Separation and characterization of clindamycin phosphate and related impurities in injection by liquid chromatography/electrospray ionization mass spectrometry

A simple high‐performance liquid chromatography/electrospray ionization tandem mass spectrometric (HPLC/ESI‐MS/MS) method has been developed for the rapid identification of clindamycin phosphate and its degradation products or related impurities in clindamycin phosphate injection. Detection was performed by quadrupole time‐of‐flight mass spectrometry (Q‐TOFMS) via an ESI source in positive mode. Clindamycin phosphate and its related substances lincomycin, 7‐epilincomycin‐2‐phosphate, lincomycin‐2‐phosphate, clindamycin B, clindamycin B‐2‐phosphate, and clindamycin were identified simultaneously by HPLC/ESI‐MS/MS results. Based on the MS/MS spectra of their quasi‐molecular ions, the fragmentation pathways of clindamycin phosphate and its related substances were compared and proposed, which are specific and useful for the identification of the lincosamide antibiotics and related impurities. The method was rapid, sensitive and specific and can be used to identify clindamycin phosphate and its related impurities in clindamycin phosphate injection without control compounds. Copyright © 2009 John Wiley & Sons, Ltd.     

Hydrophilic interaction liquid chromatography/tandem mass spectrometry for the simultaneous determination of dasatinib,imatinib and nilotinib in mouse plasma

Hydrophilic interaction liquid chromatography (HILIC) interfaced with atmospheric pressure ionization (API) sources and a tandem mass spectrometer (MS/MS) was developed for the simultaneous determination of dasatinib, imatinib and nilotinib in mouse plasma samples. The retention profiles of all analytes on several silica stationary phases under HILIC conditions were explored. The influences of experimental factors such as the compositions of mobile phases on the chromatographic performance and the ionization efficiency of all analytes in positive ion mode were investigated. The applicability of the proposed HILIC/MS/MS approach following a protein precipitation procedure for the quantitative determination of dasatinib, imatinib and nilotinib at low nano‐mole levels was examined with respect to assay specificity and linearity. The analytical results obtained by various HILIC/MS/MS approaches were found to be in good agreement with those obtained by reversed‐phase liquid chromatography/tandem mass spectrometry (RPLC/MS/MS) methods in terms of assay sample throughputs, sensitivity and accuracy. Furthermore, the potential of matrix ionization suppression on the proposed HILIC/MS/MS systems was investigated using the post‐column infusion technique. Copyright © 2009 John Wiley & Sons, Ltd.     

Derivatization reagents in liquid chromatography/electrospray ionization tandem mass spectrometry

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is one of the most prominent analytical techniques owing to its inherent selectivity and sensitivity. In LC/ESI-MS/MS, chemical derivatization is often used to enhance the detection sensitivity. Derivatization improves the chromatographic separation, and enhances the mass spectrometric ionization efficiency and MS/MS detectability. In this review, an overview of the derivatization reagents which have been applied to LC/ESI-MS/MS is presented, focusing on the applications to low molecular weight compounds.     

Assessment and validation of the MS/MS fragmentation patterns of the macrolide immunosuppressant everolimus

Everolimus (40-O-(2-hydroxyethyl)rapamycin, Certican) is a 31-membered macrolide lactone. In lymphocytes, it inhibits the mammalian target of rapamycin (mTOR) and is used as an immunosuppressant after organ transplantation. Due to its instability in pure organic solvents and insufficient HPLC separation, NMR spectroscopy analysis of its metabolite structures is nearly impossible. Therefore, structural identification based on tandem mass spectrometry (MS/MS) and MS(n) fragmentation patterns is critical. Here, we have systematically assessed the fragmentation pattern of everolimus during liquid chromatography (LC)-electrospray ionization (ESI)-MS/MS and validated the fragment structures by (1) comparison with structurally identified derivatives (sirolimus), (2) high-resolution mass spectrometry, (3) elucidation of fragmentation pathways using ion trap mass spectrometry (up to MS(5)) and (4) H/D exchange. In comparison with the structurally related immunosuppressants tacrolimus and sirolimus, our study was complicated by the low ionization efficiency of everolimus. Detection of positive ions gave the best sensitivity, and everolimus and its fragments were mainly detected as sodium adducts. LC-ESI-MS/MS of everolimus in combination with collision-induced dissociation (CID) resulted in a complex fragmentation pattern and the structures of 53 fragments were identified. These detailed fragmentation pathways of everolimus provided the basis for structural elucidation of all everolimus metabolites generated in vivo und in vitro.     

Rapid screening and identification of lycodine‐type alkaloids in Lycopodiaceae and Huperziaceae plants by ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

Lycodine‐type alkaloids have gained significant interest owing to their unique skeletal characteristics and acetylcholinesterase activity. This study established a rapid and reliable method using ultra‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UPLC‐ESI‐Q/TOF‐MS/MS) for comprehensive characterization of lycodine‐type alkaloids for the first time. The lycodine‐type alkaloids were detected successfully from Lycopodiastrum casuarinoides, Huperzia serrata and Phlegmarirus carinatus in seven plants of the Lycopodiaceae and Huperziaceae families, based on the established characteristic MS fragmentation of five known alkaloids. Furthermore, a total of 13 lycodine‐type alkaloids were identified, of which three pairs of isomers were structurally characterized and differentiated. This study further improves mass analysis of lycodine‐type alkaloids and demonstrates the superiority of UPLC with a high‐resolution mass spectrometer for the rapid and sensitive structural elucidation of other trace active compounds. Copyright © 2016 John Wiley & Sons, Ltd.     

A simultaneous determination method for 5‐fluorouracil and its metabolites in human plasma with linear range adjusted by in‐source collision‐induced dissociation using hydrophilic interaction liquid chromatography–electrospray ionization–tandem mass spectrometry

We applied a new technique for quantitative linear range shift using in‐source collision‐induced dissociation (CID) to complex biological fluids to demonstrate its utility. The technique was used in a simultaneous quantitative determination method of 5‐fluorouracil (5‐FU), an anticancer drug for various solid tumors, and its metabolites in human plasma by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC/ESI‐MS/MS). To control adverse effects after administration of 5‐FU, it is important to monitor the plasma concentration of 5‐FU and its metabolites; however, no simultaneous determination method has yet been reported because of vastly different physical and chemical properties of compounds. We developed a new analytical method for simultaneously determining 5‐FU and its metabolites in human plasma by LC/ESI‐MS/MS coupled with the technique for quantitative linear range shift using in‐source CID. Hydrophilic interaction liquid chromatography using a stationary phase with zwitterionic functional groups, phosphorylcholine, was suitable for separation of 5‐FU from its nucleoside and interfering endogenous materials. The addition of glycerin into acetonitrile‐rich eluent after LC separation improved the ESI‐MS response of high polar analytes. Based on the validation results, linear range shifts by in‐source CID is the reliable technique even with complex biological samples such as plasma. Copyright © 2016 John Wiley & Sons Ltd.     

Determination of hormones in human urine by ultra‐high‐performance liquid chromatography/triple‐quadrupole mass spectrometry

Steroid hormones play a critical role in maintaining the homeostasis of human metabolism. Urine as a noninvasive sample has been extensively used in clinical diagnosis for hormones homeostasis. In this study, the simultaneous characterization of fourteen hormones in urine was performed based on ultra‐high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPHLC/ESI(+)‐MS/MS) with multiple reaction monitoring in the positive ionization mode. The target hormones were cortisone, cortisol, 11‐deoxycortisol, aldosterone, corticosterone, 11‐deoxycorticosterone, progesterone, 17‐OH‐progesterone, pregnenolone, estrone, estradiol, estriol, testosterone and dehydreopiandrosterone. β‐Glucuronidase/sulfatase deconjugation and liquid–liquid extraction (LLE) were conducted for the determination of urinary hormones (free + conjugated forms). The limits of detection (LODs) ranged from 0.2 ng/mL (11‐deoxycortisol and testosterone) to 1 ng/mL (cortisone). The extraction recovery of the targeted compounds ranged from 87% to 127%, indicating sufficient extraction efficiency for the LLE process. Intraday precision was below 10% and the accuracy ranged from 84% to 122%. The profiling analysis of hormones in urine samples helps to understand the metabolic state of biological systems and can be employed as a diagnostic tool in diseases developed by endocrine‐disrupted systems.     

液相色谱-质谱联用方法用于严重急性呼吸系统综合症冠状病毒结构蛋白质的研究

通过高效液相反相色谱、毛细管反相色谱-串联质谱联用方法对SARS病毒攻击细胞的研究,鉴定出了N、S和M3种SARS结构蛋白质,并准确测定了N蛋白质的分子量。由分子量结果和生物信息学推断的N蛋白质的理论分子量比较,可以确定N蛋白质不存在常见的磷酸化修饰和糖基化修饰或含量很低。进一步的研究表明：N蛋白质可能存在降解现象,其降解机理尚需探索。另外,通过对样品直接酶切后进行两维毛细管分离和串联质谱鉴定与对样品先进行分离,然后再进行毛细管反相分离-串联质谱鉴定方法比较,表明两种方法在蛋白质组学研究中各具优缺点,可根据不同目的选择使用。     

Sheath liquid interface for the coupling of normal-phase liquid chromatography with electrospray mass spectrometry and its application to the analysis of neoflavonoids

A novel interface that allows normal-phase liquid chromatography to be coupled with electrospray ionization (ESI) is reported. A make-up solution of 60 mM ammonium acetate in methanol, infused at a 5 microl min(-1) flow-rate at the tip of the electrospray probe, provides a sheath liquid which is poorly miscible with the chromatographic effluent, but promotes efficient ionization of the targeted analytes. Protonated molecules generated in the ESI source were subjected to tandem mass spectrometric experiments in a triple-quadrupole mass spectrometer. The main fragmentation reactions were characterized for each analyte and specific mass spectral transitions were used to acquire chromatographic data in the multiple reaction monitoring detection mode. Results obtained during optimization of the sheath liquid composition and flow-rate suggest that the electrospray process was mainly under the control of the make-up solution, and that it forms an external charged layer around a neutral chromatographic mobile phase core. This sheath liquid interface was implemented for the analysis of some neoflavonoid compounds and its performance was evaluated. Limits of detection were established for calophillolide, inophyllum B, inophyllum P and inophyllum C at 100, 25, 15 and 100 ng ml(-1), respectively.     

Porous graphitic carbon chromatography/tandem mass spectrometric determination of cytarabine in mouse plasma

A high-performance liquid chromatography (HPLC) system using a porous graphitic carbon (PGC) stationary phase interfaced with an electrospray ionization (ESI) source and a tandem mass spectrometer (MS/MS) for the analysis of cytarabine (ara-C) in mouse plasma samples has been developed in support of a pharmacodynamic study. The graphitized carbon column was adopted for the separation of ara-C and endogenous peaks from mouse plasma samples under the reversed-phase phase mode in liquid chromatography. The retention characteristics of the PGC column and the ionization efficiencies of all analytes based on the experimental factors such as the composition of mobile phases were investigated. The potential of ionization suppression resulting from the endogenous biological matrices on the PGC column during HPLC/ESI-MS/MS was investigated using post-column infusion. The concentrations of ara-C in mouse plasma obtained by using PGC-HPLC/MS/MS and ion-pairing HPLC/MS/MS were found to be in good agreement in terms of analytical accuracy.     

Reducing glycerophosphocholine lipid matrix interference effects in biological fluid assays by using high-turbulence liquid chromatography

Matrix interferences can severely affect quantitative assays of biological samples when electrospray ionization (ESI) is employed with liquid chromatography/tandem mass spectrometry (LC/MS/MS). A major source of matrix interferences for plasma sample analyses is the presence of glycerophosphocholine (GPCho) lipids. The efficiency of online high-turbulence liquid chromatography (HTLC) extraction for eliminating these lipids is evaluated and the interfering effects of endogenous lipids on human plasma assays are measured for pharmaceutical compounds having a wide variety of chemical properties. It is found that GPCho lipids, represented by 16:0, 18:1 and 18:0 LPC (lysophosphatidylcholine) and 16:0-18:2 PC, cause variations for hydrophobic compound analyses even when optimal online HTLC extraction conditions are employed. The efficiency for lipid removal depends on the organic content of the transfer solvent, but turbulent flow loading has no significant effect.     

An alternative and fast method for determination of glyphosate and aminomethylphosphonic acid (AMPA) residues in soybean using liquid chromatography coupled with tandem mass spectrometry

A simple and specific method using reversed‐phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) was investigated, which allowed the determination of residues of glyphosate and its metabolite, aminomethylphosphonic acid (AMPA), in soybean samples. An aqueous extraction with liquid‐liquid partition followed by protein precipitation was performed before the LC/MS/MS determination. The quantitation of glyphosate and AMPA was performed in positive and negative ESI mode, respectively, using the multiple reaction monitoring (MRM) mode with three transitions for each analyte to enhance the specificity of the method and avoid false positives. The methodology reported in this work is capable of detecting residues of glyphosate and AMPA in soybean samples with limits of quantification of 0.30 and 0.34 mg kg^?1, respectively. This alternative method has throughput advantages such as simpler sample preparation and faster chromatographic analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of chemical constituents in Rhizoma Paridis Saponins and their oral administration in rat plasma by UPLC/Q‐TOF/MS

On‐line ultra‐performance liquid chromatography (UPLC) coupled with diode‐array detection (UPLC/DAD) and electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐Q‐TOF‐MS) were used for separation, identification and structural analyses of saponins in Rhizoma Paridis saponins (RPS) and rat plasma after oral administration of RPS. Thirty steroidal saponins in RPS were identified by comparing their retention time, accurate mass measurement and positive and negative mass spectrometry data with that of reference compounds. The UPLC/Q‐TOF‐MS method was proved to be rapid and efficient in that 30 steroidal saponins, including three kinds of saponins (prototype, pennogenyl and diosgenyl saponins) were tentatively characterized within 6 min. After oral administration of RPS, 21 original saponins were absorbed in RPS‐treated rat plasma. Our results indicated that UPLC/Q‐TOF‐MS is a rapid and effective tool for identification of a series of saponins at trace level. Copyright © 2010 John Wiley & Sons, Ltd.     

Liquid chromatography/negative ion atmospheric pressure photoionization mass spectrometry: a highly sensitive method for the analysis of organic explosives

Gas chromatography/mass spectrometry (GC/MS) is applied to the analysis of volatile and thermally stable compounds, while liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI‐MS) and liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS) are preferred for the analysis of compounds with solution acid‐base chemistry. Because organic explosives are compounds with low polarity and some of them are thermally labile, they have not been very well analyzed by GC/MS, LC/APCI‐MS and LC/ESI‐MS. Herein, we demonstrate liquid chromatography/negative ion atmospheric pressure photoionization mass spectrometry (LC/NI‐APPI‐MS) as a novel and highly sensitive method for their analysis. Using LC/NI‐APPI‐MS, limits of quantification (LOQs) of nitroaromatics and nitramines down to the middle pg range have been achieved in full MS scan mode, which are approximately one order to two orders magnitude lower than those previously reported using GC/MS or LC/APCI‐MS. The calibration dynamic ranges achieved by LC/NI‐APPI‐MS are also wider than those using GC/MS and LC/APCI‐MS. The reproducibility of LC/NI‐APPI‐MS is also very reliable, with the intraday and interday variabilities by coefficient of variation (CV) of 0.2–3.4% and 0.6–1.9% for 2,4,6‐trinitrotoluene (2,4,6‐TNT). Copyright © 2008 John Wiley & Sons, Ltd.     

A systematic approach toward comparing electrospray ionization efficiencies of derivatized and non‐derivatized amino acids and biogenic amines

Ionization efficiency (IE) in mass spectrometry (MS) has been studied for many different compounds, and different IE scales have been constructed in order to quantitatively characterize IE. In the case of MS, derivatization has been used to increase the sensitivity of the method and to lower the limits of detection. However, the influence of derivatization on IE across different compounds and different derivatization reagents has not been thoroughly researched, so that practitioners do not have information on the IE‐enhancing abilities of different derivatization reagents. Moreover, measuring IE via direct infusion of compounds cannot be considered fully adequate. Since derivatized compounds are in complex mixtures, a chromatographic method is needed to separate these compounds to minimize potential matrix effects. In this work, an IE measurement system with a chromatographic column was developed for mainly amino acids and some biogenic amines. IE measurements with liquid chromatography electrospray ionization mass spectrometry (LC/ESI/MS) were carried out, and IE scales were constructed with a calibration curve for compounds with and without derivatization reagent diethyl ethoxymethylenemalonate. Additionally, eluent composition effects on ionization were investigated. Results showed that derivatization increases IE for most of the compounds (by average 0.9 and up to 2‐2.5 logIE units) and derivatized compounds have more similar logIE values than without derivatization. Mobile phase composition effects on ionization efficiencies were negligible. It was also noted that the use of chromatographic separation instead of flow injection mode slightly increases IE. In this work, for the first time, IE enhancement of derivatization reagents was quantified under real LC/ESI/MS conditions and obtained logIE values of derivatized compounds were linked with the existing scale.     

Rapid characterization of the chemical constituents and rat metabolites of the Wen‐Jing decoction by ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

The Wen‐Jing decoction, a traditional Chinese medicine formula, has been used as a blood‐activating and stasis‐eliminating drug to treat gynaecological syndromes, such as dysmenorrhea, amenorrhea, and menstrual disorders. However, its pharmacodynamic material basis and mechanism of action have not been thoroughly elucidated to date. The goal of this study was to characterize and identify multiple constituents and metabolites in Wen‐Jing decoction. An ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry method was established and validated in the present study for the first time. A total of 101 compounds, including 11 monoterpene glycosides, 19 flavonoids, 49 triterpene saponins, 5 phthalides, 3 phytoecdysones, and 14 others, were unambiguously or tentatively characterized by comparing their retention times and MS data with reference standards or with data reported in the literature. After oral administration of Wen‐Jing decoction, 27 compounds, including nine prototype compounds and 18 metabolites were detected in rat plasma. Thus, the ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry method was found to be efficient for in‐depth structural elucidation of chemical compounds in complex matrices of herbal medicines, which will provide useful chemical information for quality control and mechanism‐of‐action research.     

Development and validation of a sensitive UHPLC‐MS/MS method for the simultaneous analysis of tramadol,dextromethorphan chlorpheniramine and their major metabolites in human plasma in forensic context: application to pharmacokinetics

The prerequisites for forensic confirmatory analysis by LC/MS/MS with respect to European Union guidelines are chromatographic separation, a minimum number of two MS/MS transitions to obtain the required identification points and predefined thresholds for the variability of the relative intensities of the MS/MS transitions (MRM transitions) in samples and reference standards. In the present study, a fast, sensitive and robust method to quantify tramadol, chlorpheniramine, dextromethorphan and their major metabolites, O‐desmethyltramadol, dsmethyl‐chlorpheniramine and dextrophan, respectively, in human plasma using ibuprofen as internal standard (IS) is described. The analytes and the IS were extracted from plasma by a liquid–liquid extraction method using ethyl acetate–diethyl‐ether (1:1). Extracted samples were analyzed by ultra‐high‐performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC‐ESI‐MS/MS). Chromatographic separation was performed by pumping the mobile phase containing acetonitrile, water and formic acid (89.2:11.7:0.1) for 2.0 min at a flow rate of 0.25 μL/min into a Hypersil‐Gold C₁₈ column, 20 × 2.0 mm (1.9 µm) from Thermoscientific, New York, USA. The calibration curve was linear for the six analytes. The intraday precision (RSD) and accuracy (RE) of the method were 3–9.8 and ?1.7–4.5%, respectively. The analytical procedure herein described was used to assess the pharmacokinetics of the analytes in 24 healthy volunteers after a single oral dose containing 50 mg of tramadol hydrochloride, 3 mg chlorpheniramine maleate and 15 mg of dextromethorphan hydrobromide. Copyright © 2014 John Wiley & Sons, Ltd.     

Isothiocyanates as derivatization reagents for amines in liquid chromatography/electrospray ionization–tandem mass spectrometry

The applicability of 3‐pyridyl isothiocyanate, p‐(dimethylamino)phenyl isothiocyanate and m‐nitrophenyl isothiocyanate as the derivatization reagents for amines in high‐performance liquid chromatography/electrospray ionization–tandem mass spectrometry (LC/ESI‐MS/MS) was examined. The generated derivatives of amines with these reagents were favorably separated on the reversed‐phase column and detected by ESI‐MS/MS. The C–N bond of the generated thiourea structure was efficiently cleaved by collision‐induced dissociation and gave the single and intense product ion. Among the three reagents, 3‐pyridyl isothiocyanate was the most suitable as the derivatization reagent with regard to the reactivity to amines and the detection sensitivity. Copyright © 2009 John Wiley & Sons, Ltd.     

Combination of liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry for the detection of 21 anabolic steroid residues in bovine urine

For the detection of anabolic steroid residues in bovine urine, a highly sensitive liquid chromatographic/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method was developed using both positive and negative ionization. For four compounds the ESI mode was not sensitive enough and gas chromatographic/mass spectrometric GC/MS detection was therefore still necessary as a complementary method. The sample clean-up consisted of solid-phase extraction (SPE) on a C(18) column followed by enzymatic hydrolysis and a second solid-phase extraction on a combination of a C(18) and a NH(2) column. After this last SPE clean-up, the eluate was split into two equal aliquots. One aliquot was further purified and after derivatization used for GC/MS analysis. The other aliquot was analyzed with LC/MS/MS in both ESI+ and ESI- modes. The method was validated according to the European Commission Decision 2002/657/EC. Decision limits (CCalpha) were between 0.16 and 1 ng ml(-1) for the compounds detected with the LC/MS/MS method. The developed method is used in routine analysis in our laboratory.