Assay Development in Stability Test Methods

Stability-indicating analytical methods are developed to monitor the stability of pharmaceutical dosage forms during the investigational phase of drug development, and, once the drug is marketed, for the ongoing stability studies which must be conducted. The development of these methods for pharmaceutical dosage forms forms can be approached from several avenues. Methods can be developed which measure the amount of drug remaining, the amount of drug lost (or the appearance of degradation products), or both.

Traditionally, the analytical methods used to monitor the stability of dosage forms have involved a generally non-specific spectrophotometric or titrimetric procedure for the assay of the active coupled with thin layer chromatography for the estimation of impurities and degradation products. In the last five years, this approach has changed dramatically. Currently, the method of choice for the quantitation of the active and degradation products is rapidly becoming high performance liquid chromatography. This method has obvious advantages since it both separates and measures and it lends itself well to automation. The disadvantages are that, in the absence of automation, the technique can be time-consuming, it is by no means universal, and it is relatively expensive. Recent advances in column technology have reduced some separation times to seconds and, in the next few years, this technique may find even greater utility.

HPLC, however, is not the only way to go. Other chromatographic methods still find a place, particularly gas chromatography when the stability of the component of interest does not pose a problem and thin layer chromatography for the rapid determination of degradation products. Other methods may also be used, including electrometric, e.g., polarography, and spectrophotometric, e.g., fluorimetry or NMR. The choice of an appropriate method must depend on both a scientific and practical evaluation of the drug and its dosage form.

Once an analytical method is chosen, the most important aspect of the development of a stability-indicating procedure is method validation. Validation should include evaluation of the following parameters: specificity, linearity, precision, accuracy, sensitivity, and ruggedness.

There are many other aspects to stability that could also be considered, e.g., the stability of the bulk drug and physical and organoleptic changes in a dosage form. These should be part of a separate discussion. It very often happens that, during the course of product development, analytical methods evolve. As more is learned about the drug and its dosage form, methods can be refined and revised.     

Quantitation of Cefaclor in Pharmaceutical Dosage Forms Using High Performance Liquid Chromatography

Abstract

A stability-indicating high-performance liquid chromatography for the quantitation of cefaclor in pharmaceutical dosage forms has been developed. The method is accurate and precise with a percent relative standard deviation of 1.2 based on 5 readings. A number of inactive ingredients present in the capsules and suspensions did not interfere with the assay procedure. The extraction procedure from the dosage forms is very simple. The recovery from the synthetic mixtures was quantitative. The capsules which had expired 3 years ago lost only 3% of the potency. The drug appears to be very sensitive to strong acids or bases since a 5 minute boiling caused 100% degradation of drug in both the solutions.     

A Specific Stability Indicating Hplc Method to Determine Diclofenac Sodium in Raw Materials and Pharmaceutical Solid Dosage Forms

A high performance liquid chromatography method is described for the determination of diclofenac sodium, its related compounds and degradation products in commercial sources of raw materials and solid dosage forms. This method is specific, accurate and stability indicating. The method employs a reverse-phase octylsilane (C18) column with a mobile phase composed of acetonitrile/methanol/pic B-6 (25:25:50) and detection at 229 nm. The method resolves six principal related compounds with quantitation in the range 0.3-1.5%. Assay recoveries by spiking commercial formulations with diclofenac sodium were 99.64 ± 1.30%. Drug content in several commercial formulations are reported. Accelerated stability tests were conducted on raw materials and drug products and 1-(2,6-dichlorophenyl)-2-indolin-2-one was identified for the first time as a degradation product in solid dosage forms which are stressed under humidity and heat.     

Quantitation of Cephalexin in Pharmaceutical Dosage Forms Using High-Performance Liquid Chromatography

Abstract

A high-performance liquid chromatography method has been developed to quantify cephalexin in pharmaceutical dosage forms, capsules, pediatric drops and suspensions. The method is accurate and precise with a percent relative standard deviation of 0.8 based on 6 readings. There is no interference from a variety of excipients present in the dosage forms. The procedure for the extraction of cephalexin from the dosage forms is very simple. The method is stability indicating since a sample decomposed using sodium hydroxide showed very little potency and new peaks in the chromatogram. In the powder form cephalexin appears to be very stable.     

High Pressure Liquid Chromatographic Determination of Haloperidol Stability

Abstract

Haloperidol lacks stability when exposed to elevated temperatures and light. Current assay procedures described in the USP do not permit detection or quantitation of degradation products of haloperidol. An HPLC assay for the analysis of haloperidol and the detection of degradation products has been developed. The mobile phase consisted of 40:60 THF:water with 0.75% phosphoric acid. A microbond-apak CN column was used to achieve the separation. Samples were injected using a fixed loop (200 microliter) injector and detection was by a fixed wavelength set at 254 nm. Using this mobile phase, haloperidol gave a retention time of 5.4 minutes. Samples of haloperidol treated with heat (60°C for 48 hours) gave peaks at 5.1 and 6.4 minutes as well as a haloperidol peak at 5.4 minutes. A standard curve of haloperidol concentrations was linear (r=0.99) over the range of 1 μg/ml to 100 μg/ml. The conversion of haloperidol to degradation products was noted after storage under conditions of elevated temperature, exposure to light, and as a function of pH. current USP XX assays for this drug include titration of dissolved haloperidol powder with 0.05N perchloric acid or extraction of the drug from a dose form (i.e., oral solution, injection, tablets) with subsequent ultraviolet spectrophotometric analysis (3). Neither one of these assays permits the detection or quantification of haloperidol degradation products. A high-pressure liquid chromatography (HPLC) assay was developed which overcomes the problems associated with the USP assays (3). This HPLC assay has been modified slightly for the analysis of haloperidol and detection of degradation products in drug solutions exposed to elevated temperatures, light, and as a function of pH.     

Stability Study of Lorazepam in Solid Dosage form by High Performance Liquid Chromatography

Abstract

A reverse phase column with MeOH-H₂0 as mobile phase and detection at 230 nm was employed for the determination of lorazepam and degradation products in tablet formulation. The mean coeficient variation (n=6) for the entire analytical method was 1.15%. A working calibration curve over a concentration range of 5 to 250 ng of lorazepam was obtained and the recovery (n=3) was 100.5%. Limits of detection varied from 1.6 to 3.2 ng according to the compounds. Natural and thermal stability of the drug and tablets were carried out since the method was suitable for stability indicating studies. A comparative TLC method was also performed. The effect of the type and concentration of acid and the content of methanol in reaction medium of hydrolysis of lorazepam were also investigated. Degradation products were characterized by HPLC and TLC by comparing them to authentic samples. The first degradation product that appeared was the quinazoline-carboxaldehide and 2-amino-2′,5-dichlorobenzophenone was not detected. The additives in tablets decreased drug stability and degradation pathway followed by the tablets was similar to the drug under thermal conditions.     

Statistical Evaluation of the Results Obtained with the Analytical Methods used for the Quality Control of Medicines

Abstract

To improve the way by which the quality control of medicines is carried out, general statistical concepts and methods are discussed for the evaluation of the analytical results obtained for judging the adequacy of drug substances and products for Pharmacopoeia requirements.

Statistical procedures to be adopted by the producer and the controller in the determination of characteristics are suggested when the drug substance is or is not homogeneous and when the precision of the analytical method is known or unknown.

For each of these cases, detailed numerical examples are given.     

Rapid and Sensitive Method for a Kinetic and Preformulation Study of an Investigational Antiviral Drug

Abstract

Win 51711 is an oxazoline compound investigated for antiviral activity against rhinovirus. The drug is not very soluble in water (< 1 mcg/mL) above pH 5 but is quite soluble at more acid pH's due to salt formation with the oxazoline nitrogen. Formulation of the drug depends on selective use of excipients. Acidic excipients destabilize the drug whereas neutral and basic ingredients have no adverse effect on stability. The present study reports on an HPLC method developed to separate the drug and the products under stressed conditions. The products of hydrolysis were identified and found to have little or no antiviral activity. A simple spectrophotometric method was also developed. This method was found to have an advantage of assaying solutions of Win 51711 and its hydrolysis product without separation. The HPLC method was used in selecting compatible excipients which resulted in a formulation with long term stability.     

A Solid Buffer Reagent for in Vitro Stepped-pH Dissolution Testing

Abstract

A novel method is described that simplifies in vitro dissolution tests of programmerelease drug dosage forms. It is based on a single solid reagent that affords immediate stepped-pH conditions using a single unitization to model the physiological gradient. Six pharmaceutical products were tested using two methods; the paddle method and the through flow cell method. The dissolution efficiencies obtained with the proposed reagent were identical to those obtained with classical buffers used in dissolution tests. Varying dissolution parameters (paddle rotation rate, flow rate in the through flow cell, use of surfactants) gave closely similar results for the two pH stepping methods but with the added advantage of a single medium.     

Determination of rifampicin and isoniazid in pharmaceutical formulations by HPLC

Abstract

A liquid chromatographic procedure for the analysis of Rifampicin (RIF) and Isoniazid (INH) in pharmaceutical dosage forms utilizing reverse phase chromatography was developed. Isolation of analytes was carried out under isocratic conditions with a octadecylsilane column and an aqueous mobile phase containing Methanol (75%) and 0.02 M Disodium Hydrogen Orthophosphate (25%) with pH 4.5 adjusted with orthophosphoric acid. The detection was done at 254 nm.

The method is unique in analysing Rifampicin precisely in combination with Isoniazid particularly in liquid formulation. The method is specific and can distinctly isolate the degradation product in suspension.     

Stability Indicating HPTLC Determination of Linezolid as Bulk Drug and in Pharmaceutical Dosage Form

Abstract

A simple, selective, precise, and stability-indicating high-performance thin layer chromatographic method of analysis of Linezolid both as a bulk drug and in formulations was developed and validated in pharmaceutical dosage form. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of toluene–acetone (5:5, v/v). This system was found to give compact spots for Linezolid (R_f value of 0.29 ± 0.01). Linezolid was subjected to acidic, alkali hydrolysis, oxidation, and photodegradation. The degraded products also were well separated from the pure drug. Densitometric analysis of Linezolid was conducted in the absorbance mode at 254 nm. The linear regression data for the calibration plots showed good linear relationship with r² = 0.997 ± 0.001 in the concentration range of 300–800 ng/spot. The mean value of correlation coefficient, slope, and intercept were 0.998 ± 0.003, 0.15 ± 0.009, and 19.52 ± 1.66 respectively. The method was validated for precision, accuracy, ruggedness, and recovery. The limits of detection and quantification were 20 ng/spot and 50 ng/spot, respectively. The drug undergoes degradation under acidic and basic conditions, oxidation and photo degradation. All the peaks of degraded product were resolved from the standard drug with significantly different R_f values. This indicates that the drug is susceptible to acid–base hydrolysis, oxidation, and photo degradation. Statistical analysis proves that the method is reproducible and selective for the estimation of the said drug. Because the method could effectively separate the drug from its degradation products, it can be used as a stability indicating one.     

Quantitation of Sulfacetamide,Sulfadiazine, Sulfamerazine and Sulfame Thazine in Various Cominations Dsing High-Performance Liquid Chromatography

Abstract

A reverse phase high-performance liquid chromatography method for the quantitation of sulfacetamide, sulfadiazine, sulfamerazine, and sulfamethazine in various combinations has been developed. The method is simple, accurate, precise and reproducible. The percent relative standard deviations based on 6 injections were 2.1, 0.6, 1.9, and 1.6 for sulfacetamide, sulfadiazine, sulfamerazine, and sulfamethazine, respectively. The ratio of peak heights (drug/internal standard) wer closely related (r value 0.99 or better) to concentrations (± 20% of the standard solution concentrations). The results of synthetic mixtures showed quantitative recovery and method was successfully applied to commercial dosage forms (tablets and suspension). Extraction of sulfa drugs from the dosage forms required a very simple procedure.     

The Stability of Drug Adsorbates on Silica

Colloidal and porous silicas are used as carriers in solid, semisolid and liquid dosage forms. Adsorption of active ingredients onto their large surface areas can be used to regulate drug release or for the uniform distribution of drug in single dose units with a very low drug content. In the adsorbates the contact between drug and carrier surface on the molecular level can be of great importance for the chemical stability of drug preparations. This is demonstrated by the following examples:

Hydrolytic degradation of acetyl salicylic acid in dry silica adsorbates is mainly determined by alkaline impurities of the carrier and strongly adsorbed water on the silica surface. The “catalytic” action of silicas is, therefore, directly dependent on the preparation technique of the carrier. Propantheline a cationic ester compound is adsorbed on silica from aqueous solution. In aqueous silica suspensions and in dry adsorbates the ester hydrolysis is controlled by the pH, the neutral salt content, and buffer substances, due to different adsorption mechanisms.

The oxidative degradation of butylhydroxyanisole in silica adsorbates was also found to be enhanced in the presence of alkaline impurities. The oxidation of linoleic acid methylester in oleogels of colloidal silica proved to be influenced both by carrier impurities and the specific adsorption of intermediates (peroxides) onto the surface.     

Quantitation and Stability of Verapamil Hydrochloride using High-Performance Liquid Chromatography

Abstract

A high-performance liquid chromatography method for the quantitation of verapamil hydrochloride in pharmaceutical dosage forms has been developed. The method is precise and accurate with a relative standard deviation of 0.63% based on six injections. No preliminary extraction procedure is required to assay injections and a very simple extraction procedure is needed for tablets. There is no interference from the excipients and the method appears to be stability-indicating. The optimum pH range of stability is about 3.2 to 5.6 and the phosphate buffer and ionic strength have very little effect on the stability. Verapamil hydrochloride appears to be a very stable compound since in 105 days at 50°, the aqueous solutions (0.5 mg/ml) did not decompose.     

Evaluation of Cholecystokinin (CCK-8) Peptide Thermal Stability for Use as Radiopharmaceutical by Means Isothermal and Nonisothermal Approaches

ABSTRACT

The purpose of this research was to study thethermal stability of cholecystokinin octapeptide (CCK-8) in aqueous solution at pH 12 and ionic strength 0.01M, which were kept as constants, by using isothermal and nonisothermal methods.

The isothermal decomposition of CCK-8 was investigated as a function of temperature (40°C to 70°C). Nonisothermal stability studies were performed using a linear increasing temperature program. Two different nonisothermal studies were carried out at 0.25°K and 0.5°K per hour, and the temperature interval varied from 40°C to 82°C.

The degradation of CCK-8 followed first-order kinetics, obeying the Arrhenius equation in the experimental temperature range. This indicated that the degradation mechanism of CCK-8 could be the equal within the temperature range studied. The nonisothermal approach resulted in activation energy (Ea) and shelf-life (t_90%) values that agree well with those obtained by the isothermal method. The level of uncertainty in the estimates of t_90% and Ea values is determined mainly by the extent of drug degradation and temperature change during the experiment. Therefore, nonisothermal experiments save time, labor and materials (i.e. the amount of drugs necessary to conduct the experiment) compared to the classic isothermal experiments, if they are performed using a suitable experimental design and a precise analytical method.     

Fluidized Bed Coating Technique for Production of Sustained Release Granules

Abstract

A laboratory-scale instrument for fluid-bed spray coating of granules has been constructed. Its major advantage is its suitability for development work on small batches of granules or tablets of between 50 and 300 grams. Commercial equipment available at present requires minimum loadings of 0.5-1 Kg. for effective operation.

The instrument has been successfully used for producing sustained-release products by coating granules of model drugs with EC, and data are presented which define important qualities of the products. In particular, the apparatus is capable of giving excellent batch homogeneity and reproducibility.

One of the recent successful developments in pharmaceutical technology is the production of sustained-release dosage forms. The two main approaches utilized in the design of these products are (a) the introduction of a physical barrier preventing contact between the drug and the fluids of the digestive system, the effect of which is to reduce the rate of diffusion or leaching out of the drug from the dosage form (b) the addition of selected interactants to the formulation, such as ion-exchange resins or complexants, which form weak cnemical bonds with the drug.

The present work is concerned with the first type of product which in practice may be produced using widely different technologies. The main ones are based on (a) coating techniques (b) embedding the drug in a wax or polymer matrix. Prior to 1956, coating was performed by means of the classical rotating pan-method, but in that year Spaulding (1) introduced a controlled spray technique for application of the coating solution to the pan contents, termed the rotating pan-spray technique.

A new and major step in coating technology was the introduction into pharmaceutical manufacturing of the fluidized-bed technique. The term “fluidized-bed” has been defined in a number of ways (2-4); most simply, when a solid is “fluidized” in a process, it shows in its behaviour many of the physical characteristics of a liquid.

In pharmaceutical production, fluidization methods are utilized in stages of drying (5-6), granulation (7) and coating (8-9). As a coating technique, its main advantages over the pan-coating method are as follows:

a) irregular particles may be coated directly, b) loss of material is small, c) the process may be automated and does not require learning the “art” of coating, d) it is very rapid.

The present work is a study of the preparation of sustained-release granules coated by means of the fluid-bed technique. Salicylic acid and caffeine were selected as model drugs, while ethyl cellulose (EC) with polyethylene glycol (PEG) were representative of coating materials.     

An Investigation into the use of Creep Viscometry for in Vitro Characterisation of the Raft Forming Properties of tablets Containing Alginic acid and antacid Components

Abstract

The established methods for assessing the strength of rafts formed in-vitro by alginic acid/antacid containing formulations rely on the determination of forces required to disrupt the raft structure. Creep viscometry is proposed as an alternative, non-destructive means of investigating the characteristic parameters of this type of product. Several commercial chewable tablet formulations were investigated using this technique. The instantaneous compliance and the Newtonian viscosity were calculated from the creep compliance curves obtained for these products and were used as raft strength indicators.

Differences found in the raft strength parameters of the different products were attributed to interactions between the formulation components. Additional studies conducted on experimental batches established that such interactions could be estimated using this technique. It was concluded that creep viscometry could be usefully employed in investigations of alginate rafts and, possibly, other oral dosage forms which rely on the formation of visco-elastic structures to modify drug release properties.     

Stability-Indicating HPLC Method for Betaxolol HCl and Its Pharmaceutical Dosage Forms

Abstract

The present work describes a specific, stability-indicating high-performance liquid chromatographic method for determination of betaxolol HCl and its pharmaceutical dosage forms. Betaxolol HCl was chromatographed on a microbondapak C18 column utilizing a simple mixture of methanol: acetonitrile:0.1% diethylamine (pH 3.0 adjusted using orthophosphoric acid). It was detected at 222 nm. The method is accurate and precise with a percent relative standard deviation of 0.11 based on 6 readings. A number of inactive ingredients present in the dosage forms (eye drop, tablet, gel) did not interfere in the assay procedure. The recovery from synthetic mixtures was quantitative. The extraction procedure from the dosage forms is very simple. The drug appears to be very sensitive to acids (such as sulfuric acid) since 100% of the drug decomposed on boiling for 5 min.     

Process and dosage form controlts: Formulation factors

Abstract

During the last decades, there have been considerable developments in the field of pharmaceutics, pharmaceutical technology and product manufacture.

The trend of the pharmaceutical industry is, like in most of the sophisticated industries, to produce, day after day, a better product, and as final goal, to manufacture continueously a perfect drug dosage form.

A few years ago, the defaults were counted in “percent”. After that, it was in per “thousand”. Now it is often expressed in “per million”, or even for very high series (for example empty hard gelatine capsules) the trend is “per billion”. Such an evaluation can only be achieved with a complete control of the whole manufacturing process.

The requirement for pharmaceutical dosage form are numerous (1): adequate biopharmaceutical profile, ease of manufacture, quality assurance (the dosage form must contain the correct quantity of the correct drug, and liberate it at the correct place, at the correct time, and in the correct quantity, with the correct speed), stability, …

These requirements can only be fulfilled with a perfect knowledge of the drug and the dosage form, from the beginning of the development of the dosage form (formulation) to the end of the manufacturing process (production and final product control).

It is the aim of the present lecture to show how important are the formulation factors and what is their influence on the processing and the dosage form control.