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 appropriate minimalization in the matrix composition and the sufficient concentration factor need to be achieved. Thus, the standard liquid-liquid and liquid-solid extraction is not suitable. Solid-phase extraction (SPE) is most popular for water samples, because of the high concentration factor and the exchange of the medium to organic solvent, which can be quickly evaporated and replaced into the derivatization reagent. Because of the low volume of derivatization reagent used (about 50–100 µL), the concentration factor can reach 20,000 for surface water samples for a 1–2 L volume sample. In comparison, in a case of LC/MS with about 1 mL of extract volume, a factor of 2,000 can be reached. The high concentration factor by SPE-GC/MS-based methods allows sub-traces of pharmaceuticals to be tracked in drinking and ground water [82]. The SPE is also normally applied for purification of extracts of biosolids and solids (for example for analysis of NSAIDs in mussel tissue [83]). The suppression/enhancing of the analyte signal by the matrix components (“matrix effect”) during SPE-GC/MS analysis is mainly connected with impurities accumulated in the injector and the start of the capillary column, rather than the impact on EI ionization [69], which is a crucial issue in the electrospray of LC/MS. Therefore, during analysis of pharmaceuticals by GC/MS in environmental samples, special attention needs to be given to lowering the interferents in the extract and purity of the GC system.
The pharmaceuticals can be simultaneously analysed by GC/MS with pesticides, endocrine disruptors and other semi-polar compounds [84, 85], if the extraction technique allows the efficient recovery of targets in a single extraction batch. The number of analytes in a single run is actually limited to the resolution of the capillary columns, but the effective recovery, presence of impurities and actual scope of the research limited this number to a practical 10–50 compounds in a single run. The phase I metabolites of pharmaceuticals, such as hydroxy- and carboxy-metabolites of NSAIDs, can be analysed together with the natives with the same extraction and derivatization protocols [86]. The phase II metabolites such as glucuronides cannot be analysed by GC, because of low thermal stability.
2.3.2 Liquid Chromatography and Liquid Chromatography Coupled to Mass Spectrometry
High-performance LC (HPLC) is still a widely used separation system and remains an excellent choice for the comprehensive analysis of pharmaceuticals in complex pharmaceutical samples. Currently, LC separation is most commonly achieved under reversed-phase LC (RPLC) conditions, e.g. using a C18 column [7, 13, 62, 87–89]. RPLC is a suitable choice for a wide range of compounds; however, highly polar substances are not retained and to separate mixtures of polar and highly polar compounds Hydrophilic Interaction Liquid Chromatography (HILIC) and Mixed-Mode LC (MMLC) are used [90, 91]. In recent years, there has been a significant increase in the use of ultra-high-performance LC (UHPLC) due to its reduced analysis time and chromatographic resolution and improved sensitivity (Table 2.4). Generally,
