**3. Food authentication detection**

There are several methods that can be used in food authentication process, such as electrophoretic techniques, differential scanning calorimetry (DSC), DNA-based methods (genomics, proteomics), chromatographic methods, isotopic techniques, vibrational and fluorescence spectroscopy, elemental techniques, non-chromatographics mass spectroscopy, sensory analysis, nuclear-magneticresonance spectroscopy, immunological techniques together with chemometrics and bioinformatics [40].

DNA-based technique with polymerase chain reaction [38, 44] is a common technique in food authentication testing to ensure halal and kosher brand food products adhere to the standards. However, most of these techniques still require substantial sample preparation or some have very high sensitivity to adulterants and prone to give undefined results if all procedures are not followed exactly.

**91**

fatty acid.

**3.1 Genomics**

in sausages [35].

**3.2 Electronic nose**

*Applications of Mass Spectrometry to the Analysis of Adulterated Food*

Research on vibrational spectroscopy-based food authentication techniques is getting more popular [40, 45–52]. This is partly due to the ease of sample preparation with this technique and relatively quick result and non-destructive nature of this method. Such vibrational spectroscopy is able to discriminate with high accuracy. For instance, pork meat and lard in meatball broth [45, 47], imported chocolate [50–53], and vegetable oils [48], etc. are some of the studies. Infrared-based detection techniques, such as FTIR or Fourier transform infrared spectroscopy, are capable of identifying fingerprint of compound molecules when it is incorporated with strong chemometric techniques [47]. Some research findings of lard adulterant are reported either by mixing lard with other animal fats or adulterating lard in food [53–55]. Another work on FTIR spectroscopy by Mansor et al. [56] reported an accuracy up to 100% in performing classification of lard adulterated in virgin coconut oil when the statistical technique, such as discriminant and PLS analysis, is incorporated. However, the limitation of lard detection using FTIR spectroscopy is highlighted in Rohman and Che Man [57] when identifying meat adulteration. Basically, lard has similar IR spectrum with other animal fats and vegetable oils since they are composed with (triacylglycerol) TAG, with different lengths of the

Animal fats have several chemical compositions, which mostly include TAG. In fact, fats share the same fatty acid compounds but different concentrations [58]. According to Rohman and Che Man [57], analysis of fats/oils is possible by focusing on lipid components as fats which is a part biological substance group. Triglyceride is the principal constituent of animal fat, not exception of pig fat. A triglyceride is constructed from three fatty acids and one molecule of glycerol [59]. Lard predomi-

Another popular technique that has been continually developed for lard compound detection in food is mass spectroscopy. Several MS methods have been reported, and the important ones are liquid-based chromatography and gas-based chromatography embedded with mass spectrometry (GC-MS and LC-MS).

One of the most popular food authentication methods is the genomics, where verification of foodstuff origin is done by analyzing the cells. Since DNA is similar in the whole somatic cells of a particular species, the original tissue of sample would not affect the results of the test. The advantage of this method is that it can amplify minute samples. Proteomics technique mainly depends on proteins acting as fingerprint of food products and therefore can be applied for a systematic search of new marker proteins. These methods are normally utilized to identify incorrect description and food labeling fraud, that is, detection of meats prohibited by Islamic laws

Electronic nose or e-nose is to replicate human's olfactory technique in identifying a particular substance. E-nose is commonly used metal-oxide gas sensor capable of detecting volatile organic compound (VOC) for variety detection applications including lard adulteration [60] process quality control [61, 62] and used as a formaldehyde sensor [63]. Sensing materials used in the electronic nose for metal oxide sensor are and tungsten trioxide (WO3) and tin dioxide (SnO2) because both materials are reported to be very sensitive to many types of volatile compounds. The sensor selection used in e-nose was based on the chemical compounds found in lard [58]. Decanal was the chemical compound found abundantly in lard

*DOI: http://dx.doi.org/10.5772/intechopen.84395*

nantly consists of saturated fatty acid [59].

#### *Applications of Mass Spectrometry to the Analysis of Adulterated Food DOI: http://dx.doi.org/10.5772/intechopen.84395*

Research on vibrational spectroscopy-based food authentication techniques is getting more popular [40, 45–52]. This is partly due to the ease of sample preparation with this technique and relatively quick result and non-destructive nature of this method. Such vibrational spectroscopy is able to discriminate with high accuracy. For instance, pork meat and lard in meatball broth [45, 47], imported chocolate [50–53], and vegetable oils [48], etc. are some of the studies. Infrared-based detection techniques, such as FTIR or Fourier transform infrared spectroscopy, are capable of identifying fingerprint of compound molecules when it is incorporated with strong chemometric techniques [47]. Some research findings of lard adulterant are reported either by mixing lard with other animal fats or adulterating lard in food [53–55]. Another work on FTIR spectroscopy by Mansor et al. [56] reported an accuracy up to 100% in performing classification of lard adulterated in virgin coconut oil when the statistical technique, such as discriminant and PLS analysis, is incorporated. However, the limitation of lard detection using FTIR spectroscopy is highlighted in Rohman and Che Man [57] when identifying meat adulteration. Basically, lard has similar IR spectrum with other animal fats and vegetable oils since they are composed with (triacylglycerol) TAG, with different lengths of the fatty acid.

Animal fats have several chemical compositions, which mostly include TAG. In fact, fats share the same fatty acid compounds but different concentrations [58]. According to Rohman and Che Man [57], analysis of fats/oils is possible by focusing on lipid components as fats which is a part biological substance group. Triglyceride is the principal constituent of animal fat, not exception of pig fat. A triglyceride is constructed from three fatty acids and one molecule of glycerol [59]. Lard predominantly consists of saturated fatty acid [59].

Another popular technique that has been continually developed for lard compound detection in food is mass spectroscopy. Several MS methods have been reported, and the important ones are liquid-based chromatography and gas-based chromatography embedded with mass spectrometry (GC-MS and LC-MS).

### **3.1 Genomics**

*Mass Spectrometry - Future Perceptions and Applications*

A notorious big scandal that hit Europe in 2013 related to food adulteration was the breach of true labeling due to the fraud on the beef sale that has been substituted with horsemeat [36]. The food fraud also occurred in some other part of the world when pharmaceutical preparations and chocolate were suspected to contain traces of pork in 2013 and 2014 in Malaysia [37]. In other countries, like India, it is not uncommon to sell buffalo meat adulterated with other animal meats due to financial issue and availability [38]. Such adulterated meats are very difficult to identify especially when such meats are already in the processed form. The practice of food fraud also occurs on dairy products, for example, butter is mixed with cheaper fats, such as mutton fats, chicken, and pig fats to get higher profits [39]. With these many occurrences of food adulterations around the world, ability to authenticate pure and mixed food has become a crucial aim for everybody.

Food adulteration practices not only destroy consumer trust and confidence in the products and the company reputation but also jeopardize the safety and quality of food consumed. The development of food authentication technique is necessary in food control because of the need of certain compliance in food process and the label to ensure customer confidence and trust to the food product [35, 40]. The authentication technique will also validate the food origin that includes its geographical, gene, and species source, confirming their production processes and

The need for food authentication is the result from customer concerns on the food nutrition and their health as well as an assurance of the process control and food quality purposes. Such authentication techniques will also confirm the existence of food adulteration, identify the origin of the food and its ingredients, and

For this purpose, mass spectroscopy has been very critical in validating and improving food quality and making us caution with any industrial and agriculture chemical to prevent harming our health, disturbing the food supply, and damaging the ecosystem that we depend on for our sustainability. The scientific finding in the environmental, agricultural, and food sciences has been significant to more resourceful and healthier food, improving our quality of life and better living in the

There are several methods that can be used in food authentication process, such as electrophoretic techniques, differential scanning calorimetry (DSC), DNA-based methods (genomics, proteomics), chromatographic methods, isotopic techniques, vibrational and fluorescence spectroscopy, elemental techniques, non-chromatographics mass spectroscopy, sensory analysis, nuclear-magneticresonance spectroscopy, immunological techniques together with chemometrics

DNA-based technique with polymerase chain reaction [38, 44] is a common technique in food authentication testing to ensure halal and kosher brand food products adhere to the standards. However, most of these techniques still require substantial sample preparation or some have very high sensitivity to adulterants and

prone to give undefined results if all procedures are not followed exactly.

improve the food quality and safety for pure and future mixed food.

world population that is reaching 8 billion and beyond.

**2.2 Mixed food**

**2.3 Food safety and quality**

their processing techniques [41–43].

**3. Food authentication detection**

and bioinformatics [40].

**90**

One of the most popular food authentication methods is the genomics, where verification of foodstuff origin is done by analyzing the cells. Since DNA is similar in the whole somatic cells of a particular species, the original tissue of sample would not affect the results of the test. The advantage of this method is that it can amplify minute samples. Proteomics technique mainly depends on proteins acting as fingerprint of food products and therefore can be applied for a systematic search of new marker proteins. These methods are normally utilized to identify incorrect description and food labeling fraud, that is, detection of meats prohibited by Islamic laws in sausages [35].

## **3.2 Electronic nose**

Electronic nose or e-nose is to replicate human's olfactory technique in identifying a particular substance. E-nose is commonly used metal-oxide gas sensor capable of detecting volatile organic compound (VOC) for variety detection applications including lard adulteration [60] process quality control [61, 62] and used as a formaldehyde sensor [63]. Sensing materials used in the electronic nose for metal oxide sensor are and tungsten trioxide (WO3) and tin dioxide (SnO2) because both materials are reported to be very sensitive to many types of volatile compounds.

The sensor selection used in e-nose was based on the chemical compounds found in lard [58]. Decanal was the chemical compound found abundantly in lard


#### **Table 1.**

*Decanal profile, measured in Kovats indices [58].*

but did not have significant presence in chicken fat and beef fat. **Table 1** lists the decanal content in the fats of interest in terms of Kovats indices. A set of experiments by Kohl et al. [64] revealed that both the sensing materials used in metal oxide sensors are sensitive to the presence of aldehydes. It is reported here that such sensor is expected to be more sensitive toward lard than other fats.

A scatter plot of sample dataset is shown in **Figure 2** [65]. The dataset consists of nine unique classes of three types of fat each experimented with three different temperatures. Each class consists of 10 observations. Each class is represented in the plot by a unique symbol and an abbreviation where the letter "L" represents a lard sample, "C" represents a chicken fat sample, and "B" a beef fat sample. The numbers 40, 50, and 60 after the letters represent the temperature in degree Celsius. A clear separation can be seen in the plot as except classes "L40" and "C40" where there are no overlaps. The overlap indicates the chemical structure of a chicken fat is very similar to lard, and studies conducted with other techniques have proven that as well.

**Figure 3** shows the individual plot of the three classes and their responses at different temperatures [65]. Linear regression lines in the background show an upward trend in sensor response, with lard having the highest gradient out of the three. With the increase of temperatures, the density and rate at which the odor fumes are produced must increase, thus giving rise to a higher sensor response. Besides, this lard has the lowest melting point among the three fats and will therefore melt and turn to gaseous state faster. In terms of settle point values, chicken fat scored the highest above the two as more evident from **Figure 3**. However, the higher settle point values of chicken fat can be explained by the fact that chicken fat melting points are very close to that of lard.

**93**

*Applications of Mass Spectrometry to the Analysis of Adulterated Food*

The principle of vibrational spectroscopy follows the concept that atoms in the chemical bonding within the molecule vibrate with certain frequency when it is excited. Such vibration frequency can be explained by the laws of physics and is shown in reported calculation [66]. The calculation of the lowest fundamental frequency of any two atoms that are connected by a chemical bond can be performed by assuming that the bond energy results from the vibration of diatomic harmonic

2*π* √

where, the vibrational frequency *is v*, the classical force constant is *k*, and the reduced mass of the two atoms is *μ*. In contrast to classical spring model for molecular vibration, no continuum of energy levels exists. Instead, there are levels of discrete energy that can be explained by quantum theory. Using the vibrational Hamiltonian, the time-independent Schrödinger equation can be solved for a diatomic molecule. A reduced equation of these levels can be written for the energy

> \_\_ \_\_ *k*

At certain extension of the stretch, the bond could eventually breakdown when

the vibrational energy goes beyond the dissociation energy. **Table 2** shows the different stretching frequencies. When a fast and objective analysis is required,

<sup>2</sup> )<sup>ℏ</sup> \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ 2*π* √

or by using *hv* as the quantum term, the equation can be reduced to

\_\_ \_\_ *k*

*<sup>µ</sup>* (1)

*<sup>µ</sup>* (*v* = 0, 1, 2,….) (2)

<sup>2</sup> )<sup>ℏ</sup> (*<sup>v</sup>* <sup>=</sup> 0, 1, 2,….) (3)

*DOI: http://dx.doi.org/10.5772/intechopen.84395*

**3.3 Vibrational spectroscopy**

*Response toward change in temperature [65].*

**Figure 3.**

levels of diatomic molecules as:

*Ev* <sup>=</sup> ( *<sup>v</sup>* <sup>+</sup> \_1

*Ev* <sup>=</sup> ( *<sup>v</sup>* <sup>+</sup> \_1

oscillator and follows Hooke's Law according to Eq. (1)

*v* = \_\_\_1

**Figure 2.** *Scatter plot of the entire dataset [65].*

*Applications of Mass Spectrometry to the Analysis of Adulterated Food DOI: http://dx.doi.org/10.5772/intechopen.84395*

**Figure 3.** *Response toward change in temperature [65].*

#### **3.3 Vibrational spectroscopy**

*Mass Spectrometry - Future Perceptions and Applications*

*Decanal profile, measured in Kovats indices [58].*

points are very close to that of lard.

but did not have significant presence in chicken fat and beef fat. **Table 1** lists the decanal content in the fats of interest in terms of Kovats indices. A set of experiments by Kohl et al. [64] revealed that both the sensing materials used in metal oxide sensors are sensitive to the presence of aldehydes. It is reported here that such

A scatter plot of sample dataset is shown in **Figure 2** [65]. The dataset consists of nine unique classes of three types of fat each experimented with three different temperatures. Each class consists of 10 observations. Each class is represented in the plot by a unique symbol and an abbreviation where the letter "L" represents a lard sample, "C" represents a chicken fat sample, and "B" a beef fat sample. The numbers 40, 50, and 60 after the letters represent the temperature in degree Celsius. A clear separation can be seen in the plot as except classes "L40" and "C40" where there are no overlaps. The overlap indicates the chemical structure of a chicken fat is very similar to lard, and studies conducted with other techniques have proven that as well. **Figure 3** shows the individual plot of the three classes and their responses at different temperatures [65]. Linear regression lines in the background show an upward trend in sensor response, with lard having the highest gradient out of the three. With the increase of temperatures, the density and rate at which the odor fumes are produced must increase, thus giving rise to a higher sensor response. Besides, this lard has the lowest melting point among the three fats and will therefore melt and turn to gaseous state faster. In terms of settle point values, chicken fat scored the highest above the two as more evident from **Figure 3**. However, the higher settle point values of chicken fat can be explained by the fact that chicken fat melting

sensor is expected to be more sensitive toward lard than other fats.

**92**

**Figure 2.**

**Table 1.**

*Scatter plot of the entire dataset [65].*

The principle of vibrational spectroscopy follows the concept that atoms in the chemical bonding within the molecule vibrate with certain frequency when it is excited. Such vibration frequency can be explained by the laws of physics and is shown in reported calculation [66]. The calculation of the lowest fundamental frequency of any two atoms that are connected by a chemical bond can be performed by assuming that the bond energy results from the vibration of diatomic harmonic oscillator and follows Hooke's Law according to Eq. (1)

$$\mathcal{v} = \frac{1}{2\pi} \sqrt{\frac{\overline{k}}{\mu}} \tag{1}$$

where, the vibrational frequency *is v*, the classical force constant is *k*, and the reduced mass of the two atoms is *μ*. In contrast to classical spring model for molecular vibration, no continuum of energy levels exists. Instead, there are levels of discrete energy that can be explained by quantum theory. Using the vibrational Hamiltonian, the time-independent Schrödinger equation can be solved for a diatomic molecule. A reduced equation of these levels can be written for the energy levels of diatomic molecules as:

$$E\_v = \frac{\left(\upsilon \star \frac{1}{2}\right)\hbar}{2\pi} \sqrt{\frac{k}{\mu}} \quad \text{ ( $\upsilon = 0, 1, 2, ...$ )}\tag{2}$$

or by using *hv* as the quantum term, the equation can be reduced to

$$E\_v = \left(\begin{array}{c} \upsilon + \frac{1}{2} \end{array}\right) \hbar \quad \text{( $\upsilon = 0, 1, 2, ...$ )}\tag{3}$$

At certain extension of the stretch, the bond could eventually breakdown when the vibrational energy goes beyond the dissociation energy. **Table 2** shows the different stretching frequencies. When a fast and objective analysis is required,


#### **Table 2.**

*Important IR stretching frequencies [67].*

fluorescence and absorption spectroscopies in the range of visible to infrared region are better choice. The vibrational spectroscopy is able to provide a fingerprint of the vibrational levels of molecules in the mid-infrared (MIR) radiation (4000– 400 cm<sup>−</sup><sup>1</sup> ). One of the most common IR spectroscopy techniques is the Fourier transform infrared (FTIR) spectroscopy. FTIR spectroscopy utilizes the use of mid infrared spectroscopy (4000–400 cm<sup>−</sup><sup>1</sup> ), which includes the fingerprint region.

#### *3.3.1 Meat sample preparation*

All meat samples were collected from a local slaughterhouse and were washed by distilled water. After that, the meat was cut by knife in pieces in the size of 1 cm<sup>2</sup> and stored at −20°C until it was being used. The animal fats extracted from beef, mutton, and chicken body fat as well as lard were collected by rendering the adipose tissues following the method reported by Che Man et al. [53] with little variation.

#### *3.3.2 Post-processing analysis*

Data post-processing was done using two software: Spectrograph 1.1 and MATLAB R2017b. Extracting information from spectrum results was carried out using Spectrograph 1.1, where the data are preprocessed as needed. MATLAB R2017b was used to further analyze the results from preprocessing. Principal component analysis (PCA) technique was used to analyze the quality of lard adulteration, while PLS technique was used to analyze the quantity of lard adulteration.

**Figure 4** shows FTIR spectra of pure fats. These spectra consist of four regions: 1st region ranging from 4000 to 2500 cm<sup>−</sup><sup>1</sup> , 2nd region ranging from 2500 to 2000 cm<sup>−</sup><sup>1</sup> , 3rd region ranging from 2000 to 1500 cm<sup>−</sup><sup>1</sup> , and lastly the fingerprint region ranging from 1500 to 800 cm<sup>−</sup><sup>1</sup> .

#### **3.4 Mass spectroscopy**

The mass spectroscopy methods are fast becoming popular [50, 68]. This method produces unique chemical fingerprinting that can discriminate or verify

**95**

**Figure 5.**

*the milk data set. [69].*

**Figure 4.**

*Applications of Mass Spectrometry to the Analysis of Adulterated Food*

foods. MS offers many advantages, such as the identification of mass spectral signal pattern and possible characterization of specific compounds coming from food adulterants. Additionally, MS does not easily react with water, which is different case for vibrational spectroscopy. MS can also provide the plant origin by measuring the specific chemical compounds. However, MS has disadvantages of direct contact requirement to the sample material and larger instrumentation. The spectral resolution of MS is more detail so it has higher possibility of finding fingerprint of food chemicals. MS also gives a higher versatility because of exchangeability of its ion sources. With different ion sources, MS can provide various ionization and is able to

*.*

**Figure 5** shows the spectrogram of different milks using electrospray ionization mass spectroscopy [69]. Obvious differences can be observed among the three milk, (a) cow milk, (b) goat milk, and (c) soy milk, by observing the number of peaks and peak intensities. The three pure milks and the two mixtures score plots are

*ESI mass spectroscopy using ESI spectrogram of the three milk, namely cow milk (a) goat milk, (b) soy milk, (c) Plot and (d) shows PCA score plot of the milk data set from the sum of 20 spectra. Each point in plots shows a mass spectrum of goat milk (blue), cow milk (yellow), and coy milk (red), the black-color data show 1:1 mixture of cow and soy milk, and the green shows a mixture of cow and goat milk (e) PCA loading plot of* 

perform measurement of chemically different chemical compounds.

*DOI: http://dx.doi.org/10.5772/intechopen.84395*

*Spectrogram from FTIR covering 4000–800 cm<sup>−</sup><sup>1</sup>*

*Applications of Mass Spectrometry to the Analysis of Adulterated Food DOI: http://dx.doi.org/10.5772/intechopen.84395*

**Figure 4.** *Spectrogram from FTIR covering 4000–800 cm<sup>−</sup><sup>1</sup> .*

*Mass Spectrometry - Future Perceptions and Applications*

fluorescence and absorption spectroscopies in the range of visible to infrared region are better choice. The vibrational spectroscopy is able to provide a fingerprint of the vibrational levels of molecules in the mid-infrared (MIR) radiation (4000–

N − H 3500–3300 Medium, broad N − H 3300–2700 Medium

**Wavenumber (cm<sup>−</sup><sup>1</sup>**

C ≡ N 2260–2220 Medium C ≡ C 2260–2100 Medium to weak C = C 1680–1600 Medium C = N 1650–1550 Medium

C = O 1780–1650 Strong C − O 1250–1050 Strong C − N 1230–1020 Medium O − H (alcohol) 3650–3200 Strong, broad

All meat samples were collected from a local slaughterhouse and were washed by distilled water. After that, the meat was cut by knife in pieces in the size of 1 cm<sup>2</sup> and stored at −20°C until it was being used. The animal fats extracted from beef, mutton, and chicken body fat as well as lard were collected by rendering the adipose tissues following the method reported by Che Man et al. [53] with little variation.

Data post-processing was done using two software: Spectrograph 1.1 and MATLAB R2017b. Extracting information from spectrum results was carried out using Spectrograph 1.1, where the data are preprocessed as needed. MATLAB R2017b was used to further analyze the results from preprocessing. Principal component analysis (PCA) technique was used to analyze the quality of lard adulteration, while PLS technique was used to analyze the quantity of lard adulteration. **Figure 4** shows FTIR spectra of pure fats. These spectra consist of four regions:

, 3rd region ranging from 2000 to 1500 cm<sup>−</sup><sup>1</sup>

.

The mass spectroscopy methods are fast becoming popular [50, 68]. This method produces unique chemical fingerprinting that can discriminate or verify

). One of the most common IR spectroscopy techniques is the Fourier transform infrared (FTIR) spectroscopy. FTIR spectroscopy utilizes the use of mid

), which includes the fingerprint region.

**) Intensity**

~1600 and ~1500–1430 Strong to weak

3300–2500 Strong, very broad

, 2nd region ranging from 2500 to

, and lastly the fingerprint

**94**

2000 cm<sup>−</sup><sup>1</sup>

**3.4 Mass spectroscopy**

400 cm<sup>−</sup><sup>1</sup>

**Table 2.**

O − H (carboxylic acid)

infrared spectroscopy (4000–400 cm<sup>−</sup><sup>1</sup>

1st region ranging from 4000 to 2500 cm<sup>−</sup><sup>1</sup>

region ranging from 1500 to 800 cm<sup>−</sup><sup>1</sup>

*3.3.1 Meat sample preparation*

*Important IR stretching frequencies [67].*

*3.3.2 Post-processing analysis*

foods. MS offers many advantages, such as the identification of mass spectral signal pattern and possible characterization of specific compounds coming from food adulterants. Additionally, MS does not easily react with water, which is different case for vibrational spectroscopy. MS can also provide the plant origin by measuring the specific chemical compounds. However, MS has disadvantages of direct contact requirement to the sample material and larger instrumentation. The spectral resolution of MS is more detail so it has higher possibility of finding fingerprint of food chemicals. MS also gives a higher versatility because of exchangeability of its ion sources. With different ion sources, MS can provide various ionization and is able to perform measurement of chemically different chemical compounds.

**Figure 5** shows the spectrogram of different milks using electrospray ionization mass spectroscopy [69]. Obvious differences can be observed among the three milk, (a) cow milk, (b) goat milk, and (c) soy milk, by observing the number of peaks and peak intensities. The three pure milks and the two mixtures score plots are

#### **Figure 5.**

*ESI mass spectroscopy using ESI spectrogram of the three milk, namely cow milk (a) goat milk, (b) soy milk, (c) Plot and (d) shows PCA score plot of the milk data set from the sum of 20 spectra. Each point in plots shows a mass spectrum of goat milk (blue), cow milk (yellow), and coy milk (red), the black-color data show 1:1 mixture of cow and soy milk, and the green shows a mixture of cow and goat milk (e) PCA loading plot of the milk data set. [69].*

shown in **Figure 5d**. The spectrograms of the pure milk samples are well separated in the plot, while data points for the mixture of cow and goat milk are positioned in the close proximity of those two types. The data points of cow/soy milk mixture are shown near around the data points of cow milk.

## **4. Advanced mass spectroscopy**

The recent advanced mass spectroscopy instruments offer higher speed, better resolution, higher mass accuracy, and more sensitivity to provide comprehensive qualitative investigation, rapid profiling, and better accuracy detection and quantification of chemical compounds in complex matrices. Thus, such advanced mass spectrometries such as gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) are able to investigate and analyze the complex adulterants. These advanced mass spectroscopies operate in scan mode at better spectrum resolution and accurate mass (HRAM).

This improved high-resolution mass spectroscopy is capable in identifying the chemical compounds and mass structure of pure and adulterated processed food, the presence of adulterants that create problems affecting food safety and quality, and the existence of natural toxin, food degradation and contaminations.

### **4.1 GC-MS**

Gas chromatography (GC) configured with electron capture, flame photometric detection, and nitrogen-phosphorous has been used since the early 1970s for residue analysis. The confirmation of results was done with additional use of gas chromatography equipped with a different type of column or detector. Nowadays, using GC integrated with MS, it is able to simultaneously determine and confirm the chemical residues with only one instrument in one analytical run.

Following the commercial of gas chromatography (GC) 50 years ago [70], GC has been used widely in the application involving food adulterant analysis and to perform both quantitative and qualitative analysis of food ingredients, food additives, food adulterants, and contaminants in order to discover nutritional contents, improve food safety, and introduce different food varieties. Furthermore, GC has been reported to be able to identify many organic contaminants at trace levels in complex chemical compounds of food and environmental samples.

Nowadays, gas chromatography integrated to mass spectrometry (GC-MS, GC-HRMS) utilized electron impact ionization (EI) is the most often employed in GC-based MS technique for multi residue chemical analysis in food analysis because of its high selectivity and sensitivity and its ability to screen many pesticides from different chemical compound classes in very complicated matrices in a single run [71]. Advantages of electron impact ionization mass spectroscopy are insignificant influence of molecular structure on response and vast number of characteristic fragments. GC-MS is suitable for analysis of volatile chemicals. Meanwhile, the analysis with more polar compound, LC-MS is more suitable. With the absent of chemical derivatization, GC is commonly used for the analysis of sterols, low chain fatty acids, oils, aroma components and off-flavors, and many contaminants, such as toxins, industrial pollutants, and specific of drugs in foods.

#### **4.2 LC-MS**

Liquid chromatography-mass spectrometry (LC-MS) is a combined analytical chemistry technique that separates mixtures with multiple components and

**97**

**Figure 6.**

*(B) ESI<sup>−</sup> [72].*

*Applications of Mass Spectrometry to the Analysis of Adulterated Food*

provides structural identity of the individual components with high molecular specificity and detection sensitivity. Methods based on liquid chromatography (LC) were applied later after GC, because traditional UV, diode array, and fluorescence detectors are often less selective and sensitive than GC instruments. But in the last few years, the commercial availability of atmospheric pressure ionization caused a dramatic change. Compared to traditional detectors, electrospray (ESI) or atmospheric pressure chemical ionization (APCI) in combination with MS instruments has increased the sensitivity of LC detection by several orders of magnitude.

An analytical methodology using liquid chromatography-mass spectrometry has been reported by Guijarro-Dıez et al. [72] for the detection of the adulteration of saffron samples with gardenia through the determination of geniposide as adulteration marker. **Figure 6** shows the MS spectra obtained for geniposide, and

corresponded to the adduct [M + HCOO]<sup>−</sup> (433.1384 *m/z*), and no fragmentation

The instrument of high-resolution mass spectrometry (HRMS) provides better accuracy for the analysis of food adulteration. However, due to high instrumental complexity, HRMS has previously been limited to the most critical applications, such

*Mass spectrogram geniposide spectrogram from gardenia extract investigated by LC-MS with (A) ESI+*

and NH4+

, whereas when the ESI<sup>−</sup> mode was employed, the most abundant ion

) were obtained for geniposide

 *and* 

*DOI: http://dx.doi.org/10.5772/intechopen.84395*

different MS fragments and adducts (Na+

**4.3 High resolution-mass spectroscopy**

under ESI+

was observed [72].

*Applications of Mass Spectrometry to the Analysis of Adulterated Food DOI: http://dx.doi.org/10.5772/intechopen.84395*

*Mass Spectrometry - Future Perceptions and Applications*

shown near around the data points of cow milk.

at better spectrum resolution and accurate mass (HRAM).

residues with only one instrument in one analytical run.

complex chemical compounds of food and environmental samples.

as toxins, industrial pollutants, and specific of drugs in foods.

**4. Advanced mass spectroscopy**

**4.1 GC-MS**

shown in **Figure 5d**. The spectrograms of the pure milk samples are well separated in the plot, while data points for the mixture of cow and goat milk are positioned in the close proximity of those two types. The data points of cow/soy milk mixture are

The recent advanced mass spectroscopy instruments offer higher speed, better resolution, higher mass accuracy, and more sensitivity to provide comprehensive qualitative investigation, rapid profiling, and better accuracy detection and quantification of chemical compounds in complex matrices. Thus, such advanced mass spectrometries such as gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) are able to investigate and analyze the complex adulterants. These advanced mass spectroscopies operate in scan mode

This improved high-resolution mass spectroscopy is capable in identifying the chemical compounds and mass structure of pure and adulterated processed food, the presence of adulterants that create problems affecting food safety and quality,

Gas chromatography (GC) configured with electron capture, flame photometric detection, and nitrogen-phosphorous has been used since the early 1970s for residue analysis. The confirmation of results was done with additional use of gas chromatography equipped with a different type of column or detector. Nowadays, using GC integrated with MS, it is able to simultaneously determine and confirm the chemical

Following the commercial of gas chromatography (GC) 50 years ago [70], GC has been used widely in the application involving food adulterant analysis and to perform both quantitative and qualitative analysis of food ingredients, food additives, food adulterants, and contaminants in order to discover nutritional contents, improve food safety, and introduce different food varieties. Furthermore, GC has been reported to be able to identify many organic contaminants at trace levels in

Nowadays, gas chromatography integrated to mass spectrometry (GC-MS, GC-HRMS) utilized electron impact ionization (EI) is the most often employed in GC-based MS technique for multi residue chemical analysis in food analysis because of its high selectivity and sensitivity and its ability to screen many pesticides from different chemical compound classes in very complicated matrices in a single run [71]. Advantages of electron impact ionization mass spectroscopy are insignificant influence of molecular structure on response and vast number of characteristic fragments. GC-MS is suitable for analysis of volatile chemicals. Meanwhile, the analysis with more polar compound, LC-MS is more suitable. With the absent of chemical derivatization, GC is commonly used for the analysis of sterols, low chain fatty acids, oils, aroma components and off-flavors, and many contaminants, such

Liquid chromatography-mass spectrometry (LC-MS) is a combined analytical chemistry technique that separates mixtures with multiple components and

and the existence of natural toxin, food degradation and contaminations.

**96**

**4.2 LC-MS**

provides structural identity of the individual components with high molecular specificity and detection sensitivity. Methods based on liquid chromatography (LC) were applied later after GC, because traditional UV, diode array, and fluorescence detectors are often less selective and sensitive than GC instruments. But in the last few years, the commercial availability of atmospheric pressure ionization caused a dramatic change. Compared to traditional detectors, electrospray (ESI) or atmospheric pressure chemical ionization (APCI) in combination with MS instruments has increased the sensitivity of LC detection by several orders of magnitude.

An analytical methodology using liquid chromatography-mass spectrometry has been reported by Guijarro-Dıez et al. [72] for the detection of the adulteration of saffron samples with gardenia through the determination of geniposide as adulteration marker. **Figure 6** shows the MS spectra obtained for geniposide, and different MS fragments and adducts (Na+ and NH4+ ) were obtained for geniposide under ESI+ , whereas when the ESI<sup>−</sup> mode was employed, the most abundant ion corresponded to the adduct [M + HCOO]<sup>−</sup> (433.1384 *m/z*), and no fragmentation was observed [72].

#### **4.3 High resolution-mass spectroscopy**

The instrument of high-resolution mass spectrometry (HRMS) provides better accuracy for the analysis of food adulteration. However, due to high instrumental complexity, HRMS has previously been limited to the most critical applications, such

#### **Figure 6.**

*Mass spectrogram geniposide spectrogram from gardenia extract investigated by LC-MS with (A) ESI<sup>+</sup> and (B) ESI<sup>−</sup> [72].*

#### **Figure 7.**

*The observed chromatograms (XICs) for certain signature of myoglobin proteotypic peptide-fragment pairs. The spike was noticed in the chromatograms from beef samples with 1% horse meat (blue indicates extracted blank chromatograms) [73].*

as the investigation of natural organic compounds or dioxin-related chemical compounds. The existence of modern HRMS instruments such as time-of-flight (TOF) and Orbitrap instruments has significantly changed the utilization of the equipment. Therefore, high-resolution mass spectrometry (HRMS) has gotten wider acceptance in the last decades for adulterant and residue analysis in food. This positive development is because of the availability of more versatile, robust, sensitive, and advanced instrumentation. The advantages by HRMS compared to classical unit-mass-resolution are ability to provide full-scan spectra, which offers more detail and insight into the mass composition of any sample. As a result, the analyst can measure chemical compounds without the necessity of compound-specific tuning, the need of retrospective data analysis, and has a capability performing an analysis of structural elucidations of suspected chemical compounds. HRMS is still preferable compared with classical hyphenated mass spectrometry in the investigation of quantitative multi residue methods (e.g., pesticides and veterinary drugs). It is one of the most powerful tools for identifying the unknown and non-targeted samples. Improvement of the hardware and software still needs to be addressed by the equipment manufacturers for it to be superior compared to hyphenated mass spectrometry and to be a standard trace analysis tool.

HRMS technology provides proteomic research to facilitate new discovery. The recent HRMS instruments already have the sensitivity, speed, accuracy, and selectivity to deliver comprehensive qualitative analysis, rapid chemical profiling, and high-accuracy analysis and detection of proteins in complex compounds. With these advantages, HRMS-based method was suitable specifically to perform the investigation of meat speciation and to detect food adulteration [73] and is capable to identify quite specific tryptic peptides from targeted proteins.

**99**

adulterants.

from the other animal fats.

*Applications of Mass Spectrometry to the Analysis of Adulterated Food*

Motivated by European scandal [74] in which the horse and pig DNA were detected in beef products sold from several retailers, HRMS method developed by Orduna et al. [73] were tested by mixing horse meat in beef meat at concentration 1% w/w. **Figure 7** shows the detection of adulteration of horse proteotypic myoglobin peptide using three different techniques of MS (140,000 FWHM), tMS or

GC-TOF MS instrument has two operation modes, in which one mode offers very high scan rates, allowing the segregation of overlapping spectrum peaks by automatically performing deconvolution mass spectral of overlapping spectrum signals [75]. Another type of GC-TOF MS instruments provides high mass resolution, performing data evaluation with a restricted mass window of 0.02 Da [76]. For ion separation GC-TOF MS, single-quad instruments are frequently utilized used. GC-MS systems with quadrupole ion traps integrated with time-of-flight (TOF) mass spectrometers or tandem mass spectrometers are used for the analysis

The work by Witjaksono et al. [77] was conducted for total nine meat samples of three different animal meats, that is, chicken, cow, and pig. Each animal meat type is prepared to provide three different samples. The preparation of the animal meat samples and the extraction process of these animal body fats have been done using similar method mentioned before in the FTIR measurements. After obtaining the pure fats, each animal fat (approximately of 50 mg) was dissolved in 0.8 mL hexane. Later, the mixture was stirred for 1 min using an apparatus of vortex mixer

The analysis for this food adulteration was based on GC-TOF MS to identify and study their complex chemical compounds. The equipment used is an Agilent 7693 B GC integrated with TOF MS with hp-5ms column. The analysis was performed for all nine samples, consisting of three samples each from cow, lard, and chicken fats to investigate their aromatic hydrocarbons. The result suggests that the concentration of 1,2,3-trimethyl-benzene, indane, and undecane in lard fat are higher by 250, 14.5, and 1.28 times than chicken fat's concentrations, respectively, and higher by 91.4, 2.3, and 1.24 times higher than cow fat's concentrations, respectively. This initial result promises the possibility of finding biomarkers for non-halal food

**Table 3** provides the obtained average area covered by each hydrocarbon that is coming from three samples to represent the composition weightage for the different fat types. From **Table 1**, it is obvious that lard is distinctive from the other animal fats in several hydrocarbon compositions. Here are the resulted hydrocarbons that give bigger percentage area in lard in comparison with the other fats: benzene, 1,2,3-trimethyl-; benzene, 1-methyl-3-(1-methylethyl)-; benzene, 1-methyl-4-propyl-; hexanedioic acid, bis(2-ethylhexyl)ester; p-cymene; tridecane; undecane. By using chemometric and bioinformatics analysis techniques, these results could be further analyzed to differentiate and separate the lard fat

and then stored in the dark at −18°C before going to GC-TOF MS analysis.

*DOI: http://dx.doi.org/10.5772/intechopen.84395*

DIA [73].

**5. GC-TOF MS**

of pure and mixture food.

**5.1 Sample preparation**

**5.2 GC-TOF MS results**

*Applications of Mass Spectrometry to the Analysis of Adulterated Food DOI: http://dx.doi.org/10.5772/intechopen.84395*

Motivated by European scandal [74] in which the horse and pig DNA were detected in beef products sold from several retailers, HRMS method developed by Orduna et al. [73] were tested by mixing horse meat in beef meat at concentration 1% w/w. **Figure 7** shows the detection of adulteration of horse proteotypic myoglobin peptide using three different techniques of MS (140,000 FWHM), tMS or DIA [73].
