Avanti Analytical Procedures

Qualitative and Quantitative Analysis
Analysis of phospholipids requires a "re-thinking" of typical analytical methodologies. Phospholipids derived from natural sources by their nature are bipolar and non-homogenous, precluding direct analysis by gas chromatography and reversed phase HPLC. Avanti approaches these problems with multi-technique protocols. Each technique in the protocol will yield part of the information required to define the identity, purity, and quantity of phospholipids present in a sample. These techniques offered by Avanti are described below.

Thin Layer Chromatography
Thin layer chromatography performed under a standardized procedure against appropriate standards provides a fast and accurate means to determine the identity of compounds present in the sample. This technique is typically the starting place for any sample of unknown composition. Confirmation of identity can be achieved by migration of the sample in multiple mobile phases in which it will exhibit a change in Rf or elution order.

Fourier Transform Infra-red Spectroscopy
Qualitative analysis by mid-range Fourier Transform IR (FTIR) can be performed on solids as well as liquids by either diffuse reflectance or circle cell deflection. Definitive results are best obtained on purified materials. Functional groups are readily identified to aid in the assignment of chemical structure and/or formula. Unknowns can be searched against a compendial library for identity.

Wet Chemistry Methods
A variety of wet chemistry methods are available to measure specific entities associated with phospholipid and lipid chemistry. Some of these include:

Phosphorus by UV/VIS Spectrophotometry is a quantitative method, which measures elemental phosphorus after sample digestion. The results can be used to calculate information such as % phosphorus by weight, concentration of a solution, where the molecular weight of the phospholipid is known, and % of non-phospholipid. Acid Value is a quantitative method, which measures the % of free acid in the sample. This acid can be either inorganic or organic, as in the case of free fatty acids in phospholipid samples. Peroxide Value quantitates the degree of oxidation in lipid and phospholipid preparations as % peroxide by weight. A potentiometric endpoint alleviates interference from colored solutions or solids. Water Content by Karl Fisher Titration measures the amount of water in phospholipid or lipid sample as either % by weight (w/w) of % by volume (v/v).

Nebulized Flame Atomic Absorption
Nebulized flame atomic absorption (NFAA) is useful to determine the counter-ions present on the phosphate center of acidic phospholipids. NFAA can also be used to measure composition of common trace elements. Elements currently available include: Na⁺, K⁺, Ca²⁺, Mg²⁺ and Cd²⁺.

High Performance Liquid Chromatography /
Evaporative Light Scattering Detection
High performance liquid chromatography coupled with an evaporative light scattering detector (HPLC/ELSD) provides a universal separation and detection method for phospholipids, both of saturated and unsaturated nature. Methods include normal phase and reversed phase techniques utilizing several solid phase species. Resolution and quantitation of lipids and phospholipids contained in a complex mixture or matrix is one of our specialties. Wide quantitative range calibration with matrix specific phospholipid standards diminish inaccuracies associated with ELSD concentration vs. area responses. Target compound analysis to determine % composition in a food or pharmaceutical product is available through HPLC/ELSD analysis, once method development and validation is performed.

Nuclear Magnetic Resonance Spectroscopy
Nuclear magnetic resonance spectroscopy for phosphorus is quickly becoming the definitive method for quantitation of phospholipid mixtures. The linear response, high resolution and relative speed of ³¹P NMR allows for accurate and selective analysis with higher sample throughput. ³¹P is also a superior method for determining the purity of a phospholipid on a w/w basis. ¹H and ¹³C NMR are available for molecular characterization of unknown lipid compounds.

Electrospray Mass Spectrometry
Electrospray mass spectrometry (ESMS) is a low fragmentation technique whereby molecular weight information can be obtained from a small sample either in positive or negative mode. Infusion of phospholipid solutions into the electrospray interface can identify the molecular species for each headgroup present. Quantitation can be accomplished with HPLC separation prior to mass spectral detection. Development of HPLC/ELSD/ESMS methods is also possible. Trace analysis for known substances in a sample is possible using limited range scanning and selected ion monitoring, providing sensitivity in the 10^-6 to 10^-15 gram range
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Capillary Gas Chromatography
Our experience with gas chromatography for the characterization of fatty acids and lipids is extensive. Use of modern capillary columns with a choice of several stationary phases, coupled with flame ionization detection, support a vast range of possible analytical applications. Present applications include:

Residual Organic Volatiles in final pharmaceutical or food preparations are sensitive and accurate to 10 ppm (0.001%w/w). Fatty Acid Methyl Ester (FAME) provides % composition of fatty acids present in a sample either as a total or as esterified fatty acids from phospholipids or glycerides. Data from this method can be used to calculate the ratio of unsaturated vs. saturated fatty acids present in a sample, replacing the old iodine number wet chemistry assay. Quantitation of esterified fatty acid on a w/w basis yields the information obtained from a saponification value.

Analytical Method Development and Validation
Avanti's analytical service can develop methods specific for your needs using any of the technologies listed. Our validation protocols follow Good Laboratory Practices (GLP) guidelines, or you may supply a protocol to meet internal specifications. A method development demonstrates the ability to measure a substance. Each method development is accompanied by a standard operating procedure (SOP), which outlines the steps and equipment necessary to perform the analysis. A method validation provides the statistical data to support an accurate and reliable methodology. An Analytical Validation Report (AVR) outlining the method performance is submitted for review on each method validated. An initial consultation is required, so we can determine how best to meet your needs. Following this consultation, we will provide a quotation for the method development and validation services.

Stability Testing of Lipid Related Products
Avanti can perform stability testing of lipid-related products or raw materials utilizing a combination of any of our assays. Development of a a testing protocol based on International Committee of Harmonization (ICH) Stability Testing Guidelines is available. Samples are sent to Avanti on a pre-determined schedule for analysis. Results are reported back to you upon completion of testing. Following an initial consultation, we will provide a quotation for the stability testing services.

Lipid Molecular Characterization
The favorite question of an analytical chemist is "What is this?" Our present technologies can aid you in answering this tough question. Combinational analysis provides specific data from each technology. Then this puzzle of information is put together for our best answer. Extensive consultation on approach, present knowledge, and limitations of testing is required. After consultation, we will provide a quote for molecular characterization.

Professional Consultation
Sometimes all you need is an idea to get started. Our experience in phospholipid and lipid analysis is available to you through direct conversation with one of our many technical experts. Avanti requires initial consultation on new projects and on method development / validation, or molecular characterization. This assures the best, most accurate data can be obtained in the shortest amount of time with the fewest mistakes.