Development and application of a validated HPLC method for the analysis of dissolution samples of levothyroxine sodium drug products.

A rapid, selective, and sensitive gradient HPLC method was developed for the analysis of dissolution samples of levothyroxine sodium tablets. Current USP methodology for levothyroxine (L-T(4)) was not adequate to resolve co-elutants from a variety of levothyroxine drug product formulations. The USP method for analyzing dissolution samples of the drug product has shown significant intra- and inter-day variability. The sources of method variability include chromatographic interferences introduced by the dissolution media and the formulation excipients. In the present work, chromatographic separation of levothyroxine was achieved on an Agilent 1100 Series HPLC with a Waters Nova-pak column (250 mm × 3.9 mm) using a 0.01 M phosphate buffer (pH 3.0)-methanol (55:45, v/v) in a gradient elution mobile phase at a flow rate of 1.0 mL/min and detection UV wavelength of 225 nm. The injection volume was 800 µL and the column temperature was maintained at 28°C. The method was validated according to USP Category I requirements. The validation characteristics included accuracy, precision, specificity, linearity, and analytical range. The standard curve was found to have a linear relationship (r(2)>0.99) over the analytical range of 0.08-0.8 µg/mL. Accuracy ranged from 90 to 110% for low quality control (QC) standards and 95 to 105% for medium and high QC standards. Precision was <2% at all QC levels. The method was found to be accurate, precise, selective, and linear for L-T(4) over the analytical range. The HPLC method was successfully applied to the analysis of dissolution samples of marketed levothyroxine sodium tablets.

Stability indicating validated HPLC method for quantification of levothyroxine with eight degradation peaks in the presence of excipients.

A simple, sensitive, accurate, and robust stability indicating analytical method is presented for identification, separation, and quantitation of l-thyroxine and eight degradation impurities with an internal standard. The method was used in the presence of commonly used formulation excipients such as butylated hydroxyanisole, povidone, crospovidone, croscarmellose sodium, mannitol, sucrose, acacia, lactose monohydrate, confectionary sugar, microcrystalline cellulose, sodium laurel sulfate, magnesium stearate, talc, and silicon dioxide. The two active thyroid hormones: 3,3',5,5'-tetra-iodo-l-thyronine (l-thyroxine-T4) and 3,3',5-tri-iodo-l-thyronine (T3) and degradation products including di-iodothyronine (T2), thyronine (T0), tyrosine (Tyr), di-iodotyrosine (DIT), mono-iodotyrosine (MIT), 3,3',5,5'-tetra-iodothyroacetic acid (T4AA) and 3,3',5-tri-iodothyroacetic acid (T3AA) were assayed by the current method. The separation of l-thyroxine and eight metabolites along with theophylline (internal standard) was achieved using a C18 column (25 degrees C) with a mobile phase of trifluoroacetic acid (0.1%, v/v, pH 3)-acetonitrile in gradient elution at 0.8 ml/min at 223 nm. The sample diluent was 0.01 M methanolic NaOH. Method was validated according to FDA, USP, and ICH guidelines for inter-day accuracy, precision, and robustness after checking performance with system suitability. Tyr (4.97 min), theophylline (9.09 min), MIT (9.55 min), DIT (11.37 min), T0 (11.63 min), T2 (14.47 min), T3 (16.29 min), T4 (17.60 min), T3AA (22.71 min), and T4AA (24.83 min) separated in a single chromatographic run. Linear relationship (r2>0.99) was observed between the peak area ratio and the concentrations for all of the compounds within the range of 2-20 microg/ml. The total time for analysis, equilibration and recovery was 40 min. The method was shown to separate well from commonly employed formulation excipients. Accuracy ranged from 95 to 105% for T4 and 90 to 110% for all other compounds. Precision was <2% for all the compounds. The method was found to be robust with minor changes in injection volume, flow rate, column temperature, and gradient ratio. Validation results indicated that the method shows satisfactory linearity, precision, accuracy, and ruggedness and also stress degradation studies indicated that the method can be used as stability indicating method for l-thyroxine in the presence of excipients.

Process analytical technology: non-destructive assessment of anastrozole entrapment within PLGA microparticles by near infrared spectroscopy and chemical imaging.

The objective of this study was to evaluate near-infrared (NIR) spectroscopy and imaging as approaches to assess anastrozole entrapment within PLGA microparticles. By varying the polymer concentration, three batches containing the same amount of the drug were prepared. The spectral features that allow NIR drug quantitation were evaluated and compared with a best fit line algorithm. Actual entrapment efficiencies (EEF) determined via a destructive method were used for construction of calibration models using partial least square regression (PLS) or the algorithm. On the other hand, a chemical imaging system based on array detector technology was used to rapidly collect high contrast NIR images of the formulated microparticles. A quantitative measure of anastrozole entrapped was determined by calculating the percentage standard deviation of the distribution of pixel intensities in the PLS score images and histograms. Concerning conventional NIR analysis, both methods were equivalent for the prediction of EEF over the range of polymer levels studied. Correlation coefficients of more than 0.992 were obtained for either the calibration or prediction of EEF by the two methods; 0.392% and 0.374% were the standard errors of calibration and prediction (SEC and SEP) obtained for the prediction of EEF using the fit line, respectively, whereas the prediction of the EEF by the partial least square regression showed a SEC of 0.195% and SEP of 0.179%. As a result, the spectral best fit algorithm method compared favourably to the multivariate PLS method, but was easier to develop. In contrast, NIR spectral imaging was capable of clearly differentiating the three batches, both qualitatively and quantitatively. The percentage standard deviation increased progressively by increasing the ratio of drug-to-polymer concentrations. In conclusion, both NIR approaches were capable of accurate assessment of drug entrapment within microparticles. In addition, the NIR spectral imaging system provides a rapid approach for acquiring spatial and spectral information on microparticles.

Controlled release tacrine delivery system for the treatment of Alzheimer's disease.

Alzheimer's disease is a neurodegenerative condition that affects approximately 5 million people and is the fourth leading cause of death in America. Tacrine is one of the three drugs approved by the FDA for the treatment of Alzheimer's disease. However, the drug has a short biologic half-life of 2-3 hr and gastrointestinal, cholinergic, and hepatic adverse reactions that are associated with high doses of the drug. The aim of our study was to formulate a controlled release delivery system of tacrine that could be used to minimize the side effects associated with the drug. Microparticles of tacrine were formulated using poly(D,L-lactide-co-glycolide) (PLG). PLG and tacrine were dissolved in mixed organic solvents and added to a polyvinyl alcohol solution that was stirred at a constant rate. The organic solvent was evaporated overnight and the formed microparticles were collected by filtration, dried, and sieve-sized. The effects of such formulation variables, as molecular weight of polymer, stir speed during preparation, and drug loading on encapsulation efficiency (EEF), and in vitro release profiles of tacrine were investigated. An increase in the molecular weight of polymer from 8,000 to 59,000 and 155,000 resulted in approximately 10-fold increase in EEF, but the rate of release decreased with increasing molecular weight. Stir speed during preparation had an effect on the EEF but not on the rate of release. Drug loading did not have a significant effect on the EEF but had an effect on the rate of tacrine release. The results suggest that tacrine could be delivered at controlled levels for weeks for the treatment of Alzheimer's disease.

Solubilization of liposomes by weak electrolyte drugs. II. Cefotaxime.

The solubilization of dimyristoylphosphatidylcholine (DMPC) liposomes by the weak electrolyte drug, cefotaxime (CFX), has been studied as a function of pH, DMPC, temperature, presence of cholesterol (CHOL), and method of liposome preparation. At 7.5 mM CFX the lag time for solubilization increased, the rate of solubilization decreased, and the minimum turbidity reached increased as a function of DMPC at pH 1.0 and 40 degrees C. Solubilization was most pronounced at pHs below the pKa but inhibited at least one pH unit above the pKa. The critical mole ratio of unionized CFX:DMPC, Rec, for solubilization was estimated to be 0.12. Reducing the temperature slowed the rate of solubilization as did the addition of CHOL. Encapsulation of CFX in liposomes did not significantly reduce CFX degradation,. k1 = 0.048 h-1 at 40 degrees C and a complex of DMPC and a degradation product of CFX precipitated as rectangular crystals. As a result, an increase in the turbidity of solubilized systems was observed from about 20 h to 48 h depending on the conditions. Liposomes in the gel state or with at least 20% CHOL did not undergo an apparent reversal of solubilization. It is concluded that the inclusion of weak electrolyte drugs existing predominantly as the unionized species in liquid crystalline state liposomes may undergo a slow solubilization process not necessarily recognized during characterization.

Dissolution profiles of flurbiprofen in phospholipid solid dispersions.

This study is concerned with the development of a solid dispersion formulation of flurbiprofen (FLP) and phospholipid (PL) with improved dissolution characteristics. The FLP powders were blended with PL to produce FLP-PL physical mixtures or made into solid dispersions with PL by the solvent method. The FLP exhibited significantly improved dissolution rates in PL coprecipitate (coppt) compared to the physical mixtures or FLP alone. The dissolution studies suggested that less than a 20:1 ratio of FLP to PL was required to disperse FLP completely in the carrier. The coppt yielded a ninefold greater initial dissolution rate. Also, the total amount dissolved after 60 min was twofold greater at a 10:1 ratio of FLP to L-(-dimyristoyl phosphatidylglycerol (DMPG). Similar results were observed with a ratio as low as 20:1 (FLP:DMPG). Increasing the DMPG content did not increase the rate to any significant extent. Thus, a small PL:FLP ratio improved the dissolution to a significant level. Thus, an FLP:PL dispersion may have the clinical advantages of quick release and excellent bioavailability.

Solubilization of multilamellar liposomes in the presence of non-ionized drug.

The solubilization of multilamellar liposomes by metoprolol tartrate (MPL) has been studied as a function of pH, [MPL], [dimyristoylphosphatidylcholine (DMPC)], temperature and lipid composition. The solubilization of liposomes at 37 degrees C by 7.3 nM MPL occurred at different rates at different pH values. MPL completely solubilized by 7.2 mM DMPC liposomes after about 17 h at pH 12, but only a partial solubilization occurred at pH 10 and 11. Between pH 7 and 9 no change in turbidity was observed after 1 week. Addition of cholesterol (CHOL) to DMPC (2:1 mol) had very little effect on solubilization after 24 h, however with DMPC:CHOL (5:1 mol) the decrease in turbidity was observed after 24 h, even though solubilization was much less compared with that of DMPC alone. The rate of solubilization was decreased when dipalmitoylphosphatidylcholine liposomes were employed. Addition of dicetylphosphate (DCP) to DMPC liposomes reduced the rate of solubilization significantly. The solubilization of liposomes by 7.3 mM MPL as a function of [DMPC], indicated that the lower the liposome concentration the greater the effect on solubilization. It is concluded that MPL in the non-ionized form has a solubilizing effect on liposomes, and addition of CHOL or DCP to DMPC has a stabilizing effect against solubilization.

Hydrolysis and stability of acetylsalicylic acid in stearylamine-containing liposomes.

The hydrolysis and the stabilization of acetylsalicylic acid (ASA) in liposomes at 30 degrees C were studied. The liposomes consisted of dimyristoylphosphatidylcholine (DMPC) and stearylamine. At pH 4.0 and 8.0, the pseudo-first-order rate constants (kobs) in DMPC: stearylamine (2: 1 mole ratio) liposomes were approximately 60% of the values in control solutions (kB) if ASA was incorporated via the organic phase. In contrast, when ASA was added via the aqueous phase, kobs = kB at pH 4.0 but kobs < kB at pH 8.0 and kobs increased with the fraction of stearylamine in the liposomes. However, when ASA was added via the organic phase, reactions occurred which resulted in the loss of ASA as a function of the time period between phase admixture and the point of film hydration. A product of the reactions was determined by IR and TLC to be N-stearylacetamide. Both the initial loss of ASA and the increase in stability decreased as the DMPC: stearylamine mole ratio increased. A mechanism of aminolysis occurring in the organic solvent and at liposome surfaces between ASA and stearylamine or DMPC at pH 8.0 has been suggested. It is concluded that the orientation of ASA and the ordered structural environment of the bilayers minimizes the aminolytic and hydrolytic reactions.

Photostabilization of riboflavin by incorporation into liposomes.

The influence of liposomes on the photostabilization of riboflavin in an aqueous formulation was studied under fluorescent light at various conditions. Liposomal composition, concentration, pH, and ionic strength were varied. The photostability of riboflavin was found to increase in the presence of neutral and negatively-charged liposomes but to decrease in association with positively-charged liposomes. Furthermore, increasing the concentration of dimyristoyl-phosphatidylcholine (DMPC) in the composition of the liposomes resulted in an enhancement in the photostability of riboflavin such that at 5.8mM DMPC concentration a 2.3 fold increase in photostability was observed compared to control buffer solution. The pH of the medium influenced the photostability of riboflavin. However, the ionic strength of solution appeared to demonstrate no significant effect. The photodegradation reactions appeared to follow first-order kinetics in the presence and absence of liposomes.

Effect of certain additives on photodegradation of tetracycline hydrochloride solutions.

The photostability of tetracycline hydrochloride solutions in the presence of selected potential stabilizers under various light sources was investigated. Reduced glutathione (GSH) demonstrated the greatest photostabilizing effect followed by p-aminobenzoic acid (PABA), ethylenediamine tetraacetic acid (EDTA) disodium salt, DL-cysteine, thiourea, sodium thiosulfate, and DL-methionine in a descending order. The photostabilizing effect of GSH was found to be dependent on its concentration, pH and buffer species of the medium, the light source, and the temperature of the solutions exposed to light.

Photostabilization of doxorubicin hydrochloride with radioprotective and photoprotective agents: potential mechanism for enhancing chemotherapy during radiotherapy.

p-Aminobenzoic acid (PABA), urocanic acid, and sodium urate were found to significantly enhance the photostability of doxorubicin hydrochloride [adriamycin, (ADR)]. d1-Methionine, thiourea, and glycine also increased the photostability of this drug, but to a lesser degree. Sodium thiosulfate on the other hand, was found to be detrimental to the photostability of ADR. The photostabilizing effect of PABA was found to increase with increase of its concentration and was influenced by the pH and the buffer species of the vehicle. The findings would have an impact on the enhancement of therapeutic efficacy of adriamycin when administered during radiation therapy.

Photostabilization of menadione sodium bisulfite by glutathione.

Photodecomposition of solutions of menadione sodium bisulfite (MSB) in the presence and absence of glutathione (GSH) under artificial sunlight was investigated. In presence of 0.02% (GSH), an appreciable increase in photostability was observed when clear glass vials were used. The pH and the temperature of the solution significantly influenced the stability of MSB. Photodegradation of MSB appeared to follow first-order kinetics. Photostabilization of MSB was attributed in part to complex formation between MSB and GSH and the antioxidant property of the latter. Stability constants for the various solutions were determined at several temperatures. Thermodynamic parameters were calculated and were found to support complex formation.

Complex formation between metronidazole and sodium urate: effect on photodegradation of metronidazole.

Photodegradation of solutions of metronidazole in the presence and absence of sodium urate was studied. Photodegradation appeared to follow zero-order kinetics and was found to be dependent on the pH, buffer species, sodium urate concentration, and light source. Complex formation between metronidazole and sodium urate accounted for the photostabilization of metronidazole. The dissociation constant for this complex was calculated to be 3.4 x 10(-3) M.