Determination of Cefalothin and Cefazolin in Human Plasma, Urine and Peritoneal Dialysate by UHPLC-MS/MS: application to a pilot pharmacokinetic study in humans.

An ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the analysis of cefazolin and cefalothin in human plasma (total and unbound), urine and peritoneal dialysate has been developed and validated. Total plasma concentrations are measured following protein precipitation and are suitable for the concentration range of 1-500 µg/mL. Unbound concentrations are measured from ultra-filtered plasma acquired using Centrifree(®) devices and are suitable for the concentration range of 0.1-500 µg/mL for cefazolin and 1-500 µg/mL for cefalothin. The urine method is suitable for a concentration range of 0.1-20 mg/mL for cefazolin and 0.2-20 mg/mL for cefalothin. Peritoneal dialysate concentrations are measured using direct injection, and are suitable for the concentration range of 0.2-100 µg/mL for both cefazolin and cefalothin. The cefazolin and cefalothin plasma (total and unbound), urine and peritoneal dialysate results are reported for recovery, inter-assay precision and accuracy, and the lower limit of quantification, linearity, stability and matrix effects, with all results meeting acceptance criteria. The method was used successfully in a pilot pharmacokinetic study with patients with peritoneal dialysis-associated peritonitis, receiving either intraperitoneal cefazolin or cefalothin. Copyright © 2015 John Wiley & Sons, Ltd.

Development of a novel chemiluminescence method for the determination of cefazolin sodium in injectable powder and human urine based on a luminol-Cu(III) complex reaction in alkaline medium.

A novel chemiluminescence (CL) method was developed for the determination of cefazolin sodium based on the CL reaction between the [Cu(HIO6)2](5-) Cu(III) complex and luminol in alkaline solution. Results showed that CL emission of Cu(III) complex-luminol in alkaline medium was significantly different from that in acidic medium. A possible mechanism of the enhanced effect of cefazolin on CL emission of the [Cu(HIO6)2](5-)-luminol system was proposed. The effect of the reaction conditions on CL emissions was examined. Under optimized conditions, a good linear relationship was obtained between CL intensity and concentrations of cefazolin sodium in the range of 2.0 x 10(-8) to 2.0 x 10(-6) g/mL with a correlation coefficient of R(2) = 0.9978. The limit of detection was 4.58 x 10(-9) g/mL. The proposed method was applied for the determination of cefazolin sodium in real samples with recoveries of 82.0-109% with an RSD of 0.7-2.1%. The proposed method was successfully used for the determination of cefazolin sodium in injectable powder preparations and human urine with satisfactory results.

Reduced renal clearance of a zwitterionic substrate cephalexin in MATE1-deficient mice.

Multidrug and toxin extrusion 1 (MATE1/solute carrier 47A1) mediates the transport of not only organic cations but also zwitterions such as cephalexin. However, the contribution of MATE1 to tubular secretion of cephalexin in vivo has not been elucidated. In the present study, we carried out transport experiments of cephalexin via MATE1 and performed pharmacokinetic analyses of cephalexin in Mate1 knockout [Mate1(-/-)] mice. Cephalexin uptake by human MATE1-expressing human embryonic kidney 293 cells exhibited saturable kinetics (K(m) = 5.9 +/- 0.5 mM) and a bell-shaped pH profile with a maximum at pH 7.0. We confirmed that mouse MATE1 also transported cephalexin. After a single intravenous administration of cephalexin (5 mg/kg), Mate1(-/-) mice showed higher plasma concentrations of cephalexin than wild-type [Mate1(+/+)] mice. The urinary excretion of cephalexin for 60 min was significantly reduced, and the renal concentration was markedly increased in Mate1(-/-) mice compared with Mate1(+/+) mice. The renal clearance of cephalexin in Mate1(-/-) mice was approximately 60% of that in Mate1(+/+) mice and seemed to be near the creatinine clearance. In contrast, there were no significant differences between both mice in the pharmacokinetics of anionic cefazolin, which is not a substrate for MATE1. In this study, we demonstrated that MATE1 is responsible for renal tubular secretion of a zwitterionic substrate cephalexin in vivo.

[Analysis of multiple cephalosporins in blood and urine by HPLC].

OBJECTIVE: To establish a new high performance liquid chromatography (HPLC) method for determining the concentration of cefazolin, cefradine, cefoperazone and cefotaxime in blood and urine, as well as to investigate its applicability. METHODS: Protein in blood and urine was precipitated directly by acetonitrile with acetanilide was used as the internal standard using Agilent Zorbax SB-Aq column (250 mm x 4.6 mm, 5 microm). The mixed solvents of water (triethylamine 0.12%, acetic acid 0.12%) and acetonitrile were used as the mobile phase to separate cephalosporins using gradient elution method at 1 mL/min (flow rate) and 254 nm (detection wavelength). RESULTS: The working curve of four cephalosporins showed a good correlation (r = 0.9993), with the detection limit up to 0.01 microg/mL. The recovery rate was more than 81.2%. CONCLUSION: This method is fast, easy and accurate. It is suitable for biological analysis of the 4 cephalosporins of the blood and urine in practical cases.

Analysis of multiple cephalosporins in blood and urine by HPLC / 法医学杂志

OBJECTIVE@#To establish a new high performance liquid chromatography (HPLC) method for determining the concentration of cefazolin, cefradine, cefoperazone and cefotaxime in blood and urine, as well as to investigate its applicability.@*METHODS@#Protein in blood and urine was precipitated directly by acetonitrile with acetanilide was used as the internal standard using Agilent Zorbax SB-Aq column (250 mm x 4.6 mm, 5 microm). The mixed solvents of water (triethylamine 0.12%, acetic acid 0.12%) and acetonitrile were used as the mobile phase to separate cephalosporins using gradient elution method at 1 mL/min (flow rate) and 254 nm (detection wavelength).@*RESULTS@#The working curve of four cephalosporins showed a good correlation (r = 0.9993), with the detection limit up to 0.01 microg/mL. The recovery rate was more than 81.2%.@*CONCLUSION@#This method is fast, easy and accurate. It is suitable for biological analysis of the 4 cephalosporins of the blood and urine in practical cases.

Alterations in pharmacokinetics and protein binding behavior of cefazolin in endotoxemic rats.

The possible alterations in the pharmacokinetics and protein binding behavior of the beta-lactam antibiotic cefazolin (CEZ) were investigated in endotoxemic rats induced by Klebsiella pneumoniae O3 lipopolysaccharide (LPS). LPS (250 micrograms/kg of body weight) was infused for 20 to 30 min 2 h before an intravenous administration of CEZ (20 mg/kg). Significant decreases in systemic clearance and renal clearance of CEZ were observed in LPS-treated rats without any changes in fraction of urinary excretion in unchanged CEZ (> 0.8). The volume of distribution at steady state showed a tendency to increase. The protein binding parameters of CEZ, the binding capacity, and number of binding sites on the albumin molecule were decreased by LPS, whereas the dissociation constant did not change. Significant decreases in systemic and renal clearances for unbound CEZ were observed in LPS-treated rats. The glomerular filtration rate estimated as inulin clearance was also decreased by LPS. The ratio of renal clearance of unbound CEZ to glomerular filtration rate (clearance ratio) dropped to 70% of that in control rats, and the net tubular secretion of CEZ was also dramatically reduced. The present study suggests that LPS has an effect on the pharmacokinetics of CEZ by changes which occur in renal handling and protein binding.

Determination of cefcanel in plasma and urine by high-performance liquid chromatography using coupled columns, after administration of the new cephalosporin prodrug cefcanel daloxate hydrochloride.

A rapid and sensitive high-performance liquid chromatographic method has been developed for the determination in plasma and urine of the new cephalosporin cefcanel. The method involves a simple deproteinizing step followed by separation on a coupled-column chromatographic system with ultraviolet detection. Limits of quantification were 0.2 microM for plasma samples and 2 microM for urine samples. The method has been used for the determination of cefcanel in various clinical studies.

Effect of diabetes on disposition and renal handling of cefazolin in rats.

The effects of early-stage diabetes on disposition and renal handling of cefazolin (CEZ) were investigated in streptozotocin-induced diabetic rats. Significant increase in renal clearance was found in the diabetic rats (88%) and a strong correlation was obtained between renal and systemic clearance; however, there was no change in the volume of distribution. The results suggest that systemic clearance was increased as the result of enhancement of urinary excretion rate. Unbound fraction of CEZ in plasma was also increased by 60% in the diabetic rats and the increase may be due to the increase in glycosylated protein and plasma-free fatty acids. The filtration clearance for free drug in diabetic rats, which was estimated as glomerular filtration rate, was increased by about 1.9-fold compared to the normal rats, but the secretion clearance did not change in the two groups. Since kidney hypertrophy was observed in the diabetic rats, filtration and secretion clearance for free drug were normalized by means of kidney weight. After normalization for kidney weight, the two parameters were significantly reduced, indicating that the true kidney functions were impaired under the diabetic state. The parameters for CEZ secretion, maximum velocity and Michaelis-Menten constant, were also reduced in diabetic rats, suggesting that proximal tubule cell functions for secretion were altered in the diabetic rats. These results suggest that systemic and renal clearance was apparently increased in early-stage diabetes, whereas true kidney functions were impaired.

Effect of imipenem-cilastatin and ciprofloxacin on tests for glycosuria.

The effect of the new antibiotics imipenem-cilastatin and ciprofloxacin on the accuracy of tests for glycosuria was studied. Samples of urine from two healthy volunteers were used to prepare solutions containing various concentrations of glucose and drug. Glucose concentrations were tested in triplicate by the Clinitest, Tes-Tape, and Diastix methods. As controls, samples of urine containing the antibiotics alone or various concentrations of glucose and cefazolin were tested by each of the three methods. Low concentrations of imipenem-cilastatin caused falsely low glucose results in urine samples containing 0.5% and 1% glucose analyzed by the Clinitest method. Ciprofloxacin did not interfere with determination of urine glucose concentration by the Clinitest method at any of the drug concentrations tested. Neither of the antibiotics interfered substantially with determination of urine glucose concentration by the Diastix or Tes-Tape methods regardless of the concentration of glucose or drug. At the concentrations tested, ciprofloxacin did not interfere with determination of urine glucose concentration by the Clinitest, Diastin, or Tes-Tape methods. Although imipenem-cilastatin may produce falsely low glucose measurements with the Clinitest method, this interaction is not of great clinical importance.

Nonaqueous cephalosporin suspension for parenteral administration: cefazolin sodium.

The flocculation-deflocculation behavior of cefazolin sodium (I) in nonaqueous media and the effect of surfactants as measured by zeta potential, sedimentation, and porosity were studied. A significant difference in zeta potential was observed when the particles were suspended in different nonaqueous media. The addition of surfactant produced a deflocculated state. The surfactant deflocculated the particles by a process of supersaturation and crystallization involving a surfactant-cefazolin complex. The shielding effect of the surfactant on the surface of the particles also apparently affected their electrophoretic properties. Kinetic studies on the stability of the drug as a function of temperature were conducted; it appears that the chemical stability in ethyl oleate at room temperature is adequate for a reasonable shelf life. The efficiency of absorption of the drug from the ethyl oleate suspension was evaluated after intramuscular administration in dogs. The area under the plasma concentration versus time curve and urinary recovery indicated that cefazolin was 100% bioavailable from this nonaqueous preparation.

Effects of furosemide, piretanide, and water loading on urinary excretion of cefazolin in humans.

Antibiotics and diuretics are often prescribed concomitantly for humans. We compared the effects of two potent loop diuretics, furosemide and piretanide, with those of water loading on the urinary excretion of cefazolin. During a continuous infusion of inulin and cefazolin (10 mg/kg per h), six healthy male volunteers received a single intravenous injection of furosemide (0.3 mg/kg) or piretanide (0.1 mg/kg) or again an oral water load of 15 ml/kg over a 20-min period. In vitro, furosemide at all concentrations tested significantly reduced by about 10% the percentage of cefazolin bound to serum proteins. Piretanide exhibited such an effect only at a concentration of 2 micrograms/ml. Furosemide, piretanide, and water loading significantly and similarly increased the ratio of excreted to infused cefazolin up to 2 h after the injection of diuretic or after oral water intake. In each of the three parts of the experiment, the increase of the urinary flow rate was similar when compared with the control values. Furosemide significantly increased the cefazolin filtered load during the same time. Piretanide significantly enhanced the absolute rate of net cefazolin tubular secretion. Water loading increased the urinary excretion of cefazolin, probably through a reduction in tubular reabsorption. These results suggest that (i) furosemide and piretanide as well as water loading are capable of enhancing renal excretion of cefazolin by different complex mechanisms; (ii) cefazolin undergoes a bidirectional tubular transport; (iii) piretanide might act on the proximal tubule in addition to its main site of action on Henle's loop; and (iv) the effects of both diuretics and of water loading are unlikely to affect in vivo antibiotic activity in humans.

Antibiotic concentrations in the urine from kidneys of unequal function.

Little attention has been given to whether an effective concentration of an antibiotic is obtained in the urine of a patient without azotemia who has one poorly functioning kidney and a contralateral normal kidney. This study was undertaken to measure the concentration of various antibiotics in the urine from both kidneys of 20 patients with unilateral renal disease and a radiologically and functionally normal contralateral kidney. Prior to ureteric catheterization each patient received a single parenteral dose of an antibiotic. The peak urinary drug concentration from the damaged kidney per unit of its creatinine clearance exceeded that for the normal kidney for 11/13 patients treated with an aminoglycoside and 6/7 given a cephalosporin. The most severely damaged kidney had a creatinine clearance of 0.6 ml/min. This patient received sisomicin and the peak urinary concentration was only 1.8 micrograms/ml. If a damaged kidney has a creatinine clearance less than 10-15 ml/min it would seem more appropriate to use a cephalosporin rather than an aminoglycoside antibiotic.

Comparison of the renal excretory mechanisms of cefmenoxime and other cephalosporins: effect of para-aminohippurate on renal clearance and intrarenal distribution of cephalosporins in rabbits.

The renal excretory mechanism of cefmenoxime in rabbits was compared with that of 6 other cephalosporins (cefotaxime, deacetylcefotaxime, cefotiam, cefazolin, cephaloridine, and cefsulodin). The clearance ratios (Cf-Drug/CInulin=CRf) of cefmenoxime (337) and cefazolin (73) were considerably higher than those of the 5 other cephalosporins (0.9-20). When p-amino-hippurate (PAH) was administered concurrently with each of the cephalosporins, the CRf values of the cephalosporins except for cefsulodin were significantly decreased. These findings indicate that cefmenoxime and the 5 other cephalosporins except cefsulodin are actively incorporated in the proximal tubular cells and secreted into the tubular lumen. In the case of cefotiam and cefsulodin, glomerular filtration tended to exceed urinary excretion with highest dose of PAH (40 mg/kg/minute), suggesting the possibility of tubular reabsorption of these drugs. On the other hand, glomerular filtration of cefmenoxime and the 4 other cephalosporins did not exceed urinary excretion. The drug concentration ratio of the cortex to medulla indicated that the tubular cell level of cefmenoxime was lower than, higher than, and similar to those of cephaloridine, cefotaxime, and the remaining cephalosporins, respectively. These results demonstrate that the renal excretory mechanisms of cefmenoxime is similar to that of cefazolin but not to that of the remaining cephalosporins.

Effects of phenylbutazone on extravascular diffusion, protein binding and urinary excretion of cefazolin in rabbits.

The effects of an anti-inflammatory drug, phenylbutazone, on the disposition of a commonly used cephalosporin, cefazolin, were studied in rabbits. The following investigations were made: mathematical analysis of blood levels obtained after i.v bolous injection of cefazolin, alone or combined with phenylbutazone (10 mg/kg), injection 4 hr before; protein binding by ultracentrifugation in vitro; and renal excretion and distribution in extravascular fluid obtained from s.c. tissue cages in vivo. Single i.m. injections of cefazolin (30 mg/kg) were administered either alone or in combination with phenylbutazone (10 or 100 mg/kg i.m.) or 2 or 4 hr before. The mathematical analysis disclosed a competition of phenylbutazone on protein binding of cefazolin. In vitro, phenylbutazone reduced the extent of protein binding of the antibiotic (74-80 to 47-59%). Cefazolin appeared at higher concentrations in extravascular fluid in the presence of phenylbutazone than when administered alone. Phenylbutazone appeared to be responsible for a dose-dependent effect on renal excretion of cefazolin i.e., a reduction of secretion at low doses (10 mg/kg) and a possible reduction of tubular reabsorption at high doses (100 mg/kg). A bidirectional transport of cefazolin in rabbit tubules was thus shown. The interaction of phenylbutazone on the on the disposition of cefazolin appeared also dependent on the time of injection of the former and on the mode of administration of the antibiotic.

Effect of cardiopulmonary bypass on cefazolin disposition.

Cefazolin kinetics was studied in 8 patients the day before (PREOP), during (SURG), and the day after (POSTOP) cardiopulmonary bypass (CPB) surgery. PREOP (48.6 ml/min) and POSTOP (46.6 ml/min) total body clearances were of the same order and both were greater than the SURG (27.4 ml/min) total body clearance. Since cefazolin is almost entirely eliminated by the kidney, the lower SURG clearance is a result of reduced renal elimination, as confirmed by measuring cefazolin SURG (28.7 ml/min) and POSTOP (52.9 ml/min) renal clearance. The reduction in cefazolin renal elimination was the same throughout the surgical procedure, including the period of extracorporeal circulation. Cefazolin distribution was altered by the operative procedure as evidence by a higher SURG steady-state volume of distribution. This increase in apparent cefazolin distribution volume brought about by surgery was not seen with cephalothin, which was investigated by us in a similar group of patients. The different effect of CPB surgery on cefazolin and cephalothin distribution may be due to differences in plasma protein binding.

[Pharmacokinetics and tolerance of Cefazedone compared with Cefazoline (author's transl)]. / Pharmakokinetik und Verträglichkeit von Cefazedon im Vergleich zu Cefazolin.

In a randomized cross over study Cefazoline and Cefazedone were administered i.v. as injection of 2 minutes duration and as infusion of 30 minutes duration. The concentrations in blood plasma and the excretion with the urine were similar for both substances and differed only with regard to their levels during the first 60 minutes, depending on the mode of administration.

Cefazolin in children with renal insufficiency.

Cefazolin (7 mg/kg) were administered to 11 children with renal insufficiency and to ten children on hemodialysis. The serum half-life of the drug was progressively prolonged as glomerular filtration rate fell. The serum half-life of cefazolin was variably prolonged in those children on hemodialysis, but their serum levels of cefaxolin had dropped by 35 to 65% during dialysis. Most had no measurable level prior to the next dialysis. Dosage recommendations are made for both groups of patients.

Comparative pharmacokinetics of ceforanide (BL-S786R) and cefazolin in laboratory animals and humans.

Ceforanide (BL-S786R) is a new, broad-spectrum, parenteral cephalosporin. Pharmacokinetic properties were determined in rats (100 mg/kg), rabbits (30 mg/kg), dogs (25 mg/kg), and humans (2 g or 30 mg/kg) and compared with equivalent single doses of cefazolin. Plasma half-lives for ceforanide and cefazolin were 1.1 and 0.5 h in the rat, 5 and 0.3 h in the rabbit, 1 and 0.8 h in the dog, and 2.6 and 2 h in humans, respectively. The slower elimination of ceforanide, as reflected by longer plasma half-life, larger area under the curve, and peak plasma concentrations, was due to slower body and renal clearances. The apparent volumes of distribution of ceforanide and cefazolin were comparable. Rats, dogs, and humans excreted 80 to 100% of the ceforanide dose in the 0- to 24-h urine; rabbits excreted only 50%. Tubular secretion constituted 50% of ceforanide renal excretion in rabbits, dogs, and humans and 90% in rats; the remainder was excreted by glomerular filtration. There was no apparent correlation between the extent of tubular secretion and degree of plasma protein binding in different species. There was no significant pharmacokinetic interaction between ceforanide and amikacin in the rat. The slower elimination kinetics of ceforanide are indicative of the potential for a longer dosing interval and more effective antibiotic therapy as compared with available cephalosporins.

[Experimental study of the biliary excretion of antibiotic (cefotiam) in the presence of biliary tract obstruction (author's transl)].

Excretion of a new semisynthetic analogue of cephalosporin, cefotiam (CTM), and cefazolin (CEZ) in bile was investigated in dogs with experimental obstruction of biliary tract. In the control group, peak concentrations of antibiotics in bile after 50 mg/kg intravenous administration were as follows: In CTM administrated groups, 5,302 micrograms/ml at one hour after administration; and in CEZ administrated group, 1,249 micrograms/ml after one hour. The total biliary excretory rates summed to 6 hours after administration of antibiotics were 7.8% in the CTM group, and 2.1% in the CEZ group. On the other hand, in the biliary tract obstruction group, the peak concentration in bile in CTM group was 65.7 micrograms/ml after 1 hours, and in CEZ group, 62.4 micrograms/ml after 2 hours. The total biliary excretory rate in the CTM group was 0.202%, while in the CEZ group 0.12%. The peak concentration and the excretory rate of CTM in bile was four times higher than that of CEZ in the control group. No significant difference was revealed in the peak concentration and the excretory rate in bile between CTM and CEZ in the biliary tract obstruction group.