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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, mobile phases consisting of methanol (MeOH), acetonitrile (ACN) and mixtures of them with different additives (i.e. formic acid (FA) or ammonium acetate (NH₄AC)) at different concentrations are used. The eluent type and composition are known to have a significant influence on obtaining reproducible retention times, satisfactory peak shapes and good ionization efficiencies [62, 87, 92–94]. In a publication by Tran et al. [87] the effect of different mobile phases on the separation of multiple classes of antimicrobials (AMX) was investigated. The addition of NH₄AC in the mobile phases significantly suppressed the signal intensity of almost all AMX agents and their isotopically labelled internal standards (ILIS). In contrast, the addition of formic acid improves the peak shapes and increases the detection sensitivity for most target compounds and their ILIS except triclosan, triclosan-d3, triclocarban, triclocarban-¹³C6, chloramphenicol and chloramphenicol-d5, for which a slight decrease in detection sensitivity was observed. Finally, a gradient consisting of 0.1% FA in water and 0.1% FA in a mixture of MeOH and ACN were selected as suitable mobile phases for routine HPLC-MS/MS analyses for the target compounds. A similar approach was used to determine 56 antibiotics (tetracyclines, sulfonamides, β-lactams, macrolides and quinolones) in water. ACQUITY UPLC® BEH C18 column, and a mobile phase consisting of a mixture of 0.1% formic acid in water and in acetonitrile was used for the separation of analytes. The method was implemented to assess the potential presence of antibiotics in wastewater and seawater in Tunisia [95].

Analysing the available literature data, it can be stated that sporadically a UV-VIS or diode array spectrophotometric detector is used, e.g. for analysis of sulfonamides [96] or non-steroidal anti-inflammatory drugs (NSAIDs) [97], whereas LC/UPLC in combination with a mass spectrometer having high sensitivity and specificity have been well developed in recent years and have become popular for monitoring single- or multiclass drug residues in different matrices. The preferred approach for pharmaceutical analysis is the use of triple quadrupole (QqQ) or ion trap (IT), in combination with electrospray ionization (ESI) [62, 87, 88, 93, 98]. Other hybrid techniques such as quadrupole analyser in combination with time-of-flight analyser, Orbitrap and hybrid quadrupole linear ion trap are also used [28, 62, 99, 100]. In addition to ESI ionization, other techniques, e.g. atmospheric pressure chemical ionization (APCI) or atmospheric pressure photoionisation (APPI), can be used, especially for less polar compounds that are usually not ionized by ESI [101]. In MS/MS systems, fragmentation using ESI is performed in either positive or negative mode. In general, the negative mode is preferred for acidic pharmaceuticals, while positive ionization is used for neutral and basic pharmaceuticals. A series of experiments to achieve satisfactory separation and high sensitivity for ionization of pharmaceuticals in positive and negative modes were carried out by Wu et al. [102]. The use of methanol as the organic phase resulted in increased sensitivity for most compounds compared to acetonitrile. An improvement in signal shape was observed when formic acid or ammonium acetate was added to the aqueous phase. Although 0.1% formic acid in water is very often used as a mobile phase for the simultaneous determination of both acidic and basic pharmaceuticals, it was observed that reducing the FA concentration to 0.001% improved the sensitivity by up to 14-fold for some acidic drugs detected in negative mode, including naproxen, ibuprofen, gemfibrozil and diclofenac, while this change had negligible effect for compounds analysed in the ESI + mode. Several advantages of the use of MS/MS in the analysis of pharmaceuticals in environmental samples should be highlighted. Firstly, it is not necessary to achieve full separation in chromatographic analysis for selective detection, which enables shorter analysis times using shorter LC columns. Another advantage of the MS/MS method is the ability to confirm the presence of compounds based on precursor and product ions [62, 66, 87, 89, 92, 94, 103, 104]. This is achieved by multiple reaction monitoring (MRM, also called selective reaction monitoring (SRM)) of two transitions between precursor and product ions to achieve four identification points (IPs) as a minimum requirement for positive identification and confirmation criteria as defined in EU Commission Decision 2002/657/EC. The first transition of the MRM is prone to false positive identification, therefore the results of the first MRM transition are verified by a second MRM transition leading to correct identification. Therefore, most environmental pharmaceutical studies use a quantitative approach with two MRM transitions to accurately identify target pharmaceuticals. Another advantage of LC-MS/MS, which has not yet been exploited in the analysis of pharmaceuticals in the environment, is the structure determination of unknown degradation and transformation products. In addition to the undoubted advantages of the LC-MS technique, it is widely recognized that the main drawback of electrospray ionization mass spectrometry is its susceptibility to matrix components in environmental samples [87–89, 93, 94, 105–108]. Consequently, HPLC-MS/MS analysis may be subject to signal suppression or enhancement of analytes, possibly due to the presence of co-matrix components in the samples. For this reason, assessment of matrix effects is extremely important to ensure accurate and reproducible quantitative data. Various methods to reduce the influence of matrix components, including the use of isotopically labelled standards, changes in mass spectrometer operating and chromatographic conditions and modifications to the sample extraction procedure have been described [87, 89, 108–111]. If signal suppression/enhancement cannot be sufficiently minimized by the strategies described, appropriate calibration techniques can be used to determine the value of matrix effects.

Table 2.4 LC/MS application for determination of pharmaceuticals

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