Method For Simultaneous Quantification Research Articles

Antibody-free LC-HRMS/MS method for simultaneous quantification of NGAL, CRP and SAA in serum from sepsis patients.

Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS/MS) is a valuable alternative to ligand-binding assay, enabling specific and accurate quantification of protein biomarkers. We developed a robust antibody-free LC-HRMS/MS method for the multiplex quantification of three sepsis biomarkers in serum: NGAL, CRP and SAA. The method was thoroughly optimized from sample preparation to LC-HRMS/MS analysis, alongside the calibration. Specifically, a modified trichloroacetic acid/isopropanol protein precipitation procedure combined with an optimized Parallel Reaction Monitoring acquisition allowed the quantification of the low abundant NGAL at ng/mL levels. While reference material and reference measurement procedure were available for CRP, no such standards existed for NGAL and SAA. Well-characterized peptide calibrators traceable to the international system of units were developed for NGAL and SAA. The method demonstrated suitable trueness and precision for the quantification of NGAL, CRP, and SAA with coefficients of variation (CV%) ranging from 1.6 % to 22.4 % and bias between -12.6 % and +18.0 %. Successful application to pooled serum samples illustrated the method's effectiveness. Our results pave the way toward the development of reference systems for additional sepsis biomarkers.

Gastrointestinal motility modulation efficacy-related chemical marker findings and QAMS-based quality control of Agastache rugosa.

Agastache rugosa (AR), a traditional edible and medicinal herb, is often used for treating gastrointestinal (GI) motility disorder. But little effort has been done on its gastrointestinal motility modulation (GMM) efficacy-related components and quality control of AR. In this study, a novel strategy was proposed to find GMM efficacy-related chemical markers for the quality control of AR. Firstly, network pharmacology and serum pharmacochemistry were applied to predict potential GMM efficacy-related marker components. Secondly, the GMM efficacy-related marker components were verified through literature matching, target isolation/identification and activity evaluation. Lastly, a quantitative analysis of multiple components by a single marker (QAMS)-based method for simultaneous quantification of marker components was established and validated by HPLC-DAD. The results showed that nine components in AR were screened as potential GMM related components, five of which (rosmarinic acid, tilianin, apigenin, acacetin, and cirsimaritin) were matched by literatures, and four (acacetin-7-O-(6''-O-malonyl)-β-D-glucopyranoside, agastachoside, acacetin-7-O-(2''-O-acetyl-6''-O-malonyl)-β-D-glucopyranoside, and isoagastachoside) were chemically identified and newly evaluated on zebrafish model. The nine components were used as marker compounds to develop an effective QAMS-based method for the quantitative evaluation of 26 batches of commercial AR samples.

Development and validation of a UPLC-MS/MS method for simultaneous quantification of polymyxins and caspofungin in human plasma for therapeutic drug monitoring.

To develop a rapid, convenient, accurate, and low-residual-effect ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of polymyxin B sulfate and colistin sulfate in the blood of patients with multidrug-resistant bacterial infections, as well as caspofungin acetate in the blood of patients with fungal infections, thus facilitating the rational use of antibiotics in clinical applications. All analytes were diluted with 0.2% aqueous formic acid, and plasma proteins were precipitated using acetonitrile. The selected reaction monitoring (SRM) mode was used for measurement. Separation of all analytes was completed on a Hypersil GOLD C18 column (100×2.1mm, 3.0µm). They were quantitatively analyzed using electrospray ionization on a triple quadrupole mass spectrometer in the positive ion mode. The mobile phase consisted of water (containing 0.1% formic acid) and acetonitrile, which was delivered by gradient elution at a flow rate of 0.3ml/min. The internal standard was bacitracin zinc (BcZn), and the column temperature was maintained at 25°C. The runtime for each analysis was 3.5min. The procedure was validated following the recommendations of the U.S. Food and Drug Administration, which included measurements of accuracy (ranging from 83.27% to 105.86% for within-run and between-run accuracy), precision (with coefficients of variation from 2.50% to 16.51% for within-run precision and between-run precision), and matrix effects (ranging from 88.65% to 103.94%). The extraction recoveries ranged from 38.01% to 42.76 for polymyxin B1 (PMB1), polymyxin B2 (PMB2), polymyxin E1 (PME1), polymyxin E2 (PME2), and 88.65% to 89.84% for caspofungin (CPF). Plasma samples were stable under various storage conditions, including three freeze-thaw cycles at -80°C, 24-hour periods at room temperature and 4°C, and 30days of freezing at both -20°C and -80°C, with relative standard deviations (RSD) of less than 15%. In this study, a UPLC-MS/MS method was developed to simultaneously quantify PMB1, PMB2, PME1, PME2, and CPF in human plasma. The method was validated in blood samples from patients with multidrug-resistant bacteria combined with fungal infections and is suitable for therapeutic drug monitoring.

A non-derivatized high-performance liquid chromatography method for simultaneous quantification of hydroxyl acids and amino acids in ammonolysis reactions.

A non-derivatized high-performance liquid chromatographic (HPLC) method was developed for the simultaneous quantification of hydroxyl acids and their amination products in ammonolysis reaction mixtures. By optimizing the mobile phase composition and pH (0.04 M KH2PO4-5% methanol, pH = 2.7), complete separation of hydroxyl acids and their amination products was achieved, using lactic acid as a model substrate. Lactic acid exhibited excellent linearity within the range of 0.02 to 25 mM (R2 = 0.99968), with a relative standard deviation (RSD, n = 6) of 3.51% and a recovery ratio between 96.10% and 102.23%. Similarly, alanine exhibited good linearity from 0.02 to 50 mM (R2 = 0.99999) with an RSD of 4.04% and recovery ratios between 97.30% and 100.03%. Both analytes demonstrated good intra-day precision, with RSDs of 4.23% and 1.18%, respectively. The substrate universality tests confirmed the method's ability to rapidly and effectively separate other hydroxyl acids and their corresponding amino acids in ammonolysis reactions. The results of quantitative comparison with AQC, PITC, and OPA derivatization also proved the method to be a reliable approach for the individual or simultaneous quantification of hydroxyl acids and amino acids, offering essential support for monitoring the conversion ratio and selectivity in hydroxyl acid amination processes. Moreover, it provides valuable guidance for optimizing the amination reaction of hydroxyl groups, potentially extending catalytic advantages to other related reactions.

Development and validation of an LC-MS/MS method for quantification of Osimertinib and its two metabolites AZ7550 and AZ5104 in human plasma including long-time storage.

Osimertinib (AZD9291) is a widely used tyrosine kinase inhibitor for the treatment of non-small cell lung cancer patients with activating EGFR mutations. However, the correlation between dose and efficacy has been debated for several years. For this reason, there is a need for standardized methods for routine analysis, clinical studies on pharmacokinetics and dose-response relationships, and greater understanding of preanalytical conditions, such as sample storage stability. The objective of this study was to develop and validate a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of osimertinib and its two metabolites, AZ7550 and AZ5104, in human plasma and to investigate long-term storage stability of the analytes. Samples were prepared by protein precipitation and separated on a Kinetex EVO C18 column (2.1 × 150 mm, 2.6 µm). Electrospray ionization in positive mode and multiple reaction monitoring were used to monitor the ion transitions. The validated concentration ranges were from 1.25 to 3000 ng/mL. Interassay precisions and accuracies were all ≤ 15 %. Linearity, dilution integrity, and carry-over were also examined and satisfied the validation criteria. Stability was examined under different conditions, and the analytes were found to be stable for more than 3 years at -80°C (< 15 % decline). Finally, the analytical method was successfully applied in a clinical setting on plasma samples from 30 patients with non-small cell lung cancer in treatment with osimertinib, demonstrating its suitability for use in clinical studies and its potential for therapeutic drug monitoring.

Development of a Specialized Method for Simultaneous Quantification of Functional Intestinal Metabolites by GC/MS-Based Metabolomics

Development of a Specialized Method for Simultaneous Quantification of Functional Intestinal Metabolites by GC/MS-Based Metabolomics

A Robust RP-HPLC Method for Simultaneous Quantification, Validation and Stability Studies on Vildagliptin (VILD), Dapagliflozin (DAPA) and Metformin Hydrochloride (METF) in its Combined Dosage Formulation

Background: Vildagliptin, Dapagliflozin and Metformin Hydrochloride are anti- hyperglycemic agents. Anti-hyperglycemic agents are drugs that stimulate insulin secretion and lower the glucose level in the blood. They were investigated as a medication for Diabetes Mellitus (DM). Various studies have reported the HPLC, HPTLC, LC/MS methods for the estimation of individuals of VILD, DAPA and METF. However, until date stability indicating HPLC analysis method has not been reported for the estimation of VILD, DAPA and METF in combined glucagon secretion in a glucose-dependent manner. Introduction: The objective of present work was to develop and validate the stability of RPHPLC method for the estimation of Vildagliptin (VILD), Dapagliflozin (DAPA) and Metformin Hydrochloride (METF) in combined dosage form. Method: In this research work, High Performance Liquid Chromatography (HPLC) was used for validation in the chromatography. In the HPLC, Methanol and Potassium Dihydrogen phosphate buffer (85:15 v/v) were used as mobile phases. HPLC analysis was performed at 230 nm. The method was validated with different parameters such as linearity, precision, accuracy, specificity, and robustness, limit of detection (LOD) and limit of quantitation (LOQ). The RF values of VILD, DAPA and METF were 6.24 ± 0.05 min, 8.34 ± 0.05 min. and 3.68 ± 0.05 min, respectively. The accuracies of drug recovery were 0.6398 %,0.3680 %, 0.6398 % and over the range of 1–4 μg/ml, 20-80 μg/ml and 15-85 μg/ml of VILD, DAPA and METF, respectively. Results: Degradation products found in stress conditions did not interfere with the detection of VILD, DAPA and METF; therefore, the proposed technique can be considered stable. Significant degradation products of VILD, DAPA and METF were obtained in an acid at METF at 2.05 ± 0.05 min. (MDP-1) and degradation product for oxidation of METF at 2.11 ± 0.05 min. (MDP-2). When alkaline hydrolysis was conducted, photolytic and thermal conditions were applied alone or in combination with METF, no degradation product of VILD, DAPA and METF was found. Conclusion: The developed and validation method was satisfactorily applied for the analysis of pharmaceutical preparations and proved specific and accurate for quality control of the cited drugs in their combined dosage form.

Green RP-HPLC method for simultaneous quantification of epigallocatechin-3-gallate and rosmarinic acid in lipid-based nanocarriers and biological fluids: Quality by Design-driven optimization and lean six sigma approach

This study presents the development and validation of a reverse-phase high-performance liquid chromatography (RP-HPLC) method for concurrent quantification of EGCG and RA in prepared lipid-based nanocarriers and biological fluids using a QbD approach, greenness assessment, and Six-Sigma methodology. Initially, variations in critical analytical attributes were identified using the Ishikawa fishbone diagram and Risk Priority Number scores. A Taguchi Orthogonal Array design was employed to screen the critical method parameters influencing method development. Systematic optimization of the RP-HPLC method was subsequently achieved using a Box-Behnken design, with the % organic phase, flow rate, and column temperature as the variables. The responses were retention time, tailing factor, and theoretical plates for both EGCG and RA. Chromatographic separation was accomplished with a methanol and 0.1% formic acid (60:40) isocratic flow system on a Phenomenex Luna C18 column (4.6 × 250 mm, 5 μm) at a 1 mL/min flow rate. The method was rigorously validated according to ICH guidelines. Linearity was observed over a concentration range of 2 to 10 µg/mL for both EGCG and RA, yielding correlation coefficients of 0.998 and 0.999, respectively. The LOD and LOQ were determined to be 0.51 µg/mL and 1.54 µg/mL for EGCG, and 0.35 µg/mL and 1.07 µg/mL for RA. Moreover, the %RSD for all validation parameters were consistently below 2%. Forced degradation studies were conducted under acidic, basic, oxidative, and photolytic conditions to elucidate potential degradation pathways and identify degradation products. The developed method was applied to estimate EGCG and RA in prepared lipid-based nanocarriers and biological fluids like blood plasma and urine. Recovery assays in lipid-based nanocarriers, plasma, and urine samples demonstrated excellent recoveries (96.2–102.1%). The method's greenness was assessed using various tools, and its accuracy was evaluated using Six Sigma methodology. Overall, the developed HPLC method offers a rapid, sensitive, and reliable approach for quantifying EGCG and RA, laying the groundwork for their further investigation as anticancer agents, alone and in combination therapies.

Development, validation and greenness assessment of stability indicating RP-HPLC method for simultaneous quantification of dapagliflozin and metoprolol succinate in synthetic mixture

A novel, rapid, accurate, precise and simple stability indicating RP-HPLC method has been proposed and validated for the concurrent quantitation of Dapagliflozin and Metoprolol succinate in laboratory prepared mixture. The greenness assessment of the proposed approach was accomplished utilizing GAPI and AGREE tools. The final chromatographic run was performed utilizing Methanol: 20mM Potassium Dihydrogen Phosphate: (70:30) as eluent, injected at 25°C at a rate of 1.5 mL/min. Cosmosil-MS-5 C18 column (250 mm × 4.6 mm, 5 μm) was utilised and estimation was done using 223 nm. The APIs were exposed to stress environments (oxidation, acidic and basic hydrolysis, photolysis and thermal stimuli) and stressed samples were analysed under same chromatographic condition. Dapagliflozin and metoprolol succinate had retention times of 11.6 and 2.6 minutes, correspondingly. It was demonstrated having linearity within 10-30 µg/mL for Dapagliflozin and 50–150 µg/mL for Metoprolol succinate. The % accuracy for Dapagliflozin were in the range of 99.01 - 100.56 %, while for Metoprolol succinate it was within 99.32 - 101.41 % at three levels. The outcomes for precision study were within the limits. For Dapagliflozin, the LOD and LOQ were 0.49 µg/mL and 0.59 µg/mL correspondingly and for Metoprolol succinate, values were 1.48 µg/mL and 1.79 µg/mL correspondingly. The approach is effectively suggested for stability-indicating research as well as for routine estimation of Dapagliflozin and Metoprolol succinate in bulk and laboratory prepared mixture. Furthermore, AGREE and GAPI outcomes confirmed the developed approach’s eco–friendliness and greenness.

Quantification of five antineoplastic agents in swab samples using UPLC-ESI-MS/MS: Method development and validation

BackgroundAntineoplastic agents are hazardous drugs used in cancer treatment and consequently can be present at the workplace (e.g. hospital), but also in a home-setting in case of treatment at home. Given the serious health effects, it is important to monitor environmental contamination on a regular base. As currently, no method allows for simultaneous quantification of cyclophosphamide, etoposide, mitomycin C, imatinib and 5-fluorouracil, we developed and validated a method for simultaneous quantification of multiple antineoplastic agents in swab samples. ResultsSample collection was optimised and samples were extracted using solely an extraction solvent (water or water/acetonitrile), shaking and sonicating. Ultra performance liquid chromatography coupled to tandem mass spectrometry with electrospray ionisation was used for detection of the compounds. Selectivity, linearity, sensitivity, precision and accuracy were determined. The LLOQs were 0.3 ng/swab (cyclophosphamide, mitomycin C), 1 ng/swab (etoposide) and 5 ng/swab (imatinib, 5-fluorouracil). After a spill, quantifiable concentrations of antineoplastic agents could be measured in swabs using the developed methods. After cleaning, all concentrations fell below the LLOQ. SignificanceTwo methods allowing for safe collection, rapid preparation and sensitive quantification were developed and can be applied in the field to measure environmental contamination. One for simultaneous quantification of cyclophosphamide, etoposide, mitomycin C and imatinib, one for quantification of 5-fluorouracil.

Development and Validation of a Headspace GC-MS Method for Simultaneous Quantification of Antimicrobial Preservatives in Biopharmaceutical Peptide Formulations.

The four most used antimicrobial preservatives in biopharmaceutical parenteral formulations are phenol, meta-cresol, chlorobutanol, and benzyl alcohol. Preservatives are included in various combinations in biopharmaceuticals highlighting the importance of an analytical method to quantify the four preservatives simultaneously. A headspace GC-MS method was developed to quantify phenol, chlorobutanol, meta-cresol, and benzyl alcohol. The method was validated according to USP <1225>. System suitability <USP 621> was conducted daily for retention time (%RSD < 2.0%), peak area (%RSD < 5.0%), USP tailing factor (< 2.0 and %RSD < 10.0%), and peak resolution (> 2.0). Analytical ranges were 1.5-90 μg/mL for phenol and meta-cresol, 30-240 μg/mL for benzyl alcohol, and 30-300 μg/mL for chlorobutanol. Method accuracy ranged from 94% to 108% and precision from 4% to 15 %RSD for all the tested preservatives. The method was applied to three marketed teriparatide drug products selected as a model. Preservative concentrations of the biopharmaceutical marketed products were determined and were found to be comparable with the labeled concentrations, except for an expired product with 2.5% of the label claim. The developed headspace GC-MS method can be used to evaluate the drug quality of the parenteral formulations and to support the assessment of biopharmaceutical peptide drug products.

Development and validation of an UPLC-ESI-MS/MS method for simultaneous quantification of antineoplastic agents and their metabolites in human plasma after unintentional exposure.

Cyclophosphamide, daunorubicin, epirubicin, doxorubicin and paclitaxel are commonly used drugs in cancer treatment. However, there are no methods available enabling simultaneous measurement of these compounds and their metabolites in human plasma. Our aim was to develop and validate a sensitive method for simultaneous quantification of multiple antineoplastic drugs and their major metabolites in plasma. Solid phase extraction with Oasis PRiME HLB cartridges was used for sample clean-up. The samples were separated on an Acquity UPLC BEH C18 column, ionised by electrospray ionisation and detected with tandem mass spectrometry. The method was validated based on selectivity, extraction efficiency, matrix effect, process efficiency, linearity, sensitivity, precision and accuracy. The established LLOQs were 0.05ng/mL (cyclophosphamide), 30ng/mL (4-oxo-cyclophosphamide), 0.3ng/mL (doxorubicin, daunorubicinol), 0.7ng/mL (epirubicin, epirubicinol, doxorubicinol), 1ng/mL (daunorubicin and paclitaxel) and 5ng/mL (6-alpha-hydroxypaclitaxel). Afterwards, the method was tested in a real-life, unintentional exposure setting. Twenty-two plasma samples of matched maternal and cord blood pairs from pregnant cancer patients treated with chemotherapy were analysed. This resulted in two positive samples, with cyclophosphamide concentrations up to 0.37ng/mL. The validated method is now ready to be applied in the field.

Simultaneous Quantification of Carboxylate Enantiomers in Multiple Human Matrices with the Hydrazide-Assisted Ultrahigh-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry.

Many chiral carboxylic acids with α-amino, α-hydroxyl, and α-methyl groups are concurrently present in mammals establishing unique molecular phenotypes and multiple biological functions, especially host-microbiota symbiotic interactions. Their chirality-resolved simultaneous quantification is essential to reveal the biochemical details of physiology and pathophysiology, though challenging with their low abundances in some biological matrices and difficulty in enantiomer resolution. Here, we developed a method of the chirality-resolved metabolomics with sensitivity-enhanced quantitation via probe-promotion (Met-SeqPro) for analyzing these chiral carboxylic acids. We designed and synthesized a hydrazide-based novel chiral probe, (S)-benzoyl-proline-hydrazide (SBPH), to convert carboxylic acids into amide diastereomers to enhance their retention and chiral resolution on common C18 columns. Using the d5-SBPH-labeled enantiomers as internal standards, we then developed an optimized ultrahigh-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous quantification of 60 enantiomers of 30 chiral carboxylic acids in one run. This enantiomer-resolved method showed excellent sensitivity (LOD < 4 fmol-on-column), linearity (R2 > 0.992), precision (CV < 15%), accuracy (|RE| < 20%), and recovery (80-120%) in multiple biological matrices. With the method, we then quantified 60 chiral carboxylic acids in human urine, plasma, feces, and A549 cells to define their metabolomic phenotypes. This provides basic data for human phenomics and a promising tool for investigating the mammal-microbiome symbiotic interactions.

A bioanalytically validated RP-HPLC method for simultaneous quantification of rivaroxaban, paracetamol, and ceftriaxone in human plasma: a combination used for COVID-19 management

Rivaroxaban is a direct oral anticoagulant medication that has been found to be beneficial for the management of thromboembolic events linked to the Corona virus disease 2019 (COVID-19) pandemic, which has resulted in more than 6 million deaths worldwide. Hence, a sensitive, selective, and green bioanalytically validated method was developed using RP-HPLC coupled with DAD for the simultaneous determination of rivaroxaban in human plasma, and two co-administered drugs, namely, paracetamol, an analgesic, and ceftriaxone, an antibiotic, that are used in the management of COVID-19. An Exsil 100 ODS C18 column (250 × 4.6 mm, 5 μm) was used as the stationary phase, and acetonitrile: water: methanol at a ratio of 60:30:10 (v/v/v) was used in isocratic mode as the mobile phase with a flow rate of 0.7 ml/min. The method was validated over a concentration range of 0.1–10.0 µg/mL for rivaroxaban, and 1.0–15.0 µg/mL for paracetamol and ceftriaxone. The lower limits of detection (LLODs) were found to be 0.03, 0.32, and 0.32 µg/ml for rivaroxaban, paracetamol, and ceftriaxone, respectively. Moreover, the lower limits of quantitation (LLOQs) were found to be 0.1, 0.96, and 0.98 µg/ml. The developed method showed excellent accuracy and precision for the determination of the aforementioned drugs. Four metrics were used to evaluate the greenness of the developed method. The results revealed that the suggested method is green, with values of 81 and 0.6 for the analytical eco-scale and analytical greenness assessment (AGREE), respectively.

Spectrometric method for simultaneous quantification of nadifloxacin and salicylic acid in a novel topical dosage form

Acne vulgaris is a prevalent skin disorder that affects many individuals. In the search for effective acne therapy, researchers have found that a combination of Nadifloxacin and Salicylic Acid may produce a synergistic effect. To further investigate this, a study was conducted to devise a spectrophotometric method for simultaneously measuring the concentrations of Nadifloxacin and Salicylic Acid in a novel topical formulation. Simultaneous Estimation Method was employed using methanol as the solvent. Nadifloxacin exhibited an absorbance maximum of 291 nm, while Salicylic Acid showed an absorbance maximum of 297 nm in methanol. Linearity experiments were conducted to determine the concentration ranges for both drugs, which were found to be 2–12 ppm for Nadifloxacin and 5–30 ppm for Salicylic Acid. The recovery results of Nadifloxacin ranged from 82 % to 95 %, while those of Salicylic Acid ranged from 97 % to 110 %. These results indicate the accuracy and reliability of the method. This method was further validated in accordance with the guidelines provided by the International Council for Harmonization (ICH) of Technical Requirements for Human Use. The proposed method has demonstrated its applicability for the simultaneous and quantitative assessment of Nadifloxacin and Salicylic Acid in various dosage forms. This development is significant as it provides a reliable and efficient means of measuring the concentrations of these two drugs in acne therapy formulations.

Bioanalytical method development and validation of docetaxel and carvacrol in mice plasma using LC-QqQ-MS/MS.

Present work describes the development of a liquid chromatography tandem mass spectrometry-based bioanalytical method for the reliable simultaneous quantification of docetaxel (DXL) and carvacrol (CVL) in the mice plasma. A rapid and sensitive bioanalytical method was developed and optimized in mice plasma using Paclitaxel as an internal standard. Validation of the bioanalytical method was performed according to the ICH M10 guideline covering the range of 9.62-1923.08 ng/mL in the mice plasma milleu at the low, mid, and high-quality control concentrations of 28.86 ng/mL, 961.54 ng/mL, and 1346.15 ng/mL, respectively for both the analytes. Validation parameters such as accuracy, precision, carryover-test, matrix effect, and reinjection reproducibility were carried out and were found in limits. Stability studies (Benchtop, autosampler, freeze-thaw, and long-term) were performed and found to be within limits. The developed bioanalytical method was found to be suitable for the simultaneous quantification of DXL and CVL in the mice plasma.

Validated high performance thin layer chromatographic method for simultaneous quantification of betulinic acid, β-sitosterol and lupeol in fruits, leaves, root bark and stem bark of Dillenia indica Linn

AbstractDillenia indica is one of the important medicinal plants and is extremely popular in many systems of medicine including ayurvedic, homeopathic, and siddha. Traditionally, different parts of this plant have been used to treat cancer, wound healing, diabetes, diarrhea, bone fractures, abdominal pains, cuts and burns. In the present study, a simple, precise, accurate and validated high performance thin layer chromatographic (HPTLC) method was developed for simultaneous estimation of three pharmaceutically active compounds i.e., betulinic acid (BE), β-sitosterol (BT) and lupeol (LP) in fruits, leaves, root bark and stem bark of D. indica. Standards and plant samples were applied on TLC aluminum plate precoated with 0.2 mm layer of silica gel 60F254. The plate was run in a twin glass chamber comprising toluene:methanol:chloroform (8:1:1, v/v/v) as a mobile phase which resulted in better separation of compounds. The plates were immersed in anisaldehyde-sulfuric reagent and then heated at 105 °C for 5 min in CAMAG TLC plate heater for appearance of bands. Densitometric scanning was performed at λmax = 525 nm using tungsten light source in CAMAG TLC Scanner4 armed with WinCATS software. RF values of BE, BT and LP standards and those of plant samples were found to be 0.38 ± 0.01, 0.54 ± 0.01 and 0.65 ± 0.02 respectively. The method was further validated by following the International Conference of Harmonization (ICH) guidelines. For BE, BT and LP, the linear regression data for the calibration plots revealed a satisfactory linear association with correlation coefficients (r2) = 0.9736, 0.9989 and 0.9957, respectively. Linear ranges for BE, BT and LP were 2,000–6,000 ng/band, 200–1,000 ng/band, and 200–600 ng/band respectively. Accuracy of the method was assessed by recovery study conducted at three different levels with the average recovery of 99.19, 99.69 and 100.95% for BE, BT and LP, respectively. The results exhibited the highest content of BE (0.148 ± 0.01%), BT (0.031 ± 0.01%) and LP (0.047 ± 0.01%) in stem bark. The developed method will be useful for routine and quality control analysis of fruits, leaves, root bark and stem bark of D. indica species.

Pharmacokinetic Analysis of Gatifloxacin and Dexamethasone in Rabbit Ocular Biofluid Using a Sensitive and Selective LC-MS/MS Method.

Bacterial keratitis (BK) is an infection that causes inflammation of the cornea and, if severe, can result in blindness. Topical fluoroquinolones combined with corticosteroids have been shown to be useful in the treatment of BK. A rapid, selective, and sensitive bioanalytical method for simultaneous quantification of Gatifloxacin (GAT) and Dexamethasone (DEX) has been developed and validated using tandem mass spectrometry (LC-MS/MS). Optimal separation was accomplished in under 5 min using an Agilent Zorbax C18 column (100 mm × 4.6 mm, 3.5 μm). The mobile phase was composed of a blend of 0.2% formic acid in triple distilled water and methanol with a flow rate of 0.65 mL/min in isocratic mode. GAT and DEX were detected in positive electrospray ionization multiple reaction monitoring mode (MRM), and the retention time was found to be at 1.64 and 2.93 min, respectively. The linearity of GAT and DEX was found to be in the range of 1.56-400 ng mL-1 with good precision and accuracy. The method was validated according to USFDA regulatory guidelines. The validated method was effectively utilized for preclinical pharmacokinetic analysis of GAT and DEX in rabbit tear fluid following the topical application of a commercial formulation.

Simultaneous determination of acid carnosic and carnosol in rosemary-containing foods by high-performance liquid chromatography with photodiode array detection (HPLC–PDA)

The research was conducted to develop and validate a simultaneous quantification method for acid carnosic (CA) and carnosol (CAR) in rosemary-containing foods using highperformance liquid chromatography coupled with photodiode array detection (HPLC&amp;ndash;PDA), with a simple and fast sample processing procedure. Analytical samples were extracted using a methanol&amp;ndash;phosphoric acid mixture (99.5:0.5, v/v) at room temperature for 20 minutes. The chromatographic conditions were as follows: Sunfire C18 column (4.6 mm x 250 mm; 5 &amp;micro;m), mobile phase consisting of 0.1% phosphoric acid &amp;ndash; acetonitrile (40:60, v/v), flow rate of 1 mL/min, injection volume of 10 &amp;micro;L, detection at 230 nm. The method was validated according to the Association of Official Analytical Chemists (AOAC) criteria. The results showed that the method had good specificity, and the standard curves of acid carnosic and carnosol were linear in the concentration range of 2.5 &amp;ndash; 200 &amp;micro;g/mL. The limits of detection (LOD) for acid carnosic and carnosol were 0.3 &amp;micro;g/mL and 0.25 &amp;micro;g/mL, respectively. The limits of quantification (LOQ) for acid carnosic and carnosol were 1 &amp;micro;g/mL and 0.75 &amp;micro;g/mL, respectively. The recovery rates were in the range of 98.8 &amp;ndash; 100.6% (acid carnosic) and 97.5 &amp;ndash; 102.2% (carnosol). Repeatability and internal reproducibility met the requirements of AOAC. The method was applied to evaluate the acid carnosic and carnosol levels in 20 food samples collected from the market, such as rosemary leaf powder, dried rosemary leaves, rosemary herbal tea,...

Postmortem distribution of ropivacaine and its metabolite in human body fluids and solid tissues by GC-MS/MS using standard addition method.

An analytical method was developed for determining ropivacaine and its main metabolite, 3-hydroxyropivacaine in biomedical samples using gas chromatography-tandem mass spectrometry (GC-MS/MS). Then, this established method was applied to investigate the distribution of ropivacaine and its metabolite in human fluids and solid tissues obtained from an authentic case ropivacaine involved. The fluid sample was added acetonitrile, and solid tissue was homogenized using a freezer mill and then added into acetonitrile. Then, an internal standard solution was added to the mixtures. The mixture was centrifuged at 12,000 × g for 5min, and the upper layer of acetonitrile was transferred to magnesium sulfate and octadecyl silica (C18) gel for cleaning up the sample. After centrifugation, the upper layer was then evaporated to dryness with nitrogen, and dissolved with methanol, then injected into the GC-MS/MS system. The coefficients of determination (r2) of constructed calibration curves were all greater than 0.999. The limits of detection for ropivacaine and 3-hydroxyropivacaine in target samples were 15ng/mL and 10ng/mL, respectively. The recovery rates of ropivacaine and 3-hydroxyropivacaine ranged from 97.6% to 103% and from 96.5% to 104%, respectively. The inter-day precision values of ropivacaine and 3-hydroxyropivacaine were not greater than 6.25% and 7.98%, respectively, and the inter-day trueness values were not greater than 6.90% and 8.33%, respectively; the intra-day precision and trueness values of ropivacaine and 3-hydroxyropivacaine were not greater than 3.20%, 6.78%, 7.84% and 8.99%, respectively. GC-MS/MS method for simultaneous detection and quantification of ropivacaine and 3-hydroxyropivacaine in biological samples was successfully developed. The method could also be applied to samples obtained from an authentic case; their distribution among tested fluids and solid tissues were also measured. This is the first report on the distribution of ropivacaine and its major metabolite 3-hydroxyropivacaine in a human case.