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

net/publication/259659235

Development and Validation of an RP-HPLC Method for Methionine, Cystine

and Lysine Separation and Determination in Corn Samples

Article  in  Revista de Chimie -Bucharest- Original Edition- · August 2013

CITATIONS READS

19 7,156

7 authors, including:

Iulia Varzaru Arabela Elena Untea

Institutul National de Cercetare-Dezvoltare pentru Biologie si Nutritie Animala Institutul National de Cercetare-Dezvoltare pentru Biologie si Nutritie Animala
28 PUBLICATIONS   43 CITATIONS    57 PUBLICATIONS   138 CITATIONS   

SEE PROFILE SEE PROFILE

Olteanu Margareta Tatiana Panaite

Institutul National de Cercetare-Dezvoltare pentru Biologie si Nutritie Animala Institutul National de Cercetare-Dezvoltare pentru Biologie si Nutritie Animala
53 PUBLICATIONS   139 CITATIONS    19 PUBLICATIONS   55 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Program nucleu 09- 25.01.01 – Crearea de genotipuri de lucernã tolerante la cosiri frecvente pretabile diversificãrii utilizãrilor View project

ADER 1.1.7- Maximizarea producţiilor de proteinã vegetală şi creşterea contribuţiei fixării azotului atmosferic la optimizarea rotaţiilor, prin crearea de soiuri de
leguminoase pentru boabe şi furajere mai productive, cu toleranţã îmbunãtãţitã la stres termic şi hidric şi la boli, pretabile la recoltarea mecanizată şi cu însuşiri
calitative superioare pentru diverse utilizări View project

All content following this page was uploaded by Arabela Elena Untea on 13 January 2014.

The user has requested enhancement of the downloaded file.

 Development and Validation of an RP-HPLC Method for Methionine,
Cystine and Lysine Separation and Determination in Corn Samples

IULIA VARZARU1,2*, ARABELA ELENA UNTEA2, TEODOR MARTURA3, MARGARETA OLTEANU2, TATIANA DUMITRA PANAITE2,
MARIA SCHITEA3, ILIE VAN1
1
University of Agronomic Science and Veterinary Medicine Bucharest, 59 Marasti Blv., 011464, Bucharest, Romania
2
National Research Development Institute for Animal Biology and Nutrition (INCDBNA) 1 Calea Bucuresti, 077015 Balotesti, Ilfov,
Romania
3
National Agricultural Research and Development Institute (INCDA) 1 Nicolae Titulescu Str., 915200, Fundulea, Cãlãraºi, Romania

In this paper a RP-HPLC method is developed and validated. Using a Hypersil BDS C18 column, a mobile
phase: solvent A (phosphate buffer) and solvent B (water : acetonitrile : methanol, 20:20:60 v/v/v), a 45°C
column temperature and a flow rate of 1.7 mL/min, the proposed method proved a good separation of the
investigated amino acids, in 35 minutes.

Keywords: RP-HPLC method, methionine, cystine, lysine, corn

Corn (Zea mays L.) is a worldwide grown plant which - HyperSil BDS C18 column, with silica gel, dimensions
has a high productive potential, twice as much compared 250 × 4.6 mm, particle size 5μm (Thermo-Electron
to other cereals. It is a major component of the feed Corporation, Waltham, MA);
formulations for farm animals. Like in the other cereals, - Rotary evaporator Buchi (Zurich, Switzerland);
the feeding value of the corn protein is determined by the - Dr ying stove BMT ECOCELL Blueline Comfort
presence of essential amino acids (lysine, methionine, (Neuremberg, Germany);
cystine and others). The determination of the essential - Sartorius analytical balance (Gottingen, Germany);
amino acids concentration from corn is a key-instrument - Water purification system Mili-Q Ultrapure (Millipore,
for the nutritionists, irrespective of the animal species. Billerica, MA);
Hence, the necessity to develop more accurate and precise - Laboratory mill, GRINDOMIX GM 200 Retsch (Haan,
methods to determine the amino acids from the corn used Germany);
in animal diets. - Glass vials closing in flame Termodensirom (Bucharest,
The high performance liquid chromatography (HPLC) Romania).
has been used by many authors in order to analyse the Class A glassware Schott Duran (Meinz, Germany) was
amino acid content from all kind of feed matrices: sorghum used for transfer, dilution and storage of the working
grain [1], rice grain [2], peanut meal [1], fruits and solutions.
vegetables [3-6], soybean [7], soybean meal [7],
compound feeds [8], cotton [9]. EU Regulation no. 152/ Chemicals
2009 [10], laying down the methods of sampling and Cysteic acid and methionine sulfone, reference
analysis for the official control of feed, recommends for materials for HPLC, were supplied by Sigma (Deisenhofer,
separation and quantification of amino acids the use of ion Germany).
exchange chromatography (IEC) technique with post- Lysine, aspartic acid, alanine and leucine reference
column derivatization with ninhydrine and visible spectrum materials for HPLC, were supplied by Merck (Darmstadt,
detection. Germany).
There are several methods describing the amino acid Stock solution of the standard amino acid mixture was
analysis in corn samples: IEC [11-15]; gas chromatography prepared in hydrochloric acid (0.1 M) and contained 500
coupled with mass spectrometry (GC/MS) [16]; near µg/ mL for each amino acid (cysteic acid, methionine
infrared reflectance spectroscopy (NIRS) [17, 18] and sulfone, lysine, aspartic acid, alanine, leucine).
HPLC [1, 7]. The reversed phase high performance liquid The reagents: hydrogen peroxide (30%), disodium
chromatography (RP-HPLC) was used for separation of phosphate, sodium citrate, phenol, hydrochloric acid (37%,
the amino acids from corn, on a Superpac ODS-2 column d = 1.19 kg·L-1), sodium hydroxide, boric acid, sodium
coupled to an LKB dual pump HPLC system, controlled by disulphite (all analytical reagent grade), methanol and
a gradient generator with 50 minutes time of analyse [19]. acetonitrile (HPLC grade) were supplied by Sigma
The objective of our research was to develop and (Deisenhofer, Germany).
validate a fast and sensitive RP-HPLC method for Derivatization materials: orto-phtaldehyde (OPA),
methionine, lysine and cystine separation and mercapto-propionic acid (AMP) and 9-Fluorenylmethyl
determination. The proposed method was applied on chloroformate (FMOC), formic acid and thiodiethanol (all
several corn grain samples. analytical reagent grade) were supplied by Merck
(Darmstadt, Germany).
Experimental part Buffer solutions: citrate (19.61 g/L, pH 2.2) and borate
Equipment (33.35 g/L, pH 10.2) for the samples preparation, as well
- HPLC Finningan Surveyor Plus (Thermo-Electron as phosphate (3.85 g/L, pH 7.8) for the mobile phase were
Corporation, Waltham, MA); prepared in our laboratory.
* email address: [email protected]; Tel.: 0040 21 351 20 82

REV. CHIM. (Bucharest) ♦ 64 ♦ No. 7 ♦ 2013 http://www.revistadechimie.ro 673

 Oxidation mixture: 889 g formic acid were mixed with 1 min step that raised solvent B at 100%; 3 min step at
111 g water, and 4.73 g phenol were added; 4.5 mL from 100% solvent B; 1 min step that decreased solvent B at 0%
this solution were mixed with 0.5 mL hydrogen peroxide, and 5 min step at 0% solvent B; total analysis time of 35
and incubated at 20-300C for one hour in order to form min.
performic acid, then was cooled for 15 min in a refrigerator
before it was added to the sample. Validation of RP-HPLC method for the amino acids
(methionine, lysine and cystine) separation and
Corn grain samples determination
For the amino acid determination, the corn grains The proposed RP-HPLC method has been validated
samples were prepared according to Regulation 152/2009 according to the international regulations [22, 23]. The
[10], setting the sampling and analytical methods for the evaluated validation parameters were: accuracy, precision,
official control of the forages published by the Official linearity, detection and quantification limits, selectivity,
Journal of the European Union. The official method [20, recovery.
21], also used by other authors, involves the acid hydrolysis The accuracy was calculated with equation (1) and the
for the release of amino acids from the protein molecules, bias interval was calculated with equation (2).
preceded by oxidation with performic acid, for the sulphur
amino acids. (1)
The corn samples used in this study belong to 8 different
hybrids produced by the National Agricultural Research
Development Institute (INCDA – Fundulea, Romania) for (2)
multiple utilisations, among which that of raw feed
ingredient. The sampling of the corn grains was done from where: X is the average of the ten determinations; μ is the
collective samples (0.5 kg), reduced at 100 g sub-samples reference value.
using the method of the sub-sampling quarters. The sub- Linearity was checked for all amino acids, using
samples were grounded using a laboratory mill and stored standard solutions having the concentrations between 25
in plastic box, labelled. and 200 μg/mL. According to the ICH [22], precision may
For the determination of methionine and cystine, a be performed at different levels: repeatability, intermediate
quantity of 0.3 ± 2 . 10-4 g of corn samples (dried at 65°C precision and reproducibility. In this study, the repeatability
and grounded), was weighed on a watch glass and (intraday assay) was determined by ten consecutive
quantitatively transferred into a glass tube with 5 mL of injections of ten sample solution and then by calculating
oxidation mixture. The oxidized sample was kept at 0°C the relative standard deviation (RSD %) for the repeated
for 16 h in a refrigerator; after that, the excess of oxidation measurements. Intermediate precision was studied by
reagents was decomposed by addition of 0.84 g sodium running the whole method on five different days. The
disulphite. In order to perform the hydrolysis, 20 mL 6 M precision expressed by the reproducibility (inter-days
HCl were added into the glass tube, closed in flame and assay) was determined using five samples containing 100
introduced into the stove at 110°C, for 24 h. After full cooling μg/mL from each amino acid. The determinations were
at room temperature, the sample was concentrated using performed on 5 different days by two analysts, using
the rotary evaporator system and transferred quantitatively different glassware and the same working method.
using sodium citrate buffer, pH 2.2 in volumetric flasks of Precision (RSD %) was calculated with equation (3).
10 mL, filled to the mark with the same buffer.
For the determination of lysine, a quantity of 0.3 ± 2 . (3)
10-4 g of corn sample (dried at 65°C and grounded), was where: S is the standard deviation.
weighed on a watch glass and quantitatively transferred For precision, the required performance criterion is
into a glass tube with 20 mL 6 M HCl. The glass tube was established by the Horwitz equation:
closed in flame and introduced into the stove at 110°C, for
24 h. After complete cooling at room temperature, the (4)
sample was concentrated using the rotary evaporator
system. The sample solution was transferred quantitatively In order to check method accuracy, 10 standard samples
using sodium citrate buffer, pH 2.2 in volumetric flasks of with concentration of 100 μg/mL from each amino acid,
10 mL, filled to the mark with sodium citrate buffer. were prepared from standard stock solutions. The limit of
For methionine and cystine determination, 50 μL of final detection was calculated with equation (5) and the limit
sample solution was derivatized with 50 μL OPA, 300 μL of quantification was calculated with equation (6).
borate buffer pH 10.2 and 400 μL water. For lysine
determination the final sample solution was derivatized (5)
with 50 μL FMOC, 50 μL OPA, 250μL borate buffer pH 10.2,
400 μL water. The derivatized samples were thereafter
(6)
injected into the separation column of the HPLC.
where: r is the standard deviation of the response of the
Chromatographic separation blank and S is the slope of the calibration curve.
The chromatographic separation was performed by a The recovery was evaluated using five standard samples
RP-HPLC method using a Hypersil BDS column (C18), 250 and another five standard samples to which 50 µg/mL from
x 4.6 mm, particle size 5 μm. During the separation, the each amino acid were added. The percentage of analyte
column temperature was 45°C. Mobile phase used was a recovery was calculated with formula (7).
mixture of solvent A (phosphate buffer) and solvent B
(water : acetonitrile : methanol (20:20:60 v/v/v)). The (7)
separation was carried out at a flow rate of 1.7 mL/min
and at the solvent gradient (%, v/v) as follows: 2 min step
at 0% solvent B; 23 min step that raised solvent B at 57%;

674 http://www.revistadechimie.ro REV. CHIM. (Bucharest) ♦ 64♦ No.7 ♦ 2013

where X is the average of the five sample concentrations
without added analyte and X1 is the average of the five
sample concentrations with added analyte.
Statistics
Data were acquired and processed with ChromQuest
software, and the statistical analysis was done with
STATVIEW for Windows (SAS, version 6.0).
Results and discussions
The feeding quality of the corn is influenced by the quality
of its protein, which is the result of the ratio of the composing
amino acids.
In order to develop a RP-HPLC method for the separation
and determination of cysteine, methionine, lysine from corn Fig. 1. Influence of mobile phase composition on the resolution
samples, the method [24] was used as model. This method
describes a procedure for separation and quantification of resolutions of separation of the three pairs of amino acids
24 amino acids from a standard mixture. This method is were: 1.15 for cysteine + aspartic acid, 1.60 for methionine
recommended by the authors for amino acid analysis of + alanine and 0.85 for leucine + lysine.
protein and peptide hydrolysates. Method [24] required the In order to establish the optimum composition of solvent
use of Zorbax Eclipse-AAA column, 150×4.6 mm, 5 μm B, a sample of standard mixture was analysed using the
particle size. During the separation of amino acids from following components ratio: 10:45:45 (v/v/v); 12:44:44 (v/
standard mixture, the column temperature was 40°C. The v/v); 20:40:40 (v/v/v); 20:30:50 (v/v/v); 20:20:60 (v/v/v),
used mobile phase was a mixture of solvent A (phosphate and the working conditions described in method [24] with
buffer) and solvent B (water : acetonitrile : methanol, a column with 250 x 4.6 mm. The resolution of separation
10:45:45, v/v/v). The separation was carried out at a flow for each of the three pairs of amino acids: cysteine +
rate of 2 mL/min and at the solvent gradient (%, v/v) as aspartic acid, methionine + alanine and leucine + lysine
follows: 1.9 min at 0% solvent B; 16.2 min step that raised was improved by increasing the content of water and
eluent B at 57%; 0.5 min step that raised solvent B at 100%; methanol in the mixture (fig. 1). The mixture of water :
3.7 min step at 100% solvent B; 0.9 min step that decreased ACN : MeOH with 20:20:60 (v/v/v) ratio was chosen as
solvent B at 0% and 2.8 min step at 0% solvent B; total optimal mobile phase composition due to the highest
analysis time was 26 min. separation resolution values.
Using a Hypersil Gold column, 150×4.6 mm, 5μm In a study of the influence of the mobile phase flow rate
particle size and the method [24] described above, the on the retention time and resolution of amino acids
amino acids cysteine, methionine and lysine were separation, the flow rate of the mobile phase was varied
separated but only from the standard mixtures. The method from 1 mL/min to 2 mL/min (table 1). A mobile phase flow
[24] applied on corn samples, showed a poor separation rate of 1.7 mL/min was chosen to be optimal for a good
of lysine from leucine and also for the other amino acid peaks resolution in a reasonable time.
pairs from corn samples. In order to optimize the total analysis time, the influence
In order to establish the optimal conditions for the of the gradient program was investigated. Four gradient
separation and determination of amino acids from corn programs (further noted a, b, c, d) were designed.
grain samples, the following parameters were varied: type Gradients a, b and c have 3 min at 0% solvent B at start,
of column, ratio of components of solvent B (water : ACN while gradient d has 2 min at 0% solvent B. Solvent B raised
: MeOH), mobile phase flow rate, gradient program, at 57% in 32 min for gradient a, in 27 min for gradient b and
chromatographic column temperature. c and in 23 min for gradient d. After that, it followed a 1 min
For a good separation of cysteine, methionine and lysine, step that raised solvent B at 100% - for all the gradients, a 3
the our method used a Hypersil BDS column (C18), 250 x min step at 100% solvent B for gradient a, c, d and 7 min for
4.6 mm, particle size 5 μm. The method [24], recommends gradient b. The final steps are the same for all the gradients:
another type of column with a shorter length. Using a 1 min step that decreased solvent B at 0% and 5 min step
column with 250 x 4.6 mm and the working conditions at 0% solvent B. By choosing the gradient d, the total
described by method [24], the separation and analysis time decreased from 45 min (using gradient a), to
quantification of amino acids were done in 45 min and the

Table 1
INFLUENCE OF THE
FLOW RATE ON THE
RETENTION TIME VALUES
AND ON THE
RESOLUTION FOR THE
SEPARATION OF CYSTEIC
ACID, ASPARTIC ACID,
METHIONINE SULFONE,
ALANINE, LEUCINE AND
LYSINE

REV. CHIM. (Bucharest) ♦ 64 ♦ No. 7 ♦ 2013 http://www.revistadechimie.ro 675

 Fig. 3. The obtained chromatograms after applying the gradient programs:
a. chromatogram corresponding to gradient a;
b. chromatogram corresponding to gradient b; c. chromatogram corre-
sponding to gradient c; d. chromatogram corresponding to gradient d.
Note: 1-cysteic acid; 2-aspartic acid; 3-methionice; 4-alanice; 5-leucine; 6-
Fig. 2. Graphical representation of the gradient programs. a. 45 lysine; 7-unknown compound
min gradient program; b. 44 min gradient program; c. 40 min
gradient; d. 35 min gradient program

35 min. Figures 2 and 3 show the gradient programs profile RP-HPLC Method Validation
and the corresponding chromatograms. The RP-HPLC method used for the separation and
The influence of the column temperature on the quantitative determination of methionine, cysteine and
retention time values and on the resolution of separation, lysine was validated according to the international rules
was performed by testing different levels of temperature [22, 23].
(30, 35, 40, 45 0C). As a result, the total analysis time The required performance criterion for accuracy is the
decreased proportionally with the increase of column bias in interval of -2 – 2%. For 10 repeated determinations
temperature. For 450C, the retention time of the last amino and 9 degree of freedom [25], the value given in the table
acid (lysine) on chromatogram decreased with 2.19 min. is t = 2.26. The obtained results for accuracy were
The resolution obtained for the 3 pairs of amino acids evaluated using the Student’s t test [26]. The calculated t
studied varied in the range 0.95 and 2.80 (fig. 4). values (between -2.06 and -0.80), were smaller, in all cases
In conclusion, the amino acids: cysteic acid, methionine than the theoretical one. As shown in table 2 the data
and lysine can be separated in 35 min using a Hypersil BDS belong to the same population of values as the reference
column (C18), 250 x 4.6 mm, 5 μm particle size. Mobile value, so the method can be considered validated for the
phase consists in a mixture of solvent A (phosphate buffer accuracy parameter. The correlation coefficients, R2 of the
3.85 g/L) and solvent B (water : acetonitrile : methanol, linear regression equations exceeded 0.9985,
20:20:60, v/v/v), column temperature 45°C and a flow rate demonstrating a good correlation between the measured
of 1.7 mL/min and the gradient program d. Figure 4 shows response (area of the peak) and the concentration of the
a chromatogram obtained under these conditions, where analyte. The LOD value for the investigated amino acids
the retention times are the following: 2.91 min for cysteic ranged between 0.03 and 0.34 μg/mL, and the LOQ value
acid, 3.27 min for aspartic acid, 13.28 min for methionine, between 0.10 and 1.04 μg/mL. The detection and
14.53 min for alanine, 24.47 min for leucine and 25.17 min quantification limits obtained for the proposed method are
for lysine. comparable with the values reported by other authors for

Fig. 4. Example of chromatographic separation of a

standard mixture of aminoacids using the proposed
method: 1-cysteine acid; 2-aspartic acid; 3-methion-
ine; 4-alanine; 5-leucine; 6-lysine; 7-unknown
compound

676 http://www.revistadechimie.ro REV. CHIM. (Bucharest) ♦ 64♦ No.7 ♦ 2013

 Table 2
OBTAINED RESULTS FOR THE VALIDATION OF THE RP-HPLC METHOD FOR
THE DETERMINATION OF CYSTEINE, METHIONINE AND LYSINE

Table 3
RESULTS OBTAINED FOR CYSTINE, METHIONINE AND LYSINE DETERMINATION IN CORN
GRAIN SAMPLES USING THE RP-HPLC PROPOSED METHOD AND THE METHOD [24]

the same type of analyte [27 - 29]. For a concentration of 1 Amino acids determinations in corn grain samples
mg / L, the maximum accepted RSD value is 10.72%, The proposed RP-HPLC method was applied to the
according to Horwitz equation [30]. The obtained values determination of the essential amino acids: cysteine,
for RSD were between 2.71 - 6.29% for all the investigated methionine and lysine in real samples. Eight corn grain
amino acids. The range 80 - 120% is the required samples belonging to 8 hybrids, were collected for
performance criteria for recovery. The values obtained for analytical determinations. The results obtained by the
recovery varied from 95.27 to 102.90 for all the investigated proposed method, were compared with those provided by
amino acids. The results obtained from parameters application of the method [24] but using a 250 x 4.6 mm
evaluated were in the range of performance criteria column dimensions. Table 3 shows the results obtained
required and this provided the validation of all steps in the for cysteine, methionine and lysine determinations in the
amino acid analysis. corn grain samples.
As shown in figure 5, there is a good correlation (R2 =
0.9312 for cystine, 0.8620 for methionine, and 0.804 for

REV. CHIM. (Bucharest) ♦ 64 ♦ No. 7 ♦ 2013 http://www.revistadechimie.ro 677

 Fig.5.Correlation plot for amino acids concentrations determined by the proposed method (Hypersil BDS column (C18),
250 x 4.6 mm; 5μm particle size;45oC column temperature; mobile phase: solvent A - phosphate buffer, and solvent B - water: acetonitrile :
methanol (20:20:60, v:v:v); 1.7 mL / min flow rate, non-linear gradient; time of analysis: 35 min), and by the method [24] (Hypersil BDS column
(C18); 250 x 4.6 mm; 5 μm particle size; 30oC column temperature; mobile phase: solvent A - phosphate buffer, and solvent B - water
acetonitrile : methanol (10:45:45, v:v:v); 1 mL / min flow; non-linear gradient; time of analysis: 45 min)

lysine, respectively) between the values determined with

the proposed method and those obtained by application of 4.BLANCO-GÓMIS, D., PICINELLI-LOBO, A.M., ALONSO, J. M.,
the method [24] (using a 250 x 4.6 mm column GUTIERREZ-ALVAREZ, M. D., Z. Lebensm Unters Forsch, 194, 1992,
dimensions). p. 134.
In conclusion, the proposed RP-HPLC method enables 5.BARTOK, T., SZALAI, G., LORINECZ, Z. S., BORCSOK, G., SAGI, F.,
a rapid and sensitive amino acids determination from corn J., Liq. Chromatogr., 17, 1994, p. 4391.
grain samples. 6.KELLY, M.T., BLAISE, A., LARROQUE, M., J. Chromatogr. A., 1217,
nr. 47, 2010, p. 7385.
Conclusions 7.JAJIÆ, I., KRSTOVIÆ, S., GLAMOÈIÆ, D., JAKŠIÆ, S., ABRAMOVIÆ,
This paper presents the development and validation of a B., J. Serb. Chem. Soc., 77, 2012, p. 1.
RP-HPLC method for determination of cysteine, methionine 8.KING, K.J., HUBERP, J.T., SADIK, M., BERGEN, W.G., GRANT, A.L.,
and lysine from corn grain samples. These amino acids KING, V.L., J. Dairy Sci., 73, 1990, p. 320.
can be separated using a Hypersil BDS column (C18), 250 9.MARUR, C.J., LADASLAV, S., MAGALHES, C.A., R. Bras. Fisiol. Veg.,
x 4.6 mm, 5 μm particle size. The separation and 6, nr. 2, 1994, p. 103.
determination of cysteine, methionine and lysine were 10.*** Regulations (EC) no 152/2009 laying down the methods of
done in 35 min, with a column temperature of 45°C, a mobile sampling and analysis for the official control of feed, Official Journal
phase composed by a mixture of two solvents (solvent A – of the European Union.
phosphate buffer and solvent B – water : acetonitrile : 11.DAGHIR, N.J., FARRAN, M.T., BARBOUR, G.W., BECK, M.M., Poult.
methanol, 20:20:60, v/v/v), a flow rate of 1.7 mL/min and Sci., 82, 2003, p. 267.
a non-linear gradient. 12.OPAPEJU, F.O., NYACHOTI, C.M., HOUSE, J.D., Can. J. Anim. Sci.,
The proposed method proved to be accurate and precise 87, nr. 2, 2006, p. 221.
as shown by the results of validation and of the corn grain 13.O’QUINN, P.R., NELSSEN, J.L., GOODBAND, R.D., KNABLE, D.A.,
samples analysis. The application of the RP-HPLC method WOODWORTH, J.C., TOKACH, M.D., LOHRMANN, T., J. Anim. Sci.,
on 8 corn grains samples showed good results for the 78, 2000, p. 2144.
investigated amino acids. 14.PARSONS, C.M., ZHANG, Y., ARABA, M., Poul. Sci., 77, 1998, p. 1016.
15.SCOTT, M.P., BLANCO, M., Plant Genetic Resources, 7, nr. 3, 2009,
References p. 237.
1.LI, X., REZAEI, R., LI., P., WU, G., Amino Acids, 40, 2011, p. 1159. 16.PAVLÍK, M., PAVLÍKOVÁ, D., BALÍK, J., NEUBERG, M., Plant Soil
2.LU, K., LI, L., ZHENG, X., ZHANG, Z., MOU, T., HU, Z., J. Sci. Food Environ., 56, nr. 3, 2010, p. 125.
Agric., 89, 2009, p. 2377. 17.FONTAINE, J., SCHIRMER, B., HÖRR, J. J., Agric. Food Chem., 50,
3.FABIANI, A., VERSARI, A., PARPINELLO, G.P., CASTELLARI, M., 2002, p. 3902.
GALASSI, S., J. Cromatogr. Sci., 40, 2002, p.14.

678 http://www.revistadechimie.ro REV. CHIM. (Bucharest) ♦ 64♦ No.7 ♦ 2013

18.KRYEZIU, A., BAKALLI, R.I., KAMBERI, M.A., KASTRATI, R., MESTANI, 24.HENDERSON, J.W., RICKER, R. D., BIDLINGMEYER, B. A.,
N., World Poultry Science Association Proceedings of the 16th WOODWARD, C., Agi-lent Technical Note, part no. 5980–1193E, 1999.
European Symposium on Poultry Nutrition, 2007, p. 561. 25.DUCA, R.C., BRAVIN, F., DELAFORGE, M., VLADESCU, L., BADEA,
19.GAZIOLA, S.A., ALESSI, E.S., GUIMARAES, P.E.O., DAMERVAL, C., I.A., CRISTE, R.D., J. Agric. Food Chem., 57, nr. 22, 2009, p. 10497.
AZEVEDO, R.A., J. Agric. Food Chem., 47, 1999, p. 1268. 26.***http://w.w.w.statsoft.com/textbook/sttable.html.
20.RUTHERFURD, S.M., GILANI, S.G., Curr. Protoc. Protein Sci., 58, 27.AVINO, P., CAMPANELLA, L., RUSSO, M.V., Anal. Lett., 34, nr. 6,
2009, p. 11.9.1. 2001, p. 867.
21.MOUGHAN, P.J., RUTHERFURD, S.M., J. AOAC Int., 91, nr. 4, 2008, p. 28.PETRITIS, K., PERSON, M., ELFAKIR, C., DREUX, M.,
901. Chromatographia,
.. 60, nr. 5/6, 2004, p. 293.
22.***Validation of Analytical Methods. Definitions and Terminology, 29.BUTIKOFER, U., FUCHS, D., BOSSET, J.O., GMUR, W.,
step 5. ICH Topic Q2A (CPMP/ICH/381/95) 1995 a. Chromatographia, 31, nr. 9/10, 1991, p. 441.
23.***Validation of Analytical Procedures. Methodology, step 4. ICH 30.UNTEA, A.E., CRISTE, R.D.; VLADESCU, L., Rev. Chim. (Bucharest),
Topic Q2B (CPMP/ICH/281/95) 1995 b. 63, no. 4, 2012, p. 341.

Manuscript received: 5.03.2013

REV. CHIM. (Bucharest) ♦ 64 ♦ No. 7 ♦ 2013 http://www.revistadechimie.ro 679

View publication stats