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Synthesis, characterisation and development of novel, validated methods for the detection and quantification of diphenidine-derived New Psychoactive Substances

Alkirkit, S. M. O. (2020) Synthesis, characterisation and development of novel, validated methods for the detection and quantification of diphenidine-derived New Psychoactive Substances. Doctoral thesis (PhD), Manchester Metropolitan University.


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There is a worldwide increase in the abuse of new psychoactive substances, which pose a threat to public health. The fast‐paced nature of the NPS market and increased availability is drawing international concern. There is a general lack of comprehensive evidence on the toxicity and abuse risks associated with long-term use. The rapid pace of NPSs development means that they remain as an area of concern and interest; this shows the technical challenges in terms of development of analytical methods for the detection and determination of these substances. There is a knowledge gap in terms of chromatographic methods of detection, separation and quantification of diphenidine and its derivatives, in particular HPLC approaches. Currently, there is more research applying GC methods for NPSs analysis. The aim of this project is to develop a reliable, rapid, sensitive and robust HPLC method for the analysis of this group of NPSs. Regioisomeric compounds, 2-, 3- and 4-methoxphenidine (MXP) were used to develop a robust high performance liquid chromatography (HPLC) method using mobile phase (acetonitrile:ammonium acetate 55:45% v/v) whilst the stationary phase was ACE-5 C18 AR column (150 x 4.6 mm, 5 μm particle size). The method was validated according to the International Conference on Harmonisation of Technical Requirements of Pharmaceutical for Human Use (ICH) guidelines and shown to be both selective and sensitive (Limit of Detection, LOD = 0.04-0.15 μg mL-1, Limit of Quantification, LOQ = 0.38-0.47 μg mL-1). The reference materials used for this study were characterised using 1H NMR, 13C NMR, IR, UV and GC-MS. The scope of the study was applied to the recently reported diphenidine derivatives, 2-, 3- and 4-fluephenidines, in addition to 2-, 3- and 4-fluorocyanophenidines and the halogenated diphenidine compounds. x Accuracy, precision, robustness and specificity of these substances were investigated. As an average, limit of detection (LOD) was between 0.05 and 0.60 μg mL-1, whereas, limit of quantification (LOQ) was between 0.16-1.84 μg mL-1 for all the diphenidine derived regioisomers tested in this study. Additionally, total run time of just 10 minutes with resolution values (Rs) of greater than 2 in the case of both MXP isomers and all tested halogenated diphenidine isomers indicates that the applied HPLC method was rapid and sensitive, therefore it can be implemented to examine any samples that might contain these substances.

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