Analytical Column Research Articles

Stability Indicating Reverse Phase Ultra Performance Liquid Chromatography Method Development and Validation for Amiloride and Hydrochlorothiazide Tablet in the Presence of Degradation Products and Application to Green Analytical Principles

Amiloride (AML) and hydrochlorothiazide (HCTZ) combination is used alone or with other medicines to treat high blood pressure (hypertension). The present article provides the development and validation of an reverse phase ultra performance liquid chromatography (RP-UPLC) approach for the quantification of AML, HCTZ, and related impurities in pharmaceutical dosage forms. The analytical column used for the separation of impurities is the Waters X bridge C18 3.5 μm (50 mm X 4.6 mm). Used 0.1% formic acid and ethanol as MP in an isocratic mode with 0.3 mL/min as flow rate and 3 μL as injection volume, at 254 nm as detection in UV and 12 minutes as total run time. The samples were prepared specifically to undergo forced degradation, which involved subjecting them to hydrolysis, oxidation, thermal, and photolytic conditions. The technique underwent validation by the principles set forth by the ICH Q2 guidelines. The validation confirmed that the method possesses specificity, linearity, ruggedness, robustness and accuracy. The methodology employed exhibited a linear relationship extending from 10 to 150% for all impurities. The recovery analysis was conducted throughout a range of concentrations, starting from 10 to 150% concentration. The average recovery value was determined to be within acceptable limits. The evidence of degradation and the findings of the verified study suggest that the nature of the subject under investigation is stable. Hence, this approach might be employed within the domains of pharmaceutical research and development and quality control departments. Green analytical chemistry tools are used to assess the method’s greenness and calculated using GAPI, AGREE and ecological-scale and found excellent green of >75%.

Method Development and Validation of Indigenously Isolated Caspofungin Acetate and its related Substances using RP-HPLC Method

Caspofungin is prepared from a precursor PneumocandinB0 derived from a fungus Glarea lozoyensis. Prepared crude caspofungin acetate has many impurities, namely, A, B, D, E and PneumocandinB0, and these impurities vary slightly in chemical or structural composition and need to be eliminated by purification. The present study involves the preparation of caspofungin acetate from PneumocandinB0, followed by purification and method development for detecting caspofungin and its related substances.”We used a high-tech liquid chromatograph system, which included components like the Agilent 1260 infinity quaternary pump module, a sophisticated MWD detector, and the Empower 3 data handling system. To separate and analyze substances, we employed an analytical column made of stainless steel. This column was 150 mm long and had an internal diameter of 3.0 mm. It was filled with octadecyl silane chemically bonded to porous silica particles, each with a tiny diameter of 3 micrometers. The test method underwent validation to ensure it could accurately identify specific elements, detect minimal quantities, establish the lowest measurable amounts, maintain linearity, demonstrate precision, achieve accuracy, and suitably handle sample solutions and the system.” and the developed test method is suitable and can be introduced into the routine use for the detection of caspofungin acetate and its related substances.

Development and validation of a liquid chromatography tandem mass spectrometry assay for the analysis of bedaquiline and M2 in breast milk

ObjectiveTo develop and validate an assay for the analysis of bedaquiline and its M2 metabolite in human breast milk. MethodsThe analytes were extracted using solid phase extraction following protein precipitation. Quantification was performed with liquid chromatography coupled with tandem mass spectrometry. Chromatographic separation was achieved using gradient chromatography on a Poroshell 120 SB-C18 analytical column at 40 °C, with a flow rate of 350 µL/minute and a total run time of eight minutes. An AB Sciex 3000 mass spectrometer with electrospray ionization in the positive mode was used for detection, employing multiple reaction monitoring scan mode. Bedaquiline-d6 and M2-d3-13C were used as internal standards. ResultsCalibrations curves for bedaquiline and M2 exhibited quadratic (weighted 1/x concentration) regressions over the respective concentration ranges of 0.0780 to 5.00 µg/mL and 0.0312 to 2.00 µg/mL. Inter- and intra-day validation accuracies ranged between 96.7 % and 103.5 % for bedaquiline, and 104.2 % to 106.5 % for M2, with a coefficient of variation below 9.2 % for both compounds. ConclusionThe developed assay demonstrated selectivity and robustness, enabling differentiation between bedaquiline and M2 within the context of endogenous compounds from six separate lots of breast milk samples. Successful application was observed in the analysis of breast milk samples sourced from patients treated for multidrug-resistant tuberculosis within a clinical study setting.

Development and validation of a sensitive UPLC-MS/MS analytical method for GFH009 in rat plasma and its application to toxicokinetics studies.

GFH009 is a potent, highly selective, small molecule that targets and inhibits the activity of the CDK9/cyclin T1 regulatory complex of P-TEFb. This study aimed to develop and validate a highly selective and sensitive ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for precise quantification of GFH009 in rat plasma. This method was subsequently employed for conducting toxicokinetic studies of GFH009 in rats. Plasma was prepared using a simple protein precipitation method by acetonitrile. Chromatographic separation of the analytes was achieved on a BEH C18 analytical column with a rapid 3.0 min run time and a flow rate of 0.5ml/min. The calibration curves for plasma samples exhibited excellent linearity over a wide concentration range of 1.0-1,000 ng/ml for GFH009. Intra- and inter-day accuracies were within 92.7-105.7%, and precisions were no more than 6.7%. Furthermore, the analyte demonstrated stability under four different storage conditions, with variations of <15.0%. This study pioneers a methodological innovation by introducing a highly reliable, specific and sensitive analytical method for GFH009 in rat plasma. The successful application of this method in toxicokinetic studies further underscores its significance, offering valuable insights for the methodology of clinical pharmacokinetic research.

New validated liquid chromatography‐tandem mass spectrometry method for the determination of Dacomitinib in human plasma and its application to a pharmacokinetic study

AbstractDacomitinib, a quinazoline compound, exhibits antineoplastic activity against brain metastasis activities in non‐small cell lung cancer and the central nervous system. In this study, the liquid–liquid extraction method with high‐performance liquid chromatography and tandem mass spectrometry detection method was established and validated for the determination of Dacomitinib in human plasma. Plasma samples were prepared and chromatographic separation was achieved on analytical column Discovery C18 (10 cm × 4.6 mm, 5 μm) with gradient elutes at a flow rate of 0.8 mL/min, using a mobile phase consisting of acetonitrile and ammonium formate. Dacomitinib and dacomitinib D10 (internal standard) were detected by multiple reactions. The method was fully validated according to the United States Food and Drug Administration guidelines. The calibration curve was linear with an excellent correlation coefficient (r2 ˂ 0.99). The method validation steps such as carry‐over, matrix effect, extraction recovery, dilution effect, intra‐inter accuracy, and precision were found within acceptable limits. The method was then applied to a pharmacokinetic study in human plasma. After oral administration, the plasma concentration in different volunteers reached 0.5–250.01 ng/mL. The result established can be applied to the estimation of drugs in human plasma.

Fabrication of a biochar-doped monolithic adsorbent and its application for the extraction and determination of coumarins from Angelicae Pubescentis Radix

A monolithic adsorbent was designed aiming to the structure of osthole and columbianadin, and fabricated using diallyl phthalate as the monomer and ethylene dimethacrylate as the crosslinker with the addition of bamboo biochar, via polymerization reaction in a stainless-steel tube. The prepared composite adsorbent packed in the tube was used as a solid-phase extraction column for the extraction and determination of two coumarins (osthole and columbianadin) in Angelicae Pubescentis Radix, combing with a C18 analytical column through an HPLC instrument, which show excellent matrix-removal ability and good selectivity to osthole and columbianadin. Furthermore, the present adsorbent shows good applicability, which was used for the extraction of osthole from Duhuo Jisheng Pill. Compared to the commercial C18 and phenyl adsorbent, the present adsorbent own better selectivity and higher resolution. These results attributed to the enhanced specific surface area (141 m2/g) and enriched interaction sites of the resulting composite adsorbent, due to the doping of bamboo biochar, which can produce hydrogen bond, dipole-dipole, π-π and hydrophobic force interactions with the osthole and columbianadin. The methodology validation indicated that the present method showed good precision and good accuracy, and the composite adsorbent showed good preparative repeatability, which can be reused for no less than 100 times with the relative standard deviation ≤4.6 % (n = 100). The present work provided a simple and efficient method for the extraction and determination osthole and columbianadin from Angelicae Pubescentis Radix.

A novel eco-friendly HPLC method with dual detection modes for versatile quantification of dutasteride and silodosin in pharmaceutical formulation, dissolution testing and spiked human plasma

With the increased production of new fixed combinations and cheaper generic drugs, the need for a single, sensitive and reliable analytical method is hugely evoked to cover the versatile and prerequisite tests of in-vitro and in-vivo bioequivalence studies. A new fixed dose combination, containing dutasteride (DUT) and silodosin (SLD), has been recently launched for relieving moderate and severe symptoms of benign prostatic hyperplasia. Consequently, this concurrent study was dedicated to present, for the first time, eco-friendly high-performance liquid chromatographic (HPLC) methods coupled with dual-mode detection, for the simultaneous quantification of both drugs in versatile real samples. The optimized chromatographic separation was achieved on a Waters XBridge® (150×4.6 mm, 5 μm) C18 analytical column with a mobile phase composed of methanol & phosphate buffer pH 6.0 at ratio (77: 23, v/v) and pumped at 1.0 mL/min flow rate. In the first HPLC method, drug quantification was performed at 245 nm achieving linearity range of 3–120 µg/mL, while the second one made good use of the unique features of both drugs in possessing native fluorescence for more sensitive linearity ranges (50–1000 ng/mL for DUT & 80–2500 ng/mL for SLD). Both methods were validated and successfully applied for assay dosage forms as well as testing content uniformity. In addition, the capability of the proposed HPLC-fluorometric method in detecting drugs in nano-gram scale paved the way not only for studying their dissolution behavior in various dissolution media but also analyzing spiked human plasma samples extracted by liquid-liquid extraction. Finally, sustainability of the proposed methods was clearly declared, in comparison to the official ones, via environmental, health and safety (EHS) tool and analytical greenness (AGREE) software.

Determination of quinclorac and quinclorac methyl ester in honey by online SPE-UPLC-MS/MS

A method employing online solid phase extraction (SPE) coupled to UPLC-MS/MS was developed for the determination of residues of the acid herbicide quinclorac plus its transformation product, quinclorac methyl ester, in honey. The analytical method involved dissolving the honey in a mixture of methanol:water followed by direct injection into a two-dimensional UPLC system which is used to perform an automated SPE cleanup on a reusable phenyl cartridge prior to the target analytes being transferred onto an analytical UPLC column for subsequent chromatographic separation followed by MS/MS detection. The limits of quantitation for quinclorac and quinclorac methyl ester in honey were both set at 0.5 µg kg−1 and the method detection limit was estimated to be 0.012 µg kg−1 for each compound. The working analytical range (0.5–100 µg kg−1) was validated by analysing a series of spiked replicate honey samples. The method was applied to the analysis of various honeys obtained from numerous different commercial sources. Quinclorac was detected in 9 out of 30 samples at concentrations ranging from 0.6 to 31.5 µg kg−1. Quinclorac methyl ester, which is estimated to be significantly more toxic than the parent herbicide itself, was not detected in any honey sample.

Multi-Active Method (MAM) for the Analysis of Agriculture Product Technical Ingredients and Formulated Products

In regulatory laboratories, where potentially hundreds of different types of sample and active ingredients can be evaluated every year, a unique set of conditions provided by agricultural product manufacturers in their regulatory methods are faced with the decision to purchase a new and different analytical column or not follow the enforcement method provided by the manufacturer. Because of this, a multi-analyte method has been developed at the Irish Department of Agriculture, Food and The Marine, contributed to by the regulatory laboratories in Belgium and the Czech Republic, analyzing more than 70 active ingredients using high performance liquid chromatography (HPLC) and more than 35 active ingredients by ultrahigh-pressure liquid chromatography (UHPLC).. The method has been designed for use by quality control laboratories and is suitable for determining a range of active substances in a wider range of formulated products as well as the technical AI itself. The method has been validated for linearity, precision, accuracy, and specificity for seven technical active ingredients as defined by FMC Corporation. The method and results are described in this article.

Stability Indicating HPLC Method for In-vitro Determination of Pantoprazole Sodium and its Degradation Products in Simulated Gastric and Intestinal Fluids

Background: A high-performance liquid chromatography (HPLC) method was developed for the determination of Pantoprazole Sodium (PPZ) in the presence of its degradation products. The degradation of PPZ was studied in simulated intestinal fluid (SIF) and simulated gastric fluids (SGF) in various temperature conditions. Aim: This study aimed to establish a simple, sensitive, and rapid RP HPLC method for in-vitro determination of Pantoprazole Sodium and its degradation products in simulated gastric and intestinal fluids. Objective: Pantoprazole is acid labile drug. In order to determine pantoprazole in various oral dosage forms, the stability-indicating assay of PPZ was performed in phosphate buffer (pH 6.8) representing simulated intestinal fluid (SIF) and in 0.1 molars (M) Hydrochloric acid (HCl) as simulated gastric fluid (SGF) at two different temperature conditions, i.e., 25°C and 0°C, respectively. Method: Pantoprazole sodium was obtained from the Akums laboratory in Haridwar. The analysis was performed by high-performance liquid chromatography (HPLC), Shimadzu, equipped with two LC-10 AD VP solvent-delivery modules, a SPD-10A UV–-visible detector, and a manual injector valve with 20 μL sample loop. Phenomenex ODS analytical column (150 mm × 4.6 mm i.d., 5 μm particles) was done under reversed-phase partition chromatographic conditions. The mobile phase was phosphate buffer and acetonitrile (ACN) of pH 7.4, respectively, optimized in a 70:30 (v/v) ratio followed by filtration through a 0.45 μm membrane filter and degassed by ultrasonicator before use. The mobile phase was delivered at the flow rate of 2 mL/min. The various parameters, such as linearity, accuracy and precision of the analytical method, were studied. Result: The standard curve of PPZ was linear (R2&gt;0.99) over the concentration range of 5-30 μg/mL, and the relative standard deviation (RSD) values for intra-day and inter-day variations were in the range of 1.0-1.8%. The range of RSD was within ±2. Conclusion: The stability of PPZ in aqueous solution was pH dependent. The rate of degradation increases with decreasing pH. The pH stability of pantoprazole was studied at the above-mentioned temperature conditions. The PPZ peaks were analyzed by comparing them with fresh samples and were stable in SIF solution after 24 hours elapsed time at pH 6.8. The obtained degraded peaks in SGF (pH 1) were successfully separated from the PPZ.

Novel, alternative analytical methodology for determination of antimicrobial chemicals in aquatic environments and public use assessment: Extraction sorbent, microbiological sensitivity, stability, and applicability

BackgroundAssessing antimicrobial chemicals from wastewater source to recipient water systems is crucial in planning effective, policy-related interventions for antimicrobial resistance (AMR) risk mitigation. However, the capability of related analytical methods for AMR assessment has not been explored previously. There is also a lack of knowledge on the effectiveness of alternative extraction sorbents with ion-exchange functions, and little information on chemical stability from sampling to analysis as well as preservative options. Hence, our study aims to address the clear need for advanced, broad-range and microbiologically-sensitive methodologies, paired with thorough stability assessments. ResultsOasis® WCX ion-exchange was for the first time employed in solid-phase extraction (SPE) for antibacterials, antifungals, antivirals and human metabolites in various water matrices. Analysis was performed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) on a biphenyl analytical column. The optimized and validated method provided satisfactory accuracy, precision, and recovery for 53 compounds via LC-MS/MS direct injection and for up to 35 compounds via SPE-LC-MS/MS. Method quantification limits (MQLs) were determined in groundwater (0.33–54 ng L−1), surface water (0.53–75 ng L−1), effluent wastewater (2.5–470 ng L−1), and influent wastewater (11–650 ng L−1). As a novel approach, MQLs were compared with minimum inhibitory concentrations, to confirm our method's microbiological sensitivity for studying AMR. Stability assessment revealed that most compounds remained stable in standard solution at −80 °C for six months, in various waters at −20 °C for eight weeks, and during 24-h sampling at 4 °C. Sodium azide was a better preservative than sodium metabisulfite. SignificanceOur study is an added value to the analytical methodology for water measurements of antimicrobial chemicals, in which it provides a novel, alternative method that is robust and overall more sensitive than others using generic Oasis® HLB sorbents and C18 analytical columns in SPE-LC-MS/MS. Also, the comprehensive data on antimicrobial stability helps reduce methodological uncertainty for future studies. Our method shows sufficient microbiologically-sensitive and thus is suitable for future (inter)national regulatory water monitoring of AMR.

On-line extraction and determination of coumarins compounds from mouse plasma based on a homemade phenyl-hybrid monolithic adsorbent

A phenyl-hybrid monolithic adsorbent was prepared using an organic monomer of ethylene glycol phenyl ether acrylate and inorganic monomers of tetramethoxysilane and vinyltrimethoxysilane, via polycondensation and polymerization in a stainless-steel column, which shows porous structure and multiple functional groups, according to the measurements of scanning electron microscopy, nitrogen adsorption-desorption method and infrared spectroscopy. The resulting hybrid phenyl-based monolith was used as a solid-phase extraction column, combining with an analytical column in conjunction with high-performance liquid chromatography system for the on-line extraction and determination of coumarins (praeruptorin A and praeruptorin B) in Peucedani Radix from mouse plasma. The homemade hybrid monolithic solid-phase extraction column exhibits good removal ability for the sample matrices, as well as unique selectivity for the two praeruptorins. Methodology validation results indicate that the present method is applicable for the on-line extraction and quantitative analysis of praeruptorin A and praeruptorin B in Peucedani Radix from mouse plasma with a limit of quantitation 0.06 μg/mL and a linear range 0.06–5 μg/mL (r＞0.999), thus indicating the present method is a promising and alternative method for the quantitative determination of similar target components with micro or trace concentration from complex extract solution and plasma.

A synergistic chemometric combination for whiteness and greenness assessed HPLC-DAD assay of aqueous extracts of ivy and thyme and potassium sorbate in a syrup formula

A stability-indicating fit-for-purpose method has been developed for co-determination of hederacoside C (HC), thymol (TL), and potassium sorbate (PS) combined in a syrup dosage form. Carvacrol (CL) was separated from thyme extract (THY) with demonstrated identity at high confidence. This method was based on a reversed-phase (RP) high performance liquid chromatographic (HPLC) separation of the cited analytes. The HPLC separation was performed on a RP Zorbax Eclipse XDB-C18 analytical column [250 × 4.6 mm, 5 µm (80 Å)] and SecurityGuard C18 guard column (4 × 3 mm, 5 µm) with gradient elution system of HPLC far UV grade acetonitrile and 20 mM aqueous potassium dihydrogen phosphate buffer adjusted to pH 2.1 as the mobile phase at a flow rate of 2.5 mL min−1 and column oven was set at 50 °C. Quantitation was achieved with the photodiode array detection (DAD) with interferent peak suppression. Target Measurement Uncertainty (TMU) was determined by misclassification rates approach employing 100,000 Monte Carlo simulations (MCS). The bias and imprecision standard deviation were combined as a total analytical error (TAE) and compared via an enhanced approach linked to product specifications, process variation, and shelf-life variability for proving a fit-for-purpose analytical procedure. The robustness of the method was checked using supersaturated design constructed from Plackett-Burman design (PB-SSD) which was statistically analysed via the very recent approach of autovalidation using self-validated ensemble modelling (SVEM). The calibration graphs for each analyte were rectilinear in the range of 42.12–560.00, 3.37–47.15 and 0.51–7.07 µg mL−1 for PS, HC, and TL, respectively. The proposed HPLC-DAD method was successfully applied for the determination of the investigated analytes in a syrup formula. Moreover, the whiteness and greenness of the procedure were assessed via the most recently cited approaches.

Hydrophilic Interaction Liquid Chromatography at Subzero Temperature for Hydrogen-Deuterium Exchange Mass Spectrometry.

Chromatographic separations at subzero temperature significantly improve the precision of back-exchange-corrected hydrogen-deuterium exchange mass spectrometry (HDX-MS) determinations. Our previously reported dual-enzyme HDX-MS analysis instrument used reversed phase liquid chromatography (RPLC) at -30 °C, but high backpressures limited flow rates and required materials and equipment rated for very high pressures. Here, we report the design and performance of a dual-enzyme HDX-MS analysis instrument comprising a RPLC trap column and a hydrophilic interaction liquid chromatography (HILIC) analytical column in a two-dimensional RPLC-HILIC configuration at subzero temperature. During operation at -30 °C, the HILIC column manifests greatly reduced backpressure, which enables faster analytical flow rates and the use of materials rated for lower maximum pressures. The average peptide eluted from a HILIC column during a 40 min gradient at -30 °C contained ≈13% more deuterium than peptides eluted from a tandem RPLC-RPLC apparatus using a conventional 8 min gradient at 0 °C. A subset of peptides eluted from the HILIC apparatus contained ≈24% more deuterium.

Fast HPLC-based affinity method to determine capsid titer and full/empty ratio of adeno-associated viral vectors

Recombinant adeno-associated viruses (rAAVs) are promising gene delivery vectors in the emerging field of invivo gene therapies. To ensure their consistent quality during manufacturing and process development, multiple analytical techniques have been proposed for the characterization and quantification of rAAV capsids. Despite their indisputable capabilities for performing this task, current analytical methods are rather time-consuming, material intensive, complicated, and costly, restricting their suitability for process development in which time and sample throughput are severe constraints. To eliminate this bottleneck, we introduce here an affinity-based high-performance liquid chromatography method that allows the determination of the capsid titer and the full/empty ratio of rAAVs within less than 5min. By packing the commercially available AAVX affinity resin into small analytical columns, the rAAV fraction of diverse serotypes can be isolated from process-related impurities and analyzed by UV and fluorescence detection. As demonstrated by both method qualification data and side-by-side comparison with AAV enzyme-linked immunosorbent assay results for rAAV8 samples as well as by experiments using additional rAAV2, rAAV8, and rAAV9 constructs, our approach showed good performance, indicating its potential as a fast, simple and efficient tool for supporting the development of rAAV gene therapies.

Development and Validation of Headspace-Gas Chromatography (HS-GC) Method for the Determination of Potential Genotoxic Impurities in Meloxicam

The development and validation of a specific Headspace-Gas Chromatography (HS-GC) method for the determination of genotoxic impurities (isopropyl chloride, propionaldehyde, 2-chloropropanol, and chloromethane) in Meloxicam. The chromatographic separation of these impurities was accomplished using a nitrogen carrier gas on a DB-624 (6% cyanopropyl phenyl + 94% dimethyl polysiloxane fused silica) (75 m x 0.53 mm, 3.0 µ) GC analytical column. The Flame Ionization Detector (FID) served as the detector with optimized temperatures set at 260°C for the detector and 240°C for the injector port. Additionally, an injection volume of 1.0 mL with a split ratio mode was employed. The performance of this method was assessed through parameters, including system suitability, specificity, linearity, range, accuracy, limit of detection (LOD), limit of quantification (LOQ) and precision. The limit of detection and limit of quantification for isopropyl chloride, propionaldehyde, 2-chloropropanol, and chloromethane were determined to be 2 and 4 ppm, respectively, and for chloromethane 0.8 and 1.6 respectively. The correlation coefficient of the linearity experiment was 0.999 for propionaldehyde, 2-chloropropanol, chloromethane and 1 for isopropyl chloride compounds. Furthermore, the accuracy assessment involved spiking the genotoxic impurities at various levels, demonstrating favourable recovery percentages well within the acceptable range of 80–120% of the limit level. These compelling results substantiate the suitability of the proposed HS-GC method for the accurate determination of isopropyl chloride, propionaldehyde, 2-chloropropanol, and chloromethane in Meloxicam.

Alleviation of the necessity for supernatant prefiltering in the protein a recovery of Monoclonal antibodies from Chinese hamster ovary cell cultures

Protein A (ProA) chromatography is a mainstay in the analytical and preparative scale isolation/purification of monoclonal antibodies (mAbs). One area of interest is continuous processing or continuous chromatography, where ProA chromatography is used in the large-scale purification of mAbs. However, filtration is required prior to all ProA isolations to remove large particulates in cell culture supernatant, consisting of a mixture of cell debris, host cell contaminants, media components, etc. Currently, in-line filters are used to remove particles in the supernatant, requiring replacement over time due to fouling; regardless of the scale. Here we demonstrate the ProA isolation of unfiltered Chinese hamster ovary (CHO) cell media using capillary-channel polymer (C-CP) fiber stationary phases modified with S. aureus Protein A (rSPA). The base polymer of the analytical scale C-CP columns costs ∼$5 per 30 cm column, and when modified with ProA, the base cost is ∼$25 per 30 cm column, a cost-effective option in comparison to analytical-scale commercial columns. To directly sample unfiltered media, a 5 cm gap was created at the head of the C-CP column, where the large particulates are trapped, while molecular solutes flow through the capillary channels without sacrifice in analytical performance, mAb loading capacity, or backpressure increases. The binding capacity of the gap ProA C-CP column was ∼ 2 mg mL−1 of IgG per bed volume. The same analytical column could be operated after processing a total of ∼ 56 column bed volumes of supernatant (>25 analytical cycles) without the need for caustic clean-in-place processing.

Simultaneous determination of tramadol and paracetamol in human plasma using LC-MS/MS and application in bioequivalence study of ­fixed-dose combination

The study aimed to develop a sensitive and high-throughput liquid chromatography coupled with tandem mass spectrometry method to quantify concentrations of tramadol and paracetamol simultaneously in human plasma. Sample preparation involved single-step protein precipitation using methanol and two deuterated internal standards, tramadol D6 and paracetamol D4. Agilent Poroshell 120 EC-C18 (100 × 2.1 mm, 2.1 µm) analytical column was employed to achieve chromatographic separation. Detection was in positive ion multiple reaction monitoring mode. A tailing factor (Tf) of <1.2, separation factor (K prime) of >1.5 from the column dead time and signal-to-noise (S/N) ratio >10, were obtained for analytes and internal standards. The standard curve was linear over the concentration range of 2.5–500.00 ng/mL for tramadol and 0.025–20.00 μg/mL for paracetamol. A small injection volume of 1 µL, low flow rate of 440 µL/min and short analysis time of 3.5 min reduced the solvent consumption, analysis cost and system contamination. The results of method validation parameters fulfilled the acceptance criteria of bioanalytical guidelines. The method was successfully applied to a bioequivalence study of fixed-dose combination products of tramadol and paracetamol in Malaysian healthy subjects.

Reversed phase HPLC method development and validation for the analysis of amlodipine besylate in tablets dosage form and human plasma

Introduction: Several analytical methods for quantitative determination of amlodipine either in pharmaceuticals or biological fluids have suffered different setbacks. Therefore, we aimed at developing a cost effective, simple, robust, and sensitive reversed phase high performance liquid chromatography (RP-HPLC- UV) method for quantification of amlodipine in dosage form and in human plasma. Method: The separation was performed on an analytical column 150 mm x 4.6 mm i.d., Agilent Eclipse XDB C18 Column packed with 5 μm particle size and protected by a guard column (30 mm x 4.6 mm i.d.). The mobile phase composition contained acetonitrile: phosphate buffer (25 mM KH2PO4) adjusted to pH 3.1 with orthophosphoric acid in the ratio of 45:55 v/v. The flow rate was 1.2 mL/min and the UV detector was set at a wavelength of 240 nm. The column was maintained at ambient temperature. Results: The calibration curve gave good linearity in the concentration range studied. The column efficiency was highly evident by high theoretical plates and small height equivalent to theoretical plate (HETP). The percent relative standard deviation (% RSD) was less than 3% in the inter- and intra-day o assay. Amlodipine, 50 ng/mL in plasma stored frozen at -80 C was stable There was no statistical difference in the precision of amlodipine assayed after 12 months when compared to the initial assay (P&gt;0.05). The % recovery of amlodipine in human plasma was 97.79±0.17% to 99.52±0.63%. Conclusion: The new RP-HPLC method is sensitive, reproducible, precise, accurate and robust. We conclude that the method is suitable for estimation of amlodipine besylate in human plasma for pharmacokinetic studies and assay of pharmaceutical dosage form

Urinary Free Cortisol Determination and Interferences Studies Using Liquid Chromatography Coupled to Tandem Mass Spectrometry after On-Line Solid Phase Extraction Based on TurboflowTM Chromatography.

(1) A 24 h urinary free cortisol (UFF) is one of the first-line exams recommended for the diagnosis of Cushing's syndrome. In a hospital hormonology department, this activity can exceed several hundred dosages per week. The UFF is generally determined via an immunoassay with an automate using a chemiluminescence or electrochemiluminescence detection system. To increase the cortisol concentration in the analyzed sample, the automated analysis is preceded by urine extraction, which does not prevent there from being some interferences due to other steroids with close structures. (2) This paper describes the development of on-line solid phase extraction coupled to liquid chromatography and mass spectrometry for the analysis of urinary free cortisol. The on-line extraction was based on the TurboflowTM chromatography coupled to the analytical column by two valves, easily available for the laboratories. (3) The choice of the Accucore Polar Premium® analytical column made it possible to avoid analytical interferences with exogenous or endogenous molecules having the same SRM transition (363 → 121) as cortisol. (4) The method was fully validated in the range of clinically relevant concentrations from the lower limit of quantification (LLOQ) to 411.75 nmol·L-1.