Reversed-phase C18 Column Research Articles

Development and Validation of HPLC Method for Quantification of Plumbagin in Plumbago Zeylanica L. Roots.

An RP-HPLC (Reverse Phase-High-performance liquid chromatography) method for the quantitative estimation and validation of the plumbagin in the methanolic fraction of Plumbago zeylanica L. was developed. For achieving good separation, the RP-HPLC method was carried out with reverse phase C18 column, using methanol and water as mobile phase in the ratio of 65:35 (v/v), at the flow rate of 1 mL/min. The detection wavelength was set at 265 nm. The retention time of plumbagin was found at 7.5±0.2 min. The coefficient of determination of plumbagin was found to be (r2) 0.9985 and equation Y=23148x+4327. The LOD and LOQ were found to be 34.06 and 113 ng/mL, respectively. The developed method was accurate, specific, precise, and reproducible. This RP-HPLC may be useful for quantitative estimation of the chemical constituents present in the plant extract as well as the quality assessment of the herbal product.

Validation and clinical evaluation of an ultra-performance liquid chromatography with ultraviolet detector method for plasma quantification of micafungin.

Factors associated with interindividual variability in the pharmacokinetics of micafungin have been identified. This variability can cause underexposure and loss of drug efficacy. For this reason, a simple, fast, cost-effective and sensitive ultra-performance liquid chromatography ultraviolet detector (UPLC-UV) method was developed and validated for the quantification of micafungin. The method involves simple plasma precipitation by UPLC with a reversed phase C18 column at 40°C coupled with ultraviolet detection set at a wavelength of 264nm. The mobile phase consisted of a mixture 42/58 of potassium phosphate 20mm and acetonitrile. The method was validated over the concentration range of 0.25-15.0mg/L and proved to be reliable and reproducible with an average percentage of recoveries of 101.59 ± 3.93% and inter and intraday variation coefficients lower than 15% in all cases. The method was successfully applied in determining 30 samples from 10 patients being treated with micafungin. The method proposed could be useful to facilitate the implementation of therapeutic drug monitoring for personalizing micafungin treatment in invasive fungal infections.

Can Celecoxib Assay in Preclinical Studies Be Improved?

Celecoxib, a cyclooxygenase-2 inhibitor (COX-2), is attracting considerable interest owing to its potential anticancer activity. The repurposing strategy of this drug, however, requires preclinical assessment involving the use of increasingly improved analytical methods. In this work, a rapid, accurate, precise, and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for the quantification of celecoxib in five mouse matrices (plasma, brain, spleen, liver, and kidney). Chromatographic separation was achieved within 8 min on a reversed-phase C18 column at 35 °C using a mixture of acetonitrile and 2% (v/v) acetic acid (50:50) as mobile phase, at a flow rate of 0.6 mL/min. Celecoxib and curcumin, as the internal standard, were analyzed at 425 nm and 250 nm, respectively. Linearity was observed (r2 ≥ 0.996) in the concentration ranges selected for celecoxib. Overall precision was below 14.9%, and accuracy was between −14.9% and 13.2%. The acceptance criteria specified in FDA and EMA guidelines were met. Celecoxib was reproducibly recovered (≥84%) and showed stability in all biological matrices at room temperature for 24 h. The method was then effectively applied for the quantification of celecoxib to understand in vivo biodistribution following its intraperitoneal administration in mice.

Classification and authentication of spices and aromatic herbs by means of HPLC-UV and chemometrics

Recent increase in the adulteration of spices and aromatic herbs in food industry constitutes a problem that requires exhaustive quality control. As every spice has a different composition with characteristic biomarkers, chromatographic profiles are especially valuable to authenticate these products. Thus, in this work a new high performance liquid chromatography (HPLC) method with UV–vis detection was developed for the characterization, identification and authentication of cinnamon, oregano, thyme, sesame, bay leaf, clove, cumin, and vanilla. Chromatographic separation was optimized based on the separation of six characteristic biomarkers (sesamol, eugenol, thymol, carvacrol, salicylaldehyde and vainillin) and was performed using a C18 reversed-phase column under a 35 min gradient elution based on 0.1% (v/v) formic aqueous solution and methanol by means of UV–Vis detection at 280 nm. 87 samples, purchased in local supermarkets, were analyzed and the obtained profiles were processed by chemometric techniques. First, data treatment was evaluated by principal component analysis (PCA); next soft independent modelling by class analogy (SIMCA) and partial least squares discriminant analysis (PLS-DA) were carried out in order to verify if classification according to their biomarkers was possible. The study concluded that PLS-DA (0.14–0.75% global error) classifies better the types of spice or aromatic herb than SIMCA (0.82–3.67% global error).

Development and Validation of a Simple HPLC-UV Assay Method for Determination of Levetiracetam Concentrations in Human Plasma

Levetiracetam (LEV) is a broad spectrum antiseizure medication that is used in various seizure types. There is evidence that therapeutic drug monitoring (TDM) of LEV is of value in selected patient populations, therefore determination of LEV plasma concentrations is essential. Herein we developed and validated a simple, reproducible, and practical method for the quantification of LEV concentrations in human plasma samples using high performance liquid chromatography (HPLC). Plasma samples (0.3 mL) deproteinization was done using acetonitrile. HPLC chromatographic separation of plasma samples was accomplished by reversed phase C18 column. The mobile phase constituted water and acetonitrile (90:10, v/v) ran at flow rate of 1 mL/min. Signal acquisition was conducted at a wavelength of 192 nm. Calibration curves showed excellent linearity (Correlation coefficient r2 > 0.99) over a concentration range of 3–80 μg/mL. Both inter and intraday assay accuracy and precision were less than 8% (except for the lowest limit of quantification was within 20%). Elution time was 15 min. The developed method excluded the use of buffers and utilized small volumes of plasma samples with simple mobile phase composition. Therefore, our method could be practically applied to routine TDM.

Isolation, structural characterization and quantification of impurities in bupivacaine

Bupivacaine was found to be unstable during the accelerated storage condition(40 ℃ and 75% relative humidity), and two degradation impurities with the same protonated molecular ion were observed by high performance liquid chromatography-mass spectrometry (LC-MS). A semipreparative method was used to separate and purify the two impurities, and their structures were elucidated via comprehensive HR-MSMS and NMR spectroscopy analyses. Their stereo structures were characterized through single crystal X-ray diffraction. Meanwhile, an LC-MS method was developed and validated to quantify the two degradation impurities of bupivacaine. Chromatographic separation was performed on a C18 reversed-phase column (4.6 × 150 mm, 5 µm) using an equivalent elution with water and methanol. The limits of quantitation for the two degradation impurities (named RS1 and RS2) were 0.89 and 0.65 ng, respectively, and the average recoveries were in the range of 90∼108% and relative standard deviations were less than 5.0%. The proposed LC-MS method can be used to control the quality of bupivacaine and its formulations. Data AvailabilityData will be made available on request.

Florfenicol and Florfenicol Amine Quantification in Bull Serum and Seminal Plasma by a Single Validated UHPLC-MS/MS Method.

Florfenicol is a broad-spectrum antibiotic belonging to the amphenicols class that inhibits protein synthesis by binding to bacteria's ribosomal subunits. This drug is commonly used in veterinary medicine to treat bacterial infectious diseases in cattle, swine, poultry, and fish. The proposed method uses a quick protein precipitation with acetonitrile for the extraction of florfenicol and florfenicol amine in serum and seminal plasma, followed by analysis in UHPLC-MS/MS for their simultaneous quantification. A BEH C18 reversed-phase column was chosen for analyte separation, allowing to obtaining sharp and symmetrical peak shapes in a chromatographic run of just 3.5 min under programmed conditions. Two specific transitions were observed for each analyte, and florfenicol-d3 was used as the internal standard. The approach was fully validated in each matrix over ranges suitable for field concentrations of florfenicol and florfenicol amine, showing good linearity during each day of testing (R2 always >0.99). Excellent accuracy and precision were demonstrated, for both analytes, by calculated bias always within ±15% and CV% always below 15% at all QC levels tested. The satisfactory outcomes obtained during recovery, matrix effect, and process efficiency investigations in serum and seminal plasma confirmed the strength of the method for the quantification of target compounds. To our knowledge, this is the first LC-MS/MS-validated approach for the quantification of florfenicol and florfenicol amine in serum and seminal plasma and was successfully applied for the determination of their concentration-time profiles in bulls. This paves the way to understanding the pharmacokinetics of this antibiotic and its active metabolite in bull's seminal plasma, which will enable the design of more appropriate treatment protocols.

Methodical approaches to scale-up measurement of glyphosate concentration using HPLC-FLD

Introduction. Traditionally, the hygienic assessment of working conditions when using glyphosate preparations, an extremely popular herbicide in the world, is based on the use of thin layer chromatography. In this paper, alternative approaches are considered. On the basis of experimental data, the conditions for the analysis of glyphosate content in air by high performance liquid chromatography were optimized. Material and methods. All work with glyphosate solutions was carried out in polypropylene vessels. Sampling from the air medium was carried out on a medium filtration filter (“blue ribbon”). Subsequent extraction was carried out with water. The measurements were performed by high performance liquid chromatography with a fluorimetric detector at an excitation wavelength of 270 nm and an emission wavelength of 313 nm. To transfer glyphosate to a molecule with fluorimetric properties after derivatization of glyphosate in an alkaline medium using 9-fluorenylmethyl chloroformate by heating and washing off the excess of the reagent with toluene. A C18 reverse phase column was used; ammonium acetate with the addition of acetic acid and acetonitrile were used as eluents. Results. Approbation of the developed technique was carried out on real samples taken during ground treatment of fallow fields and lands of industrial territories with glyphosate preparations. The detected levels of glyphosate did not exceed the lower limit of quantitative determination: 0.5 mg/m3 in the air of the working area and 0.025 mg/m3 in the atmospheric air (with maximum allowable concentrations of 1.0 and 0.1 mg/m3, respectively). Limitations. The study considers a limited number of chromatographic columns. The study is performed on 25 model samples of the air of the working area and atmospheric air. Conclusion. Based on the results of the work performed, methodological instructions “Measurement of glyphosate concentrations in the air by high-performance liquid chromatography” were formed and sent for metrological certification in the approved manner.

Identification and determination α and β-acids from Columbus hops using Reverse Phase High Pressure Liquid Chromatography

The present study has demonstrated the HPLC method for identification and determination a and b-acids in Columbus hops. the measurement conditions has been set up included the use of Reverse phase C18 column Chromolith, gradient elution with methanol (A) and Acetonitril acidified with phosphate buffer at pH 2.8. While the flow rate has been applied of 0.8 mL/min, and it is run for 25 minutes. The separation of standard and sample has been monitored using UV variable detector in the rang of 300-340 nm. Pretreatment sample has been done using SPE with the cartridge of C18 similar to the analyte column. The results found that a four predominant peaks for α and β-acids, namely cohumulone (6.6 min), the second peak represents a mixture of humulone and adhumulone (8.9 min), the third peak is colupulone (15.9 min) and the fourth is a mixture of lupulone and adlupulone (16.7 min). The quantity of sample has been determined according to multipoint calibration using ICE2 standard, the sample found are 0,209 g/L cohumulone, 1.087 g/L of humulone and adhumulone, 0,349 g/L of Colupulone and 0.630 g/L of mixture of lupulone and adlupulone. In addition it is high precision measurement that is indicated by the value of RSD less than 1%.

A quantification method for trace level of oxytocin in food matrices using LC-MS/MS

Backgrounds: Oxytocin is nowadays used to increase the agricultural products besides its use during the milking of cattle leading to the contamination of agricultural produce and milk with oxytocin. Monitoring of accurate oxytocin contaminations from foodstuffs is sometimes required to maintain the quality standard. The commonly used oxytocin assays in this study were interfered with by the food matrix. There is a need to develop an accurate and confirmed method for monitoring oxytocin contaminations in foodstuffs.Objective: An attempt is made to develop an accurate assay method of oxytocin from milk and agricultural produces.Methods: The acidified methanol was used for the extraction of oxytocin from target food stuff/matrices (agricultural produce and Milk). LC-MS/MS was used for its detection and quantification. In the chromatographic separation, Oxytocin concentration was optimized using selective reaction monitoring (SRM) with heated electrospray ionization (HESI) in positive polarity. The chromatographic separation was performed using a reversed-phase C18 column with gradient elution at a flow rate of 0.4 ml/min. The acidified methanol was used for the extraction of oxytocin in all target food matrices. The method performance was verified as per the SANTE 2021 guideline. After method validation, the method was applied in real food samples analysis for assessing the presence/absence of oxytocin.Results: The calibration curve offered excellent linearity (R2 = 0.999) with less than 15% residuals. The matrix effect was <20% observed for all target matrices. The mean recoveries were within 70%–115% with <11% RSD at four different levels in milk and 0.01 mg/kg in fruits and vegetables. The optimized method was applied to 50 random samples of milk, fruits, and vegetables from the market for the purposes of an established quality control approach. Based on the results, we did observe a signal of oxytocin in the random samples Therefore, this method has shown its practical suitability for the detection of oxytocin in milk, fruits, and vegetables.Conclusion: Extraction of oxytocin using acidified methanol followed by assays using LC-MS/MS is a simple, sensitive, accurate, reproducible, and practically suitable method for detection and quantification of oxytocin from milk, fruits, and vegetables.

Quantitative Analysis of Isoniazid and Its Four Primary Metabolites in Plasma of Tuberculosis Patients Using LC-MS/MS

Isoniazid and its metabolites are potentially associated with hepatotoxicity and treatment outcomes in patients who receive antituberculosis (TB) therapy. To further understand the pharmacokinetic profiles of these molecules, a method based on LC-MS/MS was developed to determine the concentration of these compounds in human plasma. Isoniazid, acetylisoniazid, and isonicotinic acid were directly analyzed, whereas hydrazine and acetylhydrazine were determined after derivatization using p-tolualdehyde. Chromatographic separation was conducted on reversed-phase C18 columns with gradient elution, and detection was carried out in multiple reaction monitoring mode. The calibration curves were linear with correlation coefficients (r) greater than 0.9947 for all analytes. The intra- and inter-day precision was less than 13.43%, and the accuracy ranged between 91.63 and 114.00%. The recovery and matrix effect of the analytes were also consistent (coefficient of variation was less than 9.36%). The developed method successfully quantified isoniazid and its metabolites in TB patients. The method has broad applications in clinical research, including isoniazid one-point-based therapeutic drug monitoring, genotype-phenotype association studies of isoniazid metabolic profile and isoniazid-induced hepatotoxicity, and the initial dose prediction of isoniazid using population pharmacokinetic modeling.

Separation and Detection of Abamectin, Ivermectin, Albendazole and Three Metabolites in Eggs Using Reversed-Phase HPLC Coupled with a Photo Diode Array Detector

An innovative and sensitive approach using high-performance liquid chromatography-photo diode array detection (HPLC-PDAD) was developed and optimized for the simultaneous determination of abamectin (ABM), ivermectin (IVM), albendazole (ABZ) and three metabolites in eggs. The samples were extracted with acetonitrile (MeCN)/water (90:10, v/v), and the extracts containing the targets were cleaned up and concentrated by a series of liquid-liquid extraction (LLE) steps. A reversed-phase C18 column and a mobile phase consisting of 0.1% trifluoroacetic acid (TFA) aqueous solution and methanol (MeOH) were utilized to perform optimal chromatographic separation. The developed method was validated on the basis of international guidelines. The limits of detection (LODs) and quantitation (LOQs) were 2.1-10.5 µg/kg and 7.8-28.4 µg/kg, respectively. Satisfactory linear relationships were observed for the targets in their corresponding concentration ranges. The mean recoveries ranged from 85.7% to 97.21% at 4 addition levels, with intraday and interday relative standard deviations (RSDs) in the ranges of 1.68-4.77% and 1.74-5.31%, respectively. The presented protocol was demonstrated to be applicable and reliable by being applied for the detection of target residues in locally sourced egg samples.

Development and validation of an analytical method using liquid chromatography-tandem mass spectrometry for the therapeutic drug monitoring of seven cardiovascular drugs in clinical usage

Therapeutic drug monitoring (TDM) of cardiovascular drugs is essential to improve treatment efficacy and minimize toxicity because of the usage of multiple drugs with a very limited therapeutic range and the high pharmacokinetic variation in patients. We developed and validated a reliable and economical liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the determination of seven cardiovascular drugs—procainamide, lidocaine, quinidine, deslanoside, digoxin, atorvastatin, and digitoxin—for clinical usage. Serum samples were prepared by simple protein precipitation with an organic solvent consisting of acetonitrile and methanol (2:1 v/v) and analyzed under optimized LC-MS/MS conditions. The chromatographic separations were accomplished within 15 min on a reversed-phase C18 column with a gradient elution of aqueous solvent and acetonitrile while maintaining 0.1 (v/v) % formic acid and 2 mM ammonium formate. The optimized MS/MS conditions in ESI-positive mode offered sufficient sensitivity for the seven cardiovascular drugs (LOQs between 0.5 and 1 ng/mL). This method was fully validated including linearity, selectivity, accuracy, precision, carry-over, and matrix effects. Additionally, stability under several conditions was tested to determine how to handle the standard solutions and serum samples. The seven cardiovascular drugs, simultaneously, were precisely and accurately analyzed in intra- and inter-day assays (RSD < 6 % and recovery between 96.3 and 102.8 %) using only two isotope-labeled internal standards (lidocaine-(diethyl-d10) and digoxin-21, 21, 22-d3). The presented method also showed good accuracy in analyzing the seven drugs in hyperlipidemia, hyperalbuminemia, and hyperglycemia serum, allowing it to be recommended as a common and routine analysis method for cardiovascular drugs in clinical practice.

New Method to Biomonitor Workers Exposed to 1,6-Hexamethylene Diisocyanate.

Isocyanates such as 1,6-hexamethylene diisocyanate (HDI), 4,4'-methylenediphenyl diisocyanate, and toluene diisocyanate are highly reactive compounds that have a variety of commercial applications, including manufacturing polyurethane foam, elastomers, paints, adhesives, coatings, insecticides, and many other products. Their primary route of occupational exposure is through inhalation. Due to their high chemical reactivity, they are toxic and have adverse effects at the cellular and subcellular levels, leading to irritative and immunological reactions associated with lung disease. High concentrations of isocyanates are strong respiratory irritants. Bronchial sensitization and asthma are among the major adverse clinical reactions associated with low-level chronic exposure to isocyanates. Albumin adducts have been linked to the mechanism of occupational asthma caused by isocyanates. Isocyanates react in vivo with albumin, which is recognized by the immune system. Albumin adducts of isocyanates trigger immune responses and are probably the antigenic basis for isocyanate asthma. Sensitization to isocyanates is the main pathway for adverse health effects. Therefore, markers for the biologically effective dose such as albumin adducts of HDI are needed. A new isocyanate adduct of HDI with lysine─Nε-[(6-amino-hexyl-amino)carbonyl]-lysine (HDI-Lys)─was synthesized and characterized by 1H-NMR, 13C-NMR, and mass spectrometry (MS). Appropriate internal standards─HDI-Lys-4,4'-5,5'-d4 (HDI-d4-Lys) and Nε-[(7-amino-heptyl-amino)carbonyl]-lysine (Hep-Lys)─were synthesized to establish a LC-MS/MS method for the analysis of HDI adducts in in vitro modified albumin and in workers. The presence of HDI-Lys was found after pronase digestion of albumin and confirmed by two independent chromatographic approaches: with a C8 reversed-phase column and with a hydrophilic interaction liquid chromatography column. Quantification was performed with positive electrospray ionization (ESI)-MS. The adduct peak found in vivo was confirmed with the less sensitive negative ESI-MS. In summary, these are new compounds and methods to determine isocyanate-specific adducts with albumin in workers exposed to HDI.

Optimized Multi-Attribute Method Workflow Addressing Missed Cleavages and Chromatographic Tailing/Carry-Over of Hydrophobic Peptides.

Peptide mapping by liquid chromatography mass spectrometry (LC-MS) and the related multi-attribute method (MAM) are well-established analytical tools for verification of the primary structure and mapping/quantitation of co- and post-translational modifications (PTMs) or product quality attributes in biopharmaceutical development. Proteolytic digestion is a key step in peptide mapping workflows, which traditionally is labor-intensive, involving multiple manual steps. Recently, simple high-temperature workflows with automatic digestion were introduced, which facilitate robustness and reproducibility across laboratories. Here, a modified workflow with an automatic digestion step is presented, which includes a two-step digestion at high and low temperatures, as opposed to the original one-step digestion at a high temperature. The new automatic digestion workflow significantly reduces the number of missed cleavages, obtaining a more complete digestion profile. In addition, we describe how chromatographic peak tailing and carry-over is dramatically reduced for hydrophobic peptides by switching from the traditional C18 reversed-phase (RP) column chemistry used for peptide mapping to a less retentive C4 column chemistry. No negative impact is observed on MS/MS-derived sequence coverage when switching to a C4 column chemistry. Overall, the new peptide mapping workflow significantly reduces the number of missed cleavages, yielding more robust and simple data interpretation, while providing dramatically reduced tailing and carry-over of hydrophobic peptides.

Synthesis and standardization of an impurity of acetaminophen, development and validation of liquid chromatographic method

One of the impurities of acetaminophen, N,N'-(oxydi-4,1-phenylene)diacetamide (ODAA), which is not specified in the organic impurities analysis method of acetaminophen by high performance liquid chromatography (HPLC) in American Pharmacopoeia Version 42 (USP 42), was synthesized, characterized and standardized. A new and optimized liquid chromatographic method for the determination of organic impurities of acetaminophen was developed using an ultra-high performance liquid chromatographic (UHPLC) system, which can separate this impurity. This new liquid chromatographic method has been optimized and validated for the simultaneous determination of acetaminophen related compound B, acetaminophen related compound C, acetaminophen related compound D, acetaminophen related compound J and ODAA, the organic impurities in acetaminophen drug substance. Acetaminophen was also subjected to stress-testing under acidic hydrolysis, alkaline hydrolysis, oxidative degradation, thermal degradation and photolytic degradation for 15 days. The impurity molecule, ODAA was synthesized using 4,4′-oxydianiline and acetic anhydride. The chemical structure of the synthesized ODAA molecule was confirmed by characterization studies. The potency of ODAA was found to be 99.64% as a result of the relevant analyses. The chromatographic separation was achieved on a C8 (150 mm × 2.1 mm; 2-µm particle size) reversed-phase column using a gradient elution, being solvent A: methanol-water-glacial acetic acid (50:950:1, v/v/v) and solvent B: methanol-water-glacial acetic acid (500:500:1, v/v/v) flowing at a rate of 0.2 mL/min. The limits of quantitation (S/N 10:1) were 1.248 µg/mL for acetaminophen, 0.373 µg/mL for acetaminophen related compound B, 1.217 µg/mL for acetaminophen related compound C, 0.369 µg/mL for acetaminophen related compound D, 0.125 µg/mL for acetaminophen related compound J and 0.373 µg/mL for ODAA. The individual mean recoveries of each impurity molecule spiked into acetaminophen samples at different concentration levels ranged from 93% to 104%. The method developed for UHPLC instrument was successfully applied to the analyses of different lots of acetaminophen. Thus, the proposed method can be used for determination of this impurity in the presence of other specified impurities of acetaminophen.

Quantitative determination of major alkaloids in areca nut products by high-performance liquid chromatography coupled with ion mobility spectrometry.

Areca nut is a popular and addictive food as well as a traditional herbal medicine in many countries. Areca nut contains alkaloids including arecoline, guvacine, and arecaidine, which are the major bioactive compounds in areca products. Areca alkaloids can be carcinogenic, and thus sensitive and specific analytical methods are urgently desired for the identification and quantification of these compounds. High-performance liquid chromatography-based methods are often preferred, but areca alkaloids do not have chromophores, and detection using a traditional UV detector can be difficult. The complexity of areca sample extracts can also lead to the co-elution of peaks leading to poor quantitative performance. We report here high-performance liquid chromatography coupled with an ion mobility spectrometer for sensitive determination of areca alkaloids in various products including areca nut, areca nut products, and herbal oral liquid. An X-Bridge reversed-phase C18 column was used in the experiment and was combined with high-performance liquid chromatography coupled to an ion mobility spectrometer system. A custom-made adjustable post-column splitter acted as an interface between the high-performance liquid chromatography and the ion mobility spectrometer; it also acted as the electrospray ionization source. The mobile phase was methanol and 0.5% ammonium hydroxide. The results demonstrate that the splitter can afford a wide range of split ratios that match the ion mobility spectrometer ionization source while keeping the separation efficiency of high-performance liquid chromatography. Three major alkaloid compounds were then accurately determined using the resulting method without dativization steps. Many coeluted high-performance liquid chromatography peaks are effectively separated in the ion mobility spectrometer dimension, which in turn improved the quantification accuracy.

Cost-Effective HPLC Method for Identification of Rosmarinic Acid in Methanol Extract of Ocimum Basilicum

Prescribing herbal remedies is now becoming common by many practitioners, who propose herbal treatments along with conventional medicine systems to treat various ailments considering their synergistic effects. Rosmarinic acid is a potent phenolic compound and caffeic acid derivative. It has many miraculous biological activities. Anti-allergic, anti-angiogenic, anti-depressant, anti-inflammatory, antitumor, anti-microbial activity, and antiviral effect of rosmarinic acid has already been documented in previous literature. It is widely present in many formulations because of its hepatoprotective and antioxidant effect. This study aimed to develop and validate a RP-HPLC method for the standardization of O. basilicum L. raw material and extracts by using rosmarinic acid as a marker. The study also aimed to quantify the rosmarinic acid content in the methanol extract of O. basilicum leaves. A simple and cost-effective High Pressure Liquid Chromatography (HPLC) method for determining rosmarinic acid in Ocimum basilicum plant extract has been developed and validated. Methanol-water-orthophosphoric acid in the ratio of 95:5:1 was used as the mobile phase using a C18 reversed-phase column for the HPLC method. Signals were detected at 254 nm. The rosmarinic acid content was found to be 4.2 mg/10 mg of the plant extract. The method is sensitive and reproducible and ideal for quick routine analysis and can be employed for detecting rosmarinic acid in other herbs. Despite the availability of literature on the medicinal properties of this plant and its chemical constituents, only a limited number of papers have been published on the determination of the rosmarinic acid in Ocimum basilicum plant using RP-HPLC. Moreover, this is the first report that quantifies rosmarinic acid in the plant extract by HPLC without using expensive columns and complex buffer systems that deteriorate the chromatographic columns and in turn, enhance the cost of the used process.

Concurrent Determination of Tigecycline, Tetracyclines and Their 4-Epimer Derivatives in Chicken Muscle Isolated from a Reversed-Phase Chromatography System Using Tandem Mass Spectrometry.

A quantitative and qualitative method using a high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) detection approach was developed and validated for the analysis of tigecycline, four tetracyclines and their three 4-epimer derivatives in chicken muscle. Samples were extracted repeatedly with 0.1 mol/L Na2EDTA–McIlvaine buffer solution. After vortexing, centrifugation, solid-phase extraction, evaporation and reconstitution, the aliquots were separated using a C8 reversed-phase column (50 mm × 2.1 mm, 5 µm) with a binary solvent system consisting of methanol and 0.01 mol/L trichloroacetic acid aqueous solution. The typical validation parameters were evaluated in accordance with the acceptance criteria detailed in the guidelines of the EU Commission Decision 2002/657/EC and the U.S. Food and Drug Administration Bioanalytical Method Validation 05/24/18. The matrix-matched calibration curve was linear over the concentration range from the limit of quantitation (LOQ) to 400 μg/kg for doxycycline, and the calibration graphs for tetracycline, chlortetracycline, oxytetracycline, their 4-epimer derivatives and tigecycline showed a good linear relationship within the concentration range from the LOQ to 200 μg/kg. The limits of detection (LODs) for the eight targets were in the range of 0.06 to 0.09 μg/kg, and the recoveries from the fortified blank samples were in the range of 89% to 98%. The within-run precision and between-run precision, which were expressed as the relative standard deviations, were less than 5.0% and 6.9%, respectively. The applicability was successfully demonstrated through the determination of residues in 72 commercial chicken samples purchased from different sources. This approach provides a novel option for the detection of residues in animal-derived food safety monitoring.

Determination and pharmacokinetic study of riociguat by UPLC-MS/MS in human plasma

Pulmonary hypertension (PH) is frequent in the general population and is linked to an increased risk of death. Riociguat is a kind of endothelin receptor antagonist that is often used to treat PH. For pharmacokinetic studies and the determination of riociguat in PH patients, a new, quick, easy, and sensitive UPLC–MS/MS approach was designed and validated. Riociguat and irbesartan (IS) were detected using ESI in positive ion and multiple reaction monitoring mode, respectively, by monitoring the mass transitions m/z 423.0 → 391.0 and 429.1 → 206.9. A reverse-phase C18 column (5 μm, 4.6 × 150 mm) was used with an isocratic mobile phase of water containing 0.1 % formic acid–acetonitrile (25:75, v/v) at a flow rate of 1 ml/min for chromatographic separation. In the range of 5–400 ng/ml, the calibration curve was linear and had a good correlation coefficient (0.9972). This is the first UPLC–MS/MS technique that has been developed and validated for determining riociguat from human plasma. The developed analytical method was extensively validated for linearity, selectivity, specificity, accuracy, precision, sensitivity, stability, matrix effect and recovery, according to FDA criteria. The devised approach was successfully used for a pharmacokinetic research and riociguat determination in PH patients.