration (g/tableta)
A 25 15.1 0.6 23.6 0.9
50 30.1 1.2 47.0 2.0
100 58.5 1.6 91.3 2.6
150 91.9 3.0 143 5
200 125 2 197 3
B 25 14.6 1.1 23.0 1.7
200 123 1 192 2
C 25 14.6 1.2 23.0 1.9
200 93 4 154 5
a
Tablet: equivalent to a tablet with mass of approximately 100 mg.
Thus, the 150 mmol L1 (NH4)2CO3 was selected as the absorbing
solution because this solution provided quantitative recoveries, even
when drugs containing high concentrations of L-T4 were digested by
the MIC. In this condition, probably, I released from the sample during
the combustion process was quantitatively converted to I-, allowing the
determination of I in all commercial L-T4 drugs by ISE. In addition, for
optimized conditions, relative standard deviations were lower than
10% and a LOD of 11.2 g/tablet for L-T4 (LOD for I-: 7.14 g/tablet)
was obtained, which is at least two times lower than concentration of L-
T4 in evaluated drugs. Thus, the proposed method can be considered
suitable for L-T4 determination at low concentrations. The LODs were
calculated from the mean of the blank values and the standard
deviation for 10 replicates, taking into account the sample mass (400
mg), the nal volume of digests (25 mL), and the dilution factor (2
times). It is important to mention that the LODs were expressed
considering 100 mg of mass of the tablet, which is the general mass of
commercial L-T4 drugs.
For comparison of results, drug samples containing 200 g of L-T4/
tablet were also analyzed by IC after the MIC using 150 mmol L1
(NH4)2CO3 as the absorbing solution. Obtained results for I- by IC
(121 3 g/tablet) did not present a statistical dierence (Student's t-
test, 95% of condence level) of those obtained by ISE (125 2 g/
tablet), indicating that the determination of I- by ISE has a suitable
accuracy.
In this context, it is also important to mention that the reported
methods in literature for L-T4 determination by HPLC may involve
several steps prior to analysis, such as dissolution, derivatization,
redissolution and ltration, and a time for L-T4 determination about
twenty minutes [2,3,14,37]. On the other hand, the proposed method
allows the sample preparation of up to 16 samples per hour, and
involves a determination technique with a very quick response. Thus,
the high throughput is another advantage of the proposed method.
3.3. Indirect determination of L-T4 in commercial drugs
The proposed method was applied for the digestion of commercial
L-T4 drugs of the same brand containing 25, 50, 100, 150 or 200 g of
L-T4 per tablet, and drugs of other brands containing low (25 g/
tablet) or high (200 g/tablet) concentrations of L-T4. These experi-
ments were performed using 400 mg of sample and 150 mmol L1
(NH4)2CO3 as an absorbing solution. Results are shown in Table 2.
For drug of brand C that contained 200 g of L-T4/tablet, the
agreement with the value reported by the manufacturer was about 80%.
On the other hand, when the other samples were evaluated, agreements
with values reported by the manufacturer were between 91% and 99%.
Thus, the obtained results indicate that most of evaluated drugs are in
agreement with the set values by the United States Pharmacopeia,
which establishes that each tablet must contain between 90% and 110%
of the amount of active substance declared by the manufacturer [5].
226
Talanta 166 (2017) 223227
4. Conclusion
The proposed MIC method was suitable for the digestion of
commercial L-T4 drugs for the subsequent determination of I by
potentiometry with ISE and the indirect quantication of L-T4. Using
the proposed method, it was possible to obtain low blank values and
LODs. Moreover, the MIC, combined with ISE, presents a high
throughput and a suitable accuracy and precision, allows the use of
an absorbing solution suitable to the analytes and the determination
technique, as well as the use of diluted solutions according to green
chemistry.
In addition, it is important to mention that the MIC enables the
complete digestion of the organic matter, ensuring total conversion of
the iodine present in L-T4 molecules to iodide (iodine specie deter-
mined by ISE). Additionally, the use of the MIC reduces the occurrence
of chemical species related to incomplete digestion of the sample,
which can cause interferences during I determination by ISE and,
consequently, in the indirect determination of L-T4. Another important
aspect is the possibility of evaluating tablets with several concentra-
tions of L-T4 in view that this characteristic allows the quality control of
these drugs, which present dosages in micrograms and a narrow
therapeutic index.
Acknowledgements
The authors are grateful to CAPES, CNPq and FAPERGS for
supporting this study.
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