Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 72 No. 1 pp.

31ñ37, 2015 ISSN 0001-6837

Polish Pharmaceutical Society

DETERMINATION OF NEOMYCIN IN THE FORM OF NEOMYCIN

DERIVATIVE WITH DABSYL CHLORIDE BY THIN LAYER
CHROMATOGRAPHY AND DENSITOMETRY
URSZULA HUBICKA, BARBARA ØUROMSKA-WITEK, JOANNA PIOTROWSKA
and JAN KRZEK*

Department of Inorganic and Analytical Chemistry, Medical College of Jagiellonian University,

Medyczna 9, 30-688 KrakÛw, Poland

Abstract: A thin layer chromatographicñdensitometric method has been developed for identification and quan-
titative determination of neomycin derivative with dabsyl chloride. The analysis of antibiotic was achieved on
the silica gel TLC plates with fluorescent indicator with n-butanol ñ 2-butanone ñ 25% ammonia ñ water (10 :
6 : 2 : 2, v/v/v/v) as the mobile phase. The densitometric measurements were made at 460 nm. Under these con-
ditions good separation of chosen aminoglycoside antibiotic from reagent used to make a complex was
obtained. The method is characterized by high sensitivity, LOD from 0.1953 µg per band and LOQ from 0.5918
µg per band, wide linearity range from 0.5918 to 2.1960 µg per band for neomycin. The precision of the method
was good; RSD varied from 1.17 to 2.05%. Satisfactory results of validation of the method were also confirmed
by determination of selected antibiotic in pharmaceutical commercial preparation. The results obtained by
TLCñdensitometric method were compared with those obtained by spectrophotometric method.

Keywords: pharmaceutical research, neomycin, dabsyl chloride, TLC, densitometry

Neomycin is an aminoglycoside antibiotic that chemical detection) (10, 11), HPLC-ELSD

is produced naturally by the actinomycete bacterium (Evaporative Light Scatering Detection) (12),
Streptomyces fradiae via the fermentation process. It HPAE-IPAD (High-performance anion-exchange
has bactericidal properties against Gram negative chromatography with integrated pulsed amperomet-
bacteria and partial also against Gram positive bac- ric detection) (13) and thin layer chromatography
teria. Neomycin in a sulfate form is a common (14, 15).
aminoglycoside indicated for treatment of gastroin- Pendela et al. described the analysis of ear drops
testinal infections. Neomycin sulfate is mainly com- containing neomycin sulfate, polymyxin B sulfate
posed of the two isometric components neomycins and dexamethasone sodium phosphate using liquid
B and C. The component B has higher antibiotic chromatography with pulsed electrochemical detec-
activity than component C (1). Neomycin is mostly tion on a gold electrode (10). The similar study was
administered in the form of powder, aerosols, performed to a formulation, which contained
creams and also drops and ointments often in com- neomycin sulfate, polymyxin B sulfate and grami-
plex preparations with bacitracin, dexamethasone, cidin (11). HPLC-ELSD method was applied suc-
polymyxin, gramicidin, hydrocortisone, fluoci- cessfully for the determination of neomycin and sul-
nolone, etc. (2, 3, 4). fate in raw materials, pharmaceutical formulations
There are several methods described for the (powder, aerosols and creams) and medicated animal
determination of neomycin in pharmaceutical prepa- feeds (12). HPAE-IPAD was used to determine
rations: colorimetric determination (5), fluorimetric neomycin B and its impurities. The method was
determination (6), near-infrared spectroscopy (7) shown to be rugged for the intended application of
and spectrophotometric determination (8, 9). Some neomycin identity, purity and assay (13). A thin layer
of the most popular methods are chromatographic chromatography with densitometric detection for
methods: liquid chromatography with varied meth- simultaneous identification and quantitative determi-
ods of detection like HPLC-PED (pulsed electro- nation of neomycin sulfate, polymyxin B sulfate, zinc

* Corresponding author: e-mail: [email protected]

31
32 URSZULA HUBICKA et al.

bacitracin and methyl and propyl hydroxybenzoate in analytical grade and were purchased either from
ophthalmic ointment was described by Krzek et al. POCh (Gliwice, Poland) or Chempur (Piekary
(14). Hubicka et al. described the analysis of ålπskie, Poland).
amikacin, gentamicin, kanamicin, neomycin, Dabsyl chloride (DBS) ñ assay = 97.5% (AT)
netilmycin and tobramycin in pharmaceutical prepa- no. 39068 (Fluka, Chemie AG Buchs, Switzerland).
rations (tobramycin ampoules, amikacin vials, tablets Solutions of DBS in acetone were prepared at con-
containing 250 mg of neomycin and eye drops con- centrations 0.3238 mg/mL and 0.1619 mg/mL.
taining 3 mg/mL of gentamicin) (15). The densito-
metric measurements were made after detection with Standard solutions and substance
a 0.2% ninhydrin solution in ethanol (14, 15). The following pharmaceutical raw material of
Anionic capillary isotachophoresis (cITP) with neomycin sulfate was used: 25 g PPH Galfarm ñ no.
conductometric detection was used for indirect 011209. Neomycin met the requirements described
determination of neomycin trisulfate as sulfate anion in a monograph in Polish Pharmacopoeia (FP VIII)
in pharmaceutical preparations (16, 17). Capillary within the scope of identity, purity and biological
elektrophoresis with direct (18) and indirect (19, 20) activity. Standard solutions in water were prepared
UV detection for simultaneous determination of at concentrations from 1.8603 to 0.1970 mg/mL cal-
neomycin sulfate and such active substances as culated on neomycin.
hydrocortisone, polymyxin in various pharmaceuti-
cal preparations have been reported (18ñ20). Pharmaceutical preparation and solution
Since 1975, dabsyl chloride (DBS) has been Neomycinum ñ tablets (250 mg neomycin sul-
used for identification of N-terminal amino acid in fate, macrogol 4000, sodium starch glycolate type
polypeptide chain during analysis of compounds A, talc, magnesium stearate, saccharose about 260
containing amine group (21). The first step in the mg in one tablet). Polfa Tarchomin S.A., Poland,
process is condensation of DBS with polypeptide series No. 1010904.
through N-terminal amino acid. The second step is A solutions of Neomycinum ñ to prepare sam-
hydrolysis in acidic environment that results in a ple solution from powdered mass of 20 tablets,
product ñ sulfonamide derivative. The final third 50.10 mg corresponding to 15.52 mg of neomycin
step is chromatographic identification of obtained was accurately weighed. Then, 15.0 mL of water
product. This reaction is highly sensitive (10-9ñ10-10 was added into the sample and the solution was
mol/L). For example, biogenic amines, bisoprolol, shaken for 15 min. Then, the solution was filled up
labetalol, and propafenone as dabsyl derivatives with water to 25.0 mL. The suspension was filtered
were determined (22ñ25). directly into a 25.0 mL volumetric flask through
The quantitative determination of bacitracin qualitative filter paper. For testing by spectrophoto-
after condensation reaction with DBS was presented metric method, the solution of neomycin at a con-
by Krzek and Piotrowska. Modification in the centration of 1.8600 mg/mL was prepared similarly.
method of N-terminal amino acid determination
with the use of DBS was done by exclusion of TLC method
hydrolysis and establishing reaction conditions to Preparation of neomycin derivative with dabsyl
enable direct spectrophotometric estimation of the chloride (NDC)
product of DBS reaction with the antibiotic (26). A product of reaction was formed when 0.5 mL
In this paper a new TLCñdensitometric method of neomycin solution was mixed with 1.0 mL of
was developed for the simultaneous identification DBS (0.1619 mg/mL) and 0.2 mL of carbonate
and quantitative determination of neomycin deriva- buffer (pH = 9.0). The samples and blank solution
tive with dabsyl chloride (NDC), directly without were heated up in water bath at 70OC for 15 min, and
the requirement of chromatogram visualization. The then cooled down and adjusted with acetone to the
developed method was validated and used for the final volume of 5.0 mL. A blank solution containing
quantitative determination of neomycin in pharma- identical volume of carbonate buffer (pH = 9.00)
ceutical preparation. and DBS concentration as in the study sample was
prepared.
EXPERIMENTAL
TLC analysis
Reagents TLC was performed on 10 ◊ 12 cm TLC plates
Acetone, sodium carbonate, sodium bicarbon- cut from 20 ◊ 20 cm aluminium foil-backed plates pre-
ate, 25% ammonia, butanol, 2-butanone were of coated with silica gel 60 F254 (Merck, Germany;
 Determination of neomycin in the form of neomycin derivative... 33

#1.05548). Solutions of the NDC obtained for standard was described in ìTLC analysisî. Linearity was
solutions and for preparation solution (30 µL) were assessed in triplicate on the basis of the relationship
applied to the plates as 0.8 cm bands, 1.0 cm from the between mean peak area and amount of neomycin in
bottom edge, 1.0 cm from the sides and 0.8 cm apart, micrograms per band. Linearity was reported as
by use of a Linomat V sample applicator (Camag, regression equations, correlation coefficients (r) and
Switzerland). Chromatograms were developed to a dis- determination coefficient (r2).
tance of 12 cm with n-butanol ñ 2-butanone ñ 25%
ammonia ñ water (10 : 6 : 2 : 2, v/v/v/v) as a mobile The limit of detection (LOD) and quantification
phase in a chromatographic chamber (18 ◊ 9 ◊ 18 cm (LOQ)
Sigma-Aldrich, USA, #Z20,415-3). The mobile phase Solutions of neomycin in the form of derivative
was chosen experimentally by checking different sol- with DBS (0.1220 ñ 0.3660 mg/mL) were applied on
vent mixtures. Plates were dried at room temperature the plates. LOD and LOQ were calculated on the
for one hour. Bands on the chromatograms retain basis of the slope (a) of the calibration line and the
durable yellow color within 24 h. Color of bands con- standard error of the estimate (Se), using formulas:
trasts with a white background of chromatogram. LOD = 3.3 Se / a and LOQ = 10 Se / a.
Bands were visible and could be used for quantitative
densitometric determination. Detection was carried out Precision
by Camag TLC Scanner 3 with winCats 1.3.4 software The repeatability of the method was deter-
at λ = 460 nm; this wavelength was selected on the mined by analysis of five replicates of NDC
basis of the recorded absorption spectra. In addition to obtained for standard solutions from individual
the RF values, identification of analyzed substances weighings. Study was done for three concentration
were done. Peak areas of NDC obtained for standard levels, 50% (0.5918 µg per band of NDC), 100%
solutions and for preparation solution were recorded (1.0980 µg per band of NDC) and 150% (1.6470 µg
directly from the chromatograms and were used for per band of NDC). Intermediate precision was
quantitative analysis. obtained by analysis of solutions at the same con-
centration by a different analyst who performed the
Method validation analysis over a period of one week.
The method was validated by checking the
specificity, linearity, precision, recovery, limits of Accuracy
detection and quantification and also robustness in Accuracy was assessed by determination of the
accordance with ICH guidelines (27). recovery (%) of the drug. Precisely known weighed
amounts of the neomycin standard (from 80 to 120%
Specificity of the declared content) were added to preparation
Specificity of the method was assessed by solutions and percentage recovery was calculated on
comparing chromatograms of NDC obtained for the basis of the determined amounts of added
standard solutions and for preparation solution, neomycin standard under conditions of developed
chromatogram of blank solution and blank chro- method in relation to the amount weighed. For each
matogram. level three determinations were done.
In obtained chromatograms, the retardation
factor (RF) values of analyzed substances, resolution Robustness
factor, peak areas, peak purity and spot color were Under conditions of the developed method,
taken into account. Resolution factor (Rs) was cal- comparison of results with change of stationary
culated according to the formula: phase from TLC (Merck, Germany) to TLC (poly-
Rs = 2 ◊ (distance between the centers of two ester sheets precoated with silica gel 60 F254
adjacent spots) / (sum of the two spots in the direc- Macherey-Nagel, Germany; #805023) plates was
tion of development). done.
The impact of small changes of 25% ammonia
Linearity in the composition of the mobile phase (± 5%) for
The calibration plot for the method was con- chromatographic separation was checked.
structed by analysis of seven solutions containing
different concentrations of neomycin in the range Determination of neomycin in pharmaceutical
0.1970 ñ 0.7320 mg/mL after reaction with dabsyl preparation
chloride. Preparation of samples working solutions Determination of neomycin in tested tablets
were described before. Further analytical procedure was carried out according to earlier described proce-
34 URSZULA HUBICKA et al.

dure. The amount of 0.9150 µg per band of NDC chloride was carried out according to earlier
obtained for standard solution and preparation solu- described procedure. For this purpose, a series of
tion were applied onto TLC plates for the determi- six solutions containing DBS with neomycin
nation of neomycin content. For each determination preparation solution at a concentration of 1.8600
three measurements were performed and the mean mg/mL were prepared. Then, the content of
value was taken for calculations. neomycin in pharmaceutical preparation was cal-
culated by comparing appropriate values of
Spectrophotometric method absorbance recorded at λmax = 492 nm for standard
In order to compare the test results obtained by and sample solutions.
TLC method for the determination of neomycin,
spectrophotometric method was performed (26). RESULTS AND DISCUSSION
The method was validated in accordance with ICH
guidelines (27). The obtained results were used for Reaction of free amine groups with DBS was
statistical evaluation. used for the development of a new TLC method for
the determination of neomycin in pharmaceutical
Formation of neomycin derivative with dabsyl chlo- preparation. In the first stage of studies, conditions
ride for the separation of NDC and DBS were estab-
A product of reaction was formed when 0.2 mL lished (Fig. 1).
of neomycin solution (0.6100 mg/mL) in water was The mobile phase n-butanol ñ 2-butanone ñ
mixed with 1.2 mL of DBS solution (0.3238 mg/mL) 25% ammonia ñ water (10 : 6 : 2 : 2, v/v/v/v)
and 0.2 mL of carbonate buffer (pH = 9.0), heated up enables good resolution of analyzed substances.
at 70OC for 15 min, then cooled down to 25OC, 0.5 NDC on TLC chromatogram appeared as a compact
mL of distilled water added and adjusted with ace- yellow band, which contrasts with the white back-
tone to the final volume of 5.0 mL. A reference mate- ground of the plate and its RF value was 0.22. The
rial containing identical volume of carbonate buffer NDC bands were stable after application to the plate
(pH = 9.00) and DBS concentration as in the studied for 24 h. Next to the NDC, band of DBS (RF 0.53)
sample was prepared for each individual sample. appeared in chromatogram and didnít interfere with
Absorbance at 492 nm was measured by use of a the band of tested complex (Fig. 2).
UV/Vis spectrophotometer (Varian Cary 100 Conc.) The application of densitometric detection
in relation to appropriate reference material. demonstrated that peaks in the chromatograms were
well resolved, symmetrical and easy to identify and
Determination of neomycin in pharmaceutical determine. Registration of peak areas for NDC was
preparation carried out at λ = 460 nm. The analytical wavelength
Determination of neomycin in tested tablets chosen for densitometric registration corresponded
after formation of neomycin derivative with dabsyl to absorption maximum for NDC.

Figure 1. Scheme of synthesis of neomycin derivative

 Determination of neomycin in the form of neomycin derivative... 35

Figure 2. Densitogram of neomycin derivative with dabsyl chloride (NDC) and dabsyl chloride (DBS)

Figure 3. Linear relationship between peak area and concentration of NDC.

In the available literature there were some For estimation of reliability of the developed
papers describing the determination of neomycin by method according to ICH recommendations, the fol-
thin-layer chromatography, where densitometric lowing parameters were determined: specificity, lin-
detection of neomycin was performed after previ- earity, limits of detection and quantitation, recovery
ously spraying with a ninhydrin solution (14, 15). and robustness (27).
Application of ninhydrin required complete evapo- The developed method was specific against the
ration of ammonia (mobile phase component) from studied components. There are no peaks in chro-
the stationary phase by heating plates at 100OC for matogram recorded for blank solution, chro-
about 1.5 h. The research presented in this paper matograms of NDC obtained for standard solutions
revealed that the developed method for the determi- and for preparation solution and blank chro-
nation of neomycin in the form of color derivative matogram where studied components occur. Good
with DBS may be an alternative to time consuming correlation between VIS spectra acquired from the
method using ninhydrin solution for the visualiza- standard and the pharmaceutical preparation indicat-
tion of chromatograms. ed that NDC spot was free of any interference that
36 URSZULA HUBICKA et al.

Table 1. The results of validation of TLC and spectrophotometric method.

Parameter TLC ñ densitometry VIS spectrophotometry

RF 0.20 ± 0.03 ñ
Limit of detection 0.1953 µg per band 1.48∑10-3 mg/mL
Limit of quantitation 0.59 µg per band 4.48∑10-3 mg/mL
Linearity range 0.59 ñ 2.20 µg per band 4.48∑10-3 ñ 2.46∑10-2 mg/mL
Regression coefficients a = 534.28 a = 23.673
P = a c + b ± Se b = 69.667 ± 55.29 b = ñ0.0357 ± 0.01679
Standard deviation of the Sa = 37.52 Sa = 0.709
regression coefficients Sb = 51.51 Sb = 0.0106
Correlation coefficient, r r = 0.9879 r = 0.9969
Determination coefficient r 2
r = 0.9759
2
r2 = 0.9938
Precision n = 5
level 50%: xm = 263.68 RSD = 1.70% xm = 95.94% RSD = 2.92%
level 100%: xm = 637.84 RSD = 1.44% xm = 96.75% RSD = 2.23%
level 150%: xm = 1026.26 RSD = 1.17% xm = 103.26% RSD = 1.89%
Intermediate precision n = 5
level 50%: xm =228.10 RSD = 2.05% xm = 100.22% RSD = 3.38%
level 100%: xm =743.42 RSD = 2.01% xm = 99.98% RSD = 3.30%
level 150%: xm =1062.44 RSD = 1.27% xm = 99.60% RSD = 2.04%
Recovery, (%) n = 3
level 80%: 101.48% RSD = 2.65% 105.31% RSD = 0.89%
level 100%: 101.26% RSD = 2.07% 97.29% RSD = 2.11%
level 120%: 100.97% RSD= 2.40% 97.75% RSD = 1.74%
P ñ peak area; c ñ concentration [µg per band]; a and b ñ regression coefficients, Se ñ standard error of the esti-
mate, Sa ñ standard deviation of the slope a, Sb ñ standard deviation of the intercept, xm ñ arithmetic mean; RSD
ñ relative standard deviation.

Table 2. The results of neomycin determination in pharmaceutical preparation with statistical evaluation.

Determined concentration
Declared
Preparation concentration Spectrophotometric
TLC method
method
xm = 253.15 xm = 254.70
SD = 5.2908 SD = 2.5145
Neomycinum RSD = 2.09% RSD = 2.47%
250 mg
(tablets) Fisher-Snedecor test:
Fobs = 4.43; F1-α = 5.05 for f = 5, α = 0.05
Fobs < F1-α statistically insignificant

f ñ number of degrees of freedom, α ñ significance level, SD ñ standard deviation, RSD ñ relative standard deviation, Fobs ñ calculated
experimental value; F1-α ñ critical value from the Snedecor law table.

might be present in the analysis. Resolution of the The regression equation of the calibration plot, val-
peaks appearing in the chromatograms was 3.94. ues of standard deviation of the slope (Sa), standard
A plot of concentration against mean peak area deviation of the intercept (Sb) and standard error of
of NDC was linear over the range from 0.5918 µg the estimate (Se) are presented in Table 1.
per band to 2.1960 µg per band. The correlation Based on parameters of the curve (P = ñ25.48
coefficient (r) and determination coefficient (r2) + 650.27C, r = 0.9846 and Se = 38.48), in low range
obtained for significance level 0.05 and n = 7 were of concentrations, the LOD and LOQ (µg per band)
close to 0.99 that proved a highly significant linear values were 0.1953 and 0.5918, respectively. These
correlation (Fig. 3). The y-intercept of the linear low values indicated satisfactory sensitivity of the
equation for NDC was statistically insignificant. method.
 Determination of neomycin in the form of neomycin derivative... 37

Accuracy of the method expressed as % recov- 5. Amin A.S., Issa Y.M.: Spectrochim. Acta A 59,
ery at three concentration levels was from 100.97 to 663 (2003).
101.48%. Good precision and intermediate precision 6. El-Shabrawy V.: Spectrosc. Lett. 35, 99 (2002).
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results are presented in Table 1. In all the deliberate- Lopes J.: Vib. Spectrosc. 56, 184 (2011).
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The usefulness of the method was examined by Anal. Lett. 23, 1005 (1990).
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no influence of additional components present in Pharm. Biomed. Anal. 36, 751 (2004).
tested preparation such as macrogol, sodium starch 11. Adams E., Schepers R., Gathu L.W., Kibaya R.,
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and the components of the substrate on the determi- Anal. 15, 505 (1997).
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determination were obtained, which were character- Chromatogr. A 1057, 125 (2004).
ized by good repeatability of measurements (RSD = 13. Hanko V.P., Rohrer J.S.: J. Pharm. Biomed.
2.09%). The concentration determined by the devel- Anal. 43, 131 (2007).
oped method was comparable to the results obtained 14. Krzek J., Starek M., KwiecieÒ A., Rzeszutko
by spectrophotometric method (Table 2). W.: J. Pharm. Biomed. Anal. 24, 629 (2001).
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obtained by TLC and spectrophotometric methods B.: J. AOAC Int. 92, 1068 (2009).
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(Table 2). The calculated experimental value Fobs for Pap. 63, 255 (2009).
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