Narrow-bore liquid chromatography-tandem mass spectrometry with simultaneous radioactivity monitoring for partially characterizing the biliary metabolites of an arginine fluoroalkyl ketone analog of d-MePhe-Pro-Arg, a potent thrombin inhibitor

Characterizing components eluting from a HPLC column is enhanced when multiple detectors are incorporated in-line. The performance of a system consisting of a combination of two detectors - electrospray ionization mass spectrometry (and tandem mass spectrometry) and radioactivity monitoring, following gradient separation with a 250×2.1mm I.D. (Vydac Protein and Peptide C₁₈, 5 μm, 300 Å) column - is evaluated with respect to chromatographic integrity and detection. The HPLC effluent was split (8:1) and a post-column make-up solvent was added to flow directed towards the radioactivity detector containing a solid glass cell. Trifluoracetic acid (0.1%) was added to the make-up flow solvent to prevent silanol interactions from degrading the profile displayed in the ¹⁴C trace. A ¹⁴C chromatographic peak representing 550 dpm was detected with signal-to-noise ratio of 3. This system was used for rapidly characterizing the biliary metabolites of an arginine fluoroalkyl ketone analog of d-MePhe-Pro-Arg, a potent thrombin inhibitor currently being evaluated as a drug candidate. These metabolites are shown to comprise of mono- and dihydroxylated drug as well as a reduced ketone form of the drug. Combining the radioactivity monitor in-line with the mass spectrometer ensured that all of the major metabolites (as evident from the ¹⁴C profile) were characterized by mass spectrometry.     

Determination of melagatran, a novel, direct thrombin inhibitor, in human plasma and urine by liquid chromatography-mass spectrometry

Analytical methods for the determination of melagatran (H 319/68) in biological samples by liquid chromatography (LC)-positive electrospray ionization mass spectrometry using multiple reaction monitoring are described. Melagatran in plasma was isolated by solid-phase extraction on octylsilica, either in separate extraction tubes or in 96-well plates. Absolute recovery of melagatran from plasma was >92%. Melagatran and the internal standard, H 319/68 D2 13C2, were separated from other sample components by LC utilizing a C18 stationary phase and a mobile phase comprising 35% acetonitrile and 0.08% formic acid in 0.0013 mol/l ammonium acetate solution. After dilution, urine was injected directly onto the LC column and subjected to gradient LC. The relative standard deviation was 1-5% for concentrations above the limit of quantification, which was estimated for plasma at 10 or 25 nmol/l for sample volumes of 500 or 200 microl, respectively, and 100 nmol/l for urine.     

Determination of ximelagatran,an oral direct thrombin inhibitor,its active metabolite melagatran,and the intermediate metabolites,in biological samples by liquid chromatography-mass spectrometry

Analytical methods for the determination of ximelagatran, an oral direct thrombin inhibitor, its active metabolite melagatran, and intermediate metabolites, melagatran hydroxyamidine and melagatran ethyl ester, in biological samples by liquid chromatography (LC) positive electrospray ionization mass spectrometry (MS) using selected reaction monitoring are described. Isolation from human plasma was achieved by solid-phase extraction on octylsilica. Analytes and isotope-labelled internal standards were separated by LC utilising a C(18) analytical column and a mobile phase comprising acetonitrile-4 mmol/l ammonium acetate (35:65, v/v) containing 0.1% formic acid, at a flow-rate of 0.75 ml/min. Absolute recovery was approximately 80% for ximelagatran, approximately 60% for melagatran ethyl ester and >90% for melagatran and melagatran hydroxyamidine. Limit of quantification was 10 nmol/l, with a relative standard deviation <20% for each analyte and <5% above 100 nmol/l. Procedures for determination of these analytes in human urine and breast milk, plus whole blood from rat and mouse are also described.     

Development of a liquid chromatography tandem mass spectrometry method for the simultaneous determination of zearalenone,deoxynivalenol and their metabolites in pig serum

A sensitive and selective liquid chromatography tandem mass spectrometry method using negative electrospray ionisation (LC-ESI-MS/MS) was developed for the simultaneous determination of zearalenone (ZEN), deoxynivalenol (DON) and their metabolites α-zearalenol, β-zearalenol, zearalanone, α-zearalanol, β-zearalanol and de-epoxy-deoxynivalenol in pig serum. For method development, different sample preparation columns were tested for their suitability for extraction and clean up. Finally, preparation of serum samples was carried out using Oasis? HLB solid-phase extraction (SPE) columns. The analyte concentrations were determined by the use of isotopically labelled internal standards (IS). The method was in-house validated for all analytes. Calibration graphs (0.3–480 ng/ml) were prepared and high degree of linearity was achieved (r?≥?0.99). Results for method precision ranged between 2.7 and 21.5 % for inter-day and between 1.1 and 11.1 % for intra-day. The recoveries were in the range of 82–131 %. Limits of detection and quantification ranged 0.03–0.71 and 0.08–2.37 ng/ml, respectively. The method has been successfully used for quantitative determination of ZEN, DON and their metabolites in pig serum from a feeding trial with practically relevant ZEN and DON concentrations. This method is precise and reproducible and can be used as a multi-biomarker method to assess animal exposure to these mycotoxins and for diagnosis of intoxications.     

Rapid method for the quantification of amoxicillin and its major metabolites in pig tissues by liquid chromatography-tandem mass spectrometry with emphasis on stability issues

A fast method for the quantitative determination of amoxicillin (AMO), amoxicilloic acid (AMA) and amoxicillin diketopiperazine-2',5'-dione (DIKETO) in pig edible tissues (kidney, liver, fat and muscle) with liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) is presented. The method uses a simple liquid-liquid extraction of the tissue matrix with a 10 mM potassium dihydrogen phosphate buffer (pH 4.5) as extraction solvent. After deproteinisation by ultrafiltration, the tissue extract was directly injected onto the LC column. Chromatographic separation of the components was performed on a PLRP-S polymeric column using 0.1% of formic acid in water and acetonitrile. The mass spectrometer was operated in the positive electrospray MS/MS mode. The method was fully validated according to EU requirements (linearity, precision, trueness, quantification limit, detection limit and specificity). The stability of the components was evaluated over the pH range from 1.2 to 8.0. Biological samples of pigs medicated with AMO and AMO/clavulanic acid were analyzed using the developed method.     

Validation of an analytical method for a potent antitumor agent, TZT-1027, in plasma using liquid chromatography–mass spectrometry

A sensitive and specific analytical method for a potent antitumor agent, TZT-1027, in plasma has been developed using liquid chromatography–mass spectrometry (LC–MS) with [²H₄]TZT-1027 as an internal standard (I.S.). A plasma sample was purified by solid-phase extraction on a C₁₈ cartridge, followed by solvent extraction with diethyl ether. The extract was then injected into the LC–MS system. Chromatography was carried out on a C₁₈ reversed-phase column using acetonitrile–0.05% trifluoroacetic acid (TFA) (55:45) as a mobile phase. Mass spectrometric analysis was performed in atmospheric pressure chemical ionization (APCI) mode with positive ion detection, and the protonated molecular ions ([M+H]⁺) of TZT-1027 and I.S. were monitored to allow quantitation. The method was applied to the determination of TZT-1027 in human, monkey, dog, rat and mouse plasma. As far as the sample preparation was concerned, good recoveries (73.5–99.1%) were obtained. The calibration curves were linear over the range of 0.25–100 ng per 1 ml of human, dog and rat plasma, per 0.5 ml of monkey plasma, and per 0.1 ml of mouse plasma. From the intra- and inter-day accuracy and precision, the present method satisfies the accepted criteria for bioanalytical method validation. TZT-1027 was stable when stored below −15°C for 6 months in human plasma and for 3 weeks in plasma from other species. TZT-1027 was also stable in plasma through at least three freeze–thaw cycles.     

Evaluation of electrospray ionisation liquid chromatography-tandem mass spectrometry for rational determination of a number of neuroleptics and their major metabolites in human body fluids and tissues

A study of liquid chromatography-triple quadrupole mass spectrometry (LC-MS-MS) with positive electrospray ionisation (ESI) for the determination of selected drugs in human tissues and body fluids such as blood, urine and hair is described. The possibility to screen for and quantify the 19 most commonly prescribed neuroleptics on the Swedish market and determine the presence of their major metabolites within a single LC-MS-MS analysis was evaluated on a PE Sciex API2000 instrument. Chromatographic conditions were optimised and the best separation, with individual retention times for most of the analytes, was obtained on a Zorbax SB-CN column within a 9-min gradient run. The MS-MS fragmentation conditions were optimised for each compound in order to obtain both specific fragments and high signal intensity. Since neuroleptics are a heterogeneous group of compounds, a markedly difference in collision energy needed to achieve fragments of the selected parent ions was seen and the number of fragments achieved varied as well. For sensitive quantification the transition of the most intense fragment of the protonated molecular ion (M+1)(+) was selected for multiple reaction monitoring analysis. More than 70 transitions were finally included in the assay. Detection levels down to the lower ng/ml level were achieved for all analytes, but between analytes more than a 10-fold difference in signal response was seen. By evaluation of extracted ion chromatograms from the analysis of authentic human blood, urine and hair sample the proposed concept for rational drug analysis was found to be both selective and sensitive for the neuroleptics included. A great number of metabolites could be determined in blood, urine and hair as well. A full method validation was not performed since the objective was to evaluate the method design rather than to validate a final method set-up.     

Development and validation of a liquid chromatography-tandem mass spectrometry for the determination of Kendine 91, a novel histone deacetylase inhibitor, in mice plasma and tissues: application to a pharmacokinetic study

A liquid chromatography-tandem mass spectrometric method (LC-MS/MS) has been developed and validated to determine the concentration of Kendine 91 in mice plasma and tissues. Simvastatin was employed as the internal standard. Separation was performed on a C8 column, with a mobile phase consisting of methanol and aqueous 10 mM formic acid (73:27 v/v). Both analyte and internal standard were determined using electrospray ionization and the MS data acquisition was via multiple-reaction monitoring (MRM) in positive scanning mode. Quantification was performed using the transitions m/z 444-->169 and 441-->325 for Kendine 91 and simvastatin, respectively. The method was validated with respect to linearity, accuracy, precision, recovery and stability. This assay has been successfully applied to a pharmacokinetic study after intravenous injection of Kendine 91 in mice in a dose of 10mg/kg.     

Development and validation of a liquid chromatography-tandem mass spectrometry for the determination of BPR0L075, a novel antimicrotuble agent, in rat plasma: application to a pharmacokinetic study

A rapid and sensitive liquid chromatography-tandem mass spectrometric method (LC-MS/MS) had been developed and validated to determine the concentrations of BPR0L075 in rat plasma. After a simple protein precipitation of plasma samples by acetonitrile, BPR0L075 was analyzed on a C(8) column at a flow rate of 0.5 mL/min. The mobile phase consisted of a mixture of 10 mM ammonium acetate containing 0.1% formic acid and acetonitrile (20:80, v/v). Both BPR0L075 (analyte) and the internal standard (BPR0L092) were determined using electro-spray ionization and the MS data acquisition was via multiple reactions monitoring (MRM) in positive scanning model. The MS/MS ion transitions monitored are m/z 342.2/195.2 and 312.5/165.2 for BPR0L075 and BPR0L092, respectively. The low limit of quantitation was 0.5 ng/mL. Each plasma sample was chromatographed within 5 min. The method was validated with respect to linearity, accuracy, precision, recovery, and stability. A good linear relationship was observed over the concentration range of 0.5-1000 ng/mL (r>0.9994). Absolute recoveries ranged from 63.45 to 68.34% in plasma at the concentrations of 2, 40, 400, and 800 ng/mL. The intra- and inter-day accuracy ranged from 92.04 to 111.80%. Intra- and inter-day relative standard deviations were 1.08-3.29% and 1.96-5.46%, respectively. This developed and validated assay method had been successfully applied to a pharmacokinetic study after intravenous injection of BPR0L075 in rats at a dose of 5mg/kg.     

Design,synthesis, and SAR of a series of activated protein C (APC) inhibitors with selectivity against thrombin for the treatment of haemophilia

A design strategy was used to identify inhibitors of activated protein C with selectivity over thrombin featured by a basic and/or aromatic functionality for binding to the S2 pocket. Our strongest inhibitor showed an IC₅₀-material value and selectivity for APC vs thrombin similar to a compound previously reported in the literature. However, in contrast to the reference compound, our compound showed a retained coagulant effect of thrombin with increasing substrate concentration in a modified Calibrated Automated Thrombogram (CAT) method. This was likely related to our compound being inactive against FVIIa, while the reference compound showed an IC₅₀ of 8.9 μM. Thus, the higher selectivity of our compound against all relevant coagulation factors likely explained its higher therapeutic potential in comparison to the reference compound. The data indicate that at least a 100-fold selectivity over other serine proteases in the coagulation cascade will be required for an effective APC inhibitor.     

Quantification of urinary o,o'-dityrosine, a biomarker for oxidative damage to proteins, by high performance liquid chromatography with triple quadrupole tandem mass spectrometry. A comparison with ion-trap tandem mass spectrometry

We recently described an isotope dilution reversed-phase liquid chromatography-atmospheric pressure chemical ionization-ion-trap-tandem mass spectrometry (HPLC-APCI-MS/MS) method for the quantitative determination of oxidized amino acids in human urine, including o,o'-dityrosine, a specific marker of protein oxidation. In the present study, we investigated the possibility to use a triple quadrupole instrument for the analysis of this biomarker in urine. The two instruments were compared in terms of sensitivity, specificity and reproducibility. Results showed that the triple quadrupole instrument reaches 2.5-fold higher sensitivity (LOD=0.01 microM) compared to the previously used ion-trap instrument. Precision of the present assay is as follows: in-day variation is 4.6% and inter-day variation is 17%. The currently developed method was applied to a group of smoker urine samples. The mean urinary o,o'-dityrosine concentration was 0.08+/-0.01 microM. Expressed per urinary creatinine concentration, this corresponds to 10.1+/-0.4 micromol/mol creatinine. This is comparable to the previously reported values of 5.8+/-0.3 micromol/mol creatinine in non-smokers night-time urines, and 12.3+/-5 micromol/mol creatinine in day-time urines measured by the ion-trap instrument.     

Use of short high-performance liquid chromatography columns and tandem-mass spectrometry for the rapid analysis of a prostaglandin analog, fluprostenol, in rat plasma

A short reversed-phase HPLC column and a tandem mass spectrometer were used to develop a stable-isotope-dilution assay for the rapid and sensitive analysis of fluprostenol, a prostaglandin analog, in rat plasma. A Waters Symmetry ODS column (2.1×10 mm) afforded rapid isocratic elution of fluprostenol (t_R=40 s) but still provided a relatively large k′ value of 4. The use of tandem mass spectrometry allowed the interference-free detection of fluprostenol under the rapid elution conditions, with a limit of quantitation of 25 pg ml⁻¹ fluprostenol, using 0.2 ml plasma sample volumes. The method was linear over three orders of magnitude, yielded accurate and precise results and allowed the pharmacokinetic profile of fluprostenol to be defined following intravenous administration in rats.     

Development of a quantification method for digoxin, a typical P-glycoprotein probe in clinical and non-clinical studies, using high performance liquid chromatography-tandem mass spectrometry: the usefulness of negative ionization mode to avoid competitive adduct-ion formation

Highly sensitive and accurate liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods have been developed and validated for measuring digoxin (DGX), a typical P-glycoprotein probe, in human plasma, rat plasma, and rat brain. We extracted DGX and deuterium-labeled DGX (as internal standard) from sample fluids under basic conditions using acetonitrile and sodium chloride-saturated 0.1 mol/L sodium hydroxide. The upper organic layer was diluted with distilled water, and the resulting solution was injected into an LC/MS/MS system in negative ionization mode. Chromatographic separation was achieved on a C(18)-ODS column in the gradient mobile phase, which comprised 0.05% (w/v) ammonium carbonate (pH 9.0) and methanol at a flow rate of 0.7 mL/min. Regardless of the type of biological matrix, intra-day and inter-day validation tests demonstrated good linearity of calibration curves within ranges of 0.1-10 ng/mL for plasma and 0.5-50 ng/g for rat brain and gave excellent accuracy and precision of quality control samples at 4 concentration levels. Unlike existing methods, our approach uses negative ionization to avoid competitive adduct formation of DGX. Our method showed higher sensitivity and wider applicability to various types of biological matrices than existing methods. Our method will support clinical and preclinical investigation of in vivo P-glycoprotein functionality using DGX.     

Simultaneous assay of morphine, morphine-3-glucuronide and morphine-6-glucuronide in human plasma using normal-phase liquid chromatography–tandem mass spectrometry with a silica column and an aqueous organic mobile phase

Morphine (MOR) is an opioid analgesic used for the treatment of moderate to severe pain. MOR is extensively metabolized to morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). A rapid and sensitive method that was able to reliably detect at least 0.5 ng/ml of MOR and 1.0 ng/ml of M6G was required to define their pharmacokinetic profiles. An LC–MS–MS method was developed in our laboratory to quantify all three analytes with the required sensitivity and a rapid turnaround time. A solid-phase extraction (SPE) was used to isolate MOR, M3G, M6G, and their corresponding deuterated internal standards from heparinized plasma. The extract was injected on a LC tandem mass spectrometer with a turbo ion-spray interface. Baseline chromatographic separation among MOR, M3G, and M6G peaks was achieved on a silica column with an aqueous organic mobile phase consisting of formic acid, water, and acetonitrile. The total chromatographic run time was 3 min per injection, with retention times of 1.5, 1.9 and 2.4 min for MOR, M6G, and M3G, respectively. Chromatographic separation of M3G and M6G from MOR was paramount in establishing the LC–MS–MS method selectivity because of fragmentation of M3G and M6G to MOR at the LC–MS interface. The standard curve range in plasma was 0.5–50 ng/ml for MOR, 1.0–100 ng/ml for M6G, and 10–1000 ng/ml for M3G. The inter-day precision and accuracy of the quality control (QC) samples were <7% relative standard deviation (RSD) and <6% relative error (R.E.) for MOR, <9% RSD and <5% R.E. for M6G, and <3% RSD and <6% R.E. for M3G. Analyte stability during sample processing and storage were established. Method ruggedness was demonstrated by the reproducible performance from multiple analysts using several LC–MS–MS systems to analyze over one thousand samples from clinical trials.     

High performance liquid chromatography with ultraviolet detection for the determination of SYUIQ-5, a novel telomerase inhibitor for cancer therapy: application to an enzyme kinetic study in rat liver microsomes

A sensitive assay for the determination of SYUIQ-5, a novel telomerase inhibitor and anti-tumor drug, in rat liver microsomes was developed by using high-performance liquid chromatography with ultraviolet detection. SYUIQ-5 was incubated in vitro with liver microsomes from rats pre-treated with control vehicle, beta-naphthofIavone, phenobarbital, 20% ethanol or dexamethasone. The analytes were extracted with diethyl ether and separated a C(18) 5-microm analytical column. Elution was conducted with 30 mM dipotassium hydrogen phosphate (pH 8.0)-methanol-triethylamine (30:70:0.05, v/v/v) at a flow-rate of 1.0 ml/min and the detection of UV absorbance was conducted at 278 nm. Intra-day and inter-day precision and accuracy of the method were within 10%. The mean analytical recoveries of SYUIQ-5 ranged from 78.8 to 95.3%. The linearity of the calibration curve was in the range of 1.0-80.0 microM. The lower limit of quantification (LOQ) was 1.0 microM. Kinetic analysis showed that beta-naphthofIavone and dexamethasone significantly induced SYUIQ-5 metabolism, suggesting that cytochrome P450 1A and 3A are the major contributor to SYUIQ-5 metabolism in rat liver microsomes.