Spectrophotometry in clinical chemistry

S.L. Upstone, UltravioletWisible Light Absorption Spectrophotometry in Clinical Chemistry, Encyclopedia of Analytical Chemistry, John WUey, Chichester, 1699-1714 (2000). 

B4. Berman, E., An application of atomic absorption spectrophotometry in clinical chemistry Determination of copper in biological materials. Proc. Intern. Congr. Clin. Chem., 5th, Detroit, 1963. 

Upstone SL. Ultraviolet/visible light absorption spectrophotometry in clinical chemistry. In Meyers RA, ed. Encyclopedia of analytical chemistry Applications, theory, and instrumentation. New York John Wiley Sons, 2000 1699-713. 

J.W. Hall, A. Pollard, Near-infrared Spectrophotometry a New Dimension in Clinical Chemistry , Clin. Chem., 38, 1623-1631 (1992). 

The activity can be measured spectrophotometri-cally. An internationally agreed reference method has been developed for routine determination in clinical chemistry. 

Reference 9 gives a review of applications of atomic absorption spectrophotometry to biological samples. Tiiis technique is widely used for metal analysis in biological fluids and tissues, in environmental samples such as air and water, and in occupational health and safety areas. Routine applications of flame emission spectrometry to biological samples are generally limited to the alkali and alkaline earth metals. Ion-selective electrode measurements (Chapter 13) have largely replaced the flame emission measurements in the clinical chemistry laboratory. 

Bannister S.J., Chang Y., Stemson L.A., Repta AJ. Atomic absorption spectrophotometry of free circulating platinum species in plasma derived from cis-dichlorodiammi-neplatinum(II). Clinical Chemistry 1978 24 877-880. 

Lykkesfeld, J. (2001). Determination of Malonaldehyde as Dithiobarbituric Acid Adduct in Biological Samples by HPLC with Fluorescence Detection Comparison with Ultraviolet-Visible Spectrophotometry, Clinical Chemistry, Vol.47, pp.l725-... 

UV-visible spectrophotometry Calcium ions form a violet-colored complex with o-cresolphthalein. Its absorbance is proportional to the calcium concentration of the sample and can be measured at 546 nm. Protein-bound calcium is released by hydrochloric acid. The interference of magnesium ions is excluded by the addition of 8-hydroxyquinoline. The method is in widespread use on mechanized analysers for clinical chemistry. 

The iron released may be measiu ed using a variety of methods based on two main techniques atomic absorption spectrometry (AAS) (flame or furnace) and ultraviolet (UV)-visible spectrophotometry. Flame AAS provides the reference method for determination of plasma iron. Protein precipitation with TCA is followed by centrifugation and measurement of iron in the supernatant by the absorption at 248.3 nm in an air-acetylene flame. While atomic absorption methods are routinely used for urine iron measurements, the need to remove protein and any hemoglobin contamination restricts the use of this technique in routine clinical chemistry for plasma iron. Electrothermal atomization AAS methods are typically used for determination of iron in tissues although inductively coupled plasma (ICP) is becoming more widely available. 

Many clinical chemistry assays are based on UVAfIS spectrophotometry, often by reaction of the chemical of interest, snch as glucose, with an enzyme and dye to create a colored complex. A novel method for determining the age of dried bloodstains at a crime scene has been developed by researchers at the National Center for Forensic Science at the University of Central Horida using the Implen NanoPhotometer Pearl (Hanson and Ballantyne). The researchers discovered a previously unidentified hypsochromic shift (a blue shift to shorter wavelengths) in the Soret band of hemoglobin =412... 

Several different methods have been utilized for measuring iron in these biological samples. However, spectrophotometry is the most widely used because it does not require unusual equipment and is readily amenable to automation. Atomic absorption spectrometry is effectively used for tissue and urine analyses [33-35], but unreliable results are obtained with serum due to sensitivity limitations as well as matrix and hemoglobin interferences [35]. Other methods utilizing inductively coupled plasma emission spectroscopy [36], coulometry [37], proton induced X-ray emission [38], neutron activation analysis [39], radiative energy attenuation [40], and radiometry with Fe [41] have been described but, with the exception of coulometry, have not become standard procedures in the clinical chemistry laboratory, inasmuch as sophisticated and expensive instrumentation is required in some instances. However, some of them, e.g., neutron activation, may be the method of choice for definitive accurate analysis. 

The techniques of flow analysis can be applied to the pharmaceutical and clinical analysis. This entails employing classical detectors and chemical strategies. Solution chemistry and ultraviolet (UV)-visible (Vis) spectrophotometry tools are frequently used. The selection of the actual procedure is determined by the type of detector used, and may in general include use of spectrometric detectors, followed by electroanalytical techniques, and separation and biochemical procedures. Special attention is paid to new devices used in flow manifolds and to detectors such as chemiluminescence detectors (in which researchers have shown increasing interest). This article discusses these procedures in some detail. 

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