Table 3.
Application of chromatographic techniques in gelatin source authentication.
| Chromatographic techniques/detectors | Targeted biomarkers | Chromatographic conditions | Main results/limitations | References |
|---|---|---|---|---|
| RP-HPLC-MS/MS | Peptides (STGISVPGPMGPSGPR and SAGISVPGPMGPSGPR) | Mobile phase of HPLC was water and acetonitrile containing acetic acid. MS analysis was conducted with mass scan range of 400–900 m/z | Possible to distinguish between bovine and porcine gelatins by identifying the marker peptides in the digested gelatins using HPLC/MS | [83] |
| RP-HPLC-MS/MS | Peptides (GPPGSAGSPGK and GPPGSAGAPGK) | Mobile phase of HPLC was water and acetonitrile. MS analysis was conducted with mass scan range of 400–900 m/z | HPLC-MS/MS identified peptides in the digested gelatin sample able to distinguish between porcine and bovine gelatin. The sequence should be checked and verified for peptides like GPPGSAGSPGK and GPPGSAGAPGK seen in digested bovine and porcine gelatin, as the mass shift brought on by proline hydroxylation might be misinterpreted with the mass difference of Serine and Alanine residues | [84] |
| RP-HPLC-Fluorescence | Aspartic acid, Hystidine, Phenylalanine, Isoleusine, Lysine Glutamic acid, Glycine, Threonine, and Tyrosine | Mobile phase was acetate buffer (pH 5.9) a mixture of methanol: Acetate buffer: tetrahydrofuran (400:75:25 v/v). The peak regions at 340 nm excitation and 450 nm emissions were measured in order to quantify the amino acid profiles | Gelatin products made from different species can be distinguished using the RP-HPLC technique | [81] |
| RP-HPLC-Fluorescence | Threonine, Serine, Methionine, Proline, Hydroxyproline, Leucine, Isoleucine, Valine, Aspartate, Glutamic acid, Lysine, and Tyrosine | AccQ.Tag™ Eluent A, concentrate (WAT052890), deionized water, and acetonitrile were utilized as the system's tertiary solvents. The peak regions at 250 nm excitation and 395 nm emissions were measured in order to quantify the amino acid markers | The grouping patterns and variable correlations were verified on the database using 12 samples from commercial gelatin-based products. In order to ascertain gelatin from diverse sources, this quantitative approach is therefore highly helpful as a screening tool | [82] |
| HPLC-UV | Asparagine and Glutamine | Mobile phase was 40 mM acetate buffer (pH 5.5) along with methanol. The peak regions at 330 nm wavelength were measured in order to quantify the amino acid markers in pulsed electric field and OPA pretreated samples | Animal-derived gelatin could be identified using O-phthalaldehyde derivatized amino acids assessed by the pulsed electric field and HPLC-UV assays | [85] |
| RP-HPLC-MS/MS | Peptides (QGPSGPSGER, GETGPAGPAGPVGPVGAR and SAGISVPGPMGPSGPR) | HPLC mobile phase was water and acetonitrile containing formic acid. MS analysis was conducted with mass scan range of 300–1800 m/z. | The reliable authentication of porcine gelatin can benefit greatly from these frequent characteristic tryptic peptides | [86] |
| UPLC-MS/MS | Peptides (AGVMGPOGSR, GETGPAGPAGPVGPVGAR, and GEOGPTGVQGPOGPAGEEGK) | HPLC mobile phase was water and acetonitrile containing formic acid. MS analysis was conducted with mass scan range of 200–1500 m/z. | The presence of porcine gelatin could be efficiently determined at a level of 0.04%. | [21] |
| UPLC-MS/MS | Peptides (GNDGATGAAGPHypGPTGPAGPHypGFHypGAVGAK, GSDGSVGPVGPAGPIGSAGPHypGFHypGAHypGPK, GSDGSVGPVGPAGPIGSAGPHypGFPGAHypGPK, GFHypGTHypGLHypGFK, and GNDGATGAAGPHypGPTGPAGPHypGFPGAVGAK) | HPLC mobile phase was water and acetonitrile containing formic acid. Analysis was conducted with mass scan range of 350–1800 m/z. | The biomarkers could be used for distinguishing the deer horn gelatin and hide gelatin, as well as calculating adulteration in commercial gelatins. | [87] |