Validation Of High-performance Liquid Chromatography Method Research Articles

Validation of HPLC and TLC analytical methods to determine radiochemical purity of 99mTc-cAbVCAM1-5, a new experimental radiotracer

Cardiovascular diseases, including fatal myocardial infarctions from atheromatous plaques, are the primary global mortality cause. Detecting stenotic atheromatous plaques is possible through coronary angiography, but vulnerable plaques with eccentric remodeling are undetectable with current diagnostic methods. Addressing this challenge, our group developed a radiopharmaceutical drug targeting vascular cell adhesion molecule 1 (VCAM-1), radiolabeled with technetium-99m. Given the absence of a monograph in the European Pharmacopoeia, and in order to draft the investigational medicinal product documentation, analytical methods had to be validated by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) to determine the radiochemical purity (RCP) of 99mTc-cAbVCAM1–5. This study therefore presents the results of the validation of analytical methods obtained in this context. The method validation followed the European Association of Nuclear Medicine (EANM) recommendations adapted from ICH Q2(R1), ensuring conformity with specificity, accuracy, repeatability and intermediate precision, linearity, robustness, quantification limit (LoQ), and range criteria. Regarding the results of specificity, both HPLC and TLC methods demonstrated excellent separation of 99mTc-cAbVCAM1–5 from impurities 99mTcO4-. Accuracy results indicated recovery percentages within the range of 99.52–101.40% for the HPLC and 99.51–101.97% for TLC, ensuring reliable measurements for each concentration of 99mTcO4-. Precision of the methods was validated by assessing repeatability and intermediate precision. Linearity was determined over the usual concentrations range and the correlation coefficient was greater than 0.99 for both methods. The limit of quantification was measured by diluting the 99mTcO4- to obtain a signal-to-noise ratio of around 10:1. Under these conditions, we obtained an LOQ of 2.10 MBq/mL for HPLC and 2Mbq/mL for TLC. In conclusion, the analytical methods developed in this study comply with EANM recommendations. This therefore allows us to correctly assess the radiochemical purity of 99mTc-cAbVCAM1–5, a new radiotracer targeting inflammation in vulnerable plaques.

Advances in High-Performance Liquid Chromatography (HPLC) Method Development and Validation: A Comprehensive Review

High-Performance Liquid Chromatography (HPLC) stands as the cornerstone technique for the detection, separation, and quantification of drugs in various matrices. Method optimization necessitates a thorough investigation of multiple chromatographic parameters, encompassing sample pretreatment, mobile phase composition, column selection, and detector configuration. This article aims to comprehensively address the processes of method development, optimization, and validation within the realm of HPLC. The inherent advantages of HPLC, including its rapidity, specificity, accuracy, precision, and automation feasibility, render it an indispensable tool for analyzing a wide spectrum of drugs in complex multi-component dosage forms. Furthermore, HPLC method development and validation hold paramount importance across the spectrum of drug discovery, development, and manufacturing, as well as in diverse human and animal studies. The validation of analytical methods during drug development and manufacturing is imperative to ensure their fitness for the intended purpose. Adhering to Good Manufacturing Practice (GMP) requirements, pharmaceutical industries must establish comprehensive validation policies outlining the validation procedures. This article primarily delves into the optimization of HPLC conditions, emphasizing the critical role of method refinement in ensuring robust and reliable analytical methods.

ISOLATION, DEVELOPMENT AND VALIDATION OF QUERCETIN FROM THE ALLIUM ASCALONICUM’S TUNIC BY HPLC METHOD

Allium ascalonicum, the tunic of which holds various bioactive compounds including including quercetin - a potent flavonoid, is renowned for its medicinal properties. This study presents the development and validation of a High-Performance Liquid Chromatography (HPLC) method for quantifying quercetin in Allium ascalonicum tunic. Validation of the method was conducted following International Conference on Harmonization (ICH) guidelines, evaluating parameters such as linearity, precision, and accuracy. This validated HPLC method offers an efficient and reliable means of quantifying quercetin in Allium ascalonicum tunic. Its application holds promise for routine analysis in pharmaceutical and nutraceutical industries, offering insights into the medicinal potential of Allium ascalonicum peels as a natural source of quercetin.

Stability assessment and content determination test of oral liquid forms of beclomethasone dipropionate prepared in hospital pharmacy

ObjectivesOral beclomethasone dipropionate (BDP) is known for its use as a therapeutic medicine for gastrointestinal graft-versus-host disease (GI-GVHD). Despite growing demand for oral BDP formulation, no commercial forms have yet...

A Review on Method Development of High Performance Liquid Chromatography (HPLC) and its Applications

The most common method for identifying, separating, and measuring the medication is HPLC.To improve the approach, a variety of chromatographic factors including sample pretreatment, mobile phase selection, column selection, and detector selection were examined. Reviewing the technique creation, optimization, and validation is the article's main goal. The chemistry of the molecules, the synthetic pathway, solubility, polarity, pH and pKa values, and the activity of the functional groups are all factors that affect the creation of an HPLC technique. Information on several stages and properties including accuracy, specificity, linearity, limit of detection, and limit of quantification are provided through the validation of HPLC methods in accordance with ICH Guidelines1.

Validation of a HPLC method for the determination of diclofenac diethylamine and three of its impurities in a gel pharmaceutic form.

Monitoring impurities in drug products is a principal requirement of pharmaceutical regulatory authorities all over the world to ensure drug safety. For this reason, there is a great need of analytical quality control of dugs products. In this study, a simple, efficient and direct high performance liquid chromatography (HPLC) method was developed for the determination of three impurities of diclofenac. The HPLC method was developed using the mobile phase which consisted of HPLC grade mixture (Acetonitrile and 0.01M phosphoric acid adjusted to pH = 2.3) in the ratio of (25:75 v/v) respectively. The separation was performed in 15 minutes. The calibrations curve of the three impurities were linear; the correlation coefficient was 0.999 at a concentration of (0.00015 - 0.003 µg/mL). The validation of this method shows that this method meets all validation criteria. This shows the reliability of the use of this method for the routine control of diclofenac impurities. The validation of a robust HPLC method for the determination of diclofenac impurities is of great importance for the pharmaceutical industry to control its products.

Advancing posaconazole quantification analysis with a new reverse-phase HPLC method in its bulk and marketed dosage form.

Introduction: Posaconazole is a widely used antifungal drug, and its accurate quantification is essential for quality control and assessment of its pharmaceutical products. This study aimed to develop and validate a reverse-phase high-performance liquid chromatography (HPLC) analytical method for quantifying Posaconazole in bulk and dosage form. Methods: The HPLC method was developed and validated based on International Conference on Harmonisation (ICH) guidelines. The developed method was then applied to quantify Posaconazole in a marketed tablet formulation. The method's specificity, linearity, precision, accuracy, robustness, and stability were evaluated. Results: The developed HPLC method showed good linearity over a 2-20 μg/mL concentration range. The percentage recovery of Posaconazole from the bulk and marketed formulations was found to be 99.01% and 99.05%, respectively. The intra-day and inter-day precisions were less than 1%, and the method was stable under different conditions. The HPLC method was successfully applied to quantify Posaconazole in the marketed formulation. Conclusion: The developed and validated HPLC method is reliable and efficient for analyzing Posaconazole in bulk and dosage forms. The method's accuracy, precision, specificity, linearity, robustness, and stability demonstrate its effectiveness. The method can be used for the quality control and assessment of Posaconazole-containing pharmaceutical products.

Discrimination of BaphicacanthisCusiae Rhizoma et radix and its adulterant species and establishment of an assay method for quality control

BackgroundBaphicacanthisCusiae Rhizoma et Radix, commonly known as Nan-Ban-Lan-Gen (NBLG), is an essential traditional Chinese medicine that possesses diverse bioactivities, particularly noteworthy for its antiviral properties. Although NBLG has been listed in the Chinese Pharmacopoeia as an independent Chinese medicine, the establishment of a comprehensive quality standard for NBLG remains elusive. The absence of assay for marker compound in its quality standards has led to the lack of corresponding quality control measures for NBLG-containing preparations, and its discrimination from adulterant species in the market which thereby can significantly impact the efficacy and safety of NBLG-containing products.MethodsUltra-high performance liquid chromatography (UHPLC) coupled with quadrupole-time-of-flight mass spectrometry (Q-TOF-MS) was employed for comprehensive profiling of the chemical constituents of NBLG, the stem of Baphicacanthus cusia (Nees) Bremek (NBLJ), and the roots of Isatis indigotica Fort. (Bei-Ban-Lan-Gen, BBLG). Additionally, multivariate analysis was conducted to compare the chemical components of NBLG with those of NBLJ and BBLG. Furthermore, we established an optimized and validated HPLC method to obtain the fingerprint of NBLG and quantify the content of 2-benzoxazolinone and acteoside in the samples.ResultsA total of 73 compounds belonging to six classes were assigned in NBLG, with alkaloids being the most abundant and diverse species. High compositional similarities with significant differences in content were observed between NBLG and NBLJ. Moreover, the chemical profile of BBLG markedly differed from that of NBLG. An informative high performance liquid chromatography (HPLC) fingerprint of NBLG comprising seven characteristic peaks that can be used for quality assessment was established. Notably, we propose a quality control standard for NBLG, stipulating that the limit of content in dry weight for both 2-benzoxazolinone and acteoside should not be less than 0.010%.ConclusionThis study provides the most comprehensive chemical information to date on NBLG, offering valuable insights into its authentication and quality control. Our findings highlight the importance of comprehensive chemical profiling to differentiate potential substitutions and adulterations of herbal medicines, particularly when the original source is scarce or unavailable. These results can aid in the development of quality control measures for NBLG-containing preparations, ensuring their safety and efficacy.

Cytotoxicity Activity of Graviola Fruit Extract with Carbamazepine and Valproic Acid Show Antagonistic and Indifferent Effects.

Graviola is a tropical fruit with medicinal properties, used for treating various diseases such as inflammation, diabetes, and cancer. Histone deacetylase inhibitors (HDACIs), including carbamazepine (CBZ) and valproic acid (VPA), have been proven strong inhibitors against cancer cell growth. This study investigated the effect of Graviola fruit extract (GFE) on CBZ in healthy rat plasma using high-performance liquid chromatography (HPLC). In addition, the effect of GFE in combination with CBZ and VPA on two human cancer cell lines (PC3 and MCF-7) was explored. The CBZ levels were analyzed using a simple validated HPLC method. The linearity was achieved at a 0.9998 coefficient of determination over a range of 75-5000 ng/mL CBZ. The MTT assay was used to quantify the percentage of viable cells. The maximum plasma concentration (Cmax) and area under the curve (AUC) for CBZ alone were 4,631 ng/mL and 49,225 ng. h/mL, respectively. However, in the presence of GFE, the values reduced significantly to 2,994 ng/mL and 26,587 ng. h/mL, while the p-value was <0.05. The 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay results for VPA showed a weak cytotoxicity activity on PC3 and MCF-7 cell lines. A simple and validated HPLC method was used to determine CBZ levels in rats' plasma. The plasma CBZ levels (Cmax) were significantly reduced in the presence of GFE, indicating the importance of drug-herb interactions. For in vitro studies, two human cancer cell lines, MCF-7 (breast cancer cells) and PC3 (prostate cancer cells), were used to screen the cytotoxicity activity of GFE, CBZ, and VPA. We observed an antagonism effect for GFE and CBZ combination in both cell lines with FIC values > 4. On the contrary, the combination of GFE and VPA showed an additive or indifferent effect.

Advancing posaconazole quantification analysis with a new reverse-phase HPLC method in its bulk and marketed dosage form

Introduction: Posaconazole is a widely used antifungal drug, and its accurate quantification is essential for quality control and assessment of its pharmaceutical products. This study aimed to develop and validate a reverse-phase high-performance liquid chromatography (HPLC) analytical method for quantifying Posaconazole in bulk and dosage form. Methods: The HPLC method was developed and validated based on International Conference on Harmonisation (ICH) guidelines. The developed method was then applied to quantify Posaconazole in a marketed tablet formulation. The method's specificity, linearity, precision, accuracy, robustness, and stability were evaluated. Results: The developed HPLC method showed good linearity over a 2-20 μg/mL concentration range. The percentage recovery of Posaconazole from the bulk and marketed formulations was found to be 99.01% and 99.05%, respectively. The intra-day and inter-day precisions were less than 1%, and the method was stable under different conditions. The HPLC method was successfully applied to quantify Posaconazole in the marketed formulation. Conclusion: The developed and validated HPLC method is reliable and efficient for analyzing Posaconazole in bulk and dosage forms. The method's accuracy, precision, specificity, linearity, robustness, and stability demonstrate its effectiveness. The method can be used for the quality control and assessment of Posaconazole-containing pharmaceutical products.

Stability indicating HPLC method development and validation of Fostemsavir in bulk and marketed formulations by implementing QbD approach

Background: Achieving a predictable degree of quality with intended and planned specifications is known as "quality by design." QbD (Quality-by-Design) is an alternative to conventional method development that places more attention on identifying and mitigating potential risks. Component of the Quality-by-Design methodology involves conducting a series of experiments to learn how various factors, including the dependant variables, affect the answers of interest. Here, we use a QbD (Quality-by-Design) loom to detail the creation and verification of a stability-indicating high-performance liquid chromatography (HPLC) method for Fostemsavir in both bulk and finished-goods forms.&#x0D; Results:&#x0D; In this work, we present a workable experimental design for optimising the RP-HPLC separation technique by identifying the optimum mobile phase concentration and flow rate. Below are the ideal chromatographic conditions as calculated by Design Expert version 13.0: Mobile phase: 80 parts acetonitrile to 20 parts formic acid (v/v), flow rate: 0.8 millilitres per minute, retention time: 3.24 minutes, column dimensions: GIST C18 (250 mm × 4.6 mm × 5.0 μm).&#x0D; Here we propose a practical experimental layout for determining the optimal mobile phase concentration and flow rate for the RP-HPLC separation technique. Using Design Expert version 13.0, the optimum chromatographic conditions were determined to be as follows: Shim-pack GIST C18 (250 mm 4.6 mm, 5.0 μ), mobile phase acetonitrile to 1% formic acid (80:20, v/v), flow rate 0.8 ml/min, and retention period 3.24 min.&#x0D; At a detection wavelength of 266 nm, it was discovered that the devised technique was linear over a concentration range of 50-90 μg/ml (r2 = 0.997). Test parameters for the system's appropriateness were determined to be 1.124 for the tailing factor and 9480 for the theoretical plates. Intraday RSD was found to range from 0.70 to 0.94, whereas interday RSD was found to range from 0.55 to 0.95 percent. Values for robustness were under 2%. The solution stability % RSD was calculated to be 0.83. The result of the assay was 100.05 percent. The created methodologies were used to studies of forced degradation, and the stressed materials were analysed. The parameters used to validate the procedure fell within the acceptable range recommended by ICH.&#x0D; Conclusion: Using Design Expert 13.0, we created a central composite design experiment that illustrates the relationships between mobile phase and flow rate across three levels, with retention duration, tailing factor, as well as theoretical plates as the responses of interest. By this work, we gain insight into the variables that affect chromatographic separation and strengthen our conviction that the HPLC method we've devised will serve our needs. Quantitative method development was applied to improve comprehension of multi-tiered method variables.

DEVELOPMENT AND VALIDATION OF HPLC METHOD ALONG WITH ANTI-AGING ACTIVITY FOR XIMENYNIC ACID IN SANTALUM ALBUM LINN.

A new analytical approach and validation study were developed to assess the quantity of ximenynic acid (XMA) in Santalum album Linn. extract. High-performance liquid chromatography (HPLC) in reverse phase is the technique that is used in this procedure. The anti-aging properties of the medicine were tested using enzymes that inhibit hyaluronidase and elastase, respectively, in order to gather information and findings. Methanol and water were used as the mobile phase for the analysis, which was carried out using a ZORBAX SB-C18 column with a size 5 µ (4.6 x 250 mm) column size. During the course of this HPLC method validation, a number of different tests that were pertinent to specificity, linearity, accuracy, precision and robustness were carried out. The technique was tested to see whether or not it would be able to fulfill the prerequisites set out by the International Conference on Harmonization (ICH). Research conducted on anti-aging found that XMA was much more efficient than catechin at decreasing the activities of elastase and hyaluronidase. Because of its use, speed and reliability, the HPLC method that was described has the potential to be effectively utilized in industry for the standardization of herbs and phytomedicines. Because XMA has the potential to be a breakthrough anti-aging treatment, the pharmaceutical and cosmetics industries may benefit from its development.

The Effect of Graviola Leaves Extract (Annona muricata L.) on Pharmacokinetic of Metformin in Rats’ Plasma and Pharmacological Activity of their Combination on Breast and Prostate Cancer Cell Lines

Objectives: Metformin is the most widely given medication for type 2 diabetes mellitus (T2DM). Annona muricata L. is a medicinal plant that belongs to the family Annonaceae, popularly known as graviola. Graviola leaves extract was found useful against diabetes, headache, insomnia, cystitis, inflammation, cancer, and other health benefits. The objectives of the current study are to investigate the effect of graviola leaves extract on metformin pharmacokinetics in rat plasma by applying high performance liquid chromatography (HPLC) method as well as its pharmacological effects on breast cancer (MCF-7) cells and prostate cancer (DU-145) cells. Methods: Wistar rats were classified into two groups; the first group (control group) received metformin (20 mg/kg) alone by oral gavage, while the second group, was administered a combination of metformin (20 mg/kg) and graviola leaves extract (20 mg/kg). Blood samples were collected at different time intervals to be analyzed using a validated HPLC method. Plasma profile and pharmacokinetic parameters were determined for each group. In addition, blood glucose levels at 0 hours and after 2 hours of metformin administration were measured in both groups. Breast cancer (MCF-7) cells and prostate cancer (DU-145) cells were used to investigate the anticancer effect of metformin (40 mg/ml), graviola leaves extract (20 mg/ml) and their combination by the standard MTT assay. Results: In the first group, metformin maximum plasma concentration (Cmax) and the area under the curve (AUC0-last) were (1509.25 ng/ml and 8705.59 h*ng/ml) respectively. In the second group, Pre-administration of graviola leaves extract significantly reduced MET (Cmax) and (AUC0-last), (701.88 ng/ml and 3467.72 h*ng/ml), respectively (P ≤0.05). Further, the use of metformin and graviola leaves extract separately showed strong anticancer activity on (MCF-7) cell lines with IC50 values of (10 and 20 mg/ml), respectively as well as on (DU-145) cell lines with IC50 value of (0.3125 and 5 mg/ml), respectively. In addition, the combination of metformin and graviola leaves extract showed a synergistic effect on (MCF-7) cells since the fractional inhibitory concentration value (FIC = 0.375) was less than 0.5, while it showed an additive effect on (DU-145) cells since the fractional inhibitory concentration value (FIC = 1.5) was between (0.5 and 4). Conclusion: In the current study, pre-administration of graviola leaves extract significantly reduced efficacy of metformin In vivo. The combination of metformin and graviola leaves extract showed a synergistic anticancer effect on breast cancer in vitro, while the combination has an additive effect on prostate cancer. The combination could be a potential therapeutic option to help treat breast cancer. The result achieved in this study is very encouraging to be considered for further investigation.

Quantitative determination of phenolic compounds in propolis samples from the Black Sea Region (Türkiye) based on HPTLC images using partial least squares and genetic inverse least squares methods

The complex chemical composition of propolis is related to the plant source to be used by honeybees. Propolis type is defined based on the plant source with the highest proportion in its composition, which is determined by chromatographic techniques as high-performance thin-layer chromatography (HPTLC). In addition to marker component identification to specify the propolis type, quantification of its proportion is also significant for prediction and reproducible pharmacological activity. One drawback for propolis marker component quantitation is that during the chromatographical analysis, not the main but the other plant sources with less proportion may cause interferences during the chemical analysis. In this study, the amounts of marker components were compared with the reference analysis data obtained by high-performance liquid chromatography (HPLC) and from HPTLC images using Partial Least Squares (PLS) and Genetic Inverse Least Squares (GILS) regression methods. Firstly, HPTLC images of propolis samples were processed by an image algorithm (developed in MATLAB) where the bands of each standard and the samples were cut same dimensional pieces as 351 × 26 pixels in height and width, respectively. Simultaneously, reference analysis of the marker components in propolis samples was performed with a validated HPLC method. Consequently, the reference values obtained from HPLC versus PLS, and GILS predicted values of the eight compounds based on the digitized HPTLC images of the chromatograms were found to be matched successfully. The results of the multivariate calibration models demonstrated that HPTLC images could be used quantitatively for quality control of propolis used as a food supplement.

Development and validation of HPLC method for ximenynic acid in semisolid dosage formulation

A novel analytical technique and validation study were developed to determine the concentration of ximenynic acid (XMA) in semisolid dosage formulations (SDF) such as cream, gel, lotion, etc. The procedure makes use of reverse-phase high-performance liquid chromatography (HPLC). A Phenomenex Luna Column 5 (4.6 x 250 mm) was used for the analysis, using acetonitrile as the mobile phase, sodium dihydrogen phosphate monohydrate as the stationary phase, and 229 nm detection at 30 degrees Celsius. In spite of the presence of additional compounds, their presence had no effect on the detection of XMA in SDF. As part of validating this HPLC technique, a number of tests were performed to evaluate its specificity, linearity, accuracy, precision, and durability. International Conference on Harmonization standards were used to determine whether the method was adequate (ICH). Because it is simple to implement and produces reproducible results, the presented HPLC approach has the potential to be utilized in industry to standardize herbs and Phytomedicines. Because XMA has the potential to be a game-changing method of treating ageing, its advancement may benefit the pharmaceutical and cosmetics sectors.

Validated HPLC-UV method for quantification of paxalisib, a pan PI3K and mTOR inhibitor in mouse plasma: Application to a pharmacokinetic study in mice.

Paxalisib is a pan-PI3K and mTOR inhibitor, currently entering into Phase II clinical trials as a potential drug to treat glioblastoma patients. We report the development and validation of a high-performance liquid chromatography (HPLC) method for the quantitation of paxalisib in mouse plasma as per the US Food and Drug Administration regulatory guidelines. From the mouse plasma, paxalisib and the internal standard (IS; filgotinib) were extracted using ethyl acetate as an extraction solvent. The chromatographic separation of paxalisib and the IS was accomplished on a Symmetry C18 (250×4.6mm, 5.0μm) column maintained at 40°C using 10mm ammonium formate and acetonitrile in gradient conditions at a 0.8ml/min flow-rate. The injection volume was 20 μl. The elution was monitored using a photo-diode array detector set at λmax 280 nm. Paxalisib and the IS eluted at 6.5 and 5.9min, respectively with a total run time of 10min. The calibration curve was linear over the range of 111-4,989 ng/ml. Inter- and intraday precision and accuracy, stability studies, dilution integrity and incurred sample reanalysis were investigated and the results met the acceptance criteria. The validated HPLC method was extended to assess the pharmacokinetic parameters of paxalisib in mice.

Studies Regarding the Detection of Sulfonamide Residues and Evaluation of the Level of Contamination in Poultry Meat

The excessive or uncontrolled use of antibiotics in poultry farming can lead to contaminated food products. Subsequently, the human consumption of products contaminated with these substances increases the global phenomenon of antibiotic resistance. The aim of this study was to develop and validate a high-performance liquid chromatography (HPLC) method suitable for the determination of sulfonamide residues in poultry meat and to check the presence of residues in random samples. The level of antimicrobial resistance was identified in order to evaluate the current stage and to estimate the tendency of this phenomenon. The HPLC method validation was performed in accordance with Commission Regulation (EC) No 657/2002 of 14 August 2002 Samples of fresh meat collected from a poultry slaughterhouse were analysed using the validated method in order to reveal the level of contamination. The data from EFSA was collected and analysed following the antimicrobial resistance for isolates of Salmonella spp., E. coli and Campylobacter spp. The method had good selectivity, linearity (R2 ≥ 0.99), precision (&lt;6%), recoverybetween 97.7-109.6% and low limits of detection (LOD) and quantification (LOQ) Sulfaquinoxaline residues were present in 2 samples and the level of contamination did not exceed the Maximum Residue Level (31.98 ± 5.18 µg/kg and 23.70 ± 3.84 µg/kg). The analysis of data from EFSA highlighted the general presence of antimicrobial resistance especially for the following antibiotics: ciprofloxacin, tetracycline, nalidixic acid, ampicillin, sulfamethoxazole and trimethoprim. The present study brings a contribution to the process of stopping antibiotic resistance through new methods of monitorization of sulfonamide residues. The statistical data shows that there is a direct correlation between the market availability of antibiotics used in poultry farming and the occurrence of antibiotic resistance.

HPLC Fingerprint Analysis of Rana chensinensis Eggs from Different Habitats and Their Antitussive Effect.

In this paper, a novel fingerprint method was established for the quality control of Rana chensinensis eggs (RE) by high-performance liquid chromatography (HPLC). Cluster analysis and principal component analysis were performed. Besides, the antitussive effect of RE was explored. The analysis was achieved on a Kromasil 100-5C18 (4.6 mm × 250 mm, 5 μm) column by gradient elution using methanol-0.1% phosphoric acid solution as the mobile phase. The influence of RE on cough latent periods and cough times of mice was investigated via an ammonia cough-inducing experiment. The validated HPLC method was precise, reproducible, and stable. The HPLC fingerprints of 10 batches of RE samples displayed 31 well-resolved common peaks in the chromatogram. Three of these peaks were identified and assigned to 1-methyl hydantoin, estradiol, and 4-cholestene-3-one. The similarities of 10 batches of samples were more than 0.95. RE from different origins could be classified into three groups via SPSS 23.0 software, suggesting RE samples from various provinces (Jilin, Liaoning, and Heilongjiang) can be well distinguished via the established method. High dose and middle dose of the RE group can significantly prolong the cough latent periods of mice (P < 0.05 or P < 0.01) and inhibit the cough times of mice (P < 0.01), indicating RE had a good antitussive effect. HPLC fingerprint combined with multicomponent determination can be an efficient and useful method for monitoring the quality of RE. This study also provided a more comprehensive strategy for the quality evaluation of RE.

Characterization and HPLC Method Validation for Determination of Dopamine Hydrochloride in Prepared Nano Particles and Pharmacokinetic Application

The emotional control monoamines neurotransmitter catecholamine group of chemicals is dopamine. It has been described and recognized that a lack of dopamine containing neurons contributes to neurological issues, such as Parkinsonism. Due to the incapability of dopamine HCl to cross the blood brain barrier, the advanced novel drug delivery of dopamine HCl is performed in the form of nano structure lipid carrier system (NLC-DOPA) to bypass the blood brain barrier using intranasal route of administration with improved compliance bioavailability and less side effect. The NLC-DOPA was prepared by the high pressure homogenizer ultra-probe sonicator and characterized by the particle size analyzer, zeta potential, transmission electron microscopy (TEM) and X-Ray diffraction (XRD). The determination of dopamine hydrochloride in formulation was evaluated by drug entrapment efficiency further it is validated by analytical method development using high performance liquid chromatography (HPLC) with C18 ODS column and 0.05 M KH2PO4 buffer pH 2.5 and Methanol (90:10) along with retention time 4.872 min at 280 nm wavelength. The method was validated for dopamine HCl in formulation with linearity, precision and recovery analysis. The study was shown that method was easy, rapid, accurate, specific and precise for quality analysis of dopamine HCl in NLC-DOPA formulation. The validated method of HPLC was further used for the biological application in term of the pharmacokinetic study to find out the concentration of drug in blood as well as brain. The pharmacokinetic parameters were calculated with help of the software PK solver 2.0 to determine the maximum concentration (Cmax), Maximum time (Tmax), Elimination half life (t1/2), Area under the curve (AUC0-t), volume of distribution (Vz/F_obs) and Clearance (Cl/F_obs) of all groups of animals (n=3). The study showed that the NLC-DOPA was producing a sustained and controlled release in appropriate amount of drug through intranasal bypass delivery into brain.

Development and Validation of Analytical RP HPLC Method for Benidipine Hydrochloride

The aim of the present study is development and validation of analytical reverse phase (RP) high performance liquid chromatography (HPLC) from Benidipine Hydrochloride. HPLC method is used for quantification, identification and purification of particular analyte or compound. The developed HPLC chromatographic technique was validated based on international conference on harmonization (ICH) Q2 guidelines for accuracy, precision, linearity, range and robustness. High performance liquid chromatography technique is rapid and precise technique. Advance for the validated of Benidipine Hydrochloride in pure and tablet dosage form. Diethyl ether was extracted from plasma in the presence of 5M NaOH (Sodium Hydroxide) Diethyl ether is used as internal standard like Benidipine and Benidipine d5. Benidipine is dihydropyridine calcium channel blocker, which is used in the treatment of hypertension and angina pectoris and is used to examine the efficacy and safety of therapy with Benidipine in elderly hypertensive patients.