Application Notes: LC

The recent development of ultra-HPLC (UHPLC) has provided a great potential for high throughput analysis achieved using small (sub-2 μm) particle size columns at increased linear velocities. The advantage of UHPLC over conventional HPLC is increased throughput without sacrificing resolution. Despite their popularity, sub-2 μm particle columns impose practical difficulties, such as high backpressure (which often requires a UHPLC system) and susceptibility to column fouling. Thus difficulty can be overcome using 2.0 to 2.5 μm particles. This study describes an example (analysis of vanilla extract) of transferring a conventional LC method to a high-throughput method using newly developed Acclaim® RSLC 2-μm columns that are based on spherical, porous, high-purity silica particles (dp = 2 μm, pore size = 120 Å, surface area = 320 m2/g).

Cephalosporins contain a four-member β-lactam ring that is inherently strained and prone to hydrolysis and photolysis, limiting stability and leading to degradation products that may be toxic (1). In addition, synthetic byproducts are generated and persist during production of these antibiotics including cefepime. Analysis of cefepime purity is particularly challenging due to the presence of such isomeric synthetic impurities. The Acclaim® 120 C18, 3 μm can be used to meet and exceed the criteria set by the USP for determining related substances and assaying the purity of cefepime.

The Fused-Core particle consists of a 1.7 micron solid core and a 0.5 micron porous shell yielding a 2.7 micron diameter. One of the benefits of the Fused-Core particle is the small diffusion path (0.5 microns) compared to conventional fully porous particles. The shorter diffusion path minimizes peak broadening. In fact, there have been many reports on the vast improvements in efficiency provided by Fused-Core particles versus conventional particles. These improvements provide sub-2 micron like performance at half of the backpressure allowing Ascentis Express columns to be used in conventional HPLC as well as UHPLC systems.

Recent developments in HRes Fast-LC systems have enabled the use of sub-3 μm stationary phases at higher flow rates and elevated pressures. HRes Fast-LC can provide significant improvements in both resolution and throughput.

In size exclusion chromatography, obtaining calibration curves over a wide range of molecular weights is a difficulty investigators often encounter when analyzing polymers with a broad molar mass distribution. To overcome this problem two procedures are typically used. One option is to use multiple columns of different pore sizes linked together in series. A second is to use a column packed with a mixed bed resin of different pore sizes at an optimized mix ratio. However, problems can occur with both of these methods, which include distortion of the chromatogram or deviations between the actual calibration curve and the calibration curve approximated from data obtained from the molecular weight standards.

This application note describes a rapid U-HPLC method for the separation of sulphorhodamine 101 (Texas Red®) and its three water-soluble derivatives using a Hypersil GOLD™ column packed with 1.9 μm particles.

A new, immobilized chiral stationary phase (CSP) based on a novel chiral selector for HPLC and SFC is described. Its unique selectivity as a complement to Daicel's other commercial immobilized polysaccharide CSPs is demonstrated.

This article describes a straightforward ion chromatographic method that uses isocratic elution and pulsed amperometric detection (PAD) to sensitively determine water-soluble polyols and sugar alcohols as well as mono-, di- and oligosaccharides in essential and nonessential foodstuffs. While carbohydrate determination of most foodstuffs requires only minimal sample pretreatment such as dilution and filtration, samples with interfering matrices such as protein-containing dairy products have to be dialyzed prior to injection.

Voglibose is an Alpha-Glucosidase inhibitor widely used for the treatment of diabetes. Alpha-glucosidase inhibitors are agents that delay the glucose absorption at the intestinal level and thereby prevent sudden surge of glucose after a meal. Voglibose is the safest and most effective drug of its class.

Cefepime is a fourth generation cephalosporin (1). During preparation and storage, cefepime degrades by release of the N-methylpyrrolidine (NMP) side chain and opening of the beta-lactam ring. An NMP concentration increase will directly affect the potency of the active component of the drug. Therefore, it is critical to determine the amount of NMP in cefepime. The US Pharmacopeia (USP) monograph specifies the limit of NMP to <0.3% in cefepime hydrochloride and <1% in cefepime for injection (2,3). The latter is a dry mixture of cefepime hydrochloride and L-arginine. The current USP method uses cation-exchange chromatography with non-suppressed conductivity detection to determine the limit of NMP in cefepime. There are several disadvantages to this method, such as the ~3-4 h time required per injection, a lack of retention time stability for NMP in standard and sample solutions, and a lack of sensitivity. In this paper, we describe an improved method using a hydrophilic, carboxylate-functionalized cation..

In general, polymer-based columns have a broad pH range (pH 2 to 13), and some have high temperature tolerance (up to 150°C or higher). Considerably large selectivity changes can be obtained by varying analysis temperature and mobile phase pH. Having control on these two parameters over wide ranges can be especially useful in method development.

Paracetamol is a major ingredient in numerous medications due to its analgesic and antipyretic properties. During its synthesis (Figure 1), a total of ten process-related impurities are observed. Several HPLC applications have been developed for the monitoring of these impurities (1, 2), including the European Pharmacopoeia which has adopted an isocratic HPLC method using a silica-based C8 column with 5 μm particle size, requiring a run time of 45 min (3). By using a gradient method and standard HPLC instrumentation, the analysis can be reduced to 7 min (4).

A large percentage of commercial and investigational pharmaceutical compounds are enantiomers and many of them show significant enantioselective differences in their pharmacokinetics and pharmacodynamics. The importance of chirality of drugs has been increasingly recognized, and the consequences of using them as racemates or as enantiomers have been frequently discussed in the pharmaceutical literature during recent years. With increasing evidence of problems related to stereoselectivity in drug action, enantioselective analysis by chromatographic methods has become the focus of intensive research of separation scientists. Most of the pharmaceutical and pharmacological studies of stereoselectivity of chiral drugs before the mid eighties involved pre-column derivatization of the enantiomers with chiral reagents forming diastereomers.

Traditionally the analysis of water-soluble vitamins by reversed-phase HPLC has been complicated by the lack of retention for these compounds on conventional silica C18 columns. Here we demonstrate efficient, multi-mode, baseline resolution of six water-soluble vitamins in 6 min using a ZirChrom®-SAX column.

The analysis of polar compounds in support of clinical and preclinical pharmacokinetic studies requires an analytical methodology capable of achieving ultra-low detection and quantification limits. The high sensitivity afforded by coupling HPLC with tandem mass spectrometry (MS–MS) has made it the technique of choice in this environment, but it is subject to the following limitations when reversed-phase liquid chromatography (RPLC) is used

An effective UHPLC–MS method for high throughput separation, identification and quantification of pseudoephedrine was developed on a Hypersil GOLD PFP 1.9 µm, 2.1 Ã- 100 mm column.

Several common birth control formulations contain both drospirenone and ethinyl estradiol. A highly selective and sensitive analytical method for the analysis of drospirenone in human plasma has been developed for use in bioequivalence studies. The solid-phase extraction (SPE) and UPLC–MS–MS methodologies are described as well as performance against validation parameters.

In March 2007, several North American manufacturers of pet food voluntarily issued nationwide recall notices for some of their products that were reportedly associated with renal failure in pets. The raw material wheat gluten, used to manufacture the pet food, was imported from China and was identified as the source of contamination.

This application note describes a fast and sensitive LC–MS method using a Hypersil GOLD column on a Thermo Scientific LC–MS system for the quantitative analysis of two widespread PFCs, perfluorooctanoic acid (PFOA) and perfluorooctansulphonate (PFOS).

Short analyses time and high resolution are in great demand from R&D and QC departments within the pharmaceutical industry. Sub-two micron ODS reversed phase columns have recently been introduced to meet these requirements, but these columns require an ultra-high pressure HPLC system to achieve optimum performance. TSK-GEL ODS-140HTP, 2.3mm columns from Tosoh Bioscience have been developed to offer a combination of short analyses time and high resolution separations that can be run at modest pressures, making these columns compatible with conventional HPLC instrumentation. The polylayer bonding chemistry of these columns results in highly efficient and physically stable columns when operated at high linear velocities. In addition, TSK-GEL ODS-140HTP, 2.3mm columns can be efficiently operated at pressures not exceeding 9000psi in UPLC® and other ultra-high pressure HPLC systems, as well as in traditional HPLC systems.

Pharmaceutical actives and impurities often contain sulfur in the form of a sulfone or sulfoxide group. Both groups have dipole moments, adding a hydrophilic character to compounds containing these functional groups. The analysis of hydrophilic compounds on traditional alkyl columns (e.g. C18) can be problematic, since alkyl columns depend on hydrophobic interactions for retention. Since the sulfone and sulfoxide groups contain π-π bonds, the biphenyl column's ability to undergo π-π interactions makes it an excellent choice when increased retention or selectivity of compounds containing these groups is desired. To demonstrate the selectivity of the biphenyl phase towards aromatic compounds containing sulfur groups, a set of target compounds was selected and analyzed on C18, phenyl, and biphenyl columns.

In pharmaceutical development, it is important to analyze small molecules or their metabolites in biological fluids. For this purpose, the analytical methods such as sample pretreatment, 2D-LC and LC–MS have been developed. However there are still problems of resolution and protein adsorption. As a result, satisfying analytical results have not always been achieved.

Several common birth control formulations contain both drospirenone and ethinyl estradiol. A highly selective and sensitive analytical method for the analysis of drospirenone in human plasma has been developed for use in bioequivalence studies. The solid-phase extraction (SPE) and UPLC®–MS–MS methodologies are described as well as performance against validation parameters.

The use of mobile phase pH to control analyte ionization states (pH-LCâ„¢) in reversed phase HPLC separations is a highly effective way to change selectivity. The ionized species of an analyte is shown to have higher polarity (less hydrophobicity) than the neutral species, which results in a loss of expected retention for that analyte. This can be attributed to less interaction with the hydrophobic stationary phase and greater affinity with the aqueous portion of the mobile phase. Ionized species also participate in ionic interactions with exposed and activated silanols, which impact peak shape and reproducibility.

Bradykinin, a 9 amino acid peptide, is a physiologically and pharmacologically active peptide of the kinin group of proteins, which is used in the development of antagonists and therapies for hereditary angioedema. In this application, 50 mg of crude bradykinin, synthesised on a StratoSpheresâ„¢ PL-Rink resin, is purified using an HPLC method that can be scaled from the laboratory through to full production.

With recent advances in technology and the use of ultra high pressure chromatography systems becoming more commonplace, it is of use to compare reproducibility of retention time and peak area of UHPLC vs HPLC.

Azo dyes are used widely in the manufacture of various consumer goods such as leather, textiles, plastics, paper, hair care products, and cosmetics. On September 11, 2003, the European Union enacted European Parliament Directive 2002/61/EC, prohibiting the manufacture and sale of consumer goods containing specified azo dyes (1). The azo dyes of concern are those that can be reduced to aromatic amines. There are 22 aromatic amines classified as carcinogenic or potentially carcinogenic to humans.

Aminoglycoside antibiotics are among those commonly used in animal feeds to manage intestinal microorganisms. The beneficial effects include improved growth and generally healthier animal populations. Use of antibiotics creates a demand for analytical procedures to verify concentrations in pre-mixes and feeds and in some instances for residue analysis in animal products.

There has been an increasing interest in the presence and availability of compounds in plant materials that may possess bioactive properties, in particular, antioxidant activity. Some of these compounds have been attributed to possess anticancer, antiaging, and antimutagenic properties as well as other health benefits (1). The types of plants that have been investigated cover a vast range from common foodstuffs to regional or exotic materials. Plant parts under study have included portions that are traditionally known to be edible, as well as sections that are considered "waste" or used for animal forage. Because most screening techniques involve lengthy separations, high throughput HPLC methods are desirable.

Urea and allantoin are added to cosmetic products for skin protection and regeneration, especially for the treatment of dry skin, and analyzed for QC purposes. As polar compounds, they are not ideal for reversed-phase HPLC separations. Neutral hydrophilic compounds like urea and allantoin are best analyzed by hydrophilic interaction chromatography (HILIC). Traditional HILIC columns use silica modified with a hydrophilic group such as diol or cyano. Analytes are adsorbed and subsequently eluted with mobile phases containing high percentages of organic solvent (>75%).