Effect of seaweed supplementation on tocopherol concentrations in bovine milk using dispersive liquid-liquid microextraction.

A dispersive liquid-liquid microextraction (DLLME) method, combined with HPLC-UV detection, was developed for the extraction and preconcentration of δ-tocopherol from bovine milk. This method was used to study the effect of supplementing cow feed with the seaweed Ascophyllum nodosum on vitamin content in milk. The optimal experimental conditions were determined: 200 µL of chloroform (extraction solvent), 1.0 mL of ethanol (dispersive solvent), 5 mL of water (aqueous phase). Under these optimal conditions the DLLME method provided linearity in the range 0.01 µg/mL to 8 µg/mL with R2 values of 0.998. Limit of detection (LOD) was 0.01 µg/mL, while the enrichment factor was 89. Cow feed that was supplemented with Ascophyllum nodosum was shown to increase δ-tocopherol levels from 3.82 µg/mL to 5.96 µg/mL.

The application of dispersive liquid-liquid microextraction in the analyses of the fatty acid profile in bovine milk in response to changes in body condition score.

Dispersive liquid-liquid microextraction (DLLME) was used prior to gas chromatography flame ionization detection (GC-FID) for the extraction of five fatty acids from milk taken from cows with different body condition scores. Optimum extraction conditions were: 300 µL of chloroform (extraction solvent), and 1 mL methanol (dispersive solvent). The procedure was optimised using Design of Experiments (DoE). The analytes were separated on a GC capillary column containing a polyethylene glycol stationary phase (15 m × 0.53 mm × 1.2 µm). Enrichment factors were in the range of 8-15 and limit of detection (LOD) was 0.04 µg/mL. Calibration graphs showed good linearity with coefficients of determination higher than 0.994% and relative standard deviations lower than 7%. This method provided a simple and rapid derivatisation and extraction method for the determination of fatty acids in bovine milk. It showed that there was a significant difference in the palmitic acid content of milk from cows that had an optimum body condition score (10.85 mg/mL) compared to cows that had a high body condition score (5.73 mg/mL).

Polystyrene-co-Divinylbenzene PolyHIPE Monoliths in 1.0 mm Column Formats for Liquid Chromatography.

The reversed phase liquid chromatographic (RP-HPLC) separation of small molecules using a polystyrene-co-divinylbenzene (PS-co-DVB) polyHIPE stationary phases housed within 1.0 mm i.d. silcosteel columns is presented within this study. A 90% PS-co-DVB polyHIPE was covalently attached to the walls of the column housing by prior wall modification with 3-(trimethoxysilyl) propyl methacrylate and could withstand operating backpressures in excess of 200 bar at a flow rate of 1.2 mL/min. Permeability studies revealed that the monolith swelled slightly in 100% acetonitrile relative to 100% water but could nevertheless be used to separate five alkylbenzenes using a flow rate of 40 µL/min (linear velocity: 0.57 mm/s). Remarkable column-to-column reproducibility is shown with retention factor variation between 2.6% and 6.1% for two separately prepared columns.

Fabrication of a GMA-co-EDMA Monolith in a 2.0 mm i.d. Polypropylene Housing.

Polymers are interesting housing materials for the fabrication of inexpensive monolithic chromatography and solid phase extraction (SPE) devices. Challenges arise when polymeric monoliths are formed in non-conical, cylindrical tubes of larger diameter due to potential monolith detachment from the housing wall resulting in loss of separation performance and mechanical stability. Here, a two-step protocol is applied to ensure formation of robust homogeneous methacrylate monolith in polypropylene (PP) tubing with a diameter of 2.0 mm. Detailed Fourier-transform infrared (FTIR) spectroscopic analysis and Scanning Electron Microscopy (SEM) imaging confirm the successful pre-modification of the tubing wall with an anchoring layer of cross-linked ethylene dimethacrylate (EDMA). Subsequent formation of an EDMA-glycidyl methacrylate (GMA) monolith in the PP tube resulted in a homogeneous monolithic polymer with enhanced mechanical stability as compared to non-anchored monoliths.

Stepped gradients on polymeric monolithic columns by photoinitiated grafting.

The surface of polymethacrylate monoliths was functionalized by a post-polymerization modification, by means of a novel photo-initiated graft procedure where the charged monomer, sulfopropyl methacrylate, was controllably grafted stepwise, i.e. with incremental graft energies. The grafting approach was optimized using scanning capacitively coupled contactless conductivity detection. The effect of the localized ion exchange capacity and resultant gradient stationary phase upon ion-exchange chromatographic retention, selectivity, and performance was investigated, and compared to a homogeneously grafted (isotropic) column. The gradient column provided reduced peak widths at half height for both cationic analytes, with a reduction of 34 and 33%, respectively, when compared to the isotropic column.

Development of a silica monolith modified with Fe3O4 nano-particles in centrifugal spin column format for the extraction of phosphorylated compounds.

In this study, citrate-stabilised iron oxide nano-particles (∼16 nm) have been immobilised on commercial silica monolithic centrifugal spin columns (MonoSpin) for the extraction of phosphorylated compounds. Two alternative strategies were adopted involving either direct electrostatic attachment to an aminated MonoSpin (single-layer method) in the first instance, or the use of a layer-by-layer method with poly(diallyldimethylammonium) chloride. Field-emission scanning electron spectroscopy and energy-dispersive X-ray spectroscopy was used for confirming notably higher coverage of nano-particles using the layer-by-layer method (2.49 ± 0.53 wt%) compared with the single-layer method (0.43 ± 0.30 wt%). The modified monolith was used for the selective separation/extraction of adenosine monophosphate, adenosine diphosphate and adenosine triphosphate with elution using a phosphate buffer. A reversed-phase liquid chromatographic assay was used for confirming that adenosine, as a non-phosphorylated control was not retained on the modified MonoSpin devices, whereas recovery of 80% for adenosine monophosphate, 86% for adenosine diphosphate and 82% for adenosine triphosphate was achieved.

Development and validation of a new stability indicating reversed phase liquid chromatographic method for the determination of prednisolone acetate and impurities in an ophthalmic suspension.

A new stability indicating reversed phase high performance liquid chromatography (RP-HPLC) method was developed and validated under current International Conference of Harmonisation (ICH) guidance for the determination of prednisolone acetate (PAC) and impurities in an ophthalmic suspension. The developed method is presented as an alternative to a modified version of the current RP-HPLC method described in the USP monograph for the assay of PAC in an ophthalmic suspension. Along with the assay of PAC, the new method is also capable of identifying and quantifying eight selected PAC impurities and degradation products in an ophthalmic suspension. Using an Agilent Poroshell 120 EC-C18 100 mm × 4.6mm (dp: 2.7 µm) column set to 60°C with step gradient elution generated using mobile phase A: acetonitrile/water (10:90) (v/v) and mobile phase B: acetonitrile delivered at 1.2 mL min(-1), all peaks of interest are eluted in 33 min with resolution of 1.5 between the critical pairs. The developed method was validated for PAC and impurities to ICH recommendations for accuracy, linearity, precision (repeatability), limit of detection, limit of quantitation, robustness and specificity.

Determination of (R)-timolol in (S)-timolol maleate active pharmaceutical ingredient: validation of a new supercritical fluid chromatography method with an established normal phase liquid chromatography method.

An enantioselective supercritical fluid chromatography (SFC) method was developed and validated to meet the current European Pharmacopoeia requirements of a limit test for the determination of S-timolol maleate enantiomeric purity in timolol maleate drug substance. The developed method is presented as an alternative to the current normal phase high performance liquid chromatography (NP-HPLC) method described in the European Pharmacopoeia (Timolol Maleate Monograph). Using a 4.6mm×250mm Chiralcel OD-H (dp: 5µm) column and a mobile phase of (93:7) CO2/0.1% (v/v) TEA in MeOH delivered at 4.0mLmin(-1) resolution of 2.0 was achieved within 5min, representing a 3-fold reduction in run-time and an 11-fold reduction in solvent consumption relative to the NP-HPLC method. Method robustness was examined by the variation of flow rate (±0.5mLmin(-1)), column temperature (±5°C) and column back-pressure (±10bar) and resolution was maintained at ≥1.9 in all cases. R-timolol was resolved from all potential impurities and the limit of detection was improved by increasing the sample concentration threefold compared to the NP-HPLC method such that the method could detect the R-timolol enantiomer at 0.5% (w/w) with respect to S-timolol maleate. Additional validation parameters demonstrated that the potential of the method to be used for routine release testing of timolol maleate raw material for drug product manufacturing in which the quantitation of R-timolol impurity in S-timolol maleate drug substance would be a requirement.

Nano-particle modified stationary phases for high-performance liquid chromatography.

This review covers the latest developments and applications of nano-materials in stationary phase development for various modes of high-performance liquid chromatography. Specific attention is placed upon the development of new composite phases, including the synthetic and immobilisation strategies used, to produce either encapsulated nano-particles, or surface attached nano-particles, layers, coatings and other structures. The resultant chromatographic applications, where applicable, are discussed with comment upon enhanced selectivity and/or efficiency of the nano-particle modified phases, where such effects have been identified. In the main this review covers developments over the past five years and is structured according to the nature of the nano-particles themselves, including carbonaceous, metallic, inorganic, and organopolymer based materials.

Iminodiacetic acid functionalised organopolymer monoliths: application to the separation of metal cations by capillary high-performance chelation ion chromatography.

Lauryl methacrylate-co-ethylene dimethacrylate monoliths were polymerised within fused silica capillaries and subsequently photo-grafted with varying amounts of glycidyl methacrylate (GMA). The grafted monoliths were then further modified with iminodiacetic acid (IDA), resulting in a range of chelating ion-exchange monoliths of increasing capacity. The IDA functional groups were attached via ring opening of the epoxy group on the poly(GMA) structure. Increasing the amount of attached poly(GMA), via photo-grafting with increasing concentrations of GMA, from 15 to 35%, resulted in a proportional and controlled increase in the complexation capacity of the chelating monoliths. Scanning capacitively coupled contactless conductivity detection (sC(4)D) was used to characterise and verify homogenous distribution of the chelating ligand along the length of the capillaries non-invasively. Chelation ion chromatographic separations of selected transition and heavy metals were carried out, with retention factor data proportional to the concentration of grafted poly(GMA). Average peak efficiencies of close to 5,000 N/m were achieved, with the isocratic separation of Na, Mg(II), Mn(II), Co(II), Cd(II) and Zn(II) possible on a 250-mm-long monolith. Multiple monolithic columns produced to the same recipes gave RSD data for retention factors of <15% (averaged for several metal ions). The monolithic chelating ion-exchanger was applied to the separation of alkaline earth and transition metal ions spiked in natural and potable waters.

Polymeric monolithic materials modified with nanoparticles for separation and detection of biomolecules: a review.

Polymer monolithic materials have found particularly utility in the field of bioanalysis, particularly in the area of separation science, for both enrichment and trapping of biomolecules and their analytical/preparative separations. Nanoparticle-modified monoliths have recently emerged as a new class of substrate, with unique characteristics and structure, and with selectively tailored surface chemistries for target molecules. Although several reviews exist on the applications of nanoparticles in analytical science, this review is the first to specifically summarise the applications in bioanalysis of nanoparticle-modified polymer monolithic materials. The review covers the range of nanoparticles being utilised in this way, their specific applications and future trends.

Separation of selected transition metals by capillary chelation ion chromatography using acetyl-iminodiacetic acid modified capillary polymer monoliths.

Capillary housed laurylmethacrylate-co-ethylene dimethacrylate (LMA-co-EDMA) polymer monoliths were fabricated, functionalised with varying amounts of vinyl azlactone, followed by immobilisation of iminodiacetic acid (IDA), forming a range of acetyl-iminodiacetic acid (AIDA) functionalised monoliths, applied to the chelation ion chromatographic separation of selected transition and heavy metals. A number of monoliths of varying length and ligand density were prepared, resulting in increased cation retention and chromatographic resolution on those displaying the highest capacity. Ligand density and related column capacity were confirmed visually using scanning capacitively coupled contactless conductivity detection (sC(4)D) techniques. Column temperature studies to determine retention mechanism and the effect of temperature on the retention of Mn(II), Cd(II) and Cu(II) was investigated, showing an increase in retention with increased temperature for Cd(II) and Cu(II), whilst a decrease in retention was obtained for Mn(II). Isocratic capillary chelation ion chromatographic separations of Mn(II), Cd(II) and Cu(II) were obtained, with dual peak detection demonstrated using combined on-column C(4)D detection and UV-Visible detection following the post-capillary column reaction of the eluted metals with 4-(2-pyridylazo) resorcinol (PAR).

Production of novel polymer monolithic columns, with stationary phase gradients, using cyclic olefin co-polymer (COC) optical filters.

Polymer monolithic columns with controlled surface ligand density, providing stationary phase gradients within monolithic capillary columns, have been developed using photo-grafting through optical filters. Utilising commercially available cyclic olefin co-polymer (COC) films, the production of an optical filter capable of attenuating UV irradiation, in a tailored manner, was investigated. This novel optical filter was successfully applied to the surface modification of poly(BuMA-co-EDMA) monolithic columns in a multi-step grafting procedure. Fabricated columns were subjected to scanning capacitively coupled contactless conductivity (sC(4)D), to determine the distribution of the grafted functional groups, axially along the column. Further modification to produce a chelating stationary phase gradient of iminodiacetic acid (IDA) was demonstrated. To demonstrate the distribution of the IDA sites, a metal cation (Cu(2+)) was complexed to the IDA forming a chelate. Upon the formation of a complex of IDA with Cu(2+), an overall drop in conductive response was observed. The COC optical filter was also used in the fabrication of a grafted gradient of strong cation exchanger (SCX), sulphopropyl methacrylate (SPM) upon a polymer monolith, demonstrating the broader applicability of such a filter.

Effect of water during the quantitation of formate in photocatalytic studies on CO2 reduction in dimethylformamide.

The effect of the water concentration on the quantitation of formate from dimethylformamide in the presence of electron-donating bases using ion chromatography is reported. This observation has important implications in the area of the photocatalytic reduction of CO(2), where formate levels are often used to calculate catalyst turnover numbers.

Pipette-tip selective extraction of glycoproteins with lectin modified gold nano-particles on a polymer monolithic phase.

The in situ preparation of ethylene dimethacrylate porous polymer monoliths within 20 µL polypropylene pipette tips, bound via surface grafted methacrylate anchor sites, is reported. Gold nano-particles (AuNPs) were immobilised onto the monolith pore surface utilising azlactone chemistry and coverage verified using field emission scanning electron microscopy. Erythrina cristagalli lectin (ECL) was immobilised upon the attached AuNPs via a bio-functional linker. The ECL-modified tip was successfully applied for the enrichment of galactosylated protein (desialylated transferrin) versus a non-galactosylated protein (ribonuclease B) due to the specificity of ECL. Reversed-phase capillary HPLC was used to validate the efficiency and selectivity of the developed micro-extraction phase which resulted in an increase in extraction recovery of ∼95% due to the AuNP enhanced surface area. Further specificity of the ECL-modified tip was demonstrated with a complex mixture of non-glycosylated and glycosylated proteins with differing terminal sugar structures. Finally, the lectin affinity phase was applied to a galactosylated glycoproteins spiked Escherichia coli cell lysate to successfully demonstrate matrix tolerance.

Versatile capillary column temperature control using a thermoelectric array based platform.

A new direct contact platform for capillary column precise temperature control based upon the use of individually controlled sequentially aligned Peltier thermoelectric units is presented. The platform provides rapid temperature control for capillary and microbore liquid chromatography columns and allows simultaneous temporal and spatial temperature programming. The operating temperature range of the platform was between 15 and 200 °C for each of 10 aligned Peltier units, with a ramp rate of approximately 400 °C/min. The system was evaluated for a number of nonstandard capillary based applications, such as the direct application of temperature gradients with both linear and nonlinear profiles, including both static column temperature gradients and temporal temperature gradients, and the formation of in-capillary monolithic stationary phases with gradient polymerization through precise temperature control.

Liquid chromatographic profiling of monosaccharide concentrations in complex cell-culture media and fermentation broths.

A solid phase extraction, liquid chromatography and fluorescence (SPE-RPLC-FL) based protocol for the determination of free monosaccharides in highly complex raw material powders and formulated fermentation feedstocks and broths has been developed. Monosaccharides within sample extracts were derivatised pre-column with anthranilic acid and the derivatives separated using reversed-phase LC with fluorescence detection. Using a 2.1 mm × 50 mm 1.8 µm Zorbax Eclipse XDB-C18 column, a flow rate of 0.4 mL min-1 and an acetonitrile gradient in a sodium acetate buffer (pH 4.3; 50 mmol L-1) the baseline resolution of glucosamine, mannosamine, galactosamine, galactose, mannose, glucose, ribose, xylose, fucose and sialic acid within 20 minutes was achieved. Pre-column derivatisation involved combining a 30 mg mL-1 solution of anthranilic acid in a 1 : 1 ratio with an aqueous standard prior to injection. Standard analytical performance criteria were used for evaluation purposes, with the method found to exhibit LOD's as low as 10 fmol, and be linear and precise (%RSD < 2.2% (n = 7). The method was applied to the analysis of a range of highly complex biopharmaceutical production samples, including yeastolate powders, chemically defined media and in-process fermentation broth samples. Sample preparation involved passing an aqueous sample through a C18 solid phase extraction cartridge to trap hydrophobic peptides and vitamins, with recovery of all test sugars exceeding 90%. Finally, standard statistical analysis was performed on samples taken from different lots in order to estimate lot-to-lot variability.

Development of a rapid and sensitive method for determination of cysteine/cystine ratio in chemically defined media.

Two rapid, sensitive and quantitative methods for the determination of the cysteine and cystine ratio in complex defined media feedstock using monolithic reversed-phase liquid chromatography (RPLC) and RPLC-MS are presented. Cysteine is pre-derivatised with purified 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) and separated from other derivatisation products on a narrow-bore 50mmx2mm I.D. monolithic C(18) column with UV detection at 355nm. For reversed-phase LC (RPLC) the separation is carried out isocratically using a mobile phase of 50mM trichloroacetic acid (TCA) adjusted to pH 2.5 with lithium hydroxide (LiOH) and acetonitrile (83:14) pumped at 1.5mL/min with an elevated column temperature. For RPLC-MS an ammonium acetate and acetonitrile gradient method was developed with a reduced flow rate of 0.3mL/min. The treatment of the samples consisted of dividing them into two aliquots, the first aliquot is analysed for cysteine and the second aliquot is analysed for cystine after its quantitative reduction to cysteine using tris(2-carboxyethyl)phosphine (TCEP). Both methods are linear, with R(2)>0.999 for 0.25-500microM for cysteine and 0.25-250microM for cystine using the LC-UV method, sensitive, with detection limit of 36nM for cysteine, and precise, with < or =1.1% RSD for both retention time and peak area (n=6). Samples (n=31) of an industry standard and supplied chemically defined media feedstock were analysed, finding cysteine ranging from 1.56 to 2.26microg/mL and cystine from 1062.02 to 1348.13microg/mL.

High-capacity gold nanoparticle functionalised polymer monoliths.

Gold nanoparticles have been immobilised on a methacrylate monolith via grafted polymer chains terminated with amine groups, resulting in a homogenous and dense surface coverage.

Micro-bore titanium housed polymer monoliths for reversed-phase liquid chromatography of small molecules.

A new method for the fixation of polymethacrylate monoliths within titanium tubing of up to 0.8 mm I.D. for use as a chromatographic column under elevated temperatures and pressures is described. The preparation of butyl methacrylate-ethylene dimethacrylate-based monolithic stationary phases with desired porous structures was achieved within titanium tubing with pre-oxidised internal walls. The oxidised titanium surface was subsequently silanised with 3-trimethoxysilylpropyl methacrylate resulting in tight bonding of butyl methacrylate porous monolith to the internal walls, providing stationary phase stability at column temperatures up to 110 degrees C and at operating column pressure drops of >28 MPa. The titanium housed monoliths exhibited a uniform and dense porous structure, which provided peak efficiencies of up to 59,000 theoretical plates per meter when evaluated for the separation of small molecules in reversed-phase mode, under optimal conditions (achieved at 15 microL/min and temperature of 110 degrees C for naphthalene with a retention factor, k=0.58). The developed column was applied to the reversed-phase isocratic separation of a text mixture of pesticides.