# Contamination with Pharmaceuticals in Aquatic Environment: Focus on Analytical Methodologies
## 3 Determination of Pharmaceuticals in Environmental Samples
### Instrumental Separation and Detection Technique  
Purified and concentrated extracts containing pharmaceuticals are further analyzed using instrumental analytical techniques such as HPLC and, less commonly, gas chromatography (GC). The main requirement for the analysis of molecules using GC is high volatility. Most pharmaceutical pollutants are not sufficiently volatile and are not directly compatible with GC. However, several examples of pharmaceutical derivatization have been recorded, allowing for their analysis using GC-MS. Some examples of derivatizing agents used for the analysis of pharmaceuticals in environmental samples by GC-MS include pentafluorobenzyl bromide, methyl chloromethane, methanol/boron trifluoride (BF3), and tetrabutylammonium salts. The main advantage of GC-MS is that the ionization techniques, such as electron ionization (EI) or chemical ionization (CI), are less affected by the sample matrix compared to the ionization techniques used in LC-MS [32]. HPLC and the more advanced UHPLC-MS/MS with ultra-high-performance rapid flow rates and MS detection are alternative methods offering several advantages, including fast separation, absence of the need for derivatization, good sample stability, and small sample size [74]. Reverse-phase HPLC is widely used for multicomponent analysis with C18-based chromatographic columns. The typical column size parameters are as follows: (i) a length ranging from 10-25 cm, (ii) an internal diameter of 2.1-4.6 mm, and (iii) a particle size of 3-5 mm [75]. Most LC-MS/MS applications use electrospray ionization (ESI), which is generally compatible with lower mobile phase flow rates, from 0.1 to 0.5 mL/min. Due to these low flow rates, LC columns with narrow diameters (i.e., 2-3 mm) are more popular compared to the conventional analytical columns with a diameter of 4.6 mm. Narrower diameter columns also provide high separation efficiency and sensitivity and are less susceptible to matrix effects. Acidic and neutral pharmaceuticals are typically analyzed using columns longer than 15 cm [76]. Gradient elution represents the most common strategy for separating multiple classes of pharmaceuticals. Organic solvents such as acetonitrile and methanol are commonly used as mobile phases. Mobile phase modifiers, buffers, and acids are often used to achieve effective analyte retention in the column and improve MS detection sensitivity. Common modifiers include ammonium acetate, ammonium formate, tri-n-butylamine (TNBA), formic acid, and acetic acid [75; 77]. Typical salt concentrations range from 2 to 20 mM, as higher concentrations have been observed to reduce signal intensity [56; 75]. Among the detection techniques, MS and MS/MS are the most commonly used. Spectrophotometric detection in the ultraviolet-visible (UV-VIS) region is used less frequently. MS is often employed due to its high specificity and sensitivity. The triple-quadrupole (QqQ) mass analyzer is frequently used in combination with LC and has become the standard for many pharmaceuticals in various environmental samples. The QqQ mass analyzer uses multiple reaction monitoring (MRM) modes to achieve high selectivity and provide very low LODs [51]. The identification and quantification of pharmaceutical residues are also performed using high-resolution MS techniques such as time-of-flight (TOF) MS and quadrupole time-of-flight (Q-TOF) MS [74]. The most frequently detected pharmaceuticals in surface and wastewater are antibiotics, analgesics, beta-blockers, and estrogens. LODs are generally in the range of ng/L to \(\mu\)g/L [77]. The detected concentrations range from a few ng/L to 100 \(\mu\)g/L [75]. However, LC-MS/MS analysis can be affected by matrix effects (i.e., co-eluting components), which influence the ionization efficiency of target analytes and reduce method sensitivity and reproducibility [78]. These effects are most frequently observed in complex matrices such as sewage, wastewater, and solid samples [75]. [PERSON] et al.