Determination of substance P in rat spinal cord by high-performance liquid chromatography electrospray quadrupole ion trap mass spectrometry

Substance P is a neuropeptide that belongs to the tachykinin neuropeptide family. It is an 11-amino acid polypeptide with the amino acid sequence: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met. It is synthesized as a larger protein and then enzymatically converted into the active undecapeptide. Substance P is widely distributed in the central and peripheral nervous systems. In the central nervous system, substance P participates in various behavioral responses and in regulating neuronal survival and degeneration. In the spinal cord, substance P participates in neurotransmission of pain and modulates autonomic reflexes. A rapid and selective method was developed for the determination of substance P concentration in rat spinal cord. The method consisted of a tissue homogenization, dilution, centrifugation and analysis by full-scan liquid chromatography electrospray quadrupole ion trap mass spectrometry (LC-ESI-QIT). The separation was achieved using a 50 x 2.1 mm C(18) analytical column combined with a gradient mobile phase composed of methanol: 0.1% formic acid in water set at a flow rate of 0.2 mL/min. An analytical range of 10-500 pmol/g was tested to analyze rat spinal cord. The LOD observed was 10 fmol injected on column. The novel method met all requirements of specificity, sensitivity, linearity, precision, accuracy and stability. In conclusion, a rapid and sensitive LC-ESI/MS/MS method was developed to identify and quantify substance P in rat spinal cord.     

Characterization of in vitro metabolites of trimethoprim and diaveridine in pig liver microsomes by liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry

Trimethoprim (TMP) and diaveridine (DVD) are used in combination with sulfonamides and sulfaquinoxlaine as an effective antibacterial agent and antiprotozoal agent, respectively, in humans and animals. To gain a better understanding of the metabolism of TMP and DVD in the food-producing animals, the metabolites incubated with liver microsomes of pigs were analyzed for the first time with high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry. Seven TMP-related and six DVD-related metabolites were characterized based on the accurate MS2 spectra and known structure of the parent drug, respectively. The metabolites of TMP were identified as two O-demethylation metabolites, a di-O-demethylation metabolite, two N-oxides metabolites, a hydroxylated metabolite on the methylene carbon and a hydroxylated metabolite on the methyl group. DVD was also biotransformed to two O-demethylation metabolites, a di-O-demethylation metabolite, an N-oxide metabolite, a hydroxylation metabolite on the methylene carbon and a hydroxylation metabolite followed by O-demethylation. The results indicate that the two compounds have similar biotransformation pathways in pigs. O-Demethylation was the major metabolic route of TMP and DVD in the pig liver microsomes. The proposed metabolic pathways of TMP and DVD in liver microsomes will provide a basis for further studies of the in vivo metabolism of the two drugs in food-producing animals.     

Quantitative determination of capsaicin, a transient receptor potential channel vanilloid 1 agonist, by liquid chromatography quadrupole ion trap mass spectrometry: evaluation of in vitro metabolic stability

Capsaicin is the most abundant pungent molecule present in red peppers and it is widely used for food flavoring, in pepper spray in self-defense devices and more recently in ointments for the relief of neuropathic pain. Capsaicin is a selective agonist of transient receptor potential channel, vanilloid subfamily member 1. A selective and sensitive quantitative method for the determination of capsaicin by LC-ESI/MS/MS was developed. The method consisted of a protein precipitation extraction followed by analysis using liquid chromatography electrospray quadrupole ion trap mass spectrometry. The chromatographic separation was achieved using a 100 x 2 mm C(18) Waters Symmetry column combined with a gradient mobile phase composed of acetonitrile and 0.1% formic acid aqueous solution at a flow rate of 220 microL/min. The mass spectrometer was operating in full-scan MS/MS mode using two-segment analysis. An analytical range of 10-5000 ng/mL was used in the calibration curve constructed in rat plasma. The interbatch precision and accuracy observed were 6.5, 6.7, 5.3 and 101.2, 102.7, 103.5% at 50, 500 and 5000 ng/mL, respectively. An in vitro metabolic stability study was performed in rat, dog and mouse liver microsomes and the novel analytical method was adapted and used to determine intrinsic clearance of capsaicin. Results suggest very rapid degradation with T(1/2) ranging from 2.3 to 4.1 min and high clearance values suggesting that drug bioavailability will be considerably reduced, consequently affecting drug response and efficacy.     

Characterization of in vitro metabolism of capsazepine,a vanilloid transient receptor potential channel antagonist,by liquid chromatography quadrupole ion trap mass spectrometry

Capsazepine is an antagonist of the transient receptor potential channel vanilloid 1 (TRPV1), which is known to play an important role in the regulation of pain and inflammation. A selective and sensitive quantitative method for the determination of capsazepine by HPLC‐ESI/MS/MS was developed. The method consisted of a protein precipitation extraction followed by analysis using liquid chromatography electrospray quadrupole ion trap mass spectrometry. The chromatographic separation was achieved using a 100 × 2 mm C₁₈ Waters Symmetry column combined with a gradient mobile phase composed of acetonitrile and 0.1% formic acid aqueous solution at a flow rate of 220 µL/min. The mass spectrometer was operating in full‐scan MS/MS mode using two‐segment analysis. An analytical range of 10–5000 ng/mL was used in the calibration curve constructed in rat plasma. The inter‐batch precision and accuracy observed were 10.1, 6.4 and 6.1% and 100.8, 98.5 and 106.2% at 50, 500 and 5 000 ng/mL, respectively. An in vitro metabolic stability using rat, dog or mouse liver microsomes was performed to determine the intrinsic clearance of capsazepine. The results suggest a very rapid degradation with T_1/2 ranging from 2.6 to 4.3 min and a high clearance, suggesting that drug bioavailability is considerably reduced following extravascular administrations, consequently affecting drug response. Three metabolites were identified by HPLC‐MS/MS. S‐hydroxylation (M + 16), oxidative desulfuration (M − 16) and desulfuration (M − 32) metabolites of capsazepine were observed following exposure to rat, dog and mouse microsomes. Copyright © 2010 John Wiley & Sons, Ltd.     

Investigation of the biotransformation pathway of verapamil using electrochemistry/liquid chromatography/mass spectrometry - a comparative study with liver cell microsomes

The biotransformation pathway of verapamil, a widely prescribed calcium channel blocker, was investigated by electrochemistry (EC) coupled online to liquid chromatography (LC) and electrospray mass spectrometry (ESI-MS). Mimicry of the oxidative phase I metabolism was achieved in a simple amperometric thin-layer cell equipped with a boron-doped diamond (BDD) working electrode. Structures of the electrochemically generated metabolites were elucidated on the basis of accurate mass data and additional MS/MS experiments. We were able to demonstrate that all of the most important metabolic products of the calcium antagonist including norverapamil (formed by N-demethylation) can easily be simulated using this purely instrumental technique. Furthermore, newly reported metabolic reaction products like carbinolamines or imine methides become accessible. The results obtained by EC were compared with conventional in vitro studies by conducting incubations with rat as well as human liver microsomes (RLMs, HLMs). Both methods showed good agreement with the data from EC/LC/MS. Thus, it can be noted that EC is very well-suited for the simulation of the oxidative metabolism of verapamil. In summary, this study confirms that EC/LC/MS can be a powerful tool in drug discovery and development when applied complementary to established in vitro or in vivo approaches.     

Multistep mass spectrometry of heterocyclic amines in a quadrupole ion trap mass analyser

The fragmentation of heterocyclic amines (HAs) in an ion trap was studied by means of the infusion of methanolic solutions containing the compounds under assay, and using an atmospheric pressure chemical ionization (APCI) as ion source. The MS(n) spectra obtained for compounds included in the same family, either aminoimidazoazaarenes (AIAs) or carbolines, were compared in order to propose fragmentation pathways for each HA. Moreover, labelled AIAs were used to establish the mechanisms. The protonated molecule was always obtained, but subsequent fragmentation was different for both families. In the case of AIAs, major product ions came from the fragmentation of the aminoimidazole moiety, thus the base peak in MS(2) corresponded to the loss of the methyl group, and losses of C(2)NH(3) and CN(2)H(2) were also observed. Further fragmentation occurred in the heterocyclic rings, mainly with losses of HCN and CH(3)CN. For carbolines, the most important product ions came from the loss of ammonia, except for harman and norharman, the loss of a methyl group for methylated carbolines or the loss of diverse fragments from the heterocyclic rings. In some cases, ion-molecule reactions into the ion trap were observed. For instance, for AalphaC or MeAalphaC one ion originating from these reactions corresponded to the base peak.     

Characterisation of selected hypnotic drugs and their metabolites using electrospray ionisation with ion trap mass spectrometry and with quadrupole time-of-flight mass spectrometry and their determination by liquid chromatography-electrospray ionisation-ion trap mass spectrometry

The electrospray ionisation-ion trap mass spectrometry (ESI-MSⁿ) of selected hypnotic drugs, i.e. zopiclone, zolpidem, flunitrazepam and their metabolites have been investigated. Sequential product ion fragmentation experiments (MSⁿ) have been performed in order to elucidate the degradation pathways for the [M+H]⁺ ions and their predominant fragment ions. These MSⁿ experiments show certain characteristic fragmentations in that functional groups are generally cleaved from the ring systems as neutral molecules such as H₂O, CO, CO₂, NO₂, amines and HF. When an aromatic entity is present in a drug molecule together with a nitrogen-containing saturated ring structure as with zopiclone and its N-desmethyl metabolite fragmentation initially occurs at the latter ring with the former being resistant to fragmentation. The structures of fragment ions proposed for ESI-MSⁿ can be supported by electrospray ionisation-quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS).These molecules can be identified and determined in mixtures at low ng/ml concentrations by the application of liquid chromatography (LC)-ESI-MSⁿ which can be used for their analysis in saliva samples.This paper includes a tabulation of mass losses/signals at low m/z values for these hypnotic drugs and many others in recent publications which will be of value in the characterisation of drug metabolites of unknown structure and also natural product pharmaceuticals isolated from plants, etc.     

High-performance liquid chromatography-tandem mass spectrometry with a new quadrupole/linear ion trap instrument

The use of a new hybrid quadrupole/linear ion trap known as the Q TRAP offers unique benefits as a LC-MS-MS detector for both small and large molecule analyses. The instrument combines the capabilities of a triple quadrupole mass spectrometer and ion trap technology on a single platform. Product ion scans are conducted in a hybrid fashion with the fragmentation step accomplished via acceleration into the collision cell followed by trapping and mass analysis in the Q3 linear ion trap. This results in triple quadrupole fragmentation patterns with no inherent low molecular mass cutoff. In-trap fragmentation is also possible in order to provide triple MS (MS3) capabilities. There are also several scan modes that are not possible on conventional instruments that enable identification of analytes within complex biological matrixes for subsequent high sensitivity product ion scans. This report will describe the new hybrid instrument and the principles of operation, and also provide examples of the unique scan modes and capabilities of the Q TRAP for LC-MS-MS detection in metabolism identification.     

离子阱颗粒质谱研究进展

随着质谱技术的不断发展,对超高质量颗粒物质的分析已经成为质谱领域研究的一个重要方向.离子阱颗粒质谱(particle ion trap mass spectrometry)作为用于完整颗粒质量分析的有利工具,拓展了质谱技术在巨大颗粒物质量分析中的应用范围.本文对离子阱颗粒质谱仪器的研究进展及其在各个领域的应用进行了综述,并展望了离子阱颗粒质谱未来的发展趋势.     

液相色谱-大气压化学电离离子阱质谱法测定烟草中的游离茄尼醇

用液相色谱/大气压化学电离离子阱质谱建立了一种分析烟草中游离茄尼醇的方法。烟草样品用甲醇振荡提取30 min,在分析前无需进行其它前处理。在1.8μm快速分离C18色谱短柱上用V(甲醇)∶V(异丙醇)=85∶15等梯度洗脱实现了茄尼醇的快速分离。用不带碰撞能量的二级质谱全扫描选择监测离子m/z 613.6进行定量,检出限为0.4μg/L,RSD为1.1%,两种添加量的回收率分别为97%和99%。方法应用于不同烟草和烟草制品样品的检测分析。     

超高效液相色谱-三重四极杆复合线性离子阱质谱法同时测定指印中36种降压药

指印中蕴含着供体摄入成分等相关信息,通过对其分析可对供体进行特征刻画,从而为案件侦查提供线索,指印分析也可用于药物摄入的定性检测,因此检验指印中的降压药具有重要的实际应用价值.建立了超高效液相色谱-三重四极杆复合线性离子阱质谱(UPLC-Q-TRAP/MS)同时检测指印中36种降压药的方法.前处理方法采用沉淀蛋白法,使...     

Trace analysis of herbicides in wastewaters by a dispersive liquid–liquid microextraction approach and liquid chromatography with quadrupole linear ion trap mass spectrometry: Evaluation of green parameters

An analytical method for determining phenylureas (monuron, isoproturon, diuron, linuron and neburon) and propanil herbicides in wastewater has been developed and validated, and the most significant parameters were compared with the corresponding ones found in the literature, thus showing the method performance. The method involves pre‐concentration by a simple, rapid, sensitive and low environmental toxicity temperature‐controlled ionic liquid dispersive liquid–liquid microextraction procedure. The herbicides were identified and determined by liquid chromatography with a hybrid triple quadrupole linear ion trap mass spectrometer. Data acquisition in selected‐reaction monitoring mode allowed the simultaneous identification and quantification of the analytes using two transitions. The information dependent acquisition scan was performed to carry out the identification of those analytes whose second transition was present at low intensity, also providing extra confirmation for the other analytes. Limits of quantification were in the range 1.0–5.0 ng/L. Good recoveries (95–103%) were obtained for the extraction of the target analytes in wastewater samples. The methodology developed was applied to analyze effluent wastewater samples from a wastewater treatment plant located in an agricultural zone of Almería (Spain) and the results indicated the presence of diuron at mean concentration levels of 73.5 ng/L.     

A comprehensive study of celastrol metabolism in vivo and in vitro using ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry

Celastrol has attracted great attention owing to its anti-arthritis, antioxidant, and anticancer activities. Nevertheless, its metabolism in vivo (rats) and in vitro (rat liver microsomes and intestinal flora) has not been comprehensively characterized. In this study, ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry was used as a rapid and sensitive approach for studying the metabolism of celastrol in vivo and in vitro. A total of 43 metabolites were identified and characterized. These include 26 metabolites in vivo, and 28 metabolites in vitro (nine metabolites in rat liver microsomes and 24 metabolites in rat intestinal flora). Additionally, the celastrol-biotransformation capacity of the intestinal tract was confirmed to exceed that of the liver. Furthermore, the metabolic profile of celastrol is summarised. The information obtained from this study may provide a basis for understanding the pharmacological mechanisms of celastrol and will be beneficial for clinical applications.     

超快速液相色谱-四极杆/线性离子阱质谱同时检测猪组织中9种β-受体阻断剂残留

建立了同时检测猪组织中9种β-受体阻断剂(BBs)残留的超快速液相色谱-四极杆/线性离子阱质谱方法。均质试样经β-葡糖醛苷酶/芳基硫酸酯酶水解,乙腈提取,硅藻土与BondElut分散固相萃取填料双重快速净化,以0.1%(v/v)甲酸水溶液-甲醇为流动相使用KinetexTMC18-XB色谱柱(150 mm×2.1 mm,2.6μm)超快速液相色谱分离,优化多反应监测(MRM)离子对后,采用预设定多反应监测(sMRM)-信息依赖性采集(IDA)-增强子离子扫描(EPI)模式检测,在线EPI谱库定性分析,内标法定量。结果表明,9种BBs在线性范围内的线性关系良好(r≥0.995);定量限(LOQ,S/N≥10)均达到0.5μg/kg;3个添加水平(0.5、1.0和5.0μg/kg)下的回收率为87.5%~111.8%;RSD为4.0%~12.5%。该方法快速、准确、灵敏,可有效用于猪组织样品中多种BBs残留的同时测定。     

Investigation of amodiaquine bulk drug impurities by liquid chromatography/ion trap mass spectrometry

Three unknown impurities in an amodiaquine bulk drug sample were detected by reversed-phase high-performance liquid chromatography with ultraviolet detection (HPLC/UV). A liquid chromatography/tandem mass spectrometry (LC/MS(n)) method is described for the investigation of these impurities. Mass spectral data were acquired on an LCQ ion trap mass analyzer equipped with an electrospray ionization (ESI) source operated in positive ion mode. The fragmentation behavior of amodiaquine and its impurities has been studied. Based on the mass spectral data and the specifics of the synthetic route, the possible structures of these impurities were elucidated as 4-[(5-chloroquinolin-4-yl)amino]-2-(diethylaminomethyl)phenol (impurity I), 4-[(7-chloroquinolin-4-yl)-amino]phenol (impurity II) and 4-[(7-chloroquinolin-4-yl)amino]-2-(diethylaminomethyl)-N(1)-oxy]phenol (impurity III). The structures were confirmed by their independent synthesis and NMR spectral assignment.     

Determination of organotins in water by micro liquid chromatography–electrospray/ion trap mass spectrometry

Due to the varying toxicity the species of organotins in their widespread applications, it is important for analytical methods to address their speciation. Traditional methods call for the hydrolysis and subsequent derivatization of the organotins before analysis. These methods can be time‐consuming, derivatization can be incomplete and high levels of background interference produce difficulties in identification and quantification. The use is described of a non‐derivatization and non‐hydrolysis micro‐liquid chromatography–electrospray/ion trap mass spectrometry for separation and detection of the organotins. Copyright © 1999 John Wiley & Sons, Ltd.     

Investigation of vancomycin and related substances by liquid chromatography/ion trap mass spectrometry

Liquid chromatography (LC) methods compatible with mass spectrometry (MS) that are suitable for impurity profiling of vancomycin mixtures have not been described in the literature. The mobile phases of the existing methods contain non-volatile additives and/or solvents that give problems in combination with MS. In this paper, a reversed-phase LC/tandem mass spectrometry method is described for the investigation of vancomycin and related substances. The LC method uses a Zorbax Extend C18 column (250 x 4.6 mm i.d.), 5 microm, and a mobile phase consisting of methanol, water and ammonium acetate solution (pH 9.0). This method allows us to separate vancomycin and its impurities. Mass spectral data are acquired on an LCQ ion trap mass spectrometer equipped with an electrospray interface operated in the positive and negative ion modes. The LCQ is ideally suited for identification of impurities and related substances because it provides on-line LC/MSn capability, which allows efficient identification without time-consuming isolation and purification procedures. Using this method, the fragmentation of vancomycin and known derivatives was studied and the structures of six substances occurring in commercial samples were elucidated.     

Multiclass,multiresidue method for the detection of antibiotic residues in distillers grains by liquid chromatography and ion trap tandem mass spectrometry

The increased production of ethanol in the US has resulted in large amounts of distillers grains (DG) which is an excellent feed supplement for livestock. However, the use of antimicrobials during ethanol fermentation has led to a growing concern over the possibility of their residues remaining in DG. To enable the detection of antimicrobial residues, a robust LC–MS/MS method was developed that included 13 antibiotics of diverse chemistries, ampicillin, penicillin G, tetracycline, oxytetracycline, chlortetracycline, bacitracin A, virginiamycin M1, chloramphenicol, erythromycin A, clarithromycin, tylosin A, monensin A and streptomycin. The residues were extracted with an aqueous solution of EDTA and trichloroacetic acid followed by methanol. The combined extract was subjected to a two-track cleanup and concentration on either hydrophilic polymeric or weak cation exchange solid phase extraction cartridges. The extracts are analyzed by LC/ion trap tandem mass spectrometry. The method was validated in dry DG matrix. Absolute recoveries of the analytes ranged from 50 to 100%. Accuracy ranged from 89 to 111% based on calibration by processed standards. The limits of detection and relative standard deviation are satisfactory to support future surveillance studies. The method was subsequently tested in three different end-products of DG: distillers dry grains, distillers wet grains and distillers grains solubles.