Quantification of piperacillin, tazobactam, cefepime, meropenem, ciprofloxacin and linezolid in serum using an isotope dilution UHPLC-MS/MS method with semi-automated sample preparation.

BACKGROUND: Recent studies have demonstrated highly variable blood concentrations of piperacillin, tazobactam, cefepime, meropenem, ciprofloxacin and linezolid in critically ill patients with a high incidence of sub-therapeutic levels. Consequently, therapeutic drug monitoring (TDM) of these antibiotics has to be considered, requiring robust and reliable routine analytical methods. The aim of the present work was to develop and validate a multi-analyte ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous quantification of the above mentioned antibiotics. METHODS: Sample preparation included a manual protein precipitation step followed by two-dimensional ultra high performance liquid chromatography (2D-UHPLC). Corresponding stable isotope-labeled substances were used as internal standards for all of the analytes, with the exception of tazobactam. The injected sample volume was 7 µL. The run time was 5.0 min. RESULTS: Inaccuracy was ≤8% and imprecision coefficient of variation (CV) was <9% for all analytes. Only minor matrix effects and negligible carry-over was observed. The method was found to be robust during the validation period. CONCLUSIONS: We were able to develop a reliable 2D-UHPLC-MS/MS method addressing analytes with highly heterogeneous physico-chemical properties. The novel assay may be an efficient tool for an optimized process workflow in clinical laboratories for important antibiotics in regards to TDM.

Determination of biapenem in a medicinal product by micellar electrokinetic chromatography with sweeping in an enhanced electric field.

The elaborated method of micellar electrokinetic chromatography (MEKC) used to separate biapenem from its related substances was successfully implemented using sweeping under an enhanced electric field, followed by UV absorption detection at 200nm. The best results were obtained with formic buffer (22.5mM) pH 4.3 and sodium dodecyl sulfate (150mM) added to the electrolyte as the sweeping agent. Neutral capillary (60/50cm; 50µm ID) with reverse polarity and voltage values of 22kV, were used throughout the investigation. The optimized method of biapenem determination, validated in terms of linearity, accuracy and precision, provides a detection limit of 0.5µg/mL at S/N=3 for biapenem. The repeatability of the CE system, expressed by relative standard deviations (RSD) in the migration times, for biapenem and its degradation products varied from 0.14 to 1.48%, whereas for the corrected peak areas RSD were about 0.68-8.43%. Satisfactory separation was achieved within 20min of electrophoresis; moreover all carbapenems (imipenem, meropenem, ertapenem, doripenem and biapenem) were separated from each other during analysis. The evaluated MEKC method was applied to the analysis of a medicinal product containing biapenem - Omegacin(®) 0.3g for intravenous drip infusion.

Reverse transsulfuration and its relationship to thienamycin biosynthesis in Streptomyces cattleya.

Cystathionine gamma-lyase (EC 4.4.1.1) was purified from Streptomyces cattleya, an actinomycete which produces the unusual beta-lactam antibiotic thienamycin. The enzyme displays broad substrate specificity and is similar to gamma-lyases purified from other microorganisms. That the gamma-lyase functions in vivo to provide cysteine for antibiotic synthesis was shown by two types of experiments. First, cystathionine and methionine, as well as cysteine itself, are efficiently utilized by S. cattleya for thienamycin biosynthesis. Second, propargylglycine, a mechanism-based inactivator of cystathionine gamma-lyase in vitro, inhibits the synthesis of thienamycin in vivo. This inhibition can be substantially reversed by providing the cells with another source of cysteine, such as cystine.

Carbapenems, a new class of beta-lactam antibiotics. Discovery and development of imipenem/cilastatin.

The discovery of Streptomyces cattleya and its antibiotic product, thienamycin, has ushered in a new era of beta-lactam agents, the carbapenems. Numerous carbapenems were subsequently discovered; however, none had the potency, broad-spectrum activity, and lack of cross-resistance exhibited by thienamycin. Chemical instability encountered with thienamycin was overcome by the N-formimidoyl derivative, imipenem. Imipenem is distinguished from other beta-lactams by its outstanding activity against gram-positive organisms as well as against Enterobacteriaceae, Pseudomonas aeruginosa, and Bacteroides. However, development was hindered by extensive renal metabolism of imipenem, resulting in low urinary concentrations of antibiotic. A renal dipeptidase, dehydropeptidase-I, was responsible for hydrolyzing imipenem and other carbapenems. To counter its action, a specific inhibitor, cilastatin, was developed. Coadministered with imipenem in a one-to-one ratio, cilastatin provides prolonged, reversible blockade of imipenem metabolism, dramatically improving urinary recoveries to therapeutically significant levels. Cilastatin also contributes to the safety of imipenem, since its coadministration prevents proximal tubular necrosis which has been observed in sensitive animals receiving imipenem alone in high doses. Thus, the combination imipenem and cilastatin overcame the pharmaceutical and metabolic challenges presented by thienamycin, and allowed for the evaluation in humans of the outstanding antimicrobial activity of this new class of beta-lactam antibiotics.

Structures of OA-6129D and E, new carbapenam antibiotics.

The structures and stereochemistry of OA- 6129D and E, new carbapenam compounds produced by Streptomyces sp. OA-6129, were determined by spectroscopic analysis and chemical transformation.

Structures of OA-6129A, B1, B2 and C, new carbapenem antibiotics produced by Streptomyces sp. OA-6129.

The chemical structures of OA-6129A, B1, B2 and C, new carbapenem antibiotics having a pantetheinyl group at C-3 were elucidated by spectroscopic analysis and chemical transformation as presented in Fig. 1.

Northienamycin and 8-epi-thienamycin, new carbapenems from Streptomyces cattleya.

Two new carbapenem antibiotics, northienamycin and 8-epi-thienamycin have been isolated from culture broth of Streptomyces cattleya grown under conditions for thienamycin production. The isolation, structure elucidation and in vitro antibacterial spectra of the new carbapenems are reported. In addition, comparison of the in vitro potency of the corresponding formamidine derivatives to that of MK787 is presented.

New beta-lactam antibiotics, carpetimycins C and D.

Two new carbapenem antibiotics, carpetimycins C and D have been isolated from the culture broth of Streptomyces sp. KC-6643, which produced carpetimycins A and B. The structures of carpetimycins C and D have been determined to be (5R,6R)-3-[2-acetamidoethyl-(R)-sulfinyl]-6-(1-hydroxy-1-methylethyl) -7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid and (5R,6R)-3-[2-acetamidoethyl-(R)-sulfinyl]-6-(1-hydroxysulfonyloxy-1 -methylethyl)-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, respectively. Studies on the fermentation, isolation and physico-chemical properties of these antibiotics are also described.

C-19393 E5, a new carbapenem antibiotic. Fermentation, isolation and structure.

A new carbapenem antibiotic, C-19393 E5, was isolated from the culture filtrate of Streptomyces griseus subsp. cryophilus C-19393 as a minor component. The chemical structure of the antibiotic was determined by comparing its spectral data with those of the known 5,6-cis carbapenem antibiotics and confirmed by partial synthesis from epithienamycin B as shown in Fig. 1. The antibiotic has a broad antimicrobial spectrum and shows strong inhibitory activity against beta-lactamases.