Analytical Methods for Environmental Contaminants of Emerging Concern. Группа авторов

Coupled to Mass Spectrometry

Gas chromatography (GC) is a powerful technique for the separation and quantification of organics in complex matrices, such as extracts of environmental water and solids [33, 68]. The simplicity of GC operations, the high resolution of the capillary columns and the lack of liquid waste are some of the advantages compared to LC. Nevertheless, for most of the pharmaceuticals derivatization is needed to improve the volatility and thermal stability in the hot injector and capillary column. The disadvantage of derivatization for GC is that this is an additional step during the analytical protocol and derivative stability is low (for example, the silylated derivatives are fragile to moisture). The artefact and impact of environmental matrix in derivatization can be obtained [69]. The advantage of derivatization is that higher MS response/lower detection limit of targets and higher capacity of columns can be achieved (for example for oestrogens [70]). Only a few pharmaceuticals can be separated by GC without derivatization, for example tri-cyclic antidepressants and oestrogens. Most of the pharmaceuticals structures have at least one polar functional group, which increases the polarity and lowers the volatility of the molecule. With the increasing number of heteroatoms and molecular mass, the probability that a pharmaceutical can be analysed by GC is decreasing. Generally, the mass limit in GC is 1000 amu, but the practical limit is even lower – about 800 amu. Thus, pharmaceuticals such as most of the tetracyclines, sulphonamides and macrolides cannot be analysed by GC, even if the derivatization would be successful, because of each substituent increase of the mass of the analyte. β-blockers, β-agonists, non-steroidal anti-inflammatory drugs (NSAIDs) and oestrogens can be analysed by GC after the single-step derivatization, because the carboxyl and hydroxyl groups are easily transformed into derivatives by commercially available reagents. The amine and amides can be derivatized as well, and the most efficient are the acylation reagent, such as perfluorinated acid anhydrites.

Derivatization is mostly performed by silylation, which aims to exchange the labile polar hydrogen in the active groups of analytes into non-polar alkyl-silyl groups [71]. The currently available reagent allows for a quick and reproducible reaction; nevertheless the conditions initially need to be optimized, especially for mixtures of pharmaceuticals [72]. The optimal reaction duration and temperature for most pharmaceuticals are 10–30 minutes and 60°C. MSTFA (N-methyl-N-(trimethylsilyl)trifluoroacetamide) and BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) with TMCS (trimethylchlorosilane) as catalyst are the most popular reagents. Two-step derivatization could be needed for pharmaceuticals with two types of active groups. For example, the derivatization of β-blockers can be performed by two steps: 1. introduction of the trifluoroacetate group to the nitrogen atom by N-methyl-bis(trifluoroacetamide) (MBTFA), and 2. silylation of the hydroxyl group by any standard silylation reagent [73]. The novel reagent DIMETRIS (dimethyl(3,3,3-trifluoropropyl)silyldiethylamine) was used for β-blockers, NSAID and oestrogens analysis in environmental samples [58, 70, 74]. This reagent is used to introduce the fluorine atoms using the silylation mechanism. The alkylation with perfluorinated reagents TFAA (trifluoroacetic anhydride) or PFPAA (pentafluoropropionic anhydride) can be performed, but the removal of acidic by-products is needed. The old-fashioned technique of methylation with unstable and very flammable diazomethane was changed into silylation by trimethylsilyldiazomethane [75]. Some special solvents and additives could be needed for the derivatization of pharmaceuticals. For example, the silylation of 17-α-ethynylestradiol needs to be performed with pyridine addition to protect the ethynyl group in the hot injector [70]. For the silylation the water needs to be removed from the extract, thus additional time needs to be added to the whole protocol. Nevertheless, there are derivatization techniques for in situ derivatization, coupled with the extraction process, presented in Table 2.3.

Mass spectrometry (MS) is actually the only appropriate technique for the detection of traces of pharmaceuticals in environmental samples. Other detectors coupled to GC, such as the Electron Capture Detector (ECD), Flame Ionization Detector (FID) and Nitrogen Phosphorus Detector (NPD), are not selective enough to sufficiently assure the chromatographic signal origin. With MS more confirmation points (m/z values, fragmentation pathways, ratios of ions) are obtained compared to the use of only retention time with other detectors. Thus, in Table 2.3 presenting the selected exemplary methods of pharmaceuticals analysis, only GC/MS can be found. The mode of the mass spectra recording used was SIM (single quadrupole) and SRM/MRM (triple quadrupole), while full spectra recording for trace analysis is not recommended due to the high detection limits caused by noise. The advantage of the GC/MS is the mass library, where the most often analysed derivatives of pharmaceutical and metabolites can be found. The mass spectra of the silylated derivatives are easy to interpret, because of the repeatable fragmentation pattern related to substituent detachment [76]. The stable isotope labelled internal standards (SILISs) can be used, but caution should be taken in the choice of molecule, because of the possible low rate of separation with the target and overlapping of some m/z [77].

Table 2.3 GC/MS application for determination of pharmaceuticals in the environmental samples.

Analytes	Matrix		Derivatization technique and reagent	Detection limit	Ref.
Aspirin, ibuprofen, tramadol, fluoxetine, metoprolol, naproxen, diclofenac, pindolol, estrone, β-estradiol, 17-α-ethinylestradiol, estriol	River water	SPE-GC/MS/MS (triple quadrupole)	In-port silylation byN-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) and N-tert-butyl dimethyl-N-methyltrifluoroacetamide (MTBSTFA)	2.71–7.31 ng/L	[118]
20 pharmaceuticals [8 NSAIDs, 5 oestrogenic hormones, 2 antiepileptic drugs, 2 β-blockers, 3 antidepressants]	Soil	UAE-GC/MS	Silylation by N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 1% trimethylchlorosilane (TMCS) in pyridine and ethyl acetate (2 : 1:1, v/v/v)	0.3–1.7 ng/g	[119]
Ibuprofen, gemfibrozil, naproxen, ketoprofen and diclofenac	Water sample	dSPE-GC/MS	In situ trimethylphenylammonium hydroxide	1–16 ng/L	[120]
Flufenamic acid, mefenamic acid, flurbiprofen, clofibrate, ketoprofen, naproxen, tolfenamic acid, gemfibrozil	River water	SPME-GC/MS	Aqueous derivatization by tetrabutylammonium hydrogen sulfate and dimethyl sulfate	0.06–1.24 ng/L	[80]
Natural and synthetic oestrogens	Wastewater	SPE-GCxGC/TOF-MS	BSTFA + 1% TMCS + pyridine	Not specified	[121]
Paracetamol, ibuprofen, flurbiprofen, naproxen, diclofenac, 4-OH-diclofenac, 5-OH-diclofenac		SPE-GC/MS	BSTFA + 1% TMCS	2–4 ng/L	[86]

The separation of pharmaceutical derivatives is obtained using the standard type “5” capillary columns, (95% dimethyl-polysiloxane with 5% phenyl-polysiloxane as the stationary phase) with dimensions of 30 m length × 0.25 mm I.D. × 0.25 μm film thickness, and this is generally the most popularly used column in GC. The chiral columns can be applied for selected pharmaceutical analysis (review in [78]). The 70 eV of the EI ion source is also a standard for GC/MS. The spitless injection is used to ensure the low detection limits. The large volume injection was tested for determination of various pharmaceutical and personal care products [79], and has shown that such introduction techniques can be used only for extracts with a low mass of matrix ingredients. SPME, as the way of sample introduction into the GC/MS system, was tested for selected acidic pharmaceuticals with aqueous derivatization by dimethyl sulfate [80]. The two-dimensional techniques (GC × GC) coupled with time-of-flight mass spectrometry was used for the quantification of pharmaceuticals in environmental samples (review in [81]). Such separation technique is especially valuable for analysis of complex matrix, such as wastewater.

The extraction of environmental samples for GC analysis can be prepared by various techniques. The