Comprehensive preclinical assessment of asenapine maleate: Advanced HPLC bioanalytical techniques

In this study, a high-performance liquid chromatographic method (HPLC) and UV spectrophotometric method were developed, validated and applied for the determination of theophylline in biological fluids. Liquid- liquid extraction is performed for isolation of the drug and elimination of plasma and saliva interferences. Urine samples were applied without any extraction. The chromatographic separation was achieved on a C18 column by using 60:40 methanol:water as mobile phase under isocratic conditions at a flow rate of 0.75 mL/min with UV detection at 280 nm in HPLC method. UV spectrophotometric analysis was performed at 275 nm.The results of HPLC analysis were as follows: the limit of quantification: 1.1 µg/mL for urine, 1.9 µg/mL for saliva, 3.1 µg/mL for plasma; recovery: 94.85% for plasma, 100.45% for saliva, 101.39% for urine; intra-day precision: 0.22–2.33%, inter-day precision: 3.17-13.12%. Spectrophotometric analysis results were as follows: the limit of quantitation: 5.23 µg/mL for plasma, 8.7 µg/mL for urine; recovery: 98.27% for plasma, 95.25% for urine; intra-day precision: 2.37 – 3.00%, inter-day precision: 5.43-7.91%.It can be concluded that this validated HPLC method is easy, precise, accurate, sensitive and selective for determination of theophylline in biological samples. Also spectrophotometric analysis can be used where it can be applicable.

The present study discusses the development of a simple, rapid, and specific HPLC method for the estimation of sodium benzoate in chlorzoxazone tablet dosage formulations. The current developed HPLC method was validated as per the current ICH guidelines. The chromatographic separation was achieved using a 0.02-M phosphate buffer adjusted to pH 3.0 with orthophosphoric acid as the buffer. Mobile phase A consisted of 100% buffer, and mobile phase B was a mixture of acetonitrile and buffer in a ratio of 80:20 (v/v). The column temperature was maintained at 30°C, the sample cooler at 25°C, and the flowrate at 0.8 mL min-1. The injection volume was 10 μL. The UV detection was performed at 230 nm for Sodium benzoate. The validated HPLC method was highly specific, with linearity ranging between 1.2 and 7.5 μg/mL for sodium benzoate, and the correlation coefficient was found to be > 0.999. The method showed high accuracy, exceeding 97%. The results demonstrate the successful applicability of the current method for the estimation of sodium benzoate in marketed formulations, which can be extended to assess other formulation systems. The developed method was validated as per international ICH guidelines with respect to specificity, linearity, precision, accuracy, and robustness. The method was applied to the analysis of stability samples.

Introduction Asenapine maleate (AM) is an atypical antipsychotic agent, that has been widely prescribed for the management of schizoaffective disorders. However, the bioavailability of AM is extremely poor due to the extensive first-pass metabolism. With the advancement in pharmaceutical technologies, significant strides have been made to create novel formulations to address the bioavailability problem of AM. Areas covered This review article provides an insight into all the formulation approaches undertaken by researchers to increase the bioavailability of AM encompassing the works utilizing ultrasound mediated transdermal delivery, nose to brain delivery, intestinal lymphatic system targeting, in situ implants, etc. All the patents associated with AM formulation have also been discussed and summarized. Expert opinion Numerous studies have been carried out on AM formulations over the recent years, many of these studies have shown significant improvement in bioavailability. We have also mentioned the unexplored domains which can be exploited for further enhancing the bioavailability of AM. Nonetheless, most of these studies are still limited to the research laboratory level and face multiple hurdles before making into the market. Attaining controllability and reproducibility for the production of novel formulations is needed to enable its transition from bench to bedside.

A simple, sensitive, selective and reproducible reversed-phase HPLC method was developed for the determination of sophoricoside in rat plasma after intravenous administration. Naringin was successfully used as internal standard (IS) for calibration. The chromatographic separation was accomplished on a reversed-phase C(18) column using acetonitrile-methanol-0.08% phosphoric acid (8:29:63, v/v/v) as mobile phase with a flow rate of 1.0 ml/min, with UV detection at 260 nm. Plasma samples were injected into the HPLC system after precipitating protein directly by methanol. Good linearity was achieved in the range of 0.0240 approximately 48.0 microg/ml (R(2)=0.9989). The limit of detection (LOD) and limit of quantification (LOQ) of this method were 0.0075 microg/ml and 0.0240 microg/ml, respectively. The absolute recoveries of sophoricoside from plasma were 95.8%, 93.2%, 98.0% at concentrations of 0.0240, 1.92, 15.0 microg/ml. The intra-day and inter-day variabilities were 3.39%~5.78% and 2.17%~4.72%, respectively. The developed method was successfully applied to the pharmacokinetic study of sophoricoside after intravenous administration of 2.5, 10 and 20 mg/kg in rats.

A simple, sensitive high performance liquid chromatographic method with UV detection was developed and validated for determination of insulin in rat plasma, using methyl paraben as an internal standard. Insulin was extracted from plasma by a liquid–liquid extraction with a mixture of dichloromethane and n-hexane (1:1, v/v) followed by an acidic back extraction. Chromatographic separation was achieved isocratically with a Phenomenex® C18 analytical column (150 × 4.6 mm ID, 5 μm) at ambient room temperature. The calibration curves were linear within a concentration range of 0.7–8.4 μg mL−1 (r2 = 0.9994). The inter-day and intra-day accuracy and precision were ≤3.33 and ≤5.55%. The limit of detection (LOD) and limit of quantification (LOQ) were 0.35 and 0.7 μg mL−1. The average recovery was 87.86% for insulin and 83.52% for methyl paraben. Insulin containing plasma samples were stable at −20 °C for 7 days. Validated HPLC method was successfully applied to a pharmacokinetic study of insulin in streptozotocin induced diabetic rats.

[(E)-3-(2-chlorophenyl)-1-(2-hydroxy-4,6-dimethoxy-3-(1,2,3,6-tetrahydropyridine-4-yl)phenyl)-2-propylene-1-one], L41, was found to be a potent cyclin-dependent kinase 1 inhibitor. To study its pharmacokinetic characteristics, a simple, specific, sensitive and reproducible HPLC method was developed and validated to quantitatively determine L41 in rat plasma. L41 and amlodipine besylate (internal standard, IS) were extracted from rat plasma by a simple liquid–liquid extraction process with ethyl acetate. Chromatographic separation was performed on an Agela C18 column and an isocratic mobile phase [methanol–water (containing 50 mM ammonium acetate), 77 : 23, v/v, pH 6.5] at a flow rate of 1 mL min−1 with a total run time of 10 min. The UV absorbance at 343 nm was recorded. L41 and IS eluted at 5.1 and 8.9 min, respectively. The calibration plot was linear over the concentration ranging of 15–1200 ng mL−1 (r > 0.998). The intra- and inter-day precisions of analysis were <12% and accuracy ranged from 93.5 to 106.3%. The validated HPLC method was successfully applied to the pharmacokinetic study of L41 in rats after a single intravenous and three oral administrations.

Determination of Z-3,5,4′-trimethoxystilbene in rat plasma by a simple HPLC method: Application in a pre-clinical pharmacokinetic study

In this study, a high-performance liquid chromatographic method (HPLC) was developed, validated and applied for the determination of raltegravir in biological sample like saliva. Liquid- liquid extraction was performed for isolation of the drug and elimination of saliva interferences. Samples of saliva was extracted with 50µL of ortho phosphoric acid and 3ml of methanol was added and spiked with raltegravir. The chromatographic separation was performed on Agilent Eclipse C18 (100 mm × 4.6 mm, 3.5µm) column, by using 80:20 v/v acetonitrile: water as a mobile phase under isocratic conditions at a flow rate of 1.0 mL/min for UV detection at 240 nm. Retention time of raltegravir was found to be 1.030 min. Linearity was found to be in the range of 25-1000 ng/mL with regression equation y = 13864x + 40495 and correlation coefficient 0.999. The low % RSD value indicates the method is accurate and precise. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.76 and 2.28 ng/mL, respectively. It can be concluded that this validated HPLC method is easy, precise, accurate, sensitive and selective for determination of raltegravir in saliva.

A simple and sensitive HPLC method was developed to simultaneously determine three active compounds, vitexin-4″-O-glucoside (VG), vitexin-2″-O-rhamnoside (VR) and hyperoside (HP), in rat plasma after administering the hawthorn leaves extract (HLE). An HPLC assay with baicalin as the internal standard was carried out using a Phenomsil C18 analytical column with UV detection at 332 nm. The mobile phase consisted of methanol–acetonitrile–tetrahydrofuran–1% glacial acetic acid (6 : 1.5 : 18.5 : 74, v/v/v/v). The calibration curves were linear over the range of 2.5–500, 0.2–25 and 0.25–12.5 µg mL–1 for VG, VR and HP, respectively. The method was reproducible and reliable, with relative standard deviations of the intra- and inter-day precision between 1.2% and 13.2% for the analysis of the three analytes. The validated HPLC method herein described was successfully applied to the pharmacokinetic study of VG, VR and HP after oral administration of HLE to rats over the dose range of 2.5–10 mL kg–1.

A simple and sensitive HPLC method using UV detection was developed to determine the concentration of protoapigenone in rat plasma. Chromatographic separation was conducted on a C18 column with a mobile phase consisting of an acetonitrile-methanol-aqueous phase (containing 0.2% acetic acid, pH 3.0) system at a flow rate of 1.0 mL/min. The UV detector was set at 248 nm. The calibration curve was linear over the range of 0.031-10.0 µg/mL. The lower limit of quantification was 31 ng/mL. The recoveries for plasma samples ranged from 70.3 to 82.5%. The intra- and inter-day accuracy and precision fulfilled the international standards. This method was successfully applied to a pharmacokinetic study of protoapigenone in rats after oral administration of protoapigenone. It was shown that protoapigenone could be absorbed rapidly after oral administration and could reach the maximum concentration within 1 h.

Summary A simple, rapid, and sensitive reversed-phase HPLC method was developed and validated for determination of metronidazole and tinidazole in human plasma samples under identical chromatographic conditions. This method involves liquid-liquid extraction using chloroform: isopropylalcohol (95:5). Chromatographic separation was performed using a μ-bondapack C18 (250 mm × 4.6 mm) column. The mobile phase consisted of potassium dihydrogen phosphate solution (0.005 M)/acetonitrile (80/20 v/v). The final pH of the mobile phase was adjusted to 4 ± 0.1 with orthophosphoric acid. The calibration curves were linear over the concentration range 0.1–15 μg/mL for metronidazole and tinidazole with the detection limit of 30 ng/mL. Within- and between-day precision and accuracy did not exceed 9.83% and 10.48%, respectively. Metronidazole and tinidazole were found to be stable in plasma samples with no evidence of degradation during 3 freeze-thaw cycles and 3 months storage in −70 °C. The current validated bio-analyti...

Berberine has been shown to have therapeutic advantages in the management of different diseases. Due to its actions and therapeutic applications, in compliance with “The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use”. (ICH) recommendations; a quick, precise and specific HPLC method was developed and validated. The chromatographic separation was carried out with Column C18 (4.6 mm X 250 mm), (5 microns) using 0.1% orthophosphoric acid: acetonitrile (60:40) as mobile phase, with a flow rate of 1 ml/min and at a wavelength of 349 nm. The developed method was also used for the estimation of berberine in various commercial preparations and also with formulated berberine-loaded cubosomal nano formulation according to ICH guidelines. Furthermore, forced degradation studies of berberine were investigated. The novel analytical technique was identified to be linear over the concentration ranging from 2 μg/ml to 14 μg/ml with R2 > 0.997. The method developed was accurate (with % recovery of 98.24% to 99.31%), precise (%RSD < 2.0%), and robust. The limit of quantification and limit of detection for berberine was found to be 0.064 μg/ml and 0.021 μg/ml respectively. In the presence of degradation products, the drug peak was obtained. The peaks for berberine were detected in the marketed formulations containing berberine and hence, the developed method could be applied to the estimation of berberine in berberine-containing formulations.

Levo-tetrahydropalmatine (l-THP) is an alkaloid isolated from Chinese medicinal herbs of the Corydalis and Stephania genera. It has been used in China for more than 40 years mainly as an analgesic with sedative/hypnotic effects. Despite its extensive use, its metabolism has not been quantitatively studied, nor there a sensitive reliable bioanalytical method for its quantification simultaneously with its metabolites. As such, the objective of this study was to develop and validate a sensitive and selective HPLC method for simultaneous quantification of l-THP and its desmethyl metabolites l-corydalmine (l-CD) and l-corypalmine (l-CP) in rat plasma and brain tissues. Rat plasma and brain samples were processed by liquid-liquid extraction using ethyl acetate. Chromatographic separation was achieved on a reversed-phase Symmetry® C18 column (4.6 × 150 mm, 5 μm) at 25°C. The mobile phase consisted of acetonitrile-methanol-10 mm ammonium phosphate (pH 3) (10:30:60, v/v) and was used at a flow rate of 0.8 mL/min. The column eluent was monitored at excitation and emission wavelengths of 230 and 315 nm, respectively. The calibration curves were linear over the concentration range of 1-10,000 ng/mL. The intra- and interday reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. The validated HPLC method was successfully applied to analyze samples from a pharmacokinetic study of l-THP in rats. Taken together, the developed method can be applied for bioanalysis of l-THP and its metabolites in rodents and potentially can be transferred for bioanalysis of human samples.

A sensitive and simple HPLC method was developed for the simultaneous quantification of berberine and lysergol in rat plasma. The chromatographic separation was achieved on a C(18) column using isocratic elution with methanol-acetonitrile-0.1% ortho-phosphoric acid (25:20:55, v/v/v), pH adjusted to 6.5 with triethylamine and detected at a UV wavelength of 230 nm. The extraction of the berberine and lysergol from the rat plasma with methylene chloride resulted in their high recoveries (82.62 and 90.17%). HPLC calibration curves for both berberine and lysergol based on the extracts from the rat plasma were linear over a broad concentration range of 50-1000 ng/mL. The limit of quantification was 50 ng/mL. Intra- and inter-day precisions were <15% and accuracy was 87.12-92.55% for berberine and 87.01-92.26% for lysergol. Stability studies showed that berberine and lysergol were stable in rat plasma for short- and long-term period for sample preparation and analysis. The described method was successfully applied to study the pharmacokinetics of berberine as well as lysergol following oral administration in Sprague-Dawley rats. The results of the study inferred that lysergol improved the oral bioavailability of berberine.

A simple and precise reverse phase high-performance liquid chromatography (RP-HPLC) method for simultaneous determination of amoxicillin, clarithromycin and esomeprazole in mice plasma after an oral administration was developed and validated. An isocratic elution was achieved on C18 column with a mobile phase containing buffer, potassium di hydrogen phosphate (KH2PO4 ) 0.05 M at pH 5 and methanol (60:40 v/v) at a flow rate of 1 mL/min, injection volume was 10 µL and UV detection was kept at 205 nm. Linearity was in the range of 0.5-100 µg/mL for amoxicillin, 50-1000 µg/mL for clarithromycin and 0.1-100 µg/mL for esomeprazole. Limit of detection (LOD) and Limit of quantification (LOQ) were 0.26 µg/mL and 0.79 µg/mL for amoxicillin, 8.97 µg/mL and 27.20 µg/mL for clarithromycin, 0.13 µg/mL and 0.39 µg/mL for esomeprazole respectively. All samples were stable at room temperature for 72 hours. The pharmacokinetic studies showed that the maximum plasma concentrations (Cmax) were 1042.17 ± 4.0, 218.67 ± 5.3 and 18.97 ± 3.6 µg/mL for amoxicillin, clarithromycin and esomeprazole respectively. Whereas, the times to reach maximum plasma concentration (Tmax) were 2.0, 4.0 and 2.0 hours respectively. Over all, the validated HPLC method may be used for the determination of such drugs in their pharmaceutical formulation and can be applied for routine quality control analysis.