HPLC gradient assay method

The case stndy of the SP development of an HPLC assay method for an OTC prodnct derived from natural materials illustrates the importance of optimizing both the extraction and filtration conditions to fully recover all the labile active components in the formulation. The HPLC gradient assay method, which separates all active ingredients from other... 

The determination of lovastatin and its hydroxyacid metabolite in plasma and bile can be accomplished by high performance liquid chromatography (25). Plasma samples are prepared for analysis by solid phase extraction and are analyzed using isocratic elution on a Cl 8 column. Bile samples do not require any sample dean-up prior to HPLC analysis, but do require the use of a gradient elution method to separate the compounds of interest. The HPLC assay has a limit of detection of 25 ng/mL... 

An HPLC assay was described as a routine method for the determination of FLU and its hydroxylated metabolite 7-OH FLU in pig kidney tissues (188). The sample was extracted with ethyl acetate after evaporation, the residue was dissolved in MeCN-oxalic acid (1 1). Analytical separation was performed using fluorimetric detection under gradient elution. The authors recommended an Ultrabase C-18 column, which allowed the work to be carried out at extreme pH values, ranging from 2 to 8. The assay was specific and reproducible within the range 50-2500 yug/kg recovery was 94.8%. 

An HPLC assay, content uniformity and related compounds method for a blockbuster new drug product, was developed that utilized concave gradient elution, a flow rate of 1.25 ml/min and no temperature control of the column. Samples were placed in 1000-ml volumetric flasks, sample diluent was added, and the flasks were sonicated for 10 min followed by 30 min of mechanical shaking. This method was to be run in a QC laboratory at the contract manufacturing facility in Puerto Rico. While the developed method worked flawlessly in the development laboratory, the QC laboratory had many problems in performing the procedure. 

Assay Procedure An aliquot (0.1 mL) is diluted to 100.0 mL with H20 and then analyzed by the following HPLC method. Instrument Spectra Physics 8800. Column 4.1 times 250 mm Ultrasphere C-8 (Altex Inc.). Eluent A H20 (0.1% v/v H3PO4). Eluent B MeCN. Gradient 97 3 to 35 65 A B over 25 min. Flow Rate 2.0 mL/min. Temperature 45°C. Injection 10.0 nL. Detection UV (230 nm). Retention Times Sulfonic Acid (cis/trans isomers) 5.0 min, Acetamidosulfone (cis isomer) 9.0 min, Acetamidosulfone (trans isomer) 10.0 min. The sulfonylation reaction was considered to be complete when less than 1% of acetamidosulfone (vs. the sulfonic acid - 6-methyl-7,7-dioxo-4,5,6,7,-... 

Potency and purity. Chromatographic methods are frequently used to measure the quantity of a single active species which is equated to the potency for most drugs. Related substances may be quantitated by similar procedures, usually incorporating gradient elution (for ion exchange or RP-HPLC methods) to insure that all species are observed. The related substance assay defines the purity of the material. The performance characteristics of these methods are crucial for the correct evaluation of potency and purity. 

In some cases, drug substance does not have chromophores with a molar absorbtivity sufficient for accurate quantitation using UV detection. If HPLC with UV detection is used as a basic quantitation technique, then MS detection as a complementary technique is desirable in most cases. LC-MS is essentially preferable in most preformulation assays. High selectivity of the MS detector allows the use of fast gradient HPLC separation methods, which does not require significant development time. Practically in all assays used in preformulation, the quantitation of only drug substance is required and MS detection provides an accurate quantitation. 

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