Bioanalysis is an essential part in drug discovery and development. Bioanalysis is related to the analysis of analytes (drugs, metabolites, biomarkers) in biological samples and it involves several steps from sample collection to sample analysis and data reporting. The first step is sample collection from clinical or preclinical studies; then sending the samples to laboratory for analysis. Second step is sample clean-up (sample preparation) and it is very important step in bioanalysis. In order to reach reliable results, a robust and stable sample preparation method should be applied. The role of sample preparation is to remove interferences from sample matrix and improve analytical system performance. Sample preparation is often labor intensive and timeconsuming. Last step is the sample analysis and detection. For separation and detection, liquid chromatography-tandem mass spectrometry (LC MS/MS) is method of choice in bioanalytical laboratories. This is due to high selectivity and high sensitivity of the LC MS/MS technique. In addition the information about the analyte chemical structure and chemical properties is important to be known before the start of bioanalytical work. This review provides an overview of bioanalytical method development and validation. The main principles of method validation will be discussed. In this review GLP and regulated bioanalysis are described. Commonly used sample preparation techniques will be presented. In addition the role of LC MS/MS in modern bioanalysis will be discussed. In the present review we have our focus on bioanalysis of small molecules.

In this work, for the first time, a method has been developed for the determination of AZD6118, a candidate drug, in dog plasma samples. The method is based on microextraction by packed sorbent (MEPS) of the drug prior to liquid chromatography-electrospray ionization tandem mass spectrometry assay. Various important factors affecting MEPS performance were optimized, and under the optimized condition, a linear calibration curve in the concentration range of 20-25,000 nmol L-1 with a coefficient of determination over 0.99 was obtained. The back-calculated values of the calibration points showed good agreement with the theoretical concentrations (coefficients of variation percent between 0.3-3.8). The lower limit of quantification and limit of detection were 20.0 and 2.9 nmol L-1, respectively. The repeatability and accuracy of the method was evaluated by determination of quality control samples at three concentration levels (low, medium and high) using the developed method, and the results (coefficients of variation values were between 1.9% and 3.2%, relative recoveries ranged between 93.5-102.1%) confirm that a powerful method has been developed for the extraction and determination of the investigated drug in dog plasma.

Liposomes were used as biomimetic models in capillary electrokinetic chromatography (EKC) for the determination of distribution constants (K-D) of certain local anesthetics and a commonly used preservative. Synthetic liposomes comprised phosphatidylcholine and phosphatidylglycerol phospholipids with and without cholesterol. In addition, ghost liposomes made from red blood cell (RBC) lipid extracts were used as pseudostationary phase to acquire information on how the liposome composition affects the interactions between anesthetics and liposomes. These results were compared with theoretical distribution coefficients at pH 7.4. In addition to 25 degrees C, the distribution constants were determined at 37 and 42 degrees C to simulate physiological conditions. Moreover, the usability of five electroosmotic flow markers in liposome (LEKC) and micellar EKC (MEKC) was studied. LEKC was proven to be a convenient and fast technique for obtaining data about the distribution constants of local anesthetics between liposome and aqueous phase. RBC liposomes can be utilized for more representative model of cellular membranes, and the results indicate that the distribution constants of the anesthetics are greatly dependent on the used liposome composition and the amount of cholesterol, while the effect of temperature on the distribution constants is less significant.

Amphetamine selective molecularly imprinted sol-gel polymer tablets, MIP-tablets, for solid-phase micro extraction of biofluid samples were prepared. An acetonitrile solution of deuterated amphetamine template and silane precursor, 3-(propylmethacrylate) trimethoxysilane, was soaked into the pores of polyethylene tablet substrates and polymerized by an acid-catalysed sol-gel process. Application of the resultant MIP-tablets to extract amphetamine from human urine samples followed by LC-MS/MS analysis was investigated. The extraction protocol was optimised with respect to pH of sample, addition of sodium chloride, extraction time, desorption solvent and desorption time. The final analysis method determined amphetamine in human urine with a limit of detection (LOD) of 1.0 ng/mL and a lower limit of quantification (LLOQ) of 5 ng/mL. Validation demonstrated accuracy of the method was 91.0-104.0% and inter-assay precision was 4.8-8.5% (RSD). Extraction recovery was 80%. The MIP-tablets could be re-used and the same tablet could be employed for more than twenty extractions.

In recent years, application of nanomaterials as sorbent has gained the attention of researchers in bioanalysis. Different nanomaterials have been utilized as the sorbent in extraction techniques such as solid phase extraction, dispersive solid phase extraction, magnetic solid phase extraction, microextraction by packed sorbent, solid phase microextraction, dispersive pt-solid phase extraction, and stir bar sorptive extraction. In the present review, different nanomaterials which have recently been utilized as sorbent for bioanalysis are classified into six main groups, namely metallic, metallic and mixed oxide, magnetic, carbonaceous, silicon, and polymer-based nanomaterials. Application of these nanomaterials in different extraction techniques for bioanalysis has been reviewed. This study shows that magnetic nanomaterials have gained significant attention owing to their magnetic separation ability. In addition, the present review shows that there is a lack in the application of nanomaterials for on-line analysis procedures, most probably due to some intrinsic properties of nanomaterials such as spontaneous agglomeration.

Microextraction by packed sorbent (MEPS) is a new miniaturized form of solid-phase extraction and it is a green sample pretreatment technology. MEPS has been widely accepted and used by several research groups online or offline as a sample preparation technique before instrument analysis. MEPS reduces the sample handling time and organic solvent consumption. MEPS is suitable for small sample volumes and can easily be connected with different chromatographic techniques without modification. The sorbent bed in MEPS is integrated into a liquid handling syringe that allows for low void volume sample manipulations either manually or in combination with laboratory robotics. MEPS is a simple, fast and robust sample preparation technique with several advantages, miniaturization, automation, fast operation course, on-line coupling with analytical instruments and low-cost operation with less solvent and low sample consumption. Sorbent type, device, and matrix are important factors in MEPS research and applications. The performance of MEPS has recently been illustrated by online with liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry assays for pharmaceutical, environmental, and food analyses. This paper deals with MEPS device-optimized sorbent, sample matrix, and application. The progress and potential development of the technique are also discussed.

A sensitive, accurate and reliable bioanalytical method for the enantioselective determination of metoprolol in plasma and saliva samples utilizing liquid chromatography-electrospray ionization tandem mass spectrometry was developed and validated. Human plasma and saliva samples were pretreated by microextraction by packed sorbent (MEPS) prior to analysis. A new MEPS syringe form with two inputs was used. Metoprolol enantiomers and internal standard pentycaine (IS) were eluted from MEPS sorbent using isopropanol after removal of matrix interferences using aliquots of 5% methanol in water. Complete separation of metoprolol enantiomers was achieved on a Cellulose-SB column (150x4.6mm, 5m) using isocratic elution with mobile phase 0.1% ammonium hydroxide in hexane-isopropanol (80:20, v/v) with a flow rate of 0.8mL/min. A post-column solvent-assisted ionization was applied to enhance metoprolol ionization signal in positive mode monitoring (+ES) using 0.5% formic acid in isopropanol at a flow rate of 0.2mL/min. The total chromatographic run time was 10min for each injection. The detection of metoprolol in plasma and saliva samples was performed using triple quadrupole tandem mass spectrometer in +ES under the following mass transitions: m/z 268.0872.09 for metoprolol and m/z 303.3154.3 for IS. The linearity range was 2.5-500ng/mL for both R- and S-metoprolol in plasma and saliva. The limits of detection and quantitation for both enantiomers were 0.5 and 2.5ng/mL respectively, in both matrices (plasma and saliva). The intra- and inter-day precisions were presented in terms of RSD values for replicate analysis of quality control samples and were <5%; the accuracy of determinations varied from 96 to 99%. The method was able to determine the therapeutic levels of metoprolol enantiomers in both human plasma and saliva samples successfully, which can aid in therapeutic drug monitoring in clinical laboratories.

In the present work molecularly imprinted sol-gel tablet (MIP-Tablet) was prepared. The MIP-sol-gel was prepared as a thin layer on polyethylene material in a tablet form. Methadone-d₉ was selected as the template and 3-(propylmethacrylate)-trimethoxysilane was used as precursor. MIP-Tablet was applied for micro-solid phase extraction (μ-SPE). The MIP-Tablet was used for the determination of methadone in human plasma samples utilizing liquid chromatography-tandem mass spectrometry; and each tablet could be used twenty times. The extraction time was 10 min while desorption time was 6 min. Factors affecting the extraction efficiency such as desorption solvents, sample pH, salt addition, extraction time, desorption time and adsorption capacity were investigated. The calibration curves were obtained within the range of 5-5000 ng/mL using methadone in human plasma samples. The coefficients of determination (r(2)) values were >= 0.999 for all runs and the extraction recovery was >80%. The accuracy values for quality control samples varied from +3.6 to +9.7% and the inter-day precision (RSD %) values were ranged from 5.0 to 8.0%. The limit of detection was 1.0 ng/mL and the lower limit of quantification was 5 ng/mL utilizing methadone in human plasma samples.

The antiviral materials or compounds required for the treatment of viruses cause some infectious diseases such as Coxsackievirus B4 (CVB4), rotavirus Wa strain, and adenovirus type 7 are indispensable and of great necessity. The aim of the present study was to synthesize a new series of substituted indazole derivatives obtained from 2-(4-substituted-benzylidene)-4-phenylcyclohexanones and 2,6-bis (4-substituted-benzylidene)-4-phenylcyclohexanone derivatives. The products formed were reacted with 4-hydrazinylbenzoic acid or 2hydrazino- 6-methylbenzothiazole in the presence of cuprous oxide and Cs2CO3 as catalysts to give rise to a variety of indazole derivatives in a simple experimental procedure in good yields and short reaction time. The new compounds were fully characterized by spectroscopic and analytical methods. The synthesized compounds were evaluated for their antiviral activity against Coxsackievirus B4, adenovirus type 7 and rotavirus Wa strain. The bioassay results showed that the synthesized compounds possessed variable antiviral bioactivity. Compound (3-Fluoro-7-(fluoromethylene)-4,5,6,7-tetrahydro-2-(6-methylbenzo[d] thiazol-2-yl)-5phenyl-2H-indazole (24) exhibited moderate activity against both Coxsackievirus B4 and rotavius Wa strain and potentially promising activity against adenovirus type 7. On the other hand, 3-Chloro-7(chloromethylene)-4,5,6,7-tetrahydro-2-(6-methylbenzo[d] thiazol-2-yl)-5-phenyl-2H-indazole (25) and 7-(2,6-Dimethoxybenzylidene)-4,5,6,7-tetrahydro-3-(2,6-dimethoxyphenyl)-2-(6-methylbenzo[d]thiazol-2-yl)-5phenyl-2H-indazole (26) revealed potential promising activity against adenovirus type 7, while compounds 25 and 26 revealed promising activities against rotavirus Wa strain.

In this work, a novel method based on in situ molecularly imprinted sol-gel for the surface modification of a polysulfone membrane (PSM) was developed. A modified molecularly imprinted sol-gel polysulfone membrane (MSM) was placed in a homemade plastic tube and coupled on-line with LC/MS/MS for the selective extraction and screening of L-Tyrosine (Tyr) as a tentative lung cancer biomarker in human plasma samples. The existence of molecularly imprinted sol-gel layers on both sides of a PSM was examined using scanning electron microscopy (SEM). To evaluate the role of precursor in the extraction performance, repeatability, and selectivity of developed method, three precursors, 3-(propylmethacrylate) trimethoxysilane (P1), 3-(triethoxysilyl)-propylamine (P2), tetraethyl orthosilicate (P3), individually and together were used for treatment of PSM. Our investigation showed that a single precursor's route is more repeatable, straightforward, precise, accurate, and selective for the extraction of Tyr in plasma samples. Moreover, to achieve the best conditions and extraction efficiency, the effect of influential parameters, including the conditioning, washing, and elution of solvents, sample flow rate, loading time, desorption time, loading sample volume, salt effect, pH, and adsorption capacity for the most efficiently prepared membranes were truly investigated. The non-molecularly imprinted sol-gel polysulfone membrane (NSM) was prepared as a blank via the same process but in the absence of the Tyr. The LOD (S/N = 3/1) was 0.1 nmol L-1 and the LOQ (S/N = 10/1) was 0.34 nmol L-1 for Tyr in the plasma samples. The linearity for the Tyr was in the range of 0.34-2000 nmol L-1 in the plasma samples. The coefficients of determination values were >= 0.998 for all runs. The extraction recovery was between 80%-85% for Tyr in the plasma samples. In addition, MSM could be used for up to 50 extractions without a significant change in recovery percentage.