NIR-Fluorescent Multidye Silica Nanoparticles with Large Stokes Shifts for Versatile Biosensing Applications.

We have synthesized and characterized of a series of single and multidye copolymerized nanoparticles with large to very large Stokes shifts (100 to 255 nm) for versatile applications as standalone or multiplexed probes in biological matrices. Nanoparticles were prepared via the Stöber method and covalently copolymerized with various combinations of three dyes, including one novel aminocyanine dye. Covalently encapsulated dyes exhibited no significant leakage from the nanoparticle matrix after more than 200 days of storage in ethanol. Across multiple batches of nanoparticles with varying dye content, the average yields and average radii were found to be highly reproducible. Furthermore, the batch to batch variability in the relative amounts of dye incorporated was small (relative standard deviations <2.3%). Quantum yields of dye copolymerized nanoparticles were increased 50% to 1000% relative to those of their respective dye-silane conjugates, and fluorescence intensities were enhanced by approximately three orders of magnitude. Prepared nanoparticles were surface modified with polyethylene glycol and biotin and bound to streptavidin microspheres as a proof of concept. Under single wavelength excitation, microsphere-bound nanoparticles displayed readily distinguishable fluorescence signals at three different emission wavelengths, indicating their potential applications to multicolor sensing. Furthermore, nanoparticles modified with polyethylene glycol and biotin demonstrated hematoprotective qualities and reduced nonspecific binding of serum proteins, indicating their potential suitability to in vivo imaging applications.

A Comparative Study of Fluorescein Isothiocyanate-Encapsulated Silica Nanoparticles Prepared in Seven Different Routes for Developing Fingerprints on Non-Porous Surfaces.

Preparation of fluorescein isothiocyanate (FITC)-encapsulated silica nanoparticles (F-SiNPs) via seven different approaches to be used as developing agents for fingerprints detection is presented in this report. In this study, the suitability of each synthesis route toward incorporation of the selected fluorophore into silica matrix and its efficiency in fingerprints detection were systematically studied. The composition of the particles was designed to examine the hydrophobic and dipole-dipole interactions between the silicate backbone and both of the fluorescent reporter molecules and the fingerprint residues. F-SiNPs were prepared with two conventional sol-gel approaches; the Stöber method and the water in oil reverse microemulsion (WORM) method. The alkoxysilane precursor, tetraethoxyorthosilicate (TEOS) and its binary mixtures with phenyltriethoxysilane (PTEOS) or 3-aminopropyl triethoxysilane (APTES) have been used in preparing the F-SiNPs to study the effect of nanoparticles composition on fingerprints development. In addition, FITC was conjugated with APTES so it can be covalently bonded to the silica matrix and to be compared with non-covalently FITC-doped SiNPs. Moreover, the enhancement effect of introducing polyvinylpyrrolidone (PVP) onto the surface of the less hydrophobic F-SiNP on fingerprints detection on different non-porous surfaces was also investigated. The mean diameters of the F-SiNPs were between 4.1 ± 0.6 and 110.4 ± 31.1 nm as obtained from the TEM size measurement for the nanoparticles prepared by the WORM and Stöber methods, respectively. The obtained results clearly highlight the advantages of using a mixture of TEOS and PTEOS alkoxysilane precursors in preparing F-SiNPs with remarkable encapsulation efficiency and clear detection of fingerprints due to efficient embedding of the fluorophore inside the silica network even without conjugation. It was also observed that both the Stöber and WORM methods can be used in preparing the F-SiNPs developing agents and that PVP coated particles did not show any significant enhancement in fingerprints visualization.

Stability-indicating RP-LC method for determination of azilsartan medoxomil and chlorthalidone in pharmaceutical dosage forms: application to degradation kinetics.

A RP-LC method was developed and validated for simultaneous determination of the active components, azilsartan medoxomil (AZL) and chlorthalidone (CLT), in their novel antihypertensive combined recipe. The chromatographic separation was achieved on an Eclipse XDB-C18 (4.6 × 150 mm, 5 µm) column using a mobile phase consisting of methanol/potassium hydrogen phosphate buffer (pH 8, 0.05 M) (40:60, v/v) in isocratic mode. The flow rate was maintained at 0.8 mL min(-1) at ambient temperature. Detection was carried out at 210 nm. The method was validated according to the ICH guidelines. Linearity, accuracy, and precision were satisfactory over the concentration range of 5.0-50.0 and 2.5-25.0 µg mL(-1) for AZL and CLT, respectively (r (2) = 0.9999). LODs for AZL and CLT were 0.90 and 0.32 µg mL(-1), whereas LOQs were 2.72 and 0.98 µg mL(-1), respectively. Both drugs were subjected to forced degradation studies under hydrolysis (neutral, acidic, and alkaline), oxidative, and photolytic extensive stress conditions. The proposed method is stability indicating by the resolution of the investigated drugs from their degradation products. Moreover, the kinetics of the acidic degradation of AZL as well as the kinetics of the alkaline degradation of CLT were investigated. Arrhenius plots were constructed and the apparent first-order rate constants, half-life times, shelf-life times, and the activation energies of the degradation processes were calculated. The method was successfully applied for the determination of the studied drugs simultaneously in their coformulated tablet. The developed method is specific and stability indicating for the quality control and routine analysis of the cited medications in their pharmaceutical preparations.

Simultaneous determination of aliskiren hemifumarate, amlodipine besylate, and hydrochlorothiazide in their triple mixture dosage form by capillary zone electrophoresis.

A novel, specific, reliable, and accurate capillary zone electrophoretic method was developed and validated for the simultaneous determination of aliskiren hemifumarate, amlodipine besylate, and hydrochlorothiazide in their triple mixture dosage form. Separation was carried out in a fused-silica capillary (57.0 cm total length and 50.0 cm effective length, 75.6 µm internal diameter) by applying a potential of 17 kV and a running buffer consisting of 40 mM phosphate buffer at pH 6.0 with UV detection at 245 nm. The method was suitably validated with respect to specificity, linearity, LOD, and LOQ, accuracy, precision, and robustness. The method showed good linearity in the ranges 1-10, 2.5-25, and 30-300 µg/mL with LODs of 0.11, 0.33, and 5.83 µg/mL for amlodipine besylate, hydrochlorothiazide, and aliskiren hemifumarate, respectively. The proposed method was successfully applied for the analysis of the studied drugs in their coformulated tablets. The results of the proposed method were statistically compared with those obtained by the RP-HPLC reference method revealing no significant differences in the performance of the methods regarding accuracy and precision.

Synchronized separation of seven medications representing most commonly prescribed antihypertensive classes by using reversed-phase liquid chromatography: Application for analysis in their combined formulations.

A reversed-phase high-performance liquid chromatography method was developed for the simultaneous determination of the diuretic, hydrochlorothiazide, along with six drugs representing the most commonly prescribed antihypertensive pharmacological classes such as atenolol, a selective ß1 blocker, amlodipine besylate, a calcium channel blocker, moexipril hydrochloride, an angiotensin-converting-enzyme inhibitor, valsartan and candesartan cilexetil, which are angiotensin II receptor blockers, and aliskiren hemifumarate, a renin inhibitor, using irbesartan as an internal standard. The chromatographic separation was achieved using acetonitrile/sodium phosphate dibasic buffer (0.02 M, pH 5.5) at a flow rate of 1 mL/min in gradient elution mode at ambient temperature on a stationary phase composed of an Eclipse XDB-C18 (4.6 × 150 mm, 5 µm) column. UV detection was carried out at 220 nm. The method was validated according to ICH guidelines. Linearity, accuracy, and precision were satisfactory over the concentration ranges of 2-40 µg/mL for hydrochlorothiazide and candesartan cilexetil, 20-120, 10-160, 5-40, 20-250, and 5-50 µg/mL for atenolol, valsartan, moexipril hydrochloride, aliskiren hemifumarate, and amlodipine besylate, respectively. The method was successfully applied for the determination of each of the studied drugs in their combined formulations with hydrochlorothiazide. The developed method is suitable for the quality control and routine analysis of the cited drugs in their pharmaceutical dosage forms.

Steady-state and synchronous spectrofluorimetric methods for simultaneous determination of aliskiren hemifumarate and amlodipine besylate in dosage forms.

Aliskiren hemifumarate (ALS) and amlodipine besylate (AML) were simultaneously determined by two different spectrofluorimetric techniques. The first technique depends on direct measurement of the steady-state fluorescence intensities of ALS and AML at 313 nm and 452 nm upon excitation at 290 and 375 nm, respectively, in a solvent composed of methanol and water (10: 90, v/v). The second technique utilizes synchronous fluorimetric quantitative screening of the emission spectra of ALS and AML at 272 and 366 nm, respectively using Δλ of 97 nm. Effects of different solvents and surfactants on relative fluorescence intensity were studied. The method was validated according to ICH guidelines. Linearity, accuracy and precision were found to be satisfactory in both techniques over the concentration ranges of 1-15 and 0.4-4 µg/mL for ALS and AML, respectively. In the first technique, limit of detection and limit of quantification were estimated and found to be 0.256 and 0.776 µg/mL for ALS as well as 0.067 and 0.204 µg/mL for AML, respectively. Also, limit of detection and limit of quantification were calculated in the synchronous method and found to be 0.293 and 0.887 µg/mL for ALS as well as 0.034 and 0.103 µg/mL for AML, respectively. The methods were successfully applied for the determination of the two drugs in their co-formulated tablets. The results were compared statistically with reference methods and no significant difference was found. The developed methods are rapid, sensitive, inexpensive and accurate for the quality control and routine analysis of the cited drugs in bulk and in pharmaceutical preparations without pre-separation.

Preparation of fluorescently labeled silica nanoparticles using an amino acid-catalyzed seeds regrowth technique: Application to latent fingerprints detection and hemocompatibility studies.

The efficiency of an amino acid catalyzed seed regrowth technique (ACSRT) in synthesizing twelve fluorescently labeled core-shell silica nanoparticles (FLSNPs) with tunable sizes, tailored hydrophobicity, low polydispersity as well as high labeling efficiency and minimized dye leakage using different combinations of organosilicate monomers and fluorophores have been systematically investigated in this report. The utilization of some of these FLSNPs in some applications that are facilitated by hydrophobicity such as developing and visualizing latent fingerprints (LFPs) on different surfaces was also investigated. The non-specific binding affinity of the developed nanoparticles to human serum albumin (HSA) and immunoglobulin G (IgG) has also been studied. Fluorescein, fluorescein isothiocyanate and its more hydrophilic butenamine derivative (WA6) have been used in this study. Also, the alkoxysilane precursor, tetraethoxyorthosilicate (TEOS) and its binary mixture with phenyltriethoxysilane (PTEOS) or 3-aminopropyl triethoxysilane (APTES) have been used in preparing the FLSNPs with tailored compositions for the core and shell of the nanoparticles. The mean diameters of the PTEOS-coated FLSNPs were between 33.4±5.9 and 42.2±10.8 nm as shown by the SEM measurements. The obtained results highlight the advantages of having a hydrophobic surface along with proper selection of the monomers forming the core to match the properties of the fluorescent reporters for clear detection of LFPs even using dyes of low hydrophobicity such as fluorescein and WA6. Furthermore, some of the developed FLSNPs were compared with bare silica nanoparticles in terms of nonspecific protein adsorption and hemolysis. The obtained results proved that the selected FLSNPs had a superior hemocompatibility in comparison with bare silica nanoparticles. These FLSNPs could also be used in some bio-related and diagnostic applications such as immunoassays and cell imaging purposes.

A new strategy utilizing electrospray ionization-quadrupole ion trap mass spectrometry for the qualitative determination of GnRH peptides.

Numerous forms of the neurotransmitter GnRH have been discovered in vertebrates and invertebrates. Methods used for identification of these peptides are laborious and often require the application of multiple, confirmatory techniques. In this study, we investigate whether HPLC-MS/MS and de novo sequencing techniques applied to whole peptide analysis can provide a simpler approach to GnRH characterization. Experiments were performed with six GnRH forms (chicken I, chicken II, lamprey III, mammalian, salmon and seabream) to determine whether MS/MS spectra would be dominated by proline-directed fragmentation to the detriment of obtaining sufficient fragmentation for sequencing. While the expected b8 fragment was prominent, sufficient ion series were obtained for the six GnRH peptides to provide sequence identification. On the basis of the patterns observed for six model peptides, similar fragmentation patterns are expected for other GnRH forms. To confirm the applicability of the method, extracts from Sprague-Dawley rat brains were examined. These experiments confirm the presence of mammalian GnRH and a posttranslationally modified form of mammalian GnRH, hydroxyproline9 GnRH, in Sprague-Dawley rat brains and demonstrate that ESI-MS/MS techniques provide a valuable addition to existing qualitative methods.

Spectroscopic study of a novel bis(heptamethine cyanine) dye and its interaction with human serum albumin.

A newly synthesized near-infrared (NIR) bis(heptamethine cyanine) dye 7 was evaluated for its utility as a non-covalent label for proteins. This dye forms inter- and intramolecular H-aggregates in polar solvents, even at very low concentrations. The intramolecular dimeric form of the dye can be described as a clam-shell complex with two interacting hydrophobic carbocyanine moieties. In this intramolecular H-aggregate, the chromophore has a low extinction coefficient and low fluorescence quantum yield. In aqueous solution, in the absence of human serum albumin (HSA), dye 7 has characteristic absorption bands at 792 and 435 nm, and its fluorescent emission is significantly diminished in comparison to that in methanol or when compared to its monomeric equivalent 5. Dye 7 seems to be more advantageous than its monomeric counterpart 5 as a non-covalent label for biomolecules. Upon addition of HSA, the H and D bands are decreased and the monomeric band is increased, with concomitant increase in fluorescence intensity, suggesting that clam-shell H-aggregates open up in the complex with HSA. The binding stoichiometry is 1:1. The main advantage of this dimeric dye as a non-covalent label is that the free dye has negligible fluorescence.

Simultaneous Determination of Aliskiren Hemifumarate, Amlodipine Besylate and Hydrochlorothiazide in Spiked Human Plasma Using UPLC-MS/MS.

A sensitive UPLC-MS/MS method was developed and validated for simultaneous estimation of aliskiren hemifumarate (ALS), amlodipine besylate (AML) and hydrochlorothiazide (HCZ) in spiked human plasma using valsartan as an internal standard (IS). Liquid-liquid extraction was used for purification and pre-concentration of analytes. The mobile phase consisted of 0.1% formic acid in ammonium acetate buffer (0.02 M, pH 3.5) and methanol (25:75, v/v), flowing through XBridge BEH (50 × 2.1 mm ID, 5 µm) C18 column, at a flow rate of 0.6 mL min(-1). Multiple reaction monitoring (MRM) transitions were measured using an electrospray source in the positive ion mode for ALS and AML, whereas HCZ and IS were measured in negative ion mode. Validation of the method was performed as per US-FDA guidelines with linearity in the range of 2.0-400.0, 0.3-25.0 and 5.0-400.0 ng mL(-1) for ALS, AML and HCZ, respectively. In human plasma, ALS, AML and HCZ were stable for at least 1 month at -70 ± 5°C and for at least 6 h at ambient temperature. After extraction from plasma, the reconstituted samples of ALS, AML and HCZ were stable in the autosampler at ambient temperature for 6 h. The LC-MS/MS method is suitable for bioequivalence and pharmacokinetic studies of this combination.

DNA and protein applications of near-infrared dyes.

The near-infrared region of the spectrum (650-1100 nm) offers distinct advantages over the traditional UV/vis region for spectroscopic measurements. In the past, the lack of commercially available equipment capable of working in the near infrared limited the utility of near-infrared techniques. However, since the advent of photodiodes and semiconductor lasers, much progress has been made in the development of near-infrared techniques. This paper discusses the use of near-infrared dyes used in DNA and protein applications.

Covalent and noncovalent labeling schemes for near-infrared dyes in capillary electrophoresis protein applications.

Capillary electrophoresis (CE) is experiencing increased use in the field of separation science. Part of its growing popularity of capillary electrophoresis can be attributed to the high efficiency of the separations achievable with the technique, making it an attractive tool for bioanalytical applications. Laser-induced fluorescence (LIF) is a common detection method for CE. One of the problems frequently experienced when using visible LIF detection is matrix autofluorescence which has the effect of degrading the overall sensitivity of the technique. However, the use of near-infrared (NIR) laser induced fluorescence nearly eliminates matrix autofluorescence, as very few molecules have intrinsic fluorescence in this region. This chapter describes the use of covalent and noncovalent labeling schemes for tagging biomolecules with near infrared dyes. To fully appreciate the advantages that the NIR LIF technique can supply, we also review applications that employ detection schemes other than NIR LIF. Specific applications to be discussed include drug-protein studies by CE, as well as capillary electrophoretic immunoassays.

Handling and detection of 0.8 amol of a near-infrared cyanine dye by capillary electrophoresis with laser-induced fluorescence detection.

We are interested in the detection of DNA adducts and other trace analytes by labeling them with a fluorescent tag followed by use of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) for high resolution and sensitivity. Towards this goal, here we report the following: (1) synthesis and handling properties of a near-IR, carboxyl-substituted heptamethine cyanine dye; (2) modification of an existing ball lens LIF detector to provide near-LIF detection with excitation at 785 nm for CE; and (3) corresponding handling and detection of as little as 0.8 amol of the dye by enrich-injection of 4.7 microl of 1 x 10(-13) mol/l dye in methanol from an 8-microl volume into a corresponding CE-LIF system. The electrolyte for the separation was methanol-40 mmol/l aqueous sodium borate (98:2, v/v). This finding encourages further exploration of the dye by functionalization of its carboxyl group for chemical labeling purposes.

High-throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography-tandem mass spectrometry.

A method has been developed for the high-throughput inhibition screening of the major human cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) using an in vitro substrate cocktail and liquid chromatography-tandem mass spectrometry (LC-MS-MS). A cocktail consisting of the selective substrates phenacetin (CYP1A2), tolbutamide (CYP2C9), omeprazole (CYP2C19), bufuralol (CYP2D6), and midazolam (CYP3A4) was incubated with human liver microsomes. The metabolic reactions were terminated with methanol containing dextrorphan as an internal standard. Following centrifugation, the supernatant was analyzed by LC-MS-MS employing a fast gradient. The concentrations of the substrate metabolites-paracetamol, 4-hydroxytolbutamide, 5-hydroxyomeprazole, 1'-hydroxybufuralol, and 1'-hydroxymidazolam-in each sample were determined by LC-MS-MS in a single assay. The method was validated by incubating known CYP inhibitors (furafylline, CYP1A2; sulfaphenazole, CYP2C9; s-mephenytoin, CYP2C19; quinidine, CYP2D6; and troleandomycin, CYP3A4) with the individual substrates they were known to inhibit and with the substrate cocktail. IC50s (microM) determined using the substrate cocktail were in good agreement with those obtained with individual substrates (furafylline, 2.9 vs. 2.0; sulfaphenazole, 0.75 vs. 0.72; s-mephenytoin, 170 vs. 180; quinidine, 0.17 vs. 0.24; troleandomycin, 2.6 vs. 3.2) and with previously reported values in the literature.

Investigation of the spectral properties of a squarylium near-infrared dye and its complexation with Fe(III) and Co(II) ions.

The spectral features of the squarylium dye NN525 in different solutions and its complexation with several metal ions were investigated. The absorbance maximum of the dye is at 669 nm in tetrahydrofuran. This value matches the output of a commercially available laser diode (650 nm), thus making use of such a source practical for excitation. The emission maximum of the dye in tetrahydrofuran is at 676 nm. The addition of either Fe(III) ion or Co(II) ion resulted in fluorescence quenching of the dye. The detection limit is 6.24 x 10(-8) M for Fe(III) ion and 1.55 x 10(-8) M for Co(II) ion. The molar ratio of the metal to the dye was established to be 1:1 for both metal ions. The stability constant Ks of the metal-dye complex was calculated to be 3.14 x 10(6) M(-1) for the Fe-dye complex and 2.64 x 10(5) M(-1) for the Co-dye complex.

Noncovalent labeling of biomolecules with red and near- infrared dyes.

Biopolymers such as proteins and nucleic acids can be labeled with a fluorescent marker to allow for their detection. Covalent labeling is achieved by the reaction of an appropriately functionalized dye marker with a reactive group on a biomolecule. The recent trend, however, is the use of noncovalent labeling that results from strong hydrophobic and/or ionic interactions between the marker and biomolecule of interest. The main advantage of noncovalent labeling is that it affects the functional activity of the biomolecule to a lesser extent. The applications of luminescent cyanine and squarylium dyes are reviewed.