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This study aimed to systematically explore compositional effects for a series of lipid systems, on the in vitro drug solubilization and in vivo bioavailability of three poorly water-soluble drugs with different physico-chemical properties. While many lipid-based drug products have successfully reached the market, there is still a level of uncertainty on the design guidelines for such drug products with limited understanding on the influence of composition on in vitro and in vivo performance. Lipid-based drug delivery systems were prepared using either single excipient systems based on partially digested triglycerides (i.e. mono- and/or di-glycerides) or increasingly complex systems by incorporating surfactants and/or triglycerides. These lipid systems were evaluated for both in vitro and in vivo behaviour. Results indicated that simple single component long chain lipid systems are more beneficial for the absorption of the weak acid celecoxib and the weak base cinnarizine compared to equivalent single component medium chain lipid systems. Similarly, a two-component system produced by incorporating small amount of hydrophilic surfactant yields similar overall pharmacokinetic effects. The lipid drug delivery systems based on medium chain lipid excipients improved the in vivo exposure of the neutral drug JNJ-2A. The higher in vivo bioavailability of long chain lipid systems compared to medium chain lipid systems was in agreement with in vitro dilution and dispersion studies for celecoxib and cinnarizine. The present study demonstrated the benefits of using mono-/di-glycerides as single component excipients in LBDDS to streamline formulation screening and improve oral bioavailability for the three tested poorly water-soluble drugs.

Rationale In liquid chromatography/mass spectrometry (LC/MS) the LC flow is often split prior to the mass spectrometer, for instance, when collecting fractions of the separated sample for other purposes or when less sensitive parallel detection is applied. The aim of this study is to optimize the actual split ratio and make-up flow composition. Methods Different types of splitters were evaluated in combination with a make-up flow. A home-made 1/10 T-piece splitter and commercial 1/10, 1/100 and 1/250 splitters were evaluated by continuous and accurate measurements of the actual split ratio throughout the LC gradient. The make-up flow composition was optimized for maximum electrospray ionization (ESI)-MS sensitivity in the positive mode using ESI efficiency measurements. Results Altogether 22 different solvent conditions were tested on 20 pharmaceutical compounds with a wide variety of functional groups and physicochemical properties (molecular weight, logP, and pKa ). Methanol/10 mM formic acid in water (90/10) provided on average the best results. Conclusions Methanol/10 mM formic acid in water (90/10) proved to be the best make-up flow composition in relation to the average sensitivity obtained. Stronger acidic conditions using oxalic acid or higher formic acid concentrations had a clear positive effect on the sensitivity of compounds with low ionization efficiency. The tested split ratios were relatively stable over the main part of the gradient but showed some variation at very low and very high organic conditions. Differences were larger with methanol compared with acetonitrile containing solvent compositions and when applied without a column or with very long connecting tubing.

Chemical derivatization allows the introduction of an “ICP-MS accessible” element into pharmaceuticals and their metabolites, thus extending the application range of HPLC-ICP-MS in pharmaceutical applications. In this study, such a pre-column derivatization approach was developed with the aim of using HPLC-ICP-MS for the quantitative metabolite profiling of pharmaceutical drugs in human plasma samples. We targeted the amino and hydroxyl groups, two of the most abundant functional groups present in drugs, and used 4-aminopyridine and 3-hydroxy-4-aminopyridine as model compounds. 4-Iodobenzoyl chloride was used as a commercially available and multi-functional derivatization reagent to introduce iodine (I) as an element that can be quantified using ICP-MS with high sensitivity. This reagent reacts readily with the amino group present in 4-aminopyridine and the amino and hydroxyl groups present in its metabolite 3-hydroxy-4-aminopyridine. The reaction conditions were systematically optimized to guarantee quantitative formation of the target derivatives. Baseline separation of the target derivatives from one another and from also derivatized endogenous compounds was achieved using reversed phase UHPLC and gradient elution. Accuracy (recovery between 95 and 107%) and precision (repeatability ≤ 6.9% RSD) were fit-for-purpose for both 4-aminopyridine and 3-hydroxy-4-aminopyridine. The limit of quantification (LOQ) is 10 μg L−1 for I, corresponding to 7.5 μg L−1 of 4-aminopyridine and 5.8 μg L−1 of 3-hydroxy-4-aminopyridine, respectively.

The development of suitable analytical methods for drug ADME (absorption, distribution, metabolism and excretion) studies is of great importance. The currently routinely applied detection techniques usually demonstrate a structure-dependent analytical response (MS-based method) or require the synthesis of a radiolabelled version of the parent drug (radiodetection) for accurate quantification. Inductively coupled plasma-(tandem) mass spectrometry (ICP-MS(/MS)) offers a promising alternative to radiolabelling followed by radiodetection due to the structure-independent nature of its analytical response. Within the context of this study, an accurate, simple and sensitive HPLC-ICP-MS/MS method for the quantitative metabolite profiling of diclofenac in human plasma based on the pre-column derivatization of the carboxylic and phenolic –OH groups present in the parent drug and its major metabolite, 4′-hydroxy-diclofenac, was developed and validated. A cost-effective and commercially available derivatization reagent, p-bromophenacyl bromide (p-BPB), was applied for the introduction of Br into the drug molecule and its major metabolite, enabling the element-selective detection and quantification based on the Br-signal. The presence of Cl in both diclofenac and 4′-hydroxy-diclofenac allowed an additional validation via simultaneous monitoring of the Cl-signal by using a state-of-art ICP-MS/MS instrument equipped with a collision/reaction cell. The reaction conditions were successfully optimized to achieve a quantitative formation of the corresponding derivatization products, while the baseline separation of the target compounds in a typical biological matrix (i.e. human plasma) was achieved using gradient reversed phase high-performance liquid chromatography (RP-HPLC). A fit-for-purpose accuracy (recovery between 85–115%) and precision (repeatability ≤7.2% RSD) were achieved. The limits of quantification (LOQ) are ≈50 μg L−1 for Br and ≈80 μg L−1 for Cl, corresponding to ≈0.2 mg L−1 and ≈0.4 mg L−1 of diclofenac, respectively.

Lack of knowledge on the expected concentration range or insufficient linear dynamic range of the analytical method applied are common challenges for the analytical scientist. Samples that are above the upper limit of quantification are typically diluted and reanalyzed. The analysis of undiluted highly concentrated samples can cause contamination of the system, while the dilution step is time consuming and as the case for any sample preparation step, also potentially leads to precipitation, adsorption or degradation of the analytes. ispartof: Journal of Chromatography A vol:1524 pages:101-107 ispartof: location:Netherlands status: published

Sensitivity is often a critical parameter in quantitative bioanalyses in drug development. For liquid-chromatography-based methods, sensitivity can be improved by reducing the column diameter, but practical sensitivity gains are limited by the reduced sample loading capacity on small internal diameter (I.D.) columns. We developed a set-up that has overcome these limitations in sample loading capacity. The set-up uses 4 columns with gradually decreasing column diameters along the flow-path (2.1 → 1 → 0.5 → 0.15 mm). Samples are pre-concentrated on-line on a 2.1 mm I.D. trapping column and back flushed to a 1 mm I.D. UHPLC analytical column and separated. The peak(s) of interest are transferred using heartcutting to a second trapping column (0.5 mm I.D.), which is back-flushed to a 0.15 mm I.D. micro-UHPLC analytical column for orthogonal separation. The proof of concept of the set-up was demonstrated by the simultaneous analysis of midazolam and 1'-hydroxy midazolam in plasma by injection of 80 μL of protein precipitated plasma. The 4-column funnel set-up proved to be robust and resulted in a 10-50 times better sensitivity compared to a trap-elute approach and 250-500 fold better compared to direct micro-UHPLC analysis. A lower limit of quantification of 100 fg/mL in plasma was obtained for both probe compounds.

High-performance liquid chromatography was used in combination with infrared ion spectroscopy for the identification of positional isomers of hydroxy-atorvastatins, the primary metabolites of the drug atorvastatin. The results demonstrate the direct applicability of infrared ion spectroscopy in the field of drug metabolism and, more generally, its promising role in state-of-the-art analytical laboratories for the identification of small molecules buried in complex mixtures. In combination with chromatographic separation, infrared spectroscopy of mass-selected ions provides a promising new route for the identification of the molecular structures of unknown m/z peaks in a mass spectrum. We demonstrate that currently existing experimental protocols allow the measurement of an IR spectrum from less than 10 ng of sample obtained in a collected HPLC fraction.

Eicosanoids, including prostaglandins and thromboxanes are lipid mediators synthetized from polyunsaturated fatty acids. They play an important role in cell signaling and are often reported as inflammatory markers. LC-MS/MS is the technique of choice for the analysis of these compounds, often in combination with advanced sample preparation techniques. Here we report a head to head comparison between an electrospray ionization source (ESI) and a new atmospheric pressure ionization source (UniSpray). The performance of both interfaces was evaluated in various matrices such as human plasma, pig colon and mouse colon. The UniSpray source shows an increase in method sensitivity up to a factor 5. Equivalent to better linearity and repeatability on various matrices as well as an increase in signal intensity were observed in comparison to ESI. publisher: Elsevier articletitle: Enhanced performance for the analysis of prostaglandins and thromboxanes by liquid chromatography-tandem mass spectrometry using a new atmospheric pressure ionization source journaltitle: Journal of Chromatography A articlelink: http://dx.doi.org/10.1016/j.chroma.2016.02.055 content_type: article copyright: Copyright © 2016 Elsevier B.V. All rights reserved. ispartof: Journal of Chromatography A vol:1440 pages:260-5 ispartof: location:Netherlands status: published

The application of two-dimensional liquid chromatography (2D-LC) is gradually growing also in the area of metabolite profiling and identification. The current contribution describes a heartcut 2D-UHPLC configuration that is applied in support of drug metabolism studies in development. The setup applies four LC columns: two analytical UHPLC columns to perform the first and second dimension separations, which are both preceded by a short HPLC column operated as trapping column. The first HPLC column allows a significant online preconcentration by large volume injection. The second short HPLC column is placed between the first and second dimension columns and enables the selection of orthogonal conditions in the second dimension independent of the first dimension making the heartcutting 2D approach more generic. The value of the setup was demonstrated with selective ultraviolet chromatograms obtained for the two major hydroxylated metabolites of atorvastatin separating them from a very high biological background, originating from an injection of 4 mL feces extract, by heartcut 2D-LC. In a second application, the main metabolite of imipramine was baseline separated from some minor metabolites that were co-eluting in the first dimension, allowing accurate and sensitive quantification. A quantification limit in the attogram/mL range was achieved thanks to the injection of 200 mL diluted urine, corresponding to 100 mL urine on column.

High-throughput simultaneous quantitative and qualitative (Quan/Qual) analysis is attractive to combine targeted with non-targeted analysis, e.g. in pharmacometabolomics and drug metabolism studies. This study aimed to investigate the possibilities and limitations of high-throughput Quan/Qual analysis by ultra-high performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS), to develop a widely applicable Quan/Qual UHPLC-HRMS method and to provide recommendations for Quan/Qual method development. A widely applicable 4.25-min UHPLC method for small-molecules was used to investigate and optimize mass spectrometric parameters of a Synapt G2S for Quan/Qual analysis. The method was applied on a rat metabolomics study investigating the effect of the fasting state and administration of a dosing vehicle on the rat plasma metabolic profile. scan mode with a short scan time of 30 ms. This 4.25-min Quan/Qual analysis method enabled quantification with accuracy and precision values ≤ 20% at the lowest quality control (QC) level and ≤15% at higher QC levels for 16 out of 19 tested analytes. It provided both parent m/z values and fragmentation spectra for compound identification with limited loss of chromatographic resolution and it revealed biologically relevant metabolites in its application to the metabolomics study. Quan/Qual method development requires balancing between the amount of qualitative data, the quality of the quantitative data and the analysis time. Recommendations are provided for MS resolution, scan mode, scan rate, smoothing and peak integration in Quan/Qual method development and analysis. BACKGROUND METHODS RESULTS CONCLUSION

Structural elucidation of metabolites is an important part during the discovery and development process of new pharmaceutical drugs. Liquid Chromatography (LC) in combination with Mass Spectrometry (MS) is usually the technique of choice for structural identification but cannot always provide precise structural identification of the studied metabolite (e.g. site of hydroxylation and site of glucuronidation). In order to identify those metabolites, different approaches are used combined with MS data including nuclear magnetic resonance, hydrogen/deuterium exchange and chemical derivatization followed by LC-MS. Those techniques are often time-consuming and/or require extra sample pre-treatment. In this paper, a fast and easy to set up tool using desorption electrospray ionization-MS for metabolite identification is presented. In the developed method, analytes in solution are simply dried on a glass plate with printed Teflon spots and then a single drop of derivatization mixture is added. Once the spot is dried, the derivatized compound is analyzed. Six classic chemical derivatizations were adjusted to work as a one drop reaction and applied on a list of compounds with relevant functional groups. Subsequently, two successive reactions on a single spot of amoxicillin were tested and the methodology described was successfully applied on an in vitro incubated alprazolam metabolite. All reactions and analyses were performed within an hour and gave useful structural information by derivatizing functional groups, making the method a time-saving and efficient tool for metabolite identification if used in addition or in some cases as an alternative to common methods.

The type and design of an ionization source can have a significant influence on the performances of a bioanalytical method. It is, therefore, of high interest to evaluate the performances of newly introduced sources to highlight their benefits and limitations in comparison to other well established sources. In this paper, liquid chromatography - mass spectrometry (LC/MS) performances of a new atmospheric pressure ionization (API) source, commercialized as UniSpray, is evaluated. The dynamic range of 24 pharmaceutical and biological compounds is compared between the new API source and electrospray ionization (ESI) for 3 different mobile phase conditions. Matrix effects are also compared with ESI on a refined selection of 19 pharmaceutical and biological compounds in 4 matrices commonly encountered in bioanalysis. A slightly better dynamic range towards lower concentrations was often observed with the new API source. Matrix effects were quite similar between the two sources with a small, but statistically significant, lower percentage of matrix effects observed for the new API source in plasma and bile in the positive ion mode, and bile in negative ion mode for ESI. Finally, the sensitivity of late eluting compounds could be improved on the new API source by post-column addition of water.

A novel quantification approach, compensating for the effect of gradient elution on the instrumental response during reversed phase high-performance liquid chromatography - inductively coupled plasma - tandem mass spectrometry analysis, has been developed and validated. As a proof of concept, diclofenac and its related compounds, including its major metabolite i.e. 4′-hydroxy-diclofenac, have been quantitatively determined in human plasma matrix. An inductively coupled plasma - tandem mass spectrometer has been applied for the interference-free determination of Cl as 35ClH2+ using H2 as a reaction gas in the collision/reaction cell. The effect of the eluent composition on the instrumental response for Cl has been thoroughly investigated for the most common organic solvents in reversed phase high-performance liquid chromatography, i.e. methanol and acetonitrile. A proper mathematical function describing the effect of the eluent composition on the sensitivity for Cl, monitored as 35ClH2+, permitted adequate correction for the otherwise detrimental effect of gradient elution for both solvents. Validation using synthetically degraded diclofenac samples spiked with its major metabolite, 4′-hydroxy-diclofenac, demonstrated appropriate accuracy (recovery for 4′-hydroxy-diclofenac between 95 and 105%) and >90% and >80% recovery for Cl using acetonitrile and methanol, respectively. When applied to spiked human plasma samples (the most important matrix in drug metabolism studies), a satisfactory accuracy (recovery of 92–98%) and precision (

An online preconcentration approach was developed allowing the injection of very high volumes of biological samples, thereby greatly increasing sensitivity while maintaining LC resolution. The approach was applied to the analysis of radioactive samples from both in vitro and in vivo metabolism studies where typically the concentration of radioactivity given is often limited, while sample volume is usually not. The described online preconcentration approach reduces sample preparation and, therefore, also the risk for degradation and recovery issues often seen with offline preconcentration methods. In addition to facilitating the identification and profiling of low level metabolites within a sample, the described approach also provides robust quantitative analysis of samples derived from a range of biological matrices. The application of this approach is illustrated on real life samples from different matrices and containing drugs and metabolites with a wide variety in polarity, more specifically the analysis of extracts derived from an in vitro hepatocyte incubation, 36mL of blood/acetonitrile (1/1, v/v; 28dpm/mL) and 72mL of urine/methanol (9/1, v/v; 208dpm/mL).

Janssen Research & Development, a Division of Janssen Pharmaceutica N.V., Belgium The role of MS in peptide quantification For quantitative analysis of small-molecule drugs, LC–MS is considered the gold standard. However, focusing on peptide drugs, the majority of peptide development programs still rely on immunoassays for evaluation of exposure of the peptide drug in study samples. The advantages of these assays are the unrivaled sensitivity and the high throughput. Selectivity issues and method development times are, on the other hand, often rate-limiting. LC–MS technology can offer an alternative to improve selectivity and reduce method development time (no reagent preparation needed), but at the expense of lower sensitivity and throughput. Therefore, in our portfolio, for early peptide drug programs, quantitative workflows with LC–MS are preferred to address the short cycle times requested at this stage. In development and also with the aim to evaluate anti-drug antibody response, immunological assays are primarily preferred. An LC–MS assay can then be requested as an orthogonal assay to confirm the selectivity of the immunoassay or to address specific issues. In rare cases, the immunological assay is confronted with considerable challenges and LC–MS(/MS) can be developed faster and with more confidence to support the program.

Quantitative determination of the candidate drug molecule and its metabolites in biofluids and tissues is an inevitable step in the development of new pharmaceuticals. Because of the time-consuming and expensive nature of the current standard technique for quantitative metabolite profiling, i.e., radiolabeling followed by high-performance liquid chromatography (HPLC) with radiodetection, the development of alternative methodologies is of great interest. In this work, a simple, fast, sensitive, and accurate method for the quantitative metabolite profiling of an amino group containing drug (levothyroxine) and its metabolites in human plasma, based on precolumn derivatization followed by HPLC-inductively coupled plasma mass spectrometry (ICPMS), was developed and validated. To introduce a suitable “heteroelement” (defined here as an element that is detectable with ICPMS), an inexpensive and commercially available reagent, tetrabromophthalic anhydride (TBPA) was used for the derivatization of free NH2-groups....

Background: Peptides represent a growing class of potential drugs. Information on metabolic clearance can be valuable for peptide drug development but different challenges are encountered with the identification of peptide metabolites in comparison to the process used for small-molecule therapeutics. Results: Enfuvirtide was selected as a test compound and dosed intravenously at 2 mg/kg to rats. Plasma samples were collected and analyzed on two different quadrupole-TOF instruments in positive and negative ion modes. Different post-acquisition processing tools were evaluated to identify the metabolites of a peptide drug in the presence of an in vivo matrix. Charge state filtering and ion mobility extraction were applied to reduce the matrix background and combined with more comprehensive software tools generally used for large molecule analyses as well as tools designed for small-molecule metabolite identification work. Conclusion: Both ion mobility spectrometry and charge state filtration proved to be successful in extracting peptide ions and significantly reducing background signals. Both small- and large-molecule software tools contain specific capabilities that could be usefully combined in a single package for peptide metabolite identification.

Background: There is considerable interest in the determination of relative abundances of human metabolites in plasma (and potentially excreta) with reasonable accuracy early on in the drug development process in order to make scientifically sound decisions with regard to the presence of potentially active or toxic disproportionate metabolites. At this point, authentic metabolite standards are generally not available. Results: A new methodology is proposed for the estimation of metabolite concentrations in the absence of authentic standards. A reference sample containing radiolabeled metabolites of interest is produced by incubating the 14C-labeled drug in vitro, and mixed with a sample to be quantitated containing the unlabeled metabolites. The 12C/14C isotope ratio is measured with high-resolution ESI–MS for each metabolite, and used as a basis for quantitation of the cold metabolite based on the concentration of radioactive metabolite, determined from independent analysis of the radioactive sample with LC-radiochemical detection. The 14C-labeled metabolite serves as an isotopically labeled internal standard, which corrects for any variations in injection volume, sample preparation, MS intensity drift, matrix effects and/or saturation of electrospray ionization. The approach was validated by the analysis of solutions containing variable amounts of the analyte with a fixed amount of radioactive standard on a QToF Synapt® G2 MS system. The same methodology was also successfully applied to first-in-human plasma samples analyzed on a LTQ-Orbitrap®. Conclusion: The metabolite abundances obtained by 12C/14C isotope ratio measurements showed suitable accuracy and precision and were very close to those obtained with matrix mixing. The parent drug concentrations also corresponded well with the bioanalytical results obtained with a validated LC–MS/MS method.

The aim of this work was speciation analysis of metabolites in feces samples collected within a clinical study during which a bromine-containing anti-tuberculosis drug (TMC207) was administered to patients with multi-drug resistant tuberculosis infection. Owing to slow elimination of the drug, no (14)C label was used within this study. Quantification of the bromine species was accomplished using high performance liquid chromatography coupled to inductively coupled plasma-mass spectrometry (HPLC/ICP-MS) in combination with on-line isotope dilution (on-line ID), while structural elucidation of the species was performed using HPLC coupled to electrospray ionization-mass spectrometry. The ICP-MS-based method developed shows a good intra- and inter-day reproducibility (relative standard deviation = 3.5%, N = 9); the limit of detection (1.5 mg TMC207 L(-1)) is of the same order of magnitude as that for HPLC/radiodetection; the dynamic range of the method covers more than two orders of magnitude. Furthermore, the column recovery was demonstrated to be quantitative (recoveries between 90.6% and 99.5%). Based on the excellent figures of merit, the "cold" HPLC/ICP-MS approach could be deployed for the actual human in vivo metabolism study, such that exposure of the human volunteers to the (14)C radiolabel was avoided.

Mass defect, neutral loss and isotope filtration techniques were applied to electrospray ionization mass spectrometry (ESI-MS) data obtained for in vivo and in vitro samples of drug metabolism studies. A combination of these post-acquisition processing techniques was shown to be more powerful than the use of one of these tools alone for the detection in complex matrices of metabolites of candidate drugs with a characteristic isotope pattern (e.g. containing bromine, chlorine, or a high proportion of radiolabeled drug (12C/14C)) or characteristic neutral losses. In combination with ‘all-in-one’ data acquisition this methodology is able to perform software-driven constant neutral loss scanning for an unlimited number of mass differences at any time after analysis. Highly selective MS chromatograms were obtained with excellent correlation with their corresponding radiochromatograms. Copyright © 2009 John Wiley & Sons, Ltd.

In this open-label, single-center study, eight healthy men each received a single 500-mg dose of [ 14 C]doripenem, containing 50 μCi of [ 14 C]doripenem, administered as a 1-h intravenous infusion. The concentrations of unchanged doripenem and its primary metabolite (doripenem-M-1) resulting from β-lactam ring opening were measured in plasma and urine by a validated liquid chromatography method coupled to a tandem mass spectrometry assay. Total radioactivity was measured in blood, plasma, urine, and feces by liquid scintillation counting. Further metabolite profiling was conducted on urine samples using liquid chromatography coupled to radiochemical detection and high-resolution mass spectrometry. Unchanged doripenem and doripenem-M-1 accounted for means of 80.7% and 12.7% of the area under the plasma total-radioactivity-versus-time curve (area under the concentration-time curve extrapolated to infinity) and exhibited elimination half-lives of 1.1 and 2.5 h, respectively. Total clearance of doripenem was 16 liters/h, and renal clearance was 12.5 liters/h. At 7 days after the single dose, 95.3% of total doripenem-related radioactivity was recovered in urine and 0.72% in feces. A total mean of 97.2% of the administered dose was excreted in the urine as unchanged doripenem (78.7% ± 5.7%) and doripenem-M-1 (18.5% ± 2.6%). Most of the urinary recovery occurred within 4 h of dosing. Three additional minor metabolites were identified in urine: the glycine and taurine conjugates of doripenem-M-1 and oxidized doripenem-M-1. These results show that doripenem is predominantly eliminated in urine as unchanged drug, with only a fraction metabolized to doripenem-M-1 and other minor metabolites.

If very-high-pressure liquid chromatography (VHPLC) is to replace conventional HPLC as the ultimate separation tool for metabolism studies in development, coupling it efficiently with online radioactivity detection (RAD) is needed. We describe the successful combination of VHPLC/RAD, facilitated by improvements in online radioactivity detection, as well as in column loading and peak capacity. The sensitivity of 14C detection was improved by the use of a variable scintillation flow achieved via a simple modification to the classical online radiochemical detection set-up. A modification of the flow-through cell design in which internal diameter of the tubing was reduced further increased the sensitivity and resolution by decreasing peak tailing. The injection of relatively large injection volumes was made possible by the use of columns packed at ultra-high pressure with 2.2 μm particles. Because of the reduced back pressure generated using these larger particle sizes, two 150 mm × 3 mm columns could be coupled, allowing 4-fold larger injection volumes and a 50% increase in theoretical plate number at a similar back pressure compared to a standard 150 mm × 2.1 mm Waters UPLC column. The value of the methodology described was demonstrated by the analysis of in vitro and in vivo metabolism samples of 3H- and 14C-labeled compounds and compared with conventional radio-HPLC. We have shown that metabolite separation can be achieved with increased efficiency while maintaining a sensitivity comparable to that of conventional HPLC/RAD.

A fast, accurate and precise method for the separation and determination of the total contents of drug-related Cl and Br in human blood plasma, based on high performance liquid chromatography - inductively coupled plasma - tandem mass spectrometry (HPLC-ICP-MS/MS), has been developed. The novel approach was proved to be a suitable alternative to the presently used standard methodology (i.e. based on a radiolabelled version of the drug molecule and radiodetection), while eliminating the disadvantages of the latter. Interference-free determination of (35)Cl has been accomplished via ICP-MS/MS using H2 as reaction gas and monitoring the (35)ClH2(+) reaction product at mass-to-charge ratio of 37. Br could be measured "on mass" at a mass-to-charge of 79. HPLC was relied on for the separation of the drug-related entities from the substantial amount of inorganic Cl. The method developed was found to be sufficiently precise (repeatability10% RSD) and accurate (recovery between 95 and 105%) and shows a linear dynamic range (R(2)0.990) from the limit of quantification (0.05 and 0.01 mg/L for Cl and Br in blood plasma, respectively) to at least 5 and 1mg/L for Cl and Br, respectively. Quantification via either external or internal standard calibration provides reliable results for both elements. As a proof-of-concept, human blood plasma samples from a clinical study involving a newly developed Cl- and Br-containing active pharmaceutical ingredient were analysed and the total drug exposure was successfully described. Cross-validation was achieved by comparing the results obtained on Cl- and on Br-basis.

Soy extracts contain a mixture of isoflavones belonging to the group of phytoestrogens. In the quality control of soy the amount of isoflavones, both aglycones and glycosides, is usually determined by means of reversed-phase HPLC–UV. On conventional C 18 -material columns, long analysis times are required in order to separate this complex mixture. In order to speed up analysis, the separation was optimized using two linked monolithic silica-based reversed-phase C 18 columns. A spectacular decrease of the analysis time, i.e. almost three-fold, was achieved by applying a flow rate of 3–4 ml/min without loosing any separation efficiency. This analysis method for determination of isoflavones in soy extracts in less than 25 min was fully validated according to the ICH guidelines.

Different reversed-phase liquid chromatography (LC) columns of conventional dimensions were coupled to an ultraviolet photodiode array detector (UV-DAD) and a magnetic sector-type spectrometer, equipped with an electrospray ionization (ESI) source, by a laboratory-made flow splitter. A mixture of three flavonoid-O-glycosides was employed to examine the effects of the solvent composition, the flow rate, the stationary phase, the pH and the organic acid added, on the chromatographic separation, the UV-DAD detection, the ESI process and the entire LC system with ESI-MS and UV-DAD detection. In the positive ion mode, methanol containing 1% acetic acid was by far the most sensitive in ESI-MS analysis, whereas an acetonitrile/water mobile phase containing 0.5% formic acid was proved to give the best sensitivity in LC/ESI-MS/UV-DAD analysis. In the negative ion mode, the highest sensitivity was obtained with a mobile phase containing 0.1% formic acid, while addition of bases decreased the sensititvity. The optimal flow rate was higher in negative ESI (20-50 micro L/min) than in positive ESI (5 micro L/min), and the percentage of organic phase had an influence on the sensitivity of ESI-MS detection. With regard to the selection of a suitable C(18) reversed-phase LC column, a column which is well end-capped is to be preferred, because residual silanol groups appear to impair the separation of flavonoid glycosides. The optimized LC/ESI-MS/UV-DAD method was applied to a commercial Crataegus extract, which is used in phytomedicine to treat cardiovascular problems and is known to be rich in flavonoids. It is demonstrated how UV spectra and first-order ESI mass spectra allow a fast characterization of flavonoids, even if reference compounds are not available or at hand.

The present study investigated the practical use of modern ultra-high performance liquid chromatography (UHPLC) separation techniques for drug metabolite profiling, aiming to develop a widely applicable, high-throughput, easy-to-use chromatographic method, with a high chromatographic resolution to accommodate simultaneous qualitative and quantitative analysis of small-molecule drugs and metabolites in biological matrices. To this end, first the UHPLC system volume and variance were evaluated. Then, a mixture of 17 drugs and various metabolites (molecular mass of 151-749Da, logP of -1.04 to 6.7), was injected on six sub-2μm particle columns. Five newest generation core shell technology columns were compared and tested against one column packed with porous particles. Two aqueous (pH 2.7 and 6.8) and two organic mobile phases were evaluated, first with the same flow and temperature and subsequently at each column's individual limit of temperature and pressure. The results demonstrated that pre-column dead volume had negligible influence on the peak capacity and shape. In contrast, a decrease in post-column volume of 57% resulted in a substantial (47%) increase in median peak capacity and significantly improved peak shape. When the various combinations of stationary and mobile phases were used at the same flow rate (0.5mL/min) and temperature (45°C), limited differences were observed between the median peak capacities, with a maximum of 26%. At higher flow though (up to 0.9mL/min), a maximum difference of almost 40% in median peak capacity was found between columns. The finally selected combination of solid-core particle column and mobile phase composition was chosen for its selectivity, peak capacity, wide applicability and peak shape. The developed method was applied to rat hepatocyte samples incubated with the drug buspirone and demonstrated to provide a similar chromatographic resolution, but a 6 times higher signal-to-noise ratio than a more traditional UHPLC metabolite profiling method using a fully porous particle packed column, within one third of the analysis time. In conclusion, a widely applicable, selective and fast chromatographic method was developed that can be applied to perform drug metabolite profiling in the timeframe of a quantitative analysis. It is envisioned that this method will in future be used for simultaneous qualitative and quantitative analysis and can therefore be considered a first important step in the Quan/Qual workflow.

Background: With an increased interest in peptides and proteins as potential new drug candidates, new approaches for sensitive and selective quantitative analysis are required. LC–MS/MS analysis provides a good alternative to immunoassays with reduced method development times and increased specificity. Results: We have evaluated two state-of-the-art triple quadrupole and high-resolution TOF mass spectrometers with respect to their performance for quantification of six peptides (glufibrinopeptide B, somatostatin, enfuvirtide, TRI1144, C34 and exenatide). The peptides were spiked into protein-precipitated plasma supernatant. Triple quadrupole quantification was performed in SRM mode, and in high-resolution, MS narrow-width extracted chromatograms were generated for quantification. Specificity, accuracy, reproducibility and robustness were found to be comparable between the two instruments. The triple quadrupole instrument is still the most sensitive instrument for quantification of peptides with a median factor of about four-times higher sensitivity (based on LLOQ evaluation). Conclusion: Based on sensitivity, the newest generation triple quadrupole MS systems are still the preferred technology for quantification of peptides. Since the sensitivity difference between triple quadrupole instruments and the new-generation high-resolution TOF-MS instruments is minor, the latter offer a useful alternative whenever additional selectivity is preferred or the use of a generic approach not requiring method optimization is advantageous.

During the development of a new drug compound, its metabolism needs to be unraveled. For quantification of the metabolites formed, the drug under investigation is traditionally synthesized with a radiolabel ((14)C or (3)H) and the metabolites present in different matrixes (blood, urine, feces) upon drug administration are determined by means of high-performance liquid chromatography (HPLC) coupled to radiodetection. This approach allows for quantification of the metabolites formed and enables a straightforward distinction between exogenous (i.e., drug-related) and endogenous species (as only the radiolabeled species are detected). However, in some cases, the use of a radiolabeled compound in human in vivo studies is not advisible, e.g., for drug compounds or their metabolites showing a long plasma or tissue half-life. In cases where the candidate drug molecule contains an element detectable by means of inductively coupled plasma mass spectrometry (ICP-MS), HPLC/ICP-MS is a promising alternative approach. However, the method lacks specificity when a distinction between drug-related species and endogenous compounds containing the same target element needs to be accomplished. As a result, we have developed an HPLC/ICP-MS-based method combined with "reverse" online isotope dilution ("reverse" online ID) for metabolite quantification. The methodology was evaluated by the analysis of feces samples from rats dosed with a (81)Br-labeled drug compound. The method allows for both (i) valid quantification of the drug metabolites and (ii) distinction among endogenous, exogenous, and "mixed" species, based on their isotopic "fingerprint". A good repeatability (relative standard deviation of 4.2%) and limit of detection (0.35 mg of drug compound L(-1) of feces extract), of the same order of magnitude as those observed for "normal" online ID HPLC/ICP-MS and HPLC/radiodetection, were achieved.

Saponins are high molecular weight glycosides which are known for their broad range of biological activities. In case of Maesa lanceolata, a tree growing in African countries, the maesasaponins showed virucidal, haemolytic, molluscicidal and anti-angiogenic activity. Since the different activities are dependent on the structure of the saponins, a method was developed and validated for the analysis of the individual saponins in this plant. Since the saponins were only present in small amounts, it was necessary to develop a very sensitive analytical method. For the fast and sensitive analysis of the extracted and purified plant samples ultra-performance liquid chromatography was coupled to a triple quadrupole mass spectrometer for MS/MS detection. A method in positive ESI mode, using sodium acetate in the mobile phase, was developed. The sodium adduct ion was selected as the precursor ion since it provided better sensitivity and a better, more stable fragmentation compared to the deprotonated and protonated ions. The intensity of the signal obtained by fragmentation of the sodium adducts of the saponins, was optimized by the addition of different concentrations of sodium acetate to the mobile phase. Reference standards were not available for all 14 saponins. Therefore, a relative MS/UV response was calculated allowing the estimation of the saponins in real samples. α-Hederin was used as external standard. The method was linear over the investigated concentration range with a good correlation coefficient (>0.99). The intra- and inter-day precisions were below 15% for most maesasaponins with the exception of maesasaponin II, which showed a precision within 20%. The recoveries of the spiked pure compounds maesasaponin IV.1 and VII.1 were 96.6% and 85.5%, respectively. The validated method can be applied in the investigation of the content of 14 saponins in transgenic and non-transgenic plant material of M. lanceolata.

In the present study, we have characterized in detail the MS(2) and MS(3) fragmentation behaviors, using electrospray ionization (ESI) in the negative ion mode, of previously identified sulfated isoprene secondary organic aerosol compounds, including 2-methyltetrols, 2-methylglyceric acid, 2-methyltetrol mononitrate derivatives, glyoxal and methylglyoxal. A major fragmentation pathway for the deprotonated molecules of the sulfate esters of 2-methyltetrols and 2-methylglyceric acid and of the sulfate derivatives of glyoxal and methylglyoxal is the formation of the bisulfate [HSO(4)](-) anion, while the deprotonated sulfate esters of 2-methyltetrol mononitrate derivatives preferentially fragment through loss of nitric acid. Rational interpretation of MS(2), MS(3) and accurate mass data led to the structural characterization of unknown polar compounds in K-puszta fine aerosol as organosulfate derivatives of photooxidation products of unsaturated fatty acids, i.e. 2-hydroxy-1,4-butanedialdehyde, 4,5- and 2,3-dihydroxypentanoic acids, and 2-hydroxyglutaric acid, and of alpha-pinene, i.e. 3-hydroxyglutaric acid. The deprotonated molecules of the sulfated hydroxyacids, 2-methylglyceric acid, 4,5- and 2,3-dihydroxypentanoic acid, and 2- and 3-hydroxyglutaric acids, showed in addition to the [HSO(4)](-) ion (m/z 97) neutral losses of water, CO(2) and/or SO(3), features that are characteristic of humic-like substances. The polar organosulfates characterized in the present work are of climatic relevance because they may contribute to the hydrophilic properties of fine ambient aerosol. In addition, these compounds probably serve as ambient tracer compounds for the occurrence of secondary organic aerosol formation under acidic conditions.

A combination of inductively coupled plasma mass spectrometry (ICP-MS) and electrospray ionization mass spectrometry (ESI-MS) was deployed for the metabolite profiling and metabolite identification of a new antituberculosis compound (R207910, also known as TMC207) that is currently in drug development. R207910 contains one bromine atom, allowing the detection by ICP-MS. Fluctuations in the Br sensitivity caused by the HPLC gradient were counteracted by the use of species-unspecific isotope dilution. In order to evaluate the method developed, the results obtained were compared with those acquired via radioactivity detection. HPLC-ESI-MS was used for the structural identification of R207910 and its metabolites. The (79)Br/(81)Br isotope ratio is also valuable in the search for metabolites in the complex background of endogenous compounds obtained using HPLC-ESI-MS analyses. Data-dependent scanning using isotope recognition with an ion trap mass spectrometer or processing of Q-Tof data provides HPLC-ICP-MS-like "bromatograms". The combination of accurate mass measurements and the fragmentation behavior in the MS(2) spectra obtained using the Q-Tof Ultima mass spectrometer or MS(n) spectra acquired using the LTQ-Orbitrap allowed structural characterization of the main metabolites of R207910 in methanolic dog and rat faeces extracts taken 0-24 h post-dose.

Inductively coupled plasma time-of-flight mass spectrometry (ICP-TOFMS) and electrospray ionization mass spectrometry (ESI-MS) were used in parallel in combination with two-dimensional chromatography (size-exclusion followed by reversed-phase HPLC) for the characterization of metal complexes with metallothionein (MT) isoforms in hepatic cytosols of Cd exposed carp. The cytosolic MT fraction was partially purified by size-exclusion (SE) HPLC, preconcentrated and de-salted by centrifugal microfiltration. The separation of the metal complexes with MT isoforms was performed by reversed-phase (RP) HPLC using a gradient up to 30% methanol in acetate buffer (pH 7.4). Two major and several minor chromatographic peaks were distinguished by RP-HPLC-ICP-TOFMS within a chromatographic run of 20 min. Applying ESI-MS as a detector for RP HPLC allowed the identification of the two major signals detected by ICP-MS on the basis of the molecular mass determined on-line for the metallocomplex(es) and for the ligand. Mass spectra taken at the peak apices indicated the co-elution of different metal (Cd, Cu, Zn and Pb) complexes with MT isoforms within each peak. A simple “in-source” protein oxidation procedure was used to obtain the ligand of the complexes (oxidized form) on arrival at the detector; direct information was obtained on the molecular mass of two MT isoforms in carp liver, allowing their identification. The efficiency and robustness of this method were demonstrated using a rabbit liver MT1 standard as test sample.

The flavonoid fraction from the seeds of Carrichtera annua was studied using high-performance liquid chromatography simultaneously coupled to a photodiode array detector (LC/UV-DAD) and a mass spectrometer equipped with an electrospray source (LC/ESI-MS). Collision-induced dissociation (CID) mass spectral data obtained off-line by nanospray (nano-ESI) analysis provided a wealth of complementary structural information, which was consistent with structures established by NMR or led to the proposal of base structures of the flavonol O-glycosides present in the Carrichtera annua seed extract. The flavonoid fraction was found to contain 12 structurally related flavonol O-glycosides. Eleven flavonoids, of which several were new compounds, were acylated with one or more benzoyl, feruloyl or sinapoyl groups. These acyl groups gave rise to characteristic product ions in the [M + H]+ and [M + Na]+ CID spectra as well as to radicalar acid-related product ions at high-energy collisional activation. In addition to the characterization of the acyl substituents, the mass spectral data allowed the identification of the aglycone, the determination of the base structure and the differentiation of several positional isomers.

Mass spectrometric methods have been developed which allow the direct stereochemical assignment of terminal monosaccharide residues in flavonoid O-glycosides without the need for chemical hydrolysis. Standards containing a glucose, galactose, mannose, xylose, arabinose or apiose residue were examined because these monosaccharides are by far the most commonly encountered in flavonoid glycosides. Following acetylation, the major peracetylated sugar related fragments, generated by fast atom bombardment (FAB) or electrospray ionization (ESI), were selected for collisional activation employing a broad range of collision energies. Both FAB and ESI proved to be useful as ionization techniques. Stereoselective fragmentation was achieved and allowed us clearly to differentiate and characterize isomeric monosaccharide residues. The method developed was successfully applied to an unknown flavonoid containing a terminal pentose and hexose residue which was isolated from Farsetia aegyptia.

An LC-MS/MS method for the simultaneous quantification of azide and azidoalanine in bacterial and mammalian cellular systems was developed to support mutagenicity evaluation of azido containing nucleoside drugs. With the aim to enhance ionization efficiency and chromatographic retention, derivatisation with dansyl chloride was optimized; study samples (diluted 1/10) were derivatised for 2 h at room temperature and subsequently analysed with reversed phase liquid chromatography in combination with electrospray mass spectrometry in the positive ion mode. Good linearity and accuracy were obtained with a lower limit of quantification of 10.0 ng mL−1 for both analytes. The method was applied in mutagenicity experiments and showed time-dependent release of azide, but no formation of azidoalanine could be detected.

Application of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) has been studied as an alternative to radioluminography (RLG) for assessing the distribution of a novel anti-tuberculosis compound containing bromine as a “hetero-element” and its metabolites over the body compartments of rat, used as a test animal. In contrast to RLG, LA-ICP-MS does not require labeling of the drug compound with a radionuclide. After administration of the Br-containing drug and a sufficient delay time to allow drug uptake and distribution, the sacrificed animal was frozen and embedded in carboxymethyl cellulose (CMC), after which thin sections were obtained using a microtome. For quantification purposes, hardened gelatin films on a glass support were used as “matrix-matched” Br standards. The limit of detection (LOD) for Br estimated using one of these in-house made standards was 0.1 μg g−1, which is sufficiently low to allow visualization of Br in the main organ of interest (lung). LA-ICP-MS analysis via single spot drilling with a wider laser beam diameter on the organ of interest was performed as well. In this case, quantification was accomplished via external calibration versus droplets of standards containing the pure drug that were co-embedded with the rat. Figures of merit achievable with LA-ICP-MS were critically evaluated and compared to those typical for the traditional technique, based on the use of radionuclides.

A multimodal mass spectrometry imaging (MSI) based approach was used to characterize the molecular content of crystal-like structures in a frozen and paraffin embedded piece of a formalin-fixed rabbit kidney. Matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) imaging and desorption electrospray ionization (DESI) mass spectrometry imaging were combined to analyze the frozen and paraffin embedded sample without further preparation steps to remove the paraffin. The investigated rabbit kidney was part of a study on a drug compound in development, in which severe renal toxicity was observed in dosed rabbits. Histological examination of the kidney showed tubular degeneration with precipitation of crystal-like structures in the cortex, which were assumed to cause the renal toxicity. The MS imaging approach was used to find out whether the crystal-like structures were composed of the drug compound, metabolites, or an endogenous compound as a reaction to the drug administration. The generated MALDI-MSI data were analyzed using principal component analysis. In combination with the MS/MS results, this way of data processing demonstrates that the crystal structures were mainly composed of metabolites and relatively little parent drug. Graphical Abstract ᅟ