Acid Mobile Phase Research Articles

Comparative Bioequivalence Studies of doxysam and doxycycline (Pfizer) by reversed phase HPLC.

Isocratic high performance liquid chromatography on reversed phase a (150x 4.6 mm I.D), 5 ?m ?-Bondapak RP-8 column (with acidic mobile phase allow the separation of doxcycycline hydrochloride with low detection limit of 0.2 µg/ml detected by UV set at 226 nm. The method was validated for Doxycycline between 0.156- to 5 µg/ml. The concentration of doxycycline was assessed in two single dose randomized crossover studies with intervals of one week between two period. In sera of 20 adults healthy male volunteers with average age of (42 + 10) year, body weight 48-85 kg, body height of (160-185cm) after a single dose of doxycycline hydrochoride 100 mg in form of capsules were orally administrated for both formulations. The blood samples (2ml) were drawn concomitantly from 0.5 – 24 hours. µg The pharmacokinetics parameter were obtained from the mean serum concentration measured at various sampling times for both formulations. The maximum peak concentration (Cmax) of doxycycline reference drug from Pfizer In serum was (3.1 +0.094 µg /ml) attained in 2 hrs. While the maximum concentration (Cmax) of test drug doxysam (2.8 +0.098 µg /ml) attained in about 2 hrs, both drug have long elimination time with half time of approximately (13 hrs.) Both test and reference drug were show no significant difference in pharmacokinetics parameters, so they were considered to be bioequivalent.

Buffer-free high pH mobile phase LC-MS/MS for determination of the alcohol biomarker phosphatidylethanol 16:0/18:1 and 20 drugs and metabolites in whole blood

BackgroundAcidic mobile phases are commonly used in reversed phase liquid chromatography tandem mass spectrometry (LC-MS/MS) bioanalysis. However, increased sensitivity, improved peak symmetry, and increased retention, especially for basic hydrophilic drugs have been observed using basic mobile phases. In our previous acidic mobile phase LC-MS/MS method we needed two injections (0.4 and 2.0 μL) of each sample for this task, which is inefficient. The aim of this study was to investigate if basic mobile phase LC-MS/MS could be used to determine phosphatidylethanol 16:0/18:1 and 20 other drugs and metabolites with satisfactory sensitivity in one single run. MethodsWhole blood was prepared by 96-well supported-liquid extraction using heptane/ethyl acetate/2-propanol (16:64:20, v:v:v). Chromatographic separation was achieved on an Acquity BEH C18 column (50 × 2.1 mm I.D.), using a mobile phase with 0.025 % ammonia, pH 10.7 (Solvent A) and methanol (Solvent B). All compounds had isotope-labelled internal standards. ResultsThe method was fully validated. Recovery was between 63 and 91 % for 20 compounds and 10 % for benzoylecgonine. Matrix effects were low, except for ion enhancement of buprenorphine and ion suppression for THC. However, internal standards compensated for these effects. Inter-assay precision and accuracy were < ± 20 % for all compounds at five tested concentrations, except for methamphetamine at the highest concentration. ConclusionAn LC-MS/MS method for simultaneous determination of PEth 16:0/18:1 and 20 drugs and metabolites in whole blood were for the first time developed and validated. Retention of PEth 16:0/18:1 was, in contrast to the other 20 compounds, largely affected by mobile phase buffer concentration. The buffer free basic mobile phase ensured that phosphatidylethanol 16:0/18:1 eluted before most of the unwanted phospholipids.

Leveraging Strengths of green chromatography in Developing eco-friendly HPLC and HPTLC methods for the simultaneous analysis of Lamivudine, Dolutegravir and tenofovir alafenamide

The development of green analytical methodologies is a critical challenge in the field of pharmaceutical analysis. The ternary mixture investigated in this study consists of the antiretroviral drugs Lamivudine (LMV), Tenofovir alafenamide fumarate (TAF), and Dolutegravir (DTG), which are all approved antiretroviral drugs for the treatment of HIV-1 infection. While LMV was approved in 1995, DTG and TAF were more recently approved in 2013 and 2016, respectively, as newer generation HIV medications with improved efficacy and safety profiles compared to earlier antiretroviral drugs. This study presents three eco-friendly approaches for the simultaneous determination of the ternary mixture. Two HPLC methods were applied: an organic-solvent free mixed-micellar HPLC technique was developed as (method A), using a Cyano column (150 × 4.6 mm,5.0 µm) with a mobile phase consisting of 0.03 M Brij-35, 0.09 M sodium dodecyl sulfate, and 0.02 M ammonium acetate in water, adjusted to pH 3.0. Green solvents were applied in (method B), namely, phosphate buffer 25 mM adjusted to pH 3.0 and ethanol (45:55, v/v) using an Inertsil-C18 column (250 × 4.6 mm,5.0 µm,). Peaks were detected at 258 nm, and the retention times for (method A) were 3.10, 3.95 and 5.03 min, and for (method B) were 2.53, 3.63 and 4.86 min for LMV, TAF and DTG, respectively. Additionally, a green thin-layer chromatographic (TLC) method was developed using a water-isopropanol-formic acid mobile phase (65/35/10,v/v/v) and RP-18 silica gel 60 HPTLC-plates. The drugs were successfully resolved, with retardation factor (Rf) values of 0.26, 0.54 and 0.82 for DTG, TAF, and LMV, respectively. The novel methods were validated and applied successfully in marketed dosage forms. Their greenness profiles were assessed using two recent metrics.

SIL-IS LC-ESI-MS/MS method for simultaneous quick detection of amoxicillin and clavulanic acid in human plasma: Development, validation and its application to a pharmacokinetics study.

A liquid chromatography electrospray ionization tandem mass spectrometry method with amoxicillin-d4 as the stable isotope-labeled internal standard for simultaneous quick detection of amoxicillin and clavulanic acid in human plasma was developed and validated. Chromatographic separations were performed on a Hedera ODS-2 column (2.1 × 150 mm, 5μm). The mobile phases for gradient elution were aqueous solution containing 0.2% acetic acid (AA) (mobile phase A) together with organic phase solution (acetonitrile and methanol mixed solution, mobile phase B). Mass spectrometry was performed using negative electrospray ionization in multiple reaction monitoring mode. The target fragment ion pairs of amoxicillin, clavulanic acid and amoxicillin-d4 were m/z 364.1 → 223.1, 198.1 → 135.9 and 368.1 → 227.1, respectively. The linear ranges of this method were 40-5,000 ng/ml for amoxicillin and 30-2,500 ng/ml for clavulanic acid, with coefficient of determination > 0.9900. This method validation included selectivity, standard curve, lower limit of quantitation, accuracy, precision, recovery, matrix effect (hemolytic matrix and hyperlipidemic matrix), carryover, stability, dilution reliability and incurred sample reanalysis study. A successful application of this method was realized in a pharmacokinetic study after administration of amoxicillin-clavulanic acid potassium granules.

New screening and quantification method by UHPLC-MS/MS of 36 new synthetic cathinones in hair. Application to real cases

A class of β-keto analogs of phenethylamine known as synthetic cathinones has been identified as the most emergent new class of psychoactive chemicals in the last ten years. Synthetic cathinones (SC) are becoming more varied, which represents a serious danger to social security and public health worldwide. In this work, an analytical technique based on UHPLC-MS/MS was developed for the simultaneously measurement of 36 synthetic cathinones and two metabolites in hair keratin samples. The separation was conducted by Atlantis Premier BEH C18 AX (2.5 μm, 2.1 × 100 mm) column using elution with 5 mM ammonium acetate in water with 0.1 % formic acid (v/v) (mobile phase A) and 2 mM ammonium formate in MeOH/Acetonitrile 50/50, +0.1 % formic acid (v/v) (mobile phase B). The analysis time was 11 min. Mass spectrometer was equipped with electrospray ionization (ESI). Multiple reaction monitoring (MRM) mode was used for the detection and quantification of the studied compounds. The methodology was successfully validated according to the Organization Scientific Area Committee guidelines, with linearity (r2 ≥ 0.99) from LOQ to 500 pg/mg concentrations for all the compounds investigated. This method was subsequently applied to nine hair samples positive for ten different synthetic cathinones. The most common SCs identified were 3,4-methylenedioxypyrovalerone (3,4-MDPV), in a concentration range 6.0–1000.0 pg/mg, along with alpha-pyrrolidinopentiophenone (α-PHP (54.0 and 554.0 pg/mg, respectively)), followed by the two positional isomers 3-MMC (556.0 and 5000.0 pg/mg) and 4-MMC (11.5 and 448.0 pg/mg). In conclusion, a validated LC-MS/MS method with high specificity was developed offering an easy and affordable sample preparation, and a run time that makes it suitable for use in a high throughput forensic laboratory for the multi-analyte quantification of 36 novel synthetic cathinones and 2 metabolites in hair.

Addressing Challenges of Membrane Clogging in AF4-UV-ICPMS Analysis for Size Determination of Trace Elements in Acidic, Organic-Rich Peat Bog Waters.

The analysis of colloid-associated trace elements (TEs) in acidic, organic-rich waters (pH 3.8-5.8) using AF4-UV-ICPMS often necessitates the use of neutral or weakly alkaline carriers (pH 7-8.6). Employing acidic mobile phases has been deemed impractical due to the substantial sorption of colloids onto a 0.3 kDa polyethersulfone (PES) membrane and greatly reduced separation performance. This has greatly restricted the determination of potentially bioavailable forms of TEs (i.e., < 1 kDa) in acidic, organic-rich waters. To address this issue, porewaters from Sphagnum moss and peat were investigated. Membrane clogging was more pronounced in peat porewaters, where a higher deposition rate of dissolved organic matter onto the membrane was observed compared to that in moss waters. This adsorption is driven by membrane-colloid interactions, with colloids in peat porewaters exhibiting weaker electrostatic repulsion due to their higher positive ζ-potentials. Considering the actual pore size and clogging tolerance of the membrane, it is advisable to employ a 5 kDa PES membrane for peat porewaters, while a 1 kDa PES membrane suits moss waters better. Employing the optimal method enables the separation of TEs within the 0.5-20 kDa size range. Operating within a metal-free, ultraclean laboratory, TEs are detectable at the ng·L-1 level. By enabling precise and accurate separation of dissolved TEs into their size species in these peat bog waters at an appropriate pH, this method addresses diverse size profiles. This information is crucial for comprehending the chemical forms, transformations, mobility, and potential bioavailability of TEs in peat bogs.

Anticandidal effect of cinnamic acid characterized from Cinnamomum cassia bark against the fluconazole resistant strains of Candida.

Candida spp., causes invasive fungal infections, especially in immune-compromised patients and the propensity of antifungal resistance against azole-based drugs need to be addressed. This study is thus aimed to characterize the anticandidal effect of the cinnamic acid extracted from the barks of Cinnamomum cassia. Five species of Fluconazole-resistant Candida sp. were retrieved from the department repertoire. The extraction of CA was performed by three different methods followed by silica gel column chromatography. Eluant was subjected to FTIR and XRD analysis for confirmation. The anticandidal activity of the CA was checked by the agar disc diffusion method and the MIC and MFC were determined. The anti-biofilm effect of CA was assessed using the CLSM technique followed by the biocompatibility check using MTT assay in normal HGF cell lines. CA was best extracted with the hot maceration method using ethanol with a maximum yield of 6.73mg. Purification by column chromatography was achieved using benzene, acetic acid, and water (6:7:3) mobile phase. CA was confirmed by FTIR with absorption peaks and by XDR based on strong intensity. CA was found to possess promising anticandidal activity at 8µg/mL with MIC and MFC values determined as 0.8µg/mL and 0.08µg/mL respectively. Antibiofilm activity by CLSM analysis revealed biofilm inhibition and was biocompatible at 8.5µg/ml concentrations in HGF cell lines until 24h. The study findings conclude that CA is the best alternative to treat candidal infection warranting further experimental preclinical studies.

A validated stability‐indicating reversed‐phase‐UPLC method for simultaneous estimation of promethazine hydrochloride, methylparaben, propylparaben and sodium benzoate assay of cough suppressant and antihistamine liquid oral dosage forms

A quick, simple, sensitive, efficient and stability-indicating reverse-phase ultraperformance liquid chromatographic method for the estimation of propylparaben, methylparaben and sodium benzoate in a pharmaceutical liquid oral formulation was developed. A Waters Acquity UPLC BEH C18, 50 × 2.1 mm, 1.7 μm i.d. column was used to perform chromatographic separation with a 0.1% perchloric acid mobile phase used as solvent A and a mixture of 0.1 % perchloric acid and methanol in the ratio 20:80 (v/v), respectively, as solvent B. The experiments were carried out at a flow rate of 0.4 ml/min and the detection wavelength was 240 nm. The compartment temperature of the column was set at 40°C and the injection volume was set at 2 μl. The main aim of the research was to develop a single UPLC assay method for promethazine (active ingredient) and preservatives in the oral solution of promethazine HCl and dextromethorphan HBr that contains promethazine (active ingredient) and methylparaben, propylparaben and sodium benzoate (preservatives). An assay of dextromethorphan HBr was developed and validated by another HPLC method. The drug and preservatives were eluted at retention times of 19.3 min for promethazine HCl, 9.3 min for methylparaben, 18.9 min for propylparaben and 8.9 min for sodium benzoate. Validation of the developed method was carried out as stated by the International Conference on Harmonization guidelines ICH Q2B and under USP<1225>. The analytical parameters verified specificity/selectivity, linearity, accuracy, ruggedness and robustness. The linearity ranges of promethazine HCL, methylparaben, propylparaben and sodium benzoate were 10-100, 10-80, 1.0-8.0 and 10-80 μg/ml, respectively, with a correlation coefficient of active ingredients and preservatives of 1.00. Percentage recoveries of promethazine, propylparaben, methylparaben, and sodium benzoate were 100.0-100.2, 99.0-100.3, 99.5-98.0 and 99.0-100.0%. The validated analytical method proves that the method is specific, precise, linear, accurate, sensitive, rugged and stable, indicating the quantification of the active ingredient and all preservatives in liquid oral formulations.

Diode Array Detector Based RP-UPLC Method for Simultaneous Estimation of Dapagliflozin Propanediol Monohydrate and Metformin

A simple, rapid, accurate, precise, and reproducible diode array detector-based RP-UPLC method for simultaneous estimation of dapagliflozin propanediol monohydrate and metformin hydrochloride in bulk and tablet dosage form has been developed and validated. The chromatographic analysis was carried out using Acquity UPLC HSS T3 C18 (50 × 2.1mm, 1.8μm) column with a mobile phase consisting of orthophosphoric acid (pH 3.00) and acetonitrile (55:45% V/V) in the isocratic mode, at a flow rate of 0.2mL/min, and eluents were the simultaneous measurement of dapagliflozin propanediol monohydrate and metformin hydrochloride in bulk and tablet dosage form using a diode array detector-based RP-UPLC technique has been developed and validated. The chromatographic analysis was carried out using an Acquity UPLC HSS T3 C18 (50 2.1mm, 1.8 m) column and an orthophosphoric acid (pH 3.00) and acetonitrile (55:45% V/V) mobile phase in the isocratic mode.at a flow rate of 0.2mL/min. Eluents were measured at 222nm. Metformin hydrochloride and dapagliflozin propanediol monohydrate were shown to have retention times of 0.845 minutes and 1.889 minutes, respectively. The linearity was established in the range of 5-15μg/mL for dapagliflozin propanediol monohydrate and 250-750μg/mL for metformin hydrochloride, which had a regression coefficient (r2) greater than 0.999. The % Recovery was found to be 98.77% - 99.57% and 99.15% -101.2% for dapagliflozin propanediol monohydrate and metformin hydrochloride respectively. In a short period,a larger number of samplescan be analyzed due to their short retention time and, therefore, will be economical for routine analysis in the pharmaceutical industry.

Assessment of pheniramine in alternative biological matrices by liquid chromatography tandem mass spectrometry.

Pheniramine is an over-the-counter antihistamine drug. Its accessibility and low cost made it more popular among drug abusers in Pakistan. In this study, pheniramine was quantified in both conventional and alternative specimens of twenty chronic drug abusers, aged 16-50years, who were positive for pheniramine in comprehensive toxicological screening for drugs by gas chromatography with mass spectral detection in positive electron impact mode. Pheniramine was extracted from biological specimens using solid phase extraction and liquid chromatography tandem mass spectrometry was employed for quantification. Chromatographic separation was carried out on a Poroshell120EC-18 (2.1mm × 50mm × 2.7µm) column using water-acetonitrile in formic acid (0.1%) mobile phase in gradient elution mode with 500μL/min flow rate. Positive electrospray ionization mode and multi-reaction monitoring with ion transitionsm/z241.3 → 195.8 and 167.1 for pheniramine andm/zm/z247.6 → 173.1 for pheniramine-d6 were employed. The quantification method showed good linear ranges of 2-1000ng/mL in blood, urine, and oral fluid; 2-1000ng/mg in hair and 5-1000ng/mg in nail with ≥ 0.985% coefficient of linearity. The retention time of pheniramine was 3.0 ± 0.1min. The detection and lower quantification limits were 1ng/mL and 2ng/mL for blood, urine, oral fluid and hair whereas 2.5ng/mg and 5ng/mg for nail, respectively. Mean extraction recovery and ionization suppression ranged 86.3-95.1% and -4.6 to -14.4% in the studied matrices. Intra-day and inter-day precision were 4.1-9.3% and 2.8-11.2%, respectively. Pheniramine levels in specimens of drug abusers were 23-480ng/mL in blood, 72-735ng/mL in urine, 25-379ng/mL in oral fluid, 10-170ng/mg in hair and 8-86ng/mg in nail specimens. Alternative specimens are of utmost significance in clinical and medico-legal cases. In this study, authors compared matrix-matched calibration curves to blood calibration curve and obtained results within ± 10%; thereby justifying the use of blood calibration curve for urine, oral fluid, hair, and nail specimens.

Mechanistic studies to understand peak tailing due to sulfinic acid- and carboxylic acid-silanophilic interactions in reversed-phase liquid chromatography

Silanophilic interactions are a primary contributor to peak tailing of acidic pharmaceutical compounds, thus a thorough understanding is especially important for reversed-phase liquid chromatography (RPLC) method development. Herein, a sulfinic acid compound that exhibited severe peak tailing in RPLC with acidic mobile phases was carefully studied. Results indicated that the neutral protonated form of the sulfinic acid is involved in the strong interaction that leads to peak tailing, but that tailing can be mitigated with a blocking effect achieved through use of acetic acid modifier in the mobile phase. Peak tailing was also observed with other structurally-similar sulfinic acids and carboxylic acids but was, in general, less severe with the latter. The Hydrophobic Subtraction Model (HSM) was applied to six commercial C18 columns that exhibited different tailing behaviors for the sulfinic acid compound in attempts to identify key sites of interaction within the stationary phase. A combination of heated acid column wash experiments and density functional theory (DFT) calculations indicate that the differential interactions of the acids with vicinal silanol pairs in the stationary phase play a major role in peak tailing.

Evaluation of Iron Chlorin e6 disappearance and hydrolysis in soil and garlic using salting-out assisted liquid-liquid extraction coupled with high-performance liquid chromatography and ultraviolet-visible detection

Iron Chlorin e6 (ICE6), a star plant growth regulator (PGR) with independent intellectual property rights in China, has demonstrated its efficacy through numerous field experiments. We innovatively employed salting-out assisted liquid-liquid extraction (SALLE) with HPLC-UV/Vis to detect ICE6 residues in water, soil, garlic seeds, and sprouts. Using methanol and a C18 column with acetonitrile: 0.1% phosphoric acid mobile phase (55:45, v:v), we achieved a low LOQ of 0.43 to 0.77 μg kg−1. Calibration curves showed strong linearity (R2 > 0.992) within 0.01 to 5.00 mg kg−1. Inter-day and intra-day recoveries (0.05 to 0.50 mg kg−1) demonstrated high sensitivity and accuracy (recoveries: 75.36% to 107.86%; RSD: 1.03% to 8.78%). Additionally, density functional theory (DFT) analysis aligned UV/Vis spectra and indicated ICE6's first-order degradation (2.03 to 4.94 days) under various environmental conditions, mainly driven by abiotic degradation. This study enhances understanding of ICE6's environmental behavior, aids in risk assessment, and guides responsible use in agroecosystems.

A UPLC method development and validation study of Upadacitinib and its impurities in extended – release oral tablet dosage forms

The primary objective was to develop a concomitant isocratic ultra-performance liquid chromatographic photo-diode array detection method to estimate Upadacitinib and its process-related impurities: impurity-1 and impurity-2. Further validation was conducted and studied for possible degradants under stress environments. All the chemicals and reagents used were of HPLC (acetonitrile, methanol) and analytical grade (trifluoro acetic acid). The ultra-performance liquid chromatography (Agilent 1290 Infinity II LC system) consists of a quaternary pump, a BEH C18 (50×2.1mm, 1.7μ) column, and photo-diode array detector. The method was developed with acetonitrile: methanol: 0.1% v/v trifluoro acetic acid (50:20:30 v/v/v) mobile phase at 0.2mL/min flow rate within a run time of 5.5min The detection was carried at 231.2nm. The respective retention times achieved were 2.289min (Upadacitinib), 0.972min (Upadacitinib impurity-1), and 3.508min (Upadacitinib impurity-2). The optimized method was validated further, and the linearity range was best fit at 15.0-180.0μg/mL for Upadacitinib and 1.0-12.0μg/mL for both Upadacitinib impurity-1 and 2 respectively. The detection and quantification limits were 4.50μg/mL, 15.00μg/mL (Upadacitinib) and 0.30μg/mL, 1.0μg/mL (Upadacitinib impurity-1 and 2). A fast, isocratic, specific, and reproducible ultra-performance liquid chromatographic method was developed and validated for various parameters according to the ICH Q2 (R1) guidelines studies. Stress studies were conducted exposing the sample dilution to various treatments (acid, alkali, peroxide, HPLC water, heat, and UV light). The degradants were well-separated apart from the peaks of the active substance. The stability indicating nature was observed during the degradation. The optimized method can be applied for the separation and estimation of Upadacitinib and its process-related impurities in pharma sector in tablet dosage forms.

Development and validation of an UPLC-MS/MS method for the determination of irinotecan (CPT-11), SN-38 and SN-38 glucuronide in human plasma and peritoneal tumor tissue from patients with peritoneal carcinomatosis

Irinotecan (CPT-11), an antineoplastic drug, is used for the treatment of colorectal and pancreatic cancer due to its topoisomerase I inhibitory activity. CPT-11 is a prodrug which is converted to its active metabolite SN-38 by carboxylesterases. SN-38 is further metabolized to its inactive metabolite SN-38 glucuronide. When evaluating the pharmacokinetic properties of CPT-11 and its metabolites, it is important to accurately assess the concentrations in both plasma as well as tumor tissues. Therefore, the aim of the current study was to develop and validate a robust and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method to quantify the concentration of CPT-11 and its metabolites (SN-38 and SN-38 glucuronide) in human plasma and peritoneal tumor tissue. The sample preparation of plasma and tumor tissue consisted of protein precipitation and enzymatic digestion/liquid–liquid extraction, respectively. Chromatographic separation was achieved with an Acquity UPLC BEH C18 column combined with a VanGuard pre-column. The mobile phases consisted of water +0.1 % formic acid (mobile phase A) and acetonitrile +0.1 % formic acid (mobile phase B). Mass analysis was performed using a Xevo TQS tandem mass spectrometer in the positive electrospray ionization mode. Method validation was successfully performed by assessing linearity, precision and accuracy, lower limit of quantification, carry over, selectivity, matrix effect and stability according to the following guidelines: “Committee for Medicinal Products for Human use, Guideline on Bioanalytical Method Validation”. A cross-validation of the developed method was performed in a pilot pharmacokinetic study, demonstrating the usefulness of the current method to quantify CPT-11 and its metabolites in the different matrices.

Optimization of ultrasound-assisted extraction of glycyrrhizic acid and glabridin from Glycyrrhiza glabra L. using response surface methodology and development of a green analytical method

In this study, ultrasound assisted extraction (UAE) were used to investigate the extraction of glycyrrhizic acid (GA) and glabridin (GD) simultaneously from Glycyrrhiza glabra L. and a green HPLC analytical approach for determining GA and GD was developed. Extraction pocess were optimizing by Response surface methodology (RSM) utilizing Box-Behnken design (BBD). According to the consequences, the ideal extraction situations were: 64% (v/v) ethanol concentration, 22 mL/g liquid-solid ratio, 51 °C and 31 min. Under these conditions, the yields of GA and GD were 2.806 ± 0.107% and 0.252 ± 0.026%, respectively, and the actual experimental extraction values were fundamentally the same as the anticipated values. Meanwhile, the effects of UAE, maceration (MAC) and heat-reflux extraction (HRE) on yeild were assessed, and the UAE possessed highest yields. A green HPLC approach was created to analyze GA and GD with combinations of ethanol and a 0.1% (v/v) trifluoroacetic acid mobile phase (following the gradient elution method). The method validated based on the guidelines of the International Conference on Harmonization (ICH) and the result indicated the method was precise, stable, reliable and valuable, could be used to analyze GA and GD from Glycyrrhiza glabra L.

Bio-analytical Method for the Pre-clinical Estimation of Donepezil Hydrochloride in Rabbit Plasma by RP-HPLC

A simple, reliable and sensitive RP-HPLC bioanalytical method with PDA as detector was developed for the estimation of donepezil HCl in the rabbit plasma and validated. The biological samples were extracted and analysed using loratadine as internal standard. The sample analysed with Inertsil ODS 150mm x 4.6mm, 5.0µm particle size column using 70:30 v/v of 0.1% trifluoroacetic acid and acetonitrile mobile phase at flow rate of 1mL/min. All the parameters validated were within the limits proposed in bioanalytical method validation guidelines. The linearity was observed in the range of 5-200ng/mL with LOD of 0.9ng/mL. The method was observed to have short analysis time of 6 min and also found to be successful in studying the pharmacokinetic parameters of donepezil HCl.

Analysis of D-amino acids secreted from murine islets of Langerhans using Marfey’s reagent and reversed phase LC-MS/MS

D-amino acids (D-AAs) are important signaling molecules due to their ability to bind ionotropic N-methyl-D-aspartate receptors. D-serine (D-Ser), D-alanine (D-Ala), and D-aspartate (D-Asp) have been found individually in the endocrine portion of the pancreas, the islets of Langerhans, and/or their secretions. However, there has been no report of a comprehensive assessment of D-AAs in islet secretions. To evaluate the release of these compounds, the effectiveness of both 1-(9-fluorenyl)-ethyl chloroformate (FLEC reagent) and 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey’s reagent, MR) in separation of D/L-AA enantiomeric pairs in islet-specific buffers were evaluated. MR-derivatized D/L AAs showed greater than baseline resolution (Rs ≥ 1.5) of 13 enantiomeric pairs when using a non-linear gradient and an acidic mobile phase system, while FLEC-derivatized AAs exhibited limited resolution on both biphenyl and C18 columns. The optimized MR method yielded highly reproducible separations with retention times less than 1% RSD. Excellent linearity between the analyte concentrations and response (R2 > 0.98) were obtained, with less than 15% RSD for all analyte responses. Most analytes had an LOD at or below 100 nM, except for L-Ala (200 nM). The optimized MR method was used to quantify D-AAs in secretions of 150 murine islets after incubation in 3- and 20-mM glucose. In response to both solutions, D-Ser and D-glutamine were tentatively identified via comparison of retention time and quantifier-to-qualifer ion ratios with standards, and from spiking experiments. Both were secreted in low quantities which did not differ significantly in either low (D-Ser: 44 ± 2 fmol islet-1h−1; D-Gln: 300 ± 100 fmol islet-1h−1) or high (D-Ser: 23 ± 1 fmol islet-1h−1; D-Gln: 120 ± 50 fmol islet-1h−1) glucose across 3 biological replicates. The method developed is robust and can be applied to further examine the release of D-AAs and their potential roles in islet physiology.

An improved ultra-high performance liquid chromatography-tandem mass spectrometry method determining hispidulin and homoplantaginin in rat plasma and associated pharmacokinetic studies

AbstractFlavonoids are the most abundant components in Salvia plebeia, with significant pharmacological antioxidant and hepatoprotective properties. Hispidulin and homoplantaginin are the main flavonoid components in S. Plebeia. In this study, we established an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to determine hispidulin and homoplantaginin in rat plasma samples, which were precipitated using acetonitrile-methanol (9:1, v/v). We used a UPLC HSS T3 (100 mm × 2.1 mm, 1.7 μm diameter) chromatographic column, an acetonitrile-water (containing 0.1% formic acid) mobile phase, and a gradient elution flow rate of 0.4 mL min−1 in an elution time of 4 min. We used electrospray ionization (ESI) detection in positive ion mode, and multiple reaction monitoring mode (MRM) for quantitative analysis: m/z 301 → 286 for hispidulin, m/z 463 → 301 for homoplantaginin, and m/z 465 → 303 for internal standard (IS). In pharmacokinetic studies, 24 rats were orally administered hispidulin and homoplantaginin (5 mg kg−1) and received sublingual intravenous injections (1 mg kg−1) at two different doses, four groups with six rats/group. Differences in hispidulin and homoplantaginin pharmacokinetics in rat plasma were evaluated. The calibration curve showed good linearity in the 0.5–1,000 ng mL−1 range, with r &gt; 0.99. Precision, accuracy, recovery, matrix effects, and stability results all met standard biological sample detection requirements. Our pharmacokinetic studies showed hispidulin bioavailability was much higher than homoplantaginin at 17.8% and 0.1%, respectively.

Challenges with retention and recovery of impurities containing acidic moieties during analytical UHPLC method development and validation for gefapixant freebase

Gefapixant citrate is a P2X3 purinergic receptor antagonist developed for the treatment of chronic cough. Gefapixant freebase is the penultimate intermediate in the commercial manufacturing route for gefapixant citrate and contains a complex impurity profile consisting of acidic and basic analytes. A UHPLC method was developed for assay and purity determination of gefapixant freebase utilizing a Waters Acquity Charged Surface Hybrid (CSH) C18 column (2.1 mm I.D. x 10 cm length, 1.7 µm particle size) with 0.1 % phosphoric acid and acetonitrile as the mobile phases. Method optimization was performed using ACD Labs LC Simulator to achieve baseline separation of all impurities and the method was successfully validated. During routine use and method transfer for gefapixant freebase, an increase in retention time for impurities containing strongly acidic functional groups, and poor recovery of a sulfinic acid impurity were observed. Subsequent investigation determined that the CSH column aging resulting from exposure of the column packing to the acidic mobile phase was the root cause for these behaviors. The mechanism of peak-shifting was further investigated using model compounds and determined to be due to an increase in ionic interactions with the CSH stationary phase with routine column use. The increase in ionic interactions was demonstrated to correlate with the charge state of the analyte. Poor recovery for the sulfinic acid impurity was attributed to increased peak tailing for this single impurity on older columns. This knowledge was leveraged to establish additional system suitability requirements to monitor column performance for the lifecycle of the analytical procedure.

UPLC-PDA Approach for Simultaneous Quantification of Elvitegravir, Tenofovir, Emtricitabine and Cobicistat in Tablets: An MS-ESI Study for Degradation Products.

A simultaneous ultra-performance liquid chromatography photodiode array detection method was developed and validated for Elvitegravir, Tenofovir and Emtricitabine and Cobicistat drugs in tablet dosage forms. Effective liquid chromatographic quantification was achieved using a Luna C18 (100 × 2.6mm, 1.6μ) column with an acetonitrile: 0.1% v/v trifluoro acetic acid (80:20% v/v) mobile phase and a photo diode array detector at 260nm for a shorter run time of 3min. The respective retention times for Elvitegravir, Tenofovir, Emtricitabine and Cobicistat were 0.311, 0.792, 1.379 and 2.045min. The range of linearity was 37.50-225.00, 2.50-15.00, 50.00-300.00and 37.50-225.00μg/mL for Elvitegravir, Tenofovir, Emtricitabine and Cobicistat, respectively. A mass spectrometer was employed for quantitative analysis using electron spray ionization multiple reaction monitoring in positive mode. The developed method was validated for different parameters to meet the acceptance criteria as per ICH Q2 (R1) guidelines. Using UPLC-ESI-MS, 12 degradants were identified and their mechanisms were studied.