Spectrophotometric determination of some penicillins with ammonium vanadate

A spectrophotometric method for determining some penicillins has been developed. A known volume of the penicillin solution-in phosphate buffer of pH 6.8 is boiled with ammonium vanadate solution-in sulphuric acid medium-for 10 min and the absorbance of the colour formed is measured at 750 nm. The excess of vanadate can also be determined volumetrically. The method has been successfully applied for the determination of penicillin G sodium, phenoxymethyl penicillin, ampicillin sodium, phenethicillin potassium, cloxacillin sodium and methicillin sodium. The procedure is also used for analysing some pharmaceutical preparations of these drugs, e.g., injections. The results obtained are in agreement with those of the B P 1973 methods.     

Flow-injection chemiluminescence determination of penicillin antibiotics in drugs and human urine using luminol-Ag(III) complex system

A chemiluminescent (CL) system based on the reaction of an Ag(III) complex with luminol in alkaline medium is presented. Gamma order of magnitude penicillin species antibiotics could dramatically enhance CL intensities. Coupled with flow injection analysis (FIA), a novel sensitive chemiluminescent analytical technique for some penicillin antibiotics in drugs and urine samples is introduced. Under optimum conditions, benzylpenicillin sodium, amoxicillin, ampicillin and cloxacillin sodium were determinated. Detection limits of this method are 70 ng/mL for benzylpenicillin sodium, 67 ng/mL for amoxicillin, 169 ng/mL for ampicillin and 64 ng/mL for cloxacillin sodium. For the spiked urine samples, the recoveries of the four drugs were in the range of 106–112% for benzylpenicillin sodium, 104–110% for amoxicillin, 104–106% for ampicillin, and 103–105% for cloxacillin sodium. Based on the fluorescence spectra, free radical trapping experiment, and chemiluminescent spectra, a possible reaction mechanism is suggested.     

Interactions of phenothiazine drugs with bile salts: Micellization and binding studies

An evaluation of the interactions of phenothiazine tranquilizer drugs (promazine hydrochloride; PMZ and promethazine hydrochloride; PMT) with bile salts viz., sodium cholate (NaC) and sodium deoxycholate (NaDC) in aqueous medium, investigated through different physicochemical measurements is presented in this work. The mixed micellization behavior and surface properties of the phenothiazine-bile salt systems have been analyzed by conductivity and surface tension measurements. Application of different theoretical approaches to all the phenothiazine-bile salt mixtures shows a non-ideal behavior. Further, the spectroscopic techniques such as UV-visible and steady state fluorescence have been employed to study the binding of phenothiazines with bile salts. The stoichiometric ratios, binding constants (K), and free energy change (ΔG) for the phenothiazine-bile salt complexes were estimated from the Benesi-Hildebrand (B-H) double reciprocal plots obtained by using the changes in spectral intensities of phenothiazines on addition of bile salts. The results are discussed in the light of use of bile salts as promising drug delivery agents for phenothiazines and hence improve their bioavailabilty.     

Extractive spectrophotometric determination of fluoroquinolones and antiallergic drugs in pure and pharmaceutical formulations.

A simple, rapid and sensitive spectrophotometric procedure has been proposed for the assay of fluoroquinolones viz., ciprofloxacin (CPF) and norfloxacin (NRF), and antiallergic drugs viz., methdilazine hydrochloride (MDH) and isothipendyl hydrochloride (IPH) in bulk and pharmaceutical formulations. The method is based on the reaction of selected drugs with Brilliant Blue G (BBG) in NaOAc-AcOH buffer of pH 4.0 for CPF and NRF or in neutral medium for MDH and IPH to give chloroform soluble ion-association complexes. The effects of various parameters have been studied. The ion-association complexes exhibited absorption maxima at 610 nm for CPF, at 614 nm for NRF and MDH, and at 612 nm for IPH. Beer's law plots were obeyed in the concentration ranges of 0.5-6.0, 0.4-8.0, 0.1-6.0 and 0.2-6.1 (mg ml(-1) for CPF, NRF, MDH and IPH, respectively, with correlation coefficients not less than 0.9969. Molar absorptivity values as calculated from the Beer's law data were found to be 2.86 x 10(4), 2.64 x 10(4), 3.13 x 10(4) and 5.51 x 10(4) mol(-1) cm(-1) for CPF, NRF, MDH and IPH, respectively. The influence of commonly employed excipients in the determination of the studied drugs has been studied. The results obtained by the proposed method were statistically validated.     

Simultaneous determination of carvedilol and ampicillin sodium by synchronous fluorimetry

The carvedilol and ampicillin sodium were simultaneously determined by the synchronous fluorimetry. With excitation wavelength at 254 nm, the maximum emission wavelengths of carvedilol and ampicillin sodium were at 357 and 426 nm, respectively. Because the emission spectra of carvedilol and ampicillin sodium were overlapped partially, carvedilol and ampicillin sodium cannot be determined directly by normal fluorimetric method. However, the synchronous fluorimetry can be used for determining both drugs simultaneously without separation procedure. The (Delta)(lambda) = 80 nm was used. Iso-propanol was selected as sensing reagent. Effects of pH, organic solvents and foreign ions on the determination of both drugs were studied. The linear relationship was obtained between the relative fluorescence intensity and concentration of carvedilol and ampicillin sodium in the range of 0.005-0.1 and 5.0-70.0 microg ml(-1), respectively. The linear regression equation of calibration graph for carvedilol is C = 0.000151F - 0.00210, and for ampicillin sodium is C = 0.0770F - 2.62. The correlation coefficient of linear regression equation is 0.9995 for carvedilol and 0.9998 for ampicillin sodium, respectively. The detection limit is 1 ng ml(-1) for carvedilol and 1 microg ml(-1) for ampicillin sodium. The relative standard deviations of carvidelol and ampicillin sodium are 2.47 and 1.61%, respectively. The recovery is from 96.0 to 103.0% for carcvedilol and from 98.0 to 105.0% for ampicillin sodium. This method was rapid, simple and highly sensitive for the determination of carvedilol and ampicillin sodium without pre-separation. The results obtained by this method agreed with those by the official methods. This method can be used for the determination of carvedilol and ampicillin sodium in the medicine dosage.     

Flow injection chemiluminescence analysis of some penicillins by their sensitizing effect on the potassium permanganate-glyoxal reaction.

A new chemiluminescence method using flow injection is described for the determination of four penicillins, namely: phenoxymethylpenicillin potassium, amoxicillin, ampicillin, and ampicillin sodium. The method is based on sensitizing effect of these drugs on the chemiluminescence reaction of potassium permanganate in sulfuric acid with glyoxal. The different experimental parameters affecting the chemiluminescence intensity were carefully studied and incorporated into the procedure. The method allows the determination of 0.1-1.0 microg/ml phenoxymethylpenicillin potassium, 0.1-1.0 microg/ml amoxicillin, 0.1-1.0 microg/ml ampicillin, and 0.1-1.0 microg/ml ampicillin sodium. The method was successfully applied to the determination of four penicillin antibiotics in pharmaceutical preparations.     

Stable competitive enzyme-linked immunosorbent assay kit for rapid measurement of 11 active beta-lactams in milk, tissue, urine, and serum

Beta-lactams are used as veterinary drugs for the treatment of food-producing animals. For consumer protection, legislation is in place to set limits for their residues. An enzyme-linked immunosorbent assay (ELISA) was developed which allowed, in a single reaction, the class-specific measurement of 11 beta-lactams, with limits of detection below European maximum residue limits. Determinations were feasible in milk, tissue, urine, and serum with simple and rapid sample preparation. In this format, the specific capture antibodies were precoated on the microtiter plate and horseradish peroxidase-labeled conjugate was used to compete with free beta-lactams. The stability of the precoated microtiter plate and conjugate was at least 1 year when stored at 2 to 8 degrees C; upon reconstitution, the conjugate was stable for 6 days at 2 to 8 degrees C. The stability of lyophilized ampicillin standards was at least 6 months when stored at 2 to 8 degrees C and at least 1 year when stored at -20 degrees C. A low cross-reactivity, 3.6%, was observed with ampicillin with open beta-lactam ring relative to 100% for intact ampicillin. Generic recognition was shown by relative cross-reactivity values ranging from 22 (penicillin V) to 144% (nafcillin). Cross-reactivity for cephalosporins was <0.1%. Intra- and interassay precisions expressed as coefficient of variation were typically 2-8%. The inhibitory concentration with 50% binding for ampicillin was typically 2 ppb. Recovery for different spiked levels was >70% with all the matrixes.     

New spectrophotometric method for the determination of some drugs with iodine and wool fast blue BL

A simple, sensitive and selective method is proposed for the spectrophotometric determination of drugs, viz. ampicillin, penicillin V, amoxycillin, cloxacillin, cefadroxil, ceftezoxime, griseofulvin, streptomycin, nicoumalone and acebutolol HCl, based on their reactivity with iodine. The method involves the addition of excess iodine of known concentration to the drug in the presence of NaOH and the unreacted iodine is determined by the measurement of the decrease in the absorbance of the dye wool fast blue BL (λ_max=540 nm) which was found to be the most suitable of several dyes tested. This method was applied for the determination of drug contents in pharmaceutical formulations and enabled the determination of the drugs in microgram quantities (0.8–9.6 μg ml⁻¹). No interferences were observed from excipients and the validity of the method was tested against reference methods.     

Liquid chromatographic determination of ampicillin residues in porcine muscle tissue by a multipenicillin analytical method: European Collaborative Study

A collaborative study involving 14 laboratories was conducted to determine residues of ampicillin in porcine muscle tissue by using a liquid chromatographic method developed for multipenicillin analysis that can quantitate 8 penicillin compounds (benzylpenicillin, phenoxymethylpenicillin, ampicillin, amoxicillin, nafcillin, oxacillin, cloxacillin, and dicloxacillin) at trace levels in muscle tissue. This method involves extraction of the penicillins with phosphate buffer, pH 9, followed by cleanup and concentration on a C18 solid-phase extraction column and reaction with benzoic anhydride at 50 degrees C and with 1,2,4-triazole and mercury(II) chloride solution, pH 9.0, at 65 degrees C. The derivatized compounds are eluted isocratically on a C8 column with a mobile phase of acetonitrile and phosphate buffer (pH 6; 0.1 M) containing sodium thiosulfate and the ion-pair reagent tetrabutylammonium hydrogen sulfate. The penicillins are detected by UV absorption at 325 nm. The limit of detection and the limit of determination (quantitation) of the method were calculated to be approximately 3-5 and 25 microg/kg, respectively, in accordance with the criteria of European Union (EU) Decision No. 93/256/EEC. In this first interlaboratory study, collaborators were instructed to monitor 4 different penicillin compounds (benzylpenicillin, phenoxymethylpenicillin, ampicillin, and amoxicillin) by analyzing 8 blind samples of muscle tissue in triplicate. These samples were prepared from 2 materials containing different concentrations of incurred ampicillin (63.5 microg/kg for material No. 1 and 358.1 microg/kg for material No. 2) and 1 blank material. The repeatability relative standard deviation and the reproducibility relative standard deviation were 10.2 and 17.4%, respectively, for material No. 1 and 7.0 and 16.0%, respectively, for material No. 2. These results demonstrate that the method is suitable for the determination of ampicillin residues in muscle tissue at the EU maximum residue limit (50 microg/kg) and above. However, the identification of positives by this procedure may need additional confirmation by techniques with greater specificity, such as liquid chromatography combined with mass spectrometry, or tandem mass spectrometry. Investigations regarding the basis of interlaboratory testing studies will further demonstrate the suitability of multiresidue methodology for detecting and quantitating other compounds in the family of penicillin antibiotics.     

Studies on residual antibacterials in foods. IV. Simultaneous determination of penicillin G, penicillin V and ampicillin in milk by high-performance liquid chromatography

A rapid and simple method for the simultaneous determination of penicillin G (PCG), penicillin V (PCV) and ampicillin (ABPC) in milk is described. The retention behaviour of these beta-lactam antibiotics in reversed-phase liquid chromatography with mobile phases containing sodium alkylsulphonate was studied. Good separations were obtained with methanol-water-0.2 M phosphate buffer (pH 4.0) (5:13:2) containing 11 mM sodium 1-heptanesulphonate and a LiChrosorb RP-18 column. The sample was pre-treated with a Sep-Pak C18 cartridge. The peaks corresponding to each beta-lactam antibiotics can be confirmed with the treatment using penicillinase. The recoveries from milk fortified with sodium PCG, potassium PCV and ABCP at levels of 0.5 and 0.1 micrograms/g each were generally better than 87% and the relative standard deviations were 1.17-4.98%. The detection limits corresponded to 0.03 microgram/g of these beta-lactam antibiotics in milk.     

Spectrophotometric determination of certain cephalosporins using molybdophosphoric acid. Part II. Determination of cefadroxil, cefapirin, ceforanide and cefuroxime

The use of molybdophosphoric acid as an oxidising agent for the spectrophotometric determination of four cephalosporin derivatives, viz., cefadroxil monohydrate (I), cefapirin sodium (II), ceforanide L-lysine (III) and cefuroxime sodium (IV), either in the pure form or in pharmaceutical formulations is described. Beer's law is obeyed up to 100 micrograms ml-1 for I, up to 60 micrograms ml-1 for II and IV and up to 80 micrograms ml-1 for III. The molar absorptivities were 4.58 X 10(3), 11.3 X 10(3), 9.8 X 10(3) and 10.9 X 10(3) l mol-1 cm-1 and the Sandell sensitivities were 83.3, 39.3, 53.0 and 41.0 ng cm-2 for I, II, III and IV, respectively. The slopes and intercepts of the equations of the regression line were calculated for each of these drugs with the following correlation coefficients: I, 0.9993; II, 0.9999; III, 1.000; and IV, 0.9999. These antibiotics were determined successfully both in the pure form and in pharmaceutical preparations. The results demonstrated that the proposed procedure is at least as accurate, precise and reproducible as the official methods, while being simpler and less time consuming. A statistical analysis indicated that there was no significant difference between the results obtained by the proposed procedure and those of the official methods.     

高效液相色谱-串联质谱法同时测定牛奶和奶粉中的青霉素类药物及其主要酶解代谢产物

建立了液相色谱-串联质谱法同时测定牛奶和奶粉中4种青霉素(青霉素G、青霉素V、阿莫西林、氨苄西林)及其4种 β-内酰胺酶酶解产物(青霉素G脱羧噻唑酸、青霉素V脱羧噻唑酸、阿莫西林脱羧噻唑酸、氨苄西林脱羧噻唑酸)残留的方法。样品采用乙腈-水提取,浓缩后经HLB柱净化,用液相色谱-串联质谱检测,外标法定量。结果表明,青霉素原药在4~200 μg/L,酶解产物在10~500 μg/L范围呈良好线性,线性相关系数均大于0.99;样品检出限为5~50 μg/kg(S/N≥3),定量限为8~100 μg/kg(S/N≥10);对牛奶和奶粉样品分别进行3个水平的加标回收实验(n=6),牛奶中青霉素及其酶解产物的平均回收率为83.48%~96.97%,相对标准偏差为3.86%~10.87%;奶粉中青霉素及其酶解产物的平均回收率为82.70%~95.14%,相对标准偏差为3.02%~9.81%。该方法稳定、可靠,适用于牛奶和奶粉中青霉素类药物及其酶解代谢产物的测定。     

Indirect determination of ampicillin sodium with sodium nitrate-ammonium thiocyanate-water system by extraction-flotation of cuprous thiocyanate

A novel method for indirect determination of ampicillin sodium by the extraction-flotation is proposed in this paper. It is indicated that the degradation of ampicillin sodium took place in the presence of 0.30 M sodium hydroxide in boiling water for 20 min. At pH 4.0, in the presence of ammonium thiocyanate, the thiol group of the degradation product of ampicillin sodium could reduce copper(II) to copper(I) due to the formation of the emulsion cuprous thiocyanate precipitation. By determining the residual amount of copper(II) in the solution and calculating the flotation yield of cuprous thiocyanate, the indirect determination of ampicillin sodium can be performed. When the concentration of cooper(II) was 5.0 μg/mL, a good linear relationship was obtained between the flotation yield of cuprous thiocyanate and the amount of ampicillin sodium in the range of 0.40～9.6 μg/mL. The linear equation is E = 4.1469 + 3.7949c with the correlation coefficient r = 0.9992, and the detection limit (3σ/K) of 0.37 μg/mL. Each parameter has been optimized and the reaction mechanism has been studied. The method has been successfully applied to the determination of ampicillin sodium in pharmaceutical, human plasma and urine samples. Analytical results obtained are satisfactory.     

The determination of some selected penicillins by enzymatic enthalpimetry

An enzymatic, enthalpimetric procedure is described for the determination of penicillin G, ampicillin sodium or phenoxymethylpenicillin in pure and dosage forms. The technique employed allows up to 20 assays with the same reagent solution. Enthalpy assignments are presented along with error (range) and precision data. The lower limit of determination is about 10^-3 mol dm^-3.     

Stability of penicillin antibiotic residues in meat during storage. Ampicillin

Incurred samples from a pig treated with ampicillin, one of the most important penicillin antibiotic drugs used in food-producing animal treatments, were analyzed at the residue level of the drug in muscle tissue (approximately 100 microg kg(-1)) during their freezing storage and using three different techniques: quantitative microbiological assay, HPLC-UV and LC-MS. Two parameters have been specifically monitored: storage temperature (-20 and -75 degrees C) and storage packaging (ground meat or bulk meat). No significant decrease was observed during the first 3 months of storage monitoring at -20 and -75 degrees C. On the contrary, the sample preparation significantly affected the drug concentration in muscle from the very beginning of the storage. Grinding the meat before storage allowed to keep the drug near the higher level of concentration (approximately 100 microg kg(-1)) when bulk meat stored frozen systematically led to a decreased value (approximately 75 microg kg(-1)). After 8 months of storage at -20 degrees C, a significant decrease arose and was never observed at -75 degrees C. All the results were similarly obtained with the three different techniques used simultaneously, which allows to indicate a good correlation between the techniques.     

Impurity-profiling UPLC methods for quantitative analysis of some antiemetics formulated with pyridoxine

Cyclizine hydrochloride (CYC) and meclozine hydrochloride (MEC) are antihistaminic drugs generally co-formulated with pyridoxine hydrochloride (PYR) to treat nausea and vomiting in pregnancy. Several analytical techniques have been applied for the determination of CYC or MEC with PYR, but determination of CYC impurity; benzhydrol (BEH) or MEC impurity; or 4-chlorobenzophenone (BEP) has not been paid attention to. Therefore, micellar UPLC method is introduced for analysis of ternary mixtures containing PYR together with both CYC and BEH (mixture I) or MEC and BEP (mixture II). Chromatographic separation was achieved using a Hypersil gold C₈ column (50 × 2.1 mm, 1.9 μm) using 0.01 M sodium dodecyl sulfate modified to pH 3.5 using phosphoric acid:acetonitrile (45:55 by volume) for mixture I and 0.1% sodium dodecyl sulfate, 0.1% sodium bicarbonate adjusted to pH 2.6 by phosphoric acid:acetonitrile (47:53 by volume) for mixture II as mobile phases. The separated peaks were detected at 230 and 245 nm for mixtures I and II, respectively. The adopted methods were validated in conformance with the International Conference on Harmonization (ICH) recommendations and were properly applied in commercial pharmaceutical formulation analysis. Comprehensive ecological comparison was achieved, confirming a higher ecological value of the presented methods compared to the earlier reported methods.     

Cold-temperature stability of five beta-lactam antibiotics in bovine milk and milk extracts prepared for liquid chromatography-electrospray ionization tandem mass spectrometry analysis

The stability of five major beta-lactam antibiotics (amoxicillin, ampicillin, cloxacillin, oxacillin, and penicillin G) in fortified milk and in milk extracts prepared for LC-ESIMS/MS analysis was studied at varying cold temperatures (4, -20, and -76 degrees C). Storage of milk samples at 4 degrees C resulted in measurable losses of all beta-lactams after 6 days (>50% in most cases). Slow degradation of penicillin G, cloxacillin, and oxacillin was observed in milk stored at -20 degrees C, but no losses were recorded at -76 degrees C over 4 weeks. All antibiotics showed good stability at all temperature tested in milk extracts prepared for LC-ESIMS/MS analysis. The results of this study emphasize adherence to adequate sample handling and storage protocols as to reflect residue levels at the time of sample submission.     

Multiresidue analytical method for the determination of eight penicillin antibiotics in muscle tissue by ion-pair reversed-phase HPLC after precolumn derivatization.

A high-performance liquid chromatographic multiresidue method was developed for the determination of 8 penicillin compounds (benzylpenicillin, phenoxymethylpenicillin, ampicillin, amoxicillin, nafcillin, oxacillin, cloxacillin, and dicloxacillin) at trace levels in muscle tissue. This method involves extraction of the penicillins with phosphate buffer pH 9 followed by cleanup and concentration on a C18 solid-phase extraction column and reaction with benzoic anhydride at 50 degrees C for 5 min and with 1,2,4-triazole and mercury(II) chloride solution pH 9 at 65 degrees C for 10 min. The derivatized compounds are eluted on a C8 column with a mobile phase containing acetonitrile and phosphate buffer (pH 6; 0.1 mol/L) loaded with sodium thiosulfate and ion-pairing tetrabutylammonium hydrogenosulphate. The method detection limit is approximately 3-11 micrograms/kg and the limit of determination was evaluated down to 25 micrograms/kg in line with the criteria of the EU decision No. 93/256/EEC.     

铁氰化钾高灵敏共振瑞利散射法测定青霉素类抗生素

在稀HCl介质中,K3[Fe(CN)6]与阿莫西林(AMO)、氨苄西林(AMP)、氯唑西林钠(CLO)、羧苄西林钠(CAR)和青霉素钠(BEN)等抗生素药物在加热条件下反应生成结合产物,会导致溶液的共振瑞利散射(RRS)强度急剧增强,并产生新的RRS光谱,5种反应产物的最大散射峰均位于330 nm附近。在一定的浓度范围内,不同的反应体系散射强度(∆I)与药物浓度成正比,反应具有很高的灵敏度,K3[Fe(CN)6]对5种药物的检出限分别在4.61至5.62 ng·mL-1之间。本文研究了RRS的光谱特征和适当的反应条件,并讨论了反应机理和散射增强的原因,还考察了共存物质的影响,表明方法具有较好的选择性,可用于胶囊、片剂和人血清及尿液中青霉素类药物的测定。     

Charm Safe-Level beta-Lactam Test for amoxicillin, ampicillin, ceftiofur, cephapirin, and penicillin G in raw commingled milk

The Charm Safe-Level beta-Lactam Test was evaluated by a U.S. Food and Drug Administration (FDA) test protocol administered by the AOAC-Research Institute. The sensitivity and selectivity of the test were evaluated with >800 negative raw commingled and drug-fortified milk samples by the manufacturer and an independent laboratory. Probit analysis by the independent laboratory determined the following 90% positive levels with 95% confidence: amoxicillin, 5.6 ppb; ampicillin, 8.5 ppb; cephapirin, 13.7 ppb; ceftiofur, 46.2 ppb; and penicillin G, 3.6 ppb. These values were within a range of +/- 20% of the manufacturer's data. Selection of negative samples met confidence specifications. Ruggedness parameters were studied and defined, and the stability of frozen milk was verified. There were no interferences from somatic cells (1,000,000 somatic cell count/mL) or bacteria (300,000 colony-forming units/mL), or from 27 other non-beta-lactam animal drugs. Test performance with raw milk samples containing incurred penicillin, ampicillin, and amoxicillin was consistent with the dose responses determined with fortified milk samples. Incurred cephalosporin in raw milk samples was detected at lower levels than was cephalosporin in fortified milk samples, presumably because of the presence of metabolite, as verified by other test methods. Quality control data support consistency in manufacture between batches and the stability of refrigerated test reagents for up to 1 year. Successful fulfillment of these criteria led to FDA certification of the test when used with a reader in U.S. milk testing programs.