High-performance Liquid Chromatography Retention Times Research Articles

Identification of Hemoglobin Variants Prevalent in China and Their Effects on Hemoglobin A1c Measurements.

We aimed to evaluate the effects of hemoglobin (Hb) variants prevalent in China on HbA1c measurements and to identify them during HbA1c measurements. We evaluated a cation-exchange high-performance liquid chromatography (HPLC) method (Bio-Rad D-100), a capillary electrophoresis (CE) method (Capillarys 3 TERA), an immunoassay (Cobas c501), and a boronate affinity method (Premier Hb9210, as a comparative method) for HbA1c measurements in the presence of Hb variants prevalent in China. The Bio-Rad D-100 and Capillarys 3 TERA gave specific retention times and numeric migration positions for each Hb variant, respectively, showing excellent interindividual reproducibility. All methods showed statistically significant differences (P < .01) for several variants. Clinically significant effects were observed for the Bio-Rad D-100 (Hb New York and Hb J-Bangkok), Capillarys 3 TERA (Hb New York and Hb J-Bangkok), and Cobas c501 (Hb New York). Among 297 samples with Hb variants, there were 75 (25.3%) unacceptable results for Bio-Rad D-100, 28 (9.4%) for Capillarys 3 TERA, and 19 (6.4%) for Cobas c501 compared with the results from Premier Hb9210. Some Hb variants prevalent in China affect HbA1c measurements. The HPLC retention time and CE migration position can aid in the presumptive identification of Hb variants.

Profiling and quantitation of alkaloids in different parts of Sophora alopecuroides L. extracts by high-performance liquid chromatography with electrospray ionisation ion mobility spectrometry detection.

Ambient pressure electrospray ionisation ion mobility spectrometry coupled to high-performance liquid chromatography (HPLC) was used to detect alkaloids from different parts of Sophora alopecuroides L. extracts. Multiplexing ion mobility spectrometry (IMS) was used to improve the signal-to-noise ratio while maintaining high resolving power for the detecting of eluents from HPLC separation. The alkaloids profile and distribution are demonstrated by retention time-drift time two-dimensional spaces, and the contents of five major alkaloids including sophoridine, sophocarpine, cytisine, aloperine, and matrine were determined in the leaf, skin, stem, seed kernel, and seed husk using the HPLC-IMS method. This method offers extra separation ability to isomers such as matrine and sophocarpine, which can be difficult to distinguish by mass spectrometry. The reduced mobilities for cytisine, sophoridine, sophocarpine, matrine, and aloperine are 0.828, 0.718, 0.731, 0.725, and 0.769 cm2 /V/s, respectively. The limits of detection are 0.553, 0.488, 0.479, 0.484, and 0.513 ug/mL. This method adds extra separation ability to HPLC to resolve co-eluted peaks and provides another qualitative parameter besides HPLC retention time.

New Insights into Detection of a Dendrobine Compound From a Novel Endophytic Trichoderma longibrachiatum Strain and Its Toxicity Against Phytopathogenic Bacteria.

Dendrobium nobile is the only plant that could produce the natural bioactive dendrobine. No other source of dendrobine has been found to date except from D. nobile and via chemical synthesis. In this study, we aimed to examine the potential fungal endophyte isolated from D. nobile stem segments using the molecular method and to detect dendrobine compound through high-performance liquid chromatography (HPLC), gas chromatography–mass spectrometry (GC-MS), and liquid chromatography with tandem mass spectrometry (LC-MS/MS) and their metabolite for their antibacterial activity. The potential dendrobine producer strain was recognized as Trichoderma longibrachiatum based on molecular DNA sequencing and GenBank databases. The T. longibrachiatum MD33 produced dendrobine and other compounds in a potato dextrose medium (PDM), as confirmed by HPLC retention time peak analysis. The HPLC results revealed that T. longibrachiatum MD33 biomass showed a peak retention time of 5.28 ± 0.2 min, similar to wild D. nobile stem dendrobine (5.32 ± 0.2 min) and standard chemical reference dendrobine (5.30 ± 0.2 min), indicating the presence of dendrobine in the fungal biomass. Results of GC-MS and LC-MS analysis revealed that T. longibrachiatum MD33 produced the same molecular weight (263 in GC-MS and 264.195 in LC-MS) of dendrobine as compared with standard chemical reference dendrobine and D. nobile dendrobine. Antibacterial activity data revealed that T. longibrachiatum MD33 produced the strongest bactericidal activity against Bacillus subtilis, Bacillus mycoides, and Staphylococcus species, and the diameter of the bacterial growth inhibition zone was 12 ± 0.2, 9 ± 0.2, and 8 ± 0.2 mm, respectively. To the best of our knowledge, this was the first study to investigate T. longibrachiatum as a dendrobine producer, and the results revealed that T. longibrachiatum was directly involved in the potential production of a similar bioactive compound to D. nobile (dendrobine). In addition, the T. longibrachiatum metabolite exhibited potent antibacterial activity and can be a potential strain for medical and industrial purposes.

Quantification of the Plasma Concentration of Apatinib by 2-Dimensional Liquid Chromatography.

Apatinib is a new oral micromolecular tyrosine kinase inhibitor, which is mainly used as a third-line treatment for chemotherapy-refractory advanced metastatic gastric cancer patients. However, apatinib has shown dose titration and severe adverse reactions in clinical practice. Quantification of plasma concentrations of apatinib may be an effective method to balance the clinical efficacy and adverse reactions. The purpose of this study was to develop and validate a 2-dimensional liquid chromatography method for the measurement of apatinib in plasma. The analysis of apatinib was performed using a 2-dimensional high-performance liquid chromatography system. We precipitated the proteins with acetonitrile. The mobile phases consisted of a first-dimensional mobile phase (acetonitrile:methanol:25 mmol·L ammonium phosphate = 25:25:50, V/V/V, pH adjusted to 7.2 using phosphoric acid) and a second-dimensional mobile phase (acetonitrile:10 mmol·L ammonium phosphate = 28:72, vol/vol, pH adjusted to 3.7 using phosphoric acid). The ultraviolet detection wavelength was set at 340 nm. The temperature of the detector cell was 40°C, and the injection volume was 500 μL. The range of calibration curve was 15.27-1491.48 ng/mL. The accuracy and imprecision were within ±2.23% and less than 10.22%, respectively (intraday and interday). The range of recovery was 97.45%-108.92%. The intraday and interday relative SDs (reproducibility) of high-performance liquid chromatography retention times were less than 0.18% and 0.46%, respectively. In the clinical assessment, the dose range of apatinib mesylate for patients with gastric cancer was 250-500 mg every day (2-60 days), resulting in trough plasma concentrations between 272.7 and 727.8 ng/mL. A simple, convenient, accurate, and robust 2-dimensional liquid chromatography method was developed and verified, which successfully determined the plasma concentrations of apatinib in patients with gastric cancer.

A validated stable HPLC method for the simultaneous determination of rifampicin and 25-O-desacetyl rifampicin – evaluation of in vitro metabolism

A simple, efficient, and stable high-performance liquid chromatography (HPLC) separation method for a combination of rifampicin (RIF), its major metabolite 25-O-desacetyl rifampicin (25ODESRIF), and neostigmine (NEO) was developed and validated. The drugs individually, and in combination, were analyzed using a Waters Alliance 2695 HPLC coupled with 2996 photodiode array detector (PDA). Successful separation of combined drugs was achieved by gradient elution on a reverse-phase C-18 Phenomenex Luna column, using a mobile phase consisting of water and methanol at detection wavelength of 254 nm. The HPLC retention times were consistent at ±7.70 min, ±8.25 min, and ±10.70 min for RIF, 25ODESRIF, and NEO, respectively. The regression data for the calibration plots exhibited linear relationship (R2 = 0.995) in the range of 0–200 μM for both RIF and 25ODESRIF, and the lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were calculated at 5.86 μM and 17.75 μM for RIF and 7.78 μM and 23.57 μM for 25ODESRIF, respectively. The method was evaluated using in vitro human liver microsomes (HLMs) assays, and linearity was established for the 15, 30, 45, and 60 min incubations (R2 = 0.99). The formation of 25ODESRIF was characterized by hyperbolic kinetics (Km 48.23 μM, Vmax 1.233 pmol/min/mg protein, and CLint 0.026 μl/min/mg protein). The method was applied in HLM assays to understand the herb–drug interaction (HDI) potential of Althaea officinalis, a popular African herb consumed by tuberculosis (TB) patients, with RIF. None of the extracts of A. officinalis inhibited the esterase-mediated metabolism pathway of RIF, compared to the positive control nelfinavir (IC50 = 9.59 μM). The method provides a tool for quantifying RIF and 25ODESRIF in in vitro drug metabolism assays as well as investigating herb– and drug–drug interactions (DDIs).

Abstract 738: Development of an HPLC method for the assessment of hydrophobicity of ADC payloads

Abstract Antibody-Drug Conjugates (ADCs) are antibodies attached to cytotoxic agents (payloads) through chemically labile linkers. The development of ADCs can fail at various stages, a common cause being a lack of reproducibility in the methods used to attach the payload to the antibody (i.e., the conjugation process) to provide a good yield and uniform drug-antibody ratio (i.e., the DAR). One of the major problems associated with conjugation is the hydrophobicity of a payload which can cause aggregation. The aim of this study was to create a hydrophobicity model to study the physicochemical properties of payloads at an early stage. This should allow their hydrophobicity to be evaluated and optimised at the design stage. A predictive model has been developed based on High Performance Liquid Chromatography (HPLC). Initial work involved development of a gradient reversed-phase HPLC method, and its use in the analysis of multiple reference compounds to establish their relative retention times. This involved the evaluation of a range of reversed-phase columns and solvent systems in order to obtain reproducibility. These initial studies, which utilized a C18 monolithic column for optimum results, demonstrated a clear relationship between the retention time (RT) of the reference compounds and their experimental LogP values obtained from the literature (r = 0.983). Next, a number of commercially available ADC payloads were analysed using the same HPLC method to determine their RT values, and their LogP values were calculated using the ChemDraw software. Payloads studied included Monomethyl Auristatin E (MMAE), Talirine and Tesirine (PBD dimers), Mertansine (DM1) and Calicheamicin. For this group of compounds, the results confirmed that their RT values correlated well with their calculated LogP values (r = 0.94). For example, the payload Talirine, which has a calculated LogP of 3.77, had an RT value of 17 min on the HPLC system equating to a LogP of 3.13 based on the reference compounds. Finally, some novel DNA-interactive payloads (e.g., the pyridinobenzodiazepine FGX-2-62) were evaluated using the same methodology. For FGX-2-62, the calculated LogP varied significantly between different software packages (i.e., 1.47, 3.38 or 4.46 for ChemSketch, MarvinSketch or ChemDraw, respectively). However, its HPLC retention time using the same method was 1.88 min, equating to a LogP of 1.63. This suggests that FGX-2-62 is significantly less hydrophobic than Talirine (e.g., RTs of 1.88 min versus 17 min, respectively), an observation consistent with the ease of conjugation of FGX-2-62 to antibodies without significant aggregation. Overall, these results suggest that HPLC retention time derived from a suitable HPLC method is more reliable than the currently available software-based LogP prediction methods in providing a realistic assessment of the hydrophobicity of novel payloads and their propensity to cause aggregation during conjugation. Citation Format: Ilona Pysz, Paul J. Jackson, Khondaker M. Rahman, David E. Thurston. Development of an HPLC method for the assessment of hydrophobicity of ADC payloads [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 738.

A silica gel orthogonal high-performance liquid chromatography method for the analyses of steviol glycosides: novel tetra-glucopyranosyl steviol

A silica gel orthogonal method using acetonitrile: water was developed for the analyses of fractions rich in very polar steviol glycosides and resolve regions of co-elution of these compounds in reversed-phase. Additionally, we also used this normal phase analytical method to scale up the purification process of steviol glycosides. Using these approaches, one novel minor tetra-glucopyranosyl diterpene glycosides together with three known compounds were purified from a commercial Stevia rebaudiana leaf extract. Compound 1 was unambiguously elucidated as 13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-(6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester (rebaudioside Y) based on high-performance liquid chromatography retention times, tandem mass spectrometry dissociation pattern and 1D and 2D NMR experiments. Known compounds were isolated in gram quantities and identified as rebaudioside D, E and M.

Structural Identification of 19 Purified Isomers of the OPV Acceptor Material bisPCBM by 13C NMR and UV-Vis Absorption Spectroscopy and High-Performance Liquid Chromatography.

The molecular structures of 19 purified isomers of bis-phenyl-C62-butyric acid methyl ester were identified by a combination of 13C NMR and UV-vis absorption spectroscopies and high-performance liquid chromatography (HPLC) retention time analysis. All 19 isomers are dicyclopropafullerenes (none are homofullerenes). There were seven isomers with C1 molecular point-group symmetry, four with C s, six with C2, one with C2 v, and one with C2 h symmetry. The C2 h, C2 v, and all five nonequatorial C1 isomers were unambiguously assigned to their respective HPLC fractions. For the other 12 isomers, the 13C NMR and UV-vis spectra placed them in six groups of two same-symmetry isomers. On the basis of the widely spaced HPLC retention times of the two isomers within each of these six groups, and the empirical inverse correlation between retention time and addend spacing, each isomer was assigned to its corresponding HPLC fraction. In addition, the missing trans-1 isomer was found, purified, and characterized.

Importance of Net Hydrophobicity in the Cellular Uptake of All-Hydrocarbon Stapled Peptides.

All-hydrocarbon stapled peptides make up a promising class of protein-protein interaction regulators; their potential therapeutic benefit arises because they have a high binding affinity and specificity for intracellular molecules. The cell permeation efficacy of these peptides is a critical determinant of their bioactivity. However, the factors that determine their cellular uptake remain an active area of research. In this study, we evaluated the effect of stapled (or cross-linked) formation on the cellular uptake of six known all-hydrocarbon stapled peptides. We found that the rate of cellular uptake of unstapled peptides (i.e., those bearing olefinic non-natural amino acids that are not subjected to olefin metathesis) was higher than that for the corresponding stapled peptides. Additionally, the insertion of these olefinic non-natural amino acids into peptide sequences significantly increased their rate of cellular uptake. According to the high-performance liquid chromatography retention times, the overall hydrophobicity of unstapled peptides was greater than that of stapled peptides, followed by that of the original peptides without olefinic non-natural amino acids. There was not a close correlation between helical content and the rate of cellular uptake of these peptides. Therefore, the increase in overall hydrophobicity resulting from the introduction of non-natural amino acids, rather than the structural stabilization resulting from staple formation, is the key driver promoting cellular uptake. Macropinocytosis, a form of fluid-phase endocytosis, was involved in the cellular uptake of all six peptides.

Bacterial cyclic β-(1,2)-glucans sequester iron to protect against iron-induced toxicity.

Cellular iron homeostasis is critical for survival and growth. Bacteria employ a variety of strategies to sequester iron from the environment and to store intracellular iron surplus that can be utilized in iron-restricted conditions while also limiting the potential for the production of iron-induced reactive oxygen species (ROS). Here, we report that membrane-derived oligosaccharide (mdo) glucan, an intrinsic component of Gram-negative bacteria, sequesters the ferrous form of iron. Iron-binding, uptake, and localization experiments indicated that both secreted and periplasmic β-(1,2)-glucans bind iron specifically and promote growth under iron-restricted conditions. Xanthomonas campestris and Escherichia coli mutants blocked in the production of β-(1,2)-glucan accumulate low amounts of intracellular iron under iron-restricted conditions, whereas they exhibit elevated ROS production and sensitivity under iron-replete conditions. Our results reveal a critical role of glucan in intracellular iron homeostasis conserved in Gram-negative bacteria.

Separation and Purification of Ombuoside from Gynostemma Pentaphyllum by Microwave-Assisted Extraction Coupled with High-Speed Counter-current Chromatography.

A rapid and efficient method for the separation and purification of ombuoside from Gynostemma pentaphyllum by microwave-assisted extraction coupled with high-speed counter-current chromatography (HSCCC) was successfully developed. Using an orthogonal array design L9 (34), the extraction conditions, including microwave power, irradiation time, solid-to-liquid ratio and extraction times, were optimized. Ombuoside was isolated and purified from the crude extraction byHSCCC with two-phase solvent system composed of n-hexane:ethyl acetate:ethanol:water (5:6:5:5, v/v) in a single run. A 210 mg quantity of the crude extract containing 2.16% ombuoside was loaded, yielding 3.9 mg of ombuoside at 96.7% purity. The chemical structure of ombuoside was determined by comparison with the high-performance liquid chromatography retention time of standard substance as well as UV, FT-IR, ESI-MS, 1H NMR and 13C NMR spectra. The purified ombuoside had strong 1,1-diphenyl-2-picrylhydrazyl and hydroxyl free radical scavenging activities.

Identification and Characterization of Metabolites of Irinotecan in-vivo and in-vitro matrixes by HPLC/LC-MS

In the present study metabolite identification and characterization has done by using HPLC and LC-MS. During method development various mobile phases have tried for identification of metabolites. The matrixes selected for in- vivo study were urine because nearly all the metabolites of irinotecan were obtained in it. The extraction mixtures have selected to retain maximum amount of analyte with less effort. During experiment four extraction solvents were used in six different concentrations out of which TBME suit our method. In-vitro study done by Human Liver microsomes by using Phosphate buffer (pH 7.4) and NADPH as co-factors for initiation of enzymatic reaction. Irinotecan is a prodrug that is converted in the liver to an active metabolite, SN-38. It is eliminate in Bile and Faeces and thus its dose reduced in Hepatic Failure. Irinotecan act by inhibiting Topoisomerase-1.It is the enzyme which nicks, introduces negative supercoils and reseals the DNA strand. Conventionally, drug metabolite identification in the past has usually been based on the comparison of ultraviolet (UV) spectral data and high-performance liquid chromatography (HPLC) retention times of isolated ‘unknown’ metabolites with those of synthesised standards. Such a method of detecting and characterising drug metabolites is an uncertain, time-consuming and expensive process, as well as affording very limited structural information. Furthermore, Phase I metabolism of a drug candidate often results in only minor structural modification of the parent compound; these minor changes can make it particularly difficult to determine suitable chromatographic conditions to effect HPLC separation of metabolites. This study describes contemporary approach to identification and characterization of xenobiotic metabolites in complex biological fluids derived from drug metabolism studies.

Optimal Hydrophobicity in Ring-Opening Metathesis Polymerization-Based Protein Mimics Required for siRNA Internalization.

Exploring the role of polymer structure for the internalization of biologically relevant cargo, specifically siRNA, is of critical importance to the development of improved delivery reagents. Herein, we report guanidinium-rich protein transduction domain mimics (PTDMs) based on a ring-opening metathesis polymerization scaffold containing tunable hydrophobic moieties that promote siRNA internalization. Structure-activity relationships using Jurkat T cells and HeLa cells were explored to determine how the length of the hydrophobic block and the hydrophobic side chain compositions of these PTDMs impacted siRNA internalization. To explore the hydrophobic block length, two different series of diblock copolymers were synthesized: one series with symmetric block lengths and one with asymmetric block lengths. At similar cationic block lengths, asymmetric and symmetric PTDMs promoted siRNA internalization in the same percentages of the cell population regardless of the hydrophobic block length; however, with 20 repeat units of cationic charge, the asymmetric block length had greater siRNA internalization, highlighting the nontrivial relationships between hydrophobicity and overall cationic charge. To further probe how the hydrophobic side chains impacted siRNA internalization, an additional series of asymmetric PTDMs was synthesized that featured a fixed hydrophobic block length of five repeat units that contained either dimethyl (dMe), methyl phenyl (MePh), or diphenyl (dPh) side chains and varied cationic block lengths. This series was further expanded to incorporate hydrophobic blocks consisting of diethyl (dEt), diisobutyl (diBu), and dicyclohexyl (dCy) based repeat units to better define the hydrophobic window for which our PTDMs had optimal activity. High-performance liquid chromatography retention times quantified the relative hydrophobicities of the noncationic building blocks. PTDMs containing the MePh, diBu, and dPh hydrophobic blocks were shown to have superior siRNA internalization capabilities compared to their more and less hydrophobic counterparts, demonstrating a critical window of relative hydrophobicity for optimal internalization. This better understanding of how hydrophobicity impacts PTDM-induced internalization efficiencies will help guide the development of future delivery reagents.

Identification and Quantification of the Major Constituents in Egyptian Carob Extract by Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry.

Background:Carob - Ceratonia siliqua L., commonly known as St John's-bread or locust bean, family Fabaceae - is one of the most useful native Mediterranean trees. There is no data about the chromatography methods performed by high performance liquid chromatography (HPLC) for determining polyphenols in Egyptian carob pods.Objective:To establish a sensitive and specific liquid chromatography–electrospray ionization (ESI)-tandem mass spectrometry (MSn) methodology for the identification of the major constituents in Egyptian carob extract.Materials and Methods:HPLC with diode array detector and ESI-mass spectrometry (MS) was developed for the identification and quantification of phenolic acids, flavonoid glycosides, and aglycones in the methanolic extract of Egyptian C. siliqua. The MS and MSn data together with HPLC retention time of phenolic components allowed structural characterization of these compounds. Peak integration of ions in the MS scans had been used in the quantification technique.Results:A total of 36 compounds were tentatively identified. Twenty-six compounds were identified in the negative mode corresponding to 85.4% of plant dry weight, while ten compounds were identified in the positive mode representing 16.1% of plant dry weight, with the prevalence of flavonoids (75.4% of plant dry weight) predominantly represented by two methylapigenin-O-pentoside isomers (20.9 and 13.7% of plant dry weight).Conclusion:The identification of various compounds present in carob pods opens a new door to an increased understanding of the different health benefits brought about by the consumption of carob and its products.SUMMARY This research proposed a good example for the rapid identification of major constituents in complex systems such as herbs using sensitive, accurate and specific method coupling HPLC with DAD and MS, which facilitate the clarification of phytochemical composition of herbal medicine for better understanding of their nature and biological activities. Abbreviation used: HPLC: High performance liquid chromatography, DAD: Diode array detector, MS: Mass spectrometry, TR: Retention time.

Microwave-Assisted Extraction and Purification of Arctiin and Arctigenin from Fructus Arctii by High-Speed Countercurrent Chromatography.

An efficient method for the rapid extraction, separation and purification of bioactive lignans, arctiin and arctigenin, from Fructus arctii by microwave-assisted extraction coupled with high-speed countercurrent chromatography was developed. The optimal extraction conditions of arctiin and arctigenin were evaluated by orthogonal array. Arctigenin could be converted from arctiin by hydrochloric acid hydrolysis. The separations were performed at a preparative scale with two-phase solvents composed of ethyl acetate-ethanol-water (5 : 1 : 5, v/v/v) for arctiin, and n-hexane-ethyl acetate-ethanol-water (4 : 4 : 3 : 4, v/v/v/v) for arctigenin. From 500 mg of crude extract sample, 122.3 mg of arctiin and 45.7 mg of arctigenin were obtained with the purity of 98.46 and 96.57%, and the recovery of 94.3 and 81.6%, respectively. Their structures were determined by comparison with the high-performance liquid chromatography retention time of standard substance as well as UV, FT-IR, electrospray ion source (ESI)-MS, (1)H-NMR and (13)C-NMR spectrum. According to the antioxidant activity assay, arctigenin had stronger 1,1-diphenyl-2-picrylhydrazyl free radicals scavenging activity.

HPLC Biogram Analysis: A Powerful Tool Used for Hit Confirmation in Early Drug Discovery

High-performance liquid chromatography (HPLC) biogram methodology is a powerful pharmaceutical screening hit confirmation strategy that couples analytical HPLC data with functional bioassay data. It is used primarily for screening hit chemical validation and triaging in support of early phase discovery programs and enables further investigation of the source of bioactivity in screening hits. The process combines semi-preparative separation technologies, automated compound handling and distribution, high-throughput biological screening, and informatics tools. The final output is an HPLC retention time versus bioactivity graphical overlay report. In this manner, biograms allow the analyst to determine which component in the sample is responsible for the biological activity, enabling decision making toward chemotype selection and prioritization from a pool of potential candidates. Another powerful aspect of the biogram assay lies in its utility in investigating biological activity in atypical samples, such as degraded samples or mixtures, for detection of minor active impurities or in addressing lot-to-lot activity discrepancies for a given test compound. Biograms are employed to track, isolate, and identify the source of biological activity in such samples, often yielding important information for program decisions.

A derivatization strategy for the detection and identification of volatile trialkylphosphites using liquid chromatography-online solid phase extraction and offline nuclear magnetic resonance spectroscopy

We demonstrate herein the application of selective derivatization method that converts volatile and labile trialkylphosphites (TAPs) into virtually non-volatile, thermally stable, and UV absorbing derivatives. After simple sample preparation, purification/enrichment of the derivatives was achieved by using high performance liquid chromatography (HPLC) coupled to on-line post column solid phase extraction (SPE) system. These derivatives were subjected to 31P{1H} NMR and 1D-selTOCSY experiments. Conclusive identification was achieved on the basis of their HPLC retention time and NMR spectral signatures (δH1,δpE1, nJH–H, and 3JP–H). This method was tested for the unambiguous identification of a mixture containing low concentrations (∼10μgmL−1) of trimethylphosphite (TMP), triethylphosphite (TEP), triisopropylphosphite (TIP), and tributylphosphite (TBP) along with a high concentration of irrelevant background chemicals. It offered a high dynamic range and good detection limit and recovery (>75%) without the need for special NMR probe heads or exotic NMR experiments.

Hydrophobic Blocks Facilitate Lipid Compatibility and Translocon Recognition of Transmembrane Protein Sequences

Biophysical hydrophobicity scales suggest that partitioning of a protein segment from an aqueous phase into a membrane is governed by its perceived segmental hydrophobicity but do not establish specifically (i) how the segment is identified in vivo for translocon-mediated insertion or (ii) whether the destination lipid bilayer is biochemically receptive to the inserted sequence. To examine the congruence between these dual requirements, we designed and synthesized a library of Lys-tagged peptides of a core length sufficient to span a bilayer but with varying patterns of sequence, each composed of nine Leu residues, nine Ser residues, and one (central) Trp residue. We found that peptides containing contiguous Leu residues (Leu-block peptides, e.g., LLLLLLLLLWSSSSSSSSS), in comparison to those containing discontinuous stretches of Leu residues (non-Leu-block peptides, e.g., SLSLLSLSSWSLLSLSLLS), displayed greater helicity (circular dichroism spectroscopy), traveled slower during sodium dodecyl sulfate–polyacrylamide gel electrophoresis, had longer reverse phase high-performance liquid chromatography retention times on a C-18 column, and were helical when reconstituted into 1-palmitoyl-2-oleoylglycero-3-phosphocholine liposomes, each observation indicating superior lipid compatibility when a Leu-block is present. These parameters were largely paralleled in a biological membrane insertion assay using microsomal membranes from dog pancreas endoplasmic reticulum, where we found only the Leu-block sequences successfully inserted; intriguingly, an amphipathic peptide (SLLSSLLSSWLLSSLLSSL; Leu face, Ser face) with biophysical properties similar to those of Leu-block peptides failed to insert. Our overall results identify local sequence lipid compatibility rather than average hydrophobicity as a principal determinant of transmembrane segment potential, while demonstrating that further subtleties of hydrophobic and helical patterning, such as circumferential hydrophobicity in Leu-block segments, promote translocon-mediated insertion.

Liquid chromatography combined with atomic and molecular mass spectrometry for speciation of arsenic in chicken liver

Human exposure to high concentrations of arsenic from water and food is an important health concern. Although 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone) was used for more than 60 years as a feed additive to feed chickens, little is known about the metabolism of this arsenic species in chicken. Determination of potential arsenic metabolites present at trace concentrations is an analytical challenge, requiring efficient separation and sensitive detection. The primary objective of this research is to develop a method that enables the identification and quantification of various arsenic species in chicken liver. This report describes a method of high performance liquid chromatography (HPLC) separation with both inductively coupled plasma mass spectrometry (ICPMS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection. Anion exchange HPLC enabled the separation of Roxarsone and other arsenic species within 12min. Detection with both ICPMS and ESI-MS/MS allowed for identification and quantification of eight arsenic species in chicken livers, including arsenobetaine, inorganic arsenite, dimethylarsinic acid, monomethylarsonic acid, inorganic arsenate, 3-amino-4-hydroxyphenylarsonic acid, N-acetyl-4-hydroxyphenylarsonic acid (N-AHPAA), and Roxarsone. The concentrations of these arsenic species, with the exception of arsenobetaine, are significantly higher in the Roxarsone-fed chickens than in the control chickens. The simultaneous detection by both ICPMS and ESIMS from the same HPLC separation allowed for comparison of peaks in both ICPMS and ESIMS chromatograms. This is advantageous over two separate analyses, particularly when HPLC retention times might fluctuate due to sample matrix effect. HPLC separation with the complementary atomic and molecular mass spectrometry detection prevented misidentification of co-eluting compounds, as demonstrated by the determination of two possible metabolites of Roxarsone, N-AHPAA and 4-amino-phenylarsonic acid (4-APAA). N-AHPAA was confirmed by HPLC separation with simultaneous arsenic-specific detection by ICPMS and multiple reaction monitoring by ESIMS. Although an arsenic-containing compound had identical retention time as 4-APAA in the HPLC–ICPMS chromatogram, it was ruled out as 4-APAA from the simultaneous detection by ESIMS. The identification and quantitation of trace arsenic species present in complex samples demonstrate the potential of HPLC separation with simultaneous ICPMS and ESIMS detection for other speciation applications.

A Study of δ-Globin Gene Mutations in the UK Population: Identification of Three Novel Variants and Development of a Novel DNA Test for Hb A′2

We report here the spectrum of δ-globin gene mutations found in the UK population. Nine different δ chain variants and two δ-thalassemia (δ-thal) mutations were characterized in a study of 127 alleles in patients with either a low Hb A2 value or a split Hb A2 peak on high performance liquid chromatography (HPLC). The most common δ chain variant was Hb A′2 (or Hb B2) [δ16(A13)Gly → Arg; HBD: c.49G > C] (77.0%), followed by Hb A2-Yialousa [δ27(B9)Ala → Ser; HBD: c.82G > T] (12.0%), Hb A2-Babinga [δ136(H14)Gly → Asp; HBD: c.410G > A] (3.0%), Hb A2-Troodos [δ116(G18)Arg → Cys; HBD: c.349C > T] (1.0%), Hb A2-Coburg [δ116(G18)Arg → His; HBD: c.350G > A] (2.0%) and Hb A2-Indonesia [δ69(E13)Gly → Arg; HBD: c.208G > C] (1.0%). Three novel variants were identified: Hb A2-Calderdale [codon 2 (CAT > AAT), His → Asn; HBD: c.7C > A], Hb A2-Walsgrave [codon 52 (GAT > CAT), Asp → His; HBD: c.157G > C] and Hb A2-St. George’s [codon 81 (CTC > TTC), Leu → Phe; HBD: c.244C > T]. In addition, two known δ-thal mutations were observed: −68 (C > T); HBD: c.-118C > T and codon 4 (ACT > ATT); HBD: c.14C > T. Amplification refractory mutation system (ARMS) primers were developed to provide a simple molecular diagnostic test for the most common variant, Hb A′2. Three of the variants had a characteristic HPLC retention time that can be used for a presumptive diagnosis.