# Lactoferrin Production: A Systematic Review of the Latest Analytical Methods
## 3 Results and Discussion
### Conclusion Remarks
#### 3.1.2 Determination and Identification of Lactoferrin  
The most common currently used methods to identify and determine the content of Lf were high-performance liquid chromatography (HPLC) with an ultra-vis (UV) [97; 105], fluorescent (FL) [99], ultraviolet (UVD) [100], or diode array detector (DAD) [109], reversed phase high-pressure liquid chromatography (RP-HPLC) [101] with a DAD [106] and HPLC coupled with dual mass spectrometry (MS/MS) [104; 111]. Only two types of chromatographic columns were used for HPLC: C4 or C18 with the mobile phase water and acetonitrile, with formic acid or trifluoroacetic acid with gradient flow. Most authors using HPLC reported method validation parameters, including LOD and LOQ. The lowest LOD and LOQ were reported using UHPLC-UV, 0.0002 g/1 g and 0.0006 g/1 g, respectively [100]. In contrast, the highest values were obtained by HPLC-FL, 0.25 mg/1 mL and 0.8 mg/1 mL, respectively [99]. However, this method can distinguish intact Lf from its denatured form compared to high-sensitivity LC-MS/MS, which does not have such capabilities [99; 104]. Despite various instrument limitations (e.g., price), liquid chromatography methods are the most popular for detecting Lf due to their high specificity and accuracy. Currently, the less popular but still used method, ion exchange chromatography, is being replaced by newer methods [101] because of the high running costs resulting in a variable and relatively low Lf yield [115]. Impurities can also be introduced to the final product, requiring more purification steps [116].  
Despite the great popularity of Lf determination by chromatographic methods, many authors used other methods:optical biosensor immunoassay [98] with an immobilised anti-Lf antibody, which offers the advantages of simpler sample preparation and estimation of intact physiologically active protein. It has been implemented in the New Zealand dairy industry for 18 years. Image J software to determine the intensity of the protein bands after SD-PAGE [107]. capillary electrophoresis, which is a good alternative for determining the content of obtained Lf, but is affected by the system pH, surfactant, organic solvent, buffer system, and auxiliary air pressure [103]. Fourier transform infrared spectroscopy (FTIR) with an attenuated total reflectance (ATR) attachment [109]. Infrared spectroscopy measures the absorption of infrared radiation by each bond in a molecule and results in a spectrum that is commonly defined as % transmittance per wavenumber (cm). The ATR mode is simple compared to the conventional transmission model [117]. spectrofluorimetry to determine the Lf content of pharmaceutical preparations (capsules, sachets) and milk powder, obtaining a low LOD: 0.00002 mg/mL and LOQ: 0.000082 mg/mL. The authors indicate that the availability, sensitivity, durability, and affordability of this method make it better than routine Lf analysis [108]. the latex agglutination assay based on the agglutination that occurs when shiny latex beads come into contact with antigens or filters. It is a very quick and simple method for determining Lf with very good validation parameters [110]. electroanalysis, electrochemical quartz crystal microbalance (EQCM), and single radial immunodiffusion (SRID). The EIS results are within a linear range with an LOD of 125 nM to 3250 \(\upmu\)M and 65.2 nM, respectively [118]. The application of impedance and single-frequency capacitance spectroscopy was successfully demonstrated for Lf determination and shortened analysis time.  
The remaining methods for determining Lf were relatively simple, such as spectrophotometry at a wavelength of 280 nm, but they required the use of more precise purification methods [102].