Quantification of glycopeptides by multiple reaction monitoring liquid chromatography/tandem mass spectrometry

Protein glycosylation has a major influence on functions of proteins. Studies have shown that aberrations in glycosylation are indicative of disease conditions. This has prompted major research activities for comparative studies of glycoproteins in biological samples. Multiple reaction monitoring (MRM) is a highly sensitive technique which has been recently explored for quantitative proteomics. In this work, MRM was adopted for quantification of glycopeptides derived from both model glycoproteins and depleted human blood serum using glycan oxonium ions as transitions. The utilization of oxonium ions aids in identifying the different types of glycans bound to peptide backbones. MRM experiments were optimized by evaluating different parameters that have a major influence on quantification of glycopeptides, which include MRM time segments, number of transitions, and normalized collision energies. The results indicate that oxonium ions could be adopted for the characterization and quantification of glycopeptides in general, eliminating the need to select specific transitions for individual precursor ions. Also, the specificity increased with the number of transitions and a more sensitive analysis can be obtained by providing specific time segments. This approach can be applied to comparative and quantitative studies of glycopeptides in biological samples as illustrated for the case of depleted blood serum sample. Copyright © 2012 John Wiley & Sons, Ltd.     

Simple and sensitive quantification of bioactive peptides in biological matrices using liquid chromatography/selected reaction monitoring mass spectrometry coupled with trichloroacetic acid clean-up

A simple and sensitive liquid chromatography/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method has been developed for the quantification of bioactive peptides in biological fluids. The method employs protein precipitation with 4% trichloroacetic acid (TCA) and selected reaction monitoring (SRM) using an immonium ion as the product ion. This method was applied to determine the synthetic parathyroid hormone (PTH) analog (MW 1721) in rat plasma and human hepcidin-25 (MW 2789) in human serum. TCA clean-up showed a sufficient recovery for peptides with a MW of less than 3000, and would be useful as a simple and rapid method because of direct injection of the supernatant without evaporation or dilution. In addition, TCA clean-up allowed us not only to reduce sample preparation time, but also to select an immonium ion as a product ion of SRM, which led to detection more sensitive than SRM using other types of product ions. The lower limits of quantitation (LLOQs) of the PTH analog and the human hepcidin-25 were 0.2 ng/mL and 5 ng/mL, respectively. This method was fully validated with acceptable linearity, intra- and inter-assay precisions, and accuracy. Furthermore, this simple and rapid method is applicable to pharmacokinetic studies.     

A rapid and sensitive method for the quantification of ganciclovir in plasma using liquid chromatography/selected reaction monitoring/mass spectrometry

A method using reversed-phase liquid chromatography coupled with electrospray ionization and selected reaction monitoring mass spectrometry has been developed for the quantitative analysis of ganciclovir in rat plasma. Acyclovir, a structurally related analog of ganciclovir, was used as the internal standard. A small volume of plasma (50 microL) was spiked with the internal standard and plasma proteins were precipitated by methanol. The supernatant was dried under nitrogen, and then reconstituted in water. The use of liquid chromatography/selected reaction monitoring/mass spectrometry effectively eliminated potential interference from endogenous constituents in the plasma. This highly selective and sensitive method made it possible to analyze plasma ganciclovir with a lower limit of quantitation of 10 ng/mL. The assay was reproducible and linear in the range 10-10,000 ng/mL. The precision and accuracy values were in the range 2.0-6.9% and 89.0-109.6%, respectively. The analyte recovery was greater than 88%. This method was successfully used to monitor the pharmacokinetic profile of ganciclovir in normal rats following intraperitoneal administration of the drug.     

CTP synthetase activity assay by liquid chromatography tandem mass spectrometry in the multiple reaction monitoring mode

Cytidine 5′‐triphosphate synthetase (CTPS) is known to be a central enzyme in the de novo synthesis of CTP. We have recently demonstrated that a deficiency in CTPS1 is associated with an impaired capacity of activated lymphocytes to proliferate leading to a combined immunodeficiency disease. In order to better document its role in immunomodulation, we developed a method for measuring CTPS activity in human lymphocytes. Using liquid chromatography‐mass spectrometry, we quantified CTPS activity by measuring CTP in cell lysates. A stable isotope analog of CTP served as internal standard. We characterized the kinetic parameters V_max and K_m of CTPS and verified that an inhibition of the enzyme activity was induced after 3‐deazauridine (3DAU) treatment, a known inhibitor of CTPS. We then determined CTPS activity in healthy volunteers, in a family whose child displayed a homozygous mutation in CTPS1 gene and in patients who had developed or not a chronic lung allograft dysfunction (CLAD) after lung transplantation. Linearity of the CTP determination was observed up to 451 μmol/L, with accuracy in the 15% tolerance range. Michaelis‐Menten kinetics for lysates of resting cells were K_m=280±310 μmol/L for UTP, V_max=83±20 pmol/min and, for lysates of activated PBMCs, K_m=230±280 μmol/L for UTP, V_max=379±90 pmol/min. Treatment by 3DAU and homozygous mutation in CTPS1 gene abolished the induction of CTPS activity associated with cell stimulation, and CTPS activity was significantly reduced in the patients who developed CLAD. We conclude that this test is suitable to reveal the involvement of CTPS alteration in immunodeficiency.     

Analysis of bioactive eicosanoids in equine plasma by stable isotope dilution reversed-phase liquid chromatography/multiple reaction monitoring mass spectrometry

Oxidative metabolites of arachidonic acid (AA) are implicated in inflammation. Thus, we evaluated cycloxygenases (COXs) and lipoxygenases (LOs) mediated metabolism of AA to eicosanoids in equine plasma. Eicosanoids were extracted from plasma by two liquid-liquid extraction (LLE) steps; first was by chloroform/isopropanol and second by methyl-tert-butyl ether. For identification and quantification of 25 eicosanoids, a highly specific, selective and sensitive stable isotope dilution liquid chromatography (LC) multiple reaction monitoring (MRM) mass spectrometric (MS) method was developed. To avoid artifact formation of eicosanoids, deferoxamine was added to plasma to chelate residual transition metal ions. The calibration curve showed excellent linearity within 0.1 to 10 ng/mL. Slopes of the calibration curves generated by adding known quantities of eicosanoids in plasma were higher than those prepared in methanol/mobile phase A. Addition of deferoxamine decreased the slope of calibration curves generated using plasma. Limit of detection (LOD) was 1-10 pg on-column for 25 different eicosanoids. Inter-day accuracy was 86-111%, whereas intra-day accuracy was from 88-110%, and precision did not exceed 15% for all quality control (QC) samples. To evaluate the formation of eicosanoids, AA was exogenously added or endogenous AA was released from esterified lipids by calcium ionophore (CI) A23187 treatment of equine whole blood. Pre-treatment of equine whole blood with dexamethasone (DEX) significantly inhibited AA or CI A23187- mediated formation of eicosanoids. The validated method is now employed in studies undertaken to better understand the mechanism of action and pharmacokinetics/pharmacodynamics of eicosanoids after administration of glucocorticoids to horses. This method is reliably reproducible.     

Analysis of phosphorylation sites on focal adhesion kinase using nanospray liquid chromatography/multiple reaction monitoring mass spectrometry

An approach based on nanospray liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) was developed in order to analyze twenty-nine phosphorylated and non-phosphorylated tryptic peptides from focal adhesion kinase (FAK). All peptides monitored were resolved and showed excellent peak shape with the exception of one doubly phosphorylated peptide. Optimization of the LC method enabled the identification and subsequent monitoring of six important tyrosine phosphorylation sites on FAK, including phosphorylated Y397 (pY397), pY407, pY576, pY577, pY861, and pY925. This technique was able to identify sites of phosphorylation on FAK as well as qualitatively differentiate between autocatalytic and Src-induced phosphorylation events. FAK was shown to have autocatalytic function, which resulted in efficient phosphorylation of Y397. FAK was also capable of autophosphorylation on residues Y407 and Y576, though apparently less effectively than autophosphorylation at Y397. Src was found to phosphorylate FAK at Y407, Y576, Y577, and Y861. The presence of Src increased the abundance of pY576 at low temperature indicating Src had particularly high kinase activity toward this residue. Furthermore, Src phosphorylated FAK at Y577 to produce FAK bis-phosphorylated at Y576 and Y577. In addition, six novel sites of phosphorylation (Y148, Y347, Y441, T503, S850, and Y1007) were identified on FAK. Interestingly, Src phosphorylated FAK to form a peptide uniquely phosphorylated on Y407, together with substantial amounts of the bis-phosphorylated pY397pY407 peptide. These findings will impact significantly on future studies of FAK activity since pY397 is often used as a measure of FAK activity and Src association.     

Liquid chromatography/triple quadrupole tandem mass spectrometry with multiple reaction monitoring for optimal selection of transitions to evaluate nutraceuticals from olive-tree materials

Optimal transitions have been selected for the identification and quantitation of the most interesting hydrophilic biophenols in extracts from olive-tree materials, which are of interest because of their nutraceutical properties. The tested materials were extra virgin olive oil, waste from oil production (known as alperujo), and olive-tree materials such as leaves, small branches and fruit stones. The identification and determination steps of the target biophenols are based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a triple quadrupole (QQQ) mass detector. The interface between the chromatograph and the QQQ was an electrospray ionization source operated in the negative ion mode. Highly selective identification of the biophenols was confirmed by multiple reaction monitoring (MRM) using the most representative transitions from the precursor ion to the different product ions. Quantitative MS/MS analysis was carried out by optimization and selection of the most sensitive transition for each analyte, which resulted in estimated detection limits of 5.10 to 11.65 ng/mL for the extracts. The biophenols were extracted from the tested samples by different methods: liquid-liquid extraction for virgin olive oil, microwave-assisted leaching for olive leaves, branches and stones, and pressurized liquid leaching for alperujo. This study provides valuable information about the most suitable source for the isolation of each nutraceutical biophenol and enables us to obtain a complete profile of them in Olea Europaea.     

Hydroxyproline quantification for the estimation of collagen in tissue using multiple reaction monitoring mass spectrometry

Collagens are highly abundant mammalian proteins that contain a high content of hydroxylated amino acids such as hydroxyproline. We have exploited the high hydroxyproline content of collagen and developed a method for hydroxyproline quantification as a measure of collagen content in muscle samples. The novel method utilizes a highly selective and sensitive method of multiple reaction monitoring (MRM) by mass spectrometry. The analytical method is simple, rapid (5min), convenient (no derivatization), precise (<17% RSD), accurate (90-108%), sensitive (4.88nmol/L) and linear (R(2)>0.999) over three orders of magnitude (5-5000nmol/L).     

Deciphering chemical interactions between Glycyrrhizae Radix and Coptidis Rhizoma by liquid chromatography with transformed multiple reaction monitoring mass spectrometry

In this study, we propose an integrated strategy for the efficient identification and quantification of herbal constituents using liquid chromatography with mass spectrometry. First, liquid chromatography with quadrupole time‐of‐flight mass spectrometry was employed for the chemical profiling of herbs, where a targeted following nontargeted approach was developed to detect trace constituents by using structural correlations and extracted ion chromatograms. Next, ion pairs and parameters of MS² of quadrupole time‐of‐flight mass spectrometry were selected to design multiple reaction monitoring transitions for the identified compounds on liquid chromatography with triple quadrupole mass spectrometry. The relative concentration of each constituent was then calculated using a semiquantitative calibration curve. The proposed strategy was applied in a study of chemical interactions between Glycyrrhizae Radix and Coptidis Rhizoma. A total of 140 compounds were identified or tentatively characterized from the herbs, 132 of which were relatively quantified. The visualized quantitative results clearly showed codecoction produced significant constituent concentration variations especially for those with a low polarity. The case study also indicated that the present methodology could provide a reliable, accurate, and labor‐saving solution for chemical studies of herbal medicines.     

Isotope dilution method for the assay of rotenone in olive oil and river waters by liquid chromatography/multiple reaction monitoring tandem mass spectrometry

A new approach for the assay of rotenone (1) by isotope dilution mass spectrometry is presented. The extreme toxicity of rotenone, a natural phytodrug with insecticidal and piscicidal activity, recently banned by national and international environmental protection agencies, calls for the development of sensitive and accurate methods of analyses. Accordingly, the proposed protocol is based on the availability of the labeled internal standard rotenone‐d₃ (3) which can be conveniently prepared by consecutive and specific mono‐O‐demethylation, and remethylation with methyl iodide‐d₃. The sensitivity of the method is confirmed by the very low limit of detection (LOD) and quantitation (LOQ) values achieved in the assay of 1 in two distinct fortified matrices, and is further supported by the observed accuracy values. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Rapid quantification of lisinopril in human plasma by liquid chromatography/tandem mass spectrometry

An assay based on protein precipitation and liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed and validated for the quantitative analysis of lisinopril in human plasma. After the addition of enalaprilat as internal standard (IS), plasma samples were prepared by one-step protein precipitation using perchloric acid followed by an isocratic elution with 10 mm ammonium acetate buffer (pH adjusted to 5.0 with acetic acid)-methanol (70:30, v/v) on a Phenomenex Luna 5 mu C(18) (2) column. Detection was performed on a triple-quadrupole mass spectrometer utilizing an electrospray ionization (ESI) interface operating in positive ion and selected reaction monitoring (SRM) mode with the precursor to product ion transitions m/z 406 --> 246 for lisinopril and m/z 349 --> 206 for enalaprilat. Calibration curves of lisinopril in human plasma were linear (r = 0.9973-0.9998) over the concentration range 2-200 ng/mL with acceptable accuracy and precision. The limit of detection and lower limit of quantification in human plasma were 1 and 2 ng/mL, respectively. The validated LC-MS/MS method has been successfully applied to a preliminary pharmacokinetic study of lisinopril in Chinese healthy male volunteers.     

Optimization for detection of flunarizine by high performance liquid chromatography/electrospray/mass spectrometry

This paper describes a newly developed high performance liquid chromatography/electrospray/mass spectrometry (HPLC/ES/MS) method for the determination of flunarizine (FZ) in artificial cerebrospinal fluid. The optimization for the detection of FZ in biological fluid by LC/ES/MS was investigated. The effects of solvent composition, the addition of modifier and flow rate on the detection of FZ by ES/MS were examined. The detection limit of this method (0.8 nM) proved to be much better than previously reported methods. Satisfactory accuracy (98.2–106.0%) of this newly developed method was obtained. The application of this method was demonstrated by analyzing FZ in rat microdialysis samples.     

Rapid and sensitive liquid chromatography tandem mass spectrometry method for the quantification of ambroxol in human plasma

A sensitive, specific and rapid high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was described and validated for the quantification of ambroxol in human plasma using enalaprilat as the internal standard (IS). Chromatographic separation was performed on a Lichrospher CN column with a mobile phase of methanol and water (containing 0.1% formic acid) (70:30, v/v). The total run time was 5.0 min for each sample. The analytes was detected by mass spectrometry with electrospray ionization source in positive selected reaction monitoring mode. The precursor-fragment ion reaction for ambroxol was m/z 378.9 --> 263.8, and for IS was m/z 349.0 --> 205.9. The linearity was established over the concentration range of 1.56-400.00 ng/mL. The inter-day and the intra-day precisions were all within 10%. A simple protein precipitation with methanol was adopted for sample preparation. The extraction recoveries of ambroxol and IS were higher than 90.80%. The validated method was successfully applied in pharmacokinetic study after oral administration of 90 mg ambroxol to 24 healthy volunteers.     

Quantification of clopidogrel in human plasma by sensitive liquid chromatography/tandem mass spectrometry

A simple, sensitive and rapid high-performance liquid chromatography/positive electrospray ionization tandem mass spectrometry method was developed and validated for the assay of clopidogrel in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M+H](+) ions, m/z 322/212 for clopidogrel and m/z 264/154 for the internal standard. The assay exhibited a linear dynamic range of 5-6000 pg/mL for clopidogrel in human plasma. The lower limit of quantification was 5 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

A new concept for isotope ratio monitoring liquid chromatography/mass spectrometry

A new interface for the on-line coupling of a liquid chromatograph to a stable isotope ratio mass spectrometer has been developed and tested. The interface is usable for (13)C/(12)C determination of organic compounds, allowing measurement of small changes in (13)C abundance in individual analyte species. All of the carbon in each analyte is quantitatively converted into CO(2) while the analyte is still dissolved in the aqueous liquid phase. This is accomplished by an oxidizing agent such as ammonium peroxodisulfate. The CO(2) is separated from the liquid phase and transferred to the mass spectrometer. It is shown that the whole integrated process does not introduce isotope fractionation. The measured carbon isotope ratios are accurate and reproducible. The sensitivity of the complete system allows isotope ratio determination down to 400 ng of compound on-column. By-passing the high-performance liquid chromatography (HPLC) separation allows bulk isotopic analysis with substantially lower sample amounts than those required by conventional elemental analyzers. The results of the first applications to amino acids, carbohydrates, and drugs, eluted from various types of HPLC columns, are presented. The wide range of chromatographic methods enables the analysis of compounds never before amenable to isotope ratio mass spectrometry techniques and may lead to the development of many new assays.