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doi: 10.1002/bmc.1130050303pmid: 1677825
Chromatographic techniques for the determination of trace amounts of neurotransmitters were reviewed. The two techniques found to be most useful were GC‐MS and the reversed‐phase mode of HPLC with an electrochemical or fluorescent detector. For structure determination or unequivocal peak identification, GC‐MS is the method of choice. In addition the limits of detection of GC‐MS were better than those obtained by HPLC. However for routine analyses, HPLC is now being used in studies of mental illness and other diseases. Good resolution, reproducibility and sensitivity can be obtained without the derivatisation steps required for GC‐MS, and catecholamines, serotonin, and their acidic metabolites can be concomitantly determined in one analysis.
Baig, S.; Halawa, I.; Qureshi, G. A.
doi: 10.1002/bmc.1130050304pmid: 1863804
In this study we report the levels of 3‐methoxy‐4‐hydroxyphenylglycol, 3,4‐dihydroxyphenylacetic acid, homovallinic acid, tryptophan, 5‐hydroxyindole‐3‐acetic acid and serotonin in lumbar cerebrospinal fluid (CSF) from patients with multiple sclerosis, cerebrovascular disease and muscular tension headache the latter, as healthy controls. The separation of these substances was performed on a reversed phase column by ion pair high performance liquid chromatography and detection was made by a glassy carbon electrode set at +900 mV vs Ag+/AgCI. The whole separation was achieved within 25 min. Concentrations of all substances (10‐1000 pmole/L) were linearly proportional to areas obtained. The system is sensitive, stable and reproducible. The significance of CSF levels of these metabolites from patient groups compared with healthy controls are discussed.
doi: 10.1002/bmc.1130050305pmid: 1863805
Chromatography has played a pivotal role in the advances made during the last 30 years in our knowledge of inborn errors of metabolism. This review discusses the application of some of these techniques to the analysis of organic acids and acylcarnitines. The separation of organic acids needed a comprehensive approach that would permit all of the many organic acids present in urine or other complex mixtures to be extracted, analysed and identified in a single run. This required analytical methods of great resolving power, wide linear range and universal detectors such as gas chromatography (GC), or GC coupled with mass spectrometry. Sample preparation was another problem that has been tackled by a variety of approaches. Organic solvents have been employed widely for the extraction of organic acids from physiological fluids. Unfortunately, recoveries of the different organic acids by this method are sometimes less than quantitative and variable depending on the compound. Other methods, such as the use of DEAE‐Sephadex columns, have the advantage of resulting in close to 100% recoveries, but are more tedious. Liquid partition chromatography on short silicic acid columns has also been recommended as a useful clean‐up step prior to GC, permitting both the identification and quantitation of organic acids in urine, plasma or amniotic fluid. Although many derivatization procedures have been used to prepare organic acids for gas chromatography, the most common is trimethylsilylation. Oxo acids are usually reacted with one of several commonly used reagents to form oximes. GC analysis of organic acids was initially done using packed columns with methylsilicone‐based, non‐polar stationary phases. In recent years most laboratories have switched to the commercially available, bonded‐phase capillary columns. Some authors have recommended the use of two columns of slightly differing polarities to improve the reliability of identification without mass spectrometry. Carnitine and acylcarnitines are substances whose measurement may yield useful diagnostic information about inborn errors and about the basic biochemical mechanisms of disease. While free carnitine is usually measured by a radioenzymatic assay, the separation of acylcarnitines requires paper chromatography or high performance liquid chromatography. The identification of various acylcarnitines following separation has been accomplished by mass spectrometric methods. Another technique relies on gas chromatographic identification of the acyl groups liberated following alkaline hydrolysis. Recently, liquid chromatography/mass spectrometry has also been utilized to separate and identify the various acylcarnitines present in urine and other biological samples.
doi: 10.1002/bmc.1130050306pmid: 1863806
Reversed phase ion pair chromatography is a highly selective separation technique for the determination of free porphyrin carboxylic acids from human materials. Isocratic and gradient elution methods can be used to analyse porphyrin isomers and to establish porphyrin profiles for the biochemical diagnosis of porphyrias. Ion pair high performance liquid chromatography led to the discovery of the atypical isomers II and IV of uroporphyrin and coproporphyrin in human urine. Advantages and limitations of the ion pair technique are discussed.
doi: 10.1002/bmc.1130050307pmid: 1863807
Many drugs are recemic and therefore much effort has to be devoted towards the stereoselective synthesis of the most effective or less harmful component of a racemic mixture. High performance liquid chromatography will play an important role in the clinical analysis of racemic drugs in anticipation of regulations that are currently being discussed and are expected to be enforced by the end of this decade. In this review a number of methods for chiral resolution are outlined. These include the formation of diastereoisomers and the use of chiral stationary phases or chiral mobile phase additives.
doi: 10.1002/bmc.1130050308pmid: 1863808
Samples of biomedical interest which have been analysed by field‐flow fractionation techniques are surveyed. The list begins with whole cells and microorganisms, going through viruses, nucleic acids, cell fragments and organelles, down to proteins and their aggregates. The principles of separation in the normal and steric mode of retention are illustrated, and instrumentation and techniques are described. The review concentrates mainly on the two systems of choice for biomedical applications: sedimentation and flow field‐flow fractionation.
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