Peaks In Chromatogram Research Articles

Speculation of hemoglobin A3 peak position in clinical cation exchange high performance liquid chromatogram of the diabetic blood sample with microarray isoelectric focusing

血红蛋白A1c(HbA1c)是糖尿病诊断的关键生物标志物,目前其常用的分析方法为阳离子交换高效液相色谱法(CX-HPLC, 5/50 mm分离柱),此方法虽然具有稳定、快捷与自动化等众多优点,但临床CX-HPLC(VARIANT Ⅱ system)谱图中仍存在未知峰,尤其干扰HbA1c准确测定的谷胱甘肽化血红蛋白A3(HbA3)在色谱图中的相对位置仍不清楚。针对这一问题,该文以人新鲜血液为样本,首先利用低分辨CX-HPLC对血样进行分析,提示未知峰P3存在。然后通过微阵列等电聚焦(IEF)电泳和高分辨阳离子交换HPLC(Mono-S 5/50 mm分离柱)对血样的主要血红蛋白(Hb)成分进行分析,提示未知峰P3为HbA3峰。随后,通过血红蛋白谷胱甘肽化实验,利用HbA1c峰降低、HbA3峰明显增强这一信息,进一步推测未知峰P3即是HbA3峰,其在CX-HPLC中的保留时间在1.50 min左右。最后,结合前期研究讨论了体内应激情况下Hb谷胱甘肽化对HbA1c检测的影响。该研究丰富了CX-HPLC的未知峰P3的色谱信息,为更精准诊断糖尿病提供了有价值的参考。

Profile of TLC-densitometry Annona squamosa and Persea americana as Antihyperlipidemic Medicinal Raw Material

Natural medicine products usually consist of two to five kinds of plants that have synergistic biological activity. The responsible constituents for the therapeutic activity in plants often involve diverse chemical components. The combination of ethanol extracts of Annona squamosa L. (EEAS) and Persea americana M. (EEPA) has been shown to have antihyperlipidemic activity. To ensure the correctness and consistency of the EEAS and EEPAs combination as the antihyperlipidemic treatments, it is necessary to characterize the plant’s secondary metabolites. This study aimed to obtain the profiles of the secondary metabolites of Annona squamosa L. and Persea americana M. using the TLC-densitometry technique. Extract samples were applied to TLC plates and screened using densitometry at 254 nm. The obtained chromatogram peak was measured at the wavelength range of 200–700 nm to obtain the maximum wavelength of each peak. The results showed that EEAS had 5 peaks with rf values of 0.00, 0.61, 0.67, 0.73, and 0.87. The TLC-densitometry analysis results of EEPA showed eight peaks with rf values of 0.00, 0.11, 0.19, 0.49, 0.56, 0.71, 0.77, and 0.90. The extract chromatograms spectra showed that 2–3 peaks at the wavelength range of 300-350 nm. Chromatogram TLC-densitometry profile data on EEAS and EEPA can be used as the indicators in the raw material combinations standardization process of EEAS and EEPA as natural medicine products to treat antihyperlipidemic.

Full Workflows for the Analysis of Gas Chromatography-Ion Mobility Spectrometry in Foodomics: Application to the Analysis of Iberian Ham Aroma.

Gas chromatography—ion mobility spectrometry (GC-IMS) allows the fast, reliable, and inexpensive chemical composition analysis of volatile mixtures. This sensing technology has been successfully employed in food science to determine food origin, freshness and preventing alimentary fraud. However, GC-IMS data is highly dimensional, complex, and suffers from strong non-linearities, baseline problems, misalignments, peak overlaps, long peak tails, etc., all of which must be corrected to properly extract the relevant features from samples. In this work, a pipeline for signal pre-processing, followed by four different approaches for feature extraction in GC-IMS data, is presented. More precisely, these approaches consist of extracting data features from: (1) the total area of the reactant ion peak chromatogram (RIC); (2) the full RIC response; (3) the unfolded sample matrix; and (4) the ion peak volumes. The resulting pipelines for data processing were applied to a dataset consisting of two different quality class Iberian ham samples, based on their feeding regime. The ability to infer chemical information from samples was tested by comparing the classification results obtained from partial least-squares discriminant analysis (PLS-DA) and the samples’ variable importance for projection (VIP) scores. The choice of a feature extraction strategy is a trade-off between the amount of chemical information that is preserved, and the computational effort required to generate the data models.

EVA: Evaluation of Metabolic Feature Fidelity Using a Deep Learning Model Trained With Over 25000 Extracted Ion Chromatograms.

Extracting metabolic features from liquid chromatography-mass spectrometry (LC-MS) data relies on the recognition of extracted ion chromatogram (EIC) peak shapes using peak picking algorithms. Unfortunately, all peak picking algorithms present a significant drawback of generating a problematic number of false positives. In this work, we take advantage of deep learning technology to develop a convolutional neural network (CNN)-based program that can automatically recognize metabolic features with poor EIC shapes, which are of low feature fidelity and more likely to be false. Our CNN model was trained using 25095 EIC plots collected from 22 LC-MS-based metabolomics projects of various sample types, LC and MS conditions. Notably, we manually inspected all the EIC plots to assign good or poor EIC quality for accurate model training. The trained CNN model is embedded into a C#-based program, named EVA (short for evaluation). The EVA Windows Application is a versatile platform that can process metabolic features generated by LC-MS systems of various vendors and processed using various data processing software. Our comprehensive evaluation of EVA indicates that it achieves over 90% classification accuracy. EVA can be readily used in LC-MS-based metabolomics projects and is freely available on the Microsoft Store by searching "EVA Metabolomics".

A new strategy for the computer-assisted development of reversed-phase liquid chromatography separation methods of unknown sample mixtures

A new strategy for the computer-assisted methods development in the reversed-phase liquid chromatographic separations of unknown sample mixtures has been developed using the latent spectral information in chromatogram raw data files of appropriately designed experiments, rather than resorting to elemental information functions (e.g., the number of peaks in chromatograms or similar criteria). The strategy developed allows unification of the approach for samples of both known and unknown composition and, thus, provide a general strategy for computer-aided tools in the chromatography laboratory. The operation principle of this strategy departs from extracting the spectra of components in the mixture chromatograms by resorting to multivariate curve resolution-alternating least squares (MCR-ALS). This technique allows the estimation of the true spectra for the individual components except when they have identical spectra or are fully overlapped. Thus, a convenient experimental design will try to perform separations of the sample mixture having at least partial resolution of components in some runs. This will allow estimating the spectra of components and, then, assign these components to the peaks in each run chromatogram. In this way, a retention model can be built for each component so computerized optimization process can be developed to provide the chromatographer with the best possible separation programs. Following this approach, strategies for sample mixtures of known and unknown composition are only different in the need of an initial spectrum discovery process for unknown mixtures and therefore a real general approach for the computer-assisted LC methods development is now available for the first time.

Quantitative and qualitative analysis of contaminating hydrocarbons in the Gulf of Santiago de Cuba by gas chromatography

The coasts of Cuba are exposed to damage caused by the contamination by petroleum hydrocarbons. The coastal strip of the Gulf of Santiago de Cuba is an example of natural resources deterioration due to this contamination, which appears as one of its main environmental problems. In the present work, using gas chromatography, quantitative and qualitative analysis were performed based on the retention time values and the area under the chromatogram curve, which allowed characterizing the degree of contamination at four points of the bay of Santiago de Cuba: playa La Estrella (P1), “Guillermón Moncada” Port (P2) “Hermanos Díaz” Oil Refinery (P3), “Antonio Maceo” Thermoelectric Power Plant RENTE (P4). Retention times of individual peaks in chromatograms of individual samples with several standards, thus obtaining the types of hydrocarbons present at each point, with points P2 and P3 being the ones with the highest pollutant and the highest concentration by peak height. Fuel oil turned out to be the most polluting agent present in the bay.

Improvement of Extractive Alkylation Gas Chromatography of Short-chain Carboxylic Acids in Aqueous Solution.

In the analysis of short-chain carboxylic acids such as formic acid and acetic acid in aqueous solution using extractive alkylation gas chromatography, tetrahexylammonium bromide (THAB) as a phase-transfer catalyst (PTC) causes a high intensity and broad peaks in the gas chromatogram, and interfere with the detection of carboxylic acid derivatives. By an easy treatment of the extractive alkylation solution with perchloric acid and n-hexane, it is possible to remove more than 95% of THAB, and to provide good gas chromatogram with a little admixture of carboxylic acid derivatives. The desensitization was 16% at the maximum, the contamination of the glass insert in gas chromatograph and liquid phase in column by THAB was minimized, and trouble in continuous measurement could be avoided.

Identification of Ethyl Para-Methoxycinnamate and Kaempferol in the Ethanol Extract of <i>Kaempferia galanga </i>L. Rhizome as Biomaterial for Drug Candidate Using Spectrophotometric and Chromatographic Analysis

Kaempferia galanga L. rhizome (KGR) has been used empirically in Asian countries, particularly Indonesia, to treat inflammation. Ethyl para-methoxycinnamate (EPMC) and kaempferol, two phytochemicals contained in KGR, are scientifically proven in playing a role as anti-inflammatory agents. Several studies have explored the pharmacology activities of EPMC and kaempferol, thus a further exploration of the physicochemical properties of the ethanol extract of KGR (EEKGR) is needed. This study aims to confirm the presence of EPMC and kaempferol in EEKGR using spectrophotometric and chromatographic analysis. The KGR was purchased from Buniayu Plantation in Subang, West Java, Indonesia and was identified at Herbarium Bandungense, School of Natural Science and Technology (SITH), Bandung, Indonesia. EEKGR was prepared by cold extraction technique 3x24 hours using 70% ethanol, then was rotary-evaporated to a viscous consistency. The yield of the extract produced was 14.55% w/w (72.7935 g viscous extract from 500 g dried powder of rhizome), with a water content of 4.37% (thermogravimetry method). Phytochemical screening revealed the presence of phenolics and flavonoids. Thin Layer Chromatography (TLC) indicated that EPMC might be present in the EEKGR (Rf = 0.92 compared to that of standard EPMC = 0.92), and kaempferol (Rf = 0.26 compared to that of standard kaempferol = 0.25). The spectrophotometric analysis of EEKGR confirmed the presence of benzoyl and cinnamoyl bands, which positively belongs to flavonoid (UV spectrum = 200-400 nm). The High-performance Liquid Chromatography (HPLC) analysis of EEKGR proved chromatogram peak at 7.2 min which is similar with the standard EPMC (detection was set at 308 nm), however, no chromatogram peak of kaempferol at 4.0 min was observed. Generally, these spectrophotometric and chromatographic analysis results proved that EPMC is present in fair amount in the EEKGR therefore, this extract is interesting to be developed as a biomaterial for drug candidate, particularly to treat inflammation.

Application of the TLC image analysis technique for the simultaneous quantitative determination of L-proline and L-lysine in dietary supplement

A simple and sensitive thin-layer chromatography (TLC) method coupled with an image analysis technique was developed for the simultaneous quantitative determination of L-proline and L-lysine in dietary supplement with good precision and accuracy. Separation was performed on silica gel plates using ethanol‒toluene (2:3, V/V) as the mobile phase. The visualization of chromatograms was based on iodine–azide reaction; therefore, pre-chromatographic derivatization reaction of amino acids with phenyl isothiocyanate was performed. Digital images of TLC plate chromatograms were converted into peak chromatograms, and quantitative analysis was conducted using TLSee software.

Synthesis of Bio-additive for Low Sulphur Diesel: Transesterification of Soybean Oil and Ethylene Glycol using K2CO3 Catalyst

The desulphurization process of diesel fuel is carried out to reduce the amount of SO2 emissions that can cause acid rain. However, the desulphurization process in diesel fuel not only removes the sulfur compounds but polyaromatic and polar compounds are also eliminated during this process. The loss of these two compounds can reduce the lubricity properties of diesel fuel. Therefore, it is necessary to add an additive compound that can increase the lubricity properties. In this research, 2-hydroxyethyl ester (HEE) was synthesized as an additive to increase the lubricity of diesel fuel. This compound was synthesized through the transesterification reaction of soybean oil and ethylene glycol with K2CO3 as the base catalyst. The composition of the synthesized additives was analyzed using the Gas Chromatography-Mass Spectrometry (GC-MS). Based on the results of GC-MS spectrum analysis, it is known that the 2-hydroxyethyl ester compound has been formed with a yield of 66.5% (relative to the area of the chromatogram peak). The HEE compound obtained is a mixture of 2 hydroxyethyl palmitate, 2 hydroxyethyl linoleate, 2 hydroxyethyl stearate, 2 hydroxyethyl arachidonate, 2 hydroxyethyl nervate, and 2 hydroxyethyl behenate.

Degradation of Nonylphenol Ethoxylate-10 (NPE-10) by Mediated Electrochemical Oxidation (MEO) Technology

Nonylphenol ethoxylate (NPE-10) is a non-ionic surfactant that is synthesized from alkylphenol ethoxylate. The accumulation of NPE-10 in wastewater will endanger the ecosystem as well as the human being. Nowadays, NPE-10 can be degraded indirectly by using an electrochemical treatment by the advancement of technology. Thus, this study is aimed to evaluate the electro-degradation potential of NPE-10 by MEO using Ce(IV) ionic mediator. In addition, the influence of Ag(I) ionic catalyst in the performance of MEO for the degradation of NPE-10 was also observed. The potency of MEO technology in the NPE-10 degradation was evaluated by voltammetry technique and confirmed by titrimetry and LC-MS analysis. The results showed that in the absence of Ag(I) ionic catalyst, the degradation of NPE-10 by MEO was 85.93%. Furthermore, when the Ag(I) ionic catalyst was applied, the performance of MEO in degradation of NPE-10 was improved to 95.12%. The back titration using Ba(OH)2 confirmed the formation of CO2 by 46.79%, whereas the redox titration shows the total of degradation organic compounds by 42.50%. It was emphasized by the formation of two new peaks in the LC-MS chromatogram. In summary, our results confirmed the potential of MEO technology for the NPE-10 degradation.

Starch bio-based composite active edible film functionalized with Carum carvi L. essential oil: antimicrobial, rheological, physic-mechanical and optical attributes.

The online version contains supplementary material available at 10.1007/s13197-021-05028-1.

Effects of Montmorillonite and Cane Sugar Composition on The Properties of Montmorillonite-Carbon Sulfonate Catalyst

The influence of montmorillonite and cane sugar mixture with composition of 1:3, 1:2, 1:1, 2:1, and 3:1 to the characteristics of the synthesized montmorillonite-carbon sulfonate composite catalyst had been investigated. The mixture of montmorillonite and cane sugar was carbonized at 400 oC for 15 hours and then sulfonated using sulfuric acid at 175 oC for 15 hours. The presence of sulfonate group was detected using FT-IR and the surface topology of the catalyst was characterized using SEM-EDX. FT-IR spectra showed the existence of sulfonate group at wave number of 1095 cm-1. SEM-EDX analysis showed that the catalyst with the montmorillonite and cane sugar ratio of 1:2 consisted of 1.06% sulfur. The catalyst activity was examined using an esterification reaction between acetic acid and ethanol, and the reaction products were characterized using GC-MS. The chromatogram peak at m/z = 88 confirmed that the product was ethyl acetate. Catalyst with the montmorillonite and cane sugar ratio of 1:2 was shown to have the highest acidity and gave the highest esterification product with 76.97% conversion.

Microscopic performance of modified asphalt based on aging

In order to clarify the effect of modified asphalt after thermal aging (aging for short) on asphalt pavement performance, five groups of asphalt samples were selected as research materials: matrix asphalt group, styrene-butadiene-styrene (SBS) group, styrene-butadiene rubber (SBR) group, stabilizer group and furfural extract oil group. The changes of macro-performance and micro-structure for asphalt were compared and analyzed before and after aging. The micro-morphology of asphalt surface was analyzed by scanning electron microscope (SEM). The cross linking and degradation of asphalt molecules was observed by gel permeation chromatography (GPC). The high temperature performance and exothermic properties were analyzed by differential scanning calorimetric (DSC). The results showed that agglomeration increased with aging and occurred in all asphalt samples, but most particles of composite modifier were evenly distributed and with a few folds being on the surface of the modified asphalt after aging. Double peaks appeared in the chromatogram of the SBS modifier before aging, but only single peak appeared after aging. The chromatogram peak of composite modifier shifted to the right after aging, and the peak of composite modifier was not obvious and dispersed widely. There was a weak exothermic peak from 350 °C to 410 °C in the DSC curve of the asphalt sample and a strong endothermic peak from 420 °C to 510 °C before and after aging. The maximum endothermic peak was at 460 °C. Except for the SBR group, the bond-breaking endothermic rate of macromolecules was higher than that of polymerization exotherm of small molecules during long-term aging, so the heat absorption of long-term aging asphalt was greater than that of short-term aging. The analysis of macro results showed that the comprehensive performance of composite modified asphalt was the best, which had a positive significance for improving the pavement performance and application of modified asphalt.

Prediction by statistical overlap theory of fraction of baseline occupied by chromatographic peaks

The average minimum resolution required for separating adjacent single-component peaks (SCPs) in one-dimensional chromatograms is an important metric in statistical overlap theory (SOT). However, its value changes with changing chromatographic conditions in non-intuitive ways, when SOT predicts the average number of peaks (maxima). A more stable and easily understood value of resolution is obtained on making a different prediction. A general equation is derived for the sum of all separated and superposed widths of SCPs in a chromatogram. The equation is a function of the saturation α, a metric of chromatographic crowdedness, and is expressed in dimensionless form by dividing by the duration of the chromatogram. This dimensionless function, f(α), is also the cumulative distribution function of the probability of separating adjacent SCPs. Simulations based on the clustering of line segments representing SCPs verify expressions for f(α) calculated from five functions for the distribution of intervals between adjacent SCPs. Synthetic chromatograms are computed with different saturations, distributions of intervals, and distribution of SCP amplitudes. The chromatograms are analyzed by calculating the sum of the widths of peaks at different relative responses, dividing the sum by the duration of the chromatograms, and graphing the reduced sum against relative response. For small values of relative response, the reduced sum approaches the fraction of baseline that is occupied by chromatographic peaks. This fraction can be identified with f(α), if the saturation α is defined with the average minimum resolution equaling 1.5. The identification is general and is independent of the saturation, the interval distribution, or the amplitude distribution. This constant value of resolution corresponds to baseline resolution, which simplifies the interpretation of SOT.

Development and validation of a fast and simple HPLC method for the simultaneous determination of bisoprolol and enalapril in dosage form

Aim. We aimed to develop and validate fast, simple, accurate, robust and rugged chromatographic method for simultaneous determination of bisoprolol fumarate and enalaril maleate in solid pharmaceutical dosage form, with using chaotropic strong chaotropic perchlorate anions in composition of mobile phase. Materials and methods. Fast simple HPLC method for simultaneous determination of bisoprolol and enalapril in solid pharmaceutical dosage forms was developed, with perfect peak symmetries eluting at 4.7 and 5.2 miutes, with mobile phase composed of methanol and diluted perchloric acid pumped with 1ml/min on Zorbax Rx C8 250x4.6mm, 5um column thermostated at 42 °C, and monitored UV signal at 214nm. Mobile phase was composed of 55%methanol and 45% perchloric acid (0.07%v/v). Results. Usage of presence of perchloric anions showed very useful role in peak shape and chromatogram view, due to distinguished chaotropic characteristics of perchlorate anions on molecules containing nitrogen atoms in molecular structures of analytes, in acidic pH environment. Linearity was examined and proven at different concentration levels in the range of working concentration of bisoprolol (20–200 ug/ml) and enalapril (20–200 ug/ml). The methods achieved very good validation parameters, with determined LOQ about 0.032 mg/ml and LOD about 0.003 mg/ml for bisoprolol, and LOQ about 0.045 mg/ml and LOD 0.005 mg/ml for enalapril. The high value of recoveries obtained for bisoprolol and enalapril indicates that the proposed method was found to be accurate. Conclusion. A new fast and simple, but selective, accurate, precise and robust HPLC method for simultaneous determination of bisoprolol and enalapril in tablets was developed and many possible variations of the same were suggested. The developed method for the simultaneous quantification of bisoprolol and enalapril from solid dosage formulations offers simplicity essential for quality control of a large number of samples in short time intervals, which is necessary for routine analysis.

Molecular Characterisation Of Growth Hormone (Gh) Gene In Indian Dromedary And Bactrian Camel

Molecular characterisation of 613 bp long growth hormone (GH) gene fragment spanning partial exon-1, intron-1, exon-2 and partial intron-2 was done in Indian (one humped) dromedary and Indian (double humped) bactrian camel through PCR amplification, sequencing and bioinformatics analysis. The sequence variations within and between single and double humped Indian camels were observed. In the Indian single humped camels of Mewari, Kachchhi and Bikaneri breed at position 264 C>T variation was seen. At this particular locus animals with single and double peaks in the sequence chromatograms were observed. Accordingly, two allele (C, T) and three genotype (CC, CT, and TT) were identified in the Indian dromedary camels. In Indian Bactrian camel at position 264 only C allele and CC genotype was identified. In double humped camel at position 242, A>G and at position 469 G>A transition variation was observed compared to single humped camel. The different sequences obtained were submitted to NCBI and Gen Bank Accession No. MT478653 for C allele Indian Dromedary, MT478654 for T allele Indian Dromedary and MT478655 for Indian Bactrian camel were obtained. Similarity ranging from 98% to 100 % was observed with available GH Sequences of camel at GenBank. Camel sequence was found to have close similarity with other Camelidae family members like Lamapacos (97.07%) and Lamaglama (96.58%). The evolutionary relationship between sequences showed close relationship between dromedary and bactrian camel followed by vicugna and llama. The domesticated species like cattle, buffalo, sheep, goat, yak and mithun were distantly related to camel. The present study showed close similarity between GH gene sequence pattern of Indian one and double humped camel except transition variation at two positions in bactrian camel.

Cloning and in silico analysis revealed a genetic variation in osmotin-encoding genes in an Indonesian local cacao cultivar

Theobroma cacao L. is an important Indonesian estate crop, which suffers from biotic and abiotic stresses. TcOSM, which encodes osmotin as a response to pathogens and environmental stresses, is, therefore, a focus of interest in this research, aiming to characterize TcOSM in an Indonesian local cacao cultivar. Bioinformatics queries for putative TcOSM were performed against the reference genome of a Criollo-type cacao cultivar. Based on nucleotide sequence determination, our results revealed two genes, TcOSM1 and TcOSM2, which have the highest similarity (≥ 90\%) to the cacao reference genes. Heterozygosity was detected in the TcOSM1-encoding gene, which contained two overlapping peaks in Sanger-sequencing chromatograms. One of the alleles resulted from a single nucleotide change (G to A), leading to a same-sense mutation that did not substitute corresponding alanine residue. Homology modeling using Phyre2 and structural alignment (superimposition) was conducted to examine the influence of genetic variations in TcOSM sequences upon the global protein structures. The result showed no significant changes (RMSD ≤ 0.206 Å, TM-score > 0.5) in tertiary protein structures. Altogether, this research succeeded in characterizing TcOSM while providing a fundamental study for future cacao biotechnology endeavors.

Study on quality evaluation method of classical prescription Mahuang Decoction primary standard substances

This study aimed to establish the HPLC characteristic chromatogram and content determination method for index components with the primary standard substances of the classical prescription Mahuang Decoction, and to provide data basis for the establishment of its quality standard and the development and utilization of compound preparations. First, HPLC was used to establish the material reference chromatograms of Mahuang Decoction, and 15 batches of standard samples of Mahuang Decoction were determined. Their similarity was calculated by the median method. Secondly, the content of the standard substances was determined and a simplecontent determination method was established by HPLC. Relevant methodology was investigated, and the extraction ratio, index component transfer rate and moisture content of 15 batches of primary standard samples were calculated. The results showed that the two sets of HPLC methods had their own characteristics. The six chromatographic peaks identified from the 10 common peaks in the former characteristic chromatogram covered all the herbal medicines in the standard substances, which can better indicate the quality characteristics of the standard substances of Mahuang Decoction. The latter method(content determination method) was simple and practical, so it was suitable for establishing the quality standard of its compound preparation. Two sets of methods were jointly used to evaluate the quality of 15 batches of Mahuang Decoction. The results were as follows: the similarity of 15 batches of samples was greater than 0.90; the average extraction ratio was 18.1%; the average moisture content was 9.7%; the average content and transfer rate of the standard ingredients ephedrine hydrochloride and total pseudoephedrine hydrochloride were 2.3% and 26.7% respectively, and the average content and transfer rate of amygdalin were 2.2% and 48.3% respectively. None of the data showed dispersion(beyond 70%-130% of the mean value), which met the application data requirements for the substance standards of ancient classical Chinese herbal compound preparations(draft for comments). Based on the above research, the primary substance quality standard of Mahuang Decoction was established in order to provide reference for the development and research of the compound preparation of Mahuang Decoction.

Technical note: Quantification of lignans in the urine, milk, and plasma of flaxseed cake–fed dairy sheep

Mammalian lignans are phytoestrogens with important bioactivities, and their concentrations in livestock milk may influence the health of consumers. This research aimed to establish a method to quantify multiple mammalian lignans in the biofluids of dairy sheep using ultra-HPLC-triple quadropole mass spectrometry with multiple-reaction monitoring. Secoisolariciresinol, 2-[(4-hydroxy-3-methoxyphenyl)methyl]-3-[(3-hydroxyphenyl)methyl]-1,4-butanediol, enterodiol (ED), enterolactone (EL), ED-sulfate (ED-S), and EL-sulfate (EL-S) were purified from the urine of flaxseed cake-fed dairy sheep. The structures of these lignans were identified by a combination of mass and nuclear magnetic resonance spectra. These purified lignans were used as standards to optimize their quantification conditions in urine, milk, and plasma of dairy sheep. On this basis, the lignan metabolites in biofluids were quantified. To improve analysis sensitivity, plasma and milk were pretreated with acetonitrile containing 1% formic acid and passed through a HybridSPE-PL 55261-U column (Supelco, Bellefonte, PA). The limit of quantification of the lignans ranged from 1.43 to 18.3 ng/mL in plasma, and from 1.01 to 18.7 ng/mL in milk. The linearity of the calibration curves ranged from their limit of quantification to at least 217 ng/mL in plasma, and 217 ng/mL in milk. Regression coefficient of the calibration curves were above 0.99 for secoisolariciresinol, 2-[(4-hydroxy-3-methoxyphenyl)methyl]-3-[(3-hydroxyphenyl)methyl]-1,4-butanediol, ED, EL, ED-S, and EL-S, indicating satisfactory relationships between the peak areas and concentrations in the quantification range. The relative concentrations of ED-glucuronide and EL-glucuronide (EL-G) in different biofluids were compared based on their chromatogram peak areas. The sheep plasma contained all forms of mammalian lignans (i.e., ED, EL, ED-S, EL-S, ED-glucuronide, and EL-G.); the urine contained ED, EL, ED-S, and EL-S; and the milk contained ED, EL, ED-S, EL-S, and EL-G. Milk-to-plasma concentration ratios of the mammalian lignans indicated that the free forms were more permeable than the sulfated conjugates. Mammalian lignans found in sheep plasma and milk may provide health benefits to the sheep and sheep-product consumers. The analytical method established in this work could be used to quantify mammalian lignans in livestock products.