Rapid High-performance Liquid Chromatography Research Articles

Determination of Tedizolid in Bacterial Growth Medium Mueller-Hinton Broth by High-Performance Liquid Chromatography and Its Application to an In Vitro Study in the Hollow-Fiber Infection Model

Pharmacokinetic/pharmacodynamic (PKPD) studies of anti-infectives are frequently performed in in vitro infection models where accurate quantification of antibiotic concentrations in bacterial growth media is crucial to establish PK/PD relationships. Here, a sensitive and rapid high-performance liquid chromatography (HPLC) method was developed to quantify tedizolid (TDZ) in the bacterial growth medium Mueller-Hinton broth (MHB). Matrix components were separated by direct protein precipitation with methanol (1:1). The chromatographic separation was carried out in a Dionex Ultimate 3000 HPLC system using an Accucore® C-18 RPMS HPLC column (2.6 µm, 100 × 2.1 mm) using isocratic elution with 25% acetonitrile and 75% of 0.1% formic acid. The lower limit of quantification was 0.03 mg/L when measured at 300 nm. Following relevant European Medicine Agency guidelines, the method was successfully validated for linearity, selectivity, recovery, inter- and intra-day precision, and accuracy and stability. When applied to in vitro PKPD studies, the method successfully quantified a range of TDZ concentration (Cmin, 0.09-Cmax, 0.65 mg/L) in MHB. The analyzed concentrations were in line with the planned PK profiles. The application of the developed method to quantify TDZ in MHB in in vitro PKPD studies is warranted.

A rapid ultra high performance liquid chromatography-tandem mass spectrometry method for the quantification of daidzein, its valine carbamate prodrug, and glucuronide in rat plasma samples: Comparison of the pharmacokinetic behavior of daidzine valine carbamate prodrugs.

Two valine carbamate prodrugs of daidzein were designed to improve its bioavailability. To compare the pharmacokinetic behavior of these prodrugs with different protected phenolic hydroxyl groups of daidzein, a rapid and sensitive method for simultaneous quantification of daidzein, its valine carbamate prodrug, and daidzein-7-O-glucuronide in rat plasma was developed and validated in this study. The samples were processed using a fast one-step protein precipitation method with methanol added to 50 μL of plasma and were analyzed by ultra-high performance liquid chromatography with tandem mass spectrometry. To improve the selectivity, peak shape, and peak elution, several key factors, especially stationary phase and the composition of the mobile phase, were tested, and the analysis was performed using the Kinetex® C18 column (100 × 2.1mm, 2.6 μm) within only 2.6min under optimal conditions. The established method exhibited good linearity over the concentration range of 2.0-1000ng/mL for daidzein, and 8.0-4000ng/mL for the prodrug and daidzein-7-O-glucuronide. The accuracy of the quality control samples was between 95.5 and 110.2% with satisfactory intra- and interday precision (relative standard deviation values<10.85%), respectively. This sensitive, rapid, low-cost, and high-throughput method was successfully applied to compare the pharmacokinetic behavior of different daidzein carbamate prodrugs.

Method Development and Validation of Salmeterol xinofoate by HPLC

A rapid and precise high performance liquid chromatography method has been developed for the validation of Salmeterol xinofoate in its pure dosage form. The separation was carried out on Agilent Zorbax Bonus RP- (250mm ×4.6mm 5μ) column with a mobile phase consisting of 0.1% Formic acid: Acetonitrile in the ratio of 64:36 v/v as a mobile phase and flow rate is 1ml/min. The detection was carried out at wavelength 234nm. The column thermostatically controlled at 30℃. The retention time of Salmeterol was found to be 1.96 min. The Salmeterol xinofoate followed linearity in the concentration range of 40-60μg/mL with r2= 0.999. The developed method was validated for sensitivity, accuracy and precision. The sample was scanned from 200- 400nm with PDA detector. The % recovery of sample was found to be. The LOD and LOQ of the Salmeterol xinofoate was found to be 2.67μg/ml and 8.08μg/ml respectively. The suitability of this HPLC method for quantitative estimation of Salmeterol xinofoate was proved by validation by the requirements of ICH guidelines.

Development and Validation of Ecofriendly HPLC-MS Method for Quantitative Assay of Amoxicillin, Dicloxacillin, and Their Official Impurity in Pure and Dosage Forms.

Novel, accurate, selective, and rapid high-performance liquid chromatography mass spectrometry method was developed for simultaneous analysis of amoxicillin trihydrate, dicloxacillin sodium, and their official impurity 6-aminopenicillanic acid. The chromatographic separation was carried out by applying the mixture on a C18 column (3.5 µm ps, 100 mm × 4.6 mm id) using acetonitrile:water (65 : 35 by volume) as a mobile phase within only 4 min. The quantitative analysis was executed using single quadrupole mass spectrometer in which electrospray ionization, selected ion monitoring, and negative mode were operated. The retention times were 1.61, 2.54, and 3.50 mins for amoxicillin, 6-aminopenicillanic acid, and dicloxacillin, respectively. The method was validated in linear ranges of 2–28 µg mL−1, 2–35 µg mL−1, and 1–10 µg mL−1 for amoxicillin, dicloxacillin, and 6-aminopenicillanic acid, respectively. The results obtained from the suggested HPLC/MS were statistically compared with those obtained from the reported HPLC method, where no significant difference appeared respecting accuracy and precision. According to the analytical eco-scale assessment method, the proposed method was proved to be greener than the reported one, where the analysis time and the amount of the wasted effluent decreased.

Study of Pharmaceutical Excipient PEG400 Alteration of Pharmacokinetics and Tissue Distribution of Main Flavonoids Metabolites of Baicalin

Background:: Polyethylene glycol 400 (PEG400), as a good traditional Chinese medicine solvent, diluent and solubilizer, is widely used as a main pharmaceutical excipient in traditional Chinese medicine compound preparations containing active ingredient baicalin. PEG400 could increase the solubility and release of baicalin in vivo, but it was unknown that PEG400 affected the absorption and distribution of baicalin or not. Objective: : At present, the effects of PEG400 on the pharmacokinetic characteristics and tissue distribution behaviors of the main flavonoid metabolites baicalin, baicalein 6-O-β-D-glucopyranoside (B6G) and baicalein after oral administration of baicalin were investigated by a rapid, efficient and sensitive ultra- high performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method. Moreover, we respectively studied the effects of PEG400 on the activities and protein expressions of two subtypes UDP-glucuronyltransferase 1 A8/A9 (UGT1A8 and UGT1A9) of UDP-glucuronosyltransferases (UGTs) in vitro and in vivo experiments to determine the partial mechanisms by which PEG400 altered the pharmacokinetics and tissue distribution behaviors of the three flavonoid metabolites. Methods: A rapid, efficient and sensitive ultra-high performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method and ELISA and so on. Results: The results showed that PEG400 significantly increased the Cmax and AUC0-t values (P &lt; 0.05 or P &lt; 0.01) of baicalin and B6G while baicalein could not be quantified due to its extremely low concentration (lower the LLOQ) in plasma. Baicalin, B6G and baicalein were mainly distributed in the stomach, small intestine, kidney and liver. PEG400 changed the distribution of three flavonoid metabolites in various tissues and also increased the activities and expressions of UGT1A8 and UGT1A9. Conclusion:: In conclusion, PEG400 significantly altered the pharmacokinetic characteristics and tissue distribution behaviors of three flavonoid metabolites may partly result from PEG400 upregulated the activities and expressions of the drug biphasic metabolic enzymes UGT1A8 and UGT1A9, which provided a material basis and useful information to reveal the mechanism of action and clinical application of PEG400.

Pharmacokinetic study of an anti-trypanosome agent with different formulations and administration routes in mice by HPLC-MS/MS.

Previously compound 12 showed great anti-trypanosome activity without toxicity in an in vivo study. In the current study, a sensitive and rapid high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated to investigate its pharmacokinetics in mouse plasma. A protein precipitation method was applied to extract the compound, and it was then separated using a Kinetex C18 column with mobile phase consisting of acetonitrile-0.1% formic acid water (50:50, v/v) at a flow rate of 300 μl/min. The analytes were detected with the multiple reaction monitoring in negative electrospray ionization source for quantitative response of the compounds. Compound 12 was detected at m/z 477.0 → 367.2, while the internal standard compound 14 was detected at m/z 499.2 → 268.2. Inter- and intra-day precision was <5.22 and 2.79% respectively, while the accuracy range was within ±9.65%. The method was successfully applied to evaluate the pharmacokinetics of compound 12 in mouse plasma with two formulations (20% Cremophor EL or sesame oil) and drug administration routes (oral and intraperitoneal injection). We observed a better drug serum concentration with the Cremophor formulation, and the two different drug administration routes did not show significant differences from the drug distribution.

Chromatographic determination of siphonodin content: A rapid and simple strategy for discriminating between Hemsleya omeiensis and other sources of Xuedan

Purpose: To develop a rapid and simple siphonodin content-based high performance liquid chromatography (HPLC) method to distinguish Hemsleya omeiensis from other sources of xuedan.&#x0D; Methods: Siphonodin was isolated from Hemsleya omeiensis and identified by x-ray crystallographic analysis. An optimized HPLC method was applied for the determination of siphonodin contents of H. omeiensis, H. dolichocarpa and H. gigantha.&#x0D; Results: Siphonodin was successfully separated by the optimized HPLC method in &lt; 10 min, and the results of validation showed that the HPLC method was stable and very accurate for the quantification of siphonodin. The mean content of siphonodin in 10 batches of H. omeiensis was 3.78 mg/g, but the compound was not detectable in H. dolichocarpa and H. gigantha using the developed HPLC method.&#x0D; Conclusion: These results indicate that the developed HPLC method is suitable for distinguishing H. omeiensis from other sources of xuedan.

Concurrent estimation of lamivudine, tenofovir disoproxil fumarate, and efavirenz in blended mixture and triple combination tablet formulation by a new stability indicating RP-HPLC method

BackgroundAn easy, defined, rapid, and accurate reverse phase high-performance liquid chromatography method was developed and subsequently validated for the concurrent estimation of lamivudine, efavirenz, and tenofovir disoproxil fumarate in their pure blend and combined tablet formulation. An efficient and appropriate separation of the three analytes was attained with Zorbax eclipse XDB-Phenyl column, with a mobile phase of methanol: buffer (0.1% v/v formic acid in water) (73:27 v/v) at a flow rate of 1mL/min and isocratic elution by using 260nm as detection wavelength. Equal ratio of acetonitrile and water was used as diluent.ResultsThe retention times of lamivudine, tenofovir disoproxil fumarate, and efavirenz were found at 2.6, 4.4, and 5.9 min respectively. The linear response for lamivudine, tenofovir disoproxil fumarate, and efavirenz was in the range of 15.0–45.0μg/mL, 15.0–45.0μg/mL, and 20.0–60.0 μg/mL respectively. The method validation was done in accordance to ICH guidelines and all validation parameters in compliance with ICH standards. The degradants produced by stress testing were well resolved from the peaks of active analytes, which stipulates the stability-indicating property of the method.ConclusionThe method has the ability to separate lamivudine, efavirenz, and tenofovir disoproxil fumarate concurrently in blended powder and their combined tablet. All degradants produced by application of stress conditions were separated with high resolution and determined with good sensitivity that ensures the stability-indicating property of the method. Thus, the projected method has high probability to adopt in the pharmaceutical industrial sector.

HPLC-UV method for quantification of favipiravir in pharmaceutical formulations

AbstractFavipiravir (FVP), a pyrazine analog, has shown antiviral activity against a wide variety of viruses. It is considered to be worth further investigation as a potential candidate drug for COVID-19. It is not officially available in any pharmacopoeia. A rapid, simple, precise, accurate, and isocratic high performance liquid chromatography (HPLC) method has been developed for routine quality control of favipiravir in pharmaceutical formulations. Separation was carried out by C18 column. The mobile phase was a mixture of 50 mM potassium dihydrogen phosphate (pH 2.3) and acetonitrile (90:10, v/v) at a flow rate of 1 mL min−1. The ultraviolet (UV) detection and column temperature were 323 nm, and 30 °C, respectively. The run time was 15 min under these chromatographic conditions. Excellent linear relationship between peak area and favipiravir concentration in the range of 10–100 μg mL−1 has been observed (r2, 0.9999). Developed method has been found to be sensitive (limits of detection and quantification were 1.20 μg mL−1 and 3.60 μg mL−1, respectively), precise (the interday and intraday relative standard deviation (RSD) values for peak area and retention time were less than 0.4 and 0.2%, respectively), accurate (recovery, 99.19–100.17%), specific and robust (% RSD were less than 1.00, for system suitability parameters). Proposed method has been successfully applied for quantification of favipiravir in pharmaceutical formulations.

Rapid size-exclusion high performance liquid chromatography method for the quality control of amyloid-β oligomers

Amyloid-β (Aβ) dysmetabolism is thought to be the main trigger for neurodegenerative events in Alzheimer's disease (AD). In particular, soluble Aβ oligomers (AβOs) are proposed as key mediators of synaptic and cognitive dysfunction in AD. Over the past few decades, AβOs prepared from synthetic Aβ have been widely applied in vitro and in vivo, the so-called chemical models of AD, uncovering their multiple neurotoxic mechanisms. However, the lack of a reliable quality control (QC) for synthetic AβOs may reflect poor experimental reproducibility. In keeping with this, we optimized and validated a rapid and reproducible SECHPLC method using fluorescence detection for the QC of synthetic AβOs. Our analytical method offers an unprecedent alternative to improve the reproducibility of AD chemical models.

Determination of finerenone – a novel, selective, nonsteroidal mineralocorticoid receptor antagonist – in human plasma by high-performance liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study in venous and capillary human plasma

A straightforward and rapid high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) assay allowing the sensitive and selective quantitation of finerenone (BAY 94–8862) in lithium heparin human plasma is described. Finerenone is a novel, selective, nonsteroidal mineralocorticoid receptor antagonist that is in phase III clinical trials for the treatment of chronic kidney disease. Finerenone quantitation is performed after addition of its stable isotope-labelled internal standard (ISTD) by protein precipitation with acidified acetonitrile followed by HPLC–MS/MS separation and detection. The determination of finerenone concentrations was validated for a plasma volume of 0.100 mL and subsequently also for a lower plasma volume of 0.010 mL, collected e.g. in paediatric studies. The analytical range was from 0.100 µg/L (lower limit of quantification) to 200 µg/L (upper limit of quantification). Inter-day accuracy was 99.7–105.0% for the plasma volume of 0.100 mL and 101.1–104.5% for the plasma volume of 0.010 mL. Inter-day precision was ≤ 7.0%, independent of the extracted plasma volume. A moderate, concentration-independent matrix effect on ionisation was observed for both finerenone and its ISTD of 0.535–0.617, which is fully compensated by the ISTD (ISTD-normalised matrix factors were 0.98–1.03). The assay was successfully applied with both validated plasma volumes to a clinical phase I study in which the pharmacokinetics of 20 mg finerenone were compared in capillary plasma (0.010 mL) and venous plasma (0.100 mL) in a concentration range from the lower limit of quantification to 310 µg/L (capillary plasma) and 252 µg/L (venous plasma). The area under the plasma concentration versus time curve was similar in both matrices, while maximum concentrations were 37% higher in capillary plasma. In conclusion, capillary sampling should not bias pharmacokinetic exposure estimates compared with venous plasma values, if limited to sampling times in the distribution and elimination phases of finerenone.

Rapid and simultaneous determination of dexamethasone and dexamethasone sodium phosphate using HPLC-UV: Application in microneedle-assisted skin permeation and deposition studies

Dexamethasone is a fluorinated derivative of the natural glucocorticoid, cortisone, with a very high systemic anti-inflammatory effect. In this study, a simple and rapid high performance liquid chromatography (HPLC) method was developed and validated to quantify dexamethasone and its prodrug dexamethasone sodium phosphate in skin permeation studies. The separation of both the compounds was achieved on a Vydac Denali C18 column(250 × 4.6 mm, 5 μm) with a mobile phase composed of 5 mM ammonium acetate buffer-acetonitrile-methanol (43:32:25, v/v) at a flow rate of 0.9 mL/min and UV detection at 240 nm. The standard curves were found to be linear in the range from 0.5 to 100 µg/mL for both the drugs and the method could successfully separate the drug peaks from interfering peaks of endogenous skin constituents. Accuracy values of both the drugs were within 98.60 to 108.60% (intra-day) and 98.70 to 107.20% (inter-day) and precision values were within 2% at the studied concentrations. The developed method was used to investigate the effect of microneedles on transdermal delivery of dexamethasone sodium phosphate. The hydrolysis of dexamethasone sodium phosphate to dexamethasone in the presence of rat skin homogenate and rat plasma was also evaluated to confirm the conversion that occurs during skin permeation and in the blood circulation. The skin permeation and deposition characteristics of microneedle-assisted diffusion were compared to those achieved by passive diffusion. The observed data demonstrated that transdermal permeation of dexamethasone is significantly enhanced with microneedle pretreatment of rat skin, showing a marked increase in flux and permeability coefficient, compared to passive diffusion. This simple isocratic HPLC method can, be effectively applied for the evaluation of skin permeation of topical/transdermal dexamethasone formulations.

Plasma d-glutamate levels for detecting mild cognitive impairment and Alzheimer's disease: Machine learning approaches.

d-glutamate, which is involved in N-methyl-d-aspartate receptor modulation, may be associated with cognitive ageing. This study aimed to use peripheral plasma d-glutamate levels to differentiate patients with mild cognitive impairment (MCI) and Alzheimer's disease (AD) from healthy individuals and to evaluate its prediction ability using machine learning. Overall, 31 healthy controls, 21 patients with MCI and 133 patients with AD were recruited. Serum d-glutamate levels were measured using high-performance liquid chromatography (HPLC). Cognitive deficit severity was assessed using the Clinical Dementia Rating scale and the Mini-Mental Status Examination (MMSE). We employed four machine learning algorithms (support vector machine, logistic regression, random forest and naïve Bayes) to build an optimal predictive model to distinguish patients with MCI or AD from healthy controls. The MCI and AD groups had lower plasma d-glutamate levels (1097.79 ± 283.99 and 785.10 ± 720.06 ng/mL, respectively) compared to healthy controls (1620.08 ± 548.80 ng/mL). The naïve Bayes model and random forest model appeared to be the best models for determining MCI and AD susceptibility, respectively (area under the receiver operating characteristic curve: 0.8207 and 0.7900; sensitivity: 0.8438 and 0.6997; and specificity: 0.8158 and 0.9188, respectively). The total MMSE score was positively correlated with d-glutamate levels (r = 0.368, p < 0.001). Multivariate regression analysis indicated that d-glutamate levels were significantly associated with the total MMSE score (B = 0.003, 95% confidence interval 0.002-0.005, p < 0.001). Peripheral plasma d-glutamate levels were associated with cognitive impairment and may therefore be a suitable peripheral biomarker for detecting MCI and AD. Rapid and cost-effective HPLC for biomarkers and machine learning algorithms may assist physicians in diagnosing MCI and AD in outpatient clinics.

STABILITY INDICATING METHOD DEVELOPMENT AND VALIDATION OF FIMASARTAN BY REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY IN BULK AND PHARMACEUTICAL DOSAGE FORM

Objective: A rapid, sensitive and specific reverse phase High performance liquid Chromatography (RP-HPLC) method was developed for the estimation of Fimasartan in bulk and pharmaceutical dosage form.&#x0D; Method: The RP-HPLC analysis was performed isocratically on a Primacel C18 column (150 mm × 4.6 mm internal diameter, 5 μm particle size) using mobile phase of composition Acetonitrile and 0.1% orthophosphoric Acid in 80:20, v/v proportions with a flow rate of 0.8 ml/min.&#x0D; Results: The analyte was monitered with UV-detector at 265 nm. In the developed method Fimasartan elutes at a typical retention time of 2.4 min. The proposed method is having linearity in the concentration ranging from 5-30 μg/ml of Fimasartan.&#x0D; Conclusion: : The method was statistically validated and had been applied to analysis of the drug in bulk and pharmaceutical dosage form.

The Differences in the Flavan-3-ol and Procyanidin Contents of the Japanese 'Fuji' and 'Orin' Apples Using a Rapid Quantitative High-Performance Liquid Chromatography Method: Estimation of the Japanese Intake of Flavan-3-ols and Procyanidins from Apple as Case Study.

Previously, we reported that apple polyphenols and their major active compounds, the flavan-3-ols and the procyanidins, can result in various health benefits in animals and humans, according to clinical studies. Here, we developed a rapid method for quantifying flavan-3-ols and procyanidins using high-performance liquid chromatography with fluorescence detection, where we investigated the amounts of flavan-3-ols and procyanidins in the Japanese major apple production centre, the Aomori Prefecture, from 2016 to 2018. The non-bagged ‘Fuji (n = 609)’, the bagged ‘Fuji (n = 1101)’, and the ‘Orin (n = 504)’ apples were evaluated in terms of their differences in flavan-3-ols and procyanidins based on apple variety and the controlled atmosphere storage. The bagging treatments of the ‘Fuji’ apples resulted in significantly higher concentrations of procyanidins, while changes in flavan-3-ols concentrations were not clearly observed by treatment. In addition, ‘Orin’ had a significantly higher concentration of procyanidins than that of ‘Fuji’. In contrast, the controlled atmosphere storage hardly caused any changes in the flavan-3-ol and procyanidin contents. Hence, we present the concentrations of flavan-3-ols and procyanidins in major Japanese apples using the rapid high-performance liquid chromatography method with fluorescence detection.

Development and Validation of a Rapid Analytical Method for the Simultaneous Quantification of Metabolic Syndrome Drugs by HPLC-DAD Chromatography

Worldwide, 25% of the population suffers from metabolic syndrome (MetS). The treatment of patients with MetS regularly includes drugs prescribed simultaneously to treat several disorders that manifest at the same time, such as hypercholesterolemia, arterial hypertension, and diabetes. To the authors’ best knowledge, there is no previous published analytical method for the simultaneous quantification of drugs used in the treatment of these diseases. In the present study, a rapid high-performance liquid chromatography with a diode-array detector HPLC-DAD methodology was developed for simultaneous quantification of carvedilol (CVD), telmisartan (TEL), bezafibrate (BZT), gliclazide (GZD), and glimepiride (GMP) in bulk and pharmaceutical form. The chromatographic separation of the five pharmaceuticals was achieved on a Hypersil GOLD C18 Selectivity (5 µm, 150 × 4.60 mm2) using a mobile phase of acetonitrile (50%) and 0.02 M KH2PO4, pH 3 (50%) at a flow rate of 1 mL/min and at 25 °C. The total separation time was 9 min. The analytical method was validated following the International Conference on Harmonization guidelines. A reproducible method was obtained with acceptable limits of detection (LOD) and quantification (LOQ) for CVD (0.012 and 0.035 μg mL−1), TEL (0.103 and 0.313 μg mL−1), BZT (0.025 and 0.076 μg mL−1), GZD (0.039 and 0.117 μg mL−1), and GMP (0.064 and 0.127 μg mL−1). The validated method allowed the determination of these drugs in commercial pharmaceutical products both individually and simultaneously. The present method was found to be suitable for simultaneous quantification of the five drugs that are most commonly used in the simultaneous treatment of the metabolic syndrome.

Rapid and Simple Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) Method for Simultaneous Quantifications of Triazole Antifungals in Human Serum.

To develop and validate a one-step, rapid and simple reversed-phase high-performance liquid chromatography (HPLC)-based protocol for the simultaneous measurement of voriconazole (VCZ), posaconazole (POSA), itraconazole (ITC) in serum/plasma. Calibration standards (CS) and quality control samples were prepared in drug-free serum by spiking with the triazoles at different concentrations. HPLC was performed with C18 column, isocratic mobile phase after extraction with cold acetonitrile. The standardized method was tested in 2693 patients' serum/plasma samples. Linearity of CS for ITC, VCZ and POSA was proportional to the nominal concentration (correlation coefficient > 0.999). Limit of detection (mg/L) for ITC, VCZ and POSA was 0.25, 0.25 and 0.125, respectively. The lower limit of quantification (mg/L) for ITC, VCZ and POSA was 0.5, 0.5 and 0.25, respectively. Precision and accuracy were in acceptable range with 100% average percentage recovery. No interferences from endogenous substances and other antimicrobial compounds were noted. In clinical samples, the therapeutic range achieved for VCZ was 39.9%. Whereas, 61.1% and 44% of samples with ITC and POSA, respectively, were in the sub-therapeutic range. We developed a rapid and simple HPLC method to quantify common triazoles in a single chromatographic run allowing simultaneous measurement of different antifungals in a small volume of serum/plasma. Thus, therapeutic drug monitoring requests can be processed in one run without changing the protocol parameters, column or column conditioning thereby improving turnaround time.

Extraction and analysis of oleanolic acid and ursolic acid from apple processing waste materials using ultrasound-assisted extraction technique combined with high performance liquid chromatography

The aim of the present study was to develop a is simple, effective, eco-friendly, reproducible and high-yield two-stage ultrasound-assisted extraction (UAE) procedure combined with quantitative determination high performance liquid chromatographic (HPLC) method for obtaining isomeric triterpene acids - oleanolic acid (OA) and ursolic acid (UA) in the crystalline dried powdered form from apple processing agro-industrial waste material. A rapid, sensitive and specific HPLC method was developed and validated with respect to robustness, specificity, linearity-range, accuracy, precision and sensitivity. The effect of the nature and the volume of the extraction solvent, the extraction time and the sample size on the extraction efficiency were investigated. The optimal conditions for high-yield extraction were found.

Bioanalytical Method Development and Validation for Determination of Sulfasalazine in Rabbit Plasma by HPLC-UV

In present study, an advanced, simple and a rapid reverse phase high performance liquid chromatography (RP-HPLC) method was developed for the quantitative determination of sulfasalazine in rabbit plasma. Sulfasalazine was separated using Chromatopak C-18 basic peerless (250 mm × 4.6 mm, 5μ) column in an isocratic mode using mobile phase consisting of the mixture of 10mM Ammonium acetate pH adjusted to 4.5 and acetonitrile (70:30 v/v) with a flow rate of about 1.0 mL/min at ambient temperature. An ultra-violet detection of sulfasalazine and the internal standard was carried out at 362 nm. Both sulfasalazine and internal standard (IS, 4-hydroxy benzoate) were extracted from plasma matrices with high efficiency using a simple protein precipitation method. The method was found to be highly selective with no carryover effects. Linearity of sulfasalazine was found with the range of 2.5-100 μg/mL with the value of r2 &gt; 0.995 a correlation coefficient. At all three quality control levels, developed bioanalytical method was found as repeatable and reproducible as well. The average recoveries of sulfasalazine from plasma were in the range of 95.59-97.16%. The bioanalytical samples showed good and acceptable stability of sulfasalazine solution at different storage, packaging and handling conditions. Hence, in conclusion, the validated and developed HPLC-UV method could be effectively utilized for determination of sulfasalazine in pharmacokinetic studies involving novel formulations.

Development and Validation of a Reversed-phase High Performance Liquid Chromatography Method for the Simultaneous Estimation of Rosuvastatin Calcium and Telmisartan in Fixed-Dose Complex Dual-Layer Tablets in Six Dosage Forms

Fixed dose complex dual-layer tablets of telmisartan and rosuvastatin calcium have been used for the treatment of hypertension and hyperlipidemia. However, the current United States Pharmacopoeia analysis method is limited by the need for twice the time and cost owing to analysis for each active pharmaceutical ingredient present separately. Due to the critical limitations in establishing simultaneous analysis methods for both active pharmaceutical ingredients, there is an urgent need for an analysis method that can be applied for immediate use by researchers. The objective of this study was to develop and validate a simultaneous quantitative assay using a rapid and selective high performance liquid chromatography method for the analysis of rosuvastatin calcium and telmisartan in fixed dose combination dual-layer tablet dosage forms. This method used Kinetex C18 (5 µm, 4.6×150 mm) columns at a flow rate of 1.0 ml/ min and Ultraviolet-visible detector set at a wavelength of 242 nm. The separation was carried out using an ammonium phosphate monobasic buffer (pH 3.0) and methanol at a ratio of 300:700 as the mobile phase. The retention times of rosuvastatin calcium and telmisartan were 2.2 and 4.3 min respectively. The system suitability was 0.41 % for rosuvastatin calcium and 0.63 % for telmisartan, and the peak of each component showed complete separation from disturbance peaks. The correlation coefficient of the active pharmaceutical ingredients was approximately 0.999 when the concentration of rosuvastatin calcium was in the range of 4.44-26.6 µg/ml and the concentration of telmisartan was 0.176-106.6 µg/ml, this indicates good linearity. The recovery rate relative to the amount added was 97.0–99.6 % for rosuvastatin calcium and 95.5-102.2 % for telmisartan. Precision and solution stability were within the permissible range. Validation acceptance criteria were met in all cases. This simultaneous quantitative analysis method was successfully applied to the quality assessment of six fixed dose combination dual-layer tablets containing various amounts of rosuvastatin calcium and telmisartan.