Analytical methods for the quantitative determination of selective serotonin reuptake inhibitors for therapeutic drug monitoring purposes in patients

Five selective serotonin reuptake inhibitors (SSRIs) have been introduced recently: citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline. Although no therapeutic window has been defined for SSRIs, in contrast to tricyclic antidepressants, analytical methods for therapeutic drug monitoring of SSRIs are useful in several instances. SSRIs differ widely in their chemical structure and in their metabolism. The fact that some of them have N-demethylated metabolites, which are also SSRIs, requires that methods be available which allow therapeutic drug monitoring of the parent compounds and of these active metabolites. Most procedures are based on prepurification of the SSRIs by liquid-liquid extraction before they are submitted to separation by chromatographic procedures (high-performance liquid chromatography, gas chromatography, thin layer chromatography) and detection by various detectors (UV, fluorescence, electrochemical detector, nitrogen-phosphorus detector, mass spectrometry). This literature review shows that most methods allow quantitative determination of SSRIs in plasma, in the lower ng/ml range, and that they are, therefore, suitable for therapeutic drug monitoring purposes of this category of drugs.     

High-performance liquid chromatographic assays for free and phosphorylated derivatives of the creatine analogues beta-guanidopropionic acid and 1-carboxy-methyl-2-iminoimidazolidine (cyclocreatine).

Creatine and phosphocreatine are substrates for creatine kinase which is a key enzyme involved in energy transfer within the cell. Analogues of creatine have been fed to animals to determine the role this enzyme plays in energy metabolism, but progress in interpretation has been hampered by the lack of quantitative techniques to determine tissue content of these compounds. We describe the separation and quantitation of substituted guanidino compounds and their phosphorylated forms by high-performance liquid chromatography. First, a cation-exchange column is used to assay free creatine and its unphosphorylated analogues, and then phosphocreatine and its phosphorylated analogues as well as adenylate content (AMP, ADP, ATP) are assayed on an anion-exchange column. These methods have proven successful in measuring the chemical contents of these compounds in neutralized perchloric acid extracts of mammalian skeletal muscles. The sensitivity of this method ranges from 50 to 200 pmol, which is adequate to provide information from tissue extracts of 5- to 10-mg samples.     

High-performance liquid chromatographic separation of fluoroquinolone enantiomers: a review

Fluoroquinolones are antibacterial agents widely used clinically. In recent years, there has been an important development of new derivatives, and more than 7000 analogues have been described today. Different fluoroquinolones (FQ) have one or two chiral centers in their chemical structure and are available as racemates, diastereoisomers, or pure enantiomers. The clinical and pharmaceutical uses of these compounds need effective analytical procedures for quality control and pharmacodynamic and pharmacokinetic studies. This review article focuses on the high-performance liquid chromatographic separation of fluoroquinolone stereoisomers by the use of derivatization methods and ligand exchange (LE) or chiral liquid chromatography.     

Advances in techniques for ecdysteroid analysis

Summary

Recent improvements in chromatographic methods for ecdysteroids are reviewed. Minor improvements in liquid chromatography have increased the separation and range of compounds which can be accomodated, some useful techniques have been introduced in thin layer chromatography, and with new radioactivity scanners and direct mass spectrometry from TLC, this technique retains an important place among the tools of the ecdysonist. Supercritical fluid chromatography has been suggested as a rapid and more sensitive alternative to liquid chromatography. In mass spectrometry, fast atom bombardment has proved most useful, though coupling HPLC through thermospray and ptasmaspray methods has been attempted, but as yet, these techniques lack sensitivity. New solid phase immunoassay methods using colorimetric rather than radioactivity measurements promise to give a better means of analysis of small amounts of ecdysteroids, capable of use in the presence of radiolabeled compounds. A solid phase separation of C-20, 22 diol ecdysteroids with immobilized phenylboronic acid has been reported.     

Chromatographic and electrophoretic methods for pharmaceutically active compounds in Rhododendron dauricum

In this review, chemical constituents present in Rhododendron dauricum L. were briefly surveyed, and the methods of pretreatment of this plant prior to analysis were also summarized. The analysis methods reported for determining pharmaceutically active compounds in R. dauricum L. include gas chromatography with mass spectrscopy, thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). In addition, both advantages and disadvantages of the above methods were mentioned.     

Methods for isolation, purification and structural elucidation of bioactive secondary metabolites from marine invertebrates

In the past few decades, marine natural products bioprospecting has yielded a considerable number of drug candidates. Two marine natural products have recently been admitted as new drugs: Prialt (also known as ziconotide) as a potent analgesic for severe chronic pain and Yondelis (known also as trabectedin or E-743) as antitumor agent for the treatment of advanced soft tissue sarcoma. In this protocol, methods for bioactivity-guided isolation, purification and identification of secondary metabolites from marine invertebrates such as sponges, tunicates, soft corals and crinoids are discussed. To achieve this goal, solvent extraction of usually freeze-dried sample of marine organisms is performed. Next, the extract obtained is fractionated by liquid-liquid partitioning followed by various chromatographic separation techniques including thin layer chromatography, vacuum liquid chromatography, column chromatography (CC) and preparative high-performance reversed-phase liquid chromatography. Isolation of bioactive secondary metabolites is usually monitored by bioactivity assays, e.g., antioxidant (2,2-diphenyl-1-picryl hydrazyl) and cytotoxicity (microculture tetrazolium) activities that ultimately yield the active principles. Special care should be taken when performing isolation procedures adapted to the physical and chemical characteristics of the compounds isolated, particularly their lipo- or hydrophilic characters. Examples of isolation of compounds of different polarities from extracts of various marine invertebrates will be presented in this protocol. Structure elucidation is achieved using recent spectroscopic techniques, especially 2D NMR and mass spectrometry analysis.     

Thiomorpholine transformation by the fungus Bjerkandera adusta

A screening of lignin-degrading basidial fungi that can grow in the presence of thiomorpholine derivatives (the mixture of 1,4-perhydrothiazines) has been performed. Strain Bjerkandera adusta VKM F-3477 was shown to have the maximal rate of growth in the presence of these compounds, and its capacity for thiomorpholine degradation was studied. The methods of quantitative analysis of thiomorpholine and its degradation products on the basis of thin layer chromatography and high-performance liquid chromatography were developed. It was shown that the B. adusta strain did not utilize thiomorpholine as a carbon source but transformed it into thiomorpholine sulfoxide that accumulated in the medium. Mn peroxidase produced by B. adusta in the course of thiomorpholine transformation is not directly involved in its oxidation.     

Ion-exchange high-performance liquid chromatography analysis of oligodeoxyribonucleotide phosphorothioates.

Oligodeoxynucleotide-containing phosphorothioate backbones have been used to regulate viral as well as cellular gene expression. The studies carried out in tissue culture have shown promising results on the use of oligonucleotide phosphorothioates as antiviral agents and, at present, study is underway to develop these oligonucleotide analogues as chemotherapeutic agents. To analyze and purify oligonucleotide analogues, high-performance liquid chromatography using weak anion exchange column has been described. The separation of oligonucleotide phosphorothioate is found to be length dependent.     

Biophysical properties and finger print of Boswellia Sp. Burseraceae

For the first time a finger print analysis via high-performance thin layer chromatography (HPTLC) of Boswellia Sp. Burseraceae was accomplished. A preliminary investigation of the Boswellia Sp. Burseraceae displayed the presence of chemical constituents that could be involved in the production of innovative pharmaceuticals for an array of antiviral, anticancer, and antibacterial uses. Moreover, the finger print analysis would deem useful for establishing HPTLC standardization for natural and herbal photochemical constituents.     

The use of high-performance liquid chromatography and diode array detection in fungal chemotaxonomy based on profiles of secondary metabolites

FRISVAD, J. C, 1989. The use of high-performance liquid chromatography and diode array detection in fungal chemotaxonomy based on profiles of secondary metabolites. Fungal chemotaxonomy (that part dealing with secondary metabolites) has often been based on thin layer chromatography (TLC) and visual or UV inspection of separated spots, before and after different chemical treatments. The identity of a small proportion of the spots can be suggested based on known internal and external standards. In most chemotaxonomical studies it is impossible to isolate, purify and identify all secondary metabolites produced, due to restraints of time and resources. High performance liquid chromatography (HPLC) of fungal extracts may have some advantages over TLC, but the problems mentioned above remain. These problems have been approached by using an alkylphenone retention time index in a reversed phase HPLC system combined with the use of a diode array UV-VIS detector. High performance thin layer chromatography is used for further confirmation of identity of the secondary metabolites. A particular advantage of this method is that the number of biosynthetic families or groups ('chemosyndromes') can be detected, as biosynthetically related metabolites usually have the same chromophores and UV-VIS spectra. Results obtained from Penicillium, Aspergillus and Fusanum species have shown that each species produces 5 to 15 different biosynthetic families of secondaiy metabolites, indicating that good chromatography data may be sufficient to identify species in the three genera. The use of the technique is exemplified by data on Aspergillus and Talaromyces species.     

Design, synthesis, biological evaluation and QSAR studies of novel bisepipodophyllotoxins as cytotoxic agents

Two moieties of epipodophyllotoxin have been linked at C4-position to provide novel bisepipodophyllotoxin analogues. These have been evaluated for their anticancer potential and DNA-topoisomerase II poisoning activity. Most of these analogues have exhibited promising in vitro anticancer activity against different human tumour cell lines and interestingly 4(')-O-methylated analogues have shown increased cytotoxic activity. Similarly, the DNA-topo II poisoning activity tested for these compounds has not only exhibited the DNA cleavage potential comparable to etoposide, but for some compounds this cleavage potential is superior to etoposide. Further, an interesting structure-activity relationship of these epipodophyllotoxin dimers have been generated on the basis of GI(50) values. The equations indicated that GI(50) activity is strongly dependent on structural and thermodynamic properties. These QSAR results are discussed in conjunction with conformational analysis from molecular modelling studies. QSAR models developed in these studies will be helpful in the future to design novel potent bispodophyllotoxin analogues by minor structural modifications.     

Chromatographic methods for the determination of toxicants in faeces

Modern chromatographic techniques and their application in the determination of toxic compounds in faeces are reviewed. Faecal analysis may be of importance in toxicokinetic studies of xenobiotics in order to determine factors such as metabolism, body burden and major routes of elimination. Compounds of interest include various food constituents, drugs and occupational or environmental factors. Further, various mutagenic or carcinogenic compounds which are excreted by faeces have been indicated to represent risk factors for colorectal cancer. In this context, the chromatographic determination of the endogenously generated fecapentaenes and bile acids, both postulated etiological factors in colorectal carcinogenesis, is reviewed. For fecapentaene determination, several high-performance liquid chromatographic (HPLC) methods are available; however, the applicability of some of these methods is limited owing to insufficient separation of various isomeric forms or discrimination between fecapentaenes and their precursors. For the determination of bile acids in faeces, many chromatographic procedures have been reported, and the characteristics of the most relevant methods are compared and discussed. It is concluded that separation by gas chromatography (GC) in combination with mass spectrometry provides the highest selectivity and sensitivity. A relatively rapid alternative analysis for the determination of total and aqueous faecal bile acids is proposed. Further, methods for the determination of polycyclic aromatic hydrocarbons (PAHs) are reviewed. Although the use of radiolabelled PAHs in animal studies has many advantages, it cannot be applied for human biological monitoring and HPLC and GC provide sensitive alternatives. An HPLC method for the determination of non-metabolized PAHs in faeces is described.     

Arsenic mononucleotides. Separation by high-performance liquid chromatography and identification with myokinase and adenylate deaminase

The spontaneous formation of arsenic mononucleotides has been detected in mixtures of arsenate and inosine or adenosine or its deoxy analogues. These compounds have been separated by high-performance liquid chromatography and identified by their behavior in the presence of myokinase and adenylate deaminase. The nucleoside 5'-arsenates are formed preferentially to the 2'- and 3'-arsenate analogues. All arsenic nucleotides detected showed similar kinetic and equilibrium constants of formation: about 8 X 10(-4) M-1 S-1 and 2 X 10(-3) M-1, respectively. These values are several orders of magnitude greater than those of their phosphoric analogues. The adenosine 5'-arsenate was able to substitute for 5'AMP in the reaction of myokinase and adenylate deaminase. The substitutions of the 2'- or 3'-hydrogen for hydroxyl groups in the ribose moiety of this compound slightly affected its suitability as substrate for myokinase but had drastic effect in the case of adenylate deaminase. The half-life of the arsenic nucleotides, at pH 7.0 and 25 degrees C, ranged from 30 to 45 min. The lability of these compounds is increased during catalysis with myokinase. Results on the reaction mechanism of myokinase with adenosine 5'-arsenate indicate that the mixed-anhydride analogue to ADP, adenosine 5'-(arsenate phosphate), is not detected either because it is not formed in the reaction with this enzyme or because it is rapidly hydrolyzed.     

Amino-terminal sequence analysis of arachin

Amino-terminal sequence analysis of arachin, a peanut protein, using a modified Edman method has yielded the sequence of the first 60 residues. The PTH-amino acids have been identified on the basis of melting point, thin layer chromatography and UV spectroscopic data. For this purpose standard PTH-amino acids were prepared. In addition, two new solvent systems for thin layer chromatography of PTH-amino acids are reported.     

Purification of Immunosuppressive Factors Extracted From Bovine Spleen (Lymphoid Chalone)

A low molecular weight immunosuppressive factor FA which is able to reduce the blastic transformation capacity of lymphoid cells from treated mice has been characterized. It was prepared from a bovine spleen acetone powder and found to be associated partly with high molecular weight carriers in the form of an active complex characterized previously as part of a ‘lymphoid chalone’ fraction. FA may be obtained by selective ultrafiltration of F followed by P-2 Biogel chromatography of the ultrafiltrate. Thymidine, deoxyinosine and deoxycytidine have been identified as the major constituents of FA by mass spectrometry, ultraviolet absorption data and thin layer chromatography. However, none of these nucleotides has the biological activity of FA.     

Purification of immunosuppressive factors extracted from bovine spleen (lymphoid chalone).

A low molecular weight immunosuppressive factor FA which is able to reduce the blastic transformation capacity of lymphoid cells from treated mice has been characterized. It was prepared from a bovine spleen acetone powder and found to be associated partly with high molecular weight carriers in the form of an active complex characterized previously as part of a 'lymphoid chalone' fraction. FA may be obtained by selective ultrafiltration of F followed by P-2 Biogel chromatography of the ultrafiltrate. Thymidine, deoxyinosine and deoxycytidine have been identified as the major constituents of FA by mass spectrometry, ultraviolet absorption data and thin layer chromatography. However, none of these nucleotides has the biological activity of FA.     

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.     

Advances of infrared spectroscopy in natural product research

Natural product's properties are related to certain classes of compounds such as alkaloids, flavonoids, essential oils and others. Traditionally, separation techniques including thin layer chromatography (TLC), liquid chromatography (LC), gas chromatography (GC) and capillary electrophoresis (CE) even hyphenated to mass spectrometry (MS) were used for the elucidation, qualitative and quantitative analysis of individual compounds.In food industry, spectroscopic investigations using infrared radiation have been used to monitor and evaluate the composition and quality already since the early sixties. During the last four decades near-infrared spectroscopy (NIR; 800–2500 nm; 12,500–4000 cm⁻¹) has become one of the most attractive and used methods for analysis for the following reasons: it represents a non-invasive analytical tool allowing a fast and simultaneous qualitative and quantitative characterization of natural products and their constituents. Additionally, the development of custom-made hand-held instruments enables in-field measurement for determining the optimum harvest time.Attenuated total reflection (ATR) and Fourier transform infrared (FTIR) spectroscopic imaging are suitable not only for the differentiation of different plant species, but also to distinct various ingredients within a plant. FTIR spectroscopic microscopy enables molecular imaging of complex botanical samples and therefore the detection and characterization of the molecular components of biological tissue.In the present contribution, the principle, technique and methodology of the different infrared spectroscopic methods are described followed by a discussion of quantitative and qualitative application possibilities in the field of natural product analysis.     

Chemotaxonomy of water beetles based on their pygidial gland constituents

The mainly aromatic pygidial gland constituents of 45 water beetle species (Hydradephaga; Dytiscidae, Haliplidae) have been investigated. Chemical data were obtained by mass spectrometry, thin layer and gas chromatography and UV-spectrometry. Investigations on Agabus melanarius showed these gland substances were species specific and constant. Seasonal quantitative variations are probably due to variations in population structure and overwintering bionomics. The phylogenetic tree based on the distribution of pygidial gland constituents shows that taxa fall into three chemical groups (sesquiterpenes, aromatic compounds, hydroxycarboxylic acids). This grouping is generally in accordance with other taxonomic conceptions. Other chemical differences correspond to the morphology of the larva and pupa (Copelatus, Hydroporus melanarius). The position of several chemically diverging taxa (Haliplidae, Laccophilinae, Hydaticus) should be clarified by using further techniques such as morphology or considering the biogenesis of the water beetle compounds.