HPLC and GC Articles

Introduction A large number of analytical techniques have been employed in the clinical diagnostics arena including liquid chromatography, gas chromatography, mass spectrometry and electrophoresis. For decades, electrophoresis has been the gold standard for clinical protein analysis (1) and has, more recently, gained popularity for clinical DNA separations such as mutation screenings. The most common format of electrophoresis used in the biomedical and clinical setting is the slab gel, which is used to separate DNA fragments solely on the basis of size. Several theories have been put forward to explain the mechanism that, in the presence of an electrical field, allows smaller DNA fragments to electrophorese through a sieving matrix faster than larger ones (2–4).

What is important for clinical purposes is that slab gel electrophoretic analysis can be performed simultaneously on multiple samples in parallel lanes with instrumentation that is relatively inexpensive.

Whether one’s approach to drug discovery involves natural products screening or the synthesis of innovative new compounds, the challenges presented to the analytical chemist are complicated when a product contains one or more chiral centres. Yet, these optically active compounds tend to make good drug candidates and, as such, require stereochemically selective assay methods in place for their analysis (1).
 
An important step towards separating enantiomers is to first create diastereomers. Diastereomers may be created through chemical derivatization with a chiral reagent or may be formed transiently through interactions with chiral selectors.

Abstract: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and inductively coupled plasma-atomic emission spectroscopy (ICP-AES) methods were developed and validated for the evaluation of motexafin lutetium (MLu, lutetium texaphyrin, PCI-0123) pharmacokinetics in human plasma. The LC-MS/MS method was specific for MLu, whereas the ICP-AES method measured total elemental lutetium. Both methods were fast, simple, precise, and accurate. For the LC-MS/MS method, a closely related analogue (PCI-0353) was used as the internal standard (IS). MLu and the IS were extracted from plasma by protein precipitation and injected into an LC–MS/MS system configured with a C18 column and an electrospray interface. The lower limit of quantitation was 0.05 µg MLu mL–1, with a signal-to-noise ratio of 15:1. The response was linear from 0.05 to 5.0 µg MLu mL–1. For the ICP-AES method, indium was used as the IS.

Abstract :  An accurate, precise, and sensitive high-performance liquid chromatography (HPLC) assay was developed for the determination of atenolol in human plasma samples to compare the bioavailability of 2 atenolol tablet (50 mg) formulations in 24 volunteers of both sexes. The study had an open, randomized, 2-period crossover design with a 1-week washout period. Plasma samples were obtained over a 24-hour interval. Atenolol concentrations were analyzed by combined reversed phase liquid chromatography and fluorescence detection (λEX = 258 nm, λEM = 300 nm). From the atenolol plasma concentration versus time curves, the following pharmacokinetic parameters were obtained: AUC0-24h, AUC0-∞, and Cmax. The geometric mean of test/reference 50-mg tablets individual percent ratio was 102.2% for AUC0-24h, and 101.6% for Cmax. The 90% confidence intervals (CI) were 100.2% to 105.4% and 100.9% to 103.5%, respectively.

Abstract : Ipratropium bromide, a bronchodilator, is used as an inhalation solution. Commercial ipratropium bromide solution products are packaged in low-density polyethylene (LDPE) vials, through which semivolatile compounds are reported to migrate. In this article, a specific reversed phase-high performance liquid chromatographic method to assay vanillin, a semivolatile compound, in ipratropium bromide solution is described. The method was validated for a concentration range for vanillin from 30 ng/mL to 1,600 ng/mL. Migration of vanillin was assessed in two commercial preparations, ATROVENT (ipratropium bromide) Inhalation Solution packaged in a secondary foil pouch and a generic ipratropium bromide inhalation solution packaged in a carton. Levels of vanillin detected in ATROVENT after 6 months of storage at 40° C and 75% RH were below the limit of detection (11 ng/mL).

There has been a wealth of recent interest in proteomics, which involves the global analysis of protein expression and function. Identification of complex protein mixtures is a difficult process that can first involve, for example, two-dimensional gel electrophoresis to resolve individual proteins. These separated proteins then can be hydrolyzed with enzymes such as trypsin to their constituent peptides, which can be identified by high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS) (1). A number of factors need to be taken into account to achieve high-resolution chromatographic separation and identification of peptides: Low pH is preferred because the ionization of silanol groups on silica-based reversed-phase columns is suppressed along with their detrimental interactions with the charged peptides.

There continues to be strong demand for improvements in sensitivity, selectivity, throughput, qualitative content, and many other performance characteristics of high performance liquid chromatography (HPLC). A major need is for methods that provide both universal detection and quantitative analysis. It is widely recognized that no single HPLC detector is capable of distinguishing all possible analytes from a given chromatographic eluent and the term "universal" often is used to describe detection of a diverse range of analytes. A primary goal for most analyses that seek universal detection is to obtain a consistent relationship between the magnitude of response and quantity injected for a range of analytes. This "consistency of response factors" allows the use of global mathematical relationships to estimate quantity (for example, use of parent drug response factor to quantify metabolites and degradants).

The possibility of gas chromatography (GC) was first mentioned in 1941, in the famous paper by A.J.P. Martin and R.L.M. Synge. When dealing with liquid-liquid partition chromatography, they predicted that Leslie S. Ettre . . . the mobile phase need not be a liquid but may be a vapour. . . . Very refined separations of volatile substances should therefore be possible in a column in which permanent gas is made to flow over gel impregnated with a nonvolatile solvent. (1) However, at that time, nobody picked up this suggestion and it was 10 years until Martin, now with A.T. James, demonstrated the possibility of gas-liquid partition chromatography (2,3). They worked in the biochemical field and demonstrated the application of the technique by separating and quantitatively determining the components of a C1-C12 fatty acid mixture.

In the last installment of "Column Watch" (1), the problem of a possible change in column selectivity was introduced, and means for selecting a different column as a replacement were considered. Ten or twenty years ago, changes in separation were not uncommon when replacing a column with one from a different production batch. Personally, we are aware of more than a few such cases within the past decade, in some cases involving columns from major manufacturers. Although batch-to-batch column reproducibility has improved greatly in recent years (2,3), it is premature to claim that all columns currently manufactured will be adequately reproducible for all possible samples and separation conditions. There also is a need to improve some older methods by substituting columns of more recent design, for which the likelihood of encountering major differences in column selectivity is increased greatly.

Reagent water is used in all aspects of liquid chromatography (LC) technology, from preparation of mobile phase to preparation of standards, blanks, and samples. Reagent water is the most widely used analytical solvent, yet it is the least characterized, especially in total organic carbon (TOC) content. TOC adversely effects performance of LC methods and hence, reagent water quality is a major issue. Organics initially present in tap water will be reduced efficiently to low parts-per-billion concentrations by combining several technologies embedded in a water purification system. Monitoring the TOC concentrations gives chromatographers added confidence in their results. High performance liquid chromatography (HPLC), used alone or as a chromatographic inlet for tandem detection analysis, is ubiquitous in pharmaceutical, e nvironmental, food and beverage, organic synthesis, biore s e a rch, proteomics, clinical, and forensic laboratories.