High-performance Liquid Chromatography With Diode Array Detection Method Research Articles

Validation of an HPLC-DAD Method for Quercetin Quantification in Nanoparticles.

The evaluation of the efficacy of incorporation of quercetin in nanoparticles is crucial, both for the development and quality control of pharmaceutical formulations. The validation of analytical methods for the precise quantification of quercetin is useful for the evaluation of various potential quercetin delivery systems and quercetin pharmacokinetics. This work aimed to validate a high-performance liquid chromatography with diode array detection (HPLC-DAD) method for quercetin detection and quantification in nanoparticles. Different mobile phase conditions and detection wavelengths (254 and 368 nm) were tested, and the major validation parameters were assessed (precision, accuracy, linearity, sensitivity, stability, and selectivity). The best peak resolution was obtained when quercetin was analyzed at 368 nm with a mobile phase of 1.5% acetic acid and a water/acetonitrile/methanol ratio of 55:40:5. Under these conditions, quercetin also eluted rapidly (retention time of 3.6 min). The method proved to be linear (R2 > 0.995), specific, and repeatable (variation coefficient between 2.4% and 6.7%) and presented intermediate precision (variation coefficient between 7.2% and 9.4%). The accuracy of the analysis ranged between 88.6% and 110.7%, and detection and quantification limits were 0.046 and 0.14 µg/mL, respectively. Quercetin solutions were more stable when stored at 4 °C than at room temperature or -20 °C. This validated method satisfied more parameters of bias assessment than most recent methods for quercetin determination and presented itself as more sensitive and efficient than general spectrophotometric methods. The method was successfully used for the analysis of quercetin incorporation in nanoparticles and will be evaluated in the future for its adequacy for the determination of quercetin in more complex matrices.

Development and Validation of an HPLC-DAD Method for the Simultaneous Analysis of Phenolic Compounds

Phenolic compounds are natural substances that exhibit different functional bioactivities and provide health-protective actions against chronic illnesses. The vast potential of these compounds in health and other sectors demands the establishment of analytical procedures for their immediate and simultaneous analysis. In this study, a high-performance liquid chromatography with diode-array detection (HPLC-DAD) method was developed and validated for the simultaneous analysis of gallic acid, catechin, epicatechin, rutin hydrate, caffeic acid, syringic acid, ellagic acid, p-coumaric acid, trans-ferulic acid, myricetin, resveratrol, and quercetin. The chromatographic separation of the selected polyphenols was carried out in a reversed-phase Inertsil ODS-3 column (250mm x 4.5mm x 5µm) at a flow rate of 0.8 mL/min, injection volume of 20 µL, and column temperature of 30°C. The detection and quantification of phenolic compounds were done at specific wavelengths (254, 275, 305, and 325 nm) using gradient elution for 40 minutes, with acidified water and acetonitrile solution as mobile phase. Validation of the established analytical procedure showed that the coefficient of determination (R2 > 0.99), limit of detection (0.01 to 0.35 µg/mL), limit of quantitation (0.03 to 1.07 µg/mL), recovery values (98.33 to 101.12%), and repeatability (RSD < 5%) respectively indicated a linear, sensitive, accurate, and precise analytical method for the simultaneous chromatographic analysis of the 12 phenolic compounds. Overall, the developed HPLC-DAD procedure can offer adequate confidence for the identification and quantification of specific polyphenols and can be modified or updated for future analysis of phenolic compounds in different plant extracts.

Development of HPLC-DAD method for the quantification of pentostatin in the fruiting body of Cordyceps militaris

Cordyceps militaris is an important medicinal mushroom in traditional medicine. It contains many bioactive compounds that have applications in medicine, such as cordycepin, adenosine... Recently, through genome analysis, C. militaris has been shown to have the ability to synthesise pentostatin. Pentostatin is an important active ingredient in the production of chemotherapy drugs used in cancer treatment. However, there have been few studies on the development of methods for quantifying pentostatin in C. militaris. In this study, a simple and sensitive high-performance liquid chromatography with diode-array detection (HPLC-DAD) method for the quantification of pentostatin in C. militaris was developed and validated. The chromatographic separations were obtained by an Agilent C18 column (250x4.6 mm i.d., 5 μm particle size) using gradient elution with acetonitrile and water. The flow rate of 0.6 ml/min and detection wavelength of 280 nm were selected. The calibration curve displayed a good linear correlation (R2>0.99). The validation results displayed great accuracy, repeatability, linearity, and selectivity according to The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline.

Determination of Coenzyme Q10 Content in Food By-Products and Waste by High-Performance Liquid Chromatography Coupled with Diode Array Detection.

Coenzyme Q10 (CoQ10) is a vitamin-like compound found naturally in plant- and animal-derived materials. This study aimed to determine the level of CoQ10 in some food by-products (oil press cakes) and waste (fish meat and chicken hearts) to recover this compound for further use as a dietary supplement. The analytical method involved ultrasonic extraction using 2-propanol, followed by high-performance liquid chromatography with diode array detection (HPLC-DAD). The HPLC-DAD method was validated in terms of linearity and measuring range, limits of detection (LOD) and quantification (LOQ), trueness, and precision. As a result, the calibration curve of CoQ10 was linear over the concentration range of 1-200 µg/mL, with an LOD of 22 µg/mL and an LOQ of 0.65 µg/mL. The CoQ10 content varied from not detected in the hempseed press cake and the fish meat to 84.80 µg/g in the pumpkin press cake and 383.25 µg/g in the lyophilized chicken hearts; very good recovery rates and relative standard deviations (RSDs) were obtained for the pumpkin press cake (100.9-116.0% with RSDs between 0.05-0.2%) and the chicken hearts (99.3-106.9% CH with RSDs between 0.5-0.7%), showing the analytical method's trueness and precision and thus its accuracy. In conclusion, a simple and reliable method for determining CoQ10 levels has been developed here.

Development of the Validated Stability-Indicating Method for the Determination of Vortioxetine in Bulk and Pharmaceutical Formulation by HPLC-DAD, Stress Degradation Kinetics Studies and Detection of Degradation Products by LC-ESI-QTOF-MS.

Vortioxetine (VOR) is a new antidepressant drug used to treat major depressive disorder. In this work, a novel, simple, rapid, accurate, precise, selective, stability-indicating, and fully validated high-performance liquid chromatography method with diode array detection (HPLC-DAD) was developed to determine VOR in bulk and pharmaceutical formulations. A Polar-RP column was used, with a mobile phase consisting of acetonitrile (ACN), methanol (MeOH), acetate buffer pH 3.5, and addition of diethylamine (DEA) in the isocratic elution mode. Assessing the stability of the VOR is fundamental to guarantee the efficacy, safety, and quality of drug products. In this study, the VOR active pharmaceutical ingredient (API) and tablets were subjected to a detailed study of forced degradation, using several degrading agents (acid, alkaline, water, heat, light, and oxidation agents). The developed HPLC-DAD method allows the collection of all the essential data to determine degradation kinetics. It was found that the decomposition of vortioxetine is fragile towards oxidative conditions and photolysis, yielding the first-order and second-order kinetic reaction in the above stress conditions, respectively. The degradation products (DPs) were identified by the high-resolution liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-QTOF-MS) method. The HPLC-DAD method was successfully applied for the quantification of VOR in tablets. Additionally, in silico toxicity prediction of the DPs was performed.

Validation and comparison of analytical methods for the determination of uric acid in pulses and cereals by salting out assisted extraction by Rapid resolution liquid chromatography

An analytical method was developed and validated for the quantitative determination of uric acid in cereals and pulses based on salting-out assisted extraction and subsequent analysis by Rapid Resolution Liquid Chromatography (RRLC). Uric acid is a degradation product of purines, which is an indicator of insect infestation and the state of stored grains and pulses. This study aims to compare and validate a high-performance liquid chromatography method with Diode-array detection (HPLC-DAD) and fluorescence detection (HPLC-FLD) for the estimation of uric acid. Protein precipitation with ammonium sulfate and acetonitrile was used for sample cleanup and pre-treatment. The addition of inorganic ions results in preferential solvation and precipitates proteins. The separation was performed on a Zorbax SB C18 column (150 × 4.6 mm, 5 µm) with an isocratic elution using water-acetonitrile containing 10 mM sodium dihydrogen phosphate (95:5, v/v), at a flow rate of 0.5 mL/min. The relative coefficient (r2) for the calibration curve was more than 0.995 over the concentration range of 25–200 mg/kg. This method's precision at concentrations of 25–150 mg/kg was within 7.25%, and the accuracy was 85.1%–92.7%. The method was validated in terms of the LOD, LOQ, repeatability, reproducibility, linearity, uncertainty, specificity & system suitability. The limit of detection and limit of quantification was 16.60 mg/kg and 50.34 mg/kg, respectively.

Phenolic profiles and antioxidant activities of exocarp, endocarp, and hypanthium of three pear cultivars grown in China

The phenolic composition of the exocarp, hypanthium, and endocarp from three pear cultivars: Jingbai, Korla and Crystal were measured by high-performance liquid chromatography method with diode array detection (HPLC-DAD), while antioxidant activities were investigated using DPPH radical andABTS radical cation scavenging activity, and FRAP. Ferulic acid, chlorogenic acid, and caffeic acid were detected as major components. The exocarp of the Jingbai pear had a relatively high chlorogenic acid content (0.691 mg kg-1) while the hypanthium and endocarp had the high chlorogenic acid content of the three pear varieties. The exocarp of the Korla pear had the highest ferulic acid and rutin content. The exocarp, hypanthium, and endocarp of the Crystal pear had the highest content of chlorogenic acid of the three pear varieties. The Crystal pear presented the highest total phenolic and flavonoid contents and had the highest antioxidant activities in terms of FRAP. The Jingbai pear exocarp presented the highest ABTS value, 83.62 ± 0.08%. The endocarp of the Crystal pear had the highest DPPH value, 87.30 ± 0.03%. Positive correlations were detected among chlorogenic acid, TPC, TFC, and FRAP. The results reveal that the different parts of Pyrus communis L. have effective antioxidant activity for the pharmaceutical application.

Extraction process and method validation for bioactive compounds from Citrus reticulata cv. Chachiensis: Application of response surface methodology and HPLC–DAD

AbstractCitrus reticulata cv. Chachiensis, a traditional Chinese herb, has extensive medicinal and edible effects. 3′,4′,5,6,7,8-Hexamethoxyflavone (HM) and 5,6,7,8,4′-pentamethoxyflavone (PM) are main bioactive compounds in Chachiensis, which have been reported to possess various biological properties. In this study, supercritical CO2 extraction (SCE) and high-speed countercurrent chromatography (HSCCC) were utilized to prepare HM and PM from Chachiensis. The contents of target compounds were determined by a high-performance liquid chromatography method with diode-array detection (HPLC-DAD), which was validated using the following parameters: linearity, sensitivity, repeatability, stability, precision and accuracy. The SCE conditions were optimized using response surface methodology with central composite design. Obtained optimum conditions were temperature of 37.9 °C, pressure of 26.3 MPa, and modifier volume of 81.0 mL. Under above conditions, the recoveries of target compounds were 92.52 ± 0.83 and 96.36 ± 0.43%, respectively. The most appropriate solvent system for HSCCC was selected as n-hexane/ethyl acetate/methanol/water (1:0.8:1:1.2, v/v). The HSCCC fractions were detected by HPLC-DAD, liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (1H NMR and 13C NMR). The results indicated that this method was successfully applied to obtain HM and PM with high purities and high recoveries from Chachiensis.

Chromatographic Separation of Fluoroquinolone Drugs and Drug Degradation Profile Monitoring through Quality-by-Design Concept.

The article reports on the development of an efficient, robust and sensitive HPLC-DAD method for the simultaneous determination of five fluoroquinolone-based antimicrobial drugs, namely ciprofloxacin, moxifloxacin, norfloxacin, ofloxacin and pefloxacin in both aquatic and tablet formulations. The robustness of the high-performance liquid chromatography with diode-array detection (HPLC-DAD) method has been evaluated through the concepts of quality-by-design (QbD) and full factorial design of experiments (DoEs), using a Minitab 17 statistical tool. The proposed method offers sequential separation with well-defined peak shape and resolution, and has also been evaluated by following international council for harmonization (ICH) pharmaceutical guidelines. A linear signal response has been achieved for the target fluoroquinolones (FQ) drugs in the concentration range of 45-20,000ng/mL, with an average correlation coefficient (r2) value of 0.9997, and a data precision and accuracy range of 99.3-100.9%, with an RSD value of ≤0.95%, for hexaplicate measurements. The methodology offers superior sensitivity for the target FQ drugs, with the limit of detection (LD) range of 10-25ng/mL, and the limit of quantification (LQ) range of 51-86ng/mL, respectively. Using the proposed method, the article carries the first of its kind report in studying the degradation profile monitoring and drug assay determination in tablet formulations and under various physiological buffer stress conditions, for pharmaceutical validation.

Development and Validation of HPLC-DAD Method for Simultaneous Determination of Seven Food Additives and Caffeine in Powdered Drinks.

The usage of food additives must respect the general legislation in force in the country and requires a reliable analytical method for surveillance. This research aimed to develop a high-performance liquid chromatography with diode array detection (HPLC-DAD) method for the simultaneous determination of seven food additives and caffeine in powdered drinks. Three factors likely to affect the chromatographic separation, namely, mobile phase composition at the beginning (x1, 0–10% of the amount of methanol in the phosphate buffer) and the end (x2, 60–100% of the amount of methanol in the phosphate buffer) of the gradient program and pH (x3, 3–7), were evaluated with the aid of a Box–Behnken Design (BBD). Subsequently, multi-response optimizations for chromatographic resolutions (Rs) and analysis time were performed using the response surface methodology (RSM) in conjunction with the desirability function (DF). Complete separation (Rs > 1.5) of seven food additives and caffeine was achieved in less than 16 min by applying 8.5% methanol in the phosphate buffer at the beginning and 90% at the end of the gradient program, in pH 6.7. The developed method was validated with low limits of detection (ranging from 1.16 mg kg−1 (sodium saccharin) to 3.00 mg kg−1 (acesulfame potassium)), low limits of quantification (ranging from 3.86 mg kg−1 (sodium saccharin) to 10.02 mg kg−1 (acesulfame potassium)), high precision (CV < 4%), and high accuracy (recoveries from 95 to 101% at 80, 100, and 120% of the target concentration). The method was successfully used to assess the seven food additives and caffeine in commercially available powdered drinks.

Analytical investigation of ternary mixture of phenylephrine hydrochloride, dimetindene maleate and benzalkonium chloride using validated stability indicating HPLC-DAD method

A stability-indicating high performance liquid chromatography method with diode array detection (HPLC-DAD) was developed and validated for simultaneous determination of phenylephrine hydrochloride (PHR), dimetindene maleate (DMD) and benzalkonium chloride (BZM) in nasal drops and gel dosage forms. Effective liquid chromatographic separation was accomplished by employing Venusil XBP Cyano column (4.6 × 250 mm, 5 µm particle size) with gradient elution of the mobile phase consisting of buffer solution of potassium dihydrogen phosphate (0.025 M) and sodium 1-butane sulfonate (SBS) (0.025 M) (adjusted to pH 6.0) and acetonitrile. Peak areas of PHR, DMD and BZM at 271, 256 and 206 nm, respectively were measured and correlated to their concentrations. Peaks of PHR and DMD eluted at retention times 3.76 and 9.06 min, respectively, while BZM eluted as a couple of peaks at 11.88 and 12.51 min. The proposed HPLC procedure was carefully validated in terms of system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection and quantification limits. The linearity range for both PHR and BZM was 10–400 μg/mL and DMD was 5–300 μg/mL with correlation coefficients >0.9999. The studied compounds were subjected to stress conditions of neutral, acidic and alkaline hydrolysis, oxidation and thermal degradation. Good resolution of the three compounds from their forced degradation products proves specificity and stability-indicating merits of the proposed method. In addition, resolution of the three drugs under investigation from some pharmaceutical compounds of different medicinal categories showed the high specificity of the described method.

A novel high-performance liquid chromatography with diode array detector method for the simultaneous quantification of the enzyme-reactivating oximes obidoxime, pralidoxime, and HI-6 in human plasma.

Oximes such as pralidoxime (2-PAM), obidoxime (Obi), and HI-6 are the only currently available therapeutic agents to reactivate inhibited acetylcholinesterase (AChE) in case of intoxications with organophosphorus (OP) compounds. However, each oxime has characteristic agent-dependent reactivating efficacy, and therefore the combined administration of complementary oximes might be a promising approach to improve therapy. Accordingly, a new high-performance liquid chromatography method with diode-array detection (HPLC-DAD) was developed and validated allowing for simultaneous or single quantification of 2-PAM, Obi, and HI-6 in human plasma. Plasma was precipitated using 5% w/v aqueous zinc sulfate solution and subsequently acetonitrile yielding high recoveries of 94.2%-101.0%. An Atlantis T3 column (150 × 2.1mm I.D., 3 μm) was used for chromatographic separation with a total run time of 15 min. Quantification was possible without interferences within a linear range from 0.12 to 120 μg/mL for all oximes. Excellent intra-day (accuracy 91.7%-98.6%, precision 0.5%-4.4%) and inter-day characteristics (accuracy 89.4%-97.4%, precision 0.4%-2.2%) as well as good ruggedness were found. Oximes in processed samples were stable for at least 12 h in the autosampler at 15°C as well as in human plasma for at least four freeze-thaw cycles. Finally, the method was applied to plasma samples of a clinical case of pesticide poisoning.

Phytochemical analysis, antipropulsive and antilymphoma activities of leaves extract of Annona cherimola miller

Background: Diarrhea and cancer are significant health problems worldwide. Annona cherimola Mill. (Annonaceae) is a fruit tree extensively used in Mexican traditional medicine for the treatment of diarrhea and cancer. Objectives: The present work reports the identification of flavonol glycosides present in the ethanol crude extract using high-performance liquid chromatography with diode array detection (HPLC-DAD). Materials and Methods: The identification of flavonoids was made by comparing their retention times and ultraviolet spectra with those standards. The crude extract, dichloromethane, and aqueous residual (AR) fractions were tested for their antipropulsive and antilymphoma activities. Results: The results revealed high levels of three flavonoid glycosides (rutin, nicotiflorin, and narcissin), especially rutin (24.5 mg/g of extract). The antipropulsive and antilymphoma activities of the leaf extract and subsequent fractions showed that the ethanol extract of the leaves of A. cherimola is cursive and AR fraction exhibited the best biological effects in both assays. Conclusion: The HPLC-DAD method used in this work enables the determination of rutin as a major flavonol glycoside of the leaves of A. cherimola . In addition, it can be suggested that rutin may play an important role in the biological properties of A. cherimola.

Determination of carotenoids in flowers and food supplements by HPLC-DAD

AbstractMarigold flowers (Tagetes patulaandCalendula officinalis) were chosen for analysis because they are the most often used source of lutein and its isomer zeaxanthin for the production of food supplements on the Czech market. Direct extraction and extraction with alkaline hydrolysis were compared to detect free or bound carotenoids. For carotenoid separation, C18 and C30 columns were used. A new method for determination of carotenoid content in food supplements in form of capsules has been developed and validated. All matrices were analysed by high-performance liquid chromatography with diode array detection (HPLC-DAD). It has been found that alkaline hydrolysis is required for both Marigold flowers and food supplements to release lutein from ester bonds to fatty acids. InCalendula officinalislutein in the concentration of 807—1472 mg·kg−1of dry matter was detected.Tagetes patulahas been identified as a better lutein source with the content of 5906—8677 mg·kg−1of dry matter. It has been found that the content of lutein and zeaxanthin in commercial food supplements (Lutein Complex PremiumandOccutein Brillant) is consistent with the declared quantity. Linearity of the HPLC-DAD method ranged from 0.1—20 μg·mL−1with the limit of quantification (LOQ) of 1.7 mg·kg−1for lutein in Marigold flowers and 200 mg·kg−1in food supplements. Repeatability was 2.3 % for lutein in all tested matrices.

HPLC-DAD method for simultaneous determination of natural polyphenols

A simple and reliable high-performance liquid chromatography with diode-array detection (HPLC-DAD) method was developed for simultaneous determination of 9 natural substances common in plants: three major catechins ((-) - epicatechin gallate, (-) - catechin, (-) - epigallocatechin), four major flavonoids (rutin, quercetin, myricetin, kaempferol), gallic and vanillic acid. The optimized method was carried out for 40 minutes with detection wavelengths of 278 and 368 nm, gradient elution system on a C18 reversed-phase column. The developed system was evaluated for several validation characteristics, as system suitability, specificity, linearity, limit of detections (LODs) and limit of quantifications (LOQs). The newly established HPLC method was proved to be specific, sensitive, linear and precise. The received results showed good chromatographic separation and assumed that the described method was validated. The HPLC-DAD method can be a useful tool for the quantitative and qualitative evaluation of the selected polyphenol compounds and can be improved in the future for the examination of the polyphenols of interests in individual herbal infusions.

Development and validation of a HPLC-DAD method for simultaneous determination of main potential ABE fermentation inhibitors identified in agro-food waste hydrolysates

Lignocellulosic agro-food wastes are regarded as interesting carbohydrate sources for acetone-butanol-ethanol (ABE) fermentation. However, the physicochemical and enzymatic pretreatments applied to release their sugars generate inhibitory compounds that hinder the fermentation. The release of inhibitory compounds in the hydrolysates of four agro-food industrial wastes [apple pomace (AP), potato peel (PP), brewers' spent grain (BSG) and coffee silverskin (CS)] obtained after various chemical pretreatments (acid, alkali, organic solvents and surfactant pretreatments) was analyzed. Sixty-seven potential inhibitors were identified by gas chromatography–mass spectrometry (GC–MS) and were classified into non-aromatic compounds (aliphatic acids, nitrogen-containing compounds, furans and fatty acids) and aromatic compounds (phenolics and non-phenolics). Then, a high performance liquid chromatography method with diode array detection (HPLC-DAD) was developed and validated for the quantification of the main potential inhibitors identified in the hydrolysates (i.e. gallic, 3,4-dihydroxybenzoic, 2,5-dihydroxibenzoic, 4-hydroxybenzoic, 3-hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric, and ferulic acids, vanillin, syringaldehyde and caffeine). The proposed HPLC-DAD method was simple, fast and robust and allowed the direct injection of samples without previous treatment, enabling the simultaneous quantification of the abovementioned compounds for the first time. The method was successfully applied to the analysis of AP, PP, BSG and CS hydrolysates.

Quantification of Herbal Mixtures Containing Cumyl-PEGACLONE—Is Inhomogeneity Still an Issue?

Synthetic cannabinoid receptor agonizts (SCRAs), also known as synthetic cannabinoids, are mostly consumed in the form of herbal mixtures available in online shops. These herbal mixtures are produced by soaking dried, crushed plant material in a solution of SCRAs or by spraying the solution on the plant material. Inhomogeneity in the distribution of the active ingredient can occur during the production process and pose a serious health risk for consumers of these drugs. In the present study 20 herbal mixtures containing Cumyl-PEGACLONE, one of the most prevalent SCRAs in Germany in 2017, were quantitatively analyzed by high-performance liquid chromatography with diode array detection (HPLC-DAD) after an initial screening by gas chromatography mass spectrometry. All investigated herbal mixtures were purchased in online shops during a systematic product monitoring carried out in the frame of the EU project "SPICE Profiling". The complete content of the packages was divided into aliquots without homogenization and extracted three times with methanol under ultrasonication. The combined extracts of each aliquot were filtered and quantified with a fully validated HPLC-DAD method using a 7-point calibration curve (1-50 μg/mL). The Cumyl-PEGACLONE content in the analyzed material ranged from 8.6 to 146 mg/g (median 29.4 mg/g, mean 38.5 mg/g). The intrapackage concentration variability was mostly below 10% RSD. Analyzed concentrations roughly correlated with product advisory (e.g., "strong") on the websites, if available. Aliquots at the bottom of a package generally tended to show higher levels of Cumyl-PEGACLONE than the upper aliquots. Packages of the same brand with different date of order did not always show the same mean concentrations. Compared to former studies, the SCRA concentrations are generally lower and the risk of extreme variation of intrapackage SCRA contents seems to have dropped.

Determination of cysteamine in animal feeds by high performance liquid chromatography with diode-array detection (HPLC-DAD)

A HPLC-DAD method for the determination of cysteamine supplementation in commercial animal feeds was developed. Samples were extracted with a mixture of 0.5 % hydrochloric acid – acetonitrile (90:10, v/v), matrix interferences were removed with a C18 cartridge, cysteamine was derivatized using 5,5'-dithiobis-(2-nitrobenzoic) acid (DTNB) as Ellman's reagent targeting to the thiol group in the molecule. Quantification of cysteamine was performed on a c18 column with DAD at 323 nm. The developed method had LOD of 1.1 mg/l, good linearity of the calibration curve (R2 ≥ 0.9998), high recoveries (&gt; 92 %), and high reproducibility (RSD &lt; 2.0%).

Determination of 5-hydroxymethylfurfural (5-HMF) in Expired Pharmaceutical Syrups by Using HPLC-DAD Method

The Maillard reaction product 5-hydroxymethylfurfural (5-HMF) is formed under acidic conditions by the dehydration of sugars in carbohydrate-based food and pharmaceutical products during heating and storage. As pharmaceutical syrup formulations contain sugar and are stored under room temperature, they provide favorable conditions for the formation of 5-HMF. The long-term storage of syrup bottles after their cap has been opened and the unintentional use of expired syrups can lead to the formation of undesirable products such as 5-HMF in medications. Although legal limits have been established for 5-HMF content in pharmaceutical preparations, these levels may exceed those limits in hot climates or under inappropriate storage conditions. The present study detects and measures 5-HMF levels in expired pharmaceutical syrups through the HPLC-DAD (High Performance Liquid Chromatography with Diode Array Detection) method, and investigates the effects on 5-HMF levels of the 72-hour storage of syrups at temperatures of 40˚C. The 5-HMF level in syrups stored at room temperature varied between 1.34 μg/mL to 15.63 μg/mL, while in syrups stored at higher temperatures, the levels ranged from 2.24 μg/mL to 18.24 μg/mL. This indicated that 5-HMF content in syrups stored at 40 ˚C was higher than those measured in syrups stored at room temperature, although the increase was not found to be statistically significant (p&amp;gt;0.05). In addition to measuring the amount of 5-HMF in pharmaceutical syrups, this study also examined the changes in the levels of this dehydration product in syrup formulations under hot climates and according to storage conditions.

Development and validation of a new HPLC-DAD method for quantification of sofosbuvir in human serum and its comparison with LC-MS/MS technique: Application to a bioequivalence study.

Although for many analyses tandem mass spectrometry (LC-MS/MS) systems have significant advantage over the high-performance liquid chromatography with diode array detection (HPLC-DAD) however, the HPLC methods are easier, cheaper and more available to perform. As no published method is available for quantitative HPLC analysis of sofosbuvir (SOF), an orally administered anti-hepatitis drug in human serum, this study was aimed to evaluate applicability of the HPLC technique to quantify sofosbuvir and comparison of the two methods for analytical performance. Following extraction of the drug and an internal standard (Hexobarbital), same chromatographic conditions were used for both the systems. After the chromatographic separation on a reverse phase C18 column using a mobile phase consisting of water (containing formic acid 0.5mL/L) and acetonitrile (57:43; v/v) at a flow rate of 0.8mL/min, the eluate was introduced into a DAD detector set at 261nm, then passed through the mass spectrometry system in single ion monitoring mode (SIM). For UV and mass spectrometry detections the calibration curves were linear over a concentration range of 25-3200 and 10-3200ng/mL, respectively and the linearity was over 0.998 for both the systems. Lower limit of quantification (LLOQ) for mass spectrometry and DAD detections were 10 and 25ng/mL, respectively. In conclusion sensitivity of DAD detection is sufficient enough to determine concentrations down to 0.5% of Cmax which achieved in bioequivalence study of sofosbuvir and meet FDA requirements for these types of studies.