A single HPLC-PAD-APCI/MS method for the quantitative comparison of phenolic compounds found in leaf, stem, root and fruit extracts of Vaccinium angustifolium

A method was developed for the analysis of Vaccinium angustifolium Ait. (Lowbush blueberry), which is a widely used natural health product, particularly for the treatment of diabetic symptoms. While the anthocyanin content of the fruit has been well characterized, the chemistry of the vegetative parts used in supportive therapy for diabetes has been largely ignored. Using a metabolomics-based approach for compound identification with an emphasis on phenolic metabolites, a single HPLC-PAD-APCI/ MS method was developed for the separation and quantitation of the major metabolites found in the 95% ethanol extracts of leaf, stem, root and fruit. The leaf extract contained high concentrations of chlorogenic acid (approximately 100 microg/mg extract) and a variety of quercetin glycosides that were also detected in the fruit and stem extracts. Flavan-3-ol monomers (+)-catechin and (-)-epicatechin were found in all plant parts but their procyanidin dimers were exclusively identified in the stem and root. The accuracy and precision of the presented method were corroborated by low intra- and inter-day variations in quantitative results in all plant part extracts. Further validation of the extraction and analytical protocols focused on identified compounds with reputed anti-diabetic activity, revealing recoveries greater than 80% and detection limits of 0.12-2.73 microg/mL.     

Identification of ginsenosides in roots of Panax ginseng by HPLC-APCI/MS

A new HPLC-APCI/MS method for the identification of ginsenosides has been developed. The analyses were performed on a reversed-phase C18 column using a binary eluent (acetonitrile and water) under gradient conditions. Although APCI is a high-temperature evaporative process, HPLC-APCI/MS could effectively identify thermo-labile ginsenosides. The [M-H]- ions and the thermal degradation ions of ginsenosides could be clearly observed under negative and positive ion conditions, respectively, and these were used to identify the molecular masses, the aglycone structures and the sugar groups of ginsenosides. APCI/MS can provide more explicit information than ESI/MS for identifying and distinguishing ginsenosides. Using the HPLC-APCI/MS method, 35 ginsenosides were identified in Panax ginseng.     

Characterisation of the phenolic profile of Boerhaavia diffusa L. by HPLC-PAD-MS/MS as a tool for quality control

Phenolic acids and flavonols of nine leaf and three root samples of Boerhaavia diffusa L., collected at different locations and subjected to several drying procedures, were characterised by reversed-phase HPLC-PAD-ESI/MS for the first time. Ten phenolic compounds were identified: 3,4-dihydroxy-5-methoxycinnamoyl-rhamnoside, quercetin 3-O-rhamnosyl(1-->6)galactoside (quercetin 3-O-robinobioside), quercetin 3-O-(2"-rhamnosyl)-robinobioside, kaempferol 3-O-(2"-rhamnosyl)-robinobioside, 3,5,4'-trihydroxy-6,7-dimethoxyflavone 3-O-galactosyl(1-->2)glucoside [eupalitin 3-O-galactosyl(1-->2)glucoside], caffeoyltartaric acid, kaempferol 3-O-robinobioside, eupalitin 3-O-galactoside, quercetin and kaempferol. Quantification was achieved by HPLC-PAD and two phenolic patterns were found for the leaves, in which quercetin 3-O-robinobioside or quercetin 3-O-(2"-rhamnosyl)-robinobioside was the major compound. Caffeoyltartaric acid was only present in the root material where it represented the main phenolic constituent. The results obtained demonstrated that the geographical origin (particularly the nature of the soil), but not the drying process, influences the phenolic composition.     

Approach to the study of C-glycosyl flavones by ion trap HPLC-PAD-ESI/MS/MS: application to seeds of quince (Cydonia oblonga)

Ion trap HPLC-PAD-ESI/MS/MS has been used to study C-glycosyl flavones in quince seeds. Comparative analysis of the ions [(M-H)-60]-, [(M-H)-90]- and [(M-H)-120]- from 6-C- and 8-C-glycosyl flavone isomers, together with their respective retention times, allowed deductions to be made about the nature of the sugar units and the positions of C-glycosylation. Vicenin-2 (6,8-di-C-glucosyl apigenin), lucenin-2 (6,8-di-C-glucosyl luteolin), stellarin-2 (6,8-di-C-glucosyl chrysoeriol), isoschaftoside (6-C-arabinosyl-8-C-glucosyl apigenin), schaftoside (6-C-glucosyl-8-C-arabinosyl apigenin), 6-C-pentosyl-8-C-glucosyl chrysoeriol and 6-C-glucosyl-8-C-pentosyl chrysoeriol were identified in quince seed.     

High-performance liquid chromatography with atmospheric pressure chemical ionization and electrospray ionization mass spectrometry for analysis of Angelica sinensis

An HPLC-PAD-API/MS method for analysing the chemical constituents of Angelica sinensis (A. sinensis) has been developed. ESI and APCI spectra, in both positive ion (PI) and negative ion (NI) modes, provided very useful information concerning the molecular weights of detected compounds. By comparing the retention times, UV spectra, mass spectra and molecular weights of detected compounds with those published in literature, 15 constituents of A. sinensis could be tentatively identified. This technique involving combined MS information may provide an objective, reliable and rapid analytical method for the quality control and database research of traditional Chinese medicines.     

Qualitative and quantitative determination of yohimbine in authentic yohimbe bark and in commercial aphrodisiacs by HPLC-UV-API/ MS methods

The development and validation of a rapid qualitative and quantitative method based on an HPLC-UV-MS technique with atmospheric pressure chemical ionisation and electrospray ionisation for the analysis of yohimbine in a number of commercial aphrodisiac products is reported. HPLC with multiple-stage mass spectrometry experiments allowed the identification of the target compound and increased the selectivity of complex analyses such as those involved with multi-botanical preparations. The precision and the robustness of the method were improved by the use of two internal standards: codeine for UV detection and deuterium-labelled yohimbine for MS detection. Twenty commercial aphrodisiac preparations were analysed and the amount of yohimbine measured and expressed as the maximal dose per day suggested on product labels ranged from 1.32 to 23.16 mg.     

Comparison of hydrolysis and HPLC/MS/MS procedure with ELISA assay for the determination of S-phenylmercapturic acid as a biomarker of benzene exposure in human urine

The present study compared three methods for the determination of S-phenylmercapturic acid (S-PMA), a metabolite of benzene, in human urine: a HPLC/MS/MS technique with two different sample treatments (strong and partial hydrolysis) and a commercial assay based on anti-S-PMA monoclonal antibodies with chemiluminescence detection. Biological monitoring was done on 126 volunteers and the results were compared for the three methods and also with benzene exposure levels (range <3.0–592.5 μg/m³). The correlation between environmental monitoring data and S-PMA levels in non-smokers (n = 73) was highly significant (p < 0.0001, Student's t-test) for both HPLC/MS/MS methods (r = 0.65 both for strong acidic hydrolysis of the urine and hydrolysis at pH 2) but not for the immunoassay, which overestimated the S-PMA levels by about 8 μg/g creatinine (creat.). Therefore the immunoassay is only useful as a semiquantitative screening test, but quantitative results need to be confirmed by a more accurate method like HPLC/MS/MS. The HPLC/MS/MS procedure with strong acid hydrolysis led to a recovery of S-PMA about double that using pH 2 hydrolysis, giving more accurate results. The difference between the results with the two methods makes it difficult to compare the strong acidic hydrolysis data with the ACGIH BEI value of 25 μg/g creat. since the BEI^® documentation is based on data collected in pH conditions that were not always controlled, which may underestimate the true S-PMA concentration. Besides, as levels of benzene exposure were high, smoking was not considered a confounding factor. The BEI for S-PMA in end of shift urine samples could be reconsidered when sufficient data are available from studies where the analyses are carried out in comparable conditions of hydrolysis and monitoring only non-smoking subjects.     

Determination of oxcarbazepine and its metabolites in postmortem blood and hair by means of liquid chromatography with mass detection (HPLC/APCI/MS)

A typical use of hair analysis in forensic toxicology is the documentation of previous drug administration. This is illustrated in a suicidal death of a 58-year-old epileptic patient who was treated with oxcarbazepine and probably with levomepromazine. The toxicological analysis carried out by HPLC/APCI/MS included also the hair (6 cm length) besides postmortem blood. The method was validated for levomepromazine, oxcarbazepine (OXCBZ) and its two metabolites, 10-hydroxycarbazepine (CBZ-10OH) and trans-diol-carbazepine (CBZ-diOH) in various biological matrices. The analysis of the postmortem blood indicated oxcarbazepine and its two main metabolites were present at therapeutic concentrations; levomepromazine was detected at a fatal concentration. In three 2-cm segments of hair, oxcarbazepine and its two metabolites were detected; however, levomepromazine was not detected in this specimen. As a result of complex chemical-toxicological investigation it was confirmed the information that the decedent. was an epileptic patient and was treated with oxcarbazepine for at least 6 months before death. In addition, he took a toxic dose of levomepromazine in order to commit suicide. The analysis revealed differences between the concentration levels of oxcarbazepine and its active metabolite CBZ-10OH in postmortem specimens and hair, suggesting different mechanisms of penetration of metabolites and their precursors into this matrix.     

Identifying citrus limonoid aglycones by HPLC-EI/MS and HPLC-APCI/MS techniques

HPLC coupled with normal phase electron ionisation (EI) and atmospheric pressure chemical ionisation (APCI)/ mass spectrometry methods has been applied to identify 17 known neutral limonoid aglycones from Citrus sources. The HPLC-MS data from the known limonoids provided chromatographic characteristics, APCI-derived molecular weight data and EI fragmentation data for each limonoid. EI fragmentation patterns for the limonoids were correlated with structural characteristics. The EI fragmentation patterns coupled with APCI-derived molecular weights were utilised as a potential method by which to discern the structural character of unknown citrus limonoids.     

Biological monitoring of bisphenol A with HLPC/FLD and LC/MS/MS assays

Biological monitoring is a necessary process for risk assessment of endocrine disrupting chemicals (EDCs), particularly, bisphenol A (BPA), in breast milk, because its human risks are not clear yet, and infants, who feed on breast milk, are highly susceptible for EDCs. Concerning biological monitoring of BPA, the HPLC/FLD has been widely used before the LC/MS/MS. However, there was no report, which simultaneously evaluated the two methods in real analyses. Therefore, we analyzed BPA with LC/MS/MS and HPLC/FLD in human breast milk and conducted comparison of two methods in analyzed BPA levels. After establishing optimal condition, e.g. linearity, recovery, reproducibility and free BPA system, we analyzed BPA levels in human breast milk samples (N = 100). The LOQs were similar in the two methods, i.e. 1.8 and 1.3 ng/mL for the HPLC/FLD and LC/MS/MS assays, respectively. There were strong associations between total BPA levels with the two methods (R² = 0.40, p < 0.01), however, only 11% of them were analyzed as similar levels with 15% CVs. In addition, the detection range of BPA was broader in the HPLC method than the LC/MS/MS method. However, the BPA levels in the HPLC/FLD analysis were lower than those in the LC/MS/MS analysis (p < 0.01). Thus, the differences in BPA levels between the two methods may come from mainly over-estimation with the LC/MS/MS method in low BPA samples and some of poor resolution with the HPLC/FLD in high BPA samples.     

Hydrolysis of diethyl diferulates by a tannase from Aspergillus oryzae

Diferulic acid forms cross-links in naturally occurring plant cell wall polymers such as arabinoxylans and pectins. We have used model ethyl esterified substrates to find enzymes able to break these cross-links. A tannase from Aspergillus oryzae exhibited esterase activity on several synthetic ethyl esterified diferulates. The efficiency of this esterase activity on most diferulates is low compared to that of a cinnamoyl esterase, FAEA, from Aspergillus niger. Of the diferulate substrates assayed, tannase was most efficient at hydrolysing the first ester bond of the 5–5- type of dimer. Importantly and unlike the cinnamoyl esterase, tannase from A. oryzae is able to hydrolyse both ester bonds from the 8–5-benzofuran dimer, thus forming the corresponding free acid product. These results suggest that tannases may contribute to plant cell wall degradation by cleaving some of the cross-links existing between cell wall polymers.     

Analysis of macamides in samples of Maca (Lepidium meyenii) by HPLC-UV-MS/MS

The macamides are a distinct class of secondary metabolites that have so far been found only in Lepidium meyenii Walp. (Maca). Using HPLC-UV-MS/MS, the main macamides have been identified as n-benzylhexadecanamide, n-benzyl-(9Z)-octadecenamide, n-benzyl-(9Z, 12Z)-octadecadienamide, n-benzyl-(9Z, 12Z, 15Z)-octadecatrienamide and n-benzyloctadecanamide. The identities of n-benzyl-(9Z)-octadecenamide and n-benzyl-(9Z, 12Z)-octadecadienamide were confirmed by comparison of chromatographic and spectral properties with synthetic analogues. Total macamides have been quantified by HPLC-UV in plant material from different vendors using n-benzylhexadecanamide as an external standard. The amount of macamides in the dried plant material ranged from 0.0016 to 0.0123%.     

Isolation and on-line identification of antioxidant compounds from three Baccharis species by HPLC-UV-MS/MS with post-column derivatisation

The aqueous extracts of aerial parts of Baccharis trimera (Less.) DC., B. crispa Spreng. and B. usterii Heering (Asteraceae) displayed significant radical scavenging activity in a diphenylpicrylhydrazole (DPPH)/TLC assay. In order to rapidly identify the active principles, the crude extracts were analysed by HPLC-UV, and an HPLC-micro-fractionation of the extract of B. usterii was performed. Six quinic acids derivatives (1-6) were isolated from B. usterii by MPLC. The fractions were monitored by DPPH/TLC assay and a series of radical-scavenging quinic acid derivatives could be identified. The comparison of the HPLC profiles of the extracts of B. usterii, B. trimera and B. crispa was performed. In order to obtain complementary on-line structural information for all peaks of interest, HPLC-MS/MS together with HPLC-UV involving post-column addition of UV shift reagents was undertaken on the crude extract. The interpretation of these data permitted the on-line identification of known compounds, some of which are reported for the first time in this plant.     

Determination of saponins in the kernel cake of Balanites aegyptiaca by HPLC-ESI/MS

The kernel cake produced from Balanites aegyptiaca fruit of Israeli origin was analysed for its saponin constituents using high-performance liquid chromatography-mass spectrometry (HPLC-MS). The HPLC was equipped with a reversed-phase C18 column and a refractive index detector (RID), and elution was isocratic with methanol and water (70:30). The MS system was equipped with electrospray ionisation (ESI). Nine compounds were chromatographically separated, their masses were determined in the negative ion mode and subsequent fragmentation of each component was carried out. From the nine components, six saponins with molecular masses of 1196, 1064, 1210, 1224, 1078 and 1046 Da were identified, with the compound of mass 1210 Da being the main saponin (ca. 36%). Saponins with masses of 1224 and 1046 Da have not been previously reported in B. aegyptiaca. In all saponins, diosgenin was found to be the sole aglycone. This study shows that HPLC-ESI/MS is a quick and reliable technique for characterizing the saponins from kernel cake of B. aegyptiaca.     

Determination of deoxynivalenol in cereals by HPLC-UV

A simple method for determination of deoxynivalenol (DON) in cereal samples is described. DON was extracted with methanol, the solvent evaporated, and the residue redissolved with water. This extract was purified on immunoaffinity columns. DON was determined by HPLC with UV-detection. The limits of detection (LOD) and quantification (LOQ) were 10 and 50 μg/kg, respectively. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003