LC ESI-MS Research Articles

Structural elucidation of in vivo metabolites of phencynonate and its analogue thiencynonate in rats by HPLC–ESI-MS n

The structural elucidation of the metabolites of phencynonate and its analogue thiencynonate in rats was performed by liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS n ) in positive ion mode, by comparing their changes in molecular masses (Δ M), retention times and spectral patterns with those of the parent drug. Phencynonate and thiencynonate were easily biotransformed in vivo by the pathways of N-demethylated, oxidative, hydroxylated and methoxylated to form seventeen metabolites that retained the some features of the two parent molecules. These metabolites included ten phencynonate metabolites (N-demethyphencynonate monoxide, N-demethyhydroxy phencynonate, phencynonate monoxide, hydroxyphencynonate, phencynonate dioxide, methoxyphencynonate, dihydroxyphencynonate, dihydroxyphencynonate, hydroxymethoxy phencynonate, trihydroxyphencynonate) and seven thiencynonate metabolites (N-demethy thiencynonate, N-demethythiencynonate monoxide, N-demethyhydroxythiencynonate, thiencynonate monoxide, hydroxythiencynonate, hydroxythiencynonate monoxide, dihydroxy thiencynonate). The described method has wide applicability to rapidly screen and provide structural information of these metabolites. The identifications of precise structures of these metabolites need to be confirmed by other techniques such as the 1H and 13C NMR.

Development and validation of a liquid chromatography–mass spectrometry method for the determination of verticinone in rat plasma and its application to pharmacokinetic study

We have developed and validated a sensitive liquid chromatography–electrospray ionization-mass spectrometric (LC–ESI-MS) method for the quantification of verticinone, a major active constituent from Fritillaria hupehensis Hsiao et KC Hsia., in rat plasma. Verticinone and the internal standard (IS), hupehenine, were extracted from plasma samples by a simple liquid–liquid extraction with ethyl acetate after being alkalified by 1 M ammonia hydroxide. Chromatographic separation was achieved on a C 18 column using a gradient elution program with methanol and water as the mobile phase. The detection was performed by selected ion monitoring (SIM) mode via positive electrospray ionization (ESI) interface. The lower limit of quantification (LLOQ) was 0.1 ng/mL. The calibration curves were linear ( r 2 > 0.998) over the concentration range of 0.1–200 ng/mL. Within- and between-run precision was less than 6.5% and accuracy was within ±10.7%. The validated method was applied to the pharmacokinetic study of verticinone in rats after a single oral administration of 1 mg/kg.

Rapid Analysis of 27 Components of Isodon serra by LC–ESI-MS–MS

A new method based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry has been developed for simultaneous analysis of 27 components (eighteen diterpenoids, six phenolic acids, and three flavonoids) of Isodon serra, a famous traditional Chinese medicine. Separation on a C18 column was achieved by gradient elution with water and methanol both containing 0.1% formic acid. Identification and quantification of the analytes were achieved by use of a hybrid quadrupole linear ion-trap mass spectrometer. Multiple-reaction monitoring (MRM) was used for quantification, with switching of electrospray ion source polarity between positive and negative modes in the same chromatographic run. An information-dependent acquisition (IDA) method was used to trigger product ion scans above the MRM signal threshold so that the 27 compounds could be identified by use of enhanced product-ion scans (EPI). The method was fully validated (for linearity, precision, accuracy, and limits of detection and quantification). The results indicated that this simple method was rapid, specific, and reliable. The method was successfully applied to analysis of 45 batches of Isodon serra samples from different sources, and quantification of the compounds in Isodon serra was achieved.

Different responses of E:Z-isomers of pesticides in liquid chromatography-electrospray ionization-mass spectrometry

The mass spectrometric behavior of four pairs of stereoisomers was investigated by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The E- and Z-isomers of the pesticides chlorfenvinphos, dimethomorph, mevinphos and phosphamidon-each with one double bond-were chosen for this study. The MS response of the individual isomers was investigated by infusing the isomers individually into the MS or after the separation of isomer mixtures via high-performance liquid chromatography (HPLC). In the case of dimethomorph, the same MS response was found for the two isomers. In contrast to that, the individual isomers of chlorfenvinphos, mevinphos and phosphamidon showed different MS response both in the single ion monitoring (SIM) mode in single quadrupole MS and multiple reaction monitoring (MRM) mode in tandem MS. The MS response of the isomers partly depends on (1) the declustering potential of the precursor ion in the SIM mode, (2) the selected transition and (3) the collision energy in the MRM mode. Consequently, quantification by summation of the peak areas of the isomers is inaccurate due to over- or underestimating of one of the stereoisomers. Accurate quantitative results can only be achieved when the compound-specific MS parameters are separately determined for each isomer. This can be done by using pure isomers or by the determination of the MS parameters after HPLC separation and the measurement of the actual isomer ratio with an independent technique.

Composition and stability of phytochemicals in five varieties of black soybeans ( Glycine max)

Phytochemical compositions of five varieties of black soybeans ( Glycine max) and their stabilities at room temperature, 4 and −80 °C over 14 months were determined by HPLC systems with electrochemical (ECD) and UV detectors. Polyphenol profiling was carried out by a liquid chromatography–electrospray ionisation-mass spectrometry (LC–ESI-MS) with orbitrap as a mass analyser in both positive and negative ion modes, and polyphenols in aglycone forms were quantified by HPLC–ECD. Five different varieties of black soybeans ( G. max) contained 249–405 μg/g dry wt of γ-tocopherol and 6.76–14.98 μg/g dry wt of lutein. Major polyphenols in black soybeans ( G. max) were daidzein (193–288 μg/g dry wt) and genistein (145–223 μg/g dry wt), mainly present as glucosides and acetyl glucosides. No significant decrease was found in total phenols of black soybeans ( G. max) stored at room temperature, 4 or −80 °C for 14 months. On the other hand, lutein and γ-tocopherol degraded significantly within a month of storage at room temperature ( p < 0.01), whereas they remained stable up to 6 months at 4 °C and up to 14 months at −80 °C. The current study indicates that black soybeans ( G. max) are rich source of γ-tocopherol and phenols (isoflavones, flavonols, flavan-3-ols, proanthocyanidins and anthocyanin) and that the levels vary depending upon varieties. In addition, storage at low temperature is recommended to reduce the loss of fat-soluble phytochemicals in black soybeans ( G. max) over an extended period of time.

Determination and quantification of active phenolic compounds in pigeon pea leaves and its medicinal product using liquid chromatography–tandem mass spectrometry

A novel method using liquid chromatography coupled to electrospray ionization mass spectrometry (LC–ESI-MS) has been optimized and established for the qualitative and quantitative analysis of ten active phenolic compounds originating from the pigeon pea leaves and a medicinal product thereof (Tongluo Shenggu capsules). In the present study, the chromatographic separation was achieved by means of a HiQ Sil C18 V reversed-phase column with a mobile phase consisting of methanol and 0.1% formic acid aqueous solution. Low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS) using the selected reaction monitoring (SRM) analysis was employed for the detection of ten analytes which included six flavonoids, two isoflavonoids and two stilbenes. All calibration curves showed excellent coefficients of determination ( r 2 ≥ 0.9937) within the range of tested concentrations. The intra- and inter-day variations were below 5.36% in terms of relative standard deviation (RSD). The recoveries were 95.08–104.98% with RSDs of 2.06–4.26% for spiked samples of pigeon pea leaves. The method developed was a rapid, efficient and accurate LC–MS/MS method for the detection of phenolic compounds, which can be applied for quality control of pigeon pea leaves and related medicinal products.

Behaviour of soyasapogenol B under optimised hydrolysis and ESI mass spec conditions

Soyasaponins are oleanane triterpenoids found in soy ( Glycine max Merr). The analysis of the main aglycone (soyasapogenol B) is hampered by inconsistent ion fragmentation patterns produced during MS analysis. The greatest ( p < 0.05) increase in soyasapogenol B during acid hydrolysis HCl (5% (v/v)) was at 100 °C (84.59 ± 4.73 μg/ml), followed by 120 °C (69.82 ± 6.20 μg/ml) and 80 °C (46.54 ± 6.41 μg/ml). The capillary temperature (LC–ESI-MS) of 220 °C produced the greatest ( p < 0.05) intensity of soyasapogenol B ion. Temperatures between 200 and 250 °C consisted the optimum temperature range for analysing soyasapogenol B. At low capillary temperatures (<200 °C), the soyasapogenol B ion ([M−H] − = 457.4) was converted to an ion m/z at 441, while, at temperatures ⩾250 °C the soyasapogenol B ion produced ions m/z at 440 and 437. We report an analysis procedure for detecting intact soyasapogenol B molecular ion without the typical fragmentation reported in the literature.

Combination of normal-phase medium-pressure liquid chromatography and high-performance counter-current chromatography for preparation of ginsenoside-Ro from panax ginseng with high recovery and efficiency

Ginsenoside-Ro which belongs to the oleanane-type saponins has anti-inflammatory, anti-platelet, anticomplementary and immunomodulatory activities. The present paper described the preparation of ginsenoside-Ro from panax ginseng with high recovery and efficiency by combination of normal-phase medium-pressure liquid chromatography (NP-MPLC) and high-performance counter-current chromatography (HPCCC). The crude sample was preliminarily chromatographed by NP-MPLC to enrich ginsenoside-Ro to the purity of 70.2% with 95.0% recovery. Then, the enriched sample was further purified by HPCCC with a solvent system composed of ethyl acetate–isopropanol–0.1% formic acid (3:1:5, v/v), where 61 mg ginsenoside-Ro was obtained from 100 mg enriched sample with 96.0% purity and 83.4% recovery. The overall recovery of ginsenoside-Ro was 79.2%, whose structure was identified by liquid chromatography–electrospray ionization mass spectrometry (LC–ESI-MS) finally.

17-Hydroxygeranyllinalool glycosides are major resistance traits of Nicotiana obtusifolia against attack from tobacco hornworm larvae

In the Great Basin Desert, Nicotiana obtusifolia (synonymous with Nicotiana trigonophylla) and Nicotiana attenuata co-occur, but the former is frequently less attacked by larvae of the tobacco hornworm than the latter, despite having lower nicotine and trypsin protease inhibitor defenses. Glycosides of the diterpene, 17-hydroxygeranyllinalool (HGL-DTGs) have recently been found to be important defenses of N. attenuata. Total HGL-DTG levels are 5-fold higher in N. obtusifolia than in N. attenuata, and we characterize the three major HGL-DTGs purified from N. obtusifolia leaves as: 3- O- α- l-rhamnopyranosyl-(1 → 4)- β- d-glucopyranosyl-17-hydroxygeranyllinalool-17- O- α- l-rhamnopyranosyl-(1 → 4)- β- d-glucopyranoside; nicotinoside III and its malonic acid conjugates. Using APCI- and ESI-LC–MS, we also identified mono- and diacetyl-nicotinoside III and quercetin glycosides. To evaluate the defensive value of these HGL-DTGs, we used virus-induced-gene silencing to reduce the transcript levels of geranylgeranyl diphosphate synthase and total HGL-DTG levels in both species. When fed on silenced plants, larvae gained up to about two times more mass than those that fed on empty vector control plants of both species. We conclude that HGL-DTGs function as the most important direct defenses for both N. attenuata and N. obtusifolia.

Simultaneous Quantitative Analysis of Shikonin and Deoxyshikonin in Rat Plasma by Rapid LC–ESI–MS–MS

The purpose of this article was to develop a rapid and robust LC–MS–MS method for quantifying shikonin and deoxyshikonin simultaneously in rat plasma using emodin as internal standard. The LC system consisted of an Agilent ZORBAX SB-C18 (1.8 μm, 250 × 4.6 mm, 20 °C) column. Elution with an isocratic mobile phase consisted of methanol/10 mM ammonium acetate in water/acetonitrile containing 0.05% formic acid (45:10:45, v/v/v) at a flow rate of 0.8 mL min−1 yielded sharp, high-resolved peaks within 12 min. The lower limits of quantitation were 0.5 ng mL−1 for shikonin, and 8 ng mL−1 for deoxyshikonin. Correlation coefficient (r) values for the linear range of two analytes were greater than 0.99. Assay precision was <13% and accuracy was 87–99%. This newly developed method was used to the pharmacokinetic studies of the shikonin analogues in rats after intravenous administration (n = 4).

Simultaneous Analysis of Zofenopril and Its Active Metabolite Zofenoprilat in Human Plasma by LC–ESI-MS Using Pre-Column Derivatization with p-Bromophenacyl Bromide

Zofenoprilat is an active metabolite of zofenopril, which is very unstable in plasma because of oxidative degradation of its thiol group. In this method, p-bromophenacyl bromide was used as derivatization reagent, immediately after plasma separation, to react with the free thiol group of zofenoprilat and form the derivative zofenoprilat-p-BPB. After acidification with 50% acetic acid, the derivatized plasma samples were extracted with methyl tert-butyl ether and separated on a C18 column with 40:60 (v/v) 10 mM ammonium acetate buffer solution containing 0.1% formic acid–acetonitrile as mobile phase. Calibration plots were linear over the concentration range 1–500 ng mL−1 for zofenopril and 2–1,800 ng mL−1 for zofenoprilat. The method was successfully used to study the bioavailability of zofenopril calcium capsules relative to that of zofenopril calcium tablets in healthy Chinese volunteers.

Reproducible Microwave-Assisted Acid Hydrolysis of Proteins Using a Household Microwave Oven and Its Combination with LC-ESI MS/MS for Mapping Protein Sequences and Modifications

A new set-up for microwave-assisted acid hydrolysis (MAAH) with high efficiency and reproducibility to degrade proteins into peptides for mass spectrometry analysis is described. It is based on the use of an inexpensive domestic microwave oven and can be used for low volume protein solution digestion. This set-up has been combined with liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI QTOF MS) for mapping protein sequences and characterizing phosphoproteins. It is demonstrated that for bovine serum albumin (BSA), with a molecular mass of about 67,000 Da, 1292 peptides (669 unique sequences) can be detected from a 2 microg hydrolysate generated by trifluoroacetic acid (TFA) MAAH. These peptides cover the entire protein sequence, allowing the identification of an amino acid substitution in a natural variant of BSA. It is shown that for a simple phosphoprotein containing one phosphoform, beta-casein, direct analysis of the hydrolysate generates a comprehensive peptide map that can be used to identify all five known phosphorylation sites. For characterizing a complex phosphoprotein consisting of different phosphoforms with varying numbers of phosphate groups and/or phosphorylation sites, such as bovine alpha(S1)-casein, immobilized metal-ion affinity chromatography (IMAC) is used to enrich the phosphopeptides from the hydrolysate, followed by LC-ESI MS analysis. The MS/MS data generated from the initial hydrolysate and the phosphopeptide-enriched fraction, in combination with MS analysis of the intact protein sample, allow us to reveal the presence of three different phosphoforms of bovine alpha(S1)-casein and assign the phosphorylation sites to each phosphoform with high confidence.

Homogeneous photodegradation study of 2-mercaptobenzothiazole photocatalysed by sodium decatungstate salts: Kinetics and mechanistic pathways

Abstract The photochemical degradation of a benzothiazole derivative, 2-mercaptobenzothiazole (MBT) has been studied in aqueous solution in the presence of a polyoxometalate (POM): sodium decatungstate salts Na 4 W 10 O 32 (DTA). In aerated conditions, the photodegradation rate of MBT clearly increased in the presence of DTA by a factor six when compared with the direct photolysis with k MBT = 0.25 h −1 and t 1/2 (MBT) = 2.8 h. For the total comprehension of the degradation mechanism, the oxygen influence has been investigated. Oxygen appeared essential for DTA regeneration, its absence induced a three times inhibition of MBT disappearance and completely stopped the photocatalytic cycle. The main photoproducts were identified with LC–ESI-MS and LC-DAD techniques and using some calculations obtained by B3LYP/6–21G method in Gaussian 4.1 software. All the results allowed to propose a mechanistic pathway. Electron transfer and H atom abstraction processes involving W 10 O 32 4−* excited state species were involved in the degradation. In the primary step of the degradation, the hydroxylation of the aromatic ring leading to four OH-MBT isomers and the formation of disulfide form of MBT were observed. For longer irradiation time, a secondary electron transfer permitted the oxidation of OH-MBT isomers and the formation of sulfoxide derivatives. For prolonged exposure (around 100 h), the complete mineralization was noticed in the presence of sodium decatungstate salts.

Structural identification of long-chain polyamines associated with diatom biosilica in a Southern Ocean sediment core

Long-chain polyamines (LCPAs) constitute a new family of natural organic compounds that have recently been isolated and characterized from the biosilicified cell walls of diatom cultures. To date, diatom-specific polyamines have not been investigated from the marine environment and their fate in the environment is entirely unknown. Here, we report a series of LCPAs in a diatom frustule-rich sediment core (TNO57-13 PC4), originating from the Atlantic sector of the Southern Ocean and spanning from the Holocene to the Last Glacial Maximum (LGM). Liquid chromatography with electrospray ionization mass spectrometry (LC–ESI–MS) revealed a complex mixture of linear polyamines with at least 28 individual molecular species. Ion trap mass fragmentation studies, combined with high resolution Time of Flight (TOF) mass spectrometry showed that the polyamine pool consisted of a series of N-methylated propylamine compounds attached to a putrescine moiety, with individual LCPAs varying in chain length and degree of methylation. The structural similarity between LCPAs extracted from the diatom-rich sediment core and those extracted from the frustules of cultured diatoms suggests that sedimentary LCPAs are derived from diatom frustules. We hypothesize that these intrinsically labile organic molecular fossils are protected from diagenesis by encapsulation within the frustule. These compounds constitute a new class of biomarkers that could potentially be indicators of diatom species distribution. Isotopic analysis of LCPAs could be used to improve age models for sediment cores that lack calcium carbonate and to improve current interpretations of diatom-based paleoproxies, including diatom-bound nitrogen isotopes.

Effect of gamma-irradiation on flavour 5′-nucleotides, tyrosine, and phenylalanine in mushrooms ( Agaricus bisporus)

The impact of gamma-irradiation on 5′-nucleotides and on the free amino acids tyrosine and phenylalanine in fresh mushrooms was studied. After irradiation the samples were freeze-dried to avoid enzyme induced chemical changes. Three 5′-nucleotides could be detected using HPLC–UV and LC–ESI-MS: adenosine 5′-monophosphate (AMP), guanosine 5′-monophosphate (GMP) and guanosine 5′-diphosphate (GDP). Irradiation significantly reduced ( p = 0.05) the GDP concentration (22%). AMP showed a marked reduction (46%) only at 5 kGy. GMP, tyrosine, and phenylalanine were not affected by gamma-irradiation.

Separation and detection of all phosphoinositide isomers by ESI-MS

Phosphoinositides (PIs) play fundamental roles as signalling molecules in numerous cellular processes. Direct analysis of PIs is typically accomplished by metabolic labelling with 3H-inositol or inorganic 32P followed by deacylation, ion-exchange chromatography and flow scintillation detection. This analysis is laborious, time-consuming, and involves massive amounts of radioactivity. To overcome these limitations we established a robust, non-radioactive LC–ESI–MS assay for the separation and analysis of deacylated PIs that allows discrimination of all isomers without the need for radioactive labelling. We applied the method to various cell types to study the PI levels upon specific stimulation.

LC-Tandem Mass Spectrometry Method for Quantification of Lumefantrine in Human Plasma and Its Application to Bioequivalence Study

A rapid, specific and robust assay based on protein precipitation and liquid chromatography-electrospray ionization tandem mass spectrometry (LC–ESI–MS–MS) has been developed and validated for the quantitative analysis of lumefantrine, an antimalarial drug, in human plasma using piperazine bis chloroquinoline as internal standard (ISTD). The precursor to product ion transitions of m/z 528.2 → 510.3 and m/z 409.2 → 205.1 were used to measure the analyte and the ISTD, respectively. Analysis was performed on C8 column (50 mm × 4.6 mm, 5 μm) with 10 mM ammonium acetate/acetonitrile/0.05% acetic acid (10/85/5, v/v) as mobile phase at a flow rate of 0.6 mL min−1. The assay exhibited a linear dynamic range of 0.21–25.05 μg mL−1. The RSD% of intra-day and inter-day assay was ≤15%. The application of this assay was demonstrated in a bioequivalence study and will be ideal for pharmacokinetic studies in pediatric population.

Determination of metoclopramide in human plasma by LC–ESI-MS and its application to bioequivalance studies

An LC–MS method for the determination of metoclopramide in human plasma was developed and validated. Sample preparation involved extraction with ethyl acetate. Chromatographic separation was performed on a Thermo Hypersil-Hypurity C 18 (150 mm × 2.1 mm, 5 μm) with the mobile phase consisting of 40 mM ammonium acetate–methanol–acetonitrile. A single-quadrupole mass spectrometer with an electrospray interface was operated in the selected-ion monitoring mode to detect the [M+H] + ions at m/ z 300 for metoclopramide and at m/ z 384 for the internal standard (prazosin). The method was validated over 0.78–50.00 ng mL −1 for metoclopramide. The recovery was 67.8–83.1%, and the limit of quantitation (LOQ) detection was 0.78 ng mL −1 for metoclopramide. The intra- and inter-day precision of the method at three concentrations was 5.0–13.6% with accuracy of 99.2–104.0%. Stability of compounds was established in a battery of stability studies. The method was successfully applied to bioequivalence studies of metoclopramide hydrochloride tablets to obtain the pharmacokinetic parameters.

Quantitative determination of helicid in rat biosamples by liquid chromatography electrospray ionization mass spectrometry

A simple liquid chromatography electrospray ionization mass spectrometry (LC–ESI–MS) method with highly improved sensitivities for the determination of helicid in rat bile, urine, feces and most tissues was developed. The tissues and feces were firstly homogenized mechanically using deionized water as the media. Bile, urine, tissues and feces homogenates were extracted by liquid–liquid extraction with n-butyl alcohol for sample preparation. The subsequent analysis procedures were performed on a Shimadzu LCMS2010A system (electrospray ionization single quadrupole mass analyzer). A Luna C 18 column (150 mm × 2.00 mm, 5 μm) was used as the analytical column, while a mixture of acetonitrile and ammonium chloride water solution was used as the mobile phase. The proportions of mobile phase were changed timely according to gradient programs. Chlorinated adducts of molecular ions [M+Cl] − at m/ z 319.00 and 363.05 were used to quantify helicid and bergeninum (internal standard), respectively. The method was validated to be accurate, precise and rugged with good linearity. The proposed method was successfully applied to the preclinical tissue distribution and excretion studies of helicid in rats.

Genetic transformation of Harpagophytum procumbens by Agrobacterium rhizogenes: iridoid and phenylethanoid glycoside accumulation in hairy root cultures

A genetic transformation method using Agrobacterium rhizogenes was developed for Harpagophytum procumbens. The influence of three factors on hairy root formation was tested: bacterial strains (A4 and ATCC 15834), various types of explants and acetosyringone (AS) (200 μM). The highest frequency of transformation (over 50% of explants forming roots at the infected sites after 6 weeks of culture on Lloyd and McCown (WP) medium) was achieved using a combination of nodal stem explants and A. rhizogenes strain A4. The addition of 200 μM AS to root induction medium was found to enhance hairy root induction but its effect varied depending on bacterial strain and explant type. Three of the most vigorously growing hairy root clones of H. procumbens were chosen and analyzed for accumulation of iridoid and phenylethanoid glycosides. The transgenic nature of these root clones was confirmed by PCR amplification; they were positive for rolB and rolC genes. Harpagoside, verbascoside and isoverbascoside were identified by HPLC and LC–ESI-MS as the major compounds from all analyzed hairy root clones. The Hp-3 root clone showed the higher harpagoside content (0.32 mg g−1 dry wt.) compared with other analyzed transformed and non-tuberized untransformed roots of H. procumbens. However, the level of the compound in the hairy root clone was lower than that detected in a sample of commercially available root tubers of H. procumbens. The Hp-3 root clone also produced high amounts of verbascoside and isoverbascoside (8.12 mg g−1 dry wt. and 9.97 mg g−1 dry wt., respectively) comparable to those found in root tubers.