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Pharmaceutics 2015, 7, 106-121; doi:10.3390/pharmaceutics7030106
pharmaceutics
ISSN 1999-4923
www.mdpi.com/journal/pharmaceutics
Article
Rapid Turbidimetric Assay to Determine the Potency of
Daptomycin in Lyophilized Powder
Eliane Gandolpho Tótoli * and Hérida Regina Nunes Salgado
School of Pharmaceutical Sciences, Universidade Estadual Paulista, Rod. Araraquara-Jaú, km 1,
CEP 14801-902 Araraquara, SP, Brazil; E-Mail: salgadoh@fcfar.unesp.br
* Author to whom correspondence should be addressed; E-Mail: eliane.totoli@gmail.com;
Tel.: +55-16-3301-6967; Fax: +55-16-3301-6900.
Academic Editor: Afzal Mohammed
Received: 18 May 2015 / Accepted: 2 July 2015 / Published: 9 July 2015
Abstract: Daptomycin is an important antimicrobial for clinical practice, mainly because it
remains very active against Gram-positive resistant strains, such as methicillin-resistant
Staphylococcus aureus and vancomycin-resistant enterococci. Development of microbiological
methods for the analysis of antimicrobials is highly recommended, since they can provide
important information about their biological activities, which physicochemical methods are
not able to provide. Considering that there are no studies in the literature describing
microbiological methods for the analysis of daptomycin, the aim of this work was to
validate a microbiological method for the quantitation of daptomycin by the turbidimetric
assay. Staphylococcus aureus was used as the test microorganism, and the brain heart
infusion broth was used as the culture medium. The validation of the method was
performed according to the ICH guidelines, and it was shown to be linear, precise, robust,
accurate and selective, over a concentration range of 8.0 to 18.0 µg mL−1. Student’s t-test
showed the interchangeability of the proposed method with a previously-validated HPLC
method. The developed turbidimetric method described in this paper is a convenient
alternative for the routine quality control of daptomycin in its pharmaceutical dosage form.
Keywords: analytical method; daptomycin; microbiological method; new antimicrobial
agent; quality control; turbidimetric assay
OPEN ACCESS
Pharmaceutics 2015, 7 107
1. Introduction
The continued emergence and global spread of multi-drug-resistant pathogenic bacteria, especially
in hospital settings, require increasingly efficient treatment regimens and the development of new
antimicrobial compounds able to overcome these mechanisms of resistance [1]. There are few new
antimicrobial agents emerging in the market, and this fact can be due to numerous reasons, such as the
high cost to introduce a new molecule in a highly competitive market and the inherent difficulty of
identifying new targets for antibiotics. Thus, the emergence of microorganisms that no longer respond
to the first-line antimicrobials is increasingly common [2,3].
As a strategy for combating resistant microorganisms, the development of drugs with novel
mechanisms of action is required. In this context, a new class of antimicrobials stands out, the cyclic
lipopeptides. Daptomycin was the first approved member of this class. This cyclic lipopeptide has a
mechanism of action distinct from all other available antimicrobials in clinical practice [4].
The mechanism of action is completely dependent on the physiological levels of calcium in the body.
For this reason, it is necessary to add calcium in the culture medium for performing in vitro
microbiological tests, in order to obtain the satisfactory antimicrobial activity of this drug [5,6].
Daptomycin (Figure 1) is globally polar and constitutes 13 amino acid residues and an n-decanoyl
fatty acid chain at the N-terminus [7]. Calcium ions act in two different steps of the mechanism of
action of this antimicrobial. In the first step, they gather together the charged amino acids of the
molecule on one side and expose its lipophilic tail on the other side, which increases its amphiphilicity.
In the second step, the calcium ions favor the oligomerization of daptomycin in micellar structures.
These structures, in the presence of negatively-charged membranes, undergo a structural transition that
enables the interaction of lipophilic tails of the molecule with the bacterial cell membrane, resulting in
its insertion [8]. After that, the molecule of daptomycin undergoes another conformational change that
causes a membrane curvature, which allows the leakage of intracellular ions (mainly potassium ions),
resulting in depolarization, loss of membrane potential and the consequent inhibition of the synthesis
of protein, RNA and DNA [7–9].
Figure 1. Chemical structure of daptomycin.
Pharmaceutics 2015, 7 108
Daptomycin is an antimicrobial selectively active against aerobic, anaerobic and facultative
Gram-positive bacteria [10]. It has activity against β-hemolytic streptococci, methicillin-resistant
Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) [11]. This antimicrobial
also shows activity against vancomycin-resistant Staphylococcus aureus (VRSA) [12]. It is recommended
for the treatment of complicated skin and soft tissues infections, as well as bloodstream infections
caused by Staphylococcus aureus, including those associated with right-sided infective endocarditis [13].
Considering that the treatment of MRSA and VRE is a challenge for medicine, daptomycin is
promising in combating these microorganisms, since resistance to this antibiotic is not frequently
reported in the literature. The successful use of daptomycin for the treatment of resistant Gram-positive
bacteria still predominates [14–16]. For this reason, daptomycin is a very important antimicrobial
agent for clinical practice nowadays.
The minimum inhibitory concentration (MIC) of daptomycin against susceptible strains of
Staphylococcus sp. and Streptococcus sp. (A, B, C and G groups) is ≤1 µg mL−1 [17] and for
Enterococcus sp. is ≤4 µg mL−1 [18].
The clinical use of daptomycin was approved by the Food and Drug Administration (FDA) in
September 2003, in the United States [2,19]. In 2005, daptomycin was also approved by the European
Medicines Agency (EMA) in Europe [17]. In 2008, it was introduced into the Brazilian market [19].
Nowadays, it is sold in more than 40 countries worldwide by the pharmaceutical companies Cubist
(patent holder), Novartis, Oryx, Sepracor, AstraZeneca, Cardinal Health, TTY Biopharm, Chiron and
Hospira [20,21]. It is estimated that more than 2.7 million people have been treated with this
antimicrobial agent (between 2003 and 2014) [13]. This fact also highlights the importance of this
antimicrobial agent for clinical practice.
In the literature, there are few published studies that show the development of analytical methods
for the analysis of daptomycin, and most of them aim at quantifying the drug in biological fluids by
high-performance liquid chromatography (HPLC) [22–29] and ultra-performance liquid chromatography
(UHPLC) [30–34]. In the same way, there are no monographs for this drug in any official
compendium. However, only one study was found in the literature in which an analytical method for
the analysis of daptomycin in the pharmaceutical dosage form using HPLC was described [35].
On the other hand, it is known that physical-chemical methods lack the ability to indicate the true
biological activity of antimicrobial agents. This fact shows the importance of microbiological methods
in the quality control of this type of drug [36,37]. A search through the literature for microbiological
methods for the analysis of the potency of daptomycin gave no results.
Considering the importance of daptomycin for the global scene and the lack of literature concerning
microbiological methods for the analysis of this antimicrobial, the aim of this work was to propose a
rapid turbidimetric method for the analysis of the potency of daptomycin in the dosage form of powder
for injectable solution. A high-performance liquid chromatography (HPLC) method, previously developed
and validated by us, was chosen as a comparative method.
Pharmaceutics 2015, 7 109
2. Experimental Section
2.1. Chemicals
Daptomycin reference standard (DPT RS) (purity of 98.00%) was purchased from Sequoia
Research Products Company (Pangbourne, UK). The samples of daptomycin in lyophilized powder for
the injectable solution (Cubicin™, Novartis, McPherson, KS, USA) containing 500 mg of the active
component were also purchased. The pharmaceutical form contains sodium hydroxide as the excipient.
Calcium chloride dihydrate (Synth, Diadema, Brazil) and sodium hydroxide (Vetec, Duque de caxias,
Brazil) werealso used.
The culture media used for the tests were a brain heart infusion (BHI) broth (Merck, Germany) and
tryptic soy agar (Difco™, Hunt Valley, MD, USA). In order to interrupt the microorganism’s growth,
analytical grade formaldehyde (Qhemis™, São Paulo, Brazil) was used. The test microorganism was
the Staphylococcus aureus ATCC 6538 IAL 2082.
All solutions and mobile phases used for carrying out both microbiological and HPLC methods were
prepared using water obtained by a Milli-Q™ Plus apparatus (Millipore™, Darmstadt, Germany). The
HPLC mobile phase was prepared using ethanol HPLC grade (JT Baker™, Ecatepec de Morelos, Mexico).
2.2. Apparatus
For the microbiological method, the microorganisms were incubated in an ECB Digital 1.2
(Odontobrás, Brodowski, Brazil) oven and in a Shaker incubator, Model MA420 (Marconi, Piracicaba,
Brazil). Culture media were sterilized in a vertical autoclave, Model AV (Phoenix Luferco,
Araraquara, Brazil), before use. The absorbance values were determined by a spectrophotometer, DU
530 (Beckman Coulter, Pasadena, CA, USA). The analytical curves were constructed using Microsoft
Excel (2007) software.
The comparative chromatographic method was carried out using a chromatograph, Model 1525
Waters (Waters Chromatography systems, Milford, MA, USA), which was connected to a Waters 2487
UV/Visible detector and a manual injector Rheodyne Breeze 7725i with a 20-µL loop (Rheodyne
Breeze, Cotati, CA, USA).
The following items were also used: H10 analytical scale (Mettler Toledo™, Greifensee,
Switzerland); B160 semi-analytical scale (Micronal™, São Paulo, Brazil) and USC2800A ultrasound
bath (Unique™, Indaiatuba, Brazil).
2.3. Solutions
2.3.1. Preparation of DPT RS Solutions
DPT RS stock solution was prepared by transferring 5.0 mg equivalent of DPT RS to a 50-mL
volumetric flask, which was filled with ultrapure water to obtain a concentration of 100 μg mL−1.
Aliquots of 0.4, 0.6 and 0.9 mL of the DPT RS stock solution were transferred to 5-mL volumetric
flasks, the volumes of which were completed with ultrapure water, for obtaining working solutions
with concentrations of 8.0, 12.0 and 18.0 µg mL−1, respectively named S1, S2 and S3.
Pharmaceutics 2015, 7 110
2.3.2. Preparation of DPT Sample Solution
The DPT sample stock solution at a theoretical concentration of 100 µg mL−1 was prepared. Three
vials of DPT powder (commercial available for the preparation of solution for injections) were
weighed, and the average weight was calculated. The contents of these vials were mixed. DPT stock
solution was prepared by transferring 5.0 mg equivalent of DPT of this mixture to a 50-mL volumetric
flask, which was filled with ultrapure water to obtain a concentration of 100 μg mL−1 DPT. Aliquots of
0.4, 0.6 and 0.9 mL of this solution were transferred to 5-mL volumetric flasks, the volumes of which
were completed with ultrapure water, for obtaining working solutions with concentrations of 8.0, 12.0
and 18.0 µg mL−1, respectively named T1, T2 and T3.
2.3.3. Preparation of Calcium Solution
A solution containing 10.0 mg mL−1 of calcium was prepared from calcium chloride dihydrate
(CaCl2·2H2O). For this, an amount of 3.66 g of CaCl2·2H2O was weighed (the equivalent of 1.0 g of
calcium), and then, it was transferred to a 100-mL volumetric flask. The volume was completed with
ultrapure water.
2.3.4. Preparation of Sodium Hydroxide Solution
A solution containing 1.0 mg mL−1 of sodium hydroxide was prepared. For this, NaOH (10 mg) was
transferred to a 10-mL volumetric flask. The volume was completed with ultrapure water.
2.4. Turbidimetric Assay
2.4.1. Preparation and Standardization of Inoculum
The strain Staphylococcus aureus ATCC 6538 IAL 2082 was inoculated in 30 mL of BHI broth and
maintained for growth in a microbiological incubator at a temperature of 35 °C ± 2 °C for 24 h before
performing the experiment. Thereafter, the inoculum was standardized at 580 nm in a spectrophotometer,
in order to obtain a transmittance of 25% ± 2% [38,39]. It is worth remembering that before the whole
procedure, the Staphylococcus aureus strain was cultivated and kept in tryptic soy agar medium in
the freezer.
2.4.2. The Bioassay
First of all, the Staphylococcus aureus inoculum was prepared and standardized as described before.
Thereafter, in three identical test tubes containing 10 mL of sterile BHI broth, 200 µL of the DPT RS
working solutions (S1, S2, S3) and 75 µL of the calcium solution were added. Subsequently, 0.5 mL of
the standardized inoculum were also added. The same procedure was carried out for the DPT sample
working solutions (T1, T2 and T3). The procedure was performed in triplicate for each DPT
concentration. Therefore, twenty test tubes were used, with nine tubes for DPT RS, nine for the DPT
samples, one for the positive control (containing BHI broth and inoculum without the addition of DPT)
and one for the negative control (containing only the BHI broth).
Pharmaceutics 2015, 7 111
After the test tubes’ preparation, they were incubated in a shaker, in a water bath, at a temperature
of 35.0 °C ± 2.0 °C for 4 h. At the end of the incubation period, the microorganisms’ growth was
interrupted by the addition of 0.5 mL of 12% formaldehyde solution in each test tube. The same
volume of formaldehyde solution was also added to the negative control tube. Thereafter, the
spectrophotometer was reset by the test tube containing the negative control, and the absorbance
readings were taken at a wavelength of 530 nm. In each test, the results were statistically analyzed, and
the DPT potency was calculated.
2.4.3. Obtaining the Analytical Curve
The analytical curve was constructed using the 3 × 3 parallel line assay design, as recommended by
the Brazilian Pharmacopoeia (2010) [38]. For this purpose, the logarithm of the DPT RS working
concentrations (8.0, 12.0 and 18.0 µg mL−1) versus their corresponding average absorbance values
were plotted on a graph, with the aid of the Microsoft Excel (2007) software. Three analytical curves
were plotted on three different days, and a final analytical curve was obtained with the average
of them.
2.4.4. Potency Calculation
The Hewitt equation was used to calculate the DPT potency [40].
2.5. Method Validation
The validation of the microbiological method was carried out according to the literature
recommendation [38,39,41]. For this purpose, some parameters were determined, such as linearity,
precision, accuracy, selectivity and robustness. The limits of detection and quantification are not
required for this category of assay.
2.5.1. Linearity
In order to assess the linearity of the method, three analytical curves performed on three different
days were analyzed. Each curve was constructed as described before in the Section 2.4.3. The results
were analyzed to obtain the equation of the line by the least squares method, and the linearity and
parallelism were assessed by analysis of variance (ANOVA).
2.5.2. Precision
Repeatability and intermediate precisions were assessed. For the repeatability, six test tubes
containing DPT RS in a concentration of 12.0 μg mL−1 were prepared as described in Section 2.4.2.
They were analyzed on the same day and at identical working conditions. The relative standard
deviation (RSD) between the absorbance values obtained from these six test tubes was calculated
and analyzed [41].
Regarding the intermediate precision, it was assessed based on two criteria: inter-assay and between
analysts. For the intra-assay precision evaluation, three bioassays were performed on three different
days, and the DPT potency was calculated for each day. After that, the relative standard deviation
Pharmaceutics 2015, 7 112
(RSD) between the obtained DPT potency values was calculated and analyzed. In order to assess the
between analysts’ precision, two bioassays were performed by two different analysts, and the DPT
potency was calculated for each one. Similarly, the relative standard deviation (RSD) between the DPT
potency values was calculated and analyzed [41].
2.5.3. Accuracy
The recovery assay was carried out in order to evaluate the accuracy of the microbiological method.
For this, known quantities of DPT RS have been added to a synthetic mixture of the drug product
excipient [41]. Three different concentrations were assessed, R1, R2 and R3. For this purpose, a stock
solution of DPR RS with a concentration of 200 µg mL−1 was prepared. An aliquot of 5 mL from this
solution was transferred to a 10-mL volumetric flask, and the volume was made up with ultrapure
water, to obtain a 100-µg mL−1 solution. From this solution, aliquots of 0.8, 1.2 and 1.8 mL were
transferred to 10-mL volumetric flasks, and the volumes were completed with ultrapure water, in order
to obtain solutions with concentrations of 8.0, 12.0 and 18.0 µg mL−1, representing P1, P2 and
P3, respectively.
For the preparation of the recovery solutions, an aliquot of 5 mL from the DPT RS stock solution of
200 µg mL−1 was transferred to a 10-mL volumetric flask. After that, to the same volumetric flask,
86 µL of the NaOH solution (1.0 mg mL−1) were added, in order to simulate the concentration of
NaOH that would be in a solution of 100 µg mL−1 of daptomycin in lyophilized powder for injectable
solution. The volume of the volumetric flask was completed with ultrapure water, resulting in a
solution with 100 µg mL−1 of DPT RS and 8.6 µg mL−1 of excipient (NaOH) (simulated DPT sample
solution). From this simulated DPT sample solution, aliquots were transferred to 10-mL volumetric
flasks, in order to obtain the recovery solutions, as is described below.
Recovery Test 1: Aliquots of 0.64, 0.96 and 1.44 mL from the simulated DPT sample solution were
transferred to 10-mL volumetric flasks, and the volumes were completed with ultrapure water, in order
to obtain solutions with the concentrations of 6.4 (R1), 9.6 (R2) and 14.4 (R3) µg mL−1, representing a
sample of 80% potency.
Recovery Test 2: Aliquots of 0.8, 1.2 and 1.8 mL from the simulated DPT sample solution were
transferred to 10-mL volumetric flasks, and the volumes were completed with ultrapure water, in order
to obtain solutions with the concentrations of 8.0 (R1), 12.0 (R2) and 18.0 (R3) µg mL−1, representing
a sample of 100% potency.
Recovery Test 3: Aliquots of 0.96, 1.44 and 2.16 mL from the simulated DPT sample solution were
transferred to 10-mL volumetric flasks, and the volumes were completed with ultrapure water, in order
to obtain solutions with concentrations of 9.6 (R1), 14.4 (R2) and 21.6 (R3) µg mL−1, representing a
sample of 120% potency.
Each simulated sample (R1, R2 and R3) was assayed in an independent trial. The percentage of
recovery (R%) was calculated by Equation 1:
R% = PF
TP×100 (1)
where PF = the potency found in the recovery samples; TP = theoretical potency.
Pharmaceutics 2015, 7 113
2.5.4. Selectivity
The selectivity of the method was performed with the aim of showing that the placebo has no
antibacterial activity against the test microorganism S. aureus under the working conditions. In this
way, a placebo solution was prepared consisting of NaOH in the same concentration that it would be in
a solution of 18.0 µg mL−1 of DPT in lyophilized powder (the higher working concentration). In this
case, the concentration is 1.56 µg/mL of NaOH.
The placebo solution (200 μL), standardized inoculum (0.5 mL) and the calcium solution
(75 μL) were all added to a test tube containing sterile BHI broth (10 mL). To the same test tube,
0.5 mL of the standardized inoculum and 75 µL of the calcium solution were also added. Then, the
turbidimetric assay was carried out as defined under Section 2.4. At the end of the test, the absorbance
values provided by the test tube containing the placebo solution were compared to the absorbance
values provided by test tubes containing just the culture medium with the inoculum in order to check if
the sodium hydroxide caused some inhibition of the bacterial growth. This assay was performed
in triplicate.
2.5.5. Robustness
The robustness of the method was evaluated by small modifications, individually, in the following
method parameters: concentration of the inoculum, calcium concentration in the culture medium, the
wavelength used to determine the results in the spectrophotometer and the period of incubation in the
shaker. In this way, bioassays were performed for each modified condition, and the DPT potency was
calculated. After that, RSD values were calculated between the responses obtained from the modified
and normal conditions.
2.6. HPLC Method
An HPLC method previously developed and validated by our research group was used as the
comparative method for DPT determination. The chromatographic conditions were: mobile phase
consisting of ethanol and water (55:45, v/v) with pH adjusted to 4.5 with glacial acetic acid; Agilent
Zorbax™ C18 analytical column (150 × 4.6 mm, 5 µm) (Agilent™, Santa Clara, CA, USA); isocratic
elution mode; volume of injection of 20 µL; flow rate of 0.6 mL min−1; and UV detection at 221 nm.
2.7. Comparison between the Microbiological and Chromatographic Methods
In order to statistically compare the proposed microbiological method with the previously validated
HPLC method, six determinations of daptomycin in the pharmaceutical dosage form were performed
using both methods. After that, the percentage contents of daptomycin were compared by
Student’s t-test, at a significance level of 5%, in order to verify whether these results were
statistically equivalent.
Pharmaceutics 2015, 7 114
3. Results and Discussion
3.1. General Aspects and Method Development
For the development of this work, the method chosen was the turbidimetric assay, mainly due to the
advantages that it offers compared to agar diffusion, which is another common microbiological method
used for the analysis of antimicrobial agents. The turbidimetric assay is faster than agar diffusion,
requiring 4 h to provide the results, while the other demands 24 h of assay [37]. This advantage makes
the turbidimetric assay more convenient for routine quality control analysis. Furthermore, the method
by agar diffusion presents another limitation, which is the fact that some drugs exhibit difficulty in
diffusing through a solid medium. This does not occur in the turbidimetric method, which employs a
liquid culture medium.
During the preliminary tests, some microorganisms considered less pathogenic than S. aureus were
tested before choosing it. The microorganisms tested were: Kocuria rhizophila ATCC 9341 IAL 636,
Bacillus atrophaeus ATCC 9372 IAL 1027 and Staphylococcus epidermidis ATCC 12228 IAL 2150.
However, it was possible to observe that K. rhizophila showed a very slow growth in the tested
culture medium (BHI broth) after 24 h of incubation. Regarding the strains of S. epidermidis and
B. atrophaeus, the first one demanded higher concentrations of daptomycin for its growth inhibition
(> 60 μg mL−1) when compared to S. aureus, and the second was not susceptible to daptomycin, even
in high concentrations (64 μg mL−1). The tested S. aureus strain showed satisfactory growth in the
culture medium, adequate linearity and reproducible results. For these reasons, this microorganism was
chosen for the development of the turbidimetric assay.
Water and phosphate buffer pH 8.0 were tested as diluents. Considering that both showed similar
results, water was chosen as the solvent due to economic reasons.
Among the various inoculum concentrations tested (from 4% to 7%), the concentration of 5%
showed the best results, due to the appropriate amount of growth of S. aureus in relation to the selected
concentrations of the antimicrobial. In the same way, among the daptomycin concentrations tested
(from 1.0 to 64.0 µg mL−1), the best concentrations were 8.0, 12.0 and 18.0 µg mL−1, which presented
better linearity and accuracy of the results.
Different concentrations of calcium in the culture medium were tested (from 0 to 100 µg mL−1), and
it was noted that the concentration of 75 mg mL−1 resulted in the better activity of daptomycin against
S. aureus. The assay performed without the addition of calcium in the culture medium showed no
inhibition of the microorganism by daptomycin, highlighting the importance of the addition of this
substance to the culture medium.
3.2. Validation of the Analytical Method
3.2.1. Linearity
By analyzing the DPT RS analytical curve, the method was shown to be linear in the range between
8.0 and 18.0 μg mL−1, with a correlation coefficient (r) of 0.9995. The obtained equation of the line
was y = −0.679ln (x) + 2.1485.
Pharmaceutics 2015, 7 115
The linearity of the method was also demonstrated by analysis of variance (ANOVA), which
showed that the analytical curve presented no deviation. This conclusion is based on the fact that the
Fcalculated for the “quadratic” parameter (0.96) was lower than Fcritical (4.96). The same occurred with the
parameter “squared difference” in which Fcalculated (0.26) was also lower than the Fcritical (4.96) value.
Usually, for microbiological methods, it is also necessary to perform an analytical curve for the
sample in order to compare it with the analytical curve obtained with the reference standard
(parallel-line model). These two curves should be linear and parallel, within the selected working
range. These parameters must to be verified by validity tests within a given significance level, which is
usually 5% [38,42,43]. This procedure was carried out, and analysis of variance ANOVA showed that
the obtained DPT analytical curves (for sample and reference standard) met these requirements.
3.2.2. Precision
Repeatability (intra-assay) and intermediate (inter-assay and between analysts) precisions were
evaluated. The RSD value calculated for the repeatability was 4.32%. Inter-assay and between-analyst
precisions provided values of 1.48% and 4.08%, respectively. Considering that the RSD values are
lower than 5%, the precision of the method was proven, according to the Brazilian legislation for
bioanalytical methods [44].
3.2.3. Accuracy
The accuracy was confirmed by the recovery test. In this test, a known amount of DPT RS was
added to a solution containing the excipient (spiked placebo). The average recovery was 101.41%, and
this fact demonstrates the ability of the method to determine pre-defined contents of DPT with accuracy.
3.2.4. Selectivity
Table 1 shows the results of the selectivity analysis of the method. Comparing the response of the
micro-organism with (positive control) and without NaOH, it is possible to observe that the excipient
did not interfere in the analysis, since this substance did not present antimicrobial activity (the absorbance
values were similar).
Table 1. Obtained results for the selectivity analysis of the developed turbidimetric assay.
Analysis Obtained absorbances for positive control Obtained absorbances in the test with NaOH
1 0.862 0.830
2 0.913 0.897
3 0.852 0.932
Average 0.876 0.886
3.2.5. Robustness
Table 2 presents the results for the robustness of the method. Considering that all of the varied
parameters presented RDS values lower than 5%, the method was shown to be robust.
Pharmaceutics 2015, 7 116
Table 2. Obtained results for the robustness analysis of the developed turbidimetric assay.
Parameter Investigated
range
Daptomycin
(g/vial)
Daptomycin
(%) RSD 1 (%)
Inoculum
concentration (%)
4.8
5.0 *
5.2
0.459
0.495
0.462
91.70
98.94
92.45
4.22
Calcium
concentration
(µg mL−1)
70
75 *
80
0.519
0.506
0.496
103.87
101.17
99.11
2.39
Wavelength (nm)
525
530 *
535
0.494
0.506
0.491
98.79
101.17
98.22
1.57
Shaker incubation
time (h)
3 h 50 min
4 h *
4 h 10 min
0.484
0.506
0.520
96.87
101.17
103.92
3.53
1 RDS: relative standard deviation; * standard working conditions.
3.3. Comparison between the Microbiological and Chromatographic Methods
A comparison between the developed turbidimetric method with a HPLC method previously
developed and validated by our group was performed in order to verify whether these two methods are
interchangeable for the analysis of daptomycin in the pharmaceutical dosage form.
Table 3 shows the percentage contents of daptomycin obtained by the microbiological and HPLC
methods. Student’s t-test performed to compare these data showed that both methods are
interchangeable, since the contents of daptomycin obtained by the two methods were statistically
equivalent, at a significance level of 5% (tcalculated = 0.82 < tcritical = 2.23).
Table 3. Values obtained in the determination of daptomycin (DPT) in powder for
injectable solution by HPLC and turbidimetric assay.
Parameters Method
HPLC a TURB
b
DPT content (%)
99.88
101.90
99.10
102.90
104.37
102.85
101.78
101.17
98.94
103.92
98.22
101.17
Average content (%) 101.83 100.87
a HPLC: high-performance liquid chromatography; b TURB: turbidimetric assay.
The most interesting aspect of this comparison is that a microbiological method is being compared
with a physicochemical method, and both have different characteristics. Microbiological methods, in
general, present disadvantages regarding the execution time and the amount of work and materials
required. For these reasons, often, these methods are being replaced by physicochemical methods in
routine analysis in the quality control of antimicrobials. However, this is not a recommended practice,
Pharmaceutics 2015, 7 117
since microbiological methods provide important information about the biological activity of
antimicrobial agents, which physicochemical methods are not able to provide. Frequently, the part of
the molecule essential for antimicrobial activity cannot be detected by physical-chemical methods,
generating false conclusions about the quality of the product [36,45]. Thus, microbiological assays
used to determine the potency of antimicrobial agents still play an essential role in manufacturing
processes and quality control of these drugs.
HPLC also presents some advantages and disadvantages when compared to the turbidimetric
method. Considering the disadvantages, this technique requires the use of a chromatograph,
chromatographic columns and high purity solvents, all costly. In addition, because it is a technique that
requires the use of organic solvents, it can harm the environment, in addition to generating costs for the
industry to treat this waste. Among the advantages, it is worth highlighting that this technique is rapid,
highly selective and ideal for the detection of degradation products and impurities of the analyzed
drug [37].
Although the turbidimetric assay is not suitable to determine this kind of impurity and the
degradation products, it is an environmentally-friendly method, which does not require the use of
organic solvents for its analysis, which is in line with the global trend. Furthermore, it is a method
increasingly used and recognized for the analysis of antimicrobial agents [37,46–50].
4. Conclusions
A microbiological method has been properly developed and validated and shown to be effective for
determining the potency of daptomycin in the pharmaceutical dosage form of lyophilized powder for
injectable solution, since it was linear, precise, accurate, robust and selective. Moreover, this method
does not require the use of organic solvents and costly equipment and materials, such as HPLC. At the
same time, the turbidimetric method described in this paper takes four hours to perform, which is
comparable to a physicochemical method. In addition, Student’s t-test showed no statistically
significant difference between the proposed turbidimetric method and a previously validated HPLC
method. Thus, considering the importance of microbiological methods for the analysis of antimicrobial
agents, the turbidimetric method developed and validated in this work becomes a convenient
alternative for the routine analysis of the quality control of this drug.
Acknowledgments
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
(São Paulo, Brazil), Fundação para o Desenvolvimento da Unesp (FUNDUNESP) (São Paulo, Brazil)
and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Brasília, Brazil).
Eliane Gandolpho Tótoli was funded by FAPESP (São Paulo, Brazil), and Hérida Regina Nunes
Salgado was funded by CNPq (Brasília, Brazil).
Author Contributions
Eliane Gandolpho Tótoli and Hérida Regina Nunes Salgado conceived of and designed the
experiments. Eliane Gandolpho Tótoli performed the experiments. Eliane Gandolpho Tótoli and Hérida
Pharmaceutics 2015, 7 118
Regina Nunes Salgado analyzed the data. Eliane Gandolpho Tótoli wrote the paper. Hérida Regina
Nunes Salgado supervised the work.
Conflicts of Interest
The authors declare no conflict of interest.
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