Developing a GC-MS Method

Developing the GC-MS method requires the preparation of a standard solution containing the pesticides to be dosed and the internal standards. In the example presented, we use two internal standards, one for the chlorinated pesticides and the other for non-chlorinated pesticides. In the standard solution, all the compounds are at the same concentration that is sufficiently high (in the range of ppm, for example) to ensure they will be detected under scanning. If some analytes and standards must be chemically derived, the standard solution should be subjected to the derivation protocol optimized in the previous step. 

From this standard solution, one optimizes the chromatographic separation (injection mode, choice of capillary column, and programming of oven temperature), generally in El because this non-selective ionization mode guarantees the detection of all compounds of interest. Remember, it is not necessary for all the compounds to be perfectly separated since detection operates only on certain characteristic ions. Nevertheless, the better the separation, the less likely the risks of interference. The point therefore is to find a compromise between the best analyte separation possible and an analysis time that is compatible with the specifications of the laboratory. 

After the chromatographic conditions are set, the next step is to determine the most efficient ionization mode (El or Cl) for the molecules to be analyzed. The signal-to-noise ratios (SNRs) obtained in El and in Cl for each compound must be compared to the currents of the main ions (not to the total ion current). The better the SNR, the better will be the limit of detection in theory. 

At this stage, the choice of ions is frequently a source of error. One type of error is comparing the intensities of the chromatographic signals obtained in El and in Cl. The point is to compare SNRs. Often, the ion current obtained in El is superior to that obtained in Cl but the comparison of the SNRs shows that in reality Cl gives better results because it renders far less background noise. Another error arises from comparing the two ionization modes on the total ion currents when the SNRs must be compared on the currents of the ions of interest (ions selected for detection in SIR or precursor ions for detection in MS/MS or MRM). A comparison must be made for each analyte. 

In summary, for a method in SIR with three ions, from the chromatograms recorded under scanning in El and in Cl, one extracts two chromatograms for each analyte. The first is reconstituted from the three chosen ions to detect the analyte in 

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Attempts to develop a GC/MS method for this metabolite were unsuccessful, no doubt because of thermal instability. An LC/MS/MS method using thermospray ionization, after derivatization to the dimethyl ester, gave a modest detection limit of 25 ng/ml, again probably due to poor thermal stability, in this case, in the thermospray ion source (12). A substantial improvement has recently been achieved (detection limit 1 ng/ml) using LC/ESI/MS/MS without derivatization (31). Concentration from acidified urine was achieved on... 

Jakubowski et al. (36) developed a GC/MS method for CVAA spiked into guinea pig urine using 1,2-ethanedithiol for derivatization, with phenyl arsine oxide as the internal standard. The same group later expanded the method to include atomic emission detection (AED) (37). CVAA was concentrated from urine (adjusted to pH 6 with 1M HC1) by SPE on Cl8. After elution with methanol and concentration to dryness, the residue was reconstituted and derivatized with ethanolic 1,2-ethanedithiol. Detection was by GC combined with arsenic selective AED and by electron impact/mass spectrometry (EI/MS) using SIM. Ions monitored were the moderately intense M+ ion at mlz 228, an intense ion [M — C2H4]+, mlz 200, and a base... 

A GC-MS method was developed for the determination of hyoscyamine and scopolamine in blood semm [91,92]. Extraction was carried out using aqueous basic solution followed by a purification step on an Extrelut column. Derivatization was done with N,0-bis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (99 1). GC-MS was performed on a HP-5 MS column (30m x 0.25 mm i.d. with a 0.25 p,m film thickness). The linearity was good between 10 and 5000ng/mL. The limit of detection (LOD) was 5ng/mL for each compound. 

Assays for several other potential urinary analytes have been developed, but the analytes have yet to be confirmed in human exposed samples. N7-(2-hydroxyethylthioethyl) guanine is a breakdown product from alkylated DNA that has been observed in animal studies. Fidder et al. (1996a) developed both a GC-MS method that requires derivatization of the analyte and also a LC-MS-MS method that can analyze the compound directly. Other possible urinary analytes are an imidazole derivative formed from the reaction of sulfur mustard with protein histidine residues (Sandelowsky et al., 1992) and sulfur mustard adducts to metallothionien (Price et al., 2000). 

The number of papers published in the field of nutritional metabo-lomics has increased rapidly over the past few years. While a detailed literature review of all papers is beyond the scope of this chapter, a more focused overview of some of the studies using functional ingredients will be presented. There are a number of studies which have investigated the effects of pol henols on metabolic effects in humans. Grun and colleagues developed a GC-MS metabolomis method to profile phenolic microbial fermentation products (Gmn et al. 2008). They successfully applied this in a cross-over human intervention trial where... 

Example 4-Hydroxynon-2-enal (4-HNE) is a major aldehydic product of lipid peroxidation (LPO), its products being indicators for oxidative stress. In order to introduce LPO products as biomarkers, a GC-MS method for 4-HNE detection in clinical studies [35] was developed using a sample volume of 50 pi of plasma. For improved GC separation and subsequent mass spectral detection the aldehyde is converted into the pentafluorobenzyl-hydroxylimine and the hydroxy group is tri-methylsilylated [36]. The TIC acquired in electron capture mode (EC, Chap. 7.4) exhibits 50 chromatographic peaks (Fig. 14.2). Those related to the target compounds can easily be identified from suitable RICs. The choice of potentially useful m/z values for RICs is made from the EC mass spectrum of the pure 4-HNE derivative (below). In this case, [M-HF], m/z 403, [M-HOSiMes] , m/z 333, and [CeFs]", m/z 167, are indicative, while [CH2C6F5] , m/z 181, is not. 

The basic approach used by Roessner et a/.39,41 has also been developed by Fiehn et al. for Arabidopsis. 43 Using GC-MS analysis, these authors distinguished some 326 distinct compounds in Arabidopsis leaves, of which roughly half could be assigned a chemical structure. They applied the GC-MS method to the analysis of four different Arabidopsis genotypes the ecotype Columbia (Col-2) and the dgdl... 

Muniz-Valencia 1 R, Ceballos-Magana SG, Gonzalo-Lumbreras R, Santos-Montes A, Izquierdo-Homillos R (2008) GC-MS method development and validation for anabolic steroids infeed samples, J. Sep. Sci. 31, 727-734... 

Gas chromatography is a powerful technique to dereplicate low to moderate polarity natural products especially when it is coupled with ESI-MS. The reproducibility of ionization and fragmentation pattern from GC/MS make it one of the most efficient and reliable techniques in structure dereplication. There are different GC/MS libraries commercially available that contain mass spectra of hundred thousands of compounds. Unfortunately, approximately 80% of all known natural compounds are nonvolatile or thermally unstable and therefore incompatible with GC/MS methods. Due to the diversity of HPLC columns and the broad selections of solvent combinations and gradients, HPLC is capable of separating almost any kind of natural products. The development of HPLC detectors and... 

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