RAPID METHOD FOR THE DETERMINATION OF AMINO RAPID METHOD FOR THE DETERMINATION OF AMINO ACIDS IN MILK BY HIGH PERFORMANCE LIQUID ACIDS IN MILK BY HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY WITH PRE-COLUMN CHROMATOGRAPHY WITH PRE-COLUMN DERIVATIZATION AND FLUORESCENCE DETECTIONDERIVATIZATION AND FLUORESCENCE DETECTIONRosaria MarinoRosaria Marino11, , Marco IammarinoMarco Iammarino22, Marilena Muscarella, Marilena Muscarella22, ,

Antonella SantilloAntonella Santillo11, Mariangela Caroprese, Mariangela Caroprese11, Marzia Albenzio, Marzia Albenzio11

11Department of Production and Innovation in Mediterranean Agriculture and Department of Production and Innovation in Mediterranean Agriculture and Food System (PrIME) University of Foggia, Via Napoli, 25 - Foggia – Italy, Food System (PrIME) University of Foggia, Via Napoli, 25 - Foggia – Italy,

22Chemistry Department - Istituto Zooprofilattico Sperimentale della Puglia e Chemistry Department - Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata – Via Manfredonia 20 - Foggia – Italydella Basilicata – Via Manfredonia 20 - Foggia – Italy

Determination of the Determination of the amino acid (AA) profile amino acid (AA) profile in milk is essential in milk is essential

for qualitative for qualitative evaluation of peptides evaluation of peptides and proteins that can and proteins that can

affect the chemical and affect the chemical and nutritional properties nutritional properties

of milk. of milk. Hydrolysis is a Hydrolysis is a critical step for this critical step for this analysis (*), being the analysis (*), being the

main source of main source of analytical errors. analytical errors. * Fountoulakis M., Lahm H. W. (1998). Hydrolysis and amino acid composition

analysis of proteins. J. Chromatogr. A 826:109–134

The official reference procedure is a The official reference procedure is a liquid-phase hydrolysis performed in liquid-phase hydrolysis performed in

constantly boiling 6 constantly boiling 6 N N HCl under vacuum at HCl under vacuum at 110°C 110°C

for 24 h (*). for 24 h (*). The time The time required for required for hydrolysis hydrolysis could be a could be a

limitation of limitation of the official the official method.method.

* AOAC. 1994. Official Methods of Analysis. 16th ed. Association of Official Analytical Chemists, Arlington, VA.

Amino acids must be derivatized before injection, which could also represent a

critical step of the protocol. In particular, no studies have been carried out on the determination of AA in milk as a food specific substrate. The definition

of an optimal volume combination of sample, buffer, and fluorescence agents is necessary to obtain an adequate final pH

value to improve the derivatization efficiency.

The aim of this work was providing a new method for the determination of AA in

milk, more rapid and accurate, compared to the reference method.

This work This work has been developed through three has been developed through three steps:steps:1)1) Definition of Definition of the correct ratio of the correct ratio of sample/buffer/derivatization agents/water sample/buffer/derivatization agents/water to mix before the injection;to mix before the injection;2)2) Comparison among dComparison among different combinations ifferent combinations of of hydrolysis agenthydrolysis agent (HCl and (HCl and methanesulfonic acid), methanesulfonic acid), analysis conditionsanalysis conditions (time of analysis (from 24 h to 45 min), (time of analysis (from 24 h to 45 min), temperaturetemperature (from 110 to 160°C)) and the (from 110 to 160°C)) and the reference protocol to define a rapid and reference protocol to define a rapid and more efficient procedure;more efficient procedure;3)3) Validation of the method.Validation of the method.

AA DERIVATIZATIONAA DERIVATIZATIONFor all trials the derivatization reaction For all trials the derivatization reaction and the chromatographic separations (HPLC) and the chromatographic separations (HPLC) were performed according to Henderson were performed according to Henderson et et alal. (*).. (*).AA were derivatized on-line using AA were derivatized on-line using oo--phthaldehyde (OPA) for primary AA and 9-phthaldehyde (OPA) for primary AA and 9-fluorenylmethyl chloroformate (FMOC) for fluorenylmethyl chloroformate (FMOC) for secondary AA.secondary AA.

* * Henderson J. W., Ricker R. D., Bidlingmeyer B. A., Woodward C. 2000. Rapid, accurate, sensitive, and reproducible HPLC analysis of amino acids. Agilent Technologies, assignee. US Pat. No. 5980-1193E.

PROTEINS HYDROLYSIS

The hydrolysis of The hydrolysis of milk proteins was milk proteins was performed in Pyrex performed in Pyrex

microcapillary tubes microcapillary tubes (Pierce Chemical (Pierce Chemical Company, Rockford, Company, Rockford,

IL) under vacuum. The IL) under vacuum. The same volumes of milk same volumes of milk

(250 μL) and (250 μL) and hydrolysis agent (250 hydrolysis agent (250 μL) were used for μL) were used for

each method.each method.

The total protein content of milk samples The total protein content of milk samples used for this trials was determined by an used for this trials was determined by an infrared spectrophotometer and it was infrared spectrophotometer and it was 3.35%, this value was used as reference 3.35%, this value was used as reference parameter to optimize the derivatization, parameter to optimize the derivatization, also considering thealso considering the reference values for reference values for bovine milk amino acids (*)bovine milk amino acids (*)..

* Davis T. A., Nguyen H. V., Garcia-Bravo R., Fioretto M. L., Jackson E. M., Lewis D. S., Lee D. R., Reeds P. J. 1994. Amino acid composition of human milk not unique. J. Nutr. 124:1126–1132.

RESULTSThe optimized The optimized procedure of procedure of

derivatization is derivatization is reported in reported in

figure 1. By this figure 1. By this volumes ratio it volumes ratio it is possible to is possible to

exalt exalt the the response of the response of the fluorescence fluorescence amino acids amino acids signals.signals.

Figure 1Figure 1 –– C Correct ratio of orrect ratio of reagents for amino acids reagents for amino acids derivatization in milkderivatization in milk

RESULTS

The hydrolysis The hydrolysis performed using performed using hydrochloric acid 6N (adding phenol hydrochloric acid 6N (adding phenol liquefied (0,02% v/v), and ß-liquefied (0,02% v/v), and ß-mercaptoethanol (0,4% v/v) to reduce mercaptoethanol (0,4% v/v) to reduce losses of some amino acids during losses of some amino acids during hydrolysis) at 160°C for 60 minutes hydrolysis) at 160°C for 60 minutes has showed values of amino acid has showed values of amino acid concentrations comparable to concentrations comparable to reference values for bovine milk and reference values for bovine milk and it was the procedure most rapid and it was the procedure most rapid and efficient.efficient.

RESULTS

Figure 2 - Chromatogram of a cow milk sample. 1: Aspartic acid; 2: Glutammic acid; 1a: Asparagine; 3: Serine; 2a: Glutammine; 4: Histidine; 5: Glycine; 6: Threonine; 7: Arginine; 8: Alanine; 9: Tyrosine; 10: Valine; 11: Methionine; 12: Thryptophan; 13: Phenylalanine; 14: Isoleucine; 15: Leucine; 16: Lysine; 17: Proline.

RESULTSThe validation procedure has showed The validation procedure has showed good analytical parameters. The good analytical parameters. The linearity for each AA was expressed as linearity for each AA was expressed as determination coefficient (>0.99) The determination coefficient (>0.99) The LOD and LOQ determined were fit for LOD and LOQ determined were fit for purpose. Trueness (CV%) and recovery purpose. Trueness (CV%) and recovery percentages of each AA were evaluated percentages of each AA were evaluated to test the accuracy of the method (6 to test the accuracy of the method (6 repeated analysis of a sample spiked repeated analysis of a sample spiked with lisozime). with lisozime).

RESULTS

The recovery percentages of AA range The recovery percentages of AA range between 95.20 and 99.84%, with a between 95.20 and 99.84%, with a mean value of 98.38%.mean value of 98.38%.

The coefficients of variation The coefficients of variation registered were < 4.6%, with a mean registered were < 4.6%, with a mean value of 2.1%; this could be value of 2.1%; this could be considered an index of high considered an index of high repeatability.repeatability.

CONCLUSIONS

Hydrolysis of milk samples using 6Hydrolysis of milk samples using 6N N HCl at HCl at 160°C for 60 mins allows a good 160°C for 60 mins allows a good quantification of the milk AA, with quantification of the milk AA, with accuracy and the possibility to analyze a accuracy and the possibility to analyze a large number of milk samples in a short large number of milk samples in a short time. The proposed modification for time. The proposed modification for derivatization procedure allowed us to derivatization procedure allowed us to obtain good chromatographic resolution and obtain good chromatographic resolution and excellent symmetry of the peaks. This excellent symmetry of the peaks. This method was also characterized by high method was also characterized by high precision and low repeatability precision and low repeatability uncertainty for all the evaluated AA.uncertainty for all the evaluated AA.

RAPID METHOD FOR THE DETERMINATION OF AMINO RAPID METHOD FOR THE DETERMINATION OF AMINO ACIDS IN MILK BY HIGH PERFORMANCE LIQUID ACIDS IN MILK BY HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY WITH PRE-COLUMN CHROMATOGRAPHY WITH PRE-COLUMN DERIVATIZATION AND FLUORESCENCE DETECTIONDERIVATIZATION AND FLUORESCENCE DETECTIONRosaria MarinoRosaria Marino11, , Marco IammarinoMarco Iammarino22, Marilena Muscarella, Marilena Muscarella22, ,

Antonella SantilloAntonella Santillo11, Mariangela Caroprese, Mariangela Caroprese11, Marzia Albenzio, Marzia Albenzio11

11Department of Production and Innovation in Mediterranean Agriculture and Department of Production and Innovation in Mediterranean Agriculture and Food System (PrIME) University of Foggia, Via Napoli, 25 - Foggia – Italy, Food System (PrIME) University of Foggia, Via Napoli, 25 - Foggia – Italy,

22Chemistry Department - Istituto Zooprofilattico Sperimentale della Puglia e Chemistry Department - Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata – Via Manfredonia 20 - Foggia – Italydella Basilicata – Via Manfredonia 20 - Foggia – Italy