The determination of raloxifene in rat tissue using HPLC

We report a rapid and reliable HPLC-UV method for determination of raloxifene, a kind of selective estrogen receptor modulator (SERM), in rat tissue. Proteins were precipitated by adding 200 microL of acetonitrile and 50 microL of methanol to 100 microL of the tissue homogenates, following vortex mixing and centrifugation. Separation was carried out on a reversed-phase C(18) column (150 x 4.6 mm, 5 microm) with a mobile phase of acetonitrile:0.05 m ammonium acetate (pH 4.0 +/- 0.1; 33:67, v/v) at a flow rate of 1.0 mL/min. The UV detection wavelength was set at 289 nm and the temperature of column was kept at 23 degrees C, without interference from endogenous tissue compounds. The calibration curve was linear from 0.0125 to 10.0 microg/mL with correlation coefficient of over 0.994, while the limit of quantification was 0.008 microg/mL. The intra- and inter-day coefficients of variation were less than 10% (RSD). The recovery of assay was between 95.8 and 104.5%. Furthermore, the method was used to measure the concentration of raloxifene in rat tissue after a simple oral dose. The highest level was observed in liver, lung, spleen, then heart and kidney. The lowest level was found in brain. These results suggest that raloxifene distributes rapidly and moderately into tissues such as liver, lung and spleen.     

Application of Multivariate Calibration Techniques to Simultaneous Spectrophotometric Determination of Copper and Iron Using 1‐(2‐Pyridylazo)‐2‐naphthol in AOT Micellar Solution

Multivariate calibration models (PCR and PLS) were developed for simultaneous determination of Fe(III) and Cu(II) with 1‐(2‐pyridylazo)‐2‐naphthol and AOT as chromogenic reagent and micellizing agent, respectively. In the presence of AOT the spectrum of Fe(III)‐PAN complex was shifted to higher wavelength and the overlapping with Cu‐PAN spectrum decreased. It seems that this anionic surfactant enters the structure of the Fe‐PAN complex to cause a shift in the absorption spectrum of it. The parameters controlling behavior of the systems were investigated and optimum conditions were selected. Sixteen ternary mixtures were selected as the calibration set. To select the number of factors in PCR and PLS algorithms, a cross validation method, leaving out one sample at a time, was employed. The calibration models were validated with 8 synthetic mixtures containing the metal ions in different proportions that were randomly designed. The best calibration model was obtained by using PLS regression. The method was successfully applied to simultaneous determination of copper and iron in biological samples.     

HPLC法同时测定乳酸菌饮料中2种防腐剂和3种甜味剂

建立了一种高效液相色谱法同时测定乳酸菌饮料中2种防腐剂（苯甲酸、山梨酸）、3种甜味剂（乙酰磺氨酸钾、天门冬酰苯丙氨酸甲酯、糖精钠）的方法。样品经12％乙酸锌溶液和12％亚铁氰化钾溶液处理后,用C18色谱柱分离-以甲醇和0．02mol／L乙酸铵溶液为流动相梯度淋洗,紫外检测波长为230,205nm,可在30min内将5种组分完全分离,测定结果的相对标准偏差为1．1％-4．3％（n=5）,加标回收率为90％-102％。     

Simultaneous determination of quinoxaline‐1,4‐dioxides in feeds using molecularly imprinted solid‐phase extraction coupled with HPLC

A simple, selective, and reproducible molecularly imprinted SPE coupled with HPLC method was developed for monitoring quinoxaline‐1,4‐dioxides in feeds. Molecularly imprinted polymers were synthesized in methanol using mequindox (MEQ) as template molecule and acrylamide as functional monomer by bulk polymerization. Under the optimum SPE conditions, the novel polymer sorbents can selectively extract and enrich carbadox, MEQ, quinocetone, and cyadox from a variety of feeds. The molecularly imprinted SPE cartridge was better than nonimprinted, C₁₈, and HLB cartridges in terms of both recovery and precision. Mean recoveries of four quinoxaline‐1,4‐dioxides from six kinds of feeds spiked at 1.0, 10, and 100 mg/kg ranged between 75.2 and 94.7% with RSDs of less than 10%. The decision limits (CCαs) and the detection capabilities (CCβs) of four analytes were 0.15–0.20 mg/kg and 0.44–0.56 mg/kg, respectively. The class selectivity of the polymers was evaluated by checking three drugs with different molecular structures to that of MEQ.     

高效液相色谱法同步检测牛奶中替米考星、泰乐菌素和螺旋霉素残留量

研究了牛奶中替米考星、泰乐菌素和螺旋霉素残留量的液相色谱同步测定方法。方法采用ZORBAX Eclipse XDB C18(5μm,150 mm×4.6 mmi.d)反相色谱柱,以甲醇为提取液,以SCX因相萃取柱为净化柱,流动相为0.05 mol/L磷酸二氢钠溶液 乙腈,梯度洗脱,流速1 mL/min,用二极管阵列检测器检测,替米考星和泰乐菌素的检测波长285 nm,螺旋霉素的检测波长232 nm,进样量100μL。替米考星、泰乐菌素和螺旋霉素的检出限分别为:30、20、40μg/kg,线性范围为20~800μg/kg,加标回收率为88.8%~99.4%,相对标准偏差为2.2%~8.9%。方法适用于牛奶中替米考星、泰乐菌素和螺旋霉素残留量的同步检测。     

Determination of paeoniflorin,calycosin‐7‐O‐β‐d‐glucoside,ononin, calycosin and formononetin in rat plasma after oral administration of Buyang Huanwu decoction for their pharmacokinetic study by liquid chromatography–mass spectrometry

The purpose of this study was to simultaneously investigate the pharmacokinetics of five bioactive compounds in rat plasma after oral administration of Buyang Huanwu decoction (BYHWD) using high‐performance liquid chromatography coupled with mass spectrometry (HPLC‐MS). The separations were performed on a Thermo Hypersil Gold C₁₈ analytical column (50 × 2.1 mm, 3 µm) with the column temperature kept at 30°C. The quantitative analysis was performed using a quadrupole mass spectrometer detector operated under selected ion monitoring mode. A linear gradient elution of A (0.1% formic acid solution) and B (100% acetonitrile) was used at a flow rate of 0.2 mL/min. The method was validated within the concentration ranges 1.8–450, 6.0–1500, 2.0–500, 1.2–300 and 1.2–150 ng/mL for paeoniflorin, calycosin‐7‐O‐β‐d ‐glucoside, ononin, calycosin and formononetin, respectively. The calibration curves were linear with correlation coefficients > 0.99. The lower limits of quantitations were < 6.0 ng/mL. The method was further applied to assess the pharmacokinetics of the five bioactive constituents of BYHWD in rat plasma. Copyright © 2010 John Wiley & Sons, Ltd.     

Lipophilicity determination of N-( benzothiazol-2-yl )-α-amino alkyl phosphonic diesters by RP-HPLC and RP-HPTLC

Using methanol-water mixtures as the mobile phase,the chro-matographic retention parameters k' and Rf were determined by reversed-phase high-performance liquid chromatography(RP-HPLC)and reversed-phase high-performance thin-layer chromatography(RP-HPTLC)for N-(benzothiazol-2-yl)-o) amino alkyl phosphonic diesters and the correlation with lipophilicity parameter(ClogP)was established.Logkw values obtained from RP-HPLC and R values obtained from RP-HPTLC can be used to evaluate the lipophilicity of this kind of compounds.Chromatographic method is a good alternative for lipophilicity measurement.     

Simultaneous quantification of anthraquinone glycosides,aglycones, and glucuronic acid metabolites in rat plasma and tissues after oral administration of raw and steamed rhubarb in blood stasis rats by UHPLC-MS/MS

Rhubarb is a widely used herbal medicine. To achieve different effects, rhubarb is usually steamed with rice wine (steamed rhubarb). This steaming treatment increases the blood-activating and stasis-removing effects of rhubarb. A specific and accurate ultra high performance liquid chromatography with tandem mass spectrometry method was established for simultaneous determination of anthraquinone glycosides, aglycones, and glucuronic acid metabolites in plasma and tissues after administration of raw rhubarb and steamed rhubarb in blood stasis rats. Chromatographic separation was performed on ACQUITY UPLC BEH Shield RP 18 column using the mobile phase consisting of water and acetonitrile both containing 0.1% formic acid. Satisfactory linearity, precision, accuracy, extraction recovery, and matrix effect have been achieved. From pharmacokinetic study, it showed that glucuronic acid metabolites were found abundantly in plasma as bioactive components. The lower area under concentration-time curve, maximum concentration, and higher apparent volume of distribution (P < 0.01), body clearance (P < 0.01) values in steamed rhubarb showed that most components of steamed rhubarb have lower bioavailability in plasma compared with raw rhubarb. But it found these components were mainly distributed in spleen and liver with large blood flow and perfusion rates. The pharmacokinetics and tissue distribution studies of anthraquinone components will provide helpful information for clinical application of steamed rhubarb and raw rhubarb.     

Stopped-flow kinetic determination of a binary mixture: Simultaneous determination of europium and cerium

A kinetic method is proposed for the simultaneous determination of europium(II) and cerium(III), based on their inductive action on the Cr(VI)-iodide redox reaction in weak acidic medium. The reaction rate was followed by the stopped-flow spectrophotometric technique using starch-iodine complex as indicator. The method can be used for the determination of europium and cerium after reduction with zinc metal powder in the concentration ranges 0–2.1 and 0–1.8 g/ml, respectively. The detection limits are 0.015 g/ml Eu and 0.010 g/ml Ce. 100 to 1000 times higher concentrations of other rare earth metal ions and Th⁴⁺, Al³⁺, Mn²⁺, Hg²⁺, Co²⁺, Ni²⁺, Cu²⁺, Pb²⁺ do not interfere. The method is applied to the determination of europium and cerium in a synthetic sample and in barium yttrium fluoride fluorescent material, with relative standard deviation of about 4%.     

Interaction of naphthalene‐labeled poly(hydrochloride quaternized 2‐norbornene‐5‐methyleneamine) with O/W microemulsion droplets

Interaction between naphthalene‐labeled poly(hydrochloride quaternized 2‐norbornene‐5‐methyleneamine), poly(HCQNBMA)/NA (luminophore), and quencher, emulsifier or coemulsifier in the aqueous or microemulsion media was studied by using steady‐state fluorescence measurements. Fluorescence experiments were carried out with poly(HCQNBMA)/NA dissolved in the aqueous solutions of ionic and nonionic emulsifiers, emulsifier/n‐hexane/water microemulsion (A) and emulsifier/n‐hexane/1‐pentanol/water microemulsion (B), respectively. The intensity of fluorescence emission of poly(HCQNBMA)/NA was much higher in the aqueous phase than in microemulsion. Furthermore, the aqueous solution of ionic emulsifier increased the monomer emission. The increase in the monomer emission can be ascribed to the shielding of the naphthalene (NA) groups by SDS micelles. This separates NA groups from each other, which depresses the deactivation of excited states. The strong decrease in monomer emission within the microemulsion media probably results from the elongated conformation structure of the polymer molecule, and higher conformation freedom of NA groups, which increases interaction between the probe and the quencher. The formation of nonfluorescence clusters is not ruled out. The quenching of NA emission by nitromethan (NM) is much stronger in microemulsion than in the aqueous phase, and the quenching is more pronounced for the low molecular weight 2‐norbornene‐5‐methoxylnaphthalene (NBMNA) than for its polymer. The extent of penetration of reactants into the interfacial layer governs quenching of hydrophobic NA probe by hydrophilic quencher. Furthermore, the quenching events are connected with the thickness and density of the interfacial layer, as well as its charge. The addition of coemulsifier (1‐pentanol) increases the total surface area of the microdroplets, the entry rate of reactants into the microdroplets, and the interaction of hydrophobic and hydrophilic reactants. The quenching events are more pronounced in the close packed o/w interfacial layer than in the loosely packed one. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 39: 101–114, 2001     

Development and validation of a single HPLC method for determination of α‐tocopherol in cell culture and in human or mouse biological samples

A straightforward and common analytical method for α‐tocopherol (αT) determination in various biological samples, including plasma, red blood cells (RBC), tissues and cultured cell lines, was developed and validated, using a reverse phase‐chromatographic method (RP‐HPLC). Even though many chromatographic methods for αT determination have been reported, most of them require readjustment when applied to different types of samples. Thus, an effective and simple method for αT determination in different biological matrices is still necessary, specifically for translational research. This method was applied using a C₁₈ column (250 × 4.6 mm, 5 µm particle size) under isocratic elution with MeOH:ACN:H₂O (90:9:1 v/v/v) at a flow rate of 1 mL/min and detected using photodiode array at 293 nm. Linearity (r >0.9997) was observed for standard calibration with inter‐ and intraday variation of standard <4%. Lower limits of detection and quantification for αT in this assay were 0.091 and 0.305 µg/mL respectively. Validation proved the method to be selective, linear, accurate and precise. The method was successfully applied in great variety of biological samples, that is, human and mouse plasma, RBCs, murine tissues and human/mouse/rat cultured cell lines. More importantly, a single protocol of extraction and detection can be applied, making this method very convenient for standardization of different types of samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous determination of phthalates and adipates in human serum using gas chromatography–mass spectrometry with solid‐phase extraction

A gas chromatography–mass spectrometry assay was developed and validated for the simultaneous determination of phthalates and adipates in human serum. The phthalates and adipates studied were dimethyl phthalate, diethyl phthalate, dibutyl phthalate, benzylbutyl phthalate, di‐2‐ethylhexyl phthalate, di‐n‐octyl phthalate, diethyl adipate, dibutyl adipate, diisobutyl adipate, bis(2‐butoxyethyl) adipate and di‐2‐ethylhexyl adipate, with diisooctyl phthalate as internal standard. The extraction and cleaning up procedure was carried out with solid‐phase extraction cartridges containing dimethyl butylamine groups, which showed extraction efficiencies over 88% for each analyte and the internal standard. The calibration curves obtained were linear with correlation coefficients greater than 0.98. For all analytes, the assay gave CV% values for intra‐day precision from 4.9 to 13.3% and mean accuracy values from 91.4 to 108.4%, while inter‐day precision was 5.2–13.4% and mean accuracy 91.0–110.2%. The limits of detection for the assay of phthalates and adipates were in the range 0.7–4.5 ng/mL. The method is simple, sensitive and accurate, and allows for simultaneous determination of nanogram levels of phthalates and adipates in human serum. It was successfully applied to an investigation on the level of phthalates and adipates in a non‐occupationally exposed population.     

HPLC法同时测定松果菊属中4种酚类化合物

松果菊属(Echinacea Moenck.)是原产于美洲的一类菊科野生花卉,也称“紫锥菊”。该属植物共有8种及数个变种,均为多年生草本,其中已开发为药品者主要为淡紫松果菊(E.palli-a)、狭叶松果菊(E.angustifolia)及紫花松果菊(E.purpurea)[1]。松果菊属植物主要用于促进机体的免疫功能     

Simultaneous determination of cobalt,nickel and iron by multi-wavelength spectrophotometry

A method for the simultaneous spectrophotometric determination of cobalt, nickel and iron based on the formation of their complexes with 1,5-bis(di-2-pyridylmethylene) thiocarbonohydrazide is proposed. The absorption curves of these complexes overlap severely in the scanning range 390–510 nm. The analyte concentrations are calculated by a least squares fit of the pure spectra to the mixture spectra, which therefore makes the simultaneous determination of these metallic ions possible without tedious pretreatment. The detection limits afforded by the proposed method range from 0.05 g/ml for Fe and Ni to 0.1 g/ml for Co. Root-mean-squared errors of prediction of 0.085 g/ml for Co, 0.048 g/ml for Ni and 0.1172 g/ml for Fe were obtained using the wavelength range 400–510 nm and 0.147 g/ml for Co, 0.107 g/ml for Ni and 0.127 g/ml for Fe using the wavelength range 420–434 nm. The effect of interferences is studied and the proposed method is applied to analysis for the above elements in synthetic samples and real samples, such as biological materials and alloys.     

Validated Method for the Simultaneous Determination of Lisinopril,Pravastatin, Atorvastatin and Rosuvastatin in API,Formulations and Human Serum by RP‐HPLC

Lisinopril is found to be useful in hypertension and statins as cholesterol lowering drug. Present work was designed for the simultaneous determination of lisinopril in presence of pravastatin, atorvastatin, and rosuvastatin using RP‐HPLC method. A Purospher star C18 (5 μm, 25×0.46 cm) column was used with mobile phase consisting of acetonitrile:water (60:40 V/V, pH 3.0) with flow rate of 1.0 mL·min^?1 and the quantitative evaluation was performed at 225 nm. The retention time of lisinopril was 2.0 min and for pravastatin, rosuvastatin and atorvastatin was found to be 3.1, 4.5 and 8.3 min respectively. Suitability of this method for the quantitative determination of the drugs was proved by validation in accordance with the requirements laid down by International Conference on Harmonization (ICH) guidelines. Application of the suggested procedures were successfully applied to the determination of these compounds in active pharmaceutical ingredient and in pharmaceutical preparations, with high percentage of recovery, good accuracy and precision.     

Simultaneous determination of caffeic acid and its major pharmacologically active metabolites in rat plasma by LC‐MS/MS and its application in pharmacokinetic study

A simple, sensitive and selective high‐performance liquid chromatography electrospray ionization tandem mass spectrometry (LC‐MS/MS) method was developed for simultaneous determination and pharmacokinetic study of caffeic acid (CA) and its active metabolites. The separation with isocratic elution used a mobile phase composed of methanol and water (containing 0.1% formic acid) at a flow rate of 0.2 mL/min. The detection of target compounds was done in selected reaction monitoring (SRM) mode. The SRM detection was operated in the negative electrospray ionization mode using the transitions m/z 179 ([M ? H]^?) → 135 for CA, m/z 193 ([M ? H]^?) → 134.8 for ferulic acid and isoferulic acid and m/z 153 ([M ? H]^?) → 108 for protocatechuic acid. The method was linear for all analytes over the investigated range with all correlation coefficients 0.9931. The lower limits of quantification were 5.0 ng/mL for analytes. The intra‐ and inter‐day precisions (relative standard deviation) were <5.86 and <6.52%, and accuracy (relative error) was between ?5.95 and 0.35% (n = 6). The developed method was applied to study the pharmacokinetics of CA and its major active metabolites in rat plasma after oral and intravenous administration of CA. Copyright © 2014 John Wiley & Sons, Ltd.     

The determination of methylmercury in biological samples by HPLC coupled to ICP‐MS detection

The use of high‐performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICP‐MS) for the determination of methylmercury (MeHg⁺) in fish tissue and hair samples is described. Analysis of these sample types is required when carrying out biomonitoring studies to determine human dietary exposure to this toxic mercurial compound. The developed method used a mobile phase containing an organic modifier and a sulfydryl compound (1:1 v/v methanol:water containing 0.01% v/v 2‐mercaptoethanol) to limit peak tailing and aid separation. The chromatographic separation was coupled to the ICP‐MS detector via a short piece of PEEK tubing, attached to the nebulizer. A cooled spraychamber and oxygen addition post‐nebulization were required to limit the solvent loading on the plasma and reduce carbon build‐up on the cones, respectively. The sample preparation procedure employed a drying step followed by digestion of the sample using tetramethylammonium hydroxide (TMAH) and heating in an open vessel microwave system. Two fish tissue certified reference materials (CRM), tuna fish CRM 463 and 464 (BCR, Brussels), a tuna fish proficiency test sample, IMEP‐20 (IRMM, Geel, Belgium) and a hair CRM NIES no. 13 (National Institute of Environmental Science, Japan), were used to evaluate the method. The recovery of MeHg⁺ for these four materials was between 83 and 100%, with precisions better than 6% for three separate extractions of the different materials. The limit of quantitation for MeHg⁺ using the developed protocol was 0.5 µg Hg g^?1. The stability of MeHg⁺ in the fish sample extracts was also assessed and losses of 14–16% were observed after storage of the extracts in a refrigerator at 5 °C, in high‐density polypropylene tubes, for 6 months. The developed protocol has been used previously with atmospheric pressure ionization mass spectrometry (API‐MS) to provide structural characterization and also with calibration via isotope dilution (IDMS) to provide high accuracy quantitation. Copyright © 2006 John Wiley & Sons, Ltd.     

Application of 10‐ethyl‐acridine‐3‐sulfonyl chloride for HPLC determination of aliphatic amines in environmental water using fluorescence and APCI‐MS

A simple, sensitive method for the determination of aliphatic amines based on a sulfonylation reaction using 10‐ethyl‐acridine‐3‐sulfonyl chloride (EASC) as pre‐column labeling reagent with fluorescence detection and APCI‐MS identification has been developed. The labeled derivatives exhibited high stability and were enough to be efficiently analyzed by HPLC with an excitation maximum at λ_ex 270 nm and an emission maximum at λ_em 430 nm. Identification of derivatives was carried out by online post‐column MS in positive‐ion mode. Comparing with the widely used 5‐dimethylaminonaphthalene‐1‐sulfonylchloride (Dansyl‐Cl), EASC‐amine derivatives not only exhibited high fluorescence but also exhibited excellent MS ionizable potential. Detection limits obtained from 0.10 pmol injection, at a S/N of 3, were 4.0–12.7 fmol. The mean intra‐ and inter‐assay precision for all aliphatic amine levels were <3.84 and 3.21%, respectively. Excellent linear responses were observed with coefficients of >0.9995.     

Simultaneous determination of nicotinamide,nicotinic acid,riboflavin, thiamin,and pyridoxine in enriched Brazilian foods by HPLC

The control of the enrichment levels in foods is difficult, due mainly to the lack of appropriate analytical methodologies. The amounts of the five B-group vitamins (nicotinamide, nicotinic acid, ribofla-vin, thiamin, and pyridoxine) have been determined in enriched Brazilian foods by a high-performance liquid chromatography (HPLC) method. Fifty products, such as biscuits, liquid and dry milks, flavored milk drinks, flour, macaroni, and cereals were analyzed. Some products showed the amounts declared on the package. Although some slight quantitative variations were shown in the biscuits, one showed levels of riboflavin 35% lower than the value declared. Of five different corn cereal brands, only one showed the declared vitamin content, the others showing levels 30 % lower than that declared. No B-group vitamins were detected in one brand of enriched macaroni, except for the nicotinic acid naturally present in the flour. On the other hand, one flavored milk drink exhibited vitamins levels 200% higher than the amounts declared and one milk drink mix presented thiamin, riboflavin, and nicotinamide levels 3 to 5 times greater than stated. These results suggest an absence of control of the amount of vitamins in enriched foods.