Table 1.

Enantioselectivity analytical methods in biological matrices for the pharmacokinetics profile.

Compound	Matrix	Enantioselectivity in Pharmacokinetics	Analytical Method	Observation	Ref.
R /S (±)-tramadol (TMD)	Human Plasma	The main metabolic pathways are O-demethylation in O-desmethyl tramadol (1st metabolite) by the polymorphic cytochrome isoenzyme P450 2D6 (CYP2D6) and N-demethylation for N-desmethyl tramadol (2nd metabolite) by CYP2B6 and CYP3A4.	LC-FD; Column: Chirapak® AGP Mobile Phase: 30 mM diammonium hydrogen phosphate buffer: ACN (98.9:1:0.1, v/v) (pH 7) Flow rates: 0.5 mL min−1	(+)-TMD has greater affinity for µ receptor and inhibits serotonin reuptake, while the (-)-TMD is a more effective inhibitor of norepinephrine reuptake.	[73]
nadolol (NAD)	Human Plasma	NAD is almost exclusively excreted unchanged in urine. As the degree of stereoselectivity in renal clearance is low, a decrease in renal function can be expected to cause proportionally equal increases in plasma concentrations of four enantiomers.	LC-UV and LC-FD; Column: Chirapak® AD-H Mobile Phase: HEX: EtOH: DEA:Trifluoroacetic (88:12:0.4:0.23 v/v/v/v) Flow rates: 1.0 mL min−1	Only one of the eight stereoisomers (R,S,R-NAD) is responsible for the therapeutic effect. The main challenges encountered when employing the CSP to analyze biological samples is the co-elution of enantiomers of interest and interfering matrix compounds.	[74,75]
R /S (±)-ketamine (KET)	Rat Plasma	(S)-KET is metabolized to (S)-norketamine, which produces rapid and sustained antidepressant-like effects and could be an alternative to (S)-KET.	LC-MS/MS Column: Chiralpak® AS-3R Mobile Phase: 1 mM ammonium bicarbonate: ACN (54:46 v/v) Flow rates: 1.0 mL min−1	(R)-KET had greater potency and longer lasting antidepressant effects than (S)-KET. However, (R)-KET has fewer detrimental side effects than either (R,S)-KET or (S)-KET.	[76,77]
R /S (±)-propranolol (PHO)	Human Urine	90% of ingested PHO was found in 12 metabolites in urine.	2D-LC-MS/MS and UV; Column: PhenylHexyl in the first dimension. Teicoplanin-based chiral column in the second dimension. Mobile phase: 10 mM ammonium formate in water (adjusted to pH 3): MeOH (gradient mode). Flow rate: 0.4 mL min−1 and the column temperature were 30 °C (first dimension).	The (S)-PHO shows more β-blocking activity than (R)-PHO.	[78]
R/S (±)-salbutamol (SBT)	Human Urine	(R)-SBT l is metabolized up to 12 times faster than (S)-SBT. Both enantiomers are actively excreted in urine.	HPLC-ESI-MS; Column: Chirobiotic® V Mobile phase: MeOH: AcOH: TEA (100:0.025:0.75 v/v/v)	(R)-SBT is 80 times more active than (S)-SBT. The inactive (S)-SBT may have undesirable actions on lung function.	[79,80]
R/S (±)-omeprazole (OMZ)	Human Plasma	The pharmacokinetic studies suggest that the efficacy of (S)-enantiomer depends on the metabolic pathway and excretion.	LC-UV; Column: Lux® Amylose-3 Mobile phase: HEX: EtOH (70:30 v/v) Flow rate: 1.0 mL min−1	The clearance of (S)-OMZ is lower than that of the (R)-OMZ.	[27,81]
R/S (±)-venlafaxine (VNF)	Rat Plasma	VNF is metabolized by CYP450 enzymes and the most abundant metabolite is O-desmethylvenlafaxine, found in plasma in high concentrations.	LC-MS/MS; Column: Chirobiotic® V Mobile phase: 30 mM AA, pH 6.0: MeOH (15:85)	The (S)-VNF inhibits serotonin reuptake, while the (R)-VNF inhibits serotonin and norepinephrine reuptake. O-desmethylvenlafaxine is a pharmacologically active metabolite which contributes to the therapeutic effect of VNF.	[44,82]
R/S (±)-ibuprofen (IBU)	Human Plasma	There is a conversion of the inactive (R)-enantiomer to its pharmacologically active (S)-enantiomer.	LC-MS/MS; Column: LUX® Cellulose-3 Mobile phase: 0.05% FA solution: MeOH (30: 70 v/v) Flow rate: 0.2 mL min−1	(S)-IBU has been reported to be 160 times more active that (R)-IBU.	[83]
R/S (±)-citalopram (CIT)	Human Plasma and breast milk	The (S)-CIT enantiomer and its ((S)-DCIT and (S)-DDCIT) metabolites are eliminated more quickly than their enantiomers.	LC-UV; Column: Phenomenex® Lux Cellulose-2 Mobile phase: AA (pH 9.0; 20 mM): ACN (gradient mode) Flow rate: 0.6 mL min−1	In the treatment of depression, (S)-CIT is over 100-fold more potent as a selective serotonin reuptake inhibitor than (R)-CIT.	[84,85]
R/S (±)-verapamil (VRP)	Human Plasma	After intravenous administration, the plasma clearance and apparent volume of distribution of (S)-VRP are almost twice as high as those of (R)-VRP.	LC-FD; Column: Chiralpak® AD Mobile phase: HEX: ISO: EtOH (85:7.5:7.5 v/v) and 0.1% TEA Flow rate: 1.5 mL min−1 with column oven temperature was 30 °C	(R)-(+)-VRP has far less cardiotoxicity than (S)-(-)VRP. However, the pharmacological potency of (S)-VRP is 10–20 times greater than its (R)-VRP in terms of negative chromotropic effect on atri-ventricular conduction and vasodilatator in man.	[55,86]
R/S (±)-felodipine (FLP)	Human Plasma	The elimination of FLP from the body depends on the metabolic clearance of CYP450. The metabolism rate of (R)-FLP was faster than that of (S)-FLP in human liver microsomes.	LC-UV Column: Chiracel® OJ Mobile phase: HEX:ISO (5:1 v/v) Flow rate: 1.0 mL min−1 with column oven temperature was 40 °C	(S)-FLP possesses the ability to antagonize the calcium channels, assuming no (inter)activity of the (R)-enantiomers.	[47,87]
R/S (±)-methadone (MTD)	Human Plasma	(R)-MTD is less bound to plasma proteins, with AGP being the predominant binding protein. Few studies to date have found the variability in protein binding in the pharmacokinetics of total (R)- and (S)-MTD.	LC-UV Column: Astec® Cyclobond Type I-Beta RSP Mobile phase: ACN: MeOH (75:25 v/v) and 1% TEA Flow rate: 0.6 mL min−1 with column oven temperature was 18 °C	(R)-MTD has been shown to be responsible for most of the analgesic activity. Elevated (R)-MTD levels can increase the risk of respiratory depression, while elevated (S)-MTD levels can increase the risk of severe cardiac arrhythmias.	[42,88]
R/S (±)-trelagliptin (TLG)	Dog plasma	The absolute bioavailability of (R)-TLG was identified to be 128.2%. No chiral bioconversion of (R)-TLG to (S)-TLG was observed.	LC-MS/MS Column: Chiralcel® OX-3R Mobile phase: 10 mmol/L ammonium bicarbonate: ACN Flow rate: 0.6 mL min−1	(R)-TLG is a highly selective and long-acting dipeptidyl peptidase IV inhibitor used for the treatment of type 2 diabetes.	[89]
R/S (±)-fexofenadine (FXF)	Human Plasma And Urine	The highest plasma concentrations of (R)-FXF are attributed to the combination of several carriers capable of chiral discrimination of enantiomers.	LC-MS/MS Column: Chirobiotic® V Mobile phase: MeOH: 7 mM AA, pH 4.25 (97:3 v/v) Flow rate: 0.7 mL min−1	(S)-FXF is a more potent human histamine H1 receptor inverse agonist and shows greater receptor occupancy than (R)-FXF.	[90]
R/S (±)-metoprolol (MET)	Human Plasma	Oral bioavailability of (S)-MET is lower after administration of the pure (S)-enantiomer solution than when the same dose of the (S)-form is administered as a racemate.	LC-ESI-MS; Column: Chirobiotic® V Mobile phase: EtOH: MeOH: AcOH (pH 6.7): TEA (50: 50: 0.225: 0.075 v/v/v/v)	(R)-MET is less effective in reducing the mean arterial blood pressure than (S)- and R/S-MET.	[91,92]
R/S (±)-amlodipine (AML)	Rat Plasma	AML has a low rate of hepatic excretion and is absorbed by liver tissue, due to high tissue affinity, and only afterwards is it redistributed to the systemic circulation. These properties result in peak plasma concentration and longer plasma clearance.	LC-UV Column: Chiralcel® OZ-RH Mobile phase: ACN: H2O (10 mM AA, 0.5% ammonia solution) (95:5 v/v) Flow rate: 0.5 mL min−1	Studies support the conclusion that there is no racemization in vivo. Only (S)-AML possesses vasodilating properties.	[93,94]
R/S (±)-asenapine (ASP)	Rat Plasma	Individual enantiomers of ASP revealed that the (+)-ASP shows a better plasma concentration compared to the (-)-ASP.	HPLC-DAD Column: Lux® Cellulose-1 Mobile phase: ACN: 50 mM ammonium bicarbonate in water (60: 40 v/v) Flow rate: 0.7 mL min−1	Only trans isomers, in the form of an enantiomer racemate R, R and S, and S, have been approved due to receptor binding. (R,R) and (S,S) enantiomers of ASP block behavioral responses mediated by 5-HT2A, 5-HT2C, 5-HT1A, D2, and D1 receptor ligands. The metabolite 11-O-sulfated-asenapine demonstrated an inability to cross the blood–brain barrier.	[95,96,97]
R/S (±)-nimodipine (NMP)	Human Plasma	(−)-(S)-NMP was more rapidly eliminated than the (+)-(R) counterpart.	LC-MS/MS Column: (S,S)-Whelk® O1 Mobile phase: MEOH: H2O (75:25 v/v) Flow rate: 0.1 mL min−1	(−)-(S)-NMP is approximately twice as potent a vasorelaxant as the racemate.	[98]
R/S (±)- butoconazole (BTZ)	Rat Plasma and tissues	The concentration of (+)-BTZ was higher than that of (-)-BTZ, indicating that (+)-BTZ tends to exist in various tissues leading to a slower metabolism. The higher concentration of (+)-BTZ in plasma can cause differences in the enantioselective distribution between (-)- and (+)- BTZ.	LC-ESI-MS/MS Column: Chiralpak® IC Mobile phase: ACN: 10mM aqueous AA (90:10 v/v) Flow rate: 0.6 mL min−1	Commercially enantiopure standards for BTZ were not available. The semi-preparative enantioseparation of the butoconazole was obtained by LC-UV. Some azole enantiomers may exhibit distinct differences in the biological activity.	[99]
R/S (±)-carbinoxamine (CAR)	Rat Plasma	It is currently unclear whether CAR enantiomers have different pharmacodynamic, toxicological, or pharmacokinetic properties. However, stereoselectivity does not occur in absorption and excretion.	LC-MS/MS Column: Chiralpak® ID Mobile phase: ACN: H2O: ammonia solution (90:10:0.1 v/v/v). Flow rate: 0.6 mL min−1	The method used for enantioseparation of CAR can be applied to other antihistamines, such as meclizine, cloperastine, azelastine, and mequitazine. (S)-CAR exert therapeutic action but (R)-CAR is inactive.	[100]
R/S (±)-fluoxetine (FLX)	Human Breast milk	FLX is administered as a racemic mixture, (R)-FLX and (S)-FLX are N-demethylated to (R)-NFLX (norfluoxetine) and (S)-NFLX.	LC-MS/MS Column: RAM-C18-BSA in the first dimension and ChirobioticTM V2 in the second dimension. Mobile phase: 10 mM aqueous AA (pH 6.8): EtOH (20:80 v/v) at 25 °C Flow rate: 0.4 mL min−1	(R)-FLX, (S)-FLX, and (S)-NFLX are equally potent selective serotonin reuptake inhibitors, while (R)-NFLX is 20-fold less potent.	[101]
R/S (±)-linagliptin (LGN)	Human Plasma, urine, and feces	The pharmacokinetics and metabolism of LGN were investigated in healthy volunteers. Unchanged LGN was the most abundant radioactive species in all matrices investigated. The metabolite was identified as a (S)-3-hydroxypiperidine derivative of LGN.	LC-MS/MS Column: Chiralpak® IA Mobile phase: MeOH/EtOH (1:1 v/v) 0.1% tetraethyl amine and MeOH/EtOH (1:1 v/v) with a column temperature of 30 °C. Flow rate: 0.7 mL min−1	LGN is a novel, orally active, highly specific, and potent inhibitor of dipeptidyl peptidase-4 that is currently used for the treatment of type 2 diabetes mellitus.	[102]

AA: Ammonium Acetate; ACN: Acetonitrile; AcOH: Acetic Acid; DAD: Diode-Array Detector; DEA: Diethylamine; ESI: Electrospray Ionization; EtOH: Ethanol; FA: Formic Acid; FD: Fluorescent Detector; HEX: Hexane; Iso: Isopropanol; MeOH: Methanol; MS/MS: Mass Spectrometry; TEA: Triethylamine; UV: Ultraviolet; 2D: Two-dimensional.