Kris Wolfs

On-line screening of matrix metalloproteinase inhibitors by capillary electrophoresis coupled to ESI mass spectrometry

Capillary electrophoresis (CE) with the use of mass spectrometry (MS) has been considered as a unique tool for microscale enzyme assay and inhibitor screening. In this study, matrix metalloproteinase-9 (MMP-9) was selected as target enzyme due to its important role in tumor invasion and metastasis. In order to define the optimal MS parameters, a two level half fraction factorial experimental design was performed. A background electrolyte consisting of 20mM ammonium acetate (pH 6.8) and a sheath liquid of water-methanol (50:50, v/v) containing 0.05% formic acid at a flow rate of 4μl/min were selected. This system was operated in the positive ion mode with a detection-limit of 10nM for the MMP reaction product and provided 60 folds enhancement of sensitivity by using selected reaction monitoring detection compared with MS full scan mode, which significantly increased the detectability of the system and therefore reduced the enzyme reaction time in both off-line and in-line mode. Both electrophoretically mediated microanalysis and pressure mediated microanalysis combined with MS detection were investigated for MMP inhibitor screening. Good repeatability (RSD of peak area and migration time were lower than 5.0%) and linearity (R(2)>0.996) were obtained for both in-capillary approaches. Several tetracycline antibiotics and natural products were selected to test the system. The results indicated an agreement on the ranking of inhibitory potency for both in-capillary approaches.

Static headspace gas chromatography of (semi-)volatile drugs in pharmaceuticals for topical use

An analytical method that allows simultaneous analysis of some (semi-)volatile drugs and additives in pharmaceuticals for topical use was developed using classical static headspace as sampling technique combined with gas chromatography (HS-GC). The capillary column used, RSL-200, showed good selectivity towards all the analytes in the samples investigated. Among the different dilution media investigated, dimethyl sulfoxide in combination with sodium chloride solution showed better sensitivity. Using the optimized headspace sample dilution medium, better sensitivities for all the analytes were achieved at low thermostatting temperature (85 degrees C). The optimized HS-GC method with flame ionization detection showed good repeatability, linearity and accuracy.

Full evaporation headspace gas chromatography for sensitive determination of high boiling point volatile organic compounds in low boiling matrices

Determination of volatile organic components (VOCs) is often done by static headspace gas chromatography as this technique is very robust and combines easy sample preparation with good selectivity and low detection limits. This technique is used nowadays in different applications which have in common that they have a dirty matrix which would be problematic in direct injection approaches. Headspace by nature favors the most volatile compounds, avoiding the less volatile to reach the injector and column. As a consequence, determination of a high boiling solvent in a lower boiling matrix becomes challenging. Determination of VOCs like: xylenes, cumene, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N,N-dimethylacetamide (DMA), N-methyl-2-pyrrolidone (NMP), 1,3-dimethyl-2-imidazolidinone (DMI), benzyl alcohol (BA) and anisole in water or water soluble products are an interesting example of the arising problems. In this work, a headspace variant called full evaporation technique is worked out and validated for the mentioned solvents. Detection limits below 0.1 μg/vial are reached with RSD values below 10%. Mean recovery values ranged from 92.5 to 110%. The optimized method was applied to determine residual DMSO in a water based cell culture and DMSO and DMA in tetracycline hydrochloride (a water soluble sample).

Optimization and validation of liquid chromatography and headspace-gas chromatography based methods for the quantitative determination of capsaicinoids, salicylic acid, glycol monosalicylate, methyl salicylate, ethyl salicylate, camphor and l-menthol in a topical formulation

Capsaicinoids, salicylic acid, methyl and ethyl salicylate, glycol monosalicylate, camphor and l-menthol are widely used in topical formulations to relieve local pain. For each separate compound or simple mixtures, quantitative analysis methods are reported. However, for a mixture containing all above mentioned active compounds, no assay methods were found. Due to the differing physicochemical characteristics, two methods were developed and optimized simultaneously. The non-volatile capsaicinoids, salicylic acid and glycol monosalicylate were analyzed with liquid chromatography following liquid-liquid extraction, whereas the volatile compounds were analyzed with static headspace-gas chromatography. For the latter method, liquid paraffin was selected as compatible dilution solvent. The optimized methods were validated in terms of specificity, linearity, accuracy and precision in a range of 80% to 120% of the expected concentrations. For both methods, peaks were well separated without interference of other compounds. Linear relationships were demonstrated with R² values higher than 0.996 for all compounds. Accuracy was assessed by performing replicate recovery experiments with spiked blank samples. Mean recovery values were all between 98% and 102%. Precision was checked at three levels: system repeatability, method precision and intermediate precision. Both methods were found to be acceptably precise at all three levels. Finally, the method was successfully applied to the analysis of some real samples (cutaneous sticks).

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.

Ethanol concentrations in the human gastrointestinal tract after intake of alcoholic beverages

INTRODUCTION The goal of this study was to monitor gastric and duodenal ethanol concentrations arising from the consumption of commonly used alcoholic beverages. MATERIALS AND METHODS In a cross-over study, five fasting volunteers were asked to drink two standard consumptions of commercially available alcoholic beverages, including beer (Stella Artois®, 500 mL, 5.2% ethanol), wine (Blanc du Blanc®, 200 mL, 11% ethanol) and whisky (Gallantry Whisky®, 80 mL, 40% ethanol). The volunteers finished drinking beer within 10 min and wine or whisky within 5 min. Ethanol concentrations in gastric and duodenal fluids, aspirated as a function of time, were analyzed by headspace gas chromatography. RESULTS In all three conditions, the average gastric profile shows a maximum ethanol concentration (Cmax) at 7 min, while the mean duodenal profiles have a Tmax at 20, 7 and 12 min for beer, wine and whisky, respectively. The median gastric ethanol Cmax (min-max) for the beer, wine and whisky conditions amounts to 4.1% (3.1-4.1), 4.1% (2.6-7.3) and 11.4% (6.3-21.1), respectively. The mean duodenal profiles follow the same pattern as their corresponding gastric profiles, albeit with lower percentages of ethanol. Median duodenal ethanol Cmax (min-max) for beer, wine and whisky are 1.97% (0.89-4.3), 2.39% (2.02-5.63) and 5.94% (3.55-17.71), respectively. Intraluminal ethanol concentrations appear to decline relatively rapidly in fasting conditions: both stomach and duodenum contained less than 0.05% of ethanol after 120 min. CONCLUSIONS This in vivo study is the first to present intraluminal ethanol concentrations in man after the intake of alcoholic beverages. Relatively low and fast declining gastric ethanol concentrations were observed, contrasting with the current Food and Drug Administration guidelines for the in vitro testing of formulations with respect to ethanol resistance. The presented gastric and duodenal ethanol concentrations and their variation may serve as reference data to design relevant models for predicting (i) ethanol resistance of drug formulations and (ii) ethanol effects on drug solubility and permeability.

Liquid paraffin as new dilution medium for the analysis of high boiling point residual solvents with static headspace-gas chromatography

Residual solvents are volatile organic compounds which can be present in pharmaceutical substances. A generic static headspace-gas chromatography analysis method for the identification and control of residual solvents is described in the European Pharmacopoeia. Although this method is proved to be suitable for the majority of samples and residual solvents, the method may lack sensitivity for high boiling point residual solvents such as N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide and benzyl alcohol. In this study, liquid paraffin was investigated as new dilution medium for the analysis of these residual solvents. The headspace-gas chromatography method was developed and optimized taking the official Pharmacopoeia method as a starting point. The optimized method was validated according to ICH criteria. It was found that the detection limits were below 1μg/vial for each compound, indicating a drastically increased sensitivity compared to the Pharmacopoeia method, which failed to detect the compounds at their respective limit concentrations. Linearity was evaluated based on the R(2) values, which were above 0.997 for all compounds, and inspection of residual plots. Instrument and method precision were examined by calculating the relative standard deviations (RSD) of repeated analyses within the linearity and accuracy experiments, respectively. It was found that all RSD values were below 10%. Accuracy was checked by a recovery experiment at three different levels. Mean recovery values were all in the range 95-105%. Finally, the optimized method was applied to residual DMSO analysis in four different Kollicoat(®) sample batches.

Evaluation of the European Pharmacopoeia method for control of residual solvents in some antibiotics

Residual solvents (RS) are volatile organic chemicals that are used or produced during the manufacturing process of drug substances or excipients. The European Pharmacopoeia (Ph. Eur.) limits the amount of RS in pharmaceuticals, considering the International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines for RS. According to the Ph. Eur. general method, water insoluble samples may be analyzed using DMF as dilution solvent at high equilibration temperatures such as 105 degrees C. This could be problematic in the case of antibiotics, many of which are water insoluble and temperature sensitive. Moreover, antibiotics are complex in nature and beside RS, one can expect several other volatile impurity peaks in the chromatogram. In this study, the Ph. Eur. method for RS analysis was evaluated for selected groups of antibiotics. An alternative dilution medium was proposed (DMSO-water), which offers optimum sensitivity while working at lower equilibration temperatures such as 80 degrees C. The optimized method was investigated for precision, accuracy, linearity and detection limits.

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.

Exploring the possibilities of capacitively coupled contactless conductivity detection in combination with liquid chromatography for the analysis of polar compounds using aminoglycosides as test case

The analysis of highly polar (often charged) compounds which lack a strong UV absorbing chromophore is really challenging. Despite the numerous analytical methods published, the demand for a simple, robust and cheap technique for their analysis still persists. Here, reversed phase (RP) liquid chromatography (LC) with capacitively coupled contactless conductivity detection (C(4)D) was explored for the first time as a possible method for separation and detection of various aminoglycoside (AMG) antibiotics which were taken as typical test compounds: tobramycin (TOB), spectinomycin, streptomycin, amikacin, kanamycin A and kanamycin B. C(4)D was performed using a commercially available as well as a laboratory made cell. As ion-pairing reagents (IPR) four perfluorinated carboxylic acids were used: pentafluoropropionic acid, heptafluorobutyric acid, nonafluoropentanoic acid (NFPA) and pentadecafluorooctanoic acid (PDFOA). 0.125 mM NFPA-acetonitrile (ACN) (90:10) or 0.125 mM PDFOA-ACN (70:30) as mobile phases were suitable to detect TOB with reasonable retention times. However, NFPA was preferred for practical reasons. Its applicable concentration range in the mobile phase was strongly restricted by loss of chromatographic performance at lower levels and excessive background conductivity at higher levels. Overall repeatability and robustness of the method were rather poor which was explained by the relatively low IPR levels. Selectivity between the tested AMGs was mainly influenced by the number of protonated amino groups per molecule making it impossible to separate compounds of equal net charges. Problems encountered with gradient elution, hydrophilic interaction liquid chromatography (HILIC) and separation at high pH without IPRs are also discussed.