A heterogeneous biotin–streptavidin-amplified enzyme-linked immunosorbent assay for detecting tris(2,3-dibromopropyl) isocyanurate in natural samples

Tris(2,3-dibromopropyl) isocyanurate (TBC) is a novel brominated flame retardant (BFR) that is widely used to substitute the prohibited BFRs throughout the world. With the development of research, the potential environmental and ecological harms of TBC have been revealed. For sensitive and selective detecting TBC, an indirect competitive biotin–streptavidin-amplified enzyme-linked immunosorbent assay (BA–ELISA) has been established in this study. The small molecular TBC–hapten was synthesized first; it mimicked the chemical structure of TBC and possessed a secondary amine group. The as-obtained hapten was then conjugated with carrier proteins to prepare artificial antigen. After immunization, the anti-TBC polyclonal antibody was obtained from separating rabbit serum. The procedures of this BA–ELISA were optimized. Under the optimal conditions, the limit of detection (IC₁₀) was 0.0067 ng/ml and the median inhibitory concentration (IC₅₀) was 0.66 ng/ml. Cross-reactivity values of the BA–ELISA with the tested TBC analogues were ?5%. This immunoassay was successfully applied to determine the TBC residue in river water samples that were collected near a BFR manufacturing plant. Satisfactory recoveries (92.1–109.2%) were obtained. The results indicated that this proposed BA–ELISA is suitable for the rapid and sensitive determining of TBC in environmental monitoring.     

Development of enzyme-linked immunosorbent assay for determination of polybrominated diphenyl ether BDE-121

Our interests are in the development of immunoassay-based fast scanning methods for persistent organic pollutants. To develop the immunoassay method of polybrominated diphenyl ether (PBDE), a model compound of PBDE, 2,3′,4,5′,6-pentabromodiphenylether (BDE-121), has been chosen to develop its antibody and the competitive indirect enzyme-linked immunosorbent assay (ELISA) is developed. The hapten of BDE-121 containing reactive carboxylic acid was synthesized and conjugated to carrier proteins (bovine serum albumin [BSA] and ovalbumin [OVA]). Anti-BDE-121 polyclonal antibody was then developed in rabbits as a result of immunization with the BDE-121–BSA conjugate. The optimal amount of coating antigen BDE-121–OVA conjugate and the dilution of antiserum needed in the ELISA were determined with the checkerboard method, and the effects of the properties of PBST (phosphate-buffered saline and Tween 20) buffer (pH and salt concentration) and chemical solvent (types and concentrations) on the ELISA were investigated to achieve a rapid robust assay with high sensitivity. Under the optimized conditions, the developed indirect ELISA shows a linear detection range from 1.74 to 84.1 ng/ml, with an IC₅₀ value of 8.07 ng/ml and a detection limit of 0.644 ng/ml. In total, 11 kinds of compounds were tested for calculating the cross-reactivity, which was less than 8% for nearly all of them. Real samples were analyzed by the proposed immunoassay and gas chromatography/mass spectrometry (GC/MS).     

Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay

3-Phenoxybenzoic acid (3-PBA) is a general metabolite of synthetic pyrethroids. It could be used as a generic biomarker for multiple pyrethroids exposure for human or pyrethroid residues in the environment. In this study, monoclonal antibodies (mAbs) against 3-PBA were developed by using PBA–bovine serum albumin (BSA) as an immunogen. In the competitive enzyme-linked immunosorbent assay (ELISA) format, the I₅₀ and I₁₀ values of purified mAbs were 0.63 and 0.13 μg/ml, respectively, with a dynamic range between 0.19 and 2.04 μg/ml. Then, the colloidal gold (CG)-based lateral flow immunoassay was established based on the mAbs. The working concentration of coating antigen and CG-labeled antibodies and the blocking effects were investigated to get optimal assay performance. The cutoff value for the assay was 1 μg/ml 3-PBA, and the detection time was within 10 min. A total of 40 river water samples were spiked with 3-PBA at different levels and determined by the lateral flow immunoassay without any sample pretreatments. The negative false rate was 2.5%, and no positive false results were observed at these levels. This lateral flow immunoassay has the potential to be an on-site screening method for monitoring 3-PBA or pyrethroid residues in environmental samples.     

Analysis of global DNA methylation by hydrophilic interaction ultra high-pressure liquid chromatography tandem mass spectrometry

We developed and validated a rapid, sensitive, and specific liquid chromatography tandem mass spectrometry (LC–MS/MS) method for determination of global DNA methylation in tissue. DNA was extracted by phenol–chloroform, hydrolyzed using 88% formic acid at 140 °C, spiked with cytosine-2,4-¹³C¹⁵N₂ as internal standard, evaporated under nitrogen, reconstituted in methanol, and analyzed by LC–MS/MS in multiple reaction monitoring mode to reflect the global DNA methylation of the tissue. The method was linear throughout the range of clinical interest and had good sensitivity, with a limit of quantification of 0.5 pg for both cytosine (Cyt) and 5-methylcytosine (5mCyt). The linear range of calibration curve was 1–50 and 1–100 ng/ml for 5mCyt and Cyt, respectively, with a correlation coefficient higher than 0.99. The relative standard deviation (RSD) was 0.70–4.09% and 0.60–4.81% for Cyt and 5mCyt, respectively. The intraday precision expressed as RSD ranged from 1.86% to 4.67%, whereas the interday values ranged from 3.72% to 4.68%. The recovery of the method varied from 86.52% to 105.14%. This yielded a simple and reliable LC–MS/MS assay for detection of Cyt and 5mCyt, thereby enabling the evaluation of global DNA methylation.     

Ultra sensitive quantitation of endogenous oxytocin in rat and human plasma using a two-dimensional liquid chromatography-tandem mass spectrometry assay

Oxytocin (OT) is a neuropeptide with an extremely low endogenous level (low pg/ml) in human plasma. It is very challenging to develop a highly sensitive assay to measure endogenous OT, including radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Electrospray ionization (ESI) liquid chromatography–tandem mass spectrometry (LC–MS/MS) can provide high-throughput and selective methods for quantification of peptides in biological samples. A novel and highly sensitive two-dimensional LC–MS/MS (2D-LC–MS/MS) assay combining solid-phase extraction (SPE) has been developed and validated for the determination of endogenous OT in both human and rat plasma. The lower limit of quantification (LLOQ) was 1.00 pg/ml for human and 50.0 pg/ml for rat. Human plasma diluted with water (1:6, v/v) was successfully optimized as a surrogate matrix for human to prepare standard curves without endogenous interference. The extraction efficiency and absolute recovery were above 65.8% using the HLB SPE procedure, and matrix effects were lower than 12%. The method was validated in the range of 1.00–250 pg/ml for human plasma and 50.0–10,000 pg/ml for rat plasma with precision less than 12.7% and accuracy less than 7%.     

An organic–inorganic hybrid nanostructure-functionalized electrode for electrochemical immunoassay of biomarker by using magnetic bionanolabels

A new electrochemical immunoassay of alpha-fetoprotein (AFP) was developed on an organic–inorganic hybrid nanostructure-functionalized carbon electrode by coupling with magnetic bionanolabels. Multi-walled carbon nanotubes (CNTs), single-stranded DNA, thionine and AFP were utilized for the construction of the immunosensor, while the core–shell Fe₃O₄-silver nanocomposites were employed for the label of horseradish peroxidase-anti-AFP conjugates (HRP-anti-AFP-AgFe). Electrochemical measurement toward AFP was carried out by using magnetic bionanolabels as traces and H₂O₂ as enzyme substrate with a competitive-type immunoassay mode. Experimental results indicated that the immunosensors with carbon nanotubes and DNA exhibited better electrochemical responses than those of without carbon nanotubes or DNA. Under optimal conditions, the electrochemical immunosensor by using HRP-anti-AFP-AgFe as signal antibodies exhibited a linear range of 0.001–200 ng mL⁻¹ AFP with a low detection limit of 0.5 pg mL⁻¹ at 3s_B. Both intra- and inter-assay coefficients of variation were 7.3%, 9.4%, 8.7% and 10.2%, 7.8%, 9.4% toward 0.01, 30, 120 ng mL⁻¹ AFP, respectively. The specificity and stability of the electrochemical immunoassay were acceptable. In addition, the methodology was validated for 12 clinical serum specimens including 9 positive specimens and 3 normal specimens, receiving a good correlation with the results obtained from the referenced electrochemiluminescence assay.     

A new reliable chemiluminescence immunoassay (CLIA) for prostaglandin E2 using enhanced luminol as substrate

A sensitive and reliable chemiluminescence immunoassay suitable for the quantitative determination of prostaglandin E₂ (PGE₂) has been developed using 96 well microtiter plates (MTP). The assay is based on a competitive reaction between a highly specific monoclonal anti-PGE₂ antibody (mouse), free antigen and solid phase bound antigen. The MTP was first coated with a bovine serum albumin (BSA)-PGE₂ conjugate. Then, after preincubating, the anti-PGE₂ antibody (Ab) and the analyte were added. The remaining amount of free antibody was captured by the solid phase bound BSA-PGE₂ conjugate. The monoclonal antibody captured on the MTP was determined using biotinylated antimouse-Ab and a complex of avidin and biotin-labelled horseradish peroxidase (HRP). Substrate for HRP was the cyclic diacyl hydrazide compound luminol, enhanced by p-iodophenol. Photons emitted during the reaction were measured using a photomultiplier tube. The assay has been validated with assay buffer and human plasma over a concentration range of 10–50,000 pglml. The lower limit of quantification is 100 pglml (2 pglwell) and 150 pglml (3 pglwell) for buffer and plasma, respectively. The intea-day coefficients of variation (CV) for the range of 100–50,000 pglml are 3.2–8.9% (buffer) and 4.2–17.7% (plasma) and inter-day CV are 2.9–19.8% (buffer) and 3.6–21.2% (plasma). The method can be used for quantification of PGE2 in biological fluids like plasma and suction blister fluid.     

Microscale high-performance liquid chromatography–electrospray tandem mass spectrometry assay for cyclosporin A in blood

To facilitate quantitative analysis of cyclosporin A in low volume blood samples we developed a sensitive and specific microscale reversed-phase HPLC–electrospray tandem mass spectrometry assay. Blood samples (100 μl) were prepared by acetonitrile precipitation and C₁₈ solid-phase extraction. Detection was by multiple-reactant monitoring. The method was linear over the range 5–1000 μg/l (r≥0.997) with accuracy between 95.4 and 102.0% over this range. Total imprecision was 11.1% at 10 μg/l and 2.8% at 800 μg/l. Absolute recovery of cyclosporin A and internal standard was 72.5 and 73.3%, respectively. When this method was evaluated against a conventional HPLC with UV detection, in patient samples, they were interchangeable (y=0.988x+10.0, r=0.996). This HPLC–ESI-MS–MS method will be applicable to therapeutic monitoring in paediatric transplant patients and multiple point pharmacokinetic studies in animals and humans.     

Discrepancy between radioimmunoassay and high performance liquid chromatography tandem-mass spectrometry for the analysis of androstenedione

The discrepancy of results for the quantification of androstenedione in human serum between a radioimmunoassay (RIA) method and high performance liquid chromatography tandem-mass spectrometry (LC–MS/MS) was investigated. RIA overestimated concentrations compared to LC–MS/MS on 59 clinical samples (RIA = 1.79 × LC–MS/MS + 0.94). RIA kit and LC–MS/MS calibrants were also determined by both methods. The RIA performed with improved accuracy on the calibrants (RIA = 1.35 × LC–MS/MS − 0.28). Lipid, protein, electrolyte content, and pH of the two sets of calibrants were further investigated. The RIA calibrants contained little lipid material, while the LC–MS/MS calibrant material contained the same levels expected in normal serum/plasma. The pH and sex hormone binding globulin (SHBG) values were different between the RIA calibrants and the LC–MS/MS calibrant material (SHBG, 31 ± 2 and 38 ± 2 nmol/l; pH, 8.27 ± 0.18 and 8.66 ± 0.03, respectively). No correlation was observed between androstenedione RIA and LC–MS/MS discrepancy and lipid or protein. LC–MS/MS sample preparation was tested for the removal of protein-bound material and recovery determined (99–108%). The corresponding RIA results overestimated androstenedione by 52–174% compared to LC–MS/MS. The results here demonstrate that LC–MS/MS is the more accurate method.     

Quantitation of amyloid beta peptides Aβ(1-38), Aβ(1-40), and Aβ(1-42) in human cerebrospinal fluid by ultra-performance liquid chromatography-tandem mass spectrometry

Critical events in Alzheimer’s disease (AD) involve an imbalance between the production and clearance of amyloid beta (Aβ) peptides from the brain. Current methods for Aβ quantitation rely heavily on immuno-based techniques. However, these assays require highly specific antibodies and reagents that are time-consuming and expensive to develop. Immuno-based assays are also characterized by poor dynamic ranges, cross-reactivity, matrix interferences, and dilution linearity problems. In particular, noncommercial immunoassays are especially subject to high intra- and interassay variability because they are not subject to more stringent manufacturing controls. Combinations of these factors make immunoassays more labor-intensive and often challenging to validate in support of clinical studies. Here we describe a mixed-mode solid-phase extraction method and an ultra-performance liquid chromatography tandem mass spectrometry (SPE UPLC–MS/MS) assay for the simultaneous quantitation of Aβ_1–38, Aβ_1–40, and Aβ_1–42 from human cerebrospinal fluid (CSF). Negative ion versus positive ion species were compared using their corresponding multiple reaction monitoring (MRM) transitions, and negative ions were approximately 1.6-fold greater in intensity but lacked selectivity in matrix. The positive ion MRM assay was more than sufficient to quantify endogenous Aβ peptides. Aβ standards were prepared in artificial CSF containing 5% rat plasma, and quality control samples were prepared in three pooled CSF sources. Extraction efficiency was greater than 80% for all three peptides, and the coefficient of variation during analysis was less than 15% for all species. Mean basal levels of Aβ species from three CSF pools were 1.64, 2.17, and 1.26 ng/ml for Aβ_1–38; 3.24, 3.63, and 2.55 ng/ml for Aβ_1–40; and 0.50, 0.63, and 0.46 ng/ml for Aβ_1–42.     

LC–MS/MS coupled with immunoaffinity extraction for determination of estrone, 17β-estradiol and estrone 3-sulfate in human plasma

Determination of estrogens in plasma is important in evaluation of effects of some anticancer drugs, such as aromatase inhibitors. However, as reported previously, high performance liquid chromatography–radio immunoassay (HPLC–RIA) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) with chemical derivatization require complicated sample preparation. In this study, a highly sensitive and simple method for determination of estrone (E1), 17β-estradiol (E2) and estrone 3-sulfate (E1S) in human plasma has been developed. Following diethylether extraction from plasma, analytes were purified by immunosorbents and then determined by LC–MS/MS using electrospray ionization (ESI). Immunosorbents were prepared by immobilization of specific antibodies raised against each analyte onto solid support. Use of selective immunosorbents in sample preparation removed interference in plasma samples that would cause ionization suppression, and markedly improved the sensitivity of LC–MS/MS for these analytes, without derivatization. Calibration curves of each analyte showed good linearity and reproducibility over the range of 0.05–50 pg/injection for E1, 0.2–50 pg/injection for E2 and 0.05–300 pg/injection for E1S, respectively. The mean values of lower limits of quantification (LLOQ) in human plasma corrected by recovery of deuterated estrogens (internal standard, I.S.) were 0.1892 pg/mL for E1, 0.7064 pg/mL for E2 and 0.3333 pg/mL for E1S, respectively. These LLOQ values were comparable to those previous reported using HPLC–RIA and LC–MS/MS. Using this method, the normal levels of three estrogens in healthy female plasma (n = 5) were determined. The mean values of E1, E2 and E1S were 38.0 pg/mL (range 24.8–53.0), 34.3 pg/mL (22.6–46.6) and 786 pg/mL (163–2080), respectively. The immunoaffinity LC–MS/MS described here allows sensitive and accurate quantification of E1, E2 and E1S without laborious sample preparation.     

Development and validation of an LC–MS/MS method for quantification of cyclic guanosine 3′,5′-monophosphate (cGMP) in clinical applications: A comparison with a EIA method

An LC–MS/MS method was developed and validated to quantify endogenous cyclic guanosine 3′,5′-monophosphate (cGMP) in human plasma. The LC–MS/MS and competitive enzyme immunoassay (EIA) assays were compared. cGMP concentrations of 20 human plasma samples were measured by both methods. For the MS-based assay, plasma samples were subjected to a simple protein precipitation procedure by acetonitrile prior to analysis by electrospray ionization LC–MS/MS. De-protonated analytes generated in negative ionization mode were monitored through multiple reaction monitoring (MRM). A stable isotope-labeled internal standard, ¹³C₁₀,¹⁵N₅-cGMP, which was biosynthesized in-house, was used in the LC–MS/MS method. The competitive EIA was validated using a commercially available cGMP fluorescence assay kit. The intra-assay accuracy and precision for MS-based assay for cGMP were 6–10.1% CV and ?3.6% to 7.3% relative error (RE), respectively, while inter-assay precision and accuracy were 5.6–8.1% CV and ?2.1% to 6.3% RE, respectively. The intra-assay accuracy and precision for EIA were 17.9–27.1% CV and ?4.9% to 24.5% RE, respectively, while inter-assay precision and accuracy were 15.1–39.5% CV and ?30.8% to 4.37% RE, respectively. Near the lower limits of detection, there was little correlation between the cGMP concentration values in human plasma generated by these two methods (R² = 0.197, P = 0.05). Overall, the MS-based assay offered better selectivity, recovery, precision and accuracy over a linear range of 0.5–20 ng/mL. The LC–MS/MS method provides an effective tool for the quantitation of cGMP to support clinical mechanistic studies of curative pharmaceuticals.     

Application of C stable isotope labeling liquid chromatography–multiple reaction monitoring–tandem mass spectrometry method for determining intact absorption of bioactive dipeptides in rats

The liquid chromatography–multiple reaction monitoring–tandem mass spectrometry (LC–MRM–MS/MS) method using ¹³C stable isotope-labeled dipeptides was newly developed to simultaneously determine the absorption of three antihypertensive peptides (Val-Tyr, Met-Tyr, and Leu-Tyr) into blood of spontaneously hypertensive rats in one run-in assay. After extracting ¹³C-labeled peptides in blood sample with a C₁₈ cartridge, the extract was applied to a ¹³C monoisotopic transition LC–MRM–MS/MS system with d-Val-Tyr included as internal standard. An excellent separation of each dipeptide in LC was achieved at the elution condition of 5–100% methanol in 0.1% formic acid at a flow rate of 0.25 ml/min. The ¹³C-labeled peptides ionized by electron spray were detected in the positive ion mode within 15 min. The established method showed high reproducibility with less than 10% coefficient of variation as well as high accuracy of more than 85%. After the administration of a mixture containing the three ¹³C-labeled dipeptides to rats at each dose of 30 mg/kg, we could successfully determine the intact absorption of each ¹³C-labeled peptide with the maximal absorption amount of 1.1 ng/ml plasma for Val-Tyr by the proposed LC–MRM–MS/MS method.     

Quantitative analysis of polychlorinated biphenyls, organochlorine insecticides, polycyclic aromatic hydrocarbons, polychlorinated hydrocarbons and polynitrohydrocarbons in spiked samples of soil, water and plasma by selected-ion monitoring gas chromatography–mass spectrometry

A broad range of pollutants such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated hydrocarbons (PCHs), polynitrohydrocarbons (PNHs), polychlorinated biphenyls (PCBs) and organochlorine (OCs) insecticides were simultaneously analyzed in spiked soil, water or plasma samples by using gas chromatography–mass spectrometry (GC–MS). Water and plasma samples containing the pollutants were extracted by a solid-phase extraction (SPE) method using florisil columns. The soil samples, fortified with the toxicants, were extracted with water, methanol or dichloromethane (DCM). The water extract was processed by the SPE method. The methanol and DCM samples were dried, dissolved in acetonitrile and subjected to the SPE extraction. The extracted samples were analyzed by GC–MS programmed to monitor selected ions. The deuterium labelled compounds were used as the internal standards. The chromatographic profile of total ions indicated complete separation of some compounds such as isophorone, naphthalene, all PCBs, most OC insecticides and PNHs; high M_r PAHs and some PCHs were partially or incompletely separated. The chromatographic profile of individual ion indicated good separation of each ion. The minimum detection limit ranged from 1 to 4 pg injected when 1 or 2 ions were monitored or from 20 to 200 pg injected when 20 ions were monitored. The SPE method that provided 60–105% recovery of pollutants from water samples, provided only 2–60% recovery from plasma samples. This may be due to the binding of pollutants to plasma proteins. Water recovered 1–30%, while methanol or DCM recovered 65–100% of the pollutants added to the soil samples. The use of internal standards corrected for the loss of pollutants from plasma or soil.     

Mass spectrometry assay for studying kinetic properties of dipeptidases: characterization of human and yeast dipeptidases

Chemical modifications of substrate peptides are often necessary to monitor the hydrolysis of small bioactive peptides. We developed an electrospray ionization mass spectrometry (ESI–MS) assay for studying substrate distributions in reaction mixtures and determined steady-state kinetic parameters, the Michaelis–Menten constant (K_m), and catalytic turnover rate (V_max/[E]_t) for three metallodipeptidases: two carnosinases (CN1 and CN2) from human and Dug1p from yeast. The turnover rate (V_max/[E]_t) of CN1 and CN2 determined at pH 8.0 (112.3 and 19.5 s⁻¹, respectively) suggested that CN1 is approximately 6-fold more efficient. The turnover rate of Dug1p for Cys-Gly dipeptide at pH 8.0 was found to be slightly lower (73.8 s⁻¹). In addition, we determined kinetic parameters of CN2 at pH 9.2 and found that the turnover rate was increased by 4-fold with no significant change in the K_m. Kinetic parameters obtained by the ESI–MS method are consistent with results of a reverse-phase high-performance liquid chromatography (RP–HPLC)-based assay. Furthermore, we used tandem MS (MS/MS) analyses to characterize carnosine and measured its levels in CHO cell lines in a time-dependent manner. The ESI–MS method developed here obviates the need for substrate modification and provides a less laborious, accurate, and rapid assay for studying kinetic properties of dipeptidases in vitro as well as in vivo.     

A method for the analysis of six thyroid hormones in thyroid gland by liquid chromatography–tandem mass spectrometry

Perchlorate can competitively inhibit iodide uptake by the thyroid gland (TG) via the sodium/iodide symporter, consequently reducing the production of thyroid hormones (THs). Until recently, the effects of perchlorate on TH homeostasis are being examined through measurement of serum levels of TH, by immunoassay (IA)-based methods. IA methods are fast, but for TH analysis, they are compromised by the lack of adequate specificity. Therefore, selective and sensitive methods for the analysis of THs in TG are needed, for assessment of the effects of perchlorate on TH homeostasis. In this study, we developed a method for the analysis of six THs: l-thyroxine (T₄), 3,3′,5-triiodo-l-thyronine (T₃), 3,3′,5′-triiodo-l-thyronine (rT₃), 3,5-diiodo-l-thyronine (3,5-T₂), 3,3′-diiodo-l-thyronine (3,3′-T₂), and 3-iodo-l-thyronine (3-T₁) in TG, using liquid chromatography (LC)–tandem mass spectrometry (MS/MS). TGs used in this study were from rats that had been placed on either iodide-deficient diet or iodide-sufficient diet, and that had either been provided with perchlorate in drinking water (10 mg/kg/day) or control water. TGs were extracted by pronase digestion and then analyzed by LC–MS/MS. The instrumental calibration range for each TH ranged from 1 to 200 ng/ml and showed a high linearity (r > 0.99). The method quantification limits (LOQs) were determined to be 0.25 ng/mg TG for 3-T₁; 0.33 ng/mg TG for 3,3′- and 3,5-T₂; and 0.52 ng/mg TG for rT₃, T₃, and T₄. Rats were placed on an iodide-deficient or -sufficient diet for 2.5 months, and for the last 2 weeks of that period were provided either perchlorate (10 mg/kg/day) in drinking water or control water. Iodide deficiency and perchlorate administration both reduced TG stores of rT₃, T₃, and T₄. In iodide-deficient rats, perchlorate exacerbated the reduction in levels of THs in TG. With the advances in analytical methodology, the use of LC–MS/MS for measurement of hormone levels in TG will allow more comprehensive evaluations of the hypothalamic-pituitary–thyroid axis.     

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.     

Histamine and tele-methylhistamine quantification in cerebrospinal fluid from narcoleptic subjects by liquid chromatography tandem mass spectrometry with precolumn derivatization

An ultra-performance liquid chromatography tandem mass spectrometry (UPLC™–MS/MS) assay was developed for the simultaneous analysis of histamine, its major metabolite tele-methylhistamine, and an internal standard (N-tele-(R)-α-dimethylhistamine) from human cerebrospinal fluid (CSF) samples. The method involves derivatization of primary amines with 4-bromobenzenesulfonyl chloride and subsequent analysis by reversed phase liquid chromatography with mass spectrometry detection and positive electrospray ionization. The separation of derivatized biogenic amines was achieved within 3.5 min on an Acquity® BEH C₁₈ column by elution with a linear gradient of acetonitrile/water/formic acid (0.1%). The assay was linear in the concentration range of 50–5000 pM for each amine (5.5–555 pg/ml for histamine and 6.25–625 pg/ml for tele-methylhistamine). For repeatability and precision determination, coefficients of variation (CVs) were less than 11.0% over the tested concentration ranges, within acceptance criteria. Thus, the developed method provides the rapid, easy, highly sensitive, and selective requirement to quantify these amines in human CSF. No significant difference was found in the mean ± standard error levels of these amines between a group of narcoleptic patients (histamine = 392 ± 64 pM, tele-methylhistamine = 2431 ± 461 pM, n = 7) and of neurological control subjects (histamine = 402 ± 72 pM, tele-methylhistamine = 2209 ± 463 pM, n = 32).     

Quantification of key folate forms in serum using stable-isotope dilution ultra performance liquid chromatography–tandem mass spectrometry

Folates act as essential coenzymes in many biological pathways. Alteration in folate form distribution might have biological significance, especially in relation to certain genetic polymorphisms. We developed a stable-isotope dilution ultra performance liquid chromatography–mass spectrometry (UPLC–MS/MS) method for quantification of the folate forms 5-methyltetrahydrofolate (5-methylTHF), 5-formylTHF, 5,10-methenylTHF, THF, and folic acid in serum. After extraction using an ion exchange and mixed mode solid-phase, samples were separated and detected using an UPLC–MS/MS system. The quantification limits were between 0.17 nmol/L (5-formylTHF) and 1.79 nmol/L (THF), and the assay was linear up to 100 nmol/L (5-methylTHF) and 10 nmol/L (5-formylTHF, 5,10-methenylTHF, THF, and folic acid). The intraassay CVs for 5-methylTHF and 5-formylTHF were 2.0% and 7.2%, respectively. Mean recoveries were between 82.3% for THF and 110.8% for 5,10-methenylTHF. Concentrations of total folate measured by the new method showed a strong correlation with those measured by an immunologic assay (r = 0.939; p < 0.001). The mean total folate from 32 apparently healthy subjects was 18.09 nmol/L, of which 87.23% was 5-methylTHF. Concentrations of homocysteine showed a better correlation to the total folate measured by the new method compared to that obtained by an immunologic assay. We also confirmed that MTHFR polymorphism has a significant effect on folate distribution in this small population of non-supplemented subjects.     

Quantitation of tetramethylene disulfotetramine in human urine using isotope dilution gas chromatography mass spectrometry (GC/MS and GC/MS/MS)

Tetramethylene disulfotetramine (tetramine) is a rodenticide associated with numerous poisonings was extracted and quantified in human urine using both gas chromatography/mass spectrometry (GC/MS) and GC/tandem mass spectrometry (MS/MS). 1200 μL samples were prepared using a ¹³C₄-labeled internal standard, a 96-well format, and a polydivinyl-benzene solid phase extraction sorbent bed. Relative extraction recovery was greater than 80% at 100 ng/mL. Following extraction, samples were preconcentrated by evaporation at 60 °C, and reconstituted in 50 μL acetonitrile. One-microliter was injected in a splitless mode on both instruments similarly equipped with 30 m × 0.25 mm × 25 μm, 5% phenyl-methylpolysiloxane gas chromatography columns. A quantification ion and a confirmation ion (GC/MS) or analogous selected reaction monitoring transitions (GC/MS/MS) were integrated for all reported results. The method was characterized for precision (5.92–13.4%) and accuracy (96.4–111%) using tetramine-enriched human urine pools between 5 and 250 ng/mL. The method limit of detection was calculated to be 2.34 and 3.87 ng/mL for GC/MS and GC/MS/MS, respectively. A reference range of 100 unexposed human urine samples was analyzed for potential endogenous interferences on both instruments—none were detected. Based on previous literature values for tetramine poisonings, this urinary method should be suitable for measuring low, moderate, and severe tetramine exposures.