Microextraction-Based Techniques for the Determination of Beta-Blockers in Biological Fluids: A Review

Abstract

Sample preparation is a critical step in the analytical process. Apart from the traditional sample cleanup approaches, microextraction-based techniques have attracted much attention in recent years and especially from researchers working in the analysis of beta-blockers. Developing sensitive and selective analytical methods is essential for detecting these compounds in complex matrices. The present review aims to provide an overview of microextraction-based analytical techniques for the determination of beta-blockers in biological samples, covering a time frame of the last decade. Detailed information on materials/coatings and instrumental configurations are provided.

1. Introduction

According to the World Health Organization (WHO), cardiovascular diseases were the most common cause of death [1,2]. Beta-blockers (β-blockers) are a distinct type of medication often prescribed for a variety of cardiovascular diseases such as angina pectoris, atrial fibrillation, cardiac arrhythmia, essential tremors, congestive heart failure, hypertension, glaucoma, hyperthyroidism, and mitral valve prolapse. Their action is based on the inhibition of the binding of norepinephrine and epinephrine to beta-adrenoceptors, which leads to a decrease in heart rate, cardiac output, and blood pressure, while also enhancing the oxygen supply to the myocardium [3,4].

Beta-blockers are categorized into three generations based on their receptor affinity. These substances control various functions based on their location in the human body: (i) β₁-receptors located in the heart, kidney and eye, (ii) β₂-receptors in the lungs, gastrointestinal tract, blood vessels, and liver and (iii) β₃-receptors in fat cells [5]. The chemical structures of these drugs are shown in Figure 1. The first-generation β-blockers non-selectively block both β1- and β2-adrenoceptors while the second-generation β-blockers are selective for β1-adrenoceptors, making them more appropriate for treating heart failure [6,7,8]. Third-generation β-blockers, such as carvedilol and labetalol, inhibit both β- and α1-receptors. The additional α-receptor blockade enhances these drugs’ blood vessel-dilating effects [9]. Besides their therapeutic uses, β-blockers are misused by athletes in sports like archery, billiards, and golf, where precise targeting is essential, as they help reduce heart rate and ease anxiety. As a result, the World Anti-Doping Agency has banned the use of β-blockers [10]. For these reasons, accurately measuring these drugs in various matrices is vital especially in the case of therapeutic monitoring, toxicology, doping control, and environmental risk assessment.

In recent years, significant advancements have been made in developing highly efficient analytical systems for drug detection in biological samples. However, a sample pretreatment step is still essential to extract and isolate the target analyte from these matrices before the final analysis [11]. This step is often the most labor-intensive and time-consuming part of the analytical process and remains a primary barrier to developing selective and sensitive analytical methods. Additionally, the sample cleanup procedure is responsible for approximately 30% of procedural errors. Ideally, sample preparation should be straightforward, affordable, fast, and capable of isolating or concentrating on the target compounds while preserving the quality of analytical data [12,13].

Since the introduction of solid-phase microextraction (SPME) by Arthur and Pawliszyn in 1990 [14,15], microextraction technologies have become promising sampling methods. These technologies generally involve minimum extraction phases (solid or liquid) to streamline the sampling process. The main advantages of these techniques include simple and rapid operation, high accuracy, improvement in sample clean-up, and less solvent consumption [16]. Both sorbent-based and liquid-phase microextraction techniques have been advanced to support diverse bioanalysis, improve compatibility with modern analytical instruments, reduce the need for hazardous chemicals, and enable efficient sampling in small-scale systems. In recent years, a variety of materials have been utilized as adsorbents in microextraction technologies, such as molecularly imprinted polymers (MIPs), carbon-based materials, restricted access material (RAM), metal–organic frameworks (MOFs), and covalent organic frameworks (COFs) [17,18].

The appropriate selection of microextraction media greatly improves the efficiency of sampling complex samples, enabling high selectivity and sensitivity in bioanalysis. Furthermore, the development of effective, biocompatible microextraction media, alongside miniaturized and automated microextraction devices, has greatly expanded their use in the analysis of biological samples and this technology will become a key focus of researchers [13,15].

A few review articles have been reported in the literature for the determination of β-blockers [5,19,20]. In 2020, a comprehensive review article has been published as an overview of the analytical methods dedicated to the determination of β-blockers covering applications, automation, and future trends [8]. The present review article focuses on microextraction-based analytical applications for the quantitation of certain drugs in biological matrices covering a timeframe of the last decade.

2. Liquid-Phase Microextraction

2.1. Theoretical Considerations

Liquid-phase microextraction (LPME) is a miniaturized version of the liquid–liquid extraction technique and it is widely used for the pretreatment of small sample volumes prior to analysis. Its principle is based on the usage of very small volumes (μL range) of an immiscible solvent (typically an organic solvent) that comes into physical contact with the sample [21]. Alternatively, the solvent can be placed in the pores of a hydrophobic membrane or separated from the donor phase by a membrane interface [22]. There are three main categories of LPME: (i) dispersive liquid–liquid microextraction (DLLME), (ii) hollow fiber LPME (HF-LPME) and single-drop microextraction (SDME). However, various modifications and alternative techniques have also been proposed such as tandem DLLME, air-assisted liquid–liquid microextraction (AALLME), three-phase solvent bar microextraction (TPSBME), which have been used for the extraction and detection of various compounds [21]. An overview of LPME applications for the determination of β-blockers is presented in Table 1.

2.2. Liquid–Liquid Microextraction

The research group of H.S. Mousa developed two methods for the determination of dorzolomide and timolol using salting-out-assisted liquid–liquid microextraction (LLME) [23,24]. After careful optimization of the microextraction parameters using experimental design, the samples were extracted using 90 μL of acetonitrile and 110 mg of (NH₄)SO₄ [23]. The 3D plots obtained from the Box–Behnken design are shown in Figure 2. The extraction procedure was performed in a 1 mL syringe where the upper layer (after phase separation) was easily collected due to capillary tube of the syringe. The mobile phase consisted of 30 mM phosphate buffer with 0.1% triethylamine (pH 3.5) and was capable of separating the analytes in a relatively short time. The method indicated adequate separation with enhanced sensitivity, with LOQs of 8.75 and 10.32 ng/mL. Two years later, a similar approach was developed by the same research group using quality-by-design principles for method development [24]. The parameters affecting the separation of the drugs were screened and optimized using Plackett–Burman and Box–Behnken experimental designs. The error propagation and the design space were estimated using Monte Carlo simulation experiments. To enhance the extractability of the drugs, an amount of 0.25–0.34 g of (NH₄)SO₄ was added into sample solution. A core–shell Kinetex XB C₁₈ analytical column was used for the rapid separation of the drugs in less than 4 min. Good recoveries were noticed ranging from 98.5% to 101.1% in all cases. The method was applied in the analysis of blood samples to support the pharmacokinetic studies of the analytes.

An application of batch UV spectrophotometry to the bioanalysis of drugs comprises the quantification of atenolol, propranolol and carvedilol in plasma and urine samples [25]. An air-assisted LLME followed by solidification of the floated organic solvent droplet was developed for the cleanup of biological matrices. The extraction procedure involved the utilization of 125 μL of 1-dodecanol. After centrifugation and phase separation, the organic solvent was solidified by placing the sample into an ice bath for few minutes. The volume of 1-dodecanol was mixed with acidified water and analyzed with UV spectrophotometer. It was found that the sample pH value of 2.0 provided the best results. To enhance the dispersion of the extraction solvent in the aqueous sample phase, the authors utilized the non-ionic surfactant Triton X-100. Statistical treatment of the results using partial least squares regression was performed to spectral discrimination of the analytes. Compared with conventional DLLME, the extraction solvent used in this approach is less toxic and no organic dispersive solvent was required.

A vortex-assisted LLME in combination with LC–MS/MS was proposed by M. A. Castro e Souza et al. for the simultaneous determination of eight cardiovascular drugs including carvedilol in human plasma [26]. A gradient elution program using 0.1% aqueous formic acid solution and methanol was employed for the separation of the drugs which quantified using tandem MS in the multiple reaction monitoring mode. Before microextraction, the proteins of the samples were precipitated using trifluoroacetic acid. It was found that the sample pH played a critical role in the extraction performance. A pH value of 6 was finally selected which resulted in the non-ionized forms of the studied molecules improving their extractability. Dichloromethane was utilized as extraction medium while a 1% m/v NaCl provided the best extraction efficiency due to the salting-out phenomenon. Weighted 1/y² or 1/x² calibration curves were constructed to prove the linearity of the method in the concentration range of 2–1000 ng/mL. The method was successfully applied to quantify the drugs in patient samples attending a hospital service.

Table 1. LPME applications for the determination of β-blockers in biological fluids.

Analyte 1	Sample	LPME Type/Extraction Solvent 2	Analytical Technique 3	LOD/LOQ (ng/mL)	Recovery (%)	Reference
ATE, MET, PRO	Human plasma	DLLME/1-butyl-3-methyl imidazolium hexa fluoro phosphate	HPLC–DAD	2.6–3.0/8.9–9.9	99.37–100.21	[27]
CAR, MET, PRO and others	Human plasma	DLLME/Dichloromethane	HPLC–UV	2–6/7–19	94–104	[28]
MET and others	Rat plasma	UA–DLLME/Chloroform	HPLC–UV	13–31/43–103	94–104	[29]
PRO	Human plasma	SFOD–DLLME/1-undecanol	HPLC–FLD	0.15/0.5	90–96.6	[30]
MET, PRO	Human plasma	TDLLME	HPLC–UV	0.8–1/2.5–3	90–91	[31]
ATE, MET, PRO	Human plasma	DLLME/tetramethylammonium chloride-terpineol	GC–MS	0.13–0.205/0.435–0.692	72–86	[32]
MET, PRO and others	Human urine	HF-LPME/methyl benzol	GC–MS	0.05–0.08/0.25	93.79–109.04	[33]
ATE, BET, PRO	Human saliva	EME/mixture of di-(2-ethylhexyl) phosphate, tris-(2-ethylhexyl) phosphate and 2-nitro phenyl octyl ether	HPLC–UV	2/10	94.8–102.7	[34]
PRO and others	Human urine, plasma	HF-LPME coupled to EME/1-octanol	HPLC–UV	0.12/2.5	65.1	[35]
PRO	Human urine, plasma	EME/2-nitro phenyl octyl ether	CE–UV	7/20	54–67	[36]
ATE, MET	Human urine	EME/thymol, coumarin, camphor	HPLC–DAD	8–9/24–29	72.6–78.6	[37]
CAR, MET, PRO and others	Human urine	MS-LLLME/toluene, acetic acid	CE–UV	0.02–0.82/0.2–0.5	91.1–114	[38]
CART, BIS, PRO, SOT	Human urine	CM-LPME/toluene	HPLC–UV	5–10/50	91.1–109.4	[39]
BIS	Human plasma	TPSBME/n-octanol	HPLC–UV	3/10	61.4–66.7	[40]

¹ ATE: atenolol, BET: betaxolol, BIS: bisoprolol, CART: carteolol, CAR: carvedilol, MET: metoprolol, PRO: propranolol, and SOT: sotalol. ² CM-LPME: carrier-mediated liquid-phase microextraction, DLLME: dispersive liquid–liquid microextraction, EME: electromembrane extraction, HF-LPME: hollow fiber liquid-phase microextraction, MS-LLLME: membrane-supported liquid–liquid–liquid microextraction, UA–DLLME: ultrasound-assisted DLLME, SFOD–DLLME: solidified organic droplet–DLLME, TDLLME: tandem DLLME, and TPSBME: three-phase solvent bar microextraction. ³ CE–UV: capillary electrophoresis-ultraviolet detector, DAD: diode array detector, FLD: fluorescence detector, HPLC: high pressure liquid chromatography, and MS: mass spectrometric detector.

Table 1. LPME applications for the determination of β-blockers in biological fluids.

Analyte 1	Sample	LPME Type/Extraction Solvent 2	Analytical Technique 3	LOD/LOQ (ng/mL)	Recovery (%)	Reference
ATE, MET, PRO	Human plasma	DLLME/1-butyl-3-methyl imidazolium hexa fluoro phosphate	HPLC–DAD	2.6–3.0/8.9–9.9	99.37–100.21	[27]
CAR, MET, PRO and others	Human plasma	DLLME/Dichloromethane	HPLC–UV	2–6/7–19	94–104	[28]
MET and others	Rat plasma	UA–DLLME/Chloroform	HPLC–UV	13–31/43–103	94–104	[29]
PRO	Human plasma	SFOD–DLLME/1-undecanol	HPLC–FLD	0.15/0.5	90–96.6	[30]
MET, PRO	Human plasma	TDLLME	HPLC–UV	0.8–1/2.5–3	90–91	[31]
ATE, MET, PRO	Human plasma	DLLME/tetramethylammonium chloride-terpineol	GC–MS	0.13–0.205/0.435–0.692	72–86	[32]
MET, PRO and others	Human urine	HF-LPME/methyl benzol	GC–MS	0.05–0.08/0.25	93.79–109.04	[33]
ATE, BET, PRO	Human saliva	EME/mixture of di-(2-ethylhexyl) phosphate, tris-(2-ethylhexyl) phosphate and 2-nitro phenyl octyl ether	HPLC–UV	2/10	94.8–102.7	[34]
PRO and others	Human urine, plasma	HF-LPME coupled to EME/1-octanol	HPLC–UV	0.12/2.5	65.1	[35]
PRO	Human urine, plasma	EME/2-nitro phenyl octyl ether	CE–UV	7/20	54–67	[36]
ATE, MET	Human urine	EME/thymol, coumarin, camphor	HPLC–DAD	8–9/24–29	72.6–78.6	[37]
CAR, MET, PRO and others	Human urine	MS-LLLME/toluene, acetic acid	CE–UV	0.02–0.82/0.2–0.5	91.1–114	[38]
CART, BIS, PRO, SOT	Human urine	CM-LPME/toluene	HPLC–UV	5–10/50	91.1–109.4	[39]
BIS	Human plasma	TPSBME/n-octanol	HPLC–UV	3/10	61.4–66.7	[40]

¹ ATE: atenolol, BET: betaxolol, BIS: bisoprolol, CART: carteolol, CAR: carvedilol, MET: metoprolol, PRO: propranolol, and SOT: sotalol. ² CM-LPME: carrier-mediated liquid-phase microextraction, DLLME: dispersive liquid–liquid microextraction, EME: electromembrane extraction, HF-LPME: hollow fiber liquid-phase microextraction, MS-LLLME: membrane-supported liquid–liquid–liquid microextraction, UA–DLLME: ultrasound-assisted DLLME, SFOD–DLLME: solidified organic droplet–DLLME, TDLLME: tandem DLLME, and TPSBME: three-phase solvent bar microextraction. ³ CE–UV: capillary electrophoresis-ultraviolet detector, DAD: diode array detector, FLD: fluorescence detector, HPLC: high pressure liquid chromatography, and MS: mass spectrometric detector.

2.3. Dispersive Liquid–Liquid Microextraction

The DLLME technique was initially introduced by M. Razaee et al. as a fast, low-cost, sensitive and broad-applicability technique offering a high preconcentration factor and high method sensitivity [41]. In this mode, a cloudy solution is formed when an appropriate mixture of extraction and dispersive solvent is injected into an aqueous sample containing the analytes of interest. Following centrifugation, the analytes in the separated phase can be analyzed using analytical techniques [42].

A DLLME protocol coupled with HPLC–DAD is applied for the determination of atenolol, metoprolol and propranolol in blood samples [27]. Plackett–Burman design (PBD) in combination with central composite design was applied to optimize the method parameters. After method optimization, good repeatability (RSD < 3.99%), linearity (r² = 0.997) and accuracy (>91%) were achieved. Analyte stability was dependent on sample solution pH while the highest extraction performance was obtained in the pH range of 9–11, where the analytes exist in their neutral forms. Compared to other ionic liquids (IL), the 1-butyl-3-methyl imidazolium hexa fluoro phosphate was selected as an extractant due to its high extraction recoveries (62.02–84.5%). The miscibility of the dispenser solvent in both water and IL was examined and it was found that methanol provided the maximum recoveries for all analytes. High method sensitivity was achieved and the LOD values ranged between 2.68 and 3 ng/mL. Five antiarrhythmic drugs—carvenolol, diltiazem, metoprolol, propranolol and verapamil—were determined in human plasma by the HPLC–UV method after DLLME [28]. The researchers utilized acetonitrile for both sample protein precipitation and as a disperser solvent in the microextraction procedure. One of the main disadvantages of the proposed method is the usage of the highly toxic dichloromethane as extraction solvent. The pH of the aqueous solution is adjusted to 11.5, containing 1% w/v NaCl. After phase separation, the sedimented phase was evaporated to dryness and redissolved in acidic solution prior to HPLC analysis. Relatively low enrichment factors were obtained (4.4–10.8) while the extraction recoveries varied between 33% and 82%. The method was linear over the dynamic range of 0.02–0.80 μg/mL (r² ≥ 0.997), repeatable (%RSD < 20), accurate and precise.

Felodipine has high plasma protein binding, which complicates its extraction from plasma. Conventional LLE methods are less effective for the determination of felodipine due to this strong binding [43]. To overcome this, S. Ahmed et al. developed an ultrasound-assisted DLLME (UA–DLLME) protocol for the simultaneous quantification of felodipine, metoprolol and ramipril in rat plasma after oral administration of two commercially available drug-containing formulations [29]. This method demonstrated that felodipine can be accurately analyzed despite the potential interference from other drugs. After the investigation of various solvents, chloroform was found to be the best extraction solvent at a volume of 500 μL. Acetonitrile was employed as protein precipitation agent and as dispersive solvent as well. A sample pH value of 10.0 was set to ensure the analytes are in their ionizable forms. The use of ultrasound irradiation speeds up the extraction process by promoting efficient phase transfer. Under optimal conditions, all drugs are separated within 13 min and the method linearity was examined between 0.05 and 2.0 µg/mL for felodipine and metoprolol and 0.1 and 2.0 µg/mL for ramipril. Low detection limits of 0.013–0.031 µg/mL were observed while the developed method showed good precision (0.63–3.85%RSD) and accuracy (92.13–100.5%).

A DLLME combined with the solidification of a floating organic droplet protocol was recently published by F. Khalil et al. for the extraction of propranolol enantiomers from human plasma [30]. Enantiomeric separation was performed using normal-phase HPLC with a mixture of hexane and ethanol (80:20 v/v) and triethylamine (0.2%) as the mobile phase. Better extraction efficiencies were recorded at a sample pH value of 13, and 1-undecanol was chosen as extracting solvent due to its low volatility and toxicity, low water miscibility and good chromatographic performance. The extraction tube was cooled with an ice bath to obtain the solidified extractant. The mean recoveries are rather poor (~14%) and linear responses were obtained in the range of 0.5–100 ng/mL for both enantiomers. Adequate repeatability (8–9.5%) and sensitivity (LOQ = 0.5 ng/mL) were also recorded.

A tandem DLLME has been introduced by M. Hemmati et al. for the cleanup of complex matrices [31]. This technique combines two sequential DLLME procedures and has been used for analyte extraction and preconcentration from complex matrices. At the first DLLME step, the sedimented extraction solvent (1,2-dichloromethane) was mixed with 45 μL of acidified water (pH 2) and the mixture was emulsified by 15 suction/injection cycles using a syringe. The analytes were then back-extracted to the aqueous phase, eliminating the problem of injection of 1,2-dichloromethane into HPLC due to its immiscibility with the mobile phase. The optimization of the extraction parameters was performed using Box–Behnken experimental design. Wide linear determination ranges were recorded for both β-blockers (metoprolol, propranolol) ranging between 2.5 and 2500 ng/mL with enrichment factors ca 100. Real human samples were analyzed with analyte recovery > 90%.

More recently, a hydrophobic deep eutectic solvent (DES) based on α-terpineol was developed for the microextraction of atenolol, metoprolol and propranolol from plasma samples [32]. Tetramethylammonium chloride (hydrogen bonding donor) and terpineol were added in the sample solution to enhance analyte extraction. Microwave irradiation at 180 W was employed to increase the temperature which then accelerated the formation of DES. After centrifugation and sample cooling, a volume of 7 μL was injected into GC–MS for analysis. The validated parameters demonstrated low LODs (0.130–0.205 ng/mL) and LOQs (0.435–0.692 ng/mL) and high extraction recoveries (72–86%). Other advantages of the method include speed, simplicity, and environmental friendliness.

2.4. Hollow Fiber Liquid-Phase Microextraction

Hollow fiber liquid-phase microextraction (HF-LPME) utilizes disposable porous hollow fibers, typically made of polypropylene (or other materials), filled with a small volume of extracting solution (acceptor phase) and immersed in an aqueous solution (donor phase) [44]. This technique offers several advantages, including low cost, effective sample cleanup, and high preconcentration factors. However, it has limitations, such as the potential adsorption of hydrophobic substances commonly present in biological samples (i.e., plasma, urine, serum) and the formation of air bubbles on the fiber surface during extraction, which can adversely affect the method’s reproducibility.

An HF-LPME technique is developed for the determination of metoprolol in human plasma using HPLC and a fluorescence detector [45]. The authors investigated the critical method’s parameters such as the concentrations of the donor phase and the acceptor phase, extraction time, temperature, stirring rate and the concentration of NaCl. The analyte was separated on a C₁₈ stationary phase using a mixture of methanol/0.1% v/v H₃PO₄, 40/60 v/v. Adequate method sensitivity was achieved with LOQ at 2 ng/mL while the analyte was detected spectrofluorimetrically at λ_ex/λ_em = 227/305 nm. The method is accurate (recovery: 87.1–92.6%) with good repeatability (RSD < 10%) and it is suitable for the quantitation of metoprolol in human plasma. A highly sensitive GC–MS method has been proposed for the determination of clenbuterol, metoprolol and propranolol in human urine samples after HF-LPME and in situ derivatization [33]. The analytes were extracted using methylbenzol and then derivatized with N-methyl-N-(trimethylsilyl) trifluoroacetamide to improve their volatility. The samples were initially basified at pH 12 and then extracted for 20 min at 35 °C. Compared to the injection-port derivatization method, the in situ derivatization procedure offered simplicity and rapidity avoiding the potential stationary damage due to the reaction of the excess of the derivatizing agent with the stationary phase. The LODs ranged between 0.05 and 0.10 ng/mL using the SCAN mode. The feasibility of the proposed method was investigated by analyzing a set of samples up to 36 h after oral administration of 50 mg drug-containing formulation.

2.5. Electromembrane Extraction

Electromembrane extraction (EME) is a membrane-based extraction technique which has been developed by the research group of Pedersen-Bjergaard [46,47]. The EME procedure incorporates two well-established concepts: partitioning (diffusive migration) and electrophoresis (electrokinetic migration). While both processes take place during EME extraction, electrophoresis serves as the primary mechanism driving the analytes from the donor solution to the acceptor solution.

An electric field across a supported liquid membrane (SLM) protocol has been proposed for the analysis of three β-blockers (atenolol, betaxolol and propranolol) [34]. Phosphoric acid in 2-nitro phenyl octyl ether was used as selective ion carrier. The method principal lies in the migration of the analytes from 3 mL sample solution into a 15 μL acidic acceptor through a thin layer of organic solvent immobilized in the pores of a porous hollow fiber. A schematic diagram of the extraction of atenolol from saliva samples is depicted in Figure 3.

A mixture containing 5% of 2-ethylhexyl phosphate and tris-(2-ethylhexyl) phosphate in 2-nitro phenyl octyl ether resulted in good selectivity as these reagents interacted with the analytes and accelerated their transport from sample solution to SLM. Performing this procedure a preconcentration factor of 74 was achieved. Almost 10 years later, Ramini et al. combined dynamic single-interface HF-LPME with EME for the isolation of some basic drugs (propranolol, diltiazem, lidocaine) from urine and plasma samples [35]. The researchers utilized polypropylene hollow fiber impregnated with 1-octanol as the acceptor phase and the alkaline sample solution (pH 12) was pumped towards to the lumen using syringe pump at a flow rate of 2 mL/min. At the 2nd step, the extracted analytes were back-extracted by a fast EME procedure using a voltage of 100 V and an acceptor phase of 0.1 M HCl solution. The intra-day and inter-day precision was lower than 10% while relatively poor recoveries were obtained in the range of 65.4–88.8%. One of the main advantages of the method is that the proposed configuration is applicable for any sample volume and common problems related to the bubble formation due to electrolysis are minimized.

A capillary electrophoresis (CE) approach has been published for the determination of enantiomeric forms of propranolol in in human body fluids [36]. The researchers took advantage of using EME with 4 mL acidified sample while the pores of the hollow fiber were impregnated with 2-nitrophenyl octyl ether. After the extraction, the extractant was transferred to a vial for CE analysis. The most critical parameters (voltage, extraction time, pH value of acceptor and donor phase) affecting the performance of the EME procedure were optimized using Box–Behnken experimental design. Good resolution between the analyte enantiomers was recorded when hydroxypropyl-β-cyclodextrin was used in the background electrolyte. Only 10 μL of organic solvent was employed for the extraction and the small pores of the hollow fiber effectively prevented the transfer of large biomolecules of biological samples to the acceptor phase.

An interesting investigation was conducted by S. Abbasi’s research group, comparing the performance of a biodegradable DES and an agarose gel for their use in EME [37]. Various drugs including atenolol and metoprolol were employed as model compounds. It was proved that EME using DES provided better extraction performance for non-polar base drugs and especially those containing coumarin and thymol. Good linearity was obtained for all tested drugs, with r² values exceeding 0.9935 in all cases. From a green chemistry point of view, gel-based and coumarin/thymol-based EME were environmentally friendly methodologies. In all cases, low LODs were obtained ranging between 5 and 10 μg/L with enrichment factors of 110–148. The applicability of the method was demonstrated by analyzing urine samples obtained from a 50-year-old man with cardiovascular disease.

2.6. Other Liquid-Phase Microextraction

A membrane-supported liquid–liquid–liquid microextraction was recently published for the isolation of six cardiovascular drugs (i.e., mexiletine, xylocaine, propafenone, propranolol, metoprolol, and carvedilol) from human urine samples [38]. According to the authors, the analytes were extracted from a 9 mL sample solution into toluene, and then back extracted into a volume of 10 μL of the acceptor phase (20 mM CH₃COOH). At the 2nd step, the acceptor phase was introduced into CE. A field-amplified sample injection (FASI) technique was used for the preconcentration of the analytes. After investigating the parameters affecting the separation, better resolution was obtained using a mixture of Tris-H₃PO₄ (pH 2.2) and methanol as background electrolyte. The developed method was effectively utilized for the real-time analysis of metoprolol in human urine samples up to 26 h after drug administration.

A carrier-mediated LPME protocol has been published by the research group of M. Ma for the extraction and preconcentration of β-blockers in human urine [39]. By adding an appropriate carrier (tetraoctyl ammonium bromide) in the organic phase, simultaneous extraction and enrichment of hydrophilic (sotalol, carteolol, and bisoprolol) and hydrophobic (propranolol) drugs have been achieved. Various organic solvents have been tested (i.e., benzene, toluene, o-xylene, n-hexane, n-octanol, and isoamyl alcohol) where the toluene provided the best extraction efficiency. Relatively high enrichment factors (up to 272) have been accomplished and resulted in low LODs (5–10 μg/L). In order to prove the applicability of the method, the authors analyzed an authentic urine sample obtained at 4 h after oral administration of propranolol-containing formulation.

M. Zhang et al. proposed a three-phase solvent bar microextraction coupled with HPLC for the study of plasma protein binding of bisoprolol [40]. Bisoprolol was extracted from a 5.6 mL basified plasma sample (donor phase) into n-octanol, which was embedded within the pores of a hollow fiber. After that, the authors transferred the extractant to an acidic solution (acceptor phase) contained within the hollow fiber’s lumen. Various parameters affecting the method’s efficiency have been examined, including the type of organic solvent (n-butanol, n-octanol, dibutyl phthalate, dihexyl ether), stirring rate, and extraction time. Stability studies (7 days at −20 °C) of the analyte in blood samples revealed that the drug was stable under these conditions. The calculated protein binding of bisoprolol was ca 32% after equilibration for at least 8 h at 37 °C.

3. Sorbent-Based Microextraction

3.1. Theoretical Considerations

The introduction of sorbent-based microextraction techniques has greatly advanced the development of sustainable sample preparation methods. Various miniaturized techniques simplify workflows and reduce both solvent consumption and waste. These techniques involve a range of approaches and operation modes offering a versatile toolkit for cost-effective, rapid, and eco-friendly sample preparation [48].

SPME was the first sorbent-based microextraction technique, originally designed as a solvent-free sample preparation protocol prior to gas chromatographic (GC) analysis [14]. Since its development, it has significantly progressed, inspiring the development of other sorbent-based microextraction techniques. Nowadays, it is a widely used green technology, available in both manual and automated versions. It encompasses a variety of extraction devices featuring expanded geometries, diverse sorbent coatings, and advanced manufacturing techniques [49].

Despite a significant breakthrough by SPME, efforts to miniaturize sorbent-based techniques trace back to the 1980s, beginning with packed device-based methods. Early attempts to downsize solid-phase extraction (SPE) involved replacing traditional cartridges with extractive discs and small syringes containing small quantity of extraction material (μ-SPE) [50]. Later innovations aimed on further minimization of sorbent quantities, leading to Disposable Pipette Extraction (DPX), where pipette tips substituted SPE cartridges [51]. Microextraction by Packed Sorbent (MEPS) was developed to automate the extraction process by using autosampler needles packed with small quantities of sorbent [52]. The minimal sorbent requirements of DPX and MEPS have significantly advanced the exploration of new extraction materials, supported broader applications and promoted the development of more cost-effective, versatile, and efficient extraction tools. Other alternatives sorbent-based microextraction techniques include Dispersive Solid-Phase Extraction (dSPE), Matrix Solid-Phase Dispersion Extraction (MSPD), QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe), and Stir Bar Sorptive Extraction (SBSE) [53,54,55,56].

The development of miniaturized sorbent-based sample preparation techniques has been accompanied by significant research and development of new extractive sorbents. The affinity of the extraction material for the analytes typically determines their extraction efficiency [50]. Thus, many efforts have been made by the researchers in recent years and especially on the development of various sorbents. Typical examples of these materials include polymers (chitosan-, silica-based polymers), porous materials (COFs, 1MOFs, MIPs, cyclodextrin based, aptamer based), carbon allotropes (carbon nanotubes), magnetic nanoparticles, ionic liquids (ILs) and green sorbents from natural sources [57,58].

3.2. Solid-Phase Microextraction

SPME was introduced by Arthur and Pawliszyn almost 30 years ago [14]. This technique combines sampling, preconcentration, and extraction into one step. The main advantages of SPME include simplicity, speed, high accuracy, improved sample clean-up, and reduced solvent use. SPME also enables simultaneous preconcentration and separation of both volatile and non-volatile compounds [16].

An interesting work has been proposed by D. Vuckovic at el. where the in vitro evaluation of new biocompatible coatings for SPME was carried out [59]. The SPME coatings were immobilized on a metal fiber core. After appropriate coating of the fiber with a biocompatible binder different silica-based material (i.e., octadecyl, polar-embedded, cyano) were finally immobilized. One of the main goals of this research work was the in vitro assessment of the fabricated fibers to use them in in vivo SPME applications. The authors examined the solvent compatibility of the biocompatible SPME fibers. It was concluded that the bonded silica-coated fibers did not swell in any tested solvent while a significant swelling was observed on Carbowax-TPR^TM commercial fibers when they were exposed to water, methanol and aqueous/organic mixtures. Thicker coatings (i.e., 45, 60 μm) showed better reproducibility because it was easy to prepare them in a uniform way using the automated batch coating procedure. The agitation speed and the elution volume was found to be critical in the desorption efficiency. The method showed adequate LOQs (5 ng/mL) for the determination of propranolol in urine and plasma samples. A potential disadvantage of the prepared coatings can be the prolonged extraction time (≥300 min for 60 μm coatings) required for equilibrium. Some years later, the same groups of researchers developed a high-throughput method based on thin-film SPME for the determination of 110 doping compounds including β-blockers after analysis with liquid chromatography-tandem mass spectrometry (LC–MS/MS) [60]. The authors took advantage of an automated 96-blade SPME system which consisted of eight rows of combs where each comb has 12 blades coated with the extraction sorbent. Each part of the system comprised four agitators for SPME blades preconditioning, sample extraction, blades washing and analytes desorption. A comprehensive review on SPME in multi-well-plate format has been published by D. Vuckovic [61]. The linearity of the method was investigated in the range of 0.05–100 ng/mL, with a R² > 0.991. Compared to liquid–liquid extraction (LLE), solid-phase extraction (SPE), the proposed analytical scheme was superior as the reported LODs were sufficiently of 166 banned compounds according to World Anti-Doping Agency (WADA). Most of the examined drugs showed no matrix effect (80–120%) and only two compounds (bendroflumethiazide, furosemide) observed ion suppression of 62–63%. Among other advantages, the method is capable of simultaneously analyze both free and glucuronidated forms of compounds through direct extraction. This feature avoids the time-consuming and laborious step of enzymatic deconjugation of the glucuronides before analysis. Two years later, W. Liu et al. proposed an environmentally friendly and sensitive method for determination of blockers and agonists using home-made sol–gel SPME coating with simultaneous on-fiber derivatization [62]. The analytes (clenbuterol, metoprolol and propranolol) were derivatized with trifluoroacetamide (MSTFA) to increase their volatility and the GC–MS responses. After optimization of the derivatization conditions the drugs are MSTFA-tagged at 40 °C for 10 min. Four different SPME fibers with different polarities were studied including Carbowax/divinylbenzene, carbowax, hydroxyl silicone oil–polymethylhydrosiloxane and polyacrylate. The preparation of these materials has been reported in a previous publication of the authors [63]. The authors concluded that the carbowax/divinylbenzene exhibited the best performance for all analytes. The sample pH played an important role in the dissociation of amino groups of the drugs and the functional groups of the SPME coating. An alkaline sample pH (pH 11) was chosen to prevent dissociation of the amino compounds providing maximum extraction efficiency. After the optimization of the SPME parameters the achieved LODs were varied between 0.2 and 0.5 ng/mL in the selected ion monitoring (SIM) mode. The method applied to the analysis of the drugs in saliva samples.

An UPLC–MS method has been proposed by K. Gorynski et al. for the quantification of eight β-blockers (namely acebutolol, atenolol, fenoterol, nadolol, pindolol, procaterol, sotalol, and timolol) in plasma and urine samples after SPME [64]. The analytes were extracted on the biocompatible extraction phase made of hydrophilic–lipophilic balance particles in a 96-well plate format. The analytes were successfully separated on a pentafluorophenyl stationary phase using a gradient elution of 0.1% v/v formic acid and methanol. A sample volume of 1200 μL under stirring rate of 850 rpm was used for the extraction of the analytes at 30 °C. An acidified methanol/acetonitrile (80/20 v/v) mixture with formic acid were employed for the drugs desorption. The optimized SPME scheme was consistent for both urine and plasma sample and permitted the parallel sample preparation of 96 samples in less than 2 min per sample. Method validation characteristics include intra-day and inter-day precision of less than 15.1% and the method accuracy ranged between 83.4 and 114.7%.

An SPME protocol has been developed by the research group of S. Ahmad for the estimation of the plasma protein binding of metoprolol and propranolol [65]. The effectiveness of SPME fibers for assessing plasma protein binding was evaluated by extracting the drugs in vitro from a protein-free matrix and rat plasma. The extracted drug amounts were compared to estimate the plasma protein binding. The unbound fraction of each drug was analyzed by LC–MS/MS in the multiple reaction monitoring mode. The results obtained from SPME experiments were compared to the well-established rapid equilibrium dialysis. The % difference ranged between −1 and 7.3% and −2.4 and 11.6% for metoprolol and propranolol, respectively. Compared to equilibrium dialysis, SPME provided several advantages, including simplicity, faster equilibration, and reduced analysis time, with the entire procedure completed in just 1 h.

A vibrating sharp-edge spray ionization device has been constructed as an interface between SPME and direct mass spectrometry for the determination of metoprolol, pindolol, acebutolol, oxprenolol, and other drugs [66]. The authors designed an interface that incorporates both desorption and ionization step in one device. It was based on the usage of a glass capillary, allowing the direct insertion of a commercial SPME probe into the capillary which serves as desorption chamber (Figure 4). After optimization of the method’s parameters, a volume of 500 μL sample was extracted for 30 min and desorbed in 3.5 μL for 3 min. Adequate linearity (R² > 0.97) was observed for all tested compounds. By utilizing the cost-effective device, the analysis throughput for multiple samples can be significantly increased to approximately one sample per min. An analogous SPME-probe electrospray ionization device has been constructed by J. Pawliszyn for the quantification of drugs of abuse in biofluids [67]. The probe was synthesized by mixing polyacrylonitrile and dimethylformamide and coated with 1.3 μm hydrophilic–lipophilic particles. A mixture consisting of isopropanol/water (50/50% v/v) containing 0.1% formic acid has been selected as the optimum desorption solvent for the tested analytes (including propranolol), as it facilitated high analyte enrichment and efficient electrospray media. The proposed method revealed adequate analytical figures of merit. The intra-day accuracy of the method was within the range of 80–120% for all drugs except for lorazepam which had an accuracy of 122% (at 30 pg/μL level). The lower LOQ of the method ranged between 5 and 10 pg/μL.

An in-tube SPME using polyacrylonitrile/MXene nanofiber yarns was developed for the determination of atenolol, propranolol and betaxolol in human urine and saliva [68]. The nanofiber yarn was prepared using electrospinning of nanofibers and characterized by Fourier-Transform Infrared Spectroscopy, Field-Emission Scanning Electron Microscopy, Energy-Dispersive X-Ray Mapping, transmission electron microscope and X-Ray Diffraction analysis. The 3D yarn sorbent exhibited high flexibility and mechanical stability, porosity and sorbent loading. The method was based on the analyte sorption in a 10 cm column packed with 20 mg of sorbent under circulation conditions using a peristaltic pump. The desorption of β-blockers was carried out using 400 μL of basified methanol. Good linearities were obtained for all analytes in the range of 5–1000 μg/L with extraction recoveries between 29.4% and 48.6%. The method was applied to the analysis of urine and saliva after oral administration of a propranolol-containing drug (20 mg/tab).

In 2019, M. A. Rosa et al. proposed a RAM-modified SPME method for the determination of three β-blockers (namely atenolol, metoprolol, propranolol) in bovine serum albumin [69]. The restricted-access nanoparticles were prepared from bovine serum albumin by coacervation. The synthesis of the sorbent was simpler and required less reagent and time compared to other extraction materials. The efficiency of RAM was proved since an almost 100% exclusion of BSA was achieved using this technology. The authors studied the adsorption isotherms of the material and Sip’s isotherm provided better fitting compared to other tested models. Low LODs (3.8–25.6 μg/L) were achieved while the prepared sorbent can be reused up to 15 sequential extraction cycles. A potential disadvantage of this method is that the RAM sorbent shows high resistance to the flow of organic solvents, such as methanol and acetonitrile, when packed and used in column-switching liquid chromatography.

3.3. Microextraction by Packed Sorbent

Microextraction by Packed Sorbent is a miniaturized version of the SPE technique, primarily used for sample purification and analyte preconcentration [70]. In this technique, the sorbent is directly integrated into the syringe, allowing it to be reused over 100 times for plasma or urine samples. MEPS is suitable for small sample volumes (i.e., 10 μL) and can accommodate volumes up to 1000 μL. It can be seamlessly connected to various separation techniques, including GC, LC, LC–MS, and GC–MS. Additionally, MEPS can be fully automated for both extraction and injection processes, serving as an on-line sampling device [52].

Two different approaches have been suggested by the research group of D. Satinsky for the on-line MEPS of β-blockers in biological samples [71,72]. In their first application, a commercial MEPS C-18 cartridge was coupled to sequential injection chromatography (SIC), which enables precise and repeatable liquid handling, to analyze betaxolol in human urine [71]. During the MEPS parameters’ optimization, it was found that 1 mL of acetonitrile/water mixture (15/85 v/v) was sufficient for the removal of matrix interference. To maintain the compatibility of the hyphenated on-line MEPS/SIC system, a monolithic C₁₈ analytical column (50 × 4.6 mm) was utilized for analyte separation. A mixture of 0.5% v/v triethylamine/acetonitrile, 70/30 v/v at pH 4.5 was used to elute the drug at 2.8 min. Some years later, the same research group proposed a column-switching chromatographic system of the on-line MEPS for the separation of metoprolol, labetalol and propranolol in human urine [72]. The MEPS cartridge was integrated into the flow system by positioning it into a PEEK tube and a joint Teflon connector. The inlet and outlet of the MEPS cartridge were then connected to a high-pressure six-port switching valve. A sample injection volume of 50 μL urine was selected as a compromise between method sensitivity and preventing MEPS column overloading. According to the authors, more than 300 injections of 10-fold diluted urine samples were repeated without any loss of extraction efficiency. A low LOD of metoprolol was achieved (1.5 ng/mL) using a fluorescence detector (λ_ex/λ_em = 220/305 nm).

A graphitic carbon (Carbon-X COS) was utilized as MEPS sorbent for the isolation of metoprolol and acebutolol from plasma samples [73]. Washing solutions containing methanol at >10% resulted in analyte desorption from MEPS sorbent. The method was validated according to FDA guidelines and found to be linear in the range of 10–2000 nM, with a r² higher than 0.999. The matrix effect was evaluated using the post extraction approach and found to be negligible as they ranged between −9.4% and 5% in all cases. The intra-day and inter-day method precision ranged from 4.4% to 14.4% (RSD) for both studied analytes. The same research group expanded their work by developing a LC–MS/MS method for the enantiomeric separation of metoprolol isomers [74]. Various sorbents were tested including C₄, C₈, C₁₈ and polysorbates. Four-fold diluted plasma or saliva samples were extracted using a C₁₈ MEPS sorbent using a water/methanol (95/5 v/v) mixture as washing solution to remove proteins and lipids. The elution of the analytes was carried out with 200 μL isopropanol to ensure compatibility with the hexane-containing mobile phase. A precursor and product ions at m/z of 268.08 and 72.09 were used for the quantitation of the analytes in the positive ionization mode. Successful resolution (R_s > 1.5) between the two isomers was achieved and a cellulose tris (3,5-dimethylphenylcarbamate) stationary phase was obtained. The analytical method permitted determination of metoprolol isomers in plasma and saliva samples at low ppb levels (1.5 ng/mL).

An alternative methodology has been published in 2020 by O.Mompo-Rosello et al. for the determination of five β-blockers in urine samples [75]. A glycidyl methacrylate-based monolith was modified with an imidazolium-based IL to serve as the stationary phase for SPE. The monolithic support was fabricated through in situ UV polymerization in a spin column format. After the optimization of method’s parameters, the drugs were efficiently retained on the modified monolith at pH 12 and were then desorbed using a water-methanol mixture. The separation of the analytes was performed using HPLC–UV. The LODs ranged from 1.4 to 40 μg/L, and the reproducibility between extraction units was lower than 8.2%. The novel sorbent was successfully applied for the extraction propranolol from urine samples, achieving recoveries greater than 90%. A MEPS procedure was employed for the determination of the enantiomers of propranolol using normal-phase LC–MS/MS [76]. A 4-fold diluted plasma has been subjected to a packed C₁₈ material in a 250 μL MEPS syringe preconditioned with methanol and water. The sample was washed with 100 μL of a mixture of H₂O/CH₃OH, 95/5% v/v to remove the potential interferences. According to the authors, the MEPS can be reused after appropriate cleaning. The authors took advantage of using a makeup solvent-assisted ionization approach to improve the ionization efficiency of the analytes.

3.4. Dispersive Solid-Phase (Micro)extraction

Dispersive solid-phase (micro)extraction (dSPE) is a simplified, miniaturized format of the SPE technique, where the target analytes are extracted by adding a small amount of dispersive nanosorbent to the bulk solution, followed by their elution into an appropriate solvent. The dSPE technique offers several advantages, including rapidity, low-cost sample cleanup and ease of use. The efficiency of the dispersion can be significantly improved with the application of auxiliary energies, such as ultrasound irradiation and air agitation [56].

An interesting application of dSPE in bioanalysis of β-blockers involves the quantitation of metoprolol, propranolol, and carvedilol in human plasma and urine [77]. A polypyrrole-sodium dodecylbenzenesulfonate/zinc oxide nanocomposite was fabricated by in situ sonochemically oxidative polymerization. The morphology and the characterization of the material were investigated by a series of techniques including Field-Emission Scanning Electron Microscopy (FESEM), X-Ray Diffraction (XRD), Energy-Dispersive X-Ray Mapping (EDX), and Thermogravimetric analysis (TGA). A central composite design was utilized for the optimization of extraction parameters including sample pH, sorbent amount, sonication time and elution volume. Compared with other SPE/SPME approaches, the developed method provides wider linear range, higher preconcentration factors and lower LODs. It was applied for the quantitation of certain drugs in biological fluids at low therapeutic levels. The simultaneous determination of metoprolol and propranolol has been reported using carboxyl-functionalized single-walled carbon nanotubes with magnetic nanoparticles in dSPE format [78]. After examining the parameters affecting the separation and the detection of the analytes, successful quantitation was achieved using isocratic elution (100 mM phosphate buffer (pH 3.14)/acetonitrile, 60/40 v/v) on a C₁₈ analytical column. The authors revealed that the extraction levelled off at 15 min while the sample pH of 5 was selected as optimum. The preconcentration factors were 278.7 and 283.1 for both compounds. The determination of the analytes has been carried out without significant matrix effect while the recoveries were in the range of 91–97.2% with an RSD < 5.5%.

A different approach was followed by J. Sara et al. for the quantitation of alprenolol, atenolol, metoprolol and propranolol in human plasma using a magnetic core–shell ionic liquid-modified nanoparticles for dSPE [79]. The silica particles have been functionalized with 1-butyl-3-methylimidazolium hexafluorophosphate. It was proved that the neutral forms of the analytes promoted their adsorption to the sorbent through hydrophobic and π–π interactions while the protonated forms interact via electrostatic interactions. The LODs varied from 0.5 to 2 ng/mL with preconcentration factors up to 18. It was proved that the dSPE showed higher extraction efficiency than magnetic effervescent powder under the same experimental conditions due to the low dispersibility of the IL magnetic nanoparticles in the latter approach. Another magnetic dSPE methodology has been proposed by M. A. Akhtiar Abadi et al. for the simultaneous determination of metoprolol, atenolol and propranolol inhuman plasma [80]. In order to provide hydrophobic properties to the sorbent, the authors modified the magnetic graphene oxide with cationic (cetyltrimethylammonium bromide), anionic (sodium dodecyl sulfate) and non-ionic (Triton X-100) surfactant. It was revealed that the formation of sodium dodecyl sulfate double layers on the magnetic Fe₃O₄ graphene oxide surface improved the drug’s extraction efficiency by facilitating appropriate interactions between the magnetic sorbent and the analytes. A proposed mechanism for the in situ sorbent modification is shown in Figure 5. Plackett–Burman and central composite experimental designs were sequentially utilized for screening and optimization of the method parameters. The preconcentration factors were between 14 and 18 while the sensitivity of the method was adequate (LODs: 0.8–2 ng/mL) for the detection of the analytes in real plasma samples. Before drug extraction, plasma samples were treated with acetonitrile and centrifuge at 4000 rpm for 10 min to precipitate the proteins.

A MIP-based dSPE protocol was developed for the selective isolation of propranolol from undiluted bovine serum albumin [81]. The preparation of MIP was carried out using the facile reversible addition-fragmentation chain transfer coupling chemistry. The authors proved that the poly (2-hydroxyethyl methacrylate crafted MIPs offered high selectivity on the analyte binding in both organic solvents and aqueous solutions. Excellent linearity was observed in the range of 0.01–100 μM with LOD/LOQ of 2 and 6.7 nM, respectively. The fabricated material can be reusable for up to 6 adsorption/desorption cycles. The applicability of the method was demonstrated by analyzing undiluted bovine serum samples spiked at 10, 20 and 50 μM while the absolute recoveries ranged between 85.2% and 97.4%.

An interesting approach has been proposed by T. Ballesteros-Estaban et al. for microextraction of two β-blockers (carvediol, propranolol) from urine samples [82]. For this purpose, the authors synthesized a magnetic-nylon terephthaloyl-based composite as sorbent and characterized by scanning electron microscopy. The extraction of propranolol and carvedilol is favored in alkaline media reaching the highest recovery at pH 12. The material integrates the excellent extraction capabilities of polyamides with the magnetic properties of Fe₃O₄ nanoparticles, streamlining the extraction process and enabling the simultaneous extraction of multiple samples.

3.5. Other Sorbent-Based Microextraction Techniques

A tapered-capillary micro-SPE combined with FASI methodology has been reported for the isolation and preconcentration of atenolol and metoprolol from human urine samples [83]. The construction of the SPE microcolumn was performed by narrowing the end of a silica capillary from 530 to 20 µm i.d., enabling the packing of 45 µm sorbent particles without a frit. The developed microextraction offered the benefits of minimum sample and sorbent volumes (<200 μL sample and 2 μL for sorbent) along with a rapid extraction time of ca 6 min. The method was successfully applied to determine atenolol and metoprolol in human urine samples, with recoveries of 93.7–105.5% and RSD < 8.5%. The proposed approach resulted in nearly a 20-fold increase in sensitivity compared to CE method. The fritless SPE microcolumn can be reused for eight consequent extractions.

Mazaraki et al. took advantage of using a fabric-phase sorptive extraction (FPSE) for the determination of eight β-blockers in human urine and serum after UHPLC––MS/MS analysis [84]. The advantages of FPSE technology have been firstly exploited by Kabir and Furton by developing a novel patented technology [85]. Eight different sol–gel “chemistries” have been evaluated and the sol–gel Carbowax 20M was selected as it provided better extraction efficiency of the drugs. A schematic representation of the selected membrane is illustrated in Figure 6. The sample was extracted in 15 min under stirring of 750 rpm. The mixture of methanol/water, 50/50 v/v was sufficient for the elution of the analytes from the FPSE membrane. High-sensitivity determinations were performed with LOD/LOQ in the range of 0.3–2 ng/mL and 50 ng/mL, respectively. The authors proved that the fabricated material is usable up to 20 times without significant loss of its performance.

A different approach has been followed by I. Ali et al. for the analysis of atenolol and atorvastatin in human plasma [86]. An iron nanocomposite adsorbent was utilized in solid-phase micromembrane tip extraction. The nanosorbent was characterized by a series of techniques including XRD, Fourier-Transform Infrared Spectroscopy, Scanning Electron Microscopy and EXD. The separation of the analytes has been carried out on a pentafluorophenyl stationary phase using a mixture of 0.1% aqueous formic acid and acetonitrile as the mobile phase. High-resolution MS was employed for the identification and quantitation of the analytes in the positive ionization mode. The maximum absorption time was 30 and 90 min for atenolol and atorvastatin, respectively. Although the authors did not validate their method, it was finally applied to the analysis of drugs in human samples.

An interesting SBSE protocol was reported using a steel pin coated with a polyaniline and multiwall carbon nanotube composite for the extraction of propranolol [87]. The nanocomposite consisting of polyaniline and multiwall carbon nanotubes was immobilized electrochemically on a steel pin. The effectiveness of this procedure was monitored using cyclic voltammetry. The surface properties of the sorbent were investigated using SEM. The extraction recovery was enhanced with the increased ionic strength of the sample. Using atenolol as an internal standard, the precision of the method was less than 10.4%.

4. Conclusions

Beta-blockers are a crucial class of medications prescribed for various health conditions. However, they can be highly toxic at elevated doses and are sometimes misused in sports as doping agents. Therefore, this review systematically compiles and carefully examines the dispersed information of the last decade on microextraction-based analytical methods for determining β-blockers in biological samples. Significant progress in sample preparation techniques (both liquid phase and sorbent based) and advanced analytical instrumentation has revolutionized the detection and quantification of the drugs in biofluids. SPME technology utilizing modern sorbents and LPME employing environmentally friendly solvents have revolutionized methodologies for the detection of these compounds. Looking at the analytical technique, HPLC–UV/DAD is widely used due to its reliability and cost-effectiveness, making it accessible for numerous laboratories.