Determination of melatonin in sleep supplements by high-performance liquid chromatography

IntroductionThe research aimed to develop a robust, high-performance liquid chromatography (HPLC) analytical method for the quantitative assessment of rebamipide encapsulated in ethosomes. Rebamipide, a quinolinone derivative, holds promise as a therapeutic agent for dry eye, but challenges such as low bioavailability and vision clouding post-installation have prompted innovative approaches. Encapsulation in ethosomes, lipid-based nanovesicles, offers a potential solution to enhance ocular bioavailability.Materials and methodsThe study focused on creating a specific, linear, accurate, precise, and robust HPLC method, addressing entrapment efficiency (%EE), drug content, and drug release of rebamipide in prepared ethosomes. Statistical validation followed International Conference of Harmonization (ICH) specifications. The method's parameters were evaluated within a concentration range of 4-24 µg/ml, with recovery rates indicating accuracy and low % relative standard deviation (RSD) values confirming precision. Limits of detection (LOD) and quantification (LOQ) for rebamipide were determined.ResultsAfter preparing the ethosome dosage form by film hydrating method for rebamipide, the rebamipide entrapment efficiency in ethosomes was established at 76% ± 7, while the drug content was found to be 93% ± 6. The drug release process demonstrated zero-order kinetics and five different models of kinetics were applied for a comprehensive analysis. The method exhibited excellent system suitability, specificity, and linearity. Recovery rates for rebamipide ranged from 90% to 100%, and repeatability was confirmed by low %RSD values. The LOD and LOQ for rebamipide were determined to be 1.04 μg/mL and 3.16 μg/mL, respectively.ConclusionThe developed HPLC method proved suitable for the quantitative determination of rebamipide in ethosomes, offering rapid and accurate analysis. The results underscore the method's specificity, accuracy, and precision within the specified concentration range. Overall, the validated method contributes to the advancement of ocular drug delivery systems, providing a reliable analytical tool for pharmaceutical research.

Context: Assessment of nisoldipine (NSP) skin flux using validated high-performance liquid chromatographic (HPLC) method. Aim: A validated HPLC method was developed for estimation of NSP in skin permeated samples from matrix type transdermal films. Materials and Methods: Chromatographic separation was done using Phenomenex Luna C18 column (150 mm × 4.6 mm i.d. 5 μm) employing acetonitrile:water:methanol (40:35:25 v/v) as the mobile phase. Settings and Design: The calibration solutions were prepared in the linearity range of 0.250-50 μg/mL. The flow rate was maintained at 0.8 mL/min at 25°C. 10 μL samples were injected and were monitored using photodiode array detector at a wavelength of 275 nm. Results: The limit of detection and limit of quantification for NSP were recorded as 0.125 μg/mL and 0.150 μg/mL, respectively. The correlation coefficient (r2) values for intraday and interday were >0.998 and % relative standard deviation values were <10%. Conclusions: The method was validated as per International Conference on Harmonization guidelines, found to be simple, sensitive, cost effective and was successfully used for ex vivo skin permeation studies of NSP from matrix type transdermal films.

A simple, sensitive and stability-indicating high-performance liquid chromatographic (HPLC) assay method was developed and validated for a bioactive peptide, lysine-proline-valine (KPV) in aqueous solutions and skin homogenates. Chromatographic separation was achieved on a reversed phase Phenomenex C18 column (4.6 × 250 mm, packed with 5 µm silica particles) with a gradient mobile phase consisting of 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in acetonitrile (B). The proposed HPLC method was validated with respect to accuracy, precision, linearity, repeatability, limit of detection (LOD) and limit of quantitation (LOQ). The calibration curve was linear with a correlation coefficient (r) of 0.9999. Relative standard deviation values of accuracy and precision experiments were <2. The LOD and LOQ of KPV were 0.01 and 0.25 µg/mL, respectively. Under stress conditions (acid, alkali and hydrogen peroxide) KPV yielded lys-pro-diketopiperazine as major degradation product, which was identified by flow injection MS analysis. The developed HPLC method was found to be efficient in separating the active peptide from its degradation products generated under various stress conditions. Also, the validated method was able to separate KPV from other peaks arising from endogenous components of the skin homogenate.

A high performance liquid chromatography (HPLC) and ultraviolet spectroscopic (UV) methods were developed and validated for the quantitative estimation of doxofylline (DF) in pharmaceutical dosage forms. HPLC was carried out using reversephase technique on RP-8 column with a mobile phase composed of 0.05M phosphate buffer pH 6 and acetonitrile (60:40, v/v). The mobile phase was pumped at a flow rate of 1mL/min, and detection was made at 230nm with PDA detector. UV method was performed with λ max at 270nm with apparent molar absorptive of 0.878x103 L mol-1 cm-1. Both the methods showed good linearity, recovery and precision. No spectral or chromatographic interferences from the tablet excipients were found in UV and HPLC methods. The various parameters such as linearity, precision, accuracy, specificity, and robustness, limit of detection and limit of quantization were studied according to ICH guidelines. Statistical analysis was done by student’s t-test and F-test, which showed no significant difference between the results of both methods. So the proposed methods could be applicable for routine analysis of DF and monitoring of the quality of marketed drugs.

A high-performance liquid chromatography (HPLC) method for the qualitative and quantitative analysis of allantoin in silk and seed of Zea mays has been developed. Allantoin separation in crude extract was achieved using a C 18 column and phosphate buffer solution (pH 3.0) as a mobile phase at ambient temperature at a flow rate of 1.0 mL/min and detected at 210 nm. The results showed that the amount of allantoin in samples was between 14 and 271 mg/100 g of dry plant material. A comprehensive validation of the method including sensitivity, linearity, repeatability, and recovery was conducted. The calibration curve was linear over the range of 0.2-200 microg/mL with a correlation coefficient of r2>0.999. Limit of detection (LOD, S/N=3) and limit of quantification (LOQ) values of the allantoin were 0.05 and 0.2 microg/mL (1.0 and 4.0 ng) respectively. The relative standard deviation (RSD) value of the repeatability was reported within 1.2%. The average recovery of allantoin added to samples was 100.6% with RSD of 1.5%.

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

Comparison of UPLC and HPLC for Determination of trans-10-Hydroxy-2-Decenoic Acid Content in Royal Jelly by Ultrasound-Assisted Extraction with Internal Standard

Various methods have been published for the analysis of these compounds in licorice, including high-performance liquid chromatography (HPLC) [14–17], high-performance thin-layer chromatography (HPTLC) [18], and capillary electrophoresis [19, 20]. However, these published methods mainly involve Glycyrrhiza glabra and G. uralensis [21–22]. There are few special studies reported on the analysis of Glycyrrhiza inflata. Based on the above-mentioned purpose, the aim of the current study was to develop a simple reversed-phase highperformance liquid chromatography (HPLC) method for simultaneous quantification of glycyrrhizin and licochalcone A. In order to validate the HPLC quantitative method, the specificity, linearity, accuracy, precision, and limits of detection and quantification of the method were investigated. The specific content of these compounds in eight samples using our technique was also examined. Each of the six standards for glycyrrhizin, liquiritin, and licochalcone A was analyzed in three replicates. Table 1 shows the results. The calibration curve was constructed by plotting the peak area against the concentration (g/mL) using linear regression analysis. The relative standard deviation (RSD) values of the peak areas of three replicate injections were between 1.52 and 3.29%. Dilute solutions of the reference compounds were further diluted to a series of concentrations with methanol to assess the limits of detection (LOD) and quantification (LOQ). The LOD and LOQ under the present chromatographic conditions were determined at a signal-to-noise (S/N) ratio of 3 and 10, respectively.The LOD values for glycyrrhizin and licochalcone A were 4.2 ng and 3.3 ng. The LOQ values were 14.3 ng and 10.5 ng, respectively.

A simple and sensitive method using first-derivative ultraviolet spectrophotometry (DS-UV) was developed, validated, and compared to the high performance liquid chromatography (HPLC) method for quantification of paclitaxel (PTX) in a liposomal formulation. Different analytical performance parameters such as linearity, accuracy, precision, specificity, detection, and quantification limits were determined according to International Conference on Harmonization (ICH) guidelines. No interference from the lipid compounds was detected in the HPLC and the DS-UV methods at 246 nm. Linearity determined for paclitaxel concentrations ranging from 6.0 to 24.0 µg mL -1 presented a correlation coefficient higher than 0.999 for both methods. Relative standard deviation (RSD) values lower than 2% for intra- and inter-day precision data could be obtained. Accuracy mean values ranged from 98.9 to 102.0%. Robustness data showed that the PTX content was unaffected by the alteration proposed. Both methods were adequate to quantify the drug in the liposomal formulation. DS-UV proved to be rapid, accurate, selective, sensitive, and, therefore, an attractive tool for routine determination of PTX.

Pharmacological treatment with antiepileptic drugs (AEDs) remains the primary choice for most patients with epilepsy, while therapeutic drug monitoring is an important strategy to ensure the efficacy and safety of AEDs. The aim of this study was to optimize and develop a green high-performance liquid chromatography (HPLC) method suitable for simultaneous detection of 8 AEDs and 2 active metabolites including primidone (PRM), lamotrigine (LTG), carbamazepine (CBZ) and its active metabolite 10,11-epoxide (CBZE), oxcarbazepine (OXC) and its active metabolite 10,11-dihydro-10-hydroxyl carbamazepine (MHD), phenobarbital (PHB), eslicarbazepine acetate (ESLA), clonazepam (CZP) and diazepam (DZP) in human serum. The results showed that using chlorzoxazone as the internal standard, ethanol as the protein precipitant of serum, a ZORBAX Eclipse Plus-C18 (150 × 4.6 mm, 3.5 μm) column as the stationary phase, and gradient ethanol and 15 mM potassium dihydrogen phosphate water solution as the green alternative mobile phase, good chromatographic separation for 10 target analytes could be obtained. The calibration curves of 10 analytes demonstrated good linearity within the test range (r > 0.996). The intra- and inter-batch precision and accuracy were all less than 15%, while extraction recoveries were in the range of 74.57-90.89% with relative standard deviation values less than 15%. The results of greenness assessment showed that the proposed method was ecofriendly. Finally, the validated method was successfully applied in the analysis of real serum samples.

Objective To make comparative analysis of the glycated hemoglobin A1c(HbA1c) values obtained by three HbA1c test methods in patients with variant hemoglobins. Methods Total of 50 blood samples of patients with different types of variant hemoglobin were collected from January 2012 to December 2012; 25 of them (14 males and 11 females) carried hemoglobin D, Q, G, J and E with a mean age of (24±3) years; the other 25 cases (11 males and 14 females) were blood samples with hemoglobin F from newborn infants. Meanwhile, 50 blood samples(25 males and 25 females) from people without variant hemoglobins were also collected as control(mean age (25±5) years). Three methods were used to test HbA1c, which were affinity high performance liquid chromatography (HPLC) method (Ultra 2 of American Primus), ion exchange HPLC method (VariantⅡ of American Bio-Rad and G8 of Japanese Tosoh) and immunization method (DCA Vantage of German Siemens). The statistic analysis were done with variance analysis and correlation analysis. Results The HbA1c of the group with normal HbA1c structure was 5.7%±1.1%, 5.7%±1.2%, 5.7%±1.2% and 5.7%±1.1% respectively when it was tested by affinity HPLC method (Ultra2), ion exchange HPLC method (G8), ion exchange HPLC (Variant Ⅱ) and immunization method (DCA Vantage), and there was no significant difference among the groups (F=0.023, P>0.05). In the 25 samples with hemoglobin F, HbA1c could not be detected by ion exchange HPLC or immunoassay method. The fasting blood glucose level correlated with HbA1c level tested by Ultra 2 method (r=0.647, P<0.05), but it didn't correlate with HbA1c level when tested with VariantⅡ and G8 as well as DCA Vantage method. The HbA1c result of affinity HPLC method was free from the disturbance of Hb D, Q, G, J and E, and had a obvious correlation with blood glucose (r=0.823, P<0.05). The HbA1c result of ion exchange HPLC method was disturbed by hemoglobin D, Q, G, J and E in varying degrees. The HbA1c measured by immunization method was associated with blood glucose (r=0.611, P<0.05). Conclusion The HbA1c value obtained by affinity HPLC method can accurately reflect the mean blood glucose level. Variant hemoglobins disturb the HbA1c result tested by ion exchange HPLC method, and the immunization test result is only disturbed by hemoglobin F. Key words: Glycated hemoglobin A1c; Variant hemoglobins; High performance liquid chromatography

A stability-indicating high performance liquid chromatography method to determine apocynin in nanoparticles

In this research caffeine content in coffee sample from Abe Dongoro, Sasiga, Gida Ayana and Sibu Sire of Wollega administrative zone of Ethiopia were determined using High Performance Liquid Chromatography (HPLC) and UV-Vis Spectrophotometry methods. Caffeine in aqueous extract of coffee samples was extracted with dichloromethane prior to analysis by UV-Vis spectrophotometry method and dichloromethane was evaporated from the extract and the extract was dissolved in water (HPLC grade) to determine caffeine contents in coffee samples using HPLC method. The linearity of the HPLC and UV-Vis spectrophotometry methods were R² = 0.9999 and R² = 0.9997 respectively. HPLC and UV-Vis spectrophotometry methods were found to be accurate with recoveries of 97.5% and 117.4%, respectively. Limits of detection (LOD) obtained were 0.148 mg/L for HPLC method and 0.284 mg/L for UV-Vis spectrophotometric method. The caffeine contents in coffee samples obtained using UV-Vis spectrophotometry method was 3.42, 2.638, 2.207 and 2.986 mg/L for Abe Dongoro, Gida Ayana, Sasiga and Sibu Sire coffee samples respectively. Similarly, using HPLC method the caffeine contents in coffee samples obtained was 1.871, 1.601, 1.307, 1.83 mg/L for Abe Dongoro, Gida Ayana, Sasiga and Sibu Sire coffee samples. There is a significant difference between the caffeine contents in coffee samples obtained by the two methods.

染发类产品中氧化型染料种类多,实际样品测定时干扰多,建立染发类产品中多种常用染料的测定方法,为该类产品的有效监管提供技术手段十分必要。该研究根据染料使用频率分组,采用能够屏蔽硅羟基和金属离子影响的C18柱,优化了《化妆品安全技术规范》(2015年版)中32种染料的高效液相色谱法(HPLC)并建立了高效液相色谱-串联质谱(HPLC-MS/MS)确证方法。样品以10 g/L亚硫酸氢钠水溶液为抗氧化剂,用无水乙醇-水(1∶1, v/v)混合溶液冰浴超声提取10 min。HPLC方法采用甲醇、乙腈和磷酸盐缓冲液为流动相分两个液相色谱条件进行梯度洗脱分离,于280 nm波长下检测,其中一个HPLC条件中的相互干扰组分均在另一个HPLC条件下完全分离,避免了实际样品检测时组分间的干扰,并排除了32种以外的其他15种常用染料的干扰。HPLC-MS/MS方法分别采用5 mmol/L乙酸铵水溶液-乙腈和5 mmol/L乙酸水溶液-乙腈为正离子和负离子模式下的流动相,电喷雾离子模式下用多反应监测(MRM)模式进行定性和定量分析。HPLC和HPLC-MS/MS两个方法中,日内精密度和48 h内稳定性的相对标准偏差(RSD)<10%,回收率为82.6%~114.9%(RSD<10%)。HPLC方法中32种染料在大约10~500 mg/L范围内线性关系良好(r2>0.99),检出限为9.7~40.1 μg/g; HPLC-MS/MS方法中氢醌线性范围为2.0~79.7 mg/L,检出限为8.0 μg/g,其他组分线性范围约为0.1~4 mg/L,检出限为0.01~0.4 μg/g。采用HPLC、HPLC-MS/MS两个方法和《化妆品安全技术规范》方法同时测定实际样品,共检出16种染料,检出含量范围为58~25160 μg/g。3个方法检测结果的RSD为1.9%~10.1%。该研究增加了HPLC-MS/MS确证方法,适应化妆品法定检验中的未知物确认程序;方法简便快速,结果准确,专属性强,具有较好的通用性和可操作性。

Objective: A new, simple, rapid, sensitive, and accurate stability-indicating high-performance liquid chromatography (HPLC) method was developed and validated for the quantitative determination of linagliptin (LNG) and empagliflozin (EMP) in pure and tablet dosage forms.&#x0D; Methods: An isocratic HPLC method, using a C18 reversed-phase column (150 mm×4.6 mm i.d., particle size 5 μm) with an isocratic binary mobile phase consisting of phosphate buffer and acetonitrile (65:35, v/v), was investigated to separate the drug from its stress degradation products. The flow rate was 1.0 mL/min at ambient temperature and photodiode array detector is used at 226 nm for detection. The developed method was validated for system suitability, linearity, accuracy, precision, limits of detection and quantitation, specificity, stability, and robustness.&#x0D; Results: The retention time of LNG and EMP was found to be 3.276±0.002 and 6.966±0.0006 min, respectively. The calibration curve was found to be linear with the equation y=158926.39X+11.139, with a correlation coefficient of R2=0.9991 for LNG and y=22688.45X+4.259, with a correlation coefficient of R2=0.9994 for EMP over a concentration range of 2.5–7.5 μg/mL and 5.0–15 μg/mL for LNG and EMP, respectively. The limits of detection were 0.29 and 0.48 μg/mL for LNG and EMP, respectively, and the limits of quantification were 0.89 and 1.5 μg/mL for LNG and EMP, respectively. The recovery values of this method are 101.11% and 101.48% for LNG and EMP, respectively, and the reproducibility is within 0.070 and 0.277 for LNG and EMP, respectively.&#x0D; Conclusion: The proposed method is a rapid stability-indicating HPLC method that can be applied for the determination of LNG and EMP in pure and tablet dosage forms.