**3. Ultra performance liquid chromatography**

*Analytical Chemistry - Advancement, Perspectives and Applications*

increasing consumer health safety concerns [3]. A very well-know proverb from nutritionists or dietitians is "we are what we eat". Definitely, it does not mean that if we eat apple we become apple, but for good or for ill, the components we eat must be incorporated, transformed, and/or excreted by our bodies. Because, food is an indispensable ingredient of life, and access to food is often the limiting factor in the size of a given populace [4]. There are several incidents of food safety outbreak, which has received major attention from all parts of the world such as occurrence of benzene in carbonated drinks (UK), foods contaminated with pesticides (Japan), presence of dioxins in milk products and pork sample (Belgium), incidence of pesticides in soft drinks (India) and occurrence of melamine in dairy products (China). Such incidents have made people distressful of their food consumption worldwide [5]. In addition to that, such contemporaneous incidents are growing concerns, mainly because of mass production of agronomic products and industrialization at a very fast pace to meet the requirement of current population. Moreover, it has been considered that mainly increasing worldwide population is making farming people to force mass production of agronomic products without giving ample consideration to the safety and quality of food produce. In addition to that, changes in life style patterns of consumers have been called responsible for food safety hazards [6]. Due to fast-paced urbanization, food products such as ready-to-eat, processed food and junk foods has increased, but due to rise in application of chemicals usage, such processed food has also come under the scanner of food safety

Moreover, the scope, relevance, and level of food safety and testing have never been in such complexity than in today's global marketplace. In recent years, a novel technology UPLC-MS has been developed to estimate the food contaminants as well as food components with better accuracy, sensitivity, precision, and high throughput. In addition to that, this advanced novel technique provided the platform to estimate different analytes at very lower levels, with better accuracy, and more importantly in less time. Moreover, the uniqueness of UPLC-MS has marked several applications to food safety. Various food safety parameters such as residual analysis, vitamins, amino acid, metabolite identification, adulteration, forensic testing, toxicity studies, phytoconstituents analysis, pesticide in agriculture, antibiotic residue, hormones, dyes and pigment analysis can be performed by using UPLC-MS [8, 9]. In addition to that, wide range of analysis makes UPLC-MS as an integral part of food safety laboratory around the globe. Moreover, in this chapter a detailed study and exploration has been made for better understanding of principles and applicability of UPLC-MS

Today, our food supply is more diverse and highly processed than ever before.

However, to ensure the nutritive value and to improve the food safety several states have disseminated regulations that states the acceptable limit for each components likewise, food additives, food residues and contaminants in food or food products. Consequently, a better and safe food can only be ensured when we have good approach to analyze such food components, contaminants, or chemical contaminants. In past few decades, chromatography has been recognized as one of important tool to identify and quantify food contaminants to ensure food safety. This novel technique allows the separation, purification, and identification from

**32**

professionals [1, 7].

in food safety.

**2. Chromatography and food safety**

UPLC is a novel technique that offers a new pathway for LC. UPLC enhances the capability of LC in four main areas like increasing speed, sensitivity, resolution and accuracy. UPLC is also known as ultra high-performance liquid chromatography (UHPLC). In comparison to high-performance liquid chromatography (HPLC), UPLC has been upgraded with column packing materials of less than 2 μm in diameter, which increases the speed, accuracy, resolution and sensitivity. Moreover, particle size used in HPLC, UPLC column ranges from 3 to 5 μm and < 2 respectively as well as mobile phase flow rate in HPLC is usually 3.0 ml/min compared to UPLC flow rate 0.6 ml/min. The basic difference in the principle of UPLC and HPLC is the column packing material, which makes a huge difference over the sensitivity and accuracy of the novel techniques. Apart from the principle involved in the LC, there is not much change in basic principle except the pressure generated or created in the instruments make it a more efficient technology. The development of UPLC techniques has urged the scientists to improve the prevailing instrumentation capability for LC, which has the advantage of improved parting performance and constant pressure. Efficiency of this technique is equivalent to the dimension of the column and inversely proportional to the radius of the atoms. As the name suggest ultra performance or ultra-pressure, UPLC works under very high pressure up to 1000 bars, however for HPLC, pump pressure not go more than 300–400 bars. A schematic diagram of UPLC and its internal diagram are presented here in **Figure 1**. In recent years, UPLC has become an integral part of any food safety laboratories, as it reduces the time of run as well as cost of analysis for any analysis [9, 12, 13].

**Figure 1.** *Flow diagram of ultra performance liquid chromatography-mass spectrometry.*

#### **3.1 Principle**

UPLC works on the van Deemter principle, which describes the correlation between the flow rate and height of chromatogram. The van Deemter states that, "the flow rate of smaller particles are much faster in compare with large particles as well as unfolding the correlation of flow rate and plate height". According to van Deemter equation, when the porous particle size reduced to less than 2.5 μm, there will be increase in efficiency; however, the efficiency does not weaken at increased flow rates or linear velocities.

The following equation describes the relationship between linear velocity (flow rate) and plate height [13, 14].

$$H = \mathbf{A} + \mathbf{B} / \boldsymbol{\upsilon} + \mathbf{C}\_{\boldsymbol{\upsilon}} \tag{1}$$

where,

A, B and C = Constants.

*v =* Linear velocity of carrier gas flow rate.

A *=* It is independent of velocity and represents "eddy" mixing. This is smallest when the packed column particles are small and uniform.

B *=* It stands for axial diffusion or the natural diffusion tendency of molecules. This effect is diminished at high flow rates and so this term is divided by *v*.

C *=* It represent kinetic resistance to equilibrium in the separation process.

According to van Deemter equation, resistance of kinetics is the time lag involved in traveling from the gas phase to the packing stationary phase and back again. Moreover, higher the gas flow, greater will be a molecule to lag behind in the mobile phase on packed stationary phase. Therefore, the term is proportional to *v.* Moreover, there will be a chance to surge throughput, and thus the rapidity of analysis without affecting the chromatographic performance [15]. However, UPLC performance is not much efficient until unless it is coupled with tandem mass spectrometry or other spectrometry techniques as it helps in molecular analysis by using mass-by-charge ratio [16, 17].

#### **3.2 Mass spectrometry**

Spectrometry method for the molecular analysis of any compound requires mass spectrometry (MS). The principle of MS was first proposed by Dr. Wien, which suggests that, refraction of charged particle in electric or magnetic field can analyzed by using MS. Mass spectrometer is an important tool to for the molecular mass analysis [18]. MS methods identifies the ionized molecules in gaseous phase in different ways


MS works on the principle of fragmentation of molecule and separation or filtration of ions on the basis of their mass-to-charge (m/z) ratio. The molecular mass resulting from mass spectrum and produced ions are a function of mass by charge ratio [19]. Consequently, fragmentation of molecular mass in MS make

**35**

the peak height:

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

it principally a very important technique over any other traditional chromatographic techniques. Notwithstanding that, on account of the capacity of MS to create m/z proportion, it considered as an exceptionally novel, straightforward, sensitive, accurate, and particular for the quantitative investigation of any

There are mainly five techniques for analyzing mass of any compound by using MS like, quadrupole mass filter (single and triple), time of flight, quadrupole ion trap and Fourier transform ion-cyclotron resonance instruments. Furthermore, MS gave a thought of molecular mass, however on the other hand it does not give authentication of molecular structure. In this way, to conquer the restriction of past mass spectrometry, improvement of couple mass spectroscopy (MS/MS) rises. This MS/MS system work into two stages, first to choose parent ions generated from parent ion cells and to disintegrate into daughter ions after the collision of parent ion into at least one daughter ions. In mass spectrometry parent ions and daughter ions gets isolated, divided, and distinguished into single ion cell. In addition to that, fast collisions of compounds performed in argon cell, where translational energy gets transformed into ion internal energy to make ions in excited state and unimolecular decay progresses [22]. The breaking of compound in ion cell of MS/MS spectrum is selected based upon parent and daughter ions. Collision of compound can be performed in in single ionization cell or triple quadrupole system (TQS). TQS is the most frequently used now a day MS/MS techniques as compared to other mass

Small-size particles not only enhance proficiency, nonetheless it also increases the flexibility to enhance linear velocity without losing efficiency of the column. Moreover, efficiency is the essential separation factor in UPLC, as it depends on the selectivity and retention activity as in HPLC. Below equation shows that: (Rs)

> 4 1 α − <sup>=</sup> α + *N k*

> > 2 <sup>1</sup> *<sup>N</sup> <sup>w</sup>* α

This demonstrates that the narrower the peaks are, the easier would be to separate from each other. Moreover, peak width height is inversely proportional to

> 1 *H w* α

Therefore, decrease in particle size increases *N* and subsequently Rs, and by virtue of which sensitivity increased, taller peak as well as narrower peak mean

However, *N* is inversely proportional to particle size (*dp*): as the particle size is lowered by a factor of three, from, for example, 5 μm (HPLC scale) to 1.7 μm (UPLC-scale), *N* is increased by three and resolution by the square root of three or

*<sup>k</sup>* (2)

(3)

(4)

<sup>1</sup> Rs

1.7. *N* is also inversely proportional to the square of the peak width:

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

**3.3 Tandem mass spectrometry (MS/MS)**

**3.4 Small-size particles and their chemistry**

resolution is directly proportional to the square root of N.

mixture or blend [20, 21].

analyzer [23].

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

it principally a very important technique over any other traditional chromatographic techniques. Notwithstanding that, on account of the capacity of MS to create m/z proportion, it considered as an exceptionally novel, straightforward, sensitive, accurate, and particular for the quantitative investigation of any mixture or blend [20, 21].

#### **3.3 Tandem mass spectrometry (MS/MS)**

*Analytical Chemistry - Advancement, Perspectives and Applications*

UPLC works on the van Deemter principle, which describes the correlation between the flow rate and height of chromatogram. The van Deemter states that, "the flow rate of smaller particles are much faster in compare with large particles as well as unfolding the correlation of flow rate and plate height". According to van Deemter equation, when the porous particle size reduced to less than 2.5 μm, there will be increase in efficiency; however, the efficiency does not weaken at increased

The following equation describes the relationship between linear velocity (flow

A *=* It is independent of velocity and represents "eddy" mixing. This is smallest

B *=* It stands for axial diffusion or the natural diffusion tendency of molecules.

Spectrometry method for the molecular analysis of any compound requires mass spectrometry (MS). The principle of MS was first proposed by Dr. Wien, which suggests that, refraction of charged particle in electric or magnetic field can analyzed by using MS. Mass spectrometer is an important tool to for the molecular mass analysis [18]. MS methods identifies the ionized molecules in gaseous phase in

MS works on the principle of fragmentation of molecule and separation or filtration of ions on the basis of their mass-to-charge (m/z) ratio. The molecular mass resulting from mass spectrum and produced ions are a function of mass by charge ratio [19]. Consequently, fragmentation of molecular mass in MS make

• Qualitative analysis of unknown compounds or mixture

• Quantitative estimation of any mixture or solution

This effect is diminished at high flow rates and so this term is divided by *v*. C *=* It represent kinetic resistance to equilibrium in the separation process. According to van Deemter equation, resistance of kinetics is the time lag involved in traveling from the gas phase to the packing stationary phase and back again. Moreover, higher the gas flow, greater will be a molecule to lag behind in the mobile phase on packed stationary phase. Therefore, the term is proportional to *v.* Moreover, there will be a chance to surge throughput, and thus the rapidity of analysis without affecting the chromatographic performance [15]. However, UPLC performance is not much efficient until unless it is coupled with tandem mass spectrometry or other spectrometry techniques as it helps in molecular analysis by

*H v* =+ + A B/ C*<sup>v</sup>* (1)

**3.1 Principle**

where,

flow rates or linear velocities.

rate) and plate height [13, 14].

A, B and C = Constants.

using mass-by-charge ratio [16, 17].

• Structure characterization

• Molecular weight determination

**3.2 Mass spectrometry**

different ways

*v =* Linear velocity of carrier gas flow rate.

when the packed column particles are small and uniform.

**34**

There are mainly five techniques for analyzing mass of any compound by using MS like, quadrupole mass filter (single and triple), time of flight, quadrupole ion trap and Fourier transform ion-cyclotron resonance instruments. Furthermore, MS gave a thought of molecular mass, however on the other hand it does not give authentication of molecular structure. In this way, to conquer the restriction of past mass spectrometry, improvement of couple mass spectroscopy (MS/MS) rises. This MS/MS system work into two stages, first to choose parent ions generated from parent ion cells and to disintegrate into daughter ions after the collision of parent ion into at least one daughter ions. In mass spectrometry parent ions and daughter ions gets isolated, divided, and distinguished into single ion cell. In addition to that, fast collisions of compounds performed in argon cell, where translational energy gets transformed into ion internal energy to make ions in excited state and unimolecular decay progresses [22]. The breaking of compound in ion cell of MS/MS spectrum is selected based upon parent and daughter ions. Collision of compound can be performed in in single ionization cell or triple quadrupole system (TQS). TQS is the most frequently used now a day MS/MS techniques as compared to other mass analyzer [23].

#### **3.4 Small-size particles and their chemistry**

Small-size particles not only enhance proficiency, nonetheless it also increases the flexibility to enhance linear velocity without losing efficiency of the column. Moreover, efficiency is the essential separation factor in UPLC, as it depends on the selectivity and retention activity as in HPLC. Below equation shows that: (Rs) resolution is directly proportional to the square root of N.

$$\text{Rs} = \frac{\sqrt{N}}{4} \left( \frac{\alpha - 1}{\alpha} \right) \left( \frac{k}{k + 1} \right) \tag{2}$$

However, *N* is inversely proportional to particle size (*dp*): as the particle size is lowered by a factor of three, from, for example, 5 μm (HPLC scale) to 1.7 μm (UPLC-scale), *N* is increased by three and resolution by the square root of three or 1.7. *N* is also inversely proportional to the square of the peak width:

$$Na \frac{1}{w^2} \tag{3}$$

This demonstrates that the narrower the peaks are, the easier would be to separate from each other. Moreover, peak width height is inversely proportional to the peak height:

$$H\alpha \frac{1}{w} \tag{4}$$

Therefore, decrease in particle size increases *N* and subsequently Rs, and by virtue of which sensitivity increased, taller peak as well as narrower peak mean

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 when migrating toward smaller particles:

$$F\_{\text{xxx}}\alpha = \frac{1}{\text{dpc}}\tag{5}$$

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 to column length.

$$Na\frac{L}{dp}\tag{6}$$

**37**

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

• Analysis of natural medicine and herbal medicine [9]

• Determination of acrylamide in food matrix [29]

• Determination of bromate in drinking water [32]

• Capsaicinoids analysis in capsicum species [35]

• Phenolic content determination in fruits and vegetables

• Determination of food-borne carcinogens heterocyclic amines [34]

Several antibiotic residues such as streptomycin (**Figure 2**), chloramphenicol, tetracycline etc. has been identified and quantified in honey by using UPLC-MS

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

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

residue at very lowest concentration in complex sample matrices [24–26].

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

• Determination of phytoconstituents

• Analysis of mycotoxin in food [30, 31]

• Pesticide in fruit and vegetables [33]

• Determination of alkaloids in cocoa

• Lactose content determination in milk

• Analysis of food based coloring agent [36]

**4.1 Determination of antibiotic residue in honey**

coupled along with electron spray ionization [37, 38].

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

• Analysis of vitamin in food

**4.3 Amino acid profiling**

mammalian milk [27].

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 separations [9, 13, 15].
