Analytical MethodsDetermination of

Analytical Methods
Determination of EDTA in feed and premix formulations by HPLC-DAD
Francesco Chiumiento ⇑
, Antonio D’Aloise, Francesca Marchegiani, Valeria Melai
Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise, Reparto Bromatologia e Residui - Campo Boario 1, 64100 Teramo, Abruzzo, Italy
article info
Article history:
Received 24 July 2014
Received in revised form 25 November 2014
Accepted 29 November 2014
Available online 6 December 2014
Keywords:
Feed safety
2002/657/EC validation
EDTA complexes
Ion-pair chromatography
abstract
A simple analytical method for the quantitative determination of ethylenediaminetetraacetic acid (EDTA)
in feed and premix formulations was developed and validated. The method involves an extraction with an
acidic ferric chloride solution, to quantitatively convert EDTA species in the samples into the Fe(III)–EDTA
complex, and its subsequent detection by Ion-Pair-Reversed Phase-High Performance Liquid Chromatography-Diode
Array Detection (IP-RP-HPLC-DAD). A robust validation procedure was performed according
to the Decision 2002/657/EC at concentrations ranging from 25 to 100 mg kg1 on sample. Good recoveries
(85.6–92.8%) were obtained; repeatability of the method was in the range of 1.3–8.0%, with an intermediate
precision ranging from 6.0% to 8.6%, both of them expressed as relative standard deviation (RSD).
No interfering species hindered the straightforward detection of EDTA. Hence, the proposed method can
be adopted for an effective and rapid routine analysis of products for livestock.
2014 Elsevier Ltd. All rights reserved.
1. Introduction
Ethylenediaminetetraacetic acid (EDTA) has a widespread use
in several laboratory and industrial applications: it is a colorless
solid aminopolycarboxylic acid, slightly soluble in water (500 mg
L1
, 25 C, 1 atm), and it is usually produced in form of several
salts, frequently disodium EDTA and calcium disodium EDTA. Its
importance is due to the ability to act as a ‘‘six-toothed’’ ligand
for a wide number of metals, i.e., EDTA sequesters cations, whose
reactivity decrease after binding.
EDTA is used as a chelating agent in various fields: in the pharmaceutical
and metal industries, it prevents the catalytic action of
metals. In the detergents industry, EDTA is used to reduce water
hardness by binding Ca(II) and Mg(II) which do not precipitate
anymore as carbonates, that may hinder an efficient action of
detergent products. In the food industry, EDTA is added as a stabilizer,
i.e., in order to prevent oxidative discoloration, while in soft
drinks containing ascorbic acid and sodium benzoate, EDTA mitigates
the formation of benzene (a carcinogen) (Food, 2006;
Nowack & VanBriesen, 2005). Within the European Union (EU),
the use of EDTA as a food additive is regulated by the Regulation
1333/2008/EC, which allows its presence only in few food categories;
conversely, the EU legislation does not approve EDTA use as a
feed additive for livestock. In the course of animal husbandry, ‘feed
materials’ shall mean various products of vegetable or animal origin,
products derived from the industrial processing thereof, and
organic or inorganic substances, whether or not containing additives,
intended for use in oral animal feeding either directly as such
or, after processing, in the preparation of compound feed.
A compound feed is a blend from various raw materials and
additives, whose main ingredients are usually corn, sorghum, oats,
barley, etc. Compound feed may also include premixes, composed
of micro-ingredients such as vitamins, minerals, chemical preservatives,
antibiotics, fermentation products with substances used
as carriers, which together assure the intake of the recommended
levels of essential nutrients to the animal. In that context, feed
safety has gained increasing attention in the scientific community,
in order to adapt to ethical issues, and to ensure the supply of
healthy food of animal origin suitable for human consumption. In
the feed industry, EDTA and its derivatives are believed to prevent
bacterial intestina1 diseases of pigs, while also increasing the
effects of antibiotics against such illnesses; moreover, it seems to
be fully harmless to the animal organism (Hutas, 2013).
EDTA faces also veal quality issues, as documented in literature
(Pommier, Lapierre, Passillé, & Gariépy, 1995): one of the considered
parameters for quality evaluation is the color of the meat,
since it is argued that veal from milk-fed calves (white) has a more
refined taste than a cheaper veal from grain-fed calves (red). A feasible
way to produce the more valuable white veal is to limit the
availability of iron in the diet, in order to reduce pigment accumulation
in muscle, and the use of chelating agents such as Ca-EDTA
has been reported to result in a lighter muscle color. As already
mentioned, within the EU EDTA presence in feed has to be
regarded as the result of a fraudulent action; meanwhile, in the
US it is intended for use in an amount not to exceed 240 mg L1
http://dx.doi.org/10.1016/j.foodchem.2014.11.159
0308-8146/ 2014 Elsevier Ltd. All rights reserved.
⇑ Corresponding author. Tel.: [phone].
E-mail address: [e-mail] (F. Chiumiento).
Food Chemistry 175 (2015) 452–456
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
in the finished product. At best of our knowledge, no validated
methods for EDTA determination in feed and premix formulations
are present in the literature, even if several approaches were
implemented for different matrices: RP-HPLC (Kemmei, Kodama,
Yamamoto, Inoue, & Hayakawa, 2013; Perez-Ruiz, MartinezLozano,
& Garcia-Martinez, 2008; Sillanpaa & Sihvonen, 1997),
gas-chromatography (Retho & Diep, 1989), ion-chromatography
(Krokidis, Megoulas, & Koupparis, 2005), capillary electrophoresis
(Laamanen, Mali, & Matilainen, 2005), and liquid chromatography–tandem
mass spectrometry (Quintana & Reemtsma, 2007).
The study presented in this manuscript provides a validated
method for an effective, rapid and simple routine determination
of EDTA in feed and premix formulations, via a procedure based
on Ion-Pair-Reversed Phase-High Performance Liquid Chromatography
and Diode Array Detection (IP-RP-HPLC-DAD). The method
exploits the well-known ability of EDTA to form the most thermodynamically
stable complexes with Fe(III) ions (Sillanpää,
Kokkonen, & Sihvonen, 1995): moreover, chelation is needed
because free EDTA does not strongly absorb in the UV–Vis region.
In the chosen experimental conditions, no interferences due to
other metal ions are present: an excess of Fe(III) ions (ferric chloride
solution) is added to the samples, in order to achieve quantitative
conversion of the several possible EDTA species into the
Fe(III)-complex.
Sample extraction must be performed in acidic conditions
(pH 3) for avoiding Fe(III) precipitation as ferric hydroxide
(Sillanpää et al., 1995). Indeed, pH for iron complexes formation
is crucial for chelation efficiency (Zheng, Watson, Tettey, &
Clements, 2008): there is a hyperbolic relationship between the
pH and the log K that give us information about the minimum
pH required to obtain a satisfactory chelation. For EDTA, this minimum
pH values for Fe(III) is below 2.0, while for Fe(II) it is higher
(>5.0) (De Luca, Dantas, & Esplugas, 2014).
After FeCl3 addition, samples solutions were kept in the dark in
order to prevent photochemical degradation of the Fe(III)-complex,
and to promote its formation sample solutions were heated at
70 C±2 C for 30 min.
The last step is necessary because although the Fe(III) complex
is thermodynamically favored over all other metal complexes,
some of them are more kinetically favored (Nowack, Kari, Hilger,
& Sigg, 1996); therefore, it seemed advisable to heat the samples
to accelerate the equilibration (Quintana & Reemtsma, 2007).
Finally, the EDTA ferric complex was spectrophotometrically determined
at its kmax, setting DAD at 254 nm (Cagnasso, López,
Rodríguez, & Valencia, 2007; Yamaguchi, Rajput, Ohzeki, &
Kambara, 1983). As regards to method validation, this is nowadays
a priority for laboratories which are responsible for carrying out
official controls of foodstuffs, and the fitness for purpose of analytical
methods applied for such routine testing is assessed through
the determination of overall method performance parameters
(Cozar-Bernal, Rabasco, & Gonzalez-Rodriguez, 2013). Many
important decisions are based on the results of quantitative analyses:
they are used, for example, to check foods against specifications
or statutory limits, thus involving legal concerns (Ellison &
Williams, 2012). As in our case, it is now a formal (frequently legislative)
requirement for laboratories to introduce quality assurance
measures, to ensure that they are capable of providing good
quality data. Such measures include, indeed, the use of validated
methods, internal quality control procedures, proficiency testing
and accreditation (International Organization for Standardization/
International Electrotechnical Commission 17025, 2005), especially
demanding when dealing with the determination of unauthorized
substances, as can be EDTA in feed and premix
formulation within the EU. Hence, in order to comply with these
requirements, the present work describes a procedure for method
validation using the following parameters: linearity, repeatability
and intermediate precision, accuracy, specificity, detection (LOD)
and quantitation (LOQ) limits, and measurement uncertainty.
2. Materials and methods
2.1. Chemicals and standards preparation
All chemicals were of analytical reagent grade: ethylenediaminetetraacetic
acid disodium salt di-hydrate, ferric chloride
hexa-hydrate, tetrabutylammonium hydroxide solution 1 M, glacial
acetic acid, ammonium acetate, and methanol were purchased
from Sigma–Aldrich. All aqueous solutions were prepared with
high-purity water (resistivity 18.2 M cm1
) obtai

ผลลัพธ์ (ไทย) 1:

วิธีวิเคราะห์ความมุ่งมั่นของ EDTA ในสูตรอาหารและ premix โดย HPLC-พ่อฟรานเชสโค Chiumiento ⇑, Antonio D'Aloise ฟรานเซสซา Marchegiani, Valeria MelaiIstituto Zooprofilattico Sperimentale dell'Abruzzo อีเดล Molise, e Reparto Bromatologia Residui - บรรดา Boario 1, 64100 Teramo ในอาบรุซโซ อิตาลีข้อมูลบทความบทความประวัติ:ได้รับ 24 2014 กรกฎาคมรับแบบฟอร์มที่ปรับปรุง 25 2014 พฤศจิกายนยอมรับ 29 2014 พฤศจิกายนมีออนไลน์ 6 2557 ธันวาคมคำสำคัญ:อาหารความปลอดภัยตรวจสอบ 2002/657/ECคอมเพล็กซ์ EDTAไอออนคู่ chromatographyบทคัดย่อวิธีวิเคราะห์อย่างง่ายสำหรับการกำหนดเชิงปริมาณของกรด ethylenediaminetetraacetic (EDTA)ในอาหารและ premix สูตรถูกพัฒนา และผ่านการตรวจสอบ วิธีการเกี่ยวข้องกับการสกัดด้วยการโซลูชั่นคคลอไรด์กรด quantitatively แปลงชนิด EDTA ในตัวอย่างเป็น Fe (III) – EDTAซับซ้อน และการตรวจสอบต่อมาโดย Ion-Pair-Reversed ระยะสูงประสิทธิภาพของเหลว Chromatography-ไดโอดตรวจแถว (IP-RP-HPLC-DAD) ขั้นตอนการตรวจสอบประสิทธิภาพมีดำเนินการตามการตัดสินใจ 2002/657/EC ที่ความเข้มข้นตั้งแต่ 25 ถึง 100 มิลลิกรัม kg1 บนตัวอย่าง Recoveries ดี(85.6 – 92.8%) ไม่รับ ทำซ้ำในวิธีการอยู่ในช่วง 1.3 – 8.0% กับกลางความแม่นยำตั้งแต่ 6.0% 8.6% ทั้งสองอย่างแสดงเป็นค่าเบี่ยงเบนมาตรฐานสัมพัทธ์ (RSD)ชนิดไม่รบกวนผู้ที่ขัดขวางการตรวจพบที่ตรงไปตรงมาของ EDTA ด้วยเหตุนี้ วิธีการนำเสนอสามารถbe adopted for an effective and rapid routine analysis of products for livestock. 2014 Elsevier Ltd. All rights reserved.1. IntroductionEthylenediaminetetraacetic acid (EDTA) has a widespread usein several laboratory and industrial applications: it is a colorlesssolid aminopolycarboxylic acid, slightly soluble in water (500 mgL1, 25 C, 1 atm), and it is usually produced in form of severalsalts, frequently disodium EDTA and calcium disodium EDTA. Itsimportance is due to the ability to act as a ‘‘six-toothed’’ ligandfor a wide number of metals, i.e., EDTA sequesters cations, whosereactivity decrease after binding.EDTA is used as a chelating agent in various fields: in the pharmaceuticaland metal industries, it prevents the catalytic action ofmetals. In the detergents industry, EDTA is used to reduce waterhardness by binding Ca(II) and Mg(II) which do not precipitateanymore as carbonates, that may hinder an efficient action ofdetergent products. In the food industry, EDTA is added as a stabilizer,i.e., in order to prevent oxidative discoloration, while in softdrinks containing ascorbic acid and sodium benzoate, EDTA mitigatesthe formation of benzene (a carcinogen) (Food, 2006;Nowack & VanBriesen, 2005). Within the European Union (EU),the use of EDTA as a food additive is regulated by the Regulation1333/2008/EC, which allows its presence only in few food categories;conversely, the EU legislation does not approve EDTA use as afeed additive for livestock. In the course of animal husbandry, ‘feedmaterials’ shall mean various products of vegetable or animal origin,products derived from the industrial processing thereof, andorganic or inorganic substances, whether or not containing additives,intended for use in oral animal feeding either directly as suchor, after processing, in the preparation of compound feed.A compound feed is a blend from various raw materials andadditives, whose main ingredients are usually corn, sorghum, oats,barley, etc. Compound feed may also include premixes, composedof micro-ingredients such as vitamins, minerals, chemical preservatives,antibiotics, fermentation products with substances usedas carriers, which together assure the intake of the recommendedlevels of essential nutrients to the animal. In that context, feedsafety has gained increasing attention in the scientific community,in order to adapt to ethical issues, and to ensure the supply ofhealthy food of animal origin suitable for human consumption. Inthe feed industry, EDTA and its derivatives are believed to preventbacterial intestina1 diseases of pigs, while also increasing theeffects of antibiotics against such illnesses; moreover, it seems tobe fully harmless to the animal organism (Hutas, 2013).EDTA faces also veal quality issues, as documented in literature(Pommier, Lapierre, Passillé, & Gariépy, 1995): one of the consideredparameters for quality evaluation is the color of the meat,since it is argued that veal from milk-fed calves (white) has a morerefined taste than a cheaper veal from grain-fed calves (red). A feasibleway to produce the more valuable white veal is to limit theavailability of iron in the diet, in order to reduce pigment accumulationin muscle, and the use of chelating agents such as Ca-EDTAhas been reported to result in a lighter muscle color. As alreadymentioned, within the EU EDTA presence in feed has to beregarded as the result of a fraudulent action; meanwhile, in theUS it is intended for use in an amount not to exceed 240 mg L1http://dx.doi.org/10.1016/j.foodchem.2014.11.1590308-8146/ 2014 Elsevier Ltd. All rights reserved.⇑ Corresponding author. Tel.: [phone].E-mail address: [e-mail] (F. Chiumiento).Food Chemistry 175 (2015) 452–456Contents lists available at ScienceDirectFood Chemistryjournal homepage: www.elsevier.com/locate/foodchemin the finished product. At best of our knowledge, no validatedmethods for EDTA determination in feed and premix formulationsare present in the literature, even if several approaches wereimplemented for different matrices: RP-HPLC (Kemmei, Kodama,Yamamoto, Inoue, & Hayakawa, 2013; Perez-Ruiz, MartinezLozano,& Garcia-Martinez, 2008; Sillanpaa & Sihvonen, 1997),gas-chromatography (Retho & Diep, 1989), ion-chromatography(Krokidis, Megoulas, & Koupparis, 2005), capillary electrophoresis
(Laamanen, Mali, & Matilainen, 2005), and liquid chromatography–tandem
mass spectrometry (Quintana & Reemtsma, 2007).
The study presented in this manuscript provides a validated
method for an effective, rapid and simple routine determination
of EDTA in feed and premix formulations, via a procedure based
on Ion-Pair-Reversed Phase-High Performance Liquid Chromatography
and Diode Array Detection (IP-RP-HPLC-DAD). The method
exploits the well-known ability of EDTA to form the most thermodynamically
stable complexes with Fe(III) ions (Sillanpää,
Kokkonen, & Sihvonen, 1995): moreover, chelation is needed
because free EDTA does not strongly absorb in the UV–Vis region.
In the chosen experimental conditions, no interferences due to
other metal ions are present: an excess of Fe(III) ions (ferric chloride
solution) is added to the samples, in order to achieve quantitative
conversion of the several possible EDTA species into the
Fe(III)-complex.
Sample extraction must be performed in acidic conditions
(pH 3) for avoiding Fe(III) precipitation as ferric hydroxide
(Sillanpää et al., 1995). Indeed, pH for iron complexes formation
is crucial for chelation efficiency (Zheng, Watson, Tettey, &
Clements, 2008): there is a hyperbolic relationship between the
pH and the log K that give us information about the minimum
pH required to obtain a satisfactory chelation. For EDTA, this minimum
pH values for Fe(III) is below 2.0, while for Fe(II) it is higher
(>5.0) (De Luca, Dantas, & Esplugas, 2014).
After FeCl3 addition, samples solutions were kept in the dark in
order to prevent photochemical degradation of the Fe(III)-complex,
and to promote its formation sample solutions were heated at
70 C±2 C for 30 min.
The last step is necessary because although the Fe(III) complex
is thermodynamically favored over all other metal complexes,
some of them are more kinetically favored (Nowack, Kari, Hilger,
& Sigg, 1996); therefore, it seemed advisable to heat the samples
to accelerate the equilibration (Quintana & Reemtsma, 2007).
Finally, the EDTA ferric complex was spectrophotometrically determined
at its kmax, setting DAD at 254 nm (Cagnasso, López,
Rodríguez, & Valencia, 2007; Yamaguchi, Rajput, Ohzeki, &
Kambara, 1983). As regards to method validation, this is nowadays
a priority for laboratories which are responsible for carrying out
official controls of foodstuffs, and the fitness for purpose of analytical
methods applied for such routine testing is assessed through
the determination of overall method performance parameters
(Cozar-Bernal, Rabasco, & Gonzalez-Rodriguez, 2013). Many
important decisions are based on the results of quantitative analyses:
they are used, for example, to check foods against specifications
or statutory limits, thus involving legal concerns (Ellison &
Williams, 2012). As in our case, it is now a formal (frequently legislative)
requirement for laboratories to introduce quality assurance
measures, to ensure that they are capable of providing good
quality data. Such measures include, indeed, the use of validated
methods, internal quality control procedures, proficiency testing
and accreditation (International Organization for Standardization/
International Electrotechnical Commission 17025, 2005), especially
demanding when dealing with the determination of unauthorized
substances, as can be EDTA in feed and premix
formulation within the EU. Hence, in order to comply with these
requirements, the present work describes a procedure for method
validation using the following parameters: linearity, repeatability
and intermediate precision, accuracy, specificity, detection (LOD)
and quantitation (LOQ) limits, and measurement uncertainty.
2. Materials and methods
2.1. Chemicals and standards preparation
All chemicals were of analytical reagent grade: ethylenediaminetetraacetic
acid disodium salt di-hydrate, ferric chloride
hexa-hydrate, tetrabutylammonium hydroxide solution 1 M, glacial
acetic acid, ammonium acetate, and methanol were purchased
from Sigma–Aldrich. All aqueous solutions were prepared with
high-purity water (resistivity 18.2 M cm1
) obtai