Lars G. Blomberg

Chiral separation of amino acids and peptides by capillary electrophoresis

Chiral separation of amino acids and peptides by capillary electrophoresis (CE) is reviewed regarding the separation principles of different approaches, advantages and limitations, chiral recognition mechanisms and applications. The direct approach details various chiral selectors with an emphasis on cyclodextrins and their derivatives, antibiotics and chiral surfactants as the chiral selectors. The indirect approach deals with various chiral reagents applied for diastereomer formation and types of separation media such as micelles and polymeric pseudo-stationary phases. Many derivatization reagents used for high sensitivity detection of amino acids and peptides are also discussed and their characteristics are summarized in tables. A large number of relevant examples is presented illustrating the current status of enantiomeric and diastereomeric separation of amino acids and peptides. Strategies to enhance the selectivity and optimize separation parameters by the application of experimental designs are described. The reversal of enantiomeric elution order and the effects of organic modifiers on the selectivity are illustrated in both direct and indirect methods. Some applications of chiral amino acid and peptide analysis, in particular, regarding the determination of trace enantiomeric impurities, are given. This review selects more than 200 articles published between 1988 and 1999.

Two new techniques for sample preparation in bioanalysis: Microextraction in packed sorbent (MEPS) and use of a bonded monolith as sorbent for sample preparation in polypropylene tips for 96-well plates

Analytical methods providing high throughput are required for the ever increasing number of samples in bioanalysis. Currently, the method of choice in bioanalysis is LC-MS-MS. This method is quite rapid and thereby the focus has been directed to sample preparation as being a bottleneck in total analysis systems. It has become necessary to develop sample preparation techniques to a new improved level. This development has been based on a systematic and scientific approach. The key factors in this development have been miniaturization, integration, and automation of the techniques. This review provides a short overview of recent developments. Special emphasis is on two techniques: microextraction in packed syringe (MEPS) and use of a monolithic acrylamide plug as sorbent in polypropylene tips primarily intended for use with 96-well plate systems.

Microextraction in Packed Syringe Online with Liquid Chromatography‐Tandem Mass Spectrometry: Molecularly Imprinted Polymer as Packing Material for MEPS in Selective Extraction of Ropivacaine from Plasma

Abstract The excellent performance of a new sample preparation method, microextraction in packed syringe (MEPS), was recently illustrated by online LC‐MS and GS‐MS assays of local anaesthetics in plasma samples. In the method, approximately 1 mg of solid packing material was inserted into a syringe (100–250 µL) as a plug. Sample preparation took place on the packed bed. The new method was easy to use, fully automated, of low cost, and rapid in comparison with previously used methods. This paper presents the use of molecularly imprinted polymers (MIPs) as packing material for higher extraction selectivity. Development and validation of a method for MIP‐MEPS online with LC‐MS‐MS using ropivacaine in plasma as model compound were investigated. A bupivacaine imprinted polymer was used. 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 2–2000 nM. The applied polymer could be used more than 100 times before the syringe was discarded. The extraction recovery was 60%. The results showed high correlation coefficients (R2>0.999) for all runs. The accuracy, given as a percentage deviation from the nominal concentration values, ranged from −6% to 3%. The precision, given as the relative standard deviation, at three different concentrations (QC samples) was consistently about 3% to 10%. The limit of quantification was 2 nM.

Microextraction in Packed Syringe/Liquid Chromatography/Electrospray Tandem Mass Spectrometry for Quantification of Acebutolol and Metoprolol in Human Plasma and Urine Samples

Abstract The aim of the present investigation was to develop a simple, fast, and sensitive method for the determination of acebutolol and metoprolol in human plasma and urine samples. The determination of acebutolol and metoprolol in plasma and urine was performed using micro extraction in packed syringe (MEPS) as a sample preparation method, online with high performance liquid chromatography and tandem mass spectrometry (LC‐MS/MS). In MEPS the sampling sorbent was 1 mg polystyrene polymer, which was inserted in a 250 µL syringe. The lower limits of quantification (LLOQ) for acebutolol and metoprolol were set to 1.0 ng/mL. The accuracy of quality control samples (QC) varied by ±10%, and precision (R.S.D.) had a deviation of 1.4–12% for plasma and urine samples. The calibration curve was obtained within the concentration range 1.0–100 ng/mL in both plasma and urine. The regression correlation coefficients (R2) for plasma and urine samples were ≥0.999 for all runs. The present method is miniaturized, fully automated, robust, and can be easily used for pharmacokinetic and pharmacodynamic studies of acebutolol and metoprolol.

Bonded dimethylacrylamide as a permanent coating for capillary electrophoresis

A method for coating capillaries for capillary electrophoresis with chemically bonded polydimethylacrylamide has been developed, and the properties of the capillaries have been evaluated. The coated capillaries provided high separation efficiency, 12 x 10(5) theoretical plates/m was obtained for cytochrome c. The electroosmotic flow at pH 8.0 was 10 x 10(-10) to 6 x 10(-10) m2 V(-1) s(-1). The coated capillaries were quite stable at high pH. At least 150 runs could be done at pH 10 without appreciable performance deterioration. The excellent performance of the coated capillaries was illustrated by separation of basic proteins, acidic proteins, 9-fluorenylmethyl chloroformate-derivatized neurotransmitter amino acids, peptide reference mixtures and peptides digested from a bacteria protein.

Drug Screening Using Microextraction in a Packed Syringe (MEPS)/Mass Spectrometry Utilizing Monolithic‐, Polymer‐, and Silica‐Based Sorbents

Abstract Micro extraction in packed syringe (MEPS) has been evaluated for drug and metabolites screening online with mass spectrometric detection. In this study, silica based (C8), polymer based (ENV+), and a methacrylate based organic monolith were used as sorbents for MEPS. Monolithic material has shown to be an effective chromatographic support for the separation of several classes of compounds. In this study, the focus is subdivided into three parts: 1) Using MEPS for drugs and metabolites screening, 2) Preparation of a monolithic material in situ in a syringe, and 3) Comparison of the monolith, ENV+, and C8 as sorbent material. The synthesis of the monolithic material was by radical polymerization of glycidyl methacrylate (GMA), ethylene glycol dimethacrylate (EGDMA), and butyl methacrylate (BMA) in porogenic solvent 1‐dodecanol and cyclohexanol. An 8xa0µL of the synthesized material was drawn into a 250xa0µL syringe and thermally polymerized at 57°C for 24xa0h. Individual syringes containing the monolithi...

Microextraction in Packed Syringe (MEPS) Utilizing Methylcyanopropyl–Silarylene as Coating Polymer for Extraction of Drugs in Biological Samples

Abstract Microextraction in packed syringe (MEPS) is a new technique for miniaturised solid‐phase extraction that can be connected online to GC or LC. In this work, a liquid polymer was used as coating polymer on a filter in a 10 µL and 250 µL syringe to handle small sample volumes (≤10 µL plasma). Ropivacaine in plasma samples was used as model substance. The validation of the methodology showed that the accuracy values of quality control samples (QC) were in the range of 103%–114% for GC‐MS, and 98–101% for LC‐MS‐MS. The precisions, given as relative standard deviation (R.S.D.) were in the range 1.9 to11% for Inter‐ and intra‐day precisions. The standard curves were obtained within the concentration ranges 5‐2,000 nM in human plasma samples. The regression correlation coefficients (R2) for plasma samples were 0.99 for all runs using GC‐MS and LC‐MS‐MS.

On-line derivatization utilizing solid-phase microextraction (SPME) for determination of Busulphan in plasma using gas chromatography-mass spectrometry (GC-MS)

Busulphan (Bu) is an alkylating agent used in preparative regimen before stem cell transplantation (SCT). Bu has a narrow therapeutic window, and underdosing or overdosing may have a fatal outcome for the patient. Therapeutic drug monitoring (TDM) combined with dose adjustment is currently used to optimize and individualize therapy with Bu. However, this approach is limited to centers with laboratory facilities. An automated and easy method for measurement of Bu plasma concentrations may facilitate TDM for Bu and thus improve the clinical outcome. A solid-phase microextraction (SPME) on line with gas chromatography (GC) and mass-spectrometric detection to quantify Bu in human plasma samples was developed using in-vial derivatization. Bu was mixed with reagent in a 2-mL vial and shaken for 15 minutes at 80°C; subsequently, the SPME fiber was immersed into the vial for 15 minutes. The fiber was washed in water for 10 seconds before injection. Several parameters influencing the extraction and recovery were studied, such as absorption and desorption times, the effects of the temperature on the reaction, and the shaking time on the derivatization yield. Carbowax–divinylbenzene, polyacrylate, and polydimethylsiloxane fibers were tested. The carbowax–divinylbenzene fiber resulted in the highest recovery in plasma samples. The validation of the method showed a high chromatographic selectivity and a good sensitivity (LOQ = 20 ng/mL). Coefficient of variation for SPME was less than 15%. The results showed good correlation between Bu concentrations and response within the range of 40 to 2500 ng/mL (R2 = 0.999). The accuracy ranged from 94% to 106%. This is well in line with the international criteria for validation. The present method was applied to patient plasma. The obtained results were comparable with the results obtained from GC with electron capture detection. The authors conclude that this method has shortened the analysis time considerably and is fully automated, which benefits TDM of Bu in SCT patients.

Increasing Sample Preparation Throughput Using Monolithic Methacrylate Polymer as Packing Material for 96-Tip Robotic Device

Abstract In this work, a laboratory robot has been used to accomplish a system for high sample cleanup throughput. The robot operating in a 96‐well format was furnished with 96 polypropylene tips packed with a chemically bonded monolithic methacrylate plug as sample adsorbent. Using this system, 96 samples could be handled in 2 minutes. Polypropylene tips were furnished with a chemically bonded monolithic methacrylate plug as sample adsorbent. Roscovitine and lidocaine in plasma samples were used as model substances. The validation of the methodology showed that the accuracy values of quality control samples (QC) were between +15%, and precision had a maximum deviation of 11%. The standard curve was obtained within the concentration range 14‐5600 nM in both plasma and water samples. The regression correlation coefficients (R2) for plasma and water samples were ≥0.999 for all runs. This paper was presented at HPLC 2005.

Evaluation of monolithic packed 96-tips and liquid chromatography–tandem mass spectrometry for extraction and quantification of pindolol and metoprolol in human plasma samples

96-well pipette tips with a chemically bonded monolithic methacrylate sorbent plug were used for solid-phase extraction (SPE) of pindolol and metoprolol in human plasma samples. The sorbent plug was formed by in situ polymerization. Monolithic packed 96-tips are a tool for miniaturized, solid-phase extraction. Using such packed 96-tips, a 96-well plate could be handled in about 2 min. The key aspect of the monolithic phase is that monolithic material can provide both relatively good binding capacity and relatively low backpressure properties. The validation of the methodology showed that the accuracy values of quality-control samples were between 101% and 103% for metoprolol, while between 94% and 114% for pindolol. The precision ranged from 4% to 15%. The standard calibration curves were obtained within the concentration range 5-5000 nM in plasma samples. The coefficients of determination (R2) for plasma samples were >or=0.99. Our prepared polymer based monolithic packed 96-tips were compared with commercial silica based 96-tips and protein precipitation.