A sensitive HPLC method was developed for the quantitative determination of isoliquiritin apioside (ILA) and isoliquiritin (IL) in rat plasma. After protein precipitation with acetonitrile, chloroform was used to separate lipid-soluble impurities from the plasma samples and remove acetonitrile. A chromatography was carried out on Diamonsil C18 (150 × 4.6 mm; 5 μm) analytical column, using a mobile phase consisting of water (containing phosphoric acid 0.1%, v/v); acetonitrile (72 : 28, v/v) at a flow rate of 1.0 mL/min. The wavelength-switching technology was performed to determine ILA and IL at 360 nm and wogonoside (internal standard, IS) at 276 nm. The calibration curves of ILA and IL were fairly linear over the concentration ranges of 0.060–3.84 μg/mL (r = 0.9954) and 0.075–4.80 μg/mL (r = 0.9968), respectively. The average extract recoveries of ILA, IL, and IS were all over 80%. The precision and accuracy for all concentrations of quality controls and standards were within 15%. The lower limit of quantification (LLOQ) was 0.060 μg/mL for ILA and 0.075 μg/mL for IL. The method was used in pharmacokinetic study after an oral administration of Zhigancao extract to rats.
Background and the purpose of the study
To develop a simple, rapid and accurate HPLC method for the measurement of the venlafaxine and its main metabolites, O-desmethylvenlafaxine and O,N-didesmethylvenlafaxine in pharmacokinetic studies and therapeutic drug monitoring.
Chromatographic separation was achieved with a ChromolithTM Performance RP-18e 100 mm×4.6 mm column equipped with a Fluorescence detectore (λex 200 nm/λem 300 nm) The mobile phase of methanol:water (35:65, v/v) adjusted to pH 2.5 by phosphoric acid was passed through the column in an isocratic mode at flow rate of 2 ml/min. The sample preparation involved a simple, one-step, extraction with ethyl acetate.
The calibration curves were linear in the concentration range of 1-300 ng/ml for all analytes (r2>0.998). The lower limit of quantification was 1 ng/ml for all analytes. Within and between day precisions in the measurement of quality control (QC) of samples were in the range of 1.8-14.1% for all analytes.
The developed procedure was used to assess the pharmacokinetics of venlafaxine and its main metabolites following oral administration of 75 mg venlafaxine to a healthy subject.
Venlafaxine; Metabolite; Pharmacokinetics; HPLC
Clinical pharmacokinetic studies of ciprofloxacin require accurate and precise measurement of plasma drug concentrations. We describe a rapid, selective and sensitive HPLC method coupled with fluorescence detection for determination of ciprofloxacin in human plasma. Internal standard (IS; sarafloxacin) was added to plasma aliquots (200 μL) prior to protein precipitation with acetonitrile. Ciprofloxacin and IS were eluted on a Synergi Max-RP analytical column (150 mm × 4.6 mm i.d., 5 μm particle size) maintained at 40 °C. The mobile phase comprised a mixture of aqueous orthophosphoric acid (0.025 M)/methanol/acetonitrile (75/13/12%, v/v/v); the pH was adjusted to 3.0 with triethylamine. A fluorescence detector (excitation/emission wavelength of 278/450 nm) was used. Retention times for ciprofloxacin and IS were approximately 3.6 and 7.0 min, respectively. Calibration curves of ciprofloxacin were linear over the concentration range of 0.02–4 μg/mL, with correlation coefficients (r2) ≥ 0.998. Intra- and inter-assay relative standard deviations (SD) were <8.0% and accuracy values ranged from 93% to 105% for quality control samples (0.2, 1.8 and 3.6 μg/mL). The mean (SD) extraction recoveries for ciprofloxacin from spiked plasma at 0.08, 1.8 and 3.6 μg/mL were 72.8 ± 12.5% (n = 5), 83.5 ± 5.2% and 77.7 ± 2.0%, respectively (n = 8 in both cases). The recovery for IS was 94.5 ± 7.9% (n = 15). The limits of detection and quantification were 10 ng/mL and 20 ng/mL, respectively. Ciprofloxacin was stable in plasma for at least one month when stored at −15 °C to −25 °C and −70 °C to −90 °C. This method was successfully applied to measure plasma ciprofloxacin concentrations in a population pharmacokinetics study of ciprofloxacin in malnourished children.
Ciprofloxacin; HPLC fluorescence detection; Plasma; Protein precipitation; Validation
A strategy for analyzing flavone C-glucosides in the leaves of different species of bamboo was developed. Firstly, the flavone C-glycosides were extracted from the bamboo leaves (51 species in 17 genera) with methanol and chromatographed on silica gel 60 plates in automatic developing chamber (ADC2), and a qualitative survey using simple derivatization steps with the NP reagent was carried out. The flavone C-glycosides were found in 40 of 51 species of bamboo examined. Secondly, an HPLC method with photodiode array and multiple wavelength detector was optimized and validated for the simultaneous determination of flavone C-glycosides, including isoorientin, isovitexin, orientin, and vitexin in the leaves of three species of bamboo and the flavone C-glycosides were confirmed by LC/MS. The optimized HPLC method proved to be linear in the concentration range tested (0.2–100 μg/mL, r2 ≥ 0.9997), precise (RSD ≤ 1.56%), and accurate (88–106%). The concentration ranges of isoorientin, isovitexin, orientin, and vitexin in three bamboo leaves samples were 1.00–2.78, 0–0.31, 0–0.07, and 0.20–0.68 mg/g, respectively. The proposed method was validated to be simple and reliable and can be a tool for quality control of bamboo leaf extract or its commercial products.
Praeruptorin D (PD), a major pyranocoumarin isolated from Radix Peucedani, exhibited antitumor and anti-inflammatory activities. The aim of this study was to investigate the pharmacokinetics and tissue distribution of PD in rats following intravenous (i.v.) administration. The levels of PD in plasma and tissues were measured by a simple and sensitive reversed-phase high-performance liquid chromatography (HPLC) method. The biosamples were treated by liquid-liquid extraction (LLE) with methyl tert-butyl ether (MTBE) and osthole was used as the internal standard (IS). The chromatographic separation was accomplished on a reversed-phase C18 column using methanol-water (75:25, v/v) as mobile phase at a flow rate of 0.8 mL/min and ultraviolet detection wave length was set at 323 nm. The results demonstrate that this method has excellent specificity, linearity, precision, accuracy and recovery. The pharmacokinetic study found that PD fitted well into a two-compartment model with a fast distribution phase and a relative slow elimination phase. Tissue distribution showed that the highest concentration was observed in the lung, followed by heart, liver and kidney. Furthermore, PD can also be detected in the brain, which indicated that PD could cross the blood-brain barrier after i.v. administration.
praeruptorin D; Radix Peucedani; HPLC; pharmacokinetics; tissue distribution
A simple, sensitive, and reproducible reversed-phase high-performance liquid chromatography (RP-HPLC) method, coupled with a photodiode array detector, was developed for the determination of rupatadine (RUPA) and its related substances in pharmaceutical dosage forms. Chromatographic separation was achieved on the Hypersil BDS (150 x 4.6 mm, 5 μm) column with a mobile phase containing a gradient mixture of a buffer (acetate buffer pH-6.0) and solvent (methanol). The eluted compounds were monitored at 264 nm for the related substances and assay, the flow rate was 1.0 mL/min, and the column oven temperature was maintained at 50°C. The developed method separated RUPA from its four known and three unknown impurities within 15.0 min. Rupatadine was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degradation. Rupatadine was found to degrade significantly under oxidative stress conditions, and degrade slightly under acid, base, hydrolytic, thermal, and photolytic stress conditions. All impurities were well-resolved from each other and from the main peak, showing the stability-indicating power of the method. The developed method was validated as per the International Conference on Harmonization (ICH) guidelines. The developed and validated RP-HPLC method is LC-MS compatible and can be explored for the identification of eluted unknown impurities of RUPA.
Rupafin; Method validation; Forced degradation; Assay; Related substances; Chromatography; Impurities
Inorganic tin (SnCl4·H2O) is toxic to microbial populations obtained from estuarine sediments plated on nutrient medium solidified with either agar or purified agar. The use of gelatin as a gelling agent decreased the apparent toxicity of tin, and toxicity was markedly reduced in medium solidified with silica gel. There was no evidence that toxic agar-tin complexes were involved. Cd, Cu, Pb, Ni, and Zn exhibited similar toxicity patterns; therefore, toxicity levels determined in the laboratory should be extrapolated to the environment with caution. The addition of cysteine to the medium had no effect on tin toxicity. Serine or 3-hydroxyflavone enhanced toxicity, while humic acids or gelatin inhibited toxicity. Replacement of SO42− with NO3− did not alter tin toxicity, but replacement of Cl− with NO3− decreased tin toxicity. Thus, the toxic effect(s) of tin depend as much on the chemical speciation of the metal as on the total concentration of the metal in the medium.
This paper deals with development and validation of a high performance liquid chromatographic method for the quantitative determination of disodium EDTA (Ethylenediaminetetraacetic acid) in Meropenem active pharmaceutical ingredient (API). EDTA was derivatized with Ferric chloride solution by heating at 70 °C in water bath for about 20 minutes and the chromatographic separation achieved by injecting 100 μL of the derivatized mixture into a Waters HPLC system with photodiode array detector using a Phenomenex Luna C18(2) column (250 × 4.6 mm), 5 μ. The mobile phase consisting of 5% methanol and 95% of 0.7 g/L solution of Tetra butyl ammonium bromide and 4.6 g/L solution of sodium acetate trihydrate in water (pH adjusted to 4.0 with the help of acetic acid glacial) and a flow rate of 1 milliliter/minute. EDTA eluted at approximately 6 minutes. The method was suitably validated with respect to specificity, linearity of response, precision, accuracy, ruggedness, stability in analytical solution, limit of quantitation and detection and robustness for its intended use.
EDTA; HPLC; precolumn derivatization; Meropenem; ferric chloride
A reliable and sensitive isocratic stability indicating RP-HPLC method has been developed and validated for assay of rosuvastatin calcium in tablets and for determination of content uniformity. An isocratic separation of rosuvastatin calcium was achieved on YMC C8, 150×4.6 mm i.d., 5 μm particle size columns with a flow rate of 1.5 ml/min and using a photodiode array detector to monitor the eluate at 242 nm. The mobile phase consisted of acetonitrile: water (40:60, v/v) pH 3.5 adjusted with phosphoric acid. The drug was subjected to oxidation, hydrolysis, photolysis and thermal degradation. All degradation products in an overall analytical run time of approximately 10 min with the parent compound rosuvastatin eluting at approximately 5.2 min. Response was a linear function of drug concentration in the range of 0.5-80 μg/ml (r2= 0.9993) with a limit of detection and quantification of 0.1 and 0.5 μg/ml respectively. Accuracy (recovery) was between 99.6 and 101.7%. Degradation products resulting from the stress studies did not interfere with the detection of rosuvastatin and the assay is thus stability-indicating.
Content uniformity; drug formulation; HPLC-PDA; method validation; Rosuvastatin calcium; stability- indicating
A rapid and specific high-performance liquid chromatography method with UV detection (HPLC-UV) for the simultaneous determination of 12 beta-lactam antibiotics (amoxicillin, cefepime, cefotaxime, ceftazidime, ceftriaxone, cloxacillin, imipenem, meropenem, oxacillin, penicillin G, piperacillin, and ticarcillin) in small samples of human plasma is described. Extraction consisted of protein precipitation by acetonitrile. An Atlantis T3 analytical column with a linear gradient of acetonitrile and a pH 2 phosphoric acid solution was used for separation. Wavelength photodiode array detection was set either at 210 nm, 230 nm, or 298 nm according to the compound. This method is accurate and reproducible (coefficient of variation [CV] < 8%), allowing quantification of beta-lactam plasma levels from 5 to 250 μg/ml without interference with other common drugs. This technique is easy to use in routine therapeutic drug monitoring of beta-lactam antibiotics.
Hydroxypropyl-sulfobutyl-β-cyclodextrin (HP-SBE-β-CD) inclusion complex was developed and used as a drug delivery system for DTX (DTX/HP-SBE-β-CD). The objective of the present study was to evaluate and compare the biological properties of DTX/HP-SBE-Β-CD with Taxotere®. The pharmacokinetics, biodistribution, antitumor efficacy in vivo and in vitro, and safety evaluation of DTX/HP-SBE-β-CD were studied. The most significant finding was that it was possible to prepare a Polysorbate-80-free inclusion complex for DTX. Studies based on pharmacokinetics, biodistribution, and antitumor efficacy indicated that DTX/HP-SBE-β-CD had similar pharmacokinetic properties and antitumor efficacy both in vitro and in vivo as Taxotere®. Fortunately, this new drug delivery system attenuated the side effects when used in vivo. As a consequence, DTX/HP-SBE-β-CD may be a promising alternative to Taxotere® for cancer chemotherapy treatment with reduced side effects. The therapeutic potential against a variety of human tumors and low toxicity demonstrated in a stringent study clearly warrant clinical investigation of DTX/HP-SBE-β-CD for possible use against human tumors.
antitumor efficacy; biodistribution; DTX/HP-SBE-β-CD; pharmacokinetics; safety evaluation
A simple, rapid, and stability-indicating reverse-phase liquid chromatographic assay method was developed for Anagrelide Hydrochloride (ANG) in the presence of its degradation products generated from forced decomposition studies. The HPLC separation was achieved on a C18 Inertsil column (250 mm × 4.6 mm i.d. particle size is 5 μm), using solution A, a mixture of 0.03 M potassium di-hydrogen phosphate pH-adjusted to 3.0 using ortho-phosphoric acid (buffer): methanol: acetonitrile (90:5:5, v/v/v), and solution B, which contains a mixture of buffer: acetonitrile (10:90, v/v). The UV detector was operated at 251 nm while column temperature was maintained at 40°C, and the gradient program had the flow rate of 1.0 mL min−1. The developed method was validated as per ICH guidelines with respect to specificity, linearity, precision, accuracy, robustness, and limit of quantification. The method was found to be simple, specific, precise, accurate, and reproducible. Selectivity was validated by subjecting the stock solution of ANG to acidic, basic, photolysis, oxidative, and thermal degradation. The calibration curve was found to be linear in the concentration range of 0.05–152 μg mL−1 (R2 = 0.9991). The peaks of degradation products did not interfere with that of pure ANG. The utility of the developed method was examined by analyzing the tablets containing ANG.
Anagrelide; Stability-indicating; Reverse phase; Validation; Forced degradation
Telomeres are coated by shelterin, a six-subunit complex that is required for protection and replication of chromosome ends. The central subunit TIN2, with binding sites to three subunits (TRF1, TRF2, and TPP1), is essential for stability and function of the complex. Here we show that TIN2 stability is regulated by the E3 ligase Siah2. We demonstrate that TIN2 binds to Siah2 and is ubiquitylated in vivo. We show using purified proteins that Siah2 acts as an E3 ligase to directly ubiquitylate TIN2 in vitro. Depletion of Siah2 led to stabilization of TIN2 protein, indicating that Siah2 regulates TIN2 protein levels in vivo. Overexpression of Siah2 in human cells led to loss of TIN2 at telomeres that was dependent on the presence of the catalytic RING domain of Siah2. In contrast to RNAi-mediated depletion of TIN2 that led to loss of TRF1 and TRF2 at telomeres, Siah2-mediated depletion of TIN2 allowed TRF1 and TRF2 to remain on telomeres, indicating a different fate for shelterin subunits when TIN2 is depleted posttranslationally. TPP1 was lost from telomeres, although its protein level was not reduced. We speculate that Siah2-mediated removal of TIN2 may allow dynamic remodeling of the shelterin complex and its associated factors during the cell cycle.
The title compound, [Sn(C6H5)2(C12H11N4S2)Cl]·CH4O, is formed during the reaction between 2-acetylthiazole 4-phenylthiosemicarbazone (Hacthptsc) and diphenyltin(IV) dichloride in methanol. In the crystal structure, the Sn atom exhibits an octahedral geometry with the [N2S] anionic tridentate thiosemicarbazone ligand having chloride trans to the central N and the two phenyl groups trans to each other. The Sn—Cl distance is 2.5929 (6), Sn—S is 2.4896 (6) and Sn—N to the central N is 2.3220 (16) Å. The MeOH molecules link the Sn complexes into one-dimensional chains via N—H⋯O and O—H⋯Cl hydrogen bonds.
A simple, precise and accurate HPLC method has been developed and validated for assay of lercanidipine hydrochloride in tablets and for determination of content uniformity. An isocratic separation was achieved using a Chromasil YMC Pack C8, 150 × 4.6 mm i.d., 5µm particle size columns with a flow rate of 1 ml/min and using a UV detector to monitor the elute at 240 nm. The mobile phase consisted of 0.02 M ammonium dihydrogen phosphate buffer:methanol (35:65, v/v) with pH 3.5 adjusted with phosphoric acid. The method was validated for specificity, linearity, pre-cision, accuracy, robustness and solution stability. The specificity of the method was deter-mined by assessing interference from the placebo and by stress testing of the drug (forced degradation). The method was linear over the concentration range of 20-80 µg/ml (r2= 0.9992) with a limit of detection and quantitation of 0.1 and 0.3 µg/ml respectively. Intraday and interday system and method precision were determined and accuracy was between 99.3-101.9 %. The method was found to be robust and suitable for assay of lercanidipine hydrochloride in a tablet formulation and for determination of content uniformity. Degradation products resulting from the stress studies did not interfere with the detection of lercanidipine hydrochloride and the assay is thus stability-indicating.
Calcium channel blocker; column liquid chromatography; degradation; stability- indicating; lercanidipine hydrochloride
Attention has shifted from the treatment of colorectal cancer (CRC) to chemoprevention using aspirin and folic acid as agents capable of preventing the onset of colon cancer. However, no sensitive analytical method exists to simultaneously quantify the two drugs when released from polymer-based nanoparticles. Thus, a rapid, highly sensitive method of high performance liquid chromatography (HPLC) analysis to simultaneously detect low quantities of aspirin (hydrolyzed to salicylic acid, the active moiety) and folic acid released from biodegradable polylactide-co-glycolide (PLGA) copolymer nanoparticles was developed. Analysis was done on a reversed phase C18 column using a photodiode array (PDA) detector at wavelengths of 233 nm (salicylic acid) and 277 nm (folic acid). The mobile phase consisted of acetonitrile − 0.1% trifluoroacetic acid mixture programmed for a 30 min gradient elution analysis. In the range of 0.1 – 100 μg/mL, the assay showed good linearity for salicylic acid (R2 = 0.9996) and folic acid (R2 = 0.9998). The method demonstrated good reproducibility, intra- and inter-day precision and accuracy (99.67, 100.1%) and low values of detection (0.03, 0.01 μg/mL) and quantitation (0.1 and 0.05 μg/mL) for salicylic acid and folic acid, respectively. The suitability of the method was demonstrated by simultaneously determining salicylic acid and folic acid released from PLGA nanoparticles.
aspirin; salicylic acid; folic acid; simultaneous detection; nano-particulate systems; HPLC
A procedure for simultaneous identification and quantification of canrenone and its biotransformed product
11-α-hydroxy-canrenone by high-performance liquid chromatography with ultraviolet detector (HPLC-UVD) and mass spectrometry (LC-MS)
methods was proposed. The optimal determination variables on the HPLC-UVD or LC-MS coupled with a ZORBAX Eclipse XDB-C18 column
(150 mm × 4.6 mm, 5 μm) were set as follows: detection wavelength of 280 nm, mobile phase of water and methanol
gradient elution, temperature for the chromatographic column of 30°C, flow rate of mobile phase of 0.8 mL/min, sample injection volume of
5 μL, and elution time of 40 min. The MS conditions were set as follows: the flow rate
of sheath gas, aux gas, and sweep gas were kept at 35 arb, 5 arb, and 0 arb, respectively. The temperature of capillary was held at
300°C, and capillary voltage was set at 30.00 V. Tube lens were performed at 100.00 V. The proposed method was validated
by linearity (r2 ≥ 0.9910), average recovery (94.93%, RSD1.21%), precision (RSD ≤ 1.31%), limit of detection, and limit of quantification (LOD 0.1~0.12 mg/L, LOQ 0.5~0.67 mg/L), which proved to be affordable for simultaneously determining canrenone and its bio-transformed product 11-α-hydroxy-canrenone.
Telomeres are maintained by three DNA-binding proteins (telomeric repeat binding factor 1 [TRF1], TRF2, and protector of telomeres 1 [POT1]) and several associated factors. One factor, TRF1-interacting protein 2 (TIN2), binds TRF1 and TRF2 directly and POT1 indirectly. Along with two other proteins, TPP1 and hRap1, these form a soluble complex that may be the core telomere maintenance complex. It is not clear whether subcomplexes also exist in vivo. We provide evidence for two TIN2 subcomplexes with distinct functions in human cells. We isolated these two TIN2 subcomplexes from nuclear lysates of unperturbed cells and cells expressing TIN2 mutants TIN2-13 and TIN2-15C, which cannot bind TRF2 or TRF1, respectively. In cells with wild-type p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere uncapping and eventual growth arrest. In cells lacking p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere dysfunction and cell death. Our findings suggest that distinct TIN2 complexes exist and that TIN2-15C–sensitive subcomplexes are particularly important for cell survival in the absence of functional p53.
Individualization of topotecan dosing reduces inter-patient variability in topotecan exposure, presumably reducing toxicity and increasing efficacy. However, logistical limitations (e.g., requirement for plasma, intensive bedside plasma processing) have prevented widespread application of this approach to dosing topotecan. Thus, the objectives of the present study were to develop and validate an HPLC with fluorescence detection method to measure topotecan lactone in whole blood samples and to evaluate its application to individualizing topotecan dosing. Plasma samples (200 μL) were prepared using methanolic precipitation, a filtration step, and then injection of 100 μL of the methanolic extract onto a Novapak® C18, 4μm, 3.9 × 150 mm column with an isocratic mobile phase. Analytes were detected with a Shimadzu Fluorescence RF-10AXL detector with an excitation and emission wavelength of 370 nm and 520 nm, respectively. This method has a lower limit of quantification of 1 ng/mL (S/N ≥5; RSD 4.9%), was validated over a linear range of 1 to 100 ng/mL, and results from a 5-day validation study demonstrated good within-day and between-day precision and accuracy. Data are presented to demonstrate that the present method can be used with whole blood samples to individualize topotecan dosing in children with cancer.
topotecan; HPLC; whole blood; pharmacokinetics
This present paper deals with the development and validation of a stability indicating high performance liquid chromatographic method for the quantitative determination of Memantine hydrochloride. Memantine hydrochloride was derivatized with 0.015 M 9-fluorenylmethyl chloroformate (FMOC) and 0.5 M borate buffer solution by keeping it at room temperature for about 20 minutes and the chromatographic separation achieved by injecting 10 μL of the derivatized mixture into a Waters HPLC system with photodiode array detector using a kromasil C18 column (150 × 4.6 mm), 5 μ. The mobile phase consisting of 80% acetonitrile and 20% phosphate buffer solution and a flow rate of 2 milliliter/minute. The Memantine was eluted at approximately 7.5 minutes. The volume of FMOC used in derivatization, concentration of FMOC and derivatization time was optimized and used. Forced degradation studies were performed on bulk sample of Memantine hydrochloride using acid (5.0 Normal (N) hydrochloric acid), base (1.0 N sodium hydroxide), oxidation (30% hydrogen peroxide), thermal (105°C), photolytic and humidity conditions. The developed LC method was validated with respect to specificity, precision (% RSD about 0.70%), linearity (linearity of range about 70–130 μg/mL), ruggedness (Overall % RSD about 0.35%), stability in analytical solution (Cumulative % RSD about 0.11% after 1450 min.) and robustness.
memantine hydrochloride; HPLC; precolumn derivatization; FMOC; assay
A simple, precise, specific, and accurate reverse phase HPLC method has been developed for the determination of pregabalin in capsule dosage form. The chromatography was set on Hypersil BDS, C8, 150×4.6 mm, 5 μm column using photodiode array detector. The mobile phase consisting of phosphate buffer pH 6.9 and acetonitrile in the ratio of 95:05 with flow rate of 1 ml/min. The method was validated according to ICH guidelines with respect to specificity, linearity, accuracy, precision and robustness. Lower limit of quantification is 0.6 mg/l. The pregabalin sample solution was found to be stable at room temperature for about 26 h.
Method development; pregabalin; RP-HPLC; validation
A high-performance liquid chromatography (HPLC) system was used in the reversed phase mode for the determination of benzalkonium chloride (BKC) in azithromycin viscous ophthalmic drops. A Venusil-XBP(L)-C18 (150 mm×4.6 mm, 5 μm) column was used at 50 °C. The mobile phase consisted of a mixture of methanol-potassium phosphate (16:5, v/v). Two sample preparation methods were compared. The results suggested that, compared with an extraction procedure, a deproteinization procedure was much quicker and more convenient. Using the deproteinization procedure for sample preparation, calibration curves were linear in the range 5.0~50 μg/ml. The within-day and inter-day coefficients of variation were less than 10%. The average recoveries were determined as 96.70%, 98.52%, and 97.96% at concentrations of 10.0, 30.0, and 50.0 μg/ml, respectively. Variability in precision did not exceed 5%. In conclusion, this HPLC method using a simple sample treatment procedure appears suitable for monitoring BKC content in azithromycin viscous ophthalmic drops.
Benzalkonium chloride; High-performance liquid chromatography; Azithromycin; Viscous ophthalmic drops
A new series of unsymmetrical macrocyclic complexes of tin(ll) has been prepared by the template process using bis(3-oxo-2-butylidene)propane-1,3-diamine as precursor. This affords a method to synthesize these complexes with various ring sizes. The tetradentate macrocyclic precursor [N4mL] reacts with SnCl2 and different diamines in a 1:1:1 molar ratio in refluxing methanol to give complexes of the type [Sn(N4mL)Cl2]. The ring expansion has been achieved by varying the diamine between the two diacetyl amino nitrogen atoms. The macrocyclic precursor and its metal complexes have been characterized on the basis of elemental analysis, molar conductance, molecular weight determinations, IR, 1H NMR,13C NMR, 119Sn NMR and electronic spectral studies. An octahedral geometry around the metal ion is suggested for these complexes. On the basis of molecular weights and conductivity measurements, their monomeric and non-electrolytic nature has been confirmed. The precursor and complexes have been screened in vitro against a number of pathogenic fungi and bacteria to assess their growth inhibiting potential. The testicular sperm density and testicular sperm morphology, sperm motility, density of cauda epididymal spermatozoa and fertility in mating trails and biochemicals parameters of reproductive organs have been examined and discussed.
Combinations of pactamycin and Salmonella typhosa 0901 endotoxin, administered simultaneously, killed more BALB/c mice than comparable doses of either agent alone The slopes of the dose-response curves for combinations of endotoxin and pactamycin were parallel to both that for endotoxin alone and the antitumor drug alone; therefore, no new mechanism of toxicity has been evoked by the combination. The synergistic toxicity of endotoxin and pactamycin was due to an in vivo interaction rather than a direct reaction between the two agents. Phenobarbital pretreatment protected the mice from toxicity of the antitumor agent alone but not from the lethal action of the combination. Pretreatment with endotoxin increased the resistance of the mice to both endotoxin alone and the combination. Third agents unable to protect mice from the synergistic toxicity of endotoxin and pactamycin were α[p-(fluoren-9-ylidenemethyl)-phenyl]-2-piperidine-ethanol, neomycin, phenylbutazone, polymyxin B, and tybamate. Prednisolone pretreatment alleviated the toxicity of the combination. For the restricted series of killed bacteria and bacterial products tested for capability to enhance the toxicity of pactamycin, only gram-negative bacterial cells were potent. These results indicated that pactamycin rendered the mice more susceptible to endotoxin and that endotoxin was the causal lethal agent.
Several high-pressure liquid chromatography procedures for the determination of flucytosine in serum or plasma have appeared. Some of these suffer from significant disadvantages, and none was applicable in our routine clinical therapeutic-drug-monitoring laboratory. A new high-pressure liquid chromatography assay for flucytosine was therefore developed. A 100-microliter sample of plasma was treated with an aqueous 5-iodocytosine internal-standard solution, and the mixture was deproteinized with trichloroacetic acid. A portion of the protein-free supernatant was diluted with 0.1 M ammonium phosphate, and an aliquot of the resulting solution was injected into the high-pressure liquid chromatography system. Chromatography was performed on a strong-cation-exchange column with a mobile phase containing aqueous ammonium phosphate, phosphoric acid, methanol, and acetonitrile. Detection was at 254 nm. The assay was shown to be linear in the 10 to 200-micrograms/ml drug-concentration range. Forty other drugs were tested for potential interference with the assay, and none was found. For routine use, a single-point working standard containing 75 micrograms of flucytosine per ml was used, giving intraassay coefficients of variation at 50 and 150 micrograms/ml of 1.8 and 2.3% respectively, whereas the day-to-day coefficient of variation at 50 micrograms/ml was 10.0%. Advantages of the procedure include the small sample size, the use of a convenient and reliable internal standard, speed, and simplicity. The assay is highly suitable for routine clinical drug-analysis laboratories.