Current Pharmaceutical Analysis - Volume 8, Issue 4, 2012

A simple reverse-phase (RP) high performance liquid chromatography (HPLC) method was developed and validated, according to the International Harmonisation Guidelines (ICH), for the determination of risperidone (RISP) from solid lipid nanoparticles (SLN). Chromatographic runs were performed on a RP-C18 column, using an isocratic mobile phase of methanol, acetate buffer (0.05 M; pH 4.6) and triethylamine (60:40:0.02, v/v/v). The flow rate was 1 ml/min, the run time was 10 min and the RISP absorbance was measured at 280 nm, using UV detection. A linear response was obtained for a RISP concentration range of 0.25 - 10.00 μg/ml (R2 = 0.9996), with a detection and quantification limits of 0.011 and 0.034 μg/ml, respectively. The method was shown to be specific, precise at the intra-day (RSD < 0.796%) and inter-day (RSD < 0.331%) levels, and accurate with recoveries between 86.86 - 100.3% (RSD < 0.613%). Method robustness was observed as well. The suitability of the method for RISP quantifications was assessed by the determination of encapsulation parameters (encapsulation efficiency and drug loading) and by studying the RISP release profile from SLN. Kinetic models (zero order, Higuchi, Korsmeyer-Peppas and Baker-Lonsdale) were used to fit the obtained release profile and to predict the in vivo performance of RISP-loaded SLN. A combined pattern of diffusion and erosion release mechanism (anomalous non-Fickian transport) was found for the RISP-loaded SLN, which shows the ability of the system for controlled drug release.

Cocaine and crack usage is a major public health concern worldwide, mainly when the target consumers are pregnant women. The purpose of this study was to develop and validate a cost-effective method for identification and quantification of cocaine, benzoylecgonine, cocaethylene, and anhydroecgonine methyl ester in meconium samples, using solid-phase extraction (SPE) and GC/MS (ion trap – full scan mode) to be employed in public hospitals in Brazil. The method was validated in the 20 - 1000 ng/g range for cocaine and cocaethylene, 40 - 1500 ng/g for benzoylecgonine and 60 - 1500 ng/g for anhydroecgonine methyl ester, using 0.5 g meconium per assay. The detector response was linear in the studied range, and the limit of detection was found to be 10 ng/g for cocaine and cocaethylene, 30 ng/g for benzoylecgonine, and 40 ng/g for anhydroecgonine methyl ester. Intra-batch coefficients of variation oscillated between 3.01% and 10.15% and inter-batch coefficients varied between 5.31% and 11.12%; accuracy was in 91.47% - 105.31% range. Recoveries were higher than 56.30%. Finally, the method was applied to meconium analysis from 20 newborns at the Hospital Clinicas da Faculdade de Medicina de Ribeirao Preto, and it identified 6 positive samples.

In this paper, a rapid and effective chromatographic procedure for determining the curcumin encapsulation efficiency in poly(lactic-co-glycolic acid) (PLGA) and poly(lactic-co-glycolic acid)-polyethyleneglycol (PLGA-PEG) nanoparticles via reversed-phase high-performance liquid chromatography (RP-HPLC) using a fluorescence detector and low flow rate is described. Chromatographic runs were performed on a RP C18 column (250 mm x 4.6 mm, 5 μm) with a mixture of ethanol, water and acetonitrile (80:10:10, v/v/v) as the mobile phase and a flow rate of 0.8 mL/min in the isocratic mode. Curcumin was detected using a fluorescence detector operating at an excitation wavelength of 365 nm and an emission wavelength of 512 nm. This method was validated in terms of the selectivity, linearity, precision, accuracy, robustness, limit of detection and limit of quantitation. The analytical curve was linear over the concentration range of 1– 50 μg/mL, and the limits of detection and quantitation were 9.65 and 50 ng/ml, respectively. The mean recovery for curcumin was 101.14 ± 2.8% (n = 9). The intra- and inter-assay coefficients of variation were less than 3.73%. The method was robust for changes in the mobile phase, column temperature and flow rate. The maximum relative standard deviation was 3.08%. The method was successfully used to determine the encapsulation efficiency of curcumin in PLGA and PLGA-PEG nanoparticles.

The construction and response characteristics of sensors based on polyvinyl chloride used for the assay of ciprofloxacin (CFX) were described. The electroactive compound was the complex between ciprofloxacin and tetraphenylborate. The wider linear concentration range 2x10-2 - 1.97x10-5mol/L was achieved when dibutylphthalate (DBP) and bis(2-ethylhexyl)sebacate (DES) were used as plasticizers, with slopes of 29.12 and 29.55 mV per decade of concentration, respectively, and detection limits of 3.687x10-6 and 7.362x10-6 mol/L, respectively. The optimized sensor had a fast response (less than 5s), with good selectivities recorded versus some inorganic and organic compounds. The sensor was successfully used for the analysis of ciprofloxacin in pharmaceutical formulation with the recoveries higher than 97.66 %, by using a direct potentiometric method which does not require tedious sample preparation.

Electrochemical behavior of zofenopril (ZOF) was studied via experimental electrochemical methods and theoretical calculations performed at B3LYP/6-31+G(d)//AM1 level. Optimum conditions for quantitative determination were investigated by several electrochemical methods such as cyclic voltammetry, square-wave voltammetry and bulk electrolysis. Electrochemical parameters like charge transfer, diffusion and surface coverage coefficients of adsorbed molecules and also number of electrons transferred in electrode mechanisms were calculated. All studies were based on the irreversible and adsorption-controlled electrochemical oxidation signal of ZOF at about 1.1 V versus Ag/AgCl at pH 5.0 in Britton-Robinson buffer (BR). This adsorptive character of ZOF was used to develop fully validated, new, rapid, selective and simple voltammetric methods for the direct determination of the molecule in pharmaceutical dosage forms and biological samples without time-consuming steps prior to drug analysis. Peak current of electrochemical oxidation of ZOF was found to change linearly with the concentration in the range from 2.0x10-6 molL-1 (0.86 mgL-1) to 1.0x10-4 molL-1 (42.9 mgL-1) in direct voltammetric methods and found to change linearly with the concentration in the range from 2.0x10-8 molL-1 (8.59 μgL-1) to 1.0x10-6 molL-1 (0.43 mgL-1) in adsorptive stripping voltammetric methods. Limit of detection (LOD) and limit of quantification (LOQ) were found to be 1.17x10-8 molL-1 (5.03 μgL-1) and 3.89x10-8 molL-1 (16.8 μgL-1) respectively in anodic adsorptive stripping voltammetry. The methods were successfully applied to assay the drug in tablets, human serum and human urine with good recoveries (between 95.0 % and 104.6 %) and relative standard deviation less than 10 %.

The HPLC-UV method previously developed for the analysis of the flavonoid 2”-O-rhamnosylswertisin (I), an active compound of dried leaf extracts of Aleurites moluccana L. Willd, was fully validated with the inclusion of another flavonoid, swertisin (II). The stability-indicating capability of the method was established by analyzing forced degradation extract samples (acid, alkali, neutral, oxidative and photolytic condition) in which the spectral purity of the markers was ascertained together with the separation of degradation products from the markers. The method proved be linear over a range of 5.89-117.8 and 1.38-27.68 μg.mL-1 for I and II, respectively. The recovery for I and II was 100.3 and 102.8%, respectively, at a level of 100%. A relative standard deviation (RSD%) < 1.0% (intra-day) and < 3.5% (inter-days) for area, and less than 0.5% for retention time indicated that the precision of the method is acceptable. Validation parameters such as selectivity and robustness were also determined. In addition, during the course of LC-MSn analysis three other flavonoids were discovered in the A. moluccana extract and their tentative structures identified.

A stability-indicating LC method for the determination of the antihyperglycemic agent sitagliptin phosphate in tablets was developed and validated using the Plackett-Burman experimental design for robustness evaluation and determination of the system suitability limits. Analytical parameters were studied according to International Conference on Harmonization (ICH) guidelines. The analytical column was operated with a solution of triethylamine 0.3% and acetonitrile (75:25, v/v), adjusted to pH 4.0 at a flow rate of 1.0 mL min-1 and detection at 207 nm, maintained at 25 °C. In forced degradation studies, the effects of acid and basic media with HCl 1M and NaOH 1M, respectively, oxidation, exposure to UV-C light and temperature 60°C were investigated, showing no interference in the drug peak, and the possible breaches of sitagliptin after degradation were suggested by mass analysis. The method was linear (r = 0.9993) at a concentration range from 70.0 to 130.0 μg mL-1 and ANOVA showed a non-significant linearity deviation (p>0.05). Adequate results were obtained for precision (inter and intra-day) and accuracy. Critical factors were selected to examine the method robustness with the two-level Plackett-Burman experimental design and no significant factors were detected (p>0.05). The sitagliptin cytotoxicity assay was determined for the degraded sample in methanolic solution, under UV-C light at 254 nm.

The present work reports a stability-indicating reversed phase ion pair liquid chromatography method for quantitative determination of metformin hydrochloride (MTF) (drug and tablets) and assessment of the cytotoxicity of degraded MTF. Chromatographic separation was performed on a C18 (Phenomenex® Luna), 5.0 μm (250 mm x 4.6 mm) column using isocratic elution. The optimized mobile phase consisted of 10 mmol L-1 heptane sulphonic acid (pH 3.0), acetonitrile and methanol (75:8:17 v/v), flow 1.0 mL.min-1, at 30 ºC. The eluted compounds were monitored at 210 nm wavelength using a DAD detector. The stability indicating capability of the developed method was established by analyzing forced degradation samples (acid, alkali, neutral, oxidative and photolytic condition) in which the spectral purity of MTF was ascertained, along with the separation of degradation products from the analyte peak. The cytotoxicity of MTF and the degraded drug was analyzed in the L929 cell test by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The 500 mg of commercial MTF tablets (Reference, Generic and Similar drugs) showed a similar chromatographic profile, but lower intensity degradation in the same conditions, in relation to the bulk drug in almost forced conditions. Neither the degraded drug samples, nor the tablet samples degraded in acid conditions, showed any cytotoxicity. The developed stability-indicating ion-pair HPLC method was fully validated according to the International Conference on Harmonization (ICH) guidelines. The degraded sample of MTF showed no affect on cell viability, compared with the non-degraded drug.

Outpatient parenteral antimicrobial therapy (OPAT) is an important clinical tool in the treatment of severe infections away from the hospital setting. The stability of amoxicillin sodium in elastomeric infusion devices has been determined for parenteral antimicrobial therapy in the outpatient setting. Firstly, stability was studied at either ambient room temperature (25°C ±1), or at refrigerated temperature (4°C). The practice followed at Auckland City Hospital, to reduce manufacturing and delivery costs, is to order the infusion devices in batches of seven – therefore patients can have up to seven devices at home. A second set of experiments was therefore conducted where stability was assessed by simulating real life conditions to which the infusion devices are exposed. These include refrigerated storage for up to 6 days, followed by 24 hours at room temperature to mimic the patient administration period. The stability of amoxicillin was found to be improved when stored at 4°C compared to room temperature. However, significant concentration dependent degradation occurred after seven days; 87% degradation at 83.3 mg/mL down to 11.0 ±0.02 mg/mL, and 54% degradation at 25 mg/mL down to 11.5 ±0.01 mg/mL. Despite this, such formulations continue to be used successfully to treat patients with severe infections. The data generated from this study has been discussed alongside current clinical practice and observed patient outcomes.

A new stability-indicating RP-HPLC method for the determination of metformin hydrochloride and nateglinide in tablets was developed. The chromatographic separation was performed on a C18 column employing 0.05 M potassium dihydrogen orthophosphate (pH 3)- acetonitrile (28:72 v/v) as the mobile phase with UV detection at 216 nm. Under the proposed chromatographic conditions, favourable retention parameters (tr, Rs) were obtained with good symmetry of the chromatographic peaks for the studied compounds. The validation studies performed as per ICH guidelines indicated high degree of accuracy, precision, with good degree of sensitivity and robustness of the proposed method. The drugs were subjected to acidic, alkaline hydrolysis, oxidation, wet heat, dry heat and photo degradation studies. The specificity of the method by forced degradation studies confirmed that the method could effectively separate the drugs in presence of their degradation products, hence it can be regarding as stability indicating.

Chemical degradation of drugs often results in altered therapeutic efficacy and can lead to toxic side effects. A single, short and sensitive GC-ECD method for the quantification of degradation and process related impurities of lindane has been developed and validated in all of its pharmaceutical topical dosage forms (shampoo, cream and lotion). Three major degradants were observed during forced degradation (stress stability) studies of lindane dosage forms under various conditions recommended by International Conference on Harmonization (ICH). These were identified and confirmed by using both chemical ionization (CI) and electron ionization (EI) techniques of GC-MS analysis. One major degradation component was identified to be the same as the one that was enhanced during the accelerated and long term stability studies of the dosage forms. A dehydrohalogenation mechanism was proposed for this degradation process, which should considerably ease the pharmaceutical development of lindane dosage forms. The chromatographic conditions were optimized using an impurity-spiked solution and the samples that were generated from forced degradation studies. The best chromatographic separation was achieved on a USP-G27 column using electron capture detector (ECD). The newly developed GC-ECD method was validated with respect to linearity, accuracy, precision and robustness. The limit of detection for the lindane and its impurities were found to be 0.004,0.005,0.005 and 0.004 μg/mL respectively. The accuracy (%recovery) was observed to be 100.0±3.0% for lindane and 100.0±5.0% for its impurities respectively in all-three dosage forms. The current method provides a significant improvement in monitoring stability, quality and therapeutic efficacy of lindane pharmaceutical dosage forms.

This article describes the development and optimization of a UV spectrophotometric method to evaluate the isomers of citral in cyclodextrins complexes by a partial least squares (PLS) regression model. The central composite design (CCD) associated with the response surface methodology (RSM) was applied to select the wavelength range that provides the best prediction results. β-cyclodextrin (β-CD) and hydroxypropyl-β-cyclodextrin (HP-β-CD) complexes with citral were prepared in ethanolic solution and spray dried. These samples were analyzed by an optimized PLS-UV model and the results compared with HPLC-UV determinations to evaluate the predictive power of the PLS-UV model. The results from the PLS-UV model showed a relative standard deviation of prediction (RSEP) ranging from 0.87% for neral up to 1.42% for geranial. The relative standard deviation (RSD) obtained in analyses of four replicates of citral-cyclodextrins complexes ranged from 2.2% to 4.1%. Geranial showed higher affinity than neral for both types of cyclodextrins evaluated. Finally, β-CD was shown to be a better complexing agent for citral than HP-β-CD.

The degradation kinetics of cefuroxime lysine in aqueous solution were investigated by a developed liquid chromatograph/mass spectrometry (LC/MS) method with pH ranging from 1.5 to 11.0. The influences of pH, temperature, ionic strength, irradiation and oxidation on the degradation rate were evaluated, and the corresponding kinetic parameters were calculated. The decomposition of cefuroxime lysine was observed to follow first order kinetics under all the experimental conditions, and the rate constant increased with an increase in the temperature, ionic strength and oxidation concentrations. The maximum stability was observed in the pH region from 3.6 to 9.0. The irradiation showed obvious effect on the stability. The activation energy was determined to be 116.0 kJ/mol, with the half-life (t1/2) and shelf-life (t0.9) to be 211.3 and 32.13 h at 25 °C, respectively. An empirical relationship between the rate constant (k) and the pH was depicted as: k=0.0241 + 2.024*10-5*exp[1.326(pH)].

This paper describes the analysis of abamectin and the characterization of its degradation products in formulation. A fast and selective reversed-phase HPLC was developed and validated for the quantitation of degradation products. Two major degradation products observed in stress study samples were isolated and characterized using chromatography and high resolution FT-MS. Abamectin and its degradation products presented similar masses at m/z 873.49848 Da, and were further distinguished by MSn studies, H/D exchange studies, 1H, 13C and 2D NMR experiments. The interpretation of analytical data positively identified unknown 1 as the stereoisomer 2-epi-abamectin and unknown 2 as the regioisomer Δ2,3-abamectin. The developed HPLC method was found to be precise, accurate and detector response was linear for the analysis of known degradation products at the identification threshold.

Due to the difficulties in plasma concentration measurement of lisinopril, it is of utmost importance to develop a new accurate analysis method for this drug. A randomized, double-blind, two-period, two-group crossover design was conducted to scrutinize the bioequivalence of a lisinopril generic product. After administration of test or reference products to each volunteer, the active ingredient was determined in plasma samples using a developed and validated HPLCUV method, and pharmacokinetic parameters, including Cmax, Tmax, AUC0—t , AUC0—∞, terminal elimination rate constant (δz), volume of distribution in steady state (Vd(ss)), mean residence time (MRT), and clearance (Cl) were determined in each subject using the standard non-compartmental approach. In this study, the developed and validated lisinopril assay protocol in human plasma was performed using a C8 analytical column and a mobile phase of 0.05M KH2PO4 (pH=2.75)- acetonitril-methanol (88:11:1, v/v/v) with the detection wavelength of 215 nm. Sample preparation consists of solid-phase extraction using commercially available C18 cartridges. The method showed significant linear response-concentration relationship throughout the lisinopril concentration range of 0.01-0.2 mcg/ml, with the average within-run and between-run variations of 3.51±4.04 and 6.82±6.51 percent throughout the linear concentration range with corresponding average accuracy values of 96.78±3.55 and 97.30±3.33 percent, respectively. The average drug recovery from plasma was 94.70±3.05 percent throughout the linear concentration range. The limits of detection (LOD) and quantitation (LOQ) of the method were 5 and 10 ng/ml, respectively. The practical applicability of the method was proven throughout a bioequivalence study.