Development and validation of a capillary electrophoresis method for the simultaneous determination of impurities of escitalopram including the R-enantiomer

A stereospecific capillary electrophoresis assay for the simultaneous determination of related substances and the enantiomeric purity of escitalopram was developed by a central composite face-centered factorial design and subsequently validated. Separations were carried out in a 50 μm, 47/40 cm fused-silica capillary. The optimized conditions included 20 mM phosphate buffer, pH 2.5, containing 0.5 mg/ml β-cyclodextrin and 22 mg/ml sulfated β-cyclodextrin as background electrolyte, an applied voltage of −20 kV and a temperature of 28 °C. Salicylic acid was used as internal standard. The assay was validated for the (R)-enantiomer of citalopram and the enantiomers of the impurity citadiol in the range of 2.5–150 μg/ml and 2.5–50 μg/ml, respectively. The limit of detection was 0.02% for all compounds, the limit of quantitation 0.05%, relative to a concentration of escitalopram of 5 mg/ml. Intraday precision of migration time and peak area ratio were in the range of 0.17–0.44% and 1.64% and 6.25%, respectively. Relative standard deviations of interday precision ranged between 0.84% and 1.85% in the case of migration times and between 5.20% and 9.28% for peak area ratio. The assay was applied to the determination of the purity of escitalopram in bulk drug and tablets. (R)-Citalopram and (S)-citadiol were detected as impurities.     

Profiling of levoamphetamine and related substances in dexamphetamine sulfate by capillary electrophoresis

A capillary electrophoresis method for the simultaneous determination of the enantiomeric purity of dexamphetamine as well as the analysis of 1R,2S-(−)-norephedrine and 1S,2S-(+)-norpseudoephedrine as potential impurities has been developed and validated. Heptakis-(2,3-di-O-acetyl-6-O-sulfo)-β-cyclodextrin was chosen as chiral selector upon a screening of neutral and charged cyclodextrin derivatives. Separation of the analytes was achieved in a fused-silica capillary at 20 °C using an applied voltage of 25 kV. The optimized background electrolyte consisted of a 0.1 M sodium phosphate buffer, pH 2.5, containing 10 mg/ml of the cyclodextrin. The assay was linear in the range of 0.06–5.0% of the impurities based on a concentration of 2.0 mg/ml dexamphetamine sulfate in the sample solution. Analysis of commercial dexamphetamine sulfate samples revealed the presence of 3–4% of levoamphetamine while norephedrine or norpseudoephedrine could not be detected, indicating that the compound was prepared by fractionated crystallization of racemic amphetamine. Comparison with polarimetric measurements indicated that dexamphetamine with an enantiomeric excess as low as 80% still passes the pharmacopeial test of specific rotation while an amount of 0.06% of levoamphetamine can be detected by capillary electrophoresis.     

In vitro antioxidant activity and total phenolic content of ethanolic leaf extract of Stevia rebaudiana Bert.

The aim of this study was to assess the in vitro potential of ethanolic leaf extract of Stevia rebaudiana as a natural antioxidant. The DPPH activity of the extract (20, 40, 50, 100 and 200 μg/ml) was increased in a dose dependent manner, which was found in the range of 36.93–68.76% as compared to ascorbic acid 64.26–82.58%. The IC₅₀ values of ethanolic extract and ascorbic acid in DPPH radical scavenging assay were obtained to be 93.46 and 26.75 μg/ml, respectively. The ethanolic extract was also found to scavenge the superoxide generated by EDTA/NBT system. Measurement of total phenolic content of the ethanolic extract of S. rebaudiana was achieved using Folin–Ciocalteau reagent containing 61.50 mg/g of phenolic content, which was found significantly higher when compared to reference standard gallic acid. The ethanolic extract also inhibited the hydroxyl radical, nitric oxide, superoxide anions with IC₅₀ values of 93.46, 132.05 and 81.08 μg/ml, respectively. However, the IC₅₀ values for the standard ascorbic acid were noted to be 26.75, 66.01 and 71.41 μg/ml respectively. The results obtained in this study clearly indicate that S. rebaudiana has a significant potential to use as a natural antioxidant agent.     

Antioxidant and antidermatophytic activities of essential oil and extracts of Magnolia liliflora Desr.

This study was carried out to assess the antioxidant and antidermatophytic activities of the essential oil and extracts of Magnolia liliflora Desr. Antioxidant activity was evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The free radical scavenging activities of the oil and ethyl acetate extract were found to be superior (IC₅₀ values = 10.11 and 16.17 μg/ml, respectively) as compared to butylatedhydreoxyanisole (BHA), (IC₅₀ value = 18.27 μg/ml). Also the ethyl acetate extract revealed the highest phenolic contents (96.13 mg/g of dry wt) as compared to the other extracts. Further, the oil (1000 μg/disc) and extracts (1500 μg/disc) revealed 42.36–63.12% and 19.07–54.14% antidermatophytic effect, respectively along with their respective MIC values ranging from 62.5 to 500 and 250 to 2000 μg/ml against the members of Trichophyton and Microsporum spp. Also the oil had strong detrimental effect on spore germination of tested fungal pathogens as well as concentration and time dependent kinetic inhibition of Microsporum canis KCTC 6348. The results of this study justify a potential role of M. liliflora to serve as a natural antioxidant and antidermatophytic agent.     

Validated bioanalytical method for the quantification of RGB-286638, a novel multi-targeted protein kinase inhibitor, in human plasma and urine by liquid chromatography/tandem triple-quadrupole mass spectrometry

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the quantitative determination of RBG-286638, a novel multi-targeted protein kinase inhibitor, in 200 μl aliquots of human potassium EDTA plasma with deuterated RGB-286638 as internal standard. The sample extraction and cleaning-up involved a simple liquid–liquid extraction with 100 μl aliquots of acetonitrile and 1 ml aliquots of n-butylchloride. Urine was accurately 5- and 10-fold diluted in blank plasma prior to extraction. Chromatographic separations were achieved on a reversed phase C₁₈ column eluted at a flow-rate of 0.250 ml/min on a gradient of 0.2 mM ammonium formate and acetonitrile both acidified with 0.1% formic acid. The overall cycle time of the method was 7 min, with RGB-286638 eluting at 1.9 min. The multiple reaction monitoring transitions were set at 546 > 402 (m/z), and 549 > 402 (m/z) for RGB-286638 and the internal standard, respectively. The calibration curves were linear over the range of 2.00 to 1000 ng/ml with the lower limit of quantitation validated at 2.00 ng/ml. The within-run and between-run precisions were within 7.90%, while the accuracy ranged from 92.2% to 99.7%. The method was successfully applied to samples derived from a clinical study.     

Development and validation of a high-performance liquid chromatographic method for determination of pinocembrin in rat plasma: Application to pharmacokinetic study

A sensitive and specific reversed-phase high-performance liquid chromatography with ultraviolet detection (RP-UV-HPLC) method has been developed and validated for the identification and quantification of pinocembrin in rat plasma using chrysin as the internal standard. Following protein precipitation with acetonitrile, the analytes were separated by the mobile phase 0.01 M ammonium acetate (pH 4.0)–methanol (35:65, v/v) with an Agilent TC-C18 column (5 μm, 4.6 mm × 150 mm) at a flow rate of 1 ml/min, column temperature 40 °C and detection wavelength 290 nm. A good linear relationship was obtained in the concentration range studied (0.07–133.33 μg/ml, r = 0.9995). The lowest limit of quantification (LLOQ) was 66.7 ng/ml and the lowest limit of detection (LLOD) was 25 ng/ml. Average recoveries ranged from 93.9 to 97.8% in plasma at the concentrations of 0.33 and 33.33 μg/ml. Intra- and inter-batch relative standard deviations were 0.15–2.03 and 1.18–9.96%, respectively. This method was successfully applied to the pharmacokinetic studies in rats after intravenous administration of pinocembrin.     

Impurity profile study of lopinavir and validation of HPLC method for the determination of related substances in lopinavir drug substance

Several related substances (RS4–RS10) were detected in lopinavir drug substance at levels ranging from 0.03% to 0.1% by employing gradient RP-HPLC. The related substances were identified by LC–MS analysis. These related substances were isolated and characterized by Mass, ¹H NMR and FT-IR spectral data. The separation was achieved on a YMC Pack ODS-AQ (250 mm × 4.6 mm, 5 μm) column thermostated at 45 °C using 0.02 M KH₂PO₄ (pH 2.5): acetonitrile as a mobile phase in gradient elution mode. A PDA detector set at 210 nm was used for detection. The investigated validation elements showed the method has acceptable specificity, accuracy, linearity, precision, robustness and high sensitivity with detection limits and quantitation limits ranging from 0.028 μg/ml to 0.063 μg/ml and 0.084 μg/ml to 0.192 μg/ml respectively. The method can be used for routine quality control analysis and stability testing of lopinavir drug substance.     

A sensitive LC–MS/MS method to quantify loureirin B in rat plasma with application to preclinical pharmacokinetic studies

A novel method for the quantification of loureirin B in rat plasma using high-performance liquid chromatography/tandem mass spectrometry (LC–MS/MS) was developed. Loureirin B and internal standard (buspirone) were extracted by liquid–liquid extraction and separated on a Agilent XDB C₁₈ column (50 mm × 4.6 mm, 5 μm). As mobile phase a binary mixture of methanol (containing 0.1% formic acid)–water (containing 0.1% formic acid) was delivered by a Shimadzu LC-20AD pump in gradient mode at a flow rate of 0.4 ml/min in a run time of 5.0 min. The detector was a Q-trap™ mass spectrometer with an electrospray ionization (ESI) interface operating in the multiple reaction monitoring (MRM) mode. The calibration curve of loureirin B in plasma showed good linearity over the concentration range of 0.08–100 ng/ml. The limit of detection and limit of quantification were 0.03 ng/ml and 0.08 ng/ml, respectively. Intra- and inter-day precisions (as relative standard deviation) in all samples were both within 15%. The validated method was successfully applied to a preliminary pharmacokinetic study of loureirin B in rats. After oral administration of 16 g/kg longxuejie to rats, the main pharmacokinetic parameters t_max, C_max, t_1/2, K_e and AUC_0–T were 0.8 h, 7.99 μg/l, 1.94 l h, 0.365/h, and 22.21 μg h/l, respectively.     

Ultra-performance liquid chromatography-tandem mass spectrometry for the determination of lacidipine in human plasma and its application in a pharmacokinetic study

A selective, rapid and sensitive ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–ESI-MS/MS) method was developed and validated for the quantification of lacidipine in human plasma. With nifedipine as an internal standard, sample pretreatment involved a simple liquid–liquid extraction with tert-butyl methyl ether of 1 ml plasma. The analysis was carried out on an Acquity™ UPLC BEH C₁₈ column (50 mm × 2.1 mm, 1.7 μm) with flow rate of 0.28 ml/min. The mobile phase was 30 mM ammonium acetate buffer–acetonitrile (18:82, v/v, pH 5.5). The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI). Linear calibration curves were obtained in the concentration range of 0.025–10.000 ng/ml, with a lower limit of quantification of 0.025 ng/ml. The intra- and inter-day precision (R.S.D.) values were below 15% and accuracy (RE) was −12.7% to 11.9% at all QC levels. The method was successfully applied to a clinical pharmacokinetic study of lacidipine in healthy volunteers following oral administration.     

Separation and determination of coumarins in Fructus cnidii extracts by pressurized capillary electrochromatography using a packed column with a monolithic outlet frit

The pressurized capillary electrochromatography (pCEC) was utilized for the separation and determination of coumarins in Fructus cnidii extracts from 12 different regions. After a thorough study of analytical parameters such as acetonitrile content of the mobile phase, the concentration and pH of the buffer, and the applied voltage, a methodology was proposed to separate and determine six coumarins of F. cnidii extracts in less than 15 min. The experiments were performed in an in-house packed column with a monolithic outlet frit under the optimal conditions: pH 4.0 ammonium acetate buffer at 10 mM containing 50% acetonitrile at −6 kV applied voltage. The calibration curves were linear in the range of 10.0–100.0 μg/mL for bergapten, 20.0–200.0 μg/mL for imperatorin, 5.0–400.0 μg/mL for osthole, 10.0–100.0 μg/mL for 2′-acetylangelicin, 10.0–200.0 μg/mL for oroselone, and 10.0–200.0 μg/mL for O-acetylcolumbianetin. The correlation coefficients were between 0.9967 and 0.9995. With this pCEC system, fingerprints of F. cnidii extracts were preliminarily established to distinguish three types of coumarins by characteristic peaks, and the quality of various sources of raw materials was evaluated by determining the contents of six coumarins.     

A stability-indicating HPLC assay with diode array detection for the determination of a benzylpenicillin prodrug in aqueous solutions

The aim of this study was to develop a stability-indicating HPLC assay for the determination of penethamate (PNT), an ester prodrug of benzylpenicillin (BP), in aqueous solutions. The method was validated by subjecting PNT to forced decomposition under stress conditions of acid, alkali, water hydrolysis and oxidation. A quenching solution was developed to limit degradation to negligible levels before and during the analysis. Both PNT and BP were simultaneously determined and separated in presence of degradation products on a C₁₈ column using a mobile phase consisting of methanol–acetonitrile–acetate buffer. Different degradation products were formed in the stress conditions. The peak purity indexes of PNT and BP obtained by diode array detection were >0.999, confirming the absence of other co-eluting substances. The assay was linear for both analytes in the concentration range 1–100 μg mL⁻¹. The LOD and LOQ of PNT were 0.03 and 0.09 μg mL⁻¹ respectively. Degradation of PNT followed pseudo-first-order kinetics with t_1/2 of 43.6 min at pH 2.01 and 4.2 min at pH 9.31. In addition, the absence of BP in the acidic solutions of PNT emphasises the futility of monitoring BP to assess the stability of PNT. In conclusion, the assay is rapid and stability-indicating with adequate precision and accuracy, and in conjunction with the quenching solution, can be used for stability studies of PNT with simultaneous quantitation of BP. The degradation studies provide useful information for formulation development of PNT.     

Simultaneous determination of senecionine, adonifoline and their metabolites in rat serum by UPLC–ESIMS and its application in pharmacokinetic studies

A rapid, selective and sensitive ultra-performance liquid chromatography–electrospray ionization mass spectrometry (UPLC–ESIMS) method was firstly developed and validated for the simultaneous determination of two hepatotoxic pyrrolizidine alkaloids (HPAs), senecionine (SEN), adonifoline (ADO), and their N-oxides (SENNOX and ADONOX), the main metabolites in rat serum. The whole analysis was achieved within 4.5 min by gradient elution on an ACQUITY UPLC BEH C18 column (50 mm × 2.1 mm, i.d. 1.7 μm) following a solid phase extraction for serum samples. Good linearity was achieved using weighted (1/x²) least squares linear regression over a 1600-fold dynamic range for SEN and ADO (LLOQ was about 0.006 μg/ml) and 800-fold dynamic range for SENNOX and ADONOX (LLOQ was about 0.012 μg/ml). The R.S.D. of intra- and inter-day precision was below 4.91% and 11.15% respectively, while the R.E. of accuracy was within 4.52%, 6.81%, 2.69%, and 7.12% for SEN, SENNOX, ADO, and ADONOX, respectively. The developed method was successfully applied to the in vivo pharmacokinetic study in rats after intravenous administration of SEN and ADO.     

Effect of P-glycoprotein inhibitor, verapamil, on oral bioavailability and pharmacokinetics of irinotecan in rats

The objective of present investigation was to study the effect of verapamil on the pharmacokinetics of irinotecan in order to evaluate the role of P-glycoprotein (P-gp) in irinotecan disposition. An in vitro study using Caco-2 intestinal cell monolayer was first carried out to determine the effect of verapamil on the function of intestinal P-gp. Verapamil (25 mg/kg) was administered orally 2 h before irinotecan oral (80 mg/kg) or intravenous (20 mg/kg) dosing in female Wistar rats. Plasma and biliary samples were collected at specified time points from control and treated animals to determine irinotecan and its metabolite, SN-38 concentrations. Bi-directional transport and inhibition studies in Caco-2 cells indicated irinotecan to be a P-gp substrate and the function of intestinal P-gp was significantly inhibited in presence of verapamil. After oral irinotecan dosing, the mean area under the plasma concentration–time curve (AUC) was found to be 14.03 ± 2.18 μg h/ml which was increased significantly, i.e. 61.71 ± 15.0 μg h/ml when verapamil was co-administered (P < 0.05). Similarly, the mean maximum plasma concentration of irinotecan increased from 2.93 ± 0.37 μg/ml (without verapamil) to 10.75 ± 1.0 μg/ml (with verapamil) (P < 0.05). There was approximately 4–5-folds increase in apparent bioavailability. On the other hand, the intravenous irinotecan administration with verapamil resulted in small but statistically significant effect on AUC (10.76 ± 2.0 to 23.3 ± 3.8 μg h/ml; P < 0.05) and systemic clearance (1206.4 ± 159.7 to 713.5 ± 78.2 ml/(h kg)). In addition, SN-38 showed significant change in oral pharmacokinetic parameters and minor changes in intravenous pharmacokinetic profile. Biliary excretion curves of both irinotecan and SN-38 were lowered by verapamil. The mean percent of irinotecan excreted into bile over 5 h following intravenous and oral administration was found to be 8% and 1%, respectively, which was further reduced to half when treated with verapamil. These results are quite stimulating for further development of a clinically useful oral formulation of irinotecan based on P-gp inhibition.     

Pharmacological analysis of the neutrophil migration induced by D. rostrata lectin: Involvement of cytokines and nitric oxide

In the present study, we investigated the involvement of resident cell and inflammatory mediators in the neutrophil migration induced by chemotactic activity of a glucose/mannose-specific lectin isolated from Dioclea rostrata seeds (DrosL). Rats were injected i.p. with DrosL (125–1000 μg/cavity), and at 2–96 h thereafter the leukocyte counts in peritoneal fluid were determined. DrosL-induced a dose-dependent neutrophil migration accumulation, which reached maximal response at 24 h after injection and declines thereafter. The carbohydrate ligand nearly abolished the neutrophil influx. Pre-treatment of peritoneal cavities with thioglycolate which increases peritoneal macrophage numbers, enhanced neutrophil migration induced by DrosL by 303%. However, the reduction of peritoneal mast cell numbers by treatment of the cavities with compound 48/80 did not modify DrosL-induced neutrophil migration. The injection into peritoneal cavities of supernatants from macrophage cultures stimulated with DrosL (125, 250 and 500 μg/ml) induced neutrophil migration. In addition, DrosL treatment induced cytokines (TNF-α, IL-1β and CINC-1) and NO release into the peritoneal cavity of rats. Finally, neutrophil chemotaxis assay in vitro showed that the lectin (15 and 31 μg/ml) induced neutrophil chemotaxis by even 180%. In conclusion, neutrophil migration induced by D. rostrata lectin occurs by way of the release of NO and cytokines such as IL-1β, TNF-α and CINC-1.     

A rapid and sensitive liquid chromatography–tandem mass spectrometric method for the determination of timosaponin B-II in blood plasma and a study of the pharmacokinetics of saponin in the rat

A rapid, sensitive and selective liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and validated for the determination of Timosaponin B-II (TB-II), a pharmacologically active constituent isolated from Anemarrhena asphodeloides. This method was used to examine the pharmacokinetics and bioavailability of TB-II in rats using ginsenoside Re as an internal standard. After simple protein precipitation of the plasma samples with acetonitrile, the analytes were separated on an ODS column (150 mm × 2.1 mm i.d., 5 μm) with the mobile phase of acetonitrile–water (35:65, v/v) containing 0.05% formic acid and detected by electrospray ionization mass spectrometry in the negative multiple reaction monitoring (MRM) mode with a chromatographic run time of 3.0 min. The calibration curves were linear over the range of 5–15,000 ng/ml and the lower limit of quantification (LLOQ) was 5 ng/ml in rat plasma. In this range, relative standard deviations (R.S.D.) were <7.4% for intra-day precision and <9.0% for inter-day precision. The accuracy was within the range of 97.7–107.3%. The method was successfully applied to assess the pharmacokinetics and oral bioavailability of TB-II after intravenous and oral administration in rats, with the oral bioavailability being only 1.1%.     

Virucidal activity of polysaccharide extracts from four algal species against herpes simplex virus

Herpes simplex virus types 1 and 2 (HSV-1, HSV-2) infections are common, but can cause serious infections in neonates and the immunocompromised. Drugs currently used to treat cutaneous or genital HSV infections are effective in limiting disease, but the emergence of drug resistant viruses in immunocompromised individuals can be problematic. While the prophylactic oral treatment with antiviral drugs can reduce virus shedding and transmission, there is a need for topical microbicides that have the potential to limit sexual transmission of the virus. Previous reports demonstrated the antiviral activity of complex sulfated polysaccharides extracted from various species of marine algae and suggested that they interfered with the attachment of virions to host cells. Here, we evaluated the antiviral activity of extracts from Undaria pinnatifida, Splachnidium rugosum, Gigartina atropurpurea, and Plocamium cartilagineum against HSV-1 and HSV-2. These extracts exhibited good activity when added during the first hour of viral infection, but were ineffective if added later. Plaque reduction assays, when the extracts were added prior to viral inoculation, yielded EC₅₀ values that ranged from 2.5–3.6 μg/ml for HSV-1 and 0.7–6.6 μg/ml for HSV-2. None of the extracts exhibited significant toxicity in a neutral red uptake assay (IC₅₀ >100 μg/ml). Subsequent assays showed that the compounds had potent virucidal activity and were active at very low concentrations. We conclude that these extracts are nontoxic and effective virucidal agents that warrant further investigation to examine their potential role in the prevention of HSV infections of humans.     

Optimization of a method for determination of phenolic acids in exotic fruits by capillary electrophoresis

In this work, the separation of nine phenolic acids (benzoic, caffeic, chlorogenic, p-coumaric, ferulic, gallic, protocatechuic, syringic, and vanillic acid) was approached by a 3² factorial design in electrolytes consisting of sodium tetraborate buffer (STB) in the concentration range of 10–50 mmol L⁻¹ and methanol in the volume percentage of 5–20%. Derringer's desirability functions combined globally were tested as response functions. An optimal electrolyte composed by 50 mmol L⁻¹ tetraborate buffer at pH 9.2, and 7.5% (v/v) methanol allowed baseline resolution of all phenolic acids under investigation in less than 15 min. In order to promote sample clean up, to preconcentrate the phenolic fraction and to release esterified phenolic acids from the fruit matrix, elaborate liquid–liquid extraction procedures followed by alkaline hydrolysis were performed. The proposed methodology was fully validated (linearity from 10.0 to 100 μg mL⁻¹, R² > 0.999; LOD and LOQ from 1.32 to 3.80 μg mL⁻¹ and from 4.01 to 11.5 μg mL⁻¹, respectively; intra-day precision better than 2.8% CV for migration time and 5.4% CV for peak area; inter-day precision better than 4.8% CV for migration time and 4.8–11% CV for peak area; recoveries from 81% to 115%) and applied successfully to the evaluation of phenolic contents of abiu-roxo (Chrysophyllum caimito), wild mulberry growing in Brazil (Morus nigra L.) and tree tomato (Cyphomandra betacea). Values in the range of 1.50–47.3 μg g⁻¹ were found, with smaller amounts occurring as free phenolic acids.     

Determination of ranolazine in human plasma by LC-MS/MS and its application in bioequivalence study

A simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for quantification of ranolazine in human plasma. The analytical method consists in the precipitation of plasma sample with methanol, followed by the determination of ranolazine by an LC–MS/MS. The analyte was separated on a Peerless Cyano column (33 mm × 4.6 mm, 3 μm) an isocratic mobile phase of methanol–water containing formic acid (1.0%, v/v) (65:35, v/v) at a flow rate of 1.0 ml/min. Protonated ions formed by a turbo ionspray in positive mode were used to detect analyte and internal standard (IS). The MS/MS detection was made by monitoring the fragmentation of m/z 428.20 → 279.50 for ranolazine and m/z 448.30 → 285.20 for internal standard on a triple quadrupole mass spectrometer. The method was validated over the concentration range of 5–2000 ng/ml for ranolazine in human plasma with correlation coefficient of 0.9937 (S.D.: ±0.00367, range: 0.9895–0.9963). The accuracy and precision values obtained from six different sets of quality control samples analyzed in separate occasions ranged from 94.53 to 117.86 and 0.14% to 4.56%, respectively. Mean extraction recovery was 82.36–94.25% for three quality control (QC) samples and 88.37% for IS. Plasma samples were stable for three freeze–thaw cycles, or 24 h ambient storage, or 1 and 3 months storage at −20 °C. Processed samples (ready for injection) were stable up to 72 h at autosampler (4 °C). The developed method was successfully applied for analyzing ranolazine in plasma samples for a bioequivalence study with 12 healthy volunteers.     

Antioxidant and antilisterial effect of seed essential oil and organic extracts from Zizyphus jujuba

Hydrodistilled volatile oil from the seeds of Zizyphus jujuba was analyzed by GC–MS. Twenty three compounds representing 91.59% of the total oil was identified. The oil and organic extracts revealed a great potential of antilisterial effect against all five strains of Listeria monocytogenes ATCC 19111, 19116, 19118, 19166 and 15313. Also the oil had strong detrimental effect on the viable count of the tested bacteria. The samples were also subjected to screening for the antioxidant activity by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radicals scavenging activities assay. In the first case, the IC₅₀ value of the Z. jujuba essential oil was determined to be 5.21 ± 0.01 μg/ml. Among the extracts, the strongest activity was exhibited by the methanol extract with an IC₅₀ value of 20.44 ± 0.18 μg/ml. In the superoxide radicals scavenging activities assay, methanol extract was superior to all other extracts (IC₅₀ = 18.60 ± 0.3 μg/ml). Furthermore, the amount of total phenolic compounds was determined. The results indicate that the essential oil and extracts of Z. jujuba could serve as natural antimicrobial and antioxidant agents for the food industry.     

In vitro analyses of the combination of high-dose doxycycline and antifungal agents against Candida albicans biofilms

The potential of antifungal agents used as antimicrobial lock therapy (ALT) for the conservative management of catheter-related candidemia has not been fully defined. We sought to determine the antifungal effect of high-dose doxycycline (DOX), alone or in combination with standard concentrations of amphotericin B (AMB), caspofungin (CAS) or fluconazole (FLC), against biofilms formed by Candida albicans in vitro. DOX alone (at 2048 μg/mL and 1024 μg/mL) demonstrated up to an 85% reduction of the metabolic activity of the C. albicans biofilm. Regardless of the concentration tested, FLC alone showed minimal activity (mean 22.9% reduction) against the C. albicans biofilm. When DOX 2048 μg/mL was used in combination with FLC, antifungal activity also increased up to 85%, suggesting an additive effect. DOX 128 μg/mL in combination with FLC demonstrated synergy (mean 58.3% reduction). The combination of DOX 2048 μg/mL or 512 μg/mL and AMB was superior to AMB alone at low concentrations (0.25–0.03125 μg/mL). However, DOX 128 μg/mL was antagonistic in combination with low concentrations of AMB. Maximal efficacy against the biofilm was observed with CAS at 8–0.25 μg/mL compared with FLC and AMB alone. A paradoxical effect (PE) occurred with CAS at 16 μg/mL, which showed a marked reduction in antifungal activity compared with lower concentrations of CAS. CAS at 16 μg/mL in combination with either DOX 2048 μg/mL or 512 μg/mL resulted in attenuation of the PE. These findings suggest that a high-dose DOX-based ALT strategy in combination with traditional antifungal agents may be useful for the treatment of C. albicans biofilms.