Application of reversed-phase liquid chromatography and prepacked C18 cartridges for the analysis of oxytetracycline and related compounds

The reversed-phase (RP) chromatographic separation of oxytetracycline (OTC) 4-epioxytetracycline (4-epiOTC), α-apooxytetracycline (α-apoOTC), and β-apooxytetracycline (β-apoOTC) has been accomplished on an Inertsil C₈ column at ambient temperature. Using the simplex method of solvent optimization, a 0.1 M ammonium acetate buffer (pH 3.0)-acetonitrile-tetrahydrofuran (72.5:12.5:15, v/v/v) mobile phase was found to give excellent separation of the compounds. OTC, 4-epiOTC, α-apoOTC and β-apoOTC were resolved in 35 min with calculated detection limits of 40, 20, 50 and 140 ng/ml, respectively. Solid-phase extraction (using RP C₁₈ cartridges) of OTC and OTC degradation compounds from distilled water and porcine muscle was tested at four concentration levels ranging from 200 to 2000 ng/ml (g); overall mean recovery of OTC from distilled water and porcine tissue was greater than 90% and 70%, respectively.     

Rapid and sensitive high-performance liquid chromatographic determination of four cephalosporin antibiotics in pharmaceuticals and body fluids

A rapid, accurate and sensitive method has been developed and validated for the quantitative simultaneous determination of four cephalosporins, cephalexin and cefadroxil (first-generation), cefaclor (second-generation) and cefataxim (third-generation), in pharmaceuticals as well as in human blood serum and urine. A Spherisorb ODS-2 250 x 4-mm, 5-microm analytical column was used with an eluting system consisting of a mixture of acetate buffer (pH 4.0)-CH(3)OH 78-22% (v/v) at a flow-rate 1.2 ml/min. Detection was performed with a variable wavelength UV-Vis detector at 265 nm resulting in limit of detection of 0.2 ng for cefadroxil and cephalexin, but only 0.1 ng for cefotaxime and cefaclor per 20-microl injection. Hydrochlorothiazide (HCT) (6-chloro-3,4-dihydro-7 sulfanyl-2H-1,2,4-benzothiadiazine-1-1-dioxide) was used as internal standard at a concentration of 2 ng/microl. A rectilinear relationship was observed up to 8, 5, 12 and 35 ng/microl for cefadroxil, cefotaxime, cefaclor, cephalexin, respectively. Analysis time was less than 7 min. The statistical evaluation of the method was examined by means of within-day repeatability (n=8) and day-to-day precision (n=9) and was found to be satisfactory with high accuracy and precision. The method was applied to the determination of the cephalosporins in commercial pharmaceuticals and in biological fluids: human blood serum after solid-phase extraction and urine simply after filtration and dilution. Recovery of analytes in spiked samples was in the range from 76.3 to 112.0%, over the range of 1-8 ng/microl.     

Search for Microorganisms Producing Cephalosporin Acylase and Enzymatic Synthesis of Cephalosporins

Microorganisms were tested for production of cephalosporin acylase. Some bacteria showed strong acylase activity for all of cephalexin, cephaloridine, cephalotin, penicillin G and ampicillin. Some showed a rather specific activity for cephalexin. Pseudomonas melanogenum KY 3987 showed specific activity only for cephalexin and ampicillin which contain a side chain of d-phenylglycine. Most of these acylase-producing bacteria had the ability to synthesize cephalexin and other cephalosporins from 7-aminocephem compounds and organic acid esters. Among them, Ktuyvera citrophila KY 7844 was one of the most promising organisms for enzymatic synthesis of cephalosporins. This organism had the ability to catalyze N-acylation of 7-aminocephem compound not only with α-amino acid ester, but also with such acid esters as 1-(1 H)-tetrazolylacetate methylester which has no α-amino group.     

Chemiluminescence immunoassay for the rapid and sensitive detection of antibody against porcine parvovirus by using horseradish peroxidase/detection antibody‐coated gold nanoparticles as nanoprobes

A rapid, simple, facile, sensitive and enzyme‐amplified chemiluminescence immunoassay (CLIA) method to detect antibodies against porcine parvovirus has been developed. Horseradish peroxidase (HRP) and the detection antibody were simultaneously co‐immobilized on the surface of gold nanoparticles using the electrostatic method to form gold nanoparticle‐based nanoprobes. This nanoprobe was employed in a sandwich‐type CLIA, which enables CL signal readout from enzymatic catalysis and results in signal amplification. The presence of porcine parvovirus infection was determined in porcine parvovirus antibodies by measuring the CL intensity caused by the reaction of HRP–luminol with H₂O₂. Under optimal conditions, the obtained calibration plot for the standard positive serum was approximately linear within the dilution range of 1:80 to 1:5120. The limit of detection for the assay was 1:10,240 (S/N = 3), which is much lower than that typically achieved with an enzyme‐linked immunosorbent assay (1:160; S/N = 3). A series of repeatability measurements using 1:320‐fold diluted standard positive serum gave reproducible results with a relative standard deviation of 4.9% (n = 11). The ability of the immunosensor to analyze clinical samples was tested on porcine sera. The immunosensor had an efficiency of 90%, a sensitivity of 93.3%, and a specificity of 87.5% relative to the enzyme‐linked immunosorbent assay results. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of 5‐hydroxytryptamine in serum by electrochemiluminescence detection with the aid of capillary electrophoresis

A rapid, sensitive and simple electrochemiluminescence method for the determination of 5‐hydroxytryptamine (5‐HT) using capillary electrophoresis was proposed. The experimental parameters, including the detection potential, the concentration of Ru(bpy)₃²⁺, the concentration and pH of phosphate buffer for separation and detection, the injection voltage and time and the separation voltage on the determination of 5‐HT, were optimized. Under the optimized conditions, the linear concentration range for 5‐HT was 3.5 × 10^‐9–5.1 × 10^‐3 mol/L, with a detection limit of 5 × 10^‐10 mol/L. The relative standard deviations (RSDs) of the ECL intensity and the migration times for six continuous injections of 1.0 µmol/L 5‐HT were 2.48% and 1.3%, respectively. The method was successfully applied to 5‐HT assay in samples of human serum in 5 min and the extraction recoveries with spiked serum samples were over 94.4%. Copyright © 2011 John Wiley & Sons, Ltd.     

In Vitro Antimicrobial Activity and Human Pharmacology of Cephalexin, a New Orally Absorbed Cephalosporin C Antibiotic

Concentrations of cephalexin (an orally absorbed derivative of cephalosporin C) in serum and urine were determined in normal volunteers and patients. The in vitro antibacterial activity was also studied. All strains of group A β-hemolytic streptococci and Diplococcus pneumoniae were inhibited by 3.1 μg/ml. Of the Staphylococcus aureus strains, 88% were inhibited by 6.3 μg/ml, and 12.5 μg/ml was inhibitory for all S. aureus, 80% of Escherichia coli, 72% of Klebsiella-Aerobacter, and 56% of Proteus mirabilis strains. About 90 to 96% of E. coli, Klebsiella Aerobacter, and P. mirabilis strains were inhibited by 25 μg of cephalexin per ml. Pseudomonas and indole-positive Proteus strains proved to be quite resistant to cephalexin. Cephalexin was well absorbed after oral administration. A peak serum concentration of cephalexin of at least 5 μg/ml was achieved in each volunteer with 250 and 500-mg doses. A mean peak serum concentration of 7.7 μg/ml was achieved with 250-mg doses; 12.3μg/ml was achieved with 500-mg doses of antibiotic. Food did not interfere with absorption. Probenecid enhanced both the peak serum concentration and the duration of antibiotic activity in the serum. Over 90% of the administered dose was excreted in the urine within 6 hr. The mean peak serum concentration of cephalexin after an oral dose of 500 mg was adequate to inhibit all group A streptococci, D. pneumoniae, and S. aureus, 85% of E. coli, and about 40 to 75% of Klebsiella-Aerobacter and P. mirabilis strains. Levels of cephalexin in urine were adequate to inhibit over 90% of E. coli, and P. mirabilis and 80 to 96% of Klebsiella-Aerobacter strains.     

Chemiluminescent immunoassay of thyroxine enhanced by microchip electrophoresis

A homogeneous chemiluminescent immunoassay of thyroxine (T4) enhanced by microchip electrophoresis separation has been developed. The method deployed the competitive immunoreaction of T4 and horseradish peroxidase (HRP)-labeled T4 (HRP-T4) with anti-T4 mouse monoclonal antibody (Ab). HRP-T4 and the HRP-T4-Ab complex were separated and quantified by using microchip electrophoresis (MCE) with chemiluminescence (CL) detection. Highly sensitive CL detection was achieved by means of HPR-catalyzed luminol-H₂O₂ reaction. Due to the effective MCE separation, the CL analytical signal was less prone to sample matrix interference. Under the selected assay conditions, the MCE separation was accomplished within 60 s. The linear range for T4 was 5-250 nM with a detection limit of 2.2 nM (signal/noise ratio = 3). The current method was successfully applied for the quantification of T4 in human serum samples. It was demonstrated that the current MCE-CL-enhanced competitive immunoassay was quick, sensitive, and highly selective. It may serve as a tool for clinical analysis of T4 to assist in the diagnosis of thyroid gland functions.     

Reactive oxygen species inhibit catalytic activity of peptidylarginine deiminase

Protein citrullination catalysed by peptidylarginine deiminase (PAD) may play an important pathogenic role in several chronic inflammatory diseases and malignancies. PAD2, PAD4, and citrullinated proteins are found in the synovium of rheumatoid arthritis patients. PAD activity is dependent on calcium and reducing conditions. However, reactive oxygen species (ROS) have been shown to induce citrullination of histones in granulocytes. Here we examine the ability of H₂O₂ and leukocyte-derived ROS to regulate PAD activity using citrullination of fibrinogen as read-out. H₂O₂ at concentrations above 40?µM inhibited the catalytic activity of PAD2 and PAD4 in a dose-dependent manner. PMA-stimulated leukocytes citrullinated fibrinogen and this citrullination was markedly enhanced when ROS formation was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium (DPI). In contrast, PAD released from stimulated leukocytes was unaffected by exogenously added H₂O₂ at concentrations up to 1000?µM. The role of ROS in regulating PAD activity may play an important part in preventing hypercitrullination of proteins.     

Liquid chromatographic determination of manidipine in serum with electrochemical detection

A highly sensitive and selective method for determining a dihydropyridine calcium antagonist, manidipine, by liquid chromatography using column switching with electrochemical detection was developed. Manidipine in serum was extracted by a rapid and simple procedure based on C₈ bonded-phase extraction and then silica extraction. Manidipine and nilvadipine as an internal standard were separated on a C₈ bonded-phase HPLC column and detected by high conversion efficiency amperometric detection at +0.7 V. Manidipine and nilvadipine (I.S.) were separated from an endogenous interference peak in serum and concentrated on a pre-column (C₁₈) by column switching using an isocratic mobile phase, and then the corresponding fractions were introduced to an analytical column with a C₈ stationary phase. Determination of manidipine was possible over the concentration range 0.5–10 ng/ml: the limit of detection was 0.3 ng/ml. The recovery of manidipine added to serum was 93.1–98.4% with coefficients of variation of less than 7.1%. The method is applicable to drug level monitoring in the serum of healthy volunteers treated with manidipine and to the analysis of pharmacokinetics.     

Antigen–antibody interaction from quartz crystal microbalance immunosensors based on magnetic CoFe<Subscript>2</Subscript>O<Subscript>4</Subscript>/SiO<Subscript>2</Subscript> composite nanoparticle-functionalized biomimetic interface

A new quartz crystal microbalance immunoassay for the detection of carcinoembryonic antigen (CEA) was developed by means of immobilizing anti-CEA onto magnetic CoFe₂O₄/SiO₂ composite nanoparticles-functionalized biomimetic interface. Under optimal conditions, the frequency shift was proportional to the CEA concentration in the range of 2.5–55 ng/mL with a detection limit of 0.5 ng/mL at a signal-to-noise ratio of 3. Moreover, the immunosensor system showed an acceptable reproducibility and stability. Clinical serum specimens were assayed with this method, and the results were in acceptable agreement with those obtained from ELISA. Compared with the conventional ELISA assay, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the developed immunoassay protocol could be further extended for the determination of other antigens.     

Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen

Introduction

Members of the peptidylarginine deiminase (PAD) family catalyse the posttranslational conversion of peptidylarginine to peptidylcitrulline. Citrullination of proteins is well described in rheumatoid arthritis (RA), and hypercitrullination of proteins may be related to inflammation in general. PAD activity has been demonstrated in various cell lysates, but so far not in synovial fluid. We aimed to develop an assay for detection of PAD activity, if any, in synovial fluid from RA patients.

Methods

An enzyme-linked immunosorbent assay using human fibrinogen as the immobilized substrate for citrullination and anti-citrullinated fibrinogen antibody as the detecting agent were used for measurement of PAD activity in synovial fluid samples from five RA patients. The concentrations of PAD2 and calcium were also determined.

Results

Approximately 150 times lower levels of recombinant human PAD2 (rhPAD2) than of rhPAD4 were required for citrullination of fibrinogen. PAD activity was detected in four of five synovial fluid samples from RA patients and correlated with PAD2 concentrations in the samples (r = 0.98, P = 0.003). The calcium requirement for half-maximal activities of PAD2 and PAD4 were found in a range from 0.35 to 1.85 mM, and synovial fluid was found to contain sufficient calcium levels for the citrullination process to occur.

Conclusions

We present an assay with high specificity for PAD2 activity and show that citrullination of fibrinogen can occur in cell-free synovial fluid from RA patients.     

Pharmacokinetics of cephalexin in sea bream, Sparus aurata (L.), after a single intraperitoneal injection

The pharmacokinetic properties of cephalexin were studied in sea bream (mean weight 77 g), Sparus aurata (L.) after a single intraperitoneal injection (200 mg kg^?1). Pharmacokinetic analysis of the serum concentrations vs time points obtained was performed using non‐compartmental analysis and a compartmental pharmacokinetic model approach. In the latter case, a two‐compartment open model with a lag time gave the best fitting. The maximum peak serum concentration was 5.018 mg ml^?1 kg^?1 1 h post‐treatment. The area under the curve (AUC) cephalexin was 17.394 mg·h kg^?1 and the elimination half‐life of 1.83 h.     

Determination of retinoids by reversed-phase capillary liquid chromatography with amperometric electrochemical detection

A method for separating and detecting retinoids by reversed-phase capillary liquid chromatography with amperometric electrochemical detection is described. Packed columns with an inner diameter of 180 μm were employed for the separation using a C₁₈ stationary phase and a mobile phase containing acetonitrile-water-methanol (65:32.5:2.5, v/v/v) with 1% tetrabutylammonium perchlorate and 0.174 M acetate buffered at pH 5. The detection cell consisted of a carbon fiber barrel electrode held at 0.9 V versus an Ag/AgCl reference. Injection volumes of 2 μl produced detection limits of 2.73, 0.472, 0.428, and 0.267 fmol (or 410, 64.1, 60.9, and 38.2 pg ml⁻¹) for 13-cis-retinoic acid, all-trans-retinoic acid, retinaldehyde, and retinol, respectively. This represents an improvement in detection limits of at least three orders of magnitude for similar analyses using liquid chromatography and UV absorbance detection. The detector signal was linear over two orders of magnitude of analyte concentration. Retinoid concentrations in bovine serum were determined and found to be in good agreement with previously reported values.     

A comparative study of PCR product detection and quantitation by electrochemiluminescence and fluorescence

Amplification and detection of target DNA sequences are made possible in a polymerase chain reaction (PCR) by using a mixture of biotinylated and ruthenium(II) trisbipyridal (Ru(bpy)₃²⁺)-end-labelled primers. In this way, biotin for capture and Ru(bpy)₃²⁺ for detection are directly incorporated into the PCR product obviating subsequent probe hybridization. PCR of a bacterial DNA template from Alteromonas species strain JD6.5 using a cocktail of biotin- and Ru(bpy)₃²⁺-labelled primers amplified a 1 kilobase region. Serial dilution of PCR product followed by magnetic separation with Streptavidin (SA)-coated magnetic beads and an electrochemiluminescence (ECL) assay using the semi-automated QPCR System 5000 demonstrated sensitive (pg range) DNA detection. ECL assay of probe hybridization to a human immunodeficiency virus (HIV) sequence also produced pg level sensitivity. Quantitative DNA determination by ECL assay correlated well with visual detection of DNA in electrophoretic gels. However, DNA detection by ECL assay was 10 to 100 times more sensitive than conventional ethidium bromide staining. The combination of DNA-based magnetic separation with ECL assay provides a very sensitive and rapid method of quantitating DNA which, owing to its rapid and facile nature, may have many applications in the research, environmental monitoring, industrial and clinical fields.     

Protein analysis by membrane preconcentration–capillary electrophoresis: systematic evaluation of parameters affecting preconcentration and separation

Fast and efficient analysis of proteins in physiological fluids is of great interest to researchers and clinicians alike. Capillary electrophoresis (CE) has proven to be a potentially valuable tool for the separation of proteins in specimens. However, a generally acknowledged drawback of this technique is the limited sample volumes which can be loaded onto the CE capillary which results in a poor concentration limit of detection. In addition, matrix components in samples may also interfere with separation and detection of analytes. Membrane preconcentration–CE (mPC–CE) has proved to be effective in overcoming these problems. In this report, we describe the systematic evaluation of parameters affecting on-line preconcentration/clean-up and separation of protein mixtures by mPC–CE. Method development was carried out with a standard mixture of proteins (lysozyme, myoglobin, carbonic anhydrase, and human serum albumin). First, using MALDI-TOF-MS, membrane materials with cation-exchange (R-SO₃H) or hydrophobic (C₂, C₈, C₁₈, SDB) characteristics were evaluated for their potential to retain proteins in mPC cartridges. Hydrophobic membranes were found most suitable for this application. Next, all mPC–CE analysis of protein samples were performed in polybrene coated capillaries and parameters affecting sample loading, washing and elution, such as the composition and volume of the elution solvent were investigated. Furthermore, to achieve optimal mPC–CE performance for the separation of protein mixtures parameters affecting postelution focusing and electrophoresis, including the composition of the background electrolyte and a trailing stacking buffer were varied. Optimal conditions for mPC–CE analysis of proteins using a C₂ impregnated membrane preconcentration (mPC) cartridge were achieved with a background electrolyte of 5% acetic acid and 2 mM ammonium acetate, 60 nl of 80% acetonitrile in H₂O as an elution solvent, and 60 nl of 0.5% ammonium hydroxide as a trailing stacking buffer. The developed method was used successfully to separate proteins in aqueous humor, which contains numerous proteins in a complex matrix of salts.     

Determination of nanomolar uric and ascorbic acids using enlarged gold nanoparticles modified electrode

Individual and simultaneous determination of 50 nM uric acid (UA) and ascorbic acid (AA) using enlarged, citrate-stabilized gold nanoparticles (AuNPs) self-assembled to 2,5-dimercapto-1,3,4-thiadiazole (DMT) monolayer modified Au (Au/DMT) electrode by an amperometric method is described for the first time. Self-assembly of AuNPs on the electrode surface was confirmed by atomic force microscopy (AFM), attenuated total reflectance FT-IR and diffuse reflectance spectral measurements. The electron transfer reaction (ETR) of [Fe(CN)₆]^3−/4− was blocked at Au/DMT electrode, whereas it was restored with a peak separation of 200 mV after the attachment of AuNPs on the Au/DMT (Au/DMT/AuNPs) electrode, which was confirmed from the ETR of the [Fe(CN)₆]^3−/4− redox couple. When the self-assembled AuNPs were enlarged by hydroxylamine seeding, the ETR of [Fe(CN)₆]^3−/4− was improved significantly with a peak separation of 100 mV. Tapping mode AFM showed that the average size of the enlarged-AuNPs (E-AuNPs) was 50-70 nm. The E-AuNPs modified electrode catalyzes the oxidation of AA and UA, separates their voltammetric signals by 200 mV, and has excellent sensitivity towards AA and UA with a detection limit of 50 nM. The practical application of the modified electrode was demonstrated by measuring the concentration of UA in blood serum and urine.     

Highly sensitive coupled-column high-performance liquid chromatographic method for the separation and quantitation of the diastereomers of leucovorin and 5-methyltetrahydrofolate in serum and urine

A column-switching chiral HPLC assay was developed that allows the separation and quantitation of the diastereomers of leucovorin (LV, 5-formyltetrahydrofolic acid) and its metabolite 5-methyltetrahydrofolate (METHF) in serum and urine by means of fluorescence detection. The analysis procedure consists of an on-line concentration of the folates in the HPLC system which is followed by the elution and separation of folates on an achiral 3-μm Microbore C₁₈ column in (6R,S)-LV and (6R,S)-LV and (6R,S)-METHF are subsequently transferred on-line onto a chiral 7-μm bovine serum albumin column through a Rheodyne valve system and are separated into their distereometers. Time of analysis is 70 min. Detection limit is 5 ng/ml for each diastereometer. The within-day variation ranges between 3.2 and 15.8% in relation to the measured concentration. Between-day variation is 4.4–12.1% for a concentration of 100 ng/ml for each diastereometer. (6R,S)-LV and (6S)-LV pharmacokinetics were assessed by analyzing serum and urine samples of four-healthy volunteers.     

PAD4 is not essential for disease in the K/BxN murine autoantibody-mediated model of arthritis

Introduction

Both murine and human genome-wide association studies have implicated peptidyl arginine deiminase (PAD4) as a susceptibility gene in rheumatoid arthritis (RA). In addition, patients with RA commonly have autoantibodies which recognize PAD4 or and/or citrullinated peptides. This study aims to evaluate the role of PAD4 in the effector phase of arthritis.

Methods

PAD4 knock out (KO) and wild type (WT) C57BL/6J mice were injected with K/BxN sera to induce disease. Progression of disease was monitored by measuring paw and ankle swelling and clinical indexes of disease, and pathogenesis was assessed by indexing of clinical progression on paws collected from WT and PAD4 KO mice injected with K/BxN serum. PAD4 activity was determined by visualization of neutrophil extracellular traps (NETs) and immunohistological analysis of histone citrullination.

Results

PAD4 activity is readily detectable in the inflamed synovium of WT but not PAD4 deficient animals, as demonstrated by histone citrullination and NET formation. However, PAD4 WT and KO animals develop K/BxN serum transfer disease with comparable severity and kinetics, with no statistically significant differences noted in clinical scores, swelling, joint erosion or joint invasion.

Conclusions

PAD4 WT and KO mice develop disease in the K/BxN serum transfer model of arthritis with similar severity and kinetics, indicating that PAD4 is dispensable in this effector phase model of disease.     

A specific radioimmunoassay for PGE2 using an antibody with high specificity and a sephadex LH-20 microcolumn for the separation of prostaglandins

Antiserum against PGE₂ was raised in rabbits following immunization with prostaglandin-hen-γ-globulin conjugate. The antiserum exhibited 14% cross reactivity with PGE₁ and far less cross-reaction with heterologous prostaglandins. A microcolumn of Sephadex LH-20 was used for a partial, but sufficient separation of PGE₂ from PGE₁ and a complete separation from heterologous prostaglandins to ensure a specific RIA for PGE₂. The precision of the method in the range 10–500 picograms showed a coefficient of variation varying between 4 and 13%. The detection limit was 10 picograms corresponding to 15 pg/ml of PGE₂ in serum.In order to demonstrate the validity of the method values obtained for non-diuretic rat renal venous serum were compared with those obtained using the isotope derivative method of Bojesen & Buckhave (1972) on the same samples. The concentrations of PGE₂ obtained were 239 ± 25 pg/ml and 250 ± 58 pg/ml, respectively.     

Comparative study of the interaction of cephalosporins with blood serum proteins and organ homogenates

Interaction of 7 semisynthetic antibiotics (cephaloridine, cephalexin, cephradine, cephazolin, cephalotin, cephacetrile and cephapirin) with proteins of human, bovine and rabbit blood serum, as well as organ and tissue homogenates of rats was studied comparatively. The study showed that binding of the cephalosporins by the blood serum depended on both the chemical structure of the antibiotic and the species affiliation of the protein substrate. The binding lvels of cephazolin and cephalotin by the blood serum proteins (except bovine serum) were the highest, while the binding level of cephaloridine was the lowest. A significant decrease in the values of binding by the serum proteins of the drugs with high percentage of binding was observed when the drug concentrations in solution were increased. Binding of the cephalosporins by the blood serum proteins was in most cases completely reversible. The activity of the cephalosporins decreased in the presence of the rat organ and tissue homogenates. The levels of the activity decrease as compared to the theoretical ones were the highest with the use of cephalotin, cephacetrile and cephapirin. The lowest values of detection of these antibiotics were noted on their incubation with the liver, kidneys and lungs.