Simultaneous determination of components released from dental composite resins in human saliva by liquid chromatography/multiple-stage ion trap mass spectrometry

Dental composite resins are widely used for fixing teeth; however, the monomers used in dental composite resins have been found to be cytotoxic and genotoxic, namely triethylene glycol dimethacrylate (TEGDMA), urethane dimethacrylate (UDMA), and bisphenol A glycol dimethacrylate (Bis-GMA). In this study, we incubated dental composite resins with human saliva for demonstrating the released monomers and biodegradation products. A simple saliva sample dilution method without purification or derivatization was used for quantification. We found that liquid chromatography coupled with multiple-stage ion trap mass spectrometry (LC-MS(n) ) operated in selected reaction monitoring (SRM) mode was able to separate the three monomers within 10 min. The calibration curves were linear (R2 >0.996) over a wide range for each monomer in saliva: TEGDMA, 5-500 ppb; UDMA, 5-100 ppb, and Bis-GMA, 5-700 ppb. Furthermore, several biodegradation products were discovered with data-dependent MS/MS scan techniques. Although TEGMA degradation products have previously been reported, we identified two previously unknown UDMA degradation products. The LC-MS/MS method developed in this study was able to successfully quantify monomers and their principal biodegradation products from dental composite resins in human saliva.     

Characterization of impurities in spiramycin by liquid chromatography/ion trap mass spectrometry

A reversed-phase liquid chromatography/tandem mass spectrometry method is described for the investigation of spiramycin and related substances. The method uses an XTerra C18 column (250 x 4.6 mm i.d.), 5 microm, and a mobile phase consisting of acetonitrile, methanol, water and ammonium acetate solution, pH 6.5. Mass spectral data were acquired on an LCQ ion trap mass spectrometer equipped with atmospheric pressure chemical ionization (APCI) operated in the positive ion mode. Using this method, the fragmentation behavior of spiramycin and its related substances was studied and the unknown impurities occurring in commercial samples were investigated. In total 17 compounds were identified, among which three reported as specified impurities in the European Pharmacopoeia. The other impurities showed mainly a modification in the forosamine sugar or in the substituent at C-3 and C-6 positions. In one impurity, the mycarose sugar is absent.     

Characterization of impurities in dirithromycin by liquid chromatography/ion trap mass spectrometry

With a recently developed liquid chromatographic (LC) method, using a phosphate buffer, several unknown impurities present in dirithromycin samples were separated. In this paper, a reversed-phase liquid chromatography-tandem mass spectrometry method is described for the investigation of dirithromycin and related substances. The method employed uses a Zorbax Extend C18 column (250 mm x 4.6 mm I.D.), 5 microm, and a mobile phase consisting of acetonitrile, 2-propanol, water and ammonium acetate solution pH 8.5. Mass spectral data are acquired on an LCQ ion trap mass spectrometer equipped with an electrospray ion (ESI) source operated in the positive ion mode. The LCQ is ideally suited for the identification of related substances because it provides on-line LC/MS(n) capability, which allows efficient identification without time-consuming isolation and purification procedures. Using this method, the fragmentation behavior of dirithromycin and its related substances was studied and the unknown impurities occurring in commercial samples were investigated. In total the structures of nine impurities were elucidated, among which three were different analogues with a modification in the side chain on the oxazine ring. Two impurities showed a different alkyl group in position C13. In two impurities the desosamine sugar was involved with changes in the degrees of methylation of the amino group. One unknown impurity was identified as dirithromycin F and another unknown was characterized as dirithromycin N-oxide.     

Quantitative determination of medroxyprogesterone acetate in plasma by liquid chromatography/electrospray ion trap mass spectrometry.

A sensitive and rapid liquid chromatography/electrospray ion trap mass spectrometry (LC/MS/MS) method has been developed for the quantitative determination of medroxyprogesterone acetate (MPA) in human plasma. Plasma samples (1.0 mL) were simply extracted with pentane and the extracts were analyzed by HPLC with the detection of the analyte in the selective reaction monitoring (SRM) mode. The determination of MPA was accurate and reproducible, with a limit of quantitation of 0.05 ng/mL in plasma. The standard calibration curve for MPA was linear (r = 0.998) over the concentration range 0.05-6.0 ng/mL in human plasma. Analysis precision over the concentration range of MPA was lower than 18.8% (relative standard deviation, RSD) and accuracy was between 96.2 and 108.7%.     

Fragmentation study of hexanitrostilbene by ion trap multiple mass spectrometry and analysis by liquid chromatography/mass spectrometry

The fragmentation pathways of three explosive compounds with similar structures, hexanitrostilbene (HNS), cyclotrimethylene trinitramine (RDX), and 2,4,6-trinitrotoluene (TNT), have been investigated by multiple mass spectrometry (MSn, n = 1, 2, 3) with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources. The electron capture mechanism for these compounds in negative ion APCI and ESI mode differs from the usual negative ion mechanism, deprotonation or addition of other species. This was shown for HNS and TNT, which both gave a [M]- anion but not a [M-H]- ion in APCI, and the [M]- anion of HNS was observed in ESI. The quantitative analysis of HNS was performed by liquid chromatography (LC)/ESI-MS, and the results obtained by the internal standard (ISTD) method were compared with those from the external standard (ESTD) method, demonstrating that both quantitation approaches are useful, with good sensitivity, reproducibility and linearity, and ESTD is preferable in routine applications.     

Simultaneous determination of etoposide and its catechol metabolite in the plasma of pediatric patients by liquid chromatography/tandem mass spectrometry

The anticancer drug etoposide is associated with leukemias with MLL gene translocations and other translocations as a treatment complication. The genotype of cytochrome P450 3A4 (CYP3A4), which converts etoposide to its catechol metabolite, influences the risk. In order to perform pharmacokinetic studies aimed at further elucidation of the translocation mechanism, we have developed and validated a liquid chromatography/electrospray/tandem mass spectrometry assay for the simultaneous analysis of etoposide and its catechol metabolite in human plasma. The etoposide analog teniposide was used as the internal standard. Liquid chromatography was performed on a YMC ODS-AQ column. Simultaneous determination of etoposide and its catechol metabolite was achieved using a small volume of plasma, so that the method is suitable for pediatric patients. The limits of detection were 200 ng ml(-1) etoposide and 10 ng ml(-1) catechol metabolite in human plasma and 25 ng ml(-1) etoposide and 2.5 ng ml(-1) catechol metabolite in protein-free plasma, respectively. Acceptable precision and accuracy were obtained for concentrations in the calibration curve ranges 0.2--100 microg ml(-1) etoposide and 10--5000 ng ml(-1) catechol metabolite in human plasma. Acceptable precision and accuracy for protein-free human plasma in the range 25--15 000 ng ml(-1) etoposide and 2.5--1500 ng ml(-1) etoposide catechol were also achieved. This method was selective and sensitive enough for the simultaneous quantitation of etoposide and its catechol as a total and protein-free fraction in small plasma volumes from pediatric cancer patients receiving etoposide chemotherapy. A pharmacokinetic model has been developed for future studies in large populations.     

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.     

高效液相色谱-离子阱质谱法测定生物体液中的痕量敌鼠和氯敌鼠

建立了中毒应急检测样品生物体液内痕量敌鼠和氯敌鼠的高效液相色谱-离子阱质谱定性定量检测方法。全血样品由甲醇/乙腈(50/50, v/v)沉淀蛋白后再经Oasis HLB固相萃取小柱净化富集,尿液样品直接经同类小柱净化富集。采用Extend C18柱分离,以乙酸铵-乙酸(0.02 mol/L, pH 5.5)缓冲溶液和甲醇(15/85, v/v)溶液为流动相,使用电喷雾电离负离子多反应监测模式检测敌鼠和氯敌鼠。对全血样品,敌鼠和氯敌鼠分别在1.0～200.0 μg/L和0.5～100.0 μg/L范围内呈良好的线性关系,回收率分别在90.1%～92.2%和87.6%～93.4%范围内,日内相对标准偏差(RSD)分别小于6.8%和7.1%,日间RSD分别小于9.9%和10.9%,定量限分别为1.0 μg/L和0.5 μg/L。对尿液样品,敌鼠和氯敌鼠分别在0.2～ 40.0 μg/L和0.1～20.0 μg/L范围内呈良好的线性关系,回收率分别在90.1%～94.5%和90.0%～ 98.0%范围内,日内RSD分别小于6.1%和7.3%,日间RSD分别小于8.9%和11.2%,定量限分别为0.2 μg/L和0.1 μg/L。本方法简便、灵敏,能满足敌鼠和氯敌鼠中毒病人的临床快速诊断要求。     

Quantitative determination of noncovalently bound acridinium in protein conjugates by liquid chromatography/electrospray ion trap mass spectrometry

A sensitive and robust liquid chromatography/electrospray ion trap mass spectrometry (LC/MS/MS) method has been developed for the quantitative determination of noncovalently bound acridinium free acid in protein-acridinium conjugates. The lower level of quantitation (LOQ) for acridinium free acid was determined to be 0.6 ng. The assay was validated with a linear concentration range of 0.6-60 ng. The method requires minimum sample handling and is specific, reproducible, and provides a new aspect for protein-acridinium conjugate characterization.     

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。     

Quantitative determination of paroxetine and its 4-hydroxy-3-methoxy metabolite in plasma by high-performance liquid chromatography/electrospray ion trap mass spectrometry: application to pharmacokinetic studies

A high-performance liquid chromatography (HPLC) method with tandem mass spectrometric detection is described for the determination of paroxetine, an antidepressant drug, and its metabolite (3S,4R)-4-(4-fluorophenyl)-3-(4-hydroxy-3-methoxyphenoxymethyl)piperidine (HM paroxetine) in human plasma. Plasma samples were hydrolysed with hydrochloric acid and then analytes were extracted with ethyl acetate at alkaline pH. Extracts were analysed by HPLC coupled to an atmospheric pressure ionisation-electrospray (ESI) interface and an ion trap mass spectrometer. Chromatography was performed on a reversed-phase column using acetonitrile/0.02% formic acid (66:34, v/v) as a mobile phase. The mass spectrometer was operated in the multiple reaction monitoring mode. The method was validated over concentration ranges of 0.75-100 microg/L and 5-100 microg/L for paroxetine and HM paroxetine, respectively. Mean recoveries of 77% for paroxetine and 76% for HM paroxetine were found, with precision always better than 15%. The limits of detection and quantification were 0.20 and 0.70 microg/L for paroxetine, and 0.70 and 2.20 microg/L for its metabolite. The method was applied to the analysis of plasma samples obtained from nine healthy male volunteers administered with a single oral dose of 20 mg paroxetine. After the 20-mg dose, the mean peak plasma concentration was 8.60 microg/L for paroxetine and 92.40 microg/L for HM paroxetine showing a tenfold ratio between the metabolite and the parent drug along the entire time-concentration curve.     

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.     

Identification and trace level determination of brominated flame retardants by liquid chromatography/quadrupole linear ion trap mass spectrometry

We describe the development of instrumental methodology for the simultaneous determination of hexabromocyclododecane (HBCD) diastereoisomers and tetrabromobisphenol A (TBBPA) and its derivatives by liquid chromatography/quadrupole linear ion trap mass spectrometry (LC-QqLIT-MS). Two different experiments were developed, optimized and compared. The first is based on a selected reaction monitoring (SRM) method in which the two most abundant transitions were selected for each analyte, as well as for the internal standards. In the second, the ion trap was used for the storage and subsequent fragmentation of precursor ions, obtaining an enhanced product ion (EPI) experiment. Both methods were validated by measuring quality parameters such as linearity, sensitivity, reproducibility and repeatability. Limits of detection (LODs) were in the range of 0.1-1.8 pg and 0.01-0.5 pg for SRM and EPI experiments, respectively, being lower than those published for the LC/QqQ-MS methods. Thus, LC-QqLIT-MS, used for quantification and confirmation, proved to be a powerful and very sensitive analytical tool.     

Rapid drug metabolite profiling using fast liquid chromatography, automated multiple-stage mass spectrometry and receptor-binding

Rapid drug metabolite profiling can be achieved using fast chromatographic separation and fast mass spectrometric scanning without compromising the separation efficiency. Fast chromatographic separations of drug and its metabolites can be achieved by eluting from a short narrow-bore guard cartridge column (20 x 2 mm I.D., 3 microns BDS Hypersil C8) at flow-rate of 1.0 ml/min and with a gradient volume greater than 90 column volumes. The need for chromatographic separation is important for automated data dependent multiple-stage mass spectrometry (MSn) experimentation. The total analysis time of 8 min permits profiling of metabolites in a 96-well plate in 13 h. The narrow chromatographic peaks resulting from the high flow-rate require the use of a mass spectrometer capable of fast scan speed due to the need to perform multiple MS experiments within the same chromatographic analysis. A method has been developed for screening potentially biologically active in vitro microsomal metabolites by affinity binding with a receptor. After separation by centrifugal ultrafiltration, the bound ligands are released and characterized by LC-MS. In vitro microsomal metabolites of tamoxifen, raloxifene and adatanserin were screened for potential biological activity using this method. The in vitro metabolites of tamoxifen captured by the receptor include N-demethyltamoxifen and three species of hydroxytamoxifen; these data are consistent with those from a conventional binding study and bioassay. In addition, both hydroxyraloxifene and dihydroxyraloxifene are also recognized by the receptor. The specificity of the molecular recognition process is illustrated by the absence of binding with control microsomal incubate and with adatanserin and its metabolites. Therefore, active metabolites can be rapidly profiled by fast LC, automated MSn, and receptor binding. This information can be obtained quickly and can add value to the drug discovery process.     

Oligosaccharide sequences in Quillaja saponins by electrospray ionization ion trap multiple-stage mass spectrometry

Ten different samples with 13 previously identified saponin structures from Quillaja saponaria Molina were investigated by electrospray ionization ion trap multiple-stage mass spectrometry (ESI-ITMS(n)) in positive and negative ion modes. Both positive and negative ion mode MS(1)-MS(4) spectra were analyzed, showing that structural information on the two oligosaccharide parts in the saponin can be obtained from positive ion mode spectra whereas negative ion mode spectra mainly gave information on one of the oligosaccharide parts. Analysis of MS(1)-MS(4) spectra identified useful key fragment ions important for the structural elucidation of Quillaja saponins. A flowchart involving a stepwise procedure based on key fragments from MS(1)-MS(3) spectra was constructed for the identification of structural elements in the saponin. Peak intensity ratios in MS(3) spectra were found to be correlated with structural features of the investigated saponins and are therefore of value for the identification of terminal monosaccharide residues.     

Determination of levamisole in feeds by liquid chromatography coupled to electrospray mass spectrometry on an ion trap

Test methods have to be developed by laboratories for official control to monitor possible misuse of veterinary drugs in animal productions, also through feeding stuff. A novel method for identification and quantification of levamisole in feeds by liquid chromatography coupled to electrospray mass spectrometry in an ion trap (LC/ESI‐MS/MS) is herein described; after a single‐step cleanup by liquid‐liquid extraction from the feed and separation by reversed‐phase liquid chromatography, levamisole was determined and unambiguously confirmed by tandem mass spectrometry, on the basis of two product ions. The method was in‐house validated, according to the Regulation 882/2004/EC, evaluating trueness, repeatability, within‐laboratory reproducibility, ruggedness, specificity, and the limit of quantification (LOQ). The method is reliable and specific for complete and complementary feeds for pigs, cattle, rabbits and poultry; very good mean recoveries (higher than 92 %) and precision (RSD values < 15.2%) were attained. The LOQ at 2.0 mg/kg was verified. Moreover, we describe how the method was developed to support Italian Police investigations regarding illegal treatments of pigs; in this case, since the drug(s) added to the feed were unknown, a preliminary untargeted analysis was performed by full scan mass spectrometry on an ion trap, from 50 up to 2000 m/z; the presence of levamisole was hypothesised, on the basis of the most abundant ion and its fragmentation pattern. Then, levamisole was unambiguously confirmed by the ion trap LC/ESI‐MS/MS method. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of atracurium and laudanosine in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method coupled with fluorometric detection has been developed for the determination of atracurium and its major end-product laudanosine in human plasma. The method enables good separation of atracurium from its metabolites after direct precipitation of plasma proteins. The assay is sensitive, reproducible and linear for atracurium concentrations ranging from 31.25 to 8000 ng/ml. In a clinical setting, drugs commonly administered during anesthesia did not interfere with the assay. This method provides a simple and time-saving alternative to existing methods.     

Simultaneous characterization of pyrrolizidine alkaloids and N‐oxides in Gynura segetum by liquid chromatography/ion trap mass spectrometry

Gynura segetum (Lour.) Merr. (Jusanqi) is a traditional herbal product used for hemostasis and detumescence in Chinese folk medicine. However, its hepatic toxicity should not be ignored. In this study, pyrrolizidine alkaloids (PAs) and their corresponding N‐oxides (PANOs) were extracted from the whole plant of G. segetum and analyzed by high‐performance liquid chromatography coupled to ion trap mass spectrometry (ITMS). Identification of eluted peaks as PANOs was indicated by virtue of their MS and MSⁿ analysis, in addition to the [M+H]⁺ adduct ion, characteristically showed a significant (usually 100% abundance) dimer adduct [2M+H]⁺ that is not observed in the MS of the parent PAs. A total of 20 compounds were identified or tentatively characterized based on their mass spectra and possible biosynthetic pathways, of which three PAs and one PANO, namely seneciphylline, senecionine, seneciphylline and seneciphyllinine N‐oxide, were unequivocally characterized, while other PAs and PANOs were tentatively assigned. Sixteen constituents were reported for the first time from G. segetum and tetrahydrosenecionine has not been previously reported as a natural product. Our results are the first comprehensive analysis of PAs and PANOs in G. segetum constituents and will be helpful for the quality control of the herb of G. segetum and its related preparations. Copyright © 2008 John Wiley & Sons, Ltd.