Simple High-performance Liquid Chromatographic Method Research Articles

Development and Validation of HPLC-FLD Analysis of Perampanel in MEPS-Processed Rat Plasma Sample

Perampanel, a novel α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, is registered for the adjunctive treatment of patients (aged ≥ 12 years) with refractory partial-onset seizures. A simple high-performance liquid chromatographic method fluorescence detection (HPLC-FLD) was developed to analyze perampanel in rats’ plasma and validated for bioanalytical purposes. Rats’ plasma (50 µL) was processed by microextraction packed sorbent (MEPS). The analytes were separated using a Hypersil Gold octadecyl silane column (250 × 4.6 mm internal diameter, 5 μm particle size) with isocratic elution. A mobile phase consisting of acetonitrile–methanol–water (275:275:450, v/v/v; containing 50 µL triethylamine and pH adjusted to 3.25 with orthophosphoric acid) was used in this analysis. The flow rate was 1.25 mL/min. Analytes were monitored at an excitation wavelength of 285 nm and an emission wavelength of 430 nm. The linearity range for this validated method was from 3.75 to 300 ng/mL. No endogenous peaks were found in the elution of analytes in drug-free rats’ plasma. Intra- and inter-batch reproducibility studies demonstrated accuracy and precision within the acceptance criteria. The results indicate that the present method is simple, selective, reproducible, and suitable for the analysis of perampanel in small volume samples. The robustness of the method was accessed using MODDE® design of experiments software version 12.5.

Stability Indicating HPLC Method for In-vitro Determination of Pantoprazole Sodium and its Degradation Products in Simulated Gastric and Intestinal Fluids

Background: A high-performance liquid chromatography (HPLC) method was developed for the determination of Pantoprazole Sodium (PPZ) in the presence of its degradation products. The degradation of PPZ was studied in simulated intestinal fluid (SIF) and simulated gastric fluids (SGF) in various temperature conditions. Aim: This study aimed to establish a simple, sensitive, and rapid RP HPLC method for in-vitro determination of Pantoprazole Sodium and its degradation products in simulated gastric and intestinal fluids. Objective: Pantoprazole is acid labile drug. In order to determine pantoprazole in various oral dosage forms, the stability-indicating assay of PPZ was performed in phosphate buffer (pH 6.8) representing simulated intestinal fluid (SIF) and in 0.1 molars (M) Hydrochloric acid (HCl) as simulated gastric fluid (SGF) at two different temperature conditions, i.e., 25°C and 0°C, respectively. Method: Pantoprazole sodium was obtained from the Akums laboratory in Haridwar. The analysis was performed by high-performance liquid chromatography (HPLC), Shimadzu, equipped with two LC-10 AD VP solvent-delivery modules, a SPD-10A UV–-visible detector, and a manual injector valve with 20 μL sample loop. Phenomenex ODS analytical column (150 mm × 4.6 mm i.d., 5 μm particles) was done under reversed-phase partition chromatographic conditions. The mobile phase was phosphate buffer and acetonitrile (ACN) of pH 7.4, respectively, optimized in a 70:30 (v/v) ratio followed by filtration through a 0.45 μm membrane filter and degassed by ultrasonicator before use. The mobile phase was delivered at the flow rate of 2 mL/min. The various parameters, such as linearity, accuracy and precision of the analytical method, were studied. Result: The standard curve of PPZ was linear (R2>0.99) over the concentration range of 5-30 μg/mL, and the relative standard deviation (RSD) values for intra-day and inter-day variations were in the range of 1.0-1.8%. The range of RSD was within ±2. Conclusion: The stability of PPZ in aqueous solution was pH dependent. The rate of degradation increases with decreasing pH. The pH stability of pantoprazole was studied at the above-mentioned temperature conditions. The PPZ peaks were analyzed by comparing them with fresh samples and were stable in SIF solution after 24 hours elapsed time at pH 6.8. The obtained degraded peaks in SGF (pH 1) were successfully separated from the PPZ.

Analytical Method Development and Validation of Perindopril and Amlodipine as Multicomponent Formulation by HPLC Method

The work aims to develop and validate a simple, precise and accurate HPLC method per ICH guidelines. The linearity, specificity, Precision, and accuracy were within limits specified by the ICH guidelines. Hence the proposed system was found to be robust and precise. The objective of this study was to validate the simultaneous assay of Perindopril and Amlodipine by using High-Performance Liquid Chromatography (HPLC). Perindopril and Amlodipine were estimated with C18 Column (2.5 cm × 4.6 mm and five μm particle size) with a VWD detector. The mobile phase consists of Buffer Solution and Acetonitrile 55:45 v/v with a flow rate of 1 ml/min. The volume injected was 20 μL. The detection wavelength was 215 nm, respectively. The retention time of Perindopril and Amlodipine was found to be 3.40 min and 4.73 min. The system suitability and all essential validation parameters were meticulously completed. The LOD and LOQ for Perindopril were found to be 2.62 μg/ml and 7.95 μg/ml, respectively, and for Amlodipine, 1.06 μg/ml and 3.22 μg/ml, respectively. The analytical curve was linear (R2 = 0.999). The system exhibits sufficient accuracy with a relative standard deviation under 2%. The method showed good duplicability and recovery with % RSD less than 2%. The linearity, specificity, Precision, and accuracy were within limits specified by the ICH guidelines. Hence the proposed system was found to be robust and precise. The method has to be such that it takes less time development and the best accurate and complete results have been obtained. Hence the proposed method for simultaneous estimation of Perindopril and Amlodipine by HPLC was found to be robust and precise.

Simple HPLC-UV Method for Piperacillin/Tazobactam Assay in Human Plasma

Piperacillin (Pip)/tazobactam (Taz) is a broad-spectrum antimicrobial agent that has been commonly used in the intensive care unit for severe and life-threatening infections. Recent evidence suggests that therapeutic drug monitoring (TDM) for Pip could be beneficial in clinical practice to facilitate dose optimization and increase the odds of treatment success. The aim was to develop and validate a sensitive and simple high-performance liquid chromatography (HPLC) method for the simultaneous quantification of Pip and Taz in human plasma. Samples (0.3 mL) were deproteinized with acetonitrile. The supernatant was evaporated and then reconstituted and injected into the HPLC. The chromatographic analysis was carried out by using the C18 column and gradient elution with the acetonitrile:water mobile phase mixture with 0.1% trifluoracetic acid at a flow rate of 0.8 mL/min using a UV detector at 218 nm. The method had acceptable linearity (r2 > 0.99) over the concentration ranges of 0.5-400 μg/mL and 1-100 μg/mL for Pip and Taz, respectively. The method demonstrated acceptable inter- and intra-day precision and accuracy within ±20% with adequate stability results. The developed method is sensitive and simple and utilizes simple sample preparation and elution steps, making it suitable and practical for Pip/Taz TDM.

Development and Validation of a Rapid HPLC-DAD Method for Determination of Favipiravir in Pharmaceutical Formulation

Objective: The aim of this work was to develop and validate a rapid and simple high-performance liquid chromatography method with a diode- array detector (HPLC-DAD) for determination of favipiravir in bulk and tablet formulations.
 
 Methods: The chromatographic analysis was performed at 30 °C with a Poroshell 120EC-C18 column (4.6 x 50 mm, 2.7 µm). The mobile phase was a mixture of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (90:10, v/v). The run time was 5 min at a flow rate of 0.5 mL/min.
 
 Results: The proposed method was successfully validated in terms of precision, accuracy, linearity, robustness, limits of detection (LOD) and quantification (LOQ) parameters. The calibration plot was linear over a concentration range of 10-100 µg/mL. The LOD and LOQ values were found to be 0.58 µg/mL and 2.03 µg/mL, respectively. The average recovery values were found to vary from 99.45 percent to 104.29 percent.
 
 Conclusion: As a result, it was concluded that the developed method can be used successfully in the determination of favipiravir in pharmaceutical preparations.

Development and validation of a reverse phase High Performance Liquid Chromatographic method, using standard addition calibration, for determination of risperidone in human plasma

Risperidone is a drug of choice in the management of psychosis and high performance liquid chromatography (HPLC) is regarded as the most sensitive and accurate method for quantitative analysis of risperidone, as well as other drugs having low plasma concentrations in biological fluids. A simple and accurate HPLC method was developed for the determination of risperidone in human plasma using the standard addition calibration technique to augment the weak signals due to low plasma concentrations of risperidone. Risperidone was eluted using isocratic mode with a mobile phase of methanol: acetonitrile (60:40) on a Chemisil ODS C18 column (250 mm×4.6 mm i.d., 5 μ particle size). The sample injection volume, flow rate, column temperature and wavelength of detection were set at 10 µl, 1.0 ml/min, 35 ˚C and 280 nm respectively. The run time for the method was 7 minutes, with risperidone having a retention time of 3.566 minutes. The method was linear within the concentration 10-50 ng/ml (R2= 0.998), with LOD and LOQ of 0.13 and 0.40 ng/ml respectively. The precision and relative error was 3.44 % and 3.38 % respectively. The developed method can be employed in the quantitative determination of risperidone in pharmacokinetic and bioequivalence studies.

Development and validation of novel HPLC method for analytical evaluation of Lemborexant in tablet dosage form

A simple and novel isocratic high-performance liquid chromatography (HPLC) method was developed for quantitative determination of lemborexant in bulk as well as in its tablet dosage form. The developed HPLC method was validated as per ICH (Q2R1) guideline. The results of validation parameters indicate that developed HPLC method was specific, accurate, precise, rapid, reliable and reproducible. Therefore, it can be applied for routine quality control analysis of Lemborexant in bulk and tablet dosage form. Materials and Methods: The chromatographic separation was achieved on Thermoscientific BDS Hypersil (C18, 15 cm × 4.6 mm id, 5μ) column. The mobile phase consisting of buffer (triethylamine (0.1% v/v) adjusted to pH 3.0 with orthophosphoric acid) and acetonitrile in the ratio of (60:40) was passed through the column maintained at 40 °C with a flow rate of 1 ml/min. Approximately 20 µl of the solution was injected and the analyte was eluted at 265 nm. Results: The retention time of lemborexant was around 7.77 min. The percentage RSD of each parameter was found within the limit. The recovery of lemborexant was found to be 100.71%. LOD and LOQ values of lemborexant were found to be 0.54 µg/ml and 1.6 µg/ml respectively. The method was linear over the range of 10-70 µg/ml with a regression coefficient 0.9996. All the verification parameters were within the range according to ICH guidelines. Conclusion: The developed RP-HPLC method is economical, simple, and practical and useful in routine analysis of Lemborexant in bulk and tablet dosage form.

Simultaneous detection of residual diazepam, ketamine, nimetazepam, and xylazine by high-performance liquid chromatography: application in drug-spiked beverages for forensic investigation

ABSTRACT Drug facilitated crimes (DFCs) involve the incapacitation of a victim due to the administration of a single drug or a combination of drugs. Traditionally, biological samples are collected from a victim and analysed to provide evidence of drug administration. However, the rapid metabolism of many such drugs together with delays in analysis can compromise the detection of such substances. This study investigated the capacity of a high-performance liquid chromatography (HPLC) method to detect, simultaneously, four sedative-hypnotic drugs, diazepam, ketamine, nimetazepam, and xylazine, recovered from spiked beverages. A HPLC method was developed and validated and was then applied to the detection of the target substances in simulate forensic case scenarios. The four target substances were well-separated (correlation coefficient, 0.99) while the limit of detection was determined to be 39.1 ng/mL (xylazine), 78.1 ng/mL (diazepam and nimetazepam), and 156.3 ng/mL (ketamine), respectively. Precision (%RSD <7.8%) and accuracy (95.3%–106.6% recovery) were also acceptable and good recoveries were achieved from the drug-spiked samples. In conclusion, a simple and direct HPLC method was successfully developed and applied to the recovery of sedative-hypnotic drugs from spiked beverages.

Validation of a simple HPLC method to quantify methotrexate concentrations in human plasma

Methotrexate (MTX) is a chemotherapy and immunosuppressive agent widely used to treat cancer, autoimmune diseases in children and adult patients, and ectopic pregnancy. However, MTX is highly toxic to the liver, kidney, and nervous system. This study aimed to quantify the concentration of MTX in human plasma using high-performance liquid chromatography (HPLC). MTX and its internal standard (para aminoacetophenone-PAPA) in plasma samples were extracted simultaneously with methanol. Sample purity was performed using the 1 cc OASIS HLB cartridges. Sample injection volume of 10 μL was analyzed on a Lichrocart Supersil 125-4 column C18 maintained at 40 °C on a Waters 2695 XE equipped with a PDA detector set at 303 nm. The mobile phase contained phosphate buffer (pH 6.0) and methanol at a ratio of 80:20 (v/v) and was maintained at a flow rate of 1 ml/min. The results showed that the total time of chromatographic analysis was 15 min. MTX and PAAP were found in the chromatograms at retention times of 2.3 and 5.2 min, respectively. The linear range of the MTX from 0.5 to 25 μg/mL. Intra-day and inter-day imprecision for MTX ranged from 3.42 to 8.128%. LLOQ of MTX was 0.5 μg/mL and the extraction effects were above 77%. In conclusion, we developed and validated a simple HPLC method to determine the MTX concentrations in human plasma.

NEW VALIDATED STABILITY-INDICATING RP-HPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF METFORMIN HYDROCHLORIDE, LINAGLIPTIN AND EMPAGLIFLOZIN IN BULK AND PHARMACEUTICAL DOSAGE FORMS

Objective: The purpose of the present study is to develop simple, fast, accurate, precise, and robust stability-indicating reverse phase high-performance liquid chromatographic (RP-HPLC) method for the simultaneous determination of metformin HCl, empagliflozin, and linagliptin in their combinations. Methods: Separation was performed on Agilent Eclipse XDB-C18 (250 mm x 4.6 mm, 5 µm) column with a mobile phase consisting of 0.1 % triethylamine (pH =3) buffer and acetonitrile in the ratio 40: 60 (v/v) at a flow rate of 1 ml/min. Detection of the analytes was carried out at a wavelength of 240 nm with a photodiode array detector. The developed method was validated as per the International Conference on Harmonization (ICH) guidelines. Results: The retention time values under the optimized condition were 2.660 min, 3.586 min, and 5.412 min for metformin HCl, linagliptin, and empagliflozin, respectively. The method was linear over a concentration range of 100 µg/ml-1500 µg/ml, 0.5 µg/ml-7.5 µg/ml, and 2.5 µg/ml-37.5 µg/ml for metformin HCl,linagliptin and empagliflozin respectively. The limit of detection (LOD) of the method was found to be 4.00 µg/ml, 0.02 µg/ml, and 1.00 µg/ml for metformin HCl, linagliptin, and empagliflozin, respectively. The degradation peaks were clearly resolved from the parent drug peaks in the chromatograms of forced degradation studies. Conclusion: The validated method was successfully applied for the determination of metformin HCl, linagliptin, and empagliflozin in their combined tablet dosage forms and hence can be used for the routine quality control of the drugs in pharmaceutical bulk, and dosage forms.

Two different spectrophotometric methods for simultaneous determination of binary mixture of atenolol and amlodipine in commercial formulation

Two different spectrophotometric methods for simultaneous determination of binary mixture of atenolol and amlodipine in commercial formulation

A simple and robust HPLC method to analyze lutein in wheat

A method for the determination of lutein, the main component of carotenoid in wheat by high performance liquid chromatography (HPLC) was established. The samples were extracted with acetone and methanol (acetone : methanol 7 : 3, v / v, 0.1 % BHT w / v), and fully dissolved in methanol / acetonitrile / n-hexane (7 : 2 : 1, v / v / v) mixed solution. Poroshell 120 EC-C18 column (4.6 mm× 150 mm, 4μm) was used as the separation column, acetonitrile,methanol and n-hexane solution were used as mobile phase with gradient elution, flow rate was 1.0 mL·min−1, column temperature was 25 ℃, injection volume was 50 μL, and detection wave length was 450 nm. The mass concentration of lutein had a good linear relationships with the chromatographic peak area in the range of 0.5-5.0 μg·mL−1, the correlation coefficients were all not less than 0.999 8, and the detection limits was 12 ng·mL−1. The recoveries rate of standard addition in sampling were 95.50%-97.33%, and the relative standard deviations of determination results were 1.20%-1.66%(n=5). The method is sensitive, precise, rapid, accurate and efficient. The advantages of this study can be summarized in the next bullet points:•Can be used for the determination of lutein in wheat.•The detection time is short.•Limit of detection (LOD) and limit of quantification (LOQ) for lutein was 12 ng·mL−1 and 42 ng·mL−1.

Evaluation of intraocular penetration of levofloxacin by high performance liquid chromatography

AIM: To evaluate the levofloxacin eye drop into human eye penetration, levofloxacin eye drop concentrations in human ocular aqueous of 33 patients undergoing cataract surgery were measured by high performance liquid chromatography (HPLC). METHODS: Totally 33 volunteer patients who scheduled for phacoemulsification surgery received one drop of levofloxacin every 6h for 3d before and on the day of surgery, administration of drug was stopped 1h before surgery. Levofloxacin concentration in aqueous humor was measured by HPLC method with fluorescence detector. RESULTS: A simple, effective and sensitive HPLC method for determination of levofloxacin in human ocular aqueous was validated. Linearity was shown for levofloxacin concentration over a wide range of 1.95×10-3-1.50 µg/mL. The mean aqueous level of levofloxacin was 0.3399±0.03405 µg/mL. CONCLUSION: Results from the present study demonstrate that topical administration of levofloxacin 0.5% before cataract surgery with routine dose (one drop every 6h) unable to reach MIC90 for most common microorganism causing acute bacterial endophthalmitis.

Validation of a simple HPLC method to quantify mycophenolic acid concentrations in human plasma

Mycophenolic acid (MPA) is an active metabolite of mycophenolate mofetil and mycophenolate sodium which are widely prescribed to prevent organ rejection after solid organ transplantations. However, MPA induced many side effects on gastrointestinal tract and haematological system. The purpose of this study is to establish a high-performance liquid chromatography (HPLC) method to determine the MPA concentration in plasma in order to optimize the treatment efficacy of MPA or apply to bioequivalence studies. MPA and visnadine (as an internal standard) were extracted from plasma samples with methanol by solid phase extraction using Osis HLB 1cc cartridge. 10 μL of sample extract was injected onto LiChroCART®125-4 (C18 reversed-phase column) at 43 °C on a Waters 2695 XE system. The signals were detected by PDA detector (photodiodes array) at 254 nm. The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 3) with a flow rate of 1 mL/min. The validation criteria included: selectivity, linearity, accuracy, precision, recovery, lower limit of quantification. Total chromatographic runtime was 15 min. MPA and visnadine were found at 6.45 and 10.79 min, respectively. MPA concentrations were in the linear range from 0.25 to 50 μg/mL. The coefficient of variation (CV) of mean intra-day and inter-day precision levels for MPA was less than 7.5%. The lower limit of quantification was 0.25 μg/mL. No interference was found in the assay. A simple and reliable HPLC method was developed to quantify the MPA concentration in plasma.

Rapid Determination of Metronidazole and 2-Hydroxymetronidazole in Murine Blood Plasma.

Metronidazole is a drug used to treat bacterial and protozoan infections. Nowadays, it is one of the most frequently prescribed drugs worldwide. The main aim of this paper is to present a rapid, reliable and simple high-performance liquid chromatography (HPLC) method to determine metronidazole along with its primary metabolite, 2-hydroxymetronidazole, in plasma or serum using paracetamol as an internal standard. A total of 100% methanol was used to denature plasma proteins. After centrifugation, the supernatant was evaporated under nitrogen flow. The samples were dissolved in the mobile phase and injected into a Li-Chrospher RP-18 column. A total of 10mmol/L NaH2PO4: acetonitrile (90:10, v/v) solution with a flow rate of 1mL/min was used as the mobile phase. Metronidazole and 2-hydroxymetronidazole were detected at two different wavelengths at 320nm and 311nm, respectively. The method is characterized by high precision (relative standard deviation % < 6). The method was used for the determination of metronidazole and 2-hydroxymetronidazole in murine blood using small amounts of plasma (≤100μL).

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC ESTIMATION METHOD FOR TRANSDERMAL DIFFUSION STUDY OF GRANISETRON

Objective: The study is aimed to develop a simple, isocratic and sensitive reversed-phase high-performance liquid chromatography method for the analysis of granisetron during transdermal permeation studies through porcine ear epithelium.&#x0D; Methods: A reversed-phase (C18) column was used with ultraviolet detection at 210 nm. Selected mobile phase contained 25% (v/v) of acetonitrile and 75% (v/v) of 0.25 mM potassium dihydrogen orthophosphate solution pH adjusted to 3.0 using 1% orthophosphoric acid solution.&#x0D; Results: Calibration curve showed good linearity over 0.1–30 μg/mL concentration range of porcine ear permeates in 80% ethanol and 20% water (blank permeates). The applicability of the method was demonstrated by the analysis of diffusion study samples of granisetron through porcine ear epithelium and the steady-state permeability flux (J) from ethanolic solution was found to be 4.707 µg/square cm/h.&#x0D; Conclusion: The developed method can be used in the analysis of diffusion study samples of granisetron transdermal formulations through the porcine ear epithelium.

Determination of ceftazidime in plasma by RP-HPLC and ultraviolet detection.

A simple high-performance liquid chromatography method for the determination of ceftazidime in plasma has been developed. Using an ultrafiltration technique samples were separated by reverse-phase high-performance liquid chromatography on a Symmetry C18 4.6 × 250 mm column (5.0μm) and ultraviolet absorbance was measured at 260 nm. The mobile phase was a mixture of 10mm potassium phosphate monobasic pH2.5 with phosphoric acid and acetonitrile (90:10). The standard curve ranged from 0.1 to 100 μg/ml. Intra- and inter-assay variability for ceftazidime was <12%, and the average recovery was 89%. The lower limit of quantification was 0.1μg/ml. This method has been used successfully to analyze frog plasma samples at this institution and it could be applied to other small volume samples in a clinical or research setting.

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.

Isocratic high-performance liquid chromatography (HPLC) for simultaneous quantification of curcumin and piperine in a microparticle formulation containing Curcuma longa and Piper nigrum

Poor bioavailability has been reported as a major challenge in the development of curcumin as a pharmaceutical agent. However, co-administration of curcumin with piperine has been shown to improve curcumin bioavailability. Therefore, to assure product control quality, an analytical method needs to be developed for the determination of curcumin and piperine content in a dosage form formulation. The objective of this study was to develop a simple isocratic reversed-phase HPLC (RP-HPLC) method to simultaneously quantify curcumin and piperine content in solid dispersion based microparticle formulation containing Curcuma longa and Piper nigrum extracts. The method was validated according to the International Council for Harmonization (ICH) guideline. Chromatographic separation of three curcuminoids and piperine could be achieved using acetonitrile-methanol-water of 65:5:35 %, at a flow rate of 1 mL/min and a wavelength of 353 nm for detection. Resolution (Rs) of 3.57 and 1.68 for piperine and curcumin, respectively, a theoretical plate number (N) > 8000 and a tailing factor (T) < 1.5 indicate a satisfactory separation of the compounds. The calibration curve was linear from 1.25-15 μg/mL and 2.5–30 μg/mL for piperine and curcumin, respectively, with the correlation coefficient of >0.999. The intra-day/inter-day accuracy and precision demonstrated a recovery of 99.54–101.50%/99.38–99.89% and 100.78–102.51%/101.15–102.47% with a Relative Standard Deviation (RSD) of 0.53–0.95%/0.13–1.44 % and 0.28–1.62%/0.46–1.14% for piperine/curcumin. The limit of detection (LOD) were 0.27 and 0.42 μg/mL, for piperine and curcumin, which reveals an adequate sensitivity. A solid dispersion based microparticle formulation containing C. longa and P. nigrum extracts confirmed the validity of the developed method as a recovery of 91.14% and 99.14% for piperine and curcumin, respectively. In conclusion, all the tested parameters confirm the precision, accuracy, and reliability of the method for the simultaneous analysis of curcumin and piperine within a microparticle formulation containing C. longa and P. nigrum extracts.

Simple HPLC-DAD-based method for determination of ergosterol content in lichens and mushrooms

A novel yet simple high-performance liquid chromatography (HPLC) method with diode array detector has been developed and validated for the quantification of ergosterol (Pro-vitamin D2) in commercially available lichens and cultivated edible mushrooms as per International Conference on Harmonization (ICH) guidelines. The extraction of ergosterol in the lichens and mushrooms was carried out with alcoholic KOH via reflux, and the extracts were processed for HPLC analysis. The chromatographic identification of ergosterol from the extracts was achieved using C18 column (5 µm, 4.6 × 250 mm) thermostatted at 30 °C using an isocratic elution of 100% methanol at a flow rate of 1.5 mL/min. The elution was monitored at 280 nm using a diode array detector. In this method, ergosterol was separated from the extract within a short interval of time (7.0 (±0.1) min). The developed method exhibited good linearity (r 2>0.9998), limit of detection (0.03 µg/mL), limit of quantification (0.095 µg/mL), interday and intraday precision and accuracy. A total of five lichen samples and three mushroom samples were analyzed using this method to quantify the amount of ergosterol present in them. This work, thus, highlights the possible use of lichens as an excellent source of ergosterol.