See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/348649819

Development and Validation of RP-HPLC method for determination of

Ethylenediaminetetraacetic acid (EDTA) in Cosmetics with UV detection using
precolumn derivatization technique

Article · July 2020

CITATIONS READS
0 714

2 authors, including:

Rakhi Singh
Shriram Institute for Industrial Research
9 PUBLICATIONS   5 CITATIONS   

SEE PROFILE

All content following this page was uploaded by Rakhi Singh on 21 January 2021.

The user has requested enhancement of the downloaded file.

 Singh Rakhi et al., IJSRR 2020, 9(3), 73-81

Research Article Available online www.ijsrr.org ISSN: 2279–0543

International Journal of Scientific Research and Reviews

Development and Validation of RP-HPLC method for determination of
Ethylenediaminetetraacetic acid (EDTA) in Cosmetics with UV detection using
precolumn derivatization technique

Manjusha, Singh Rakhi*, Aggarwal M. L. and Chacko K. M.

Shriram Institute for Industrial Research, 19 University Road, Delhi-110007, India

ABSTRACT
Analytical method for the quantitative determination of EDTA in Cosmetics has been developed
and validated by high-performance liquid chromatography (HPLC) with UV detection at wavelength
280 nm. The analysis was performed in gradient mode on a reversed phase C18 column, 4.6mm x
250mm using mobile phase consisted of a 25mM tetrabutyl ammonium hydrogen sulphate, in water as
mobile phase A and acetonitrile as mobile phase B. The developed method was validated for various
parameters such as Specificity, Linearity, Precision, Recovery, Repeatability and Ruggedness by
employing HPLC. A linear calibration curve was observed in the range of 0.5-10.0 mg/kg with r2 values
≥ 0.99. The limits of detection and quantification were 0.25 mg/kg and 0.5 mg/kg respectively.
Cosmetics samples were spiked at 0.5, 1.0 and 2.0mg/kg fortification levels. Better recoveries between
70% to 120% were obtained with the acceptable relative standard deviation (% RSD) i.e. <20%. The
validated method has been satisfactorily applied to the analysis of EDTA in Cosmetics samples due to
its high sensitivity and selectivity.

KEYWORDS: Cosmetics, EDTA, Method Validation, Pre-column Derivatization, HPLC

*
Corresponding Author
Rakhi Singh
Shriram Institute for Industrial Research, 19 University Road, Delhi-110007, India
Email: rakhis1973@gmail.com
Phone: 9891858095

IJSRR, 9(3) July – Sep., 2020 Page 73

 Singh Rakhi et al., IJSRR 2020, 9(3), 73-81

1. INTRODUCTION
Ethylenediaminetetraacetic acid (EDTA) (Figure No. 1) and its salts (collectively known as
Edetates) are substituted diamines. EDTA is a powerful chelating agent, forming stable complexes
with most metal ions1.EDTA is less stable than its salts. Disodium edetate is disodium salt of EDTA
and is commonly used in pharmaceutical formulations, cosmetics, and foods as chelating agents.
These ingredients function as chelating agents in cosmetic formulations by combining with
polyvalent metal cations in solutions to form soluble ring structures2.

Cosmetics, detergents, and pharmaceutical preparations require protection against microbial

growth to ensure the safety of their use and to extend the length of their shelf life3. EDTA increases
the permeability of cell membranes and make them more sensitive to antimicrobial agents. In
addition, chelating agents block the iron required for metabolism and microbial growth, and can
enhance the antimicrobial efficacy of the used preservatives4; 5.The binding of metal ions helps
prevent deterioration of cosmetics and personal care products. It also protects fragrance compounds
and prevents rancidity. It form complexes with calcium, magnesium and iron which allow for better
foaming and cleaning performance of cosmetics and personal care products. By binding with metal
ions, these ingredients prevent metals from being deposited onto the hair, scalp and skin6.

EDTA Disodium EDTA

Figure No. 1: Structure of EDTA and Disodium EDTA

Disodium EDTA (Figure No. 1) primarily works as a preservative, chelator and stabilizer, but
has also been shown to enhance the foaming and cleaning capabilities of a cosmetic solution7. It
stabilizes or helps preserve all kinds of cosmetics products like creams, lotions, shampoos,
conditioners, make-up products, sunscreen products. Disodium EDTA, as a stabilizer in cosmetics is

IJSRR, 9(3) July – Sep., 2020 Page 74

 Singh Rakhi et al., IJSRR 2020, 9(3), 73-81

used to prevent ingredients in a given formula from binding with trace elements that can be present
in water. It stabilizes emulsions, surfactants and foam builders. In shampoos, cleaners and other
personal care products. EDTA acts as co-preservative that enhances efficacy of preservatives and
other anti-bacterial agents. EDTA does not contain any chromophoric group. Hence, it is very
difficult to determine EDTA by direct UV detection8. The quantification of the residual EDTA is
essential as per regulatory requirements.

Various analytical methods have been proposed for the determination of EDTA in a wide
variety of sample matrices9. These include titrimetry10, spectrophotometry11, electrochemistry
polarography12, differential pulse olorography13, catalytic potential tirtrimetry14, differential pulse
anodic stripping voltammetry15, amperometry16, capillary electrophoresis17, and chromatography.
Gas chromatography and HPLC (reverse phase) are the prevailing techniques. The gas
chromatographic methods always include a time consuming derivatization steps, in which EDTA is
converted into methyl, ethyl, propyl and butyl esters to obtainvolatility18, 19.

Up to date, different analytical methods have been reported for determination of Edetates based
on high-performance liquid chromatography (HPLC) in commercial pharmaceutical products. To
our knowledge, there is not much report about determination of Edetates in Cosmetic products which
is particularly important for both quality assurance and consumer protection.

The aim of this work was to develop fast, simple, selective, and easy-to-use method for control
of EDTA in Cosmetics. The present study deals with the development and validation of HPLC
derivatized method for quantitative determination of EDTA in Cosmetics products utilizing trivalent
iron for complex formation with ferric chloride solution and its subsequent determination by
reversed phase HPLC.

2. EXPERIMENTAL
2.1. Chemicals and Reagents
Cosmetics samples were purchased from market. Di-sodium salt of Ethylenediaminetetraacetic
acid dihydrate (Disodium EDTA) and Ferric chloride anhydrous (AR grade) were purchased from
Qualigens. Tetra butyl ammonium hydrogen sulphate (AR grade) was procured from Spectrochem,
Acetonitrile (HPLC grade) was purchased from Rankem. Deionized water (Millipore-Advantage
A10) was used for the preparation of standard and sample solutions.

IJSRR, 9(3) July – Sep., 2020 Page 75

 Singh Rakhi et al., IJSRR 2020, 9(3), 73-81

2.2. Preparation of Stock and Working Standard Solution

Diluent: 25mM Tetrabutyl Ammonium Hydrogen Sulphate Solution. Standard and sample
solutions were prepared in the diluent.

A standard stock solution of 1000mg/Kg was prepared by dissolving accurately weighed

63.5mg of disodium salt of EDTA dihydrate (equivalent to 50 mg of EDTA)2 in 2.5ml of acetonitrile
followed by derivatization process carried out by adding 25ml of 1mM ferric chloride solution and
final volume was made up to 50 mL with diluent.

Working standard solutions were prepared by serial dilution of the standard stock solution
into 100mL volumetric flasks containing 5mL of acetonitrile and 50mL of 1mM Ferric chloride
solution to achieve the desired linearity range of 0.5-10.0 mg/Kg of EDTA. Finally the volume was
made up to the mark with diluent, shaken well and allowed to stand for 15 minutes at room
temperature. The solution was filtered through 0.45 μm syringe filter prior to injection on HPLC.

2.3. Extractionprocedure
About 1g of the cosmetic sample was weighed into 100mL capacity volumetric flask.
Derivatization was carried out by adding 5mL of acetonitrile and 50ml of 1mM Ferric chloride
solution into the flask. The final volume was made up with the diluent, shaken well and allowed to
stand for 15minutes at room temperature. The solution was centrifuged at 5000rpm for 5 minutes
and then filtered through 0.45 μm syringe filter prior to injection on HPLC.

2.4. Instrumentation and Conditions

The analytical separations were carried out on HPLC-Agilent 1200 Technologies, equipped
with UV detector and a Prominence autosampler. The analytical column, Agilent C18column
(250mm × 4.6 mm), 5 μm was used for chromatographic analysis. The mobile phase consisted of
25mM tetra butyl ammonium hydrogen sulphate, in water as mobile phase A and acetonitrile as
mobile phase B. The mobile phase was degassed and filtered through a 0.45 μm membrane filter.
The gradient profile was 0-7 minutes 2% B; 7-15 minutes 80% B; 15-20 minutes 80% B, 20-
25minutes 2% B and 25-30 minutes2% B. The flow rate was 1 mL/min and total runtime was 30
minutes. Column temperature was maintained at 25°C. UV detection was measured at 280 nm and
the volume of sample injected on to the column was 25 μL.

IJSRR, 9(3) July – Sep., 2020 Page 76

 Singh Rakhi et al., IJSRR 2020, 9(3), 73-81

3. RESULTS AND DISCUSSION

3.1. Method Validation
The developed analytical method for the determination of EDTA was validated for their
performance characteristics such as Specificity, Linearity, Precision, Recovery, Repeatability and
Ruggedness.

3.2. Specificity
Chromatograms of blank and spiked samples were analyzed to examine interference, if any. No
peak from the blank was observed at the retention time of EDTA peak ensuring that the peak is pure
and there were no interferences in the retention time of the target analyte. Hence it can be said that
the proposed analytical method is specific and selective for the determination of EDTA in cosmetic
products.

3.3. Linearity
To establish the linearity of the proposed method, a series of disodium EDTA solution (0.5 to
10.0 mg/Kg) were prepared from the stock solution and analyzed. Linearity was evaluated by
plotting calibration curves between peak areas versus concentration at five fortification levels.
Linear calibration curves with correlation coefficients (r2) > 0.99 were obtained and proved linearity
of the method in defined concentration ranges. The linearity data is shown in Table 1 whereas
Linearity graph is shown in Figure 2.

Table No. 1: Linearity Data for EDTA in Cosmetics

S.No. Concentration (mg/kg) Area

1 0.5 31825

2 1.0 61006

3 2.0 130215

4 5.0 318370

5 10.0 647021

IJSRR, 9(3) July – Sep., 2020 Page 77

 Singh Rakhi et al., IJSRR 2020, 9(3), 73-81

Linearity
800000
y = 64760x - 1926
600000 R² = 0.9999

Area
400000

200000

0
0 5 10
Conc. (mg/kg)

Figure No. 2: Linearity Graph of EDTA

3.4. Limit of Detection (LOD) and Limit of Quantification (LOQ)

The sensitivity of the method was expressed as LOD and LOQ. The limits of detection (LOD)
and quantification (LOQ) were found by adding decreasing concentrations of standard solution in
the samples, and then subjected to extraction and quantification, up to the lowest detectable
concentration (LOD) and the lowest quantifiable concentration (LOQ), under suitable conditions of
repeatability (n = 5, RSD < 20%). The limits of detection and quantification found were 0.25µg/kg
and 0.5µg/kg, respectively, for determination of EDTA in cosmetics.

3.5. Accuracy
Typically, accuracy is represented and determined by recovery studies. The accuracy was
evaluated by spiking the samples at three fortification levels i.e. 0.5, 1.0, and 2.5 µg/kg with six
replicates at each level. The relative standard deviation (% RSD) for all spiked levels was found
lower than 20%. The recovery data shown in Table 2 indicates that the method has an acceptable
level of accuracy.

Table No. 2: Recovery Data for EDTA in Cosmetics

Fortification Mean Recovery

Compound % Recovery ± SD
Level (mg/kg) (mg/kg)

0.5 0.58 115.5 ± 1.9

EDTA 1.0 1.07 107.3 ± 0.2

2.0 2.18 108.9 ± 0.4

IJSRR, 9(3) July – Sep., 2020 Page 78

 Singh Rakhi et al., IJSRR 2020, 9(3), 73-81

3.6. Precision
Precision in terms of repeatability (Intra-day precision) and ruggedness (Intermediate
precision) was evaluated at three concentration levels i.e. 0.5, 1.0 and 2.5µg/kg with six replicates at
each level. The acceptance criterion found was within 20% relative standard deviation (% RSD). The
values shown in Table 3were found well within the acceptable range indicating that the proposed
method has an excellent repeatability and intermediate precision. These results also suggested that
the proposed method may be considered validated in term of precision.

Table No. 3: Repeatability and Ruggedness Data for EDTA in Cosmetics

Mean Recovery (mg/Kg) % Recovery ± SD

Fortification
Compound
Level (mg/Kg)
Repeatability Ruggedness Repeatability Ruggedness

0.5 0.58 0.57 116.6 ± 1.7 114.8 ± 1.4

EDTA 1.0 1.07 1.07 107.2 ± 0.5 106.6 ± 1.1

2.0 2.18 2.18 108.9 ± 0.4 108.8 ± 0.8

4. CONCLUSION
The reversed-phase RP-HPLC method developed was found to be convenient for the
simultaneous determination of EDTA. The validated method is found to be highly sensitive
therefore; it could be used for routine analysis of EDTA in cosmetics. Obtained validation
parameters proved that the suggested method is convenient enough for routine determination of
EDTA in quality control laboratories. This method also has advantages over other techniques as in
this method EDTA response is measured by direct UV detection with enhanced sensitivity and
method is simpler, highly reproducible, specific and accurate. The proposed method showed good
precision and reproducibility with acceptable linearity and accuracy range. Hence, the proposed
method can be applied to the analysis of EDTA in commercially available cosmetics.

5. ACKNOWLEDGEMENT
The authors are thankful to the Department of Analytical Science Division, Shriram Institute
for Industrial Research, Delhi for providing the facilities for this research work.

IJSRR, 9(3) July – Sep., 2020 Page 79

 Singh Rakhi et al., IJSRR 2020, 9(3), 73-81

REFERENCES
1. Bhavil Narola, Singh A.S., Mitra M., Santhakumar P.R. and Chandrashekhar T.G. “A validated
reverse phase HPLC method for the determination of Disodium EDTA in Meropenem Drug
substance with UV-Detection using Pre-column derivatization technique”. Analytical Chemistry
Insights 2011; 6: 7-14, doi: 10.4137/ACI.S5953.
2. Lanigan RS, Yamarik TA. “Report Safety Assessment of EDTA, Calcium Disodium EDTA,
Diammonium EDTA, Dipotassium EDTA, Disodium EDTA, TEA-EDTA, Tetrasodium EDTA,
Tripotassium EDTA, Trisodium EDTA, HEDTA and Trisodium HEDTA. Cosmetic Ingredient
Review”. International Journal of Toxicology 2002; 21(2): 95-142.
3. Irena Baranowska, Iwona Wojciechowska. “The Determination of Preservatives in Cosmetics
and Environmental Waters by HPLC”. Pol. J. Environ. Stud. 2013; 22(6): 1609-1625.
4. Varvaresou A., Papageorgiou S.,Tsirivas E.,Protopapa E.,Kintziou H.,Kefala V.,Demetzos C.
“Self-preserving Cosmetics”. Int. J. Cosmet. Sci. 2009; 31: 163-175.
5. Siegert W. Boosting. “The Antimicrobial efficiency of multifunctional additives by chelating
agents”. Int. J. Appl. Sci. 2014; 140: 1-6.
6. https://cosmeticsinfo.org/ingredient/disodium-edta.
7. https://www.truthinaging.com/ingredients/disodium-edta.
8. Shripad Deshpande, Mazahar Farooqui, Gajanan Ganap, Vishal Khadke, Kayande D. D.
“Development and Validation of a gradient HPLC method for quantification of Edetate
Disodium in lyophilized injectable drug product”. Int. J. Curr. Pharm. Res., ISSN- 0975-7066:
2019; 11(3): 38-41.
9. Sillanpaa, M, Sihvonen, ML. “Analysis of EDTA and DTPA”. Talanta 1997; 44: 1487-97.
10. Clinkemaille GG. “Determination of Nitrilotriacetic acid and Ethylenediaminetetraacetic acid in
granular detergent formulations”. Anal Chim Acta. 1968; 43:520-2.
11. Hamano T, Mitsuhashi Y, Kojima N, et al. “Sensitive Spectrophotometric method for the
determination of Ethylenediaminetetraacetic Acid”. Analyst 1993; 118: 909-12.
12. Nomura T, Nakagava G. Tensammetric. “Determination of Microgram Amounts of EDTA”. J
Electroanal Chem. 1980; 111: 319-24.
13. Stolzberg RJ. “Determination of Ethylenediaminetetraacetate and Nitrilotriacetate by Differential
Pulse Polorography. Anal Chim Acta. 1977; 92: 139-48.

IJSRR, 9(3) July – Sep., 2020 Page 80

 Singh Rakhi et al., IJSRR 2020, 9(3), 73-81

14. Hadjiioannou TP, Koupparis MA, Efstathiou CE. “Semiautomatic indirect titration of alkaline
earth-ions with catalytic end point indication”. Anal Chim Acta. 1977; 88: 281-7.
15. Voulgaropoulos A, Tzivanakis N. “Use of Ion Exchangers for the Voltammetric determination of
NTA and EDTA in Natural Waters”. Electroanalysis. 1992; 4: 647-51.
16. Fogg AG, Fernandez-Arciniega MA, Alonso RM. “Amperometric flow injection determination
of Ethylenediaminetetraacetic Acid (EDTA) at an electrochemically pre-treated glassy carbon
electrode”. Analyst 1985; 110: 1201-4.
17. Pozdniakova S, Ragauskas R, Dikcius A, Padarauskas A. “Determination of EDTA in used
fixing solutions by capillary electrophoresis. Fres J Anal Chem. 1999; 363: 124-5.
18. Sorvari J, Sillanpaa M, Sihvonen ML. “Development of a Gas Chromatographic method for the
simultaneous determination of trace amounts of Ethylenediaminetetraacetic Acid and
Diethylenetriaminepentaacetic Acid in Natural Waters”. Analyst 1996; 121: 1335-9.
19. Nishikawa Y, Okumura T. “Determination of Nitrilotriacetic Acid and
Ethylenediaminetetraacetic Acid in Environmental samples as their methyl ester derivatives by
Gas Chromatography-Mass Spectrometry”. J. Chromatog A. 1995; 690: 109-18.
____________________________________________________________________________________

IJSRR, 9(3) July – Sep., 2020 Page 81

View publication stats