Quantitative liquid chromatographic and tandem mass spectrometric determination of vitamin D3 in human serum with derivatization: a comparison of in-tube LLE, 96-well plate LLE and in-tip SPME.

Sensitive and selective methods based on high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection were developed for the determination of vitamin D(3) in human serum. Derivatization of vitamin D(3) and its stable isotope labeled internal standard provided highly sensitive quantification and selective detection from endogenous compounds. Samples were prepared using the in-tube liquid-liquid extraction (LLE), 96-well plate LLE, and in-tip solid phase micro-extraction (SPME) in 96-well format. In all methods, the MS/MS detection was performed using Applied Biosystems-Sciex API 3000 tandem mass spectrometers interfaced with a heated nebulizer probe and operated in the positive ionization mode. Both tube and plate LLE methods achieved a lower limit of quantitation (LLOQ) of 0.5 ng/mL when 1.0 and 0.4 mL of human serum was processed, respectively, and were validated in the concentration range of 0.5-25 ng/mL; while for the in-tip SPME method, LLOQ was 5 ng/mL with only 0.1 mL of human serum required. Comparisons were made among three different methods, including precision and accuracy, sample throughput, recovery and matrix effects.

Automation of in-tip solid-phase microextraction in 96-well format for the determination of a model drug compound in human plasma by liquid chromatography with tandem mass spectrometric detection.

Studies using in-tip solid phase microextraction (in-tip SPME) in a 96-well plate format are conducted to investigate the feasibility of SPME automation. The sample preparation process, including extraction and desorption, was fully automated and coupled with currently commercially available automated liquid handling systems. Several process parameters including extraction time and speed, and desorption time were investigated. An LC-MS/MS method has been developed and validated to determine the levels of a drug compound (MK-0533) in human plasma that demonstrates the suitability of this new approach. The developed method has a lower limit of quantitation (LLOQ) of 5 ng/mL when 0.25 mL of human plasma is processed and is validated in the concentration range of 5-2, 000 ng/mL. The successful application of the assay in clinical sample analysis indicates that in-tip SPME can be easily automated and has great potential to be used for high throughput quantitative determination of drugs in pharmaceutical industry.

Comparison of solid-phase microextraction and liquid-liquid extraction in 96-well format for the determination of a drug compound in human plasma by liquid chromatography with tandem mass spectrometric detection.

Two sensitive and selective methods based on solid phase microextraction (SPME) and liquid-liquid extraction (LLE) in 96-well format, in combination with high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection have been developed to determine a model drug compound in human plasma. Both assays were performed on an Applied Biosystems-Sciex API 4000 tandem mass spectrometer interfaced with a turbo ion-spray probe and operated in the negative ionization mode. A lower limit of quantitation (LLOQ) of 1 ng/mL achieved when 0.25 mL of human plasma was processed. In both methods, a stable isotope labeled internal standard was utilized. The methods were validated in the concentration range of 1-500 ng/mL. The intraday precision (%C.V.) of the method using LLE was 0.8% at LLOQ, and was equal to or lower than 3.3% at all other concentrations, while the intraday precision (%C.V.) of the method using SPME was 6.9% at LLOQ, and was equal to or lower than 5.7% at all other concentrations. Based on the direct comparison of the two methods and their successful applications in clinical sample analysis, it may be concluded that SPME may be considered and used as an alternative approach for quantitative determination of drugs in pharmacokinetic studies.

Monitoring of drugs and metabolites in whole blood by restricted-access solid-phase microextraction coupled to liquid chromatography-mass spectrometry.

Robust biocompatible solid-phase microextraction (SPME) devices were prepared using various alkyldiol-silica (ADS) restricted-access materials (RAM) as the SPME coating. The ADS-SPME approach was able to simultaneously fractionate the protein component from a biological sample, while directly extracting diazepam and the major metabolites N-desmethyldiazepam, oxazepam and temazepam, and overcame the present disadvantages of direct sampling in biological matrices by SPME. The devices were interfaced with an LC-MS system and an isocratic mobile phase was used to desorb, separate, and quantify the analytes. The calculated diazepam, nordiazepam, temazepam, and oxazepam detection limits were 20, 20, 30, and 35 ng/ml in heparinized blood, respectively. The method was confirmed to be linear over the range of 50-1000 ng/ml with an average linear coefficient (R2) value of 0.996. The injection repeatability and intra-assay precision of the method were evaluated over ten injections at concentrations of 50, 200, and 500 ng/ml, resulting in a R.S.D. of ca. 10%. The robustness of the ADS-SPME device was evaluated for future use in in vivo studies, providing many direct extractions and subsequent determination of benzodiazepines in blood. For the extraction of the peptides angiotensin I, II, and III from blood, a novel restricted access material with cation exchange properties was evaluated. The ion-exchange diol silica improved the extraction efficiency of peptides relative to the conventional ADS material with reversed phase extraction centers.

Electrochemically controlled solid-phase microextraction based on conductive polypyrrole films.

Solid-phase microextraction (SPME) fiber coatings based on conductive polypyrrole films were prepared for the electrochemical extraction and desorption of ionic analytes. Simple preparation of each of the PPY extraction coatings on a platinum wire was possible with a constant potential method, but more importantly, cycling of the film between oxidation and reduction potentials facilitated the extraction and desorption of ionic analytes. The analytes were desorbed into a sample aliquot of water and were determined by flow injection analysis using a mass spectrometer. The fiber coatings and the developed electrochemical SPME method were found to be stable and reproducible (RSD < 5%; N = 5) and could be extended to several cations and anions, confirming the versatility of the approach. Preconcentration of the analyte on the fiber was also possible by repeating the processes to increase the amount of analyte extracted.

Verapamil drug metabolism studies by automated in-tube solid phase microextraction.

Verapamil is a common calcium antagonist described with antianginal, antihypertensive and antiarrythmic properties. The metabolites of verapamil have also shown pharmacological properties and therefore sample preparation and analysis techniques capable of metabolic screening for verapamil are important. In-tube SPME is a relatively new method integrating sample extraction, concentration and introduction into one single step without the use of organic solvents. The capability of in-tube SPME in bioanalysis has been reviewed but there has been no application described in the field of drug metabolism. Since automation and interfacing of in-tube SPME coupled to liquid chromatography-mass spectrometry (LC-MS) is possible, we confirm in this study that it is a powerful method to monitor the main metabolites of verapamil in various biological matrices like plasma, urine and cell culture media. Further, we show that it could also be used in routine pharmacokinetics measurements. An in-tube SPME LC-MS method was developed to extract and analyze the metabolic profile of verapamil from biological matrices. The detection limit for verapamil, gallopamil, norverapamil and PR22 were 52, 53, 65 and 83 ng/ml (UV detection) and 5, 6, 6 and 8 ng/ml (MS detection), respectively. The precision of the method was calculated in various biological matrices and the average % R.S.D. (N=5) for verapamil, gallopamil, norverapamil and PR22 was 3.9, 3.7, 3.8 and 4.3% (MS detection), respectively. The linear dynamic range was determined to be 100-800 ng/ml (UV detection) with a total sample preparation and analysis time of 34 min.

Direct LC analysis of five benzodiazepines in human urine and plasma using an ADS restricted access extraction column.

An alkyl-diol-silica (ADS) precolumn was used for the direct and on-line extraction of several benzodiazepines from serum and urine. The protein component of the biological sample was flushed through the ADS column, while simultaneously extracting the benzodiazepine compounds in the pores of the ADS stationary phase. The role of hydrophobic interactions in the extraction mechanism was confirmed. Column switching was employed to elute the extracted analytes from the ADS column into a high-performance liquid chromatography reverse-phase C18 column for the isocratic separation and UV detection of the benzodiazepines. Sample preconcentration via large volume injections was possible, improving the limits of detection. The calculated clonazepam, oxazepam, temazepam, nordazepam and diazepam detection limits were 38.8, 24.2, 31.7, 31.3, 45.0 ng/ml in serum, respectively, and 48.4, 24.5, 31.7, 33.1, 52.9 ng/ml for urine, respectively. The method was linear over the range of 50-10000 ng/ml in both matrices with an average linear coefficient (R(2)) value of 0.9918. The injection repeatability and intra-assay precision of the method were evaluated over ten injections, resulting in a percent relative standard deviation <5%. The ADS extraction column was robust, providing many direct injections of biological fluids for the extraction and subsequent determination of benzodiazepines.

Biological sample analysis with immunoaffinity solid-phase microextraction.

A theophylline antiserum was covalently immobilized on the surface of a fused silica fiber, modified with 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde, and used as a selective and sensitive extraction medium for the immunoaffinity solid-phase microextraction (SPME) determination of theophylline in serum samples. The specificity of the immunoaffinity SPME fiber was first investigated using a fixed concentration of [3H]theophylline together with various amounts of interference, possessing no cross-reactivity with the theophylline antibody. No significant non-specific binding was observed. The reproducibility of the fiber preparation and the immunoaffinity SPME analysis was also investigated, resulting in a relative standard deviation of 6.1% for five analyses of the same fiber. The antigen-antibody binding isotherm was obtained by analyzing theophylline standards of various concentrations (0.1-5 ng mL(-1)) until saturation values were reached. Initial binding of theophylline was linear with a r2 = 0.968. The cross-reactivity of the theophylline immunoaffinity SPME fiber for the structural analog caffeine was investigated by adding various amounts of caffeine in the presence of theophylline at a saturation concentration and produced a low cross-reactivity value of 0.1%. Finally. spiked serum samples (10 and 50 ng mL(-1)) were successfully analyzed with an excellent correlation with the standard binding isotherm, thus confirming the performance of the immunoaffinity SPME coating for improved bioanalysis.

In-tube moleculary imprinted polymer solid-phase microextraction for the selective determination of propranolol.

A molecularly imprinted polymer (MIP) material was synthesized for use as an in-tube solid-phase microextraction (SPME) adsorbent. The inherent selectivity and chemical and physical robustness of the MIP material was demonstrated as an effective stationary-phase material for in-tube SPME. An automated and on-line MIP SPME extraction method was developed for propranolol determination in biological fluids. This simplified the sample preparation process and the chromatographic separation of several beta-blocker compounds. The method developed for propranolol showed improved selectivity in comparison to alternative in-tube stationary-phase materials, overcoming the limitations of existing SPME coating materials. Preconcentration of the sample by the MIP adsorbent increased the sensitivity, yielding a limit of detection of 0.32 microg/mL by UV detection. Excellent method reproducibility (RSD < 5.0%) and column reusability (> 500 injections) were observed over a fairly wide linear dynamic range (0.5-100 microg/mL) in serum samples. To our knowledge, this is the first report on the automated application of a MIP material for in-tube SPME. The method was inexpensive, simple to set up, and simplified the choice of SPME adsorbent for in-tube extraction. The approach can potentially be extended to other MIPs for the determination of a wide range of chemically significant analytes.

Surface plasmon resonance-based immunoassays.

Surface plasmon resonance (SPR) has been successfully incorporated into an immunosensor format for the simple, rapid, and nonlabeled assay of various biochemical analytes. Proteins, complex conjugates, toxins, allergens, drugs, and pesticides can be determined directly using either natural antibodies or synthetic receptors with high sensitivity and selectivity as the sensing element. Immunosensors are capable of real-time monitoring of the antigen-antibody reaction. A wide range of molecules can be detected with lower limits ranging between 10(-9) and 10(-13) mol/L. Several successful commercial developments of SPR immunosensors are available and their web pages are rich in technical information. This review highlights many recent developments in SPR-based immunoassay, functionalizations of the gold surface, novel receptors in molecular recognition, and advanced techniques for sensitivity enhancement. Furthermore, it describes the challenge of current problems and provides some insights toward the future technologies.

Rapid determination of theophylline in serum by selective extraction using a heated molecularly imprinted polymer micro-column with differential pulsed elution.

Molecular imprinting of theophylline in poly(methacrylic acid ethylene dimethacrylate) form binding sites with complementary size, shape and chemical functionalities to theophylline. This molecularly imprinted polymer (MIP) can be packed into a micro-column for selective solid phase extraction (SPE) of theophylline from 20 microl of sample solution. Its chemical inertness and thermal stability allow the use of various organic solvents and elevated column temperatures for effective binding of theophylline. Non-specific adsorption of interfering drugs on the MIP surface is eliminated by an intermediate wash with 20 microl of acetonitrile, prior to quantitative desorption of the bound theophylline by 20 microl of methanol for in-line UV spectrophotometric determination. In this differential pulsed elution (DPE) technique, both the column temperature and solvent flow rate can be optimized to enhance selectivity. Application of this micro-analytical method, molecularly imprinted solid phase extraction DPE (MISPE-DPE), is demonstrated for accurate determination of theophylline in human blood serum. The method is validated over a linear range from 2 microg/ml to at least 20 microg/ml.

Determination of theophylline in serum by molecularly imprinted solid-phase extraction with pulsed elution.

The technique of molecular imprinting is used to produce an extensively cross-linked poly(methacrylic acid-co-ethylene dimethacrylate) material that contains theophylline as a print molecule. After Soxhlet extraction of the theophylline, binding sites are formed in the polymer with complementary size, shape, and positioning of chemical functionalities. The molecularly imprinted polymer's (MIP) high theophylline selectivity, chemical stability, and physically robust nature make it an ideal stationary-phase material in columns for HPLC separation of theophylline from other structurally related drug compounds. Mobile-phase tests confirm that a retention mechanism typical of normal-phase chromatography governs the separation, and selectivity of the MIP column can be controlled by a combination of the mobile phase and the sample solvent. Under optimal conditions, the MIP column functions like a solid-phase sorbent for theophylline extraction. Rapid elution of the bound theophylline can be accomplished in a pulsed format through injection of 20 µL of a solvent that has the proper polarity and protic nature to disrupt the electrostatic interactions and hydrogen bonding between theophylline and binding sites. A concentration detection limit of 120 ng/mL is obtained using direct UV absorption detection at 270 nm, which corresponds to a mass detection limit of 2.4 ng. This new technique, molecularly imprinted solid-phase extraction with pulsed elution (MISPE-PE), permits on-line preconcentration of theophylline from a large volume of dilute sample solution. Using a sample volume of 300 µL, a 40 ng/mL standard solution produces a detectable peak signal. Application of MISPE-PE in serum analysis further demonstrates the high capability of the MIP column to selectively isolate theophylline from other matrix components for fast, accurate determination.