Determination of H2O2 and organic peroxides by high-performance liquid chromatography with post-column UV irradiation, derivatization and fluorescence detection

A method for the determination of hydrogen peroxide and several organic peroxides by high-performance liquid chromatography with post-column UV irradiation, derivatization and fluorescence detection is described. By means of post-column UV irradiation in the presence of water organic peroxides are converted into hydrogen peroxide and organic hydroperoxides, which react rapidly with the post-column derivatization agent p-hydroxyphenylacetic acid (PHPAA) under catalysis of horseradish peroxidase to yield the fluorescent PHPAA dimer that is detected at excitation and emission wavelengths of 285 and 400 nm, respectively. The detection limit for hydrogen peroxide is 14 ng/mL, for organic peroxides between 34 ng/mL and 5 μg/mL. No interference by other compounds was observed when their concentrations were below 10 mg/mL except ethers and phenols. Received: 6 August 1997 / Revised: 11 December 1997 / Accepted: 15 December 1997     

Detection of volatile organic peroxides in indoor air

A supercritical fluid extraction cell filled with adsorbent (Carbotrap and Carbotrap C) was used directly as a sampling tube to enrich volatile organic compounds in air. After sampling, the analytes were extracted by supercritical fluid CO2 with methanol as modifier. Collected organic peroxides were then determined by a RP-HPLC method developed and validated previously using post-column derivatization and fluorescence detection. Some volatile organic peroxides were found in indoor air in a new car and a newly decorated kitchen in the lower microg m(-3) range. tert-Butyl perbenzoate, di-tert-butyl peroxide, and tert-butylcumyl peroxide could be identified.     

Determination of artemisinin in human serum by high-performance liquid chromatography with on-line UV irradiation and peroxyoxalate chemiluminescence detection

Artemisinin is an antimalarial drug containing an internal endoperoxide linkage in its structure. A simple, selective and sensitive high-performance liquid chromatography (HPLC)-peroxyoxalate chemiluminescence (PO-CL) method for the determination of artemisinin was developed. This method is based on the fact that endoperoxide in artemisinin structure can be converted to hydrogen peroxide (H(2)O(2)) under ultraviolet (UV) irradiation and the generated hydrogen peroxide can be measured using PO-CL detection. The HPLC-PO-CL system was optimized on a mobile phase, post column chemiluminescence reagent, UV source and irradiation time. In addition, the system was combined with simple liquid-liquid extraction using n-hexane that allowed selective and sensitive determination of artemisinin in serum. The limit of detection using 0.5 mL of blood was 0.062 micromol/L (17.5 ng/mL) at a signal-to-noise ratio of 3. Calibration curve obtained for artemisinin in human serum 4-80 micromol/L (1.1-22.6 microg/mL) showed a good linearity (r = 0.999).     

高效液相色谱-荧光检测法测定环境样品中的过氧化物

对高效液相色谱-荧光检测法测定过氧化氢和有机过氧化物的方法进行了改进,从而提高了方法的检测灵敏度。以氯化血红素(hemin)作催化剂进行柱后衍生反应,过氧化物将对羟基苯乙酸氧化生成能吸收荧光的二聚物,然后用荧光检测器检测。实验确定了最佳反应管温度和荧光检测波长。应用该法测定了大气和雨水样品中过氧化物的浓度。     

Peroxide generation and decomposition on polymer surface

Polymer peroxides were generated onto polypropylene and polyurethane films, and polyester fibers by ozonization, UV irradiation, and plasma treatment. Their thermal and redox decompositions were studied using two assays: peroxidase and iodide. They may differ in the sensitivity to access the peroxides, depending on their localization in the polymer substrates. It was found that the generated peroxides are likely to be distributed not only on the outermost surface, but also in the subsurface of the polymer substrates. Plasma treatment provided polyurethane film with easily accessible peroxides, while UV irradiation and ozonization generated peroxides that were mostly incapable of reacting with aqueous solution of peroxidase. Redox decomposition of the peroxide groups by ferrous ions at 25°C showed that less than 50% of peroxides could react with ferrous ions with a rate constant similar to that of hydrogen peroxide in aqueous solution. Thermal decomposition of the peroxides did not obey the first-order kinetics, probably due to formation of not single peroxide species with different decomposition rate constants. The lowest decomposition rate constant observed at 65°C was 3 × 10^?3 min^?1, which did not depend on the polymer substrate nature and the method of peroxide generation. © 1995 John Wiley & Sons, Inc.     

Automated pre-column derivatization of amines in biological samples with dansyl chloride and with or without post-column chemiluminescence formation by using TCPO-H2O2.

On-line automation of two different liquid chromatographic procedures, a pre-column derivatization system and a pre- and post-column system, in order to generate chemiluminescence is reported. Dansyl chloride (Dns-Cl) was used as a pre-column reagent to form fluorophores and bis(2,4,6-trichlorophenyl) oxalate (TCPO) and hydrogen peroxide (H2O2) as a post-column reagent to generate chemiluminescence. This procedure is based on the employment of a primary column packed with C18 material inserted in a multi-dimensional assembly for sample clean-up and derivatization with Dns-Cl. The dansyl derivatives formed are transferred and separated in a LiChrospher 100 RP18 analytical column (125 x 4 mm id, 5 microns film thickness) using acetonitrile-imidazole buffer (pH 6.8) (70 + 30) as eluent. The separated derivatives were transferred to the detector for fluorescence detection or to the post-column system where the chemiluminescence response was generated by using TCPO-H2O2 and the products were detected by chemiluminescence. The procedure was optimised for amphetamine and related compounds. A comparison between the on-line pre-column and pre- and post-column systems was performed. The results show that the sensitivity of chemiluminescence detection can be higher than that of fluorescence detection. The recoveries obtained ranged from 98 +/- 8 up to 108 +/- 8% for amphetamine and methamphetamine, respectively. The accuracy and precision of these methods were evaluated.     

Design and evaluation of a multi-detection system composed of ultraviolet, evaporative light scattering and inductively coupled plasma mass spectrometry detection for the analysis of pharmaceuticals by liquid chromatography

Reversed-phase liquid chromatography was coupled to a multi-detection system composed of ultraviolet (UV) detection, evaporative laser scattering detection (ELSD) and inductively coupled plasma mass spectrometry (ICP-MS). By applying the principle of post-column solvent compensation, the organic modifier content was kept constant in ELSD and ICP-MS under gradient elution. Chlorine ((35)Cl), bromine ((79)Br and (81)Br) and sulfur ((34)S) were monitored in several pharmaceutical compounds. The limit of quantitation (LOQ) was 80 ng/mL for chlorine (chlorpropamide) and 2 ng/mL for bromine (bromazepam). Calibration graphs were linear from 1.0 microg/mL to 100 microg/mL for chlorpropamide (r(2) 0.990) and from 10 ng/mL to 500 ng/mL for bromazepam (r(2) 0.996). The low LOQ value for bromine allows to quantify bromine in pharmaceutical samples below the 0.05% level of the active pharmaceutical ingredient.     

Determination of idebenone in plasma by HPLC with post-column fluorescence derivatization using 2-cyanoacetamide

Idebenone is a benzoquinone analog that is used in the treatment of several neurological disorders including Friedreich's ataxia. It was found that the reaction of idebenone with 2-cyanoacetamide under alkaline conditions generates fluorescent products, and the reaction was considered to proceed via Craven's reaction. The reaction mixture from idebenone gave fluorescence with excitation and emission maximum wavelengths at 358 nm and 409 nm, respectively. It was adopted for HPLC with post-column fluorescence derivatization of idebenone. Idebenone in the plasma showed a linear response in the range of 0.5-32 ng (25-1600 ng/mL), and the quantitation limit (S/N=10) was 12.5 ng/mL. The detection limit (S/N=3) of the standard solution of idebenone was 0.1 ng. The HPLC system was applied to the human blood plasma obtained by finger prick. The plasma sample obtained by finger prick gave a similar chromatogram to that of venous blood obtained by venipuncture.     

Spectrophotometric reaction rate method for the determination of molybdenum by its catalytic effect on the oxidation of pyrogallol red with hydrogen peroxide

A kinetic spectrophotometric method for the determination of molybdenum is based on its catalytic effect on the oxidation of pyrogallol red with hydrogen peroxide. The decrease of the absorbance of pyrogallol red in the presence of hydrogen peroxide with time from 0.5 to 4.5 min is proportional to the concentration of Mo(VI) over the range 0.010–0.500 μg/mL. The limit of detection is 0.008 μg Mo/mL. The precision and the effect of the presence of more than forty ions on the molybdenum determination are reported. Probable interferences are completely removed by a cation exchange resin. The procedure was successfully applied to the determination of molybdenum in plant materials and steels. Received: 28 April 1997 / Revised: 16 June 1997 / Accepted: 18 June 1997     

Spectrophotometric reaction rate method for the determination of molybdenum by its catalytic effect on the oxidation of pyrogallol red with hydrogen peroxide

A kinetic spectrophotometric method for the determination of molybdenum is based on its catalytic effect on the oxidation of pyrogallol red with hydrogen peroxide. The decrease of the absorbance of pyrogallol red in the presence of hydrogen peroxide with time from 0.5 to 4.5 min is proportional to the concentration of Mo(VI) over the range 0.010–0.500 μg/mL. The limit of detection is 0.008 μg Mo/mL. The precision and the effect of the presence of more than forty ions on the molybdenum determination are reported. Probable interferences are completely removed by a cation exchange resin. The procedure was successfully applied to the determination of molybdenum in plant materials and steels. Received: 28 April 1997 / Revised: 16 June 1997 / Accepted: 18 June 1997     

Determination of halofantrine and its main metabolite desbutylhalofantrine in rat plasma by high-performance liquid chromatography with on-line UV irradiation and peroxyoxalate chemiluminescence detection

A sensitive, selective and reliable method has been developed and validated for the determination of halofantrine and its metabolite desbutylhalofantrine in rat plasma using 9,10-diphenylanthracene as an internal standard. The method is based on peroxyoxalate chemiluminescence detection of hydrogen peroxide produced from fused aromatic rings in the structures of halofantrine and desbutylhalofantrine upon UV irradiation. Using spiked rat plasma, good linear relationships were obtained for both halofantrine and desbutylhalofantrine between peak height ratios (vs internal standard) and their corresponding concentrations over a range of 0.01-0.8 microg/mL with correlation coefficients of at least 0.997. The detection limits at signal-to-noise ratio of 3 using 0.2 mL of rat plasma were 1.5 and 1.4 ng/mL for halofantrine and desbutylhalofantrine, respectively. Relative standard deviations (n = 3) intra- and inter-day were between 0.5 and 5.4% for all the studied concentrations. Using this method with simple sample treatment, halofantrine and desbutylhalofantrine in rat plasma could be precisely determined without interference from endogenous substances. The method was successfully applied to the measurement of the time courses of plasma halofantrine concentration after oral administration of the drug (7 mg/kg) to rats.     

柱后衍生-高效液相色谱-荧光检测法测定面粉中的过氧化苯甲酰

利用过氧化苯甲酰能够在氯化血红素的催化下将底物对羟基苯乙酸氧化成具有荧光的二聚体的性质,建立了柱后衍生-高效液相色谱-荧光检测法测定面粉中过氧化苯甲酰的方法。优化了色谱分离条件和柱后衍生条件,结果表明:催化剂氯化血红素的浓度为8μmol/L,底物对羟基苯乙酸的浓度为80μmol/L,衍生化试剂的pH 10.5,衍生温度为35℃时衍生效率最高。在优化条件下,过氧化苯甲酰的线性范围为0.5～100 mg/L;样品的检出限为1 mg/kg,样品的加标回收率为98.5%～99.5%。本方法与现有的高效液相色谱法相比,检测面粉中过氧化苯甲酰具有抗干扰能力强,灵敏度高。     

Derivatization of Xanthomegnin for Fluorometric Determination

Abstract

A derivatization procedure was developed for the fluorometric determination of purified xanthomegnin. A highly fluorescent product is formed by reaction of xanthomegnin with ammonia and hydrogen peroxide under defined conditions. The derivative is a light brown compound that can easily be dissolved in water or polar organic solvents. Its excitation and emission wavelengths in methanol are 340 and 445 nm, respectively. As little as 0.1 ng of xanthomegnin can be detected using high pressure liquid chromatography in a reverse phase system with fluorescence detection.     

Improved detection of leucovorin in mixed folates and antifolates by reversed-phase liquid chromatography and on-line post-column UV irridiation

Summary An improved, reversed-phase, high-performance liquid chromatography (HPLC) method with dual UV-fluorescence detection for simultaneous determination of the antifolate methotrexate, the folate leucovorin and their two main metabolites 7-hydroxymethotrexate and 5-methyltetrahydrofolate, respectively is presented. The fluorescence intensity of leucovorin could be significantly increased by on-line, post-column irradiation with UV at 254 nm thus lowering the limit of detection for leucovorin to 0.2 ng absolute at a signal-to-noise ratio 31.     

Direct separation and detection of biogenic amines by ion-pair liquid chromatography with chemiluminescent nitrogen detector

Analysis of biogenic amines is critical to pharmaceutical and food industry due to their biological importance. For many years, the determination of biogenic amines has relied on high performance liquid chromatography (HPLC) coupling with pre-, on-, or post-column derivatization procedures to enable UV or fluorescent detections. In this study, 14 biogenic amines were separated on a Phenomenex Luna Phenyl-Hexyl column by an ion-pair liquid chromatography method using perfluorocarboxylic acids as ion-pair reagents and detected by a chemiluminescent nitrogen detector (CLND). This direct separation and detection HPLC method eliminated the time consuming and cumbersome derivatization procedures. Compared with HPLC-UV (post-column derivatization with ninhydrin) and HPLC-charged aerosol detector (CAD) methods, this HPLC-CLND technique provided narrower peaks, better baselines, and improved separations and detections. Excellent linearity was acquired by CLND for each of the 14 biogenic amines ranging from less than 1 ng to about 1000 ng (on-column weights). The relative response factors determined by this LC-CLND method were proportional to the numbers of nitrogen atoms in each compound, which has been the characteristic of the equimolar determinations by CLND. In addition, a number of samples including beer, dairy beverage, herb tea, and vinegar were analyzed by the LC-CLND method with satisfactory precision and accuracy.     

Optimization of two methods for the analysis of hydrogen peroxide: high performance liquid chromatography with fluorescence detection and high performance liquid chromatography with electrochemical detection in direct current mode

Two complementary methods were optimized for the separation and detection of trace levels of hydrogen peroxide. The first method utilized reversed-phase high-performance liquid chromatography with fluorescence detection (HPLC-FD). With this approach, hydrogen peroxide was detected based upon its participation in the hemin-catalyzed oxidation of p-hydroxyphenylacetic acid to yield the fluorescent dimer. The second method utilized high performance liquid chromatography with electrochemical detection (HPLC-ED). With this approach, hydrogen peroxide was detected based upon its oxidation at a gold working electrode at an applied potential of 400 mV vs. hydrogen reference electrode (Pd/H(2)). Both methods were linear across the range of 15-300 μM, and the electrochemical method was linear across a wider range of 7.4-15,000 μM. The limit of detection for hydrogen peroxide was 6 μM by HPLC/FD, and 0.6 μM by HPLC/ED. A series of organic peroxides and inorganic ions were evaluated for their potential to interfere with the detection of hydrogen peroxide. Studies investigating the recovery of hydrogen peroxide with three different extraction protocols were also performed. Post-blast debris from the detonation of a mixture of concentrated hydrogen peroxide with nitromethane was analyzed on both systems. Hydrogen peroxide residues were successfully detected on this post-blast debris.     

UV-induces formation of hydrogen peroxide based on the photochemistry of ketoprofen

Ketoprofen (KP) is a potent nonsteroidal anti-inflammatory drug. However, application to the skin is problematic because the photosensitizing properties of the benzophenone moiety may cause phototoxic effects when the treated skin region is exposed to UVA light. Using capillary electrophoresis with electrochemical detection we are able to differentiate the peroxides formed during illumination of KP-containing solutions of linoleic acid. Contrary to other profens a high amount of hydrogen peroxide was found among the reaction products. For investigation of the skin damaging effect human keratinocytes were used as models. Cell viability, DNA synthesis efficiency and intracellular concentration of peroxides were determined. Viability and proliferation behavior was not altered under the influence of KP. While lower concentrations of KP (10-100 nM) led to a protection against the UVA-induced (8 J/cm2) cell proliferation damage, higher concentrations (10-100 microM) led to an amplification of the proliferation decrease. With UVB irradiation at relevant doses the effects were lower than using UVA. Furthermore, intracellular peroxide content was increased after UV irradiation and KP addition. In conclusion some efforts have to be done to avoid these side effects in the use of KP for topical or transdermal application.     

HPLC-fluorescence determination of equilin and equilenin in postmenopausal women's urine

A high-performance liquid chromatographic (HPLC) method with fluorescence detection (lambda(ex) = 280 nm; lambda(em) = 410 and 312 nm) in combination with a post-column on-line photochemical derivatization is described for the determination of equilin and equilenin in urine from normal postmenopausal women after therapy with conjugated oestrogens. The column effluents were subjected on-line to UV irradiation (254 nm) and the photo-induced modifications were useful for the identification of the analytes. The conjugated (sulphate and glucuronide) forms were analysed after enzymatic or chemical hydrolysis and extracted with chloroform. Solid-phase extraction using strong anion-exchange sorbent was applied to the analysis of unconjugated oestrogen fraction to obtain a practical and reliable sample clean-up. The HPLC separations were achieved using ODS columns with a mobile phase consisting of 0.05 M triethylamine phosphate buffer (pH 4.0)-acetonitrile (64:36, v/v) at a flow rate of 1.0 mL/min. The method was accurate and reproducible; for the equilin and equilenin separation isocratic conditions were satisfactory, allowing a sensitive detection in urine samples with a detection limit of about 50 fmol for equilin (lambda(ex) = 280 nm; lambda(em) = 312 nm, after photoderivatization) and 10 fmol for equilenin (lambda(ex) = 280 nm; lambda(em) = 410 nm).     

Fluorescamine Post-Column Derivatization for the HPLC Determination of Cephalosporins in Plasma and Urine

Abstract

A post-column fluorescamine derivatization procedure is proposed for the determination of cephalosporins having an α-primary amino group in their side chain (cefaclor, cephalexin, cephradine, cefroxadine, cefaloglycine and cefadroxil). The linearity, repeatability and detection limits of fluorescence emission and UV absorption detection are compared under the same chromatographic conditions. Fluorescence detection is about two times more sensitive than UV absorption detection. Application to the determination of these cephalosporins in plasma and urine shows an improved selectivity by comparison with UV detection.     

高效液相色谱-柱后衍生法检测养殖水体及沉积物中11种磺胺药物残留

建立了养殖水体及沉积物中11种磺胺化合物的高效液相色谱-柱后衍生分析方法。养殖水体过滤后采用HLB固相萃取柱进行净化、富集;沉积物采用甲醇/EDTA-Mcllvaine缓冲液(1:1, v/v)提取,HLB固相萃取柱净化富集。经高效液相色谱分离,用荧光胺衍生试剂进行柱后衍生,荧光检测器检测。对柱后衍生系统参数进行了优化,确定了荧光胺溶液的浓度、流速和反应温度分别为0.2 g/L、0.15 mL/min和50 ℃,磺胺化合物在0.01~1.0 mg/L范围内线性显著,其相关系数r²值大于0.99995。11种磺胺类药物在养殖水体和沉积物中的加标回收率分别为79.3%~100.7%和74.6%~95.3%,相对标准偏差为2.2%~11.0%和2.6%~10.3%,检出限(LOD, S/N=3)为0.9~5.5 ng/L和0.3~1.3 μg/kg,定量限(LOQ, S/N=10)为3.0~18.1 ng/L和1.0~4.4 μg/kg。该法可应用于养殖环境中磺胺类药物的定性定量检测,具有较好的实用性。