Study of polyphenols in grape berries by reversed-phase high-performance liquid chromatography

Several polyphenols have been tested in grape berries from Spain. The flavonoid content is important because of the pharmacological properties of these compounds, whereas resveratrol has been proved to be an antifungal, antiinflammatory and an anticarcinogenic compound. A reversed-phase HPLC method has been developed and applied to determine resveratrol, quercetine, quercitrine and rutine content in several grape berries samples in a single analysis. Covering the grapes with a preservative paper yields a healthier product, but one which has a lower polyphenol content than unprotected grapes.     

High-resolution liquid chromatography/electrospray ionization time-of-flight mass spectrometry combined with liquid chromatography/electrospray ionization tandem mass spectrometry to identify polyphenols from grape antioxidant dietary fiber

Grape antioxidant dietary fiber (GADF) is a dietary supplement that combines the benefits of both fiber and antioxidants that help prevent cancer and cardiovascular diseases. The antioxidant polyphenolic components in GADF probably help prevent cancer in the digestive tract, where they are bioavailable. Mass spectrometry coupled to liquid chromatography is a powerful tool for the analysis of complex plant derivatives such as GADF. We use a combination of MS techniques, namely liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) on a triple quadrupole, for the identification of the polyphenolic constituents of the soluble fraction of GADF. First, we separated the mixture into four fractions which were tested for phenolic constituents using the TOF system in the full scan mode. The high sensitivity and resolution of the TOF detector over the triple quadrupole facilitate the preliminary characterization of the fractions. Then we used LC/ESI-MS/MS to identify the individual phenols through MS/MS experiments (product ion scan, neutral loss scan, precursor ion scan). Finally, most of the identities were unequivocally confirmed by accurate mass measurements on the TOF spectrometer. LC/ESI-TOF-MS combined with MS/MS correctly identifies the bioactive polyphenolic components from the soluble fraction of GADF. High-resolution TOF-MS is particularly useful for identifying the structure of compounds with the same LC/ESI-MS/MS fragmentation patterns.     

Azithromycin extraction from municipal wastewater and quantitation using liquid chromatography/mass spectrometry

Azithromycin is a broad spectrum antimicrobial agent that is approved in the United States for use in humans. Due to azithromycin's low rate of metabolism it is likely to be found in wastewater treatment plants, where its broad spectrum of antimicrobial activity could lead to development of resistance in bacteria. A liquid-liquid extraction using K2CO3 and methyl-t-butyl ether (MTBE) was used to extract azithromycin from 10 ml aliquots of wastewater. Liquid chromatography was performed using a Luna C18(2) (30 mm x 2.0 mm) column with a mass spectrometer friendly mobile phase containing 50:24:2:24 acetonitrile, methanol, tetrahydrofuran, and 0.04 M ammonium hydroxide. The mass spectrometer used an electrospray source with positive ionization and an ion trap detector. A linear standard curve from 5 to 200pg/ml was validated and used to quantitate azithromycin in wastewater.     

Oligosaccharide relative quantitation using isotope tagging and normal-phase liquid chromatography/mass spectrometry

The growing interest in the conversion of plant biomass into biofuels has recently highlighted the lack of analytical techniques that are able to profile the fine structures of plant cell-wall polysaccharides. Here we present a new liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) platform called Oligosaccharide Quantitation using Isotope Tagging (OliQuIT) developed for profiling the oligosaccharides derived from glycosyl hydrolase digestion of polysaccharides. The method is demonstrated using different arabinoxylan-derived oligosaccharide samples, which are reductively aminated with either the light (12C6) or heavy (13C6) form of aniline. The complex oligosaccharide mixtures are analysed by capillary normal-phase (NP)-LC and ESI-MS. Importantly, arabinoxylan oligosaccharide isomers are separated by NP-LC and their relative abundance in different samples can be determined from the intensities of ions labeled with the different isotopes. OliQuIT will be of use in multiple applications, including screening for plant varieties with improved saccharification properties, characterizing glycosyl hydrolase specificities and analysing plant glycosyl transferase mutants.     

Evaluation of propolis polyphenols absorption in humans by liquid chromatography/tandem mass spectrometry

Propolis has various biological activities such as antibacterial, antiviral, antioxidant, immunostimulating and antiinflammatory, which are generally ascribed to the polyphenolic fraction. The aim of this study was to evaluate the absorption of the main polyphenols [caffeic acid (CA), pinobanksin-5methyl ether (P-5ME), pinobanksin (Pb), chrysin (C), pinocembrin (P), galangin (G), pinobanksin-3-acetate, pinobanksin esters and caffeic acid phenylethyl ester (CAPE)] from a dewaxed and standardized extract of propolis (EPID). Fifteen healthy volunteers consumed 5 mL EPID in water, corresponding to 125 mg of flavonoids. Blood samples were collected before, each hour for 8 h and 24 h after EPID intake. After deconjugation by beta-glucuronidase/sulfatase the plasma samples were analyzed by a selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method using morin as internal standard (I.S.). A kinetic profile characterized by two t(max), respectively at 1 h and about 5 h post-ingestion, was observed in all the subjects. The two peaks may be due to enterohepatic cycling. Among the various polyphenols ingested, only P-5ME, Pb, C, P and G were detected in plasma and C(max)t(1h) were 65.7 +/- 13.3, 46.5 +/- 12.7, 79.5 +/- 18.6, 168.1 +/- 16.3 and 113.7 +/- 16.8 ng/mL, respectively. These levels decreased significantly after 8 h and were no longer detectable 24 h after EPID intake. The recovery of the extraction for CA, Pb, C, P, G and I.S. from spiked plasma was 95.2 +/- 3.1, 93.1 +/- 3.6, 91 +/- 2.5, 96.4 +/- 4.2, 93.4 +/- 2.4 and 85.5 +/- 2.4%, respectively. The results of this study evidence that flavonoids from EPID are absorbed, metabolized and Pb-5ME and G seem to have apparent absorption, measured as (AUC/dose), higher than C, P and Pb.     

高效液相色谱-串联质谱法测定葡萄果实中的乙酰辅酶A

建立了高效液相色谱-三重串联四极杆质谱法(HPLC-MS/MS)测定葡萄果实中重要中间代谢产物乙酰辅酶A的分析方法。样品用纯水提取后离心,经C18固相萃取小柱净化,以5 mmol/L甲酸铵和乙腈（20:80,v/v）为流动相等度洗脱,经C18柱分离后,在电喷雾正离子化(ESI+)模式下,通过多反应监测(MRM)模式进行定性,以n-丙酰辅酶A为内标进行定量。该方法在1～2000 μg/L范围内线性关系良好,相关系数大于0.99,检出限(以信噪比(S/N)为3计)为0.1 μg/L,定量限(以S/N=10计)为0.5 μg/L;添加水平分别为50、500、1000 μg/L的3个样品的加标回收率为82.87%～89.67%,相对标准偏差均小于10%。该方法简便、快速、灵敏,可明显减少乙酰辅酶A在实验过程中的损失,适用于葡萄果实中乙酰辅酶A的检测分析。     

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.     

Improved characterization of tomato polyphenols using liquid chromatography/electrospray ionization linear ion trap quadrupole Orbitrap mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry

Tomato (Lycopersicon esculentum Mill.) is the second most important fruit crop worldwide. Tomatoes are a key component in the Mediterranean diet, which is strongly associated with a reduced risk of chronic degenerative diseases. In this work, we use a combination of mass spectrometry (MS) techniques with negative ion detection, liquid chromatography/electrospray ionization linear ion trap quadrupole‐Orbitrap‐mass spectrometry (LC/ESI‐LTQ‐Orbitrap‐MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) on a triple quadrupole, for the identification of the constituents of tomato samples. First, we tested for the presence of polyphenolic compounds through generic MS/MS experiments such as neutral loss and precursor ion scans on the triple quadrupole system. Confirmation of the compounds previously identified was accomplished by injection into the high‐resolution system (LTQ‐Orbitrap) using accurate mass measurements in MS, MS² and MS³ modes. In this way, 38 compounds were identified in tomato samples with very good mass accuracy (<2 mDa), three of them, as far as we know, not previously reported in tomato samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Trace-level quantitation of iralukast in human plasma by microbore liquid chromatography/tandem mass spectrometry

Iralukast (CGP 45715A) is a potent peptido-leukotriene antagonist that is active in various in vitro and animal models for the treatment of asthma. An analytical challenge was to develop a sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method with a lower limit of quantitation (LLOQ) of 10 pg/mL for the analysis of iralukast when administered at low doses during clinical trials. Several issues had to be addressed in order to devise a LC/MS/MS assay for the above compound. First, iralukast appeared to be light sensitive and unstable at room temperature under acidic conditions. Second, a LLOQ of 10 pg/mL was needed to support several clinical trials. Third, positive electrospray ionization of iralukast did not yield the necessary sensitivity required for studies in humans. Consequently, LC/MS/MS conditions were optimized for the negative ion mode of detection. Fourth, sample preparation steps proved to be critical to reduce the possibility of microbore HPLC column (50 mm x 1.0 mm i.d.) obstruction, chromatographic deterioration, and matrix-mediated electrospray ion suppression. While our validated method addressed the above challenges, its major drawback was limited sample throughput capability. Nonetheless, plasma concentration-time profiles for patients with moderate asthma after oral administration of 200, 500, 1000, and 5000 microgram/kg/day of iralukast were successfully obtained.     

Validation of liquid chromatography and liquid chromatography/tandem mass spectrometry methods for the determination of etoricoxib in pharmaceutical formulations

Reversed-phase liquid chromatography (LC) and LC/tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the determination of etoricoxib in pharmaceutical dosage forms. The LC method was performed by reversed-phase chromatography on a Synergi fusion C18 column (150 x 4.6 mm id) maintained at ambient temperature. The mobile phase consisted of 0.01 M phosphoric acid, pH 3.0-acetonitrile (62 + 38, v/v) at a flow rate of 1.0 mL/min, and photodiode array detection at 234 nm was used. The chromatographic separation was obtained within 7.0 min, and calibration curves were linear in the concentration range of 0.02-150 microg/mL. The LC/MS/MS method was performed on a Luna C18 column (50 x 3.0 mm id). The mobile phase consisted of acetonitrile-water (95 + 5)-0.1% acetic acid (90 + 10, v/v). Detection was performed by positive electrospray ionization in the multiple reaction monitoring mode, monitoring the transitions 359.3 > 280.0 and 332.0 > 95.0 for etoricoxib and piroxicam (internal standard), respectively. The chromatographic separation was obtained within 2.0 min, and calibration curves were linear in the concentration range of 1-5000 ng/mL. Validation parameters, such as specificity, linearity, precision, accuracy, and robustness, were evaluated, which gave results within the acceptable range for both methods. Moreover, the proposed methods were successfully applied for routine quality control analysis of pharmaceutical products and showed significant correlation (r = 0.9999) of the results.     

Quantitative determination of coenyzme Q10 by liquid chromatography and liquid chromatography/mass spectrometry in dairy products

The dietary sources of CoQ10 and the evaluation of CoQ10 in dairy products were characterized. For quantitation of CoQ10 in food samples, 2 liquid chromatography (LC) methods with UV and mass spectrometry (MS) detections were developed. LC with UV detection was performed at 25 degrees C on a Hyperclone ODS 5 microm 150 x 4.6 mm column with mobile phase consisting of methanol-ethanol-2-propanol (70 + 15 + 15, v/v/v). Flow rate was 1.0 mL/min. Retention time of CoQ10 was 10.9 +/- 0.1 min. The method was sensitive [limit of detection (LOD) = 0.2 mg/kg], reproducible [relative standard deviation (RSD) = 3:0%), and linear up to 25 mg/kg (R > 0.999). LC/MS analysis was performed on a LUNA C18 3 microm, 150 x 4.6 mm column, using mobile phase consisting of ethanol-dioxane-acetic acid (9 + 1 + 0.01, v/v/v), flow rate was 0.6 mL/min, and the retention time of CoQ10 was 4.1 +/- 0.1 min. Identification and quantitation were performed with a Finnigan-LCQ mass detector in positive atmospheric pressure chemical ionization mode. Mass spectra were obtained in selected-ion monitoring mode; molecular mass (M+H)+ m/z 863.4 +/- 1 was used for quantitative determination. MS detection is more sensitive than UV detection (LOD = 0.1 mg/kg), less reproducible (RSD = 4.0%), and linear in selected range. Analytical recoveries are 75-90% and depend on the ratio between the amount of fat in the matrix and the concentration of CoQ10 in the sample. Some soybean milk products were analyzed together with different cow, goat, and sheep milk products. Concentrations obtained with LC and LC/MS were compared with a few accessible results available from the literature. Concentrations varied from 0 ppm in soybean milk to nearly 2 ppm in fresh milk from local farms.     

Different quantitation approaches for xenobiotics in human urine samples by liquid chromatography/electrospray tandem mass spectrometry

The potential of liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) for the determination of pesticide metabolites in human urine at the sub-ppb level is explored. Metabolites from two organophosphorous pesticides, 4-nitrophenol (from parathion and parathion-methyl) and 3-methyl-4-nitrophenol (from fenitrothion), are taken as model analytes to conduct this study. After direct injection of the urine sample (10 microL), different approaches were evaluated in order to achieve correct quantitation of analytes using an electrospray ionisation (ESI) interface. Thus, the feasibility of using external calibration was checked versus the use of different isotope-labeled internal standards. The advantages of applying coupled-column liquid chromatography (LC/LC) as an efficient clean-up without any type of sample manipulation are also discussed. The combination of LC/LC with ESI-MS/MS allows the direct analysis of free metabolites in urine, as the automated clean-up performed by the coupled-column technique is sufficient for the removal of interferences that suppress the ionisation of analytes in the ESI source. Using this procedure with external calibration, good precision and recoveries, and detection limits below 1 ng/mL are reached with analysis run times of around 8 min. The hyphenated technique LC/LC/ESI-MS/MS is proved to be a powerful analytical tool, allowing the rapid, sensitive and selective determination of 4-nitrophenol and 3-methyl-4-nitrophenol in human urine without any sample treatment.     

Quantitative determination of ochratoxin A in kidneys by liquid chromatography/mass spectrometry

A sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for the quantitative determination of ochratoxin A (OTA) in kidney samples was developed. Ochratoxin B (OTB) was used as internal standard. Extraction of homogenized kidney samples was done by adding a chloroform/phosphoric acid mixture. Due to restriction of the sample size, less chloroform could be used than in previously described methods. Two different columns for clean-up were compared: strong anion exchange (Bond Elut SAX) and Extrelut NT columns. The high-performance liquid chromatography (HPLC) was used with gradient elution consisting of variable mixtures of formic acid (0.3%) in acetonitrile and formic acid (0.3%) in water. The mass spectrometer was operated in the positive ESI mode using multiple reaction monitoring. For OTA the precursor ion was m/z 404 while the product ions were at m/z 239 and m/z 341. For OTB the precursor ion was m/z 370 while the product ions were at m/z 205 and at m/z 324. A calibration curve of fortified kidney samples was used to quantify OTA. Method validation was performed according to Commission Decision 2002/657/EC. Decision limit (CCalpha), detection capability (CCbeta), precision, bias, trueness, specificity and measurement uncertainty were determined. In general, the best results were obtained using SAX clean-up. CCalpha and CCbeta were 0.11 and 0.25 microg kg(-1), respectively. Within-laboratory reproducibility (% CV) was 9, 9, and 5% for OTA-fortified kidney samples of 0.5, 1, and 2.5 microg kg(-1), respectively. Trueness (%) was 75, 69, and 57% for OTA-fortified kidney samples at 0.25, 0.5, and 1 microg kg(-1), respectively. Measurement uncertainty and expanded uncertainty were 14.85 and 29.70%, respectively. All criteria as laid down in Commission Decision 2002/657/EC were fulfilled. Therefore, this LC/ESI-MS/MS method can be used to monitor kidneys for OTA in the framework of Council Directive No. 96/23/EC.     

Application of ion trap technology to liquid chromatography/mass spectrometry quantitation of large peptides

Triple quadrupole mass spectrometers are generally considered the instrument of choice for quantitative analysis. However, for the analysis of large peptides we have encountered some cases where, as the data presented here would indicate, ion trap mass spectrometers may be a good alternative. In general, specificity and sensitivity in bioanalytical liquid chromatography/mass spectrometry (LC/MS) assays are achieved via tandem MS (MS/MS) utilizing collision-induced dissociation (CID) while monitoring unique precursor to product ion transitions (i.e. selected reaction monitoring, SRM). Due to the difference in CID processes, triple quadrupoles and ion traps often generate significantly different fragmentation spectra of product ion species and intensities. The large peptidic analytes investigated here generated fewer fragments with higher relative abundance on the ion trap as compared to those generated on the triple quadrupole, resulting in lower limits of detection on the ion trap.     

Fast analysis of isobaric grape anthocyanins by Chip‐liquid chromatography/mass spectrometry

In a previous work, direct‐infusion electrospray ionization ion trap tandem mass spectrometry (ESI‐IT‐MS/MS) was applied to the study of anthocyanins in extracts from the skins of Clinton grapes, a non‐Vitis vinifera red grape variety qualitatively and quantitatively rich in anthocyanins. A good characterization of anthocyaninins was obtained, but it was impossible to differentiate some compounds with the same nominal mass but with different elemental composition. In this work, the capabilities of quadrupole time‐of‐flight mass spectrometry (QTOF‐MS) coupled with Chip‐liquid chromatography (LC‐Chip) were applied to the study of Clinton anthocyanins and this method provided the complete sample anthocyanin fingerprint in less than 5 min. Multi‐stage mass spectrometry (MSⁿ; n >2) was not necessary to identify isobaric compounds, nor were deuterium‐exchange experiments necessary to distinguish between compounds containing the same aglycone. The fast separation bypasses the problem of petunidin‐3‐O‐(6‐O‐acetyl)monoglucoside and delphinidin‐3,5‐O‐diglucoside quantification, present in the direct‐infusion ESI‐ITMS approach, due to overlapping with matrix interferences. Copyright © 2009 John Wiley & Sons, Ltd.     

Sensitive high-performance liquid chromatography/mass spectrometry method for determination of steviol in rat plasma

The main toxicological concern of stevioside, a highly potent sweetener from S. rebaudiana, is its main metabolite, steviol. To determine very low levels of steviol in in vivo experiments, a sensitive liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS) method was developed for quantifying steviol in rat plasma after oral administration of a single dose of stevioside (0.5 g/kg). The sample preparation uses liquid-liquid extraction with tert-butyl methyl ether in an acidic environment. The retention time of steviol was 10.5 min. The assay was linear over the range 2-1000 ng/mL with a lower limit of detection of 1 ng/mL. The intra- and inter-day precision were <5 and <7%, respectively, and the accuracy was in the range 95-108%. The steviol concentration profile in rat plasma was determined.     

Identification and quantitation of auxins in plants by liquid chromatography/electrospray ionization ion trap mass spectrometry

Auxin is an important phylohormone, which regulates specific physiological responses such as division, elongation and differentiation of cells. A new method using liquid chromatography/electrospray ionization ion trap mass spectrometry (LC/ESI-ITMS) has been developed for identification and quantitation of four auxins. Under the optimum conditions, four auxins (indole-3-acetic acid, indole-3-propionic acid, indole-3-butyric acid and 1-naphthylacetic acid) were completely separated and quantitated within 7 min with a minimum detection limit of 8.0 ng mL(-1) with relative standard deviations lower than 5.0%. This method also has been applied to analysis of auxins in Chinese cabbage where, even with a complicated serious background perturbation due to the natural biological matrix, the mean recoveries ranged from 77.5% to 99.8%. Finally, we discuss the MS-relevant properties of the identified auxins in detail.     

Hydrophilic interaction liquid chromatography/mass spectrometry for determination of domoic acid in Adriatic shellfish

This paper describes a new method for sensitive, specific and direct determination of domoic acid (DA), the causative toxin of amnesic shellfish poisoning (ASP) syndrome, in shellfish. It is based on combination of hydrophilic interaction liquid chromatography with mass spectrometry (HILIC/MS). The high percentage of organic modifier in the mobile phase and the omission of ion-pairing reagents, both favoured in HILIC, result in enhanced detection limits with MS detection. The new method was set up either on an ionspray ion trap MS instrument operating in MS and MS/MS scanning acquisition modes, or on a turboionspray triple-quadrupole MS system operating in selected ion monitoring (SIM) and multiple reaction monitoring (MRM) acquisition modes. Positive and negative ion experiments were performed. MRM experiments are recommended for screening contaminated shellfish tissue and for quantitative analyses due to highest sensitivity and selectivity. The minimum detection levels for the toxin in tissue were found to be 63 and 190 ng/g in positive and negative MRM experiments, respectively, which are well below the regulatory limit for DA in tissue (20 microg/g). Application to shellfish samples collected in the Adriatic Sea (Italy) in the period 2000-2004 demonstrated for the first time in Italy the presence of DA as a new toxin that has entered the Adriatic Mytilus galloprovincialis toxin profile.     

Protein open-access liquid chromatography/mass spectrometry

Each year increasing numbers of proteins are submitted for routine characterization by liquid chromatography/mass spectrometry (LC/MS). This paper reports a solution that transforms routine LC/MS analysis of proteins into a fully automated process that significantly reduces analyst intervention. The solution developed, protein open-access (OA) LC/MS, consists of web-enabled sample submission and registration, automated data processing, data interpretation, and report generation. Sample submissions and results are recorded in a LIMS that utilizes an Oracle database. The protein sequence is captured during the sample submission process, stored in the database, and utilized to determine the theoretical protein molecular weight. This calculated mass is used to set the parameters for transformation of the mass-to-charge spectra to the mass domain and evaluate the presence or absence of the desired protein. Three protein OA-LC/MS instruments have been deployed in our facility to support protein characterization, purification, and modification efforts.