Quantification of three beta-lactam antibiotics in breast milk and human plasma by hydrophilic interaction liquid chromatography/positive-ion electrospray ionization mass spectrometry.

The use of cephalosporins during breast feeding raises several issues, including the risk of drug exposure through breast milk for the infant. In this paper, a hydrophilic interaction liquid chromatography/positive ion electrospray mass spectrometric assay (HILIC/ESI-MS) was developed for the quantitation of cefuroxime, cefoxitin, and cefazolin in breast milk and human plasma. The assay was based on the use of small sample size, 25 µL of biological samples, following acetonitrile precipitation of proteins and filtration that enabled injection into the HILIC/ESI-MS system. All analytes and the internal standard, alfuzosin, were separated by using a ZIC®-HILIC analytical column (150.0 × 2.1 mm i.d., particle size 3.5 µm, 200 Å) with isocratic elution. The mobile phase was composed of a 6% 12.5 mM ammonium acetate water solution in acetonitrile and pumped at a flow rate of 0.25 mL min-1 . The assay was linear over a concentration range of 0.2 to 5 µg mL-1 and 0.4 to 20 µg mL-1 for all the analytes in breast milk and in human plasma, respectively. Intermediate precision was found to be less than 4.2% over the tested concentration ranges. A run time of less than 12 min for each sample made it possible to analyze a large number of biological samples per day. The method is the first reported application of HILIC in the analysis of antibiotics in breast milk and human plasma and it can be used to support a wide range of clinical studies. Copyright © 2016 John Wiley & Sons, Ltd.

Comparison of ChromID C. difficile agar and cycloserine-cefoxitin-fructose agar for the recovery of Clostridium difficile.

AIM: The rapidly changing epidemiology of Clostridium difficile infection highlights the need for improved and continuing surveillance involving stool culturing to enable molecular tracking. Culture of C. difficile can be difficult and time consuming. In this report ChromID C. difficile agar (CDIF) was compared to cycloserine-cefoxitin-fructose-egg-yolk agar which contained 0.1% sodium taurocholate (TCCFA) as a germinant. RESULTS: All ribotypes of C. difficile tested (n=90) grew well on CDIF within 24 h and most gave characteristic small irregular black colonies with a raised umbonate profile. Counts from standard suspensions of C. difficile at 24 h (p<0.005) and 48 h (p=0.01) were significantly higher on CDIF than on TCCFA. Similar results were achieved after alcohol shock. When temperature shock was used to differentiate vegetative cells and spores, the total number of culturable and vegetative cells on CDIF was significantly higher than on TCCFA (culturable cells, p=0.003 at 24 h and p=0.002 at 48 h; vegetative cells, p=0.0003 at 24 h and p=0.0002 at 48 h). CONCLUSIONS: These data suggest that CDIF is a better medium for the recovery of vegetative C. difficile than TCCFA and equal to TCCFA for spore recovery.

A comparative study of capillary zone electrophoresis and pH-potentiometry for determination of dissociation constants.

Acidity constants of six cephalosporin antibiotics, cefalexin, cefaclor, cefadroxil, cefotaxim, cefoperazon and cefoxitin are determined using capillary zone electrophoresis (CZE) and pH-potentiometric titrations. Since CZE is a separation method, it is not necessary for the samples to be of high purity and known concentration because only mobilities are measured. The effect on determination of dissociation constants of different matrices (serum, 0.9% NaCl, fermentation matrix) was examined. The advantages of CZE can be utilized in those fields where potentiometry has limitations (sample quantity, solubility, purity, simultaneous determinations), although pK(a) values that are close to each other can be determined by potentiometry with more accuracy.

Microbiological assay for cefoxitin sodium in dosage form.

A microbiological assay applying the cylinder-plate method is described for determination of the activity of cefoxitin sodium in injectables. Using a strain of Staphylococcus epidermidis ATCC 12226 as the test organism, cefoxitin sodium was measured in concentrations ranging from 50.0 to 200.0 microg/mL. The validation showed that the method was linear (r = 0.9998), precise (RSD = 0.81%), and accurate. It was concluded that the microbiological assay is satisfactory for quantitation of cefoxitin sodium in injectables.

Determination of cefoxitin in serum and tissue.

A simple, rapid and sensitive method for the clean-up and analysis of cefoxitin in serum and tissue is described. Serum (0.5 ml) and tissue (100 mg) samples after homogenization underwent high speed centrifugation. Chromatography was performed on a muBondapak C18 cartridge using a mobile phase of 0.005 M potassium dihydrogen phosphate-acetonitrile-glacial acetic acid (77.5:22:0.5, v/v/v) with a flow-rate of 2.0 ml/min. Ultraviolet detection occurred at 235 nm. The procedure produced a linear curve for the concentration range 100-5000 ng/ml. The assay produced accurate, repeatable and rapid results for both tissue and serum samples without the need for chemical extraction.

Analytical investigation of beta-lactam antibiotics in pharmaceutical preparations. IX. Colorimetric determination of six cephalosporins of second and third generation in the range of micromolar concentrations.

A sensitive, accurate, precise and the same time simple and rapid method for the colorimetric determination of some cephalosporins of the second and third generations, such as: cefoxitin sodium (CFXT), cefaclor (CFCL), cefamandole nafate (CFMD), ceforanide l-lysine (CFRN), cefotaxime sodium (CFTX), and cefurozime sodium (CFRX) was described. The new method proposed is based: a) On the reduction of Fe(III) to Fe(II) by the drug analysed and b) On complexation of Fe(II) formed with o-Phenanthroline (O-Phen) consistently the formation of the well known highly stable orange-red coloured chelate complex [Fe(II)-(o-Phen)3]2+ which exhibits an absorption maximum at lambda = 510 nm (pH 4.50 +/- 0.2). Beer's law is obeyed for: 1.0 - 37.5 microgram mL-1 for CFX, 1.0 - 25.0 microgram mL-1 for CFMD, CFRN, and CFTX and 2.0 - 37.5 microgram mL-1 for CFTX and CFCL, while the apparent molar absorptivity ( epsilon in L mol-1cm-1) and the Sandell's sensitivity in (ngcm-2) both referred to the drug analyzed, are 1.29 x 10(4); 34.7 (CFXT), 7.61 x 10(3); 50.7 (CFCL), 3.33 x 10(4); 15.4 (CFMD), 2.60 x 10(4); 17.6 (CFRN) respectively. The regression line equation for each one of the above studied cephalosporins were calculated with a correlation coefficient 0.9997 < r < 1.0000; the accuracy and the precision of the method was considered as very satisfactory, while the results of a statistical analysis by means of the Student's t-test and the variance ratio F-test prove that no significant difference was observed between the results of the proposed method and those of official one.

Analysis of binary mixtures of cephalothin and cefoxitin by using first-derivative spectrophotometry.

A method for the analysis of two-component mixtures of cephalothin and cefoxitin using zero-crossing first-derivative spectrophotometry is described. This technique permits the quantification of these drugs with closely overlapping spectral bands without any separation step. Linear calibration graphs of first-derivative values at 235.00 and 236.75 nm for cephalothin and cefoxitin, respectively, with negligible intercepts were obtained versus concentration in the range 4.0-32.0 micrograms ml-1 for both antibiotics. This paper presents a systematic examination of the experimental data by applying an exhaustive statistical analysis to demonstrate the validity of the method. The results of the determination of these antibiotics in mixtures of injectable dosage forms are also presented, together with their determinations in physiological serum and glucosed physiological serum.

Relationship between drug absorption enhancing activity and membrane perturbing effects of acylcarnitines.

Acylcarnitines with chain lengths of 2 to 18 carbon atoms were tested for their effects on rat intestinal brush border membrane order (S) by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). These results were compared to the previously reported effectiveness of the acylcarnitines as absorption enhancers of the poorly absorbed antibiotic cefoxitin. Acylcarnitines with fatty acids less than 12 carbon units in length were ineffective in increasing drug absorption and perturbing brush border membrane order. Long-chain acylcarnitines (12-18 carbons) significantly increased the bioavailability of cefoxitin and decreased the lipid order of brush border membranes. The results suggest that, in order to promote drug absorption, the acylcarnitines must surpass a critical chain length (10 carbon units) to partition effectively into the membrane and, in addition, must perturb the lipid order beyond a threshold value (15-20%). Membrane perturbing capacity may serve as an indicator of the absorption enhancing potential of other aliphatic-type compounds.

Stability of cefazolin sodium, cefoxitin sodium, ceftazidime, and penicillin G sodium in portable pump reservoirs.

The stability of cefazolin sodium, cefoxitin sodium, ceftazidime, and penicillin G sodium in prefilled drug reservoirs that were stored at -20 degrees C for 30 days, thawed at 5 degrees C for four days, and pumped at 37 degrees C for one day was studied. Each antimicrobial agent was diluted with sterile water for injection to a concentration representative of the most common dosage when administered via a portable infusion pump. Ten milliliters of each drug solution was placed in individual glass vials to serve as controls, and volumes appropriate to deliver the designated dosages were loaded into the drug reservoirs. Triplicate reservoirs were prepared for each drug. One-milliliter samples from all containers were taken on days 0, 30, 31, 32, 33, 34, 34.5, and 35. All solutions were observed for color change and precipitation. Drug concentrations were determined using high-performance liquid chromatography. Leaching of the plasticizer diethylhexyl phthalate (DEHP) was analyzed by packed-column gas chromatography on days 0 and 35. No color change or precipitation was observed. No DEHP concentrations above 1 ppm were detected. More than 90% of the initial concentrations of each drug remained, except penicillin G sodium, which had a mean concentration of 83.9 +/- 0.5% at the end of the study. Cefazolin sodium, cefoxitin sodium, and ceftazidime in admixtures with sterile water for injection are stable under the conditions of this study. Penicillin G sodium should not be administered for more than 12 hours after such a cycle of freezing and thawing.

Measurement of cefoxitin levels in tissue using high-pressure liquid chromatography.

A high-pressure liquid chromatography (HPLC) method that successfully measured cefoxitin (a modification of the antibiotic cephamycin C) levels in subcutaneous tissue, muscle, and aortic and peripheral arterial walls has been developed. Samples were obtained from 11 patients submitted to prosthetic aortic replacement. All patients received an intravenous bolus dose of cefoxitin 2 g just before induction of anaesthesia. Blood and tissue samples were taken at various intervals intra-operatively. The tissue samples were mechanically homogenised. Both plasma and the tissue homogenates were deproteinated with trichloracetic acid. The cefoxitin was separated by HPLC and measured by ultraviolet absorbance. The results show that the tissue concentration of the drug fell over a 4-hour period and that all levels exceeded the MIC90 (minimum inhibitory concentration that inhibits growth of bacteria at the 90% level) for most aerobic and anaerobic pathogens for at least 3 hours.

Cefoxitin disposition in colorectal surgery. Implications for the effective use of prophylactic antibiotics.

Failure of antibiotic prophylaxis to prevent infectious complications following colorectal operations is reported to occur in 5 to 10% of all cases. Factors such as the length of surgery and inadequate antibacterial coverage or duration predispose patients to higher rates of infectious complications. The pharmacokinetics of cefoxitin in eight patients undergoing colectomy, mucosal proctectomy, and endorectal ileal pouch-anal anastomosis (IPAA) for chronic ulcerative colitis or familial polyposis coli were examined. Peak plasma concentrations were 40% higher than values reported in healthy volunteers. During the first dose, the plasma half-life of cefoxitin was similar to plasma half-lives reported in healthy volunteers; however, total body clearance was reduced five- to eightfold. These data suggest that the volume of distribution of cefoxitin was also markedly reduced. Fluid replacement during the operation produced lower cefoxitin peak plasma concentrations after the second dose. Cefoxitin clearance increased during the second dose after fluid replacement, but remained below that reported in healthy volunteers. These data suggest that fluid depletion resulting in decreased kidney perfusion contributed to a reduction in drug clearance. The amount of cefoxitin recovered in the urine during the 12-hour study period averaged 53%. Tissue concentrations of cefoxitin in proximal colon, distal colon, rectal mucosa, and rectal muscle tissue ranged from 0.2 to 9.3 mcg/g of tissue. The preoperative fluid status of the patient, the time of drug administration, and the amount of extra-renal drug elimination appear to be important factors, affecting the disposition of parenterally administered prophylactic antibiotics in patients undergoing colorectal operations.

Stability of intravenous admixtures of aztreonam and cefoxitin, gentamicin, metronidazole, or tobramycin.

The stability of aztreonam and cefoxitin, gentamicin, metronidazole, or tobramycin in intravenous admixtures containing aztreonam and one of the other drugs was studied. Admixtures of aztreonam and gentamicin, aztreonam and tobramycin, and aztreonam and cefoxitin were each prepared in four different concentrations in both 0.9% sodium chloride injection and 5% dextrose injection. Admixtures of aztreonam and metronidazole were prepared in two different concentrations using a commercially available solution of metronidazole 5 mg/mL in a phosphate-citrate buffer. One of each of these admixtures was stored at 25 degrees C for 48 hours and at 4 degrees C for seven days. At various storage times, 1-mL samples of the admixtures were tested for pH and assayed using high-performance liquid chromatography or fluorescence polarization immunoassay. The pH of all admixtures except admixtures of aztreonam and cefoxitin decreased only slightly during storage. Concentrations of aztreonam and tobramycin under both storage conditions decreased by less than 10%. Concentrations of cefoxitin and aztreonam decreased by more than 10% at 25 degrees C, and concentrations of gentamicin decreased by more than 10% under both storage conditions. Visual inspection of admixtures of aztreonam and metronidazole revealed an incompatibility between the two drugs, as evidenced by the appearance of a cherry-red color. Admixtures of aztreonam 10 and 20 mg/mL and tobramycin 0.2 and 0.8 mg/mL in 5% dextrose injection or 0.9% sodium chloride injection are stable for 48 hours at 25 degrees C or seven days at 4 degrees C. Admixtures of aztreonam 10 and 20 mg/mL and gentamicin 0.2 and 0.8 mg/mL in 5% dextrose injection or 0.9% sodium chloride injection are stable for eight hours at 25 degrees C and 24 hours at 4 degrees C. Admixtures of aztreonam 10 and 20 mg/mL and cefoxitin 10 and 20 mg/mL in 5% dextrose injection or 0.9% sodium chloride injection are stable for 12 hours at 25 degrees C and seven days at 4 degrees C. Aztreonam and metronidazole should be administered separately.

Antibiotic prophylaxis in vascular surgery: pharmacokinetic study of four commonly used cephalosporins.

Plasma levels of antibiotics often do not correlate well with their tissue levels. To determine optimal antibiotic coverage for prophylactic effect in vascular surgery, we studied the tissue pharmacokinetics of four cephalosporins in dogs: cefazolin, cefoxitin, cefamandole, and moxalactam for 3 hours after a single (25 mg/kg) intravenous injection. The minimal inhibitory concentration (MIC) of these antibiotics for the three most common pathogens involved in graft infections (Staphylococcus aureus, S. albus, and Escherichia coli) and their tissue concentration (TC) in the plasma, muscle, subcutaneous tissue, and aortic wall were assayed. The data are presented as TC/MIC ratio. Cefoxitin and moxalactam failed to achieve an effective therapeutic TC/MIC ratio (greater than 10) for S. aureus and S. albus in all the tissues studied whereas cefoxitin and cefamandole were above therapeutic levels. All antibiotics achieved an effective therapeutic ratio against E. coli, but cefamandole performed better (p less than 0.05) than cefoxitin; the latter reached effective levels at 3 hours. Cefamandole attained the most effective bioactive aortic tissue levels when the three most common pathogens were considered together and should therefore be considered as an antibiotic agent of choice for prophylaxis in vascular surgery.

Stability of clindamycin phosphate and ceftizoxime sodium, cefoxitin sodium, cefamandole nafate, or cefazolin sodium in two intravenous solutions.

The stability and compatibility of clindamycin phosphate and ceftizoxime sodium, cefoxitin sodium, cefamandole nafate, or cefazolin sodium in two intravenous solutions were studied. Each antibiotic alone as well as each of the four two-drug combinations were examined when mixed in duplicate 100-mL glass bottles of 5% dextrose and 0.9% sodium chloride injections. Antibiotic concentration, pH, and visual appearance were recorded at the time of preparation and at 1, 4, 8, 12, 24, and 48 hours. Antibiotic concentrations were assessed with drug-specific high-performance liquid chromatographic assays. Decreases in concentration of 10% or more from the original concentration were considered to indicate instability. All the single antibiotic solutions were stable for 48 hours. Clindamycin was stable in all combinations except with ceftizoxime in 0.9% sodium chloride injection, which measured 89.3% of its original clindamycin concentration at 48 hours. All the cephalosporins mixed with clindamycin were stable for 48 hours. Clindamycin is stable for at least 48 hours when mixed with cefoxitin sodium, cefamandole nafate, or cefazolin sodium in either 5% dextrose or 0.9% sodium chloride injections and for at least 24 hours when mixed with ceftizoxime sodium in 0.9% sodium chloride injection.

Cefoxitin concentration in wound fluid.

The concentration of cefoxitin was determined in fluid obtained from human surgical wounds during the first postoperative day. Intravenous administration of cefoxitin sodium at a dosage of 1 or 2 g every six hours rapidly produced wound-fluid concentrations greater than the minimal inhibitory concentration for most susceptible organisms. After three hours, wound-fluid concentrations surpassed the serum concentrations of cefoxitin. The higher dosage resulted in higher wound-fluid levels.

Rapid chromatographic determination of cefotaxime and its metabolite in biological fluids.

A reversed-phase high-performance liquid chromatographic assay for the simultaneous determination of cefotaxime and its metabolite desacetylcefotaxime in plasma and urine was developed. Plasma was deproteinized with small amounts of acetonitrile. After separation of the proteins the supernatant was extracted with a mixture of chloroform and 1-butanol. A phase separation was obtained leaving the cephalosporin and its metabolite in the aqueous part and extracting most of the interfering endogenous material. The aqueous phase was injected directly into the chromatograph. As part of the plasma water was dissolved in the acetonitrile--1-butanol--chloroform layer, the concentration of the cephalosporin in the aqueous phase was significantly higher than in the original plasma sample. Therefore, the usual diluting effect of the deproteinization could be avoided. In a similar way the assay was applicable to measure cefotaxime and its metabolite in urine. Calibration curves were set up and were linear up to 25 micrograms/ml for desacetylcefotaxime and 250 micrograms/ml for cefotaxime. The assay was applied to study the pharmacokinetics of cefotaxime and its metabolite in a healthy volunteer. In a similar way this deproteinization and extraction method was also applied to assay for ceftazidime, cephalexin, cephazolin and cefoxitin.

Cefoxitin and cefamandole levels in the human bile.

The bile levels of cefoxitin and cefamandole were studied in 75 patients. The two antibiotics were shown to be present at high concentration and to undergo a rapid metabolization. It is concluded that they represent the antibiotics of choice in the prophylactic and therapeutic treatment of biliary tract infections.