Penalty points for the determination of the analytical eco-scale score of the proposed method.

Parameters		Developed HPLC methoda	Penalty points
Reagents	PP of solvent = subtotal PP × number of pictogram × signal word	0.5 mM KH 2 PO 4 solution
		Consumed volume = 8.29 mL	0
		Subtotal PP = 1 [solvent <10 mL]
		Signal word= 0 none
		No. of pictogram = 0
	Consumed volume = run time × flow rate × solvent percentage in system	Methanol
		Consumed volume = 4.46 mL	6
		Subtotal PP = 1 [solvent <10 mL]
		Signal word = 2 danger [more severe hazard = 2]
		No. of pictogram =3
Instruments	Energy	≤1.5 kW h per sample	1
	Occupational hazard	Analytical process hermetization	0
	Centrifuge		1
	Wastes	>10 mL	5
Total penalty points		12
Analytical eco-scale total score		87

^aMobile phase consisted of 0.5 mM KH₂PO₄ solution : methanol (65 : 35, v/v).