Mobile Phase Ratio Research Articles

Development and Validation ofa High-Performance Liquid Chromatography Method for the Estimation of Desogestrel and Ethinyl Estradiol in Bulk & Dosage Forms

A new method was established for estimation of Tizanidine and Ibuprofen by RP-HPLC method. The chromatographic conditions were successfully developed for the separation of Tizanidine and Ibuprofen by using Intersil ODS C18 column (250×4.6mm) 5.0µm, flow rate was 1.0ml/min, mobile phase ratio was (40:60 v/v) Potassium di hydrogen Phosphate Buffer: Acetonitrile pH 3.2 (pH was adjusted with Ortho-phosphoric acid), detection wavelength was 269 nm. The instrument used was Shimadzu HPLC, UV detector 2450, Spinchrom -software version-2. The method shows linearity between the concentration range of 0.6-1.4µg / ml for Tizanidine and 120-280 for Ibuprofen. The % recovery of Tizanidine and Ibuprofen were found to be in the range of 98.0 % - 102.0 %. Keywords: Intersil ODS C18 column, Tizanidine and Ibuprofen and RP-HPLC.

Chiral separation of new chiral insecticide pyraquinil isomers and establishment of analytical methods in vegetables

以全新手性杀虫剂唑虫酯为研究对象,通过筛选手性色谱柱和优化流动相比例,建立了唑虫酯及其氧化代谢物异构体的拆分方法,在此基础上开发利用高效液相色谱-串联质谱(HPLC-MS/MS)同时测定小白菜和蕹菜中唑虫酯及其氧化产物手性异构体的分析方法。以纤维素-三(3,5-二氯苯基氨基甲酸酯)共价键合手性柱(Chiral INC)(250 mm×4.6 mm, 5 μm)为分析柱,乙腈和2 mmol/L甲酸铵水溶液作为流动相进行梯度洗脱分离,在多反应监测负离子模式下进行检测,唑虫酯4个异构体分离度分别为1.63、2.83和1.74,唑虫酯氧化产物异构体分离度为5.82。通过衍生化的方法进一步确定出峰顺序为RS-唑虫酯、SS-唑虫酯、RR-唑虫酯、SR-唑虫酯、S-唑虫酯氧化产物和R-唑虫酯氧化产物。唑虫酯和其氧化产物的手性异构体分别在1.25~1250 μg/L和2.5~2500 μg/L范围内具有良好的线性关系,相关系数(R2)大于0.99。在蕹菜和小白菜样品中同时添加唑虫酯和唑虫酯氧化产物消旋体进行添加回收试验,添加水平为1、20、400 μg/kg(即唑虫酯异构体为0.25、5、100 μg/kg;唑虫酯氧化代谢产物异构体为0.5、10、200 μg/kg),回收率为72.6%~110.6%,相对标准偏差(RSD)均在9.4%以下,其中日内重复性的RSD在0.5%~9.4%之间;日间重复性的RSD在1.0%~8.6%之间,表明该方法具有良好的回收率和精密度。该研究可为唑虫酯这一新型手性农药的环境行为研究及后续质量控制、药效评价等提供相应的分析技术,为新农药开发应用提供有力的技术支撑。

Optimization and Validation of HPLC-DAD Method for the Quantification of Curcuminoids in Turmeric (Curcuma Longa L.)

Curcumin and its derivatives named curcuminoids are naturally occurring polyphenolic compounds extensively marketed as a nutraceutical. The wide therapeutic potential of curcumin is highly explored by the authors. Therefore, pharma industries made many clinical inventions to bring it to the market as a drug. In this study, a simple, rapid, and sensitive High Performance Liquid chromatographic (HPLC) method for simultaneous determinations of curcumin, desmethoxycurcumin (DMC), and bis-desmethoxycurcumin (BDMC) was optimized and validated. Separation of curcumin, DMC, and BDMC was performed using the C18 column. Six factors, including the solvent type, ratio of mobile phase, flow rate of mobile phase, column temperature, injection volume, and wavelength, were investigated. The LOD and LOQ were observed to be 0.001 and 0.003 mg/L, R2 value < 0,99, respectively. RSD (%) is 0.974 for inter-day, and 1,312 for intra-day. Taking into consideration the retention time, peak area, resolution, and tailing factor, the optimum conditions were preferred: methanol as solvent, THF/Citric Acid (40/60) as mobile phase, flow rate of mobile phase at 0.5 mL/min, temperature of column at 30oC, injection volume at 20 µL and wavelength at 425 nm. The method is further used to determine ppm levels of curcumin derivatives, and recovery was found to be 90%. The proposed analytical method can be used for the quantification of curcumin in medicinal plants.

Stress Degradation Studies and Development of a Validated RP-UHPLC Method of Ledipasvir

The intent of the research work was to develop and validate a simple, selective and precise reversed phase ultra-high performance liquid chromatography (RP-UHPLC) method for the determination of ledipasvir. A forced degradation study was performed as per International Conference on Harmonisation (ICH) guidelines Q1A (R2) and Q1B. Ledipasvir was found to be well separated from degradation products using an analytical C18 column (150 mm × 4.6 mm i.d., 5 μm particle size) with a ratio of mobile phase (75:25 v/v) consisting of methanol and 0.1% trifluoroacetic acid (TFA) kept at ambient temperature. The average retention time of ledipasvir was found 4.45 min at 254 nm wavelength with 1.6 mL/min isocratic flow rate and 10 µL injection volume. Linearity, accuracy, precision, sensitivity, robustness, and ruggedness were studied according to ICH guideline Q2 (R1) to validate the method. Then, this validated method was applied for forced degradation studies of ledipasvir. In conclusion, the developed method has been successfully used to study degradation behaviour of ledipasvir and may be useful to quantify the drug in different pharmaceutical dosage forms.

A Comprehensive Review of the Analytical Development and Validation of Alprazolam in Bulk and Pharmaceutical Dosage Form

Alprazolam is approved in 2003 by US FDA. Alprazolam is available as an extended-release tablet, a mouth dissolving tablet (a tablet that dissolves rapidly in the mouth), as well as a concentrated solution (liquid). For the treatment of severe anxiety and panic disorder, alprazolam is one of the most commonly prescribed benzodiazepines. IPUAC name of Alprazolam is 8-Chloro-1-methyl-6-phenyl-4H-[1,2,4]triazolo[4,3-a] [1, 4] benzodiazepine. The molecular formula and molecular Weight is C17H12ClFN4 and 326.75 g/mol. These articles may serve as an overview of Alprazolam with their drug profile, pharmacology, pharmacokinetics, and analytical HPLC procedures that are commonly employed in determining common provision issues. These reviews cover topics such as mobile phase, mobile phase ratio, column, retention time, flow rate, UV detector wavelength, run time, and more. Linearity, percent recovery, detection limit, and quantification limit are all validating parameters. The pharmaceutical analysis serves as an internal control for pure and pharmaceutical dosage forms, which is critical for quality assurance. The development of analytical methods has emerged as a crucial study activity.

A Validated Ultra Performance Liquid Chromatography Method for Simultaneous Estimation of Diacerein and Aceclofenac in Bulk and Pharmaceutical Formulation

A simple, exceptionally cost-effective, extremely accurate, quite precise and highly reproducible analytical method was developed and validated for simultaneous estimation of Diacerein and Aceclofenac in bulk and Pharmaceutical formulation. The new Ultra Performance Liquid Chromatography technique was developed. Method- The separation has been done on column ACQUITY UPLC BEH Shield C18 (50 x 2.1mm), 1.7µm (40°C temperature). The mobile phase contained Buffer and Acetonitrile (Buffer: ACN) (55:45 V/V).. The flow rate was set at 0.4ml/min, and detected at 268nm with PDA detector. Mobile phase was sonicated for 15 min. before use. 5μl of samples of standard stock solution and tablet solution were injected. Different trials were performed to separate diacerein and aceclofenac. The total run time of the detection was 4 min. The developed method was validated against different validation parameters. Results - The retention times were obtained at 1.762min. and 2.891 min. for Diacerein and Aceclofenac respectively. Accuracy study was observed in the range of 100.33% to 101.67% with less than 2% RSD. LOQ was found to be 2.98μg/ml and 5.9041μg/ml. similarly LOD was found to be 0.9841μg/ml and 1.9467μg/ml. for Diacerein and Aceclofenac respectively. Precision %RSD for intraday and interday were found to be 0.19 and 0.18 for Diacerein , 0.44 and 0.16 for Aceclofenac respectively. Linearity was found in the range of 2.5-17.5μg/ml and 5-35μg/ml for Diacerein and Aceclofenac respectively. The method was found robust by deliberate changes in the flow rate, ratio of mobile phase and detection wavelength. Conclusion-The method was found to be satisfactory and can be used successfully for determination of Diacerein and Aceclofenac simultaneously in bulk and pharmaceutical dosage form.

A Simple and Sensitive HPLC-MS/MS Assay for the Quantitation of Blonanserin and N-Desethyl Blonanserin in Rat Plasma and Its Application to Pharmacokinetic Study.

A high-performance liquid chromatographic method coupled with triple quadrupole mass spectrometry (LC-MS/MS) for the analysis of blonanserin and its active metabolite, N-desethyl blonanserin, in rat plasma has been developed and validated. The biological samples were treated by simple direct protein precipitation before separation on an Agilent Eclipse Plus C18 column (4.6 × 100 mm, 3.5 μm) with a column temperature of 35°C at a flow rate of 0.5 mL/min. The mobile phase A is a mixture of methanol and water (75 : 25, v/v, 5 mM ammonium formate), and the mobile phase B is acetonitrile containing 0.1% formic acid. The ratio of mobile phase A to mobile phase B is 15 : 85. Electrospray ionization (ESI) multiple reaction monitoring modes are used for detection, which are m/z 368.10 ⟶ 296.90 (blonanserin), m/z 340.15 ⟶ 297.05(N-desethyl blonanserin), and m/z 348.15⟶ 302.05 (N-desethyl blonanserin-d8). The linear response range was 0.1–100.0 ng/mL for blonanserin and N-desethyl blonanserin. The lower limit of quantification (LLOQ), calibration curves, carryover, and matrix effects were sufficiently accurate and precise according to the National Medical Products Administration (NMPA) guidelines for bioanalytical method validation. This analytical method was successfully applied in a blonanserin-poloxamer thermosensitive gel pharmacokinetic study in rats.

Analytical Method Development and Validation for the Simultaneous Estimation of Sulfadiazine and Pyrimethamine by RP-HPLC Method

Background Sulfadiazine and Pyrimethamine is a combination drug of choice to treat malaria and used in those living in or will be travelling to an area where there is chance of getting malaria. Objective The main objective of the study was to validate the simultaneous estimation and the method established with resisting small changes in flow rate and ratio of organic compounds and proposed method ensures its use in routine quality control analysis of pharmaceutical formulation. MethodologyA HPLC AllianceWater 2695 with UVVIS DetectorPDA detector UV lab India UV 3000 series and Zodiac C18 250 mm times 4.6 mm times 5 microm column were used. A new method was established for simultaneous estimation of Sulfadiazine and Pyrimethamine by RP-HPLC method. The chromatographic conditions were successfully developed for the separation of Sulfadiazine and Pyrimethamine by using Zodiac sil C18 column 4.6times150 mm 5micro flow rate was 1mLmin mobile phase ratio was 7030 vv methanolphosphate buffer KH2 PO4 and K2 HPO4 phosphate pH 3 pH was adjusted with orthophosphoric acid detection wavelength was 240 nm. Results The results were in good agreement with those obtained with official HPLC with maximum absorption of 240 nm by preparing mobile phase 7030 methanolphosphate buffer with flow rate 1 mLmin and was run for 10 minutes by selecting column Zodiac silica RP C18 4.5times100 mm 3.0 microm of ambient temperature. All the results were obtained with good precision accuracy and robustness as per ICH guidelines. Conclusion It can be concluded that the proposed RPHPLC method was accurate precise sensitive specific robust and reproducible for the simultaneous analysis of Sulfadiazine and Pyrimethamine with less tailing factor and also economical. Zodiac sil C18 column 4.6times150mm5micro flow rate was 1mLmin mobile phase ratio was 7030 vv methanol phosphate buffer KH2 PO4 and K2 HPO4 phosphate pH 3 pH was adjusted with orthophosphoric acid detection wavelength was 240 nm.

Analytical Method Development and Validation for the Simultaneous Estimation of Rifampicin and Isoniazid in its Bulk and Pharmaceutical Dosage Forms by RP-HPLC

Background A combination of Rifampicin and Isoniazid is the drug of choice to treat tuberculosis patients in sustenance or those travelling to a region where there is a probability of being infected with tuberculosis.Objective The main objective of the simultaneous estimation of combined drug was to establish identity purity physical characteristics and potency of the drugs.Methodology The chromatographic environment was fortunately evolved for the partition of Rifampicin and Isoniazid by using column thermosil RPC18 4.5times100 nm 5.0 microm flow rate was 1 mLmin mobile phase ratio was 7030 vv methanolsodium acetate buffer. A pH of 3 was adjusted with Ortho phosphoric acid wavelength was detected at 240 nm. Column dimensions were Thermosil C184.6times100mm 5.0 microm.Results The results were in good agreement with those obtained with official HPLC with absorption maximum at 240 nm by preparing mobile phase 7030 methanolsodium acetate buffer with a flow rate 1 mLmin and it ran for 5 minutes by selecting thermosil RPC18 4.5times100 nm of ambient temperature. All the results obtained were precise accurate and robust as per international conference on Harmonization ICH guidelines.Conclusion It can be concluded that the proposed Reverse Phase High Performance Liquid Chromatographic RPHPLC method is accurate precise sensitive specific robust and reproducible for the simultaneous analysis of Rifampicin and Isoniazid with less tailing factor and is also economical. Thermosil RPC18 4.5times100 nm 5.0 microm flow rate was 1 mLmin. Both the samples were in the range of 200 to 400 nm and maximum wavelength was identified at 240 nm.

Development and Validation of an Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Quantitative Determination of N-((3S,4S)-4-(3,4-Difluorophenyl)piperidin-3-yl)-2-fluoro-4-(1-methyl-1H-pyrazol-5-yl)benzamide in Dog Plasma

The PI3K/AKT/MTOR signalling pathway plays an important role in the growth and proliferation of tumour cells. N-((3S,4S)-4-(3,4-Difluorophenyl)piperidin-3-yl)-2-fluoro-4-(1-methyl-1H-pyrazol-5-yl)benzamide (Hu7691) is a new-generation selective AKT inhibitor developed at Zhejiang University. In this study, we developed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the measurement of Hu7691 in dog plasma. Plasma was precipitated with acetonitrile and then separated on a trifunctionally bonded alkyl column. Excellent separation efficiency and selectivity were achieved by adjusting the mobile phase ratio, with a total running time of only 5 min. The linear dynamic range of the calibration curve was 5–1000 ng/mL. The method was fully validated, and all performance metrics met the criteria. The validated method was used for the pharmacokinetic monitoring and bioavailability assessment of Hu7691 in dogs. The results showed that the area under the curve and peak plasma concentration of Hu7691 increased with increasing dose (oral 5, 10, 20 mg/kg, intravenous 10 mg/kg), and oral bioavailabilities were 86.7%, 50.8%, and 50.5%, respectively, indicating a high bioavailability of Hu7691 in dogs. This provides a test basis for the clinical application of the compound.

RP-HPLC Method for Quantification of Emtricitabine and Tenofovir Alafenamide Fumarate Drug Substance and Tablet Dosage Form

Aim: The primary objective of the research work is to develop a effective, sensitive, economical and simple reverse phase HPLC method to estimate Emtricitabine and tenofovir alafenamide fumarate in its pure and binary mixture of tablets.
 Study Design: HPLC based Quantification Studies.
 Place and Duration of Study: 1Department of Chemistry, Acharya Nagarjuna University,Guntur, Andhra Pradesh between April 2019 and August 2020.
 Methodology: Separation of the analytes were done by using Eclipse XDB-Phenyl (250 x 4.6mm, 5µ,100 A0) column and a mobile phase ratio of 30:10:70 percentage of 0.1% trifluoro acetic acid: acetonitile: methanol at a flow rate of 1 ml/min. The injected standard and sample solutions were detected 260nm wavelength.
 Results: The retention time of Emtricitabine and tenofovir alafenamide fumarate were found at 2.3min and 2.8 min respectively. The method has good linearity range about 50 to 150µg/ml of Emtricitabine and 6.5 to 19.5 µg/ml of tenofovir alafenamide fumarate. The method has validated as per ICH guidelines and all the validation parameterwere satisfy the ICH Q2 specification acceptance limits
 Conclusion: The developed method said to be highly sensitive, accurate, specific and robust, therefore this method has high probability to adopt in pharmaceutical industry for regular analysis of Emtricitabine and tenofovir alafenamide.

UPLC-MS/MS Method Validation for Estimation of Resveratrol in Rat Skin from Liposphere Gel Formulation and Its Application to Dermatokinetic Studies in Rats.

For the quantification of resveratrol (RV) in the Wistar rat skin, an ultra-performance liquid chromatography-mass spectrometric (UPLC-MS/MS) method was developed and validated on ACQUITY UPLC BEH C18 column (1.7 μm). The mobile phase ratio of methanol (A) and 2% formic acid (B) (ratio 10: 90% v/v, 80: 20 v/v) at isocratic elution with flow rate 0.3 mL/min, and run time 3 min was used for analysis. In addition, the use of multiple reaction monitoring (MRM)/ES+ mode to detect the analytes and to track parents to daughter ion transition of 229.17 > 107.04 m/z (time scan 3 min, retention time 1.48) for RV and curcumin as an internal standard shows 369.16 > 176.93m/z (scan time is 2.80 min, retention time is 1.11), respectively. Linearity was observed in the range of 2.5 to 2,000 ng/mL (R2 = 0.987). Precision and accuracy on rat skin were within the acceptability range (RE%: ±15; RSD%: ±15). Moreover, it showed a good percentage recovery and found within acceptance limit 90-110%. Lower limit of detection and quantitation for the method observed to be 2.5 and 20 ng.mL-1, respectively. Method application indicated successful determination of dermatokinetics parameters of RV from lipospheres gel and suspension in the rats.

QBD APPROACH TO ANALYTICAL METHOD DEVELOPMENT AND ITS VALIDATION FOR ESTIMATION OF LENVATINIB IN BULK AND PHARMACEUTICAL FORMULATION

Objective: The objective of this research was to develop a simple, very rapid, sensitive, accurate, precise reverse phase High-Performance Liquid Chromatography (RP-HPLC) technique for the estimation of Lenvatinib in bulk and its dosage form. Methods: To perform this study, we employed a central composite design (CCD) to make method robust and effective to create chromatographic database. The factor screening studies were performed using 2-factor 10-runs. The factors were selected as the mobile phase ratio and buffer pH. Results: The desirability value of the optimized model was found to be 0.869 and The optimized chromatographic condition was achieved on Enable C18 analytical column with 0.01M Ammonium acetate buffer pH 3.84: methanol (33.17:66.83 v/v) as the mobile phase and flow rate of 1 ml min-1 and detection wavelength was set to 240 nm. The retention time of Lenvatinib was found to be 5.122 min. Linearity was established for Lenvatinib in the range of 10-50 µg/ml with a correlation coefficient (r2=0.9995). The accuracy values were found to be in the range of 98–102%. Intraday precision and Interday precision were in prescribed (Less than 0.98% RSD). Robustness was found to be less than 1.22% RSD. Conclusion: The proposed method was useful for best analysis of Lenvatinib in Bulk pharmaceutical dosage forms. Central Composite Design was an effective tool for the proposed RP-HPLC method.

Analytical Method Development and Validation for Simultaneous Determination of Simvastatin and Mupirocin Using Reverse-Phase High-pressure Liquid Chromatographic Method

This study was aimed to develop and validate the use of reverse-phase high pressure liquid chromatographic method for the estimation of simvastatin (SIM) and mupirocin (MUP) simultaneously. The chromatographic method developed is optimized for flow rate with the column, solvent, and buffer used and mobile phase ratio, molarity, and pH. The validation of the optimized method and the forced degradation studies of both drugs (under acidic, alkaline, oxidation, heat, light, and neutral conditions) were conducted following the The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. Kromasil C18 column (250 mm × 4.6 mm, 5 µm) with ultraviolet detection at 224 nm and acetonitrile/phosphate buffer (30 mM, 70:30 v/v, pH 3.5; adjustment done using orthophosphoric acid) as mobile phase at a flow rate of 1.1 mL/min were observed to provide a good resolution for MUP and SIM at retention times of 2.32±0.008 and 13.55±0.254 min, respectively, with high accuracy (percent recovery was 99.69±0.82 for MUP and 101.10±0.02 for SIM) and linearity in the range of 5-30 µg/mL (r2: 0.9969 for MUP and r2: 0.9959 for SIM). The diagnostic limit and the lower limit of determination were 0.771±0.234 and 2.338±0.246 μg/mL for MUP and 0.595±0.282 and 1.803±0.334 µg/mL for SIM, respectively. The validated method was used to understand the degradation behavior of both drugs after the forced degradation studies. The analytical method developed is determined to be specific, sensitive, precise, and accurate for the estimation of MUP and SIM simultaneously in the combined dosage form.

Method Development and Validation of Canagliflozin by using RP-HPLC in Pure and Tablet Dosage Form

A new method was established for simultaneous estimation of Canagliflozin by RP-HPLC method. The chromatographic conditions were successfully developed for the separation of Canagliflozin by using INERTSIL column, C18 (150x4.6)5µm column, flow rate was 1ml/min, mobile phase ratio was Water: Acetonitrile (70:30), detection wavelength was 264nm. The instrument used was Hitachi HPLC Auto Sampler, Separation module 1575. The analytical method was validated according to ICH guidelines (ICH, Q2 (R1)). The linearity study for Canagliflozin was found in concentration range of 1μg-5μg and 100μg-500μg and correlation coefficient (r2) was found to be 0.999 and 0.999, %mean recovery was found to be 100% and 100.5%, %RSD for repeatability was 0.2 and 0.4, %RSD for intermediate precision was 0.5 and 0.1 respectively.

Analytical Method Development and Validation of Etravirine by RP-UFLC

An ultra-fast liquid chromatography (RP-UFLC) approach was processed for the estimation of ETR, which is quick, responsive, reliable, and inexpensive. Work on the Phenomenex Kinetex C18 column (250x 4,6mm. 5μ) was carried out with MeOH and acetonitrile in the mobile phase ratio of (60:40v/v) at 1.0mL/min. With a PDA detection system, the eluent was tracked at 311nm. ETR elutes at a persistence time of 3.226 min. The proposed method gives linearity of concentration from 1 to 5μg/mL, with the value of R2 at 0.9942%. For the proposed method, LOD and LOQ are measured as 0.02 and 0.073μg/mL. The pharmaceutical drug included acidic, alkaline, corrosion, Ultraviolet, and heat stress stipulations. The deterioration material was nicely resolved from ETR peaks, which showed the reliability of the process. The approach has been tested with respect to process adequacy, linearity, reliability, and robustness, as per ICH guidelines.

A simple stability indicating RP-HPLC-DAD method for concurrent analysis of Tenofovir Disoproxil Fumarate, Doravirine and Lamivudine in pure blend and their combined film coated tablets

The main aim of the study was to develop an economical, insightful, accurate and simple RP-HPLC-DAD method with high precision and good sensitivity for concurrent determination of Tenofovir disoproxil fumarate, Doravirine and Lamivudine in blended bulk form and their combined tablet form. A method with Ascentis C18 (150×4.6mm, 5μm) column, mobile phase ratio of 0.1% ortho phosphoric acid and Acetonitrile in 70:30 (v/v), 1mL/min flow rate and detection wavelength of 260nm was highly proficient in effective separation of all three drugs. The developed method was validated in accordance with ICH specifications. The retention times of Doravirine, Lamivudine and Tenofovir disoproxil fumarate observed were 2.4, 2.9, and 3.6min, respectively. The linear responses were observed for Doravirine, Lamivudine and Tenofovir disoproxil fumarate in the range of 12.5-75μg/mL, 75-225μg/mL and 75-225μg/mL, respectively. The limit of detection and quantification values were calculated to be 0.36μg/mL and 0.11μg/mL for Lamivudine, 0.55μg/mL and 1.66μg/mL for Tenofovir disoproxil fumarate and 0.03μg/mL and 0.09μg/mL for Doravirine. The % RSD values of the intra-day and inter-day precision were calculated in the range of 0.134-1.749. The mean percentage recovery of all three analytes was in the range of 98.85-100.18%. The statistical results of the validation parameters ensured that the method was accurate, specific, and precise with good sensitivity. Investigation of analytes under different stressed conditions ensures the stability of analytes reflecting the stability indication of the method. The developed method has high proficiency in separation of Tenofovir disoproxil fumarate, Doravirine and Lamivudine. The degradation products generated due to stress conditions also separated with good resolution. The current method is a stability-indicating assay method consisting of appropriate specificity, accuracy, precision and sensitivity. The developed method has a good potential to be adopted by the pharmaceutical industrial sector.

Implementation of Analytical Quality-by-Design for Developing a Robust HPLC Method for Quantitative Estimation of Voriconazole: Application in Drug Formulations

The current studies endeavour to develop and validate a simple, sensitive and robust reversed- phase (RP) high-performance liquid chromatography (HPLC) methodology for quantitative estimation of voriconazole (VRC), following systematic principles of Analytical Quality-by-Design (AQbD). A comprehensive risk assessment plan, followed by factor screening studies employing Taguchi design, was executed to select “vital few” critical method parameters, influencing critical analytical attributes. Subsequently, response surface optimisation studies were conducted on the identified critical method parameters, viz., mobile phase ratio and flow rate, with chosen critical analytical attributes, viz., peak area, theoretical plate count, retention time and peak tailing, employing a Central Composite Design. The design space, demarcating the optimal method operating conditions, was identified using graphical optimisation and subsequently validated employing Monte Carlo simulations. The optimal solution consisted of acetonitrile and acetic acid solution (0.05 %, pH 4) as mobile phase (50:50 v/v), flow rate of 1 mL/ min with a detection wavelength of 256 nm, exhibiting linearity between 0.1 and 50 μg/mL in methanol and Hanks balanced salt solution. Validation studies confirmed precision (97.78-103.42 %), accuracy (98.99-102.34 %), and suitability of the developed analytical method for effective quantification of VRC with high sensitivity (LOD: 0.031 μg/mL; LOQ: 0.093 μg/mL). The validated analytical technique was successfully ratified for quantification of VRC in drug nanoformulations too. In a nutshell, the application of AQbD and Monte Carlo simulations aided in developing a robust analytical method for the estimation of VRC per se as well as in drug nanoformulations.

The Storage Effect against Vitamin C Content in crystal guava (Psidium guajava L.) Juice

Guava fruit (Psidium guajava L.) has many varieties, one of which is crystal guava. Crystal guava contains vitamin C which is easily oxidized by temperature. Therefore, this study aimed to determine the levels of vitamin C in crystal guava juice at different storage. Crystal guava samples were obtained from Bogor and then stored at different places and temperatures. Hence, guava fruits were placed in the freezer, refrigerator, and room temperature for 3 days, and all samples were juiced. Based on visual evaluation, guava fruit color is different. Moreover, the quantification method of vitamin C levels in guava juice by HPLC can be carried out with a flow rate of 1 mL/minute using the mobile phase ratio (methanol/water: 5/95, v/v), λmax: 265 nm and C18 as a stationary phase. Retention time of vitamin C in guava is around 2.765 minutes. The effect of storage of vitamin C content in guava juice from fresh samples, and guava stored in the freezer, in the refrigerator, and at room temperature are 3.28 mg/mL, 3.20 mg/mL, 1.65 mg/mL, and 0.82 mg/mL. Therefore, it can be concluded that the best storage of guava fruit is in the freezer because vitamin C levels in crystal guava do not have a significant difference after treatment.

Developing a Validated HPLC Method for Quantification of Ceftazidime Employing Analytical Quality by Design and Monte Carlo Simulations.

Ceftazidime, a third-generation cephalosporin, is widely used in the treatment of lung infections, often given as "off-label" nebulization. There is a need to develop a sensitive and robust analytical method to compute aerodynamic properties of ceftazidime following nebulization. The current study entails development of a simple, accurate, and sensitive HPLC method for ceftazidime estimation, employing the principles of analytical quality-by-design (AQbD) and Monte Carlo simulations. Selection of critical material attributes (CMAs) affecting method performance was accomplished by factor screening exercises. Subsequently, the influential CMAs, i.e., mobile phase ratio and flow rate, were systemically optimized using a face-centered cubic design for the chosen critical analytical attributes (CAAs). The factor relationship(s) between CMAs and CAAs was explored employing a 3 D-response surface and 2 D-contour plots, followed by numerical as well as graphical optimization, for establishing the optimal chromatographic conditions. The obtained method operable design region was validated by Monte Carlo simulations for defect rate analysis. The optimized HPLC conditions for estimating ceftazidime were acetonitrile to acetic acid solution (75:25) as mobile phase at a flow rate of 0.7 mL/min, leading to Rt of 4.5 min and peak tailing ≤2. Validation studies, as per International Conference on Harmonization Q2(R1) guidance, demonstrated high sensitivity, accuracy, and efficiency of the developed analytical method with an LOD of 0.075 and LOQ of 0.227 µg/mL. Application of this chromatographic method was extrapolated for determining aerodynamic performance by nebulizing ceftazidime at a flow rate of 15 L/min using a next-generation impactor. The study indicated superior performance, sensitivity, and specificity of the developed analytical system for quantifying ceftazidime. Application of an AQbD approach, coupled with Monte Carlo simulations, aided in developing a robust HPLC method for estimationof ceftazidime per se and on various stages of impactor. (i) QbD-enabled development of robust RP-HPLC method for ceftazidime quantification, (ii) Analytical method optimization employing Risk Assessment and Design of Experiments, (iii) Design space verification and defect rate analysis using Monte Carlo simulations, (iv) Chromatographic method validation as per ICH Q2 R1 guidelines and (v) Quantitative estimation of ceftazidime on various stages of impactor.