Rapid and sensitive LC‐MS method for pharmacokinetic study of vinorelbine in rats

A rapid and sensitive LC‐electrospray ionization‐MS method was developed for determining vinorelbine in rat plasma. A 100 µL plasma sample was treated using a protein precipitation procedure and was chromatographed within 4 min using an Inertsil ODS‐3 C₁₈ (2.1 × 50 mm, 5 µm) column. The selected ion monitoring ions [M + H]⁺ were m/z 779 and m/z 811 for vinorelbine and vinblastine (internal standard), respectively. The method validation showed that the calibration curve for vinorelbine was linear over a concentration range of 1–1000 ng/mL with lower limit of quantification at 1 ng/mL. The method has been successfully applied to pharmacokinetics in rat plasma. Copyright © 2009 John Wiley & Sons, Ltd.     

Estimation of peptide N–Cα bond cleavage efficiency during MALDI‐ISD using a cyclic peptide

Matrix‐assisted laser desorption/ionization in‐source decay (MALDI‐ISD) induces N–C_α bond cleavage via hydrogen transfer from the matrix to the peptide backbone, which produces a c′/z? fragment pair. Subsequently, the z? generates z′ and [z + matrix] fragments via further radical reactions because of the low stability of the z?. In the present study, we investigated MALDI‐ISD of a cyclic peptide. The N–C_α bond cleavage in the cyclic peptide by MALDI‐ISD produced the hydrogen‐abundant peptide radical [M + 2H]⁺? with a radical site on the α‐carbon atom, which then reacted with the matrix to give [M + 3H]⁺ and [M + H + matrix]⁺. For 1,5‐diaminonaphthalene (1,5‐DAN) adducts with z fragments, post‐source decay of [M + H + 1,5‐DAN]⁺ generated from the cyclic peptide showed predominant loss of an amino acid with 1,5‐DAN. Additionally, MALDI‐ISD with Fourier transform‐ion cyclotron resonance mass spectrometry allowed for the detection of both [M + 3H]⁺ and [M + H]⁺ with two ¹³C atoms. These results strongly suggested that [M + 3H]⁺ and [M + H + 1,5‐DAN]⁺ were formed by N–C_α bond cleavage with further radical reactions. As a consequence, the cleavage efficiency of the N–C_α bond during MALDI‐ISD could be estimated by the ratio of the intensity of [M + H]⁺ and [M + 3H]⁺ in the Fourier transform‐ion cyclotron resonance spectrum. Because the reduction efficiency of a matrix for the cyclic peptide cyclo(Arg‐Gly‐Asp‐D‐Phe‐Val) was correlated to its tendency to cleave the N–C_α bond in linear peptides, the present method could allow the evaluation of the efficiency of N–C_α bond cleavage for MALDI matrix development. Copyright © 2016 John Wiley & Sons, Ltd.     

Ambient ionization and direct identification of volatile organic compounds with microwave‐induced plasma mass spectrometry

An innovative method of volatile organic compounds analysis by using microwave‐induced plasma ionization (MIPI) source in combination with an ambient ion trap mass spectrometer is presented here. Using MIPI for direct sample vapor, analysis was achieved without any sample preparation or subsequent heating. The relative abundance of the target compounds can be obtained almost instantly within a few seconds. The ionization processes of different volatile compounds was optimized, and the limits of detection were identified in the range of 0.15–4.5 pptv or 0.73–8.80 pg ml^?1. The relative standard deviation (RSD) is in the range of 4–14%, while correlation coefficients of the working curves (R²) are better than 0.98. The new method possesses advantages of ease operation, time‐saving, high sensitivity and inexpensive setup. In addition, the ionization processes of short n‐alkane chains were investigated with the MIPI technique, and a unique [M + 13]⁺ was detected, which has not been reported in detail by any other related ionization techniques. An ionization mechanism was proposed on the basis of the experimental results obtained in this work and available information in literatures, in which the n‐alkanes in the plasma environment possibly generate protonated cyclopentadiene [M – 5]⁺ or alkyl‐substituted analogues as well as hydrous ions [M + 13]⁺ and [M + 13 + 18]⁺, as shown in Scheme 1 in the main text. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of an LC‐ESI‐MS/MS method for the quantitation of hemslecin A in rhesus monkey plasma and its application in pharmacokinetics

In this study, a new LC‐ESI‐MS/MS‐based method was validated for the quantitation of hemslecin A in rhesus monkey plasma using otophylloside A as internal standard (IS). Hemslecin A and the IS were extracted from rhesus monkey plasma using liquid–liquid extraction as the sample clean‐up procedure, and were subjected to chromatography on a Phenomenex Luna CN column (150 × 2.0 mm, 3.0 µm) with the mobile phase consisting of methanol and 0.02 mol/mL ammonium acetate (55:45, v/v) at a flow rate of 0.2 mL/min. Detection was performed on an Agilent G6410B tandem mass spectrometer by positive ion electrospray ionization in multiple reaction monitoring mode, monitoring the transitions m/z 580.5 [M + NH₄]⁺ → 503.4 and m/z 518.2 [M + NH₄]⁺ → 345.0 for hemslecin A and IS, respectively. The assay was linear over the concentration range of 0.5–200 ng/mL and was successfully applied to a pharmacokinetic study in rhesus monkeys. Copyright © 2013 John Wiley & Sons, Ltd.     

Pharmacokinetic studies of soyalkaloid A from Portulaca oleracea L. using ultra high‐performance liquid chromatography electrospray ionization quadrupole–time of flight mass spectrometry and its antioxidant activity

Soyalkaloid A was isolated from Portulaca oleracea L. for the first time in our laboratory and then a rapid and sensitive ultra‐high‐performance liquid chromatography electrospray ionization quadrupole–time of flight mass spectrometry (UHPLC–ESI–Q–TOF/MS) method with hesperidin as internal standard (IS) was developed and validated to investigate the pharmacokinetics of soyalkaloid A in rats after oral and intravenous administrations. The analysis was achieved on an Agilent Zorbax Eclipse Plus C₁₈ Column (2.1 × 50 mm, 1.8 μm) by elution with acetonitrile and water (containing 0.1% formic acid), at a flow rate of 0.3 mL/min. The MS analysis was performed in the positive ion mode with monitored ion m/z 227.0814 [M + H]⁺ and 611.1971 [M + H]⁺ for soyalkaloid A and IS, respectively. The linear range was established over the concentration range 7.5–6000 ng/mL (r = 0.9951). The intra‐ and inter‐assay accuracy and precision were between ?4.86‐4.49 and 1.93–9.66, respectively. The lower limits of detection and quantitation observed were 2.1 and 7.4 ng/mL, respectively. The rapid, sensitive and specific UHPLC–ESI–Q–TOF/MS method was successfully applied to a pharmacokinetic study of soyalkaloid A. Moreover, its antioxidant was studied via a 1,1‐diphenyl‐2‐picryl‐hydrazyl radical scavenging assay, the IC₅₀ value being 20.73 ± 0.51 μM.     

Three pairs of enantiomers bearing mitochondria‐targeted TPP+ groups as potential anti‐cancer agents

Six complexes with chiral Schiff‐base ligands containing TPP⁺ groups, [VO L ^R,R/^S,S](ClO₄)₂( 1 for RR, 2 for SS), [Ni L ^R,R/S,S](ClO₄)₂·C₂H₅OH ( 3 for RR, 4 for SS) and [CuL^R,R/S,S](ClO₄)₂·CHCl₃·CH₃CH₂OH ( 5 for RR, 6 for SS) ( L ^R,R/S,S = N,N′‐Bis{5‐[(triphenylphosphonium)‐methyl]salicylidine}‐(1R,2R/1S,2S)‐diphenylethane‐1,2‐diamine, were synthesized to serve as mitochondrion‐targeting anticancer drugs. The introduction of TPP⁺ group(s) might markedly influence the properties of complexes. Compounds 3 and 5 were structurally characterized by X‐ray crystallography. Complexes 1–6 could be moderate intercalating agents to CT‐DNA which is determined by several spectroscopy methods. DNA cleavage experiments revealed that all compounds could promote oxidative cleavage of pBR322 plasmid DNA in the presence of H₂O₂. MTT assay indicated 1–6 exhibited effective cytotoxicity on A549 and MCF‐7 cell lines. Notably, the IC₅₀ values of 5 (1.24 ± 0.33 μM) or 6 (1.47 ± 0.52 μM) were approximately 9–11 fold lower than that of cisplatin (IC₅₀ = 13.56 ± 0.88 μM) on A549 cells. 5 and 6 were picked for further study, which indicated that the cytotoxicity seems to result from multiple mechanisms of action, including effectively suppress the growth and proliferation of A549 cells, generation of reactive oxygen species, dissipation of mitochondrial membrane potential, cell cycle perturbation and apoptosis induction. Compounds 1–6 could highly accumulate in the mitochondria by means of ICP‐MS assay. This study demonstrates that 1–6 with mitochondrion‐targeting function could be efficient anticancer drugs.     

Simultaneous Determination Baicalin and Forsythin in Shuanghuanglian Oral Liquid by HPLC/DAD and LC‐ESI‐MS

Rapid, simple and reliable HPLC/DAD and LC‐ESI‐MS methods for the simultaneous determination of baicalin and forsythin in the traditional Chinese medicinal preparation Shuanghuanglian oral liquid were described and validated. The separation condition for HPLC/DAD was optimized using a BDS hypersil C18 column (Thermo, 2.1 × 150 mm, particle size 5 μm) by gradient elution using methanol‐0.2 % ammonium acetate as the mobile phase. The suitable detection wavelength was set at 277 nm for the quantitative analysis of baicalin and forsythin in this method. Some operational parameters of the ESI interface were optimized, negative m/z 445[M?H]^? for baicalin and negative m/z 593[M+CH₃COO]^? for forsythin, positive m/z 447[M+H]⁺ for baicalin and positive m/z 552[M+NH 4]⁺ for forsythin, respectively. These HPLC/DAD and LC‐ESI‐MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). These methods can be used as a complementary method for the commercial quality control of Shuanghuanglian oral liquid and its pharmaceutical preparations.     

Synthesis and characterization of water‐soluble copper(II), cobalt(II) and zinc(II) complexes derived from 8‐hydroxyquinoline‐5‐sulphonic acid: DNA binding and cleavage studies

Three water‐soluble complexes, [Cu₂L₂Cl₂] ( 1 ), [CoL₂(im)₂] ( 2 ) and [ZnLClH₂O] ( 3 ) (HL = 8‐hydroxyquinoline‐5‐sulphonic acid; im = N ‐methylimidazole), were prepared and characterized using various spectral techniques. The DNA binding behaviour of complexes 1 – 3 was studied using UV–visible and circular dichroism (CD) spectra and cyclic voltammetry. All three complexes exhibit hypochromism but complexes 1 and 3 alone give a red shift of 4 nm with a significant binding constant of K _b = 2.1 × 10⁴ and 1.0 × 10⁴ M⁻¹, respectively, but complex 2 shows no red shift with lower K _b of 4.1 × 10³ M⁻¹. The voltammetric E _1/2 of complex 1 on interaction with herring sperm DNA shifts to a more positive potential, as expected, than complex 2 due to higher DNA affinity. Additionally, analysis of electrochemical data yields a value of K ₊/K ₂₊ greater than one suggesting that complex 1 binds to DNA through intercalation in the M(I) state. Evidently in CD spectral analysis, complex 1 exhibits a decrease in molar ellipticity with a red shift of 10 nm and a significant decrease in intensity compared to complexes 2 and 3 . This clearly indicates that complex 1 induces the B → A transition to a greater extent than 2 and 3 . Oxidative cleavage using circular plasmid pUC18 DNA with complex 1 was investigated using gel electrophoresis. Interestingly, complex 1 displays a strong DNA binding affinity and is efficient in cleaving DNA in the presence of H₂O₂ at pH = 8.0 at 37 °C.     

Determination and toxicokinetics comparison of ketamine and S(+)‐ketamine in dog plasma by HPLC‐MS method

;A simple and reproducible method was developed for the quantification of ketamine and S(+)‐ketamine in dog plasma using a high‐performance liquid chromatography system coupled to a positive ion electrospray mass spectrometric analysis. Solid‐phase extraction was used for extracting analytes from dog plasma samples. The analytes were separated on a Zorbax SB C₁₈ column (100 × 2.1 mm, 3.5 μm) with acetonitrile–formate buffer (10 mM ammonium formate and 0.3% formic acid) (17 : 83, v/v) as mobile phase at a flow‐rate of 0.2 mL/min. Detection was operated under selected ion monitoring mode. [M + H]⁺ at m/z 238 for ketamine and S(+)‐ketamine and [M + H]⁺ at m/z 180 for phenacetin (internal standard) were selected as detecting ions, respectively. The method was linear in the concentration range 51.6–2580 ng/mL. The intra‐ and inter‐day precisions (RSD %) were within 11.3% and the assay accuracies ranged from 80.0 to 101.4%. Their average recoveries were greater than 91.1% at all test concentrations. The analytes were proved to be stable during all sample storage, preparation and analysis procedures. The method was successfully applied to the toxicokinetics study and comparison of ketamine and S (+)‐ketamine following intravenous administration to dogs. Copyright © 2009 John Wiley & Sons, Ltd.     

Sodiation‐based in‐source collision for profiling of pyranocoumarins in Radix Peucedani (Qianhu): utility of sodium adducts' stability with in‐source collision

Full scan mode of liquid chromatography‐mass spectrometry equipped with an electrospray ionization source offers a chance on global detection of complicated components; however, the scan mode carries significant challenges in rapidly capturing information of analysts. Sodiation‐based in‐source collision was proposed here, as a technique for rapid detecting untargeted analytes in full scan analysis, which was based on the stability of sodium adducts and the nonselectivity of in‐source collision. Then the technique was applied to profile of angular‐type pyranocoumarins (APs) in Radix Peucedani, with full scan analysis performed at two specific in‐source collision energy: a high energy 50 V that is tolerated by the sodium adducts of APs, and a low energy 10 V, at which abundant adducts were offered. The spectra list of two average mass spectra was exported, and stable ions were selected based on the intensity ratio of standards at the two collision energy. Then 27 plausible [M + Na]⁺ m/z values of APs were acquired after filtering the fragment ion and isotope ions and validating with [M + NH₄]⁺. Eighty‐two APs finally were tentatively identified based on their accurate spectral data of MSⁿ, fragmentation rules, and elution order regardless of their absolute configuration, which included 25 reported APs from Peucedanum praeruptorum Dunn. The technique provided a novel application of sodium adduct in qualitative analysis. And it was valuable for rapidly capturing information of analytes in full scan analysis, not only for APs but also for other compounds that could form sodium adducts. Copyright © 2017 John Wiley & Sons, Ltd.     

Simple and sensitive LC‐ESI‐MS method for the quantitation of sildenafil in plasma samples

Sildenafil is a selective inhibitor of phosphodiesterase type 5 enzyme. A simple and sensitive LC‐MS assay was developed to determine the concentration of sildenafil in human plasma. Sildenafil and omeprazole (internal standard) were extracted from the plasma with diethyl ether. The extract was evaporated under nitrogen and the residue was constituted with ACN and injected onto Novapak C₁₈ column (75×3.9 mm, 4 μm). The mobile phase consisted of 90% ACN plus 10% ammonium acetate (20 mM) containing 0.02% formic acid and was delivered isocratically at a flow rate of 0.2 mL/min. Sildenafil and omeprazole were monitored using a positive electrospray mode with single‐ion recording set at m/z 475 and 346, respectively, which are consistent with [M+H]⁺ molecular ions, and the run time was less than 5 min. The detection limit of sildenafil was 0.5 ng/mL, and the calibration curve was linear between 0.5 and 2000 ng/mL (R²>0.99). Within‐ and between‐day coefficients of variation were less than 7%. This method has been successfully used to measure sildenafil plasma concentrations in a beagle dog model following an oral administration of the drug.     

HPLC‐DAD and HPLC‐ESI‐MS separation,determination and identification of the spin‐labeled diastereoisomers of podophyllotoxin

Spin‐labeled nitroxide derivatives of podophyllotoxin had better antitumor activity and less toxicity than that of the parent compounds. However, the 2‐H configurations of these spin‐labeled derivatives cannot be determined by nuclear magnetic resonance (NMR) methods. In the present paper, a high‐performance liquid chromatography‐diode array detection (HPLC‐DAD) and a high‐performance liquid chromatography‐electrospray ionization tandem mass spectrometry (HPLC‐ESI/MS/MS) method were developed and validated for the separation, identification of four pairs of diastereoisomers of spin‐labeled derivatives of podophyllotoxin at C‐2 position. In the HPLC‐ESI/MS spectra, each pair of diastereoisomers of the spin‐labeled derivatives in the mixture was directly confirmed and identified by [M+H]⁺ ions and ion ratios of relative abundance of [M‐ROH+H]⁺ (ion 397) to [M+H]⁺. When the [M‐ROH+H]⁺ ions (at m/z 397) were selected as the precursor ions to perform the MS/MS product ion scan. The product ions at m/z 313, 282, and 229 were the common diagnostic ions. The ion ratios of relative abundance of the [M‐ROH+H]⁺ (ion 397) to [M+H]⁺, [A+H]⁺ (ion 313) to [M‐ROH+H]⁺, [A+H‐OCH₃]⁺ (ion 282) to [M‐ROH+H]⁺ and [M‐ROH‐ArH+H]⁺ (ion 229) to [M‐ROH+H]⁺ of each pair of diastereoisomers of the derivatives specifically exhibited a stereochemical effect. Thus, by using identical chromatographic conditions, the combination of DAD and MS/MS data permitted the separation and identification of the four pairs of diastereoisomers of spin‐labeled derivatives of podophyllotoxin at C‐2 in the mixture.     

Positive chemical ionization triple‐quadrupole mass spectrometry and ab initio computational studies of the multi‐pathway fragmentation of phthalates

We report the first positive chemical ionization (PCI) fragmentation mechanisms of phthalates using triple‐quadrupole mass spectrometry and ab initio computational studies using density functional theories (DFT). Methane PCI spectra showed abundant [M + H]⁺, together with [M + C₂H₅]⁺ and [M + C₃H₅]⁺. Fragmentation of [M + H]⁺, [M + C₂H₅]⁺ and [M + C₃H₅]⁺ involved characteristic ions at m/z 149, 177 and 189, assigned as protonated phthalic anhydride and an adduct of phthalic anhydride with C₂H₅⁺ and C₃H₅⁺, respectively. Fragmentation of these ions provided more structural information from the PCI spectra. A multi‐pathway fragmentation was proposed for these ions leading to the protonated phthalic anhydride. DFT methods were used to calculate relative free energies and to determine structures of intermediate ions for these pathways. The first step of the fragmentation of [M + C₂H₅]⁺ and [M + C₃H₅]⁺ is the elimination of [R? H] from an ester group. The second ester group undergoes either a McLafferty rearrangement route or a neutral loss elimination of ROH. DFT calculations (B3LYP, B3PW91 and BPW91) using 6‐311G(d,p) basis sets showed that McLafferty rearrangement of dibutyl, di(‐n‐octyl) and di(2‐ethyl‐n‐hexyl) phthalates is an energetically more favorable pathway than loss of an alcohol moiety. Prominent ions in these pathways were confirmed with deuterium labeled phthalates. Copyright © 2010 John Wiley & Sons, Ltd.     

Ionization and collision induced dissociation of steroid bisglucuronides

Studies on steroid metabolism are of utmost importance to improve the detection capabilities of anabolic androgenic steroids (AASs) misuse in sports drug testing. In humans, glucuronoconjugates are the most abundant phase II metabolites of AAS. Bisglucuronidation is a reaction where two separated functional groups on the same molecule are conjugated with glucuronic acid. These metabolites have not been studied in depth for steroids and could be interesting markers for doping control. The aim of the present work was to study the ionization and collision‐induced dissociation of steroid bisglucuronides to be able to develop mass spectrometric analytical strategies for their detection in urine samples after AAS administration. Because steroid bisglucuronides are not commercially available, 19 of them were qualitatively synthesized to study their mass spectrometric behavior. Bisglucuronides ionized as [M+NH₄]⁺ in positive mode, and as [M–H]⁻ and [M–2H]²⁻ in negative mode. The most specific product ions of steroid bisglucuronides in positive mode resulted from the neutral losses of 387 and 405 Da (corresponding to [M+NH₄–NH₃–2gluc–H₂O]⁺ and [M+NH₄–NH₃–2gluc–2H₂O]⁺, respectively, being “gluc” a dehydrated glucuronide moiety), and in negative mode, the fragmentation of [M–2H]²⁻ showed ion losses of m /z 175 and 75 (gluc⁻ and HOCH₂CO₂⁻, respectively). On the basis of the common behavior, a selected reaction monitoring method was developed to detect bisglucuronide metabolites in urine samples. As a proof of concept, urines obtained after administration of norandrostenediol were studied, and a bisglucuronide metabolite was detected in those urines. The results demonstrate the usefulness of the analytical strategy to detect bisglucuronide metabolites in urine samples, and the formation of these metabolites after administration of AAS.     

Rapid simultaneous determination of codeine and morphine in plasma using LC‐ESI‐MS/MS: Application to a clinical pharmacokinetic study

A rapid and sensitive high‐performance LC‐MS/MS method was developed and validated for the simultaneous quantification of codeine and its metabolite morphine in human plasma using donepezil as an internal standard (IS). Following a single liquid‐liquid extraction with ethyl acetate, the analytes were separated using an isocratic mobile phase on a C₁₈ column and analyzed by MS/MS in the selected reaction monitoring mode using the respective [M+H]⁺ ions, mass‐to‐charge ratio (m/z) 300/165 for codeine, m/z 286/165 for morphine and m/z 380/91 for IS. The method exhibited a linear dynamic range of 0.2–100/0.5–250 ng/mL for codeine/morphine in human plasma, respectively. The lower LOQs were 0.2 and 0.5 ng/mL for codeine and its metabolite morphine using 0.5 mL of human plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated LC‐MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 30 mg codeine phosphate.     

Bioanalytical assay development and validation for simultaneous quantification of five schisandra lignans in rat primary hepatocytes based on LC‐MS/MS: application to a real‐time uptake study for Schisandra Lignan Extract

Schisandra lignans, mainly including schizandrol A, schizandrol B, schisantherin A, schizandrin A, schizandrin B, etc., are the major active ingredients of Schisandra chinensis . In the present study, a robust liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method was developed for the simultaneous quantification of schisandra lignans in rat primary hepatocytes. Lovastatin was used as an internal standard, and chromatographic separation was achieved on a Shimadzu C₁₈ column with a gradient elution at the flow rate of 0.2 mL/min. All of the analytes were detected in multiple reaction monitoring mode with positive electrospray ionization since the sodium adduct ion [M + Na]⁺ was observed as the most intensive peak in the MS spectrum. For schizandrol A, schisantherin A and schizandrin A, the dynamic range was within 2–1000 ng/mg protein, and the linear range of schizandrol B and schizandrin B was from 5 to 1000 ng/mg protein. The intra‐ and inter‐day precision was <15% and the accuracy (relative error) ranged from −15 to 15%. No significant variation was observed in the stability tests. The validated method was then successfully applied to the time‐dependent uptake study for the Schisandra Lignan Extract in rat primary hepatocytes.     

Characterization of N‐Boc/Fmoc/Z‐N′‐formyl‐gem‐diaminoalkyl derivatives using electrospray ionization multi‐stage mass spectrometry

N‐Boc/Fmoc/Z‐N′‐formyl‐gem‐diaminoalkyl derivatives, intermediates particularly useful in the synthesis of partially modified retro‐inverso peptides, have been characterized by both positive and negative ion electrospray ionization (ESI) ion‐trap multi‐stage mass spectrometry (MSⁿ). The MS² collision induced dissociation (CID) spectra of the sodium adduct of the formamides derived from the corresponding N‐Fmoc/Z‐amino acids, dipeptide and tripeptide acids show the [M + Na‐NH₂CHO]⁺ ion, arising from the loss of formamide, as the base peak. Differently, the MS² CID spectra of [M + Na]⁺ ion of all the N‐Boc derivatives yield the abundant [M + Na‐C₄H₈]⁺ and [M + Na‐Boc + H]⁺ ions because of the loss of isobutylene and CO₂ from the Boc protecting function. Useful information on the type of amino acids and their sequence in the N‐protected dipeptidyl and tripeptidyl‐N′‐formamides is provided by MS² and subsequent MSⁿ experiments on the respective precursor ions. The negative ion ESI mass spectra of these oligomers show, in addition to [M‐H]^?, [M + HCOO]^? and [M + Cl]^? ions, the presence of in‐source CID fragment ions deriving from the involvement of the N‐protecting group. Furthermore, MSⁿ spectra of [M + Cl]^? ion of N‐protected dipeptide and tripeptide derivatives show characteristic fragmentations that are useful for determining the nature of the C‐terminal gem‐diamino residue. The present paper represents an initial attempt to study the ESI‐MS behavior of these important intermediates and lays the groundwork for structural‐based studies on more complex partially modified retro‐inverso peptides. Copyright © 2013 John Wiley & Sons, Ltd.     

A liquid chromatography–mass spectrometric method for the quantification of azithromycin in human plasma

A liquid chromatographic mass spectrometric assay for the quantification of azithromycin in human plasma was developed. Azithromycin and imipramine (as internal standard, IS) were extracted from 0.5 mL human plasma using extraction with diethyl ether under alkaline conditions. Chromatographic separation of drug and IS was performed using a C₁₈ column at room temperature. A mobile phase consisting of methanol, water, ammonium hydroxide and ammonium acetate was pumped at 0.2 mL/min. The mass spectrometer was operated in positive ion mode and selected ion recording acquisition mode. The ions utilized for quantification of azithromycin and IS were m/z 749.6 (M + H) ⁺ and m/z 591.4 (fragment) for azithromycin, and 281.1 m/z for internal standard; retention times were 6.9 and 3.4 min, respectively. The calibration curves were linear (r² > 0.999) in the concentration ranges of 10–1000 ng/mL. The mean absolute recoveries for 50 and 500 ng/mL azithromycin and 1 µg/ mL IS were >75%. The percentage coefficient of variation and mean error were <11%. Based on validation data, the lower limit of quantification was 10 ng/mL. The present method was successfully applied to determine azithromycin pharmacokinetic parameters in two obese volunteers. The assay had applicability for use in pharmacokinetic studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Change of the favored routes of EI MS fragmentation when proceeding from N1, N1‐dimethyl‐N2‐arylformamidines to 1,1,3,3‐tetraalkyl‐2‐arylguanidines: substituent effects

Although series of N¹, N¹‐dimethyl‐N²‐arylformamidines and of 1,1,3,3‐tetraalkyl‐2‐arylguanidines are structurally analogous and similar electron‐ionization mass spectral fragmentation may be expected, they display important differences in the favored routes of fragmentation and consequently in substituent effects on ion abundances. In the case of formamidines, the cyclization‐elimination process (initiated by nucleophilic attack of the N‐amino atom on the 2‐position of the phenyl ring) and formation of the cyclic benzimidazolium [M‐H]⁺ ions dominates, whereas the loss of the NR₂ group is more favored for guanidines. In order to gain information on the most probable structures of the principal fragments, quantum‐chemical calculations were performed on a selected set. A good linear relation between log{I[M‐H]⁺I [M]^+?} and σ_R⁺ constants of substituent at para position in the phenyl ring occurs solely for formamidines (r = 0.989). In the case of guanidines, this relation is not significant (r = 0.659). A good linear relation is found between log{I[M‐NMe₂]⁺/I [M]^+?} and σ_p⁺ constants (r = 0.993). Copyright © 2010 John Wiley & Sons, Ltd.     

Binding of Cu+ and Cu2+ with peptides: Peptides = oxytocin,Arg8‐vasopressin,bradykinin, angiotensin‐I,substance‐P,somatostatin, and neurotensin

The intrinsic binding ability of 7 natural peptides (oxytocin, arg⁸‐vasopressin, bradykinin, angiotensin‐I, substance‐P, somatostatin, and neurotensin) with copper in 2 different oxidation states (Cu^I/II) derived from different Cu^+/2+ precursor sources have been investigated for their charge‐dependent binding characteristics. The peptide‐Cu^I/II complexes, [M − (n‐1)H + nCu^I] and [M − (2n‐1)H + nCu^II], are prepared/generated by the reaction of peptides with CuI solution/Cu‐target and CuSO₄ solution and are analyzed by using matrix‐assisted laser desorption/ionization (MALDI) time‐of‐flight mass spectrometry. The MALDI mass spectra of both [M − (n‐1)H + nCu^I] and [M − (2n‐1)H + nCu^II] complexes show no mass shift due to the loss of ─H atoms in the main chain ─NH of these peptides by Cu⁺ and Cu²⁺ deprotonation. The measured m/z value indicates the reduction of Cu^I/II oxidation state into Cu⁰ during MALDI processes. The number and relative abundance of Cu⁺ bound to the peptides are greater compared with the Cu²⁺ bound peptides. Oxytocin, arg⁸‐vasopressin, bradykinin, substance‐P, and somatostatin show the binding of 5Cu⁺, and angiotensin‐I and neurotensin show the binding of 7Cu⁺ from both CuI and Cu targets, while bradykinin shows the binding of 2Cu²⁺, oxytocin, arg⁸‐vasopressin, angiotensin‐I, and substance‐P; somatostatin shows the binding of 3Cu²⁺; and neurotensin shows 4Cu²⁺ binding. The binding of more Cu⁺ with these small peptides signifies that the bonding characteristics of both Cu⁺ and Cu²⁺ are different. The amino acid residues responsible for the binding of both Cu⁺ and Cu²⁺ in these peptides have been identified based on the density functional theory computed binding energy values of Cu⁺ and the fragment transformation method predicted binding preference of Cu²⁺ for individual amino acids.