Development Of High-performance Liquid Chromatography Method Research Articles

Development of HPLC method for determination of phenolic compounds on a core shell column by direct injection of wine samples

Phenolic compounds are frequently present in various natural products, and they can have different beneficial biological potentials. The most widely used method for determination of individual phenolic compounds is high-performance liquid chromatography (HPLC). In this paper, a method for simultaneous determination of 16 phenolic compounds (gallic acid,p-hydroxybenzoic acid, catechin, syringic acid,trans-cinnamic acid, hesperetin, naringenin, vanillic acid, benzoic acid, coumaric acid, resveratrol, chlorogenic acid, caffeic acid, rutin, quercetin, and kaempferol) on a core–shell column was developed. The separation method conducted on a standard ODS (250 mm) column was transferred to Poroshell column and optimized using non-ultra-high-performance liquid chromatography (UHPLC) apparatus. Phenolic compounds were separated fast and efficiently during 30-min analysis, and validation parameters were determined. The developed method was successfully applied on the analysis of phenolic content after direct injection of red wines from three different grape varieties.

Development of a rapid HPLC method for quantification of the copper tetramibi tetraflourborate in a kit for preparation of Technetium 99 m Sestamibi Injection.

Radiopharmaceuticals are radioactive compounds that can be used for diagnostic and therapeutic purposes. Technetium (99mTc) Sestamibi is the most commercialized radiopharmaceutical in the world. It includes a coordination complex consisting of the radioisotope 99 m technetium bound to six copper tetramibi tetrafluorborate ligands, and is mainly used to image the myocardium via scintigraphy. As radiopharmaceuticals are regarded as drugs, they are subject to the same regulations; therefore, the objective of this study was to develop a quantification method for the active pharmaceutical ingredient before their complexation with the radioisotope by employing high-performance liquid chromatography (HPLC) methodology. A simple and efficient method (retention time = 2.5 min) was developed and validated for copper tetramibi tetrafluorborate in the final product using a buffer and organic solvent mixtures (ACN-methanol-ammonium sulfate buffer) and a C18 column. The analytical protocol was fast, taking around 30 min until evaluation of results. The validation parameters were evaluated with satisfactory results: in terms of linearity r > 0.99 (160-240 μg/mL) and no deviation was observed. The RSD of precision was <5%, and an average recovery of 99% was observed for accuracy. The proposed method was thus considered adequate for routine analysis in the pharmaceutical industries.

Development and validation of a reverse phase HPLC method for SHetA2, a novel anti-cancer drug, in mouse biological samples

SHetA2 is a flexible heteroarotinoid that has the potential to prevent and treat lung, ovarian and cervical cancer without significant toxicity. A simple and reliable high performance liquid chromatographic (HPLC) method was developed to determine SHetA2 concentrations in the lungs, reproductive organs and plasma of mice. SHetA2 was extracted from these biological matrices by solid phase and liquid-liquid extraction in the presence of 4% H3PO4 and acetonitrile followed by filtration through a Captiva® filtration plate. Drug concentrations in the filtrates were quantified by a Waters HPLC Alliance system coupled with XBridge® C18 column, guard column and UV detection at 361 nm. The mobile phase consisted of methanol and 0.25 N sodium acetate buffer (80:20, v/v) at pH: 3. SHetA2 was eluted after 5.35 and 6.14 min for tissues and plasma, respectively. Recovery of SHetA2 from biological samples was more than 95% of the spiked amount in tissues and more than 80% of the spiked amount in plasma. The limit of detection (LOD) was 0.005 μg/mL and the limit of quantitation (LOQ) was 0.025 μg/mL, which were 280 and 56 times lower than the predicted therapeutic concentration of SHetA2, respectively. The method was suitable to quantify SHetA2 concentrations in biological matrices from animal studies administering the drug by the vaginal, pulmonary and oral routes that had the purpose of determining the pharmacokinetic parameters of drug disposition. The HPLC method developed meets the ICH Harmonized Tripartite Guideline of a reliable, sensitive, reproducible and accurate method to be used in the determination of drug concentrations in biological samples

Quantification of α-Acids in Fresh Hops by Reverse-Phase High-Performance Liquid Chromatography.

This publication describes a method for the quantification by high-performance liquid chromatography (HPLC) of resinous compounds known as α-acids found in freshly harvested, unprocessed hops. This method provides consistent, efficient, and accurate results as well as the theories and rationale involved in HPLC method development. A system of quality checks was utilized as well as the validation of numerous developmental variables. By starting with a theoretical approach in preparation, extraction, and instrumental techniques and then further developing these practices by experimentation, a reproducible method was developed. Following the validation, fresh cascade hops grown in Sonoma County were analyzed during the 2017 harvest season and found to be within the predicted range specific to this cultivar. This method encompasses the techniques necessary to analyze fresh or dried hops, considering variability between different laboratories.

Reversed-phase high-performance liquid chromatographic method development and validation for allyl isothiocyanate estimation in phytosomes of Brassica nigra extract.

A simple, specific, accurate, precise, and robust reverse-phase high-performance liquid chromatographic method has been developed and validated for the estimation of allyl isothiocyanate (AITC) in phytosomes of black mustard extract (Brassica nigra). The method was validated with respect to specificity, linearity, accuracy, precision, and robustness. The linearity was achieved over a range of 10–18 μg/mL and regression coefficient was obtained 0.9961. Accuracy of chromatographic method was evaluated by standard addition method; percentage recovery attained was 97.07 ± 0.008–103.66 ± 0.013. Relative standard deviation for intraday and interday precision was 0.02% and 0.22%, respectively. The limit of detection and limit of quantification of the AITC were found to be 0.0043 μg/mL and 0.01324 μg/mL, respectively. This result shows that the method was well validated. In the present study, the AITC content was found 0.0009% ± 0.014% in black mustard. This study reveals that the proposed high-performance thin-layer chromatography method is accurate, fast, and cost-effective for the routine estimation of AITC in the phytosome formulation.

Development of stability-indicating HPLC method and accelerated stability studies for osmotic and pulsatile tablet formulations of Clopidogrel Bisulfate

The purpose of the present study is to develop a simple, rapid and sensitive stability-indicating high-performance liquid chromatography (HPLC) method for Clopidogrel Bisulfate (CBS) and further extending it for assessment of CBS stability in osmotic and pulsatile tablet formulations tested under accelerated conditions. A stability-indicating HPLC method for quantitative determination of CBS in gastro-retentive formulations is developed by using a C18 HPLC column, acetonitrile and 0.1% formic acid (60:40 v/v) as mobile phase, with a flow rate of 0.9 mL/min, UV detection at 222 nm and subsequently validated. The key objective was to analyze the stability profile of formulations under accelerated conditions. The retention time (Rt) of CBS was observed as 5.9 min with the linearity range between 0.06–1.95 μg/mL. Forced degradation studies were performed on bulk samples of CBS using acidic, basic, oxidative, thermal (80 °C) and photolytic (under sunlight) conditions. The resulting method was validated as per ICH Q2(R1) guidelines. Moreover, an attempt has been made to identify the degradation products by Liquid chromatography-mass spectrometry (LC–MS) analysis. The proposed method was successfully applied to novel gastro-retentive tablet formulations (osmotic tablet and pulsatile tablet) for assessment of stability under accelerated conditions.

Analysis of scopoletin and mangiferin in botanicals and formulations of Shankhpushpi by HPLC

Summary Introduction: Shankhpushpi has been widely used in traditional Indian systems of medicine as a brain and memory boosting tonic. There are a variety of botanicals reported to be used as sources of Shankhpushpi in various parts of India. For instance, Canscora decussata Schult, Clitorea ternatea Linn., Convolvulus pluricaulis Choisy. and Evolvulus alsinoides Linn. are most commonly used as sources of Shankhpushpi by practitioners of Ayurveda in different parts of the country. Objective: When it comes to using Shankhpushpi in herbal formulations, qualitative and quantitative analysis of the correct botanicals in the formulation decides its pharmacological effectiveness. Scopoletin and mangiferin are proven bioactive markers identified in Shankhpushpi botanicals in our previous studies. Hence the study is aimed at providing a simple analytical method for the identification of the correct variety of Shankhpushpi using proven markers. Methods: In this study, a High Performance Liquid Chromatographic (HPLC) method has been developed for the estimation of scopoletin and mangiferin levels in four botanicals of Shankhpushpi and their marketed formulations. Result: A simple analytical method was developed which proved to be very crucial in estimating concentrations of mangiferin and scopoletin in various test samples. This method can be used to identify the correct botanicals of Shankhpushpi present in any Ayurvedic formulation or raw material or processed powder by evaluating the content of scopoletin or mangiferin as markers. Conclusion: The developed HPLC method is a quick and reliable method for the quantitative monitoring of mangiferin and scopoletin in herbal extracts and marketed formulations of Shankhpushpi.

Development of an Innovative Quality by Design (QbD) Based Stability-Indicating HPLC Method and its Validation for Clofazimine from its Bulk and Pharmaceutical Dosage Forms

The present research work discusses the systematic Quality by Design (QbD) enabled development of a simple, rapid, economical, and stability-indicating high-performance liquid chromatography (HPLC) method for effective analysis of clofazimine (CFZ). An Ishikawa fish-bone diagram was constructed for initial risk assessment. Analytical target profile (ATP) was defined and critical analytical attributes (CAAs) were assigned to meet these ATP requirements. Taguchi design was applied for screening critical material attributes (CMAs) and critical process parameters (CPPs) making an impact on the assigned CAAs. The major contributing factors were finely tuned using 33 Box-Behnken design with numerical and graphical optimization. Further, the method was validated as per the ICH guidelines and force degradation studies were carried out under different stress conditions. The optimum chromatographic separation was accomplished using 75:25% v/v ratio of methanol and ammonium acetate buffer (0.01 mol/L) as the mobile phase at flow rate 1.0 mL/min, and UV detection at 284 nm. The developed HPLC method was found highly sensitive, specific with linearity ranging between 2 and 10 µg/mL, and correlation coefficient (R2) 0.9995. It showed high accuracy with % recovery between 99.68 and 100.44%. It depicted detection limit and quantitation limit of 0.0066 µg/mL and 0.0199 µg/mL, respectively. In force degradation studies the drug was found to be highly susceptible in alkaline stress conditions. The results reveal successful applicability of the method for the estimation of CFZ from its marketed formulation which can be wisely extrapolated to assess the CFZ from its other formulation systems and different biological samples.

Standardization and xanthine oxidase inhibitory potential of Zanthoxylum armatum fruits

Ethnopharmacological relevanceTejovati (Zanthoxylum armatum DC; Family- Rutaceae) popularly known as toothache tree is widely distributed in sub-tropical Himalaya region. Traditionally, The Southeast Asian population of Indo-Nepal origin uses it to treat asthma, gout, pain, and inflammation. The Ayurvedic action of the plant includes the balancing of Vata-Kapha in the body. Which lead to various ailments related to the circulation of blood and water, digestion, immunity, and skin. Therefore, in-vitro xanthine oxidase (XO) inhibition potential of the extract could be worth to explore prospect in the prevention/treatment of gouty affections of the joints and other diseases. Aim of studyAnti-inflammatory and antioxidant potential of Z. armatum fruit (ZAF) has been reported. To date, no scientific study to validate the claim for gout treatment/management has been attempted so far. The present study deals with the xanthine oxidase inhibitory potential of a various extract of ZAF and marker-based high-performance liquid chromatography (HPLC) standardization of most active fraction. Materials and methodsLiquid-liquid partioning of crude methanol extract of the ZAF followed by repeated column chromatography of most active fraction has resulted in the isolation of seven compounds. Five distinct groups of compounds were isolated, purified, and identified. We have investigated the therapeutic action of ZAF in the management of gout through in-vitro assay of XO, a key enzyme involved in gout pathogenesis. ResultsPhytochemical investigation of ZAF has resulted in the isolation of seven compounds of diverse nature. It is noteworthy to mention that out of seven, five compounds have shown the xanthine oxidase inhibitory action. The ethyl acetate fraction was most potent to inhibit XO. The XO inhibitory activity (IC50 values) of isolated marker chemical was ranging from 5.62 to 41.21 µM. Three compounds viz. acetyl phenyl acetate (ZA-2), prudomestin (ZA-6), and tambulin (ZA-7) showed the most potent XO inhibitory activity (IC50 ≈ 6 µM) comparable with a positive control (Allopurinol, IC50, 3.38 µM). This is the first validated HPLC-PDA method for simultaneous analysis and accurate quantification of seven compounds (phenolic acid, acetyl phenyl acetate, xylopyranoside, diphenyl ether and three flavones) in ZAF as well as their distribution in other tissues of the plant. ConclusionMost potent three chemicals (ZA-2, 6 and 7) could be considered as bioactive to ensure the robust quality of the enriched fraction of ZAF with defined XO inhibition potential. Therefore, either single purified component or their enriched fraction could be a better choice for the management of gout than the crude extract of ZAF. Developed HPLC method is suitable for quality assurance analysis and process control of ZAF derived product intended for gout management. XO inhibitory potential exhibited by the characterized compounds validate the traditional use of this ZAF for the treatment of gout. Further, a detailed study is required to assess the effect of ZAF chemicals on serum uric acid and mechanism of XO inhibition.

High-performance liquid chromatographic determination of 2-aminoethylphosphonic acid and 2-amino-3-phosphonopropionic acid in seawater matrix using precolumn fluorescence derivatization with o-phthalaldehyde-ethanethiol

2-Aminoethylphosphonic acid (2-AEP) and 2-amino-3-phosphonopropionic acid (2-AP3) are two types of abundant and ubiquitous naturally occurring phosphonates used as sources of phosphorus by many prokaryotic lineages. The potential utilization mechanism of 2-AEP and 2-AP3 in eukaryotic phytoplankton is currently under investigation. However, the lack of suitable analytical methods in saline samples are the limitation of such researches. Herein, a high-performance liquid chromatography (HPLC) method for monitoring 2-AEP and 2-AP3 using precolumn fluorescence derivatization with o-phthalaldehyde-ethanethiol (OPA-ET) in seawater matrix was developed. The derivatization procedure and HPLC conditions were carefully examined, which included optimization of the fluorescence excitation and emission wavelengths, the ammonium acetate concentration and pH of the mobile phase, the OPA-ET reagent content and composition and derivatization time. Because increasing salinity was observed to lower the derivatization efficiency, working standards were freshly prepared in artificial seawater with the same salinity as that of the samples for the quantification of 2-AEP and 2-AP3. The developed HPLC method showed a wide linear response with high linearity (R2 > 0.999) and high repeatability at three concentration levels. The relative standard deviation was less than 4.1% for 2-AEP and less than 1.7% for 2-AP3 (n = 7). The limits of detection for 2-AEP and 2-AP3 in artificial seawater matrix were both 12.0 μg/L. The recoveries were 83.0–104% for 2-AEP and 72.6–98.6% for 2-AP3 in different aqueous samples, including algal culture medium prepared with filtered seawater. These results indicated the matrix effect of this method was insignificant.

Rapid monitoring of proline accumulation in paprika leaf sap relative to leaf position and water stress

The present study evaluated the pattern of proline accumulation in paprika leaf sap using a modified high-performance liquid chromatography (HPLC) method. Leaf sap samples were extracted using five different solvents and two extraction procedures, and were analyzed using modified HPLC analytical conditions. Enhanced extraction and HPLC analytical conditions were applied to analyze paprika leaf sap obtained from different plant parts (petiole and lamina sap) under different environmental conditions. The results showed that pure water extraction without heating or organic solvent performed best. This newly developed HPLC method was validated and confirmed suitable for analyzing proline content in leaf sap. The proline concentration in both the petiole and lamina sap showed a similar accumulation pattern, exhibiting the lowest proline concentration in the arbitrary node group I (1–5 nodes) and the highest concentration in the VI node group (21–30 nodes). In addition, sap from both the petiole and lamina of side stem leaves showed statistically higher proline content than the main stem leaves. The proline concentration also increased with increasing water stress during both the fruit enlargement and coloring stages, but primarily during the fruit coloring stage. The overall results suggest that the proline concentration in paprika leaves differentially accumulates throughout the plant and can be analyzed by water extraction using a modified HPLC method.

Abstract 738: Development of an HPLC method for the assessment of hydrophobicity of ADC payloads

Abstract Antibody-Drug Conjugates (ADCs) are antibodies attached to cytotoxic agents (payloads) through chemically labile linkers. The development of ADCs can fail at various stages, a common cause being a lack of reproducibility in the methods used to attach the payload to the antibody (i.e., the conjugation process) to provide a good yield and uniform drug-antibody ratio (i.e., the DAR). One of the major problems associated with conjugation is the hydrophobicity of a payload which can cause aggregation. The aim of this study was to create a hydrophobicity model to study the physicochemical properties of payloads at an early stage. This should allow their hydrophobicity to be evaluated and optimised at the design stage. A predictive model has been developed based on High Performance Liquid Chromatography (HPLC). Initial work involved development of a gradient reversed-phase HPLC method, and its use in the analysis of multiple reference compounds to establish their relative retention times. This involved the evaluation of a range of reversed-phase columns and solvent systems in order to obtain reproducibility. These initial studies, which utilized a C18 monolithic column for optimum results, demonstrated a clear relationship between the retention time (RT) of the reference compounds and their experimental LogP values obtained from the literature (r = 0.983). Next, a number of commercially available ADC payloads were analysed using the same HPLC method to determine their RT values, and their LogP values were calculated using the ChemDraw software. Payloads studied included Monomethyl Auristatin E (MMAE), Talirine and Tesirine (PBD dimers), Mertansine (DM1) and Calicheamicin. For this group of compounds, the results confirmed that their RT values correlated well with their calculated LogP values (r = 0.94). For example, the payload Talirine, which has a calculated LogP of 3.77, had an RT value of 17 min on the HPLC system equating to a LogP of 3.13 based on the reference compounds. Finally, some novel DNA-interactive payloads (e.g., the pyridinobenzodiazepine FGX-2-62) were evaluated using the same methodology. For FGX-2-62, the calculated LogP varied significantly between different software packages (i.e., 1.47, 3.38 or 4.46 for ChemSketch, MarvinSketch or ChemDraw, respectively). However, its HPLC retention time using the same method was 1.88 min, equating to a LogP of 1.63. This suggests that FGX-2-62 is significantly less hydrophobic than Talirine (e.g., RTs of 1.88 min versus 17 min, respectively), an observation consistent with the ease of conjugation of FGX-2-62 to antibodies without significant aggregation. Overall, these results suggest that HPLC retention time derived from a suitable HPLC method is more reliable than the currently available software-based LogP prediction methods in providing a realistic assessment of the hydrophobicity of novel payloads and their propensity to cause aggregation during conjugation. Citation Format: Ilona Pysz, Paul J. Jackson, Khondaker M. Rahman, David E. Thurston. Development of an HPLC method for the assessment of hydrophobicity of ADC payloads [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 738.

Stability-indicating high-performance liquid chromatography (HPLC) method development and validation for the determination of quetiapine fumarate in bulk and dosage form by HPLC

Objective: The purpose of the realistic study is the determination of Quetiapine fumarate in bulk and tablet dosage form utilizing a basic, particular, exact, and formalize stability signifying high-performance liquid chromatography (HPLC) method. Materials and Methods: The assessment of Quetiapine fumarate in tablet dosage form performed using a validated stability-indicating HPLC method. HPLC Binary Gradient System of model no. HPLC 3000 Series having Greece C18 (250 mm × 4.6 ID, Particle size: 5 micron) column using mobile phase composition of methanol:water (80:20) is used and pH was adjusted to 3. Flow rate was maintained at 0.9 ml/min at room temperature. Evaluation was accomplished with ultraviolet detection at 213 nm. Results and Discussion: The retention time acquired for Quetiapine fumarate was at 3.3 min. The result acquired with the detector response was found to be linear which is in the concentration range of 20–100 μg/ml. This technique has been accepted and appeared to be particular, delicate, exact, straight, precise, rough, vigorous, and quick. Quetiapine fumarate was subjected to different accelerated stress conditions. The degradation products when anywhere well resolved from the pure drug with significantly different retention time values. Conclusion: It is inferred that this system can be connected for routine quality control of Quetiapine fumarate in tablet dosage forms as well as in bulk drug.

Stability of 5 mg/mL Nitrendipine Oral Suspension in Syrspend® SF PH4

Abstract Background Nitrendipine is prescribed to children for the treatment of primary hypertension (off-label use). Available specialties (Nidrel®, Baypress® and others generic drugs) are only marketed in tablet form, which is unsuitable for pediatric use. A hospital preparation of nitrendipine oral suspension at 5 mg/mL was developed. The aim of the study was to determine physicochemical and microbiological stability of the nitrendipine oral suspension in order to set a shelf life for the preparation. Methods A validated high-performance liquid chromatographic (HPLC) method was developed for the assay of nitrendipine. Nitrendipine oral suspensions were prepared using 20 mg Nidrel® tablets and suspending vehicle Syrspend® SF PH4. These preparations were packaged in amber glass bottles and stored at room temperature. The physicochemical (pH, osmolality, nitrendipine concentration, macroscopic changes) and microbiological stability of the preparation was tested over 90 days. Nitrendipine concentration at day 0 was considered as 100 % and nitrendipine concentration in subsequent samples greater than 95 % were considered stable. Results The developed HPLC method was validated in terms of linearity, accuracy, precision and specificity. After 90 days, no significant pH and osmolality variation was observed. No microbial growth was noted. Concentrations of nitrendipine were found to be always higher 95 % of the initial concentration. Conclusions Nitrendipine oral suspensions 5 mg/mL are stable for at least 90 days when stored at temperature room and in amber glass bottles. This suspension is more suitable for children than tablets and allows obtaining accurate doses based on patient’s body weight.

Development of an in-house HPLC method for the analysis of ecstasy-laced beverages

BackgroundPills of ecstasy continue to be the major drug of abuse among nightclub patrons. This stimulant type of substance can be taken orally in many ways, one of which is by drink spiking where the tablet or its powder is deliberately added to some beverage before consumption.MethodA high performance liquid chromatographic (HPLC) method was developed to quantify three analytes (methamphetamine, 3,4-methylenedioxymethamphetamine and ketamine) extracted from beverages associated with drink spiking. Ribena drink, Lipton tea drink and Pepsi soft drink were employed as the target sample matrices for validation studies.ResultsThe HPLC method was found precise (RSD < 3.2%) and accurate (mean recovery between 97 and 107%) regardless of the matrix type used. The linearity curve for each analyte presented a good regression line with a coefficient of determination, R2 > 0.994. The method was sensitive to detect at least 0.004 mg/mL spiked drugs and precise to quantify the lowest limit of analytes at 0.02 mg/mL.ConclusionAnalysis of beverages which were contrivedly spiked with genuine ecstasy substances substantiated that the method is fit for the intended purpose.

HPLC Method Development for Quantification of Doxorubicin in Cell Culture and Placental Perfusion Media.

Assessment of drug transport across the placenta is important in understanding the effect of drugs on placental and fetal health. These phenomena can be studied in both in vitro cell lines and ex vivo placental perfusions. We have successfully developed a sensitive yet simple high performance liquid chromatography (HPLC) method coupled with fluorescence detection to determine the concentration of doxorubicin (DXR) in cell culture media for transport studies in human trophoblast cells (BeWo, b30 clone) and in fetal media for placental perfusion experiments. The method was developed based on a protein precipitation technique and was validated in both media types for linearity, intra-day, and inter-day precision and accuracy. The relationship of peak area to concentration was linear with R2 values of 0.99 or greater obtained over the concentration range of 1.5 to 15,000 ng/mL. Despite the high concentrations of albumin in fetal perfusion media (30 mg/mL), the lower limits of detection and quantification for DXR were found to be 1.5 and 5 ng/mL, respectively. This analytical method may be used to study the transport of DXR across BeWo cells and human placenta during placental perfusion studies.

Separation and Characterization of New Forced Degradation Products of Macitentan: A Dual Endothelin Receptor Antagonist

Macitentan (MCT) is an endothelin receptor antagonist used for the treatment of pulmonary arterial hypertension. In the present study, MCT was subjected to forced degradation as per ICH guidelines. The drug degraded extensively in acidic, basic as well as neutral hydrolytic conditions and seven degradation products (DPs) were formed. All these DPs were selectively separated using high-performance liquid chromatography (HPLC) with a stationary phase of Inertsil C18 column (150 × 4.6 mm, 5 μm) and a mobile phase consisting of gradient mixture of 0.02% trifluoroacetic acid (TFA) and acetonitrile (ACN). The developed HPLC method was transferred to LC–ESI–QTOF–MS/MS for identification of DPs. The final mass spectrometric conditions were optimized for better ionization of drug and DPs with optimum mass signal sensitivity. All the formed DPs were new and well separated with sufficient resolution. The developed HPLC method was validated as per ICH-guidelines and can be used in drug testing labs for determination of quality of MCT in bulk and finished formulations.

Method Development and Validation for the Determination of Caffeine: An Alkaloid from Coffea arabica by High-performance Liquid Chromatography Method.

Objective:The present study was investigated to develop and validate a reversed phase high performance liquid chromatography method for the determination of caffeine from bean material of Coffee arabica.Materials and Methods:The separation was achieved on a reversed-phase C18 column using a mobile phase composed of water: methanol (50:50) at a flow rate of 1.0 mlmin-1. The detection was carried out on a UV detector at 272 nm. The developed method was validated according to the requirements for International Conference on Harmonisation (ICH) guidelines, which includes specificity, linearity, precision, accuracy, limit of detection and limit of quantitation.Results:The developed method validates good linearity with excellent correlation coefficient (R2 > 0.999). In repeatability and intermediate precision, the percentage relative standard deviation (% RSD) of peak area was less than 1% shows high precision of the method. The recovery rate for caffeine was within 98.78% - 101.28% indicates high accuracy of the method. The low limit of detection and limit of quantitation of caffeine enable the detection and quantitation of caffeine from C. arabica at low concentrations.Conclusion:The developed HPLC method is a simple, rapid, precisely, accurately and widely accepted and it is recommended for efficient assays in routine work.SUMMARYA simple, accurate, and sensitive high-performance liquid chromatography (HPLC) method for caffeine from Coffea arabica has been developed and validated. The developed HPLC method was validated for linearity, specificity, precision, recovery, limits of detection, and limits of quantification by the International Conference on Harmonization guidelines. The results revealed that the proposed method is highly reliable. This method could be successfully applied for routine quality work analysis.Abbreviation Used: C. arabica: Coffee arabica, ICH: International Conference on Harmonisation, % RSD: Percentage Relative Standard Deviation, R2: Correlation Coefficient, ppm: Parts per million, LOD: Limits of detection, LOQ: Limits of quantification, SD: Standard deviation, S: Slope, RP-HPLC: Reverse phase high performance liquid chromatography, v/v: Volume per volume.

Validation of high-performance liquid chromatography method for quantification of costunolide content in Laurus nobilis

Objective: To establish a method to determine the content of costunolide in Laurus nobilis leaves by high-performance liquid chromatography (HPLC). Materials and Methods: The separation was performed on a reversed-phase C18 column (100 A, 150 mm × 4.6 mm, 5 μm pore size) using a mobile phase composed of water:acetonitrile (40:60) at a flow rate of 1.0 ml/min. The detection was carried out on a ultraviolet detector at 210 nm. The developed method was validated according to the requirements for International Conference on Harmonisation guidelines. Results: The proposed method for costunolide was validated for linearity with excellent correlation coefficient (r2 > 0.999). The relative standard deviation (RSD) is less than 1% in precision (i.e repeatability and intermediate) of the method. The recovery rate for costunolide was within 100.54%–102.62%. The limit of detection and limit of quantitation were 2.29 and 6.64 parts per million, respectively. Conclusion: The developed HPLC method is simple, rapid, precisely, accurately, and widely accepted and it is recommended for efficient assays in routine work. Abbreviation Used: HPLC: High-performance liquid chromatography; UV: Ultraviolet; ICH: International Conference on Harmonisation; r2: Correlation coefficient; WHO: World Health Organization; ppm: Parts per million; % RSD: Percentage relative standard deviation

FORMULATION DEVELOPMENT AND EVALUATION OF GASTRO-RETENTIVE DOSAGE FORM OF ATAZANAVIR SULPHATE

Objective: To improve dissolution properties of atazanavir sulphate by preparing gastro-retentive granules by solid dispersion method and development of RP-HPLC method for estimation of this drug.Methods: Estimation of atazanavir sulphate was done using high performance liquid chromatography (HPLC) on inertsil column (5 µm, 250x4, 6 mm) with a mobile phase consists of methanol: water (91:9 v/v), at 0.5 ml/min flow rate and 249 nm UV detection. The method was validated as per ICH guidelines. Selection of the carrier for gastro-retentive formulation was based on phase solubility study of the drug. Solid dispersions of gastro-retentive granules of different composition of drug and carrier, were prepared by the kneading, heating and solvent evaporation. A 32factorial design was applied to optimize the gastro-retentive formulation. The amounts of polyethylene glycol 6000 (PEG 6000) (X1) and hydroxypropyl methyl cellulose (HPMC) (X2) were selected as independent variables and in vitro-release at 5, 9 h and total floating time was selected as dependent variables. Results: HPLC method was found to be linear in a concentration range of 10-60 μg/ml of the drug (r2= 0.999). The low value of % RSD in precision study indicates reproducibility of the method. The low value of LOD and LOQ suggests the sensitivity of the method. The solubility enhancement study of drug with various carriers followed descending order of solubility [Gelucire 44/14&gt;PEG 6000&gt;polyvinyl pyrrilidone (PVP)]. Highest % cumulative release was observed for the heating method at drug polymer (PEG 6000) ratio 1:5. Hence, this ratio has been selected for preparation of solid dispersion. From comparison of dissolution profile of formulated batches, formulation F4 [containing PEG6000 (1.6 g) and HPMC (200 mg)] showed promising dissolution parameters with desired floating properties.Conclusion: Results obtained by validation studies suggested that the developed HPLC method is simple, accurate, precise and can be used for routine analysis of atazanavir sulphate formulation. Results of evaluation of prepared batches indicate that batch F4 is a promising formulation for gastro-retentive dosage form of drug.