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Gentamicin Sulphate: A Current Review of Analytical Methods

Priyanka S. Malani 1*, Dr.Hasumati A. Raj1, Dr.Vinit C. Jain1, Bhagyashree M. Patel1

Shree Dhanvantary Pharmacy College,Kim, Surat, Gujarat


Address For correspondence:

Department of Quality Assurance

Shree Dhanvantary Pharmacy College

Near Railway Station, Kudsad Road.

At: Kim, Taluka: Olpad, Dist: Surat, Pin code: 394110

Mobile No: 9924860850

Number of Figures: 2

Number of Tables: 3

Gentamicin is a broad-spectrum amino glycoside antibiotics drug which is available in the
different pharmaceutical dosage forms through various routes of administration, such as oral,
topical, systemic and ophthalmic. The drug is a broad spectrum amino glycoside antibiotic and
used to treat many types of bacterial infections, particularly those caused by Gram-
negative organisms infections. This article reviews the current analytical methods for
identification and quantitative determination of Gentamicin in samples. The clinical and
pharmaceutical analysis of this drug requires effective analytical procedures for quality control
and pharmacodynamic and pharmacokinetic studies as well as stability study. An extensive
survey of the literature published in various analytical and pharmaceutical chemistry related
journals has been conducted and the instrumental analytical methods which were developed
and used for determination of Gentamicin as single or combination with other drugs in bulk
drugs, formulations and biological fluids have been reviewed. This review covers the time
period from 1980 to 2012 during many analytical methods including Enzyme-Linked
Immunosorbent Assay, Immunochromatographic Assay, spectrophotometric methods like
UV and derivative; and chromatographic method including HPLC and alternative method like
Electrochemical Detection method were reported. The application of these methods for the
determination of Gentamicin in pharmaceutical formulations and biological samples has also
been discussed.

Keyword: Gentamicin, Analytical method, Spectrophotometry, Chromatography, Microbiological


1. Introduction:

Gentamicin [(3R,4R,5R)-2{[(1S,2S,3R,4S,6R)-4,6-diamino-3-{[(2R,3R,6S)-3-amino-6-[(1R)
3,5 diol ] is a lipophilic aminogiycoside derivative appears as white to off white crystalline
powder (Figure 1). The drug is freely soluble in Water, insoluble in Methanol.1, 2, 3 pKa4 values of
Gentamicin sulphate in strongest acidic condition is 12.55 and in basic condition 10.18.
Gentamicin sulphate melts at 218-237 C 5
Figure 1: chemical structures of Gentamicin.
It is a broad-spectrum amino glycoside antibiotic that has been shown to be efficient in the
treatment of human bacterial infections. The drug is active against Infections caused by
staphylococci, pseudomonas, klebsiella, enterobacter & serratia. Active against a wide range of
human bacterial infections, mostly Gram-negative bacteria including Pseudomonas,
Proteus, Serratia, and the Gram-positive Staphylococcus.[6,7] The therapeutic and pharmacologic
action of Gentamicin in the treatment of Meningitis, Endocarditis, Urinary Tract Infections,
Otitis & Ocular infections, Infections of burns & Skin ulcers. [8] pharmaceutical dosage forms are
cream, powder, eye drops.

Gentamicin is a broad spectrum aminoglycoside antibiotic which acts by binding to the bacterial
30s ribosomal subunit, causing misreading of t-RNA leaving the bacterium unable to synthesize
proteins vital to its growth. [7, 8]

Figure 2: Mechanism of Gentamicin.

The use of the Gentamicin as a drug essential in pharmaceutical formulations highlights the
requirement for its determination and quantification with appropriate analytical methods. This
paper gives an overview of the analytical techniques that are available and nowadays have been
used for determination of Gentamicin in pharmaceutical and biological samples.


The analytical methods that are currently used for determination of Gentamicin in
Pharmaceutical samples (Cream, Ointment, Powder, eye preparation, and oral solution) and
Biological samples (plasma, serum, urine, saliva, tissues of lung, liver, muscles) are:
antimicrobial assay, Enzyme Linked Immunosorbent Assay, Immunochromatographic
Assay, spectrophotometric methods like uv and derivative; and chromatographic method
including HPLC and alternative method like Electrochemical Detection. The analytical methods
which are already published usually require sample preparation, including extraction and clean-
up, as well as the subsequent Instrumental determination of Gentamicin from the matrixes with
the other azole derivatives. The Developed method for determination of the Gentamicin should
be Selective, Sensitive and Reproducible, with Less Consumption of solvent and time for
analysis, as well as they should be Validated in order to prove that the Developed procedure is
suitable for intended analytical purpose and give accurate results.

a) Sample Procedures:

A number of Extraction techniques are available for isolation of the Gentamicin

from the pharmaceutical and biological matrixes.

All samples were derivatized prior to analysis using the following method: 440 L of
isopropanol and 160 L of Reagent 2 (1.0 g Phthaldialdehyde in 5 mL Methanol, 95
mL Reagent 1 [0.4 M Boric acid adjusted to pH 10.4 using 8 N Potassium hydroxide]
and 2 mL Thioglycolic acid, the resulting solution was adjusted to pH 10.4 using 8 N
Potassium hydroxide) were added to each sample (0.4 mL sample in 2 mL auto sampler
vial). Each sample was then vortexed for 10 seconds 1 second and heated in an oven
at 60C 3C for 15 minutes 1 minute. The samples were allowed to return to room
temperature prior to analysis. [9]

Aminoglycoside Gentamicin in hospital wastewater via liquid chromatography

electrospray-tandem mass spectrometry by solid-phase extraction (SPE) procedure, this
method is perfomed on weak cation exchanger; Filteration is avoided to loss of
Gentamicin sulphate. [10]

Different method of sample preparation in ELISA and in immunoassay in different

biological fluids such as (plasma, milk) [11]

b) Compendial Method:

Gentamicin is official in Indian pharmacopoeia, British Pharmacopoeia and United

State Pharmacopoeia and for that Microbiological assay is described for quantitative
Table-1: Summary of Compendial methods:

Standard Stock Solution
Assay method Gel Clot Limit Test Method
Prior drying Yes
Initial solvent Phosphate buffer pH 8.0
Final Stock conc 1mg
Use before no of days 30 days

Test Dilution
Final dilution Phosphate buffer pH 8.0
Median dose 0.1 g
Incubation temp(C) 36 - 37.5


Solvent Water
Stock Soln Conc. 1 mg/ml

Test dilution
Final dilution Water
Median dose 0.1 mcg

Solvent Phosphate Buffer
Stock Soln Conc 1 mg/ml
Within Day 30 days
Final Conc 0.1 mcg
c) Spectrophotometric method:

Derivative Spectrophotometry

A new derivative spectrophotometric method was developed for determination of

Gentamicin beside methyl and propyl hydroxy benzoates in injection solutions. The
determination was carried out after modify ing the Gentamicin molecule by reaction with
o-phthalaldehyde. The obtained spectrum of product in methanol solution was converted
into a third-derivative spectrum. The measurements were made at wavelength(281 nm),
where a linear relationship between D3 and antibiotic concentration occurs, when no
coexisting constituents are present. The method is of high specificity to Gentamicin in the
presence of methyl and propyl hydroxy benzoates and has good accuracy. The recovery is
from 99.38% to 100.16%, and wide linearity ranges from 0.004% to 0.008%. The method
has satisfactory precision (RSD = 2.52%) as well as high sensitivity (LOD = 1 .66 F
104% and LOQ = 5.04 F 1 04%).

Table-2: Summary of Spectrophotometry

Drug Method Wave Calibratio Recovery Reference

length n range
Gentamicin Third derivative 281 nm 004% to 99.38% to 12
Spectrophotometry 0.008%. 100.16%

d) Chromatographic Methods:

The High-pressure Liquid Chromatography (HPLC):

Determination of Gentamicin in urine samples after inhalation by Reversed-Phase High-

performance Liquid Chromatography using pre-column derivatisation with o-
phthalaldehyde for Gentamicin and Netilmicin (internal standard) were extracted from
urine using C18 solid-phase extraction cartridges (94.3% recovery) and then derivatised
with o-phthalaldehyde and 3-mercaptopropionic acid. The derivative was stable for
>6 h [13]

Micro determination of Gentamicin in serum by High-Performance Liquid

Chromatography with ultraviolet detection, here The serum proteins are precipitated with
Acetonitrile and the Gentamicin components in the supernatant are derivatized with 1-
fluoro-2, 4-dinitrobenzene [14]

A reversed-phase high performance liquid chromatographic (RP-HPLC) method has

been developed and validated to determine the composition of Gentamicin sulfate and to
estimate its related substances (without any pre- or post-column derivatization) in a
pharmaceutical cream. As Gentamicin has a weak UV chromophore, it is not possible to
detect low levels of known and unknown related substances of Gentamicin using a UV
detector. In this method, a Charged Aerosol Detector (CAD) was used to obtain high
sensitivity that was necessary for the intended purpose of the method. This method can
separate all the analogues of Gentamicin including all known and unknown related
substances of the API. [15]

Table-3: Summary of Chromatographic Methods

Drug Method Mobile phase Stationar Wavelengt Refer

y phase h ence

Gentamicin in RP-HPLC Methanolglacial C18colu Fluoresce 13

urine samples METHOD Acetic Acidwater mn nce
after (800:20:180, V/V), Detection
inhalation by Contained (Excitatio
0.02 M Sodium n 340 nm,
Heptanesulfonic Emission
Acid, pH 3.4 418 nm)

Microdetermi High- C18 rever 365 nm 14

nation of Performance sed-
Gentamicin in Liquid phase
serum Chromatography column
with Ultraviolet
Gentamicin Novel HPLC Methanol-Water- C18colu 16
Released from Acetate Buffer mn
Gentamicin HPLC Assay Sodium 1-hepta C18- 10
Sulfate and suiphonate column
Leucine From monohydrate
a Novel Dry And glacial acetic
Powder For acid
Gentamicin in liquid Methanol, Water C18- 9
hospital chromatography and 20 column
wastewater electrospray- Mmol/L Heptafluor
tandem mass o butyric Acid
spectrometry Solution
Gentamicin RP HPLC Heptafluoro Pentaflu 15
sulfate and its Method using a butyric orophen
related short Acid: yl
substances in pentafluorophen Water:Aceto column
a yl column and a nitrile
pharmaceutica Charged Aerosol (0.025:95:5,
l cream Detector v/v/v)
tic acid:

e) Alternative Methods:

I. ELISA and Immunochromatographic Assay

Competitive direct Enzyme-Linked Immunosorbent Assay and the

Immunochromatographic assay were developed using a monoclonal
antibody to detect Gentamicin in the animal plasma and milk. No cross-
reactivity of the antibody was observed with other Aminoglycosides based
on competitive direct ELISA, indicating that the antibody is highly
specific for Gentamicin. On the basis of the standard curves, the detection
limits were determined to be 0.9 ng/ml in Phosphate-Buffered Saline
(PBS), 1.0 ng/ml in plasma, and 0.5 ng/ml in milk, respectively.[16]

An Enzyme-Linked Immunosorbent Assay (ELISA) for the quantitative

detection of Gentamicin in human blood serum was developed.
Peculiarities of the adsorption on the micro titer plate Surface of the
Gentamicin-protein conjugate were investigated.. The method permits
Gentamicin concentrations to be determined in human blood serum,
diluted 1/1000, in the linear range from 1 to 30 ng/ml. The assay is
characterized by high sensitivity(0.5 ng/ml), good reproducibility (CV <
12%) and good correlation with PFIA (r2 =0.943)[17]

II. Electrochemical detection:

Tobramycin and Gentamicin are two Aminoglycosidic antibiotics used in
lung infection, ophthalmic treatments as well as in skin infections.
Pharmaceutical companies which produce remedies containing
Tobramycin and Gentamicin need an analytical method for their internal
quality control. For several years a simple chromatographic method based
on anion exchange separation coupled with Amperometric detection was
proposed for aminoglycosides. This analytical approach was partially used
in the last edition of the European Pharmacopoeia (EP) for Tobramycin
and Gentamicin analysis. In fact they use integrated pulsed Amperometric
detection (IPAD) on a gold electrode while the separation is obtained on a
polymeric wide pore reversed phase instead of anion exchange in alkaline
conditions. Such coupling seems to be cumbersome and not so easy to
realize and to reproduce from one laboratory to another. Besides, the
described method lacks some of the details as important as the waveform
steps duration. Unfortunately the quality control (QC) laboratories have to
use exactly the method described in the EP, so they complained about the
troubles. Therefore, the EP authors published recently a paper regarding
the guidelines for good practice in the method application, but the
suggestion was not yet resolute. In our work we evaluated the eluent
composition and the kind of Amperometric cell, work electrode diameter
and cell volume. Mainly we optimized the Amperometric waveform. In
addition, for Tobramycin analysis another chromatographic phase was
explored in order to achieve better efficiency and to separate all the
impurities confirming the effectiveness of the detection. The conditions
described in the paper seem to allow the analyst to operate in conformity
with the EP method.[18]


Presented systematic review covers the current analytical methods for the determination of
Gentamicin in pharmaceutical and biological samples. The limitation of the reported methods
requires developing new optimized method which would be suitable for intended analytical
purpose for analyzing the content of Gentamicin in pharmaceutical, as well as in biological
samples. The new trends and advances for quantification of Gentamicin are based on using high-
pressure liquid chromatography which is widely available and flexible method with the ability
of coupling with mass spectrometry. The HPLC method could be automated; there are different
column fillings; different solvents with different polarity as mobile phases and different
detection modes. As Gentamicin has a weak UV chromophore derivation of its done with
different chemical such as o-phthalaldehyde and 1-fluoro-2, 4-dinitrobenzene and detection of
its done with UV detector. As Gentamicin has a weak UV chromophore, it is not possible to
detect low levels of known and unknown related substances of Gentamicin using a UV detector.
so using, a Charged Aerosol Detector (CAD) was used to obtain high sensitivity that was
necessary for the intended purpose of the method. For High sensitive ELISA and
Immunochromatographic Assay in different biological fluid such as milk, blood, serum and in
the plasma.


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