A validated LC-MS/MS method for simultaneous quantitation of piperacillin, cefazolin, and cefoxitin in rat plasma and twelve tissues.

BACKGROUND: The investigation of drug disposition in tissues is critical to improving dosing strategy and maximizing treatment effectiveness, yet developing a multi-tissue bioanalytical method could be challenging due to the differences among various matrices. Herein, we developed an LC-MS/MS method tailored for the quantitation of piperacillin (PIP), cefazolin (CFZ), and cefoxitin (CFX) in rat plasma and 12 tissues, accompanied by validation data for each matrix according to the FDA and EMA guidelines. RESULTS: The method required only a small sample volume (5 µL plasma or 50-100 µL tissue homogenates) and a relatively simple protocol for simultaneous quantitation of PIP, CFZ, and CFX within different biological matrices. Mobile phase A was composed of 5 mM ammonium formate and 0.1 % formic acid in water, while mobile phase B contained 0.1 % formic acid in acetonitrile. The mobile phase was pumped through a Synergi Fusion-RP column equipped with a guard column with a gradient elution program at a 0.3 mL/min flow rate. The mass spectrometer was operated in positive ionization mode (ESI+) using multiple reaction monitoring. SIGNIFICANCE: The validated method has been successfully applied to quantify PIP, CFZ, and CFX from the plasma and tissue samples collected in a pilot rat study and will further be used in a large pharmacokinetic study. To our knowledge, this is also the first report presenting long-term, freeze-thaw, and autosampler stability data for PIP, CFZ, and CFX in rat plasma and multiple tissues.

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.

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.

Bioactive antibiotic levels in the human aorta.

A new bioassay was used to determine the level of active antibiotic within the aortic wall in 24 patients undergoing elective aortic surgery involving prosthetic grafts. The patients were divided into three groups and received either cefazolin, clindamycin, or cefoxitin intravenously at the induction of general anesthesia. Cefazolin and cefoxitin attained satisfactory tissue levels. Clindamycin did not reach therapeutic levels in the aortic wall. Blood levels did not correlate well with tissue levels.

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.

Cefoxitin levels in human aqueous humor.

We administered 2 g of cefoxitin to 25 patients before cataract surgery. An additional 23 patients received 500 mg of probenecid 30 minutes before the 2-g doses of cefoxitin. Average aqueous humor levels of 1.54, 3.16, 2.46, 1.22, and 0.82 microgram/ml were achieved at 30 minutes and one, two, four, and six hours, respectively, after the 2-g dose. The addition of probenecid prolonged significant aqueous humor levels. Therapeutic levels against common gram-positive pathogens were consistently achieved at two hours after injection without probenecid and from two to four hours after injection with probenecid. Therapeutic levels effective against Enterobacteriaceae were inconsistent.

Cefoxitin sodium: solution and solid-state chemical stability studies.

Studies were undertaken to provide the basic physicochemical information necessary for preparing a suitable parenteral formulation of cefoxitin sodium. Emphasis was placed on the physico-chemical properties of the compound in solution and in the solid state. Cefoxitin sodium is very soluble in water and exhibits apparent first-order decomposition in this medium at pH 3-9. Maximum stability in water is at pH 5-7. Under these pH conditions, cefoxitin sodium loses about 10% of its activity in 2 days at 25 degrees. Thermal decomposition rates for amorphous and crystalline cefoxitin sodium samples were determined. Amorphous cefoxitin sodium was considerably less stable than its corresponding crystalline form. Solid-state decomposition plots are biphasic, displaying initial rapid losses followed by a slower decay period. The extent of loss in the crystalline solid at the end of the more rapid initial phase can be correlated with the water content of the solid.

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.

[Clinical studies of cefoxitin for the treatment of chronic respiratory tract infections (author's transl)].

1. Serum and sputum concentrations of cefoxitin were measured at fixed intervals following a one hour drip infusion of 2 g to a total of three patients with chronic respiratory tract diseases. The peak levels in serum were found to be 142.1-273.6 microgram/ml at the end of the infusion and those in sputum were found to be 0.92-2.30 microgram/ml at 2 to 4 hours after initiation of the infusion. 2. Cefoxitin was administered to a total of five patients with chronic respiratory tract infections who had failed to respond to the previous treatments with conventional antibiotics. The results were that in one case the therapeutic effect was judged as excellent, in three cases as good, and in one case it was difficult to evaluate. In all cases, there was no significant abnormality indicative of side effects regarding clinical symptoms and laboratory tests of renal and hepatic functions. In a view of the findings stated above, it is considered that cefoxitin is a new antibiotic which can be used for the treatment of chronic respiratory tract infections.

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.

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.

Rapid HPLC analysis of cefoxitin in plasma and urine.

A rapid, specific and precise method is described for the analysis of cefoxitin in plasma and urine by reversed-phase, high performance liquid chromatography (HPLC). Cefoxitin and the internal standard, 3-isobutyl-l-methyl xanthine are eluted after 5.3 and 7.5 min, respectively. The assay sensitivity limit is 1 to 2 mg/l of cefoxitin sodium at 254 nm. Commonly prescribed antibiotics do not interfere. The assay is suitable for routine monitoring and pharmacokinetic studies of cefoxitin.

Determination of cefoperazone concentration in serum and muscle tissue with a versatile high-pressure liquid chromatographic method.

A rapid, specific, and reproducible high-pressure liquid chromatographic method was developed for the determination of cefoperazone concentration in serum and tissue. The assay uses a simple methanol extraction, with cefoxitin as the internal standard. The limits of detection are 1 to 150 micrograms/ml; the maximum coefficient of variation is 7.4%. Using the same chromatography column, muBondapak phenyl, and mobile-phase 0.005 M tetrabutylammonium buffer-acetonitrile (80:20), the method can be easily adapted for the analysis of cefoxitin and moxalactam.

New rapid method of analysis of cefoxitin in serum and bone, by high-performance liquid chromatography.

A method for the extraction and quantification of cefoxitin in blood and bone samples is described in this paper. The procedure, which prepares biological material for reversed-phase high-performance liquid chromatographic analysis is convenient, rapid and reproducible. It also allows for use of cephalothin as an internal standard in measuring serum cefoxitin levels. Conventional extraction procedures, involving use of organic solvents, generally yield drug recoveries of 60-80%. Use of Baker--10 SPE disposable extraction columns allowed us to consistently obtain greater than 98% recovery of both cefoxitin and cephalothin. Methods for quantification of the extracted drugs include comparison of peak ratios (for serum) or peak heights (for bone) to first-order equations obtained from regression analyses.

Cefoxitin sodium compatibility with intravenous infusions and additives.

The compatibility and stability of cefoxitin sodium in solution with a series of frequently used intravenous infusion fluids and injectable additives were studied. Cefoxitin sodium's stability in various solutions was measured by ultraviolet spectrophotometry, iodometry, thin-layer chromatography, high-pressure liquid chromatography, ion-exchange chromatography and microbiological assay. Cefoxitin sodium was shown to maintain 90% of its initial concentration in aqueous solution for 40 hours at room temperature (25 C) and about 30 days at 5 C. The stability of cefoxitin sodium in common i.v. infusion fluids was independent of the concentrations (1 mg/ml to 400 mg/ml) and containers used, and was retained after 30 weeks storage at -20 C. Similar stability patterns were demonstrated for cefoxitin sodium in protein hydrolysate solutions and multivitamin formulations. Cefoxitin sodium was chemically and visually compatible with amikacin sulfate, gentamicin sulfate, kanamycin sulfate and tobramycin sulfate when admixed with normal saline or 5% dextrose in water injections. Cefoxitin sodium (397 mg/ml) in 0.5% lidocaine hydrochloride was stable after 26 weeks of storage at -20 C. Sodium cefoxitin is compatible with a wide variety of commonly used infusion solutions. Its stability is independent of concentration or pH within the ranges studied, and of types of common containers.

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.

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.