5. Derivatization for improving GC/MS qualitative and quantitative analysis

The most powerful tool used for compound identification purposes is very likely mass spectrometry (spectroscopy). This technique is capable to provide information from very low amounts of material such as that eluting from a chromatographic column and can be easily coupled with a gas chromatograph. Most analyses performed with MS detection (GC/MS or GC/MS/MS) are using EI+ ionization mode with electron impact at 70 eV. The electrons interact with the molecule A to eject an additional electron leaving a positively charged species (with an odd number of electrons) of the type A▪<sup>+</sup> . The ions also receive energy during electron impact and the excess of energy induces fragmentation. For most molecules, this process can be written as follows:

$$\mathbf{A} + \mathbf{e}^- \to \mathbf{A}^{\bullet+} + 2\mathbf{e}^- \text{ and } \mathbf{A}^{\bullet+} \to \mathbf{B}\_i^+ + \mathbf{C}\_i^{\bullet} \tag{6}$$

The fragments Bi <sup>+</sup> are commonly but not always with an even number of electrons. The formation of molecular ions takes place with a range of internal energies, and more than one fragmentation path is possible for a given molecule. Also, the fragments can suffer further fragmentations. In general, the most abundant fragment ion results from the fragmentations that form the most stable products (ion and neutral radical). The abundance of a fragment ion is affected by its stability. For this reason, the intensity of the response of a mass spectrometric detector can be very different for different molecular species, and the prediction of this intensity is difficult. As a result, the improvements in the sensitivity in EI + �type mass spectrometry (in GC/MS using EI+ ionization) are not usually sought (but not impossible) through derivatization.

Derivatization for enhancing sensitivity is, however, frequently applied in NCI-MS. In this technique, the electrons interact with the molecules of the CI gas which is lowering their energy but without forming ions. The ionization of analyte molecules takes place by interaction with the low-energy electrons or with already formed negative ions by electron capture, dissociative electron capture, ion pair formation, or ion molecule reaction. The ionization process with the formation of

negative ions is efficient only for molecules with positive electron affinities. For this reason, the sensitivity in NCI-MS is highly dependent on the electron affinity of the analyte, similarly to the sensitivity in ECD. For enhancing the electron affinity, the derivatization with reagents containing, for example, fluorinated moieties (indicated in Figures 4–6) is practiced. The sensitivity of the analytical methods where such derivatization is applicable can have very good sensitivity. For example, derivatization with heptafluorobutyric anhydride of aromatic amines that are present at low trace level in cigarette smoke leads to limit of detection (LOD) values as low as 0.05 ng/cig. for compounds such as 4-aminobiphenyl [10, 11].

The fragmentation pattern generated by EI+ ionization mode that generates a specific mass spectrum of a molecule is very likely the most utilized technique for the identification of the molecular species. For this identification, large libraries of mass spectra are available, and computer algorithms are used for automatic searches. The identification of compounds using mass spectroscopy is not a simple process even with the capabilities offered by the electronic searches in the mass spectral libraries. This is particularly true for analysis of complex mixtures or when the analyzed compound is present in traces. Some compounds do not have a very characteristic mass spectrum, or during the chromatographic process, the separation is not achieved, and it is difficult to make an identification due to the spectra overlapping. Also, numerous compounds may have a mass spectrum that matches more than one compound (with a good quality fit). In such cases, a derivatization with the purpose of obtaining a compound that forms more informative fragments in the mass spectrum can be very useful.

The fragments from derivatized compounds can be used for the identification of unknown compounds using library searches and even when the mass spectrum is not available in the libraries. As an example, the derivatization by silylation allowed the identification of a new pentacyclic triterpenoid present in several bioactive botanicals [12]. An unidentified compound with MW = 456.7 was detected by LC/ MS/MS in a rosemary extract. The structure of the compound was elucidated after silylation of the plant material based on the comparison of mass spectrum of the unidentified compound with that of silylated betulinic acid. The new compound was identified as (3β)-3-hydroxy-lupa-18,20(29)-dien-28-oic acid (or betul-18-enoic acid). The mass spectra of the two acids are shown in Figure 7.

The two mass units difference between different fragments from the mass spectra of the two compounds allowed the identification of the new compound structure. Neither free betulinic acid nor betul-18-en-oic acid are volatile, such that the use of GC/MS for identification was possible only after derivatization.

derivatization is involved in the analysis, this can be done with a non-labeled reagent for the analytes in the sample, while the internal standards are obtained by derivatization of standards with the same reagent but isotopically labeled. Such technique has been proven to be very successful, for example, in the analysis of

6. General comments regarding the main types of chemical reactions

Derivatizations as chemical reactions can be classified as follows: (1) reactions with formation of alkyl or aryl derivatives, (2) silylation reactions, (3) reactions with formation of acyl derivatives, (4) reactions of addition to carbon-hetero multiple bonds, (5) reactions with formation of cyclic compounds, and (6) other reactions specific to a certain analysis. The selection of the derivatization reaction is typically done based on the desired property to be brought to the analyte and its possible reactivity. For this reason, the reagent is selected to have moieties that add the desired property to the analyte and also to have the capability to react with the specific functional group of the analyte. The matrix of the sample also has a role in

multiple amino acids (but using an LC/MS/MS procedure [13]).

Mass spectrum of silylated betulinic acid and that of silylated betul-18-en-oic acid.

used in derivatization

Derivatization Methods in GC and GC/MS DOI: http://dx.doi.org/10.5772/intechopen.81954

Figure 7.

19

Another special procedure that may be utilized for compound identification based on mass spectra is the use of two parallel derivatizations, one of them being done with an isotope-labeled reagent. Common labeling isotopes are 2 H (deuterium, d), 13C, 15N, etc. One such isotopic labeling can be done, for example, using silylation with d18-N,O-bis(trimethylsilyl)-trifluoroacetamide (d18- BSTFA). Derivatization of an aliquot of sample with regular BSTFA and another with d18-BSTFA provides a pairing chromatogram with peaks at retention times that have only small differences from the first but with spectra differing by a number of units. The comparison of the spectra for corresponding peaks (based on retention time) of a given compound allows the calculation of the number of silyl groups attached to that compound. In addition, the fragmentation in the spectra can be better interpreted allowing easier compound identification.

Derivatization in GC/MS analysis may have multiple other utilizations and benefits. For example, quantitative analysis frequently utilizes isotopically labeled internal standards. In an analysis with multiple analytes, addition of an isotopically labeled internal standard for each analyte may become a complex process. When a

## Derivatization Methods in GC and GC/MS DOI: http://dx.doi.org/10.5772/intechopen.81954

negative ions is efficient only for molecules with positive electron affinities. For this reason, the sensitivity in NCI-MS is highly dependent on the electron affinity of the analyte, similarly to the sensitivity in ECD. For enhancing the electron affinity, the derivatization with reagents containing, for example, fluorinated moieties (indicated in Figures 4–6) is practiced. The sensitivity of the analytical methods where such derivatization is applicable can have very good sensitivity. For example, derivatization with heptafluorobutyric anhydride of aromatic amines that are present at low trace level in cigarette smoke leads to limit of detection (LOD) values as

The fragmentation pattern generated by EI+ ionization mode that generates a specific mass spectrum of a molecule is very likely the most utilized technique for the identification of the molecular species. For this identification, large libraries of mass spectra are available, and computer algorithms are used for automatic searches. The identification of compounds using mass spectroscopy is not a simple process even with the capabilities offered by the electronic searches in the mass spectral libraries. This is particularly true for analysis of complex mixtures or when the analyzed compound is present in traces. Some compounds do not have a very characteristic mass spectrum, or during the chromatographic process, the separation is not achieved, and it is difficult to make an identification due to the spectra overlapping. Also, numerous compounds may have a mass spectrum that matches more than one compound (with a good quality fit). In such cases, a derivatization with the purpose of obtaining a compound that forms more informative fragments

The fragments from derivatized compounds can be used for the identification of unknown compounds using library searches and even when the mass spectrum is not available in the libraries. As an example, the derivatization by silylation allowed the identification of a new pentacyclic triterpenoid present in several bioactive botanicals [12]. An unidentified compound with MW = 456.7 was detected by LC/ MS/MS in a rosemary extract. The structure of the compound was elucidated after silylation of the plant material based on the comparison of mass spectrum of the unidentified compound with that of silylated betulinic acid. The new compound was identified as (3β)-3-hydroxy-lupa-18,20(29)-dien-28-oic acid (or betul-18-en-

low as 0.05 ng/cig. for compounds such as 4-aminobiphenyl [10, 11].

Gas Chromatography - Derivatization, Sample Preparation, Application

oic acid). The mass spectra of the two acids are shown in Figure 7.

be better interpreted allowing easier compound identification.

The two mass units difference between different fragments from the mass spectra of the two compounds allowed the identification of the new compound structure. Neither free betulinic acid nor betul-18-en-oic acid are volatile, such that the use of GC/MS for identification was possible only after derivatization. Another special procedure that may be utilized for compound identification based on mass spectra is the use of two parallel derivatizations, one of them being done with an isotope-labeled reagent. Common labeling isotopes are

H (deuterium, d), 13C, 15N, etc. One such isotopic labeling can be done, for example, using silylation with d18-N,O-bis(trimethylsilyl)-trifluoroacetamide (d18- BSTFA). Derivatization of an aliquot of sample with regular BSTFA and another with d18-BSTFA provides a pairing chromatogram with peaks at retention times that have only small differences from the first but with spectra differing by a number of units. The comparison of the spectra for corresponding peaks (based on retention time) of a given compound allows the calculation of the number of silyl groups attached to that compound. In addition, the fragmentation in the spectra can

Derivatization in GC/MS analysis may have multiple other utilizations and ben-

efits. For example, quantitative analysis frequently utilizes isotopically labeled internal standards. In an analysis with multiple analytes, addition of an isotopically labeled internal standard for each analyte may become a complex process. When a

in the mass spectrum can be very useful.

2

18

Figure 7. Mass spectrum of silylated betulinic acid and that of silylated betul-18-en-oic acid.

derivatization is involved in the analysis, this can be done with a non-labeled reagent for the analytes in the sample, while the internal standards are obtained by derivatization of standards with the same reagent but isotopically labeled. Such technique has been proven to be very successful, for example, in the analysis of multiple amino acids (but using an LC/MS/MS procedure [13]).
