Development and Validation of an RP–LC–UV Method for the Determination of Ondansetron: Application to Pharmaceutical Dosage Forms

A new, simple, rapid, sensitive and specific isocratic RP–LC–UV method was developed and validated for the determination of ondansetron in pharmaceutical dosage forms of orally disintegrating tablets, oral solution and injection. The LC separation was achieved on a Hypersil C4 column (250 × 4.6 mm, 5 μm) using a mobile phase of 50 mM potassium dihydrogen phosphate anhydrous adjusted to pH 3.5 with orthophosphoric acid and acetonitrile (30:70, v/v) at a flow rate of 1.0 mL min^?1 and UV detection at 310 nm. The method was validated for specificity, linearity, precision, accuracy, limit of quantification, limit of detection, robustness and solution stability. The calibration curve was linear over a concentration range of 100–1,000 ng mL^?1 (r ²  = 0.9996) with limit of detection and limit of quantification 50 and 100 ng mL^?1, respectively. The intra-day and inter-day precision and accuracy were between 0.79 and 2.37% and ?0.64 and 1.65%, respectively. The method was successfully applied for analysis of ondansetron in the presence of excipients in commercially available pharmaceutical dosage forms.     

高效液相色谱法测定大鼠血浆中芳香异羟肟酸二丁基锡的含量以及它在动力学研究中的应用

[(n-Bu)2Sn[{4-ClC6H4C(O)NHO}2] (DBDCT) 是课题组自主设计合成的一种新型芳香异羟肟酸二丁基锡化合物(已获国际国内发明专利),有较高的体内和体外抗肿瘤活性,小鼠急毒实验揭示其具有较低的毒性作用,初步动物实验提示DBDCT还具有升高白细胞的功能,在肿瘤化疗治疗中将产生重要的影响。本文首次建立了HPLC法测定化合物在血浆中的动力学参数。用甲醇直接沉淀血浆蛋白,乙酰苯胺为内标, Diamonsil ODS（4.6 mm × 200 mm, 5 μm）色谱柱,甲醇:0.5%三氟乙酸水溶液（30:70,pH 3.0,v/v）为流动相,检测波长238 nm。方法在0.1～25 µg·mLl-1范围内线性关系良好(r = 0.9992),定量限和检测限分别为50 和10 ng·mL-1。该方法用来测定单次静脉注射不同剂量(2,5,12mg·kg-1) DBDCT后大鼠体内的浓度-时间曲线,并采用3p97软件对动力学参数和房室模型进行估计,结果表明DBDCT在大鼠体内的动力学符合二室模型,方法简便快速,专属性好,其动力学研究中的应用为制剂的质量控制和临床前动物合理用药以及临床研究提供了实验基础。     

A New Nanogold-Labeled Immunoresonance Scattering Spectral Probe for Determination of Trace IgG

A kind of 9 nm gold nanoparticles was prepared with the trisodium citrate and used to label goat anti-human IgG to obtain an IgG immunoresonance scattering spectral probe. In pH 5.8 buffer solution and in the presence of polyethylene glycol （PEG）, the immune reaction between gold-labeled goat anti-human IgG and IgG took place, and the resonance scattering intensity at 580 nm （I580nm） was enhanced greatly. The enhanced intensity AIRS is pro- portional to the IgG concentration from 1.3 to 1.5 X 10^3 ng.mL^-1, with a detection limit of 0.78 ng.mL ^-1. This assay showed high sensitivity and good selectivity for quantitative determination of IgG in human serum, with satisfactory results.     

Study on the Interaction between a Novel Ionic Liquid Probe and Anionic Surfactants Using Resonance Light Scattering Spectra and Its Analytical Application

The interaction between anionic surfactants (AS) and 1‐hexadecyl‐3‐methylimidazolium bromide [C₁₆mim]Br was studied by using resonance light scattering (RLS) technique, UV‐Vis spectrophotometry and fluorometric methods. In Britton Robinson (BR) buffer (pH 6.0), [C₁₆mim]Br reacted with AS to form supermolecular complex which resulted in enhancement in RLS intensity. Their maximum RLS wavelengths were all at 390 nm. Some important interacting experimental variables, such as the solution acidity, [C₁₆mim]Br concentration, salt effect and addition order of the reagents, were investigated and optimized. Under the optimum conditions, quantitative determination ranges were 0.001–7 μg·mL^?1 for dodecyl sodium sulfate (SDS), 0.001–6 μg·mL^?1 for sodium dodecylbenzene sulfonate (SDBS) and 0.005–7 μg·mL^?1 for sodium lauryl sulfonate (SLS), respectively, while the detection limits were 1.3 ng·mL^?1 for SDS, 1.0 ng·mL^?1 for SDBS and 5.1 ng·mL^?1 for SLS, respectively. Based on the ion‐association reaction, a highly sensitive, simple and rapid method has been established for the determination of AS.     

Study on the Supramolecular Interaction of β-Cyclodextrin with Gemfibrozil by Spectrofluorimetry and Its Analytical Application

The supramolecular interaction of gemfibrozil with β-cyclodextrin （β-CD） was studied by spectrofluorimetry. The mechanism of the inclusion was discussed by spectrofluoremetry, infrared spectrum and ^1H NMR spectrum. The results showed that a 1 ： 1 （β-CD ： gemfibrozil） complex was formed with an apparent association constant of 3.844 × 10^3 L·mol^-1. Based on the enhancement of the fluorescent intensity of gemfibrozil, a spectrofluorimetric method for the determination of gemfibrozil in bulk aqueous solution in the presence of β-CD was developed. The linear range was 3.30 ng·mL^- 1 -6.00 ug·mL^-1 with the detection limit of 0.980 ng·mL^-1. There was no interference from the excipients normally used in tablet composition and the serum main compositions. The proposed method was then successfully applied to the determination of gemfibrozil in capsules and serum.     

Determination of Potassium Ferrocyanide in Foods by Resonance Rayleigh Scattering Method with Double-Charged Triaminotriphenylmethane Dyes

In pH 1.0 acidic medium, double-charged triaminotriphenylmethane dyes such as methyl green （MEG） and iodine green （IG） react with potassium ferrocyanide to form 2 ： 1 ion-association complexes by virtue of electrostatic forces and hydrophobic interaction. It results in the change of absorption and the great enhancement of resonance Rayleigh scattering （RRS） and the appearance of new RRS spectra. Two systems have similar spectral characteristics and their maximum RRS wavelengths are all located at 276 nm and smaller peaks are located at 332 and 457 nm, respectively. The intensity of RRS is directly proportional to the concentration of [Fe（CN）6]^4- in the range of 0.03-5.7 μg·mL^-1 （MeG system） or 0.04-5.9 μg·mL^-1 （IG system）. The RRS method has high sensitivity and the detection limit （3σ） for potassium ferrocyanide is 9.3 ng·mL^-1 （MeG system） or 11.2 ng·mL^-1 （IG system）. The optimum conditions, influencing factors and effects of foreign substances are investigated. The method also has a good selectivity. A sensitive, rapid and simple RRS method for the determination of potassium ferrocyanide in salinized food and table salt has been developed.     

免疫纳米金催化共振散射光谱法测定痕量IgM

用粒径为10 nm的金纳米微粒标记羊抗人免疫球蛋白M（IgM）,制备了IgM的免疫纳米金共振散射光谱探针。在pH4.49的KH2PO4-Na2HPO4缓冲溶液及PEG存在下,金标羊抗人IgM与IgM发生特异性结合生成胶体金免疫复合物,离心分离,获得未反应的金标抗上层清液。以此纳米金标抗作为催化剂,在pH 1.93的盐酸-柠檬酸钠缓冲溶液,催化NH2OH·HCl还原吸附在免疫纳米金表面的金络离子物种（AuCl4-）生成粒径更大的金纳米微粒,导致580 nm 处金纳米微粒的共振散射强度急剧增大。结果表明,随着IgM浓度增大,离心上层液中金标抗降低,I 580 nm线性降低,其△I580 nm与IgM浓度在0.06～4.80 ng· ml-1范围内呈良好的线性关系,其回归方程为ΔI580 nm=14.5cIgM + 1.8,检出限为0.03 ng·ml-1。本法具有灵敏、快速和较高的特异性,用于定量分析人血清中IgM,结果满意。     

Solid Phase Extraction of Uranium by Naphthalene‐Methyltrioctylammonium Chloride and Arsenazo(III) Adsorbent and Subsequent Spectrophotometric Determination

A simple and effective method has been presented for the preconcentration of uranium by solid phase extraction. For this purpose arsenazo(III) supported on naphthalene‐methyltrioctylammonium chloride was used as an adsorbent and uranium solution at pH 3.5 with flow rate of 1 mL·min⁻¹ was passed through the column. Therefore, uranium‐arsenazo(III) complex was formed onto column. Uranium was quantitatively eluted with 5 mL of a 0.1% ammonium tetraphenylborate and determined by spectrophotometric method at 652 nm. Several parameters such as pH, amount of reagents, sample volume, etc. were investigated. The effect of diverse ions on the preconcentration has also been studied and the optimized conditions developed have been utilized for the trace determination of uranium. A preconcentration factor of 100 was achieved. The relative standard deviation (N=8) was 0.5% for 3 ng· mL⁻¹ of uranium. The three sigma detection limit (36) was 0.045 ng·mL⁻¹     

HPLC Determination of Letrozole in Plasma Using Fluorescence Detection: Application to Pharmacokinetic Studies

A simple, rapid and sensitive HPLC method has been developed and validated for the analysis of letrozole in human plasma. The separation was achieved on a monolithic silica column using acetonitrile–phosphate buffer. A fluorescence detector was used for the quantitation with excitation and emission wavelengths at 230 and 295 nm. The assay enables the measurement of letrozole for therapeutic drug monitoring with a minimum quantification limit (LOQ) of 0.5 ng mL^?1. The method involves a simple, one-step extraction procedure with complete recovery. Calibration was linear over the concentration range 0.5–80 ng mL^?1. The coefficients of variation for inter-day and intra-day assay were found to be less than 8%.     

Determination of Ferulic Acid in Rat Plasma by Liquid Chromatography–Tandem Mass Spectrometry Method: Application to a Pharmacokinetic Study

Abstract

A rapid, sensitive, and specific liquid chromatography–tandem mass spectrometric (HPLC-MS/MS) method has been developed for quantification of ferulic acid in rat plasma. The analyte and docetaxel (internal standard) were extracted from plasma samples with diethyl ether and analyzed on a C₁₈ column. The chromatographic separation was achieved within 3.5 min by using acetonitrile-water as the mobile phase and the flow rate was 0.2 mL · min^?1. The method was linear within the range of 0.5 ? 800 ng · mL^?1. The lower limit of quantification (LLOQ) was 0.5 ng · mL^?1. Finally, the method is successfully applied for the pharmacokinetic study of ferulic acid in rats following intravenous administration.     

A Spectrophotometric Study of Streptomycin Effect on the Clinical Urea Determination

The results of studying inhibitory effect of streptomycin on the modified Berthelot reaction were presented in this paper and a new kinetic method for determining streptomycin in pharmaceutical preparations and human urine was developed on the basis of the obtained results. The rates of catalytic and catalytic‐inhibitory reaction were monitored at 700 nm (t=25±0.1°C) using UV/vis spectrophotometer. By analyzing the spectra and experimental dependences of the catalytic and catalytic‐inhibitory reaction rates on the reactant concentrations, it was noticed that streptomycin attacked nitroprusside and hypochlorite causing the inhibition of the production of 2,2′‐dicarboxylindophenol. According to this effect, an analytical decrease for determination of urea by modified Berthelot reaction appeared in the presence of small amounts of streptomycin. Beer's law was obeyed in the interval of streptomycin sulfate concentration from 18.2 to 182 µg·mL^?1. The detection limit calculated by two methods was obtained at 11.75 µg·mL^?1 and 8.54 µg·mL^?1. The relative standard deviation of 0.55%–8.83% and the recovery of 109.10% were determined. The obtained results were validated using the referent HPLC method.     

钯（II）-克林霉素-卤代荧光素体系的共振瑞丽散射光谱及其分析应用

在pH为5.0-5.4的乙酸-乙酸钠缓冲溶液中,克林霉素（Clin）与钯(Ⅱ)形成螯合阳离子,它能进一步与二碘荧光素（DIF）,赤藓红（Ery）,曙红Y（EY）等卤代荧光素类染料反应形成1：1：1的三元离子缔合物,此时将引起吸收光谱变化和荧光猝灭,同时还导致共振瑞利散射(RRS)的急剧增强并产生新的RRS光谱,钯(Ⅱ)-克林霉素与DIF,Ery和EY形成产物的最大散射波长分别位于285,287,32 1nm处,另外还有些较弱的散射峰存在。散射增强（ΔI）与克林霉素浓度在一定范围内成正比,可用于克林霉素的定量测定。对于DIF,Ery和EY体系的线性范围和检出限分别为0.025-2.1μg•mL-1和7.8 ng•mL-1,0.053-2.4μg•mL-1和16.0 ng•mL-1;以及0.038-2.4μg•mL-1和11.0 ng•mL-1。本文研究了适宜的反应条件,考察了共存物质的影响,表明方法有较好的选择性,基于三元离子缔合物的RRS光谱,发展了一种高灵敏、简便快速测定克林霉素的新方法。文中还对离子缔合物的组成,结构和反应机理,以及离子缔合物对吸收,荧光和RRS光谱的影响进行了讨论。     

Second‐order Scattering and Frequency Doubling Scattering Spectra of Thallium(III)‐Methotrexate System and Its Analytical Application

In pH 4.9 Britton-Robinson buffer solution, methotrexate (MTX) reacted with thallium(III) to form a 3∶1 chelate. This resulted in great enhancement of second-order scattering (SOS) spectra and frequency doubling scattering (FDS) spectra and appearance of new SOS and FDS spectra. Their maximum wavelengths were located at 520 and 390 nm, respectively. The increments of scattering intensities (ΔI) were directly proportional to the concentrations of MTX in the ranges of 0.022—2.0 μg•mL－1 (SOS method) and 0.008—2.5 μg•mL－1 (FDS method). The methods exhibited high sensitivities. The detection limits for MTX were 7.4 ng•mL－1 (SOS method) and 2.3 ng•mL－1 (FDS method), respectively. The optimum conditions of the reaction, the influencing factors and the effects of coexisting substances were investigated. A highly sensitive, simple and fast method for the determination of MTX has been developed. The method can be applied satisfactorily to the determination of MTX in human serum samples. In this work, the charge distribution of MTX was calculated by a CNDO quantum chemistry method. In addition, the reaction mechanism was discussed.     

Study of Spectrophotometric Characteristics of the Charge Transfer Reaction of Aminomethylbenzoic Acid with 7,7,8,8‐Tetracyanoquinodimethane

A new spectrophotometric method was developed for the determination of aminomethylbenzoic acid (PAMBA) using 7,7,8,8‐tetracyanoquinodimethane (TCNQ). The method was based on the formation of charge transfer (CT) complex of this drug as n‐electron donor with the π‐acceptor TCNQ. TCNQ was found to react with PAMBA to produce a kind of yellow complex. The CT reaction proceeded quantitatively in pH 8.5 buffer solution. Different variables affecting the reaction were carefully studied and optimized. Under optimal reaction conditions, the stoichiometric ratio of the reaction, maximum absorption wavelength and the value of molar absorptivity were measured to be 1:1, 425 nm, and 1.9×10⁴ L·mol^?1·cm^?1, respectively. Beer′s law was obeyed in the range of 1–9 µg·mL^?1 of PAMBA. The data have been filled to a linear regression equation A=?0.2612+0.1123c (µg·mL^?1), with a correlation coefficient of 0.9996. The detection limit was 0.4 µg·mL^?1, R.S.D. was less than 1.9%, and average recovery was over 97.6%. The formation of the CT complex was also confirmed by both infrared and ¹H NMR measurements. The thermodynamic property, kinetic property and reaction mechanism have also been discussed. The method developed was applied successfully to the determination of the subject drug in its pharmaceutical dosage forms with good precision and accuracy compared to official method revealed by t‐ and F‐tests.     

An Experimental Design Approach to Selecting the Optimum LC Conditions for the Determination of Local Anaesthetics

A sensitive high performance liquid chromatographic (HPLC) method has been developed and validated for the simultaneous determination of four local anaesthetics: lidocaine, proparacaine, bupivacaine and oxybuprocaine. A full factorial design was used. The chromatographic separation was achieved using a Bondesil C₈ (4.6 × 2.5 mm i.d., particle size 5 μm) analytical column. An optimised mobile phase consisted of acetonitrile and sodium dihydrogen phosphate (pH = 3.0, 20 mM) (30:70, v/v) at a flow rate of 1.2 mL min^?1. Local anaesthetics detection was performed by UV-Vis detector at 220 nm. The retention times for lidocaine, proparacaine, bupivacaine and oxybuprocaine were 5.74, 9.28, 16.84 and 26.26 min, respectively. HPLC-UV-Vis method was linear in the range of 50–5,000 ng mL^?1 for lidocaine and proparacaine and 100–5,000 ng mL^?1 for bupivacaine and oxybuprocaine. The limit of detection (LOD) was 25 ng mL^?1 for lidocaine, proparacaine and 30 ng mL^?1 for bupivacaine and oxybuprocaine. The limit of quantification (LOQ) was found to be 50 ng mL^?1 for lidocaine, proparacaine and 100 ng mL^?1 for bupivacaine, oxybuprocaine. In intra-day and inter-day precision and accuracy analysis, the relative standard deviation was found to be less than 8%.     

Quantification of Thiram in Honeybees: Development of a Chemiluminescent ELISA

Abstract

A Chemiluminescence Enzyme‐Linked Immuno‐Sorbent Assay (CL‐ELISA) for determination and quantification of the fungicide thiram in honeybees was developed in an indirect competitive format. The assay was optimized by determining: the optimal coating conjugate concentration and anti‐thiram antiserum dilution, the effect of the incubation time on the competitive step, the tolerance to organic solvents. The IC₅₀ and the limit of detection (LOD) values were 60 ng mL^?1 and 9 ng mL^?1, respectively, similar to those of colorimetric ELISA with a calibration range of 9–15,000 ng mL^?1. Cross reactivity of some related compounds such as some dithiocarbamates, a thiocarbamate, the ethylenethiourea and the tetramethylthiourea were tested. The assay was then applied to honeybees sample extracts obtained by using the liquid‐liquid extraction or the graphitized carbon‐based solid phase extraction.

The calibration curves in honeybee extracts from liquid‐liquid procedure gave an IC₅₀ of 141 ng mL^?1 and a LOD of 17 ng mL^?1. In case of extracts obtained by SPE these values were 139 ng mL^?1 and 15 ng mL^?1, respectively. The average recovery value from honeybee extracts spiked with 75 ng mL^?1 of thiram was 72% for SPE, higher than for liquid‐liquid extraction (60%). On the opposite, when the honeybees were directly spiked with 2 and 10 ppm the average recovery was higher for liquid‐liquid extraction (54%), than for SPE (31%). Finally, the assay was applied to honeybee samples collected during monitoring activities in Italy and Russia.     

GC Determination of Prilocaine HCl in Human Plasma: Analytical Application to Real Samples

A gas chromatographic (GC) method with a rapid and simple sample preparation was developed and validated for determination of prilocaine in human plasma. The validation parameters of linearity, precision, accuracy, recovery, specificity, limit of detection and limit of quantification were studied. The range of quantification for the GC method was 50–300 ng mL^?1 in plasma. Intra- and inter-day precision, expressed as the relative standard deviation (RSD) were less than 4.5%, and accuracy (relative error) was better than 8.0% (n = 6). The analytical recovery of prilocaine HCl from plasma has averaged 96.5% and the recovery of internal standard (lidocaine HCl) reached 96.8%. The limit of quantification (LOQ) and the limit of detection (LOD) of the method for plasma were 50 and 40 ng mL^?1, respectively. Also the developed and validated method was applied to three healthy volunteers to whom a local anaesthesia with citanest was administered.     

Determination of Danofloxacin and Marbofloxacin in Milk Samples by Micellar Liquid Chromatography with Fluorescence Detection

Abstract

A simple, rapid, and sensitive analytical method has been developed for the determination of two fluoroquinolones, danofloxacin and marbofloxacin, in bovine milk samples. Separation and quantification were performed by micellar liquid chromatography with fluorescence detection (MLC?FD), using sodium dodecyl sulfate (SDS) as a surfactant. The influence of the principal factors, namely, the micelle concentration, the amount of organic modifier, tail‐reducing agents, the pH, and the temperature were studied. The suitable condition was found to be 75 mM SDS?10 mM phosphate buffer–18 mM tetrabutylammonium bromide/3% (v/v) 1‐propanol at pH 3.0 for the separation of marbofloxacin, danofloxacin, and tosufloxacin (internal standard) in about 20 min. The linear concentration range of application was 1.8–30.0 ng · mL^?1 for danofloxacin and 16–120 ng · mL^?1 for marbofloxacin, and the relative standard deviation ranged between 4.9 and 2.7%. The limit of detection found for danofloxacin was 0.5 ng · mL^?1 and 5 ng · mL^?1 for marbofloxacin. These values were lower than the maximum residue limits (MRLs) established by the European Union for these compounds in bovine milk. It was applied to check the eventual existence of these compounds above these limits on commercial milk samples. The validation method was completed with spiked milk samples. Recovery levels obtained were 90.3–108.2%.     

RP-LC Analysis and Hydrolytic Degradation Profile of Racecadotril

A sensitive, stability-indicating liquid-chromatographic method for analysis of racecadotril in the presence of its degradation products has been developed and validated. Efficient chromatographic separation was achieved on a C₁₈ column with a simple isocratic mobile phase—60:40 methanol–water. Quantification was by photo-diode array (PDA) detection at 220 nm. The linearity of the method was excellent over the range 1–32 μg mL^?1. The method was sensitive, with low limits of detection (20 ng mL^?1) and quantification (100 ng mL^?1). The recovery of the method was consistently good (98.7–100.9%), with low (<1%) intra-day and inter-day relative standard deviation. Robustness studies confirmed that peak area was unaffected by small changes in temperature, and mobile phase composition and flow rate. Both alkaline and acidic hydrolytic degradation were performed in methanolic solution. In alkaline medium the drug was degraded immediately; it was degraded within 90 min in acidic medium. The validated, stability-indicating, method was used for analysis of racecadotril in pharmaceutical dosage form and also to reveal the hydrolytic degradation profile of the racecadotril.     

Combination of Solid‐Phase Micro‐Extraction and Direct Analysis in Real Time‐Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Sensitive and Rapid Analysis of 15 Phthalate Plasticizers in Beverages

A method for rapid identification and quantification of phthalate plasticizers in beverages was developed. A number of 15 phthalate plasticizers which covered all the phthalates concerned in the US Consumer Product Safety Improvement Act (CPSIA), European Union legislations and Chinese national standards (GB) were analyzed. By a combined solid‐phase micro‐extraction (SPME) and direct analysis in real time mass spectrometry (DART‐MS) approach, phthalates at sub‐ng·mL^?1 levels can be qualitatively and quantitatively analyzed in a short time. The use of ultrahigh‐resolving power and the accurate mass measurement capacity naturally provided by Fourier transform ion cyclotron resonance mass spectrometry (FT‐ICR‐MS) minimizes the matrix interferences and thus enables the evaluation of phthalates in a complex matrix without extensive sample handlings or preparations. The limits of quantification (LOQs) were estimated to be at 0.3–5.0 ng·mL^?1, lower than the Maximum Residue Limit (MRL) regulated by the European Union legislations (2007/19/EC) in foods, beverages, food packaging and toys (0.3–30 ng·mL^?1). This rapid and easy‐to‐use SPME‐DART‐FT‐ICR‐MS method provided a relatively high‐throughput and powerful analytical approach for quick testing and screening phthalates in beverages and water samples to ensure food safety.