Other Fluorometric Methods

Sensitive fluorometric assays for double-stranded (native) DNA in tissue extracts are based on the noncovalent interaction of intercalating dyes (Fig. 1.7 such as ethi-dium bromide, 4,6 -diamidino-2-phenylindole (DAPI), or 2-(2-[4-hydroxyphenyl]-6-benzimidazolyl)-6- (1-methyl-4-piperazyl)benzimidazol trihydrochloride (Hoechst 33258), with double-stranded DNA in neutral, aqueous solutions. 

When free in aqueous solution, these dyes exhibit limited fluorescence, but upon binding DNA, their fluorescence increases markedly. Ethidium binds to RNA and DNA while DAPI and Hoechst 33258 selectively interact with DNA. Hoechst 33258 at a final concentration of 1 pg/mL in 0.05 M phosphate, pH 7.4, with 2 M NaCl has been used to quantitate as little as 10-ng double-stranded DNA.18 Because these dye-binding methods involve intercalation between the bases of double-stranded DNA they are not useful for the quantitation of single-stranded DNA. 

Single-stranded DNA containing deoxyguanosine residues can be quantitated in the presence of terbium(III). Ten micromolar Tb3+ in a pH 6 cacodylate buffer shows no detectable fluorescence, with excitation at 290 nm and emission measured at 488 nm. In the presence of single-stranded DNA, Tb3+ coordinates with deoxy-guanosine-5-phosphate nucleotides, and a linear dependence of fluorescence on concentration has been observed over the 1-10-pg/mL range of thermally denatured rat liver DNA.19 

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A fluorometric method was developed for determination of atmospheric H2O2 simultaneously with other species present at ppbv or lower levels, avoiding chromatographic separation. H2O2 is selectively collected by diffusion through a Nafion membrane, and is carried by a water stream into a reactor where it oxidizes thiamine hydrochloride (117) to a fluorescent ionic form of thiochrome (118), catalyzed by bovine hematin (75b) in alkaline solution, as shown in equation 40. The end solution containing 118 is passed through... 

This is one vitamin that most laboratories can measure. There are a number of old-fashioned approaches that use 2,6-dichloroindophenol in a titrimetric method such as AOAC 985.33. This works well in some systems but can give rise to false positive results if there are other reducing substances present. It will not detect dehydroascorbic acid (DHA) and so it may well underestimate the actual vitamin C activity if a product contains a significant level of DHA. However, even with for these shortcomings, it is often used as a quick and rough method. In the AOAC there is also a fluorometric method (AOAC 984.26) where ascorbic acid is oxidised to DHA and this is reacted with o-phenylenediamine to give a fluorometric compound which can be detected. This is a robust method that has general applicability. 

Determination of oxidized amino acids in urine is usually performed by isotope dilution gas chromatography-mass spectrometry (L9). DOPA is estimated by HPLC separation of acid protein hydrolysates with fluorescence detection (excitation 280 nm, emission at 320 nm) (A15). Other methods are based on borate-hydrochloric acid difference spectroscopy (this method suffers interference from tyrosine and tryptophan) (W2), derivatization of DOPA with nitrite and subsequent coulometric determination (W3), and fluorometric detection after derivatization with ethylenediamine (A15). 3-Hydroxylysine is quantitated by HPLC with 9-fluorenylmethyl chloroformate precolumn derivatization (M25) of amino acids obtained by gas-phase hydrolysis of proteins (F21). Other general methods to detect amino acid damage are mass spectometry methods applied to protein hydrolysates, such as tandem mass spectrometry (F6). 

Although most of the elements have been determined by XRF (21), some other methods were used. The fluorometric method for selenium uses diaminonaphthalene (32). The colorimetric method for molybdenum uses potassium thiocyanate (33). The uranium analyses were done by delayed neutron activation analysis (34). For the XRF analyses of the oil and water, a blank value implies that there were no x-rays above background for that element. Two elements conspicuously missing from Table IV are cadmium and mercury. Preliminary analyses for these two elements have not yielded reproducible results. Further work is needed before we can make definitive statements about cadmium and mercury. 

Other problems of differential diagnosis in which chemical measurements are of crucial value all involve moderately sensitive, or at most, highly sensitive methods, with the exception of the acute obstetric problems mentioned previously. In many of these problems a battery of simple and well-established colorimetric and fluorometric methods not involving chromatography could reasonably be used. Thus cortisol could be measured by a variant of the Silber and Busch method (see B17) or an isotope-displacement method (M7, N6) 17-ketosteroids and 17-keto-... 

Wide use of photometric scanning for high-precision measurements of steroids will depend on the introduction of a commercially available instrument with satisfactory specifications. This is probable in the next six months. Meanwhile it should be noted that many fluorometric methods, whether by direct photometric scanning or by more conventional techniques, are strictly competitive with all other existing methods in the highly sensitive range. Their future successful extension to the ultrasensitive range is not unlikely. 

Macro quantities of selenium can be determined gravimetrically after reduction to the elemental form by various reagents such as tin (II) chloride, potassium iodide, or ascorbic acid (I). Ooba described a technique whereby the element is precipitated from perchloric acid solution with hydrazine (2). Selenium may be titrated with standard solutions of sodium thiosulfate, iodide, and ferrous, chromous, or trivalent titanium salts after oxidation to Se(VI) (I). Photometric and fluorometric methods based on formation of the piaselenol with diaminobenzidine or 2,3-diaminonaphthalene has been used for the determination of selenium (I, 3,4,5). Interfering elements such as As, Co, Cr, Cu, Fe, Hg, and Ni, are masked with EDTA or other chelating agents. 

As has been previously said, 2,3-butanodione (diacetyl) is an important aroma of alcoholic beverages, it has not been studied and measured extensively in the past because of analytical difficulties in the quantitation caused by its highly volatile nature, chemical instability, and interference of other compounds. Colorimetric methods to measure diacetyl have been widely used in the past. These methods involve steam distillation to isolate diacetyl from the matrix. However, distillation has the disadvantage of incomplete isolation of diacetyl from other closely related compounds that will result in an overestimation of its concentration. A fluorometric method was developed to improve upon the lengthy distillation methods that involve derivatization. Although acetaldehyde and its acetal can be determined by direct injection GC-FID in spirit drinks (EU reference method for spirits), most chromatographic methods for minor aldehydes implicate also derivatization. While a very sensitive and accurate method based on SMPE without derivatization and MS detection has been developed, it requires the use of... 

Comparison studies of MS/MS with other NBS methods demonstrated its equivalence and versatility for detection of PKU. It was shown that it was more clinically sensitive and accurate in the detection of PKU than other methods such as fluorometry [3]. A reduction of the false result rates without the addition of several new separate assays, that is, tyrosine (Tyr) analysis, demonstrated the advantages of using an MS/MS approach. To better understand metabolic disease, its detection, consider Figure 13.1, in which screening for PKU is illustrated. From the illustration, the advantages in using a multiple metabolite approach of MS/MS versus single analyte for fluorometric assays should be clear. 

The detection limit depends on the method used. Huorometric detection is much more sensitive than its spectrophotometric counterpart. The latter method is suitable for analysis of large quantities of thiamine in pharmaceutical preparations and foods the detection limit is approximately 2 ng or 6 pmol as thiamine hydrochloride. On the other hand, the detection limit by the fluorometric method is <17 pg or 0.05 pmol as thiamine hydrocholoride. The lowest detection limit so far reported for thiamine is 5 fmol, using fluorescence (21,22). Huorescence detection is therefore more suitable for the analysis of thiamine in biological materials such as cells, blood, and urine. 

In a study by spectrofluorimetry, Duggan, Bowman, Brodie and Uden-friend found that in phosphate buffer pH 7, cyanocobalamin had an activation maximum at 275 m t and a fluorescence maximum at 305 m/bc and that a practical sensitivity of 0-003 jug of cyanocobalamin per ml was obtainable. Since fluorometric methods usually have high sensitivity and specificity, these considerations may enable a valuable method to be developed for the determination of cyanocobalamin, in particular since it may overcome interference by analogues and other cobalamins. 

The recognition of their structure permits the determination of vitamins by the tools of analytical chemistry, but while such methods are widely used in industrial production, the minute quantities in body fluids and tissues limit the purely chemical approach to a few members of this group present in relatively high concentration, e.g., vitamin C (K5). Microchemical methods are in use for the determination of thiamine, riboflavin, and some of the fat-soluble vitamins, based on the most sensitive colorimetric and, in particular, fluorometric techniques. Vitamin D, on the other hand, is determined by animal assay. 

Acid dye method for the analysis of thiamin, 18A, 73 electrophoretic separation and fluorometric determination of thiamin and its phosphate esters, 18A, 91 catalytic polarography in the study of the reactions of thiamin and thiamin derivatives, 18A, 93 preparation of thiamin derivatives and analogs, 18A, 141 preparation of the mono- and pyrophosphate esters of 2-methyl-4-amino-5-hydroxymethylpyrimidine for thiamin biosynthesis, 18A, 162 formation of the pyrophosphate ester of 2-methyl-4-amino-5-hydroxymethylpyrimidine by enzymes from brewers yeast in thiamin biosynthesis, 18A, 203 resolution, reconstitution, and other methods for the study of binding of thiamin pyrophos-... 

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