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RATIONALE Refined cottonseed oil has widespread applications in the food and chemical industries. Although the major lipids comprising cottonseed oil (triacylglycerols) are well known, there are many diverse lipid species in cotton seeds that occur at much lower levels and have important nutritional or anti-nutritional properties. METHODS The lipid technical samples were prepared in chloroform. The biological samples were extracted using a mixture of isopropanol/chloroform/H2 O (2:1:0.45). The data were collected using high and low collision energy with simultaneous data collection on a time-of-flight (TOF) mass spectrometer which allowed the characterization of lipids by precursor and product ion alignment. The supercritical fluid chromatography methodology is flexible and can be altered to provide greater retention and separation. The comprehensive method was used to screen seed lipid extracts from several cotton genotypes using multivariate statistical analysis. RESULTS Method variables influencing the peak integrity and chromatographic separation for a mixture of lipids with different degrees of polarity were explored. The experiments were designed to understand the chromatographic behavior of lipids in a controlled setting using a variety of lipid extracts. Influences of acyl chain length and numbers of double bonds were investigated using single moiety standards. CONCLUSIONS The methodology parameters were examined using single moiety lipid standards and standard mixtures. The method conditions were applied to biological lipid extracts, and adjustments were investigated to manipulate the chromatography. Insights from these method variable manipulations will help to frame the development of targeted lipid profiling and screening protocols. Copyright © 2017 John Wiley & Sons, Ltd.

Highlights • An SFC method has been developed for the analysis of polar urinary metabolites. • 12 stationary phases, 9 modifier additives and 3 temperatures were evaluated. • DIOL and 2-PIC columns provide highest peak capacity and overall resolution. • Ammonium formate, ammonium hydroxide and water considerably improved separation. • Alkylamine additives should be strongly considered for polar SFC-UV analysis., Supercritical fluid chromatography (SFC) is frequently used for the analysis and separation of non-polar metabolites, but remains relatively underutilised for the study of polar molecules, even those which pose difficulties with established reversed-phase (RP) or hydrophilic interaction liquid chromatographic (HILIC) methodologies. Here, we present a fast SFC-MS method for the analysis of medium and high-polarity (−7 ≤ cLogP ≤ 2) compounds, designed for implementation in a high-throughput metabonomics setting. Sixty polar analytes were first screened to identify those most suitable for inclusion in chromatographic test mixtures; then, a multi-dimensional method development study was conducted to determine the optimal choice of stationary phase, modifier additive and temperature for the separation of such analytes using SFC. The test mixtures were separated on a total of twelve different column chemistries at three different temperatures, using CO2-methanol-based mobile phases containing a variety of polar additives. Chromatographic performance was evaluated with a particular emphasis on peak capacity, overall resolution, peak distribution and repeatability. The results suggest that a new generation of stationary phases, specifically designed for improved robustness in mixed CO2-methanol mobile phases, can improve peak shape, peak capacity and resolution for all classes of polar analytes. A significant enhancement in chromatographic performance was observed for these urinary metabolites on the majority of the stationary phases when polar additives such as ammonium salts (formate, acetate and hydroxide) were included in the organic modifier, and the use of water or alkylamine additives was found to be beneficial for specific subsets of polar analytes. The utility of these findings was confirmed by the separation of a mixture of polar metabolites in human urine using an optimised 7 min gradient SFC method, where the use of the recommended column and co-solvent combination resulted in a significant improvement in chromatographic performance.

This paper describes the fabrication of a new porous monolith, prepared in 100μm i.d. capillaries by the co-polymerisation of hexyl methacrylate with 1,6-hexanediol ethoxylate diacrylate, poly (HMA-co-1,6 HEDA), in the presence of azobisisobutyronitrile, 1, 4-butanediol and 1-propanol were used as porogens for the monoliths; the monoliths were then used as a stationary phase for capillary liquid chromatography. Two cross linkers namely 1,6 HEDA and EDMA were utilised in order to investigate the effects of cross linker length on the separation efficiency of small molecules, and it was found that the efficiency of the separation improved tenfold when using the longer cross linker, 1,6 HEDA. This improvement is associated with the increase in number of methylene groups which resulted in an increased number of mesopores, less than 50nm. The 1,6 HEDA based monolith showed a high porosity (90%) and no evidence of swelling or shrinking with the use of organic solvents. Moreover, the 1,6 HEDA monolith demonstrated high reproducibility for the separation of the retained compounds anisole and naphthalene; these showed retention time RSDs of 1.79% and 2.74% respectively. The fabricated monolith also demonstrated high selectivity for neutral non-polar molecules, weak acids, and basic molecules. The asymmetry factors for basic molecules (nortriptyline and amitriptyline) were 1.5 and 1.3 respectively, indicating slight tailing, which is often noticeable on silica based phases due to secondary interactions between basic moieties and the hydroxyl groups of the silica.

The use of small scale renewable sorbent material for automated solid phase extraction of multi-residue pharmaceuticals in environmental samples exploiting the sequential injection analysis-bead injection with direct coupling to liquid chromatography-electrospray ionization tandem mass spectrometry (SIA-BI-μSPE-LC-ESI-MS/MS) is presented to determine beta-blockers, namely atenolol, sotalol, pindolol, acebutolol, timolol, metoprolol, labetalol, carazolol, propranolol and betaxolol. These compounds yielded the same product ions, therefore were affected in terms of quantification when flow injection analysis-mass spectrometry (FIA-MS) was used. Thus, analytes and matrix present in the sample travel together into the ionization source which can seriously affect the ionization efficiency and analyte signals due to monitoring over a short time period. Graphical abstract A two-dimensional analysis involving a time dimension (retention time) and an m/z dimension (fragmentation ion) is promising for the various sample types. Using the developed method, absolute recoveries percentages of 10 mL of sample loading volume were >91% for all β-blockers with enrichment factor of 62-74, limits of detection of 0.005-0.07 μg L(-1), limits of quantification of 0.01-0.23 μg L(-1), enrichment factor of 62-72 and repeatability within range 7-12%. This developed method is suggested to be used as quantitative screening technique for drugs of abuse or persistent contamination using different kinds of sorbent materials and complex matrix such as biological fluid sample as well.

An integrated capillary scale (300 μm id) ceramic microfluidic LC system combined with MS/MS has been successfully employed for the quantitative analysis of pharmaceutical compounds in human plasma. The capillary ceramic microfluidic LC/MS/MS system showed an approximate 20-fold (range 11-38-fold) increase in sensitivity compared with a standard 2.1 mm scale UPLC/MS/MS system for a broad range of analytes. The loading capacity of the devices capillary separations channel allowed injection of 2 μL of an aqueous solution, and up to 1.2 μL of a typical protein-precipitated plasma sample, onto the reversed-phase chromatography system. The system also showed excellent chromatographic performance and robustness, with no deleterious effects on the chromatography observed over the course of 1000 injections of protein-precipitated plasma. The ability of the ceramic microfluidic LC/MS/MS system to deliver this level of sensitivity and performance enables the routine quantification of pharmaceutical compounds from small format samples, such as those obtained by dried blood spot or other blood microsampling approaches, to be performed.

Systematic in-vitro studies have been conducted to determine the ability of a range of 10 potential hydrotropes to improve the apparent aqueous solubility of the poorly water soluble drug, indomethacin. Solubilisation of the drug in the presence of the hydrotropes was determined experimentally using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. These experimental data, together with various known and computed physicochemical properties of the hydrotropes were thereafter used in silico to train an artificial neural network (ANN) to allow for predictions of indomethacin solubilisation. The trained ANN was found to give highly accurate predictions of indomethacin solubilisation in the presence of hydrotropes and was thus shown to provide a valuable means by which hydrotrope efficacy could be screened computationally. Interrogation of the network connection weights afforded a quantitative assessment of the relative importance of the various hydrotrope physicochemical properties in determining the extent of the enhancement in indomethacin solubilisation. It is concluded that in-silico screening of drug/hydrotrope systems using artificial neural networks offers significant potential to reduce the need for extensive laboratory testing of these systems, and could thus provide an economy in terms of reduced costs and time in drug formulation development.

Roman and German chamomile are widely used throughout the world. Chamomiles contain a wide variety of active constituents including sesquiterpene lactones. Various extraction techniques were performed on these two types of chamomile. A packed-column supercritical fluid chromatography-mass spectrometry method was designed for the identification of sesquiterpenes and other constituents from chamomile extracts with no derivatization step prior to analysis. Mass spectrometry detection was achieved by using electrospray ionization. All of the compounds of interest were separated within 15 min. The chamomile extracts were analyzed and compared for similarities and distinct differences. Multivariate statistical analysis including principal component analysis and orthogonal partial least squares-discriminant analysis (OPLS-DA) were used to differentiate between the chamomile samples. German chamomile samples confirmed the presence of cis- and trans-tonghaosu, chrysosplenols, apigenin diglucoside whereas Roman chamomile samples confirmed the presence of apigenin, nobilin, 1,10-epioxynobilin, and hydroxyisonobilin.

Ultra high resolution SFC-MS (on sub-2μm particles) coupled to mass spectrometry has been evaluated for the metabolic profiling of rat and dog bile. The selectivity of the SFC separation differed from that seen in previous reversed-phase UPLC-MS studies on bile, with the order of elution for analytes such as e.g., the bile acids showing many differences. The chromatography system showed excellent stability, reproducibility and robustness with relative standard deviation of less than 1% for retention time obtained over the course of the analysis. SFC showed excellent chromatographic performance with chromatographic peak widths in the order of 3s at the base of the peak. The use of supercritical fluid carbon dioxide as a mobile phase solvent also reduced the overall consumption of organic solvent by a factor of 3 and also reduced the overall analysis time by a factor of 30% compared to reversed-phase gradient LC. SFC-MS appear complementary to RPLC for the metabolic profiling of complex samples such as bile.

Inhaled nanomaterials present a challenge to traditional methods and understanding of respiratory toxicology. In this study, a non-targeted metabolomics approach was used to investigate relationships between nanoparticle hydrophobicity, inflammatory outcomes and the metabolic fingerprint in bronchoalveolar fluid. Measures of acute lung toxicity were assessed following single-dose intratracheal administration of nanoparticles with varying surface hydrophobicity (i.e. pegylated lipid nanocapsules, polyvinyl acetate nanoparticles and polystyrene beads; listed in order of increasing hydrophobicity). Broncho-alveolar lavage (BAL) fluid was collected from mice exposed to nanoparticles at a surface area dose of 220 cm(2) and metabolite fingerprints were acquired via ultra pressure liquid chromatography-mass spectrometry-based metabolomics. Particles with high surface hydrophobicity were pro-inflammatory. Multivariate analysis of the resultant small molecule fingerprints revealed clear discrimination between the vehicle control and polystyrene beads (p

This work provides a multidimensional method for the simultaneous, direct quantification of intact human insulin and five insulin analogs in human plasma. This investigation solves both the selectivity and sensitivity problems encountered for accurate quantification of insulins in plasma since the former is not possible with conventional assays and the latter with conventional LC-MS/MS. The method uses a mixed-mode SPE and a multidimensional LC method including a solid-core particle column containing an anion exchange stationary phase. Matrix factors for all analogs were calculated in 6 sources of human plasma and CVs of the matrix factors were15% in all cases supporting the selectivity of the method, while achieving LLOQs of 50-200 pg/mL (1.4-5.6 μIU/mL) for each insulin from 250 μL of human plasma. The average accuracy for the standard curve points in extracted human plasma was 99-100%. Average inter- and intraday accuracies for QC samples were 98% and 94%, respectively. Average inter- and intraday precisions for QC samples were 7.5 and 5.3%, respectively. Patient samples were analyzed in a blind study and results concurred with their diabetes multidosing regimes. The study also demonstrated that the presence of high levels of human insulin and bovine insulin does not interfere with quantification of any of the analyzed analogs. We propose this method for the accurate pharmacokinetic monitoring of diabetic patients, for sport antidoping and forensic toxicology analysis.

A major toxicological challenge is distinguishing whether morphine in urine, in the absence of 6-monoacetylmorphine (6-MAM), originates from 'street' heroin use or poppy seed ingestion. Manufacturing byproducts from the synthesis of illicit heroin include those that originate from the reaction of acetic anhydride with the alkaloid impurity, thebaine, which undergoes skeletal rearrangement, resulting in compounds with a 2-(N-methylacetamido)ethyl side-chain. The hypothesis that the tertiary amide in this side-chain is resistant to endogenous hydrolysis was supported from in-vitro experiments; a glucuronide metabolite (designated 'ATM4G') was identified that may be used as a marker of 'street' heroin administration. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for this metabolite was then performed on selected urine specimens from 22 known heroin users, these being negative on routine testing for 6-MAM by gas chromatography-mass spectrometry (GC-MS), using the generally applied reporting threshold of 10 ng/mL, but positive for the presence of morphine. Peaks corresponding to the retention time for the metabolite marker were clearly observed for 16 of the 22 samples, with variations of the ratios of its three dependent ions being within ± 30% of that produced in vitro. Conversely, 6-MAM was detected in only 3 samples, but at concentrations Language: en

Teriparatide, the 1-34 fragment of human parathyroid hormone, is used to treat osteoporosis patients with a high risk of fracture by stimulating new bone formation. Routinely teriparatide is quantified using radioimmunoassay however the LC-MS/MS described here has the potential to achieve greater accuracy and precision, higher specificity, and is readily implemented in routine bioanalytical laboratories. Hence a complete method combining effective sample prep with appropriate LC separation and selected reaction monitoring (SRM) MS detection was developed to selectively separate teriparatide from closely related endogenous peptides and to reduce interferences. Samples were concentrated without evaporation, minimizing the risk of adsorptive losses. Chromatography was performed on a sub 2μm particle charged surface hybrid column, which provided significantly higher peak capacity than a traditional C18 column when formic acid was used as the mobile phase modifier. Total LC cycle time was 6min. An LOD of 15pg/mL (3.6fmol/mL) from 200μL of human plasma was readily achieved and standard curves were accurate and precise from 15pg/mL to 500pg/mL. Mean QC accuracies ranged from 90% to 106%. Mean QC precision was better than 7%. The CV of matrix factors across 6 sources of human plasma was 5%. The assay presented here is the first LC-MS method which reaches clinically relevant detection limits for teriparatide.

The ability to link criminal activity and identity using validated analytical approaches can be of great value to forensic scientists. Herein, the factors affecting the recovery and detection of inorganic and organic energetic material residues within chemically or physically enhanced fingermarks on paper and glass substrates are presented using micro-bore anion exchange chromatography with suppressed conductivity detection. Fingermarks on both surfaces were enhanced using aluminium powder or ninhydrin after spiking with model test mixtures or through contact with black-powder substitutes. A quantitative study of the effects of environmental/method interferences, the sweat matrix, the surface and the enhancement technique on the relative anion recovery of forensically relevant species is presented. It is shown that the analytical method could detect target analytes at the nanogram level even within excesses of enhancement reagents and their reaction products when using solid phase extraction and/or microfiltration. To our knowledge, this work demonstrates for the first time that ion chromatography can detect anions in energetic materials within fingermarks on two very different surfaces, after operational enhancement techniques commonly used by forensic scientists and police have been applied.

In order to prepare zwitterionic HILIC monolithic columns with high polarity, the highly hydrophilic monomer N,N-dimethyl-N-acryloyloxyethyl-N-(3-sulfopropyl)ammonium betaine (SPDA) and crosslinker N,N'-methylenebisacrylamide (MBA) were employed for developing a novel sulfoalkylbetaine type stationary phase. The polymerization parameters were systematically optimized in order to obtain a satisfactory performance for column permeability, mechanical stability, hydrophilicity, efficiency and selectivity. Compared to the previously reported poly(N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine-co-ethylene dimethacrylate) (poly(SPE-co-EDMA)) monolith and the poly(SPDA-co-EDMA) monolith that we developed, a significantly enhanced hydrophilicity was obtained on the poly(SPDA-co-MBA) monolithic column, illustrated by the lowered critical composition of the mobile phase corresponding to the transition from the HILIC to the RP mode. Excellent permeability, reproducibility and stability were achieved on this optimized poly(SPDA-co-MBA) monolith. A column efficiency of 70,000plates/m was obtained for the analysis of bases at a linear velocity of 1.95mm/s. As expected, by studying the influence of mobile phase pH and salt concentration on their retention, a weak electrostatic repulsion interaction for negatively charged analytes was also observed at low organic solvent content on the poly(SPDA-co-MBA) monolithic column. The final optimized poly(SPDA-co-MBA) monolith exhibited good selectivity for a series of polar compounds, such as phenols, bases, benzoic acid derivatives, small peptides, urea and allantoin.

Background: Intact insulins are difficult to analyze by LC–MS/MS due to nonspecific binding and poor sensitivity, solubility and fragmentation. This work aims to provide a simpler, faster LC–MS method and focuses on solving the above issues. Results: A novel charged-surface chromatographic column produced peak widths for insulin that were significantly narrower than traditional C18 columns when using formic acid as mobile phase. Mass spectral fragments m/z >700 provided greater specificity, significantly reducing endogenous background. Detection limits in human plasma were 0.2 ng/ml for insulin glargine, glulisine and detemir, and 0.5 ng/ml for insulin aspart. Average accuracy for standard curve and QC samples was 93.4%. Conclusion: A simple SPE LC–MS analysis was developed for direct, simultaneous quantification of insulin glargine, detemir, aspart and glulisine.

Refined cottonseed oil has widespread applications in the food and chemical industries. Although the major lipids comprising cottonseed oil (triacylglycerols) are well known, there are many diverse lipid species in cotton seeds that occur at much lower levels and have important nutritional or anti-nutritional properties.The lipid technical samples were prepared in chloroform. The biological samples were extracted using a mixture of isopropanol/chloroform/HMethod variables influencing the peak integrity and chromatographic separation for a mixture of lipids with different degrees of polarity were explored. The experiments were designed to understand the chromatographic behavior of lipids in a controlled setting using a variety of lipid extracts. Influences of acyl chain length and numbers of double bonds were investigated using single moiety standards.The methodology parameters were examined using single moiety lipid standards and standard mixtures. The method conditions were applied to biological lipid extracts, and adjustments were investigated to manipulate the chromatography. Insights from these method variable manipulations will help to frame the development of targeted lipid profiling and screening protocols. Copyright © 2017 John WileySons, Ltd.

A novel hydrophilic interaction liquid chromatography (HILIC) stationary phase was prepared by the co-polymerisation of zwitterionic N,N'-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl) ammonium betaine (SPE) and the crosslinker 1,2-bis(p-vinylphenyl) ethane (BVPE) in the presence of the porogens, toluene and methanol. Monolithic columns were produced by carrying out the α,α'-azoisobutyronitrile (AIBN) initiated reaction for 1, 2, 4, 8 and 12 h inside a 200 μm i.d. fused silica capillary at 75°C (water bath). The optimum polymerisation time was shown to be 2 h, as this resulted in good porosity, due to enlarged flow-channels and the presence of a higher proportion of mesopores provided a relatively larger surface area than the other columns. The chromatographic properties of the optimised poly (SPE-co-BVPE) monolithic column were evaluated with test mixtures containing both basic and neutral compounds in the HILIC gradient separation mode. This produced relatively sharp peaks (average peak width at half height=0.1 min) with average asymmetry factors of 1.4 and baseline resolution was obtained for all the compounds. Using the isocratic separation of the test mixture, the number of theoretical plates (N) per metre calculated was between 26,888 and 35,930 by using average values obtained for triplicate injections of the compounds thiourea, toluene and acrylamide.

Gunshot residue (GSR) is commonly analysed in forensic casework using either scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDX) or gas chromatography-mass spectrometry (GC-MS). Relatively little work has been reported on the post-discharge GSR content of non-metallic inorganic or low molecular weight organic anions to distinguish between different ammunition types. The development of an analytical method using suppressed micro-bore anion exchange chromatography (IC) is presented for the analysis of GSR. A hydroxide gradient was optimised for the separation of 19 forensically relevant organic and inorganic anions in23min and sensitivities of the order of 0.12-3.52ng of anion detected for all species were achieved. Along with an optimised extraction procedure, this method was applied to the analysis of post-ignition residues from three selected ammunition types. By profiling and comparing the anionic content in each ammunition residue, the possibility to distinguish between each type using their anionic profiles and absolute weight is presented. The potential for interference is also discussed with respect to sample types which are typically problematic in the analysis of GSR using SEM-EDX and GC-MS. To the best of our knowledge this represents the first study on the analysis of inorganic anions in GSR using suppressed ion chromatography.

The sensitivity and accuracy of a bioanalytical method is critical in defining the pharmacokinetic (PK) parameters of a potential new chemical entity (NCE). Inhaled therapeutics and low dose NCEs present one of the most significant analytical challenges to the bioanalyst, due to their low systemic concentration. The sensitivity of a bioanalytical LC/MS/MS based assay can be influenced by multiple parameters, including: mobile phase composition, extraction efficiency and chromatographic performance. In this work, we discuss the influence of acidic (pH 3), and basic (pH 10) aqueous mobile phases in conjunction with the two most common organic modifiers used in HPLC, acetonitrile and methanol, on the assay sensitivity of twenty-four probe pharmaceuticals in solvent and biological fluid extract. The study showed that when the test probe pharmaceuticals were analyzed with basic aqueous mobile phases compared to standard acidic conditions the following results were observed: increases in chromatographic peak area ranging from 1.2 to 9.6 fold for twenty-one of the test compounds as well as increased signal-to-noise for greater than seventy percent of the compounds. This observed increase in the MS response was not necessarily related to the later elution of the analyte in a higher organic composition under basic conditions. This was demonstrated as seven out of the twenty-four (approximately thirty percent) of the probe pharmaceuticals tested, eluted earlier, or with the same retention time, under basic conditions, and still produced a greater signal-to-noise when analyzed under these basic conditions. Also observed were decreases in chromatographic peak width, and increases in the retention time of very hydrophilic pharmaceutical compounds. The effect of the mobile phase combinations on the retention and MS response of the choline-containing phospholipids present in precipitated plasma was also investigated, as these analytes are a major source of interference when developing a bioanalytical assay.

Although enantiomers of 2-phenylpropionic acids (2-PPAs), or profens are important group of nonsteroidal anti-inflammatory drugs (NSAIDs) and have been in clinical use for many years, there is no literature covering its binding interaction in particular with cyclodextrins. NSAIDs are marketed as racemates, chiral discrimination and knowledge of enantiomeric bioavailability is essential. Circular dichroism (CD) spectroscopy is the technique of choice for elucidating chirality and monitoring and characterizing molecular recognition phenomena in solution. Methods em-ploying the fundamentals of the simultaneous measurements of absorbance and CD and a novel efficient titration method have been developed to study the binding of β-Cyclodextrin (β-CyD) and the two enantiomers of 2-PPA as a function of pH. The effect on physicochemical properties and bioavailability was investigated. The binding constant, stoichiometry and pKa for both the free and the bound drugs were determined using a Levenburg-Marquadt non-linear equation. The exact nature of the enantiomer discriminating interactions by cyclodextrins (CyDs) is not well understood. In this work, the interactions and co-conformations of both enantiomers of 2-PPA with β-CyD were explained and es-timated using spectroscopic variations upon complexation. The results indicated a change in the physicochemical prop-erties of 2-PPAs upon complexation and highlighted the enantioselective binding of β-CyD as a function of pH. The charge on the guest molecule and its stereochemistry are of great importance in regulating the stability of the guest/β-CyD complexes; hence the bioavailability of drugs. This work elucidates 2-PPAs/β-CyD binding interactions and highlights the effect of β-CyD on drugs with an effective novel method for binding titration and the potential of the simultaneous measurements of absorbance and CD in future chiral drug interactions studies.

Sub-2-µm particle size hydrophilic interaction liquid chromatography [HILIC] combined with mass spectrometry has been increasing in popularity as a complementary technique to reversed-phase LC for the analysis of polar analytes. The organic-rich mobile phase associated with HILIC techniques provides increases in compound ionization, due to increased desolvation efficiency during electrospray ionisation mass spectrometric (ESI-MS) analysis. Although recent publications illustrated selectivity and response comparisons between reversed-phase LC/MS and HILIC LC/MS, there are limited discussions evaluating the optimisation of the mass spectrometry parameters regarding analytes and alternative mobile phases. The use of acetone as an alternative organic modifier in HILIC has been investigated with respect to signal-to-noise in ESI-MS for a variety of polar analytes. Analyte reponses were measured based on a variety of cone and capillary voltages at low and high pH in both acetone and acetonitrile. In order to visualise compound behaviour in the ESI source, surface plots were constructed to assist in interpreting the observed results. The use of acetone in ESI is complicated at low m/z due to the formation of condensation products. Favourable responses were observed for certain analytes and we envisage offering an insight into the use of acetone as an alternative to acetonitrile under certain analytical conditions for particular compound classifications for small molecule analysis. We also highlight the importance of optimising source voltages in order to obtain the maximum signal stability and sensitivity, which are invariably, highly solvent composition dependent parameters. Copyright © 2011 John Wiley & Sons, Ltd.

The advent of liquid chromatography-tandem mass spectrometry (LC-MS/MS), with the sensitivity it confers, permits the analysis of both phase I and II drug metabolites that in the past would have been difficult to target using other techniques. These metabolites may have relevance to current analytical toxicology employing LC-MS/MS, and lorazepam was chosen as a model drug for investigation, as only the parent compound has been targeted for screening purposes. Following lorazepam administration (2 mg, p.o.) to 6 volunteers, metabolites were identified in urine by electrospray ionization LC-MS/MS, aided by the use of deuterated analogues generated by microsomal incubation for use as internal chromatographic and mass spectrometric markers. Metabolites present were lorazepam glucuronide, a quinazolinone, a quinazoline carboxylic acid, and two hydroxylorazepam isomers, one of which is novel, having the hydroxyl group located on the fused chlorobenzene ring. The quinazolinone, and particularly the quinazoline carboxylic acid metabolite, provided longer detection windows than lorazepam in urine extracts not subjected to enzymatic hydrolysis, a finding that is highly relevant to toxicology laboratories that omit hydrolysis in order to rapidly reduce the time spent on gas chromatography-mass spectrometry (GC-MS) analysis. With hydrolysis, the longest windows of detection were achieved by monitoring lorazepam, supporting the targeting of the aglycone with free drug for those incorporating hydrolysis in their analytical toxicology procedures.

A simple CE-C 4 D method has been developed for the determination of glucosamine by direct injection of human serum and pharmaceutical samples. Glucosamine was electrokinetically injected and analysed in its protonated form using 20 mM MES/His (pH 6) as background electrolyte in order to separate it from the matrix and to provide a better response to the C 4 D detector. Separation of glucosamine in human serum and pharmaceutical samples was performed in 3 min without the need for protein precipitation or matrix removal. Good precision in terms of %RSD for the migration time and peak area were less than 1.91% ( n = 10). The conductivity signal was linear with glucosamine concentration in the range 0.10–2.50 mg/mL, with a detection limit of 0.03 mg/mL. Recoveries of glucosamine in serum and pharmaceutical samples were 86.5–104.78%. The method was successfully applied for the determination of the glucosamine content in pharmaceutical formulations and validated with high performance liquid chromatography (HPLC). Good agreements were observed between the developed method, label values and the HPLC method. Glucosamine could be detected in spiked serum sample by direct injection. This was not possible by HPLC due to co-eluting interferences.

This work investigated the repeatability of column preparation for a reversed-phase C18 monolith, namely stearyl methacrylate-co-ethylene glycol dimethacrylate (SMA-EDMA). The columns were thermally polymerised using three commonly available heating devices (GC oven, hot air oven and water bath) and their chromatographic performance evaluated using micro-liquid chromatography for separation of five test compounds. Precision in terms of %RSD of retention times were 9.0, 6.5, and 12.5 using GC oven, hot air oven and water bath, respectively. Between-batch precision for the hot air oven ( n = 3 days) was less than 10.4% for retention time. The SMA-EDMA monolith was applied to the separation of tocopherol homologues by capillary electrochromatography. Usually tocopherol homologues cannot be completely separated by conventional reversed-phase C8- or C18-packed bed or C18-silica based monolithic columns. Polymer monolith has been shown to give remarkable selectivity towards the tocopherols compared to the conventional microparticulate phase and silica based monolith. Successful separation of the tocopherol isomers was achieved on the SMA-EDMA monolith without any column modification.

A rapid UPLC-MS/MS quantitative assay for the quantification of quinine and (3S)-3-hydroxyquinine requiring minimal sample pre-treatment - dilute-and-shoot type approach - has been developed. The assay was run at 0.6mL/min using gradient elution with (pH 10; 10mM) ammonium bicarbonate and methanol with a total cycle time of 2.5min on a 50mm×2.1mm ID, 1.7μm Acquity BEH column. Peak shapes were highly symmetrical allowing for accurate peak integration. Calibration curves for both analytes were constructed from 1.00 to 20.00ng/mL, yielding R(2) values >0.995. Intra- and inter-batch assay precision and accuracy were evaluated using 6 injections of QC solutions on 3 separate days (n=18) and were found to be within ±10% and 90-110% respectively. The method was shown to be suitable for quantitatively determining the ratio of quinine to (3S)-3-hydroxyquinine for a cohort of samples from an epidemiological study.

The activity of Cytochrome P450 3A4 (CYP3A4) enzyme is associated with many adverse or poor therapeutic responses to drugs. We used (1)H NMR-based metabonomics to identify a metabolic signature associated with variation in induced CYP3A4 activity. A total of 301 female twins, aged 45--84, participated in this study. Each volunteer was administered a potent inducer of CYP3A4 (St. John's Wort) for 14 days and the activity of CYP3A4 was quantified through the metabolism of the exogenously administered probe drug quinine sulfate (300 mg). Pre- and postintervention fasting urine samples were used to obtain metabolite profiles, using (1)H NMR spectroscopy, and were analyzed using UPLC--MS to obtain a marker for CYP3A4 induction, via the ratio of 3-hydroxyquinine to quinine (3OH-Q:Q). Multiple linear regression was used to build a predictive model for 3OH-Q:Q values based on the preintervention metabolite profiles. A combination of seven metabolites and seven covariates showed a strong (r = 0.62) relationship with log(3OH-Q:Q). This regression model demonstrated significant (p < 0.00001) predictive ability when applied to an independent validation set. Our results highlight the promise of metabonomics for predicting CYP3A4-mediated drug response.

Hydrophilic interaction chromatography (HILIC) has experienced increasing attention in recent years. Much research has been carried out in the area of HILIC separation mechanisms, column techniques and applications. Because of their good permeability, low resistance to mass transfer and easy preparation within capillaries, hydrophilic monolithic columns represent a trend among novel HILIC column techniques. This review attempts to present an overview of the preparation and applications of HILIC monolithic columns carried out in the past decade. The separation mechanism of various hydrophilic monolithic stationary phases is also reviewed.

Solvent systems for use with LC–MS often result in a compromise between chromatographic performance and mass spectrometric detection, exemplified here by a LC–MS/MS method development for the analysis of ephedrines in doping control. Ephedrines, frequently found in therapeutic and nutritional preparations, are among the most commonly administered doping agents in competitive sport. Improved separation of these hydrophilic, basic compounds, some of which are diastereoisomers, is achieved in reversed-phase LC by the use of a high pH mobile phase in order to suppress analyte ionisation, and thus alter their polarity, resulting in reduced peak tailing and enhanced retention. However, when coupled to an ESI-MS detector, this eluent composition generated a non-linear and poorly reproducible signal. APCI yielded greater stability and reproducibility and is here presented as an ion source for the analysis of basic compounds under conditions that suppress their ionisation. Errors as large as 49.3% were observed with ESI, compared with 15.4% generated using APCI, for pseudoephedrine over the calibration range (25–400 μg/mL) in urine with a simple dilution and injection of samples. These data highlight the importance of suitable MS conditions for stable performance, necessary for accurate quantification, without undue compromise to the LC separation.

In order to overcome the drawbacks associated with particle-based fluorinated stationary phases, a fluorinated cationic monolith was successfully synthesized by the in situ copolymerization of a 45% monomer mixture and 55% porogens with thermal initiation using 0.1% initiator with respect to the monomer mixture. The monomer mixture was composed of perfluorododecyl acrylate (49.75%), ethylene dimethacrylate (49.75%), and 0.5% of the quaternary amine acrylic monomer supporting an anodal electroosmotic flow. A tertiary porogenic solvent system consisting of 40% 1-propanol, 40% 1-octanol, and 20% 1-dodecanol was used. The resulting monolith column showed good characteristics in terms of morphology, permeability (pressure drop was 4.8-25.7 MPa between 5 and 25 μL/min), and the electroosmotic mobility (3.68 (±0.05)×10(-4) cm(2) v(-1) s(-1)). The monolith was tested for the separation of four phenolic compounds, three aromatic hydrocarbons, and one fluorine-containing compound. Good efficiencies (up to 135,000 (±4000) plate/m and resolution up to 2.6 (±0.1)) with short analysis time (< 9 min for tau-fluvalinate

The aim was to simultaneously extract, separate and detect not only the opioid methadone and its primary metabolites (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine and 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline), but also creatinine from urine, plasma and fingerprints. Creatinine is highly polar and analysis by RP chromatography using conventional stationary phases such as silica-bonded C8 and C18 is unsuitable. Hydrophilic stationary phases are increasingly being applied for the analysis of highly polar analytes, this chemistry being investigated as a suitable alternative. A hydrophilic interaction liquid chromatography phase column successfully retained creatinine and permitted the co-analysis of methadone and its metabolites by LC-MS/MS. Prior to analysis, an extraction protocol for urine and plasma was required but for fingerprint deposits this was not necessary. Alteration of sample pH, necessary to extract methadone, and its metabolites led to difficulties associated with the extraction of creatinine. This problem was addressed by first performing an SPE incorporating a hydrophilic interaction phase to extract creatinine, and the eluent then combined with the opioid extract from a mixed-mode cation exchange phase. The assay for creatinine, methadone and its primary phase I metabolites met validation criteria. LC-MS/MS analysis of creatinine and drug compounds together offers considerable advantages over traditional approaches that necessitate the quantification of creatinine using spectrophotometric approaches.

A novel zwitterionic hydrophilic porous poly(SPV-co-MBA) monolithic column was prepared by thermal co-polymerisation of 1-(3-sulphopropyl)-4-vinylpyridinium-betaine (4-SPV) and N,N'-methylenebisacrylamide (MBA). An HILIC/RP dual separation mechanism was observed on this optimised poly(SPV-co-MBA) monolithic column and the composition of the mobile phase corresponding to the transition from the HILIC to the RP mode was around 30% ACN in water. Higher hydrophilicity was achieved on this novel monolithic column compared to the poly(N,N-dimethyl-N-methacryloxyethyl-N-(3-sulphopropyl)ammonium betaine-co-ethylene dimethacrylate) monolithic column. Permeability studies showed slight swelling and/or shrinking with mobile phases of different polarity. As might be anticipated, a weak electrostatic interaction for charged analytes was also observed by studying the influence of mobile phase pH and salt concentration on their retention on the poly(SPV-co-MBA) monolithic column. The final optimised poly(SPV-co-MBA) monolith showed comparable selectivities to commercial ZIC-pHILIC phases for polar test analytes. Fast separation of five pyrimidines and purines was achieved in less than 1 min due to the high permeability of the monolithic column. Additionally, baseline separation of nine benzoic acid derivatives was also observed using either a pH or ACN gradient.

The use of fingerprints as an alternative biological matrix to test for the presence of drugs and/or their metabolites is a novel area of research in analytical toxicology. This investigation describes quantitative analysis for the benzodiazepine lorazepam and its 3-O-glucuronide conjugate in fingerprints following the oral administration of a single 2 mg dose of lorazepam to five volunteers. Creatinine was also measured to investigate whether the amount of drug relative to that of creatinine would help to account for the variable amount of secretory material deposited. Fingerprints were deposited on glass cover slips and extracted by dissolving them in a solution of dichloromethane/methanol, containing tetradeuterated lorazepam as an internal standard. The samples were evaporated, reconstituted with mobile phase and analysed by LC-MS/MS. Chromatography was achieved using an RP (C18) column for the analysis of lorazapem and its glucuronide, and a hydrophilic interaction column (HILIC) for the analysis of creatinine. Lorazepam and its glucuronide were only detected where ten prints had been combined, up to 12 h following drug administration. In every case, the amount of lorazepam glucuronide exceeded that of lorazepam, the peak amounts being 210 and 11 pg, respectively. Adjusting for creatinine smoothed the elimination profile. To our knowledge, this represents the first time a drug glucuronide has been detected in deposited fingerprints.

Fritless columns were fabricated by encapsulating packing material within monolith for capillary electrochromatography. They were applied for separation of 3,4-methylenedioxymethamphetamine and metabolites. The packing material was suspended in polymerisation mixture and then forced into a pre-treated capillary following by polymerisation. This made the preparation much easier, saved time, cost and showed less tendency towards bubble formation with better resolution than with packed column. Using this fritless column, migration time of all analytes increased with buffer concentration and amount of organic component in mobile phase, but decreased on increasing the pH from 3.0 to 4.5 and then increased above pH 4.5.

In-line solid-phase extraction (SPE) for capillary electrophoresis (CE) was investigated using a synthesized monolith and a commercial packing material. Terbutaline (TER) and 4-hydroxy-3-methoxy-methamphetamine (HMMA) with benzyl alcohol as the electro-osmotic flow marker were employed as model compounds. Two types of methacrylate-based monoliths, namely methacrylic acid-ethylene dimethacrylate and butylmethacrylate-ethylene dimethacrylate were examined. Preliminary results indicated that a non-aqueous separating medium is more suitable for these methacrylate monoliths than a purely aqueous medium (non-reproducible elution). However, coupling of the methacrylic acid-ethylene dimethacrylate with non-aqueous capillary electrophoresis could not provide good precision for the three model compounds. A packed-silica C18 SPE was also adopted by simply packing the C18 particles in situ in the separation capillary. Using an aqueous running buffer (10 mM phosphate buffer (PPB), pH 7), acceptable precision could be obtained with this type of SPE material. With a 10 min loading time and 20 min total analysis time, the pre-concentration factors were 333 and 1000 for TER and HMMA, respectively. The %RSD were less than 4.5 and 0.3 for the peak areas and migration times, respectively, for both HMMA and TER (n = 20).

Capillary electrochromatography suffered in its development because of difficulty in producing stable columns with good permeability. Variability in frit characteristics gave rise to non-reproducible capillaries whose fabrication was extremely difficult and time consuming. Monolithic stationary phases gained popularity in the early 1990s due to the fact that they were easy to fabricate and required no retaining frits. They were also able to be manufactured in a wide variety of chemistries which made them very interesting to the analytical chemist who is constantly looking for materials with different selectivity to the popular silica-based stationary phases.

In cancer patients, pharmacokinetic variations between individuals and within individuals due to impairments in organs’ function and other reasons such as genetic polymorphisms represent a major problem in disease management, which can result in unpredictable toxicity and variable antineoplastic effects. Addressing pharmacokinetic variations in cancer patients with liver dysfunction and their implications on anticancer and analgesic drugs, in addition to the use of advanced analytical techniques such as metabolomics and pharmacometabolomics, to monitor altered kinetic and discover metabolic biomarkers during therapeutic intervention will help in understanding and reducing pharmacokinetic variations of drugs in cancer patients as a step forward towards personalised medicine. Reviewing published literature addressing and/or related to complications resulting from altered pharmacokinetics (PKs) in cancer patients with liver dysfunction, anticancer and analgesic drugs, evaluating recent advances of pharmacokinetic detection using metabolomics/pharmacometabolomics and the challenges that are currently facing these techniques. The current situation presents a pressing need to reduce pharmacokinetic variations of drugs in cancer patients. Although most of the omics technologies are not entirely focussed on the study of pharmacokinetic variations and some studies are met with uncertainty, the use of pharmacometabolomics combined with other omics technology such as pharmacogenomics can provide clues to personalised cancer treatments by providing useful information about the cancer patient’s response to medical interventions via identification of patients’ dependent variables, understanding of correlations between individuals and population PKs, and therapy outcomes to achieve optimum therapeutic effects with minimum toxicity. We also propose an approach for PKs' evaluation using pharmacometabolomics.

In this study, aristolochic acid in different herbal medicines containing a mixture of varying herb species was identified through fingerprint pattern similarities. Aristolochic acid I and II are nephrotoxic compounds naturally present in the Aristolochia plant species that are commonly used in Chinese herbal medicines. Twenty-four commercially available herbal formulations were extracted into an aqueous solution and injected into a UPLC-MS system. All the samples were analysed by multiple reaction monitoring (MRM) to check for the presence of aristolochic acids I and II. The same samples were then fingerprinted using two different gradient methods and the chromatograms deconvoluted into retention time (RT) and masses for the chemicals present taking concentration into account. Statistical analysis of this data revealed that samples were highly heterogeneous, and that the main differences between the preparations were concentrations of polar compounds. A model was constructed where the samples could be separated into two groups differentiated by the presence of the two forms of aristolochic acid.

Background: Increased pressure to obtain more, higher sensitivity data from less sample is especially critical for large peptides, whose already optimized LC–MS methods are heavily challenged by traditional ligand-binding assays. Results: Critical bioanalytical assays were adapted to integrated microscale LC to reduce sample volumes while increasing sensitivity. Assays for teriparatide, glucagon and human insulin and five analogs were transferred from 2.1 mm analytical scale LC to a 150 µm scale system. This resulted in a 15–30 fold overall improvement in sensitivity derived from increased signal to noise, three to six fold reduction in injection volumes, and a two to five fold reduction in sample consumption. Conclusion: Integrated microscale LC reduces sample consumption while enabling single picomolar quantification for therapeutic and endogenous peptides.

A porous zwitterionic monolith was prepared by thermal copolymerization of N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine and ethylene dimethacrylate inside a 100-μm-i.d. capillary. The resulting monolith was evaluated as a hydrophilic liquid chromatography (HILIC) stationary phase. No evidence of swelling or shrinking of the monolith in different polarity solvents was observed. A typical HILIC mechanism was observed at higher organic solvent content (ACN% > 60%). The poly(SPE-co-EDMA) monolith showed very good selectivity for neutral, basic, and acidic polar analytes. For charged analytes, both hydrophilic interactions and electrostatic interactions contributed to their retention, which provide chromatographers more choice to optimize the separations.

A polar-embedded stationary phase (ULTIMA C18) has been investigated for the separation of alpha-, beta-, gamma- and delta-tocopherols by CEC in comparison with commercially available C(18) and C(30) n-alkyl RPs. The behavior of this stationary phase was tested for different mobile phases based on methanol, ACN, or mixtures thereof and different separation parameters such as retention factors and resolution were evaluated. The main feature of this stationary phase is the improved selectivity for the separation of beta- and gamma-tocopherols (positional isomers) when compared with the pure n-alkyl C(18) material, which was unable to resolve these compounds. Additionally, it is possible to observe a reversal in the elution order of the beta- and gamma-tocopherol isomers with respect to that obtained on the C(30) column. The resulting data indicate that the enhanced selectivity obtained with the polar-embedded stationary phase, with respect to the conventional C(18) material, is due to the participation of both hydrophobic and polar interactions: these latter are of the hydrogen bridge type with the amide group of the polar-embedded stationary phase, which increases the retention of the tocopherols and facilitates the discrimination between the beta- and gamma-isomers. Adequate separation of the four tocopherols was obtained by CEC using the polar-embedded stationary phase and 95:5 v/v methanol/water (5 mM Tris, final concentration) as the mobile phase.

Using normal phase solvents with small percentages of water, it was shown that an electroosmotic flow (EOF) could be generated and these solvents can be used to separate a normal phase test mixture on a silica stationary phase. Run times were reduced further by pressure assisted capillary electrochromatography (CEC). This knowledge was then transferred to the chiral stationary phase Whelk-O 1 where it was used successfully to separate of a number of chiral analytes. The effects of the different contributions of pressure and voltage on the linear velocity were investigated. Altering the applied pressure and reducing the length of the packed bed then optimized the separation of the enantiomers of the chiral compound benzoin. For the column with the shorter packed bed, run times of 6 minutes were observed, whilst still retaining good resolution.

Capillary electrochromatography (CEC) is classed as a hybrid technique between CE and HPLC and it combines the advantages of both these techniques. However, in some cases the disadvantages are also brought to light and some of these are difficult to resolve. For example the analysis of basic compounds using CEC. The problems of tailing peaks during HPLC analysis of basic compounds was resolved by end capping the residual silanol groups, but in CEC these are the groups that generate the electroosmotic flow. The analysis of basic compounds is crucial within the pharmaceutical industry where a high percentage of the drug actives are basic. Specially designed Continuous Beds stationary phases (CB) can mean that each application can have a specific stationary phase. In order to overcome the problem associated with the analysis of basic compounds using electrochromatography, we have designed a CB stationary phase with a positive charge, which could be operated using negative voltage. The resulting chromatography showed almost gaussian peaks for bases like nortriptyline which tail significantly using stationary phase typically used in CEC.

The use of wide-pore stationary phases in capillary electrochromatography has shown exceptional increases in separation efficiency in conjunction with high electroosmotic flow. These effects are due to the perfusive flow mechanism which is primarily controlled by the ionic strength of the mobile phase. Good correlation between calculated values of electrochemical double-layer thickness and efficiency data have also been obtained. Reduced plate height values of0.5 have been observed with pore sizes of 4000 A. In addition, electroosmotic flow mobility twice that of 3 microm Spherisorb ODS-1 has been obtained.

A system is presented that demonstrates the principle of on-line and on-chip post-column derivatisation reactions in micro-high-performance liquid chromatography (micro-HPLC) hyphenated to electrospray time-of-flight mass spectrometry (ESI-TOFMS). In this micro-HPLC-chip-MS set-up, the analytes are separated using gradient micro-HPLC and subsequently derivatised on-chip and detected. One of the major limitations of MS detection is its dependency on the degree of ionisation, which is widely variable and compound-specific. Optimising and controlling the degree of ionisation in a simple manner would allow MS detection to be truly generic. One way of achieving this is by pre-ionisation of analytes using simple derivatisation procedures that are both rapid and quantitative. Performing this in situ on the system described here overcomes issues of sample handling and efficiency losses when time-consuming "bench chemistry" is necessary prior to analysis. The power of the system is demonstrated by the separation of primary and secondary amines, which are subsequently derivatised with a positively charged phosphonium complex and detected in an enhanced manner. Typically, molecular cations (M(+)) are detected showing that the ionisation process is dominated by the phosphonium species, leading to more constant ionisation for a variety of compounds. In addition, stable isotopically labelled ((12)C/(13)C)-phosphonium reagent is used for the reactions, allowing for inherent signal/noise (S/N) improvement and automated data processing using cluster analysis. A similar reaction scheme is used for the derivatisation of ketones and aldehydes, also demonstrating dramatic increases in sensitivity, especially with increasing temperature. Minimal loss in chromatographic fidelity in terms of retention times is observed by the introduction of the micromixer chip into the system. Optimal flow rates in micro-HPLC and ESI-MS are compatible with flow rates for the chip as well as a multitude of in-line optical detectors including UV and fluorescence. In addition, the micromixer chip can be positioned pre-column if preferred. The system is robust, easily fully automated and applicable to a wide variety of reactions. The system has a major advantage in its simple robust connection to the "normal scale" outside world.

There has recently been increased interest in coupling ion chromatography (IC) to high resolution mass spectrometry (HRMS) to enable highly sensitive and selective analysis. Herein, the first comprehensive study focusing on the direct coupling of suppressed IC to HRMS without the need for post-suppressor organic solvent modification is presented. Chromatographic selectivity and added HRMS sensitivity offered by organic solvent-modified IC eluents on a modern hyper-crosslinked polymeric anion-exchange resin (IonPac AS18) are shown using isocratic eluents containing 5-50 mM hydroxide with 0-80% methanol or acetonitrile for a range of low molecular weight anions (

Our aim was to develop a fast and efficient on-line method using micro-reactors for the digestion and deglycosylation of recombinant human erythropoietin extracted from equine plasma. The trypsin digestion micro reactors were fabricated using fused silica capillaries with either a dextran-modified coating or a porous monolith that was able to immobilise the enzyme. These were both found to be reasonably robust and durable, with the trypsin immobilised on dextran-modified fused silica capillaries offering better reproducibility than the micro-reactor based upon covalent attachment of this enzyme to the polymer. It is also evident that the enzyme attached micro reactors produced some tryptic peptides in a greater yield than in-solution digestion. A peptide-N-glycosidase F reactor was also fabricated and, when coupled with the trypsin reactor, the deaminated peptides T5 DAM and T9 DAM from recombinant human erythropoietin could also be detected by LC–ESI–MS/MS analysis. These results were better than those achieved using off-line digestion plus deglycosylation reactions and the analysis required far less time and effort to complete. The use of this on-line approach improved the sensitivity, efficiency and speed of our confirmation methodology that is based upon detecting the unique peptide segments of recombinant human erythropoietin that has been affinity extracted from positive equine plasma samples.

Capillary electrochromatography (CEC) is a versatile new technique used to complement other separation techniques like HPLC. The main advantage of CEC over HPLC is the electrically driven flow, which generates a flat flow profile and provides sharper peaks and better efficiencies. In CEC the flow profiles within the channels between the particles are plug-like (as in CE), In HPLC (pressure driven flow) the flow profiles within similar channels are parabolic leading to band broadening. Hence efficiencies seen in HPLC are not as good as those obtained from CEC. This advantage along with the suitability of CEC with miniaturisation has led to extensive development of the technique. CEC has progressed significantly in recent years and numerous examples of CEC applications have been published. However most of the work has been carried out in a research environment. In order to progress the technique in the pharmaceutical industry it is necessary to demonstrate that CEC can be a robust and reliable technique. In this article we discuss the validation and robustness testing of a capillary electrochromatography method used for the determination of impurities in a potential drug substance. The CEC method was developed and validated in accordance with the ICH guidelines. The data generated was compared with the data from the existing HPLC method. Overall results show that CEC can be used in a highly regulated environment.

Separations of compounds in plasma were performed on a Hypersil Duet C18/SCX capillary electrochromatography (CEC) column, utilizing an automated injection system combined with a short in-house designed and fabricated micro-electrospray CEC mass spectrometer interface. Protein precipitation was used prior to the CEC separations. More than two hundred separations of the corticosteroids Dexamethasone and Betamethasone 17-valerate, and Fluticasone Propionate in complex plasma matrix were performed on a single column under isocratic conditions. The method demonstrated good reproducibility, selectivity, sensitivity and high efficiencies. Linear calibration with good correlation was typical. Estimated detection limits in the low micromolar and nanomolar range for all compounds were obtained using UV-Vis absorbance (UV) and Electrospray Mass Spectrometric (ES/MS) detection respectively. Efficiencies for all compounds were typically 87,500 plates on a 25 cm column (350,000 plates m−1) and increased with the number of plasma samples injected, up to 250,000 plates per column (1,000,000 plates m−1). These very high observed plate counts may be artificially enhanced by the inadequate scan possibilities of the MS over very narrow chromatographic peaks.

Over 95% of all papers published to the present time on capillary electrochromatography (CEC) have reported on the use of C 18 and to a much lesser extent, C 8 stationary phases. The relationship between the electroosmotic flow and pH is of considerable importance in CEC because it allows the analyst to develop methods under optimum conditions, especially with respect to analysis times. Although the electroosmotic flow dependence on pH for C 18 phases has been reported, there have been very few comparable studies carried out on other phases, and for this reason a systematic study of the performance of a wide range of stationary phases was initiated. The phases studied were Waters 3 μm Spherisorb ODS-1, propyl SCX, phenyl SCX, Symmetry SCX and a C 6 /SCX mixed-mode. Important new knowledge regarding the performance of CEC columns has been realised through this study, the first results of which are now reported.

The separation and quantification of hydrophilic basic compounds continues to challenge reversed-phase chromatography. Ephedrines are an example where the optimal separation of their isomers and related substances is complicated due to both their hydrophilicity and basic nature. Here we study two potential ultra-high pressure liquid chromatography (UHPLC) methods and present the merits and limitations of a high pH reversed-phase and a hydrophilic interaction liquid chromatography (HILIC) approach for the separation and quantification of ephedrines for doping control analysis. The study compares a hybrid silica material used for the HILIC separations with a C18 reversed-phase material produced from the same hybrid silica. While both analytical approaches provide good retention and resolution, HILIC offers benefits in terms of peak shape, sample loading capacity and enhanced sensitivity with electrospray ionisation-mass spectrometry (ESI-MS). HILIC permits favourable kinetic performance owing to the low viscosity mobile phase and hence better mass transfer characteristics. Common problems associated with HILIC including retention shifts and undesirable peak shapes are investigated and overcome using a suitable re-equilibration time and injection solvent. Validation data show both approaches provide good linearity (r>0.995), accuracy (RPLC