Identification of phenolic compounds from pollen extracts using capillary electrophoresis–electrospray time-of-flight mass spectrometry

In this work, a new, easy and rapid method of analyzing phenolic compounds in pollen extract, based on capillary electrophoresis coupled with electrospray ionization time-of-flight-mass spectrometry (CE–ESI–TOF–MS), has been developed. A systematic investigation of separation parameters has been performed with respect to resolution, sensitivity, analysis time and peak shape. The electrophoretic parameters and electrospray conditions must be optimized to obtain reproducible analyses. Using this method, several important phenolic compounds such as acetin-glucoside, 7-O-methylherbacetin-3-sophoroside, galloyl-glucose, quercetin-3-sophoroside, apigenin-6,8-di-C-glycoside, quercetin-3-rutinoside, genistein-7-O-β-D-glucoside, luteolin-7-O-glucoside, apigenin-7-O-glucoside and 2′,4′,6′-trihydroxy-3′-formyldihydrochalcone have been determined directly from pollen extract. The efficiency, the rapidity, the small amounts of sample required, and the high resolution of CE coupled with the sensitivity, the selectivity, the accurate masses and the true isotopic patterns obtained using TOF-MS point to the potential of this approach for identifying the phenolic compounds present in pollen.     

Use of high‐conductivity sample solution with sweeping‐micellar electrokinetic capillary chromatography for trace‐level quantification of paliperidone in human plasma

Paliperidone is a new antipsychotic drug with a relatively low therapeutic concentration of 20–60 ng/mL. We established an accurate and sensitive CE method for the determination of paliperidone concentrations in human plasma in this study. To minimize matrix effect caused by quantification errors, paliperidone was extracted from human plasma using Oasis HLB SPE cartridges with three‐step washing procedure. To achieve sensitive quantification of paliperidone in human plasma, a high‐conductivity sample solution with sweeping‐MEKC method was applied for analysis. The separation is performed in a BGE composed of 75 mM phosphoric acid, 100 mM SDS, 12% acetonitrile, and 15% tetrahydrofuran. Sample solution consisted of 10% methanol in 250 mM phosphoric acid and the conductivity ratio between sample matrix and BGE was 2.0 (γ, sample/BGE). The results showed it able to detect paliperidone in plasma samples at concentration as low as 10 ng/mL (S/N = 3) with a linear range between 20 and 200 ng/mL. Compared to the conventional MEKC method, the sensitivity enhancement factor of the developed sweeping‐MEKC method was 100. Intra‐ and interday precision of peak area ratios were less than 6.03%; the method accuracy was between 93.4 and 97.9%. This method was successfully applied to the analysis of plasma samples of patients undergoing paliperidone treatment.     

Determination of flavonoids in the flowers of <Emphasis Type="Italic">Paulownia tomentosa</Emphasis> by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) technique coupled with photodiode array (PDA) detection was developed for the simultaneous determination of four flavonoids, i.e. apigenin (AP), diplacone (DI), mimulone (MI) and 5,4′-dihydroxy-7,3′-dimethoxyflavanone (DDF) in extracts of the flowers of Paulownia tomentosa. The optimized method was proposed for the separation and detection of the selected constituents, using methanol-1% acetic acid as the mobile phase at a flow rate of 0.8 mL/min, 290 and 267 nm as the detection wavelengths. All the flavonoids showed good linearity in a relatively wide concentration range (r > 0.9999). The detection limits for the analytes ranged from 0.2 to 4.0 ng, at a signal-to-noise ratio of 3. Inter- and intra-assay accuracy and precision were all lower than 5.0%. The recovery of the method was 95.9–101.9%. Moreover, the optimized HPLC method was employed to analyze the flowers of Paulownia tomentosa.     

Development of a micellar electrokinetic capillary chromatography method for the determination of four naphthalenediols in cosmetics and a comparison with a HPLC method

A micellar electrokinetic capillary chromatography (MEKC) method with ultraviolet visible (UV) detection was used for the determination of 1,7-naphthalenediol, 2,3-naphthalenediol, 1,5-naphthalenediol, and 2,7-naphthalenediol in cosmetics. The current method for their determination in various cosmetics is high-performance liquid chromatography (HPLC). Separation conditions affecting the MEKC method were optimized as 20 mM Na₂B₄O₇–50mM SDS, pH 9.8, with 22 kV applied voltage and UV detection at 230 nm. Under optimal conditions, electrophoretic analysis was completed in less than 6 min, with limit of detection (LOD) of 0.070–0.19 μg/mL and limit of quantitation (LOQ) of 0.23–0.63 μg/mL. A good linear relationship (r² > 0.99) was obtained at the range of 0.75–20 μg/mL. Recoveries for the four naphthalenediols in lotion, loose powder, and sun cream are between 91.2–107.2% with relative standard deviation (RSD) less than 4.04%. The method has been successfully applied to the determination of the four naphthalenediols in different kinds of cosmetics. A comparison with HPLC-UV method was also carried out according to the National Standards of the People's Republic of China. The results obtained by MEKC and HPLC methods are comparable, but the proposed MEKC method can help us obtain a much shorter detection time and low cost.     

Sensitive and fast determination of Sudan dyes in chili powder by partial-filling micellar electrokinetic chromatography-tandem mass spectrometry

A fast and sensitive method for the simultaneous determination of Sudan dyes (I, II, III, and IV) in food samples was developed for the first time using partial filling micellar electrokinectic chromatography-mass spectrometry (MEKC-MS). The use of MEKC was essential to achieve the separation of these neutral analytes, while the partial filling technique was necessary to avoid the contamination of the ion source with non-volatile micelles. MEKC separation and MS detection conditions were optimized in order to achieve a fast, efficient, and sensitive separation of the four dyes. Filling 25% of the capillary with an MEKC solution containing 40 mM ammonium bicarbonate, 25 mM SDS, and 32.5% (v/v) acetonitrile, a baseline separation of the four azo-dyes was obtained in 10 min. Tandem MS was investigated in order to improve the sensitivity and selectivity of the analysis. Limits of detection (LOD) values 5, 8, 15, and 29 times better were obtained for Sudan III, I, II, and IV, respectively, using partial filling MEKC-MS/MS instead of partial filling MEKC-MS. Under optimized conditions, LOD from 0.05 to 0.2 μg/mL were obtained. The suitability of the developed method was demonstrated through the fast and sensitive determination of Sudan I, II, III, and IV in spiked chilli powder samples. This determination could not be achieved by MEKC-UV due to the existence of several interfering compounds from the matrix.     

Development and application of UHPLC-MS/MS method for the determination of phenolic compounds in Chamomile flowers and Chamomile tea extracts

UHPLC-MS/MS method using BEH C18 analytical column was developed for the separation and quantitation of 12 phenolic compounds of Chamomile (Matricaria recutita L.). The separation was accomplished using gradient elution with mobile phase consisting of methanol and formic acid 0.1%. ESI in both positive and negative ion mode was optimized with the aim to reach high sensitivity and selectivity for quantitation using SRM experiment. ESI in negative ion mode was found to be more convenient for quantitative analysis of all phenolics except of chlorogenic acid and kaempherol, which demonstrated better results of linearity, accuracy and precision in ESI positive ion mode. The results of method validation confirmed, that developed UHPLC-MS/MS method was convenient and reliable for the determination of phenolic compounds in Chamomile extracts with linearity >0.9982, accuracy within 76.7-126.7% and precision within 2.2-12.7% at three spiked concentration levels. Method sensitivity expressed as LOQ was typically 5-20 nmol/l.Extracts of Chamomile flowers and Chamomile tea were subjected to UHPLC-MS/MS analysis. The most abundant phenolic compounds in both Chamomile flowers and Chamomile tea extracts were chlorogenic acid, umbelliferone, apigenin and apigenin-7-glucoside. In Chamomile tea extracts there was greater abundance of flavonoid glycosides such as rutin or quercitrin, while the aglycone apigenin and its glycoside were present in lower amount.     

Development and validation of a high performance liquid chromatographic method for the simultaneous determination of potassium clavulanate and cefadroxil in synthetically prepared tablets

A simple, sensitive and rapid high performance liquid chromatographic method was developed and validated for the simultaneous determination of potassium clavulanate and cefadroxil in synthetically prepared tablets. Chromatographic separation and detection was carried out on a C-18 column using 0.05 M potassium dihydrogen phosphate buffer (pH 5.0) and acetonitrile in the ratio of 94: 06 (v/v) as mobile phase at wavelength of 225 nm. The method was linear in the concentration range of 3.75–22.5 μg/mL for potassium clavulanate and 15–90 μg/mL for cefadroxil. The flow rate was 1.0 mL/min and the total analysis time was less than 10 min. The mean recoveries was found to be greater than 99% with RSD less than 1.0%. The proposed method was validated by performing linearity, recovery, specificity, robustness, LOD/LOQ and within-day and between-day precision. The chromatographic results obtained from the synthetically prepared tablets show that the method is highly precise and accurate for the simultaneous quantitation of clavulanate potassium and cefadroxil.     

Quantitative reversed-phase high-performance liquid chromatography of major and modified nucleosides in DNA

Improved, highly accurate high-performance liquid chromatographic methods for the measurement of the major and modified nucleosides in enzymatic digests of DNA using a single column are described. Four high resolution separation protocols (isocratic, binary, ternary and high speed) with specifically improved selectivity for 5-methyldeoxycytidine (m5dCyd) from Ade, dIno and Guo are presented. From a detailed study of the various factors contributing to the precision and accuracy of the measurement, optimized conditions and quantitative protocols were established. The ternary buffer allows for the first time the determination of N6-methyldeoxyadenosine (m6dAdo) in the same chromatographic analysis with the other deoxyribonucleosides. The binary system allows quantitation of the absolute amounts of each ribo- and deoxyribonucleoside as well as the mole % of each as the second buffer elutes 5'dA and the internal standard 8-bromoguanosine. The isocratic system allows precise quantitation of the mole % of each ribo- and deoxyribonucleoside while eliminating the need for buffer change valves, buffer cycling and column re-equilibration. Also, a high-speed isocratic system is described which permits separation of the deoxyribonucleosides in 6 min. The quantitative, enzymatic hydrolysis of DNA was evaluated by comparing a 40-h, three-enzyme system with a 4-h, two-enzyme procedure. The latter protocol proved to be an excellent hydrolysis method. These high resolution liquid chromatography techniques provide the most precise, sensitive and accurate measurement of m5dCyd available, in a straightforward method using as little as 1 microgram of DNA, and have allowed us to demonstrate: the existence of tissue-specific differences in levels of m5dCyd in DNA of humans, monkeys, rats and mice; that m5dCyd levels in DNA change during fetal development; that genomic undermethylation of DNA is correlated with cancer and the presence of m6dAdo in DNA of thermophilic organisms.     

Quantitative TLC Analysis of Sterol (24β-Ethylcholesta-5,22E,25-triene-3β-ol) in Agnimantha (Clerodendrum phlomidis Linn)

A quantitative method using silica gel 60F₂₅₄ high performance thin layer chromatography plates, automated bandwise sample application, and automated visible mode densitometric method has been developed for the determination of 24β-ethylcholesta-5,22E,25-triene-3β-ol (ECTO) in the aerial part of Clerodendrum phlomidis. ECTO was used as a chemical marker for the standardization of C. phlomidis plant extracts. The separation was performed on silica gel 60F₂₅₄ TLC plates using chloroform-methanol (98.5: 1.5, v/v) as mobile phase. The quantitation of ECTO was carried out using the densitometric reflection/absorption mode at 650 nm after post chromatographic derivatization with anisaldehyde reagent. A precise and accurate quantification can be performed in the linear working concentration range of 150–400 ng band⁻¹ with good correlation (r ² = 0.996). The method was validated for peak purities, precision, robustness, limit of detection (LOD) and quantitation (LOQ), etc. as per ICH guidelines.     

Optimization and validation of a nonaqueous micellar electrokinetic chromatography method for determination of polycyclic musks in perfumes

A nonaqueous micellar electrokinetic chromatography method was developed for determination of Tonalide®, Galaxolide®, and Traseolide® polycyclic musks (PCMs). These compounds are widely used as fragrance ingredients in cosmetics. The method was optimized by using a three variable Box‐Behnken experimental design and response surface methodology. A modified chromatographic response function was defined in order to adequately weigh the terms in the response function. After optimization of experimental conditions, an electrolyte solution of 195 mM SDS and 40 mM NaH₂PO₄ in formamide was selected for the separation of the three PCMs, and the applied voltage was fixed at 30 kV. The nonaqueous MEKC method was then checked in terms of linearity, limits of detection and quantification, repeatability, intermediate precision and accuracy, providing appropriate values (i.e. RSD values for precision never exceeding 7%, and accuracy 96–107%). Nonaqueous MEKC for determination of the selected compounds was successfully applied to the analysis of commercial perfume samples.     

Simultaneous determination of acetaminophen, dextromethorphen hydrobromide and pseudoephedrine hydrochloride in a new drug formulation for cold treatment by HPLC

Summary A rapid and accurate HPLC method is described for the simultaneous determination of acetaminophen, dextromethorphen hydrobromide and pseudoephedrine hydrochloride in a new cold formulation. Chromatographic separation of the three pharmaceuticals was performed on a Hypersil CN column (150×5.0 mm, 5 μm) with a mobile phase mixture of an ion-pairing solution, methanol and acetonitrile (25:57:18, v/v), at a flow rate of 1.0 mL min⁻¹, with detection at 220 nm. Separation was complete in less than 10 min. The method was validated for linearity, accuracy, precision, limit of quantitation and robustness. Linearity, accuracy, and precision were found to be acceptable over the ranges of 2.06∼20.6 μg·mL⁻¹ for acetaminophen, 0.202∼2.02 mg·mL⁻¹ for pseudoephedrine hydrochloride and 0.042∼1.06 mg·mL⁻¹ for dextromethorphen hydrobromide.     

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.     

A comparative study of micellar and microemulsion EKC for the analysis of benzoylurea insecticides and their analogs

An investigation of the basic factors which govern the microemulsion EKC (MEEKC) and MEKC for the separation of four benzoylurea (BU) insecticides and their four analogs was carried out. In MEEKC, the separation of eight BU compounds was optimized by changing the microemulsion composition, such as concentration of SDS, octane, n-butanol, and isopropanol percentages, as well as capillary temperature. Separation optimization was also carried out for MEKC, showing that ACN and a high level of another additive gamma-CD were needed to achieve effective separation of these analytes. Although separation with baseline resolution was achieved by either MEEKC or MEKC methods, the separation selectivity resulting from the proposed MEEKC method was completely different from that of MEKC. In addition, analytical time in MEEKC was longer than that in MEKC, but in view of theoretical plate numbers, detection limits, and reproducibility, both methods were effective for the analysis of BU insecticides and their analogs.     

Analysis of genomic methylation level using micellar electrokinetic chromatography with UV detection

Analytical methods for quantification of 5′‐methylcytosine in genomes are important tools to investigate epigenetic changes in gene expression during development, differentiation, aging, or cancer. Here, we report a novel genomic methylation content assay based on enzymatic hydrolysis of DNA and MEKC separation of 5′‐deoxyribonucleoside monophosphates (dNMP) using the cationic surfactant CTAB as pseudostationary phase. Calf Thymus DNA was used during method development to determine electrophoretic parameters and electrolyte composition for a complete separation between 2′‐deoxycytosine‐5′‐monophosphate and 2′‐deoxy‐5′‐methylcytosine 5′‐monophosphate (d5mCMP). Methylated and not methylated oligonucleotides were used to confirm the identity of each peak and evaluate analytical parameters of the method. The LOD of the method was found to be 12.5 pmol/μL for d5mCMP.     

Simple HPLC method for simultaneous determination of acetaminophen, caffeine and chlorpheniramine maleate in tablet formulations

Summary A simple, rapid and accurate, routine-HPLC method is described for simultaneous determination of acetaminophen, caffeine and chlorpheniramine maleate in a new tablet formulation Chromatographic separation of the three pharmaceuticals was achieved on a Hypersil CN column (150×5.0 mm, 5 μm) using a mobile phase comprising a mixture of acetonitrile, an ion-pair solution and tetrahydrofuran (13:14:87, v/v,pH4.5). The flow-rate was changed from 1.0 mL min⁻¹ (in 0≈7.5 min) to 1.8 mL min⁻¹ (after 3.5 min). was complete in <10 min. The method was validated for system suitability, linearity, accuracy, precision, limits of detection and quantitation, and robustness. Linearity, accuracy and precision were found to be acceptable over the ranges 31.6≈315.8 μg mL⁻¹ for acetaminophen, 9.5≈94.6 μg mL⁻¹ for caffeine and 1.4≈13.8 μg mL⁻¹ for chlorpheniramine maleate.     

Aptasensor for ampicillin using gold nanoparticle based dual fluorescence–colorimetric methods

A gold nanoparticle based dual fluorescence–colorimetric method was developed as an aptasensor to detect ampicillin using its single-stranded DNA (ssDNA) aptamer, which was discovered by a magnetic bead-based SELEX technique. The selected aptamers, AMP4 (5′-CACGGCATGGTGGGCGTCGTG-3′), AMP17 (5′-GCGGGCGGTTGTATAGCGG-3′), and AMP18 (5′-TTAGTTGGGGTTCAGTTGG-3′), were confirmed to have high sensitivity and specificity to ampicillin (K _d, AMP7 = 9.4 nM, AMP17 = 13.4 nM, and AMP18 = 9.8 nM, respectively). The 5′-fluorescein amidite (FAM)-modified aptamer was used as a dual probe for observing fluorescence differences and color changes simultaneously. The lower limits of detection for this dual method were a 2 ng/mL by fluorescence and a 10 ng/mL by colorimetry for ampicillin in the milk as well as in distilled water. Because these detection limits were below the maximum residue limit of ampicillin, this aptasensor was sensitive enough to detect antibiotics in food products, such as milk and animal tissues. In addition, this dual aptasensor will be a more accurate method for antibiotics in food products as it concurrently uses two detection methods: fluorescence and colorimetry.     

Micellar electrokinetic capillary chromatographic method for simultaneous determination of drugs used to treat advanced breast cancer

Summary A micellar electrokinetic capillary chromatography (MEKC) method has been developed for quantification of four drugs-tamoxifen, anastrozole, letrozole, and methotrexate—used to treat advanced breast cancer. Separation was performed at 25°C and 25kV, with 20mm borate buffer (pH 9.2) containing 40mm sodium dodecylsulfate as electrolyte solution. Under these conditions analyses were performed in 12 min. The linearity of the response was investigated for the concentration range 2.0–20.0 mg L⁻¹. The intra-day residual standard deviation (n=4 graphs) between the slopes of the calibration graphs was acceptable for the four drugs studied. Detection limits (signal-to-noise ratio=3) were below 1 mg L⁻¹ for all the compounds. The simplicity, precision, and sensitivity of MEKC proved suitable for quality control of pharmaceutical preparations used to treat advanced breast cancer. Six different pharmaceutical preparations, each containing one of the above-mentioned drugs, were successfully analyzed.     

Capillary electrophoresis for the direct determination of cephalosporins in clinical samples

Summary The possibility of determination of four cephalosporin antibiotics in clinical samples by capillary electrophoresis has been investigated. The separation conditions for capillary zone electrophoresis (CZE) were studied in detail. The precision of migration times measured by use of the optimized method was satisfactory (RSD<1%) and response was linearly dependent on concentration over the approximate range 2–150 mg L⁻¹ for all the compounds studied (cefuroxime, cefotaxime, ceftriaxone, and ceftazidime). Complete separation could be achieved within 5 min. The CZE method was found to be highly suitable for direct determination of the antibiotics in clinical samples such as wound drainage, cerebrospinal fluid, and urine; for serum, however, the use of micellar electrokinetic capillary chromatography (MECC) was more advantageous. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Simultaneous determination of Δ9-tetrahydrocannabinol and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol in oral fluid using isotope dilution liquid chromatography tandem mass spectrometry

The detection and confirmation of cannabinoids in oral fluid are important in forensic toxicology. Currently, the presence of Δ⁹-tetrahydrocannabinol (THC) is used for the detection of cannabis in oral fluid. A low concentration of 11-nor-9-carboxy-Δ⁹-tetrahydrocannabinol (THC-COOH) is found in oral fluid, which suggested a convenient and low-sensitivity confirmation assay can be used in a routine forensic laboratory. In this study, a highly sensitive isotope dilution liquid chromatography–tandem mass spectrometry method following dansylation was successfully developed for simultaneous determination of THC and THC-COOH in oral fluid. The dansylated derivatives dramatically demonstrated and enhanced the sensitivity of THC and THC-COOH. To avoid signal influenced by the matrix, a 5-min liquid chromatography gradient program was evaluated and optimized, which reduced the sample diffusion and caused sharp peaks (less than 12 s) and thus helped to achieve detection at a low level. The sensitivity, accuracy, and precision were also evaluated, and high quantitative accuracy and precision were obtained. The limit of quantitation of this approach was 25 pg/mL for THC and 10 pg/mL for THC-COOH in oral fluid. Finally, the method was successfully applied to eight suspected cannabis users. Among them, in six oral fluid samples THC-COOH was determined at a concentration from 13.1 to 47.2 pg/mL.     

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.