An improved narrow-bore LC method for quantification of alfuzosin in pharmaceutical formulations

A simple rapid and stability-indicating LC method using a narrow-bore column has been developed, fully validated, and applied to the quantification of alfuzosin in pharmaceutical formulations. Chromatography was achieved isocratically on a narrow-bore, 5-μm particle size, C8 analytical column. The mobile phase was a 35:65 (v/v) 0.0125 m ammonium formate-acetonitrile at a flow rate of 0.35 mL min-1. Detection was by UV absorption at 245 nm. Evaluation over the range 200-800 ng mL-1 revealed linearity was good. Limits of detection and quantification for alfuzosin were 22.9 and 69.5 ng mL-1, respectively. Intra-day and inter-day RSD were less than 6.4%, and the relative percentage error was less than -1.7% (n = 5). Accelerated degradation performed under different stress conditions including oxidation, hydrolysis, and heat, proved the selectivity of the procedure. The method was successfully used for quality-control and content-uniformity testing of commercial tablets. © 2008 Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH

Hydrophilic interaction liquid chromatography/positive ion electrospray mass spectrometry for the quantification of deferasirox, an oral iron chelator, in human plasma

A rapid hydrophilic interaction liquid chromatography/positive ion electrospray mass spectrometric assay (HILIC/ESI-MS) was developed, validated and applied to the determination of deferasirox, in human plasma. The sample preparation process involved liquid-liquid extraction of 50μL plasma sample using ethyl acetate as an extraction solvent. Chromatographic separation was performed on an XBridge ®-HILIC analytical column (150.0mm×2.1mm i.d., particle size 3.5μm, 135Å) under isocratic elution. The mobile phase was composed of a 10% 8.0mM ammonium acetate water solution pH=5.0, adjusted with formic acid, in a binary mixture of acetonitrile/methanol (50:50, v/v) and pumped at a flow rate of 0.20mL/min. Quantitation of deferasirox was performed with selected ion monitoring (SIM) in positive ionization mode using electrospray ionization interface. The assay was found to be linear in the concentration range of 0.20-120.0μg/mL for deferasirox. Intermediate precision was found less than 3.9% over the tested concentration ranges. A run time of less than 6.0min for each sample made it possible to analyze a large number of human plasma samples per day. The method can be used to support a wide range of clinical studies concerning deferasirox monitoring and it was applied to the analysis of human plasma samples obtained from patients with β-thalassemia major. © 2012 Elsevier B.V

Development and validation of a liquid chromatographic/electrospray ionization mass spectrometric method for the determination of benazepril, benazeprilat and hydrochlorothiazide in human plasma

A new method was developed and fully validated for the quantitation of benazepril, benazeprilat and hydrochlorothiazide in human plasma. Sample pretreatment was achieved by solid-phase extraction (SPE) using Oasis HLB cartridges. The extracts were analysed by high-performance liquid chromatography (HPLC) coupled to a single-quadrupole mass spectrometer (MS) with an electrospray ionization interface. The MS system was operated in selected ion monitoring (SIM) modes. HPLC was performed isocratically on a reversed-phase porous graphitized carbon (PGC) analytical column (2.1 × 125.0 mm i.d., particle size 5 μm). The mobile phase consisted of 55% acetonitrile in water containing 0.3% v/v formic acid and pumped at a flow rate of 0.15 ml min -1. Chlorthalidone was used as the internal standard (IS) for quantitation. The assay was linear over a concentration range of 5.0-500 ng ml-1 for all the compounds analysed, with a limit of quantitation of 5 ng ml-1 for all the compounds. Quality control (QC) samples (5, 10, 100 and 500 ng ml-1) in five replicates from three different runs of analyses demonstrated intra-assay precision (coefficient of variation (CV) ≤14.6%), inter-assay precision (CV ≤ 5.6%) and overall accuracy (relative error less than -8.0%). The method can be used to quantify benazepril, benazeprilat and hydrochlorothiazide in human plasma, covering a variety of pharmacokinetic or bioequivalence studies. Copyright © 2006 John Wiley & Sons, Ltd

Direct injection LC/ESI-MS horse urine analysis for the quantification and identification of threshold substances for doping control. I. Determination of hydrocortisone

Two simple and rapid LC/MS methods with direct injection analysis were developed and validated for the quantification and identification of hydrocortisone in equine urine using the same sample preparation but different mass spectrometric systems: ion trap mass spectrometry (IT-MS) and time-of-flight mass spectrometry (TOF-MS). The main advantage of the proposed methodology is the minimal sample preparation procedure, as particle-free diluted urine samples were directly injected into both LC/MS systems. Desonide was used as internal standard (IS). The linear range was 0.25-2.5 μg ml -1 for both methods. Matrix effects were evaluated by preparing and analyzing calibration curves in water solutions and different horse urine samples. A great variation of the signal both for hydrocortisone and the internal standard was observed in different matrices. To overcome matrix effects, the unavailability of blank matrix and the excessive cost of the isotopically labeled internal standard, standard additions calibration method was applied. This work is an exploration of the performance of the standard additions approach in a method where neither nonisotopic internal standards nor extensive sample preparation is utilized and no blank matrix is available. The relative standard deviations of intra and interday analysis of hydrocortisone in horse urine were lower than 10.2 and 5.4%, respectively, for the LC/IT-MS method and lower than 8.4 and 4.4%, respectively, for the LC/TOF-MS method. Accuracy (bias percentage) was less than 9.7% for both methods. Copyright © 2008 John Wiley & Sons, Ltd

Insights into the mechanism of separation of bisphosphonates by zwitterionic hydrophilic interaction liquid chromatography: Application to the quantitation of risedronate in pharmaceuticals

Bisphosphonates are used to treat various skeletal disorders, as they modulate bone metabolism by inhibition of the osteoclast-mediated bone resorption. These compounds are both polar and ionic, and therefore, by using reversed phase liquid chromatography are eluted rapidly. Hydrophilic interaction liquid chromatography (HILIC) is an advantageous technique for the separation and analysis of polar molecules. As the elution order in HILIC is reversed to reversed phase liquid chromatography, a reasonable retention and selectivity for polar compounds is expected. In this work the retention mechanism of three bisphosponates, namely risedronate, tiludronate and zoledronate, was investigated under zwitterionic HILIC conditions. The key factors influencing the retention of the analytes on a zwitterionic ZIC®-pHILIC column (150.0 × 2.1 mm i.d., 200 Å, 3.5 µm) have been systematically investigated. It was found that apart from partition, electrostatic repulsions play an important role in the retention of bisphosphonates. Peak tailing of risedronate and zoledronate was improved by the addition of sodium pyrophosphate in the mobile phase. A zwitterionic hydrophilic interaction liquid chromatography-photodiode array (HILIC-PDA) method was further optimized and fully validated to quantitate risedronate in commercial film-coated tablets. The calibration curves for risedronate showed good linearity (r ≥ 0.9991) within the calibration range tested. The intra-and inter-day coefficient of variation (CV) values was less than 0.6%, while the relative percentage error (%Er) was less than −2.3%. Accelerated stability studies of risedronate conducted under several degradation conditions including hydrolysis, oxidation and heat demonstrated the selectivity of the procedure. A short-run analysis of not more than 6 min allowed the analysis of large samples per day. The applicability of the method for the quantitation of risedronate was demonstrated via the analysis of commercial tablets containing this compound. © 2019 by the authors. Licensee MDPI, Basel, Switzerland

Direct injection liquid chromatography/electrospray ionization mass spectrometric horse urine analysis for the quantification and confirmation of threshold substances for doping control. II. Determination of theobromine

In equine sport, theobromine is prohibited with a threshold level of 2 μgmL-1 in urine, hence doping control laboratories have to establish quantitative and qualitative methods for its determination. Two simple liquid chromatography/mass spectrometry (LC/MS) methods for the identification and quantification of theobromine were developed and validated using the same sample preparation procedure but different mass spectrometric systems: ion trap mass spectrometry (ITMS) and time-of-flight mass spectrometry (TOFMS). Particle-free diluted urine samples were directly injected into the LC/MS systems, avoiding the time-consuming extraction step. 3-Propylxanthine was used as the internal standard. The tested linear range was 0.75-15 μgmL -1. Matrix effects were evaluated analyzing calibration curves in water and different fortified horse urine samples. A great variation in the signal of theobromine and the internal standard was observed in different matrices. To overcome matrix effects, a standard additions calibration method was applied. The relative standard deviations of intra-and inter-day analysis were lower than 8.6 and 7.2%, respectively, for the LC/ ITMS method and lower than 5.7 and 5.8%, respectively, for the LC/TOFMS method. The bias was less than 8.7% for both methods. The methods were applied to two case samples, demonstrating simplicity, accuracy and selectivity. Copyright © 2009 John Wiley & Sons, Ltd

Direct injection horse urine analysis for the quantification and identification of threshold substances for doping control. III. Determination of salicylic acid by liquid chromatography/quadrupole time-of-flight mass spectrometry

In equine sport, salicylic acid is prohibited with a threshold level of 750∈μg mL-1 in urine; hence, doping control laboratories have to establish quantitative and qualitative methods for its determination. A simple and rapid liquid chromatographic/mass spectrometric method was developed and validated for the quantification and identification of salicylic acid. Urine samples after 900-fold dilution and addition of the internal standard (4-methylsalicylic acid) were directly injected to the liquid chromatography/quadrupole time-of-flight mass spectrometry system. Electrospray ionization in negative mode with full scan acquisition mode and product ion scan mode were chosen for the quantification and identification of salicylic acid, respectively. Run time was 2.0 min. The tested linear range was 2.5-50∈μg mL-1 (after 100-fold sample dilution). The relative standard deviations of intra- and inter-assay analysis of salicylic acid in horse urine were lower than 2.5% and 2.8%, respectively. Overall accuracy (relative percentage error) was less than 3.3%. Method was applied to two real samples found to be positive for salicylic acid, demonstrating simplicity, accuracy, and selectivity. © 2009 Springer-Verlag

Direct injection horse-urine analysis for the quantification and confirmation of threshold substances for doping control. IV. Determination of 3-methoxytyramine by hydrophilic interaction liquid chromatography/quadrupole time-of-flight mass spectrometry

Levodopa and dopamine have been abused as performance-altering substances in horse racing. Urinary 3-methoxytyramine is used as an indicator of dopaminergicmanipulation resulting fromdopamine or levodopa administration and is prohibitedwith a urinary threshold of 4 μ gmL-1 (free and conjugated). A simple liquid chromatographic (LC)/mass spectrometric (MS) (LCMS) method was developed and validated for the quantification and identification of 3-methoxytyramine in equine urine. Sample preparation involved enzymatic hydrolysis and protein precipitation. Hydrophilic interaction liquid chromatography (HILIC) was selected as a separation technique that allows effective retention of polar substances like 3-methoxytyramine and efficient separation from matrix compounds. Electrospray ionization (ESI) in positive mode with product ion scan mode was chosen for the detection of the analytes. Quantification of 3-methoxytyramine was performed with fragmentation at low collision energy, resulting in one product ion,while a second run at high collision energywas performed for confirmation (at least three abundant ions). Studies on matrix effects showed ion suppression depending on the horse urine used. To overcome the variability of the results originating from the matrix effects, isotopic labelled internal standard was used and linear regression calibration methodology was applied for the quantitative determination of the analyte. The tested linear range was 1-20 μ gmL-1. The relative standard deviations of intra- and inter- assay analysis of 3-methoxytyramine in horse urine were lower than 4.2% and 3.2%, respectively. Overall accuracy (relative percentage error) was less than 6.2%. The method was applied to case samples, demonstrating simplicity, accuracy and selectivity. Copyright © 2009 John Wiley & Sons, Ltd

Direct injection human plasma analysis for the quantification of antihypertensive drugs for therapeutic drug monitoring using hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry

The concept of personalized medicine is related to the development of new sensitive, precise and accurate analytical methods for therapeutic drug monitoring. In this article a rapid, sensitive and specific method was developed for the quantification of aliskiren, losartan, valsartan and hydrochlorothiazide in human plasma. Sample preparation was performed by protein precipitation with acetonitrile followed by filtration. All analytes and the internal standard (tiamulin) were separated by hydrophilic interaction liquid chromatography using an X-Bridge-HILIC analytical column (150.0×2.1mm i.d., particle size 3.5μm) under isocratic elution. The mobile phase was composed of a 10% 5mM ammonium formate water solution pH 4.5, adjusted with formic acid, in acetonitrile and pumped at a flow rate of 0.25mLmin-1. The assay was linear over the concentration range of 5-500ngmL-1 for all the analytes. Intermediate precision was less than 5.2% over the tested concentration ranges. The method is the first reported application of HILIC in the analysis antihypertensives in human plasma. With a small sample size (50μL human plasma) and a run time less than 6.0min for each sample the method can be used to support a wide range of clinical studies and therapeutic drug monitoring. © 2015 Elsevier B.V