Indirect detection of organic acids in non-aqueous capillary electrophoresis.

Non-aqueous capillary electrophoresis (NACE) with indirect detection has been applied to the determination of fatty acids (FAs) and ascorbic acid (AA), respectively. C2-C18 FAs have been separated in less than 12 min using 8-hydroxy-7-iodoquinoline sulfonic acid as chromophores in NACE with indirect absorbance. The dissociation constant (pKa) values of C8-C18 FAs obtained from the slope of the linear plot -log [(mu 0/mu)-1] vs. pH, using 20% isopropanol and 40% acetonitrile as the organic modifier in NACE, are all above about two units than those obtained in aqueous solution. NACE with indirect laser-induced fluorescence, using merocyanine 540 (MC540) as fluorophores, has been performed to the analysis of AA and its stereoisomer, isoascorbic acid (IAA), and the limits of detection of AA and IAA are 0.30 microM and 0.17 microM, respectively. This method has been applied to the determination of AA in a lemon juice spiked with IAA as the internal standard in less than 3 min and its concentration is 76.7 +/- 0.4 mM.     

Photoisomerization of merocyanine 540 in polymer-surfactant aggregate

Photoisomerization of merocyanine 540 (MC540) in a polymer-surfactant aggregate is studied using picosecond time resolved emission spectroscopy. The aggregate consists of the polymer, poly(vinylpyrrolidone) (PVP) and the surfactant, sodium dodecyl sulphate (SDS). With increase in the concentration of SDS in an aqueous solution of MC540 containing PVP, the emission quantum yield and lifetime of MC540 increase markedly. This indicates marked retardation in the nonradiative photoisomerization process of MC540, when it binds to the polymer-surfactant aggregate. The critical association concentration of SDS for binding to PVP has been found to be 0.5 mM. This is about 16 times lower than the CMC of SDS in pure water (8 mM).     

Analysis of triacylglycerol and fatty acid isomers by low-temperature silver-ion high performance liquid chromatography with acetonitrile in hexane as solvent: limitations of the methodology

Silver ion HPLC (Ag-HPLC), utilizing columns containing silver ions bonded to a silica substrate and acetonitrile in hexane as solvent, has proven to be a powerful technology for the analysis of geometric (cis or trans) or positional fatty acids, fatty acid ester (primarily methyl ester; FAME), or triacylglycerol (TAG) isomers. Previous studies had demonstrated that, unlike gas chromatography, samples eluted more rapidly at lower temperatures (at 20 degrees C versus 40 degrees C, for example). A low-temperature bath [dual-column Ag-HPLC; isocratic solvent systems of 0.3 to 0.7% acetonitrile (ACN) in hexane] was utilized to study the application of this system at low (below 0 degrees C) temperatures for analysis of FAME (zero to six double bonds) and TAG [SSS, OOO and LLL, where S=stearic acid (18:0), O=oleic acid (9c-18:1), and L=linoleic acid (9c, 12c-18:2)] standards. While FAME elution times continued to decrease from 0 degrees C to -10 degrees C, they began to increase at -20 degrees C. A similar situation was noted for the TAG isomers, except that retention times began to increase below 0 degrees C. The lower temperature limit of the Ag-HPLC/ACN in hexane system is thus ca. -25 degrees C. Increasing sample elution times and pump head pressures upon sample injection were noted at temperatures of -25 degrees C to -40 degrees C. Equilibration times at each temperature could be reduced to ca. 15 min without loss of resolution and with retention times of +/-2%. Temperature, rather than solvent composition, can therefore be utilized with the Ag-HPLC/ACN in hexane solvent system to optimize elution times and resolution(s) of FAME and TAG isomers.     

Characterization and quantitation of mixtures of alkyl ether sulfates and carboxylic acids by capillary electrophoresis with indirect photometric detection

The separation, characterization, and determination of mixtures of alkyl ether sulfates (AES) and fatty acids (C10-C16) in background electrolytes (BGEs) containing acetonitrile (ACN)-water mixtures is addressed. Due to inhibition of the ionization of the carboxylate groups, the migration time and the resolution between the fatty acids decreased when the water content of the BGE was reduced, but efficiency and resolution between the AES oligomers improved. The migration times increased and resolution improved by substituting 5% ACN by an equivalent amount of dioxane. A complete separation of the two surfactant classes, up to the AES oligomers with 8 ethylene oxide units (EOs) with respect to C10, with excellent resolution between the AES oligomers, while preserving a satisfactory resolution between the fatty acids, was achieved with a BGE containing 5 mM trimethoxybenzoic acid, 7 mM dipentylamine, 85% ACN, 5% dioxane, and 10% water. The two surfactant classes were increasingly resolved by further reducing the water content of the BGE. Thus, C2 (acetate) was resolved from the AES oligomers up to 7 EOs using 90% ACN and 5% dioxane, but the resolution between the heavier fatty acids was poor with this BGE. Identification of the AES oligomers was eased by the excellent regularity of the successive migration times; thus, within each AES subclass or series of oligomers with the same number of carbon atoms in the alkyl chain, the migration times decreased following a mild curve as the number of EOs increased. The way how the data obtained by indirect photometry (corrected peak areas that are proportional to the molar concentrations) should be managed to avoid systematic error when the calibration curve is constructed using an AES standard with an oligomer distribution different from that of the samples is discussed and equations are given. Decyl sulfate was successfully used as internal standard. The detection limits (S/N = 3) were of ca. 2 microM for individual AES oligomers.     

pH,serum proteins and ionic strength influence the uptake of merocyanine 540 by WiDr cells and its interaction with membrane structures

It has been suggested that selective uptake of photosensitizers is due to significantly lower pH of the interstitial fluid in tumors compared to normal tissue. Therefore, the cellular uptake of merocyanine 540 (MC 540) was examined at two pH values: 6.8+/-0.1 and 7.4+/-0.1. There was no difference in spectral properties (absorption and fluorescence maxima positions, fluorescence intensity) of the drug in the presence of increasing amounts of either human blood plasma or FCS (0-2%) at the two pH values investigated. Nevertheless, significantly higher amounts of the drug were taken up by WiDr cells at pH 6.8+/-0.1, both in the presence of 10% FCS and in the absence of FCS. The absorption spectra of MC 540 in the presence of egg phosphatidylcholine (PC) liposomes turned out to be NaCl concentration-dependent (0.00-0.30 mol l(-1)). Membrane fluidity, as measured by fluorescence anisotropy of diphenylhexatriene (DPH), was unchanged within the experimental error in the NaCl concentration range 0.01-0.30 mol l(-1). The spectral changes indicated an enhancement of the incorporation of MC 540 into lipid membranes with increasing ionic strength. Such a salt concentration dependence suggests a possible involvement of the surface potential in the interaction of MC 540 with lipid membranes. The results might provide an explanation of the pH dependency of the cellular uptake of MC 540 observed in this study.     

Photosensitized lipid peroxidation and enzyme inactivation by membrane-bound merocyanine 540: reaction mechanisms in the absence and presence of ascorbate

The lipophilic photosensitizing dye merocyanine 540 (MC540) is being studied intensively as an antitumor and antiviral agent. Since plasma membranes are believed to be the principal cellular targets of MC540-mediated photodamage, we have studied membrane damage in a well characterized test system, the human erythrocyte ghost. When irradiated with white light, MC540-sensitized ghosts accumulated lipid hydroperoxides (LOOHs derived from phospholipids and cholesterol) at a rate dependent on initial dye concentration. Neither desferrioxamine nor butylated hydroxytoluene inhibited LOOH formation, suggesting that Type I (iron-mediated free radical) chemistry is not important. By contrast, azide inhibited the reaction in a dose-dependent fashion, implicating a Type II (singlet oxygen, 1O2) mechanism. Stern-Volmer analysis of the data gave a 1O2 quenching constant approximately 50 times lower than that determined for an extramembranous target, lactate dehydrogenase (the latter value agreeing with literature values). This suggests that 1O2 reacts primarily at its membrane sites of origin and that azide has limited access to these sites. Using [14C]cholesterol-labeled membranes and HPLC with radiodetection, we identified 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide as the major cholesterol photoproduct, thereby confirming 1O2 intermediacy. Irradiation of MC540-sensitized membranes in the presence of added iron and ascorbate resulted in a large burst of lipid peroxidation, as shown by thiobarbituric acid reactivity and appearance of 7-hydroperoxycholesterol and 7-hydroxycholesterol as major oxidation products. Amplification of MC540-initiated lipid peroxidation by iron/ascorbate (attributed to light-independent reduction of nascent photoperoxides, with ensuing free radical chain reactions) could prove useful in augmenting MC540's phototherapeutic effects.     

PHOTODYNAMIC ACTION OF MEROCYANINE 540 IN ARTIFICIAL BILAYERS AND NATURAL MEMBRANES: ACTION SPECTRA AND QUANTUM YIELDS

The action spectra and quantum yields for singlet oxygen (1O2) generation by merocyanine 540 (MC540) in liposomes and isolated erythrocyte membranes were obtained using electron spin resonance techniques. Oxygen consumption was measured by spin label oximetry in the presence of histidine for fully-saturated dimyristoylphosphatidylcholine vesicles, mono-unsaturated 1-palmitoyl-2-oleoylphosphatidylcholine vesicles and erythrocyte membranes. The quantum yield for the photogeneration of 1O2 by membrane-bound MC540 in aqueous buffer was determined to be 0.065 +/- 0.005, which is approx. 1/10 of the value determined for Rose Bengal under similar conditions. Using unilamellar liposomes and isolated erythrocyte membranes containing MC540 at different monomer/dimer ratios, we have observed that the action spectra of 1O2 generation closely overlap the absorption spectra of the monomeric dye in these systems. It is likely that factors which affect the monomer-dimer equilibrium of MC540 will influence the production of 1O2. These findings have important implications for the phototherapeutic efficacy of MC540.     

Nonaqueous capillary electrophoresis with laser-induced fluorescence detection: a case study of comparison with aqueous media

A novel method based on separation by nonaqueous capillary electrophoresis (NACE) combined with laser-induced fluorescence (LIF) detection was developed and compared with classic aqueous modes of electrophoresis in terms of resolution of solutes of interest and sensitivity of the fluorescence detection. Catecholamines derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) were chosen as test analytes for their subtle fluorescence properties. In aqueous systems, capillary zone electrophoresis (CZE) was not suitable for the analysis of test analytes due to complete fluorescence quenching of NBD-labeled catecholamines in neat aqueous buffer. The addition of micelles or microemulsion droplets into aqueous running buffer can dramatically improve the fluorescence response, and the enhancement seems to be comparable for micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC). As another alternative, NACE separation was advantageous when performing the analysis under the optimum separation condition of 20 mM sodium tetraborate, 20 mM sodium dodecyl sulfate (SDS), 0.1% (v/v) glacial acetic acid, 20% (v/v) acetonitrile (ACN) in methanol medium after derivatization in ACN/dimethyl sulfoxide (DMSO) (3:2, v/v) mixed aprotic solvents containing 20 mM ammonium acetate. Compared with derivatization and separation in aqueous media, NACE-LIF procedure was proved to be superior, providing high sensitivity and short migration time. Under respective optimum conditions, the NACE procedure offered the best fluorescence response with 5-24 folds enhancement for catecholamines compared to aqueous procedures. In addition, the mechanisms of derivatization and separation in nonaqueous media were elucidated in detail.     

Super-resolution microscopy of lipid bilayer phases

Sub-diffraction optical imaging with nanometer resolution of lipid phase-separated regions is reported. Merocyanine 540, a probe whose fluorescence is sensitive to the lipid phase, is combined with super-resolution imaging to distinguish the liquid- and gel-phase nanoscale domains of lipid bilayers supported on glass. The monomer-dimer equilibrium of MC540 in membranes is deemed responsible for the population difference of single-molecule fluorescence bursts in the different phase regions. The extension of this method to other binary or ternary lipid models or natural systems provides a promising new super-resolution strategy.     

CHOLESTEROL CONTENT BUT NOT PLASMA MEMBRANE FLUIDITY INFLUENCES THE SUSCEPTIBILITY OF L1210 LEUKEMIA CELLS TO MEROCYANINE 540-SENSITIZED IRRADIATION

This paper examines the relationship between lipid composition, plasma membrane fluidity, expression of dye binding sites, and susceptibility to merocyanine 540 (MC540)-sensitized irradiation in L1210 leukemia cells. Reducing the cells' cholesterol content by exchange diffusion with phosphatidylcholine liposomes or by inhibiting its biosynthesis with 25-hydroxycholesterol enhanced plasma membrane fluidity, the expression of dye binding sites, and the cells' susceptibility to MC540-sensitized irradiation. Conversely, if the cholesterol content was enhanced by exchange diffusion with cholesterol:phosphatidylcholine liposomes, the cells' susceptibility to MC540-sensitized irradiation was decreased. However, contrary to expectations, dye-binding was slightly enhanced and plasma membrane fluidity remained unchanged. Growing the cells in fatty acid-supplemented medium had profound effects on their lipid composition. Cells enriched in polyunsaturated fatty acids had more fluid plasma membranes. However, dye-binding was not significantly affected and photosensitivity was slightly reduced. These results suggest that cholesterol is one, but probably not the only, determinant of the expression of cellular dye binding sites and, consequently, the cell's susceptibility to MC540-sensitized irradiation. By contrast, plasma membrane fluidity does not appear to play a major role in the regulation of dye-binding site expression.     

Comparison of the performance of different reversed-phase columns for liquid chromatography separation of 11 pollutant phenols

A systematic optimization of the HPLC separation of a mixture containing 11 pollutant phenols (PPs) using a Hypersil ODS (250 mm x 4.6 mm, 5 microm) column and UV-DAD detection has been carried out. The binary mobile phases used were obtained by mixing 50 mM phosphate (pH = 3.0) and methanol, ACN, or THF as organic modifiers. After selecting ACN as an organic modifier, the effects of pH and temperature on PPs separation were studied. A mobile phase of 50 mM acetate (pH = 5.0)-ACN (60:40 v/v) at 50 degrees C allowed the separation of 11 phenols but not to baseline in 17 min. To improve the performance of this separation, the following RP columns were tested: Luna C18 (2), Purospher C18, Synergi C12, Synergi Fusion C18, Gemini C18, Luna Cyano, Lichrospher C8, and Envirosep-PP (polymeric). In all the cases, the performance (analysis time, retention, selectivity, resolution, asymmetry factors, and efficiency) was evaluated. A further reoptimization of the mobile phase was carried out for all the columns by studying the ACN content and pH, with the aim of improving the above-mentioned separations and selecting the most suitable one for PPs analysis.     

Simultaneous separation of nitrofuran antibiotics and their metabolites by using micellar electrokinetic capillary chromatography

Mixtures comprising nitrofuran antibiotics (NFA) and nitrofuran metabolites (NFM) were resolved for the first time by using MEKC. Sodium deoxycholate (SDC) was chosen as the micelle-forming surfactant. Optimization of separation conditions was achieved by using a central composite experimental design (CCD) approach. Experimental parameters such as concentration ratio of borate to phosphate in the buffer, pH of the running electrolyte and voltage were investigated. The effect of concentration of the surfactant on resolution was significant. Under optimal conditions of 80 mM SDC, pH 9.0, (20 mM borate + 20 mM phosphate) and 16 kV, the resolution between eight consecutive peak pairs ranged from 1.9 to 11.8. Due to the absence of a UV-active chromophore in the metabolites, they were derivatized with 2-nitrobenzaldehyde (2-NBA). In order to mimic a proposed extraction procedure for the analysis of both NFA and/or derivatized NFM in a sample, aqueous samples (prederivatized with 2-NBA) were extracted by using C(18) SPE cartridges. After washing with H(2)O, the cartridges were eluted with a small portion of organic solvent with weak elution characteristics to remove excess 2-NBA (hexane was chosen). Target analytes were then recovered with ACN. Excellent reproducibility of migration time (t(mig)) was achieved for all analytes using the developed MECC approach, with absolute t(mig) <1% RSD and t(mig) ratio <0.2% RSD, and peak area ratio was 4% RSD. The LOD for each compound, calculated by extrapolating to an S/N of 3, were found to be 0.19-2.0 microg/mL.     

Chiral capillary electrophoretic determination of the enantiomeric purity of homocamptothecin derivatives, promising antitumor topoisomerase I inhibitors, using highly sulfated CDs and fluorescence detection

EKC methods for the enantiomeric resolution of homocamptothecin derivatives, potent anticancer agents targeting DNA topoisomerase I selected for clinical trials, were developed using highly sulfated beta-CD as chiral selectors at acidic pH. Optimal electrophoretic conditions, with migration times under 15 min, were as follows: for the neutral homocamptothecin analog 1, a BGE of 75 mM phosphate buffer pH 2.5 (H(3)PO(4) + triethanolamine)/ACN - 95/5 v/v, with 7.5% w/v highly S-beta-CD, an applied field of 0.2 kV/cm and a fused capillary temperature control of 30 +/- 0.1 degrees C (typical current approximately 175 microA); for the cationic homocamptothecin 2, a BGE of 25 mM phosphate buffer pH 2.5 (H(3)PO(4) + TEA)/ACN - 90/10 v/v, with 2.5% w/v highly S-beta-CD, an applied field of 0.15 kV/cm and a fused capillary temperature control of 25 +/- 0.1 degrees C (typical current approximately 45 muA), and both are validated. The best results in terms of LOQ were obtained by EC with fluorescence detection: 10 ng/mL and 20 ng/mL for 1 and 2, respectively (LOQ divided by 150 for 1 and 5 for 2 with respect to UV), thus making this method particularly convenient for enantiomeric purity determination of galenic forms. UV detection appears to be an alternative to fluorescence for the analysis of the main component either for the control of galenic forms or for therapeutic adaptation. Moreover, this method exhibits better performances than HPLC.     

Photoisomerization of cyanine derivatives in 1-butyl-3-methylimidazolium hexafluorophosphate and aqueous glycerol: influence of specific interactions

Photoisomerization of two cyanine derivatives, 3,3(')-diethyloxadicarbocyanine iodide (DODCI) and merocyanine 540 (MC 540), has been investigated in an ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate and aqueous glycerol (93 wt % glycerol +7 wt % water) by measuring fluorescence lifetimes and quantum yields. The aim of this work is to understand how the rates of photoisomerization of DODCI and MC 540 are influenced by specific solute-solvent interactions besides the viscosity of the medium. For DODCI, it has been observed that the nonradiative rate constants, which represent the rates of photoisomerization, are almost identical in the ionic liquid and aqueous glycerol at given temperature, indicating that viscosity is the sole parameter that governs the rate of photoisomerization. In contrast, the photoisomerization rate constants of MC 540 have been found to be a factor of 2 higher in aqueous glycerol compared to the ionic liquid. The observed behavior is due to the zwitterionic character of MC 540, a consequence of which, the twisted state gets stabilized by the solute-solvent hydrogen bonding interactions in aqueous glycerol, thus lowering the barrier for isomerization.     

Micellar and aqueous‐organic liquid chromatography using sub‐2 μm packings for fast separation of natural phenolic compounds

The objective of the present work was to investigate the chromatographic behavior of natural phenolic compounds in micellar and aqueous‐organic LC using a short column packed with 1.8 μm particles. Firstly, the effect of ACN and SDS on elution strength and selectivity was examined by isocratic submicellar (0–30% ACN/5% 1‐butanol/1–6 mM SDS) and micellar (0–30% ACN/5% 1‐butanol/40–60 mM SDS) systems. The varied concentrations of two modifiers in the mobile phases revealed different eluting power. Then, the application of organic modifier gradient was discussed in both submicellar and micellar LC using mobile phases of 4 mM SDS/5% 1‐butanol or 50 mM SDS/5% 1‐butanol containing ACN gradient from 0 to 30%, respectively. For micellar system, the separation was found to be better in gradient than isocratic elution. Additionally, the sensitivity of aqueous‐organic LC was examined. The mobile phase was a mixture of ACN and water employing gradient elution at a flow rate of 0.5 mL/min, with analysis time below 9 min. It was found that separation efficiency was significantly better compared with micellar LC. Besides, the aqueous‐organic LC has been applied to separation of various phenolic compounds in Yangwei granule or Radix Astragali samples.     

Capillary electrophoresis of boron cluster compounds in aqueous and nonaqueous solvents

The electrophoretic properties of boron cluster compounds were determined in water, methanol and ACN as solvents of the BGE and discussed based on the principles of ion migration. Two types of boron cluster compounds were investigated. One type consisted of derivatives of the nido-7,8-dicarbaundecaborate cluster, the other types are derivatized cobalt bis(dicarbollide) ions (COSANs) whose central cobalt atom is sandwiched by two 7,8-dicarbaundecaborate clusters. The BGE in all solvents was acetate/acetic acid buffer with pH 4.75 in water, 9.7 in methanol and 22.3 in ACN, respectively, at different ionic strength between 5 and 30 mM. The dependence of the mobility on ionic strength could not be explained by the theory of Debye, Hückel and Onsager, but good agreement was found upon considering an ion size parameter. Limiting mobilities were derived by curve fitting, and by the aid of the solvent viscosities the hydrodynamic radii of the analyte anions were calculated. They are between 0.25 and 0.48 nm, and were nearly independent of the solvent. Electrophoresis of the analytes in a BGE consisting of 6 mM perchloric acid in ACN allows the conclusion that the present boron cluster compounds behave as stronger acids than perchloric acid.     

DIFFERENTIAL INACTIVATION OF SURROGATE VIRUSES WITH MEROCYANINE 540

Bacteriophages may be useful as surrogates for animal viruses when the virucidal properties of different photosensitizing compounds are initially investigated. We studied photoinactivation of four bacteriophages, phi X174, T7, PRD1, and phi 6, by the dye merocyanine 540 (MC540) (15 micrograms/mL). Merocyanine 540 (MC540) should be most effective with lipid-containing viruses, since it is primarily lipophilic (but also binds to proteins). Two of the phages, PRD1 and phi 6 contain lipid, with only phi 6 having an external lipoprotein envelope. Filtered radiation (450-600 nm) from a 750 W projector was used at 16-100 W/m2. The survival curves of the different viruses clearly demonstrated different levels of sensitivity to photoinactivation by MC540, with phi 6 (Do = 1.5 kJ/m2) being the most sensitive, followed by T7 (21-fold less sensitive). While both PRD1 and phi 6 have lipid components, only phi 6 was photoinactivated by MC540. Thus the internal lipid components of PRD1 were not sufficient to allow photoinactivation by this dye, at fluences up to 300 kJ/m2. For comparison, we also photoinactivated Herpes simplex virus (Do = 0.053 kJ/m2) and found it to be 28-fold more sensitive than phi 6 to photoinactivation by the same concentration of MC540. Thus phi 6 may be used as a surrogate for enveloped human viruses for photoinactivation by lipophilic dyes, but the results may only be useful qualitatively.     

Optimization of the separation conditions of tetracyclines on a preselected reversed-phase column with embedded urea group

The use of a C12 stationary phase with embedded polar group has been investigated for the separation of seven tetracyclines. The influence of pH, organic modifier, buffer, and temperature on the peak shape and analyte separation was discussed. It appears that all the chromatographic conditions had a great effect on both the resolution and peak shape whereas the elution order was not affected. The baseline separation with symmetrical peaks of the seven tetracyclines can be obtained with a mobile phase containing either 5 mM phosphate buffer pH 2.5/ACN (84:16 v/v) or 5 mM perchlorate buffer pH 2.5/ACN (75:25 v/v) at a temperature not exceeding 20 degrees C. This study reveals that the retention mechanism is ion-pairing.     

Interaction of merocyanine 540 with charged membranes.

In this work, phospholipid liposomes were used to investigate the influence of lipid negative charge on the interaction of merocyanine 540 (MC540) with model membranes. Liposomes were prepared from a mixture of neutral dimyristoyl lecithin (DMPC) and negatively charged dimyristoyl phosphatidic acid (DMPA). A strong dependence between the presence of charges on the membrane and dye association was found. The affinity of the dye to liposomes was decreased with an increasing content of DMPA in liposomes. Changes in absorption spectra of MC540 suggest that the decrease in affinity of MC540 to charged membranes is accompanied by a hypsochromic solvatochromic shift and changes in monomer/dimer equilibrium of MC540 incorporated in the membrane.     

TUMOR CELL SPECIFIC DARK CYTOTOXICITY OF LIGHT-EXPOSED MEROCYANINE 540: IMPLICATIONS FOR SYSTEMIC THERAPY WITHOUT LIGHT

Merocyanine 540 (MC540) was activated by exposure to 514 nm laser light. The light-exposed MC540 was then mixed (in the dark) with tumor cells and normal cells to determine the antiproliferative activity. Treatment with light-exposed MC540 resulted in 70-90% tumor cell kill from different cell lines, while 85% of the normal human mononuclear cells and 41% of the granulocyte-macrophage colony forming cells (CFU-GM) survived the treatment. The observed cytotoxicity of light-exposed MC540 to the tumor cells was significantly greater (P less than 0.05) than the native MC540. Results show that tumor cell specificity and cytotoxicity in the light activated dye are retained for at least 30 days. Addition of catalase and mannitol decreased the cell kill by light-exposed compound, indicating that the observed effects may be due to reactive oxygen species. The electron micrographs of treated cells show a progression towards apoptosis in a majority of the cells. The life span of L1210 leukemia-bearing mice treated with light-exposed MC540 was prolonged compared to the untreated and native MC540 treated mice. High pressure liquid chromatography (HPLC) analysis of light-exposed material shows a completely different elution profile compared to the native compound. Results presented here show that light-exposed photoactive compounds can be used without further illumination and may have significant clinical applications. Photoactive mechanisms dependent on events other than short-lived transient elevations in energy or singlet oxygen must be invoked to explain the reported cytotoxicity.