Highly sensitive method for determination of N-nitrosamines using high-performance liquid chromatography with online UV irradiation and luminol chemiluminescence detection

A new method for the measurement of N-nitrosamines in part-per-trillion concentrations from water samples without preconcentration steps has been developed. This method is based on online UV irradiation after high-performance liquid chromatographic separation and subsequent luminol chemiluminescence detection without addition of an oxidant. It was confirmed that N-nitrosamines in basic aqueous solution were transformed to peroxynitrite by UV irradiation. The detection limits for this method were 1.5 ng/L, 2.9 ng/L, 3.0 ng/L, and 2.7 ng/L for N-nitrosodimethylamine, N-nitrosomorpholine, N-nitrosomethylethylamine, and N-nitrosopyrrolidine, respectively, at a signal-to-noise ratio of 3. The calibration graphs were linear in the range of 5–1000 ng/L for these N-nitrosamines. This method was used for the determination of N-nitrosamines in tap water, river water, and industrial plant effluent samples. The recoveries of N-nitrosodimethylamine, N-nitrosomorpholine, N-nitrosomethylethylamine, and N-nitrosopyrrolidine present in tap water sample at a concentration of 10 ng/L (mean ± standard deviation, n = 4) were (94.8 ± 2.7)%, (102.0 ± 6.9)%, (99.3 ± 3.9)%, and (102.8 ± 2.5)%, respectively. These results indicate that our proposed method can be applied satisfactorily to the determination of N-nitrosamines in water samples.     

Selective determination method for measurement of nitrite and nitrate in water samples using high-performance liquid chromatography with post-column photochemical reaction and chemiluminescence detection

A simple, sensitive and selective method for the simultaneous determination of nitrite and nitrate in water samples has been developed. The method is based on ion-exchange separation, online photochemical reaction, and luminol chemiluminescence detection. The separation of nitrite and nitrate was achieved using an anion-exchange column with a 20 mM borate buffer (pH 10.0). After the separation, these ions were converted to peroxynitrite by online UV irradiation using a low-pressure mercury lamp and then mixed with a luminol solution prepared with carbonate buffer (pH 10.0). The calibration graphs of the nitrite and nitrate were linear in the range from 2.0 × 10⁻⁹ to 2.5 × 10⁻⁶ M and 2.0 × 10⁻⁸ to 2.5 × 10⁻⁵ M, respectively. Since the sensitivity of nitrite was about 10 times higher than that of nitrate, the simultaneous determination of nitrite and nitrate in the water samples could be efficiently achieved. This method was successfully applied to various water samples – river water, pond water, rain water, commercial mineral water, and tap water – with only filtration and dilution steps.     

纳米银增敏鲁米诺液相化学发光分析法测定4-乙酰氨基酚的研究

研究发现在碱性条件下,纳米银对鲁米诺-铁氰化钾液相化学发光体系发光信号具有明显的增敏作用,而4-乙酰氨基酚对该体系具有强烈的抑制作用。结合流动注射技术,建立了流动注射化学发光分析法测定对乙酰氨基酚的新方法。该方法测定对乙酰氨基酚的线性范围为9.0×10-12～1.0×10-10g/mL(0.9947)和1.0×10-1...     

Simple and sensitive method for determination of glycoalkaloids in potato tubers by high-performance liquid chromatography with chemiluminescence detection

A novel, simple and sensitive high-performance liquid chromatographic method for the determination of the potato glycoalkaloids, alpha-solanine and alpha-chaconine, based on the chemiluminescent reaction of tris(2,2'-bipyridine)ruthenium(III) has been developed. The calibration graph was linear in the range of 5 ng/ml-10 microg/ml for both alpha-solanine and alpha-chaconine. The detection limits of alpha-solanine and alpha-chaconine were 1.2 and 1.3 ng/ml, respectively. This method was successfully applied to a potato tuber sample without cleanup, pre-concentration, and derivatization steps. The recoveries (mean +/- standard deviation, %) of alpha-solanine and alpha-chaconine spiked in tuber pith at 10 microg/g (n = 6) were 101.0 +/- 4.4% and 103.6 +/- 7.1%, respectively.     

Determination of vitamin K homologues by high-performance liquid chromatography with on-line photoreactor and peroxyoxalate chemiluminescence detection

A sensitive and highly selective high-performance liquid chromatography (HPLC) method was developed for the determination of vitamin K homologues including phylloquinone (PK), menaquinone-4 (MK-4) and menaquinone-7 (MK-7) in human plasma using post-column peroxyoxalate chemiluminescence (PO-CL) detection following on-line ultraviolet (UV) irradiation. The method was based on ultraviolet irradiation (254 nm, 15 W) of vitamin K to produce hydrogen peroxide and a fluorescent product at the same time, which can be determined with PO-CL detection. The separation of vitamin K by HPLC was accomplished isocratically on an ODS column within 35 min. The method involves the use of 2-methyl-3-pentadecyl-1,4-naphthoquinone as an internal standard. The detection limits (signal-to-noise ratio = 3) were 32, 38 and 85 fmol for PK, MK-4 and MK-7, respectively. The recoveries of PK, MK-4 and MK-7 were greater than 82% and the inter- and intra-assay R.S.D. values were 1.9-5.4%. The sensitivity and selectivity of this method were sufficient for clinical and nutritional applications.     

A post-column extraction system for the determination of tertiary amines by liquid chromatography with chemiluminescence detection

Summary The combination of an ion-pair extraction detection system with peroxyoxalate chemiluminescence detection has been investigated. Ion-pairs of protonated tertiary amines with a chemiluminescent counter ion are on-line post-column extracted to 1,2-dichloroethane containing bis(2,4,6-trichlorophenyl)oxalate (TCPO). Hydrogen peroxide is added to the organic phase by means of a solid-state perhydrit reactor. The influence of the base catalyst (imidazole) on the chemiluminescence reaction in apolar solvents was studied. Some sulfonated chemiluminophores were compared with respect to their chemiluminescence, ion-pairing and extraction properties, and 5-dimethylaminonaphthalene-1-sulfonate was found to be the most suitable reagent. The detection limit of the potential drug secoverine is in the sub-nanogram range.     

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).     

An improved method for acetaldehyde determination in blood by high-performance liquid chromatography and solid-phase extraction

This study reports on an improved method for acetaldehyde (ACH) determination in blood by high-performance liquid chromatography (HPLC). In the case of HPLC analysis, ACH is generally converted to derivatives for ultraviolet detection (for example 2,4-dinitrophenylhydrazine [DNPH] derivative). Nevertheless, elevation of the background during protein precipitation, hydrazone synthesis, or both frequently results in a serious loss of accuracy and precision of the analysis. The method in this study is developed to minimize the increase in nonspecific ACH-DNPH with a view to optimize mainly the synthetic condition of ACH-DNPH. The background is decreased dramatically by gentle deproteination, optimization of the DNPH amount and reaction pH, and reversed-phase solid extraction for the elimination of excess DNPH reagent. The standard curves show good linearity between 0 and 100 microM and minimal background is observed, indicating that the method is useful for monitoring the ACH concentration in blood.     

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.     

Simple, stable and sensitive electrogenerated chemiluminescence detector for high-performance liquid chromatography and its application in direct determination of multiple fluoroquinolone residues in milk

A simple, stable and sensitive electrogenerated chemiluminescence (ECL) detector was developed. It was based on tris(2,2-bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺) immobilized on the surface of a Pt wire with Nepem-105D ion exchange solution. The detector was prepared by inserting a Pt wire with immobilized Ru(bpy)₃²⁺ (working electrode) into a capillary tube, followed by inserting another Pt wire (counter electrode) in this tube and sealing. ECL behavior was investigated using ofloxacin as an analyte. Under optimal conditions, stable ECL intensity was obtained. This detector has been used in HPLC-ECL for the determination of multiple target fluoroquinolone residues in milk. There is no post column reagent addition, which would dilute the analytes, potentially leading to chromatographic band-broadening. The system is very simple with low dead volume, low baseline and background noise, together with high sensitivity and stability. The as-prepared ECL detector, when was used for the determination of ofloxacin, pefloxacin, enrofloxacin and difloxacin in milk, demonstrated adequate sensitivity to allow quantification of trace FQ levels in commercial milk samples. One or more of the target FQ analytes were present at levels above the LOD of the new ECL detector in each and every one of the 22 milk samples analysed.     

An optimized method for determination of intracellular glutathione in mouse macrophage cultures by fluorimetric high-performance liquid chromatography

We have optimized a method for the determination of intracellular glutathione by high-performance liquid chromatography, using fluorimetric detection. To minimize artifacts and provide an accurate determination of intracellular glutathione, cell extracts were prepared using extraction conditions specifically designed to inhibit autoxidation and enzymatic degradation of glutathione. The sensitivity of the method was enhanced by adjusting the dansyl chloride derivatization reaction with regard to parameters such as pH, reaction time and dansyl chloride concentration. Both oxidized and reduced forms of glutathione were quantified using the refined method in extracts of oxidatively stressed J774A.1 mouse macrophage cells and reflected an expected shift in cellular redox status.     

Facile and sensitive method for the determination of mesna in plasma by high-performance liquid chromatography with ultraviolet detection

The use of 2-chloro-1-methylquinolinium tetrafluoroborate, an ultraviolet tagging reagent, for the ion-pair, reversed-phase high-performance liquid chromatography of mesna in human plasma is reported. In order to achieve this objective optimization of the two-step procedure, derivatization and separation of mesna S-quinolinium derivative from that of other thiols present in plasma and internal standard, was investigated. The derivatization was optimized in terms of pH, reagent excess and time of the reaction, and the mobile phase in terms of ion-pairing reagent concentration, pH, organic modifier content and temperature. Baseline separation was achieved on an analytical Waters Nova-Pak C18 (150x3.9 mm, 5 microm) column with a mobile phase consisting of pH 2.3 0.05 M trichloroacetic acid-acetonitrile (89:11, v/v) pumped at 1.2 ml/min. The peak height ratios of the mesna derivative to that of the internal standard (thiomalic acid) varied linearly with the concentration of the analyte added to normal plasma with a correlation coefficient of 0.9997. The lower limits of detection and quantitation were 40 pmol/ml (0.8 pmol on-column) and 160 pmol/ml (3.2 pmol on-column), respectively. The intra-run imprecision and inaccuracy were from 1.3 to 2.4 and from 1.3 to 2.0%, respectively.     

An amobarbital molecularly imprinted microsphere for selective solid-phase extraction of phenobarbital from human urine and medicines and their determination by high-performance liquid chromatography

A 40-60 microm amobarbital molecularly imprinted microsphere, used as a solid-phase selective sorbent for extracting phenobarbital from human urine and medicines, was prepared by a suspension polymerization method. A series of binding studies was performed in order to find optimal loading, washing and eluting conditions for solid-phase extraction. Under optimal conditions, good recoveries of phenobarbital in samples were obtained. Normally, molecularly imprinted polymers, prepared in bulk, require laborious work. Significant losses occur during the procedure of grinding and washing. In this work all molecularly imprinted polymers made into microsphere could be utilized, and the cost of the template was reduced too (the price of phenobarbital is twice that of amobarbital). As the phenobarbital to be extracted was different from the template molecule amobarbital, the interference caused by template leaking could be avoided in the assay.     

Development and optimization of an analytical method for the determination of Sudan dyes in hot chilli pepper by high-performance liquid chromatography with on-line electrogenerated BrO- -luminol chemiluminescence detection

The determination of four Sudan dyes by means of high-performance liquid chromatography (HPLC) with chemiluminescence (CL) detection was proposed. The method was based on the enhancement effect of Sudan dyes on the chemiluminescence reaction between luminol and BrO-, which was on-line electrogenerated by constant current electrolysis. The separation was carried out on Nucleosil RP-C18 column (250 mm x 4.6 mm i.d., 5 microm, pore size, 100 A) at 35 degrees C. The mobile phase consisted of a V (methanol): V (0.2% aqueous formic acid) = 90:10 solution. At a flow-rate of 1.0 ml min(-1), the total run time was 25 min. The effects of several parameters on the HPLC resolution and CL emission were studied systematically. For the four Sudan dyes, the limits of detection (LOD) at a signal-to-noise of 3 ranged from 4 to 8 microg kg(-1) and the limits of quantification (LOQ) at a signal-to-noise of 10 ranged from 13 to 27 microg kg(-1). The relative standard deviations (RSD) of intra-and inter-day precision were below 4.4%. The average recoveries for all four Sudan dyes (spiked at the levels of 1.0 and 1.5 mg kg(-1)) in chilli tomato sauce and hot chilli pepper ranged from 94% to 105%, and the relative standard deviations of the quantitative results were from 2.5 to 4.2%. The proposed method had been successfully applied to the determination of four Sudan dyes in hot chilli products.     

Microdialysis sampling and high-performance liquid chromatography with chemiluminescence detection for in-vivo on-line determination and study of the pharmacokinetics of levodopa in blood

A simple, reliable, and reproducible method for in-vivo on-line separation and determination of levodopa has been based on microdialysis then high-performance liquid chromatography with chemiluminescence detection. The perfusate is perfused at a flow rate of 5 L min^–1. The concentration of levodopa in the dialysate is determined on line with a chemiluminescence system. The dialysate sample volume is approximately 20 L. The response of the system is linearly related to the concentration of levodopa in the range 1×10^–8 to 1×10^–6 g mL^–1 (r²=0.9995) with a detection limit (3) of 3×10^–9 g mL^–1 and sample throughput of 12 h^–1; RSD is 2.8% (n=11). The method has been successfully used to study the pharmacokinetics of levodopa in vivo; the values of the pharmacokinetics parameters C_max, AUC_0–t and T_max were 16.60, 20.92 ng mL^–1, and 90 min, respectively.     

Preliminary evaluation of monolithic column high-performance liquid chromatography with tris(2,2′-bipyridyl)ruthenium(II) chemiluminescence detection for the determination of quetiapine in human body fluids

High-performance liquid chromatography (HPLC) with tris(2,2′-bipyridyl)ruthenium(II) chemiluminescence detection methodology is reported for the determination of the atypical antipsychotic drug quetiapine and the observation of its major active and inactive metabolites in human urine and serum. The method uses a monolithic chromatographic column allowing high flow rates of 3 mL min⁻¹ enabling rapid quantification. Flow injection analysis (FIA) with tris(2,2′-bipyridyl)ruthenium(II) chemiluminescence detection and HPLC time of flight mass spectrometry (TOF-MS) were used for the determination of quetiapine in a pharmaceutical preparation to establish its suitability as a calibration standard. The limit of detection achieved with FIA was 2 × 10⁻¹¹ mol L⁻¹ in simple aqueous solution. The limits of detection achieved with HPLC were 7 × 10⁻⁸ and 2 × 10⁻¹⁰ mol L⁻¹ in urine and serum, respectively. The calibration range for FIA was between 5 × 10⁻⁹ and 1 × 10⁻⁶ mol L⁻¹. The calibration ranges for HPLC were between 1 × 10⁻⁷-1 × 10⁻⁴ and 1 × 10⁻⁸-1 × 10⁻⁴ mol L⁻¹ in urine and serum, respectively. The quetiapine concentrations in patient samples were found to be 3 × 10⁻⁶ mol L⁻¹ in urine and 7 × 10⁻⁷ mol L⁻¹ in serum. Without the need for preconcentration, the HPLC detection limits compared favourably with those in previously published methodologies. The metabolites were identified using HPLC-TOF-MS.     

Validation and pharmacokinetic application of a method for determination of doxazosin in human plasma by high-performance liquid chromatography

A simple high-performance liquid chromatographic method for the determination of doxazosin in human plasma was developed and validated. Prazosin was used as internal standard. After extraction twice with ethyl acetate, chromatographic separation of doxazosin in human plasma was carried out using a reversed-phase Apollo C18 column (250 x 4.6 mm, 5 microm) with mobile phase of methanol-acetonitrile-0.04 m disodium hydrogen orthophosphate (22:22:56, v/v/v) adjusted to pH 4.9 with 0.9 m phosphoric acid and quantified by fluorescence detection operated with an excitation wavelength of 246 nm and an emission wavelength of 389 nm. The lower limit of quantification (LLOQ) of this assay was 1 ng/mL using 500 microL human plasma. Linearity was established over the range 1-25 ng/mL (r2 > 0.9994). The intra- and inter-day accuracy ranged from 90.5 to 104.4% and the coefficient of variation were not more than 8.6% for both intra- and inter-day precision, over the range of the calibration curve. The absolute recoveries of doxazosin and prazosin from human plasma were more than 91%. Doxazosin demonstrated acceptable short-term, long-term and freeze-thaw stability in human plasma. The assay has been successfully applied to plasma sample ana-lysis for pharmacokinetic study.