Dichloroindophenol Vitamin C

Dichloroindophenol Vitamin C

2,6 Dichlorophenolindophenol

If DCPIP is used in the assay, the complex II enzyme-catalyzed reduction of DCPIP is measured by following the decrease in absorbance due to the oxidation of DCPIP at 600nm (extinction coefficient for DCPIP 19.1mM−1cm−1).

From: Methods in Cell Biology , 2020

Ascorbic Acid: Properties, Determination and Uses

S.K. Chang , ... Z.A.M. Daud , in Encyclopedia of Food and Health, 2016

Oxidation–reduction methods

2,6-Dichloroindophenol (DCIP) titration is an established method to determine l-AA content. DCIP works on the principle of l-AA reduction to a colorless solution from the deep blue color of the oxidized dye. Subsequently, l-AA is oxidized to l-DHAA and any excess dye is pink in the acidic solution, forming a visual end point for the titration. The end point can be determined visually (518   nm). There are a few drawbacks to this method, the most important being titration is limited to quantitation of l-AA only. l-DHAA cannot be measured, unless it is first reduced to l-AA. The titration is also unable to differentiate between l-AA and isoascorbic acid, meaning this method cannot be used with processed and cured meats containing isoascorbic acid. DCIP titration is suitable for fresh juices and multivitamin supplements that do not contain significant quantities of copper or iron. However, highly colored extracts from fruits and vegetables, for example, can mask color changes at the end point. In this respect, solid-phase extraction (SPE) can extend DCIP titration to highly colored samples such as multivitamins, soft drinks, fruits, and vegetables, because cleanup removes copper, iron, sulfite, and other interfering reducing substances, such as cysteine and glutathione. The method can be adapted for l-DHAA by reducing it to l-AA with cysteine before cleanup. This relatively simple approach increases the sensitivity of DCIP, and inclusion of SPE would decrease limitations of the established method.

AOAC Official Method 967.21, Ascorbic Acid in Vitamin Preparations and Juices, 2,6-Dichloroindophenol Titrimetric Method, AOAC Official Methods of Analysis 45.1.14 (AOAC Method 967.21) has been recommended for the analysis of l-AA in beverages and juices for the purpose of nutrition labeling. The AOAC Official Method 985.33, Vitamin C ((Reduced AA) in Ready-to-Feed Milk-Based Infant Formula) (Chapter 50.1.09), is also based on the DCIP titration. The method differs from Method 967.21 at the extraction stage of the method. AOAC International has updated the change of method for vitamin C determination in infant formula and adult/pediatric nutritional formula (AOAC SMPR 2012.012) in its newest edition (19th edn. 2012).

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Rare-Earth Element Biochemistry: Methanol Dehydrogenases and Lanthanide Biology

Jing Huang , ... Ludmila Chistoserdova , in Methods in Enzymology, 2021

3.5.1 Quantitative assay

Alcohol dehydrogenase activity was measured by monitoring the phenazine methosulfate (PMS)-mediated reduction of 2,6-dichlorophenolindophenol (DCPIP; ɛ₆₀₀  =   21.0   mM^{−   1}  cm^{−   1}). Initially, all assays were carried out at pH 9.0 in accordance with the classic assay (Anthony & Zatman, 1967), the standard reaction mixture containing: 100   mM Tris-HCl buffer pH 9.0, 45   mM NH₄Cl, 1   mM PMS, 150   μM DCPIP, 10   mM methanol and 10–20   μL of crude cell extract (20–35   mg   mL^{−   1} protein) or 5–15   μL of pure protein preparation (0.5–5   mg   mL^{−   1} protein). Assays were performed at room temperature (approximately 26   °C), in a total volume of 0.8   mL in plastic cuvettes (1   cm path length). One unit (U) of specific enzyme activity was defined as 1   μM DCPIP reduced per minute (determined at 600   nm) and was expressed as U per milligram of protein. While this assay is straightforward in cases of the purified enzymes, in crude extracts it measures cumulative activities of multiple enzymes, both Ln^{3   +}-dependent and non-Ln^{3   +}-dependent, if multiple enzymes are active.

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Mitochondria, 3rd Edition

Ann E. Frazier , ... Robert W. Taylor , in Methods in Cell Biology, 2020

4.2.2 Definition

Complex II activity can be measured as succinate:ubiquinone₁ oxidoreductase, either by monitoring the reduction of ubiquinone₁ as performed in Melbourne, or linked to the artificial electron acceptor 2,6-dichlorophenolindophenol (DCPIP), as measured in Newcastle, which may improve sensitivity (Fig. 2). To measure complex II activity, the absorbance is monitored at 280   nm to detect reduction of ubiquinone₁ (extinction coefficient for ubiquinone₁ 12   mM^{−   1}  cm^{−   1}). If DCPIP is used in the assay, the complex II enzyme-catalyzed reduction of DCPIP is measured by following the decrease in absorbance due to the oxidation of DCPIP at 600   nm (extinction coefficient for DCPIP 19.1   mM^{−   1}  cm^{−   1}).

Reaction conditions

25   mM potassium phosphate buffer, 5   mM MgCl₂, pH 7.2

20   mM sodium succinate

2   mM KCN

2   μg/mL antimycin A

2   μg/mL rotenone in ethanol

65   μM ubiquinone₁

(±   50   μm DCPIP)

Sample: Mitochondrial extract diluted in hypotonic buffer (Medium E, Table 1) and frozen in liquid nitrogen or dry ice/ethanol slurry and thawed three times.

Procedure

1.

Make up the assay reagent in a 1-mL cuvette, or if using multiple cuvettes, make up an assay reagent sufficient for 10   ×   1-mL cuvettes consisting of:

1   mL 250   mM potassium phosphate, 50   mM MgCl₂, pH 7.2

200   μL 1   M sodium succinate

20   μL 1   M KCN

20   μL 1   mg/mL antimycin A

20   μL 1   mg/mL rotenone

(±   100   μL 5   mM DCPIP)

2.

To each cuvette, add 136   μL (for a 1-mL reaction) or 63   μL (for a 0.5-mL reaction) assay reagent, then adjust volume to either 1   mL or 0.5   mL, allowing for addition of sample volume.

3.

Equilibrate cuvettes at 30   °C for 5–10   min, then add mitochondrial sample (10–50   μg protein) and incubate for an additional 10   min at 30   °C.

4.

Record the baseline rate (absorbance at 280   nm) for 3   min.

5.

Start the reaction by adding 1   μL/mL of 65-mM ubiquinone₁ and monitoring absorbance at 280   nm for 3–5   min. Rates measured using a blank lacking ubiquinone₁ should be insignificant.

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CITRUS FRUITS | Limes

R.E. Berry , in Encyclopedia of Food Sciences and Nutrition (Second Edition), 2003

Storage

Because of their extremely high acidity and low pH, limes are more stable than many other citrus fruits. However, problems may be encountered at certain temperatures and humidities with growth of acid-tolerant surface fungi. Control has been accomplished using biological growth regulators. In Persian limes, 2,4-dichlorophenolindophenol (24D) and gibberellic acid (GA) have been successful in reducing the growth of 'Penicillium'-type molds, yellowing, mottling and discoloration of the rinds during storage. Such problems have not been solved through the use of controlled-atmosphere storage, but can be greatly controlled through the use of optimum storage conditions for fresh fruit, i.e., 10   °C and 90–95% relative humidity where a storage life of up to 4 weeks or longer has been reported without any notable injury, pitting, or discoloration of fruit.

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FOOD AND NUTRITIONAL ANALYSIS | Fruits and Fruit Products*

R.J. Pither , in Encyclopedia of Analytical Science (Second Edition), 2005

Vitamin C

There are two standard (AOAC) procedures for vitamin C (ascorbic acid) determination in fruits and fruit products. The sample preparation is common to both and involves maceration/dilution of the sample in a stabilizing solution such as 5% metaphosphoric acid or trichloroacetic acid followed by filtration. The ascorbic acid can then be determined by titration with 2,6-dichloroindophenol, where the ascorbic acid reduces the redox indicator dye to a colorless solution, or by fluorimetric detection, in which the ascorbic acid is oxidized to dehydroascorbic acid, which then reacts with o-phenylenediamine to produce a fluorophore. The latter method has the advantages that it is suitable for colored solutions and can also be used to measure levels of naturally occurring dehydroascorbic acid as well as ascorbic acid. Many other methods are available for determination of vitamin C, with the use of LC techniques currently the subject of much interest. Reversed-phase LC techniques can be used to determine both dehydroascorbic acid, ascorbic acid and their isomers.

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ASCORBIC ACID | Properties and Determination

M.A. Kall , in Encyclopedia of Food Sciences and Nutrition (Second Edition), 2003

Determination

The classic chemistry associated with spectroscopic methods can be divided into two general categories: methods using redox indicators and methods involving formation of chromofor or flourofor derivatives. The principal reactions for some classical methods of direct determination of vitamin C are briefly reviewed, but HPLC methods will be discussed in detail.

Redox Reactions

2,2'-Dipyridyl

This method is based on the reduction of Fe(III) to Fe(II) by AA. Fe(II) forms a complex with 2,2'-dipyridyl that can be quantified colorimetrically. Other chromogen formation Fe(II) complexing agents exist, e.g., ferrozine and 2,4,6-tripyridyl-S-triazine.

2,6-Dichloroindophenol

This method is based on reduction of 2,6-dichloroindophenol by AA. In acidic solution, 2,6-dichloroindophenol has an absorption maximum at 518   nm and, when reduced by AA, this chromophor disappears. The specificity of this method is limited owing to the presence of naturally occurring reductants of 2,6-dichloroindophenol in fruits and vegetables. This method will typically determine 5–10% higher amounts of AA in fruit samples than a HPLC method.

Derivatization Reactions

2,4-Dinitrophyl hydrazine

Ascorbic acid has to be oxidized to DHAA prior to the derivatization. The reaction between DHAA and 2,4-dinitrophenyl hydrazine results in the formation of the bis-2,4-dinitrophenylhydrazone derivative, which can be measured at 520   nm. The specificity of the method is limited, owing to unspecific reactions of 2,4-dinitrophenyl hydrazine with 5- and 6-carbon, sugar-like, nonascorbic compounds.

o-Phenyldiamine

Ascorbic acid has to be oxidized to DHAA prior to the derivatization. The reaction between DHAA and o-phenyldiamine (OPD) results in the formation of an intensive fluorophore: 3(1,2-dihydroxyethyl)furo[3,4-b]quinooxaline-1-one (DFQ) (see Figure 2). The fluorophore can be measured by the emission wavelength at 430   nm when excited at 350   nm. This reaction can be applied to direct measurement or to a chromatographic method. Some interference should be expected from naturally occurring fluorescing compounds if the method is used for direct measurement.

Figure 2. Reaction of DHAA with o-phenyldiamin (OPD) under formation of 3(1,2-dihydroxyethyl)furo[3,4-b]quinooxaline-1-one (DFQ).

HPLC Determination of Vitamin C

For common foodstuff analysis and control analysis, the amount of vitamin C usually exceeds 1   mg per 100   g of sample, and in this case, a HPLC system coupled with ultraviolet (UV) detection provides adequate sensitivity. In foodstuffs with amounts less than 1   mg per 100   g and in physiological samples, where the concentration often is below 1   μg ml⁻¹, it may be necessary either to use an electrochemical detector or to transform AA into a fluorescent compound in order to increase the sensitivity by fluorometric measurement. In addition, the electrochemical and fluorescent detection are more specific than the UV detection.

In principle, three different strategies for determination of vitamin C by HPLC exist:

1.

reduction of DHAA to AA followed by measurement of total AA;

2.

oxidation of AA to DHAA followed by measurement of total DHAA; and

3.

simultaneous determination of AA and DHAA, by pre- or postcolumn derivatization of DHAA, perhaps with additional postcolumn oxidation of AA.

As mentioned previously, extraction with MPA provides the most efficient protection of AA during extraction and subsequent analysis. However, MPA may cause serious analytical interactions with silica-based column materials. Ion-pair reversed-phase chromatography with C18 column material or pseudonormal-phase chromatography with an aminopropyl column material provide separation of AA and IAA and potential coeluting compounds with high efficiency and selectivity, but the use of MPA in the extraction buffer may affect the robustness of the chromatographic performance. The interactions may result in incomplete separation of the four compounds and cause a variation in retention time of the compound peaks. Furthermore, the use of EDTA in extraction buffers may result in a time-consuming analysis owing to a long retention time of the ligand. The interference problems with the silica-based polymer column materials caused by injection of MPA may be solved by using a polystyrene divinyl benzene (PLRP-S) polymer column material. An effluent based on 0.2   M phosphate buffer applied to the PLRP-S column performs adequate separation of AA and IAA. The PLRP-S column is compatible with injection of MPA in high concentrations in sample extracts and provides a very stable system for separation of the compounds without baseline- and retention drift. However, the efficiency and selectivity of the PLRP-S column are low with regard to AA and coeluting compounds and particularly DHAA and DHIAA, and the column should be used in combination with electrochemical or fluorescence detection only (see Figure 5).

Determination of vitamin C as total ascorbic acid

Dehydroascorbic acid is reduced to AA at neutral pH in the presence of thiol-containing reducing agents such as cysteine, homocysteine, 2-mercaptoethanol, and DTT. During incubation of the sample extract with a reductant, DHAA should be transformed quantitatively into AA. Addition of a reducing agent to the sample extract will prolong the stability of vitamin C in the sample extract during the extraction procedure as well as the storage period in the autosampler during HPLC analysis. Ascorbic acid is protected against oxidation, and since DHAA is reduced to AA, DHAA is protected from hydrolyses to 2,3-diketogulonic acid. Opposite AA and DHAA, thiol-containing reductants have both a low stability and a low reducing capacity in acidic environments. Sample preparation is often carried out by MPA extraction at low pH followed by adjustment of the pH to neutral, and incubation with a reductant. Subsequently pH has to be reduced, to ensure the stability of the AA. This procedure is more laborious, and as a result of the degradation of reductants at a low pH, the reducing sample environment, and thus stabilization of the AA in extracts, is maintained for less than 24   h. It should be noted that a similar stability of AA and DHAA without addition of a reductant can be obtained by optimizing the extraction buffer, regarding pH and temperature, and the concentrations of MPA, and chelator.

Recently, tris[2-carboxyethyl]phosphine (TCEP) has been described as a promising reductant of DHAA in physiological samples. The major advantage of this compound is a high stability and a high reductive capacity at a low pH, in contrast to the commonly used reductants. It has been shown that the stabilization of AA in sample extracts can be maintained for at least 96   h and that TCEP may not cause any interference with the chromatographic system (see Figure 3).

Figure 3. Chromatographic separation of AA and IAA on a C18 column with electrochemical detection. From Lykkesfeldt J (2000) Determination of ascorbic acid and dehydroascorbic acid in biological samples by high-performance liquid chromatography using subtraction method: Reliable reduction with Tris[2-carboxyethyl]phosphine hydrochloride. Analytical Biochemistry 282: 89–93 with permission.

Determination of vitamin C as total dehydroascorbic acid

This principle is based on oxidation and derivatization prior to HPLC analysis. The quinoxaline derivatives are more hydrophobic than AA and DHAA, and it is possible to obtain a reversed-phase HPLC method with a high efficiency, selectivity, and baseline separation of the two quinoxaline derivatives corresponding to AA/DHAA and IAA/DHIAA. In order to obtain adequate retention times, an organic modifier has to be added to the mobile phase in concentrations that will eliminate the interaction between injected MPA and the column material, as described previously. Preparative oxidation of AA to DHAA during extraction may be accomplished by addition of iodine or heavy metal ions at a low pH or by addition of ascorbate oxidase at neutral pH. Dehydroascorbic acid is much less stable than AA, and therefore the derivatization should be carried out directly after the oxidation. The stability of the quinoxaline derivatives, however, is also limited. A stability of 8–12   h is described for the quinoxaline derivatives in sample extracts. Further, the preparation of the quinoxaline derivative during extraction requires additional labor compared with the postcolumn derivatization.

In spite of the high selectivity, lack of interaction with MPA, and high sensitivity and specificity, the method is not recommended for purposes involving assay of many samples because of the low stability of the quinoxaline derivatives in the extracts (see Figure 4).

Figure 4. Chromatographic separation of DFQ and IDFQ on a C18 column with fluorometric detection. From Speek AJ, Schrijver J, and Schreurs WHP (1984) Fluorometric determination of total vitamin C and total isovitamin C in foodstuffs and beverages by high-performance liquid chromatography with precolumn deravitization. Journal of Agricultural and Food Chemistry 32: 352–355 with permission.

Simultaneous determination of ascorbic acid and dehydroascorbic acid

In general, simultaneous determination of AA and DHAA requires more equipment than the other two principles, but it may require less instrumentation. Under the right conditions, oxidation of AA to DHAA is an instantaneous reaction in contrast to reduction of DHAA to AA, which, even under optimal conditions, is a relative slow reaction. In order to determine AA and DHAA simultaneously, it is therefore necessary to perform online, postcolumn oxidation of AA to DHAA and subsequently measure both compounds as DHAA. The oxidation may be performed by addition of a solution containing Cu(II), Hg(II), bromine, or iodine to the effluent after the HPLC column. Alternatively, the effluent can be led through an electrochemical cell or through a solid-state oxidation column packed with active charcoal. In a second postcolumn step, DHAA is mixed with a solution of OPD to form the fluorophore derivative, DFQ (see Figure 2). Another possibility is to measure AA directly and only use the second postcolumn step to measure the original DHAA (see Figures 5 and 6).

Figure 5. Simultaneous determination of AA, IAA, DHAA, and DHIAA on a PLRP-S column coupled to a postcolumn oxidation and postcolumn derivatization with fluorometric detection. From Vanderslice JT and Higgs DJ (1991) Vitamin C content of foods: sample variability. American Journal of Clinical Nutrition 51: 1323S–1327S with permission.

Figure 6. Separation of DHAA and DHIAA on a C18 column followed by postcolumn derivatization and fluorometric detection. From Kall MA and Andersen C (1999) Improved method for simultaneous determination of ascorbic acid and dehydroascorbic acid, isoascorbic acid and dehydroisoascorbic acid in food and biological samples. Journal of Chromatography B 730: 101–111, with permission.

In contrast to the subtraction method, the simultaneous determination procedure provides data on AA as well as DHAA from a sample in the same analytical run. The procedure needs no reduction, oxidation, or derivatization prior to the HPLC analysis. However, DHAA spontaneously hydrolyzes to 2,3-diketogulonic acid at pHs as low as 3.2–3.5, and it may be difficult to preserve DHAA in the extracts for 24   h. Furthermore, the concentration of DHAA in most foodstuffs and physiological samples is low compared with AA, and even small degree of oxidation of AA during extraction or in autosampler vials will result in a pronounced increase in the DHAA concentration. Consequently, this analytical principle requires careful handling of samples during extraction and storage.

Ascorbic acid has a number of chemical properties responsible for the biological reactions that characterize vitamin C. Usually, these characteristics are positively associated with the benefits provided by this vitamin. Paradoxically, these properties provide difficulties in the analysis of ascorbic acid in foods and physiological samples.

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Amino Acids, Peptides and Proteins

William T. Self , in Comprehensive Natural Products II, 2010

5.05.3.4.3 Purine hydroxylase of Clostridium purinolyticum

Upon purification of the XDH from C. purinolyticum, a separate ⁷⁵Se-labeled peak appeared eluting from a DEAE sepharose column. This second peak also appeared to contain a flavin based on UV–visible spectrum. This peak did not use xanthine as a substrate for the reduction of artificial electron acceptors (2,6 dichloroindophenol, DCIP), and based on this altered specificity this fraction was further studied. Subsequent purification and analysis showed the enzyme complex consisted of four subunits, and contained molybdenum, iron selenium, and FAD. The most unique property of this enzyme lies in its substrate specificity. Purine, hypoxanthine (6-OH purine), and 2-OH purine were all found to serve as reductants in the presence of DCIP, yet xanthine was not a substrate at any concentration tested. The enzyme was named purine hydroxylase ³¹⁵ to differentiate it from similar enzymes that use xanthine as a substrate. To date, this is the only enzyme in the molybdenum hydroxylase family (including aldehyde oxidoreductases) that does not hydroxylate the 8-position of the purine ring. This unique substrate specificity, coupled with the studies of Andreesen on purine fermentation pathways, suggests that xanthine is the key intermediate that is broken down in a selenium-dependent purine fermentation pathway. ^181,183

In an effort to better define the role of selenium in catalysis, EPR studies were carried out on PH. ³¹⁶ It was determined that the FeS centers and molybdenum cofactor could be reduced using NADPH through the flavin site. Removal of selenium by cyanide completely blocked electron transfer to FeS centers and FAD with hypoxanthine as a reductant (forward reaction), but the Mo(V) signal was present. However, cyanide inactivated PH reduced by NADPH displayed reduction of FAD and both 2Fe–2S centers and yet no reduction of the Mo site. Taken together, these data strongly suggest that selenium cofactor is somehow involved in the transfer of electrons from the Mo site to the first of the two FeS centers. However, when stable isotope (⁷⁷Se) enriched PH was examined, no significant hyperfine coupling was observed with the reduced Mo site. ³¹⁶ This is contradictory to the data obtained for the NAH using a similar experimental analysis with EPR. This may put into question the exact chemical form of the selenium cofactor.

One underlying question remains: why does this small class of microorganisms require a labile selenium cofactor in these enzymes? Few have speculated on this in the published literature. Yet one key comparison between selenium and non-selenium-dependent hydroxylases may be quite telling. The well-studied bovine XDH has a turnover rate of approximately 5   s⁻¹, ³⁰⁷ while the PH enzyme from C. purinolyticum has a far faster catalysis of hypoxanthine with a turnover of 450   s⁻¹. ³¹⁶ Since these enzymes catalyze very similar reactions, it is likely that the presence of the labile selenium cofactor results in a significant improvement in catalysis of a slow step. Since this organism was isolated on adenine as a sole carbon and nitrogen source, the conversion of hypoxanthine into xanthine, subsequent to production of critical nitrogen and carbon intermediates, is probably a key and limiting step in growth. One can also speculate that this form of selenoenzyme may have arisen independent of selenoproteins that contain selenocysteine, but until we understand more about the nature and synthesis of this labile cofactor these questions will remain unanswered.

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Marine Enzymes and Specialized Metabolism - Part A

Robin Teufel , in Methods in Enzymology, 2018

7 Evidence for the Flavin-N5-Oxide Cofactor of EncM

In the beginning of the in-depth investigation of EncM, the presence of the Fl_N5ox cofactor was by no means clearly obvious due to several reasons:

1.

The highly similar UV–vis spectra of Fl_N5ox and Fl_ox and the fact that the Fl_N5ox spectrum—although having been reported in the late 1970s—was not commonly known.

2.

The absence of any visible modifications of the flavin in the EncM crystal structure.

3.

The sheer unlikelihood of an unrecognized flavin redox state and oxygenating species after many decades of flavoprotein studies.

At first, we suspected that EncM harbors a second redox center which may interact with the flavin, possibly explaining why four electrons are required to fully reduced EncM and accounting for the slight spectral changes observed for EncM oxidized with O₂ vs DCIP. The only redox active residue close to the flavin was a methionine, which may conceivably form a catalytically active methionine sulfoxide. However, mutation of this residue neither altered the EncM UV–vis spectrum nor abolish activity (Teufel et al., 2015). After ruling out other possibilities, the flavin cofactor itself had to be considered, also because denatured EncM mostly retained its spectrum. The common flavin-C4a-peroxide was ruled out based on spectroscopic features and stability. Several lines of evidence eventually led to the proposal of a Fl_N5ox species in EncM. It is generally known that the N5-C4a locus of the flavin serves as entry/exit points for electrons and formation of covalent adducts, and we thus focused our attention on this region. The Fl_N5ox seemed the most plausible possibility, as nitroxyl groups are commonly relatively stable and are applied in organic synthesis, e.g., in radical oxidation reactions. Indeed, chemically synthesized Fl_N5ox exhibited the same key spectral features as EncM, in particular after denaturation of EncM that minimized the effects of the protein surroundings on the flavin cofactor (Fig. 3). Further evidence came from digestion of EncM with proteinase K, which was required due to the covalent link of the flavin with His78 (Teufel et al., 2015):

•

EncM was digested for 4–6   h at 22°C in the dark with 2   mg/mL fungal proteinase K, 5   mM CaCl₂, and 25   mM Tris–HCl (pH 7.8).

•

Peptide fragments were separated from undigested protein using Nanosep spin columns (10   kDa cutoff, Pall Corp.).

•

Peptides were analyzed by high-resolution electrospray ionization tandem mass spectrometry (HR-ESI-LCMS²) experiments in positive mode using a 1290 Infinity LC system with a Luna 5   μm C18E (2) column (Phenomenex, Torrence, CA) (150   ×   4.6   mm) coupled to a 6530 Accurate-Mass Q-TOF MS system (Agilent Technologies, Santa Clara, CA). An acetonitrile gradient of 2%–100% (v/v) was used over 25   min in 0.1% (v/v) formic acid. Collision-induced dissociation for tandem MS spectra was set to 35 with a fragmentor voltage of 175   V. Pseudo-MS³ measurements were conducted by increasing the in-source fragmentor voltage to 430   V.

•

MS, MS², and pseudo-MS³ data analysis revealed an ion of a pentapeptide fragment containing His78 with covalently bound flavin as well as an additional oxygen, consistent with the proposed Fl_N5ox.

Notes

(1)

Proteins under investigation should not be overdigested, as this will result in partial or complete decay of the Fl_N5ox. A few hours at ambient temperature should suffice.

(2)

The flavin cofactor significantly facilitates identification of relevant fragments when MS analysis is combined with UV–vis detection of the flavin at ~   450   nm, e.g., by using a diode array detector, which allows facile determination of the retention time of flavinylated peptides.

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KINETIC METHODS | Noncatalytic Techniques

M. Silva , in Encyclopedia of Analytical Science (Second Edition), 2005

Methods of Monitoring Reactions

Kinetic-based determinations involving noncatalytic reactions use instruments of variable complexity for monitoring reactions. The great developments in the so-called intelligent instrumentation have fostered automation to a greater or lesser extent of the three essential steps involved in measuring reaction rates (Figure 4), the first of which is the most relevant to automation on account of the high complexity of the operations involved (complete automation of this step, however, is still impossible). Thus, the choice of the type of instrument to be used should be dictated not only by its technical features (mainly related to the second and third steps) but also by the half-life of the reaction concerned (closely related to the first step).

Figure 4. Basic steps of reaction rate-based determinations. (Reproduced with permission from Silva M (1993) Recent strategies in automated reaction rate-based determinations. Tutorial Review. Analyst 118: 681–688.)

Manual mixing of sample and reagents in the reaction vessel, which may be a spectrophotometer cuvette or an electrode cell, is usually adequate for monitoring slow reactions, whether in batch or closed systems. Flow systems avoid some manipulation, and so they result in increased precision in the determinations.

Flow systems, both continuous and discrete, are used in kinetic-based determinations for monitoring fast reactions mainly. To this end (1) the dead time in the mixing system should be several orders of magnitude lower than the half-life of the reaction concerned; and (2) nearly the whole kinetic curve must be recorded in order to implement reaction rate-based determinations and perform fundamental kinetic studies (e.g., the determination of reaction orders and rate constants). The advent of stopped-flow mixing and the continuous-addition-of-reagent technique has made noncatalytic reactions competitive with equilibrium methods in practical terms.

The Stopped-Flow Technique

The stopped-flow technique is the most commonly used for studying and implementing application of fast reactions with half-lives between a few milliseconds and a few seconds. The special features of this technique, in which reactants are driven at a high rate into a mixing and/or observation cell, the flow is abruptly stopped, and the extent of reaction monitored (Figure 5), have facilitated studies on the kinetics and mechanism of fast reactions and enabled the development of reaction rate-based determinations.

Figure 5. Typical instrumental setup used and signal–time profile provided by the stopped-flow technique. The shaded area denotes reaction development during the stop time. (Reproduced with permission from Silva M (1993) Recent strategies in automated reaction rate-based determinations. Tutorial Review: Analyst 118: 681–688.)

The stopped-flow technique was introduced by Chance in 1940. Earlier applications to kinetic analysis were concerned with studies on kinetics and reaction mechanisms (e.g., the formation of the iron(III)–thiocyanate complex, that of 12-molybdophosphoric acid, the redox reaction between 2,6-dichlorophenolindophenol and ascorbic acid, etc.) as well as the resolution of mixtures of metal ions using substitution reactions. On the other hand, the inception of commercially available stopped-flow instruments and inexpensive modular mixing systems for adaptation to existing detectors have led to a broad use of this technique in routine kinetic determination of individual species and mixtures in a variety of samples of clinical, pharmaceutical, nutritional, and environmental interest. The analytical features of the methods developed for this purpose usually surpass those of the equilibrium counterparts, as shown by the selected examples given in Table 2. In addition, stopped-flow systems accelerate some slow reactions relative to the conventional kinetic technique as a result of mixing taking place at a higher pressure. Hence the stopped-flow technique can also be advantageously applied to this type of reaction, thereby saving time and reagents and simplifying handling of the reaction ingredients.

Table 2. Selected applications illustrating the advantages of the stopped-flow technique over equilibrium methods

Analyte/chemical system	Remarks
Urea/biacetyl monoxime	Conventional equilibrium method: reaction time ∼20–30   min; uses a high-temperature and a concentrated acid medium.
	Stopped flow: 2–3   min for acquisition of analytical data; avoids the use of drastic experimental conditions
Carbaryl/diazotized sulfanilic acid	Wider determination range; higher selectivity factors for other N-methylcarbamate pesticides; no blank is required for the stopped-flow technique
Psychotropic drugs/hydrogen peroxide	Addition of an oxidant is unnecessary in the stopped-flow technique since oxidation is effected by dissolved oxygen itself

The Continuous-Addition-of-Reagent Technique

This technique is a major alternative to the stopped-flow technique for performing rate measurements on fast reactions as it uses simpler instrumentation (Figure 4, previous article) to add the reagent (R) continuously at a constant rate, u, over a volume V ₀ of sample containing the analyte (A). The overall reaction rate of the process, which depends on the rate of the reaction and the dilution of the species present in the reaction vessel, can be expressed in integral form as

[14] $\ln \left(\left[\mathrm{A}\right]/{\left[\mathrm{A}\right]}_0\right)=-k{\left[\mathrm{R}\right]}_{0}t+\left(k{\left[\mathrm{R}\right]}_0{V}_0/u-1\right)\text{ln}\left(V_0+ut/V_0\right)$

where [A]₀ and [A] are the analyte concentrations at time zero and time t, respectively; [R]₀ is the reagent concentration in the addition unit; and k is the pseudo-second-order reaction rate constant. The special way in which sample and reagents are mixed in this technique allows reaction half-lives to be altered through changes in the reagent concentration and its rate of addition.

This technique provides a full kinetic profile (Figure 6A) that can be used to implement two reaction rate methodologies, namely (1) the initial-reaction method, based on the initial concave portion of the curve, along which the analytical signal is directly proportional to t ² and (2) the maximum-reaction method, which relies on the linear intermediate portion of the curve. Based on reported results, the maximum-reaction method is preferable. In addition, it offers several advantages over traditional pseudo-first-order initial-rate methods, particularly a much wider linear portion for measurements to be made, where instrumental errors are much smaller.

Figure 6. Kinetic profiles provided by continuous-addition-of-reagent technique. (A) Typical kinetic curve. (B) Recording obtained for a chemical system involving an undesirable interaction between the reagent and some reaction ingredient. (Reproduced with permission from Silva M (1993) Recent strategies in automated reaction rate-based determinations. Tutorial Review: Analyst 118: 681–688.)

This technique is currently being used for individual and multiple determinations of species with interesting results. However, it is particularly suitable for reactions where excess reagent might have undesirable effects on the analytical signal (e.g., the determination of sulfonamides using the Bratton–Marshall reaction). Even though the kinetic profile obtained in such a case is somewhat special (Figure 6B), the maximum-reaction method can be accurate over the linear portion of the kinetic curve.

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FLUORESCENCE | Food Applications

A. Gómez-Hens , in Encyclopedia of Analytical Science (Second Edition), 2005

Vitamins

Vitamins are the foodstuff components most often quantified using fluorimetric means. There are several official fluorimetric methods for the determination of three water-soluble vitamins: vitamin B₁ (thiamine) (AOAC 942.23 and 957.17), B₂ (riboflavine) (AOAC 970.65 and 981.15), and C (ascorbic acid) (AOAC 984.26). Thiamine is determined by oxidation to fluorescent thiochrome with alkaline hexacyanoferrate(III) or an alternative oxidant (Figure 1). The method is quite simple, reproducible, and selective and provides good recoveries. Many LC methods for thiamine determination in foods have been developed using pre- or postcolumn conversion to thiochrome, which entail a wide variety of chromatographic modes and elution techniques.

Figure 1. Formation of fluorescent derivatives of vitamins B₁ and C.

Riboflavin is commonly determined fluorimetrically, for instance in milk, by its strong native fluorescence at pH 7, which arises from the extended conjugation and rigidity of the nonribose portion of the molecule. Another fluorimetric method involves conversion of riboflavin into its fluorescent derivative lumiflavin using ultraviolet (UV) irradiation. Mixtures of thiamin and riboflavin in foods such as cereal products have been resolved using LC with fluorimetric detection.

Vitamin C is another soluble vitamin frequently determined in foodstuffs using fluorimetric detection. It is most often oxidized to dehydroascorbic acid with HgCl₂ or 2,6-dichloroindophenol, which is subsequently reacted with p-phenylenediamine to form a fluorescent quinoxaline derivative (Figure 1). The method can be implemented in an automated fashion. The determination of total vitamin C (ascorbic and dehydroascorbic acid) in foods (vegetables and fruits) is also of great interest. The recommended method uses reversed-phase ion-pair LC and fluorimetric detection. Total vitamin C and total isovitamin C (erythorbic and dehydroerythorbic acids) can be determined similarly by prior extraction with trichloroacetic acid and precolumn derivatization, wherein ascorbic and erythorbic acids are oxidized enzymatically and total dehydroascorbic and dehydroerythorbic acids are treated with o-phenylenediamine.

The fluorimetric determination of vitamin B₆ compounds (pyridoxine, pyridoxamine, pyridoxal, pyridoxamine 5′-phosphate, pyridoxal 5′-phosphate, and 4-pyridoxic acid) in foods by measurement of their native fluorescence entails a prior LC isolation. Some of these determinations involve postcolumn derivatization with sodium bisulfite in a phosphate buffer. Mixtures of riboflavin and pyridoxine in infant formula products can be determined simultaneously using ion-pair LC with fluorescence detection.

Fluorimetric LC methods for the determination of fat-soluble vitamins in foods have been described for vitamins A (retinol), E, and K. These methods usually entail saponification and extraction steps. The determination of vitamin A, which shows intrinsic fluorescence, in foods containing large amounts of carotenoids, requires chromatographic purification. This technique has also been used in studying the extent of vitamin A isomerization during manufacture and storage of liquid and dried milk products, which involves prior isolation of the different isomers using LC.

Estimation of true vitamin E in foods requires quantitative determination of all its components since they vary in their biological potency. This vitamin consists of four tocopherols (α, β, γ, and δ) and four tocotrienols (α, β, γ, and δ), but the three major constituents responsible for vitamin E activity are the α-, β-, and γ-tocopherols. While these compounds are fluorescent, their esters must be reduced to free alcohols for total tocopherol assays. Total vitamin E can be directly obtained through fluorimetry, but the determination of individual components is carried out using LC with fluorimetric detection. This procedure has been used to determine the composition of vitamin E in seed oils from maize, olives, soya beans, sesame, safflower, and sunflower by measuring the content of all the four tocopherols plus α-tocotrienol. The simultaneous determination of tocopherols, carotenes, and retinol in cheese has been carried out using LC with two programmable detectors connected in series, a spectrophotometer and a fluorimeter. Carotenes have been determined photometrically, and fluorimetric measurements have been obtained for tocopherol and retinol.

The vitamin K group is usually determined in foods such as fats, oils, and milk through LC with fluorimetric detection after postcolumn zinc reduction. Samples are usually digested with lipase and extracted into hexane.

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Dichloroindophenol Vitamin C

Source: https://www.sciencedirect.com/topics/medicine-and-dentistry/2-6-dichlorophenolindophenol