# Lactoferrin Production: A Systematic Review of the Latest Analytical Methods
## 3 Results and Discussion  
Obtaining Lf is becoming more popular due to its beneficial health properties. The methods of obtaining and determining Lf differ in their applications; some of them can be used for bovine Lf, others for camel Lf, and human Lf. Typically, bovine Lf is used on the largest scale due to good and cheap access to cow's milk or by-products used to produce dairy based on cow's milk, i.e., whey. Lf from human milk can be used to treat premature  
Figure 3: Search strategy. babies, whereas Lf from camel milk is used in areas of camel milk abundance. Nevertheless, obtaining and determining Lf depends on the source material: liquid milk, powdered milk, Lf supplements, and whey. Each of these matrices has its specification, distinct Lf concentration, structural composition, and pH; therefore, the production methods differ. Over the last five years, new analytical methods have been developed to improve Lf production and determination by reducing production costs and its toxicity, increasing the Lf yield, or improving the accuracy of Lf determination. Table 2 presents the latest purification and determination methods depending on the matrix. Out of 17 studies, 11 covered bovine Lf, but it was not the only source. Lf was mainly prepared from milk, its powdered form [97; 98; 99; 100; 101; 102; 103] or milk-based infant formula [97; 98; 99; 104; 105]. Some articles also used whey or whey protein concentrate [106; 107], human milk [108; 109], colostrum [110], camel milk [111], or lactoferrin powder [112]. [PERSON] et al. used Boron-doped titania (inorganic boric acid and highly hydrophilic titanium dioxide) to detect Lf in dairy products. Parameters like extraction time, pH, desorption time, and solution had to be optimised to achieve an adsorption capacity of 63.9 mg/g. It was confirmed by UHPLC-UV and made a cheap and efficient determination technique [97]. [PERSON] et al. developed and validated a method for quantifying Lf in infant formulae using ultra-high performance LC-tandem mass spectrometry (MS/MS). All validation parameters were met with a precision RSDR (mean recovery, repeatability) ranging from 2.1 to 7.1 and an intermediate RSDR ranging from 7.0 to 10.4 across types of infant formulae. Accuracy with certified reference material resulted in mean recoveries of 91.7-96.4%. This study supports manufacturing specifications and nutrient labelling requirements [104]. [PERSON] et al. described and evaluated the analytical performance of an optical biosensor immunoassay to determine Lf in infant formulae and other nutritional powdered formulae. This method is rapid, sensitive, precise, accurate, and simple to implement with an analytical range of 0-200 mg/hg, detection limit of 0.8 mg/hg, recovery of 96.1-109.2%, and repeatability of 1.0-5.3%. Additionally, it provides physiological analytical information because the method is specific for intact Lf, and thermally denatured Lf generates no measurable binding response [98]. [PERSON] et al. described a method using immunoaffinity magnetic purification coupled with high-performance liquid chromatography-fluorescence (HPLC-FL) detection for the determination of Lf in dairy products (pasteurised milk, infant formula powder, whey protein concentrate). This method is more sensitive (curve range 0.8-30 mg/mL) than the HPLC-UV method (10-1200 mg/mL). Additionally, in comparison to LC-MS/MS, where the sensitivity is higher (1-1000 nM), it is possible to distinguish intact Lf from its denatured form. This method is simple, specific, and sensitive, but not suitable for yoghurt because its pH is not conducive to immunoaffinity binding [99]. This method is shown in Figure 4.  
[PERSON] et al. used an aptamer affinity column combined with high-performance liquid chromatography HPLC-UVD for the purification and enrichment of LF in milk. This purification method is a good alternative to the heparin affinity column due to reduced cost and improved stability.