Sylwia Magiera

Application of statistical experimental design to the optimisation of microextraction by packed sorbent for the analysis of nonsteroidal anti-inflammatory drugs in human urine by ultra-high pressure liquid chromatography.

A new approach based on microextraction by packed sorbent (MEPS) and a reversed-phase ultra-high pressure liquid chromatography (UHPLC) method was developed and validated for the determination and quantification of nonsteroidal anti-inflammatory drugs (NSAIDs) (acetylsalicylic acid, ketoprofen, diclofenac, naproxen and ibuprofen) in human urine. The important factors that could influence the extraction were previously screened using the Plackett-Burman design approach. The optimal MEPS extraction conditions were obtained using C18 phase as a sorbent, small sample volume (20μL) and a short time period (approximately 5min) for the entire sample preparation step. The analytes were separated on a core-shell column (Poroshell 120 EC-C18; 100mm×3.0mm; 2.7μm) using a binary mobile phase composed of aqueous 0.1% trifluoroacetic acid and acetonitrile in the gradient elution mode (4.5min of analysis time). The analytical method was fully validated based on linearity, limits of detection (LOD), limits of quantification (LOQ), inter- and intra-day precision and accuracy, and extraction yield. Under optimised conditions, excellent linearity (R(2)>0.9991), limits of detection (1.07-16.2ngmL(-1)) and precision (0.503-9.15% RSD) were observed for the target drugs. The average absolute recoveries of the analysed compounds extracted from the urine samples were 89.4-107%. The proposed method was also applied to the analysis of NSAIDs in human urine. The new approach offers an attractive alternative for the analysis of selected drugs from urine samples, providing several advantages including fewer sample preparation steps, faster sample throughput and ease of performance compared to traditional methodologies.

Development and validation of UHPLC-ESI-MS/MS method for the determination of selected cardiovascular drugs, polyphenols and their metabolites in human urine.

A sensitive ultra-performance liquid chromatography tandem mass spectrometry method with electrospray ionisation (UHPLC-ESI-MS/MS) was developed for the simultaneous determination of 52 compounds: β-blockers, polyphenols (antioxidants) and their metabolites in mixture of standards and after addition the 52 standard solutions to human urine samples. The analyses of urine samples obtained from patients treated with β-blockers were also carried out. The separation of analytes was performed on a Hypersil GOLD™ column (100 mm × 2.1mm, 1.9 μm) using a gradient elution profile for 10 min and mobile phase consisting of 0.1% formic acid in water and acetonitrile. In these conditions, some of the tested compounds were not separated, but this was compensated by the use of MS/MS detection. The drugs, polyphenols and their metabolites were detected with a tandem mass spectrometer after being ionised positively or negatively (depending on the molecule) using an electrospray ionisation (ESI) source. The MS system was operated in the selected reaction monitoring (SRM) mode, where one quantitation and one confirmation transition was done for each analyte. The quantitative method was validated for selectivity, linearity, low limits of quantitation, accuracy, precision, recovery, matrix effect and analyte stability. The LLOQ varied from 0.01 to 0.40 ng mL(-1) for β-blockers and from 0.05 to 40.0 ng mL(-1) for polyphenols. The linear range was 0.08-1000 ng mL(-1) for the drugs and 0.10-2300 ng mL(-1) for the polyphenols. Intra-day and inter-day precision was less than 8%, and the accuracy ranged from -4.40 to 2.23% for all analytes. The average recoveries for all compounds analysed were better than 90%. The developed method can be successfully used to monitor cardiovascular drugs and their metabolites in urine samples of patients treated with β-blockers and can also be used to study the effect of polyphenols on the metabolism of drugs.

Fast, simultaneous quantification of three novel cardiac drugs in human urine by MEPS-UHPLC-MS/MS for therapeutic drug monitoring.

A sensitive and selective ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method was developed for the fast, simultaneous quantification of three novel cardiac drugs (aliskiren, prasugrel and rivaroxaban) in human urine. Sample preparation was performed with microextraction with packed sorbent (MEPS), which is a miniaturization of solid phase extraction. The optimal conditions for MEPS extraction were obtained using C8 sorbent, small sample volumes and a short time period (about 3min for the entire sample preparation step). Chromatographic separation of the selected compounds was achieved in less than 1.5min on a Zorbax Rapid Resolution High Definition SB-C18 column using isocratic elution with 0.1% formic acid and acetonitrile (70:30, v/v) at a flow rate of 0.8mLmin(-1). The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via an electrospray ionization source with positive ionization mode. The method was fully validated according to the latest recommendations of international guidelines. The lower limit of quantification was 5.0pgmL(-1) for aliskiren and rivaroxaban and 0.5pgmL(-1) for prasugrel. The intra- and inter-day precision was within 7.12% and the accuracy ranged from -7.54% to 4.17%. The mean extraction recoveries of the MEPSC8 methodology were found to be 98.3% for aliskiren, 100.3% for rivaroxaban and 99.9% for prasugrel. This MEPSC8-UHPLC-MS/MS method offers a fast, simple and precise way to determine selected novel cardiac drugs in human urine that could be applied to therapeutic drug monitoring and pharmacokinetic studies.

UHPLC-UV method for the determination of flavonoids in dietary supplements and for evaluation of their antioxidant activities.

A simple and rapid ultra-high performance liquid chromatographic (UHPLC) method coupled with an ultraviolet detector (UV) has been developed and validated for the separation and determination of 14 major flavonoids ((±)-catechin, (-)-epicatechin, glycitin, (-)-epicatechin gallate, rutin, quercitrin, hesperidine, neohesperidine, daidzein, glycitein, quercetin, genistein, hesperetin, and biochanin A) in herbal dietary supplements. The flavonoids have been separated on a Chromolith Fast Gradient Monolithic RP-18e column utilizing a mobile phase composed of 0.05% trifluoroacetic acid in water and acetonitrile in gradient elution mode. Under these conditions, flavonoids were separated in a 5 min run. The selectivity of the developed UHPLC-UV method was confirmed by comparison with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The validation parameters such as linearity, sensitivity, precision, and accuracy were found to be highly satisfactory. The optimized method was applied to determination of flavonoids in different dietary supplements. Additionally, the developed HPLC-UV method combined with 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay was used in the evaluation of antioxidant activity of the selected flavonoids.

Clinical applications of fast liquid chromatography: A review on the analysis of cardiovascular drugs and their metabolites

One of the major challenges facing the medicine today is developing new therapies that enhance human health. To help address these challenges the utilization of analytical technologies and high-throughput automated platforms has been employed; in order to perform more experiments in a shorter time frame with increased data quality. In the last decade various analytical strategies have been established to enhance separation speed and efficiency in liquid chromatography applications. Liquid chromatography is an increasingly important tool for monitoring drugs and their metabolites. Furthermore, liquid chromatography has played an important role in pharmacokinetics and metabolism studies at these drug development stages since its introduction. This paper provides an overview of current trends in fast chromatography for the analysis of cardiovascular drugs and their metabolites in clinical applications. Current trends in fast liquid chromatographic separations involve monolith technologies, fused-core columns, high-temperature liquid chromatography (HTLC) and ultra-high performance liquid chromatography (UHPLC). The high specificity in combination with high sensitivity makes it an attractive complementary method to traditional methodology used for routine applications. The practical aspects of, recent developments in and the present status of fast chromatography for the analysis of biological fluids for therapeutic drug and metabolite monitoring, pharmacokinetic studies and bioequivalence studies are presented.

Ultrasound-assisted emulsification microextraction combined with ultra-high performance liquid chromatography–tandem mass spectrometry for the analysis of ibuprofen and its metabolites in human urine

In this study, a fast, simple and efficient method based on ultrasound-assisted emulsification-microextraction (USAEME) coupled with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was successfully developed for the determination of ibuprofen (IBU) and its four metabolites (1-hydroxyibuprofen (1-HIBU), 2-hydroxyibuprofen (2-HIBU), 3-hydroxyibuprofen (3-HIBU), carboxyibuprofen (CIBU)) in human urine. For this purpose, the influence of the different parameters affecting the USAEME procedure was evaluated in order to optimize the efficiency of the process. The optimum conditions were found to be: 100μL of 1-octanol as extraction solvent, 2mL of urine sample, 15% (w/v) NaCl to control the ionic strength, ultrasonication for 10min; and centrifugation for 5min at 6500rpm. After sample preparation, chromatographic separation was achieved on a Zorbax Rapid Resolution High Definition (RRHD) SB-C18 column using the mobile phase consisting of 0.1% formic acid in water and acetonitrile in an elution gradient. Detection was performed in a triple quadrupole tandem mass spectrometer using the multiple reaction monitoring (MRM) mode and negative ionization. The proposed method showed satisfactory linearity over a wide concentration range (correlation coefficients over 0.9994). The lower limit of quantification (LLOQ) was 0.0005ng/mL for IBU and its metabolites. The intra- and inter-day precisions were in the range of 2.19-10.8% and the accuracies were between -5.93% and 6.29%. The mean recovery of analytes ranged from 90.7 to 104%. As a result, this method has been successfully applied for the sensitive determination of IBU and its metabolites in human urine samples.

Simultaneous chiral separation and determination of carvedilol and 5′-hydroxyphenyl carvedilol enantiomers from human urine by high performance liquid chromatography coupled with fluorescent detection

AbstractA sensitive and specific high performance liquid chromatography coupled with fluorescent detection (HPLC-FL) and tandem mass spectrometry detection (HPLC-MS/MS) methods for separation and determination of carvedilol (CAR) enantiomers and 5′-hydroxyphenyl carvedilol (5′-HCAR) enantiomers has been developed and validated. The analysed compounds were extracted from human urine by solid phase extraction. Good enantioseparation of the studied enantiomers was achieved on CHIRALCEL® OD-RH column using 0.05% trifluoroacetic acid and 0.05% diethylamine in water and acetonitrile in a gradient elution. The mass spectrometric data were acquired using the multiple reaction monitoring mode by positive electrospray ionisation. The method was validated over the concentration range from 25.0 ng mL−1 to 200 ng mL−1 for the analysed compounds. The limit of quantification varied from 14.2 ng mL−1 to 24.2 ng mL−1. Both the repeatability and inter-day precisions were below 10.0%, and the accuracy varied from −13.2% to 3.77%. The extraction recoveries ranged from 79.2% to 108%. The present paper reports the method for the simultaneous determination of CAR enantiomers and their metabolite enantiomers (5′-HCAR) in human urine samples. This newly developed method was successfully used to analyse the aforementioned analytes in human urine samples obtained from patients suffering from cardiovascular disease.

Determination of carnitine and acylcarnitines in human urine by means of microextraction in packed sorbent and hydrophilic interaction chromatography–ultra-high-performance liquid chromatography–tandem mass spectrometry

A method using semi-automatic microextraction by packed sorbent (eVol®-MEPS) and hydrophilic interaction chromatography-ultra-high-performance liquid chromatography-tandem mass spectrometry (HILIC-UHPLC-MS/MS) was described for the simultaneous determination of carnitine and acylcarnitines in human urine. The optimal conditions of MEPS extraction were obtained using C2 of M1 (C8+SCX) phase as a sorbent. Chromatographic separation of the analytes was achieved within 2.5min on Acquity UPLC BEH HILIC column using a gradient elution program with water containing 5mM ammonium acetate and acetonitrile as the mobile phase. The detection was performed on a triple-quadrupole tandem mass spectrometer in a positive ion mode via electrospray ionization (ESI). The linearity of the calibration curves for all compounds was found over a range from 0.1ng/mL to 500ng/mL. The method afforded satisfactory results in terms of sensitivity, specificity, precision, accuracy, recovery as well as stability of the analyte under various conditions. The method was used successfully for determination of carnitine and acylcarnitines in human urine.

Comparison of different sorbent materials for solid-phase extraction of selected drugs in human urine analyzed by UHPLC-UV

A procedure based on solid-phase extraction (SPE) followed by ultra-high-performance liquid chromatography (UHPLC) with UV detection has been developed for the analysis of multiple drugs in human urine. The compounds evaluated were aliskiren, prasugrel, rivaroxaban, prednisolone, propranolol, ketoprofen, nifedipine, naproxen, terbinafine, ibuprofen, diclofenac, sildenafil and acenocoumarol. Seventeen different solid phase extraction (SPE) cartridges were tested to evaluate their applicability for the isolation of drugs from human urine. Comparison were recovery of different drugs and reproducibility. The samples were analyzed by UHPLC using a Poroshell 120 EC-C18 column and acetonitrile -0.05% TFA in water as the mobile phase under gradient elution conditions. SPE combined with UHPLC-UV allowed the determination of drugs over a linear range of 0.01-30.0μg/mL, with limits of detection at 0.003-0.217μg/mL and precision of 0.8-7.1%. Phenyl (C6H5) sorbent was found to provide the most effective clean-up, removing the greatest amount of interfering substance and simultaneously ensuring analyte recoveries higher than 85.5% with relative standard deviations (RSD) <10%. The method was applied with good accuracy and precision in the determination of drugs in human urine obtained from patients treated with selected drugs.

Fast, simple and efficient salting-out assisted liquid-liquid extraction of naringenin from fruit juice samples prior to their enantioselective determination by liquid chromatography.

In this study, an easy, simple and efficient method for the determination of naringenin enantiomers in fruit juices after salting-out-assisted liquid-liquid extraction (SALLE) and high-performance liquid chromatography (HPLC) with diode-array detection (DAD) was developed. The sample treatment is based on the use of water-miscible acetonitrile as the extractant and acetonitrile phase separation under high-salt conditions. After extraction, juice samples were incubated with hydrochloric acid in order to achieve hydrolysis of naringin to naringenin. The hydrolysis parameters were optimized by using a half-fraction factorial central composite design (CCD). After sample preparation, chromatographic separation was obtained on a Chiralcel® OJ-RH column using the mobile phase consisting of 10mM aqueous ammonium acetate:methanol:acetonitrile (50:30:20; v/v/v) with detection at 288nm. The average recovery of the analyzed compounds ranged from 85.6 to 97.1%. The proposed method was satisfactorily used for the determination of naringenin enantiomers in various fruit juices samples.