Journal of Chromatography B (v.817, #1)
Coupled-column systems in the biosciences by Ad (G.J.) de Jong; Udo A.Th. Brinkman (1).
Bioanalysis of drugs by liquid-phase microextraction coupled to separation techniques by Stig Pedersen-Bjergaard; Knut Einar Rasmussen (3-12).
The demand for automation of liquid–liquid extraction (LLE) in drug analysis combined with the demand for reduced sample preparation time has led to the recent development of liquid-phase microextraction (LPME) based on disposable hollow fibres. In LPME, target drugs are extracted from aqueous biological samples, through a thin layer of organic solvent immobilised within the pores of the wall of a porous hollow fibre, and into an μl volume of acceptor solution inside the lumen of the hollow fibre. After extraction, the acceptor solution is subjected directly to a final analysis either by high performance liquid chromatography (HPLC), capillary electrophoresis (CE), mass spectrometry (MS), or capillary gas chromatography (GC) without any further treatments. Hollow fibre-based LPME may provide high enrichment of drugs and excellent sample clean-up, and probably has a broad application potential within the area of drug analysis. This review focuses on the principle of LPME, and recent applications of three-phase, two-phase, and carrier mediated LPME of drugs from plasma, whole blood, urine, and breast milk.
Keywords: Liquid-phase microextraction; Hollow fibres; Drug analysis; Plasma; Whole blood; Urine; Breast milk;
Solid-phase extraction or liquid chromatography coupled on-line with gas chromatography in the analysis of biological samples by Tuulia Hyötyläinen; Marja-Liisa Riekkola (13-21).
This review provides an overview of the on-line coupling of solid-phase extraction or liquid chromatography with gas chromatography for the analysis of biological samples. Principles relevant to techniques are briefly presented and selected applications are described. Benefits of the coupled systems are discussed.
Keywords: Reviews; Biological samples; On-line coupling;
Structure specific chromatographic selection in targeted proteomics by Hamid Mirzaei; Fred Regnier (23-34).
The whole proteome of any organism is too complicated to be analyzed in a simple one-step process and direct attempts for the entire proteome analysis normally lead to considerable loss of information. A practical approach is the targeting of the specific structural feature of interest using chromatography. This approach simplifies the proteome while preserving most of the vital information necessary for analysis. Selection of peptides with specific amino acids (cysteine, histidine and methionine) or N- or C-terminal peptides is an accepted procedure for proteome simplification when general analysis is desired. While selection of enzymatically and non-enzymatically modified proteins and peptides is used when post-translational modifications are targeted. Protein interaction with small molecules as well as other proteins also has been studied using chromatographic selection methods.
Keywords: Proteomics; Amino acid; Chromatography; Affinity selection; Glycosylation; Phosphorylation; Oxidation; Nitration; Glycation; Specific amino acid selection;
Multidimensional separation of peptides for effective proteomic analysis by Haleem J. Issaq; King C. Chan; George M. Janini; Thomas P. Conrads; Timothy D. Veenstra (35-47).
Current solution based proteomic analysis methods are generally based on enzymatic digestion of a protein mixture followed by separation using multidimensional liquid chromatography and/or electrophoresis where peptide identification is typically accomplished by tandem mass spectrometry (MS/MS). It is generally accepted that no single chromatographic or electrophoretic procedure is capable of resolving the complex mixture of peptides that results from a global proteolytic digest of a proteome. Therefore, combining two or more orthogonal (multimodal) separation procedures dramatically improves the overall resolution and results in a larger number of peptides being identified from complex proteome digests. Separation of a proteome digest is a particularly challenging analytical problem due to the large number of peptides and the wide concentration dynamic range. While it has been demonstrated that increasing the number of dimensions of separation prior to MS analysis increases the number of peptides that may be identified, a balance between the time invested and the overall results obtained must be carefully considered. This manuscript provides a review of two- and three-dimensional peptide separation strategies combined with MS for the analysis of complex peptide mixtures.
Keywords: Multidimensional separation; Peptides; Proteomic analysis;
On-line multidimensional liquid chromatography and capillary electrophoresis systems for peptides and proteins by Thom Stroink; Mireya Castillo Ortiz; Auke Bult; Henk Lingeman; Gerhardus J. de Jong; Willy J.M. Underberg (49-66).
Peptides and proteins are gaining increasing attention in biosciences and, consequently, in analysis. This overview highlights the different approaches to couple on-line various separation techniques for the determination of proteins and peptides. The first section discusses the liquid chromatography (LC)–LC coupling, the second one reviews the on-line LC–capillary electrophoresis (CE) coupled systems and the third section summarizes the strategies for on-line CE–CE. The advantages, disadvantages, most relevant difficulties and particular systems for on-line coupling are discussed. Special attention is paid to the interface between the two dimensions. Applications are summarized in tables and a few typical examples are discussed. Many multidimensional separation methods are available, and it is demonstrated that peptide and protein mapping, or quantitation of proteins or peptides in various samples (aqueous solutions, cells, plasma) require different coupled systems. For mapping a semi-quantitative detection is often sufficient, while comprehensiveness is very important. For quantitation of a certain peptide or protein at a low concentration level a validated method should be used, while a heart-cut transport of the first dimension to the second one can offer sufficient selectivity. The combination with mass spectrometry as part of the total system is stressed and illustrated.
Keywords: Capillary electrophoresis; Comprehensive; Heart-cut; Liquid chromatography; Multidimensional separations; On-line coupling; Peptides; Proteins;
HPLC-MS-based methods for the study of metabonomics by Ian D. Wilson; Robert Plumb; Jennifer Granger; Hilary Major; Rebecca Williams; Eva M. Lenz (67-76).
The development and use of HPLC-MS for the study of metabonomics is reviewed. To date the technique has been applied to the analysis of urine samples obtained from studies in rodents in investigations of physiological variation (e.g., factors such as strain, gender, diurnal variation, etc.) and toxicity. Examples are provided of the use of conventional HPLC, capillary methods and the recently introduced high-resolution systems based on a combination of high pressure and small particle size (“UPLC”). Comparison is also made of the use of 1H NMR spectroscopy and HPLC-MS for the analysis of biofluid samples and the advantages and limitations of the two approaches are assessed. Likely future developments are considered.
Keywords: Metabolite profiling; Metabonomics; Metabolomics; HPLC-MS; Capillary HPLC-MS; High-resolution HPLC-MS; UPLC;
96-Well solid-phase extraction: a brief history of its development by Richard F. Venn; James Merson; Susan Cole; Paul Macrae (77-80).
This communication describes the invention and further development of the first 96-well solid-phase extraction system and the original purposes to which it was put. We also describe the adaption of this system for bioanalysis of pharmaceutically active small molecules and the needs underlying it. The system has become a world-wide standard for high-throughput bioanalysis and has been extended by others to include, for example, disk-phase extraction and supported liquid-liquid extraction, as well as 384-well systems. The factors that enabled this leap forward in productivity are discussed.
Keywords: Solid-phase extraction; High throughput; Bioanalysis; SPE systems;
Reduction of non-specific binding in Ga(III) immobilized metal affinity chromatography for phosphopeptides by using endoproteinase glu-C as the digestive enzyme by Erin H. Seeley; Larry D. Riggs; Fred E. Regnier (81-88).
The selectivity of immobilized metal affinity chromatography (IMAC) systems for the purification of phosphopeptides is poor. This is particularly a problem with tryptic digests of proteins where a large number of acidic peptides are produced that also bind during IMAC. The hypothesis examined in this work was that the selectivity of IMAC columns for phosphopeptides could be increased by using endoproteinase glu-C (glu-C) for protein digestion. Glu-C cleaves proteins at acidic residues and should reduce the number of acidic residues in peptides. This method was successfully applied to a mixture of model proteins and bovine milk. The percentage of phosphorylated peptides selected from proteolytic digests of the milk sample was increased from 40% with trypsin to 70% with glu-C. Additionally, this method was coupled with stable isotope coding methods to quantitatively compare the concentration of phosphoproteins between samples.
Keywords: Phosphopeptides; Endoproteinase glu-C;
Quantification of phosphoproteins with global internal standard technology by Larry Riggs; Erin H. Seeley; Fred E. Regnier (89-96).
Global internal standard technology (GIST) is being developed for the quantification of all primary structure and post-translational variants of proteins in a proteome. This paper is directed at an analysis of phosphorylation, primarily of serine and threonine. Quantification was achieved by acylation of primary amino groups in peptide cleavage fragments of proteins with isotopically coded derivatizing agents. Peptides from controls were globally coded with an isotopically “light” form of the reagent while those from experimental samples were coded with a “heavy” form of the reagent. The two types coding reagents used in this work were N-hydroxyl succinimide derivatives of acetate and 4-trimethylammoniumbutyrate. Heavy isotope forms were produced by deuteration of methyl groups. Subsequent to coding and mixing, the two samples were passed through a Ga(III) immobilized metal affinity chromatography (IMAC) column and the selected peptide fraction was further resolved by reversed-phase chromatography (RPC) and analyzed by mass spectrometry (MS). Relative differences in phosphopeptide concentration between samples were derived from isotope ratio measurements of the peptide isoforms observed in mass spectra. The method was validated with model peptides.
Keywords: Global internal standard technology; Phosphoproteins;
Analysis of strawberry volatiles using comprehensive two-dimensional gas chromatography with headspace solid-phase microextraction by Angela Williams; Danielle Ryan; Alexandra Olarte Guasca; Philip Marriott; Eddie Pang (97-107).
The aims of the current study were to develop an enantioselective multi-dimensional gas chromatography (GC × GC) method for the examination of strawberry volatiles and to use this method to make comparisons between the volatile profiles of different cultivars and between fresh picked and post-harvest berries of the same cultivar. Strawberry volatiles were sampled using solid-phase microextraction (SPME), and the repeatability and reproducibility of this method was examined. Semi-quantitative analysis of the volatiles was conducted using the relatively new technique of comprehensive multi-dimensional gas chromatography, using enantioselective (chiral) columns for the differentiation of analyte enantiomers. Chiral GC × GC facilitated the detection of key enantiomers in strawberry flavour. The (−)-enantiomer of 2,5-dimethyl-4-hydroxy-(2H)-furan-3-one (DMHF) and the S-enantiomer of linalool were tentatively identified as the predominant forms in both the cultivars Selva and Adina. The compounds benzaldehyde and methyl hexanoate were shown to decrease in post-harvest berries, whilst DMHF and nerolidol increased upon storage.
Keywords: Solid-phase microextraction; Enantiomer separation; Gas chromatography, comprehensive two-dimensional;
Automated method for the determination of a new matrix metalloproteinase inhibitor in ovine plasma and serum by coupling of restricted access material for on-line sample clean-up to liquid chromatography by P. Chiap; M. Piette; B. Evrard; F. Frankenne; B. Christiaens; G. Piel; D. Cataldo; J.-M. Foidart; L. Delattre; J. Crommen; Ph. Hubert (109-117).
A fully automated liquid chromatographic method was developed for the determination of Ro 28-2653, a new synthetic inhibitor of matrix metalloproteinases (MMPs), in ovine serum and plasma. The method was based on the coupling of a pre-column packed with restricted access material, namely LiChrospher RP-8 ADS (alkyl diol silica), for sample clean-up to an analytical column containing octyl silica stationary phase. One hundred μl of biological sample, to which 2-propanol was automatically added, were injected onto the ADS pre-column, which was then washed with a washing liquid consisting of a mixture of 25 mM phosphate buffer (pH 7.0) and acetonitrile (90:10; v/v) for 10 min. By rotation of the switching valve, the analyte was then eluted in the back-flush mode with the LC mobile phase composed of a mixture of acetonitrile and 25 mM phosphate buffer (pH 7.0) (57:43; v/v). The UV detection was performed at 395 nm. The main parameters likely to influence the sample preparation technique were investigated. The method was then validated over a concentration range from 17.5 to 1950 ng/ml, the first concentration level corresponding to the lower limit of quantitation. At this concentration level, the mean bias and the R.S.D. value for intermediate precision were −2.4% and 4.2%, respectively.
Keywords: Matrix metalloproteinase inhibitor; Ro 28-2653; Plasma; Serum; Sample preparation; Column-switching; Restricted access material; Liquid chromatography;
On-line combination of capillary isoelectric focusing and capillary non-gel sieving electrophoresis using a hollow-fiber membrane interface: a novel two-dimensional separation system for proteins by Hechun Liu; Chun Yang; Qing Yang; Weibing Zhang; Yukui Zhang (119-126).
A novel two-dimensional (2D) separation system for proteins was reported. In the system, a piece of dialysis hollow-fiber membrane was employed as the interface for on-line combination of capillary isoelectric focusing (CIEF) and capillary non-gel sieving electrophoresis (CNGSE). The system is similar equivalent to two-dimensional polyacrylamide gel electrophoresis (2D PAGE), by transferring the principal of 2D PAGE separation to the capillary format. Proteins were focused and separated in first dimension CIEF based on their differences in isoelectric points (pIs). Focused protein zones was transferred to the dialysis hollow-fiber interface, where proteins hydrophobically complexed with sodium dodecyl sulfate (SDS). The negatively charged proteins were electromigrated and further resolved by their differences in size in the second dimension CNGSE, in which dextran solution, a replaceable sieving matrix instead of cross-linked polyacrylamide gel was employed for size-dependent separation of proteins. The combination of the two techniques was attributed to high efficiency of the dialysis membrane interface. The feasibility and the orthogonality of the combined CIEF–CNGSE separation technique, an important factor for maximizing peak capacity or resolution elements, were demonstrated by examining each technique independently for the separation of hemoglobin and protein mixtures excreting from lung cancer cells of rat. The 2D separation strategy was found to greatly increase the resolving power and overall peak capacity over those obtained for either dimension alone.
Keywords: Capillary isoelectric focusing; Capillary non-gel sieving electrophoresis; Hollow-fiber membrane interface; Proteins;
Dynamic electric field assisted multi-dimensional liquid chromatography of biological samples by T.P. Hennessy; M. Quaglia; O. Kornyšova; B.A. Grimes; D. Lubda; K.K. Unger (127-137).
Complex biological samples require very high resolution separation strategies. The platform introduced here capitalises on the hyphenation of liquid chromatographic (LC) and electric potential gradient electrochromatographic multi-dimensional separation genres. First-dimension selectivity is provided by simultaneous size exclusion (SEC) and strong cation exchange (SCX) chromatography modes, while the second dimension comprises reversed phase (RP) characteristics in a dynamic (time-variant) electric field. The time-variant potential gradient with reversal of polarity is applied across the second dimension monolithic capillary throughout the duration of the solvent strength gradient elution. Hence, the platform offers comprehensive on-line sample clean-up (matrix depletion, analyte enrichment), fractionation (first dimention LC), and separation (second dimension LC) with the prospect of altering selectivity via polarity reversal dynamic electric field tuning.
Keywords: Multidimensional liquid chromatography; Size exclusion chromatography; Strong cation exchange chromatography; Reversed phase chromatography; Affinity chromatography; Tailor-made sorbents; Monolithic capillary columns; On-line sample clean-up; Human hemofiltrate; Human plasma; Electrically assisted liquid chromatography; Polarity reversal; Dynamic electric field; Transport mechanism;