Conclusion Of Capillary Electrophoresis

In conclusion, CZE analysis of heavy metals is still under research and development. Although promising, the techniques that have been used still do not have adequate sensitivity for routine applications in the clinical analysis of biological samples. Depending on the CE technique used, an additional one to two orders of magnitude increase in sensitivity is still needed. Perhaps with the newer electrochemical techniques that are being developed or by using indirect fluorescence techniques...

Basic Principles Of Capillary Electrophoresis

Fundamentals of Electrophoresis Capillary electrophoresis (CE) is a special case of using an electrical field to separate the components of a mixture. Electrophoresis in a capillary is differentiated from other forms of electrophoresis in that it is carried out within the confines of a narrow tube. To understand the behavior of molecules under the influence of an electrical field inside a capillary it is essential to understand the phenomena that result from the geometry of a capillary. It...

References

S. (1994) Felbamate measured in serum by two methods HPLC and capillary electrophoresis method. Clin. Chem. 40, 1904-1908. 2. Friedberg, M. A. and Shihabi, Z. K. (1997) Ketoprofen analysis in serum by capillary electrophoresis. J. Chromatogr. B 695, 193-198. 3. Scholl, J. B. and DeZwaan, J. (1997) Micellar electrokinetic chromatography as a generalized alternative to high-performance liquid chromatography for purity determination of a class of investigational...

Cems Interfaces For Peptide And Protein Analysis

A number of elegant but different approaches have been reported in coupling off-line and interfacing on-line CE with MS. This subject area has been reviewed extensively (16,17,28-32,92) and is also discussed in some detail in Chapter 15. In this section we will briefly discuss coupling and interfacing strategies as they pertain to analysis of peptides and proteins. In Fig. 3. (opposite page) Nonaqueous-CE-MS (NACE-MS) analysis of gramicidin S and bacitracin. The separation buffer is 20 mM...

Lipoprotein And Apolipoprotein Analysis In Research And Clinical Settings By Capillary Electrophoresis

The matrices of the samples analyzed for lipoprotein and apolipoprotein determination are frequently very complex, e.g., a high total protein concentration, a high content of lipids, a high ionic strength, and so on. Thus, when developing methods it is necessary to focus not only on component separation but also on possible matrix effects. Thus, methods quantifying lipopro-teins and apolipoproteins in or isolated from biological specimens have to be sensitive, selective, reproducible, robust,...

Monitoring Glycemic Control In Diabetic Patients

Our research with Hb variants clearly indicated that the same cIEF methodology used for the analysis of structural Hb variants could also be used for the analysis of posttranslational or chemically modified Hb variants. Post-translational hemoglobins include oxidation products (8) like methemoglo-bins and Hb A3 (glutathione adduct), and glycation products like Hb A1c. Adding additional tests to an existing platform and reagent system is attractive because of the potential for decreasing the...

Info

Chol, cholesterol PL, phospholipid TG, triglyceride Prot, protein. aThe apolipoproteins in brackets are acquired after secretion. hand, plays an important role in reverse cholesterol transport. A gradient of reverse cholesterol transport is maintained, either by the liver taking up HDL or by transfer via cholesterol ester transferase protein (CETP) of HDL cholesterol ester to Apo B-containing particles, which are also subject to rapid uptake by the liver. Lipoprotein (a) (Lp a ), whose function...

Biomedical Background

Structure, Classes, and Metabolism of Lipoproteins Medieval physicians first observed lipoproteins (milky serum) in association with diabetes mellitus, nephrotic syndrome, and overindulgence of alcohol. In the early 1900s studies focused on the metabolism of plasma cholesterol and triglycerides. This approach quickly gave way to the study of the various lipoprotein classes after it was recognized that lipids and cholesterol were merely cargo and that apolipoproteins on the surface of...

Analysis Of Urinary Drugs And Metabolytes By Ce With Optical Detection

Assays with No or Minimal Sample Detection When using CE, urine specimens can often be injected directly into the separation column or may require only minimal pretreatment, such as dilution, centrifugation, or filtration. The data depicted in Fig. 1 represents MECC data of a urine from a patient with suspected salicylate intoxication. This sample tested markedly positive (> 1 mM) for salicylate employing the modified spectrophotometric method of Trinder (21). Using a fluorescence...

Kargain In 1992

P. (1994) Incidence of heavy metal toxicity in a clinical population (Abstract). 4th International conference on Therapeutic Drug Monitoring and Toxicology, Vienna, Austria. 2. Ash, O. K. and Komaromy-Hitler, G. L. (1997) Analysis of clinical specimens using inductively coupled plasma mass spectrometry, in Handbook of Analytical Therapeutic Drug Monitoring and Toxicology. (Wong, S. H. Y. and Sunshine, I., eds.), CRC Press, Boca Raton, FL pp. 109-125. 3. Nuttall, K. L. and Gordon W. H....

Chiral Ce Of Urinary Drugs And Metabolites Assays For Enantiomer Screening Of Misused Abused And Banned Substances

Drug Separation Enantiomers

CE has proven to provide a simple, inexpensive, and effective approach for the separation of enantiomers after the addition of a chiral selector to the buffer. The separation by CE of the enantiomers of several drugs of interest in toxicology and doping control has been investigated. By using various cyclodextrins it was possible to effect the chiral differentiation of the optical isomers of racemethorphan and racemorphan (45), amphetamines, cathine, cathinone, cocaine, and others (68), as well...

Urine Proteins

Urine Capillary Electrophoresis

Currently, no CZE methods have been approved for the separation of urine proteins by the FDA. Various authors, however, have developed techniques that have used CZE to separate urine proteins. These reports may well help in the eventual introduction of such methods. A limiting factor appears to be salt concentration of urine as well as degraded protein fragments, which can present a very confusing picture. Fig. 2. C Immunosubtraction patterns of an IgM kappa monoclonal gammopathy. Fig. 2. C...

Introduction Analysis Of Urinary Drugs In Clinical And Forensic Toxicology

Clinical and forensic toxicology is concerned with the detection, identification, and measurement of toxic compounds and their metabolites in human body fluids and tissues. Most often the toxic compounds are drugs taken either accidentally or intentionally in quantities sufficient to cause an adverse reaction or death. Analysis and identification of a possible drug or drug combinations, toxicological drug screening and confirmation should encompass as many different classes of drugs as...

Applications For The Clinical Laboratory

The applications discussed in this chapter start with methylmalonic acid, since this is the one with which the authors have the most experience. An assay for urine methylmalonic acid has been operating on a routine basis in the first author's clinical laboratory since 1994. Several other applications share many characteristics with methylmalonic acid, and are discussed next, including succinic acid, oxalic and citric acids, and the simple short-chain organic acids. Most of these assays use...

Capillary Isotachophoresis cITP

In this technique the sample plug is introduced between two different buffers. One of these, the leading electrolyte, has the highest mobility in the separation. The second, trailing electrolyte, has a mobility lower than anything else does. The sign of the charge on the analytes and the buffers must be the same. When the voltage is applied the ions in the sample form discrete zones that are not separated into peaks one zone is adjacent to the next. The concentration of the analyte within a...

Introduction

Dna Migration Ogston Reptation Model

The development of methods for the amplification and detection of specific regions of the DNA molecule using the polymerase chain reaction PCR has resulted in rapid and dramatic advances in biochemical analysis 1 . With the advent of the PCR it is now possible to easily produce analytically significant amounts of a specified DNA product. A typical PCR reaction can produce microgram quantities of target DNA, allowing rapid and efficient screening of genetic defects, cancer susceptibility, and...

Methods Of Separation

Capillary Electrophoresis Amino Acids

Micellar Electrokinetic Capillary Chromatography MEKC Pure mixtures have been used in the great majority of the studies involving separation of amino acids. However, low-peak capacity of the methods used limited the use of CE in separating more complex mixtures. Using micellar electrokinetic capillary chromatography MEKC , separation of neutral along with charged components was possible based on their partitioning between the aqueous phase and a pseudo-stationary phase 46 . MEKC was shown...

Serum and Urine Paraprotein Capillary Electrophoresis

Most requests for serum and urine electrophoresis occur because of the reversal of albumin globulin ratios in the general chemistry profile or the presence of protein in the urine. The suspicion of possible myelomatosis by the physician also alerts him her to rule out the presence of a monoclonal gammopathy in the serum or light chain in the urine. Because of the increased sensitivity afforded by the newer electrophoresis systems, paraproteins are found more often. Their appearance in a number...

Congenital Adrenal Hyperplasia

Hydroxyprogesterone

Congenital adrenal hyperplasia CAH also known as the adrenogenital syndrome can be considered as a family of inborn error of steroidogensis see Fig. 1 . All CAH variants are inherited as autosomal recessive traits. Each member of this family is characterized by a specific enzyme deficiency that impacts cortisol production by the adrenal cortex, and if severe enough can lead to sexual ambiguity in both males and females. The enzymes usually affected are 21-hydroxylase types I and II , beta...

Capillary Electrophoresis Advantage And Disadvantage

CZE offers an alternative way of evaluating serum samples for paraproteinemias. The two automated instruments available and approved by the FDA offer a time saving and labor saving approach to these assays. There are a number of other instruments that have greater flexibility with regard to various parameters such as temperature, time of separation, changes in buffer, and capillary size. However, these instruments are considered research platforms and as such can offer a great number of...

Quirino J.p. Terabe S. Anal. Chem. 1998 70 1893-1901.

M. and Matthew, N. 1988 in Physiology, 3rd ed. Mosby Year Book, St. Louis, MO. 2. Rumsy, G. and Avery, C. J. 1998 Genotype-phenotype analysis in late onset 21-hydroxylase deficiency in comparison to the classical forms. Clin. Endocrinol. 48, 707. 3. Ko, T. M. and Kao, C. H. 1999 Congenital hyperplasia. Molecular characterization. J. Reprod. Med. 43, 379-386. 4. Bose, H. S., Baldwin, S., and Miller, W. L. 1998 Incorrect folding of steroidogenic acute regulatory protein StAR in...

Historical Perspective

Beckman Pace 2100 Fullerton

In 1937 Tiselius 1 reported on a methodology that was to become one of the most novel ways by which proteins could be separated from one another. His apparatus, although large in size relative to current systems, established the serum protein profile still used today. The apparatus, a U-tube, separated the serum proteins in the liquid phase into five fractions, albumin, a-1, a-2, P, and y globulins. Through the years chemical pathologists and clinical chemists have refined both the methodology...

Physiological Effects Of Glucocortioids

Of the naturally occurring steroids only cortisol, corticosterone, cortisone, and 11-dehydrocorticosterone have appreciable glucocorticoid activity. Cortisol, which is found in the highest concentration, accounts for most of this activity. About 75 of plasma cortisol is bound to cortisol binding globulin CBG, an alpha globulin , 15 is bound to plasma albumin, and 10 is unbound free , representing the physiologically active portion. CBG also has a high binding affinity for progesterone,...

Comparison Of Cze With Hrage

Electrophoresis Pattern Paraproteinemia

Chapter 4 discussed the classification of serum protein dyscrasias using CZE and HRAGE. This chapter covers paraproteinemia and the comparison of CZE vs HRAGE. CZE appears to offer an alternative to HRAGE for the detection of paraproteinemias in serum. When we used the Paragon CZE 2000, we obtained a 100 concordance between CZE and HRAGE in the detection of monoclonal gammopathies 3 . However this was only when the paraproteins were within the detection limits of HRAGE IgG 0.5 g L, IgA 0.75 g...

Electrophoresis Methods

Oligoclonal Band Serum Electrophoresys

Multiple methods such as agarose gel 4 , polyacrylamide 8 , isoelectric focusing 8,9 , and two-dimensional 10 electrophoresis have been described to measure the protein profiles associated with the various diseases of the CNS. All of these methods are labor intensive and require an approx 40-fold preconcentration of the CSF in order for the protein bands to be visualized with the stains used e.g., Coomassie brilliant blue . Because of problems associated with preconcentration, a method using...

Adrenal Gland

Enzyme Deficiencies Adrenal Gland

The adrenal glands are paired structures situated above the kidneys that are approx 2-3 cm wide and 6 cm long and weigh approx 5 g. The glands consists of a yellow, outer cortex that constitutes approx 80 of the adrenal gland and a gray, inner medulla 1 . The adrenal cortex consists of three distinct layers or zones of cells. The outermost layer, the zona glomerulosa, is the site of aldosterone synthesis, the principal mineralocorticoid produced by the human adrenal cortex, and corticosterone...

Cebased Immunoassay

CE evolved from the investigation of column electrophoresis by Hjerten 1 and Castimpoolas 2 . Later, Martin and Everaets developed a capillary isotachophoresis system that became commercially available in the mid-1970s. Subsequently, Jorgen and coworkers demonstrated the analytical potential of CE using 75- m fused silica capillaries 3-5 leading to the adaptation and modification of CE for specific applications. The main advantage of CE when used as a separation technique is the small...

Au

Biofocus 3000

Capillary electropherogram of a mixture of apolipoprotein A-I and A-II. Capillary fused silica 50 cm x 50 m running conditions 20 kV buffer BioRad LLV evaluation buffer detection UV at X 220 nm equipment Bio-Rad BioFocus 3000 capillary electrophoresis system. Fig. 4. Capillary electropherogram of a mixture of apolipoprotein A-I and A-II. Capillary fused silica 50 cm x 50 m running conditions 20 kV buffer BioRad LLV evaluation buffer detection UV at X 220 nm equipment Bio-Rad BioFocus...

Cze Instrumentation

CZE instrumentation offers a semi-automated Bio-Focus 2000 or completely automated Paragon CZE 2000 system. These instruments elimi- Fig. 4. Dysproteinemias as identified by CE. Fig. 4. Dysproteinemias as identified by CE. nate many of the errors mentioned in the previous section that are associated with conventional HRAGE methods. Labor costs are also decreased with CZE since there is no time spent in specimen application, fixing, staining, washing, and drying of the gels. Both methods,...

Immunofixation Electrophoresis Vs Immunosubtraction

Immunofixation electrophoresis IFE is in all probability the gold standard for the detection of monoclonal gammopathies in the clinical laboratory and to identify the type as well as the heavy- and light-chain class. It has also replaced the classical immunoelectrophoresis because of its simplicity of interpretation. In addition, it is also an extremely sensitive procedure because of the combination of two proteins, antigen and antibody, which increases the amount of dye uptake almost 10-fold....