Henk Lingeman

Coupling of biological sample handling and capillary electrophoresis

The analysis of biological samples (e.g., blood, urine, saliva, tissue homogenates) by capillary electrophoresis (CE) requires efficient sample preparation (i.e., concentration and clean-up) procedures to remove interfering solutes (endogenous/exogenous and/or low-/high-molecular-mass), (in)organic salts and particulate matter. The sample preparation modules can be coupled with CE either off-line (manual), at-line (robotic interface), on-line (coupling via a transfer line) or in-line (complete integration between sample preparation and separation system). Sample preparation systems reported in the literature are based on chromatographic, electrophoretic or membrane-based procedures. The combination of automated sample preparation and CE is especially useful if complex samples have to be analyzed and helps to improve both selectivity and sensitivity. In this review, the different modes of solid-phase (micro-) extraction will be discussed and an overview of the potential of chromatographic, electrophoretic (e.g., isotachophoresis, sample stacking) and membrane-based procedures will be given.

Development of a Selective ESI-MS Derivatization Reagent: Synthesis and Optimization for the Analysis of Aldehydes in Biological Mixtures

In LC-MS, derivatization is primarily used to improve ionization characteristics, especially for analytes that are not (efficiently) ionized by ESI or APCI such as aldehydes, sugars, and steroids. Derivatization strategies are then directed at the incorporation of a group with a permanent charge. A compound class that typically requires derivatization prior to LC-MS is the group of small aliphatic aldehydes that are, for instance, analyzed as the key biomarkers for lipid peroxidation in organisms. Here we report the development of a new tailor-made, highly sensitive, and selective derivatization agent 4-(2-(trimethylammonio)ethoxy)benzenaminium halide (4-APC) for the quantification of aldehydes in biological matrixes with positive ESI-MS/ MS without additional extraction procedures. 4-APC possesses an aniline moiety for a fast selective reaction with aliphatic aldehydes as well as a quaternary ammonium group for improved MS sensitivity. The derivatization reaction is a convenient one-pot reaction at a mild pH (5.7) and temperature (10 degrees C). As a result, an in-vial derivatization can be performed before analysis with an LC-MS/MS system. All aldehydes are derivatized within 30 min to a plateau, except malondialdehyde, which requires 300 min to reach a plateau. All derivatized aldehydes are stable for at least 35 h. Linearity was established between 10 and 500 nM and the limits of detection were in the 3-33 nM range for the aldehyde derivatives. Furthermore, the chosen design of these structures allows tandem MS to be used to monitor the typical losses of 59 and 87 from aldehyde derivatives, thereby enabling screening for aldehydes. Finally, of all aldehydes, pentanal and hexanal were detected at elevated levels in pooled healthy human urine samples.

Capillary electrophoresis as a versatile tool for the bioanalysis of drugs - a review

This review article presents an overview of current research on the use of capillary electrophoretic techniques for the analysis of drugs in biological matrices. The principles of capillary electrophoresis and its various separation and detection modes are briefly discussed. Sample pretreatment methods which have been used for clean-up and concentration are discussed. Finally, an extensive overview of bioanalytical applications is presented. The bioanalyses of more than 200 drugs have been summarised, including the applied sample pretreatment methods and the achieved detection limits.

Recent Advancements in the LC- and GC-Based Analysis of Malondialdehyde (MDA): A Brief Overview.

Malondialdehyde (MDA) is an end-product of lipid peroxidation and a side product of thromboxane A2 synthesis. Moreover, it is not only a frequently measured biomarker of oxidative stress, but its high reactivity and toxicity underline the fact that this molecule is more than “just” a biomarker. Additionally, MDA was proven to be a mutagenic substance. Having said this, it is evident that there is a major interest in the highly selective and sensitive analysis of this molecule in various matrices. In this review, we will provide a brief overview of the most recent developments and techniques for the liquid chromatography (LC) and gas chromatography (GC)-based analysis of MDA in different matrices. While the 2-thiobarbituric acid assay still is the most prominent methodology for determining MDA, several advanced techniques have evolved, including GC–MS(MS), LC–MS(MS) as well as several derivatization-based strategies.

Online magnetic bead dynamic protein-affinity selection coupled to LC-MS for the screening of pharmacologically active compounds

The online, selective isolation of protein-ligand complexes using cobalt(II)-coated paramagnetic affinity beads (PABs) and subsequent liquid chromatography-mass spectrometry (LC-MS) determination of specifically bound ligands is described. After in-solution incubation of an analyte mixture with His-tagged target proteins, protein-analyte complexes are mixed with the Co(II)-PABs and subsequently injected into an in-house built magnetic trapping device. Bioactive ligands bound to the protein-Co(II)-PABs are retained in the magnetic field of the trapping device while inactive compounds are removed by washing with a pH 7.4 buffer. Active ligands are online eluted toward the LC-MS system using a pH shift. In the final step of the procedure, the protein-Co(II)-PABs are flushed to waste by temporarily lowering the magnetic field. The proof-of-principle is demonstrated by using commercially available Co(II)-PABs in combination with the His-tagged human estrogen-receptor ligand-binding domain. The system is characterized with a number of estrogenic ligands and nonbinding pharmaceutical compounds. The affinities of the test compounds varied from the high micromolar to the subnanomolar range. Typical detection limits are in the range from 20 to 80 nmol/L. The system is able to identify binders in mixtures of compounds, with an analysis time of 9.5 min per mixture. The standard deviation over 24 h is 9%.

Evaluation of different column chemistries for fast urinary metabolic profiling

Fast analytical methodologies are mandatory for large scale metabolic profiling. Here, we present a thorough evaluation of different column chemistries in combination with different mobile phases for fast LC-MS urinary metabolic profiling. Three porous HILIC materials were investigated, next to core-shell C18-, XB-C18- and PFP-RPLC material. The performance of the selected column chemistries was tested in a non-targeted manner with pooled urine samples and in a targeted manner with a set of 54 common urinary metabolites. In order to evaluate the differential behaviour of the tested columns in a targeted manner, we applied a peak scoring algorithm. This algorithm takes into account several quality criteria such as retention time, dead time, peak height and peak shape. In general, HILIC columns generate more retention for polar metabolites. Our results show that the diol-HILIC column outperforms the RPLC columns. However, because of their opposite nature, comprehensive behaviour is observed as well, which was shown by investigating gender differences in a small urinary sample set. All applied column chemistries enabled sufficient peak capacity within a short gradient time.

Online Fluorescence Enhancement Assay for the Acetylcholine Binding Protein with Parallel Mass Spectrometric Identification

The acetylcholine binding protein (AChBP) is considered an analogue for the ligand-binding domain of neuronal nicotinic acetylcholine receptors (nAChRs). Its stability and solubility in aqueous buffer allowed the development of an online bioaffinity analysis system. For this, a tracer ligand which displays enhanced fluorescence in the binding pocket of AChBP was identified from a concise series of synthetic benzylidene anabaseines. Evaluation and optimization of the bioaffinity assay was performed in a convenient microplate reader format and subsequently transferred to the online format. The high reproducibility has the prospect of estimating the affinities of ligands from an in-house drug discovery library injected in one known concentration. Furthermore, the online bioaffinity analysis system could also be applied to mixture analysis by using gradient HPLC. This led to the possibility of affinity ranking of ligands in mixtures with parallel high-resolution mass spectrometry for compound identification.

On-line SPE-CE for the determination of insulin derivatives in biological fluids

An on-line SPE-CE system is described for the determination of insulin derivatives in urine, serum and plasma. By combining techniques based on different separation mechanisms, in this case reversed-phase SPE and CE, a more selective sample clean-up is obtained. The described on-line SPE-CE procedure is able to desalt and clean biological samples, resulting in more repeatable electrophoretic results as well as a good linearity for urine, serum and plasma samples spiked with insulin derivatives, thus proving the elimination of detrimental effects caused by the sample matrix. The on-line SPE-CE system was linear for urine, serum and plasma samples spiked with insulin derivatives between 5 and 80 mg/l. The repeatability in migration time was below 1% relative standard deviation (R.S.D.). The repeatability of the peak was better (<2.4% R.S.D.) when no off-line precipitation reaction (<6.2% R.S.D.) was used, proving the beneficial characteristics of on-line sample pretreatment procedures over off-line sample pretreatment procedures which are prone to sample losses and contamination.

At-line solid-phase extraction for capillary electrophoresis: application to negatively charged solutes.

The analysis of complex biological samples with capillary electrophoresis (CE) requires proper sample pretreatment. In this paper the applicability of solid-phase extraction (SPE) coupled at-line with CE is studied, by using a laboratory-made interface. A fresh (disposable) SPE cartridge is used for each sample to prevent carry-over effects. The sample handling procedure is performed parallel with the analysis of the previous sample, to improve sample throughput. Using this set-up, negatively charged test compounds (some non-steroid anti-inflammatory drugs) can be determined in serum and urine. The method is linear over at least two decades and detection limits are around 40 microg/l. A single capillary, flushed only once a week with a sodium hydroxide solution, was used without problems for the analysis of ca. 900 samples during 1 year. The robustness of the system was very good: no blocking of loop, interface or capillary was found during this period. Furthermore, the system was successfully used for overnight runs.

Derivatization of carboxylic acids with 4-APEBA for detection by positive-ion LC-ESI–MS(/MS) applied for the analysis of prostanoids and NSAID in urine ☆

In order to develop a generic positive ionization ESI LC-MS method for a variety of interesting substance classes, a new derivatization strategy for carboxylic acids was developed. The carboxylic acid group is labeled with the bromine containing 4-APEBA reagent based on carbodiimide chemistry. The derivatization reaction can be carried out under aqueous conditions, thereby greatly simplifying sample preparation. In this paper, the derivatization of carboxylic acids is exemplified for the determination of prostanoids and non-steroidal anti-inflammatory drugs (NSAID). Optimization of the derivatization conditions was studied. In order to prove the applicability of the presented approach, we applied the described protocol to urine samples from complex regional pain syndrome (CRPS) patients and were able to detect several prostanoids not visible in the urine of healthy volunteers. Further, the determination of the non-steroidal anti-inflammatory drug ibuprofen in a urine sample was possible.