Gentamicin Sulphate: A Current Review of Analytical Methods
Priyanka S. Malani 1*, Dr.Hasumati A. Raj1, Dr.Vinit C. Jain1, Bhagyashree M. Patel1
1
Shree Dhanvantary Pharmacy College,Kim, Surat, Gujarat
*
Email: priyankamalani2000@gmail.com
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
Abstract:
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 Gramnegative 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
assay
1. Introduction:
Gentamicin [(3R,4R,5R)-2{[(1S,2S,3R,4S,6R)-4,6-diamino-3-{[(2R,3R,6S)-3-amino-6-[(1R)
1(methylamino)ethyl]oxan-2-yl]oxy}-2hydroxycyclohexyl]oxy}5-methyl-(methylamino)oxane3,5 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.
2. METHODS FOR THE DETERMINATION OF GENTAMICIN
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 cleanup, 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 μL of
isopropanol and 160 μL 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 60°C ± 3°C 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
analysis.
Table-1: Summary of Compendial methods:
PHARMACOPOEIA METHOD
IP(1)
MICROBIOLOGICAL ASSAY
GEL CLOT LIMIT TEST METHOD
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
Median dose
Incubation temp(ºC)
B.P(2)
Phosphate buffer pH 8.0
0.1 μg
36 - 37.5
MICROBIOLOGICAL ASSAY
STOCK SOLUTION
Solvent
Stock Soln Conc.
Water
1 mg/ml
Test dilution
Final dilution
Median dose
Water
0.1 mcg
USP(3)
MICROBIOLOGICAL ASSAY
CYLINDER-PLATE METHOD
STOCK SOLUTION
Solvent
Stock Soln Conc
Within Day
TEST SOLUTION
Final Conc
Phosphate Buffer
1 mg/ml
30 days
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
10−4% and LOQ = 5.04 F 1 0−4%).
Table-2: Summary of Spectrophotometry
Drug
Method
Gentamicin Third derivative
Spectrophotometry
Wave
length
281 nm
Calibratio
n range
004% to
0.008%.
Recovery
99.38%
100.16%
Reference
to 12
d) Chromatographic Methods:
The High-pressure Liquid Chromatography (HPLC):
Determination of Gentamicin in urine samples after inhalation by Reversed-Phase Highperformance Liquid Chromatography using pre-column derivatisation with ophthalaldehyde 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 1fluoro-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
Gentamicin in RP-HPLC
urine samples METHOD
after
inhalation by
Microdetermi
nation
of
Gentamicin in
serum
Gentamicin
Released from
Orthopedic
Carrier
System
Gentamicin
Sulfate
and
Leucine From
a Novel Dry
Powder For
Inhalation
Gentamicin in
hospital
wastewater
Mobile phase
Stationar
y phase
Methanol–glacial
C18colu
Acetic Acid–water mn
(800:20:180, V/V),
Contained
0.02 M Sodium
Heptanesulfonic
Acid, pH 3.4
Wavelengt
h
Refer
ence
Fluoresce 13
nce
Detection
(Excitatio
n 340 nm,
Emission
418 nm)
HighPerformance
Liquid
Chromatography
with Ultraviolet
detection
Novel HPLC
Methanol-WaterAcetate Buffer
C18 reve
rsedphase
column
C18colu
mn
16
HPLC Assay
Sodium 1-hepta
suiphonate
monohydrate
And glacial acetic
acid
C18column
10
Methanol, Water
and 20
Mmol/L Heptafluor
o butyric Acid
Solution
 Heptafluoro
butyric
C18column
9
Pentaflu
orophen
15
liquid
chromatography
–electrospraytandem
mass
spectrometry
Gentamicin
RP
HPLC
sulfate and its Method using a
365 nm
14
related
substances in
a
pharmaceutica
l cream
short
pentafluorophen
yl column and a
Charged Aerosol
Detector

Acid:
Water:Acet
onitrile
(0.025:95:5,
v/v/v)
Trifluoroace
tic acid:
Water:
Acetonitrile
(1:95:5,
v/v/v).
yl
column
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 crossreactivity 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]
3. CONCLUSION:
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 highpressure 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 it’s done with different
chemical such as o-phthalaldehyde and 1-fluoro-2, 4-dinitrobenzene and detection of it’s 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.
4. REFERENCE:
1) Indian Pharmacopoeia, the Indian Pharmacopoeia Commission, Ghaziabad, Govt. of
India Ministry of Health and Family Welfare, Volume II, P.P-536, 2007
2) British Pharmacopoeia 2009, The Department of Health, London, The Stationary
Office;2008, vol-2,P.P-8374, 2008.
3) The United States Pharmacopoeia (USP 30 NF 25) Asian Edition, Rockville, MD: USP
Convention Rockville, MD, USP Convention Inc, P.P- 2219, 2007.
4) Gentamicin sulphate http://www.drugbank.ca/drugs/DB00798
5) Neil MJ, Smith A, “The Merck index”, 13thedition, published by Merck reasearch
laboratory,P.P-4406, 2001.
6) Gentamicin spectrum of bacterial susceptibility and Resistance". Retrieved 15 May 201
7) Finkel R, Cubeddu L, Michelle C. “lippincott lllustrated review”,pharmacology,
edition, Wolters Kluwer Publication, P.P - 411,374.
4th
8) Tripathi KD, “Essentials of Medical Pharmacology’’, 6thedition, Jaypee Brother medical
Publisher(P) Ltd; P.P-721,2001.
9) Kuehl PJ, et al. Development and Validation of an HPLC Assay for Dual Detection of
Gentamicin Sulfate and Leucine from a Novel Dry Powder for Inhalation. Analytical &
Bioanalytical Techniques, 3(6): 100-152, 2012
10) Löffler D, Ternes TA. Analytical method for the determination of the aminoglycoside
Gentamicin in Hospital Wastewater via Liquid Chromatography-Electrospray-Tandem
Mass Spectrometry. Journal of Chromatography, 1000 (1-2): 583-8, 2003.
11) Jin Yong, Jang Jin-Wook, Chang-Hoon Han ,and Mun-Han Lee. Development of ELISA
and Immunochromatographic Assay for the Detection of Gentamicin. J. Agric. Food
Chem., 53 (20): 7639–7643, 2005.
12) Krzek,Hubicka.Determination of Gentamicin Sulphate in Injection
Solutions by Derivative Spectrophotometry. Analytical Letters, 42(3): 473-482, 2009.
13) Amoud AI, Clark BJ, Chrystyn H. Determination of Gentamicin in urine samples after
inhalation by Reversed-Phase High-Performance Liquid Chromatography using precolumn derivatisation with o-phthalaldehyde, J Chromatogr B Analyt Technol Biomed
Life Sci., 769(1): 89-95, 2002.
14) Barends DM, van der Sandt JS, Hulshoff A. Micro determination of Gentamicin in serum
by High-Performance Liquid Chromatography with Ultraviolet detection. Journal of
Chromatography, 182(2):201-10,1980
15) Joseph A, Rustum A. Development and Validation of a RP-HPLC method for the
determination of Gentamicin sulfate and its related substances in a Pharmaceutical cream
using a short Pentafluorophenyl column and a Charge Aerosol Detector. Journal of
Pharmaceutical Biomedical Analysis, 5, 51(3): 521-31, 2010.
16) Laki M,et al. Determination of Gentamicin Released from Orthopedic Carrier System by
a Novel HPLC Method. Journal of Chromatographic Science, 492011, 2011.
17) Jin Yong, Jang Jin-Wook, Chang-Hoon Han, and Mun-Han Lee. Development of ELISA
and Immunochromatographic Assay for the Detection of Gentamicin. J. Agric. Food
Chem., 53 (20):7639–7643, 2005.
18) Kolosova AY, Blintsov AN, Samsonova JV. Development of an Enzyme-Linked
Immunosorbent Assay for Gentamicin in human blood serum. Fresenius' Journal of
Analytical Chemistry, 361(3):329-330, 1998.
19) Ghinami C.et al.,Electrochemical Detection of Tobramycin or Gentamicin according to
the European Pharmacopoeia Analytical Method. Journal of Chromatography,
1139(1):53-6, 2007.