Tigeciclina -Método turbidimétrico

1. Rapid turbidimetric assay to potency evaluation of tigecycline in
lyophilized powder
Lucélia Magalhães da Silva a,
⁎, Hérida Regina Nunes Salgado b
a
Universidade Federal de Alfenas — UNIFAL, Alfenas, MG, Brazil
b
Faculdade de Ciências Farmacêuticas de Araraquara — UNESP, Araraquara, SP, Brazil
a b s t r a c ta r t i c l e i n f o
Article history:
Received 5 November 2014
Received in revised form 22 January 2015
Accepted 22 January 2015
Available online 22 January 2015
Keywords:
Tigecycline
Turbidimetric assay
Quality control
Tigecycline, a first-in-class glycylcycline and an analog of the semisynthetic antibiotic minocycline, is a potent,
broad-spectrum antibiotic that acts by the inhibition of protein translation in bacteria. This glycylcycline inhibits
Gram-positive, Gram-negative, atypical, anaerobic and antibiotic-resistant organisms. There is no microbiological
analytical method for tigecycline in lyophilized powder reported yet. Thus, this paper reports the development
and validation of a simple, sensitive, accurate and reproducible turbidimetric method to quantify tigecycline in
lyophilized powder, using Staphylococcus aureus as microorganism test and 3 × 3 parallel line assay design,
with twenty tubes for each assay. The validated method showed good results of linearity in the concentration
range from 3 to 4.32 μg/mL (r2
= 0.9999), selectivity, precision, robustness and accuracy of 99.74%. The results
demonstrated the validity of the proposed bioassay, which allows reliable quantitation of tigecycline in pharma-
ceutical samples and therefore can be used as a useful alternative methodology for the routine quality control of
this medicine.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
Glycylcyclines, discovered in 1993, are structural analogues of
tetracycline designed to avoid resistance mediated by efflux and ribo-
somal protection (Chopra, 2001). Tigecycline, a novel, first-in-class
glycylcycline, is a potent, broad-spectrum antibiotic that acts by the in-
hibition of protein translation in bacteria (Hoffmann et al., 2007). Tige-
cycline is structurally derived from minocycline by adding a tert-butyl-
glycylamido side chain to carbon 9 of the D ring of the tetracycline
backbone (Pankey, 2005). Chemically, tigecycline is [(4S,4aS,5aR,12aS)-
9-(2-tert-butylaminoacetylamino)-4,7-bis-dimethylamino-3,10,12,12a-
tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydronaphthacene-2-
carboxamide]. Its chemical formula is C29H39N5O8, and its molecular
weight is 585.65 Da (Hoffmann et al., 2007; Doan et al., 2006).
Tigecycline exhibits robust activity against bacterial isolates resistant
to other antibiotic classes, including beta-lactams and fluoroquinolones,
while resisting deactivation by most of the known tetracycline resistance
mechanisms found in clinically significant bacteria (Bauer et al., 2004).
This antibiotic has been shown to be as effective and safe as standard an-
timicrobial therapy for the treatment of adults with complicated intra-
abdominal infections, complicated skin and skin structure infections,
and community acquired bacterial pneumonia (Stein and Babinchak,
2013). It has also been evaluated as monotherapy for other serious
infections in human clinical trials as a result of its microbiological, phar-
macodynamic and pharmacokinetic properties (Chopra, 2001; Zhanel
et al., 2004).
There currently exists a disturbing global trend that suggests the
coming of a new post-antibiotic era, in which there are few antimicro-
bials available to treat new and emerging pathogens, fuelled by the
use, overuse, and misuse of antibiotic therapy (Peterson, 2008). There
is a need to develop new agents that overcome existing mechanisms
of resistance displayed by multidrug-resistant bacteria (Bhattacharya
et al., 2009). In this context, the development of new antimicrobials
with activity against resistant pathogens and the study of analytical
methodology to assure its quality, represent important clinical practice
advance.
There are few methods described to analyze tigecycline and its me-
tabolites, using techniques such as spectrophotometry (Silva et al.,
2012) and HPLC-UV and HPLC/MS/MS (Li et al., 2004; Bradford et al.,
2005; Muralidharan et al., 2005; Conte Jr et al., 2005; Ji et al., 2007,
2008; Hoffmann et al., 2007; Silva and Salgado, 2012; D'Avolio et al.,
2013; Ozcimen et al., 2014; Xie et al., 2014). It is already known that liq-
uid chromatography methods are more accurate, precise and specific
than microbiological assays (Ródenas et al., 1995), however, the low
cost and simple procedures of the bioassays have allowed them to be-
come an alternative methodology for the drug potency assessment in
pharmaceutical formulations, in addition, this assay can reveal subtle
changes not demonstrable by conventional chemical methods (The
United States Pharmacopoeia, 2011). Physicochemical methods used
to quantify antimicrobial agents, although accurate, are not able to
Journal of Microbiological Methods 110 (2015) 49–53
⁎ Corresponding author at: Universidade Federal de Alfenas — UNIFAL, Rua Gabriel
Monteiro da Silva, n.700, CEP 37130-000, Alfenas, MG, Brazil.
E-mail address: lucmsil@yahoo.com.br (L.M. da Silva).
http://dx.doi.org/10.1016/j.mimet.2015.01.017
0167-7012/© 2015 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Journal of Microbiological Methods
journal homepage: www.elsevier.com/locate/jmicmeth

2. indicate the true biological activity of the drug. For this reason, microbi-
ological methods are used to determine the potency of antimicrobial
agents and they play an essential role in the manufacturing processes
and quality control of these drugs (Moreno and Salgado, 2007; Vieira
et al., 2012). Thus, it was considered important to develop an alternative
method for tigecycline determination for application in routine quality
control of these pharmaceutical dosage forms.
Additionally, the possibility to use alternative analytical methods
fully validated for antibiotics, such as turbidimetric microbiological
assay, which is a simple and operationally inexpensive one, represents
a great advantage for quality control laboratories that do not have spe-
cialized and sophisticated instruments (Souza et al., 2006; Schmidt
et al., 2009). Standard plate diffusion assays for antibiotics, although ad-
equate, often do not provide the rapid and accurate assay information
for the large number of test samples generated by development and
control needs. Excellent precision can be achieved with a manual turbi-
dimetric microbiological assay provided special care is taken in all of the
operational details of the assay (Pitkin et al., 1974; Vieira et al., 2014).
Studies with tigecycline for microbiological testing showed that this
antimicrobial agent should be incorporated in the culture medium on
the day of use, or when conducted in liquid media, it must be freshly
prepared and degassed in order to reduce the amount of oxygen in
the medium. An alternative to fresh medium prepared is the addition
of oxirase enzyme to the culture medium, which also decreases the
concentration of oxygen present (Hope et al., 2005). It was observed
that the activity of tigecycline against diverse population of bacteria
was higher when compared to fresh media with different culture
media preparation times (Petersen and Bradford, 2005).
Tigecycline is commercially available, but at the moment, there is no
microbiological method for the analysis of this drug described in the lit-
erature. Considering that the turbidimetric assay has the advantage of
reduced analysis time when compared to the agar diffusion method,
where the analysis time is 24 h, the aim of this work was to propose a
rapid turbidimetric method for the analysis of tigecycline in lyophilized
powder, contributing to improve the quality control and assuring the
therapeutic efficacy.
2. Experimental
2.1. Chemicals
The tigecycline reference substance was purchased from Sequoia Re-
search Products (Oxford, UK). The batches of Tygacil® (Wyeth, USA) ly-
ophilized powder, containing 50 mg of tigecycline were obtained from
commercial sources within their shelf-life period. The samples contain
lactose as the unique excipient. The culture media tryptic soy broth
(TSB) and tryptic soy agar (Acumedia Manufacturers, Mi, EUA) were
used for the method. Analytical grade formaldehyde (Qhemis, SP,
Brazil) was used to interrupt the growth of microorganisms.
2.2. Apparatus
Incubation of microorganisms was performed using a Shaker incu-
bator MA420 model (Marconi, SP, Brazil). A photometer Q-798DRM
(Quimis, SP, Brazil) was used to determine the culture absorbance.
The software Microsoft Excel (2007) was used to construct the
calibration curves. The RP-LC method was performed on a Waters
LC system (Waters Corporation, Milford, Massachusetts, USA)
equipped with a Waters® 1525 binary pump, a Rheodyne Breeze
7725i manual injector and a Waters® 2487 UV detector. The peak
areas were integrated automatically by computer using an Empower 2
software program. The chromatographic separation was carried out
on a reversed-phase Phenomenex (Torrance, USA) Luna C18 column
(250 mm × 4.6 mm I.D.).
2.3. Preparation of reference substance solution
The stock solution was prepared by weighing accurately, 12.50 mg of
tigecycline reference substance, transferred to 100 mL volumetric flask
and diluted to volume with ultrapure water, obtaining a concentration
of 125 μg/mL of tigecycline. This solution was diluted with ultrapure
water to a concentration of 25 μg/mL. Aliquots of 3.0, 3.6 and 4.32 mL
of this solution were transferred to 25 mL volumetric flasks, the vol-
umes of which were completed with ultrapure water in order to obtain
working solutions with concentrations of 3.0, 3.6 and 4.32 μg/mL,
respectively.
2.4. Preparation of sample solutions
To prepare the sample solution, vials containing 50 mg of tigecycline
were accurately weighed and mixed. An appropriated amount was
transferred into an individual 50 mL volumetric flask and diluted to vol-
ume with ultrapure water to concentration of 125 μg/mL of the active
pharmaceutical ingredient. After, the same reference substance dilu-
tions were carried out.
2.5. Preparation of culture medium
The medium was prepared as indicated in their respective label being
dissolved in water under heating, distributed in test tubes (10 mL/tube)
and autoclaved (conditions: 121 °C, 1 atm) for 15 min. After autoclaving,
the tubes were cooled and immediately used in the bioassay.
2.6. Turbidimetric assay
For the preparation and standardization of inocula, the strain
Staphylococcus aureus was inoculated, with a platinum loop, into tryptic
soy broth and incubated at 35 °C ± 2 °C, for 23 h before the assay, for the
growth of S. aureus. The bacteria, previously incubated in tryptic soy
broth, were diluted with pure tryptic soy broth to achieve a suspension
turbidity of 25% ± 2% (transmittance), using a photometer with a wave-
length of 580 nm and a 10 mm absorption cell, against tryptic soy broth
as blank. The bioassay was performed using the 3 × 3 parallel line assay
design (three doses of the standard and three doses of the sample).
800 μL of the standardized S. aureus suspension was added to twenty
test tubes containing 10 mL of tryptic soy broth. In nine of these tubes,
200 μL of standard working solutions was added (at the concentrations
of 3.0 (S1), 3.6 (S2) and 4.32 (S3) μg/mL, respectively), and each con-
centration was performed in triplicate. In the other nine tubes, the
same was carried out with the working sample solutions (T1, T2 and
T3). After that, the test tubes were incubated with shaking at a temper-
ature of 35.0° ± 2.0 °C for 3 h. After the incubation period, the microbial
growth was interrupted by the addition of 0.5 mL of 12% formaldehyde
solution to each tube. Then, the photometer was reset by the test tube
containing a negative control (10 mL of tryptic soy broth containing
0.5 mL of the formaldehyde solution) and the absorbance values were
determined for each tube at a wavelength of 530 nm. As a positive con-
trol of the test, one tube containing 10 mL of tryptic soy broth, 800 μL of
the standardized microorganism suspension and, after incubation,
0.5 mL of the formaldehyde solution was performed.
2.7. Calculation of activity and method validation
To calculate the activity of tigecycline, the Hewitt equation was used
(Hewitt, 2003). The assays were calculated statistically by the linear
parallel model and regression analysis and verified using analysis of
variance (ANOVA). The method was validated using samples of
pharmaceutical formulations with the label claim of 50 mg by determi-
nations of the following parameters: specificity, linearity, precision,
accuracy and robustness following the International Conference on
Harmonisation (ICH) guidelines (ICH, 2005).
50 L.M. da Silva, H.R.N. Salgado / Journal of Microbiological Methods 110 (2015) 49–53

3. 2.7.1. Linearity
The analytical curve was constructed by plotting the logarithm of the
concentration versus the average of the absorbance values, with the av-
erage absorbance value of each concentration of the tigecycline refer-
ence substance. Three curves were obtained on three different days.
The data obtained from the analytical curve were analyzed by the
least squares and the verification of linearity and parallelism was done
by the analysis of variance (ANOVA).
2.7.2. Precision
The precision of the method was determined by repeatability and in-
termediate precision and was expressed as the relative standard deviation
(RSD). The repeatability was examined by assaying six times the interme-
diary concentration of reference substance solution (3.6 μg/mL) on the
same day (intraday) and under the same experimental conditions. The in-
termediate precision of the method was evaluated through the perfor-
mance of the assay on four days (interday) in the same laboratory.
2.7.3. Accuracy
To determine the accuracy of the proposed method, the test was per-
formed assaying simulated samples over three potency levels, 80 (R1),
100 (R2) and 120% (R3). Aliquots of 4, 5 and 6 mL of the reference
substance solution (125 μg/mL) were accurately transferred into three
25 mL volumetric flasks, respectively, and the volumes of which flask
were completed with placebo solution (lactose solution at 0.5 mg/mL).
After that, aliquots of 3.0, 3.6 and 4.32 mL of each one of these three solu-
tions, were transferred to 25 mL volumetric flasks and the volumes of
which were completed with ultrapure water in order to obtain working
solutions of simulated samples with following concentrations:
R1: 2.4, 2.88 and 3.46 μg/mL, representing a sample of 80% potency.
R2: 3.0, 3.6 and 4.32 μg/mL, representing a sample of 100% potency.
R3: 3.6, 4.32 and 5.18 μg/mL, representing a sample of 120% potency.
Each simulated samples (R1, R2 and R3) were assayed in indepen-
dent experiments.
The recovery percentage of the simulated samples was calculated by
the following equation: R% = (Recovered Potency / Theoretical
Potency) × 100.
2.7.4. Robustness
The robustness of the method was determined by assaying the same
sample under a variety of conditions. The factors considered were incu-
bation time (2 h 45 min; 3 h; 3 h 15 min), wavelength (525, 530 and
535 nm) and inoculum added volume (780, 800, 820 μL).
2.8. HPLC method
The HPLC method, chosen as a comparative method in the determi-
nation of tigecycline in lyophilized powder, was previously developed
and validated by our study group (Silva and Salgado, 2012). The LC
method was carried out on a Luna C18 column (250 mm × 4.6 mm I.D.),
maintained at room temperature. The mobile phase consisted of buffer
containing sodium phosphate monobasic (0.015 M) and oxalic acid
(0.015 M) (pH 7.0): acetonitrile (75:25, v/v), run at a flow rate of
1.0 mL/min and using ultraviolet (UV) detection at 280 nm.
2.9. Comparison of methods
The results of the determinations obtained by the microbiological
assay were statistically compared with those obtained with the HPLC
method, using the Student's t-test, to evaluate the difference between
the two methods at a level of significance of 5%.
3. Results and discussion
Taking into account that the potency of an antibiotic may be evaluat-
ed through the comparison of the inhibition of growth of a susceptible
microorganism induced by known concentrations of the antibiotic and
its respective reference standard (European Pharmacopoeia, 2008; The
United States Pharmacopoeia, 2011), a 3 × 3 microbiological assay
was proposed for determining the tigecycline concentration in lyophi-
lized powder. Biological methods are advantageous because the param-
eters that are measured with these techniques and the properties for
the drug used are the same. Thus, impurities and the related substances
do not interfere, maintaining the precision of the analytical method
(Hodjes, 2001). Therefore, microbial or biological assays remain, in gen-
eral as the standard for resolving doubt with respect to possible loss of
activity (The United States Pharmacopoeia, 2011).
3.1. Validation of the analytical method
3.1.1. Linearity
For the linearity, the experimental mean absorbance values and RSD
values (in parentheses) for standard solutions were 0.810 (0.78%), 0.692
(1.25%) and 0.573 (1.90%) for doses of 3.00; 3.60 and 4.32 μg/mL, respec-
tively (Table 1), showing low variability in the intradose response. The
calibration curve of tigecycline was constructed by plotting the logarithm
of concentrations (μg/mL) versus mean absorbance values; good linearity
was found in the range of 3.0–4.32 μg/mL. The representative linear equa-
tion was y = −0.6524 ln(×) + 1.5274. The high value of the determina-
tion coefficient obtained (r2 = 0.9999) was considered highly significant
for the method. For this research, a parallel-line model has been chosen, in
which two curves are constructed, one of them for tigecycline RS and the
other for the sample of lyophilized powder, and these two curves must be
parallel and linear over the working range chosen. These parameters
must be verified by validity tests, considering a given probability, which
is usually p = 0.05 (The United States Pharmacopoeia, 2011). The tests
performed in this study were validated through the analysis of variance
(ANOVA), as described in official guidelines. Through this analysis, it
was found that there was no deviation in the linearity and parallelism
of the curves.
3.1.2. Precision
The method precision in terms of repeatability (intra-assay) was
evaluated by analyzing, on the same day, six times the intermediate
concentration of reference substance solution (3.6 μg/mL) and the
Table 1
Absorbances of tigecycline standard solutions obtained for the standard curve.
Absorbancesa
S1
(3.0 μg/mL)
S2
(3.6 μg/mL)
S3
(4.32 μg/mL)
T1
(3.0 μg/mL)
T2
(3.6 μg/mL)
T3
(4.32 μg/mL)
0.809 0.687 0.560 0.715 0.595 0.491
0.805 0.702 0.579 0.699 0.598 0.494
0.817 0.687 0.579 0.746 0.609 0.497
Mean 0.810 0.692 0.573 0.720 0.600 0.494
RSD (%) 0.78 1.25 1.90 3.35 1.22 0.64
a
Mean of three tubes.
51L.M. da Silva, H.R.N. Salgado / Journal of Microbiological Methods 110 (2015) 49–53

4. intermediate precision was determined by analyzing the same sample
on four days (between-day) with obtained RSD values of 1.80% and
0.37%, respectively. The lower RSD values achieved confirm that the
proposed method has the capacity to generate, for the same sample, re-
producible results with low response variation between independent
assays.
3.1.3. Accuracy
The accuracy of the method was evaluated at 80, 100 and 120% of the
nominal analytical concentration in the specified range of 3.0–4.32 μg/mL.
The mean accuracy was 99.74 and RSD was 0.95% (Table 2), which con-
firms the ability of the method to determine with accuracy the tigecycline
concentration within the range of 80–120% and, in the same way, shows
that the results obtained from the bioassay were close to the true concen-
tration values of the samples.
3.1.4. Robustness
The robustness was evaluated by small modifications, individually,
in the following method parameters: incubation time, wavelength and
inoculum added volume. The results are presented in Table 3. The RSD
values obtained are lower than 5%, showing the robustness of the turbi-
dimetric assay for the analysis of tigecycline in lyophilized powder.
3.2. Comparison of methods
In order to establish a comparison between the proposed microbiolog-
ical method and physicochemical method by HPLC, Student's t-test of the
average contents of tigecycline in lyophilized powder obtained by both
methods, was performed, considering a significance level of 5%. The
percentage contents of tigecycline calculated by both methods were
118.87% and 123.04% to HPLC and 115.67% and 127.43% to microbiologi-
cal method. Statistical analysis of these values showed no significant dif-
ference between the methods, with t calculated = 0.095 b t critical =
4.303.
The results obtained in this study were very satisfactory, and the per-
formed validation proved that microbiological assay is a good alternative
methodology for pharmaceutical analysis of tigecycline in lyophilized
powder. It is a useful analytical tool as a supplement or substitution for
the physicochemical method.
There are no official specifications for the content of tigecycline in
pharmaceutical product. Taking into account that the range normally
recommended by the pharmacopeias for determining the drug content
in pharmaceutical dosage forms is from 90.0 to 110.0% (USP 33, 2010),
the tigecycline samples would be out of specification. However, for
this, additional studies should be conducted in order to establish an of-
ficial reference pattern to content and quality assured for this drug.
4. Conclusions
The results indicated that the turbidimetric assay demonstrated
good linearity, precision and accuracy at concentration ranging from
3.0 to 4.32 μg/mL, and, therefore, being an acceptable alternative meth-
od for the routine quality control of tigecycline in pharmaceutical forms.
The method uses simple reagents, with minimum sample preparation
procedures, and no toxic residues, encouraging its application in routine
analysis. This method also has the advantage of being faster than the
agar diffusion, a widely used microbiological method.
Acknowledgments
The authors wish to thank the FAPESP (Fundação de Amparo à
Pesquisa do Estado de São Paulo, project 2009/53434-2).
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Table 3
Robustness of the microbiological assay for tigecycline in lyophilized powder.
Condition Studied range Found potency (%) RSD (%)
Incubation time 2 h 45 min 111.53 0.60
3 h 110.30
3 h 15 min 111.35
Wavelength (nm) 525 110.93 0.31
530 110.30
535 110.37
Inoculum added volume (μL) 780 111.92 1.55
800 109.35
820 112.63
Table 2
Accuracy of the microbiological assay for tigecycline in lyophilized powder.
Theoretical potency
(%)
Found potency
(%)
Accuracy
(%)
Mean accuracy
(%)
RSD
(%)
R1 80 78.92 98.65 99.74 0.95
R2 100 100.18 100.18
R3 120 120.45 100.38
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