An HPLC method was being developed to analyse nitrazepam (a benzodiazepine drug) in blood. Nitrazepam from the samples was extracted and reconstituted in HPLC eluent ready for analysis. (a) To determine the detection limit ten blank samples were run. The standard deviation of the ten absorbance readings was 0.003. A 1.00 mg L-1 standard had an absorbance of 0.82. Using a value of 3xstandard deviation for the detection limit response, determine the detection limit, showing how you arrived at your answer. (3 marks) b) Determine the quantitation limit, showing how you arrived at your answer. (3 marks) (c) The expected maximum concentration of nitrazepam in the samples after extraction and reconstituting in eluent was 0.2 mg L-1. Suggest appropriate values for the calibration standards. (3 marks) (d) Explain what is meant by repeatability. To determine the repeatability, several replicate samples of the same concentration were analysed and gave a standard deviation of 0.008 mg L-1. State the minimum number of replicates required to determine repeatability. Calculate the repeatability, showing how you do this. (4 marks) (e) The accuracy of the method was checked using a certified reference material (CRM). Explain what a certified reference material is. State two types of CRM. (3 marks) Accuracy was also checked by determining the recoveries of spiked samples. Explain what spiking is. Explain what precautions are necessary to ensure that spiking will give a good result. (2 marks) (g) Explain what is meant by "working range". How are the limits of the working range usually set? (2 marks) An HPLC method was being developed to analyse nitrazepam (a benzodiazepine drug) in blood. Nitrazepam from the samples was extracted and reconstituted in HPLC eluent ready for analysis. (a) To determine the detection limit ten blank samples were run. The standard deviation of the ten absorbance readings was 0.003. A 1.00 mg L-1 standard had an absorbance of 0.82. Using a value of 3xstandard deviation for the detection limit response, determine the detection limit, showing how you arrived at your answer. (3 marks) b) Determine the quantitation limit, showing how you arrived at your answer. (3 marks) (c) The expected maximum concentration of nitrazepam in the samples after extraction and reconstituting in eluent was 0.2 mg L-1. Suggest appropriate values for the calibration standards. (3 marks) (d) Explain what is meant by repeatability. To determine the repeatability, several replicate samples of the same concentration were analysed and gave a standard deviation of 0.008 mg L-1. State the minimum number of replicates required to determine repeatability. Calculate the repeatability, showing how you do this. (4 marks) (e) The accuracy of the method was checked using a certified reference material (CRM). Explain what a certified reference material is. State two types of CRM. (3 marks) Accuracy was also checked by determining the recoveries of spiked samples. Explain what spiking is. Explain what precautions are necessary to ensure that spiking will give a good result. (2 marks) (g) Explain what is meant by "working range". How are the limits of the working range usually set? (2 marks)