End-Point Method

This procedure is reliable when the reaction proceeds virtually to completion. If the substrate is only partly consumed, the equilibrium is displaced in favor of the products by increasing the concentration of reactant or by removing one of the products of the reaction. If it is not possible to achieve this, a standard curve must 

L-Lactate assay is achieved by a reversed reaction of d), and o-lactate assay with a dehydrogenase specific for o-enantiomer. Creatininase 

Substrate Enzyme Km (mol/1) Enzyme concen- tration (gcat/1) 

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Assays using equiUbrium (end point) methods are easy to do but the time requited to reach the end point must be considered. Substrate(s) to be measured reacts with co-enzyme or co-reactant (C) to produce products (P and Q) in an enzyme-catalyzed reaction. The greater the consumption of S, the more accurate the results. The consumption of S depends on the initial concentration of C relative to S and the equiUbrium constant of the reaction. A change in absorbance is usually monitored. Changes in pH and temperature may alter the equiUbrium constant but no serious errors are introduced unless the equihbrium constant is small. In order to complete an assay in a reasonable time, for example several minutes, the amount and therefore the cost of the enzyme and co-factor maybe relatively high. Sophisticated equipment is not requited, however. 

End-point methods are often not based on kinetic-ally optimum conditions. However, an end-point method is often the only convenient one available. In this case, the method should have been validated by showing that the catalysis of the substrate follows well defined kinetics, rate of reaction is proportional to enzyme concentration, blanks and interfering substances are corrected for, and that appropriate standards are available. 

The reverse titration of thiosulphate with iodine is depicted in Fig. 3.81 and is often called the "reversed dead-stop end-point method . 

One of the most important applications of the dead-stop end-point method is the Karl Fischer titration of water the titrant usually consists of I2 amd S02 with pyridine in methanol, which reacts with H20 as follows ... 

Cano A, Hemandez-Ruiz J, Garcia-Canovas F, Acosta M and Arnao MB. 1998. An end-point method for estimation of the total antioxidant activity in plant material. Phytochem Anal 9(4) 196-202. 

Lebel [224] has described an automated chelometric method for the determination of sulfate in seawater. This method utilises the potentiometric end-point method for back titration of excess barium against EDTA following precipitation of sulfate as barium sulfate. An amalgamated silver electrode was used in conjunction with a calomel reference electrode in an automatic titration assembly consisting of a 2.5 ml autoburette and a pH meter coupled to a recorder. Recovery of added sulfate was between 99 and 101%, and standard deviations of successive analyses were less than 0.5 of the mean. 

End-point Detection The end-point of the Karl Fischer titration may be determined quite easily by adopting the electrometric technique employing the dead-stop end-point method. When a small quantum of e.m.f. is applied across two platinum electrodes immersed in the reaction mixture, a current shall tend to flow till free iodine exists, to remove hydrogen and ultimately depolarize the cathode. A situation will soon arise when practically all the traces of iodine have reacted completely thereby setting the current to almost zero or veiy close to zero or attain the end-point. 

Hi) Amperometric titrations with twin-polarized microelectrodes (or Biamperometric Titrations or Dead-stop-end-point method). 

AMPEROMETRIC TITRATIONS WITH TWIN-POLARIZED MICROELECTRODES (BIAMPEROMETRIC TITRATIONS OR DEAD-STOP-END-POINT METHOD)... 

Dead-stop-end-point method was first introduced by Foulk and Bawden in 1926. Evidently, this particular technique is a modification of the classical amperometric titration. This technique is specifically applicable to only such systems where the phenomenon of oxidation-reduction exists both before as well as after the equivalence point has been duly accomplished. 

Part—III exclusively treats Electrochemical Methods invariably and extensively used in the analysis of pharmaceutical substances in the Official Compendia. Two important methods, namely potentiometric methods (Chapter 16) deal with various types of reference electrodes and indicator electrodes, automatic titrator besides typical examples of nitrazepam, allopurinol and clonidine hydrochloride. Amperometric methods (Chapter 17) comprise of titrations involving dropping-mercury electrode, rotating—platinum electrode and twin-polarized microelectrodes (i.e., dead-stop-end-point method). 

Amperometric Titrations with Twin-Polarized Microelectrodes (Biamperometric Titrations or Dead-Stop-End-Point Method)... 

End-point methods for the quantitation of substrates are not appropriate when the assay involves coupled reaction systems BECAUSE... 

Detn of N in NC by ferrous-titanous titrimetric method) 37)S.Sandi G.Flanquart, ChimAnal(Paris) 39, 20-4(1957) CA 51, 7943 (1957)(Detn of N in NC, NG, PETN, etc by titration with ferrous sulfate soln using the dead-stop end point method) 38)Y.Lacroix er al, MP 39, 459-68(1957) CA 52, 19688(1958)... 

Under these conditions, a study of the antioxidant potential of pure compounds could be carried out. Table 1 shows the values of antioxidant activity (expressed as TEAC) of different compounds of interest, determined by the on-hne method (HPLC-ARTS method) and compared with the values obtained by our conventional photometric end-point method. As can be observed, the two most important standard antioxidants, trolox and ascorbic acid, presented similar TEAC using either method. Thus, either can be used as reference to express antioxidant activity, except that trolox has the advantage because it can be used in both hydrophilic and lipophilic assays. The TEAC values of phenolic compounds were underestimated by approximately half when the HPLC-ARTS method was used as compared to the end-point method. This was due to the different reactivities of antioxidants with ARTS, and because, unfortunately, the time dependence of online scavenging activity determinations made it very difficult to obtain the total reaction for the slowest antioxidants, resulting in a partial estimation of this activity. Nevertheless, the HPLC-ARTS method provided important additional information in the form of correlation between the different peaks of a sample and their antioxidant activities. 

Table 1 Antioxidant activities of different compounds determined by the HPLC-ABTS and by the end-point method...

Determinations of antioxidant activity are widely used in phytochemistry, nutrition, food chemistry, clinical chemistry, as well as in human, animal, and plant physiology, etc. Methods adapted to HPLC have appeared only recently but can be expected to have multiple apphcations in the future. ARTS" is an excellent metastable chromogen for the detection and quantification of the HAA and LAA of biological samples. Thus, using a simple photometer (end-point method), a microplate reader (multisample titration method), or HPLC equipment, a broad range of possibilities are available for the characterization of diverse samples (animal- or plant-derived). Some apphcations of special interest could include ... 

The samples of products are incubated with Limulus amoebocyte lysate at 37°C. If endotoxins are present a solid gel forms, indicating the presence of endotoxins. The British Pharmacopoeia (2002) describes six separate methodologies for the test for endotoxin. These are (A) gel-clot limit test (B) gel-clot semi quantitative (C) turbidimetric kinetic method (D) chromogenic kinetic method (E) chro-mogenic end-point method and (F) turbidimetric end-point method. There are checks for interfering factors. Any validated method may be used, but the gel-clot method is the referee test in the case of dispute. Coloured products cannot be tested by... 

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