Principles and applications of luminescence spectrometry coupled to liquid chromatographic techniques

An overview is presented of the physicochemical basis of luminescence, and its application to the detection of chemicals (drugs, biomedically important compounds, environmentally active substances) in liquid chromatographic systems.     

Partial purification of egg development neurosecretory hormone with reverse-phase liquid chromatographic techniques

We report the use of reverse-phase liquid chromatographic techniques for the isolation of a steroidogenic neuropeptide (EDNH) from mosquito heads. Activity of fractions was assayed by measuring the ability of ovaries to produce ecdysteroid $\text{in}$$\text{vitro}$. Dose response profiles using crude head extracts or partially purified EDNH were nearly identical, indicating that the methods of preparation did not alter biological activity. EDNH activity eluted from a reverse-phase HPLC (RP-HPLC) column primarily near 35 percent acetonitrile using a linear gradient. Methods developed with an analytical RP-HPLC column were successfully adapted for preparative work. Active fractions from the preparative RP-HPLC were further purified on a second analytical column under isocratic conditions at 30% acetonitrile. Two adjacent UV absorbing peaks were found, each with EDNH activity. Activity was sensitive to proteolysis.     

Neuroimaging techniques in modern forensic psychiatry

Applied neuroscientific knowledge such as brain neuroimaging has widespread application in the medical diagnostic and treatment areas. Neuroscientific progress such as cognitive neuroscience has strong implications in specific medical fields such as forensic psychiatry. Significant progress in forensic psychiatry has affected the practice of law, in which an understanding of the complex relationship among mind, brain, and behavior is becoming necessary. Forensic psychiatry is concerned with the relationship between psychiatric abnormalities and legal violations and crimes. Due to the lack of available biological criteria, assessment, evaluation and therapy in forensic psychiatry have so far been restricted to psychosocial and mental criteria of offender personality. Recent advances in nuclear radiology such as brain imaging techniques (fMRI, DT-MRI, PET SPECT) allow a closer approach to the neural correlates of personality, moral judgments and decision-making. Introduction of neurobiological criteria (based on advanced neuroimaging techniques) in the field of forensic psychiatry and establishing the rules to what extent such biological criteria will be more reliable choice in evaluating mentally ill offenders would be of fundamental value in the modern forensic psychiatry. Psychosocial and subjective criteria in forensic evaluation will be more accomplished by biopsychosocial and objective criteria. Advances in the neuroimaging techniques bring specificity to the problems underlying the application of neuroscience to criminal law.     

High-pressure liquid chromatographic analysis of amino acid phenylthiohydantoins: Comparison with other techniques

High-pressure liquid chromatography, utilizing reverse phase μ Bondapak C₁₈ columns and elution with increasing acetonitrile concentrations, has been used to resolve amino acid phenylthiohydantoins obtained from the automated Edman degradation of proteins. Assignment of identity to residues which are difficult to distinguish or identify conclusively by other conventional techniques is easily achieved by high-pressure liquid chromatographic techniques. The use of high-pressure liquid chromatography, in parallel with gas-liquid and polyamide thin-layer chromatography, allows unequivocal assignments of identity to amino acid phenylthiohydantoins obtained in protein sequencing. Single protein sequence determinations can be extended by 20 to 100% by the use of high-pressure liquid chromatography with rapid, accurate, and quantitative identifications of amino acid phenylthiohydantoins.     

Isocratic reversed-phase liquid chromatographic of all-trans-retinoic acid and its major metabolites in new potential supplementary test systems for developmental toxicology

An isocratic reversed-phase high-performance liquid chromatographic procedure for the determination of all-trans-retinoic acid (all-trans-RA) and its metabolites, all-trans-4-oxo-RA, 5,6-epoxy-RA, 9-cis-RA and13-cis-RA, in mouse plasma and embryo and in new in vitro potential test systems for development toxicology has been developed. These compounds, their biological precursor retinol (vitamin A) and the internal standard were resolved on a Spherisorb ODS-2 (5 μm) column (250×4.6 mm I.D.) with acetonitrile-water-methanol-n-butyl alcohol (56:37:4:3, v/v) containing 100 mM ammonium acetate and 70 mM acetic acid as the elution system with a total run time of 23 min. The assay was linear over a wide range, with a lower limit of quantitation of 50 ng/ml or 10 ng/ml of protein for all-trans-RA, 13-cis-RA and 9-cis-RA and of 25 ng/ml or 5 ng/ml protein for the 4-oxo- and 5,6-epoxy-metabolites. At these concentrations, intra-assay coefficients of variation (C.V.) of the retinoids were 3–9%. Mean intra-assay C.V. averaged 5–7% in the tissues studied. Its use is discussed for RA measurements in some of the new test systems — Drosophila melanogaster, sea urchin embryos and cultured human keratinocytes — that have to be evaluated in toxicological testing, supplementary to standard assays in mammals.     

Application of gas chromatography-surface ionization organic mass spectrometry to forensic toxicology

Surface ionization (SI), which consists in the formation of positive and negative ions along the course of thermal desorption of particles from a solid surface, was first applied as a detector for gas chromatography (GC), GC-surface ionization detection (SID); we developed many new sensitive methods for the determination of abused and other drugs by GC-SID. Recently, Fujii has devised a combination of SI and a quadrupole mass spectrometer and named this system a surface ionization organic mass spectrometer (SIOMS), which is highly selective and sensitive for organic compounds containing tertiary amino groups. We have tried to apply this mass spectrometer to forensic toxicological study; so far we have succeeded in determining important drugs-of-abuse and toxic compounds, such as phencyclidine (PCP), pethidine, pentazocine, MPTP and its derivatives from human body fluids with high sensitivity and selectivity. In this review, we describe our recent studies on the application of GC-SIOMS to forensic toxicology.     

Role of chromatographic techniques in proteomic analysis

Proteomics, the characterization of the proteome, is conceptually simple but technically challenging. Development of such technologies as mass spectrometry, multidimensional protein separation, and DNA sequencing has allowed the new field of proteomics to flourish. Proteomic analysis relies on a set of techniques chosen on the basis of the biological question. In any proteomic analysis, the first and most important task is the separation of a complex protein mixture, i.e. the proteome. Chromatography, one of the most powerful methods of separation, employs one or more inherent characteristics of a protein-its mass, isoelectric point, hydrophobicity or biospecificity. This review emphasizes high-performance liquid chromatography as an integrated part of technologies used to study the proteome, discusses the capabilities and limitations of current instruments, and highlights the potential of multidimensional liquid chromatography in proteomic analysis.     

High-performance liquid chromatographic and gas—liquid chromatographic determination of diazepam and nordiazepam in plasma

A one-step method for extraction of diazepam, nordiazepam, and internal standard into toluene is followed by chromatographic separation and detection with either dual-wavelength high-performance liquid chromatography or electron-capture gas—liquid chromatography. Agreement between the two methods was excellent for diazepam (r = 0.99, n = 38) and good for nordiazepam (r = 0.96, n = 79) over a concentration range that included subtherapeutic, therapeutic, and toxic plasma levels.     

Role of advances in chromatographic techniques in phytochemistry

Chromatography is the lynchpin of phytochemistry and is the key to obtaining pure compounds for structure elucidation, for pharmacological testing or for development into therapeuticals. It also plays a fundamental role as an analytical technique for quality control and standardisation of phytotherapeuticals. Although liquid chromatography is barely 100 years old, an extraordinary variety of instrumental and ancillary equipment is available, notably in the domain of high-performance liquid chromatography. It is impossible to touch all areas of chromatography in such a review but certain areas are worthy of mention: HPLC, HPTLC, UPLC and countercurrent chromatography. Another important addition has been the development of hyphenated techniques involving HPLC: LC/UV, LC/MS, LC/MS(n) and LC/NMR. These are indispensable nowadays for the early detection and identification of compounds in crude plant extracts.     

Extraction, pre-high-performance liquid chromatographic purification, and high-performance liquid chromatographic analysis of plant nucleotides

Problems encountered in obtaining reliable analytical data by HPLC for the free nucleotide constituents of plant tissues are considered and methods of overcoming them experimentally assessed. Major problems include suppression of residual phosphatase activity during extraction, and removal of pigments, phenolics, alkaloids, and other uv-absorbing nonnucleotides, prior to HPLC. An optimal combination of extraction and pre-HPLC purification techniques is discussed which, in combination with HPLC by anion exchange, yields quantitatively reliable data. The optimized procedure involves extraction with a monophasic mixture of methanol: chloroform:formic acid:water and purification of the nucleotide extract by a batch treatment with poly-N-vinylpyrrolidone, followed by ligand-exchange chromatography. The main HPLC separation uses mu Bondapak NH2 in a linear phosphate gradient and gives good resolution of all the commonly occurring plant nucleotides in a single chromatographic run.     

Dual ultraviolet wavelength high-performance liquid chromatographic method for the forensic or clinical analysis of seventeen antidepressants and some selected metabolites

A sensitive method suitable for the determination of tricyclic and other antidepressants in postmortem and clinical specimens is presented. The procedure, which utilizes reversed-phase HPLC combined with dual ultraviolet wavelength detection, enables the separation of 17 commonly prescribed antidepressants and some selected metabolites in a single extraction. Peak purity was confirmed using absorbance ratios at 220 nm and 254 nm wavelengths and revealed little interference from other eluting analytes. The blood detection limit for most antidepressants was 50 ng/ml. The most commonly observed antidepressants in 281 forensic cases analysed over a two-year period with the described method were dothiepin, amitriptyline, nortriptyline and doxepin.     

Chiral separation principles in chromatographic and electromigration techniques

Almost half of the drugs in use today are chiral. It is well established that the pharmacological activity is mostly restricted to one of the enantiomers (eutomer). There can be qualitative and quantitative differences in the activity of the enantiomers. In many cases, the inactive enantiomer (distomer) shows unwanted side effects or even toxic effects. Even if the side effects are not that drastic, the distomer has to be metabolized and this represents an unnecessary burden for the organism. Therefore, the development of methods for the separation of enantiomers, both on analytical and preparative scale, has become increasingly important. Chromatographic techniques such as thin layer chromatography (TLC), gas chromatography (GC), supercritical fluid chromatography (SFC), and above all high-performance liquid chromatography (HPLC) have been used for enantiomer separation for about two decades. More recently, electromigration techniques, such as capillary electrophoresis and capillary electrochromatography, have been shown to be powerful alternatives to chromatographic methods. This review gives a short overview of different chiral separation principles and their application. Several new developments are discussed.     

Method for the isolation and purification of pyridinoline and deoxypyridinoline crosslinks from bone by liquid chromatographic techniques

Significant progress has been made in recent years in the development of new bone resorption markers, based principally on the urinary excretion of pyridinoline (Pyd) and deoxypyridinoline (Dpd) crosslinks. For their measurement, in spite of the recent development of immunoassays, HPLC remains the method of reference. However, the lack of an appropriate internal standard requires large amounts of pure crosslinks for external standardisation. Herein, we describe an efficient method for the isolation of both crosslinks from bone of adult turkey by isocratic semi-preparative HPLC. Demineralized bone is hydrolysed in hydrochloric acid, 9 M. A first liquid extraction step in butanol allowed to eliminate less polar compounds. The aqueous phase was concentrated and separated by gel filtration on Biogel P2 and eluted by acetic acid solution (10%). Fractions containing pyridinoline were pooled, concentrated, and purified on a CF1 cellulose column. Pyd and Dpd crosslinks were then separated isocratically by HPLC on a C18 reversed phase column (Vydac 218 TP 1010, 250x10 mm) and eluted with HFBA as the ion-pairing agent. Retention times of Pyd and DPD were 23.6 and 28.7 min, respectively. Both crosslinks prepared by HPLC were then transformed as hydrochloride to cellulose phosphate and desalted on Sephadex G-10 columns. These two further steps yielded highly purified compounds (the purity was greater than 98% evaluated by aminoacid analysis). In conclusion, the efficiency of this method allows to obtain rapidly Pyd and Dpd without interfering compounds as proven by spectral studies (NMR and mass spectroscopy).     

High-performance liquid chromatographic analysis of corticosteroids

This review presents recent developments in high-performance liquid chromatographic (HPLC) analysis of corticosteroids for the determination of clinically important steroids in biological specimens. Various sample preparation techniques are described.     

Rapid method for the isolation of hydroxylysylpyridinoline and lysylpyridinoline from concentrated bone hydrolysates by liquid chromatographic techniques

A rapid method for the isolation of hydroxylysylpyridinoline and lysylridinoline from bone by liquid chromatographic methods is described. Decalcified bone is hydrolysed in 7 M hydrochloric acid. After evaporation of the acid, the high molecular mass dark coloured degradation products are removed by adsorption on non-polar adsorbents. The pyridinolines are separated from the majority of the amino acids by adsorption on cellulose. Separation of HP and LP is performed either by cation-exchange chromatography or by reversed-phase ion-pari chromatography. The pyridinoline containing fractions are desalted by size-exclusion chromatography. The progress of the hydrolytic cleavage of collagen and the optimal parameters for purification and separation were examined. As a result the existing method allows the isolation of high amounts of pyridinolines with low amounts of adsorbents and chemical within a short time.     

Determination of the stereochemical composition of the major metabolites of verapamil in dog urine with enantioselective liquid chromatographic techniques

The stereochemical composition of verapamil and seven of its basic-extractable metabolites, isolated from the urine of dogs administered oral racemic verapamil, was determined by HPLC, using an Ultron OVM (ovomucoid) column. One dog was given oral (R)-verapamil alone in order to discriminate the (R)- and (S)-enantiomers of the metabolites. Structure identification of the isolated verapamil metabolites was accomplished using a combination of HPLC-MS and FAB-MS-MS techniques. Six of the urinary verapamil metabolites, including verapamil, were predominantly of the (R)-configuration, whereas one of the metabolites was predominantly in the (S)-form. The remaining isolated metabolite was comprised of approximately equal amounts of the two forms.