Overview of novel sample pretreatment methods and their pharmaceutical applications in bioanalysis

1. INTRODUCTION

Sample pretreatment is an integral part of bioanalysis for the extraction of analytes from different matrices and their quantitation using analytical tools [1]. Biological matrices are often complicated due to the presence of endogenous (e.g., Proteins, lipids, or the target analyte’s metabolites) or exogenous chemicals (additives used in analysis) [2]. Besides, matrix components that co-elute with target analytes during chromatographic separation affected the mass spectrometry (MS) detection of the analyte’s response, either positively (ion enhancement impact) or negatively (ion suppression effect) [3].

Among the sample pretreatment methods available, protein precipitation is a straightforward and affordable method. The proteins in the sample are denatured by a high concentration of organic solvent (preferably acetonitrile) and eventually precipitate out of the sample by centrifugation [4]. Liquid–liquid extraction (LLE) is another commonly used pretreatment method, which involves the selection of two immiscible solvents for the extraction of the desired analyte. According to their partition coefficients, immiscible organic solvents separate the target analyte into the organic layer and can be extracted [5,6]. The advantage of this technique is that highly clean extracts with strong selectivity for the desired analyte can be produced with the careful selection of solvents, pH of the aqueous phase, polarity, and analyte solubility in the organic solvent. However, LLE suffers from certain limitations such as the use of large amounts of organic solvent, lack of automation, and time consumption [7]. Another important pretreatment method is solid phase extraction (SPE), which involves chemically separating the components of a sample by using chromatographic packing material, usually in cartridge-type devices. The selection of a suitable SPE extraction sorbent depends upon the interaction between the sorbent and the analyte of interest. SPE generally retains materials by van der Waals forces (nonpolar interactions) [8]. The extraction steps of SPE include conditioning, equilibration, sample load, washing, and elution [9]. SPE is widely employed in the LC-MS/MS applications to quantify molecules in biofluids [10].

Overall, the traditional pretreatment methods are time-consuming, labor-intensive, and poorly suited for large as well as very small samples. Many conventional multistep procedures do not have adequate sensitivity, selectivity, and reliability. In addition, assays for metabolites or biomarker detection in clinical samples at very low concentrations face a similar set of challenges. In addition, improvements in sample preparation methods are increasingly focused on improving the clean-up procedure and exploring the possibility of automating, miniaturising, and improving their specificity [1]. Therefore, sample pretreatment has made significant progress in miniaturized and solvent-free extraction techniques, and faster separation methods designated as novel pretreatment methods [11]. A glimpse of the different available sample pretreatment methods is shown in Figure 1.

Figure 1. Overview of the traditional and novel sample pretreatment methods. [Click here to view]

Recent comprehensive reviews on bioanalytical sample preparation techniques emphasize that matrix effects remain one of the most significant challenges in achieving accurate and reproducible analytical results [12]. Biological matrices such as blood, plasma, urine, and tissues contain various endogenous substances—proteins, lipids, and salts—that can interfere with the detection and quantification of target analytes. These interferences can suppress or enhance analytical signals, leading to compromised data quality. As a result, modern sample preparation methods increasingly focus on minimizing matrix effects through improved extraction efficiency, selectivity, and compatibility with advanced analytical platforms like LC-MS/MS.

This review focuses on the recent developments in novel pretreatment methods, covering their working principle, applications (mainly pharmaceutical uses), and recent updates. Much effort has been made to cover the maximum case studies that involve novel pretreatment methods for analyte extraction and hyphenated techniques for their detection. In addition, different matrices (blood, urine, saliva, and so on) treated with novel methods have been covered to understand their overall applicability. Besides, a comparative analysis of different novel methods has been performed to assess their overall performance based on critical parameters. Furthermore, a separate section dealing with recent trends in the bioanalytical pretreatment methods provides the innovative strategies being developed and explored in bioanalysis. The recent use of pretreatment methods for the separation of biomarkers and their diagnostic uses has also been covered.

2. NOVEL SAMPLE PRETREATMENT METHODS

Novel sample pretreatment methods are becoming more popular to overcome the limitations of traditional methods. These methods are the modified versions of either SPE or LLE with the advantages of miniaturization, automation, and less time-consuming with good efficiency. ICH Q2(R²) guideline emphasizes the integration of method validation within the broader analytical procedure lifecycle, aligning with ICH Q14. Pretreatment methods largely affect the limit of detection (LOD) and limit of quantitation (LOQ) of the analytical method and overall affect the sensitivity of the technology employed. A literature survey in the scientific database (PubMed) has been done for the last 10 years and showed a surge in the publications using these novel methods (Fig. 2). Among them, solid phase microextraction (SPME) showed the highest usage. Another survey in terms of the application of these novel methods in diverse applications. The trend showed that environment and food analysis are among the top, followed by pharmaceutical and clinical applications (Fig. 3). These surveys demonstrate the growing interest among the scientific community in novel methods to overcome the critical issues encountered in the traditional methods.

Figure 2. Trends of novel sample pretreatment methods in the scientific database (PubMed) in the last 10 years [till December 2024]. [Click here to view]

Figure 3. Applications of novel sample pretreatment methods in diverse areas based on the scientific database (PubMed). [Click here to view]

This section deals with different novel methods along with their working principle and is complemented by pharmaceutical applications. Prominent case studies under each novel method have been briefly described. Finally, all the case studies collectively are summarized in tabular form (Table 1) based on the target analyte, novel methods, matrix type, recovery, and hyphenated methods used, along with their detection limits.

Table 1. Extraction of different analytes using novel pretreatment methods from biological fluids.

SLE, Supported Liquid Extraction; PRP, Phospholipid Removal Plate; MEPs, Microextraction by Packed Sorbents; MIPs, Molecular Imprinted Polymers; SPME, Solid Phase Microextraction; SERS, Surface Enhance Raman Spectroscopy; LC-MS/MS, Liquid-Chromatography Tandem Mass Spectrometry; GC-MS/MS, Gas Chromatography Tandem Mass Spectrometry; UPLC-MS/MS, Ultra-High Performance Liquid Chromatography-Tandem Mass Spectroscopy; ICP-OES, Inductively Coupled Plasma-Optical Emission Spectroscopy; HPLC-DAD, High Performance Liquid Chromatography-Diode Array Detector; MOI-MS/MS, Microfluidic Open Interface- Mass Spectroscopy; ESI-IMS, Electron Spray Ionization- Ion Mobility Spectrometry.

2.2. Phospholipid removal plate

A phospholipid removal plate is a specialized SPE device designed to selectively eliminate phospholipids from biological samples such as plasma and serum before analytical procedures like LC-MS. The plate contains a sorbent bed made of zirconia-coated silica particles, positioned between two filtration layers: an upper 5 µm frit and a lower 0.2 µm hydrophobic filter, which act as depth filters to maintain optimal flow rates and prevent clogging (Fig. 4).

Figure 4. Mechanism of phospholipid removal plate-based extraction showing zirconia-coated silica sorbent with integrated filters enabling efficient flow that binds through Lewis acid-base interactions. (Adapted from Ahmad et al. [27]. [Click here to view]

The zirconium atoms on the silica surface act as Lewis acids due to their empty d orbitals, forming strong electron donor-acceptor complexes with the phosphate groups in phospholipids, which serve as Lewis bases. As the biofluid sample passes through the plate, phospholipids are selectively retained by the zirconia surface, while other components, such as proteins and metabolites, flow through unretained. This selective extraction significantly reduces matrix effects, enhances analyte recovery, and improves sensitivity and reproducibility in downstream analytical workflows [25]. Phospholipids are a major cause of ion suppression in blood and plasma analysis. To address this, Hybrid SPE precipitation plates have been developed wherein the plasma or serum samples are first treated with acetonitrile for protein precipitation, then passed through the Hybrid SPE precipitation plates packed bed for phospholipid removal. Structurally, phospholipids consist of a zwitterionic phosphonate moiety as the polar head group and two hydrophobic fatty acyl chains as tails. The phosphate group in all phospholipids interacts strongly with the zirconium atoms functionalized on the surface of the particles, enabling selective retention [26,27].

Phospholipid removal plates have been found to have potential applications in the plasma extraction of several drugs that are used in various indications, such as asthma, allergic rhinitis, and autoimmune hepatitis. Nilsson et al. [28] have implemented phospholipid removal plates in the bioanalysis of budesonide and quantified using UPLC/MS/MS and achieved a good LOD of 24 pg/ml. The developed method was applied to compute various pharmacokinetic parameters. Another interesting study reported the selective extraction of (S)-enantiomers of metoprolol (β blocker) in blood samples using LC-MS/MS [29]. Samples were extracted from the biological matrix using phospholipid removal micro elution-SPE plates, and eluates were reconstituted in acetonitrile with methyl benzyl isocyanate for chiral derivatization. The method offers maximum recovery (69.4%–78.7%) and a good LOQ of 0.5 ng/ml along with minimal matrix effect. In addition, this pretreatment method has been widely used in various areas, viz. cosmetic products, pesticides, as well as forensic sciences [30–35].

Technically, these plates streamline workflows by combining protein precipitation and phospholipid filtration in a single step, making them compatible with 96-well formats and automated systems. However, their selectivity can vary depending on analyte chemistry, and some compounds may be unintentionally retained or co-eluted. Despite its outperforming traditional methods in phospholipid removal; however, all matrix effects in complex samples may not be fully eliminated. Besides, relatively higher cost and assay compatibility can limit its routine adoption in analytical laboratories.

2.3. Magnetic solid phase extraction (MSPE)

The complexities associated with traditional SPE methods, such as sorbent selection, isolation, and enrichment of the analyte, can be overcome by using magnetic nanoparticles (MNPs). The core of the nanoparticles comprises iron, nickel, cobalt, and their oxides, while the inorganic materials (such as silica, alumina, manganese oxide (IV), or graphene) are used for the surface coating. MNPs are distinguished by a large surface area, high sorption capacity, and good selectivity for analytes because of their small particle size, together with a shorter duration in the extraction process [36–39]. MSPE operates through a sequence of well-defined steps that leverage the unique properties of MNPs. The process begins with the dispersion of MNPs into a sample containing target analytes. These nanoparticles are engineered with surface functional groups that facilitate selective binding. During the adsorption phase, analytes interact with the nanoparticle surfaces primarily through hydrophobic and van der Waals interactions, as well as hydrogen bonding, electrostatic forces, or specific chemical affinities (Fig. 5). Once adsorption is complete, an external magnetic field is applied to perform magnetic separation, efficiently isolating the analyte-bound nanoparticles from the rest of the sample without the need for filtration or centrifugation. In the elution step, a suitable solvent is introduced to desorb the analytes from the nanoparticles, transferring them into a clean solution for downstream analysis such as LC-MS or inductively coupled plasma MS (ICP-MS). Finally, the sorbent regeneration step allows the magnetic nanoparticles to be washed and reused, making MSPE a cost-effective, rapid, and sustainable method for sample preparation [40].

Figure 5. Illustration of the magnetic solid phase extraction process involving functionalized magnetic nanoparticles selectively capturing target analyte through adsorption, followed by magnetic separation, and elution. (Adapted from Plastiras et al. [39] . [Click here to view]

Recent advancements in MSPE have focused on the integration of metal-organic frameworks (MOFs) with MNPs to enhance selectivity and efficiency in pharmaceutical analysis. A notable development involves the use of NH₂-MIL-101(Cr)-based magnetic nanocomposites, which combine the high surface area and porosity of MOFs with the magnetic responsiveness of Fe₃O₄ nanoparticles. These hybrid materials exhibit excellent adsorption capacity and photocatalytic activity, making them effective for the removal and analysis of pharmaceutical contaminants such as acebutolol from aqueous environments. The amino-functionalized MIL-101(Cr) enhances light absorption and charge transfer, while the magnetic core facilitates rapid separation and reusability, supporting sustainable and high-throughput analytical workflows [41].

Asgharinezhad and Ebrahimzadeh [42] extracted nonsteroidal anti-inflammatory drugs, viz. naproxen, diclofenac, and ibuprofen from urine samples by the MSPE method. Good recovery of analytes was observed (85%–90%) with a LOD in the range of 0.007–2000 µg/ml, which indicates the high efficiency of the extraction method. A preclinical study conducted by Wang et al. [43] in which the bioactive compound puerarin was efficiently extracted with the MSPE method based on C18 magnetic silica nanoparticles in rat plasma, and its bioanalysis was performed by HPLC. Another interesting study on hair samples pretreatment by MSPE for simultaneous determination of elements, viz. Cr(III), Cu(II), Pb(II), and Zn(II) and quantified by inductively coupled plasma-optical emission spectrometry [44]. The method was found highly efficient with good recovery (85%–104.5%) and a detection limit of 0.01–0.06 µg/ml, and can be suitable for trace element determination. Besides, the MSPE method has been extensively employed for the extraction of secondary metabolites of aflatoxins in food products, drug metabolites and phthalate detection in industrials and cosmetic products [45–50].

Overall, MSPE is a modern sample pretreatment method that employs functionalized magnetic nanoparticles to selectively capture and concentrate target analytes from complex matrices such as biological fluids, environmental samples, and food extracts. It offers notable analytical benefits, including high selectivity, rapid processing, and reduced solvent consumption, making it particularly effective for trace-level detection. From a technical standpoint, MSPE involves dispersing MNPs into the sample, allowing analytes to bind to their surfaces, and then using an external magnet to isolate the analyte-bound particles, streamlining the workflow and enabling easy automation. However, its performance can be hindered by nanoparticle aggregation, reduced efficiency in viscous or particulate-rich samples. In addition, there is a need for precise surface functionalization to maintain specificity and stability across diverse analyte types [51].

2.4. Microextraction by packed sorbent (MEPS)

The miniaturisation of SPE modified into MEPS, in which the sorbent is integrated into the syringe; therefore, a separate robotic accessory is not required. MEPS may link online to different chromatographic systems (capillary, gas chromatography, and liquid chromatography) and can be fully automated [52]. MEPS comprises of syringe and barrel insert and needle (BIN) to perform sample extraction, and cleanup (Fig. 6). Thermo-packed sorbent is filled in a little tube of BIN, and target analytes are eluted in sequential phases after the syringe feeds the material through this BIN, enabling sample purification or speciation [53]. In addition, various commercial sorbents, including new varieties of graphitic sorbent, polyamide, polyaniline nanowires, functionalized silica monoliths, and so on, have been successfully used in MEPS devices to extract various groups of analytes [54,55].

Figure 6. Schematic representation of the workflow of the microextraction by packed sorbent involving selective analyte retention, washing, elution, and cleaning steps. (Adapted from Kabir et al. [50]. [Click here to view]

MEPS is extensively used for the extraction of several drugs in biological fluids (blood, saliva, and urine) and their quantification with different analytical tools. Ferrone et al. [56] employed MEPS in the clinical study for the bioanalysis of ciprofloxacin and linezolid among pneumonia patients using the UHPLC-PDA technique. Good recovery (92.4%–97.4%) of target analytes was achieved with a detection limit of 0.007 and 0.004 ng/ml, respectively. Compared with the conventional SPE method, MEPS was found to be simpler, low-cost, requires a small sample volume (10 µl), as well as reduced sampling time. In another reported study [57], simultaneous extraction of the enantiomeric form (-R) and (-S) of metoprolol in blood plasma and saliva samples was accomplished with the help of the MEPS method. The recovery of the study was in the range of 93.1%–97.8% and bioanalysis was performed using LC-MS/MS with high sensitivity. Besides, MEPS has been used for the detection of drugs of abuse such as opiates and cannabis as well as CNS-acting drugs in blood samples using hyphenated tools [58–63]. This pretreatment method offers rapid and effective clean up sample preparations, satisfactory recovery, and minimum matrix effect.

In a nutshell, MEPS is a miniaturized, syringe-based solid-phase extraction technique that integrates sample extraction, cleanup, and preconcentration into a single, reusable device. It offers significant analytical advantages such as reduced sample and solvent consumption, high-throughput capability, and compatibility with automation and direct coupling to LC-MS or GC-MS systems. Technically, MEPS uses a small amount of sorbent (1–4 mg) packed within a syringe or barrel insert, allowing for efficient analyte interaction through repeated aspiration cycles, followed by washing and elution steps all within microliter volumes. Recent advancements in sorbent materials for MEPS are the integration of molecularly imprinted polymers (MIPs), which act as synthetic receptors with high affinity for specific target molecules. These MIPs are tailored through imprinting techniques that create selective binding sites complementary in shape and functionality to the analyte, enabling precise extraction even in trace concentrations. Despite several stellar benefits of MEPS, its performance is highly dependent on sorbent selection, and manual operation in flow rate control can introduce variability due to user-dependent handling. In addition, the issue of sorbent degradation over multiple cycles can affect reproducibility and sensitivity [64].

2.8. Affinity sorbent extraction

The most current developments in the field of sample preparation include frameworks modified with various biomolecules (such as proteins, nucleobases, amino acids, aptamers, and so on) as ligands to create affinity-based sorbents. These affinity materials’ synthesis and inclusion methods, as well as their use as sorbents for the selective extraction of molecules and the cleanup of intricate samples. These sorbents have been used in sample treatment directly, or they have been specifically created to satisfy the needs of analytical applications [83]. Immunoextraction, molecular imprinted polymer, and aptamers are the methods that work on the principle of affinity sorbent extraction. The literature survey revealed that the application of the affinity sorbent extraction methods is primarily in the diagnosis of diseases by detecting the biomarkers in the blood samples. Representative case studies have been summarized in Table 2.

Table 2. Application of novel pretreatment methods for the extraction of biomarkers.

Biomarker	Extraction technique	Matrix	Volume (µl)	Sorbent	Solvent	Recovery	Quantitation range (Technique used)	Significance	References
Neopterin, tryptophan, creatinine, uric acid, kynurenine (Inflammatory biomarkers)	Immunosorbent	Saliva	200 µl	Microcon centrifugal filter device (Merck)	Sodium hydroxide	90%–114%	0.00036– 0.44 µmol/l (HPLC-FID/DAD)	− Simultaneous extraction of inflammatory biomarkers − Simple and time saving process − This method considered as gold standard for extraction	[86]
Human chorionic gonadotropin (Biomarker)	Immunosorbent	Serum	5 µl	Biotinylated antibodies Covalently linked to HEMA-VDM-functionalized paper.	PBS with Tween 20, ammonium bicarbonate buffer	33%	65 pg/ml (LC-MS)	− Convenient storage and transport − Analyte stability − Low sample amount − Minimal invasive sample collection	[88]
Cytokeratin fragment Cyfra 21.1 (cancer biomarker)	Immunosorbent	Saliva	5 µl	Photographic paper / Ag ink / antibody	[Fe (CN)6]3-/4-/ KCl solution	_	0.001–2.5 ng/ml (High performance biosensors)	− Practical, sensitive, fast, selective method − Portable and cost-effective	[89]
Hypoxia-inducible factor-1 (HIF-1α) (Myocardial infarction biomarker)	Aptamer	Blood serum	50 µl	AuNP-Coated Gold nanospheres functionalized with HIF-1α-binding aptamer	PBS buffer, gold seed growth solution, Na2HPO4, citric acid, H2O2, TMB	≥ 80%	0.2 ng/ml (colorimetry)	− Good stability − Not susceptible to environmental factors like pH temperature − Easy preparation − Low cost	[109]
Cortisol (stress biomarker)	Aptamer	Saliva	900 µl	Au-NP-duplex DNA	Ethanol	89%–114%	0.37 ng/ml (Lateral flow Assay Biosensor)	− Higher stability − Low cost − Potential improvement with the future SELEX procedure	[110]
Amyloid-β oligomers (Alzheimer’s disease biomarker)	Aptamer	Blood serum	100 µl	AβO-specific aptamer immobilized on the surface of core-shell nanoparticles (silver and silica)	Phosphate buffer saline, pH 7.4	93%–107%	1.22 pg/ml (Transmission electron microscopy)	− Portable − Simplicity of sample preparation − Design versatility, facial modification − Low mol. wt, chemical stability − High binding affinity − High selectivity	[111]
Exosome (Colorectal cancer biomarker)	Aptamer	Blood serum	5 µl	Graphene oxide-DNA aptamer (cy3 conjugated CD63-aptamer)	Tris HCl buffer, DNase-1 reaction buffer, EDTA	45%– 65%	2.4 × 104 particles/ µl (Fluorescence spectroscopy)	− Steps are simple and can be mixed with fluorescent dye-conjugated aptamers − No need of isolation step. − Time saving − Joint detection of two exosome biomarkers is possible − Improve the storage stability	[112]
Immunoglobin E (biomarker)	Aptamer	Blood serum	5 µl	Fabrication of zwitterionic peptide and IgE aptamer onto a microporous Au substrate.	Phosphate buffer saline, water	97%101%	42 pg/ml (Differential pulse voltammetry)	− Microporous Au greatly increases the surface area for sensitivity − Satisfying antifouling ability	[113]
Insulin (biomarker)	Aptamer	Blood plasma	5 µl	Poly-orthophenylene diamine ornate gold nanoparticles (GNPs) with single stranded DNA aptamer immobilized of pencil graphite electrode.	Trichloroacetic acid	88% –103%	1.0–1,000.0 nmol/l (Electrochemical impedance spectroscopy)	− Specific and sensitive tools for the extraction and detection of plasma insulin in presence of proteins − Mechanical and chemical stability − Modification at the terminal end of the aptamer sequence is possible − Reversibility under thermal denaturation	[114]
Thrombin (biomarker)	Aptamer	Blood serum	50 µl	Au-mNP functionalized core shell magnetic microspheres.	Tyrosine for direct digestion	≥ 99%	0.36 ng/ml (MALDI-TOF-MS)	− Simpler than other methods. − Combining easy immobilization − Fast separation − High throughput and high selectivity and sensitivity − Stability in harsh environment	[116]

SLE, Supported Liquid Extraction; PRP, Phospholipid Removal Plate; MEPs, Microextraction by Packed Sorbents; MIPs, Molecular Imprinted Polymers; SPME, Solid Phase Microextraction; MSPE, Magnetic Solid Phase Extraction.

2.8.1. Immunosorbent

In this technique, antibodies have been immobilised on a solid phase, also known as immunoaffinity extraction sorbents. It offers good selectivity and high affinity due to the antigen–antibody interaction. The robust procedures allow one-step selective extraction and enrichment of specific compounds. Solid support is decorated with antibodies and packed in an SPE cartridge to create the immunosorbents. The formation of the antibody–antigen complex during this step is predominantly influenced by electrostatic forces. Desorption will only take place by significantly altering the experimental conditions due to the high energies of the biological interactions. However, matrix materials and sample additives can have an impact on analyte-antibody interactions, which can also reduce extraction recoveries [84]. Immunocapture methods are gaining renewed attention in pharmaceutical and biomedical analysis due to their high specificity and adaptability for complex biological matrices. These techniques utilise antibodies or antibody fragments to selectively bind and isolate target molecules such as therapeutic proteins, biomarkers, or drug metabolites. When coupled with advanced analytical platforms like LC-MS, immunocapture enables sensitive and selective quantification, particularly useful in pharmacokinetic and pharmacodynamic studies. Recent innovations have focused on hybrid immunocapture LC-MS workflows that combine ligand-binding specificity with mass spectrometric sensitivity, streamlining assay development and improving throughput. These methods are especially valuable in early-stage drug development, where rapid and reliable bioanalytical tools are essential [85].

Immunosorbent-based extraction methods largely find their application in the screening of biomarkers for disease diagnosis and their progression [86,87]. For instance, human chorionic gonadotropin, a biomarker, has been successfully extracted and analysed by Johannsen et al. [88] using the combination of paper-bound streptavidin as an anchor for biotinylated antibodies and quantified by LC-MS. Similarly, cytokeratin fragments are a high-potential biomarker in many epithelial cell cancers. A high-performance immunosorbent-based biosensor technique was employed for the extraction of this biomarker [89]. It involves the affinity of an antigen towards antibodies mechanism using Photographic paper/Ag ink/antibody as a capturing plate and [Fe (CN)₆]^3-/4-/KCl solution as a solvent. In addition, a rapid colourimetry sensing system using competitive enzyme-linked immunosorbent assay (ELISA) on a microfluidic paper-based analytical device has been developed for the detection and determination of ketamine in oral fluid (3 µl) using a coated ketamine antibody [90].

2.8.2. Molecular imprinted polymers

MIP is a selective sorbent for SPE, which has gained popularity recently for selective extraction of analytes [91]. Functional polymers were grafted (Fig. 7) on the surface of silica particles and then cross-linked with the templates sustained by dipole interactions or hydrogen bonding [92]. MIPs are of tremendous interest because they can be produced more cheaply, have greater chemical and physical stability, and can be reused. However, molecular imprinting takes a long time and can have issues like heterogeneous imprinting, whereas macromolecule imprinting is challenging [93–95].

Figure 7. Mechanistic understanding of molecular imprinted polymer, where functional monomers are polymerized around a template molecule and subsequently removed to create selective binding sites complementary to the target analyte. (Adapted from Sajini and Mathew [95]. [Click here to view]

Several studies have reported for the use of MIPs-based extraction procedures and be highly robust, chemically stable, have less extraction time, and have high selectivity [96]. Magnetic multiwalled carbon nanotubes have been developed by Madrakian et al. [97] via synthetic amidoamine as the monomer and naproxen as the template. This method has been used for the bioanalysis of naproxen in human urine and detected by spectrofluorometric methods. Yang et al. [98] have performed a test on a simple Pickering emulsion polymerization method used to prepare bisphenol A imprinted polymer microspheres and used for the extraction from the human urine sample. Similarly, several analytes have also been extracted by MIPs in different biological matrices with good [99–103].

MIPs are synthetic materials engineered to mimic natural molecular recognition systems, such as antibodies, by creating highly specific binding sites tailored to a target molecule. Their appeal lies in their robustness, chemical stability, and adaptability across various formats, making them valuable in applications such as sensors, separation, drug delivery, and environmental monitoring. Technically, MIPs are formed by polymerizing functional monomers around a template molecule, which is later removed to leave behind complementary cavities. This lock-and-key mechanism enables selective recognition even in complex matrices. However, a few challenges associated with MIPs are template removal, limited binding site accessibility, and reduced performance in aqueous environments due to disrupted noncovalent interactions. In addition, their commercial translation is hindered by difficulties in reproducibility, scalability, and integration into real-world devices. Finally, the innovative application of conducting polymer-based MIPs and stimuli-responsive designs is helping bridge the gap between laboratory research and practical deployment [104].

2.8.3. Aptamers

Aptamers are synthetic, single-stranded oligonucleotides that are short (up to 110 base pairs), fold into distinctive shapes with high specificity. Aptamers use conformational complementarity to identify and bind their target. Furthermore, adjustments in the buffering system (salt composition and ionic strength) cause the release of the bound target molecules. Aptamers also aid in reducing the utilization of research animals, unlike antibodies, which frequently need animals for both initial discovery and manufacturing. Finally, SELEX (Systematic Evolution of Ligands by Exponential Enrichment) can be used to create specific aptamers [105] for a given target analyte, as shown in (Fig. 8). Aptamer functionalized materials (AFMs) are created by immobilizing aptamers on a solid support using a linking agent (linker). Compared to free aptamers, AFMs have higher chemical, biological, and mechanical stability in addition to their intrinsic high selectivity and strong affinity [106]. The stellar benefits of aptamers, such as high thermal stability, tailored specificity, good affinity, and scalability of production, have resulted in large-scale use in rapid diagnostic applications [107]. Besides, they can be explored in various immunoassays such as ELISA, western blot, immunohistochemistry, and flow cytometry in biomedical research and clinical diagnostic applications [108].

Figure 8. Representation of aptamer-based recognition mechanism, binding to the target, and subsequently target release. (Adapted from Mayol et al. [117]. [Click here to view]

Hypoxia-inducible factor-1 (HIF-1α) is a transcription factor that has been proven to be widely involved in hypoxia metabolism and has developed into an essential regulator of myocardial injury. A highly sensitive colorimetry technique has been created to measure HIF-1α in a circulating rat serum exosome, which is an early indicator for myocardial infarction. Wang et al. [109] investigated the growth of gold nanospheres functionalized with HIF-1α binding aptamer using seed-mediated culture. Na₂HPO₄, citric acid, H₂O₂, and TMB (coloring agent) were used as solvents in the detection and extraction of HIF-1α biomarker in 50 µl of serum sample. This method was able to offer good recovery (≥ 80%) of biomarkers, and the lowest LOD was found to be 0.2 ng/l. Dalirirad et al. [110] reported the detection of cortisol (stress biomarker) using duplex deoxyribonucleic acid (DNA) aptamer conjugated to the surface of gold nanoparticles by Au-S bonds as the sensor probe in a lateral flow assay. Similarly, the aptamer method of extraction is widely used in the diagnosis of biomarkers for diseases like cancer, diabetes, and diseases related to the central nervous system and analytical application in biological samples reported in studies [111–117].

Aptamers are short, single-stranded DNA or RNA molecules selected through the SELEX process for their ability to bind specific targets with high affinity and specificity, offering a promising alternative to antibodies in diagnostics, therapeutics, and biosensing. Their key analytical advantages include low immunogenicity, ease of chemical synthesis, and the ability to function under a wide range of conditions, making them ideal for applications in complex biological environments. Technically, aptamers can be engineered and modified post-SELEX to enhance stability, binding strength, and resistance to nucleases, with strategies such as truncation, multivalent integration, and chemical modification improving their performance. However, aptamers face notable limitations: they are prone to rapid degradation in biological fluids, often exhibit poor in vivo stability, and their small size leads to fast renal clearance. In addition, the SELEX process can be labor-intensive and may yield aptamers with suboptimal binding characteristics, requiring further optimization to ensure reproducibility and clinical viability [118].

In summary, all the novel sample pretreatment methods are found to offer stellar benefits of automation, good recovery, and faster cleanup (Table 3). However, they differ in terms of their versatile application and cost-effectiveness. Among all, immunosorbents and aptamers were found to be relatively costly due to frequent changes in the sorbent and have very specific applications in biomarker studies. On the other hand, other novel methods possess wide applications in drug analysis and are found to be less costly. Despite several advantages, there are some associated limitations, for instance, clogging of the sorbent, carryover effect, issues about hydrophobic analytes, template bleeding, and so on. However, these limitations have been overcome by applying innovative interventions in the pretreatment procedure to some extent.

Table 3. Comparative analysis of different novel sample pretreatment methods.

Novel pretreatment method	Miniaturization	Automation	Cleanup speed	Recovery	Matrix effect	Cost	Application	Reusable	Limitation
SLE	Yes	Yes	Good	Not satisfactory	Yes	Medium cost	Versatile	One time use	Single use consumable requirement
PRP	Yes	Yes	Good	Satisfactory	No	Low cost	Versatile	Reusable	Limitations for Hydrophobic analyte
MSPE	Yes	Yes	Fast	Satisfactory	No	Low cost	Versatile	Reusable	-Set magnetic beads apart from other methods of sample isolation, which might have different protocols for different types of samples.
MEPS	Yes	Yes	Fast	Satisfactory	No	High cost	Versatile	Reusable	Clogging of Sorbent, large volume sample performing problem, & carry over effect
SPME	Yes	Yes	Fast	Satisfactory	No	Low cost	Versatile	Reusable	Low effectiveness of process due to small amount of PDMS coated on fiber
IMMUNOSORBENT	Yes	Yes	Time consuming	Satisfactory	No	High cost	Specific	One time use	Animal requirement, specific antigen require, time consuming, & risk of cross reactivity
MIPS	Yes	No	Fast	Satisfactory	No	Medium cost	Specific	Reusable	Possible template bleeding, sometimes tedious synthesis procedures, & problematic application to aqueous samples.
APTAMERS	Yes	No	Fast	Satisfactory	No	Medium cost	Specific	One time use	Susceptible to nuclease degradation, limited building block diversity, PCR bias in SELEX method

3. RECENT TRENDS IN BIOANALYTICAL PRETREATMENT METHODS

Bioanalytical laboratories are increasingly prioritizing green sample preparation methods to align with sustainability goals and reduce environmental impact. In addition, bioanalytical pretreatment methods for biofluids have undergone a significant transformation, driven by the need for higher sensitivity, sustainability, and automation. For instance, solvent-free or solvent-reducing techniques such as microextraction and direct chromatographic methods that eliminate or minimize the need for hazardous solvents. The integration of biocompatible materials, miniaturized devices, and multitarget extraction systems—capable of isolating diverse analytes in a single step—further supports eco-friendly practices [119].

In drug analysis, green sample preparation is gaining traction through innovations that combine sustainability with high analytical performance, reflecting a broader commitment to environmentally responsible science. Techniques such as solidified droplet microextraction, DESs, and natural DESs are replacing traditional solvent-intensive methods. These approaches reduce environmental impact, minimize sample volume requirements, and improve analyte stability, aligning with sustainability goals and decentralized healthcare models. Similarly, the development of advanced microextraction strategies like dispersive liquid–liquid microextraction, hollow fiber liquid-phase microextraction, and electro-membrane extraction offers improved analyte recovery, reduced solvent usage, and compatibility with high-throughput workflows. Microsampling technologies such as volumetric absorptive microsampling, microneedle-based sampling, and dried blood spot methods are also gaining huge attraction among pharmaceutical scientists. These technologies enable decentralized and offer minimally invasive sample collection while maintaining analyte stability [50,120].

In recent years, a major innovation has been the integration of artificial intelligence (AI) and online automation into bioanalytical workflows [121]. AI is being used to optimize sample processing, predict analyte behavior, and enhance data interpretation, particularly in metabolomics and personalized medicine. Automation technologies such as robotic liquid handlers and online SPE systems are streamlining sample preparation, improving reproducibility, and enabling high-throughput analysis [122]. These systems not only reduce human error but also significantly increase sample throughput, as compared to manual methods. Predictive analytics and real-time monitoring are also being incorporated to support adaptive trial designs and faster decision-making in clinical research.

Overall, the adoption of green sampling technologies together with the convergence of AI and automation not only reduces waste and energy consumption but also enhances analytical efficiency. These developments have a significant impact in making bioanalytical science more accessible, eco-conscious, and adaptable to modern healthcare needs.

4. CONCLUSION

The present review comprehensively explores the landscape of novel sample pretreatment methods and their transformative role in pharmaceutical bioanalysis, particularly in the extraction of drugs and biomarkers from complex biological matrices such as plasma, serum, and urine. These innovative techniques are designed to address the persistent challenges of matrix effects, low recovery, and labor-intensive workflows that are common in conventional sample preparation methods.

A central focus of the review is the working principles of various emerging techniques, including SPME, MSPE, and novel sorbent-based methods such as immunosorbents and aptamer-functionalized materials. These methods are evaluated not only for their analytical performance but also for their compatibility with miniaturization and automation, two critical factors for high-throughput pharmaceutical analysis. The review systematically compiles case studies that detail the analytical parameters of these methods, such as recovery rates, LOD, and the detection techniques employed (e.g., LC-MS/MS, GC-MS, and HPLC). Immunosorbents and aptamers have shown exceptional promise in clinical diagnostics due to their high specificity and binding affinity, making them ideal for the selective extraction of biomarkers. Despite their advantages, the widespread adoption of these novel methods in routine pharmaceutical workflows is currently limited by higher initial costs and the need for specialized materials or instrumentation. However, ongoing research and technological advancements are steadily reducing these barriers, paving the way for broader implementation.

In a nutshell, the review underscores that while traditional methods still hold value, the integration of novel pretreatment techniques is essential for improving the efficiency, sensitivity, and reliability of pharmaceutical assays. These advancements are expected to significantly influence the future of bioanalytical workflows, especially as the demand for personalized medicine and rapid diagnostics continues to grow.

5. AUTHOR CONTRIBUTIONS

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.

6. FINANCIAL SUPPORT

There is no funding to report.

7. CONFLICT OF INTEREST

The authors report no financial or any other conflicts of interest in this work.

8. ETHICAL APPROVALS

This study does not involve experiments on animals or human subjects.

9. DATA AVAILABILITY

All data generated and analyzed are included in this research article.

10. PUBLISHER’S NOTE

All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.

11. USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED TECHNOLOGY

The authors declare that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI.

REFERENCES

1. Bylda C, Thiele R, Kobold U, Volmer DA. Recent advances in sample preparation techniques to overcome difficulties encountered during quantitative analysis of small molecules from biofluids using LC-MS/MS. Analyst. 2014;139(10):2265–76. CrossRef

2. Singleton C. Recent advances in bioanalytical sample preparation for LC–MS analysis. Bioanalysis. 2012;4(9):1123–40. CrossRef

3. Kole PL, Venkatesh G, Kotecha J, Sheshala R. Recent advances in sample preparation techniques for effective bioanalytical methods. Biomed Chromatogr. 2011;25(1-2):199–217. CrossRef

4. Van de Merbel N. Sample Preparation for LC-MS Bioanalysis of Proteins. Sample Preparation in LC-MS Bioanalysis. 2019;304–18.. CrossRef

5. Mulvana DE. Critical topics in ensuring data quality in bioanalytical LC–MS method development. Bioanalysis. 2010;2(6):1051–72. CrossRef

6. Aubry AF. LC–MS/MS bioanalytical challenge: ultra-high sensitivity assays. Bioanalysis. 2011;3(16):1819–25. CrossRef

7. Zhao L, Lucas D, Long D, Richter B, Stevens J. Multi-class multi-residue analysis of veterinary drugs in meat using enhanced matrix removal lipid cleanup and liquid chromatography-tandem mass spectrometry. J Chromatogr A. 2018;1549:14–24. CrossRef

8. Vaghela A, Patel A, Patel A, Vyas A, Patel N. Sample preparation in bioanalysis: a review. Int J Sci Technol Res. 2016;5(05):6–10.

9. Côté C, Bergeron A, Mess JN, Furtado M, Garofolo F. Matrix effect elimination during LC–MS/MS bioanalytical method development. Bioanalysis. 2009;1(7):1243–57. CrossRef

10. Lahaie M, Mess JN, Furtado M, Garofolo F. Elimination of LC–MS/MS matrix effect due to phospholipids using specific solid-phase extraction elution conditions. Bioanalysis. 2010;2(6):1011–21. CrossRef

11. Hyötyläinen T. Novel sample extraction and chromatographic techniques in environmental analysis. LCGC Eur. 2009;22(4):172–9.

12. Ingle RG, Zeng S, Jiang H, Fang WJ. Current developments of bioanalytical sample preparation techniques in pharmaceuticals. J Pharm Anal. 2022;12(4):517–29. CrossRef

13. Zhang Y, Cao H, Jiang H. Supported liquid extraction versus liquid–liquid extraction for sample preparation in LC–MS/MS-based bioanalysis. Bioanalysis. 2013;5(3):285–8. CrossRef

14. Svanström C, Hansson GP, Svensson LD, Sennbro CJ. Development and validation of a method using supported liquid extraction for the simultaneous determination of midazolam and 1′-hydroxy-midazolam in human plasma by liquid chromatography with tandem mass spectrometry detection. J Pharm Biomed Anal. 2012;58:71–7. CrossRef

15. Rositano J, Harpas P, Kostakis C, Scott T. Supported liquid extraction (SLE) for the analysis of methylamphetamine, methylenedioxymethylamphetamine and delta-9-tetrahydrocannabinol in oral fluid and blood of drivers. Forensic Sci Int. 2016;265:125–30. CrossRef

16. Sauve EN, Langødegård M, Ekeberg D, Øiestad AML. Determination of benzodiazepines in ante-mortem and post-mortem whole blood by solid-supported liquid–liquid extraction and UPLC–MS/MS. J Chromatogr B. 2012;883:177–88. CrossRef

17. Wu J, Lu J, Wilson C, Lin Y, Lu H. Effective liquid–liquid extraction method for analysis of pyrethroid and phenylpyrazole pesticides in emulsion-prone surface water samples. J Chromatogr A. 2010;1217(41):6327–33. CrossRef

18. Zheng M, Zhang C, Wang L, Wang K, Kang W, Lian K, et al. Determination of nine mental drugs in human plasma using solid-phase supported liquid-liquid extraction and HPLC-MS/MS. Microchem J. 2021;160:105647. CrossRef

19. Meunier C, Blondelle D, Faure P, Baguet JP, Le Goff C, Chabre O, et al. Development and validation of a method using supported liquid extraction for aldosterone determination in human plasma by LC-MS/MS. Clin Chim Acta. 2015;447:8–15. CrossRef

20. Kohira T, Kita Y, Tokuoka SM, Shiba M, Satake M, Shimizu T. Characterization of supported liquid extraction as a sample pretreatment method for eicosanoids and related metabolites in biological fluids. J Chromatogr B. 2019;1124:298–307. CrossRef

21. Shang T, Zhao LJ, Li P, Zeng XY, Yu ZQ. Determination of ten kinds of monohydroxylated polycyclic aromatic hydrocarbons in human urine by supported liquid extraction followed by liquid chromatography-tandem mass spectrometry. Chin J Anal Chem. 2019;47(6):876–882. CrossRef

22. Jiang J, Ip HSS, Zhou J, Guan Y, Zhang J, Liu G, et al. Supported-liquid phase extraction in combination with isotope-dilution gas chromatography triple quadrupole tandem mass spectrometry for high-throughput quantitative analysis of polycyclic aromatic hydrocarbon metabolites in urine. Environ Pollut. 2019;248:304–11. CrossRef

23. Chen R, Ning Z, Zheng C, Yang Y, Zhang C, Ou X, et al. Simultaneous determination of 16 alkaloids in blood by ultrahigh-performance liquid chromatography-tandem mass spectrometry coupled with supported liquid extraction. J Chromatogr B. 2019;1128:121789. CrossRef

24. Doctor EL, Mccord B. The application of supported liquid extraction in the analysis of benzodiazepines using surface enhanced Raman spectroscopy. Talanta. 2015;144:938–43. CrossRef

25. Ferreiro-Vera C, Priego-Capote F, Luque De Castro MD. Comparison of sample preparation approaches for phospholipids profiling in human serum by liquid chromatography–tandem mass spectrometry. J Chromatogr A. 2012;1240:21–8. CrossRef

26. Neville D, Houghton R, Garrett S. Efficacy of plasma phospholipid removal during sample preparation and subsequent retention under typical UHPLC conditions. Bioanalysis. 2012;4(7):795–807. CrossRef

27. Ahmad S, Kalra H, Gupta A, Raut B, Hussain A, Rahman MA. HybridSPE: a novel technique to reduce phospholipid-based matrix effect in LC–ESI-MS Bioanalysis. J Pharm Bioallied Sci. 2012;4(4):267–75. CrossRef

28. Nilsson K, Andersson M, Beck O. Phospholipid removal combined with a semi-automated 96-well SPE application for determination of budesonide in human plasma with LC–MS/MS. J Chromatogr B. 2014;970:31–5. CrossRef

29. Meloche M, Jutras M, St-Jean I, De Denus S, Leclair G. Isocyanate derivatization coupled with phospholipid removal microelution-solid phase extraction for the simultaneous quantification of (S)-metoprolol and (S)-α-hydroxymetoprolol in human plasma with LC–MS/MS. J Pharm Biomed Anal. 2021;204:114263. CrossRef

30. Guo H, Wang J, Wu Y, Liu W, Bu J, Zhao Q. Sensitive and simultaneous determination of nine anticoagulant rodenticides in human blood by UPLC–MS-MS with phospholipid removal pretreatment. J Anal Toxicol. 2018;42(7):459–66. CrossRef

31. Pilli NR, Narayanasamy S, Florian J, Zusterzeel R, Patel V, Strauss DG, et al. Novel simultaneous method for the determination of avobenzone and oxybenzone in human plasma by UHPLC-MS/MS with phospholipid removal pretreatment: an application to a sunscreen clinical trial. J Chromatogr B. 2021;1169:122615. CrossRef

32. Gao X, Sun Y, You H, Dai Y, Guo H, Zhao Q. Simultaneous determination of amitraz, chlordimeform, formetanate and metabolites in human blood by liquid chromatography tandem mass spectrometry with phospholipid-removal pretreatment. Biomed Chromatogr. 2019;33(4):4477. CrossRef

33. Sidqey D, Liane VH, Kristoffersen L. Quantitative determination of ethyl glucuronide and ethyl sulfate in postmortem and antemortem whole blood using phospholipid removal 96-well plate and UHPLC–MS-MS. J Anal Toxicol. 2021;45(4):378–88. CrossRef

34. Zidekova N, Nemcek A, Sutovska M, Mokry J, Kertys M. Development of sensitive and high-throughput liquid chromatography–tandem mass spectrometry method for quantification of haloperidol in human plasma with phospholipid removal pretreatment. J Anal Toxicol. 2021;45(6):573–80. CrossRef

35. Žideková N, Pršo K, Brisudová K, Babálová L, Bolek T, Sivák S, et al. A high-throughput liquid chromatography-tandem mass spectrometry method for simultaneous determination of direct oral anticoagulants in human plasma. J Sep Sci. 2023;46(13):e2300084. CrossRef

36. Wierucka M, Biziuk M. Application of magnetic nanoparticles for magnetic solid-phase extraction in preparing biological, environmental and food samples. Trends Anal Chem. 2014;59:50–8. CrossRef

37. Lu Z, Dai J, Song X, Wang G, Yang W. Facile synthesis of Fe3O4/SiO2 composite nanoparticles from primary silica particles. Colloids Surf A. 2008;317(1-3):450–6. CrossRef

38. Giakisikli G, Anthemidis AN. Magnetic materials as sorbents for metal/metalloid preconcentration and/or separation. A review. Anal Chim Acta. 2013;789:1–6. CrossRef

39. Plastiras OE, Deliyanni E, Samanidou V. Applications of graphene-based nanomaterials in environmental analysis. Appl Sci. 2021;11(7):3028. CrossRef

40. Dmitrienko SG, Apyari VV, Tolmacheva VV, Gorbunova MV, Furletov AA. Dispersive and magnetic solid-phase extraction of organic compounds: review of reviews. J Anal Chem. 2024;79(2):105–18. CrossRef

41. Waleng NJ, Munonde TS, Mpupa A, Moremedi T, Zhang Y, Nomngongo PN. Photoelectrochemical properties of magnetic amine-based MIL-101(Cr) hybrid material and its application in the degradation of acebutolol in water. RSC Adv. 2025;15(23):17986–99. CrossRef

42. Asgharinezhad AA, Ebrahimzadeh H. Poly (2-aminobenzothiazole)-coated graphene oxide/magnetite nanoparticles composite as an efficient sorbent for determination of non-steroidal anti-inflammatory drugs in urine sample. J Chromatogr A. 2016;1435(2):18–29. CrossRef

43. Wang Q, Huang L, Yu P, Wang J, Shen S. Magnetic solid-phase extraction and determination of puerarin in rat plasma using C18-functionalized magnetic silica nanoparticles by high performance liquid chromatography. J Chromatogr B. 2013;912:33–7. CrossRef

44. Cheng G, He M, Peng H, Hu B. Dithizone modified magnetic nanoparticles for fast and selective solid phase extraction of trace elements in environmental and biological samples prior to their determination by ICP-OES. Talanta. 2012;88:507–15. CrossRef

45. Rastkari N, Ahmadkhaniha R. Magnetic solid-phase extraction based on magnetic multi-walled carbon nanotubes for the determination of phthalate monoesters in urine samples. J Chromatogr A. 2013;1286:22–8. CrossRef

46. Eskandari H, Naderi-Darehshori A. Preparation of magnetite/poly (styrene-divinylbenzene) nanoparticles for selective enrichment-determination of fenitrothion in environmental and biological samples. Anal Chim Acta. 2012;743:137–44. CrossRef

47. Bylda C, Velichkova V, Bolle J, Thiele R, Kobold U, Volmer DA. Magnetic beads as an extraction medium for simultaneous quantification of acetaminophen and structurally related compounds in human serum. Drug Test Anal. 2015;7(6):457–66. CrossRef

48. Wang X, Niessner R, Knopp D. Magnetic bead-based colorimetric immunoassay for aflatoxin B1 using gold nanoparticles. Sensors. 2014;14(11):21535–48. CrossRef

49. Huang W, Ding J, Feng YQ. Magnetic solid phase extraction followed by high performance liquid chromatography for determination of urinary 1-hydroxypyrene. Chin J Anal Chem. 2012;40(6):830–4. CrossRef

50. Kabir A, Locatelli M, Ulusoy H. Recent trends in microextraction techniques employed in analytical and bioanalytical sample preparation. Separations. 2017;4(4):36. CrossRef

51. Zhao MM, Wu HZ, Deng XK, Yi RN, Yang Y. The application progress of magnetic solid-phase extraction for heavy metal analysis in food: a mini review. Anal Methods. 2024;16(3):333–43. CrossRef

52. Moein MM, Abdel-Rehim A, Abdel-Rehim M. Microextraction by packed sorbent (MEPS). Trends Anal Chem. 2015;67:34–44. CrossRef

53. Pereira J, Gonçalves J, Alves V, Câmara JS. Microextraction using packed sorbent as an effective and high-throughput sample extraction technique: recent applications and future trends. Sample Preparation. 2013;5(1):38–53. CrossRef

54. Ribeiro C, Ribeiro AR, Maia AS, Gonçalves VM, Tiritan ME. New trends in sample preparation techniques for environmental analysis. Crit Rev Anal Chem. 2014;44(2):142–85. CrossRef

55. Yang L, Said R, Abdel-Rehim M. Sorbent, device, matrix and application in microextraction by packed sorbent (MEPS): a review. J Chromatogr B. 2017;1043:33–43. CrossRef

56. Ferrone V, Carlucci M, Cotellese R, Raimondi P, Cichella A, Marco LD, et al. Development and validation of a fast micro-extraction by packed sorbent UHPLC-PDA method for the simultaneous determination of linezolid and ciprofloxacin in human plasma from patients with hospital-acquired pneumonia. Talanta. 2017;164:64–8. CrossRef

57. Elmongy H, Ahmed H, Wahbi AA, Amini A, Colmsjö A, Abdel-Rehim M. Determination of metoprolol enantiomers in human plasma and saliva samples utilizing microextraction by packed sorbent and liquid chromatography–tandem mass spectrometry. Biomed Chromatogr. 2016;30(8):1309–17. CrossRef

58. Prata M, Ribeiro A, Figueirinha D, Rosado T, Oppolzer D, Restolho J, et al. Determination of opiates in whole blood using microextraction by packed sorbent and gas chromatography-tandem mass spectrometry. J Chromatogr A. 2019;1602:1–10. CrossRef

59. Malaca S, Rosado T, Restolho J, Rodilla JM, Rocha PMM, Silva L, et al. Determination of amphetamine-type stimulants in urine samples using microextraction by packed sorbent and gas chromatography-mass spectrometry. J Chromatogr B. 2019;1120:41–50. CrossRef

60. Ventura S, Rodrigues M, Pousinho S, Falcão A, Alves G. Determination of lamotrigine in human plasma and saliva using microextraction by packed sorbent and high performance liquid chromatography -diode array detection: an innovative bioanalytical tool for therapeutic drug monitoring. Microchem J. 2017;130:221–8. CrossRef

61. Ares AM, Fernández P, Regenjo M, Fernández AM, Carro AM, Lorenzo RA. A fast bioanalytical method based on microextraction by packed sorbent and UPLC–MS/MS for determining new psychoactive substances in oral fluid. Talanta. 2017;174:454–61. CrossRef

62. Sergi M, Montesano C, Odoardi S, Mainero Rocca L, Fabrizi G, Compagnone D, et al. Micro extraction by packed sorbent coupled to liquid chromatography tandem mass spectrometry for the rapid and sensitive determination of cannabinoids in oral fluids. J Chromatogr A. 2013;1301:139–46. CrossRef

63. Vieira Diniz ML, Batista JM, Da Silva PHR, Fernandes C. Microextraction by packed sorbent and high-performance liquid chromatography for determination of benznidazole in human plasma. J Chromatogr B. 2023;1219:123640. CrossRef

64. Martins RO, Borsatto JVB, Will C, Lanças FM. Advancements in microextraction by packed sorbent: insights into sorbent phases and automation strategies. Separations. 2025;12(1):11. CrossRef

65. Ferreira L, Perestrelo R, Caldeira M, Câmara JS. Characterization of volatile substances in apples from rosaceae family by headspace solid-phase microextraction followed by GC-qMS. J Sep Sci. 2009;32(11):1875–88. CrossRef

66. Silva C, Cavaco C, Perestrelo R, Pereira J, Câmara J. Microextraction by packed sorbent (MEPS) and solid-phase microextraction (SPME) as sample preparation procedures for the metabolomic profiling of urine. Metabolites. 2014;4(1):71–97. CrossRef

67. Spietelun A, Pilarczyk M, Kloskowski A, Namiesnik J. Current trends in solid-phase microextraction (SPME) fibre coatings. Chem Soc Rev. 2010;39(11):4524–37. CrossRef

68. Pawliszyn J. Theory of solid-phase microextraction. Ontario, Canada: Elsevier; 2012. 13 p. CrossRef

69. Gorynski K, Bojko B, Kluger M, Jerath A, Wasowicz M, Pawliszyn J. Development of SPME method for concomitant sample preparation of rocuronium bromide and tranexamic acid in plasma. J Pharm Biomed Anal. 2014;92:183–92. CrossRef

70. Yang R, Xie W. Determination of cannabinoids in biological samples using a new solid phase micro-extraction membrane and liquid chromatography–mass spectrometry. Forensic Sci Int. 2006;162(1-3):135–9. CrossRef

71. Mirzajani R, Kardani F, Ramezani Z. Preparation and characterization of magnetic metal–organic framework nanocomposite as solid-phase microextraction fibers coupled with high-performance liquid chromatography for determination of non-steroidal anti-inflammatory drugs in biological fluids and tablet formulation samples. Microchem J. 2019;144:270–84. CrossRef

72. Xie Y, Zhang L, Hou W, Cheng Y, Luo F, Liu Z, et al. A novel method for monitoring N-nitrosamines impurities using NH2-MIL-101 (Fe) mediated dispersive micro-solid phase extraction coupled with LC-MS/MS in biopharmaceuticals. J Pharm Sci. 2023;112(11):2783–989. CrossRef

73. Nazdrajic E, Tascon M, Rickert DA, Gómez-Ríos GA, Kulasingam V, Pawliszyn JB. Rapid determination of tacrolimus and sirolimus in whole human blood by direct coupling of solid-phase microextraction to mass spectrometry via microfluidic open interface. Anal Chim Acta. 2021;1144:53–60. CrossRef

74. Ponce-Rodríguez HD, García-Robles AA, Sáenz-González P, Verdú-Andrés J, Campíns-Falcó P. On-line in-tube solid phase microextraction coupled to capillary liquid chromatography-diode array detection for the analysis of caffeine and its metabolites in small amounts of biological samples. J Pharm Biomed Anal. 2020;178:112914. CrossRef

75. Argente-García A, Moliner-Martínez Y, López-García E, Campíns-Falcó P, Herráez-Hernández R. Application of carbon nanotubes modified coatings for the determination of amphetamines by in-tube solid-phase microextraction and capillary liquid chromatography. Separations. 2016;3(1):7. CrossRef

76. Jalili V, Barkhordari A, Ghiasvand A. A comprehensive look at solid-phase microextraction technique: a review of reviews. Microchem J. 2020;152:104319. CrossRef

77. Kalhor P, Ghandi K. Deep eutectic solvents for pretreatment, extraction, and catalysis of biomass and food waste. Molecules. 2019;24(22):4012. CrossRef

78. Procentese A, Raganati F, Olivieri G, Russo ME, Rehmann L, Marzocchella A. Deep eutectic solvents pretreatment of agro-industrial food waste. Biotechnol For Biofuels. 2018;11(1):37. CrossRef

79. Affat S. A review of deep eutectic solvents (DESs), preparation, classification, physicochemical properties, advantages and disadvantages. UTJSCI. 2024;11(1):167–75. CrossRef

80. Nan L, Zhang H, Weitz DA, Shum HC. Development and future of droplet microfluidics. Lab Chip. 2024;24(5):1135–53. CrossRef

81. Berlanda SF, Breitfeld M, Dietsche CL, Dittrich PS. Recent advances in microfluidic technology for bioanalysis and diagnostics. Anal Chem. 2021;93(1):311–31. CrossRef

82. El-Deen AK, Shimizu K. Deep eutectic solvents as promising green solvents in dispersive liquid–liquid microextraction based on solidification of floating organic droplet: recent applications, challenges and future perspectives. Molecules. 2021;26(23):7406. CrossRef

83. Pérez-Cejuela HM, Herrero-Martínez JM, Simó-Alfonso EF. Recent advances in affinity MOF-based sorbents with sample preparation purposes. Molecules. 2020;25(18):4216. CrossRef

84. Pichon V, Chapuis-Hugon F, Hennion M-C. Bioaffinity sorbents. In: Comprehensive sampling and sample preparation, Academic Press; 2012. 359–88 pp.

85. Werth EG, Roos D, Philip ET. Immunocapture LC–MS methods for pharmacokinetics of large molecule drugs. Bioanalysis. 2024;16(7):165–77. CrossRef

86. Vernerová A, Krcmová LK, Heneberk O, Radochová V, Strouhal O, Kašparovský A, et al. Chromatographic method for the determination of inflammatory biomarkers and uric acid in human saliva. Talanta. 2021;233:122598. CrossRef

87. Li X, Wang W, Wang L, Wang Q, Pei X, Jiang H. Rapid determination of phenylethanolamine A in biological samples by enzyme-linked immunosorbent assay and lateral-flow immunoassay. Anal Bioanal Chem. 2015;407:7615–24. CrossRef

88. Johannsen C, Haq AU, Reubsaet L, Halvorsen TG. On the spot immunocapture in targeted biomarker analysis using paper-bound streptavidin as anchor for biotinylated antibodies. Anal Bioanal Chem. 2022;414(19):5979–89. CrossRef

89. Tofighi FB, Saadati A, Kholafazad-Kordasht H, Farshchi F, Hasanzadeh M, Samiei M. Electrochemical immunoplatform to assist in the diagnosis of oral cancer through the determination of CYFRA 21.1 biomarker in human saliva samples: preparation of a novel portable biosensor toward non-invasive diagnosis of oral cancer. J Mol Recognit. 2021;34(12):2932. CrossRef

90. Chen CA, Wang PW, Yen YC, Lin HL, Fan YC, Wu SM, et al. Fast analysis of ketamine using a colorimetric immunosorbent assay on a paper-based analytical device. Sensors Actuat B Chem. 2019;282:251–8. CrossRef

91. Turiel E, Martín-Esteban A. Molecularly imprinted polymers for sample preparation: a review. Anal Chim Acta. 2010;668(2):87–99. CrossRef

92. Gao B, Li Y, Zhang Z. Preparation and recognition performance of creatinine-imprinted material prepared with novel surface-imprinting technique. J Chromatogr B. 2010;878(23):2077–86. CrossRef

93. Bitas D, Samanidou V. Molecular imprinting for sample preparation. LCGC Eur. 2018;31(12):660–4. doi:https://doi.org/10.3390/separations5020031.

94. Díaz-Bao M, Barreiro R, Regal P, Cepeda A, Fente C. Evaluation of molecularly imprinted polymers for the simultaneous SPE of six corticosteroids in milk. Chromatographia. 2012;75:223–31. CrossRef

95. Sajini T, Mathew B. A brief overview of molecularly imprinted polymers: Highlighting computational design, nano and photo-responsive imprinting. Talanta Open. 2021;4:100072. CrossRef

96. El-Beqqali A, Abdel-Rehim M. Molecularly imprinted polymer-sol-gel tablet toward micro-solid phase extraction: i. Determination of methadone in human plasma utilizing liquid chromatography–tandem mass spectrometry. Anal Chim Acta. 2016;936:116–22. CrossRef

97. Madrakian T, Ahmadi M, Afkhami A, Soleimani M. Selective solid-phase extraction of naproxen drug from human urine samples using molecularly imprinted polymer-coated magnetic multi-walled carbon nanotubes prior to its spectrofluorometric determination. Analyst. 2013;138(16):4542–949. CrossRef

98. Yang J, Li Y, Wang J, Sun X, Cao R, Sun H, et al. Molecularly imprinted polymer microspheres prepared by Pickering emulsion polymerization for selective solid-phase extraction of eight bisphenols from human urine samples. Anal Chim Acta. 2015;872:35–45. CrossRef

99. Sadeghi S, Motaharian A, Moghaddam AZ. Electroanalytical determination of sulfasalazine in pharmaceutical and biological samples using molecularly imprinted polymer modified carbon paste electrode. Sens Actuat B Chem. 2012;168:336–44. CrossRef

100. Arabi M, Ghaedi M, Ostovan A, Wang S. Synthesis of lab-in-a-pipette-tip extraction using hydrophilic nano-sized dummy molecularly imprinted polymer for purification and analysis of prednisolone. J Colloid Interface Sci. 2016;480:232–9. CrossRef

101. Soledad-Rodríguez B, Fernández-Hernando P, Garcinuño-Martínez RM, Durand-Alegría JS. Effective determination of ampicillin in cow milk using a molecularly imprinted polymer as sorbent for sample preconcentration. Food Chem. 2017;224:432–8. CrossRef

102. Behbahani M, Bagheri S, Amini MM, Sadeghi Abandansari H, Reza Moazami H, Bagheri A. Application of a magnetic molecularly imprinted polymer for the selective extraction and trace detection of lamotrigine in urine and plasma samples. J Sep Sci. 2014;37(13):1610–6. CrossRef

103. Deng DL, Zhang JY, Chen C, Hou XL, Su YY, Wu L. Monolithic molecular imprinted polymer fiber for recognition and solid phase microextraction of ephedrine and pseudoephedrine in biological samples prior to capillary electrophoresis analysis. J Chromatogr A. 2012;1219:195–200. CrossRef

104. Kim W, Cha YL, Kim DJ. Advances and challenges in molecularly imprinted conducting and non-conducting polymers for selective and sensitive electrochemical sensors. ECS Sens Plus. 2025;4(1):15201. CrossRef

105. Augusto F, Hantao LW, Mogollón NGS, Braga SCGN. New materials and trends in sorbents for solid-phase extraction. Trends Anal Chem. 2013;43:14–23. CrossRef

106. Du F, Wei Z, Zeng Q, Ruan G. Aptamer-based sample preparation in LC/MS bioanalysis. Sample Preparation in LC/MS Bioanalysis. 2019:174–87. CrossRef

107. Bruno J, Sivils J. Aptamer ‚Western‘ blotting for E. coli outer membrane proteins and key virulence factors in pathogenic E. coli serotypes. Aptamers Synth Antibodies. 2016;2(1):29–35.

108. Bauer M, Strom M, Hammond DS, Shigdar S. Anything you can do, I can do better: can aptamers replace antibodies in clinical diagnostic applications?. Molecules. 2019;24(23):4377. CrossRef

109. Wang QL, Huang WX, Zhang PJ, Chen L, Lio CK, Zhou H, et al. Colorimetric determination of the early biomarker hypoxia-inducible factor-1 alpha (HIF-1α) in circulating exosomes by using a gold seed-coated with aptamer-functionalized Au@Au core-shell peroxidase mimic. Microchim Acta. 2020;187:1–1. CrossRef

110. Dalirirad S, Han D, Steckl AJ. Aptamer-based lateral flow biosensor for rapid detection of salivary cortisol. ACS Omega. 2020;5(51):32890–98. CrossRef

111. You M, Yang S, An Y, Zhang F, He P. A novel electrochemical biosensor with molecularly imprinted polymers and aptamer-based sandwich assay for determining amyloid-β oligomer. J Electroanal Chem. 2020;862:114017. CrossRef

112. Wang H, Chen H, Huang Z, Li T, Deng A, Kong J. DNase I enzyme-aided fluorescence signal amplification based on graphene oxide-DNA aptamer interactions for colorectal cancer exosome detection. Talanta. 2018;184:219–26. CrossRef

113. Wang Y, Cui M, Jiao M, Luo X. Antifouling and ultrasensitive biosensing interface based on self-assembled peptide and aptamer on macroporous gold for electrochemical detection of immunoglobulin E in serum. Anal Bioanal Chem. 2018;410:5871–8. CrossRef

114. Ensafi AA, Khoddami E, Rezaei B. Aptamer@ Au-o-phenylenediamine modified pencil graphite electrode: a new selective electrochemical impedance biosensor for the determination of insulin. Colloids Surf B. 2017;159:47–53. CrossRef

115. Aslipashaki SN, Khayamian T, Hashemian Z. Aptamer based extraction followed by electrospray ionization-ion mobility spectrometry for analysis of tetracycline in biological fluids. J Chromatogr B. 2013;925:26–32. CrossRef

116. Zhang X, Zhu S, Deng C, Zhang X. Highly sensitive thrombin detection by matrix assisted laser desorption ionization-time of flight mass spectrometry with aptamer functionalized core–shell Fe3O4@ C@ Au magnetic microspheres. Talanta. 2012;88:295–302. CrossRef

117. Mayol B, Qubbaj IZ, Nava-Granados J, Vasquez K, Keene ST, Sempionatto JR. Aptamer and Oligonucleotide-Based Biosensors for Health Applications. Biosensors. 2025;15(5):277. CrossRef

118. Yu H, Zhu J, Shen G, Deng Y, Geng X, Wang L. Improving aptamer performance: key factors and strategies. Microchimica Acta. 2023;190(7):255. CrossRef

119. Jain R, Jain B, Al-Khateeb LA, Alharthi S, Ghoneim MM, Abdelrahman M, et al. Advances in green sample preparation methods for bioanalytical laboratories focusing on drug analysis. Bioanalysis. 2025;17(7):489–508. CrossRef

120. Protti M, Milandri E, Di Lecce R, Mercolini L, Mandrioli R. New trends in bioanalysis sampling and pretreatment: how modern microsampling is revolutionising the field. Adv Sample Prep. 2025;13:100161. CrossRef

121. Chen WF. Integrating artificial intelligence with bioanalytical techniques for predictive modeling. J Bioanal Biomed. 2024;16(4):441.

122. Thurow K. Strategies for automating analytical and bioanalytical laboratories. Anal Bioanal Chem. 2023;415(21):5057–66. CrossRef