Charged Aerosol Detector Research Articles

Novel MS Solutions Inspired by MIST

To improve patient safety and to help avoid costly late-stage failures, the pharmaceutical industry, along with the US FDA and International Committee on Harmonization (ICH), recommends the identification of differences in drug metabolism between animals used in nonclinical safety assessments and humans as early as possible during the drug-development process. LC-MS is the technique of choice for detection and characterization of metabolites, however, the widely different LC-MS response observed for a new chemical entity (NCE) and its structurally related metabolites limits the direct use of LC-MS responses for quantitative determination of NCEs and metabolites. While no method provides completely accurate universal response, UV, corona charged aerosol detection (CAD), radioactivity, NMR and low-flow (< 20 µl/min) nanospray approaches provide opportunities to quantify metabolites in the absence of reference standards or radiolabeled material with enough precision to meet the needs of early clinical development.

Assessment of microcystin purity using charged aerosol detection

The increasing occurrence of toxic cyanobacterial blooms has led to a requirement for robust monitoring strategies and whilst several validated procedures have been developed these can be limited by the lack of high quality calibration standards. High quality standards must have confirmation of identity, purity and concentration by multiple methods. One aspect, purity, is rarely addressed but is essential. This is the first evaluation of the charged aerosol detector (CAD) to determine the benefits of incorporating a universal detector for more accurate purity determination of these peptides. Microcystins were detected at 5–10 ng on the column using the CAD, providing comparable quantification limits to those obtained using traditional UV detection. Purity determination of test compounds that had been partially purified, had showed that highest purity was at 238 nm > UV TIC > ESI TIC > CAD indicating that increased impurities could be detected using the CAD thus providing a more accurate indication of compound quality. Compounds purified by preparative HPLC were shown to have relative purities between 97% and 99%, however, when evaluated by CAD this dropped to 90–94% supporting the multi-detector strategy as essential for production of high quality compounds.

Alternatives to amino acid analysis for the purity control of pharmaceutical grade L‐alanine

The quality control of drugs is important for safety of patients and consumers. International pharmacopoeias mostly employed HPLC with UV detection for this purpose. This is always a problem when the drugs, such as most amino acids, are lacking a chromophor. Instead of UV detection, the impurity control of alanine was performed using charged aerosol detector, evaporative light-scattering detector, nano quantity analyte detector, MS detector, and as an orthogonal method, quantitative NMR spectroscopy. The charged aerosol detector, MS detector, and quantitative NMR spectroscopy provided similar and precise results, whereas the evaporative light-scattering detector and nano quantity analyte detector revealed some problems regarding sensitivity, linearity, and robustness.

SEC Assay for Polyvinylsulfonic Impurities in 2-(N-Morpholino)ethanesulfonic Acid Using a Charged Aerosol Detector

A quantitative method for measuring polyvinylsulfonic acid (PVS) concentrations in 2-(N-morpholino)ethanesulfonic acid (MES) is described here. Utilizing a TSK-GEL G2500PWxl SEC column, PVS elutes in the void volume as a sharp peak, yielding group separation between PVS and MES. 20 mM formic acid is used as the mobile phase, as its volatility is compatible with a charged aerosol detector. The low pH mobile phase also suppresses residual negative charges on the stationary phase, reducing the ion exclusion effect commonly observed in aqueous SEC. This method was qualified to measure PVS concentration in MES per International Conference on Harmonization guidelines, using multiple MES lots and other Good’s buffers such as N-(2-acetamido)-2-aminoethanesulfonic acid and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

Simultaneous Determination of Triterpenoid Saponins from Pulsatilla koreana using High Performance Liquid Chromatography Coupled with a Charged Aerosol Detector (HPLC-CAD)

Several triterpenoid saponins from root of Pulsatilla koreana Nakai (Ranunculaceae) were studied and their biological activities were reported. It is difficult to analyze triterpenoid saponins using HPLC-UV due to the lack of chromophores. So, evaporative light scattering detection (ELSD) is used as a valuable alternative to UV detection. More recently, a charged aerosol detection (CAD) has been developed to improve the sensitivity and reproducibility of ELSD. In this study, we developed and validated a novel method of high performance liquid chromatography coupled with a charged aerosol detector for the simultaneous determination of four triterpenoid saponins: pulsatilloside E, pulsatilla saponin H, anemoside B4 and cussosaponin C. Analytes were separated by the Supelco Ascentis ® Express C18 column (4.6 mm × 150 mm, 2.7 µm) with gradient elution of methanol and water at a flow rate of 0.8 mL/min at 30 o C. We examined various factors that could affect the sensitivity of the detectors, including various concentrations of additives, the pH of the mobile phase, and the CAD range. Linear calibration curves were obtained within the concentration ranges of 2 - 200 µg/mL for pulsatilloside E, anemoside B4 and cussosaponin C, and 5 - 500 µg/mL for pulsatilla saponin H with correlation coefficient (R 2 ) greater than 0.995. The limit of detection (LOD) and quantification (LOQ) were 0.04 - 0.2 and 2 - 5 µg/mL, respectively. The validity of the developed HPLC-CAD method was confirmed by satisfactory values of linearity, intra- and inter-day accuracy and precision. This method could be successfully applied to quality evaluation, quality control and monitoring of Pulsatilla koreana.

Determination of Pancuronium and Its Impurities in Pharmaceutical Preparation by LC with Charged Aerosol Detection

A novel, sensitive and simple liquid chromatography method with charged aerosol detection (LC-CAD) for the simultaneous assay determination of pancuronium and its impurities (dacuronium and vecuronium) has been developed. The limit of quantitation for vecuronium and dacuronium was 1.64 and 3.42 μg mL−1, respectively. The elaborated method was validated for linearity, precision and accuracy. This LC-CAD method is expected to have potential application in routine analysis of pancuronium in substance and in pharmaceutical preparations. All impurities were confirmed using time-of-flight mass spectrometry with electrospray ionization.

Comparison between evaporative light scattering detection and charged aerosol detection for the analysis of saikosaponins

Saikosaponins are triterpene saponins derived from the roots of Bupleurum falcatum L. (Umbelliferae), which has been traditionally used to treat fever, inflammation, liver diseases, and nephritis. It is difficult to analyze saikosaponins using HPLC-UV due to the lack of chromophores. Therefore, evaporative light scattering detection (ELSD) is used as a valuable alternative to UV detection. More recently, a charged aerosol detection (CAD) method has been developed to improve the sensitivity and reproducibility of ELSD. In this study, we compared CAD and ELSD methods in the simultaneous analysis of 10 saikosaponins, including saikosaponins-A, -B 1, -B 2, -B 3, -B 4, -C, -D, -G, -H and -I. A mixture of the 10 saikosaponins was injected into the Ascentis ® Express C18 column (100 mm × 4.6 mm, 2.7 μm) with gradient elution and detection with CAD and ELSD by splitting. We examined various factors that could affect the sensitivity of the detectors including various concentrations of additives, pH and flow rate of the mobile phase, purity of nitrogen gas and the CAD range. The sensitivity was determined based on the signal-to-noise ratio. The best sensitivity for CAD was achieved with 0.1 mM ammonium acetate at pH 4.0 in the mobile phase with a flow rate of 1.0 mL/min, and the CAD range at 100 pA, whereas that for ELSD was achieved with 0.01% acetic acid in the mobile phase with a flow rate at 0.8 mL/min. The purity of the nitrogen gas had only minor effects on the sensitivities of both detectors. Finally, the sensitivity for CAD was two to six times better than that of ELSD. Taken together, these results suggest that CAD provides a more sensitive analysis of the 10 saikosaponins than does ELSD.

Analysis of Terpene Lactones in a Ginkgo Leaf Extract by High-Performance Liquid Chromatography Using Charged Aerosol Detection

A new HPLC method using charged aerosol detection was developed for the determination of terpene lactones in a Ginkgo leaf extract. The linearity of the standard curves was excellent (r>0.999). The repeatability of the method was less than 3%, and its reproducibility was less than 5% for each analyte. The limit of detection was between 0.087 and 0.45 microg/ml. The developed method was applied to the analysis of terpene lactones in Ginkgo leaf products distributed in the Japanese market. The results suggest that some health food products contained approximately equivalent amounts of terpene lactones to those in the medical product and that the proportion of terpene lactones varied in each health product.

Fast and sensitive determination of Polysorbate 80 in solutions containing proteins

A fast and sensitive method has been developed for the specific determination of Polysorbate 80 (Tween 80) in liquid formulations in the presence of proteins and excipients. The quantitative determination is based on a fast liquid chromatographic (HPLC) separation and charged aerosol detection (CAD). The method was validated using a Poroshell 300SB-C18 column packed with 5 microm shell particles (75 mm x 2.1 mm) and acetonitrile-methanol-water-trifluoroacetic acid mobile phase at a flow rate of 0.65 ml/min. The rapid LC-CAD method is suitable for quantifying Polysorbate 80 in the range of 10-60 microg/ml in protein solutions within good manufacturing practices (GMPs) of the pharmaceutical industry.

QUANTITATIVE STUDY OF THE STRATUM CORNEUM LIPID CLASSES BY NORMAL PHASE LIQUID CHROMATOGRAPHY: COMPARISON BETWEEN TWO UNIVERSAL DETECTORS

The stratum corneum owns a barrier function, which can be altered by a modification of its lipid composition. In this work, a new simple method was developed in normal phase liquid chromatography to separate the three major lipid classes of the stratum Corneum, fatty acids, ceramides, and cholesterol. A quantitative investigation was carried out, comparing an internal normalisation and an external standardisation with two universal detectors: an evaporating light scattering detector and a charged aerosol detector, the Corona. The study points out the fact that the external standardisation is suitable for a quantitative study of the different lipid classes, using a representative standard for each class. Moreover, the use of the Corona is more adapted for further investigation, due to a better repeatability, accuracy, and precision. A test made on a lipid extract from the forearm of volunteers shows that the present method is appropriate for biological extract analysis.

Simultaneous Analysis for Carbohydrates in Drinks by High Performance Liquid Chromatography with Charged Aerosol Detector

An HPLC method with a charged aerosol detector (CAD) was optimized and validated for the quantification of six carbohydrates (xylose, fructose, glucose, sucrose, lactose, maltose) and sugar alcohol (erythritol) in drinks. Chromatographic separation was achieved using gradient elution with a mixture of water-methanol (7 : 3) and acetonitril. Carbohydrates were cleaned up using a strong cation exchange cartridge (Bond Elut SCX, Varian). Although an interfering peak appeared without any clean up, it was confirmed that interfering substance had be removed by a treatment using this cartridge and its peak had disappeared. The determinations were performed in the linear range of 5 ∼ 10000 μg/mL for six carbohydrates and sugar alcohol, the correlation coefficients (R) were 0.998 ∼ 1.000, the relative standard deviations (RSDs) ranged over 0.4 ∼ 4.5% (spiked level at 500 μg/mL, n = 5), recoveries ranged between 84 ∼ 113% (spiked level at 500 μg/mL, n = 3), and the limits of quantification were 0.4 ∼ 19.3 μg/mL. In addition, we performed analysis of carbohydrates with CAD, an evaporative light scattering detector (ELSD) and a refractive index detector (RID) to compare and investigate the sensitivity and analysis precision in the each optimum condition. As for the CAD, high sensitivity and good analysis precision were provided. Because it is not necessary to optimize the conditions, CAD is the simplest and easiest device in these detectors. It became clear that CAD was the most suitable to perform correct quantitative analysis of carbohydrates in drinks.

Determination of atracurium, cisatracurium and mivacurium with their impurities in pharmaceutical preparations by liquid chromatography with charged aerosol detection

The Corona CAD (charged aerosol detection) is a new type of detector introduced for LC applications that has recently become widely applied in pharmaceutical analysis. The Corona CAD measures a physical property of analyte and responds to almost all non-volatile species, independently of their nature and spectral or physicochemical properties. The LC method with charged aerosol detection was developed for the determination of three isomers of atracurium, cisatracurium and also three isomers of mivacurium with their impurities. The limit of quantitation for laudanosine was 1 microg ml(-1). The elaborate method for the analysis of those active substances and laudanosine proved to be fast, precise, accurate and sensitive. All other impurities were identified using time-of-flight mass spectrometry with electrospray ionization.

The effect of composting on the degradation of a veterinary pharmaceutical

Composting has been identified as a viable means of reducing the environmental impact of antibiotics in manure. The focus of the present study is the potential use of composting on the degradation of salinomycin in manure prior to its field application. Manure contaminated with salinomycin was collected from a poultry farm and adjusted to a C:N ratio of 25:1 with hay material. The manure was composted in three identical 120 L plastic containers, 0.95 m height × 0.40 m in diameter. The degradation potential for salinomycin was also ascertained under open heap conditions for comparison (control). Salinomycin was quantified on HPLC with a Charged Aerosol Detector, at an interval of every 3 days. The salinomycin level in the compost treatment decreased from 22 mg kg −1 to 2 × 10 −5 μg kg −1 over 38 days. The corresponding decrease in the control was from 27.5 mg kg −1 to 24 μg kg −1. The changes in pH, EC (dS m −1), temperature, total kjeldahl nitrogen (TKN), total potassium (TK), total phosphorus (TP) and carbon content in both the composting and the control samples were monitored and found to be different in compost as compared to the control. During the composting process, the loss of TKN was 36%, which was substantially lower than corresponding loss of 60% in the control. The loss of carbon was 10% during composting, whereas the loss in the control was 2%. In composting, the temperature modulated from 27 °C (initially) to a high of 62.8 °C (after 4 days), and then declined to 27.8 °C at the end of 38 days. On the basis of the results obtained in this study, it appears that the composting technique is effective in reducing salinomycin in manure.

Control of impurities in l-aspartic acid and l-alanine by high-performance liquid chromatography coupled with a corona charged aerosol detector

In this study a reversed phase ion-pair high-performance liquid chromatography (HPLC) method using charged aerosol detection (CAD) was developed and fully validated for the pharmaceutical quality control of l-aspartic acid (Asp). With a slight modification, the method also allows the evaluation of related substances in l-alanine (Ala). The method enables simultaneous control of related amino acids and of possibly occurring organic acids contaminants. A minimum limit of quantification of 0.03% could be achieved for all occurring related substances. Moreover, the detector sensitivity of the CAD was compared with an evaporative light scattering detector (ELSD). Depending on the analyte the CAD was found to be 3.6–42 times more sensitive than the ELSD. The HPLC method was applied to the purity testing of 8 samples of pharmaceutical grade and reagent grade Asp and of 12 samples of Ala supplied by various manufacturers. Both substances were found to be of high purity (greater than 99.8% for Asp and greater than 99.9% for Ala). Malic acid and Ala were the major impurities in Asp. Asp and glutamic acid (Glu) were the only detectable impurities in Ala.

Optimization of pressurized liquid extraction for spicatoside A in Liriope platyphylla

Method development based on pressurized liquid extraction technique was investigated for the extraction of spicatoside A from Liriope platyphylla. Determination of spicatoside A was carried out by high performance liquid chromatography coupled with charged aerosol detector. Central composite design consisting of 15 experimental points in triplicate was applied to evaluated the effects of three independent variables, namely extraction time (min), extraction temperature (°C) and ethanol concentration (%) on the acquired content of spicatoside A. Response surface analysis indicated that the highest predicted content of spicatoside A might be 0.0161% of the optimized extract at extraction time 20 min, extraction temperature 130 °C and ethanol concentration 86%. Under this optimal condition, the experimental value was 0.0157%, which agrees with the predicted one by analysis of variance.

Comparison of ultraviolet detection, evaporative light scattering detection and charged aerosol detection methods for liquid-chromatographic determination of anti-diabetic drugs

Recently, charged aerosol detection (CAD), a new kind of universal detection method, has been widely employed in the HPLC system. In the present study, four kinds of anti-diabetic drug standards, glipizide, gliclazide, glibenclamide and glimepiride were determined by ultraviolet (UV) detection, evaporative light scattering detection (ELSD) and the aforementioned CAD. The results were compared with reference to linearity, accuracy, precision and limit of detection (LOD). All of the experiments were performed on a reverse phase column with water and acetonitrile as the mobile phase. Separations were achieved under the same chromatographic conditions for each detection method. As a result, CAD generated nearly uniform responses compared with UV detection and ELSD. It showed the best accuracy and LOD among 3 detectors and had similar precision with UV detection at higher concentrations while UV detection showed a better precision at lower concentrations than did CAD or ELSD. The LOD of CAD, in fact, can be up to two times higher than that of ELSD. The UV and ELSD linearity was satisfactory at R 2 > 0.99, though in the case of CAD, a log–log transformation was needed. The proposed methods were also applied to the real anti-diabetic drugs and diabetes-related dietary supplements.

Comparison of the sensitivity of evaporative universal detectors and LC/MS in the HILIC and the reversed-phase HPLC modes

It was hypothesized that the hydrophilic interaction liquid interface chromatography (HILIC) mode should produce more response than the reversed-phase HPLC mode on detectors with an evaporative component to the detection process. HILIC mobile phases are mostly composed of polar organic solvent and are more volatile than reversed-phase mobile phases. Therefore the more easily evaporated HILIC mobile phases should produce greater sensitivity for those detectors that remove mobile phase by evaporation. The responses of 12 compounds were measured in the reversed-phase mode and the HILIC mode with three detectors: evaporative light scattering detector (ELSD), corona charged aerosol detector (cCAD), and electrospray mass spectrometry (ESI-MS). The compounds studied were very polar compounds that were retained in the HILIC mode. Generally, the HILIC mode was able to achieve greater sensitivity than the reversed-phase mode for these compounds. The increases in sensitivity observed can be attributed to the more volatile HILIC mobile phase. For the ELSD, the HILIC mode produced slightly greater sensitivity than the reversed-phase mode. The cCAD was approximately 10 times more sensitive in the HILIC mode and the ESI-MS was approximately 5–10 times more sensitive in the HILIC mode. There was one instance in the study where a compound produced more response in the reversed-phase mode. Thymine yielded more sensitivity in the reversed-phase mode with the ESI-MS detector. In a given mode of operation, there was significant variation in the measured response factors for all compounds on each detector. While this is not unexpected for the ESI-MS detector, variation in the response factors between compounds indicates that the cCAD and ELSD are not truly universal detectors in the sense that all compounds have identical responses.

Development and validation of a RP-HPLC method for the determination of gentamicin sulfate and its related substances in a pharmaceutical cream using a short pentafluorophenyl column and a Charged Aerosol Detector

Gentamicin sulfate is a potent broad spectrum aminoglycoside antibiotic which is used as an active pharmaceutical ingredient (API) against both Gram-positive and Gram-negative bacteria. A reversed-phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated to determine the composition of gentamicin sulfate and to estimate its related substances (without any pre- or post-column derivatization) in a pharmaceutical cream. As gentamicin has a weak UV chromophore, it is not possible to detect low levels of known and unknown related substances of gentamicin using a UV detector. In this method, a Charged Aerosol Detector (CAD) was used to obtain high sensitivity that was necessary for the intended purpose of the method. This method can separate all the analogues of gentamicin including all known and unknown related substances of the API. A short (5 cm × 4.6 mm) pentafluorophenyl HPLC column from Restek (Allure PFP) was used with an ion-pair gradient mobile phase consisting of (A) heptafluorobutyric acid:water:acetonitrile (0.025:95:5, v/v/v) and (B) trifluoroacetic acid:water:acetonitrile (1:95:5, v/v/v).

Lipid analysis via HPLC with a charged aerosol detector

AbstractMost lipid extracts are a mixture of saturated and unsaturated molecules. Therefore, the most successful high performance liquid chromatography (HPLC) detectors for the quantitative analysis of lipids have involved the use of “universal” or “mass” detectors such as flame ionization detectors and evaporative light scattering detectors. Recently a new type of HPLC “universal” detector, a charged aerosol detector (CAD), was developed and is now commercially available. This detection method involves nebulizing the HPLC column effluent, evaporating the solvents, charging the aerosol particles and measuring the current from the charged aerosol flux. During the approximately four years that the charged aerosol detector has been commercially available, several publications have described HPLC‐CAD methods for lipid analysis. The most common lipids can be quantitatively analyzed via HPLC‐CAD except for some volatile lipids such as common fatty acid methyl esters and short chain free fatty acids (&lt;C16). The major results of these publications will be summarized in this report.

Evaluation of Methods for the Simultaneous Analysis of Cations and Anions Using HPLC with Charged Aerosol Detection and a Zwitterionic Stationary Phase

This paper describes the development and qualification of a method capable of analyzing inorganic ions as salts and counter-ions of both active pharmaceutical ingredients and other compounds such as lysine. The use of a polymeric zwitterionic column with a binary high-performance liquid chromatography gradient enables the separation of several anions and cations in a single run. A generic gradient (method #1) was developed and validated with respect to specificity, correlation, intermediate precision, accuracy, and sensitivity (limits of quantitation and detection) for four anions and two cations. Furthermore, the ability to alter chromatographic selectivity by simple gradient manipulation (without altering the mobile phase composition or column type) is demonstrated for nine anions and three cations (method #2). The simultaneous measurement of cations and anions at the parts per billion level using the Corona charged aerosol detector with zwitterionic chromatography-polymeric hydrophilic interaction chromatography is a viable alternative to traditional techniques used for ion analysis.