A UHPLC-MS/MS method for the simultaneous determination of piperacillin and tazobactam in plasma (total and unbound), urine and renal replacement therapy effluent.

Piperacillin-tazobactam is a beta-lactam/beta-lactamase combination antibiotic used in patients with moderate to severe infection. Dosing of piperacillin-tazobactam requires an understanding of this patient group to maximise the effectiveness of this antibiotic and limit a further emergence of resistant pathogens. This is the first method that measures piperacillin and tazobactam simultaneously, across this range of clinically-relevant biological matrices. The calibration line was linear across the concentration range of 0.5-500µg/mL for piperacillin and 0.625-62.5µg/mL for tazobactam. All validation testing for matrix effects, precision and accuracy, specificity and stability were within 15%. A calibration equivalence study was performed to investigate the suitability of applying calibration curves prepared in an alternative matrix, with a mean bias of -10.8% identified for the application of a calibration line prepared for tazobactam in plasma only. Bias for all other calibration lines prepared in alternate matrices was within the 5% acceptance criteria. The method was successfully applied to a pharmacokinetic study of a critically ill patient receiving renal replacement therapy, with the results included.

Pharmacokinetics of piperacillin and tazobactam in plasma and subcutaneous interstitial fluid in critically ill patients receiving continuous venovenous haemodiafiltration.

This prospective pharmacokinetic study aimed to describe plasma and interstitial fluid (ISF) pharmacokinetics of piperacillin and tazobactam in critically ill patients on continuous venovenous haemodiafiltration (CVVHDF). Piperacillin/tazobactam (4g/0.5g) was administered every 8h and CVVHDF was performed as a 3-3.5L/h exchange using a polyacrylonitrile filter with a surface area of 1.05m(2). Serial blood (pre- and post-filter), filtrate/dialysate, urine and ISF concentrations were measured. Subcutaneous tissue ISF concentrations were determined using microdialysis. A total of 407 samples were collected. Median peak plasma concentrations were 210.5 (interquartile range=161.5-229.0) and 29.4 (27.9-32.0) mg/L and median trough plasma concentrations were 64.3 (49.0-68.9) and 12.3 (7.7-13.7) mg/L for piperacillin and tazobactam, respectively. The plasma elimination half-life was 6.4 (4.6-8.7) and 7.3 (4.6-11.8) h, volume of distribution 0.42 (0.29-0.49) and 0.32 (0.24-0.36) L/kg, total clearance 5.1 (4.2-6.2) and 3.8 (3.3-4.2) L/h and CVVHDF clearance 2.5 (2.3-3.1) and 2.5 (2.3-3.2) L/h for piperacillin and tazobactam, respectively. The tissue penetration ratio or ratio of area under the concentration-time curve of the unbound drug in ISF to plasma (unbound AUCISF/AUCplasma) was ca. 1 for both piperacillin and tazobactam. This is the first report of concurrent plasma and ISF concentrations of piperacillin and tazobactam during CVVHDF. For the CVVHDF settings used in this study, a dose of 4.5g piperacillin/tazobactam administered evry 8h resulted in piperacillin concentrations in plasma and ISF >32mg/L throughout most of the dosing interval.

Population pharmacokinetics of piperacillin at two dose levels: influence of nonlinear pharmacokinetics on the pharmacodynamic profile.

Piperacillin in combination with tazobactam is one of the most commonly used intravenous antibiotics. There is evidence for a possible saturable elimination of piperacillin. Therefore, the saturable elimination and its impact on the choice of optimal dosage regimens were quantified. In a randomized crossover study, 10 healthy volunteers received 1,500 mg and 3,000 mg of piperacillin as 5-min intravenous infusion. Population pharmacokinetics based on plasma and urine data were determined utilizing NONMEM and S-ADAPT. Probabilities of target attainment (PTAs) were compared for different models and dosage regimens, based on the target time of the non-protein-bound concentration above the MIC of at least 50% of the dosing interval. Total clearance of piperacillin was 18% (geometric mean ratio, 90% confidence interval, 11 to 24%) lower (P < 0.01), and renal clearance was 24% (9 to 37%) lower (P = 0.02) at the high compared to the low dose. The final model included first-order nonrenal elimination and parallel first-order and mixed-order renal elimination. Nonrenal clearance was 5.44 liter/h (coefficient of variation, 18%), first-order renal clearance was 4.42 liter/h (47%), and the maximum elimination rate of mixed-order renal elimination was 219 mg/h (84%), with a Michaelis-Menten constant of 36.1 mg/liter (112%). Compared to models with saturable elimination, a linear model predicted up to 10% lower population PTAs for high-dose short-term infusions (6 g every 8 h) and up to 4% higher population PTAs for low-dose continuous infusions (6 g/day). While renal elimination of piperacillin was saturable at therapeutic concentrations, the extent of saturation of nonrenal clearance was small. The influence of saturable elimination on PTAs for clinically relevant dosage regimens was relatively small.

[The impact of different renal function measuring methods on the dosages of meropenem, piperacillin/tazobactam and cefepime in critically ill patients]. / Impacto de distintos métodos de estimación de la función renal en la dosificación de meropenem, piperacilina/tazobactam y cefepima en pacientes críticos.

OBJECTIVE: Assesment of dosage deviations of three ss-lactam antibiotics eliminated through the kidneys (meropenem, piperacillin/tazobactam and cefepime) by comparison of two prediction formulae, Cockroft-Gault (CG) and Modification of Diet in Renal Disease (MDRD) with 24 h urinary creatinine clearance (CrCl(24h)), as a reference method. METHOD: 125 samples of 61 critically ill patients (each one with CG, MDRD y CrCl(24h) values) were classified in one of the five stages of the National Kidney Foundation (NKF) according to CrCl(24h). Dosage discrepancies for each antibiotic based on CG y MDRD were studied in reference to CrCl(24h) by percentage agreement and weighted kappa. At each of the NKF stages, daily dosage differences (Delta=DosisCG-DosisCrCl(24h); Delta=DosisMDRD-DosisCrCl(24h)) and percentage of samples with dosage discrepancies by CG and MDRD in reference to CrCl(24h) were calculated. RESULTS: There were no statistically significant differences between the two prediction formulae in respect to CrCl(24h), achieving good degrees of concordance. Deviation percentages fluctuated between 15.2% and 28% and occurred mainly by underdosing on stages 1 and 2 and by overdosing on stages 4 and 5. CONCLUSIONS: The two renal function prediction formulae can be indistinctly used to optimize the ss-lactam antibiotics dose regimen, CG being the easiest one.

Influence of pregnancy on the pharmacokinetic behaviour and the transplacental transfer of the piperacillin-tazobactam combination.

The safety/acceptability, blood pharmacokinetics and urinary excretion of the piperacillin-tazobactam (PPR-TZB) combination were studied in six patients between 25 1/7 and 31 5/7 weeks of amenorrhea. The combination was given for a materno-fetal infection due to susceptible organisms i.e. 4/0.5 g/6 h. Whenever possible, the trans-placental transfer (TPT) of the combination was assessed in several sub-compartments of the feto-placental unit i.e. maternal blood sample, cord blood, amniotic fluid, placenta tissue and fetal urine. Two series of nine blood samples were scheduled for each patient, i.e. on D1 (first dose) and D3 (at plateau). Samples were assayed by HPLC and data were analyzed by a non-compartmental method. Safety/acceptability of the treatment proved to be good. The kinetic behavior of both beta-lactams appeared to be identical. Evidence was found during pregnancy of an increase in Vss and Cl of the combination. These increases can be linked to a notable decrease in AUCs. The TPT of the combination was significant. Regarding other accessible compartments (i.e. placenta tissue, amniotic fluid and fetal urine), the ratio of PPR-TZB concentrations was invariably about 8. Maternal circulating levels of PPR-TZB were, by 4 h, less than the MIC of target organisms (i.e. < or = 8 micrograms/ml), both on D1 and at steady state. This raises the question of the pertinence of the dosage regimen. Regarding PPR, it is accepted that antibacterial protection is satisfactory when circulating concentrations are kept at a Css (steady state concentration) of the order of 20 micrograms/ml or more. PPR-TZB combination would be administered by continuous infusion i.e. 8 mg/min to obtain 3 h later a Css of more than 20 micrograms/ml. The daily dosage would then be 12/1.5 g instead of 16/2 g, which is also more satisfactory from a pharmaco-economic standpoint. This proposal must be validated in a sufficient number of patients and, could avoid disqualification of the combination PPR-TZB in the treatment of serious infections during certain pathological pregnancies.

High-performance liquid chromatographic determination of tazobactam and piperacillin in human plasma and urine.

A high-performance liquid chromatographic (HPLC) method with ultraviolet (UV) absorbance was developed for the analysis of piperacillin-tazobactam (tazocillin), in plasma and urine. The detection was performed at 218 nm for tazobactam and 222 nm for piperacillin. The procedure for assay of these two compounds in plasma and of piperacillin in urine involves the addition of an internal standard (ceftazidime for tazobactam and benzylpenicillin for piperacillin) followed by a treatment of the samples with acetonitrile and chloroform. To quantify tazobactam in urine, diluted samples were analysed using a column-switching technique without internal standard. The HPLC column, LiChrosorb RP-select B, was equilibrated with an eluent mixture composed of acetonitrile-ammonium acetate (pH 5). The proposed technique is reproducible, selective, and reliable. The method has been validated, and stability tests under various conditions have been performed. Linear detector responses were observed for the calibration curve standards in the ranges 5-60 micrograms/ml for tazobactam, and 1-100 micrograms/ml for piperacillin and spans what is currently though to be the clinically relevant range for tazocillin concentrations in body fluids. The limit of quantification was 3 micrograms/ml for tazobactam and 0.5 microgram/ml for piperacillin in plasma and urine. Extraction recoveries from plasma proved to be more than 85%. Precision, expressed as C.V., was in the range 0.4-18%.

Inhibition of the renal excretion of tazobactam by piperacillin.

A 1:4 by weight of combination of tazobactam, a new beta-lactamase inhibitor, and piperacillin, is now under development in Japan. After bolus iv administration of the combination to beagle dogs, piperacillin both significantly raised the area under plasma concentration time curve (AUC0 approximately infinity) and significantly decreased the total body clearance (Cltot) of tazobactam. The percentage binding of tazobactam and piperacillin to dog and human serum protein was the same for the combination as for the individual compounds. Piperacillin significantly decreased the renal clearance (Clr) and the clearance ratio (Cr) of tazobactam in dogs. Further, probenecid significantly decreased Clr of both tazobactam and piperacillin, and the Cr of tazobactam and piperacillin approximately reached unity. These results indicate that piperacillin inhibits the renal excretion of tazobactam. Both tazobactam and piperacillin are secreted by a tubular anion transport system which is identical to the probenecid secretion system.

Determination of tazobactam and piperacillin in human plasma, serum, bile and urine by gradient elution reversed-phase high-performance liquid chromatography.

A gradient elution high-performance liquid chromatographic method is described for the analysis of the beta-lactamase inhibitor tazobactam (YTR-830H) and a semi-synthetic parenteral penicillin, piperacillin, in human plasma, serum, bile and urine. The assay for plasma, serum and bile involves deproteinization with acetonitrile and the removal of lipids with dichloromethane; urine is diluted with buffer. Separation and quantitation are achieved using a mobile phase based on ion-suppression chromatography on a C18 reversed-phase column with ultraviolet detection at 220 nm. The limit of quantitation for both compounds is 1.0 microgram/ml in plasma, serum and bile using a 0.2-ml sample and 50.0 micrograms/ml in urine using a 0.1-ml sample. The method has been validated by preparing and analyzing a series of fortified samples (range 1.0-200 micrograms/ml for each compound in plasma, serum and bile and 50.0-10,000 micrograms/ml for each compound in urine). Excellent linearity, accuracy, precision and recovery were obtained. The method was not interfered with by other endogenous components, nor by other commonly administered antibiotics such as amoxicillin, mezlocillin, cefometazole and cefotaxime. The assay has been successfully applied to the analysis of samples from pharmacokinetic studies in man and animals.

High-performance liquid chromatographic determination of a new beta-lactamase inhibitor and its metabolite in combination therapy with piperacillin in biological materials.

[2S-(2 alpha,3 beta,5 alpha)]-3-Methyl-7-oxo-3-(1H-1,2,3-triazol-1-yl- methyl)-4-thia-1-azabicyclo [3.2.0]-heptane-2-carboxylic acid 4,4-dioxide (YTR-830H) is a new beta-lactamase inhibitor and the combination therapy of this compound with piperacillin is now under study. For the determination of the beta-lactamase inhibitor and piperacillin in biological materials, plasma and visceral tissue homogenates were deproteinized, whereas diluted urine and filtered faeces homogenates were treated with a Sep-Pak C18 cartridge. In order to assay the inactive metabolite of beta-lactamase inhibitor, each sample was treated with a Sep-Pak C18 cartridge. Aliquots of each preparation were chromatographed using ion-pair and reversed-phase chromatographic techniques on a high-performance liquid chromatograph equipped with a UV detector, set at 220 nm. The detection limits of beta-lactamase inhibitor and piperacillin were 0.2 microgram/ml in plasma, 2.5-5.0 micrograms/ml in urine and 0.2-0.5 microgram/g in visceral tissue and faeces. Those of the metabolite were 1.0 microgram/ml in plasma, 2.5-5.0 micrograms/ml in urine and 1.0 microgram/g in visceral tissue and faeces. A precise and sensitive assay for the determination of the beta-lactamase inhibitor, its metabolite and piperacillin is described, and their stabilities in several media are reported.

High-pressure liquid chromatographic assay of sulbactam in plasma, urine, and tissue.

A reverse-phase high-pressure liquid chromatography method for the quantitation of sulbactam in plasma, urine, and tissue is described. The assay used the formation of an imidazole derivative followed by extraction with acetonitrile and dichloromethane and used UV absorbance for detection. The mobile phase consisted of acetonitrile, tetrabutylammonium hydroxide, and phosphate buffer. The assay was linear from 100 micrograms/ml (g of tissue) to 1 microgram/ml (g). Within- and between-batch recovery was greater than 90%. The coefficient of variation was generally less than 15%. There were no interfering peaks in the quantitation of sulbactam.

High-performance liquid chromatographic assay of sulbactam using pre-column reaction with 1,2,4-triazole.

A high-performance liquid chromatographic (HPLC) method for the determination of sulbactam in human and rat plasma and urine has been developed. Sulbactam was reacted with 1,2,4-triazole to yield a product having an ultraviolet absorption maximum at 326 nm. The product was separated using reversed-phase HPLC from the regular components of plasma and urine with an ion-pair buffer at 50 degrees C and detected at the ultraviolet maximum. The limits of accurate determination were 0.2 and 1.0 micrograms/ml in plasma and urine, respectively. The coefficients of variation of inter- and intra-assays in human plasma spiked at 4.0 micrograms/ml (n = 5) were 1.02 and 3.05%, respectively. Coexisting cefoperazone, penicillins, or the alkaline degradation product(s) of sulbactam did not interfere in the sulbactam assay. The pharmacokinetic behaviour of sulbactam and cefoperazone coadministered to rats was estimated by moment analysis.

Effect of clavulanic acid on inactivated amoxicillin excretion in urinary tract infection.

Oral amoxicillin was taken with and without clavulanic acid by normal subjects and by patients with chronic complicated urinary tract infection to examine the in vitro protective effect of clavulanic acid on amoxicillin degradation. When amoxicillin alone was taken, urinary excretion of the penicilloic acid of amoxicillin in bacteria-positive patients was higher than that in bacteria-negative patients and in normal subjects. There was no comparable change in urinary penicilloic acid excretion in the presence of clavulanic acid. There were significant in vitro protective effects of clavulanic acid on beta-lactamases in the urine.

Pharmacokinetics of sulbactam and ampicillin following oral administration of sultamicillin with probenecid.

Eight volunteers each received lg of probenecid followed immediately by 1.5 g sultamicillin, both given by the oral route. Plasma concentrations of ampicillin and sulbactam were measured using a differential bioassay method. An ampicillin mean peak plasma concentration of 23.1 mg/l was found typically 1.5 h after dosing; the mean peak plasma concentration of sulbactam (10.0 mg/l) occurred at the same time. The mean plasma half-life of ampicillin was 1.45 h and that of sulbactam 1.3 h. The mean urinary recovery of ampicillin was 65% and that of sulbactam 62%. Five of the volunteers reported minor changes in bowel habits.

In vitro effect of penicillins and aminoglycosides on commonly used tests for glycosuria.

The effect of penicillins (ampicillin, carbenicillin, penicillin G, and penicilloic acid) and aminoglycosides (amikacin, gentamicin, streptomycin, and tobramycin) on the accuracy of Clinitest, Diastix, and TesTape determinations of glycosuria was studied. Solutions of each of the drugs were prepared in urine in a range of clinically obtainable drug concentrations. In addition, urine solutions were prepared that contained the same drug concentrations and sufficient glucose to give final concentrations of 0.5, 1, and 2%. All solutions were tested in triplicate using the five-drop Clinitest method, Diastix, and TesTape. Falsely elevated and falsely decreased Clinitest readings were obtained with the penicillins. These readings were influenced by the concentration of the penicillins and of glucose. The aminoglycosides had no effect of Clinitest determinations. Neither drug class interfered with Diastix or TesTape readings. The Clinitest-penicillin interaction is unpredictable. If Clinitest is used as a quantitative test for glycosuria in patients receiving penicillins, the results should be rechecked using a qualitative method such as the glucose oxidase tests, Diastix or TesTape. All three tests can be used to test for glycosuria in patients receiving aminoglycosides.

Effect of urine concentration versus tissue concentration of ampicillin and mecillinam on bacterial adherence in the rat bladder.

We investigated the effect of tissue and urine concentrations of ampicillin and mecillinam on bacterial bladder wall adherence in rats. Escherichia coli 02 labeled with 3H-1-leucine was inoculated into the bladder for 2 hr. With the ureters transected bilaterally, antibiotic was instilled into the bladder lumen or administered intravenously only. No bacteria survived after the presence of antibiotic in the bladder lumen for 1.5 hr. After intravenous administration of ampicillin, with no antibiotic in the bladder lumen, measured by viable counts, decreased significantly as compared to controls. Adherence, measured by radioactive counts, decreased significantly after intravenous administration of mecillinam, possibly indicating prevention of tissue invasion. This finding was explained by entrapment of bacterial debris in the bladder lumen and invasion of bacteria into the bladder wall. Our results support the importance of tissue concentrations in the treatment of urinary tract infections.

The metabolism of carfecillin in rat, dog and man.

1. The absorption of the phenol moiety of [phenol-14C]carfecillin following oral administration to rat, dog and man was extensive, since 95%, 73% and 99% of the administered radioactivity respectively was recovered in the urine. In contrast, less than half of the carbenicillin moiety of carfecillin was absorbed after oral administration, as judged by excretion studies using [carbenicillin-14C]carfecillin in intact and bile-duct cannulated animals. 2. The patterns of radiometabolites in the urines of rat, dog and man following single oral administration of [phenol-14C]carfecillin were determined by chromatography and radioassay. In two men, the majority of a dose was excreted as phenylsulphate (71%) and phenylglucuronide (16%) with the sulphate and glucuronic acid conjugates of quinol representing small amounts of the urinary radioactivity. Similar metabolic patterns were observed in the rat and dog following oral administration of either [14C]phenol or [phenol-14C]carfecillin, although some saturation of sulphate conjugation was apparent at the dose levels employed.

Urinary excretion of mecillinam by volunteers receiving film-coated tablets of pivmecillinam hydrochloride.

Mecillinam is a new amidinopenicillin which is taken orally as its ester pivmecillinam. 10 healthy volunteers were each given 200 mg of pivmecillinam hydrochloride four times daily and all urine was collected and cooled to -20 degrees C to minimize antibiotic degradation. 43% of the ingested antibiotic was recovered from the urine in its microbiologically active form, during the first 12 h and 34% during the subsequent 24 h. The corresponding figures for 10 volunteers receiving double this dose of antibiotic was 30 and 34%. The mean half life of mecillinam in urine due to spontaneous breakdown at 37 degrees C was 17 h. There was evidence that stearate-film-coated pivmecillinam hydrochloride tablets may be less likely to cause dyspepsia than gelatine capsules.