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The determination of the relative amounts of the B and C components of neomycin sulphate by thin-layer chromatography using silica gel plates from Whatman as the stationary phase is described. The mobile phase consisted of methanol-20% (m/v) sodium chloride solution (15:85). Fluorescence detection was performed after derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. The influence of different parameters on the separation was investigated. A number of commercial samples was analysed using this method and the results were compared with results obtained with ion-exchange chromatography and ninhydrin colorimetric detection, which is the official method prescribed by the European Pharmacopoeia. The described method is much easier to perform than the official method.

A liquid chromatography method using poly(styrene-divinylbenzene) as the stationary phase was used as the analytical method to investigate the stability of the macrolide antibiotic erythromycin in neutral and alkaline solutions (pH 7.5–11). Decomposition of erythromycin A was monitored up to 20% of degradation. Three degradation products were detected: pseudoerythromycin A enol ether, pseudoerythromycin A hemiketal and a hydrolysis product of erythromycin A, i.e., the product with an opened lactone structure. The decomposition kinetics have been studied as a function of the ionic strength, the concentration of the buffer, the amount of methanol in the solvent and the temperature.

An isocratic liquid chromatographic method for the assay and purity control of josamycin using wide-pore poly(styrene-divinylbenzene) as the stationary phase is presented. Josamycin has been well separated from all the related leucomycins, present as impurities in commercial samples. Wide-pore PLRP-S 8 μm 1000 A has shown the best selectivity. The separation has been carried out at 65°C with a mobile phase of 2-methyl-2-propanol/0.2 mol/l potassium phosphate buffer pH 10.5/water (24.5: 5: 70.5, V/V) and a flow rate of 1.0 ml/min. UV detection has been performed at 232 nm. The total analysis time has been about 40 min. The method has been used to analyse commercial bulk samples and tablets containing josamycin base.

A liquid chromatographic method that can separate each of a series of diamino analogues of 2′- or 3′-deoxyadenosine from their main degradation product, adenine, is described. A PLRP-S 1000 A (8 μm) 250 mm × 4.6 mm. I.D. column was used at 60°C. The method was developed by systematic evaluation of the influence of the mobile phase pH, the type and concentration of the organic modifier, the concentration of the ion-pairing agent and the buffer. The mobile phase consisted of tetrahydrofuran-0.2 M sodium octanesulphonate-0.2 M potassium phosphate buffer (pH 2.0)-water (15:25:30:30, v/v).

A simple, robust and fast high-performance liquid chromatographic method is described for the analysis of oxytetracycline and its related impurities. The principal peak and impurities are all baseline separated in 20 min using an Inertsil C₈ (150 mm × 4.6 mm, 5 μm) column kept at 50 °C. The mobile phase consists of a gradient mixture of mobile phases A (0.05% trifluoroacetic acid in water) and B (acetonitrile-methanol-tetrahydrofuran, 80:15:5, v/v/v) pumped at a flow rate of 1.3 ml/min. UV detection was performed at 254 nm. The developed method was validated for its robustness, sensitivity, precision and linearity in the range from limit of quantification (LOQ) to 120%. The limits of detection (LOD) and LOQ were found to be 0.08 μg/ml and 0.32 μg/ml, respectively. This method allows the separation of oxytetracycline from all known and 5 unknown impurities, which is better than previously reported in the literature. Moreover, the simple mobile phase composition devoid of non-volatile buffers made the method suitable to interface with mass spectrometry for further characterization of unknown impurities. The developed method has been applied for determination of related substances in oxytetracycline bulk samples available from four manufacturers. The validation results demonstrate that the method is reliable for quantification of oxytetracycline and its impurities.

Tobramycin is one of the aminoglycoside antibiotics that lack a UV absorbing chromophore. However, the application of pulsed electrochemical detection (PED) has been used successfully for the analysis of this and similar antibiotics. This work describes an improved liquid chromatographic (LC) method combined with PED, which is able to separate much more impurities than before. Using a Discovery C-18 RP column (250 mm×4.6 mm i.d., 5 μm), isocratic elution was carried out with a mobile phase, containing sodium sulfate (35 g/L), sodium octanesulphonic acid (1 g/L), tetrahydrofuran (14 mL/L) and 0.2 M phosphate buffer pH 3.0 (50 mL/L). Using these experimental conditions, the limit of quantification (LOQ, S/N=10) was 5 ng. The linearity was examined in the range LOQ-60 μg/mL and the coefficient of determination was 0.998. The method also proved to be repeatable and the recovery was close to 100%. The influence of the different chromatographic parameters on the separation was investigated by means of an experimental design. The proposed method is useful in quality control of tobramycin drug substances and drug products. ispartof: Journal of Pharmaceutical Analysis vol:3 issue:3 pages:161-167 ispartof: location:China status: published

Investigation of unknown impurities in a tylosin sample was performed using liquid chromatography coupled to mass spectrometry (LC/MS). Separation was performed according to the recently described LC-UV method of Ashenafi et al. (2011) [14]. This method was reported to have a good selectivity as it was able to separate the four main components of tylosin from the already known and 23 unknown impurities. However, as this method uses a mobile phase with non-volatile constituents, direct characterization of these impurities using LC/MS was not possible. The impurity fractions were therefore first collected and then desalted before sending them to the MS. Identification of the impurities in the tylosin sample was performed with a quadruple ion trap (IT) MS, with an electrospray ionization (ESI) source in the positive ion mode. The structure of the impurities was deduced by comparing their fragmentation pattern with those of the main components of tylosin. As several peaks in the LC-UV method contained multiple compounds, using this method in total 41 new impurities were (partly) characterized.

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.

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.

A reversed phase (RP)-LC method using a C 18 column resistant to basic pH and an alkaline (pH 10) aqueous mobile phase was developed and coupled to MS with an electrospray ionization (ESI) source in the positive ion mode which provides MS n capability. In total, 26 impurities were detected in a commercial sample. The structures of the impurities were proposed based on comparison of their fragmentation patterns with those of the available reference substances, the synthetic route and literature data. Starting material and its residual impurities, intermediates, synthetic by-products and degradation products were the main sources of those impurities. 14 impurities described in this work were newly identified.

The characterization of unknown (UNK) impurities in capreomycin (CMN) using liquid chromatography coupled to mass spectrometry (LC/MS) has been described. The ion-pair liquid chromatography method coupled to ultraviolet detection (LC-UV) described by Mallampati et al. was used for the separation of CMN from its related substances. As the method uses non-volatile reagents it could not be directly coupled to mass spectrometry (MS) for impurity characterization. So, these UNK impurities were collected and desalted before sending to MS for structural characterization. As no information about the fragmentation of the main components of CMN, except for CMN IB, was available in the literature, they were studied first. Next, the structures of the impurities were deduced by comparing their fragmentation to that of the main components of CMN. Fourteen UNK impurities that were never described before, were (partly) characterized.

Cefpiramide is susceptible to many factors, including high temperature. Two solid states were observed in cefpiramide: a crystalline form and an amorphous one, and both the accelerated (40°C ± 2°C /75% ± 5% relative humidity (RH)) and stress (60°C ± 2°C / 75% ± 5% RH) stability study demonstrated that the crystalline form of cefpiramide was much more stable than its amorphous form, with related substances, high molecular mass polymers and assay as critical stability indicating parameters. Thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and water content results indicated that each molecule of crystalline cefpiramide may contain two molecules of crystal water, which can explain its high thermal stability. This study provides valuable information for cefpiramide and its drug products with respect to the improvement of its thermal stability through crystallization.

A capillary zone electrophoretic method was developed for monitoring the stability of hexopyranosyi analogues of thymidine. The final analytical conditions adopted were as follows: capillary, fused silica, 70 cm (62 cm to detector) × 75 μm I.D.; background electrolyte, 20 mM sodium tetraborate (pH 10.5); voltage, 30 kV; detection, UV at 262 nm; and temperature. 15°C. The performance of this analytical system is discussed. The stability study at three pH values allows to compare the stability of the analogues with that of thymidine and to investigate the influence of the configuration of the N-glycosidic bond on the rate constant of degradation.

The development of a selective and sensitive method for analysis of erythromycin A (EA) and its metabolites in biological fluids (urine and plasma) is reported here. A mobile phase consisting of acetonitrile - 2-methyl-2-propanol - 0.2 M phosphate buffer pH 9.0 - water (3:19:5:73, v/v) at 1.5 ml/min enables the separation of the main component (EA) from all of its potential metabolites on a 250 × 4.6 mm I.D. poly(styrene-divinylbenzene) (PLRP-S 8 μm, 1000 A) column at 70°C. To improve the sensitivity of the method, a post-column ion-pair extraction with the strongly fluorescent 9, 10-dimethoxy-anthracene-2-sulphonate was used. The ion-pairs were extracted in chloroform for on-line fluorescence detection. Analytical recoveries for erythromycin and its metabolites after extraction of the plasma with tertiary butyl methyl ether were better than 90 %. The calibration curves of EA and its potential metabolites in plasma and urine were linear over the concentration range studied. The detection limits w...

An impurity of unknown identity was isolated from commercial cefradine by liquid chromatography on poly (styrene-divinylbenzene) with HOAc (0.01 M)-CH3CN (94:6, v/v) as the mobile phase. The structure was elucidated as 4',5'-dihydrocefradine using nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). The structure was confirmed by comparison with the chromatographic retention characteristics and photodiode-array detected ultraviolet spectrum of the synthetic compound and with its infrared, NMR and MS spectra. The presence of 4',5'-dihydrocefradine in cefradine has not been described previously.

A liquid chromatographic method was developed for the separation of thymidine from its anomer and isomers. The method was implemented during a comparative stability study of thymidine and 1- (2-deoxy-α-D-erythro-pentofurano-syl)thymine.

The synthesis of some impurities of the cephalosporin antibiotics cefalexin and cefradine is described. These impurities which may be present in commercial samples are formed during the semi-synthetic preparation of these antibiotics or upon their degradation. The preparation of these compounds enables the validation of selective quantitative analytical methods, such as column liquid chromatography and thin layer chromatography.

Reversed-phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was used to characterize the components of bitespiramycin, a group of 4"-acylated spiramycins produced by bioengineered strains. In total 38 components were characterized in commercial samples, including 12 impurities that had never been reported before and 12 other that were partially characterized. The structures of these unknown compounds were deduced by comparison of their fragmentation patterns with those of known major components. Their ultraviolet spectra were used to confirm the presence of an α-, β-, γ-, δ-unsaturated butadiene in the macrocyclic lactone. Compared with the classical method, LC/ESI-MS/MS is particularly advantageous in terms of sensitivity and efficiency to characterize minor components at trace levels in multi-component antibiotics.

The European Pharmacopoeia (Ph. Eur.) prescribes a selective and sensitive liquid chromatography/ultraviolet (LC–UV) method for the separation of the 16-membered ring macrolide josamycin and its related compounds. Since josamycin is obtained by fermentation, several closely related substances can be found in the sample. Several impurities have already been identified using reference substances. However, many peaks in the chromatogram cannot be correlated with known compounds or correspond to structures which were not described previously. The hyphenation of LC to mass spectrometry (MS) is a very useful tool for the characterization of impurities. The existing LC–UV method however uses non-volatile buffers, while for LC/MS a volatile mobile phase is required. In this study, each peak from the non-volatile system was collected separately and reinjected into a LC system using volatile mobile phase constituents. This way, the analyte could be separated from the buffer salts. Mass spectral data of this macrolide antibiotic were acquired on a LCQ ion trap mass spectrometer, equipped with an electrospray ionization (ESI) probe operating in the positive ion mode. The identity of the unknown compounds was deduced using the MS/MS and MS n collision-induced dissociation spectra of reference substances, combined with knowledge about the nature of functional group fragmentation behavior. The impurity profiling was done on 30 peaks in a josamycin bulk sample. This way, 12 compounds reported in the literature and Ph. Eur. were found in the bulk sample. Furthermore, 12 novel related substances were characterized and 18 compounds were partially characterized.

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.

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.

Gas chromatography-mass spectrometry is a well established analytical technique. However, mass spectrometers with electron ionization sources may suffer from signal drifts, hereby negatively influencing quantitative performance. To demonstrate this phenomenon for a real application, a static headspace-gas chromatography method in combination with electron ionization-quadrupole mass spectrometry was optimized for the determination of residual dichloromethane in coronary stent coatings. Validating the method, the quantitative performance of an original stainless steel ion source was compared to that of a modified ion source. Ion source modification included the application of a gold coating on the repeller and exit plate. Several validation aspects such as limit of detection, limit of quantification, linearity and precision were evaluated using both ion sources. It was found that, as expected, the stainless steel ion source suffered from signal drift. As a consequence, non-linearity and high RSD values for repeated analyses were obtained. An additional experiment was performed to check whether an internal standard compound would lead to better results. It was found that the signal drift patterns of the analyte and internal standard were different, consequently leading to high RSD values for the response factor. With the modified ion source however, a more stable signal was observed resulting in acceptable linearity and precision. Moreover, it was also found that sensitivity improved compared to the stainless steel ion source. Finally, the optimized method with the modified ion source was applied to determine residual dichloromethane in the coating of coronary stents. The solvent was detected but found to be below the limit of quantification.

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.

Previous stability studies on 2′-deoxyuridine (dUrd) made use of UV spectrophotometry. However, this technique does not allow dUrd to be determined separately from its anomer and pentopyranosyl isomers formed during acid degradation. The anomerization isomerization have been monitored by TLC before, but the separation between dUrd and its α-anomer was not optimal. Therefore, the aim of this study was to determine the rate constants of the degradation of dUrd, using a recently developed liquid chromatographic method. Rate constants of degradation of dUrd were determined at several pH values in the range 1–12. Studies at different temperatures showed that the Arrhenius equation is applicable at pH 1 and 7. To elucidate further the mechanism of hydrolysis in acidic media, some degradation tests were performed at pH 1 on the α-anomer and pentopyranosyl isomers of dUrd.

An established method for the analysis of chlortetracycline by liquid chromatography using octylsilylated silica gel as the stationary phase was examined in a multicentre study involving five laboratories and a total of six columns. Three chlortetracycline hydrochloride samples were analysed. The main component and the impurities were determined. An analysis of variance, treating each column as a different laboratory, showed an absence of consistent between-laboratory bias and the presence of a significant laboratory-sample interaction. The repeatability and the reproducibility of the method, expressed as relative standard deviations of the result of the determination of chlortetracycline hydrochloride, were calculated to be 0.9% and 1.1% respectively.

A comparative study of three isocratic liquid Chromatographic methods for the analysis of cefalexin is described. Two methods, taken from the European Pharmacopoeia and the US Pharmacopeia, use classical alkyl bonded phases (C18) as the stationary phase. The third method uses poly(styrene-divinylbenzene). Poor reproducibility of the selectivity towards cefalexin and its related substances was observed for the C18 columns. In both C18 methods, none of the columns was able to separate all the potential related substances from cefalexin under the prescribed LC conditions. The copolymer columns on the other hand showed good selectivity, independent of the origin or age of the columns. Four bulk samples of cefalexin were analysed by these three methods and the results were compared. Although the selectivity of polymer columns was better, for the samples examined no significant differences were observed for the assay results obtained with the three methods.

Anomerisation and isomerisation take place during acidic degradation of 2′-deoxyuridine (dUrd) and thymidine (Thd) due to the opening and reclosure of the furanose ring. A capillary electrophoretic method was developed which was able to separate dUrd and Thd from their respective anomers and pentopyranosyl isomers. Using a fused-silica capillary (70 cm × 75 μm I.D.) the influence of the type, pH and concentration of the buffer was systematically investigated as well as the effect of voltage and temperature. With a 20 mM sodium tetraborate buffer of pH 9.5 and 10 for dUrd and Thd, respectively, good resolution between anomers and isomers was obtained at 20 kV and 50°C.

A previously developed method for the analysis of tetracycline by liquid chromatography on poly(styrene-divinylbenzene) packing materials was examined in a collaborative study involving five laboratories and a total of seven columns. The main compound and the impurities of three tetracycline samples were determined. An analysis of variance, considering each column as a different laboratory, proved absence of consistent laboratory bias. The laboratory-sample interaction was significant. Estimates for the repeatability and the reproducibility of the method, expressed as relative standard deviations of the result of the determination of tetracycline, were calculated to be 0.9 and 1.5 % respectively

Oligonucleotides containing acyclic nucleosides with a 3( S ),5-dihydroxypentyl ( 1a–e ) or 4( R )-methoxy-3( S ),5-dihydroxypentyl ( 2a ) side chain were prepared and their hybridization properties as well as their stability towards degradation with snake venom posphodiesterase were studied. Attachment of an acyclic nucleoside at the 3′-end of an oligonucleotide makes it extremely resistant against enzymatic breakdown. Whereas oligonucleotides consisting completely of acyclic 2′-deoxyadenosine analogues ( 1a or 2a ) can still hybridize with an unmodified oligothymidylate, completely modified oligothymidylates or hetero-oligomers do not hybridize with their unmodified complementary oligonucleotide. This can be explained by the favourable enthalpy change on hybridization for the oligomers with adenine bases because of their higher degree of stacking and the ability to form T- A * .T triplets. In base-pairing with the natural DNA-nucleosides (dA,dC,dG,T), the acyclic nucleoside analogues ( 1a–e ) discriminate less compared to the natural 2′-deoxynucleosides. 9-(3( S ),5-Dihydroxypentyl)hypoxanthine shows the least spreading in melting temperature on hybridization with the four natural 2′-deoxynucleosides. Because of their conformational flexibility, acyclic nucleosides can be considered as universal nucleoside for the design of probes with ambiguous positions.

The keto and enol tautomers of doxycycline were separated by liquid chromatography on poly(styrene-divinylbenzene). The keto-enol tautomerism occurred between C-11a and C-12. The chemical structure of the tautomers was elucidated by on-line and off-line UV spectrophotometry and by 13 C-NMR spectrometry. The kinetics of the equilibrium were investigated in the pH range 1–12. The equilibrium was subject to base catalysis. The tautomerism was enhanced by protonation of doxycycline. The assignment of the groups responsible for the micro-ionization of doxycycline was discussed. Activation parameters were determined at pH 4.0.

A previously established method for analysis of erythromycin and related substances by liquid chromatography using a wide-pore poly(styrene-divinylbenzene) stationary phase was examined in a multicentre study involving 6 laboratories and a total of 7 columns. Three erythromycin samples were analysed. The main component and the impurities were determined. An analysis of variance treating each column as a different laboratory, showed absence of consistent laboratory bias and of laboratory-sample interaction. Estimates for the repeata-bility and reproducibility of the method, expressed as relative standard deviation (RSD) of the result of the determination of erythromycin A, were calculated to be 1.1 % and 1.3 % respectively.

A liquid chromatographic method that can separate each of the monoamino analogues of dideoxyadenosine and 9-(-dideoxy-β-D-lyxo-pentofuranosyl)adenine from their main degradation product, adenine, is described. The influence of the pH of the mobile phase, the type and concentration of the organic modifier and the concentration of the silanol masking agent and the buffer have been investigated. Several reversed phases were examined. Samples were finally analyzed on a 10 μm Spherisorb ODS1 column (250 mm×4.6 mm I.D.) using acetonitrile −0.2 M potassium phosphate buffer pH 6.0–0.2 M tetramethylammonium phosphate pH 6.0-water (5∶5∶0.5∶89.5, v/v) as the mobile phase. The interactions between the solute and the stationary phase are discussed in view of the basic properties of the compounds under study.

A comparative study of two isocratic liquid chromatographic methods for the determination of cefadroxil is described. The first method, prescribed in the monograph of the European Pharmacopoeia for the assay of cefadroxil, uses a classical alkyl-bonded phase (C18) as the stationary phase. This method is very similar to that prescribed by the United States Pharmacopeia. The other method uses poly(styrene- divinylbenzene). Poor reproducibility of the selectivity towards cefadroxil and related substances was observed when the first method was examined on different C18 columns. Copolymer columns, on the other hand, gave the same elution order on stationary phases from different manufacturers and of different age. Four bulk samples of cefadroxil were analysed following both methods and the results were compared.

Dimer building blocks (5a-j) with an N substituted guanideine function were prepared by reaction of 5'-amino-5'-deoxythymidine (1) with S,S -dimethyl- N substituted iminodithicarbonimidates (2a-j) followed by reaction of the isothiourea (3a-j) with 3'-amino-3'-deoxythymidine (4). All dimers were protected at the primary hydroxyl group with dimethoxytrityl group, converted to the phosphoramidite building blocks and incorporated into DNA.

The suitability of eleven silanol-deactivated reversed phases for the liquid chromatography of erythromycin was investigated. The selectivity and efficiency of each stationary phase were examined. The performance was compared to that of a non-deactivated C18 silica-based reversed-phase material, Hypersil C18 (5 μm). Two types of mobile phases were used, one containing no tetrabutylammonium (TBA) and the other containing TBA. Addition of TBA as a silanol-blocking agent improved the theoretical plate number and symmetry factor of the peaks corresponding to erythromycin A (EA) and erythromycin A enol ether for all the deactivated reversed phases. These results are an indication of the presence of some residual silanol activity in these phases. Separation of erythromycin E and EA was achieved on only two of the eleven phases. The selectivity was always poorer than that obtained in a previously described method using a poly(styrene-divinylbenzene) stationary phase.

A column liquid chromatographic method suitable for the quality control of tylosin A is described. The determination can be carried out on different C8 or C18 columns, using a mobile phase containing acetonitrile, 0.2 M tetrabutylammonium hydrogensulphate, 0.2 M phosphoric acid and water. The flow-rate is 1 ml/min and detection is performed at 280 nm. The method shows good selectivity towards the major components tylosin A, B, C and D and demycinosyltylosin. Minor degradation products, mainly observed in solutions, are also separated. The compositions of several standards are compared and results for a number of commercial samples are presented.

Isocratic column liquid chromatography on a poly(styrene-divinylbenzene) stationary phase (PLRP-S, 25 cm × 0.46 cm I.D.) at 50°C allowed the separation of cefadroxil from related substances. The mobile phase was acetonitrile-0.02 M sodium 1-octanesulphonate-0.2 M phosphoric acid-water (10.5:20:5:up to 100, v/v). The flow-rate was 1.0 ml/min and UV detection was performed at 254 nm. Official standards were compared and a number of commercial bulk samples and specialities were analysed.

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.

The European Pharmacopoeia (Ph. Eur.) describes liquid chromatography-ultraviolet (LC-UV) methods using C(18) stationary phases for the analysis of polymyxin B and colistin. Several unknown impurities were detected in commercial samples of those polypeptide complexes. However, the Ph. Eur. does not specify any related substances for polymyxin B and colistin. Since both methods use non-volatile buffers, the mobile phases were incompatible with mass spectrometry (MS). For the identification of related substances in bulk samples by LC/MS, volatile mobile phase systems were developed. However, the LC/MS methods (with volatile additives) showed inferior chromatographic separation compared to the LC-UV method (with non-volatile additives). Moreover, previously identified impurities by LC/MS could not be assigned in LC-UV methods as the separation in both systems was different. In this study, known impurities were traced in the LC-UV methods and new impurities present in polymyxin B and colistin bulk samples were characterized. To achieve this, each peak from the non-volatile system was collected separately and reinjected into an LC system with a volatile mobile phase coupled to MS. This way, collected impurity peaks were rechromatographed on a reversed phase column in order to separate the analyte from the buffer salts. Using this method, out of 39 peaks, five novel related substances were characterized in a polymyxin B bulk sample. Fourteen impurities, which were already reported in the literature were traced as good as possible in the LC-UV method. In the case of colistin, a total of 36 peaks were investigated, among which four new compounds. Additionally, 30 known impurities were traced in the LC-UV method.

The synthesis of some impurities of the cephalosporin antibiotics cefalexin and cefradine is described. These impurities which may be present in commercial samples are formed during the semi-synthetic preparation of these antibiotics or upon their degradation. The preparation of these compounds enables the validation of selective quantitative analytical methods, such as column liquid chromatography and thin layer chromatography.

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.

A method was developed to determine traces of trifluoroacetic acid as impurity in synthetic or semi-synthetic drugs as antibiotics, macropeptides, etc. Capillary electrophoresis in combination with capacitively coupled contactless conductivity detection (CE-C(4)D) was used due to lack of UV absorbance property of trifluoroacetic acid (TFA). The optimized method took less than 1 min with good linearity (R(2)=0.9995) for trifluoroacetic acid concentration from 2 to 100 ppm. It also has a good repeatability expressed by the relative standard deviation (% RSD) which is 1.2 and 2.1% for intraday and interday precision, respectively, at 50 ppm TFA, and good sensitivity with 0.34 ppm, 1.2 ppm LOD and LOQ, respectively. In addition, the content of TFA in synthetic drug, was determined using the validated method which gave good linearity (R(2)=0.9996) for trifluoroacetic acid spiked into drug in a concentration range of 2-80 ppm, with good intraday repeatability of 2.0%. The analysis is performed in a background electrolyte composed of 20mM morpholinoethane-sulfonic acid (Mes) and 20mM L-histidine (L-His) pH 6.1. Cetyltrimethylammonium bromide (CTAB) was added as flow modifier in a concentration (0.2mM) lower than the critical micellar concentration. Ammonium formate 6 ppm was used as internal standard. The applied voltage was 30 kV in reverse polarity. A fused silica capillary with 75 μm internal diameter and total length 47 cm (31 cm to C(4)D detector and 37 cm to DAD detector) was used.

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.

Investigation of acetylspiramycin (ASPM) and its related substances was carried out using a reversed-phase liquid chromatography/tandem mass spectrometry method. The identification of impurities in the ASPM complex was performed with a quadrupole ion trap mass spectrometer, with an electrospray ionization (ESI) source in the positive ion mode which provides MSn capability. A total of 83 compounds were characterized in commercial samples, among which 31 impurities that had never been reported and 31 partially characterized impurities were deduced using the collision-induced dissociation (CID) spectra of major ASPM components as templates. Most of the major impurities arise from the starting materials and the synthesis process. This work provides very useful information for quality control of ASPM and evaluation of its synthesis process.