A novel method for the quality control of saffron through the simultaneous analysis of authenticity and adulteration markers by liquid chromatography-(quadrupole-time of flight)-mass spectrometry.

A liquid chromatography-(quadrupole-time of flight)-mass spectrometry methodology was developed to assess the authenticity of saffron through the analysis of a group of kaempferol derivatives recently proposed as novel authenticity markers as a result of a metabolomic study of saffron (kaempferol 3-O-glucoside, kaempferol 3-O-sophoroside, kaempferol 3,7-O-diglucoside, kaempferol 3,7,4'-O-triglucoside, kaempferol 3-O-sophoroside-7-O-glucoside). Geniposide was also studied as an adulteration marker of saffron with gardenia. The optimized chromatographic conditions enabling the simultaneous separation of glycosylated kaempferols and geniposide consisted of the use of a C18 column and an elution gradient with acetonitrile and water as mobile phases (both with formic acid at 0.1%). A strategy was proposed to evaluate the minimum quantifiable adulteration percentage which was established at a 0.2% regardless of the adulterant employed. The analysis of nineteen commercial samples showed the method to be specific and suitable for saffron quality control.

Metabolomic fingerprinting of saffron by LC/MS: novel authenticity markers.

An untargeted metabolomic approach using liquid chromatography coupled to electrospray ionization time-of-flight mass spectrometry was developed in this work to identify novel markers for saffron authenticity which is an important matter related to consumer protection, quality assurance, active properties, and also economical impact (saffron is the most expensive spice). Metabolic fingerprinting of authentic and suspicious saffron samples from different geographical origin was obtained and analyzed. Different extracting protocols and chromatographic methodologies were evaluated to obtain the most adequate extracting and separation conditions. Using an ethanol/water mixture at pH 9.0 and an elution gradient with a fused core C18 column enabled obtaining the highest number of significant components between authentic and adulterated saffron. By using multivariate statistical analysis, predictive classification models for authenticity and geographical origin were obtained. Moreover, 84 and 29 significant metabolites were detected as candidates for markers of authenticity and geographical origin, respectively, from which only 34 metabolites were tentatively identified as authenticity markers of saffron, but none related to its geographical origin. Six characteristic compounds of saffron (kaempferol 3-O-glucoside, kaempferol 3-O-sophoroside, kaempferol 3,7-O-diglucoside, kaempferol 3,7,4'-O-triglucoside, kaempferol 3-O-sophoroside-7-O-glucoside, and geranyl-O-glucoside) were confirmed by comparing experimental MS/MS fragmentation patterns with those provided in scientific literature being proposed as novel markers of authenticity. Graphical Abstract Metabolomic fingerprinting of saffron.

Off-line two dimensional isoelectrofocusing-liquid chromatography/mass spectrometry (time of flight) for the determination of the bioactive peptide lunasin.

Progress in liquid chromatography and mass spectrometry technologies offers a great opportunity for the determination of bioactive peptides. Nevertheless, in many cases, the direct application of this technology does not enable the detection of the investigated peptides due to serious signal suppression. This is the case of lunasin, a cancer preventive, anti-inflammatory, and cholesterol-reducing peptide originally isolated from soybean and later found in some cereals. Most methods applied for the quantitation of this peptide were immunological and based on the detection of just a fragment of the lunasin sequence. At this regard, there is a peptide commercially available with a sequence similar to lunasin but differing in just one amino acid that has been wrongly used for the quantitation of lunasin. The use of high resolution mass spectrometry has enabled to be aware of this issue and of the need for new methods enabling the reliable identification and determination of lunasin. However, when different approaches were evaluated in this work for the reduction of the interferences originating signal suppression, such as matrix dilution, previous lunasin purification by reversed-phase or ion-exchange solid-phase extraction, and use of different chromatographic columns, no one resulted successful in the case of soybean. Just a one-dimensional separation of the soybean extract by isoelectrofocusing followed by a second dimension separation by reversed-phase liquid chromatography enabled a significant reduction of matrix interferences and the detection of lunasin in soybean products by high resolution mass spectrometry with a time of flight (TOF) analyzer. After method optimization, selectivity, linearity, accuracy, precision, and limits of detection and quantitation were evaluated, being possible to quantitate as low as 25ng/mL (1.5µg lunasin/g protein). Concentration of lunasin in the analyzed soybean flour and textured soybean ranged from 14.0 to 22.5mg lunasin/g protein.

New approaches in sensitive chiral CE.

CE has shown to have a big potential for chiral separations, with advantages such as high efficiency, high resolution, and low sample and reagents consumption. Nevertheless, when UV detection is employed, CE has some drawbacks, especially the low sensitivity obtained due to the short optical path length. Notwithstanding, sensitivity improvements can be achieved when different approaches are employed, such as sample treatment strategies (off-line or on-line), in-capillary sample preconcentration techniques, and/or alternative detection systems to UV-Vis (such as fluorescence, conductimetry, electrochemiluminiscence, MS, etc.). This article reviews the most recent methodological and instrumental advances reported from June 2011 to May 2013 for enhancing the sensitivity in chiral analysis by CE. The sensitivity achieved for the enantioseparated analytes and the applications carried out using the developed methodologies are also summarized.

LC-ESI-TOF MS method for the evaluation of the immunostimulating activity of soybeans via the determination of the functional peptide soymetide.

Bioactive peptides content in foodstuffs can seriously vary with many factors such as crop variety, food processing, animal breeding, etc. Because of this variability, quantitative methodologies are required to evaluate the content of bioactive peptides in foodstuffs. Progress in liquid chromatography and mass spectrometry technologies offer a great opportunity for the quantitation of bioactive peptides. This study undertook the development of a liquid chromatography-electrospray ionization-time-of-flight mass spectrometry method using a fused-core technology column for the sensitive and unambiguous determination of the immunostimulating peptide soymetide in soybean varieties. Soymetide precursor protein (α' subunit of ß-conglycinin) was extracted with a Tris-HCl buffer (pH 8.0) containing urea and digested with trypsin. Soymetide separation conditions by reversed phase liquid chromatography (ion-pairing reagent, organic modifier, separation column, and elution gradient) and detection by MS were optimized, and a study of soymetide stability was also conducted. The method selectivity having been demonstrated, the linearity, accuracy, precision, and limits of detection and quantitation were evaluated. The developed method enabled the detection and quantitation of soymetide concentrations in the ppb range (7.5 ng/mL and 25 ng/mL, respectively), and about 30 times lower than those detected and determined in a previous work by capillary liquid chromatography with UV detection. These values could allow the quantitation of only 17 µg of soymetide per gram of soybean. The developed methodology was applied to the quantitation of soymetide in different soybean varieties from Europe, Japan, and USA observing great differences in soymetide content that ranged from 40 to 600 µg per gram of soybean depending on the soybean variety.

Molecularly imprinted SPE and MEKC with in-capillary sample preconcentration for the determination of digoxin in human urine.

Molecularly imprinted solid-phase extraction (MISPE) combined with MEKC was used for clean-up, preconcentration and determination of digoxin in the presence of its aglycon digoxin (digoxigenin) in human urine samples. In addition, the use of an in-capillary sample concentration electrophoretic technique by sweeping was investigated to enhance the concentration sensitivity in MEKC. The highly selective, fast and effective sample pretreatment by MISPE along with the preconcentration by sweeping could overcome the low sensitivity of the highly efficient capillary electrophoresis separation with UV detection. The optimization of the variables affecting the separation as well as MISPE conditions procedure was carried out to select the best conditions of selectivity and sensitivity to determine digoxin at low concentration levels in urine. To demonstrate the suitability of the developed method several analytical characteristics (selectivity, linearity, accuracy, precision, and LOD) were evaluated. Satisfactory results were obtained in terms of linearity (r > 0.99), recovery (95.4-96.5% with RSD from 1.3% to 2.6%), precision (RSD from 0.3% to 1.7% for migration times and from 2.1% to 7.3% for corrected peak areas), and sensitivity (LODs of 6 µg/L with 5 mL of sample or 1.2 µg/L with 25 mL). The proposed MISPE-MEKC method was satisfactorily applied to the analysis of spiked human urine samples achieving a concentration factor up to 7500-fold.