Comparison of proanthocyanidins in commercial antioxidants: grape seed and pine bark extracts

The major constituents in grape seed and pine bark extracts are proanthocyanidins. To evaluate material available to consumers, select lots were analyzed using high-performance liquid chromatography, gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), gel permeation chromatography (GPC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Atmospheric pressure chemical ionization (APCI) LC/MS was used to identify monomers, dimers, and trimers present. GC/MS analyses led to the identification of ethyl esters of hexadecanoic acid, linoleic acid, and oleic acid, as well as smaller phenolic and terpene components. The GPC molecular weight (MW) distribution indicated components ranging from approximately 162 to approximately 5500 MW (pine bark less than 1180 MW and grape seed approximately 1180 to approximately 5000 MW). MALDI-TOF MS analyses showed that pine bark did not contain oligomers with odd numbers of gallate units and grape seed contained oligomers with both odd and even numbers of gallate. Reflectron MALDI-TOF MS identified oligomers up to a pentamer and heptamer, and linear MALDI-TOF MS showed a mass range nearly double that of reflectron analyses.     

Determination of zeranol/zearalenone and their metabolites in edible animal tissue by liquid chromatography with electrochemical detection and confirmation by gas chromatography/mass spectrometry

A sensitive method is described for the determination and confirmation of zeranol and zearalenone, as well as their isomers and metabolites, in edible animal tissue. The analytes are extracted from tissue with methanol, hydrolyzed enzymatically, cleaned up by acid-base partitioning, determined by liquid chromatography (LC) with electrochemical (EC) detection, and confirmed by gas chromatography/mass spectrometry (GC/MS). LC analysis is performed by isocratic elution with a buffered mobile phase using a Nova-Pak reverse-phase C18 column with amperometric EC detection at +0.90 V. Capillary GC/MS analysis of the trimethylsilyl derivatives provides mass spectral confirmations.     

Phenolic acids in foods: an overview of analytical methodology

Phenolic acids are aromatic secondary plant metabolites, widely spread throughout the plant kingdom. Existing analytical methods for phenolic acids originated from interest in their biological roles as secondary metabolites and from their roles in food quality and their organoleptic properties. Recent interest in phenolic acids stems from their potential protective role, through ingestion of fruits and vegetables, against oxidative damage diseases (coronary heart disease, stroke, and cancers). High performance liquid chromatography (HPLC) as well as gas chromatography (GC) are the two separation techniques reviewed. Extraction from plant matrixes and cleavage reactions through hydrolysis (acidic, basic, and enzymatic) are discussed as are the derivatization reagents used in sample preparation for GC. Detection systems discussed include UV-Vis spectroscopy, mass spectrometry, electrochemical, and fluorometric detection. The most common tandem techniques are HPLC/UV and GC/MS, yet LC/MS is becoming more common. The masses and MS fragmentation patterns of phenolic acids are discussed and tabulated as are the UV absorption maxima.     

Efficient and sensitive method for quantitative analysis of alkaloids in hardinggrass (Phalaris aquatica L.)

An efficient high-performance thin-layer chromatography (HPTLC) method for the analysis of alkaloids in hardinggrass (Phalaris aquatica L.) was developed. The method employed HPTLC glass plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of ethyl acetate/chloroform/7 N NH4OH in methanol (8:2:1, v/v/v). Using unidimensional double-development, bands were well separated for 10 alkaloid standards as well as alkaloids observed in hardinggrass plant extracts. Identities of compounds observed using HPTLC were validated by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Software was used to quantify individual alkaloids in plant samples based on HPTLC retention factors and intensities relative to standards of known concentration. Correlation coefficients of 0.99 were obtained between estimated and actual concentrations for four standards (methyltyramine, hordenine, gramine, and 5-methoxydimethyltryptamine), with linearity in the range of 120-3840 ng/spot. The HPTLC method is repeatable and specific for beta-carboline, tryptamine, gramine, and tyramine type alkaloids in mixed standard and plant extracts. Initial results indicate substantial variation in alkaloid composition among and within hardinggrass populations.     

The power of hyphenated chromatography/time-of-flight mass spectrometry in public health laboratories

Laboratories devoted to the public health field have to face the analysis of a large number of organic contaminants/residues in many different types of samples. Analytical techniques applied in this field are normally focused on quantification of a limited number of analytes. At present, most of these techniques are based on gas chromatography (GC) or liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS). Using these techniques only analyte-specific information is acquired, and many other compounds that might be present in the samples would be ignored. In this paper, we explore the potential of time-of-flight (TOF) MS hyphenated to GC or LC to provide additional information, highly useful in this field. Thus, all positives reported by standard reference targeted LC-MS/MS methods were unequivocally confirmed by LC-QTOF MS. Only 61% of positives reported by targeted GC-MS/MS could be confirmed by GC-TOF MS, which was due to its lower sensitivity as nonconfirmations corresponded to analytes that were present at very low concentrations. In addition, the use of TOF MS allowed searching for additional compounds in large-scope screening methodologies. In this way, different contaminants/residues not included in either LC or GC tandem MS analyses were detected. This was the case of the insecticide thiacloprid, the plant growth regulator paclobutrazol, the fungicide prochloraz, or the UV filter benzophenone, among others. Finally, elucidation of unknowns was another of the possibilities offered by TOF MS thanks to the accurate-mass full-acquisition data available when using this technique.     

Improved liquid chromatography methods for the separation and quantification of biotin in NIST standard reference material 3280: multivitamin/multielement tablets

Two independently developed liquid chromatography (LC) methods for the quantitative determination of biotin in multivitamin/multielement tablets (NIST Standard Reference Material 3280 (SRM 3280)) are described. The methods use distinctly different tablet extraction solvents (methanol vs 1.5% aqueous formic acid) and analyte detection principles (mass spectrometry (MS) versus evaporative light-scattering detection (ELSD)) to ensure quantitative reliability. The use of different extraction and detection procedures allows cross-validation of the methods and enhances confidence in the final quantitative results. Both methods yield highly comparable results for the mean level of biotin (LC/MS = 26.5 mg/kg +/- 0.3 mg/kg (n = 12); LC/ELSD = 24.7 mg/kg +/- 1.7 mg/kg (n = 12)) in SRM 3280, yet the methods differ considerably in their analytical characteristics. The isotope-dilution LC/MS method exhibits excellent linearity from 0.02 ng to 77 ng biotin on-column with a method limit of detection (LOD) and limit of quantification (LOQ) of 0.02 ng (S/N > 3) and 0.06 ng (S/N > 10) biotin on-column, respectively. The LC/ELSD method exhibits good linearity from 155 ng to 9900 ng biotin on-column with a method LOD and LOQ of 155 ng (S/N > 3) and 310 ng (S/N > 10) biotin on-column, respectively. Method performance data indicates that the LC/MS method is analytically superior to the LC/ELSD method; however, either method in combination with SRM 3280 should provide quality assurance, accuracy, and traceability for biotin levels in multivitamin/multielement dietary supplements.     

Determination of ethyl carbamate in distilled alcoholic beverages by gas chromatography with flame ionization or mass spectrometric detection

Quantitative methods are detailed for determination of ethyl carbamate in distilled alcoholic beverages by capillary gas chromatography with flame ionization detection (GC/FID) and by packed-column gas chromatography/mass spectrometry (GC/MS) using selected ion monitoring. Five g samples of distillate of known ethanol concentration are diluted with water to 25% ethanol (v/v), washed with petroleum ether, and extracted with dichloromethane prior to GC/FID or GC/MS analysis. As necessary, sample extracts that exhibit GC/FID interference are passed through alumina for additional cleanup. When internal standards (tert-butyl carbamate and n-butyl carbamate for GC/FID, or ethyl 13C-15N-carbamate for GC/MS) were used for quantitation, the limit of detection for ethyl carbamate was in the range of 5-25 ppb. Coefficients of variation ranged from 3.5 to 6.0% for GC/FID determinations, and from 1.4 to 3.2% for GC/MS. Correlation between methods for 22 random distillate samples ranging in concentration from approximately 40 to 800 ppb gave a correlation coefficient (r) of 0.996.     

Two new Monascus metabolites with strong blue fluorescence isolated from red yeast rice

Red yeast rice obtained as cultures of Monascus AS3.4444 on rice was extracted and analyzed by high-performance liquid chromatography (HPLC). Two new Monascus metabolites with similar fluorescence spectra (lambda ex = 396 nm, lambda em = 460 nm) and UV absorption spectra (lambda max = 386 nm) were detected. They were isolated by rechromatography on a silica gel column and semipreparative HPLC, and two strong blue fluorescent compounds were obtained. Their structures were elucidated by electrospray ionization mass spectrometry (ESI-MS), electrospray ionization tandem mass spectrometry (ESI-MS/MS), intensive ESI-MS, and nuclear magnetic resonance spectroscopy ( (1)H NMR, (13)C NMR, COSY, and HMBC) studies. High-resolution mass spectrometry indicated the molecular formulas C 21H 24O 5 and C 23H 28O 5. The two new compounds, named monasfluore A and monasfluore B, respectively, contain a alkyl side chain, gamma-lactone, and propenyl group, whereas the more lipophilic compound, monasfluore B, is a higher homologue of monasfluore A, with the more lipophilic octanoyl instead of the hexanoyl side chain.     

Ion-trap tandem mass spectrometry for the analysis of polychlorinated dibenzo-p-dioxins, dibenzofurans, and dioxin-like polychlorinated biphenyls in food

This paper reports on the applicability of gas chromatography coupled to ion-trap tandem mass spectrometry (GC/ITMS/MS) for the analysis of polychlorinated dibenzo- p-dioxins (PCDDs), dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (dl-PCBs) in food. MS/MS parameters were selected to achieve the high sensitivity and selectivity required for food analysis. Good precision (RSD=5-18% for PCDD/Fs and 6-14% for dl-PCBs) and low limits of detection for PCDD/Fs (0.1-0.93 pg/g of fat) and dl-PCBs (0.1-0.89 pg/g of fat) were obtained. A comparative study of the congener-specific determination using both GC/ITMS/MS and GC-high resolution mass spectrometry (GC/HRMS) was performed by analyzing several matrices such as milk, fish oil, chicken, pork, fish, eggs, and a chicken compound feed, at low pg/g levels. The results using GC/ITMS/MS were in good agreement with those obtained by GC/HRMS. Consequently, GC/ITMS/MS is proposed for the analysis of PCDD/Fs and dl-PCBs in food and feed samples.     

Determination of oxyfluorfen herbicide and oxyfluorfen amine residues in garbanzo beans by liquid chromatography

Oxyfluorfen and oxyfluorfen amine were determined by liquid chromatography (LC) with ultraviolet (UV) and photoconductivity detection (PCD). A simple extraction procedure acceptably recovered both analytes from garbanzo beans over a wide range of fortifications (0.05 to 20 ppm) (83 +/- 4 for oxyfluorfen; 85 +/- 4 for oxyfluorfen amine). Percent recoveries decreased slightly as the fortification level decreased. Both analytes could be determined simultaneously at a concentration greater than 0.2 ppm in garbanzo beans. Detection limits were 3 ng for oxyfluorfen and 100 ng for oxyfluorfen amine using LC/UV, and 12 ng for both oxyfluorfen and oxyfluorfen amine with LC/PCD. Different knitted reaction coils and photoreactors were evaluated. Photoproduct yields and identification were determined by ion chromatography. The LC/PCD method measures oxyfluorfen and oxyfluorfen amine separately and has a shorter analysis time, while the standard method using gas chromatography measures total residues and is more sensitive.     

LC/MS/MS characterization of phenolic constituents in dried plums

Dried plums are known as a healthy food in the West and used as medicine in India. They have been characterized by high concentrations of phenolic compounds, which are believed to play a crucial role in protection against various age-related diseases. Liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) with four different conditions was used to analyze the phytochemicals in commercial dried plums. The major components were neochlorogenic acid and cryptochlorogenic acid. Forty minor components were characterized by their MS/MS spectra and LC retention time. Six of them are novel ester isomers formed by two caffeic acids and one quinic acid. The diagnostic fragmentation patterns of different phenolics are presented on the basis of electrospray ionization (ESI) MS/MS data of components in dried plums and fourteen authentic standards.     

Development of liquid chromatography mass spectrometry method for analysis of organic N monomers in soil

The aim of this study was to develop an analytical procedure based on liquid chromatography-mass spectrometry (LC–MS) for analysis of monomeric organic N compounds in soil extracts. To benchmark the developed LC–MS method it was compared with a capillary electrophoresis–mass spectrometry (CE–MS) method recently used for analysis of small organic N monomers in soil. The separation was optimized and analytical performance assessed with 69 purified standards, then the LC–MS method was used to analyse soil extracts. Sixty-two out of 69 standards were analysable by LC–MS with separation on a hydrophilic interaction liquid chromatography column. The seven compounds that could not be analysed were strongly cationic polyamines. Limits of detection for a 5 μL injection ranged between 0.002 and 0.38 μmol L⁻¹, with the majority (49 out of 62) having limits of detection better than 0.05 μmol L⁻¹. The overall profile and concentration of small organic N monomers in soil extracts was broadly similar between LC–MS and CE–MS, with the notable exception of four ureides that were detected by LC–MS only. In soil extracts that had been concentrated ten-fold the detection and quantification of (some) organic N compounds was compromised by the presence of large amounts of inorganic salts. The developed LC–MS method offered advantages and disadvantages relative to CE–MS, and a combination of the two methods would achieve the broadest possible coverage of organic N in soil extracts.     

Liquid chromatographic determination of vitamin K1 trans- and cis-isomers in infant formula

The normal phase liquid chromatographic (LC) method for determination of trans- and cis-isomers of vitamin K1 (phylloquinone) in infant formula described here uses an Apex silica column, isocratic elution, and UV absorption detection at 254 nm. Vitamin K1 is extracted quantitatively from the product matrix by pretreating the as-fed liquid with concentrated ammonium hydroxide and methanol, and then extracting it with a 2:1 mixture of dichloromethane and isooctane. The extract is cleaned up by silica open-column chromatography and concentrated for LC analysis. For trans-vitamin K1, the method precision is less than or equal to 3.3% RSD (relative standard deviation), and the spike recovery is 98 +/- 4%. For cis-vitamin K1, the precision is less than or equal to 12% RSD, determined at levels near the detection limit, and the spike recovery is 95 +/- 9%. The detection limit is 0.3 ng for both isomers at signal/noise = 3.     

Gas chromatography/electron-capture negative ion mass spectrometry for the quantitative determination of 2- and 3-hydroxy fatty acids in bovine milk fat

2- and 3-hydroxy fatty acids (2- and 3-OH-FAs) are bioactive substances reported in sphingolipids and bacteria. Little is known of their occurrence in food. For this reason, a method suitable for the determination of OH-FAs at trace levels in bovine milk fat was developed. OH-FAs (and conventional fatty acids in samples) were converted into methyl esters and the hydroxyl group was derivatized with pentafluorobenzoyl (PFBO) chloride to give PFBO- O-FA methyl esters. These derivatives with strong electron affinity were determined by gas chromatography interfaced to mass spectrometry using electron-capture negative ion in the selected ion monitoring mode (GC/ECNI-MS-SIM). This method proved to be highly sensitive and selective for PFBO-O-FA methyl esters. For the analysis of samples, two internal standards were used. For this purpose, 9,10-dideutero-2-OH-18:0 methyl ester (ISTD-1) from 2-OH-18:1(9 c) methyl ester as well as the ethyl ester of 3-PFBO-O-12:0 (ISTD-2) was synthesized. ISTD-1 served as a recovery standard whereas ISTD-2 was used for GC/MS measurements. The whole-sample cleanup consisted of accelerated solvent extraction of dry bovine milk, addition of ISTD 1, saponification, conversion of fatty acids into methyl esters by use of boron trifluoride, separation of the methyl esters of OH-FAs from nonsubstituted FAs on activated silica, conversion of OH-FAs methyl esters into PFBO-O-FA methyl esters, addition of ISTD-2, and measurement by GC/ECNI-MS-SIM. By this method, ten OH-FAs were quantified in bovine milk fat with high precision in the range from 0.02 +/- 0.00 to 4.49 +/- 0.29 mg/100 g of milk fat.     

Enzymatic conversion of precarthamin to carthamin by a purified enzyme from the yellow petals of safflower

Precarthamin, a yellow precursor of carthamin, was efficiently isolated from the yellow petals of safflower (Carthamus tinctorius L. ) with Sephadex LH-20 column chromatography and preparative HPLC, and identified with UV-vis and NMR spectrometry. The UV-vis spectrum of precarthamin showed lambda(max) of 238 and 406 nm in MeOH. The molar extinction coefficients of precarthamin at 406 nm in MeOH and 50 mM citrate buffer (pH 5.0) were 59 000 M(-)(1) cm(-)(1) and 45 400 M(-)(1) cm(-)(1), respectively. The isolated and structurally identified precarthamin was converted to a red pigment by a homogeneously purified enzyme from the immature petals of safflower in 50 mM citrate buffer (pH 5.0). The enzymatically converted red pigment was identified as carthamin, a red pigment of safflower by TLC, HPLC, and UV-vis spectral analysis.     

Mass spectrometric determination of the predominant adrenergic protoalkaloids in bitter orange (Citrus aurantium)

The predominant adrenergic protoalkaloid found in the peel and fruit of bitter orange, Citrus aurantium, is synephrine. Synephrine is reputed to have thermogenic properties and is used as a dietary supplement to enhance energy and promote weight loss. However, there exists some concern that the consumption of dietary supplements containing synephrine or similar protoalkaloids may contribute to adverse cardiovascular events. This study developed and validated a positive-ion mode liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the quantitative determination of the major (synephrine) and minor (tyramine, N-methyltyramine, octopamine, and hordenine) adrenergic protoalkaloids in a suite of National Institute of Standards and Technology (NIST) bitter orange Standard Reference Materials (SRMs): SRM 3258 Bitter Orange Fruit, SRM 3259 Bitter Orange Extract, and SRM 3260 Bitter Orange Solid Oral Dosage Form. The limit of quantitation (LOQ) for all protoalkaloids is approximately 1 pg on-column, except for octopamine (20 pg on-column). Additionally, the method has a linear dynamic range of > or =3 orders of magnitude for all of the protoalkaloids. Individual, as well as "total", protoalkaloid levels (milligrams per kilogram) in the NIST SRMs were determined and compared to the levels measured by an independent liquid chromatography/fluorescence detection (LC/FD) method. Satisfactory concordance between the LC/MS/MS and LC/FD protoalkaloid measurements was demonstrated. LC/MS/MS analysis of the protoalkaloids in the SRMs resulted in mean measurement imprecision levels of < or =10% coefficient of variation (% CV).     

Kinetics of light-induced cis-trans isomerization of four piperines and their levels in ground black peppers as determined by HPLC and LC/MS

The pungent compounds piperine and isomers thereof, secondary metabolites present in black and white pepper fruit, undergo light-induced isomerizations. To facilitate studies in this area, an HPLC method has been developed for analysis and isolation of the following four possible piperine-derived photoinduced isomers: piperine, isopiperine, chavicine, and isochavicine. The limits of detection (LOD) estimated from calibration plots were approximately 15-30 ng for each isomer. Reproducibilities of the analyses were excellent, and recoveries of spiked samples were as follows (average +/- SD; n = 3): chavicine, 98.4 +/- 2.1%; isopiperine, 96.2 +/- 3.2%; piperine, 104 +/- 3.8%; isochavicine, 98.9 +/- 3.0%. To determine the kinetics of these isomerizations, fluorescent light, sunlight, and UV radiation at 254 nm was used to induce cis-trans geometric isomerization as a function of light intensities and time of exposure determined with the aid of high-performance liquid chromatography (HPLC) and liquid chromatography with diode array UV detection-mass spectrometry (LC-DAD/MS). HPLC was also used to determine the distribution of the isomers in four commercial ground black pepper products used as spices in culinary practice. Isomerization increased with light intensities and time of exposure and leveled off at the so-called photostationary phases. The piperine levels of the four products were quite similar, ranging (in wt %) from 10.17 to 11.68. The amounts of the other three isomers ranged from 0.01 to 0.07 of the total for chavicine; from 0.15 to 0.23 for isopiperine; and from 0.37 to 0.42 for isochavicine. The results establish the utility of the HPLC method for simultaneous analysis of the four isomers both in pure form and in black pepper extracts. The dietary significance of the results is discussed.     

Separation and characterization of phenolic compounds in fennel (Foeniculum vulgare) using liquid chromatography-negative electrospray ionization tandem mass spectrometry

Liquid chromatography (LC) diode array detection (DAD) coupled to negative electrospray ionization (ESI) tandem mass spectrometry (MS/MS) was used for the rapid and sensitive identification of water-soluble phenolic compounds in fennel waste. The plant material was first extracted and then chromatographed on Sephadex LH-20 to afford seven fractions, each of them being subjected to LC-MS analysis. Identification of the compounds was carried out by interpretation of UV, MS, and MS/MS spectra. Forty-two phenolic substances were identified, 27 of which had not previously been reported in fennel, including hydroxycinnamic acid derivatives, flavonoid glycosides, and flavonoid aglycons.     

Development of multiclass methods for drug residues in eggs: hydrophilic solid-phase extraction cleanup and liquid chromatography/tandem mass spectrometry analysis of tetracycline, fluoroquinolone, sulfonamide, and beta-lactam residues

A method was developed for detection of a variety of polar drug residues in eggs via liquid chromatography/tandem mass spectrometry (LC/MS/MS) with electrospray ionization (ESI). A total of twenty-nine target analytes from four drug classes-sulfonamides, tetracyclines, fluoroquinolones, and beta-lactams-were extracted from eggs using a hydrophilic-lipophilic balance polymer solid-phase extraction (SPE) cartridge. The extraction technique was developed for use at a target concentration of 100 ng/mL (ppb), and it was applied to eggs containing incurred residues from dosed laying hens. The ESI source was tuned using a single, generic set of tuning parameters, and analytes were separated with a phenyl-bonded silica cartridge column using an LC gradient. In a related study, residues of beta-lactam drugs were not found by LC/MS/MS in eggs from hens dosed orally with beta-lactam drugs. LC/MS/MS performance was evaluated on two generations of ion trap mass spectrometers, and key operational parameters were identified for each instrument. The ion trap acquisition methods could be set up for screening (a single product ion) or confirmation (multiple product ions). The lower limit of detection for screening purposes was 10-50 ppb (sulfonamides), 10-20 ppb (fluoroquinolones), and 10-50 ppb (tetracyclines), depending on the drug, instrument, and acquisition method. Development of this method demonstrates the feasibility of generic SPE, LC, and MS conditions for multiclass LC/MS residue screening.     

Analysis of acrylamide in a complex matrix of polyacrylamide solutions treated by heat and ultraviolet light

A recently developed headspace/solid-phase microextraction/gas chromatograph (GC) equipped with a nitrogen-phosphorus detector (NPD) (HS/SPME/GC/NPD) method was used to analyze acrylamide formed in an aqueous polyacrylamide solution (25%) treated by heat or photo-irradiation. Original polyacrylamide contained 0.43 +/- 0.11 microg/mL of acrylamide. When polyacrylamide solution was heated at 70 degrees C for 16 h with 0.5% potassium persulfate, the amount of acrylamide increased to 1.02 +/- 0.11 microg/mL. When polyacrylamide solution was irradiated by UV (lambda = 300 nm) for 16 h with 0.05% 2-anthraquinone sulfate sodium salt, the amount of acrylamide increased to 1.14 +/- 0.54 microg/mL. Polyacrylamide has been used in cosmetic formulations. The present study, therefore, suggests that there is another route of acrylamide exposure to humans in addition to foods and beverages.