Simultaneous Determination of Xanthopterin and Isoxanthopterin in Human Urine by Synchronous Fluorescence Spectroscopy

A simple, rapid, sensitive and selective method for simultaneously determining xanthopterin and isoxanthopterin content in human urine has been developed using synchronous fluorescence spectroscopy based on their intrinsic fluorescence. The synchronous fluorescence spectra were obtained with Δλ = 65 nm in a pH 8.5 KH₂PO₄-NaOH buffer solution. The detected wavelengths of quantitative analysis were set at 410 nm for xanthopterin and 325 nm for isoxanthopterin, respectively. Pretreatment of urine samples only was filtrated through a 0.45 μm membrane filter, which was free from the tedious separation procedures. Under optimized conditions, the limits of detection (LOD) were 0.94 ng/mL for xanthopterin and 0.48 ng/mL for isoxanthopterin. The recoveries ranged from 88.0% to 103.8 % for healthy and cancer urine samples, with coefficient of variation between 2.09% and 7.06%. The proposed method has been successfully applied to the simultaneous analysis for xanthopterin and isoxanthopterin in human urine. The results showed that the average level of isoxanthopterin was significantly elevated in urine excreted by stomach cancer patients (P < 0.01), while no significant change of xanthopterin level was found between stomach cancer patients and healthy individuals. This potentially indicates that an increase in amounts of isoxanthopterin can be associated with the presence of stomach cancer.     

First Derivative Synchronous Fluorescence Spectroscopy for the Simultaneous Determination of Sulpiride and Mebeverine Hydrochloride in Their Combined Tablets and Application to Real Human Plasma

A rapid, simple and highly sensitive first derivative synchronous fluorometric method has been developed for the simultaneous analysis of binary mixture of sulpiride (SUL) and mebeverine hydrochloride (MEB). The method is based upon measurement of the synchronous fluorescence intensity of these drugs at ∆λ = 100 nm in water. The different experimental parameters affecting the fluorescence of the two drugs were carefully studied and optimized. The fluorescence-concentration plots were rectilinear over the range of 0.05–1 μg/mL and 0.2–3.2 μg/mL for SUL and MEB respectively with lower detection limits (LOD) of 0.006 and 0.01 μg/mL and quantification limits (LOQ) of 0.0.02 and 0.05 μg/mL for SUL and MEB, respectively. The proposed method was successfully applied for the determination of the two compounds in synthetic mixtures and in commercial tablets. The high sensitivity attained by the proposed method allowed the determination of both of SUL and MEB metabolite (veratic acid) in real human plasma samples applying second derivative synchronous fluorometric technique. The mean% recoveries (n = 3) for both MEB metabolite (veratic acid) and SUL were 99.82 ± 2.53 and 98.84 ± 6.20 for spiked human plasma respectively, while for real human plasma, the mean% recoveries (n = 3) were 91.49 ± 4.25 and 91.36 ± 8.46 respectively.     

Second-derivative Synchronous Fluorometric Method for the Simultaneous Determination of Cinnarizine and Domperidone in Pharmaceutical Preparations. Application to Biological Fluids

A rapid, simple and highly sensitive second derivative synchronous fluorometric method has been developed for the simultaneous analysis of binary mixture of cinnarizine (CN) and domperidone (DOM). The method is based upon measurement of the native fluorescence of these drugs at Δλ = 80 nm in aqueous methanol (50% V/V). The different experimental parameters affecting the native fluorescence of the studied drugs were carefully studied and optimized. The fluorescence-concentration plots were rectilinear over the range of 0.1 to 1.3 μg mL⁻¹ and 0.1–3.0 μg mL⁻¹ for CN and DOM, respectively with lower detection limits of 0.017 and 5.77 × 10⁻³ μg mL⁻¹ and quantification limits of 0.058 and 0.02 μg mL⁻¹ for CN and DOM. The proposed method was successfully applied for the determination of the studied compounds in synthetic mixtures and in commercial tablets. The results obtained were in good agreement with those obtained with reference methods. The high sensitivity attained by the synchronous fluorometric method allowed the determination of CN in real and spiked human plasma. The mean % recoveries in case of spiked human plasma (n = 3) were 96.39 ± 1.18 while that in real human plasma (n = 3) was 104.67 ± 4.16.     

恒能量同步荧光法快速同时测定蒽和9, 10-二甲基蒽

建立了蒽和9,10-二甲基蒽的同时恒能量同步荧光分析法。它们的恒能量同步荧光光谱和常规荧光光谱相比,分辨力明显提高。蒽和9,10-二甲基蒽的线性范围分别为0～2 μg·mL-1和0～5 μg·mL-1,检出下限分别为2.2 ng·mL-1和1.7 ng·mL-1, 相对标准偏差不大于2%。水样中蒽和9,10-二甲基蒽的回收率为85%～103％。该方法简便快速,无需预分离。     

同步扫描荧光光谱法同时测定阿司匹林和水杨酸

建立了同步扫描荧光光谱法单次扫描同时测定阿司匹林和水杨酸双组分的方法。荧光光度计同步扫描时,得到两组分互不干扰的荧光峰,借此分别测定两组分的含量。阿司匹林和水杨酸浓度分别在4.0×10-6～1.0×10-4 mol·L-1, 8.0×10-7～1.0×10-4 mol·L-1范围内与荧光强度呈线性关系,相关系数分别为0.994 9和0.997 5,水杨酸的检测限为4.0×10-7 mol·L-1。该法简单、快速准确、易操作,可用于药物制剂中阿司匹林和水杨酸的同时测定。     

低温恒能量同步荧光法同时快速检测食品中多种多环芳烃

恒能量同步荧光法应用于多环芳烃的检测可以提高选择性,低温可使谱带呈指纹特征,提供常温光谱无法获得的光谱细节信息,有助于实现对复杂基体中多环芳烃的检测。文章结合恒能量同步荧光扫描技术与斯波斯基低温技术,建立了食品中多种多环芳烃的低温恒能量同步荧光同时快速分析方法。对低油脂样品直接用正辛烷浸泡,高油脂样品也只需要增加皂化萃取,即可进行光谱扫描来检测食品样中的多种多环芳烃。对两种类型的实际样进行加标回收实验,回收率为80.2%～98.9%,定量工作曲线线性较好(r≥0.993 8)。该方法选择性好、操作简便快捷、费用低廉。     

卟啉全内反射同步荧光法测定蛋白质

应用全内反射与同步荧光相结合的技术,建立了利用蛋白质吸附在液-固界面上的同步荧光信号来测定本体蛋白质溶液浓度的新方法。其原理为meso-四(4-磺酸基苯基)卟啉(TPPS)标记牛血清蛋白(BSA)在石英界面的吸附后信号强度随本体溶液浓度的增加呈线性关系。方法的检出限是94 ng·mL-1。用于实际人血清蛋白样品的测定,结果令人满意。     

Spectrophotometric Determination of Pyrithioxin in Pharmaceutical Preparations

Rapid direct and induced difference spectrophotometric methods for determination of pyrithioxin in single dosage forms (tablets and syrups) are reported. The direct methods depend upon measurement of the absorbance of pyrithioxin in different media at λ_max i-e at 296 nm in 0.1 M hydrochloric acid, at 328 nm in citric acid-phosphate buffer of pH 7 and at 314 nm in 0.1 M sodium hydroxide. The mean percentage recovery of the authentic samples were 100.55±0.43, 101.21±0.58 and 100.29±0.64 respectively (P=0.05). The absorbance difference methods are based upon either measurement of the difference between the acid and the alkaline solutions i-e. Δ A (Alk-Acid) at 318 nm with an accuracy of 100.72±0.88 or the absorbance difference between the acid and neutral solutions i-e Δ A (pH 7-acid) at 328 nm with an accuracy of 100.31±0.68.     

二维相关光谱探测荧光能量转移现象

以EuCl₃和NdCl₃混合水溶液为研究对象,按正交浓度序列以浓度为外部扰动构建紫外可见-荧光二维相关光谱。在混合溶液的二维相关光谱中,观察到了Eu3+的荧光发射谱峰与Nd^{3+的吸收谱峰之间存在交叉峰。交叉峰的出现表明Eu3+和Nd3+的荧光发射与吸收之间存在能量传递。二维相关光谱中交叉峰的产生并非由于溶剂水分子与溶质(Eu3+或Nd3+)之间相互作用;若以单一溶质的EuCl₃和NdCl₃的水溶液构造模拟的“混合溶液”的拟合光谱构建二维紫外可见-荧光相关光谱,由于Eu3+和Nd3+在空间上相互分离,无相互作用发生,交叉峰并不存在。二维相关光谱的交叉峰可为从光谱学角度探测复杂体系能量传递及其相关机制提供一条新思路。}     

同步荧光光谱比率法检测过氧亚硝酸根及其清除剂的研究

利用同步荧光光谱建立了一种新的荧光比率法检测过氧亚硝酸根(ONOO－)及评价其清除剂的方法。酪氨酸自身有荧光,在pH 8.5的磷酸缓冲溶液中,酪氨酸与CO₂和ONOO－反应生成强 荧光的酪氨酸二聚体,利用同步荧光光谱技术, 在荧光发射光谱中能同时获得酪氨酸单体(λ_em=364 nm)与二聚体的荧光峰(λ_em=406 nm),且两峰的荧光强度的比值与ONOO－的浓度呈计量相 关。结果表明荧光强度的比值不受实验参数改变的影响,与传统的荧光法相比,有利于扩大检测的线性范围,提高灵敏度。荧光强度的比值与ONOO－的浓度在1.60×10－7 mol·L-1～6.00× 10－6 mol·L-1范围内呈线性关系,线性相关系数为0.999,检测限为1.84×10－8 mol·L-1。对浓度为1.00×10－6 mol·L-1 ONOO－ 平行测定8次,其相对标准偏差为2.4％。利用该法测得抗癌药米托蒽醌的IC₅₀为0.065 μg·mL-1。方法简便易行,试剂廉价易得,体系稳定,可作为一种检测ONOO-及筛选其清除剂的方法。     

基于同步荧光光谱法的鸭肉中西维因残留含量检测研究

为了快速测定鸭肉中西维因残留含量,提出应用同步荧光光谱法检测鸭肉中西维因残留含量,同时运用遗传算法-支持向量回归(GA-SVR)建立了鸭肉中西维因残留含量的回归预测模型.首先,通过荧光分光光度计分别采集了西维因水解物和含有西维因的鸭肉的三维同步荧光光谱图,经过分析确定了最佳波长差Δλ都为140 nm;其次,分析了鸭肉中西维因的浓度猝灭现象;最后采用GA进行同步荧光光谱的优化和选择,根据交互验证均方根误差(RMSECV)选择出了21个特征波长,并分别用全波长和21个特征波长作为SVR回归预测模型的输入特征变量,发现通过GA选择的特征波长可以得到更好的预测效果,并且其预测集的相关系数(R2)达到0.9764,均方根误差(RMSECP)为12.2322.试验结果表明利用同步荧光技术结合GA-SVR方法能有效、快速的检测鸭肉中西维因残留含量.     

Simultaneous Surface-Near and Solution Fluorescence Correlation Spectroscopy

We report the first simultaneous measurement of surface-confined and solution fluorescence correlation spectroscopy (FCS). We use an optical configuration for tightly focused excitation and separate detection of light emitted below (undercritical angle fluorescence, UAF) and above (supercritical angle fluorescence, SAF) the critical angle of total internal reflection of the coverslip/sample interface. This creates two laterally coincident detection volumes which differ in their axial extent. While detection of far-field UAF emission producesa standard confocal volume, near-field-mediated SAF produces a highly surface-confined detection volume at the coverslip/sample interface which extends only ~200 nm into the sample. A characterization of the two detection volumes by FCS of free diffusion is presented and compared with analytical models and simulations. The presented FCS technique allows to determine bulk solution concentrations and surface-near concentrations at the same time.     

Synchronous Fluorescence as a Green and Selective Method for the Simultaneous Determination of Cetirizine and Azelastine in Aqueous Humor

A green, simple, quick and economical method is implemented for the first time for the simultaneous estimation of cetirizine (CTZ) and azelastine (AZE) as co-administered eye drops. The method relies on synchronous spectrofluorimetry with ?λ?=?60 nm. Cetirizine can be estimated at 231 nm and AZE can be measured at 294 nm, each at the other’s zero crossing point. All factors affecting the method were studied and properly optimized. Good correlation was obtained in the range of 0.1–2 µg mL^?1 for both drugs. The limits of detection were 0.014 and 0.010 µg mL^?1 and limits of quantitation were 0.043 and 0.029 µg mL^?1 for CTZ and AZE, respectively. Moreover, ICH guidelines were carried out to validate the adopted method. The method was suitable for the analysis of CTZ and AZE in synthetic mixtures, eye drops and aqueous humor. The mean percentage of recoveries of CTZ and AZE in spiked aqueous humor were 99.83 and 99.37, respectively. Furthermore, Green Analytical Procedure Index (GAPI) and analytical Eco-scale approaches were used to evaluate the greenness of the suggested method.

     

Simultaneous Determination of Acetylsalicylic Acid and Caffeine in Pharmaceutical Formulation by First Derivative Synchronous Fluorimetric Method

A sensitive, rapid, and specific assay has been developed for the simultaneous determination of acetylsalicylic acid and caffeine in commercial tablets based on their natural fluorescence. The mixture of these drugs was resolved by first derivative synchronous fluorimetric technique using two scans. At Δλ=106 nm, using first derivative synchronous scanning, only acetylsalicylic acid yields a detectable signal at 316 nm (peak to zero method) which is unaffected by caffeine. At Δλ=30 nm, the signal of caffeine at 288 nm (peak to zero method) is not affected by acetylsalicylic acid. The range of application is between 0.021 and 41.62 μg ml⁻¹ (correlation coefficient, R=0.9995) for acetylsalicylic acid and between 0.4486 and 44.86 μg ml⁻¹ (correlation coefficient, R=0.99786) for caffeine. The recovery range of 98.40–102% for acetylsalicylic acid and 90–100.5% for caffeine from their synthetic mixture was reported. Overall recovery of both compounds about 97–99% for acetylsalicylic acid and 97–98% for caffeine was obtained from real sample analysis. The detection limits are 0.0013 μg ml⁻¹ and 0.0306 μg ml⁻¹ for acetylsalicylic acid and caffeine, respectively. The relative standard deviation (n=10) for 20 μg ml⁻¹ of acetylsalicylic acid is 2.75% and for 2.2 μg ml⁻¹of caffeine is 1.7%.     

Fluorescence Determination of Azithromycin in Pharmaceutical Formulations by Using the Synchronous Scanning Approach After its Acid Derivatization

In this present work, a fluorescence method for azithromycin (9-deoxo-9a-aza-9a-methyl-9a-homoerythromycin) determination in pharmaceutical formulations is proposed. The method is based on the synchronous fluorescence (Δλ?=?30 nm, 482 nm) produced when azithromycin is derivatized in strong acidic medium (9.0 mol L^?1 HCl). The influence of the derivatization conditions (acid concentration, reaction time and temperature) was studied. Also, the possible reaction mechanism was discussed. In the optimized conditions, the method presented a limit of detection of 0.23 mg L^?1 and a limit of quantification of 0.76 mg L^?1. The developed procedure was successfully applied in the determination of azithromycin in pharmaceutical formulations.     

Spectrofluorimetric Determination of Etodolac, Moxepril HCl and Fexofenadine HCl Using Europium Sensitized Fluorescence in Bulk and Pharmaceutical Preparations

A simple, selective and sensitive luminescence method has been developed for the assay of etodolac (I), moxepril HCl (II) and fexofenadine HCl (III) in bulk drug and pharmaceutical formulations. The method is based on the luminescence sensitization of europium (Eu³⁺) by complexation with the studied drugs. The fluorescence intensities of the products were measured at 667 nm for (I) and at 615 for (II) and (III) while exciting at 276 for all the studied drugs. The fluorescence intensity was directly proportional to the concentration over the range (20–280), (40–240) and (30–80) ng/ml with limits of detection (LOD) = 0.93, 0.92 and 0.95 μg/ml for drugs I, II and III respectively. Optimum conditions for the formation of the complex in methanol were carefully studied. The proposed method was successfully applied for the assay of the studied drugs in pharmaceutical formulations with excellent recovery.     

Spectrofluorometric Determination of Methocarbamol in Pharmaceutical Preparations and Human Plasma

A simple, sensitive and rapid spectrofluorometric method for determination of methocarbamol in pharmaceutical formulations and spiked human plasma has been developed. The proposed method is based on the measurement of the native fluorescence of methocarbamol in methanol at 313 nm after excitation at 277 nm. The relative fluorescence intensity-concentration plot was rectilinear over the range of 0.05–2.0 μg/mL, with good correlation (r = 0.9999), limit of detection of 0.007 μg/ mL and a lower limit of quantification of 0.022 μg/ mL. The described method was successfully applied for the determination of methocarbamol in its tablets without interference from co-formulated drugs, such as aspirin, diclofenac, paracetamol and ibuprofen, The results obtained were in good agreement with those obtained using the official method (USP 30).The high sensitivity of the method allowed the determination of the studied drug in spiked human plasma with average percentage recovery of 99.42 ± 3.84.     

Conventional and First Derivative Synchronous Fluorometric Determination of Ethamsylate in Pharmaceutical Preparations and Biological Fluids. Application to Stability Studies

Two simple, accurate and highly sensitive spectrofluorometric methods were developed for the determination of ethamsylate (ETM). Method I is based on measuring the native fluorescence of ethamsylate in water at 354 nm after excitation at 302 nm. The calibration plot was rectilinear over the range of 0.05–1 μg/mL for ETM with limits of detection and quantitation of 7.9 and 26 ng/mL, respectively. Method II involved synchronous and first derivative synchronous fluorometric methods for the simultaneous determination of ethamsylate (ETM) and hydroquinone (HQ) which is considered as an impurity and/or acidic degradation product. The synchronous fluorescence of both the drug and its impurity were measured in methanol at Δ λ of 40 nm. The peak amplitudes (¹D) were estimated at 293.85 or 334.17 nm for ETM and at 309.05 nm for HQ. Good linearity was obtained for ETM over the ranges 0.1–1.4 μg/mL and 0.1–1.0 μg/mL at 293.85 and 334.17 nm, respectively. For HQ, the calibration plot was rectilinear over the range of 0.01–0.14 μg/mL at 309.05 nm. Limits of detection were 20, 2.01 ng/mL and limits of quantitation were 60, 6.7 ng/mL for ETM and HQ by method II, respectively. Both methods were successfully applied to commercial ampoules and tablets. The results were in good agreement with those obtained by the reference method. Method I was utilized to study the stability of ETM and its degradation kinetics using peroxide. The apparent first-order rate constant, half-life times and activation energy of the degradation process were calculated. Method I was further extended to the in-vitro and in-vivo determination of ETM in spiked and real plasma samples. The mean% recoveries were 99.57 ± 3.85 and 89.39 ± 5.93 for spiked and real human plasma, respectively.     

利用同步荧光光谱快速鉴别潲水油

为快速鉴别潲水油,采用三维同步荧光光谱结合平行因子法解析潲水油的特征波长差(Δλ),并利用支持向量机建立潲水油鉴别模型。结果表明,潲水油的特征Δλ为60 nm;特征Δλ下的样品原始同步荧光光谱经过主成分分析提取5个主成分,以径向基函数(RBF)为核函数,利用网格搜索和6-fold交叉验证优化建模参数,得到惩罚因子C=512、核参数g=0.5,该条件下建立的模型对训练集和预测集的判别率均达到100%。采用同步荧光光谱可以快速、准确地鉴别潲水油。