© 2017 IJSRSET | Volume 3 | Issue 8 | Print ISSN: 2395-1990 | Online ISSN : 2394-4099

Themed Section : Engineering and Technology

A Sensitive Method for Determination of Residual EDTA in

Diuretic Drugs, Furosemide by Reversed Phase High
Performance Liquid Chromatography
Rohidas Gilbile*,1,2, Dr. Saleem Shaikh2, Dr. Ritu Vyas1
1
Department of Chemistry, Pacific Academy of Higher Education and Research University, Udaipur, Rajasthan, India
2
Ullmann Laboratories, Five Star Shendra ,MIDC Aurangabad, Maharashtra State, India.
*Corresponding author E-mail: [email protected]

ABSTRACT

A sensitive method for the determination of residual EDTA by high-performance liquid chromatographic method
with UV detection has been developed. The analysis was performed in gradient mode on a reversed phase C18
column, 150 mm x 4.6 mm x 5 m. Gradient mobile phase consisted of a 25mM tetrabutyl ammonium hydrogen
sulphate, in water as mobile phase A and acetonitrile as mobile phase B. The linear gradient conditions 0-7 minutes
2%B; 7-10 minutes 80%B; 10-15 minutes 80%B, 15-17 minutes 2%B and 17-32 minutes2%B was set for the
determination of residual EDTA . Chromatograms were recorded by using flow rate 1.0 mL/minute and the injection
volume was 25 µL. The detection wavelength was set at 258 nm. The proposed liquid chromatographic method was
successfully applied to the determination of residual EDTA in furosemide. A linear calibration graph was obtained
for 25% to 150%of specification limit (200 ppm) with a correlation coefficient of 0.9999 for EDTA. The
quantification limit for EDTA in furosemide drug substances was found to be 50 ppm. The recovery for EDTA was
found to be within the acceptance criteria (75-125%) from QL level to 150% of the specification limit. Limit of
quantitation was found to be 50ppm. The precision of the method at 100% level for EDTA was within the
acceptance criteria. The method developed in this study is sensitive and selective and can be applied to routine
quality control
Keywords : Routing, non-repudiation, Byzantine failure, MANET, Security, Authentication, Integrity, Non-
repudiation, Confidentiality, Key and Trust Management(KTM).

I. INTRODUCTION blood circulation problems such as intermittent

claudication and Raynaud's syndrome. Other
EDTA is a prescription medicine, given by injection intravenous uses include treatment of cancer,
into the vein (intravenously) or into the muscle rheumatoidarthritis, osteoarthritis, an eye condition
(intramuscularly). Intravenous EDTA is used to treat called macular degeneration, diabetes, Alzheimer's
lead poisoning and brain damage caused by lead disease, multiple sclerosis, Parkinson's disease, and skin
poisoning; to evaluate a patient's response to therapy for conditions.
suspected lead poisoning; to treat poisonings by
radioactive materials such as plutonium, thorium, EDTA is also used intramuscularly for lead poisoning
uranium, and strontium; for removing copper in patients and related brain damage. EDTA is sometimes used as
with Wilson’s disease; and for treating high levels of an ointment for skin irritations produced by metals such
calcium. EDTA is also used intravenously for heart and as chromium, nickel, and copper. Eye drops containing
blood vessel conditions including irregular heartbeat EDTA are used to treat calcium deposits in the eye.
due to exposure to chemicals called cardiac glycosides,
“hardening of the arteries” (atherosclerosis), chest pain In foods, EDTA bound to iron is used to “fortify” grain-
(angina), high blood pressure, high cholesterol, and based products such as breakfast cereals and cereal bars.

IJSRSET1738209 | Received : 20 Nov 2017 | Accepted : 18 Dec 2017 | November-December-2017 [(3) 8 : 720-727] 720
 EDTA is also used in calcium and sodium compounds EDTA is safe when used as a prescription medicine, as
to preserve food; and to promote the color, texture, and eye drops, and in small amounts as a preservative in
flavor of food [1]. foods however the safety of large amounts is unknown
[5]. EDTA can cause nausea, vomiting, diarrhea,
In manufacturing, EDTA is used in calcium and sodium headache, low blood pressure, skin problems, and fever.
compounds to improve stability in pharmaceutical Nebulizer solutions containing disodium EDTA as a
products, detergents, liquid soaps, shampoos, preservative can cause the breathing tubes to narrow in
agricultural chemical sprays, oil emulsion devices, some people with asthma. The size of the dose
contact lens cleaners and cosmetics. It is also used in determines the amount of the narrowing. EDTA might
certain blood collection tubes used by medical make heart rhythm problems worse. EDTA might
laboratories. interfere with blood sugar control because it can interact
with insulin. EDTA can decrease serum calcium levels,
Ethylenediminetetraacetic acid is a powerful chelating making hypocalcemia worse. EDTA can bind with
agent, forming stable complexes with most metal ions. potassium and increase the amount of potassium that is
EDTA is a chemical that binds and holds on to (chelates) flushed out in the urine. This might cause potassium
minerals and metals such as chromium, iron, lead, levels to drop too low, especially in people who have
mercury, copper, aluminum, nickel, zinc, calcium, low levels to begin with. EDTA can bind with
cobalt, manganese, and magnesium. When they are magnesium and increase the amount of magnesium that
bound, they can't have any effects on the body and they is flushed out in the urine. This might cause magnesium
are removed from the body. Due to its ability to levels to drop too low, especially in people who have
sequester metal ions, EDTA is widely used in medicine, low levels to begin with. EDTA might make liver
chemical industry, food technology, agriculture and disease worse.
pharmaceutical technology. EDTA in its disodium salt
or calcium disodium salt form is frequently used in EDTA can harm the kidney and might make kidney
pharmaceuticals because of its stability, compatibility disease worse. EDTA doses should be reduced in
and low toxicity. The most common use of EDTA in patients with kidney disease. There is some concern that
analytical chemistry is in complexometric titrations EDTA might increase the risk of seizure in people with
[2,3]. In analytical techniques EDTA is being used in epilepsy or in people who tend to have seizures. EDTA
the ligands for the complexation of metals, which can cause severe decreases in blood levels of calcium,
enables for the chromatographic separations [4]. and this can cause a seizure [6].

Administering EDTA intravenously and intramuscularly EDTA can decrease blood sugar. Insulin is also used to
is effective for treating lead poisoning and brain damage decrease blood sugar. By taking EDTA along with
caused by lead exposure. The calcium disodium form of insulin can cause serious decreases in your blood sugar.
EDTA is approved by the U.S. Food and Drug So closely monitoring of blood sugar is necessary and
Administration (FDA) for these uses. Treatment with accordingly the dose of insulin might need to be
calcium disodium EDTA improves symptoms of lead changed. As interaction of EDTA with insulin and
poisoning such as abdominal pain, fatigue, constipation, Warfarin is major so as per literature it is advisable not
and loss of appetite. It also seems to slow progression of to take in combination. Warfarin (Coumadin) is used to
kidney failure in patients who have had long-term lead slow blood clotting. EDTA has been reported to
poisoning. However, EDTA does not seem to be decrease the effectiveness of warfarin (Coumadin).
effective for diagnosing lead poisoning. Emergency Decreasing the effectiveness of warfarin (Coumadin)
treatment of life-threatening high calcium levels might increase the risk of clotting. It is unclear why this
(hypercalcemia), when given intravenously. The interaction might occur. Be sure to have your blood
disodium form of EDTA is approved by the FDA for checked regularly. The dose of your warfarin
this use, but healthcare providers generally prefer other (Coumadin) might need to be changed [7, 8].
methods of treatment that are less likely to cause kidney
side effects. Water pills (Diuretic drugs) interacts with EDTA: Large
amounts of EDTA can decrease potassium levels in the
body. "Water pills" can also decrease potassium in the

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 body. Taking EDTA along with "water pills" might isocratic for 8 min, the acetonitrile ratio finally being
decrease potassium in the body too much. returned to 2% in 1 min.

Some "water pills" that can deplete potassium include In this paper, the development and validation of a
chlorothiazide (Diuril), chlorthalidone (Thalitone), reproducible RP-HPLC method intended to quantify
furosemide (Lasix), hydrochlorothiazide (HCTZ, ethylenediaminetetraacetic acid content in Furosemide
HydroDiuril, Microzide), and others. Hence the caution for routine laboratory use are described, according to
shall be taken when it will be used in combination. International Conference of Harmonisation (ICH)
guidelines [24, 25].
Furosemide is a potent diuretic (water pill) that is used II. EXPERIMENTAL
to eliminate water and salt from the body. The onset of
action after injection is five minutes and the duration of 2.1 Instrumentation
diuresis is two hours. The diuretic effect of furosemide
can cause depletion of sodium, chloride, body water and HPLC analysis was carried out using a Agilent 1200
other minerals. EDTA is being used in the synthesis of Series HPLC system (Agilent, USA), which is equipped
Furosemide. Therefore the quantification of residual with a degasser, a quaternary pump, an auto injector, a
EDTA is essential as per regulatory requirement [9, 10, temperature controlled column compartment and photo
11]. diode array detector connected to EZ-Chrome software.

There are several analytical methods have been Agilent technologies USA was used. The column
proposed for the determination of EDTA in wide variety temperature was maintained at 25ºC. The standard and
of sample matrices. They include spectrophotometry samples solutions are analysed using gradient elution
[12], electrochemistry [13], differential pulse mode using 25mM tetra butyl ammonium hydrogen
polorography [14], voltametric determination [15], sulphate (A) and acetonitrile (B). The linear gradient
amperometry [16], capillary electrophoresis [17] and conditions were 0-7 minutes 2%B; 7-10 minutes 80%B;
chromatography [18, 19]. Among these HPLC (ion pair 10-15 minutes 80%B, 15-17 minutes 2%B and 17-32
or ion exchange retention mechanism) appear to be the minutes2%B. The flow rate was set at 1.0 mL/minute
prevailing techniques, despite the fact that EDTA lacks and the injection volume was 25 µL. The detection
volatility and exhibits low UV/visible absorptivity [20, wavelength was set at 258 nm.
21]. The gas chromatographic methods always include a
time consuming derivatization steps, in which EDTA is 2.2 Reference substance, reagents and chemical
converted into methyl, ethyl, propyl and butyl esters to
obtain volatility [22, 23]. Furosemide drug substance samples were obtained from
DK Pharma Chem Pvt Ltd, Mumbai. EDTA di-sodium
The present study deals the development and validation salt, Hydrochloric acid, Tetra butyl ammonium
of HPLC method for determination of EDTA in hydrogen sulphate, Ferric Chloride Hexahydrate and
Furosemide drug substance utilizing trivalent iron for Acetonitrile (HPLC grade) was purchased from Merck
complex formation and its subsequent determination Chemicals India Pvt Ltd. HPLC grade water used of a
based on ion pair chromatography. EDTA being UV Millipore, Milli-Q water purification system (Millipore)
inactive was first complexed with trivalent iron Milford, MA, USA.
resulting in the formation of strong UV active complex.
In order to retain the EDTA-Fe+++ complex on to a 2.3. Chromatographic Conditions
C18 reverse phase column, tetra butyl ammonium
hydrogen sulfate was used as ion pairing agent. A well Gradient mobile phase consisted of a 25mM tetrabutyl
separated peak was obtained for EDTA-Fe+++ complex ammonium hydrogen sulphate, in water as mobile phase
on a C18 column with an acetonitrile and 25mM tertiary A and acetonitrile as mobile phase B. The gradient
butyl ammonium hydrogen sulfate mobile phase (1 profile was Time - %B, 0-7min 2%, 10-15min 80%, 17-
ml/min).A gradient pump program was used with 2% 32min 2%. The Mobile phase was filtered and degassed
acetonitrile for an initial 7 min which was increased through membrane filter of 0.45 µm porosity under
linearly to 80% over 10 min and then maintained vacuum. HPLC column Zorbax XDB C-18,150 mm x

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722
 was used as stationary 100 ml volumetric flask, about 10 mL of diluent was
phase. A constant flow rate of 1.0mL/min was added, shaken to dissolve the sample and diluted to
employed throughout the analysis. Variable UV-Vis volume with diluent and mixed. The solution was
detector was set at 258 nm. All pertinent analyses were filtered through 0.45µm membrane filter and 25µl was
injected directly on to the column.
column was 25 µl. The diluents blank was used by
transferring 5mL of Acetonitrile and 50mL of 1mM 2.7. Quantitation
ferric chloride into 100mL volumetric flask, sonicate for
10min and make up to the mark with 25mM tetrabutyl Peak areas were recorded for all peaks. Peak areas were
ammonium hydrogen sulfate. taken into account to quantitate the EDTA disodium
content in drug substance by using the following
2.4. Samples formula:

The Furosemide drug substance was obtained from DK

Pharma Chem Pvt. Ltd, Mumbai -A Furosemide API
Manufacturer Company.
III. RESULTS AND DISCUSSION
2.5. Solution preparation
3.1. Chromatography
2.5.1. EDTA disodium standard solution
HPLC method development started with the search for
Accurately weighed 20 mg of EDTA di-sodium in to a the suitable column and mobile phase. Chromatographic
100 mL volumetric flask, transferred 5 mL of system comprising0.05M sodium dihydrogen
acetonitrile and 50 mL of 1 mM ferric chloride solution, orthophosphate : acetonitrile (80 : 20 v/v), as mobile
sonicate for 10 minutes and make up to the mark with phase at a constant flow rate of l.0mL/min, Luna C-18
25 mM tetra butyl ammonium hydrogen sulphate. The
solution was allowed to stand for 15 minutes. This is column as stationary phase and detector wavelength at
EDTA di-sodium stock solution. 258nm resulted in no peak elution even after 60 minutes
of run time. Mobile phase consisting of 0.05M aqueous
Transferred 1.0 mL of EDTA di-sodium standard stock ammonium dihydrogen phosphate and acetonitrile in the
solution in to a 100 mL standard flask, added 5 mL of ratio (80:20) v/v, were tried in isocratic conditions on
acetonitrile and 50 mL of 1 mM ferric chloride solution, the InertsilODS-
sonicate for 10 minutes and make up to the mark with symmetrical peak shapes, clear separation of the signal
25 mM tetra butyl ammonium hydrogen sulphate. The peaks from the solvent front peaks.
solution was allowed to stand for 15 minutes and then
filtered through 0.45 µm membrane filter and 25 µL After investigation of following two chromatographic
was injected. system containing 25mM tetra butyl ammonium
hydrogen sulphate and acetonitrile as mobile phase (90 :
2. 6. Estimation from drug substance 10) at a constant flow rate of l.0mL/min, on Agilent
HPLC column XDB C-
Accurately weighed 1000 mg of furosemide sample in and50mM aqueous diammonium hydrogen phosphate
to 100 mL volumetric flask, transferred 5 mL of and acetonitrile as mobile phase (80 : 20) on Waters C-
acetonitrile and 50 mL of 1 mM ferric chloride solution,
sonicate for 10 minutes and make up to the mark with flow rate of 1.0mL/min and detector wavelength at
25 mM tetra butyl ammonium hydrogen sulphate. The 258nm resulted in peak elution at about 2.6 minutes
solution was allowed to stand for 15 minutes. which is very close to peaks of blank diluent. First
investigation resulted in EDTA disodium peak eluted
Six test preparations of drug substances were prepared very close to blank peak (peak from diluent) as shown
of 10.0 mg/mL concentration and mixed. About figure 1.
1000mg of test sample was weighed accurately into a

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 Further, in order to develop a suitable and robust LC
method for the determination of EDTA disodium by UV
detection different mobile phases and columns were
employed (table 1) to achieve the best signal response
and retention time.

Finally, the mobile phase consisting of 25mM tetrabutyl

ammonium hydrogen sulphate and acetonitrile with
gradient programme, at a constant flow rate of 1.0
mL/min and detector wavelength set at 258 nm, using
Agilent XDB, C-
column was found to be appropriate, allowing good
signal response of EDTA disodium.

3.2 Optimization of HPLC

The gradient mobile phase use can affect the analyte’s

retention time as well as the detection sensitivity. No
peak elution of Furosemide was observed due to
isocratic use of mobile phase as 25mM 3.3. Method validation
tetrabutylammonium hydrogen sulphate and acetonitrile
(90:10), so the gradient use of mobile phase was chosen Test method for the determination of EDTA disodium
for the determination of EDTA disodium and was validated to include the essential demands of
Furosemide elution with appropriate retention time. International conference on Harmonization (ICH)
guidelines. Parameters like specificity, linearity,
Finally gradient mobile phase chromatographic accuracy, precision, range and system suitability were
conditions were optimized to have good retentions and examined.
detection response for EDTA di-sodium and elution of
Furosemide peak. The EDTA di-sodium is eluted at 3.3.1. Specificity
retention time at about 5.6 minutes and Furosemide at
about 14.5 minutes, the well resolved peaks between No interferences were observed due to obvious presence
EDTA di-sodium and Furosemide is shown in figure 2. of mobile phase.
Therefore, gradient mobile phase 25mM tetrabutyl
ammonium hydrogen sulphate and acetonitrile was 3.3.2. Linearity
chosen for estimation of EDTA disodium in Furosemide
drug substance, typical chromatogram of test solution is Peak areas versus concentration in milligram per
shown in Figure. 3. milliliter were plotted for EDTA disodium at the
concentration range between LOQ level to150 percent
of target level. EDTA disodium showed linearity in the

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724
 range of 0.5-3.0µg/mL with a correlation coefficient injections. Results obtained from five replicate
(r2) of 0.9999. injections of standard solution observed relative
standard deviation less than 2.
3.3.3. Accuracy
3.4. Application of the proposed method
Accuracy of the proposed HPLC determination was
evaluated from the EDTA disodium content In-house prepared samples were evaluated for content
determination results of the components. Accuracy was of EDTA disodium. The method gave reproducible
done by performing the samples and calculated the peak results for all the samples tested for EDTA disodium.
area responses of different samples by component The probable degradation products of test sample and
recovery method. EDTA disodium did not interfere with the estimation of
the component. The EDTA disodium content, of the test
Stock solution samples are summarized in Table 3.

Stock Solution was prepared by dissolving accurately IV. CONCLUSION

weighed about 19.95mg of EDTA disodium in portions
mobile phase and diluted to produce 100 mL solution. A sensitive HPLC method based on UV detection has
Appropriate portions of stock solution were spiked into been developed and validated for determination of
blank diluent to produce concentrations of 25, 50, 75, EDTA disodium in Furosemide drug substance. The
100, 125 and 150% of target level. Mean recovery of method is simple, rapid, specific, accurate (error
spiked samples was 103.9 % for EDTA disodium (Table ±4.9%), precise, (RSD 2.0%) robust and linear
2). (r2=0.9999). The described method is suitable for
routine analysis and quality control for determination of
3.3.4. Precision EDTA disodium content in Furosemide drug substance
and drug product.
Instrumental precision was determined by five replicate
determinations of standard solution and the relative
V. Acknowledgement
standard deviation was 0.2% for EDTA disodium.
Author would like to thank to the Research guide and
Method precision or intra-precision was performed by
Director, Ullmann Laboratories Pvt. Ltd for their
preparing six different samples involving different
helpful suggestions and constant encouragement.
weighing. Each solution was injected in under same
conditions and peak area responses for each solution
VI. REFERENCES
were taken. Corrections in area were made for each
weight taken to prepare six sample solutions. The
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samples by two different instruments. Standard solution ChimActa. 1977;88:281-7.
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were prepared. The EDTA disodium content in all 12 Analysis of EDTA and DTPA. Talanta, 44, 1487-
preparations was found nil. 1497.
[4]. Buchbergr W, Haddad PR, Alexander PW.
3.3.5. System suitability Separation of metal complexes of
ethylenediaminetetraacetic acid in environmental
System suitability tests were performed to water samples.J chromatogr. 1991;558:181-6.
chromatograms obtained from standard solutions to [5]. Oser BL, Oser M, Spencer HC (1963) Safety
check the relative standard deviation of five replicate evaluation studies of calcium EDTA. Toxicology
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 [6]. Foreman, H., Trujillo, T. The metabolism of C14 fixing solutions by capillary electrophoresis. Fres
labelled ethylenediaminetetraacetic acid in human J AnalChem. 1999;363:124-5.
beings. J Lab Clin Med, 1954, 43: 566-571. [18]. Chawla S, Ghosh S, Sihorkar V, Nellore R,
[7]. American Diabetes Association. Standards of Kumar TRS, Srinivas NR. High-perfomance
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2013;36:S11-66. validation for simultaneous determination of five
[8]. Ramirez-Bosca, A., Soler, A., Carrion-Gutierrez, model compounds, antipyrine metoprolol,
M.A., et al. Antioxidant curcuma extracts ketoprofen, furosemide and phenol red, as a tool
decrease the blood lipid peroxide levels of human for standardization of rat in situ intestinal
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[9]. Perez-Ruiz, T., Martinez-Lozano, C., & Garcia, detection. Biomedical
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[11]. Retho, C., & Diep, L. (1989). Low-level [21]. Krokidis, A. A., Megoulas, N. C., & Koupparis,
determination of ethylenediaminetetraacetic acid M. A. (2005). EDTA determination in
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Untersuchung und -Forschung, 188, 223-226. based on ion chromatography with suppressed
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Analyst. 1993;118:909-12. chromatographic determination of
[13]. Nomura T, Nakagava G. Tensammetric ethylenediaminetetraacetic acid in pickled
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[15]. Voulgaropoulos A, Tzivanakis N. Use of ion chromatography-mass spectrometry. J Chromatog
exchangers for the voltammetric determination of A. 1995;690:109-18
NTA and EDTA in natural waters. [24]. ICH. Validation of analytical procedures:
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 Table 1. Employed mobile phases, columns and elution time during the investigation of EDTA

Column Mobile phase Elution time

Luna C-18 column, 250 mm x 4.6 0.05M sodium dihydrogen orthophosphate : *No peak eluted
mm, 5m acetonitrile (80 : 20 v/v)

Inertsil ODS-1, 250 mm x 4.6 mm, 0.05M ammonium dihydrogen phosphate : *No peak eluted
5m acetonitrile, (75 : 25 v/v)

Waters, C-18, 250 mm x 4.6 mm, 50mM aqueous diammonium hydrogen ** Peak eluted
5m phosphate : acetonitrile, (80 : 20 v/v) close to blank
peak
Agilent XDB, C-18, 150 x 4.6 mm, 25mM Tetrabutyl ammonium hydrogen sulphate ** Peak eluted
5m. and acetonitrile (90:10) close to blank
peak
Agilent XDB, C-18, 150 x 4.6 mm, 25mM Tetrabutyl ammonium hydrogen sulphate About 5.6
5m. and acetonitrile (Gradient programme) minutes.

* No peak EDTA disodium eluted after 60 minutes time

** EDTA disodium peak eluted near very close to blank peak (peak from diluents)

Table 2. Accuracy data (analyte recovery): EDTA

Amount std. Amount std. Determined Recovered (%) Bias (%)

added(µg/mL) recovered (µg/mL) (% of target level)
1 0.527* 0.561 25.07 102.3 2.3

2 2.108* 2.125 104.7 104.7 4.7

4 3.204* 3.222 154.3 104.9 4.9

*Average of three determination.

Table 3: Application of the developed HPLC method for the determination of EDTA content in Furosemide drug
substance and drug product

Sr. Test sample ETDA Content found to be

No.
1 Furosemide Drug substance Nil
2 Lasix ® Furosemide tablets Nil
3 Furosemide tablets Nil

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