**4. Application of ultra performance liquid chromatography in food safety**

In recent years, the demand of UPLC-MS/MS in food analysis has increased, because of the novel characteristics of UPLC with good resolution, better accuracy and sensitivity and reproducibility. Since its inception, it has reduces the time of food scientists as well as cost of the analysis because of its capability of producing more valuable, reliable, and reproducible data. The UPLC sensitivity has reached to ppb and ppt levels by virtue of which a food analyst would be more confident in ensuring safe food for consumption. Analysis of several food components as well as food contaminants has been performed using UPLC-MS/MS technique. By using this technique, below-mentioned food matrices can be tested for ensuring better food safety and we can also get more accurate qualitative and quantitative data of samples with high standards [11].


*UPLC-MS: An Emerging Novel Technology and Its Application in Food Safety DOI: http://dx.doi.org/10.5772/intechopen.92455*

• Determination of phytoconstituents

*Analytical Chemistry - Advancement, Perspectives and Applications*

when migrating toward smaller particles:

to column length.

separations [9, 13, 15].

**in food safety**

with high standards [11].

• Amino acid profiling [9, 27]

• Metabolomics study in food safety [9]

more peak capacity per unit time in gradient separations, as per the requirement in several food safety application notes. Moreover, another equation comes into play

> 1 dpc

*F* α = (5)

(6)

xxx

Van Deemter equation revealed that, as particle size decreases, the optimum flow *F* opt to reach maximum *N* increases. However, flow rate is directly proportional to back pressure as smaller particle sizes needed much higher operating pressures. Efficiency is inversely proportional to the particle size however proportional

> *<sup>L</sup> <sup>N</sup> dp* α

Moreover, the column can be shortened by the same factor as the particle size without loss of resolution. Although non-porous, high-efficiency 1.5-μ particles are easily available in market, but these non-porous particles suffer poor loading capacity as well as poor retention because of low surface area. However, silica-based column have good mechanical strength nonetheless, it can undergo to a number of disadvantages, such as limited pH range and tailing of basic analytes. In addition to that, polymeric columns can overcome pH limitations. Moreover, packed column bed and their uniformity are also important, mainly if shorter columns have to uphold resolution while achieving the objective of faster

**4. Application of ultra performance liquid chromatography** 

• Determination of antibiotic residue in food matrices

• Quantification of pesticides residues in food [24–26]

• Multi-drug residue quantitation in food matrix [28]

• Analysis of food contaminants in food matrices

In recent years, the demand of UPLC-MS/MS in food analysis has increased, because of the novel characteristics of UPLC with good resolution, better accuracy and sensitivity and reproducibility. Since its inception, it has reduces the time of food scientists as well as cost of the analysis because of its capability of producing more valuable, reliable, and reproducible data. The UPLC sensitivity has reached to ppb and ppt levels by virtue of which a food analyst would be more confident in ensuring safe food for consumption. Analysis of several food components as well as food contaminants has been performed using UPLC-MS/MS technique. By using this technique, below-mentioned food matrices can be tested for ensuring better food safety and we can also get more accurate qualitative and quantitative data of samples

**36**


#### **4.1 Determination of antibiotic residue in honey**

Several antibiotic residues such as streptomycin (**Figure 2**), chloramphenicol, tetracycline etc. has been identified and quantified in honey by using UPLC-MS coupled along with electron spray ionization [37, 38].

#### **4.2 Multi pesticide residue analysis in cereal grains**

Pesticides are chemicals widely used against plant pests in agriculture and farming to increase crop production, either against plant diseases or prophylactic usage. Currently, more than 350 pesticides are known, which are used to protect plants or plant products; however these pesticide are not allowed more than the permitted level. In addition to that, these chemicals could be dangerous to human health. The function of full scan UHPLC-Orbitrap-MS/UPLC-MS is adequate enough to enable detection and accurate analysis of mass measurement of a broad range pesticides residue at very lowest concentration in complex sample matrices [24–26].

#### **4.3 Amino acid profiling**

Amino acid profiling is one of the important proximate analyses parameter in food safety, as it contributes major portion of protein and an essential component of human diet. However, among the several protein food resources mammalian milk is purest food available over the globe. However, free amino acids are calculated from total nitrogen present in milk. UPLC coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS) system has been estimated for free amino acid analysis in milks of human, rat, and cow as presented in **Figure 3**. Moreover, UPLC-ESI-MS/MS allowed the quantitation of 21 free amino acids in 10-minute run time using labeled amino acids as internal standard in mammalian milk [27].

**Figure 2.**

*Chromatogram showing blank honey sample (A) vs. spiked honey sample (B).*

**Figure 3.**

*Free amino acid ion chromatogram obtained in human milk (cumulative).*

#### **4.4 Metabolomics study in food safety**

In recent years, the performance of UPLC has set the stage for a myriad of metabolomics analysis in plants and plant products. UPLC along with qTOF (quadrupole time of flight) system has been applied for semi-polar metabolite analysis in tomato fruit model. Moreover, UPLC coupled with qTOF mass spectrometer produces high-resolution and mass accuracy, good dynamic range, and a fast spectral acquisition capacity, which makes UPLC one of the most appropriate techniques for extensive profiling of many plant metabolites. In addition to that UPLC-MS

**39**

*UPLC-MS: An Emerging Novel Technology and Its Application in Food Safety*

along with multivariate data analysis has been used for metabolomics profiling of Trignella seed. Metabolomic study of all the three Trigonella species *T. caerulea*, *T. corniculata*, and *T. foenum-graecum* identified 93 metabolites including 26 saponins, 5 peptides, 22 C/O-flavonoid conjugates, and 9 fatty acids as determined in **Figure 4**. Out of which, various novel compounds such as dipeptides, flavonoids

*Metabolomic analysis of* T. caerulea*,* T. corniculata*, and* T. foenum-graecum *using UPLC-qTOF-MS.*

In recent year, poultry industries have become million dollar industries due to higher consumption among the world population. However, multi-drug residue is very common in poultry muscles as poultry husbandry people illegally feed several drugs such as quinolones, amantadine, sulfonamides, tetracycline, amoxicillin, lincomycin, and so on. UHPLC-ESI-MS/MS has been used to analyses such veterinary drug residues in poultry muscle ranging from very polar to nonpolar compounds. UHPLC-ESI-MS/MS operating in positive multiple reactions monitoring (MRM) has been operated to quantify most of the multi-drug residue in sample [28].

Method development plays a great role in concluding for any analytical method. In quantitative evaluation, development of method can roughly divided into three parts

Depending upon physical or chemical characteristics of analyzing components method development could be easily performed considering the following factors

*DOI: http://dx.doi.org/10.5772/intechopen.92455*

were reported for first time [39–41].

**Figure 4.**

**4.6 Method development and validation**

• Mass spectrometry parameters

• Preparation of sample

• Optimization of chromatography conditions

**4.5 Multi-drug residue quantitation in poultry muscle**

*UPLC-MS: An Emerging Novel Technology and Its Application in Food Safety DOI: http://dx.doi.org/10.5772/intechopen.92455*

**Figure 4.** *Metabolomic analysis of* T. caerulea*,* T. corniculata*, and* T. foenum-graecum *using UPLC-qTOF-MS.*

along with multivariate data analysis has been used for metabolomics profiling of Trignella seed. Metabolomic study of all the three Trigonella species *T. caerulea*, *T. corniculata*, and *T. foenum-graecum* identified 93 metabolites including 26 saponins, 5 peptides, 22 C/O-flavonoid conjugates, and 9 fatty acids as determined in **Figure 4**. Out of which, various novel compounds such as dipeptides, flavonoids were reported for first time [39–41].

### **4.5 Multi-drug residue quantitation in poultry muscle**

In recent year, poultry industries have become million dollar industries due to higher consumption among the world population. However, multi-drug residue is very common in poultry muscles as poultry husbandry people illegally feed several drugs such as quinolones, amantadine, sulfonamides, tetracycline, amoxicillin, lincomycin, and so on. UHPLC-ESI-MS/MS has been used to analyses such veterinary drug residues in poultry muscle ranging from very polar to nonpolar compounds. UHPLC-ESI-MS/MS operating in positive multiple reactions monitoring (MRM) has been operated to quantify most of the multi-drug residue in sample [28].

#### **4.6 Method development and validation**

Method development plays a great role in concluding for any analytical method. In quantitative evaluation, development of method can roughly divided into three parts


Depending upon physical or chemical characteristics of analyzing components method development could be easily performed considering the following factors

*Analytical Chemistry - Advancement, Perspectives and Applications*

**38**

**Figure 3.**

**Figure 2.**

**4.4 Metabolomics study in food safety**

*Free amino acid ion chromatogram obtained in human milk (cumulative).*

*Chromatogram showing blank honey sample (A) vs. spiked honey sample (B).*

In recent years, the performance of UPLC has set the stage for a myriad of metabolomics analysis in plants and plant products. UPLC along with qTOF (quadrupole time of flight) system has been applied for semi-polar metabolite analysis in tomato fruit model. Moreover, UPLC coupled with qTOF mass spectrometer produces high-resolution and mass accuracy, good dynamic range, and a fast spectral acquisition capacity, which makes UPLC one of the most appropriate techniques for extensive profiling of many plant metabolites. In addition to that UPLC-MS

like selection of column, mobile phase, pH, and particle size and flow rate in any chromatographic setting.

The benefits of using UPLC-MS method over others were better recovery, good repeatability, and amount of extraction solvent volume. The selection of ionization techniques is depending on analytical results with pretreated samples. UPLC-MS/ MS tuning parameters and scan modes are decided by uninterrupted infusion of standard solution, depending on the sensitivity and specificity needed. Few key elements for method development are sample pre-treatment, chromatography, internal standard, choice between electrospray ionization (ESI) and APCI, and mass spectrometry [42]. On the other hand, method validation results support for new analytical procedures or new drug development such as Carnosol, Carnosic acid, and Rosmarinic acid in food matrices. Validation required defining performance of developed method and reliability of obtained results. The analytical developed method could be utilized for quantitation application then it would be better to be validated to ensure minimum requirement of validation experiments along with satisfactory results [43].

#### **4.7 Determination of acrylamide in food matrices**

Acrylamide as a risk factor come to scientists attention recently, as its discovery in food was accidental. Formation of acrylamide in different types of cooked food or processed food at high temperatures reported recently. Several researchers have validated an analytical method for the analysis of acrylamide in food by UPLC-MS/MS as determined in **Figure 5**. Various reports suggests that processed food such as potato, coffee, bakery and other human dietary products contain acrylamide. One of the study carried out in Cyprus found that potato crisp had highest amount of acrylamide (642 ppb), followed by French fries and biscuits. Concurrently, regular consumption of such food products may lead to carcinogenicity [29].

#### **4.8 Determination of phytoconstituents**

Determination of phytoconstituents analysis involves usage of several analytical techniques for the isolation and characterization of phytoconstituents. Primitive techniques basically involved usage of UPLC-MS for the isolation and determination of phytoconstituents. Analysis and identification of chemical constituents of fenugreek by UPLC-MS and UPLC-Q-TOF-MS revealed that, 57 saponins and 19 flavonoid components. In addition to that, characterizations and quantitation of phytoconstituents has been reported in *Piper betle*. Moreover, quantitative data revealed significant variances in the contents of the major bioactive components in *Piper betle* species [44, 45].

**41**

**Figure 6.**

*(100 ng/mL) using UPLC-MS method.*

*UPLC-MS: An Emerging Novel Technology and Its Application in Food Safety*

In current years, various food such as legumes, cereals, potatoes, eggs, aquatic foods, dairy products, vegetables, fruits, and beverages reported to have several mycotoxins such as beauvericin, enniatin A, enniatin B, alternariol, tentoxin, and tenuazonic acid (**Figure 6**). These mycotoxins have been considered as a major food contaminates. In recent years, UPLC-MS has emerged as one of the most suitable method for the determination of these food contaminants. UPLC-MS has advantages over other instruments because of having better detection level, fast and accurate. UPLC-MS has emerged as a powerful tool for monitoring and measuring

It's been well-know that antioxidant has ability to fight against free radicals since free radicals are considered as a causative agent for several diseases. However, use of antioxidant has increased in food industry due to its antimicrobial property. Nowadays, natural as well as synthetic antioxidant such as butylated hydroxyanisole (BHA), butylated hydroxytoluene has been extensively used in food industry. However, the safety and toxicity of synthetic antioxidant is still a matter of concern for human health. On the other hand, several phenolic compounds have been well known for human nutrition. Moreover, these components are used for retarding microbial growth, increasing shelf life, reducing undesirable fragrances, enhancing nutritional value as well as delaying the formation of toxic oxidation. Phenolic profiling as well as antioxidant activities can be analyzed UPLC-ESI-MS/MS in Salvia species in some of the medicinal plants from South West Anatolia, Turkey. Moreover, it is assumed that, it was first reported for the analysis of individual phenolic profiles

Most of the drinking water contains bromide, as the primary source of bromide is soils containing bromide or sea water containing excess amount of

*Chromatogram (100 ng/mL) showing complete separation of a mixture of 10 mycotoxin standards at* 

**4.9 Determination of food contaminants in food matrices**

dietary exposure assessment of such mycotoxins [30].

of *S. potentillifolia, S. albimaculata,* and *S. nydeggeri* [46].

**4.11 Bromate in drinking water**

**4.10 Analysis of antioxidant and phenolic compound using UPLC-MS**

*DOI: http://dx.doi.org/10.5772/intechopen.92455*

**Figure 5.** *UPLC-MS chromatogram for acrylamide standard solution at 500 ng ml<sup>−</sup><sup>1</sup> .*
