High-performance Liquid Chromatographic Method For Analysis Research Articles

Development and Validation of a Single HPLC Method for Analysis of Purines in Fish Oil Supplements

Gout is one of the most common forms of inflammatory arthritis, affecting over 8 million adults in the US. Individuals with gout are advised to avoid the habitual intake of purine-rich foods such as meats, seafood, purine-rich vegetables, and animal protein. An increased risk of developing or having subsequent attacks of gout is particularly associated with the consumption of seafood. However, clinical studies have shown that certain seafood and fish oil supplements that contain large amounts of omega-3 fatty acids provide important cardiovascular benefits. Individuals who might benefit from omega-3 fatty acid supplementation may therefore avoid fish oil products because they contain purines. Currently, there are no distinct high-performance liquid chromatography (HPLC) methods available in the literature that are validated as per the International Conference on Harmonisation (ICH) guidelines for the analysis of omega-3 fatty acid oils or fish oil containing products for purine content. A robust, fast, and efficient reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the specific analysis of the naturally occurring purines guanine, purine, theobromine, and adenine. These purines are often found in fish oil and seafood. The analytical method reported herein quantifies all four purines in fish oil in about 20 minutes.

A single extraction and HPLC procedure for simultaneous analysis of phytosterols, tocopherols and lutein in soybeans

A saponification/extraction procedure and high performance liquid chromatography (HPLC) analysis method were developed and validated for simultaneous analysis of phytosterols, tocopherols and lutein (a carotenoid) in soybeans. Separation was achieved on a phenyl column with a ternary, isocratic solvent system of acetonitrile, methanol and water (48:22.5:29.5, v/v/v). Evaporative light scattering detection (ELSD) was used to quantify β-sitosterol, stigmasterol, campesterol, and α-, δ- and γ-tocopherols, while lutein was quantified with visible light absorption at 450nm. Peak identification was verified by retention times and spikes with external standards. Standard curves were constructed (R2>0.99) to allow for sample quantification. Recovery of the saponification and extraction was demonstrated via analysis of spiked samples. Also, the accuracy of results of four soybeans using the described saponification and HPLC analytical method was validated against existing methods. This method offers a more efficient alternative to individual methods for quantifying lutein, tocopherols and sterols in soybeans.

Development of HPLC analytical method for determination of anthranilamide into food simulants from polyethylene terephthalate

An analytical method for the determination of specific migration levels of anthranilamide from polyethylene terephthalate (PET) bottles into food simulant has been developed. The conditions of ultrasound-assisted liquid-liquid extraction (LLE) for the simulant of vegetable oil have been optimized. The analysis have been carried out by reversed-phase high performance liquid chromatography (HPLC) coupled with a fluorescence detector, which present suitable detection limits and good recovery.

Competitive binding between 4,4′-diphenylmethane-bis(methyl) carbamate and RAGE ligand MG-H1 on human umbilical vein endothelial cell by cell membrane chromatography

The compound 4,4′-diphenylmethane-bis(methyl) carbamate (CM1) has a protective activity on AGEs-induced endothelial dysfunction on human umbilical vein endothelial cell (HUVEC) in our previous study. It suggested that CM1 which may act as a competitive antagonist to the blockade of AGEs to receptor of AGEs (RAGE) and attenuate the HUVEC damage. In order to testify that hypothesis, the cell membrane chromatography (CMC) combined with high performance liquid chromatography (HPLC) was developed for analyzing the competitive binding properties on RAGE of HUVEC between CM1 and MG-H1, the agonist of RAGE. The results from saturation binding of CM1 and MG-H1 on cells demonstrated that dissociation equilibrium constants ( K d) of CM1 and MG-H1 were 3.653 nM and 4.12 nM, respectively; while maximum binding capacity ( B max) of CM1 and MG-H1 were 30.08 and 18.72 fmol/mg protein, respectively. In competition experiments, IC 50 of CM1 with pre-incubation 10 −10 M and 10 −9 M MG-H1 were 1.37 × 10 −9 M and 4.56 × 10 −8 M, respectively. The present findings indicated that CM1 conjugated competitively to cells with RAGE ligand MG-H1. The primary study illustrated that CMC combined with HPLC analysis method could be an alternative, rapid and efficient approach for the interaction of drug molecule and receptor, and that CM1 intervene the AGEs inducing HUVEC damage may via the competitively block the AGEs–RAGE path way.

Qualitative and Quantitative Analyses of Flavonoids in Spirodela polyrrhiza by High‐performance Liquid Chromatography Coupled with Mass Spectrometry

Spirodela polyrrhiza (L.) Schleid. is a traditional Chinese herbal medicine for the treatment of influenza. Despite its wide use in Chinese medicine, no report on quality control of this herb is available so far. To establish qualitative and quantitative analytical methods by high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) for the quality control of S. polyrrhiza. The methanol extract of S. polyrrhiza was analysed by HPLC/ESI-MS(n). Flavonoids were identified by comparing with reference standards or according to their MS(n) (n = 2-4) fragmentation behaviours. Based on LC/MS data, a standardised HPLC fingerprint was established by analysing 15 batches of commercial herbal samples. Furthermore, quantitative analysis was conducted by determining five major flavonoids, namely luteolin 8-C-glucoside, apigenin 8-C-glucoside, luteolin 7-O-glucoside, apigenin 7-O-glucoside and luteolin. A total of 18 flavonoids were identified by LC/MS, and 14 of them were reported from this herb for the first time. The HPLC fingerprints contained 10 common peaks, and could differentiate good quality batches from counterfeits. The total contents of five major flavonoids in S. polyrrhiza varied significantly from 4.28 to 19.87 mg/g. Qualitative LC/MS and quantitative HPLC analytical methods were established for the comprehensive quality control of S. polyrrhiza.

Development and Validation of a Confirmatory HPLC Method for Simultaneous Determination of Sudan Dyes in Animal Tissues and Eggs

A simple and sensitive high-performance liquid chromatography (HPLC) analytical method for the simultaneous determination of six Sudan dyes (Sudan Red G, Sudan I, Sudan II, Sudan III, Sudan Red 7B, Sudan IV) in animal tissues and eggs was developed. Samples were extracted with acetonitrile followed by a cleanup using a C(18) solid-phase extraction column. Chromatographic separation was achieved on a Zorbax SB-C(18) column under gradient conditions. The analytes were detected at 510 nm by HPLC with diode array detection. Specificity, decision limit (CCalpha), detection capacity (CCbeta), accuracy, and precision were determined during validation process. Recoveries for six Sudan dyes from three animal tissues and eggs were 77.2-98.0% with excellent relative standard deviations. CCalpha and CCbeta were in the range of 7.7-9.0 microg/kg and 9.1-10.3 microg/kg, respectively. The limits of quantitations were between 12.8 microg/kg and 15.0 microg/kg.

Study on Determination of Pentacyclic Triterpenoids in Chaenomeles by HPLC-ELSD

A new direct high-performance liquid chromatography analytical method using evaporative light scattering detection (ELSD) for the simultaneous determination of the major pentacyclic triterpenoids in Chaenomeles (Chinese medicinal herb) has been developed. The simultaneous separation of the seven pentacyclic triterpenoids was achieved on a Zorbax Stable Bound (4.6 mm x 100 mm i.d., 1.8 microm) C(18) column by gradient elution with acetonitrile-water as the mobile phase and detected with ELSD. This method provides good reproducibility and sensitivity for the quantification of seven major pentacyclic triterpenoids, namely erythodiol, betulin, acetyl ursolic acid, ursolic acid, oleanolic acid, betulinic acid, and pomolic acid, respectively. The relative standard deviation (RSD) of overall intra-day variations was less than 2.2%, and the RSD of inter-day variations was less than 2.8%. The standard recoveries (three different concentrations of markers: 0.1, 0.5, and 2.0 mg) ranged from 96-104%. The results demonstrate that this method is simple, sensitive, selective, and suitable for the quality control of this commonly used Chinese medicinal herb.

Development and validation of HPLC method for analysis of some antiretroviral agents in their pharmaceutical dosage forms

Anew analytical method using reversed-phase high performance liquid chromatography (HPLC) has been developed for analysis of four antiretroviral molecules Lamivudine, Abacavir, Zidovudine, and Efavirenz. The proposed method was validated in accordance with the ICH Q2 (R1) validation guidelines by determining its selectivity, linearity, accuracy, and precision. Based on the results, the method is an effective choice to analyze these drugs in their single and combined pharmaceutical dosage forms.

Analytical HPLC Method Validation of Amiloride and Its Pharmacokinetic Study in Humans

This study was aimed to validate a reliable analytical method for the pharmacokinetic study of amiloride in human plasma by a high performance liquid chromatography (HPLC) system with UV detection. Triamterene was used as an internal standard. After extraction with ethylacetate, the supernatant was evaporated. Then, the residue was reconstituted and an aliquot was injected onto the HPLC system. Separation was performed on a Capcell Pak C18 UG120 column (4.6 mm × 150 mm, 5 µm particles) with a mobile phase of 12% acetonitrile containing 0.4% glacial acetic acid and UV detection at a wavelength of 360 nm. The intra- and inter-day precision expressed as the relative standard deviation was less than 15%. The flow rate of mobile phase was 1 mL/min and the retention time of amiloride and internal standard, triamterene, was found to be 3.06 and 8.80 min, respectively. The lower limit of quantification (LOQ) was 0.2 ng/mL of amiloride using 1 mL of plasma. The calibration curve was linear in the concentration range of 0.2–50 ng/mL (r 2 = 1.0000). The mean accuracy was 85.1–101.7%. The coefficient of variation (precision) in the intra- and inter-day validation was 2.1–12.5 and 2.4–13.7%, respectively. The pharmacokinetics of amiloride was evaluated after a single oral administration of 10 mg to healthy volunteers. The AUC0–72hr, Cmax, Tmax, and T1/2 were 267.7 ± 60.0 ng·hr/mL, 22.9 ± 5.4 ng/mL, 3.0 ± 0.8 hr, and 13.6 ± 2.4 hr, respectively. These results demonstrated that this method was highly feasible and reproducible for pharmacokinetic studies of amiloride in eight volunteers after oral adminis-tration (10 mg as amiloride HCl).

Hydrolytic degradation profile and RP-HPLC estimation of cilostazol in tablet dosage form

A simple, selective, precise and stability-indicating high-performance liquid-chromatographic method of analysis of cilostazol in pharmaceutical dosage form was developed and validated. The solvent system consisted of 10 mM phosphate buffer (pH 6.0):acetonitrile:methanol (20:40:40). Retention time of cilostazol in C18 column was 5.7 ± 0.1 min at the flow rate 1.3 ml/min. Cilostazol was detected at 248 nm at room temperature. The linear regression analysis data for the calibration plots showed good linear relationship with correlation coefficient value, r 2 =0.9998 in the concentration range 100–3200 ng/ml with slope 43.45 intercept 156.75. The method was validated for linearity, range, accuracy, precision and specificity. Cilostazol was determined in tablet dosage form in range of 99.58-100.67% with 0.4600 standard deviation. Stress studies were conducted in acid and alkali hydrolysis with gradual increasing concentration. Cilostazol was found to be stable in various concentrations of acidic and alkaline.

Validation of a dissolution method with HPLC analysis for lasofoxifene tartrate low dose tablets

A dissolution method with high performance liquid chromatography (HPLC) analysis was validated for an immediate release low dose tablet formulation. The method was validated to meet requirements for a global regulatory filing and this validation included specificity, precision, linearity, accuracy and range. Validation of precision included an intermediate precision study using an experimental design in order to satisfy Japanese regulatory requirements. In addition, filter suitability, standard and sample solution stability and method robustness were demonstrated. A statistical design of experiments was used for the robustness evaluation of both the dissolution method and the HPLC analysis method. All results were acceptable and confirmed that the method is suitable for its intended use.

Plasma and Gingival Crevicular Fluid Phenytoin Concentrations as Risk Factors for Gingival Overgrowth

Gingival enlargement is one of the side effects associated with the administration of phenytoin. The mechanism by which phenytoin induces gingival enlargement is not well understood. This study was conducted to investigate the relationship between plasma and gingival crevicular fluid (GCF) phenytoin concentrations and the degree of gingival overgrowth in patients with similar gingival and plaque indices and also to determine the risk factors for gingival enlargement. Eighteen patients taking phenytoin in regular doses > or =6 months prior to the investigation participated in the study. Gingival enlargement was evaluated with two indices to score vertical and horizontal overgrowth. The gingival index (GI), plaque index (PI), gingival bleeding time index (GBTI), probing depth (PD), and clinical attachment level (CAL) were also evaluated. GCF and plasma phenytoin concentrations were determined by using high-performance liquid chromatography (HPLC). There was no significant difference between responders and non-responders for PD, CAL, PI, GI, and GBTI. Phenytoin was detected in all of the GCF and plasma samples using the HPLC analysis method. The mean concentration of phenytoin in GCF was significantly greater than the concentration of phenytoin in plasma. No significant difference was observed for the concentration of GCF phenytoin between responders and non-responders. However, the concentration of plasma phenytoin was significantly higher in responders than non-responders. This study showed that plasma phenytoin level appeared to be a risk factor for phenytoin-induced gingival overgrowth.

Sampling Hazelnuts for Aflatoxin: Uncertainty Associated with Sampling, Sample Preparation, and Analysis

The variability associated with the aflatoxin test procedure used to estimate aflatoxin levels in bulk shipments of hazelnuts was investigated. Sixteen 10 kg samples of shelled hazelnuts were taken from each of 20 lots that were suspected of aflatoxin contamination. The total variance associated with testing shelled hazelnuts was estimated and partitioned into sampling, sample preparation, and analytical variance components. Each variance component increased as aflatoxin concentration (either B1 or total) increased. With the use of regression analysis, mathematical expressions were developed to model the relationship between aflatoxin concentration and the total, sampling, sample preparation, and analytical variances. The expressions for these relationships were used to estimate the variance for any sample size, subsample size, and number of analyses for a specific aflatoxin concentration. The sampling, sample preparation, and analytical variances associated with estimating aflatoxin in a hazelnut lot at a total aflatoxin level of 10 ng/g and using a 10 kg sample, a 50 g subsample, dry comminution with a Robot Coupe mill, and a high-performance liquid chromatographic analytical method are 174.40, 0.74, and 0.27, respectively. The sampling, sample preparation, and analytical steps of the aflatoxin test procedure accounted for 99.4, 0.4, and 0.2% of the total variability, respectively.

Validation of HPLC analysis method of a novel antihypertensive agent MS23 in rat plasma

MS23 is a vasodilator with unique dual action pharmacological profile to inhibit type 4 PDE and antagonize L-type calcium channels. We validated an analytical protocol for MS23 in rat plasma using high performance liquid chromatography (HPLC). A C18 column and a phosphate/acetonitrite buffer were used for chromatographic separation. UV detection was performed at 307 nm. The calibration curve for MS23 was linear in the range from 50 to 10,000 ng/ml. The limit of quantification (LOQ) was 50 ng/ml. The results demonstrate that the method has linearity (R = 0.9989), specificity, and acceptable precision/accuracy. This method is simple, economic, and sufficient for in vivo pharmacokinetic studies on the compound.

Validation of a high-performance liquid chromatography analytical method for ochratoxin A quantification in cocoa beans

A validated high-performance liquid chromatography (HPLC) method with fluorescence detection for the quantitative analysis of ochratoxin A (OTA) in cocoa beans is described. OTA was extracted with methanol–3% sodium hydrogen carbonate solution and then purified with immunoaffinity columns before its analysis by HPLC. The validation of the analytical method was based on the following criteria: selectivity, linearity, limit of detection and quantification, precision (within- and between-day variability) and recovery, robustness and uncertainty. Detection and quantification limits were 0.04 and 0.1 μg kg−1, respectively. Recovery was 88.9% (relative standard deviation = 4.0%). This method was successfully applied to the measurement of 46 cocoa bean samples of different origins. A total of 63% of cocoa bean samples was contaminated with a level greater than the limit of detection. The means and medians obtained for cocoa bean were 1.71 and 1.12 μg kg−1, respectively. Surveillance controls should be set up in both crops and factories involved in transformation processes to avoid this mycotoxin in final products.

Measurement of triclabendazole and its metabolites in liver flukes: method development and full validation

The trematode parasite Fasciola hepatica is still the cause of considerable loss in livestock production all over the world. The knowledge of the pharmacological properties of the available flukicidal drugs is critical to control this parasite. Triclabendazole is an halogenated benzimidazole anthelmintic with potent flukicidal activity. A simple reversed-phase high-performance liquid chromatographic analytical method has been developed, validated and applied for the quantitative determination of the flukicidal drug triclabendazole and its sulpho- and hydroxy-metabolites in the liver fluke, F. hepatica. Triclabendazole, triclabendazole sulphoxide, triclabendazole sulphone, hydroxy-triclabendazole, hydroxy-triclabendazole sulphoxide and hydroxy-triclabendazole sulphone were measured simultaneously in this trematode parasite. Linearity, resolution, precision, accuracy, recovery, limits of detection and quantification of the method were determined. Drug extraction from the parasite’s tissue homogenate was effectively performed using liquid extraction (acetonitrile), avoiding clean up by solid phase extraction, prior to analysis by reversed-phase high-performance liquid chromatography. The resolution of all the halogenated benzimidazole thiol molecules assayed was obtained on a C 18 reversed-phase (5 μm, 250 mm×4.6 mm) column using acetonitrile and ammonium acetate as the mobile phase and ultraviolet detection at 300 nm. Regression analyses were linear over the concentration range examined (from 0.272 to 16.331 nmol/100 mg trematode protein) and the correlation coefficients of the calibration curves ranged between 0.996 and 1.000. The calculated limits of detection of the proposed method for the parent drug and its metabolites ranged between 0.007 and 0.079 nmol/100 mg trematode protein. The extraction efficiency for the different analytes from the parasite material was greater than 71%. The results obtained indicated that the developed chromatographic method was selective, accurate and easy to reproduce. The developed procedure was successfully applied to quantify triclabendazole/metabolites in F. hepatica incubated under ex vivo conditions, demonstrating to be efficient for the determination of the most extensively used flukicidal drug available for veterinary medicine, and its metabolites. The analytical method described here is an useful tool for the measurement of this flukicidal compound in different studies addresses to evaluate drug influx/eflux and metabolism in its main target parasite.

Trihydroxyflavones from Scutellaria baicalensis: Separation by a Facile MEKC Technique and Comparison to an Analytical HPLC Method

Phenolic were extracted from the roots of Scutellaria baicalensis Georgi (Labiatae) using methanol. The phenolics of the crude extract were examined by high‐performance liquid chromatography (HPLC) using an analytical C18 column coupled with ultraviolet‐diode array detection (UV‐DAD). Chromatograms were compared with those acquired by micellar electrokinetic chromatography (MEKC) with UV‐DAD. A good separation of the phenolics from the crude extract was achieved by the electrophoretic technique, and in a shorter time than by HPLC. Two dominant flavones, believed to be 5,6,7‐trihydroxyflavone and 5,6,7‐trihydroxyflavone‐7‐O‐β‐D‐glucopyranosiduronate, which are commonly referred to as baicalein and baicalin, respectively, were then isolated from the crude extract using a semi‐preparative HPLC method on a RP‐18 column. The identities of the separated trihydroxyflavones were confirmed by NMR spectroscopies and mass spectrometry as being baicalein (1) and baicalin (2). The employment of MEKC coupled with UV‐DAD as a technique to separate and to identify phenolic compounds, or their classes in natural products research, is expected to expand over the next decade.

Quantitative liquid chromatographic determination of sanguinarine in cell culture medium and in rat urine and plasma

Sanguinarine is a quaternary benzo[ c]phenanthridine alkaloid, extracted from the argemone oil, which produced severe human intoxications. To investigate the sanguinarine biotransformation, we develop a simple extraction process and a high performance liquid chromatographic separation coupled to a sensitive fluorometric detection of sanguinarine in cell culture medium, as well as in rat urine and plasma. After extraction with an acidified organic solvent, sanguinarine elution is performed within 15 min on a Nucleosil C18 column with a gradient using 0.2% formic acid/water/acetonitrile as mobile phase. Extracted and standard sanguinarine are characterized by mass spectrometry. The extraction recovery of sanguinarine is about 80% in cell culture medium and in rat urine, but lower in plasma. This convenient high performance liquid chromatography (HPLC) method allows to quantify sanguinarine over concentrations ranged 10–2000 ng ml −1. The limit of fluorometric detection is 0.5 ng. Under these conditions, the lower limit of quantification of sanguinarine is 50 ng ml −1 in cell culture medium and in rat urine and 100 ng ml −1 in rat plasma. This analytical HPLC method is specific, linear and reproducible in all media and is suitable for quantitative determination of sanguinarine in biological fluids.

Purification, analysis, and preservation of reduced nicotinamide adenine dinucleotide 2 ′-phosphate

Nicotinamide-containing cofactors are ubiquitous in biological systems. Consequently, numerous assays have been developed to study such systems that involve a variety of derivatives and isotopically labeled forms of these cofactors. Often the nicotinamide ring is labeled at the C-4 position which is directly involved in the hydride transfer chemistry catalyzed by nicotinamide-dependent enzymes. A label remote from the reactive center is often also required to follow the course of a reaction or the location of the cofactor. Since the necessary labeling pattern can be as unique as the designed experiment, these cofactors need to be synthesized, analyzed, and, most preferably, preserved. The micro-scale preservation of reduced nicotinamides has long been a challenge due to the inherent lability of the reduced cofactors. Furthermore, it has been found that the reduced 2 ′-phosphorylated cofactor is even less stable (i.e., reduced nicotinamide adenine dinucleotide phosphate (NADPH) is more labile than reduced nicotinamide adenine dinucleotide). Presented here are methods that were established to purify nicotinamide cofactors via reverse-phase high-performance liquid chromatography (HPLC) and, most importantly, to stabilize NADPH under optimal conditions for long-term storage. Additionally, an analytical HPLC method which achieves 7-min resolution between oxidized and reduced cofactors was developed. This method also results in over 4-min resolution of these nicotinamide cofactors from various derivatives of folic acid. This analysis affords a new analytical assay for the dihydrofolate reductase-catalyzed reaction and several dehydrogenases involved in folic acid metabolism.

Development and validation of an automated extraction method (accelerated solvent extraction®) and a reverse-phase HPLC analysis method for assay of ivermectin in a meat-based chewable formulation

A new method for monitoring ivermectin content in HEARTGARD CHEWABLES® has been developed and validated. The method consists of the automated extraction of ivermectin from the meat-based formulation under conditions of elevated temperature and pressure (accelerated solvent extraction, ASE®), and determination of the active by reverse-phase high performance liquid chromatography (HPLC). The method resolves both active species of ivermectin (components H 2B 1a and H 2B 1b) from the formulation matrix.