INTRODUCTION:

Amino acids are the basic building blocks of proteins and one of the most important elements of the food pyramid^1,2,3,4, as they are important and necessary elements in the composition of the tissues of the human body^5,6. They also contribute directly to the flavour of food, as they are precursors of aromatic compounds with odours and colours formed as a result of thermal or enzymatic reactions during meat production and processing^7,8. Meat, which is the most important source of amino acids, is exposed to high temperatures during various cooking processes, which leads to the formation of heterocyclic amines (HCA) that are mutagenic and carcinogenic compounds^7,8,9.

The latter also results in a decrease in the amount of amino acids, as they represent the main substrates of the Maillard reaction (non-enzymatic browning reaction) in addition to the sugars and creatinine^7,8,10,11. On the other hand, heterocyclic amines are formed on the outer surfaces of meat and fish at concentrations of parts per billion when exposed to temperatures above 150°C^12,13, and more than 25 specific compounds are known^7,8,14,15.

Studies have shown that the most common polar heterocyclic amines in meat are PHIP (2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), MeIQx (2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline) and 4,8DiMeIQx (2 -Amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline)^7,8,16. These polar heterocyclic amines are listed in the Report of Carcinogens as reasonably anticipated to be human carcinogens^12,13,16.

Recently, chemical model systems have been used to study the formation of HCAs in vitro, where these compounds were first identified^14,15,17, and later in meat⁷. These systems are useful in studying the formation of heterocyclic amines and the factors and conditions that may influence their formation since many of the reactions and side products, formed in high-protein foods, are eliminated^18,19,20.

Amino acids are polar, non-volatile compounds that are zwitterionic in nature and exhibit a small ultraviolet (UV) absorbance²¹. Traditionally, they have been analyzed using amino acid analyzers with post-column derivatization or liquid chromatography with pre- and/or post-column derivatization²¹. Most of these methods are based on derivatization to enable amino acids to be detected with fluorescence or UV detectors^22,23. Several reagents were used for derivatization, such as orthophthaldialdehyde^22,23, ninhydrin, phenyl isothiocysnate, 9-fluorenymethyl-chloroformate, dansyl chloride, and aminoquinolyl-N-hydroxyl succinimidyl carbamate^24,25. Some of the derivatization reagents, such as orthophthaldialdehyde, can only react with the primary amines, while others, such as 1-(9-fluorenyl)ethyl chlorofomate, can interact with both primary and secondary amino acids^22,23.

Some studies showed the ability to analyze amino acids without derivatization using liquid chromatography, hydrophilic interaction chromatography (HILIC) and silica gel columns^26,27^,28. Although silica gel columns are usually used in normal phase chromatography, these columns enable the separation of underivatized amino acids using a mobile phase of acetonitrile and dipotassium phosphate, and those are detected using spectroscopy at a wavelength of 200 nanometers. Sherif et al. succeeded in the separation of 10 amino acids using a C18 column and UV detection at 225 nm without derivatization^29,³⁰.

High-performance liquid chromatography (HPLC) is the most widely used technique for assaying heterocyclic amines using UV absorbance, fluorescence, or UV absorption detectors connected to a mass spectrometer³¹^,32,33. Usually, the use of the detector depends on the method of sample preparation³⁴. A system that combined HPLC with capillary electrophoresis and mass spectrometry was used to identify 16 heterocyclic amino compounds^35,36. Moreover, ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) technology made it possible to separate 16 compounds within 20 minutes from meat extracts³⁷.

In order to preserve the nutritional value of meat and its products, this research aims to study the decrease in amino acids under different conditions in chemical model systems (water ratio, temperature, heating time), as well as monitor the reaction products that are given by more amino acids among the studied amino acids. Few studies demonstrate the analysis of amino acids in Maillard chemical model systems, whereas polar heterocyclic amines were addressed. While amino acids and heterocyclic amines co-exist in chemical model systems, this study indented to develop a simple RP-HPLC methodology for the simultaneous identification and quantification of amino acids (glycine, lysine and phenyl alanine) and resulted polar heterocyclic amines (MeIQx, 4,8 DiMeIQx and PHIP) in chemical model systems. In addition to study the effect of some conditions on amino acid decreasing and heterocyclic amine formation in these model systems.

MATERIALS AND METHODS:

Chemicals, standards and solvents:

Standard PHIP, MeIQx, and 4,8 DiMeIQx were purchased from Clear Synth Company. Phenylalanine (99%), glycine (99%), lysine (99%), creatinine (99%), and glucose (99%) were obtained from Sigma Aldrich. Diethylene glycol (100%) from the German Roth company, acetonitrile (HPLC grade), methanol (HPLC grade) from the Scharlau company, double-distilled water, and potassium dihydrogen phosphate from the HiMedia Leading BioSciences company. A 200 parts per million (ppm) separated stock solution of PHIP, MeIQx, and 4,8 DiMeIQx was prepared using methanol, and a 200ppm separated stock solution of phenylalanine, glycine, and lysine was prepared with water. Calibration standards for each compound are shown in Table 1.

Table 1: Calibration standards for each compound

Mix standards	Compound	Linearity range (ppm)
Phenylalanine and PHIP	Phenylalanine	25 - 200
Phenylalanine and PHIP	PHIP	0.1 – 4
Glycine and MeIQx	Glycine	20-300
Glycine and MeIQx	MeIQx	0.5- 20
Lysine and 4,8 DiMeIQx	Lysine	30- 400
Lysine and 4,8 DiMeIQx	4,8 DiMeIQx	0.1-5

Equipment and conditions

The (phenylalanine and PHIP) and (lysine and 4,8 DiMeIQx) mixes were separated in an HPLC system (Shimadzu, Japan) equipped with a rheodyne injector (model 77251), a UV-visible detector (model SPD-10AVP), and CLASS-VP software (version 6.14). Chromatographic analysis was performed using an ODS column (250 × 4.6mm × 5µm). The mobile phase is isocratic (acetonitrile: 25mM sodium dihydrogen phosphate) (40:60), and the flow rate was set to 0.85 ml/minute with an injection volume of 10µl and column temperature of 25°C. The eluents were detected by monitoring their absorbance at 214nm. Whereas mixed glycine and MeIQx were separated by the same apparatus and column with a different isocratic mobile phase (acetonitrile: 25mM sodium dihydrogen phosphate) (30:70), the flow rate was set to 0.7 ml/minute with an injection volume of 10µl and column temperature of 25°C. The eluents were detected by monitoring their absorbance at 200nm. The injections were performed in less than 20minutes, including column regeneration and stabilization during the last 10 minutes.

Preparing chemical model systems:

The model systems were prepared from the reaction substrates based on the proportion [amino acid: creatinine: glucose equals 0.4mM : 0.4mM : 0.2mM]. While the amounts of substrate (mole) were based on previous studies^8,9,20,34.

The effect of [deionized water: diethylene glycol] (V/V%) was tested on phenylalanine chemical model systems by weighing [0.066 g, 0.046g and 0.036g] in a row, then dissolving the latter using solutions of [25:75, 20:80, 15:85, 10:90, 5:95, 0:100] (V/V%). These model systems were heated in the oven at 150°C for 45 minutes, then cooled in an ice bath and kept in the refrigerator until used for the desired analysis.

The effect of time and temperature was tested by preparing chemical model systems of phenylalanine, glycine and lysine that contain the proportions mentioned above of reaction substrates using [deionized water: diethylene glycol] (20:80) as a solvent.

Model systems prepared to test time and temperature effects were heated in the oven at [150, 175, 200, and 225°C] for [15, 30, 45, 60, and 90minutes, respectively]. Then cooled in an ice bath and kept in the refrigerator until used for the desired analysis.

The chemical models were diluted ten times with the mobile phase, filtered with 0.45µm filters, and injected into the HPLC system.

Calibration curves and linearity:

The linearity of the calibration curve was evaluated by linear regression analysis using Microsoft Excel 2010 (Microsoft Corporation, Redmond, WA, USA). All of the standard compounds were injected three times, and their standard deviations were determined to assess the repeatability of the calibration curves.

Limits of detection and quantification:

The linear regression approach was used to estimate the limit of detection (LOD) and limit of quantification (LOQ) of each standard trial's unique peak. The LOD and LOQ values were computed using Equations 1 and 2 according to the International Conference on Harmonisation (ICH) recommendations. Where R and S are the regression line's residual standard deviation and the calibration curve's slope, respectively.

3.3R 10R

LOD = ------ ---(1) LOQ = -------- ….. (2)

S S

Precision and accuracy:

The precision of the suggested HPLC method was assessed by intra-day and inter-day changes using the relative standard deviation (RSD) value. The low RSD% (<2%) suggests that the approach is accurate enough for the analysis. The accuracy of the method was measured based on its capacity to quantify sample concentration and by comparing the theoretical (T) concentration of the samples to the experimental (E) concentration. For each concentration, three samples were produced, and each was tested three times. All of the analyses were completed on the same day, with the same equipment, and by the same analyst. Equation 3 was used to calculate accuracy, where P refers to the predicted value and A is the actual value.

Accuracy (%) = --------- 100 …… (3)

Statistical analysis:

All experiments were done in triplicate, and the results are represented as the mean ± standard deviation. One-way analysis of variance (ANOVA) was used for statistical analysis in Microsoft Excel 2010, where (p<0.05) refers to statistically significant differences and (p>0.95) proves that the null hypothesis is true and there are no differences between means.

RESULT:

Method validation:

ICH criteria were used to validate the chromatographic separation³⁰. The retention times of phenylalanine and PHIP standard solutions, glycine and MeIQx standard solutions, and lysine and 4,8-DiMeIQx were used to identify the sample analytes. The reference standards different mixes were injected singly to determine retention times. The external approach was used to quantify the eluted phenylalanine and PHIP, glycine and MeIQx, and lysine and 4,8DiMeIQx based on peak regions. In this part, we analyse the outcomes in terms of (1) chromatographic separation system efficiency; (2) curve calibration; (3) precision; and (4) accuracy.

a) Chromatographic separation:

All system suitability parameters (Table 2) showed acceptable values according to ICH, which was estimated from chromatograms (figure 1). The chromatogram of phenylalanine and PHIP, glycine and MeIQx, and lysine and 4,8 DiMeIQx shows a retention time of 2.625 and 4.869, 2.483 and 3.363, and 2.273 and 3.232 minutes, respectively, with a tailing factor of less than (1.7).

c) Precision:

The robustness of the chromatographic separation was evaluated by nine consecutive injections of the same sample during a working day. We used RSD% to measure the standard deviation of the mean value, which is considered precision (see Table 4). All compounds show an RSD% of less than 2% of peak area and retention time, thus proving the robustness and high reproducibility of the proposed method.

Table 4: The performance of repeated chromatographic separations is represented by the mean and RDS of area and retention time.

Compound	Area		Retention time
Compound	Mean ± SD	RSD%	Mean ± SD	RSD%
Phenylalanine	1386115 ± 8875	0.6403	2.539 ± 0.011	0.4331
PHIP	65798 ± 616.4	0.9368	4.895 ± 0.0303	0.6189
Glycine	614463 ± 1661	0.2702	2.478 ± 0.005	0.2117
MeIQx	305835 ± 2175	0.7110	3.333 ± 0.023	0.9605
lysine	122453 ± 483.1	0.394	2.276 ± 0.013	0.5516
4,8DiMeIQx	1123503 ± 5804	0.516	3.346 ± 0.028	0.9491

d) Accuracy:

The accuracy was estimated based on the mean values of the recovery tests. For the evaluation of the recovery rate, a phenylalanine chemical model system was spiked with (1 ppm of PHIP and 15 ppm of phenylalanine), (2 ppm of PHIP and 25 ppm of phenylalanine), and (3ppm of PHIP and 50 ppm of phenylalanine), respectively, and calculated. The accuracy rate averages were acceptable, with values ranging from (99.01 to 99.25%) for phenylalanine and (98.4 to 99.1%) for PHIP, as shown in (Table 5).

The lysine chemical model system was spiked with (0.25 ppm of 4,8 DiMeIQx and 10 ppm of lysine), (0.5 ppm of 4,8 DiMeIQx and 20 ppm of lysine), and (0.75 ppm of 4,8 DiMeIQx and 40 ppm of lysine), respectively, and calculated. The accuracy rate averages were acceptable, with values ranging from (98.47 to 99.15%) for lysine and (99.3 to 99.76%) for 4,8 DiMeIQx (Table 5).

The glycine chemical model system was spiked with (0.25 ppm of MeIQx and 25 ppm of glycine), (0.5 ppm of MeIQx and 50 ppm of glycine), and (0.75 ppm of MeIQx and 75 ppm of glycine), respectively, and calculated. The accuracy rate averages were acceptable, with values ranging from (98.23 to 99.55%) for glycine and (98.77 to 99.15%) for MeIQx, as shown in (Table 5).

Table 5: Accuracy of separation process based on actual (AV) and predicted value (PV) of compounds.

	PHIP (ppm)			Phenylalanine (ppm)
Before addition	1.825			25.78
AV (ppm)	AV (ppm)	PV (ppm)	Accuracy (%)	AV (ppm)	PV (ppm)	Accuracy (%)
1^st addition: 1 µg PHIP and 15 µg phenylalanine	2.825	2.794	98.9	40.78	40.48	99.25
2^nd addition: 2 µg PHIP and 25 µg phenylalanine	3.825	3.793	99.1	50.78	50.27	99.01
3^rd addition: 3 µg PHIP and 50 µg phenylalanine	4.825	4.746	98.4	75.78	75.21	99.2
	MeIQx (ppm)			Glycine (ppm)
Before addition	0.548			18.75
	AV (ppm)	PV (ppm)	Accuracy (%)	AV (ppm)	PV (ppm)	Accuracy (%)
1^st addition: 0.25 µg MeIQx and 10 µg glycine	0.798	0.789	98.87	28.75	28.62	99.55
2^nd addition: 0.5 µg MeIQx and 20 µg glycine	1.048	1.032	98.47	38.75	38.45	99.23
3^rd addition: 0.75 µg MeIQx and 40 µg glycine	1.298	1.287	99.15	58.75	58.39	99.39
	4,8 DiMeIQx (ppm)			Lysine (ppm)
Before addition	0.286			357.596
	AV (ppm)	PV (ppm)	Accuracy (%)	AV (ppm)	PV (ppm)	Accuracy (%)
1^st addition: 0.25 µg 4,8 DiMeIQx and 25 µg Lysine	0.536	0.529	99.36	60.76	60.34	99.3
2^nd addition: 0.5 µg 4,8 DiMeIQx and 75 µg Lysine	1.286	1.276	99.23	85.76	85.45	99.64
3^rd addition: 0.75 µg 4,8 DiMeIQx and 75 µg Lysine	2.786	2.767	99.32	110.76	110.5	99.76

Lysine and 4,8 DiMeIQx: the results show a noticeable decrease in the lysine content (figure 5.A) of the samples after 15minutes of heating at the different temperatures used. The temperature of 225°C showed the greatest decrease in the amount of lysine with the passage of time. The greater formation of 4.8 DiMeIQx (figure 5.B) was clearly noticed at temperatures of 200°C and 225°C with heating time, while the amounts formed at temperatures of 150°C and 175°C were relatively small.

DISCUSSION:

Maillard chemical model system have an important role in studying the effects of some conditions in phenylalanine, glycine, and lysine amino acids on decreasing and conjugated (PHIP, MeIQx, and 4,8 DiMeIQx) HCA formation in these model systems^18,19. In this study, we proposed a method for simultaneous identification and quantification of phenylalanine and PHIP in maillard chemical model systems. The method was validated using different criteria involving linearity (Figure 2), LoQ and LoD (Table 3), precision (Table 4), and accuracy (Table 5) based on ICH guidelines²⁹.

The calibration curves of standard amino acids and conjugated HCA were determined using the optimum chromatographic conditions, and then the linearity was evaluated using linear regression (figure 2 and Table 3). The results showed good residuals of curve regression of 0.9991 and 0.9985 for phenylalanine and PHIP, 0.9977 and 0.9973 for glycine and MeIQx, and 0.9950 and 0.9965 for lysine and 4,8 DiMeIQx, respectively, with less than 0.02 of a standard deviation. These results indicate the high sensitivity of the developed method towards the analytes.

The precision of intra-sample and intra-day phenylalanine and PHIP in chemical model systems was determined using three identical samples from three consecutive days. The results showed that there were no differences between the averages of the replicates between different days (p>0.95) with a low RSD% (<2%) as shown in (Table 4). The values of accuracy (Table 5) prove the high efficiency and reliability of the proposed method, where the accuracies of different compounds range between 97.95% and 99.96%.

Concerning the effects of water ratio on the model system, the results showed that a 20% water ratio led to higher formation of PHIP and a decrease in phenylalanine³⁸. Those outcomes refer to the lower solubility of the chemical model system's components in different solvents. Furthermore, the same outcomes are mainly observed at 0% and 5% of water ratio, which lead to fewer amounts of reaction substrates that can go through the maillard reaction that can be influenced by water activity. Those findings are related to the role of water as the main solvent for dissolving the reaction substrates of phenylalanine, creatinine, and glucose, which are necessary for the formation of PHIP. In contrast, the full solubility of these substrates was not observed except with water in excess of 20% of the medium of interaction.

Based on our findings, the most degraded amino acid during the Maillard reaction in model systems is phenylalanine, with a degradation rate of up to 99%, followed by lysine and then glycine, which was not accompanied by a proportional formation of conjugate heterocyclic amines in these systems. MeIQx was the most highly formed among the other heterocyclic amines, with a higher temperature and longer heating time. All the amino acids showed a clear decrease during the 15 minutes of the heating period, but this decrease was not associated with a clear formation of heterocyclic amines. Moreover, heterocyclic amines were not formed in a significant amount until after 45 minutes of the reaction time, which may explain the need for the time required to form these amines.

Regarding the changes in outcomes based on temperature and heating time, the content of the prepared systems of phenylalanine and PHIP decreased with the increase in temperature and heating time. These results can be explained by its participation in side interactions that lead to the formation of high-molecular weight polymers. Additionally, it was observed visually that an insoluble precipitate was formed in most of these systems³⁹. On the other hand, in the chemical model systems prepared from lysine and glycine, this matter was observed only at a temperature of 225 °C and a heating time of 90 minutes for the glycine systems.

CONCLUSION:

The method used in the simultaneous analysis of both amino acids and heterocyclic amines in Maillard model systems was easy and accurate. Through this method, by determining the content of these model systems of amino acids and conjugated heterocyclic amines, the optimal conditions for water, temperature, and time required for the depletion of amino acids and the formation of conjugated cyclic amines were determined. These conditions must be taken into account when studying the effect of some food additives on the Maillard reaction in model systems. The percentage of water, temperature, and heating time affect the Maillard reaction, as the amino acids decrease by a large percentage after a relatively short heating time. While heterocyclic amines known for their mutagenic effect and predisposing to cancer require a longer time and a higher temperature, so it is necessary to choose a technique to cook meat that does not require high temperatures or a long heating time, and it is also necessary to reduce the activity of the water.

CONFLICT OF INTEREST:

The authors declare that they have no conflicts of interest.

FUNDING:

Funding information: This research is funded by Damascus University- funder No.501100020595.

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Received on 04.07.2023 Modified on 17.08.2023

Research J. Pharm. and Tech 2024; 17(2):538-546.

DOI: 10.52711/0974-360X.2024.00084

Compound	Retention time (minute)	k	α	Theoretical plates	resolution	Tailing factor
Phenyl alanine	2.625	7.75	1.97	2646.655	--	1.698
PHIP	4.869	15.23	1.97	4860.657	9.285	1.504
Glycine	2.483	3.96	1.44	2732	--	1.325
MeIQx	3.363	5.73	1.44	4043	4.172	1.522
Lysine	2.273	6.58	1.49	2067	--	1.321
4,8 DiMeIQx	3.232	9.77	1.49	4178	4.795	1.533

Compound	Phenylalanine	PHIP	Glycine	MeIQx	Lysine	4,8 DiMeIQx
Retention time (minute)	2.539	4.895	2.483	3.363	2.483	3.363
Calibration range (ppm)	25 - 200	0.1 - 4	30- 300	0.5- 20	30- 300	0.1- 5
Regression equation	Y = 25184*X + 62054	Y = 127203*X + 5008	Y = 11722*X - 10594	Y = 60002*X + 2681	Y = 3768*X - 5411	Y = 209843*X + 8949
R² + SD	0.9999 ± 0.01	0.9999 ± 0.01	0.9997 ± 0.02	0.9999 ± 0.01	0.9998 ± 0.02	0.9995± 0.01
LoD (ppm)	2.92	0.029	4.379	0.065	6.35	0.029
LoQ (ppm)	9.73	0.098	16.63	0.28	21.19	0.091

Water ratio	Phenylalanine (mg/sample)	PhIP (µg/samples)
0	8.6767	0.0713
5	6.6413	0.0733
10	10.8314	0.3874
15	10.5498	0.3323
20	7.8167	4.0697
25	12.0407	3.46035