Table 5.

Chromatographic methods for the analysis of the drug substance in different biological matrices.

Matrix	Objective of analysis	Stationary phase	Mobile phase	Gradient		Detection	Flow rate	Column temperature	Reference
Urine (Human)	Drug content	Zorbax SB-C8 Column 2.1 × 100mm,1.8 μm	Mobile phase A-10 mM ammonium formate and acetic acid (pH 4) Mobile B- Acetonitrile	Time	%B	Triple quadrupole mass spectrometer (Waters) Positive mode	400 μL/min	20 °C	[31]
					10
				8	55
				8.1	100
				8.6	10
				10.6	10
Urine (human)	Drug content	Zorbax XDB-C8 Column 2.1 × 150mm,5.0 μm	Mobile phase A-10 mM ammonium formate and acetic acid (pH 4) Mobile B- Acetonitrile	Time	%B	TSQ Quantum Ultra triple quadrupole mass spectrometer With positive mode	250 μL/min
				0	40
				5	90
				9.5	90
				9.6	40
				15	40
Plasma and urine (human)	Drug and metabolite analysis	Biphenyl (100 × 2.1 mm, 2.7 μm	Mobile phase A-25 mM ammonium formate with 0.1% formic acid Mobile B- Acetonitrile	Time	%B	Triple quadrupole mass spectrometer	400 μL/min	40 °C	[7]
				0	14
				0.5	70
				6.5	70
				6.6	95
				7.5	95
				7.6	14
				9.0	14
Human hepatocyte	Metabolites study	Waters, BEH C18 Column 2.1 × 50mm,1.7 μm	Mobile phase A-5 mM ammonium acetate pH 3.5 Mobile B- Acetonitrile	Time	%B	Water Xevo TQ-S triple quadrupole mass spectrometer	300 μL/min	50 °C	[33]
				0	0.5
				0.5	5
				6.0	50
				8.0	50
				10.0	70
				12.0	95
				14	95
				16.5	5
Liver microsome and fungal sample		Acquity UPLC BEH C18 Column 2.1 × 100 mm,1.7 μm	Mobile phase A-0.1% Formic acid B- methanol	Time	%B		500 μL/min	65 °C
				0	5
				1.0	5
				6.0	95
				8.0	95
				8.1	5
				10.0	5
Plasma and urine(mice)	Metabolites study	Acquity HSS T3; C18, 150mmx2.1 mm,1.8 μm	Mobile phase A-0.01% Formic acid B- Acetonitrile with 0.01% Formic acid	Time	%B	Waters synapt G2-Si QTOF MS with ESI	400 μL/min	40 °C	[34]
				0	1
				1.0	1
				8.0	99
				13	85
				10.0	1
Plasma (human)	Pharmacokinetics and drug-drug interaction study	C18 Column	Mobile phase A-0.1% Formic acid B- acetonitrile 0.1% Formic acid	Isocratic – A: B (45:55)					[37]
Urine	Antidoping analysis	Synchronis C18 (100 × 2.1 mm, 1.7 μm)	Mobile phase A-0.2% Formic acid B- acetonitrile 0.2% Formic acid	Time	%B	Thermo-scientific Q Exactive plus tandem mass spectrometer	500 μL/min		[38]
				0	2
				0.5	2
				8.5	95
				10.0	95
				12.0	2
Urine (human)	Simultaneous analysis of drug	Supelco AscentisR C18 (150 mm × 2.1mm × 2.7 μm)	Mobile phase A-0.1% Formic acid B- acetonitrile 0.1% Formic acid	Time	%B	QExactive focus benchtop Orbitrap-based mass spectrometer	250 μL/min	30 °C	[39]
				0	5
				7	65
				8	100
				11.0	100
				13.0	5
Urine (Horse)	Metabolites study	Eclipse plus C18 (4.6 × 150mm, 3.5 μm)	Mobile phase A-5Mm ammonium acetate or 0.2% formic in water B- acetonitrile	Time	%B	QExactive high-resolution accurate mass spectrometer	600 μL/min		[40]
				0	2
				5	95
				8.0	95
				8.5	50
				13.5	50
				14.0	50
				15.5	2
Liposome formulation	HPLC assay	Supelco Discovery BIO wide pore C5, 250mmx4.6 mm,5 μm	Mobile phase A-5% acetonitrile and 0.1% trifluoroacetic acid in water Mobile phase B-95% acetonitrile and 0.1% trifluoroacetic acid in water	Time	%B	UV 348 nm	1 mL/min		[41]
				0	30
				20	100
				21	30
Active pharmaceutical substance	Purity analysis	Kinetex C18 150mmX3.0 mm,2.6 μm	Mobile phase A-10 mM ammonium formate pH 6.3 Mobile Phase B-acetonitrile	Time	%B	LTQ XL Orbitrap Mass spectrometer (Thermo)	0.6 mL/min		[42]
				0	30
				4	30
				18	100
				23	100
				25	30
				30	30
		Supelco- Ascentis Express C18	Mobile phase A-0.01 M pH 3.0 Phase B-Acetonitrile	Time	%B	UV 225 nm
				0	10
				1	10
				10	90
				12	90
				13	10
				14	10