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HPLC of small molecules — A practical approach
121
Optimal Use of the Clinical Laboratory 0. Zinder (Editor) Karger, Basel, 1986, 140 pp., Price S. Fr.89.00
The title of this slim 140-page book does not accurately reflect the content; the subtitle ‘5th International Meeting on Clinical Laboratory Management, Haifa 1985’ does, because this is a collection of papers presented at that meeting. The contents is not well balanced, being that there are eight papers on the economics of clinical laboratory utilization, four on the use of computers, three on chronobiology and timing of clinical laboratory testing and two on decentralized laboratory testing. As with many such collections of papers presented orally at meetings, most of the contributions do not provide useful critical overviews, reviews, or even annotations of current work, but provide details of the work of particular individuals or groups. This is amply demonstrated by perusal of the references; the paper of Albert et al has 13 self-citations of the 16 references and that of Hrushesky 9 of 10. Certain papers presented here have been given in more or less identical form elsewhere. The editing appears to have been done with a very light hand and many of the phrases used are either jargon ‘we first standardize the chemistries’ concentrations’ or much more suited to oral presentation ‘the seasonality of many physiologic functions is profoundly felt by those of us inhabiting latitudinal extremes’. Some of the most interesting aspects of such books are often found in the edited transcripts of the discussion sessions. Professor Zinder states, in his preface, that the subjects were discussed in depth, and at times debated heatedly and with vigour. Yet, dissapointingly, the short two-page and one-paragraph ‘General Discussion’ section, which is at the end of the book, simply alludes some of the problems of provision of biochemistry tests closer to the patient, a subject which has been dealt with in detail many times before. The details provided on the back cover state that this volume will be read with interest by all clinical laboratory professionals, and those involved in hospital administration; this is doubted. The contents are of relevance to the interests of some clinical biochemists, who would be better served by study of these subjects in the format of published refereed papers in learned journals. Hospital administrators would be unlikely to have the perspicacity to dissect the truly relevant gems from the detail concerning clinical chemistry. Every year new texts, journals and proceedings of conferences compete for the institutional, departmental and personal resources allocated for their purchase. This book would have a very low priority. Callum
G. Fraser
* * * * *
HPLC of small molecules -
A Practical Approach
C.K. Lim (Editor) IRL Press, Oxford, 1986, 350 pp., El7.00 soft cover, E27.00 hard cover
Almost any analysis requiring resolution and quantitation of complex mixtures can now be carried out by HPLC and workers are faced with a bewildering range of chromatographic conditions reported in diverse publications. This book provides the answer. After a brief but pertinent introduction to the basic equations used to define column performance, the entire book is dedicated to practical techniques, and, of particular value, provides detailed procedures for sample preparation. (How often does a publication detail the chromatography but lightly pass over problematical areas in the equally essential purification procedures?)
122
The first chapter describes the different modes of separation and introduces basic steps in sample preparation while the following ten chapters deal with analysis of specific groups of compounds. The compounds selected can be criticised for omissions or inclusions according to personal taste but the broad range of molecules included give insight into the analytical approach even if the assay is not of direct interest. For example, the chapter on bile acids arguably dwells too long on results of the assay in liver disease but the use of fluorimetric measurement of NADH generated by an immobilised enzyme as a post-column reaction has great potential for other molecules. Other chapters include sections on trouble-shooting the electrochemical detector, peak identification, column care and a discussion of the effect of end-capping of silanol groups. The practical approach to HPLC includes discussion of the effect of different column types on resolution, information which allows assays to be ‘finely tuned’ for individual needs and also imparts the theory in a painless manner! For example the resolution of carbohydrates, including sulphated, sialylated and phosphorylated oligosaccharides is compared on different columns and incorporated into a scheme for carbohydrate compositional analysis. The chapter on biogenic amines compares reverse phase and ion-pair reverse phase chromatography and the effect solvent changes have on resolution. Vitamins comprise such a diverse group of chemical structures that each vitamin is treated independently while lipids are broken into fatty acids, prostaglandins, triglycerides and phospholipids. Cholesteryl esters are included with fatty acids as this section details a transesterification reaction which produces fatty acid methyl esters although the detection is not as sensitive as other derivatives described for fatty acids. The value of HPLC for fractionation of triglycerides and phospholipids species is shown, which allows further analysis of the acyl groups present in these molecules, using the power of capillary column GC or HPLC-mass spectroscopy. The preparative role of HPLC is also demonstrated in the chapter on steroids which gives an interesting discussion of the limitations of peak identification and quantitation. For example, the increased specificity by use of a photodiode array detector at several wavelengths and problems with mass spectroscopy detection are highlighted. Sensitivity of detection can be overcome by using radioimmunoassay after HPLC fractionation, a technique which also reduces potential problems of cross-reaction in the immunoassay. The high standard of the text is maintained by the chapter on nucleotides, nucleosides and bases, with particularly useful discussion of the merits of the different detectors. The extraction procedures include sections on validation, stability and sources of error, all areas which are usually discovered the hard way! The book is completed by two excellent chapters which consider the extraction and analysis of porphyrins and bile pigments, describing in detail the precautions and difficulties of analysing these compounds. I have no doubt that this book will become a standard to be found beside each HPLC and used by beginner and expert developing a new assay. P.E. Ross
Optimal Use of the Clinical Laboratory 0. Zinder (Editor) Karger, Basel, 1986, 140 pp., Price S. Fr.89.00
The title of this slim 140-page book does not accurately reflect the content; the subtitle ‘5th International Meeting on Clinical Laboratory Management, Haifa 1985’ does, because this is a collection of papers presented at that meeting. The contents is not well balanced, being that there are eight papers on the economics of clinical laboratory utilization, four on the use of computers, three on chronobiology and timing of clinical laboratory testing and two on decentralized laboratory testing. As with many such collections of papers presented orally at meetings, most of the contributions do not provide useful critical overviews, reviews, or even annotations of current work, but provide details of the work of particular individuals or groups. This is amply demonstrated by perusal of the references; the paper of Albert et al has 13 self-citations of the 16 references and that of Hrushesky 9 of 10. Certain papers presented here have been given in more or less identical form elsewhere. The editing appears to have been done with a very light hand and many of the phrases used are either jargon ‘we first standardize the chemistries’ concentrations’ or much more suited to oral presentation ‘the seasonality of many physiologic functions is profoundly felt by those of us inhabiting latitudinal extremes’. Some of the most interesting aspects of such books are often found in the edited transcripts of the discussion sessions. Professor Zinder states, in his preface, that the subjects were discussed in depth, and at times debated heatedly and with vigour. Yet, dissapointingly, the short two-page and one-paragraph ‘General Discussion’ section, which is at the end of the book, simply alludes some of the problems of provision of biochemistry tests closer to the patient, a subject which has been dealt with in detail many times before. The details provided on the back cover state that this volume will be read with interest by all clinical laboratory professionals, and those involved in hospital administration; this is doubted. The contents are of relevance to the interests of some clinical biochemists, who would be better served by study of these subjects in the format of published refereed papers in learned journals. Hospital administrators would be unlikely to have the perspicacity to dissect the truly relevant gems from the detail concerning clinical chemistry. Every year new texts, journals and proceedings of conferences compete for the institutional, departmental and personal resources allocated for their purchase. This book would have a very low priority. Callum
G. Fraser
* * * * *
HPLC of small molecules -
A Practical Approach
C.K. Lim (Editor) IRL Press, Oxford, 1986, 350 pp., El7.00 soft cover, E27.00 hard cover
Almost any analysis requiring resolution and quantitation of complex mixtures can now be carried out by HPLC and workers are faced with a bewildering range of chromatographic conditions reported in diverse publications. This book provides the answer. After a brief but pertinent introduction to the basic equations used to define column performance, the entire book is dedicated to practical techniques, and, of particular value, provides detailed procedures for sample preparation. (How often does a publication detail the chromatography but lightly pass over problematical areas in the equally essential purification procedures?)
122
The first chapter describes the different modes of separation and introduces basic steps in sample preparation while the following ten chapters deal with analysis of specific groups of compounds. The compounds selected can be criticised for omissions or inclusions according to personal taste but the broad range of molecules included give insight into the analytical approach even if the assay is not of direct interest. For example, the chapter on bile acids arguably dwells too long on results of the assay in liver disease but the use of fluorimetric measurement of NADH generated by an immobilised enzyme as a post-column reaction has great potential for other molecules. Other chapters include sections on trouble-shooting the electrochemical detector, peak identification, column care and a discussion of the effect of end-capping of silanol groups. The practical approach to HPLC includes discussion of the effect of different column types on resolution, information which allows assays to be ‘finely tuned’ for individual needs and also imparts the theory in a painless manner! For example the resolution of carbohydrates, including sulphated, sialylated and phosphorylated oligosaccharides is compared on different columns and incorporated into a scheme for carbohydrate compositional analysis. The chapter on biogenic amines compares reverse phase and ion-pair reverse phase chromatography and the effect solvent changes have on resolution. Vitamins comprise such a diverse group of chemical structures that each vitamin is treated independently while lipids are broken into fatty acids, prostaglandins, triglycerides and phospholipids. Cholesteryl esters are included with fatty acids as this section details a transesterification reaction which produces fatty acid methyl esters although the detection is not as sensitive as other derivatives described for fatty acids. The value of HPLC for fractionation of triglycerides and phospholipids species is shown, which allows further analysis of the acyl groups present in these molecules, using the power of capillary column GC or HPLC-mass spectroscopy. The preparative role of HPLC is also demonstrated in the chapter on steroids which gives an interesting discussion of the limitations of peak identification and quantitation. For example, the increased specificity by use of a photodiode array detector at several wavelengths and problems with mass spectroscopy detection are highlighted. Sensitivity of detection can be overcome by using radioimmunoassay after HPLC fractionation, a technique which also reduces potential problems of cross-reaction in the immunoassay. The high standard of the text is maintained by the chapter on nucleotides, nucleosides and bases, with particularly useful discussion of the merits of the different detectors. The extraction procedures include sections on validation, stability and sources of error, all areas which are usually discovered the hard way! The book is completed by two excellent chapters which consider the extraction and analysis of porphyrins and bile pigments, describing in detail the precautions and difficulties of analysing these compounds. I have no doubt that this book will become a standard to be found beside each HPLC and used by beginner and expert developing a new assay. P.E. Ross