Acid Mobile Phase Research Articles

LC-MS/MS determination of a human mAb drug candidate in rat serum using an isotopically labeled universal mAb internal standard.

We report the application of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical method for the determination of a recombinant human immunoglobulin G1 (hIgG1), NVSMAb-1, in rat serum. A stable isotopically labeled universal monoclonal antibody (SILuMab), instead of stable isotopically labeled surrogate peptide, was employed as the internal standard. The internal standard was added to the sample matrix in the first step of the sample preparation process, which involved protein precipitation and pellet digestion. The digestion of the resulting pellet with trypsin was performed prior to analysis of surrogate peptides of both NVSMAb-1 and SILuMab using LC-MS/MS. Precipitation reagents (1% TCA in IPA, 75% MeOH and 14% PEG) and digestion conditions (50°C for 2h and 60°C for 0.5h) were evaluated by monitoring LC-MS/MS responses of GPS and VVS in the resulting sample extracts. Overall, the use of 1% TCA in IPA appeared to be more effective as compared to 75% methanol in protein precipitation and removal of unwanted matrix components, e.g., albumin, and more appealing than 14% PEG as it avoided additional steps that are necessary to remove PEG or reduce PEG to a negligible level. The yield (LC-MS/MS response) of GPS is less sensitive than VVS to the changes of digestion conditions (time and temperature). The results obtained using SILuMab over SIL surrogate peptide as the internal standard appeared unaffected by the suboptimal sample processing method. For the current assay, surrogate peptide GPSVFPLAPSSK (GPS) was selected as surrogate peptide over VVSVLTVLHQDWLNGK (VVS) for quantitative analysis of NVSMAb-1. The optimal chromatographic separation was achieved on a Waters Cortecs C18 (100×2.1mm, 2.7μm) column using gradient elution with a total cycle time of approximately 8min. The mobile phases were water containing 0.1% formic acid (mobile phase A) and acetonitrile containing 0.1% formic acid (mobile phase B). The current method was validated for specificity, sensitivity, matrix effect, recovery, linearity, accuracy and precision, dilution integrity, and stability. The validated assay dynamic range was 10-5000μg/mL using 20μL of rat serum. The accuracy and precision for the LLOQs (10μg/mL) were within ±6.0% bias and ≤6.5% CV, respectively. From the intra-day and inter-day assay performance evaluations, the precision of the other QC sample (30, 300, 2500 and 3750μg/mL) results were ≤6.8% CV and the accuracy within ±4.8% bias, respectively. Additional assessment of incurred sample reanalysis (ISR) was conducted to demonstrate the ruggedness and robustness of the assay method. The validated method was successfully implemented in support of a toxicity study in rats administered 30, 150 and 750mg/kg/week intravenous infusion and 150mg/kg/week subcutaneous injection of NVSMAb-1.

Hydrophilic interaction liquid chromatography-mass spectrometry for cinnamic acid determination and its use to evaluate components of dried hot peppers (Capsicum annum ) associated with growth inhibition of Salmonella enterica serovars

A rapid and selective hydrophilic interaction chromatography-mass spectrometry (HILIC-MS) method was developed to measure cinnamic acids in dried chili peppers (Capsicum annum) for the purpose of investigating the association between cinnamic acid levels and inhibition of Salmonella growth in the peppers. Trans-cinnamic and hydroxy-cinnamic acids were quantified in 15 varieties of chili peppers by the HILIC-MS method, using electrospray ionization in negative mode with multiple reaction monitoring for detection. Trans-cinnamic acid and 4-OH-cinnamic acid were found in all samples; highest concentrations were 0.81 and 7.15 ppm, respectively. The compound, 2-OH-cinnamic acid was not detected. Mean recoveries of the target compounds from spiked samples were greater than 79%. The pepper samples were evaluated for Salmonella growth inhibition after addition of moisture to the dried samples. No correlation was found between Salmonella growth inhibition and cinnamic acid content, suggesting that these cinnamic acids are not major inhibitory compounds in these peppers. Practical applications The HILIC-MS method developed for the analysis of dried chili peppers is a simple, sensitive method for organic acids and is applicable to any food matrix. Only extraction and automated filtration are employed to produce extracts for analysis. Ammonium formate buffer in the mobile phase, pH 6, enhances ionization of acids for negative ESI-MS detection relative to the degree of ionization from use of acidic mobile phases containing formic or acetic acid modifiers. The use of 4-Cl-benzoic acid as internal standard and evaluation of matrix effects from the dried pepper extracts are important for future research in similar applications. The HILIC chromatographic separation may separate critical peak pairs in pepper extracts that are not resolvable by reversed-phase HPLC.

Trace analysis of amines in cheese serum with liquid chromatographic potentiometric detection by using amine-selective electrode

A trace quantification liquid chromatographic-potentiometric method for the determination of amines in cheese serum is described. An all solid-state contact polyvinyl chloride membrane amine-selective electrode was prepared and used as a detector in a low-dead volume flow through detection cell. Potentiometric performance characteristics of the proposed electrodes were then examined. A slope of 52.4±3.8mV per decade toward amines was received at the concentration range of 1.0×10−2–8.0×10−6M (r2=0.99) under dynamic conditions. Six amine compounds were separated, with a good resolution in the chromatographic system by using acetonitrile-methane sulfonic acid mobile phase gradient at the flow-rate of 1mLmin−1 in 30min. The potentiometric detector was successfully applied to the analysis of amines in cheese serum samples. The relative standard deviations were ranged from 2.2% to 4.0% and recoveries were ranged from 88% to 101% for the determination. The average concentrations of ethylamine, butylamine and hexylamine in cheese serum samples calculated were 6.46, 4.51 and 8.37μgL−1 respectively.

Functionalized nanoparticles based solid-phase membrane micro-tip extraction and high-performance liquid chromatography analyses of vitamin B complex in human plasma.

Iron nanoparticles were prepared by a green method following functionalization using 1-butyl-3-methylimidazolium bromide. 1-Butyl-3-methylimidazole iron nanoparticles were characterized using FTIR spectroscopy, energy dispersive X-ray fluorescence, X-ray diffraction, scanning electron microscopy and transmission electron microscopy. The nanoparticles were used in solid-phase membrane micro-tip extraction to separate vitamin B complex from plasma before high-performance liquid chromatography. The optimum conditions obtained were sorbent (15 mg), agitation time (30 min), pH (9.0), desorbing solvent [water (5 mL) + methanol (5 mL) + sodium hydroxide (0.1 N) + acetic acid (d = 1.05 kg/L, pH 5.5), desorbing volume (10 mL) and desorption time (30 min). The percentage recoveries of all the eight vitamin B complex were from 60 to 83%. A high-performance liquid chromatography method was developed using a PhE column (250×4.6 mm, 5.0 μm) and water/acetonitrile (95:5, v/v; pH 4.0 with 0.1% formic acid) mobile phase. The flow rate was 1.0 mL/min with detection at 270 and 210 nm. The values of the capacity, separation and resolution factor were 0.57-39.47, 1.12-6.00 and 1.84-26.26, respectively. The developed sample preparation and chromatographic methods were fast, selective, inexpensive, economic and reproducible. The developed method can be applied for analyzing these drugs in biological and environmental matrices.

Qualitative and quantitative two-dimensional thin-layer chromatography/high performance liquid chromatography/diode-array/electrospray-ionization-time-of-flight mass spectrometry of cholinesterase inhibitors

Recently launched thin-layer chromatography-mass spectrometry (TLC–MS) interface enabling extraction of compounds directly from TLC plates into MS ion source was unusually extended into two-dimensional thin-layer chromatography/high performance liquid chromatography (2D, TLC/HPLC) system by its a direct connection to a rapid resolution 50×2.1mm, I.D. C18 column compartment followed by detection by diode array (DAD) and electrospray ionisation time-of-flight mass spectrometry (ESI-TOF-MS). In this way, even not separated bands of complicated mixtures of natural compounds could be analysed structurally, only within 1–2min after development of TLC plates. In comparison to typically applied TLC–MS interface, no ion suppression for acidic mobile phases was observed. Also, substantial increase in ESI-TOF-MS sensitivities and quality of spectra, were noticed. It has been utilised in combination with TLC- based bioautographic approaches of acetylcholinesterase (AChE) inhibitors, However, it can be also applied in any other procedures related to bioactivity (e.g. 2,2-Diphenyl-1-picryl-hydrazyl—DPPH screen test for radicals). This system has been also used for determination of half maximal inhibitory concentration (IC50 values) of the active inhibitor—galanthamine, as an example. Moreover, AChE inhibitory potencies of some of purified plant extracts, never studied before, have been quantitatively measured. This is first report of usage such the 2D TLC/HPLC/MS system both for qualitative and quantitative evaluation of cholinesterase inhibitors in biological matrices.

Use of basic mobile phase to improve chromatography and boost sensitivity for quantifying tetrahydrocurcumin in human plasma by LC-MS/MS.

Tetrahydrocurcumin (THC), a major metabolite of curcumin, is often quantified by LC-MS or LC-MS/MS using acidic mobile phases due to the concern of its instability in a basic medium. However, acidic mobile phases often lead to poor chromatography (e.g. split or double peaks) and reduced detection sensitivity in the commonly used negative ionization mode. To overcome these shortcomings, a basic mobile phase was used for the first time in the LC-MS/MS quantification of THC. In comparison with the acidic mobile phases, a single symmetrical chromatographic peak was obtained and the sensitivity increased by 7-fold or more under the equivalent conditions. The new LC-MS/MS method using the basic mobile phase has been successfully validated for the quantification of THC in human EDTA plasma over the concentration range of 5-2500ng/ml. The within-batch accuracy (% nominal concentration) was between 88.7 and 104.9 and the between-batch accuracy ranged from 96.7 to 108.6. The CVs for within- and between-batch precisions were equal to or less than 5.5% and 9.1%, respectively. No significant matrix interference or matrix effect was observed from normal or lipemic and hemolytic plasma matrices. In addition, the common stabilities with adequate durations were established, including up to 5days of post-preparative stability. Furthermore, when the validated method was applied to a clinical study, the passing rate of ISR samples was 83%, indicating the good reproducibility of the method. The success of the unconventional approach presented in this article demonstrates that a mobile phase could be selected based mainly on its merits to facilitate LC separation and/or MS detection. There is no need for excessive concern about the stability of the compound(s) of interest in the selected mobile phase because the run time of modern LC-MS or LC-MS/MS methods is typically only a few minutes.

Validated methods for determination of neurotransmitters and metabolites in rodent brain tissue and extracellular fluid by reversed phase UHPLC-MS/MS.

Fast and sensitive methods for simultaneous determination of dopamine (DA), the two DA-metabolites homovanillic acid (HVA) and 3-methoxytyramine (3-MT), serotonin (5-HT) and the 5-HT-metabolite 5-hydroxyindoleacetic acid (5-HIAA), norepinephrine (NE), acetylcholine (ACh), glutamic acid (Glu) and γ-aminobutyric acid (GABA) in rodent brain tissue (1.0-4000nM) and extracellular fluid (ECF) (0.5-2000nM) based on ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) have been developed. Of the three different sample preparation methods for brain tissue samples tested, a simple and rapid protein precipitation procedure with formic acid was found to give the best results. The neurotransmitters (NTs) and NT metabolites were separated using UHPLC with an Acquity UPLC HSS T3 C18 column (2.1×100mm, 1.8μm particle size) with acidic mobile phase. Gradient elution with methanol was used and quantification was performed using multiple reaction monitoring (MRM). The total run time was 5.2min including equilibration time. The methods were validated by determining calibration model, intra- and inter-day precision and accuracy, limit of detection (LOD), lower limit of quantification (LLOQ), matrix effects (ME), carry-over and stability. Surrogate analytes were used to enable determination of the recovery and ME of the endogenous analytes in brain tissue. The methods were applied for determination of NTs at basal levels in rodent brain ECF and brain tissue homogenate. The developed methods are valuable tools in the studies of mechanisms of drugs of abuse, and neurologic and psychiatric disease.

Characterization of IgG2 Disulfide Bonds with LC/MS/MS and Postcolumn Online Reduction

The complication of IgG2 disulfide connections demands advances in techniques for disulfide bond determination. We have developed a new LC/MS/MS method for improved disulfide analysis. With postcolumn introduction of dithiothreitol (DTT) and ammonium hydroxide, each disulfide-containing peptide eluted out of LC in an acidic mobile phase can be rapidly reduced prior to MS analysis. The reduction can be driven to near completion. The reagents are MS-friendly, and the reaction occurs at no cost of separation (little is added to the postcolumn dead volume of the LC system). Comparing LC/MS data with and without online reduction, a direct correlation can be established between a disulfide peptide and its composing peptides using retention time. With disulfide online removal, high-quality MS/MS fragmentation data can be acquired and allows for definitive determination of the disulfide peptide. This technique is especially valuable in determining the disulfide bond linkage of complicated molecules such as the hinge-containing disulfide peptides produced from IgG2 disulfide isoforms. Due to over/under enzymatic cleavages, multiple hinge-containing disulfide peptides are produced from each isoform. Twenty-two hinge-containing disulfide peptides in total have been confidently identified with this technique. Without the method, successful identification to many of these peptides would have become extremely difficult.

Simultaneous quantitative determination of 5-aza-2′-deoxycytidine genomic incorporation and DNA demethylation by liquid chromatography tandem mass spectrometry as exposure-response measures of nucleoside analog DNA methyltransferase inhibitors

The epigenetic and anti-cancer activities of the nucleoside analog DNA methyltransferase (DNMT) inhibitors decitabine (5-aza-2'-deoxycytidine, DAC), azacitidine, and guadecitabine are thought to require cellular uptake, metabolism to 5-aza-2'-deoxycytidine triphosphate, and incorporation into DNA. This genomic incorporation can then lead to trapping and degradation of DNMT enzymes, and ultimately, passive loss of DNA methylation. To facilitate measurement of critical exposure-response relationships of nucleoside analog DNMT inhibitors, a sensitive and reliable method was developed to simultaneously quantitate 5-aza-2'-deoxycytidine genomic incorporation and genomic 5-methylcytosine content using LC-MS/MS. Genomic DNA was extracted and digested into single nucleosides. Chromatographic separation was achieved with a Thermo Hyperpcarb porous graphite column (100mm×2.1mm, 5μm) and isocratic elution with a 10mM ammonium acetate:acetonitrile with 0.1% formic acid (70:30, v/v) mobile phase over a 5min total analytical run time. An AB Sciex 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for the detection of 5-aza-2'-deoxycytidine, 2'-deoxycytidine, and 5-methyl-2'-deoxycytidine. The assay range was 2-400ng/mL for 5-aza-2'-deoxycytidine, 50-10,000ng/mL for 2'-deoxycytidine, and was 5-1000ng/mL for 5-methyl-2'-deoxycytidine. The assay proved to be accurate (93.0-102.2%) and precise (CV≤6.3%) across all analytes. All analytes exhibited long-term frozen digest matrix stability at -70°C for at least 117 days. The method was applied for the measurement of genomic 5-aza-2'-deoxycytidine and 5-methyl-2'-deoxycytidine content following exposure of in vitro cell culture and in vivo animal models to decitabine.

Effects of starvation for 4–20 days on the neutral lipid and phospholipid content of Biomphalaria glabrata as determined by high performance thin layer chromatography-densitometry

ABSTRACTThe effects of 4, 7, 10, 14, and 20 days of starvation on the neutral and phospholipid levels of Biomphalaria glabrata were determined by high performance thin layer chromatography (HPTLC)-densitometry. Snails were maintained in artificial spring water in jars at 25 ± 1°C. The digestive gland–gonad complex was extracted with chloroform–methanol (2:1), and Analtech laned preadsorbent silica gel HPTLC plates were used with petroleum ether–diethyl ether–glacial acetic acid (80:20:1) mobile phase, phosphomolybdic acid detection reagent, and scanning at 610 nm for neutral lipid analysis, and chloroform–methanol–water (65:25:4.5) mobile phase, cupric sulfate–phosphoric acid reagent, and scanning at 370 nm for phospholipids. Significant differences were observed in the triacylglycerol and steryl ester levels in the starved versus the control snails.

Micellar and sub-micellar ultra-high performance liquid chromatography of hydroxybenzoic acid and phthalic acid positional isomers

Micellar liquid chromatography (MLC) has been used primarily for the separation of neutral analytes of varying polarities, most commonly phenols and polyaromatic hydrocarbons, but does not seem to have been used to study aromatic hydroxy acids in detail. We have studied the separation of hydroxybenzoic acid mixtures, including monohydroxybenzoic and dihydroxybenzoic acid positional isomers by MLC. Sodium dodecylsulfate (SDS) is investigated as the modifying surfactant on a C18 ultra-high performance liquid chromatography (UHPLC) column (100×2.1mm, 1.8μm). The addition of only SDS (no organic solvent) to the mobile phase reduced the influence of hydrophobic interactions while improving the retention times, resolution, and peak shapes, even at concentrations below the critical micellization concentration (CMC). The UHPLC separation of 7 hydroxybenzoic acids, including 6 dihydroxybenzoic acid positional isomers and one trihydroxybenzoic acid, is achieved with high efficiency using 0.1% SDS in 1.84mM sulfuric acid (pH 2.43) mobile phase, in less than 6min with a flow rate of 0.3mLmin−1, and in less than four min with a flow rate of 0.7mLmin−1. Six monohydroxybenzoic acid isomers are also effectively separated by MLC, using a 0.5% SDS mobile phase modifier, in less than 20min with a flow rate of 0.3mLmin−1, and in less than 14min with a flow rate of 0.7mLmin−1. The 3 phthalic acid isomers could be separated using a similar mobile phase and flow rates in less than 6 and 4min. Solute-micelle equilibrium constants and partition coefficients are calculated for 6 monohydroxybenzoic acids based on a plot of MLC retention factor vs. mobile phase micelle concentration. All aromatic acid isomers studied can be classified as binding solutes in the MLC retention mechanism. Less effective separations are observed with shorter chain surfactants, leading to higher retention times and poor peak shapes. It is concluded that increasing chain length led to more efficient MLC separations, and SDS is the preferred modifying surfactant for the examined separation.

High Performance Liquid Chromatography Tandem Mass Spectrometry Measurement of Bimatoprost, Latanoprost and Travoprost in Eyelash Enhancing Cosmetic Serums

Most common prostaglandin analogs, bimatoprost, latanoprost and travoprost, are licensed for the reduction of elevated intraocular pressure in patients with open angle glaucoma and ocular hypertension, but their non approved use as eyelash enhancers is becoming popular, especially in patients with eyelashes hypotrichosis. A fast and sensitive high performance liquid chromatography tandem mass spectrometry method was developed for the measurement of bimatoprost, latanoprost and travoprost in cosmetic serums freely web-sold to increase eyelash length, thickness and darkness. The analytes and the internal standard (reserpine) were separated by reversed phase chromatography with 5 mM ammonium acetate with 0.02% formic acid (mobile phase A) and 5 mM ammonium acetate in acetonitrile/water (95/5; v/v) with 0.02% formic acid (mobile phase B) by gradient elution and detected with tandem mass spectrometry operated in multiple reaction monitoring mode. Linearity between 1 and 500 μg/g shows good correlation coefficients (r2 = 0.99) for all substances. Analytical recovery of analytes under investigation were always higher than 90% and intra-assay and inter-assay precision and accuracy always better than 11%. This method was successfully applied to analyze cosmetic serums freely sold on the Internet websites.

High-performance thin-layer chromatography and pressurized planar electrochromatography of some diastereomeric amino acid derivatives in reversed-phase system with carboxylic acid mobile phase buffers

The separation of 1-fluoro-2,4-dinitrophenyl-5-l-valine amide (FVDA) diastereomeric derivatives of aspartic acid, cysteine, and histidine by means of high-performance thin-layer chromatography (HPTLC) as well as pressurized planar electrochromatography (PPEC) techniques in systems with HPTLC RP-18W plates and the various acetonitrile—buffer mobile phases is presented. The influence of the mobile phase components, i.e., acetonitrile concentration and buffer kind on migration distance of the solute zones, was investigated. The effect of mono (formic) and various dicarboxylic acids (oxalic, malonic, maleic, malic, succinic, tartaric, and pimelic) as the mobile phase buffer components on the solute retention was studied. It is observed that an increase of acetonitrile content of the mobile phase affects the solute zone migration and retention in PPEC and HPTLC. What is more, the separation selectivity in the latter and former techniques differs. The PPEC mode presents a higher efficiency in comparison with HP...

Analysis of Linear Alkylbenzene Sulfonate in Laundry Wastewater by HPLC–UV and UV–Vis Spectrophotometry

AbstractA lack of natural water resources and an increase in the demand for fresh potable water has shifted focus to the possible reuse of recycled laundry wastewater water that is considered to be relatively clean. Organic components such as linear alkylbenzene sulfonates (LAS) are the major and most abundant contributing anionic surfactant constituents found in laundry detergents. The development and reliability of treatment methods targeted at purification of laundry wastewater necessitates a fast and accurate method for quantification of LAS. This paper focuses on a comparative study for the quantification of LAS based on traditional liquid–liquid extraction (LLE) and HPLC–UV methods. In the case of LLE, the anionic surfactant LAS complexes via ion association to a methylene blue (MB) cationic dye resulting in the formation of an anionic surfactant–methylene blue (AS–MB) complex. The AS–MB complex extracted with chloroform absorbs at a λ max of 653 nm. Optimized conditions for quantification of a single eluted LAS peak using HPLC–UV were obtained by isocratic elution on a C18 column with a 95 % acetonitrile and 5 % 0.7 M acetic acid mobile phase. Both methods displayed percentage recoveries >90 % and statistically showed reproducibility and precision in the quantitation of LAS. HPLC–UV prevailed over UV–Vis as the method of choice for LAS determinations given the ease of sample preparation and applicability to a wider range of samples. Typical levels of LAS in laundry samples assessed in this study ranged between 116 and 454 mg L−1.

Quantitative analysis of pasireotide (SOM230), a cyclic peptide, in monkey plasma using liquid chromatography in combination with tandem mass spectrometry.

A novel liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the determination of Pasireotide (SOM230) was developed and validated with a dynamic range of 0.5-250ng/ml using 50μl of monkey plasma. SOM230 and the internal standard, [M+6]SOM230, were extracted from monkey plasma via μElution SPE. The acidified sample matrix was loaded onto the preconditioned Waters SPE plate for further processing. The analyte was eluted from the SPE plate using freshly prepared elution solvent, which was followed by dilution and LC-MS/MS analysis. By eliminating a step of evaporation of the SPE eluent, instead, injecting the eluent after a simple dilution, compound loss due to non-specific binding to the 96-well materials was prevented. Furthermore, freshly prepared elution solution was found a key to optimal extraction recovery of the analyte from monkey plasma. The optimal chromatographic separation was achieved on an Atlantis dC18 (50×2.1mm, 5μm particle size) column using gradient elution with a total analysis cycle time approximately 4min per injection. The mobile phases were water containing 0.5% acetic acid and 0.05% trifluoroacetic acid (TFA) (mobile phase A) and acetonitrile containing 0.5% acetic acid and 0.05% TFA (mobile phase B). The incorporation of TFA (0.05%, v/v) and acetic acid (0.5%, v/v) into the mobile phases was accompanied by the improved chromatography and minimized carryover due to the HPLC column. The current method was validated for specificity, sensitivity, matrix effect, recovery, linearity, accuracy and precision, dilution integrity, batch size and stability. The accuracy and precision for the LLOQs (0.5ng/ml) were within ±5.6% bias and ≤7.8% CV, respectively. From the intra-day and inter-day evaluations, the precision of the other QC samples (1.5, 7.5, 75 and 190ng/ml) ranged from 2.7 to 4.9% CV and the accuracy (% bias) from -1.3 to 7.3%, respectively. Additional assessment of incurred sample reanalysis (ISR) was conducted to demonstrate the ruggedness and robustness of the assay method. The validated method was successfully implemented to support a toxicity study in monkeys administered with 5 and 30mg of SOM230 in a single intramuscular injection of a long acting release (LAR) formulation.

Determination of Growth-Phase Dependent Influences Exerted by Prions on Yeast Lipid Content Using HPTLC-Densitometry.

Prions of the baker's yeast Saccharomyces cerevisiae allow for the inheritance of complex traits based solely on the acquisition of cytoplasmic protein aggregates and confer distinctive phenotypes to the cells which harbor them, creating heterogeneity within an otherwise clonal cell population. These phenotypes typically arise from a loss-of-function of the prion-forming protein that is unable to perform its normal cellular function(s) while sequestered in prion amyloid aggregates, but the specific biochemical consequences of prion infection are poorly understood. To begin to address this issue, we initiated a direct investigation into the potential control that yeast prions exert over fungal lipid content by utilizing the prions [URE3] and [PSI+], the first two prions discovered in yeast. We utilized silica gel high-performance thin-layer chromatography (HPTLC)-densitometry to conduct pair-wise quantifications of the relative levels of free sterols, free fatty acids, and triacylglycerols [petroleum ether-diethyl ether-acetic acid (80:20:1) mobile phase, phosphomolybdic acid (PMA) detection reagent]; steryl esters and squalene (hexane-petroleum ether-diethyl ether-acetic acid (50:20;5:1), PMA]; and phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol (chloroform-diethyl ether-acetic acid (65:25:4.5), cupric sulfate-phosphoric acid) in otherwise clonal prion-infected ([PSI+] or [URE3]) and prion-free ([psi-] or [ure-o]) cells in two growth phases: log-phase and stationary phase. Our analysis revealed multiple statistically significant differences (p < 0.00625) between prion-infected and prion-free cells. Interestingly, prion-induced changes varied dramatically by growth phase, indicating that prions exert differential influences on cell physiology between log and stationary growth. Further experimental replication and extension of the analysis to other prions is expected to resolve additional physiological effects of prion infection. This investigation demonstrates that HPTLC-densitometry is an effective method for studying prion-induced alterations in lipid content in yeast.

Bioprofiling of unknown antibiotics in herbal extracts: Development of a streamlined direct bioautography using Bacillus subtilis linked to mass spectrometry

Working in the field of profiling and identification of bioactive compounds in herbal extracts is faced with the challenge that common chromatographic methods do not directly link to bioactive compounds. Direct bioautography, the combination of TLC/HPTLC with bioassays, linked to structure elucidating techniques is demonstrated to overcome this challenge. The combination of TLC and Bacillus subtilis bioassay was already demonstrated to detect the antibiotics in samples. However, previous studies in this field were faced with some challenges, like being time-consuming, leading not to a homogenous plate background or being restricted to a non-acidic mobile phase. In this study, these aspects were investigated and a streamlined HPTLC-B. subtilis bioassay was developed that generated a homogenous plate background, which was crucial to yield a good baseline for biodensitometry. Two commonly used broths for B. subtilis and a self-designed medium were compared with regard to their capability of detection and baseline noise. The workflow developed allowed the use of acidic mobile phases for the first time. To prove this, 20 herbal extracts were screened for antimicrobial substances developed in parallel with an acidic mobile phase. The main antimicrobial substance in Salvia officinalis tincture detected was further characterized by microchemical reactions, Aliivibrio fischeri, β-glucosidase and acetylcholinesterase (bio)assays as well as mass spectrometry. Scientists looking for new herbal-based medicine may benefit from this time-saving and streamlined bioactivity profiling.

A robust and rapid liquid chromatography tandem mass spectrometric method for the quantitative analysis of 5-azacytidine.

The DNA methyltransferase inhibitor 5-azacytidine is being evaluated clinically as an oral formulation to treat various solid tumors. A sensitive, reliable method was developed to quantitate 5-azacytidine using LC-MS/MS to perform detailed pharmacokinetic studies. The drug of interest was extracted from plasma using Oasis MCX ion exchange solid-phase extraction 96-well plates. Chromatographic separation was achieved with a YMC J'sphere M80 C18 column and isocratic elution with a methanol-water-formic acid (15:85:0.1, v/v/v) mobile phase over a 7 min total analytical run time. An AB Sciex 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for the detection of 5-azacytidine. The assay range was 5-500 ng/mL and proved to be accurate (97.8-109.1%) and precise (CV ≤ 9.8%). Tetrahydrouridine was used to stabilize 5-azacytidine in blood/plasma samples. With the addition of tetrahydrouridine, long-term frozen plasma stability for 5-azacytidine at -70°C has been determined for at least 323 days. The method was applied for the measurement of total plasma concentrations of 5-azacytidine in a cancer patient receiving a 300 mg oral daily dose.

Alleviating nonlinear behavior of disulfide isoforms in the reversed-phase liquid chromatography of IgG2

Reversed-phase chromatography is an established method for characterizing the disulfide isoforms of IgG2. This work explores the effect of mobile phase gradient profile and sample concentration on the separation of disulfide isoforms. The acidic mobile phase can alter the relative proportions of disulfide isoforms, but only when the level of the reactive A1 isoform is much higher than for typical conditions of separation and typical IgG2 samples. Otherwise, there is minimal disulfide scrambling. A slower gradient and flow rate modestly improve resolution, but the peaks remain heavily overlapped. Resolution is further improved and nonlinear chromatography lessened when injection is performed under non-stacking conditions. Non-stacking conditions also keep the concentration from spiking at the head of the column, reducing noncovalent associations that can promote disulfide scrambling. The higher resolution from non-stacking injection reveals the presence of at least seven species.

Simultaneous Determination of Trigonelline, Caffeine, Chlorogenic Acid and Their Related Compounds in Instant Coffee Samples by HPLC Using an Acidic Mobile Phase Containing Octanesulfonate.

In order to analyze trigonelline, caffeine, chlorogenic acid, and their related compounds simultaneously, an HPLC method using an InertSustain C18 column and a mobile phase containing octanesulfonate as an ion-pairing reagent under an acidic condition was developed. The optimum mobile phase conditions were determined to be 0.1% phosphoric acid, 4 mM octanesulfonate, and 15% methanol at 35°C. Using the proposed method, trigonelline, nicotinic acid, caffeine, theophylline, chlorogenic acid, and caffeic acid in ten instant coffee samples were analyzed. These analytes except for theophylline were detected in all samples. An increase in the caffeine content in instant coffee samples tended to decrease in both trigonelline and chlorogenic acid contents, and the trigonelline content was found to be correlated well with the chlorogenic acid content (R(2) = 0.887).