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An HPLC method for the separation of six target alkaloids from Papaver somniferum L. (morphine, codeine, oripavine, thebaine, papaverine, and noscapine) was developed, optimized, and validated. The chromatographic behavior of these alkaloids was investigated using a reversed-phase chromatography at acidic and alkaline pH. The effects of ion-pairing agents, pH value of the mobile phase, concentration of the buffer components, mobile phase organic modifier, and column temperature were studied. Regardless of the large differences in their pKa values, all alkaloids were separated within a close retention window, and good peak shape was achieved for each of the six alkaloids. The proposed method has adequate selectivity, linearity, accuracy, precision, and reproducibility and is applicable for poppy straw.

The excessive and continuously growing interest in the simultaneous determination of poppy alkaloids imposes the development and optimization of convenient high-throughput methods for the assessment of the qualitative and quantitative profile of alkaloids in poppy straw. Systematic optimization of two chromatographic methods (gas chromatography (GC)/flame ionization detector (FID)/mass spectrometry (MS) and reversed-phase (RP)-high-performance liquid chromatography (HPLC)/diode array detector (DAD)) for the separation of alkaloids from Papaver somniferum L. (Papaveraceae) was carried out. The effects of various conditions on the predefined chromatographic descriptors were investigated using chemometrics. A full factorial linear design of experiments for determining the relationship between chromatographic conditions and the retention behavior of the analytes was used. Central composite circumscribed design was utilized for the final method optimization. By conducting the optimization of the methods in very rational manner, a great deal of excessive and unproductive laboratory research work was avoided. The developed chromatographic methods were validated and compared in line with the resolving power, sensitivity, accuracy, speed, cost, ecological aspects, and compatibility with the poppy straw extraction procedure. The separation of the opium alkaloids using the GC/FID/MS method was achieved within 10 min, avoiding any derivatization step. This method has a stronger resolving power, shorter analysis time, better cost/effectiveness factor than the RP-HPLC/DAD method and is in line with the "green trend" of the analysis. The RP-HPLC/DAD method on the other hand displayed better sensitivity for all tested alkaloids. The proposed methods provide both fast screening and an accurate content assessment of the six alkaloids in the poppy samples obtained from the selection program of Papaver strains.

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The global need for changing the processes in order to meet the green analytical chemistry (GAC) criteria is a great challenge for the pharmaceutical industry. High-performance liquid chromatography (HPLC), as one of the most frequently used techniques in various stages in the pharmaceutical industry, generates huge amounts of organic toxic waste. Therefore, the implementation of the GAC principles in pharma analysis is highly required. Although the number of published papers concerning green chromatography approaches is constantly increasing, the use of eco-friendly HPLC methods in the pharma industry has not been widely implemented. The reasons for this mainly include the need for adaptation of the conventional HPLC instruments, lack of time, lack of experience, or uncertainty of the analysts regarding fulfillment of the method criteria. In this chapter, an overview of green strategies that can be easily applied to conventional instruments for liquid chromatography (LC) in developing eco-friendly HPLC methods in pharma analysis is given. The aim is to emphasize that the green method development in pharma analysis can be easily accomplished and to encourage the analytical community in the pharmaceutical industry not only to develop but also to transfer the already established conventional HPLC methods into green ones.

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