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 5330 plates m⁻¹ and 1.25 μg mL⁻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.     

An investigation into detector limitations using evaporative light-scattering detectors for pharmaceutical applications

Evaporative light-scattering detection (ELSD) high-performance liquid chromatography (HPLC) is an alternative technology to low-wavelength UV analysis that is often employed when compounds lack sufficient absorptivity. Although ELSD provides an additional detector option for liquid chromatographers, studies in our laboratory indicate analyte properties may adversely affect the ability to detect certain molecules. In this investigation, a series of low-molecular-weight compounds of pharmaceutical interest are evaluated with two commercially available ELSDs. It is observed that melting point is a useful analyte property to consider in optimizing ELSD detectors. The melting point of the analyte should be significantly higher than what the compound will experience in the nebulizer/evaporator chambers to achieve the best analyte response. It is found that some analytes could not be distinguished from the evaporated mobile phase background when ELSD temperatures exceed the melting point of the compound. Though useful for many applications and of particular interest for compounds that are weak chromophores, ELSD falls short of being a "universal detector" technology. In addition to boiling points of mobile phase components, scientists should also consider the melting point and volatility of the analyte(s) when optimizing ELSD response.     

Comparison of two aerosol-based detectors for the analysis of gabapentin in pharmaceutical formulations by hydrophilic interaction chromatography

Comparison of hydrophilic interaction chromatography (HILIC) columns coupled with an evaporative light scattering detector (ELSD) or charged aerosol detector (CAD) was done for the detection of gabapentin in pharmaceutical formulations. The chromatographic separations were achieved on four HILIC columns: ZIC HILIC, ZIC pHILIC, Luna HILIC, and Atlantis HILIC. Experimental factors such as mobile phase composition, acetonitrile content, and mobile phase pH were evaluated. Validation of method was done in terms of linearity, sensitivity, accuracy, and precision. The performance of ELSD detection method is comparable to that of CAD. The intra-day and inter-day variations were below 1.7% and 3.2% for CAD and 2.8%, and 3.4% for ELSD, respectively. In addition, detection sensitivities of ELSD, CAD, and UV detectors were also compared for HILIC and reversed phase (RP) modes and the highest sensitivities were obtained in the HILIC mode when connected with CAD and ELSD. The developed HILIC aerosol based detection methods were successfully applied to the analysis of gabapentin in commercial tablets and capsules.     

Universal response model for a corona charged aerosol detector

The universality of the response of the Corona Charged Aerosol Detector (CoronaCAD) has been investigated under flow-injection and gradient HPLC elution conditions. A three-dimensional model was developed which relates the CoronaCAD response to analyte concentration and the mobile phase composition used. The model was developed using the response of four probe analytes which displayed non-volatile behavior in the CoronaCAD and were soluble over a broad range of mobile phase compositions. The analyte concentrations ranged from 1μg/mL to 1mg/mL, and injection volumes corresponded to on-column amounts of 25ng to 25μg. Mobile phases used in the model were composed of 0-80% acetonitrile, mixed with complementary proportions of aqueous formic acid (0.1%, pH 2.6). An analyte set of 23 compounds possessing a wide range of physicochemical properties was selected for the purpose of evaluating the model. The predicted response was compared to the actual analyte response displayed by the detector and the efficacy of the model under flow-injection and gradient HPLC elution conditions was determined. The average error of the four analytes used to develop the model was 9.2% (n=176), while the errors under flow-injection and gradient HPLC elution conditions for the evaluation set of analytes were found to be 12.5% and 12.8%, respectively. Some analytes were excluded from the evaluation set due to considerations of volatility (boiling point <400°C), charge and excessive retention on the column leading to elution outside the eluent range covered by the model. The two-part response model can be used to describe the relationship between response and analyte concentration and also to offer a correction for the non-linear detector response obtained with gradient HPLC for analytes which conform to the model, to provide insight into the factors affecting the CoronaCAD response for different analytes, and also as a means for accurately determining the concentration of unknown compounds when individual standards are not available for calibration.     

Comparison of universal detectors for high-temperature micro liquid chromatography

This study compares, through micro high-temperature liquid chromatography (microHTLC), three commercial universal detectors that allow a direct detection of lipids. The detectors are: the charged aerosol detector (CAD), the evaporative light-scattering detector (ELSD) and the ion trap mass spectrometer with atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) sources (APCI-MS and ESI-MS). This study shows the feasibility to use the high temperature with these detectors and hybrid behavior between concentration and mass flow rate detector in microHTLC. The detectors were compared in terms of response intensity, linearity and limit of detection for different high temperatures. The charged aerosol detector shows a linear response from 5 to 500 microg/mL and the correlation coefficients (r(2)) obtained for squalene, cholesterol and ceramide IIIB exceed 0.99.     

Operational parameters of voltammetric high-performance liquid chromatographic detectors with copper electrodes and application to a determination of some fodder biofactors

Two versions of an amperometric detector with a copper working electrode have been constructed and tested for high-performance liquid chromatography (HPLC). The performance of the detectors was studied using selected amino acids. The dependence of the detector response on the mobile phase flow-rate was studied in the range common in both macro- and microcolumn HPLC (5 microliter/min to 1.0 ml/min). It has been found that the detection sensitivity generally increases with decreasing flow-rate, i.e., the detector response is governed by the rate of the complexation reaction between the cupric ions and the solutes. This fact makes amperometric detection with a copper electrode especially useful for microcolumn separations. For all 20 amino acids studied, calibration curve parameters and detection limits have been determined; the latter vary from 0.4 to 18 ng in the injected volume. The amino acids can also be sensitively detected in a medium of 0.1-1.0 M ammonia, which is promising for the use of strong anion exchangers in amino acid separations. Choline can also be detected at a copper electrode, with a detection limit of 40 ng. An HPLC method with amperometric detection at a copper electrode has been developed for the determination of lysine, methionine and choline in fodder biofactors.     

Simple and precise detection of lipid compounds present within liposomal formulations using a charged aerosol detector

In recent decades the use of liposomal preparations as drug delivery systems has become very attractive in pharmaceutical development. Therefore, thorough characterization and quantification of the lipids which form liposomes is wished from both investigators and regulatory authorities when the application in humans is being considered. In this study a new HPLC method for the detection of lipids in liposomal formulations was established using corona charged aerosol detection (CAD) which has the advantage to be independent of the chemical properties of the analytes. The superiority of this method over UV detection was demonstrated. Compared to UV detection no absorption effects of the organic solvent in the mobile phase interfering with the lipid signals were observed with CAD. CAD showed good linearity (R² > 0.990) for all liposomal compounds. The acceptance criteria for precision including repeatability were met. The average recovery for each of the excipients of the liposomal formulation was in the range of 90.0–110%.     

A Comparison of the Properties of Contactless Conductivity and Diode‐Array Photometric Detectors in Analyses of Low‐Molecular,Biologically Active Substances by Capillary Electrophoresis in Acetic Acid Solutions

The performance of the contactless conductivity (C⁴D) and diode array photometric (DAD) detectors has been compared for CE separations of creatinine, arginine and 3‐methylhistidine in acetic acid background electrolytes. The contactless conductivity detector response has also been modeled. It has been found that the two detectors provide similar responses and can readily be used for dual CE detection. Changes in the acetic acid concentration affect the C⁴D noise less than the DAD noise, but their effect on the C⁴D response to the analytes is greater than with DAD. In general, C⁴D provides better detection results at higher acetic acid concentrations, while DAD is more sensitive and reliable at very low ones. Capillaries with greater internal diameters are preferable for both detectors, provided that the separation efficiency is not adversely affected. Acetic acid is a suitable background electrolyte for CE separations of small, basic organic molecules.     

Size exclusion chromatography with Corona charged aerosol detector for the analysis of polyethylene glycol polymer

A technique of using size exclusion chromatography (SEC) with the Corona charged aerosol detector (CAD) was developed and evaluated in comparison with refractive index (RI) and evaporative light scattering detection (ELSD) for fast screening of polyethylene glycol (PEG), a polymer used in preparing pegylated pharmaceutical compounds. These detection techniques were used in the analysis of multiple lots of PEG reagents. CAD was found to provide more accurate impurity and polydispersity profiles of PEG reagents that better differentiate their quality, while RI was not suitable for this application due to its low sensitivity and ELSD led to underestimation of the impurity and polydispersity. The accuracy of polydispersity determination by SEC-CAD was validated against a commercial reference standard of known polydispersity. The SEC-CAD technique and the observed differences between the three detectors can also be applied to polymer analysis in general.     

Direct separation and detection of biogenic amines by ion-pair liquid chromatography with chemiluminescent nitrogen detector

Analysis of biogenic amines is critical to pharmaceutical and food industry due to their biological importance. For many years, the determination of biogenic amines has relied on high performance liquid chromatography (HPLC) coupling with pre-, on-, or post-column derivatization procedures to enable UV or fluorescent detections. In this study, 14 biogenic amines were separated on a Phenomenex Luna Phenyl-Hexyl column by an ion-pair liquid chromatography method using perfluorocarboxylic acids as ion-pair reagents and detected by a chemiluminescent nitrogen detector (CLND). This direct separation and detection HPLC method eliminated the time consuming and cumbersome derivatization procedures. Compared with HPLC-UV (post-column derivatization with ninhydrin) and HPLC-charged aerosol detector (CAD) methods, this HPLC-CLND technique provided narrower peaks, better baselines, and improved separations and detections. Excellent linearity was acquired by CLND for each of the 14 biogenic amines ranging from less than 1 ng to about 1000 ng (on-column weights). The relative response factors determined by this LC-CLND method were proportional to the numbers of nitrogen atoms in each compound, which has been the characteristic of the equimolar determinations by CLND. In addition, a number of samples including beer, dairy beverage, herb tea, and vinegar were analyzed by the LC-CLND method with satisfactory precision and accuracy.     

"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 D₃ in rice bran oil, diluted to 100 mL, were analyzed without the need for lengthy saponification and extraction. Vitamin D₃ 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 MS⁴, an ELSD, and a CAD. The method proved to be very sensitive for vitamin D₃, 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.     

Sequential optimisation of the separation of a complex mixture of plastics additives by HPLC with a quaternary gradient and a dual detection system

Summary The separation of two families of plastics additives (phenolic antioxidants and UV absorbers) has been achieved by high performance liquid chromatography with a quaternary gradient. A methodology of separation based on a sequential optimization is described. After a preliminary study of the effects of solvents on retention of compounds, the mobile phase is first chosen for each class of additives (Irganox and Tinuvin), then for the mixture of all the compounds and the separation is finally optimized. The importance of the column phase ratio is also reported. The use of two detectors, UV absorbance and light-scattering, enables all the compounds to be detected. The performance of the detectors has been compared and the effects of the nebulization temperature on the detection of low molecular mass compounds is reported.     

Peak dispersion and contributions to plate height in nonaqueous capillary electrophoresis at high electric field strengths: propanol as background electrolyte solvent

Peak dispersion effects in nonaqueous capillary electrophoretic separations of aromatic anionic analytes were investigated in a propanolic background electrolyte solution. Poly(glycidylmethacrylate-co-N-vinylpyrrolidone) coating was applied to the capillary to suppress the electroosmotic flow and to improve the repeatability of the migration times. Electrical field strengths up to 2000 Vcm(-1) were applied in separations and the separation efficiencies were compared with theoretical values calculated on the basis of plate height theory. The contributions to the total plate height were calculated for injection plug length, diffusion, Joule heating, electromigration dispersion, analyte adsorption to the capillary wall, and detector slit aperture length. Analyte diffusion coefficients were measured by Taylor dispersion method, while distribution constants were measured chromatographically. Agreement between the calculated and empirical results was fairly good even though some approximations were required. In most cases the longitudinal diffusion contribution governed the total plate height, while the contribution of Joule heating was insignificant even at exceptionally high field strengths used. The relatively long detection slit aperture was found to influence the separation efficiency strongly, while the other dispersion sources that were investigated were of minor importance, except for adsorption in the case of one analyte. With all analytes, the dispersive effect of longitudinal diffusion was reduced as the field strength was increased, leading to enhanced migration velocities and faster separations.     

Performance evaluation of evaporative light scattering detection and charged aerosol detection in reversed phase liquid chromatography

In pharmaceutical industry ultraviolet (UV) detection is often used as the preferred detection technique in HPLC analysis since most pharmaceutical compounds possess a UV-absorbing chromophore. However, in case the active pharmaceutical ingredient (API) does not have a UV-absorbing chromophore, or if some of the impurities present lack a chromophore, they will not be detected in routine HPLC analysis employing only a UV detector and alternative detection schemes have to be used. Refractive index detection or mass spectroscopy (MS) can be used but these detectors have their intrinsic weaknesses, such as lack of sensitivity or high cost. With the appearance of semi-universal techniques such as evaporative light scattering detection (ELSD), and more recent, charged aerosol detection (CAD), detection of non-UV-absorbing compounds became feasible without having to resort to such complex or costly detection methods. This paper evaluates the different performance characteristics such as sensitivity, linearity, accuracy and precision of both the ELSD and CAD detector coupled to HPLC. One disadvantage of this type of detector is the non-linear response behaviour which makes direct linear regression for making calibration curves inaccurate.     

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 μg 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.     

Graphene quantum dots for enhancement of fluorimetric detection coupled to capillary electrophoresis for detection of ofloxacin

A new CE method with fluorescence detection is reported on the determination of ofloxacin in milk samples using graphene quantum dots (GQDs) for sensitivity enhancement. Injection of GQDs prior the standards/sample is crucial to increase the antibiotic fluorescence response. Clean‐up and preconcentration steps allowed for a good linear correlation in a concentration range between 50 and 1000 ng/mL for the ofloxacin, detection and quantification limits being 10.7 and 35.5 ng/mL, respectively. Optimal CE conditions for the seven‐fluoroquinolone separation method were assessed in terms of buffer type, pH, and voltage. The selective interaction of GQDs with ofloxacin as model analyte was subsequently studied finding a significant sensitivity improvement; therefore, the analytes would be detected at low concentrations by means of a commercial CE device equipped with a multi‐wavelength photoluminescence detector. Due to the different maximum emission wavelengths of the target compounds and the limitations shown by the single‐wavelength photoluminescence detector coupled to the CE system, we demonstrated the usefulness of the GQD‐assisted sensitivity‐enhanced CE method to determine ofloxacin in milk samples. This work opens an interesting possibility of using GQDs in separation techniques combined with photoluminescence detectors for lowering sensitivity levels typically provided by the mere device.     

Microfabricated refractive index gradient based detector for reversed-phase liquid chromatography with mobile phase gradient elution

Typical refractive index (RI) detectors for liquid chromatography (LC) are not well suited to application with mobile phase gradient elution, due to the difficulty in correcting for the detected baseline shift during the gradient. We report a sensitive, highly reproducible, microfabricated refractive index gradient (micro-RIG) detector that performs well with mobile phase gradient elution LC. Since the micro-RIG signal remains on-scale throughout the mobile phase gradient, one can apply a baseline correction procedure. We demonstrate that by collecting two mobile phase gradient blanks and subtracting one of them from the other, a reproducible, flat baseline is achieved. Therefore, subtracting a blank from a separation provides a baseline corrected chromatogram with reasonably high signal-to-noise ratio for eluting analytes. The micro-RIG detector uses a collimated diode laser beam to optically probe a RIG formed perpendicular to the laminar flow direction within a microfabricated borosilicate glass chip. The chip-based design of the detector is suitable for either traditional bench-top or LC-on-a-chip technologies. We report reversed phase high performance liquid chromatography (RP-HPLC) separations of proteins and polymers, over mobile phase gradient conditions of 67% A:33% B to 3% A:97% B by volume, where A is 96% methanol:3.9% water:0.1% trifluoroacetic acid (TFA), and B is 3.9% methanol:96% water:0.1% TFA. The separations were performed on a Jupiter 5 mu C4 300 A 150 mm x 1.0 mm Phenomenex column at a flow rate of 20 microl/min. Viscosity changes during the mobile phase gradient separation are found to shift the on-chip merge position of the detected concentration gradient (i.e., RIG), in a reproducible fashion. However, this viscosity effect makes detection sensitivity vary throughout the mobile phase gradient, due to moving the optimized position of the probe beam in relation to the analyte concentration gradient being probed. None-the-less, consistent limits of detection (LODs) were achieved. The 3-sigma deflection angle LOD was 16 microrad for micro-RIG detection, corresponding to an injected concentration LOD of 7 ppm (mass/mass) for cytochrome c.     

Investigating the performance characteristics of the barrier discharge ionization detector and comparison to the flame ionization detector for the gas chromatographic analysis of volatile and semivolatile organic compounds

Abstract

The response behavior and performance characteristics of the recently introduced barrier discharge ionization detector (BID) for gas chromatography (GC-BID) were investigated by analyzing different classes of organic compounds such as alcohols, alkanes, cycloaliphatic compounds, polycyclic aromatic hydrocarbons (PAHs), and others. The results obtained by GC-BID were compared with those of gas chromatography with flame ionization detection (GC-FID), aiming to demonstrate the particular merits of the new BID detector over the well-established FID. The response of the BID not only was found to be strongly dependent on the detector settings, but also shows a high dependence on the analyte class and the individual analyte. The sensitivity of the BID detector compared to the FID was higher by a factor of ca. 4 on average when considering all compounds analyzed. The relative standard deviation (RSD) was better than 5% for the majority of the cases. The BID detector showed better precision (lower RSD) in comparison with the FID for the investigated compounds. Linear calibrations were obtained for the analytes over more than four orders of magnitude with coefficients of determination typically higher than 0.999 and the limits of detection varied from 0.04 to 1.48?ng/s for the GC-BID.     

Systematic HPLC Study of a New Series of 4-Methyl-1,4-dihydropyridines with Antitrombotic Activity

Abstract

A systematic study of a new series of 4-methyl-1,4-dihydropyridine derivatives by HPLC by both photodiode array and electrochemical detectors is reported.

The optimum mobile phase was acetonitrile 0.05 M phosphate buffer pH 3 (55/45, v/v) at a flow-rate of 1.5 mL/min.

Compounds I–VIII were determined with UV detection at 250 nm and by electrochemical detection at + 1200 mV.

Retention times varied between 3.0 and 14.0 minutes in the optimal experimental conditions.

Good linear relationships between the peak areas and concentration were found, with limits of quantification ranging between 1 x 10^?8 M and 1 x 10^?6 M. Repeatability studies showed average variation coefficients lower than 1% for a photodiode array detector. No significant differences between the detectors in sensitivity and selectivity were found.

Also, we used different degradation trials to test the selectivity of the methods. Results of these experiments revealed that the developed methods exhibited a good selectivity.     

Analysis of the Cinchona alkaloids by high-performance liquid chromatography and other separation techniques

The Cinchona alkaloids, which include the pharmaceuticals quinine and quinidine, continue to have a wide variety of important uses. A number of different chromatographic procedures have been developed for the qualitative and quantitative analysis of these compounds in a variety of sample matrices. Reversed-phase HPLC using ODS columns in combination with acidic mobile phases, and UV detection, is the most widely used method. Nevertheless, precautions need to be taken due to the strong silanophilic interactions which can occur with these analytes and the column surface, which can lead to poor peak shape and resolution. Different selectivity may be achieved in HPLC separations by use of alternative stationary phases, or by varying mobile phase pH. The specificity of detection systems may be improved by use of photodiode array UV detectors, or especially mass spectrometers. Thin-layer chromatography (TLC) provides a cheap alternative analytical method, which is especially useful for qualitative analysis. High-performance TLC, gas chromatography, capillary electrophoresis and capillary electrochromatography are all methods which after some development, could prove useful for Cinchona alkaloid separations.