Determination of small quantities of atropine in commercial preparations by liquid chromatography with fluorescence detection

A method is presented for the determination of small quantities of atropine in commercial preparations by liquid chromatography (LC) with fluorescence detection. The sample is extracted with CHCl3 from basic suspension, the CHCl3 is evaporated on the steam bath, and the dry residue is dissolved in a small volume of CH3OH. A reverse phase column is used for the LC analysis; the eluting solvent is prepared by mixing 950 mL CH3OH with 50 mL water containing 1 g of the sodium salt of 1-pentanesulfonic acid. The fluorescence detector is set at an excitation wavelength of 255 nm and an emission wavelength of 285 nm. Several commercial tablets and injections containing atropine in combination with other ingredients and a commercial sample of belladonna extract were analyzed by the proposed method. Recoveries of atropine sulfate from aqueous solutions averaged 100.7% with a relative standard deviation (RSD) of 3.35% for atropine sulfate levels of 0.12 mg. Recoveries of atropine sulfate from synthetic injection formulations were 99.8 and 100.0% with RSDs of 2.03 and 2.35%, respectively; the atropine sulfate concentrations of commercial injections with the same formulations were found to be 97.0 and 100.0% of the labeled amounts with RSDs of 0.53 and 1.46%, respectively.     

Determination of reserpine and hydrochlorothiazide in commercial tablets by liquid chromatography with fluorescence and UV absorption detectors in series

A procedure is presented for the determination of reserpine and hydrochlorothiazide in commercial tablets by liquid chromatography (LC). Reference and sample solutions are prepared in methanol. For LC, a normal phase column is used, methanol is the eluting solvent, and 2 detectors are arranged in series. A fluorescence detector set at an excitation wavelength of 280 nm and emission wavelength of 360 nm quantitates reserpine, and a UV absorption detector set at 345 nm determines hydrochlorothiazide. Several synthetic mixtures containing the 2 ingredients in the amounts approximately present in commercial tablets were analyzed by the proposed method. Two samples of commercial tablets were also analyzed; for each sample, 5 determinations were made on a ground composite of 20 tablets; 10 individual tablets were also analyzed. For comparison, some of the solutions were analyzed for each ingredient by an alternative procedure.     

Collaborative study of a spectrofluorometric assay for Rauwolfia serpentina tablets and powdered root.

Reserpine-rescinnamine group alkaloids are extracted from Rauwolfia serpentina preparations into a dimethylsulfoxide (DMSO)-methanol mixture and diluted with 0.5N H2SO4. The chloroform extract of this solution is passed through a 0.1N NaOH-Celite column and then through a silica gel column. The weakly basic alkaloids trapped on the latter column are eluted with a methanol mixture; a portion of the eluate is treated with nitrous acid and the reserpine-rescinnamine content is determined by measuring the intensity of fluorescence of the oxidation product. The following means and standard deviations (11 collaborators) were obtained for the determination of reserpine-rescinnamine group alkaloids in 4 samples of Rauwolfia serpentina (NF reference powder, 100 mg and 50 mg commercial tablets, and a 45 mg synthetic tablet formulation) : 0.174% +/- 0.0112, 0.131% +/- 0.0047, 0.160% +/- 0.0100, and 0.153% +/- 0.0083, respectively.     

Sensitive determination of ethopabate residues in chicken tissues by liquid chromatography with fluorometric detection

A liquid chromatographic (LC) method is described for determination of ethopabate residues in chicken tissues. The drug is extracted from tissues with acetonitrile, and the extract is concentrated to 2-3 mL. This aqueous solution is rinsed with ethyl acetate and cleaned up by Florisil column chromatography. LC analysis is carried out on a Zorbax ODS column, and ethopabate is quantitated by using a fluorometric detector set at 306 nm (excitation) and 350 nm (emission). Recoveries of ethopabate added to chicken tissues at levels of 0.01 and 0.05 ppm were 87.8 and 92.7%, respectively. The detection limit was 100 pg for ethopabate standard, and 0.5 ppb in chicken tissues.     

Determination of ergotamine tartrate in tablets by liquid chromatography with fluorescence detection

A method is presented for the liquid chromatographic (LC) determination of ergotamine tartrate in tablets that is applicable even in the presence of other ingredients such as phenobarbital, belladonna alkaloids, and caffeine. The sample is transferred to a volumetric flask, a small volume of formic acid is added to dissolve and stabilize the ergotamine, and the solution is diluted to volume with methanol. The solution is mixed and filtered through paper. The LC system consists of a Rheodyne injector fitted with a 20 microL loop and a C18 reverse phase column; the mobile phase is acetonitrile-water-triethylamine (700 + 300 + 0.5). Ergotamine tartrate is determined fluorometrically at an excitation wavelength of 250 nm and an emission wavelength of 430 nm. Recovery studies were conducted at the 0.3 and 1.0 mg levels. Average recoveries were 99.6 and 100.8%, respectively; relative standard deviations (RSDs) were 1.08 and 2.21%, respectively. Some commercial preparations containing ergotamine tartrate in combination with other ingredients were also analyzed. The RSDs for 5 determinations of each of 2 ground composites were 0.09 and 0.34%.     

Liquid chromatographic determination of amprolium in chicken tissues, using post-column reaction and fluorometric detection

A method is presented for determination of amprolium residues in chicken muscles by a liquid chromatographic post-column reaction system. The drug is extracted from muscles with methanol, and the extract is concentrated to 3-4 mL. This aqueous solution is rinsed with n-hexane and cleaned up by alumina column chromatography. The drug is separated from the interferences on a LiChrosorb RP-8 column, reacted with ferricyanide in alkaline solution, and quantitated by fluorometric detection at 367 nm (excitation) and 470 nm (emission). Recoveries of amprolium added to chicken muscles at levels of 0.1 and 0.2 ppm were 74.9 and 80.9%, respectively. The detection limit was 1 ng for amprolium standard and 0.01 ppm in chicken muscles.     

Determination of reserpine in commercial tablets by liquid chromatography with fluorescence detection

A procedure is presented for the determination of reserpine in commercial tablets by liquid chromatography (LC). The sample is extracted with methanol if only reserpine is present. If the sample contains other ingredients, CHCI3 is used for extraction from aqueous suspension; the CHCI3 is subsequently completely evaporated in the presence of methanol. For LC, a normal phase column, methanol as the eluting solvent, and a fluorometric detector are used. A recovery study indicated that no measurable degradation of reserpine occurs during evaporation of the CHCI3 extract. Several commercial tablets containing reserpine alone or in combination with other ingredients were analyzed by the proposed method, and the results were compared with those obtained by the current official USP methods for reserpine.     

基于特征荧光信号的去囊衣带芯橘瓣分选

为实现去囊衣带芯橘瓣的机器视觉分选,给去囊衣带芯橘瓣的机器识别提供直接、准确的判别信号,采用荧光分光光度计对50颗去囊衣带芯橘瓣的橘芯和砂囊分别进行三维荧光光谱扫描,通过对橘芯、砂囊平均三维荧光光谱分析,确定了橘芯相对砂囊的特征荧光信号,据此对带芯橘瓣荧光图像识别的可行性进行了验证。检测发现,在370~390 nm紫外光激发下,橘芯和砂囊在440 nm处的荧光强度差异较大;橘芯与砂囊在370 nm紫外光激发下,440 nm处荧光强度分布的箱线图表明两者荧光强度分布存在明显差异,且以橘芯在440 nm处荧光强度的下四分位数(Q1)与砂囊在440 nm处荧光强度的上四分位数(Q3)的平均值作为分类标准,去囊衣带芯橘瓣检出准确率可达85%。对(370±2)nm激发下得到的单色(440±5)nm荧光图像进行二值化及形态学处理后,可在以砂囊为背景的橘瓣图形中显现橘芯亮斑。利用橘芯与砂囊的荧光特性差异进行机器视觉成像分析,可作为识别去囊衣带芯橘瓣的一种有效方法。     

用于污染黄曲霉毒素花生分选的荧光信号研究

为在加工前将黄曲霉毒素超限的带衣花生米从原料中剔除,参照已有的色选系统,提出一种依据黄曲霉毒素含量超限带衣花生米的专属荧光信号进行逐粒分选的技术构想。采用Cary Eclipse荧光分光光度计测定100粒外观具有代表性的带衣花生米表面的紫外-荧光规律,通过与免疫亲和层析净化荧光光度法(GB/T18979-2003)检测结果对比,判定了黄曲霉毒素超限带衣花生米的荧光光谱特征;通过绘制450/490、460/490荧光强度比值的箱线图,评估了表面荧光法判断黄曲霉毒素超限带衣花生米的准确率;在搭建的荧光成像系统上,对黄曲霉毒素超限带衣花生米进行了荧光成像。检测发现,在365 nm波长激发下,黄曲霉毒素超限带衣花生米在420~460 nm处有荧光峰;以450/490荧光强度比值为依据剔除超限值带衣花生米的判断准确率为81%;a.u.40的带衣花生米可在图像中呈现亮蓝荧光光斑。表明表面荧光信号可作为带衣花生米在线、无损、逐粒分选的专属光学信号,用于黄曲霉毒素超限带衣花生米的剔除。     

Fluorescence Tracing of Diffuse Landfill Leachate Contamination in Rivers

Landfill leachates are composed of a complex mixture of degradation products which include a wide range of potentially fluorescent organic molecules and compounds. Here we investigate the use of fluorescence excitation–emission matrix (EEM) analysis in detecting diffuse landfill leachate contamination in rivers. Landfill leachates from three unlined landfill sites adjacent to our study river are characterised by intense fluorescence at excitation wavelength 220–230 nm, and emission wavelength 340–370 nm, which derives from fluorescent components of the xenobiotic organic matter fraction. Seven surface water sample sites on an adjacent polluted river system were analysed for fluorescence and water quality properties. The 220–230 nm excitation wavelength, 340–370 nm emission wavelength fluorescent centre was also detected in this river system at the sample locations downstream of the landfills, but not at upstream control sites, demonstrating its use as a tracer of landfill leachate contamination. Negative correlations are observed between this fluorescence centre and dissolved oxygen in the river water samples, demonstrating the water quality implications of leachate contamination at this study site. The fluorescence intensity at the 220–230 nm excitation wavelength, 340–370 nm emission wavelength fluorescent centre in landfill leachates is such that it remains detectable at dilutions of 10²–10³, and the fluorescence EEM technique is rapid and cost-effective for use by river managers and water quality regulators.     

High pressure liquid chromatographic determination of naphthaleneacetic acid residues in apples.

An ion-suppression reverse phase high pressure liquid chromatographic method is described for determining naphthaleneacetic acid (NAA) residues in apples. Samples are extracted with acidic chloroform, filtered through pre-acidified Hy-Flo Supercel, and cleaned up by acid-base partitioning. The extract can be successfully chromatographed on either a muLiChrosorb NH2 or muBondapak C18 column and quantitated by using a variable wavelength ultraviolet detector set at 220 nm. The mobile phase is acetonitrile-water (20 + 80) buffered to pH 3.5 (MULiChrosorb column) or pH 5.2 (MUBondapak column) and flowing at 1.0--2.0 ml/min. Recoveries ranged from 86 to 98%. The minimum detectable amount was 0.5 ng, which easily permitted the quantitation of 0.01 ppm NAA in 50 g sample. A fluorometric detector was 4 times as sensitive, using an excitation wavelength of 220 mm and monitoring the emission at 340 nm. For this detector, the minimum detectable amount was 0.12 ng NAA.     

Multiresidue method for determination of eight neutral beta-lactam penicillins in milk by fluorescence-liquid chromatography

A method of determining total penicillins begins with an enzymatic hydrolysis of the beta-lactam ring to form their respective penicilloate product. Acetonitrile precipitates much of the casein and protein, which are then separated from the liquid by centrifugation. The lipids are removed from the aqueous fraction with methylene chloride. Mercuric chloride is added, which reacts with the penicilloate to liberate the side chain that has a terminal aldehyde. These penilloaldehyde products are extracted with methylene chloride and are subsequently reacted with dansyl hydrazine. The resulting fluorolabeled side chains are separated by liquid chromatography on a C18 column with acetonitrile-water as mobile phase. The fluorescence is measured by the mercury line at 254 nm excitation wavelength and a 500 nm filter on the emission side. The overall average recoveries from milk spiked at 25, 50, and 100 ppb are benzyl penicillin 79.4%; phenoxymethyl penicillin 59.7%; phenethicillin 75.9%; nafcillin 87.7%; methacillin 47.5%; oxacillin 57.6%; cloxicillin 37.3%; and dicloxicillin 26.4%.     

SPE-HPLC/FLD法同时测定水中4种雌激素

研究建立了固相萃取（SPE）-高效液相色谱仪（HPLC）-荧光检测器（FLD）测定水体中4种雌激素（雌三醇、17β-雌二醇、炔雌醇和双酚A）的分析方法。水样过C18固相萃取柱净化浓缩,用5.00mL超纯水淋洗,15.00mL甲醇洗脱,洗脱液经氮气吹干后用50%甲醇溶解经HPLC-FLD测定;4种雌激素以甲醇/乙腈/水为流动相（体积比为25：30：45）,经InertsilODS-SP-C1（8150mm×4.6mm,5μm）反相色谱柱分离,激发和发射波长分别为280nm和310nm,流速1.0mL.min-1,柱温40℃,进样量20μL,以保留时间定性、外标法定量。该方法的线性范围为5.00～1000.00μg.L-1,且相关性良好（R〉0.9999）,4种雌激素的仪器检出限为0.107～0.271μg.L-1,方法检出限为0.214～0.540ng.L-1。在自来水中添加不同浓度的雌激素混合标准溶液,测得溶液中4种物质的加标回收率除炔雌醇为55.71%～66.78%外,其余雌激素的加标回收率均大于85%,相对标准偏差RSD（n=5）均小于4%。该方法灵敏度高、检出限低、重复性和精密性良好,能有效去除基质干扰,可用于水体中痕量雌激素的分析测定。     

Steviol quantification at the picomole level by high-performance liquid chromatography

A simple and highly sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) has been developed for the determination of steviol (SV) using dihydroisosteviol (DHISV) as an internal standard (IS). SV and DHISV were derivatized by reaction of the acids with 4-(bromomethyl)-7-methoxycoumarin in an aprotic solvent (DMF or acetone). The resulting ester derivatives were separated on an ODS column (250 x 4.6 mm i.d., 5 microm particle size) using fluorescence detection with excitation at 321 nm and emission at 391 nm. The mobile phase consisted of acetonitrile/water (80:20 v/v) with a flow rate of 1 mL min(-)(1). A linear relationship was observed for concentrations between 0.5 and 50 microg/mL of SV, and the detection limit was 100 pg. For application of this method to samples of beer fortified with stevioside, a simple procedure for extraction of the beer with diethyl ether and derivatization in DMF was applied. Whereas beer samples spiked with SV gave a linear response over the range 0.1-15 microg/mL beer, no SV could be detected in beer samples enriched in stevioside that had been stored for over 3 years. The application of the method to plant samples involved preparation of an acid fraction containing the SV analyte, derivatization, and sample cleanup using small silica columns and thin-layer chromatography. A sensitive determination of 594 ng of steviol present in 100 mg of dry plant material was performed with high precision and accuracy.     

Fluorescence, browning index, and color in infant formulas during storage

Free and total fluorescent compounds, browning index, and color formation were measured in milk-based powdered infant formulas (IF) during 2 years of storage at 20 and 37 degrees C. The excitation spectra from 415 nm emission show three peaks (ex lambda1 = 270 nm, lambda2 = 325/315 nm, lambda3 = 350 nm) and from 347 nm excitation two emission peaks (415 and 520 nm), and no wavelength shifts were observed. Temperature and time of storage exert in general no significant effect on the development of fluorescence emission intensity and browning index. However, an important increase in pentodilysine was recorded-probably because of the iron and ascorbic acid contents of the samples-as well as in browning index in adapted IF. In both IF a color increase (deltaE) throughout storage was observed, this increase being greater in samples stored at 37 degrees C than in those stored at 20 degrees C. The increase in color with time fitted a linear regression model. Color appeared to be an indicator of sufficient sensitivity to measure the effect of temperature or storage time.     

Liquid chromatographic determination of multiresidue fluorescent derivatives of ionophore compounds, monensin, salinomycin, narasin, and lasalocid, in beef liver tissue

A liquid chromatographic (LC) method with fluorometric detection was developed to quantitatively determine residue levels of monensin, salinomycin, narasin, and lasalocid in beef liver tissue. The ionophores are extracted from the tissue, purified by both alumina and Sephadex LH-20 column chromatography, and then derivatized. Lasalocid was directly esterified with 9-anthryldiazomethane (ADAM), but monensin, salinomycin, and narasin were first acetylated with acetic anhydride and then esterified with ADAM. The ADAM derivatives were purified on a silica gel column and separated by LC using an RP C-8 5 micron column. A fluorescence detector set at 365 nm (excitation) and 418 nm (emission) was used to monitor the column effluent. The detection limits were 0.15 ppm, and the calibration curves were linear between 0.5 and 5.0 ppm for all 4 ionophores. Mean recoveries were 57, 70, 75, and 90% for lasalocid (5 ppm), monensin (2.5 ppm), salinomycin (2.5 ppm), and narasin (2.5 ppm), respectively. The ionophores were also separated and semiquantitated by using bioautography and thin layer chromatography with a vanillin spray.     

Simultaneous stopped-flow determination of butylated hydroxyanisole and propyl gallate using a T-format luminescence spectrometer

A simple and fast luminescent method is used for the first time to resolve a mixture of two synthetic antioxidants, propyl gallate (PG) and butylated hydroxyanisole (BHA), by the joint use of the stopped-flow mixing technique and a T-format luminescence spectrometer. The determination of these compounds involves two different and independent reactions. On the one hand, PG determination is based on an energy transfer process that involves the formation of a lanthanide chelate with terbium in the presence of Triton X-100 and tri-n-octylphosphine oxide. On the other hand, BHA is determined using a reaction between the oxidized form of Nile Blue and the antioxidant. Both systems are excited at the same excitation wavelength (310 nm), and the emission wavelengths are 545 and 665 nm for PG and BHA, respectively. The absence of overlap in the emission spectra makes it possible to measure separately the analytes in each channel of the instrument. Initial rate and equilibrium signal are used as analytical parameters and measured in 0.1 and 1 s for PG and BHA, respectively. Calibration graphs are linear over the range 0.09-3.5 microg mL(-)(1) for PG and 0.3-15 microg mL(-)(1) for BHA. The relative standard deviations of both systems are close to 2%. The proposed method is applied to the determination of these two antioxidants in several commercial food samples with recoveries ranging between 94.8 and 102.9% for PG and between 94.1 and 102.1% for BHA.     

Immunosorbents coupled on-line with liquid chromatography for the determination of fluoroquinolones in chicken liver.

Four fluoroquinolones were analyzed in fortified chicken liver using an automated, on-line immunoaffinity extraction method. The fluoroquinolones were extracted from the liver matrix using an immunoaffinity capture column containing anti-sarafloxacin antibodies covalently cross-linked to protein G. After interfering liver matrix components had been washed away, the captured fluoroquinolones were automatically eluted directly onto a reversed phase column. Liquid chromatographic analyses were performed by isocratic elution using 2% acetic acid/acetonitrile (85:15) as the mobile phase and an Inertsil phenyl column with fluorescence detection at excitation and emission wavelengths of 280 and 444 nm, respectively. No significant interferences from the sample matrix were observed, indicating good selectivity with the immunoaffinity column. Overall recoveries from fortified liver samples (20, 50, and 100 ng/g) ranged between 85.7 and 93.5% with standard deviations of <5%. The limit of quantification for each fluoroquinolone was 1 ng/mL. The limits of detection, based on a signal-to-noise ratio of 5:1, were 0.47, 0.32, 0.87, and 0.53 ng/mL for ciprofloxacin, enrofloxacin, sarafloxacin, and difloxacin, respectively.     

Simple Measurement of 4,4’-bis(2-sulfostyryl)-biphenyl in River Water by Fluorescence Analysis and Its Application as an Indicator of Domestic Wastewater Contamination

A characteristic peak of fluorescent whitening agents (FWAs) was detected by fluorescence excitation spectrum (FES) measurement of river water samples. The main causative chemical was 4,4’-bis(2-sulfostyryl)-biphenyl (DSBP), which is commonly added to household detergents in Japan. As the fluorescence of DSBP overlaps with that of fulvic-like organic matter in the spectral fluorescent signatures, DSBP concentration was determined by the newly proposed calculation method, which uses fluorescence intensity at three excitation wavelengths of 320, 345 and 360 nm at emission wavelength of 430 nm for baseline correction. The concentration of DSBP calculated using this method showed strong correlation (correlation coefficient: r = 0.992) with that obtained by high-performance liquid chromatography analysis. The concentrations of DSBP detected in river water samples were 0.28 to 1.84 μg l^?1, with high concentrations observed at the stations with relatively high flow rates of upstream sources of treated domestic wastewater and untreated gray water (domestic wastewater excluding flush toilet wastewater). It was proved that the concentration of DSBP in river water is useful for giving rough estimation of the magnitude of domestic wastewater contamination in river water.     

Liquid chromatographic determination of levodopa, levodopa-carbidopa, and related impurities in solid dosage forms

A rapid reverse phase liquid chromatographic method was developed for the determination of levodopa and levodopa-carbidopa in tablets and capsules. The method also separates these drugs from 3-(3,4,6-trihydroxyphenyl)alanine and methoxytyrosine, impurities of levodopa, and from methyldopa and 3-O-methylcarbidopa, impurities of carbidopa. The mobile phase was 3% acetic acid and the detection wavelength was 280 nm. The method was linear over the concentration range 0.05-0.40 mg levodopa/mL, 0.01-0.06 mg carbidopa/mL, 0.9-12.8 micrograms 3-(3,4,6-trihydroxyphenyl)alanine/mL, 0.7-3.1 micrograms methyldopa/mL, 5-20 micrograms methoxytyrosine/mL, and 0.5-3.3 micrograms 3-O-methylcarbidopa/mL. Mean recoveries (%) for spiked commercial tablets were: levodopa 100.3, carbidopa 100.4, 3-(3,4,6-trihydroxyphenyl)alanine 99.1, methoxytyrosine 100.0, methyldopa 100.0, and 3-O-methylcarbidopa 99.4.