Journal of Applied Pharmaceutical Science Vol. 5 (12), pp.

008-013, December, 2015

Available online at http://www.japsonline.com
DOI: 10.7324/JAPS.2015.501202
ISSN 2231-3354

Determination of the Chemical Stability of Various Formulations of

Tobramycin Eye-Drops by HPLC Method and Data Analysis by R-
GUI Stability Software
María Ana Rosasco, Adriana Inés Segall*
Cátedra de Calidad de Medicamentos, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, CONICET, Junín 956, 1113 Buenos Aires,
Argentina.

ARTICLE INFO ABSTRACT

Article history: Tobramycin, is a water soluble aminoglycoside antibiotic produced by the fungus Streptomyces tenebrarius and
Received on: 28/09/2015 used in a variety of pharmaceutical applications including ophthalmic solutions, suspensions and ointments;
Revised on: 10/10/2015 inhalation solutions and intravenous administration. There are commercially available eye drops formulations in
Accepted on: 22/10/2015 the Argentinian market that have different conservation conditions. We formulated six eye drops solutions,
Available online: 27/12/2015 studied their stability at 2-8°C, 25°C, and 40°C, 75% RH and quantified using USP method. Only half of the
formulations studied were found to be stable for two years at ambient temperature which is their expected expiry
Key words: date and only one for three years in the same condition. One system was unstable in the three conservation
Tobramycin, eyes-drops, conditions studied, including appearance and pH.
stability, software generated
shelf life, formulations.

INTRODUCTION Tobramycin oxidizes giving several products including nebramine,

deoxystreptamine and deoxystreptamine-kanasaminide (Fig 1).
Tobramycin, D-streptamine, O-3-amino-3-deoxy-α-D- According to these results we formulated six solutions and studied
glucopyranosyl-(1→6)-O-[´2,6-diamino-2,3,6-trideoxy-α-D- their stability at 2-8°C, 25°C, and 40°C, 75% RH. Direct analysis
ribo-hexopyranosyl-(1→4)]-2-deoxy-, is a water soluble of tobramycin is not simple. This is due to the polar basic nature
aminoglycoside antibiotic produced by the fungus Streptomyces and the lack of UV absorbing chromophore in the molecule.
tenebrarius and used in a variety of pharmaceutical applications Several methods have been described to determine tobramycin by
including ophthalmic solutions, suspensions and ointments; adsorptive stripping voltammetric method (Sun et al., 2005),
inhalation solutions and intravenous administration (Hanko and electrophoresis capillary (El-Attug et al., 2012; Ahmed and Ebeid,
Rohrer, 2006; Manyanga et al., 2013). It is active against a broad 2015) and ion pair LC methods (Hankoand Rohrer, 2006; Szúnyog
spectrum of gram-negative bacteria. There are commercially et al., 2000; Valentini et al., 2008; Hanko et al., 2008; Chopra et
available eye drops formulations in the Argentinian market that al., 2010). A literature survey revealed some reversed phase liquid
have different conservation conditions (ambient temperature and chromatographic methods for the quantitation of tobramycin
refrigerator). Brandl and Gu studied the stability of Tobramycin employing mass detector (Keevil et al., 2003; Guo et al., 2006),
in aqueous solution (Brandl and Gu, 1992). They concluded that ELSD (Megoulas et al., 2005; Pfeifer et al., 2015) and PED
although hydrolysis of tobramycin occurs at the pH extremes, it (Manyanga et al., 2013). The literature showed also reversed phase
is not an important degradation pathway at neutral pH values. HPLC methods with pre column derivatization with 1 Naphthyl
The major degradation pathway for tobramycin at neutral pH isothiocyanate (Feng et al., 2002), Fluorescein isothiocyanate
values where the drug is formulated (pH 5.8-7.4) is oxidation. (Mashat et al., 2008), 2,4,6-Trinitrobenzenesulphonic acid (Dash
and Suryanarayanan, 1991a; Dash and Suryanarayanan, 1991b), 4-
* Corresponding Author Chloro-3,5-dinitrobenzotrifluoride (He et al., 2011),
Email: [email protected] 9-Fluorenylmethyl chloroformate (Zhang and Peng, 2012) and

© 2015 Maria Ana Rosasco and Adriana Ines Segall. This is an open access article distributed under the terms of the Creative Commons Attribution License -
NonCommercial-ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/).
 Rosasco and Segall / Journal of Applied Pharmaceutical Science 5 (12); 2015: 008-013 09

A B

C D
Fig. 1: A) Tobramycin, B) Deoxystreptamine, C) Deoxystreptamine-kanasaminide, D) Nebramine.

Table 1: Quantitative composition of formulations (g %)

Materials (g/100g) System A SystemB System C SystemD SystemE SystemF
Tobramicyn 0.3 0.3 0.3 0.3 0.3 0.3
Excipients:Benzalkonium chloride 1.0 x 10-2
Sodium chloride 1.0 x 10-2 0.1 7.0 x 10-2 2.0 x 10-2
Dibasic sodium phosphate 0.6
Monobasic sodium phosphate 0.4
Boric acid 1.24 x 10-2 1.26 x 10-3 1.9 1.9
Anhydrous sodium sulfate 1.52 x 10-3 2.26 x 10-4
Tyloxapol 1.0 x 10-3 0.1
Edetate disodium 0.1
Sodium metabisulfite 0.5
Thimerosal 1.0 x 10-3
Sodium hyaluronate 0.3 0.3
Potassium sorbate 0.18
Water for injection csp 100 100 100 100 100 100

o-Phthalaldehyde pre column derivatization (Caturla et al., 1992) (Argentine), Tyloxapol Sigma-Aldrich (India), Edetate disodium
and post column derivatization (He and Yang, 2010). This work Merck (Germany), Sodium hydroxide Anedra (Argentine),
describes the analysis of tobramycin in six formulations Sodium metabisulfite J.T. Baker (Mexico), Thimerosal Ningbo
maintained in stability studies and quantitated using USP method Hi-Tech (China), Sodium hyaluronate CPN SPOL SRO (Czech
(The United States Pharmacopeia, 2015). This method use 2,4- Republic), Potassium sorbate granular Merck (Germany) and
Dinitrofluorobenzene as pre- derivatizating agent. Russ and distilled water.
coworkers use a modification of this method for the quantitation of All chemicals used were of analytical grade, sulphuric
tobramicyn in an ophthalmic suspension (Russ et al., 1998). acid Merck (Germany), Buffer TRIS (Tris(Hidroxymethyl)
aminomethane) Biopack (Argentine), 2,4-Dinitrofluorobenzene
MATERIALS AND METHODS Sigma-Aldrich (USA), p-naphtholbenzein Sigma-Aldrich (USA).
Acetonitrile and water were of HPLC grade. Solvents were filtered
Materials and reagents through a 0.45 µm membrane and degassed.
Tobramicyn (955,9 µg/g calculated with reference to the -
dried substance) pharmaceutical grade was provided by Cross Preparation of the eye-drops
Chem (China), Benzalkonium chloride Merck (Germany), Sodium The six systems were prepared with the same drug
chloride Merck (USA), Dibasic sodium phosphate Anedra substance of Tobramycin. Formulations are described in Table 1.
(Argentine), Monobasic sodium phosphate Anedra (Argentine), In a glass flask containing 350 mL of distilled water at 25 °C, the
Boric acid J.T Baker (Mexico), Anhydrous sodium sulfate Anedra salts and other excipients were dissolved once each and stirring
010 Rosasco and Segall / Journal of Applied Pharmaceutical Science 5 (12); 2015: 008-013

vigorously. A nitrogen flow was placed before the Tobramycin This reagent was used within 4 hs. To separate 50 mL volumetric
was added. Then, the pH was corrected around 7.5 with NaOH 1N flask, 4.0 mL of standard preparation, 4.0 mL of sample
or HCl 3N. The solutions were diluted with distilled water to 500 preparation and 4.0 mL of water were transferred and added 10
mL. The solutions were passed through a 0.22 µm nylon mL of 2,4-Dinitrofluorobenzene solution and 10 mL of TRIS
membrane filter before injection (25 mm disposable filter; Cat. N° solution each. The volumetric flask were kept in a water bath at 60
R04SP02500 Osmonics Inc., Minnesota, USA). Approximately 5 ± 2°C during 50 ± 5 minutes. The flask were removed from the
mL of Tobramycin solutions were transfer in plastic bottles of bath, and allowed to stand for 10 minutes. Added acetonitrile to
HDPE/LDPE and placed in stability conditions: 2-8 ºC, 25 ºC and about 2 mL below the 50 mL mark, allowed to cool to room
40 ºC, 75% HR. temperature, then diluted with acetonitrile to volume, and mixed.
The resolution solution was prepared transferring 2 mL
Instrumentation of a fresh solution of p-naphtholbenzein in acetonitrile (containing
The HPLC system consisted of a dual piston 0.24 mg per mL) to a 10 mL volumetric flask and diluted with
reciprocating Thermo Finnigan pump (Waltham, Massachusetts, derivatized standard preparation for volume and used promptly.
United States, Model P2000), a Rheodyne injector (Model 7125), a It was injected a blank of mobile phase, another with the
UV-Vis KONIK detector (Barcelona, Spain, Model KNK-027- derivatization solution diluted as the standard solution and the
757) with operating software WinPCC Chrom XY (Buenos Aires, resolution solution. The relative retention times were about 0.6 for
Argentine) was used during the study. p-naphtholbenzein and 1.0 for tobramycin, and the resolution, R,
between the two peaks was not less 4.0.The derivatized standard
HPLC conditions preparation was chromatographed, recorded the responses until the
The experiment was performed on a reversed phase C18 relative standard deviation for replicate was not more than 2.0 %.
column (Phenomenex, Torrance, CA, USA) 300 x 4.6mm, 10µm. Separately were injected equal volumes of the derivatized standard
The separation was carried out under isocratic elution with preparation and the derivatized assay preparation into the
acetonitrile:buffer TRIS. For the preparation of the mobile phase, chromatograph, recorded the chromatograms and measured the
2 g of buffer TRIS were dissolved in 800 ml of water. To this area responses for the major peaks and calculated the quantity.
solution, 20 ml of Sulfuric acid was added and diluted to 2000 ml
with acetonitrile. The flow rate was 1.1 mL/min. The wavelength pH Determinations
was monitored at 365 nm, and the injection volume was 20 µL. The pH data for all the systems were obtained with
The HPLC was operated at ambient temperature. In these model Altronix TPX I (Saen S.R.L., Buenos Aires, Argentina).
conditions the retention time (tR) was roughly 6 minutes. The pH was measured as directed in USP 38 <791>, using an
indicator glass electrode. The buffer solutions for standardization
Preparation of standard solution were from Merck (Darmstadt, Germany) at pH 4.00 and 7.01.
An accurately weighed quantity of 33 mg of tobramycin
was transferred to a 50 mL volumetric flask, added 1 mL of 1 N Software
sulfuric acid and dissolved in 20 ml of water and then taken to To evaluate the shelf life of Tobramycin eye drops, an R-
volume with water. Then, 10.0 mL were withdraw in a 50 mL package “stab” software version 3.0.1 was used (Lee et al., 2010;
volumetric flask, diluted with water to volume, and mixed. The Hassan et al., 2015). The software meets ICH Q1E specifications
solutions were passed through a 0.45 µm nylon membrane filter (Guidance for the industry: ICH 2003).
before injection (25 mm disposable filter; Cat. N° Y02025WPH
icroclar, Buenos Aires, Argentina). RESULTS AND DISCUSSION
In the present study, the stability of different eye drops
Sample Preparation formulations containing Tobramycin were studied. The eye drops
Approximately 1 mL of eye drops tobramycin solution produced in this work are similar to those it can be found in the
0.3% (3 mg/mL) were exactly weighed, placed into a 25 mL Argentinian market. The trouble is that the products have different
volumetric flask and taken to volume with water. conservation conditions.
The solutions were passed through a 0.45 µm nylon The stability studies are executed to estimate the
membrane filter before injection (25 mm disposable filter; Cat. N° mechanism of the active pharmaceutical ingredients´ (APIs)
Y02025WPH icroclar, Buenos Aires, Argentina). degradation in crude and in dosage forms.
The preparations were analyzed by HPLC with a
Derivatization previous derivatization reaction and UV detection. The stability
A solution of 2,4-Dinitrofluorobenzene containing 10 mg results were analysed with R-package “stab” software version
per mL in alcohol was prepared and maintained during 5 days in 3.0.1., having a single-factor analysis, for single-batch based on
refrigerator. A stock solution of TRIS was prepared in water ICH Q1E specifications (Guidance for the industry: ICH 2003).
containing 15 mg per mL. 40 mL were transferred to a 200 mL The assay results are mentioned in Table 2. With reference to pH
volumetric flask and diluted to volume with dimethyl sulfoxide. for tobramycin eye-drops solutions, USP 38 (The United States
 Rosasco and Segall / Journal of Applied Pharmaceutical Science 5 (12); 2015: 008-013 011

Pharmacopeia, 2015) sets a range from 7 to 8. The appearance and temperature (25 °C), systems B was the most stable with 4.58
pH results are indicated in Table 3, in bold pH data that are out of years and systems D and E greater than 24 months. At the
specification. Only system C does not meet this requirement in any accelerated condition (40 °C and 75% RH), only system B was
of the storage conditions. First order analysis was made at one stable at 1.75 years. System B was found most stable whereas
sided lower control analysis at 90% confidence interval (Fig. 2-4). system C was the least stable. With regard to appearance, System
The results are presented in Table 4. In refrigerator (2-8 °C) C seems to turn to yellowish at 6 months at ambient temperature.
systems A, B, D and E shown a shelf life of 5 years or more while The same occur to System F since 9 months and System A since
system F present 4.83 years and system C 1.58 years. At ambient 18 months.

Fig. 2: Estimation of shelf life of Tobramycin System D al 40 °C, 75% RH Fig. 3: Estimation of shelf life of Tobramycin System D al 25 °C.

Fig. 4: Estimation of shelf life of Tobramycin System C al 2-8 °C.

012 Rosasco and Segall / Journal of Applied Pharmaceutical Science 5 (12); 2015: 008-013

Table 2: The remaining percentage content of the active ingredient tobramycin in eye-drops solutions.
Time (months)
System A 0 1 3 6 9 12 18 24 30 36
40 ºC, 75% HR 97.65 93.08 88.19 88.32 74.52 62.92
25 °C, 60% HR 97.65 99.92 96.24 95.11 95.84 94.77 91.50 91.45 85.95 85.30
2-8ºC 97.65 98.48 96.84 96.71 97.50 97.24 97.25 98.47 98.24 99.54
SystemB
40 ºC, 75% HR 99.42 97.09 98.87 95.90 96.70 96.47
25 °C, 60% HR 99.42 100.94 97.56 95.67 97.63 98.08 97.38 99.12 95.29 94.51
2-8ºC 99.42 97.41 96.72 96.66 97.73 98.15 98.09 98.48 99.86 98.99
SystemC
40 ºC, 75% HR 100.09 82.42 80.11 75.40 73.82 68.78
25 °C, 60% HR 100.09 94.32 84.70 80.53 80.82 79.56 77.70 75.87 76.06 69.97
2-8ºC 100.09 98.08 96.78 94.84 93.55 93.18 89.65 89.48 86.62 85.43
SystemD
40 ºC, 75% HR 99.04 99.48 99.71 97.30 97.19 93.78
25 °C, 60% HR 99.04 99.09 96.52 95.84 95.16 94.00 94.95 93.27 93.12 90.72
2-8ºC 99.04 97.41 97.12 95.77 96.73 97.17 97.53 97.46 97.53 96.56
SystemE
40 ºC, 75% HR 99.24 97.20 97.54 88.70 85.77 81.49
25 °C, 60% HR 99.24 100.77 96.29 96.31 96.24 93.47 95.56 95.88 90.03 87.55
2-8ºC 99.24 97.58 96.75 96.34 96.53 96.52 97.27 99.04 97.87 100.74
SystemF
40 ºC, 75% HR 98.6 92.18 93.61 86.68 81.30 69.08
25 °C, 60% HR 98.6 98.70 94.62 92.79 93.24 91.88 90.48 84.73 85.73 83.82
2-8ºC 98.6 96.04 96.07 95.30 95.80 94.99 95.45 94.80 93.12 95.52

Table 3: Appearance and pH of the formulations.

System A System B System C SystemD SystemE SystemF
Time 2-8 25°C 40ºC 2-8 25°C 40ºC 2-8 25°C 40ºC 2- 25°C 40ºC 2- 25 40ºC 2- 25°C 40ºC
(month) °C °C °C 8°C 8°C °C 8°C
0 Appearance a a a a a a a a a a a a a a a a a a
pH 7.52 7.52 7.52 7.50 7.50 7.50 7.55 7.55 7.55 7.40 7.40 7.40 7.42 7.42 7.42 7.40 7.40 7.40
1 Appearance a a a a a a a a c a a a a a a a a c
pH 7.69 7.69 7.68 7.50 7.50 7.49 7.48 7.23 6.98 7.37 7.33 7.37 7.47 7.44 7.50 7.40 7.41 7.41
3 Appearance a a a a a a a a a a a a a a a a a a
pH 7.69 7.68 7.68 7.51 7.53 7.52 7.40 7.17 6.97 7.33 7.34 7.34 7.49 7.49 7.50 7.43 7.42 7.40
6 Appearance a a a a a a a c d a a a a a a a a c
pH 7.70 7.70 7.69 7.52 7.56 7.55 7.37 6.99 6.92 7.26 7.37 7.36 7.50 7.55 7.50 7.42 7.43 7.42
9 Appearance a a c a a a a d d a a a a a a a c c
pH 7.69 7.67 7.59 7.44 7.42 7.45 7.33 6.90 6.78 7.16 7.19 7.23 7.52 7.45 7.47 7.44 7.39 7.42
12 Appearance a a c a a a a d d a a c a a c a c c
pH 7.63 7.65 7.49 7.30 7.39 7.40 7.19 6.85 6.62 7.11 7.17 7.15 7.47 7.47 7.44 7.40 7.41 7.34
18 Appearance a b - a a - a d - a a - a a - a c -
pH 7.67 7.65 - 7.51 7.41 - 7.15 6.85 - 7.24 7.17 - 7.57 7.44 - 7.48 7.34 -
24 Appearance a c - b a - a d - a a - a a - a c -
pH 7.67 7.65 - 7.45 7.52 - 7.03 6.96 - 7.14 7.27 - 7.53 7.50 - 7.42 7.41 -
30 Appearance a c - c a - a d - a a - a a - a c -
pH 7.66 7.63 - 7.36 7.47 - 6.84 6.86 - 7.05 7.24 - 7.44 7.48 - 7.38 7.41 -
36 Appearance a c - c a - b d - a a - a a - a c -
pH 7.65 7.61 - 7.32 7.43 - 6.77 6.75 - 7.08 7.19 - 7.51 7.48 - 7.48 7.42 -
a: limpid solution, colorless., b: limpid solution, slightly yellowish., c: limpid solution, yellowish., d: limpid solution, yellow

Table 4: Software originated results for apparent First-Order Rate Constant (kobs) for the degradation of tobramycin eye-drops.
System K obs. month-1 Correlationcoefficient t90 years (months)
A40 ºC, 75% RH 2.655 0.9648 0.08 (1 month)
25 °C 0.368 0.9679 1.67 (20 months)
2-8ºC 0.045 0.6497 >5
B 40 ºC, 75% RH 0.204 0.6814 1.75 (21 months)
25 °C 0.101 0.6401 4.58 (55 months)
2-8ºC 0.049 0.5786 >5
C40 ºC, 75% RH 1.970 0.8503 0.08 (<1 month)
25 °C 0.598 0.8380 0.08 (<1 month)
2-8ºC 0.378 0.9757 1.58 (19 months)
D40 ºC, 75% RH 0.431 0.9116 1.42 (17 months)
25 °C 0.190 0.9197 2.83 (34 months)
2-8ºC 0.013 0.1944 >5
E40 ºC, 75% RH 1.530 0.9807 0.42 (5 months)
25 °C 0.275 0.8811 2.17 (26 months)
2-8ºC 0.063 0.5495 >5
F40 ºC, 75% RH 2.169 0.9661 0.17 ( 2 months)
25 °C 0.404 0.9574 1.25 (15 months)
2-8ºC 0.070 0.6477 4.83 (58 months)
 Rosasco and Segall / Journal of Applied Pharmaceutical Science 5 (12); 2015: 008-013 013

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integrated pulsed amperometric detection. J Pharm Biomed Anal, 2008;
47:828–833. Rosasco MA, Segall AI. Determination of the Chemical Stability of
Hassan S, Zaheer E, Muhammad IN, Hassan A, Ali M, Qadri Various Formulations of Tobramycin Eye-Drops by HPLC Method
M. Determination of chemical stability of various Famotidine and Data Analysis by R-GUI Stability Software. J App Pharm Sci,
dosage forms by UV-Visible spectrophotometric method and data 2015; 5 (12): 008-013.
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