Current Pharmaceutical Analysis - Volume 7, Issue 4, 2011

The carbapenems are a class of a β-lactam antibiotics with a broad spectrum of antibacterial activity. In all carbapenems, a β-lactam ring is required for their antibacterial activity. In addition, the bi-cyclic 4:5 fused ring is one of the main reasons for the instability of carbapenems. The 2C, 3C and 6C substituents affect the spectrum of microbiological activity, pharmacokinetic parameters, enzymatic and chemical stability. The early carbapenems (imipenem, panipenem) were degraded by renal dihydropeptidase (DHP-I) and required co-administration with DHP-I inhibitors to prevent this inactivation. The newer carbapenems (meropenem, ertapenem, doripenem and biapenem) with a 1β-methyl group at 4C are stable to DHP-I hydrolysis. The carbapenems are still susceptible to chemical degradation under the influence of different stress factors (temperature, humidity, light, oxidizing, hydrogen concentration) in aqueous solutions and solid state. This paper provides an overview of recent observations on the degradation kinetics of carbapenems used in therapy, their mechanisms of degradation, general and specific acid-base hydrolysis, catalytic effect of buffers and thermodynamic parameters describing the degradation of carbapenems in aqueous solutions and solid state. As during the degradation of carbapenems depending on stress factors (solvents, pH, drug concentration, temperature, time) various degradation products are formed, their chemical structures and pathways of their formation are also compared.

Cannabidiol (CBD) is a cannabinoid found in the Cannabis sativa plant and it is devoid of the typical cognitive and psychological effects of Delta 9-tetrahydrocannabinol (Delta 9-THC). However, studies with animals suggest that CBD presents anxiolytic properties. In the face of evidence of a cannabinoid system in humans, and the growing interest in the therapeutic use of CBD, the purpose of the present study is to develop and validate an analytical methodology for determination of CBD in human plasma. The methodology developed is based on liquid-liquid extraction and gas chromatography/mass spectrometry, and it is linear in the concentration range from 2.5 to 250 ng/mL of plasma, with the limit of detection of 1.0 ng/mL. The precision of inter and intra assays was respectively in the range from 7.5% to 8.2% and from 2.5% to 7.0%. Accuracy of the values of inter and intra assays varied respectively from 1.3% to 8.6% and from 1.2% to 4.4%. Extraction efficiency obtained was in the range of 54.6% to 75.3%. The validated methodology was demonstrated to be rapid, sensitive and suitable for application in clinical studies, as determined by monitoring patients in a controlled administration trial with CBD.

In this paper, we describe a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method used to detect the antipsychotic drug bromperidol, in biological post-mortem fluids (urine, blood and vitreous humour). The method was used to investigate an unusual case of death which occurred after bromperidol ingestion. A 44 year-old woman arrived at the emergency department in cardiac arrest. Her sister reported that the woman had taken several drops of a medicine containing bromperidol. An autopsy was performed and the biological fluid samples were submitted to a full toxicological work-up first and then to the target analysis of bromperidol. The analytical results obtained from the biological fluids collected at autopsy supported the hypothesis of death due to bromperidol intoxication.

A high-performance liquid chromatographic assay with tandem mass spectrometric detection has been developed to simultaneously quantify lenalidomide (LEN) and dexamethasone (DEX) in human plasma to facilitate their combined clinical development. The analytes and the internal standard (IS) fluconazole were extracted from 250 μL aliquots of human plasma via liquid-liquid extraction in ethyl acetate. Chromatographic separation was achieved in a run time of 2.0 min on XTerra RP18 column (50 mmx4.6 mm, 5μm) using isocratic mobile phase, consisting of methanolwater containing 0.1% formic acid (90:10, v/v), at a flow-rate of 0.5 mL/min. Detection of analytes and IS was performed by electrospray ionisation tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for LEN, DEX and IS were m/z 260.1→148.8, 393.3→147.0 and 307.1→238.0, respectively. The method was validated over the concentration range of 9.99 to 1009.65 ngmL-1 for LEN and 1.99 -500.99 ngmL-1 for DEX in human plasma. The intra-batch and inter-batch precision (% CV) across four quality control levels was ≤ 6.4% for both the analytes. In conclusion, a simple and sensitive analytical method was developed and validated in human plasma. This method is suitable for measuring accurate concentration in clinical trial samples following combined administration of lenalidomide and dexamethasone in patients with multiple myeloma.

A stability-indicating liquid chromatographic method was developed and validated for the simultaneous determination of enalapril maleate and hydrochlorothiazide in drug substances and dosage forms. The method was developed using a RP-18 column with mobile phase containing methanol - tetrahydrofuran - phosphate buffer (pH 2.2; 0.01M) (32:5:63, v/v/v) at a flow rate of 1.0 mL min-1 and UV detection at 210 nm. Enalapril and hydrochlorothiazide were subjected to hydrolytic, oxidative, thermal and photolytic stress conditions. The degradation products were well resolved from main peaks, proving the stability-indicating power of the method. The assay was linear for enalapril and hydrochlorothiazide concentrations of 40-140 μg/mL and 100-350 μg/mL respectively. The developed method was selective, accurate and precise for enalapril and hydrochlorothiazide determination. This method was successfully applied for determination of enalapril and hydrochlorothiazide in combined commercial tablets. The proposed method was found to be suitable for quantitative determination and the stability study of enalapril and hydrochlorothiazide in pharmaceutical preparations.

Three EPMEs based on maltodextrins with different dextrose equivalent (DE) values [I (4.0-7.0), II (13.0-17.0) and III (16.5-19.5)], were designed for the determination of L-vesamicol. The linear concentration ranges of the proposed electrodes were 10-4 - 10-2 mol/L for maltodextrin I-based electrode, 10-5 -10-3 mol/L for maltodextrin II-based electrode and 10-9 - 10-7 mol/L for maltodextrin III-based electrode. The detection limits of the proposed electrodes were 1.34x10-5, 5.0x10-6 and 3.33x10-10 mol/L for maltodextrin I, II and III based electrodes, respectively. The selectivity of EPMEs for L-vesamicol was tested over D-vesamicol, creatine, creatinine and some inorganic ions. The surfaces of the electrodes can be easily renewed by simply polishing on alumina.

Four enantioselective, potentiometric membrane electrodes based on carbon paste impregnated with α-, β-, 2- hydroxyl-3-trimethylammoniopropyl -β- (as chloride salt) and γ-cyclodextrins are proposed for the enantioanalysis of Lcysteine. The proposed electrodes exhibited near-Nernstian slopes in a wide linear concentration range: 10-10 - 10-3 mol/L, with very low detection limits, near 10-11 mol/L. The surfaces of the electrodes are easily renewable by simply polishing on an alumina paper.

Treatments fail to eliminate Toxoplasma gondii due to low drug brain penetration. Spiramycin is an Mrp2, Pglycoprotein substrate, active in acute and chronic murine toxoplasmosis. Metronidazole is a CYP3A4 inhibitor and Pglycoprotein substrate. We developed a simple HPLC method to analyze spiramycin and metronidazole simultaneously. Male Balb/c mice were randomly selected in three groups and were dosed orally either 500 mg/kg metronidazole (group 1, n=4) or 400 mg/kg spiramycin (group 2, n=4) or coadministered 500 mg/kg metronidazole 30 min prior to 400 mg/kg spiramycin (group 3, n=4). Mice were euthanized 2 hours after spiramycin administration. Metronidazole and spiramycin brain and plasma concentrations were measured by HPLC using a Phenomenex® C-18 (150x3.8 mm, 5 μm) column and acetonitrile-phosphate buffer (pH 2.5) gradient elution (20/80 to 30/70 in 3 min) at 1 mL/min flow, 29°C and 232 nm. The method was linear (0.25-50.0 μg/mL), the LLOQ was 0.25 μg/mL, intra- and interday variability, precision and accuracy were within 15%. Recoveries were above 75% and there was no matrix interference. Metronidazole eluted at 3 min and spiramycin at 5 min. Spiramycin did not affect plasma metronidazole concentration (6.93±0.48 μg/mL in combination vs. alone 7.65±0.55 μg/mL) or brain (2.96±0.60 μg/g after coadministration vs. control, 4.02±0.78 μg/g). Metronidazole did not change spiramycin plasma concentration (coadministration: 3.94±1.30 μg/mL, control: 3.71±0.94 μg/mL). However, spiramycin brain concentration increased 2-fold after metronidazole coadministration from 2.44±0.33 μg/g to 4.83±1.25 μg/g (P < 0.05). Metronidazole increased spiramycin brain uptake, probably due to P-glycoprotein inhibition, which may improve toxoplasmosis encephalitis treatments.

The thermal stability and thermal decomposition behavior of sodium salts of some penicillins and cephalosporins have been studied using a differential scanning calorimetry technique under a stream of air with a linear heating rate up to 873 K. A “model-free” kinetic method based on the Kissinger equation was applied to decomposition processes of several penicillin and cephalosporin sodium salts to determine activation energy and the pre-exponential factor, following a simplified approach. The values of these parameters referring either to the first or to the slowest decomposition process were used in an attempt to calculate the lifetime for a 1.0% conversion degree of carbenicillin and cephalosporin C, extrapolated at 298.15, 323.15, 373.15 and 423.15 K. Lastly, a comparison of stability data and different calorimetric curve profiles of several antibiotics belonging to the cephalosporin and penicillin classes led to the conclusion that the presence of penicillanic and cephalosporanic rings in all the molecular structures considered does not seem to noticeably affect the overall decomposition mechanisms of the various antibiotics.

A simple, precise, rapid, and low-cost conductometric method for verapamil hydrochloride determination in pharmaceutical formulations is described. Verapamil present in pharmaceuticals containing known quantities of the drug was conductometrically titrated in aqueous solution with silver nitrate using a conductometric cell coupled to an autotitrator. The method is based on the precipitation of chloride ions coming from verapamil hydrochloride with Ag(I) ions yielding AgCl(s) and the conductance of the solution is measured as a function of the volume of titrant. Under optimized experimental conditions the method was applied with success for verapamil determination in three pharmaceutical formulations in the concentration level of 1.00 x 10-2 mol L-1. The relative standard deviation for six successive measurements was smaller than 0.5 % and no interferences were observed in the presence of common components of the tablets such as sodium monophosphate, magnesium stearate and lactose. Recoveries of verapamil from various tablet dosage formulations ranging from 97.1 to 102.8% were obtained. These results are in good accordance with the declared values of manufacturer and an official method based on spectrophotometric analysis.

Non-transferrin bound iron (NTBI) is known to facilitate reactive oxygen species generation which contributes to the development of tissue damage observed in various disorders like hemolytic anemia, hemochromatosis, diabetes mellitus or many forms of neonatal central nervous system damage. Here we are summarizing information about biochemical nature of NTBI and review methods developed for its detection in biological material. Ability to detect NTBI concentration is likely to become an essential tool when it comes to diagnosis of diseases characterized by excessive iron accumulation. In contrast to routinely used parameters such as transferrin saturation or ferritin concentration, NTBI is not affected by inflammatory responses which makes it a reliable indicator. Unfortunately, we are still lacking validated laboratory methods to detect NTBI in biological fluids.

Imidazoacridinones and triazoloacridinones are acridinone derivatives characterized by potent antitumor activity. From those, two of the most active compounds are C-1305 and C-1311. C-1305 was selected for extended preclinical trials, and C-1311 underwent phase II clinical trials for colon and breast cancers. These compounds exhibit biological (cytotoxic and/or antitumor) activity against various tumors including leukemia, melanoma, colon adenocarcinoma, lung carcinoma, breast carcinoma, and colon carcinoma. There are several suggested mechanisms of action that could be responsible for acridinone's cytotoxic and antitumor actions, most of which are associated with the interactions with DNA and its proper functionality. It has been shown that triazoloacridinones and imidazoacridinones inhibit the interactions between cleavable complexes of topoisomerase II with DNA. They also inhibit nucleic acid or protein synthesis induced by G2 block of cell cycle, which is followed by apoptosis or mitotic catastrophe, intercalating to DNA, binding in minor groove, and forming of interstrand DNA crosslinks. In the literature, there is not enough convincing evidence indicating that only one particular mechanism of action is responsible for the biological activity of presented acridinone derivatives. This article is a review of the information concerning imidazoacridinones' and triazoloacridinones' mechanisms of action in view of their biological activity. Further information concerning the potential pharmacological activity of acridinone derivatives in view of the quantitative structure-activity relationships studies (QSAR) is presented.