Determination of Curcuminoids, Piperine, Boswellic Acids and Andrographolides in Food and Dietary Supplements by HPLC.

Research background: As use of functional food and herbal combination products is ever increasing, methods for quality control of such preparations are necessary. Moreover, low quality of products can cause either lack of benefit or harm to the consumer. In this work, determination of three curcuminoids, piperine, six boswellic acids and three andrographolides, often used in combination products, was carried out in raw materials and dietary supplements. Experimental approach: After extraction optimization using Box-Behnken design, maximum active substance yields were obtained using 81.5% ethanol in hydroethanolic extraction solvent, 30 min sonication time and 60 °C extraction temperature. Afterwards, a high-performance liquid chromatography method was developed and validated, with special attention paid to selectivity, precision and robustness of the method. Lastly, 54 food and dietary supplement samples were analyzed. Results and conclusions: Most products were bought locally, from credible vendors and they all complied with relevant regulatory requirements. However, products obtained on the Internet contained little to no active substances (24% of samples contained less than 20% declared content), presumably showing no efficacy, or were either found to be likely adulterated or contained very high amounts of active substances, compromising safety in terms of dose-dependent adverse effects (one sample containing andrographolides) and pharmacokinetic interactions (one sample containing piperine). In conclusion, consumers should refrain from purchasing such products from the Internet and obtain them only from verified suppliers such as local pharmacies or health stores. Novelty and scientific contribution: This work demonstrates the first developed method for the analysis of aforementioned combination products, which are on the rise today. The method is simple and robust and can be adapted by most laboratories for routine quality control of the said products. Moreover, the work sheds light on the low quality of several products and signifies the need for increased consumer awareness of dangers of taking such products.

Compatibility investigation for a new antituberculotic fixed dose combination with an adequate drug delivery.

The compatibility of formulation components is crucial for safe and high-quality medicines. To detect the potential for incompatibility and to assess formulation stability, it is beneficial to conduct a compatibility study during the drug development phase. The therapy of tuberculosis normally consists of two or more medicines taken together. Consequently, different antituberculotic fixed-dose combination (FDC) formulations have been developed. Isoniazid is first-line medicine and present in several FDCs. Low bioavailability due to the active substances' incompatibility in acidic medium was reported for some of these FDC forms. Rifabutin, also a first-line antituberculotic, is available in the market as a single component formulation. This study presents compatibility testing of these two active substances for a new FDC and evaluates the impact of the most common solid dosage forms' excipients on the stability of two active substances. The potential for interaction between the formulation components was analyzed by the UHPLC method. One degradation product and one interaction product were observed and further characterized by high-resolution mass spectrometry. Still, significant degradation of two active substances, such as reported in marketed FDC formulations was not detected for this combination. The stability and drug delivery of the proposed combination were confirmed by the dissolution test in acidic medium.

Lipophilicity and bio-mimetic properties determination of phytoestrogens using ultra-high-performance liquid chromatography.

This paper presents lipophilicity and bio-mimetic property determination of 15 phytoestrogens, namely biochanin A, daidzein, formononetin, genistein, genistein-4,7-dimethylether, prunetin, 3,4,7-trihydroxyisoflavon, 4,6,7-trihydroxyisoflavon, 4,6,7-trimethoxyisoflavon, daidzin, genistin, ononin, sissotrin, coumestrol and coumestrol dimethylether. High-performance liquid chromatography with fast gradient elution and Caco-2 cell line were used to determine the physicochemical properties of selected phytoestrogens. Lipophilicity was determined on octadecyl-sylane stationary phase using pH 2.0 and pH 7.4 buffers. Immobilized artificial membrane chromatography was used for prediction of interaction with biological membranes. Protein binding was measured on human serum albumin and α-1-acid-glycoprotein (AGP) stationary phases. Caco-2 assay was used as a gold standard for assessing in vitro permeability. The obtained results differentiate phytoestrogens according to their structure where aglycones show significantly higher lipophilicity, immobilized artificial membrane partitioning, AGP binding and Caco-2 permeability compared with glucosides. However, human serum albumin binding was very high for all investigated compounds. Furthermore, a good correlation between experimentally obtained chromatographic parameters and in silico prediction was obtained for lipophilicity and human serum albumin binding, while the somewhat greater difference was obtained for AGP binding and Caco-2 permeability.

Pharmacokinetic Profiling and Simultaneous Determination of Thiopurine Immunosuppressants and Folic Acid by Chromatographic Methods.

With the increase in the number of medicines patients have to take, there has been a rapid rise of fixed-dose combinations (FDCs) in the last two decades. Prior to FDC development, pharmacokinetic properties of active pharmaceutical ingredients (APIs) have to be evaluated, as well as methods for their determination developed. So as to increase patient compliance in inflammatory bowel disease, three novel FDCs of thiopurine immunosuppressants and folic acid are proposed; physico-chemical and pharmacokinetic properties such as hydrophobicity, lipophilicity and plasma protein binding of all APIs are evaluated. Moreover, experimental results of different properties are compared to those computed by various on-line prediction platforms so as to evaluate the viability of the in silico approach. A simultaneous method for their determination is developed, optimized, validated and applied to commercial tablet formulations. The method has shown to be fast, selective, accurate and precise, showing potential for reliable determination of API content in proposed FDCs during its development.

Rapid Electroanalytical Method for Determination of Nebivolol at a Boron-Doped Diamond Electrode.

A boron-doped diamond electrode provided a sensitive and cost-effective sensing platform for detection and quantitative determination of novel beta(1)-adrenergic receptor antagonist nebivolol. The net square-wave voltammetric response at 1.31 V related to the oxidation of nebivolol was obtained in Britton-Robinson buffer solution at pH 8. It increased linearly with the drug concentration in the range of 2.5×10(-7) to 1.5×10(-5) M. The LOD attained was 3.2×10(-8) M. The practical analytical approach was illustrated by high speed quantification of nebivolol in a commercial pharmaceutical formulation. The RP-HPLC was selected as a comparative method for evaluating the proposed electroanalytical method. The newly developed method at the unmodified electrode surface was faster and simpler in comparison with HPLC (the retention time was 17.6 min), and only 6 s was necessary for direct voltammetric measurement in the potential range from 0.5 to 1.7 V with a 2 mV potential step and pulse frequency of 100 Hz.

Blackberry wines mineral and heavy metal content determination after dry ashing: multivariate data analysis as a tool for fruit wine quality control.

This study brings out the data on the content of 21 mineral and heavy metal in 15 blackberry wines made of conventionally and organically grown blackberries. The objective of this study was to classify the blackberry wine samples based on their mineral composition and the applied cultivation method of the starting raw material by using chemometric analysis. The metal content of Croatian blackberry wine samples was determined by AAS after dry ashing. The comparison between an organic and conventional group of investigated blackberry wines showed statistically significant difference in concentrations of Si and Li, where the organic group contained higher concentrations of these compounds. According to multivariate data analysis, the model based on the original metal content data set finally included seven original variables (K, Fe, Mn, Cu, Ba, Cd and Cr) and gave a satisfactory separation of two applied cultivation methods of the starting raw material.

Phenolic content and antioxidant activities of burr parsley (Caucalis platycarpos L.).

Since C. platycarpos contains a wide variety of antioxidants, in the present study total flavonoid and phenolic acid content as well as antioxidative activity of various C. platycarpos extracts were investigated. The results obtained show a significant polyphenol content and antioxidant activity of the investigated plant. Moreover, a positive correlation between antioxidant activity and content of flavonoids and phenolic acids was found, indicating the responsibility of these compounds for the antioxidant effectiveness of C. platycarpos extracts and making C. platycarpos a good potential source of natural antioxidants.

Size exclusion chromatography as green support for forced degradation study of adalimumab.

Size exclusion chromatography (SEC) has become a powerful tool for analysing size variants of biologic drugs in their native form. Modern SEC can be defined by the use of chromatographic columns packed with sub-3 µm particles, allowing an increase in method throughput compared to that of conventional SEC. We performed the forced degradation study of adalimumab, the first genetically engineered fully humanised immunoglobulin G1 monoclonal antibody, and evaluated tha possibilities of an advanced SEC column packed with sub-3 µm particles for elucidation of the degradation pathway. Our results revealed the main adalimumab degradation products to be antibody fragments. Acidic and basic conditions had the most intensive effect on the degradation of the adalimumab while the drug exhibits relative stability under thermal and photolytic stress conditions. The AGREE and AGREEprep calculators were used for the evaluation of the environmental performance of the forced degradation procedure. The results of the green score evaluation are presented as round pictograms with a circle in the centre that shows the overall score of 0.81 and 0.61, respectively. Both pictograms are highlighted in green, indicating the eco-friendly conditions.

Assessment of Physicochemical Parameters and Contaminants in Herbal Dietary Supplements Used in the Treatment of Inflammatory Bowel Disease.

Inflammatory bowel disease is a complex disorder characterized by chronic gastrointestinal inflammation. Thus, patients prefer to use herbal dietary supplements containing turmeric, Indian frankincense, green chiretta, and black pepper in an attempt to cope better with their chronic condition. The dietary supplements' dosage forms and herbal ingredients were assessed in terms of the products' physicochemical parameters (weight uniformity, friability, disintegration, rupture test, tablet's breaking force, and powder flowability) in view of the USP-NF requirements. In addition, contaminants such as organic solvents and ethylene oxide were evaluated using gas chromatography. Assessment of gluten via an Enzyme-Linked Immunosorbent Assay was also performed. Most of the products met USP requirements. The high average weight of one multicomponent tablet sample with a high breaking force value can explain the observed negative results of the disintegration test. A total of 26% of samples tested positive for gluten, but the most alarming fact is that the ethylene oxide levels found in two samples were up to 30 times higher than the EU limit. Accordingly, dietary supplement quality control is of fundamental importance.

Oleuropein in olive leaf, branch, and stem extracts: stability and biological activity in human cervical carcinoma and melanoma cells.

Olive leaves as a main byproduct of olive oil and fruit industry are a valuable source of phytochemicals such as polyphenols, with multiple biomedical effects. Apart from leaves, olive branches and stems make up a significant amount of olive waste. It is well known that the drying process and long-term storage affect the stability and concentration of polyphenols present in raw materials. For that matter, two different means of storing olive waste, at room temperature and +4 °C, were compared by determining the content of the polyphenol oleuropein (OLE) in olive leaf, branch, and stem extracts (LE, BE, and SE) by HPLC-DAD method. Total phenols (TPC), o-diphenols (o-DPC), and total flavonoids (TFC) content in extracts were assessed by UV-Vis measurements. LE prepared from leaves stored at +4 °C had the highest OLE content, 30.7 mg g-1 of dry extract (DE). SE from stems stored at +4 °C was the richest in TPC and TFC (193 mg GAE/g DE and 82.9 mg CE/g DE, respectively), due to the higher purity of the extract. The biological activity of extracts was determined on cervical cancer (HeLa), melanoma (A375), metastatic melanoma (A375M) tumor cell lines, and on spontaneously immortalized cell line of keratinocytes (HaCaT), using the MTT assay. The data show that all extracts had a similar dose-dependent effect on cell viability in HeLa cells, while the effect of LE on melanoma A375 and A375M, and HaCaT cells was cell-line dependent.

Simultaneous analysis of mitotane and its main metabolites in human blood and urine samples by SPE-HPLC technique.

Adrenocortical carcinoma (ACC) is a rare malignancy with an incompletely understood pathogenesis and a poor prognosis. The adrenalytic activity of mitotane has made it the most important single drug in the treatment of ACC. Unfortunately, the exact mechanism of mitotane action is still unknown. It is believed that mitotane belongs to the class of drugs that require metabolic transformation by cytochrome P450 for therapeutic action; therefore determination of plasma levels of not only mitotane but also its metabolites would help in carrying out the treatment. The objective of this work was to develop and validate an SPE-HPLC method for simultaneous determination of mitotane and its metabolites in different biological fluids. The sample preparation consisted of a solid-phase extraction on a Discovery DSC(18) cartridge, while analysis of extracts was performed on a Symmetry C(18) column. The usefulness of the proposed method was confirmed by analysis of plasma, red cell and urine samples from patient chronically treated with 1.5 g of mitotane. The patient involved in this study had a high plasma concentration of mitotane and none of the investigated metabolites were found. In order to investigate whether the polymorphism of CYP2C9 and CYP2C19 enzymes could be related to the metabolism of mitotane, RT-PCR analysis was performed.

Antioxidant Activity of Pharmaceuticals: Predictive QSAR Modeling for Potential Therapeutic Strategy.

Since oxidative stress has been linked to several pathological conditions and diseases, drugs with additional antioxidant activity can be beneficial in the treatment of these diseases. Therefore, this study takes a new look at the antioxidant activity of frequently prescribed drugs using the HPLC-DPPH method. The antioxidative activity expressed as the TEAC value of 82 drugs was successfully determined and is discussed in this work. Using the obtained values, the QSAR model was developed to predict the TEAC based on the selected molecular descriptors. The results of QSAR modeling showed that four- and seven-variable models had the best potential for TEAC prediction. Looking at the statistical parameters of each model, the four-variable model was superior to seven-variable. The final model showed good predicting power (r = 0.927) considering the selected descriptors, implying that it can be used as a fast and economically acceptable evaluation of antioxidative activity. The advantage of such model is its ability to predict the antioxidative activity of a drug regardless of its structural diversity or therapeutic classification.

Accessing Lipophilicity and Biomimetic Chromatography Profile of Biologically Active Ingredients of Botanicals Used in the Treatment of Inflammatory Bowel Disease.

In the present study, various procedures have been compared for the determination of lipophilicity, hydrophobicity, and plasma protein binding of curcuminoids, boswellic acids, andrographolides, and piperine as biologically active ingredients of botanicals used in IBD treatment. Our results have shown that IAM-HPLC assay is the most suitable one for lipophilicity determination of all analytes regardless of their class and botanical source. HSA-HPAC and AGP-HPAC assays revealed that all investigated compounds have a higher affinity for HSA which is the most abundant protein in human plasma. The high affinity of biologically active compounds to all biological structures (phospholipids and proteins) admonishes that their small portion is available for therapeutic effects in IBD patients. Our experimental research is complemented by various theoretical approaches based on different algorithms for pharmacokinetic properties prediction. The similarities between experimental and calculated values were evaluated using PCA and CA as a statistical tool. The statistical analysis implies that plasma protein binding is a complex process, and theoretical approaches still cannot fully replace experimental ones.

Polyphenol content and antioxidant activity of phytoestrogen containing food and dietary supplements: DPPH free radical scavenging activity by HPLC.

Soy, red clover, chaste tree, hop and flax have all been found to contain a wide range of phytoestrogenic compounds, and a large number of dietary supplements contain their extracts as principal ingredients. This study is aimed to evaluate the total polyphenolic content and antioxidant activity of phytoestrogen-containing food and formulated dietary supplements. The HPLC-DPPH method was applied for DPPH free radical scavenging activity testing of various phytoestrogen-containing samples. Polyphenol content and antioxidant activity in dietary supplements were higher than in functional food samples; multiple-botanical-source preparations showed higher polyphenol content and antioxidant activity than the mono-botanical counterparts. Furthermore, the correlation between polyphenol content and anti-oxidant activity was strongly statistically significant, so it might be concluded that antioxidant activity is proportional to the content of these secondary metabolites. The most striking batch-to-batch deviations were represented by one chaste berry-based product (RSD 41.3 %) and one red clover derived product (RSD 57.9 %). The results of this study contribute to a better understanding of the phenolic profile and antioxidant properties of phytoestrogen containing food and dietary supplements.

A Comprehensive Approach to Compatibility Testing Using Chromatographic, Thermal and Spectroscopic Techniques: Evaluation of Potential for a Monolayer Fixed-Dose Combination of 6-Mercaptopurine and Folic Acid.

In this work, a systematical compatibility investigation of 6-mercaptopurine and folic acid, two commonly used medications in the treatment of inflammatory bowel disease, for the needs of a fixed-dose combination development strategy is shown. Various techniques and approaches, such as differential scanning calorimetry, isothermal stress testing, attenuated total reflectance-Fourier-transform infrared spectroscopy, dissolution medium stability and forced degradation studies, were used to elucidate the possible interactions from different aspects. The results predominantly point to the absence of physicochemical interactions between the examined substances in a variety of possible conditions. However, the forced degradation of the blend of substances and excipients in basic conditions showed a drastic degradation of 6-mercaptopurine, signifying that attention needs to be directed to the careful selection of the excipients for the formulation. To sum up, our findings indicate that a fixed-dose combination of 6-mercaptopurine and folic acid could be produced using one formulation blend, immensely simplifying its manufacture.

Development of a HPLC-DAD stability-indicating method and compatibility study of azathioprine and folic acid as a prerequisite for a monolayer fixed-dose combination.

Adherence in chronic diseases is a major problem which can be combated by prescribing fixed-dose combinations in the therapy of the disease. Thus, a combination of azathioprine and folic acid in the treatment of inflammatory bowel disease is highly required, but prior to formulation development, chemical compatibility of the two drugs needs to be investigated. In this work, differential scanning calorimetry, isothermal stress testing, in vitro dissolution and forced degradation studies were utilized to investigate compatibility. Moreover, a stability-indicating HPLC-DAD method for the determination of parent drugs and five of their impurities was developed, validated and applied to the in-house sample. Compatibility testing revealed no noteworthy interactions of the two drug substances. Furthermore, forced degradation showed no substantial differences between the degradation profiles of each active pharmaceutical ingredient, their mixture and the in-house sample, further reinforcing the claim of compatibility. Lastly, the in-house sample was analyzed: it was shown to conform to the requirements of relevant regulatory documents for all the investigated analytes, demonstrating the method's viability for use in formulation and process development. Our results give way to the possibility of realization of said fixed-dose combination.

Drug-Drug Compatibility Evaluation of Sulfasalazine and Folic Acid for Fixed-Dose Combination Development Using Various Analytical Tools.

The simultaneous administration of sulfasalazine and folic acid is regular practice in the therapy of inflammatory bowel diseases in order to maintain sufficient folate concentration in patients. Having multiple drugs in the therapy increases the possibility of patients failing adherence, thus unintentionally endangering their health. A fixed-dose combination of sulfasalazine and folic would simplify the classical polytherapeutic approach; however, the physicochemical compatibility investigation of two active pharmaceutical ingredients plays an important role in the development of such a product. In this work, various analytical tools were used to determine the physicochemical compatibility of sulfasalazine and folic acid. For the evaluation of chemical compatibility, infrared spectroscopy in combination with advanced statistical methods, such as the principal component analysis and cluster analysis, were used, whilst a simultaneous thermogravimetry/differential thermal analysis gave us an insight into the physical compatibility of two drugs. Isothermal stress testing, forced degradation and dissolution studies, followed by the analysis with a developed chromatographic method for the monitoring of folic acid, sulfasalazine and two of its related impurities, sulfapyridine and salicylic acid, gave us an insight into its chemical compatibility. The combination of the results obtained from the used techniques implies a satisfactory physicochemical compatibility between sulfasalazine and folic acid, which opens the path to the development of the proposed fixed-dose combination.

Physicochemical Compatibility Investigation of Mesalazine and Folic Acid Using Chromatographic and Thermoanalytical Techniques.

Inflammatory bowel disease is a common name for Crohn's disease and ulcerative colitis. These inflammatory states cause damage in the sidewalls of the gastrointestinal tract, resulting in malabsorption of food and vitamins. Folic acid (Vitamin B9) is often associated with inflammatory bowel diseases since reduced overall folate concentration in the human body may lead to the development of colorectal cancer and megaloblastic anaemia. However, its deficiency is easily compensated by taking an additional folic acid pill during regular therapy. At the moment, there are no studies that have examined the compatibility of folic acid with 5-aminosalicylate drugs used in the treatment of inflammatory bowel diseases. In this work, differential scanning calorimetry, forced degradation studies, isothermal stress testing and dissolution stability testing were used to determine the stability of folic acid and one of the most commonly used 5-aminosalicylates, mesalazine, when present in the same solution or blend. To monitor the assay of folic acid, mesalazine and nine of its related impurities, a single HPLC method was developed. Results of compatibility studies showed that no physicochemical interaction between mesalazine and folic acid occurs when combined, opening the path to the development of new formulations, such as a mesalazine/folic acid fixed-dose combination.

Assessment of Bioactive Phenolic Compounds and Antioxidant Activity of Blackberry Wines.

Blackberry wine is a natural source of bioactive phenolic compounds that have profound antioxidant potential. The objectives of the present research were to assess the phenolic compounds and antioxidant activity of blackberry wines (BW), and to use the chemometric analysis to differentiate among the two groups of samples, i.e., conventional and organic. Fifteen BW samples were analyzed for their total polyphenol index, total polyphenols, total flavonoids, total tannins, total monomeric anthocyanins and antioxidant activity by the appropriate spectrophotometric methods. The concentrations of individual phenolic acids (gallic acid, chlorogenic acid, caffeic acid, p-coumaric acid and cinnamic acid) and trans-resveratrol were determined by high-performance liquid chromatography. A comparison between the two groups of investigated BW samples revealed a statistically significant difference in the concentration of caffeic acid and p-coumaric acid, both being higher in the organic BW samples. Furthermore, the results showed a series of statistically highly significant relationships between the analyzed constituents (caffeic acid and p-coumaric acid). The antioxidant activity of the investigated wines was proportional to the concentrations of bioactive phytochemicals.

A chromatographic approach to development of 5-aminosalicylate/folic acid fixed-dose combinations for treatment of Crohn's disease and ulcerative colitis.

Medication adherence is an important factor in inflammatory bowel disease therapy, which includes regular supplementation of malabsorbed vitamins. Absorption of folic acid is limited due to the damaging of the gastrointestinal tract, which can increase the chances to develop megaloblastic anaemia and colorectal cancer. In this work, 5-aminosalicylates (mesalazine, balsalazide, sulfasalazine and olsalazine) and folic acid were characterized regarding their pharmacokinetic related properties (hydrophobicity, phospholipid and plasma protein binding) using the biomimetic chromatographic approach. Despite the high binding percentage of 5-aminosalicylates for human serum albumin (> 61.44%), results have shown that folic acid binding to human serum albumin protein is far greater (69.40%) compared to α1-acid-glycoprotein (3.45%). Frontal analysis and zonal elution studies were conducted to provide an insight into the binding of folic acid to human serum albumin and potential competition with 5-aminosalicylates. The analytical method for the simultaneous determination of assay in proposed fixed-dose combinations was developed and validated according to ICH Q2 (R1) and FDA method validation guidelines. Separation of all compounds was achieved within 16 min with satisfactory resolution (Rs > 3.67) using the XBridge Phenyl column (150 × 4.6 mm, 3.5 µm). High linearity (r > 0.9997) and precision (RSD < 2.29%) was obtained, whilst all recoveries were within the regulatory defined range by British (100.0 ± 5.0%) and United States Pharmacopeia (100.0 ± 10.0%).