Current Pharmaceutical Analysis - Volume 12, Issue 3, 2016

Profens (aryl propionic acid) are non-steroidal anti-inflammatory drugs. These are broadly used for treating swelling, arthritis and fever. This article presents the comprehensive ordinary and enantioselective analyses of the profens by liquid chromatography. Liquid-liquid extraction, solid phase extraction, solid phase micro-extraction and other sample preparation techniques have been discussed. Most widely used high-performance liquid chromatographic columns are of octadecyl silica gels for ordinary analyses and polysaccharides and cyclodextrins for the enantioselective analyses. Many normal and reversed mobile phases have been used. The profens analyses are discussed in the biological and water samples and pharmaceuticals formulations. The optimization strategies, recent trends in the analyses, future challenges and prospectives have also been discussed.

The development and general performance of the PVC membrane sensors for tizanidine HCl (Tz) are evaluated. The method included two ion -pair complexes of tizanidine cation and tetraphenyl borate (TPB) or phosphotungstate (PT) as electroactive sensing materials. The characteristic performance of the two sensors was evaluated according to the IUPAC guidelines, which revealed that both sensors show fast, stable and near-Nernstian response over a relatively wide tizanidine concentration range with detection limits of 3 x 10-6 and 2 x 10-6 M for Tz-TPB (sensor 1), and Tz-PT (sensor 2), respectively over a wide pH range of 3.0 - 7.0. The sensors show good selectivity for tizanidine as compared with different ions. The direct determinations of Tz show an average recovery 97.5 and 98.0% and a mean relative standard deviation of 1 . 7 a n d 1 . 5% at 500.0 μg/ml for sensor 1 and sensor 2, respectively. The investigated sensors have been applied for direct determination of Tz in some pharmaceutical preparations. The proposed methods were applied for the determination of Tz in tablets, and the results are compared with those obtained by spectrophotometric method.

Background: A fast and reliable method for the quantification of ursodeoxycholic acid (UDCA) in hepatic function medicines was developed. Objective: To develop and validate a simple cleanup procedure based on a solid-phase extraction using an OASIS HLB cartridge for extracting ursodeoxycholic acid and its internal standard, dehydrocholic acid, from pharmaceutical formulations. Methods: Resoluble separation of both analytes through isocratic ultra-performance liquid chromatography with a reversed- phase ACQUITY CSH C18 column was carried out within 5 min. Results: The recovery rates of the newly developed procedure were 95 ± 4% for ursodeoxycholic acid and 91 ± 4% for dehydrocholic acid. Using electrospray ionization-ion trap tandem mass spectrometry, the intra- and inter-day precision values were determined to be <4.5%, whereas accuracy ranged from 90.4 to 95.3%. The ursodeoxycholic acid contents in three different samples (two from a soft capsule and one from a tablet) obtained by the established analytical tool were analogous to the ursodeoxycholic acid contents indicated on the labels of the pharmaceuticals. In addition, analysis of ursodeoxycholic acid in human plasma with the newly developed method was validated, revealing the method to be the most sensitive of all reported UDCA bio-analytical tools. Finally, the validated protocol was successfully applied to a comparative bioavailability study of ursodeoxycholic acid tablets. Conclusion: The newly developed UPLC-ESI-MS/MS method is the most sensitive bio-analytical tool available for UDCA analysis. The UPLC-ESI-MS/MS method can be utilized in studies of the pharmacokinetics and bioavailability of bile acids such as UDCA.

Furosemide is a potent high ceiling (loop) diuretic mainly used in the treatment of edematous states associated with cardiac, renal, and hepatic failure, and the treatment of hypertension. Furosemide has been classified as a class IV drug as per the biopharmaceutical classification system (BCS). D-Glucosamine HCl was used to increase the aqueous solubility of furosemide. The objective of the present work is to improve de dissolution profile of furosemide by formation of a physical mixture and a solid dispersion with D-glucosamine HCl. The solid dispersion was prepared by solvent method using acetone/water. The dissolution properties and physicochemical properties of furosemide: D-glucosamine HCl physical mixture and solid dispersion were investigated by dissolution test, X-ray diffractometry, infrared spectrometry, differential scanning calorimetry (DSC), microscopy (SEM) and 35Cl Nuclear Quadrupole Resonance (NQR). The diuretic activity was proved comparing the solid dispersion with pure furosemide. This study shows that the dissolution rate of furosemide can be enhanced considerably by formulating it with D-glucosamine HCl, as a physical mixture or as a solid dispersion although cristallinity was maintained. Solid dispersion and pure furosemide showed significant increase in diuresis in rats as compared to the control group.

Background: Orlistat is commonly as a potent and long-lasting specific inhibitor of gastric and pancreatic lipase. Seven potential process-related impurities and degradation products were discovered in orlistat capsule out of which two were novel. However, no analytical methods reported in the literature could separate all the impurities presented in this work. Therefore, it is necessary to develop a simple, rapid and reliable HPLC method to detect potential impurities in orlistat pharmaceutical dosage forms and identify the novel impurities. Methods and Results: The drug was subjected to the stress conditions of oxidative, acid, base, thermal and photolytic degradation as per International Conference on Harmonization (ICH). A gradient reverse phase highperformance liquid chromatographic (RP-HPLC) method with UV spectrophotometric detection was developed for separating the impurities and degradation products in orlistat capsules. Results showed that the main peak and the degradation products were well resolved. Five degradation impurities were characterized rapidly and two novel impurities were found. Liquid chromatography coupled with electrospray ionization, quadrupole-time of flight mass spectrometry (LC-ESIMS/ Q-TOF) was used to identify the novel impurities. After comparing their retention time and mass spectrometric data with that of available standards and references, initial structural confirmation of these impurities were obtained. Based on the spectrometric data and other assistant experiments, the novel impurities were characterized as (2S, 3R, 5S)-5-((formyl- D-leucyl)oxy)-2-hexyl-3-hydroxyhexadecanoic acid and (2S, 3S, 5S)-5-((formyl-D-leucyl)oxy)-2-hexyl-3- hydroxyhexadecanoic acid. The method was validated for specificity, accuracy, precision, linearity, limit of detection, limit of quantification, and robustness according to the present ICH guidelines. Conclusion: A novel, sensitive and selective gradient reverse phase high-performance liquid chromatographic (RPHPLC) method with UV spectrophotometric detection was developed and validated to detect seven impurities out of which two were novel and not reported in earlier literature. The two novel impurities were identified by LC-ESI-MS/QTOF. The proposed HPLC method was suitable for routine analysis of orlistat in pharmaceutical dosage forms and assessed the stability of samples of orlistat.

Background: Methods reported so far in literature, considered to be less sensitive, uneconomical and time consuming; overall run time more than 10 minutes. Objective: From economic point of view and for the purpose of routine analysis, it was decided to develop a simple, more sensitive, rapid and economical HPLC method for estimation of EM. Method: A HPLC analytical method has been developed for the quantification of eprosartan in bulk drugs and pharmaceutical formulations at 235 nm using HPLC-UV detector. Chromatographic separation was performed on a Waters® C18 column (μBondapakTM5 μm, 150 mm x 3.9 mm i.d). The acetonitrile and potassium dihydrogen orthophosphate buffer (20mM, pH 3) in ratio of 35%: 65% respectively was used as mobile phase; which pumped at a flow rate of 1.2 ml/min. The developed analytical method was validated following ICH guidelines taking linearity, accuracy, precision, sensitivity, selectivity, robustness and stability of method into consideration. Result: The calibration curves were found linear with regression coefficient (r2) of 0.99. The developed method was found to be rapid and sensitive; as the retention time for eprosartan was found to be lesser than 5 min and the LOD and LOQ were found to be 0.014 μg/ml and 0.042 μg/ml, respectively. Additionally, the developed method was successfully applied for the estimation of eprosartan in pharmaceutical formulations (bulk drug, tablet and ultradeformable lipid based formulation). Conclusion: The developed method was found simple, rapid, accurate, and reproducible for the determination of eprosartan in bulk, tablet and in lipid based formulation.

Background: Humans are exposed continuously to a large number of chemicals from various sources and via multiple routes. One of the most common sources is the use of cosmeceuticals like lipsticks and sunscreen which contain significant amount of heavy metals like Lead (Pb) and Cadmium (Cd). Introduction: The present study was undertaken with an objective to investigate the content of heavy metals especially Pb and cadmium Cd from commonly used brands of cosmetic products (lipsticks and sunscreen creams) in India. Methods: Sixty samples of lipsticks (5 colors in 6 brands) and sunscreen creams (3 colors in 10 brands) were chosen for the present investigation, procured from cosmetic shop centre, Moradabad (India). The Pb and Cd concentrations of the formulations were analyzed using atomic absorption spectroscopy. Result: The results indicated that the concentrations of Pb and Cd in the lipstick samples were within the range of 0.12– 4.9 μg/g and 3.48–46.38 μg/g, respectively. In sunscreen creams, the concentration of Pb and Cd were found to be in the range of 0.96–4.50 μg/g and 9.54–22.41 μg/g respectively. The observed concentrations of the heavy metals in the lipsticks samples were lower than that of the sunscreen creams. Conclusion: The significant difference between the average of Pb content in the different brands of the sunscreen creams and the lipsticks was recorded. Therefore, the frequent use of such cosmetics may culminate enhanced absorption of heavy metals, especially Pb and Cd, in the body through absorption of cosmetics over skin surface as well as by swallowing of lipsticks during its application. Pb and Cd as heavy metal may be harmful, if these are present in cosmeceuticals in excess quantity. Therefore, attempts must be made to familiarize the cosmetic users and the public about the harmful consequences of heavy metal present in cosmetics.

Background: In the last years, the toxicity of the waters due to the presence of antibiotics is well studied. Introduction: The Microtox test, based on the measurement of bioluminescence from the bacterium Vibrio fischeri, was applied on samples of river water spiked with several antibiotics to assess acute toxicity. Thirteen antibiotics, presenting different mechanism of action, were selected as the most widely used in anti-infective therapy. Method: Toxicity was evaluated for drug concentrations distributed on six levels ranging from 1.0 - 50.0 μg/ml on one-component solutions and real samples spiked with drug mixtures. Bioluminescence was measured after half-hour and 24 h of incubation. The results were reported as no observable effect concentration, lowest observable effect concentration or half-effective concentration. The light output from the test samples was compared to that of a standard phenol solution as a control. Results: Chlortetracycline, Oxytetracycline, Ofloxacin, Norfloxacin, Oxolinic Acid, Nalidixic Acid and Streptomycin were found to be very toxic after just 30 min of incubation at low concentration levels. Clindamycin, Neomycin, Ampicillin and Amoxicillin induced bioluminescence decrease but below the toxicity limits permitted by law. In contrast, Erythromycin and Sulphamethoxazole showed hormesis, a phenomenon increasing bioluminescence. The mixtures of antibiotics showed toxicity amplified with additive or synergic activity. Conclusion: The bioluminescence test was proved to be effective in measuring toxicity of drugs in environmental waters, highlighting also the difficulties in predicting the toxicity in presence of several drugs or other pollutants.

Background: Given that pharmacopoeia pyrogen tests, rabbit pyrogen test, and limulus amoebocyte lysate (LAL) test have some shortcomings, supplements for pyrogen tests are necessary. Objective: This study aimed to develop a novel monocyte-based pyrogen test. Method: The human promyelocytic leukemia cell line (HL-60), which was selected for its high sensitivity to pyrogens, such as lipopolysaccharide (LPS), zymosan, and lipoteichoic acid (LTA), was evaluated for interleukin- 6 (IL-6) production with IL-6 ELISA kit. The important parameters of the assay were researched, and a novel pyrogen test was developed. The linearity, reproducibility, and accuracy of the assay were verified, and the assay was compared with the LAL test by assessing the response to samples containing pyrogen. Results: HL-60/IL-6 assay was developed as a novel monocyte-based pyrogen test. The linearity between the pyrogen concentration and IL-6 amount was good (R2 > 0.96), and the RSD of the assay was not more than 20%. The recovery of pyrogens in biological products, such as monoclonal antibody, vaccine, and blood products, ranged from 50% to 200%. The pyrogen level detected by the assay was similar to that detected by LAL. Conclusion: The developed HL-60/IL-6 assay was a good supplement to traditional pyrogen tests for the detection of pyrogens, such as LPS, zymosan, and LTA.

Introduction: Predictability of partial least squares regression (PLSR) method is improved by four multivariate signal processing modelling approaches; including genetic algorithm PLS (GAPLS), net analyte processing PLS (NAP-PLS), orthogonal signal correction PLS (OSC-PLS) and direct orthogonal signal correction PLS (DOSC-PLS). The objective of the introduced work is to establish a comparison among proposed chemometric models; indicating the constituent algorithm of each and setting a comparison of analysis results. Method: The proposed models are compared through stability indicating analysis of mixtures of metopimazine (MTP) and its reported degradation products via handling spectrofluorimetric data, at excitation wavelength of 268 nm and working emission range of 420-520 nm. The degradation products include the oxidative degradation product (OMTP) and the alkaline hydrolysis degradation product (AMTP). A 3-factor 5-level experimental design was set; ending up with a training set of 25 mixtures containing different ratios of the interfering components. A test set composed of 12 mixtures was implemented to validate the predictive ability of the proposed models. Leave one out- cross validation procedure (LOO-CV) was used to predict optimum number of PLS components. Result: The 4 introduced models were successfully applied to assay MTP in raw material and the best results were given by GA-PLSR (test set 99.00% ± 2.980). Conclusion: The 4 models were applied to analysis of pharmaceutical tablets, then statistically compared to a reported spectrofluorimetric method; showing no significant difference regarding accuracy and precision; indicating the ability of the suggested models to be trusted for routine quality control analysis of pharmaceutical product.

An accurate and selective high-performance liquid chromatography with ultraviolet detector (HPLC-UV) method was developed for quantification of alibendol in human plasma. The sample was prepared by one-step liquid-liquid extraction (LLE) using ethyl acetate from plasma. The chromatographic retention times of alibendol and carvedilol (the internal standard; IS) were 4.3 and 3.5 min, respectively, on a reverse phase C18 CAPCELL PAK (250 mm x 4.6 mm I.D., 5 μm) column. The isocratic mobile phase consisted of acetonitrile-10 mM sodium phosphate (45:55, v/v; adjusted to pH 3.0 with phosphoric acid). The lower limit of quantitation (LLOQ) was 0.3 μg/mL and no interferences were detected in chromatograms. The developed HPLC method was validated by evaluating its inter- and intra-day precisions and accuracies for a linear concentration range of 0.3 and 20.0 μg/mL. Stability testing showed that alibendol was stable in human plasma during sample processing and storage. The proposed method was successfully applied to the pharmacokinetic study of the single-dose oral administration of 100 mg alibendol tablet in healthy Korean male volunteers. The Cmax, T1/2 and AUC0-t of alibendol were 5.82 ± 1.40 μg/mL, 1.47 ± 0.43 h, 10.62 ± 2.40 μg·h/mL, respectively.

A series of 16 Daphniphyllum alkaloids isolated from Daphniphyllum macropodum were investigated by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESIQTOF- MS/MS) in positive-ion mode. Deuterium labeling experiments were carried out to identify the major product ions. Although compounds have similar structures, the profiles of their MS/MS spectra vary greatly. For compounds 1 and 2, abundant product ions both in high and low mass range can be detected. A six-member H rearrangement and the cleavage of C1-C2 bond connecting rings A, C and D play an important role in providing the product ions in low mass range. For compounds 3-9, the relative abundance of product ions in low mass range is low. This may result from the loss of HCOOCH3 occurring readily because C4-C5 double bond prevents six-member H rearrangement. Main fragmentation patterns of compounds 10-14 are the loss of the different side-chain groups and the cleavage of pyrrol and pyridine rings. A series of ion clusters detected in low mass range are characteristic ions for compounds 10-14. Fragmentation patterns of compounds 15 and 16 were elucidated. Four groups of isomers in these compounds were distinguished by characteristic product ions or fragmentation patterns.

Background: Tamsulosin is used for the treatment of difficult urination, a common symptom of an enlarged prostate. Objective: In the present paper, for the first time a microextraction technique was introduced for the detection and determination of tamsulosin in urine and plasma samples. Method: Procedure for extraction of tamsulosin consists of hollow fiber based liquid phase microextraction (HF-LPME) followed by high performance liquid chromatography (HPLC) coupled with ultraviolet (UV) detection. The organic liquid membrane consists of 1-Octanol immobilized in the pores of a hollow fiber. A pH gradient was applied to migrate analytes from the sample solution with pH 10.5, through the organic liquid membrane into an acidic acceptor solution with pH 3.0 which was located inside the lumen of hollow fiber. Results: Extraction recoveries greater than 80% were obtained in different biological matrices which resulted in preconcentration factors greater than 102 and acceptable repeatability (2.5 < RSD% <3.5). Conclusion: The method offers good linearity with estimation of coefficacy higher than 0.9990. Finally, it was applied for the determination and quantification of tamsulosin in human plasma and urine samples.

Background: Febuxostat is xanthin oxidase inhibitor and Diclofenac Potassium is potent prostaglandin inhibitor. A fixed dose combination of febuxostat and diclofenac potassium is used for the treatment of gout. Objective: The presented work explains the development and validation of simple accurate TLC/Densitometric method for simultaneous estimation of Febuxostat (Feb) and Diclofenac potassium (Dic) in tablet formulation. Method: Precoated silica gel 60F254S TLC aluminium plates were employed as a stationary phase and the eluent cyclohexane- dichlorometane-ethyl acetate and formic acid (8/1/1/0.3, v/v/v/v) was uesd. Densitometric analysis of Feb and Dic was achieved at λmax 275 nm. The method validation was done by robustness, precision and accuracy. Stress degradation of Feb and Dic was carried out under different stress conditions like acid, base, oxidation, photo-degradation and dry heating. Result: This mobile phase system was observed to give compact spots for both Feb and Dic and the Rf values were 0.49±0.02 and 0.69±0.02 for Feb and Dic, respectively. Linear regression analysis data of Feb and Dic showed a good linear relationship in range of 200 –1200 ng/ spot and 500 - 3000 ng/spot, respectively. LOD and LOQ was found to be 15.39 and 46.63ng for Feb and 45.66 and 138.37ng for Dic. Conclusion: Statistical analysis confirmed that the developed method was repeatable and selective for the estimation of Feb and Dic in bulk, in formulation and in presence of their degradation products. As the method could efficiently separate the drug from its degradation products, it could be used as a stability- indicating method.