Reversed Phase Liquid Chromatography of Phenacyl Esters of Some Natural Penicillins

Abstract

The present work demonstrates a method by which natural, non-UV absorbing penicillins can be derivatized to form phenacyl esters. The reaction is rapid and essentially completed in twenty minutes. A straight line correlation was found between the peak height of a phenacyl ester derivative and the amount of the penicillin, a fact which attests to the applicability of the method for quantisation. The phenacyl esters of the penicillins studied here were separated on a reversed phase column.     

An Improved Spectrophotometric Method for Determination of Penicillins Alone and in Pharmaceutical Dosage Forms

Abstract

A new spectrophotometric method for determining penicillins has been developed. A known volume of the penicillin solution is heated at 95[ddot]C for 70 min with ammonium molybdate, in sulphuric acid medium and the absorbance of the blue species formed is measured at 670 nm. The method has been successfully applied for the determination of thirteen commercially available penicillins. Beer's law is valid for up to 35-90 μg/ml of antibiotic (according to the penicillin). The procedure has been used for analysing some pharmaceutical preparations for these drugs, e.g., injections and capsules. The sensitivity of the method is largely increased with respect to the vanadate method previously described.     

A NEW METHOD FOR PREPARATION OF β-LACTAM SULFOXIDES

Abstract

Penicillin sulfoxides (II) are key intermediates in chemical transformations of penicillins to cephalosporins. Oxidation of the sulfur atom in penicillin nucleus (I) to the sulfoxide (II) has been studied and a new economically feasible method developed.     

Spectrodensitometric Analysis of Penicillins by Detection with Iodine

Abstract

A rapid, sensitive and selective method is described for the determination of some penicillin derivatives and their additive and degradation products in the presence of each other. Penicillin derivatives are separated from their degradation and additive products on high performance thin layer silica gel G plates. The plates were developed in a linear chamber, air dried, exposed to iodine vapour and measured on a spectrodensitometer at 290 nm. Procaine and procaine penicillin are measured at 360 nm. The results of the analysis show good agreement with the method of the USP XXI. For the reaction of penicillins with iodine, the formation of charge-transfer complexes is considered.     

Potentiometric Biosensing of Penicillins Using a Flow-Through Reactor with Penicillinase or Penicillin Amidase Immobilized by Gamma-Irradiation

Abstract

Penicillinase and penicillin amidase were immobilized, with similar yield, by physical entrapment in porous beads of poly-2-hydroxyethyl methacrylate matrix formed by gamma-irradiation. Flow-injection analysis of penicillins was performed by using a column reactor filled with enzyme-loaded polymer beads and a wall-jet pH-metric micro-cell with a combined flat-membrane glass electrode as the sensing element. With both enzymes it was possible to have either a linear response dynamic range or a logarithmic one by appropriate selection of buffer capacity and flow rate of the carrier solution.     

Determination of eight penicillin antibiotics in pharmaceuticals,milk and porcine tissues by nano-liquid chromatography

This study describes the ability of nanoscale liquid chromatography (nano-LC) coupled with UV or mass spectrometry (MS) for the simultaneous determination of eight common penicillin antibiotics (amoxicillin, ampicillin, penicillin G, penicillin V, oxacillin, cloxacillin, nafcillin and dicloxacillin) in commercial samples (pharmaceuticals, milk, porcine tissues (liver and kidney)) for the first time. Material types of the on-column polymeric frits (polystyrene-based and polymethacrylate-based monoliths) and the packed stationary phase materials (C8 and C18 particles of 3 μm) used in the nano-LC for the influence of penicillin separation were evaluated. The nano-LC and MS parameters such as the composition and flow rate of mobile phase, capillary voltage and temperature of dry gas were examined in order to acquire high separation resolution and detection sensitivity for penicillin analyses. Furthermore, a home-made in-line filter (a nylon membrane of 0.2 μm pore size), was first used to connect with the flow cell of high sensitivity UV detector or the nanoelectrospray needle in MS detection. The result indicated it could effectively improve the reproducibility of penicillin mass signals or prolong the lifetime of the flow cell. The nano-LC methods provided good quantitative precisions in the range of 89.5–111.2% for UV detection at 0.5 μg/mL penicillins, and 83. 1–94.9% for MS detection at 5 μg/L penicillins), respectively, as well as offered stable retention repeatabilities (the relative standard deviation (RSD) of retention time was lower 0.30% in both the UV and MS detections). Compared to other LC–MS methods, the proposed nano-LC systems provided better detection sensitivity for these penicillins (the limits of detection (LOD) was of 2.27–4.06 μg/L for UV mode, and 0.01–0.51 μg/L for MS mode) when either UV or MS detector was employed.     

The determination of penicillins by titrations with mercury(II) solution.

A simple technique, involving two titrations with mercury(II) solutions, is described for the determination of penicillins and their degradation products. The first titration, at pH 4–5 on an untreated penicillin solution, gives the amount of degradation products; the second titration, on a hydrolysed solution at the same pH, gives the sum of the degradation products and penicillin degraded during the hydrolysis. Enzymic hydrolysis is superior to alkaline hydrolysis for penicillinase-sensitive penicillins. Enzyme-resistant penicillins should be hydrolysed with alkali at optimum conditions, e.g. for cloxacillin at pH 13.5 for 5 min. A standard deviation of less than 0.5 % was obtained for the penicillins investigated. The method is absolute; calibration with standard penicillin is not necessary.     

Direct HPLC Separation of Carazolol Enantiomers on a Cellulose Tris-3, 5-Dimethylphenyl Carbamate Column

Abstract

Direct liquid chromatographic resolution and optimization of carazolol are accomplished without any derivatizations on a cellulose tris-3, 5-dimethylphenyl carbamate column (known as OD-CHIRALCEL). Maximum stereochemical resolution (R) of 2.2 is obtained. The effects of 2-propanol and diethylamine content in mobile phase and effect of temperature on capacity factor (k), stereochemical separation factor (α) and stereochemical resolution factor (R) is demonstrated.     

Kinetic Analysis of Single or Multi-Step Decomposition Processes; Limits introduced by statistical analysis

A kinetic study on decomposition processes of some penicillin and some commercial drugs was carried out. As expected by the complex structures of penicillins, several steps with different activation energies occurred in their decomposition processes. Model-fitting and model-free kinetic approach were applied to non-isothermal and isothermal data. In the model-fitting methods the kinetic triplets (f(α), A and E _a) that defines a single reaction step resulted in being at variance with the multi-step nature of penicillins decomposition. The model-free approach represented by isothermal and non-isothermal isoconversional methods, gave dependences of the activation energies on the extent of conversion. The complex nature of the multi-step process of the studied compounds was more easily revealed using a broader temperature range in non-isothermal isoconversional method. The failure in the model fitting method did not allow calculating storage times. Model-fitting and model-free methods, both isothermal and non-isothermal, showed that F1 mechanism is able to describe decomposition processes for drugs (having Phosphomycin salts as active component) for which a single decomposition process occurs. Statistical analysis allowed us to select reliable kinetic parameters related to the decomposition processes for these last compounds. This procedure showed that the values obtained by extrapolation, outside the temperature range where the processes occurred must be used with caution. Indeed half-life and shelf-life values, commonly extrapoled at room temperature, seemed to be unrealistic. This revised version was published online in August 2006 with corrections to the Cover Date.     

Study of Interactions Between Sugammadex and Penicillins Using Affinity Capillary Electrophoresis

Sugammadex, a new modified gamma cyclodextrin, reverses the neuromuscular blockage induced by rocuronium by forming a strong complex with this muscle relaxant. To evaluate possible interactions with potentially co-administered drugs, interactions between sugammadex and penicillins were investigated using the affinity capillary electrophoresis method for the first time. Capillary electrophoresis coupled to an ultra violet detector was used as an analytical device for the analysis and detection of cyclodextrin inclusion complexes. Changes in the effective mobility of guest drugs (amoxicillin, ampicillin, oxacillin, dicloxacillin and azlocillin) were correlated with the increasing concentration of host molecules in background electrolyte, and successfully fitted into a non-linear curve equation; assuming 1:1 stoichiometric interaction. The calculated association constants (K _a) were: 383.44, 184.54, 265.34 and 95.06 M⁻¹ for amoxicillin, ampicillin, oxacillin and dicloxacillin, respectively. No complex formation with sugammadex could be detected for both penicillin G and piperacillin. The difference in the chemical structure of the penicillins, especially in the (R) side chains, is suggested to be responsible for the variety of binding strength between penicillins and sugammadex. The displacement study demonstrated that interactions between sugammadex and co-administered penicillins could reduce the pharmacological effects of both.

     

Optimization of culture medium and conditions for penicillin acylase production by streptomyces lavendulae ATCC 13664

The culture medium for Streptomyces lavendulae ATCC 13664 was optimized on a shake-flask scale by using a statistical factorial design for enhanced production of penicillin acylalse. This extracellularenzyme recently has been reported to bea penicillin Kacylase, presenting also high hydrolytic activity against penicillin V and other natural aliphatic penicillins such as penicillin K, penicillin F, and penicillin dihydroF,. The factorial design indicated that the main factors that positively affect penicillin acylase production by S. lavendulae were the concentration of yeast extract and the presence of oligoelements in the fermentation medium, whereas the presence of olive oil in the medium had no effect on enzyme production. An initial concentration of 2.5% (w/v) yeast extract and 3 μg/mL of CuSO₄·5H₂O was found to be best for acylase production. In such optimized culture medium, fermentation, of the microorganism yielded 289 IU/L of enzyme in 72 h when employing a volume medium/volume flask ratio of 0.4 and a 300-rpm shaking speed. The presence of copper, alone and in combination with other metals, stimulated biomass as well as penicillin acylase production. The time course of penicillin acylase production was also studied in the optimized medium and conditions. Enzyme production showed catabolite repression by different carbon sources such as glucose, lactose, citrate, glycerol, and glycine.     

Separation of Labile Terpenoids by Low Temperature HPLC

Abstract

Semipreparative low temperature HPLC was used to isolate two sesquiterpene hydrocarbons, i. e. anastreptene and β-barbatene, that could not be separated at ambient temperature. A linear relationship was observed between the logarithm of capacity factor and the reciprocal of column temperature. This was consistent with previous reports (4, 5, 8). It could be demonstrated that differences in enthalpy and entropy may cause a temperature-dependent resolution for both compounds. Low temperature H-NMR revealed that separation was not due to a fixation of conformation.     

Evaluation of Certain Pharmaceuticals with Dibromohydantion. V. Determination of Penicillins

Abstract

A new bromometric assay procedure for penicillins was developed, using an organic brominating agent. 1,3-Dibromo, 5,5-dimethylhydantion (DBH). The experimental conditions, the molar ratio as well as the identification of the reaction products were thoroughly studied. The end point could be detected visually or potentiometrically. Spectrophotometric titration could also be performed. The suggested procedure could be successfully applied to pharmaceutical preparations containing penicillins. The percentage recoveries for different penicillins were satisfactory.     

高效毛细管电泳法检测牛奶中的青霉素中间体以及3种青霉素类药物

建立了高效毛细管电泳(HPCE)同时检测牛奶中青霉素类抗生素中间体6-氨基青霉烷酸(6-APA)以及3种青霉素类药物青霉素钾(PEN)、氨苄青霉素(AMP)和阿莫西林(AMO)的方法。利用正交实验设计,对HPCE中的缓冲液离子浓度和pH值、分离电压、分离温度等分离条件进行了优化。结果表明: 在采用40 mmol/L磷酸二氢钾-20 mmol/L硼砂缓冲体系(pH 7.8)、分离电压为28 kV、分离温度为30 ℃的电泳条件下,4.5 min内可以实现上述4种青霉素类药物的快速分离检测。各组分在1.56～100 mg/L范围内有良好的线性,相关系数(r2)为0.9979～0.9998,加标回收率为84.91%～96.72%,相对标准偏差(RSDs)为1.11%～9.11% (n=6)。该方法简便、快速,可以应用于市售牛奶中4种青霉素类药物的快速检测。     

Study of Interactions Between Sugammadex and Penicillins Using Affinity Capillary Electrophoresis

Sugammadex, a new modified gamma cyclodextrin, reverses the neuromuscular blockage induced by rocuronium by forming a strong complex with this muscle relaxant. To evaluate possible interactions with potentially co-administered drugs, interactions between sugammadex and penicillins were investigated using the affinity capillary electrophoresis method for the first time. Capillary electrophoresis coupled to an ultra violet detector was used as an analytical device for the analysis and detection of cyclodextrin inclusion complexes. Changes in the effective mobility of guest drugs (amoxicillin, ampicillin, oxacillin, dicloxacillin and azlocillin) were correlated with the increasing concentration of host molecules in background electrolyte, and successfully fitted into a non-linear curve equation; assuming 1:1 stoichiometric interaction. The calculated association constants (K _a) were: 383.44, 184.54, 265.34 and 95.06 M^?1 for amoxicillin, ampicillin, oxacillin and dicloxacillin, respectively. No complex formation with sugammadex could be detected for both penicillin G and piperacillin. The difference in the chemical structure of the penicillins, especially in the (R) side chains, is suggested to be responsible for the variety of binding strength between penicillins and sugammadex. The displacement study demonstrated that interactions between sugammadex and co-administered penicillins could reduce the pharmacological effects of both.     

Simultaneous Determination of Procaine Penicillin G and Benzathine Penicillin G by Second Derivative Spectrophotometry

Abstract

A new method for determining the binary mixtures of procaine penicillin G and benzathine penicillin G using “zero-crossing” second derivative spectrophotometry is described. Calibration graphs were linear up to 8.80 10^?5 M for procaine penicillin G and 4.40 10^?5 M for benzathine penicillin G. The method was realized in ethanol/water medium (30 % v/v). A complete and exhaustive statistical analysis of the experimental data was realized to demonstrate the validity of method. The method was applied for determining procaine penicillin G and benzathine penicillin G synthetic mixtures with good results. The procedure does not require any separation step.     

Determination of Some Penicillin Derivativies Using High Performance Liquid Chromatography

Abstract

A rapid, precise and selective method is described for determination of penicillin derivatives. Penicillin derivatives are separated from closely related degradation products by high performance liquid chromatography at ambient temperature using 10cm column packed with 5um Nucleosil RP₁₈ and buffered aqueous acetonitrile as mobile phase. The eluate is monitored at 254nm. The procedure is suitable for determination of 8 penicillin derivatives either in raw material or phamaceutical dosage forms.     

Influence of Bonded-Phase Column Type,Mobile Phase Composition,Temperature and Flow-Rate in the Analysis of Triglycerides by Reverse-Phase High Performance Liquid Chromatography

Abstract

Two chromatographic systems for RP-HPLC analysis of triglycerides, operating under isocratic conditions using octadecylsilane and octylisilane bonded phases, are described.

The influence of such chromatographic factors as bonded phase column type, mobile phase composition, temperature and flow rate on retention, analysis selectivity and efficiency, and separation of mixtures of homogeneous triglycerides was assessed. Linear relationships were established for the logarithm of the capacity factor and selectivity for each triglyceride in relation to temperature, the proportion of certain mobile phase components and flow rate.

The octadecylsilane bonded phase was more selective when analyzing triglycerides with a partition number below 48, while octylsilane was appropiate for separating mixtures of long chain saturated triglycerides to the detriment of the resolution of triglycerides with low partition numbers. ACN/ACE/THF (58/38/4) was a suitable mobile phase for use with the octadecylsilane bonded phase, and ACN/THF /H₂O (60/40/1) for the octylsilane bonded phase. A column temperature of 30°C and a flow rate of 1.5 mL/min resulted in acceptable resolution and analysis time in both systems.     

Effect of Temperature and Mobile Phase Composition on RP-HPLC Separation of Cephalosporins

Abstract

The effects of mobile phase composition and column temperature on resolution in reversed-phase high performance liquid chromatography (RP-HPLC) were used to separate the following cephalosporins: cefonicid, cefaclor, cephazolin, cefodizime, cephaloridine, cephamandole and cephalotin, from a single sample. The capacity factor (k') was described as a function of temperature and mobile phase composition. Semi-empirically estimated values of k' were determined using a small number of experimental data for different temperatures between 20°C and 60°C, and mobile phase compositions (acetate buffer/isopropanol). The capacity factor of each cephalosporin was observed to decrease with increasing temperature and the volume fraction (V) of isopropanol in the mobile phase; linear relationships were obtained for plots of In k' versus 1/T and log k' versus log V. The method developed, while simple, reveals the optimal isocratic elution conditions for column temperature and eluent concentration for the complete separation and rapid analysis of the cephalosporins studied.     

Quantitative analysis of penicillins in porcine tissues, milk and animal feed using derivatisation with piperidine and stable isotope dilution liquid chromatography tandem mass spectrometry

Penicillins are used universally in both human and veterinary medicine. The European Union (EU) has established maximum residue levels (MRLs) for most ß-lactam antibiotics in milk and animal tissues and included them in the National Residue Monitoring Programs. In this study, a novel method is described for the determination and confirmation of eight penicillins in porcine tissues, milk and animal feed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). To prevent degradation of penicillin residues during workup, a derivatisation procedure was developed, by which penicillins were converted to stable piperidine derivatives. Deuterated piperidine derivatives were synthesised for all relevant penicillins, enabling the use of isotope dilution for accurate quantification. Penicillin residues were derivatised in the crude extract with piperidine and isolated using solid-phase extraction. The penicillin piperidine derivatives were determined by LC–MS/MS. The method was validated at the current MRLs, which range from 25–300 µg kg^?1 in muscle and kidney to 4–30 µg kg^?1 in milk as well as at the target value of 100 µg kg^?1 chosen for animal feed, according to the EU requirements for a quantitative confirmatory method. Accuracy ranged from 94–113% (muscle), 83–111% (kidney) and 87–103% (milk) to 88–116% (animal feed). Intra-day precision (relative standard deviation (RSD)_r) ranged from 5–13% (muscle, n?=?18), 4–17% (kidney, n?=?7) and 5–18% (milk, n?=?7) to 11–32% (animal feed, n?=?18). Inter-day precision (RSD_RL, n?=?18) ranged from 6–23% (muscle) to 11–36% (animal feed). From the results, it was concluded that the method was fit for purpose at the target MRLs in animal tissue and target levels for animal feed.