Reaction Monitoring-information Dependent Acquisition-enhanced Product Research Articles

Simultaneous determination of the residues of anesthetics and sedatives in fish using LC-QLIT-MS/MS combined with DSPE

Liquid chromatography coupled with quadrupole linear ion trap tandem mass spectrometry (LC-QLIT-MS/MS) technology operated in electrospray ionization (ESI) was developed for tracing anesthetic (AETs) and sedatives (SDTs) in fish. Sampling procedure was achieved by using acetonitrile extraction followed by dispersive solid phase extraction (DSPE) clean-up. Under the optimized laboratory conditions, reliable qualitative confirmation was obtained through the multiple reaction monitoring-information dependent acquisition-enhanced product ion (MRM-IDA-EPI) mode. Results indicated a favorable linear in the concentration range of 1–100 μg∙kg−1 (0.1–10 μg∙kg−1 for MS-222), with regression coefficient not less than 0.9997. The detection limit ranges from 0.03 to 0.4 μg∙kg−1 (S/N = 3). The validated method was applied to determine AETs and SDTs in fish with satidfied recoveries of 86.3 %–111.7 % and the relative standard deviations (RSD) of 1.9 %–8.9 % (n = 6). Practical samples analysis indicated that the proposed method is simple, rapid, sensitive and accurate for identification of AETs and SDTs.

Simultaneous determination of 36 hypotensive drugs in fingerprints by ultra performance liquid chromatography-triple quadrupole composite linear ion trap mass spectrometry

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,使用3×3 cm滤纸采集指印,将滤纸剪碎置于2 mL塑料离心管中,加入0.50 mL甲醇,涡旋混合1 min,超声振荡3 min,取出后以12000 r/min离心5 min,取上清液进样分析。采用ACQUITY UPLC® BEH C18柱(100 mm×3.0 mm, 1.7 μm)分离,以0.01%甲酸水溶液和甲醇作为流动相进行梯度洗脱。质谱分析采用可编程多反应监测-信息关联采集-增强子离子(SMRM-IDA-EPI)扫描方式,在高灵敏度分析的同时进行二级谱库检索,增加定性结果的准确性。36种药物在0.05~50.00 ng/fingerprint范围内线性关系良好,相关系数(r)大于0.99,检出限和定量限分别为0.001~0.045 ng/fingerprint和0.002~0.050 ng/fingerprint,在0.25、2.50、25.00 ng/fingerprint 3个加标水平下的基质效应为79.0%~119.2%,回收率为79.3%~116.2%,日内精密度为0.2%~18.3%,日间精密度为1.6%~19.1%。使用该方法检测了87名高血压患者指印中的降压药,大部分样本中可检出其所服用的药物。该方法操作简单,灵敏度高,选择性好,适用于指印中降压药的筛查检验。

Metabolite Profiling and Characterization of LW6, a Novel HIF-1α Inhibitor, as an Antitumor Drug Candidate in Mice.

A novel HIF (hypoxia-inducible factor)-1α inhibitor, the (aryloxyacetylamino)benzoic acid derivative LW6, is an anticancer agent that inhibits the accumulation of HIF-1α. The aim of this study was to characterize and determine the structures of the metabolites of LW6 in ICR mice. Metabolite identification was performed using a predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (pMRM-IDA-EPI) method in negative ion mode on a hybrid triple quadrupole-linear ion trap mass spectrometer (QTRAP). A total of 12 metabolites were characterized based on their MS/MS spectra, and the retention times were compared with those of the parent compound. The metabolites were divided into five structural classes based on biotransformation reactions: amide hydrolysis, ester hydrolysis, mono-oxidation, glucuronidation, and a combination of these reactions. From this study, 2-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)acetic acid (APA, M7), the metabolite produced via amide hydrolysis, was found to be a major circulating metabolite of LW6 in mice. The results of this study can be used to improve the pharmacokinetic profile by lowering the clearance and increasing the exposure relative to LW6.

Simultaneous determination and quantitation of hypolipidemic drugs in fingerprints by UPLC-Q-TRAP/MS

An ultra-performance liquid chromatography tandem triple quadrupole compound linear ion trap mass spectrometry (UPLC-Q-TRAP/MS) method was developed and validated for the detection of hypolipidemic drugs in fingerprints. 13 hypolipidemic drugs were well separated by the gradient elution of 0.01% formic acid in water and methanol at a flow rate of 0.4 mL/min within 11 min. The analytes were detected in positive (ESI+) and negative (ESI-) modes and scanned using scheduled multiple reaction monitoring-information dependent acquisition-enhanced product ion (SMRM-IDA-EPI) for best selectivity and sensitivity. The calibration curves showed good linearity in the range of 0.050–50.000 ng/patch with coefficients (r2) higher than 0.9904 for all analytes. Meantime, the LODs and LLOQs were in ranges of 0.001–0.034 and 0.003–0.050 ng/patch. The accuracies, intra-day and inter-day precision ranged from −13.3 to 0.3%, 1.1–10.4% and 3.7–14.5%, respectively. The recoveries ranged from 79.9 to 114.8%, while the absolute and relative matrix effects were in the range of 83.0–107.2% and 2.2–9.7%. By comparing the non-spiked fingerprints from healthy volunteers with the fingerprints obtained from patients, demonstrated that the method was competent for determination and quantitation of hypolipidemic drugs in fingerprints.

Enhanced identification of the in vivo metabolites of Ecliptae Herba in rat plasma by integrating untargeted data-dependent MS2 and predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion scan

Detection and identification of the in vivo metabolites of traditional Chinese medicine by untargeted profiling strategies are often confronted with severe interference from complex endogenous substances. Here we developed an integral approach, by combining untargeted data-dependent MS2 (dd-MS2) of Q-Orbitrap mass spectrometry and predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion scan (pMRM-IDA-EPI) of triple quadrupole-linear ion trap (QTRAP) mass spectrometry, aiming to detect and identify more extensive metabolites in bio-samples. Ecliptae Herba (EH) is a widely consumed medicinal herb with the effects of nourishing liver/kidney, but its metabolites in vivo have not been fully elucidated. Firstly, after UHPLC separation on an HSS T3 column, chemical fingerprinting of 70% ethanolic extract of EH was performed by untargeted dd-MS2 in negative ion mode. We could characterize 41 compounds from EH, and 24 were detectable in the plasma of rats (prototypes) after oral administration of EH extract (1 g/kg). Secondly, using echinocystic acid (triterpene), wedelolactone (coumarin), and apigenin (flavonoid) as the different parent templates, an MRM list containing 150 predicted ion-pairs was established to enhance MS2 scan by pMRM-IDA-EPI, which enabled the primary identification of up to 200 metabolites. The biotransformations mainly involve oxidation, hydrogenation, methylation, glucuronidation, sulfonation etc. Thirdly, the rat plasma samples obtained after oral administration of three pure compounds (echinocystic acid, wedelolactone and apigenin) were analyzed to verify the reliability of metabolites identification, and 11, 4, and 10 metabolites were found individually. This is the first comprehensive research on the metabolism of EH in vivo.

Development of a highly sensitive liquid chromatography with tandem mass spectrometry method for the qualitative and quantitative analysis of high-intensity sweeteners in processed foods

A new method for the simultaneous determination of two sweeteners (Advantame and Neotame) in processed foods using liquid chromatography (LC) with tandem mass spectrometry(MS/MS) was developed herein. Chromatographic separations were performed using an ACQUITY UPLC CSH C18 column at 40 °C via a mobile phase comprising 10-mmol/L ammonium formate and methanol. Samples were prepared via rapid dialysis using 30% methanol solution in a thermostatic shaker set at 160 rpm and 50 °C for 1 h. The matrix in the test solution had no effect on the identification and quantification of the compound without a clean-up step using solid-phase extraction (SPE). This method satisfied all validation criteria with a limit of quantification (LOQ) of 0.01 μg/g for all samples. Using this method, the amounts of Advantame and Neotame in 24 processed foods were subsequently investigated, with the results indicating their detection beyond the lower LOQ. Moreover, a multiple reaction monitoring information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) method was developed and described to further enhance product-identification ability.

Simultaneous rapid determination of 84 toxic plant constituents in plasma and urine by ultra-performance liquid chromatography- triple quadrupole/linear ion trap mass spectrometry

An ultra-performance liquid chromatography-triple quadrupole/linear ion trap mass spectrometry (UPLC-Qtrap MS) method was developed for the determination of 84 toxic plant constiuents in plasma and urine. Plasma was precipitated by acetonitrile to remove proteins and then passed through a Prime HLB SPE column to remove phospholipids, while urine was diluted with methanol. Chromatographic separation of the analytes was achieved on an Acquity BEH C18 column (100 mm×2.1 mm, 1.7 μm) by gradient elution using the mobile phase of 0.1% (v/v) formic acid and 2 mmol/L ammonium formate both in 97% (v/v) acetonitrile aqueous solution and water. Electrospray ionization mass spectrometry was carried out in the positive ion mode with multiple reaction monitoring-information dependent acquisition-enhanced product ion scan mode (MRM-IDA-EPI). The 84 analytes were quantified by the matrix working standard curve internal standard method, and a good linear relationship was observed, with correlation coefficients of ≥ 0.9911. The limits of detection (LODs) in plasma and urine were 0.01-1 μg/L and 0.03-2 μg/L, respectively. The intra- and inter-day precisions of these analytes were 0.7%-18.4% and 1.1%-18.5%, and the accuracy of all analytes ranged from 70.6% to 124.5%. This method is simple, sensitive, and accurate for the measurement of these analytes in plasma and urine for both clinical and forensic applications.

Identification and quantification of cereulide in cow’s milk using liquid chromatography-tandem mass spectrometry

ABSTRACTIn this study, the presence of cereulide in cow’s milk was identified and quantified using our validated method with liquid chromatography-tandem mass spectrometry. Cereulide was concentrated using protein acid-precipitation and extracted from the precipitate by using acetonitrile twice. The combination of protein acid-precipitation and extraction sufficiently eliminated the matrix compounds from the milk and a further clean-up step utilising solid-phase extraction could be omitted. For robustly measuring the samples and keeping the MS devices clean, the extraction solution was diluted 10-fold using methanol. Owing to the minimisation of the interferences caused by fragmentation patterns, multiple reaction monitoring information-dependent acquisition-enhanced product ion spectra enabled the characterisation and identification of cereulide. Besides the matrix effect (−4%), an external solvent calibration curve was adapted for accurate quantification. The method was validated using fortified recovery tests, at two concentrations (10 and 50 µg kg−1), using three samples daily on five different days based on the Japanese guidelines. This new method exhibited good accuracy ranging from 91% to 94%. The relative standard deviations of repeatability ranged from 2% to 5%, and the relative standard deviation of within-laboratory reproducibility ranged from 5% to 6%. These standard deviations satisfied the criteria for the Japanese validation guidelines. The limit of quantification (LOQ) was estimated to be 2 μg kg−1. On the product ion spectra at the LOQ level, the library match was satisfactory with a purity fit value of >70%. The method was applied to 14 raw milk and three milk samples pasteurised using the low-temperature, long-time process and collected in Tokyo. None of the samples was found to contain the target toxin.

Simultaneous determination of amantadine and rimantadine in feed by liquid chromatography-Qtrap mass spectrometry with information-dependent acquisition.

A sensitive method for simultaneous determination of amantadine and rimantadine in feed was developed using an ultra-high-performance liquid chromatography-triple quadrupole linear ion trap mass spectrometry (UHPLC-Qtrap-MS) in the multiple reaction monitoring information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) mode, and employing the mixed cation exchange (MCX) solid-phase extraction column as sample cleanup and amantadine-d15 and rimantadine-d4 as internal standards, respectively. Compared to traditional MRM mode, for the targeted drugs in feed simultaneously both the secondary mass spectra and MRM information can be obtainedusing UHPLC-Qtrap-MS with MRM-IDA-EPI mode, and thus more accurate qualitative confirmation results achievedeven at lower concentration of 0.2μg/L in acceptable purity fit values. After optimization of sample preparation, good linearities (R > 0.9994) were obtained over the concentration range from 1 to 200μg/L for amantadine and rimantadine. The precision was validated by intra-day and inter-day, and the relative standard deviations were all within 9.61%. Mean recoveries ranged from 76.1 to 112% at spiked concentrations of 0.5-100 μg/kg in three types of feed samples, including formula feed and complex concentrated feed for pigs and premix feed for chicken. The limits of detection (LODs) and quantification (LOQs) were 0.2 and 0.5μg/kg for both drugs, respectively. The application in real feed samples further proved the accuracy and reliability of the developed method. This method provides an important tool to detect illegal uses of amantadine and rimantadine in feed. Graphical abstract Simultaneous quantitation and qualitative confirmation of amantadine and rimantadine in feed by MRM-IDA-EPI.

Rapid Simultaneous Screening and Detection of 12 Anticoagulant Rodenticides in Food by Ultra-performance Liquid Chromatography-Triple Quadrupole/Linear Ion Trap Tandem Mass Spectrometry

A rapid analytical method for the simultaneous screening and detection of 12 anticoagulant rodenticides in food samples was established based on modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) sample preparation method using an ultra-performance liquid chromatography-triple quadrupole/linear ion trap tandem mass spectrometry (UPLC-QTrap-MS/MS). Food samples were extracted and purified with modified QuEChERS method. The 12 anticoagulant rodenticides (warfarin, brodifacoum, difethialone, coumachlor, coumatetralyl, bromadiolone, chlorophacinone, difenacoum, diphacinone, pindone, valone, and flocoumafen) and the internal standard (warfarin-D5) were separated within 6 min using an ACQUITY UPLC BEH-C18 column (1.7 μm, 2.1 mm × 50 mm) and gradient elution with the mobile phase consisting of 5 mM ammonium acetate formate buffer and acetonitrile. The multiple reaction monitoring-information dependent acquisition-enhanced product ion (MRM-IDA-EPI) scanning was employed for detection. The calibration curves were linear (R2 > 0.99) for all the compounds. The mean recoveries for the 12 analytes at three spiked levels (1× LOQ, 5× LOQ, and 10× LOQ) were in the range of 79.5–113.2% with RSDs of 1.8–13.2%.The limits of detection (LOD) for the 12 rodenticides ranged from 0.01 (warfarin) to 0.05 μg/kg (diphacinone). The limit of quantification (LOQ) for the 12 rodenticides was between 0.02 (warfarin) and 0.10 μg/kg (diphacinone). The developed method was more straightforward, less time and labor intensive, and more sensitive, selective, and accurate for screening multiple anticoagulant rodenticides, and it was successfully used in several poisoning cases.

Simultaneous qualitative and quantitative analysis of 21 mycotoxins in Radix Paeoniae Alba by ultra-high performance liquid chromatography quadrupole linear ion trap mass spectrometry and QuEChERS for sample preparation.

A high-throughput method for simultaneous qualitative and quantitative analysis of 21 mycotoxins in Radix Paeoniae Alba (RPA) was developed by coupling the modified QuEChERS method with ultra-high performance liquid chromatography quadrupole linear ion trap mass spectrometry (UHPLC-QqLIT-MS). The 21 mycotoxins were extracted and cleaned up using QuEChERS-based procedure, then further separated on a C18 column and detected by a hybrid triple quadrupole linear ion trap mass spectrometer equipped with electrospray ionization source in the multiple reaction monitoring-information dependent acquisition-enhanced product ion (MRM-IDA-EPI) mode. Under this technique, 13 mycotoxins were detected using acetonitrile and water containing 0.1% formic acid as the mobile phase in positive mode while the other 8 mycotoxins were detected using acetonitrile and water containing 0.1% ammonia as the mobile phase in negative mode. The calibration curves of all analytes showed good linearity (r(2)>0.995) within test ranges. The limits of detection and quantification ranged from 0.031 to 5.4μg/kg and 0.20 to 22μg/kg, respectively. Additionally, recoveries were all above 75.3% with relative standard deviations within 15%. The method proposed herein with significant advantages including simple pretreatment, rapid determination as well as high sensitivity, accuracy and throughput would be a preferred candidate for the determination and quantification of multi-class mycotoxin contaminants in real samples.

Characterization of fimasartan metabolites in human liver microsomes and human plasma

1. The metabolites of fimasartan (FMS), a new angiotensin II receptor antagonist, were characterized in human liver microsomes (HLM) and human subjects.2. We developed a method for a simultaneous quantitative and qualitative analysis using predictive multiple reaction monitoring information-dependent acquisition-enhanced product ion scanning. To characterize metabolic reactions, FMS metabolites were analyzed using quadrupole-time of flight mass spectrometer in full-scan mode.3. The structures of metabolites were confirmed by comparison of chromatographic retention times and mass spectra with those of authentic metabolite standards.4. In the cofactor-dependent microsomal metabolism study, the half-lives of FMS were 56.7, 247.9 and 53.3 min in the presence of NADPH, UDPGA and NADPH + UDPGA, respectively.5. The main metabolic routes in HLM were S-oxidation, oxidative desulfuration, n-butyl hydroxylation and N-glucuronidation.6. In humans orally administered with 120 mg FMS daily for 7 days, the prominent metabolites were FMS S-oxide and FMS N-glucuronide in the 0–8-h pooled plasma sample of each subject.7. This study characterizes, for the first time, the metabolites of FMS in humans to provide information for its safe use in clinical medicine.

Determination of species-difference in microsomal metabolism of amitriptyline using a predictive MRM–IDA–EPI method

We investigated to compare species differences in amitriptyline (AMI) metabolism among mouse, rat, dog, and human liver microsomes. We developed a method for simultaneous determination of metabolic stability and metabolite profiling using predictive multiple reaction monitoring information-dependent acquisition-enhanced product ion (MRM–IDA–EPI) scanning. In the cofactor-dependent microsomal metabolism study, AMI was metabolized more rapidly in rat and human liver microsomes incubated with NADPH than UDPGA. AMI incubated with NADPH+UDPGA in rat, dog, or mouse liver microsomes disappeared rapidly with a half-life of 3.5, 8.4, or 9.2min, respectively, but slowly in human liver microsomes with a half-life of 96min. In total, 9, 10, 11, and 6 putative metabolites of AMI were detected in mouse, rat, dog, and human liver microsomes, respectively, based on mass spectrometric analyses. Kinetic analysis of metabolites in liver microsomes from each species over 120min showed common metabolic routes of AMI, such as N-demethylation, hydroxylation, and glucuronidation, and subtle interspecies differences in AMI metabolism. The main metabolic routes in mouse, rat, dog, and human liver microsomes were hydroxylation followed by glucuronide conjugation, methyl hydroxylation, and N-demethylation, respectively. The MRM–IDA–EPI method can provide quantitative and qualitative information about metabolic stability and metabolite profiling simultaneously. Moreover, time course analysis of metabolites can not only eliminate false identification of metabolites, but also provide a rationale for proposed metabolic pathways. The MRM–IDA–EPI method combined with time course analysis of metabolites is useful for investigating drug metabolism at the early drug discovery stage.

In vitro enantioselective metabolism of TJ0711 hydrochloride by human liver microsomes using a novel chiral liquid chromatography–tandem mass spectrometry method

A novel liquid chromatography–tandem mass spectrometry (LC–MS/MS) method employing chiral analytical techniques was developed and validated for in vitro enantioselective metabolic stability study of racemic 1-[4-(2-methoxyethyl) phenoxy]-3-[[2-(2-methoxyphenoxy) ethyl]amino]-2-propanol hydrochloride (TJ0711 HCl), a newly developed vasodilatory β-blocker. Robust enantiomeric separations were achieved on a chiral SUMICHIRAL OA-2500 column using ethanol and hexane (40:60, v/v) as a mobile phase. Metabolic stability results demonstrated that both TJ0711 enantiomers underwent a rapid phase I metabolism, but preferential metabolism of R-TJ0711 was observed. Our previously reported ultra-performance liquid chromatography-multiple reaction monitoring-information dependent acquisition-enhanced product ion (UPLC-MRM-IDA-EPI) method was finally chosen for metabolite profiling study of TJ0711 enantiomers, because the newly developed HPLC-based method resulted in compromised chromatographic separation, particularly for TJ0711 metabolites. A number of metabolic products were detected and the structures of formed metabolites were predicted. Similar to racemic TJ0711 HCl, demethylation and hydroxylation were proposed to be the principle metabolism pathways during in vitro incubations of each enantiomer with human liver microsomes.

Tentative identification of new metabolites of epimedin C by liquid chromatography–mass spectrometry

Epimedin C is one of the major bioactive constituents of Herba Epimedii. The aim of this study is to characterize and elucidate the structure of metabolites in the rat after administration of epimedin C. Metabolite identification was performed using a predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (pMRM-IDA-EPI) scan in positive ion mode on a hybrid triple quadrupole-linear ion trap mass spectrometer. A total of 18 metabolites were characterized by the changes in their protonated molecular masses, their MS/MS spectrum and their retention times compared with those of the parent drug. The results reveal possible metabolite profiles of epimedin C in rats; the metabolic pathways including hydrolysis, hydroxylation, dehydrogenation, demethylation and conjugation with glucuronic acid and different sugars were observed. This study provides a practical approach for rapidly identifying complicated metabolites, a methodology that could be widely applied for the structural characterization of metabolites of other compounds.