A simultaneous determination method for 5‐fluorouracil and its metabolites in human plasma with linear range adjusted by in‐source collision‐induced dissociation using hydrophilic interaction liquid chromatography–electrospray ionization–tandem mass spectrometry

We applied a new technique for quantitative linear range shift using in‐source collision‐induced dissociation (CID) to complex biological fluids to demonstrate its utility. The technique was used in a simultaneous quantitative determination method of 5‐fluorouracil (5‐FU), an anticancer drug for various solid tumors, and its metabolites in human plasma by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC/ESI‐MS/MS). To control adverse effects after administration of 5‐FU, it is important to monitor the plasma concentration of 5‐FU and its metabolites; however, no simultaneous determination method has yet been reported because of vastly different physical and chemical properties of compounds. We developed a new analytical method for simultaneously determining 5‐FU and its metabolites in human plasma by LC/ESI‐MS/MS coupled with the technique for quantitative linear range shift using in‐source CID. Hydrophilic interaction liquid chromatography using a stationary phase with zwitterionic functional groups, phosphorylcholine, was suitable for separation of 5‐FU from its nucleoside and interfering endogenous materials. The addition of glycerin into acetonitrile‐rich eluent after LC separation improved the ESI‐MS response of high polar analytes. Based on the validation results, linear range shifts by in‐source CID is the reliable technique even with complex biological samples such as plasma. Copyright © 2016 John Wiley & Sons Ltd.     

Automatic analyzer for highly polar carboxylic acids based on fluorescence derivatization–liquid chromatography

A sensitive, versatile, and reproducible automatic analyzer for highly polar carboxylic acids based on a fluorescence derivatization–liquid chromatography (LC) method was developed. In this method, carboxylic acids were automatically and fluorescently derivatized with 4‐(N,N‐dimethylaminosulfonyl)‐7‐piperazino‐2,1,3‐benzoxadiazole (DBD‐PZ) in the presence of 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholinium chloride by adopting a pretreatment program installed in an LC autosampler. All of the DBD‐PZ‐carboxylic acid derivatives were separated on the ODS column within 30 min by gradient elution. The peak of DBD‐PZ did not interfere with the separation and the quantification of all the acids with the exception of lactic acid. From the LC‐MS/MS analysis, we confirmed that lactic acid was converted to an oxytriazinyl derivative, which was further modified with a dimethoxy triazine group of 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholinium chloride (DMT‐MM). We detected this oxytriazinyl derivative to quantify lactic acid. The detection limits (signal‐to‐noise ratio = 3) for the examined acids ranged from 0.19 to 1.1 µm , which correspond to 95–550 fmol per injection. The intra‐ and inter‐day precisions of typical, highly polar carboxylic acids were all <9.0%. The developed method was successfully applied to the comprehensive analysis of carboxylic acids in various samples, which included fruit juices, red wine and media from cultured tumor cells. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of a hydrophilic interaction ultra‐high‐performance liquid chromatography with triple quadrupole MS/MS for the absolute and relative quantification of amino acids in Sophora alopecuroides L.†

Amino acids are naturally occurring compounds in many edible or medicinal plants, which possess a variety of pharmacological effects on humans. The aim of this study is to develop and validate a hydrophilic interaction LC coupled with MS/MS method for the absolute and relative quantification of amino acids without derivatization. The application of this method has been proven through 20 naturally occurring amino acids in 21 samples from different parts and phenological growth stage of Sophora alopecuroides. The method was performed on an ultra‐high performance LC separation system coupled with ESI‐MS on a triple quadrupole mass spectrometer. The proposed absolute quantitative method was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery. The analysis results showed that S. alopecuroides is rich in free amino acids. In addition, relative quantitative determination of amino acids with several amino acids selected for the best accuracy was investigated. The accuracies of relative quantitative method for amino acids determinations suggest that it is feasible to quantify amino acids by the proposed relative quantitative determination method, which contributes to breaking through the choke point of lack of standards.     

Characterization of lemon (Citrus limon) polar extract by liquid chromatography–tandem mass spectrometry in high resolution mode

Eighty four metabolites (32 flavonoids, 15 amino acids, nine carboxylic acids, six coumarins, six sugars, five phenolic acids and 11 unclassified compounds) have been tentatively identified in a polar extract from lemon, without reference standards, based on their liquid chromatography–quadrupole‐time‐of‐flight MS/MS spectra and the comparison with databases. Despite information in databases for some families of plant compounds is poor, tentative identification based on MS/MS information (mass of the precursor ion and their fragments, together with neutral mass loss) was possible with the help of known fragmentation patterns for the given families of compounds. Both positive and negative ionization modes and at least two collision energies were always applied to obtain as much information as possible from each molecular entity, thus helping for identification. As the tentatively identified metabolites are the same regardless of the organism they belong, their fragmentation patterns are useful for identification with independence of the sample nature. Copyright © 2015 John Wiley & Sons, Ltd.     

Material basis studies of anti‐Influenza A active ingredients in Tanreqing Injection

Tanreqing Injection (TRQ) has been used primarily in treating infections of the upper respiratory tract and serious influenza in China, as a classical compound herbal recipe. TRQ had been demonstrated to have effects of clearing heat, eliminating phlegm, detoxification, reducing inflammation and alleviating cough. The survival rate, histopathology of lungs and viral titers in mice were evaluated in this study to verify the curative effect of TRQ. However, there is not enough information about the components. In the present study, a high‐performance and practical LC/QTOF/MS method was developed for characterization and identification of the natural ingredients in TRQ. A total of 60 compounds, including 10 amino acids, 10 iridoid glucosides, 14 flavonoids, 13 other phenolic compounds, 10 steroid acids and three other compounds, were characterized and identified. We also confirmed the material basis of anti‐Influenza A active ingredients in TRQ. Therefore, we have developed an accurate analytical method. LC/QTOF/MS could be applied for identification the complex components in traditional Chinese medicine.     

ESI‐MS/MS analysis of protonated N‐methyl amino acids and their immonium ions

Methylation is one of the important posttranslational modifications of biological systems. At the metabolite level, the methylation process is expected to convert bioactive compounds such as amino acids, fatty acids, lipids, sugars, and other organic acids into their methylated forms. A few of the methylated amino acids are identified and have been proved as potential biomarkers for several metabolic disorders by using mass spectrometry–based metabolomics workstation. As it is possible to encounter all the N‐methyl forms of the proteinogenic amino acids in plant/biological systems, it is essential to have analytical data of all N‐methyl amino acids for their detection and identification. In earlier studies, we have reported the ESI‐MS/MS data of all methylated proteinogenic amino acids, except that of mono‐N‐methyl amino acids. In this study, the N‐methyl amino acids of all the amino acids ( 1 ‐ 21 ; including one isomeric pair) were synthesized and characterized by ESI‐MS/MS, LC/MS/MS, and HRMS. These data could be useful for detection and identification of N‐methyl amino acids in biological systems for future metabolomics studies. The MS/MS spectra of [M + H]⁺ ions of most N‐methyl amino acids showed respective immonium ions by the loss of (H₂O, CO). The other most common product ions detected were [MH‐(NH₂CH₃]⁺, [MH‐(RH)]⁺ (where R = side chain group) ions, and the selective structure indicative product ions due to side chain and N‐methyl group. The isomeric/isobaric N‐methyl amino acids could easily be differentiated by their distinct MS/MS spectra. Further, the MS/MS of immonium ions inferred side chain structure and methyl group on α‐nitrogen of the N‐methyl amino acids.     

Matrix‐less laser desorption/ionisation mass spectrometry of polyphenols in red wine

Matrix‐assisted laser desorption/ionisation (MALDI) of small molecules is challenging and in most cases impossible due to interferences from matrix ions precluding analysis of molecules <300–500 Da. A common matrix such as ferulic acid belongs to an important class of compounds associated with antioxidant activity. If the shared phenolic structure is related to the propensity as an active MALDI matrix then it follows that direct laser desorption/ionisation should be possible for polyphenols. Indeed matrix‐less laser desorption/ionisation mass spectrometry is achieved whereby the analyte functions as a matrix and was used to monitor low molecular weight compounds in wine samples. Sensitivity ranging from 0.12–87 pmol/spot was achieved for eight phenolic acids (4‐coumaric, 4‐hydroxybenzoic, caffeic, ferulic, gallic, protocatechuic, syringic, vanillic) and 0.02 pmol/spot for trans‐resveratrol. Additionally, 4‐coumaric, 4‐hydroxybenzoic, caffeic, ferulic, gallic, syringic, vanillic acids and trans‐resveratrol were identified in wine samples using accurate mass measurements consistent with reported profiles based on liquid chromatography (LC)/MS. Minimal sample pre‐treatment make the technique potentially appropriate for fingerprinting, screening and quality control of wine samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Use of high‐pH (basic/alkaline) mobile phases for LC–MS or LC–MS/MS bioanalysis

High‐pH or basic/alkaline mobile phases are not commonly used in LC–MS or LC–MS/MS bioanalysis because of the deeply rooted concern with column instability and reduced detection sensitivity for basic compounds in high‐pH mobile phases owing to charge neutralization. With the advancement of LC column technology and the wide recognition of the “wrong‐way‐round” phenomena, high‐pH mobile phases are more and more used in LC–MS or LC–MS/MS bioanalysis to improve chromatographic peak shape, retention, selectivity, resolution, and detection sensitivity, not only for basic compounds, but also for many other compounds. In this article, the benefits, practical considerations, application examples and cautions for using high‐pH mobile phases in LC–MS or LC–MS/MS bioanalysis are reviewed, with a focus on quantification. Furthermore, the future trends in this field are also envisaged. A total of 84 references are cited in this review.     

Hydrophilic interaction chromatography/tandem mass spectrometry for the simultaneous determination of three polar non‐structurally related compounds,imipenem, cilastatin and an investigational β‐lactamase inhibitor,MK‐4698, in biological matrices

A method coupling hydrophilic interaction chromatography (HILIC) with tandem mass spectrometry (MS/MS) has been developed for the simultaneous determination of three polar non‐structurally related compounds – a carbapenem antibiotic, imipenem (IMP), a renal dehydropeptidase inhibitor, cilastatin (CIL), and an investigational β‐lactamase inhibitor, MK‐4698 (BLI), in rat plasma, monkey plasma and mouse blood. The analytes were extracted through protein precipitation, chromatographed on a Waters Atlantis HILIC column, and detected on a Sciex API4000 mass spectrometer using a Turbo‐Ion Spray ion source in positive ionization mode following multiple‐reaction monitoring (MRM). The assay dynamic range was 0.1–100 µg/mL for IMP, CIL and BLI, respectively, using a total of 20–25 µL biologic samples, and the total HPLC/MS/MS run time was 4 min/injection. The assay was found to be sensitive, selective and reproducible. The challenges, namely, sample stability, blood sample processing, matrix effect in monkey study samples, and dilution re‐assays for the limited mouse blood samples, are resolved and discussed. This technique allowed rapid analysis of polar compounds in biologic matrixes with satisfactory chromatographic retention and increased throughput. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of β‐blockers in groundwater using large‐volume injection coupled‐column reversed‐phase liquid chromatography with fluorescence detection and liquid chromatography time‐of‐flight mass spectrometry

Atenolol, nadolol, metoprolol, bisoprolol and betaxolol were simultaneously determined in groundwater samples by large‐volume injection coupled‐column reversed‐phase liquid chromatography with fluorescence detection (LVI‐LC‐LC‐FD) and liquid chromatography‐time‐of‐flight mass spectrometry (LC‐TOF‐MS). The LVI‐LC‐LC‐FD method combines analyte isolation, preconcentration and determination into a single step. Significant reductions in costs for sample pre‐treatment (solvent and solid phases for clean up) and method development times are also achieved. Using LC‐TOF‐MS, accurate mass measurements within 3 ppm error were obtained for all of the β‐blockers studied. Empirical formula information can be obtained by this method, allowing the unequivocal identification of the target compounds in the samples. To increase the sensitivity, a solid‐phase extraction step with Oasis MCX cartridge was carried out yielding recoveries of 79–114% (n=5) with RSD 2–7% for the LC‐TOF‐MS method. SPE gives a high purification of β‐blockers compared with the existing methods. A 100% methanol wash was allowed for these compounds with no loss of analytes. Limit of quantification was 1–7 ng/L for LVI‐LC‐LC‐FD and 0.25–5 ng/L for LC‐TOF‐MS. As a result of selective extraction and effective removal of coextractives, no matrix effect was observed in LVI‐LC‐LC‐FD and LC‐TOF‐MS analyses. The methods were applied to detect and quantify β‐blockers in groundwater samples of Almería (Spain).     

Butelase‐Mediated Macrocyclization of d‐Amino‐Acid‐Containing Peptides

Macrocyclic compounds have received increasing attention in recent years. With their large surface area, they hold promise for inhibiting protein–protein interactions, a chemical space that was thought to be undruggable. Although many chemical methods have been developed for peptide macrocyclization, enzymatic methods have emerged as a promising new economical approach. Thus far, most enzymes have been shown to act on l ‐peptides; their ability to cyclize d ‐amino‐acid‐containing peptides has rarely been documented. Herein we show that macrocycles consisting of d ‐amino acids, except for the Asn residue at the ligating site, were efficiently synthesized by butelase 1, an Asn/Asp‐specific ligase. Furthermore, by using a peptide‐library approach, we show that butelase 1 tolerates most of the d ‐amino acid residues at the P1′′ and P2′′ positions.     

Electrospray ionization mass spectrometric detection of low polar compounds by adding NaAuCl4

Liquid chromatography electrospray ionization mass spectrometry (LC/ESI/MS) has been widely used for various analyses. However, it is difficult to use LC/ESI/MS for the analysis of low polar compounds, such as polycyclic aromatic hydrocarbons. It is well known that AuCl₄^? ion decomposes to AuCl₃ by heating, and AuCl₃ is a strong π‐electrophilic Lewis acid. Low polar compounds (pyrene, benzo[a]pyrene, perylene, benzo[ghi]perylene, dibenzothiophene and p‐dimethoxybenzene) were detected by ESI/MS in the positive ion mode by adding NaAuCl₄. The low polar compound interacts with AuCl₃ formed at the ESI interface, and undergoes electron transfer to AuCl₃. The radical cation of the low polar compound was then detected by MS. In addition, the LC/ESI/MS determination of polycyclic aromatic hydrocarbons by the post‐column addition of NaAuCl₄ was studied. © 2016 The Authors Journal of Mass Spectrometry Published by John Wiley & Sons Ltd     

Quantification of glycated N‐terminal peptide of hemoglobin using derivatization for multiple functional groups of amino acids followed by liquid chromatography/tandem mass spectrometry

A novel method of amino acid analysis using derivatization of multiple functional groups (amino, carboxyl, and phenolic hydroxyl groups) was applied to measure glycated amino acids in order to quantify glycated peptides and evaluate the degree of glycation of peptide. Amino and carboxyl groups of amino acids were derivatized with 1‐bromobutane so that the hydrophobicities and basicities of the amino acids, including glycated amino acids, were improved. These derivatized amino acids could be detected with high sensitivity using LC‐MS/MS. In this study, 1‐deoxyfructosyl‐VHLTPE and VHLTPE, which are N‐terminal peptides of the β‐chains of hemoglobin, were selected as target compounds. After reducing the peptide sample solution with sodium borohydride, the obtained peptides were hydrolyzed with hydrochloric acid. The released amino acids were then derivatized with 1‐bromobutane and analyzed with LC‐MS/MS. The derivatized amino acids, including glycated amino acids, could be separated using an octadecyl silylated silica column and good sharp peaks were detected. We show a confirmatory experiment that the proposed method can be applied to evaluate the degree of glycation of peptides, using mixtures of glycated and non‐glycated peptide. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis of new racemic and optically active N‐phosphonoalkyl bicyclic β‐amino acids via the kabachnik–fields reaction as potential biologically active compounds

Novel racemic and optically active constrained N‐phosphonoalkyl bicyclic β‐amino acids have been synthesized via the Kabachnik–Fields reaction of the (RS) or (R)‐1‐aminobicyclo[2.2.2]octane‐2‐carboxylic acids with paraformaldehyde and benzaldehyde, and the dimethyl‐H‐phosphonate. The structure of obtained compounds was characterized by NMR (¹H, ¹³C, and ³¹P), LC/MS, and X‐ray diffraction analyses. © 2012 Wiley Periodicals, Inc. Heteroatom Chem 23:123–130, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/hc.20759     

Potential of Flame Ionization Detection Coupled On‐Line with Microcolumn Liquid Chromatography Using Aqueous Eluents and an Eluent‐Jet Interface

The potential of the on‐line coupling of microcolumn liquid chromatography (μLC) using aqueous eluents with a flame ionization detector (FID) was evaluated. An eluent‐jet interface was modified to allow the efficient introduction of the eluent into the FID. The potential of the method is demonstrated by the μLC–FID determination of lower alcohols and bis(2‐hydroxyethylthio)alkanes on porous and non‐porous stationary phases, respectively. Flow injection analysis (FIA)–FID experiments with highly polar, thermolabile, semi‐volatile and non‐volatile compounds like amino acids, organic acids, alkylphosphonic acids, and carbohydrates showed the developed configuration to be a promising approach for the detection of a wide range of analytes. Compared with a nebulization interface, the eluent‐jet interface showed 4–10 times higher peaks for citric acid. Detection limits by FIA for all compounds were in the range of 0.2–5 ng injected. With ribose as test compound, plots of peak height vs. amount injected showed good linearity (r² > 0.999) in the range of 75–12,000 μg/mL. The repeatability showed relative standard deviations of less than 5%.     

Structural elucidation of amino amide‐type local anesthetic drugs and their main metabolites in urine by LC‐MS after derivatization and its application for differentiation between positional isomers of prilocaine

The demand for clinical toxicology analytical methods for identifying drugs of abuse and medicinal drugs is steadily increasing. Structural elucidation of amino amide‐type local anesthetic drugs and their main metabolites by GC‐EI‐MS and LC‐ESI‐MS/MS is of great analytical challenge. These compounds exhibit only/mostly fragments/product ions representing the amine‐containing residue, while the aromatic amide moiety remains unidentified. This task becomes even more complicated when discrimination between positional isomers of such compounds is required. Here, we report the development of a derivatization procedure for the differentiation and structural elucidation of a mixture of local anesthetic drugs and their metabolites that possess tertiary and secondary amines in water and urine. A method based on two sequential “in‐vial” instantaneous derivatization processes at ambient temperature followed by LC‐ESI‐MS/MS analysis was developed. 2,2,2‐Trichloro‐1,1‐dimethylethyl chloroformate (TCDMECF) was utilized to selectively convert the secondary amines into their carbamate derivatives, followed by hydrogen peroxide addition to produce the corresponding tertiary amine oxides. The resulting derivatives exhibited rich fragmentation patterns, enabling improved structural elucidation of the original compounds. The developed method was successfully applied to the differentiation and structural elucidation of prilocaine and its four positional isomers, which all possess similar GC and LC retention times and four of them exhibit almost identical EI‐MS and ESI‐MS/MS spectra, enabling their structural elucidation in a single LC‐ESI‐MS/MS analysis. The developed technique is fast and simple and enables discrimination between isomers based on different diagnostic ions/fragmentation patterns.     

Multi‐mycotoxin analysis in complex biological matrices using LC‐ESI/MS: Experimental study using triple stage quadrupole and LTQ‐Orbitrap

In the present study, we report the application of LC‐MS based on two different LC‐MS systems to mycotoxin analysis. The mycotoxins were extracted with an ACN/water/acetic acid mixture and directly injected into a LC‐MS/MS system without any dilution procedure. First, a sensitive and reliable HPLC‐ESI‐MS/MS method using selected reaction monitoring on a triple quadrupole mass spectrometer (TSQ Quantum Ultra AM) has been developed for determining 32 mycotoxins in crude extracts of wheat and maize. This method was operated both in positive and in negative ionization modes in two separate chromatographic runs. The method was validated by studies of spiked recoveries, linearity, matrix effect, intra‐assay precision and sensitivity. Further, we have developed and evaluated a method based on accurate mass measurements of extracted target ions in full scan mode using micro‐LC‐LTQ‐Orbitrap as a tool for fast quantitative analysis. Both instruments exhibited very high sensitivity and repeatability in positive ionization mode. Coupling of micro‐LC to Orbitrap technology was not applicable to the negatively ionizable compounds. The LC triple quadrupole MS method has proved to be stable in quantitation, as it is with respect to the matrix effects of grain samples.     

Serial coupling of reversed‐phase and zwitterionic hydrophilic interaction LC/MS for the analysis of polar and nonpolar phenols in wine

In the present study, an easy and efficient method based on the serial coupling of analytical reversed‐phase and zwitterionic hydrophilic interaction liquid chromatography was developed for the simultaneous separation of polar and nonpolar phenols occurring in wine. The zwitterionic hydrophilic column was connected in series to the reversed‐phase one via a T‐piece, with which the ACN content in eluent of the second dimension was increased, in order to cope the solvent strength incompatibility between the two columns. The final mobile phase at low‐flow rate (≤0.5 mL/min), high‐ACN content (90%), and low‐salt concentration was directed to an ESI‐TOF‐MS , for high accurate mass detections. The developed method was applied for the identification of target phenols in several wines. Retention time and peak width intra‐ and interday repeatability studies proved the reliability of the method for the simultaneous analysis of all the polar and nonpolar analytes in wine. The serial reversed‐phase/zwitterionic hydrophilic interaction liquid chromatography coupling offered the possibility to enlarge the number of identified compounds and it represents a valid approach for nontarget analysis of complex samples by a single injection.     

HILIC LC‐MS for the determination of 2′‐C‐methyl‐cytidine‐triphosphate in rat liver

A very accurate and selective LC‐MS/MS method was developed and validated for the quantification of 2′‐C‐modified nucleoside triphosphate in liver tissue samples. An efficient pretreatment procedure of liver tissue samples was developed, using a fully automated SPE procedure with 96‐well SPE plate (weak anion exchange sorbent, 30 mg). Nucleotide hydrophilic interaction chromatography has been performed on an aminopropyl column (100 mm×2.0 mm, 3 μm) using a gradient mixture of ACN and ACN/water (5:95 v/v) with 20 mM ammonium acetate at pH 9.45 as mobile phase at 300 μL/min flow rate. The 2′‐C‐modified nucleoside triphosphate was detected in the negative ESI mode in multiple reaction monitoring (MRM) mode. Calibration curve was linear over the 0.05–50 μM concentration range. Satisfying results, confirming the high reliability of the established LC‐MS/MS method, were obtained for intraday precision (CV = 2.5–9.1%) and accuracy (92.6–94.8%) and interday precision (CV = 9.6–11.5%) and accuracy (94.4–102.4%) as well as for recovery (82.0–112.6%) and selectivity. The method has been successfully applied for pharmacokinetic studies of 2′‐C‐methyl‐cytidine‐triphosphate in liver tissue samples.     

Towards the Optimization of Microwave-Assisted Extraction and the Assessment of Chemical Profile,Antioxidant and Antimicrobial Activity of Wine Lees Extracts

Wine lees, a sub-exploited byproduct of vinification, is considered a rich source of bioactive compounds, such as (poly)phenols, anthocyanins and tannins. Thus, the effective and rapid recovery of these biomolecules and the assessment of the bioactive properties of wine lees extracts is of utmost importance. Towards this direction, microwave-assisted extraction (MAE) factors (i.e., extraction time, microwave power and solvent/material ratio) were optimized using experimental design models in order to maximize the (poly)phenolic yield of the extracts. After optimizing the MAE process, the total phenolic content (TPC) as well as the antiradical, antioxidant and antimicrobial activity of the extracts were evaluated. Furthermore, Fourier transform infrared spectroscopy (FTIR) was employed to investigate the chemical profile of wine lees extracts. Red varieties exhibited higher biological activity than white varieties. The geographical origin and fermentation stage were also considered as critical factors. The white variety Moschofilero presented the highest antioxidant, antiradical and antimicrobial activity, while Merlot and Agiorgitiko samples showed noteworthy activities among red varieties. Moreover, IR spectra confirmed the presence of sugars, amino acids, organic acids and aromatic compounds. Thus, an efficient, rapid and eco-friendly process was proposed for further valorization of wine lees extracts.