Reversed-phase Ultra-performance Liquid Chromatography Research Articles

FGL2-Associated Transcriptional and Histopathological Features of Immunological Preeclampsia

Podocytes are highly differentiated visceral cells, and several related specific proteins, such as podocalyxin and podocin are potential tools for the evaluation of podocyturia. However, precise quantitation of podocyturia-related proteins is complex and often unreliable.A reversed-phase ultra-performance liquid chromatography coupled to tandem mass spectrometry method was developed and validated to quantify podocalyxin and podocin levels in urine supernatant by using specific cleavable peptides and standards. Urine samples from women with normotensive or hypertensive pregnancies, gestational diabetes and preeclampsia, as well as treated and untreated Fabry patients, and gender-matched controls were investigated.The multiplex analysis shows that podocalyxin levels were higher than podocin levels in patients, the former being particularly higher in pregnant women. Women with preeclampsia had abnormal urine levels of both proteins with a higher sensitivity for podocalyxin. Slightly increased levels of podocin were also observed in Fabry males, while both proteins were increased in untreated Fabry females. Correlations were established between podocalyxin and podocin levels and clinical parameters associated with Fabry disease and preeclampsia.This methodology makes possible the precise, simultaneous and reliable analysis of podocalyxin and podocin levels, and offers a valuable tool for the evaluation of podocyturia.

Characterisation of the Urinary Metabolic Profile of Liver Fluke-Associated Cholangiocarcinoma

BackgroundHuman infection with Opisthorchis viverrini, a carcinogenic liver fluke inhabiting the biliary tree, is endemic in Southeast Asia. Chronic infection is associated with a fatal complication, cholangiocarcinoma (CCA), a late-presenting and aggressive malignancy. Currently, annual mortality rates from CCA mirror trends in incidence, due in part to limited availability of efficient prognostic and early diagnostic biomarkers. With ability to detect thousands of urinary metabolites using metabonomics, the urine metabolome holds great potential in providing an insight into system-level alterations in carcinogenesis and in identifying metabolic markers altered in response to disturbed homoeostasis.MethodsGlobal molecular profiling using reversed-phase ultraperformance liquid chromatography mass spectrometry was utilised to acquire the urinary spectral profile of 137 Thai subjects (48 at high risk of infection, 41 with O. viverrini infection, 34 periportal fibrosis and 14 CCA) from Khon Kaen, Thailand.ResultsMultivariate statistical analysis identified perturbation in several molecular classes related to purine metabolism and lipid metabolism in the CCA urine metabolome. These markers mainly reflect changes in energy metabolism to support proliferation (increased fatty acid oxidation and purine recycling), DNA methylation and hepatic injury.ConclusionsSeveral metabolites of biological interest were discovered from this proof-of-principle dataset. Augmenting these findings is essential to accelerate the development of urinary metabolic markers in CCA.

Effect of high-nitrogen fertilizer on gliadin and glutenin subproteomes during kernel development in wheat (Triticum aestivum L.)

Nitrogen (N), a macronutrient essential for plant growth and development, is needed for biosynthesis of protein and starch, which affect grain yield and quality. Application of high-N fertilizer increases plant growth, grain yield, and flour quality. In this study, we performed the first comparative analysis of gliadin and glutenin subproteomes during kernel development in the elite Chinese wheat cultivar Zhongmai 175 under high-N conditions by reversed-phase ultra-performance liquid chromatography and two-dimensional difference gel electrophoresis (2D-DIGE). Application of high-N fertilizer led to significant increases in gluten macropolymer content, total gliadin and glutenin content, and the accumulation of individual storage protein components. Of 126 differentially accumulated proteins (DAPs) induced by high-N conditions, 24 gliadins, 12 high-molecular-weight glutenins, and 27 low-molecular-weight glutenins were significantly upregulated. DAPs during five kernel developmental stages displayed multiple patterns of accumulation. In particular, gliadins and glutenins showed respectively five and six accumulation patterns. The accumulation of storage proteins under high-N conditions may lead to improved dough properties and bread quality.

Characterization of unknown impurities in Coenzyme Q10 using LC–MS and NMR

During studies on related substances in coenzyme Q10 (CoQ10) active pharmaceutical ingredient (API) and capsules, two impurities (Impurity 1 and Impurity 2) with each content more than 0.1% attracted our attention. Accelerated testing (40 °C /75%RH) and forced degradation of CoQ10 API and drug products in different stress conditions (base and heat) gave rise to the two unknown impurities. These unknown impurities were separated by reverse phase ultra-performance liquid chromatography (UPLC), where they were eluted at 0.77 (Impurity 1) and 1.10 (Impurity 2) relative retention times to CoQ10 peak. Impurity 2 was separated into two peaks (Impurity 2-1 and Impurity 2-2) by normal phase high performance liquid chromatography (NP-HPLC). In this study, an ultra-performance liquid chromatography complied with quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF/MS) via atmospheric pressure chemical ionization (APCI) was used to identify the proposed structure of the three impurities (Impurity 1, Impurity 2-1 and Impurity 2-2). We got the three impurities through preparative high performance liquid chromatography and their structures were further confirmed by 1D and 2D nuclear magnetic resonance (NMR). Based on the significance of these results, Impurity 1 was the impurity E referred in British Pharmacopoeia (BP), Impurity 2-1 and Impurity 2-2 were a pair of isomers which had not been reported before.

Analysis of polyhexamethylene biguanide and alexidine in contact lens solutions using capillary electrophoresis, ultra-performance liquid chromatography and quadrupole time of flight mass spectrometry

Polymeric biguanides, as well as quaternary ammonium compounds, are ubiquitous antimicrobial agents in healthcare. Due to the highly cationic and polymeric nature of these compounds and the complex matrices in which they are found, the analytical characterization of products containing them remains challenging. In this work an efficient, sensitive, and high-resolution separation protocol was developed to perform quantitative measurements (sub-mg L−1) of alexidine dihydrochloride (ADH) and polyhexamethylene biguanide (PHMB) in commercial multipurpose contact lens solutions (MPS). Initially, contactless conductivity (C4D) detection was explored, but lacked adequate selectivity and sensitivity to quantify PHMB or ADH in commercial MPS. To overcome these limitations, an alternative approach using solid phase extraction (SPE) followed by separation with reversed phase ultra-performance liquid chromatography (RP-UPLC) was developed for both ADH and PHMB separation and detection. The most sensitive and reliable method investigated utilized standard additions to compensate for matrix effects. For ADH, concentration values measured with the presented method were consistent with data provided by the MPS manufacturer (1.6 mg L−1) within 0.10 mg L−1. PHMB quantification in MPS products was successful at concentrations <1 mg L−1 with quantitative reproducibility better than 2% RSD. Comparison of blind sample testing using the RP-UPLC method showed strong correlation (R2 = 0.939) of PHMB concentrations with results obtained by the United States Food and Drug Administration using a published HPLC-Evaporative light scattering detection (ELSD) assay. A significant advantage of this method is the ability to partially resolve PHMB polydispersity, which to date has been minimally studied and explained. By coupling with electrospray mass spectrometry (MS), a general trend was observed for increased retention as a function of PHMB chain length. The improved robustness and reproducibility of UV detection versus ELSD coupled with the superior resolving power of UPLC is an asset to the detection and characterization of PHMB and ADH. In addition to quality control of MPS, this method has potential application to the analyses skin wipes, wound dressings and other medical products where understanding how manufacturing processes lead to differences in polydispersity is important to maximize the antimicrobial properties while minimizing toxicologic effects.

Utility of S-FLT1 / PLGF ratio for the diagnosis of preeclampsia: A systematic review

Podocytes are highly differentiated visceral cells, and several related specific proteins, such as podocalyxin and podocin are potential tools for the evaluation of podocyturia. However, precise quantitation of podocyturia-related proteins is complex and often unreliable.A reversed-phase ultra-performance liquid chromatography coupled to tandem mass spectrometry method was developed and validated to quantify podocalyxin and podocin levels in urine supernatant by using specific cleavable peptides and standards. Urine samples from women with normotensive or hypertensive pregnancies, gestational diabetes and preeclampsia, as well as treated and untreated Fabry patients, and gender-matched controls were investigated.The multiplex analysis shows that podocalyxin levels were higher than podocin levels in patients, the former being particularly higher in pregnant women. Women with preeclampsia had abnormal urine levels of both proteins with a higher sensitivity for podocalyxin. Slightly increased levels of podocin were also observed in Fabry males, while both proteins were increased in untreated Fabry females. Correlations were established between podocalyxin and podocin levels and clinical parameters associated with Fabry disease and preeclampsia.This methodology makes possible the precise, simultaneous and reliable analysis of podocalyxin and podocin levels, and offers a valuable tool for the evaluation of podocyturia.

들깨 유전자원의 지방산 변이 및 rosmarinic acid와 caffeic acid의 함량에 따른 항산화 활성 비교

Background: The aim of this study was to compare the fatty acid composition, antioxidant activity, and the content of two major phenolic acids, caffeic and rosmarinic acids, of 45 Perilla accessions collected from Russia.BR Methods and Results: A total of 45 accessions of the genus Perilla were used in this study. The antioxidant activities of these accessions were analyzed using a spectrophotometer, and their caffeic and rosmarinic acid contents were determined using a reversedphase ultraperformance liquid chromatography (UPLC) system. The Perilla seed oil was recovered using hexane in a soxhlet extraction method. The fatty acid compositions were analyzed using a Shimadzu QP2010 gas chromatography-mass spectoroscopy system. The results showed that accessions IT226732 and IT274300 had the highest content of caffeic and rosmarinic acid, respectively. A cluster analysis based on antioxidant assay results and concentration of phenolic acids led to the categorization of Perilla accessions into three major groups. The accessions in Group I were characterized by higher caffeic and rosmarinic acid content and antioxidant activity, compared to the accessions in the other two groups (p 0.05). The total oil content ranged between 28.39 and 46.89%. The compositions of oleic, linoleic, and linolenic acid ranged from 11.83 to 19.55%, 11.92 to 16.71%, and 59.19 to 67.28%, respectively.BR Conclusions: The results of this study indicated that accessions IT274300, IT226732, IT274293, IT235818, and IT235820 could be used as a source of functional materials.

Tandem mass spectrometry analysis of urinary podocalyxin and podocin in the investigation of podocyturia in women with preeclampsia and Fabry disease patients

BackgroundPodocytes are highly differentiated visceral cells, and several related specific proteins, such as podocalyxin and podocin are potential tools for the evaluation of podocyturia. However, precise quantitation of podocyturia-related proteins is complex and often unreliable. MethodA reversed-phase ultra-performance liquid chromatography coupled to tandem mass spectrometry method was developed and validated to quantify podocalyxin and podocin levels in urine supernatant by using specific cleavable peptides and standards. Urine samples from women with normotensive or hypertensive pregnancies, gestational diabetes and preeclampsia, as well as treated and untreated Fabry patients, and gender-matched controls were investigated. ResultsThe multiplex analysis shows that podocalyxin levels were higher than podocin levels in patients, the former being particularly higher in pregnant women. Women with preeclampsia had abnormal urine levels of both proteins with a higher sensitivity for podocalyxin. Slightly increased levels of podocin were also observed in Fabry males, while both proteins were increased in untreated Fabry females. Correlations were established between podocalyxin and podocin levels and clinical parameters associated with Fabry disease and preeclampsia. ConclusionsThis methodology makes possible the precise, simultaneous and reliable analysis of podocalyxin and podocin levels, and offers a valuable tool for the evaluation of podocyturia.

UPLC/FT-ICR MS-based high-resolution platform for determining the geographical origins of raw propolis samples

In this study, we demonstrate a high-resolution 15 Tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry coupled with a reverse-phase ultra-performance liquid chromatography (RP-UPLC) system for determining the geographical origins of raw propolis samples. The UPLC/FT-ICR MS-based high-resolution platform was validated on the ethanol-extracted propolis (EEP) from various propolis raw materials originating from different countries (i.e., Argentina, Brazil, China, and Korea) to determine the geographical origins of the propolis and the origin-specific key compounds. Based on approximately 8000 molecular features extracted from UPLC/FT-ICR MS datasets, a partial least squares-discriminant analysis (PLS-DA) plot showed distinct separations among propolis samples from four different origins. Key propolis components contributing to the discrimination of Korean propolis from Brazilian and Chinese propolis were identified and classified into five subgroups (i.e., flavonoids, phenols, terpenoids, fatty acids, and others). This analysis revealed the characteristic features of the different propolis samples, and this analytical platform can be further used to determine the geographical origins and to assess the quality of the commercial products.

Lipidomic Analysis of Cancer Cell and Tumor Tissues.

Due to their role in cellular structure, energetics, and signaling, characterization of changes in cellular and extracellular lipid composition is of key importance to understand cancer biology. In addition, several mass spectrometry-based profiling as well as imaging studies have indicated that lipid molecules may be useful to augment existing biochemical and histopathological methods for diagnosis, staging, and prognosis of cancer. Therefore, analysis of lipidomic changes associated with cancer cells and tumor tissues can be useful for both fundamental and translational studies. Here, we provide a high-throughput single-extraction-based method that can be used for simultaneous lipidomic and metabolomic analysis of cancer cells or healthy or tumor tissue samples. In this chapter, a modified Bligh-Dyer method is described for extraction of lipids followed by analysis of fatty acid composition by gas chromatography-mass spectrometry (GC-MS) or untargeted lipidomics using electrospray ionization mass spectrometry (ESIMS) coupled with reverse-phase (RP) ultraperformance liquid chromatography (UPLC) followed by multivariate data analysis to identify features of interest.

Immunomodulatory activity of 5 kDa permeate fractions of casein hydrolysates generated using a range of enzymes in Jurkat T cells and RAW264.7 macrophages

The in vitro bioactivity of 5 kDa ultrafiltration permeate fractions of casein hydrolysates produced using different enzymes were compared. Reverse phase ultra-performance liquid chromatography and gel permeation chromatography showed that the permeates had different physicochemical properties (molecular mass and degree of hydrolysis). The Flavourzyme® permeate had the highest activity in the 2,2’-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay. Cellular antioxidant and immunomodulatory assays showed that none of the permeates exhibited in vitro antioxidant activity, while all permeates significantly (P < 0.05) decreased interleukin-6 (IL-6) production in ConA-stimulated Jurkat T cells at 0.50% (w/v) and LPS-stimulated RAW264.7 cells at 0.05 and 0.50% (w/v). Three permeates, obtained using Flavourzyme®, Flavorpro Whey and trypsin, also significantly (P < 0.05) decreased IL-1β production at 0.05% (w/v) in RAW264.7 cells. Western blot analysis showed that all permeates significantly decreased the expression of the NF-κB subunit, p65, in RAW264.7 cells indicating that anti-inflammatory activity may be associated with this pathway.

Deletion of high-molecular-weight glutenin subunits in wheat significantly reduced dough strength and bread-baking quality

BackgroundHigh-molecular-weight glutenin subunits (HMW-GS) play important roles in the elasticity of dough made from wheat. The HMW-GS null line is useful for studying the contribution of HMW-GS to the end-use quality of wheat.MethodsIn a previous work, we cloned the Glu-1Ebx gene from Thinopyrum bessarabicum and introduced it into the wheat cultivar, Bobwhite. In addition to lines expressing the Glu-1Ebx gene, we also obtained a transgenic line (LH-11) with all the HMW-GS genes silenced. The HMW-GS deletion was stably inherited as a dominant and conformed to Mendel’s laws. Expression levels of HMW-GS were determined by RT-PCR and epigenetic changes in methylation patterns and small RNAs were analyzed. Glutenins and gliadins were separated and quantitated by reversed-phase ultra-performance liquid chromatography. Measurement of glutenin macropolymer, and analysis of agronomic traits and end-use quality were also performed.ResultsDNA methylation and the presence of small double-stranded RNA may be the causes of post-transcriptional gene silencing in LH-11. The accumulation rate and final content of glutenin macropolymer (GMP) in LH-11 were significantly lower than in wild-type (WT) Bobwhite. The total protein content was not significantly affected as the total gliadin content increased in LH-11 compared to WT. Deletion of HMW-GS also changed the content of different gliadin fractions. The ratio of ω-gliadin increased, whereas α/β- and γ-gliadins declined in LH-11. The wet gluten content, sedimentation value, development time and stability time of LH-11 were remarkably lower than that of Bobwhite. Bread cannot be made using the flour of LH-11.ConclusionsPost-transcriptional gene silencing through epigenetic changes and RNA inhibition appear to be the causes for the gene expression deficiency in the transgenic line LH-11. The silencing of HMW-GW in LH-11 significantly reduced the dough properties, GMP content, wet gluten content, sedimentation value, development time and stability time of flour made from this wheat cultivar. The HMW-GS null line may provide a potential material for biscuit-making because of its low dough strength.

Forced Degradation Study for Tenofovir DF, Lamivudine and Efavirenz in Triple Combination Anti-Retroviral Tablets and Development of Validated Stability Indicating Assay Method by UPLC

Introduction: Tenofovir Disoproxil Fumarate/Lamivudine/Efavirenz Tablets are a pharmaceutical dosage form indicated for the treatment of Human Immunodeficiency Virus (HIV) infection. Methods: A simple, time efficient and stability indicating Reverse Phase Ultra Performance Liquid Chromatography (RP – UPLC) method was developed for the simultaneous determination of the three drugs present in the tablets. Mobile phase-A is 0.1% Trifluoro Acetic acid in water and mobile phase-B is acetonitrile in gradient elution mode. Flow rate at 0.4 mL/min was fixed in the method. Acquity BEH Phenyl (100 mm × 2.1 mm), 1.7 µ column was used for the separation of analytes at 260 nm wavelength. The developed method has been validated as specified in International Conference on Harmonization (ICH) guideline. Forced degradation study was conducted for Tenofovir Disoproxil fumarate (TDF), Lamivudine (LAM) and Efavirenz (EFA) pharmaceutical tablets to identify the degradation behaviour of individual drugs under stress study. Results: The method was observed to be linear from 6-90 µg/mL concentration for TDF &amp; LAM and 12-180 µg/mL for EFA with correlation coefficient values of more than 0.999. The method was found to be precise with RSD (Relative standard deviation) values of below 2% for replicate measurements. Recovery results were found to be between 99.0 and 100.8%. LOQ values for TDF, LAM &amp; EFA were 0.041 µg/mL, 0.034 µg/mL &amp; 0.053 µg/mL, respectively. Specificity of the analytical method was checked by injecting the stressed samples and evaluating the homogeneity of peaks using Photo Diode Array (PDA) detector. Peak purity results indicated that no interference was observed from degradation impurities for the quantification of TDF, LAM and EFA in the dosage form. The method was found to be robust under the deliberately modified chromatographic parameters. Conclusion: The developed UPLC method is stability indicating, successfully validated and suitable for quantification purpose.

Triazene salts: Design, synthesis, ctDNA interaction, lipophilicity determination, DFT calculation, and antiproliferative activity against human cancer cell lines

Synthesis, characterization and investigation of antiproliferative activity of nine triazene salts against human cancer cells lines (MV-4-11, MCF-7, JURKAT, HT-29, Hep-G2, HeLa, Du-145 and DAUDI), and normal human mammary epithelial cell line (MCF7-10A) is presented. The structures of novel compounds were determined using 1H and 13C NMR, and GC-APCI-MS analyses. Among the derivatives, compound 2c, 2d, 2e and 2f has very strong activity against biphenotypic B myelomonocytic leukemia MV4-11, with IC50 values from 5.42 to 7.69 µg/ml. The cytotoxic activity of compounds 2c-2f against normal human mammary gland epithelial cells MCF-10A is 6–11 times lower than against cancer cell lines. Our results also show that compounds 2c and 2f have very strong activity against DAUDI and HT-29 with IC50 4.91 µg/ml and 5.59 µg/ml, respectively. Their lipophilicity was determined using reversed-phase ultra-performance liquid chromatography and correlated with antiproliferative activity. Our UV–Vis spectroscopic results indicate also that triazene salts tends to interact with negatively charged DNA phosphate chain. To support the experiment, theoretical calculations of the 1H NMR shifts were carried out within the Density Functional Theory.

Quality Comparison of Biosimilar and Copy Filgrastim Products with the Innovator Product

PurposeFilgrastim, a recombinant human granulocyte-colony stimulating factor, is widely used to treat congenital and acquired neutropenia. Following patent expiration of the innovator filgrastim product, biosimilar filgrastim products have been approved in the EU and shown to be comparable with the innovator with respect to quality, safety and efficacy. In less regulated markets, copy filgrastim products are available but data about their quality are scarce. In the present study, we provide a head-to-head comparative study on the quality of biosimilar and copy filgrastim products.MethodsInnovator filgrastim product, Neupogen®, two EU-licensed biosimilars, Zarzio® and Tevagrastim®, and two copy filgrastim products, Biocilin® and PDgrastim®, were subjected to peptide mapping, circular dichroism spectroscopy, fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance size-exclusion chromatography, reversed-phase ultra-performance liquid chromatography, endotoxin test, flow imaging microscopy and in vitro potency assay.ResultsZarzio® and Tevagrastim® have comparable quality to Neupogen®, while Biocilin® showed a significantly lower and PDgrastim® a higher specific activity. Moreover, PDgrastim® showed a higher level of impurities and a lower thermo stability than the other products.ConclusionsExcept for the deviating specific activities of the two copy filgrastim products, we found no substantial differences in product quality between the filgrastim products studied.

Development and Validation of an Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for the Concurrent Measurement of Gabapentin, Lamotrigine, Levetiracetam, Monohydroxy Derivative of Oxcarbazepine, and Zonisamide Concentrations in Serum in a Clinical Setting.

Therapeutic drug monitoring of antiepileptic drugs (AEDs) is often necessary to prevent associated destructive toxicities. Tandem mass spectrometry (MS/MS) with stable-isotope-labeled internal standards is considered the gold standard for the measurement of AEDs. This study presents the development and validation of a clinical ultra-performance liquid chromatography-MS/MS method for the concurrent measurement of gabapentin, lamotrigine, levetiracetam, monohydroxy derivative of oxcarbazepine, and zonisamide in human serum. To determine the optimal assay analyte range, one year of AED therapeutic drug monitoring results (n = 1825) were evaluated. Simple protein precipitation with acetonitrile containing isotopically labeled internal standards was used. Reverse-phase ultra-performance liquid chromatography chromatographic separation was used, having a total run time of 3 minutes. Quantification of analytes was accomplished using electrospray ionization in positive ion mode and collision-induced dissociation MS. Assay parameters were evaluated per Food and Drug Administration bioanalytical guidelines. After evaluating internal patient data, the analytical measuring range (AMR) of the assay was established as 0.1-100 mcg/mL. All AEDs were linear across the AMR, with R values ranging from 0.9988 to 0.9999. Imprecision (% coefficient of variation) and inaccuracy (% difference) were calculated to be <20% for the lower limit of quantitation and <15% for the low, mid, and high levels of quality controls across the AMR. All AEDs demonstrated acceptable assay parameters for carryover, stability under relevant storage conditions, matrix effects, recovery, and extraction and processing efficiency. In addition, the assay displayed acceptable concordance to results obtained from a national reference laboratory, with Deming regression R of 0.99 and slope values ranging from 0.89 to 1.17. A simple, cost-effective, and robust ultra-performance liquid chromatography-tandem mass spectrometry method for monitoring multiple AEDs was developed and validated to address the clinical needs of patients at our institution.

Development and Validation of UPLC–PDA Method for Quality Control of Dikamali gum—A Natural Tablet Binder: Fast Simultaneous Quantitation of Six Polymethoxyflavones

Dikamali gum (Gardenia lucida Roxb., Family—Rubiaceae) in wet and dry form is used as one of the popular tablet binders in the Indian traditional herbal formulations. Recently, its application as a binder in modern drug delivery has also been reported. However, its quality specification is yet to be established. To the best of our knowledge, no validated method is reported so far for the quality assurance of Dikamali. A new reverse-phase ultraperformance liquid chromatography–photodiode array (UPLC–PDA) method was developed for the simultaneous quantification of six polymethoxyflavones (PMFs), viz., gardenin E, gardenin D, xanthomicrol, 5-desmethynobiletin, gardenin A, and gardenin B, in Dikamali gum using a Waters X-select C18 (2.5 µm, 2.1 × 100 mm) column. The influence of acid additive in mobile phase on peak response and column temperature on the peak resolution was also investigated. ICH guideline was followed for method validation of analytical procedures for the compliance of precision, accuracy, linearity, etc. The method is about three times faster than classical HPLC analysis and completed within 15 min. The limits of detection (LOD) and limit of quantitation (LOQ) were in the range 0.02–0.06 and 0.08–0.20 µg mL− 1, respectively. Calibration curves were linear from 0.18 to 1.78 µg mL− 1 with acceptable correlation coefficients ranged 0.9993–0.9999. The percent mean recoveries of the PMFs were ranged from 96.07 to 101.69%. Successful analysis of three G. lucida samples from different locations demonstrated the method to be reproducible and convenient. The present work also provides some references for quality specification of G. lucida based on polymethoxyflavones.

An impurity characterization based approach for the rapid development of integrated downstream purification processes.

In this study, we describe a new approach for the characterization of process-related impurities along with an in silico tool to generate orthogonal, integrated downstream purification processes for biological products. A one-time characterization of process-related impurities from product expression in Pichia pastoris was first carried out using linear salt and pH gradients on a library of multimodal, salt-tolerant, and hydrophobic charge induction chromatographic resins. The Reversed-phase ultra-performance liquid chromatography (UPLC) analysis of the fractions from these gradients was then used to generate large data sets of impurity profiles. A retention database of the biological product was also generated using the same linear salt and pH gradients on these resins, without fraction collection. The resulting two data sets were then analyzed using an in silico tool, which incorporated integrated manufacturing constraints to generate and rank potential three-step purification sequences based on their predicted purification performance as well as whole-process "orthogonality" for impurity removal. Highly ranked sequences were further examined to identify templates for process development. The efficacy of this approach was successfully demonstrated for the rapid development of robust integrated processes for human growth hormone and granulocyte-colony stimulating factor.

Serum metabolomics using ultra performance liquid chromatography coupled to mass spectrometry in lactating dairy cows following a single dose of sporidesmin

IntroductionPhotosensitization is a common clinical sign in cows suffering from liver damage caused by the mycotoxin sporidesmin. This disease, called facial eczema (FE), is of major importance in New Zealand. Current techniques for diagnosing animals with subclinical sporidesmin-induced liver damage (i.e. without photosensitization) are nonspecific. In addition, little is known of the mechanisms involved in sporidesmin resistance, nor the early effects seen following low-dose sporidesmin intoxication.ObjectiveThe objective of this study was to identify individual metabolites or metabolic profiles that could be used as serum markers for early stage FE in lactating cows.MethodsResults are presented from a 59-day sporidesmin challenge in Friesian-cross dairy cows. Serum metabolite profiles were obtained using reversed phase ultra-performance liquid chromatography (UPLC) electrospray ionization mass spectrometry (MS) and UPLC tandem MS. Multivariate and time series analyses were used to assess the data.ResultsStatistical analysis, both with and without the temporal component, could distinguish the profiles of animals with clinical signs from the others, but not those affected subclinically. An increase in the concentrations of a combination of taurine- and glycine-conjugated secondary bile acids (BAs) was the most likely cause of the separation. This is the first time that MS methods have been applied to FE and that bile acids changes have been detected in cattle exposed to sporidesmin.ConclusionsIt is well known that BA concentrations increase during cholestasis due to damage to bile ducts and leakage of the bile. This is the first study to investigate metabolomic changes in serum following a sporidesmin challenge. Further work to establish the significance of the elevation of individual BAs concentrations in the serum of early-stage sporidesmin-poisoned cows is necessary.

Effects of water deficit on breadmaking quality and storage protein compositions in bread wheat (Triticum aestivum L.).

Water deficiency affects grain proteome dynamics and storage protein compositions, resulting in changes in gluten viscoelasticity. In this study, the effects of field water deficit on wheat breadmaking quality and grain storage proteins were investigated. Water deficiency produced a shorter grain-filling period, a decrease in grain number, grain weight and grain yield, a reduced starch granule size and increased protein content and glutenin macropolymer contents, resulting in superior dough properties and breadmaking quality. Reverse phase ultra-performance liquid chromatography analysis showed that the total gliadin and glutenin content and the accumulation of individual components were significantly increased by water deficiency. Two-dimensional gel electrophoresis detected 144 individual storage protein spots with significant accumulation changes in developing grains under water deficit. Comparative proteomic analysis revealed that water deficiency resulted in significant upregulation of 12 gliadins, 12 high-molecular-weight glutenin subunits and 46 low-molecular-weight glutenin subunits. Quantitative real-time polymerase chain reaction analysis revealed that the expression of storage protein biosynthesis-related transcription factors Dof and Spa was upregulated by water deficiency. The present results illustrated that water deficiency leads to increased accumulation of storage protein components and upregulated expression of Dof and Spa, resulting in an improvement in glutenin strength and breadmaking quality. © 2018 Society of Chemical Industry.