Development and Validation of a Sensitive LC-MS/MS Method for the Simultaneous Analysis of Three-Tropane Alkaloids in Blood and Urine

A simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous analysis of three tropane alkaloids in blood and urine. After 1 mL of a blood or urine sample was extracted using a liquid–liquid extraction method with ethyl acetate at pH 8 and homatropine as the internal standard, the tropane alkaloids were separated. An Allure PFP propyl column (50 mm × 2.1 mm, 5 µm) separated the tropane alkaloids using an acetonitrile-buffer solution (20 mmol/L ammonium acetate and 0.1% formic acid, pH 4) (70:30) as the mobile phase at a flow-rate of 0.2 mL/min in isocratic mode, with the LC-MS/MS in the positive ionization mode. For each compound, detection was related to two daughter ions (scopolamine: m/z 304.4 → 138.1 and 155.9; atropine: m/z 290.3 → 124.0 and 93.1; anisodamine: m/z 306.3 → 140.1 and 91.1; and homatropine: m/z 276.3 → 124.3 and 142.1). The tropane alkaloids exhibited excellent linearity in the range of 0.05–100 ng/mL in blood and 0.2–100 ng/mL in urine, with a limit of detection range from 0.02 to 0.05 ng/mL for biological materials. The extraction recoveries of atropine, scopolamine, and anisodamine were more than 53% in the blood and urine; the interday and intraday RSDs were less than 10%; the within-day and between-day accuracy were between ?9.8% and +8.8%. The present method is simple and rapid, as shown by its application to a clinical case. This method is useful for routine analysis of tropane alkaloids in cases of suspected tropane alkaloid poisoning.     

Development and Validation of a Bioanalytical Method for 3′- and 6′-Sialyllactose in Minipig Liver and Kidney Using Liquid Chromatography-Tandem Mass Spectrometry and Its Application to Analysis of Tissue Distribution

Breast milk contains human milk oligosaccharides (HMOs), including sialyllactose (SL). SL is composed of sialic acid and lactose, and is divided into 3′-SL and 6′-SL according to the binding position. SL has immunoprotective effects against bacteria and viruses, and acts as a probiotic in the gastrointestinal tract. In this study, we developed a bioanalytical method for simultaneous analysis of 3′-SL and 6′-SL in liver and kidney tissues of Yucatan minipigs using liquid chromatography–tandem mass spectrometry (LC-MS/MS) under conditions optimized in our previous study. LC-MS/MS was performed using a hydrophilic interaction liquid chromatography (HILIC) column (50 mm × 2.1 mm, 3 μm) with a mobile phase consisting of 10 mM ammonium acetate in water (pH 4.5) and acetonitrile with gradient elution at a flow rate of 0.3 mL/min. A surrogate matrix method using water was applied for analysis of endogenous SL. The developed method was validated with regard to selectivity, linearity, precision, accuracy, the matrix effect, recovery, parallelism, dilution integrity, carryover, and stability according to the US Food and Drug Administration guidelines. We performed a tissue distribution study of minipigs, and analyzed liver and kidney tissues using the developed method to determine the tissue distribution of 3′-SL and 6′-SL. The tissue concentrations of 3′-SL and 6′-SL were readily measurable, suggesting that the method would be useful for evaluating the tissue distributions of these compounds in minipigs.     

Development and Validation of a Micellar Liquid Chromatographic Method with UV Detection for Determination of Azithromycin in Tablets and Capsules

A rapid and simple micellar liquid chromatographic method that does not require use of specific chromatographic columns has been developed and validated for azithromycin determination. The method uses a Hypersil C₁₈ column at 60 °C, 1-butanol–pH 6.86 phosphate buffer solution–water, 15:25:60 (v/v), containing 0.10 M sodium dodecyl sulfate, as mobile phase, and UV-detection at 215 nm. Different characteristics of the method were validated satisfactorily. The specificity, accuracy, linearity, precision (repeatability), and robustness of the method were demonstrated. The method proved suitable for determination of the azithromycin content of capsules and uncoated tablets.Revised: 5 February and 11 March 2004     

Development of an Accurate Mass Retention Time Database for Untargeted Metabolomic Analysis and Its Application to Plasma and Urine Pediatric Samples

Liquid-chromatography coupled to high resolution mass spectrometry (LC-HRMS) is currently the method of choice for untargeted metabolomic analysis. The availability of established protocols to achieve a high confidence identification of metabolites is crucial. The aim of this work is to describe the workflow that we have applied to build an Accurate Mass Retention Time (AMRT) database using a commercial metabolite library of standards. LC-HRMS analysis was carried out using a Vanquish Horizon UHPLC system coupled to a Q-Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Milan, Italy). The fragmentation spectra, obtained with 12 collision energies, were acquired for each metabolite, in both polarities, through flow injection analysis. Several chromatographic conditions were tested to obtain a protocol that yielded stable retention times. The adopted chromatographic protocol included a gradient separation using a reversed phase (Waters Acquity BEH C18) and a HILIC (Waters Acquity BEH Amide) column. An AMRT database of 518 compounds was obtained and tested on real plasma and urine samples analyzed in data-dependent acquisition mode. Our AMRT library allowed a level 1 identification, according to the Metabolomics Standards Initiative, of 132 and 124 metabolites in human pediatric plasma and urine samples, respectively. This library represents a starting point for future metabolomic studies in pediatric settings.     

Development and Validation of the UPLC-DAD Methodology for the Detection of Triterpenoids and Phytosterols in Fruit Samples of Vaccinium macrocarpon Aiton and Vaccinium oxycoccos L.

Cranberries are used in the production of medicinal preparations and food supplements, which highlights the importance of triterpene compounds determination in cranberry fruit raw material. The aim of our study was to develop and validate for routine testing suitable UPLC-DAD methodology for the evaluation of triterpene acids, neutral triterpenoids, phytosterols, and squalene content in cranberry samples. The developed and optimized UPLC-DAD methodology was validated according to the guidelines of the International Council for Harmonization (ICH), evaluating the following parameters: range, specificity, linearity (R² > 0.999), precision, LOD (0.27–1.86 µg/mL), LOQ (0.90–6.18 µg/mL), and recovery (80–110%). The developed and validated technique was used for the evaluation of triterpenic compounds in samples of Vaccinium macrocarpon and Vaccinium oxycoccos fruits, and their peels, pulp and seeds. The studied chromatogram profiles of Vaccinium macrocarpon and Vaccinium oxycoccos were identical but differed in the areas of the analytical peaks. Ursolic acid was the dominant compound in fruit samples of Vaccinium macrocarpon and Vaccinium oxycoccos. The highest amounts of triterpenic compounds were detected in the cranberry peels samples. The developed method for the detection of triterpene compounds can be applied in further studies for routine testing on the qualitative and quantitative composition of fruit samples of Vaccinium macrocarpon and Vaccinium oxycoccos species and cultivars.     

Development and validation of an RP‐HPLC method for the quantitation of Orteronel (TAK‐700), a CYP17A1 enzyme inhibitor,in rat plasma and its application to a pharmacokinetic study

A novel, simple, specific, sensitive and reproducible high‐performance liquid chromatography assay method has been developed and validated for the estimation of Orteronel in rat plasma. The bioanalytical procedure involves extraction of Orteronel and phenacetin (internal standard) from rat plasma with a simple liquid–liquid extraction process. The chromatographic analysis was performed on a Waters Alliance system using a gradient mobile phase conditions at a flow rate of 1 mL/min and a C₁₈ column maintained at ambient room temperature. The eluate was monitored using a photodiode array detector set at 242. Orteronel and internal standard eluted at 4.8 and 6.2 min, respectively and the total run time was 9 min. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 100–3149 ng/mL (r² = 0.995). The intra‐ and inter‐day precisions were in the ranges of 0.31–7.87 and 3.97–6.35, respectively, in rat plasma. The validated HPLC method was successfully applied to a pharmacokinetic study of Orteronel in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Validated Method for the Simultaneous Determination of Lisinopril,Pravastatin, Atorvastatin and Rosuvastatin in API,Formulations and Human Serum by RP‐HPLC

Lisinopril is found to be useful in hypertension and statins as cholesterol lowering drug. Present work was designed for the simultaneous determination of lisinopril in presence of pravastatin, atorvastatin, and rosuvastatin using RP‐HPLC method. A Purospher star C18 (5 μm, 25×0.46 cm) column was used with mobile phase consisting of acetonitrile:water (60:40 V/V, pH 3.0) with flow rate of 1.0 mL·min^?1 and the quantitative evaluation was performed at 225 nm. The retention time of lisinopril was 2.0 min and for pravastatin, rosuvastatin and atorvastatin was found to be 3.1, 4.5 and 8.3 min respectively. Suitability of this method for the quantitative determination of the drugs was proved by validation in accordance with the requirements laid down by International Conference on Harmonization (ICH) guidelines. Application of the suggested procedures were successfully applied to the determination of these compounds in active pharmaceutical ingredient and in pharmaceutical preparations, with high percentage of recovery, good accuracy and precision.     

A Simple,Cost‐effective,and Environmentally Friendly Method for Determination of Ciprofloxacin in Drugs and Urine Samples Based on Electrogenerated Chemiluminescence

Ciprofloxacin is an antibiotic that belongs to the class of drugs known as quinolones and it is frequently used to treat a variety of bacterial infections. The present work aims the development of a simple, cost‐effective, and environmentally friendly method for the determination of ciprofloxacin in drugs and artificial urine samples due to the high importance of this antibiotic for the human health. The proposed method is based on the electrogenerated chemiluminescence (ECL) resulting from the reaction between the ciprofloxacin and the tris(2,2′‐bipyridyl)ruthenium(II) complex. This method exploits a screen‐printed carbon electrode positioned in an ECL cell with capacity to 50 μL of electrolytic solution. The ECL intensity was monitored with the aid of a photodiode. The ECL signal was simultaneously registered to the voltammetric measurements. Under optimized experimental conditions, the ECL method presented a linear response range for ciprofloxacin between 0.5 and 500 μmol L^?1 (or 0.0005 and 0.5 mmol L^?1). The proposed method presented a detection limit of 0.5 μmol L^?1 and it was successfully applied for the ciprofloxacin determination in drugs and artificial urine samples, with good accuracy and precision.     

Development and validation of a fast isocratic liquid chromatography method for the simultaneous determination of norfloxacin,lomefloxacin and ciprofloxacin in human plasma

A simple and fast liquid chromatographic method coupled with fluorescence detection (LC‐FD) is reported, for the first time, for the simultaneous quantification of norfloxacin (NOR), ciprofloxacin (CIP) and lomefloxacin (LOM) in human plasma, using levofloxacin as internal standard (IS). Sample preparation consists of a single‐step precipitation of plasma proteins followed by vortex‐mixing and centrifugation. Chromatographic separation was achieved within 7 min on a reversed‐phase C₁₈ column with a mobile phase consisting of 0.1% aqueous formic acid (pH = 3.0, triethylamine)–methanol (82:18, v/v) pumped isocratically at 1.2 mL/min. The detector was set at excitation/emission wavelengths of 278/450 nm. Calibration curves were linear (r² ≥ 0.994) in the range of 0.02–5.0 µg/mL, and the limit of quantification was established at 0.02 µg/mL for all analytes (NOR, CIP and LOM). The overall precision did not exceed 8.19% and accuracy was within ±10.91%. NOR, CIP and LOM were extracted from human plasma with an overall mean recovery ranged from 90.1 to 111.5%. No interferences were observed at the retention times of the analytes and IS. This novel LC‐FD method enables the reliable determination of NOR, CIP and LOM in a single chromatographic run, which may be suitable to support human pharmacokinetic‐based studies with those antimicrobial agents. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and Validation of a Method for Determining the Quercetin-3-O-glucuronide and Ellagic Acid Content of Common Evening Primrose (Oenothera biennis) by HPLC-UVD

Toward the standardization of common evening primrose (Oenothera biennis) sprout extract (OBS-E), we aimed to obtain indicator compounds and use a validated method. HPLC-UVD allowed simultaneous quantification of the indicator compounds quercetin-3-O-glucuronide and ellagic acid. The method was validated in terms of specificity, linearity, precision, accuracy, and limit of detection/limit of quantification (LOD/LOQ). High specificity and linearity was demonstrated, with correlation coefficients of 1.0000 for quercetin-3-O-glucuronide and 0.9998 for ellagic acid. The LOD/LOQ values were 0.486/1.472 μg/mL for quercetin-3-O-glucuronide and 1.003/3.039 μg/mL for ellagic acid. Intra-day and inter-day variability tests produced relative standard deviation for each compound of <2%, a generally accepted precision criterion. High recovery rate were also obtained, indicating accuracy validation. The OBS-E prepared using various concentrations of ethanol were then analyzed. The 50% ethanol extract had highest content of quercetin-3-O-glucuronide, whereas the 70% ethanol extract possessed the lowest. However, the ellagic acid content was highest in the 70% ethanol extract and lowest in the 90% ethanol extract. Thus, quercetin-3-O-glucuronide and ellagic acid can be used industrially as indicator compounds for O. biennis sprout products, and our validated method can be used to establish indicator compounds for other natural products.     

Lab-in-Syringe,a Useful Technique for the Analysis and Detection of Pollutants of Emerging Concern in Environmental and Food Samples

Lab-in-syringe is a new approach for the integration of various analytical extraction steps inside a syringe. Fully automated dispersive liquid–liquid microextraction is carried out in-syringe using a very simple instrumental setup. Dispersion is achieved by aspiration of the organic phase and then the watery phase into the syringe as rapidly as possible. After aggregation of the solvent droplets, the organic phase is pushed towards the detector allowing a highly sensitive spectrophotometric or fluorimetric detection. This technique is very useful not only for the preconcentration of analyte, but also for the elimination of their interferences. In this work, its application is described using solvents that are lighter and denser than water. The magnetically assisted variant and its coupling to different instruments has been also described with the aim of increasing the resolution of complex samples, especially useful for the determination of emerging contaminants.     

Development of an LC-MS/MS Method for ARV-110, a PROTAC Molecule,and Applications to Pharmacokinetic Studies

ARV-110, a novel proteolysis-targeting chimera (PROTAC), has been reported to show satisfactory safety and tolerability for prostate cancer therapy in phase I clinical trials. However, there is a lack of bioanalytical assays for ARV-110 determination in biological samples. In this study, we developed and validated an LC-MS/MS method for the quantitation of ARV-110 in rat and mouse plasma and applied it to pharmacokinetic studies. ARV-110 and pomalidomide (internal standard) were extracted from the plasma samples using the protein precipitation method. Sample separation was performed using a C18 column and a mobile phase of 0.1% formic acid in distilled water–0.1% formic acid in acetonitrile (30:70, v/v). Multiple reaction monitoring was used to quantify ARV-110 and pomalidomide with ion transitions at m/z 813.4 → 452.2 and 273.8 → 201.0, respectively. The developed method showed good linearity in the concentration range of 2–3000 ng/mL with acceptable accuracy, precision, matrix effect, process efficiency, and recovery. ARV-110 was stable in rat and mouse plasma under long-term storage, three freeze-thaw cycles, and in an autosampler, but unstable at room temperature and 37 °C. Furthermore, the elimination of ARV-110 via phase 1 metabolism in rat, mouse, and human hepatic microsomes was shown to be unlikely. Application of the developed method to pharmacokinetic studies revealed that the oral bioavailability of ARV-110 in rats and mice was moderate (23.83% and 37.89%, respectively). These pharmacokinetic findings are beneficial for future preclinical and clinical studies of ARV-110 and/or other PROTACs.     

Development of a liquid chromatography assay for the determination of opicapone and BIA 9–1079 in rat matrices

Opicapone is a novel potent, reversible and purely peripheral third generation catechol‐O‐methyltransferase inhibitor, currently under clinical trials as an adjunct to levodopa therapy for Parkinson's disease. To support additional nonclinical pharmacokinetic studies, a novel high‐performance liquid chromatographic method coupled to a diode array detector (HPLC‐DAD) to quantify opicapone and its active metabolite (BIA 9–1079) in rat plasma and tissues (liver and kidney) is herein reported. The analytes were extracted from rat samples through a deproteinization followed by liquid‐liquid extraction. Chromatographic separation was achieved in less than 10 min on a reversed‐phase C₁₈ column, applying a gradient elution program with 0.05 M monosodium phosphate solution (pH 2.45 ± 0.05) and acetonitrile. Calibration curves were linear (r² ≥ 0.994) within the ranges of 0.04‐6.0 µg/mL for both analytes in plasma, 0.04‐4.0 µg/mL for opicapone in liver and kidney homogenates, and 0.07‐4.0 µg/mL and 0.06‐4.0 µg/mL for BIA 9–1079 in liver and kidney homogenates, respectively. The overall intra‐ and inter‐day accuracy ranged from ?12.68% to 7.70% and the imprecision values did not exceed 11.95%. This new HPLC‐DAD assay was also successfully applied to quantify opicapone and BIA 9–1079 in a preliminary pharmacokinetic study. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of a RP‐HPLC method for the quantitation of tofacitinib in rat plasma and its application to a pharmacokinetic study

A novel, simple, specific, sensitive and reproducible high‐performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of tofacitinib in rat plasma. The bioanalytical procedure involves extraction of tofacitinib and itraconazole (internal standard, IS) from rat plasma with a simple liquid–liquid extraction process. The chromatographic analysis was performed on a Waters Alliance system using a gradient mobile phase conditions at a flow rate of 1.0 mL/min and C₁₈ column maintained at 40 ± 1 °C. The eluate was monitored using an UV detector set at 287 nm. Tofacitinib and IS eluted at 6.5 and 8.3 min, respectively and the total run time was 10 min. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 182–5035 ng/mL (r² = 0.995). The intra‐ and inter‐day precisions were in the range of 1.41–11.2 and 3.66–8.81%, respectively, in rat plasma. The validated HPLC method was successfully applied to a pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and Validation of a Bioanalytical LC-MS/MS Method for Simultaneous Determination of Sirolimus in Porcine Whole Blood and Lung Tissue and Pharmacokinetic Application with Coronary Stents

Sirolimus is a hydrophobic macrolide compound that has been used for long-term immunosuppressive therapy, prevention of restenosis, and treatment of lymphangioleiomyomatosis. In this study, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for the simultaneous determination of sirolimus in both porcine whole blood and lung tissue. Blood and lung tissue homogenates were deproteinized with acetonitrile and injected into the LC-MS/MS system for analysis using the positive electrospray ionization mode. The drug was separated on a C18 reversed phase column with a gradient mobile phase (ammonium formate buffer (5 mM) with 0.1% formic acid and acetonitrile) at 0.2 mL/min. The selected reaction monitoring transitions of m/z 931.5 → 864.4 and m/z 809.5 → 756.5 were applied for sirolimus and ascomycin (the internal standard, IS), respectively. The method was selective and linear over a concentration range of 0.5–50 ng/mL. The method was validated for sensitivity, accuracy, precision, extraction recovery, matrix effect, and stability in porcine whole blood and lung tissue homogenates, and all values were within acceptable ranges. The method was applied to a pharmacokinetic study to quantitate sirolimus levels in porcine blood and its distribution in lung tissue following the application of stents in the porcine coronary arteries. It enabled the quantification of sirolimus concentration until 2 and 14 days in blood and in lung tissue, respectively. This method would be appropriate for both routine porcine pharmacokinetic and bio-distribution studies of sirolimus formulations.     

Development and validation of an LC‐ESI‐MS/MS method for the quantification of D‐84, reboxetine and citalopram for their use in MS Binding Assays addressing the monoamine transporters hDAT,hSERT and hNET

MS Binding Assays represent a label‐free alternative to radioligand binding assays. In this study, we present an LC‐ESI‐MS/MS method for the quantification of (R,R)‐4‐(2‐benzhydryloxyethyl)‐1‐(4‐fluorobenzyl)piperidin‐3‐ol [(R,R)‐D‐84, (R,R)‐ 1 ], (S,S)‐reboxetine [(S,S)‐ 2 ], and (S)‐citalopram [(S)‐ 3 ] employed as highly selective nonlabeled reporter ligands in MS Binding Assays addressing the dopamine [DAT, (R,R)‐D‐84], norepinephrine [NET, (S,S)‐reboxetine] and serotonin transporter [SERT, (S)‐citalopram], respectively. The developed LC‐ESI‐MS/MS method uses a pentafluorphenyl stationary phase in combination with a mobile phase composed of acetonitrile and ammonium formate buffer for chromatography and a triple quadrupole mass spectrometer in the multiple reaction monitoring mode for mass spectrometric detection. Quantification is based on deuterated derivatives of all three analytes serving as internal standards. The established LC‐ESI‐MS/MS method enables fast, robust, selective and highly sensitive quantification of all three reporter ligands in a single chromatographic run. The method was validated according to the Center for Drug Evaluation and Research (CDER) guideline for bioanalytical method validation regarding selectivity, accuracy, precision, calibration curve and sensitivity. Finally, filtration‐based MS Binding Assays were performed for all three monoamine transporters based on this LC‐ESI‐MS/MS quantification method as read out. The affinities determined in saturation experiments for (R,R)‐D‐84 toward hDAT, for (S,S)‐reboxetine toward hNET, and for (S)‐citalopram toward hSERT, respectively, were in good accordance with results from literature, clearly demonstrating that the established MS Binding Assays have the potential to be an efficient alternative to radioligand binding assays widely used for this purpose so far.     

Development and validation of a rapid reversed-phase HPLC method for the determination of insulin from nanoparticulate systems

A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of insulin in nanoparticulate dosage forms. Its application for the development and characterization of insulin-loaded nanoparticulates composed of polyelectrolytes has also been carried out. A reversed-phase (RP) C18 column and gradient elution with a mobile phase composed of acetonitrile (ACN) and 0.1% aqueous trifluoroacetic acid (TFA) solution at a flow rate of 1 mL/min was used. Protein identification was made by UV detection at 214 nm. The gradient changed from 30:70 (ACN:TFA, v/v) to 40:60 (v/v) in 5 min followed by isocratic elution at 40:60 (v/v) for a further five minutes. The method was linear in the range of 1-100 microg/mL (R2 = 0.9996), specific with a good inter-day and intra-day precision based on relative standard deviation values (less than 3.80%). The recovery was between 98.86 and 100.88% and the detection and quantitation limits were 0.24 and 0.72 microg/mL, respectively. The method was further tested for the determination of the association efficiency of insulin to nanoparticulate carriers composed of alginate and chitosan, as well as its loading capacity for this protein. Encapsulant release under simulated gastrointestinal fluids was evaluated. The method can be used for development and characterization of insulin-loaded nanoparticles made from cross-linked chitosan-alginate.     

Development,Validation and Application of an ICP-SFMS Method for the Determination of Metals in Protein Powder Samples,Sourced in Ireland,with Risk Assessment for Irish Consumers

A method has been developed, optimised and validated to analyse protein powder supplements on an inductively coupled plasma-sector field mass spectrometer (ICP-SFMS), with reference to ICH Guideline Q2 Validation of Analytical Procedures: Text and Methodology. This method was used in the assessment of twenty-one (n = 21) elements (Al, Au, Ba, Be, Bi, Cd, Co, Cr, Cu, Fe, Hg, Li, Mg, Mn, Mo, Pb, Pt, Sn, Ti, Tl, V) to evaluate the safety of thirty-six (n = 36) protein powder samples that were commercially available in the Irish marketplace in 2016/2017. Using the determined concentrations of elements in samples (µg·kg⁻¹), a human health risk assessment was carried out to evaluate the potential carcinogenic and other risks to consumers of these products. While the concentrations of potentially toxic elements were found to be at acceptable levels, the results suggest that excessive and prolonged use of some of these products may place consumers at a slightly elevated risk for developing cancer or other negative health impacts throughout their lifetimes. Thus, the excessive use of these products is to be cautioned, and consumers are encouraged to follow manufacturer serving recommendations.     

Development and validation of an LC-MS/MS-ESI method for the determination of lorglumide, a CCK-1 antagonist in mouse plasma: application to a pharmacokinetic study

A highly sensitive, rapid assay method was developed and validated for the estimation of lorglumide in mouse plasma using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in positive‐ion mode. The assay procedure involves extraction of lorglumide and phenacetin (internal standard, IS) from mouse plasma with simple protein precipitation. Chromatographic separation was achieved using an isocratic mobile (0.2% formic acid solution–acetonitrile, 20:80, v/v) at a flow‐rate of 0.5 mL/min on an Atlantis dC₁₈ column maintained at 40 °C with a total run time of 4.0 min. The MS/MS ion transitions monitored were 459.2 → 158.4 for lorglumide and 180.1 → 110.1 for IS. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.42 ng/mL and the linearity range extended from 0.42 to 500 ng/mL. The intra‐ and inter‐day precisions were in the ranges of 1.47–10.9 and 3.56–7.53, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Development of a Fast and Robust UHPLC Method for Apixaban In-Process Control Analysis

In-process control (IPC) is an important task during chemical syntheses in pharmaceutical industry. Despite the fact that each chemical reaction is unique, the most common analytical technique used for IPC analysis is high performance liquid chromatography (HPLC). Today, the so-called “Quality by Design” (QbD) principle is often being applied rather than “Trial and Error” approach for HPLC method development. The QbD approach requires only for a very few experimental measurements to find the appropriate stationary phase and optimal chromatographic conditions such as the composition of mobile phase, gradient steepness or time (tG), temperature (T), and mobile phase pH. In this study, the applicability of a multifactorial liquid chromatographic optimization software was studied in an extended knowledge space. Using state-of-the-art ultra-high performance liquid chromatography (UHPLC), the analysis time can significantly be shortened. By using UHPLC, it is possible to analyse the composition of the reaction mixture within few minutes. In this work, a mixture of route of synthesis of apixaban was analysed on short narrow bore column (50 × 2.1 mm, packed with sub-2 µm particles) resulting in short analysis time. The aim of the study was to cover a relatively narrow range of method parameters (tG, T, pH) in order to find a robust working point (zone). The results of the virtual (modeled) robustness testing were systematically compared to experimental measurements and Design of Experiments (DoE) based predictions.