Molecularly imprinted polymer-sol-gel tablet toward micro-solid phase extraction: II. Determination of amphetamine in human urine samples by liquid chromatography-tandem mass spectrometry.

Amphetamine selective molecularly imprinted sol-gel polymer tablets, MIP-tablets, for solid-phase microextraction 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 1.0ng/mL and a lower limit of quantification (LLOQ) of 5ng/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.

Bioanalytical method development and validation: Critical concepts and strategies.

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 time consuming. 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.

Determination of AZD6118 in dog plasma samples utilizing microextraction by packed sorbent and liquid chromatography-electrospray ionization tandem mass spectrometry.

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 20-25,000nmolL-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 2.9nmolL-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.

Molecularly imprinted polymer-sol-gel tablet toward micro-solid phase extraction: I. Determination of methadone in human plasma utilizing liquid chromatography-tandem mass spectrometry.

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.

On-line detection of hippuric acid by microextraction with a molecularly-imprinted polysulfone membrane sorbent and liquid chromatography-tandem mass spectrometry.

Destruction of sorbents during consecutive extractions using the micro-extraction 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 (LOD) and quantification (LOQ) for HA in both plasma and urine samples were 0.30nmolL-1 and 1.0nmolL-1, respectively. Standard calibration curves were obtained over the range of 1-1000nmolL-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.

Determination of amphetamine and methadone in human urine by microextraction by packed sorbent coupled directly to mass spectrometry: an alternative for rapid clinical and forensic analysis.

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.

Preparation of monolithic molecularly imprinted polymer sol-gel packed tips for high-throughput bioanalysis: extraction and quantification of L-tyrosine in human plasma and urine samples utilizing liquid chromatography and tandem mass spectrometry.

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.

Microextraction by packed sorbent for LC-MS/MS determination of drugs in whole blood samples.

BACKGROUND: Microextraction by packed sorbent (MEPS) is used as an online sample-preparation method. The determination of local anesthetics lidocaine, ropivacaine and bupivacaine directly in human blood was performed using MEPS online with LC-MS/MS. RESULTS: The range of the calibration curves in whole blood was 10-10000 nmol/l. The lower limit of quantification was set to 10.0 nmol/l. The accuracy of the quality control samples ranged from 85 to 97%. The interday precision of the studied analytes was within the range 1-5%. The regression correlation coefficient (r(2)) was over 0.995 for all runs. The present method is rapid, reliable and robust and may be used for therapeutic drug monitoring of studied analytes in whole blood. CONCLUSION: This assay allows the analysis of drugs in human blood directly. Sample preparation is simple and automated. The assay reduced the handling time and the cost, and could handle small volumes of whole blood samples (25 µl).

Quantitative analysis of methadone in human urine samples by microextraction in packed syringe-gas chromatography-mass spectrometry (MEPS-GC-MS).

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 (r(2)) were over 0.99 in all experiments.

Determination of dopamine and serotonin in human urine samples utilizing microextraction online with liquid chromatography/electrospray tandem mass spectrometry.

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 inverse of the concentration: 1/x for the calibration range 50-4000 microg/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 > 0.999) 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.

Fast and sensitive environmental analysis utilizing microextraction in packed syringe online with gas chromatography-mass spectrometry: determination of polycyclic aromatic hydrocarbons in water.

A new sensitive, selective, fast and accurate technique for online sample preparation was developed. Microextraction in a packed syringe (MEPS) is a new miniaturised, solid-phase extraction (SPE) technique that can be connected online to GC or LC without any modifications. In MEPS approximately 1mg of the solid packing material is inserted into a syringe (100-250 ml) as a plug. Sample preparation takes place on the packed bed. The bed can be coated to provide selective and suitable sampling conditions. The new method is very promising. It is very easy to use, fully automated, of low cost and rapid in comparison with previously used methods. The determination of polycyclic hydrocarbons (PAHs) in water was performed using MEPS as sample preparation method online with gas chromatography and mass spectrometry (MEPS-GC-MS). The results from MEPS as sample preparation were compared with other techniques such as stir bar sorptive extraction (SBSE) and solid-phase microextraction (SPME). The method was validated and the standard curves were evaluated by the means of quadratic regression and weighted by inverse of the concentration: 1/x for the calibration range 5-1,000 ng/L. The MEPS applied polymer (silica-C8) could be used more than 400 times before the syringe was discarded. The extraction recovery was about 70%. The results showed close correlation coefficients (R>0.998) for all analytes in the calibration range studied. The accuracy of MEPS-GC-MS was between 90 and 113% and the inter-day precision (n=3 days), expressed as the relative standard deviation (RSD%), was 8-16%. MEPS reduced the handling time by 30 and 100 times compared to SPME and SBSE, respectively.