Determination of limonin in dog plasma by liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

A highly sensitive liquid chromatography–tandem mass spectrometry method was developed and validated for the determination of limonin in beagle dog plasma using nimodipine as internal standard. The analyte and internal standard (IS) were extracted with ether followed by a rapid isocratic elution with 10 mm ammonium acetate buffer–methanol (26:74, v/v) on a C₁₈ column (150 × 2.1 mm i.d.) and subsequent analysis by mass spectrometry in the multiple reaction monitoring mode. The precursor to product ion transitions of m/z 469.4 → 229.3 and m/z 417.2 → 122.0 were used to measure the analyte and the IS. The assay was linear over the concentration range of 0.625–100 ng/mL for limonin in dog plasma. The lower limit of quantification was 0.312 ng/mL and the extraction recovery was >90.4% for limonin. The inter‐ and intra‐day precision of the method at three concentrations was less than 9.9%. The method was successfully applied to pharmacokinetic study of limonin in dogs. Copyright © 2012 John Wiley & Sons, Ltd.     

Dye sensitized luminescent europium nanoparticles and its time-resolved fluorometric assay for DNA

High performance liquid chromatography (HPLC) method has been developed for simultaneous quantification of limonoid aglycones and glucosides on a reversed phase C₁₈ column using a binary solvent system, coupled with diode array detector. Seven limonoids such as limonin, nomilin, isolimonic acid, ichangin, isoobacunoic acid, limonin 17-β-d glucopyranoside and deacetyl nomilinic acid 17-β-d glucopyranoside were separated and detected at 210 nm. Furthermore, limonoids were separated, identified and quantified in four varieties of citrus fruits and seeds using developed method. Limonin and limonin glucoside were found to be the predominant limonoid aglycone and glucoside, respectively, in all tested samples. The sensitivity of the method was found to be 0.25–0.50 μg for tested limonoids.     

Application of matrix solid-phase dispersion in the determination of dibenzo[a,l]pyrene content of experimental animal diets used in a large-scale tumor study.

A method utilizing matrix solid-phase dispersion (MSPD) was developed for isolation and determination of dibenzo[a,l]pyrene (DBP) in experimental rainbow-trout diets used in a large-scale carcinogenesis study. A 0.5 g sample of moist ration containing 0-225 ppm DBP (dry basis) was mixed with 2 g C18 sorbent and benzo[a]pyrene internal standard was added to the mixture. Extraction and clean-up were accomplished in a single step by extracting the sample mixture with hexane-benzene 4:1 from a cartridge containing 2 g Florisil. DBP was quantified by HPLC on a C5 bonded phase column with fluorescence detection. Mean analytical recovery of DBP from control diet spiked at three concentration levels was 101 to 107% with relative standard deviations of 1 to 7%. The limit of detection of DBP was equivalent to 0.014 ppm in the ration. Application of the method to verification of DBP levels in trout rations from the carcinogenesis study is described. Control ration (0 ppm DBP) was screened for possible DBP contamination and none was found. This is the first report on analysis of DBP in experimental animal diets.     

Quantitative determination of limonin in citrus juices by HPLC using computerized solvent optimization

The commercially available computer program, Drylab, for optimization of separations by high-performance liquid chromatography (HPLC) using binary solvent mixtures is used to improve an HPLC method for separation of the bitter principle, limonin, in grapefruit and navel orange juices. Best conditions for separation of limonin in a reasonable time are 30 to 32% acetonitrile in water at 0.9 mL/min using a 5-micron C18 column 10 cm long. These conditions are used to analyze grapefruit and navel orange juice samples, and these HPLC results are compared with values determined by enzyme immunoassay or thin-layer chromatography (TLC) on the same samples.     

高效液相色谱法测定柑橘汁中的柠檬苦素和柚皮苷

柑橘汁的苦味主要是由于柚皮苷和柠檬苦素的存在所致,其含量的测定可用于控制柑橘类果汁的质量。采用高效液相色谱法在KR100-5C18(4.6 mm i.d.×250 mm,5 μm)上,分别以乙腈-四氢呋喃-水(体积比为17.5∶17.5∶65)和甲醇-冰醋酸-水(体积比为40∶1∶59)为流动相（流速均为1 mL/min）,在207 nm和283 nm检测波长下分别测定了柠檬苦素和柚皮苷。实验结果表明,柠檬苦素在1.00～50.00 mg/L时线性关系良好(r=0.9992),检出限为0.07 μg,平均加标回收率为98.69%,相对标准偏差（RSD）为2.5%;柚皮苷在20.00～160.00 mg/L时线性关系良好(r=0.9988),检出限为0.14 μg,平均加标回收率为100.13%,RSD为1.5%。用该法检测柑橘汁样品中的柠檬苦素与柚皮苷,方法简便、快速、准确。     

Liquid chromatography/atmospheric pressure chemical ionization-mass spectrometric analysis of benzoylurea insecticides in citrus fruits

A liquid chromatography (LC) method for the quantitative determination of three benzoylurea insecticide residues (diflubenzuron, flufenoxuron and hexaflumuron) in citrus fruits is described. Residues were successfully separated on a C18 column by methanol/water gradient elution. Detection was by negative-ion, selected-ion monitoring atmospheric pressure chemical ionization-mass spectrometry (APCI-MS); the main ions were [M - H]-, and the secondary fragment ions were [M - H - HF]-. Useful confirmatory information can thus be obtained at low extraction voltages from losses of HF. Detection limits for standard solutions were 10 fg injected and good linearity and reproducibility were obtained. The optimum LC/APCI-MS conditions were applied to the analysis of benzoylureas in oranges. Samples were extracted using matrix solid phase dispersion (MSPD), in which orange samples were homogenized with Cs, placed onto a glass column and eluted with dichloromethane. Detection limits of 2 microg kg(-1) in the crop were obtained. Average recoveries from citrus fortified with approximately (25-1000 microg kg(-1)) ranged from 87 to 102%. The method was applied to field-treated orange samples and benzoylureas were sometimes detected at concentration levels lower than maximum residue limits.     

高效液相色谱法测定柑橘皮中柠檬苦素

提出了高效液相色谱法测定柑橘皮中柠檬苦素含量的方法。样品用体积分数为70%乙醇溶液于50℃超声提取75min。以Sinochrom ODS-BP(250mm×4.6mm,10μm)为分离柱,以乙腈-水(45+55)溶液作为流动相,用紫外检测器在波长210nm处进行测定。柠檬苦素的质量浓度在12.0～384mg.L-1范围内与其峰面积呈线性关系,方法的平均回收率为99.9%,相对标准偏差(n=5)为1.8%。     

Separation and determination of aminohalogenbenzophenones by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method with electrochemical detection has been developed for the determination of three aminohalogenbenzophenones: 2-amino-2',5-dichlorobenzophenone, 2-amino-5-chlorobenzophenone and 2-amino-5-bromo-2'-fluorobenzophenone, metabolites of benzodiazepinooxazoles and other psychotropic drugs. A mobile phase of methanol-water (65:35), containing 5 mM KH2PO4 appeared to be the optimal when a 4-microns, 60-A Nova-Pak C18 column and a flow-rate of 0.75 ml/min (130 bar) were used. The temperature was optimized at 30 degrees C. The amperometric detector, equipped with glassy carbon electrode, was operated at 1.3 V versus Ag/AgCl in the DC mode. The method was applied to the determination of these compounds at two concentration levels: ppm and ppb (ng/cm3) using 2-amino-5-chlorobenzophenone as internal standard. The limit of determination was 750 pg/ml of biological fluid for each compound, and recoveries greater than 97% were obtained for spiked samples of urine and serum, using C18 Sep-Pak cartridges in the sample clean-up procedure.     

Determination of abamectin in citrus fruits by liquid chromatography-electrospray ionization mass spectrometry

Liquid chromatography coupled to electrospray mass spectrometry (LC-ES-MS) with positive ion detection was used to determine abamectin in oranges. MS conditions were optimized to achieve maximum sensitivity. The main ion for abamectin was [M+Na]+ at a fragmentor voltage of 180 V. Abundant structural information can be obtained at different fragmentor voltages. The detection limit for the standard solution was 12 pg injected, and good linearity and reproducibility were observed. Abamectin residues were extracted using matrix solid-phase dispersion. Orange samples were homogenized with C18 bonded silica placed onto a glass column and eluted with dichloromethane. Recoveries of the abamectin from oranges fortified with approximately 0.01-10 mg/kg ranged from 94 to 99% with an overall average recovery of 96%. The quantification limit is 0.0025 mg/kg, which means detection limit for this analyte could be set at a few hundred picograms per gram of fruit. The presence in the electrosprayed solution of numerous citrus constituents did not interfere significantly with the ionization process of abamectin. The assay procedure provides a simple, rapid, and sensitive method for monitoring residues in oranges. The method was applied to field treatment orange samples.     

Debittering of Tangerine Citrus Reticulata Blanco Juice by β-Cyclodextrin Polymer

The use of insoluble β-cyclodextrin polymer for the reduction of limonin in Thai tangerine juice by batch, column and fluidized bed processes were studied and compared. Direct correlation between complexation of limonin and amount of β-cyclodextrin polymer was observed. The binding was not affected by temperature when tested at cold room and room temperatures. Using 3 g% β-CD polymer at room temperature, limonin reduction by the batch and column processes were 70 and 94% respectively. In a fluidized bed column (50 × 3 cm i.d.) under the condition of 15 g β-cyclodextrin polymer with a feed flow rate of 100 mL/min at room temperature, the initial efficiency of debittering was about 90% and gradually decreased. Increasing the flow rate or amount of the cyclodextrin bed did not improve the efficiency at the present study condition.     

Simultaneous Determination of Five Antibacterials in Swine Muscle by High Performance Liquid Chromatography

Abstract

A high performance liquid chromatographic method (HPLC) for the determination of olaquindox, morantel, furazolidone, nitrofurazone and carbadox residues in swine muscles was developed. The drugs were extracted from muscles with acetonitrile and cleaned up by alumina column. HPLC analysis was carried out on an Inertsil C8 column with a mobile phase of acetonitrile-water-acetic acid (3:97:1), and the drugs were detected at 340 nm. The average recoveries of all drugs added to muscles at 0.1 ppm level were more than 75% and the detection limit of each drug was 0.03 ppm in muscles.     

Simultaneous determination of three alkaloids,four ginsenosides and limonin in the plasma of normal and headache rats after oral administration of Wu‐Zhu‐Yu decoction by a novel ultra fast liquid chromatography‐tandem mass spectrometry method: application to a comparative pharmacokinetics and ethological study

A novel, sensitive and reliable ultra fast liquid chromatography‐tandem mass spectrometry (UFLC‐MS/MS) method has been developed and validated for simultaneous quantitation of eight main active ingredients (evodiamine, rutaecarpine, dehydroevodiamine, limonin, ginsenoside Rb₁, Rd, Re and Rg₁) in rat plasma after oral administration of Wu‐Zhu‐Yu (WZY) decoction, which is a celebrated and widely used Traditional Chinese Medicine formula for the treatment of headache. The analytes and internal standard (IS) were separated on a SHIM‐PACK XR‐ODS II column, and the detection was performed on a UFLC‐MS/MS system with turbo ion spray source. The lower limits of quantification were 1.5, 0.5, 1.0, 2.0, 2.0, 1.0, 0.5 and 0.2 ng ml^?1 for evodiamine, rutaecarpine, dehydroevodiamine, limonin, gensenoside Rb₁, Rd, Re and Rg₁, respectively. Linearity, accuracy, precision and absolute recoveries of the eight analytes were all within satisfaction. The IS‐normalized matrix factor was adopted for assessing the matrix effect and accompanied with a satisfactory result. The validated method has been successfully applied to compare pharmacokinetic profiles of the eight active ingredients in rat plasma between normal and headache rats after administration. Exact pharmaceutical effect of WZY decoction on headache was demonstrated by the ethological response of headache rats induced by nitric oxide donor after administration. The results indicated that the absorption of evodiamine, rutaecarpine, gensenoside Rb₁, Re and Rg₁ in headache group were significantly higher than those in normal group with similar concentration–time curves while no significant differences existed in limonin and ginsenoside Rd between the two groups. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitative determination of potential genotoxic impurity 3-aminopyridine in linagliptin active pharmaceutical ingredient using HILIC–UV

This study aimed to develop an analytical method to determine the quantity of the impurity 3-aminopyridine (3AP). 3-Aminopyridine is a reactive reagent in the synthesis of linagliptin. The method was sensitive at level of 30.0 ppm of 3AP relative to linagliptin. The analysis was carried out using hydrophilic interaction liquid chromatography. The analytical column was Tracer Extrasil Silica (150 × 4.0 mm, 3 μm). A mobile phase of water–acetonitrile (10:90, v/v) containing 10.0 mM ammonium acetate was prepared and adjusted to pH 6.0. A UV detector was used to detect the amount of 3AP at a wavelength of 298 nm. Validation of the method was performed as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use in terms of detection limit, quantitation limit, linearity, accuracy, precision, specificity and robustness. The calibration curve was linear (r² = 0.999) for 3AP concentration in the range of 30.0–450.0 ppm. This method showed a good sensitivity with a detection limit and a quantitation limit of 7.5 and 25.0 ppm, respectively.     

Permeation-solid adsorbent sampling and GC analysis of formaldehyde

A passive method with membrane permeation sampling for the determination of time-weighted-average (TWA) concentration of formaldehyde in air is described. The sampling device was constructed by affixing an unbacked dimethyl silicone membrane to the base of a glass tube and by sealing the top with a rubber stopper. Formaldehyde permeates the membrane and reacts with 2-(hydroxymethyl)piperidine (2-HMP) coated on the surface of XAD-2. Sampling times from 15 min to 8 hr have been used. The formaldehyde—oxazolidine produced is thermally desorbed and determined by a packed column gas chromatograph equipped with a flame ionization detector (FID). The response of the monitor is directly proportional to the external concentration of formaldehyde over the concentration range 0.050–100 ppm. The permeation constant (the slope of the permeation curve) of the membrane is 0.333 μg ppm⁻¹. hr, and the detection limit of the method is 0.03 ppm for an 8-hr sampling period. Relative humidity (RH) (35–94%), temperature (0–82°) and storage period (0–25 days) do not affect the permeation process for sample collection. Moreover, potential chemical interferences, 10 ppm acetone or acrolein, respectively, have no detectable effect on the process. The method gives TWA concentration directly from the measurements, and the equipment is economical and convenient for personal or multi-location sample collections.     

Extraction and isolation of acetylcholinesterase inhibitors from Citrus limon peel using an in vitro method

A simple and efficient ultrafiltration–liquid chromatography–mass spectrometry–based method was developed for the rapid screening and identification of ligands from Citrus limon peel, which are suitable acetylcholinesterase inhibitors. Subsequently, the anti‐Alzheimer's activity of these compounds was assessed using a PC12 cell model. Six major compounds, viz. neoeriocitrin, isonaringin, naringin, hesperidin, neohesperidin, and limonin, were identified as potent acetylcholinesterase inhibitors. A continuous and efficient online method, which involved the use of a microwave‐assisted extraction device, solvent concentration tank, and centrifugal partition chromatography column, was developed for the scale‐up of these compounds, and the obtained compounds presented high purity. Next, their bioactivity was evaluated using a PC12 cell model. This novel approach, which was based on ultrafiltration–liquid chromatography–mass spectrometry, microwave‐assisted extraction online coupled with solvent concentration tank, and centrifugal partition chromatography along with in vitro evaluation, could represent a powerful tool for the screening and extraction of acetylcholinesterase inhibitors from complex matrices, and could be a useful platform for the large‐scale production of bioactive and nutraceutical ingredients.     

A new method of analysis of peroxydisulfate using ion chromatography and its application to the simultaneous determination of peroxydisulfate and other common inorganic ions in a peroxydisulfate matrix

A new method for the determination of peroxydisulfate using ion chromatography has been developed. Elution of peroxydisulfate was effected by isocratic elution using 200 mM NaOH at 40°C. A modification of the method using gradient elution was able to simultaneously determine other common inorganic ions (nitrate, nitrite, sulfate and chloride) down to significantly low concentrations in a peroxydisulfate matrix. The relative standard deviations (RSD) were in the range of 0.5-5%, for peak areas and <0.2% for peak retention times. The recoveries were between 95% and 120% for a concentration range of about 0.5-42 ppm. The limit of detection for peroxydisulfate ion was 0.2 ppm and for the other ions were ≤2×10(-2) ppm. The calibration curves were linear with slope and intercepts close to 1 and 0, respectively.     

Determination of thiamphenicol residues in chicken muscles by column liquid chromatography

A column liquid chromatographic (CLC) method for the determination of thiamphenicol residues in chicken muscles was developed. The drug is extracted from minced muscles with ethyl acetate and the extract is evaporated to dryness. The residue is dissolved in 10% sodium chloride solution and partitioned with n-hexane. Thiamphenicol is extracted with ethyl acetate and, after evaporation of the solvent, the residue is cleaned up by alumina column chromatography. CLC analysis is carried out on a Nucleosil C18 column with ultraviolet detection of thiamphenicol at 230 nm. The average recoveries of thiamphenicol added to muscles at 0.2 and 0.1 ppm were 92.8 and 90.0%, respectively. The detection limit was 5 ng for thiamphenicol standard, which corresponds to 0.05 ppm in muscles.     

Determination of deoxynivalenol in whole wheat flour and wheat bran

A liquid chromatographic (LC) method was developed for determining deoxynivalenol (DON) in whole wheat flour and wheat bran. A 15 g test sample was extracted with acetonitrile-water (84 + 16, v/v) and applied to a Romer MycoSep cleanup column. The eluate was dried and then reconstituted in a 0.1 M phosphate buffer, pH 7.0, and applied to a Vicam DONtest-LC cleanup column. The methanol eluate was chromatographed with a methanol-water (17 + 83, v/v) mobile phase on a C18 column with UV detection at 220 nm. Five replicates at each of 5 fortification levels (0.25, 0.50, 1.0, 2.0, and 4.0 ppm), plus 5 controls, were determined for both whole wheat flour and wheat bran. For flour, the average recoveries were 72.2-91.5% with relative standard deviations (RSDs) of 4.9-18.4%. The intra-assay flour recovery was 82.4% with 9.8% RSD. A 5 replicate sample of naturally incurred wheat had an average of 1.1 ppm DON with 6.7% RSD. For bran, average recoveries of fortified samples were 69.5-99.7% with RSDs of 1.7-18.8%. The intra-assay bran recovery was 81.5% with 8.9% RSD. The limit of detection (about 3x noise) for the method is 0.05 ppm; the correlation coefficient (linearity) was >0.9995. The DON peak was clearly identified and easily integrated in the chromatograms.     

A simple and selective sensor for the determination of ascorbic Acid in vitamin C tablets based on paptode.

A method for the determination of ascorbic acid in vitamin C tablets based on a very simple paptode design on TLC strips is described. This procedure is based on the reduction of iron(III) with ascorbic acid and the formation of a colorful red complex with immobilized 2,2'-dipyridyl (dipy) on TLC strips. The linear range of the system was 20-200 ppm with a detection limit of 1 ppm and a relative standard deviation of 1.5% (n = 28). The parameters, such as pH, concentration of iron(III), concentration of dipy and the volume of dipy per 1 cm(2) of TLC strips, were optimized. The proposed sensor was successfully applied for the determination of ascorbic acid in vitamin C tablets.     

High-performance liquid chromatographic method for determining trichothecene mycotoxins by post-column fluorescence derivatization

The method described here is based on a separation of deoxynivalenol, nivalenol and fusarenon-X on a C18 column using aqueous acetonitrile, and successive post-column fluorescence derivatization involving an alkaline decomposition to form formaldehyde and modified Hantzsch reaction with methyl acetoacetate and ammonium acetate (lambda ex = 370 nm and lambda em = 460 nm). By this method, 5-10 ng of the standard trichothecenes could be determined. By employing a clean-up procedure with a florisil column and a Sep-Pak CN cartridge, 61.4-96.9% recoveries were obtained for deoxynivalenol and nivalenol added to corn, wheat and barley at concentration levels of 0.05-1 ppm.