Quantitative analysis of xanthohumol and related prenylflavonoids in hops and beer by liquid chromatography-tandem mass spectrometry

A method for quantitation of six prenylflavonoids (xanthohumol, isoxanthohumol, desmethylxanthohumol, 6- and 8-prenylnaringenins and 6-geranylnaringenin) in hops and beer by HPLC-tandem mass spectrometry has been developed. The method allows direct analysis of beer and crude methanolic extracts of hops. After HPLC separation, prenylflavonoids were detected by positive ion multiple-reaction monitoring using a triple-quadrupole mass spectrometer equipped with a heated nebulizer--atmospheric pressure chemical ionization interface. The accuracy and precision were evaluated by replicate analyses of (spiked) samples. Thirteen commercial beers were analysed with the method. Isoxanthohumol, formed by isomerization of xanthohumol during the brewing process, was the most abundant flavonoid in hopped beers, ranging from 0.04 to 3.44 mg/l.     

Liquid chromatographic separation and thermodynamic investigation of stereoisomers of darunavir on Chiralpak AD‐Hcolumn

Liquid chromatographic separation of stereoisomers of darunavir on Chiralpak AD‐H, a column containing the stationary phase coated with amylose tris(3,5‐dimethylphenylcarbamate) as a chiral selector, was studied under normal‐phase conditions at different temperatures between 20 and 50°C. The effect of quality and quantity of different polar organic modifiers viz: methanol, ethanol, 1‐propanol, and 2‐propanol in the mobile phase as well as column temperature on retention, separation, and resolution was investigated and optimized. The optimum separation was accomplished using a mobile phase composed of n‐hexane/ethanol/diethyl amine (80:20:0.1 v/v/v) at 40°C. Apparent thermodynamic parameters ΔH⁰ and ΔS* were derived from the Van't Hoff plots (lnk′ versus 1/T) and used to explain the strength of interactions between the stereoisomers and amylose tris(3,5‐dimethylphenylcarbamate) coated chiral stationary phase.     

Characterization of prenylflavonoids and hop bitter acids in various classes of Czech beers and hop extracts using high-performance liquid chromatography–mass spectrometry

Hops contain a wide range of polyphenolic compounds with antioxidant properties divided in various chemical classes. These compounds are detected in hop extracts and also in beer as its main product. Based on the careful optimization of column type, column packing, mobile phase composition and gradient steepness, two high-performance liquid chromatography–mass spectrometry (HPLC/MS) methods have been developed. The first method using Purospher Star RP-8e column and the gradient of aqueous acetonitrile containing 0.3% formic acid is optimized for the separation of low polar polyphenolic compounds, while the second one with Zorbax SB-CN column is used for more polar hops and beer components. In this work, more compounds are detected in comparison to previous reports. In total, 49 low polar and 37 polar compounds are detected in studied samples and their molecular weights are determined based on atmospheric pressure chemical ionization (APCI) mass spectra. Some compounds are identified based on the interpretation of their full scan and tandem APCI mass spectra, retention behavior and UV spectra, while the full structure elucidation of other species still requires further research. The quantitation of xanthohumol related prenylflavonoids and bitter acids is done with two detection techniques (APCI-MS and UV detection) providing comparable results. Both compound classes (i.e., prenylflavonoids and bitter acids) are separated and quantitated in a single HPLC run, where numerous other polyphenolics are detected as well.     

Determination of the purity of phenoxymethylpenicillin by micellar electrokinetic chromatography and reversed phase liquid chromatography on a monolithic silica column

A micellar electrokinetic chromatographic and a fast reversed‐phase liquid chromatographic method have been developed for determination of the purity of phenoxymethylpenicillin. The optimized running buffer composition was 40 mM phosphate–borate–125 mM SDS–3.5% (v/v) methanol. The HPLC method employed a monolithic silica C18 column and a mobile phase composed of phosphate buffer, pH 3.5, and ACN, the flow‐rate being 3.5 mL/min. Both methods were successfully validated. Linearity, intermediate precision, limits of quantitation, accuracy, and a good correlation of the HPLC and MEKC results were demonstrated. Both methods proved to be fast and reliable and sufficiently sensitive. A combination of the two methods can be very useful in impurity profiling.     

An efficient synthesis of the phytoestrogen 8-prenylnaringenin from isoxanthohumol with magnesium iodide etherate

Xanthohumol was isolated from supercritical carbon dioxide-spent hop and transformed into isoxanthohumol. The demethylation of isoxanthohumol with the best yield 93% occurred when MgI₂ etherate in anhydrous THF was applied. Salts such as MgBr₂, MgCl₂, CaI₂, Mg(OAc)₂, Mg(OMe)₂ were also investigated. A convenient method for the xanthohumol isolation from supercritical carbon dioxide-spent hop is also described.     

Enantioselective separation of twelve pairs of enantiomers on polysaccharide‐based chiral stationary phases and thermodynamic analysis of separation mechanism

The enantioseparation of twelve pairs of structurally related 1‐aryl‐1‐indanone derivatives was studied in the normal‐phase mode using three different polysaccharide‐type chiral stationary phases, namely Chiralpak IB, Chiralpak IC, and Chiralpak ID. n‐Hexane/2‐propanol and n‐hexane/ethanol were employed as mobile phases. Among all the investigated chiral columns, Chiralpak IC exhibited the most universal and the best enantioseparation ability toward all the racemates, particularly with the mobile phase composed of n‐hexane/2‐propanol (90/10, v/v). Then the effects of column temperature on retention and enantioselectivity were examined in the range of 25–40°C. Satisfactory enantioseparation was obtained at ambient temperature. The natural logarithm of retention and separation factors (ln k and ln α) versus the reciprocal of absolute temperature (1/T) (Van't Hoff plots) were found to be linear for all racemates, indicating that the retention and separation mechanisms were independent of temperature in the range investigated. Then, the thermodynamic parameters (ΔΔH°, ΔΔS°, and ΔΔG°) were calculated from Van't Hoff plots. These values indicated that the solute transfer from the mobile to stationary phase was enthalpically favorable, and the process of enantioseparation was mainly enthalpy controlled. At last, the impact of small changes in molecular structures of the tested 1‐indanone derivatives on enantioseparation was also discussed.     

Chiral separation of helical chromenes with chloromethyl phenylcarbamate polysaccharide‐based stationary phases

Two chloromethyl phenylcarbamate‐based chiral stationary phases, one containing an amylose‐type chiral selector (Lux Amylose 2, from Phenomenex) and the other a cellulose‐type one (Lux Cellulose‐4, from Phenomenex), were successfully used for the chiral resolution of three helical chromenes featuring a helicene‐like structure. The compound bearing a phenyl substituent on the helicene‐like structure was enantioresolved at 25°C with Lux Cellulose‐4 and a n‐hexane/1‐propanol 99:1 v/v eluent. With a n‐hexane/2‐propanol 99.8:0.2 v/v mobile phase, the same column (operated at 35°C) provided the separation of the four isomers of the compound having a hexyl residue on the helicene‐like motif and an additional asymmetric carbon. Lux Amylose‐2 was necessary for the enantioseparation of the compound having the sole hexyl residue on the helical scaffold. For the last compound a n‐hexane/2‐propanol 99.8:0.2 v/v eluent was used, and the column temperature was fixed at 5°C. The enantiomer elution order was appraised by using electronic circular dichroism and theoretical calculations. Notably, different thermodynamics of retention and enantioseparation were observed for molecules with pronounced structural similarity, that is, the enantiomer pairs of the compound containing the additional asymmetric carbon atom. Indeed, both entropically and enthalpically controlled adsorption and separation processes were observed.     

High-Performance Liquid Chromatographic Determination of SAZ-VII-23, A Novel Antiarrhythmic Agent,in Dog Plasma and Urine

Abstract

A HPLC method has been developed to determine the concentrations of SAZ-VII-23 (3-benzoyl-7-isopropyl-3,7-diazabicyclo[3.3.1]nonane HClO₄), a novel antiarrhythmic agent, in dog plasma and urine. Plasma treated with acetonitrile and alkalinized urine were extracted with chloroform- propanol (9:1). An aliquot was injected on to HPLC system using a C₆ reversed-phase column and acetonitrile-methanol-37.5 mM phosphate buffer, pH 6.8 (28.5:28.5:43 v/v) containing 4.0 mM triethylamine as mobile phase. Detection wavelength was 255 nm. The linear range were 0.04–8 μg/ml, and the lower limit of quantitation was 0.04 μg/ml in plasma and urine, respectively. The method was applied to determine plasma and urine concentrations and preliminary pharmacokinetic profiles of SAZ-VII-23 in a dog.     

Preparation of Fritless Packed Silica Columns for Capillary Electrochromatography

Fritless packed silica gel columns were prepared using sol‐gel technology. A part of a 75 μm i.d. fused silica capillary was filled with a mixture of tetramethoxysilane and poly(ethylene glycol). After gelling at 40°C and heating at 300°C, the resultant silica gel was derivatized with dimethyloctadecylchlorosilane. A scanning electron micrograph of a cross‐section of the capillary column showed that the gel took the form of a spherical particle aggregate and adhered to the column inner wall. The column performance was evaluated for electrochromatography using acetonitrile–50 mM HEPES buffer (pH 6.6) (60/40 or 40/60, v/v) as the mobile phase. An electroosmotic flow of 1.0 mm/s was generated with (60/40, v/v) acetonitrile/HEPES buffer at a field strength of 546 V/cm. Using a sol‐gel‐derived packed column at an electroosmotic flow of 0.5 mm/s, efficiencies of up to 1.1×10⁵ plates/m were obtained for retained solutes.     

Development and validation of HPTLC and green HPLC methods for determination of furosemide,spironolactone and canrenone,in pure forms,tablets and spiked human plasma

Two selective and accurate chromatographic methods are presented for simultaneous quantitation of spironolactone (SP) and furosemide (FR) and canrenone (CN), the main degradation product and the main active metabolite of SP. Method A was HPTLC, where separation was completed on silica gel HPTLC F₂₅₄ plates using ethyl acetate–triethylamine–acetic acid (9:0.7:0.5, by volume) as a developing system and UV detection at 254 nm. Method B was a green isocratic RP‐HPLC utilizing a C₁₈ (4.6 × 100 mm) column, the mobile phase consisting of ethanol–deionized water (45: 55, v/v) and UV estimation at 254 nm. Adjustment of flow rate at 1 mL/min and pH at 3.5 with glacial acetic acid was done. Regarding the greenness profile, the proposed RP‐HPLC method is greener than the reported one. ICH guidelines were followed to validate the developed methods. Successful applications of the developed methods were revealed by simultaneous determination of FR, SP and CN in pure forms and plasma samples in the ranges of 0.2–2, 0.05–2.6 and 0.05–2 μg/band for method A and 5–60, 2–60 and 2–60 μg/mL for method B for FR, SP and CN, respectively.     

Chiral determination of a novel acaricide cyflumetofen enantiomers in cucumber,tomato, and apple by HPLC

A simple chiral analytical method was developed for the enantiomeric determination of cyflumetofen in cucumber, tomato, and apple by normal‐phase HPLC. The effects of mobile phase composition and column temperature on the enantioseparation were evaluated. Excellent separation was achieved at 25°C on a Chiralpak AD‐H column, with a mixture of n‐hexane and 2‐propanol (95:5, v/v) as mobile phase at a flow rate of 1.0 mL/min detecting at 234 nm. The resolution of cyflumetofen enantiomers was up to 5.5. The elution order of the enantiomers was determined by an online OR‐2090 detector, which was performed under the same chromatographic conditions. The first eluted enantiomer was (–)‐cyflumetofen and the second eluted one was (+)‐cyflumetofen. The method was validated for linearity, repeatability, accuracy, LOD, and LOQ. LOD ranged from 0.1 to 0.15 mg/kg, with the LOD varying from 0.33 to 0.5 mg/kg for each enantiomer, respectively. The average recoveries of the pesticide ranged from 71.4 to 102.0% at all fortification levels. The precision values associated with the analytical method, expressed as RSD values, were below 14.8% in all matrices. The method was then successfully applied to detect cyflumetofen enantiomers in real samples.     

Quantification of xanthohumol, isoxanthohumol, 8-prenylnaringenin, and 6-prenylnaringenin in hop extracts and derived capsules using secondary standards

Hop is a well-known and already frequently used estrogenic phytotherapeutic, containing the interesting prenylflavonoids, xanthohumol (XN), isoxanthohumol (IXN), 8- and 6-prenylnaringenin (8-PN and 6-PN). Since the use of secondary standards can form a solution whenever the determination is required of certain components, not commercially available or too expensive, it was decided to develop an accessible HPLC-DAD method for the determination of these prenylflavonoids. The amounts were determined in hop extract and capsules, using quercetin and naringenin as secondary standards. After optimization of the sample preparation and HPLC conditions, the analysis was validated according to the ICH guidelines. The response function of XN, 8-PN, quercetin and naringenin showed a linear relationship. For the determination of XN, a calibration line of at least three concentrations of quercetin has to be constructed. The correction factors for XN (quercetin) and for 8-PN (naringenin) were validated and determined to be 0.583 for XN, and 1.296 for IXN, 8-PN and 6-PN. The intermediate precision was investigated and it could be concluded that the standard deviation of the method was equal considering time and concentration (RSD of 2.5-5%). By means of a recovery experiment, it was proven that the method is accurate (recoveries of 96.1-100.1%). Additionally, by analysing preparations containing hop extracts on the Belgian market, it was shown that the method is suitable for its use, namely the determination of XN, IXN, 8-PN and 6-PN in hop extract and capsules, using quercetin and naringenin as secondary standards.     

Development and comparison of HPLC and MEKC methods for the analysis of cyclic sulfur mustard degradation products

In this study, novel, fast, and simple methods based on RP‐HPLC and MEKC with DAD are developed and validated for the qualitative and quantitative determination of five cyclic sulfur mustard (HD) degradation products (1,4‐thioxane, 1,3‐dithiolane, 1,4‐dithiane, 1,2,5‐trithiepane, and 1,4,5‐oxadithiepane) in water samples. The HPLC method employs a C18 column and an isocratic water‐ACN (55:45, v/v) mobile phase. This method enables separation of all five cyclic compounds within 8 min. With the CE method, the baseline separation of five compounds was achieved in less than 11 min by applying a simple BGE composed of a 10 mM borate buffer and 90 mM SDS (pH 9.15). Both methods showed good linear correlation (R ² > 0.9904). The detection limits were in the range of 0.08–0.1 μM for the HPLC method and 10–20 μM for MEKC. The precision tests resulted in RSDs for migration times and peak areas less than 0.9 and 5.5%, respectively, for the HPLC method, and less than 1.1 and 7.7% for the MEKC method, respectively. The developed methods were successfully applied to the analysis of five cyclic HD degradation products in water samples. With the HPLC method, the LODs were lowered using the SPE for sample purification and concentration.     

Preparation and evaluation of amphiphilic silica‐based monolithic column having surface‐bound octanoyl‐aminopropyl moieties for capillary electrochromatography

A novel amphiphilic silica‐based monolithic column having surface‐bound octanoyl‐aminopropyl moieties was successfully prepared by a one‐step in situ derivatization process. As expected, the amphiphilic monolithic column exhibited RP chromatographic behavior toward non‐polar solutes (e.g., alkyl benzenes) with high column performance. As the pH of the buffer inside the column increases, the EOF changed from −2.65×10⁻⁸m² V⁻¹s⁻¹ at pH 3.0 to 1.20×10⁻⁸ m² V⁻¹s⁻¹ at pH 8.0 with the reversion of EOF at about pH 6.4. Using acidic mobile phase, five aromatic acids can be efficiently separated in less than 6 min under co‐EOF conditions. For basic compounds, symmetrical peaks were obtained due to the existence of hydrophilic acyl amide group, which can effectively minimize the adsorption of the positively charged basic analyte to the silica‐based surface of the capillary column.     

A simple,rapid and fully validated HPLC method for simultaneous quantitative bio‐analysis of rosuvastatin and candesartan in rat plasma: Application to pharmacokinetic interaction study

A simple, precise and accurate HPLC method was developed, optimized and validated for simultaneous determination of rosuvastatin and candesartan in rat plasma using atorvastatin as an internal standard. Solid‐phase extraction was used for sample cleanup and its subsequent optimization was carried out to achieve higher extraction efficiency and to eliminate matrix effect. A quality by design approach was used, wherein three‐level factorial design was applied for optimization of mobile phase composition and for assessing the effect of pH of the mobile phase using Design Expert Software. Adequate separation for both analytes was achieved with a Waters C₁₈ column (250 × 4.6 mm, 5 μm) using acetonitrile–5 mm sodium acetate buffer (70:30, v/v; pH adjusted to 3.5 with acetic acid) as a mobile phase at a flow rate of 1.0 mL/min and wavelength of 254 nm. The calibration curves were linear over the concentration ranges 5–150 and 10–300 ng/mL for rosuvastatin (ROS) and candesartan (CAN), respectively. The validated method was successfully applied to a pharmacokinetic study in Wistar rats and the data did not reveal any evidence for a potential drug–drug interaction between ROS and CAN. This information provides evidence for clinical rational use of ROS and CAN.     

Simplified liquid-chromatographic determination of residues of tetracycline antibiotics in eggs

Summary A high-performance liquid chromatographic (HPLC) procedure is described for the identification and quantification of residues of tetracycline antibiotics (TCA) (oxytetracycline, tetracycline, chlortetracycline, and doxycycline), in eggs. Spiked and blank samples were prepared by homogenization with 1∶1 (v/v) acetonitrile-mixed Mcllvaine buffer and EDTA solution (pH 4.0) then centrifugal ultrafiltration. HPLC was performed on a reversed-phase column with acetonitrile-5% (v/v) aqueous acetic acid, 35∶65 (v/v), as mobile phase and photo-diode array detection. Average recoveries (each drug spiked at 0.1, 0.2, 0.3, 0.5 and 1.0 μg g⁻¹) were >-77% with standard deviations (SD) between 1.5 and 3.5%. The inter-assay variabilities and theirSD were <3.4% and <0.7%, respectively, and intra-assay variability was between 2.0 and 3.9%. The limits of quantitation (LOQ) were 0.064 0.087, 0.121, and 0.131 μg g⁻¹ for OTC, TC, CTC, and DC, respectively. The total time required for the analysis of one sample was less than 30 min.     

Developing a robust LC–MS/MS method to quantify Zn‐DTPA,a zinc chelate in human plasma and urine

Quantitation of Zn‐DTPA (zinc diethylenetriamene pentaacetate, a metal chelate) in complex biological matrix is extremely challenging on account of its special physiochemical properties. This study aimed to develop a robust and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for determination of Zn‐DTPA in human plasma and urine. The purified samples were separated on Proteonavi (250 × 4.6 mm, 5 μm; Shiseido, Ginza, Tokyo, Japan) and a C₁₈ guard column. The mobile phase consisted of methanol–2 mm ammonium formate (pH 6.3)–ammonia solution (50:50:0.015, v/v/v), flow rate 0.45 mL/min. The linear concentration ranges of the calibration curves for Zn‐DTPA were 1–100 μg/mL in plasma and 10–2000 μg/mL in urine. The intra‐ and inter‐day precisions for quality control (QC) samples were from 1.8 to 14.6% for Zn‐DTPA and the accuracies for QC samples were from −4.8 to 8.2%. This method was fully validated and successfully applied to the quantitation of Zn‐DTPA in plasma and urine samples of a healthy male volunteer after intravenous infusion administration of Zn‐DTPA. The result showed that the concentration of Zn‐DTPA in urine was about 20 times that in plasma, and Zn‐DTPA was completely (94.7%) excreted through urine in human.     

Simple Liquid Chromatographic Determination of Minocycline in Brain and Plasma

A new high-performance liquid chromatography assay was developed for the determination of minocycline in plasma and brain. A solid–liquid extraction procedure was coupled with a reversed-phase HPLC system. The system requires a mobile phase consisting of acetonitrile:water:perchloric acid (26:74:0.25, v/v/v) adjusted to pH 2.5 with 5 M sodium hydroxide for elution through a RP8 column (250 × 3.0 mm, i.d.) with UV detection set at 350 nm. The method proved to be accurate, precise (RSD < 20%) and linear between 0.15–20 μg mL⁻¹ in plasma and 1–20 μg mg⁻¹ in brain. The method was successfully applied to a blood-brain barrier minocycline transport study.     

Determination of Visnagin inAmmi visnagaHairy Root Cultures Using Solid-Phase Extraction and High-Performance Liquid Chromatography

A high-performance liquid chromatographic method was developed for separation of the furochromone fraction and for determination of visnagin inAmmi visnagahairy root cultures. Lyophilized samples were extracted with chloroform:methanol (1:1, v/v) and purified on solid-phase extraction cartridges. HPLC analyses were performed on a Eurospher 100-C₈Knauer column and the mobile phase was 29:28:526:417 (v/v/v/v) acetonitrile:tetrahydrofuran:30 mM citric acid (pH 3.0):methanol. Quercetin was used as internal standard. Peaks were identified by addition of authentic standards and/or by diode-array detection.