Synchrotron Radiation Microcomputed Tomography Guided Chromatographic Analysis for Displaying the Material Distribution in Tablets.

One unusual and challenging scientific field that has received only cursory attention to date is the three-dimensional (3D) microstructure and spatial distribution of drug(s) and formulation materials in solid dosage forms. This study aims to provide deeper insight into the relationships between the microstructure of multiple-unit pellet system (MUPS) tablets and the spatial distribution of the active pharmaceutical ingredient (API) and excipients to facilitate the design of quantitative models for drug delivery systems. Synchrotron radiation X-ray microcomputed tomography (SR-µCT) was established as a 3D structure elucidation technique, which, in conjunction with liquid chromatography coupled to mass spectrometry (LC-MS) or liquid chromatography with evaporative light-scattering detector (LC-ELSD) enables chemical analysis of tablets. On the basis of the specific interior construction of theophylline MUPS tablets, the spatial distribution of materials was acquired by quantifying microregion samples that had been validated by SR-µCT for their locations in the MUPS tablets. The 3D structure of the MUPS tablets was catalogued as three structural domains: a matrix layer (ML), a protective cushion layer (PCL), and pellets (PL). Compared with the components in the ML, components in the PL had a larger proportion of theophylline, sucrose, and diethyl phthalate and a smaller proportion of lactose and sodium lauryl sulfate, whereas glyceryl monostearate was found to account for a large portion of the PCL. Microstructural characterization-guided zonal chemical determination represents a new approach for quality assessment and the development of drug delivery systems with in-depth insight into their constituent layers on a new scale.

Enhanced stability of vitamin A palmitate microencapsulated by γ-cyclodextrin metal-organic frameworks.

γ-Cyclodextrin metal-organic frameworks (γ-CD-MOFs) are highly porous and bio-friendly novel materials formed by γ-CD as an organic ligand and potassium ion as an inorganic metal centre. The aim of this study was to enhance the stability of vitamin A palmitate (VAP) using γ-CD-MOFs as the carrier. Herein, γ-CD-MOFs displayed VAP microencapsulating capacity of 9.77 ± 0.24% with molar ratio as nMOFs:nVAP = 3.2:1.0. It was important to find that the improved stability of VAP microencapsulated by γ-CD-MOFs without addition of any antioxidant(s) was better than that of the best available reference product in the market, with 1.6-fold elongated half-life. The protecting mechanism of γ-CD-MOFs for VAP contributed that VAP molecules preferentially curled inside the cavities of dual γ-CD pairs in γ-CD-MOFs. It was proved that γ-CD-MOFs were an efficient new carrier to deliver and protect VAP for food and pharmaceutical applications.

A stable hydrocortisone nanosuspension for improved dissolution: Preparation, characterization and in vitro evaluation.

Drug nanosuspensions have gained tremendous attraction as a platform in drug delivery. In the present work, a nanosuspension was prepared by a wet milling approach in order to increase saturation solubility and dissolution of the water insoluble drug, hydrocortisone. Size of the generated particeles was 290 nm ± 9 nm having a zeta potential of -1.9 mV ± 0.6 mV. Nanosized particles were found to have a rod shape with a narrow particle size distribution (PDI =0.17). Results of differential scanning calorimetry and X-ray diffraction analyses revealed minor modifications of crystallinity of hydrocortisone following the milling process. Solubility of hydrocortisone was enhanced by nanonization to 875µg/ml ±2.5, an almost 2.9-fold compared to the raw hydrocortisone. Moreover, the nanosuspension formulation substabtially enhanced the dissolution rate of hydrocortisone where >97% of the hydrocortisone was dissolved within 10 minutes opposed to 22.3% for the raw 50% for the raw hydrocortisone and the commercial tablet, respectively. The bioavailability study resulted in AUC 0-9h for HC nanosuspensions (31.50±2.50), which is significantly (p<0.05) higher compared to the AUC 0-9h (14.85±3.25) resulted for HC solution. The nanosuspension was physically stable at room temperature for 24 months.

Prediction of Rate Constant for Supramolecular Systems with Multiconfigurations.

The control of supramolecular systems requires a thorough understanding of their dynamics, especially on a molecular level. It is extremely difficult to determine the thermokinetic parameters of supramolecular systems, such as drug-cyclodextrin complexes with fast association/dissociation processes by experimental techniques. In this paper, molecular modeling combined with novel mathematical relationships integrating the thermodynamic/thermokinetic parameters of a series of isomeric multiconfigurations to predict the overall parameters in a range of pH values have been employed to study supramolecular dynamics at the molecular level. A suitable form of Eyring's equation was derived and a two-stage model was introduced. The new approach enabled accurate prediction of the apparent dissociation/association (k(off)/k(on)) and unbinding/binding (k-r/kr) rate constants of the ubiquitous multiconfiguration complexes of the supramolecular system. The pyronine Y (PY) was used as a model system for the validation of the presented method. Interestingly, the predicted k(off) value ((40 ± 1) × 10(5) s(-1), 298 K) of PY is largely in agreement with that previously determined by fluorescence correlation spectroscopy ((5 ± 3) × 10(5) s(-1), 298 K). Moreover, the k(off)/k(on) and k-r/kr for flurbiprofen-ß-cylcodextrin and ibuprofen-ß-cyclodextrin systems were also predicted and suggested that the association processes are diffusion-controlled. The methodology is considered to be especially useful in the design and selection of excipients for a supramolecular system with preferred association and dissociation rate constants and understanding their mechanisms. It is believed that this new approach could be applicable to a wide range of ligand-receptor supramolecular systems and will surely help in understanding their complex mechanism.

Two-way effects of surfactants on Pickering emulsions stabilized by the self-assembled microcrystals of α-cyclodextrin and oil.

The influence of surfactants on the stability of cyclodextrin (CD) Pickering emulsions is not well understood. In this study, we report two-way effects of Tween 80 and soybean lecithin (PL) on the long term stability of Pickering emulsions stabilized by the self-assembled microcrystals of α-CD and medium chain triglycerides (MCT). The CD emulsions in the absence and presence of Tween 80 or PL at different concentrations were prepared and characterized by the droplet size, viscosity, contact angle, interfacial tension and residual emulsion values. After adding Tween 80 and PL, similar effects on the size distribution and contact angle were observed. However, changes of viscosity and interfacial tension were significantly different and two-way effects on the stability were found: (i) synergistic enhancement by Tween 80; (ii) inhibition at low and enhancement at high concentrations by PL. The stability enhancement of Tween 80 was due to the interfacial tension decrease caused by the interaction of Tween 80 with CD at the o/w interface at lower concentrations, and significant viscosity increase caused by the Tween 80-CD assembly in the continuous phase. For PL at low concentrations, the replacement of α-CD/MCT by α-CD/PL particles at the o/w interface was observed, leading to inhibitory effects. High concentrations of PL resulted in an extremely low interfacial tension and stable emulsion. In conclusion, the extensive inclusion of surfactants by CD leads to their unique effects on the stability of CD emulsions, for which the changes of viscosity and interfacial tension caused by host-guest interactions play important roles.

A quality by design approach using artificial intelligence techniques to control the critical quality attributes of ramipril tablets manufactured by wet granulation.

Quality by design (QbD) is an essential part of the modern approach to pharmaceutical quality. This study was conducted in the framework of a QbD project involving ramipril tablets. Preliminary work included identification of the critical quality attributes (CQAs) and critical process parameters (CPPs) based on the quality target product profiles (QTPPs) using the historical data and risk assessment method failure mode and effect analysis (FMEA). Compendial and in-house specifications were selected as QTPPs for ramipril tablets. CPPs that affected the product and process were used to establish an experimental design. The results thus obtained can be used to facilitate definition of the design space using tools such as design of experiments (DoE), the response surface method (RSM) and artificial neural networks (ANNs). The project was aimed at discovering hidden knowledge associated with the manufacture of ramipril tablets using a range of artificial intelligence-based software, with the intention of establishing a multi-dimensional design space that ensures consistent product quality. At the end of the study, a design space was developed based on the study data and specifications, and a new formulation was optimized. On the basis of this formulation, a new laboratory batch formulation was prepared and tested. It was confirmed that the explored formulation was within the design space.

Prediction of the mechanical behaviour of crystalline solids.

PURPOSE: To explore the use of crystal inter-planar d-spacings and slip-plane interaction energies for predicting and characterising mechanical properties of crystalline solids. METHODS: Potential relationships were evaluated between mechanical properties and inter-planar d-spacing, inter-planar interaction energy, and dispersive surface energy as determined using inverse gas chromatography (IGC) for a set of pharmaceutical materials. Inter-planar interaction energies were determined by molecular modelling. RESULTS: General trends were observed between mechanical properties and the largest inter-planar d-spacing, inter-planar interaction energies, and IGC dispersive surface energy. A number of materials showed significant deviations from general trends. Weak correlations and outliers were rationalised. CONCLUSIONS: Results suggest that the highest d-spacing of a material could serve as a first-order indicator for ranking mechanical behaviour of pharmaceutical powders, but with some reservation. Inter-planar interaction energy normalised for surface area shows only a weak link with mechanical properties and does not appear to capture essential physics of deformation. A novel framework linking mechanical properties of crystals to the distinct quantities, slip-plane energy barrier and inter-planar interaction (detachment) energy is proposed.

Quality by design approach: application of artificial intelligence techniques of tablets manufactured by direct compression.

The publication of the International Conference of Harmonization (ICH) Q8, Q9, and Q10 guidelines paved the way for the standardization of quality after the Food and Drug Administration issued current Good Manufacturing Practices guidelines in 2003. "Quality by Design", mentioned in the ICH Q8 guideline, offers a better scientific understanding of critical process and product qualities using knowledge obtained during the life cycle of a product. In this scope, the "knowledge space" is a summary of all process knowledge obtained during product development, and the "design space" is the area in which a product can be manufactured within acceptable limits. To create the spaces, artificial neural networks (ANNs) can be used to emphasize the multidimensional interactions of input variables and to closely bind these variables to a design space. This helps guide the experimental design process to include interactions among the input variables, along with modeling and optimization of pharmaceutical formulations. The objective of this study was to develop an integrated multivariate approach to obtain a quality product based on an understanding of the cause-effect relationships between formulation ingredients and product properties with ANNs and genetic programming on the ramipril tablets prepared by the direct compression method. In this study, the data are generated through the systematic application of the design of experiments (DoE) principles and optimization studies using artificial neural networks and neurofuzzy logic programs.

Antioxidant Biodegradable Covalent Cyclodextrin Frameworks as Particulate Carriers for Inhalation Therapy against Acute Lung Injury.

Drug therapies for acute lung injury (ALI) are far from satisfactory, primarily because drugs cannot specifically target the lungs. Direct delivery of drugs to the deep alveolar regions by inhalation administration is crucial for the treatment of ALI. However, conventional inhalable carriers such as lactose and mannitol are generally inactive. Therefore, the use of a novel pharmacologically active carrier for pulmonary delivery may produce synergetic effects in treating ALI. Considering the pathophysiological environment of ALI, which typically featured excessive reactive oxygen species (ROS) and acute inflammation, we synthesized a novel kind of biodegradable and ROS-sensitive cross-linked covalent cyclodextrin frameworks (OC-COF) with uniform inhalable particle size to treat ALI. OC-COF was devised to incorporate H2O2-scavenging peroxalate ester linkages, which could hydrolyze and eliminate ROS generated in inflammatory sites. Ligustrazine (LIG), an antioxidant and anti-inflammatory natural compound, was loaded into OC-COF and evaluated as a dry powder inhaler (LIG@OC-COF) in vitro and in vivo, showing favorable aerodynamic properties and prominent antioxidant and anti-inflammatory capacities for the synergistic effects of OC-COF and LIG. In ALI rats, inhalation of LIG@OC-COF with a one-fifth LIG dose significantly alleviated the inflammation, oxidant stress, and lung damage. Western blot analysis demonstrated that LIG@OC-COF protected the lungs by regulating the Nrf2/NF-κB signaling pathway. In summary, this study provides a novel ROS-responsive material as an inhalable particulate carrier for the improved treatment of ALI and other medical conditions.

Redefinition to bilayer osmotic pump tablets as subterranean river system within mini-earth via three-dimensional structure mechanism.

Defining and visualizing the three-dimensional (3D) structures of pharmaceuticals provides a new and important tool to elucidate the phenomenal behavior and underlying mechanisms of drug delivery systems. The mechanism of drug release from complex structured dosage forms, such as bilayer osmotic pump tablets, has not been investigated widely for most solid 3D structures. In this study, bilayer osmotic pump tablets undergoing dissolution, as well as after dissolution in a desiccated solid state were examined, and visualized by synchrotron radiation micro-computed tomography (SR-µCT). In situ formed 3D structures at different in vitro drug release states were characterized comprehensively. A distinct movement pattern of NaCl crystals from the push layer to the drug layer was observed, beneath the semi-permeable coating in the desiccated tablet samples. The 3D structures at different dissolution time revealed that the pushing upsurge in the bilayer osmotic pump tablet was directed via peripheral "roadways". Typically, different regions of the osmotic front, infiltration region, and dormant region were classified in the push layer during the dissolution of drug from tablet samples. According to the observed 3D microstructures, a "subterranean river model" for the drug release mechanism has been defined to explain the drug release mechanism.

Bridging the structure gap between pellets in artificial dissolution media and in gastro-intestinal tract in rats.

Changes in structure of oral solid dosage forms (OSDF) elementally determine the drug release and its therapeutic effects. In this research, synchrotron radiation X-ray micro-computed tomography was utilized to visualize the 3D structure of enteric coated pellets recovered from the gastrointestinal tract of rats. The structures of pellets in solid state and in vitro compendium media were measured. Pellets in vivo underwent morphological and structural changes which differed significantly from those in vitro compendium media. Thus, optimizations of the dissolution media were performed to mimic the appropriate in vivo conditions by introducing pepsin and glass microspheres in media. The sphericity, pellet volume, pore volume and porosity of the in vivo esomeprazole magnesium pellets in stomach for 2 h were recorded 0.47, 1.55 × 108 µm3, 0.44 × 108 µm3 and 27.6%, respectively. After adding pepsin and glass microspheres, the above parameters in vitro reached to 0.44, 1.64 × 108 µm3, 0.38 × 108 µm3 and 23.0%, respectively. Omeprazole magnesium pellets behaved similarly. The structural features of pellets between in vitro media and in vivo condition were bridged successfully in terms of 3D structures to ensure better design, characterization and quality control of advanced OSDF.

Microrobotic laser steering for minimally invasive surgery.

The creation of multiarticulated mechanisms for use with minimally invasive surgical tools is difficult because of fabrication, assembly, and actuation challenges on the millimeter scale of these devices. Nevertheless, such mechanisms are desirable for granting surgeons greater precision and dexterity to manipulate and visualize tissue at the surgical site. Here, we describe the construction of a complex optoelectromechanical device that can be integrated with existing surgical tools to control the position of a fiber-delivered laser. By using modular assembly and a laminate fabrication method, we are able to create a smaller and higher-bandwidth device than the current state of the art while achieving a range of motion similar to existing tools. The device we present is 6 millimeters in diameter and 16 millimeters in length and is capable of focusing and steering a fiber-delivered laser beam at high speed (1.2-kilohertz bandwidth) over a large range (over ±10 degrees in both of two axes) with excellent static repeatability (200 micrometers).

Multiscale Co-reconstruction of Lung Architectures and Inhalable Materials Spatial Distribution.

The effective pulmonary deposition of inhaled particulate carriers loaded with drugs is a prerequisite for therapeutic effects of drug delivery via inhalation route. Revealing the sophisticated lung scaffold and intrapulmonary distribution of particles at three-dimensional (3D), in-situ, and single-particle level remains a fundamental and critical challenge for dry powder inhalation in pre-clinical research. Here, taking advantage of the micro optical sectioning tomography system, the high-precision cross-scale visualization of entire lung anatomy is obtained. Then, co-localized lung-wide datasets of both cyto-architectures and fluorescent particles are collected at full scale with the resolution down to individual particles. The precise spatial distribution pattern reveals the region-specific distribution and structure-associated deposition of the inhalable particles in lungs, which is undetected by previous methods. Overall, this research delivers comprehensive and high-resolution 3D detection of pulmonary drug delivery vectors and provides a novel strategy to evaluate materials distribution for drug delivery.

Factors affecting the stability of nanoemulsions--use of artificial neural networks.

PURPOSE: The aim of this study was to identify the dominant factors affecting the stability of nanoemulsions, using artificial neural networks (ANNs). METHODS: A nanoemulsion preparation of budesonide containing polysorbate 80, ethanol, medium chain triglycerides and saline solution was designed, and the particle size of samples with various compositions, prepared using different rates and amounts of applied ultrasonic energy, was measured 30 min and 30 days after preparation. Using ANNs, data were modelled and assessed. The derived predictive model was validated statistically and then used to determine the effect of different formulation and processing input variables on particle size growth of the nanoemulsion preparation as an indicator of the preparation stability. RESULTS: The results indicated that the data can be satisfactorily modelled using ANNs, while showing a high degree of complexity between the dominant factors affecting the stability of the preparation. CONCLUSION: The total amount of applied energy and concentration of ethanol were found to be the dominant factors controlling the particle size growth.

Cocrystalization and simultaneous agglomeration using hot melt extrusion.

PURPOSE: To explore hot melt extrusion (HME) as a scalable, solvent-free, continuous technology to design cocrystals in agglomerated form. METHODS: Cocrystal agglomerates of ibuprofen and nicotinamide in 1:1 ratio were produced using HME at different barrel temperature profiles, screw speeds, and screw configurations. Product was characterized for crystallinity by XRPD and DSC, while the morphology was determined by SEM. Dissolution rate and tabletting properties were compared with ibuprofen. RESULTS: Process parameters significantly affected the extent of cocrystallization which improved with temperature, applied shear and residence time. Processing above eutectic point was required for cocrystallization to occur, and it improved with mixing intensity by changing screw configuration. Product was in the form of spherical agglomerates, which showed directly compressible nature with enhanced dissolution rate compared to ibuprofen. This marks an important advantage over the conventional techniques, as it negates the need for further size modification steps. CONCLUSIONS: A single-step, scalable, solvent-free, continuous cocrystallization and agglomeration technology was developed using HME, offering flexibility for tailoring the cocrystal purity. HME being an established technology readily addresses the regulatory demand of quality by design (QbD) and process analytical technology (PAT), offering high potential for pharmaceuticals.

Evaluation of a nanoemulsion-based formulation for respiratory delivery of budesonide by nebulizers.

The aim of this work was to evaluate the in vitro performance of a nebulized nanoemulsion formulation which had been optimised previously. To do so, a transparent nanoemulsion preparation containing 1.5 mg/ml of budesonide was prepared and diluted to achieve concentrations of 250 and 500 µg/ml budesonide. The in vitro characteristics of the diluted nanoemulsions were then compared with the commercially available suspension of budesonide (Pulmicort Respules®) when nebulized using a jet and a vibrating mesh nebulizer. A smaller MMAD with improved aerosol output was observed in the nanoemulsion preparations compared with the corresponding suspension formulations indicating an improved in vitro performance for the nanoemulsion-based preparations.

Dynamic structure model of polyelectrolyte complex based controlled-release matrix tablets visualized by synchrotron radiation micro-computed tomography.

Hydrophilic matrix tablets are the most commonly used dosage forms to fabricate oral controlled-release systems. It is highly desirable to design delivery system with novel mechanism to achieve sustained drug release through a simplified preparation process. The chitosan-anionic polymers based matrix tablets is assumed to produce self-assembly in the gastrointestinal tract, then transferring into film-coated tablets from original matrix type. But its dynamic behavior during dissolution process and the on-going internal microstructural changes during drug release were still in the dark. In this study, by using synchrotron radiation X-ray micro-tomography (SR-µCT) with phase contrast imaging, the micro-structure characteristics of chitosan-λ-carrageenan (CS-λ-CG) matrix based tablets during the dissolution were successfully elucidated for the first time. The qualitative and quantitative analyses of intensity distribution distinguished a hydrated CS-λ-CG layer from a solid core. Visualization based on 3D models provided quantitative details on the micro-structural characteristics of hydration dynamics. After CS-λ-CG matrix tablets were immersed in simulated gastric fluid (SGF) pH 1.2 medium for 0.5-2.0 h, the hydrated layer transformed into a gel layer and a solid swollen layer. The erosion front, swelling front, and solvent penetration front were also defined from the distinguishable micro-structures. More importantly, once the matrix tablet was transferred from SGF to the simulated intestinal fluid (SIF) pH 6.8 medium, a new layer with the enhanced strength and compactness in comparison to common gels was formed on the surface of tablets. The temporal and spatial variation of 3D models further provided direct evidence for this cross-linking behavior, the new layer was composed of CS-λ-CG polyelectrolyte complexes (PEC) which subsequently dominated release mechanisms. In summary, the phase contrast SR-µCT technique was utilized to investigate the hydration dynamics of CS-λ-CG matrix tablets which was supposed to provide a novel drug release mechanism. Based on the structure feature obtained from the high contrast image, different hydration region was distinguished and the cross-linked film was identified and visualized directly for the first time.

Static and dynamic structural features of single pellets determine the release behaviors of metoprolol succinate sustained-release tablets.

The multiple-unit sustained-release (MUSR) dosage forms containing numerous sustained-release subunits present a reliable choice for oral formulation of controlled release systems. As a typical MUSR, the metoprolol succinate sustained-release tablet is an advanced system with limited researches devoted to relating its structure to the drug release phase other than the preparation process and modulation to the release behaviors. This research details a three-dimension method to image the internal structure and detail drug release features of commercial metoprolol succinate sustained-release tablets and component individual single pellets. As such, a new perspective for MUSR dosage form is provided. Using high energy synchrotron radiation X-ray microcomputed tomography (SR-µCT), the in-situ structure parameters were obtained nondestructively. It was demonstrated that the average number of spherical pellets in a tablet was 853 ± 12 (n = 3). The average volume of the pellets was 0.09 ± 0.01 mm3, the diameter was 0.55 ± 0.03 mm, and the sphericity was 0.87 ± 0.06. These data reflected the numerical features of pellets in MUSR tablets, which were helpful for reverse engineering to MUSR. Based on the three dimensional model generated by image processing and analysis software, the pellet structures were divided into three layers of typical depot sustained release system: pellet core, drug-containing layer and outer film. The dynamic structural features determined refer to the changes of structures in pellets during in vitro drug release, with evidence that the coating layer on the pellets maintained a spherical morphology whilst numerous valleys appeared on the surface. The material constitution and distribution in coating layer were evaluated by synchrotron radiation-based Fourier transform infrared mapping and results indicated a composition of hydroxypropyl methylcellulose dispersed in ethyl cellulose. Knowledge of these structural characteristics confirmed that the mechanism of sustained drug release was membrane controlled and consistent with the drug release profiles. In conclusion, the structural investigation provided knowledge of the intrinsic quality of metoprolol succinate sustained-release tablets and offers guidance for reverse engineering of MUSR.

Comparison between monolithic and particle-packed platinum C18 columns in HPLC determination of acidic and basic test mixtures.

Herein we report the results of a comparative study on the performance of Monolithic RP-18e and Platinum C(18) 3 mum columns for isocratic separation of acidic and basic test compounds. The inter- and intraday precision of different practical parameters including number of theoretical plates (N), capacity factor (K'), tailing factor (T(0.05)), and resolution (R(s)) were determined for both columns. Two different production batches were used for each column and batch to batch reproducibility of both columns was evaluated. The column backpressure drop over flow rate range 0.5-2 mL/min at the monolithic columns was two- to three-times lower than that on the platinum column without loss of the column efficiency. The plate heights were used to estimate the columns efficiency using Van Deemter plots. Both types of columns were able to separate the tested compounds well with sufficient resolution and peak symmetry but they differ in the analysis time and column backpressure, significantly. Monolithic column was more convenient as it enables the analytical run under low backpressure at shorter time with sufficient separation efficiency.

Development and validation of a rapid and efficient method for simultaneous determination of methylxanthines and their metabolites in urine using monolithic HPLC columns.

A new rapid, sensitive and validated HPLC method has been developed for the determination of methylxanthines and their metabolites in asthmatic patients. The method was initiated by using spiked urine samples on a silica monolithic column as a novel packing material. The mobile phase consisted of 10 mM potassium dihydrogen phosphate buffer/methanol (87.5:12.5 v/v), at a flow rate 1 mL/min. Detection was set at 274 nm. The LOQ for all the compounds ranged from 14 to 41 ng/mL. Excellent linearity was achieved over the studied range of concentration with correlation coefficients 0.9991-0.9998 (n = 6). The developed method was validated by precision and accuracy with RSD <2.55%. On extraction of the drugs and metabolites from the urine samples high recoveries were achieved ranging from 82.06 to 98.34% w/w on RP18 cartridges and methanol/chloroform (20:80 v/v) as the extraction solvent. This method has advantages over other methods using conventional C18 packings.