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

 

 

Table 2. Validation report for HPLC method for the determination of EDTA:

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.

 

EXPERIMENTAL:

Several conventional and chromatographic methods are reported for the determination of EDTA in water and wastewater [6-14]. In presented method ion pair reagent is used with copper sulphate a metal ion for the determination of EDTA when EDTA reacts with metal ion present in mobile phase it gets stabilized and dose not react with the metallic part of the instrument and column. In absence of metal ion EDTA reacts with the metallic part on instrument and column, gets distributed in a larger part of mobile phase and dose not appear as a single peak.

 

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.

 

RESULT AND DISCUSSION:

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).

 

Linearity Different concentration solution from 0.6µg/ml to 3.0µg/ml of EDTA was prepared in diluent and linear regression was driven (Table2).

 

Precision Intra-day and inter-day precision of method was studied by spiking standard EDTA solution in samples and % rsd of results was calculated (Table2).

 

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)

 

CONCLUSION:

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     

© A & V Publication all right reserved

Research Journal of Pharmaceutical Dosage Forms and Technology. 1(2): Sept.-Oct. 2009, 116-118