Simultaneous determination of water-soluble vitamins in a vitamin-enriched drink by an in-capillary enzyme reaction method

The in-capillary enzyme reaction method was used to determine riboflavin phosphate in a vitamin-enriched drink based on its conversion to riboflavin (vitamin B2) with alkaline phosphatase. Simultaneously, three water-soluble vitamins [thiamine nitrate (vitamin B2 mononitrate), pyridoxine hydrochloride (vitamin B6 hydrochloride) and nicotinamide (vitamin PP)] and anhydrous caffeine in the drink were subjected to quantitative analysis. In the system, electrophoretic migration was used to mix zones containing the substrate (riboflavin phosphate) and the enzyme (alkaline phosphatase). The reaction was then allowed to proceed in the presence of a weak electric field and, finally, the product (riboflavin) of enzyme reaction and other water-soluble vitamins migrated under the influence of an applied electric field to the detector. All the active ingredients and the formulation excipients were successfully separated by micellar electrokinetic chromatography with 135 mM sodium dodecyl sulfate. To prevent inhibition of enzyme reaction by the addition of sodium dodecyl sulfate to the reaction zone, sandwich mode injection, in which plugs of sandwich solution without sodium dodecyl sulfate were introduced into the capillary on both sides of the reaction zone, was utilized as a barrier to protect the enzyme reaction from the inhibitor. The relationship between the peak area of the product and the concentration of the substrate was calculated in the in-capillary enzyme reaction method. Excellent linearity was obtained, with correlation coefficients of 0.9999. The established method was validated and demonstrated to be applicable to the determination of the five active ingredients, including riboflavin phosphate, in a commercial vitamin-enriched drink. No interference from the formulation excipients was observed. Good linearities were obtained, with correlation coefficients above 0.999. Recoveries and precisions ranged from 99.3 to 101.8%, and from 0.1 to 2.5% RSD, respectively. Good agreement was obtained between the established method and traditional high-performance liquid chromatographic methods. These results suggest that the in-capillary enzyme reaction method can be used for the simultaneous determination of riboflavin phosphate and other water-soluble vitamins in pharmaceuticals.     

Spectrophotometric determination of Diclofenac sodium with Methylene Blue

A sensitive spectrophotometric method was established for the determination of Diclofenac sodium (DS) with Methylene Blue (MB) as analytical reagent. It was found that DS reacts with an excess of MB in the pH range 9.2-9.4, to form a chloroform-extractable blue ion-association complex. Good agreement with Beer's law was found in the range of DS concentrations of 0.8-6.4 mug/ml with a detection limit of 0.37 mug/ml. The method was applied for the determination of DS in various tableted forms with a good precision.     

Development and validation of a solid-phase extraction and gas chromatography-tandem mass spectrometry method for the determination of isopropyl-9H-thioxanthen-9-one in carton packaged milk

A simple and efficient method for the determination of isopropyl-9H-thioxanten-9-one (ITX) in different fat content milk samples and baby milk samples stored in packaged cartons was developed and validated. Samples were extracted using solid-phase extraction (SPE) and analysed by gas chromatography-tandem mass spectrometry operated in selected reaction monitoring mode (SRM). Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values in the low microg/L were achieved, whereas linearity was established within 0.5-500 microg/L range. Good precision was obtained both in terms of intra-day repeatability and inter-day precision on two concentration levels (RSD% lower than 2%). Good percentage recoveries were obtained (92.0-102.0%) even in the presence of high amount of fat. Finally, the developed method was successfully applied to analyse a number of commercial milk samples with different fat content and baby milk samples.     

Characterization of the quantitative relationship between signal-to-noise (S/N) ratio and sample amount on-target by MALDI-TOF MS: Determination of chondroitin sulfate subsequent to enzymatic digestion

Chondroitin sulfate (CS) is an important glycosaminoglycan of the extracellular matrix and its quantitative detection is of interest in different pathologies. Although there are already methods of quantitative CS determination, many of them are laborious, require time-consuming sample workup and/or suffer from low sensitivity. It will be shown here that the CS content of biological samples can be easily assessed in the picomole range subsequent to enzymatic digestion. MALDI-TOF MS (matrix-assisted laser desorption and ionization time-of-flight mass spectrometry) was used to determine the concentrations of the unsaturated disaccharide of CS obtained by enzymatic digestion of native CS with chondroitin ABC lyase. The signal-to-noise (S/N) ratio can be used as a quantitative measure: amounts of CS (measured as the disaccharide) down to at least 500 fmol could be detected and there is a direct correlation between the S/N ratio and the amount of CS between about 2 and 200 pmol although the curve per se is sigmoidal. The influence of different parameters such as the used matrix, the applied laser intensity and different methods of data analysis were also tested. Advantages and drawbacks of this approach are critically discussed. Finally, the method was validated by the determination of the CS content in samples of known concentration as well as in enzymatically digested bovine nasal cartilage and compared with two further established methods of CS determination (Carbazole and Alcian Blue method).     

Interaction between sulfonephthalein dyes and chitosan in aqueous solution and its application to the determination of surfactants.

A spectrophotometric method for the determination of ionic surfactants with Bromophenol Blue (BPB) based on incorporation into a precipitated chitosan was studied. Cationic surfactants (CS+), such as a quaternary ammonium ion containing a long-chain alkyl group, associate with BPB2- buffered at about pH 9 to form the ion associate (CS+)2 x BPB2-. CS+ associates with anionic surfactants (AS-). In the presence of a definite amount of CS+, an increase in the amount of AS- leads to a decrease in the amount of excess CS+, and therefore to a decrease in the amount of the ion associate of CS+ with BPB2-. The addition of a chitosan dissolved in acetic acid to a solution containing these ion associates leads immediately to precipitation of the chitosan and the incorporation of the ion associates (CS+)2 x BPB2- or CS+ x AS- into the precipitated chitosan. After centrifuging, ionic surfactants can be determined by the following two methods: (1) the absorbance of the supernatant solution is measured at 590 nm. (2) After the supernatant solution is separated, the precipitate is dissolved in an acetic acid solution and the absorbance is measured at 625 nm. Because the color of the precipitate is judged by the naked eye, this can be applied to the visual method. This is a simple and rapid method for the determination of a 10(-6) M order of ionic surfactants.     

Enzyme-linked immunosorbent assay for detection of organophosphorylated butyrylcholinesterase: a biomarker of exposure to organophosphate agents

Lowering of the detection limit of solid-state lead-selective electrodes was achieved by using the tuned galvanostatic polarization method. A Nernstian response was obtained down to nanomolar concentrations (low detection limit 10(-9) mol dm(-3)Pb(2+)). Good repeatability of the calibration curves was achieved by using a well established measuring procedure. Relatively high cathodic current densities were applied to the solid-state membrane in order to shorten the measurement time. Successful determination of lead in a synthetic sample (pPb(2+)=7.97±0.08) was achieved by introducing an analytical protocol and favourably compared to inductively coupled plasma mass spectrometry (pPb=7.93). By applying this method, a significant improvement in the detection limit of solid-state lead-selective electrodes was attained.     

用结晶紫测定DNA 的分光光度法

研究了结晶紫与ＤＮＡ的可见吸收光谱和提出了测定ＤＮＡ的分光光度法。在ｐＨ９．５６的条件下,加入ＤＮＡ后结晶紫在５８９ｎｍ的最大吸收峰强度显著下降,下降程度与ＤＮＡ的含量呈线性关系,确定了实验的最佳条件,ＤＮＡ线性响应范围为０～５ｍｇ／Ｌ,检出限１９．５μｇ／Ｌ。该法简便,快速,具有较高的选择性,对合成样品中ＤＮＡ的测定结果令人满意。     

Spectrophotometric determination of trace iron by using its catalytic effect on the N-phenylanthranilic acid-potassium periodate reaction.

The analytical features of the reaction between N-phenylanthranilic acid (PAA) and potassium periodate in acidic medium are explored with the aim of improving the catalytic kinetic determination of iron in water samples. In the presence of Fe(II, III), PAA is oxidized by potassium periodate in a formic acid medium to form a violet-colored compound. The reaction is followed spectrophotometrically by measuring the increase in the absorbance of the oxidation product at 525 nm. The variables that affected the reaction rate were investigated and the reaction conditions were established. Calibration graphs are linear in the range of concentrations 2 - 500 ng mL(-1). As low as 10(-8) mol L(-1) Fe(II, III) can be easily determined by the fixed time method. The established catalytic method was successfully applied to the determination of iron in tap water and in pharmaceutical samples.     

荧光光度法测定血清中碱性磷酸酶

合成了一种新的底物水杨酸磷酸酯 (SP),用于荧光光度法测定血清中碱性磷酸酶(ALP)活力.在37.0 ℃的Tris-HCl缓冲液(pH 9.0)条件下, 碱性磷酸酶作用于荧光底物SP,水解生成强荧光产物水杨酸(SA),生成的SA的量与参与反应的ALP 的活力成正比.据此建立了荧光光度法测定血清中碱性磷酸酶活力的新方法.测定碱性磷酸酶的线性范围是0.03～6.00 U/L,检测限为7.04 mU/L.本方法适用于血清中碱性磷酸酶的测定.测定结果与临床上常用的以对硝基苯磷酸酯为底物的分光光度法相比,无显著性差异.     

Inhibition-based determination of metrifonate in liquid and solid samples using the triple integration chemical hydrolysis-pervaporation-enzymic derivatisation

An innovative continuous flow approach consisting of the triple integration of chemical hydrolysis, analytical pervaporation and enzyme inhibition-based reaction for the determination of metrifonate is presented. The method is based on chemical degradation of the pesticide, a separation step consisting of analytical pervaporation of its volatile metabolite and inhibition action of this on the acetylcholinesterase catalysis. The subsequent derivatisation reaction is a two-step reaction involving choline oxidase (ChOD) and horseradish peroxidase (POD) with fluorimetric detection (lambda (ex )=310 nm and lambda (em )=415 nm ) of the dimer formed by the action of hydrogen peroxide. The efficiency of the inhibitory effect was increased using an open-closed flow system. Applied to liquid samples, the method has a linear determination range of 0.0025-0.15 g l(-1)(n=8, r=0.9993) with a precision, expressed as RSD, of 3.2-6.7% and a sampling frequency of 3 h(-1). When applied to solid samples the method shows a linear determination range of 0.0026-0.13 g kg(-1) (n=5, r(2)=0.9981, RSD 2.7-7.7%) and a sampling frequency of 2h(-1). The approach has been applied to the determination of metrifonate in natural water and spiked soil samples with recoveries ranging between 94.3 and 107.8% for liquid samples and between 86.5 and 99.6% for solid samples.     

Determination of trace anions in liquefied petroleum gas using liquid absorption and electrokinetic migration for enrichment followed by ion chromatography

A simple sample enrichment technique, electrokinetic migration enrichment in single phase using a designed device, coupled with ion chromatography is presented for the determination of four anions (H(2)PO(4)(-), Cl(-), NO(3)(-), and SO(4)(2-)) in liquefied petroleum gas by liquid adsorption. The electrokinetic migration enrichment is based on the phenomenon of ion electrokinetic migration to the opposite electrode. When the anions migrated to the anode in a smaller volume chamber under the electric field, the concentration was realized. The main parameters affecting enrichment efficiency of applied voltage and enrichment time were investigated. The ion chromatography condition for anions separation was also studied. Under the optimal electrokinetic migration enrichment and ion chromatography conditions, the four anions were detected simultaneously with good linear relationship (r(2) = 0.9908-0.9968) and high precisions (less than 5% of the relative standard deviations of peak areas). The limits of detection of anions (S/N of 3) were in the range of 8-600 μg L(-1). The enrichment factors of the four anions ranged from 3.1 to 5.8. The established method was successfully applied to the analysis of the trace anions in liquefied petroleum gas by liquid adsorption with satisfactory results. The advantages of this method are simple operation and low cost.     

Evaluation of a fluorometric-enzymatic method based on 3alpha-hydroxysteroid dehydrogenase for the mycotoxin zearalenone determination in corn

A new method for the fluorometric determination of zearalenone (ZEN) based on its reaction with βNADH in the presence of the enzyme 3α-hydroxysteroid dehydrogenase (3α-HSD) is described. The procedure is based on the change in fluorescence intensity that takes place during the enzymatic reaction (excitation at 340 nm and emission at 454 nm). The optimum reaction conditions and the analytical characteristics were studied; linear response range (1-10 mg l⁻¹) and reproducibility (8 mg l⁻¹, 2.7%, n=7). Moreover, a mathematical model explaining the analytical signal is proposed. The method has been applied to zearalenone determination in a spiked corn sample.     

在线自动化柱前衍生-高效液相色谱法测定食品中组胺的研究

建立了在线自动化柱前衍生.高效液相色谱法测定食品中组胺的新方法.通过对测定过程中各个影响因素进行优化,如自动化衍生程序的设定,衍生试剂的用量,衍生体系pH影响等,确立了适宜的测定条件.在该条件下,对于组胺的检出限为0.01 μg/mL,在0.05～100 λg/mL范围内,线性关系良好(r2＞0.999).通过对样品基质进行加标,检出限为0.20 mg/kg.将所建立的方法应用于金枪鱼罐头,烟熏鲣鱼,冻鲭鱼等样品中组胺的测定,测得的组胺含量为0.59～167 mg/kg,加标回收率均大于97%,测定值的相对标准偏差均小于5%.所建立的方法适用于大量样品的常规分析测定.     

Spectrophotometric determination of microgram amounts of chlorpromazine with chloranil

A simple spectrophotometric method for the determination of small amount of chlorpromazine has been developed. The reaction of chloranil as π-acceptor reagent with chlorpromazine was studied. The method is rapid, simple, and relatively sensitive and precise (RSD 1.8%). Beer's law was followed within the approximate concentration range of 50–900 μg in a final volume of 25 ml. Negative deviation was abserved beyond these limits. The general analytical and physical parameters were also established.     

Determination of Lead in Lipstick by Direct Solid Sampling High-Resolution Continuum Source Graphite Furnace Atomic Absorption Spectrometry: Comparison of Two Digestion Methods

A new analytical procedure for the determination of lead in lipstick has been developed using direct solid sampling high resolution continuum source graphite furnace atomic absorption spectrometry (SS HR CS GFAAS). The performance of this method has been compared to acid digestion methods for sample preparation, with or without hydrofluoric acid (HF) with inductively coupled plasma mass spectrometry (ICP-MS) detection. Good reliability was obtained for all three methods; the results obtained for certified reference materials with concentrations between 1 and 20 ppm were in agreement with the certified values. However, for materials with complex matrices, such as pearl or Ca-Na borosilicate, only ICP-MS with HF sample digestion or AAS with direct solid sampling allowed complete recovery of lead. To avoid the use of hazardous acids, the development of SS HR CS GFAAS is an interesting alternative. With the AAS method, a characteristic mass of 13.2 pg of lead was obtained, and the limit of detection was 0.005 µg/g. The performance of the method was evaluated by determining lead in lipstick. The use of the solid sampling technique constitutes a good alternative for accurate and rapid determination of lead content in lipstick and cosmetic raw materials, with a suitable limit of detection and a reduced risk of contamination or of analyte loss. Another alternative would be to use ICP-MS determination in conjunction with microwave-assisted acid digestion without the use of HF, which implies accepting a quantification of “nearly total” lead, closer to a “bio-extractible” fraction.     

A hybrid triazine‐imine core‐shell magnetic covalent organic polymer for analysis of pesticides in fruit samples by ultra high performance liquid chromatography with tandem mass spectrometry

A novel hybrid triazine‐imine core‐shell magnetic covalent organic polymer with high adsorption capacity and excellent stability was fabricated by surface‐assisted in situ growth technique. The composite possesses porous and extended π‐conjugated system, and was applied as the magnetic sorbent for efficient enrichment and rapid separation of pesticides. A new analytical method for simultaneous determination of eight pesticides in fruit samples was developed by magnetic solid phase extraction combined with ultra high performance liquid chromatography and tandem mass spectrometry. The effect of extraction time, desorption time, and the type of desorption solvent on the extraction efficiency were evaluated. The established method shows good repeatability and high sensitivity. The repeatability of this method was estimated with relative standard deviations in the range of 0.7–7.0% (n = 5) for the same batch, and 1.7–10% (n = 3) for batch to batch. Good linearity for eight pesticides was obtained with coefficient of determination in the range of 0.9942–0.9990. Limit of detections ranged from 0.4 to 1.2 ng/L. Real sample determination showed that four and two pesticides were detected in strawberry and grape, respectively. The results demonstrated that the established method was efficient, sensitive, and convenient for trace determination of pesticides in fruit samples.     

Chemiluminescence Determination of Molybdenum by on-Line Reduction with a Flow Injection System

ThereactionsofLuwithhydrogenperoxidecatalysedbymetalionsandalsoreactionofLuwithorganicreductantssuchasglucoseandascorbicacidhavebeenreported"=.OurrecentstudiesshowedthatsomeinorganicreductantssuchasMo(Ill),V(11),U(ill),W(ill),Cr(II),Ti(ill)andFe(11)etc.canreactwithLuinstrongalkalinemediumtogenerateCL.Inthispaper,theCLreactionbetweenLuandMo(ill)producedbyaJonesreductor3hasbeenstudiedindetailforthefirsttime,andtheCLreactionhasbeensuccessfullyappliedtodeterminemolybdenumwithexcellentsensi…     

Determination of trace glucose and forecast of human diseases by affinity adsorption solid substrate room temperature phosphorimetry based on Triticum valgaris lectin labeled with 4.0-generation dendrimers

A new phosphorescence labeling reagent Triton-100X-4.0G-D (4.0G-D refers to 4.0-generation dendrimers) was found. Quantitative specific affinity adsorption (AA) reaction between Triton-100X-4.0G-D-WGA and glucose (G) was carried out on the surface of nitrocellulose membrane (NCM), and the DeltaI(p) of the product of AA reaction was linear correlation to the content of G. Based on the facts above, a new method for the determination of trace G was established by WGA labeled with Triton-100X-4.0G-D affinity adsorption solid substrate room temperature phosphorimetry (Triton-100X-4.0G-D-WGA-AA-SS-RTP). This research showed that AA-SS-RTP for either direct method or sandwich method could combine very well the characteristics of both the high sensitivity of SS-RTP and the specificity of the AA reaction. Detection limits (LD) were 0.24 fg spot(-1) for direct method and 0.18 fg spot(-1) for sandwich method, indicating both of them were of high sensitivity. The method has been applied to the determination of the content of G in human serum, and the results were coincided with those obtained by glucose oxidize enzyme method. It can also be applied to forecast accurately some human diseases, such as primary hepatic carcinoma, cirrhosis, acute and chronic hepatitis, transfer hepatocellular, etc. Meanwhile, the mechanism for the determination of G with AA-SS-RTP was discussed.     

5-（5-溴-2-吡啶偶氮）-2，4-二氨基甲苯催化光度法测定钯

在H2SO4介质和室温条件下,微量钯（Ⅱ）对KBrO3氧化5-（5-溴-2-吡啶偶氮）-2,4-二氨基甲苯（5-Br-PA—DAT）褪色反应具有显著的催化作用,据此建立了测定微量钯的催化光度法．方法的检测限为0．034mg／L,线性范围0．2～0．75mg／L．已用于钯碳催化剂及水样中钯含量的测定,标准加入回收率为97．2％～99．0％,结果满意．     

双氧水活化-面粉酶抑制法测定蔬菜中有机磷农药

基于α-乙酸萘酯和固蓝B在面粉酶的催化作用下生成紫红色的偶氮化合物,而有机磷农药在双氧水的活化下会抑制酶的催化作用,其抑制程度与农药的浓度在一定范围内呈线性关系,由此建立了一种快速测定有机磷农药的方法。探讨了最佳的反应条件,测定甲基对硫磷和甲胺磷的线性范围分别为1~10μg/mL,1~50μg/mL,最低检出限分别为0.1136μg/mL,0.3525μg/mL。该法具有试剂廉价易得、操作简便、迅速的特点,适合果蔬中常见有机磷农药的快速检测。