Evaluation of Different Light Conditions in the Working Environment for Handling Photosensitive and Thermolabile Compounds.

Lighting in the working environment plays a significant role on the degree of degradation of photosensitive, thermolabile compounds and on working efficiency. Light emitting diodes (LEDs) are semiconductor light emitting devices that are promising artificial light sources with easy modulation of light wave signals and are also known for low heat generation. Therefore, the effect of polychromatic LED light was tested in the working environment using the drug compounds montelukast, nifedipine, and clavulanic acid, which are known to be photosensitive or thermolabile. As a control, other lighting sources like a sodium lamp, a classic (incandescent, tungsten) lamp, and indirect sunlight were also used in this study. All the experiments were carried out with methanolic solutions at room temperature. An Acquity UPLC/MS/MS system was used for quantification of the main analytes and degradation products. Under the tested conditions, LED lighting proved to be more suitable for handling photosensitive and thermolabile compounds.

Development and validation of a reversed-phase liquid chromatographic method for analysis of demeclocycline and related impurities.

A simple, robust, and rapid reversedphase high-performance liquid chromatographic method for the analysis of demeclocycline and its impurities is described. Chromatographic separations were achieved on a Symmetry Shield RP8 (75 mm × 4.6 mm, 3.5 µm) column kept at 40°C. The mobile phase was a gradient mixture of acetonitrile, 0.06 M sodium edetate (pH 7.5), 0.06 M tetrapropylammonium hydrogen sulphate (pH 7.5) and water, A (2:35:35:28 v/v/v/v) and B (30:35:35:0 v/v/v/v) pumped at a flow rate of 1 mL/min. UV detection was performed at 280 nm. The developed method was validated according to the ICH guidelines for specificity, limit of detection, limit of quantification, linearity, precision, and robustness. An experimental design was applied for robustness study. Results show that the peak shape, chromatographic resolution between the impurities, and the total analysis time are satisfactory and better than previous methods. The method has been applied for the analysis of commercial demeclocycline bulk samples available on the market.

Identification of the oxidation products of cysteamine and cystamine by CE-MS interfaced by a noncommercial electrospray ionization source.

Sodium cysteamine phosphate is a prodrug derivative of cysteamine that can be used in cystinosis treatment. Although titrimetric assays are very well established and precise, iodimetric determination of sodium cysteamine phosphate requires considerably more carefulness and time from the analyst than usual. The possibility to assess sodium cysteamine phosphate by CE was evaluated by means of the quantification of its oxidation product, cystamine, which is a more suitable substance to be used as primary standard than sodium cysteamine phosphate. Apparently, this approach should be straightforward, but systematic differences between the results obtained with CE and titrimetric assays were noticed. MS and CE-MS were employed to aid in the investigation of the possible causes of imprecision of the sodium cysteamine phosphate titration and CE determination. For this purpose, a simple and inexpensive ESI source was constructed. It was observed that cystamine is not the final product of the cysteamine and/or sodium cysteamine phosphate iodine-oxidation and other species besides cystamine may be formed depending on the reaction conditions, which explains the difficulties observed in the sodium cysteamine phosphate quantification.

Identification of the major photodegradant in metronidazole by LC-PDA-MS and its reveal in compendial methods.

Metronidazole in aqueous solution is sensitive to light and UV irradiation, leading to the formation of N-(2-hydroxyethyl)-5-methyl-l,2,4-oxadiazole-3-carboxamide. This is revealed here by liquid chromatography with tandem photo diode array detection and mass spectrometry (LC-PDA-MS) and further verified by comparison with the corresponding reference substance and proton nuclear magnetic resonance (1H-NMR). However, in current compendial tests for related substances/organic impurities of metronidazole, the above photolytic degradant could not be detected. Thus, when photodegradation of metronidazole occurs, it could not be demonstrated. In our study, an improved LC method was developed and validated, which includes a detection at a wavelength of 230 nm and optimization of mobile phase composition thereby a better separation was obtained.

In-capillary screening of matrix metalloproteinase inhibitors by electrophoretically mediated microanalysis with fluorescence detection.

A capillary electrophoresis-based method with enzymatic reaction inside the capillary for the screening of matrix metalloproteinase (MMP) inhibitors has been developed. MMP-2 and MMP-9, which have been considered as promising targets for cancer therapy, were selected as the model enzymes. The hydrolysis of a fluorogenic substrate catalyzed by MMPs was determined by measuring the increase in fluorescence. For high-throughput screening, the short-end injection was employed. The enzyme, substrate containing inhibitors, and enzyme solutions were injected from the outlet of the capillary via the sandwich mode. They were mixed by alternating the potential at positive and negative polarities. Online hydrolysis, separation, and detection were achieved in 70 s with approximately 0.87 fmol of MMP required for each assay. The applicability of electrophoretically mediated microanalysis (EMMA) with fluorescence detection to estimate the inhibitory mechanism and to determine the IC(50) values was evaluated for two natural inhibitors, epigallocatechin gallate and oleic acid. A few other natural compounds such as resveratrol, quercetin, caffeic acid, glucosamine, and doxycycline were also screened to test their inhibitory potency. The results obtained were compared with those obtained by offline enzyme assay and confirm the effectiveness of the present method. A rapid, cost-effective, and fully automated method for MMP inhibitor screening is proposed.

An improved liquid chromatographic method for the analysis of tylosin and its impurities.

A selective reversed-phase (RP) liquid chromatographic (LC) method coupled with UV for the determination of tylosin and its related substances is described. The gradient method uses a Capcell pak C18 ACR column (25 cm×4.6 mm id, 5 µm) maintained at a temperature of 60°C. The mobile phases consist of acetonitrile, phosphate buffer pH 5.5 and water: (A; 27.5:10:62.5 v/v/v) and (B; 50:10:40 v/v/v). The flow rate is 1.0 mL/min and UV detection is performed at 280 nm. It allows the separation of all known and 22 other unknown related substances (≥0.02%) from the main compound and from one another. The method shows good precision, sensitivity, linearity (between 0.2 µg/mL and 1.25 mg/mL) and robustness. The limit of quantification is 0.2 µg/mL, corresponding to 0.020%. Seven bulk tylosin samples containing a large number of impurities were examined using this method.

Capacitively coupled contactless conductivity detection as an alternative detection mode in CE for the analysis of kanamycin sulphate and its related substances.

A method was developed to determine simultaneously kanamycin, its related substances and sulphate in kanamycin sulphate using capacitively coupled contactless conductivity detection. Kanamycin is an aminoglycoside antibiotic that lacks a strong UV-absorbing chromophore. Due to its physicochemical properties, CE in combination with capacitively coupled contactless conductivity detection was chosen. The separation method uses a BGE composed of 40 mM 2-(N-morpholino)ethanesulphonic acid monohydrate and 40 mM L-histidine, pH 6.35. A 0.6 mM N-cetyltrimethyl ammonium bromide (CTAB) solution was added as electroosmotic flow modifier in a concentration below the critical micellar concentration (CMC). Ammonium acetate 50 mg/L was used as internal standard. In total, 30 kV was applied in reverse polarity on a fused-silica capillary (65/41 cm; 75 µm id). The optimized separation was obtained in less than 6 min with good linearity (R(2)=0.9999) for kanamycin. It shows a good precision expressed as RSD on the relative peak areas equal to 0.3 and 1.1% for intra-day and inter-day precision, respectively. The LOD and LOQ are 0.7 and 2.3 mg/L, respectively. Similarly, for sulphate, a good linearity (R(2)=0.9996) and precision (RSD 0.4 and 0.6% for intra-day and inter-day, respectively) were obtained.

Mixed aqueous solutions as dilution media in the determination of residual solvents by static headspace gas chromatography.

Static headspace (HS) sampling has been commonly used to test for volatile organic chemicals, usually referred to as residual solvents (RS) in pharmaceuticals. If the sample is not soluble in water, organic solvents are used. However, these seriously reduce the sensitivity in the determination of some RS. Here, mixed aqueous dilution media (a mixture of water and an organic solvent like dimethyl formamide, dimethyl sulfoxide or dimethyl acetamide) were studied as alternative media for static HS-gas chromatographic analysis. Although it has been known that mixed aqueous dilution media can often improve sensitivity for many RS, this study used a systematic approach to investigate phase volumes and the organic content in the HS sampling media. Reference solutions using 18 different class 1, 2 and 3 RS were evaluated. The effect of salt addition was also studied in this work. A significant increase in the peak area was observed for all RS using mixed aqueous dilution media, when compared with organic solvents alone. Matrix effects related to the mixed aqueous dilution media were also investigated and reported. Repeatability and linearity obtained with mixed aqueous dilution media were found to be similar to those observed with pure organic solvents.

Characterization and improvement of signal drift associated with electron ionization quadrupole mass spectrometry.

Quadrupole mass spectrometry with electron ionization (EI-QMS) is a very popular detection technique in combination with gas chromatography. It is deployed for the analysis of volatile and semivolatile analytes in many industry domains. Although a very important factor for quantitative analysis, little is known about the stability of ion source performance. Only a few papers and patents report possible signal instabilities due to sample adsorption, degradation, or insulating deposits on the hot stainless steel surface of the ion source. In this study, a conventional stainless steel ion source was used to investigate possible signal drifts. It was observed that the EI-QMS instrument indeed suffered from continuous signal instability. It was found that the key parts which are responsible for the signal instabilities are those that regulate the ion beam toward the mass analyzer: the repeller, exit plate, and focusing lenses. The voltage of the repeller was found to have a major influence on the signal stability. The surface of the repeller, exit plate, and focusing lenses was modified by applying a gold coating. It was demonstrated that the signal stability of the MS dramatically improved when using the gold-coated parts. The contribution of each part to the stability improvement was quantitatively determined and compared with the standard stainless steel source performance. It was assumed that the signal drift observed with the stainless steel EI source originated from charge buildup on the surfaces. This hypothesis was supported by software simulations.

Recent developments and applications of EMMA in enzymatic and derivatization reactions.

This review covers the time period of 2007 until mid-2009 and describes new developments in the field of electrophoretically mediated microanalysis. The review is subdivided in two parts dealing with (i) enzymatic and (ii) derivatization or chemical reactions. A compilation of the relevant literature is given for each part.

Application of carbon nanotubes for in-capillary incubations with cytochrome P450 enzymes.

The utility of multi-walled carbon nanotubes (MWNTs) to decrease the interaction between cytochrome P450 enzymes and the capillary wall during in-capillary enzymatic incubation was investigated. First, 18 surfactants were screened to determine their MWNT-dispersing capacity. A probe sonication procedure was developed in order to attain homogeneous MWNT dispersions within a reasonable time. Next, the influence of surfactants and MWNTs on P450 activity was studied, employing verapamil and CYP3A4 as model substrate and P450 isoform, respectively. MWNTs dispersed in Brij 35 did not affect CYP3A4 activity significantly and were selected for subsequent in-capillary tests. An in-line CE assay, involving electrophoretic mixing of reagents and zero potential amplification in the thermostatted part of the capillary, was developed. In-capillary incubations without MWNTs caused adsorption of enzyme to the capillary wall and a concomitant decline of capillary lifetime, even when extensive between-run rinsing was applied. Addition of MWNTs to the enzyme solution entailed substantial improvement of migration time and peak shape repeatability. The performance of three types of MWNTs was compared.

On-line drug metabolites generation and their subsequent target analysis by capillary zone electrophoresis with UV-absorption detection.

This study presents the in-capillary enzymatic biotransformation of dextromethorphan, an antitusive drug and opioid receptor antagonist, and subsequent electrophoretic separation of its products. The study includes the optimization of separation parameters to fulfill the requirements of an online microreaction. The analyses were performed in a bare fused-silica capillary using 100 mM sodium tetraborate (pH 10.0) mixed with linear polyacrylamide (20%, v/v) and 2-propanol (10%, v/v). This BGE was suitable for monitoring both off-line and in-capillary incubations. The partial filling technique enabled the enzymatic reaction to be carried out in its optimal environment (20 mM sodium phosphate, pH 7.4). Finally, in-capillary microreaction in the presence of cytochrome P450 3A4 gave satisfactory outcomes.

A rapid and sensitive CE method with field-enhanced sample injection and in-capillary derivatization for selenomethionine metabolism catalyzed by flavin-containing monooxygenases.

A rapid and sensitive electrophoretically mediated microanalysis method with field-enhanced sample injection (FESI) for in-capillary derivatization was developed to determine selenomethionine (SeMet) and selenomethionine selenoxide (SeOMet). Phthalic anhydride (PA) was selected as the derivatization reagent due to the fast reaction at room temperature and the stability of derivatives. The in-capillary derivatization was accomplished by electrophoretically mixing PA and sample plugs. PA reagent was introduced hydrodynamically into the capillary, whereas the sample solution was injected electrokinetically, thus allowing a selective preconcentration of the analytes by FESI. For FESI, the optimum sample solvent was 2 mM borate solution. The borate buffer was suitable for both in-capillary derivatization and separation of the derivatives. The combination of electrophoretically mediated microanalysis with FESI for in-capillary derivatization was successfully achieved with about 800-fold concentration sensitivity enhancement compared to direct CE-UV detection in the same setup. The present method is miniaturized and fully automated, which ensures the on-line derivatization, stacking, separation and detection in 10 min. Finally, the developed method was successfully applied to measure enzyme activities by analyzing the reaction mixtures of SeMet with human flavin-containing monooxygenases (FMO). The results showed that both FMO1 and FMO3, but not FMO5 could catalyze the Se-oxygenation of SeMet.

Development and validation of an improved reversed-phase liquid chromatographic method combined with pulsed electrochemical detection for the analysis of netilmicin.

Netilmicin is one of the aminoglycoside antibiotics that lacks a strong UV absorbing chromophore. However, the application of pulsed electrochemical detection has been used successfully for the direct analysis of aminoglycoside antibiotics. This study describes an improved LC method combined with pulsed electrochemical detection for the analysis of netilmicin. Using a Zorbax SB C-18 column (250 mm x 4.6 mm id, 5 microm), isocratic elution was carried out with a mobile phase containing sodium sulfate (20 g/L), sodium octanesulfonate (0.3 g/L), THF (20 mL/L), and 0.2 M phosphate buffer pH 3.0 (50.0 mL/L). The robustness of the method was examined by means of an experimental design. The method proved to be sensitive, repeatable, linear, and robust. The method has also been used to analyze some commercial netilmicin samples.

Development of a validated liquid chromatographic method for the determination of related substances and assay of tenofovir disoproxil fumarate.

The present study describes the development and validation of a selective liquid chromatographic (LC) method for the analysis of tenofovir disoproxil fumarate (TDF) and its related substances. The gradient method uses a base deactivated C18 column (Hypersil BDS column; 25 cmx4.6 mm I.D.) maintained at a temperature of 30 degrees C. The mobile phases consist of acetonitrile, tetrabutylammonium/phosphate buffer pH 6.0 and water: (A; 2:20:78 v/v/v) and (B; 65:20:15 v/v/v). The flow rate is 1.0 mL/min and UV detection is performed at 260 nm. Good separation of TDF and 21 impurities was achieved. A system suitability test (SST) to check the quality of separation is also specified. The developed method was further validated with respect to robustness, precision, sensitivity and linearity. The method is proved to be robust, precise, sensitive and linear between 0.1 microg/mL and 0.15 mg/mL. The limit of detection and limit of quantification are 0.03 and 0.1 microg/mL, respectively. The method was successfully applied to the quantification of related substances and assay of commercial TDF samples (bulk substances and tablets).

Enantioselective in-line and off-line CE methods for the kinetic study on cimetidine and its chiral metabolites with reference to flavin-containing monooxygenase genetic isoforms.

An in-line screening and an off-line chiral CE method were developed to determine the stereoselectivity of flavin-containing monooxygenase (FMO) isoforms using cimetidine (CIM) as a substrate. The S-oxygenation of CIM was investigated using achiral chemical oxidants and (human supersomes) enzymatic metabolism procedures. In the off-line setup, the chiral selector sulfobutylether-beta-CD was chosen to separate the CIM S-oxide (CSO) metabolites. The electrophoretic migration order of CSO was confirmed to be (+) before (-) through the use of single enantiomers obtained by preparative chromatography. For the electrophoretically mediated microanalysis method, the in-line enzymatic reaction was performed in 100 mM phosphate reaction buffer (pH 8.3), whereas 50 mM phosphate buffer with 30 mM chiral selector (pH 2.5) was used as a BGE. During the screening of FMO isoenzymes by the electrophoretically mediated microanalysis method, formation of the new chiral center on the CIM sulfur was found to be stereoselective. FMO1 produces more (-)-CSO-enantiomer, while FMO3 generates mainly (+)-CSO-enantiomer. On the other hand, FMO5 shows no activity. The kinetic constants of FMO1 and FMO3 were measured by the off-line method. A K(m)=4.31 mM for the formation of the (+)-CSO-enantiomer and a K(m)=4.56 mM for the (-)-CSO-enantiomer are reported for the first time for FMO1.

Characterization of dihydrostreptomycin-related substances by liquid chromatography coupled to ion trap mass spectrometry.

Dihydrostreptomycin sulphate (DHS) is a water-soluble, broad-spectrum aminoglycoside antibiotic. For quantitative analysis, the European Pharmacopoeia (Ph. Eur.) prescribes an ion-pairing liquid chromatography/ultraviolet (LC/UV) method using a C18 stationary phase. Several unknown compounds were detected in commercial samples. Hence, for characterization of these unknown peaks in a commercial DHS sample, the Ph. Eur. method was coupled to mass spectrometry (MS). However, since the Ph. Eur. method uses a non-volatile mobile phase, each peak eluted was collected and desalted before introduction into the mass spectrometer. The desalting procedure was applied to remove the non volatile salt, buffer and ion-pairing reagent in the collected fraction. In total, 20 impurities were studied and 14 of them were newly characterized. Five impurities which are already reported in the literature were also traced in this LC/UV method.

Capillary zone electrophoresis method development for the analysis of Hippeastrum hybrid agglutinin samples.

A capillary zone electrophoresis (CZE) method was developed aiming the analysis of Hippeastrum hybrid agglutinin (HHA) samples. HHA is presently being tested as a vaginal microbicide to prevent HIV transmission. It acts by direct binding to mannose residues that are abundantly present on the HIV gp120 envelope and so interrupts the virus entry process. The final CZE method employs 50mM sodium tetraborate (pH 9.9) as background electrolyte. In this condition, a cluster of about 30 isoform peaks is obtained, with very repeatable patterns. RSDs in the order of 0.2% for the migration time and detection sensitivity in the order of 70microgml(-1) were achieved.

Development and validation of an LC method for the determination of emtricitabine and related compounds in the drug substance.

A robust, precise, sensitive, linear, and simple RP LC method coupled with UV for the determination of emtricitabine or 2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC) and its related substances is described. The method uses an RP C18 column (25 cm x 4.6 mm i.d.), 5 microm kept at a temperature of 35 degrees C. The mobile phases for gradient elution consist of ACN, phosphate buffer (pH 4.4), and water. The flow rate is 1.0 mL/min and UV detection is performed at 280 nm. A system suitability test (SST) was developed to verify the adequate performance of the chromatographic system. The developed method was further validated with respect to robustness, precision, sensitivity, and linearity. A central composite design was applied to examine the robustness of the method. The method shows good precision, sensitivity, linearity, and robustness. Three commercial FTC samples were examined using this method. This method is suitable to be used for the determination of related substances and assay of FTC.

LC-MS of streptomycin following desalting of a nonvolatile mobile phase and pH gradient.

Streptomycin (SM) is composed of streptidine, streptose and N-methyl glucosamine sugar moieties. For the determination of SM and its related substances, an ion-pair LC-UV detection method using a Supelcosil LC-ABZ column was developed previously. While analyzing commercial samples, several unknown compounds were detected. Most of these compounds are not yet characterized. In this study, the above LC method was coupled to MS for impurity profiling in a selected commercial sample. However, it could not be directly coupled to MS due to the presence of the nonvolatile salt, buffer and ion-pair reagent in the mobile phase. So, for structural characterization, each peak eluted from the nonvolatile eluent system was collected and transferred to MS after the desalting process. In total, 16 compounds were studied, 15 compounds including 12 unknowns could be identified.