Metabolomic Studies Using High Performance Liquid Chromatography and Tandem Mass Spectrometry to Discover Quality Markers for Oriental Beauty (Dongfang Meiren) Tea

In this study, a high-performance liquid chromatograph and an electrospray ionization (ESI) triple quadrupole mass spectrometry (TQ MS) detector were used to scan Oriental Beauty tea of different grades and prices. Principle component analysis (PCA) of the profiling data was performed for pattern recognition, clearly showing that the proposed MS profiling method was able to classify Oriental Beauty tea into different grades. The component mass ions primarily responsible for the separation were selected with high loading strength in the PCA for subsequent identification with tandem mass (MS/MS). Caffeine, citrate and salicylate were verified, whereas certain other compounds remained ambiguous. Regression analysis considering caffeine, citrate and salicylate showed a linear relationship between the prices of the Oriental Beauty tea with an adjusted R2 of 0.84. If all the selected marker ions (in addition to caffeine, citrate and salicylate) could have been identified and incorporated into regression analysis, a stronger relationship could have been confirmed. These results suggest that metabolomics can facilitate the determination of real markers in the quality control of Oriental Beauty tea, and may lead to the further application of metabolomics in other food quality controls.

Simultaneous Determination of Ultraviolet Absorbers and Antibacterial Agents in Textiles by Ultra-High Performance Liquid Chromatography/Orbitrap High Resolution Mass Spectrometry

This paper reported a new analytical method for the simultaneous determination of seven benzotriazole ultraviolet absorbers and seven antibacterial agents in textiles. After ultrasonic extraction for the textile samples in methanol, the solutions were analyzed by ultra-high performance liquid chromotagraphy/orbitrap high resolution mass spectrometry (UPLC/Orbitrap HRMS). It showed that a good chromatographic separation for these target compounds was achieved by a Hypersil GOLD column (100 mm × 2.1 mm × 1.9 μm) with a gradient elution of methanol and 0.1% aqueous formic acid solution (containing 0.5 mmol/L ammonium acetate). Triclosan and 4-chloro-3,5-dimethyl phenol (PCMX) were detected by the orbitrap HRMS in an electrospray ionization (ESI) negative mode while the other twelve target compounds were detected by orbitrap HRMS in ESI positive mode. Full scan experiment was performed over the range from m/z 100 to m/z 500. These target compounds were routinely detected with mass accuracy below 2 × 10-6 (2 ppm) at the optimized conditions. The results showed that the limits of detection (LODs) were in the range from 0.1 to 0.3 μg/kg. The blank samples were spiked at three levels and their average recoveries varied from 80.5% to 96.3% while the relative standard deviation (RSD) changed from 3.2% to 9.9%. The present method was also applied for the determination of those ultraviolet absorbers and antibacterial agents in the commercial textiles.

Cation-exchange high-performance liquid chromatography for variant hemoglobins and HbF/A2:What must hematopathologists know about methodology?

Cation-exchange high-performance liquid chromatography(CE-HPLC) is a widely used laboratory test to detect variant hemoglobins as well as quantify hemoglobins F and A2 for the diagnosis of thalassemia syndromes. It's versatility, speed, reproducibility and convenience have made CE-HPLC the method of choice to initially screen for hemoglobin disorders. Despite its popularity, several methodological aspects of the technology remain obscure to pathologists and this may have consequences in specific situations. This paper discusses the basic principles of the technique, the initial quality control steps and the interpretation of various controls and variables that are available on the instrument output. Subsequent sections are devoted to methodological considerations that arise during reporting of cases. For instance, common problems of misidentified peaks, totals crossing 100%, causes of total area being above or below acceptable limits and the importance of pre-integration region peaks are dealt with. Ultimately, CE-HPLC remains an investigation, the reporting of which combines in-depth knowledge of the biological basics with more than a working knowledge of the technological aspects of the technique.

Trace Determination of Tamoxifen in Biological Fluids Using Hollow Fiber Liquid-Phase Microextraction Followed by High-Performance Liquid Chromatography-Ultraviolet Detection

The applicability of hollow fiber liquid-phase microextraction (HF-LPME) combined with high-performance liquid chromatography-ultraviolet detection (HPLC-UV) was evaluated for the extraction and determination of tamoxifen (TAM) in biological fluids including human urine and plasma. The drug was extracted from a 15 mL aqueous sample (source phase;SP) into an organic phase impregnated in the pores of the hollow fiber (membrane phase;MP) followed by the back-extraction into a second aqueous solution (receiving phase;RP) located in the lumen of the hollow fiber. The effects of several factors such as the nature of organic solvent, compositions of SP and RP solutions, extraction time, ionic strength and stirring rate on the extraction efficiency were examined and optimized. An enrichment factor of 360 along with substantial sample clean up was obtained under the optimized conditions. The calibration curve showed linearity in the range of 1 - 500 ng?mL–1 and the limit of detection was found to be 0.5 ng?mL–1 in aqueous medium. A reasonable relative recovery (≥89%) and satisfactory intra-assay (3.7% - 4.2%, n = 3) and inter-assay (7.5% - 7.8%, n = 3) precision illustrated good performance of the analytical procedure in spiked human urine and plasma samples.

Modernization of Chinese herbal compound and the high performance liquid chromatography tandem mass spectrometry (HPLC-MS)

Chinese herbal compound is playing an important role on curing human diseases.And it has been a trend that Chinese herbal compound is being used all over the world in 21 century.However,our Chinese herbal compound is facing serious challenge for the lack of canonical system of quality criterion for Chinese herbal compound so it has been a urgent problem to set up the quality control standards and reveal therapeutic basis of Chinese herbal compound.In order to give full play to the advantages of Chinese herbal compound,modern scientific and technological is used to research of Chinese herbal compound,especially the high performance liquid chromatography tandem mass spectrometry(HPLC-MS),because it is high sensitive,rapid,and obtain more information.It is very necessary that HPLC-MS is uesed to elucidate the effective components of basic substances of Chinese Herbal Compound,and endow traditional Chinese medicine with modern scientific connotation.

Chemical Components of Achyranthes bidentata Leaves by Ultra High Performance Liquid Chromatography-Mass Spectrometry

[Objectives] To study the chemical components and relative content of Achyranthes bidentata leaves and provide a scientific basis for further development and utilization of A. bidentata leaves.[Methods] The chemical components of A. bidentata leaves were rapidly analyzed using the ultra high performance liquid chromatography-time of flight-high resolution mass spectrometry (UHPLC-TOF-MS).[Results] Thirty eight chemical compounds were identified in samples of A. bidentata leaves collected from Wen County of Henan Province, in which seven chemical compounds had the relative content higher than 5%, linoleic acid reached 25.7% and inokosterone A reached 13.8%.[Conclusions] A. bidentata leaves contain many kinds of chemical compounds. This study is expected to provide a certain basis for further extraction of linoleic acid and inokosterone A.

Stress Degradation Studies on Lisinopril Dihydrate Using Modified Reverse Phase High Performance Liquid Chromatography

A simple, precise, accurate and sensitive reverse phase high performance liquid chromatographic method for simultaneous estimation of lisinopril dihydrate and its degradation products occuring under different ICH prescribed stress conditions has been modified. Drug was resolved on a C18 column, utilizing modified mobile phase of tetra butyl ammonium hydroxide solution and acetonitrile. Ultra violet detection was carried out at 210 nm. The method was modified with respect to linearity, precision, accuracy, selectivity, specificity and ruggedness. The results obtained revealed that lisinopril dihydrate was an active product slightly changed under stress conditions.

A Sensitive Reversed-Phase High-Performance Liquid Chromatography Method for the Quantitative Determination of Milk Xanthine Oxidase Activity

A new reversed-phase high performance liquid chromatography method was developed to quantitate the activity of xanthine oxidase involved in milk fat globule membrane with xanthine as the substrate and the separation of product (uric acid). The increment of uric acid in the reaction system was used to calculate the total activity of XO. The optimized assay conditions, linearity of detection, recovery of uric acid and chromatogram were developed in text, indicating this method is simple, rapid and efficient. It is an alternative potential method for the determination of the activity of XO in milk.

Analysis of Amino Acids in Houttuynia Cordata Thunb. by High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD)

[Objectives] The biologically active compounds of Houttuynia cordata Thunb. as a medicinal and edible plant are well documented. However, amino acid as an important indicator of the value of H. cordata is little reported. [Methods] Amino acids of the H. cordata were analysed by pre-column derivatization with 2,4-dinitrochlorobenzne(CDNB) coupled to HPLC/DAD. The optimized conditions of the amino acids derivatized by CDNB were followed: 110 ℃ of hydrolysis temperature, 1∶12 of the equivalent proportion between amino acid and CDNB, 80 min of derivatization reaction time and 80 ℃ of derivatization reaction temperature. The method was used to determine the amino acids of H. cordata samples from 11 different geographical sites in Guizhou. [Results] The results suggested that the contents of 17 amino acids were quite variable at different H. cordata samples. [Conclusions] This study will provide a basis for the quality evaluation of H. cordata nutrition.

Simultaneous Determination of Amlodipine with H₁-Receptor Antagonists by Reversed Phase High Performance Liquid Chromatography and Application to Interaction Studies

A rapid, fast and precise method has been developed and validated for the simultaneous determination of amlodipine with H1-receptor antagonists (cetirizine, fexofenadine, and buclizine) from dosage forms. The chromatography was performed on a Purospher? Star, C18 (5 mm, 250 × 4.6 mm) column using acetonitrile: buffer (0.01 mM) (40:60, v/v, pH adjusted to 3.0), as a mobile phase. The mobile phase was pumped at a flow rate of 1.0 mL·min-1 and UV detection was performed at 240 nm. The method was validated for linearity, accuracy, precision and specificity. The method was applied to study the interaction between amlodipine and H1-receptor antagonists. These interactions were carried out in simulated gastric juice (pH 1), simulated full stomach (pH 4), blood pH (pH 7.4) and simulating GI (pH 9). The interacting drugs were heated at 37℃ with intermit-tent shaking and the samples were withdrawn every thirty minutes for three hours and drug contents were analyzed by RP-HPLC techniques. In most cases the in vitro availability of amlodipine was decreased. It was observed that the change in in vitro availability was pH dependent.

Isolation and purification of carbazole contained in anthracene slag by extraction combined with medium pressure liquid chromatography

Ultrasonic-assisted extraction(UAE)combined with medium pressure liquid chromatography(MPLC)was designed for carbazole separation from anthracene slag(AS).The effects of liquid/solid ratio,temperature,and extraction times on carbazole separation were investigated.When using CC14 and ethyl acetate as extraction solvents and combining with MPLC,carbazole recovery and purity are 75.1%and 95.4%,respectively.The mechanism for carbazole separation were presumed by examining intermolecular interactions such as N-H…π,π-π,and C-Cl…πinteractions.These results demonstrate that UAE/MPLC has a considerable potential as a green and promising strategy for separating and purifying carbazole and other chemicals from AS.

Quantitative analysis by reversed-phase high-performance liquid chromatography and retinal neuroprotection after topical administration of moxonidine

AIM:To determine moxonidine in aqueous humor and iris-ciliary body by reversed-phase high performance liquid chromatography(RP-HPLC),and to evaluate the retinal neuroprotective effect after topical administration with moxonidine in a high intraocular pressure(IOP)model.METHODS:The eyes of albino rabbits were administered topically and ipsilaterally with 0.2%moxonidine.A RPHPLC method was employed for the identification and quantification of moxonidine between 2 and 480 min,which presented in the aqueous humor and iris-ciliary body.Flash electroretinography(F-ERG)amplitude and superoxide dismutase(SOD)level were measured between day 1 and day 15 after topical administration with moxonidine in a rabbit model of high IOP.Histological and ultrastructural observation underwent to analyze the changes of retinal morphology,the inner retinal layers(IRL)thickness,and retinal ganglion cell(RGC)counting.RESULTS:Moxonidine was detectable between 2 and 480 min after administration,and the peak concentration developed both in the two tissues at 30 min,0.51μg/m Lin aqueous humor and 1.03μg/g in iris-ciliary body.In comparison to control,F-ERG b-wave amplitude in moxonidine eyes were significantly differences between day 3 and day 15(P<0.01)in the high IOP model;SOD levels were significantly higher at all time-points(P<0.01)with a maximum level of 20.29 U/mgprot at day 15;and RGCs were significantly higher(P<0.05).CONCLUSION:Moxonidine is a viable neuroprotective agent with application to high IOP model.All layers of retina,including RGC layer,retinal nerve fiber layer and INL,are more preserved after moxonidine administration.SOD plays a neuroprotective role in ocular hypertension-mediated RGC death.

Determination of okadaic acid related toxins from shellfish (<i>sinonovacula constricta</i>) by high performance liquid chromatography tandem mass spectrometry

Consumption of shellfish contaminated with algal toxins produced by marine dinoflagellates can lead to diarrhetic shellfish poisoning (DSP). It was therefore essential that there are analytical techniques to identify and quantify DSP toxins in shellfish. This new methodology could facilitate DSP monitoring and create a means of rapidly responding to incidents threatening public health. In the last years there were different analytical methods for DSP, such as mouse bioassay and LC-FLD. With the development of instrument, Liquid chromatography-mass spectrometry was substituted for other analytical methods with its good sensitivity and selectivity and without derivatization for the determination of DSP. In this report, a high performance liquid chromatogra-phytandem mass spectrometric(HPLC-MS/MS)method was developed for the simultaneous determination of okadaic acid (OA) and dinophysistoxins(DTX1) in Sinonovacula constricta. Optimization of pretreatment experiment was carried out to maximize recoveries and the effectiveness. The analytes were determined under multi-reactions monitoring (MRM) scan type with tandem mass analyzer using negative ion electrospray ionization (-ESI) mode .Finally, the detection and identification of OA and DTX-1 were based upon their retention times (RT) and the fragmentation patterns of their mass spectra. The method of LOQ for the two poisons was 0.02 mg·kg-1.The real sample test showed that this method could be used for sensitive, fast, and accurate determination of the two diarrheic shellfish poisons in shellfish.

Development of a Fast and Facile Analytical Approach to Quantify Radiometabolites in Human Plasma Samples Using Ultra High Performance Liquid Chromatography

Introduction: Conventional metabolite analyses often require manual sample preparation, generating variability of measurements. This study describes a new method to quantify radiometabolites in blood, combining ultra high performance liquid chromatography (UHPLC) and turbulent flow chromatography, an alternative fully automated process allowing analyte’s extraction. Methods: A new radiotracer for dopamine transporter imaging, namely LBT-999, was used to demonstrate the method’s robustness. Matrix effect, Turboflow column loading, linearity, specificity and precision were evaluated with in vitro samples of LBT-999 in human plasma. Radiodetector sensitivity and preliminary evaluation were respectively determined by analysis of calibrated samples of [18F]LBT-999 and blood samples from 4 healthy subjects injected with [18F]LBT-999, withdrawn at 5, 15, 30 and 45 min pi. Results: With three sequential loadings (3 × 100 μL) of the Turboflow column, mean coefficients of variation were 1%, below 2%, 2% and 30.9% for matrix effect, specificity, repeatability and intermediate precision, respectively. Correlation coefficients for linearity were superior to 0.97. Limits of detection and quantification of the radiodetector were fixed at 3 and 9 c/s. Retention times for [18F]LBT-999 and the two radiometabolites detected by radio-UHPLC were 6.5, 4.8 and 9.6 min. Forty-five min after the injection, parent fraction was still predominant with 57.8% ± 25% of the total radioactivity. Conclusions: An innovative approach, allying UHPLC and Turboflow column, was developed and its sensitivity, linearity, specificity and repeatability validated. Preliminary results of the clinical trial are in accordance with literature data, demonstrating its efficiency in radiometabolites quantification.

Determination of 24 Allergens in Perfume with High Performance Liquid Chromatography Tandem Mass Spectrometry

A method of 24 allergens determination in cosmetics were established with high performance liquid chromatography tandem mass spectrometry. The targeted compounds were extracted with acetonitrile and determined with LC-MS/MS (MRM mode) with external method. The linearity between concentrations and peak area ratio was obtained from 1.0~5.0 mg/L. The limits of detection were 1.0 mg/L for the instrument and 5.0 mg/kg for the method respectively. The LOQ was 15.0 mg/L. The average recoveries of 24 allergens were between 85.9% and 110.0% at spiked levels of 5, 10 and 20 mg/kg with relative standard derivation (RSDs) of 5.5%~12.0%(n=10). The method could be used as a reliable means for simultaneous quantitative determination of allergens in cosmetics.

Determination of thyreostats in bovine urine using ultra-high performance liquid chromatography-tandem mass spectrometry

Five thyreostats(TSs),namely tapazole,thiouracil,methylthiouracil,propylthiouracil,and phenylthiouracil,were determined in bovine urine using ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS)in positive electrospray ionization mode.Extraction and clean-up were achieved using a ChemElut cartridge with tert-butyl methyl ether,without a derivatization step.Separation was achieved on an Acquity UPLC SS T3 column.The mobile phase was acetonitrile and water containing 0.2%(v/v)formic acid.The mass spectrometer was operated in multiple reaction monitoring mode.Urine samples were spiked with TS solution at levels corresponding to 5,10,15,and 20μg/L.The accuracy(internal standard corrected)ranged from 92%to 107%,with a repeatability precision(relative standard deviation,RSD)less than 15%for all five analytes.The RSDs within-laboratory reproducibility was less than 26%.The decision limits(CCα)and detection capabilities(CCβ)were obtained from a calibration curve and were in the ranges of 3.1-6.1μg/L and 4.0-7.4μg/L,respectively.The CCαand CCβvalues were below the recommended concentration,which was set at 10μg/L.The results show that the described method is suitable for the direct detection of TSs in bovine urine.This method can also be used to determine TSs in porcine urine.

Establishing a protein expression profile database for the normal human pituitary gland using two-dimensional high-performance liquid chromatography combined with LTQ-Orbitrap mass spectrometry

In this study, we selected adult normal pituitary gland tissues from six patients during operations for pituitary microadenomas via the transsphenoidal approach for extended normal pituitary tissue resection around the tumor, and analyzed the protein expression of human normal pituitary using two-dimensional high-performance liquid chromatography combined with LTQ-Orbitrap mass spectrometry proteomics technology. The ten most highly expressed proteins in normal human pituitary were： alpha 3 type VI collagen isoform 5 precursor （abundance among tall pituitary proteins 1.30%）, fibrinogen beta chain preproprotein （0.99%）, vimentin （0.73%）, prolactin （0.69%）, ATP synthase, H~ transporting and mitochondrial F1 complex beta subunit precursor （0.52%）, keratin I （0.49%）, growth hormone （0.45%）, carbonic anhydrase I （0.40%）, heat shock protein 90 kDa I （0.31%）, and annexin V （0.30%）. Based on the biological function classifications of these proteins, the top three categories by content were neuroendocrine proteins （abundance among all pituitary proteins, 40.1%）, catalytic and metabolic proteins （28.3%）, and cell signal transduction proteins （9.8%）. Based on cell positioning classification, the top three categories were cell organelle （24.5%） membrane （20.8%）, and cytoplasm （13.0%）. Based on biological process classification, the top three categories of proteins are involved in physiological processes （42.9%）, cellular processes （40.4%）, and regulation of biological processes （9.1%）. Our experimental findings indicate that a protein expression profile database of normal human pituitary can be precisely and efficiently established by proteomics technology.

Analysis of Pesticide Raid^(█) in Feed of Wistar Rat by High-Pressure Liquid Chromatography (HPLC)

The distribution of pesticide by-product in tissues of wistar rats were analyzed using high pressure liquid chromatography. The limit of detection of the HPLC was 0.1 μg. Results show bioaccumulation factor of pesticide “Raid?” in lipid, up to three times that of the feed at the first concentration and gradually decreased as the concentration increased in the muscle > (0.7), brain > (0.5) and liver > (0.3) as indicated in the text. At higher concentration of 961 μg/g, bioaccumulation factor decreased in the lipid to 1.2 and 0.6 in the muscle, 0.03 in the brain and 0.08 in the liver respectively. High Pressure Liquid Chromatography (HPLC) analysis of raid extract suggests the presence of micprothrin and palethrin. The implications are numerous, but simply put that accidental ingestion of chlorinated hydrocarbon as in “Raid?” may involve convulsions, collapse and coma after only brief excitation and ataxia at the onset.

Determination of 17<i>α</i>-Methyltestosterone in Freshwater Samples of Tilapia Farming by High Performance Liquid Chromatography

17α-methyltestosterone is used to induce the sex reversal of Tilapia sp. to obtain cultures mono-sex to an economically viable. This practice may lead to environmental contamination and problems in human health. Therefore methods need to be developed to detect residues of 17α-methyltestosterone in aqueous matrices. A simple high-performance liquid chromatographic method using ultraviolet detection (245 nm) and testosterone as internal standard has been developed for the monitoring 17α-methyltestosterone in freshwater samples of tilapia aquaculture. The method described involves limited sample preparation as it includes a filtration followed by a single solid-phase extraction step using C18 cartridge. Validation data indicated that the HPLC-UV method for 17α-methyltestosterone determination in the concentration range of 50 - 2000 μg/L provided good linearity, sensitivity, accuracy and precision. Method performance was efficiently applied to monitoring the freshwater samples of fish ponds and the surrounding aquatic channels.

High Performance Liquid Chromatography Analysis of Sugars and Acid Components in ’Xintai Tianhong’ Hawthorn Fruit

[Objectives]This study was conducted to provide basic information for the utilization,quality control and deep processing of‘Xintai Tianhong’hawthorn fruit resources.[Methods]The contents of sugar and acid components in‘Xintai Tianhong’hawthorn fruit were determined by high performance liquid chromatography(HPLC).[Results]‘Xintai Tianhong’hawthorn fruit contained such four kinds of sugar components as fructose,sorbitol,glucose and sucrose,and such six kinds of organic acid components as oxalic acid,tartaric acid,malic acid,acetic acid,citric acid and succinic acid.The fruit had the highest sucrose content,accounting for 70.53%of the total sugar content,followed by fructose,glucose and sorbitol.For the organic acid components,the content of succinic acid was the highest,accounting for 47.32%of the total acid content,followed by citric acid,malic acid,oxalic acid and tartaric acid,and acetic acid had the lowest content.[Conclusions]‘Xintai Tianhong’hawthorn should be a succinic acid-type hawthorn variety,which can be used as a parent in genetic research and breeding practice.