Simple High-performance Liquid Chromatography Research Articles

Establishment of an HPLC method for testing acetylcholinesterase inhibitory activity and compared with traditional spectrophotometry

Acetylcholinesterase (AChE) inhibitory activity test is one of the important areas of natural product research, which is traditionally tested by spectrophotometry. In this work, a simple high-performance liquid chromatography (HPLC) method was developed for testing AChE inhibitory activity in several natural products. Using galanthamine hydrobromide and huperzine A as positive controls, it was found that the optimal conditions for sample preparation are 7.0–8.0 for pH, 30 min for reaction time, 37 °C for reaction temperature, 1.1 × 10−3 U mL−1 for concentration of AChE, and 0.625 mM for acetylthiocholine iodide in the final reaction mixture with total volume 2 mL. The Diamonsil C18 column was used and the chromatographic conditions were mobile phase methanol–water–triethylamine (40:60:0.05, v/v/v), flow rate 1.0 mL min−1, injection volume 5 μL, run time 5 min, column temperature 37 °C, and wavelength 405 nm. The AChE inhibitory activity of anthocyanin with concentration of 10, 20, and 50 μg mL−1, blueberry extract and purple potato extract with concentration of 20, 100, and 500 μg mL−1 were further determined under this conditions. The results showed the inhibition ratios obtained from spectrophotometry were nearly adjacent and approximately lower than 10% and the values of absorbance mostly exceed 0.8, but the absorbance should ensure in the range of 0.15–0.8 to guarantee smaller relative error for spectrophotometry. While the inhibition ratios gained from HPLC method were higher than 20% and had greater difference, they had better comparability to the different concentrations of anthocyanin and its extracts. It is revealed that the HPLC method can be used for the evaluation of the AChE inhibitory activity in natural product extracts with deep color or not.

Development and validation of a rapid LC–MS/MS method for simultaneous quantification of arecoline and its two active metabolites in rat plasma and its application to a pharmacokinetic study

Arecoline is the primary active and toxic constituent of areca nut. Arecaidine and arecoline N-oxide are two major active metabolites of arecoline. In this work, an accurate and simple high performance liquid chromatography tandem mass spectrometry method for simultaneous quantification of arecoline, arecaidine and arecoline N-oxide in rat plasma was developed and fully validated to study their pharmacokinetic behaviors in rats. After extracted from rat plasma by protein precipitation with methanol and then concentrated, the analytes were chromatographic separated on a Sepax Sapphire C18 analytical column. The mobile phase consisted of methanol and 2 mM ammonium acetate buffer solution containing 0.2% (v/v) formic acid (8:92, v/v) under isocratic elution. The analytes were detected by multiple reaction monitoring (MRM) with an electrospray ionization source in the positive ion mode. The transitions of m/z 156.2 → 53.2, m/z 142.2 → 44.2 and m/z 172.2 → 60.2 were selected for arecoline, arecaidine and arecoline N-oxide, respectively. The method was linear over the concentration range of 0.5–100 ng/mL for arecoline, 5–5000 ng/mL for arecaidine and arecoline N-oxide with no carry-over effect. The accuracies and intra- and inter-batch precisions were all within the acceptance limits. No matrix effect and potential interconversion between the analytes and other metabolites were observed in this method. The validated method was further employed to a preclinical pharmacokinetic study of arecoline, arecaidine and arecoline N-oxide after oral treatment with 20 mg/kg arecoline to rats.

Comprehensive determination of nine polyphenols in Polygoni Avicularis Herba with a new HPLC–DAD method and their correlation with the antioxidant activities

A simple high performance liquid chromatography (HPLC) method was established for identification and quantification of nine polyphenols (gallic acid, chlorogenic acid, caffeic acid, myricetrin, hyperoside, luteoloside, quercitrin-3-rhamnoside, quercetin and kaempferol) in Polygoni Avicularis Herba. The analysis was performed on a Pinnacle II C18 column (250 × 4.6 mm, 5.0 µm) with a gradient elution of an aqueous mobile phase (containing 0.3% acetic acid) modified by acetonitrile. Diode-array detector (DAD) was used to detect the analytes at four wavelengths (270, 326, 355, and 370 nm). The established method was validated with good linearity, precision, accuracy and reproducibility to determine the polyphenols in Polygoni Avicularis Herba. The applicability of this analytical method was confirmed by successful analysis of the herb samples. The results indicated that the contents of these polyphenols varied with different locations of the samples. The anti-oxidation activities of Polygoni Avicularis Herba were further evaluated based on DPPH, ABTS and superoxide anion free radical scavenging assay. The herb exhibited obvious radical scavenging capacity, and gallic acid, chlorogenic acid, myricetrin and hyperoside played important roles in this activity.

Antioxidant and antiplatelet potential of different methanol fractions and flavonols extracted from onion (Allium cepa L.).

In this study, we reported the potential of various methanol fractions of onion (MFO) and its components quercetin and quercetin glucosides on platelet aggregation and antioxidant activity. The onion extracts were separated into several fractions using methanol and water. Further, these extracts were analyzed using simple high-performance liquid chromatography-diode array detector method to separate quercetin (Q), quercetin-4'-O-monoglucoside and quercetin-3, 4'-O-diglucoside from onion sample. It was observed that the bioactive compounds were accumulated in the non-polar portions rather than in the polar one. MFO and flavonol glucosides inhibited collagen-induced platelet aggregation, and the anti-aggregatory effects were comparatively studied using rat PRP (platelet-rich plasma). Among the extracted compounds, quercetin was found as a potent inhibitor of platelet aggregation compared to quercetin glucosides, whereas quercetin glucosides showed high antioxidant activities. These results suggest that MFO, quercetin and quercetin glucosides have powerful antiplatelet and antioxidant activities. These studies provide possible information leading to the intake of onions rich in these flavonols as a dietary supplement and functional food ingredient to prevent thrombosis and cardiovascular and oxidative stress-related diseases and might be utilized as a real source of valuable phytochemicals for the pharmaceutical and food industries for the development of antioxidant, anticoagulant and antiplatelet agents.

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF DILTIAZEM HYDROCHLORIDE IN BULK AND PHARMACEUTICAL DOSAGE FORM BY RP- HPLC

A simple, specific, precise and accurate Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method was developed and validated for the estimation of Diltiazem Hydrochloride in bulk and pharmaceutical dosage form. The chromatographic determination was performed on isocratic high performance liquid chromatography system of Agilent model no.1220. The separation was conducted by using column of Zorbax [C8 (5µ, 4.6 mm×250)] with mobile phase consisting of buffer and Acetonitrile in the ratio of (60:40). The mobile phase was delivered at the flow rate of 1.0ml/min. The eluent was monitored at wavelength 240 nm and found a sharp and symmetrical peak with retention time of 4.66min. The method was validated for linearity, accuracy, precision, specificity, robustness. Recovery of Diltiazem Hydrochloride was found to be in the range of 98%-102%. The method was found to be linear over the concentration range 50-150 µg/ml with coefficient correlation r2 = 0.995. After developing method, validation parameters were carried out successfully and obtained results were complied with USP monograph.

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

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

Determination of 2,4-Dichlorophenoxyacetic acid (2,4-D) in rat serum for pharmacokinetic studies with a simple HPLC method.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a chlorophenoxy herbicide used worldwide. We describe a high-performance liquid chromatography (HPLC) method with UV detection for the determination of 2,4-D in female and male rat serum. This allows to observe the change of serum 2,4-D concentration in rats with time and its pharmacokinetics characteristics with a simple, rapid, optimized and validated method. The serum samples are pretreated and introduced into the HPLC system. The analytes are separated in a XDB-C18 column with a mobile phase of acetonitrile (solvent A) and 0.02 M ammonium acetate (containing 0.1% formic acid) (solvent B) using a gradient elution at a flow rate of 1.0 mL/min. The wavelength for UV detection was set at 230 nm. Calibration curve for 2,4-D was constructed over a range of 0.1–400 mg/L. The method was successfully applied to study the pharmacokinetics of 2,4-D in rats in this study. After oral administration of 300 mg/kg and 60 mg/kg 2,4-D, the mean Cmax values were 601.9 and 218.4 mg/L, the AUC0→∞ values were 23,722 and 4,127 mg×h/L and the clearance (Cl) were 1.10 and 0.02 L/(h×kg), respectively. The developed method was found to be specific, precise, reproducible and rapid.

Development and Validation of a HPLC-based Bioanalytical Method for Lorcaserin using Solid Phase Extraction and Application to a Pharmacokinetic Study in Rats

A rapid, precise, accurate, specific and simple high performance liquid chromatography method for estimation of lorcaserin hydrochloric in human plasma, using metoprolol as an internal standard, was developed and validated as per the regulatory requirements. Sample preparation included solid phase extraction and chromatographic separation was performed using a Phenomenex Luna C18 column (250×4.6 mm i.d, 5 µ particle size), with phosphate buffer (pH 3):acetonitrile:methanol (65:20:15) as the mobile phase at a flow rate of 1.0 ml/min. Wavelength of detection was 222 nm. Retention times of internal standard and lorcaserin HCl were found to be 5.15 and 7.19 min, respectively. The method was developed and tested in the linearity range of 500 to 3000 ng/ml. The method was validated for accuracy, precision, linearity, recovery and stability in compliance to international regulatory guidelines.

Strategic Combination of Isocratic and Gradient Elution for Simultaneous Separation of Polar Compounds in Traditional Chinese Medicines by HPLC.

A simple high-performance liquid chromatography (HPLC) method for the simultaneous separation of the highly polar and weakly polar components of traditional Chinese medicines was developed via a strategic combination of isocratic and gradient elution methods. Liu-Shen-Wan and Liu-Wei-Di-Huang-Wan were used as representative examples of traditional Chinese medicines. This is the first time that 6 components of varying degrees of polarity in Liu-Shen-Wan had been successfully resolved in a single chromatographic run using an ultraviolet-visible detector with a fixed wavelength of 296 nm. In contrast to conventional gradient separation methods, this novel method offered a viable route for separation of the highly and weakly polar fractions simultaneously, thus greatly reducing the time and cost of analysis. This method therefore provides a more efficient way to determine the polar components present in traditional Chinese medicines. It would find potential application in drug screening, drug authentication, and product quality control.

Development and Validation of a Simple High Performance Liquid Chromatography/UV Method for Simultaneous Determination of Urinary Uric Acid, Hypoxanthine, and Creatinine in Human Urine.

Uric acid and hypoxanthine are produced in the catabolism of purine. Abnormal urinary levels of these products are associated with many diseases and therefore it is necessary to have a simple and rapid method to detect them. Hence, we report a simple reverse phase high performance liquid chromatography (HPLC/UV) technique, developed and validated for simultaneous analysis of uric acid, hypoxanthine, and creatinine in human urine. Urine was diluted appropriately and eluted with C-18 column 100 mm × 4.6 mm with a C-18 precolumn 25 mm × 4.6 mm in series. Potassium phosphate buffer (20 mM, pH 7.25) at a flow rate of 0.40 mL/min was employed as the solvent and peaks were detected at 235 nm. Tyrosine was used as the internal standard. The experimental conditions offered a good separation of analytes without interference of endogenous substances. The calibration curves were linear for all test compounds with a regression coefficient, r2 > 0.99. Uric acid, creatinine, tyrosine, and hypoxanthine were eluted at 5.2, 6.1, 7.2, and 8.3 min, respectively. Intraday and interday variability were less than 4.6% for all the analytes investigated and the recovery ranged from 98 to 102%. The proposed HPLC procedure is a simple, rapid, and low cost method with high accuracy with minimum use of organic solvents. This method was successfully applied for the determination of creatinine, hypoxanthine, and uric acid in human urine.

Comparative Assessment of Extraction Methods and Quantitative Estimation of Thymoquinone in the Seeds of Nigella sativa L By HPLC

The objective of the present study was to find out the best organic solvent and extraction method for the isolation of thymoquinone from Nigella sativa L. seeds. The seeds of N. sativa were authenticated by a taxonomist, washed, dried and powdered. Different extraction techniques such as maceration, soxhlet, reflux and ultrasound assisted extraction were employed for extraction of thymoquinone from seeds by using three solvents of different polarity like petroleum ether, hexane and methanol. Thymoquinone was quantified in each sample extract of seeds by simple high performance liquid chromatography technique. The content of thymoquinone was found to be higher in methanolic extract among the various techniques and solvents tried. Ultrasound assisted extraction technique was observed the best for extraction of thymoquinone. The calibration curve of standard thymoquinone demonstrated linear relationship in the concentration range of 1 to 0.0625 µg/ml with correlation coefficient, r2 of 0.9976. It can be concluded on the basis of HPLC finding that ultrasound assisted extraction technique is the best for extraction of thymoquinone . This method will be preffered in industy or academia for extraction of thymoquine from N. sativa seeds.

Determination of Tangeretin in Rat Plasma: Assessment of Its Clearance and Absolute Oral Bioavailability.

Tangeretin (TAN) is a dietary polymethoxylated flavone that possesses a broad scope of pharmacological activities. A simple high-performance liquid chromatography (HPLC) method was developed and validated in this study to quantify TAN in plasma of Sprague-Dawley rats. The lower limit of quantification (LLOQ) was 15 ng/mL; the intra- and inter-day assay variations expressed in the form of relative standard deviation (RSD) were all less than 10%; and the assay accuracy was within 100 ± 15%. Subsequently, pharmacokinetic profiles of TAN were explored and established. Upon single intravenous administration (10 mg/kg), TAN had rapid clearance (Cl = 94.1 ± 20.2 mL/min/kg) and moderate terminal elimination half-life (t1/2 λz = 166 ± 42 min). When TAN was given as a suspension (50 mg/kg), poor but erratic absolute oral bioavailability (mean value < 3.05%) was observed; however, when TAN was given in a solution prepared with randomly methylated-β-cyclodextrin (50 mg/kg), its plasma exposure was at least doubled (mean bioavailability: 6.02%). It was obvious that aqueous solubility hindered the oral absorption of TAN and acted as a barrier to its oral bioavailability. This study will facilitate further investigations on the medicinal potentials of TAN.

Quantitative analysis of triterpenoids in different parts of Aralia elata (Miq.) Seem using HPLC–ELSD and their inhibition of human umbilical vein endothelial cell ox-LDL-induced apoptosis

ABSTRACTA simple and accurate method coupling high-performance liquid chromatography–evaporative light scattering detector was developed to simultaneously quantify the five triterpenoids in different parts of Aralia elata (Miq.) Seem (A. elata). for the first time. Samples were extracted using 70% methanol by ultrasonication. Separation of the analytes was performed on a Welchrom C18 column with acetonitrile and water as the mobile phase. All calibration curves had good linearity over relatively wide range of concentrations. This method showed good average recovery and precision, also high sensitivity with limits of detection and quantification. This method was successfully applied to quantify triterpenoid saponins in 14 samples collected from different areas of China. This study demonstrate that there is significant variation in the amounts of the five triterpenoids of interest in A. elata. Furthermore, 70% methanol extracts of roots had the strongest effect on ox-low-density lipoprotein-induced human umbilical vein endothelial cell apoptosis.

Therapeutic drug monitoring of ponatinib using a simple high-performance liquid chromatography method in Japanese patients

A simple and highly sensitive high-performance liquid chromatography (HPLC) method was developed for the quantification of ponatinib in human plasma. The developed HPLC method was validated based on International D.S. Food and Drug Administration guidelines. This technique utilized a solid-phase extraction step and required only 200μL plasma for a single analysis. The lower limit of quantification for ponatinib was 1.0ng/mL. Coefficients of variation and accuracies for intra- and interday assays were less than 10.8% and within 13.7%, respectively. The precision and accuracy of our HPLC assay was suitable for pharmacokinetic studies of ponatinib. On day 8 after beginning ponatinib therapy with an initial dose of 15mg, patients having a ponatinib C0 of less than 23ng/mL by HPLC may require a dose adjustment to 30mg to obtain a C0 of 23ng/mL of more. The median ponatinib C0 in 6 Japanese patients taking a 15mg daily dose was 24.6ng/mL, which was greater than the target concentration of 23ng/mL, and that of patients taking 30mg increased to a plasma concentration of 48.0ng/mL. This novel treatment strategy using the HPLC method developed herein may be useful for routine ponatinib therapy.

Simple chromatographic detection modes for antitumor agent and its degradants

ABSTRACTDegradation of active ingredients is common during the production, transportation, and storage of the pharmaceutical preparations. The resulted degradation products can affect the pharmaceuticals’ therapeutic effect. Also, they may have some toxic properties that make them have deleterious effects on patients. Nifuroxazide, antitumor, antimetastasis, and antidiarrheal agent, has been detected in pharmaceutical formulation by two sensitive, selective, and cheap methods. The first method was densitometric thin-layer chromatography technique. The compound was detected in the presence of its degradation products without any interference. Limits of quantification and detection values were 200 and 94.3 ng/band, respectively. The method was linear in the range 0.2–12 µg/band with mean recovery% ±relative standard deviation (RSD)% = 99.97% ± 1.414. The second method was developed using simple high-performance liquid chromatography technique. The method was successfully able to separate the proposed compound from its degradation forms in the presence of pentoxiftylline as an internal standard without any interference in less than 5 min. The method also detected nifuroxazide degradation products down to 500 ng/mL. Both methods were statistically compared to a reported method with no significant difference in performance.

Qualitative and quantitative analysis of chemical constituents in Ardisiae Japonicae Herba.

Ardisiae Japonicae Herba is a well-known traditional Chinese medicine for the treatment of bronchitis conjunctivitis, pneumonia, and trauma. In this work, a high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry method was first established for the separation and structural identification of the chemical constituents in Ardisiae Japonicae Herba. A total of 15 compounds including coumarins, flavonoid glycosides, and catechins were identified or tentatively characterized based on their chromatographic behaviors and mass spectral fragmentation and by comparisons with the reference standards. Furthermore, a simple high-performance liquid chromatography with diode array detection method was developed for the simultaneous determination of five major constituents. Results obtained from method validation, including linearity, precision, repeatability, stability, and recovery, showed that the established method was reliable and accurate. Bergenin and quercitrin were found to be the most abundant constituents and could be served as chemical markers for quality control of Ardisiae Japonicae Herba.

A rapid and simple HPLC–FLD screening method with QuEChERS as the sample treatment for the simultaneous monitoring of nine bisphenols in milk

A specific, precise and accurate high performance liquid chromatographic (HPLC) analytical method with a fluorescence detector (FLD) was established for the simultaneous determination of nine bisphenols (BPs) in milk samples. Samples were extracted ultrasonically with acetonitrile and cleaned using the QuEChERS technique. Under the optimized conditions, good linearity was obtained for the nine BPs and the correlation coefficients (R2) ranged from 0.9942 to 0.9997. Recovery values for the nine bisphenols in spiked samples were 75.82–93.86% with intra-day and inter-day relative standard deviations (RSDs) from 2.6 to 11.1%. The limits of detection (LODs) and quantification (LOQs) were 1.0–3.1 μg/kg and 3.5–9.8 μg/kg, respectively. The results demonstrated clearly that the approach developed provides reliable, simple, rapid and environmentally-friendly quantification and identification of nine bisphenols in a fatty matrix and could be used for monitoring bisphenols in milk.

Development of Bioanalytical HPLC Method for Estimation of Milnacipran Hydrochloride in Rabbit Plasma Using Solid Phase Extraction Technique and its Application in Pharmacokinetic Investigation

Background: The availability of a low-cost bioanalytical method, easy to transfer and to set up, represents an advantage in therapeutic drug monitoring and industrial research. Objective: A simple, sensitive and rapid high performance liquid chromatographic (HPLC) method is developed and validated for quantitative determination of milnacipran hydrochloride in rabbit plasma. Method: Simple and effective solid phase extraction technique was performed for sample treatment and it has resulted in consistent and high recoveries (93–95%) at all concentrations studied. Efficient chromatographic separation has been performed on a LiChrospher® C18 column using a mobile phase consisting of a mixture of phosphate buffer, acetonitrile and methanol (65:25:10; v/v/v) at a flow rate of 0.8 mL/min. Results: The method has demonstrated linearity from 25 ng to 2000 ng/mL with a regression coefficient of 0.9998. The accuracy was found to be very high (99.31 to 101.44 %). %RSD values for inter-day and intra-day variation were not more than 3.58. The method has demonstrated high sensitivity with a lower limit of quantification of 25 ng/mL and excellent stability of milnacipran in rabbit plasma. The method was applied for pharmacokinetic investigation of immediate release and controlled release milnacipran tablets. The in-vivo study results indicated that developed HPLC method was sensitive to accurately quantify the concentration of milnacipran in plasma, which is an essential requirement for determination of pharmacokinetic parameters for drug formulation studies as well as future bioavailability or bioequivalence studies. Conclusion: A simple and sensitive bioanalytical HPLC method was developed, validated and applied for pharmacokinetic studies. Keywords: Milnacipran, liquid chromatography, solid phase extraction, rabbit plasma, pharmacokinetic investigation.

Development and validation of a simple high-performance liquid chromatography analytical method for simultaneous determination of phytosterols, cholesterol and squalene in parenteral lipid emulsions.

A simple analytical method for simultaneous determination of phytosterols, cholesterol and squalene in lipid emulsions was developed owing to increased interest in their clinical effects. Method development was based on commonly used stationary (C18 , C8 and phenyl) and mobile phases (mixtures of acetonitrile, methanol and water) under isocratic conditions. Differences in stationary phases resulted in peak overlapping or coelution of different peaks. The best separation of all analyzed compounds was achieved on Zorbax Eclipse XDB C8 (150 × 4.6 mm, 5 μm; Agilent) and ACN-H2 O-MeOH, 80:19.5:0.5 (v/v/v). In order to achieve a shorter time of analysis, the method was further optimized and gradient separation was established. The optimized analytical method was validated and tested for routine use in lipid emulsion analyses.

Determination of hydroxyurea in human plasma by HPLC-UV using derivatization with xanthydrol.

A simple and rapid high performance liquid chromatography (HPLC) method using ultraviolet (UV) detection was developed to determine hydroxyurea (HU) concentration in plasma sample after derivatization with xanthydrol. Two hundred microliters samples were spiked with methylurea (MeU) as internal standard and proteins were precipitated by adding methanol. Derivatization of HU and MeU was immediately performed by adding 0.02M xanthydrol and 1.5M HCl in order to obtain xanthyl-derivatives of HU and MeU that can be further separated using HPLC and quantified using UV detection at 240nm. Separation was achieved using a C18 column with a mobile phase composed of 20mM ammonium acetate and acetonitrile in gradient elution mode at a flow rate of 1mL/min. The total analysis time did not exceed 18min. The method was found linear from 5 to 400μM and all validation parameters fulfilled the international requirements. Between- and within-run accuracy error ranged from -4.7% to 3.2% and precision was lower than 12.8%. This simple method requires small volume samples and can be easily implemented in most clinical laboratories to develop pharmacokinetics studies of HU and to promote its therapeutic monitoring.