Journal of Chromatography B (v.878, #25)

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.
Keywords: Ticagrelor; AZD6140; P2Y12 receptor inhibitor; Platelets; Incurred sample reproducibility; Incurred sample stability;

Accelerating high quality bioanalytical LC/MS/MS assays using fused-core columns by Ethan R. Badman; Richard L. Beardsley; Zhenmin Liang; Surendra Bansal (2307-2313).
High quality, ultra-fast bioanalytical LC/MS/MS methods were developed using short columns packed with fused-core particles and high (1.0–3.0 mL/min) flow rates. For more than two years, at flow rates up to 3.0 mL/min, using 0.33 min non-ballistic gradients, these methods were shown to provide comparable or better performance than slower assays for accuracy, precision, sensitivity, specificity, and ruggedness, and met all criteria required by the bioanalytical regulatory guidance.
Keywords: High throughput; Bioanalysis; LC/MS/MS; Quantitation; Fused-core column;

We characterized the two-dimensional electrophoretic patterns of fibrinogen chains α, β, and γ from the plasma of six animal species –Bos taurus, Canis familiaris, Equus caballus, Felis catus, Gallus domesticus and Sus scrofa. Comparing the spots resolved from serum and plasma samples, or exploiting the cross-reactivity of animal fibrinogen with an antiserum raised against the human protein could detect only some of the fibrinogen chains. Conversely, the analysis of the precipitate obtained by heating plasma for some minutes at 56 °C was adequate for the recognition of all fibrinogen chains in all samples. Physicochemical properties of the homologous proteins were found to extensively vary across species, with complete separation among the mapping areas for α, β and γ chains and maximal heterogeneity among β chains.
Keywords: Fibrinogen; Two-dimensional electrophoresis; Animal species; Serum/plasma;

BMS-708163 is a γ-secretase inhibitor that is being developed for the treatment of Alzheimer's disease. Several LC–MS/MS methods have been developed for the determination of BMS-708163 in both plasma and cerebrospinal fluid in support of dog, rat, mouse and human studies. To support non-clinical studies, an LC–MS/MS method with a lower limit of quantitation (LLOQ) of 5 ng/mL, was developed and validated in dog, rat, and mouse plasma by using the deprotonated ion as the precursor ion. To support clinical studies, an LC–MS/MS method with LLOQ of 0.1 ng/mL, was developed and validated in human plasma by using the formate adduct as the precursor ion. Formic acid (0.01%) in water and acetonitrile was found to be the most favorable mobile phases for both deprotonated and formate adduct ions in negative electrospray ionization mode. A combination of a 3M Empore™ C18 plate for SPE and a Waters Atlantis dC18 analytical column for separation was used to achieve a highly selective solid phase extraction and chromatographic procedure from plasma without dry down and reconstitution steps. In the development of an assay for BMS-708163 in cerebral spinal fluid (CSF), significant non-specific binding of BMS-708163 was observed and resolved with pre- or post-spike of 0.2% Tween 20 into CSF samples. A dilute-and-shoot LC–MS/MS method with LLOQ of 0.1 ng/mL was developed and validated to assess BMS-708163 exposure in human CSF.
Keywords: LC–MS; Fomate adduct; Non-specific binding;

Validated GC/MS method for the simultaneous determination of clozapine and norclozapine in human plasma. Application in psychiatric patients under clozapine treatment by I. Vardakou; A. Dona; C. Pistos; G. Alevisopoulos; S. Athanaselis; C. Maravelias; C. Spiliopoulou (2327-2332).
A sensitive and specific GC/MS method for the determination of clozapine (CLZ) and its major metabolite norclozapine (NCLZ), in plasma has been developed, optimized and validated. Specimen preparation includes solid-phase extraction of both analytes using Bond-Elut Certify cartridge and further derivatization with TFAA. Clozapine-d8 was used as internal standard for the determination of CLZ and NCLZ. Limits of detection were 0.45 ng/mL for CLZ and 1.59 ng/mL for NCLZ, while limits of quantification were 1.37 ng/mL for CLZ and 4.8 ng/mL for NCLZ, as calculated by the calibration curves. The calibration curves were linear up to 600 ng/mL for CLZ and NCLZ. Absolute recovery ranged from 82.22% to 95.35% for both analytes. Intra- and interday accuracy was less than 7.13% and −12.52%, respectively, while intra- and interday precision was between 9.47% and 12.07%, respectively, for CLZ and NCLZ. The method covers all therapeutic range and proved suitable for the determination of CLZ and NCLZ not only in psychiatric patients but also in forensic cases with clozapine implication.
Keywords: Clozapine; Norclozapine; GC/MS; Plasma; Patients;

Molecularly-imprinted polymers in the form of microspheres were synthesized using the dispersion polymerization protocol; cyromazine was used as dummy template, while methacrylic acid, ethylene glycol dimethacrylate and acetonitrile (MeCN) were used as functional monomer, cross-linker, and porogen, respectively. When compared with the non-imprinted polymer, the molecularly-imprinted polymers (MIPs) showed outstanding affinity toward melamine in MeCN with a maximum binding concentration (B max) of 53.20 nmol mg−1 MIPs, imprinting effect of 4.6, and a dissociation constant (K d) of 90.45 μM. After optimization of the molecularly-imprinted solid-phase extraction conditions, a new method was developed to determine the melamine in milk and feed with gas chromatography–mass spectrometry. The performance of this method has been evaluated in the tainted milk and feed in terms of recovery, precision, linearity, the limit of detection (LOD) and limit of quantitation (LOQ). Recovery ranged in samples from 93.1 to 101.3% with intra-day and inter-day relative standard deviation values below 5.34%. The LOD and LOQ of melamine in milk and feed were 0.01 μg mL−1 (μg g−1) and 0.05 μg mL−1 (μg g−1), respectively.
Keywords: Molecularly-imprinted polymers; Microsphere; Melamine; Solid-phase extraction; Gas chromatography–mass spectrometry;

A novel, simple and fast reversed-phase HPLC/UV method was developed, optimized for various chromatographic conditions, and validated according to international guidelines for simultaneous determination of all-trans-retinol and α-tocopherol in human serum using retinyl acetate as internal standard in the concentration of 0.5 μg/ml. A liquid-phase extraction was applied to the 250 μl of serum with n-hexane–dichloromethane mixture (70:30, v/v), in two steps, using ethanol–methanol mixture (95:5, v/v) for protein precipitation and BHT (butylated hydroxy toluene) as stabilizer for sample preparation. Both analytes were analyzed on Kromasil 100 C18 column (150 mm × 4.6 mm, 5 μm), Brownlee analytical (Perkin Elmer) C18 column (150 mm × 4.6 mm, 5 μm), and Supelco (Supelcosil) LC-18 column (150 mm × 3 mm, 3 μm), protected by a Perkin Elmer C18 (30 mm × 4.6 mm, 10 μm; Norwalk, USA) pre-column guard cartridge, at 292 nm wavelength, using methanol–water (99:1, v/v), in isocratic mode as mobile phase applied at flow rate of 1.5 ml/min and 1 ml/min for both 5 μm and 3 μm columns, respectively. Complete separation of all the analytes was achieved in 3 and 6 min on 3 μm and 5 μm columns, respectively by injecting 20 μl of sample into the HPLC system by autosampler, keeping column oven temperature at 25 °C. Different particulate reversed-phase chromatographic columns were evaluated in order to select the best column in terms of sensitivity, selectivity, resolution and short run time of both the analytes and it was concluded that 3 μm columns are better to be used in clinical set up as well as in laboratories for the separation of these analytes in a shorter time as compared with 5 μm columns. The method was validated and applied for the analysis of all-trans-retinol and α-tocopherol in the serum of human volunteers.
Keywords: Validation; All-trans-retinol; α-Tocopherol; Isocratic mode; Chromatographic columns; Resolution;

Determination of the γ-secretase inhibitor MK-0752 in human plasma by online extraction and electrospray tandem mass spectrometry (HTLC–ESI-MS/MS) by Feng Bai; Michael Tagen; Courtney Colotta; Laura Miller; Maryam Fouladi; Clinton F. Stewart (2348-2352).
A sensitive and rapid HTLC–ESI-MS/MS method with an advanced online sample preparation was developed for determination of the γ-secretase inhibitor MK-0752 in human plasma using an internal standard. Plasma samples (100 μL) were diluted and injected directly onto an online extraction column (Cohesive Cyclone MAX 0.5 mm × 50 mm, >30 μm), the sample matrix was washed out with an aqueous solution, and retained analytes were eluted out and transferred directly to the analytical column (Phenomenex Gemini 3μ C18 110A, 50 mm × 2.0 mm at 50 °C) for separation using a gradient mobile phase. The eluted analytes were then detected on an API-3000 LC–MS/MS System with ESI and a negative multiple reaction monitoring mode. The monitored ion transitions were m/z 441 → 175 for MK-0752 and 496 → 175 for the internal standard. Online extraction recoveries were 81%. The method was validated and was linear in the range of 0.05–50 μg/mL. Within-day and between-day precisions were < 8.6%, and accuracies were 0.7 and 7.1%. This method was applied to the measurement of plasma MK-0752 levels in a Phase I study of pediatric patients with recurrent or refractory brain tumors.
Keywords: MK-0752; Online sample preparation; High turbulence liquid chromatography (HTLC); Electrospray tandem mass spectrometry (ESI-MS/MS);

The development and validation of a bioanalytical assay is described for the simultaneous analysis of 2-amino-1-methyl-6-phenylimidazo[4-5-b]pyridine (PhIP) and its main metabolite 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) in plasma, urine, faeces, bile, liver, kidney, testis, spleen, brain, as well as colon-, cecum- and small intestinal tissue and contents from mice. The effect of the matrix on the accuracy of the method was extensively investigated. The bioanalytical assay is based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using multiple reaction monitoring for analyte quantification. The assay is validated from 1 to 250 ng/mL and the sample pretreatment consists of protein precipitation with acetonitrile using only 100 μL matrix (plasma, bile diluted in 4% (m/v) BSA, intestinal contents, faeces and tissue samples homogenized in 4% (m/v) BSA). The measured concentrations of PhIP and N-OH-PhIP in homogenates were expressed in ng/mL. Based on the weight of the isolated intestinal contents, faeces or tissue the amount of PhIP and N-OH-PhIP per mass unit intestinal content, faeces or tissue was calculated. The validated range for PhIP in urine is from 10 to 1000 ng/mL using 20 μL urine. For N-OH-PhIP quantification, mouse urine was diluted 100× in blank human urine to compensate for matrix effects. The developed method is simple, robust and reproducible. The applicability of the method was demonstrated and the assay could be successfully used to support in vivo toxicokinetics studies of PhIP and N-OH-PhIP in mice.
Keywords: Heterocyclic amines; Liquid chromatography; Mice; Matrix effects; Metabolites; N-OH-PhIP; PhIP; Tandem mass spectrometry; Tissue;

Measurement of urinary oxypurinol by high performance liquid chromatography–tandem mass spectrometry by Sophie L. Stocker; Michael E. Franklin; Jacqueline M. Anderson; Peter I. Pillans; Kenneth M. Williams; Andrew J. McLachlan; Richard O. Day; Paul J. Taylor (2363-2368).
Oxypurinol is the active metabolite of allopurinol which is used to treat hyperuricaemia associated with gout. Both oxypurinol and allopurinol inhibit xanthine oxidase which forms uric acid from xanthine and hypoxanthine. Plasma oxypurinol concentrations vary substantially between individuals and the source of this variability remains unclear. The aim of this study was to develop an HPLC-tandem mass spectrometry method to measure oxypurinol in urine to facilitate the study of the renal elimination of oxypurinol in patients with gout. Urine samples (50 μL) were prepared by dilution with a solution of acetonitrile/methanol/water (95/2/3, v/v; 2 mL) that contained the internal standard (8-methylxanthine; 1.5 mg/L), followed by centrifugation. An aliquot (2 μL) was injected. Chromatography was performed on an Atlantis HILIC Silica column (3 μm, 100 mm × 2.1 mm, Waters) at 30 °C, using a mobile phase comprised of acetonitrile/methanol/50 mM ammonium acetate in 0.2% formic acid (95/2/3, v/v). Using a flow rate of 0.35 mL/min, the analysis time was 6.0 min. Mass spectrometric detection was by selected reactant monitoring (oxypurinol: m/z 150.8 → 108.0; internal standard: m/z 164.9 → 121.8) in negative electrospray ionization mode. Calibration curves were prepared in drug-free urine across the range 10–200 mg/L and fitted using quadratic regression with a weighting factor of 1/x (r 2  > 0.997, n  = 7). Quality control samples (20, 80, 150 and 300 mg/L) were used to determine intra-day (n  = 5) and inter-day (n  = 7) accuracy and imprecision. The inter-day accuracy and imprecision was 96.1–104% and <11.2%, respectively. Urinary oxypurinol samples were stable when subjected to 3 freeze–thaw cycles and when stored at room temperature for up to 6 h. Samples collected from 10 patients, not receiving allopurinol therapy, were screened and showed no significant interferences. The method was suitable for the quantification of oxypurinol in the urine of patients (n  = 34) participating in a clinical trial to optimize therapy of gout with allopurinol.
Keywords: Oxypurinol; Urine; HPLC; Mass spectrometry; Gout;

A sensitive and selective liquid chromatography/tandem mass spectrometry method for determination of MLN8237 in human plasma by Emily Lipsitz; Ganesh Moorthy; Yael Mosse; Elizabeth Fox; Peter C. Adamson (2369-2373).
We describe a selective and a highly sensitive high-performance liquid chromatography–electron spray ionization-collision induced dissociation-tandem mass spectrometry (HPLC–ESI-CID-MS/MS) assay for the Aurora A kinase inhibitor MLN8237 in human plasma. The intra-day precision based on the standard deviation of replicates of quality control samples ranged from 0.2 to 4% and with accuracy ranging from 96 to 102%. The inter-day precision ranged from 0.5 to 7% and the accuracy ranged from 93 to 105%. Stability studies showed that MLN8237 was stable both during the expected conditions for sample preparation and storage. The lower limit of quantification for MLN8237 was 5 ng/mL. The analytical method showed excellent sensitivity, precision, and accuracy. This method is robust and is being successfully employed in a Children's Oncology Group Phase 1 Consortium study of MLN8237 in children with cancer.
Keywords: MLN8237; LC–ESI-CID-MS/MS; Plasma; Pediatric; Cancer;