A simplified guide for charged aerosol detection of non-chromophoric compounds-Analytical method development and validation for the HPLC assay of aerosol particle size distribution for amikacin.

Amikacin, an aminoglycoside antibiotic lacking a UV chromophore, was developed into a drug product for delivery by inhalation. A robust method for amikacin assay analysis and aerosol particle size distribution (aPSD) determination, with comparable performance to the conventional UV detector was developed using a charged aerosol detector (CAD). The CAD approach involved more parameters for optimization than UV detection due to its sensitivity to trace impurities, non-linear response and narrow dynamic range of signal versus concentration. Through careful selection of the power transformation function value and evaporation temperature, a wider linear dynamic range, improved signal-to-noise ratio and high repeatability were obtained. The influences of mobile phase grade and glassware binding of amikacin during sample preparation were addressed. A weighed (1/X2) least square regression was used for the calibration curve. The limit of quantitation (LOQ) and limit of detection (LOD) for this method were determined to be 5µg/mL and 2µg/mL, respectively. The method was validated over a concentration range of 0.05-2mg/mL. The correlation coefficient for the peak area versus concentration was 1.00 and the y-intercept was 0.2%. The recovery accuracies of triplicate preparations at 0.05, 1.0, and 2.0mg/mL were in the range of 100-101%. The relative standard deviation (Srel) of six replicates at 1.0mg/mL was 1%, and Srel of five injections at the limit of quantitation was 4%. A robust HPLC-CAD method was developed and validated for the determination of the aPSD for amikacin. The CAD method development produced a simplified procedure with minimal variability in results during: routine operation, transfer from one instrument to another, and between different analysts.

A general static-headspace gas chromatographic method for determination of residual benzene in oral liquid pharmaceutical products.

Sodium benzoate is used in oral liquid pharmaceutical products for its anti-microbial properties. The benzoate salts present in liquid pharmaceutical products can potentially generate residual levels of free benzene during manufacturing of the drug product and or during the shelf-life of the product under its storage conditions. To ensure the safety and quality of the pharmaceutical products (containing benzoate in the formulation), a selective and sensitive analytical method is required to monitor residual benzene in oral liquid pharmaceutical products. In this paper, we report the development and validation of a general static-headspace gas chromatographic (SH-GC) method to determine residual benzene in oral liquid pharmaceutical products. The liquid pharmaceutical drug product sample is dissolved in dimethylsulfoxide (DMSO) in a GC headspace vial. A DB-624 capillary column (30 m x 0.32 mm I.D. and 1.8 µm film thickness) was used under isothermal conditions with a flame ionization detection (FID). The benzene peak was well separated from all other volatile compounds that are present in the formulation of a number of liquid drug products. This method was successfully validated using a representative oral liquid pharmaceutical drug product. The limit of detection of the method for benzene is 0.5 ppm which met the 2 ppm limit of current ICH guideline for residual benzene in pharmaceutical products.

Development and validation of a stability-indicating RP-HPLC method for simultaneous assay of betamethasone dipropionate, chlorocresol, and for the estimation of betamethasone dipropionate related compounds in a pharmaceutical cream and ointment.

A new stability-indicating reversed-phase HPLC (RP-HPLC) method has been developed and validated for simultaneous assay of betamethasone dipropionate (BD) and chlorocresol and also for the estimation of BD related compounds in a pharmaceutical cream matrix. In addition, this newly developed RP-HPLC method was also demonstrated as suitable for a pharmaceutical ointment product that does not contain chlorocresol. The RP-HPLC method uses a Waters SymmetryShield RP18 analytical column (150 × 4.6 mm). Water (mobile phase A) and acetonitrile (mobile phase B) were used in the gradient elution with a flow rate of 1.5 mL/min and detection wavelength at 240 nm. A Waters XBridge Shield RP18 analytical column (150 × 4.6 mm) was identified as an alternate column. The limit of detection (LOD) and the limit of quantitation (LOQ) are 0.02 µg/mL and 0.05 µg/mL, respectively. The precision of the method for BD is less than 0.3% RSD, and the accuracy of BD ranged from 99.5% to 102.6%. The stability-indicating capability of this method has been demonstrated by analyzing aged samples of the product. This RP-HPLC method was successfully validated per ICH guidelines and proved to be suitable for routine quality control use.

Development and validation of a novel stability-indicating reversed-phase high-performance liquid chromatography method for assay of loratadine and determination of its related compounds.

Loratadine is an important active pharmaceutical ingredient used in a wide variety of prescription and over-the-counter products for the treatment and relief of allergy symptoms. A novel stability-indicating gradient ion-pair RP-HPLC method for assay of loratadine and determination of both of its degradation compounds and process impurities has been developed. This method can separate loratadine from its eight structurally related compounds; it can also separate all of the related compounds from each other in less than 20 min. The stability-indicating capability of this method has been demonstrated by analyzing aged stability samples of loratadine. A 15 cm x 4.6 mm id YMC-Pack Pro C18 HPLC column was the primary column and a 15 cm x 4.6 mm id SunFire C18 column has been identified as an alternate (truly equivalent) column for this method. This gradient method uses mobile phases consisting of acetonitrile and an aqueous solution of 10 mM sodium acetate and 5 mM sodium dodecyl sulfate at pH 5.5. The new HPLC method was validated according to International Conference on Harmonization guidelines and proved to be suitable for routine QC use.

Development and validation of a stability-indicating RP-HPLC method for assay of betamethasone and estimation of its related compounds.

Betamethasone (9alpha-fluoro-16beta-methylprednisolone) is one of the members of the corticosteriod family of active pharmaceutical ingredient (API), which is widely used as an anti-inflammatory agent and also as a starting material to manufacture various esters of betamethasone. A stability-indicating reverse-phase high performance liquid chromatography (RP-HPLC) method has been developed and validated which can separate and accurately quantitate low levels of 26 betamethasone related compounds. The stability-indicating capability of the method was demonstrated through adequate separation of all potential betamethasone related compounds from betamethasone and also from each other that are present in aged and stress degraded betamethasone stability samples. Chromatographic separation of betamethasone and its related compounds was achieved by using a gradient elution at a flow rate of 1.0mL/min on a ACE 3 C18 column (150mmx4.6mm, 3microm particle size, 100A pore size) at 40 degrees C. Mobile phase A of the gradient was 0.1% methanesulfonic acid in aqueous solution and mobile phase B was a mixture of tert-butanol and 1,4-dioxane (7:93, v/v). UV detection at 254nm was employed to monitor the analytes. For betamethasone 21-aldehyde, the QL and DL were 0.02% and 0.01% respectively. For betamethasone and the rest of the betamethasone related compounds, the QL and DL were 0.05% and 0.02%. The precision of betamethasone assay is 0.6% and the accuracy of betamethasone assay ranged from 98.1% to 99.9%.

Comparison study of porous, fused-core, and monolithic silica-based C18 HPLC columns for Celestoderm-V Ointment analysis.

In this paper, three C18 columns with different substrates (i.e., porous ACE-3 C18, 3 microm, fused-core Halo C18, 2.7 microm, and monolithic Chromolith C18 were compared for the analysis of a pharmaceutical product, Celestoderm-V Ointment, that contains one active pharmaceutical ingredient, betamethasone-17-valerate and one critical pair of low level impurities, betamethasone-E-enolaldehyde and betamethasone-Z-enolaldehyde. Key column performance for the analysis of pharmaceutical products including selectivity, efficiency, separation impedance, resolution factor, sample loading capacity, linearity and lifetime from the three columns were determined. The potential applications of these three C18 columns for different methods for Celestoderm-V Ointment analysis are also recommended.

Development and validation of a stability-indicating HPLC method for simultaneous determination of salicylic acid, betamethasone dipropionate and their related compounds in Diprosalic Lotion.

Diprosalic Lotion is an anti-inflammatory drug product that contains salicylic acid and betamethasone dipropionate as active pharmaceutical ingredients (APIs). A reversed-phase high performance liquid chromatography (RP-HPLC) method was developed for simultaneous determination of salicylic acid, betamethasone dipropionate, and their related compounds in Diprosalic Lotion. A 150 mm x 4.6 mm I.D. YMC J'sphere ODS-H80 column at 35 degrees C and UV detection at 240 nm was used. A gradient elution was employed using 0.05% (v/v) methanesulfonic acid solution and acetonitrile as mobile phases. A total of thirty three compounds from Diprosalic Lotion samples were separated in 38 min. The stability-indicating capability of this method has been demonstrated by the adequate separation of all the impurities and degradation products in expired stability samples of Diprosalic Lotion. The method was validated as per the current ICH guidelines.

Development of a high performance size exclusion chromatography method to determine the stability of Human Serum Albumin in a lyophilized formulation of Interferon alfa-2b.

Intron Powder for Injection is a lyophilized formulation of Interferon alfa-2b marketed for treatment of Hepatitis C and some cancer indications. Human Serum Albumin (HSA) is used as a lyoprotectant and cryoprotectant at 1.0 mg/mL in the product formulation. No stability-indicating method, which can quantitate HSA and its dimer or oligomer aggregates in the formulated product, has been published to date. This paper describes the development and validation of a stability-indicating high performance size exclusion chromatography (HPSEC) method for the assay of HSA and estimation of HSA related compounds in lyophilized Intron Powder for Injection. The method employs a YMC-Pack Diol-200 column (7.8 mm x 30 cm, 5 microm porous particles with 250 A pore size), UV detection at 214 nm, and a mobile phase of 0.1 M phosphate buffer at pH 7.0 with 0.1 M sodium sulfate. The mobiles phase runs in an isocratic mode at 1.0 mL/min and the total chromatographic run time is 30 min. The method was validated for specific, linearity, accuracy, sensitivity, and robustness. It was shown to be specific for HSA and HSA aggregates (dimer and oligomers) with a limit of quantitation of 0.0005 mg/mL or 0.05% of HSA label claim in the presence of active therapeutic protein, Interferon alfa-2b, and the other pharmaceutical excipients, glycine, sodium phosphate dibasic, sodium phosphate monobasic. The method is stability indicating and is suitable for assay of HSA from 0.0005 mg/mL to 1.5mg/mL. (0.05-150% of HSA label claim) and for estimation of HSA related aggregates (dimer, and oligomer) from 0.0005 mg/mL to 0.15 mg/mL (0.05-15% of HSA label claim). The method is robust for routine use in product quality control. The method was applied to the analysis of batches of lyophilized Intron Powder for Injection of low, middle and high strength from the beginning, middle and end of shelf-life. The results indicated that HSA is stable in the product through out its shelf-life.

Development and validation of a stability-indicating reversed-phase high performance liquid chromatography method for assay of betamethylepoxide and estimation of its related compounds.

Betamethylepoxide (16beta-methyl-Delta(1,4)-pregnadiene-9beta-11beta-oxide-17alpha,21-diol-3,20-dione) is a key intermediate for the synthesis of various active pharmaceutical ingredients (APIs) of steroid compounds. A stability-indicating reversed-phase HPLC method for assay of betamethylepoxide and estimation of its related compounds has been developed and validated. This method can accurately quantitate betamethylepoxide in the presence of numerous structurally related compounds (including the alpha-epimer, known as alphamethylepoxide). This method can also adequately separate most of the impurities from each other and estimate their quantities in betamethylepoxide samples. The stability-indicating capability of this method has been demonstrated by adequate separation of the degradation products from betamethylepoxide in stress degraded and aged stability samples. The HPLC column used in the method was a 5 cm YMC Hydrosphere C(18) column (4.6 mm I.D.) and the mobile phase consisted of (A) water and (B) acetonitrile:methanol (8:25, v/v).