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14 QUALITY CONTROL OF MEDICINAL AND AROMATIC PLANTS AND THEIR EXTRACTED PRODUCTS BY HPLC AND HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY 14.3.3.5.1 Repeatability Repeatability is precision under the same operating conditions over a short interval of time (intra-day precision). It is demonstrated by a minimum of 9 determinations covering the specifi ed range for the procedure (e.g. 3 concentrations/3 replicates each) or a minimum of 6 determinations at 100% of the test concentration. 14.3.3.5.2 Intermediate Precision Intermediate precision expresses intra-laboratory variations: different days, different analysts, different equipment, etc. 14.3.3.5.3 Reproducibility Reproducibility is precision at the inter-laboratory level. It is especially important if the analytical procedure is to be used in different laboratories, for instance, a pharmacopoeial procedure. 14.3.3.6 Detection Limit Detection limit (DL) is the lowest amount of analyte in a sample which can be detected, but not necessarily quantitated as an exact value. It is determined by analyzing samples containing known concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected. Other methods of determining DL are based on the signal-to-noise ratio and on the standard deviation of the response and the slope. A signal-to-noise ratio of 3:1 or 2:1 is a fair estimate of DL. Using the standard deviation of the response (σ) and slope of the calibration curve (S), DL=3.3σ/S. 14.3.3.7 Quantitation Limit Quantitation limit (QL) is the lowest concentration of the analyte that can be determined with acceptable precision and accuracy under the stated experimental conditions. It is generally determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be quantifi ed with acceptable accuracy and precision. QL can also be defi ned from the signal-to-noise ratio (a typical signal-to-noise ratio is 10:1), or from the standard deviation of the response (σ) and the slope (S) of the calibration curve (QL=10σ/S). 14.3.3.8 Robustness Robustness of an analytical method ensures that it performs well and has few variables affecting its performance. For example, a robust HPLC analytical procedure does not show variation when columns of differ- 246
EXTRACTION TECHNOLOGIES FOR MEDICINAL AND AROMATIC PLANTS ent lots or from different manufacturers are used, when there is a slight variation in pH or composition of the mobile phase, or when temperature and fl ow rate vary. Steps of extraction, purifi cation or enrichment of an analyte in herbal material should be simple and time effective. Solutions used in the analysis should be stable over a reasonable period of time. Lastly, the procedure should be feasible in most laboratories. If measurements are prone to variations with small changes in the test conditions then such conditions should be suitably controlled or a precautionary statement should be included in the procedure. 14.4 Thin Layer Chromatography in Quality Control of Plant Products Thin layer chromatography (TLC), also called planar chromatography, is a widely accepted and extensively used separation technique that is over 65 years old. The technique is simple, cost effective, versatile, and useable in all laboratories around the globe. It can be easily adapted to any given situation of qualitative, quantitative or preparative separation. Despite the great variety and complete automation of the technique, it still lags behind other chromatographic techniques when it comes to its use as analytical technique. However, there is no substitute for this technique for situations requiring qualitative analyses of plant extracts. TLC has nearly become indispensable for the standardization of plant materials, be it the fingerprint profiling or analysis of a marker. The advantages of the technique over other analytical techniques are many when handling plant materials. The samples can be applied without undertaking tedious, time-consuming processes of sample preparation. The loss in sensitivity is far compensated by the gain on several fronts, including ease of assays, multiple sample analyses and low cost per sample. The two prominent uses of TLC in the standardization of plant materials include fi ngerprint profi ling for the assessment of chemical constituents of a drug and quantitative analysis of markers in plant drugs. A typical TLC procedure involves sample preparation, selection of the chromatographic layer and the mobile phase, sample application, development and drying of the plate, derivatization (if required) and chromatogram evaluation. 14.4.1 Sample Preparation Methods of sample preparation for fi ngerprinting and estimation of marker differ signifi cantly. Whereas in fi ngerprinting, only proportionate quantities of components must be extracted, in assaying the marker complete and exhaustive extraction has to be ensured. Correspondingly, particle size of the crude drug, the solute-solvent ratio, extraction period and 247
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CONTRIBUTORS Chapter 6 Aqueous Alco
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7 DISTILLATION TECHNOLOGY FOR ESSEN
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9 SOLID PHASE MICRO-EXTRACTION AND
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