Quality control dilution linearity

A simple and rapid RP-HPLC method was developed for the determination of retinoid in galenicals. Commercial preparations were diluted, filered and used for separation. Measurements were carried out in an ODS column (150 X 4.6 mm i.d. particle size 3 /xm). Solvents A and B were methanol-10 mM ammonium acetate (75 25, v/v) and methanol-THF (84 16, v/v), respectively. The flow rate was 0.8ml/min. Gradient conditions were 0-25 min, 0 per cent B 35 min, 100 per cent B, isocratic for 10 min. Typical chromatograms are shown in Fig. 2.37. The repeatability of peak area ranged between 0.48 -3.2 per cent for UV-DAD and 0.57 - 3.1 per cent for fluorescence detection. The reproducibility varied between 0.26 - 4.6 per cent. It was found that the method is precise, selective, sensitive and linear, therefore, it can be employed for the routine quality control of this class of drags [85],... 

Murakami et al. [82] developed and validated a sensitive HPLC technique to quantify omeprazole in delayed release tablets. The analysis was carried out using a RP-Cig column with UV-VIS detection at 280 nm. The mobile phase was diluted with phosphate buffer (pH 7.4) and acetonitrile (70 30) at a flow-rate of 1.5 ml/min. The parameters used in the validation process were linearity, range, quantification limit, accuracy, specificity, and precision. The retention time of omeprazole was about 5 min with symmetrical peaks. The linearity in the range of 10-30 ng/ml presented a correlation coefficient of 0.9995. The excipients in the formulation did not interfere with the analysis and the recovery was quantitative. Results were satisfactory and the method proved to be adequate for quality control of omeprazole delayed-release tablets. 

Dilution linearity is also normally monitored during the study sample analysis phase by using dilution linearity quality control (DLQCs) samples. These samples are prepared at concentrations above the assay range and then diluted into range at the same time as the study samples. The dilutions performed for these DLQCs should be representative of how the samples are diluted. 

Linearity of a method should be established or a series of standards selected for use with non-linear-method calibration. This can be checked by preparing and analyzing serial dilutions of aqueous reference standard solutions, quality control materials, enzyme solutions, or commercially available materials for demonstrating linearity (again, these are designed for use in human medicine) and comparing the determined values with the theoretical values calculated for the dilutions. The serial dilutions used for linearity checks can also help establish the analytical sensitivity when defined as the minimal detectable change from one concentration to another. 

The linearity of the calibration curve was ascertained by adding known concentrations of manufactured lactose to cow milk over the concentration range of interest. Intraday precision was determined by analyzing 15 replicates of low- and high-quality control samples. Interday precision was determined by analyzing qnal-ity control samples over 15 consecutive days, and the coefficient of variation for lactose over two levels was calculated. The low limit of quantification was condncted by additional dilution until indication of peak-to-peak signal-to-noise ratio as 5 1 was achieved. To determine the recovery rate, lactose was added to cow milk samples. Absolute recovery was indicated by a ratio of the observed value to the corresponding expected value. 

The specific and robust nature of the pups response to milk allowed Keil et al. (1990) to develop a behavioural bioassay for use in characterizing the actual pheromonal substance or substances. Using the bioassay it was found that pups responsiveness declines linearly with the exponent of dilution, and that cues contained in the milk are so potent that milk diluted by as much as 10 4 still elicits significantly more responses than cow s milk or other control substances (Keil et al. 1990). However, when left at room temperature milk loses most of its behaviour-releasing quality within about 30 minutes, but retains it for several weeks when stored at -40°C (Muller 1978 Keil et al. 1990). 

---