The analytical GREEnness metric (AGREE) tool is widely used as a reliable greenness assessment method for chromatographic analyses. The AGREE tool has the ability to determine the greenness of analytical methods in terms of both quality and quantity, whereas other commonly used methods assess the greenness either quantitatively or qualitatively. Greenness profiles of six chromatographic methods for simultaneous estimations of serotonin, dopamine, acetylcholine, GABA and glutamate were assessed using AGREE and NEMI tools as a case study. The AGREE assessment tool proved to be user-friendly, and provides a full profile of assessment, hence it can be described as the tool of choice for the assessment of LC-MS chromatographic methods. For optimum application, the weights of 4 of the 12 assessment criteria were set high (weight of four) due to their importance, namely criteria number 7 (waste), number 8 (analysis thruput/number of analytes per run), number 11 (toxicity) and number 12 (operator’s safety). Setting proper weights of the assessment criteria contributed significantly to the discrimination of greenness of the compared methods. The selected greenest method for the analysis of the proposed quinary mixture showed an AGREE tool pictogram with a 0.66 score. Additionally, the selected method allows simultaneous estimation of seven constituents in total. It offers high sensitivity, allowing detection of acetylcholine, serotonin and glutamate at levels as low as 2 pg, and dopamine, norepinephrine, GABA and glycine at levels as low as 10 pg, and finally offers fast analysis where all components can be analyzed within 5 min.

Numerous agents with anxiolytic or stimulant effects have the potential to be overused, and their misuse is associated with serious side effects. In Saudi Arabia, the estimated percentage of Saudis who abuse drugs is around 7–8% and the age range is 12–22 years. Methamphetamine, captagon, tramadol, heroin, and cannabis/cannabinoids have been proven to be the most commonly abused drugs in Saudi Arabia, with methamphetamine being at the top of the list. The present study focuses on the chromatographic analytical methods used for the analysis of methamphetamine in combination with commonly abused drugs, aiming to point out the greenest among them. These mixtures have been chosen as they are analyzed periodically and frequently in criminal evidence and forensic medicine. Therefore, the chances of hazards for analysts and the environment are high if the mixtures are not handled appropriately. This study aims to compare 23 chromatographic methods used for the analysis of methamphetamine mixtures in four major combinations, and to assess their greenness by using three greenness assessment tools, namely, NEMI, ESA and AGREE, to recommend the greenest analytical method. The NEMI results were proven to have low discriminating abilities and, accordingly, the comparisons are based on ESA and AGREE scores. The analysis results show that the safest methods with the most eco-friendly results (based on ESA and AGREE) are the GC-MS method proposed by Mohammed et al. to analyze methamphetamine and captagon mixtures (ESA = 79 and AGREE = 0.57), the UHPLC–MS-MS method proposed by Busardò et al. to analyze methamphetamine and cannabis/cannabinoid mixtures (ESA = 78 and AGREE = 0.57), the LC-MS method proposed by Herrin et al. to analyze methamphetamine and tramadol mixtures (ESA = 81 and AGREE = 0.56), and the LC-MS method proposed by Postigo-et al to analyze methamphetamine and heroin mixtures (ESA = 76 and AGREE = 0.58).