Reference standards chromatographic methods

The standard chemical and biological methods of analysis are those accepted by the JnitedStates Pharmacopeia XXIII as well as the ones accepted by the AO AC in 1995 (81—84). The USP method involves saponification of the sample (dry concentrate, premix, powder, capsule, tablet, or aqueous suspension) with aqueous alcohoHc KOH solvent extraction solvent removal chromatographic separation of vitamin D from extraneous ingredients and colormetric deterrnination with antimony trichloride and comparison with a solution of USP cholecalciferol reference standard. 

The external standard method requires that the standard is chromatographed separately from the sample and thus, the chromatographic conditions must be maintained extremely constant. The great advantage of the external standard method is that the reference standard (or standards) can be identical to the solute (or solutes) of interest in the sample. Thus, a synthetic mixture can be made up in which the concentration of the components is closely similar to those of the sample. 

A number of analytical methods for the separation of organic mercury compounds use an initial extraction of the organic materials with an organic solvent. Klisenko and Shmigidina [83] then converted both the inorganic mercury held in the aqueous fraction and the organic mercury in the chloroform extract to dithizonate, separated the components on chromatographic columns, and determined the concentration of the various fractions by comparison with reference standards. This method is semi-quantitative at best. 

Tsuji and Goetz24 developed a quantitative high performance liquid chromatographic method for separating and measuring erythromycins A, B, and C, their epimers and degradation products. This method uses a /iBondapak Ci 8 reverse column with acetonitrile-methanol-O.2m ammonium acetate-water (45 10 10 25) as solvent. The pH and composition of the mobile phase may be adjusted to optimize resolution and elution volume. The authors utilized the procedure on USP reference standard and report a relative standard deviation of 0.64%. 

The preferred method of analysis for imparity qnantification is through the comparison to an external standard. External standard methods rely on a stable chromatographic system and the ability to obtain a reference standard for the imparity nnder test. 

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 third category of drugs are phytotherapeutical preparations 80% of the world population use exclusively plants for the treatment of illnesses [11]. Chromatography is relied on to guarantee preparations contain therapeutically effective doses of active drug and maintain constant batch composition. A quantitative determination of active principles is performed when possible, using pure reference standards. In many phytotherapeutic preparations, the active constituents are not known, so marker substances or typical constituents of the extract are used for the quantitative determination [11]. The Applications chapter of this book (Chapter 8) contains numerous references to the use of chromatographic methods in the control of plant extracts. 

A more complex situation is a multi-component mixture where all pure standards are available, such as a mixture of four pharmaceuticals.3 It is possible to control the concentration of the reference compounds, so that a number of carefully designed mixtures can be produced in the laboratory. Sometimes the aim is to see whether a spectrum of a mixture can be employed to determine individual concentrations, and, if so, how reliably. The aim may be to replace a slow and expensive chromatographic method by a rapid spectroscopic approach. Another rather different aim might be impurity monitoring,4 how well the concentration of a small impurity may be determined, for example, buried within a large chromatographic peak. 

Reliable quantitative results are obtained by external calibration if automatic injectors or autosamplers are used. This method involves direct comparison of the peak responses obtained by separately chromatographing the test and reference standard solutions. If syringe injection, which is irrepro-ducible at the high pressures involved, must be used, better quantitative results are obtained by the internal calibration procedure where a known amount of a noninterfering compound, the internal standard, is added to the test and reference standard solutions, and the ratios of peak responses of the analyte and internal standard are compared. 

There are two procedures for using reference standards in the first method a weighed amount of the standard can be added directly to the sample and the area or heights of the peaks of interest compared with that of the standard. This procedure is called the internal standard method. In the second procedure a weighed amount of the standard can be made up in a known volume of solvent and chromatographed under exactly the same conditions as the unknown sample, but as a... 

One cannot fail to note the vast expansion of the collection in the last few decades. Surely this was not fueled by additional biologic assays. Underlying the initial growth phase was the widespread utilization of spectrophotometry for identification and assay. Separation science was the second phase in pharmaceutical industry control laboratories. As a corollary, USP and NF method selection moved in the same direction. Spectrophotometric identity tests and assays are more reliable, especially for compliance testing, when performed in the relative mode, which uses a reference standard, rather than the absolute mode, which is the norm in titrimetry. There is some residual difference of opinion in other countries on this point, but that is rendered moot by the widespread adoption of separation science by the pharmaceutical industry and, thus, by the compendia. It is a characteristic of chromatographic methods that a reference standard be required, sometimes more than one for a procedure. The accumulation of modern tests and assays results in 5 to 10 uses for many reference standards. 

Titration The determination of assay values for reference standards, counter-ions, or impurities can often be independently determined via titration. While titration assays generally have less selectivity in comparison to chromatographic methods, the advantages of a broad spectrum of classical titration techniques that exist for organic functional groups is often overlooked. The methodologies include not only classical potentiometric acid/base titrations but also nonaqueous, redox, indirect, precipitation, and derivatization titrations.76-79... 

This method relies on the mass selectivity of the mass spectrometer in addition to the LC separation. No interferences were detected with any of these selected ions. The ions selected for quantitation were the most intense ions to obtain good sensitivity. Structure confirmation is inherent in an LC/MS method due to die structural information of the selected ion and their relative abundance. In addition LC/MS offers the LC chromatographic confirmation based on retention time relative to the reference standard. 

Along with the matrix RMs, a series of other quality control tools such as calibrants and standard solutions was also produced. On the occasion of the first set of reference materials, calibrants for trace elements and major components were also prepared and sent to the laboratories in order to minimize the differences in results produced by the use of different calibrants. For the other two sets of materials, multi component standard solutions for PAHs and pesticides were prepared and distributed in order to check the analytical performance of the chromatographic methods used. 

Standard test methods should be described in detail and should provide sufficient information to allow properly trained analysts to perform the analysis in a reliable manner. As a minimum, the description should include the chromatographic conditions (in the case of chromatographic tests), reagents needed, reference standards, the formulae for the calculation of results and system suitability tests. 

A reference standard batch of the drug substance is used to conduct structure elucidation and confirmation studies. The elucidation and confirmation of structure should include physical and chemical information derived from applicable analyses, such as (1) elemental analysis (2) functional group analysis using spectroscopic methods (i.e., mass spectrometry, nuclear mag netic resonance) (3) molecular weight determinations (4) degradation stud-ies (5) complex formation determinations (6) chromatographic study methods using HPLC, GC, TLC, GLC (7) infrared spectroscopy (8) ultra-violet spectroscopy (9) stereochemistry and (10) others, such as optical rotatory dispersion (ORD) or x-ray diffraction. 

A qualitative method is primarily an identification test that confirms the presence (or absence) of a certain analyte(s) in the sample by matching retention time with that of a reference standard. UV spectral data from a photodiode array detector are often used as a secondary confirmation technique. This type of method can be a limit test to evaluate whether the level of the analyte is above or below a certain preset limit or to generate a chromatographic profile for comparative purposes. 

The ultimate goal of an assay or an analytical procedure is to measure accurately a quantity or a concentration of an analyte, or to measure a specific activity, as in some assays for biomarkers. However, many activity assays, such as cell-based and enzyme activity assays, may not be very sensitive, may lack precision, and/or do not include the use of definitive reference standards. Assays based on measurements of physicochemical (such as chromatographic methods) or biochemical (such as ligand-binding assays) attributes of the analyte assume that these quantifiable characteristics are reflective of the quantities, concentration, or biological activity of the analyte. For the purpose of bioanalytical method validation, we will follow the recently proposed classifications for assay data by Lee et al. [4,5]. These classifications, as summarized below, provide a clear distinction with respect to analytical validation practices and requirements. 

More specific characterization of the identity of a DS is performed (if suitable) by measurement of its melting point, the specific rotation (in solution), and by determination of the IR- or UV-spectrum of the substance (as compared to the spectrum of a reference standard). All chromatographic methods - for example, thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and gas chroma-... 

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