Lumichrome Research Articles

Multicomponent spectrofluorimetric method for the assay of formylmethylflavin and its hydrolytic products: Kinetic applications

A multicomponent spectrofluorimetric method has been developed for the simultaneous assay of formylmethylflavin (FMF), an intermediate product in the photolysis of riboflavin (vitamin B2), and its side-chain hydrolytic products, lumichrome (LC) in acidic solution and LC and lumiflavin (LF) in the alkaline solution as well as its ring cleavage products, 1,2-dihydro-1-methyl-2-keto-3-quinoxaline carboxylic acid (KA) and 1,2,3,4-tetrahydro-1-methyl-2,3-dioxo-quinoxaline (DQ) in alkaline solution. The assay method also takes into account an oxidation product of FMF, i.e. carboxymethylflavin (CMF), in both acid and alkaline solutions. The method involves adjustment of the pH of hydrolysed solution to 2.0 to convert FMF to its protonated form, extraction of LC (acid solution) or LC and LF (alkaline solution) with chloroform and their simultaneous assay by fluorescence measurement at 478 and 530 nm, respectively. The aqueous phase is readjusted to pH 6.5, extracted with chloroform to remove undegraded FMF and used for the assay of CMF, KA and DQ at 530, 443 and 420 nm, respectively. The chloroform extract is used for the assay of FMF at 530 nm. The proposed method has been validated and applied to the study of the kinetics of a hydrolysis reaction of FMF at pH 11.0. The calibration curves for FMF and degradation products are linear in the range of 0.1–1.0 × 10−6 M. The limit of detection (LOD) and limit of quantification (LOQ) range from 2.54–5.75 × 10−8 M and 0.78–1.74 × 10−7 M, respectively, for these compounds. The mean recovery ranges from 99.3–102.1% with a RSD of 0.14–0.35%. Judging from the molar balance of FMF and the hydrolytic products, uniformity of analytical data during the reactions and linearity of kinetic plot, the method gives accurate results for the assay of FMF and all of its degradation products. It can be conveniently used for the assay of these compounds and for the kinetics and stability studies of FMF.

Stability-indicating spectrofluorimetric method for the assay of riboflavin and photoproducts: Kinetic applications.

A stability-indicating spectrofluorimetric method has been developed for the simultaneous assay of riboflavin (RF) and photoproducts, formylmethylflavin (FMF), lumichrome (LC) and lumiflavin (LF) in aqueous solution. The method is based on the extraction of LC formed in acid solution and LC and LF formed in alkaline solution with chloroform at pH2.0 and their assay by fluorescence measurements at 478 and 530nm, respectively. The aqueous phase, on readjustment of the pH to 6.5, is used to extract FMF with chloroform and its assay is carried out at 530nm. The aqueous phase is then used for the assay of RF at 530nm. The proposed method gives more accurate results for the assay of RF compared to those of the United States Pharmacopeia (USP) spectrofluorimetric method which does not take into account the presence of RF photoproducts having similar fluorescence characteristics. The proposed method along with the USP method has been applied to the study of the kinetics of photolysis of RF, assay of stored commercial vitamin preparations and their radiated samples. The results show that the USP method does not distinguish between the fluorescence of RF and its photoproducts, and, therefore, gives erroneous results with about 11% excess in the quantity of the vitamin compared to that of the proposed method. This is due to the interference of the fluorescence of photoproducts in the assay of RF. The method has been validated for various analytical parameters according to the guideline of the International Council for Harmonization (ICH).

Development of a HPLC method for the simultaneous analysis of riboflavin and other flavin compounds in liquid milk and milk products

A high-performance liquid chromatography method with fluorescence detection was developed for the quantification of riboflavin (RF), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and their photodegradation products, lumichrome (LC) and lumiflavin (LF), in liquid milk and milk products. Both sample preparation and chromatographic separation were studied to avoid acidic conditions that proved to affect flavin stability and degrade FAD into FMN. The sample preparation includes centrifugal skimming and ultrafiltration steps and is suitable for routine application. Linear response was obtained for individual flavins in the respective concentration ranges of interest and relative standard deviation (RSD) was lower than 5%, except for FAD (RSD 11%). The recovery ranged between 80–100%. The proposed method proved to be suitable for assessing flavins in commercial liquid milk and fermented milk products, and for monitoring the degradation of FAD, FMN and RF and the formation of LF and LC in bottled milk exposed to light during shelf storage.

Metal ion mediated photolysis reactions of riboflavin: A kinetic study

The effect of metal ion complexation on the photolysis of riboflavin (RF) using various metal ions (Ag+, Ni2+, Co2+, Fe2+, Ca2+, Cd2+, Cu2+, Mn2+, Pb2+, Mg2+, Zn2+, Fe3+) has been studied. Ultraviolet and visible spectral and fluorimetric evidence has been obtained to confirm the formation of metal-RF complexes. The kinetics of photolysis of RF in metal-RF complexes at pH7.0 has been evaluated. The apparent first-order rate constant (kobs) for the photolysis of RF and the formation of lumichrome (LC) and lumiflavin (LF) (0.001M phosphate buffer) and LC, LF and cyclodehydroriboflavin (CDRF) (0.2–0.4M phosphate buffer) have been determined. The values of kobs indicate that the rate of photolysis of RF is promoted by divalent and trivalent metal ions. The second-order rate constants (kʹ ) for the interaction of metal ions with RF are in the order: Zn2+>Mg2+>Pb2+>Mn2+>Cu2+>Cd2+>Fe2+>Ca2+>Fe3+>Co2+>Ni2+>Ag+. In phosphate buffer (0.2–0.4M), an increase in the metal ion concentration leads to a decrease in the formation of LC compared to that of CDRF by different pathways. The photoproducts of RF have been identified and RF and the photoproducts have simultaneously been assayed by a multicomponent spectrometric method. The mode of photolysis of RF in metal-RF complexes has been discussed.

Intriguing Tautomerism of Lumichrome in Binary Aqueous Solvent Mixtures: Implications for Probing Microenvironments.

The water-assisted tautomerism of lumichrome (LC) in binary aqueous-organic solvent mixtures exhibits intriguing spectroscopic features. An especially striking and novel observation is the occurrence of an induction period or lag time for the evolution of the isoalloxazine form of LC from the excited alloxazine form. It is inferred that the observed lag time provides an overview of the changing H-bond network of the surrounding water molecules, at various compositions of the mixed solvent systems. The unique spectral changes for the water-assisted tautomerization of LC, along with the observed lag times, can offer exciting prospects for the use of LC as a promising site-specific probe, to examine complex aqueous microenvironments and active biological systems.

Interaction of Biologically Active Flavins inside Bile Salt Aggregates: Molecular Level Investigation.

In this work we have studied the photophysics of biologically active flavin molecule lumichrome (LCM) in different bile-salt aggregates. With alteration of the functional groups of the bile salts, the photophysics of confined fluorophore is largely affected and shows difference in their spectral behavior. This study also reveals the selective prototropic species of LCM present in bile salt aggregates. In the presence of the bile salt aggregates, LCM molecule shows excitation and emission wavelength-dependent emission properties, indicating switch over of the structural change of different prototropic form of the LCM molecule. The observation of higher rotational relaxation time in NaDC aggregates compared to NaTC aggregates clearly reflects that NaDC aggregates are more rigid due to its greater hydrophobicity and large in size, which is capable to bind the guest molecule more into their nanoconfined medium. Moreover, due to less acidic nature, NaDC aggregates have more ability to accept hydrogen bond from the LCM molecule and show the selective formation of isoalloxazine N10 anion (A1 monoanionic form) of LCM.

Ionic strength effects on the photodegradation reactions of riboflavin in aqueous solution

A study of the effect of ionic strength on the photodegradation reactions (photoreduction and photoaddition) of riboflavin (RF) in phosphate buffer (pH7.0) has been carried out using a specific multicomponent spectrometric method. It has been found that the rates of photodegradation reactions of RF are dependent upon the ionic strength of the solutions at different buffer concentrations. The apparent first-order rate constants (kobs) for the photodegradation of riboflavin at ionic strengths of 0.1–0.5 (0.5M phosphate) lie in the range of 7.35–30.32×10−3min−1. Under these conditions, the rate constants for the formation of the major products, lumichrome (LC) by photoreduction pathway, and cyclodehydroriboflavin (CDRF) by photoaddition pathway, are in the range of 3.80–16.03 and 1.70–6.07×10−3min−1, respectively. A linear relationship has been observed between log kobs and √μ/1+√μ. A similar plot of log k/ko against √μ yields a straight line with a value of ~+1 for ZAZB showing the involvement of a charged species in the rate determining step. NaCl appears to promote the photodegradation reactions of RF probably by an excited state interaction. The implications of ionic strength on RF photodegradation by different pathways and flavin–protein interactions have been discussed.

Formulation and stabilization of riboflavin in liposomal preparations

A study of the formulation of liposomal preparations of riboflavin (RF) with compositional variations in the content of phosphatidylcholine (PC) and their entrapment efficiency (26–42%) have been conducted. Light transmission characteristics of the liposomal preparations have been determined to evaluate their effect on the amount of light passing through the system to initiate a photochemical reaction. Dynamic light scattering (DLS) and atomic force microscopy (AFM) have been used to study the physical characteristics of liposomes. The liposomal preparations of RF have been subjected to photolysis using visible light and the apparent first- order rate constant, kobs, for the degradation of RF have been determined. The values of kobs (1.73–2.29×10−3min−1) have been found to decrease linearly with an increase in PC concentration in the range of 12.15 to 14.85mM. Thus, an increase in PC concentration of liposomes leads to an increase in the stability of RF. RF and its main photoproduct, lumichrome (LC), formed in liposomes have been assayed by a two-component spectrometric method at 356 and 445nm using an irrelevant absorption correction to compensate for the interference of liposomal components. The fluorescence measurements of RF in liposomes indicate excited singlet state quenching and the formation of a charge-transfer complex between RF and PC. It results in electron transfer from PC to RF to cause photoreduction and stabilization of RF.

Stability-Indicating Photochemical Method for the Assay of Riboflavin: Lumichrome Method

A stability-indicating photochemical method for the assay of riboflavin (RF) in photodegraded samples and aged vitamin preparations has been developed. It is based on photochemical conversion of RF to lumichrome (LC) in alkaline solution under controlled conditions of light intensity, temperature, pH, time of exposure, and distance. Under these conditions about two-thirds of RF is converted to LC and on the basis of the RF : LC ratio the concentration of RF can be determined in degraded solutions. The method involves the extraction of photolyzed solutions of RF (pH 2.0) with chloroform and determination of LC along with lumiflavin (LF) by a two-component spectrometric method at 356 and 445 nm. The method has been validated and the results of the assay of RF in photodegraded solutions compare well with those of the standard USP fluorimetric method. The recovery of the method is 99–101% and the precision is within 2%. The method is stability-indicating and can be applied to the assay of RF in photodegraded solutions and aged vitamin preparations. The method is specific compared to that of the USP fluorimetric method in which the degraded LC may interfere with the fluorescence emission of RF.

Effect of phosphate buffer on the complexation and photochemical interaction of riboflavin and caffeine in aqueous solution: A kinetic study

A study of the photodegradation of 5 × 10−5 M riboflavin (RF) in 0.2–1.0 M phosphate buffer in the presence and absence of 2.50 × 10−4 M caffeine at pH 6.0–8.0 has been carried out. RF in phosphate buffer is photodegraded simultaneously by normal photolysis (photoreduction) and photoaddition reactions giving rise to lumichrome (LC) and cyclodehydroriboflavin (CDRF) as the main final products, respectively. RF and its photoproducts, formylmethylflavin (FMF), lumiflavin (LF), LC and CDRF in degraded solution have been determined by a specific multicomponent spectrophotometric method with an accuracy of ±5%. The apparent first-order rate constants for the photodegradation of RF and for the formation of LC and CDRF are 5.47–15.05 × 10−3 min−1, 1.06–8.30 × 10−3 min−1 and 4.31–8.05 × 10−3 min−1, respectively. An increase in phosphate concentration leads to an increase in the rate of formation of CDRF and alters the photodegradation of RF in favor of the photoaddition reaction. This photoaddition reaction is further enhanced in the presence of caffeine which results in a further decrease of the fluorescence of RF in phosphate buffer. Caffeine may facilitate the photoaddition reaction by suppression of the photoreduction pathway of RF.

Riboflavin's Time-Dependent Degradation Rate Induced by Ultraviolet a Irradiation

To evaluate the time-dependent degradation rate of riboflavin after ultraviolet A (UVA) irradiation. Two solutions of commercially available riboflavin solution (0.1%) were used; one served as control, while the second was irradiated using UVA light at 370 nm wavelength. Four samples of riboflavin solution were retrieved prior to irradiance and at 1, 5, 15, 30, and 60 minutes after irradiation (group A); at the same time points samples of riboflavin were retrieved from the control solution in order to assess environmental time-induced degradation of riboflavin (group B). All samples were immediately analyzed using liquid chromatograph mass spectrometry to detect riboflavin and its 2 subproducts, lumiflavin (LF) and lumichrome (LC). Mean percentage of riboflavin degradation was 0.0, 5.3, 9.1, 15.3, 20.6, and 33.3 at 0, 1, 5, 15, 30, and 60 minutes after UVA irradiation, respectively (group A). The time-dependent riboflavin degradation was statistically significant (p<0.05), while for group B there was no change in riboflavin concentration at all time intervals. In group A, mean LC concentration demonstrated a gradual concentration increase, reaching 2.386±1.526 ppm after 60 minutes of UVA exposure. The time-dependent degradation of riboflavin solution is significant, reaching 20.6% after 30 minutes of UVA exposure. It seems that only a small fraction of the overall riboflavin molecules break down since more than 65% remain intact even after 1 hour of UVA irradiation. Control riboflavin solution seems to be stable, as no degradation is evident even after 60 minutes.

Fluorescence Solvatochromism in Lumichrome and Excited-State Tautomerization: A Combined Experimental and DFT Study

Fluorescence solvatochromism of lumichrome (LC) was studied by steady-state and time-resolved fluorescence spectroscopy. The excited-state properties of LC do not show any correlation with solvent polarity, however, reasonably good correlation with solvent E(T)(30) parameter was observed. A quantitative estimation of contribution from different solvatochromic parameters, like solvent polarizability (π*), hydrogen bond donor (α), and hydrogen bond acceptor (β) ability of the solvent, was made using linear free energy relationship on the basis of Kamlet-Taft equation. The analysis reveals that hydrogen bond donating ability (acidity) of the solvent is the most important parameter that characterizes the excited-state behavior of lumichrome. Quantum mechanical calculations using density functional theory (DFT) were done to study the most stable structure and excited-state tautomerization process of LC toward the formation of isoalloxazines. Charge localization in the excited state and formation of hydrogen-bonded cluster through solvent hydrogen bond donation on the N10 atom of alloxazine moiety were predicted to be the key step toward this water-catalyzed tautomerization process.

Fluorescence quenching and the binding interaction of lumichrome with nucleic acids

The interaction of lumichrome (LC) as an endogenous fluorescence probe with nucleobases, nucleosides and nucleic acids has been studied by UV-visible absorption, fluorescence spectroscopy, polarized fluorescence and viscosity. The fluorescence of LC was strongly quenched by a series of nucleic acids and their precursors. The influences of the medium, temperature and salt effects on LC-nucleobase and LC-nucleic acid complexes were investigated. The influences of polarized fluorescence, thermal denaturation and viscosity on LC-ctDNA interaction were examined. The results demonstrate that the main binding model of LC-ctDNA includes strong intercalating into the DNA helix chain.

Practically Efficient QM/MM Alchemical Free Energy Simulations: The Orthogonal Space Random Walk Strategy.

The difference between free energy changes occurring at two chemical states can be rigorously estimated via alchemical free energy (AFE) simulations. Traditionally, most AFE simulations are carried out under the classical energy potential treatment; then, accuracy and applicability of AFE simulations are limited. In the present work, we integrate a recent second-order generalized ensemble strategy, the orthogonal space random walk (OSRW) method, into the combined quantum mechanical/molecular mechanical (QM/MM) potential based AFE simulation scheme. Thereby, within a commonly affordable simulation length, accurate QM/MM alchemical free energy simulations can be achieved. As revealed by the model study on the equilibrium of a tautomerization process of hydrated 3-hydroxypyrazole and by the model calculations of the redox potentials of two flavin derivatives, lumichrome (LC) and riboflavin (RF) in aqueous solution, the present OSRW-based scheme could be a viable path toward the realization of practically efficient QM/MM AFE simulations.

Effect of divalent anions on photodegradation kinetics and pathways of riboflavin in aqueous solution

The present investigation is based on a study of the effect of buffer and non-buffer divalent anions (phosphate, sulphate, tartrate, succinate, malonate) on the kinetics, product distribution and photodegradation pathways of riboflavin (RF) at pH 6.0–8.0. RF solutions (5 × 10 −5 M) were photodegraded in the presence of divalent anions (0.2–1.0 M) using a visible light source and the photoproducts, cyclodehydroriboflavin (CDRF), formylmethylflavin (FMF), lumichrome (LC) and lumiflavin (LF) were assayed by a specific multicomponent spectrophotometric method. RF degradation in the presence of divalent anions follows parallel first-order kinetics to give CDRF and LC as the final products through photoaddition and photoreduction reactions, respectively. The divalent anion-catalysed CDRF formation is affected in the order: phosphate > sulphate > tartrate > succinate > malonate, showing maximum activity of the anions around pH 7. The divalent anions cause deviation of the photoreduction pathway in favour of the photoaddition pathway to form CDRF. The first- and second-order rate constants for the reactions involved in the photodegradation of RF have been determined and the rate–pH profiles and pathway relationships discussed. The catalytic activity of the divalent anions appears to be a function of the relative strength and chemical reactivity of the RF–divalent anion complex acting as a mediator in the photoaddition reaction.

Effect of borate buffer on the photolysis of riboflavin in aqueous solution

The photolysis of riboflavin (RF) in the presence of borate buffer (0.1–0.5 M) at pH 8.0–10.5 has been studied using a specific multicomponent spectrophotometric method for the determination of RF and photoproducts, formylmethylflavin (FMF), lumichrome (LC) and lumiflavin (LF). The overall first-order rate constants for the photolysis of RF (1.55–4.36 × 10 −2 min −1) and the rate constants for the formation of FMF (1.16–3.52 × 10 −2 min −1) and LC (0.24–0.84 × 10 −2 min −1) have been determined. The values of all these rate constants decrease with an increase in buffer concentration suggesting the inhibition of photolysis reaction by borate species. The kinetic data support the formation of a RF-borate complex involving the ribityl side chain to cause the inhibition of photolysis. The second-order rate constants for the borate inhibited reaction range from 1.17–3.94 × 10 −2 M −1 min −1. The log k-pH profiles for the reaction at various buffer concentrations indicate a gradual increase in rate, with pH, up to 10 followed by a decrease in rate at pH 10.5 probably due to ionization of RF and quenching of fluorescence by borate species. A graph of second-order rate constants against pH is a sigmoid curve showing that the rate of photolysis increases with an increase in pH. The results suggest the involvement of excited singlet state, in addition to excited triplet state, in the formation of LC.

Study on electron transfer of fluorescent probe lumichrome and nucleic acid by laser flash photolysis

Laser flash photolysis (LFP) of lumichrome (LC) has been studied upon laser excitation at 355 nm. The transient absorption spectra of LC in aqueous solution displayed two bands at 380 nm and 540–700 nm for the triplet 3LC, and a wide band at 430–530 nm for the radical anion LC.. The triplet-state lifetime was measured up to 12 µs. It was found that the self-quenching of 3LC by the ground state of LC occurred to produce LC. during LFP. The transient spectra of LC in the presence of electron-donating triphenylamine (TPA), nucleosides and nucleic acids showed that the absorbance of 3LC was strongly quenched by these substrates and the formation of LC. was enhanced in a great extent simultaneously. The bimolecular quenching kinetics were studied and the rate constants of 3LC by a series of quenchers, including TPA, LC, nucleosides and nucleic acids were determined to be 108–109 M−1 s−1. The photo-induced electron-transfer mechanism for these processes involving the triplet 3LC was proposed.

Photolysis of formylmethylflavin in aqueous and organic solvents

The photolysis of formylmethylflavin (FMF), a major intermediate in the photodegradation sequence of riboflavin, has been carried out in water (pH 7.0) and in several organic solvents. FMF produces lumichrome (LC) in organic solvents and LC and lumiflavin (LF) in aqueous solution. FMF and its photoproducts have been analysed using a specific multicomponent spectrophotometric method. FMF undergoes a bimolecular redox reaction on photolysis. The second-order rate constants for the reaction range from 0.66 (chloroform) to 2.44 M(-1) s(-1) (water) and are a linear function of the solvent dielectric constant. A plot of ln k against 1/epsilon is linear for the reactions in 1-butanol, 1-propanol, ethanol, methanol, acetonitrile and water (epsilon approximately 17-79) and non-linear in chloroform and dichloroethane (epsilon approximately 5-10) suggesting a change in reaction mechanism in the two regions. This may be explained on the basis of the existence of a dipolar intermediate along the reaction pathway. The rate of photolysis is governed by the solvation of the intermediate and is thus influenced by the dielectric constant of the medium. The solvent effect on the rate of photolysis of FMF has been expressed in terms of the solvent acceptor number. A linear relationship has been found between ln k and the solvent acceptor number.

Effect of light intensity and wavelengths on photodegradation reactions of riboflavin in aqueous solution

A study of the effect of light intensity and wavelengths on photodegradation reactions of riboflavin (RF) solutions in the presence of phosphate buffer using three UV and visible radiation sources has been made. The rates and magnitude of the two major photodegradation reactions of riboflavin in phosphate buffer (i.e., photoaddition and photoreduction) depend on light intensity as well as the wavelengths of irradiation. Photoaddition is facilitated by UV radiation and yields cyclodehydroriboflavin (CDRF) whereas photoreduction results from normal photolysis yielding lumichrome (LC) and lumiflavin (LF). The ratios of the photoproducts of the two reactions at 2.0 M phosphate concentration, CDRF/RF (0.09–0.22) and CDRF/LC (0.54–1.75), vary with the radiation source and are higher with UV radiation than those of the visible radiation. On the contrary, the ratios of LF/LC (0.15–0.25) increase on changing the radiation source from UV to visible. The rate is much faster with UV radiation causing 25% degradation of a 10 −5 M riboflavin solution in 7.5 min compared to that of visible radiations in 150–330 min.

Effect of phosphate buffer on photodegradation reactions of riboflavin in aqueous solution

The effect of phosphate buffer on aerobic photodegradation reactions of riboflavin (RF) at pH 7.0 has been studied. The photoproducts of the two major reactions, viz., intramolecular photoreduction and intramolecular photoaddition, have been determined by a specific multicomponent spectrophotometric method. The overall photodegradation of riboflavin in the presence of phosphate buffer involves the participation of both H 2 PO 4 - and HPO 4 2 - species. The second-order rate constants for the H 2 PO 4 - -catalysed photodegradation of riboflavin (normal photolysis) to lumichrome (LC) and HPO 4 2 - -catalysed photodegradation of riboflavin (photoaddition) to cyclodehydroriboflavin (CDRF) are 0.93 × 10 −4 and 4.0 × 10 −4 M −1 s −1, respectively. The addition of 0.25–2.00 M phosphate to RF solutions at pH 7.0 gives rise to RF – HPO 4 2 - complex and hence the quenching of 4–36% fluorescence, respectively. This results in the suppression of normal photolysis leading to the formation of LC in favour of photoaddition to yield CDRF. The present study shows the involvement of H 2 PO 4 - anions in the base-catalysed degradation of riboflavin by normal photolysis vis-a-vis the involvement of HPO 4 2 - anions in photoaddition reactions of riboflavin suggested earlier [M. Schuman Jorns, G. Schollnhammer, P. Hemmerich, Intramolecular addition of the riboflavin side chain. Anion-catalysed neutral photochemistry, Eur. J. Biochem. 57 (1975) 35–48].