Quantitative determination of plasma vitamin K1 by high-performance liquid chromatography coupled to isotope dilution tandem mass spectrometry

Plasma vitamin K1 (phylloquinone) determination is commonly used for the diagnosis of vitamin K deficiency in patients suffering from lipid malabsorption. Moreover, current evidence that adequate vitamin K intake, and correspondingly adequate plasma vitamin K1 concentration, could also be of importance in relation to bone and brain diseases emphasizes the need to improve the current analytical methods. We developed a liquid chromatography coupled to tandem mass spectrometry method using a stable isotope ring-D4-labeled internal standard of vitamin K1 and operating in the multiple reaction monitoring mode by the selection of a precursor and product ions. The atmospheric pressure chemical ionization (APCI) method was shown to be more sensitive than electrospray ionization. After a single-step extraction with cyclohexane, chromatographic separation was performed on a C18 column with an isocratic mobile phase. The linearity was up to 5400 ng/L, and the limit of detection was 14 ng/L. Intra- and interrun precision were 2.4% and 8.3%, respectively, for the lower limit of the reference range. Recovery was better than 98%. The method is simple and reliable, allowing accurate vitamin K1 measurement in plasma samples from healthy subjects and patients suffering from vitamin K deficiency.     

Determination of the toluene diisocyanate binding sites on human serum albumin by tandem mass spectrometry

Diisocyanates are highly reactive chemical compounds widely used in the manufacture of polyurethanes. Although diisocyanates have been identified as causative agents of allergic respiratory diseases, the specific mechanism by which these diseases occur is largely unknown. To better understand the chemical species produced when diisocyanates react with protein, tandem mass spectrometry was employed to unambiguously identify the binding sites of the industrially important isomers, 2,4- and 2,6-toluene diisocyanate, on human serum albumin at varying diisocyanate/protein ratios. The 2,4-isomer results in approximately 2-fold higher conjugation product ion abundances than does the 2,6-isomer, suggesting that the 2,4-isomer has a higher reactivity toward albumin. Both isomers preferentially react with the N-terminal amine of the protein and the ε-NH₂ of lysine. At a low (1:2) diisocyanate/protein ratio, five binding sites are identified, whereas at a high (40:1) ratio, near-stoichiometric conjugation is observed with a maximum of 37 binding sites identified. Binding sites observed at the lowest conjugation ratios are conserved at higher binding ratios, suggesting a subset of 5–10 preferential binding sites on albumin. Diisocyanate–protein conjugation results in a variety of reaction products, including intra- and intermolecular crosslinking, diisocyanate self-polymerization, and diisocyanate hydrolysis.     

Determination of anandamide and other fatty acyl ethanolamides in human serum by electrospray tandem mass spectrometry

We developed a new selective liquid chromatography-electrospray ionization-tandem mass spectrometry method for the identification and quantification of anandamide (AEA), an endogenous cannabinoid receptor ligand, and other bioactive fatty acid ethanolamides (FAEs) in biological samples. Detection limit (0.025 pmol for AEA and 0.1 pmol for palmitoylethanolamide (PEA) and oleoylethanolamide (OEA)) and quantification limit (0.2 pmol for AEA and 0.4 pmol for OEA and PEA) were in the high fmol to low pmol range for all analytes. Linear correlations (r(2)=0.99) were observed in the calibration curves for standard AEA over the range of 0.025-25 pmol and for standard PEA and OEA over the range of 0.1-500 pmol. This method provides a time-saving and sensitive alternative to existing methods for the analysis of FAEs in biological samples.     

A proteomic view of Bifidobacterium infantis generated by multi-dimensional chromatography coupled with tandem mass spectrometry

Bifidobacteria are Gram-positive prokaryotes that naturally colonize the human gut where they exert several health-promoting effects. The present paper reports the use of a strong cation exchange-reversed-phase-tandem mass spectrometry strategy to catalogue the most abundantly expressed proteins of a probiotic Bifidobacterium infantis strain. A global view of the B. infantis proteome was obtained. The bimodal representation of the proteins identified by mass spectrometry provides the first theoretical two-dimensional map of protein distribution for this organism. Among the 136 proteins identified by multidimensional protein identification technology (MudPIT) analysis, 118 showed the highest similarity with the translated sequences of B. longum genome, two proteins were similar to other Bifidobacterium species and the remaining 16 were similar to different genera. Specific biological activities have been assigned to 115 identified proteins, whereas 21 have been referred to the group of hypothetical proteins. The MudPIT approach allowed us to identify high mass and basic isoelectric point proteins that are generally challenging to visualize using the traditional two-dimensional electrophoresis technique. Redundancy in peptide and protein identification using the double chromatography technique was also evaluated.     

Optimization and performance of a rapid gas chromatography–mass spectrometry analysis for methylmalonic acid determination in serum and plasma

We have developed a rapid and sensitive GC–MS assay for methylmalonic acid determination in serum and plasma utilizing an anion exchange solid-phase extraction and trimethylsilyl derivatization. Each step of the procedure was optimized by the experimental design methods to assure the assay reliable performance. The limit of detection and limit of quantitation were 0.025 and 0.1 μmol/l. The total coefficient of variation for the method was 9.8, 4.4, and 4.6% at the concentration of 0.2, 3.1, and 6.2 μmol/l methylmalonic acid concentration, respectively. The assay are linear up to 9.0 μmol/l, and showed good correlation with a reference method. The method has proven to be reliable in routine production, producing clean chromatography, unique ion fragments, and consistent ion mass ratio.     

Determination of misoprostol acid in human plasma by liquid chromatography coupled to tandem mass spectrometry

A rapid and sensitive liquid chromatographic/tandem mass spectrometric method for determination of misoprostol acid, the active metabolite of misoprostol, was developed and validated. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a C(18) column. An API 4000 tandem mass spectrometer equipped with Turbo IonSpray ionization source was used as detector and was operated in the negative ion mode. Multiple reaction monitoring using the precursor to product ion combinations of m/z 367-249 and 296-269 was performed to quantify misoprostol acid and the internal standard hydrochlorothiazide, respectively. The method was linear in the concentration range of 10.0-3000 pg mL(-1) using 200 microL plasma. The lower limit of quantification was 10.0 pg mL(-1). The intra- and inter-day relative standard deviation over the entire concentration range was less than 8.3%. Accuracy determined at three concentrations (25.0, 200 and 2700 pg mL(-1) for misoprostol acid) ranged from -0.5 to 1.2% in terms of relative error. Each plasma sample was chromatographed within 3.5 min. The method was successfully used in a pharmacokinetic study of misoprostol in human plasma after an oral administration of 0.6 mg misoprostol.     

Development of a novel method for triterpenoidal saponins in rat plasma by solid-phase extraction and high-performance liquid chromatography tandem mass spectrometry

A novel method using high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) has been developed for the quantification of four triterpenoidal saponins (anemoside B4, pulsatilloside B, anemoside A3, and 23-hydroxybetulinic acid) in rat plasma following solid-phase extraction (SPE). The optimized procedure utilized off-line extraction of the analytes from plasma using polymeric (Strata-X) SPE cartridges. Detection and quantitation were performed by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in a novel multiswitching monitoring mode. The analytes and internal standard (scutellarin) were analyzed using a Sapphire C18 column (250 × 4.6 mm, 5 μm) with a linear gradient elution. The mass transition ion pairs of the triterpenoidal saponins were executed as follows: m/z 1219.7/749.4 for anemoside B4, m/z 819.4/347.2 for pulsatilloside B, m/z 749.6/471.2 for anemoside A3, m/z 471.4/471.4 for 23-hydroxybetulinic acid, and m/z 461.1/285.0 for the internal standard. The specificity, linearity, accuracy, precision, recovery, matrix effect, and stabilities were validated for all analytes in the plasma samples. In conclusion, the validation results demonstrate that this method is robust and specific. This validated method is a novel technique for sample preparation and quantitation and was successfully applied to estimate the pharmacokinetics of triterpenoidal saponins.     

Detection of chlorinated DNA and RNA nucleosides by HPLC coupled to tandem mass spectrometry as potential biomarkers of inflammation

Upon inflammation, activated neutrophils secrete myeloperoxidase, an enzyme able to generate hypochlorous acid (HOCl) from hydrogen peroxide and chloride ions. An analytical method, involving HPLC coupled to electrospray tandem mass spectrometry, has been set-up to detect low levels of HOCl-induced nucleic acids lesions, including both ribo and 2'-deoxyribonucleoside derivatives of 8-chloroguanine, 8-chloroadenine and 5-chlorocytosine. Validation of the developed method was achieved using isolated cells treated with HOCl. The method was found to be sensitive enough to allow the measurement of background levels of 5-chloro-2'-deoxycytidine in the DNA of human white blood cells isolated from 7 mL of blood.     

Detection and characterization of N-alpha-chloramines by electrospray tandem mass spectrometry

Hypochlorous acid (HOCl) is a major product of activated neutrophils and may be important in antimicrobial activities of cells by oxidation or chlorination of susceptible amino acids. Three major peaks separated using C18 reverse phase-high-performance liquid chromatography RP-HPLC after incubation of leucine enkephalin (LeuEnk) with HOCl. Electrospray mass spectrometry showed masses of m/z 556.2, 590.2, and 624.4 corresponding to unmodified LeuEnk and peptides altered by addition of one or two chlorines (Cl). Formation of stable N-alpha-chloramines was indicated because the chlorinated peptides were readily reduced with the physiological reductants glutathione and ascorbic acid to LeuEnk (m/z 556.2) within 10 min. Sequence-specific ions observed in product ion spectra of single-charged monochlorinated and dichlorinated peptides were consistent with modification of the N-terminal amine. There was no evidence for chlorination of the Tyr aromatic ring in any spectra. Similar RP-HPLC profiles were obtained after oxidation of des-Tyr1-LeuEnk (GGFL) with the masses of the major products being m/z 393.3, 427.2, and 461.1. These were identified as unmodified GGFL, N-alpha-Cl-GGFL, and N-alpha-Cl2-GGFL based on comparison of tandem mass spectra. Oxidation of Met and formation of disulfide dimers was observed after incubation of either N-alpha-Cl-LeuEnk or N-alpha-Cl2-LeuEnk with a protein, indicating that both peptide N-alpha-chloramines were able to readily modify sulfur-containing amino acids within proteins. These data indicate initial formation of stable N-alpha-chorinated peptides after incubation with HOCl and suggest that N-alpha-chlorinated peptides may exist for some hours in the absence of physiological reducing agents or sulfur-containing amino acids.     

Determination of iodide in urine using electrospray ionization tandem mass spectrometry

A rapid and sensitive electrospray ionization (ESI) tandem mass spectrometry (MS–MS) procedure was developed for the determination of iodide (I⁻). A gold (Au) and I⁻ complex was formed immediately after the addition of the chelating agent NaAuCl₄ to I⁻ solution, and was extracted with methyl isobutyl ketone. One to five microliters of the extract were injected directly into an ESI–MS–MS instrument. I⁻ quantification was performed by selecting reaction monitoring of the product ion I⁻ at m/z 127 derived from the precursor ion ¹⁹⁷AuI₂⁻ at m/z 451. I⁻ concentration was measured in the quantification range from 10⁻⁷ to 10⁻⁵ M using 50 μL of solution within 10 min. Iodate was reduced to I⁻ with ascorbic acid and determined. I⁻ concentration in reference urine 2670a was measured after treatments.     

Determination of LSD and N-demethyl-LSD in urine by liquid chromatography coupled to electrospray ionization mass spectrometry

A sensitive and highly specific method for the determination of LSD and N-demethyl-LSD in urine, using combined liquid chromatography and mass spectrometry (LC-MS) with electrospray ionization, has been developed. Extrelut-3 extraction cartridges were used for a basic sample clean-up. Elution was obtained by toluene-diethyl ether (60:40, v/v). A Nucleosil C₁₈ (150×1 mm I.D.) reversed-phase column was used for the chromatographic separation, together with a mixture of 2 mM ammonium formate buffer (pH 3) and acetonitrile (70:30, v/v) as mobile phase. Recoveries were 93 and 80%, detection limits 0.025 and 0.035 ng/ml for LSD and N-demethyl-LSD, respectively. Intra-assay precision, studied at four concentrations, was better than 9% at the ng/ml range and better than 14% at 0.10 ng/ml for both compounds. Limits of quantitation were 0.05 and 0.10 ng/ml for LSD and N-demethyl-LSD, respectively. Reproducibility was good and linearity excellent for LSD in the range from 0.05 to 20 ng/ml (r>0.9999, N=7).     

High-performance liquid chromatography coupled with tandem mass spectrometry applied for metabolic study of ginsenoside Rb1 on rat

Liquid chromatography coupled with mass spectrometry and tandem mass spectrometry has been applied to investigate the in vivo metabolism of ginsenoside Rb(1) in rat. Both positive electrospray ionization mass spectrometry and negative electrospray ionization mass spectrometry were used to identify the Rb(1) and its metabolites in rat plasma, urine, and feces samples. Oxygenation and deglycosylation were found to be the major metabolic pathways of Rb(1) in rat. A total of nine metabolites were detected in urine and feces samples collected after intravenous and oral administration. Deglycosylated metabolism of Rb(1) generated other ginsenosides as the major metabolites, such as Rd, Rg(3) or F(2), Rh(2), or C-K. This result indicates that the ginsenoside Rb(1) has many pharmacological activities and could be used as a prodrug.     

Characterization of wheat gliadin proteins by combined two-dimensional gel electrophoresis and tandem mass spectrometry

A proteomics-based approach was used for characterizing wheat gliadins from an Italian common wheat (Triticum aestivum) cultivar. A two-dimensional gel electrophoresis (2-DE) map of roughly 40 spots was obtained by submitting the 70% alcohol-soluble crude protein extract to isoelectric focusing on immobilized pH gradient strips across two pH gradient ranges, i.e., 3-10 or pH 6-11, and to sodium dodecyl sulfate-polyacrylamide electrophoresis in the second dimension. The chymotryptic digest of each spot was characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano electrospray ionization-tandem mass spectrometry (MS/MS) analysis, providing a "peptide map" for each digest. The measured masses were subsequently sought in databases for sequences. For accurate identification of the parent protein, it was necessary to determine de novo sequences by MS/MS experiments on the peptides. By partial mass fingerprinting, we identified protein molecules such as alpha/beta-, gamma-, omega-gliadin, and high molecular weight-glutenin. The single spots along the 2-DE map were discriminated on the basis of their amino acid sequence traits. alpha-Gliadin, the most represented wheat protein in databases, was highly conserved as the relative N-terminal sequence of the components from the 2-DE map contained only a few silent amino acid substitutions. The other closely related gliadins were identified by sequencing internal peptide chains. The results gave insight into the complex nature of gliadin heterogeneity. This approach has provided us with sound reference data for differentiating gliadins amongst wheat varieties.     

Determination of ticagrelor and two metabolites in plasma samples by liquid chromatography and mass spectrometry

Rapid and sensitive analytical methods using liquid chromatography with tandem mass spectrometry (LC/MS/MS) were developed for the determination of ticagrelor, the first reversible oral platelet P2Y₁₂ receptor inhibitor, and its metabolites AR-C124910XX and AR-C133913XX in human plasma. Ticagrelor and its metabolites were extracted using protein precipitation with acetonitrile. Chromatographic separations were performed on reversed phase columns and detection using atmospheric pressure chemical ionization (APCI). Ticagrelor and AR-C124910XX were analyzed in the same assay, with the internal standard, d7-ZD6140, on a C18 column using negative ionization; AR-C133913XX analyzed separately on a phenyl column using positive ionization. Full validation of the methods was performed including selectivity, lower limit of quantification, accuracy, precision stability and incurred sample reproducibility and incurred sample stability. Total analytical run time was short (2 min). Calibration curves were established in the range 5–5000 ng/mL for ticagrelor, 2.5–2500 ng/mL for AR-C124910XX and 2–1000 ng/mL for AR-C133913XX. Lower limits of quantification for ticagrelor, AR-C124910XX and AR-C133913XX were determined to be 5, 2.5 and 2.0 ng/mL, respectively from 100 μL of human plasma. For ticagrelor, AR-C124910XX and AR-C133913XX, mean intra-batch accuracy was 91.9–109.0%, 86.8–109.2% and 100.5–112.0%, respectively; intra-batch precision was 4.0–8.4%, 5.2–16.9% and 3.9–12.3%, respectively. The methods were also applied to quantification of ticagrelor, AR-C124910XX and AR-C133913XX in rabbit, rat, mouse and marmoset, using 25 μL of animal plasma. A modified methodology was developed to quantify ticagrelor and AR-C124910XX in plasma from dog and cynomolgus monkey. Human incurred samples were found to generate consistent reproducibility and stability results. This method was successfully applied to determine plasma concentrations following administration of ticagrelor in human volunteers and patients, and animal safety evaluation studies. This validated methods has the advantages of being straightforward, robust and allows a fast throughput of samples.     

High-performance liquid chromatography coupled with tandem mass spectrometry for the detection of amyloid Beta peptide related with Alzheimer's disease.

Recent studies show that quantitative and qualitative differences in amyloid beta (Abeta ) peptides may be implicated in the development of Alzheimer's disease. New evidence seems to support the existence of a dynamic equilibrium between Abeta peptide in the brain and peripheral blood circulation. The quantitation of Abeta in the blood may allow the development of the potential value of Abeta peptides as a biomarker in the development of Alzheimer's disease. In this communication, quantitation of Abeta peptides using high-performance liquid chromatography coupled with tandem mass spectrometry in a linear ion trap mode is presented. RP-HPLC was performed using a Waters Xterra MS C8 column (3.0 mm x 150 mm). Abeta(1-40) peptide was eluted using a gradient elution program. Eluate from the RP-HPLC column was split to both the UV detector and electrospray ionization MS source. The product ion scan was performed in a linear ion trap mode utilizing the transition of a multiply charged molecular ion of Abeta(1-40) to a singly charged product ion. The detection limit of 31.25 ng in column load using a 3.0-mm-diameter conventional C8 column was achieved. The Abeta(1-40) standard calibration curves show excellent linearity from 34 ng to 2500 ng Abeta(1-40) of column sample load. The product ion scan enhances sensitivity 10 times compared with the best previously achieved by a single-quadrupole instrument in the selective ion monitoring mode. Moreover, the product ion scan of Abeta(1-40) provides superior selectivity and specificity, which is very important in the quantitation of Abeta(1-40) in a complex biological matrix.     

Determination of endogenous and supplied deuterated abscisic acid in plant tissues by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry with multiple reaction monitoring

A specific, sensitive, and accurate method for determination of abscisic acid (ABA) in plant tissues is described. The method employs reversed-phase high-performance liquid chromatography and electrospray ionization-tandem mass spectrometry for multiple reaction monitoring of underivatized ABA and deuterated ABA analogs. Specific analogs were used to study the mechanism of ABA fragmentation, to select appropriate standards, and to identify compounds suitable for metabolic studies involving the supply of differentially labeled ABA. Limits of detection and quantification of 1.9 and 4.7 pg, respectively, were obtained over a linear calibration range of 0-1.5 ng ABA (on-column injected) using 5.8', 8', 8'-d(4) ABA as the internal standard. Accuracy and precision were within 15% for routine quality control samples. The method of standard additions, as applied to Arabidopsis thaliana seed extracts, was also used to validate the method for analysis of plant tissue samples. The utility of the method was further demonstrated by determining levels of ABA in western white pine seeds and of ABA and supplied 8', 8', 8', 9', 9', 9'-d(6) ABA in Brassica napus tissues, using 5.8', 8', 8'-d(4) ABA or 8', 8', 8'-d(3) ABA as the internal standard. Limits of quantification as low as 0.89 ng/g were achieved by optimizing the extraction procedure for each type of plant tissue.     

Determination of benzimidazole residues in bovine milk by ultra-high performance liquid chromatography-tandem mass spectrometry

A method based on ultra-high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UHPLC–MS/MS) for the simultaneous determination of benzimidazole residues in bovine milk has been optimized and validated. Rapid chromatographic separation of 13 analytes in 8 min was obtained by means of UHPLC. The samples were subject to Oasis MCX solid-phase extraction cartridges for extraction and clean-up. Matrix-matched calibration curves were performed to compensate for the matrix effect and loss in sample preparation. Mean recoveries ranged from 80% to 101% and inter-day precision was lower than 14%. Limit of detection and limit of quantification of the method ranged from 0.01 to 0.5 μg L⁻¹ and from 0.1 to 1.0 μg L⁻¹, respectively.     

Identification of phenylbutyrate-generated metabolites in Huntington disease patients using parallel liquid chromatography/electrochemical array/mass spectrometry and off-line tandem mass spectrometry

Oral sodium phenylbutyrate (SPB) is currently under investigation as a histone deacetylation (HDAC) inhibitor in Huntington disease (HD). Ongoing studies indicate that symptoms related to HD genetic abnormalities decrease with SPB therapy. In a recently reported safety and tolerability study of SPB in HD, we analyzed overall chromatographic patterns from a method that employs gradient liquid chromatography with series electrochemical array, ultraviolet (UV), and fluorescence (LCECA/UV/F) for measuring SPB and its metabolite phenylacetate (PA). We found that plasma and urine from SPB-treated patients yielded individual-specific patterns of approximately 20 metabolites that may provide a means for the selection of subjects for extended trials of SPB. The structural identification of these metabolites is of critical importance because their characterization will facilitate understanding the mechanisms of drug action and possible side effects. We have now developed an iterative process with LCECA, parallel LCECA/LCMS, and high-performance tandem MS for metabolite characterization. Here we report the details of this method and its use for identification of 10 plasma and urinary metabolites in treated subjects, including indole species in urine that are not themselves metabolites of SPB. Thus, this approach contributes to understanding metabolic pathways that differ among HD patients being treated with SPB.     

Determination of dansylated polyamines in red blood cells by liquid chromatography-tandem mass spectrometry

The concentration of polyamines in red blood cells (RBCs) is considered to be an index of cell proliferation. This index has been demonstrated to be of clinical importance for the follow-up and treatment of some cancer patients. The concentration of polyamines in RBCs is usually determined by high-performance liquid chromatography (HPLC) with fluorescence detection. In the current work, we present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of putrescine, spermidine, and spermine, the three major polyamines in RBCs. The polyamines were dansylated and analyzed by an LC gradient of 20-min duration on a C18 column on-line with a tandem mass spectrometer. An internal standard (1,8-diaminooctane) was used for quantification. This method exhibited excellent linearity for the three polyamines with regression coefficients higher than 0.99. The limits of detection for putrescine, spermidine, and spermine were 0.10, 0.75, and 0.50 pmol/ml, respectively. The intrarun precision values for putrescine, spermidine, and spermine all were better than 10%, and the interrun precision values were 13%, 9%, and 20%, respectively. The LC-MS/MS method is sufficiently simple and reliable enough to replace the currently used HPLC method with fluorescence detection in which putrescine is not always detectable.