Discovery HS F5 Column Research Articles

HPLC method development, validation, and impurity characterization of a potent antitumor indenoisoquinoline, LMP776 (NSC 725776)

An HPLC method for the assay of a DNA topoisomerase inhibitor, LMP776 (NSC 725776), has been developed and validated. The stress testing of LMP776 was carried out in accordance with International Conference on Harmonization (ICH) guidelines Q1A (R2) under acidic, alkaline, oxidative, thermolytic, and photolytic conditions. The separation of LMP776 from its impurities and degradation products was achieved within 40min on a Supelco Discovery HS F5 column (150mm×4.6mm i.d., 5μm) with a gradient mobile phase comprising 38–80% acetonitrile in water, with 0.1% trifluoroacetic acid in both phases. LC/MS was used to obtain mass data for characterization of impurities and degradation products. One major impurity was isolated through chloroform extraction and identified by NMR. The proposed HPLC assay method was validated for specificity, linearity (concentration range 0.25–0.75mg/mL, r=0.9999), accuracy (recovery 98.6–100.4%), precision (RSD≤1.4%), and sensitivity (LOD 0.13μg/mL). The validated method was used in the stability study of the LMP776 drug substance in conformance with the ICH Q1A (R2) guideline.

Chromatographic separation of arsenic species with pentafluorophenyl column and application to rice

Arsenic species, including arsenous acid, arsenic acid, methylarsonic acid, and dimethylarsinic acid, were determined using HPLC–ICPMS. The species were separated with a Discovery HS F5 column and a simple, volatile, and isocratic mobile phase of 0.1% (v/v) formic acid and 1% (v/v) methanol. The Discovery HS F5 column with a pentafluorophenyl (PFP) stationary phase gave sharp peaks and full separation of the arsenic species in 5min, and other PFP columns showed lower performance. This separation method was applied to arsenic species analysis in rice. The extraction of arsenic from rice samples was performed using 0.15M nitric acid. The methodology was validated by use of certified reference materials, NMIJ CRM 7503-a and NIST SRM 1568a, and extremely low arsenic rice samples as blank samples.

Simultaneous determination of testosterone, cortisol, and dehydroepiandrosterone in saliva by stable isotope dilution on-line in-tube solid-phase microextraction coupled with liquid chromatography–tandem mass spectrometry

We have developed a simple and sensitive method for the simultaneous determination of testosterone (TES), cortisol (CRT), and dehydroepiandrosterone (DHEA) in saliva by automated online in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Discovery HS F5 column. The optimum in-tube SPME conditions were 25 draw/eject cycles of 40 μL of sample at a flow rate of 200 μL/min using a Supel-Q PLOT capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. The in-tube SPME LC-MS/MS method showed good linearity with correlation coefficients r ≥ 0.9998 for TES, CRT, and DHEA using their respective stable isotope-labeled internal standards. The intra-day and inter-day precisions (relative standard deviations) were below 4.9 and 8.5 % (n = 5), respectively. This method was successfully utilized to analyze TES, CRT, and DHEA in saliva samples without any other pretreatment or interference peaks, and the quantification limits (S/N = 10) of TES, CRT and DHEA were about 0.01, 0.03 and 0.29 ng/mL saliva, respectively. The recoveries of these compounds spiked into saliva samples were each above 94%. This method was applied to analyze changes in salivary TES, CRT, and DHEA levels resulting from stress and fatigue load.

Automated analysis of salivary stress-related steroid hormones by online in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry

We have developed a simple and sensitive method for the simultaneous determination of stress-related hormones such as cortisol (CRT) and dehydroepiandrosterone (DHEA) in saliva by automated online in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). CRT and DHEA were separated within 5 min on a Discovery HS F5 column, with 0.2% formic acid/acetonitrile (65/35, v/v) as the mobile phase at a flow rate of 0.2 mL min−1. Electrospray ionization conditions in the positive ion mode were optimized for MS/MS detection of CRT and DHEA. The optimum in-tube SPME conditions were 25 draw/eject cycles of 40 μL of sample at a flow rate of 200 μL min−1 using a Supel-Q PLOT capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME LC-MS/MS method, we obtained good linearity of the calibration curve (r ≥ 0.9990) for CRT (0.005–1.0 ng mL−1) and DHEA (0.05–10.0 ng mL−1) using a stable isotope-labeled internal standard. The limits of detection (S/N = 3) for CRT and DHEA were 0.9 and 13 pg mL−1, respectively. This method had sensitivity more than 30-fold higher than that of the direct injection method. The intra-day and inter-day precisions (relative standard deviations) were below 4.3% and 9.6% (n = 5), respectively. This method was successfully utilized to analyze CRT and DHEA in saliva samples without any other pretreatment or interference peaks, and the recoveries of CRT and DHEA spiked into saliva samples were each above 94%. This method was used to analyze changes in salivary CRT and DHEA concentrations in response to stress.

Simplified extraction of tetracycline antibiotics from milk using a centrifugal ultrafiltration device

A simplified method was developed for the simultaneous determination of oxytetracycline, tetracycline, chlortetracycline, and doxycycline in milk. Isolation of the target compounds was performed using an Ultrafree-MC/PL centrifugal ultrafiltration device without prior sample preparation. Analyses were carried out via high-performance liquid chromatography using a Discovery HS F5 column with a gradient mobile phase which consisted of 30mM citric acid solution (pH 3, in water) and acetonitrile (60:40→40:60, v/v). Recovery of the target compounds from spiked samples at four levels (0.05, 0.1, 0.2, and 0.5μg/mL) was higher than 87%, with relative standard deviations of less than 6%. Limits of quantification ranged from 0.01 to 0.04μg/mL.

Advantages of pentafluorophenylpropyl stationary phase over conventional C18 stationary phase—Application to analysis of triamcinolone acetonide

A pentafluorophenylpropyl (PFPP) stationary phase was for the first time tested for the simultaneous determination of triamcinolone acetonide, its degradation product triamcinolone and two preservatives, methylparaben, and propylparaben. A new simple isocratic reversed phase HPLC method with UV detection, using estradiol hemihydrate as an internal standard, has been developed and validated. Chromatography was performed on a Discovery HS F5 column (150 mm × 4.6 mm, 5 μm) using a binary mobile phase composed of acetonitrile and water 45:55 (v:v). The flow-rate was 0.6 mL/min, the column temperature 25 °C and the UV detection was accomplished at 240 nm. The chromatography results using PFPP stationary phase were compared with those obtained using conventional C18 columns.

Determination of ephedrine alkaloids in Ephedra natural products using HPLC on a pentafluorophenylpropyl stationary phase

In this study a pentafluorophenylpropyl (PFPP) stationary phase was applied to the fast and reliable qualitative and quantitative analysis of ephedrine alkaloids in Ephedra plant material and derivatives. A Discovery HS F5 column (150 mm × 4.6 mm i.d., 5 μm) was used, with an isocratic mobile phase composed of ammonium acetate (7 mM) in acetonitrile–water (90:10, v/v), at a flow rate of 1.0 ml/min. The column temperature was set at 45 °C. UV detection was set at 215 and 225 nm. The total analysis time was 16 min. The validation parameters, such as linearity, sensitivity, accuracy, precision and specificity, were found to be highly satisfactory. Sonication and microwave extractions were compared in order to optimize the yield of the target analytes. Under the optimized extraction conditions (based on two cycles of sonication with methanol at 40 °C for 15 min), different matrices containing Ephedra were successfully analyzed for their alkaloid content. The method was applied to the analysis of standard reference materials (SRMs) containing Ephedra. Furthermore, the developed technique allowed the simultaneous determination of ephedrine alkaloids and synephrine, the main phenethylamine alkaloid of Citrus aurantium, that has replaced Ephedra in dietary supplements used in the treatment of obesity. The results indicated that this procedure is suitable for the phytochemical analysis of Ephedra plant material and extracts, and can be applied to demonstrate the label claims for product content, including the absence of ephedrine alkaloids in Ephedra-free products.

Fast high-performance liquid chromatography analysis of phenethylamine alkaloids in Citrus natural products on a pentafluorophenylpropyl stationary phase

In this study, the chromatographic performance of a pentafluorophenylpropyl (PFPP) stationary phase was evaluated for the rapid separation of phenethylamine alkaloids (i.e. (±)-octopamine, (±)-synephrine, tyramine, N-methyltyramine and hordenine) in Citrus aurantium plant material (fruits and peel), various Citrus species, extracts and dietary supplements claiming to contain C. aurantium. The problems of phenethylamine alkaloid separation, such as peak tailing, low retention and low resolution, were successfully solved with this stationary phase. The parameters used for the method optimization included the mobile phase counter ion concentration and column temperature. A Discovery HS F5 column (150 mm × 4.6 mm i.d., 5 μm) was used, with an isocratic mobile phase composed of 10 mM ammonium acetate in 90:10 ACN–H 2O (v/v), at a flow rate of 1.0 mL/min. The column temperature was set at 20 °C. The photodiode array detector monitored the eluent at 225 nm. The total analysis time was 10 min. The validation parameters, such as linearity, sensitivity, accuracy, precision and specificity, were found to be highly satisfactory. With a simple sample preparation procedure, different matrices were successfully analyzed for their alkaloid content. The results indicated that the products on sale, labeled as dietary supplements, vary widely in the quantitative composition of the active constituents: the amount of (±)-synephrine, the major alkaloid, in such products ranged from 0.65 to 27.41 mg/g. The other compounds were either not detected or were present at low levels. The developed method can be considered suitable for the quality control of Citrus plant material and commercial products.

High-performance liquid chromatographic method with UV photodiode-array, fluorescence and mass spectrometric detection for simultaneous determination of galantamine and its phase I metabolites in biological samples

Galantamine, an alkaloid isolated from the bulbs and flowers of Caucasian snowdrop (Galanthus woronowii, Amaryllidaceae) and related species, is employed in human medicine for the treatment of various neuromuscular and neurodegenerative diseases. After the administration, the products of oxidative biotransformation (O-desmethyl-galantamine, N-desmethyl-galantamine, galantamine-N-oxide) and chiral conversion (epigalantamine) are formed in various concentrations from parent compound. For the identification and determination of galantamine and its phase I metabolites in blood plasma and tissues, a new bioanalytical method based on a reversed-phase high-performance liquid chromatography with UV photodiode-array, fluorescence and mass spectrometric detection was developed, validated and applied to pharmacokinetic and biotransformation studies. Sample preparation included a homogenization of the rat tissues (liver, brain, hypophysis) in a phosphate buffer 0.05mol/L pH 7.4. Plasma samples and tissue homogenates were purified using a mixed-mode solid-phase extraction (Waters Oasis MCX cartridges). Galantamine, its above-mentioned metabolites and the internal standard codeine were separated on a Discovery HS F5 column (Supelco, 150mm×4.6mm I.D., 5μm) at flow rate of 1mL/min using a linear gradient elution. UV photodiode-array and mass spectrometric detection were employed for the identification of individual galantamine metabolites in various biomatrices, the fluorescence detection (λexcit=280nm/λemiss=310nm) was chosen for the quantification of galantamine and its metabolites. The developed method was applicable in liver tissue in the range from 0.50 to 63.47nmol/g of galantamine, from 0.32 to 41.42nmol/g of O-desmethyl-galantamine, from 0.54 to 69.40nmol/g of N-desmethyl-galantamine and from 0.70 to 89.03nmol/g of epigalantamine. Limit of detection was found to be 0.04nmol/g for galantamine, 0.19nmol/g for O-desmethyl-galantamine, and 0.07nmol/g for N-desmethyl-galantamine and epigalantamine.

Development and validation of HPLC-DAD methods for the analysis of two novel iron chelators with potent anti-cancer activity

Di-2-pyridylketone isonicotinoyl hydrazone (PKIH) and di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) novel iron chelators which possess marked anti-cancer activity in vivo. However, further progress in the development of these drug candidates requires precise and convenient methods for their qualitative and quantitative analysis. The aim of this study was to develop and validate HPLC methods suitable for the purity and stability evaluation of Dp44mT and PKIH and subsequently to employ these methods in stress tests addressing their chemical stability. The chromatographic analyses of both chelators were accomplished via HPLC using a Discovery HSF5 column (25 cm × 4 mm; 5 μm). For separation of Dp44mT and its synthetic precursors, the mobile phase was composed of a mixture of 2 mM EDTA and acetonitrile in a ratio 60:40 (v/v). A desirable separation of PKIH from its synthetic precursors was achieved with a mixture of 0.01 M phosphate buffer (pH 3.0), methanol and acetonitrile in a ratio of 65:21:14 (v/v/v) with the addition of EDTA (2 mM). In order to confirm the utility of these HPLC methods for measuring these drugs and their stability, Dp44mT and PKIH were subjected to chemical stress tests. These experiments showed that Dp44mT was relatively stable against hydrolytic degradation, but quite sensitive to oxidation. On the other hand, PKIH was slightly sensitive to acid-catalyzed hydrolysis, but it was relatively stable under other tested conditions. Furthermore, these studies confirmed the utility of these methods not only for appropriate evaluation of purity but also stability. The analytical methods developed and validated in this study, as well as the basic data on the chemical stability, should further support the development of both these novel anti-cancer chelators as promising drug candidates.

Nature of the main contaminant in the drug primaquine diphosphate: SFC and SFC–MS methods of analysis

The drug primaquine diphosphate is used for causative treatment of malaria. Using HPLC–MS and GC–MS, this research group was previously able to show that the main contaminant of primaquine is the positional isomer quinocide [I. Brondz, D. Mantzilas, U. Klein, D. Ekeberg, E. Hvattum, M.N. Lebedeva, F.S. Mikhailitsyn, G.D. Soulimanov, J. Roe, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 800 (2004) 211–223; I. Brondz, U. Klein, D. Ekeberg, D. Mantzilas, E. Hvattum, H. Schultz, F. S. Mikhailitsyn, Asian J. Chem. 17 (2005) 1678–1688]. Primaquine and quinocide are highly toxic substances which can have a number of side effects upon use in medical treatment. A standard for quinocide is not typically commercially available. In the present work, supercritical fluid chromatography–mass spectrometry (SFC–MS) with two different columns was used to achieve a shorter analysis time for the separation between the positional isomers quinocide and primaquine in primaquine diphosphate and to elucidate additional information about differences in their MS fragmentation. Unlike using HPLC–MS, it was possible to achieve the differential fragmentation of positional isomers at branching points using the SFC–MS technique. The desired short analysis time was achieved using SFC equipped with a Discovery HS F5 column and the differential fragmentation of positional isomers during SFC–MS provides information on the differences in the structure of these substances. Using a Chiralpak AD-H chiral column, it was possible to resolve the enantiomers in primaquine and separate quinocide from those enantiomers.