Identification of the Maillard reaction intermediates as divalent iron complexes in alanine/glucose/FeCl2 model system using ESI/qTOF/MS/MS and isotope labelling technique.

Due to their high reactivities and short half-lives, the detection of Maillard reaction intermediates is relatively difficult to achieve in a single analytical run. In this study, the formation of Maillard reaction intermediates from heated alanine/glucose mixtures (110 ​°C for 2 ​h) was investigated through their complexation with divalent iron using electrospray ionization/quadrupole time-of-flight mass spectrometry and isotope labeling techniques. Analysis of the mixtures indicated that this approach allows the simultaneous detection of many important labile and reactive Maillard reaction intermediates along with unreacted alanine and glucose in addition to various other Maillard reaction products, such as glyceraldehyde, erythrose, ribose, acetol, glycolaldehyde, fructosamine, glucosone, osones, deoxyosones, and Amadori products. Some osones and deoxyosones also formed their corresponding Schiff bases with alanine. The above mentioned Maillard reactions intermediates were detected either as binary metal complexes with alanine or with other enediol generating species including self-complexation adducts and they formed positively charged ions such as [M + H]+, [M + Na]+, [M + K]+, [M ​+ ​Fe35Cl]+, and [M ​+ ​Fe37Cl]+, that can be detected using the positive ionization mode.

Introduction

In the thermal processing of food, Maillard reaction intermediates (MRIs), resulting from the degradation of sugars and Amadori rearrangement products (ARPs) are considered important precursors for the development of color, flavor, and thermally generated toxicants (Yaylayan, 1997). Analysis of the MRIs and sugar degradation products (SDPs) has been achieved through the use of a range of time-consuming analytical technique (Yaylayan and Huyghues-Despointes, 1994; Davidek et al., 2002; Gensberger et al., 2013) that required elaborate procedures. Thus, various systems have been developed for the discrimination and determination of MRIs and ARPs. For example, volatile Maillard reaction products, including ARPs were analyzed by gas chromatography (GC) after derivatization step; however, this system has achieved limited success in analysis of the MRIs and their degradation products due to their low volatility (Yaylayan and Huyghues-Despointes, 1994). High-performance liquid chromatography (HPLC) and high-performance anion-exchange chromatography (HPAEC) based methods reported in the literature (Davidek et al., 2002; Gensberger et al., 2013) have been focused on the detection of nonvolatile water-soluble compounds of the Maillard products by using either refractive index or UV detection (Davidek et al., 2002; Gensberger et al., 2013). However, chemical derivatization steps (Davidek et al., 2002; Gensberger et al., 2013; Page et al., 1982) are essential for their analysis. Infrared (IR) spectroscopy was applied to quantify the open chain or keto forms of ARPs (Tamic and Hartman, 1983). In addition, Fourier transformed infrared (FTIR) spectroscopy has provided a more useful method to study the effect of environmental factors, such as pH and temperature, on the concentration of the keto form (Wnorowski and Yaylayan, 2003). Furthermore, nuclear magnetic resonance (NMR) spectroscopy, including 1D-1H NMR, 13C-NMR, DEPT-2D 1H–1H and 13C–1H correlational spectroscopy (COSY), and 2D nuclear Overhauser enhancement spectroscopy (NOESY) has also been employed for structural elucidation of the ARPs (Li et al., 2014; Kaufmann et al., 2016).

However, rapid analytical procedures for the simultaneous profiling of MRIs and SDPs have yet to be reported in the literature. In a previous study (Kim and Yaylayan, 2020), a convenient analytical procedure for profiling of SDPs through complexation with divalent metal ions combined with ESI/qTOF/MS was developed and applied for the analysis of honey. Here we demonstrate the utility of this technique to detect iron (II) catalyzed Maillard reaction intermediates of alanine and glucose using a methodology that most researchers already utilize, the qTOF/LC/MS with additional step of adding metal salts to the solution being analyzed. This step facilitates the detection of not only hard-to-identify and labile products but at the same time enhances the detection of nitrogen containing MRIs due to the ability metal ions to coordinate equally with nitrogen and oxygen atoms.

Materials and methods

Materials and reagents

L-alanine (98%), D-glucose, copper(II) chloride (CuCl2) (99.9%) and iron(II) chloride (FeCl2) (99%) were purchased from Sigma-Aldrich Chemical Co. (Oakville, Ontario, Canada). Alanine-3-13C (13CH3CH(NH2)CO2H) (98%) and glucose 13C–U (13C6H12O6) (99%) were purchased from Cambridge Isotope Laboratories (Andover, MI). Liquid chromatography-mass spectrometry (LC-MS)-grade water and methanol (OmniSolv, > 99%) were obtained from VWR International (Mississauga, Ontario, Canada).

Sample preparation

Test model systems were prepared by heating glucose (18 ​mg), alanine (9 ​mg), and FeCl2 (6.4 ​mg) in methanol or water (1 ​mL) in tightly closed stainless-steel reactors at 110 ​°C for 2 ​h. Control model systems were prepared by heating glucose (18 ​mg) and alanine (9 ​mg) with or without CuCl2 (5.6 ​mg) in methanol at 110 ​°C for 2 ​h. All samples were analyzed at least in two replicates, as indicated in Table 1.

Table 1

Composition of the model systemsa.

Model System
Control Model	Alanine was added to glucose solution and heated in the absence of metal ions - Ala/Glu
System	Alanine was added to glucose solution and heated in the presence of CuCl2 - Ala/Glu/CuCl2
Test Model	Alanine was added to glucose solution and heated in the presence of FeCl2 - Ala/Glu/FeCl2
System
Isotope Labelling Model System	Alanine was added to glucose13C–U solution and heated in the presence of FeCl2 - Ala/Glu[13C–U]/FeCl2
	Alanine-3-13C was added to glucose solution and heated in the presence of FeCl2 -Ala[13C-3]/Glu/FeCl2

All the Model systems were prepared in 1:1 ​M ratio and heated at 110 ​°C for 2 ​h in water or methanol by using a sealed stainless-steel reactor and analyzed in at least two replicates.

ESI/qTOF/MS

The dry reaction mixtures were dissolved in liquid chromatography (LC)-grade methanol to a concentration of 1 ​mg/mL. The samples were then diluted 10-fold in 10% methanol prior to analysis by ESI/qTOF/MS in positive mode. The ESI/qTOF/MS system was comprised of a Bruker Maxis Impact quadrupole-time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany) operated in positive-ion mode. Samples (1 ​μL) were injected directly into ESI/qTOF/MS. Instrument calibration was performed using sodium formate clusters. The electrospray interface settings were the following: nebulizer pressure, 0.6 ​bar; drying gas, 4 ​L/min; temperature, 180 ​°C; and capillary voltage, 4500 ​V. The scan range was from m/z 90 to 1000. Molecular formulae were assigned to all the observed peaks based on their exact m/z values by using the online software “ChemCalc” (Institute of Chemical Sciences and Engineering, Lausanne, Switzerland) (Patiny and Borel, 2013). ESI/qTOF/MS/MS was carried out in the multiple reaction monitoring (MRM) mode using a collision energy of 10.0 ​eV for the ions at m/z 252, 342, and 395.

Structural elucidation

Evidence for the proposed structures was provided through ESI/qTOF/MS analysis of their elemental composition, MS/MS analysis, and isotope-labeling. Furthermore, the incorporation of chlorine and copper in the identified complexes was also confirmed through the detection of their specific isotopic signatures; for chlorine the [M ​+ ​2] peaks accounted for ​~ ​25% of the peak intensity for the M ions, while for copper, the [M ​+ ​2] peaks accounted for ​~ ​30% of the peak intensity. Isotope labelling techniques was also used to generate the corresponding isotopically-labelled counterparts from [13C–U]-labelled glucose and [13C-3]-labelled alanine. The proposed structures represent only one possible isomeric form out of many possible forms for a particular nominal molecular weight, and are based on the most commonly reported structures in the literature.

Results and discussion

Sugar degradation products formed during the Maillard reaction are normally more amenable for analysis under negative ionization mode when analyzed by mass spectrometry (Figure S1). However, the addition of metal ions prior to analysis allows these mixtures to be analyzed in the positive ionization mode (Kim and Yaylayan, 2020), where nitrogen-containing MRPs are also readily detectable (Figure S2). Furthermore, the formation of metal complexes can prevent the degradation or further reactions of these reactive intermediates, while at the same time providing structural features for the development of the positive charge necessary for their detection under the positive ionization mode of the ESI/qTOF/MS system (Kim and Yaylayan, 2020). In the presence of metal ions, sugars, amino acids, MRIs (i.e., ARPs), and SDPs (i.e., 3-deoxyglucosone (3-DG), α-hydroxyl carbonyl, and α-dicarbonyl compounds) have the ability to undergo self- or random complexation to generate various metal-centered binary complexes, as listed in Table 2. In this study, the alanine/glucose model system was heated at 110 ​°C for 2 ​h in the presence of metal ions (FeCl2 or CuCl2) in water or methanol, and analyzed by ESI/qTOF/MS in the positive ionization mode. The heating of glucose and alanine in the absence of metal ions was also performed as a control, and it was found that the control system also produced alanine Amadori compound with glucose as the dominant product, but with reduced formation of other MRPs, as analyzed under positive ionization mode.

Table 2

Possible binary complexes of divalent metal ions with Maillard reaction precursors and intermediates.a

Identification of the Maillard reaction intermediates through complexation with Iron(II) using ESI/qTOF/MS and ESI/qTOF/MS/MS analysis

The Maillard reaction intermediates obtained (see Tables S1 and S2) in all the model systems heated at 110 ​°C for 2 ​h could be categorized into four groups (1) metal complexes with free amino acids/and or intact sugars, (2) ARPs and their corresponding metal complexes, (3) amino sugars and their corresponding metal complexes, and (4) reactive SDPs and their metal complexes. Table 2 shows selected examples of the above complexes. Previously (Kim and Yaylayan, 2020), we demonstrated that SDPs acting as bidentate ligands were converted into stable metal complexes, and were easily profiled by ESI/qTOF/MS in the positive ionization mode. In this study, the alanine/glucose model system was reacted in the presence or absence of FeCl2 or CuCl2 as metal catalysts to enhance the formation of MRPs, and at the same time to provide the metal ions needed for the formation of stable binary complexes for detection by ESI/qTOF/MS in positive ionization mode.

Detection of intact amino acid and intact sugar metal complexes

The amino acid metal complexes were detected as mono(alaninate)- and bis(alaninato)iron(II) complexes and were observed as [M]+ ions at m/z values of 143.9744 (C3H6FeNO2) and 233.0224 (C6H13FeN2O4), respectively. These structures were confirmed by observing the incorporation of one or two carbon atoms from [13C-3] alanine, but no carbon atoms from [13C–U] glucose. Furthermore, mono(alaninate)iron(II) was found to conjugate with glucose to give a signal as [M + H]+ at m/z 324.0378 (C9H18FeNO8) that was found to incorporate six carbon atoms from [13C–U] glucose and one C-3 atom from [13C-3] alanine. The ions corresponding to the free alanine or glucose were observed as [M + H]+ ions at m/z 90.055 (C3H9NO2) or [M + K]+ ions at m/z 219.0266 (C6H12KO6) not shown in Tables 2 and S1.

Detection of Amadori rearrangement products

The Amadori product of alanine with glucose, namely N-(1-deoxy-D-fructose-1-yl)-L-alanine, was observed as the dominant peak in both the Ala/Glu and the Ala/Glu/FeCl2 model systems, being detected in its free form [M]+ at m/z 252.1074 (C9H18NO7). It was also detected as [M + Na]+ at m/z 274.0891 (C9H17NNaO7) and [M + K]+ at m/z 290.0662 (C9H17NKO7). All structures were confirmed by observing the incorporation of six carbon atoms from [13C–U] glucose and one C-3 atom from [13C-3] alanine. The Amadori product was observed to undergo three dehydration reactions generating [M ​− ​H2O]+ at m/z 234.0967 (C9H16NO6) and [M ​− ​2H2O]+ at m/z 216.0863 (C9H14NO5), and [M ​− ​3H2O]+ at m/z 198.0755 (C9H12NO4). In addition, the hydrated form [M ​+ ​H2O]+ appeared at m/z 270.1175 (C9H20NO8). All three dehydrated ions and the hydrated ion were found to incorporate six carbon atoms from [13C–U] glucose and one carbon atom from [13C-3] alanine. Furthermore, the ARP was also observed as a bis-ARP iron(II) complex as [M + H]+ at m/z 557.1295 (C18H33FeN2O14), where twelve carbon atoms from glucose and two C-3 atoms from alanine were incorporated in the structure. In addition, the ARP was able to form iron complexes with alanine at m/z 395.0752 (C12H23FeN2O9) and with glucose at m/z 486.0917 (C15H28FeNO13), wherein the former was confirmed by detecting the incorporation of six carbons from [13C–U] glucose and two C-3 atoms from [13C-3] alanine, while the latter contained twelve carbon atoms from [13C–U] glucose and one C-3 atoms from [13C-3] alanine (see Tables S1 and 2). In methanol, the methyl ester of the ARP was also observed as the second dominant peak in the form of [M + H]+ at m/z 266.1229 (C10H20NO7), as well as [M + Na]+ at m/z 288.1059 (C10H19NNaO7). These structures were confirmed by observing the incorporation of six carbon atoms from [13C–U] glucose and one C-3 atom from [13C-3] alanine, respectively. Dehydrated ARP esters were detected as [M ​+ ​H ​− ​H2O]+ at m/z 248.1125 (C10H18NO6) and [M ​+ ​H ​− ​2H2O]+ at m/z 230.1019 (C10H16NO5) (see Tables 3 and S1). Both dehydrated ions were found to incorporate six carbon atoms from [13C–U] glucose and one carbon atom from [13C-3] alanine.

Table 3

Elemental composition and/or isotope incorporation of the common Maillard reaction intermediates obtained in the Ala/Glu/CuCl2, and Ala/Glu/FeCl2 model system in methanol (see Table S1).

Ala/Glu/CuCl2			Ala/Glu/FeCl2			Ala/Glu[13C–U]/FeCl2			Ala[13C-3]/Glu/FeCl2
[M ​+ ​X]	Elemental Compositiona	Error PPMb	[M ​+ ​X]	Elemental Composition	Error PPM	[M ​+ ​X]	Elemental Composition	Error PPM	[M ​+ ​X]	Elemental Composition	Error PPM
127.0386	C6H7O3	7.235	127.0389	C6H7O3	4.873	133.0585	[13C]6H7O3	8.629	127.0384	C6H7O3	8.809
ndc			143.9744	C3H6FeNO2	2.747	143.9741	C3H6FeNO2	4.831	144.9769	C2[13C]H6FeNO2	8.625
180.0862	C6H14NO5	5.539	180.0867	C6H14NO5	2.763	186.1058	[13C]6H14NO5	8.203	180.0857	C6H14NO5	8.316
162.0757	C6H12NO4	5.756	162.0761	C6H12NO4	2.671	168.0952	[13C]6H12NO4	9.292	162.0751	C6H12NO4	9.458
144.0645	C6H10NO3	10.885	144.0656	C6H10NO3	4.638	150.085	[13C]6H10NO3	7.977	144.0644	C6H10NO3	11.579
126.0545	C6H8NO2	7.961	126.0543	C6H8NO2	3.994	132.0751	[13C]6H8NO2	4.032	126.0544	C6H8NO2	8.754
202.0701	C6H13NNaO5	4.74	202.071	C6H13NNaO5	9.194	nd			202.068	C6H13NNaO5	5.653
206.1018	C8H16NO5	5.083	206.102	C8H16NO5	4.113	212.1201	C2[13C]6H16NO5	13.56	207.1042	C7[13C]H16NO5	9.669
188.0914	C8H14NO4	4.694	188.0917	C8H14NO4	3.099	194.1109	C2[13C]6H14NO4	7.789	189.0939	C7[13C]H14NO4	9.19
240.0159	C6H13[63Cu]N2O4	5.137	233.0224	C6H13FeN2O4	0.318	233.0205	C6H13FeN2O4	8.471	235.0253	C4[13C]2H13FeN2O4	16.524
242.013	C6H13[65Cu]N2O4d	9.609	nae			na			na
nd			234.9907	C6H11FeO6	0.85	241.0089	[13C]6H11FeO6	7.196	234.9897	C6H11FeO6	3.427
nd			216.9802	C6H9FeO5	1.196	222.9981	[13C]6H9FeO5	8.832	nd
nd			198.9686	C6H7FeO4	3.899	204.9872	[13C]6H7FeO4	11.24	198.9668	C6H7FeO4	12.946
nd			270.9673	C6H12[35Cl]FeO6	0.45	276.9861	[13C]6H12[35Cl]FeO6	4.157	270.9652	C6H12[35Cl]FeO6	−7.3
nd			272.9646	C6H12[37Cl]FeO6f	1.37	278.983	[13C]6H12[37Cl]FeO6	– 4.86	272.9629	C6H12[37Cl]FeO6	−4.86
252.1074	C9H18NO7	3.677	252.1082	C9H18NO7	1.297	258.1266	C3[13C]6H18NO7	7.19	253.1099	C8[13C]H18NO7	7.039
234.0967	C9H16NO6	4.538	234.0976	C9H16NO6	1.547	240.1161	C3[13C]6H16NO6	7.46	235.0994	C8[13C]H16NO6	7.304
216.0863	C9H14NO5	4.154	216.087	C9H14NO5	1.84	222.1056	C3[13C]6H14NO6	7.774	ndc
198.0755	C9H12NO4	5.719	198.0765	C9H12NO4	1.68	204.0963	C3[13C]6H12NO4	2.263	199.077	C8[13C]H12NO4	15.008
270.1175	C9H20NO8	5.152	270.1188	C9H20NO8	1.08	296.0822	C3[13C]6H20NO8	7.22	271.1204	C8[13C]H20NO8	6.81
274.0891	C9H17NNaO7	4.274	274.0908	C9H17NNaO7	0.626	276.1391	C3[13C]6H17NnaO7	0.287	275.0087	C8[13C]H17NnaO7	7.366
290.0662	C9H17KNO7	6.865	290.0673	C9H17KNO7	10.657	280.1084	C3[13C]6H17NKO7	7.142	291.0641	C8[13C]H17NKO7	11.9
266.1229	C10H20NO7	4.047	266.1237	C10H20NO7	1.041	272.1423	C4[13C]6H20NO7	6.636	267.1255	C9[13C]H20NO7	6.858
248.1125	C10H18NO6	3.677	248.1132	C10H18NO6	0.856	nd			249.115	C9[13C]H18NO6	7.094
230.1019	C10H16NO5	4.118	230.1026	C10H16NO5	1.076	236.1213	C4[13C]6H16NO5	7.101	231.1046	C9[13C]H16NO5	6.934
288.1059	C10H19NNaO7	0.075	288.1071	C10H19NNaO7	4.09	294.1239	C4[13C]6H19NnaO7	7.312	289.1074	C9[13C]H19NnaO7	6.491
nd			284.9825	C7H14[35Cl]FeO6	−1.15	291.0013	C[13C]6H14[35Cl]O6	– 5.69	284.9819	C7H14ClFeO6	−3.25
nd			286.9816	C7H14[37Cl]FeO6	6	nd			nd
331.0317	C9H18[63Cu]NO8	3.452	324.0378	C9H18FeNO8	1.184	330.0561	C3[13C]6H18FeNO8	6.704	325.0384	C8[13C]H18FeNO8	9.656
333.0304	C9H18[65Cu]NO8	– 1.9	na			na			na
360.1468	C12H26NO11	10.511	360.1503	C12H26NO11	0.793	372.1868	[13C]12H26NO11	10.87	360.1483	C12H26NO11	6.346
342.1387	C12H24NO10	3.861	342.14	C12H24NO10	0.061	354.1779	[13C]12H24NO10	6.717	342.1381	C12H24NO10	5.615
324.1281	C12H22NO9	4.184	324.1292	C12H22NO9	0.791	336.1675	[13C]12H22NO8	6.587	324.1275	C12H22NO9	6.036
306.1176	C12H20NO8	4.219	306.1188	C12H20NO8	0.299	318.157	[13C]12H20NO7	6.757	306.1172	C12H20NO8	5.526
nd			288.107	C12H18NO7	4.606	300.1432	[13C]12H18NO6	17.94	288.1051	C12H18NO7	11.2
402.0686	C12H23[63Cu]N2O9	3.374	395.0752	C12H23FeN2O9	0.247	401.0928	C6[13C]6H23FeN2O9	6.548	397.0789	C10[13C]2H23FeN2O9	7.825
386.058	C12H21[65Cu]N2O8	1.09	na			na			na
424.0504	C12H22[63Cu]N2NaO9	3.54	417.0581	C12H22FeN2NaO9	2.057	nd			419.0609	C12[13C]2H21FeN2O9	13.022
426.0495	C12H22[65Cu]N2NaO8	– 1.38	na			na			na
nd			415.054	C12H23FeO12	0.257	427.0929	[13C]12H23FeO12	2.93	415.0516	C12H23FeO12	5.525
nd			451.0306	C12H24[35Cl]FeO12	0.63	463.0713	[13C]12H24[35Cl]FeO12	1.03	451.0283	C12H24[35Cl]FeO12	−5.02
nd			453.0294	C12H24[37Cl]FeO12	3.94	465.0634	C12H24[37Cl]FeO12	– 9.62	453.0289	C12H24[37Cl]FeO12	2.84
493.0852	C15H28[63Cu]NO13	0.945	486.0917	C15H28FeNO13	1.425	498.1289	C3[13C]12H28FeNO13	4.748	487.0918	C14[13C]H28FeNO13	5.26
495.0837	C15H28[65Cu]NO13	0.321	na			na			na
475.0761	C15H26[63Cu]NO12	2.102	468.0798	C15H26FeNO12	0.2	480.116	C3[13C]12H26FeNO12	9.79	nd
564.121	C18H33[63Cu]N2O14	3.155	557.1295	C18H33FeN2O14	2.475	569.1658	C6[13C]12H33FeN2O14	4.531	559.1323	C16[13C]2H33FeN2O14	4.526
566.1197	C18H33[65Cu]N2O14	2.248	na			na			na

All of the ions listed in Table S1 are included in this table.

Error (in ppm) in calculating the elemental composition.

nd: not detected.

[M ​+ ​2] represents copper isotopes 65Cu.

na: not available.

[M ​+ ​2] represents chlorine isotopes37Cl

In addition to glucose, smaller sugars, such as glycolaldehyde, glyceraldehyde, and erythrose were also found to form Amadori products with alanine as either free or as mono(alaninato)iron(II) complexes. More specifically, the free glycolaldehyde Amadori compound was observed as [M + H]+ at m/z 132.0656 (C5H10NO3) and the iron complex was observed as [M]+ at m/z 185.9848 (C5H8FeNO3). Both structures incorporated two carbon atoms from glucose and one C-3 atom from alanine. Similarly, the glyceraldehyde and acetol Amadori compounds of mono(alaninate)iron(II) were observed at m/z 215.9958 (C6H10FeNO4) and m/z 200.001 (C6H10FeNO3), respectively, where three carbon atoms from glucose and one C-3 atom from alanine were incorporated in both structures. Moreover, the erythrose Amadori compound of alanine was also observed at m/z 246.0064 (C7H12FeNO5), which was found to incorporate four carbon atoms from glucose and one C-3 atom from alanine. Interestingly, 3-deoxyerythrosone was observed as the mono(alaninate)iron(II) complex of its Schiff base as [M]+ at m/z 227.9968 (C7H10FeNO4), whereas, 3-deoxyerythrose was observed at m/z 230.0117 (C7H12FeNO4) most likely as the Amadori compound. These structures were confirmed by detecting the incorporation of four carbon atoms from [13C–U] glucose and one C-3 atom from [13C-3] alanine. Similar to the case of 3-deoxyerythrosone, glycerosone (hydroxymethylglyoxal) was also observed as the mono(alaninato)iron(II) complex of its Schiff base at m/z 231.9913 (C6H10FeNO5), where three carbon atoms from glucose and one C-3 atom from alanine were found incorporated. Furthermore, the Schiff base of glucosone with methyl ester of alanine was detected as [M + H]+ at m/z 264.1085 (C10H18NO7) along with its dehydrated form [M ​+ ​H ​− ​H2O]+ at m/z 246.0979 (C10H16NO6). Both structures incorporated six carbon atoms from glucose and one C-3 atom from alanine.

MS/MS fragmentations of the Amadori product (m/z 252), the Amadori product-iron complex (m/z 395), and Amadori product of fructosamine (m/z 342) using a collision energy of 10 ​eV

To further confirm the structures of the glucose/alanine Amadori products, the free ARP, the Amadori product of fructosamine, and the ARP(alaninate)iron(II) complex observed at m/z 252, 342, and 395, respectively, were analyzed using ESI/qTOF/MS/MS, and the MS/MS fragmentations are shown in Fig. 1 that the free ARP and the Amadori product of fructosamine formed with glucose generated a greater number of fragment ions under a 10 ​eV ionization energy compared to the ARP(alaninate)iron(II) complex (m/z 395), which generated only four fragment ions, thereby indicating the stability imparted by metal ion complexation to the Amadori product (see Table 4). As shown in Fig. 1, the fragment ions are consistent with the proposed structures, and the MS/MS fragmentations of the free ARP (Fig. 1A) generated the expected diagnostic ions at m/z 88 and 97 (Xing et al., 2020) in addition to dehydrated ions at m/z 234 and 216 characteristic of the Amadori compounds in positive ionization mode. (Xing et al., 2020).

Fig. 1

Proposed MS/MS fragmentation pathways of (A) Amadori products (m/z 252), (B) Amadori product conjugated (alaninate)iron(II) their derivatives (m/z 395), and (C) glucose conjugated amino sugar (m/z 342) in the Ala/Glu/FeCl2 model system.

Table 4

MS/MS fragmentations of the ions observed at m/z 252, 342, and 395 generated in the Ala/Glu/FeCl2 model system using 10 ​eV collision energy (see Fig. 1).

Product ions of m/z 252
Structure	m/z	Elemental compositiona	Error PPMb	Glu [13C–U]	Error PPM	Ala [13C-3]	Error PPM
Image 2[M + H]+ ​= ​252	88.0386	C3H6NO2	– 14.24	0	– 14.24	ndc
	90.0547d	C3H8NO2	– 8.92	0	– 8.92	1	9.426
	97.028	C5H5O2	– 9.84	5	– 6.16	0	– 9.84
	99.0439	C5H7O2	– 7.11	Nd		0	– 7.11
	102.0546	C4H8NO2	– 8.85	1	8.329	1	7.358
	104.0705	C4H10NO2	– 6.28	1	– 26.73	1	7.694
	112.0386	C3H7NNaO2	10.28	0	– 4.89	1	– 7.10
	126.0546	C4H9NNaO2	11.92	Nd		nd
	146.0804	C6H12NO3	– 9.02	6	8.2	0	8.34
	168.0651	C8H10NO3	– 5.76	6	24.72	1	10.332
	216.0866	C9H14NO5	– 2.77	6	7.774	1	14.68
	234.0984	C9H16NO6	2.72	6	7.46	1	7.304
Product ions of m/z 342
Structure	m/z	Elemental composition	Error PPMa	Glu [13C–U]	Error PPMa	Ala [13C-3]	Error PPMa
Image 3 [M + H]+ ​= ​342	90.0548d	C3H8NO2	– 7.81	3	28.281	0	8.922
	104.0703	C4H10NO2	– 8.2	nd		0	8.202
	144.0659	C6H10NO3	– 1.17	6	7.977	0	11.579
	146.0812	C6H12NO3	– 3.55	6	8.2	0	8.34
	162.0762	C6H12NO4	– 2.67	6	9.292	0	9.458
	164.0921	C6H14NO4	– 1.11	6	– 10.06	0	– 10.26
	174.077	C7H12NO4	2.11	7	– 16.66	0	2.68
	288.1094	C12H18NO7	3.72	12	17.941	0	11.2
	306.1207	C12H20NO8	5.91	12	6.757	0	5.526
	324.1319	C12H22NO9	7.54	12	6.587	0	6.036
	342.1424	C12H24NO10	6.95	12	6.717	0	5.615
Product ions of m/z 395
Structure	m/z	Elemental composition	Error PPMa	Glu[13C–U]	Error PPMa	[13C-3] Ala	Error PPMa
Image 4 [M + H]+ ​= ​395	90.0552d	C3H8NO2	– 3.37	0	8.922	1	9.426
	215.9955	C6H10FeNO4	– 1.94	3	3.148	1	12.81
	246.0074	C7H12FeNO5	3.72	4	– 7.61	1	– 6.63
	306.0292	C9H16FeNO7	5.19	6	7.204	1	8.383

All of the ions listed in Figure 1 are included in this table.

Error (in ppm) in calculating the elemental composition.

Detection of amino sugars and their complexes

Amino sugars, such as fructosamine, are known to be formed in Maillard model systems containing metal ions (Nashalian and Yaylayan, 2015). They originate from the oxidative decarboxylation of glucose-conjugated bis(amino acid) metal complexes. As expected, fructosamine was observed only in the metal ion containing model systems, and was detected in the form of [M + H]+ at m/z 180.0867 (C6H14NO5) or as [M + Na]+ at m/z 202.071 (C6H13NNaO5). This ion, which is considered to the Amadori product of ammonia, underwent three characteristic dehydration reactions, generating [M ​+ ​H ​− ​H2O]+ at m/z 162.0757 (C6H12NO4), [M ​+ ​H ​− ​2H2O]+ at m/z 144.0645 (C6H10NO3), and [M ​+ ​H ​− ​3H2O]+ at m/z 126.0543 (C6H8NO2). All of the above ions incorporated six carbon atoms from glucose and no C-3 atoms from alanine, further supporting the proposed structures. Furthermore, the Schiff base formed between fructosamine and the Strecker aldehyde of alanine (acetaldehyde) along with its dehydration product were also observed at m/z 206.102 (C8H16NO5) and m/z 188.0917 (C8H14NO4), respectively. In the iron(II)-containing model systems (see Tables 3 and S1), both of the above ions incorporated six carbon atoms from glucose and one C-3 atom from alanine. In addition, the Amadori product formed between fructosamine and glucose was observed at m/z 342.14 (C12H24NO10), along with its three dehydration products at m/z 324.1292 (C12H22NO9), m/z 306.1188 (C12H20NO8), and m/z 288.107 (C12H18NO7). Moreover, the monohydrated product [M ​+ ​H ​+ ​H2O]+ was also detected at m/z 360.1503 (C12H26NO11) (Table 3). All the five ions, including the three dehydrated ions and one hydrated ion, were found to incorporate twelve carbon atoms from glucose and no C-3 atoms from alanine.

Detection of iron (II) complexes of sugar degradation products

The formation pathways of the reactive sugar degradation products, such as glyoxal and methylglyoxal have been previously reported in the literature (Kerler et al., 2010a; Hodge, 1953), and these compounds are up to 20,000-fold more reactive than glucose (Hofmann, 1999; Usui et al., 2007). As a result, they have been widely studied in model systems (Marceau and Yaylayan, 2009; Yan et al., 2019; Kerler et al., 2010b; Scalone et al., 2015; Thornalley, 2005; Wang and Ho, 2012); however, the profiling of SDPs is complicated due to their high reactivities and their ability to undergo further reactions prior to detection. In this study, it was found that these reactive intermediates, when generated in the presence of metal ions, can act as bidentate ligands and be converted into stable metal complexes that can be easily profiled by ESI/qTOF/MS. Furthermore, the ability of various SDPs to undergo self- or random complexation with other SDPs can generate multiple metal-centered binary complexes of the same SDPs; for example, 3-DG was found to form metal complexes with alanine and with itself, thereby providing several opportunities for their identification. In the absence of metal ions, no SDPs were detected due to their high reactivities. A total of 37 degradation products of the MRIs (including their dehydration products) were detected, as confirmed by isotope labelling experiments (see Tables 3 and S2). In this context, alanine was able to form iron(II) complexes with SDPs, such as glycolaldehyde, acetol, glyceraldehyde, 3-deoxyerythrose, and erythrose, which were observed at m/z 203.9955 (C5H10FeNO4), m/z 218.011 (C6H12FeNO4), m/z 234.0079 (C6H12FeNO5), m/z 248.0247 (C7H14FeNO5), and m/z 264.0162 (C7H12FeNO6), respectively. Supporting evidence for these structures were provided by observing the incorporation of expected number of carbon atoms from [13C–U] glucose and [13C-3] alanine. For example, the binary complex of glycolaldehyde with alanine was found to incorporate two carbon atoms from glucose and one C-3 atom from alanine, while acetol and glyceraldehyde complexes incorporated three carbon atoms from glucose, and 3-deoxyerythrose and erythrose complexes incorporated four carbon atoms from glucose with one C-3 atom originating from alanine. Glyceraldehyde and erythrose were also observed as their respective iron(II) complexes. More specifically, the glyceraldehyde complex was detected as [M]+ at m/z 144.9585 (C3H5FeO3) and erythrose was observed in the form of [M]+, [M ​+ ​35Cl]+, and [M ​+ ​37Cl]+, at m/z 174.969 (C4H7FeO4), 210.9473 (C4H8[35Cl]FeO4), and 212.9434 (C4H8[37Cl]FeO4), respectively. All structures were confirmed by detecting the incorporation of expected number of carbon atoms from [13C–U] glucose. For example, glyceraldehyde and erythrose incorporated three and four carbon atoms from [13C–U] glucose, respectively, but no C-3 carbon atom from alanine. Other SDPs, such as dideoxypentosone, erythritol, 3-deoxy-glucoson-5-one, rhamnose, and ribose were also observed as their corresponding iron(II) complexes. Some of these SDPs such as dideoxypentosone and 3-deoxy-glucoson-5-one were associated with solvent molecules, i.e., water or methanol. More specifically, the former was detected in its hydrated form associated with an iron(II) complex to give [M]+ at m/z 188.9843 (C5H9FeO4), where five carbon atoms from glucose were incorporated, while the latter was observed in its methanolated form complexed with iron(II) to give [M + Na]+ at m/z 268.9734 (C7H10FeNaO6) where six carbon atoms from glucose and no C-3 carbon atoms from alanine were incorporated in the structure. In addition, erythritol was observed as an iron complex with dehydrated erythrosone at m/z 295.0113 (C8H15FeO8). Supporting information was provided by observing the incorporation of eight carbon atoms from [13C–U] glucose (Kim and Yaylayan, 2020). Furthermore, rhamnose was found to form an iron complex with glyceraldehyde and was detected as [M + H]+ at m/z 309.0278 (C9H17FeO8), where nine carbon atoms from glucose was incorporated in the structure. Moreover, pentose was detected as a binary iron(II) complex i.e., [M + H]+ at m/z 355.0327 (C10H19FeO10), with its dehydrated form [M ​+ ​H ​− ​H2O]+ at m/z 337.0247 (C10H17FeO9). All of the above ions incorporated as expected ten carbon atoms from glucose and no C-3 atoms from alanine (Table S2).

Along with ARP, the 3-DG was also found to be one of the highest intensity peaks, and was associated with solvent molecules (i.e., water and/or methanol) in the forms of [M ​+ ​Fe35Cl]+, [M ​+ ​Fe37Cl]+, and [M + H]+ in the Ala/Glu/FeCl2 system. The monohydrated 3-DG iron(II) complex was detected in the form of [M ​+ ​Fe35Cl]+ at m/z 270.9673 (C6H12[35Cl]FeO6) and [M ​+ ​Fe37Cl]+ at m/z 272.9646 (C6H12[37Cl]FeO6), and also in the form of [M + H]+ at m/z 234.9907 (C6H11FeO6) (Kim and Yaylayan, 2020). Furthermore, its dehydration peaks were observed as [M ​+ ​H ​− ​H2O]+ at m/z 216.9802 (C6H9FeO5) and [M ​+ ​H ​− ​2H2O]+ at m/z 198.9686 (C6H7FeO4) (Table 3). All the five ions corresponding to 3-DG were found to incorporate six carbon atoms form glucose and no carbon atoms from alanine. In addition, 3-DG was detected as a binary iron(II) complex in the form of [M ​+ ​Fe35Cl]+ at m/z 451.0306 (C12H24[35Cl]FeO12) and [M ​+ ​Fe37Cl]+ at m/z 453.0294 (C12H24[37Cl]FeO12), as well as [M + H]+ at m/z 415.054 (C12H23FeO12). These structures were found to incorporate twelve carbon atoms from glucose, but no carbon atoms from alanine. Furthermore, 3-DG was also detected as methanolated iron(II) complex corresponding to [M ​+ ​Fe35Cl]+ at m/z 284.9825 (C7H14[35Cl]FeO6) and [M ​+ ​Fe37Cl]+ at m/z 286.9816 (C7H14[37Cl]FeO6). These hydrated- and methanolated 3-DG iron(II) complexes were confirmed based on their MS/MS fragmentations (Kim and Yaylayan, 2020), and by observing the incorporation of six carbon atoms from glucose. Moreover, Hydroxymethylfurfural (HMF) was detected as [M + H]+ at m/z 127.0389 (C6H7O3); both in the presence and absence of iron(II). The peak intensity of HMF in the Ala/Glu/FeCl2 system was at least 2-fold higher than in the Ala/Glu model system (Table 3). As expected, the ion observed at m/z 127.0389 incorporated six carbon atoms from glucose. Finally, 3-DG was detected also as alanine-iron(II) complex [M + H]+ at m/z 306.0274 (C9H16FeNO7), in addition to its corresponding dehydrated product [M ​+ ​H ​− ​H2O]+ at m/z 288.0174 (C9H14FeNO6) and [M ​+ ​H ​− ​2H2O]+ at m/z 270.0067 (C9H12FeNO5). Furthermore, 3-DG conjugated with mono(alaninato)iron(II) was also observed at m/z 342.0044 and m/z 344.0026, corresponding to [M ​+ ​Fe35Cl]+ (C9H17Fe[35Cl]NO7) and [M ​+ ​Fe37Cl]+ (C9H17Fe[37Cl]NO7), respectively (Table S2). All the five ions corresponding to 3-DG conjugated with mono(alaninate)iron(II) were found to incorporate six carbon atoms from glucose and one C-3 carbon atom from alanine.

Conclusions

The addition of metal ions to Maillard model systems before heating not only enhances the reaction and generates metal specific products, such as fructosamine, but also stabilizes many of the reactive enediol-containing moieties through the formation of binary metal complexes, which renders them easily detectable by electrospray ionization/quadrupole time-of-flight mass spectrometry (ESI/qTOF/MS).

CRediT authorship contribution statement

Eun Sil Kim: Data curation, Formal analysis, Methodology, Validation, and, Writing – original draft. Varoujan Yaylayan: Supervision, Conceptualization, Project administration, Writing – review & editing, and, Funding acquisition.

Declaration of competing interest

The authors declare no conflict of interest.