Microfabricated electrochemical detector for high-performance liquid chromatography

A microfabricated electrochemical cell has been developed as a disposable detector for flow injection analysis (FIA) and high-performance liquid chromatography (HPLC). The simplicity of the fabrication process allows this detector to be used as a low-cost, disposable device that can be replaced easily if its performance degrades rather than disassembling the detector and polishing the electrode surface, which is the usual procedure. The detector consists of thin film-metal electrodes-platinum working electrode, platinum auxiliary electrode, and silver/silver chloride coated on Pt reference electrode-deposited on a polyimide substrate with a locking layer of chromium in between. A microfluidic cover made of polyimide directs the solution flow across the electrodes. The detector was evaluated with FIA of ferrocyanide and HPLC of ascorbic acid and acetaminophen and a mixture of two pharmaceutical compounds-dextrorphan and levallorphan-with acetaminophen internal standard. The HPLC calibration curves showed good linearity (R(2) > 0.99). Limits of detection were 1 nM for acetaminophen, 1 nM for ascorbic acid, 50 nM for dextrorphan, and 80 nM for levallorphan. When detected with a commercial detector dextrorphan and levallorphan had lower limits of detection, 3 and 5 nM, respectively. Chromatograms of the mixture were comparable to those obtained with a commercial detector. The detector could be used continuously for about 48 h with FIA and about 10-20 h with HPLC after which performance gradually degraded as the AgCl on the reference electrode dissolved causing loss of potential control.     

Fully automated 96-well liquid-liquid extraction for analysis of biological samples by liquid chromatography with tandem mass spectrometry

A fully automated high-throughput liquid-liquid extraction (LLE) methodology has been developed for preparation of biological samples using a 96-well LLE plate and a 96-channel robotic liquid handling workstation. The 96-well LLE plate is made of a 96-well filter plate filled with inert diatomaceous earth particles, allowing continuous and efficient extraction of analytes between the aqueous biological sample and the organic extraction solvent. Two carboxylic acid-based protease inhibitor compounds with high and low levels of plasma protein binding were chosen for the development and application of the automated methodology. The LLE extracts of the plasma samples of the two compounds were analyzed by high-performance liquid chromatography with electrospray (ESI) tandem mass spectrometry (LC-MS/MS). The LC-MS/MS method was developed using a rapid gradient LC separation, followed by sample introduction through an ionspray interface in the negative ion mode and tandem mass spectrometric detection with selected reaction monitoring. In the optimized LLE method, a formate buffer solution was first loaded into a 96-well filter plate packed with inert diatomaceous earth material. Then crude plasma samples and a water-immiscible organic solvent, methyl ethyl ketone, were sequentially added to the LLE plate so that LLE would occur in the interface between the two liquid phases on the surface of individual particles in each well. The organic eluate containing extracted analytes was evaporated and reconstituted for LC-MS/MS analysis. This fully automated LLE methodology avoids several disjointed steps involved in a manual or semiautomated LLE method, leading to significantly reduced sample preparation time, increased sample throughput, and clean sample extracts for improved ESI-MS/MS detection. The automated LLE methodology is universal and can be employed for sample preparation of other biological fluids. The complete bioanalytical method, based on the automated LLE and fast gradient LC-MS/MS, was validated and successfully applied to the quantitative analysis of protease inhibitors in rat plasma.     

Nanoflow gradient generator for capillary high-performance liquid chromatography

A novel method of generating a nanoflow gradient elution for a capillary high-performance liquid chromatography (HPLC) system has been developed. An important feature of this system is that any gradient (GR) profile generated by a conventional microflow GR pump can be asymptotically traced and converted as a corresponding nanoflow GR profile simply by using a 10-port switching valve with two injection loops installed. Consequently, it has been called an "asymptotic trace 10-port valve" (AT10PV) nanoflow GR generator. Performance of the AT10PV nanoflow GR generator was tested in the range of flow rates from 50 to 500 nL/min. The test demonstrated that the AT10PV nanoflow GR generator can asymptotically trace the original gradient profile with good reproducibility. A capillary HPLC system using the AT10PV nanoflow GR generator provides reasonably good repeatability of peak retention times on the chromatogram of the tryptic digest of a BSA sample, RSD of less than 0.3% at a flow rate of 200 nL/min. It also enables sequential running of a series of sample injections in the same manner as conventional analysis at microflow rates.     

An improved high-performance liquid chromatography assay for spironolactone analysis

This study prepared an extemporaneously formulated liquid suspension dosage form (5 mg/ml) from commercially available 25 mg tablets. Stability-indicating HPLC assay procedures were established and utilized to analyze the concentration of the drug. The method proved to be a simple model since it does not contain a buffer system. The mobile phase used was the same as that suggested by the manufacturer for the storage of the column. Therefore, the solvent system saves analytical processing time, since it does not require a change in the mobile phase before or after the analysis. The analytical method has been shown to be stability-indicating. The results have shown that there is no interference from any of the degradation products obtained from stressing spironolactone by heat and extremes in pH or with the internal standard, hydrocortisone 21-acetate.     

A high-performance liquid chromatography assay for yohimbine HCl analysis

The analysis used yohimbine HCl solution prepared from commercially available yohimbine HCl powder. Stability-indicating high-performance liquid chromatographic (HPLC) assay procedures were established and utilized to analyze the concentration of the drug. The method proved to be a simple model since it does not contain a buffer system. The mobile phase used, a methanol:water 70:30 ratio, was similar to that suggested by the manufacturer for the storage of the column. Therefore, the solvent system saves analytical processing time since it does not require a change in the mobile phase before and after the analysis. The analytical method has been shown to be stability indicating. The assay method showed a retention time for yohimbine of 4.2 min; for caffeine, the internal standard, it was 2.3 min. The standard deviation and the coefficient of variation were under acceptable limits of 2% and were specifically 1.51% and 1.35% for within-day and between-day samples, respectively. The results showed that the degradation products obtained from stressing yohimbine HCl by heat and extremes in pH did not interfere with the yohimbine HCl peak, although the internal standard, caffeine, did show some interference due to having a retention time similar to the degradation products.     

Overload for ionized solutes in reversed-phase high-performance liquid chromatography

Overloading occurs for submicrogram quantities of ionized solutes particularly when using low ionic strength mobile phases at low pH (e.g., formic acid), even with highly inert silica RP-HPLC columns of normal dimensions. Much higher loads can produce a sharp L-shaped peak with retention above the column void volume, in line with the hypothesis that a small number of high-energy sites fill first and are rapidly overloaded, followed by a much larger number of weaker sites. However, charged acids and bases show identical overloading behavior; overloading is reduced as the mobile-phase ionic strength is increased. These findings raise questions about the physical nature of the strong sites. The rapid overloading of silica and purely polymeric phases could be explained by mutual repulsion of ionic species or their inability to fully penetrate the hydrophobic structure of the phase. However, these alternative hypotheses cannot readily explain the high total saturation capacities obtained using frontal analysis. Ion pairing with trifluoroacetic acid may reduce overload, while the effect is less important for formate or phosphate buffers. A surface layer of acetonitrile is not a prerequisite for rapid overloading, as shown by studies using purely aqueous buffers.     

Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography of proteins

A comprehensive two-dimensional (2-D) liquid chromatographic separation system is presented. The system uses a microbore cation exchange column, operated under gradient conditions, as the first dimension separation. Effluent from this first column alternately fills one of two loops on a computer-controlled eight-port valve. A second pump then forces loop material onto a second column, a size exclusion column. UV detection is used, and the system is applied to the separation of protein standards and serum proteins. The 2-D system has a higher resolving power and peak capacity than either of the two columns used alone. The entire first column effluent is analyzed on the second column in virtually the same time it takes to complete the first column separation, without the use of stopped flow methods. The entire system is automated and operated under computer control. Three-dimensional (3-D) data representation provides a means of viewing peak profiles in either separation dimension and contour mapping of the 3-D data provides a more reliable means of peak identification from run to run than that provided by single-column elution times.     

Analysis of oligosaccharides by microbore high-performance liquid chromatography

A 1-mm microbore hydrophilic interaction column has been used for the separation of 2-aminoacridone (2-AMAC)-derivatized glycan mixtures, released from naturally occurring and recombinant proteins. Primary structure identification of the 2-AMAC glycan derivatives was carried out by HPLC using fluorescence and mass spectrometric detection. In some cases, enzymatic digestion of the 2-AMAC glycans was applied to confirm glycan structure. This strategy is considerably more rapid than methods normally used in glycan analysis, which involves manual collection of separated oligosaccharide derivatives and analysis of individual fractions by mass spectrometry.     

固相萃取-高效液相色谱串联质谱测定牛奶中9种性激素残留

采用固相萃取-高效液相色谱质谱联用法(SPE-HPLC/MS),建立牛奶中9种性激素(雌酮、雌三醇、17α-雌二醇、诺龙、甲睾酮、丙酸睾酮、醋酸氯地孕酮、醋酸甲地孕酮、醋酸甲羟孕酮)残留的检测方法。样品经甲醇提取,过固相萃取柱净化,氮气吹干,残留物甲醇溶解后测定。其中雌激素(雌酮、雌三醇、17α-雌二醇)采用负模式;其余性激素为正模式,进行多反应监测(MRM)模式定性定量分析。雌激素检出限(LODs)为1.1~1.2μg/L,定量限(LOQs)为3.63~3.96μg/L;其余性激素检出限(LODs)为0.1~0.5μg/L,定量限(LOQs)为0.33~1.65μg/L;分别在20.0~500.0μg/L及2.5~100.0μg/L范围内线性关系良好(r20.994 4)。在50.0~100.0μg/L的添加水平上,9种性激素的平均回收率在82.7%~98.2%之间,变异系数(CV)为4.2%~11.2%。该法操作简单、灵敏度高,可用于牛奶中9种性激素的测定。     

Geometrical model for the retention of fullerenes in high-performance liquid chromatography

In high-performance liquid chromatography (HPLC) using a poly(octadecylsiloxane) as a stationary phase, methanol as a mobile phase, C(60) and C(70) fullerenes as solutes, and water as a mobile phase modifier, a study on the surface tension effect of water on fullerene retention was carried out by varying the water concentration [W] and the column temperature T. The thermodynamic parameters for fullerene transfer from the mobile to the stationary phase were determined from linear van't Hoff plots. An enthalpy-entropy compensation revealed that the types of interactions between fullerenes and the stationary phase were independent of the fullerene structure and the mobile phase composition. An analysis of the experimental variations of the retention factor and the selectivity values with [W] was performed using a novel geometrical model. It was shown that the increase in fullerene retention accompanying the water concentration was due to the increased effects of surface tension. This brought about an increase in the interactions between fullerene and the stationary phase, explaining the observed thermodynamic parameter trends over the water concentration range. The theoretical model provided an estimation of the radius of fullerene which was found for C(60) to be equal to 3.3 ? and an activation energy during the transfer equal to 9.8 kJ/mol.     

Silica xerogel as a continuous column support for high-performance liquid chromatography

A preliminary study of the chromatographic performance and permeability of a continuous silica xerogel column under reversed-phase HPLC conditions was performed. A porous chromatographic support was synthesized inside a 0.32 mm i.d. × 13 cm fused silica tube from potassium silicate solution and derivatized with dimethyloctadecylchlorosilane. The plate height at 0.01 cm/s (0.5 μL/min), near the apparent optimum linear velocity, was about 65 μm. The column efficiencies in terms of numbers of plates per meter were 5000 and 13?000 for ethyl benzoate (k = 0.8) and naphthalene (k = 2.0), respectively, at 0.5 μL/min. The major parameter affecting column efficiency was the heterogeneous morphology of the xerogel, modifications to which are expected to improve chromatographic performance. The column provided efficiencies comparable to those reported for continuous polymeric columns but less than that previously reported for a continuous silica column. Gradient elution mode was demonstrated with a mixture of polycyclic aromatic hydrocarbons. The column was highly permeable, exhibiting a linear dependence of pressure to flow rate and a back pressure of only 632 psi at 10 μL/min when a 95% aqueous mobile phase was used.     

Validation of tablet dissolution method by high-performance liquid chromatography

Dissolution is a qualitative and quantitative tool that can provide valuable information about biological availability of a drug, as well as batch-to-batch consistency. It is considered one of the most important quality control tests performed on pharmaceutical dosage forms, and validation of dissolution methods is an important part of good manufacturing practices (GMP). Hydroxypropylcellulose (HPC) was formulated with acetaminophen (APAP) and hydrochlorothiazide (HCTZ). Dissolution methods and limits are reported in the USP/NF. Standard operating procedures (SOP) for the HP 8452A spectrophotometer and Vanderkamp 600/6010 Dissolution Tester were followed according to the GMP Manual. A dissolution method was developed for each formulation based on the above. The only discrepancy between high-performance liquid chromatography (HPLC) and standard dissolution testing occurred when comparing the results of the HPC/HCTZ formulation. The ultraviolet (UV) samples were filtered through a 10-μm filter, and the HPLC samples were filtered through a 0.2-μm filter. When the HPC/HCTZ samples were filtered through a 10-μm filter for both UV and liquid chromatography (LC), the results were equal. Filter pore size and area have a large effect on concentration of HPC/HCTZ. The smaller the pore size and the smaller the diameter of the filter, the more HPC/HCTZ is filtered out. HCTZ has a greater tendency to interact with HPC in the filter than other active ingredients tested. HPC and HCTZ levels have little or no effect on the amount of HCTZ lost