On-line chemiluminescence determination of mitoxantrone by capillary electrophoresis

A novel capillary electrophoresis (CE) with chemiluminescence (CL) detection method for the determination of mitoxantrone (MTX) has been developed, which based on the CL reaction of potassium ferricyanide with luminol in sodium hydroxide medium sensitized by MTX. Under optimum analytical conditions, MTX is determined over the range of 7.0 × 10⁻⁸–1.0 × 10⁻⁶ M with a detection limit of 1.0 × 10⁻⁸ M. The relative standard deviation (RSD) was 3.7%, 2.6% and 3.0% for 7.0 × 10⁻⁸, 5.0 × 10⁻⁷ and 1.0 × 10⁻⁶ M MTX (n = 11), respectively. In laboratory-built CE–CL apparatus, the proposed method has been applied to determination of MTX in commercial drug and spiked in human urine and plasma with satisfactory results.     

Optimization of ascorbic and uric acid separation in human plasma by free zone capillary electrophoresis ultraviolet detection

In this paper we propose a new fast free zone capillary electrophoresis method for the simultaneous determination of ascorbic acid (AA) and uric acid (UA) in human plasma. We investigated the effect of analytical parameters, such as concentration and pH of borate running buffer, cartridge temperature, and sample treatment, on resolution, migration times, corrected peak areas, and efficiency. A good separation was achieved using a 60.2-cmx75-microm uncoated silica capillary and 100 mmol/L sodium borate buffer, pH 8, when metaphosphoric acid was employed as protein precipitant, in less than 4 min. These conditions gave a good reproducibility of migration times (CV 0.35 and 0.34%) and peak areas (CV 3.2 and 3.1%) for ascorbate and urate, respectively. The limit of detection was 0.5mg/L for both analytes when the detection was performed at 254 nm for AA and at 292 nm for UA. We compared the present method with a validated capillary electrophoresis assay by measuring plasma urate and ascorbate in 32 normal subjects and the obtained data were analyzed by the Passing and Bablok regression.     

Simultaneous determination of aminomethylbenzoic acid,cefminox sodium and etamsylate in human urine by capillary electrophoresis

A sensitive and selective capillary electrophoresis method is developed, for the first time, for effective separation and simultaneous determination of aminomethylbezoic acid (PAMBA), cefminox sodium (CMNX) and etamsylate (ETM). The electrophoresis conditions were investigated and optimized. A 25 mM phosphate solution (pH 8.5) was used as a buffer and the peak area was determined with UV detection at 216 nm wavelength under 18 kV separation voltage. Under optimal conditions, the three drugs can be separated effectively. Good linearity was achieved in 3.13–150 μg/mL for PAMBA, 6.25–150 μg/mL for CMNX and 3.13–150 μg/mL for ETM, with the correlation coefficients of >0.999. The limit of detection (LOD) for PAMBA, CMNX and ETM was 1.04, 2.08 and 1.04 μg/mL, respectively. Their recoveries in human urine were in the range from 90.2% to 101% with the RSD (n = 5) of 0.7–3.1%. The proposed method is simple, rapid and accurate, and provides the sensitivity and linearity necessary for analysis of the test drugs in human urine at clinically relevant concentrations.     

Identification and quantitative determination of uric acid in human urine and plasma by capillary electrophoresis with amperometric detection

Application of capillary zone electrophoresis with electrochemical detection to the identification and quantitative determination of uric acid in human urine as well as plasma is described. This work was carried out in a 30 cm×25 μm I.D. fused-silica capillary with tricine buffer and a carbon fiber bundle was employed as a working electrode, the working voltage in amperometric detection was set at +0.80 V (vs. SCE). The sample constituent is identified by stopped flow-linear sweep voltammetry. Under optimal conditions, a lower detection limit of 0.48 fmol was obtained for uric acid.     

Determination of octopamine in human plasma by capillary electrophoresis with optical fiber light-emitting diode-induced fluorescence detection

A new analytical method based on capillary electrophoresis (CE) separation and optical fiber light-emitting diode (LED)-induced fluorescence detection has been developed for the determination of octopamine. Naphthalene-2,3-dicarboxaldehyde (NDA) was used for precolumn derivatization of octopamine. The separation and determination of the derivative was performed using a laboratory-built CE system with an optical fiber LED-induced fluorescence detector. Optimal separation was obtained at 20 kV using a background electrolyte solution consisting of 25 mM sodium borate (pH 9.2). High sensitivity detection was achieved by the optical fiber LED-induced fluorescence detection using a purple LED as the excitation source. The limit of detection (signal/noise=3) for octopamine was 5.0 x 10(-9)M. A calibration curve ranging from 1.0 x 10(-8) to 5.0 x 10(-7)M was shown to be linear. Using this method, the levels of octopamine in human plasma from healthy donors were determined.     

Determination of uric acid in human saliva by high-performance liquid chromatography with amperometric electrochemical detection

The aim of the present study is to establish a highly sensitive method for the determination of uric acid (UA) in human saliva. The monitoring of UA levels in less invasive biological samples such as saliva is suggested for the diagnosis and therapy of gout, hyperuricemia, and the Lesch-Nyhan syndrome, and for detecting such conditions as alcohol dependence, obesity, diabetes, high cholesterol, high blood pressure, kidney disease, and heart disease. Reversed-phase high-performance liquid chromatography with electrochemical detection (HPLC-ED) was employed for the determination of UA obtained by solid-phase extraction from saliva. To quantify UA, we compared the ED efficiencies of an amperometric ED (Ampero-ED) with a single electrode and a coulometric ED (Coulo-ED) with a multiple electrode array. The results showed that the detection limits (S/N=3) were 3 nM for Ampero-ED and 6 nM for Coulo-ED, and the linearity of the calibration curves of 60-6000 nM had correlation coefficients exceeding 0.999. In addition, the total analytical time was 10 min. In the sample preparation of UA in saliva, an Oasis MAX solid-phase cartridge was used. The recoveries of UA spiked at 0.6 and 3 microM in saliva were above 95% with a relative standard deviation (RSD) of less than 15%. Therefore, the present method may be used in the routine and diagnostic determination of UA in human saliva.     

Simultaneous determination of isoniazid and p‐aminosalicylic acid by capillary electrophoresis using chemiluminescence detection

It was found that isoniazid (ISO) or p‐aminosalicylic acid (PAS) could enhance the chemiluminescence (CL) emission from Cu (II)‐luminol‐hydrogen peroxide system, and the increased chemiluminescence signals were proportional to their concentrations, respectively. Based on this phenomenon, a chemiluminescence method coupled to capillary electrophoresis (CE) was established for simultaneous determination of ISO and PAS. The CE conditions including running buffer and running voltage were investigated in detail. The effects of the pH of H₂O₂ solution and the concentrations of luminol, H₂O₂ and Cu (II) on the CL signal were also investigated carefully. Under the optimized conditions, the analysis could be accomplished within 10 min, with the limits of detection of 0.3 µg mL^–1 for ISO and 1.1 µg mL^–1 for PAS, corresponding to 7.2 and 26.4 pg per injection (24 nL), respectively. Finally, the method was validated by determining the two analytes in pharmaceutical preparation and spiked human serum samples. The results of pharmaceutical tablet analysis were in good agreement with the labeled amounts. The recoveries for ISO and PAS in human serum were in the range of 92–104% and 90–113%, respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

Monitoring the formation of Maillard reaction products of glucosamine with fibrinogen and human serum albumin using capillary electrophoresis

Formation of Maillard reaction products (MRP) of glucosamine (GlcN) with fibrinogen and human serum albumin (HSA), under simulated physiological conditions, was detected by fluorescence (excitation/emission: 340/420 nm) and UV/Vis (max. 275 nm) spectroscopy. The application of polyacrylamide gel electrophoresis demonstrated the generation of high-molecular-weight fibrinogen and HSA MRP by GlcN. A simple and rapid capillary electrophoresis method was developed to separate MRP formed by the reaction of GlcN with proteins from GlcN autocondensation products.     

Non‐competitive immunoassay for luteinizing hormone in human serum using capillary electrophoresis with chemiluminescence detection

A non‐competitive immunoassay based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of luteinizing hormone (LH) in human serum. The work involved the development of separation and CL conditions, allowing for routine analysis of serum samples. In this study, horseradish peroxidase (HRP)‐labelled monoclonal anti‐LH can catalyse the luminol–hydrogen peroxide reaction. The determined LH can react with excessive amount of HRP‐labelled anti‐LH. Within 14 min, free enzyme conjugate and immune complex could be separated in alkaline borate buffer by means of a high voltage (15 kV). To improve sensitivity, a series of measures were adopted, including the choice of para‐iodophenol as a CL enhancer, unique design in detect window. Under the optimal conditions, the calibration curve for LH was established in the concentration range 1–200 mIU/mL and the detection limit was 0.08 mIU/mL. Compared with ELISA, this method decreased the detection limit by about 12 times, and it has been successfully employed in the determination of LH in human serum. Copyright © 2007 John Wiley & Sons, Ltd.     

Capillary electrophoretic determination of methotrexate, leucovorin and folic acid in human urine

A simple, rapid and sensitive procedure using capillary zone electrophoresis (CZE) to measure methotrexate, folinic acid and folic acid in human urine has been developed and validated. Optimum separation of methotrexate, folinic acid and folic acid was obtained on a 60 cm x 75 microm capillary using a 15 mM phosphate buffer solution (pH 12.0), temperature and voltage 20 degrees C and 25 kV, respectively and hydrodynamic injection. Under these conditions the analysis takes approximately 9.0 min. Good results were obtained for different aspects including stability of the solutions, linearity, accuracy and precision. Before CZE determination, the urine samples were purified and enriched by means of a solid phase extraction step with a preconditioned C(18) cartridge and eluting the compound with a mixture 1:1 of methanol:water. A linear response over the urine concentration range 1.0-6.0 mgL(-1) for MTX and 0.5-6.0 mgL(-1) for folinic acid and folic acid was observed. Detection limits for the three compound in urine were 0.35 mgL(-1). CZE was shown to be a good method with regard to simplicity, satisfactory precision, and sensitivity.     

Determination of amphetamine-derived designer drugs in human urine by SPE extraction and capillary electrophoresis with mass spectrometry detection

In recent years, a number of newer designer drugs have entered the illicit drug market. The methylenedioxy-derivates of amphetamine represent the largest group of designer drugs. This paper describes a method for screening for and quantification of ten 2,5-methylenedioxy-derivates of amphetamine and phenylethylamine in human urine, using capillary electrophoresis coupled to electrospray ionisation-mass spectrometry (CE-ESI-MS). Prior to CE-MS analysis, a simple solid-phase extraction (SPE) was used for sample cleanup. The method was validates according to international guidelines.     

Simultaneous quantification of sialyloligosaccharides from human milk by capillary electrophoresis

The acidic oligosaccharides of human milk are predominantly sialyloligosaccharides. Pathogens that bind sialic acid-containing glycans on their host mucosal surfaces may be inhibited by human milk sialyloligosaccharides, but testing this hypothesis requires their reliable quantification in milk. Sialyloligosaccharides have been quantified by anion exchange high-performance liquid chromatography (HPLC), reverse- or normal-phase HPLC, and capillary electrophoresis (CE) of fluorescent derivatives; in milk, these oligosaccharides have been analyzed by high pH anion exchange chromatography with pulsed amperometric detection and, in our laboratory, by CE with detection at 205nm. The novel method described here uses a running buffer of aqueous 200mM NaH2PO4 (pH 7.05) containing 100mM sodium dodecyl sulfate (SDS) mixed with 45% (v/v) methanol to baseline resolve 5 oligosaccharides and separate all 12. This allows automated simultaneous quantification of the 12 major sialyloligosaccharides of human milk in a single 35-min run. This method revealed differences in sialyloligosaccharide concentrations between less and more mature milk from the same donors. Individual donors also varied in expression of sialyloligosaccharides in their milk. Thus, the facile quantification of sialyloligosaccharides by this method is suitable for measuring variation in expression of specific sialyloligosaccharides in milk and their relationship to decreased risk of specific diseases in infants.     

Determination of three major catecholamines in human urine by capillary zone electrophoresis with chemiluminescence detection

A sensitive simple method is presented for the determination of three major catecholamines in human urine by capillary electrophoresis (CE) with on-line chemiluminescence (CL) detection. This was also the first time that the luminol-Ag(III) complex CL system was used for CE detection. This method was based on the enhancing effect of epinephrine (EP), norepinephrine (NE), and dopamine (DA) on the CL reaction between luminol and the Ag(III) complex in alkaline solution. The separations and determinations were performed with an electrophoretic buffer consisting of 20.0mM sodium borate and 1.0mM luminol. Under optimized conditions, the three catecholamines were baseline separated and detected in less than 8 min. Detection limits of 7.9 × 10(-8)M, 1.0 × 10(-7)M, and 6.9 × 10(-8)M were observed for EP, NE, and DA, respectively. Relative standard deviation (RSD) values for the peak height were 4.7% to 5.4% (n = 5). Our proposed method was applied to the determinations of the catecholamines in urine samples from 12 healthy individuals and 26 pheochromocytoma patients. Our results suggest that this method might be useful to monitor the catecholamine levels in routine screening and to diagnose pheochromocytoma.     

Determination of salicylic acid in human serum with capillary zone electrophoresis

The determination of salicylic acid (SA), a metabolite of aspirin, in human serum was developed using capillary zone electrophoresis (CZE) with diode array detection. The reproducibility of separation and quantification with CZE analysis of the extract of SA from human serum was appropriate for the intra- and inter-day assay coefficients. A high correlation was revealed between the serum SA levels in volunteers determined by CZE and those determined by a fluorescence polarization immunoassay (r=0.973, n=12), although the former values were slightly higher than the latter. There were no peaks interfering with the assay of SA by internal standard method. This CZE method could provide a simple and efficient method for monitoring SA in patients.     

Development of a novel capillary electrophoresis chemiluminescence system for amino acid analysis

In the present study, a novel peroxyoxalate CE–CL system was developed to achieve high signal stability and sensitivity based on a design of a new interface including a new mixing mode and a new grounding electrode mode. Amino acids fluorescently tagged with dansyl chloride and naphthalene‐2,3‐dicarboxaldehyde(NDA) were used for the study. Experiment results show this new system is quite effective to separate and detect amine acid with high stability and resolute. The detection limits were 1.1 nmol/L for dansyl‐leucine (Leu) and 2.0 nmol/L for dansyl‐aspartic acid (Asp). The relative standard deviations of peak height and migration time were in the ranges of 2.3–3.8% and 1.2–1.5%, respectively. Copyright © 2008 John Wiley & Sons, Ltd.     

Electrochemical profiles of Herba Saussureae Involucratae by capillary electrophoresis with electrochemical detection

A high-performance capillary electrophoresis with electrochemical detection method has been developed for the determination of the pharmacologically active ingredients, acacetin, rutin, umbelliferone, kaempferol, apigenin, luteolin and quercetin, in Herba Saussureae Involucratae. Under optimum conditions, the seven analytes could be completely separated within 19 min in a 75 cm length capillary at a separation voltage of 16 kV in a 50 mM borax running buffer (pH 9.2). A 300 microm diameter carbon disk electrode, positioned opposite the outlet of the capillary in a wall-jet configuration at a potential of +950 mV (vs a saturated calomel electrode) was used as the working electrode. A good linear relationship was established between peak current and concentration of the analytes over two orders of magnitude with detection limits (signal-to-noise ratio = 3) ranging from 1.2 x 10(-7) to 4.1 x 10(-8) g/mL for all analytes. The proposed method has been successfully applied to the analyses of bio-active components of Herba Saussureae Involucratae samples after a relatively simple extraction procedure. The assay results show that the resultant electrochemical profiles are indicative of the content diversity of each electrochemically active ingredient in the various samples, and may also offer some evidence for phytotaxonomy.     

Paraoxonase activity against nerve gases measured by capillary electrophoresis and characterization of human serum paraoxonase (PON1) polymorphism in the coding region (Q192R)

An analytical method for determining paraoxonase activity against sarin, soman and VX was established. We used capillary electrophoresis to measure directly the hydrolysis products: alkyl methylphosphonates. After enzymatic reaction of human serum paraoxonase (PON1) with nerve gas, substrate was removed with dichloromethane, and alkyl methylphoshphonates were quantified by capillary electrophoresis of reversed osmotic flow using cationic detergent and sorbic acid. This method was applied to the characterization of human serum PON1 polymorphism for nerve gas hydrolytic activity in the coding region (Q192R). PON1-192 and PON1-55 genotypes were determined by their gel electrophoretic fragmentation pattern with restriction enzymes after polymerase chain reaction (PCR) of blood leukocyte genomic DNA. Frequencies of genotypes among 63 members of our institutes with PON1-192 and PON1-55 were 9.5% (¹⁹²QQ), 30.1% (¹⁹²QR) and 44.4% (¹⁹²RR), and 82.5% (⁵⁵LL), 17.5% (⁵⁵LM) and 0% (⁵⁵MM), respectively. ¹⁹²Q and ¹⁹²R enzymes were purified from the respective genotype human plasma, using blue agarose affinity chromatography and diethyl amino ethane (DEAE) anion exchange chromatography. V_max and K_m were measured using Lineweaver-Burk plots for hydrolytic activities against sarin, soman and VX at pH 7.4 and 25 °C. For sarin and soman, the V_max for ¹⁹²Q PON1 were 3.5- and 1.5-fold higher than those for ¹⁹²R PON1; and k_cat/K_m for ¹⁹²Q PON1 were 1.3- and 2.8-fold higher than those for ¹⁹²R PON1. For VX, there was little difference in V_max and k_cat/K_m between ¹⁹²Q and ¹⁹²R PON1, and VX hydrolyzing activity was significantly lower than those for sarin and soman. PON1 hydrolyzed sarin and soman more effectively than paraoxon.     

Study on the multiple sites binding of human serum albumin and porphyrin by affinity capillary electrophoresis

Equations to describe the two sites binding between proteins and ligands were deduced. According to these equations, not only the binding constants, but also the mole fraction of proteins in different forms could be obtained. Using the published data on the interaction between human serum albumin (HSA) and three kinds of porphyrin (coproporphyrin (CP), uroporphyrin I (UP) and protoporphyrin (PP)), a further study on their binding was carried out. It was concluded that there may exist two binding sites with the binding constants at the first site, proved to be the preferential one, being 6.50 x l0(5), 1.94 x 10(6) and 8.94 x 10(5), respectively. In addition, it was also demonstrated that the two binding sites of HSA with CP and UP might be of different kinds, though those of HSA and PP were of the same kind but at different positions.     

Determination of puerarin by capillary electrophoresis with chemiluminescence detection

A new analytical method for puerarin using capillary electrophoretic (CE) separation and chemiluminescence (CL) detection has been developed. The detection was based on the enhanced CL intensity of the reaction between luminol and potassium ferricyanide by puerarin in alkaline solution. A laboratory-built CE–CL apparatus was deployed for the puerarin detection. Under the optimal conditions, a linear range from 5.0 × 10^?8 to 2.5 × 10^?6 M and a detection limit of 1.0 × 10^?8 M (S/N = 3) for puerarin were achieved. The determination of puerarin was achieved in less than 5 min, and the proposed method was applied to the determination of puerarin in pharmaceutical, human urine and human plasma samples.     

Rapid analysis of amplified double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection

DNA amplification technology has been applied to clinical diagnosis of infectious disease, genetic disorder, and cancer. After in vitro amplification of a particular DNA region, the methods of analysis for these amplified samples play a pivotal role in clinical diagnosis. Conventional gel electrophoresis has been routinely used in the lab for checking DNA. The whole procedure is time consuming and requires more than 1 ng of DNA for detection. To achieve greater performance in DNA diagnosis, we demonstrated capillary electrophoresis with laser induced fluorescence detection for analysis of amplified DNA. The analysis of DNA could be completed within 3 min and the data is directly entered into the computer. Considering the automatic and rapid process, we believe that this method could be routinely utilized for the clinical diagnosis of amplified DNA products.