Study of tocopherol content and its potential antioxidant activity in commercial lipid emulsions for parenteral nutrition.

The objective of this study was to determine the content and evaluate the potential antioxidant effect of tocopherols in commercially available lipid emulsions, using a simple validated method adequate for further routine use. During the study, variability between manufacturers as well as between three non-consecutive batches of the same emulsion was observed. Furthermore, addition of α-tocopherol to lipid emulsions as excipient yields more stable emulsions and potentially a beneficial clinical effect. It was concluded that the variation of the tocopherol content between batches implies the importance of control and specification of tocopherol content by the manufacturers.

Chiral separations by non-aqueous capillary electrophoresis in DMSO-based background electrolytes.

Capillary electrophoresis (CE) is a powerful technique for enantioseparations due to its high separation efficiency, high versatility, speed of analysis and low consumption of samples and reagents. Non-aqueous capillary electrophoresis (NACE) appears as a promising technique to perform enantioseparations when the drugs, chiral selectors or samples are non-water soluble. Chiral separations have been performed by NACE mainly using alcoholic solvents as BGEs, with problems of current breakdowns and changes in the BGE composition, due to their high volatility. In this work, the suitability of DMSO as BGE in NACE has been evaluated. Different experimental variables affecting the enantioresolution of three drugs have been evaluated, finally achieving complete enantioresolution of two drugs (verapamil, Rs=1.5 and pindolol, Rs=2.0) and partial resolution of the third one (fenfluramine, Rs=1.2). DMSO has been demonstrated to be a good alternative to methanolic BGEs in NACE.

Mechanisms of cell migration in the adult brain: modelling subventricular neurogenesis.

Neurogenesis has been the subject of active research in recent years. Although the majority of neurons form during the embryonic period, neurogenesis continues in restricted regions of the mammalian brain well into adulthood. In rodent brains, neuronal migration is present in the rostral migratory stream (RMS), connecting the subventricular zone to the olfactory bulb (OB). The migration in the RMS is characterised by a lack of dispersion of neuroblasts into the surrounding tissues and a highly directed motion towards the OB. This study uses a simple mathematical model to investigate several theories of migration of neuroblasts through the RMS proposed in the literature, including chemo-attraction, chemorepulsion, general inhibition and the presence of a migration-inducing protein. Apart from the general inhibition model, all the models were able to provide results in good qualitative correspondence with the experimental observations.

A mechanobiological model of orthodontic tooth movement.

Orthodontic tooth movement is achieved by the process of repeated alveolar bone resorption on the pressure side and new bone formation on the tension side. In order to optimize orthodontic treatment, it is important to identify and study the biological processes involved. This article presents a mechanobiological model using partial differential equations to describe cell densities, growth factor concentrations, and matrix densities occurring during orthodontic tooth movement. We hypothesize that such a model can predict tooth movement based on the mechanobiological activity of cells in the PDL. The developed model consists of nine coupled non-linear partial differential equations, and two distinct signaling pathways were modeled: the RANKL-RANK-OPG pathway regulating the communication between osteoblasts and osteoclasts and the TGF-ß pathway mediating the differentiation of mesenchymal stem cells into osteoblasts. The predicted concentrations and densities were qualitatively validated by comparing the results to experiments reported in the literature. In the current form, the model supports our hypothesis, as it is capable of conceptually simulating important features of the biological interactions in the alveolar bone-PDL complex during orthodontic tooth movement.

Mechanobiological modeling can explain orthodontic tooth movement: three case studies.

Progress in medicine and higher expectation of quality of life has led to a higher demand for several dental and medical treatments. This increases the occurrence of situations in which orthodontic treatment is complicated by pathological conditions, medical therapies and drugs. Together with experiments, computer models might lead to a better understanding of the effect of pathologies and medical treatment on tooth movement. This study uses a previously presented mechanobiological model of orthodontic tooth displacement to investigate the effect of pathologies and (medical) therapies on the result of orthodontic treatment by means of three clinically relevant case studies looking at the effect of estrogen deficiency, the effect of OPG injections and the influence of fluoride intake. When less estrogen was available, the model predicted bone loss and a rise in the number of osteoclasts present at the compression side, and a faster bone resorption. These effects were also observed experimentally. Experiments disagreed on the effect of estrogen deficiency on bone formation, while the mechanobiological model predicted very little difference between the pathological and the non-pathological case at formation sites. The model predicted a decrease in tooth movement after OPG injections or fluoride intake, which was also observed in experiments. Although more experiments and model analysis is needed to quantitatively validate the mechanobiological model used in this study, its ability to conceptually describe several pathological conditions is an important measure for its validity.

Combined use of liquid chromatography with mass spectrometry and nuclear magnetic resonance for the identification of degradation compounds in an erythromycin formulation.

A commercial erythromycin formulation containing erythromycin A (EA) as the major compound showed the presence of an unknown degradation compound that was co-eluted with erythromycin E (EE) in the European Pharmacopoeia (Ph. Eur.) liquid chromatographic (LC) method. The amount of the degradation compound increased with respect to time. To separate this unknown (UNK1), investigation was performed with different LC methods coupled to ultraviolet detection (LC-UV). With the present Ph. Eur. method, the degradation compound could not be well separated. However, with the most selective LC-UV method (XTerra method), two more degradation products (UNK2 and UNK3) were found in the formulation which could not be observed using other methods because of their poor separation. By combining the results obtained with LC-UV, LC/MS and LC/NMR, the degradation products were identified as pseudoerythromycin A hemiketal (PsEAHK), erythromycin A enol ether carboxylic acid and erythromycin C enol ether carboxylic acid. PsEAHK is known to be a base-catalysed degradation product of EA, whereas the other two degradation products were newly identified.

Quantification of amikacin in bronchial epithelial lining fluid in neonates.

Amikacin efficacy is based on peak concentrations and the possibility of reaching therapeutic levels at the infection site. This study aimed to describe amikacin concentrations in the epithelial lining fluid (ELF) through bronchoalveolar lavage (BAL) in newborns. BAL fluid was collected in ventilated neonates treated with intravenous (i.v.) amikacin. Clinical characteristics, amikacin therapeutic drug monitoring serum concentrations, and the concentrations of urea in plasma were extracted from the individual patient files. Amikacin and urea BAL fluid concentrations were determined using liquid chromatography with pulsed electrochemical detection (LC-PED) and capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C(4)D), respectively. ELF amikacin concentrations were converted from BAL fluid concentrations through quantification of dilution (urea in plasma/urea in BAL fluid) during the BAL procedure. Twenty-two observations in 17 neonates (postmenstrual age, 31.9 [range, 25.1 to 41] weeks; postnatal age, 3.5 [range, 2 to 37] days) were collected. Median trough and peak amikacin serum concentrations were 2.1 (range, 1 to 7.1) mg/liter and 39.1 (range, 24.1 to 73.2) mg/liter; the median urea plasma concentration was 30 (8 to 90) mg/dl. The median amikacin concentration in ELF was 6.5 mg/liter, the minimum measured concentration was 1.5 mg/liter, and the maximum (peak) was 23 mg/liter. The highest measured ELF concentration was reached between 6 and 14.5 h after i.v. amikacin administration, and an estimated terminal elimination half-life was 8 to 10 h. The median and highest (peak) ELF amikacin concentrations observed in our study population were, respectively, 6.5 and 23 mg/liter. Despite the frequent use of amikacin in neonatal (pulmonary) infections, this is the first report of amikacin quantification in ELF in newborns.

Mass spectrometric characterization of gentamicin components separated by the new European Pharmacopoeia method.

Liquid chromatography combined with pulsed electrochemical detection (LC-PED) is the method of choice in the European Pharmacopoeia for the determination of gentamicin and its related substances. A recently approved improved LC-PED method, with a reversed-phase C(18) column and a mobile phase consisting of trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA), sodium hydroxide and acetonitrile, showed better separation and more sensitive detection of the gentamicin components than the previous method using a polymer column. More unknown peaks can be separated from the main components and from each other. As the LC-PED method cannot be directly coupled to a mass spectrometer (MS), the unknown substances were collected after the LC column, desalted and analyzed by MS. The structures of the unknown compounds were deduced based on comparison of their fragmentation patterns with those of reference substances investigated by MS(n) experiments using an electrospray ion trap mass spectrometer. A comparison was also made with an already previously published LC-MS method using a volatile mobile phase.

Characterization of impurities in tobramycin by liquid chromatography-mass spectrometry.

The characterization of unknown impurities present in tobramycin by liquid chromatography (LC) coupled with mass spectrometry (MS) is described. A reversed-phase (RP)-LC method using a volatile mobile phase containing a perfluorinated ion-pair reagent was developed and coupled with an ion trap mass spectrometer. The structures of the unknown impurities were deduced by comparison of their fragmentation patterns with those of the available reference substances obtained by LC-MS(n) experiments.

Characterization of impurities in dirithromycin by liquid chromatography/ion trap mass spectrometry.

With a recently developed liquid chromatographic (LC) method, using a phosphate buffer, several unknown impurities present in dirithromycin samples were separated. In this paper, a reversed-phase liquid chromatography-tandem mass spectrometry method is described for the investigation of dirithromycin and related substances. The method employed uses a Zorbax Extend C18 column (250 mm x 4.6 mm I.D.), 5 microm, and a mobile phase consisting of acetonitrile, 2-propanol, water and ammonium acetate solution pH 8.5. Mass spectral data are acquired on an LCQ ion trap mass spectrometer equipped with an electrospray ion (ESI) source operated in the positive ion mode. The LCQ is ideally suited for the identification of related substances because it provides on-line LC/MS(n) capability, which allows efficient identification without time-consuming isolation and purification procedures. Using this method, the fragmentation behavior of dirithromycin and its related substances was studied and the unknown impurities occurring in commercial samples were investigated. In total the structures of nine impurities were elucidated, among which three were different analogues with a modification in the side chain on the oxazine ring. Two impurities showed a different alkyl group in position C13. In two impurities the desosamine sugar was involved with changes in the degrees of methylation of the amino group. One unknown impurity was identified as dirithromycin F and another unknown was characterized as dirithromycin N-oxide.

Investigation of vancomycin and related substances by liquid chromatography/ion trap mass spectrometry.

Liquid chromatography (LC) methods compatible with mass spectrometry (MS) that are suitable for impurity profiling of vancomycin mixtures have not been described in the literature. The mobile phases of the existing methods contain non-volatile additives and/or solvents that give problems in combination with MS. In this paper, a reversed-phase LC/tandem mass spectrometry method is described for the investigation of vancomycin and related substances. The LC method uses a Zorbax Extend C18 column (250 x 4.6 mm i.d.), 5 microm, and a mobile phase consisting of methanol, water and ammonium acetate solution (pH 9.0). This method allows us to separate vancomycin and its impurities. Mass spectral data are acquired on an LCQ ion trap mass spectrometer equipped with an electrospray interface operated in the positive and negative ion modes. The LCQ is ideally suited for identification of impurities and related substances because it provides on-line LC/MSn capability, which allows efficient identification without time-consuming isolation and purification procedures. Using this method, the fragmentation of vancomycin and known derivatives was studied and the structures of six substances occurring in commercial samples were elucidated.

Interlaboratory study of a liquid chromatography method for erythromycin: determination of uncertainty.

Erythromycin is a mixture of macrolide antibiotics produced by Saccharopolyspora erythreas during fermentation. A new method for the analysis of erythromycin by liquid chromatography has previously been developed. It makes use of an Astec C18 polymeric column. After validation in one laboratory, the method was now validated in an interlaboratory study. Validation studies are commonly used to test the fitness of the analytical method prior to its use for routine quality testing. The data derived in the interlaboratory study can be used to make an uncertainty statement as well. The relationship between validation and uncertainty statement is not clear for many analysts and there is a need to show how the existing data, derived during validation, can be used in practice. Eight laboratories participated in this interlaboratory study. The set-up allowed the determination of the repeatability variance, s(2)r and the between-laboratory variance, s(2)L. Combination of s(2)r and s(2)L results in the reproducibility variance s(2)R. It has been shown how these data can be used in future by a single laboratory that wants to make an uncertainty statement concerning the same analysis.

Solid-phase synthesis of polymyxin B1 and analogues via a safety-catch approach.

As part of a program towards the development of novel antibiotics, a convenient method for solid-phase synthesis of the cyclic cationic peptide polymyxin B1 and analogues thereof is described. The methodology, based on cleavage-by-cyclization using Kenner's safety-catch linker, yields crude products with purities ranging from 37-67%. Antibacterial assays revealed that analogues 23-26, in which the (S)-6-methyloctanoic acid moiety is replaced with shorter acyl chains, exhibit distinct antimicrobial activity. The results suggest that the length of the acyl chain is rather critical for antimicrobial activity. On the other hand, substitution of the hydrophobic ring-segment D-Phe-6/Leu-7 in polymyxin B1 with dipeptide mimics (i.e. analogues 27-33) resulted in almost complete loss of antimicrobial activity.

Analysis of unknown compounds in azithromycin bulk samples with liquid chromatography coupled to ion trap mass spectrometry.

A selective reversed-phase liquid chromatography/mass spectrometry (LC/MS(n)) method is described for the identification of azithromycin impurities and related substances in commercial azithromycin samples. Mass spectral data are acquired on an LCQ ion trap mass spectrometer equipped with an atmospheric pressure chemical ionization interface operated in positive ion mode. The LCQ provides on-line LC/MS(n) capability, making it ideally suited for identification purposes. In comparison with UV detection, this hyphenated technique provides as its main advantage efficient identification of novel substances without time-consuming isolation and purification procedures. Using this technique, six novel related substances detected in commercial azithromycin samples have been studied.

Determination of kanamycin in serum by solid-phase extraction, pre-capillary derivatization and capillary electrophoresis.

An effective method based on solid-phase extraction (SPE) and capillary electrophoresis (CE) for the determination of kanamycin in human serum was developed and validated. Off-line SPE was employed for the isolation of kanamycin from serum on a carboxypropyl-bonded phase (CBA) weak cation-exchange cartridge. A mixture of 0.2 M borate (pH 10.5)-methanol (50:50, v/v) was used as analyte eluting solvent. After pre-capillary derivatization with o-phthalaldehyde/mercaptoacetic acid reagent, the sample was analyzed by CE with a separation buffer of 30 mM borax, pH 10.0, containing 16% (v/v) methanol. A linear response over the concentration range 5-40 microgram/ml was obtained with a detection limit of 2 microgram/ml. Intra-day and inter-day precision were 6.2 and 10.3% RSD, respectively. Recoveries of approximately 90% were found. For the determination of lower levels of kanamycin (<5 microgram/ml), NH(4)OH (25%, w/v)-methanol (30:70, v/v) was used for analyte elution. After evaporation, reconstitution and derivatization, the sample was analyzed by on-line field-amplified sample stacking (FASS) CE. Good linearity in the concentration range 0.4-5 microgram/ml was obtained with a detection limit of 0.1 microgram/ml. Intra-day and inter-day RSD were 3.4 and 11.2%, respectively. Recoveries of approximately 60% were found. The method was successfully applied to the analysis of kanamycin in sera of tuberculosis patients at peak level and trough level concentrations.

Study of the stability of polymyxins B(1), E(1) and E(2) in aqueous solution using liquid chromatography and mass spectrometry.

Polymyxins B(1), E(1) (colistin A) and E(2) (colistin B) were subjected to degradation in aqueous solutions of different pH values (1.4, 3.4, 5.4 and 7.4) and at different temperatures (37, 50 and 60 degrees C) in order to investigate the characteristics of decomposition. The progress of decomposition was followed by reversed-phase liquid chromatography on YMC-Pack Pro, C-18 stationary phase. The degradation curves showed (pseudo) first order kinetics. The pH-rate profiles indicate that colistin is more susceptible to degradation in solutions of pH above 5 and is more stable in acidic media. The degradation of polymyxin B(1) was most rapid at pH 7.4. Qualitative analysis of the degradation products by LC/MS reveals that racemization is the major mechanism of degradation in both acidic and neutral media.

Analysis of clindamycin by micellar electrokinetic chromatography with a mixed micellar system.

This study details the development and validation of an optimized method with micellar electrokinetic chromatography for the analysis of clindamycin. The method uses a mixed micellar phase containing anionic sodium dodecylsulfate (SDS) and non ionic Brij 35 on an untreated fused-silica capillary. The influences of buffer concentration, pH, SDS, Brij 35 and organic modifier were investigated. Special attention was given to the role of the non ionic Brij 35 in the mixed micellar system. Optimization with a central composite design resulted in optimal separation conditions: background electrolyte containing 25 mM sodium tetraborate pH 7.75, 90 mM SDS, 14 mM Brij 35 and 21% acetonitrile. The applied voltage was 15 kV and the capillary temperature 15 degrees C. The method was robust and gave good linearity and repeatability. The limits of detection and quantitation were 0.05 and 0.15%, respectively, relative to a 2.5 mg/ml clindamycin solution. Two commercial bulk products were analysed with this system.

Electrophoretically mediated microanalysis of gentamicin with in-capillary derivatization and UV detection.

This paper describes a system for integration of a one-step-microscale chemical derivatization and analysis by a methodology known as electrophoretically mediated microanalysis (EMMA). Differential electrophoretic mobility between an analyte, reagent, and their product offers EMMA a unique capability to selectively carry out electrophoretic mixing, control product formation, and separation. This system was successfully applied to perform derivatization and separation of the multicomponent aminoglycoside antibiotic gentamicin using 1,2-phthalic dicarboxaldehyde and mercaptoacetic acid as labeling reagents. A multivariate approach based on central composite experimental design was used to optimize the derivative yield. Full automation of the derivatization and analytical procedure, high derivatization efficiency, high sample throughput, and precision are the excellent features of the present method. In addition, this methodology offers short analysis time, as well as selectivity and sensitivity suitable for impurities determination. Separation of gentamicin C1, C1a, C2, C2a, C2b, sisomicin, and several minor components was achieved. For the first time separation and identification of three impurities, namely garamine, 2-deoxystreptamine, and paromamine are described.

Isolation and structural characterization of colistin components.

Preparative-scale separation of colistin sulphate bulk sample was carried out on a preparative poly(styrene-divinylbenzene) stationary phase. Isocratic elution with acetonitrile-sodium sulphate solution (0.7% m/v; pH adjusted to 2.5 with TFA) - water (16:50:34, % v/v/v) was carried out at a flow rate of 4.0 ml min(-1). Six colistin components were isolated and characterized using 1H and 13C NMR. The molecular weights were confirmed by mass spectrometry. The structures of 2 components were determined for the first time. Polymyxin E7 was identified as having the same composition as polymyxin E1, except that the fatty acid moiety was 7-methyloctanoic acid. Isoleucine polymyxin E8 was characterized as having the same composition as isoleucine polymyxin E1 with 7-methylnonanoic acid as the fatty acid moiety.

Analysis of vancomycin and related impurities by micellar electrokinetic capillary chromatography. Method development and validation.

A fast and highly selective micellar electrokinetic capillary chromatography (MEKC) method for quantitative analysis of vancomycin and related impurities is described. Among the tested surfactants, cetyltrimethylammonium chloride (CTAC) offered the best selectivity. Another important parameter, which strongly influenced the selectivity, was buffer pH. It was found that the selectivity increased with buffer pH decreasing from 9 to 5. Using Tris-phosphate buffer containing CTAC, satisfactory separation could be obtained in the pH range from 5.0 to 5.5. Excellent repeatability in terms of migration time and peak area could be obtained when the capillary was carefully washed between two runs. In order to obtain optimal conditions and to evaluate the method robustness, a central composite experimental design was carried out. The optimal conditions were: 44 cm length of fused-silica capillary with 50 microm ID, 120 mM Tris-phosphate buffer (pH 5.2) containing 50 mM CTAC, -15 kV applied voltage, UV detection at 210 nm, and a column temperature of 25 degrees C. Under the optimal conditions, more than 20 peaks could be separated within 8 min. The method has a linearity range from 0.004 to 1.2 mg/ml (concentration of vancomycin B, active component). The limit of detection (LOD) and limit of quantitation (LOQ) were 0.4 microg/mL vancomycin, equivalent to 0.3 microg/mL vancomycin B (0.04%) and 1.1 microg/mL vancomycin, equivalent to 0.9 microg/mL vancomycin B (0.1%), respectively.