LC-MS/MS method for quantitative profiling of ketone bodies, α-keto acids, lactate, pyruvate and their stable isotopically labelled tracers in human plasma: An analytical panel for clinical metabolic kinetics and interactions.

An important area within clinical research is in vivo metabolism of ketone bodies (ß-hydroxybutyrate and acetoacetate) and in connection metabolites that may affect their production and/or cellular transport such as the keto-acids from the branched-chain amino acids, lactate and pyruvate. To determine in vivo metabolite turnover, availability of accurate and sensitive methods for analyzing the plasma concentrations of these metabolites and their stable isotopically labeled enrichments is mandatory. Therefore, the present study describes a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous analysis of ketone bodies, α-keto acids, lactate, pyruvate, and their tracer enrichments in humans using 2 different derivatization techniques with 4-bromo-N-methylbenzylamine and O-benzylhydroxylamine as derivatization reagents, and 1-ethyl-3-dimethylaminopropyl carbodiimide as coupling compound followed by a single LC-MS/MS run. The method was validated for matrix effects, linearity, accuracy, precision, recovery, stability, and enrichment (ratio) analysis of a stable isotopically labelled analytes (tracers) continuously infused in humans divided by the unlabeled endogenous analyte (tracee) that makes it possible to quantify the analyte in vivo synthesis and degradation rates. The applied parallel derivatization procedure yielded good sensitivity for all analytes of interest and their tracers. Despite the double derivatization method, mixing the ethyl acetate portions at the final stage made it possible to simultaneously analyze all compounds in a single LC-MS/MS run. Moreover, the liquid chromatography method was optimized to robustly quantify the keto acids derived from leucine (α-keto-isocaproic acid) and isoleucine (α-keto-ß-methylvaleric acid), the compounds with similar chemical structure and identical molecular weights. The presented method is designed and validated for human plasma. However, care should be taken in blood sampling and processing procedures as well as quick freezing and storage at -80 °C due to the instability of especially acetoacetate.

Pharmaceutical and clinical studies of celecoxib topical hydrogel for management of chemotherapy-induced hand-foot syndrome.

Hand-foot syndrome (HFS) can be categorized as a frequent dose-limiting side effect following administration of chemotherapeutic agents, which needs an effective medication to avoid dose reduction or discontinuation. Oral celecoxib has been proved to be the best pharmacological intervention to ameliorate the skin lesions. However, due to reported gastrointestinal and cardiovascular toxicity following its long-term administration, celecoxib topical application would be a safe alternative for skin disorders. In this work, first, we formulated and optimized a topical hydrogel of celecoxib (1%) and then we investigated its efficacy in the management of chemotherapy-induced HFS in cancer patients. Optimized hydrogel showed acceptable results for drug content, pH, rheology, and stability. Analyzing in vitro drug release study by various mathematical models, the optimized hydrogel showed a zero-order release pattern with 93.27 ± 1.56% cumulative celecoxib release within 8 h. Ex vivo permeation studies across Wistar rat skin indicated suitable skin retention of celecoxib for topical delivery. Twenty-nine patients suffering from HFS were randomized to receive celecoxib and the placebo hydrogels 2 times a day for 3 weeks. At the baseline and at the end of the trial, HFS grades were determined. No serious adverse events occurred in patients who completely followed the instructions. No statistically significant differences between two arms were observed at the baseline (p value = 0.38). By contrast, Wilcoxon signed-rank test showed significant differences when secondary grades (p value = 0.05) and grade differences (p values < 0.001) were analyzed. Overall, the study proved that celecoxib hydrogel could be a promising intervention to manage HFS side effect.

Efficacy of topical Lawsonia inermis L. (Henna) hydrogel in fluorouracil-induced hand-foot syndrome: a pilot randomized double-blind placebo-controlled clinical trial.

PURPOSE: Hand-foot syndrome (HFS) is a frequent dose-limiting adverse reaction of fluoropyrimidine drugs like capecitabine and 5-flourouracil (5-FU) in breast and gastrointestinal cancers. It has been shown that conventional application of Lawsonia inermis L. (Henna) is effective in ameliorating of the skin lesions. To increase the patient compliance, in this study we formulated a standardized topical hydrogel (H.gel) containing the hydroalcoholic extract (10%) of Henna and evaluated its clinical efficacy for the management of fluorouracil associated HFS. MATERIAL AND METHODS: The topical dosage form was standardized based on its Lawsone content. Eighteen patients suffering from HFS were randomized to receive H.gel and the placebo four times a day for 2 weeks. At the baseline and at the end of the trial, HFS grades were determined. RESULTS AND CONCLUSIONS: Allergic reactions following administration of H.gel were observed in one patient, while no serious adverse events occurred in the others. No statistically significant differences between two arms were observed at the baseline (p-value = 0.133), after treatment (p-value = 0.590) and grade differences (p-value = 0.193). The applied hydrogel showed less efficacy compared to the traditional method of using Henna, meaning that Lawsone may not be a good indicator for standardizing the topical dosage form.

Effect of monoterpenes on ex vivo transungual delivery of itraconazole for the management of onychomycosis.

BACKGROUND: Onychomycosis, a fungal nail infection, is an important problem as it may cause local pain, paresthesia, difficulties in performing activities of daily life, and impair social interactions. Systemic treatment of onychomycosis presents safety issues due to possible drug-drug interactions and severe side effects. Although topical therapy of onychomycosis is advantageous due to its localized effect, the efficacy of such therapy depends on achieving effective concentrations of antifungal agents at the infection site. An approach to reach to this end would be driving benefit from synergic activity of antifungal agents for example itraconazole and monoterpenes. However, because of low transungual penetration of itraconazole, a molecule with high molecular weight and very low water-solubility, the effect of the latter compounds on itraconazole nail delivery should be investigated, which was the aim of this study. METHODS: Ex vivo permeation experiments were carried out through soaking the nail clippings of ten healthy volunteers in control and working solutions containing itraconazole (1 mg mL-1 ) and itraconazole (1 mg mL-1 ) plus 6% of each monoterpene including camphor, eucalyptol, menthol, and thymol, respectively, for 36 hours. The amount of itraconazole in nail clippings was quantified hereafter using a validated HPLC method. RESULTS: Statistical analysis showed that itraconazole transungual permeation was not influenced by the studied monoterpenes (P value > .05). CONCLUSION: These results provided a new perspective for designing topical dosage forms for the treatment of onychomycosis.

Development and Validation of A Fast, Simple And Specific Stability Indicating RP-HPLC Method for Determination of Dexpanthenol in Eye Gel Formulation.

In this study a simple and reliable stability-indicating RP-HPLC method was developed and validated for analysis of Dexpanthenol in an artificial tear formulation. The chromatographic separation was performed on a HPLC C18 column (25.0 cm ´4.6 mm, 5 mm) using a mixture of 0.037 M monobasic potassium phosphate in water (adjusted with 0.1% (v/v) phosphoric acid to a pH of 3.2) and methanol (90:10). The flow rate was set at 1.5 mL/min and Dexpanthenol concentration was determined at λmax = 205 nm. The HPLC analysis method was validated in terms of linearity, precision, accuracy, specificity, and sensitivity according to International Conference on Harmonization (ICH) guidelines. The results indicated that the retention time was 8 min and no interferences were observed from the formulation excipients and stress degradation products. Linear regression analysis of data for the calibration plot showed a linear relationship between peak area and concentration over the range of 10 - 100 µg/mL; the regression coefficient was 0.996 and the linear regression equation was y = 20.011 x + 146.83. This HPLC method was precise and accurate in the range of 10 - 100 µg/mL. Also, the dexpanthenol concentration in artificial tear formulation was determined by this HPLC method, which was in accordance with the label claimed. This validated HPLC method could be used for routine analysis, quality control and the stability of analysis of eye gel containing dexpanthenol formulations.

Topical hydrogel containing Fumaria vaillantii Loisel. extract enhances wound healing in rats.

BACKGROUND: Fumaria species (Fumariacea) has traditionally been used in wound healing in Iranian folk medicine. However, with the discovery of newer agents, its use has faded off into total obscurity. This study explored the wound healing potential of a gel containing 10% Fumaria vaillantii Loisel through topical application of total extract in a model of excisional as well as incisional wound healing in albino Wistar rats. METHODS: Rats were anesthetized, and excisional skin wound was established using a sterilized surgical scissors. The animals were then treated with 10% F.vaillantii topical gel formulation along with the gel base. The treatments were administered once a day after the injury for 21 days. For topical treatment, the hydrogel was formulated and evaluated for chemical and physical characteristics. Histopathological analysis with hematoxylin and eosin (H&E) was used for microscopic examination of the skin tissues on 21-day-old sections of excision wound. To verify collagen formation, hydroxyproline determination was performed 21 days post wound healing. Breaking strength was determined in a 10-day-old incision wound by the uniaxial tensile test. RESULTS: Topical administration of F.vaillantii gel formulation significantly enhanced skin wound closure on the 6th post-wounding day compared to both gel base and the negative control, indicating an accelerated wound healing process, while a significant difference was observed on 10th and 14th post -wound days in F.vaillantii treatment compared to the negative control groups. Gel formulation prepared with a 10% F. vaillantii extract exhibited a response in terms of wound epithelialization, angiogenesis and number of hair follicles at wound area better than the gel base on the 21st post-wound day. Application of gel base produced further advantages by increasing hydroxyproline content and collagen fiber thickness. Our results on incision wound model were supported by histopathological data indicating the role of gel base in the enhancement of breaking strength. CONCLUSION: Traditional use of Fumaria species in the skin diseases was justified in this study by revealing the increase in wound healing activity after hydrogel containing F. vaillantii total extract administration.

Effect of CYP2D6 polymorphisms on the pharmacokinetics of propafenone and its two main metabolites.

AIM OF THE STUDY: Propafenone (PPF) is an antiarrhythmic drug, metabolized mainly by CYP2D6 to 5-hydroxypropafenone (5OH-PPF) and by CYP3A4 to norpropafenone (NOR-PPF). CYP2D6 shows a high degree of genetic polymorphism which is associated with diminished antiarrhythmic efficacy or cardiac seizures/cardiotoxicity. This study aimed to investigate the effect of the CYP2D6 polymorphism on the pharmacokinetics of PPF and its two main metabolites. The usefulness of PPF/5OH-PPF ratio for CYP2D6 phenotyping in healthy adults was also evaluated. METHODS: Twelve healthy volunteers, 3 poor metabolizers (PM), 2 intermediate metabolizers (IM) and seven extensive metabolizers (EM) received an oral dose of PPF. Concentrations of PPF and its metabolites were analyzed in serum samples over 27h. RESULTS: The PPF/5OH-PPF ratio distinguished EMs from PMs, but not from IMs. In PMs, the mean transit time (MTT) values were almost the same for PPF and NOR-PPF and much higher than those of EMs and IMs. 5OH-PPF was not detected in EMs. Mean MTT values of 5OH-PPF and NOR-PPF in IMs were 5.27- and 1.52-fold higher than those of EMs. CONCLUSION: A single time point serum PPF-MR approach is a useful tool to identify PMs. CYP2D6 polymorphism significantly affects the pharmacokinetics of PPF and its two metabolites.

Topical Turmeric Microemulgel in the Management of Plaque Psoriasis; A Clinical Evaluation.

Psoriasis is an autoimmune and recurrent chronic inflammatory skin disease. About 1-3% of the world wide populations are affected. The characteristic features are hyperprolifration of keratinocytes leading to redness, thickening and scaling of epidermis followed with itching and appearance of the lesions which in most cases bother the patients medically and psychologically. Psoriasis is symptomatically treated by the range of oral and topical medications, however, major side effects in some cases are associated with them. Based on several studies, Curcuma longa can inhibit several inflammatory enzymes mainly involved in the inflammatory process of Psoriasis. Therefore, we decided to target this well-known herbal agent with fantastic safety profile to be formulated as a novel topical microemulgel. The clinical and therapeutic benefit of this novel topical formulation was evaluated on 34 patients with mild to moderate plaque psoriasis in a randomized, prospective intra-individual, right-left comparative, placebo-controlled, double-blind clinical trial. The Dermatology Life Quality Index (DLQI) Questionnaire and Psoriasis area & severity index (PASI) score as well as photos before and after treatment was used to evaluate the outcomes. The results show that the clinical and quality of life parameters in treated lesions in comparison with untreated lesions have improved (P<0.05). The reported side effects were also recorded and were trivial. Based on our findings, the proposed microemulgel may well be considered as an alternative in some patients and most likely as an add-on therapeutic option for many patients suffering with plaque psoriasis.

Validation of a Stability-Indicating RP-HPLC Method for Determination of l-Carnitine in Tablets.

A rapid and stability-indicating RP-HPLC method was developed for determination of l-carnitine in tablets. The separation was based on a C18 analytical column using a mobile phase which consisted of 0.05 M phosphate buffer (pH = 3): ethanol (99 : 1), including 0.56 mg/mL of sodium 1-heptanesulfonate. Column temperature was set at 50°C and quantitation was achieved by UV detection at 225 nm. In forced degradation studies, the drug was subjected to oxidation, hydrolysis, photolysis, and heat. Among the different stress conditions, the exposure to acidic and basic conditions was found to be an important adverse stability factor. The method was validated for specificity, selectivity, linearity, precision, accuracy, and robustness. The applied procedure was found to be linear in l-carnitine concentration range of 84.74-3389.50 µg/mL (r (2) = 0.9997). Precision was evaluated by replicate analysis in which relative standard deviation (RSD) values for areas were found below 2.0%. The recoveries obtained (100.83%-101.54%) ensured the accuracy of the developed method. The expanded uncertainty (3.14%) of the method was also estimated from method validation data. Accordingly, the proposed validated and rapid procedure was proved to be suitable for routine analyzing and stability studies of l-carnitine in tablets.

Risks to health professionals from hazardous drugs in Iran: a pilot study of understanding of healthcare team to occupational exposure to cytotoxics.

Ongoing concerns exist regarding the dangers inherent when handling cytotoxics, particularly drugs which are in parenteral formulations. On occasions, nurses and medical doctors have been preparing and administrating these drugs in the open spaces of wards in the absence of suitable personal protective equipment (PPE) and safety cabinets. To explore further into the severity of occupational hazards, we conducted our research in order to evaluate the healthcare's understanding of occupational exposure to cytotoxics and occurrence of any side effects. A cross-sectional study using a self-administered questionnaire was distributed amongst oncology nurses in nine specialized cancer centers in Tehran. The questionnaire was based on most reputable international guidelines, aiming to evaluate the attitude, knowledge and safe practices of nurses' handling cytotoxic drugs. The gathered data and reported side effects were compared between "oncology/hematology" and "non-oncology" participants. The majority of nurses from oncology wards were aware of the potential hazards associated with handling of chemotherapy and reported high levels of compliance with the use of PPE during reconstitution of antineoplastic agents. Almost all nurses reported the use of a safety cabinet during preparation, however only 55 % reported that they have annual medical checkups and 45 % reported having received specialized training. This work was also to evaluate the experimental procedures as well as cleaning solutions used to reduce the level exposure. While the level of knowledge about antineoplastic agents is high among nurses, along with the level of PPE use, medical surveillance and employee training seems to be lagging behind.

Validation and uncertainty estimation of an ecofriendly and stability-indicating HPLC method for determination of diltiazem in pharmaceutical preparations.

A green, simple, and stability-indicating RP-HPLC method was developed for the determination of diltiazem in topical preparations. The separation was based on a C18 analytical column using a mobile phase consisted of ethanol: phosphoric acid solution (pH = 2.5) (35 : 65, v/v). Column temperature was set at 50°C and quantitation was achieved with UV detection at 240 nm. In forced degradation studies, the drug was subjected to oxidation, hydrolysis, photolysis, and heat. The method was validated for specificity, selectivity, linearity, precision, accuracy, and robustness. The applied procedure was found to be linear in diltiazem concentration range of 0.5-50 µ g/mL (r (2) = 0.9996). Precision was evaluated by replicate analysis in which % relative standard deviation (RSD) values for areas were found below 2.0. The recoveries obtained (99.25%-101.66%) ensured the accuracy of the developed method. The degradation products as well as the pharmaceutical excipients were well resolved from the pure drug. The expanded uncertainty (5.63%) of the method was also estimated from method validation data. Accordingly, the proposed validated and sustainable procedure was proved to be suitable for routine analyzing and stability studies of diltiazem in pharmaceutical preparations.

Capillary electrophoretic investigation of the enantioselective metabolism of propafenone by human cytochrome P-450 SUPERSOMES: Evidence for atypical kinetics by CYP2D6 and CYP3A4.

An enantioselective CE method was used to identify the ability of CYP450 enzymes and their stereoselectivity in catalyzing the transformation of propafenone (PPF) to 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF). Using in vitro incubations with single CYP450 enzymes (SUPERSOMES), 5OH-PPF is shown to be selectively produced by CYP2D6 and N-dealkylation is demonstrated to be mediated by CYP2D6, CYP3A4, CYP1A2, and CYP1A1. For the elucidation of kinetic aspects of the metabolism with CYP2D6 and CYP3A4, incubations with individual PPF enantiomers and racemic PPF were investigated. With the exception of the dealkylation in presence of R-PPF only, which can be described by the Michaelis-Menten model, all CYP2D6-induced reactions were found to follow autoactivation kinetics. For CYP3A4, all NOR-PPF enantiomer formation rates as function of PPF enantiomer concentration were determined to follow substrate inhibition kinetics. The formation of NOR-PPF by the different enzymes is stereoselective and is reduced significantly when racemic PPF is incubated. Clearance values obtained for CYP3A4 dealkylation are stereoselective whereas those of CYP2D6 hydroxylation are not. This paper reports the first investigation of the PPF hydroxylation and dealkylation kinetics by the CYP2D6 enzyme and represents the first report in which enantioselective CE data provide the complete in vitro kinetics of metabolic steps of a drug.

Validated capillary electrophoresis assay for the simultaneous enantioselective determination of propafenone and its major metabolites in biological samples.

A robust, inexpensive, and fully validated CE method for the simultaneous determination of the enantiomers of propafenone (PPF), 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF) in serum and in in vitro media is described. It is based upon liquid-liquid extraction at alkaline pH followed by analysis of the reconstituted extract by CE in presence of a pH 2.0 running buffer composed of 100 mM sodium phosphate, 19% methanol, and 0.6% highly sulfated beta-CD. For each compound, the S-enantiomers are shown to migrate ahead of their antipodes, and the overall run time is about 30 min. Enantiomer levels between 25 and 1000 ng/mL provide linear calibration graphs, and the LOD for all enantiomers is between 10 and 12 ng/mL. The assay is shown to be suitable for the determination of the enantiomers of PPF and its metabolites in in vitro incubations comprising human liver microsomes or single CYP450 enzymes (SUPERSOMES). Incubations with CYP2D6 SUPERSOMES revealed, for the first time, the simultaneous formation of the enantiomers of 5OH-PPF and NOR-PPF with that enzyme. CE data can be used for the evaluation of the enzymatic N-dealkylation and hydroxylation rates.

Dextromethorphan metabolic phenotyping in an Iranian population.

OBJECTIVE: CYP2D6 polymorphism of drug metabolism represents an important source of interindividual and interethnic variation in drug response. Since this polymorphism has not been studied in an Iranian population, the present study was undertaken. METHODS: Two hundred healthy unrelated Iranian subjects participated in this study. Phenotyping was based on high-performance liquid chromatography determination of the dextromethorphan/total dextrorphan molar ratios as metabolic ratios (MRs) in plasma samples collected at 3 h after oral administration of 30 mg dextromethorphan hydrobromide. Since the dextromethorphan detection limit of 5 ng/ml achieved in the simultaneous assay for dextromethorphan and its metabolites was not adequate to identify intermediate metabolizers (IMs), 80 of 200 samples selected randomly were also assayed using a modified, more sensitive procedure with a dextromethorphan detection limit of 1 ng/ml. RESULTS: Poor and extensive metabolizers (EMs) could be identified distinctly. A 520-fold interindividual variation in dextromethorphan MRs was observed in this study. In contrast to undetectable dextrorphan and hydroxymorphinan concentrations, clearly determinable dextromethorphan concentrations higher than 10 ng/ml were observed in plasma samples of poor metabolizers (PMs). Considering the antimode of 0.3, five (2.5%, 95% confidence interval of 0.34-4.66) volunteers were identified as PMs. Using the more sensitive method, dextromethorphan was quantified in 4 (one PM) of 80 samples. Excluding the PM, a Shapiro-Wilk test indicated a non-normal distribution of MRs (P < 0.01) in the latter population. CONCLUSIONS: From this study it can be concluded that the frequency of PMs in an Iranian population is 2.5% (95% confidence interval of 0.34-4.66). IMs could be identified using dextromethorphan plasma assays with detection limits of at least 1 ng/ml. However, the phenotype-genotype relationships in this respect remain to be established.

A rapid HPLC assay for the simultaneous determination of propafenone and its major metabolites in human serum.

A rapid and specific HPLC method has been developed and validated for the simultaneous determination of propafenone, an antiarrhythmic agent, and its major metabolites in human serum. The sample preparation was a simple deproteinization with a mixture of ZnSO4 and methanol, yielding almost 100% recoveries of three compounds. Separation was developed on a reverse-phase tracer excel C18 column (25 x 0.46 cm i.d., 5 microm), using an acetonitrile-phosphate buffer gradient at a flow rate of 1.7 ml min(-1), and UV detection of 210 nm. The calibration curves were linear (r2 > 0.999) in the concentration range of 10 - 750 ng ml(-1). The lower limit of quantification was 10 ng ml(-1) for all of the compounds studied. The within and between day precisions in the measurement of QC samples at four tested concentrations were in the range of 1.4 - 8.1% and 4.2 - 11.5% RSD, respectively. The developed procedure was applied to assess the pharmacokinetics of propafenone and its major metabolites following administration of a single 300 mg oral dose of propafenone hydrochloride to three healthy male volunteers.

Simple chromatography method for simultaneous determination of dextromethorphan and its main metabolites in human plasma with fluorimetric detection.

Dextromethorphan, the innocuous non-narcotic antitussive agent, is the most widely used probe drug to assess CYP2D6 function both in vivo and in vitro. For this reason a simple and selective high performance liquid chromatography method with fluorimetric detection for simultaneous quantitation of dextromethorphan, and its main metabolites in human plasma was developed and validated. The method involved a simple and rapid protein precipitation protocol, using a mixture of ZnSO(4) and methanol. The analysis was performed on a 3 microm, C(18) Tracer Excel 15 cm x 0.4 cm i.d. column by gradient elution in which Mobile phase A consisted of potassium dihydrogen phosphate buffer (pH = 3, 0.01 M):methanol:tetrahydrofuran (68.5:31:0.5), and mobile phase B consisted of methanol:tetrahydrofuran (93.25:6.75). Linear calibration curves were obtained in the range of 10-500 ng/ml for dextromethorphan, dextrorphan and hydroxymorphinan. The limit of quantitation (LOQ) was 10 ng/ml for each compound. The maximum within and between days precisions were 7.4 and 7.8%, respectively. The accuracies at four different concentration levels ranged from 88.2 to 111.5%. The recoveries were between 88.0 and 108.6%. The assay method was successfully applied to determine dextromethorphan metabolic ratio after an oral dose of 30 mg of dextromethorphan hydrobromide.