Charged Aerosol Detector Research Articles

Hydrophilic interaction chromatography with aerosol-based detectors (ELSD, CAD, NQAD) for polar compounds lacking a UV chromophore in an intravenous formulation

In this work, a high performance liquid chromatography (HPLC) method is reported for the separation and quantitation of a drug substance that is highly polar and lacking a chromophore in a mannitol intravenous (IV) formulation. Three polar stationary phases operated in hydrophilic interaction chromatography (HILIC) mode were evaluated in conjunction with an Alltech 800 ELSD detector. These columns were evaluated with respect to chromatographic properties such as buffer, pH and organic concentrations to identify the best stationary phase. The chromatographic method was then validated for the determination of mGlu2/3 receptor agonist (−)-(1R, 4S, 5S, 6S)-4-Amino-2-sulfonylbicyclo [3.1.0] hexane-4,6-dicarboxylic acid (LY404039) content in a mannitol IV formulation with respect to linearity (R2 of 0.9997), repeatability (%RSD of 0.36%), accuracy, solution stability (99.56% after 24h), specificity, intra-assay precision (%RSD 0.48%) and limit of detection (LOD, ∼50μg/mL). In addition to the Alltech 800 ELSD detector, several other aerosol-based detectors were investigated for reproducibility, linearity and LOD. These additional detectors consisted of an Alltech 3300 evaporative light scattering detector (ELSD), a nano quantity analyte detector (NQAD) and a charged aerosol detector (CAD). Based on the data from this report, a feasible isocratic LC method was achieved using a TSKgel Amide-80 column with mobile phase conditions of 30% water with 0.2% trifluoroacetic acid (TFA) and 70% acetonitrile (ACN) using any of the four aerosol-based detectors for detection and quantitation.

A comparative study on the analytical performance of a charged aerosol detector and an ultraviolet detector for the RP-LC analysis of dabigatran etexilate in capsules

The reversed-phase liquid chromatography (RP-LC) method was validated for the assessment of dabigatran etexilate (DAB) in capsules using an ultraviolet detector (UV) and a charged aerosol detector (CAD) connected in series. The RP-LC method was carried out on a GL Sciences Inc. Inertsil ODS-2 column (250 mm × 4.6 mm I.D.). The mobile phase consisted of acetonitrile and ammonium acetate buffer (pH 5.5; 10 mM) (65 : 35, v/v) (flow rate 1.0 mL min−1). CAD detection was performed using a nitrogen pressure of 35 psi and a range of 100 pA and the UV detection at 225 nm. The chromatographic separation was obtained with a retention time of 6.7 min. Validation parameters such as specificity, linearity, precision, accuracy and robustness were evaluated. The method was linear over the concentration range of 1–70 μg mL−1 in both detectors. The specificity and stability-indicating capability of the method were proven through the degradation studies, which also show that there was no interference from the excipients. The method was precise and accurate, with the Plackett–Burman experimental design used to evaluate robustness, delivering results within the acceptable range. The method was successfully applied for DAB analysis and the results of the CAD and UV detectors were compared, showing a non-significant difference (P > 0.05).

Determination of neomycin and related substances in pharmaceutical preparations by reversed-phase high performance liquid chromatography with mass spectrometry and charged aerosol detection

A new, simple and repeatable liquid chromatographic method with charged aerosol detection (LC-CAD) for determination of neomycin and related substances has been developed. Analysis of neomycin or other aminoglycosides is problematic due to a lack of chromophores. Universal response of CAD enables direct quantification of neomycin and related substances, for which no reference standard are available. Separation was performed on C18 Hypersil® Gold aQ column using water, methanol and heptaflurobutyric acid as mobile phase. Under developed chromatographic conditions all impurities were well separated from neomycin B. Peaks identification was evaluated by electrospray ionization mass spectrometry. The proposed method was validated according to ICH guidelines and applied to the content determination of neomycin and related substances in pharmaceutical preparations.

Quantification aspects of constant pressure (ultra) high pressure liquid chromatography using mass-sensitive detectors with a nebulizing interface

The present contribution investigates the quantitation aspects of mass-sensitive detectors with nebulizing interface (ESI-MSD, ELSD, CAD) in the constant pressure gradient elution mode. In this operation mode, the pressure is controlled and maintained at a set value and the liquid flow rate will vary according to the inverse mobile phase viscosity. As the pressure is continuously kept at the allowable maximum during the entire gradient run, the average liquid flow rate is higher compared to that in the conventional constant flow rate operation mode, thus shortening the analysis time. The following three mass-sensitive detectors were investigated: mass spectrometry detector (MS), evaporative light scattering detector (ELSD) and charged aerosol detector (CAD) and a wide variety of samples (phenones, polyaromatic hydrocarbons, wine, cocoa butter) has been considered. It was found that the nebulizing efficiency of the LC-interfaces of the three detectors under consideration changes with the increasing liquid flow rate. For the MS, the increasing flow rate leads to a lower peak area whereas for the ELSD the peak area increases compared to the constant flow rate mode. The peak area obtained with a CAD is rather insensitive to the liquid flow rate. The reproducibility of the peak area remains similar in both modes, although variation in system permeability compromises the ‘long-term’ reproducibility. This problem can however be overcome by running a flow rate program with an optimized flow rate and composition profile obtained from the constant pressure mode. In this case, the quantification remains reproducibile, despite any occuring variations of the system permeability. Furthermore, the same fragmentation pattern (MS) has been found in the constant pressure mode compared to the customary constant flow rate mode.

Lokesh Bhattacharyya and Jeffrey S. Rohrer (Eds): Applications of Ion Chromatography for Pharmaceutical and Biological Products

Ion chromatography revolutionized the analysis of small inorganic ions by replacing tedious individual gravimetric and titration methods with a single automated process, capable of analysing a number of ions simultaneously. Today the range of potential applications has grown to include a wider range of ionic and ionizable materials such as biopolymers. This book focuses on a segment of those applications arising from the use of ion chromatography in the pharmaceutical industry, and does so very well. The book is a little gem, and those readers attracted to the book by its title will find that it delivers exactly what it promises. The book contains 24 chapters under four general section headings with an appendix containing acid dissociation constants for compounds of pharmaceutical interest. Part I describes instrumentation, retention mechanisms, and operating principles in five chapters. These are quite general and useful to novice and more experienced chromatographers alike. There is a good discussion of ion exchange and ion exclusion mechanisms, modelling and method development, and instrumentation (focused on the design of ion suppressors and the pulsed electrochemical detector). The pulsed electrochemical detector is discussed further in later chapters, particularly its applications in biotechnology, where it has played a major role in the expansion of ion chromatography from its origins. Later chapters also discuss the use of the charged aerosol detector and mass spectrometry for the detection and identification of compounds difficult to determine by conductivity and electrochemical methods. These are novel detection methods at present, but have the potential to play a significant role in the development of ion chromatography for the analysis of unconventional samples. The coupling of ion chromatography to mass spectrometry (electrospray ionization of the desalted eluent) tackles the problem of how to identify compounds for which standards are not available, particularly impurities in high molecular weight biopolymers or the totally unexpected compound in a chromatogram. Part II covers pharmaceutical applications in 11 chapters. As well as the expected identification of small anions and cations used in drug formulations, the analysis of aminoglycoside antibiotics and cholinergic compounds is described. About half of all drugs are formulated as salts so the assay of active ingredients and excipients, determination of impurity profiles, identification of contaminants, and forensic source identity of raw materials can be performed by ion chromatography. This section also contains chapters on sample preparation, validation and pharmacopeial methods. The chapters are copiously illustrated with chromatograms, appreciated by this old chromatographer. There is also a focus on regulatory issues of interest to those who work according to the dictates of good laboratory practice. Part III covers biotechnology applications in five chapters. Topics covered include glycan, monosaccharide, oligosaccharide, glycoprotein, amino acid, and carbohydrate C. F. Poole (&) Wayne State University, Detroit, MI, USA e-mail: cfp@chem.wayne.edu

Determination of pharmaceutically related compounds by suppressed ion chromatography: IV. Interfacing ion chromatography with universal detectors

This work forms the final part of a study investigating gradient elution ion-exchange chromatography of pharmaceutically relevant compounds, aiming at achieving complementary selectivity to reversed-phase HPLC. In this study the coupling of three universal detectors (electro-spray ionisation mass spectrometer (ESI-MS); corona charged aerosol detector (CAD); and evaporative light scattering detector (ELSD)) to suppressed IC using complex elution profiles with potassium hydroxide eluents is demonstrated. The non-volatile ions were removed from the eluent by the suppressor prior to detection, thus allowing a stable detector response, especially with the prototype electrolytic suppressor. The detector response for ten weakly anionic pharmaceuticals followed the expected models and the limits of detection obtained were not compromised by the use of a suppressor, yielding values below 50ng/mL with MS, low to sub μg/mL levels with CAD and 2–20μg/mL with ELSD (25μL injections). When coupled to MS and CAD, the prototype electrolytic suppressor showed percentage relative standard deviations (%RSDs) in peak areas of 0.4–2.5% on average, compared to the chemical suppressor which yielded 1.5–3 fold higher %RSD values for the test analytes. The prototype electrolytic suppressor also generally exhibited wider linear response ranges than the chemical suppressor.

Analysis of Sucrose Esters by HPLC Using Charged Aerosol Detector

A high performance liquid chromatographic method has been developed for separation and quantitation of sucrose esters using charged aerosol detection (CAD) combined with mobile phase compensation. Two Acclaim120 C18 columns (75×3.0mm, 3μm) and the gradient composition (0 min – 72% A + 25% B + 3%C, 7.5 min – 75% A + 25% B + 3%C, 19.5 min – 97% A + 3%C, 30 min – 97% A + 3%C, where A is methanol, B is water and C is tetrahydrofuran) were applied. A precisely inverse gradient composition (0 min – 97% A + 3%C, 7.5 min – 97% A + 3%C, 19.5 min – 72% A + 25% B + 3%C, 30 min – 72% A + 25% B + 3%C) was also used. The mobile phase compensation was performed by mixing of the column effluent with the mobile phase of exactly reverse composition provided by a second pump before introduction into the CAD. Introduction

Novel, universal approach for the measurement of natural products in a variety of botanicals and supplements

Botanicals contain a great diversity of compounds that exhibit wide variation in their physicochemical properties. Although no single analytical method is available to measure all potentially active components, HPLC with charged aerosol detection is a nearly universal approach that nonselectively measures any non-volatile and many semi-volatile compounds; that is, CAD does not require that analytes be ionizable (as required for mass spectrometry) or contain a chromophore (as required for UV spectrophotometry). A number of isocratic and gradient HPLC-charged aerosol detection methods were developed and evaluated for the measurement of phytochemicals extracted from a variety of botanicals including: flavonolignans from milk thistle (Silybum marianum); triterpene glycosides from black cohosh (Cimicifuga racemosa); ginkgolides and bilobalides from ginkgo (Ginkgo biloba); ginsenosides from ginseng (Panax ginseng); phytoestrogens from soy; ursane and oleanane triterpenes from gotu kola (Centella asiatica); and diterpene glycosides from stevia (Stevia rebaudiana). Analytes showed consistent response independent of chemical structure (typically <10% variability between compounds corrected for gradient elution). All methods had a wide dynamic range (˜four orders of magnitude), good sensitivity (typically low ng levels of detection), and excellent reproducibility (RSDs typically <2%) even at low detection levels. Comparative data from ELSD and UV detection will also be discussed.

Investigation of polar organic solvents compatible with Corona Charged Aerosol Detection and their use for the determination of sugars by hydrophilic interaction liquid chromatography

A range of organic solvents (ethanol, isopropanol and acetone) has been investigated as alternatives to acetonitrile and methanol when used in conjunction with Corona Charged Aerosol Detection (Corona CAD). These solvents have been evaluated with regard to their effect on the response of the Corona CAD. Three dimensional response surfaces were constructed using raw data showing the relationship between detector response, analyte concentration and percentage of organic solvent in the mobile phase, using sucralose or quinine as the test analyte. The detector response was non-linear in terms of analyte concentration for all solvents tested. However, detector response varied in an approximately linear manner with percentage of organic solvent over the range 0–40% for ethanol or isopropanol and 0–80% for acetone and methanol. The chromatographic performance of the various solvents when used as aqueous–organic mobile phases was evaluated for isocratic and gradient separations of sugars and sugar alcohols by hydrophilic interaction liquid chromatography (HILIC) using an Asahipak NH2P-504E column coupled with Corona CAD detection. It was found that whilst acetonitrile provided the highest column efficiencies and lowest detection limits of the solvents studied, acetone also performed well and could be used to resolve the same number of analytes as was possible with acetonitrile. Typical efficiencies and detection limits of 5330platesm−1 and 1.25μgmL−1, respectively, were achieved when acetone was used as the organic modifier. Acetone was utilised successfully as an organic modifier in the HILIC separation of carbohydrates in a beer sample and also for a partially digested dextran sample.

Poster #212 RISK OF RELAPSE AND HOSPITALIZATION IN THE 2-YEAR OPEN-LABEL TREATMENT OF OUTPATIENTS WITH SCHIZOPHRENIA RANDOMIZED TO OLANZAPINE LONG-ACTING INJECTION OR ORAL OLANZAPINE

Two new stability-indicating liquid chromatographic methods using two detectors, an ultraviolet (UV) and a charged aerosol detector (CAD) simultaneously connected in series were validated for the assessment of lisdexamfetamine dimesylate (LDX) in capsule. The method was optimized and the influence of individual parameters on UV and CAD response and sensitivity was studied. Chromatography was performed on a Zorbax CN column (250 mm × 4.6 mm, 5 μm) in an isocratic elution mode, using acetonitrile and 20 mM ammonium formate at pH 4.0 (50:50, v/v) as mobile phase and UV detection at 207 nm. The developed method was validated according to ICH guidelines and the parameters’ specificity, limit of detection, limit of quantitation, linearity, accuracy, precision and robustness were evaluated. CAD is designated to be a non-linear detector in a wide dynamic range, however, the method was linear over the concentration range of 70–130 μg mL−1 in both detectors. The method was precise and accurate. Robustness study was performed by a Plackett–Burman design, delivering results within the acceptable range. Neither the excipients nor the degradation products showed interference in the method after studies of specificity as well as under stress conditions. The results of the LC-UV and LC-CAD methods were statistically compared through ANOVA and showed no significant difference (p > 0.05). Both proposed methods could be considered interchangeable and stability-indicating, and can be applied as an appropriate quality control tool for routine analysis of LDX in capsule.

Charged aerosol detection in pharmaceutical analysis

Due to its wide field of application, sensitivity, wide range of linearity and the applicability to gradient elution, the most common detection technique for HPLC nowadays is UV/vis spectrophotometry. However, UV/vis detection comes to its limits when the analytes are lacking suitable chromophors or exhibit very different UV responses. In the past years, different types of evaporation/aerosol based HPLC detectors have been developed to fill this gap in HPLC detection. Amongst those, the corona-charged aerosol detector (CAD) is one of the most powerful and versatile representatives. In the recent past a variety of papers have been published, demonstrating the potential of the CAD in different fields of analytical chemistry. This paper is intended to provide an overview of the key performance characteristics and manifold applications for HPLC-CAD in the field of pharmaceutical analysis.

Strategies for the Analysis of Pharmaceutical Cocrystals Using HPLC with Charged Aerosol Detection

Several active pharmaceutical ingredients are currently being developed as pharmaceutical cocrystals as these systems often have superior properties compared to traditional pharmaceutical forms. Pharmaceutical cocrystal formers typically used are polar, small molecule acids or bases which often lack a UV chromophore. Their polar nature results in almost no reversed phase retention and their detection typically cannot be done with UV. Here we discuss approaches for the analysis of pharmaceutical cocrystals using HPLC columns designed for polar retention, ion pairing chromatography (IPC), and hydrophilic interaction chromatography (HILIC) using model cocrystal formers. Corona charged aerosol detection (CAD) was used to monitor the cocrystal formers. l-alanine was used as a model basic cocrystal former, and succinic acid and glutaric acid were used as model acidic cocrystal formers. The acidic cocrystal formers were adequately retained on a C18 column. Heptafluorobutyric acid was used as the ion-pairing reagent for l-alanine as it was unretained without the ion-pairing reagent. HILIC, a newer approach for polar compound retention, was also investigated. Using the HILIC mode, all three model cocrystal formers were retained adequately. Of all the approaches studied for the analysis of the cocrystal formers, HILIC appears to be the best choice as the same column can be used for both acidic and basic cocrystal formers. With IPC, the ion-pairing reagent permanently alters the column chemistry and dedicated columns are required for each ion-pairing reagent used. CAD detection provided a linear response in the 80–100% test concentration range for the analytes studied here.

Analysis of volatile bases by high performance liquid chromatography with aerosol-based detection

The development and performance of two methods are described whereby low levels of volatile bases are quantified by HPLC using either a charged aerosol detector (CAD) or a nano-quantity analyte detector (NQAD). A test set of 12 volatile bases (ammonia, hydrazine, methylamine, ethylamine, diethylamine, triethylamine, isobutylamine, N,N-diisopropylethylamine, morpholine, piperazine, ethylenediamine, and 1,4-diazabicyclo[2.2.2]octane) were analyzed. The studied compounds all exhibit poor UV chromophores and are typically undetectable by aerosol-based detection when using conventional volatile mobile phases. The ability to detect these analytes by CAD or NQAD depended on their propensity towards formation of a low volatility salt between the target analyte and mobile phase modifier. Trifluoroacetic acid (TFA) was found to significantly improve detection of most volatile bases. A low concentration (0.2mM) of hydrochloric acid was additionally needed to enable detection of ammonia. The compounds were separated under hydrophilic interaction liquid chromatography (HILIC) conditions on a zic-pHILIC column. For all analytes, limits of detection (LOD) were measured in the range of 1–27ng on column, which is comparable to previously reported detection limits for non-volatile analytes.

Comprehensive impurity profiling of nutritional infusion solutions by multidimensional off-line reversed-phase liquid chromatography × hydrophilic interaction chromatography–ion trap mass-spectrometry and charged aerosol detection with universal calibration

A new analysis strategy was employed for the establishment of a comprehensive qualitative and quantitative impurity profile of a stressed multi-constituent pharmaceutical drug formulation, namely a nutritional infusion solution composed of amino acids and dipeptides. To deal with the highly complex samples a multidimensional analysis approach was developed which made use of an off-line two-dimensional reversed-phase liquid chromatography (RPLC)×hydrophilic interaction chromatography (HILIC) separation and combination of complementary detection involving ion trap mass spectrometry (IT-MS) and a charged aerosol detector (CAD). The CAD is a mass-sensitive universal detector for non-volatile compounds with relatively consistent detector response. A universal calibration function was set up with a set of standards. This universal calibration function was then employed to quantify unknown impurities allowing their classification into those that need to be reported (>0.05% relative to the precursor compound), identified (>0.1%), and quantified (>0.15%). The dilemma of unavailability of authentic standards at this stage of research for quantification could thereby be circumvented. Relevant impurities above the reporting threshold were identified by IT-MS. Impurities detected comprised di-, tri- and tetrapeptides, cyclic dipeptides (diketopiperazines), pyroglutamic acid derivatives and their condensation products. Cross-validation with HPLC–MS/MS methods using synthesized authentic standards confirmed the results obtained by the presented multidimensional analysis assay.

Non-Derivatization Method for the Determination of Gabapentin in Pharmaceutical Formulations, Rat Serum and Rat Urine using High Performance Liquid Chromatography Coupled with Charged Aerosol Detection

A simple and sensitive isocratic reverse-phase high performance liquid chromatography method coupled with charged aerosol detection (HPLC-CAD) was developed and validated for the determination of gabapentin concentrations in a pharmaceutical formulation and in biological samples (rat serum and urine). Biological samples were simply treated with acetonitrile and directly injected into the HPLC without any further extraction or derivatization procedures. The separation was achieved on a GraceSmart RP-C18 packed column (250 × 4.6 mm, 5 μm) using a methanol-water (55:45, v/v) mobile phase at a flow rate of 1.1 mL/min at 30 °C. The total run time was less than 8 min. The standard calibration curves were linear (r2 > 0.998) over the range of 2-300 μg/mL for the pharmaceutical formulation, 25-800 μg/mL for rat serum and 50-1600 μg/mL for rat urine. The limits of detection (signal/noise = 3) were 0.5 μg/mL, 10 μg/mL and 15 μg/mL for pharmaceutical formulation, serum and urine, respectively. The intra-day and inter-day precisions in the pharmaceutical formulation analysis ranged between 2.7-3.9% and 3.1-4.4%, respectively. In the rat serum analysis, the intra-day and inter-day precision ranges were 3.5-4.7% and 3.0-9.0%, respectively, while the rat urine analysis intra-day and inter-day precision ranges were 1.9-7.6% and 4.2-9.9%, respectively. The developed HPLC-CAD method successfully determined gabapentin concentrations in commercial gabapentin tablets, as well as in rat serum and urine samples after drug administration.

Simultaneous Determination of Caffeine and Aspartame in Diet Supplements and Non-Alcoholic Beverages Using Liquid-Chromatography Coupled to Corona CAD and UV-DAD Detectors

A method for the simultaneous determination of caffeine and aspartame in various diet supplements and non-alcoholic beverages is presented. The analytes were analysed by high-performance liquid chromatography coupled to a charged aerosol detector (Corona CAD) and ultraviolet-diode array detector (UV-DAD) simultaneously connected in series. The method was validated using a Thermo Hypersil Gold-C18 column packed with 5 μm shell particles (150 × 4.6 mm) and acetonitrile–water (15/85% v/v) mobile phase at a flow rate of 1.00 mL/min. The elaborated method was validated for linearity, precision and accuracy. The rapid HPLC–CAD–UV-DAD technique is suitable for quantifying caffeine and aspartame in the range of 0.25–75 μg/mL in diet supplements and non-alcoholic beverages. The limit of detection for caffeine and aspartame was 62 and 43 ng/mL for Corona CAD and 31 and 30 ng/mL for UV-DAD detector, respectively. Each analyte calibration curve had a correlation coefficient of at least 0.999 and was linear in the defined range. The accuracies of CAD and UV-DAD detection were all acceptable, with the mean value of 100% for aspartame and 98.3% for caffeine. Precision values ranged from 0.09% to 1.12%. The work has demonstrated that charged aerosol detector can be successfully used in a rapid screening technique for biologically active substances in non-alcoholic beverages and diet supplements.

“Dilute-and-shoot” triple parallel mass spectrometry method for analysis of vitamin D and triacylglycerols in dietary supplements

A method is demonstrated for analysis of vitamin D fortified dietary supplements that eliminates virtually all chemical pretreatment prior to analysis, which is referred to as a "dilute-and-shoot" method. Three mass spectrometers, in parallel, plus a UV detector, an evaporative light-scattering detector (ELSD), and a corona charged aerosol detector (CAD) were used to allow a comparison of six detectors simultaneously. Ultraviolet data were analyzed using internal standard, external standard, and response factor approaches. The contents of gelcaps that contained 2,000 IU (50 μg) vitamin D(3) in rice bran oil, diluted to 100 mL, were analyzed without the need for lengthy saponification and extraction. Vitamin D(3) was analyzed using UV detection, extracted ion chromatograms, selected ion monitoring (SIM) atmospheric pressure chemical ionization mass spectrometry (APCI-MS), and two transitions of multiple reaction monitoring (MRM) APCI-MS. The internal standard, external standard, and response factor methods gave values of 0.5870 ± 0.0045, 0.5893 ± 0.0041, and 0.5889 ± 0.0045 μg/mL, respectively, by UV detection. The values obtained by MS were 0.6117 ± 0.0140, 0.6018 ± 0.0244, and 0.5848 ± 0.0146 μg/mL by SIM and two transitions of MRM, respectively. The triacylglycerols in the oils were analyzed using full-scan APCI-MS, electrospray ionization (ESI) MS, up to MS(4), an ELSD, and a CAD. The method proved to be very sensitive for vitamin D(3), as well as triacylglycerols (TAGs), allowing identification of intact TAGs containing fatty acids up to 28 carbons in length. LC-ESI-MS of glycerin polymers is also demonstrated.

Quantitation of Underivatized Omega-3 and Omega-6 Fatty Acids in Foods by HPLC and Charged Aerosol Detection

The omega fatty acids are a group of compounds that include essential n-3 (omega-3, e.g., a-linolenic acid [ALA]), n-6 (omega-6, e.g., linoleic and arachidonic acids), and nonessential n-9, (omega-9, e.g., oleic and erucic acids) analytes. The omega-3 fatty acids, which also include eicosapentanoic acid (EPA) and docosahexanoic acid (DHA), are required for normal growth. Their consumption is purported to have a number of health benefits: e.g., cancer prevention, cardiovascular disease prevention, and improved immune function. Although both omega-3 and -6 fatty acids can give rise to eicosanoid-signaling molecules (prostaglandins, prostacyclins, thromboxanes, and leukotrienes), the omega-6 eicosanoids are generally pro-inflammatory and may play a role in cardiovascular disease, high blood pressure, and arthritis. It appears that the amounts and balance of omega fatty acids in a person’s diet affect their eicosanoid-controlled functions. A proper balance of omega fatty acids in the diet is important. Traditionally, omega fatty acids are measured using gas chromatography (GC). For foods, analytes are extracted from the samples prior to hydrolysis to release the fatty acids from their triglycerides, then converted to their volatile methyl esters prior to analysis by GC. This approach is tedious, time-consuming, and the high temperatures can affect polyunsaturated fatty acid stability. The Thermo Scientific Dionex Corona charged aerosol detector provides a univeral massbased approach that is sensitive, has a wide dynamic range, and has a major advantage in that all nonvolative analytes give similar response independent of chemical structure. No derivatization is required, and unlike UV detection, the analyte does not need to contain a chromophore. Presented here is a simple and direct high-performance liquid chromatography and charged aerosol detection (HPLC-charged-aerosol-detector) method that can be used to measure omega-3, -6 and -9 fatty acids in traditional and commercially produced meat, fish, and oils, as well as over-the-counter supplements. Quantitation of Underivatized Omega-3 and Omega-6 Fatty Acids in Foods by HPLC and Charged Aerosol Detection Ian Acworth, Marc Plante, Bruce Bailey, and Christopher Crafts Thermo Fisher Scientific, Chelmsford, MA, USA Introduction The common determination of omega lipids in foods comprises several steps, including extraction, hydroysis, and derivitization for measurement by GC. GC works well as a standard analytical tool for these determinations but requires alteration of the sample, and it can also adversely affect temperature-sensitive functional groups on specialized lipids. HPLC with ultraviolet detection requires use of low wavelengths, which limits solvent selection and increases the likelihood of matrix interference. Shown here is a reversed-phase HPLC method for the determination of omega fatty acids in oil/food samples using a dual-gradient method and charged aerosol detection. This combination enables determination of many fatty acids in a single analysis, and without the sample derivatization that is required for GC analysis. Several fatty acids were analyzed, including six omega-3 fatty acids (stearidonic acid [SDA], eicosapentanoic acid [EPA], a-linolenic acid [ALA], docosahexanoic acid [DHA], docosapentanoic acid [DPA], eicosatrienoic acid [ETA]), five omega-6 fatty acids (γ-linolenic acid [GLA], arachidonic acid [Arach.], linoleic acid [LLA], adrenic acid, and eicosadienioic acid [EDA]), and two omega-9 fatty acids (oleic and erucic acids). An omega-7 fatty acid, 9E,14Z-conjugated linoleic acid (CLA), was also included due to its cited importance as a nutrient.1,2 Charged aerosol detection is mass sensitive and can be added to HPLC or ultra HPLC (UHPLC) platforms. The detector provides the most consistent response for all nonvolatile and some semivolatile analytes of all HPLC detection techniques.3 It works by charging particles as shown in Figure 1, and is not dependent on light scattering, which has large variability and generally lower sensitivity. Charged aerosol detection has been successfully used to characterize lipids of all classification,4 including phospholipids (reversed phase5 and normal phase6,7), acylglycerides, phytosterols, free fatty acids, and free fatty alcohols. This method complements the free fatty acids method, using higher specificity for these analytes and a Thermo Scientific Acclaim C30 reversed-phase column.

Simple and Direct Analysis of Phytosterols in Red Palm Oil by Reverse Phase HPLC and Charged Aerosol Detection

Phytosterols (PSs) are a group of naturally occurring steroid alcohols found in plants. There is considerable interest in PSs as dietary supplements as they are reported to lower cholesterol levels and also have a positive impact on cardiovascular diseases. However, recent research suggests that PS supplementation may aggravate atherosclerosis and lead to aortic valve stenosis. PSs are typically measured by gas chromatography (GC), but this approach is time-consuming since it requires saponification of the sample, several extractions, and derivatization. We developed a simplified method using reversed-phase, HPLC and charged aerosol detection (CAD). CAD is sensitive, has a dynamic range of >4 orders of magnitude, can measure any non-volatile species, and analytes shows similar response independent of their chemical structure. Samples were prepared by simple dilution prior to analysis. Five PSs, campesterol, cholesterol, stigmasterol, beta-sitosterol, and stigmastanol, were resolved in <35min. Calibration curves showed linear correlation coefficients >0.997. The LOD was <=5ng (on column). Analysis of red palm oil is used as an example. The method is simple to use, has good linearity and sensitivity, and is capable of measuring numerous PSs in plant extracts. This approach can be used to examine product purity, supplement content, and adulteration.

The Corona Charged Aerosol Detector – A „Universal“ Detector for the Measurement of Non-volatile Components in Food, Natural Products and Supplements

The Corona® Charged Aerosol Detector® is a „universal“ mass-sensitive detector capable of measuring any non-volatile and many semi-volatile analytes. Its response is independent of chemical structure and does not require the analyte to possess a chromophore (a prerequisite for UV and fluorescence detection) or to be ionized (essential for MS detection). The Corona CAD is compatible with gradient elution, has a wide dynamic range (4+ orders of magnitude), high sensitivity (sub-nanogram), good reproducibility, and a predictable response independent of chemical structure. As discussed in this presentation, charged aerosol detection overcomes many of the limitations seen with other „universal“ detectors including evaporative light scattering, condensation nucleation light scattering, refractive index, and UV. Charged aerosol detection has great applicability to the measurement of foods, supplements, botanicals and natural products. Some of the topics I will discuss include the measurement of carbohydrates, profiling lipids, measurement of fat-soluble vitamins, analysis of antibiotics, examination of potential bioactives in foods and supplements, and the evaluation of the composition of supplements and botanicals.