Analytical Services
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Analysis of lipid compounds requires alternate approaches to those used in classical non-polar compounds. Phospholipids, sphingolipids and neutral lipids derived from natural sources are polar, heterogenous in molecular weight and diverse in structure and species. Normal phase HPLC methods provide lipid class separation and quantitation on a variety of sample types. Reversed phase HPLC and gas chromatography methods are employed for analysis within a lipid class. Classical methods such as thin-layer chromatography, elemental phosphorus, acid value, peroxide value and moisture content are available for qualitative and quantitative information on lipid containing samples. The modern techniques of mass spectrometry and nuclear magnetic resonance provide molecular information and definitive identification of a lipid compound or extract. Discovery and quantitation of periodic elements within a sample can be performed using inductively coupled plasma-mass spectrometry or ion chromatography. Descriptions of available methodologies are listed below. |
Extraction of Lipids for Analysis
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Unless a submitted sample is directly and completely soluble in a non-polar solvent or solution, it will require extraction prior to analysis. The most common extraction methods for lipid samples are the modified Folch and the Bligh and Dyer methods. Extraction recovery is assumed to provide 100% recovery of lipids from a submitted matrix. Additional spike recovery procedures after discussion with Avanti’s technical staff may be required to document actual recovery. |
Thin Layer Chromatography
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Thin layer chromatography performed against appropriate standards provides a fast and accurate means to determine the identity of lipids classes present in a sample. This technique is typically the starting place for any sample of unknown composition. |
Fourier Transform Infra-red Spectroscopy
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Qualitative analysis by mid-range Fourier Transform IR (FTIR) can be performed on solids as well as liquids by ATR. Best results are obtained on purified materials. Functional groups are readily identified to aid in the assignment of chemical structure and/or formula. Spectral matching can be performed against any of Avanti’s lipid compounds for confirmation. |
Wet Chemistry Methods
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A variety of wet chemistry methods are available to measure specific entities associated with phospholipid and lipid compounds.
Some of these include: |
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Phosphorus by UV/VIS Spectrophotometry
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is a quantitative method, which measures elemental phosphorus after sample digestion. The results can be used to calculate information such as % phosphorus and concentration of a phospholipid solution. Weight % determination of a purified lipid may be assigned based on measurements relative to theoretical phosphorus content. |
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Acid Value
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is a semi-quantitative titration method which measures the free acid content in a sample. The method is based on American Oil Chemist’s Society method Cd 3d-63. Results are reported as the milligrams of KOH necessary to neutralize free acids in 1 gram of sample. |
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Peroxide Value
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is a semi-quanitative measurement of the degree of early oxidation events in lipid and phospholipid preparations. The method is based on American Oil Chemist’s Society method Ja 8-87. Results are reported as mEq peroxide / Kg of sample. |
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Water Content by Karl Fisher Titration
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measures the amount of water in sample as either % by weight (w/w) of % by volume (v/v). Sample must be soluble in non-polar solvents such as alcohols, alkanes and chlorinated hydrocarbon solvents. Acetone and other ketone solvents are not acceptable for apparatus. |
High Performance Liquid Chromatography / Evaporative Light Scattering Detection
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High performance liquid chromatography coupled with an evaporative light scattering detector (HPLC/ELSD) performs separation and quantitation of lipid compounds in a sample. Methods include normal phase techniques for class separation and reversed phase techniques for measurement of known compounds within a formulation. Separation and quantitation of lipids and phospholipids in a complex mixture or matrix is one of our specialties. Wide quantitative range calibration with matrix specific standards diminish inaccuracies associated with an ELSD’s non-linear response. Target compound analysis to determine % composition in a food or pharmaceutical product is available through HPLC/ELSD analysis upon method development and validation. |
Nuclear Magnetic Resonance Spectroscopy
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Nuclear magnetic resonance (NMR) spectroscopy for phosphorus is a method for quantitation of phospholipid mixtures. The linear response, high resolution and relative speed of 31P NMR allows for accurate and selective analysis. 31P – NMR 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. Customers should inquire on available services and technical aspects of performing sample analysis prior to submission of samples. Method development and / or validation is available.
31P-NMR brochure. |
Electrospray Mass Spectrometry
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Electrospray mass spectrometry (ESMS) is a low fragmentation technique whereby molecular weight and structural information can be obtained from a small sample amount. Infusion of lipid solutions into the electrospray or atmospheric chemical ionization interface can identify the heterogenous molecular species present. Structural information / confirmation can be obtained utilizing tandem mass spectral (MS/MS) or MS/ion trap techniques. Quantitative trace analysis for targeted analytes and degradation products in a sample is possible at sensitivities of microgram to picograms with HPLC /MS/MS. Customers should inquire on available services and technical aspects of performing sample analysis prior to submission of samples. Method development and / or validation is available.
LC/MS / MS brochure. |
Capillary Gas Chromatography
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Our experience with gas chromatography for the characterization of fatty acids is extensive. Use of modern capillary columns with a choice of stationary phases, coupled with flame ionization detection or mass spectrometry 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 20 ppm. |
Fatty Acid Methyl Ester (FAME)
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provides % composition of fatty acids present in a sample. Sample preparation techniques allow for assay of esterified fatty acids only or total fatty acids (esterified and free). Standardization is available for fatty acids ranging from C4 through C24. Quantitation of fatty acids on a weight percent basis employs internal standards spiked into sample matrix upon extraction / preparation.
GC/MS brochure. |
Elemental Analysis
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Quantitation of periodic elements in lipid samples is available using inductively coupled plasma – mass spectrometry (ICP-MS). Applications for this technique include heavy metal and earth metal analysis. Cation and anion analysis is also available utilizing ion chromatography (IC) coupled with polarity specific conductivity detection. |
Analytical Method Development and Validation
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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, to determine how best to meet your needs. Following this consultation, we provide a quotation for the method development and validation services. |
Stability Testing of Lipid Related Products
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Avanti can perform stability testing of lipid-related products or raw materials utilizing a combination of any of our assays. Development of 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 at each time point. Following an initial consultation, we provide a quotation for the stability testing services. |
Lipid Molecular Characterization
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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 provide a quote for molecular characterization. |
Professional Consultation
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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. |
Instrumentation
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• Bruker Avance™ III 400mHz with BBFO Probe NMR spectrometer. This new milestone in NMR
detection fidelity features second-generation digital receiver technology.
• Binary or quaternary HPLC systems with Sedex evaporative light scattering detectors
(UV available).
• Binary HPLC system with chemiluminescent nitrogen detector.
• Capillary gas chromatographs with flame ionization detector.
• GC-Mass Spectrometer with direct injection and headspace autosampler.
• 4000 QTrap™ mass spectrometry system with Agilent model 1100 binary system.
• 4000 QTrap™ mass spectrometry system with Dionex Ultimate 3000 dual flow (macro & micro)
capability and dual 10 port valve adaptability.
• ICP-DRC2 mass spectrometer.
All instrumentation is IQ/OQ Qualified upon installation and Performance Qualified through scheduled maintenance agreements.
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Analytical Services