Simultaneous Determination of Eight ß-Lactam Antibiotics, Amoxicillin, Cefazolin, Cefepime, Cefotaxime, Ceftazidime, Cloxacillin, Oxacillin, and Piperacillin, in Human Plasma by Using Ultra-High-Performance Liquid Chromatography with Ultraviolet Detection.

A simple and rapid ultra-high-performance liquid chromatography (UHPLC) method using UV detection was developed for the simultaneous determination of eight ß-lactam antibiotics in human plasma, including four penicillins, amoxicillin (AMX), cloxacillin (CLX), oxacillin (OXA), and piperacillin (PIP), and four cephalosporins, cefazolin (CFZ), cefepime (FEP), cefotaxime (CTX), and ceftazidime (CAZ). One hundred-microliter samples were spiked with thiopental as an internal standard, and proteins were precipitated by acetonitrile containing 0.1% formic acid. Separation was achieved on a pentafluorophenyl (PFP) column with a mobile phase composed of phosphoric acid (10 mM) and acetonitrile in gradient elution mode at a flow rate of 500 µl/min. Detection was performed at 230 nm for AMX, CLX, OXA, and PIP and 260 nm for CFZ, FEP, CTX, and CAZ. The total analysis time did not exceed 13 min. The method was found to be linear at concentrations ranging from 2 to 100 mg/liter for each compound, and all validation parameters fulfilled international requirements. Between- and within-run accuracy errors ranged from -5.2% to 11.4%, and precision was lower than 14.2%. This simple method requires small-volume samples and can easily be implemented in most clinical laboratories to promote the therapeutic drug monitoring of ß-lactam antibiotics. The simultaneous determination of several antibiotics considerably reduces the time to results for clinicians, which may improve treatment efficiency, especially in critically ill patients.

Cloxacillin concentrations in serum, subcutaneous fat, and muscle in patients with chronic critical limb ischemia.

BACKGROUND: Patients suffering from critical limb ischemia (CLI) have poor wound healing in the ankle and foot areas. Secondary wound infections are frequent and often treated with prolonged courses of antibiotics. PURPOSE: This study set out to investigate to what extent the unbound fraction of 4 g of cloxacillin i.v. reaches its target organ in poorly vascularized tissues, i.e., the calf and foot of patients suffering from CLI. METHODS: Cloxacillin concentrations were measured by HPLC in serum and in microdialysis samples from skin and muscle of the lower part of the calf and as reference subcutaneously at the pectoral level in eight patients suffering from CLI (four males, four females, mean age 78 years, range 66-85 years) and in three healthy controls (two females, one male, mean age 67, range 66-68 years). RESULTS: In patients suffering from CLI, the tissue penetration of cloxacillin after a single 4 g dose was comparable to that of healthy controls, despite impaired blood circulation. CONCLUSIONS: The reduced blood flow in the peripheral vessels of the CLI patients presented here apparently is not the rate-limiting factor for delivery or tissue penetration of cloxacillin.

Saliva versus plasma pharmacokinetics: theory and application of a salivary excretion classification system.

The aims of this work were to study pharmacokinetics of randomly selected drugs in plasma and saliva samples in healthy human volunteers, and to introduce a Salivary Excretion Classification System. Saliva and plasma samples were collected for 3-5 half-life values of sitagliptin, cinacalcet, metformin, montelukast, tolterodine, hydrochlorothiazide (HCT), lornoxicam, azithromycin, diacerhein, rosuvastatin, cloxacillin, losartan and tamsulosin after oral dosing. Saliva and plasma pharmacokinetic parameters were calculated by noncompartmental analysis using the Kinetica program. Effective intestinal permeability (Peff) values were estimated by the Nelder-Mead algorithm of the Parameter Estimation module using the SimCYP program. Peff values were optimized to predict the actual average plasma profile of each drug. All other physicochemical factors were kept constant during the minimization processes. Sitagliptin, cinacalcet, metformin, tolterodine, HCT, azithromycin, rosuvastatin and cloxacillin had salivary excretion with correlation coefficients of 0.59-0.99 between saliva and plasma concentrations. On the other hand, montelukast, lornoxicam, diacerhein, losartan and tamsulosin showed no salivary excretion. Estimated Peff ranged 0.16-44.16 × 10(-4) cm/s, while reported fraction unbound to plasma proteins (fu) ranged 0.01-0.99 for the drugs under investigation. Saliva/plasma concentrations ratios ranged 0.11-13.4, in agreement with drug protein binding and permeability. A Salivary Excretion Classification System (SECS) was suggested based on drug high (H)/low (L) permeability and high (H)/low (L) fraction unbound to plasma proteins, which classifies drugs into 4 classes. Drugs that fall into class I (H/H), II (L/H) or III (H/L) are subjected to salivary excretion, while those falling into class IV (L/L) are not. Additional data from literature was also analyzed, and all results were in agreement with the suggested SECS. Moreover, a polynomial relationship with correlation coefficient of 0.99 is obtained between S* and C*, where S* and C* are saliva and concentration dimensionless numbers respectively. The proposed Salivary Excretion Classification System (SECS) can be used as a guide for drug salivary excretion. Future work is planned to test these initial findings, and demonstrate SECS robustness across a range of carefully selected (based on physicochemical properties) drugs that fall into classes I, II or III.

Effect of multidrug resistance gene-1 (ABCB1) polymorphisms on the single-dose pharmacokinetics of cloxacillin in healthy adult Chinese men.

BACKGROUND: Plasma concentrations of cloxacillin have been found to vary as much as 20-fold among individuals receiving the same oral dose. There is evidence that cloxacillin may be a substrate for P-glycoprotein, suggesting that polymorphisms in the ABCB1 gene may be a contributing factor to the observed variability in plasma cloxacillin concentrations. OBJECTIVE: This study investigated the effect of ABCB1 polymorphisms on the pharmacokinetic profile of cloxacillin in healthy subjects. METHODS: A single oral dose of cloxacillin 500 mg was administered to healthy Chinese male subjects under fasting conditions. Serial blood and urine samples were collected for up to 6 hours after administration. A high-performance liquid chromatography method was used to determine plasma cloxacillin pharmacokinetics and urinary excretion. A polymerase chain reaction technique was used for genotyping of 3 single nucleotide polymorphisms (SNPs) of the ABCB1 gene: exon 12 C1236T, exon 21 G2677T/A, and exon 26 C3435T. Cloxacillin pharmacokinetic parameters and urinary excretion were then compared according to genotype and haplotype groups. RESULTS: The study included 18 healthy Chinese male subjects who ranged in age from 21 to 26 years, had a mean weight ranging from 55.6 to 70.6 kg, and had normal renal function at baseline (mean [SD] serum creatinine, 93.4 [11.0] micromol/L). Plasma concentrations of cloxacillin were generally lower in the group carrying the 1236CC genotype (n = 3) compared with those carrying the 1236CT genotype (n = 9) or the 1236TT genotype (n = 6). Compared with the other groups, carriers of the 1236CC genotype had a significantly lower mean Cmax (-53%; P = 0.013) and AUC(0-infinity) (-40%; P = 0.044), and a significantly higher mean apparent oral clearance (35%; P = 0.013). They also had significantly lower urinary excretion of cloxacillin over 6 hours (-52%; P = 0.027). There were no significant differences in cloxacillin t(1/2) or renal clearance between the 3 C1236T genotypes, nor was the G2677T or C3435T SNP associated with any significant changes in the cloxacillin pharmacokinetic profile. Among subjects with 1 of the 3 major haplotype pairs, those carrying the CGC/CGC pair had a significantly lower C(max) (P = 0.017), AUC (P = 0.032), and urinary excretion of cloxacillin (P = 0.026) compared with those carrying the CGC/TGC and TTT/TTT pairs. CONCLUSIONS: In this small population of healthy Chinese men, the C1236T variant of ABCB1 appeared to be an important contributor to interindi-vidual differences in plasma cloxacillin exposure, most likely through an effect on oral absorption rather than on disposition. Studies of multiple doses in larger sample sizes are needed to confirm these findings.

On-line clean-up and screening of oxacillin and cloxacillin in human urine and plasma with a weak ion exchange monolithic column.

A weak ion exchange monolithic column prepared by modifying the GMA-MAA-EDMA (glycidyl methacrylate-methacrylic acid-ethylene glycol dimethacrylate) monoliths with ethylenediamine was applied to remove matrix compounds in biological fluid. Using this monolithic column, on-line clean-up and screening of oxacillin and cloxacillin in human urine and plasma samples had been investigated. Chromatography was performed by reversed-phase HPLC on a C(18) column with ultraviolet detection at 225 nm. Results showed that the ion exchange monolithic column could be used for deproteinization and retaining oxacillin and cloxacillin in human urine and plasma, which provided a simple and fast method for assaying drugs in human urine and plasma.

Development and validation of a fully automated LC method for the determination of cloxacillin in human plasma using anion exchange restricted access material for sample clean-up.

In the framework of a preliminary investigation on the plasma profile of cloxacillin after oral administration, a simple and rapid LC method was developed for the direct determination of this compound in human plasma. The on-line sample clean-up was carried out using a weak anion exchanger (diethylaminoethyl groups) as restricted access material (RAM). The effects of the washing liquid pH, the ionic strength and the addition of organic modifier to the washing liquid were studied in order to obtain an efficient sample clean-up and a high recovery of cloxacillin. The separation was achieved on octadecylsilica stationary phase using a mobile phase consisting in a mixture of phosphate buffer (pH 4.0; 25 mM) and acetonitrile (72:28, v/v). The UV detection was performed at 215 nm. The most appropriate regression model of the response function as well as the limit of quantitation (LOQ) were first selected during the pre-validation step. These criteria were then assessed during the formal validation step. The LOQ was 50 ng/ml. The method was also validated with respect to analyte recovery, precision, trueness, accuracy and linearity. Finally, it was successfully applied for the analysis of the first plasma samples obtained from patients having taken an oral dose of 500 mg cloxacillin.

Comparison of human serum, parotid and mixed saliva levels of phenoxymethylpenicillin, ampicillin, cloxacillin and cephalexin.

1. A study has been made of serum, mixed and parotid salivary levels attained in normal volunteers following oral dosage of 500 mg phenoxymethylpenicillin tablets, 500 mg crushed phenoxymethylpenicillin tablets in capsules, 500 mg ampicillin, 500 mg cloxacillin and 500 mg cephalexin.2. High mixed saliva levels were obtained with phenoxymethylpenicillin tablets and it is considered that these were due to rapid intra-oral dissolution of surface powder from friable tablets. No saliva levels were detected when tablets from the same batch were put into capsules.3. Low or no saliva levels were achieved with ampicillin, cloxacillin and cephalexin.4. The mode of action of antibiotics in oral infections is discussed.

The distribution of antibacterial agents between plasma and lymph in the dog.

1. Plasma, peripheral and thoracic lymph concentrations of penicillin V, phenethicillin, carbenicillin, ampicillin, cloxacillin, penicillin G, chloramphenicol and sulphadiazine were determined at various time intervals up to 6 h following intramuscular administration of 50 mg/kg to dogs.2. Peak plasma concentrations of the penicillins occurred within half an hour after administration with the peak lymphatic concentrations occurring 1.5 to 3 h afterwards. For the remaining period of the test the concentration in the lymph exceeded the corresponding concentration in the plasma. Sulphadiazine gave concentrations in thoracic lymph equal to the plasma concentration, but the peripheral lymph concentrations were lower while the concentrations of chloramphenicol in both peripheral and thoracic lymph were always lower than the plasma concentrations.3. After the peak concentrations were reached, the concentration curves for penicillins in lymph followed the same pattern as found in plasma, the penicillin concentrations declining exponentially. Sulphadiazine produced more persistent levels both in lymph and in plasma while the concentrations of chloramphenicol were still rising 6 h after administration.4. The free concentrations of penicillin in lymph were equal to the free concentrations in plasma, whereas the concentrations of free sulphadiazine and chloramphenicol in lymph were less than those in the plasma.

Absorption of antibiotics during peritoneal dialysis in patients with renal failure.

Cloxacillin, ampicillin, cephaloridine, kanamycin, and polymyxin B were administered singly in the dialysis fluid of patients having peritoneal dialysis for chronic renal failure. Therapeutic blood levels could be attained with all the drugs by the end of 12 hours of dialysis, using a concentration in the dialysis fluid which did not produce any local toxic symptoms. Absorption rates across the peritoneum were higher for cephaloridine, ampicillin, and cloxacillin than for kanamycin and polymyxin B. The serum half-lives after termination of dialysis were in excess of 11 hours, except that for cloxacillin which had a half life of about 2.5 hours.

Comparison of ex-vivo serum bactericidal activity of cefepime, ceftazidime and cloxacillin against Staphylococcus aureus.

Cefepime (1 g), ceftazidime (1 g), and cloxacillin (2 g) were administered intravenously to 10 volunteers each. After infusion of a single dose over 30 min, blood samples were obtained at 0.5, 1, 2, 3, 4, 5, 6, 8, 10, and 12 h (for ceftazidime at 0.5 and 4 h) after dosing. Drug levels were determined by the bioassay method. Serum bactericidal activity against five clinical isolates of cloxacillin-susceptible Staphylococcus aureus were determined by the microdilution method according to the National Committee for Clinical Laboratory Standards guidelines. The mean peak serum level was 76.88 +/- 24.71 mg/L for cefepime, 42.8 +/- 15.98 mL/L for ceftazidime, and 92.81 +/- 24.7 mg/L for cloxacillin. Concentrations of cefepime were detected during the whole testing period (mean trough level, 1.43 +/- 0.9 mg/L at 12 h), whereas concentrations of cloxacillin were measurable up to 5 h after administration (mean trough level, 0.90 +/- 0.97 mg/L). The mean peak reciprocal bactericidal titers were 29.41 for cefepime, 5.6 for ceftazidime, and 377 for cloxacillin. Effective bactericidal titers were detected as long as 5 h for cefepime (approximately 40% of the dosing interval) and 3 h for cloxacillin (at least 50% of the dosing interval). For ceftazidime, serum bactericidal activity was markedly lower compared with that of cefepime. Although cefepime has demonstrated an improved antistaphylococcal bactericidal activity compared with ceftazidime, it was somewhat lower than that of cloxacillin.

Bactericidal activity and pharmacology of flucloxacillin.

Flucloxacillin, a recent addition to the group of isoxazolyl penicillins, was studied in vitro and in normal volunteers. The bactericidal activity of the drug against most strains of gram-positive bacteria including penicillin-resistant Staphylococcus aureus was similar to that of oxacillin and approximately fourfold greater than that of cloxacillin. Each of the three penicillins was administered orally to a group of ten volunteers for eight days in a dose of 500 mg four times a day. The mean concentrations of flucloxacillin in the serum were two- to sixfold higher than those of the other two agents on the first, fourth and eighth days of therapy. The percentage of flucloxacillin bound by serum protein was 94.6 per cent; for cloxacillin and oxacillin the values were 93.5 and 91.5 per cent, respectively. Using these data, the concentrations of free flucloxacillin in serum were found to be twice as high as those of cloxacillin and oxacillin. These findings suggest that, when administered orally, this new agent may offer some therapeutic advantage over oxacillin and cloxacillin.

Comparative bioavailability and half-lives of cloxacillin and flucloxacillin.

The bioavailability of cloxacillin and flucloxacillin were compared after the oral administration, in capsule form, of 500 mg cloxacillin or 250 mg flucloxacillin to healthy volunteers. There were no significant differences observed in time to peak concentrations, peak concentrations or area under the serum concentration/time curve. However, flucloxacillin had a significantly longer serum elimination half-life. Serum levels of cloxacillin and flucloxacillin were determined using a high-performance liquid chromatographic assay.

Ocular and serum disposition kinetics of cloxacillin after topical administration of benzathine cloxacillin and intravenous administration of sodium cloxacillin to calves.

Disposition kinetics of cloxacillin were examined in calves after topical administration of benzathine cloxacillin and single IV administration of sodium cloxacillin, and the susceptibility of 17 field isolates of Moraxella bovis was measured. For the IV pharmacokinetic phase, sodium cloxacillin was administered at dosage of 10 mg/kg of body weight to male Holstein calves (n = 6, weighing 146 to 170 kg), and serum concentration of cloxacillin was measured thereafter for 10 hours. For the ocular pharmacokinetic phase, 6 calves were given either of 4 benzathine cloxacillin topical formulations consisting of 50-, 125-, 250-, or 375-mg doses. Treatment was repeated every 10 days until all 4 benzathine cloxacillin dosages were tested in the same 6 calves. Blood and tears were collected for 72 hours after each benzathine cloxacillin formulation was administered, and the concentration of cloxacillin in each specimen was measured, using a bioassay. The minimal inhibitory concentration of cloxacillin for 17 field isolates of M bovis was determined by use of an agar pour-plate dilution assay. After single IV administration of sodium cloxacillin, its half-life, body clearance, and volume of distribution were 19.5 +/- 12.8 minutes, 18.3 +/- 2.2 ml/min.kg, and 496 +/- 290 ml/kg, respectively. After topical administration of benzathine cloxacillin, cloxacillin concentration in lacrimal fluid peaked between 30 and 45 minutes and ranged between 963 micrograms/ml and 3,256 micrograms/ml for the 125- and 375-mg doses, respectively. There was no detectable cloxacillin activity in the lacrimal fluid of any calf by 36 hours after topical administration of benzathine cloxacillin, and cloxacillin was not detected in the serum at any time.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of interindividual variability of plasma concentrations after administrazion of high doses of intravenous amoxicillin or cloxacillin in critically ill patients.

The aim of the present retrospective observational clinical study was to assess the interindividual pharmacokinetic variability of plasma concentrations of amoxicillin or cloxacillin administered in high doses intravenously in critically ill patients, related to renal function or administration method.Four hundred and two plasma concentrations were measured at steady-state with a high performance liquid chromatography technique in 162 patients treated with 100 - 300 mg/kg/day of intravenous amoxicillin or cloxacillin.For both drugs and administration methods, plasma concentrations were significantly higher for patients with creatinine clearance below 60 ml/min, even though doses were adapted for renal impairment. the correlations calculated between plasma concentrations and creatinine level, creatinine clearance or doses were all low. There were fewer outlying drug concentrations in patients receiving continuous rather than intermittent regimens.Our results are in favor of adapting dosages of these beta-lactam antibiotics based on plasma concentrations, especially in cases of renal impairment.