Drug substance , analyzed with

Figure 8.2. The first HPLC chromatogram of a drug substance analyzed with generic broad gradient conditions. The active pharmaceutical ingredient (API) elutes at %B of 35-40% of acetonitrile, showing presence of several impurities between 5 and 9min.

The excipients present in pharmaceutical formulations can and often do interfere with quantitation of APIs, limiting the applications of direct UV-vis measurement for analyzing formulated products due to its lack of specificity. To minimize the interference of excipients, colorimetric methods based on chemical reactions have been used for rapid determination of drug substances in pharmaceutical formulations although their role in pharmacopoeias has been greatly reduced.117-122... 

Except for bulk drug substances, samples can rarely be analyzed directly following simple preparation steps. In many cases, an active ingredient is found in a formulated form or in a physiological fluid or tissue. Likewise, interferences associated with the matrix and the presence of possible degradation products, metabolites, and other closely related compounds mean the target analyte is often highly diluted. Also, an analyte may be difficult to detect. Effective sample preparation steps serve up to three broad purposes (1) eliminate and/or minimize possible interferences, (2) concentrate the sample, and (3) render the analyte of interest into a more easily detectable form. 

C. Eckers,N. Haskins and J. Langridge.The use of liquid chromatography combined with a quadrupole time-of-flight analyzer for the identification of trace impurities in drug substance. Rapid Communications in Mass Spectrometry, 1997,11(17), 1916-1922. 

Rao et al. reported a high performance liquid chromatographic method to determine diloxanide furoate and metronidazole in single and in combined dosage forms [41]. A 30 mg equivalent of diloxanide furoate and 25 mg of metronidazole (either as the bulk drug substances or in powdered tablets) was dissolved in methanol, amidopyrine added as the internal standard, and the mixture analyzed by HPLC at room temperature. The analytical column (30 cm x 3.9 mm) consisted of p-Bondapak Cig, with 9 9 1 1 methanol water 0.05 M KH2PO4 0.05 M NaH2P04 as the mobile phase. The flow rate was 1 mL/min), and detection was performed at 254 nm. 

A modem monograph is devoted to a drug substance and starts with a concise description of the product. This is followed by instructions for packaging and storage, relevant USP reference standards, identification, and procedures for the assay of the drag or the product components. There is sufficient information for those skilled in the arts to be able to analyze the drug and its components, making the compendium... 

Intrinsic Accuracy. Intrinsic accuracy indicates the bias caused by sample matrix and sample preparation. In this approach, a stock solution is prepared by using known quantities of related substance and drug substance. The stock solution is further diluted to obtained solutions of lower concentrations. These solutions are used to generate linearity results. In addition, these linearity solutions of different concentrations are spiked into placebo. The spiked solutions are prepared according to the procedure for sample analysis. The resulting solutions, prepared from the spiked solution, are then analyzed. If the same stock solution is used for both linearity and accuracy and all of these solutions are analyzed on the same HPLC run, the response of linearity (without spike into matrix) and accuracy (with spike into matrix) can be compared directly. Any differences in response indicate the bias caused by matrix interference or sample preparation. To determine the intrinsic accuracy at each concentration level, one can compare the peak area of accuracy (with matrix) with that of linearity (without matrix) at the same concentration (Figure 3.11). This is the simplest approach, and one would expect close to 100% accuracy at all concentration levels. 

A liquid chromatographic method is utilized for the determination of clopidogrel bisulfate in samples of the bulk drug substance. The method uses a column (L57 column size 15 cm x 4.6 mm) packed with ovomucoid (a chiral-recognition protein) that is chemically bonded to silica particles of 5 /im diameter and a pore size of 120 A. Both the reference standard and the sample to be analyzed are dissolved in methanol, and then diluted with mobile phase. The mobile phase is 75 25 0.01 M phosphate buffer /acetonitrile, and the flow rate is adjusted to 1.0 ml/min. Observation is made on the basis of the UV absorbance at 220 nm, and the clopidogrel peak has a relative retention time about 1.0 min. 

The routine studies for potential mutagenic activity normally performed on chemical drugs do not apply to biopharmaceuticals, as the administration of large amounts of peptides or proteins makes the results difficult to analyze. There is no evidence that these substances interact with DNA or other chromosomal material. In some instances the tests verify the effect of new excipients added to the formulated product. 

These have also been applied to the analysis of drug substances because they have advantages over equilibrium techniques, especially when mixtures of closely related compounds, compounds that react slowly, or catalytically acting compounds are to be analyzed. The selectivity and sensitivity of kinetic methods of analysis combined with the selectivity and sensitivity of ion-selective electrodes provide a versatile combination that may lead to new analytical schemes. ... 

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