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.

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 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.

Sample preparation (sample pre-treatment) is the initial step and an essential part in bioanalysis procedure. The main role of sample preparation is to extract and transfer the analyte(s) of interest from a complex matrix to a purified media such as a pure solvent for analysis and quantification. Biological fluids are complex and contain, in addition to the target analyte(s), many different unwanted compounds from salts to proteins. Thus, the analysis of these samples requires an effective sample preparation method prior to the liquid chromatography-mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS) assays. The aim of the present work was to evaluate micro-extraction by packed sorbent (MEPS) and develop new sample preparation techniques for the extraction of neurotransmitters and biomarkers from biological samples. Moreover, two new sample preparation techniques were developed. The first developed technique is molecular imprinting on polysulfone membrane (MIPM), and the second one is molecularly imprinted polymer in tablet form (MIPT).

MEPS is a well-known sample preparation technique that can be used online with analytical instruments without any modifications. In this thesis, MEPS was used online with liquid chromatography-tandem mass spectrometry (LC/MS/MS) for the quantification of dopamine and serotonin in human urine samples (Study I) and with GC/MS for the analysis of methadone in humane urine (Study II). Polystyrene polymer and silica-C8 were used as sorbent in Study I and Study II, respectively. In both studies, small sample volumes (50 µL) were used and full method validation was performed. In both studies (I and II), MEPS enhanced the limit of detection (LOD) and reduced the extraction time compared to the previously published methods. In Study I, the mean accuracies of quality control (QC) samples of dopamine and serotonin were 99–101% while the precision values (RSD) were 6–11%. In Study II, the accuracy values of methadone were between 97 and 107% while the precisions (RSD) were between 11 and 15%.

MIP-sol-gel on polysulfone membrane (MIPM) was developed and used in combination with MEPS for the extraction of hippuric acid (HA) in plasma and urine samples (Study III). A good selectivity was obtained using plasma and urine samples. The precision of QC samples in plasma and urine samples were 2.2–4.8% and 1.1–6.7%, respectively. The method recovery was above 90%.

In Study IV, a new technique was developed using a tablet form of molecularly imprinted sol-gel (MIPT) for the extraction of methadone from human plasma samples. Methadone-d9 was selected as the template for accurate recovery, and 3-(propyl methacrylate) trimethoxysilane (3PMTMOS) was used as a precursor. The extraction recovery was higher than 80%. The LOD and LLOQ were 1.0 and 5.0 ng mL^-1, respectively. The validation showed good selectivity, accuracy and precision.

In Study I, III and IV, LC/MS/MS system was used while GC/MS was used in Study II for the separation and detection of target analytes.

There will always be a high demand for rapid, selective, reliable and sensitive techniques for sample preparation. It is convenient to use molecular dynamics simulations as a theoretical tool for the optimization of molecularly imprinted system. Suitable monomers and cross-linkers are crucial to synthesize the new membrane and tablet molecular imprinted polymer platforms for all kinds of molecules.

Speed and low cost, together with regulatory approval, are the most important requirements of clinical assays. Therefore, a fast and automated on-line sample preparation method is essential for the routine analysis of biological samples. Microextraction by packed sorbent is an option for optimal sample preparation due to its easy automation, minimal requirements for the sample and elution solvent volumes, elimination of evaporation and reconstitution steps, and ability to integrate sample preparation and injection into one step. The use of effective sample preparation steps circumvents the need for chromatographic separation and therefore allows more rapid and less expensive sample analysis in clinical and forensic practice. Two biologically active compounds, amphetamine and methadone, were chosen as representative drugs of abuse for the application of microextraction by packed sorbent coupled directly to mass spectrometry. The developed method was validated, with the results confirming the suitability of the combination of these techniques for the analysis of biological samples. The approach was confirmed to be appropriate for use in clinical and forensic practice with regard to cost and time requirements for analysis.

Destruction of sorbents during consecutive extractions using the microextraction by packed sorbent (MEPS) technique is a serious problem. In MEPS the complex matrix such as plasma and blood can affect the sorbent physical properties and the sorbent can be deteriorated after handling of few samples. To overcome this problem, the surface of a polysulfone membrane (PSM) was modified by a molecularly imprinted sol-gel and utilized for online extraction of a lung cancer biomarker, hippuric acid (HA), in biological matrices. The molecularly imprinted polymer membrane provided fast, sensitive, selective and robust sample preparation method for HA in biological fluids. In addition, MIP membrane could be used for up to 50 extractions without a significant change in extraction recovery. To achieve the best results, the parameters that influenced the extraction efficiency were thoroughly investigated. Moreover, for evaluating the performance of the molecularly imprinted sol-gel membrane (MISM), a non-molecularly imprinted sol-gel membrane (NISM) as a blank was prepared. The limits of detection CLOD) and quantification (LOQ) for HA in both plasma and urine samples were 0.30 nmol L^-1 and 1.0 nmol L^-1, respectively. Standard calibration curves were obtained over the range of 1-1000 nmol L^-1 for HA in plasma and urine samples. The coefficients of determination (R²) were ≥ 0.997. The extraction recoveries of HA from human plasma and urine samples were higher than 91%. The precision values for HA in plasma and urine samples were 2.2-4.8% and 1.1-6.7%, respectively.

In situ monolithic molecularly imprinted polymer sol-gel packed tips (MMSTs) were prepared and evaluated for the extraction of lung cancer biomarker L-tyrosine (Tyr) from human plasma and urine samples. Several extraction parameters such as the conditioning, washing and elution solutions, pH and time were investigated. The enrichment factor (EF) and extraction recovery (ER) were studied. MMST showed good selectivity and a high extraction recovery, and MMST as a sorbent showed good stability and repeatability. The method validation showed good regression correlation coefficients for plasma and urine samples (R-2 >= 0.996) within the concentration range of 5-1000 and 1-1000 nmol L-1 in plasma and urine samples, respectively. The lower limits of quantification (LLOQ) in the plasma and urine samples were 5 and 1 nmol L-1, respectively. The between-batch precision for Tyr in plasma ranged from 1.0 to 6.0%, and in urine it was from 1.0 to 7.0%. The results show that the developed method has more facility, stability, durability and repeatability compared with previous similar methods. To the best of our knowledge, this is the first study aimed at the selective separation of Tyr as a lung cancer biomarker by MMSTs from biological matrixes and detection by LC/MS/MS.

A specific LC-MS-MS method for the determination of dopamine and serotonin (5-hydroxytryptamine; 5HT) in human urine is described. The analytes were extracted from urine and preconcentrated by microextraction in a packed syringe (MEPS). The new method is very promising, very easy to use, fully automated, of low cost, and rapid in comparison to previously used methods. The method was validated and the standard curves were evaluated by means of quadratic regression and weighted by the inverse of the concentration: 1/x for the calibration range 50–4000 μg/L. The MEPS applied polymer (silica-C8) could be used more than 300 times. The extraction recovery was about 50%. The results showed close correlation coefficients (r² A0.999) for all analytes in the calibration range studied. The accuracy of MEPS-LC-MS-MS was 100–101% for dopamine and 99–100% for 5HT. The interday precision (n = 3 days), expressed as the RSD%, was 6.0–7.7% for dopamine and 6.1–11% for 5HT. MEPS reduced the handling time by 12 times compared to a published method.

A method for the simultaneous analysis of methadone in urine samples by microextraction in a packed syringe online with GC-MS (MEPS-GC-MS) is described. The new method reduced the sample handling and the detection limit by two- to seven-fold compared to published methods. Using a quantitation method based on the calculation of analyte concentration by comparison to an internal standard, we were able to measure methadone levels consistent with values reported for healthy individuals. The intra-assay precisions (RSD) of the method using quality control (QC) samples at three different concentration levels were about 11-14% (n = 6). The interassay precisions (RSD) were 11–15% for methadone in urine samples (n = 18). The accuracy varied from 89 to 109% for intra-assay (n = 6), and 97 to 107% for inter-assay (n = 18). The regression correlation coefficients (r²) were over 0.99 in all experiments.