A Validated Method
for the Estimation of EDTA in Drug Substances and their Intermediates by using
Reversed Phase High Performance Liquid Chromatography
Dharmendra Kumar Kushwah*, Prakash Yashwant Kohle, Bimal Kumar Srivastava, Manoj Parmar and Dhaval Mehta
CTX
Life sciences Pvt. Ltd., 251-252, Sachin Magdalla Road, Sachin, Surat, Gujarat-394230, India
ABSTRACT
Ethylenediaminetetraacetic acid (EDTA) is widely used for the different purposes in pharmaceutical (bulk
drug and formulations) industry; recent reports shows that EDTA exhibits low toxicity hence the
detremination of content of EDTA is of prime importance. The method has been developed for the determination of EDTA contents
accurately and precisely on HPLC using reversed phase C-18 HPLC column, by UV
at 300 nm wavelength for detection. EDTA was determined by using its chelating
property; mobile phase was mixed with a small amount of metal salt equivalent
to about 50-70µg per ml of metal ion. The method is validated for its
specificity, precision, accuracy, linearity, ruggedness and robustness. EDTA is
linear from 0.6µg/ml to 3.0µg/ml. Limit of quantitation
for EDTA is 0.60 µg/ml and Limit of Detection is
0.30µg/ml.
Keywords: EDTA, C-18 column,
Wavelength, Limit of quantitation, and Limit
of Detection.
INTRODUCTION
Ethylenediaminetetraacetic acid (EDTA) is an important industrial
chemical, is used as chelating agent in drug substances to remove metallic
impurities by bonding metal ions, which may come from
catalyst and raw material. Metal ions cause detrimental effects in several
industrial processes and in the formulation of many products. Earth alkaline
divalent cations such Ca, Mg and Ba
form insoluble precipitates with carbonates, sulfates and phosphates. In
addition, the presence of transition metal ions such as those of copper, iron,
zinc and manganese may trigger chemical processes of corrosion, catalytic
degradation, polymerization inhibition, redox
reactivity and changes in the coloring of products. In industrial processes
these metal ions may come from the process waters, raw materials, equipment
erosion and corrosion. They may also be added as a specific metal species, but
they may later suffer undesired alterations due to changes in concentration,
pH, oxidation, or reactions with other ingredients during the process. EDTA is
a chelate ligand with a
high affinity constant to form metal-EDTA complexes, being deliberately added
to sequester metal ions. EDTA makes a complex with such impurities and is
washed off from the product.
Some
characteristic of ligand which are generally
recognized as influencing the stability of its complexes are, its basic
strength, its chelating properties and steric
effects. The term chelate effect refers to the fact
that bidentate and multidentate
ligands are more stable than monodentate
ligand, greater the point of attachment of ligand to the metal ion greater the stability. The
stability constant on complex K; can be calculated as follows; [1]
Mn+ + Y4- = (MY)(n-4)+
K=
[(MY)(n-4)+]/[Mn+][Y4-]
Stability
constant (as log K) of Cu2+ is 18.8, Mg2+
is 8.7 and Ca2+ is 10.7 [1], copper is
preferred over other available metal ions because of its higher stability
constant.
Table 1. Gradient program:
Time (minutes) |
Buffer solution (%) |
Acetonitrile (%) |
0 |
92 |
8 |
10 |
92 |
8 |
15 |
80 |
20 |
20 |
60 |
40 |
22 |
92 |
8 |
30 |
92 |
8 |
Parameters
|
Result
|
Linearity
Regression
parameters
Slope
Intercept
Correlation Coefficient (r)
Precision
± %RSD
Intra-day (n=6)
Inter-day (n=6)
Ruggedness (n=12)
Accuracy (n=9)
Limit of quantitation
Limit of Detection
|
0.6 –3.0 µg/ml
9461
-1451
0.99709
99.51µg/g±3.95%
100.13µg/g ±4.50%
99.82µg/g ±4.05
102.75%±4.94%
0.60µg/ml
0.30µg/ml
|
EDTA is such
widespread use that it has emerged as a persistent organic pollutant[2].
On degradation EDTA produces ethylenediaminetriacetic
acid, which then cyclizes. EDTA exhibits low toxicity with LD50 (rat) of 2.0 – 2.2
g/kg[3].It has been found to be both cytotoxic and weak genotoxic in
laboratory animals. Oral exposures have been noted to cause reproductive and
developmental effects, Free EDTA has been shown to produce adverse
reproductive and developmental effects in mammals. They trace
the effects of chronic exposure to low levels of EDTA (< 100 mM) in cultured cells of rat kidney, resulting in high
rates of cellular death. In addition, Gabard [4] reported inhibition of DNA, RNA and protein synthesis due to the chelation of zinc and manganese in rat liver cells after
EDTA-Ca (II) administration.
Instrumentation Waters
2695 Separation Module with PDA detector with Empower 2 software, Shimadzu
LC-2010 with PDA detector and LC solution software and Mettler
Toledo XS 205 analytical balance. Kromasil C18 (250 x
4.6) mm, 5µm HPLC column was used for separation and quantification
during method development and validation.
Figure 2. EDTA metal
complex
Figure 3.
Representative chromatogram of EDTA
Materials
and reagent: Ethylenediaminetetraacetic acid disodium salt dihydrate
AR grade Merck, Sodium acetate anhydrous (LR grade), Tetrabutylammonium
bromide (LR grade), Copper sulfate (II) penthydrate
(GR grade), Glacial acetic acid (HPLC grade), Acetonitrile
(HPLC grade), Purified water (HPLC grade or equivalent)
Preparation of diluent Mix water
and Acetonitrile in the ratio of (50:50).
Preparation of
standard solution Prepare a solution in diluent
having known concentration of EDTA disodium salt equivalent to EDTA about 0.002
mg per ml.
Sample
preparation Prepare a solution of test
sample in diluent having a known concentration of
sample about 20mg per ml.
Chromatographic conditions
Gradient
Mode is used for the determination of EDTA, detection wavelength 300 nm, and
Flow rate 1.0 ml/min, Injection volume 20ml, Column oven temp. 30°C and Run time 30 min. Buffer
solution 6.5 g of tetrabutylammonium bromide, 4.1 g
of sodium acetate anhydrous, 250 mg of copper sulfate (II) penthydrate
and 2.0 ml of glacial acetic acid in 1 litter of water. Acetonitrile
is used as organic phase.
In initial method
development EDTA solution in diluent water: Acetonitrile (50:50) was injected in the chromatograph
without metal ion in mobile phase, no prominent peak of EDTA was observed. Upon
addition of copper (II) ion about 65 µg/ml in mobile phase the EDTA forms a
complex and gets stabilized and appears as a single peak at RT about 10 min.
Chromatograms were extracted at 210nm and 300 nm, 300nm is finalized as
wavelength of detection because less noise at this wavelength instead of 210nm.
To optimize HPLC method different composition of mobile phases, pH, gradient
and columns were studied. A satisfactory separation was obtained with a mobile
phase containing Buffer solution 6.5 g of tetrabutylammonium
bromide, 4.1 g of sodium acetate anhydrous, 250 mg of copper sulfate (II) penthydrate and 2.0 ml of glacial acetic acid in 1 litre of water with the gradient elution (Table 1).
Accuracy of the
method was studied by recovery and amount recovered was calculated (Table2).
Accuracy of method was performed on 6-chloro-1,1-dioxo-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide.
Specificity of the
method was determined by calculating the peak purity of EDTA peak in spiked
sample, EDTA peak is pure.
Ruggedness of
the method is verified by injecting spiked sample solutions, spiked with
100µg/g of EDTA, the results are indicating that the method is rugged for
analyst-to-analyst, instrument-to instrument, column-to-column and day-to-day
(Table 2)
The
presented work describes that developed HPLC method is specific, Rugged,
robust, linear accurate, precise, and rugged. The method can be applied for the
quantification of EDTA in a wide range of drug substances and their
intermediates in the present form or after small modifications.
ACKNOLEDGEMENT:
We are highly thankful to
the CTX Life Sciences management for granting permission for the publication of
this work.
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Received on
08.06.2009
Accepted on
16.08.2009
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Publication all right reserved
Research
Journal of Pharmaceutical Dosage Forms and Technology. 1(2): Sept.-Oct. 2009,
116-118