UPLC-MS/MS quantitative analysis and structural fragmentation study of five Parmotrema lichens from the Eastern Ghats.

Comparative phytochemical analysis of five lichen species [Parmotrema tinctorum (Delise ex Nyl.) Hale, P. andinum (Mull. Arg.) Hale, P. praesorediosum (Nyl.) Hale, P. grayanum (Hue) Hale, P. austrosinense (Zahlbr.) Hale] of Parmotrema genus were performed using two complementary UPLC-MS systems. The first system consists of high resolution UPLC-QToF-MS/MS spectrometer and the second system consisted of UPLC-MS/MS in Multiple Reaction Monitoring (MRM) mode for quantitative analysis of major constituents in the selected lichen species. The individual compounds (47 compounds) were identified using Q-ToF-MS/MS, via comparison of the exact molecular masses from their MS/MS spectra, the comparison of literature data and retention times to those of standard compounds which were isolated from crude extract of abundant lichen, P. tinctorum. The analysis also allowed us to identify unknown peaks/compounds, which were further characterized by their mass fragmentation studies. The quantitative MRM analysis was useful to have a better discrimination of species according to their chemical profile. Moreover, the determination of antioxidant activities (ABTS+ inhibition) and Advance Glycation Endproducts (AGEs) inhibition carried out for the crude extracts revealed a potential antiglycaemic activity to be confirmed for P. austrosinense.

Reversed-phase liquid chromatography with electrospray mass detection and 1H and 13C NMR characterization of new process-related impurities, including forced degradants of efavirenz: related substances correlated to the synthetic pathway.

In this study, a stability-indicating reversed-phase liquid chromatographic electrospray mass spectrometric method was developed and validated for the determination of process-related impurities and forced degradants of Efavirenz in bulk drugs. Efavirenz was subjected to acid, alkaline hydrolysis, H2O2 oxidation, photolysis, and thermal stress. Significant degradation was observed during alkaline hydrolysis, and the degradants were isolated on a mass-based purification system and characterized by high-resolution mass spectrometry, positive electrospray ionization tandem mass spectrometry, and (1)H and (13)C NMR spectroscopy. Accurate mass measurement and NMR spectroscopy revealed the possible structure of process-related impurities and degradant under stress conditions. The acceptable separation was accomplished on Waters bondapak C18 column (250 mm × 4.6 mm; 5 µm), using 5 mM ammonium acetate and acetonitrile as a mobile phase in a gradient elution mode at a flow rate of 1.0 mL/min. The eluents were monitored by diode array detector at 247 nm and quantitation limits were obtained in the range of 0.1-2.5 µg/mL for Efavirenz, degradants, and process-related impurities. The liquid chromatography method was validated with respect to accuracy, precision, linearity, robustness, and limits of detection and quantification as per International Conference on Harmonization guidelines.

Comparison of conventional and supported liquid extraction methods for the determination of sitagliptin and simvastatin in rat plasma by LC-ESI-MS/MS.

Three extraction methods were compared for their efficiency to analyze sitagliptin and simvastatin in rat plasma by LC-MS/MS, including (1) liquid-liquid extraction (LLE), (2) solid phase extraction (SPE) and (3) supported liquid extraction (SLE). Comparison of recoveries of analytes with different extraction methods revealed that SLE was the best extraction method. The detection was facilitated with ion trap-mass spectrometer by multiple reactions monitoring (MRM) in a positive ion mode with ESI. The transitions monitored were m/z 441.1→325.2 for simvastatin, 408.2→235.1 for sitagliptin and 278.1→260.1 for the IS. The lower limit of quantification (LLOQ) was 0.2 ng/mL for sitagliptin and 0.1 ng/mL for simvastatin. The effective SLE offers enhanced chromatographic selectivity, thus facilitating the potential utility of the method for routine analysis of biological samples along with pharmacokinetic studies.

LC-ESI-MS/MS determination of paclitaxel on dried blood spots.

A simple and rapid high-performance liquid chromatography-tandem mass spectrometric assay for determination of paclitaxel on rat dried blood spots was developed and validated. The extracted sample was chromatographed without further treatment using a reverse-phase Oyster ODS3, 4.6 × 50 mm, 3 µm column with mass spectrometry detection. The mobile phase comprised of acetonitrile-water, 60:40 v/v, with a flow rate of 0.4 mL/min was used. The calibration was linear over the range 0.2-20 ng/mL. The limits of detection and quantification were 0.08 and 0.2 ng/mL, respectively. The intra- and inter-day precision (CV%) and accuracy (relative error %) were less than 10 and 12%, respectively.

Differentiation of positional isomers of hybrid peptides containing repeats of ß-nucleoside derived amino acid (ß-Nda-) and L-amino acids by positive and negative ion electrospray ionization tandem mass spectrometry (ESI-MSn).

A new class of positional isomeric pairs of -Boc protected oligopeptides comprised of alternating nucleoside derived ß-amino acid (ß-Nda-) and L-amino acid residues (alanine, valine, and phenylalanine) have been differentiated by both positive and negative ion electrospray ionization ion-trap tandem mass spectrometry (ESI-MS(n)). The protonated dipeptide positional isomers with ß-Nda- at the N-terminus lose CH(3)OH, NH(3), and C(2)H(4)O(2), whereas these processes are absent for the peptides with L-amino acids at the N-terminus. Instead, the presence of L-amino acids at the N-terminus results in characteristic retro-Mannich reaction involving elimination of imine. A good correlation has been observed between the conformational structure of the peptides and the abundance of y(n)(+) and b(n)(+) ions in MS(n) spectra. In the case of tetrapeptide isomers that are reported to form helical structures in solution phase, no y(n)(+) and b(n)(+) ions are observed when the corresponding amide -NH- participates in the helical structures. In contrast, significant y(n)(+) and b(n)(+) ions are formed when the amide -NH- is not involved in the H-bonding. In the case of tetra- and hexapeptides, it is observed that abundant b(n)(+) ions are formed, presumably with stable oxazolone structures when the C-terminus of the b(n)(+) ions possessed L-amino acid and the ß-Nda- at the C-terminus appears to prevent the cyclization process leading to the absence of corresponding b(n)(+) ions.

Decolorization and biotransformation of triphenylmethane dye, methyl violet, by Aspergillus sp. isolated from Ladakh, India.

Methyl violet, used extensively in the commercial textile industry and as a biological stain, is a hazardous recalcitrant. Aspergillus sp. strain CB-TKL-1 isolated from a water sample from Tsumoriri Lake, Karzok, Ladakh, India, was found to completely decolorize methyl violet within 24 h when cultured under aerobic conditions at 25 degrees C. The rate of decolorization was determined by monitoring the decrease in the absorbance maxima of the dye by UV-visible spectroscopy. The decolorization of methyl violet was optimal at pH 5.5 and 30 degrees C when agitated at 200 rpm. Addition of glucose or arabinose (2%) as a carbon source and sodium nitrate or soyapeptone (0.2%) as a nitrogen source enhanced the decolorization ability of the culture. Furthermore, the culture exhibited a maximum decolorization rate of methyl violet after 24 h when the C:N ratio was 10. Nine N-demethylated decolorized products of methyl violet were identified based on UV-visible spectroscopy, Fourier transform infrared (FTIR), and LC-MS analyses. The decolorization of methyl violet at the end of 24 h generated mono-, di-, tri-, tetra-, penta-, and hexa-Ndemethylated intermediates of pararosaniline. The variation of the relative absorption peaks in the decolorized sample indicated a linear decrease of hexa-N-demethylated compounds to non-N-demethylated pararosaniline, indicating a stepwise N-demethylation in the decolorization process.

Diastereomeric differentiation of norbornene amino acid peptides by electrospray ionization tandem mass spectrometry.

A new class of diastereomeric pairs of non-natural amino acid peptides derived from butyloxycarbonyl (Boc-)protected cis-(2S,3R)- and trans-(2S,3S)-beta-norbornene amino acids including a monomeric pair have been investigated by electrospray ionization (ESI) tandem mass spectrometry using quadrupole time-of-flight (Q-TOF) and ion-trap mass spectrometers. The protonated cis-BocN-beta-nbaa (2S,3R) (1) (betanbaa = beta-norbornene amino acid) eliminates the Boc group to form [M+H-Boc+H](+), whereas an additional ion [M+H-C(4)H(8)](+) is formed from trans-BocN-beta-nbaa (2S,3S) (2). Similarly, it is observed that the peptide diastereomers (di-, tri- and tetra-), with cis-BocN-beta-nbaa (2S,3R)- at the N-terminus, initially eliminate the Boc group to form [M+H-Boc+H](+) which undergo further fragmentation to give a set of product ions that are different for the peptides with trans-BocN-beta-nbaa (2S,3S)- at the N-terminus. Thus the Boc group fragments differently depending on the configuration of the amino acid present at the N-terminus. It is also observed that the peptide bond cleavage in these peptides is less favoured and most of the product ions are formed due to retro-Diels-Alder fragmentation. Interestingly, sodium-cationized peptide diastereomers mainly yield a series of retro-Diels-Alder fragment ions which are different for each diastereomer as they are formed starting from [M+Na-Boc+H](+) in peptides with cis-BocN-beta-nbaa (2S,3R)- at the N-terminus, and [M+Na-C(4)H(8)](+) in peptides with trans-BocN-beta-nbaa (2S,3S)- at the N-terminus. All these results clearly indicate that these diastereomeric pairs of peptides yield characteristic product ions which help distinguish the isomers.

Compositional analysis and rheological properties of gum kondagogu (Cochlospermum gossypium): a tree gum from India.

Gum kondagogu ( Cochlospermum gossypium) is a tree exudate gum that belongs to the family Bixaceae. Compositional analysis of the gum by HPLC and LC-MS revealed uronic acids to be the major component of the polymer ( approximately 26 mol %). Furthermore, analysis of the gum by GC-MS indicated the presence of sugars such as arabinose (2.52 mol %), mannose (8.30 mol %), alpha- d-glucose (2.48 mol %), beta- d-glucose (2.52 mol %), rhamnose (12.85 mol %), galactose (18.95 mol %), d-glucuronic acid (19.26 mol %), beta- d-galactouronic acid (13.22 mol %), and alpha- d-galacturonic acid (11.22 mol %). Gum kondagogu, being rich in rhamnose, galactose, and uronic acids, can be categorized on the basis of its sugar composition as a rhamnogalacturonan type of gum. The rheological measurements performed on the gum suggest that above 0.6% (w/v) it shows a Newtonian behavior and shear rate thinning behavior as a function of gum concentration. The viscoelastic behavior of gum kondagogu solutions (1 and 2%) in aqueous as well as in 100 mM NaCl solution exhibits a typical gel-like system. The G' (viscous modulus)/ G'' (elastic modulus) ratios of native gum kondagogu (1 and 2%) in aqueous solution were found to be 1.89 and 1.85 and those in 100 mM NaCl to be 1.54 and 2.2, respectively, suggesting a weak gel-like property of the polymer. Crossover values of G' and G'' were observed to be at frequencies of 0.432 Hz for 1% and 1.2 Hz for 2% for native gum in aqueous condition, indicating a predominantly liquid- to solid-like behavior, whereas crossover values of 2.1 Hz for 1% and 1.68 Hz for 2% gum in 100 mM NaCl solution suggest a larger elastic contribution.