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.     

A reversed phase liquid chromatographic method for the simultaneous determination of several common penicillins in human serum

A rapid and sensitive high performance liquid chromatographic method is described for the simultaneous determination of benzylpenicillin, ampicillin, phenoxymethylpenicillin, cloxacillin, dicloxacillin and nafcillin in small samples of human serum. The chromatographic system involves the use of a Spherisorb ODS reversed phase column and a gradient elution with 1 mM ammonium acetate buffer/acetonitrile (from 90:10 to 75:25 in 15 min). Detection and quantification are monitored by UV absorption at 208 nm. The compounds are extracted with dichloromethane, using tetrabutylammonium hydrogen sulfate neutralized with sodium hydroxide and buffered with borate as an ion pairing reagent; beta-hydroxyethyltheophylline is added as an internal standard. Our results show that the method is accurate and reproducible, allowing quantification of serum levels of assayed penicillins (0.5-50 micrograms/mL) without interference from other drugs commonly used in therapy. Recoveries were generally greater than 79.4%.     

Application of capillary electrophoresis to the determination of various benzylpenicillin salts

The application of capillary electrophoresis for separation of benzylpenicillin, procaine, benzathine and clemizole was investigated. Phosphate-borate buffer supplemented with sodium dodecyl sulphate 14.4 g/l and electrophoresis voltage 18 kV seem to provide optimal conditions for micellar electrokinetic chromatographic assay of penicillin salts. This method is selective and precise. The results obtained from CE method recovery assay (above 98% for all but procaine-97% substances) and from determination of benzylpenicillin by CE compared with HPLC results, confirmed good accuracy.     

Determination of Benzylpenicillin,Ampicillin, and Carbenicillin by Paper Chromatography

A mobile phase was developed for selective determination of benzylpenicillin, carbenicillin, and ampicillin in drugs by paper chromatography.__________Translated from Zhurnal Prikladnoi Khimii, Vol. 78, No. 4, 2005, pp. 613–615.Original Russian Text Copyright © 2005 by Alekseev, Nersesova, Khalyapina.     

A theoretical study of the aminolysis reaction of lysine 199 of human serum albumin with benzylpenicillin: consequences for immunochemistry of penicillins

Herein, we present results of a computational study on benzylpenicillin attachment to Lys199 of human serum albumin via an aminolysis reaction. The internal geometry of the reactive part of the system was taken from previous work at the B3LYP/6-31+G* level on the water-assisted aminolysis reaction of a penicillin model compound (Díaz, N.; Suárez, D.; Sordo, T. L. J. Am. Chem. Soc. 2000, 122, 6710--6719). The protein environment around Lys199, the 6-acylamino side chain, and the 2-methyl groups of benzylpenicillin were relaxed by carrying out geometry optimizations with a hybrid QM/MM method (PM3/AMBER). Two different mechanistic routes were explored: a one-step water-assisted process and a carboxylate and water-assisted route in which the beta-lactam carboxylate and the ancillary water molecule mediate the proton transfer from the epsilon-amino group of Lys199 to the beta-lactam leaving N atom. The corresponding energy profiles in the protein combine the B3LYP/6-31+G* and PM3 energies of the reactive subsystem (benzylpenicillin + Lys199 side chain + the ancillary water molecule) and semiempirical PM3 energies of the entire system evaluated with a "divide and conquer" linear-scaling method. It is observed that penicillin haptenation to HSA can proceed through the water-assisted concerted mechanism which is calculated to have a high energy barrier of approximately 38 kcal/mol, in agreement with the experimentally observed slow reaction kinetics.     

Potentiometric determination of penicillins with ion-selective electrodes

Quaternary ammonium, phosphonium and arsonium membrane electrodes sensitive to benzylpenicillin, ampicillin and oxacillin have been investigated. The order of merit of electrode performance is cetyltrioctylammonium> cetyltrioctylphosphonium> cetyltrioctylarsonium. The electrodes are suggested for use in rapid determination of penicillin drugs by direct potentiometry.     

Calixarenes in a membrane environment: a monolayer study on the miscibility of three p-tert-butylcalix[4]arene beta-lactam derivatives with 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine

Literature data indicate that some calixarene derivatives with antimicrobial activities may be useful as drugs; one of the aspects of the biological activity of different classes of antibiotics concerns interactions with lipid membranes. Here, the possibility of incorporation and/or translocation of three amphiphilic p-tert-butylcalix[4]arene derivatives across membranes was studied using lipid monolayers. The derivatives used have 6-aminopenicillanic acid or benzylpenicillin moieties grafted in alternate positions at the calixarene lower rim; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), a model bacterial membrane lipid, was used to prepare the monolayers. The miscibility of calixarene-antibiotic conjugates with lipid films was studied using surface pressure and surface potential measurements, as well as Brewster angle microscopy. The results obtained show that the miscibility is significantly different for the 6-aminopenicillanic acid and the two benzylpenicillin derivatives. Molecular modeling allowed the assessment of the lowest energy conformations of the calixarene derivatives and gave more insight into the interactions with the DMPE films.     

荷移荧光光谱法测定牛奶中苄青霉素残留

采用荧光光谱法研究了电子供体苄青霉素降解产物与电子受体四氯苯醌(TCBQ)之间的荷移反应,结果表明TCBQ与苄青霉素的酸性降解产物在甲醇-水介质中易发生荷移反应,生成稳定的络合物,其荧光强度较苄青霉素降解产物本身有了显著的增强。据此建立一种测定苄青霉素的新方法,在最佳条件下,苄青霉素浓度在0.30～8.0 mg/L范围...     

Development and validation of a reference measurement procedure for certification of phenytoin, phenobarbital, lamotrigine, and topiramate in human serum using isotope-dilution liquid chromatography/tandem mass spectrometry

Phenytoin (PHT), phenobarbital (PHB), lamotrigine (LTG), and topiramate (TPM) are some of the most widely used antiepileptic drugs (AEDs). Monitoring of their concentrations in serum is important for the treatment of epilepsy. A reference measurement procedure (RMP) for certification of PHT, PHB, LTG, and TPM in serum has been developed and critically evaluated. Isotopically labeled compounds of PHT, PHB, LTG, and TPM are used as internal standards for the four AEDs. The four drugs and their respective labeled internal standards are simultaneously extracted from serum using solid-phase extraction prior to reversed-phase liquid chromatography–tandem mass spectrometry (LC-MS/MS). Chromatographic separation was performed using a C₁₈ column. Electrospray ionization (ESI) in the positive ion mode for PHT and LTG, and in the negative ion mode for PHB and TPM were used. The recovery of AEDs added to serum (accuracy of the extraction method) was evaluated by recovery studies of measuring the four drugs in spiked samples with known drug levels. The recoveries of the added drugs ranged from 98.6% to 102.0%. The absolute recoveries (extraction efficiencies) of the four drugs with this method ranged from 97% to 100%. Excellent repeatability was obtained for the four drugs with between-set coefficients of variation (CVs) within 1%. The type B components estimates are conservatively large and are considerably larger than the type A component. Therefore, we use the usual metrological expansion factor of 2 to provide an approximate 95% coverage interval. The relative expanded uncertainties for the four AEDs ranged from 2.3% to 2.4%. This LC-MS/MS RMP for PHT, PHB, LTG, and TPM in serum demonstrating good accuracy and precision can be used to assess the accuracy of routine methods used in clinical laboratories.     

Determination of antimigraine compounds rizatriptan, zolmitriptan, naratriptan and sumatriptan in human serum by liquid chromatography/electrospray tandem mass spectrometry

Development of a rapid, sensitive and selective method for the determination of antimigraine drugs from human serum is essential for understanding the pharmacokinetics of these drugs when administered concurrently. Solid phase extraction (SPE) using Oasis HLB was used to extract the drugs (sumatriptan, naratriptan, zolmitriptan and rizatriptan) and the internal standard bufotenine from serum. A method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated to simultaneously quantitate these antimigraine drugs from human serum. The precursor and major product ions of the analytes were monitored on a triple quadrupole mass spectrometer with positive ion electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The base peak in all the analytes is formed by alpha cleavage associated with protonation of the secondary amine. Mechanisms for the formation of the collision-induced dissociation products of these antimigraine compounds are proposed. Linear calibration curves were generated from 1-100 ng/mL with all coefficients of determination greater than 0.99. The inter- and intraday precision (%RSD) were less than 9.3% and accuracy (%error) was less than 9.8% for all components. The limits of detection (LOD) for the method were 250 pg/mL for sumatriptan and 100 pg/mL for the remaining analytes based on a signal-to-noise ratio of 3.     

Synthesis of a mixed-functional silica support for direct injection analysis of drugs in serum by liquid chromatography

Summary A new mixed-functional silica material has been developed for direct injection analysis of drugs in serum. The mixed-functional material is synthesized from porous silica by three steps; introduction of 3-glycidoxypropyl groups, introduction of phenyl groups, and hydrolysis of the oxirane ring to diol groups. The prepared column packing can be used for direct injection analysis of hydrophobic and hydrophilic drugs in serum over the eluent pH range employed for ordinary siloxane-bonded silica. The recovery of drugs from serum was almost 100%, regardless of the difference in their protein bindings.     

Antidepressants in serum determined by isolation with two on-line sorbent cartridges and liquid chromatography

A selective method for measuring tricyclic antidepressants in serum is reported. A single assay can be done within ca. 30 min and eight samples can be assayed in less than 150 min. A 1-ml serum sample was diluted and the drugs were extracted from it by passage through a graphitized carbon black (Carbopack B) cartridge. After one washing, this cartridge was connected on line to another one containing a silica-based strong acid exchanger. The tricyclics were removed from the Carbopack surface and selectively readsorbed onto the cation-exchange surface by passing 4 ml of methylene chloride-methanol (60:40, v/v) through the two cartridges. After another wash, the drugs were desorbed from the cation-exchange surface with 0.8 ml of acetonitrile-methanol-water (72:18:10, v/v) saturated with potassium chloride. An aliquot of this solution was chromatographed on a cyano column, and the absorbance of the effluent was measured at 215 nm. The mean analytical recoveries of tricyclic antidepressants added to serum within the range 10-200 micrograms/l exceeded 90%, except for 8-hydroxyamoxapine (mean recovery 85.3%) and amoxapine (mean recovery 83.8%) at the lowest serum concentration considered.     

Separation and determination of selected psychotropic drugs in human serum by SPE/HPLC/DAD on C18 and Polar-RP columns

A simple and sensitive high-performance liquid chromatographic method with diode array detection is described for the quantification of some psychotropic drugs: fluoxetine, sertraline, alprazolam, perphenazine, zolpidem, and hydroxyzine in fortified human serum samples. The test compounds were extracted from human serum by solid-phase extraction using C18 extraction column and injected into C18 or Polar-RP analytical columns for separation. A mobile phase was composed of methanol, water, acetate buffer at pH 3.5, and diethylamine. The method was validated for the concentration range 0.4–5?µg?mL^?1 for zolpidem and 0.5–6?µg?mL^?1 for other drugs. Mean recoveries were from 87.79% to 107.94% with adequate precision (% RSD ≤2.1%). The full separation of all investigated drugs, good peaks’ symmetry, and simultaneously high system efficiency were obtained on Polar-RP column, which was used for the first time to analyze these drugs. System efficiency obtained on the column was significantly higher compared to that obtained on commonly used C18 column. The method seems to be suitable for the analysis of investigated drugs in human plasma for psychiatric patients in multiple drug overdoses as well as for control of pharmacotherapy, particularly in combination therapy.     

Intramolecular general acid catalysis in the aminolysis of beta-lactam antibiotics

The rate of aminolysis of benzylpenicillin and cephaloridine by hydroxylamine, unlike other amines, shows only a first order dependence on amine concentration. The rate enhancement compared with that predicted from a Bronsted plot for other primary amines with benzylpenicillin is greater than 10(6). This is much more than an alpha-effect and is compatible with rate-limiting formation of the tetrahedral intermediate due to a rapid intramolecular general acid catalysed breakdown of the intermediate. For cephaloridine, the rate enhancement is greater than 10(4) which demonstrates that beta-lactam C-N bond fission and expulsion of the leaving group at C3' are not concerted.     

A micro liquid column chromatography procedure for twelve anticonvulsants and some of their metabolites

Solvent extracts of 50 microliter volumes of serum were sufficient for the determination of twelve anticonvulsant drugs. The liquid chromatography procedure utilized a C-18 reversed-phase column and isocratic elution with 15% acetonitrile in water. No derivatization was required. Eluted anticonvulsants were detected by UV absorption at 195 nm and quantitated by drug--internal standard peak area ratios. The procedure provided linear working curves over the concentration range from 1 to 100 mg/l of drug in the serum. The procedure for serum provided recoveries of the drugs from 92 to 101%. Within-day precision was about 4% and day-to-day precision was about 6.5%. The procedures has been applied to urine samples to facilitate bioavailability studies. Data are also given for several metabolites. There is a discussion of many practical aspects of the procedure to improve the reliability of the results.     

Synthesis,isolation, and purification of benzylpenicillin β-diethylaminoethyl ester hydroiodide

Processes of synthesis, isolation, and purification of benzylpenicillin β-diethylaminoethyl ester hydroiodide were studied. The optimal conditions of benzylpenicillin esterification with chloroethyldiethylamine in dimetylformamide and acetonitrile were found. Processes for isolation of the target product were developed and its physicochemical properties were studied. Product stabilization conditions providing a stable pH value in solutions of benzylpenicillin β-diethylaminoethyl ester hydroiodide were found.     

Zur Kenntnis der Benzylpenicilloinsäure

The molar penamaldate value of benzylpenicilloic acid and the influence of large amounts of benzylpenicillin on the estimation of penamaldate values and penamaldate stabilities of benzylpenicilloic acid and ε-(benzylpenicilloyl-α-amido)-caproic acid have been determined. The molar penamaldate values of purified benzylpenicilloate and of benzylpenicillin hydrolyzed in alkali were equal, indicating that benzylpenicillin upon alkaline hydrolysis is converted to penicilloic acid exclusively, with no side reactions occurring to any considerable degree. Benzylpenicilloate mutarotates in neutral solution as well as in alkali, the reaction being unaffected by metal ion complexing with ethylene diamine tetraacetate. ε-(Benzylpenicilloyl-α-amido)-caproate also mutarotates in alkali; no mutarotation occurs however in neutral solution. Different mechanisms of epimerisation have therefore to be considered for the mutarotations of penicilloates and of α-amides of penicilloic acid respectively.     

Spectrophotometric determination of penicilloates in penicillins

A procedure has been developed for the determination of penicilloates based on the reduction of CuII and detection of CuI using neocuproine. The sensitivity range is 5.6-56 microns penicilloate and the standard deviation was found to be between 1 and 2%. All the penicilloates tested reduced CuII under the conditions of the assay and a slight modification allowed a reasonably accurate determination of the level of this biologically inactive ingredient in a mixture with the parent penicillin. The deterioration of solutions of benzylpenicillin was also studied.     

高效液相色谱法同时测定血清和尿中厚朴酚与和厚朴酚

 建立了大鼠服用厚朴提取物后的血清中及尿中厚朴酚与和厚朴酚的高效液相色谱测定法。色谱柱填料为SpherisorbC18,流动相为甲醇-水-冰醋酸(体积比为70∶30∶1),UV检测波长为294nm,灵敏度0.005AUFS。样品用甲醇沉淀蛋白,上清液酸化后用乙酸乙酯-乙醚萃取,然后测定其中的药物浓度。血清和尿中的药物浓度与峰面积的线性关系良好,线性范围分别为0.05～2mg/L(厚朴酚)、0.025～1mg/L(和厚朴酚);精密度和重现性良好。血清中厚朴酚与和厚朴酚的平均加样回收率分别为95.6%(RSD=3.85%)和93.8%(RSD=3.95%),尿中分别为96.0%(RSD=3.83%)和94.9%(RSD=3.54%)。     

Determination of benzylpenicillin, oxacillin, cloxacillin, and dicloxacillin in cows' milk by ion-pair high-performance liquid chromatography after precolumn derivatization

A high-performance liquid-chromatographic method has been developed for the determination of five penicillin compounds (benzylpenicillin, phenoxymethylpenicillin, oxacillin, cloxacillin, and dicloxacillin) at trace levels in commercially available milk samples. This method comprises extraction of the lipids with ethyl acetate, clean-up and concentration on a C-18 solid-phase extraction column, and derivatization with 1,2,4-triazole and mercury(II) chloride solution, pH 8, at 65 degrees C for 10 min. The derivatized compounds are eluted from a C-2 column with a mobile phase containing acetonitrile and phosphate buffer loaded with sodium thiosulfate and tetrabutylammonium hydrogen sulfate as ion-pairing reagent. The limit of determination was found to be 4 microg L(-1) milk for benzylpenicillin and 10 microg L(-1) for the others. This meets EU criteria according to decision No. 93/256/EEC.