Validation Of High Performance Liquid Chromatography Research Articles

A bioanalytical assay for estimation of thymoquinone in rats cerebrospinal fluid and brain tissues of nasally administrated thymoquinone loaded lipo-polymeric nanoshells and its pharmacokinetic profiling

Thymoquinone (TH) has been one of the major phytochemical used in the treatment of cancers since long time, especially in the management of glioblastoma multiforme (GBM). The formulation of lipo-polymeric nanoshells (LPNs) and their nasal delivery are fascinating approaches for overcoming the drawbacks of low solubility and poor bioavailability of TH. Hence targeting LPNs to the brain requires a validated bioanalytical method for the assessment of TH concentration in Cerebrospinal fluid (CSF) and brain tissue homogenates (BTH). Therefore, the current work focuses on the development and validation of high-performance liquid chromatography (HPLC) method in CSF by employing nasal simulated fluid (NSF) as one of the major components of the mobile phase. The developed method was checked for linearity in the range of 0.05 to 1.6 μg/mL, having an r2 value of 0.999 with mean % recovery >95% and % RSD values below <2.0%. The developed method gave a clear separation of TH at 6.021 ± 0.17 min with an internal standard at 4.102 ± 0.09 min and a CSF spike at 2.170 ± 0.12 min. The developed method assisted in determining the in-vitro and in-vivo drug release study of LPNs, pharmacokinetic profiling, qualitative in-vivo brain uptake study, in-vitro cellular uptake, and generating stability data of formulated LPNs proposed for intranasal administration in rats.

Optimization and validation of high-performance liquid chromatography using modified QuEChERS to determine anthelmintic drugs in mutton.

The aim of this study was to rapidly determine the presence of anthelmintic drugs in sheep meat using the optimized high-performance liquid chromatography-ultraviolet (HPLC-UV) method with modified QuEChERS (quick, easy, cheap, effective, rugged, safe) technology. Fifty fresh sheep meat samples from different slaughterhouses were collected. A double extraction procedure (QuEChERS/HPLC-UV technology) was used to extract the target analytes. A multilevel calibration curve from 1 to 1000 g/kg was used to establish instrument linearity for rafoxanide, albendazole, and closantel, whereas 0.1-100 μg/kg was used for ivermectin, levamisole, and oxyclozanide to find the lowest concentration, maximum residue limit (MRL), and occupied range for targeted analytes. The concentration levels were used to investigate the linearity, whereas several certified reference materials were applied to determine accuracy. The process was linear for all combinations, from the limit of quantification (LOQ) to the maximum concentration. The LOQ was established at 0.5μg/kg for ivermectin, levamisole, and oxyclozanide and 10μg/kg for rafoxanide, albendazole, and closantel. Recovery values were 70%-120%, and repeatability/reproducibility stated in relative standard deviation was obtained at less than 20%. QuEChERS method revealed that most meat samples contained anthelmintic drug residues, of which the majority exceeded the MRLs. Thus, the drugs should be used correctly in animals to avoid residues in food for human consumption.

Validation of high-performance liquid chromatography coupled with LTQ orbitrap mass spectrometry for analysis of acrylamide

Validation of high-performance liquid chromatography coupled with LTQ orbitrap mass spectrometry for analysis of acrylamide

Development and validation of high-performance liquid chromatography method for determination of clarithromycin in pharmaceutical tablets.

Clarithromycin is a very important macrolide antibiotic used to treat bacterial infections in human and veterinary medicine. This study reports the development and validation of cost-effective, simple, precise, accurate, and robust high-performance liquid chromatography (HPLC) for the determination of clarithromycin (CLA) in tablets. Reversed-phase chromatography was conducted using a standard column at 55°C with ultraviolet detection at 215nm. A mobile phase consisting of acetonitrile -2-methyl-2-propanol -potassium phosphate buffer was used at a flow rate of 1.0mL/min. The proposed method displayed good linearity, precision, accuracy, robustness, and specificity. The present HPLC was compared with capillary electrophoresis and bioassay methods and the results indicated that there was no significant difference between these methods. Moreover, the obtained results demonstrated the validity of the isocratic HPLC, which allows reliable quantitation of CLA in pharmaceutical samples. Thus, it can be used as a substitute alternative methodology for the routine quality control of this medicine, in situations where other methods are less accessible in the laboratory.

Validation of High-Performance Liquid Chromatography (HPLC) Method for Quantification of Ethyl p-Methoxycinnamate in Kaempferia galanga Extract

Validation of High-Performance Liquid Chromatography (HPLC) Method for Quantification of Ethyl p-Methoxycinnamate in Kaempferia galanga Extract

Development and Validation of High-Performance Liquid Chromatography for Identification and Quantification of Phytoecdysteroids Ecdysterone and Turkesterone in Dietary Supplements

Phytoecdysteroids are a group of naturally occurring ecdysteroid hormones found in certain plant families and used for centuries for their adaptogenic, tonifying, and antioxidant properties. Ecdysterone and turkesterone are the dominant phytoecdysteroids found in the plant species Rhaponticum carthamoides Willd., Cyanotis arachnoidea C.B.Clarke and Ajuga turkestanica (Regel) Briq., and have been recognized for their adaptogenic potential and ability to enhance physical performance. However, over the past few years, supplementation with ecdysterone by professional athletes has raised some concerns about its safety and quality, leading to its inclusion in the World Anti-Doping Agency monitoring program. This study proposes a simple and reliable method for quality control of ecdysterone- and turkesterone-containing products using high-performance liquid chromatography (HPLC) analysis. The HPLC method is based on reversed-phase chromatography and gradient elution to achieve a superior separation of phytoecdysteroids. The method has been shown to qualify both phytoecdysteroids at low concentrations such as 10.98 μg/mL for ecdysterone and 11.43 μg/mL for turkesterone. This method could be successfully used in research programs and routine quality control of dietary supplements to ensure their safety in professional athletes’ nutrition.

Simultaneous Determination of Nirmatrelvir and Ritonavir in Human Plasma by HPLC-MS/MS

Introduction. SARS-CoV-2 (severe acute respiratory syndrome-related coronavirus 2) is expected to remain a persistent global threat. Therefore, development of coronavirus disease 2019 (COVID-19) drugs is the most urgent global issue. Nirmatrelvir and ritonavir combination is an oral antiviral drug combination with activity against SARS-CoV-2. Nirmatrelvir and ritonavir combination is highly efficacious in reducing the risk of COVID-19. The study describes development and validation of high-performance liquid chromatography – tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous determination of nirmatrelvir and ritonavir in human blood plasma. The method could be applied in pharmacokinetic study of nirmatrelvir and ritonavir.Aim. The aim of this study is to develop and validate a HPLC-MS/MS bioanalytical method for the determination of nirmatrelvir and ritonavir in human plasma.Materials and methods. The determination of nirmatrelvir and ritonavir in human plasma by HPLC-MS/MS. The samples were processed by acetonitrile protein precipitation. Internal standard: promethazine. Mobile phase: 0.1% formic acid solution in water (Eluent A), 0.1% formic acid in acetonitrile (Eluent B). Column: Phenomenex Luna C18 50 × 2.0 mm, 5 μm. Analytical range: 50.00–10000.00 ng/mL for nirmatrelvir, 5.00–1000.00 ng/mL for ritonavir in human plasma. Ionization source and ionization: electrospray ionization, positive. Detection conditions: 499.90 → 110.10 m/z, 499.90 → 319.20 m/z (nirmatrelvir), 720.90 → 426.00 m/z, 720.90 → 296.20 m/z, 720.90 → 268.10 m/z, 720.90 → 197.10 m/z, 720.90 → 139.90 m/z (ritonavir), 285.15 → 198.05 m/z (promethazine).Results and discussion. This method was validated for selectivity, matrix effect, calibration curve, accuracy, precision, spike recovery, the lower limit of quantification, carry-over effect and stability.Conclusion. The HPLC-MS/MS method for quantitative determination of nirmatrelvir and ritonavir in human plasma was developed and validated. The analytical range was 50.00–10000.00 ng/mL for nirmatrelvir, 5.00–1000.00 ng/mL for ritonavir in human plasma. This method was applied to investigate the pharmacokinetics of nirmatrelvir and ritonavir.

Simultaneous Determination of Major Molnupiravir Metabolite (β-D-N4-hydroxycytidine) and Favipiravir in Human Plasma by HPLC-MS/MS

Introduction. Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the SARS-CoV-2 virus (severe acute respiratory syndrome-related coronavirus 2). COVID-19 is now expected to stay with us for many years as a recurring disease. Molnupiravir and favipiravir are oral antiviral drugs with anti-RNA polymerase activity. The Russian Health Ministry has approved molnupiravir and favipiravir for the treatment of COVID-19. The study describes development and validation of high-performance liquid chromatography – tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous determination of β-D-N4-Hydroxycytidine and favipiravir in human blood plasma. The method could be applied in pharmacokinetic study of molnupiravir and favipiravir.Aim. The aim of this study is to develop and validate a HPLC-MS/MS bioanalytical method for the determination of β-D-N4-Hydroxycytidine and favipiravir in human plasma.Materials and methods. The determination of β-D-N4-Hydroxycytidine and favipiravir in human plasma by HPLC-MS/MS. The samples were processed by 0.1 % formic acid in acetonitrile. Internal standard: promethazine. Mobile phase: 0.01 mol/L Ammonium formate buffer solution (Eluent A), 0.1 % formic acid and 0.08 % aqueous ammonia in water/acetonitrile 10 : 90 (Eluent B). Column: Shim-pack GWS C18, 150 × 4.6 mm, 5 μm. Analytical range: 50.00–10000.00 ng/mL for β-D-N4-Hydroxycytidine, 250.00–20000.00 ng/mL for favipiravir in human plasma. Ionization source: electrospray ionization. Detection conditions: 260.00 m/z → 82.10 m/z, 260.00 m/z → 111.00 m/z, 260.00 m/z → 127.95 m/z (β-D-N4-Hydroxycytidine); 156.15 m/z → 65.95 m/z, 156.15 m/z → 85.00 m/z, 156.15 m/z → 113.10 m/z (favipiravir); 285.05 m/z → 198.05 m/z (promethazine).Results and discussion. This method was validated by selectivity, suitability of reference standard, matrix effect, calibration curve, accuracy, precision, spike recovery, the lower limit of quantification, carry-over effect and stability.Conclusion. The HPLC-MS/MS method for quantitative determination of β-D-N4-Hydroxycytidine and favipiravir in human plasma was developed and validated. The analytical range was 50.00–10000.00 ng/mL for β-D-N4-Hydroxycytidine, 250.00–20000.00 ng/mL for favipiravir in human plasma. This method was applied to investigate the pharmacokinetics of molnupiravir and favipiravir.

Analytical method validation of reversed phase HPLC for quantitative analysis of tartrazine and auramine o in powder drinks

This study was aimed to perform analytical method validation of high-performance liquid chromatography (HPLC) technique using photo-diode array detector for the simultaneous determination of Tartrazine (TAR) and Auramine O (AUO) in powder drink products. TAR and AUO were analysed using Waters Shield C18 column (250 mm x 4.6 mm i.d., 5 µm) using PDA detector at 300-650 nm. The mobile phase used was acetonitrileammonium acetate 19 mM (86:14 v/v) delivered isocratically at a flow rate of 1.2 mL/ min. The optimized HPLC condition was subjected to analytical method validation by assessing some performance characteristics as guided by International Conference on Harmonization (ICH). The method was linear over the studied concentration ranges with the coefficient of determination (R2 ) of 0.999 and 0.997 for TAR and AUR with % intercept less than 2%, respectively. The developed method was sensitive with a limit of detection value of 0.0325 μg/mL and 0.1052 μg/mL for TAR and AUO, respectively. The method is also accurate and precise as indicated with acceptable recovery values of 99.0- 100.7% for TAR and 102.1-106.5% for AUO with relative standard deviation (RSD) values lower than those required by Association of Official Analytical Chemists’ (AOAC). The developed method is simple and can be used for routine analysis of TAR and AUO for quality assurance purposes of powder drinks.

Validation of High-Performance Liquid Chromatography for Determination of Bromelain in Pineapple (Ananas comosus (L) Merr) Water

Objective: The aim of the present study was to validate HPLC method for analysis bromelain levels in pineapple water and application that method to determine the storage time of pineapple water. Methods: The reversed phase of HPLC method was tested and optimized before it is validated. The composition and the flow rate of the mobile phase is the optimized parameter. The analytical parameters validated were detection limits, linearity, accuracy and precision. Pineapple water was stored for 8 h at 10ºC and bromelain was determined using the validated HPLC method. Result: The optimum mobile phase composition was methanolwater (70:30) with a flow rate of 1 ml/min. The detector concentration-response was linear with coefficient of determination of 0.993. The accuracy of HPLC method at a recovery of 1 and 2% bromelain was 106.37 ± 1.94% and 98.12 ± 1.29% (n = 5), respectively. The precision, expressed as the coefficients of variation (CV), at 1 and 2% bromelain were 1.83 and 1.32% (n = 5), respectively. Bromelain level at zero time was 81.53%. After storage for 8 h at 10ºC, bromelain levels in pineapple juice appeared to decrease not statistically significant (p > 0.05), with to mean value of 78.46 ± 2.88%. Conclusion: The HPLC method developed was valid to analyze accurately concentrations of bromelain in pineapple water and it can be used to study the shelf life of pineapple water based on bromelain content. The bromelain content in pineapple juice was not statistically significantly different after 8 h storage at 10°C.

Development and validation of high-performance liquid chromatography method for determination of some pesticide residues in table grape

This study presents the development and validation of a new reversed-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous determination of captan, folpet, and metalaxyl residues in table grape samples with ultraviolet–diode array detection (UV–DAD). Successful separation and quantitative determination of analytes was carried out on LiChrospher 60 RP-select B (250 × 4 mm, 5 μm) analytical column. Mixture of acetonitrile–0.1% formic acid in water (65:35, v/v) was used as a mobile phase, with flow rate of 1 mL/min, constant column temperature at 25 °C, and UV detection at 220 nm. The target residues were extracted with acetone by ultrasonication, followed by a cleanup using liquid–liquid extraction (LLE) and solid-phase extraction (SPE). The obtained values for multiple correlation coefficients (R2 &gt; 0.90), relative standard deviation (RSD) of retention times, peak areas and heights (RSD ≤ 2.25%), and recoveries ranging from 90.55% to 105.40%, with RSD of 0.02% to 5.37%, revealed that the developed method has a good linearity, precision, and accuracy for all analytes. Hence, it is suitable for routine determination of investigated fungicides in table grape samples.

Analytical Method Validation of High-Performance Liquid Chromatography and Stability-Indicating Study of Medroxyprogesterone Acetate Intravaginal Sponges.

Medroxyprogesterone acetate is widely used in veterinary medicine as intravaginal dosage for the synchronization of breeding cycle in ewes and goats. The main goal of this study was to develop reverse-phase high-performance liquid chromatography method for the quantification of medroxyprogesterone acetate in veterinary vaginal sponges. A single high-performance liquid chromatography/UV isocratic run was used for the analytical assay of the active ingredient medroxyprogesterone. The chromatographic system consisted of a reverse-phase C18 column as the stationary phase and a mixture of 60% acetonitrile and 40% potassium dihydrogen phosphate buffer as the mobile phase; the pH was adjusted to 5.6. The method was validated according to the International Council for Harmonisation (ICH) guidelines. Forced degradation studies were also performed to evaluate the stability-indicating properties and specificity of the method. Medroxyprogesterone was eluted at 5.9 minutes. The linearity of the method was confirmed in the range of 0.0576 to 0.1134 mg/mL (R2 > 0.999). The limit of quantification was shown to be 3.9 µg/mL. Precision and accuracy ranges were found to be %RSD <0.2 and 98% to 102%, respectively. Medroxyprogesterone capacity factor value of 2.1, tailing factor value of 1.03, and resolution value of 3.9 were obtained in accordance with ICH guidelines. Based on the obtained results, a rapid, precise, accurate, sensitive, and cost-effective analysis procedure was proposed for quantitative determination of medroxyprogesterone in vaginal sponges. This analytical method is the only available method to analyse medroxyprogesterone in veterinary intravaginal dosage form.

Validation of high-performance liquid chromatography (HPLC) method for quantitative analysis of histamine in fish and fishery products

AbstractA high-performance liquid chromatography method is described for quantitative determination and validation of histamine in fish and fishery product samples. Histamine is extracted from fish/fishery products by homogenizing with tri-chloro acetic acid, separated with Amberlite CG-50 resin and C18-ODS Hypersil reversed phase column at ambient temperature (25°C). Linear standard curves with high correlation coefficients were obtained. An isocratic elution program was used; the total elution time was 10 min. The method was validated by assessing the following aspects; specificity, repeatability, reproducibility, linearity, recovery, limits of detection, limit of quantification and uncertainty. The validated parameters are in good agreement with method and it is a useful tool for determining histamine in fish and fishery products.

Development and validation of high-performance liquid chromatography and high-performance thin-layer chromatography methods for the quantification of khellin in Ammi visnaga seed.

Objective:The present study was used to design simple, accurate and sensitive reversed phase-high-performance liquid chromatography RP-HPLC and high-performance thin-layer chromatography (HPTLC) methods for the development of quantification of khellin present in the seeds of Ammi visnaga.Materials and Methods:RP-HPLC analysis was performed on a C18 column with methanol: Water (75: 25, v/v) as a mobile phase. The HPTLC method involved densitometric evaluation of khellin after resolving it on silica gel plate using ethyl acetate: Toluene: Formic acid (5.5:4.0:0.5, v/v/v) as a mobile phase.Results:The developed HPLC and HPTLC methods were validated for precision (interday, intraday and intersystem), robustness and accuracy, limit of detection and limit of quantification. The relationship between the concentration of standard solutions and the peak response was linear in both HPLC and HPTLC methods with the concentration range of 10–80 μg/mL in HPLC and 25–1,000 ng/spot in HPTLC for khellin. The % relative standard deviation values for method precision was found to be 0.63–1.97%, 0.62–2.05% in HPLC and HPTLC for khellin respectively. Accuracy of the method was checked by recovery studies conducted at three different concentration levels and the average percentage recovery was found to be 100.53% in HPLC and 100.08% in HPTLC for khellin.Conclusions:The developed HPLC and HPTLC methods for the quantification of khellin were found simple, precise, specific, sensitive and accurate which can be used for routine analysis and quality control of A. visnaga and several formulations containing it as an ingredient.

Development and Validation of High-Performance Liquid Chromatography for Simultaneous Determination of Seven Bioactive Compounds in Flos Lonicerae japonicae

Development and Validation of High-Performance Liquid Chromatography for Simultaneous Determination of Seven Bioactive Compounds in Flos Lonicerae japonicae

Development and Validation of High-Performance Liquid Chromatography–Diode Array Detector Method for the Determination of Tramadol in Human Saliva

A high-performance liquid chromatography–diode array detector method (HPLC-DAD) was developed and validated for the determination of tramadol in human saliva. Samples of saliva were prepared utilizing liquid–liquid extraction with hexane-ethyl acetate (4:1, v/v). Propranolol was used as an internal standard (IS). The linear range and average recovery of tramadol were 0.25–4.00 μg/mL and 94.70%, respectively. The intra- and inter-assay precisions at low, intermediate and high concentrations were within 1.40–9.79 % and 0.77–3.98%, respectively. Intra- and inter-assay accuracies were within 98.50–107.13% and 100.44–103.89%, respectively. The proposed method was successfully applied to determine tramadol in saliva concentrations in three healthy volunteers for 24 hours after administration of 100 mg oral doses of tramadol.

Validation of a high-performance liquid chromatography assay for quantification of caffeine and paraxanthine in human serum in the context of CYP1A2 phenotyping.

In this study the validation of a reversed-phase high-performance liquid chromatography (HPLC) method, with UV-detection, for both caffeine and paraxanthine in human serum is described. This method is feasible for cytochrome P450 1A2 (CYP1A2) phenotyping, according to the results of a pilot study. With this HPLC method caffeine and paraxanthine can be determined selectively and specifically. In the expected concentration range, caffeine recoveries were 98-108% (within-run variation 4.0-6.4%, between-run variation 6.4-8.8%), paraxanthine recoveries were 96.6-97.5% (within-run variation 5.0-7.2%, between-run variation 7.2-10.8%). The limits of detection for caffeine and paraxanthine using this HPLC system were 0.3 and 0.1 mg/L, respectively. Linear calibration curves for both caffeine and paraxanthine were obtained in the concentration range 0.5-30 mg/L (r > 0.9999. Serum samples were stable for a week, when stored at -20 and +4 degrees C.