Anion-exchange high-performance liquid chromatography with post-column detection for the analysis of phytic acid and other inositol phosphates

The use of gradient anion-exchange HPLC, with a simple post-column detection system, is described for the separation of myo-inositol phosphates, including "phytic acid" (myo-inositol hexaphosphate). Hexa-, penta-, tetra-, tri- and diphosphate members of this homologous series are clearly resolved within 30 min. This method should facilitate analysis and quantitation of "phytic acid" and other inositol phosphates in plant, food, and soil samples.     

高效液相色谱法同时测定血清中的犬尿氨酸和色氨酸

建立了一种能同时检测血清中的犬尿氨酸(kynurenine,Kyn)和色氨酸(tryptophan,Trp)的高效液相色谱-紫外检测法。采用的色谱柱为Symmetry Shield RP-C18柱(150 mm×3.9 mm i.d.,5 μm),流动相为15 mmol/L乙酸钠-乙酸溶液(含2.7%乙腈,pH 3.6),流速为1.0 mL/min,紫外检测波长为225 nm。血清标本经5.0%(体积分数)高氯酸溶液去除蛋白质后取上清液直接进样分析测定。研究结果表明,Kyn保留时间为3.5 min,线性范围为0.098~49 μmol/L,最低检出浓度为0.02 μmol/L,回收率为90.82%~93.45%;Trp保留时间为8.1 min,线性范围为4.9~490 μmol/L,最低检出浓度为0.20 μmol/L,回收率为95.51%~98.67%。Kyn和Trp日内、日间测定的相对标准偏差均小于4%,苯丙氨酸、酪氨酸、5-羟色胺和犬尿喹啉酸等物质对该法均无干扰。该方法简便、快速、稳定、可行,可应用于临床和科研工作。     

A rapid urine creatinine assay by capillary zone electrophoresis.

Using capillary zone electrophoresis, the urine creatinine (uCr) assay was validated in extemporaneous diluted urine, both in healthy subjects and athletes, with the uCr concentration as a reference value to compare excretion rates of other metabolites in the same samples. The electrokinetic sample injection was carried out at 10 kV per 10 s; UV absorbance detection was at 254 nm. Using standard samples, the creatinine migration mean time in 100 mmol/L acetate buffer, pH 4.4, was 3.3+/-0.2 min; the repeatability for absolute migration mean time was 0.6% and peak height repeatability was 2.9%. The correlation coefficient of the standard curve was r = 0.999 and the detection limit was 23.1 micromol/L. Intra- and interassay coefficients of variation (CV) were 3.0 and 3.6%, respectively; recovery was 99+/-3% and linearity was r= 0.98. Normal urine samples were diluted 1:80 in run buffer. The present CE urine creatinine assay showed a good correlation with HPLC and with Jaffe methods (r = 0.98 and r = 0.97, respectively; p < 0.0001). The uCr in the morning urine samples of 34 healthy males (M), 38 healthy females (F), and 83 male athletes (A) was 10.4+/-6.1 mmol/L, 10.8+/-8.1 mmol/L and 13.2+/-6.5 mmol/L, respectively. The uCr difference (p < 0.02) between M and A and a correlation (p < 0.05) with age in A were observed.     

High-throughput capillary electrophoretic method for determination of total aminothiols in plasma and urine.

Increased interest in the analysis of aminothiols in body fluids during the last years results in a request for high-throughput analytical methods for their determination. We report here a novel, high-throughput method for the determination of total concentrations of biogenous aminothiols - homocysteine, cysteine, glutathione, cysteinylglycine, gamma-glutamylcysteine, and of penicilamine, mercaptopropionylglycine, and cysteamine, three compounds used to treat disorders of aminothiol metabolism in plasma and urine. Samples were reduced with tris(carboxyethyl)phosphine and labeled with 5-(bromomethyl)fluorescein. Capillary electrophoretic separations were performed in 60 mmol/L borate - 15 mmol/L sodium dodecyl sulfate - 2-amino-2-methyl-1-propanol, pH 10.0, with laser-induced fluorescence detection. Analysis time was less than 2 min. The assay is linear (r > 0.999) up to 500 micromol/L. Reproducibilities of migration times (coefficient of variation, CV) were < 0.5%. Interassay repeatabilities (CV, n = 10) were 5.08% and 6.09% for 5 micromol/L addition of homocysteine and 0.60% and 3.78% for 100 micromol/L addition of cysteine in plasma and urine, respectively. Recovery values were within 94-106% and sensitivity was better than 0.19 micromol/L for all analyzed compounds. Results agreed well with a standard high-performance liquid chromatography (HPLC) method. The diagnostic usefulness of the method has been proven on 79 samples of cystinuric patients and 12 samples of homocystinuric patients. We report here a novel method for the determination of aminothiols in body fluids by capillary electrophoresis (CE). Determination is fast and sensitive enough for diagnostic purposes.     

Separation of phytic acid and other related inositol phosphates by high-performance ion chromatography and its applications

A high-performance anion-exchange chromatographic method was developed for the separation of phytic acid and other inositol phosphates (myo-inositol bis-, tris-, tetrakis-, and pentakisphosphates) with gradient elution and ultraviolet absorbance detection after post-column derivatization. With the acidic eluents, the combination of anion-exchange and ion suppression retention mechanisms led to the separation of 35 inositol phosphates (excluding enantiomers) into 27 peaks for the first time, and the retention behaviors of all myo-inositol bis- to hexakisphosphate isomers were studied. The whole separation procedure was completed within 65 min. Based on the investigations of nonenzymatic hydrolysis of phytic acid under different conditions by using this method, an in-house reference standard solution was produced, which can be used for method development. In addition, by applying this method to in vitro kinetic studies, at least one new enzymatic hydrolysis pathway of phytic acid was found, and one rule of enzymatic dephosphorylation of inositol phosphates (position effect) was proposed and another one (neighboring effect) was confirmed. The principle of the proposed identification approach for several inositol phosphate isomers based on hydrolysis products study will be applicable to other natural products analysis, for which standards are very expensive or not available.     

Determination of low cadmium concentrations in wine by on-line preconcentration in a knotted reactor coupled to an inductively coupled plasma optical emission spectrometer with ultrasonic nebulization

An on-line cadmium preconcentration and determination system implemented with inductively coupled plasma optical emission spectrometry (ICP-OES) associated to flow injection (FI) with ultrasonic nebulization system (USN) was studied. The cadmium was retained as the cadmium-2-(5-bromo-2-pyridylazo)-5-diethylaminophenol, Cd-(5-Br-PADAP), complex, at pH 9.5. The cadmium complex was removed from the knotted reactor (KR) with 3.0 mol/L nitric acid. A total enhancement factor of 216 was obtained with respect to ICP-OES using pneumatic nebulization (12 for USN and 18 for KR) with a preconcentration time of 60 s. The value of the detection limit for the preconcentration of 5 mL of sample solution was 5 ng/L. The precision for 10 replicate determinations at the 5 microg/L Cd level was 2.9% relative standard deviation (RSD), calculated from the peak heights obtained. The calibration graph using the preconcentration system for cadmium was linear with a correlation coefficient of 0.9998 at levels near the detection limits up to at least 1,000 microg/L. The method was successfully applied to the determination of cadmium in wine samples.     

Study of low-molecular weight organic acids in maize roots under the stress of cadmium using capillary zone electrophoresis

An indirect CE method was developed for the study of low-molecular weight organic acids (LMWOAs) in maize under the stress of cadmium. The influences of indirect reagents (phthalate, salicylic acid, and benzoic acid), coion concentration, and pH were studied. A buffer composed of 15 mmol/L benzoate and 0.2 mmol/L CTAB (pH 5.7) was used in the organic acid determination. The detection limit ranged between 0.5 and 6 micromol/L. The RSD (n = 14) of the method was found to be in the range of 0.11-0.49% for migration time and 1.25-4.72% for peak area. In the maize roots, obvious increases of LMWOAs were observed when the plants were under the stress of cadmium and/or organic acids. The recovery of standard organic acids added in real samples ranged from 85 to 116%.     

Capillary electrophoresis for determination of free and albumin-bound bilirubin and the investigation of drug interaction with bilirubin-bound albumin

Capillary electrophoresis (CE) is a promising technique for assessment of free bilirubin and its interaction with albumin, as it requires only a small sample volume and provides a rapid and efficient separation of free bilirubin from its albumin-bound complex in a one-phase system. In order to maintain the equilibrium without dissociation of bilirubin from the albumin/bilirubin complex as in real clinical conditions, the coupling of CE with frontal analysis (FA) was investigated. A very large sample plug was introduced hydrodynamically into the capillary (36 cm length, 50 microm inner diameter) at 15 psi x s to develop the frontal conditions during CE separation. The working conditions for CE/FA separation of bilirubin and albumin were optimized as follows: +20 kV; running buffer, 10 mmol/L phosphate and 1 mmol/L ethylenediaminetetraacetic acid (EDTA), pH 7.4. The working range for bilirubin was found to vary from 5 to 206 micromol/L; precision with relative standard deviation (RSD) <2.0% for n = 3 and detection limit (signal to noise, S/N = 2) was 2 micromol/L. The residual binding capacity of a simulated cord blood serum for bilirubin was 26 mg/100 mL at pH 7.4. Bilirubin was shown to be displaced from albumin when aspirin was added. The free bilirubin concentration was found to increase to clinical significant concentrations from 11.9 to 21.1% when increasing aspirin was added in the range of 5-50 mg/100 mL, respectively. Thus, the investigation of aspirin displacement of bilirubin from albumin is clinically important and the CE/ FA method is a suitable procedure for this purpose.     

Direct determination of alkyl phosphates in human urine by liquid chromatography/electrospray tandem mass spectrometry

A novel and rapid procedure based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the determination of dialkyl phosphates (metabolites of organophosphorus pesticides) in human urine has been developed. After addition of 40 mM tetrabutylammonium acetate, 10 microL of urine sample were directly injected into the LC/MS/MS system. The method was validated yielding calibration curves with correlation coefficients greater than 0.997 and repeatability coefficient of variation (CV) lower than 9%. The accuracy and precision were evaluated by direct injection of spiked samples at 10 and 100 microg/L obtaining recoveries between 78 and 119% with coefficients of variation below 12%. Limits of detection of 1 microg/L for diethyl phosphate (DEP), diethylthiophosphate (DETP) and diethyldithiophosphate (DEDTP) and 2 microg/L for dimethyldithiophosphate (DMDTP) were achieved, all the analytes being detected in negative ion mode. The fragmentation pathway of dialkyl phosphates allowed us the use of an additional transition for confirmation in order to improve their identification in real-world samples. The applicability of the LC/MS/MS method was demonstrated by applying it to the analysis of urine samples of farmers exposed to the organophosphorus pesticide chlorpyrifos. Good correlation between application of the product in the field (citrus orchards), concentration levels of dialkyl phosphates and levels of the chlorpyrifos-specific metabolite (1,3,5-trichloro-2-pyridinol) was obtained.     

On-line complexation of zinc with 5-Br-PADAP and preconcentration using a knotted reactor for inductively coupled plasma atomic emission spectrometric determination in river water samples

An on-line zinc preconcentration and determination system implemented with inductively coupled plasma atomic emission spectrometry (ICP-AES) associated with flow injection (FI) was studied. The zinc was retained as zinc-2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (Zn-(5-Br-PADAP)) complex at pH 9.2. The zinc complex was removed from the knotted reactor (KR) with 30% v/v nitric acid. An enrichment factor of 42 was obtained for the KR system with respect to ICP-AES using pneumatic nebulization. The detection limit for the preconcentration of 10 mL of aqueous solution was 0.09 microg/L. The precision for 10 replicate determinations at the 5 microg/L Zn level was 2.3% relative standard deviation (RSD), calculated with the peak heights obtained. The calibration graph using the preconcentration system for zinc was linear with a correlation coefficient of 0.9997 at levels near the detection limits up to at least 100 microg/L. The method was succesfully applied to the determination of zinc in river water samples.     

Sensitive determination of norepinephrine, synephrine, and isoproterenol by capillary electrophoresis with indirect electrochemiluminescence detection

A new and sensitive method for the determination of norepinephrine (NE), synephrine, and isoproterenol was developed by CE separation and indirect electrochemiluminescence detection (ECL) based on their quenching effects on the tris(2,2'-bipyridyl)-ruthenium(II)/tripropylamine (TPA) system. The conditions for CE separation and ECL detection were investigated in detail. Under the optimum conditions, the three analytes were well separated within 9 min. The LODs (S/N = 3) in standard solution are 2.6 x 10(-8) mol/L for NE, 6.6 x 10(-9) mol/L for synephrine and 8.4 x 10(-8) mol/L for isoproterenol, respectively. The precisions of intraday and interday are less than 4.4 and 6.1%, respectively. The LOQs (S/N = 10) in real human urine samples are 2.6 x 10(-7) mol/L for NE, 8.8 x 10(-8 ) mol/L for synephrine, and 8.8 x 10(-7) mol/L for isoproterenol, respectively. The applicability of the proposed method was illustrated in the determination of 20 human urine samples from diabetic patients and healthy persons. The results obtained indicated that the level of NE in patients (mean value 0.41 micromol/L) was higher than that in healthy persons (mean value 0.24 micromol/L).     

Application of a novel micro-injector in the determination of indole derivatives in the rat pineal gland by capillary electrophoresis with electrochemical detection

A novel micro-injector has been fabricated for capillary electrophoresis (CE). It was successfully used for the determination of some indole derivatives for example melatonin (MT), serotonin (5-HT), tryptophan (Trp), and 5-hydroxy-tryptophane (5-HTrp) in the rat pineal gland by capillary electrophoresis with electrochemical detection (CE-EC). CE was performed in 0.20 mol L(-1) phosphate buffer (pH 8.0). The compounds investigated can be well separated and detected within 15 min. The working electrode used was a 300-microm diameter carbon electrode positioned opposite the outlet of the capillary. The relationship between peak current and analyte concentration was highly linear in the range from 0.10 to 500 micromol L(-1); detection limits (S/N = 3) were 0.03-0.13 micromol L(-1). The proposed method has been successfully used to analyze real biological samples.     

Amperometric sensor for nitrite based on copper tetrasulphonated phthalocyanine immobilized with poly-L-lysine film

In this work, an amperometric sensor for nitrite detection based on a glassy carbon electrode modified with copper tetrasulphonated phthalocyanine immobilized by polycationic poly-L-lysine film is presented. The modified electrode showed an excellent catalytic activity toward nitrite oxidation. A linear response range from 0.12 up to 12.20 micromol L(-1) was obtained with a sensitivity of 0.83 microA L micromol(-1). The detection limit for nitrite was 36 nmol L(-1). The repeatability of the proposed sensor, evaluated in terms of relative standard deviation, was 1% for 10 measurements of 10 micromol L(-1) nitrite solution. Finally, the developed sensor was applied for nitrite determination in water samples and the results were in agreement to the comparative method. The average recovery for the samples was 101 (+/-4)%.     

Determination of myo-inositol (free and bound as phosphatidylinositol) in infant formula and adult nutritionals by liquid chromatography/pulsed amperometry with column switching: first action 2011.18

Myo-inositol is a 6-carbon cyclic polyalcohol also known as meso-inositol, meat sugar, inosite, and i-inositol. It occurs in nature in both free (myo-inositol) and bound (inositol phosphates and phosphatidylinositol) forms. For the determination of free myo-inositol, samples are mixed with dilute hydrochloric acid to extract myo-inositol and precipitate proteins, diluted with water, and filtered. For the determination of myo-inositol bound as phosphatidylinositol, samples are extracted with chloroform, isolated from other fats with silica SPE cartridges, and hydrolyzed with concentrated acid to free myo-inositol. Prepared samples are first injected onto a Dionex CarboPac PA1 column, which separates myo-inositol from other late-eluting carbohydrates. After column switching, myo-inositol is further separated on a CarboPac MA1 column using a 0.12% sodium hydroxide mobile phase; strongly retained carbohydrates are eluted from the PA1 column with a 3% sodium hydroxide mobile phase. Eluant from the CarboPac MA1 analytical column passes through an electrochemical detector cell where myo-inositol is detected by pulsed amperometry using a gold electrode. The method showed appropriate performance characteristics versus selected established standard method performance requirement parameters for the determination of myo-inositol: linear response; repeatability (RSDr) of 2%; and intermediate precision (RSDir) of 2.5%. Instrument LOD and LOQ were 0.0004 and 0.0013 mg/100 mL, respectively, and correspond to a free myo-inositol quantitation limit of 0.026 mg/100 g and a phosphatidylinositol quantitation limit of 0.016 mg/100 g. Correlation with the reference microbiological assay was good. The proposed method has been accepted by the Expert Review Panel as an AOAC First Action Method, suitable for the routine determination of myo-inositol in infant formula and adult nutritionals.     

On-line complexation of zinc with 5-Br-PADAP and preconcentration using a knotted reactor for inductively coupled plasma atomic emission spectrometric determination in river water samples

An on-line zinc preconcentration and determination system implemented with inductively coupled plasma atomic emission spectrometry (ICP-AES) associated with flow injection (FI) was studied. The zinc was retained as zinc-2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (Zn-(5-Br-PADAP)) complex at pH 9.2. The zinc complex was removed from the knotted reactor (KR) with 30% v/v nitric acid. An enrichment factor of 42 was obtained for the KR system with respect to ICP-AES using pneumatic nebulization. The detection limit for the preconcentration of 10 mL of aqueous solution was 0.09 μg/L. The precision for 10 replicate determinations at the 5 μg/L Zn level was 2.3% relative standard deviation (RSD), calculated with the peak heights obtained. The calibration graph using the preconcentration system for zinc was linear with a correlation coefficient of 0.9997 at levels near the detection limits up to at least 100 μg/L. The method was succesfully applied to the determination of zinc in river water samples. Received: 27 December 1999 / Revised: 14 March 2000 / Accepted: 15 March 2000     

磁敏传感器竞争免疫法检测牛奶中的氯霉素残留量

利用直接竞争免疫原理和巨磁致电阻效应,建立了磁敏生物传感器检测氯霉素的方法。制备氯霉素半抗原芯片,依次加入待测样品、生物素化抗体、链霉亲和素磁颗粒偶联物,使之发生竞争免疫反应,再利用传感器检测芯片上结合的磁颗粒数目。通过对检测条件的优化,建立了氯霉素浓度与磁颗粒数目的标准工作曲线。本方法的检测范围为0.05～100.0μg/L;检出限为50 ng/L;用于牛奶检测,回收率为95.97%～99.36%;批内相对标准偏差为0.8%～3.9%,批间相对标准偏差为1.1%～1.7%;与ELISA方法的一致相关系数达到0.98。本方法可在30 min内快速完成定量检测,为快速多靶标磁敏竞争免疫检测体系的建立提供了可行性。     

微波消解一氢化物原子荧光光谱法测定罐头食品中锡

为建立微波消解-氢化物原子荧光光谱法测定罐头食品中锡含量的方法。样品在HNO3＋HC1或HN03＋HCl＋H2O2体系中经微波消解处理后,以硫脲-抗坏血酸混合液作预还原剂,用氢化物原子荧光光谱法测定罐头食品中锡。结果表明,在优化的条件下,锡质量浓度在0～100μg／L范围内线性关系良好,相关系数为0．9998,方法最低检出限为0．067mg／kg,加标回收率在99．7％～108．3％之间,方法变异系数为2．67％（n=6）。该法具有准确度高、精密度好、灵敏快速且安全环保等优点,适用于罐头食品中锡的测定。     

Flow injection spectrophotometric determination of reducing sugars using a focalized coiled reactor in a domestic microwave oven

A flow injection (FI) spectrophotometric procedure for determining reducing sugars content in sugar cane juices using a focalized PTFE coiled reactor positioned at the output antenna of a domestic microwave oven at 700 W is proposed. In this system, sample solution converge to 1.0 mol l(-1) NaOH and 5.2 mmol l(-1) K(3)Fe(CN)(6) solutions previously mixed and the decrease of hexacyanoferrate(III) concentration was monitored at 420 nm. Under best analytical conditions, there was a direct relationship between absorbance decrease and reducing sugar content (fructose plus glucose concentrations) in the concentration range from 50 to 1200 micromol l(-1) with a detection limit of 15 micromol l(-1). The relative standard deviations (rds) were less than 1.4% for ten injection of 400 and 800 micromol l(-1) fructose solution and the analytical frequency was 70 h(-1). A paired t-test showed that all results obtained for sugar cane juices using this FI procedure and the Somogyi-Nelson batch procedure agree at the 95% confidence level.     

Combination of flow injection with capillary electrophoresis: 8. Miniaturized capillary electrophoresis system with flow injection sample introduction and electrogenerated chemiluminescence detection

The combined flow injection (FI)-capillary electrophoresis (CE) system was further exploited by coupling to an electrogenerated chemiluminescence (ECL) detection system. A low-cost miniaturized CE system was developed on a chip platform to provide easy interface both with FI sample introduction and with ECL detection. A falling-drop interface was employed to perform FI split-flow sample introduction while achieving electrical isolation from the CE high voltage. A plexiglas reservoir at the capillary outlet served as both the reaction and detection cell for the ECL reaction, with Ru(bpy)₃²⁺ reagent continuously flowing through the cell. An optical fiber was positioned within the reservoir close to the capillary outlet for transferring the ECL emission to the PMT. The relative positions of the capillary outlet, working electrode and optical fiber as well as reagent renewal flow-rate were optimized to achieve both good sensitivity and separation efficiency under non-interrupted sampling conditions, involving large numbers of samples. An on-column joint often used in other works for isolating the ECL detection system from the CE separation voltage was not found necessary. The performance of the system was illustrated by the baseline separation of proline, valine and phenylalanine with a high throughput of 50 h⁻¹ and plate height of 14 μm for proline under 147 V cm⁻¹ field strength. Detection limits (3σ) were 1.2, 50 and 25 μM and peak height precisions were 1.4, 5.4 and 4.3% R.S.D. (n=9) for proline, valine and phenylalanine, respectively.     

NACE for the analysis of acrylamide in food

An NACE method was developed for the quantitative determination of acrylamide in processed food. The method is premised on the modification of the aqueous acid-base character of acrylamide in an organic solution. Acrylamide, which is a polar molecule in aqueous solution, in a low-pH environment in ACN acquires a proton, and thereby migrates under its own electrophoretic mobility in CE. Thus, nonaqueous separation of acrylamide was achieved by employing 30 mmol/L HClO(4) in ACN as the running electrolyte. The detection limit of the method for acrylamide was found as 0.041 mg/L using UV detection at 200 nm. The run-to-run and day-to-day precisions for the corrected peak areas were calculated as 1.65 and 3.90%, respectively. The applicability of the method has been demonstrated by analyzing acrylamide in the samples of potato chips and French fries. The method is simple, rapid, inexpensive, and widely applicable for the determination of acrylamide in food samples.