Fluids, biological

The analysis of drugs and metabolites in biological fluids, particularly plasma or serum is one of the most demanding but one of the most common uses of HPLC. Blood, plasma or serum contain numerous endogenous compounds often present at concentrations much greater than 

Liquid-liquid or solid phase extraction are also very commonly carried out on plasma or serum, either directly or on a protein-free solution. A single solvent extraction of plasma may not provide a clean enough extract for trace analysis. An extra step whereby charged analytes are back extracted into an acidic or basic aqueous solution is one means of further cleaning up in the sample. In some methods the pH of the aqueous solution is then altered again and subsequent re-extraction takes place. One other feature of blood, plasma or urine is that only low volumes are available for analysis. 

The other biological fluid commonly assayed is mine. Unlike plasma or serum this contains only low concentrations of protein so protein removal is not necessary. The composition of urine varies widely and so does the volume passed by different individuals. Generally speaking large volumes are available for analysis. One of the major problems with urine is the large number of small organic compounds that are present. Liquid-liquid extraction (often preceded by conjugate hydrolysis) is often the sample preparation procedure used for urine analysis. 

There are several other biological samples less commonly analysed these include bile, sweat, saliva, faeces, lung and bone. Of these, saliva analysis has gained in popularity, chiefly because this fluid is easy to collect. Volumes are low, however. The concentration of analyte in saliva is often quite close to that in plasma (although the pH difference between saliva and plasma means that for some ionised compounds it is significantly different). From the viewpoint of sample preparation, saliva can 

Microfluidic devices have gained importance and utility for analyses of various molecules, including drugs and their metabolites. Vrouwe et al. [151] developed NCE for point-of-care testing of lithium in blood samples. The device consisted of a glass chip coupled with a conductivity detector. The authors tested this system for lithium analysis in five patients in the hospital. Furthermore, the authors reported that sodium, lithium, magnesium, and calcium were separated in 20 seconds. The authors claimed that the NCE system provided a convenient and rapid method for point-of-care testing of electrolytes in serum and whole blood. 

Zhuang et al. [152] derivatized glycan samples with 8-aminopyrene-l,3,6-trisulfonic acid to get a charge for electrophoresis and a fluorescent label for detection. They analyzed glycan derivatives in the blood of cancer patients 

The endogenous levels of amino acids in blood and urine have been measured using GC-MF (Schulman et al., 1975 Petty et al., 1976 Mee et al., 1977 Kingston et al., 1978 Irving et al, 1978, Finlayson, 1980) 

As a result of the ethical problems associated with the clinical use of radioisotopes, the use of amino acids labeled with stable isotopes also has made great inroads in clinical studies (Halliday and Rennie, 1982). Such studies have been able to define the level of ammo acid transamination and oral bioavailability in man (Irving et al., 1978, Haymond et al., 1980 Matthews et al, 1981a). In addition, the use of dual labels (e.g., [ N, C-leucine) also has enabled the simultaneous evaluation of ammo aad transamination and decarboxylation (Matthews et al, 1981b). 

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Foams are used industrially and are important in rubber preparations (foamed-latex) and in fire fighting. The foam floats as a continuous layer across the burning surface, so preventing the evolution of inflammable vapours. Foams are also used in gas absorption and in the separation of proteins from biological fluids. See anti-foaming agents. 

The third method is of limited application and is used only in special cases, The second is the most accurate and rapid method, and is of considerable technical importance. The chemical method (described below), although less accurate than the polarimetric method, is of great value for the estimation of sugars in biological fluids. In fact, for such purposes, it is often to be preferred to the polarimetric method owing to the probable presence of other substances having high optical rotations. 

This difference in behavior for acetic acid in pure water versus water buffered at pH = 7 0 has some important practical consequences Biochemists usually do not talk about acetic acid (or lactic acid or salicylic acid etc) They talk about acetate (and lac tate and salicylate) Why Its because biochemists are concerned with carboxylic acids as they exist in di lute aqueous solution at what is called biological pH Biological fluids are naturally buffered The pH of blood for example is maintained at 7 2 and at this pH carboxylic acids are almost entirely converted to their carboxylate anions... 

Validation Considerations. Mechanisms other then size exclusion maybe operative ia the removal of vimses from biological fluids. Thus vims removal must be vaUdated within the parameters set forth for the production process and usiag membrane material representative of the product line of the filter. 

Monobasic acids are determined by gas chromatographic analysis of the free acids dibasic acids usually are derivatized by one of several methods prior to chromatographing (176,177). Methyl esters are prepared by treatment of the sample with BF.—methanol, H2SO4—methanol, or tetramethylammonium hydroxide. Gas chromatographic analysis of silylation products also has been used extensively. Liquid chromatographic analysis of free acids or of derivatives also has been used (178). More sophisticated hplc methods have been developed recentiy to meet the needs for trace analyses ia the environment, ia biological fluids, and other sources (179,180). Mass spectral identification of both dibasic and monobasic acids usually is done on gas chromatographicaHy resolved derivatives. 

J. Chamberlain, Mnalysis of Drugs in Biological Fluids, CRC Press, Boca Raton, Fla., 1985. 

A review pubHshed ia 1984 (79) discusses some of the methods employed for the determination of phenytoia ia biological fluids, including thermal methods, spectrophotometry, luminescence techniques, polarography, immunoassay, and chromatographic methods. More recent and sophisticated approaches iaclude positive and negative ion mass spectrometry (80), combiaed gas chromatography—mass spectrometry (81), and ftir immunoassay (82). 

For more specific analysis, chromatographic methods have been developed. Using reverse-phase columns and uv detection, hplc methods have been appHed to the analysis of nicotinic acid and nicotinamide in biological fluids such as blood and urine and in foods such as coffee and meat. Derivatization techniques have also been employed to improve sensitivity (55). For example, the reaction of nicotinic amide with DCCI (AT-dicyclohexyl-0-methoxycoumarin-4-yl)methyl isourea to yield the fluorescent coumarin ester has been reported (56). After separation on a reversed-phase column, detection limits of 10 pmol for nicotinic acid have been reported (57). 

Ion Selective Electrodes Technique. Ion selective (ISE) methods, based on a direct potentiometric technique (7) (see Electroanalytical techniques), are routinely used in clinical chemistry to measure pH, sodium, potassium, carbon dioxide, calcium, lithium, and chloride levels in biological fluids. 

Sample Handling System. Venous or capillary blood, urine, and cerebrospinal fluid are specimens routinely used in medical diagnostic testing. Of these biological fluids, the use of venous blood is by far the most prevalent. Collection devices such as syringes and partial vacuum test tubes, eg, Vacutainer, are used to draw ten milliliters or less of venous blood. At collection time, the test tubes are carefully labeled for later identification. 

Until the early 1960s, laboratory iavestigators rehed on dialysis for the separation, concentration, and purification of a wide variety of biologic fluids. Examples iaclude removal of a buffer from a proteia solution or concentrating a polypeptide with hyperosmotic dialysate. Speciali2ed fixtures were sometimes employed alternatively, dialysis tubes, ie, cylinders of membrane about the si2e of a test tube and sealed at both ends, were simply suspended ia a dialysate bath. In recent years, dialysis as a laboratory operation has been replaced largely by ultrafiltration and diafiltration. 

The realization of sensitive bioanalytical methods for measuring dmg and metaboUte concentrations in plasma and other biological fluids (see Automatic INSTRUMENTATION BlosENSORs) and the development of biocompatible polymers that can be tailor made with a wide range of predictable physical properties (see Prosthetic and biomedical devices) have revolutionized the development of pharmaceuticals (qv). Such bioanalytical techniques permit the characterization of pharmacokinetics, ie, the fate of a dmg in the plasma and body as a function of time. The pharmacokinetics of a dmg encompass absorption from the physiological site, distribution to the various compartments of the body, metaboHsm (if any), and excretion from the body (ADME). Clearance is the rate of removal of a dmg from the body and is the sum of all rates of clearance including metaboHsm, elimination, and excretion. 

DIRECT POTENTIOMETRIC DETERMINATION OF SULFATE CONTENT IN BIOLOGICAL FLUIDS (URINE)... 

The separation of proteins and peptides mixtures is the objective of protein biochemisdy. Albumin (Mr 66 000) concentration in a biological fluid (seaim, urine or cerebrbrospinal fluid) is assayed as markers for a series disease, such as nephritic syndrome or chronic glomuleronephritis. In diabetic patients the progression of microalbuminuria is accompanied by an increase in urinary concentrations of human semm albumen. In normal the excretion of albumin is 20 (tg/ml, in pathology - 20-200 p.g/ml. 

One of the important problems in the diagnosis of different disease in their early stages is the determination of bio-active substances in biological fluids. We are currently interested in applying capillary electrophoresis (CE) as technique for the rapid and highly efficient separation of corticosteroids in semm and urine. Steroids can analyze by MEKC. 

We have developed the method of the determination of steroids in biological fluids (semm and urine) by MEKC with on-line concentration (sweeping) with detection limit for about 3 ng/ml (S/N=3). 

The pur pose of work is to develop the technique of separ ation of purine bases (caffeine, theophylline, theobromine) and the technique of detection of purine bases in biological fluid by TLC using micellar mobile phases containing of different surfactants. 

On the basis of the received data, simple, expressive procedures for the coulometric determination of the antioxidants mentioned above in biological fluids and pharmaceuticals have been proposed. 

Histamine is the biological amine, playing an important role in living systems, but it can also cause unnatural or toxic effects when it is consumed in lai ge amounts. It can occur with some diseases and with the intake of histamine-contaminated food, such as spoiled fish or fish products, and can lead to undesirable effects as headache, nausea, hypo- or hypertension, cai diac palpitations, and anaphylactic shock syndrome. So, there is a need to determine histamine in biological fluids and food. 

The pH scale is widely used in biological applications because hydrogen ion concentrations in biological fluids are very low, about 10 M or 0.0000001 M, a value more easily represented as pH 7. The pH of blood plasma, for example, is 7.4 or 0.00000004 M H. Certain disease conditions may lower the plasma pH level to 6.8 or less, a situation that may result in death. At pH 6.8, the H concentration is 0.00000016 M, four times greater than at pH 7.4. 

Most biotin-dependent carboxylations (Table 18.5) use bicarbonate as the carboxylating agent and transfer the carboxyl group to a substrate earbanion. Bicarbonate is plentiful in biological fluids, but it is a poor electrophile at carbon and must be activated for attack by the substrate earbanion. 

The aromatic portion of the molecules discussed in this chapter is frequently, if not always, an essential contributor to the intensity of their pharmacological action. It is, however, usually the aliphatic portion that determines the nature of that action. Thus it is a common observation in the practice Ilf medicinal chemistry that optimization of potency in these drug classes requires careful attention to the correct spatial orientation of the functional groups, their overall electronic densities, and the contribution that they make to the molecule s solubility in biological fluids. These factors are most conveniently adjusted by altering the substituents on the aromatic ring. 

Supercritical fluid extraction (SFE) and Solid Phase Extraction (SPE) are excellent alternatives to traditional extraction methods, with both being used independently for clean-up and/or analyte concentration prior to chromatographic analysis. While SFE has been demonstrated to be an excellent method for extracting organic compounds from solid matrices such as soil and food (36, 37), SPE has been mainly used for diluted liquid samples such as water, biological fluids and samples obtained after-liquid-liquid extraction on solid matrices (38, 39). The coupling of these two techniques (SPE-SFE) turns out to be an interesting method for the quantitative transfer... 

Immunoaffinity extraction combined on-line with LC in conjunction with MS (108 -110) or tandem MS (111, 112) has also been demonstrated for the determination of analytes in biological fluids. Obviously, such systems offer a very high... 

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