Determination of phosphorus using capillary electrophoresis and micro-high-performance liquid chromatography hyphenated with inductively coupled plasma mass spectrometry for the quantification of nucleotides

We performed the quantification of phosphorus in deoxynucleotides using capillary electrophoresis (CE) and micro-HPLC (μHPLC) hyphenated with inductively coupled plasma mass spectrometry (ICP-MS). DNA and its component units have conventionally been determined by photometry; however, more selective and sensitive methods are needed for small biological samples. CE and μHPLC offer the advantages of good separation and small consumption of samples, and ICP-MS is a highly sensitive technique for the determination of a chemical element. Therefore, we have developed an interface device for combining CE and μHPLC with ICP-MS for quantifying nucleotides based on phosphorus content. The interface utilizes 4.5 μL/min for nebulizing and effective introduction of the sample into ICP. The samples of nucleotides and free phosphoric acid were well separated in the CE–ICP-MS measurement, and the calibration curves (1–100 μg/mL) of the nucleotides showed a linear (R² > 0.999) increase in intensity. Similarly, the samples of nucleotides were baseline separated using μHPLC–ICP-MS, and the calibration curves of the nucleotides were linear (R² > 0.998). The detection limits of these species and phosphorus in nucleotides using CE–ICP-MS and μHPLC–ICP-MS were 0.77–6.5 ng/mL and 4.0–6.5 ng/mL, respectively. These values were about one or two orders lower than those in a previous report. The sample volumes of these experiments were calculated to be about 10 nL and 50 nL per analysis. Therefore, these analytical methods have the potential to be useful for the determination of biological samples, such as DNA and RNA molecules.     

Lambda genomic DNA quantification using ultrasonic treatment followed by liquid chromatography–isotope dilution mass spectrometry

Quantification of genomic DNA that is traceable to the SI was performed successfully by measuring the individual nucleotides. Specifically, ultrasound was used to shear lambda genomic DNA into fragments of less than 200 base pairs, followed by deoxyribonuclease Ι and phosphodiesterase Ι digestion and liquid chromatography–isotope dilution mass spectrometry (LC-IDMS) quantification to estimate the mass fraction of the lambda DNA, based on the constituent deoxynucleotide monophosphates (dNMPs) within the molecule. Digital PCR (dPCR) was employed to quantify the same lambda DNA solution to provide independent data for comparing the performance of two quantitative methods. On the basis of the LC-IDMS measurement after ultrasonic treatment of the sample, the concentration of lambda DNA was 273.1 ± 9.8 μg/g (expanded uncertainty at the 95% confidence interval). This shows good agreement with the data from dPCR. Additionally, the result calculated on the basis of the sum of the concentrations of the four dNMPs is the same as that calculated on the basis of the sequence, which indicates that knowledge of the DNA sequence and length is unnecessary to measure the total DNA concentration when applying ultrasonic treatment–LC-IDMS.     

微波消解-原子荧光光谱法测定海洋生物体中汞

应用原子荧光光谱法测定海洋生物体中汞的含量。试样用盐酸、硝酸和过氧化氢混合溶液在微波消解仪中消解处理,经试验选定消解程序为:①消解功率1 600 W;②5min升温至120℃,保持5min;③5min升温至160℃,保持10min。分析时仪器工作条件为负高压260V,灯电流为40mA,载气流量为800mL.min-1。汞的质量浓度在0.80μg.L-1以内与相应的荧光强度呈线性关系,方法检出限(3s/k)为0.002mg.kg-1。方法用于测定海洋生物体中汞的含量,测定值的相对标准偏差(n=6)在2.7%～6.0%之间,加标回收率在90%～104%之间。     

Synthesis of fluorescently labeled alkylated DNA adduct standards and separation by capillary electrophoresis

DNA adducts are regarded as individual internal dosimeters for the exposure to chemical carcinogens. To date, the most sensitive method for DNA adduct analysis is the radioactive 32P-postlabeling method, which allows the detection of one adduct in 10(10) unmodified nucleotides in microg amounts of DNA. However, this technique suffers from disadvantages such as working with radioactive phosphorus and time-consuming chromatographic separation procedures. In addition, the simultaneous detection of adducts from different classes of carcinogens in a DNA sample is difficult. In order to overcome these drawbacks, we are developing a new detection method, comprising fluorescence labeling of DNA adducts, capillary electrophoretic (CE) separation, and on-line detection by monitoring laser-induced fluorescence (LIF). So far, we have evaluated the separation power and the detection limit of CE with fluorescently labeled standard compounds such as unmodified nucleotides or alkylated thymidines. For this purpose, we developed a universal method for labeling 5'-OH-mononucleosid-3'-dicyanoethyl-phosphates with fluorescent dyes based on the phosphoramidite technology for DNA synthesis. The separation of N3-methylated, N3-, O2- and O4-butylated thymidines from the unmodified nucleotide within a few minutes recommends CE-LIF as a powerful method for DNA adduct analysis.     

Investigation of a liposomal oxaliplatin drug formulation by capillary electrophoresis hyphenated to inductively coupled plasma mass spectrometry (CE-ICP-MS)

A capillary electrophoresis-inductively coupled plasma mass spectrometry (CE-ICP-MS) method was developed for separation of the free oxaliplatin drug substance from liposome-entrapped oxaliplatin. Simultaneous determination of phosphorous and platinum opened the possibility to simultaneously monitor the liposomes (phospholipids) and platinum-based drug. In order to suppress the interferences, argon gas was used as a collision gas in ICP-MS. A detection limit of 29 ng/mL of platinum and a precision of 2.9% (for 10 μg/mL of oxaliplatin standard) were obtained. Measurement of the total concentration of free and encapsulated oxaliplatin by CE-ICP-MS was compared with total determination by ICP-MS after microwave digestion and showed a good agreement. A liposomal formulation of oxaliplatin based on PEGylated liposomes was used as a model drug formulation. Studies of accelerated drug release induced by sonication and phospholipase A₂ catalyzed hydrolysis were performed. It was demonstrated that the CE-ICP-MS was an efficient in vitro characterization method in the development and quality assurance purposes of lipsome-based formulation of metallodrugs.     

Analysis of mono-phosphate nucleotides as a potential method for quantification of DNA using high performance liquid chromatography–inductively coupled plasma-mass spectrometry

The determination of total deoxyribonucleic acid (DNA) concentration is of great importance in many biological and bio-medical analyses. The quantification of DNA is traditionally performed by UV spectroscopy; however the results can be affected greatly by the sample matrix. The proposed method quantifies phosphorus in digested calf thymus DNA and human DNA by high performance liquid chromatography (HPLC) combined with inductively coupled plasma mass spectrometry (ICP-MS). The method presented showed excellent baseline separation between all four DNA mono-nucleotides and 5′UMP. The ability of LC-ICP-MS to provide an internal check that only DNA derived phosphorus was counted in the assay was demonstrated by establishing a mass balance between the total phosphorous signal from undigested DNA and that from the speciated DNA. Column recoveries ranging from 95% to 99% for phosphorus resulted in a mass balance of 95% ± 0.5% for standard nucleotides, determined by LC-ICP-MS, compared to total DNA determined by flow injection coupled to ICP-MS (FI-ICP-MS). The method for quantification was validated by analysis of NIST SRM 2,372; a total speciated DNA recovery of 52.1 ng/μL, compared with an expected value of 53.6 ng/μL, was determined by external calibration. From repeat measurements, a mass balance of 97% ± 0.5% for NIST DNA was achieved. The method limits of detection for individual nucleotides were determined between 0.8 and 1.7 μg L⁻¹ (³¹P) for individual nucleotides by LC-ICP-MS, and 360 ng L⁻¹ for 5′AMP by direct nebulisation.     

微波消解-氢化物发生-原子荧光光谱法测定中药材中痕量砷

采用微波消解法用硝酸-过氧化氢(3+1)溶液消解样品,以硫脲及抗坏血酸混合溶液作为预还原剂,利用氢化物发生-原子荧光光谱法测定中药材当归、丹参、甘草、三七和黄芪中痕量砷的含量。在盐酸(3+97)溶液中加入溶于20g.L-1氢氧化钾溶液中的10g.L-1硼氢化钾溶液使与溶液中砷离子反应生成氢化物。分析中采用载气流量为800mL.min-1。砷的质量浓度在12μg.L-1以内与其对应的荧光强度呈线性关系,方法的检出限(3s/k)为0.020μg.L-1。应用此法对国家标准物质人发(GBW 09101)进行分析,测定值与认定值一致。     

Method Development and Quantitative Elemental Analysis of Mentha Longifolia L. Leaves from Saudi Arabia by Total Reflection X-Ray Fluorescence

A method has been developed for the quantitative elemental analysis of Mentha longifolia L. leaves collected from different cities in Saudi Arabia using total reflection X-ray fluorescence. Using a microwave digestion system, 100?mg of each sample was completely digested using 3?mL of nitric acid and 2?mL of hydrogen peroxide. The stabilization of the digested samples on the silicon reflectors was studied using a silicone solution and polyvinyl alcohol. 5?µL of either silicone solution or polyvinyl alcohol (1% m/v) was pipetted and dried on the silicon reflector prior to the deposition of the digested samples. It was recognized that there is some enhancement on the intensity of the peak area with the silicone solution at photon energies less than 11?keV. However, the obtained results confirm the ability of using silicone solution or polyvinyl alcohol (1% m/v) as the stabilizer prior to the deposition of the sample droplet on the quartz reflector. However, the silicone solution was more applicable. Based on the developed method, 15 elements were quantified, namely, P, S, Cl, K, Ca, Ti, Cr, Mn, Fe, Cu, Zn, Br, Rb, Sr, and Ba. Based on the present quantitative analysis results, M. longifolia samples collected from Al-Madina city had the highest concentration of P, Cl, K, Ti, Fe, Ni, and Cu. The lowest concentrations of P, S, Cl, K, Mn, Ni, and Br were found in Taif-Shafa.     

Rapid and accurate determination of deoxyribonucleoside monophosphates from DNA using micellar electrokinetic chromatography with a cationic surfactant additive

A micellar electrokinetic chromatography (MEKC) method for rapid and accurate determination of 2′-deoxyribonucleoside 5′-monophosphates (dNMPs), four structural elements of DNA, is described. MEKC separation at an optimized pH enabled complete separation of four dNMPs. The use of a cationic surfactant additive for MEKC led to the reversal of EOF, which enhanced the migration velocities of the negatively charged dNMPs. Under the optimized condition, full-baseline separation of the four dNMPs assuring accurate peak integration was obtained within 5 min. For the given separation condition, pH-mediated on-column sample stacking was optimized and applied to enhance sensitivity up to 6-fold. Analytical precision was improved by spiking iothalamate as an internal standard. The accuracy of dNMP quantitation was ensured with dNMP standard solutions determined by inductively coupled plasma-optical emission spectroscopy that measured phosphorous quantity. Performance of the proposed method was ultimately proven by accurate quantitation of a DNA oligonucleotide that was enzymatically hydrolyzed prior to dNMP analysis. The proposed MEKC method turned out to be a reliable analytical method for dNMPs that features high speed, high sensitivity, and high precision, and could be utilized for high-accuracy determination of the amount of DNA as well as the base composition of DNA.     

Determination of ultratrace bismuth in milk samples by atomic fluorescence spectrometry

A sensitive procedure was developed for determination of bismuth (Bi) in milk samples by hydride generation atomic fluorescence spectrometry (HG-AFS) after microwave-assisted sample digestion with HNO3 and H2O2. The method provides a sensitivity of 1832 fluorescence units (ng/mL) with a detection limit of 0.01 ng/mL, which corresponds to 20 pg absolute limit of detection, equivalent to 0.50 ng/g in the original sample. Application of the methodology to cow milk samples from the Spanish market showed the presence of Bi at a concentration of 11.8-28.8 ng/g, which compared well with data obtained after dry ashing of samples and with data obtained by inductively coupled plasma-mass spectrometry after microwave-assisted digestion.     

Preconcentration and separation of double-stranded DNA fragments by electrophoresis in plastic microfluidic devices

We have evaluated double-stranded DNA separations in microfluidic devices which were designed to couple a sample preconcentration step based on isotachophoresis (ITP) with a zone electrophoretic (ZE) separation step as a method to increase the concentration limit of detection in microfluidic devices. Developed at ACLARA BioSciences, these LabCard trade mark devices are plastic 32 channel chips, designed with a long sample injection channel segment to increase the sample loading. These chips were designed to allow stacking of the sample into a narrow band using discontinuous ITP buffers, and subsequent separation in the ZE mode in sieving polymer solutions. Compared to chip ZE, the sensitivity was increased by 40-fold and we showed baseline resolution of all fragments in the PhiX174/HaeIII DNA digest. The total analysis time was 3 min/sample, or less than 100 min per LabCard device. The resolution for multiplexed PCR samples was the same as obtained in chip ZE. The limit of detection was 9 fg/microL of DNA in 0.1xpolymerase chain reaction (PCR) buffers using confocal fluorescence detection following 488 nm laser excitation with thiazole orange as the fluorescent intercalating dye.     

A new quantitative method for circulating DNA level in human serum by capillary zone electrophoresis with laser-induced fluorescence detection

We present a method for the quantification of circulating DNA in serum by capillary zone electrophoresis (CZE) with laser-induced fluorescence detection (LIF). The serum was digested by proteinase to release free DNA. SYBR Gold was utilized as DNA intercalating dye, fluorescein as internal standard (ISTD). CZE-LIF was applied for the separation and quantification of total circulating DNA. Good linearity (R = 0.9992) in the low range of DNA concentrations (0.5-40 ng/mL) and a detection limit of 0.5 ng/mL for DNA (S/N = 3) were obtained. Our data demonstrated that CZE-LIF system has a good linearity with excellent sensitivity and satisfactory reproducibility in the quantification of circulating DNA in serum. This method was successfully used for the quantification of circulating DNA levels in serum. We observed that the circulating DNA levels in certain cancer patients were significantly higher than that in healthy individuals. Compared to current methods, our protocol does not need the extraction of DNA from serum. Our preliminary results have illustrated that CZE-LIF system is simple, rapid, and sensitive, and it is well suitable for large-scale quantification of circulating DNA levels in clinical diagnosis.     

Quantitative determination of DNA adducts using liquid chromatography/electrospray ionization mass spectrometry and liquid chromatography/high-resolution inductively coupled plasma mass spectrometry

The quantitative determination of nucleotides from DNA modified by styrene oxide is described using a combination of inductively coupled plasma high-resolution mass spectrometry (ICP-HRMS) and electrospray ionization mass spectrometry (ESI-MS), both interfaced to reversed-phase high-performance liquid chromatography (HPLC). LC/ICP-MS (resolution > 1500 to discriminate against 15N16O+ and 14N16OH+) was employed to determine quantitatively the content of modified nucleotides in standard solutions based on the signal of phosphorus; phosphoric acid served as an internal standard. By means of the standard addition technique the sensitivity of the LC/ESI-MS approach was subsequently determined. Since a comparison of UV, ICP and ESI-MS data suggested that in ESI-MS the ionization efficiency of the adducts is identical within the error limits, quantitative determination of all adducts is possible. For LC/ESI-MS with single ion monitoring, the detection limit for styrene oxide adducts of nucleotides was determined to be 20 pg absolute or 14 modified in 10(8) unmodified nucleotides in a 5 micrograms DNA sample, which comes close to the best methods available for the detection of chemical modifications in DNA.     

Quantitave determination of losartan and its metabolite E-3174 in rat urine

A technique for the quantitative determination of losartan and its metabolite E-3174 in biological liquids (rat urine) has been developed. The analysis was carried out using high-performance liquid chromatography coupled with fluorescence detection. The technique??s detection limit for losartan and E-3174 was equal to 2.5 ng/mL and 5 ng/mL, respectively.     

离子色谱法测定海产品中磷酸盐、焦磷酸盐、偏磷酸盐和总磷

建立了离子色谱法测定海产品中磷酸盐、焦磷酸盐、偏磷酸盐和总磷的分析方法。样品经100 mmol/L NaOH溶液浸提,固相萃取柱去除有机物、阳离子、中和OH~后用于海产品中磷酸盐、焦磷酸盐和偏磷酸盐的测定;样品经干灰化法消化,固相萃取柱净化后用于总磷测定。考察了提取溶液的pH、有机物和共存离子对测定结果的影响。该方法的线性范围为0.3～60 mg/L,检出限为2.1～2.3 mg/kg,相对标准偏差为1.6%～2.6%。海鱼和虾仁样品中目标物的加标回收率为81.8%～100.0%。该方法选择性好,灵敏度高,用于实际样品测定结果令人满意。     

Towards minimization of chlorinated solvents consume in Fourier transform infrared spectroscopy determination of Propamocarb in pesticide formulations

A method has been developed for Fourier transform infrared (FTIR) spectroscopy determination of Propamocarb in emulsifiable pesticide concentrate formulations. Five microliter sample was directly injected without any pretreatment in a CHCl3 stream at 2 mL min(-1) into a closed system and the FTIR spectra of sample and standard solutions were obtained using a nominal resolution of 4 cm(-1) from 4000 to 900 cm(-1) spectral region and accumulating 2 scans per spectrum. Propamocarb determination was based on the measurement of flow injection analysis (FIA) recording height established from FTIR peak area measurements from 1713 to 1703 cm(-1) corrected using a baseline defined at 2000 cm(-1). The concentration of Propamocarb in samples was calculated by interpolation in an external calibration line obtained from several injections of 2 microL of a 47% (w/v) standard solution into the CHCl3 closed system. This procedure provided a limit of detection of 0.8% (w/v) in the original sample, a sensitivity of 0.3190 absorbance units mL mg(-1) for a path length of 0.11 mm and a relative standard deviation of 0.2% for five independent measurements at 0.74 mg mL(-1) concentration level. The maximum sampling frequency of the whole procedure was 34 h(-1) and the waste generation was reduced to only 2 mL of CHCl3 solution per sample and additional 2 mL for the whole calibration line.     

A laser-induced fluorescence dual-fiber optic array detector applied to the rapid HPLC separation of polycyclic aromatic hydrocarbons

A multi-channel detection system utilizing fiber optics has been developed for the laser-induced fluorescence (LIF) analysis of chromatographic eluents. It has been applied to the detection of polycyclic aromatic hydrocarbons (PAH) in a chromatographically overlapped standard mixture and to a complex soil sample extract obtained during fieldwork. The instrument utilizes dual-fiber optic arrays, one to deliver multiple excitation wavelengths (258-342 nm) generated by a Raman shifter, and the other to collect fluorescence generated by the sample at each excitation wavelength; the collected fluorescence is dispersed and detected with a spectrograph/CCD combination. The resulting data were arranged into excitation emission matrices (EEM) for visualization and data analysis. Rapid characterization of PAH mixtures was achieved under isocratic chromatographic conditions (1.5 mL min(-1) and 80% acetonitrile in water), with mid microg L(-1) detection limits, in less than 4 minutes. The ability of the instrument to identify co-eluting compounds was demonstrated by identifying and quantifying analytes in the rapid analysis of a 17 component laboratory-prepared PAH mixture and a soil extracted sample. Identification and quantification were accomplished using rank annihilation factor analysis (RAFA) using pure component standards and the EEMs of mixtures measured during the rapid high-performance liquid chromatography (HPLC) method as the unknowns. The percentage errors of the retention times (RTs) determined using RAFA compared to the known RTs measured with a standard absorbance detector were between 0 and 11%. For the standard PAH mixture, all 17 components were identified correctly and for the soil extracted sample, all 8 analytes present were correctly identified with only one false positive. Overall, the system achieved excellent qualitative performance with semi-quantitative results in the concentration predictions of both the standard mixture and the real-world sample. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00216-001-1125-6.     

Comparison between transient isotachophoretic capillary zone electrophoresis and reversed-phase liquid chromatography for the determination of peptides in plasma.

Low levels of peptide drugs in human plasma can be determined employing off-line solid-phase extraction, followed by capillary zone electrophoresis with UV detection. A bioanalytical procedure is presented, using gonadorelin and angiotensin II in human plasma as model compounds. The solid-phase extraction method, based on a weak cation exchange mechanism, is able to remove interfering endogenous components from the plasma sample, extract the model peptides quantitatively, and give a possibility of concentrating the sample at the same time. Transient isotachophoretic conditions were kept to increase the sample loadability by about two orders of magnitude. Up to about 70% of the capillary was filled with the reconstituted extract, whereafter the peptides were selectively concentrated during the first 15 min. Subsequently, the concentrated sample zones were separated under capillary zone electrophoresis conditions, showing the technique's high resolution. For the model cationic peptides (gonadorelin, angiotensin II) good linearity and reproducibility was observed in the 20-100 ng/mL concentration range. A more extensive washing procedure permits quantitation of gonadorelin at the 5 ng/mL level. In comparison with a liquid chromatography analysis, superior mass sensitivity and separation are obtained with the transient isotachophoretic capillary zone electrophoresis method. Moreover, in this case equivalent sensitivity is achieved when it is directly compared with a liquid chromatography method with UV detection, keeping in mind that 60 times more sample is needed for the latter method. A further gain in sensitivity can be obtained when the analysis is combined with native fluorescence detection, as is demonstrated by combining liquid chromatography separation with fluorescence detection.     

甲基异丁基甲酮萃取-火焰原子吸收光谱法测定氧化锑中的磷

研究了MIBK萃取-火焰原子吸收光谱法测定氧化锑中的磷。在盐酸介质中,磷酸根与钼酸铵形成磷钼物质的量之比为1∶12磷钼杂多酸,用酒石酸钾钠掩蔽锑,以甲基异丁基甲酮为萃取试剂,用火焰原子吸收光谱法测定有机相中磷钼杂多酸中的钼,据此建立了一种间接测定氧化锑中微量磷的新方法。在优化的实验条件下,当磷的含量在0.055~1.00 mg/L范围内时,其吸光度与浓度呈良好的线性关系;方法的检出限为0.011 mg/L;对4个样品进行测定的回收率在97.8%~106.2%之间;相对标准偏差为2.6%~4.2%。用于氧化锑中微量磷的测定,结果满意。     

Paper test strip for silver ions detection in drinking water samples based on combined fluorometric and colorimetric methods

In this study, a portable silver ion (Ag⁺) sensor was fabricated based on a dual signal output system using black phosphorus quantum dots (BPQDs) as probes. It is the first work for Ag⁺ detection using paper test strip based on BPQDs. The color change of BPQDs paper sensor for the determination of Ag⁺ was easily identified by naked eye. BPQDs were synthesized from bulk black phosphorus (BP) by mechanical exfoliation combined with a solvothermal method. BPQDs exhibited blue fluorescence with a quantum yield of 8.82 %. The fluorescence of BPQDs can be quenched by Ag⁺, and the absorbance of BPQDs is increased with increasing Ag⁺ concentration. The mechanism of the interaction between BPQDs and Ag⁺ involving fluorescence quenching and bonding was investigated by experimental and computational methods. The detection limit of Ag⁺ was 1.56 μg/mL and 0.19 μg/mL using fluorometry and colorimetry methods, respectively. A portable visual sensor based on paper test strip was constructed for Ag⁺ detection using the colorimetric approach. The strategy was employed to determine Ag⁺ successfully in drinking water samples. Therefore, the proposed portable Ag⁺ sensor can be potentially utilized for the lab-free analysis of drinking water and even dietary samples.