Application Notes: LC

Simulated Moving Bed technology (SMB) is a continuous chromatography technique widely used in the pharmaceutical/biotechnology industry. The continuous chromatographic separation of sodium sulphate (Na2 SO4) from a mixture of glucose and mannose was established by using the Orochem Zuccheroâ„¢ columns and the SMB technology. The main advantages of SMB over batch chromatography includes better yields, higher purity, and decreased solvent usage, which together make the SMB technology economically viable and ideal for desalting applications.

Many polar compounds are difficult to retain by reversed-phase analysis without the use of ion-pair chromatography. We have overcome this disadvantage with a revolutionary phase structure called "Multi-mode ODS." This technology uses uniformly blended packing material consisting of two types of porous silica particles: 3 μm silica substituted with ODS+cation ligands and 3 μm silica substituted with ODS+anion ligands (Figure 1). This novel multi-mode ODS column, named Scherzo SM-C18, enables a multi-separation mode, consisting of: anion exchange, cation exchange, normal phase, and reversed-phase. In this article, we will validate this multi-separation mode by separating compounds which are difficult to separate on ODS column.

Although supercritical fluid chromatography (SFC) is generally regarded as a technique to separate enantiomers, it has also been proven in many laboratories as a fast approach for the purification of impurities in achiral samples.

Polynuclear aromatic hydrocarbons (PAHs) are carcinogenic condensed ring aromatic compounds widely found as trace pollutants in waters, wastes, air particulates, soil, and foods. PAHs can be monitored routinely using HPLC with a combination of UV and fluorescence detection as prescribed in EPA methods 550.1, 610, and 8310. Conventional HPLC analysis of 19 PAHs typically requires 20 min and uses 25 mL of acetonitrile. However, there is a continual drive to improve productivity and reduce solvent consumption and waste in chemical analysis. Using ultra high pressure LC (UHPLC) with sub-2 μm particle-size columns, we demonstrate a 3-fold improvement in throughput and a 90% reduction of mobile phase solvent in the determination of 19 PAHs.

Iodine is an essential nutrient in seawater, seafood, and iodine-enriched foods, such as iodized table salt. The most common forms of iodine in the diet are iodide and iodate, with additional iodo-organic compounds providing a small fraction of the bio-available iodine. Iodine deficiency affects thyroid hormone production and leads to developmental diseases, goiter, and paralysis (1).

Since the introduction of high-performance liquid chromatography (HPLC) nearly 40 years ago, many improvements have been made to column stationary phases to achieve faster, more efficient separations. HPLC columns containing superficially porous (sometimes called fused-core) particles have recently gained increasing attention. Though this technology is not entirely new, it has been improved to the point where rapid, highly efficient separations can be achieved for some applications.

Measure UV-absorbing compounds in consumer products and characterize the UV absorption spectra of the individual components by employing high-speed liquid chromatography with photodiode array detection.

Salt formation is a critical aspect in drug development (1) and HPLC is an important tool for determination of pharmaceutical counterions. Reversed-phase columns often fail to retain very hydrophilic counterions. Ion chromatography (IC) is preferred for selective and sensitive screening of cationic and anionic pharmaceutical counterions (2), usually with multiple runs. Positive and negative counterions can be separated simultaneously using a zwitterionic HILIC column using an evaporative light scattering detector (3). However, the zwitterionic column method has limitations: 1) limited to HILIC mode; 2) ions are retained as salts rather than via ion-exchange, making method development difficult; and 3) the method is not suitable for simultaneous separation of acidic, basic, and neutral analytes.

This application note describes a fast LC method for the analysis of water-soluble vitamins using a Thermo Scientific Hypersil GOLD aQ column.

Since the USA Patriot Act* has been enacted, many pharmaceutical companies have reformulated their over the counter (OTC) drug products with phenylephrine (a nasal decongestant) as a substitute for pseudoephedrine. Phenylephrine comes as a tablet, a liquid, or a dissolving strip to take orally - all as a treatment for cold symptoms (1). Besides phenylephrine, most pharmaceutical formulations for common cold and sinus medications often contain multiple active ingredients to treat different types of symptoms in addition to numerous excipients. From an analytical perspective, the challenge is to develop chromatographic conditions that allow quantitative analysis of a variety of excipients that vary widely in hydrophobic properties.

Since September 2008, 294 000 infants and young children suffered urinary problems as a result of the contamination of melamine in infant milk powder and were hospitalized. This hospitalization was required to treat the symptoms caused by the ingestion of melamine contaminated infant formula and related dairy products. Previously in 2007, pet food, animal feed wheat gluten and other protein-based foods were found to contain residues of melamine and its degradation product cyanuric acid.

Using HILIC with highly efficient ethylene bridged hybrid (BEH) particles results in faster methods that exhibit improved polar retention, higher sensitivity, enhanced chromatographic resolution and significantly improved column lifetime.

Hydrophilic interaction liquid chromatography (HILIC) offers unique advantages for the separation of very polar compounds when compared to reversed-phase chromatography. A new silica based HILIC phase was developed to provide additional selectivity options in HILIC separations. The separation of water soluble vitamins on the new TSKgel NH2-100 HILIC column and on the well known TSKgel Amide-80 HILIC column demonstrates the differences in selectivity.

Forensic laboratories face a daunting task to identify trace amounts of controlled substances in small samples of seized evidence. Unambiguous identification is required to meet the stiff challenge that is sure to be raised in the courtroom. Positive proof is especially difficult to establish if the controlled substance is hidden in a complex food matrix with a high content of sugars, fats, fatty acids, proteins and alkaloids.

Penicillins are regulated substances in food for human consumption. The minimum residue limits range from 1 ng/g to as high as 50 ng/g depending on the compound. Because of the complexity of food matrices measuring the level of contaminants requires a total solution composed of sample extraction, sample clean-up, chromatographic separation and detection. In this paper, a solid-phase extraction method with a high performance liquid chromatography tandem mass spectrometry method (HPLC–MS–MS) is shown for the simultaneous determination of six antibiotic residues.

Phenols are frequently present in water because of their widespread use in commercial products and because they are by-products of processes in petrochemical, pulp and paper, plastic, and glue manufacturing industries (1,2). The concentration of phenolic compounds in the waste discharges can be as high as 20 mg/L (2); however, phenol-containing pesticides and wood preservatives may cause significant health hazards even at mg/L levels (1). Consequently, it is important to monitor phenols and substituted phenols in environmental and biological samples. Liquid chromatography with electrochemical detection is one of the widely used methods due to its high selectivity and sensitivity for phenolic compounds. However, glassy carbon working electrodes, used in the electrochemical detection of phenols, often require polishing (3). This time-consuming and often poorly reproducible polishing can be avoided with disposable carbon electrodes, which offer comparable or better analytical performance (4).

In HPLC method development, screening of various para-meters such as stationary phase, eluents, and temperatures is conducted to find optimal resolution. However, method development can be a time consuming and inefficient process. UHPLC technology can be applied to significantly shorten both the analysis and development times. Here we describe an integrated and ultrafast automated method scouting solution that provides fast and efficient method development processes.

Development of cephalosporin antibiotics has led to compounds with a broad spectrum of activity against both Gram-positive and Gram-negative bacteria with low toxicity profiles. Cefepime, a fourth-generation cephalosporin, is a commonly prescribed broad spectrum antibiotic with improved activity against Gram-negative bacteria compared to other commercially available cephalosporins (1). Despite extensive research on this class of drugs, quantitative analysis and purity assays remain problematic (2).

This method is rapid and sensitive for the analysis of melamine and cyanuric acid simultaneously in infant formula. Using two Oasis solid-phase extraction protocols and the ACQUITY UPLC, the results are consistent with the published US FDA interim method, while demonstrating a reduced analysis time.

Carbonyl compounds, including low molecular weight aldehydes and ketones, have environmental and health concerns; for example, short-term exposure to aldehydes can irritate the eyes, skin, and upper respiratory tract. Motor vehicles emit reactive hydrocarbons that undergo photochemical oxidation in the atmosphere, which generates formaldehyde and other carbonyls. In addition, formaldehyde contributes to the formation of photochemical ozone. California Air Resources Board (CARB) Method 1004 (1) provides an analytical method for the automotive industry to monitor 13 carbonyl compounds in engine exhaust. US EPA Method TO-11A (2) and Method 8315 (3) monitor atmospheric formaldehyde and 14 other carbonyl compounds and are used for a variety of environmental and occupational health purposes. In these methods, carbonyl compounds are trapped as the dinitrophenylhydrazine (DNPH) derivatives before analysis by HPLC.

Increased temperature has been used to assist the elimination of organic modifier required in the mobile phase, to achieve analyte separation in pure aqueous mobile phases.

Using HILIC with highly efficient ethylene bridged hybrid (BEH) particles results in faster methods that exhibit improved polar retention, higher sensitivity, enhanced chromatographic resolution, and significantly improved column lifetime.

The new reversed-phase ProSwift® 1 mm i.d. column is a divinylbenzene-based monolithic column for routine chromatography of proteins and other biomolecules. It is available in two different lengths. The shorter (1 Ã- 50 mm) format is designed for fast separations and the longer (1 Ã- 250 mm) format is intended for high resolution analytical separations. However, depending on the application, either can be used for separation of proteins and for coupling with mass spectrometry.

Goal: Positively identify trace amounts of cannabinoids in a complex food matrix quickly, with minimal sample preparation and no chemical derivatization.

A highly sensitive analytical method for the analysis of tamsulosin in human plasma has been developed for use in bioanalytical studies. The solid-phase extraction (SPE) and UPLC–MS–MS methodologies are described, as well as performance against validation parameters.

An ultrafast gradient LC separation method was developed to separate a 5-component drug mixture in 30 seconds, with peak widths of 1 second. maXis mass accuracy at sub-ppm levels and true isotopic pattern of the spectra from the peaks lead to a confident elemental formula assignment for each drug compound with the SmartFormula algorithm.

The separation and quantitation of drug substances and counterions are two important determinations in the pharmaceutical industry (1). Drug substances and counterions are determined by HPLC (often on reversed-phase columns) and by ion chromatography (IC), respectively. IC is the preferred method for selective and sensitive screening of both cationic and anionic pharmaceutical counterions (2). To increase the analysis throughput, it is desirable that for analysis of drug formulation both drug substance and counterions can be determined within a single run. Naproxen is a non-steroidal anti-inflammatory drug commonly used for treating moderate to severe pain, fever, inflammation, and stiffness. Naproxen sodium was approved by the U.S. Food and Drug Administration (FDA) as an over-the-counter drug. No reports were found describing one technique for the simultaneous determinations of both naproxen and the counterion (Na+).

Improve the separation of 14 drugs of abuse in a complex mixture by employing a ternary solvent gradient.

Additional studies were undertaken to better understand the chromatographic behavior of PEGylated proteins in an effort to improve purification and characterization techniques of such proteins. Proteins were PEGylated using larger (20 KDa and 40 KDa) PEGylation reagents that are commonly used in pharmaceutical drug development. Generated PEGylated proteins were separated from unmodified proteins using different reversed phase medias (Jupiter® C4 and Jupiter® C18). In these studies it was found that the Jupiter C18 media provided the best separation of PEGylated proteins from their unmodified counterparts. Such results further clarify good method starting points for developing analytical and preparative separations of PEGylated proteins.

Transforming "standard" gradient HPLC systems into extremely fast gradient systems is readily achievable with proper application of chromatographic principles, particularly temperature control, combined with utilization of advanced HPLC columns. Bottom line: You don't have to buy a new LC to achieve ultra high quality and speed!